Regulation of Expression and Activity of the Bhlh-PAS Transcription

Regulation of Expression and Activity of

the bHLH-PAS Transcription Factor NPAS4

David Christopher Bersten

B.Sc. (Biomedical Science), Honours (Biochemistry)

A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy

Discipline of Biochemistry

School of Molecular and Biomedical Science

University of Adelaide, Australia

June 2014

Contents

Abstract ...... 3 PhD Thesis Declaration ...... 5 Acknowledgements ...... 6 Publications ...... 8 Conference oral presentations ...... 9 Additional publications ...... 9 Chapter 1: ...... 10 Introduction ...... 10 bHLH-PAS transcription factor structure and mechanism ...... 10 bHLH-PAS dimerization, DNA binding and signal transduction mechanisms ...... 16 bHLH-PAS transcription factor function ...... 19 Neuronal PAS Factor 4 ...... 19 Drosophila Dysfusion ...... 20 NPAS4 Expression and Regulation ...... 21 NPAS4–dependent Transcriptional Regulation ...... 24 NPAS4 Function ...... 26 Chapter 2: Results ...... 31 2.1 Elucidation of molecular mechanisms which restrict NPAS4 expression to the brain...... 31 2.2 Human variants in the NPAS4/ARNT2 transcriptional complex can disrupt function ...... 32 2.3 Generation and validation of Inducible and reversible systems for lentiviral and ES cell (Col1a1-RMCE) mediated manipulation of NPAS4 levels...... 33 Chapter 3: Discussion ...... 36 References...... 45 Appendix 1 ...... 103 Appendix 2 ...... 104 Appendix 3 ...... 105 Appendix 4 ...... 106

Abstract

Development of the Central Nervous System (CNS) relies on complex transcriptional programs to specify distinct neuronal areas/cell types, and guide the formation of neuronal networks.

Synaptic activity during post-natal brain development dictates the number and strength of synapses as well as promoting neuronal cell survival through activation of transcriptional programs. The establishment of synapses during this “critical period” of post-natal neuronal development and the local rearrangement, fine tuning and maintenance of synaptic connections into adulthood contributes to synaptic plasticity, memory, learning and cognitive function, while dysfunction in these processes is thought to contribute to a number of neuropsychiatric diseases. Studying transcription which underlies these events and disease states has been technically challenging due to the lack of gain and loss of gene expression systems to interrogate complex biological questions in primary neurons or the developing nervous system of rodents. As a result, despite clinical and anatomical data, the molecular mechanisms underlying neuropsychiatric disease or memory and learning remain poorly understood.

The basic-Helix-Loop-Helix (bHLH) – Per/Arnt/Sim (PAS) (bHLH-PAS) homology domain transcription factor Neuronal PAS factor 4 (NPAS4) is tightly coupled to neuron function by homeostatically regulating neuronal activity via stimulating formation of inhibitory synapses.

NPAS4 expression is brain restricted and highly induced following neuronal depolarisation, paradigms of learning, seizure or ischemia. NPAS4 null mice are prone to seizures, hyperactivity, have defects in memory formation, social interaction, cognitive impairments, as well as age related neurodegeneration.

This thesis shows that NPAS4 expression is highly restricted to the CNS, in particular the cortex, by repressive activity of RE-1 Silencing Transcription Factor/Neuron-Restrictive Silencer

Factor (REST/NRSF) in non-neuronal cells and stem cells. In addition, we also provide evidence

that microRNA-224 targets the NPAS4 3’UTR, which may contribute to regionalised NPAS4 expression in the brain. We identify human variants within NPAS4 and ARNT2 which disrupt

NPAS4 function, which may have implications for neuropsychiatric disease. Using structural modelling and biochemical experiments we show that one of these variants disrupts dimerisation, providing insight into bHLH-PAS dimerisation mechanisms.

We also describe a new system for knockdown and ectopic expression which is broadly applicable for reliable, flexible and temporal control of gene expression to facilitate investigating gene function. This system incorporates single gateway compatible vector systems for lentiviral infection and Recombination Mediated Cassette Exchance (RMCE), the latter targeting the Collagen 1a1 (Col1a1) locus in germline competent embryonic stem cells.

Using an optimised reverse tetracycline transactivator (rtTA) system with reduced background expression and increased sensitivity to doxycycline, we have shown that we can rapidly generate inducible overexpression and short hairpin RNA (shRNA) mediated knockdown cell lines with homogenous, inducible expression.

The work encompassed within this thesis investigates the molecular mechanisms underlying the restricted expression pattern of NPAS4, the contribution of human non-synonymous variants to NPAS4/ARNT2 transcription factor function, and the development of flexible, inducible and reversible gene expression systems for studying NPAS4 function in vitro and in vivo.

PhD Thesis Declaration

This thesis contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution to David Christopher Bersten and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. I give consent to this copy of my thesis when deposited in the University Library, being made available for loan and photocopying, subject to the provisions of the Copyright Act 1968. The author acknowledges that copyright of published works contained within this thesis (as listed below*) resides with the copyright holder(s) of those works. I also give permission for the digital version of my thesis to be made available on the web, via the University’s digital research repository, the

Library catalogue, and also through web search engines, unless permission has been granted by the University to restrict access for a period of time.

David Christopher Bersten

June 2014

Acknowledgements

Without the support, patience, generosity, friendship and love of colleagues, mentors, friends, and family the PhD journey would not have been as enjoyable or rewarding. For these people I must acknowledge.

Ass. Prof. Murray Whitelaw, you are truly an inspiring scientist and educator. I am forever indebted to you for nurturing and supporting my interest in science, and guiding my development as an independent researcher. I aspire to be as purely principled in scientific pursuit, as kind, as patient and as enthusiastic as you are.

Dr. Daniel Peet, you have been a fantastic co-supervisor and mentor for me over the years.

Your door has always open for scientific discussion or a beer on a Friday afternoon, the former being invaluable to me.

The biochemistry discipline – I would like especially thank all the past and present researchers and educators in the biochemistry discipline for creating and maintaining an environment of excellence and scientific rigor within the discipline.

For all those who have helped be along the way, colleagues and friends thank you for all the help and support. There are many to name but I would specifically like to thank Adrienne

Sullivan, Pete McCarthy, Jo Wright, James Hughes, Paul Thomas, Sandy Piltz, Stephen Bent, and Andrew (Nan) Hao.

Jayne, the love and support you have given me is above and beyond, thank you for being so understanding. I would also like to thank the McConachy’s and my siblings for their support and patience.

Lastly, I must acknowledge my parents Libby and Andrew. This was not possible without your unfailing support and gentle guidance. Thank you for having confidence in me and allowing me to pursue my interests.

Publications

This thesis is based on the following publications and referred to in the text:

I. Bersten DC, Sullivan AE, Peet DJ, Whitelaw ML. bHLH-PAS proteins in cancer. Nat Rev

Cancer. 2013 Dec;13(12):827-41.

II. Bersten DC, Wright JA, McCarthy PJ, Whitelaw ML. Regulation of the neuronal

transcription factor NPAS4 by REST and microRNAs. Biochim Biophys Acta. 2014

Jan;1839(1):13-24.

III. Bersten DC, Bruning JB, Peet DJ, Whitelaw ML. Human Variants in the Neuronal Basic

Helix-Loop-Helix/Per-Arnt-Sim (bHLH/PAS) Transcription Factor Complex

NPAS4/ARNT2 Disrupt Function. PLOS one. 2014 Jan, DOI:

10.1371/journal.pone.0085768

IV. Bersten DC, Sullivan AE, Bhakti V, Li D, Thomas PQ, Bent S, Whitelaw ML, Inducible

and reversible lentiviral and recombination mediated cassette exchange (RMCE)

systems for controlling gene expression. Manuscript submitted Nucleic Acids Research

Conference oral presentations

REGULATION OF EXPRESSION AND ACTIVITY OF THE NEURONAL TRANSCRIPTION FACTOR

NPAS4

Bersten D.C., Peet D.J. and Whitelaw M.L. Australian Society of for Biochemistry and

Molecular Biology annual conference (ComBio 2012)

Additional publications

Bonnefond A, Raimondo A, Stutzmann F, Ghoussaini M, Ramachandrappa S, Bersten DC,

Durand E, Vatin V, Balkau B, Lantieri O, Raverdy V, Pattou F, Van Hul W, Van Gaal L, Peet DJ,

Weill J, Miller JL, Horber F, Goldstone AP, Driscoll DJ, Bruning JB, Meyre D, Whitelaw ML,

Froguel P.

Loss-of-function mutations in SIM1 contribute to obesity and Prader-Willi-like features.

J Clin Invest. 2013 Jul 1;123(7):3037-41.

All publications have been reproduced with the permission from the copyright holders

Chapter 1: Introduction

NPAS4 is a member of the basic-Helix-Loop-Helix (bHLH) – Per/Arnt/Sim (PAS) (bHLH-PAS) transcription factor family which is almost exclusively expressed within the central nervous system (CNS) and is tightly regulated by neuronal activity. Like other members of this family it must heterodimerise with a class II bHLH-PAS transcription factor (Aryl hydrocarbon receptor nuclear translocator (Arnt) or Arnt2) to bind DNA and activate transcription. The heterodimer binds to asymmetric E-BOX like elements (NNCGTG) within the promoters and enhancers of target genes to modulate gene expression. The function of NPAS4 is primarily to homeostatically regulate the activity state of excitatory neurons to keep a balance between excitatory and inhibitory inputs within the neuron. bHLH-PAS transcription factor structure and mechanism

bHLH-PAS proteins are a conserved family of proteins which contain two major functional domain structures, the bHLH domain and the PAS domain (or PAS repeats)(Crews 1998; Yun,

Maecker et al. 2002; Kewley, Whitelaw et al. 2004; McIntosh, Hogenesch et al. 2010).

Transcription factors containing basic Helix-Loop-Helix (bHLH) motifs contribute to gene expression profiles critical for many development processes such as myogenesis, haematopoiesis, neurogenesis, heart and pancreatic development (Massari and Murre 2000).

This motif mediates dimerisation and DNA binding to a degenerate E-box CANNTG to regulate transcription (Blackwell, Kretzner et al. 1990; Blackwell and Weintraub 1990; Baxevanis and

Vinson 1993). The N-terminal basic region of this motif contacts bases in the major groove of

DNA, while the HLH domain primarily contributes to dimmer stability (Ma, Rould et al. 1994).

The interaction between the two amphipathic helices of two proteins allows the formation of homo- or heterodimers, which pairs the basic regions to facilitate binding to their cognate

half sites (Ferre-D'Amare, Prendergast et al. 1993; Ma, Rould et al. 1994). Over 240 HLH proteins have been described in almost all eukaryotes ranging from Saccharomyces cerevisiae to Caenorhabditis elegans, Drosophila melanogaster to mammals (Atchley and Fitch 1997;

Massari and Murre 2000).

There are two sub-families of bHLH transcription factors which utilize secondary dimerisation domains adjacent to the bHLH domain; these are the bHLH leucine zipper (Zip) transcription factor family and the bHLH-Per/Arnt/Sim (bHLH-PAS) homology domain family of transcriptional regulators (Massari and Murre 2000). The bHLH-PAS proteins are characterized by two N-terminal PAS domains denoted PAS A and PAS B. The PAS domain or the PAS repeats are also ancient sensor and protein interaction domain structures found in organisms from bacteria to humans (Crews and Fan 1999; McIntosh, Hogenesch et al. 2010; Henry and Crosson

2011). The PAS domain was initially defined as a ~275 amino acid motif with shared sequence homology between the Drosophila melanogaster clock protein Period (Per), the midline expressed neurogenesis factor Single Minded (Sim) and vertebrate ARNT (Reddy, Zehring et al.

1984; Crews, Thomas et al. 1988; Hoffman, Reyes et al. 1991). However, recent structural insights have indicated that isolated PAS domains encompass an ~110 amino acid generic PAS fold which is structurally highly conserved but poorly conserved in amino acid sequence

(Figure 1)(Huang, Chelliah et al. 2012; Bersten, Sullivan et al. 2013).

The PAS domains are present in thousands of proteins and are most highly represented in bacterial proteins (Moglich, Ayers et al. 2009). A general mechanism of PAS domain sensing coupled to transduction effector output domains, such as protein kinase, catalytic, dimerisation, DNA binding or ion channel domains, has been proposed for PAS containing proteins. Indeed PAS domain containing proteins are involved in sensing and responding to light, voltage, oxygen, xenobiotics, redox potential and biological time keeping (McIntosh,

Hogenesch et al. 2010; Henry and Crosson 2011). A classic signal sensing mechanism exists in

Photoactive Yellow Protein (PYP), providing an example of a blue light sensing PAS protein which acts negatively on phototaxis (Genick, Borgstahl et al. 1997; Henry and Crosson 2011).

In contrast to prokaryotes, few ligands or stimuli directly targeting the PAS domains have been described for the mammalian PAS containing proteins. The best example lies with planar aromatic hydrocarbons and dioxins, ligands which have been shown to directly bind to the PAS

B repeat in the Aryl hydrocarbon Receptor (AhR), thereby stimulating translocation to the nucleus and activation of genes encoding xenobiotic metabolising enzymes (Whitelaw,

Gottlicher et al. 1993; Antonsson, Whitelaw et al. 1995; Whitelaw, McGuire et al. 1995). It has also been reported that the NPAS2 PAS A domain can bind heme and carbon monoxide, resulting in inhibition of an interaction with its bHLH/PAS partner, BMAL1 (Dioum, Rutter et al.

2002). Per2 has also been shown to bind heme, which mediates degradation of the protein.

As Per is an inhibitor of CLOCK/BMAL1, reduced Per levels result in increased CLOCK/BMAL1 dimer formation and function (Yang, Kim et al. 2008). CLOCK and NPAS2 have overlapping function and can both bind nicotinamide adenine dinucleotide (NAD), which inhibits DNA binding in vitro (Rutter, Reick et al. 2001). Recent structural resolution of the PAS B domain of the hypoxia inducible factor HIF2 reveal its ability to bind small synthetic ligands and X-ray crystallography of the bHLH-PAS A-PAS B regions of the CLOCK-BMAL1 dimer show a CLOCK cavity which could potentially be targeted by small molecules (Scheuermann, Tomchick et al.

2009; Huang, Chelliah et al. 2012). While ligands or stimuli that directly act on the PAS domains have not been described for most bHLH-PAS proteins there is generally a signal regulated role for most PAS containing proteins.

Across all phyla, the PAS domain is frequently used as a protein-protein interaction interface and ligand binding domain and forms a conserved structural module despite poor primary sequence homology (Taylor and Zhulin 1999; Moglich, Ayers et al. 2009). Conformity of the

PAS domain structure has been elucidated over the last decade, mostly from crystallisation of

bacterial PAS containing proteins. The overall PAS fold and structure comprises five central stranded anti-parallel -sheets (denoted A, B, G, H and I) which are flanked by alpha helices (denoted C, D, E, and F) and connected by flexible linkers ( Figure 1). This forms the PAS core or PAS fold which presents an ideal pocket for binding natural or synthetic ligands (exploited by antagonist discovery for HIF2(Scheuermann, Tomchick et al. 2009)).

When dual PAS repeats are present they are usually separated by a variable, unstructured linker domain. Initially overlaying the structures from the Light, Oxygen and Voltages (LOV)

PAS domain containing sensors PYP, FixL and HERG demonstrated the conservation of the overall PAS fold structure. As additional structures have been solved the overall conservation of the PAS fold has held true even between kingdoms of life. However, the notion of signal regulated domains and specificity of protein-protein interactions between PAS domains or PAS domain contain proteins suggests a significant degree of conformation flexibility or surface specific interactions. Recently the concept of surface specific interactions which confer specificity has been described for the ARNT/AhR transcriptional complex in a screen for PAS A domain residues critical for dimerisation (Hao, Whitelaw et al. 2011). Furthermore, recent structures of the bHLH-PAS (PAS A and PAS B) of CLOCK and BMAL1 support the notion of conserved PAS fold structure but divergent conformation. For example, while PAS A domains of CLOCK and BMAL interact through reciprocal -helical and -sheet interactions, which is also observed for NifL and Per/CLOCK PAS interactions, the CLOCK/BMAL1 PAS B domain interactions fundamentally differ from those seen in HIF2/ARNT PASB interactions (Huang,

Chelliah et al. 2012).

Figure 1 | Roles and structures of class I and class II bHLH–PAS family members. a | Examples of heterodimeric basic

HLH (helix-loop-helix)–PER–ARNT–SIM (bHLH–PAS) transcription factors are shown. Dimers are formed between a class I factor and a class II factor (AHR nuclear translocator (ARNT), ARNT2, brain muscle ARNT-like 1 (BMAL)). A class I factor may be tissue-restricted in expression (such as single-minded homologue (SIM) and neuronal PAS domain-containing protein

(NPAS)) or active in response to a stimulus (such as a ligand for aryl hydrocarbon receptor (AHR) and hypoxia for hypoxia- inducible factor (HIF)). The heterodimers bind to class I-specific variations of the canonical E-box sequence and regulate specific target genes, thereby mediating various developmental or homeostatic processes and/or responses to environmental or physiological stresses. Class II factors are capable of forming dimers with more than one class I factor, although some combinations may be limited in vivo; for example, ARNT2 expression is mostly restricted to neurons, and it functions as the obligate partner in vivo for SIM1 and probably for NPAS4. HIF1 can form active dimers with either ARNT or ARNT2 (REF. 37), but the HIF–ARNT dimer is more abundant overall owing to both proteins being ubiquitously expressed. Conversely, ligand-bound AHR can dimerize with either ARNT or ARNT2 in vitro, but only AHR–ARNT dimers can activate target genes171. b | The HLH and PAS folds constitute the interface of dimerization between class I and class II bHLH–PAS factors. Comparison of the limited available structural information indicates that the tertiary structure of the

PAS fold is well conserved. However, this does not preclude different orientations and surfaces from being involved in

PAS–PAS interactions throughout the family. The PASB regions of BMAL1 and circadian locomotor output cycles kaput

(CLOCK) associate in roughly parallel orientations, whereas isolated PASB folds of HIF2 and ARNT show antiparallel orientation and a -sheet interface8 65. These different interfaces may contribute to the specificity of binding and partner selection, and they present different surfaces for specific interactions with accessory signalling proteins or transcription co-regulators. The period proteins, which lack a bHLH, show promiscuous binding with other family members and, similar to ARNT, can homodimerize172. Nuclear receptor co-activator 1 (NCOA1) is one of a small class of co-activator proteins that contain typical PAS folds but that do not seem to function as obligate heterodimers. Structures shown: HIF2 PASB, ARNT

PASB (Protein Data Bank (PDB) ID: 4GHI); CLOCK PASB, BMAL1 PASB (PDB ID: 4F3L); PER2 PASB (PDB ID: 3GDI); NCOA1

PASB (PDB ID: 1OJ5). ARC, activity-regulated cytoskeleton-associated protein; BDNF, brain-derived neurotrophic factor;

CME, central midline element; CRY, cryptochrome; CYP1A1, cytochrome P4501A1; EGR1, early growth response 1; EPO, erythropoietin; GLUT1, glucose transporter 1; GST, glutathione S-transferase; HRE, hypoxia response element; PER, period circadian protein homologue; ROR, retinoic acid-related orphan receptor; VEGF, vascular endothelial growth factor;

XRE, xenobiotic response element.

Reproduced from Paper I Bersten et al, Nat Rev Cancer. 2013.

bHLH-PAS dimerization, DNA binding and signal transduction mechanisms

The bHLH-PAS class of proteins can be generally separated into transcription factors or coactivator proteins which contain a shared domain structure (Kewley, Whitelaw et al. 2004;

Partch and Gardner 2010). bHLH-PAS proteins are characterised by an invariant N-terminal bHLH domain which is involved in primary dimerisation and DNA binding (Reisz-Porszasz,

Probst et al. 1994; Fukunaga, Probst et al. 1995) followed by a PAS domain which encompasses two PAS repeats denoted PAS A and PAS B, and a PAC domain (Ponting and

Aravind 1997) or S2 boxes (Zhulin, Taylor et al. 1997) which are structurally associated with the

PAS domain (Hefti, Francoijs et al. 2004). The bHLH-PAS transcription factors are further divided into class I or class II factors, to form functional transcriptional complexes (Figure 1).

Class I factors (AhR, HIF1-3, Sim1-2, NPAS1-4, and CLOCK) must heterodimerise with class II factors (ARNT, ARNT2, BMAL1, BMAL2) to enable DNA binding and transcriptional activation.

Unlike the bHLH and bHLH/Zip transcription factors which bind the classic CANNTG E-box, the bHLH-PAS transcription factors utilize an atypical E-box which has variable 5’ sequence

(Massari and Murre 2000; Kewley, Whitelaw et al. 2004). It has been shown in vitro and in cultured cells that class II factors such as ARNT can homodimerise to activate canonical E-box elements, although the biological significance of this has not been addressed (Sogawa, Nakano et al. 1995; Swanson and Yang 1999).

The PAS A domain of bHLH-PAS transcription factors also contributes to dimerisation strength

(Erbel, Card et al. 2003; Chapman-Smith, Lutwyche et al. 2004) as wells as mediating dimerisation specificity between bHLH-PAS family members (Pongratz, Antonsson et al. 1998).

Interestingly, the PAS A domain has also been implicated in direct DNA contact and DNA bending, downstream of the primary bHLH-major groove contact (Chapman-Smith and

Whitelaw 2006). While PAS B also provides a dimerisation interface between bHLH-PAS

proteins (Card, Erbel et al. 2005; Huang, Chelliah et al. 2012), in selected cases it also directly binds ligands (Whitelaw, Pongratz et al. 1993; Scheuermann, Tomchick et al. 2009), coactivator proteins (Partch, Card et al. 2009; Partch and Gardner 2010; Partch and Gardner

2011) and chaperone proteins to control signal transduction (Pongratz, Mason et al. 1992;

Coumailleau, Poellinger et al. 1995).

Importantly, the PAS domains can also contribute to target gene specificity, for example, in

Drosophila the bHLH/PAS members Trachealess (Trh) and Single Minded (SIM) are able to bind to the same DNA recognition sequence in collaboration with the obligate bHLH-PAS partner factor, Tango (Tgo; equivalent to Aryl Hydrocarbon Nuclear Translocator (ARNT) in mammals).

While the DNA response elements of these two bHLH/PAS heterodimers are identical, target gene induction can be switched by swapping the PAS domains between Trh and SIM, suggesting that in this case target gene specificity is conferred by the PAS domain (Zelzer,

Wappner et al. 1997). How they achieve this is unclear, but may involve ancillary factors recruited by the PAS domains or direct contact between the PAS domain and DNA elements adjacent to the primary bHLH contact (Kimura, Weisz et al. 2001; Pearson, Watson et al. 2012).

This is somewhat supported by the ability of some bHLH-PAS transcription factors to bind to the same DNA binding motifs yet have distinct target gene profiles or the capacity to bind to several variants of the asymmetric E-box core (Swanson, Chan et al. 1995; Woods and

Whitelaw 2002; Ooe, Saito et al. 2004; Jiang and Crews 2007; Farrall and Whitelaw 2009;

Bersten, Sullivan et al. 2013).

The DNA binding specificity of bHLH-PAS transcription factors is conferred by the basic region within the bHLH (Davis and Weintraub 1992). As with other bHLH containing transcription factors, basic residues make direct contact with nucleotides to guide DNA binding specificity

(Ma, Rould et al. 1994). bHLH-PAS transcription factors utilising ARNT or ARNT2 bind to a asymmetric E-Box like element where Class I and Class II factors contribute to three nucleotide

half site specificity. For example AhR/ARNT heterodimers bind to a Xenoboitic Response

Element (XRE) (TNGCGTG) where the AhR binds to NGC half site and ARNT binds to GTG half site. In this context the ARNT DNA binding specificity is fixed to GTG whereas the class I partner protein sequence specificity can vary. Although ARNT2 can also bind to many of the class I factors the DNA binding specificity appears to be the same as for ARNT. This is in contrast to various heterodimers formed with either BMAL1 or BMAL2, or BMAL homodimers or ARNT homodimers, all of which bind to canonical E-Box CACGTG (Hogenesch, Gu et al.

1998; Reppert and Weaver 2002).

Many of the class I bHLH-PAS transcription factors dimerise with a common class II factor, which can create competition for that Class II factor within the cell (Chan, Yao et al. 1999;

Woods and Whitelaw 2002). In addition, overlapping DNA binding specificities can allow for further competition between heterodimeric complexes (Woods and Whitelaw 2002; Farrall and Whitelaw 2009). Transcriptionally inactive or DNA binding defective forms of Class I bHLH-

PAS transcription factors can also compete for Class II factors to regulate the output transcriptional pathways (Mimura, Ema et al. 1999; Makino, Cao et al. 2001; Makino, Kanopka et al. 2002). The extent to which cross talk occurs in vivo or in pathological scenarios is yet to be explored, however it is expected that ARNT levels will be limiting, supporting the concept of competition for ARNT by class I bHLH-PAS factors (Semenza, Jiang et al. 1996; Holmes and

Pollenz 1997).

