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Transcriptional Regulation by the Estrogen-Related Receptors

Annie Tremblay

A thesis submitted to the faculty of Graduate studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy

© Annie Tremblay, November 2009

Department of Biochemistry McGill University Montréal, Québec, Canada

ABSTRACT

The Estrogen-Related receptors (ERRα, ERRβ and ERRγ) are ubiquitous, constitutively active, orphan nuclear receptors and little is known concerning post-translational modifications affecting their transcriptional activity. We observed that the conserved -dependent sumoylation motif (PDSM) within the N-terminal domain of the ERRs represses their transcriptional activity on compound promoters via a synergy control mechanism. We also identified inhibitor of activated stat y (PIASy), a SUMO E3 ligase, as a new interacting partner of ERRα which promotes the sumoylation of ERRα and represses its transcriptional activity in a PDSM-dependent manner. Furthermore, by showing that an ERRα phosphoS19-specific antibody, but not a polyclonal ERRα antibody with a minimal affinity for phosphorylated S19, allows detection of endogenous sumoylated ERRα in mouse liver extract, we confirmed that the ERRα phospho-sumoyl switch is functional in vivo. ERRα is highly expressed in the kidney, but its role in this organ is unknown. Therefore, we used a combination of physiological studies, expression and genome-wide location analysis to explore the role of ERRα in the kidney. A defect in sodium and potassium was observed in the ERRα null mice, which correlated with the ERRα renal transcriptional program comprising key sodium and potassium channels. Furthermore, telemetry monitoring revealed that the ERRα null mice display a significantly reduced blood pressure at nighttime and this correlated the renal transcriptional program of ERRα comprising involved in blood pressure regulation. In addition, we identified the Renin-Angiotensin pathway genes as direct ERRα target genes in the kidney. These results identify a role for ERRα in renal sodium/potassium handling, intra-renal renin-angiotensin pathway, blood pressure regulation and possibly .

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RÉSUMÉ Les récepteurs reliés aux récepteurs de l’estrogène (ERRs) sont ubiquitaires et constitutivement actifs et le rôle joué par les modifications post- traductionnelles sur leur activité transcriptonnelle est peu connu. Nous avons démontré qu’un motif consensus de sumoylation phospho-dependente (PDSM) situé dans le domaine N-terminal diminue l’activité transcriptionnelle des ERRs sur des promoteurs à élements de réponses multiples grâce à un mécanisme de contrôle de la synergie. Nous avons aussi établi que les ERRs intéragissent avec la E3-SUMO-ligase PIASy et que cette dernière promouvoit la sumoylation du ERRα de manière phospho-dépendante. De plus, en montrant que la forme sumoylée endogène de ERRα dans de l’extrait de foie de souris n’était détectable qu’avec un anticorps spécifique dirigé contre la sérine 19 phoshorylée nous avons confirmé la validité de l’interrelation phosphorylation-sumoylation dans un contexte physiologique in vivo. Même si le haut niveau d’expression de ERRα dans les reins est reconnu, son rôle dans cet organe est inconnu. Nous avons donc utilisé une approche combinatoire d’analyses physiologiques, d’expression génique et d’identification de sites spécifiques de liaison à l’ADN au niveau génomique afin d’explorer plus avant le rôle physiologique de ERRα dans le rein. Nous avons observé que les souris knock-out pour le gène de ERRα présentent un problème au niveau de l’homéostasie sodique et potassique corrèlant directement avec le programme transcriptionnel rénal qui comprends plusieurs canaux sodiques et potassiques importants. De plus, la mesure des paramètres cardiovasculaires par télémétrie a révélé que les souris knock-out pour le gène du ERRα ont une pression sanguine nocturne plus faible qui corrèle avec le programme transcriptionnel comprenant plusieurs gènes influençant la pression sanguine. Nous avons aussi identifié les promoteurs de certains gènes composant le système rénine-angiotensine comme gènes cibles potentiels de ERRα dans les reins. Nos résultats suggèrent une implication du ERRα dans le contrôle de la pression sanguine basale et possiblement dans l’hypertension.

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ACKNOWLEDGEMENTS

I am thankful to my supervisor, Vincent Giguère, for giving me the opportunity to work in his laboratory. I am sincerely grateful not only for the advice, constructive criticism and directions, but especially for the freedom and latitude for research, which adequately prepared me for the future challenges of independent research.

I wish to thank all the members of the laboratory, past and present, for the interesting discussions, scientific or not, which rendered more pleasant spending a lot of time in the laboratory, and especially to my bench neighbor Brian for his friendship, all the laughs as well as scientific collaboration. I also want to thank Majid and Geneviève for their suggestions and encouragements, their positive spirit and friendship.

I also wish to express gratitude to our skillful technician Cathy for generating the in vivo ChIP-on-chip lists and sharing some of her precious mouse tissue collection. I want to extend my gratitude to Anna, Yoshi and other past members of the lab, for passing on their technical expertise of the molecular biology basics.

I am grateful to the members of my advisory committee, Michel Tremblay and Xiang-Jiao Yang, for their valuable advice and suggestions. I also want to thank Serge for the interesting discussions and technical advice in the SUMO project. I am thankful to Carlo for his help and interest into my mice projects, as well as to our collaborators Tim Reudelhuber and Chantale for the telemetry and plasma renin experiments.

Last but not least, I wish to express my profound gratitude to my family and friends for their endless and priceless support along this journey and, most of all, for believing in me.

I am also thankful to Canderel and CIHR for funding.

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LIST OF PUBLICATIONS

Arising From work of the thesis

- Tremblay AM, Wilson BJ, Yang XJ and Giguère V. Phosphorylation- Dependent Sumoylation Regulates Estrogen-Related -α and -γ Transcriptional Activity through a Synergy Control Motif. (2008) Mol Endocrinol 22(3): 570–584. First Published Online December 6, 2007.

- Tremblay AM, Dufour CR, Ghahremani M, Reudelhuber TL and Giguère V. Physiological Genomics Identifies Estrogen-Related Receptor α as a Regulator of Renal Sodium and Potassium Homeostasis and the Renin-Angiotensin Pathway. Mol Endocrinol. In Press. Epub November 9th 2009.

Other publications

- Grégoire S, Tremblay AM, Xiao L, Yang Q, Ma K, Nie J, Mao Z, Wu Z, Giguère V and Yang XJ. Control of transcriptional activity by coordinated phosphorylation and sumoylation. (2006) J Biol Chem 281, 4423 –4433.

- Tremblay AM and Giguère V. The NR3B subgroup: an ovERRview. (2007) Signaling 5, e009. Review.

- Wilson BJ, Tremblay AM, Giguère V. An Switch Modulates the Function of Estrogen Related Receptor α. To be submitted shortly.

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CONTRIBUTIONS OF AUTHORS

Vincent Giguère: Supervised all aspects of the projects and edited the manuscripts.

Xiang-Jiao Yang: Provided the wild-type Ubc9 and SUMO and E3 ligases plasmids; Discussed the results and revised the manuscript.

Timothy L. Reudelhuber: Generated the telemetry and plasma renin concentration results. Discussed these results and revised the manuscript.

Brian J. Wilson: Generated the following serine mutants: ERRα S19D, KR/S19A, KR/S19D, ERRγ S45A, S45D, KR/S45A, KR/S45D; performed the reporter gene assays presented in panels 3G, 3H, 5D and 6D; immortalized my primary ERRα null MEFs by SV40 large T viral infection.

Catherine R. Dufour: Performed the ChIP-on-chip on mouse kidneys and target validations comprising those shown in panel 5A; provided the frozen circadian kidneys.

Majid Ghahremani: Technical assistance and qRT-PCR validation.

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TABLE OF CONTENTS

ABSTRACT ...... ii RÉSUMÉ ...... iii ACKNOWLEDGEMENTS ...... iv LIST OF PUBLICATIONS ...... v CONTRIBUTIONS OF AUTHORS...... vi TABLE OF CONTENTS ...... vii LIST OF FIGURES...... x LIST OF TABLES...... xii LIST OF ABBREVIATIONS...... xiii CHAPTER I: Literature Review...... 1 1.1 The Nuclear Receptor Superfamily ...... 1 1.1.1 Classical Mode of Action and Hormone Response Elements ...... 1 1.1.2 Nomenclature...... 5 1.1.3 Nuclear Receptor Anatomy ...... 6 1.1.3.1 The A/B domain: N-terminal domain ...... 7 1.1.3.2 The C domain: DNA-binding domain ...... 8 1.1.3.3 The E/F domain: Ligand-binding domain...... 10 1.2 The NR3B subgroup: Estrogen-Related Receptors...... 12 1.2.1 Discovery and Cloning ...... 12 1.2.2 Regulation of Transcriptional Activity...... 14 1.2.2.1 DNA ...... 15 1.2.2.2 Coactivators ...... 17 1.2.2.3 ...... 20 1.2.2.4 Synthetic ligands ...... 21 1.2.2.5 Post-translational modifications...... 24 1.2.3 Tissue Expression of the ERRs...... 25 1.2.4 Target Genes of the ERRs ...... 26 1.2.5 Biological Roles of the ERRs ...... 28 1.2.5.1 ERRs in energy metabolism: role in and heart failure ...... 28 1.2.5.2 ERRs in cancer...... 32 1.2.5.3 ERRα in bone ...... 33 1.2.5.5 ERRβ in development...... 35 1.3 Post-Translational Modifications in Transcriptional Regulation...... 36 1.3.1 Phosphorylation...... 36 1.3.1.1 General mechanism...... 36 1.3.1.2 Phosphorylation of Nuclear Receptors ...... 37 1.3.1.3 Phosphorylation of the ERRs...... 39

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1.3.2 Sumoylation ...... 40 1.3.2.1 General mechanism...... 43 1.3.2.2. Biological Roles of sumoylation...... 47 1.3.3 Sumoylation in Transcriptional Regulation...... 48 1.3.3.1 Sumoylation of Nuclear Receptors and coregulators...... 49 1.3.3.3 Sumoylation, Synergy control and DNA binding...... 53 1.3.4 Interplay between SUMO and other post-translational modifications ...... 57 1.4 Nuclear receptors and blood pressure regulation ...... 60 1.4.1 General principles of blood pressure regulation ...... 61 1.4.2 Transcriptional regulation of Renin and nuclear receptors...... 64 1.4.3 Insights from mouse models ...... 67 1.5 Goals of the research ...... 69 BIBLIOGRAPHY...... 70 Chapter II: Phosphorylation-dependent sumoylation regulates ERRα and γ transcriptional activity through a synergy control motif...... 94 PREFACE ...... 94 ABSTRACT ...... 95 INTRODUCTION ...... 96 RESULTS...... 100 DISCUSSION...... 109 MATERIALS AND METHODS...... 112 ACKNOWLEDGEMENTS...... 118 REFERENCES ...... 135 Chapter III: Physiological genomics identifies estrogen-related receptor α as a regulator of renal sodium and potassium homeostasis and the renin- angiotensin pathway...... 140 PREFACE ...... 140 ABSTRACT ...... 141 INTRODUCTION ...... 142 RESULTS...... 144 DISCUSSION...... 150 MATERIALS AND METHODS...... 155 ACKNOWLEDGEMENTS...... 159 REFERENCES ...... 194 Chapter IV: General Discussion ...... 197 4.1 Context Summary...... 197 4.2 ERRα sumoylation and synergy control: physiological relevance ...... 198 4.2.1 The ERRα phospho-sumoyl switch to fine tune ERRα expression levels ... 198 4.2.2 Identification of additional ERRα synergy control responsive promoters ... 199 4.2.3 Breaking the code of the ERRα NTD...... 200 4.2.4 Signaling pathways targeting ERRα Ser19: insights from cancer cell lines 202 4.3 Physiological cues modulating ERRα expression levels may involve the PDSM: potential biological roles of the phospho-sumoyl switch ...... 205 4.3.1 Fine tuning the metabolic rate via regulation of ERRα expression...... 205 4.3.2 Cardiac hypertrophy and heart failure ...... 206 4.3.3 Cell proliferation and cell progression ...... 206

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4.3.4 -regulated ERRα expression...... 207 4.4 The similarity between ERRs and PPARγ regulation ...... 208 4.5 ERRα in blood pressure regulation...... 209 4.6 Conclusion: ERRα as a therapeutic target...... 214 REFERENCES ...... 216 Chapter V: Contribution to original research...... 219 APPENDIX I...... 220 APPENDIX II ...... 224

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LIST OF FIGURES

Chapter I

Figure 1.1: Model of the Nuclear Receptor modes of action ...... 2 Figure 1.2: Typical NR anatomy ...... 7 Figure 1.3: The regions of the NR DBD...... 8 Figure 1.4: Crystal structure of monomer and dimer DNA-bound NR DBD...... 9 Figure 1.5: Model crystal structure of NR LBD inactive and active forms ...... 11 Figure 1.6: Schematics of the major ERR isoforms ...... 14 Figure 1.7: DNA binding modes of the ERRs ...... 16 Figure 1.8: Nuclear receptors typical LBD conformations...... 22 Figure 1.9: The sumoylation pathway ...... 44 Figure 1.10: The Synergy control hypothesis...... 55 Figure 1.11: Interplay between SUMO and other modifications ...... 59 Figure 1.12: The heart-kidney connection ...... 62 Figure 1.13: Graphical representation of the systemic RAAS...... 63

Chapter II

Figure 2.1: ERRα and γ contain consensus sites for sumoylation and are modified by SUMO2...... 120 Figure 2.2: The NTDs of ERRα and γ harbor the main SUMO attachment sites .122 Figure 2.3: Arginine substitution at the major sumoylation site of ERRα and γ increases their transcriptional activity ...... 124 Figure 2.4: ERRα subcellular localization, DNA binding and interaction with PGC- 1α are not affected by the K14R mutation...... 126 Figure 2.5: Phospho-mimetic mutants display elevated sumoylation and reduced transcriptional activity ...... 128 Figure 2.6: PIASy enhances ERRα sumoylation in a phosphorylation-dependent manner ...... 130 Figure 2.7: Coupled phosphorylation and sumoylation of ERRα in mouse liver ...132 Figure 2.S1: Antibodies-antigenic peptide affinity comparison ...... 134

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Chapter III

Figure 3.1: Blood pressure parameters and heart rate in ERRα null mice...... 163 Figure 3.2: ERRα target genes are enriched for specific biological functions ...... 165 Figure 3.3: Genome-wide identification of ERRα-bound segments within extended regions in mouse kidneys ...... 167 Figure 3.4: profiling in ERRα null kidneys ...... 169 Figure 3.5: Regulation of genes involved in the RAAS by ERRα ...... 171 Figure 3.S1: Plasma renin and aldosterone concentrations in wild-type and ERRα null mice...... 173

Appendix I

Figure A.1: Additional mouse endogenous promoters displaying a synergy control effect ...... 221 Figure A.2: ERRα in the cell cycle: insights from the kidney microarray analysis222 Figure A.3: Plasma renin levels in ERRα wild-type and null mice after Angiotensin II administration...... 223

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LIST OF TABLES

Chapter I

Table 1.1: The mammalian Nuclear Receptor superfamily...... 4 Table 1.2: ERRα target genes identified individually...... 26 Table 1.3: Nuclear receptors sumoylation summary...... 50

Chapter III

Table 3.1: Blood and urinary parameters in wild type and ERRα null mice...... 160 Table 3.2: ERRα ChIP-on-chip target genes associated with Na+ and/or K+ transport ...... 161 Table 3.S1: List of ERRα ChIP-on-chip target genes in mouse kidney ...... 174 Table 3.S2: List of the downregulated genes in the ERRα null kidney...... 184 Table 3.S3: List of the upregulated genes in the ERRα null kidney...... 190

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LIST OF ABBREVIATIONS

Å Ångström A or Ala alanine ACTR activator of thyroid and receptors AF-1 activation function-1 AF-2 activation function-2 Agt angiotensinogen AIB1 amplified in breast cancers-1 Ang angiotensin ANS autonomic nervous system Aos1 SUMO-activating subunit 1 AP-1 activator protein-1 AR AREs androgen response elements ATP adenosine triphosphate BMD bone mass density BMI body mass index bp BP blood pressure cAMP cyclic adenosine monophosphate CAR constitutive androstane receptor CARM1 -associated arginine methyltransferase-1 CBP CREB-binding protein CDK cyclin-dependent kinase ChIP chromatin immunoprecipitation CNRE cAMP and negative response element CoA coactivator CoR CoRNR corepressor nuclear receptor box CREB cAMP response element-binding CTE carboxy-terminal extension D or Asp aspartic acid

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DAPI 4’,6-diaminido-2-phenylindole DBD DNA-binding domain DES diethystilbestrol DMSO dimethyl sulfoxide DNA deoxyribonucleic acid DR direct repeat ECF extracellular fluid EGF epidermal growth factor EGFR epidermal growth factor receptor ER ErbB2 Human Epidermal growth factor Receptor 2 (HER-2) ERE estrogen response element ERK extracellular signal-regulated kinase ERR estrogen-related receptor ERRE estrogen-related response element F FXR GFP green fluorescent protein GR GR glucocorticoids receptor GRIP1 glucocorticoid receptor interacting protein 1 GST glutathione-S-transferase HA hemagglutinin HAT histone acetyltransferase HDAC histone deacetylase HEK human embryonic kidney HRE hormone response element HSF-1 1 I isoleucine IFN interferon IR inverted repeat K or Lys lysine kDa kilodalton L LBD ligand binding domain

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LPS lipopolysaccharide LUC luciferase LXR M methionine MAPK mitogen-activated protein kinase MCAD medium-chain acyl-CoA dehydrogenase MEFs mouse embryonic fibroblasts MR mineralocorticoid receptor NDSM negatively charged -dependent sumoylation motif NF-kB nuclear factor-kB NMR nuclear magnetic resonance NR nuclear receptor NTD aminoterminal domain OHT hydroxytamoxifen OXPHOS oxidative phoshorylation p/CAF p300/CBP-associated factor PAC plasma aldosterone concentration Pal palindrome PDSM phosphorylation-dependent sumoylation motif PGC-1 PPAR-g coactivator 1 PIAS protein inhibitor of activated STAT PKA protein kinase A PKC protein kinase C PML promyelocytic leukemia gene PNRC proline rich nuclear receptor coactivator PPAR peroxisome proliferator–activated receptor PR PR progesterone receptor PRC plasma renin concentraion PRMT1 protein arginine methyltransferase 1 PTM post-translational modification RAAS renin-angiotensin-aldosterone system RAR Ren1 renin RIP140 receptor interacting protein 140

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RLU relative luciferase units ROR retinoic acid receptor-related orphan receptor ROS reactive oxygene species RT-PCR reverse transcriptase polymerase chain reaction RXR S or Ser serine SAPK stress activated protein kinase SC synergy control SCC synergy control complex SCF synergy control factor SENP SUMO-specific proteases SERM selective ER modulators SF-1 steroidogeninc factor -1 SHP small hetorodimer partner SIM SUMO interacting motif siRNA short interfering RNA SMRT silencing for retinoic acid and thyroid hormone receptors SNS sympathetic nervous system SRC steroid receptor coactivator STAT signal transducer and activator of SUMO small -like modifier SV40 simian virus 40 T-rep transrepression TAC Trans-aortic constriction TBL-1 transducin b–like protein TCA tricarboxilic acid THRE thyroid hormone response elements TIF2 transcriptional intermediary factor 2 TK thymidine kinase TR thyroid receptor UAS upstream activating sequence Uba2 SUMO acivating enzyme Ubc9 SUMO conjugating enzyme V valine VDR

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CHAPTER I: Literature Review

1.1 The Nuclear Receptor Superfamily

The human nuclear receptor (NR) superfamily is composed of DNA-binding transcription factors that orchestrate the transcriptional responses crucial for a diverse array of physiological and pathophysiological processes, ranging from embryonic development to adult homeostasis. The NRs were originally defined as a group of structurally related transcription factors controlling gene expression in response to small lipophilic ligands which can penetrate the plasma membrane of the cell and enter the cytoplasm and the nucleus (Evans 1988). The NRs can respond to various ligands of endocrine, paracrine, or intracrine origin as well as several synthetic ligands and xenobiotics.

1.1.1 Classical Mode of Action and Hormone Response Elements

In humans, the NR superfamily comprises 48 receptors encoded by different genes. The classical model of the NR mode of action predominantly applies to the classical steroid hormone receptors (SHRs) which bind steroid hormones like estrogen (ER), androgen (AR), progesterone (PR), glucocorticoids (GR) and mineralocorticoids (MR). In the absence of their cognate ligand, the aporeceptors are bound to chaperones (the heat-shock (HSPs)) in the cytosol or in the nucleus for ER. Ligand binding induces the release of the HSPs as well as receptor conformational changes, homodimerization and translocation into the nucleus where the holoreceptors bind to DNA on a specific hormone response element (HRE). The HRE of SHRs is composed of an inverted palindrome of two

1 Chapter I – Literature Review hexameric core motifs AGAACA (referred to as a half-site) separated by a three- nucleotide spacer, except for ER for which the half-site sequence is AGGTCA (Truss and Beato 1993; Beato, Herrlich et al. 1995; Zilliacus, Wright et al. 1995). The DNA-bound SHR then recruits coregulator proteins and modulates the expression of its target genes (Figure 1.1).

Figure 1.1: Model of the Nuclear Receptor modes of action.

1- Upon entrance of a lipophilic hormone, the steroid nuclear receptors are released from the heat shock proteins (HSP), dimerize, enter the nucleus and bind their cognate DNA response elements in order to regulate gene expression. 2- Alternatively, the DNA-bound NR dimer modulates target gene expression upon ligand binding-induced coregulator exchange. 3- In addition, NRs can act as heterodimers or 4- monomers, and 5- their activity as well as that of their coregulators can be modulated by phosphorylation induced by signaling cascades originating from the plasma membrane.

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On the other hand, the classical non-steroidal NRs, which respond to non- steroid ligands such as thyroid hormone (T3R) as well as vitamin D (VDR) and retinoic acid (RAR), are not bound to HSPs in the cytosol. It is believed that, in the absence of ligand, these NRs are already bound to their DNA response elements along with a corepressor (or a corepressor complex) inducing a basal repression of the target gene. Upon ligand binding, dissociation of the corepressor complex occurs followed by the recruitment of a coactivator (or a coactivator complex) allowing transcriptional activation (Chen and Evans 1995; Horlein, Naar et al. 1995). This regulatory mechanism is called coregulator exchange (Glass and Rosenfeld 2000). The non-steroidal NRs can bind their response elements as homodimers, but preferentially bind DNA as heterodimers with the promiscuous partner retinoid X receptor (RXR), which responds to 9-cis retinoic acid, an oxidized form of vitamin A (Kersten, Kelleher et al. 1995). The different RXR heterodimers bind to two AGGTCA half-sites arranged in various configurations (inverted or everted palindrome but preferentially direct repeats (DR)) with variable spacing ranging from 1 to 5 nucleotides (Table 1.1).

The HRE configuration and nucleotide spacing determines the specificity for the various RXR heterodimers (Glass 1994; Mangelsdorf and Evans 1995). In addition, DR0 and half-sites with broader spacing can also act as binding sites for several NRs (Kato, Sasaki et al. 1995; Aranda and Pascual 2001). The NRs can also accommodate some sequence variability from the perfect consensus half- sites, and these slight variations further participate in the binding selectivity and discrimination between NRs (Mader, Leroy et al. 1993).

As opposed to the classical NRs for which the ligands were known before the receptor was identified, the RXRs and the other members of this superfamily were identified before their ligand, and were therefore initially categorized as orphan receptors. This dichotomy in NR classification (liganded versus orphans) quickly became confusing (and conceptually inadequate) with the discovery of the naturally occurring agonists associated with some of the orphans, such as fatty acids for the peroxisome proliferator-activated receptors (PPARs), 9-cis retinoic

3 Chapter I – Literature Review acid for the RXRs, oxysterols for the liver X receptors (LXRs) and bile acids for the farnesoid X receptors (FXRs) (Gottlicher, Widmark et al. 1992; Kersten, Pan et al. 1995; Janowski, Willy et al. 1996; Makishima, Okamoto et al. 1999; Parks, Blanchard et al. 1999). These (and others) are referred to as the adopted orphan receptors (Table 1.1). There is no consensus as to whether the remaining orphans possess a yet unknown associated endogenous ligand or not (Benoit, Cooney et al. 2006). The hunt for endogenous ligands continues, as the discovery of endogenous ligands for the orphans would considerably help to better understand their physiological roles and would facilitate the development of novel therapeutic compounds (Xu and Li 2008).

Table 1.1: The mammalian Nuclear Receptor superfamily.

Group Official Name Category Major ligand DNA binding sites Mode of DNA I NR1A1name THRα Nby- SHRligand Thyroid hormone Pal,type DR-4, IP Hbinding status NR1A2 THRβ N-SHR Thyroid hormone Pal, DR-4, IP H

NR1B1 RARα N-SHR Retinoic acid Pal, IP, DR-2, DR-5 H NR1B2 RARβ N-SHR Retinoic acid Pal, IP, DR-2, DR-5 H NR1B3 RARγ N-SHR Retinoic acid Pal, IP, DR-2, DR-5 H

NR1C1 PPARα adopted TZDs; fatty acids DR-1 H NR1C2 PPARβ/δ adopted TZDs; fatty acids DR-1 H NR1C3 PPARγ adopted TZDs; fatty acids DR-1 H

NR1D1 Rev-Erbα orphan unknown DR-2, HS M, D NR1D2 Rev-Erbβ orphan unknown DR-2, HS M, D

NR1F1 RORα orphan unknown HS M NR1F2 RORβ orphan unknown HS M NR1F3 RORγ orphan unknown HS M

NR1H2 LXRβ adopted Oxysterols DR-4 H NR1H3 LXRα adopted Oxysterols DR-4 H NR1H4 FXR adopted Bile acids DR-4, IR-1 H

NR1I1 VDR N-SHR Vitamin D DR-3, IP-9 H NR1I2 PXR adopted Pregnanes DR-3 H NR1I3 CARβ adopted Androstanes DR-5 H NR1I4 CARα adopted Androstanes DR-5 H

II NR2A1 HNF4α adopted Fatty acids DR-1, DR-2 D NR2A2 HNF4γ adopted Fatty acids DR-1, DR-2 D NR2A3 HNF4β adopted Fatty acids DR-1, DR-2 D

NR2B1 RXRα adopted 9-cis retinoic acid Pal, DR-1 D

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NR2B2 RXRβ adopted 9-cis retinoic acid Pal, DR-1 D NR2B3 RXRγ adopted 9-cis retinoic acid Pal, DR-1 D

NR2C1 TR2α orphan unknown DR-1 to DR5 D, H NR2C2 TR2β orphan unknown DR-1 to DR5 D, H

NR2E1 TLX orphan unknown DR-1, HS M, D

NR2F1 COUP-TF2 orphan unknown Pal, DR-5 D, H NR2F2 COUP-TF orphan unknown Pal, DR-5 D, H NR2F6 EAR2 orphan unknown Pal, DR-5 D, H

III NR3A1 ERα SHR Estradiol Pal D NR3A2 ERβ SHR Estradiol Pal D

NR3B1 ERRα orphan unknown Pal, HS M, D, H NR3B2 ERRβ orphan unknown Pal, HS M, D, H NR3B3 ERRγ orphan unknown Pal, HS M, D, H

NR3C1 GR SHR Glucocorticoids Pal D NR3C2 MR SHR Mineralocorticoids Pal D NR3C3 PR SHR Progestins Pal D NR3C4 AR SHR Androgens Pal D

IV NR4A1 NUR77 orphan unknown Pal, DR-5 M, D, H NR4A2 NURR1 orphan unknown Pal, DR-5 M, D, H NR4A3 NOR1 orphan unknown Pal, DR-5 M, D, H

V NR5A1 SF1 adopted phospholipids HS M NR5A2 LRH-1 adopted phospholipids HS M

VI NR6A1 GCNF orphan unknown DR-0 D

0 NR0B1 DAX-1 atypical unknown no DBD - NR0B2 SHP atypical unknown no DBD - Adapted from (Aranda and Pascual 2001) and (Benoit, Cooney et al. 2006). SHR, steroid ; N-SHR, non-steroidal hormone receptor; Pal, palindrome; DR, direct repeat; IP, inverted palindrome; HS, half-site; M, monomer; D, homodimer; H, heterodimer; TZD, thiazolidinedione.

1.1.2 Nomenclature

Originally, the NRs were cloned by different groups and from different species and were, consequently, named by the groups which found them. This produced considerable confusion due to the discrepancies in the naming process (Committee 1999). By 1999, the entire family had been described in humans.

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Upon the completion of the sequence, a uniform nomenclature was established based on grouping by sequence identity and borrowing from the existing nomenclature for the cytochrome p450 superfamily (Nebert, Adesnik et al. 1987). NR nomenclature was normalized as follows: First, the NRs were clustered into groups and subgroups according to their sequence identity within the C domain and/or within the E domain. Subfamily’s were assigned a relevant name based on ligand or on the phylogenetic proximity to a liganded receptor. Then, for members of a subfamily composed of structurally related receptors, a Greek letter was assigned to each isotype encoded by a different gene. The isoforms of a given isotype, generated either by alternative splicing or differences in promoter usage, were appended an Arabic numeral.

For the official nomenclature, the gene group number (0 to 6) is given the prefix NR and is followed by capital letters for the group and with a second Arabic numeral for individual genes (refer to Table 1.1).

1.1.3 Nuclear Receptor Anatomy

All members of the NR superfamily possess similar typical domain architecture. The NRs are composed of five modular domains which fold independently of one another and play distinct although interrelated roles (Giguère, Hollenberg et al. 1986; Green and Chambon 1987; Bain, Heneghan et al. 2007). The folding particularities and roles of each domain are described below. It is now well recognized that NRs are subjected to allosteric regulation by ligands, DNA and inter-domain interactions (Gronemeyer and Bourguet 2009). The domains of the NRs are the N-terminal domain (NTD; A/B domain) containing the ligand-independent activation function-1 (AF-1) domain, the DNA binding domain (DBD; C domain), the hinge region (D domain) and the ligand- binding domain (LBD; E/F domain) in which the activation function-2 (AF-2) is embedded (Figure 1.2) (Giguère 1999; Kumar and Thompson 1999; Owen and Zelent 2000; Renaud and Moras 2000; Aranda and Pascual 2001).

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Figure 1.2: Typical NR anatomy. The NR domains are modular and fold independently of one another. NTD, N-terminal domain; DBD, DNA-binding domain; H, hinge region; LBD, ligand-binding domain; AF-2, activation function-2.

1.1.3.1 The A/B domain: N-terminal domain

The N-terminal domain is a hypervariable region and, consequently, represents the least conserved region among NRs from a sequence point of view (Giguère, Tini et al. 1994; Chambon 1996). However, the role and the structural properties of the region are well conserved among the families. The NTD of many NRs, mostly the steroid receptors, contains a ligand-independent AF-1 responsible for the ligand-independent positive transcriptional modulation in response to phosphorylation (Weigel and Zhang 1998).

The A/B domain can also play a dual role for the transcriptional activity of the NRs. Indeed, the NTD of some NRs harbors determinants for both activation and repression (Pearce and Yamamoto 1993; Iniguez-Lluhi, Lou et al. 1997). In general, the A/B domains are poorly structured. They display a partially folded state, called the molten-globule-like structure, and the native structure of the NTD is induced allosterically through post-translational modifications, interaction with coregulator proteins or through interaction of the DBD with DNA. Therefore, the transcriptional modulatory role of the NTD is expected to be promoter- or cell context-dependent (Lavery and McEwan 2005; McEwan, Lavery et al. 2007).

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1.1.3.2 The C domain: DNA-binding domain

The DBD of the NRs is a typical feature of the superfamily. Along with the LBD, it is the most conserved domain among the NRs and these domains have played a fundamental role for the discovery and primary characterization of the members of the family (Glass 1994; Evans 2005). The NRs can bind DNA either as monomers, homodimers or heterodimers. The DBD confers the DNA binding specificity of a NR for its cognate response element via two key regions, the N- terminal zinc fingers and the C-terminal extension (CTE) (Figure 1.3) (Glass 1994; Giguère 1999).

Figure 1.3: The regions of the NR DBD.

Schematics of the linear organization of the ERRα DBD sections with Zn (zinc) fingers (containing the P-box and D-box) and the c-terminal extension (CTE).

In each of the two zinc fingers, four cysteine residues coordinate the zinc ion. The fingers are implicated in the formation of both protein-DNA (via the ‘‘P- box’’) and protein-protein interactions (via the ‘‘D-box’’) (Freedman, Luisi et al. 1988; Hard, Kellenbach et al. 1990; Freedman 1992; Baumann, Paulsen et al. 1993). In addition to their role in DNA response element specificity, the two modules are also involved in DNA-induced dimerization (Perlmann, Rangarajan et al. 1993; Zechel, Shen et al. 1994; Moraitis and Giguère 1999). The CTE stabilizes the binding to DNA by making contact with the 5’ extension

8 Chapter I – Literature Review of the DNA response element. The CTE is therefore crucial for the NRs that bind DNA as monomers, such as the RORs and the ERRs (Table 1.1; Figure 1.4A) (Wilson, Paulsen et al. 1992; Lee, Kliewer et al. 1993; Giguère, McBroom et al. 1995; Gearhart, Holmbeck et al. 2003). For dimers, the CTE also participates in the selectivity between DNA response elements with various spacings, and is therefore implicated in the homo- or heterodimerization process (Lee, Kliewer et al. 1993; Rastinejad, Perlmann et al. 1995; Zhao, Khorasanizadeh et al. 1998).

The first crystal structures of a DNA-bound DBD to be resolved were those of GR, RARβ and ERα (Figure 1.4B) (Luisi, Xu et al. 1991; Katahira, Knegtel et al. 1992; Schwabe, Chapman et al. 1993). Since then, these receptors are often referred to as the typical models of dimeric DNA binding for the NRs. In addition to the two zinc finger modules, the NR DBD also contains two perpendicular α-helices. Helix 1 contacts the major groove of DNA via the ‘‘P- box’’ while the second helix and the ‘‘D-box’’ mediate dimerization (Figure 1.3 and 1.4) (Glass 1994; Giguère 1999; Bain, Heneghan et al. 2007).

Figure 1.4: Crystal structure of monomer and dimer DNA-bound NR DBD.

A, Crystal structure of DNA-bound ERRβ DBD monomer (PDB 1LO1) (Gearhart, Holmbeck et al. 2003). CTE: C-terminal extension B, Crystal structure of DNA-bound ERα DBD dimer (PDB1HCQ) (Schwabe, Chapman et al. 1993).

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1.1.3.3 The E/F domain: Ligand-binding domain

There are two principal structural regulatory determinants within the LBD of NRs, the ligand binding pocket and the AF-2 function (Moras and Gronemeyer 1998; Aranda and Pascual 2001; Bain, Heneghan et al. 2007). The first crystal structure of a nuclear receptor LBD was that of RXR (Bourguet, Ruff et al. 1995). The LBD of the NRs generally consists of 200 to 250 amino acids and folds into a globular domain composed of 11-13 α-helices (and one β-turn) forming three antiparallel helical sheets. Therefore, the tertiary structure of the NR LBD is often described as an α-helical sandwich, and contains the ligand binding pocket at its core. The AF-2 function relies on a conserved sequence stretch, called AF-2 (see Figure 1.5), which is located on helix 12. This helix is therefore referred to as the AF-2 helix. The AF-2 helix is mobile and is responsible for the inactive versus active conformation adopted by the LBD, mostly in response to ligand binding (Figure 1.5). In the aporeceptor, the AF-2 helix is found in an extended conformation, located away from the core LBD and preventing the formation of the coactivator binding hydrophobic cleft (Wurtz, Bourguet et al. 1996; Johnson, Wilson et al. 2000). Upon agonist ligand binding, the LBD adopts a more compact structure where the AF-2 helix is repositioned closer to the LDB to form the hydrophobic cleft (mediating AF-2 function) destined for coactivator interaction (Feng, Ribeiro et al. 1998; Shiau, Barstad et al. 1998).

Interaction of the coactivator with the NR involves hydrogen bonds between the LxxLL motif of the coactivator and the hydrophobic cleft. In addition, two highly conserved residues of the LBD on each side of the groove (a positively charged lysine residue on helix 3 and a negatively charged glutamic acid residue on helix 12) form a charge clamp which locks the coactivator in the groove of the holoreceptor (Nolte, Wisely et al. 1998; Perissi, Staszewski et al. 1999; Wu, Chin et al. 2003). Different ligands induce slightly different conformations, and consequently, the type of ligand can influence the coregulator binding specificity (Norris, Joseph et al. 2009).

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Figure 1.5: Model crystal structure of NR LBD inactive and active forms.

Left: unliganded RXRα LBD (inactive with the AF2 helix extended); right: liganded RARγ LBD (active with the coactivator binding pocket formed) Used with permission from (Wurtz, Bourguet et al. 1996) and presented with permission as reprinted in (Aranda and Pascual 2001).

Alternatively, the binding of antagonists provokes different rearrangements of the LBD structure that interferes with coactivator binding or favors the binding of a corepressor in the hydrophobic cleft instead of a coactivator. Antagonist ligands can inhibit coactivator binding by several mechanisms. They can prevent helix 12 from folding back into the active conformation and thus prevent the formation of the coactivator binding groove, or they can induce the binding of helix 12 into the coactivator binding cleft causing steric interference for coactivator binding (Brzozowski, Pike et al. 1997; Shiau, Barstad et al. 1998; Xu, Stanley et al. 2002). Alternatively, antagonists can stabilize the LBD structure to form a larger hydrophobic cleft favoring the binding of corepressors over coactivators (Xu, Stanley et al. 2002). In addition to its role in ligand binding, the LBD, also mediates dimerization of the LBD via a constitutive dimerization interface. Along with the dimerization interface of the

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DBD, LBD dimerization stabilizes DNA binding of dimers (Perlmann, Umesono et al. 1996; Jiang, Lee et al. 1997).

The interaction of two NR dimers into a tetramer is another mechanism for the inactivation of NRs, although this mechanism is not well characterized. In this configuration, the formation of a functional hydrophobic coactivator groove is prevented. Tetramers have been proposed to constitute the storage form of free NRs. Upon agonist binding, the tetramer is separated into dimers, ready for DNA binding, coactivator cleft formation and coactivator interaction (Gampe, Montana et al. 2000; Figueira, Dias et al. 2006). The formation of NR tetramers on DNA has also been shown to induce looping (Yasmina, Yeunga et al. 2004). This mode of regulation by oligomerization of the NRs is less studied than the standard monomeric or dimeric forms but recent advances suggest that oligomerization may constitute a more widespread mechanism in the NR superfamily than previously thought (Gampe, Montana et al. 2000) (Fischer, Dias et al. 2003; Gu, Morgan et al. 2005; Mengeling, Pan et al. 2005; Figueira, Dias et al. 2006; Tocchini-Valentini, Rochel et al. 2009).

1.2 The NR3B subgroup: Estrogen-Related Receptors

1.2.1 Discovery and Cloning

The NR3B subgroup includes three receptors referred to as ERRα (NR3B1, ERR1, ESRRA), ERRβ (NR3B2, ERR2, ESRRB) and ERRγ (NR3B3, ERR3, ESRRG). The founding member of the subgroup (ERRα) was originally identified owing to the significant sequence similarity that it shared with the estrogen receptor α (NR2B1) gene and protein while ERRβ was identified using the ERRα cDNA as a probe (Giguère, Yang et al. 1988). ERRα was the first orphan nuclear receptor identified, while ERRγ was the last NR to be discovered.

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This last isoform was identified during a search for potential candidate genes for the Usher syndrome type IIa (USH2a), an autosomal recessive disorder associated with moderate to severe sensorineural hearing loss and with retinal degeneration leading to blindness (retinitis pigmentosa) (Eudy, Yao et al. 1998). Although found in the same region as the gene associated with the disease, ERRγ is unlikely to be the USH2a gene due to the absence of mutations in ERRγ in the affected patients included in the study. Independently, another group reported the identification of ERRγ through a yeast two-hybrid screen using the coactivator glucocorticoid receptor-interacting protein 1 (GRIP1) as a bait and, a year later, Heard et al. (2000) reported the cloning of the human ERRγ (Hong, Yang et al. 1999; Heard, Norby et al. 2000).

Although the existence of ERR homologs in invertebrates such as Drosophila (Sullivan and Thummel 2003) and amphioxus (Branchiostoma floridae) (Bardet, Schubert et al. 2005) suggests an ancient origin for the ERRs, it is not yet possible to identify the ancestral member of the NR3B subgroup (Bardet, Laudet et al. 2006). The genomic organization of the three ERR loci also shares a structural characteristic unique among receptor isotypes within a subgroup of the superfamily. In most NRs, the NTD and first zinc finger are encoded by separate . In contrast, for ERRs, a single encodes for both regions. This constitutes a genetic link between the two domains that could account the unusually high level of amino acid sequence identity between the NTDs of the ERRs (Tremblay and Giguère 2007).

In humans, a single transcript encodes for the 423 amino acid ERRα. In contrast, several splice variants of ERRβ and γ have been identified, the major ones being the ERRβ2 variant containing an extension of its LBD (Cheng, Zhu et al. 1999) and the ERRγ2 splice variant harboring an additional 23 amino acids within its NTD (Figure 1.6) (Heard, Norby et al. 2000; Süsens, Hermans- Borgmeyer et al. 2000).

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Figure 1.6: Schemas of the major ERR isoforms. The length of the major ERR isoforms are shown at the right with a different colour for each isotype. The paler square represents the AF-2.

Also, an ERRβ2Δ10 transcript variant lacking exon 10 and encoding a different carboxy-terminal region has been described in vitro (Zhou, Liu et al. 2006). The ERRγ3 splice variant lacking 39 amino acid residues of the second zinc finger of the DNA-binding domain has been identified in adipocytes and the prostate (Kojo, Tajima et al. 2006). This isoform has been shown to regulate the activity of other NRs instead of binding DNA directly. The determination of the relevance of these newly identified transcripts in terms of endogenous protein expression, relative abundance in comparison to the classical ERR transcripts as well as physiological roles awaits further study.

1.2.2 Regulation of Transcriptional Activity

The three ERRs display constitutive transcriptional activity with ERRα usually displaying a weaker activity than ERRβ or ERRγ. These factors activate

14 Chapter I – Literature Review transcription independently of any exogenously added natural ligand and their relative potency is cell context- and promoter-dependent (Mangelsdorf, Thummel et al. 1995).

1.2.2.1 DNA response element

Although the ERRs can bind DNA as monomers or dimers (both heterodimers and homodimers) to a single NR half-site sequence AGGTCA, they preferentially bind to the Estrogen-related response element (ERRE), a 9 base- pair extended NR half-site originally identified as TNAAGGTCA (Figure 1.7). Experimental unbiased binding site testing revealed a binding preference for C, G or A at the N (-2) position while binding to elements with a T at this position is much less frequent (Johnston, Liu et al. 1997; Sladek, Bader et al. 1997; Vega and Kelly 1997; Vanacker, Pettersson et al. 1999; Gearhart, Holmbeck et al. 2003; Huppunen and Aarnisalo 2004; Barry, Laganière et al. 2006; Dufour, Wilson et al. 2007).

The identity of the base at the N (-2) position has been shown to dictate the mode of binding of ERRα. Barry et al. (2006) have demonstrated that ERRα preferentially binds DNA as a monomer on ERRE elements bearing a C or a G, while the dimer mode of binding was preferred on the elements bearing an A or T (Barry, Laganière et al. 2006). This DNA-element determinant for binding mode has functional consequences regarding the transcriptional activity of ERRα since it affects the binding of the preferred ERRα coactivator PGC-1α (see section below). The PGC-1α coactivator was shown to interact with ERRα dimers but not monomers on DNA, therefore the identity of the N (-2) nucleotide will indirectly determine the efficiency of the transcriptional activity of ERRα (Barry and Giguère 2005; Barry, Laganière et al. 2006).

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Figure 1.7: DNA binding modes of the ERRs.

The ERRs preferentially bind to their cognate element, the ERRE, an extended NR response element (TNAAGGTCA). They can bind to this element as monomers, dimers or heterodimers. The ERRs can also bind to the ER response element, the ERE, an inverted palindrome of the hexameric AGGTCA sequence with a 3 nucleotide spacer.

The ERRs have also been shown to recognize the same response element as the ERs, referred to as estrogen response element (ERE), and to bind this element as homodimers (Yang, Shigeta et al. 1996; Vanacker, Pettersson et al. 1999; Zhang and Teng 2000). Although two studies suggested the heterodimerization of ERα and ERRα in vitro, the physiological significance of the potential ER-ERR heterodimers and of ERR binding to ERE element in vivo requires further experimental proof (Yang, Shigeta et al. 1996; Johnston, Liu et al. 1997). This is well exemplified by the TFF1 gene (pS2) which harbors both an ERRE and an ERE in its promoter. Deletion of the ERRE abolishes the transactivation by ERR while deletion of the ERE does not. This suggests that on this particular promoter, the ERRE is the main functional binding site for ERRα (Lu, Kiriyama et al. 2001).

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Recently, a novel response element for the ERRs was identified (Deblois, Hall et al. 2009). This ERRE/ERE consists of an 18-bp element formed by an ERRE embedded within an ERE. It can be assumed that the ERRs will retain the same DNA binding properties on this novel element, although this has not been experimentally tested yet. Furthermore, this study also demonstrated that among the common ERα/ERRα target genes identified by ChIP-on-chip, the occurrence of this novel element (the ERRE embedded in the ERE) is of much greater importance than the individual ERRE and ERE. This strongly suggests that the presence of an ERRE is an absolute requirement for ERR binding in vivo, even in the case of the binding of ERRα to an ERE. This study also demonstrated that the proportion of overlapping target genes between ERα and ERRα is indeed small compared to the proportion of specific ERRα targets. This suggests that the interplay between ERR and ER may not be as general as initially anticipated. Nevertheless, competition for binding between ERRα and ERα is likely to occur at the promoters bearing such ERE/ERRE elements (Deblois, Hall et al. 2009).

1.2.2.2 Coactivators

Within the NR superfamily, coactivator exchange is a common mechanism for the regulation of NR transcriptional activity. The concept is that in their inactive state, the NRs are generally bound to corepressor complexes which are exchanged for coactivator complexes upon ligand binding (Glass and Rosenfeld 2000). The three ERR isotypes bind to a number of coactivator proteins which also act as coactivators for the other NR family members.

The p160 family. The members of the steroid receptor coactivator (SRC) family (SRC-1, TIF-2/SRC-2, AIB1/ACTR/SRC-3), also known as the p160 family, are well-studied coactivators with intrinsic histone acetyltransferase (HAT) activity, which is associated with histone hyperacetylation and transcriptionally active chromatin. The SRCs coactivate the steroid receptors, as

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indicated by their name, but also coactivate other NRs and were the first identified coactivators for the ERRs (Hong, Yang et al. 1999; Xie, Hong et al. 1999; Zhang and Teng 2000). They interact with the NRs via well-described coactivator interaction determinants, the LXXLL motifs, where L is leucine and X is any amino acid. These motifs (also known as NR boxes), are often found in one to three copies within the receptor-interacting domain (RID) and the activation domain (AD) regions of the SRC. In total, the SRCs possess 7 NR boxes, which interact in a specific manner with each NR. Following their recruitment to chromatin via direct interaction with the NRs, the SRCs recruit other HATs (ex. CBP/p300, p/CAF) and coactivator complexes (ex. the large DRIP/TRAP complex) and are, therefore, at the core of a coactivator complex which finally recruits the basal transcription machinery as well as proteins involved in initiation (Leo and Chen 2000). In addition, the p160 coactivators synergize with other coactivators such as the methyltransferases PRMT1 and CARM1 to further increase the transcriptional activity of the NRs, including the ERRs. This synergistic coactivation of NRs is dose dependent; such that the cooperation is most efficient at lower levels of NR expression (Koh, Chen et al. 2001).

The PGC-1 family. The transcriptional activity of the ERRs is also stimulated by the members of the peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1) family, principally PGC-1α and PGC-1β (Huss, Kopp et al. 2002; Kamei, Ohizumi et al. 2003; Schreiber, Knutti et al. 2003; Laganière, Tremblay et al. 2004; Sonoda, Laganière et al. 2007). Similarly to the SRCs, the PGC-1 coactivators play a general role since they coactivate several other NRs.

However, the specificity of the interaction between ERRα and PGC-1α highlights the central importance of the PGC-1 family for the transcriptional activity of the ERRs over the other NRs. The PGC-1α coactivator interacts with NRs via 3 NR boxes (designated L1 to L3) located within its activation domain region. The L2 and L3 NR boxes have been shown to mediate the interaction between most of the NRs and PGC-1α (Tcherepanova, Puigserver et al. 2000;

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Vega, Huss et al. 2000). However, although the ERRs also interact with PGC-1α via the L2 and L3 NR boxes, the L2 has been shown to be dispensable for interaction with ERRα, while the L3 constitutes the major ERR interacting box contacting specifically the AF-2 coactivator binding region of ERRα.