While the bHLH-PAS transcription factors primarily act to modulate gene expression by direct

DNA binding, alternate functions for the bHLH-PAS transcription factors have also been described. The most notable are the direct control of hypoxic induced translation by HIF2 by incorporation into the translational machinery (Uniacke, Holterman et al. 2012) and the regulation of ubiquitin ligase protein degradation mediated by ligand activated AhR (Ohtake,

Baba et al. 2007; Kawajiri, Kobayashi et al. 2009).

bHLH-PAS transcription factor function

bHLH/PAS proteins are involved in a diverse array of physiological and pathological processes, such as the cellular response to hypoxia (Hypoxia Inducible Factors

(HIF1/HIF2/HIF3IPAS)), the maintenance of circadian rhythms (circadian locomotor output cycles kaput (CLOCK), Period (PER1/PER2/PER3),Neuronal PAS domain protein 2

(NPAS2)), the response to environmental pollutants (Aryl hydrocarbon Receptor (AhR)/ Dioxin receptor (DR), AhR repressor AhRR), neurogenesis and lung development (NPAS1, and NPAS3) and appetite control (Single minded 1 (Sim1))(Reppert and Weaver 2002; Kewley, Whitelaw et al. 2004; Semenza 2012).

In addition to canonical roles in normal physiology, emerging roles in T cell immunity, cancer progression and metastasis, diabetes, and behaviour for bHLH-PAS transcription factors are being discovered (Gunton, Kulkarni et al. 2005; Marcheva, Ramsey et al. 2010; Dang, Barbi et al. 2011; Semenza 2012; Bersten, Sullivan et al. 2013; Hao and Whitelaw 2013). Normal and cancer related functions of the bHLH-PAS transcription factors was reviewed recently in Paper

I (Bersten, Sullivan et al. 2013).

Neuronal PAS Factor 4

The mammalian NPAS4 gene (also known as Limbic enhanced-PAS (lePAS and NXF), was discovered from screening a human fetal brain cDNA library with a PAS domain oligonuceotide probe (Ooe et al, 2004). Human, rat and mouse NPAS4 were cloned and have significant degree of homology in the bHLH and PAS domains to each other and to drosophila gene

Dysfusion (Dys), Zebrafish (Danio rerio) NPAS4 and the Caenorhabditis Elegans (C.Elegans) gene C15C8.2 (Cky-1) (Jiang and Crews 2003; Ooe, Saito et al. 2004; Ooe, Saito et al. 2007;

Ooe, Saito et al. 2009; Baxendale, Holdsworth et al. 2012). While NPAS4 is clustered in the bHLH-PAS transcription factor family phylogenetic analysis of mouse PAS domain containing

proteins suggests it has little similarity to any other bHLH-PAS factors (Ooe, Saito et al. 2004;

McIntosh, Hogenesch et al. 2010). This is also observed in C.elegans and drosophila (Jiang and

Crews 2003; Ooe, Saito et al. 2007). Within the bHLH and PAS A domain NPAS4 is most similar to AhR/SIM2/NPAS1 (43%, 33% and 33% amino acid identity respectively) and within in the

PAS B domain is most similar to HIF1/PER/BMAL1 (29%/27%/24% amino acid identity).

While the C-terminal region of the mammalian NPAS4 members is highly conserved, there is little or no sequence homology to either C.elegans (Cky-1), Drosphila (Dys), or any of the mammalian bHLH-PAS C-termini.

Drosophila Dysfusion

Drosophila SIM and trachealess (Trh) control transcription and development in the CNS midline cells and tracheal cells, respectively, and both appear to do so through a common DNA regulatory element known as the Central Midline Element (CME; ACGTG)(Crews 1998). The

NPAS4 related gene Dys was discovered by screening for bHLH containing proteins in drosophila (Ledent and Vervoort 2001; Peyrefitte, Kahn et al. 2001). Expression of Dys was subsequently shown in embryonic tissues such as foregut precursors, CNS cells which are either part of the medial brain or frontal ganglion, tracheal fusion cells, the epidermal leading edge, the hind gut and the anal pad from stage 11-12 of development (Peyrefitte, Kahn et al.

2001; Jiang and Crews 2003). Dys is expressed prior to tracheal fusion events and RNAi knockdown or loss of function (LoF) Dys mutants results in aberrant tracheal fusion (Jiang and

Crews 2003; Jiang and Crews 2006). Development of the tracheal system is critical to the complete formation of the oxygen delivering tubular network (Ghabrial, Luschnig et al. 2003) and as such, inhibition of tracheal fusion by loss of Dys results in a lethal phenotype (Jiang and

Crews, 2003). The tracheal fusion cells where Dys is expressed extend actin-rich filopodia, which are guided by cues in a similar fashion to that of axon growth cone guidance (Tanaka-

Matakatsu, Uemura et al. 1996; Englund, Steneberg et al. 2002; Ribeiro, Ebner et al. 2002).

Tracheal branches undergo systematic migration from adjacent hemi-segments until they are in close enough contact for the fusion cells to adhere to each other. Over expression of Dys throughout the trachea results in inhibition of migration and ectopic branch fusion, implicating

Dys in the migration and fusion of actin-rich filopodia of the Drosophila tracheal system (Jiang and Crews 2006). Like other members of the Drosophila bHLH-PAS family, Dys appears to heterodimerise with Tgo to regulate target genes, in this case shotgun, CG13196, and members only, all of which contribute to cell adhesion or other aspects of tracheal fusion or migration (Wilk, Weizman et al. 1996; Jiang and Crews 2003; Jiang and Crews 2006). However, due to the low degree of sequence similarity between NPAS4 and Dys, especially in the C- terminus, and the absence of any reported expression of NPAS4 in the lung, NPAS4 may have diverged in function in mammals.

NPAS4 Expression and Regulation

NPAS4 expression is highly restricted to the brain in rodents and humans, with a several reports of weak expression within the testes (Flood, Moyer et al. 2004; Moser, Knoth et al.

2004; Ooe, Saito et al. 2004; Ramamoorthi, Fropf et al. 2011). Within the CNS, NPAS4 expression appears to be further restricted to neurons within the hippocampus, cortex, olfactory bulb, and amygdala with very low or no expression seen in other brain areas or the spinal cord (Moser, Knoth et al. 2004; Lin, Bloodgood et al. 2008; Ramamoorthi, Fropf et al.

2011). Unlike NPAS3 or Sim2, NPAS4 does not appear to be expressed in the developing mouse embryo. Appreciable levels begin to appear perinatally and increase as SIM2 gradually decreases up to 4 weeks post natal (Brunskill, Witte et al. 1999; Ooe, Saito et al. 2004).

A common theme among bHLH-PAS transcription factors is signal or stress responsiveness, for example with HIF activated in hypoxia, AhR responsive to xenobiotics and SIM1 responsive to appetite signals. The NPAS4 gene is also a signal or stress responsive, as expression has been shown to be upregulated by seizure (Flood, Moyer et al. 2004; Baxendale, Holdsworth et al.

2012) ischemia (Shamloo, Soriano et al. 2006; Leong, Klaric et al. 2013), several psychostimulants and opioid drugs (Piechota, Korostynski et al. 2010; Guo, Xue et al. 2012;

Martin, Jayanthi et al. 2012). In addition, physiological stimuli such as contextual fear conditioning (CFC) (Ploski, Monsey et al. 2011; Ramamoorthi, Fropf et al. 2011), light stimulation (Lin, Bloodgood et al. 2008; Maya-Vetencourt, Tiraboschi et al. 2012) or neuronal depolarisation (Lin, Bloodgood et al. 2008; Ramamoorthi, Fropf et al. 2011) induce expression of NPAS4.

At a mechanistic level these stimuli are thought to act primarily through stimulation of voltage gated calcium channels which initiates, calcium dependent gene expression. NPAS4 induction can be blocked by calcium chelation, calcium channel blockade, NMDA receptor blockade or

AMPA receptor blockade but does not appear to be upregulated by neurotrophin signals (Lin,

Bloodgood et al. 2008; Ramamoorthi, Fropf et al. 2011). Neuronal depolarisation results in an extremely rapid (within 5 mins for CFC), robust and transient increase in NPAS4 mRNA and protein through activation of VDCC channels in a protein synthesis independent manner

(Ramamoorthi, Fropf et al. 2011). NPAS4 induction following depolarisation peaks at 0.5-2 hrs post depolarisation and is rapidly down regulated to near basal levels by 4-6hrs (Lin,

Bloodgood et al. 2008; Ramamoorthi, Fropf et al. 2011).

Unlike other genes transiently stimulated by neuronal activity, NPAS4 protein is not activated by neurotrophic factors, growth factors, or forskolin (a protein kinase A activator) making it uniquely situated to respond only to learning and memory cues (Lin, Bloodgood et al. 2008;

Ramamoorthi, Fropf et al. 2011; Ebert and Greenberg 2013). While the molecular mechanisms underlying both the restricted brain and activity-dependent expression of NPAS4 remain unexplored, it is clear that the latter requires calcium dependent signalling events. Activation of NPAS4 expression, which has been suggested to be mediated by calmodulin dependent kinase IV (CaMK IV) and cAMP-response element binding protein (CREB), can be blocked by

inhibition of nuclear calcium signalling (Greer and Greenberg 2008; Zhang, Zou et al. 2009). In addition, it has also been suggested that PI3K and ERK/MAPK may also play a role in upregulation of NPAS4 following NMDA receptor activation (Coba, Valor et al. 2008; Ooe,

Kobayashi et al. 2009). Activity dependent up regulation of NPAS4 is initially protein synthesis independent and may involve other activity induced transcription factors such as CREB and

MEF2A/D (Flavell, Kim et al. 2008; Ramamoorthi, Fropf et al. 2011; Ebert and Greenberg

2013). Following the initial up regulation of NPAS4 following depolarisation, it’s proposed that

NPAS4 may strengthen its own expression through a feed forward pathway. Indeed, NPAS4 has been shown to bind and activate its own promoter, however the ramifications of this are unknown (Ooe, Saito et al. 2004; Lin, Bloodgood et al. 2008; Kim, Hemberg et al. 2010). While there is cursory information about stimulus dependent activation, developmental regulation of NPAS4 expression has not been investigated. Furthermore, it is not clear how NPAS4 protein and mRNA are down regulated following initial induction.

In other contexts an increase of intracellular calcium through endoplasmic reticulum stressors can also induce NPAS4 expression (Ooe, Motonaga et al. 2009). Recently it has been shown that pancreatic beta cells are capable of depolarisation induced NPAS4 expression (as demonstrated by qRT-PCR and immunofluorescence experiments), indicating that NPAS4 may act more broadly outside the nervous system as a calcium induced transcription factor in a limited number of tissues (Sabatini, Krentz et al. 2013). However, the pervasiveness or consequences of NPAS4 upregulation outside the nervous system or in response to ER stress or calcium induction have yet to be fully elucidated.

Very little is known about the mechanisms which regulate NPAS4 expression, however recently it has been shown that the NPAS4 promoter is methylated at several CpG sites and mRNA expression may be repressed by this methylation (Rudenko, Dawlaty et al. 2013). Ten- eleven translocation 1 (Tet1) is a dioxygenase enzyme which catalyses the addition of a

hydroxy group onto methylated CpGs to mediate demethylation (Wu and Zhang 2011). It has been proposed that demethylation may be important for regulation of activity inducible genes and indeed knockout of Tet1 in mice leads to hypermethylated and suppressed NPAS4 expression in the hippocampus and cortex (Kaas, Zhong et al. 2013; Rudenko, Dawlaty et al.

2013). Interestingly, all bHLH-PAS transcription factor binding sites contain a CpG dinucleotide between the Class I and Class II half sites which could potentially be methylated, however the effect of non-methyl versus methyl-CpG bHLH-PAS response elements on binding of heterodimers has not been explored.

NPAS4–dependent Transcriptional Regulation

NPAS4 is able to heterodimerise with the partner proteins ARNT and ARNT2 to bind to many bHLH-PAS regulatory sequences by in vitro DNA binding assays and luciferase reporter experiments (Ooe, Saito et al. 2004). Due to overlapping expression between NPAS4 and

ARNT2 within the CNS, and the suggestion that NPAS4 may interact more strongly with ARNT2 than ARNT, NPAS4 is anticipated to function in vivo with ARNT2 (Hirose, Morita et al. 1996;

Aitola and Pelto-Huikko 2003; Ooe, Saito et al. 2004; Ooe, Saito et al. 2009). bHLH-PAS regulatory elements which NPAS4/ARNT2 have been shown to bind contain the core CME /

HRE (ACGTG) or the XRE (TNGCGTG) (Moser, Knoth et al. 2004; Ooe, Saito et al. 2004), however the preferred DNA binding sequence of NPAS4/ARNT2 heterodimers are unknown.

Comparison of activities of NPAS4 on bHLH-PAS responsive reporters and in vitro DNA binding suggests that NPAS4 binds most strongly to TCGTG and GCGTG containing sites (Ooe, Saito et al. 2004). Analysis of NPAS4 Chromatin immunoprecipitation -sequencing (ChIP-Seq) experiments in depolarised mouse cortical neurons, together with in vitro site selection experiments to identify the preferred NPAS4/ARNT DNA binding motif, also supports this notion (Kim, Hemberg et al. 2010)( D.C. Bersten, V. Bhakti, and M.L. Whitelaw unpublished results).

Initial functional experiments suggested that NPAS4 may control expression of the cytoskeletal binding protein Drebrin and the apoptosis inducing gene Bax (Ooe, Saito et al. 2004; Hester,

McKee et al. 2007). However, examination of NPAS4 dependent target genes during neuronal depolarisation suggest that these maybe either context dependent or artefacts of overexpression in cell lines (Lin, Bloodgood et al. 2008; Kim, Hemberg et al. 2010; Pruunsild,

Sepp et al. 2011; Ramamoorthi, Fropf et al. 2011). ChIP-seq and microarray analysis of NPAS4 bound and differentially regulated target genes following KCl-depolarisation has been performed and reveals extensive occupancy of NPAS4 at promoters and enhancers to regulate many activity dependent genes involved in inhibitory synapse development (Lin, Bloodgood et al. 2008; Kim, Hemberg et al. 2010; Ramamoorthi, Fropf et al. 2011; Bloodgood, Sharma et al.

2013). NPAS4 occupancy at enhancer sites correlates well with CBP occupancy and supports existing data suggesting CBP/p300 acts as an NPAS4 transactivator (Ooe, Saito et al. 2004; Kim,

Hemberg et al. 2010). A number of confirmed NPAS4 target genes have been examined including BDNF, c-Fos, Egr1 and Arc (Lin, Bloodgood et al. 2008; Kim, Hemberg et al. 2010;

Pruunsild, Sepp et al. 2011; Ramamoorthi, Fropf et al. 2011). It is proposed that NPAS4 predominantly acts through enhancer recruitment of RNA Pol II to activate transcription of its target genes (Kim, Hemberg et al. 2010; Ramamoorthi, Fropf et al. 2011). NPAS4 binds directly to BDNF promoter I, promoter III and promoter IV to act as one of the main drivers of activity induced expression of BDNF (Lin, Bloodgood et al. 2008; Pruunsild, Sepp et al. 2011;

Bloodgood, Sharma et al. 2013). Deletion of promoter IV NPAS4 response element significantly weakens activity induced expression of BDNF although it does not alter the initial responsiveness of BDNF to depolarisation (Pruunsild, Sepp et al. 2011). It has therefore been proposed that NPAS4 acts to enhance or strengthen activity induced gene expression

(Pruunsild, Sepp et al. 2011; Ramamoorthi, Fropf et al. 2011). In addition, it has been suggested by several groups that NPAS4 may act directly on its own promoter to strengthen its activation (Ooe, Saito et al. 2004; Lin, Bloodgood et al. 2008). Importantly, ARNT2 has also

been shown to bind to BDNF promoters and regulate BDNF expression, although the extent of its contribution to activity dependent or NPAS4 dependent gene expression remains unexplored (Pruunsild, Sepp et al. 2011).

NPAS4 Function Activity-Dependent Transcription in the CNS

Neuronal activity plays key roles in neuron survival, proliferation, migration, axon/dendrite guidance and growth, synapse formation, maintenance and elimination, and neurotransmitter specification (Spitzer 2006; Greer and Greenberg 2008; Spitzer 2012). These processes guide neuronal activity profiles in the prospective central nervous system and underlie the cellular and molecular basis of memory and learning. In addition, genetic aberrations in neuronal activity are thought to be critical to many neuropsychiatric disease states and may be the underlying cause of autism and schizophrenia (Ramocki and Zoghbi 2008; Ebert and Greenberg

2013). Importantly, over excitation due to disruption of neuron homeostasis may also underlie the pathogenesis of many neurodegenerative disorders such as dementia,

Alzheimer’s disease, stroke and seizure(Saxena and Caroni 2011).

Sensory experience drives synaptic plasticity and learning in animals. One seminal experiment outlining this phenomena showed that ocular occlusion during critical periods around birth, when neuron development and synapse formation occur, leads to cortical circuit rearrangements to favour the non-occluded eye (Wiesel 1982). It is now well established that sensory experience and neuronal activity drive synaptic plasticity during critical periods and throughout adulthood. Electrical activity in neurons can take many forms, of which the best established being activation of voltage-gated calcium channels leading to action potentials

(Greer and Greenberg 2008). During early stages of neuron development GABA and glycine are able to generate depolarising chloride currents due to efflux of intracellular chloride ions

(Spitzer 2006). Later in neuron development this phenomena is reversed and leads to chloride

influx and inhibition of neuronal activity. In addition, glutamate generates depolarisation via activation of voltage gated calcium and sodium channels. Calcium influx through voltage gated calcium channel triggers rapid activation of gene expression, which in turn acts to modulate synapse formation and plasticity. Calcium activated gene expression in neurons is mediated by a number of activity-dependent transcription factors including CREB, MEF2A/D,

USF1/2, NFB, MeCP2, SRF and NPAS4, many of which are regulators of synapse formation and function (Greer and Greenberg 2008).

NPAS4 and synapse formation

Initial experiments in cultured hippocampal neurons showed that NPAS4 was able to regulate the number and strength of inhibitory inputs on excitatory pyramidal neurons both perisomatically and dendritically (Lin, Bloodgood et al. 2008). In contrast, NPAS4 seemed not to have any effect on excitatory synapse number or dendritic patterning of the neurons (Lin,

Bloodgood et al. 2008). More recently, using conditional knockout NPAS4 mice where NPAS has been sparsely ablated in CA1 hippocampal neurons, recording of inhibitory post synaptic potentials in layers containing either dendrites or soma has revealed that NPAS4 has opposing actions on inhibitory synapse formation in the two compartments, increasing inhibition within the soma and decreasing inhibition in dendrites (Bloodgood, Sharma et al. 2013). The function of compartment specific regulation of inhibitory synapses is as yet unclear, however, somatic inhibition may be more effective at limiting action potential spikes than dendritic inhibition, while increased activity in dendrites may aid more effective dendritic synapse plasticity

(Sylwestrak and Scheiffele 2013). The downstream mechanism(s) by which NPAS4 imparts compartment specific neuron inhibition is also unclear. It has been shown that specific transcripts of BDNF are regulated by NPAS4 binding and activation at promoter I, III and IV

(Lin, Bloodgood et al. 2008; Pruunsild, Sepp et al. 2011; Ramamoorthi, Fropf et al. 2011;

Bloodgood, Sharma et al. 2013). While BDNF has been shown to be involved in inhibitory

synapse formation (Marty, Wehrle et al. 2000; Lin, Bloodgood et al. 2008) and some transcripts can be can be targeted to different cellular compartments (Pattabiraman, Tropea et al. 2005; An, Gharami et al. 2008; Dean, Liu et al. 2012), the contribution of NPAS4 to regulating subcellular levels of BDNF through transcript specific regulation is yet to be addressed.

NPAS4 in memory, learning and behaviour.

The selective and transient induction of NPAS4 following sensory experience and neuron depolarisation prompted several groups to investigate the function NPAS4 in memory and learning in mice (Ploski, Monsey et al. 2011; Ramamoorthi, Fropf et al. 2011; Coutellier, Beraki et al. 2012; Maya-Vetencourt, Tiraboschi et al. 2012). In independent mouse knockout models

NPAS4 has been shown to be involved in contextual memory formation in adult mice. Either global or conditional knockout of NPAS4 in the CA3 region of the hippocampus attenuated long-term memory formation in mice, which is dependent on NPAS4 transcriptional activity

(Ramamoorthi, Fropf et al. 2011; Coutellier, Beraki et al. 2012). However, short-term memory was affected in only in the global NPAS4 knockout mice (Ramamoorthi, Fropf et al. 2011).

Additional behavioural deficits such as increased aggression and hyperactivity are observed in the NPAS4 null mice and decreased contact with unfamiliar mice are observed in animals heterozygous null for NPAS4 (Coutellier, Beraki et al. 2012). Both heterozygous and homozygous null NPAS4 mice also have defects in sensorimotor gating which may reflect impaired cognitive processing of sensory information (Coutellier, Beraki et al. 2012). Taken together this outlines the importance of NPAS4 in sensory induced memory, learning and cognitive abilities which results in behavioural and social defects in NPAS4 null mice.

NPAS4 in Neuroprotection

NPAS4 null mice have been shown to have much reduced life span, with only 20-30% of mice surviving to 16 months (Ooe, Motonaga et al. 2009). This reduction in life span was associated with a marked increase in age related neurodegeneration in knockout animals in addition to increased susceptibility of NPAS4 null mice to glutamate induced neurotoxicity (Ooe,

Motonaga et al. 2009). It is therefore hypothesised that induction of NPAS4 expression may confer neuroprotection during hyperexcitation or ischemia. This is supported by experiments where overexpression of NPAS4 (Hester, McKee et al. 2007) increased cell survival or knockdown decreased cell survival in post-mitotic neurons induced to undergo apoptosis

(Zhang, Zou et al. 2009). While priming post-mitotic neurons with elevated NPAS4 by overexpression or increased neuron depolarisation may provide neuroprotection, sustained overexpression in cultured cell lines appears to induce apoptosis (Hester, McKee et al. 2007).

Taken together, this suggests that moderate transient upregulation of NPAS4 maybe protective while sustained high level NPAS4 expression maybe detrimental to cell survival.

Project Rationale

NPAS4 expression is mostly restricted to the brain and exhibits transient increases to dampen neuronal activity by upregulating inhibitory synapse number and strength on excitatory neurons. This homeostatic regulation of inhibitory synapses implicates NPAS4 in various neuropsychiatric diseases including autism as well as neurodegenerative diseases in which excitotoxicity plays a role in the aetiology of the disease. While these are of key interest in the field, the mechanisms of regulation of NPAS4 gene expression, the potential contribution to human disease and effective modelling of behaviour in mice are yet to be explored. These aspects were therefore the focus of the research in this thesis, with the aims of;

1. Exploring the molecular mechanisms which control and restrict NPAS4 expression to

the brain

2. Investigating the contribution of human single nucleotide variants to the function of

the NPAS4/ARNT2 transcriptional dimer

3. Generating technological platforms to effectively model NPAS4 function in vitro and in

mice

Chapter 2: Results

2.1 Elucidation of molecular mechanisms, which restrict NPAS4 expression to the brain.

NPAS4 is an activity dependent bHLH-PAS transcription factor that controls the expression of genes involved in inhibitory synapse formation and maintenance. NPAS4 expression is predominantly restricted to cortical and hippocampal regions of the brain and is highly induced following neuronal depolarisation, paradigms of learning, seizure or ischemia. The molecular mechanisms underlying restricted expression pattern were addressed in paper II, entitled “ Regulation of the neuronal transcription factor NPAS4 by REST and microRNAs”.

For the study in paper II we generated antibodies against NPAS4 protein and confirmed both activity-induced expression in primary cultured neurons and restriction of mRNA expression to predominantly cortical/hippocampal brain regions. Paper II identified conserved binding sites for the transcriptional repressor RE-1 silencing transcription factor (REST) within the promoter and Intron I of the NPAS4 gene. We found REST to be strongly bound to these sites to repress

NPAS4 expression in non-neuronal and undifferentiated cells. Deletion of these REST binding sites derepressed NPAS4 expression in embryonic stem cells, and reporter gene experiments in HEK293T cells showed that both intron I and promoter RE-1 sites are important for repression. We also showed that REST binding correlates with CTCF occupancy, known to induce DNA looping and mediate insulator function, this suggesting that CTCF may aid silencing of NPAS4.

In addition, we also showed that the highly conserved 3’UTR of NPAS4 might also play a role in regulating NPAS4 expression. We established that NPAS4 can be repressed by miR-224 and

miR-203 and went on to show that miR-224 is enriched with the hypothalamic/midbrain regions; being expressed from an intron of the GABA A receptor epsilon gene along with miR-

452. We propose that miR-224 may fine tune NPAS4 expression to restrict expression to cortical regions of the brain. The work describes the first mechanistic explanation of the brain- restricted expression of NPAS4.

2.2 Human variants in the NPAS4/ARNT2 transcriptional complex can disrupt function

Neuropsychiatric disease has been suggested to derive, in part, from defects which disrupt neuronal excitation/inhibition balance (Rubenstein and Merzenich 2003; Kehrer, Maziashvili et al. 2008; Rubenstein 2010; Yizhar, Fenno et al. 2011). Loss of function mutations in synapse effectors also suggests synapse dysfunction may play a key role in disrupting this homeostatic balance in these diseases (Ramocki and Zoghbi 2008; Sudhof 2008; Ebert and Greenberg

2013). In addition, glutamatergic hyperexcitation, which may also arise from disruption of the excitatory/inhibitory balance or other neuronal stressors, may promote the progression of neurodegenerative disorders (Bezprozvanny and Mattson 2008; Saxena and Caroni 2011).

Given the links between NPAS4 function and neurological disease like phenotypes in mice, we sought to investigate whether non-synonymous single nucleotide variants found within human

NPAS4, or its heterodimeric partner protein ARNT2, may disrupt function of the dimer. If so, we reasoned that a rationale for exome sequencing of NPAS4 in neurological disease affected patient cohorts might emerge.

In paper III, “Human Variants in the Neuronal Basic Helix-Loop-Helix/Per-Arnt-Sim (bHLH/PAS)

Transcription Factor Complex NPAS4/ARNT2 Disrupt Function” we found that several variants in NPAS4 or ARNT2 were able to reduce or ablate a bHLH-PAS responsive luciferase reporter gene. We also showed that one variant, NPAS4.F147S, failed to upregulate endogenous BDNF expression due to disruption of dimerisation with ARNT2. We also showed that an ARNT2

variant, ARNT2.R46W, had reduced activity due to disrupted nuclear localisation. Structural modelling of the bHLH-PAS regions of the NPAS4/ARNT2 heterodimer, based on the related

CLOCK/BMAL structure (Huang, Chelliah et al. 2012), indicated that the conserved F147 residue lies at the PAS A dimer interface and may directly participate in heterodimer formation. This is also supported by the finding that mutations which weaken AhR-ARNT dimerisation lie within this region (Hao, Whitelaw et al. 2011). Paper III shows the potential for de novo discovery of disease relevant variants by selecting polymorphisms from databases for screening in function based assays.

2.3 Generation and validation of Inducible and reversible systems for lentiviral and ES cell (Col1a1-RMCE) mediated manipulation of NPAS4 levels.

Inducible and reversible systems for modulating gene expression are critical for investigating biological pathways. These systems are of particular importance when studying genes critical for essential cell processes. For example, removal or knockdown of genes critical for cell survival or proliferation results in cell death, making generation of animal models and cell lines redundant. In addition, inducible and reversible manipulation of gene expression is of particular interest in neuroscience, immunology, developmental and cancer biology disciplines where induction and reversal of genes proposed to underpin a phenotype would be advantageous for studying disease progression or biological processes.

Several tools for inducible and reversible control of gene expression have been developed in the past and have been used with limited success. The best studied example being the tetracycline inducible system combined with either shRNA-mediated knockdown or cDNA mediated ectopic or over expression. The use of the tetracycline inducible system has been hampered for several technical reasons. Initial systems employed tetracycline repressor TetR variants, which repressed transcription from a constitutive promoter containing tet operator

elements, but these systems were generally very leaky in cell lines and animals. While the use of Tet activator variants and modified tet operator elements reduced background expression in the absence of doxycycline, most of the variants still have poor sensitivity to Dox, which is a limitation for in vivo experiments, eg in tissues where DOX transport is poor (i.e. brain). Most currently used systems still contain significant background expression and generally the tet responsive promoter and tet activator are delivered on separate plasmids, leading to mosaic inducible expression in cell lines.

Reliable and reproducible generation of inducible cell lines and mice has been inadequate due to technical limitations such as cell lines being refractory to transfection and, particularly in mice, random integration effects. Furthermore, the use of shRNA systems for use in generating inducible and reversible knockdown in cells and mice relies heavily on efficient selection of potent shRNA sequences, which are able knockdown gene expression in single copy.

In manuscript IV, “Inducible and reversible lentiviral and recombination mediated cassette exchange (RMCE) systems for controlling gene expression”, we attempt to overcome many of the limitations of previous inducible and reversible systems for ectopic expression and knockdown of gene expression. We design and test single inducible and reversible lentiviral constructs for reliable generation of cell lines, which utilise Tet activator variants with dramatically improved sensitivity to doxcycline and no detectable background expression.

These constructs allow for uniform inducible and reversible expression in cell lines. In addition, we overcome limitations of reliable targeting into embryonic stem cells for the generation of mice by adapting a Recombination Mediated Cassette Exchange (RMCE) strategy which results in ~100% efficient targeting of single inducible and reversible constructs into the

Col1a1 locus. We also design and test constructs which allow for tissue specific Dox inducible

expression either by Cre mediated gain of Tet activator expression or using tissue specific promoters.

Using published high throughput data which tested the efficiency of ~60,000 shRNA sequences, we built an algorithm to predict shRNA sequences with high knockdown potency.

We demonstrate that this is a potentially useful tool for predicting shRNA targets by selecting and testing sequences for knockdown of ARNT and ARNT2, using the single integration RMCE embryonic stem cells. We find that the majority of predicted shRNA sequences against either

ARNT or ARNT2 are able to efficiently knockdown endogenous protein expression in embryonic stem cells.

Chapter 3: Discussion

NPAS4 has recently been revealed as a critical gene in the regulation and maintenance of neuronal activity in excitatory neurons. NPAS4 protein and mRNA are transiently upregulated by neuronal activity to homeostatically regulate inhibitory synapse inputs, preferentially in the soma of neurons, to dampen neuronal activity. The signal regulated nature of NPAS4 has many similarities with other bHLH-PAS domain containing proteins such as the AhR, which is signal regulated by binding to polyaromatic hydrocarbons, resulting in up regulation of genes involved in cellular detoxification. In response to hypoxic signals, the HIF proteins are stabilised and become transcriptionally active to regulate genes, which respond to low oxygen tension by altering metabolism and promoting increased angiogenesis. Responding to physiological or stress signals to induce genetic programs for protection, adaption or maintaining homeostasis seems to be unifying theme for bHLH-PAS transcription factors.

Tight temporal and spatial control of NPAS4 expression is likely to be critical for the correct specification of inhibitory synapses and the homeostatic activity balance of neurons within the

CNS. Sustained overexpression in cell lines and by pathological inducers such as seizure and ischemia may indeed be detrimental to neuronal cellular function. Therefore the precise mechanisms which control NPAS4 expression are likely to be highly regulated and may have importance in disease states. In paper II we describe a mechanism that restricts NPAS4 expression to the brain by finding the repressor REST silences expression in non-neuronal and undifferentiated tissues (Figure 2.). REST binds strongly to multiple sites within the NPAS4 gene to mediate silencing of NPAS4 expression outside the CNS, implying that repression of

NPAS4 expression may be functionally important during development. Silencing the NPAS4 gene outside the CNS may also be aided by CTCF, which flanks the NPAS4 gene and overlaps

with REST binding sites, perhaps implying that looping may enhance silencing. This possibility remains to be explored. Interestingly, CTCF binding at sites flanking the NPAS4 gene has been reported for brain tissue and CTCF may act to insulate activity inducible expression of NPAS4 from adjacent genes, which are not depolarisation regulated.

We also show that miR-224 may play role in restricting NPAS4 expression to cortical brain regions by targeting the NPAS4 3’UTR (Paper II). Given that NPAS4 can influence the number of inhibitory inputs, miR-224 may allow for an altered activity profile in certain neuronal subtypes and it would therefore be of interest to investigate the in vivo function of loss of miR-

224 in mice. Interestingly, we found that inhibition of both methyltransferases and histone deacetylases in embryonic stem cells leads to an upregulation of NPAS4 expression, which is supported by recent evidence that the NPAS4 promoter is methylated and regulated by the demethylase promoting enzyme Tet1 (Rudenko, Dawlaty et al. 2013). While the mechanism of how Tet1 is regulated in response to neuronal activation in unknown, it has been shown that the methyl-CpG-Binding protein 2 (MeCP2) can be phosphorylated in response to depolarisation, leading to dissociation of MeCP2 from methyl-CpGs and corepressors. This may allow Tet1 access to catalyse demethylation and depression of transcription (Chen, Chang et al. 2003; Ebert, Gabel et al. 2013).

Figure 2. Schematic diagram of the regulation of NPAS4 expression by REST and miR-224. RE-

1 Silencing Transcription factor (REST) represses NPAS4 expression in embryonic stem cells and non-neuronal cells by binding to multiple RE-1 sites in the promoter and intron I of the

NPAS4 gene. In unstimulated primary neurons, REST expression is down regulated derepressing NPAS4 expression. microRNA 224 (miR-224) is expressed in midbrain and hypothalamic brain regions and is able to repress NPAS4 expression, which may restrict NPAS4 expression to cortical and hippocampal neurons. Upon depolarisation of neurons, NPAS4 expression rapidly increases and binds its own promoter, which may further increase its own expression. Reproduced from paper II Bersten et al, Biochim Biophys Acta. 2014.

Significant questions still remain in the regulation of NPAS4 expression. For example, while strong binding of NPAS4 to its own promoter is present following depolarisation and NPAS4 has been shown to activate its own promoter in luciferase reporter gene assays, it is still unclear what involvement NPAS4 has in stimulating its own expression ((Ooe, Saito et al.

2004)Paper II). For example, initial upregulation of NPAS4 mRNA following depolarisation is unhindered by cyclohexamide treatment, implying that other proteins may be responsible for the initial wave of NPAS4 expression (Ramamoorthi, Fropf et al. 2011). In addition, Cre mediated knockout of NPAS4 in neurons does not affect depolarisation mediated NPAS4 promoter driven luciferase induction (Ramamoorthi, Fropf et al. 2011), but induction kinetics have not been investigated in NPAS4 knockout GFP reporter mice . As such there is a critical need to identify proteins, which initiate the transient induction of NPAS4 following neuronal depolarisation. As with the BDNF promoter, NPAS4 may not be responsible for the initial induction of its own expression, but rather strengthening the response after gene induction

(Pruunsild, Sepp et al. 2011). In addition, NPAS4 protein is rapidly down regulated after being

initially induced by depolarisation, peaking in expression at ~2 hrs and almost absent after

6hrs. The mechanism by which this down regulation occurs remains unexplored.

Elucidation of NPAS4 function has revealed a critical homeostatic role in regulating neuronal activity by controlling the balance between excitatory and inhibitory synaptic inputs. Mice lacking NPAS4 have many phenotypic characteristics similar to those seen in both neuropsychiatric diseases such as autism and schizophrenia and in neurodegenerative disease.

Hypotheses for the aetiology of both of these disease states include dysfunction in excitatory/inhibitory balance within the brain, leading to cognitive dysfunction. At least in the case of autism like behaviours, this has been elegantly shown in mice using optogenetic techniques to disrupt excitatory/inhibitory balance in cortical neurons (Yizhar, Fenno et al.

2011). For aforementioned reasons NPAS4 and the regulation of NPAS4 expression may have a role either directly or indirectly in these diseases.

In Paper III we investigate the potential contribution of human non-synonymous single nucleotide variants on the function of the NPAS4/ARNT2 transcriptional complex. We find that several naturally occurring variants have the ability to disrupt NPAS4/ARNT2 transcriptional function and find biochemical mechanisms for the defective complex include either attenuating heterodimer formation or nuclear localisation. The location of variants showing loss of heterodimer formation was consistent with previous loss of dimerisation mutations discovered by bacterial two hybrid screening (Hao, Whitelaw et al. 2011). We also recently published a collaborative work describing loss of function for single nucleotide variants in

SIM1, which were associated with severe obesity (Bonnefond, Raimondo et al. 2013;

Ramachandrappa, Raimondo et al. 2013). The location of loss of function variants described in these manuscripts is consistent with our data in Paper II in that they are commonly clustered

N-terminally in domains critical for heterodimerisation, DNA binding and nuclear localisation.

Many of the variants described in paper III were discovered from high throughput genome sequencing efforts, the implication of which is that many have not been independently validated. In addition, while many of the variants studied in paper III are common variants within the human population, those which display altered activity appear to be rare or de novo variants. Anonymous donation of samples in these sequencing efforts makes linking genotype to phenotype difficult. As such we cannot draw conclusions about the relevance of these variants to disease, suffice to say that common variations do not appear to significantly alter

NPAS4 function in reporter experiments.

While links with disease phenotype was beyond the scope of Paper III, it suggests that the identified loss of function variants may have implications for neuropsychiatric or neurodegenerative disease. Furthermore, it also outlines the potential use of de novo functional screening of human variants in genes with already defined roles in disease.

Following our observations regarding the ability of naturally occurring variants to disrupt

NPAS4/ARNT2 function outlined in paper III and the tightly regulated mechanisms to control

NPAS4 expression outlined in paper II, we propose that screening for disease associated single nucleotide and copy number variants within coding and regulatory regions of NPAS4 is warranted, within neuropsychiatric or neurodegenerative disease populations.

As discussed above, bHLH-PAS transcription factors often act transiently to respond to signals and perform protective or homeostasis related functions. Homeostatic regulation of key signal response pathways is consistent with bHLH-PAS family members having important developmental and disease related roles. Targeted disruptions of many of the bHLH-PAS genes result in embryonic lethality and many have distinct roles in different tissues. It would therefore be advantageous to study function following gene manipulation in an inducible and reversible manner. Specifically, development of inducible, reversible and tissue specific gain or

loss of HIF1/ HIF2mouse models would significantly aid the elucidation and distinction for roles of the HIFs in cancer initiation, progression and metastasis. In addition, An Inducible and reversible loss of NPAS4 mouse model would be invaluable to the advancement of the study of the basic mechanisms underlying synapse formation, memory and learning formation and the investigation and modelling of neuropsychiatric disease.

We therefore embarked on designing and testing generic platforms in which bHLH-PAS transcription factors could be manipulated in an inducible, reversible and tissue specific fashion. Utilising optimised Tet-On variants with improved sensitivity to Dox and reduced background we constructed lentiviral and recombination mediated cassette exchange plasmids, which contained all the components necessary for Dox-inducible expression (see paper IV and Figure 3.).

We demonstrated that lentiviral generation of cell lines affords homogeneous inducible and reversible manipulation of gene expression with no detectable background. We also generated Col1a1 targeted RMCE FLP-In embryonic stem cells and mice for efficient FLP mediated targeting of FLP-INDUCER Dox regulated constructs into the Col1a1 locus. We demonstrate tight, inducible and reversible expression from mES-FLP-INDUCER cells able to knockdown gene expression or ectopically express cDNA similar to endogenous levels.

Modular design of FLP-INDUCER plasmids allow for promoter exchange to enable tissue specific promoters to be used to gain tissue/cell specific expression. We also show that LoxP-

STOP-LoxP mES FLP-INDUCER cells may, in principle, be used to limit inducible expression to certain tissues or cells (Figure 3.).

While there are other established (i.e. Cre/loxP) and emerging (CRISPR/TALEN) techniques to allow temporal and/or spatial gene ablation, some of which can employ inducible knockout strategies such as tamoxifen inducible nuclear translocation of oestrogen receptor (ER)-CRE recombinase to remove flox’d genes. These approaches irreversibly knockout the gene and require time consuming generation of flox’d genes and crossing into CRE driver lines.

Furthermore, in many disease models it may be advantageous to reversibly modulate gene expression to monitor the progression/reversion of disease. We note that the FLP-INDUCER system may be well suited to these specific disease model applications while also being compatible with established CRE-mediated tissue specific and/or temporal control of gene expression.

Alternative RMCE targeting loci have also been successfully used in the past for transgenic mouse experiments including inducible Tet-On expression systems (Tchorz, Suply et al. 2012; Haenebalcke, Goossens et al. 2013). Several of these systems would be compatible with the FLP-INDUCER system with minimal modification, presenting an opportunity to generate multi allelic inducible mouse models (Tasic, Hippenmeyer et al. 2011; Tchorz, Suply et al. 2012). While the Rosa26 locus has been used extensively for generating site-specific transgenesis, expression from this locus appears to result in significant mosaicsim and lower inducible transgene induction as compared to the Col1a1 locus (Haenebalcke, Goossens et al. 2013). In addition to the Col1a1 and rosa26 sites of integration the Hipp11 and hprt loci have successfully been used to drive ubiquitous expression in mouse models and may represent useful sites for RMCE mediated FLP-INDUCER insertion (Bronson, Plaehn et al. 1996; Tasic, Hippenmeyer et al. 2011).

Figure 3. Lentiviral and Collagen 1a1 Recombination Mediated Cassette Exchange (RMCE) systems for inducible and reversible knockdown or overexpression. A schematic showing the single construct modular design of lentiviral and FLP-INDUCER vector systems used to

generate stable cell lines or site-specific integration into germline competent mouse embryonic stem cells.

In an attempt to more accurately predict high potency miR30 shRNAs able to knockdown gene expression using the FLP-INDUCER system, we also designed and tested an shRNA prediction tool. This tool incorporated experimental data assessing the potency of ~60,000 shRNAs from high throughput experiments as well as mRNA secondary structure to score shRNA sequences based on their putative targeting efficiency. We show by cloning and testing 10 shRNA targets against ARNT or ARNT2 using the mES-FLP-INDUCER system that the prediction tool can effectively identify high potency shRNA sequences.

We are now well situated to rapidly generate inducible and reversible knockdown mouse models for studying the function of the bHLH-PAS transcription factors. As discussed above this will be invaluable to studying the more complex adult roles for the bHLH-PAS transcription factors in normal physiology and disease.

References

Aitola, M. H. and M. T. Pelto-Huikko (2003). "Expression of Arnt and Arnt2 mRNA in developing murine tissues." The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 51(1): 41-54. An, J. J., K. Gharami, et al. (2008). "Distinct role of long 3' UTR BDNF mRNA in spine morphology and synaptic plasticity in hippocampal neurons." Cell 134(1): 175-187. Antonsson, C., M. L. Whitelaw, et al. (1995). "Distinct roles of the molecular chaperone hsp90 in modulating dioxin receptor function via the basic helix-loop-helix and PAS domains." Molecular and cellular biology 15(2): 756-765. Atchley, W. R. and W. M. Fitch (1997). "A natural classification of the basic helix-loop-helix class of transcription factors." Proceedings of the National Academy of Sciences of the United States of America 94(10): 5172-5176. Baxendale, S., C. J. Holdsworth, et al. (2012). "Identification of compounds with anti- convulsant properties in a zebrafish model of epileptic seizures." Disease models & mechanisms 5(6): 773-784. Baxevanis, A. D. and C. R. Vinson (1993). "Interactions of coiled coils in transcription factors: where is the specificity?" Current opinion in genetics & development 3(2): 278-285. Bersten, D. C., A. E. Sullivan, et al. (2013). "bHLH-PAS proteins in cancer." Nature reviews. Cancer 13(12): 827-841. Bezprozvanny, I. and M. P. Mattson (2008). "Neuronal calcium mishandling and the pathogenesis of Alzheimer's disease." Trends in neurosciences 31(9): 454-463. Blackwell, T. K., L. Kretzner, et al. (1990). "Sequence-specific DNA binding by the c-Myc protein." Science 250(4984): 1149-1151. Blackwell, T. K. and H. Weintraub (1990). "Differences and similarities in DNA-binding preferences of MyoD and E2A protein complexes revealed by binding site selection." Science 250(4984): 1104-1110. Bloodgood, B. L., N. Sharma, et al. (2013). "The activity-dependent transcription factor NPAS4 regulates domain-specific inhibition." Nature 503(7474): 121-125. Bonnefond, A., A. Raimondo, et al. (2013). "Loss-of-function mutations in SIM1 contribute to obesity and Prader-Willi-like features." The Journal of clinical investigation 123(7): 3037-3041. Brunskill, E. W., D. P. Witte, et al. (1999). "Characterization of npas3, a novel basic helix-loop- helix PAS gene expressed in the developing mouse nervous system." Mechanisms of development 88(2): 237-241. Card, P. B., P. J. Erbel, et al. (2005). "Structural basis of ARNT PAS-B dimerization: use of a common beta-sheet interface for hetero- and homodimerization." Journal of molecular biology 353(3): 664-677. Chan, W. K., G. Yao, et al. (1999). "Cross-talk between the aryl hydrocarbon receptor and hypoxia inducible factor signaling pathways. Demonstration of competition and compensation." The Journal of biological chemistry 274(17): 12115-12123. Chapman-Smith, A., J. K. Lutwyche, et al. (2004). "Contribution of the Per/Arnt/Sim (PAS) domains to DNA binding by the basic helix-loop-helix PAS transcriptional regulators." The Journal of biological chemistry 279(7): 5353-5362.

Chapman-Smith, A. and M. L. Whitelaw (2006). "Novel DNA binding by a basic helix-loop-helix protein. The role of the dioxin receptor PAS domain." The Journal of biological chemistry 281(18): 12535-12545. Chen, W. G., Q. Chang, et al. (2003). "Derepression of BDNF transcription involves calcium- dependent phosphorylation of MeCP2." Science 302(5646): 885-889. Coba, M. P., L. M. Valor, et al. (2008). "Kinase networks integrate profiles of N-methyl-D- aspartate receptor-mediated gene expression in hippocampus." The Journal of biological chemistry 283(49): 34101-34107. Coumailleau, P., L. Poellinger, et al. (1995). "Definition of a minimal domain of the dioxin receptor that is associated with Hsp90 and maintains wild type ligand binding affinity and specificity." The Journal of biological chemistry 270(42): 25291-25300. Coutellier, L., S. Beraki, et al. (2012). "Npas4: a neuronal transcription factor with a key role in social and cognitive functions relevant to developmental disorders." PloS one 7(9): e46604. Crews, S. T. (1998). "Control of cell lineage-specific development and transcription by bHLH- PAS proteins." Genes & development 12(5): 607-620. Crews, S. T. and C. M. Fan (1999). "Remembrance of things PAS: regulation of development by bHLH-PAS proteins." Current opinion in genetics & development 9(5): 580-587. Crews, S. T., J. B. Thomas, et al. (1988). "The Drosophila single-minded gene encodes a nuclear protein with sequence similarity to the per gene product." Cell 52(1): 143-151. Dang, E. V., J. Barbi, et al. (2011). "Control of T(H)17/T(reg) balance by hypoxia-inducible factor 1." Cell 146(5): 772-784. Davis, R. L. and H. Weintraub (1992). "Acquisition of myogenic specificity by replacement of three amino acid residues from MyoD into E12." Science 256(5059): 1027-1030. Dean, C., H. Liu, et al. (2012). "Distinct subsets of Syt-IV/BDNF vesicles are sorted to axons versus dendrites and recruited to synapses by activity." The Journal of neuroscience : the official journal of the Society for Neuroscience 32(16): 5398-5413. Dioum, E. M., J. Rutter, et al. (2002). "NPAS2: a gas-responsive transcription factor." Science 298(5602): 2385-2387. Ebert, D. H., H. W. Gabel, et al. (2013). "Activity-dependent phosphorylation of MeCP2 threonine 308 regulates interaction with NCoR." Nature 499(7458): 341-345. Ebert, D. H. and M. E. Greenberg (2013). "Activity-dependent neuronal signalling and autism spectrum disorder." Nature 493(7432): 327-337. Englund, C., P. Steneberg, et al. (2002). "Attractive and repulsive functions of Slit are mediated by different receptors in the Drosophila trachea." Development 129(21): 4941-4951. Erbel, P. J., P. B. Card, et al. (2003). "Structural basis for PAS domain heterodimerization in the basic helix--loop--helix-PAS transcription factor hypoxia-inducible factor." Proceedings of the National Academy of Sciences of the United States of America 100(26): 15504- 15509. Farrall, A. L. and M. L. Whitelaw (2009). "The HIF1alpha-inducible pro-cell death gene BNIP3 is a novel target of SIM2s repression through cross-talk on the hypoxia response element." Oncogene 28(41): 3671-3680. Ferre-D'Amare, A. R., G. C. Prendergast, et al. (1993). "Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain." Nature 363(6424): 38-45. Flavell, S. W., T. K. Kim, et al. (2008). "Genome-wide analysis of MEF2 transcriptional program reveals synaptic target genes and neuronal activity-dependent polyadenylation site selection." Neuron 60(6): 1022-1038. Flood, W. D., R. W. Moyer, et al. (2004). "Nxf and Fbxo33: novel seizure-responsive genes in mice." The European journal of neuroscience 20(7): 1819-1826. Fukunaga, B. N., M. R. Probst, et al. (1995). "Identification of functional domains of the aryl hydrocarbon receptor." The Journal of biological chemistry 270(49): 29270-29278.