Furthermore, the PGC-1α L3 box has been shown to be dedicated solely to the interaction with the ERRs. Indeed, none of other the other NRs (except for HNF4α) have been shown to directly interact with the L3 box (Huss, Kopp et al. 2002; Schreiber, Knutti et al. 2003; Gaillard, Dwyer et al. 2007). This suggests that the interaction between PGC-1α and ERR is not subject to competition by other NRs. Therefore, the interaction with ERRs is privileged and likely to be favored over the other NRs (Villena and Kralli 2008). In addition to the potent induction of ERRα transcriptional activity, PGC-1α also induces the expression of ERRα at the transcriptional level via a positive autoregulatory loop involving the ERRs bound to the polymorphic ERRE elements of the ERRα promoter (Schreiber, Knutti et al. 2003) (Laganière, Tremblay et al. 2004; Mootha, Handschin et al. 2004; Liu, Zhang et al. 2005). The transcriptional activity as well as expression of ERRα is dramatically increased by the PGC-1 coactivators, highlighting the importance of these coactivators for the transcriptional activity of the ERRs. The PGC-1 coactivators are even considered as protein ligands for the ERRs (Kamei, Ohizumi et al. 2003; Villena and Kralli 2008). PGC-1α also interacts with other coactivators such as SRC-1, p300, PRMT1 as well as with the transcriptional machinery (Puigserver, Adelmant et al. 1999; Lin, Handschin et al. 2005; Teyssier, Ma et al. 2005).

Other coactivators. Other coactivators for the ERRs have been reported, but they are not as well studied in terms of ERR coactivation as the two aforementioned coactivator families. They are the proline-rich nuclear receptor co-regulatory protein (PNRC), PNRC2, transducin-like enhancer of split 1 and Troponin 1 (Zhou, Quach et al. 2000; Zhou and Chen 2001; Hentschke and Borgmeyer 2003; Li, Chen et al. 2008).

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1.2.2.3 Corepressors

The NR corepressor (NCoR) and the related silencing mediator of retinoid and thyroid hormone receptors (SMRT) are the earliest discovered and best characterized corepressors of the NR superfamily (Chen and Evans 1995; Horlein, Naar et al. 1995) (Perissi, Staszewski et al. 1999). In a similar way to coactivators interacting with NRs via NR boxes, NCoR and SMRT typically interact with unliganded NRs via CoRNR (corner) boxes and serve as docking platforms for corepressor complexes containing histone deacetylases (HDACs), the transducin β-like protein 1 and related family member (TBL1 and TBLR1) among others (Hu and Lazar 1999; Nagy, Kao et al. 1999; Perissi, Staszewski et al. 1999; Guenther, Lane et al. 2000; Li, Wang et al. 2000). The corepressors of the ERRs are less characterized than the coactivators. While the interaction between NCoR and the ERRs has never been demonstrated experimentally, the details of the interaction with SMRT have been revealed by the crystal structure of the complex (Wang, Zuercher et al. 2006).

The transcriptional activity of the ERRs is also modulated by the nuclear receptor corepressor RIP140/Nrip1 (Sanyal, Matthews et al. 2004; Augereau, Badia et al. 2006; Castet, Herledan et al. 2006; Debevec, Christian et al. 2007; Giguere 2008). RIP140 is the prototype of an atypical corepressor group, the corepressors of agonist-bound NRs. RIP140 represses the activity of several agonist-bound NRs through binding to the NR coactivator binding groove, and it harbors 9 LxxLL coactivator NR boxes which discriminate between the NRs (Gurevich, Flores et al. 2007). In a similar manner to the PGC-1 family being the main ERR coactivators, RIP140 is so far the main known corepressor of the ERRs. Initially, RIP140 was identified as a transcriptional coactivator for ERα (Cavaillès, Dauvois et al. 1995; Henttu, Kalkhoven et al. 1997). In addition, the positive effect of RIP140 on transcriptional activity of ERα, RARα and GR was consistently observed in yeast (Joyeux, Cavailles et al. 1997; Nephew, Sheeler et al. 1998; Windahl, Treuter et al. 1999; Sheeler, Dudley et al. 2000). A positive

20 Chapter I – Literature Review effect of RIP140 on the transcriptional activity of ERRα and ERRγ, but not ERRβ was also observed. This positive effect of RIP140 was specifically associated with promoters where the ERRs cooperate with the Sp1 , and was proposed to activate the ERRs via an indirect mechanism involving titration of HDACs (Castet, Herledan et al. 2006). However, RIP140 also exerts a direct repressive role on other promoters and is a bona fide repressor of the ERRs (Augereau, Badia et al. 2006; Augereau, Badia et al. 2006; Castet, Herledan et al. 2006; Debevec, Christian et al. 2007).

In addition, all three ERRs interact with the orphan nuclear receptor small heterodimer partner (SHP; NR0B2) (Sanyal, Kim et al. 2002). SHP (Table 1.1) is an atypical NR that does not interact directly with DNA because it lacks a conventional DBD. Instead of direct DNA binding, SHP represses the transcriptional activity of other DNA-bound NRs. (Sanyal, Kim et al. 2002).

1.2.2.4 Synthetic ligands

Despite the absence of endogenous ligand, several synthetic compounds can modulate the transcriptional activity of the ERRs. Although the LBD of the ERRs are relatively similar to the LBD of the ERs, the ERRs do not respond to natural estrogens. However, the synthetic estrogen analog (DES) inhibits the transcriptional activity of all ERR isoforms (Coward, Lee et al. 2001; Tremblay, Kunath et al. 2001) while the selective estrogen receptor modulator (SERM) 4-hydroxytamoxifen (OHT) represses the transcriptional activity of ERRβ and ERRγ, but not of ERRα (Coward, Lee et al. 2001; Tremblay, Bergeron et al. 2001). Indeed, the binding of DES and 4-OHT to ERRγ has been shown to induce the displacement of the Helix12 to an inactive conformation that fills the coactivator interaction region of the LBD and therefore sterically interferes with coactivator binding (Figure 1.8) (Greschik, Flaig et al. 2004; Wang, Zuercher et al. 2006)

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Several other synthetic compounds have been shown to modulate the transcriptional activity of the ERRs. Interestingly, consistent with the LBD sequence kinship between the ERs and ERRs, most of the ERR modulating compounds identified possess estrogenic or anti-estrogenic potential. For example, bisphenol A, a ubiquitous environmental contaminant with estrogenic activity, was shown to bind to ERRγ and antagonize the OHT-mediated repression of ERRγ (Takayanagi, Tokunaga et al. 2006) while toxaphene and chlordane, two organochlorine pesticides with estrogen-like activity, both acted as weak antagonists for ERRα (Yang and Chen 1999). However, the direct binding of the organochlorine pesticide molecules to ERRα could not be experimentally demonstrated (Tremblay, Bergeron et al. 2001).

Figure 1.8: Nuclear receptors typical LBD conformations.

Ribbon representation of the crystal structures of A) ERRα in complex with a PGC-1α peptide (PDB 1xb7) (Kallen, Schlaeppi et al. 2004), B) ERRγ LBD in complex with 4-OHT showing the repressive position of Helix12 sterically hindering the coactivator binding region (PDB 2gpu) (Wang, Zuercher et al 2006), and C) apo-ERRγ LBD showing the helix12 in the active conformation and the coactivator binding region in the unliganded ERRs (PDB 2zbs) (Matsushima, Teramoto et al. 2008). The helix3 position is shown on the 3 panels for comparative positional purpose.

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In addition, the isoflavones genistein, daidzein, and biochanin A and the flavone 6,3',4'-trihydroxyflavone were identified as agonists of the ERRs by mammalian two-hybrid experiments under comparable conditions to those for the activation of ERα and ERβ (Suetsugi, Su et al. 2003). The majority of the ERR ligands identified so far are estrogenic compounds, and although synthetic, they are physiologically relevant to human health and physiology. The ERRβ/ERRγ inhibitor 4-OHT is a first line of treatment for breast cancer while the other estrogenic compounds are found in many plastic products destined for human utilization and are often referred to as endocrine disruptors. They are thought to cause several human health issues and concern is given to their potential carcinogenic effects in humans. Their role as ligands for the ERRs suggests that the ERRs might be involved in mediating some of the potentially deleterious health effects of these compounds. They strengthen the existing link between ERRs and cancer and they add up to the yet unconfirmed physiological link between the ERRs and the ERs. It is indeed possible that these compounds modulate the parameters regulating the interplay of the ERRs with the ERs as well as the of ERR binding to EREs. Therefore, these compounds, which are not specific for the ERRs since they modulate the activity of other nuclear receptors such as the ERs, constitute nevertheless useful tools to study the regulation of the ERRs, at least from a mechanistic point of view.

ERR-specific synthetic ligands have also been identified. For example, XCT790 has been described as a highly specific inverse agonist for ERRα. The disruption of the interaction between ERRα and PGC-1α was proposed as the mechanism, however this experimental proof was weak (Willy, Murray et al. 2004; Busch, Stevens et al. 2004). It has since been reported that the effect of this compound is to increase the degradation rate of ERRα rather than strictly affecting the interaction with PGC1α, suggesting a more complex mechanism of action (Lanvin, Bianco et al. 2007). GSK5182, a tamoxifen analog, displays improved inverse agonist selectivity for ERRγ while the structurally related phenolic acyl hydrazones GSK4716 and DY131 were reported to effectively and

23 Chapter I – Literature Review selectively activate ERRβ and ERRγ (Yu and Forman 2005; Zuercher, Gaillard et al. 2005; Chao, Collins et al. 2006).

The existence or the need for an endogenous activating ligand for the ERRs is a subject of controversy. The crystal structures of ERRα and ERRγ ligand-binding domains strongly suggest that the ERRs do not require a ligand. Indeed, evidence from several studies for ligand-independent transactivation by the ERRs has shown that the apo-ERRs are in a permanent active configuration and are ready to interact with coactivator proteins (Figure 1.8) (Greschik, Wurtz et al. 2002; Kallen, Schlaeppi et al. 2004; Wang, Zuercher et al. 2006; Kallen, Lattmann et al. 2007). In addition, structure-based predictions suggested that the ligand binding pockets of ERRα and ERRγ are indeed too small (approximately 100 and 280 Å, respectively) to allow the binding of molecules composed of more than four to five non-hydrogen atoms (Greschik, Wurtz et al. 2002; Kallen, Schlaeppi et al. 2004). However, the crystal structures of the ERRα LBD bound to the inverse agonist cyclohexylmethyl-(1-p-tolyl-1H-indol-3-ylmethyl)-amine as well as the ERRγ LBD bound to the agonist GSK4716 revealed a compound- induced rearrangement of the ERR ligand binding pockets. These results suggest that larger compounds could act as natural ligands in an induced-fit manner owing to the plasticity of the ERR ligand binding pockets (Wang, Zuercher et al. 2006; Kallen, Lattmann et al. 2007; Matsushima, Teramoto et al. 2008).

1.2.2.5 Post-translational modifications

In addition to the physiological cue provided by their ligands, the NRs also integrate the signals arising from signaling pathways. Post-translational modifications of NRs (and of their coregulators) enhance or inhibit their activity via the regulation of nuclear localization, ligand binding and recruitment of coregulators (Rochette-Egly 2003; Faus and Haendler 2006; Han, Lonard et al. 2009).

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Although this is a well-studied aspect of NR function, little is known concerning the post-translational modifications that target the ERRs. Section 1.3 (See below) is entirely dedicated to post-translational modifications, chiefly phosphorylation and sumoylation, and their impact on transcriptional regulation.

1.2.3 Tissue Expression of the ERRs

In general, the ERRs display a similar tissue distribution in both mice and humans. They are ubiquitously expressed and generally, ERRα is more abundantly expressed than ERRγ, which in turn is more abundant than ERRβ. The three isotypes are expressed at elevated levels in tissues subjected to high energy demand, such as the heart and kidney. ERRα is also expressed at high levels in the intestine, brown adipose tissue and while ERRγ mRNA can be found in abundance in the brainstem and the spinal cord. ERRβ can be found at relatively high levels in a subset of extra-embryonic, ectoderm-derived structures in the developing placenta as well as in undifferentiated trophoblast stem cell lines. This form is also found in the adult eye and inner ear (Giguère, Yang et al. 1988; Pettersson, Svensson et al. 1996; Luo, Sladek et al. 1997; Tremblay, Kunath et al. 2001; Bookout, Jeong et al. 2006; Chen and Nathans 2007).

Furthermore, consistent with their preferential interaction and transcriptional partnership, the tissue expression pattern of ERRα correlates with that of the PGC-1α coactivator. In fact, the levels of ERRα have been shown to be higher in tissues expressing high levels of the PGC-1 coactivators (such as brown adipose tissue, heart, kidney, small intestine and skeletal muscle). Furthermore, ERRα levels increase along with physiological stressors known to induce the expression PGC-1α, notably fasting in liver, cold exposure in brown adipose tissue and muscle, exercise in skeletal muscle and long-term caloric restriction in several tissues (Ichida, Nemoto et al. 2002; Schreiber, Knutti et al. 2003; Cartoni, Leger et al. 2005; Villena, Hock et al. 2007; Ranhotra 2009).

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1.2.4 Target Genes of the ERRs

The investigation of the physiological roles played by NRs can be best achieved by the identification and study of their target genes. After the identification of the ERR binding site, the first insights into the physiological roles of the ERRs emerged via the identification of individual ERRα target genes. Several groups have identified target genes based on the discovery of a putative ERRE in the promoters of genes expressed in various tissues and associated with specific cellular functions(Table 1.2).

Table 1.2: ERRα target genes identified individually.

Target gene ERR Reg References

SV40 late promoter α - (Wiley, Kraus et al. 1993); (Zuo and Mertz 1995) Medium-chain acyl coenzyme α + (Sladek, Bader et al. 1997);(Vega and A Dehydrogenase (MCAD) Kelly 1997) Osteopontin (OPN) α + (Bonnelye, Vanacker et al. 1997);(Vanacker, Delmarre et al. 1998) Thyroid receptor α (TRα) α + (Vanacker, Bonnelye et al. 1998)

Aromatase α + (Yang, Zhou et al. 1998)

Lactoferrin α + (Yang, Shigeta et al. 1996)

Small heterodimer partner α + (Sanyal, Kim et al. 2002), (SHP) Endothelial nitric oxide α + (Sumi and Ignarro 2003) synthase (eNOS) peroxisome proliferator- α + (Huss, Pineda Torra et al. 2004) activated receptor α (PPARα) pyruvate dehydrogenase α + (Araki and Motojima 2006); (Wende, kinase 4 (PDK4) Huss et al. 2005); (Zhang, Ma et al. 2006) Monoamine oxidase B (MAO- α + (Willy, Murray et al. 2004); (Zhang, Chen B) et al. 2006) Estrogen-related receptor α α, γ + (Laganière, Tremblay et al. 2004); (Liu, (ERRα) Zhang et al. 2005); (Mootha, Handschin et al. 2004)

26 Chapter I – Literature Review

Apolipoprotein A4 (ApoA4) α + (Carrier, Deblois et al. 2004)

Specificity protein 1 (Sp1) α + (Sumi and Ignarro 2005) dehydroepiandrosterone α + (Seely, Amigh et al. 2005) sulfotransferase 2A1 (Sult2a1) Phosphoenolpyruvate α - (Herzog, Cardenas et al. 2006) carboxykinase (PEPCK) Surfactant protein A (SP-A) α + (Liu, Hinshelwood et al. 2006)

RIP-140/Nrip1 α + (Nichol, Christian et al. 2006)

Mitofusin 2 α + (Soriano, Liesa et al. 2006)

Polo-like kinase 2 (Plk2) α + (Park, Kim et al. 2007)

Uncoupling protein 1 (UCP-1) α + (Debevec, Christian et al. 2007)

Vascular endothelial growth α + (Stein, Gaillard et al. 2009) factor (Vegf) Cytochrome P450 11A1 α + (Grasfeder, Gaillard et al. 2009) (CYP11A1) Cytochrome P450 17A1 α + (Seely, Amigh et al. 2005);(Grasfeder, (CYP17A1) Gaillard et al. 2009)

In addition to the above analysis of individual genes, methods exploiting the potent coactivation of ERRα by PGC-1α such as stable overexpression of PGC-1α (or an ERR-specific PGC-1α mutant), coupled with microarray analysis, has allowed a high-throughput identification of mitochondrial biogenesis and the OXPHOS pathways as potentially regulated by the ERRα/PGC-1α partnership (Mootha, Handschin et al. 2004; Schreiber, Emter et al. 2004; Gaillard, Grasfeder et al. 2006). Further studies involving the combined use of computational biology, ERRα-specific small interfering RNAs (siRNAs), specific antagonists as well as DNA binding assays and co-transfection of reporter genes, have established a small subset of bona fide ERRα targets (ATP synthase b, somatic cytochrome c, COX4, GABPA, adenine nucleotide translocator 1 and carnitine

27 Chapter I – Literature Review palmytoyltransferase 1A). These studies support the view that ERRα/PGC-1α are key regulators of mitochondrial biogenesis and the OXPHOS pathway.

The greatest advance in ERR target gene identification have come from the development of the chromatin immunoprecipitation (ChIP) and genomic DNA arrays (ChIP-on-chip) which have allowed the determination of the occupancy of several NRs and transcription factors on a genome-wide scale (Odom, Zizlsperger et al. 2004; Carroll, Liu et al. 2005; Laganière, Deblois et al. 2005; Carroll, Meyer et al. 2006; Deblois and Giguere 2008). When applied to the ERRs, the ChIP-on- chip technique not only greatly extended the repertoire of ERR target genes, but also led to the identification of the important roles of ERRα and ERRγ in the adult mouse heart (Dufour, Wilson et al. 2007), of ERRγ in the newborn heart (Alaynick, Kondo et al. 2007), as well as the roles of ERRα in bone marrow- derived macrophages (Sonoda, Laganière et al. 2007) and in breast cancer cell lines (Deblois, Hall et al. 2009). These studies, which played a major role in uncovering the biological roles of the ERRs, are described in greater detail in the following section.

1.2.5 Biological Roles of the ERRs

1.2.5.1 ERRs in energy metabolism: role in obesity and heart failure

The identification of the first ERRα target gene, MCAD, readily pointed towards a role for ERRα in fatty acid metabolism (Sladek, Bader et al. 1997). Furthermore, the definition of the close partnership between ERRα and PGC-1α, a known regulator of mitochondrial biogenesis and fatty acid β-oxidation, further linked ERRα to energy metabolism (Lehman, Barger et al. 2000; Knutti and Kralli 2001; Huss, Kopp et al. 2002; Schreiber, Knutti et al. 2003; Laganière, Tremblay et al. 2004; Giguere 2008). While phenotypic analysis of ERRα null mice confirmed the status of the ERRs as important metabolic regulators, large-

28 Chapter I – Literature Review scale location analysis by ChIP-on-chip expanded the ERRα metabolic transcriptional program (Luo, Sladek et al. 2003; Alaynick, Kondo et al. 2007; Dufour, Wilson et al. 2007; Sonoda, Mehl et al. 2007; Giguere 2008).

The ERRα null mice are viable and fertile without any major anatomical difference from wild-type mice, except for their smaller body weight (Luo, Sladek et al. 2003). Subsequent physiological analyses revealed that the reduction in body weight was attributable to a decrease in peripheral fat deposits. The ERRα null mice displayed a similar number of adipocytes in the peripheral fat pads, but the adipocytes were smaller and had a reduced lipid content (Luo, Sladek et al. 2003). In addition, the adult ERRα null mice are resistant to high fat diet-induced obesity and subsequent analysis revealed the enterocytes of the adult ERRα null mice have a decreased capacity for β-oxidation. In addition, the ERRα null pups showed lipid malabsorption and decreased fat transport (Carrier, Deblois et al. 2004).

The latter observation partially explains the genotype-phenotype paradox of the ERRα null mice by suggesting a complex and tissue-specific role for ERRα in the control of energy metabolism in the whole animal. The paradox was uncovered when the first metabolic target genes of ERRα were identified. The changes in expression of the lipid metabolism and OXPHOS ERRα target genes in several tissues of the ERRα null mice (adipose tissue, muscle and small intestine), would have correlated better with a decreased fat burning and energy expenditure than with the observed phenotype of leanness and resistance to high- fat diet obesity (Luo, Sladek et al. 2003; Carrier, Deblois et al. 2004; Huss, Pineda Torra et al. 2004).

In addition to this obesity-related phenotype in mice the genomic location and promoter organization of the ERRα gene (ESRRA) in humans also suggest a possible role for ERRα in obesity. The ESRRA gene maps to the chromosome 11q13 and this region is associated with body mass index (BMI) and fat content in humans (Shi, Shigeta et al. 1997; Sladek, Beatty et al. 1997;

29 Chapter I – Literature Review

Pérusse, Rankinen et al. 2005). The ESRRA promoter harbors a polymorphic autoregulatory ERRE sequence of 23bp, referred to as the ESRRA23 element, found in 1 to 4 copies among the population, the 2.3 genotype being the most frequent (Laganière, Tremblay et al. 2004). Therefore, this polymorphism has been tested for correlation with obesity, type II and BMI. However, the association between the ESRRA23 polymorphism or the ERRα Pro116Pro variant and physiological parameters such as BMI, obesity or type II diabetes was not confirmed by all epidemiological studies. In one study, it was found that the 2.3 genotype of ESRRA23 was associated with a higher BMI in the Japanese population. On the other hand, another study found no association with the ESRRA23 or ERRα Pro116Pro variants with obesity or type II diabetes in Danish whites (Kamei, Lwin et al. 2005; Larsen, Rose et al. 2007). ERRγ is also linked to obesity, although indirectly, through the repressive atypical DNA-binding deficient NR SHP. Indeed, mutations in SHP that are associated with moderate obesity were shown to alleviate its repressive effect on ERRγ transcriptional activity (Sanyal, Kim et al. 2002).

Studies in mouse heart were fundamental to substantiate the metabolic role of ERRα and ERRγ. Genome-wide location analysis of ERRα and ERRγ in mouse heart by ChIP-on-chip revealed that both isotypes shared the same transcriptional program, and identified the ERRs as pleiotropic regulators of cardiac energy metabolism. The cardiac ERRs’ transcriptional program includes subunits of all the complexes of the electron transport chain as well as genes involved in cardiac contractility, calcium handling and fuel sensing (Dufour, Wilson et al. 2007).

The application of a physiological stressor is necessary to uncover the physiological impact of ERRα gene ablation. Indeed, ERRα is thought to mediate the fine-tuning and the adaptive response to physiological conditions associated with increased energy demands. Considering the metabolic transcriptional program of ERRα and ERRγ in the heart identified by ChIP-on- chip and the importance of energy metabolism for cardiac physiology and disease,

30 Chapter I – Literature Review the role of ERRα was explored in the trans-aortic constriction (TAC)-induced cardiac pressure overload model of hypertrophy and heart failure. The expression of ERRα is decreased in the heart of wild-type mice subjected to TAC and the ERRα null TAC mice displayed cardiac chamber dilation, left ventricular fraction shortening, depletion of the cardiac phosphocreatine pool and reduced ATP synthesis in the heart leading rapidly to heart failure (Huss, Imahashi et al. 2007). In addition, a recent gene expression study of failing human hearts identified downregulation of ERRα and PGC-1α as part of a gene expression signature of heart failure (Sihag, Cresci et al. 2009).

The ERRγ null mice die shortly after birth from a cardiac defect. Consistent with the cardiac energy metabolism transcriptional program regulated by ERRα and ERRγ in the adult heart, the role of ERRγ in the fetal and post-natal heart is also metabolic (Alaynick, Kondo et al. 2007). The ERRγ null pups have a smaller ventricular mass and the metabolic shift from fetal carbohydrate energy metabolism to post-natal oxidative energy metabolism is impaired. Consequently, the ERRγ null pups showed significant lactatemia, electrocardiogram abnormalities, high mitochondrial genome number and altered electron transport chain biochemical activities (Alaynick, Kondo et al. 2007).

The metabolic role of ERRα is not restricted to the heart. Indeed, the ERRα null mice showed an impaired thermogenic response to cold. They developed hypothermia faster than wild type and displayed a slower recovery to normal body temperature. This physiological defect correlates with a reduction in mitochondria number in brown adipose tissue (Villena, Hock et al. 2007).

Furthermore, the ERRα null mice were shown to be more susceptible to infection by Listeria monocytogenes and studies using primary ERRα null macrophages showed that ERRα is required for the induction of the mitochondrial reactive oxygen species (ROS) production and efficient clearance of Listeria monocytogenes in response to interferon γ (IFN-γ) in a PGC-1β-dependent manner (Sonoda, Laganière et al. 2007). In animal models, administration of

31 Chapter I – Literature Review lipopolysaccharide (LPS) or turpentine oil mimics the immune acute phase response. The metabolic effects of acute phase response have been associated with ERRα and PGC-1α, as these two metabolic regulators were shown to be downregulated in parallel to MCAD in LPS- and turpentine oil-induced acute phase response in mice (Kim, Shigenaga et al. 2005). These studies suggest a potential role for ERRα in the immune system, particularly in host defense and acute phase response.

1.2.5.2 ERRs in cancer

The implication of all three ERR isotypes in cancer is becoming clearer as the experimental evidence is accumulating. The first insight into the link between ERRα and breast cancer came from the similarity with ERα in sequence identity and in vitro DNA binding properties. The regulation of the ERRs by synthetic estrogenic compounds such as DES, 4-OHT and endocrine disruptors further added to the concept (Giguère 2002). In addition, the ERRs are expressed in various cell lines and primary tumors related to several types of cancer such as breast, ovary, endometrium prostate and colon (Lu, Kiriyama et al. 2001; Ariazi, Clark et al. 2002; Suzuki, Miki et al. 2004; Cavallini, Notarnicola et al. 2005; Cheung, Yu et al. 2005; Sun, Sehouli et al. 2005; Gao, Sun et al. 2006; Sun, Gao et al. 2006; Watanabe, Kinoshita et al. 2006; Zhou, Liu et al. 2006; Fujimura, Takahashi et al. 2007).

ERRα is considered a bad prognosis indicator for breast cancer because its expression in breast tumors correlates with unfavorable biomarkers (such as ERα negativity and high levels of ErbB2 expression), while ERRγ is considered as a better prognosis indicator since its expression in breast tumors is associated with more favorable biomarkers (such as anti-estrogen treatment responsiveness due to estrogen/progesterone receptor positivity and expression of ErbB4). In addition, a positive correlation was established between ERRα levels and the risk of

32 Chapter I – Literature Review recurrence and poor clinical outcome in human breast carcinoma (Ariazi, Clark et al. 2002; Suzuki, Miki et al. 2004). In addition, the removal of ERRα from the aggressive ERα-negative breast carcinoma MDA-MB-231 cell line by stably expressed siRNA did not affect the proliferation rate in vitro but significantly reduced their migratory potential and their of tumor growth rate when these cells were implanted as xenografts (Stein, Chang et al. 2008).

At the molecular level, the phosphorylation status of ERRα has been associated to EGF/ErbB2 signaling (Barry and Giguère 2005; Ariazi, Kraus et al. 2007). Breast cancer cell lines, like the tumors they originate from, display various degrees of ErbB2 overexpression/amplification. The ERRα phosphorylation levels as well as its transcriptional activity were found to be higher in BT474, a breast cancer cell line with high levels of ErbB2. In these cells, inhibition of ErbB2 signaling abrogated ERRα transcriptional activity (Ariazi, Kraus et al. 2007). Furthermore, studies using breast cancer cell lines expressing different levels of ERα also helped to clarify the role of ERRα in breast cancer as well as the presumed ERα/ERRα crosstalk. The transcriptional program of ERRα in breast cancer cell lines was recently identified. This study showed that indeed the overlap between ERα and ERRα target genes was smaller than expected, suggesting that ERα and ERRα are likely to play distinct roles in vivo, at least in cancer. Furthermore, this study identified an ERRα target gene signature associated with breast cancer subtype progression and ErbB2 status (Deblois, Hall et al. 2009).

1.2.5.3 ERRα in bone

From its identification via similarity with ERα to the finding of the capacity to bind a ERE in vitro as well as transcriptional modulation with several synthetic estrogenic compounds, ERRα has been thought to impinge upon the estrogen axis. Given the key role of estrogens in bone formation and maintenance

33 Chapter I – Literature Review as well as osteoporosis, the physiological role of ERRα in bone constitutes an active axis of research (Giguère 2002; Bonnelye and Aubin 2005).

Also, among the first ERRα target genes identified, several were related to bone physiology, such as osteopontin, lactoferrin, aromatase, TRα and more recently, eNOS (Yang, Shigeta et al. 1996; Vanacker, Bonnelye et al. 1998; Vanacker, Delmarre et al. 1998; Yang, Zhou et al. 1998; Sumi and Ignarro 2003). In addition, not only has ERRα been shown to be expressed in cultured bone cells but its expression has been shown to be regulated by estradiol in rat calvaria cells in culture and in vivo (Bonnelye, Vanacker et al. 1997; Sladek, Bader et al. 1997; Bonnelye, Merdad et al. 2001; Bonnelye and Aubin 2002; Bonnelye, Kung et al. 2002). In this regard, the ERRα promoter contains an ERE and the polymorphism of the ERRα promoter has been associated with bone mass density (BMD) in white premenopausal women (Laganière, Tremblay et al. 2004; Laflamme, Giroux et al. 2005). The study showed that higher BMD was significantly associated with more copies of the ESRRA23 element, while the lower copy number carriers had a decreased BMD and an increased risk of bone fracture (Laflamme, Giroux et al. 2005).

Recently, a role for ERRα in cartilage formation was also reported. ERRα is expressed in the rat chondrogenic cell line C5.18 cells in vitro, as well as in fetal and adult rat chondrocytes in growth plate and articular cartilage (Bonnelye, Zirngibl et al. 2007). In C5.18 cells, overexpression of ERRα induces the expression of SOX9, a key regulator of cartilage formation, while downregulation of ERRα by siRNA inhibited cartilage formation and maturation of proliferating chondrocytes into hypertrophic chondrocytes in vitro (Bonnelye, Zirngibl et al. 2007).

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1.2.5.5 ERRβ in development

The ERRβ null mice die in utero at 10.5 days post-coitum due to an impairment in placental formation. Early phenotypic analysis of the ERRβ null embryos revealed chorionic developmental abnormalities associated with a deficiency in diploid trophoblasts number and an overabundance of trophoblast giant cells (Luo, Sladek et al. 1997). Similar placental defects were observed in pregnant wild-type mice treated with DES, a synthetic estrogen that promotes coactivator release from ERRβ and inhibits its transcriptional activity (Tremblay, Kunath et al. 2001).

The embryonic lethality of the ERRβ null model can be successfully rescued by the aggregation of diploid embryos from the ERRβ heterozygote mice intercrossed with four-cell stage tetraploid wild-type embryos followed by transfer to foster mothers. The ERRβ null embryos produced by tetraploid rescue develop to adulthood and are fertile despite a reduced number of primordial germ cells in their gonads. However, the ERRβ null rescued mice harbor severe behavioral abnormalities starting 10 days after birth such as circling behavior, head-tossing, tottering and falling down while walking intertwined with some backward walking stretches (Mitsunaga, Araki et al. 2004). These behavioral abnormalities suggest a role for ERRβ in the brain and central nervous system. It was recently shown that the ERRβ null mice display an inner ear defect that could account partially for the equilibrium and balance defect phenotype. Indeed, the ERRβ null mice display abnormal development of the stria vascularis and endolymph production. Gene expression profiling of this inner ear structure revealed differential expression of ion channels involved in endolymph production. This study suggests that potential ERRβ ligands could be used to treat some disorders of hearing and balance (Chen and Nathans 2007).

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1.3 Post-Translational Modifications in Transcriptional Regulation

Following transcription, the newly formed messenger RNA is spliced and translated into protein. After translation and until their degradation, proteins are subjected to post-translational modifications (PTMs). PTMs occur on many proteins and regulate a myriad of, if not all, cellular processes from transcriptional regulation, translation, regulation of signal transduction pathways, trafficking and localization as well as protein stability or degradation. Numerous types of PTMs coexist within a cell and it is not uncommon for multiple modifications (same or different types) to occur on one protein, and even to compete for the same target site. As mentioned above, the NRs integrate the signals from signaling cascades and translate them into modulation of gene expression. Although almost all types of post-translational modifications have been shown to affect NRs, the focus of the first part of this thesis concerns the interplay between sumoylation and phosphorylation on the ERRα and ERRγ NTD and, therefore, only these two types of modifications and their role in transcriptional regulation specifically are described herein.

1.3.1 Phosphorylation

1.3.1.1 General mechanism

Phosphorylation started to be recognized and widely accepted as a general regulatory mechanism during the 1970s and early 1980s. It constitutes the addition of a phosphate group from a phosphate donor (generally ATP) onto a serine, threonine or residue within the target proteins (Cohen 2002). Phosphorylation, like almost all PTMs, is a reversible process. The addition of the phosphate group is performed by a group of , the kinases, while the

36 Chapter I – Literature Review phosphatases are responsible for its removal. The occurrence of phosphorylation by a specific kinase is determined by the cellular context (activation or inactivation of the kinase), as well as the amino acid sequence surrounding the targeted amino acid (serine/threonine or tyrosine), the consensus site.

The concept of substrate specificity among phosphatases is starting to emerge and is much less characterized than that of the kinases. So far, specificity of the phosphatases was associated with the modified amino acid in the target protein, mainly giving rise to a general classification of phosphatases into the three major groups: tyrosine phosphatases, the serine/threonine phosphatases and the dual/specificity phosphatases. This concept is evolving toward a more specific classification and role of protein phosphatase complexes. Indeed, to consider the phosphatases as non-specific and promiscuous is a misconception on the verge of being dismissed (Virshup and Shenolikar 2009).

Phosphorylation events generally originate from the cellular membrane in response to a physiological cue and are transduced to the nucleus via cascades of kinase activation, which ultimately affect the transcriptional regulators (transcription factors, nuclear receptors and their coregulators) to modulate gene expression.

1.3.1.2 Phosphorylation of Nuclear Receptors

Every domain of the NRs can be targeted by phosphorylation events. However, at least for the classical steroid receptors, the majority of the identified phosphorylation sites are clustered in NTDs with very few identified phosphorylation sites in DBDs (Faus and Haendler 2006). Several signaling pathways are involved in NR phosphorylation, but the mitogen-activated protein kinase (MAPK) pathway is by far the most studied and best-defined pathway targeting the NRs (Weigel and Zhang 1998; Kyriakis 2000). In general, phosphorylation of the steroid NR NTDs is associated with hormone-independent

37 Chapter I – Literature Review activation, with the exception of GR. For example, phosphorylation of the PR NTD by protein kinase A (PKA) and of the ERs’ NTDs by MAPK are associated with ligand-independent transcriptional activation (Denner, Weigel et al. 1990; Zhang, Bai et al. 1994; Kato, Endoh et al. 1995; Patrone, Ma et al. 1996; Tremblay, Tremblay et al. 1999). Phosphorylation also modulates the activity of the liganded NRs. In the case of GR, the PKC and PKA pathways have been shown to potentiate the stimulatory effect of its ligand (Rangarajan, Umesono et al. 1992; Nordeen, Moyer et al. 1994). The MAPK pathway has been shown to induce the nuclear translocation of ERα and facilitate its DNA binding (Katzenellenbogen 1996; Lu, Ebling et al. 2002). In addition, the phosphorylation of the antagonist-bound ERα NTD by MAPK has been shown to disrupt its interaction with NCoR (Lavinsky, Jepsen et al. 1998).

The non-steroidal NRs also are modulated by phosphorylation of the NTDs. Phosphorylation of the AF-1 domain of PPARα and SF-1 by the MAPK and PKA pathways, respectively, has been shown to enhance their transcriptional activity (Hammer, Krylova et al. 1999; Juge-Aubry, Hammar et al. 1999). On the other hand, phosphorylation of the NR NTD can also repress their activity. For example, phosphorylation of the AF-1 domain of PPARγ by the stress-activated protein kinase (SAPK)/ERK/MAPK pathway decreases its ligand binding capability and represses its transcriptional activity (Hu, Kim et al. 1996; Camp and Tafuri 1997).

Signaling also affects the NRs indirectly via phosphorylation of their coregulators. The MAPK pathway has an activating effect on NRs at multiple levels. For example, MAPK pathway activation has been shown to induce a redistribution of the SMRT corepressor to the nuclear membrane or cytoplasm, while phosphorylation of the p160 coactivators SRC-1 and SRC-3 induces their nuclear translocation (Hong and Privalsky 2000; Wang, Rose et al. 2000; Amazit, Pasini et al. 2007). Phosphorylation of coregulators also affects their enzymatic activity as well as their protein-protein interactions. For example, the methyltransferase activity of CARM1 is inhibited by phosphorylation, and

38 Chapter I – Literature Review phosphorylation of SRC-1 increases its interaction with the progesterone receptor (Narayanan, Adigun et al. 2005; Higashimoto, Kuhn et al. 2007) Of course, PTMs other than phosphorylation such as methylation, acetylation, ubiquitination and sumoylation, also affect the same properties of the NRs and their coactivators. Many of these coactivators with various modification activity, such as methyltransferase or acetyltransferase activity, act in concert and reciprocally modulate their PTM status as well as that of the NR they bind to (Koh, Chen et al. 2001). The post-translational modification of coactivators and NRs adds another layer of complexity to transcriptional regulation (Han, Lonard et al. 2009).

Recently, a role for phosphorylation in regulating the DNA binding and dictating the target gene specificity of LXR and GR has been shown using combinations of mRNA level quantification by real-time PCR and ChIP techniques on cell lines stably expressing the studied NR or its phosphorylation site mutant (Chen, Dang et al. 2008; Torra, Ismaili et al. 2008).

1.3.1.3 Phosphorylation of the ERRs

Although the modulation of ERR activity by PTMs is still largely understudied, there is a growing body of evidence showing that phosphorylation plays a central role for ERRα transcriptional activity. The first suggestion of the phosphoprotein status of ERRα was provided by the observation that phosphatase treatment accelerated the migration of ERRα in lysates from transfected COS-1 cells (Sladek, Bader et al. 1997). Later, the ERRα DBD was shown to be phosphorylated in MCF-7 breast cancer cells after epidermal growth factor (EGF) treatment. In silico phosphorylation site analysis of the ERRα sequence revealed 2 high confidence PKC consensus sites within the ERRα DBD (S118 and T124), thus the phosphorylation event was mapped to the ERRα DBD using an in vitro phosphorylation assay with recombinant protein kinase C delta (PKCδ). In addition, both EGF treatment and PKCδ signaling increased the DNA binding and

39 Chapter I – Literature Review transcriptional activity of ERRα in a promoter-specific manner (Barry and Giguère 2005). While the S118A mutation did not affect the DNA binding properties and transcriptional activity of ERRα, the T124A mutant showed decreased monomeric DNA binding on an ERRE element with a T or A at position N as well as defective DNA-dependent dimerization, decreased DNA binding on ERE element and decreased coactivation by PGC-1α on ERRE elements with a C or G at position N (Barry, Laganière et al. 2006). Consistent with this, ERRα phosphorylation downstream of ErbB2 signaling was reported to increase its transcriptional activity in breast cancer cells (Ariazi, Kraus et al. 2007).

Recently, ERRα was shown to be responsive to cyclic AMP/PKA signaling and this response was abrogated by alanine mutation of three serine residues (S87, S114 and S277) located within the DBD. Cyclic AMP/PKA signaling was associated with an increase in ERRα expression, nuclear localization and transcriptional activity at the SP-A promoter. Furthermore, PKA signaling was also shown to increase the coactivation of ERRα by SRC-2 as well as the recruitment of both proteins at the SP-A promoter (Liu, Hinshelwood et al. 2006; Liu, Benlhabib et al. 2009).

1.3.2 Sumoylation

Sumoylation is the reversible covalent attachment of the small ubiquitin- like modifiers (SUMO) proteins to a target Lysine (K) embedded into the core consensus site ΨKxE/D, where Ψ represents a large hydrophobic residue (mostly V, L, I, M or F), x represents any amino acid, and E/D represents glutamic acid or aspartic acid (Iniguez-Lluhi and Pearce 2000; Rodriguez, Dargemont et al. 2001). The SMT3 gene (also referred to as SUMO), encoding for a small protein of approximately 10 kDa, was first discovered in yeast (Seufert, Futcher et al. 1995). The SUMO-1 protein was the first isoform discovered in mammals and reported

40 Chapter I – Literature Review to be covalently conjugated to the GTPase activating protein RanGAP1, the first identified SUMO substrate (Matunis, Coutavas et al. 1996; Mahajan, Delphin et al. 1997).

While a single SUMO gene (SMT3) is expressed in lower eukaryotes (such as insects, nematodes and yeast), there are four SUMO paralogues expressed in mammals while plants (Arabidopsis Thaliana) express up to eight SUMO isoforms (Kurepa, Walker et al. 2003; Lois, Lima et al. 2003). The mammalian SUMO paralogues are SUMO-1 (Smt3c, PIC1, GMP1, sentrin, Ubl1), SUMO-2 (Smt3a, Sentrin3), SUMO-3 (Smt3b, Sentrin2), and the most recently identified intronless SUMO-4 gene, which was initially thought to be a non-expressed pseudogene, but was found to be expressed in kidney cells (Saitoh and Hinchey 2000; Tatham, Jaffray et al. 2001; Su and Li 2002). The SUMO-4 isoform has 84% sequence identity with SUMO-2. The SUMO-1 isoform has 18% sequence identity with ubiquitin and 50% identity with the SUMO-2 and SUMO-3 isoforms, while the latter two are 97% identical in sequence. Due to the high level of sequence identity between SUMO-2 and SUMO-3, these two paralogues are considered as a subfamily and often referred to as SUMO-2/3 (Hay 2005).

Although very similar in terms of their conjugation mechanism (see below), there are several differences between the three principal mammalian SUMO paralogues. The three SUMO proteins are ubiquitously expressed and display a partially similar cellular localization. All three paralogues are found in the cytoplasm and nucleus, but within some specific substructures. SUMO-1 is uniquely distributed to the nuclear envelope and to the nucleolus while SUMO-2 and -3 are distributed throughout the nucleoplasm but excluded from the nucleolus (Ayaydin and Dasso 2004). In addition, the SUMO paralogues display some subcellular localization specificity during mitosis. SUMO-1 preferentially localizes to the mitotic spindle and spindle midzone whereas SUMO-2/3 localize to centromeres and condensed (Zhang, Goeres et al. 2008).

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In addition to the relative specificity observed in subcellular localization, the capacity of forming polychains is restricted to the SUMO-2/3 isoforms owing to the presence of a SUMO conjugation consensus motif in their sequence which is absent in SUMO-1 (Tatham, Jaffray et al. 2001). The role of SUMO-1 in these structures has been associated with polychain termination (Matic, van Hagen et al. 2008). Also, the SUMO-1 paralogue is found almost exclusively conjugated to substrates within the cell, while free pool of SUMO-2/3 exist and their conjugation has been shown to be induced by various cellular stresses such as osmotic and oxidative stress or heat shock (Saitoh and Hinchey 2000; Manza, Codreanu et al. 2004). The induction of global sumoylation by ethanol and oxidative stress has also been demonstrated in yeast (Zhou, Ryan et al. 2004). Sumoylation, at least by SUMO-2 and SUMO-3, is therefore associated with stress response. In vivo, protein sumoylation levels are greatly increased in the brain, liver and kidney of hibernating ground squirrels (Spermophilus tridecemilineatus) (Lee, Miyake et al. 2007). There is also functional distinctions between the SUMO isoforms: SUMO-2 and SUMO-3 are better transcriptional repressors than SUMO-1 (Holmstrom, Van Antwerp, et al. 2003).

In addition, there is an apparent specificity for substrate among the SUMO paralogues. The first suggestion of substrate specificity was from the observation that RanGAP1 is preferentially modified by SUMO-1 (Saitoh and Hinchey 2000). However, the extent of paralogue specificity is currently unknown. Even if the potential for paralogue specific modifications has been raised by a proteomic study using tagged versions of each SUMO paralogue (Vertegaal, Andersen et al. 2006), the recently reported biology seems to speak otherwise. The SUMO-1 -/- mice are viable and show no obvious defect except for a mislocalization of RanGAP1 and impaired PML body formation. These mice show that SUMO-1 is dispensable and suggest that compensation by SUMO-2/3 is sufficient for normal mouse development (Evdokimov, Sharma et al. 2008; Zhang, Mikkonen et al. 2008).

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1.3.2.1 General mechanism

The basic mechanisms of SUMO conjugation are relatively well defined, although new findings are constantly improving our understanding of this actively growing field of research (Gill 2003; Seeler and Dejean 2003; Gill 2005; Hay 2005). The SUMO proteins are conjugated to their target lysine by a set of enzymes that is mechanistically analogous but distinct from that acting on the ubiquitin pathway (IL Kim, Baek et al. 2002; Verger, Perdomo et al. 2003; Johnson 2004; Matunis and Pickart 2005).

While the sequence identity between the SUMO proteins and ubiquitin is only about 18-20%, structure determination by nuclear magnetic resonance (NMR) revealed that SUMO proteins and ubiquitin share the ββαββαβ ubiquitin- fold structure (Bayer, Arndt et al. 1998; Jin, Shiyanova et al. 2001; Huang, Ko et al. 2004). The only differences reside in the presence of an extended N-terminal tail in SUMO but absent in ubiquitin as well as the charge of the exposed surface, which is highly negatively charged in SUMO and positive in ubiquitin (Bayer, Arndt et al. 1998; Liu, Jin et al. 1999). This difference in the exposed surface charge provides the mechanism for the specificity of the enzymes between the SUMO and ubiquitin conjugation pathways by electrostatic attraction or repulsion (Giraud, Desterro et al. 1998).

The first step of the SUMO conjugation pathway is the maturation of the SUMO protein by C-terminal hydrolases, also called Sentrin/SUMO-specific proteases (SENPs), removing the C-terminal end to expose a diglycine (GG) motif necessary for conjugation. The mature SUMO protein is then conjugated to its target lysine in a three step sequence of enzymatic reactions involving a heterodimeric E1 activating enzyme SAE1/SAE2 (Aos1/Uba2), a unique E2 conjugating enzyme (Ubc9; UBE2l) and several E3 ligases (RanBP2, the polycomb group protein Pc2, PIAS proteins) (Figure 1.9).

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Figure 1.9: The sumoylation pathway. The SUMO protein, after the maturation step by Sentrin/SUMO specific proteases (SENPs) exposing the GG motif for conjugation, is activated by the E1 heterodimer Aos1/Uba2 via ATP-dependent adenylation of the C- terminal glycine residue of SUMO and thioester bond formation with a cysteine residue on Uba2. Then, the SUMO moiety is transferred on to the Ubc9 also forming a thioester bond with cysteine 93 which in turn conjugates the SUMO moiety through formation of a covalent isopeptide bond between the C- terminus of SUMO and the ε-amino group of the target lysine with the help of E3 ligase. The SENPs also remove the SUMO moiety from the target. Adapted from Hay 2005.

The E3 ligases are dispensable in vitro, but greatly enhance the efficiency of SUMO conjugation and contribute to substrate specificity in vivo. The RanBP2 and the PIAS SUMO E3 ligases interact with Ubc9 to promote the transfer of SUMO to the target lysine on the substrate protein (Johnson and Gupta 2001; Kahyo, Nishida et al. 2001; Takahashi, Kahyo et al. 2001; Takahashi, Toh-e et al. 2001; Pichler, Gast et al. 2002). The E3 ligase activity of RanBP2 has been

44 Chapter I – Literature Review shown to be specific for RanGAP1 and Sp100 while inactive towards (Pichler, Gast et al. 2002).

The distinct subcellular localization of the SUMO E3 ligases is likely to contribute to the observed substrate specificity. For example, RanBP2 is localized to the nuclear pore complex while the PIAS proteins are found in the cytoplasm and nuclear bodies and Pc2 is localized to a distinct type of nuclear bodies called the polycomb group bodies (Sachdev, Bruhn et al. 2001; Kotaja, Karvonen et al. 2002; Pichler, Gast et al. 2002; Kagey, Melhuish et al. 2003). In addition to substrate selectivity, a role for RanBP2 in SUMO paralogue selectivity has also been proposed (Tatham, Kim et al. 2005).

Ubc9, the unique SUMO E2 conjugating enzyme, was initially thought to be non-specific. However Ubc9 has recently been shown to participate in the substrate specificity of sumoylation as well. While nothing is known concerning the phosphorylation status of Ubc9, several groups have reported that Ubc9 was sumoylated but the exact role of Ubc9 sumoylation had remain elusive until recently (Wohlschlegel, Johnson et al. 2004; Zhao, Kwon et al. 2004; Zhou, Ryan et al. 2004; Hannich, Lewis et al. 2005). Knipscheer et al. (2008) reported that Ubc9 autosumoylation was a determinant of substrate specificity. Sumoylated Ubc9 greatly enhances the sumoylation of sp100. On the other hand, SUMO modified Ubc9 is unable to promote the sumoylation of RanGap1 while the sumoylation of Ubc9 has no effect on its activity towards other SUMO substrates such as histone deacetylase 4, promyelocytic leukemia (PML) protein or thymine DNA glycosylase (Knipscheer, Flotho et al. 2008).

Simultaneously as the E3 ligases act as enhancers for the efficiency of the sumoylation event, several SENP cysteine proteases work actively to remove the SUMO protein from its substrate. The balance between the sumoylation and de- sumoylation rate contributes to the highly dynamic nature of this modification. The SENPs are responsible for both the prerequisite maturation step of the SUMO proteins as well as the removal from the target lysine. Similarly to the SUMO E3 ligases, substrate specificity is also observed among the SENPs and has also been

45 Chapter I – Literature Review associated with specific subcellular localization of the enzymes (Gill 2005). For example, SENP1 is found principally in the nucleoplasm and nuclear bodies, SENP2 is localized to the nuclear pore while SENP3 is found in the nucleolus and SENP6 in the cytoplasm (Gong, Millas et al. 2000; Kim, Baek et al. 2000; Nishida, Tanaka et al. 2000; Bailey and O'Hare 2002; Hang and Dasso 2002). To date, several SENPs have been identified in mammals and the characterization of each isoform awaits further study (Mukhopadhyay and Dasso 2007; Kim and Baek 2009).