Genick, U. K., G. E. Borgstahl, et al. (1997). "Structure of a protein photocycle intermediate by millisecond time-resolved crystallography." Science 275(5305): 1471-1475. Ghabrial, A., S. Luschnig, et al. (2003). "Branching morphogenesis of the Drosophila tracheal system." Annual review of cell and developmental biology 19: 623-647. Greer, P. L. and M. E. Greenberg (2008). "From synapse to nucleus: calcium-dependent gene transcription in the control of synapse development and function." Neuron 59(6): 846- 860. Gunton, J. E., R. N. Kulkarni, et al. (2005). "Loss of ARNT/HIF1beta mediates altered gene expression and pancreatic-islet dysfunction in human type 2 diabetes." Cell 122(3): 337-349. Guo, M. L., B. Xue, et al. (2012). "Upregulation of Npas4 protein expression by chronic administration of amphetamine in rat nucleus accumbens in vivo." Neuroscience letters 528(2): 210-214. Hao, N. and M. L. Whitelaw (2013). "The emerging roles of AhR in physiology and immunity." Biochemical pharmacology 86(5): 561-570. Hao, N., M. L. Whitelaw, et al. (2011). "Identification of residues in the N-terminal PAS domains important for dimerization of Arnt and AhR." Nucleic acids research 39(9): 3695-3709. Hefti, M. H., K. J. Francoijs, et al. (2004). "The PAS fold. A redefinition of the PAS domain based upon structural prediction." European journal of biochemistry / FEBS 271(6): 1198- 1208. Henry, J. T. and S. Crosson (2011). "Ligand-binding PAS domains in a genomic, cellular, and structural context." Annual review of microbiology 65: 261-286. Hester, I., S. McKee, et al. (2007). "Transient expression of Nxf, a bHLH-PAS transactivator induced by neuronal preconditioning, confers neuroprotection in cultured cells." Brain research 1135(1): 1-11. Hirose, K., M. Morita, et al. (1996). "cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt)." Molecular and cellular biology 16(4): 1706-1713. Hoffman, E. C., H. Reyes, et al. (1991). "Cloning of a factor required for activity of the Ah (dioxin) receptor." Science 252(5008): 954-958. Hogenesch, J. B., Y. Z. Gu, et al. (1998). "The basic-helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors." Proceedings of the National Academy of Sciences of the United States of America 95(10): 5474-5479. Holmes, J. L. and R. S. Pollenz (1997). "Determination of aryl hydrocarbon receptor nuclear translocator protein concentration and subcellular localization in hepatic and nonhepatic cell culture lines: development of quantitative Western blotting protocols for calculation of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein in total cell lysates." Molecular pharmacology 52(2): 202-211. Huang, N., Y. Chelliah, et al. (2012). "Crystal structure of the heterodimeric CLOCK:BMAL1 transcriptional activator complex." Science 337(6091): 189-194. Jiang, L. and S. T. Crews (2003). "The Drosophila dysfusion basic helix-loop-helix (bHLH)-PAS gene controls tracheal fusion and levels of the trachealess bHLH-PAS protein." Molecular and cellular biology 23(16): 5625-5637. Jiang, L. and S. T. Crews (2006). "Dysfusion transcriptional control of Drosophila tracheal migration, adhesion, and fusion." Molecular and cellular biology 26(17): 6547-6556. Jiang, L. and S. T. Crews (2007). "Transcriptional specificity of Drosophila dysfusion and the control of tracheal fusion cell gene expression." The Journal of biological chemistry 282(39): 28659-28668.

Kaas, G. A., C. Zhong, et al. (2013). "TET1 controls CNS 5-methylcytosine hydroxylation, active DNA demethylation, gene transcription, and memory formation." Neuron 79(6): 1086- 1093. Kawajiri, K., Y. Kobayashi, et al. (2009). "Aryl hydrocarbon receptor suppresses intestinal carcinogenesis in ApcMin/+ mice with natural ligands." Proceedings of the National Academy of Sciences of the United States of America 106(32): 13481-13486. Kehrer, C., N. Maziashvili, et al. (2008). "Altered Excitatory-Inhibitory Balance in the NMDA- Hypofunction Model of Schizophrenia." Frontiers in molecular neuroscience 1: 6. Kewley, R. J., M. L. Whitelaw, et al. (2004). "The mammalian basic helix-loop-helix/PAS family of transcriptional regulators." The international journal of biochemistry & cell biology 36(2): 189-204. Kim, T. K., M. Hemberg, et al. (2010). "Widespread transcription at neuronal activity-regulated enhancers." Nature 465(7295): 182-187. Kimura, H., A. Weisz, et al. (2001). "Identification of hypoxia-inducible factor 1 ancillary sequence and its function in vascular endothelial growth factor gene induction by hypoxia and nitric oxide." The Journal of biological chemistry 276(3): 2292-2298. Ledent, V. and M. Vervoort (2001). "The basic helix-loop-helix protein family: comparative genomics and phylogenetic analysis." Genome research 11(5): 754-770. Leong, W. K., T. S. Klaric, et al. (2013). "Upregulation of the neuronal Per-Arnt-Sim domain protein 4 (Npas4) in the rat corticolimbic system following focal cerebral ischemia." The European journal of neuroscience 37(11): 1875-1884. Lin, Y., B. L. Bloodgood, et al. (2008). "Activity-dependent regulation of inhibitory synapse development by Npas4." Nature 455(7217): 1198-1204. Ma, P. C., M. A. Rould, et al. (1994). "Crystal structure of MyoD bHLH domain-DNA complex: perspectives on DNA recognition and implications for transcriptional activation." Cell 77(3): 451-459. Makino, Y., R. Cao, et al. (2001). "Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression." Nature 414(6863): 550-554. Makino, Y., A. Kanopka, et al. (2002). "Inhibitory PAS domain protein (IPAS) is a hypoxia- inducible splicing variant of the hypoxia-inducible factor-3alpha locus." The Journal of biological chemistry 277(36): 32405-32408. Marcheva, B., K. M. Ramsey, et al. (2010). "Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes." Nature 466(7306): 627-631. Martin, T. A., S. Jayanthi, et al. (2012). "Methamphetamine causes differential alterations in gene expression and patterns of histone acetylation/hypoacetylation in the rat nucleus accumbens." PloS one 7(3): e34236. Marty, S., R. Wehrle, et al. (2000). "Neuronal activity and brain-derived neurotrophic factor regulate the density of inhibitory synapses in organotypic slice cultures of postnatal hippocampus." The Journal of neuroscience : the official journal of the Society for Neuroscience 20(21): 8087-8095. Massari, M. E. and C. Murre (2000). "Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms." Molecular and cellular biology 20(2): 429-440. Maya-Vetencourt, J. F., E. Tiraboschi, et al. (2012). "Experience-dependent expression of NPAS4 regulates plasticity in adult visual cortex." The Journal of physiology 590(Pt 19): 4777-4787. McIntosh, B. E., J. B. Hogenesch, et al. (2010). "Mammalian Per-Arnt-Sim proteins in environmental adaptation." Annual review of physiology 72: 625-645. Mimura, J., M. Ema, et al. (1999). "Identification of a novel mechanism of regulation of Ah (dioxin) receptor function." Genes & development 13(1): 20-25. Moglich, A., R. A. Ayers, et al. (2009). "Structure and signaling mechanism of Per-ARNT-Sim domains." Structure 17(10): 1282-1294.

Moser, M., R. Knoth, et al. (2004). "LE-PAS, a novel Arnt-dependent HLH-PAS protein, is expressed in limbic tissues and transactivates the CNS midline enhancer element." Brain research. Molecular brain research 128(2): 141-149. Ohtake, F., A. Baba, et al. (2007). "Dioxin receptor is a ligand-dependent E3 ubiquitin ligase." Nature 446(7135): 562-566. Ooe, N., K. Kobayashi, et al. (2009). "Dynamic regulation of bHLH-PAS-type transcription factor NXF gene expression and neurotrophin dependent induction of the transcriptional control activity." Biochemical and biophysical research communications 378(4): 761- 765. Ooe, N., K. Motonaga, et al. (2009). "Functional characterization of basic helix-loop-helix-PAS type transcription factor NXF in vivo: putative involvement in an "on demand" neuroprotection system." The Journal of biological chemistry 284(2): 1057-1063. Ooe, N., K. Saito, et al. (2009). "Characterization of functional heterodimer partners in brain for a bHLH-PAS factor NXF." Biochimica et biophysica acta 1789(3): 192-197. Ooe, N., K. Saito, et al. (2004). "Identification of a novel basic helix-loop-helix-PAS factor, NXF, reveals a Sim2 competitive, positive regulatory role in dendritic-cytoskeleton modulator drebrin gene expression." Molecular and cellular biology 24(2): 608-616. Ooe, N., K. Saito, et al. (2007). "Characterization of Drosophila and Caenorhabditis elegans NXF-like-factors, putative homologs of mammalian NXF." Gene 400(1-2): 122-130. Partch, C. L., P. B. Card, et al. (2009). "Molecular basis of coiled coil coactivator recruitment by the aryl hydrocarbon receptor nuclear translocator (ARNT)." The Journal of biological chemistry 284(22): 15184-15192. Partch, C. L. and K. H. Gardner (2010). "Coactivator recruitment: a new role for PAS domains in transcriptional regulation by the bHLH-PAS family." Journal of cellular physiology 223(3): 553-557. Partch, C. L. and K. H. Gardner (2011). "Coactivators necessary for transcriptional output of the hypoxia inducible factor, HIF, are directly recruited by ARNT PAS-B." Proceedings of the National Academy of Sciences of the United States of America 108(19): 7739-7744. Pattabiraman, P. P., D. Tropea, et al. (2005). "Neuronal activity regulates the developmental expression and subcellular localization of cortical BDNF mRNA isoforms in vivo." Molecular and cellular neurosciences 28(3): 556-570. Pearson, J. C., J. D. Watson, et al. (2012). "Drosophila melanogaster Zelda and Single-minded collaborate to regulate an evolutionarily dynamic CNS midline cell enhancer." Developmental biology 366(2): 420-432. Peyrefitte, S., D. Kahn, et al. (2001). "New members of the Drosophila Myc transcription factor subfamily revealed by a genome-wide examination for basic helix-loop-helix genes." Mechanisms of development 104(1-2): 99-104. Piechota, M., M. Korostynski, et al. (2010). "The dissection of transcriptional modules regulated by various drugs of abuse in the mouse striatum." Genome biology 11(5): R48. Ploski, J. E., M. S. Monsey, et al. (2011). "The neuronal PAS domain protein 4 (Npas4) is required for new and reactivated fear memories." PloS one 6(8): e23760. Pongratz, I., C. Antonsson, et al. (1998). "Role of the PAS domain in regulation of dimerization and DNA binding specificity of the dioxin receptor." Molecular and cellular biology 18(7): 4079-4088. Pongratz, I., G. G. Mason, et al. (1992). "Dual roles of the 90-kDa heat shock protein hsp90 in modulating functional activities of the dioxin receptor. Evidence that the dioxin receptor functionally belongs to a subclass of nuclear receptors which require hsp90 both for ligand binding activity and repression of intrinsic DNA binding activity." The Journal of biological chemistry 267(19): 13728-13734.

Ponting, C. P. and L. Aravind (1997). "PAS: a multifunctional domain family comes to light." Current biology : CB 7(11): R674-677. Pruunsild, P., M. Sepp, et al. (2011). "Identification of cis-elements and transcription factors regulating neuronal activity-dependent transcription of human BDNF gene." The Journal of neuroscience : the official journal of the Society for Neuroscience 31(9): 3295-3308. Ramachandrappa, S., A. Raimondo, et al. (2013). "Rare variants in single-minded 1 (SIM1) are associated with severe obesity." The Journal of clinical investigation 123(7): 3042- 3050. Ramamoorthi, K., R. Fropf, et al. (2011). "Npas4 regulates a transcriptional program in CA3 required for contextual memory formation." Science 334(6063): 1669-1675. Ramocki, M. B. and H. Y. Zoghbi (2008). "Failure of neuronal homeostasis results in common neuropsychiatric phenotypes." Nature 455(7215): 912-918. Reddy, P., W. A. Zehring, et al. (1984). "Molecular analysis of the period locus in Drosophila melanogaster and identification of a transcript involved in biological rhythms." Cell 38(3): 701-710. Reisz-Porszasz, S., M. R. Probst, et al. (1994). "Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT)." Molecular and cellular biology 14(9): 6075-6086. Reppert, S. M. and D. R. Weaver (2002). "Coordination of circadian timing in mammals." Nature 418(6901): 935-941. Ribeiro, C., A. Ebner, et al. (2002). "In vivo imaging reveals different cellular functions for FGF and Dpp signaling in tracheal branching morphogenesis." Developmental cell 2(5): 677-683. Rubenstein, J. L. (2010). "Three hypotheses for developmental defects that may underlie some forms of autism spectrum disorder." Current opinion in neurology 23(2): 118-123. Rubenstein, J. L. and M. M. Merzenich (2003). "Model of autism: increased ratio of excitation/inhibition in key neural systems." Genes, brain, and behavior 2(5): 255-267. Rudenko, A., M. M. Dawlaty, et al. (2013). "Tet1 is critical for neuronal activity-regulated gene expression and memory extinction." Neuron 79(6): 1109-1122. Rutter, J., M. Reick, et al. (2001). "Regulation of clock and NPAS2 DNA binding by the redox state of NAD cofactors." Science 293(5529): 510-514. Sabatini, P. V., N. A. Krentz, et al. (2013). "Npas4 is a novel activity-regulated cytoprotective factor in pancreatic beta-cells." Diabetes 62(8): 2808-2820. Saxena, S. and P. Caroni (2011). "Selective neuronal vulnerability in neurodegenerative diseases: from stressor thresholds to degeneration." Neuron 71(1): 35-48. Scheuermann, T. H., D. R. Tomchick, et al. (2009). "Artificial ligand binding within the HIF2alpha PAS-B domain of the HIF2 transcription factor." Proceedings of the National Academy of Sciences of the United States of America 106(2): 450-455. Semenza, G. L. (2012). "Hypoxia-inducible factors in physiology and medicine." Cell 148(3): 399-408. Semenza, G. L., B. H. Jiang, et al. (1996). "Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1." The Journal of biological chemistry 271(51): 32529-32537. Shamloo, M., L. Soriano, et al. (2006). "Npas4, a novel helix-loop-helix PAS domain protein, is regulated in response to cerebral ischemia." The European journal of neuroscience 24(10): 2705-2720. Sogawa, K., R. Nakano, et al. (1995). "Possible function of Ah receptor nuclear translocator (Arnt) homodimer in transcriptional regulation." Proceedings of the National Academy of Sciences of the United States of America 92(6): 1936-1940.

Spitzer, N. C. (2006). "Electrical activity in early neuronal development." Nature 444(7120): 707-712. Spitzer, N. C. (2012). "Activity-dependent neurotransmitter respecification." Nature reviews. Neuroscience 13(2): 94-106. Sudhof, T. C. (2008). "Neuroligins and neurexins link synaptic function to cognitive disease." Nature 455(7215): 903-911. Swanson, H. I., W. K. Chan, et al. (1995). "DNA binding specificities and pairing rules of the Ah receptor, ARNT, and SIM proteins." The Journal of biological chemistry 270(44): 26292-26302. Swanson, H. I. and J. H. Yang (1999). "Specificity of DNA binding of the c-Myc/Max and ARNT/ARNT dimers at the CACGTG recognition site." Nucleic acids research 27(15): 3205-3212. Sylwestrak, E. and P. Scheiffele (2013). "Neuroscience: Sculpting neuronal connectivity." Nature 503(7474): 42-43. Tanaka-Matakatsu, M., T. Uemura, et al. (1996). "Cadherin-mediated cell adhesion and cell motility in Drosophila trachea regulated by the transcription factor Escargot." Development 122(12): 3697-3705. Taylor, B. L. and I. B. Zhulin (1999). "PAS domains: internal sensors of oxygen, redox potential, and light." Microbiology and molecular biology reviews : MMBR 63(2): 479-506. Uniacke, J., C. E. Holterman, et al. (2012). "An oxygen-regulated switch in the protein synthesis machinery." Nature 486(7401): 126-129. Whitelaw, M., I. Pongratz, et al. (1993). "Ligand-dependent recruitment of the Arnt coregulator determines DNA recognition by the dioxin receptor." Molecular and cellular biology 13(4): 2504-2514. Whitelaw, M. L., M. Gottlicher, et al. (1993). "Definition of a novel ligand binding domain of a nuclear bHLH receptor: co-localization of ligand and hsp90 binding activities within the regulable inactivation domain of the dioxin receptor." The EMBO journal 12(11): 4169- 4179. Whitelaw, M. L., J. McGuire, et al. (1995). "Heat shock protein hsp90 regulates dioxin receptor function in vivo." Proceedings of the National Academy of Sciences of the United States of America 92(10): 4437-4441. Wiesel, T. N. (1982). "Postnatal development of the visual cortex and the influence of environment." Nature 299(5884): 583-591. Wilk, R., I. Weizman, et al. (1996). "trachealess encodes a bHLH-PAS protein that is an inducer of tracheal cell fates in Drosophila." Genes & development 10(1): 93-102. Woods, S. L. and M. L. Whitelaw (2002). "Differential activities of murine single minded 1 (SIM1) and SIM2 on a hypoxic response element. Cross-talk between basic helix-loop- helix/per-Arnt-Sim homology transcription factors." The Journal of biological chemistry 277(12): 10236-10243. Wu, H. and Y. Zhang (2011). "Mechanisms and functions of Tet protein-mediated 5- methylcytosine oxidation." Genes & development 25(23): 2436-2452. Yang, J., K. D. Kim, et al. (2008). "A novel heme-regulatory motif mediates heme-dependent degradation of the circadian factor period 2." Molecular and cellular biology 28(15): 4697-4711. Yizhar, O., L. E. Fenno, et al. (2011). "Neocortical excitation/inhibition balance in information processing and social dysfunction." Nature 477(7363): 171-178. Yun, Z., H. L. Maecker, et al. (2002). "Inhibition of PPAR gamma 2 gene expression by the HIF- 1-regulated gene DEC1/Stra13: a mechanism for regulation of adipogenesis by hypoxia." Developmental cell 2(3): 331-341. Zelzer, E., P. Wappner, et al. (1997). "The PAS domain confers target gene specificity of Drosophila bHLH/PAS proteins." Genes & development 11(16): 2079-2089.

Zhang, S. J., M. Zou, et al. (2009). "Nuclear calcium signaling controls expression of a large gene pool: identification of a gene program for acquired neuroprotection induced by synaptic activity." PLoS genetics 5(8): e1000604. Zhulin, I. B., B. L. Taylor, et al. (1997). "PAS domain S-boxes in Archaea, Bacteria and sensors for oxygen and redox." Trends in biochemical sciences 22(9): 331-333.

Aitola, M. H. and M. T. Pelto-Huikko (2003). "Expression of Arnt and Arnt2 mRNA in developing murine tissues." The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 51(1): 41-54. An, J. J., K. Gharami, et al. (2008). "Distinct role of long 3' UTR BDNF mRNA in spine morphology and synaptic plasticity in hippocampal neurons." Cell 134(1): 175-187. Antonsson, C., M. L. Whitelaw, et al. (1995). "Distinct roles of the molecular chaperone hsp90 in modulating dioxin receptor function via the basic helix-loop-helix and PAS domains." Molecular and cellular biology 15(2): 756-765. Atchley, W. R. and W. M. Fitch (1997). "A natural classification of the basic helix-loop-helix class of transcription factors." Proceedings of the National Academy of Sciences of the United States of America 94(10): 5172-5176. Baxendale, S., C. J. Holdsworth, et al. (2012). "Identification of compounds with anti- convulsant properties in a zebrafish model of epileptic seizures." Disease models & mechanisms 5(6): 773-784. Baxevanis, A. D. and C. R. Vinson (1993). "Interactions of coiled coils in transcription factors: where is the specificity?" Current opinion in genetics & development 3(2): 278-285. Bersten, D. C., A. E. Sullivan, et al. (2013). "bHLH-PAS proteins in cancer." Nature reviews. Cancer 13(12): 827-841. Bezprozvanny, I. and M. P. Mattson (2008). "Neuronal calcium mishandling and the pathogenesis of Alzheimer's disease." Trends in neurosciences 31(9): 454-463. Blackwell, T. K., L. Kretzner, et al. (1990). "Sequence-specific DNA binding by the c-Myc protein." Science 250(4984): 1149-1151. Blackwell, T. K. and H. Weintraub (1990). "Differences and similarities in DNA-binding preferences of MyoD and E2A protein complexes revealed by binding site selection." Science 250(4984): 1104-1110. Bloodgood, B. L., N. Sharma, et al. (2013). "The activity-dependent transcription factor NPAS4 regulates domain-specific inhibition." Nature 503(7474): 121-125. Bonnefond, A., A. Raimondo, et al. (2013). "Loss-of-function mutations in SIM1 contribute to obesity and Prader-Willi-like features." The Journal of clinical investigation 123(7): 3037-3041. Bronson, S. K., E. G. Plaehn, et al. (1996). "Single-copy transgenic mice with chosen-site integration." Proceedings of the National Academy of Sciences of the United States of America 93(17): 9067-9072. Brunskill, E. W., D. P. Witte, et al. (1999). "Characterization of npas3, a novel basic helix-loop- helix PAS gene expressed in the developing mouse nervous system." Mechanisms of development 88(2): 237-241. Card, P. B., P. J. Erbel, et al. (2005). "Structural basis of ARNT PAS-B dimerization: use of a common beta-sheet interface for hetero- and homodimerization." Journal of molecular biology 353(3): 664-677.

Chan, W. K., G. Yao, et al. (1999). "Cross-talk between the aryl hydrocarbon receptor and hypoxia inducible factor signaling pathways. Demonstration of competition and compensation." The Journal of biological chemistry 274(17): 12115-12123. Chapman-Smith, A., J. K. Lutwyche, et al. (2004). "Contribution of the Per/Arnt/Sim (PAS) domains to DNA binding by the basic helix-loop-helix PAS transcriptional regulators." The Journal of biological chemistry 279(7): 5353-5362. Chapman-Smith, A. and M. L. Whitelaw (2006). "Novel DNA binding by a basic helix-loop-helix protein. The role of the dioxin receptor PAS domain." The Journal of biological chemistry 281(18): 12535-12545. Chen, W. G., Q. Chang, et al. (2003). "Derepression of BDNF transcription involves calcium- dependent phosphorylation of MeCP2." Science 302(5646): 885-889. Coba, M. P., L. M. Valor, et al. (2008). "Kinase networks integrate profiles of N-methyl-D- aspartate receptor-mediated gene expression in hippocampus." The Journal of biological chemistry 283(49): 34101-34107. Coumailleau, P., L. Poellinger, et al. (1995). "Definition of a minimal domain of the dioxin receptor that is associated with Hsp90 and maintains wild type ligand binding affinity and specificity." The Journal of biological chemistry 270(42): 25291-25300. Coutellier, L., S. Beraki, et al. (2012). "Npas4: a neuronal transcription factor with a key role in social and cognitive functions relevant to developmental disorders." PloS one 7(9): e46604. Crews, S. T. (1998). "Control of cell lineage-specific development and transcription by bHLH- PAS proteins." Genes & development 12(5): 607-620. Crews, S. T. and C. M. Fan (1999). "Remembrance of things PAS: regulation of development by bHLH-PAS proteins." Current opinion in genetics & development 9(5): 580-587. Crews, S. T., J. B. Thomas, et al. (1988). "The Drosophila single-minded gene encodes a nuclear protein with sequence similarity to the per gene product." Cell 52(1): 143-151. Dang, E. V., J. Barbi, et al. (2011). "Control of T(H)17/T(reg) balance by hypoxia-inducible factor 1." Cell 146(5): 772-784. Davis, R. L. and H. Weintraub (1992). "Acquisition of myogenic specificity by replacement of three amino acid residues from MyoD into E12." Science 256(5059): 1027-1030. Dean, C., H. Liu, et al. (2012). "Distinct subsets of Syt-IV/BDNF vesicles are sorted to axons versus dendrites and recruited to synapses by activity." The Journal of neuroscience : the official journal of the Society for Neuroscience 32(16): 5398-5413. Dioum, E. M., J. Rutter, et al. (2002). "NPAS2: a gas-responsive transcription factor." Science 298(5602): 2385-2387. Ebert, D. H., H. W. Gabel, et al. (2013). "Activity-dependent phosphorylation of MeCP2 threonine 308 regulates interaction with NCoR." Nature 499(7458): 341-345. Ebert, D. H. and M. E. Greenberg (2013). "Activity-dependent neuronal signalling and autism spectrum disorder." Nature 493(7432): 327-337. Englund, C., P. Steneberg, et al. (2002). "Attractive and repulsive functions of Slit are mediated by different receptors in the Drosophila trachea." Development 129(21): 4941-4951. Erbel, P. J., P. B. Card, et al. (2003). "Structural basis for PAS domain heterodimerization in the basic helix--loop--helix-PAS transcription factor hypoxia-inducible factor." Proceedings of the National Academy of Sciences of the United States of America 100(26): 15504- 15509. Farrall, A. L. and M. L. Whitelaw (2009). "The HIF1alpha-inducible pro-cell death gene BNIP3 is a novel target of SIM2s repression through cross-talk on the hypoxia response element." Oncogene 28(41): 3671-3680. Ferre-D'Amare, A. R., G. C. Prendergast, et al. (1993). "Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain." Nature 363(6424): 38-45.

Flavell, S. W., T. K. Kim, et al. (2008). "Genome-wide analysis of MEF2 transcriptional program reveals synaptic target genes and neuronal activity-dependent polyadenylation site selection." Neuron 60(6): 1022-1038. Flood, W. D., R. W. Moyer, et al. (2004). "Nxf and Fbxo33: novel seizure-responsive genes in mice." The European journal of neuroscience 20(7): 1819-1826. Fukunaga, B. N., M. R. Probst, et al. (1995). "Identification of functional domains of the aryl hydrocarbon receptor." The Journal of biological chemistry 270(49): 29270-29278. Genick, U. K., G. E. Borgstahl, et al. (1997). "Structure of a protein photocycle intermediate by millisecond time-resolved crystallography." Science 275(5305): 1471-1475. Ghabrial, A., S. Luschnig, et al. (2003). "Branching morphogenesis of the Drosophila tracheal system." Annual review of cell and developmental biology 19: 623-647. Greer, P. L. and M. E. Greenberg (2008). "From synapse to nucleus: calcium-dependent gene transcription in the control of synapse development and function." Neuron 59(6): 846- 860. Gunton, J. E., R. N. Kulkarni, et al. (2005). "Loss of ARNT/HIF1beta mediates altered gene expression and pancreatic-islet dysfunction in human type 2 diabetes." Cell 122(3): 337-349. Guo, M. L., B. Xue, et al. (2012). "Upregulation of Npas4 protein expression by chronic administration of amphetamine in rat nucleus accumbens in vivo." Neuroscience letters 528(2): 210-214. Haenebalcke, L., S. Goossens, et al. (2013). "The ROSA26-iPSC mouse: a conditional, inducible, and exchangeable resource for studying cellular (De)differentiation." Cell Rep 3(2): 335-341. Haenebalcke, L., S. Goossens, et al. (2013). "Efficient ROSA26-based conditional and/or inducible transgenesis using RMCE-compatible F1 hybrid mouse embryonic stem cells." Stem Cell Rev 9(6): 774-785. Hao, N. and M. L. Whitelaw (2013). "The emerging roles of AhR in physiology and immunity." Biochemical pharmacology 86(5): 561-570. Hao, N., M. L. Whitelaw, et al. (2011). "Identification of residues in the N-terminal PAS domains important for dimerization of Arnt and AhR." Nucleic acids research 39(9): 3695-3709. Hefti, M. H., K. J. Francoijs, et al. (2004). "The PAS fold. A redefinition of the PAS domain based upon structural prediction." European journal of biochemistry / FEBS 271(6): 1198- 1208. Henry, J. T. and S. Crosson (2011). "Ligand-binding PAS domains in a genomic, cellular, and structural context." Annual review of microbiology 65: 261-286. Hester, I., S. McKee, et al. (2007). "Transient expression of Nxf, a bHLH-PAS transactivator induced by neuronal preconditioning, confers neuroprotection in cultured cells." Brain research 1135(1): 1-11. Hirose, K., M. Morita, et al. (1996). "cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt)." Molecular and cellular biology 16(4): 1706-1713. Hoffman, E. C., H. Reyes, et al. (1991). "Cloning of a factor required for activity of the Ah (dioxin) receptor." Science 252(5008): 954-958. Hogenesch, J. B., Y. Z. Gu, et al. (1998). "The basic-helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors." Proceedings of the National Academy of Sciences of the United States of America 95(10): 5474-5479. Holmes, J. L. and R. S. Pollenz (1997). "Determination of aryl hydrocarbon receptor nuclear translocator protein concentration and subcellular localization in hepatic and nonhepatic cell culture lines: development of quantitative Western blotting protocols

for calculation of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein in total cell lysates." Molecular pharmacology 52(2): 202-211. Huang, N., Y. Chelliah, et al. (2012). "Crystal structure of the heterodimeric CLOCK:BMAL1 transcriptional activator complex." Science 337(6091): 189-194. Jiang, L. and S. T. Crews (2003). "The Drosophila dysfusion basic helix-loop-helix (bHLH)-PAS gene controls tracheal fusion and levels of the trachealess bHLH-PAS protein." Molecular and cellular biology 23(16): 5625-5637. Jiang, L. and S. T. Crews (2006). "Dysfusion transcriptional control of Drosophila tracheal migration, adhesion, and fusion." Molecular and cellular biology 26(17): 6547-6556. Jiang, L. and S. T. Crews (2007). "Transcriptional specificity of Drosophila dysfusion and the control of tracheal fusion cell gene expression." The Journal of biological chemistry 282(39): 28659-28668. Kaas, G. A., C. Zhong, et al. (2013). "TET1 controls CNS 5-methylcytosine hydroxylation, active DNA demethylation, gene transcription, and memory formation." Neuron 79(6): 1086- 1093. Kawajiri, K., Y. Kobayashi, et al. (2009). "Aryl hydrocarbon receptor suppresses intestinal carcinogenesis in ApcMin/+ mice with natural ligands." Proceedings of the National Academy of Sciences of the United States of America 106(32): 13481-13486. Kehrer, C., N. Maziashvili, et al. (2008). "Altered Excitatory-Inhibitory Balance in the NMDA- Hypofunction Model of Schizophrenia." Frontiers in molecular neuroscience 1: 6. Kewley, R. J., M. L. Whitelaw, et al. (2004). "The mammalian basic helix-loop-helix/PAS family of transcriptional regulators." The international journal of biochemistry & cell biology 36(2): 189-204. Kim, T. K., M. Hemberg, et al. (2010). "Widespread transcription at neuronal activity-regulated enhancers." Nature 465(7295): 182-187. Kimura, H., A. Weisz, et al. (2001). "Identification of hypoxia-inducible factor 1 ancillary sequence and its function in vascular endothelial growth factor gene induction by hypoxia and nitric oxide." The Journal of biological chemistry 276(3): 2292-2298. Ledent, V. and M. Vervoort (2001). "The basic helix-loop-helix protein family: comparative genomics and phylogenetic analysis." Genome research 11(5): 754-770. Leong, W. K., T. S. Klaric, et al. (2013). "Upregulation of the neuronal Per-Arnt-Sim domain protein 4 (Npas4) in the rat corticolimbic system following focal cerebral ischemia." The European journal of neuroscience 37(11): 1875-1884. Lin, Y., B. L. Bloodgood, et al. (2008). "Activity-dependent regulation of inhibitory synapse development by Npas4." Nature 455(7217): 1198-1204. Ma, P. C., M. A. Rould, et al. (1994). "Crystal structure of MyoD bHLH domain-DNA complex: perspectives on DNA recognition and implications for transcriptional activation." Cell 77(3): 451-459. Makino, Y., R. Cao, et al. (2001). "Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression." Nature 414(6863): 550-554. Makino, Y., A. Kanopka, et al. (2002). "Inhibitory PAS domain protein (IPAS) is a hypoxia- inducible splicing variant of the hypoxia-inducible factor-3alpha locus." The Journal of biological chemistry 277(36): 32405-32408. Marcheva, B., K. M. Ramsey, et al. (2010). "Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes." Nature 466(7306): 627-631. Martin, T. A., S. Jayanthi, et al. (2012). "Methamphetamine causes differential alterations in gene expression and patterns of histone acetylation/hypoacetylation in the rat nucleus accumbens." PloS one 7(3): e34236. Marty, S., R. Wehrle, et al. (2000). "Neuronal activity and brain-derived neurotrophic factor regulate the density of inhibitory synapses in organotypic slice cultures of postnatal

hippocampus." The Journal of neuroscience : the official journal of the Society for Neuroscience 20(21): 8087-8095. Massari, M. E. and C. Murre (2000). "Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms." Molecular and cellular biology 20(2): 429-440. Maya-Vetencourt, J. F., E. Tiraboschi, et al. (2012). "Experience-dependent expression of NPAS4 regulates plasticity in adult visual cortex." The Journal of physiology 590(Pt 19): 4777-4787. McIntosh, B. E., J. B. Hogenesch, et al. (2010). "Mammalian Per-Arnt-Sim proteins in environmental adaptation." Annual review of physiology 72: 625-645. Mimura, J., M. Ema, et al. (1999). "Identification of a novel mechanism of regulation of Ah (dioxin) receptor function." Genes & development 13(1): 20-25. Moglich, A., R. A. Ayers, et al. (2009). "Structure and signaling mechanism of Per-ARNT-Sim domains." Structure 17(10): 1282-1294. Moser, M., R. Knoth, et al. (2004). "LE-PAS, a novel Arnt-dependent HLH-PAS protein, is expressed in limbic tissues and transactivates the CNS midline enhancer element." Brain research. Molecular brain research 128(2): 141-149. Ohtake, F., A. Baba, et al. (2007). "Dioxin receptor is a ligand-dependent E3 ubiquitin ligase." Nature 446(7135): 562-566. Ooe, N., K. Kobayashi, et al. (2009). "Dynamic regulation of bHLH-PAS-type transcription factor NXF gene expression and neurotrophin dependent induction of the transcriptional control activity." Biochemical and biophysical research communications 378(4): 761- 765. Ooe, N., K. Motonaga, et al. (2009). "Functional characterization of basic helix-loop-helix-PAS type transcription factor NXF in vivo: putative involvement in an "on demand" neuroprotection system." The Journal of biological chemistry 284(2): 1057-1063. Ooe, N., K. Saito, et al. (2009). "Characterization of functional heterodimer partners in brain for a bHLH-PAS factor NXF." Biochimica et biophysica acta 1789(3): 192-197. Ooe, N., K. Saito, et al. (2004). "Identification of a novel basic helix-loop-helix-PAS factor, NXF, reveals a Sim2 competitive, positive regulatory role in dendritic-cytoskeleton modulator drebrin gene expression." Molecular and cellular biology 24(2): 608-616. Ooe, N., K. Saito, et al. (2007). "Characterization of Drosophila and Caenorhabditis elegans NXF-like-factors, putative homologs of mammalian NXF." Gene 400(1-2): 122-130. Partch, C. L., P. B. Card, et al. (2009). "Molecular basis of coiled coil coactivator recruitment by the aryl hydrocarbon receptor nuclear translocator (ARNT)." The Journal of biological chemistry 284(22): 15184-15192. Partch, C. L. and K. H. Gardner (2010). "Coactivator recruitment: a new role for PAS domains in transcriptional regulation by the bHLH-PAS family." Journal of cellular physiology 223(3): 553-557. Partch, C. L. and K. H. Gardner (2011). "Coactivators necessary for transcriptional output of the hypoxia inducible factor, HIF, are directly recruited by ARNT PAS-B." Proceedings of the National Academy of Sciences of the United States of America 108(19): 7739-7744. Pattabiraman, P. P., D. Tropea, et al. (2005). "Neuronal activity regulates the developmental expression and subcellular localization of cortical BDNF mRNA isoforms in vivo." Molecular and cellular neurosciences 28(3): 556-570. Pearson, J. C., J. D. Watson, et al. (2012). "Drosophila melanogaster Zelda and Single-minded collaborate to regulate an evolutionarily dynamic CNS midline cell enhancer." Developmental biology 366(2): 420-432. Peyrefitte, S., D. Kahn, et al. (2001). "New members of the Drosophila Myc transcription factor subfamily revealed by a genome-wide examination for basic helix-loop-helix genes." Mechanisms of development 104(1-2): 99-104.

Piechota, M., M. Korostynski, et al. (2010). "The dissection of transcriptional modules regulated by various drugs of abuse in the mouse striatum." Genome biology 11(5): R48. Ploski, J. E., M. S. Monsey, et al. (2011). "The neuronal PAS domain protein 4 (Npas4) is required for new and reactivated fear memories." PloS one 6(8): e23760. Pongratz, I., C. Antonsson, et al. (1998). "Role of the PAS domain in regulation of dimerization and DNA binding specificity of the dioxin receptor." Molecular and cellular biology 18(7): 4079-4088. Pongratz, I., G. G. Mason, et al. (1992). "Dual roles of the 90-kDa heat shock protein hsp90 in modulating functional activities of the dioxin receptor. Evidence that the dioxin receptor functionally belongs to a subclass of nuclear receptors which require hsp90 both for ligand binding activity and repression of intrinsic DNA binding activity." The Journal of biological chemistry 267(19): 13728-13734. Ponting, C. P. and L. Aravind (1997). "PAS: a multifunctional domain family comes to light." Current biology : CB 7(11): R674-677. Pruunsild, P., M. Sepp, et al. (2011). "Identification of cis-elements and transcription factors regulating neuronal activity-dependent transcription of human BDNF gene." The Journal of neuroscience : the official journal of the Society for Neuroscience 31(9): 3295-3308. Ramachandrappa, S., A. Raimondo, et al. (2013). "Rare variants in single-minded 1 (SIM1) are associated with severe obesity." The Journal of clinical investigation 123(7): 3042- 3050. Ramamoorthi, K., R. Fropf, et al. (2011). "Npas4 regulates a transcriptional program in CA3 required for contextual memory formation." Science 334(6063): 1669-1675. Ramocki, M. B. and H. Y. Zoghbi (2008). "Failure of neuronal homeostasis results in common neuropsychiatric phenotypes." Nature 455(7215): 912-918. Reddy, P., W. A. Zehring, et al. (1984). "Molecular analysis of the period locus in Drosophila melanogaster and identification of a transcript involved in biological rhythms." Cell 38(3): 701-710. Reisz-Porszasz, S., M. R. Probst, et al. (1994). "Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT)." Molecular and cellular biology 14(9): 6075-6086. Reppert, S. M. and D. R. Weaver (2002). "Coordination of circadian timing in mammals." Nature 418(6901): 935-941. Ribeiro, C., A. Ebner, et al. (2002). "In vivo imaging reveals different cellular functions for FGF and Dpp signaling in tracheal branching morphogenesis." Developmental cell 2(5): 677-683. Rubenstein, J. L. (2010). "Three hypotheses for developmental defects that may underlie some forms of autism spectrum disorder." Current opinion in neurology 23(2): 118-123. Rubenstein, J. L. and M. M. Merzenich (2003). "Model of autism: increased ratio of excitation/inhibition in key neural systems." Genes, brain, and behavior 2(5): 255-267. Rudenko, A., M. M. Dawlaty, et al. (2013). "Tet1 is critical for neuronal activity-regulated gene expression and memory extinction." Neuron 79(6): 1109-1122. Rutter, J., M. Reick, et al. (2001). "Regulation of clock and NPAS2 DNA binding by the redox state of NAD cofactors." Science 293(5529): 510-514. Sabatini, P. V., N. A. Krentz, et al. (2013). "Npas4 is a novel activity-regulated cytoprotective factor in pancreatic beta-cells." Diabetes 62(8): 2808-2820. Saxena, S. and P. Caroni (2011). "Selective neuronal vulnerability in neurodegenerative diseases: from stressor thresholds to degeneration." Neuron 71(1): 35-48.

Scheuermann, T. H., D. R. Tomchick, et al. (2009). "Artificial ligand binding within the HIF2alpha PAS-B domain of the HIF2 transcription factor." Proceedings of the National Academy of Sciences of the United States of America 106(2): 450-455. Semenza, G. L. (2012). "Hypoxia-inducible factors in physiology and medicine." Cell 148(3): 399-408. Semenza, G. L., B. H. Jiang, et al. (1996). "Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1." The Journal of biological chemistry 271(51): 32529-32537. Shamloo, M., L. Soriano, et al. (2006). "Npas4, a novel helix-loop-helix PAS domain protein, is regulated in response to cerebral ischemia." The European journal of neuroscience 24(10): 2705-2720. Sogawa, K., R. Nakano, et al. (1995). "Possible function of Ah receptor nuclear translocator (Arnt) homodimer in transcriptional regulation." Proceedings of the National Academy of Sciences of the United States of America 92(6): 1936-1940. Spitzer, N. C. (2006). "Electrical activity in early neuronal development." Nature 444(7120): 707-712. Spitzer, N. C. (2012). "Activity-dependent neurotransmitter respecification." Nature reviews. Neuroscience 13(2): 94-106. Sudhof, T. C. (2008). "Neuroligins and neurexins link synaptic function to cognitive disease." Nature 455(7215): 903-911. Swanson, H. I., W. K. Chan, et al. (1995). "DNA binding specificities and pairing rules of the Ah receptor, ARNT, and SIM proteins." The Journal of biological chemistry 270(44): 26292-26302. Swanson, H. I. and J. H. Yang (1999). "Specificity of DNA binding of the c-Myc/Max and ARNT/ARNT dimers at the CACGTG recognition site." Nucleic acids research 27(15): 3205-3212. Sylwestrak, E. and P. Scheiffele (2013). "Neuroscience: Sculpting neuronal connectivity." Nature 503(7474): 42-43. Tanaka-Matakatsu, M., T. Uemura, et al. (1996). "Cadherin-mediated cell adhesion and cell motility in Drosophila trachea regulated by the transcription factor Escargot." Development 122(12): 3697-3705. Tasic, B., S. Hippenmeyer, et al. (2011). "Site-specific integrase-mediated transgenesis in mice via pronuclear injection." Proceedings of the National Academy of Sciences of the United States of America 108(19): 7902-7907. Taylor, B. L. and I. B. Zhulin (1999). "PAS domains: internal sensors of oxygen, redox potential, and light." Microbiology and molecular biology reviews : MMBR 63(2): 479-506. Tchorz, J. S., T. Suply, et al. (2012). "A modified RMCE-compatible Rosa26 locus for the expression of transgenes from exogenous promoters." PloS one 7(1): e30011. Uniacke, J., C. E. Holterman, et al. (2012). "An oxygen-regulated switch in the protein synthesis machinery." Nature 486(7401): 126-129. Whitelaw, M., I. Pongratz, et al. (1993). "Ligand-dependent recruitment of the Arnt coregulator determines DNA recognition by the dioxin receptor." Molecular and cellular biology 13(4): 2504-2514. Whitelaw, M. L., M. Gottlicher, et al. (1993). "Definition of a novel ligand binding domain of a nuclear bHLH receptor: co-localization of ligand and hsp90 binding activities within the regulable inactivation domain of the dioxin receptor." The EMBO journal 12(11): 4169- 4179. Whitelaw, M. L., J. McGuire, et al. (1995). "Heat shock protein hsp90 regulates dioxin receptor function in vivo." Proceedings of the National Academy of Sciences of the United States of America 92(10): 4437-4441.

Wiesel, T. N. (1982). "Postnatal development of the visual cortex and the influence of environment." Nature 299(5884): 583-591. Wilk, R., I. Weizman, et al. (1996). "trachealess encodes a bHLH-PAS protein that is an inducer of tracheal cell fates in Drosophila." Genes & development 10(1): 93-102. Woods, S. L. and M. L. Whitelaw (2002). "Differential activities of murine single minded 1 (SIM1) and SIM2 on a hypoxic response element. Cross-talk between basic helix-loop- helix/per-Arnt-Sim homology transcription factors." The Journal of biological chemistry 277(12): 10236-10243. Wu, H. and Y. Zhang (2011). "Mechanisms and functions of Tet protein-mediated 5- methylcytosine oxidation." Genes & development 25(23): 2436-2452. Yang, J., K. D. Kim, et al. (2008). "A novel heme-regulatory motif mediates heme-dependent degradation of the circadian factor period 2." Molecular and cellular biology 28(15): 4697-4711. Yizhar, O., L. E. Fenno, et al. (2011). "Neocortical excitation/inhibition balance in information processing and social dysfunction." Nature 477(7363): 171-178. Yun, Z., H. L. Maecker, et al. (2002). "Inhibition of PPAR gamma 2 gene expression by the HIF- 1-regulated gene DEC1/Stra13: a mechanism for regulation of adipogenesis by hypoxia." Developmental cell 2(3): 331-341. Zelzer, E., P. Wappner, et al. (1997). "The PAS domain confers target gene specificity of Drosophila bHLH/PAS proteins." Genes & development 11(16): 2079-2089. Zhang, S. J., M. Zou, et al. (2009). "Nuclear calcium signaling controls expression of a large gene pool: identification of a gene program for acquired neuroprotection induced by synaptic activity." PLoS genetics 5(8): e1000604. Zhulin, I. B., B. L. Taylor, et al. (1997). "PAS domain S-boxes in Archaea, Bacteria and sensors for oxygen and redox." Trends in biochemical sciences 22(9): 331-333.

Blackwell, T. K. and H. Weintraub (1990). "Differences and similarities in DNA-binding preferences of MyoD and E2A protein complexes revealed by binding site selection." Science 250(4984): 1104-1110. Bloodgood, B. L., N. Sharma, et al. (2013). "The activity-dependent transcription factor NPAS4 regulates domain-specific inhibition." Nature 503(7474): 121-125. Bonnefond, A., A. Raimondo, et al. (2013). "Loss-of-function mutations in SIM1 contribute to obesity and Prader-Willi-like features." The Journal of clinical investigation 123(7): 3037-3041. Bronson, S. K., E. G. Plaehn, et al. (1996). "Single-copy transgenic mice with chosen-site integration." Proceedings of the National Academy of Sciences of the United States of America 93(17): 9067-9072. Brunskill, E. W., D. P. Witte, et al. (1999). "Characterization of npas3, a novel basic helix-loop- helix PAS gene expressed in the developing mouse nervous system." Mechanisms of development 88(2): 237-241. Card, P. B., P. J. Erbel, et al. (2005). "Structural basis of ARNT PAS-B dimerization: use of a common beta-sheet interface for hetero- and homodimerization." Journal of molecular biology 353(3): 664-677. Chan, W. K., G. Yao, et al. (1999). "Cross-talk between the aryl hydrocarbon receptor and hypoxia inducible factor signaling pathways. Demonstration of competition and compensation." The Journal of biological chemistry 274(17): 12115-12123. Chapman-Smith, A., J. K. Lutwyche, et al. (2004). "Contribution of the Per/Arnt/Sim (PAS) domains to DNA binding by the basic helix-loop-helix PAS transcriptional regulators." The Journal of biological chemistry 279(7): 5353-5362. Chapman-Smith, A. and M. L. Whitelaw (2006). "Novel DNA binding by a basic helix-loop-helix protein. The role of the dioxin receptor PAS domain." The Journal of biological chemistry 281(18): 12535-12545. Chen, W. G., Q. Chang, et al. (2003). "Derepression of BDNF transcription involves calcium- dependent phosphorylation of MeCP2." Science 302(5646): 885-889. Coba, M. P., L. M. Valor, et al. (2008). "Kinase networks integrate profiles of N-methyl-D- aspartate receptor-mediated gene expression in hippocampus." The Journal of biological chemistry 283(49): 34101-34107. Coumailleau, P., L. Poellinger, et al. (1995). "Definition of a minimal domain of the dioxin receptor that is associated with Hsp90 and maintains wild type ligand binding affinity and specificity." The Journal of biological chemistry 270(42): 25291-25300. Coutellier, L., S. Beraki, et al. (2012). "Npas4: a neuronal transcription factor with a key role in social and cognitive functions relevant to developmental disorders." PloS one 7(9): e46604. Crews, S. T. (1998). "Control of cell lineage-specific development and transcription by bHLH- PAS proteins." Genes & development 12(5): 607-620. Crews, S. T. and C. M. Fan (1999). "Remembrance of things PAS: regulation of development by bHLH-PAS proteins." Current opinion in genetics & development 9(5): 580-587. Crews, S. T., J. B. Thomas, et al. (1988). "The Drosophila single-minded gene encodes a nuclear protein with sequence similarity to the per gene product." Cell 52(1): 143-151. Dang, E. V., J. Barbi, et al. (2011). "Control of T(H)17/T(reg) balance by hypoxia-inducible factor 1." Cell 146(5): 772-784. Davis, R. L. and H. Weintraub (1992). "Acquisition of myogenic specificity by replacement of three amino acid residues from MyoD into E12." Science 256(5059): 1027-1030. Dean, C., H. Liu, et al. (2012). "Distinct subsets of Syt-IV/BDNF vesicles are sorted to axons versus dendrites and recruited to synapses by activity." The Journal of neuroscience : the official journal of the Society for Neuroscience 32(16): 5398-5413.

Dioum, E. M., J. Rutter, et al. (2002). "NPAS2: a gas-responsive transcription factor." Science 298(5602): 2385-2387. Ebert, D. H., H. W. Gabel, et al. (2013). "Activity-dependent phosphorylation of MeCP2 threonine 308 regulates interaction with NCoR." Nature 499(7458): 341-345. Ebert, D. H. and M. E. Greenberg (2013). "Activity-dependent neuronal signalling and autism spectrum disorder." Nature 493(7432): 327-337. Englund, C., P. Steneberg, et al. (2002). "Attractive and repulsive functions of Slit are mediated by different receptors in the Drosophila trachea." Development 129(21): 4941-4951. Erbel, P. J., P. B. Card, et al. (2003). "Structural basis for PAS domain heterodimerization in the basic helix--loop--helix-PAS transcription factor hypoxia-inducible factor." Proceedings of the National Academy of Sciences of the United States of America 100(26): 15504- 15509. Farrall, A. L. and M. L. Whitelaw (2009). "The HIF1alpha-inducible pro-cell death gene BNIP3 is a novel target of SIM2s repression through cross-talk on the hypoxia response element." Oncogene 28(41): 3671-3680. Ferre-D'Amare, A. R., G. C. Prendergast, et al. (1993). "Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain." Nature 363(6424): 38-45. Flavell, S. W., T. K. Kim, et al. (2008). "Genome-wide analysis of MEF2 transcriptional program reveals synaptic target genes and neuronal activity-dependent polyadenylation site selection." Neuron 60(6): 1022-1038. Flood, W. D., R. W. Moyer, et al. (2004). "Nxf and Fbxo33: novel seizure-responsive genes in mice." The European journal of neuroscience 20(7): 1819-1826. Fukunaga, B. N., M. R. Probst, et al. (1995). "Identification of functional domains of the aryl hydrocarbon receptor." The Journal of biological chemistry 270(49): 29270-29278. Genick, U. K., G. E. Borgstahl, et al. (1997). "Structure of a protein photocycle intermediate by millisecond time-resolved crystallography." Science 275(5305): 1471-1475. Ghabrial, A., S. Luschnig, et al. (2003). "Branching morphogenesis of the Drosophila tracheal system." Annual review of cell and developmental biology 19: 623-647. Greer, P. L. and M. E. Greenberg (2008). "From synapse to nucleus: calcium-dependent gene transcription in the control of synapse development and function." Neuron 59(6): 846- 860. Gunton, J. E., R. N. Kulkarni, et al. (2005). "Loss of ARNT/HIF1beta mediates altered gene expression and pancreatic-islet dysfunction in human type 2 diabetes." Cell 122(3): 337-349. Guo, M. L., B. Xue, et al. (2012). "Upregulation of Npas4 protein expression by chronic administration of amphetamine in rat nucleus accumbens in vivo." Neuroscience letters 528(2): 210-214. Haenebalcke, L., S. Goossens, et al. (2013). "Efficient ROSA26-based conditional and/or inducible transgenesis using RMCE-compatible F1 hybrid mouse embryonic stem cells." Stem Cell Rev 9(6): 774-785. Hao, N. and M. L. Whitelaw (2013). "The emerging roles of AhR in physiology and immunity." Biochemical pharmacology 86(5): 561-570. Hao, N., M. L. Whitelaw, et al. (2011). "Identification of residues in the N-terminal PAS domains important for dimerization of Arnt and AhR." Nucleic acids research 39(9): 3695-3709. Hefti, M. H., K. J. Francoijs, et al. (2004). "The PAS fold. A redefinition of the PAS domain based upon structural prediction." European journal of biochemistry / FEBS 271(6): 1198- 1208. Henry, J. T. and S. Crosson (2011). "Ligand-binding PAS domains in a genomic, cellular, and structural context." Annual review of microbiology 65: 261-286.