Sumoylation induces conformational changes and creates a different interaction interface, which was defined functionally through extensive mutagenesis (Chupreta, Holmstrom, et al. 2005) and later shown to be site for interaction with SUMO interacting motifs (SIMs) by which the new interacting partner of SUMO modified proteins interacts non-covalently with the SUMO moiety. Since the discovery of SUMO modification, efforts have been directed toward the identification of partners non-covalently interacting with SUMO via SUMO interacting motifs (SIMs) (Minty, Dumont et al. 2000; Song, Durrin et al. 2004; Hecker, Rabiller et al. 2006). In addition to their role in complex formation with sumoylated proteins, the SIMs are also central to the SUMO conjugation pathway itself since the enzymes of the SUMO pathway, such as Ubc9 and E3 ligases, must bind both the substrate as well as the SUMO protein to achieve conjugation. For example, the E2 enzyme Ubc9 interacts with the target protein SUMO acceptor site Ψ KxE while the catalytic cysteine forms a thioester bond with the SUMO moiety. Simultaneously, SUMO modification of an adjacent sumoylation consensus site on Ubc9 allows for the formation of another interaction interface with the SUMO targets bearing a SIM. Indeed, the SIM is not the only determinant for the sumoylation-induced substrate discrimination since differences in target sumoylation efficiency by Ubc9 sumoylation have been observed also between non-SIM bearing substrates. The other determinants of substrate specificity are thought to be more complex and are not well defined yet (Knipscheer, Flotho et al. 2008).

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1.3.2.2. Biological Roles of sumoylation

SUMO conjugation affects a wide variety of biological processes and is required for cell viability in yeast and nematodes as well as in higher eukaryotes (Seufert, Futcher et al. 1995; Fraser, Kamath et al. 2000; Hayashi, Seki et al. 2002). A better definition of the roles of SUMO conjugation emerged with the increasing amount of identified SUMO substrates. Besides the individual target identification by manual consensus site detection within the proteins of interest, the greatest advance in the identification of SUMO substrates came through proteomics studies. The majority of the SUMO substrates identified by these studies were involved in transcriptional regulation, chromatin organization and RNA metabolism (Li, Evdokimov et al. 2004; Vassileva and Matunis 2004; Zhao, Kwon et al. 2004; Vertegaal, Andersen et al. 2006). Sumoylation has also been shown to be involved in cell cycle progression, mitosis, DNA repair and genome stability, and cancer (Seeler and Dejean 2003; Nacerddine, Lehembre et al. 2005; Kim, Choi et al. 2006; Mukhopadhyay and Dasso 2007; Seeler, Bischof et al. 2007; Zhang, Goeres et al. 2008).

Insights into the biological roles of sumoylation and its physiological importance, notably in development, also came from mice models. While the SUMO-1 null mice show no obvious developmental defect, the Ubc9 null mice die at the early postimplantation stage. A role for sumoylation in nuclear organization and integrity, PML nuclear body formation, chromosome segregation and condensation as well as RanGAP1 nuclear pore localization was identified by the study of the Ubc9 null embryo-derived cells (Nacerddine, Lehembre et al. 2005). Consistent with their dispensability in vitro, the PIAS family of E3 ligases also seems nonessential or redundant in vivo as PIASx or PIASy gene ablation in mice resulted in only minor defects in testis weight or interferon and Wnt signaling, respectively (Roth, Sustmann et al. 2004; Santti, Mikkonen et al. 2005). Also, inactivation of the SENP1 gene in mice results in placental abnormality leading to embryonic lethality. In addition, the levels of

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SUMO-1 conjugates were elevated in the mutant embryos, while the levels of SUMO-2/3 seemed unchanged (Yamaguchi, Sharma et al. 2005).

At the molecular level, the functions of sumoylation are diverse. They include modulation of protein localization and trafficking, protein stability by competition with ubiquitin-mediated degradation or by sequestration to nuclear bodies, but the common ground of all SUMO modification effects appears to involve a modulation of inter- or intramolecular interactions between proteins or with DNA (Chauchereau, Amazit et al. 2003; Bae, Jeong et al. 2004; Floyd and Stephens 2004; Chalkiadaki and Talianidis 2005; Desterro, Rodriguez et al. 1998).

1.3.3 Sumoylation in Transcriptional Regulation

A great proportion of identified SUMO substrates are involved in transcription (Golebiowski, Matic et al. 2009). Indeed, sumoylation being a specific, reversible, and highly dynamic process, it provides a potent means to rapidly regulate assembly and disassembly of transcriptional complexes. The role of sumoylation in transcriptional regulation is generally repressive, although direct and indirect activating effects have been reported as well. In several cases, the enzymes mediating sumoylation or de-sumoylation, such as Ubc9, E3 ligases or SENPs, have been shown to increase the transcriptional activity of various NRs, and even of their non-sumoylatable mutants, independently of their SUMO conjugating functions or via indirect effects on coregulators (Le Drean, Mincheneau et al. 2002; Kobayashi, Shibata et al. 2004; Sentis, Le Romancer et al. 2005; Zheng, Cai et al. 2006; Yokota, Shibata et al. 2007; Kaikkonen, Jaaskelainen et al. 2009).

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1.3.3.1 Sumoylation of Nuclear Receptors and coregulators

The list of sumoylated NRs is constantly growing and it is clear that sumoylation is an important and general mechanism of transcriptional regulation of NRs. As the molecular mechanisms for the repressive effect of NR sumoylation are just beginning to be identified, a lot of contradictory results are reported for the same NR. The differences in experimental conditions or cell lines used are likely to account for these discrepancies and suggest a more complex regulation of sumoylation than presently estimated. Indeed, sumoylation simultaneously affects several transcriptional properties of the same NR, and sometimes in a subtle manner that can easily go undetected in settings of overexpression. In addition, with so many sumoylated proteins involved in transcriptional regulation, and so far no other means other than overexpression to study the molecular mechanisms involved, the study of sumoylation is complex and can be easily misleading. The development of pharmacological tools such as specific inhibitors of SUMO E3 ligases or SENPs will be of great use for sumoylation studies.

However, there are some well-defined and consistently observed facts about NR sumoylation. These are described in the test, and the summary and NR- specific references are given in Table 1.3. For almost all sumoylated NRs, the sumoylation event has been associated with transcriptional repression upon mutation of the targeted lysines to arginines, while reports of activating effects of sumoylation for NRs are based on the overexpression of SUMO proteins and chances of indirect effects are high (Le Drean, Mincheneau et al. 2002). However, when the activating effects are lost upon mutation of the target lysine, this is good indication that the effect is direct as shown for the transcription factor p53 (Gostissa, Hengstermann et al. 1999; Rodriguez, Desterro et al. 1999).

In most cases, the sumoylation events occur within the NTD of NRs, except for ERα and SF-1, which are sumoylated in the hinge region. Of interest

49 Chapter I – Literature Review is the absence of a SUMO consensus motif in ERα, while the RXRs possess an atypical consensus ΨKPx in their NTD (Choi, Chung et al. 2006).

Table 1.3: Nuclear receptors sumoylation summary.

NR SUMO Sites Regulation Assay Effect Mechanism References isoform ERα SUMO-1 hinge: ligand-dependent in vitro; - unknown (Sentis, Le no endogenous Romancer et al. consens 2005) us site AR SUMO-1 NTD: ligand promoted in vitro; - synergy (Poukka, K386 in cells control Karvonen et al. 2000) PR SUMO-1 NTD: ligand promoted in vitro; - synergy (Chauchereau, K388 phosphorylation- in cells control Amazit et al. inhibited? 2003; Daniel, Faivre et al. 2007; Abdel-Hafiz, Dudevoir et al. 2009) GR all NTD: phosphorylation- in vitro; - synergy (Tian, Poukka et K297, induced in cells control al. 2002), K313 (Holmstrom, Van Antwerp et al. 2003), (Davies, Karthikeyan et al. 2008) MR SUMO-1 NTD: unknown in vitro; - synergy (Tallec, Kirsh et K89, in cells control al. 2003; Tirard, K399, Almeida et al. K494, 2007) K953R LBD: K953 LRH-1 SUMO-1 hinge: phosphorylation in vitro; - NB (Chalkiadaki and K173 in cells localization Talianidis 2005) K224 K289 K350 SF-1 SUMO-1 Hinge: unknown in vitro; - synergy (Komatsu, K119, in cells control, NB Mizusaki et al. K194 localization 2004; Lee, Lebedeva et al. 2005) PPARγ NTD: phosphorylation- In vitro - synergy (Ohshima, Koga K107 induced In cells control et al. 2004; Yamashita, Yamaguchi et al. 2004) LBD: Ligand-dependent In vitro, - T-rep (Pascual, Fong et K395 In cells al. 2005)

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Tr2 SUMO-1 NTD: phosphorylation- in vitro; - NB (Park, Hu et al. K238 induced in cells; localization, ê 2007; Gupta, Ho endogenous CoA and é et al. 2008) (testis, CoR brain) recruitment RXR SUMO-1 NTD unknown in cells - unknown (Choi, Chung et al. 2006) LXR SUMO-2 LBD Ligand-dependent In vitro; - T-rep (Ghisletti, Huang In cells et al. 2007) PNR SUMO-1 NTD unknown in vitro; - unknown (Onishi, Peng et in vivo al. 2009) (endogenous) NTD, N-terminal domain; LBD, ligand binding domain; -, repression; T-rep, transrepression; NB, nuclear bodies; CoA, coactivator; CoR, corepressor.

Mostly, the repressive effect of NR sumoylation is dependent on the presence of multiple response elements within the promoter, an effect referred to as the synergy control mechanism (described below). At the moment, the synergy control motifs seem to be associated principally, although not exclusively, with NTDs of the NRs, while the positively modified SUMO sites located within the LBDs are likely to be associated with a transrepression mechanism (Pascual, Fong et al. 2005; Ghisletti, Huang et al. 2007; Holmstrom, Chupreta et al. 2008). The latter mechanism was elegantly described for PPARγ and LXRs downstream of inflammatory response. In addition to its N-terminal sumoylation consensus motif, PPARγ harbors a C-terminal motif as well. However, in the absence of ligand, this site is buried into the LBD and therefore inaccessible for sumoylation. Upon agonist ligand binding, the conformational change allows the LBD SUMO site target lysine to become exposed and subsequently SUMO modified, resulting in interaction with the NCoR/HDAC3 corepressor complex and target gene transrepression by preventing the corepressor promoter clearance. LXRs are subjected to a similar transrepression mechanism. The difference residing in that HDAC4 promotes the modification of LXRs by SUMO-2, while it is PIAS1 which promotes the modification of PPARγ by SUMO-1 (Pascual, Fong et al. 2005; Ghisletti, Huang et al. 2007).

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In general, NR ligands promote their sumoylation. For example, sumoylation levels of the unliganded and antagonist-bound AR are low while the sumoylation levels of the agonist-bound AR are higher. This is also the case for PR and MR, while ERα sumoylation levels have been shown only in the presence of ligand (Poukka, Karvonen et al. 2000; Chauchereau, Amazit et al. 2003; Tallec, Kirsh et al. 2003; Sentis, Le Romancer et al. 2005; Man, Li et al. 2006; Yokota, Shibata et al. 2007; Kaikkonen, Jaaskelainen et al. 2009). On the other hand, sumoylation can affect the NR ligand sensitivity and the timing of ligand-induced responses (Abdel-Hafiz, Dudevoir et al. 2009).

The levels of a sumoylated protein (at steady-state), for example a NR, have been shown to be as low as 5% of the total pool of that NR, while the effect induced by mutation of the NR acceptor lysine to arginine are sometimes dramatic and would suggest that a bigger pool of the NR should be modified to match the intensity of the effect. Models that reconcile the outsized effect considering the low steady-state levels of sumoylated transcription factors and NRs have now been described. These include the recruitment of corepressors with chromatin- remodeling activity, such as HDACs, either directly or indirectly. For example, the CoRNR-like motif containing corepressor Daxx indirectly recruits HDACs specifically to sumoylated transcription factors or NRs (Girdwood, Bumpass et al. 2003; Yang and Sharrocks 2004; Kuo, Chang et al. 2005; Lin, Huang et al. 2006).

Corepressors with the same properties as Daxx are likely to serve as a platform to initiate the NR sumoylation-driven formation of larger corepressor complexes and the repressive state of the chromatin induced by the recruitment of these factors is thought to remain, even if the SUMO moiety is removed (Hay 2005). However, these interactions are most likely cell context- and NR-specific. Indeed, the repressive effect of Daxx on GR transcriptional activity is sumoylation independent (Holmstrom, Chupreta et al. 2008).

In addition, dissociation of coactivator complexes, sequestration of transcription factors to nuclear bodies, export from the nucleus or impairment of nuclear import, increased stability by competition with ubiquitin-mediated

52 Chapter I – Literature Review proteasomal degradation, modulation of sensitivity to ligand, synergy control and transrepression have also been described as mechanisms for the repressive effect of SUMO on transcription (IL Kim, Baek et al. 2002; Yang and Sharrocks 2004; Wu, Sun et al. 2006; Li and Shang 2007; Abdel-Hafiz, Dudevoir et al. 2009).

In addition, several NR coregulators are SUMO modified, such as the 3 members of the p160 coactivators family and the acetyltransferase p300 as well as the corepressors NCoR and RIP140 (Tiefenbach, Novac et al. 2006; Wu, Sun et al. 2006; Li and Shang 2007; Rytinki and Palvimo 2008). Sumoylation of coregulators was shown to induce changes in localization and nuclear mobility, and to affect complex formation and NR selectivity. For example, sumoylated SRC-1 displays an increased interaction with PR resulting in an increased PR transcriptional activity while sumoylation of SRC-2 facilitates its interaction with AR and thus enhances AR-mediated transcription (Kotaja, Karvonen et al. 2002; Kotaja, Karvonen et al. 2002; Chauchereau, Amazit et al. 2003). In contrast, sumoylation of the third SRC family member, SRC-3, has been shown to decrease its coactivator activity. Also, sumoylation of NCoR and RIP140 has been associated with an increase in their corepressor activity (Tiefenbach, Novac et al. 2006; Rytinki and Palvimo 2008).

The field of sumoylation is relatively young and a better picture of the mechanisms of sumoylation in transcriptional regulation of the NRs and their coregulators is emerging at an exponential speed. A better understanding of this actively evolving field can be expected in the near future.

1.3.3.3 Sumoylation, Synergy control and DNA binding

The discovery of the synergy control motif and its mechanism for repressing NR-mediated transcription preceded the identification of its role as a consensus site for sumoylation (Iniguez-Lluhi and Pearce 2000; Holmstrom, Chupreta et al. 2008).

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This mechanism of repression depends on the presence of multiple DNA response elements on the promoter for efficient repression and the repressive effect linearly correlates to the number of consecutive DNA response elements. The recruitment of a yet unknown synergy control factor by multiple SUMO moieties is at the core of the synergy control hypothesis (Figure 1.10) (Iniguez- Lluhi and Pearce 2000).

The synergy control factor is expected to interact non-covalently with the SUMO modified NR, potentially via one or more SIMs. Interestingly, Holmstrom et al. (2003) demonstrated, using SUMO-GAL4 fusions, that the recruitment of the SUMO moiety to the promoter is sufficient for synergy control and that the distance between the multiple elements was a determinant of the mechanism. Indeed, increasing the distance between the DNA elements alleviates the effect possibly by disrupting the interaction of the synergy control factor with multiple SUMO moieties (Holmstrom, Van Antwerp et al. 2003).

The synergy control mechanism is usually demonstrated by the mutation of the NR SUMO target lysine to arginine. Another demonstration of the synergy control mechanism was recently reported via desumoylation of AR by SENP1. SENP1 desumoylates AR and increases its transcriptional activity only on compound promoters while no effect of SENP1 is observed on the transcriptional activity of AR sumoylation site mutants. In addition, siRNA against SENP1 increased the endogenous AR transcriptional activity in prostate cancer cells and attenuated the androgen-induced expression of some endogenous AR target genes without affecting the AR promoter occupancy (Kaikkonen, Jaaskelainen et al. 2009). However, SENP1 was previously shown to increase AR transcriptional activity via desumoylation of HDAC1 rather than direct desumoylation of the AR itself (Cheng, Wang et al. 2004). These contradictory results suggest that other determinants such as promoter specificity, signaling or DNA-induced allosteric effects are potentially involved.

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Figure 1.10: The Synergy control hypothesis.

ERRα was used as a model. Signaling induces phosphorylation and subsequent phosphorylation of ERRα via a PDSM (see Chapter II). The sumoylated receptor recruits a synergy control factor (or maybe complex) (SCF/SCC) which decreases the promoter activation. The presence of multiple elements allows the recruitment of several SUMO moieties which are required for the interactions with the SIMs of the SCF/SCC. Whether or not the binding of coactivators is disrupted is unknown. (Adapted from Iñiguez-Lluhí and Pearce, 2000).

Consistent with this idea, the sequence of the DNA response elements has been shown to affect the synergy control mechanism. AR can bind to canonical inverted palindromic elements shared by several NRs. In addition, direct repeat elements specifically bound by AR exist and are referred to as selective androgen response elements (AREs). On the latter type of element, the AR synergy control mechanism is not observed; the AR sumoylation site mutants do not display an increased transcriptional activity relative to wild-type AR in the presence of

55 Chapter I – Literature Review multiple consecutive elements (Callewaert, Verrijdt et al. 2004). Indeed, it was demonstrated that the sole presence of multiple properly spaced DNA elements is not sufficient for synergy control, as stable DNA binding and an intact DBD and dimerization interface in the LBD are also crucial determinants to the synergy control effect (Iniguez-Lluhi and Pearce 2000; Holmstrom, Chupreta et al. 2008). That the binding of AR to canonical elements is more stable and the dimeric binding mode is favored is consistent with the absence of the synergy control effect on the non-canonical elements (on which NR binding does not require the DBD dimerization interface) (Schoenmakers, Verrijdt et al. 2000; Verrijdt, Schoenmakers et al. 2000). As none of these determinants are sufficient by themselves to support synergy control, and the combination of these is required, the synergy control mechanism is thought to be highly NR-, cell context- and promoter-dependent (Holmstrom, Chupreta et al. 2008).

Conversely, and in a similar manner as other PTMs, sumoylation can affect the DNA binding capacity and/or specificity of NRs. This effect has been recently described for SF-1. Using cell lines stably expressing tagged SF-1 or its K194R non-sumoylatable mutant, Yang et al. (2009) showed that the more active K194R mutant displayed elevated promoter occupancy without alteration of the cyclic pattern of SF-1 recruitment and clearance, suggesting that sumoylation of SF-1 impairs its DNA-binding capacity (Yang, Heaton et al. 2009). In addition, studies by the Ingraham’s group suggest that sumoylation of the SF-1 DBD at Lys119 impairs its DNA binding specifically at noncanonical SF-1 promoters (SUMO sensitive DNA binding) and suggest that sumoylation of SF-1 would result in the selective binding to a SUMO resistant subset of target gene promoters harboring canonical sites (Campbell, Faivre et al. 2008).

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1.3.4 Interplay between SUMO and other post-translational modifications

Post-translational modifications of proteins provide the cell with infinite possibilities for the regulation of a function or multiple functions accomplished by one protein. These regulatory possibilities further increase when multiple modification events occur on the same protein. Moreover, the interplay between multiple PTMs can be seen as a code dictating the transcriptional outcome in the same manner as multiple modifications of the histone tails are hypothesized to constitute the histone epigenetic code.

The interplay between sumoylation and phosphorylation is now well recognized (Yang, Jaffray et al. 2003; Guo, Yang et al. 2007). The first report of interplay between phosphorylation and sumoylation was by Hietakangas et al. (2003) for the transcription factor heat shock factor 1 (HSF-1). They showed that phosphorylation of serine 303, a phosphorylation event previously identified for its repressive effect on HSF-1 transcriptional activity, was a prerequisite for its sumoylation (Hietakangas, Ahlskog et al. 2003). This phosphorylation-dependent sumoylation mechanism was also found true for GR, PPARγ and the myocyte enhancer factor 2 family members, also consistent with the repressive effect previously associated with the phosphorylation event (Yamashita, Yamaguchi et al. 2004; Flavell, Cowan et al. 2006; Grégoire, Tremblay et al. 2006; Kang, Gocke et al. 2006; Shalizi, Gaudilliere et al. 2006; Davies, Karthikeyan et al. 2008).

This mechanism of phosphorylation-induced sumoylation was generalized by the discovery of an extended SUMO consensus site, the phospho-sumoyl switch motif Ψ KxExxSP also called phosphorylation-dependent sumoylation motif (PDSM) (Grégoire, Tremblay et al. 2006; Hietakangas, Anckar et al. 2006; Yang and Gregoire 2006). Also, another SUMO consensus subset was identified: the negatively charged amino acid-dependent motif (NDSM) (Yang, Galanis et al.

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2006). This motif acts in a similar manner as the PDSM, the difference residing in the constitutive presence of the negatively charged amino acid cluster (acidic patch) versus the regulatory possibilities provided by the PDSM. These two motifs are now considered powerful determinants for bona fide SUMO target prediction (Figure 1.11) (Guo, Yang et al. 2007).

Alternatively, phosphorylation can also prevent sumoylation. This phenomenon has been first demonstrated for the activator protein-1 (AP-1) transcription factors c-Jun and c-Fos. Phosphorylation of serine residues known to induce their transcriptional activity has been linked to reduced sumoylation levels (Muller, Berger et al. 2000; Bossis, Malnou et al. 2005). This phosphorylation- inhibiting effect of sumoylation was also observed for p53, ELK1 and PR (Yang, Jaffray et al. 2003; Lin, Ohshima et al. 2004; Daniel, Faivre et al. 2007). However, PR sumoylation was recently reported to be uncoupled from the phosphorylation event. Furthermore, Abdel-Hafiz et al. (2009) attributed the apparent effect of phosphorylation-repressed sumoylation reported by Daniels et al. (2008) to the different degradation rates of the various mutants in response to ligand rather than to interplay between the two modifications (Abdel-Hafiz, Dudevoir et al. 2009). The interplay between phosphorylation and sumoylation also regulates the activity of coregulators. The phosphorylation of AIB1 (SRC-3) has been reported to inhibit its sumoylation (Wu, Sun et al. 2006).

Recently, an opposite interplay was shown for the orphan NR SF-1 (Campbell, Faivre et al. 2008; Yang, Heaton et al. 2009). The hinge region of SF- 1 harbors a variation of the standard PDSM ΨKxExxSP motif with a spacing of 6 amino acids between the E and the SP (K194-S203) instead of two. This larger spacing in the SF-1 motif most likely accounts for the lack of K194 sumoylation- promoting effect of S203 phosphorylation. However, loss of sumoylation on K194 (by mutation to arginine) increases recruitment of cdk7 and phosphorylation of S203, a modification previously shown to have a stimulatory effect on SF-1 activity (Yang, Heaton et al. 2009). This study reports a mechanism for sumoylation-induced repression of SF-1 whereby sumoylation of SF-1 on K194

58 Chapter I – Literature Review impairs the recruitment of cdk7 and decreases the levels of the activating S203 phosphorylation, leading to a decrease in SF-1 transcriptional activity. The interplay or competition with other lysine PTMs is also important for the effects of sumoylation on their target function. The competition with ubiquitin is involved in increasing stability by preventing ubiquitin-mediated degradation while competition with acetylation is another means for sumoylation to repress transcriptional activity (Figure 1.11) (Bae, Jeong et al. 2004; Floyd and Stephens 2004).

Figure 1.11: Interplay between SUMO and other modifications. 1- The target lysine of the consensus sumoylation site can be sumoylated, but also 2- ubiquitinated or 3-acetylated. The presence of a proline residue favors acetylation. 4- Phosphorylation at various distances from the sumoylation site can impair its sumoylation, and alternatively 5- the phosphorylation within the PDSM and 6- the negatively charged acidic cluster composing the NDSM motifs are associated with promotion of sumoylation.

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For example, MEF2 is acetylated by CBP at the lysine residue within the SUMO consensus site. While acetylation of this lysine has a positive role on its transcriptional activity, sumoylation has a repressive role. Interestingly, MEF2 sumoylation is increased by HDAC4 and SIRT1 which, in addition to recruiting a kinase to phosphorylate the MEF2 PDSM, may also deacetylate the motif to allow sumoylation (Grégoire, Tremblay et al. 2006; Shalizi, Gaudilliere et al. 2006). In the PDSM, the presence of a proline residue in the core motif (ΨKxEP) has been identified as an important determinant of the phospho-SUMO-acetyl switch (Stankovic-Valentin, Deltour et al. 2007). This potential for competition between lysine modifications constitutes one of the main concerns about results obtained with the lysine to arginine mutants because the effect attributed to the lack of sumoylation might as well be caused by the alleviation of acetylation or ubiquitination, adding another layer of complexity to sumoylation studies.

1.4 Nuclear receptors and blood pressure regulation

High blood pressure affects one in three adults in the United States and one in five in Canada. In general, in only 5-10% of the cases of high blood pressure the direct cause can be identified, and this condition is referred to as secondary hypertension. In the remaining 90–95% of the cases, the cause is unknown and this condition is referred to as essential hypertension. Hypertension is the primary risk factor for stroke and a major risk factor for cardiovascular diseases, which altogether represent the first cause of death in the elderly and the second cause of death among 25-75 years old. From 1995 to 2005 the death rate from high blood pressure increased 25.2% and the prevalence of hypertension is predicted to further increase by 24% in developed countries and by 80% in developing regions by 2025 (Bakris and Ritz 2009; Lloyd-Jones, Adams et al. 2009; Statistics Canada 2009). The need for development of novel therapies and for the identification of new targetable players involved blood pressure control are vital. Therefore, dynamic research is dedicated to the identification of genes

60 Chapter I – Literature Review involved in blood pressure control, both to help in deciphering causes of essential hypertension as well as to ameliorate treatments of high blood pressure via identification of promising therapeutic targets. Recently, members of the NR superfamily received attention in this regard and their implication in the control of blood pressure at the molecular level in several organs is currently an active subject of research (Kuipers, van der Harst et al. 2008).

1.4.1 General principles of blood pressure regulation

The regulation of blood pressure (BP) is particularly complex and involves the coordinated response of multiple organs, but chiefly heart, vasculature and kidney. The role of the heart and kidney in blood pressure control is interrelated and, should the function of one of these organs fail, the function of both organs becomes affected over time in a spiral effect leading to the worsening of each others’ dysfunction. This mutual impact of cardiovascular and kidney disease constitutes the newly delineated Cardiorenal Syndrome and relates to the hemodynamic and humoral interrelation between heart and kidney, which impacts on blood pressure regulation (Sekhar, Lalmalsawma et al. 2009). In addition to the hemodynamic effects, determinants of the heart-kidney connection include NO-ROS balance, the RAAS, the immune system and the nervous system (Bongartz, Cramer et al. 2005).

The main hemodynamic determinants of BP regulation are cardiac output, vascular resistance and blood volume, and several organs control a part of the equation (Figure 1.13) (Ackermann 2004; Suzuki and Saruta 2004). The heart pumps the blood into the whole body and its role is therefore central to the cardiovascular system and in the establishment of BP levels. Defects in the ability of the heart to properly contract and generate enough force to drive the circulation of the blood to all organs (cardiac output), such as cardiac hypertrophy and heart failure, lead to a fall in BP levels which trigger hormonal/humoral adaptive cascades involving other organs in an attempt to restore BP. The vasculature and

61 Chapter I – Literature Review the central nervous system also participate in the tight regulation of systemic BP levels. Vasodilatation or vasoconstriction mechanisms are triggered in response to BP variations, and their role is to rapidly modulate vascular tone and resistance to adjust BP levels (Cain and Khalil 2002; Ponnuchamy and Khalil 2009). In addition, the central nervous system, principally the autonomic nervous system (ANS), regulates individual organ function and homeostasis. The ANS is involved in blood pressure control via reflex arcs originating from the baroreceptors located in the carotid sinus and aortic arch and regulating heart rate, cardiac contractility and coronary vascular tone. The vasculature of other organs, such as the kidneys and liver, is also subjected to nervous input (Lohmeier 2001; Ackermann 2004; Thrasher 2006).

Figure 1.12: The heart-kidney connection

Schematics of the heart-kidney connection representing the Guytonian hemodynamic parameters and the four major players of the kidney/heart connection (boxed). SNS, sympathetic nervous system; RAAS, Renin- Angiotensin-Aldosterone System; NO, nitric oxide; ROS, reactive oxygen species. Adapted from (Bongartz, Cramer et al. 2005).

Last but not least, the kidneys sense blood pressure variations, mostly the renal perfusion pressure via mechanical distension sensors, and are responsible for the adjustments of electrolyte balance, principally of salt and water retention/excretion, in order to maintain proper extracellular fluid and blood

62 Chapter I – Literature Review volumes. In addition, the kidneys are responsible for the secretion of renin, the rate-limiting enzyme of the Renin-Angiotensin-Aldosterone System (RAAS) (Figure 1.13).

Figure 1.13: Graphical representation of the systemic Renin- Angiotensin-Aldosterone System. The primary role of the systemic RAAS is to regulate blood pressure. The signals leading to an increase in BP are in red and to those leading to a decrease in BP are in green. The RAAS regulates the three major determinants of BP, cardiac output, vascular tone and resistance and extracellular fluid (ECF) volume (boxed). Agt, angiotensinogen; Ang, angiotensin; Ace, angiotensin-converting enzyme.

The RAAS is a key player in both long-term and short-term BP regulation, and the most effective antihypertensive therapies so far are those targeting the RAAS such as inhibitors of the angiotensin-converting enzyme, of the AT1 receptor and, more recently, direct renin inhibitors. Aldosterone is secreted by the adrenals in response to angiotensin II, the main effector peptide of the RAAS, and regulates renal salt handling either via activation of its receptor, the MR, or by triggering rapid non-genomic effects. The classical view of the RAAS has

63 Chapter I – Literature Review recently been updated. Indeed, prorenin and other angiotensin peptides (angiotensin III, IV and 1-7) were shown to be biologically active. In addition, a second ACE, called ACE2, was recently identified and is believed to antagonize the effects of ACE (Ryan, Black et al. 2000; Der Sarkissian, Huentelman et al. 2006; Fyhrquist and Saijonmaa 2008).

1.4.2 Transcriptional regulation of Renin and nuclear receptors

As the rate-limiting enzyme of the RAS, the regulation of renin has been one of the main focal points of attention of the RAS, along with the angiotensin converting enzyme and the angiotensin receptors, in terms of anti-hypertensive therapies (Berra and Miller 2009). Renin is produced and secreted principally by the renal juxtaglomerular cells although the existence of individual intra-organ local RAS regulating the local levels of Angiotensin II in several organs is a fairly novel concept gaining attention (Der Sarkissian, Huentelman et al. 2006; Raizada, Skipper et al. 2007). However, the circulating renin concentrations originate mostly from the kidney juxtaglomerular apparatus and are controlled at multiple levels, from transcription, translation, mRNA or protein storage to vesicular secretion. Indeed, there are two pathways for renin secretion. There is the constitutive pathway, in which the inactive and unprocessed form of renin, the prorenin, is constantly and continuously secreted to maintain a minimal availability at all times, and the regulated pathway, in which the processed active renin contained in the storage vesicles is rapidly released when BP falls (Hackenthal, Paul et al. 1990; Persson 2003; Persson, Skalweit et al. 2003; Persson, Ollerstam et al. 2004).

The main regulatory element for transcriptional regulation of renin is the renin kidney enhancer. This 242-bp regulatory element is located 2.9kb upstream of the mouse Ren1 gene and 11kb upstream the REN gene in humans (Yan, Jones et al. 1997; Shi, Black et al. 1999). This element is well characterized and several transcription factors and nuclear receptors regulating renin transcription are now

64 Chapter I – Literature Review identified. The renin enhancer is responsible principally for the basal renin levels but not for the tissue-specific expression of renin, and it is critical for the long- term control of basal BP as well as for replenishment of renin stores and response following a chronic in vivo stimulus of renin secretion (Markus, Goy et al. 2007). Surprisingly, deletion of this element in mice produces only mild hypotension and salt-sensitivity (Adams, Head et al. 2006). Although the renin enhancer is highly conserved between mice and humans, some differences exist between mice and men concerning the transcriptional regulation of renin.

In addition to the further upstream localization of the enhancer from the transcriptional start site in humans, one of the key mouse regulatory sites (DR10) is mutated in the human enhancer, leading in a lower activity of the human enhancer sequence in transiently transfected As4.1 cells (Yan, Jones et al. 1997). This is consistent with the higher plasma renin levels observed in mice versus humans. Indeed, a mutation in the human corresponding site transforms the sequence of the downstream half-site of the DR10 from AGGTCA to AGGCCA and the distance between it and the half-sites is 8 bp, creating an imperfect DR8 element instead of a DR10 in the human enhancer (Shi, Black et al. 1999).

In addition, some mouse strains such as FVB and Sv129 harbor two renin genes, Ren1 and Ren2, while other strains such as C57BL/6, similarly to humans, only possess one renin gene. This difference in the number of renin gene directly translates into strain differences observed in basal BP but inversely in terms of plasma renin levels and intra-renal renin content. The regulatory determinants of the Ren2 gene expression are completely unknown and this renders the use of mice strains with 2 renin genes inadequate for cardiovascular or renal studies (Lum, Shesely et al. 2004).

Several NRs regulating the mouse renin enhancer have been identified so far. RARα and EAR2 have been identified to regulate renin transcription via DR10 element on the renin enhancer. RARα, consistent with the positive effect exerted by retinoids on renin transcription, is an activator of renin transcription via the DR10 site and the orphan NR EAR2 has been identified as a repressor via

65 Chapter I – Literature Review the same site (Shi, Gross et al. 2001; Liu, Huang et al. 2003). The LXRs have been shown to positively regulate the expression of renin in response to cAMP/PKA signaling. However, their effect has been associated with a conserved element located in the proximal promoter region at approximately -600 bp, rather than to the DR10 site on the enhancer. This element is distinct from the classical LXR response element and is constituted by an overlapping cAMP and negative response element (CNRE) (Morello, de Boer et al. 2005).

VDR, consistent with the negative effect of vitamin D on renin expression, represses renin expression. Although initially thought to bind to the DR10 site, VDR has been recently shown to exert its negative effect via recruitment of NCoR to a CREB/CREM site a few bp upstream of the DR10, and also by interfering with the CNRE (Li, Kong et al. 2002; Yuan, Pan et al. 2007).

PPARγ has also been shown to regulate renin expression. Activation of the mouse Ren1 gene has been shown following treatment with the PPARγ ligand rosiglitazone in mouse primary juxtaglomerular cells. Although the DR10 element of the enhancer drives the maximal activity of the enhancer, the maximal activation of the renin gene by PPARγ has been mapped to a conserved Pal3 element located in the proximal promoter region (Petrovic, Black et al. 1996; Todorov, Desch et al. 2007; Todorov, Desch et al. 2008).

The cardiovascular effects of steroid hormones and thyroid hormone are well known. As such, the ERs, AR and MR may participate in the transcriptional regulation of the RAAS gens, notably renin (Kuipers, van der Harst et al. 2008). While the roles of these receptors on renin transcription were not directly addressed, the fluctuations of renin levels during the oestrus cycle and the decrease in renin expression after ovariectomy in rats suggest that the ERs and PRs are involved in regulating renin transcription. In addition, treatment of human chorionic cells with steroid hormones such as estradiol, progesterone, testosterone or aldosterone affects renin expression (Klar, Vitzthum et al. 2004).

The TRs were also described as positive regulators of renin transcription.

66 Chapter I – Literature Review

In addition, hyperthyroidism induces cardiac hypertrophy accompanied by an increase in renal renin mRNA and activation of the cardiac RAAS. Also, hyperthyroid rats show an increase in renin levels while conversely, renal renin expression and plasma renin activity is decreased in hypothyroid rats. In addition to the 3 putative thyroid hormone response elements (THRE) identified within the human renin promoter, the TR seems to function as an activator of renin transcription (Kobori, Ichihara et al. 1997; Ichihara, Kobori et al. 1998; Kobori, Hayashi et al. 2001).

1.4.3 Insights from mouse models

PPARγ has been highly studied regarding blood pressure regulation in terms of genetically modified mouse models. The reason for this is that the sensitizers PPARγ agonists rosiglitazone (Avandia®) and pioglitazone (Actos®), used in the treatment of type II diabetes, have been shown to lower BP in mice and human, although one clinical trial reported an increase in BP levels by pioglitazone (Willson, Lambert et al. 2001; Dobrian, Schriver et al. 2004; Ryan, Didion et al. 2004; Cho and Momose 2008; Zanchi, Chiolero et al. 2004). However, despite the positive transcriptional regulation of renin by PPARγ demonstrated in vitro and the lowering effects on blood pressure by the PPARγ agonists in vivo, the PPARγ null mice rescued from lethality show hypotension accompanied by an increase in plasma renin activity without changes in renal renin mRNA levels (Duan, Ivashchenko et al. 2007). In another model, where one allele of PPARγ has been replaced by a knock-in strategy with a PPARγ gene carrying the P465L dominant-negative mutation, a modest increase in blood pressure accompanied with an increase in angiotensinogen expression in inguinal adipose tissue and in Angiotensin II type 1 receptor expression in gonadal adipose was reported, while no change in renal or adipose renin mRNA were observed (Tsai, Kim et al. 2004). Whether PPARγ truly regulates renin expression in vivo is still an unresolved question. The effects of PPARγ on BP have been recently associated with vascular effects (Beyer, Baumbach et al. 2008; Halabi, Beyer et

67 Chapter I – Literature Review al. 2008). Indeed, in a vascular smooth muscle specific PPARγ mouse model, the hypotensive effects of the PPARγ deletion were shown to be associated with an impairment of vascular reactivity. These mice showed a decreased vasoconstriction response to norepinephrine and a concomitant increased vasorelaxation response to beta-adrenergic receptor agonists in vitro. This effect was associated with and an elevated beta2-adrenergic receptor expression and a more pronounced BP reduction in response to beta-adrenergic receptor agonists in vivo. In addition, the hypotension in the vascular smooth muscle cell specific PPARγ KO mice was more pronounced at rest and the circadian regulator BMAL1 was shown to be involved in the cyclic effect of PPARγ on BP circadian oscillations (Halabi, Beyer et al. 2008; Wang, Yang et al. 2008; Chang, Villacorta et al. 2009).

In addition to PPARγ mice models, very few other NR mice models were reported to display a basal blood pressure phenotype so far. The AR null mice, despite the known role of testosterone in the cardiovascular system and on renin transcription, showed no differences in BP or heart rate (Ikeda, Aihara et al. 2005). However, the method employed to reach these conclusions was the tail cuff method, suggesting that there could be BP differences at nighttime. For that reason, the use of telemetry is superior; the expression of the vast majority of the NRs varies according to the circadian rhythm in several organs (Yang, Downes et al. 2006). Contradicting observations are reported concerning the participation of ERα in blood pressure regulation. Although the effects of estradiol are mostly hypotensive, the ERα null mice show no difference in BP levels, while the ERβ display an elevated BP (Skavdahl, Steenbergen et al. 2005; Jazbutyte, Arias-Loza et al. 2008).

Little is known about the molecular mechanisms of transcriptional regulation of BP. Normal BP regulation and its related defects are complex, multigenic and involve multiple organs. Although the physiological regulation of blood pressure is relatively well understood at the hormonal/humoral level and from a mechanistic standpoint, the molecular determinants of the role of the NRs

68 Chapter I – Literature Review in BP regulation are largely unknown. Further studies on the role of the NR superfamily members in BP regulation are required, but the existing studies already suggest that the NRs may constitute interesting therapeutic targets for pharmacological interventions in essential hypertension as well as in secondary hypertension associated with obesity, metabolic syndrome or diabetes.

1.5 Goals of the research

Little is known concerning the regulation of the ERRs by post‐ translational modifications and their roles in several organs remain unknown. As these receptors constitute an interesting avenue for pharmacological treatment of several human diseases, it is crucial to substantiate and increase our understanding of the regulation of and by the ERRs. One goal of this study was to broaden the understanding of how post‐ translational modifications regulate the transcriptional activity of the ERRs. In parallel, considering the lack of knowledge surrounding the role of ERRα in the kidney, an organ in which ERRα is highly expressed, our other goal was to explore the physiological role of ERRα in this particular organ via a physiological genomics approach.

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Chapter II: Phosphorylation-dependent sumoylation regulates ERRα and γ transcriptional activity through a synergy control motif

PREFACE

Little is known concerning the regulation of the ERRs by post-translational modifications. As I undertook this study, no PTMs were yet associated to the N- terminal domains of the ERRs. This chapter describes the characterization of the ERRα and ERRγ phosphorylation-dependent sumoylation motif (PDSM) and its repressive role for the regulation of their transcriptional activity via synergy control. The SUMO E3 ligase PIASy was identified as an interacting partner of ERRα, which promotes its sumoylation in a PDSM dependent manner. Importantly, the ERRα phospho-sumoyl switch is functional in vivo, as shown by the dependency on an antibody specific for phosphorylated S19 in the PDSM to detect the endogenous sumoylated form of ERRα in mouse liver.

This chapter is a manuscript published in Molecular Endocrinology.

Chapter II - Manuscript

ABSTRACT

Interplay between different post-translational modifications of transcription factors is an important mechanism to achieve an integrated regulation of gene expression. For the estrogen-related receptors (ERR) α and γ, regulation by post-translational modifications is still poorly documented. Here we show that transcriptional repression associated with the ERR amino-terminal domains is mediated through sumoylation at a conserved phospho-sumoyl switch, ψKxEPxSP, that exists within a larger synergy control motif. Arginine substitution of the sumoylatable lysine residue or alanine substitution of a nearby phosphorylatable serine residue (serine 19 in ERRα) increased the transcriptional activity of both ERRα and γ. In addition, phospho-mimetic substitution of the serine residue with aspartate restored the sumoylation and transcriptional repression activity. The increased transcriptional activity of the sumoylation-deficient mutants was more pronounced in the presence of multiple adjacent ERR response elements. We also identified PIASy [protein inhibitor activated STAT (signal transducer and activator of transcription) y] as an interacting partner and a SUMO E3 ligase for ERRα. Importantly, analysis with a phosphospecific antibody revealed that sumoylation of ERRα in mouse liver requires phosphorylation of serine 19. Taken together, these results show that the interplay of phosphorylation and sumoylation in the amino-terminal domain provides an additional mechanism to regulate the transcriptional activity of ERRα and γ.

95 Chapter II - Manuscript

INTRODUCTION

Nuclear hormone receptors (NRs) play essential roles in the regulation of a wide array of developmental and physiological pathways. NRs are regulated by specific ligands with the exception of orphan members, for which no known natural ligands have been identified to date (Giguère 1999). The estrogen-related receptors α and β (ERRα and β, NR3B1 and NR3B2) were the first orphan nuclear receptors identified based on their high level of sequence identity with the estrogen receptor α (ERα, NR3A1) (Giguère, Yang et al. 1988). The ERR subfamily also contains a third isoform (ERRγ, NR3B3) (Eudy, Yao et al. 1998; Hong, Yang et al. 1999), and all three proteins possess the typical functional domains of nuclear receptors. The amino acid sequences of the three ERR isoforms are highly similar, sharing as expected the highest identity in the DNA- binding domain (DBD) and ligand-binding domain (LBD). However, unlike most related nuclear receptors, the three ERR isoforms also share considerable amino acid sequence similarity in their respective amino-terminal domain (NTD), suggesting that the NTD may influence the transcriptional acitivity of the three ERRs by common mechanisms.

Although the ERRs have no known natural ligand, the three receptors can be activated in a ligand-independent manner in the presence of coactivator proteins, most notably by members of the steroid receptor coactivator (SRC) and the peroxisome proliferator-activated receptor (PPAR) -γ coactivator 1 (PGC-1) families (Hong, Yang et al. 1999; Xie, Hong et al. 1999; Huss, Kopp et al. 2002; Schreiber, Knutti et al. 2003). Indeed, the elucidation of the crystal structures of ERRα LBD bound to a PGC-1α peptide, as well as that of ERRγ LBD bound to a SRC-1 or RIP-140 peptides have shown that the two ERRs assume the conformation of ligand-activated NRs in the apparent absence of a ligand, again suggesting that the presence of an agonist ligand may not be an obligatory requirement for the activation of the receptors (Greschik, Wurtz et al. 2002; Kallen, Schlaeppi et al. 2004; Wang, Zuercher et al. 2006). Thus, post-

96 Chapter II - Manuscript translational modifications could play a major role in the control of ERR transcriptional activity. Although ERRα has been shown to be a phosphoprotein (Sladek, Bader et al. 1997; Barry and Giguère 2005; Ariazi, Kraus et al. 2007), the current knowledge about ERR post-translational modifications is still very limited.

Phosphorylation of NRs as well as their coactivators is a well-documented mechanism involved in the control of their activities (reviewed in Shao and Lazar 1999; Rochette-Egly 2003; Weigel and Moore 2007). Similarly, sumoylation, the process of conjugating the small ubiquitin-related modifier (SUMO) protein, has been reported for NRs and coregulators, namely the androgen (Poukka, Karvonen et al. 2000), glucocorticoid (Tian, Poukka et al. 2002), progesterone (Chauchereau, Amazit et al. 2003; Man, Li et al. 2006), estrogen (Sentis, Le Romancer et al. 2005) and mineralocorticoid receptors (Tallec, Kirsh et al. 2003; Tirard, Almeida et al. 2007) as well as SF-1 (Chen, Lee et al. 2004; Lee, Lebedeva et al. 2005), PPARγ (Ohshima, Koga et al. 2004; Pascual, Fong et al. 2005; Ghisletti, Huang et al. 2007), LRH-1 (Chalkiadaki and Talianidis 2005), LXRs (Ghisletti, Huang et al. 2007), Tr2 (Park, Hu et al. 2007), the SRC coactivators (Kotaja, Karvonen et al. 2002; Chauchereau, Amazit et al. 2003; Wu, Sun et al. 2006), the histone acetyltransferase p300 (Girdwood, Bumpass et al. 2003), and the corepressor N-CoR (Tiefenbach, Novac et al. 2006). The exact function of sumoylation is still unknown although a growing body of evidence now supports the role of SUMO proteins in the negative regulation of transcription, mainly through corepressor recruitment or clearance related mechanisms (Seeler and Dejean 2003; Verger, Perdomo et al. 2003; Gill 2005; Hay 2005; Pascual, Fong et al. 2005; Ghisletti, Huang et al. 2007).

SUMO proteins are conjugated to a lysine residue within the core consensus site ψKxE, where ψ represents a hydrophobic residue and x is any residue. Sumoylation is carried out by a set of enzymes that are distinct from those acting on the ubiquitin pathway (IL Kim, Baek et al. 2002; Schwartz and Hochstrasser 2003) and consist of a SUMO-activating heterodimeric complex

97 Chapter II - Manuscript consisting of Aos1 and Uba2 (E1), the single E2-type conjugating enzyme UBC9, and E3-like proteins, which serve to increase the affinity between UBC9 and the substrates by bringing them in a close proximity to UBC9 with a catalytically favorable orientation. In vitro, however, the E3-like activity is not necessary for sumoylation to occur, as E1 and UBC9 are sufficient to induce sumoylation. Three types of SUMO E3 ligases have been described: RanBP2 (a component of nuclear pore complex), the Polycomb protein Pc2, and members of the protein inhibitor of activated signal transducer and activator of transcription (PIAS) family (reviewed in Melchior, Schergaut et al. 2003).

A subset of consensus SUMO conjugation motifs has recently been extended to include ψKxExxSP, establishing new sumoylation sites that are phosphorylation-dependent and thus referred to as phosphorylation-dependent sumoylation motifs (PDSMs) or phospho-sumoyl switches (Grégoire, Tremblay et al. 2006; Hietakangas, Anckar et al. 2006; Yang and Gregoire 2006). In addition, this sequence also corresponds to the synergy control (SC) motif Px(0-

3)[I/V]K[Q/T/S/L/E/P]Ex(0-3)P, a protein determinant that was identified before the sumoylation consensus and initially proposed to modulate higher-order interactions among transcription factors, including nuclear receptors and their coregulators (Iniguez-Lluhi and Pearce 2000; Subramanian, Benson et al. 2003). The efficiency of the transcriptional repression exerted by sumoylation of transcription factors within a SC motifs has been proposed to depend on the number of consecutive DNA response elements present in the target promoter (Holmstrom, Van Antwerp et al. 2003).

Here we present evidence that ERRα and γ are sumoylated within their respective NTDs and that this modification is induced by phosphorylation of a functional PDSM. Our results show that sumoylation negatively affects ERRα and γ transcriptional activity without altering subcellular localization, DNA binding properties or interactions with the coactivator PGC-1α. The PDSM within the NTD of the ERRs also controls synergy in the presence of multiple ERR response element (ERRE) and consequently, sumoylation-deficient receptor

98 Chapter II - Manuscript variants are more potent activators of transcription on the polymorphic ESRRA promoter containing multiple copies of the ERRE. We have also demonstrated that PIASy interacts with and possess an E3-ligase activity towards ERRα. Using a phosphorylation-specific antibody, we found that phosphorylation of serine 19 is required for sumoylation of endogenous ERRα in mouse liver. Thus, the interplay of phosphorylation and sumoylation at the SC motif provides a novel mechanism to regulate the transcriptional activities of ERRα and γ.