Hester, I., S. McKee, et al. (2007). "Transient expression of Nxf, a bHLH-PAS transactivator induced by neuronal preconditioning, confers neuroprotection in cultured cells." Brain research 1135(1): 1-11. Hirose, K., M. Morita, et al. (1996). "cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt)." Molecular and cellular biology 16(4): 1706-1713. Hoffman, E. C., H. Reyes, et al. (1991). "Cloning of a factor required for activity of the Ah (dioxin) receptor." Science 252(5008): 954-958. Hogenesch, J. B., Y. Z. Gu, et al. (1998). "The basic-helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors." Proceedings of the National Academy of Sciences of the United States of America 95(10): 5474-5479. Holmes, J. L. and R. S. Pollenz (1997). "Determination of aryl hydrocarbon receptor nuclear translocator protein concentration and subcellular localization in hepatic and nonhepatic cell culture lines: development of quantitative Western blotting protocols for calculation of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein in total cell lysates." Molecular pharmacology 52(2): 202-211. Huang, N., Y. Chelliah, et al. (2012). "Crystal structure of the heterodimeric CLOCK:BMAL1 transcriptional activator complex." Science 337(6091): 189-194. Jiang, L. and S. T. Crews (2003). "The Drosophila dysfusion basic helix-loop-helix (bHLH)-PAS gene controls tracheal fusion and levels of the trachealess bHLH-PAS protein." Molecular and cellular biology 23(16): 5625-5637. Jiang, L. and S. T. Crews (2006). "Dysfusion transcriptional control of Drosophila tracheal migration, adhesion, and fusion." Molecular and cellular biology 26(17): 6547-6556. Jiang, L. and S. T. Crews (2007). "Transcriptional specificity of Drosophila dysfusion and the control of tracheal fusion cell gene expression." The Journal of biological chemistry 282(39): 28659-28668. Kaas, G. A., C. Zhong, et al. (2013). "TET1 controls CNS 5-methylcytosine hydroxylation, active DNA demethylation, gene transcription, and memory formation." Neuron 79(6): 1086- 1093. Kawajiri, K., Y. Kobayashi, et al. (2009). "Aryl hydrocarbon receptor suppresses intestinal carcinogenesis in ApcMin/+ mice with natural ligands." Proceedings of the National Academy of Sciences of the United States of America 106(32): 13481-13486. Kehrer, C., N. Maziashvili, et al. (2008). "Altered Excitatory-Inhibitory Balance in the NMDA- Hypofunction Model of Schizophrenia." Frontiers in molecular neuroscience 1: 6. Kewley, R. J., M. L. Whitelaw, et al. (2004). "The mammalian basic helix-loop-helix/PAS family of transcriptional regulators." The international journal of biochemistry & cell biology 36(2): 189-204. Kim, T. K., M. Hemberg, et al. (2010). "Widespread transcription at neuronal activity-regulated enhancers." Nature 465(7295): 182-187. Kimura, H., A. Weisz, et al. (2001). "Identification of hypoxia-inducible factor 1 ancillary sequence and its function in vascular endothelial growth factor gene induction by hypoxia and nitric oxide." The Journal of biological chemistry 276(3): 2292-2298. Ledent, V. and M. Vervoort (2001). "The basic helix-loop-helix protein family: comparative genomics and phylogenetic analysis." Genome research 11(5): 754-770. Leong, W. K., T. S. Klaric, et al. (2013). "Upregulation of the neuronal Per-Arnt-Sim domain protein 4 (Npas4) in the rat corticolimbic system following focal cerebral ischemia." The European journal of neuroscience 37(11): 1875-1884. Lin, Y., B. L. Bloodgood, et al. (2008). "Activity-dependent regulation of inhibitory synapse development by Npas4." Nature 455(7217): 1198-1204.

Ma, P. C., M. A. Rould, et al. (1994). "Crystal structure of MyoD bHLH domain-DNA complex: perspectives on DNA recognition and implications for transcriptional activation." Cell 77(3): 451-459. Makino, Y., R. Cao, et al. (2001). "Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression." Nature 414(6863): 550-554. Makino, Y., A. Kanopka, et al. (2002). "Inhibitory PAS domain protein (IPAS) is a hypoxia- inducible splicing variant of the hypoxia-inducible factor-3alpha locus." The Journal of biological chemistry 277(36): 32405-32408. Marcheva, B., K. M. Ramsey, et al. (2010). "Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes." Nature 466(7306): 627-631. Martin, T. A., S. Jayanthi, et al. (2012). "Methamphetamine causes differential alterations in gene expression and patterns of histone acetylation/hypoacetylation in the rat nucleus accumbens." PloS one 7(3): e34236. Marty, S., R. Wehrle, et al. (2000). "Neuronal activity and brain-derived neurotrophic factor regulate the density of inhibitory synapses in organotypic slice cultures of postnatal hippocampus." The Journal of neuroscience : the official journal of the Society for Neuroscience 20(21): 8087-8095. Massari, M. E. and C. Murre (2000). "Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms." Molecular and cellular biology 20(2): 429-440. Maya-Vetencourt, J. F., E. Tiraboschi, et al. (2012). "Experience-dependent expression of NPAS4 regulates plasticity in adult visual cortex." The Journal of physiology 590(Pt 19): 4777-4787. McIntosh, B. E., J. B. Hogenesch, et al. (2010). "Mammalian Per-Arnt-Sim proteins in environmental adaptation." Annual review of physiology 72: 625-645. Mimura, J., M. Ema, et al. (1999). "Identification of a novel mechanism of regulation of Ah (dioxin) receptor function." Genes & development 13(1): 20-25. Moglich, A., R. A. Ayers, et al. (2009). "Structure and signaling mechanism of Per-ARNT-Sim domains." Structure 17(10): 1282-1294. Moser, M., R. Knoth, et al. (2004). "LE-PAS, a novel Arnt-dependent HLH-PAS protein, is expressed in limbic tissues and transactivates the CNS midline enhancer element." Brain research. Molecular brain research 128(2): 141-149. Ohtake, F., A. Baba, et al. (2007). "Dioxin receptor is a ligand-dependent E3 ubiquitin ligase." Nature 446(7135): 562-566. Ooe, N., K. Kobayashi, et al. (2009). "Dynamic regulation of bHLH-PAS-type transcription factor NXF gene expression and neurotrophin dependent induction of the transcriptional control activity." Biochemical and biophysical research communications 378(4): 761- 765. Ooe, N., K. Motonaga, et al. (2009). "Functional characterization of basic helix-loop-helix-PAS type transcription factor NXF in vivo: putative involvement in an "on demand" neuroprotection system." The Journal of biological chemistry 284(2): 1057-1063. Ooe, N., K. Saito, et al. (2009). "Characterization of functional heterodimer partners in brain for a bHLH-PAS factor NXF." Biochimica et biophysica acta 1789(3): 192-197. Ooe, N., K. Saito, et al. (2004). "Identification of a novel basic helix-loop-helix-PAS factor, NXF, reveals a Sim2 competitive, positive regulatory role in dendritic-cytoskeleton modulator drebrin gene expression." Molecular and cellular biology 24(2): 608-616. Ooe, N., K. Saito, et al. (2007). "Characterization of Drosophila and Caenorhabditis elegans NXF-like-factors, putative homologs of mammalian NXF." Gene 400(1-2): 122-130. Partch, C. L., P. B. Card, et al. (2009). "Molecular basis of coiled coil coactivator recruitment by the aryl hydrocarbon receptor nuclear translocator (ARNT)." The Journal of biological chemistry 284(22): 15184-15192.

Partch, C. L. and K. H. Gardner (2010). "Coactivator recruitment: a new role for PAS domains in transcriptional regulation by the bHLH-PAS family." Journal of cellular physiology 223(3): 553-557. Partch, C. L. and K. H. Gardner (2011). "Coactivators necessary for transcriptional output of the hypoxia inducible factor, HIF, are directly recruited by ARNT PAS-B." Proceedings of the National Academy of Sciences of the United States of America 108(19): 7739-7744. Pattabiraman, P. P., D. Tropea, et al. (2005). "Neuronal activity regulates the developmental expression and subcellular localization of cortical BDNF mRNA isoforms in vivo." Molecular and cellular neurosciences 28(3): 556-570. Pearson, J. C., J. D. Watson, et al. (2012). "Drosophila melanogaster Zelda and Single-minded collaborate to regulate an evolutionarily dynamic CNS midline cell enhancer." Developmental biology 366(2): 420-432. Peyrefitte, S., D. Kahn, et al. (2001). "New members of the Drosophila Myc transcription factor subfamily revealed by a genome-wide examination for basic helix-loop-helix genes." Mechanisms of development 104(1-2): 99-104. Piechota, M., M. Korostynski, et al. (2010). "The dissection of transcriptional modules regulated by various drugs of abuse in the mouse striatum." Genome biology 11(5): R48. Ploski, J. E., M. S. Monsey, et al. (2011). "The neuronal PAS domain protein 4 (Npas4) is required for new and reactivated fear memories." PloS one 6(8): e23760. Pongratz, I., C. Antonsson, et al. (1998). "Role of the PAS domain in regulation of dimerization and DNA binding specificity of the dioxin receptor." Molecular and cellular biology 18(7): 4079-4088. Pongratz, I., G. G. Mason, et al. (1992). "Dual roles of the 90-kDa heat shock protein hsp90 in modulating functional activities of the dioxin receptor. Evidence that the dioxin receptor functionally belongs to a subclass of nuclear receptors which require hsp90 both for ligand binding activity and repression of intrinsic DNA binding activity." The Journal of biological chemistry 267(19): 13728-13734. Ponting, C. P. and L. Aravind (1997). "PAS: a multifunctional domain family comes to light." Current biology : CB 7(11): R674-677. Pruunsild, P., M. Sepp, et al. (2011). "Identification of cis-elements and transcription factors regulating neuronal activity-dependent transcription of human BDNF gene." The Journal of neuroscience : the official journal of the Society for Neuroscience 31(9): 3295-3308. Ramachandrappa, S., A. Raimondo, et al. (2013). "Rare variants in single-minded 1 (SIM1) are associated with severe obesity." The Journal of clinical investigation 123(7): 3042- 3050. Ramamoorthi, K., R. Fropf, et al. (2011). "Npas4 regulates a transcriptional program in CA3 required for contextual memory formation." Science 334(6063): 1669-1675. Ramocki, M. B. and H. Y. Zoghbi (2008). "Failure of neuronal homeostasis results in common neuropsychiatric phenotypes." Nature 455(7215): 912-918. Reddy, P., W. A. Zehring, et al. (1984). "Molecular analysis of the period locus in Drosophila melanogaster and identification of a transcript involved in biological rhythms." Cell 38(3): 701-710. Reisz-Porszasz, S., M. R. Probst, et al. (1994). "Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT)." Molecular and cellular biology 14(9): 6075-6086. Reppert, S. M. and D. R. Weaver (2002). "Coordination of circadian timing in mammals." Nature 418(6901): 935-941.

Ribeiro, C., A. Ebner, et al. (2002). "In vivo imaging reveals different cellular functions for FGF and Dpp signaling in tracheal branching morphogenesis." Developmental cell 2(5): 677-683. Rubenstein, J. L. (2010). "Three hypotheses for developmental defects that may underlie some forms of autism spectrum disorder." Current opinion in neurology 23(2): 118-123. Rubenstein, J. L. and M. M. Merzenich (2003). "Model of autism: increased ratio of excitation/inhibition in key neural systems." Genes, brain, and behavior 2(5): 255-267. Rudenko, A., M. M. Dawlaty, et al. (2013). "Tet1 is critical for neuronal activity-regulated gene expression and memory extinction." Neuron 79(6): 1109-1122. Rutter, J., M. Reick, et al. (2001). "Regulation of clock and NPAS2 DNA binding by the redox state of NAD cofactors." Science 293(5529): 510-514. Sabatini, P. V., N. A. Krentz, et al. (2013). "Npas4 is a novel activity-regulated cytoprotective factor in pancreatic beta-cells." Diabetes 62(8): 2808-2820. Saxena, S. and P. Caroni (2011). "Selective neuronal vulnerability in neurodegenerative diseases: from stressor thresholds to degeneration." Neuron 71(1): 35-48. Scheuermann, T. H., D. R. Tomchick, et al. (2009). "Artificial ligand binding within the HIF2alpha PAS-B domain of the HIF2 transcription factor." Proceedings of the National Academy of Sciences of the United States of America 106(2): 450-455. Semenza, G. L. (2012). "Hypoxia-inducible factors in physiology and medicine." Cell 148(3): 399-408. Semenza, G. L., B. H. Jiang, et al. (1996). "Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1." The Journal of biological chemistry 271(51): 32529-32537. Shamloo, M., L. Soriano, et al. (2006). "Npas4, a novel helix-loop-helix PAS domain protein, is regulated in response to cerebral ischemia." The European journal of neuroscience 24(10): 2705-2720. Sogawa, K., R. Nakano, et al. (1995). "Possible function of Ah receptor nuclear translocator (Arnt) homodimer in transcriptional regulation." Proceedings of the National Academy of Sciences of the United States of America 92(6): 1936-1940. Spitzer, N. C. (2006). "Electrical activity in early neuronal development." Nature 444(7120): 707-712. Spitzer, N. C. (2012). "Activity-dependent neurotransmitter respecification." Nature reviews. Neuroscience 13(2): 94-106. Sudhof, T. C. (2008). "Neuroligins and neurexins link synaptic function to cognitive disease." Nature 455(7215): 903-911. Swanson, H. I., W. K. Chan, et al. (1995). "DNA binding specificities and pairing rules of the Ah receptor, ARNT, and SIM proteins." The Journal of biological chemistry 270(44): 26292-26302. Swanson, H. I. and J. H. Yang (1999). "Specificity of DNA binding of the c-Myc/Max and ARNT/ARNT dimers at the CACGTG recognition site." Nucleic acids research 27(15): 3205-3212. Sylwestrak, E. and P. Scheiffele (2013). "Neuroscience: Sculpting neuronal connectivity." Nature 503(7474): 42-43. Tanaka-Matakatsu, M., T. Uemura, et al. (1996). "Cadherin-mediated cell adhesion and cell motility in Drosophila trachea regulated by the transcription factor Escargot." Development 122(12): 3697-3705. Tasic, B., S. Hippenmeyer, et al. (2011). "Site-specific integrase-mediated transgenesis in mice via pronuclear injection." Proceedings of the National Academy of Sciences of the United States of America 108(19): 7902-7907. Taylor, B. L. and I. B. Zhulin (1999). "PAS domains: internal sensors of oxygen, redox potential, and light." Microbiology and molecular biology reviews : MMBR 63(2): 479-506.

Tchorz, J. S., T. Suply, et al. (2012). "A modified RMCE-compatible Rosa26 locus for the expression of transgenes from exogenous promoters." PloS one 7(1): e30011. Uniacke, J., C. E. Holterman, et al. (2012). "An oxygen-regulated switch in the protein synthesis machinery." Nature 486(7401): 126-129. Whitelaw, M., I. Pongratz, et al. (1993). "Ligand-dependent recruitment of the Arnt coregulator determines DNA recognition by the dioxin receptor." Molecular and cellular biology 13(4): 2504-2514. Whitelaw, M. L., M. Gottlicher, et al. (1993). "Definition of a novel ligand binding domain of a nuclear bHLH receptor: co-localization of ligand and hsp90 binding activities within the regulable inactivation domain of the dioxin receptor." The EMBO journal 12(11): 4169- 4179. Whitelaw, M. L., J. McGuire, et al. (1995). "Heat shock protein hsp90 regulates dioxin receptor function in vivo." Proceedings of the National Academy of Sciences of the United States of America 92(10): 4437-4441. Wiesel, T. N. (1982). "Postnatal development of the visual cortex and the influence of environment." Nature 299(5884): 583-591. Wilk, R., I. Weizman, et al. (1996). "trachealess encodes a bHLH-PAS protein that is an inducer of tracheal cell fates in Drosophila." Genes & development 10(1): 93-102. Woods, S. L. and M. L. Whitelaw (2002). "Differential activities of murine single minded 1 (SIM1) and SIM2 on a hypoxic response element. Cross-talk between basic helix-loop- helix/per-Arnt-Sim homology transcription factors." The Journal of biological chemistry 277(12): 10236-10243. Wu, H. and Y. Zhang (2011). "Mechanisms and functions of Tet protein-mediated 5- methylcytosine oxidation." Genes & development 25(23): 2436-2452. Yang, J., K. D. Kim, et al. (2008). "A novel heme-regulatory motif mediates heme-dependent degradation of the circadian factor period 2." Molecular and cellular biology 28(15): 4697-4711. Yizhar, O., L. E. Fenno, et al. (2011). "Neocortical excitation/inhibition balance in information processing and social dysfunction." Nature 477(7363): 171-178. Yun, Z., H. L. Maecker, et al. (2002). "Inhibition of PPAR gamma 2 gene expression by the HIF- 1-regulated gene DEC1/Stra13: a mechanism for regulation of adipogenesis by hypoxia." Developmental cell 2(3): 331-341. Zelzer, E., P. Wappner, et al. (1997). "The PAS domain confers target gene specificity of Drosophila bHLH/PAS proteins." Genes & development 11(16): 2079-2089. Zhang, S. J., M. Zou, et al. (2009). "Nuclear calcium signaling controls expression of a large gene pool: identification of a gene program for acquired neuroprotection induced by synaptic activity." PLoS genetics 5(8): e1000604. Zhulin, I. B., B. L. Taylor, et al. (1997). "PAS domain S-boxes in Archaea, Bacteria and sensors for oxygen and redox." Trends in biochemical sciences 22(9): 331-333.

Atchley, W. R. and W. M. Fitch (1997). "A natural classification of the basic helix-loop-helix class of transcription factors." Proceedings of the National Academy of Sciences of the United States of America 94(10): 5172-5176. Baxendale, S., C. J. Holdsworth, et al. (2012). "Identification of compounds with anti- convulsant properties in a zebrafish model of epileptic seizures." Disease models & mechanisms 5(6): 773-784. Baxevanis, A. D. and C. R. Vinson (1993). "Interactions of coiled coils in transcription factors: where is the specificity?" Current opinion in genetics & development 3(2): 278-285. Bersten, D. C., A. E. Sullivan, et al. (2013). "bHLH-PAS proteins in cancer." Nature reviews. Cancer 13(12): 827-841. Bezprozvanny, I. and M. P. Mattson (2008). "Neuronal calcium mishandling and the pathogenesis of Alzheimer's disease." Trends in neurosciences 31(9): 454-463. Blackwell, T. K., L. Kretzner, et al. (1990). "Sequence-specific DNA binding by the c-Myc protein." Science 250(4984): 1149-1151. Blackwell, T. K. and H. Weintraub (1990). "Differences and similarities in DNA-binding preferences of MyoD and E2A protein complexes revealed by binding site selection." Science 250(4984): 1104-1110. Bloodgood, B. L., N. Sharma, et al. (2013). "The activity-dependent transcription factor NPAS4 regulates domain-specific inhibition." Nature 503(7474): 121-125. Bonnefond, A., A. Raimondo, et al. (2013). "Loss-of-function mutations in SIM1 contribute to obesity and Prader-Willi-like features." The Journal of clinical investigation 123(7): 3037-3041. Bronson, S. K., E. G. Plaehn, et al. (1996). "Single-copy transgenic mice with chosen-site integration." Proceedings of the National Academy of Sciences of the United States of America 93(17): 9067-9072. Brunskill, E. W., D. P. Witte, et al. (1999). "Characterization of npas3, a novel basic helix-loop- helix PAS gene expressed in the developing mouse nervous system." Mechanisms of development 88(2): 237-241. Card, P. B., P. J. Erbel, et al. (2005). "Structural basis of ARNT PAS-B dimerization: use of a common beta-sheet interface for hetero- and homodimerization." Journal of molecular biology 353(3): 664-677. Chan, W. K., G. Yao, et al. (1999). "Cross-talk between the aryl hydrocarbon receptor and hypoxia inducible factor signaling pathways. Demonstration of competition and compensation." The Journal of biological chemistry 274(17): 12115-12123. Chapman-Smith, A., J. K. Lutwyche, et al. (2004). "Contribution of the Per/Arnt/Sim (PAS) domains to DNA binding by the basic helix-loop-helix PAS transcriptional regulators." The Journal of biological chemistry 279(7): 5353-5362. Chapman-Smith, A. and M. L. Whitelaw (2006). "Novel DNA binding by a basic helix-loop-helix protein. The role of the dioxin receptor PAS domain." The Journal of biological chemistry 281(18): 12535-12545. Chen, W. G., Q. Chang, et al. (2003). "Derepression of BDNF transcription involves calcium- dependent phosphorylation of MeCP2." Science 302(5646): 885-889. Coba, M. P., L. M. Valor, et al. (2008). "Kinase networks integrate profiles of N-methyl-D- aspartate receptor-mediated gene expression in hippocampus." The Journal of biological chemistry 283(49): 34101-34107. Coumailleau, P., L. Poellinger, et al. (1995). "Definition of a minimal domain of the dioxin receptor that is associated with Hsp90 and maintains wild type ligand binding affinity and specificity." The Journal of biological chemistry 270(42): 25291-25300. Coutellier, L., S. Beraki, et al. (2012). "Npas4: a neuronal transcription factor with a key role in social and cognitive functions relevant to developmental disorders." PloS one 7(9): e46604.

Crews, S. T. (1998). "Control of cell lineage-specific development and transcription by bHLH- PAS proteins." Genes & development 12(5): 607-620. Crews, S. T. and C. M. Fan (1999). "Remembrance of things PAS: regulation of development by bHLH-PAS proteins." Current opinion in genetics & development 9(5): 580-587. Crews, S. T., J. B. Thomas, et al. (1988). "The Drosophila single-minded gene encodes a nuclear protein with sequence similarity to the per gene product." Cell 52(1): 143-151. Dang, E. V., J. Barbi, et al. (2011). "Control of T(H)17/T(reg) balance by hypoxia-inducible factor 1." Cell 146(5): 772-784. Davis, R. L. and H. Weintraub (1992). "Acquisition of myogenic specificity by replacement of three amino acid residues from MyoD into E12." Science 256(5059): 1027-1030. Dean, C., H. Liu, et al. (2012). "Distinct subsets of Syt-IV/BDNF vesicles are sorted to axons versus dendrites and recruited to synapses by activity." The Journal of neuroscience : the official journal of the Society for Neuroscience 32(16): 5398-5413. Dioum, E. M., J. Rutter, et al. (2002). "NPAS2: a gas-responsive transcription factor." Science 298(5602): 2385-2387. Ebert, D. H., H. W. Gabel, et al. (2013). "Activity-dependent phosphorylation of MeCP2 threonine 308 regulates interaction with NCoR." Nature 499(7458): 341-345. Ebert, D. H. and M. E. Greenberg (2013). "Activity-dependent neuronal signalling and autism spectrum disorder." Nature 493(7432): 327-337. Englund, C., P. Steneberg, et al. (2002). "Attractive and repulsive functions of Slit are mediated by different receptors in the Drosophila trachea." Development 129(21): 4941-4951. Erbel, P. J., P. B. Card, et al. (2003). "Structural basis for PAS domain heterodimerization in the basic helix--loop--helix-PAS transcription factor hypoxia-inducible factor." Proceedings of the National Academy of Sciences of the United States of America 100(26): 15504- 15509. Farrall, A. L. and M. L. Whitelaw (2009). "The HIF1alpha-inducible pro-cell death gene BNIP3 is a novel target of SIM2s repression through cross-talk on the hypoxia response element." Oncogene 28(41): 3671-3680. Ferre-D'Amare, A. R., G. C. Prendergast, et al. (1993). "Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain." Nature 363(6424): 38-45. Flavell, S. W., T. K. Kim, et al. (2008). "Genome-wide analysis of MEF2 transcriptional program reveals synaptic target genes and neuronal activity-dependent polyadenylation site selection." Neuron 60(6): 1022-1038. Flood, W. D., R. W. Moyer, et al. (2004). "Nxf and Fbxo33: novel seizure-responsive genes in mice." The European journal of neuroscience 20(7): 1819-1826. Fukunaga, B. N., M. R. Probst, et al. (1995). "Identification of functional domains of the aryl hydrocarbon receptor." The Journal of biological chemistry 270(49): 29270-29278. Genick, U. K., G. E. Borgstahl, et al. (1997). "Structure of a protein photocycle intermediate by millisecond time-resolved crystallography." Science 275(5305): 1471-1475. Ghabrial, A., S. Luschnig, et al. (2003). "Branching morphogenesis of the Drosophila tracheal system." Annual review of cell and developmental biology 19: 623-647. Greer, P. L. and M. E. Greenberg (2008). "From synapse to nucleus: calcium-dependent gene transcription in the control of synapse development and function." Neuron 59(6): 846- 860. Gunton, J. E., R. N. Kulkarni, et al. (2005). "Loss of ARNT/HIF1beta mediates altered gene expression and pancreatic-islet dysfunction in human type 2 diabetes." Cell 122(3): 337-349. Guo, M. L., B. Xue, et al. (2012). "Upregulation of Npas4 protein expression by chronic administration of amphetamine in rat nucleus accumbens in vivo." Neuroscience letters 528(2): 210-214.

Haenebalcke, L., S. Goossens, et al. (2013). "Efficient ROSA26-based conditional and/or inducible transgenesis using RMCE-compatible F1 hybrid mouse embryonic stem cells." Stem Cell Rev 9(6): 774-785. Hao, N. and M. L. Whitelaw (2013). "The emerging roles of AhR in physiology and immunity." Biochemical pharmacology 86(5): 561-570. Hao, N., M. L. Whitelaw, et al. (2011). "Identification of residues in the N-terminal PAS domains important for dimerization of Arnt and AhR." Nucleic acids research 39(9): 3695-3709. Hefti, M. H., K. J. Francoijs, et al. (2004). "The PAS fold. A redefinition of the PAS domain based upon structural prediction." European journal of biochemistry / FEBS 271(6): 1198- 1208. Henry, J. T. and S. Crosson (2011). "Ligand-binding PAS domains in a genomic, cellular, and structural context." Annual review of microbiology 65: 261-286. Hester, I., S. McKee, et al. (2007). "Transient expression of Nxf, a bHLH-PAS transactivator induced by neuronal preconditioning, confers neuroprotection in cultured cells." Brain research 1135(1): 1-11. Hirose, K., M. Morita, et al. (1996). "cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt)." Molecular and cellular biology 16(4): 1706-1713. Hoffman, E. C., H. Reyes, et al. (1991). "Cloning of a factor required for activity of the Ah (dioxin) receptor." Science 252(5008): 954-958. Hogenesch, J. B., Y. Z. Gu, et al. (1998). "The basic-helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors." Proceedings of the National Academy of Sciences of the United States of America 95(10): 5474-5479. Holmes, J. L. and R. S. Pollenz (1997). "Determination of aryl hydrocarbon receptor nuclear translocator protein concentration and subcellular localization in hepatic and nonhepatic cell culture lines: development of quantitative Western blotting protocols for calculation of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein in total cell lysates." Molecular pharmacology 52(2): 202-211. Huang, N., Y. Chelliah, et al. (2012). "Crystal structure of the heterodimeric CLOCK:BMAL1 transcriptional activator complex." Science 337(6091): 189-194. Jiang, L. and S. T. Crews (2003). "The Drosophila dysfusion basic helix-loop-helix (bHLH)-PAS gene controls tracheal fusion and levels of the trachealess bHLH-PAS protein." Molecular and cellular biology 23(16): 5625-5637. Jiang, L. and S. T. Crews (2006). "Dysfusion transcriptional control of Drosophila tracheal migration, adhesion, and fusion." Molecular and cellular biology 26(17): 6547-6556. Jiang, L. and S. T. Crews (2007). "Transcriptional specificity of Drosophila dysfusion and the control of tracheal fusion cell gene expression." The Journal of biological chemistry 282(39): 28659-28668. Kaas, G. A., C. Zhong, et al. (2013). "TET1 controls CNS 5-methylcytosine hydroxylation, active DNA demethylation, gene transcription, and memory formation." Neuron 79(6): 1086- 1093. Kawajiri, K., Y. Kobayashi, et al. (2009). "Aryl hydrocarbon receptor suppresses intestinal carcinogenesis in ApcMin/+ mice with natural ligands." Proceedings of the National Academy of Sciences of the United States of America 106(32): 13481-13486. Kehrer, C., N. Maziashvili, et al. (2008). "Altered Excitatory-Inhibitory Balance in the NMDA- Hypofunction Model of Schizophrenia." Frontiers in molecular neuroscience 1: 6. Kewley, R. J., M. L. Whitelaw, et al. (2004). "The mammalian basic helix-loop-helix/PAS family of transcriptional regulators." The international journal of biochemistry & cell biology 36(2): 189-204.

Kim, T. K., M. Hemberg, et al. (2010). "Widespread transcription at neuronal activity-regulated enhancers." Nature 465(7295): 182-187. Kimura, H., A. Weisz, et al. (2001). "Identification of hypoxia-inducible factor 1 ancillary sequence and its function in vascular endothelial growth factor gene induction by hypoxia and nitric oxide." The Journal of biological chemistry 276(3): 2292-2298. Ledent, V. and M. Vervoort (2001). "The basic helix-loop-helix protein family: comparative genomics and phylogenetic analysis." Genome research 11(5): 754-770. Leong, W. K., T. S. Klaric, et al. (2013). "Upregulation of the neuronal Per-Arnt-Sim domain protein 4 (Npas4) in the rat corticolimbic system following focal cerebral ischemia." The European journal of neuroscience 37(11): 1875-1884. Lin, Y., B. L. Bloodgood, et al. (2008). "Activity-dependent regulation of inhibitory synapse development by Npas4." Nature 455(7217): 1198-1204. Ma, P. C., M. A. Rould, et al. (1994). "Crystal structure of MyoD bHLH domain-DNA complex: perspectives on DNA recognition and implications for transcriptional activation." Cell 77(3): 451-459. Makino, Y., R. Cao, et al. (2001). "Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression." Nature 414(6863): 550-554. Makino, Y., A. Kanopka, et al. (2002). "Inhibitory PAS domain protein (IPAS) is a hypoxia- inducible splicing variant of the hypoxia-inducible factor-3alpha locus." The Journal of biological chemistry 277(36): 32405-32408. Marcheva, B., K. M. Ramsey, et al. (2010). "Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes." Nature 466(7306): 627-631. Martin, T. A., S. Jayanthi, et al. (2012). "Methamphetamine causes differential alterations in gene expression and patterns of histone acetylation/hypoacetylation in the rat nucleus accumbens." PloS one 7(3): e34236. Marty, S., R. Wehrle, et al. (2000). "Neuronal activity and brain-derived neurotrophic factor regulate the density of inhibitory synapses in organotypic slice cultures of postnatal hippocampus." The Journal of neuroscience : the official journal of the Society for Neuroscience 20(21): 8087-8095. Massari, M. E. and C. Murre (2000). "Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms." Molecular and cellular biology 20(2): 429-440. Maya-Vetencourt, J. F., E. Tiraboschi, et al. (2012). "Experience-dependent expression of NPAS4 regulates plasticity in adult visual cortex." The Journal of physiology 590(Pt 19): 4777-4787. McIntosh, B. E., J. B. Hogenesch, et al. (2010). "Mammalian Per-Arnt-Sim proteins in environmental adaptation." Annual review of physiology 72: 625-645. Mimura, J., M. Ema, et al. (1999). "Identification of a novel mechanism of regulation of Ah (dioxin) receptor function." Genes & development 13(1): 20-25. Moglich, A., R. A. Ayers, et al. (2009). "Structure and signaling mechanism of Per-ARNT-Sim domains." Structure 17(10): 1282-1294. Moser, M., R. Knoth, et al. (2004). "LE-PAS, a novel Arnt-dependent HLH-PAS protein, is expressed in limbic tissues and transactivates the CNS midline enhancer element." Brain research. Molecular brain research 128(2): 141-149. Ohtake, F., A. Baba, et al. (2007). "Dioxin receptor is a ligand-dependent E3 ubiquitin ligase." Nature 446(7135): 562-566. Ooe, N., K. Kobayashi, et al. (2009). "Dynamic regulation of bHLH-PAS-type transcription factor NXF gene expression and neurotrophin dependent induction of the transcriptional control activity." Biochemical and biophysical research communications 378(4): 761- 765.

Ooe, N., K. Motonaga, et al. (2009). "Functional characterization of basic helix-loop-helix-PAS type transcription factor NXF in vivo: putative involvement in an "on demand" neuroprotection system." The Journal of biological chemistry 284(2): 1057-1063. Ooe, N., K. Saito, et al. (2009). "Characterization of functional heterodimer partners in brain for a bHLH-PAS factor NXF." Biochimica et biophysica acta 1789(3): 192-197. Ooe, N., K. Saito, et al. (2004). "Identification of a novel basic helix-loop-helix-PAS factor, NXF, reveals a Sim2 competitive, positive regulatory role in dendritic-cytoskeleton modulator drebrin gene expression." Molecular and cellular biology 24(2): 608-616. Ooe, N., K. Saito, et al. (2007). "Characterization of Drosophila and Caenorhabditis elegans NXF-like-factors, putative homologs of mammalian NXF." Gene 400(1-2): 122-130. Partch, C. L., P. B. Card, et al. (2009). "Molecular basis of coiled coil coactivator recruitment by the aryl hydrocarbon receptor nuclear translocator (ARNT)." The Journal of biological chemistry 284(22): 15184-15192. Partch, C. L. and K. H. Gardner (2010). "Coactivator recruitment: a new role for PAS domains in transcriptional regulation by the bHLH-PAS family." Journal of cellular physiology 223(3): 553-557. Partch, C. L. and K. H. Gardner (2011). "Coactivators necessary for transcriptional output of the hypoxia inducible factor, HIF, are directly recruited by ARNT PAS-B." Proceedings of the National Academy of Sciences of the United States of America 108(19): 7739-7744. Pattabiraman, P. P., D. Tropea, et al. (2005). "Neuronal activity regulates the developmental expression and subcellular localization of cortical BDNF mRNA isoforms in vivo." Molecular and cellular neurosciences 28(3): 556-570. Pearson, J. C., J. D. Watson, et al. (2012). "Drosophila melanogaster Zelda and Single-minded collaborate to regulate an evolutionarily dynamic CNS midline cell enhancer." Developmental biology 366(2): 420-432. Peyrefitte, S., D. Kahn, et al. (2001). "New members of the Drosophila Myc transcription factor subfamily revealed by a genome-wide examination for basic helix-loop-helix genes." Mechanisms of development 104(1-2): 99-104. Piechota, M., M. Korostynski, et al. (2010). "The dissection of transcriptional modules regulated by various drugs of abuse in the mouse striatum." Genome biology 11(5): R48. Ploski, J. E., M. S. Monsey, et al. (2011). "The neuronal PAS domain protein 4 (Npas4) is required for new and reactivated fear memories." PloS one 6(8): e23760. Pongratz, I., C. Antonsson, et al. (1998). "Role of the PAS domain in regulation of dimerization and DNA binding specificity of the dioxin receptor." Molecular and cellular biology 18(7): 4079-4088. Pongratz, I., G. G. Mason, et al. (1992). "Dual roles of the 90-kDa heat shock protein hsp90 in modulating functional activities of the dioxin receptor. Evidence that the dioxin receptor functionally belongs to a subclass of nuclear receptors which require hsp90 both for ligand binding activity and repression of intrinsic DNA binding activity." The Journal of biological chemistry 267(19): 13728-13734. Ponting, C. P. and L. Aravind (1997). "PAS: a multifunctional domain family comes to light." Current biology : CB 7(11): R674-677. Pruunsild, P., M. Sepp, et al. (2011). "Identification of cis-elements and transcription factors regulating neuronal activity-dependent transcription of human BDNF gene." The Journal of neuroscience : the official journal of the Society for Neuroscience 31(9): 3295-3308. Ramachandrappa, S., A. Raimondo, et al. (2013). "Rare variants in single-minded 1 (SIM1) are associated with severe obesity." The Journal of clinical investigation 123(7): 3042- 3050.

Ramamoorthi, K., R. Fropf, et al. (2011). "Npas4 regulates a transcriptional program in CA3 required for contextual memory formation." Science 334(6063): 1669-1675. Ramocki, M. B. and H. Y. Zoghbi (2008). "Failure of neuronal homeostasis results in common neuropsychiatric phenotypes." Nature 455(7215): 912-918. Reddy, P., W. A. Zehring, et al. (1984). "Molecular analysis of the period locus in Drosophila melanogaster and identification of a transcript involved in biological rhythms." Cell 38(3): 701-710. Reisz-Porszasz, S., M. R. Probst, et al. (1994). "Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT)." Molecular and cellular biology 14(9): 6075-6086. Reppert, S. M. and D. R. Weaver (2002). "Coordination of circadian timing in mammals." Nature 418(6901): 935-941. Ribeiro, C., A. Ebner, et al. (2002). "In vivo imaging reveals different cellular functions for FGF and Dpp signaling in tracheal branching morphogenesis." Developmental cell 2(5): 677-683. Rubenstein, J. L. (2010). "Three hypotheses for developmental defects that may underlie some forms of autism spectrum disorder." Current opinion in neurology 23(2): 118-123. Rubenstein, J. L. and M. M. Merzenich (2003). "Model of autism: increased ratio of excitation/inhibition in key neural systems." Genes, brain, and behavior 2(5): 255-267. Rudenko, A., M. M. Dawlaty, et al. (2013). "Tet1 is critical for neuronal activity-regulated gene expression and memory extinction." Neuron 79(6): 1109-1122. Rutter, J., M. Reick, et al. (2001). "Regulation of clock and NPAS2 DNA binding by the redox state of NAD cofactors." Science 293(5529): 510-514. Sabatini, P. V., N. A. Krentz, et al. (2013). "Npas4 is a novel activity-regulated cytoprotective factor in pancreatic beta-cells." Diabetes 62(8): 2808-2820. Saxena, S. and P. Caroni (2011). "Selective neuronal vulnerability in neurodegenerative diseases: from stressor thresholds to degeneration." Neuron 71(1): 35-48. Scheuermann, T. H., D. R. Tomchick, et al. (2009). "Artificial ligand binding within the HIF2alpha PAS-B domain of the HIF2 transcription factor." Proceedings of the National Academy of Sciences of the United States of America 106(2): 450-455. Semenza, G. L. (2012). "Hypoxia-inducible factors in physiology and medicine." Cell 148(3): 399-408. Semenza, G. L., B. H. Jiang, et al. (1996). "Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1." The Journal of biological chemistry 271(51): 32529-32537. Shamloo, M., L. Soriano, et al. (2006). "Npas4, a novel helix-loop-helix PAS domain protein, is regulated in response to cerebral ischemia." The European journal of neuroscience 24(10): 2705-2720. Sogawa, K., R. Nakano, et al. (1995). "Possible function of Ah receptor nuclear translocator (Arnt) homodimer in transcriptional regulation." Proceedings of the National Academy of Sciences of the United States of America 92(6): 1936-1940. Spitzer, N. C. (2006). "Electrical activity in early neuronal development." Nature 444(7120): 707-712. Spitzer, N. C. (2012). "Activity-dependent neurotransmitter respecification." Nature reviews. Neuroscience 13(2): 94-106. Sudhof, T. C. (2008). "Neuroligins and neurexins link synaptic function to cognitive disease." Nature 455(7215): 903-911. Swanson, H. I., W. K. Chan, et al. (1995). "DNA binding specificities and pairing rules of the Ah receptor, ARNT, and SIM proteins." The Journal of biological chemistry 270(44): 26292-26302.

Swanson, H. I. and J. H. Yang (1999). "Specificity of DNA binding of the c-Myc/Max and ARNT/ARNT dimers at the CACGTG recognition site." Nucleic acids research 27(15): 3205-3212. Sylwestrak, E. and P. Scheiffele (2013). "Neuroscience: Sculpting neuronal connectivity." Nature 503(7474): 42-43. Tanaka-Matakatsu, M., T. Uemura, et al. (1996). "Cadherin-mediated cell adhesion and cell motility in Drosophila trachea regulated by the transcription factor Escargot." Development 122(12): 3697-3705. Tasic, B., S. Hippenmeyer, et al. (2011). "Site-specific integrase-mediated transgenesis in mice via pronuclear injection." Proceedings of the National Academy of Sciences of the United States of America 108(19): 7902-7907. Taylor, B. L. and I. B. Zhulin (1999). "PAS domains: internal sensors of oxygen, redox potential, and light." Microbiology and molecular biology reviews : MMBR 63(2): 479-506. Tchorz, J. S., T. Suply, et al. (2012). "A modified RMCE-compatible Rosa26 locus for the expression of transgenes from exogenous promoters." PloS one 7(1): e30011. Uniacke, J., C. E. Holterman, et al. (2012). "An oxygen-regulated switch in the protein synthesis machinery." Nature 486(7401): 126-129. Whitelaw, M., I. Pongratz, et al. (1993). "Ligand-dependent recruitment of the Arnt coregulator determines DNA recognition by the dioxin receptor." Molecular and cellular biology 13(4): 2504-2514. Whitelaw, M. L., M. Gottlicher, et al. (1993). "Definition of a novel ligand binding domain of a nuclear bHLH receptor: co-localization of ligand and hsp90 binding activities within the regulable inactivation domain of the dioxin receptor." The EMBO journal 12(11): 4169- 4179. Whitelaw, M. L., J. McGuire, et al. (1995). "Heat shock protein hsp90 regulates dioxin receptor function in vivo." Proceedings of the National Academy of Sciences of the United States of America 92(10): 4437-4441. Wiesel, T. N. (1982). "Postnatal development of the visual cortex and the influence of environment." Nature 299(5884): 583-591. Wilk, R., I. Weizman, et al. (1996). "trachealess encodes a bHLH-PAS protein that is an inducer of tracheal cell fates in Drosophila." Genes & development 10(1): 93-102. Woods, S. L. and M. L. Whitelaw (2002). "Differential activities of murine single minded 1 (SIM1) and SIM2 on a hypoxic response element. Cross-talk between basic helix-loop- helix/per-Arnt-Sim homology transcription factors." The Journal of biological chemistry 277(12): 10236-10243. Wu, H. and Y. Zhang (2011). "Mechanisms and functions of Tet protein-mediated 5- methylcytosine oxidation." Genes & development 25(23): 2436-2452. Yang, J., K. D. Kim, et al. (2008). "A novel heme-regulatory motif mediates heme-dependent degradation of the circadian factor period 2." Molecular and cellular biology 28(15): 4697-4711. Yizhar, O., L. E. Fenno, et al. (2011). "Neocortical excitation/inhibition balance in information processing and social dysfunction." Nature 477(7363): 171-178. Yun, Z., H. L. Maecker, et al. (2002). "Inhibition of PPAR gamma 2 gene expression by the HIF- 1-regulated gene DEC1/Stra13: a mechanism for regulation of adipogenesis by hypoxia." Developmental cell 2(3): 331-341. Zelzer, E., P. Wappner, et al. (1997). "The PAS domain confers target gene specificity of Drosophila bHLH/PAS proteins." Genes & development 11(16): 2079-2089. Zhang, S. J., M. Zou, et al. (2009). "Nuclear calcium signaling controls expression of a large gene pool: identification of a gene program for acquired neuroprotection induced by synaptic activity." PLoS genetics 5(8): e1000604.

Zhulin, I. B., B. L. Taylor, et al. (1997). "PAS domain S-boxes in Archaea, Bacteria and sensors for oxygen and redox." Trends in biochemical sciences 22(9): 331-333.

Coba, M. P., L. M. Valor, et al. (2008). "Kinase networks integrate profiles of N-methyl-D- aspartate receptor-mediated gene expression in hippocampus." The Journal of biological chemistry 283(49): 34101-34107. Coumailleau, P., L. Poellinger, et al. (1995). "Definition of a minimal domain of the dioxin receptor that is associated with Hsp90 and maintains wild type ligand binding affinity and specificity." The Journal of biological chemistry 270(42): 25291-25300. Coutellier, L., S. Beraki, et al. (2012). "Npas4: a neuronal transcription factor with a key role in social and cognitive functions relevant to developmental disorders." PloS one 7(9): e46604. Crews, S. T. (1998). "Control of cell lineage-specific development and transcription by bHLH- PAS proteins." Genes & development 12(5): 607-620. Crews, S. T. and C. M. Fan (1999). "Remembrance of things PAS: regulation of development by bHLH-PAS proteins." Current opinion in genetics & development 9(5): 580-587. Crews, S. T., J. B. Thomas, et al. (1988). "The Drosophila single-minded gene encodes a nuclear protein with sequence similarity to the per gene product." Cell 52(1): 143-151. Dang, E. V., J. Barbi, et al. (2011). "Control of T(H)17/T(reg) balance by hypoxia-inducible factor 1." Cell 146(5): 772-784. Davis, R. L. and H. Weintraub (1992). "Acquisition of myogenic specificity by replacement of three amino acid residues from MyoD into E12." Science 256(5059): 1027-1030. Dean, C., H. Liu, et al. (2012). "Distinct subsets of Syt-IV/BDNF vesicles are sorted to axons versus dendrites and recruited to synapses by activity." The Journal of neuroscience : the official journal of the Society for Neuroscience 32(16): 5398-5413. Dioum, E. M., J. Rutter, et al. (2002). "NPAS2: a gas-responsive transcription factor." Science 298(5602): 2385-2387. Ebert, D. H., H. W. Gabel, et al. (2013). "Activity-dependent phosphorylation of MeCP2 threonine 308 regulates interaction with NCoR." Nature 499(7458): 341-345. Ebert, D. H. and M. E. Greenberg (2013). "Activity-dependent neuronal signalling and autism spectrum disorder." Nature 493(7432): 327-337. Englund, C., P. Steneberg, et al. (2002). "Attractive and repulsive functions of Slit are mediated by different receptors in the Drosophila trachea." Development 129(21): 4941-4951. Erbel, P. J., P. B. Card, et al. (2003). "Structural basis for PAS domain heterodimerization in the basic helix--loop--helix-PAS transcription factor hypoxia-inducible factor." Proceedings of the National Academy of Sciences of the United States of America 100(26): 15504- 15509. Farrall, A. L. and M. L. Whitelaw (2009). "The HIF1alpha-inducible pro-cell death gene BNIP3 is a novel target of SIM2s repression through cross-talk on the hypoxia response element." Oncogene 28(41): 3671-3680. Ferre-D'Amare, A. R., G. C. Prendergast, et al. (1993). "Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain." Nature 363(6424): 38-45. Flavell, S. W., T. K. Kim, et al. (2008). "Genome-wide analysis of MEF2 transcriptional program reveals synaptic target genes and neuronal activity-dependent polyadenylation site selection." Neuron 60(6): 1022-1038. Flood, W. D., R. W. Moyer, et al. (2004). "Nxf and Fbxo33: novel seizure-responsive genes in mice." The European journal of neuroscience 20(7): 1819-1826. Fukunaga, B. N., M. R. Probst, et al. (1995). "Identification of functional domains of the aryl hydrocarbon receptor." The Journal of biological chemistry 270(49): 29270-29278. Genick, U. K., G. E. Borgstahl, et al. (1997). "Structure of a protein photocycle intermediate by millisecond time-resolved crystallography." Science 275(5305): 1471-1475. Ghabrial, A., S. Luschnig, et al. (2003). "Branching morphogenesis of the Drosophila tracheal system." Annual review of cell and developmental biology 19: 623-647.

Greer, P. L. and M. E. Greenberg (2008). "From synapse to nucleus: calcium-dependent gene transcription in the control of synapse development and function." Neuron 59(6): 846- 860. Gunton, J. E., R. N. Kulkarni, et al. (2005). "Loss of ARNT/HIF1beta mediates altered gene expression and pancreatic-islet dysfunction in human type 2 diabetes." Cell 122(3): 337-349. Guo, M. L., B. Xue, et al. (2012). "Upregulation of Npas4 protein expression by chronic administration of amphetamine in rat nucleus accumbens in vivo." Neuroscience letters 528(2): 210-214. Haenebalcke, L., S. Goossens, et al. (2013). "Efficient ROSA26-based conditional and/or inducible transgenesis using RMCE-compatible F1 hybrid mouse embryonic stem cells." Stem Cell Rev 9(6): 774-785. Hao, N. and M. L. Whitelaw (2013). "The emerging roles of AhR in physiology and immunity." Biochemical pharmacology 86(5): 561-570. Hao, N., M. L. Whitelaw, et al. (2011). "Identification of residues in the N-terminal PAS domains important for dimerization of Arnt and AhR." Nucleic acids research 39(9): 3695-3709. Hefti, M. H., K. J. Francoijs, et al. (2004). "The PAS fold. A redefinition of the PAS domain based upon structural prediction." European journal of biochemistry / FEBS 271(6): 1198- 1208. Henry, J. T. and S. Crosson (2011). "Ligand-binding PAS domains in a genomic, cellular, and structural context." Annual review of microbiology 65: 261-286. Hester, I., S. McKee, et al. (2007). "Transient expression of Nxf, a bHLH-PAS transactivator induced by neuronal preconditioning, confers neuroprotection in cultured cells." Brain research 1135(1): 1-11. Hirose, K., M. Morita, et al. (1996). "cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt)." Molecular and cellular biology 16(4): 1706-1713. Hoffman, E. C., H. Reyes, et al. (1991). "Cloning of a factor required for activity of the Ah (dioxin) receptor." Science 252(5008): 954-958. Hogenesch, J. B., Y. Z. Gu, et al. (1998). "The basic-helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors." Proceedings of the National Academy of Sciences of the United States of America 95(10): 5474-5479. Holmes, J. L. and R. S. Pollenz (1997). "Determination of aryl hydrocarbon receptor nuclear translocator protein concentration and subcellular localization in hepatic and nonhepatic cell culture lines: development of quantitative Western blotting protocols for calculation of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein in total cell lysates." Molecular pharmacology 52(2): 202-211. Huang, N., Y. Chelliah, et al. (2012). "Crystal structure of the heterodimeric CLOCK:BMAL1 transcriptional activator complex." Science 337(6091): 189-194. Jiang, L. and S. T. Crews (2003). "The Drosophila dysfusion basic helix-loop-helix (bHLH)-PAS gene controls tracheal fusion and levels of the trachealess bHLH-PAS protein." Molecular and cellular biology 23(16): 5625-5637. Jiang, L. and S. T. Crews (2006). "Dysfusion transcriptional control of Drosophila tracheal migration, adhesion, and fusion." Molecular and cellular biology 26(17): 6547-6556. Jiang, L. and S. T. Crews (2007). "Transcriptional specificity of Drosophila dysfusion and the control of tracheal fusion cell gene expression." The Journal of biological chemistry 282(39): 28659-28668.