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RESULTS

Sumoylation of ERRα and γ

Scanning of the amino acid sequence of the ERR isoforms led to the identification of two consensus attachment sites for SUMO proteins (lysines 14 and 403) in ERRα and three (lysines 40, 360 and 439) in ERRγ (Figure 2.1A). Interestingly, the NTD sumoylation sites (lysine 14 in ERRα and 40 in ERRγ) were found to be embedded within a PDSM motif (Grégoire, Tremblay et al. 2006; Hietakangas, Anckar et al. 2006; Yang, Galanis et al. 2006) (Figure 2.1B). To determine whether the two ERR isoforms are targets of sumoylation, we first cotransfected HEK293 cells with either a -ERRα or a Flag-ERRγ tagged expression vector along with an HA-SUMO2 plasmid. Immunoprecipitation with either anti-myc or anti-Flag antibodies followed by immunoblotting using an anti-HA polyclonal antibody suggested that both ERRα (Figure 2.1C) and γ (Figure 2.1D) are modified by SUMO2.

Identification of sumoylation sites

To identify the sumoylation sites in ERRα and γ, potential target lysines were mutated to arginines and the point mutants were subjected to in vitro sumoylation assays with recombinant SUMO1 and SUMO3 proteins. As shown in Figure 2.2A and B respectively, only the ERRα K14R and ERRγ K40R mutants displayed significantly decreased levels of sumoylation while the remaining point mutants (ERRα K403R, ERRγ K360R and K439R) were sumoylated to a level similar to the wild-type receptors. In the absence of the recombinant E1 activating enzyme, the sumoylated forms were totally absent. The absence of one band in the K403R

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mutant in comparison to wild-type ERRα suggests residual sumoylation of lysine 403 (Figure 2.2A).

Endogenous SUMO proteins are subjected to a maturation step before they can be conjugated to the acceptor protein. The extreme carboxy-terminal end is cleaved by SUMO-specific proteases to expose a diglycine motif necessary for conjugation and the removal of the diglycine motif prevents the conjugation (Tatham, Jaffray et al. 2001). To determine if the same site would be subjected to sumoylation in cells, the KR point mutants of ERRα and γ were transfected in HEK293 cells, along with an HA-SUMO2GG activated form and UBC9 or with HA-SUMO2ΔGG and UBC9-C93S dominant negative forms and 80 µg of extracts were subjected to Western blot analysis using a anti-ERRα or anti-Flag M2 antibody. As shown in Figure 2.2C and D, slower migrating bands were present when the activated form of SUMO2 and UBC9 were introduced in the HEK293 cells. Consistent with this, the bands were absent when the dominant negative forms were used, demonstrating that the slower migrating bands were the sumoylated forms of the receptors. Moreover, the slower migrating band was significantly decreased for ERRα K14R (Figure 2.2C) and absent for ERRγ K40R mutants (Figure 2.2D), confirming that the NTD of both ERR isoforms is the main SUMO attachment site. The residual sumoylation in the ERRα K14R lane may suggest a possible modification of lysine 403 (Figure 2.2C).

Increased transcriptional activity of sumoylation-deficient mutants of ERRα and γ requires multiple DNA response elements

To assess whether sumoylation of the two ERR isoforms affects their transcriptional activity, we next transfected HeLa cells with either ERR wild-type forms and NTD KR mutants in the presence or the absence of the coactivator PGC-1α together with the reporter construct 3xESRRApromoter-LUC. This

101 Chapter II - Manuscript reporter is driven by the promoter of the gene encoding ERRα (ESRRA) which is a known target of both ERRα (Laganière, Tremblay et al. 2004) and γ (Liu, Zhang et al. 2005). As shown in Figure 2.3A, in the presence of PGC-1α, the mutant ERRα K14R displays a greater transcriptional activity than its wild-type counterpart at both 50 and 100 ng. In Figure 2.3B, ERRα induced only a modest transcriptional response in the absence of PGC-1α. However, a more significant induction of basal luciferase activity was observed with the ERRα K14R mutant. As previously reported (Huss, Pineda Torra et al. 2004; Laganière, Tremblay et al. 2004), introduction of PGC-1α in HeLa cells stimulated the basal activity of the ESRRA promoter due to the presence of endogenous ERRs in these cells, but also stimulated the activity of exogenous ERRα. In the presence of PGC-1α, the K14R mutant displayed a marked increase in transcriptional activity as compared to the wild-type receptor. Similar results were obtained with ERRγ and the K40R mutant (Figure 2.3C). The increased activity of NTD KR mutants was not caused by differences in protein expression, as wild-type and mutant constructs were expressed at similar levels for both ERRα and γ (Figure 2.3B and C, insets).

In order to avoid interference by endogenous ERRα in the transfection assay, we performed the same experiment using ERRα-null mouse embryonic fibroblasts immortalized with SV40 large T antigen (ERRα-null MEFs-T). In these cells, introduction of PGC-1α had only a minor effect on the basal activity of the reporter construct (Figure 2.3D) and both wild-type ERRα and the K14R mutant failed to display basal transcriptional activity on ESRRA driven reporter. However, both proteins showed a strong transcriptional response to the presence of PGC-1α, and the K14R mutant displayed much higher transcriptional activity than the wild-type receptor. To rule out any other interference by endogenous ERR isoforms on the ESRRA driven reporter, we also assessed the activity of Gal4 DBD-ERRα and -K14R mutant fusion proteins on a 2 copy UAS-tk-LUC reporter (Figure 2.3E). In this context, the sumoylation-deficient ERRα K14R mutant displayed a synergistic response to the presence of PGC-1α.

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As depicted in Figure 2.1C, the consensus motif for sumoylation in both ERRα and γ also overlaps with an SC motif responsible for regulation of synergy control in the presence of multiple response elements (Iniguez-Lluhi and Pearce 2000). We therefore wanted to determine whether the synergy control mechanism was regulating the transcriptional activity of ERRα. As shown in Figure 2.3F, the greater transcriptional activity of the ERRα K14R mutant was observable solely on the 3xERRE-TK-LUC reporter constructs, while on the reporter bearing only a single copy of the ERRE, the transcriptional activity was lower and similar for both ERRα and ERRα K14R. Related to this observation, we have previously demonstrated that a naturally occuring polymorphism within the ESRRA promoter changes the number of consecutive ERREs present in the distal region of the promoter from 1 to 4 (Laganière, Tremblay et al. 2004).

We next tested whether changing the sumoylation status of the ERRs affects the regulation of the polymorphic ESRRA promoter. Results presented in Figure 2.3F and G show that, in the presence of PGC-1α, the KR NTD mutants were more potent on the reporter construct driven by the ESRRA promoter containing 3 copies of the ERR response elements than the reporter containing only 2 copies of the element. We next tested whether the mutations of lysine 14 and 40 to arginines could, by themselves, change the interaction between the coactivator PGC-1α and the receptor proteins. As shown in Figure 2.3I, all four proteins interacted with PGC-1α to a similar level when assayed by GST- pulldowns using equal amounts of immobilized GST and GST-PGC-1α/1-250. Taken together, these results demonstrate that sumoylation of ERRα and γ represses the transcriptional activity of the receptors. Therefore, the regulatory effect of ERR sumoylation would be of greater importance for the individuals expressing promoters bearing multiple copies of the ERRE.

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Sumoylation site mutants display wild-type nuclear localization and DNA binding properties

Sumoylation has been shown to affect subcellular localization of transcription factors, an effect that has been associated with repression due to sequestration within nuclear bodies (Gill 2003; Verger, Perdomo et al. 2003). To verify if sumoylation of ERRα affects its cellular localization, we transfected HeLa cells with GFP constructs for ERRα (Figure 2.4A, top row) and the K14R mutant (Figure 2.4A, middle row). The nuclear localization remained unchanged between the ERRα wild-type and K14R mutant (Figure 2.4A, first column). Furthermore, no targeting to nuclear bodies was observed for both ERRα variants as shown by the absence of colocalization between the HA-SUMO2 induced nuclear bodies (Figure 2.4A, second and third columns). Sumoylation is also known to modulate the DNA binding properties of certain transcription factors (Gill 2003; Seeler and Dejean 2003; Verger, Perdomo et al. 2003). Thus, we assessed whether sumoylation affects the DNA binding properties of ERRα. Using nuclear extracts from HEK293 cells transfected with ERRα wild-type, K14R or K403R mutants, we observed no change in the DNA binding pattern as well as in supershift generated by an ERRα antibody or a purified GST-PGC- 1α/1-250 fusion protein (Figure 2.4B).

We also compared the DNA binding pattern of in vitro sumoylated ERRα, with both SUMO1 and SUMO3 (Figure 2.4C). We observed that sumoylated ERRα binding to DNA was as efficient than that of the K14R mutant. The same experiment was then reproduced with the addition of GST-PGC-1α in the DNA binding reaction, and the result showed that the sumoylated form of ERRα was similarly supershifted by PGC-1α on DNA (Figure 2.4D). As the synergy control mechanism is dependent on the presence of more than one copy of the response element, we assessed the DNA binding properties of non-sumoylated and sumoylated ERRa on a tandem element probe. We observed no difference in the

104 Chapter II - Manuscript binding pattern (Figure 2.4E) or the intensity of binding (Figure 2.4F) with increasing amount of the non-sumoylated and sumoylated ERRa.

Phosphorylation of ERRα on serine 19 is essential for sumoylation of lysine 14

Interplay with phosphorylation events on a target protein has been shown to modulate sumoylation (Hietakangas, Ahlskog et al. 2003; Yang, Jaffray et al. 2003; Yamashita, Yamaguchi et al. 2004). Moreover, a PDSM has recently been proposed to be present in sumoylated proteins and to constitute a general mechanism for regulating sumoylation (Grégoire, Tremblay et al. 2006; Hietakangas, Anckar et al. 2006; Yang and Gregoire 2006). As described above, the NTD sumoylation sites of ERRs possess this consensus motif (Figure 2.1B). We then assessed the effect of ERRα S19A and S19D mutations (Figure 2.5A and 2.5B) as well as the ERRγ S45A and S45D mutations (Figure 2.5C) on the transactivation properties of the receptors. Using the 3xESRRApromoter-LUC reporter construct in HeLa cells (Figure 2.5A and 2.5C) or the 2 copy UAS-tk- LUC reporter in COS-1 cells (Figure 2.5B), we observed that the ERRα S19A and ERRγ S45A mutants displayed increased transcriptional activity compared to their wild-type counterparts. In contrast, the phospho-mimetic mutants ERRα S19D and ERRγ S45D had similar transcriptional activity as the wild-type receptors.

This result is in agreement with the sumoylation level of wild-type and mutant ERRs. HEK293 cells were transfected with the ERR point mutants along with an HA-SUMO2GG activated form and UBC9 or with HA-SUMO2ΔGG and UBC9-C93S dominant negative forms and 80 µg of extracts were subjected to western blot analysis using a anti-ERRα or to an anti-FlagM2 agarose beads immunoprecipitation and western analysis with anti-flag antibody. We observed that the sumoylation capacity was impaired in a similar manner as the KR mutants and after alanine mutations of serine 19 for ERRα (Figure 2.5D) and of serine 45

105 Chapter II - Manuscript for ERRγ (Figure 2.5E). Conversely, the phospho-mimetic mutations of these serines to aspartic acid residues restored the capability for sumoylation for both ERRα (Figure 2.5D) and γ (Figure 2.5E). Taken together, these results support a phosphorylation-dependent sumoylation mechanism of the ERRα and γ NTDs.

PIASy interacts with and induces sumoylation of ERRα

We next sought to identify the E3 ligase that generates ERRα sumoylation. Of potential SUMO E3 ligase candidates tested, we observed that PIASy was the most effective in promoting the sumoylation of ERRα in cultured cells (Figure 2.6A). HEK293 were cotransfected with pCMV5-Flag-PIASy and myc-ERRα WT or K14R mutant expression plasmid. The coexpression of Flag- PIASy with ERRα resulted in covalent modification of myc-ERRα with endogenous SUMO. Considering that in vitro, both SUMO1 and SUMO3 were able to modify the ERRs, the endogenous SUMO modifier in this setting could be either one of the SUMO isoforms. Following immunoprecipitation on FlagM2 agarose, PIASy was shown to interact with both the non-sumoylated and the sumoylated form of ERRα (Figure 2.6A). In a similar manner, PIASy also interacted with an apparent similar affinity with the ERRα sumoylation-deficient mutant K14R (Figure 2.6A). The cotransfection of flag-PIASy along with the ERRα and phospho-sumoyl switch mutants markedly stimulated the sumoylation of ERRα. In agreement with the role associated with serine 19, the S19A mutant showed no modified form in the presence of the ligase, while the S19D mutation restored the modification (Figure 2.6B). Considering the high potential for ERRα multi site phosphorylation events suggested by large-scale studies of HeLa cells and mouse liver nuclear phosphoproteins (Beausoleil, Jedrychowski et al. 2004; Villen, Beausoleil et al. 2007), mutants of the adjacent serine 22 (S22A and S22D) were also tested for sumoylation levels and show no difference in comparison to the wild-type receptor (Figure 2.6B). Interestingly, the

106 Chapter II - Manuscript transcriptional activity of the mutants shows good correlation with the levels of sumoylation observed in Figure 2.6B (Figure 2.5A and 2.6C).

Phosphorylation-dependent sumoylation of ERRα in vivo

We next wanted to determine whether the ERRα phospho-sumoyl switch was functional in vivo. Therefore, we generated a custom made rabbit antiserum directed against the phosphorylated S19 of ERRα. The relative affinity of the anti-ERRα and the anti-ERRα pS19 antisera was assessed by dot blot against the immobilized phosphorylated synthetic peptide antigen and its non-phosphorylated homolog. We observed that the anti-ERRα recognized this particular epitope very weakly in comparison to the anti-ERRα pS19 antisera. Also, the anti-ERRα pS19 antisera, although detecting slightly the non-phosphorylated antigen, displayed a strong preference for the immobilized phospho-peptide (Figure 2.S1).

Mouse liver extracts prepared in a phosphorylation and sumoylation preserving buffer were subjected to parallel immunoprecipitations using the anti-ERRα as well as the anti-ERRα pS19 antisera along with corresponding pre-immune serum. Subsequent Western blot analysis with the anti-ERRα antiserum confirmed the high levels of ERRα protein in mouse liver and the effective immunoprecipitation of the non-sumoylated form of ERRa by the anti-ERRa antiserum but not as effectively, although present, by the anti-ERRa pS19 antibody (Figure 2.7A). In both immunoprecipitation conditions (Figure 2.7A, left side with anti-ERRα and right side with anti-ERRα pS19), the higher band corresponding to sumoylated ERRα could not be detected by the anti-ERRα. This is because the anti-ERRα displays a weaker recognition of the serine 19 epitope, involved in the present mechanism (Figure 2.7A). To overcome this, a commercially available anti-ERRα antibody recognizing the C-terminus of hERRα (amino acids 339-364) was used to validate the identity of the different bands. Western blot analysis with this antibody revealed a weak higher band after

107 Chapter II - Manuscript immunoprecipitation with the anti-ERRα antibody (Figure 2.7B, left side). Interestingly, the higher band corresponding to sumoylated ERRα was more abundant when the immunoprecipitation was performed using the anti-ERRα pS19 antibody (Figure 2.7B, right side).

Remarkably, subsequent Western blot analyses with anti-ERRα pS19 antiserum (Figure 2.7C) and anti-SUMO2 (Figure 2.7D) antibodies revealed only a SUMO2 modified form of ERRα when immunoprecipitation was performed with the phospho-specific antisera and not with the anti-ERRα. Also, in agreement with the sumoylation promoting role of serine 19, Figure 2.7C (right side) demonstrates that the pool of serine 19 phosphorylated ERRα is completely sumoylated, as depicted by the shift in molecular weight observed. The relative abundance of the sumoylated versus the non-sumoylated species is usually low. The detection of endogenously sumoylated proteins is therefore difficult. The anti-ERRα pS19 antiserum also slightly detected the lower ERRα band in the input lanes (Figure 2.7C). The reason for this is the slight recognition of the non- phosphorylated ERRα, in agreement with the much lower affinity for the non- phosphorylated peptide (Figure 2.S1). However, after enrichment for the serine 19 phosphorylated ERRα by immunoprecipitation with the anti-ERRα pS19, the only band detected is the sumoylated ERRα.

Furthermore, the higher molecular weight band observed in Figure 2.7B and 2.7C (right side) co-migrates with the one observed when Western blot analysis was preformed with the anti-SUMO2 antibody (Figure 2.7D, right side). Taken together these results not only indicate that ERRα is sumoylated in vivo but demonstrate the requirement of serine 19 phosphorylation for efficient sumoylation of the NTD.

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DISCUSSION

In this report, we identified ERRα and γ as bona fide targets of sumoylation. We showed that the NTDs of ERRα and γ contain a phosphorylation-dependent sumoylation consensus motif ψKxExxSP, also known as PDSM (Grégoire, Tremblay et al. 2006; Hietakangas, Anckar et al. 2006; Yang and Gregoire 2006), that is conserved from Drosophila to humans and is also present in the ERRβ isoform. Moreover, the PDSM is embedded within a SC motif (Figure 2.1B), an extended motif determinant proposed to regulate higher- order interactions among transcription factors (Iniguez-Lluhi and Pearce 2000). Our study demonstrated that the three overlapping motifs are functional, placing the ERR isoforms in a unique category within the superfamily of NRs.

Sumoylation is generally associated with transcriptional repression. In agreement with this observation, we have shown that mutation of the main SUMO acceptor sites in the ERRα and γ NTDs increases their transcriptional activities. Importantly, the greater transcriptional activity of the NTD KR mutants can be observed only in the presence of PGC-1α. This reinforces the notion that ERRα is a major conduit of PGC-1α activity (Huss, Kopp et al. 2002; Schreiber, Knutti et al. 2003; Laganière, Tremblay et al. 2004; Mootha, Handschin et al. 2004; Schreiber, Emter et al. 2004; Willy, Murray et al. 2004). In fact, the presence of a PGC-1 coactivator family member appears to be crucial for ERRα activation and has been proposed to act as a “protein ligand” for the ERRs (Kamei, Ohizumi et al. 2003). The apparent dependency on PGC-1α for the effect observed in this study mirrors the importance of this cofactor in ERR function. Furthermore, since PGC-1α activates ERR function via the LDB (Gaillard, Dwyer et al. 2007), our results suggest that intramolecular interactions between the NTD and the LBD may play an important role in controlling ERR transcriptional activity.

SUMO conjugation has been shown to negatively regulate the activity of transcription factors through the regulation of different molecular properties of the target protein. These effects seem to be specific to each target protein, and vary

109 Chapter II - Manuscript from reduction of DNA binding, to alteration of protein stability or sequestration to subnuclear bodies (Seeler and Dejean 2003; Verger, Perdomo et al. 2003; Gill 2005). For the ERRs, it seems that properties such as localization, coactivator recruitment, DNA binding, and protein stability are not affected by sumoylation. Therefore, these may not account for the repressive effect of sumoylation suggested by the increased transcriptional activity of the mutated ERR proteins (Figure 2.3 and 2.5). Instead, we showed that sumoylation regulates ERR transcriptional activity via a SC mechanism. Indeed, the increased transcriptional activity of the ERR NTD sumoylation-deficient mutants on the ESRRA promoter containing 3 copies of the ERR response element suggests that sumoylation of the SC motif could have a direct impact on the expression of ERRα itself. Thus, sumoylation of ERRα may be an important component for the fine tuning of the autoregulatory loop regulating ERRα expression in the presence of PGC-1α. This regulatory mechanism is also likely to influence the expression of other ERR target genes that harbor multiple ERREs in their promoter/regulatory regions.

In the absence of a known natural ligand, the regulation of ERRα and γ transcriptional activities by post-translational modifications becomes of crucial importance. To our knowledge, the only identified phosphorylation site within the ERRs so far is threonine 124 of ERRα, which lies within a consensus PKCδ phosphorylation site (Barry and Giguère 2005). In addition, ERRα has also been shown to be phosphorylated following epidermal growth factor treatment in MCF-7 cells (Barry, Laganière et al. 2006) and hyperphosphorylated in BT-474 cells, a human breast cancer cell line over-expressing the oncogene ErbB2 (Ariazi, Kraus et al. 2007). Moreover, a large-scale characterization of HeLa cell nuclear phosphoproteins confirmed the phosphorylation status of the endogenous ERRα, identifying serine 19 as one of multiple phosphorylated residues within the NTD by tandem mass spectroscopy (Beausoleil, Jedrychowski et al. 2004). Using phosphorylation mimicking mutants of ERRα and γ, we have provided evidence for the importance of the phosphorylation status of serines 19 and 45 for sumoylation of the ERRα lysine 14 and ERRγ lysine 40, respectively (Figure

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2.6). The negative charges created by the phosphorylation events close to the SUMO acceptor site can be compared to the recently identified negatively charged amino acid-dependent sumoylation motif (NDSM). The NDSM mechanism relies on the presence of negatively charged amino acids in close proximity to the sumoylation site to enhance the sumoylation. The ERR isoforms also possess such an acidic patch, although it is located outside of the limit of the identified NDSM (Yang, Galanis et al. 2006). The main difference with the ERRs occurs in the possibilities offered by a regulated phosphorylation event as opposed to the fixed enhancement provided by the presence of a negatively charged glutamate residue.

Our data, together with other recent reports showing phosphorylation- dependent sumoylation of MEF2 family members (Flavell, Cowan et al. 2006; Grégoire, Tremblay et al. 2006; Kang, Gocke et al. 2006; Shalizi, Gaudilliere et al. 2006), HSF-1 (Hietakangas, Ahlskog et al. 2003), and PPARγ (Yamashita, Yamaguchi et al. 2004) strengthen this model as a common signaling-dependent regulating mechanism for sumoylation of transcription factors and demonstrate the functionality of the phospho-sumoyl switch in vivo. Furthermore, the observation that PPARγ is the only member of the nuclear receptor family sharing this PDSM with the three ERR isoforms also suggests a potential link between phosphorylation-dependent sumoylation and metabolic control by NRs. As both ERRα and γ have emerged as essential regulators of energy metabolism (Sladek, Bader et al. 1997; Vega and Kelly 1997; Carrier, Deblois et al. 2004; Huss, Pineda Torra et al. 2004; Mootha, Handschin et al. 2004; Schreiber, Emter et al. 2004; Wende, Huss et al. 2005; Herzog, Cardenas et al. 2006; Alaynick, Kondo et al. 2007; Dufour, Wilson et al. 2007; Huss, Imahashi et al. 2007; Villena, Hock et al. 2007), the identification of the signaling pathways regulating ERR sumoylation and the study of how phosphorylation-dependent sumoylation specifically affect the expression of metabolic genes will be of importance for our understanding of pathologies such as cardio-vascular diseases, diabetes and obesity.

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MATERIALS AND METHODS

Plasmids and constructs

The ΔESRRApromoter-LUC, 2xESRRApromoter-LUC and the 3xESRRApromoter-LUC reporter constructs as well as the pCMX-ERRα expression plasmid have been previously described (Laganière, Tremblay et al. 2004). The expression vectors for wild-type SUMO2 and wild-type UBC9 were previously described (Tatham, Jaffray et al. 2001; Bernier-Villamor, Sampson et al. 2002). The shorter forms of SUMO2, one terminated with the diglycine motif (SUMO2GG) acting as a constitutively activated protein as well as one non- conjugatable form terminated before the diglycine motif (SUMO2ΔGG) acting as a dominant negative, were generated by PCR and the purified products were cloned into pcDNA3.1 derivatives (Invitrogen). Point mutants of ERRα (K14R, K403R, S19A, S19D, S22A, S22D), ERRγ (K40R, K360R, K439R, S45A, S45D) and UBC9 (C93S) were made by site-directed mutagenesis. The DNA fragments were sequenced and subcloned into pCMX-myc for ERRα constructs, pCMX- Flag for ERRγ constructs and pcDNA-HA for UBC9 (C93S). The wild-type and mutant versions of ERRα and γ were also subcloned into pCMX-Gal4 and pEGFP-C1 (Clontech). The expression vector pcDNA3/HA-hPGC-1α was provided by A. Kralli (Kressler, Schreiber et al. 2002). The plasmids pGEX2T- PGC-1α/1-250 and pCMV5-Flag-PIASy, -PIASxα, -PIASxβ, and -PIAS3 were described previously (Long, Matsuura et al. 2003; Long, Zuo et al. 2005; Barry, Laganière et al. 2006).

Cell culture and transient transfections

COS-1, HeLa and HEK293 cells were cultured in Dulbecco's Modified Eagle Medium (Invitrogen) supplemented with 10% fetal bovine serum. Cells were

112 Chapter II - Manuscript plated in 12-well or 10-cm plates 16-18 h before transfection with Fugene 6 (Roche). Mouse embryonic fibroblasts were freshly isolated as previously described (Laganière, Deblois et al. 2005) using ERRα-null mice embryos (Luo, Sladek et al. 2003), and were immortalized by inducing a stable expression of the SV40 large T antigen (ERRα-null MEF-T).

Reporter gene assays

48 h post-transfection, cells were harvested on ice for determination of luciferase and β-galactosidase activities. Each transfection was performed in duplicate at least three times.

Fluorescence microscopy

HeLa cells were plated on glass coverslips 16 h before transfection with GFP and HA-SUMO2 expression constructs for 48 h. Cells were washed three times with PBS, fixed and permeabilized on ice with 2% paraformaldehyde/0.2% Triton X100 in PBS for 15 min. The cells were then washed three times in PBS, quenched with 50 mM NH4Cl in 1X PBS for 10 min, rinsed twice with PBS and blocked with 5% BSA in PBS for 2 h at room temperature. Following a 30 min incubation at room temperature with α-HA monoclonal antibody (Roche), the cells were washed three times with 1X PBS and then incubated with α-mouse Alexa555 (Molecular Probes) for 30 min at room temperature and rinsed three times with PBS. The cells were then subjected to staining with 4'6-diamidino-2- phenylindole (DAPI, Sigma) rinsed again and mounted on glass slides with Immu-Mount (Thermo Shandon). Cells were analyzed under a Zeiss epifluorescence confocal microscope.

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GST pull-down assay

Equal amounts of bacterially expressed GST or GST-PGC-1α/1-250 protein containing the nuclear receptor interaction motifs immobilized on glutathione sepharose beads were combined with 10 µl of [35S]-labeled ERRα as well as ERRγ and NTD KR mutant proteins produced with the TNT®T7 coupled reticulocyte lysate system (Promega) in 500 µl of GST binding buffer (20 mM Tris, pH 7.5, 100 mM KCl, 0.1 mM EDTA, 0.05% Nonidet P-40, 10% glycerol, 1 mg/ml bovine serum albumin, 1 mM phenylmethylsulfonyl fluoride, protease inhibitor tablet complete mini (Roche) for 1 h at 4 °C. The beads were washed five times with cold binding buffer and the immobilized proteins were eluted by boiling in 2x sample buffer. The eluted proteins were resolved on SDS-PAGE and the fixed and dried gels were visualized by autoradiography.

In vitro sumoylation assay

[35S]-myc-ERRα, K14R, K403R and [35S]-flag-ERRγ, K40R, K360R, K439R proteins were produced using the TNT®T7 coupled reticulocyte lysate system (Promega) and subjected to in vitro sumoylation reactions with E1 and E2 purified enzymes along with SUMO1 or SUMO3 purified proteins (LAE Biotech). Briefly, 2 µl of [35S]-myc-ERRα, [35S]-flag-ERRγ and KR variant IVT reactions were combined to 150 ng of purified human SAE1/SAE2 (E1), 1 µg of purified human UBC9 and 1 µg of purified human SUMO1 or SUMO3 proteins, then incubated in a sumoylation buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1 mM dithiothreitol, and 2.5 mM ATP at 37°C for 1 h. The control reaction was performed under the same conditions but the purified E1 enzyme

114 Chapter II - Manuscript was omitted to prevent sumoylation from occurring. Reactions were stopped by boiling in reducing SDS sample buffer for separation by SDS-PAGE and detection by autoradiography.

In cells and in vivo sumoylation assay

HEK293 cells were transfected with the specified ERRα or γ constructs along with the activated HA-SUMO2GG and UBC9 to favor the sumoylation of targets or with the dominant negative forms HA-SUMO2ΔGG and UBC9 (C93S) to inhibit sumoylation. About 48 h after transfection, cells were lysed in buffer S (15 mM Tris-HCl pH 6.7, 0.5% SDS, 3% glycerol, 0.8 x PBS, 4% NP-40, 0.1% β- mercaptoethanol) containing 25 mM N-ethylmaleimide, 20 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 50 mM sodium fluoride and 1X complete mini protease inhibitor tablet (Roche). The lysates were sonicated at power 5 for 15 sec using a VirSonic 100 (Virtis) sonicator. Protein concentration was determined by Bradford assay and 80 µg of lysates were used for western blot analysis or 250 µg of lysates were used for immunoprecipitation with a monoclonal anti-myc antibody (Roche) for ERRα constructs and an anti-flag M2 resin (Sigma) for ERRγ constructs. For endogenous ERRα sumoylation, 2 month old male C57/BL6 mouse liver extract was prepared in buffer K (20 mM phosphate buffer pH 7, 150 mM NaCl, 0.1% NP-40, 5 mM EDTA) containing 25 mM N-ethylmaleimide, 20 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 50 mM sodium fluoride, 25 mM β-glycerol phosphate and 1X complete mini protease inhibitor tablet (Roche). Briefly, mouse livers were homogenized in buffer K for 5 seconds with a polytron-type homogenizer. The crude lysate incubated for 1 h at 4°C with rotation followed by centrifugation at 13 000 rpm for 5 minutes at 4°C. The supernatant was then quantified by Bradford and 2 mg of whole cell liver extract

115 Chapter II - Manuscript was used for immunoprecipitation with a previously described anti-hERRα polyclonal antiserum raised in our laboratory against the whole N-terminus (first 74 amino acids) (Dufour, Wilson et al. 2007) or with an anti-ERRα pS19 (described below). The anti-SUMO2/3 antibody was obtained from Chemicon International (#AB3876).

Electromobility shift assay

EMSAs were performed as previously described (Tremblay, Tremblay et al. 1997) using the consensus ERR response element (ERRE) probe (5’- TCGACGCTTTCAAGGTCATATCCG-3’) and a tandem probe containing two ERRE elements from the ERRa promoter endogenous sequence (5'- CCGTGACCTTCATTCGGTCACCGCAGT GACCTTCAT-3'). The ERRE sequences are underlined. HEK293 cells were transfected with 10 µg of pCMX- myc-ERRα, K14R or K403R expression vectors for 48 h then nuclear extract were prepared as previously described (Andrews and Faller 1991). About 2 µg of extract was used per EMSA reaction. For PGC-1α supershift experiments, 2 µg of nuclear extracts was mixed with 2 µg of purified GST-PGC-1α/1-250. EMSA with in vitro sumoylated proteins was performed as described above for in vitro sumoylation and then the complete reaction (20 µl) or increasing amount of the reaction (5, 10, 20 µl) was used for the EMSA reaction.

Generation of the pS19 phospho-specific antibody

The anti-ERRα pS19 rabbit antiserum was custom-generated by Chemicon International (Temecula, CA) against the phosphorylated Ser19 peptide (CPLYIKAEPApSPD) conjugated to keyhole limpet hemocyanin (KLH). To

116 Chapter II - Manuscript assess specificity of the antisera for the phosphorylated epitope, a dot blot analysis was performed by spotting 0.2 to 1 µg of synthetic phosphorylated and the correponding non-phosphorylated peptides on nitrocellulose membranes followed by Western blot analysis using the previously described general anti- ERRα antiserum and the new anti-ERRα pS19 phospho-antiserum.

Statistics

One-way ANOVA followed by Bonferonni post-test analysis were performed using the GraphPad InStat software. Where indicated, *** is p < 0.0001 and ** is p < 0.001.

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ACKNOWLEDGEMENTS

We thank Serge Grégoire for helpful discussions and A. Kralli, R.T. Hay and C.D. Lima for expression vectors.

This work was funded through the support of the Canadian Institutes for Health Research (CIHR) and the Canadian Cancer Society through the National Cancer Institute of Canada. A.M.T. is a recipient of a CIHR graduate scholarship.

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Figure 2.1: ERRα and γ contain consensus sites for sumoylation and are modified by SUMO2.

(A) Schematic representation of the domain organization of ERRα and γ along with their putative sumoylation sites. (B) Sequence alignment of the ERRα N- terminal domain (NTD) showing the conservation of the N-terminal sumoylation site in different species, and among the human ERRα, β and γ isoforms. ψ, hydrophobic residue; SC, synergy control; h, human; m, mouse, r; rat; d, Drosophila. The correspondence of this sumoylation site with the phosphorylation-dependent sumoylation motif (PDSM) and the SC (SC) motif is also depicted. (C) HEK293 cells were transfected with expression plasmids for HA-SUMO2 and myc-ERRα as specified. Whole cell lysates were prepared and used for immunoprecipitation with anti-myc or -Flag antibody, followed by Western blotting with anti-HA, -ERRα or -Flag antibody as indicated. Ab, antibody; i, input. (D) Same as (C) except that Flag-ERRγ was expressed and analyzed. The asterisk denotes a non-specific band.

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Figure 2.1

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Figure 2.2: The NTDs of ERRα and γ harbor the main SUMO attachment sites.

(A) ERRα and its mutants were translated and [35S]-labeled in vitro then subjected to in vitro reconstituted sumoylation assays with (+) or without (-) the E1 recombinant activating enzyme along with recombinant SUMO1 or SUMO3. The single asterisk (*) denotes a missing band for the ERRα K403R mutant. (B) Same as (A) except that ERRγ and its mutants were analyzed. (C) HEK293 cells were transfected with expression plasmids for ERRα and its mutants, along with constructs for SUMO2GG and UBC9 (SUMO +; lanes 1, 3, 5 & 7) or for SUMO2ΔGG and UBC9(C93S) (SUMO D; lanes 2, 4, 6 & 8). Whole cell lysates (80 µg) were prepared for Western blot analysis with the indicated antibodies. (D) Same as (C) except that ERRγ and its mutants were analyzed along with constructs for SUMO2GG and UBC9 (SUMO +; lanes 1, 3, 5, 7 & 9) or for SUMO2ΔGG and UBC9(C93S) (SUMO D; lanes 2, 4, 6, 8 & 10). Sumoylated ERRα (S-ERRα) and ERRγ (S-ERRγ) are labelled with open arrowheads, whereas the non-sumoylated forms are marked by filled arrowheads. The double asterisks (**) represent a non-specific band.

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Figure 2.2

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Figure 2.3: Arginine substitution at the major sumoylation site of ERRα and γ increases their transcriptional activity.

(A) HeLa cells were transfected with 50 or 100 ng of the indicated ERRα expression plasmid, along with the expression construct pCDNA3.1-HA-PGC-1α (250 or 500 ng respectively) on the 3xESRRApromoter-LUC reporter (250 ng). The luciferase activity has been adjusted relative to the CMVβGAL internal control. Results are presented in relative luciferase units (RLU). (B and C) HeLa cells were transfected with 100 ng of the indicated ERR expression plasmid and 500 ng of PGC-1α expression plasmid or pCDNA-HA along with the 3xESRRApromoter-LUC reporter. Results are presented in fold activation relative to the control condition (vector). Whole cell lysates were analyzed by Western blot with anti-ERRα or anti-Flag antibody to determine the expression levels of the different ERR mutants. (D) Same as (B) except that ERRα-null MEFs-T were used. (E) COS-1 cells were transfected with 50 ng the expression plasmid for Gal4-ERRα or Gal4-ERRα K14R mutant on a 2 copies UAS-tk-LUC reporter plasmid in the presence of 250 ng of the PGC-1α or its control vector. Whole cell lysates were analyzed by Western blot with the anti-ERRα antibody to examine the expression levels of the different ERR mutants. (F) COS-1 cells were transfected with 100 ng of the indicated expression plasmid for ERRα on the synthetic 1xERRE-TK-LUC or the 3xERRE-TK-LUC reporter plasmid in the presence of 500 ng of the PGC-1α expression plasmid. (G) ERRα-null MEFs-T were transfected with 100 ng of the indicated ERR expression plasmids in the presence of the PGC-1α expression plasmid or its vector. The reporter activities of the ΔESRRA-LUC, 2xESRRA-LUC and 3xESRRApromoter-LUC reporter plasmids were compared. (H) Same as (E) except that the activity of the Flag- ERRγ or Flag-ERRγ K40R expression plasmids was analyzed. (I) [35S]-labeled in vitro translated ERRα or γ and their respective NTD mutants were subjected to pull-down analysis with bacterially expressed GST-PGC-1α (a.a. 1-250) protein. V, vector; WT, wild-type; KR, N-terminal ERRα K14R or ERRγ K40R mutant.

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Figure 2.3

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Figure 2.4: ERRα subcellular localization, DNA binding and interaction with PGC-1α are not affected by the K14R mutation.

(A) HeLa cells were transfected with the expression plasmid for GFP tagged ERRα (top row) or GFP tagged ERRα K14R mutant (middle row) along with the HA-SUMO2 expression plasmid. The empty GFP expression vector (bottom panel) was used as control. The corresponding GFP fluorescent images (green, first column) and anti-HA antibody staining images (red, second column) were merged together (merged, third column), with the last column representing the DAPI staining (blue). (B) Nuclear extracts of HEK293 cells transfected with the control vector (V) or the indicated expression plasmid for ERRα or its mutants were subjected to EMSA analysis with the consensus ERRE probe. Ab: anti- ERRα antibody; M: ERRα monomer; D: ERRα dimer; SS: supershift ; SSAb: antibody supershift; P: GST-PGC-1α 1-250 purified protein; SSPGC-1: supershift with GST-PGC1α (1-250) purified protein. (C) [35S]-labeled in vitro translated ERRα and K14R proteins were sumoylated in vitro with (+) or without (-) the recombinant activating E1 enzyme in the presence of recombinant SUMO1 or SUMO3 and subjected to EMSA on the ERRE consensus probe. SUMO-D, dimer of sumoylated ERRα. Sumoylated ERRα (S-ERRα) complexes are represented by open arrowheads and ERRα complexes are represented by filled arrowheads. (D) [35S]-labeled in vitro translated ERRα and K14R proteins were sumoylated in vitro with (+) or without (-) the E1 enzyme with recombinant SUMO1 and were subjected to EMSA in the presence or absence of GST-PGC-1α/1-250 fusion protein. (E) Increasing amounts of in vitro translated ERRα protein were sumoylated in vitro using recombinant SUMO1 with (+) or without (-) the E1 enzyme and subjected to EMSA on a [32P]-labelled tandem ERRE probe. (F) Graphical representation of the total binding intensities for each lane (all bands) of the tandem probe EMSA gel (in E) quantified by phosphorimager.

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Figure 2.4

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Figure 2.5: Phospho-mimetic mutants display elevated sumoylation and reduced transcriptional activity.

(A) HeLa cells were transfected with 100 ng of the expression plasmid for ERRα or the ERRα S19A and ERRα S19D mutants in the presence of 500 ng of the PGC-1α expression construct or its control vector on the 3xESRRApromoter- LUC reporter (250 ng). The luciferase activity has been adjusted relative to the CMVβGAL internal control. Results are presented in fold activation relative to the control condition (vector). Whole cell lysates were analyzed by Western blot with anti-ERRα antibody to examine the expression levels of the different ERR mutants. (B) Same as (A) except that COS-1 cells were transfected with Gal4- tagged ERRα expression construct or the indicated mutant on a 2 copies UAS- TK-LUC reporter. (C) The ERRγ, S45A or S45D mutant expression plasmids were transfected as in (A). Whole cell lysates were analyzed by Western blot with anti-Flag antibody to compare the expression levels of the different ERR mutants. (D) HEK293 cells were transfected as Figure 2.2C with the expression plasmid for ERRα or the indicated mutant and sumoylation levels were determined by Western blot analysis of whole cell lysate (80 µg) with anti-ERRa. (E) Same as in Figure 2.2D with the indicated ERRγ expression plasmid except that sumoylation levels were determined by immunoprecipitation (250 µg) of whole cell lysate on anti-flagM2 agarose followed by Western blot analysis with anti-Flag antibody.

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Figure 2.5

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Figure 2.6: PIASy enhances ERRα sumoylation in a phosphorylation- dependent manner.

(A) HEK293 cells were transfected with the expression constructs for Flag- PIASxa, xb, 3 or y along with the ERRα expression plasmid. Whole cell lysates (80 µg) were subjected to Western blot analysis with the indicated antibody. Sumoylated ERRα (S-ERRα) is indicated by open arrowheads and ERRα by filled arrowheads. (B) HEK293 cells were transfected with the expression plasmid for Flag-PIASy along with the ERRa or ERRa K14R expression plasmids. Whole cell lysates (250 µg) were subjected to immunoprecipitation on anti-FlagM2 agarose beads followed by Western blot analysis with the indicated antibody. (B) HEK293 cells were transfected with the expression plasmids for Flag-PIASy along with the indicated ERRα expression plasmid. Whole cell lysates (80 µg) were sujected to Western blot analysis with the indicated antibody. (C) ERRα- null MEFs-T were transfected with the 3xESRRApromoter-LUC reporter plasmid and the indicated ERRα mutant expression plasmid in the presence or absence of PGC-1α construct.

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Figure 2.6

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Figure 2.7: Coupled phosphorylation and sumoylation of ERRa in mouse liver.

(A-D) 2 mg of mouse liver total extracts was subjected to immunoprecipitation with the anti-ERRα antibody (αERRα) or the anti-ERRα phospho serine 19 (αpS19) antisera followed by Western blot analysis with anti-ERRα (A), anti- ERRα (B; Upstate, Cat No. 07-662), anti-ERRα pS19 (C) or anti-SUMO2 (D). Sumoylated ERRα (S-ERRα) is represented by open arrowheads and ERRα by filled arrowheads. i: input; P: pre-immune serum; Ab: antibody; N-S: non- specific; LC: immunoglobulins G light chain; HC: immunoglobulins G heavy chain.

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Figure 2.7

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Figure 2.S1. Supplemental figure: Antibodies-antigenic peptide affinity comparison.

The indicated amount of synthetic phosphopeptide (pS19 antigen) and its non- phosphorylated homolog (0.2, 0.5 and 1 µg) were spotted on nitrocellulose membranes and subjected to Western blot analysis with anti-ERRα (A) or anti- ERRα pS19 (B). The panel A has been over-exposed to allow for the detection of the peptides by the anti-ERRα.

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Figure 2.S1

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Chapter III: Physiological genomics identifies estrogen- related receptor α as a regulator of renal sodium and potassium homeostasis and the renin-angiotensin pathway

PREFACE

Despite the known high levels of expression of ERRα in the kidney, the knowledge surrounding its role in this particular organ is lacking. This chapter describes the physiological and functional genomics studies undertook to explore the physiological role of ERRα in the kidney. The results of this study unraveled a role for ERRα in electrolyte balance and blood pressure regulation, and identified ERRα as a novel transcriptional regulator of the renin-angiotensin- aldosterone genes pathway genes.

This chapter is a manuscript published in Molecular Endocrinology. Chapter III - Manuscript

ABSTRACT

Estrogen-related receptor α (ERRα) is an orphan nuclear receptor highly expressed in the kidney, an organ playing a central role in blood pressure regulation through electrolyte homeostasis and the renin-angiotensin system. Physiological analysis revealed that, relative to wild-type mice, ERRα null mice are hypotensive despite significant hypernatremia, hypokalemia and slight hyperreninemia. Using a combination of genome-wide location analysis and expression profiling, we demonstrate that ERRα regulates the expression of channels involved in renal Na+ and K+ handling (Scnn1a, Atp1a1, Atp1b1) and altered in Bartter syndrome (Bsnd, Kcnq1). In addition, ERRα regulates the expression of receptors implicated in the systemic regulation of blood pressure (Ghr, Gcgr, Lepr, Npy1r) and of genes within the renin-angiotensin pathway (Ren1, Agt, Ace2). Our study thus identifies ERRα as a pleiotropic regulator of renal control of blood pressure, renal Na+/K+ homeostasis and renin-angiotensin pathway and suggests that modulation of ERRα activity could represent a potential avenue for the management of hypertension.

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INTRODUCTION

The kidneys play a central role in blood pressure regulation through the production of renin and maintenance of electrolyte homeostasis (Suzuki and Saruta 2004). Defects in the expression or activity of renal ion channels and electrolyte transporters induce a panel of physiological conditions and symptoms, notably Bartter syndrome, a genetically inherited disorder of renal electrolyte transport caused by the loss-of-function mutations in various ion channels (Jentsch, Hubner et al. 2004). Although considerable progress has been made in understanding ion channel function and associated channelopathies of the kidney, knowledge about the molecular mechanisms underlying their coordinated expression in this tissue is still lacking. Estrogen-related receptor α (ERRα; NR3B1) is an orphan member of the nuclear receptor superfamily (Giguère, Yang et al. 1988). In the absence of a cognate ligand, ERRα is a constitutive transcription factor whose activity is dependent on the presence of coregulatory proteins and post-translational modifications generated in response to external cues (Huss, Kopp et al. 2002; Kamei, Ohizumi et al. 2003; Schreiber, Knutti et al. 2003; Laganière, Tremblay et al. 2004; Mootha, Handschin et al. 2004; Barry and Giguère 2005; Ariazi, Kraus et al. 2007; Tremblay, Wilson et al. 2008). ERRα is ubiquitously expressed but its highest expression levels are detected in tissues with elevated energy demands such as the heart, brown adipose tissue and the kidneys (Sladek, Bader et al. 1997; Bookout, Jeong et al. 2006; Villena, Hock et al. 2007). The expression of ERRα is also known to be under circadian regulation in the liver, skeletal muscle, bone, uterus and adipose tissues (Horard, Rayet et al. 2004; Yang, Downes et al. 2006). Whether the expression of ERRα is subjected to the same circadian regulation in the kidney is still unknown. Acting as a transcription factor, ERRα has been shown to control the expression of genes involved in all aspects of bioenergetics including fat and glucose metabolism, energy production by the mitochondria as well as intracellular fuel sensing (Giguère 2008). In agreement with these findings,

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phenotypic analyses of mice lacking ERRα have shown that, while ERRα is dispensable for normal development, growth and reproduction (Luo, Sladek et al. 2003), ERRα-deficient mice are unable to respond to physiological challenges requiring energy demands above levels necessary to maintain basic physiological functions (Giguère 2008; Villena and Kralli 2008). Recently, the combination of chromatin immunoprecipitation (ChIP) and genomic DNA arrays (ChIP-on-chip) allowed the identification of nuclear receptor binding sites in both promoters and more distal enhancers of genes as well as the construction of extensive regulatory networks (Carroll and Brown 2006; Deblois and Giguère 2008). For example, the combination of ChIP-on-chip, microarray gene expression profiling and phenotypic analysis of genetically modified mice has led to the discovery of comprehensive ERRα-dependent transcriptional programs in the heart, macrophages and breast cancer cells that were associated with biological processes specific to each tissue (Alaynick, Kondo et al. 2007; Dufour, Wilson et al. 2007; Huss, Imahashi et al. 2007; Sonoda, Laganière et al. 2007; Giguère 2008; Deblois, Hall et al. 2009). In this work, we used the powerful mix of a physiological genomics-based approach together with phenotypic studies of ERRα null mice to explore the role of ERRα in the kidneys. Taken together, our results revealed that ERRα null mice display a phenotype reminiscent of a Bartter-like syndrome and identify ERRα as an important transcriptional regulator of two important components of blood pressure control by the kidney, namely Na+/K+ homeostasis and the renin- angiotensin pathway.

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RESULTS

Hypernatremia and hypokalemia in the ERRα knockout mice To test the role of ERRα in the kidneys, we initially compared the overall renal function and electrolyte handling in wild-type versus ERRα null mice by measuring the urine and blood constituents (Table 1). ERRα null mice tend to drink slightly less, although not in a statistically significant manner, but are excreting significantly less urine volume than their wild-type siblings. The urinary measures retained the same differences and level of significance when corrected for body weight which, as previously observed, is slightly smaller due to a lower fat content (Luo, Sladek et al. 2003 and data not shown). The urinary excretion of creatinine was found to be significantly decreased in the ERRα null mice. However, the blood concentrations of creatinine were similar between the wild type and the ERRα null mice and the glomerular filtration rate was slightly reduced but not significantly. The blood Na+ concentrations were in the normal range for both groups, but were significantly higher in the ERRα null mice resulting in a mild hypernatremia relative to the wild-type animals. Also, the blood concentrations of K+ in the ERRα null mice, although still in the normal range, were significantly lower resulting in a relative hypokalemia compared to the wild-type mice. No significant difference was observed in regards to blood calcium and chloride content. ERRα null mice also excreted the same amount of chloride and Ca2+ as the wild-type mice, suggesting a normal chloride and Ca2+ handling under basal physiological conditions. Taken together, these results suggest that the ERRα null mice possess a relatively normal renal filtration capacity, but also indicate a deficiency in Na+/K+ handling with a mechanism favoring Na+ retention.

ERRα knockout mice are hypotensive Because electrolyte homeostasis can affect blood pressure, we used telemetry to determine whether the ERRα null mice showed differences in blood

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pressure parameters. Continuous measurements over a 72 h are presented in Fig. 1A and B. Telemetry analysis revealed that the ERRα null mice display a significantly lower diastolic and systolic blood pressure between zeitgeber time (ZT) 12 and ZT24, the activity period of nocturnal animals (Fig. 1A and B, respectively). Heart rate was identical between the ERRα null and wild type mice during this period (Fig. 1C). However, during the resting period (between ZT24 and ZT12), the heart rate of the ERRα null mice tends to remain slightly higher (Fig. 1C). In addition, the ERRα null mice showed a decreased activity state than the wild-type during the nocturnal period (Fig. 1D). As noted above, the expression of ERRα has been shown to cycle according to a circadian rhythm in a tissue specific manner in the liver, skeletal muscle, brown and white adipose tissues as well as in estrogen-responsive tissues (Horard, Rayet et al. 2004; Yang, Downes et al. 2006). Therefore, we tested whether there existed a circadian regulation of ERRα expression in the kidneys. We found that the level of expression of renal ERRα mRNA was indeed cycling according to the circadian rhythm (Fig. 1E), and that the cycling of ERRα closely paralleled that of blood pressure. These results indicate that the presence of ERRα is required to sustain elevated blood pressure of mice during the period of nocturnal activity.

Identification of ERRα target genes in the kidney by ChIP-on-chip To determine whether the transcriptional program governed by ERRα in the kidneys could provide a molecular basis for the phenotypic differences observed in the ERRα null animals, we performed a ChIP-on-chip experiment using tiled genomic DNA arrays covering the extended promoter regions (~-5.5 to ~+2.5 kb from transcriptional start sites) of approximately 17,000 mouse genes and chromatin isolated from wild-type mouse kidneys. Analysis of the ChIP-on- chip dataset identified 1391 high-confidence ERRα binding sites mapping to 1366 different genes (Table S1). The ERRα target gene list was then analyzed using FatiGO. In agreement with the known function of ERRα in energy homeostasis (Giguère 2008), genes involved in the oxidative phosphorylation

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process (as reflected in the GO term oxidoreductase (0016491), mainly containing mitochondrial enzymes) were enriched in the analysis (Fig. 2A). Other categories of interest enriched for ERRα targets included transcriptional repressors (Ncor2, Sin3A, Hdac5) and activators (Ncoa1, PPARa) and enzymes with transferase activity (Got1, Pdk4, Stk11). In addition and consistent with the differences observed in electrolyte handling by the ERRα null mice, the analysis also revealed that the “ion transporter activity’’ category (GO:0015075) was significantly enriched among the ERRα targets (Fig. 2A). We further dissected the ion specificity within the “ion transporter activity’’ category and found that a great proportion of target genes within this category were implicated in Na+ and K+ handling (Fig. 2B). The binding profiles of a set of representative ERRα targets of the ion transporter activity category (GO: 0015075) are presented in Fig. 3A and details on the identified bindings sites of the genes of interest are listed in Table 2. We observed that genes crucial for Na+ handling and, more precisely Na+ reabsorption were target genes of ERRα. Indeed, ERRα was bound to the regulatory regions of the Na+/K+ ATPase α, β and γ subunits (Atp1a1, Atp1a3, Atp1b1, Atp1b2, Fxyd2) and the key epithelial Na+ channel ENaC (Scnn1a) (Zacchia, Trepiccione et al. 2008). ERRα was also found to be bound to the regulatory region of the K+ channels KvLQT1 (Kcnq1) and Kv5.1 (Kcnj16), Bartter syndrome related K+/chloride channel β subunit Barttin (Bsnd), the chloride/carbonate anion exchanger 1 (AE1; Slc4a1) as well as the K+/chloride cotransporters KCC3 (Slc12a6) and KCC4 (Slc12a7). We also noticed that genes implicated in Na+ handling, without being ion transporters themselves, were also ERRα target genes. The serum/glucocorticoid-regulated kinase 2 (Sgk2) plays a crucial role in activating and prolonging the surface expression of K+ and Na+ channels, notably ENaC (Scnn1a) (35). Also, the genes encoding the transcription factor NFAT5 (also known as TonEBP) and the receptor for growth hormone (Ghr) were also identified as ERRα targets (Fig. 3B). NFAT5 is crucial for the response to osmotic stress and growth hormone, through activation of its receptor Ghr, affects blood pressure through regulation of peripheral vascular tone in addition to its

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direct antinatriuretic effects on tubular Na+ and water reabsorption (Kamenicky, Viengchareun et al. 2008). Finally, ERRα target genes other than ion transporters but known to play a role in electrolyte handling or showing an association to blood pressure regulation or hypertension include Agt, Add3, Aldoa, Anxa5, ApoA4, ApoC3, Fgf1, Fgf9, Gclc and Lnpep (Table S1).

Identification of differentially expressed genes in the ERRα null kidney We next wanted to further explore the interconnection between the observed phenotype and the transcriptional program in the ERRα null kidney. We found 652 differentially expressed genes in the kidneys of the ERRα null mice compared to wild-type mice (276 upregulated genes, 428 downregulated genes; fold change (log2) cut-off of ±0.3 and a p-value cut-off of <0.05) (GEO:16623). Manual inspection of those genes revealed that several of them, involved in different biological processes, have been implicated in blood pressure regulation (Fig. 4A). Of interest, several ion transporters and channels known to play a role in electrolyte homeostasis are differentially expressed in the kidney of the ERRα null mice versus wild-type mice (Fig. 4A). As can be expected, genes identified by the microarray analysis include several primary ERRα target genes identified by our ChIP-on-chip analysis (e.g. Bsnd, Kcnq1, Slc4a1, Aldoa, Fgf1, Fgf9 and Gclc). The differential expression of Avpr1a, Ptgds, Atp4a, Slc2a5, Kcnq1 and Kcnj1 (ROMK) observed from the microarray was validated by quantitative real- time PCR (qRT-PCR) (Fig. 4B). In addition, the expression levels of Scnn1a and Atp1a1, two key genes responsible for Na+ reabsorption, identified by the ChIP- on-chip analysis as ERRα target genes but not by the microarray analysis were assessed by qRT-PCR (Fig. 4C). The levels of expression of both genes were found significantly upregulated in the kidneys of ERRα null mice.

ERRα regulates the expression of the renin-angiotensin-aldosterone system (RAAS) genes in the kidney The renin-angiotensin-aldosterone system (RAAS) is intertwined with Na+/K+ homeostasis in the regulation of blood pressure (Carey and Siragy 2003).

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We first measured the plasma renin concentration (PRC) and plasma aldosterone concentration (PAC) (Fig. S1). The PRC was slightly increased in the ERRα null mice and no difference was observed in the PAC. Aldosterone is a regulator of sodium and potassium homeostasis via its activating effect on the mineralocorticoids receptor (MR). The expression levels of the MR and of the hydroxysteroid dehydrogenases-11β1 and -11β2 enzymes responsible for the conversion of cortisone to cortisol which can bind the MR, were also unchanged in the ERRα null kidneys at ZT15 (Fig. S1). Considering that the angiotensinogen gene (Agt) was identified as an ERRα target gene in the kidney by ChIP-on-chip analysis (Table S1), we then investigated whether ERRα was regulating other components of the renin- angiotensin system at the transcriptional level. Since the region corresponding to the main regulatory element of Ren1, a ~240 bp region located 2.9 kb upstream of the transcriptional start site and referred to as the renin kidney enhancer (Petrovic, Black et al. 1996), was not included on the array used in our ChIP-on-chip analysis, we probed this region by standard PCR ChIP for possible ERRα binding. As shown in Fig. 5A, the Ren1 enhancer was enriched with the ERRα antiserum. In the same manner, we identified ERRα-bound segments within the promoters of both Ace2 and Agt (Fig. 5A). In agreement with ERRα binding, we observed that the expression of both Ace2 and Agt (Fig. 5B) as well as Ren1 (Fig. 5C) was upregulated at the mRNA level in the kidneys of ERRα null mice, suggesting a repressive effect of ERRα on the expression of these genes. Also, the difference in Ren1 mRNA levels between wild-type and ERRα null mice was exacerbated when the mice were fed a Na+-deficient diet. In addition, the repression of renin expression usually induced by high salt was not as efficient in the ERRα null mice (Fig. 5C). Finally, in order to confirm the direct repressive effect of ERRα on renin expression suggested by the differences in the effect of Na+ diets on renin mRNA levels in vivo, we introduced three different siRNA duplexes targeting mouse ERRα mRNA in the immortalized juxtaglomerular As4.1 cell line (Fig. 5E). The As4.1 cells retain renin expression in culture and are

148 Chapter III - Manuscript thus recognized as a good model to study renin expression (Sigmund, Okuyama et al. 1990). All 3 duplexes induced a 2.5-3 fold increase in renin mRNA levels (Fig. 5D) as well as renin protein levels (Fig. 5F). This is consistent with a complex regulatory role of ERRα on Na+ channels expression and/or activity and suggests a repressive role for ERRα on renin expression.

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DISCUSSION

The kidney is involved in the regulation of blood pressure via Na+ and K+ handling as well as the production of renin, the rate-limiting step of the renin- angiotensin pathway. Here, using a combination of genome-wide location analysis, expression profiling and physiological studies, we showed that the blood pressure of ERRα null mice is abnormal and that ERRα controls genes involved in the regulation of this function in the kidney. The ERRα-dependent transcriptional program in the kidney comprises several genes involved directly in K+ and Na+ homeostasis (Bsnd, Kcnq1, Kcnj16, Kcnj11, Atp1a1, Atp1a3, Atp1b1, Atp1b2, Scnn1a, Slc12a6, Slc12a7) or indirectly through regulation of the activity of these channels (Sgk2), or through impaired response to systemic cues regulating Na+ handling (Ghr, Gcgr, Lepr, Npy1r) as well as targets of the renin- angiotensin pathway (Agt, Ace2, Ren1). The ERRα null mice are viable and show no obvious defects besides their smaller size, previously associated to a reduced fat mass (Luo, Sladek et al. 2003). Indeed, the basal phenotype of the ERRα null mice is inconspicuous in many aspects and the application of a physiological stressor is often necessary to uncover the role of ERRα in vivo (Giguère 2008; Villena and Kralli 2008). Despite their adapted phenotype, the ERRα null mice showed significant alterations in electrolyte handling at basal level, suggesting a role of ERRα in this pathway. Indeed, the renal transcriptional program of ERRα correlates with the observed phenotype as the deregulated expression of several genes involved in controlling electrolyte balance are ERRα targets in the kidney. Aldosterone is known to increase the expression and/or activity of ionic channels (Shigaev, Asher et al. 2000; Loffing, Zecevic et al. 2001; Musch, Lucioni et al. 2008). The hormone can also trigger Na+ retention through the stimulation of ENaC expression. Aldosterone also activates the kinase Sgk2 which in turn influences the activity and surface residency time of ENaC and other ion channels (Friedrich, Feng et al. 2003). In the ERRα null mice,

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aldosterone levels, measured at ZT4, were not different from wild-type animals although variation of aldosterone concentrations at a later time point cannot be excluded. The global physiological effects observed in the ERRα null mice seems to imply that ERRα plays a role in the cardiorenal axis. Indeed, the dissection of the renal-specific mechanisms involved in the regulation of natremia and blood pressure by ERRα is excluded in our total knockout mouse model as a systemic effect arising from the cardiac (or other organ) defect cannot be ruled out in the current setting. However, the global regulation of genes involved in these processes, demonstrated by the renal transcriptional program and microarray analysis of the ERRα null kidneys, validates our hypothesis of a general role of ERRα in the control of natremia and blood pressure and awaits futher studies concerning protein expression and activity of the various channels identified by our ChIP-on-chip. From a general point of view, ERRα could affect natremia and blood pressure in a direct manner by favoring Na+ excretion as well as by participating in the repression of the renin-angiotensin pathway genes. In addition, ERRα directly regulates the expression of Sgk2, Fxyd2 (γ subunit of the NaK- ATPase) and indirectly regulates the expression of Tsc22d (also known as GILZ), thus potentially affecting in a broader manner the activity and stability of Na+ and K+ channels (Friedrich, Feng et al. 2003). Also, the protective role of ERRα could also involve a better response to the natriuretic effects of glucagon, leptin and NP- Y considering that Gcgr, Lepr and Npy1r are downregulated in the ERRα null kidney. The response to is also likely to be impaired in the ERRα null mice considering vasopressin receptors, Avpr1a and Avpr2, are downregulated in the ERRα null kidneys. Therefore, the hypernatremia of the ERRα null mice cannot be attributed to the effects of vasopressin. In light of our current model, the hypernatremia does not seem attributable to aldosterone effects either, as the expression of the mineralocorticoids receptor (MR) and of the enzymes regulating the levels of cortisol, which could activate the MR, are unchanged in the ERRα null kidneys at nighttime (Fig. S1). Indeed, the in-depth study of these potential renal mechanisms, and of how the various players of electrolyte balance regulated by ERRα interplay together in regulating natremia and blood pressure via ERRα

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will require the generation of kidney-specific ERRα null mice. Furthermore, genes involved in vascular reactivity, such as the prostaglandin pathway (Ptgds, Hpgd) and others (Guca2a), were found to be downregulated. Given these findings, together with the previous report of the upregulation of the eNOS gene by ERRα (Sumi and Ignarro 2003), our study constitutes a proper rationale for future studies on the role of ERRα in vasculature. From a cardiac point of view, ERRα could ameliorate overall blood pressure homeostasis via an improvement of cardiac function. It was recently shown that the ERRα null mice have a slightly decreased cardiac output and contractile force correlating with deregulated expression of genes regulating cardiac calcium handling and contractility (Dufour, Wilson et al. 2007; Huss, Imahashi et al. 2007). This latter study however, did not identify the differences in blood pressure most likely because measurements were performed during the day. In addition, the ERRα null mice were also demonstrating an impaired cardiac remodeling response to pressure overload induced by transaortic constriction (Huss, Imahashi et al. 2007). Given the global picture brought by the published cardiac phenotype of the ERRα null mice with our renal and blood pressure phenotype, we can speculate that ERRα impinges on the cardiorenal axis for homeostatic maintenance of blood pressure. Taken together, the renal transcriptional program and the expression profile in the ERRα null kidneys suggest a role for ERRα in many aspects of electrolyte handling. Despite the absence of salt-wasting and metabolic alkalosis under standard housing conditions, ERRα null mice display a Bartter-like phenotype, principally owing to the simultaneous presence of a lower blood pressure along with hypokalemia, an elevated plasma level of renin and to the downregulation of channels associated with the etiology of Bartter syndrome (Bsnd, Kcnj1, Slc12a1 encoding respectively Barttin, ROMK and NKCC2). In individuals affected by Bartter syndrome, the symptoms are thought to derive from a cascade of adaptive physiological reactions in response to the salt wasting caused by the loss-of-function of one or more ion channels. In the ERRα null mice, however, the final phenotype is likely caused by an accumulation of minor deregulations of multiple genes within the same pathway. As ERRα also regulates

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the expression of Agt and Ren1, the adaptive cascades are likely to be impaired by the absence of ERRα. The fact that the removal of ERRα by siRNA in As4.1 cells induces a more pronounced increase in renin mRNA levels than that observed in vivo suggests that other mechanisms come into play and potentially counteract the effect of the absence of ERRα on renin transcription in vivo. Indeed, the ERRα null mice are hypernatremic and this condition itself could diminish the increase of renin expression in the ERRα null kidney. This explanation could also apply to the plasma renin concentration. Further studies using tissue-specific ERRα null mice would be helpful to distinguish the causes from the consequences for the observed differences in blood pressure and to precisely assess the contribution of the kidneys in the observed phenotype. ERRβ, an ERR isoform closely related to ERRα (Giguère, Yang et al. 1988), has recently been implicated in the development of the endolymph- producing cells of the inner ear and the regulation of inner ear fluid homeostasis (Chen and Nathans 2007). Several differentially expressed genes in the stria vascularis of the ERRβ null mice (Kcne1, Atp1b2 and Kcnq1 among others) are modulated by ERRα in the kidney. Also the ERRγ null mice die at birth from heart defects that have been associated to a shorter depolarization phase and decreased expression of voltage-dependent cardiac Na+ channels (Alaynick, Kondo et al. 2007). The ERRγ null mice displayed a prolonged QT interval on ECG. QT prolongation is typically associated to a longer repolarization phase of the cardiac action potential, which rely principally on rectifying K+ currents, suggesting that ERRγ could also be involved in K+ channels regulation in the heart. Indeed, we have previously shown that ERRα and ERRγ can bind as heterodimers and share the same target genes in the heart (Dufour, Wilson et al. 2007). Taken together with these studies, our results substantiate the implication of all members of the NR3B subgroup as regulators of the electrolyte handling transcriptional program. In conclusion, this study connects ERRα with long-term blood pressure regulation in part via a global regulation of renal electrolyte handling program and the renin-angiotensin pathway suggesting that ERRα, and potentially other

153 Chapter III - Manuscript isoforms of the ERR family, could be targeted for pharmacological regulation of blood pressure disorders. In addition, taken together with the previously shown cardiac phenotypic studies of the ERRα null mice, our study suggests a role of ERRα in the systemic homeostasis of blood pressure through the cardiorenal axis.

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MATERIALS AND METHODS

Mice and diets The ERRα null mice (C57/BL6 background) were previously described (Luo, Sladek et al. 2003). The animals were housed on a 12 h light-dark cycle (lights off at 7 PM). 2-3 months old male mice were used throughout the study. For the salt response analysis the mice were fed ad libitum with a standard diet containing 0.49% NaCl (TD.90208, Harlan Laboratories, Madison, WI) or a Na+- deficient diet (TD.90228, Harlan Laboratories, Madison, WI) for 5 days. At ZT12, the mice were euthanized, the tissues removed, snap frozen in liquid nitrogen and stored at -80 0C until subsequent analysis. All the manipulations were performed in accordance with the McGill Animal Care Committee guidelines.

Telemetry monitoring The telemetry transmitters were surgically implanted and mice (3 per genotype) were allowed 7 days of post-surgery recovery before telemetry data acquisition. Data were collected for 72 consecutive hours. Statistical significance was reached for the nighttime period only (two way Anova with Bonferroni post- test, p<0.05) for all parameters except heart rate and activity state (n.s.).

Urine and blood constituents analysis Briefly, mice were housed in metabolic cages (5 cages of 3-5 mice per cage for a total of 19 mice per genotype) and were fed standard diet and water ad libitum. Mice were allowed a week adaptation period before urine collection over a 24 h period. For plasma aldosterone measurements, non-stressed blood samples were collected at ZT4. The plasma was separated by centrifugation. Plasma aldosterone concentration was measured by RIA. The rest of the blood was collected by cardiac puncture at ZT4 for the electrolytes, creatinine and BUN measurements. For the plasma renin concentration determination, mice were housed in standard conditions. Non-stressed blood samples were collected before

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and following a low-salt diet-feeding period. The plasma was separated by centrifugation and PRC was measured by non-commercial RIA (n=7-13 per group).

ChIP assays and ChIP-on-ChIP on extended promoter arrays Chromatin was prepared from C57/Bl6 from a pool of kidneys taken around ZT4 from a total of 85 mice. ChIP labeling was performed as previously described (Weinmann, Yan et al. 2002) on chromatin immunoprecipitated with an antiserum against ERRα and non-immunoprecipitated as control. The labeled samples were hybridized according to the Agilent Mammalian ChIP-on-chip Protocol (v.9.2) in duplicate on mouse extended promoter microarrays (Agilent 2X 244 K array format) covering approximately 8 kb around the transcriptional start sites of ~17,000 genes. The slides were air-dried and scanned immediately using the Agilent scanner (G2565BA) according to the protocol provided (Agilent Mammalian ChIP-on-chip Protocol (v.9.2)). The data was then extracted using the Agilent Feature Extraction Software (v.9.5.1) using the grid template for mm8 mouse genome assembly, according to the protocol provided. The extracted data was subsequently analyzed with the ChIP Analytics Software (v.1.3.1, Agilent). The data was normalized using the Lowess normalization method and significant binding peaks were detected using the Whitehead Peak Detection method. Complete datasets and bed files are available upon request. For standard ChIP assays, quantification of DNA enrichment was achieved by qRT-PCR with specific primers designed to amplify the regions of interest. Quantification by qRT-PCR were carried out using the LightCycler®2.0 (Roche).

Microarray analysis Triplicate microarray analyses of wild-type and ERRα-null kidneys (collected at ZT4) were conducted at the McGill University and Génome Québec Innovation Centre using 10 µg of total RNA and hybridized to Affymetrix MOE430 2.0 GeneChIP arrays. In this study, the mean of the expression data obtained from the individual wild-type and ERRα-null mice was determined to

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identify genes with altered levels of expression in the knockout mice versus their wild-type littermates obtained using the Genespring X software (Agilent). A p value cutoff of <0.05 and a relative fold change (log2) cutoff of +/- 0.3 were used and genes were classified by biological function based on GO annotation (http://fatigo.org/), NCBI gene descriptions and UniprotKB annotations.

siRNA knockdown assays As4.1 cells were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin. The cells were plated in 6-cm dishes 16-18 h before transfection with HiPerfect (Qiagen) according to the manufacturer’s instructions. Briefly, the cells were transfected with 30 nM of a scrambled siRNA in parallel with 3 different siRNA duplexes targeting Esrra (IDT). After 48 h, the cells were collected and frozen until subsequent analysis.

RNA isolation, reverse transcription and real-time quantitative PCR Prior to RNA isolation, the frozen kidneys were pulverized using a frozen mortar and pestle. Liquid nitrogen was added regularly to the powder during the pulverization procedure to prevent thawing. Total RNA was isolated from frozen cell pellets or grinded mouse kidneys using the RNeasy miniprep or midiprep kit (Qiagen) according to the manufacturer’s instructions. For each sample, 2 µg of total RNA was reverse transcribed to cDNA using the SuperScript First-Strand Synthesis system (Invitrogen) according to the manufacturer’s protocol. The abundance of mRNA transcripts was assessed by LightCycler®480 (Roche) and LightCycler®480 SYBR Green I Master mix (Roche) according to the manufacturer instructions. Values were normalized normalized to Arbp control gene.

Protein extraction and Western blotting As4.1 cells were lysed in Modified Ripa containing mini protease inhibitor tablet (Roche) for 30 min at 4 oC, then the cellular debris were pelleted at 10,000 rpm

157 Chapter III - Manuscript for 10 min at 4 oC. Protein concentration was determined with Bradford Ultra reagent (Expedeon) according to the manufacturer’s instructions. 20 or 100 µg of total protein extracts were resolved by SDS-PAGE and subjected to Western blot analysis using an in-house rabbit polyclonal anti-ERRα antibody (Laganière, Tremblay et al. 2004) or goat anti-renin antibody (R&D Biosciences). Equal loading was assessed by Ponceau red staining of the corresponding membrane.

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ACKNOWLEDGEMENTS

We thank Carlo Ouellet and Chantale Mercure for skillful technical assistance and Dr. Christian Deschepper for the As4.1 cell line. This work was supported by grants from the Canadian Institutes for Health Research (CIHR). A.M.T. was supported by a Canada Graduate Scholarship from CIHR.

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Table 3.1: Blood and urinary parameters in wild type and ERRα null mice.

WT ERRα KO pValue mean SEM mean SEM Body weight (g) 24.5 0.3 22.2 0.6 0.002 ** Water intake (mL) 4.6 0.7 3.4 0.1 0.100 Urine volume (mL) 1.07 0.12 0.54 0.12 0.008 ** GFR (mL/d) 1.00 0.11 0.75 0.12 0.129 Urinary parameters (mmol/d) Creatinine 4.0 0.3 2.4 0.4 0.025 * Na+ 166.7 24.4 94.6 14.5 0.035 * K+ 323.5 38.3 206.1 40.6 0.069 Na/K ratio 0.51 0.04 0.49 0.05 0.816 Chloride 270.1 46.2 160.8 28.6 0.097 Ca2+ 2.1 0.5 1.9 0.5 0.971 Blood parameters Urea 8.32 0.53 8.23 0.30 0.885 Creatinine 18.21 1.87 17.40 1.95 0.760 Na+ 140.79 0.90 144.80 1.00 0.005 ** K+ 8.29 0.21 6.73 0.15 <0.001 *** Na+/K+ ratio 16.04 0.65 20.30 0.62 <0.001 *** Chloride 107.95 0.63 108.60 0.63 0.455 Ca2+ 2.17 0.07 2.22 0.03 0.462

GFR, glomerular filtration rate. Values are presented as mean ± SEM. Unpaired Student t-test was used for statistical analysis. Measurements were made on 5 metabolic cages each housing 3 to 5 mice per cage. A total of 19 mice per genotype were used for the analysis.

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Table 3.2: ERRα ChIP-on-chip target genes associated with Na+ and/or K+ transport

Gene Transport Peak probe Location Binding site

Chr Start End

Atp1a1 Na+/K+ 3 101732164 101732220 intron TCAAGGTCA

Atp1a3 Na+/K+ 7 24718010 24718054 promoter TGAAGGTCA

Atp1b1 Na+/K+ 1 166292531 166292588 intron cCAAGGgCA

1 166293675 166293734 intron TTAAGGTCA

1 166295022 166295066 promoter AGGTCA

1 166295318 166298801 promoter TCAAGGTCA

1 166297763 166297820 promoter gGAAGGTCA

Atp1b2 Na+/K+ 11 69420940 69422229 intron AGGTCA

Bsnd Cl-/K+ 4 105994809 105994867 promoter TCAAGGTCA

+ + Fxyd2 Na /K 9 45149761 45151240 promoter cCAAGGTCA

9 45150607 45150651 promoter TCAAGGTCA

9 45152194 45152248 promoter cTAAGGTCA

9 45157922 45157971 intron TCAAGGTCA

Hcn3 K+/Na+ 3 89245442 89246567 intron -

Kcnc3 K+ 7 44458383 44459841 promoter -

Kcnh2 K+ 5 23861900 23861944 promoter TAAAGGTCc

Kcnj11 K+ 7 45967896 45969085 intron AGGTCA

Kcnj16 K+ 11 110783890 110783949 promoter TGAAGGTCA

+ Kcnq1 K 7 142917727 142917784 intron TCAAGGgCA

Kcnq3 K+ 15 66116269 66116313 promoter -

Kctd15 K+ 7 34362551 34363715 promoter - Grey shading: gene associated to blood pressure. Kctd8 K+ 5 69619598 69620713 intron TGAAGGaCA

Scnn1a Na+ 6 125283488 125283545 promoter TCAAGGTCA

6 125286119 125286172 promoter TCAAGGTCA

6 125287189 125287233 promoter TAcAGGTCA

Slc12a6 Cl-/K+/Na+ 2 112065842 112067213 promoter AGGTCA161

Slc12a7 Cl-/K+/Na+ 13 74230441 74230485 intron cCAAGGTCA Chapter III - Manuscript

Figure 3.1: Blood pressure parameters and heart rate in ERRα null mice. A, Telemetry analysis of diastolic blood pressure over a 72 h period. B, Telemetry analysis of systolic blood pressure over a 72 h period. C, Heart rate of wild-type and ERRα null mice over a 72 h period. Grey shading, nighttime periods (ZT12-ZT24); Grey line, wild-type mice; black line, ERRα null mice; ZT, zeitgeber time; bpm, beats per min. D, Activity levels (counts per minutes) of wild-type and ERRα null mice over a 72 h period. E, Analysis of the temporal expression of ERRα in the kidney over a 24 h period. *, P<0.05.

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Figure 3.1

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Figure 3.2: ERRα target genes are enriched for specific biological functions. A, Comparison between the functionally enriched biological functions associated with ERRα targets and the overall genomes. (**, p<0.01; ***, p<0.001). B, Sub- division of ERRα target genes within the ion transporter category shows a large proportion of genes encoding proteins implicated in Na+ and K+ handling.

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Figure 3.2

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Figure 3.3: Genome-wide identification of ERRα-bound segments within extended promoter regions in mouse kidneys.

A, Enrichment ratio profiles for ERRα at extended promoter regions of genes encoding ion channels. B, Enrichment ratio profiles for ERRα at extended promoter regions of genes encoding diverse proteins implicated in blood pressure regulation. Arrows indicate the transcriptional start sites for each gene. Black boxes represent exons.

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Figure 3.3

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Figure 3.4. Gene expression profiling in ERRα null kidneys.

A, Schematic representation of a subset of differentially regulated genes involved in blood pressure regulation expressed in relative fold change (log2). B, Real-time quantitative PCR validation performed on RNA isolated from adult mouse kidneys used in the microarray analysis. Relative fold (log2) expression levels were normalized to Arbp levels and bars represent mean (±S.E.M.). C, Relative expression of Scnn1a and Atp1a1 in kidneys of wild type and ERRα mice by real-time quantitiative PCR. (*, p<0.05)

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Figure 3.4

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Figure 3.5: Regulation of genes involved in the RAAS by ERRα.

A, Standard ChIP validation of ERRα binding to targets not identified in the original ChIP-on-chip experiment and involved in the RAAS pathway. Results shown are of one experiment based on two independent immunoprecipitations prepared from a pool of 85 mice kidneys. B, Ace2 and Agt are upregulated in the kidneys of ERRα null kidneys as assessed by real-time quantitiative PCR. C, Relative expression by quantitative real-time PCR of Ren1 in kidneys of wild type and ERRα null mice fed a diet low (LS) or high (HS) in Na+ as compared to mice fed standard chow (STD). D, Knockdown of Esrra in As4.1 cells using 3 different siRNA duplexes increases Ren1 expression as assessed by real-time quantitiative PCR. E, Western blot shows the level of ERRα in As4.1 cells transfected with the three siRNA duplexes. F, Western blot shows the level of renin in As4.1 cells transfected with the three siRNA duplexes. (*, p<0.05; **, p<0.01; ***, p<0.001). Equal loading was assessed by Ponceau red staining of the corresponding membrane.

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Figure 3.5

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FIG. 3.S1: Plasma renin, plasma aldosterone concentrations and expression of mineralocorticoid receptor and cortisol metabolizing enzymes in wild-type and ERRα null mice. A, RIA measurement of the plasma renin concentration (PRC) in wild-type and ERRα null mice under standard and low-sodium diet. (mean +/- S.E.M., n=7-13 per group). B, RIA measurements of the plasma aldosterone concentrations (PAC) in wildtype and ERRα null mice under standard diet measured by radioimmunoassay (mean +/- S.E.M., n=19 per group). C, Western blot analysis of mineralocorticoid receptor (MR) expression levels at ZT15 in wild-type and ERRα null mice kidneys. Anti-Tubulin of the corresponding membrane is shown as loading control. D, Fold mRNA levels in wild-type and ERRα null mice kidneys at ZT15.

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Figure 3.S1

173 Chapter III – Manuscript – ChIP-on-chip gene list

Table 3.S1: List of ERRα ChIP-on-chip target genes in mouse kidney

Gene name Description Ppp1cb , catalytic subunit, beta isoform Amino acid and derivative metabolism Ppp1cc protein phosphatase 1, catalytic subunit, gamma isoform Abhd1 abhydrolase domain containing 1 Rbks ribokinase Adc arginine decarboxylase St8sia5 ST8 alpha-N-acetyl-neuraminide alpha-2,8- Alas1 aminolevulinic acid synthase 1 St8sia6 ST8sialyltransferase alpha-N-acetyl 5 -neuraminide alpha-2,8- Ass1 argininosuccinate synthetase 1 Sulf2 sulfatasesialyltransferase 2 6 Ddah1 dimethylarginine dimethylaminohydrolase 1 Tpi1 triosephosphate isomerase 1 Ddo D-aspartate oxidase Xylb xylulokinase homolog (H. influenzae) Fech ferrochelatase Cell adhesion and communication Gm237 2-aminoethanethiol (cysteamine) dioxygenase Cdh1 cadherin 1 Got1 glutamate oxaloacetate transaminase 1, soluble Cdh2 cadherin 2 Got2 glutamate oxaloacetate transaminase 2, mitochondrial Cdh22 cadherin 22 Mat2a methionine adenosyltransferase II, alpha Cldn12 claudin 12 Mcee methylmalonyl CoA epimerase Cobl cordon-bleu Oaz2 ornithine decarboxylase antizyme 2 Dock4 dedicator of cytokinesis 4 Sardh sarcosine dehydrogenase Efs embryonal Fyn-associated substrate Shmt1 serine hydroxymethyltransferase 1 (soluble) Fblim1 filamin binding LIM protein 1 Apoptosis Hs6st2 heparan sulfate 6-O-sulfotransferase 2 Aifm1 apoptosis-inducing factor, mitochondrion-associated 1 Igsf4a cell adhesion molecule 1 Atxn2 ataxin(AKA 2AIF) Mucdhl mucin-like protocadherin Bbc3 Bcl-2 binding component 3 Nell1 NEL-like 1 (chicken) Cbx4 chromobox homolog 4 (Drosophila Pc class) Nrp1 neuropilin 1 Ciapin1 cytokine induced apoptosis inhibitor 1 Nrp2 neuropilin 2 Dffa DNA fragmentation factor, alpha subunit Nrxn2 neurexin II Fastkd1 FAST kinase domains 1 Pcdh24 protocadherin 24 Gcgt Gamma-glutamylcyclotransferase Pcdh8 protocadherin 8 Hipk3 homeodomain interacting protein kinase 3 Pkp4 plakophilin 4 Ltbr lymphotoxin B receptor Pvrl2 poliovirus receptor-related 2 Mal myelin and lymphocyte protein, T-cell differentiation Rac1 RAS-related C3 botulinum substrate 1 Mcl1 myeloidprotein cell leukemia sequence 1 Spp1 secreted phosphoprotein 1 Mtp18 mitochondrial protein 18 kDa Tyro3 TYRO3 protein tyrosine kinase 3 Naif1 nuclear apoptosis inducing factor 1 Zyx zyxin Prdx2 peroxiredoxin 2 Gja12 gap junction protein, gamma 2 Tmem166 transmembrane protein 166 Gja9 gap junction membrane channel protein alpha 9 Tnfsf5ip1 proteasome (prosome, macropain) assembly chaperone 2 Cell cycle Yars tyrosyl-tRNA synthetase Appl1 adaptor protein, phosphotyrosine interaction, PH domain Carbohydrate metabolism Aurkb auroraand leucine kinase zipper B containing 1 Aldoa aldolase 1, A isoform Banp Btg3 associated nuclear protein B4galt5 UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, Cables1 Cdk5 and Abl enzyme substrate 1 Dlat dihydrolipoamidepolypeptide 5 S-acetyltransferase (E2 component of Ccnl1 cyclin L1 Fut8 fucosyltransferasepyruvate dehydrogenase 8 complex) Ccnl2 cyclin L2 G6pc glucose-6-phosphatase, catalytic Cdc73 Vcell division cycle 73, Paf1/RNA polymerase II Gale galactose-4-epimerase, UDP Clip1 CAPcomplex-GLY component, domain containing homolog linker(S. cerevisiae) protein 1 Galt galactose-1-phosphate uridyl transferase Dnaja2 DnaJ (Hsp40) homolog, subfamily A, member 2 Gapdh glyceraldehyde-3-phosphate dehydrogenase Dsn1 DSN1, MIND kinetochore complex component, Glb1 galactosidase, beta 1 Esco2 establishmenthomolog (S. cerevisiae) of cohesion 1 homolog 2 Gloxd1 4-hydroxyphenylpyruvate dioxygenase-like Heca headcase homolog (Drosophila) Gyk glycerol kinase Itgb1 integrin beta 1 (fibronectin receptor beta) Hisppd2a acid phosphatase domain containing 2A Jmy junction-mediating and regulatory protein Ldhb lactate dehydrogenase B Khdrbs1 KH domain containing, RNA binding, signal Man1b mannosidase, alpha, class 1A, member 2 Lats2 largetransduction tumor suppressor associated 21 Oxct1 3-oxoacid CoA transferase 1 Nudc nuclear distribution gene C homolog (Aspergillus) Pck1 phosphoenolpyruvate carboxykinase 1, cytosolic Pard3 par-3 (partitioning defective 3) homolog (C. elegans) Pdha1 pyruvate(PEPCK) dehydrogenase E1 alpha 1 Pmf1 polyamine-modulated factor 1 Pdk2 pyruvate dehydrogenase kinase, isoenzyme 2 Rabgap1 RAB GTPase activating protein 1 Pdk4 pyruvate dehydrogenase kinase, isoenzyme 4 Rb1cc1 RB1-inducible coiled-coil 1 Pfkl phosphofructokinase, liver, B-type Rcc1 regulator of chromosome condensation 1 Pfkp phosphofructokinase, platelet Sept8 septin 8 Pgam1 phosphoglycerate mutase 1 Strn3 striatin, calmodulin binding protein 3 Pgk1 phosphoglycerate kinase 1 Sycp3 synaptonemal complex protein 3 Pgp phosphoglycolate phosphatase Terf1 telomeric repeat binding factor 1 Pmm1 phosphomannomutase 1 Cell differentiation Pofut2 protein O-fucosyltransferase 2 Cend1 cell cycle exit and neuronal differentiation 1 Ppp1ca protein phosphatase 1, catalytic subunit, alpha isoform Csrp2 cysteine and glycine-rich protein 2

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Dock7 dedicator of cytokinesis 7 Aldh1a3 aldehyde dehydrogenase family 1, subfamily A3 Enah enabled homolog (Drosophila) Aldh3b1 aldehyde dehydrogenase 3 family, member B1 Gpm6b glycoprotein m6b Gstm5 glutathione S-transferase, mu 5 Ntng2 netrin G2 Unc93b1 unc-93 homolog B1 (C. elegans) Ppp1r9a protein phosphatase 1, regulatory (inhibitor) subunit 9A Cbln1 cerebellin 1 precursor protein Slit1 slit homolog 1 (Drosophila) Ebf3 early B-cell factor 3 Slit2 slit homolog 2 (Drosophila) Epas1 endothelial PAS domain protein 1 Slit3 slit homolog 3 (Drosophila) Evx2 even skipped homeotic gene 2 homolog/homeo box D13 Cell growth Fbn2 fibrillin 2 Gdf6 growth differentiation factor 6 Gja7 gap junction protein, gamma 1 Ing1 inhibitor of growth family, member 1 Hes6 hairy and enhancer of split 6 (Drosophila) Vat1 vesicle amine transport protein 1 homolog (T Hoxa1 homeo box A1 Cell motility californica) Hoxa5 homeo box A5 Actc1 actin, alpha, cardiac Hoxa9 homeo box A9 Actg1 actin, gamma, cytoplasmic 1 Hoxb9 homeo box B9 Cmtm3 CKLF-like MARVEL transmembrane domain Hoxd11 homeo box D11 Kif26a kinesincontaining family 3 member 26A Hoxd13 homeo box D13 Myh9 myosin, heavy polypeptide 9, non-muscle Hoxd9 homeo box D9 Tspan1 tetraspanin 1 Lnp limb and neural patterns Tspan3 tetraspanin 3 Mbnl3 muscleblind-like 3 (Drosophila) Tspan4 tetraspanin 4 Mesdc1 mesoderm development candidate 1 Vasp vasodilator-stimulated phosphoprotein Mesdc2 mesoderm development candiate 2 Cell proliferation Nog noggin (AKA SYM1; SYNS1) Cd81 CD81 antigen (AKA Tspan28) Plxna1 plexin A1 Clec11a C-type lectin domain family 11, member a Rai1 retinoic acid induced 1 Ctbp2 C-terminal binding protein 2 Rai2 retinoic acid induced 2 Fgf3 fibroblast growth factor 3 Shroom4 shroom family member 4 Gab1 growth factor receptor bound protein 2-associated Smo smoothened homolog (Drosophila) Gfer growthprotein 1factor, erv1 (S. cerevisiae)-like (augmenter of Tnni1 Troponin I, slow skeletal muscle Gpc4 glypicanliver regeneration) 4 (AKA ALR) Zmym3 zinc finger, MYM-type 3 Hdgf hepatoma-derived growth factor DNA repair Hdgfrp3 hepatoma-derived growth factor, related protein 3 Aste1 asteroid homolog 1 (Drosophila) Hells helicase, lymphoid specific Msh2 mutS homolog 2 (E. coli) Park7 Parkinson disease (autosomal recessive, early onset) 7 Msh6 mutS homolog 6 (E. coli) Pdgfa platelet derived growth factor, alpha Electron transport/Oxphos Pdgfb platelet derived growth factor, B polypeptide 2610507B1 RIKEN cDNA 2610507B11 gene Pdgfc platelet-derived growth factor, C polypeptide Aco21Rik aconitase 2, mitochondrial Prdx1 peroxiredoxin 1 Atp5a1 ATP synthase, H+ transporting, mitochondrial F1 Tob1 transducer of ErbB-2.1 Atp5b ATPcomplex, synthase, alpha H+ subunit, transporting isoform mitochondrial 1 F1 Vegfa vascular endothelial growth factor A Atp5c1 ATPcomplex, synthase, beta subunitH+ transporting, mitochondrial F1 Cytoskeleton Atp5d ATPcomplex, synthase, gamma H+ polypeptide transporting, 1 mitochondrial F1 Actrt2 actin-related protein T2 Atp5g1 ATPcomplex, synthase, delta H+subunit transporting, mitochondrial F0 Add3 adducin 3 (gamma) Atp5g3 ATPcomplex, synthase, subunit H+ c transporting,(subunit 9), isoform mitochondrial 1 F0 Cdc42ep3 CDC42 effector protein (Rho GTPase binding) 3 Atp5k ATPcomplex, synthase, subunit H+ c transporting,(subunit 9), isoform mitochondrial 3 F1F0 Diap2 diaphanous homolog 2 (Drosophila) Atp5l ATPcomplex, synthase, subunit H+ e transporting, mitochondrial F0 Eml2 echinoderm microtubule associated protein like 2 Atp5o ATPcomplex, synthase, subunit H+ g transporting, mitochondrial F1 Epb4.1 erythrocyte protein band 4.1 Ckmt1 creatinecomplex, kinas O subunite, mitochondrial 1, ubiquitous Epb4.9 erythrocyte protein band 4.9 Cox10 COX10 homolog, cytochrome c oxidase assembly Espn espin Cox11 COX11protein, hemehomolog, A: farnesyltransferase cytochrome c oxidase (yeast) assembly Fmnl1 formin-like 1 Cox4i1 cytochromeprotein (yeast) c oxidase subunit IV isoform 1 Fscn1 fascin homolog 1, actin bundling protein Cox5a cytochrome c oxidase, subunit Va Hook2 hook(Strongylocentrotus homolog 2 (Drosophila) purpuratus) Cox6b1 cytochrome c oxidase, subunit VIb polypeptide 1 Mprip myosin phosphatase Rho interacting protein Cox6c cytochrome c oxidase, subunit VIc Mtap2 microtubule-associated protein 2 Cox7a2 cytochrome c oxidase, subunit VIIa 2 Mtap4 microtubule-associated protein 4 Cox7b cytochrome c oxidase subunit VIIb Nefm neurofilament, medium polypeptide Cs citrate synthase Odf2 outer dense fiber of sperm tails 2 Cycs cytochrome c, somatic Plekhc1 pleckstrin homology domain containing, family C (with Cyp2s1 cytochrome P450, family 2, subfamily s, polypeptide 1 Rdx radixinFERM domain) member 1 Cyp4b1 cytochrome P450, family 4, subfamily b, polypeptide 1 Sync syncoilin Etfa electron transferring flavoprotein, alpha polypeptide Defense response Foxred1 FAD-dependent oxidoreductase domain containing 1 Ahsa2 AHA1, activator of heat shock protein ATPase homolog Gfod1 glucose-fructose oxidoreductase domain containing 1 Ars2 arsenate2 (yeast) resistance protein 2 Idh2 isocitrate dehydrogenase 2 (NADP+), mitochondrial Banf1 barrier to autointegration factor 1 Idh3a isocitrate dehydrogenase 3 (NAD+) alpha Ncf1 neutrophil cytosolic factor 1 Mdh1 malate dehydrogenase 1, NAD (soluble) Sod2 superoxide dismutase 2, mitochondrial Mdh2 malate dehydrogenase 2, NAD (mitochondrial) Srxn1 sulfiredoxin 1 homolog (S. cerevisiae) Me3 malic enzyme 3, NADP(+)-dependent, mitochondrial Umod uromodulin Ndufa12 NADH dehydrogenase (ubiquinone) 1 alpha Detoxification Ndufa2 NADHsubcomplex, dehydrogenase 12 (ubiquinone) 1 alpha subcomplex, 2

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Ndufa4 NADH dehydrogenase (ubiquinone) 1 alpha Pla2g2c phospholipase A2, group IIC Ndufa6 NADHsubcomplex, dehydrogenase 4 (ubiquinone) 1 alpha Plscr2 phospholipid scramblase 2 Ndufb2 NADHsubcomplex, dehydrogenase 6 (B14) (ubiquinone) 1 beta subcomplex, Ptdss2 phosphatidylserine synthase 2 Ndufb4 NADH2 dehydrogenase (ubiquinone) 1 beta subcomplex Rdh14 retinol dehydrogenase 14 (all-trans and 9-cis) (AKA Ndufb5 NADH4 dehydrogenase (ubiquinone) 1 beta subcomplex, Stard5 StARPan2)- related lipid transfer (START) domain containing Ndufs1 NADH5 dehydrogenase (ubiquinone) Fe-S protein 1 Meiosis 5 Ndufs2 NADH dehydrogenase (ubiquinone) Fe-S protein 2 Klhdc3 kelch domain containing 3 Ndufs4 NADH dehydrogenase (ubiquinone) Fe-S protein 4 Metabolism Ndufs7 NADH dehydrogenase (ubiquinone) Fe-S protein 7 Abhd11 abhydrolase domain containing 11 Ndufv2 NADH dehydrogenase (ubiquinone) flavoprotein 2 Cmbl carboxymethylenebutenolidase-like (Pseudomonas) Nqo2 NAD(P)H dehydrogenase, quinone 2 Cryzl1 crystallin, zeta (quinone reductase)-like 1 Ogdh oxoglutarate dehydrogenase (lipoamide) Fahd1 fumarylacetoacetate hydrolase domain containing 1 Oxnad1 oxidoreductase NAD-binding domain containing 1 Hdhd3 haloacid dehalogenase-like hydrolase domain containing Prom2 prominin 2 Miox myo3 -inositol oxygenase Sdha succinate dehydrogenase complex, subunit A, Nmral1 NmrA-like family domain containing 1 Sdhc succinateflavoprotein dehydrogenase (Fp) complex, subunit C, integral Ppa2 pyrophosphatase (inorganic) 2 Sdhd succinatemembrane dehydrogenase protein complex, subunit D, integral microRNA Uqcr10 RIKENmembrane cDNA protein 111002 0P15 gene 148a mmu-mir-148a Uqcrb ubiquinol-cytochrome c reductase binding protein Nucleic acid metabolism Uqcrc1 ubiquinol-cytochrome c reductase core protein 1 2610101N1 RIKEN cDNA 2610101N10 gene Uqcrc2 ubiquinol cytochrome c reductase core protein 2 Adcy10Rik adenylate cyclase 1 Uqcrfs1 ubiquinol-cytochrome c reductase, Rieske iron-sulfur Ak1 adenylate kinase 1 Immune responsepolypeptide 1 Ak3 adenylate kinase 3 Cx3cl1 chemokine (C-X3-C motif) ligand 1 Ak3l1 adenylate kinase 3 alpha-like 1 Gtpbp1 GTP binding protein 1 Apex2 apurinic/apyrimidinic endonuclease 2 Hcst hematopoietic cell signal transducer Aptx aprataxin Il25 interleukin 25 Bat1a HLA-B-associated transcript 1A Il3ra interleukin 3 receptor, alpha chain Cmpk cytidylate kinase Nfkbid nuclear factor of kappa light polypeptide gene enhancer Cpsf6 cleavage and polyadenylation specific factor 6 Prdx5 peroxiredoxinin B-cells inhibitor, 5 delta Cugbp1 CUG triplet repeat, RNA binding protein 1 Tnfrsf9 receptor superfamily, member 9 D11Bwg05 DNA segment, Chr 11, Brigham & Women's Genetics Lipid metabolism Ddx2017e DEAD0517 expressed (Asp-Glu -Ala-Asp) box polypeptide 20 Acadm acyl-Coenzyme A dehydrogenase, medium chain Ddx24 DEAD (Asp-Glu-Ala-Asp) box polypeptide 24 Acox1 acyl-Coenzyme A oxidase 1, palmitoyl Ddx51 DEAD (Asp-Glu-Ala-Asp) box polypeptide 51 Acsl4 acyl-CoA synthetase long-chain family member 4 Dnmt3b DNA methyltransferase 3B Agpat9 1-acylglycerol-3-phosphate O-acyltransferase 9 Enpp4 ectonucleotide pyrophosphatase/phosphodiesterase 4 Apoa4 apolipoprotein A-IV Enpp6 ectonucleotide pyrophosphatase/phosphodiesterase 6 Apoc3 apolipoprotein C-III Es2el expressed sequence 2 embryonic lethal Arsa arylsulfatase A G430022H RIKEN cDNA G430022H21 gene C130090K2 RIKEN cDNA C130090K23 gene Grsf121Rik G-rich RNA sequence binding factor 1 Cerk3Rik ceramide kinase Gtpbp3 GTP binding protein 3 Cpt1b carnitine palmitoyltransferase 1b, muscle Heyl hairy/enhancer-of-split related with YRPW motif-like Crat carnitine acetyltransferase Hnrpa2b1 heterogeneous nuclear ribonucleoprotein A2 Dbi diazepam binding inhibitor Hnrpa3 heterogeneous nuclear ribonucleoprotein A3 Dhcr7 7-dehydrocholesterol reductase Hnrpab heterogeneous nuclear ribonucleoprotein A/B Ech1 enoyl coenzyme A hydratase 1, peroxisomal Hnrpf heterogeneous nuclear ribonucleoprotein F Elovl7 ELOVL family member 7, elongation of long chain fatty Inoc1 INO80 complex homolog 1 (S. cerevisiae) Faah fattyacids acid(yeast) amide hydrolase Ints6 integrator complex subunit 6 Fabp3 fatty acid binding protein 3, muscle and heart Larp2 La ribonucleoprotein domain family, member 2 Fads3 fatty acid desaturase 3 Larp7 La ribonucleoprotein domain family, member 7 Fdx1 ferredoxin 1 Lsm11 U7 snRNP-specific Sm-like protein LSM11 Gal3st1 galactose-3-O-sulfotransferase 1 Lsm8 LSM8 homolog, U6 small nuclear RNA associated (S. Glt28d2 glycosyltransferase 28 domain containing 2 Msi2h Musashicerevisiae) homolog 2 (Drosophila) Hadhb hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl- N6amt1 N-6 adenine-specific DNA methyltransferase 1 Hao3 hydroxyacidCoenzyme A oxidasethiolase/enoyl (glycolate-Coenzyme oxidase) A 3 hydratase Nadsyn1 NAD(putative) syntheta se 1 Lipe lipase,(trifunctional hormone protein), sensitive beta (AKA subunit HSL) Nmnat1 nicotinamide nucleotide adenylyltransferase 1 Lpin1 lipin 1 Nola1 nucleolar protein family A, member 1 (H/ACA small Lrp10 low-density lipoprotein receptor-related protein 10 Nudt12 nudixnucleolar (nucleoside RNPs) (AKA diphosphate GAR1) linked moiety X)-type Lypla1 lysophospholipase 1 Paics phosphoribosylaminoimidazolemotif 12 carboxylase, Mir16 membrane interacting protein of RGS16 Pcbp1 poly(rC)phosphoribosylaminoribosylaminoimidazole, binding protein 1 Pafah2 platelet-activating factor acetylhydrolase 2 Pcbp2 poly(rC)succinocarboxamide binding protein synthetase 2 Pank1 pantothenate kinase 1 Pold3 polymerase (DNA-directed), delta 3, accessory subunit Perld1 -like domain containing 1 Polg polymerase (DNA directed), gamma Piga phosphatidylinositol glycan anchor biosynthesis, class A Prpf40a PRP40 pre-mRNA processing factor 40 homolog A Pigc phosphatidylinositol glycan anchor biosynthesis, class C Psmc3ip proteasome(yeast) (prosome, macropain) 26S subunit, ATPase Pigp phosphatidylinositol glycan anchor biosynthesis, class P Ptbp1 polypyrimidine3, interacting protein tract binding protein 1 Pigw phosphatidylinositol glycan anchor biosynthesis, class Qk quaking Pik3c2g phosphatidylinositolW 3-kinase, C2 domain containing, Rad17 RAD17 homolog (S. pombe) Pla2g12a phospholipasegamma polypeptide A2, group XIIA Raly hnRNP-associated with lethal yellow

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Rbm17 RNA binding motif protein 17 Gga1 golgi associated, gamma adaptin ear containing, ARF Rbm4b RNA binding motif protein 4B Gtpbp4 GTPbinding binding protein protein 1 4 Rbms1 RNA binding motif, single stranded interacting protein 1 Herpud1 homocysteine-inducible, stress- Rbmxrt RNA binding motif protein, retrogene Hs3st3b1 heparaninducible, sulfate ubiquitin (glucosamine)-like domain 3- Omember-sulfotransferase 1 3B1 Rcl1 RNA terminal phosphate cyclase-like 1 Hs6st1 heparan sulfate 6-O-sulfotransferase 1 Rrp1b ribosomal RNA processing 1 homolog B (S. cerevisiae) Hsdl1 hydroxysteroid dehydrogenase like 1 Sart3 squamous cell carcinoma antigen recognized by T-cells Hspa9 heat shock protein 9 Serbp1 Serpine13 mRNA binding protein 1 Htra1 HtrA serine peptidase 1 Sfrs2 splicing factor, arginine/serine-rich 2 (SC-35) Kcmf1 potassium channel modulatory factor 1 Sfrs3 splicing factor, arginine/serine-rich 3 (SRp20) Kifc3 kinesin family member C3 Sfrs7 splicing factor, arginine/serine-rich 7 Klk15 kallikrein related-peptidase 15 Smc3 structural maintenace of chromosomes 3 Lnpep leucyl/cystinyl aminopeptidase Srpk1 serine/arginine-rich protein specific kinase 1 Lonrf3 LON peptidase N-terminal domain and ring finger 3 Srrm1 serine/arginine repetitive matrix 1 Map1lc3a microtubule-associated protein 1 light chain 3 alpha Srrm2 serine/arginine repetitive matrix 2 Map3k5 mitogen activated protein kinase kinase kinase 5 Syncrip synaptotagmin binding, cytoplasmic RNA interacting Map3k7ip1 mitogen-activated protein kinase kinase kinase 7 Thex1 threeprotein prime histone mRNA exonuclease 1 Mapkapk3 mitogeninteracting-activated protein protein1 kinase-activated protein Upp1 uridine phosphorylase 1 march11 membranekinase 3 -associated ring finger (C3HC4) 11 Wbp11 WW domain binding protein 11 March5 membrane-associated ring finger (C3HC4) 5 Rsad1 radical S-adenosyl methionine domain containing 1 Mfn2 mitofusin 2 Perception of sound Mgea5 meningioma expressed antigen 5 (hyaluronidase) Coch coagulation factor C homolog (Limulus polyphemus) Mgrn1 mahogunin, ring finger 1 Peroxisome metabolism Mid1ip1 Mid1 interacting protein 1 (gastrulation specific G12- Pex5 peroxisome biogenesis factor 5 Midn midnolinlike (zebrafish)) Pxmp3 peroxisomal membrane protein 3 Mlk4 Mitogen-activated protein kinase kinase kinase (mixed Protein modification and metabolism Nek8 NIMAlineage (neverkinase in4) mitosis gene a)-related expressed kinase Fbxo18 F-box protein 18 Nmt2 N8 -myristoyltransferase 2 Fbxo21 F-box protein 21 Nuak1 NUAK family, SNF1-like kinase, 1 Fbxo3 F-box protein 3 Ogt O-linked N-acetylglucosamine (GlcNAc) transferase Fkbp14 FK506 binding protein 14 Pacsin3 protein(UDP-N kinase-acetylglucosamine:polypeptide C and casein kinase substrate-N- in neurons 3 Fkbp2 FK506 binding protein 2 Parp12 polyacetylglucosaminyl (ADP-ribose) polymerase transferase) family, member 12 Iscu IscU iron-sulfur cluster scaffold homolog (E. coli) Parp3 poly (ADP-ribose) polymerase family, member 3 Ahsa1 AHA1, activator of heat shock protein ATPase homolog Pcnp PEST proteolytic signal containing nuclear protein Ap3s1 adaptor1 (yeast)-related protein complex 3, sigma 1 subunit Pcolce procollagen C-endopeptidase enhancer protein Atg4d autophagy-related 4D (yeast) Pctk1 PCTAIRE-motif protein kinase 1 Brap BRCA1 associated protein Pctk2 PCTAIRE-motif protein kinase 2 Bre brain and reproductive organ-expressed protein Pdik1l PDLIM1 interacting kinase 1 like Cd37 CD37 antigen (AKA Tspan26) Pdss1 prenyl (solanesyl) diphosphate synthase, subunit 1 Clk4 CDC like kinase 4 Pepd peptidase D Csnk1d , delta Phpt1 phosphohistidine phosphatase 1 Csnk1e casein kinase 1, epsilon Pim3 proviral integration site 3 D8Ertd82e DNA segment, Chr 8, ERATO Doi 82, expressed Pkn2 protein kinase N2 (AKA PAK2) Dcamkl3 doublecortin-like kinase 3 Plk3 polo-like kinase 3 (Drosophila) Derl3 Der1-like domain family, member 3 Ppia peptidylprolyl isomerase A Dnajb1 DnaJ (Hsp40) homolog, subfamily B, member 1 Ppib peptidylprolyl isomerase B Dnajc16 DnaJ (Hsp40) homolog, subfamily C, member 16 Ppif peptidylprolyl isomerase F (cyclophilin F) Dnajc6 DnaJ (Hsp40) homolog, subfamily C, member 6 Ppm1d protein phosphatase 1D magnesium-dependent, delta Dpm1 dolichol-phosphate (beta-D) mannosyltransferase 1 Ppm1l proteinisoform phosphatase 1 (formerly 2C)-like (AKA PP2CE) Dusp18 dual specificity phosphatase 18 Ppm1m protein phosphatase 1M Dusp6 dual specificity phosphatase 6 Ppp1r10 protein phosphatase 1, regulatory subunit 10 Dusp9 dual specificity phosphatase 9 Ppp5c protein phosphatase 5, catalytic subunit Dzip3 DAZ interacting protein 3, zinc finger Pptc7 PTC7 protein phosphatase homolog (S. cerevisiae) Edem1 ER degradation enhancer, mannosidase alpha-like 1 Psma3 proteasome (prosome, macropain) subunit, alpha type 3 Eml4 echinoderm microtubule associated protein like 4 Rhobtb3 Rho-related BTB domain containing 3 Epha10 Eph receptor A10 Ripk2 receptor (TNFRSF)-interacting serine-threonine kinase 2 Epha7 Eph receptor A7 Ripk5 receptor interacting protein kinase 5 Faf1 Fas-associated factor 1 Rnf128 ring finger protein 128 Fbxl10 F-box and leucine-rich repeat protein 10 Rnf138 ring finger protein 138 Fbxl14 F-box and leucine-rich repeat protein 14 Rnf144 ring finger protein 144 Fbxl15 F-box and leucine-rich repeat protein 15 Rnf44 ring finger protein 44 Fbxo28 F-box protein 28 Rnpepl1 arginyl aminopeptidase (aminopeptidase B)-like 1 Fbxo45 F-box protein 45 Rock2 Rho-associated coiled-coil containing protein kinase 2 Fbxw11 F-box and WD-40 domain protein 11 Rwdd1 RWD domain containing 1 Fkbp8 FK506 binding protein 8 Samd4 sterile alpha motif domain containing 4 Fut11 fucosyltransferase 11 Sbk1 SH3-binding kinase 1 Fyn Fyn proto-oncogene Senp8 SUMO/sentrin specific peptidase 8 Galnt14 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- Sgk2 serum/glucocorticoid regulated kinase 2 Galnt2 UDPacetylgalactosaminyltransferase-N-acetyl-alpha-D-galactosamine:polypeptide 14 N- Slk STE20-like kinase (yeast) (AKA Stk2) Galnt7 UDPacetylgalactosaminyltransferase-N-acetyl-alpha-D-galactosamine: 2 polypeptide N- Smap1l stromal membrane-associated protein 1-like acetylgalactosaminyltransferase 7

177 Chapter III – Manuscript – ChIP-on-chip gene list

Smurf2 SMAD specific E3 ubiquitin protein ligase 2 Cnksr2 connector enhancer of kinase suppressor of Ras 2 Spcs3 signal peptidase complex subunit 3 homolog (S. Cntln centlein, centrosomal protein Spg7 spasticcerevisiae) paraplegia 7 homolog (human) Crk v-crk sarcoma virus CT10 oncogene homolog (avian) Sppl2b Signal peptide peptidase-like 2B Ctxn1 cortexin 1 Src Rous sarcoma oncogene Cxxc5 CXXC finger 5 St3gal3 ST3 beta-galactoside alpha-2,3-sialyltransferase 3 D430028G RIKEN cDNA D430028G21 gene Stk11 serine/threonine kinase 11 (AKA LKB1) Dgka21Rik diacylglycerol kinase, alpha Stk35 serine/threonine kinase 35 Dkk2 dickkopf homolog 2 (Xenopus laevis) Stub1 STIP1 homology and U-Box containing protein 1 Dll4 delta-like 4 (Drosophila) Styxl1 serine/threonine/tyrosine interacting-like 1 Efna1 ephrin A1 Tbc1d10a TBC1 domain family, member 10a Egfr epidermal growth factor receptor Tbc1d2 TBC1 domain family, member 2 Ephb4 Eph receptor B4 Tesk1 testis specific protein kinase 1 Eps8l2 EPS8-like 2 Timm10 translocase of inner mitochondrial membrane 10 Erbb2 v-erb-b2 erythroblastic leukemia viral oncogene Timm8b translocasehomolog (yeast) of inner mitochondrial membrane 8 homolog Errfi1 ERBBhomolog receptor 2, neuro/glioblastoma feedback inhibitor derived 1 oncogene Trap1 TNFb (yeast) receptor -associated protein 1 Fam13b familyhomolog with (avian) sequence similarity 13, member B Tubb5 tubulin, beta 5 Fgf1 fibroblast growth factor 1 Uba52 ubiquitin A-52 residue ribosomal protein fusion product Fgf9 fibroblast growth factor 9 Ubc ubiquitin1 C Fgfr2 fibroblast growth factor receptor 2 Ube2b ubiquitin-conjugating enzyme E2B, RAD6 homology (S. Fzd4 frizzled homolog 4 (Drosophila) Ube2i ubiquitincerevisiae)-conjugating enzyme E2I Gabrb3 gamma-aminobutyric acid (GABA-A) receptor, subunit Ube2n ubiquitin-conjugating enzyme E2N Garnl1 GTPasebeta 3 activating RANGAP domain-like 1 Ube2o ubiquitin-conjugating enzyme E2O Gfra4 glial cell line derived neurotrophic factor family receptor Usp1 ubiquitin specific peptdiase 1 Ghr growthalpha 4 hormone receptor Usp10 ubiquitin specific peptidase 10 Gipc1 GIPC PDZ domain containing family, member 1 Usp2 ubiquitin specific peptidase 2 Gng12 guanine nucleotide binding protein (G protein), gamma Usp3 ubiquitin specific peptidase 3 Gpr120 G12 protein -coupled receptor 120 Usp43 ubiquitin specific peptidase 43 Gpr124 G protein-coupled receptor 124 Wee1 wee 1 homolog (S. pombe) Gpr173 G-protein coupled receptor 173 Yod1 YOD1 OTU deubiquitinating enzyme 1 homologue (S. Gpr56 G protein-coupled receptor 56 Zdhhc17 zinccerevisiae) finger, DHHC domain containing 17 Igf1r insulin-like growth factor I receptor Zdhhc2 zinc finger, DHHC domain containing 2 Igf2bp1 insulin-like growth factor 2 mRNA binding protein 1 Zfp364 zinc finger protein 364 Il10rb interleukin 10 receptor, beta Znrf1 zinc and ring finger 1 Il11ra1 interleukin 11 receptor, alpha chain 1 Akap1 A kinase (PRKA) anchor protein 1 Impa2 inositol (myo)-1(or 4)-monophosphatase 2 Aktip thymoma viral proto-oncogene 1 interacting protein Iqgap1 IQ motif containing GTPase activating protein 1 Bmp2k BMP2 inducible kinase Irs4 insulin receptor substrate 4 Calu calumenin Jag1 jagged 1 Cct7 chaperonin subunit 7 (eta) Kras v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog Cdc42bpb Cdc42 binding protein kinase beta Letm1 -EF-hand containing transmembrane Clk3 CDC-like kinase 3 Magi1 membraneprotein 1 associated guanylate kinase, WW and PDZ Coq7 demethyl-Q 7 Magi3 membranedomain containing associated 1 guanylate kinase, WW and PDZ Dnaja4 DnaJ (Hsp40) homolog, subfamily A, member 4 Map3k1 mitogendomain containing activated protein 3 kinase kinase kinase 1 Dnajc3 DnaJ (Hsp40) homolog, subfamily C, member 3B Map3k11 mitogen activated protein kinase kinase kinase 11 Dusp3 dual specificity phosphatase 3 (vaccinia virus Mapk1ip1 mitogen activated protein kinase 1 interacting protein 1 march7 membranephosphatase-associated VH1-related) ring finger (C3HC4) 7 Mapk6 mitogen-activated protein kinase 6 (AKA ERK3) Signal transduction Mcf2l mcf.2 transforming sequence-like 1300014I06 RIKEN cDNA 1300014I06 gene Nck1 non-catalytic region of tyrosine kinase adaptor protein 1 Acvr2aRik activin receptor IIA Nfkbia nuclear factor of kappa light chain gene enhancer in B- Agt angiotensinogen (serpin peptidase inhibitor, clade A, Ngfb nervecells inhibitor, growth factor, alpha beta Anxa5 annexinmember A58) Nrtn neurturin Arhgap29 Rho GTPase activating protein 29 Oxt oxytocin Arhgap8 Rho GTPase activating protein 8 P2ry13 purinergic receptor P2Y, G-protein coupled 13 Arhgef12 Rho guanine nucleotide exchange factor (GEF) 12 Pde10a phosphodiesterase 10A Arl2 ADP-ribosylation factor-like 2 Pde4c phosphodiesterase 4C, cAMP specific Arl5a ADP-ribosylation factor-like 5A Pde8a phosphodiesterase 8A Arl8a ADP-ribosylation factor-like 8A Pdzrn3 PDZ domain containing RING finger 3 Azi2 5-azacytidine induced gene 2 Pik3r1 phosphatidylinositol 3-kinase, regulatory subunit, Baiap2l1 BAI1-associated protein 2-like 1 Pim1 proviralpolypeptide integration 1 (p85 alpha)site 1 Baiap2l2 BAI1-associated protein 2-like 2 Plekhg2 pleckstrin homology domain containing, family G (with Bambi BMP and activin membrane-bound inhibitor, homolog Plxnd1 plexinRhoGef D1 domain) member 2 Bmper BMP(Xenopus-binding laevis) endothelial regulator Ppp1r1a protein phosphatase 1, regulatory (inhibitor) subunit 1A Camk2b calcium/calmodulin-dependent protein kinase II, beta Ppp1r1b protein phosphatase 1, regulatory (inhibitor) subunit 1B Cd164 CD164 antigen Ppp2r2d protein phosphatase 2, regulatory subunit B, delta Centg1 centaurin, gamma 1 Ppp2r5e proteinisoform phosphatase 2, regulatory subunit B (B56), Chrna4 cholinergic receptor, nicotinic, alpha polypeptide 4 Prkcd proteinepsilon kinaseisoform C, delta Cish cytokine inducible SH2-containing protein Prkce protein kinase C, epsilon Cnih2 cornichon homolog 2 (Drosophila) Prkch protein kinase C, eta Cnih4 cornichon homolog 4 (Drosophila) Pscd1 pleckstrin homology, Sec7 and coiled-coil domains 1

178 Chapter III – Manuscript – ChIP-on-chip gene list

Psd pleckstrin and Sec7 domain containing Elavl2 ELAV (embryonic lethal, abnormal vision, Drosophila)- Ralgps1 Ral GEF with PH domain and SH3 binding motif 1 Elf1 E74like -2like (Hu factor antigen 1 B) Rap1gap Rap1 GTPase-activating protein Eno1 enolase 1, alpha non-neuron Rap2b RAP2B, member of RAS oncogene family Epc1 enhancer of polycomb homolog 1 (Drosophila) Rasip1 Ras interacting protein 1 Esrra estrogen related receptor, alpha Rasl10b RAS-like, family 10, member B Esx1 extraembryonic, spermatogenesis, 1 Rassf10 Ras association (RalGDS/AF-6) domain family (N- Ets1 E26 avian leukemia oncogene 1, 5' domain Rassf3 Rasterminal) association member (RalGDS/AF 10 -6) domain family 3 Ewsr1 Ewing sarcoma breakpoint region 1 Rassf5 Ras association (RalGDS/AF-6) domain family 5 Fev FEV (ETS oncogene family) Rgs12 regulator of G-protein signaling 12 Fhl2 four and a half LIM domains 2 Rgs14 regulator of G-protein signaling 14 Fosb FBJ osteosarcoma oncogene B Rhot2 ras homolog gene family, member T2 Foxd2 forkhead box D2 Rhpn2 rhophilin, Rho GTPase binding protein 2 Foxe1 forkhead box E1 (thyroid transcription factor 2) Rspo2 R-spondin 2 homolog (Xenopus laevis) Foxh1 forkhead box H1 Sh2b1 SH2B adaptor protein 1 Foxn2 forkhead box N2 Sh2b3 SH2B adaptor protein 3 Foxo3a forkhead box O3a Shank3 SH3/ankyrin domain gene 3 Foxq1 forkhead box Q1 She src homology 2 domain-containing transforming protein Gabpa GA repeat binding protein, alpha Sipa1l1 signalE -induced proliferation-associated 1 like 1 Gabpb1 GA repeat binding protein, beta 1 Socs1 suppressor of cytokine signaling 1 Gata2 GATA binding protein 2 Socs3 suppressor of cytokine signaling 3 Gbx2 gastrulation brain homeobox 2 Spry2 sprouty homolog 2 (Drosophila) Gpbp1 GC-rich promoter binding protein 1 Sqstm1 sequestosome 1 Grlf1 glucocorticoid receptor DNA binding factor 1 Tacstd2 tumor-associated calcium signal transducer 2 Gtf3c4 general transcription factor IIIC, polypeptide Tbl1x transducin (beta)-like 1 X-linked H1fx H1 histone family, member X Tenc1 tensin like C1 domain-containing phosphatase Hand2 heart and neural crest derivatives expressed transcript 2 Ticam1 toll-like receptor adaptor molecule 1 Hdac5 histone deacetylase 5 Tmem11 transmembrane protein 11 Hes1 hairy and enhancer of split 1 (Drosophila) Tnfrsf1a tumor necrosis factor receptor superfamily, member 1a Hic2 hypermethylated in cancer 2 Ttrap Traf and Tnf receptor associated protein Hist1h1c histone cluster 1, H1c Ush1c Usher syndrome 1C homolog (human) Hist1h2bc histone cluster 1, H2bc Vav2 vav 2 oncogene Hist2h2ab histone cluster 2, H2ab Vipr1 vasoactive intestinal peptide receptor 1 Hivep1 human immunodeficiency virus type I enhancer binding Wnt4 wingless-related MMTV integration site 4 Hivep2 humanprotein immunodeficiency1 virus type I enhancer binding Wsb2 WD repeat and SOCS box-containing 2 Hmga1 highprotein mobility 2 group AT-hook 1 Xpr1 xenotropic and polytropic retrovirus receptor 1 Hmgb3 high mobility group box 3 Ywhab tyrosine 3-monooxygenase/tryptophan 5- Hsf2 heat shock factor 2 Transcriptional monooxygenaseregulation activation protein, beta polypeptide Id2 inhibitor of DNA binding 2 Arid4b AT rich interactive domain 4B (Rbp1 like) Id3 inhibitor of DNA binding 3 Asxl1 additional sex combs like 1 Ilf3 interleukin enhancer binding factor 3 Atf1 activating transcription factor 1 Insm1 insulinoma-associated 1 Bach2 BTB and CNC homology 2 Irx2 Iroquois related homeobox 2 (Drosophila) Bapx1 bagpipe homeobox gene 1 homolog (Drosophila) (AKA Jarid2 jumonji, AT rich interactive domain 2 Barhl2 BarHNkx3-like2) 2 (Drosophila) Jun Jun oncogene Barx1 BarH-like homeobox 1 Junb Jun-B oncogene Bcl6 B-cell leukemia/lymphoma 6 Jund1 Jun proto-oncogene related gene d1 Bhlhb2 basic helix-loop-helix domain containing, class B2 Khdrbs3 KH domain containing, RNA binding, signal Bnc1 basonuclin 1 Klf11 Kruppeltransduction-like associatedfactor 11 3 Brwd1 bromodomain and WD repeat domain containing 1 Klf13 Kruppel-like factor 13 Cbfa2t2 core-binding factor, runt domain, alpha subunit 2, Klf16 Kruppel-like factor 16 Cbfb coretranslocated binding to,factor 2 (human) beta (AKA PEBP2B) Lhx1 LIM homeobox protein 1 Cbx3 chromobox homolog 3 (Drosophila HP1 gamma) Lmo4 LIM domain only 4 Cbx8 chromobox homolog 8 (Drosophila Pc class) Maf avian musculoaponeurotic fibrosarcoma (v-) AS42 Cebpd CCAAT/enhancer binding protein (C/EBP), delta Mafa voncogene-maf musculoaponeurotic homolog fibrosarcoma oncogene Cggbp1 CGG triplet repeat binding protein 1 Mafb vfamily,-maf musculoaponeurotic protein A (avian) fibrosarcoma oncogene Cited1 Cbp/p300-interacting transactivator with Glu/Asp-rich Maged1 melanomafamily, protein antigen, B (avian) family D, 1 Cited4 Cbp/p300carboxy-terminal-interacting domain transactivator, 1 with Glu/Asp-rich Med13l mediator complex subunit 13-like Clock circadiancarboxy-terminal locomoter domain, output 4 cycles kaput Med22 mediator complex subunit 22 Cnot3 CCR4-NOT transcription complex, subunit 3 Mllt6 myeloid/lymphoid or mixed lineage-leukemia Creb1 cAMP responsive element binding protein 1 Mxd4 Maxtranslocation dimerization to 6 homologprotein 4 (Drosophila) Crsp2 Med14: mediator complex subunit 14 Mycbp c-myc binding protein (AKA AMY-1) Crsp3 Med23: mediator complex subunit 23 Ncoa1 nuclear receptor coactivator 1 (AKA SRC1; RIP160) Crsp7 Med26: mediator complex subunit 26 Ncoa5 nuclear receptor coactivator 5 (AKA CIA) Cutl1 cut-like 1 (Drosophila) (AKA CDP; CUX) Ncor2 nuclear receptor co-repressor 2 (AKA SMRT) Dbp D site albumin promoter binding protein Neud4 neuronal d4 domain family member (AKA DPF1 in Ddit3 DNA-damage inducible transcript 3 Nfat5 nuclearhumans) factor of activated T-cells 5 Deaf1 deformed epidermal autoregulatory factor 1 (Drosophila) Nfatc3 nuclear factor of activated T-cells, cytoplasmic, Dmrta2 doublesex and mab-3 related transcription factor like Nfil3 nuclearcalcineurin factor,-dependent interleukin 3 3, regulated Egr3 earlyfamily growth A2 response 3 Nfx1 nuclear transcription factor, X-box binding 1

179 Chapter III – Manuscript – ChIP-on-chip gene list

Nkx2-4 NK2 transcription factor related, locus 4 (Drosophila) Ybx2 Y box protein 2 Nkx6-2 NK6 transcription factor related, locus 2 (Drosophila) Yy1 YY1 transcription factor Nr0b2 nuclear receptor subfamily 0, group B, member 2 (AKA Zbtb1 zinc finger and BTB domain containing 1 Nr2f2 nuclearSHP) receptor subfamily 2, group F, member 2 (AKA Zbtb37 zinc finger and BTB domain containing 37 Nr2f6 nuclearCOUPTFII) receptor subfamily 2, group F, member 6 (AKA Zbtb43 zinc finger and BTB domain containing 43 Otx1 orthodenticleEAR2) homolog 1 (Drosophila) Zbtb7a zinc finger and BTB domain containing 7a Pax7 paired box gene 7 Zfp143 zinc finger protein 143 Pax8 paired box gene 8 Zfp219 zinc finger protein 219 Pbx2 pre B-cell leukemia transcription factor 2 Zfp238 zinc finger protein 238 Pcgf2 polycomb group ring finger 2 Zfp27 zinc finger protein 27 Per3 period homolog 3 (Drosophila) Zfp326 zinc finger protein 326 Phf12 PHD finger protein 12 Zfp516 zinc finger protein 516 Phf20 PHD finger protein 20 Zfp60 zinc finger protein 60 Phf6 PHD finger protein 6 Zfp629 zinc finger protein 629 Pitx1 paired-like homeodomain transcription factor 1 Zfp655 zinc finger protein 655 Pnrc1 proline-rich nuclear receptor coactivator 1 Zfp668 zinc finger protein 668 Pnrc2 proline-rich nuclear receptor coactivator 2 Zfp687 zinc finger protein 687 Pogz pogo transposable element with ZNF domain Zfp691 zinc finger protein 691 Polr3d polymerase (RNA) III (DNA directed) polypeptide D Zfp710 zinc finger protein 710 Pou2f1 POU domain, class 2, transcription factor 1 (AKA Zfp768 zinc finger protein 768 Ppara peroxisomeOCT1) proliferator activated receptor alpha Zfp784 zinc finger protein 784 Ppargc1b peroxisome proliferative activated receptor, gamma, Zfp787 zinc finger protein 787 Prdm16 PRcoactivator domain 1containing beta 16 Zfp91 zinc finger protein 91 Prdm4 PR domain containing 4 Zfx zinc finger protein X-linked Rarb retinoic acid receptor, beta Zic3 zinc finger protein of the cerebellum 3 Rbbp7 retinoblastoma binding protein 7 Vezf1 vascular endothelial zinc finger 1 Rqcd1 rcd1 (required for cell differentiation) homolog 1 (S. Translation Safb2 scaffoldpombe) attachment factor B2 Dus3l dihydrouridine synthase 3-like (S. cerevisiae) Sall3 sal-like 3 (Drosophila) Eef2 eukaryotic translation elongation factor 2 Satb2 special AT-rich sequence binding protein 2 Eif1ad eukaryotic translation initiation factor 1A domain Sin3a transcriptional regulator, SIN3A (yeast) Eif3s10 eukaryoticcontaining translation initiation factor 3, subunit 10 Six5 sine oculis-related homeobox 5 homolog (Drosophila) Eif4g2 eukaryotic(theta) translation initiation factor 4, gamma 2 Smad3 MAD homolog 3 (Drosophila) Eif4g3 eukaryotic translation initiation factor 4 gamma, 3 Smad7 MAD homolog 7 (Drosophila) Eif5 eukaryotic translation initiation factor 5 Smarcd1 SWI/SNF related, matrix associated, actin dependent Eif5a eukaryotic translation initiation factor 5A Smarce1 SWI/SNFregulator of related, chromatin, matrix subfamily associated, d, memberactin dependent 1 Etf1 eukaryotic translation termination factor 1 Sox12 SRYregulator-box ofcontaining chromatin, gene subfamily 12 e, member 1 Fau Finkel-Biskis-Reilly murine sarcoma virus (FBR- Sox18 SRY-box containing gene 18 Mrpl12 mitochondrialMuSV) ubiquitously ribosomal expressed protein (fox L12 derived) Sp3 trans-acting transcription factor 3 Mrpl23 mitochondrial ribosomal protein L23 Sp8 trans-acting transcription factor 8 Mrpl36 mitochondrial ribosomal protein L36 Sp9 trans-acting transcription factor 9 Mrpl4 mitochondrial ribosomal protein L4 Spen SPEN homolog, transcriptional regulator (Drosophila) Mrps18b mitochondrial ribosomal protein S18B Spib Spi-B transcription factor (Spi-1/PU.1 related) Mrps6 mitochondrial ribosomal protein S6 Ssbp3 single-stranded DNA binding protein 3 Mtrf1l mitochondrial translational release factor 1-like Suv420h2 suppressor of variegation 4-20 homolog 2 (Drosophila) Pelo pelota homolog (Drosophila) Taf7l TAF7-like RNA polymerase II, TATA box binding Phf5a PHD finger protein 5A Taf9 TAF9protein RNA (TBP) polymerase-associated II, factor TATA box binding protein Rpl7 ribosomal protein L7 Tal1 T(TBP)-cell -acuteassociated lymphocytic factor leukemia 1 Rpl7a ribosomal protein L7a Tbl1xr1 transducin (beta)-like 1X-linked receptor 1 Rplp0 ribosomal protein, large, P0 Tbx2 T-box 2 Rps20 ribosomal protein S20 Tbx21 T-box 21 Snrpd3 small nuclear ribonucleoprotein D3 Tcf2 transcription factor 2 Transport Tcf7l2 transcription factor 7-like 2, T-cell specific, HMG-box Abcb10 ATP-binding cassette, sub-family B (MDR/TAP), Tcfcp2l1 transcription factor CP2-like 1 Abcd1 ATPmember-binding 10 cassette, sub-family D (ALD), member 1 Tcfe3 transcription factor E3 Abcd3 ATP-binding cassette, sub-family D (ALD), member 3 Tead3 TEA domain family member 3 Aqp3 aquaporin 3 Tgif2 TGFB-induced factor 2 Arf1 ADP-ribosylation factor 1 Thrap3 thyroid hormone receptor associated protein 3 Arf4 ADP-ribosylation factor 4 Tigd5 tigger transposable element derived 5 Arf6 ADP-ribosylation factor 6 Tle1 transducin-like enhancer of split 1, homolog of Atg16l1 autophagy-related 16-like 1 (yeast) Tle3 transducinDrosophila- likeE(spl) enhancer of split 3, homolog of Atp10d ATPase, Class V, type 10D Tle4 transducinDrosophila- likeE(spl) enhancer of split 4, homolog of Atp11a ATPase, class VI, type 11A Trim28 tripartiteDrosophila motif E(spl) protein 28 Atp11c Atpase, class VI, type 11C Trps1 trichorhinophalangeal syndrome I (human) Atp13a1 ATPase type 13A1 Twist1 twist gene homolog 1 (Drosophila) Atp1a1 ATPase, Na+/K+ transporting, alpha 1 polypeptide Uimc1 ubiquitin interaction motif containing 1 Atp1a3 ATPase, Na+/K+ transporting, alpha 3 polypeptide Usf2 upstream transcription factor 2 Atp1b1 ATPase, Na+/K+ transporting, beta 1 polypeptide Wt1 Wilms tumor homolog Atp1b2 ATPase, Na+/K+ transporting, beta 2 polypeptide Wwtr1 WW domain containing transcription regulator 1 Atp2b1 ATPase, Ca++ transporting, plasma membrane 1

180 Chapter III – Manuscript – ChIP-on-chip gene list

AU042651 expressed sequence AU042651 Slc25a5 solute carrier family 25 (mitochondrial carrier, adenine Bet1l blocked early in transport 1 homolog (S. cerevisiae)-like Slc26a4 solutenucleotide carrier translocator), family 26, membermember 45 (Pds/pendrin)(Ant2) Bsnd Bartter syndrome, infantile, with sensorineural deafness Slc2a2 solute carrier family 2 (facilitated glucose transporter), C1qtnf5 C1q(Barttin) and tumor necrosis factor related protein 5 Slc30a1 solutemember carrier 2 (Glut2) family 30 (zinc transporter), member 1 C1qtnf6 C1q and tumor necrosis factor related protein 6 Slc30a4 solute(Znt1) carrier family 30 (zinc transporter), member 4 Cacna1g calcium channel, voltage-dependent, T type, alpha 1G Slc35a1 solute(Im/Znt4) carrier family 35 (CMP-sialic acid transporter), Cacna1h calciumsubunit channel, voltage-dependent, T type, alpha 1H Slc35e1 solutemember carrier 1 family 35, member E1 Cacna2d2 calciumsubunit channel, voltage-dependent, alpha 2/delta Slc38a2 solute carrier family 38, member 2 (ATA2/SAT2) Cacna2d3 calciumsubunit 2channel, voltage-dependent, alpha2/delta Slc3a2 solute carrier family 3 (activators of dibasic and neutral Cadps Ca<2+>dependentsubunit 3 activator protein for secretion (AKA Slc41a3 soluteamino carrieracid transport), family 41, member member 2 3 Car2 carbonicCAPS) anhydrase 2 (AKA CA2) Slc43a2 solute(4F2/Cd98/Ly10/Mdu1/NACAE) carrier family 43, member 2 (Lat4) Chchd4 coiled-coil-helix-coiled-coil-helix domain containing 4 Slc47a1 solute carrier family 47, member 1 Clic4 chloride intracellular channel 4 (mitochondrial) Slc4a1 solute carrier family 4 (anion exchanger), member 1 Cnnm1 cyclin M1 (Metal transporter CNNM1 ) Slc4a2 solute(Ae1/CD233/Empb3) carrier family 4 (anion exchanger), member 2 Copz2 coatomer protein complex, subunit zeta 2 Slc4a3 solute(Ae2/B3RP) carrier family 4 (anion exchanger), member 3 Cplx1 complexin 1 Slc4a8 solute(Ae3) carrier family 4 (anion exchanger), member 8 Dnm1 dynamin 1 Slc6a17 solute carrier family 6 (neurotransmitter transporter), Dscr3 Down syndrome critical region gene 3 Slc6a2 solutemember carrier 17 family 6 (neurotransmitter transporter, Eif4enif1 eukaryotic translation initiation factor 4E nuclear import Slc6a8 solutenoradrenalin), carrier family member 6 (neurotransmitter 2 (Net/NE-T) transporter, Erp29 endoplasmicfactor 1 reticulum protein 29 Slc7a14 solutecreatine), carrier member family 8 (CRT/CT1/CRTR/Creat)7 (cationic amino acid transporter, Exoc3 exocyst complex component 3 Snx13 sortingy+ system), nexin member 13 14 Exoc8 exocyst complex component 8 Stau1 staufen (RNA binding protein) homolog 1 (Drosophila) Fmr1 fragile X mental retardation syndrome 1 homolog Steap3 STEAP family member 3 Fxyd2 FXYD domain-containing ion transport regulator 2 Stx17 syntaxin 17 G3bp2 GTPase activating protein (SH3 domain) binding protein Stxbp1 syntaxin binding protein 1 Golga2 golgi2 autoantigen, golgin subfamily a, 2 Sypl2 synaptophysin-like 2 Grin2d glutamate receptor, ionotropic, NMDA2D (epsilon 4) Tfrc transferrin receptor Hcn3 hyperpolarization-activated, cyclic nucleotide-gated K+ Timm9 translocase of inner mitochondrial membrane 9 homolog Hpxn hemopexin3 Tloc1 SEC62(yeast) homolog (S. cerevisiae) Itpr1 inositol 1,4,5-triphosphate receptor 1 Tm9sf2 transmembrane 9 superfamily member 2 Kcnc3 potassium voltage gated channel, Shaw-related Tmem37 transmembrane protein 37 Kcnh2 potassiumsubfamily, voltagemember-gated 3 channel, subfamily H (eag- Tomm20 translocase of outer mitochondrial membrane 20 Kcnj11 potassiumrelated), member inwardly 2 rectifying channel, subfamily J, Tpcn1 twohomolog pore channel(yeast) 1 Kcnj16 potassiummember 11 inwardly -rectifying channel, subfamily J, Trpc5 transient receptor potential cation channel, subfamily C, Kcnq1 potassiummember 16 voltage -gated channel, subfamily Q, member Trpm7 transientmember 5receptor potential cation channel, subfamily M, Kcnq3 potassium1 voltage-gated channel, subfamily Q, member Tst thiosulfatemember 7 sulfurtransferase, mitochondrial Kctd15 potassium3 channel tetramerisation domain containing 15 Txlng gamma taxilin Kctd8 potassium channel tetramerisation domain containing 8 Vdac1 voltage-dependent anion channel 1 Klhl2 kelch-like 2, Mayven (Drosophila) Vdac2 voltage-dependent anion channel 2 Le51 Protein fucU homolog Wipi1 WD repeat domain, phosphoinositide interacting 1 Lman2 lectin, mannose-binding 2 Visual perception Mfsd4 major facilitator superfamily domain containing 4 Rdh10 retinol dehydrogenase 10 (all-trans) Mtch2 mitochondrial carrier homolog 2 (C. elegans) Gclc glutamate-cysteine ligase, catalytic subunit Osbpl11 oxysterol binding protein-like 11 Pdxp pyridoxal (pyridoxine, vitamin B6) phosphatase Osbpl2 oxysterol binding protein-like 2 Vkorc1l1 vitamin K epoxide reductase complex, subunit 1-like 1 Osbpl5 oxysterol binding protein-like 5 Ppcs phosphopantothenoylcysteine synthetase Osbpl6 oxysterol binding protein-like 6 Other or Unknown Osbpl8 oxysterol binding protein-like 8 0610012H0 RIKEN cDNA 0610012H03 gene Pctp phosphatidylcholine transfer protein 0610025P13Rik RIKEN cDNA 0610025P10 gene Plekha8 pleckstrin homology domain containing, family A 1110007M00Rik RIKEN cDNA 1110007M04 gene Pttg1ip pituitary(phosphoinositide tumor-transforming binding specific) 1 interacting member protein 8 1110008F14Rik RIKEN cDNA 1110008F13 gene Rab21 RAB21, member RAS oncogene family 1110014K03Rik family with sequence similarity 100, member B Rab34 RAB34, member of RAS oncogene family 1110019N18Rik RIKEN cDNA 1110019N10 gene Rab35 RAB35, member RAS oncogene family 1110036O00Rik RIKEN cDNA 1110036O03 gene Ranbp2 RAN binding protein 2 1110058L13Rik RIKEN cDNA 1110058L19 gene Ranbp6 RAN binding protein 6 1190005I069Rik RIKEN cDNA 1190005I06 gene Rilp Rab interacting lysosomal protein 1190017O1Rik RIKEN cDNA 1190017O12 gene Rnuxa RNA U, small nuclear RNA export adaptor 1700011J12Rik RIKEN cDNA 1700011J10 gene Scnn1a sodium channel, nonvoltage-gated, type I, alpha 1700021F00Rik RIKEN cDNA 1700021F05 gene Seh1l SEH1-like (S. cerevisiae) 1700029G05Rik RIKEN cDNA 1700029G01 gene Slc10a7 solute carrier family 10 (sodium/ cotransporter 1700040L01Rik RIKEN cDNA 1700040L02 gene Slc12a6 solutefamily), carrier member family 7 12, member 6 (KCC3) 1700067K02Rik RIKEN cDNA 1700067K01 gene Slc12a7 solute carrier family 12, member 7 (KCC4) 1700120B01Rik RIKEN cDNA 1700120B06 gene Slc16a11 solute carrier family 16 (monocarboxylic acid 1810020D16Rik RIKEN cDNA 1810020D17 gene Slc22a15 solutetransporters), carrier familymember 22 11 (organic anion/cation 1810048J17Rik RIKEN cDNA 1810048J11 gene Slc22a5 solutetransporter), carrier member family 22 15 (organic cation transporter), 2210020M01Rik RIKEN cDNA 2210020M01 gene Slc25a28 solutemember carrier 5 (Jvs/Lstpl/Octn2) family 25, member 28 (Mfrn2/Mrs3/4) 2310003L21Rik RIKEN cDNA 2310003L22 gene Slc25a39 solute carrier family 25, member 39 2310008M12Rik RIKEN cDNA 2310008M10 gene Slc25a4 solute carrier family 25 (mitochondrial carrier, adenine 2310035K20Rik RIKEN cDNA 2310035K24 gene nucleotide translocator), member 4 (Ant1) 4Rik

181 Chapter III – Manuscript – ChIP-on-chip gene list

2310045A2 RIKEN cDNA 2310045A20 gene Ccdc56 coiled-coil domain containing 56 2310057M20Rik RIKEN cDNA 2310057M21 gene Ccdc68 coiled-coil domain containing 68 2610019F01Rik RIKEN cDNA 2610019F03 gene Ccdc92 coiled-coil domain containing 92 2610110G13Rik RIKEN cDNA 2610110G12 gene Ccdc96 coiled-coil domain containing 96 2610528J12Rik RIKEN cDNA 2610528J11 gene Cep110 RIKEN cDNA 6330503K22 gene 2700081O11Rik RIKEN cDNA 2700081O15 gene Cep68 centrosomal protein 68 2810046L05Rik RIKEN cDNA 2810046L04 gene Chchd3 coiled-coil-helix-coiled-coil-helix domain containing 3 4632411B14Rik RIKEN cDNA 4632411B12 gene Clmn calmin 4930402H22Rik RIKEN cDNA 4930402H24 gene Cobll1 Cobl-like 1 4930452B04Rik RIKEN cDNA 4930452B06 gene Commd3 COMM domain containing 3 4930542C16Rik RIKEN cDNA 4930542C12 gene Cops7a COP9 (constitutive photomorphogenic) homolog, 5730410E12Rik RIKEN cDNA 5730410E15 gene Coq10b coenzymesubunit 7a Q10(Arabidopsis homolog thaliana) B (S. cerevisiae) 5730419I095Rik RIKEN cDNA 5730419I09 gene Cpeb3 cytoplasmic polyadenylation element binding protein 3 5830415L2Rik RIKEN cDNA 5830415L20 gene Creld1 cysteine-rich with EGF-like domains 1 6330416G10Rik RIKEN cDNA 6330416G13 gene Ctage5 CTAGE family, member 5 6330512M03Rik RIKEN cDNA 6330512M04 gene Cyhr1 cysteine and histidine rich 1 6330578E14Rik RIKEN cDNA 6330578E17 gene D10Jhu81e DNA segment, Chr 10, Johns Hopkins University 81 6430527G17Rik RIKEN cDNA 6430527G18 gene D12Ertd64 DNAexpressed segment, Chr 12, ERATO Doi 647, expressed 9530058B08Rik RIKEN cDNA 9530058B02 gene D130007C7e RIKEN cDNA D130007C19 gene A030009H02Rik RIKEN cDNA A030009H04 gene D17Wsu9219Rik DNA segment, Chr 17, Wayne State University 92, A530082C14Rik RIKEN cDNA A530082C11 gene D230039L0e RIKENexpressed cDNA D230039L06 gene A730020M1Rik RIKEN cDNA A730020M07 gene D3Ertd7516Rik DNA segment, Chr 3, ERATO Doi 751, expressed A830073O207Rik RIKEN cDNA A830073O21 gene D430041B1e RIKEN cDNA D430041B17 gene A830093I21Rik RIKEN cDNA A830093I24 gene D4Ertd22e7Rik DNA segment, Chr 4, ERATO Doi 22, expressed A930011G24Rik predicted gene, OTTMUSG00000007392 D4Wsu53e DNA segment, Chr 4, Wayne State University 53, Abhd124 abhydrolase domain containing 12 D630039A0 RIKENexpressed cDNA D630039A03 gene AI413782 expressed sequence AI413782 D630044L23Rik RIKEN cDNA gene D630044L22 gene AI837181 expressed sequence AI837181 D830046C2Rik RIKEN cDNA D830046C22 gene AI854703 expressed sequence AI854703 D930015E022Rik RIKEN cDNA D930015E06 gene Alkbh7 alkB, alkylation repair homolog 7 (E. coli) D9Ertd2806Rik DNA segment, Chr 9, ERATO Doi 280, expressed Ankrd34a ankyrin repeat domain 34A Dact2e dapper homolog 2, antagonist of beta-catenin (xenopus) Ankrd34b ankyrin repeat domain 34B Dcun1d1 DCUN1D1 DCN1, defective in cullin neddylation 1, Ankrd39 ankyrin repeat domain 39 Dcun1d3 DCN1,domain defectivecontaining in 1 cullin (S. cerevisiae) neddylation 1, domain Ankrd47 KN motif and ankyrin repeat domains 3 Dennd2a DENN/MADDcontaining 3 (S. domain cerevisiae) containing 2A Ankrd9 ankyrin repeat domain 9 Dis3l2 DIS3 mitotic control homolog (S. cerevisiae)-like 2 Aof1 amine oxidase, flavin containing 1 Dnajc25 DnaJ (Hsp40) homolog, subfamily C , member 25 Arl6ip6 ADP-ribosylation factor-like 6 interacting protein 6 Dock8 dedicator of cytokinesis 8 Atad2b ATPase family, AAA domain containing 2B Dpy30 dpy-30 homolog (C. elegans) Atl3 atlastin GTPase 3 E030041M UHRF1 (ICBP90) binding protein 1-like Atxn2l ataxin 2-like E130114P121Rik RIKEN cDNA E130114P18 gene AW544981 expressed sequence AW544981 E130306M8Rik sterile alpha motif domain containing 5 AW549877 expressed sequence AW549877 E130308A117Rik RIKEN cDNA E130308A19 gene B330016D1 RIKEN cDNA B330016D10 gene E130309D09Rik RIKEN cDNA E130309D02 gene B3bp0Rik NEDD4 binding protein 2 E130309D12Rik RIKEN cDNA E130309D14 gene B430319F0 RIKEN cDNA B430319F04 gene E230019M4Rik RIKEN cDNA E230019M04 gene B4galnt44Rik beta-1,4-N-acetyl-galactosaminyl transferase 4 Fam108c04Rik family with sequence similarity 108, member C B630019K0 RIKEN cDNA B630019K06 gene Fam109a family with sequence similarity 109, member A Bahd16Rik bromo adjacent homology domain containing 1 Fam176b family with sequence similarity 176, member B Basp1 brain abundant, membrane attached signal protein 1 Fam20a family with sequence similarity 20, member A BC019143 RIKEN cDNA 1300010F03 gene Fam40a family with sequence similarity 40, member A BC023744 cDNA sequence BC023744 Fam53b family with sequence similarity 53, member B BC031748 cDNA sequence BC031748 Fam54b family with sequence similarity 54, member B BC051227 cDNA sequence BC051227 Fam60a family with sequence similarity 60, member A BC055791 RIKEN cDNA 2310028O11 gene Fam82a expressed sequence AW061290 BC061194 cDNA sequence BC061194 Fcho1 FCH domain only 1 BC107398 RIKEN cDNA C130092O11 gene Fundc2 FUN14 domain containing 2 Bend3 BEN domain containing 3 Gcap14 granule cell antiserum positive 14 Brp44 brain protein 44 Gipc2 GIPC PDZ domain containing family, member 2 C030014K2 RIKEN cDNA C030014K22 gene Glod5 glyoxalase domain containing 5 C230071H2Rik RIKEN cDNA C230071H18 gene Gm129 gene model 129, (NCBI) C230078M18Rik RIKEN cDNA C230078M08 gene Gm1568 gene model 1568, (NCBI) C530044N108Rik RIKEN cDNA C530044N13 gene Gm691 V-set and transmembrane domain containing 2-like Cab393Rik calcium binding protein 39 Gm75 gene model 75, (NCBI) Cabp7 calcium binding protein 7 Gm854 transmembrane protein 211 Cachd1 cache domain containing 1 Gm962 gene model 962, (NCBI) Casd1 CAS1 domain containing 1 Grwd1 glutamate-rich WD repeat containing 1 Cc2d1b coiled-coil and C2 domain containing 1B Gse1 genetic suppressor element 1 Ccdc102a coiled-coil domain containing 102A Higd1b HIG1 domain family, member 1B Ccdc124 coiled-coil domain containing 124 Hint2 histidine triad nucleotide binding protein 2

182 Chapter III – Manuscript – ChIP-on-chip gene list

Htf9c HpaII tiny fragments locus 9c Rhbdd3 rhomboid domain containing 3 Iffo1 intermediate filament family orphan 1 Rilpl2 Rab interacting lysosomal protein-like 2 Immt inner membrane protein, mitochondrial Rmi1 RMI1, RecQ mediated genome instability 1, homolog Irgq immunity-related GTPase family, Q Rmnd1 required(S. cerevisiae) for meiotic nuclear division 1 homolog (S. Islr2 immunoglobulin superfamily containing leucine-rich Rnf38 ringcerevisiae) finger protein 38 Kbtbd4 kelchrepeat repeat 2 and BTB (POZ) domain containing 4 Rtn4ip1 reticulon 4 interacting protein 1 Kdelc1 KDEL (Lys-Asp-Glu-Leu) containing 1 Rusc1 RUN and SH3 domain containing 1 Klhdc7a kelch domain containing 7A Rusc2 RUN and SH3 domain containing 2 Klhl24 kelch-like 24 (Drosophila) S100pbp S100P binding protein Lactb2 lactamase, beta 2 Sacm1l SAC1 (suppressor of actin mutations 1, homolog)-like Letm2 leucine zipper-EF-hand containing transmembrane Scoc short(S. cerevisiae) coiled-coil protein Limd2 LIMprotein domain 2 containing 2 Sestd1 SEC14 and spectrin domains 1 LOC43287 predicted gene, EG432870 Setd5 SET domain containing 5 Loh12cr10 loss of heterozygosity, 12, chromosomal region 1 Sfi1 Sfi1 homolog, spindle assembly associated (yeast) Lrfn4 leucinehomolog rich (human) repeat and fibronectin type III domain Slmo2 slowmo homolog 2 (Drosophila) Lrrc24 leucinecontaining rich 4 repeat containing 24 Smek1 SMEK homolog 1, suppressor of mek1 (Dictyostelium) Lrrc4b leucine rich repeat containing 4B Smtnl2 smoothelin-like 2 Lrrc58 leucine rich repeat containing 58 Snx29 sorting nexin 29 Lrrc66 leucine rich repeat containing 66 Spata17 spermatogenesis associated 17 Lrrc8a leucine rich repeat containing 8A Spats1 spermatogenesis associated, serine-rich 1 Lrrc8b leucine rich repeat containing 8 family, member B Spats2 spermatogenesis associated, serine-rich 2 Luc7l2 LUC7-like 2 (S. cerevisiae) Spats2l RIKEN cDNA 2810022L02 gene Lynx1 Ly6/neurotoxin 1 Specc1 sperm antigen with calponin homology and coiled-coil Lyrm1 LYR motif containing 1 St7 Suppressiondomains 1 of tumorigenicity 7 Lyrm5 LYR motif containing 5 Stard7 START domain containing 7 Magt1 magnesium transporter 1 Surf4 surfeit gene 4 Mal2 mal, T-cell differentiation protein 2 Susd3 sushi domain containing 3 Mbd6 methyl-CpG binding domain protein 6 Tada1l transcriptional adaptor 1 (HFI1 homolog, yeast) like Mcph1 microcephaly, primary autosomal recessive 1 Tanc1 tetratricopeptide repeat, ankyrin repeat and coiled-coil Metrnl meteorin, glial cell differentiation regulator-like Tatdn2 TatDcontaining DNase 1 domain containing 2 Mff mitochondrial fission factor Tctn3 tectonic family member 3 Mllt10 myeloid/lymphoid or mixed lineage-leukemia Tlcd1 TLC domain containing 1 Mobkl1b MOB1,translocation Mps Oneto 10 Binder homolog kinase (Drosophila) activator- like 1B (yeast) Tlcd2 TLC domain containing 2 Mobkl2b MOB1, Mps One Binder kinase activator-like 2B (yeast) Tmco4 transmembrane and coiled-coil domains 4 Mp68 RIKEN cDNA 2010107E04 gene Tmem106b transmembrane protein 106B Msx1 This gene encodes a protein containing two conserved Tmem127 transmembrane protein 127 N4bp2l1 NEDD4tandem RNA binding recognition protein 2 motifs.-like 1 Similar proteins in Tmem136 transmembrane protein 136 Nat11 Nother-acetyltransferase species function 11 as RNA-binding proteins and play Tmem161b transmembrane protein 161B central roles in posttranscriptional gene regulation. Two Ndg2 Nur77 downstream gene 2 Tmem163 transmembrane protein 163 transcript variants encoding distinct isoforms have been Nipsnap1 identified4-nitrophenylphosphatase for this gene domain and non-neuronal Tmem168 transmembrane protein 168 Nkap NFKBSNAP25 activating-like protein protein homolog 1 (C. elegans) Tmem171 transmembrane protein 171 No66 nucleolar protein 66 Tmem30b transmembrane protein 30B Noc4l nucleolar complex associated 4 homolog (S. cerevisiae) Tmem57 transmembrane protein 57 Notum notum pectinacetylesterase homolog (Drosophila) Tmem80 transmembrane protein 80 Nsun4 NOL1/NOP2/Sun domain family, member 4 Tmem82 transmembrane protein 82 Nudt19 nudix (nucleoside diphosphate linked moiety X)-type Tmie transmembrane inner ear Nudt8 nudixmotif 19(nucleoside diphosphate linked moiety X)-type Tor1aip1 torsin A interacting protein 1 Nufip2 nuclearmotif 8 fragile X mental retardation protein interacting Trabd TraB domain containing Oaf OAFprotein homolog 2 (Drosophila) Trap1a tumor rejection antigen P1A Oplah 5-oxoprolinase (ATP-hydrolysing) Trim44 tripartite motif-containing 44 ORF61 open reading frame 61 Trim46 tripartite motif protein 46 Pcm1 pericentriolar material 1 Trim59 tripartite motif-containing 59 Pdzd7 PDZ domain containing 7 Trim62 tripartite motif-containing 62 Peli2 pellino 2 Trp53inp2 transformation related protein 53 inducible nuclear Phf15 PHD finger protein 15 Ttc36 tetratricopeptideprotein 2 repeat domain 36 Phf23 PHD finger protein 23 Tusc1 tumor suppressor candidate 1 Phf8 PHD finger protein 8 Uap1l1 UDP-N-acteylglucosamine pyrophosphorylase 1-like 1 Pld5 phospholipase D family, member 5 Ugt3a1 UDP glycosyltransferases 3 family, polypeptide A1 Pnkd paroxysmal nonkinesiogenic dyskinesia Usmg5 upregulated during skeletal muscle growth 5 Ppm1h protein phosphatase 1H (PP2C domain containing) Vwa5b2 von Willebrand factor A domain containing 5B2 Ppp1r16a protein phosphatase 1, regulatory (inhibitor) subunit 16A Wapal wings apart-like homolog (Drosophila) Ppp4r4 protein phosphatase 4, regulatory subunit 4 Wdr32 WD repeat domain 32 Ptcd3 Pentatricopeptide repeat domain 3 Wdr34 WD repeat domain 34 Rad54l2 Rad54 like 2 (S. cerevisiae) Wdr72 WD repeat domain 72 Raver2 ribonucleoprotein, PTB-binding 2 Wdr77 WD repeat domain 77 Rbm47 RNA binding motif protein 47 Wdtc1 WD and tetratricopeptide repeats 1 Rbpms2 RNA binding protein with multiple splicing 2 Wfdc16 WAP four-disulfide core domain 16 Rdh13 retinol dehydrogenase 13 (all-trans and 9-cis) Wtip WT1-interacting protein Rftn1 raftlin lipid raft linker 1 Wwc1 WW, C2 and coiled-coil domain containing 1

183 Chapter III – Manuscript – Microarray downregulated genes

Table 3.S2: List of the downregulated genes in the ERRα null kidney

Gene Symbol LOG2 p-value Gene Title Esrra -2.930 3.20766E-06 estrogen related receptor, alpha Top2a -1.278 0.004494077 topoisomerase (DNA) II alpha Col5a2 -1.211 0.000176391 procollagen, type V, alpha 2 Col3a1 -1.189 0.001675718 procollagen, type III, alpha 1 Klk1b5 -1.024 0.001535858 kallikrein 1-related peptidase b5 Cdc2a -0.996 0.011516477 cell division cycle 2 homolog A (S. pombe) Ptgds -0.994 0.008707223 prostaglandin D2 synthase (brain) Mki67 -0.994 0.00230932 antigen identified by monoclonal antibody Ki 67 Rrm2 -0.982 0.013745613 ribonucleotide reductase M2 4933439C20Rik -0.959 0.02946438 RIKEN cDNA 4933439C20 gene Asb11 -0.919 0.004029305 ankyrin repeat and SOCS box-containing protein 11 Spag5 -0.900 0.033560738 sperm associated antigen 5 R3hcc1 -0.896 0.02526563 R3H domain and coiled-coil containing 1 Mogat2 -0.855 0.002143854 monoacylglycerol O-acyltransferase 2 Birc5 -0.841 0.012052809 baculoviral IAP repeat-containing 5 Hmgcr -0.839 0.037641395 3-hydroxy-3-methylglutaryl-Coenzyme A reductase 1200016E24Rik -0.829 0.011554615 RIKEN cDNA 1200016E24 gene /// similar to gag protein Pbk -0.823 0.006878275 PDZ binding kinase 2310076G05Rik -0.818 0.013581776 RIKEN cDNA 2310076G05 gene Luc7l2 -0.802 0.006857663 LUC7-like 2 (S. cerevisiae) Enpp6 -0.766 7.96852E-05 ectonucleotide pyrophosphatase/phosphodiesterase 6 Ccnb2 -0.766 0.035319928 cyclin B2 Cdc20 -0.762 0.020209845 cell division cycle 20 homolog (S. cerevisiae) Ugt2b37 -0.755 0.0006278 UDP glucuronosyltransferase 2 family, polypeptide B37 Asns -0.752 0.041903887 asparagine synthetase Figf -0.739 0.005440182 C-fos induced growth factor Fst -0.733 0.015425351 follistatin Ctsc -0.726 0.001316615 cathepsin C Btc -0.722 0.020271216 betacellulin, epidermal growth factor family member EG622782 -0.719 0.018757777 similar to LOC635138 protein /// predicted gene Slc4a1 -0.719 0.000780748 solute carrier family 4 (anion exchanger), member 1 Arntl -0.714 0.00299459 aryl hydrocarbon receptor nuclear translocator-like Tubb2a -0.707 0.002494503 tubulin, beta 2a C730036D15Rik -0.705 0.003102167 RIKEN cDNA C730036D15 gene AI314604 -0.701 0.000270045 expressed sequence AI314604 RP23-480B19.10 -0.700 0.004079151 similar to histone 2a 2610205E22Rik -0.694 0.028103707 RIKEN cDNA 2610205E22 gene Ybx2 -0.676 0.036506735 Y box protein 2 /// similar to Y box protein 2 Npas2 -0.675 0.01618803 neuronal PAS domain protein 2 Hells -0.675 0.011987744 helicase, lymphoid specific Pla2g7 -0.674 0.007909319 phospholipase A2, group VII Ccna2 -0.674 0.013576182 cyclin A2 Tiam2 -0.673 0.002653435 T-cell lymphoma invasion and metastasis 2 Slc12a1 -0.667 0.01771239 sodium/potassium/chloride transporter, member 1 Nisch -0.666 0.016268492 nischarin Gclc -0.665 0.007659256 glutamate-cysteine ligase, catalytic subunit Etnk1 -0.664 0.025538927 ethanolamine kinase 1 Cck -0.660 0.029055856 cholecystokinin C86595 -0.659 0.013010301 expressed sequence C86595 1600015H20Rik -0.656 0.004624009 RIKEN cDNA 1600015H20 gene Cfd -0.656 0.044101454 complement factor D (adipsin) 4932438A13Rik -0.643 0.028102577 RIKEN cDNA 4932438A13 gene Lpl -0.640 0.004431058 lipoprotein lipase Col1a1 -0.639 0.002722232 procollagen, type I, alpha 1 Mapk8 -0.637 0.025689488 mitogen activated protein kinase 8 Calb1 -0.633 0.037370343 calbindin-28K Cdca3 -0.629 0.033459697 cell division cycle associated 3 1700007B13Rik -0.628 0.001928683 RIKEN cDNA 1700007B13 gene Tcf19 -0.622 0.01024405 transcription factor 19 Col1a2 -0.622 0.000762443 procollagen, type I, alpha 2 4632434I11Rik -0.622 0.0048302 RIKEN cDNA 4632434I11 gene Fgf1 -0.618 0.008093536 fibroblast growth factor 1 Lynx1 -0.618 0.000289255 Ly6/neurotoxin 1 Aqp6 -0.618 0.008169765 aquaporin 6 Plod2 -0.614 0.004610593 procollagen lysine, 2-oxoglutarate 5-dioxygenase 2

184 Chapter III – Manuscript – Microarray downregulated genes

Ypel2 -0.611 0.007390768 yippee-like 2 (Drosophila) Emp1 -0.607 0.000990991 epithelial membrane protein 1 Brd8 -0.604 0.036934923 bromodomain containing 8 Rnps1 -0.602 0.035006188 ribonucleic acid binding protein S1 Elovl2 -0.591 0.010346867 elongation of very long chain fatty acids 2 Olfml1 -0.591 0.011859082 olfactomedin-like 1 Cbfb -0.588 0.010572866 beta Avpr1a -0.586 0.009319612 arginine vasopressin receptor 1A 6720475J19Rik -0.585 0.037746564 RIKEN cDNA 6720475J19 gene Osmr -0.582 0.049935885 oncostatin M receptor Smc2 -0.580 0.03360241 structural maintenance of chromosomes 2 Sertad4 -0.579 0.020180812 SERTA domain containing 4 Gclc -0.577 0.012437161 glutamate-cysteine ligase, catalytic subunit Sdf2l1 -0.576 0.03200801 stromal cell-derived factor 2-like 1 Hsd17b14 -0.575 0.010329599 Hydroxysteroid (17-beta) dehydrogenase 14 Acsl1 -0.572 0.04956432 acyl-CoA synthetase long-chain family member 1 Slc38a1 -0.564 0.020336002 solute carrier family 38, member 1 A430110A21Rik -0.560 0.02102864 RIKEN cDNA A430110A21 gene Gpc6 -0.557 0.027113399 glypican 6 /// similar to Glypican 6 Itga6 -0.556 0.008826129 integrin alpha 6 AI317158 -0.555 0.004460394 expressed sequence AI317158 BC014805 -0.553 0.009109807 cDNA sequence BC014805 Adssl1 -0.549 0.014266484 adenylosuccinate synthetase like 1 4930402H24Rik -0.543 0.016254611 RIKEN cDNA 4930402H24 gene AI987662 -0.542 0.0009872 expressed sequence AI987662 5730469M10Rik -0.541 0.011074062 RIKEN cDNA 5730469M10 gene BC003993 -0.537 0.01925689 cDNA sequence BC003993 Fignl1 -0.537 0.005133999 fidgetin-like 1 Aurka -0.536 0.01906949 aurora kinase A Zbtb20 -0.534 0.002964494 zinc finger and BTB domain containing 20 Cdca8 -0.533 0.021126691 cell division cycle associated 8 Trim35 -0.533 0.002686886 tripartite motif-containing 35 4930502E18Rik -0.529 0.038361125 RIKEN cDNA 4930502E18 gene Ccnb1 -0.528 0.027141333 cyclin B1, related sequence 1 /// cyclin B1 Sost -0.528 0.048348885 sclerostin B230216N24Rik -0.523 0.016320813 RIKEN cDNA B230216N24 gene Cml5 -0.520 0.022754848 camello-like 5 Ncapg2 -0.520 0.012077962 non-SMC condensin II complex, subunit G2 Shcbp1 -0.518 0.011784596 Shc SH2-domain binding protein 1 Rbm39 -0.514 0.023314232 RNA binding motif protein 39 Atad2 -0.514 0.010262549 ATPase family, AAA domain containing 2 Tspan1 -0.511 0.004082562 tetraspanin 1 C330001K17Rik -0.511 0.03630525 RIKEN cDNA C330001K17 gene Slc34a2 -0.511 0.001363737 solute carrier family 34 (sodium phosphate), member 2 Sostdc1 -0.508 0.016752638 sclerostin domain containing 1 Ifi44 -0.506 0.002246303 interferon-induced protein 44 2610301F02Rik -0.503 0.000630384 RIKEN cDNA 2610301F02 gene Lox -0.499 0.011208286 lysyl oxidase Cd55 -0.497 0.010870564 CD55 antigen Igf2bp1 -0.494 0.026579063 insulin-like growth factor 2 mRNA binding protein 1 Mcm5 -0.493 0.002307328 minichromosome maintenance deficient 5) 1200009I06Rik -0.489 0.007427462 RIKEN cDNA 1200009I06 gene Ccl21a -0.489 0.000159785 chemokine (C-C motif) ligand 21a Ccl21b -0.489 0.000159785 chemokine (C-C motif) ligand 21b Ccl21c -0.489 0.000159785 chemokine (C-C motif) ligand 21c Aspm -0.486 0.010249169 asp (abnormal spindle)-like, microcephaly associated) Itgb6 -0.485 0.013407179 integrin beta 6 Chka -0.483 0.04581158 choline kinase alpha D0H4S114 -0.482 0.005036883 DNA segment, human D4S114 6030400A10Rik -0.482 0.007273938 RIKEN cDNA 6030400A10 gene Tnrc15 -0.481 0.010223223 trinucleotide repeat containing 15 Mcm4 -0.481 0.04210305 minichromosome maintenance deficient 4 homolog Tnfaip2 -0.481 0.038322218 tumor necrosis factor, alpha-induced protein 2 Ptpla -0.478 0.01703382 protein tyrosine phosphatase-like member a C86807 -0.476 0.0164032 Expressed sequence C86807 Cspp1 -0.472 0.029895147 Centrosome and spindle pole associated protein 1 Fabp5 -0.469 0.00635181 fatty acid binding protein 5, epidermal Fgf9 -0.469 0.037860945 fibroblast growth factor 9 Prkcm -0.467 0.021005971 Protein kinase C, mu

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Postn -0.466 0.014290248 periostin, osteoblast specific factor Ftcd -0.466 0.040300332 formiminotransferase cyclodeaminase Wsb1 -0.465 0.011375793 WD repeat and SOCS box-containing 1 D16H22S680E -0.465 0.013565078 DNA segment, Chr 16, human D22S680E, expressed Hpgd -0.461 0.001983529 hydroxyprostaglandin dehydrogenase 15 (NAD) Nrbp2 -0.458 0.03030011 nuclear receptor binding protein 2 Hspa4l -0.458 0.000405505 heat shock protein 4 like Dap -0.457 0.002804909 death-associated protein Ap2a2 -0.457 0.02816052 adaptor protein complex AP-2, alpha 2 subunit Dpt -0.457 0.04631873 dermatopontin Jarid2 -0.457 0.035274707 jumonji, AT rich interactive domain 2 Ckb -0.456 0.004130294 creatine kinase, brain Pla1a -0.455 0.012917724 phospholipase A1 member A Phf20l1 -0.452 0.03909935 PHD finger protein 20-like 1 Serpinh1 -0.452 0.001513745 serine (or cysteine) peptidase inhibitor, clade H, 1 Hs2st1 -0.450 0.021461809 Heparan sulfate 2-O-sulfotransferase 1 Mycn -0.450 0.007562848 v-myc myelocytomatosis viral related oncogene Prpf38b -0.450 0.024397776 PRP38 pre-mRNA processing factor 38 B Mdfic -0.447 0.000404008 MyoD family inhibitor domain containing Cxcr7 -0.446 0.012329036 chemokine (C-X-C motif) receptor 7 Col4a2 -0.445 0.002154103 procollagen, type IV, alpha 2 Nfkbiz -0.444 0.023961574 nuclear factor of kappa light polypeptide zeta Cd14 -0.442 0.032622118 CD14 antigen Dnaja2 -0.442 0.030577077 DnaJ (Hsp40) homolog, subfamily A, member 2 Tnfsf10 -0.439 0.01137728 tumor necrosis factor (ligand) superfamily, member 10 Rai1 -0.439 0.015947979 retinoic acid induced 1 Tmem45a -0.439 0.009554327 transmembrane protein 45a Senp7 -0.438 0.016401755 SUMO1/sentrin specific peptidase 7 C130032J12Rik -0.438 0.019551149 RIKEN cDNA C130032J12 gene Casp7 -0.437 0.01014446 caspase 7 Zzef1 -0.437 0.023534153 zinc finger, ZZ-type with EF hand domain 1 2310007B03Rik -0.436 0.002647403 RIKEN cDNA 2310007B03 gene Plau -0.435 0.041397788 plasminogen activator, urokinase Ncapd2 -0.434 0.028670518 non-SMC condensin I complex, subunit D2 Alcam -0.433 0.01466488 activated leukocyte cell adhesion molecule Ucp2 -0.433 0.024799397 uncoupling protein 2 (mitochondrial, proton carrier) Mdk -0.432 0.018191248 midkine Gas5 -0.432 0.03417363 growth arrest specific 5 Cenpa -0.432 0.009806776 centromere protein A Macrod2 -0.431 0.002244704 MACRO domain containing 2 Nfxl1 -0.431 0.005445412 nuclear transcription factor, X-box binding-like 1 Crot -0.430 0.000869773 carnitine O-octanoyltransferase Rad51 -0.429 0.008528647 RAD51 homolog (S. cerevisiae) Malat1 -0.429 0.03129835 metastasis associated lung adenocarcinoma transcript 1 Gm540 -0.428 0.005433566 gene model 540, (NCBI) Tnrc15 -0.427 0.024330186 trinucleotide repeat containing 15 Rgs19 -0.426 0.00734045 regulator of G-protein signaling 19 Mcm9 -0.426 0.004577688 minichromosome maintenance complex component 9 Anln -0.426 0.043580335 anillin, actin binding protein Rai14 -0.425 0.048107963 retinoic acid induced 14 Parp11 -0.425 0.03351411 poly (ADP-ribose) polymerase family, member 11 Zadh1 -0.425 0.009590652 zinc binding alcohol dehydrogenase, domain containing 1 Ttc14 -0.424 0.013585802 tetratricopeptide repeat domain 14 Slc40a1 -0.424 0.0469517 solute carrier 40 (iron-regulated transporter), member 1 Zwint -0.423 0.001007847 ZW10 interactor Col4a1 -0.422 9.01294E-05 procollagen, type IV, alpha 1 Ecm1 -0.421 0.000757392 extracellular matrix protein 1 Arg2 -0.421 0.024502482 arginase type II Racgap1 -0.420 0.022577105 Rac GTPase-activating protein 1 Foxm1 -0.419 0.011043419 forkhead box M1 Aldoc -0.417 0.020869555 aldolase 3, C isoform A430081F14Rik -0.416 0.004533467 RIKEN cDNA A430081F14 gene Uhrf1 -0.416 0.025787562 ubiquitin-like, PHD and RING finger domains, 1 Kcnq1 -0.416 0.003545546 potassium voltage-gated channel, subfamily Q, member 1 Ptger3 -0.416 0.011664568 prostaglandin E receptor 3 (subtype EP3) Tuba1a -0.414 0.0204708 tubulin, alpha 1A 2010109K11Rik -0.412 0.035568092 RIKEN cDNA 2010109K11 gene Wwox -0.412 0.00732308 WW domain-containing oxidoreductase Casr -0.412 0.011750091 calcium-sensing receptor

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Slc13a4 -0.409 0.04608179 solute carrier 13 (sodium/sulfate symporters), member 4 Slc6a15 -0.408 0.026672145 solute carrier 6 (neurotransmitter transporter), member 15 Zfat1 -0.407 0.030462436 ZFAT zinc finger 1 Npy1r -0.405 0.026249994 neuropeptide Y receptor Y1 Lonrf3 -0.404 0.020012131 LON peptidase N-terminal domain and ring finger 3 Whsc1 -0.403 0.008468427 Wolf-Hirschhorn syndrome candidate 1 (human) Defb1 -0.402 0.014130419 defensin beta 1 Gpsm2 -0.402 0.027415931 G-protein signalling modulator 2 (AGS3-like, C. elegans) Shmt1 -0.402 1.52827E-05 serine hydroxymethyltransferase 1 (soluble) Gas2l3 -0.401 0.015967077 growth arrest-specific 2 like 3 Serpinb1a -0.401 0.03176764 Serine peptidase inhibitor, clade B, member 1a E430014L09Rik -0.401 0.01999124 RIKEN cDNA E430014L09 gene Gcgr -0.400 7.63952E-05 glucagon receptor Cd24a -0.399 0.003841465 CD24a antigen Arhgap11a -0.399 0.033158172 Rho GTPase activating protein 11A Cml2 -0.399 0.010561771 camello-like 2 Sult1a1 -0.398 0.038372956 sulfotransferase family 1A, phenol-preferring, member 1 Pgam2 -0.398 0.02231596 phosphoglycerate mutase 2 Trim2 -0.397 0.04604618 tripartite motif protein 2 Vav3 -0.397 0.036591616 vav 3 oncogene Ifi27 -0.394 0.008980501 interferon, alpha-inducible protein 27 Glrx -0.393 0.01709708 glutaredoxin 6430510B20Rik -0.393 0.001071442 RIKEN cDNA 6430510B20 gene Tmem169 -0.392 0.04413129 transmembrane protein 169 Lpl -0.391 0.007579874 lipoprotein lipase Ttll10 -0.390 0.03446203 tubulin tyrosine ligase-like family, member 10 Lepr -0.390 0.030073304 leptin receptor Klk1b22 /// -0.389 0.00287117 kallikrein 1-related peptidase b22 /// kallikrein 1-related Asb9Klk1b9 -0.389 0.008502516 ankyrinpeptidase repeat b9 and SOCS box-containing protein 9 2510017J16Rik -0.388 0.021886475 RIKEN cDNA 2510017J16 gene D2Ertd93e -0.388 0.037837103 DNA segment, Chr 2, ERATO Doi 93, expressed Car4 -0.388 0.008238088 carbonic anhydrase 4 Ogt -0.386 0.013270709 O-linked N-acetylglucosamine (GlcNAc) transferase Chek1 -0.386 0.04567527 checkpoint kinase 1 homolog (S. pombe) Slain1 -0.385 0.008814648 SLAIN motif family, member 1 Cep55 -0.385 0.043280255 centrosomal protein 55 Mtr -0.384 0.007133653 5-methyltetrahydrofolate-homocysteine methyltransferase C130068B02Rik -0.384 0.002659104 RIKEN cDNA C130068B02 gene Prox1 -0.384 0.011444218 prospero-related homeobox 1 Dsg2 -0.384 0.024441551 desmoglein 2 Tnc -0.383 0.022205094 tenascin C Tmem58 -0.383 0.000737147 transmembrane protein 58 Tarsl2 -0.383 0.004110504 threonyl-tRNA synthetase-like 2 Prc1 -0.383 0.038993713 protein regulator of cytokinesis 1 Akr1b7 -0.382 0.020880273 aldo-keto reductase family 1, member B7 Dck -0.382 0.04145801 deoxycytidine kinase Slit2 -0.381 0.02484797 slit homolog 2 (Drosophila) Slc5a3 -0.380 0.049571346 solute carrier family 5 (inositol transporters), member 3 B230114P17Rik -0.380 0.003845975 RIKEN cDNA B230114P17 gene Gpr116 -0.379 0.019668337 G protein-coupled receptor 116 Slc1a5 -0.379 0.004294737 solute carrier family 1 (neutral amino acid transporter), Pi4k2b -0.379 0.001165901 phosphatidylinositolmember 5 4-kinase type 2 beta Slc4a9 -0.378 0.02331996 solute carrier 4, sodium bicarbonate cotransporter, 9 AA407107 -0.378 0.0172826 expressed sequence AA407107 Serpinb8 -0.378 0.03064605 serine (or cysteine) peptdiase inhibitor, clade B, 8 Rdh14 -0.378 0.001850953 retinol dehydrogenase 14 (all-trans and 9-cis) Gbp3 -0.377 0.04031473 guanylate nucleotide binding protein 3 Rnpepl1 -0.377 0.015564182 arginyl aminopeptidase (aminopeptidase B)-like 1 Cldn16 -0.377 0.001140138 claudin 16 Anxa9 -0.377 0.014559681 annexin A9 Pscdbp -0.377 0.000686949 pleckstrin homology, Sec7 and coiled-coil domains bp Ccdc28b -0.374 0.000172541 coiled coil domain containing 28B Klhdc8a -0.373 0.021655375 kelch domain containing 8A Stat3 -0.371 0.030328223 Signal transducer and activator of transcription 3 Papss1 -0.371 0.021224096 3'-phosphoadenosine 5'-phosphosulfate synthase 1 ENSMUSG000000715 -0.370 0.016316315 predicted gene, ENSMUSG00000071543 Cdca543 -0.369 0.015518386 cell division cycle associated 5 Rragd -0.369 0.007871183 Ras-related GTP binding D Guca2a -0.368 0.004866536 guanylate cyclase activator 2a (guanylin)

187 Chapter III – Manuscript – Microarray downregulated genes

Ifitm3 -0.367 0.03957456 interferon induced transmembrane protein 3 Prss23 -0.367 0.001175518 protease, serine, 23 Mcm3 -0.365 0.00782564 minichromosome maintenance deficient 3 Nav2 -0.365 0.041591186 Neuron navigator 2 Dgke -0.365 0.015400705 diacylglycerol kinase, epsilon Nusap1 -0.365 0.036337346 nucleolar and spindle associated protein 1 Ccne2 -0.365 0.003268744 cyclin E2 Gbp6 -0.365 0.01738675 guanylate binding protein 6 LOC100046740 -0.364 0.000559285 similar to Secreted acidic cysteine rich glycoprotein Paqr5 -0.363 0.003073849 progestin and adipoQ receptor family member V Pole2 -0.361 0.044290926 polymerase (DNA directed), epsilon 2 (p59 subunit) Mcm6 -0.361 0.043857012 minichromosome maintenance deficient 6 BC025076 -0.361 0.029145123 cDNA sequence BC025076 Tinagl -0.361 0.000131681 tubulointerstitial nephritis antigen-like 2310076L09Rik -0.360 0.007293886 RIKEN cDNA 2310076L09 gene Ccdc66 -0.360 0.003052076 coiled-coil domain containing 66 Birc6 -0.360 0.026508568 baculoviral IAP repeat-containing 6 Ckap2 -0.360 0.009148508 cytoskeleton associated protein 2 2310035C23Rik -0.359 0.002460121 RIKEN cDNA 2310035C23 gene Nrg1 -0.359 0.046881106 neuregulin 1 Slc39a14 -0.357 0.001918896 solute carrier family 39 (zinc transporter), member 14 Iigp2 -0.357 0.048998956 interferon inducible GTPase 2 Ndrg1 -0.356 0.012780646 N-myc downstream regulated gene 1 Osbpl1a -0.356 0.00641937 oxysterol binding protein-like 1A 2200001I15Rik -0.355 0.002596329 RIKEN cDNA 2200001I15 gene Chn2 -0.355 0.004060412 chimerin (chimaerin) 2 Prpf4b -0.355 0.043323297 PRP4 pre-mRNA processing factor 4 homolog B (yeast) Spire1 -0.355 0.001082462 spire homolog 1 (Drosophila) Ddit4l -0.354 0.00934407 DNA-damage-inducible transcript 4-like Slc19a2 -0.353 0.02900738 solute carrier family 19 (thiamine transporter), member 2 Iars -0.353 0.001076109 isoleucine-tRNA synthetase -0.353 0.001907005 Transcribed locus Il13ra1 -0.353 0.033206552 interleukin 13 receptor, alpha 1 Bsnd -0.352 0.021701127 Bartter syndrome, infantile, with sensorineural deafness Crispld2 -0.352 0.001394694 cysteine(Barttin)- rich secretory protein LCCL domain 2 Dbn1 -0.351 0.003633648 drebrin 1 Malt1 -0.351 0.039302547 mucosa associated lymphoid tissue lymphoma 4930478M09Rik -0.350 0.003880548 RIKENtranslocation cDNA gene 4930478M09 1 gene Cutl1 -0.349 0.046826143 Cut-like 1 (Drosophila) Avpr2 -0.349 0.043733098 arginine vasopressin receptor 2 Rtp4 -0.349 0.047518402 receptor transporter protein 4 AW049765 -0.349 0.04386337 expressed sequence AW049765 LOC667118 -0.349 0.017280031 similar to Zinc finger BED domain containing protein 4 EG434179 -0.347 0.044520568 predicted gene, EG434179 5730494M16Rik -0.347 0.003153656 RIKEN cDNA 5730494M16 gene Cyfip2 -0.346 0.043322068 cytoplasmic FMR1 interacting protein 2 Smpd4 -0.346 0.03811073 sphingomyelin phosphodiesterase 4 Ccbe1 -0.346 0.028774012 collagen and calcium binding EGF domains 1 Sh2d4a -0.344 0.024913518 SH2 domain containing 4A Dyx1c1 -0.343 0.045517225 dyslexia susceptibility 1 candidate 1 homolog (human) Sbno2 -0.342 0.049351837 strawberry notch homolog 2 (Drosophila) Fgf18 -0.342 0.0381965 fibroblast growth factor 18 Arhgap6 -0.342 0.007921773 Rho GTPase activating protein 6 Tshr -0.342 0.010475235 thyroid stimulating hormone receptor Oxgr1 -0.341 0.028437434 oxoglutarate (alpha-ketoglutarate) receptor 1 Tpx2 -0.341 0.002437215 TPX2, microtubule-associated protein homolog Sox17 -0.340 0.021215595 SRY-box containing gene 17 Tubb2b -0.340 0.015396258 tubulin, beta 2b 130004C03 -0.340 0.013462928 hypothetical LOC403343 Usp40 -0.340 0.03890758 ubiquitin specific peptidase 40 Tcf2 -0.339 0.049273394 transcription factor 2 Ctso -0.339 0.011855003 cathepsin O Umod -0.339 0.037687156 uromodulin Krt7 -0.338 0.012321201 keratin 7 4930519N06Rik -0.338 0.008214357 RIKEN cDNA 4930519N06 gene Vpreb1 -0.338 0.02992729 pre-B lymphocyte gene 1 A230106N23Rik -0.337 0.013341224 RIKEN cDNA A230106N23 gene Mllt10 -0.337 0.022957904 myeloid/lymphoid or mixed lineage-leukemia transloc 10 Isg20 -0.335 0.003148172 interferon-stimulated protein

188 Chapter III – Manuscript – Microarray downregulated genes

9530028C05 -0.335 0.008224008 hypothetical protein 9530028C05 Chrna7 -0.334 0.042973753 cholinergic receptor, nicotinic, alpha polypeptide 7 Pdgfrb -0.333 0.043131594 platelet derived growth factor receptor, beta polypeptide 2310035P21Rik -0.333 0.035911452 RIKEN cDNA 2310035P21 gene Skp2 -0.332 0.011157136 S-phase kinase-associated protein 2 (p45) Dnase2a -0.332 0.02069925 deoxyribonuclease II alpha Ifi203 -0.332 0.00369177 interferon activated gene 203 2210011C24Rik -0.331 0.018730458 RIKEN cDNA 2210011C24 gene Xpr1 -0.331 0.046270013 xenotropic and polytropic retrovirus receptor 1 Sytl2 -0.331 0.008496755 synaptotagmin-like 2 Hmmr -0.330 0.015949575 hyaluronan mediated motility receptor (RHAMM) Kifap3 -0.330 0.010432779 kinesin-associated protein 3 Kcnj1 -0.330 0.011229395 potassium inwardly-rectifying channel, subfamily J, Eif4ebp1 -0.330 0.029471558 membereukaryotic 1 translation initiation factor 4E binding protein Tagln -0.330 0.008903767 transgelin1 6720422M22Rik -0.330 0.048454195 RIKEN cDNA 6720422M22 gene Vti1a -0.330 0.046495933 vesicle transport through interaction with t-SNAREs Tspan8 -0.330 0.03758837 tetraspaninhomolog 1A 8 (yeast) Tmem34 -0.329 0.015985303 transmembrane protein 34 Aldh18a1 -0.329 0.02356159 aldehyde dehydrogenase 18 family, member A1 Mapre2 -0.329 0.012314819 microtubule-associated protein, RP/EB family, member 2 Mtr -0.329 0.002138615 5-methyltetrahydrofolate-homocysteine methyltransferase ENSMUSG0000 -0.329 0.03881831 predicted gene, ENSMUSG00000073000 Rab230073000 -0.329 0.019697499 RAB23, member RAS oncogene family Sparc -0.328 0.009382077 secreted acidic cysteine rich glycoprotein /// similar to Zfp276 -0.328 0.024525007 ZincSecreted finger acidic protein cysteine (C2H2 rich type) glycoprotein 276 Sema6d -0.327 0.020397482 sema domain, transmembrane domain (TM), and Slc13a3 -0.327 0.007885597 cytoplasmicsolute carrier domain, family 13(semaphorin) (sodium-dependent 6D dicarboxylate Jam2 -0.327 0.017605966 junctiontransporter), adhesion member molecule 3 2 10-Sep -0.326 0.047938515 septin 10 Rcan2 -0.326 0.008625793 regulator of calcineurin 2 B230120H23Rik -0.326 0.005545706 RIKEN cDNA B230120H23 gene Tmem181 -0.324 0.048106644 Transmembrane protein 181 Smg5 -0.324 0.008029153 Smg-5 homolog, nonsense mediated mRNA decay factor A130022J15Rik -0.324 0.002822909 RIKEN(C. elegans) cDNA A130022J15 gene Col6a2 -0.324 0.039858427 procollagen, type VI, alpha 2 Ankrd47 -0.323 0.027386872 ankyrin repeat domain 47 C1qc -0.323 0.010283837 complement component 1, q subcomponent, C chain Hsp90aa1 -0.322 0.01738718 heat shock protein 90kDa alpha (cytosolic), class A member 1

189 Chapter III – Manuscript – Microarray upregulated genes

Table 3.S3: List of the upregulated genes in the ERRα null kidney by Microarray analysis

Gene Symbol LOG2 p-value Gene Title 2010205A11Rik 2.547 0.010895215 ENSMUSG00000076577 Igh 2.386 0.028074874 Ig heavy chain H Cr1/Ig-kC 2.288 0.016036948 Ig immunoglobulin kappa Igj 2.236 0.034426954 immunoglobulin joining chain ENSMUSG00000 2.108 0.029799795 predicted gene, ENSMUSG00000076577 076577Cyp2f2 1.537 0.017562639 cytochrome P450, family 2, subfamily f, polypeptide 2 Atp4a 1.529 2.49468E-05 ATPase, H+/K+ exchanging, gastric, alpha polypeptide 9030605E09Rik 1.462 0.006099211 RIKEN cDNA 9030605E09 gene Car3 1.378 0.000765531 carbonic anhydrase 3 Cyp2d9 1.361 0.000591743 cytochrome P450, family 2, subfamily d, polypeptide 9 Unc13b 1.339 0.000351473 unc-13 homolog B (C. elegans) Neurog2 1.310 0.000138259 neurogenin 2 Cadm4 1.286 0.000167155 cell adhesion molecule 4 Azgp1 1.132 0.00028329 alpha-2-glycoprotein 1, zinc Dbp 1.119 0.001075206 D site albumin promoter binding protein Apoe 1.105 0.007681383 apolipoprotein E A030009H04Rik 1.087 0.00521953 RIKEN cDNA A030009H04 gene Scd1 1.083 0.013891418 stearoyl-Coenzyme A desaturase 1 Cox8b 1.060 0.003432227 cytochrome c oxidase, subunit VIIIb Per2 1.045 0.002682192 period homolog 2 (Drosophila) Egr1 0.999 0.005314172 early growth response 1 Hspa1a 0.963 0.011076395 heat shock protein 1A Rhox6 0.915 0.001144269 reproductive homeobox 6 Slc25a25 0.903 0.012193385 solute carrier family 25 (mitochondrial carrier, Pfkp 0.902 0.000988923 phosphatephosphofructokinase, carrier), member platelet 25 Abcc4 0.879 0.008728534 ATP-binding cassette, sub-family C (CFTR/MRP), Ndrg2 0.859 0.000915665 memberN-myc downstream 4 regulated gene 2 Hao3 0.842 0.004011897 hydroxyacid oxidase (glycolate oxidase) 3 Cyp2a4 0.840 0.001685787 cytochrome P450, family 2, subfamily a, polypeptide 4 Cyp2a5 0.840 0.001685787 ///cytochrome cytochrome P450, P450, family family 2, subfamily2, subfamily a, polypeptidea, polypeptide 4 Cyp26b1 0.819 0.043813415 5///cytochrome /// cytochrome similar toP450, CytochromeP450, family family 26, P450, 2, subfamily subfamily family b, 2,a, polypeptide subfamilypolypeptide a, 1 Klf10 0.782 0.013485912 polypeptide5Kruppel /// similar-like to4 factor Cytochrome 10 P450, family 2, subfamily a, BC089597 0.782 0.013747534 polypeptidecDNA sequence 4 BC089597 Apoc3 0.782 0.04107193 apolipoprotein C-III Rbm11 0.775 0.011246929 RNA binding motif protein 11 Hspa1b 0.751 0.01968848 heat shock protein 1B Pla2g5 0.723 0.007924081 phospholipase A2, group V Cdh2 0.708 0.031045543 cadherin 2 Ugt2b34 0.706 0.000435193 UDP glucuronosyltransferase 2 family, polypeptide B34 Rarb 0.699 0.01939532 retinoic acid receptor, beta 1190003J15Rik 0.686 0.003498537 RIKEN cDNA 1190003J15 gene Sc5d 0.676 0.000787976 sterol-C5-desaturase (fungal ERG3, delta-5-desaturase) Ccrn4l 0.675 0.003236306 homologCCR4 carbon (S. cerevisae) catabolite repression 4-like (S. cerevisiae) Uroc1 0.673 0.014643159 ///urocanase similar todomain carbon containing catabolite 1 repression 4 protein 6430502M16Rik 0.666 0.002967643 homologRIKEN cDNA 6430502M16 gene Thap4 0.657 0.000217294 THAP domain containing 4 Rnf190 0.652 0.002464516 ring finger protein 190 Sardh 0.645 0.000105375 sarcosine dehydrogenase Rap2ip 0.644 0.004192348 Rap2 interacting protein Cox6a2 0.634 0.011038981 cytochrome c oxidase, subunit VI a, polypeptide 2 Apob 0.629 0.018740289 apolipoprotein B A530016O06Rik 0.622 0.002563247 RIKEN cDNA A530016O06 gene Cyp2d22 0.616 0.023516338 cytochrome P450, family 2, subfamily d, polypeptide 22 Gm128 0.608 0.01431508 gene model 128, (NCBI) EG226654 0.595 0.004791328 predicted gene, EG226654 Gm129 0.589 0.003446722 gene model 129, (NCBI) Pmaip1 0.587 0.03926047 phorbol-12-myristate-13-acetate-induced protein 1 Dusp9 0.585 0.001292688 dual specificity phosphatase 9 Nefl 0.570 0.014999639 neurofilament, light polypeptide Atp1a2 0.568 0.005592687 ATPase, Na+/K+ transporting, alpha 2 polypeptide H2-DMb1 0.568 0.018973265 histocompatibility 2, class II, locus Mb1 Cyp2c44 0.564 0.005382089 cytochrome P450, family 2, subfamily c, polypeptide 44 Slc2a5 0.558 0.000220968 solute carrier family 2 (facilitated glucose transporter), Prkag3 0.552 0.002951045 memberprotein kinase, 5 AMP-activated, gamma 3 non-catatlytic 1700040L02Rik 0.548 0.00880312 subunitRIKEN cDNA 1700040L02 gene Xtrp3s1 0.548 0.008060679 X transporter protein 3 similar 1 gene Mex3d 0.546 0.003673911 mex3 homolog D (C. elegans) AW011956 0.540 0.012250933 expressed sequence AW011956

190 Chapter III – Manuscript – Microarray upregulated genes

Coro2a 0.539 0.021850107 coronin, actin binding protein 2A Aldoa 0.535 0.00054946 aldolase 1, A isoform Hist2h2aa1 0.532 0.023374708 histone cluster 2, H2aa1 D630023F18Rik 0.532 0.004408546 RIKEN cDNA D630023F18 gene Mtac2d1 0.527 0.001151909 membrane targeting (tandem) C2 domain containing 1 Rasl2-9 0.526 0.006395691 RAS-like, family 2, locus 9 Cd1d1 0.522 0.01509105 CD1d1 antigen Dnajb4 0.522 0.024163231 DnaJ (Hsp40) homolog, subfamily B, member 4 Sergef 0.519 0.00879073 secretion regulating guanine nucleotide exchange factor Npb 0.517 0.011037832 neuropeptide B Sectm1b 0.515 0.00011322 secreted and transmembrane 1B Cd81 0.511 4.12903E-05 CD 81 antigen Dnajb1 0.508 0.031601958 DnaJ (Hsp40) homolog, subfamily B, member 1 Agmat 0.505 0.0101146 agmatine ureohydrolase (agmatinase) Cbs 0.505 0.00265151 cystathionine beta-synthase Nqo1 0.502 0.000935231 NAD(P)H dehydrogenase, quinone 1 Olfm4 0.502 0.001217352 olfactomedin 4 2810439F02Rik 0.500 0.008455272 RIKEN cDNA 2810439F02 gene Usp2 0.500 0.007121511 ubiquitin specific peptidase 2 Lipg 0.496 0.025965862 lipase, endothelial Ttyh3 0.496 0.002392039 tweety homolog 3 (Drosophila) Plk3 0.494 0.018853465 polo-like kinase 3 (Drosophila) Dnajc6 0.493 0.019514326 DnaJ (Hsp40) homolog, subfamily C, member 6 Ddit3 0.488 0.031228887 DNA-damage inducible transcript 3 1500015O10Rik 0.487 0.01127486 RIKEN cDNA 1500015O10 gene Osr2 0.486 0.022030046 odd-skipped related 2 (Drosophila) Kcnab2 0.486 0.002417105 potassium voltage-gated channel, shaker-related E030010A14Rik 0.484 0.011186022 subfamily,RIKEN cDNA beta E030010A14 member 2 gene Rundc3a 0.484 0.018104026 RUN domain containing 3A Arrdc4 0.482 0.028018696 arrestin domain containing 4 Npr2 0.481 0.002865892 natriuretic peptide receptor 2 Cpn1 0.480 0.001022688 carboxypeptidase N, polypeptide 1 Tmtc4 0.479 0.000589413 transmembrane and tetratricopeptide repeat containing 4 H2-DMb2 0.479 0.025871575 histocompatibility 2, class II, locus Mb1 /// Edg4 0.477 0.016971244 histocompatibilityendothelial differentiation, 2, class II,lysophosphatidic locus Mb2 acid G- Dnajc6 0.475 0.005657907 proteinDnaJ (Hsp40)-coupled homolog, receptor subfamily4 C, member 6 Chrdl1 0.475 0.008632536 chordin-like 1 E130012A19Rik 0.472 0.020275958 RIKEN cDNA E130012A19 gene Cxcl14 0.471 0.038169 chemokine (C-X-C motif) ligand 14 Nr1d2 0.470 0.010853028 nuclear receptor subfamily 1, group D, member 2 C920025E04Rik 0.467 0.016180554 RIKEN cDNA C920025E04 gene Steap1 0.464 0.018049136 six transmembrane epithelial antigen of the prostate 1 C130090K23Rik 0.458 0.001447546 RIKEN cDNA C130090K23 gene Glod5 0.458 0.000229998 glyoxalase domain containing 5 C330050A14Rik 0.457 0.003931483 RIKEN cDNA C330050A14 gene Dock9 0.452 0.007663136 dedicator of cytokinesis 9 Glb1 0.452 0.013049388 galactosidase, beta 1 Armet 0.448 0.038731236 arginine-rich, mutated in early stage tumors Gabrb3 0.447 0.019122135 gamma-aminobutyric acid (GABA-A) receptor, subunit Hist3h2a 0.447 0.002521851 betahistone 3 cluster 3, H2a Pfkfb3 0.445 0.001712064 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 Ltc4s 0.444 0.003934991 leukotriene C4 synthase Park2 0.443 0.030261591 parkin Dmkn 0.441 0.018698819 dermokine Prr15 0.441 0.022133403 proline rich 15 2810408A11Rik 0.436 0.03975736 RIKEN cDNA 2810408A11 gene Epor 0.435 0.013656638 erythropoietin receptor Cxcr4 0.434 0.002342884 chemokine (C-X-C motif) receptor 4 Gnmt 0.432 0.003671326 glycine N-methyltransferase ENSMUSG00000 0.431 0.041787267 predicted gene, ENSMUSG00000073738 073738Tef 0.429 0.009077595 thyrotroph embryonic factor BB120497 0.428 0.008826814 expressed sequence BB120497 Qprt 0.427 0.00156916 quinolinate phosphoribosyltransferase LOC245350 /// 0.425 0.004416572 similar to Ubiquitin-conjugating enzyme E2Q (putative) LOC634012Agpat9 0.423 0.013224824 2RIKEN /// similar cDNA to CG2924 A230097K15-PC, isoform gene C Cend1 0.422 0.027064458 cell cycle exit and neuronal differentiation 1 Dio2 0.420 0.003229387 deiodinase, iodothyronine, type II C130038G02Rik 0.420 0.04957683 RIKEN cDNA C130038G02 gene Cry2 0.419 0.025420945 2 (photolyase-like) Pitpnc1 0.417 0.000814036 phosphatidylinositol transfer protein, cytoplasmic 1 Upp2 0.416 0.033710275 uridine phosphorylase 2 A2bp1 0.415 0.000797732 Ataxin 2 binding protein 1 Mcm10 0.414 0.002864179 minichromosome maintenance deficient 10

191 Chapter III – Manuscript – Microarray upregulated genes

C730027J19Rik 0.413 0.008832176 RIKEN cDNA C730027J19 gene Nab2 0.413 0.001759497 Ngfi-A binding protein 2 Sms 0.412 0.0074672 spermine synthase /// similar to spermine synthase Acaa2 0.411 0.005318354 acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3- Slc7a2 0.410 0.014147026 oxoacylsolute carrier-Coenzyme family A 7 thiolase)(cationic amino acid transporter, Dhtkd1 0.409 0.009264322 y+dehydrogenase system), member E1 and 2 transketolase domain containing Fabp7 0.408 0.014394432 1fatty acid binding protein 7, brain H2-Ab1 0.405 0.03086636 histocompatibility 2, class II antigen A, beta 1 Per3 0.405 0.039196726 period homolog 3 (Drosophila) C1ql3 0.405 0.035411216 C1q-like 3 Cox7a1 0.404 0.014810749 cytochrome c oxidase, subunit VIIa 1 Adora1 0.401 0.02288178 adenosine A1 receptor Hspa12a 0.400 0.00429407 heat shock protein 12A Mknk2 0.400 0.03721637 MAP kinase-interacting serine/threonine kinase 2 Sema5b 0.399 0.006416416 sema domain, seven thrombospondin repeats (type 1 and Slc5a9 0.398 0.015377804 typesolute 1 -carrierlike), transmembrane family 5 (sodium/glucose domain (TM) cotransporter), and short Acss1 0.397 0.003107267 cytoplasmicmemberacyl-CoA 9 synthetase domain, short(semaphorin)-chain family 5B member 1 1110054O05Rik 0.397 0.00695083 RIKEN cDNA 1110054O05 gene Emilin2 0.396 0.016863614 elastin microfibril interfacer 2 Plekhb1 0.396 0.009156316 pleckstrin homology domain containing, family B Ceacam2 0.394 0.001614051 (evectins)CEA-related member cell adhesion 1 molecule 2 C030033M12Rik 0.394 0.026924508 RIKEN cDNA C030033M12 gene Ropn1l 0.394 0.04746063 ropporin 1-like Tsc22d3 0.393 0.02286649 TSC22 domain family 3 GILZ Tnfrsf10b 0.388 0.033069357 tumor necrosis factor receptor superfamily, member 10b Herpud1 0.388 0.003961689 homocysteine-inducible, endoplasmic reticulum stress- Mosc2 0.388 0.00240309 inducible,MOCO sulphurase ubiquitin C-like-terminal domain domain member containing 1 2 Pdxp 0.388 0.008894611 pyridoxal (pyridoxine, vitamin B6) phosphatase Prnp 0.388 0.00349043 prion protein Angptl2 0.387 0.016866064 angiopoietin-like 2 E330017L17Rik 0.385 0.02816581 RIKEN cDNA E330017L17 gene Brwd1 0.385 0.015273278 bromodomain and WD repeat domain containing 1 Trib1 0.385 0.03323489 tribbles homolog 1 (Drosophila) Defb35 0.384 0.04967427 defensin beta 35 H2-DMa 0.384 0.003435823 histocompatibility 2, class II, locus DMa Epb4.1l3 0.383 0.002783289 erythrocyte protein band 4.1-like 3 4930435H24Rik 0.383 0.00068425 RIKEN cDNA 4930435H24 gene Spns3 0.383 0.006397854 spinster homolog 3 (Drosophila) C8g 0.381 0.011049402 complement component 8, gamma subunit 4930500O05Rik 0.379 0.006838011 RIKEN cDNA 4930500O05 gene Krt10 0.378 0.003845315 keratin 10 Mmd 0.377 0.00794665 to macrophage differentiation-associated /// Rbks 0.374 0.02813766 similarribokinase to monocyte to macrophage differentiation- 1700060J05Rik 0.374 0.04613995 associatedRIKEN cDNA 1700060J05 gene Gypc 0.373 0.015676815 glycophorin C Ltb4dh 0.373 0.00955705 leukotriene B4 12-hydroxydehydrogenase A630072M18Rik 0.372 0.035137173 RIKEN cDNA A630072M18 gene Gm1123 0.371 0.000376346 gene model 1123, (NCBI) 4921505C17Rik 0.371 0.008266114 RIKEN cDNA 4921505C17 gene Dnase1l3 0.371 0.006312139 deoxyribonuclease 1-like 3 Gpr146 0.370 0.030431524 G protein-coupled receptor 146 Gcc1 0.370 0.007426515 golgi coiled coil 1 Slc22a4 0.370 0.012908334 solute carrier family 22 (organic cation transporter), Tufm 0.370 0.006586881 memberTu translation 4 elongation factor, mitochondrial AI853106 0.369 0.004828093 expressed sequence AI853106 Kcnd3 0.369 0.030706024 potassium voltage-gated channel, Shal-related family, Cdkn1c 0.369 0.013160556 membercyclin-dependent 3 kinase inhibitor 1C (P57) Pbx2 0.369 0.023012409 pre B-cell leukemia transcription factor 2 Ctse 0.369 0.015653659 cathepsin E Cd209a 0.368 0.028492806 CD209a antigen Gpi1 0.366 0.001441991 glucose phosphate isomerase 1 Lbh 0.366 0.002012487 limb-bud and heart /// similar to limb-bud and heart Usp37 0.365 0.002299541 ubiquitin specific peptidase 37 6330419J24Rik 0.363 0.026230074 RIKEN cDNA 6330419J24 gene 1810010H24Rik 0.363 0.019658215 RIKEN cDNA 1810010H24 gene Dqx1 0.363 0.003115896 DEAQ RNA-dependent ATPase Dnase1 0.362 0.013644775 deoxyribonuclease I Lrp8 0.361 0.011088161 low density lipoprotein receptor-related protein 8, Nt5c2 0.360 0.036150046 apolipoprotein5'-nucleotidase, e cytosolic receptor II Osbpl5 0.359 0.009631692 oxysterol binding protein-like 5 1500012D20Rik 0.359 0.019928304 RIKEN cDNA 1500012D20 gene Fmnl1 0.358 0.013303026 formin-like 1 Wfikkn2 0.358 0.000182374 WAP, follistatin/kazal, immunoglobulin, kunitz and

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netrin domain containing 2 Gstm6 0.358 0.046344116 glutathione S-transferase, mu 6 Cldn9 0.357 0.004634659 claudin 9 Cd44 0.356 0.02007828 CD44 antigen Tnfrsf21 0.356 0.002795775 tumor necrosis factor receptor superfamily, member 21 Spon2 0.355 0.032307044 spondin 2, extracellular matrix protein Slc32a1 0.355 0.024294661 Solute carrier family 32 (GABA vesicular transporter), Ldlr 0.354 0.010234967 memberlow density 1 lipoprotein receptor 4930434J08Rik 0.354 0.042712636 RIKEN cDNA 4930434J08 gene Hes6 0.353 0.001088143 hairy and enhancer of split 6 (Drosophila) Nxph4 0.353 0.018113442 neurexophilin 4 Slc6a2 0.352 0.02371048 solute carrier family 6 (neurotransmitter transporter, Nox4 0.352 0.003334541 noradrenalin),NADPH oxidase member 4 2 Tcrb-V13 0.351 0.024496006 T-cell receptor beta, variable 13 Anks1b 0.351 0.012468496 ankyrin repeat and sterile alpha motif domain containing Clybl 0.351 0.001776778 1Bcitrate lyase beta like Ccs 0.350 0.001805382 copper chaperone for superoxide dismutase 5033411D12Rik 0.350 0.007259302 RIKEN cDNA 5033411D12 gene Slc25a36 0.348 0.011715898 solute carrier family 25, member 36 B3galnt1 0.348 0.021455653 UDP-GalNAc:betaGlcNAc beta 1,3- 9030605I04Rik 0.347 0.020100895 galactosaminyltransferase,RIKEN cDNA 9030605I04 polypeptide gene /// RIKEN 1 cDNA Pla2g10 0.347 0.014116185 9930109F21phospholipase gene A2, /// group expressed X sequence AI987692 9130024F11Rik 0.347 0.03890657 RIKEN cDNA 9130024F11 gene Klf1 0.346 0.04159954 Kruppel-like factor 1 (erythroid) Ces3 0.346 0.001400172 carboxylesterase 3 4930526F13Rik 0.345 0.03821458 RIKEN cDNA 4930526F13 gene Bbox1 0.344 0.022675667 butyrobetaine (gamma), 2-oxoglutarate dioxygenase 1 Gnrhr 0.343 0.000903462 (gammagonadotropin-butyrobetaine releasing hydroxylase)hormone receptor Crim2 0.342 0.009745179 cysteine rich BMP regulator 2 (chordin like) Hlf 0.342 0.00724313 hepatic leukemia factor Agps 0.341 0.006101891 alkylglycerone phosphate synthase Cadm1 0.340 0.025182888 cell adhesion molecule 1 Fcgbp 0.340 0.035492517 Fc fragment of IgG binding protein Rbm35a 0.338 0.008194817 RNA binding motif protein 35A Kit 0.338 0.010649825 kit oncogene Dnaja1 0.338 0.028122123 DnaJ (Hsp40) homolog, subfamily A, member 1 Traf3ip1 0.337 0.009210521 TNF receptor-associated factor 3 interacting protein 1 9130019P16Rik 0.335 0.008701323 RIKEN cDNA 9130019P16 gene Psat1 0.335 0.013190094 phosphoserine aminotransferase 1 Uchl5ip 0.333 0.015844116 UCHL5 interacting protein /// similar to UCH37- Muc20 0.332 0.00475692 interactingmucin 20 protein 1 Kitl 0.331 0.02269104 kit ligand Pdk2 0.330 0.007759296 pyruvate dehydrogenase kinase, isoenzyme 2 BC040758 0.330 0.005418083 cDNA sequence BC040758 Rnf133 0.329 0.013671065 ring finger protein 133 Coq10b 0.328 0.00772865 coenzyme Q10 homolog B (S. cerevisiae) Rdh16 0.328 0.003736345 retinol dehydrogenase 16 Mll1 0.328 0.007316871 myeloid/lymphoid or mixed-lineage leukemia 1 B230208B08Rik 0.328 0.0337995 RIKEN cDNA B230208B08 gene Aldh7a1 0.327 0.040674422 aldehyde dehydrogenase family 7, member A1 4933433G19Rik 0.327 0.005579239 RIKEN cDNA 4933433G19 gene Chrdl2 0.326 0.026690524 chordin-like 2 Msn 0.326 0.008865764 moesin Fbxw13 0.326 0.008074204 F-box and WD-40 domain protein 13 Cxcl16 0.325 0.001984904 chemokine (C-X-C motif) ligand 16 Vasn 0.324 0.010602863 vasorin Nsdhl 0.324 0.006386003 NAD(P) dependent steroid dehydrogenase-like 6530401N04Rik 0.324 0.028796082 RIKEN cDNA 6530401N04 gene D12Ertd647e 0.323 0.001474331 DNA segment, Chr 12, ERATO Doi 647, expressed Naaladl1 0.323 0.03555202 N-acetylated alpha-linked acidic dipeptidase-like 1 Syk 0.323 0.019528735 spleen tyrosine kinase Spty2d1 0.322 0.034012858 SPT2, Suppressor of Ty, domain containing 1 (S. cerevisiae)

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REFERENCES

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Chapter IV: General Discussion

4.1 Context Summary

The ERRs are ubiquitous orphan nuclear receptors (NRs) showing their highest level of expression in tissues of high energy demand. All ERR isoforms are similar in terms of sequence and DNA binding capacity; each of them can bind to their cognate response element as monomers or homodimers. The ERRα and ERRγ isotypes also possess the ability to heterodimerize, a property that is inferred to ERRβ. Our group studies the ERRα and ERRγ isotypes. These two metabolic regulators are involved, along with PGC1-α, in the transcriptional regulation of genes controlling fatty acid β-oxidation, mitochondrial biogenesis, TCA cycle and OXPHOS pathways. Physiological genomics studies have identified them as having a role in cardiac hypertrophy and heart failure, innate immunity, obesity and cancer. The ERRs are pharmacologically targetable with synthetic compounds making them interesting potential therapeutic targets for several human diseases.

The present chapter is dedicated to ERRα; however, considering the high level of similarity between the isoforms, especially the common occurrence of a phosphorylation-induced sumoylation motif PDSM within their NTD, the mechanisms described herein can potentially apply to ERRγ as well (Tremblay, Wilson et al. 2008; Hentschke, Susens et al. 2009). In addition, the other ERR isotypes can contribute or compensate for some dysfunctions in the ERRα null mice. Nevertheless, differences between them definitely exist since they display a different level of constitutive activity, they are associated with cancer prognosis in an opposite manner, they show a different circadian expression pattern in several organs and the null mice of each isotype exhibit major differences regarding viability and severity of the phenotype (Ariazi, Clark et al. 2002; Luo, Sladek et

197 Chapter IV – General Discussion al. 2003; Mitsunaga, Araki et al. 2004; Yang, Downes et al. 2006; Alaynick, Kondo et al. 2007).

4.2 ERRα sumoylation and synergy control: physiological relevance

4.2.1 The ERRα phospho-sumoyl switch to fine tune ERRα expression levels

ERRα sumoylation on Lys14 is induced by phosphorylation of Ser19. The resulting repressive effect on its transcriptional activity is directly proportional to the number of consecutive response elements found within the target gene promoter; this effect is referred to as synergy control (SC). The natural polymorphism of the ERRα promoter in humans, which relates to the copy number of ERREs located within the proximal promoter region, strengthens the physiological relevance of the SC effect of ERRα sumoylation.

The intensity of the repressive effect produced by ERRα sumoylation likely varies according to the ESRRA promoter genotype. The number of ERREs within the polymorphic region can vary between one and four copies (spaced by 14 bp), with approximately 80% of the population harboring 2 or 3 copies. In addition, there is another ERRE, located 14 bp downstream of the polymorphism, adding one extra element for the SC effect (Laganière, Tremblay et al. 2004). This suggests that a total absence of response to ERRα sumoylation is unlikely, as even the carriers of the one-copy element genotype should respond to ERRα sumoylation-induced synergy control with the additional ERRE, although much less efficiently than the bearers of the four-copy genotype. Whether the ESRRA promoter polymorphism will dictate the efficiency of metabolic adjustments in response to physiological stressors, such as exercise or exposure to cold, is suggested by the role of ERRα in these processes. Similarly, the recently identified implication of ERRα in cardiac hypertrophy and heart failure, in addition to our findings that ERRα is also involved in blood pressure regulation,

198 Chapter IV – General Discussion suggests a potential role for the polymorphism in the observed heterogeneity of cardiovascular responses to physiological and pathophysiological stresses among the population. If this proves true via epidemiological meta-analysis, the ERRα phospho-sumoyl switch will be a likely candidate involved in regulating these differences. As the number of response elements also dictates the responsiveness to PGC-1α coactivation, the SC mechanism is integral to the ERRα-PGC-1α conduit and can be seen as a signaling-induced break on the ERRα-PGC-1α autoregulatory loop.

4.2.2 Identification of additional ERRα synergy control responsive promoters

In addition to the ERRα promoter, we have identified additional ERRα target genes, Atp5a1, Nadk and Cend1, each harboring three consecutive ERREs within their proximal promoter regions and displaying a positive SC response (Appendix 1, Figure A.1). Therefore, the mechanism is not unique to the ERRα promoter and it is likely that a subset of SC responsive ERRα target genes exist. However, the occurrence of three consecutive ERREs does not ensure a SC response. For example, the proximal promoter of the Ndufb4 gene, harboring three consecutive perfect dimer-favoring ERREs (containing a C or a G at position N (-2)) with a spacing highly similar to that of the 3-copy ESRRA gene promoter, is not SC responsive, as the ERRα Lys14Arg mutant displayed the same activity as the wild-type on the Ndufb4 promoter by reporter gene assay. This is consistent with the fact that the number of DNA elements, stable dimer formation and spacing, although important, are not the only determinants for SC mechanism to occur (Iniguez-Lluhi and Pearce 2000; Holmstrom, Chupreta et al. 2008). The exact mechanism behind the observed promoter specificity is yet unknown, but is likely to involve differences in coregulators interactions.

On the other hand, only one of the single element containing promoters tested displayed a SC response, suggesting that this mechanism could also occur

199 Chapter IV – General Discussion on other single ERRE promoters. Indeed, heterotypic SC effects were reported between SF-1 and Sox9, as well as for the Kruppel-type zinc finger protein ZBP- 89 and for the transcription factor MITF (Komatsu, Mizusaki et al. 2004; Murakami and Arnheiter 2005; Chupreta, Brevig et al. 2007). As the repressive effect of sumoylation via a SC mechanism affects the transcriptional activity of numerous NRs, it is tempting to speculate that heterotypic SC effects could be involved in transcriptional regulation between and by the ERR family members in cooperation with adjacent sumoylated NRs or other transcription factors.

Furthermore, the determinants identified so far concern linear SC, but it is also appealing to ask whether the SC mechanism could occur in an inter- chromosomal manner. The 3-D structure of the genome and the possibility for regulatory elements to be located several kb away from a promoter, and even on a different chromosome, are well recognized (Kininis, Chen et al. 2007; Deblois and Giguere 2008). Therefore, promoters bearing a single ERRE could also be subjected to SC if multiple elements bound by sumoylated factors were to come in close proximity by looping. Experimental evidence of this hypothesis is lacking and technically challenging to obtain at this moment. Indeed, it may be difficult to identify this single element SC responsive promoter by a standard reporter gene assay as, if inter-chromosomal, the effect might not be observable in the context of a circular plasmid. In vivo analysis using the recently described Combined 3C- ChIP-Cloning (6C) technique could potentially be used to answer this question (Tiwari, Cope et al. 2008). However, to identify the signaling pathways involved in the regulation of the ERRα phospho-sumoyl switch in a given physiological context, and even to uncover the identity of the ERRα-specific SC factor(s), may constitute a prerequisite for a successful SC responsive promoter hunt.

4.2.3 Breaking the code of the ERRα NTD

ERRα Ser19 phosphorylation was identified in vivo by the Gygi group’s large-scale proteomics studies of HeLa cells and mouse liver phosphoproteomes

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(Beausoleil, Jedrychowski et al. 2004; Villen, Beausoleil et al. 2007). A peptide comprising the ERRα NTD was identified and showed several patterns of paired phosphorylation events, suggesting that the ERRα is indeed regulated via multisite phosphorylation of the NTD in a seemingly conserved manner between mouse and human. There is potential for these sites to interplay with the PDSM function. The multisite phosphorylation events and the different pairing suggest that these phosphorylation events can interplay together and may even constitute a code of ordered events influencing each other. It is also possible that they interplay with other types of post-translational modifications potentially occurring on the ERRα NTD.

Indeed, the PDSM of ERRα corresponds to the ΨKxEPxSP subset of consensus sites, recently associated to acetylation/sumoylation/phosphorylation events (Stankovic-Valentin, Deltour et al. 2007). Acetylation of ERRα Lys14 could possibly prevent sumoylation by competition. It is unknown whether Ser19 phosphorylation event would be prevented as well or left unaffected. The latter possibility could potentially provide a means to dissociate Ser19 phosphorylation from Lys14 sumoylation, and allow transcriptional activation by Ser19 phosphorylation in certain cases. Although Lys14 acetylation has not been detected in the conditions tested, the possibility exist that Lys14 acetylation is a regulated event as well. Signaling events leading to other modifications occurring along the NTD could influence the recruitment of the acetyltransferase specific for Lys14. In the case of MEF2, however, acetylation was promoted by dephosphorylation of the PDSM serine suggesting that phosphorylation was not dissociated from the sumoylation event in that setting (Shalizi, Gaudilliere et al. 2006). However, the data available on the phosphorylation events targeting the PDSM of PPARγ suggest that dissociation of the sumoylation and phosphorylation event on the PDSM is possible, although no mechanisms have been identified (Beekum, Fleskens et al. 2009).

Further studies are required to delineate the transcriptional roles of the ERRα NTD phosphorylated residues, and to ultimately break the putative post-

201 Chapter IV – General Discussion translational modification code of the ERRα NTD. The regulation of ERRα activity by post-transcriptional modifications provides a means to regulate the transcriptional activity of ERRα in the presumed absence of a ligand. Also, the possible ERRα NTD phosphorylation code is likely to display some cell- or disease-specific particularities which could ultimately be targeted for specific ERRα modulation.

4.2.4 Signaling pathways targeting ERRα Ser19: insights from cancer cell lines

Most of the signals linked to ERRα phosphorylation that have been identified to date, such as the EGFR/ErbB2-triggered signaling cascades activating PKC, MAPK/ERK1-2 or PI3K/Akt pathways, have been in the context of cancer (Barry and Giguère 2005; Ariazi, Kraus et al. 2007). Phosphorylation of ERRα downstream of EGF/ErbB2 signaling is positively correlated with ERRα levels and activity.

Therefore, at first glance, these EGF/ErbB2 pathways seem not likely candidates for the induction of ERRα sumoylation via Ser19 phosphorylation. In addition, ERRα is expressed at higher levels and shows a greater constitutive activity in the high-ErbB2 expressing BT-474 cells than in the lower ErbB2 expressing MCF-7 breast cancer cells. Considering the role of the phospho- sumoyl switch as a signaling induced break on ERRα expression, this may suggest that the phospho-sumoyl switch is not activated by ErbB2 signaling via MAPK/ERK1-2 or PI3K/Akt pathways. PKCδ is unlikely to be the Ser19 kinase as the NTD harbors no PKC consensus site and this kinase was shown not to phosphorylate the ERRα NTD in vitro (Barry and Giguère 2005).

However, recent results, presented by the Mertz group, at the Nuclear Receptors: Orphan Brothers meeting of the Keystone Symposia series in March 2008, seem to indicate the reverse. They have put forward that the levels of

202 Chapter IV – General Discussion sumoylated ERRα in MCF-7 cells were higher than in BT474. This correlates with the role of the ERRα phospho-sumoyl switch in regulating the ERRα expression levels as well as with the differences in expression levels and transcriptional activity of ERRα between the two cell lines - there is more sumoylated ERRα in the cell line with the lowest ERRα activity and total levels. And again, this seems against the promotion of sumoylation by the MAPK/ERK1- 2 or PI3K/Akt pathways downstream of ErbB2 as MCF-7 express less ErbB2 than BT474.

At this same conference, the McDonnell group reported an increase in ERRα activity associated with Ser19 phosphorylation by ERK2. We have observed that the role of Ser19 was solely to induce sumoylation, hence repression on compound promoters, since the activity of the double mutants ERRα K14R-S19A and K14R-S19D was the same as the K14R mutant on the ESRRA promoter. Indeed, the cellular and promoter element contexts may play key roles in these observed differences. Also, as proposed above, another post- translational modification, such as acetylation or another phosphorylation along the NTD, could dissociate the sumoylation from the phosphorylation in a cell- and context- specific manner.

Another point to consider that may partially reconcile some of these effects is that the levels of ERK2 and phosphorylated ERK2 can vary independently of ErbB2 signaling. Therefore, despite the lower level of ErbB2 in MCF-7 cells, an increase in ERK2 activity is not excluded. Indeed, a recent report suggested that ERK2 phosphorylation levels (and activity) could indeed be as high, if not higher, in MCF-7 cells than in BT-474, as shown by an apparent increased ratio of phospho-ERK/total ERK in MCF-7 cells compared to BT-474. This finding was associated with cooperation between ErbB2 and IGFIR signaling (Chakraborty, Liang et al. 2008). This argument is consistent with the fact that ERK2 is a nodal signaling intersection downstream of several growth factor receptor pathways. In addition, the duration of the ERK2 signal is also to be taken into account as phosphatase activity may differ between cell lines.

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Therefore, it is possible that ERK2 would be the Ser19 kinase inducing Lys14 sumoylation despite the contradiction between levels of ErbB2 expression and ERRα sumoylation in MCF-7 versus BT474. The ErbB2 and IGFIR synergy presumably leads to an increase in ERK2 activity in MCF-7 cells relatively to BT- 474, hence elevating the level of sumoylated ERRα in MCF-7, in comparison to BT-474, despite the lower level of ErbB2 overexpression/amplification. This, however, does not explain the activation by Ser19 phosphorylation observed by the McDonnell group, and further studies are required to determine the mechanisms leading to the disruption of the phospho-sumoyl switch in certain cellular contexts.

On the other hand, our results and the Gygi group’s results were obtained in HeLa cells, an ErbB2-negative cell line originating from cervical cancer, and this demonstrates that ErbB2 signaling, although important in cancer cells, may not be the only growth factor receptor pathway targeting ERRα on Ser19. Hence, it is possible that the activation, and potential interaction, of diverse cell-specific signaling pathways impinge in a different manner upon the Lys14-Ser19 relationship by targeting other serine residues on the ERRα NTD, affecting the function of the phospho-sumoyl switch in a cell-specific manner. The phosphorylation of Ser19 by other kinases is also to be considered, as for PPARγ, the S112 of the PDSM is targeted by several kinases.

The kinase prediction algorithm NetphosK returned four potential kinases targeting ERRα on Ser19, which are cdk5, cdc2, GSK3 and p38MAPK (Blom, Sicheritz-Ponten et al. 2004). Some of these kinases are associated with specific cellular functions and physiological conditions and this can point towards other potential roles of the ERRα phospho-sumoyl switch, providing they are bona fide kinases for Ser19.

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4.3 Physiological cues modulating ERRα expression levels may involve the PDSM: potential biological roles of the phospho-sumoyl switch

4.3.1 Fine tuning the metabolic rate via regulation of ERRα expression

ERRα sumoylation is likely to regulate ERRα expression in various physiological states, not only disease. Notably, the ERRα phospho-sumoyl switch is likely to be modulated in response to signaling triggered by alterations in the metabolic demand, and hence be involved in the fine-tuning of metabolic rate. As such, exposure to cold induces ERRα expression to increase the metabolic rate and restore the body temperature to prevent hypothermia. It is therefore possible that Lys14 sumoylation could be decreased by exposure to cold in order to allow the increase in ERRα levels by PGC-1α, and that, upon recovery of the proper body temperature, the levels of ERRα Lys14 sumoylation would increase back to steady state levels and rapidly reduce the PGC1-α coactivation. Given that exposure to cold, in a similar fashion to other stressors like heat shock, is generally associated with an increase in sumoylation, this hypothesis may seem unlikely at first glance. However, the increase in SUMO-2 conjugates under heat shock stress has recently been shown to involve a redistribution of sumoylation among the SUMO-2 substrate proteome (Golebiowsy, Matic et al, 2009). In addition, this study revealed ERRα modification by SUMO-2 was decreased after heat shock. It could therefore be possible that an effect such as this would be observed under other stress conditions, including exposure to cold.

Since the proportion of sumoylated form versus non-sumoylated form of a given factor is estimated to be 5% at steady state, it can be supposed that this allows a rapid adjustment of the levels of sumoylation in response to signaling and that this regulatory control mechanism could go both ways. Although the modulation of ERRα expression levels is a physiological mechanism used for adaptation to normal increases in energy demand, the regulation of the metabolic

205 Chapter IV – General Discussion rate by ERRα seems also important in disease state, such as cardiac hypertrophy and heart failure.

4.3.2 Cardiac hypertrophy and heart failure

The expression of ERRα was shown to be decreased in the heart of wild- type mice subjected to transverse aortic constriction (TAC) over 7 and 14 days, while the ERRα null mice showed an enhanced hypertrophic response and a more pronounced deterioration of the cardiac function by this cardiac stressor (Huss, Imahashi et al. 2007). The Kelly group performed a gene expression analysis of the TAC hearts and observed that the expression levels of the E3 SUMO ligase PIASy were increased in the heart of the wild-type mice after TAC. As PIASy was the only E3 ligase enhancing ERRα sumoylation in our screening, it appears that ERRα sumoylation might be stimulated by cardiac hypertrophic stress, via PIASy, thus potentially accounting for the lower ERRα expression in the cardiac pressure overload model. The activity of p38 MAPK, among other kinases, is induced in the TAC model of cardiac pressure overload (Wang, Huang et al. 1998). The p38 MAPK stress kinase was predicted by the NetphosK phosphorylation site prediction algorithm to be one of the ERRα Ser19-targeting kinases. Subsequently, this pathway is a likely candidate to regulate stress- induced ERRα sumoylation in the heart.

4.3.3 Cell proliferation and cell cycle progression

Several recent lines of evidence now point towards a role of the ERRs in cell proliferation and cycle progression. ERRα was reported to display a cell cycle-regulated pattern of expression, similar in HeLa cells and mouse embryonic fibroblasts (van der Meijden, Lapointe et al. 2002). Moreover, inhibition of

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ERRα by siRNA was shown to reduce the growth rate of MDA-MB-231 breast cancer cells grown as xenografts (Stein, Chang et al. 2008). This is in accordance with the previous association of ERRα and poor prognosis (Ariazi, Clark et al. 2002). In addition, our gene expression profiling of the ERRα-null kidneys revealed that several key cell cycle genes are downregulated in comparison to the wild-type kidneys, while GO analysis of the microarray returned M-phase as the most significantly enriched biological process (Appendix 1, Figure A.2). Among those genes, the downregulation of Cdc2a and MKi67 suggests a lower proliferative index and slower cell cycle in the ERRα null kidneys, which is in harmony with the slower tumor growth rate caused by the removal of ERRα via siRNA in cancer xenografts.

Interestingly, a large-scale siRNA screen in the lung adenocarcinoma cell line A549 identified a role for ERRα in G2/M transition (Cheng, Beauchamp et al. 2005). Taken together, the data currently available suggest that ERRα could be involved in G2/M transition. Also, cdc2 kinase, a known M-phase promoting kinase, was predicted to target ERRα Ser19 by the NetphosK algorithm. It is therefore possible that ERRα Ser19 phosphorylation by cdc2, assuming cdc2 does indeed target ERRα, could link the ERRα phospho-sumoyl switch to G2/M transition. In addition, the involvement of the pro-apoptotic SUMO E3 ligase PIASy in cancer cell apoptosis, and the fact that progression to the more aggressive myeloid leukemia from pre-malignant state involves the downregulation of PIASy expression, adds another reason to look upon the ERRα phospho-sumoyl switch, or its disruption, as a potentially important mechanism in the regulation of cancer cell proliferation and/or tumor growth (Ohmine, Ota et al. 2001)

4.3.4 Circadian rhythm-regulated ERRα expression

The expression of ERRα is also regulated in a circadian manner. While the expression levels of the SUMO isoforms remain stable throughout the

207 Chapter IV – General Discussion circadian cycle in liver, the levels of BMAL1 sumoylation vary with the circadian rhythm (Cardone, Hirayama et al. 2005). Whether the levels of ERRα sumoylation also vary in a circadian rhythm-regulated manner, but opposite to the ERRα expression, and account for the circadian pattern of expression of ERRα is an interesting possibility.

4.4 The similarity between ERRs and PPARγ regulation

The fact that both the ERRs and PPARγ are the only members of the NR superfamily to harbor a PDSM suggests that this mechanism might be important for energy metabolism, as both receptors share PGC-1α as a principal coactivator and both participate in the regulation of similar pathways (Giguere 2008; Hummasti and Tontonoz 2008).

In addition to the possible physiological interplay between ERRs and PPARγ, there seem to be some resemblance in their mode of regulation (Beekum, Fleskens et al. 2009). Indeed, the signaling pathways targeting PPARγ S112 of the PDSM may also be similar between the ERRs and PPARγ. Indeed, PPARγ S112 is phosphorylated by ERK1/2, p38MAPK/JNK pathways which are repressing PPARγ adipogenic gene program. On the other hand, phosphorylation of S112 by cdk7 or cdk9 is associated with activation of the adipogenic gene program. This suggests, although still unproven, that the phosphorylation of S112 might be dissociated from Lys107 sumoylation depending on the implicated kinase, thereby supporting our hypothesis concerning the potential phosphorylation code of the ERRα NTD as a mechanism for the dissociation of Ser19 phosphorylation from Lys14 sumoylation in ERRα as well as of the existence of a ERRα synergy control responsive program. It also strengthens the potential for p38MAPK to be a bona fide kinase for ERRα in the heart and implies that a similar dichotomy in the Ser19 kinases could dictate a phosho-sumoyl switch-dependent transcriptional program exchange for ERRα.

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4.5 ERRα in blood pressure regulation

Consistent with the sharing of metabolic pathways between PPARγ and ERRα, our physiological studies identified a novel common biological role between ERRα and PPARγ: blood pressure regulation. The PPARγ null mice were recently shown to be slightly hypertensive during the day and hypotensive at night, as the circadian oscillations of blood pressure are blunted in the PPARγ null mice models. This was associated with a vascular effect involving the circadian regulator BMAL1, which is a target gene of PPARγ in the vasculature (Wang, Yang et al. 2008; Chang, Villacorta et al. 2009).

Our study unraveled a role for ERRα as a blood pressure regulator as the ERRα null mice are also hypotensive at night. However, the circadian oscillations are kept intact as the blood pressure levels start to rise and decrease at the same time in both wild-type and ERRα null mice. This suggests a potentially different mechanism for ERRα in blood pressure regulation than that of PPARγ.

The phenotype of the ERRα null mice seems paradoxical at first glance. While the mice are hypernatremic and hypokalemic relative to their wild-type counterparts, they also show an increase in renin mRNA in the kidney and a slightly increased plasma renin concentration, with no change in the plasma aldosterone levels. The set of promoters bound by ERRα in the kidney correlates with the phenotype in terms of hypernatremia, hypokalemia and elevated renin levels, as ERRα it comprises key channels responsible for sodium reabsorption, the epithelial sodium channel ENaC and the Na+/K+-ATPase subunits as well as genes of the RAAS such as renin (Ren1), angiotensinogen (Agt) and the angiotensin converting enzyme 2 (Ace2). Taken separately, the hypernatremia and slightly elevated renin, should lead to hypertension or at least not associate to hypotension. However, the blood pressure of the ERRα null mice is lower suggesting that the normal chain of adaptive event is broken. This could be a vascular effect due to the imbalance between vasodilating and vasoconstricting

209 Chapter IV – General Discussion factors in the ERRα null mice vasculature as suggested by the downregulation of genes responsible for prostaglandins production (Ptgds, Hpgd) or action (Ptger3) (Boushel, Langberg et al. 2004). A decrease in prostaglandins could contribute to the salt retention phenotype. Indeed, the Bartter syndrome is sometimes referred to as the hyperprostaglandin E2 syndrome as the EP3 receptor (Ptger3) contributes to the PGE2-enhanced salt and water losses (Nusing, Treude et al. 2005).

Indeed, the simultaneous occurrence of hypotension and elevated levels of renin is typically observed in Bartter syndrome. The term Bartter syndrome encompasses a variety of genetically inherited disorders of renal electrolyte transport classified into 5 subtypes on the basis of the gene bearing the loss-of- function mutations (Rodriguez-Soriano 1998). Types I and II, or antenatal Bartter syndrome, are associated to mutations in the sodium-potassium-chloride symporter NKCC2 (Slc12a1) and the potassium channel ROMK (Kcnj1), respectively. Type III (classic Bartter syndrome) relates to mutations in the CLC- Kb (Clcnkb) chloride channel, while type IV (Bartter’s syndrome with sensorineural deafness) is caused by mutations in the Barttin chloride channel beta-subunit (Bsnd), which is known to interact with and modulate the activity of Clcnkb. Type V is associated with mutations in both CLC-Ka (Clcnka) and CLC- Kb (Clcnkb) chloride channels although some experts classify it as being associated with autosomal dominant hypocalcemia caused by a gain-of-function mutation in the calcium-sensing receptor Casr. Finally, the related Gitelman syndrome is induced by mutations in the sodium-chloride symporter NCCT (Slc12a3) (Naesens, Steels et al. 2004; Seyberth 2008). Consistent with the genetic heterogeneity associated with these renal ion transport defects, the panel of symptoms is wide although constantly related to a dysregulation of blood and urine electrolyte homeostasis, mostly hyponatremia and salt wasting, and often accompanied with hypokalemic metabolic alkalosis (Bartter, Pronove et al. 1962). However, in some atypical cases of Bartter syndrome, hypernatremia and metabolic acidosis have been reported and the genotype-phenotype correlations within these complex hereditary tubular disorders are quite variable (Bettinelli,

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Ciarmatori et al. 2000). In the ERRα null kidney, Bsnd, Kcnj1, Kcnq1 and Slc12a1 are downregulated at the mRNA level. As the loss-of-function of only one of these channels causes salt wasting, the downregulation of almost all channels associated to the Bartter syndrome in the ERRα null kidney is suggestive of a salt wasting phenotype (Ji, Foo et al. 2008).

We observed no salt wasting in the ERRα null mice under basal condition, but on the contrary, the ERRα null mice are hypernatremic and show salt retention. However, it is possible that compensation of the salt wasting occurs considering the widespread binding of ERRα on promoters genes in the electrolyte handling program. Indeed, background potassium channels, such as Kcnh2 and Kcnc3, which are normally not expressed in the kidney, are bound by ERRα in this organ and were upregulated at the mRNA level in the ERRα null kidney. In addition, several of the potassium channels mutated in Bartter syndrome co-transport chloride ions, an effect participating in the acid-base imbalance associated to this syndrome. The ERRα null mice do not show chloride imbalance nor acid-base imbalance as the urinary and blood chloride concentrations as well as blood pH are similar in both genotypes. Indeed, the dysregulation of chloride channels (Abcc4, Ttyh3, Clic3) and of the chloride/carbonate exchanger Slc4a1 as well as the carbonic anhydrase Car3 in the ERRα null kidneys suggests a possible compensation of salt wasting and acid- base balance.

Compensatory mechanisms in mice are very strong and one supporting example is that mice null for the key potassium channel Kcnq1, did not show signs of renal potassium wasting until subjected to low potassium diet, owing to the upregulation of background renal potassium channels and fecal losses (Vallon, Grahammer et al. 2005). This provides the rationale to suppose that the ERRα null mice could show signs of salt wasting under low sodium diet. The monitoring of electrolyte handling under salt deficient diets could better support the notion that the ERRα null mice display an atypical Bartter-like syndrome phenotype. Another resemblance to Bartter syndrome, in addition to the Bartter syndrome

211 Chapter IV – General Discussion genes downregulation and the simultaneous occurrence of hypotension with elevated renin, is the shorter stature of the ERRα mice. Indeed, the antenatal Type IV Bartter Syndrome (Bsnd mutation involving sensorineural deafness) is often associated with a failure to thrive. Tough, in our case, the binding of ERRα to the growth hormone receptor (Ghr) gene promoter could be another likely explanation for this phenotypic characteristics of the ERRα null mice. The presence of ERRα on the Ghr gene promoter suggests that ERRα could play a broader role in development than initially suspected.

Other cases where hypotension was associated to elevated renin is when AngII production (Agt and Ace null mice) or action (Agtr1a null mice) was blunted (Tanimoto, Sugiyama et al. 1994; Krege, John et al. 1995; Sugaya, Nishimatsu et al. 1995). In our mice, we did not assess the plasma AngII levels, but our hypothesis is that impairment of AngII actions could be participating in the observed hypotensive phenotype. AngII is the main vasoconstrictor peptide of the RAAS and the ERRα null mice could show a decreased level of AngII expression or an impaired sensitivity to AngII. Indeed, although the plasma levels of AngII were not measured, we did assess the response to AngII in terms of renin levels. From that result, we can suppose that the response to AngII is kept in the ERRα null mice. Ren1 expression is under the negative control of AngII via a tubuloglomerular feedback loop and exogenous administration of AngII repressed the plasma renin levels in both the wild-type and the ERRα null mice, even slightly more efficiently in the ERRα null mice (Appendix I, Figure A.3). However, this does not guarantee that the vasomotor effects of AngII are preserved and further testing on isolated arterial rings is warranted to this end.

Assuming the response to AngII is maintained in the blood vessels as well as it is in the kidney, this could suggest that AngII production could be the broken link in the adaptive cascade of blood pressure control in the ERRα null mice. Yet, in the liver as in the kidney, ERRα binds to the promoter of Agt and the levels of Agt mRNA are elevated in the ERRα null mice in both organs. This suggests that maybe the AngII production is not impaired as substrate availability

212 Chapter IV – General Discussion may be increased, leaving the other possibility of a faster AngII degradation rate. Indeed, the increased expression level of Ace2 mRNA supports the latter hypothesis as Ang(1-7) is a potent vasodilatator. The balance between ACE and ACE2 activity is a key factor for the duration of AngII effects (Burrell, Johnston et al. 2004). While the effects of ACE2 are local it is possible that the plasma levels of AngII would be similar, since ACE cleaves AngI to produce circulating levels of AngII in the lungs, but that the effects at the target site would be pushed towards vasodilatation by Ang(1-7) owing to the increased expression of Ace2.

This constitutes one hypothesis concerning the mechanisms involved in the hypotensive phenotype of the ERRα null mice, but a plethora of alternative effects may also contribute. For example, a role for tonicity regulating factors Nfat5 and Hspa4l, or the ENaC regulators Sgk2 and Gilz (Tsc22d3). Also, several identified deregulated genes in the ERRα null kidney, such as Adora1, Aldoa, Casr, Cldn16, Egr1, Fgf1, Fgf9, Gcgr, Gclc, Ghr, Lepr, Lpl or Npy1r, have been linked to hypertension in epidemiological and animal studies. Several of these gene promoters (underlined) are bound by ERRα in the kidney. The overlap between binding event (ChIP-on-chip) and expression (microarray) is This is in addition to the possible role played by energy metabolism on the activity of the Na+/K+-ATPases (ATP levels) or by other organs such as intestine and muscles in electrolyte homeostasis, which could as well involve ERRα. Further studies using organ-specific ERRα null mice are required to put the pieces of the ERRα-blood pressure control puzzle together, but our study is nonetheless the first to link ERRα to blood pressure control and possibly hypertension.

ERRα may also be involved in blood pressure regulation in the setting of secondary hypertension, often observed in obesity, diabetes or the metabolic syndrome. Our results also suggest that ERRα could be playing a role in the cardiorenal syndrome. Indeed, roles of ERRα have been reported in 3 or of the 4 players of the cardiorenal connection. The renal function of the ERRα null mice is apparently not impaired as shown by a normal blood urea nitrogen and glomerular filtration rate. However, the mice show decreased cardiac output and

213 Chapter IV – General Discussion shorter ejection fraction after TAC (Huss, Imahashi et al. 2007). The cardiac defect may contribute to the hypotension, while it is probable that the renal electrolytes handling defect could contribute to the impaired cardiac adaptation to TAC. Indeed, water retention seems to occur along with salt retention as the ERRα null mice excrete less volume relative to their water intake than the wild- types. The blood volume would need to be compared and normalized to body weight in the ERRα null mice, but an increase in blood volume and the hypernatremia could likely partake in an impairment of cardiac function in the long-term or in response to hemodynamic cardiac stressors.

4.6 Conclusion: ERRα as a therapeutic target

We have identified a role for ERRα in blood pressure regulation and this suggests a role for ERRα in hypertension – either primary essential hypertension or secondary to other co-morbidities such as metabolic syndrome, cardiorenal syndrome or diabetes. In addition to the previously identified physiological roles of ERRα, this clearly demonstrates that, prior to pharmacologically targeting ERRα, more research is needed to identify the determinants for organ- and disease-specificity as well as the relative contributions of other ERR isotypes.

Our results on the ERRα null mice phenotype, taken together with the phenotypic observations of other groups, if translated directly to humans, suggest that the inhibition of all ERRα activity in a systemic manner might potentially be beneficial in cancer or cardiovascular diseases, but only in the absence of any of the common physiological stressors encountered on a daily basis, such as exercise, high fat diet or high salt diet and exposure to cold. It is dangerous to evaluate the benefits versus risks at fist glance based on the phenotypic alterations in mice, but it is possible to wonder: would the cancer patient under general ERRα inhibitor therapy develop cardiac hypertrophy or heart failure, hypothermia in the winter, hypotension and hypernatremia like the ERRα null mice? Or would

214 Chapter IV – General Discussion the salt sensitive hypertensive patient on a general ERRα inhibitor see his blood pressure lowered but his cardiac function and stress response deteriorate? And would an ERRα agonist be cardioprotective but make you obese and at higher cancer risk at the same time?

The best strategy to achieve a successful ERRα-based treatment is, as it is currently done, to first establish the transcriptional programs of ERRα in all organs and to assess their level of organ-specificity and its determinants. This could include, for example, the ERRα NTD post-translational modification code and its specific coregulators favoring one SC responsive program or the other, in a similar manner as the PPARγ phospho-sumoyl switch, which may direct the adipocytes differentiation program. The following step being to precisely target a modified form or interaction of ERRα specifically, at the proper time of the day or night. As diverse ligand classes for AR induce different AR-coactivator pairs, it might be possible to achieve the same with ERRα. Many unknowns remain and the road might be long before holding the first generation of selective ERR modulators (or SERRMs) in our hands, but it appears that the most interesting years of the research on ERRs are yet to come.

215 Chapter IV – General Discussion

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Chapter V: Contribution to original research

Chapter II

I have identified a mechanism of regulation of the transcriptional activity of the ERRs by post-translational modifications, namely phosphorylation-induced sumoylation regulated by a phosphorylation-dependent sumoylation motif (PDSM).

I have established that ERRα expression is regulated via a synergy control mechanism that could possibly be involved in the fine-tuning of ERRα levels in response to physiological cues.

I have identified a novel interacting partner of ERRα, the E3-ligase PIASy, and demonstrated that PIASy promotes the sumoylation of ERRα in a PDSM- dependent manner.

I have achieved the detection of sumoylated ERRα at the endogenous level and demonstrated the functionality of the ERRα phospho-sumoyl switch in vivo via the comparison between a general ERRα antibody and the one that I have generated, which is directed against phosphorylated ERRα Ser19.

The regulation of ERRα sumoylation by signaling events targeting the phospho- sumoyl switch is likely to be important for multiple physiological and pathophysiological conditions and could eventually be targeted for therapy.

Chapter III

I have identified novel physiological roles for ERRα in the kidney:

• sodium and potassium handling • regulation of genes of the renin-angiotensin-aldosterone-system • regulation of several genes involved in blood pressure control or hypertension

I have also identified that ERRα most likely involved in blood pressure control at multiple levels and that this receptor is subjected to circadian regulation in the kidney. My results provide a rationale to study ERRα in the vasculature and in development.

These results, along with the identified roles of ERRα, suggest that this receptor may be involved in essential hypertension or in secondary hypertension in the setting of diabetes, metabolic syndrome or cardiorenal syndrome.

219

APPENDIX I

220

Figure A.1

Figure A.1 Additional mouse endogenous promoters displaying a synergy control effect

Luciferase assays of three other endogenous compound promoters in mouse. Atp5a1 has 3 sites within 134 bp. Cend1 has 4 sites within 261 bp. Nadk has 4 sites within 325 bp. (Of note is that Esrra has 3 sites within 69 bp while the non- SC responsive Ndufb4 (not shown) has 3 sites within 64 bp.)

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Figure A.2: ERRα in the cell cycle: insights from the kidney microarray analysis

Figure A.2: ERRα in the cell cycle: insights from the kidney microarray analysis

A, Pie charts representing the classification of the ERRα null kidney microarray analysis. B, Significant GO biological processes related to cell cycle returned by FatiGO analysis of the differentially expressed genes in the ERRα null mice kidney by microarray. *** , p < 0.001.

222

Figure A.3

Figure A.3: Plasma renin levels in ERRα wild-type and null mice after Angiotensin II administration.

The mice were treated with Angiotensin II for 14 days with a constant dosage of 350 ng/Kg/min with a subcutaneous osmotic mini-pump. Of note is that the basal values of PRC (see Figure 3.S1) were all higher than 115 ng/mL/h of Ang I, showing the effective repression of renin levels following the AngII.

223

APPENDIX II

224