Spitzer, N. C. (2006). "Electrical activity in early neuronal development." Nature 444(7120): 707-712. Spitzer, N. C. (2012). "Activity-dependent neurotransmitter respecification." Nature reviews. Neuroscience 13(2): 94-106. Sudhof, T. C. (2008). "Neuroligins and neurexins link synaptic function to cognitive disease." Nature 455(7215): 903-911. Swanson, H. I., W. K. Chan, et al. (1995). "DNA binding specificities and pairing rules of the Ah receptor, ARNT, and SIM proteins." The Journal of biological chemistry 270(44): 26292-26302. Swanson, H. I. and J. H. Yang (1999). "Specificity of DNA binding of the c-Myc/Max and ARNT/ARNT dimers at the CACGTG recognition site." Nucleic acids research 27(15): 3205-3212. Sylwestrak, E. and P. Scheiffele (2013). "Neuroscience: Sculpting neuronal connectivity." Nature 503(7474): 42-43. Tanaka-Matakatsu, M., T. Uemura, et al. (1996). "Cadherin-mediated cell adhesion and cell motility in Drosophila trachea regulated by the transcription factor Escargot." Development 122(12): 3697-3705. Tasic, B., S. Hippenmeyer, et al. (2011). "Site-specific integrase-mediated transgenesis in mice via pronuclear injection." Proceedings of the National Academy of Sciences of the United States of America 108(19): 7902-7907. Taylor, B. L. and I. B. Zhulin (1999). "PAS domains: internal sensors of oxygen, redox potential, and light." Microbiology and molecular biology reviews : MMBR 63(2): 479-506. Tchorz, J. S., T. Suply, et al. (2012). "A modified RMCE-compatible Rosa26 locus for the expression of transgenes from exogenous promoters." PloS one 7(1): e30011. Uniacke, J., C. E. Holterman, et al. (2012). "An oxygen-regulated switch in the protein synthesis machinery." Nature 486(7401): 126-129. Whitelaw, M., I. Pongratz, et al. (1993). "Ligand-dependent recruitment of the Arnt coregulator determines DNA recognition by the dioxin receptor." Molecular and cellular biology 13(4): 2504-2514. Whitelaw, M. L., M. Gottlicher, et al. (1993). "Definition of a novel ligand binding domain of a nuclear bHLH receptor: co-localization of ligand and hsp90 binding activities within the regulable inactivation domain of the dioxin receptor." The EMBO journal 12(11): 4169- 4179. Whitelaw, M. L., J. McGuire, et al. (1995). "Heat shock protein hsp90 regulates dioxin receptor function in vivo." Proceedings of the National Academy of Sciences of the United States of America 92(10): 4437-4441. Wiesel, T. N. (1982). "Postnatal development of the visual cortex and the influence of environment." Nature 299(5884): 583-591. Wilk, R., I. Weizman, et al. (1996). "trachealess encodes a bHLH-PAS protein that is an inducer of tracheal cell fates in Drosophila." Genes & development 10(1): 93-102. Woods, S. L. and M. L. Whitelaw (2002). "Differential activities of murine single minded 1 (SIM1) and SIM2 on a hypoxic response element. Cross-talk between basic helix-loop- helix/per-Arnt-Sim homology transcription factors." The Journal of biological chemistry 277(12): 10236-10243. Wu, H. and Y. Zhang (2011). "Mechanisms and functions of Tet protein-mediated 5- methylcytosine oxidation." Genes & development 25(23): 2436-2452. Yang, J., K. D. Kim, et al. (2008). "A novel heme-regulatory motif mediates heme-dependent degradation of the circadian factor period 2." Molecular and cellular biology 28(15): 4697-4711. Yizhar, O., L. E. Fenno, et al. (2011). "Neocortical excitation/inhibition balance in information processing and social dysfunction." Nature 477(7363): 171-178.

Yun, Z., H. L. Maecker, et al. (2002). "Inhibition of PPAR gamma 2 gene expression by the HIF- 1-regulated gene DEC1/Stra13: a mechanism for regulation of adipogenesis by hypoxia." Developmental cell 2(3): 331-341. Zelzer, E., P. Wappner, et al. (1997). "The PAS domain confers target gene specificity of Drosophila bHLH/PAS proteins." Genes & development 11(16): 2079-2089. Zhang, S. J., M. Zou, et al. (2009). "Nuclear calcium signaling controls expression of a large gene pool: identification of a gene program for acquired neuroprotection induced by synaptic activity." PLoS genetics 5(8): e1000604. Zhulin, I. B., B. L. Taylor, et al. (1997). "PAS domain S-boxes in Archaea, Bacteria and sensors for oxygen and redox." Trends in biochemical sciences 22(9): 331-333.

Chapman-Smith, A., J. K. Lutwyche, et al. (2004). "Contribution of the Per/Arnt/Sim (PAS) domains to DNA binding by the basic helix-loop-helix PAS transcriptional regulators." The Journal of biological chemistry 279(7): 5353-5362. Chapman-Smith, A. and M. L. Whitelaw (2006). "Novel DNA binding by a basic helix-loop-helix protein. The role of the dioxin receptor PAS domain." The Journal of biological chemistry 281(18): 12535-12545. Chen, W. G., Q. Chang, et al. (2003). "Derepression of BDNF transcription involves calcium- dependent phosphorylation of MeCP2." Science 302(5646): 885-889. Coba, M. P., L. M. Valor, et al. (2008). "Kinase networks integrate profiles of N-methyl-D- aspartate receptor-mediated gene expression in hippocampus." The Journal of biological chemistry 283(49): 34101-34107. Coumailleau, P., L. Poellinger, et al. (1995). "Definition of a minimal domain of the dioxin receptor that is associated with Hsp90 and maintains wild type ligand binding affinity and specificity." The Journal of biological chemistry 270(42): 25291-25300. Coutellier, L., S. Beraki, et al. (2012). "Npas4: a neuronal transcription factor with a key role in social and cognitive functions relevant to developmental disorders." PloS one 7(9): e46604. Crews, S. T. (1998). "Control of cell lineage-specific development and transcription by bHLH- PAS proteins." Genes & development 12(5): 607-620. Crews, S. T. and C. M. Fan (1999). "Remembrance of things PAS: regulation of development by bHLH-PAS proteins." Current opinion in genetics & development 9(5): 580-587. Crews, S. T., J. B. Thomas, et al. (1988). "The Drosophila single-minded gene encodes a nuclear protein with sequence similarity to the per gene product." Cell 52(1): 143-151. Dang, E. V., J. Barbi, et al. (2011). "Control of T(H)17/T(reg) balance by hypoxia-inducible factor 1." Cell 146(5): 772-784. Davis, R. L. and H. Weintraub (1992). "Acquisition of myogenic specificity by replacement of three amino acid residues from MyoD into E12." Science 256(5059): 1027-1030. Dean, C., H. Liu, et al. (2012). "Distinct subsets of Syt-IV/BDNF vesicles are sorted to axons versus dendrites and recruited to synapses by activity." The Journal of neuroscience : the official journal of the Society for Neuroscience 32(16): 5398-5413. Dioum, E. M., J. Rutter, et al. (2002). "NPAS2: a gas-responsive transcription factor." Science 298(5602): 2385-2387. Ebert, D. H., H. W. Gabel, et al. (2013). "Activity-dependent phosphorylation of MeCP2 threonine 308 regulates interaction with NCoR." Nature 499(7458): 341-345. Ebert, D. H. and M. E. Greenberg (2013). "Activity-dependent neuronal signalling and autism spectrum disorder." Nature 493(7432): 327-337. Englund, C., P. Steneberg, et al. (2002). "Attractive and repulsive functions of Slit are mediated by different receptors in the Drosophila trachea." Development 129(21): 4941-4951. Erbel, P. J., P. B. Card, et al. (2003). "Structural basis for PAS domain heterodimerization in the basic helix--loop--helix-PAS transcription factor hypoxia-inducible factor." Proceedings of the National Academy of Sciences of the United States of America 100(26): 15504- 15509. Farrall, A. L. and M. L. Whitelaw (2009). "The HIF1alpha-inducible pro-cell death gene BNIP3 is a novel target of SIM2s repression through cross-talk on the hypoxia response element." Oncogene 28(41): 3671-3680. Ferre-D'Amare, A. R., G. C. Prendergast, et al. (1993). "Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain." Nature 363(6424): 38-45. Flavell, S. W., T. K. Kim, et al. (2008). "Genome-wide analysis of MEF2 transcriptional program reveals synaptic target genes and neuronal activity-dependent polyadenylation site selection." Neuron 60(6): 1022-1038.

Flood, W. D., R. W. Moyer, et al. (2004). "Nxf and Fbxo33: novel seizure-responsive genes in mice." The European journal of neuroscience 20(7): 1819-1826. Fukunaga, B. N., M. R. Probst, et al. (1995). "Identification of functional domains of the aryl hydrocarbon receptor." The Journal of biological chemistry 270(49): 29270-29278. Genick, U. K., G. E. Borgstahl, et al. (1997). "Structure of a protein photocycle intermediate by millisecond time-resolved crystallography." Science 275(5305): 1471-1475. Ghabrial, A., S. Luschnig, et al. (2003). "Branching morphogenesis of the Drosophila tracheal system." Annual review of cell and developmental biology 19: 623-647. Greer, P. L. and M. E. Greenberg (2008). "From synapse to nucleus: calcium-dependent gene transcription in the control of synapse development and function." Neuron 59(6): 846- 860. Gunton, J. E., R. N. Kulkarni, et al. (2005). "Loss of ARNT/HIF1beta mediates altered gene expression and pancreatic-islet dysfunction in human type 2 diabetes." Cell 122(3): 337-349. Guo, M. L., B. Xue, et al. (2012). "Upregulation of Npas4 protein expression by chronic administration of amphetamine in rat nucleus accumbens in vivo." Neuroscience letters 528(2): 210-214. Haenebalcke, L., S. Goossens, et al. (2013). "Efficient ROSA26-based conditional and/or inducible transgenesis using RMCE-compatible F1 hybrid mouse embryonic stem cells." Stem Cell Rev 9(6): 774-785. Hao, N. and M. L. Whitelaw (2013). "The emerging roles of AhR in physiology and immunity." Biochemical pharmacology 86(5): 561-570. Hao, N., M. L. Whitelaw, et al. (2011). "Identification of residues in the N-terminal PAS domains important for dimerization of Arnt and AhR." Nucleic acids research 39(9): 3695-3709. Hefti, M. H., K. J. Francoijs, et al. (2004). "The PAS fold. A redefinition of the PAS domain based upon structural prediction." European journal of biochemistry / FEBS 271(6): 1198- 1208. Henry, J. T. and S. Crosson (2011). "Ligand-binding PAS domains in a genomic, cellular, and structural context." Annual review of microbiology 65: 261-286. Hester, I., S. McKee, et al. (2007). "Transient expression of Nxf, a bHLH-PAS transactivator induced by neuronal preconditioning, confers neuroprotection in cultured cells." Brain research 1135(1): 1-11. Hirose, K., M. Morita, et al. (1996). "cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt)." Molecular and cellular biology 16(4): 1706-1713. Hoffman, E. C., H. Reyes, et al. (1991). "Cloning of a factor required for activity of the Ah (dioxin) receptor." Science 252(5008): 954-958. Hogenesch, J. B., Y. Z. Gu, et al. (1998). "The basic-helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors." Proceedings of the National Academy of Sciences of the United States of America 95(10): 5474-5479. Holmes, J. L. and R. S. Pollenz (1997). "Determination of aryl hydrocarbon receptor nuclear translocator protein concentration and subcellular localization in hepatic and nonhepatic cell culture lines: development of quantitative Western blotting protocols for calculation of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein in total cell lysates." Molecular pharmacology 52(2): 202-211. Huang, N., Y. Chelliah, et al. (2012). "Crystal structure of the heterodimeric CLOCK:BMAL1 transcriptional activator complex." Science 337(6091): 189-194.

Jiang, L. and S. T. Crews (2003). "The Drosophila dysfusion basic helix-loop-helix (bHLH)-PAS gene controls tracheal fusion and levels of the trachealess bHLH-PAS protein." Molecular and cellular biology 23(16): 5625-5637. Jiang, L. and S. T. Crews (2006). "Dysfusion transcriptional control of Drosophila tracheal migration, adhesion, and fusion." Molecular and cellular biology 26(17): 6547-6556. Jiang, L. and S. T. Crews (2007). "Transcriptional specificity of Drosophila dysfusion and the control of tracheal fusion cell gene expression." The Journal of biological chemistry 282(39): 28659-28668. Kaas, G. A., C. Zhong, et al. (2013). "TET1 controls CNS 5-methylcytosine hydroxylation, active DNA demethylation, gene transcription, and memory formation." Neuron 79(6): 1086- 1093. Kawajiri, K., Y. Kobayashi, et al. (2009). "Aryl hydrocarbon receptor suppresses intestinal carcinogenesis in ApcMin/+ mice with natural ligands." Proceedings of the National Academy of Sciences of the United States of America 106(32): 13481-13486. Kehrer, C., N. Maziashvili, et al. (2008). "Altered Excitatory-Inhibitory Balance in the NMDA- Hypofunction Model of Schizophrenia." Frontiers in molecular neuroscience 1: 6. Kewley, R. J., M. L. Whitelaw, et al. (2004). "The mammalian basic helix-loop-helix/PAS family of transcriptional regulators." The international journal of biochemistry & cell biology 36(2): 189-204. Kim, T. K., M. Hemberg, et al. (2010). "Widespread transcription at neuronal activity-regulated enhancers." Nature 465(7295): 182-187. Kimura, H., A. Weisz, et al. (2001). "Identification of hypoxia-inducible factor 1 ancillary sequence and its function in vascular endothelial growth factor gene induction by hypoxia and nitric oxide." The Journal of biological chemistry 276(3): 2292-2298. Ledent, V. and M. Vervoort (2001). "The basic helix-loop-helix protein family: comparative genomics and phylogenetic analysis." Genome research 11(5): 754-770. Leong, W. K., T. S. Klaric, et al. (2013). "Upregulation of the neuronal Per-Arnt-Sim domain protein 4 (Npas4) in the rat corticolimbic system following focal cerebral ischemia." The European journal of neuroscience 37(11): 1875-1884. Lin, Y., B. L. Bloodgood, et al. (2008). "Activity-dependent regulation of inhibitory synapse development by Npas4." Nature 455(7217): 1198-1204. Ma, P. C., M. A. Rould, et al. (1994). "Crystal structure of MyoD bHLH domain-DNA complex: perspectives on DNA recognition and implications for transcriptional activation." Cell 77(3): 451-459. Makino, Y., R. Cao, et al. (2001). "Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression." Nature 414(6863): 550-554. Makino, Y., A. Kanopka, et al. (2002). "Inhibitory PAS domain protein (IPAS) is a hypoxia- inducible splicing variant of the hypoxia-inducible factor-3alpha locus." The Journal of biological chemistry 277(36): 32405-32408. Marcheva, B., K. M. Ramsey, et al. (2010). "Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes." Nature 466(7306): 627-631. Martin, T. A., S. Jayanthi, et al. (2012). "Methamphetamine causes differential alterations in gene expression and patterns of histone acetylation/hypoacetylation in the rat nucleus accumbens." PloS one 7(3): e34236. Marty, S., R. Wehrle, et al. (2000). "Neuronal activity and brain-derived neurotrophic factor regulate the density of inhibitory synapses in organotypic slice cultures of postnatal hippocampus." The Journal of neuroscience : the official journal of the Society for Neuroscience 20(21): 8087-8095. Massari, M. E. and C. Murre (2000). "Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms." Molecular and cellular biology 20(2): 429-440.

Maya-Vetencourt, J. F., E. Tiraboschi, et al. (2012). "Experience-dependent expression of NPAS4 regulates plasticity in adult visual cortex." The Journal of physiology 590(Pt 19): 4777-4787. McIntosh, B. E., J. B. Hogenesch, et al. (2010). "Mammalian Per-Arnt-Sim proteins in environmental adaptation." Annual review of physiology 72: 625-645. Mimura, J., M. Ema, et al. (1999). "Identification of a novel mechanism of regulation of Ah (dioxin) receptor function." Genes & development 13(1): 20-25. Moglich, A., R. A. Ayers, et al. (2009). "Structure and signaling mechanism of Per-ARNT-Sim domains." Structure 17(10): 1282-1294. Moser, M., R. Knoth, et al. (2004). "LE-PAS, a novel Arnt-dependent HLH-PAS protein, is expressed in limbic tissues and transactivates the CNS midline enhancer element." Brain research. Molecular brain research 128(2): 141-149. Ohtake, F., A. Baba, et al. (2007). "Dioxin receptor is a ligand-dependent E3 ubiquitin ligase." Nature 446(7135): 562-566. Ooe, N., K. Kobayashi, et al. (2009). "Dynamic regulation of bHLH-PAS-type transcription factor NXF gene expression and neurotrophin dependent induction of the transcriptional control activity." Biochemical and biophysical research communications 378(4): 761- 765. Ooe, N., K. Motonaga, et al. (2009). "Functional characterization of basic helix-loop-helix-PAS type transcription factor NXF in vivo: putative involvement in an "on demand" neuroprotection system." The Journal of biological chemistry 284(2): 1057-1063. Ooe, N., K. Saito, et al. (2009). "Characterization of functional heterodimer partners in brain for a bHLH-PAS factor NXF." Biochimica et biophysica acta 1789(3): 192-197. Ooe, N., K. Saito, et al. (2004). "Identification of a novel basic helix-loop-helix-PAS factor, NXF, reveals a Sim2 competitive, positive regulatory role in dendritic-cytoskeleton modulator drebrin gene expression." Molecular and cellular biology 24(2): 608-616. Ooe, N., K. Saito, et al. (2007). "Characterization of Drosophila and Caenorhabditis elegans NXF-like-factors, putative homologs of mammalian NXF." Gene 400(1-2): 122-130. Partch, C. L., P. B. Card, et al. (2009). "Molecular basis of coiled coil coactivator recruitment by the aryl hydrocarbon receptor nuclear translocator (ARNT)." The Journal of biological chemistry 284(22): 15184-15192. Partch, C. L. and K. H. Gardner (2010). "Coactivator recruitment: a new role for PAS domains in transcriptional regulation by the bHLH-PAS family." Journal of cellular physiology 223(3): 553-557. Partch, C. L. and K. H. Gardner (2011). "Coactivators necessary for transcriptional output of the hypoxia inducible factor, HIF, are directly recruited by ARNT PAS-B." Proceedings of the National Academy of Sciences of the United States of America 108(19): 7739-7744. Pattabiraman, P. P., D. Tropea, et al. (2005). "Neuronal activity regulates the developmental expression and subcellular localization of cortical BDNF mRNA isoforms in vivo." Molecular and cellular neurosciences 28(3): 556-570. Pearson, J. C., J. D. Watson, et al. (2012). "Drosophila melanogaster Zelda and Single-minded collaborate to regulate an evolutionarily dynamic CNS midline cell enhancer." Developmental biology 366(2): 420-432. Peyrefitte, S., D. Kahn, et al. (2001). "New members of the Drosophila Myc transcription factor subfamily revealed by a genome-wide examination for basic helix-loop-helix genes." Mechanisms of development 104(1-2): 99-104. Piechota, M., M. Korostynski, et al. (2010). "The dissection of transcriptional modules regulated by various drugs of abuse in the mouse striatum." Genome biology 11(5): R48. Ploski, J. E., M. S. Monsey, et al. (2011). "The neuronal PAS domain protein 4 (Npas4) is required for new and reactivated fear memories." PloS one 6(8): e23760.

Pongratz, I., C. Antonsson, et al. (1998). "Role of the PAS domain in regulation of dimerization and DNA binding specificity of the dioxin receptor." Molecular and cellular biology 18(7): 4079-4088. Pongratz, I., G. G. Mason, et al. (1992). "Dual roles of the 90-kDa heat shock protein hsp90 in modulating functional activities of the dioxin receptor. Evidence that the dioxin receptor functionally belongs to a subclass of nuclear receptors which require hsp90 both for ligand binding activity and repression of intrinsic DNA binding activity." The Journal of biological chemistry 267(19): 13728-13734. Ponting, C. P. and L. Aravind (1997). "PAS: a multifunctional domain family comes to light." Current biology : CB 7(11): R674-677. Pruunsild, P., M. Sepp, et al. (2011). "Identification of cis-elements and transcription factors regulating neuronal activity-dependent transcription of human BDNF gene." The Journal of neuroscience : the official journal of the Society for Neuroscience 31(9): 3295-3308. Ramachandrappa, S., A. Raimondo, et al. (2013). "Rare variants in single-minded 1 (SIM1) are associated with severe obesity." The Journal of clinical investigation 123(7): 3042- 3050. Ramamoorthi, K., R. Fropf, et al. (2011). "Npas4 regulates a transcriptional program in CA3 required for contextual memory formation." Science 334(6063): 1669-1675. Ramocki, M. B. and H. Y. Zoghbi (2008). "Failure of neuronal homeostasis results in common neuropsychiatric phenotypes." Nature 455(7215): 912-918. Reddy, P., W. A. Zehring, et al. (1984). "Molecular analysis of the period locus in Drosophila melanogaster and identification of a transcript involved in biological rhythms." Cell 38(3): 701-710. Reisz-Porszasz, S., M. R. Probst, et al. (1994). "Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT)." Molecular and cellular biology 14(9): 6075-6086. Reppert, S. M. and D. R. Weaver (2002). "Coordination of circadian timing in mammals." Nature 418(6901): 935-941. Ribeiro, C., A. Ebner, et al. (2002). "In vivo imaging reveals different cellular functions for FGF and Dpp signaling in tracheal branching morphogenesis." Developmental cell 2(5): 677-683. Rubenstein, J. L. (2010). "Three hypotheses for developmental defects that may underlie some forms of autism spectrum disorder." Current opinion in neurology 23(2): 118-123. Rubenstein, J. L. and M. M. Merzenich (2003). "Model of autism: increased ratio of excitation/inhibition in key neural systems." Genes, brain, and behavior 2(5): 255-267. Rudenko, A., M. M. Dawlaty, et al. (2013). "Tet1 is critical for neuronal activity-regulated gene expression and memory extinction." Neuron 79(6): 1109-1122. Rutter, J., M. Reick, et al. (2001). "Regulation of clock and NPAS2 DNA binding by the redox state of NAD cofactors." Science 293(5529): 510-514. Sabatini, P. V., N. A. Krentz, et al. (2013). "Npas4 is a novel activity-regulated cytoprotective factor in pancreatic beta-cells." Diabetes 62(8): 2808-2820. Saxena, S. and P. Caroni (2011). "Selective neuronal vulnerability in neurodegenerative diseases: from stressor thresholds to degeneration." Neuron 71(1): 35-48. Scheuermann, T. H., D. R. Tomchick, et al. (2009). "Artificial ligand binding within the HIF2alpha PAS-B domain of the HIF2 transcription factor." Proceedings of the National Academy of Sciences of the United States of America 106(2): 450-455. Semenza, G. L. (2012). "Hypoxia-inducible factors in physiology and medicine." Cell 148(3): 399-408.

Semenza, G. L., B. H. Jiang, et al. (1996). "Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1." The Journal of biological chemistry 271(51): 32529-32537. Shamloo, M., L. Soriano, et al. (2006). "Npas4, a novel helix-loop-helix PAS domain protein, is regulated in response to cerebral ischemia." The European journal of neuroscience 24(10): 2705-2720. Sogawa, K., R. Nakano, et al. (1995). "Possible function of Ah receptor nuclear translocator (Arnt) homodimer in transcriptional regulation." Proceedings of the National Academy of Sciences of the United States of America 92(6): 1936-1940. Spitzer, N. C. (2006). "Electrical activity in early neuronal development." Nature 444(7120): 707-712. Spitzer, N. C. (2012). "Activity-dependent neurotransmitter respecification." Nature reviews. Neuroscience 13(2): 94-106. Sudhof, T. C. (2008). "Neuroligins and neurexins link synaptic function to cognitive disease." Nature 455(7215): 903-911. Swanson, H. I., W. K. Chan, et al. (1995). "DNA binding specificities and pairing rules of the Ah receptor, ARNT, and SIM proteins." The Journal of biological chemistry 270(44): 26292-26302. Swanson, H. I. and J. H. Yang (1999). "Specificity of DNA binding of the c-Myc/Max and ARNT/ARNT dimers at the CACGTG recognition site." Nucleic acids research 27(15): 3205-3212. Sylwestrak, E. and P. Scheiffele (2013). "Neuroscience: Sculpting neuronal connectivity." Nature 503(7474): 42-43. Tanaka-Matakatsu, M., T. Uemura, et al. (1996). "Cadherin-mediated cell adhesion and cell motility in Drosophila trachea regulated by the transcription factor Escargot." Development 122(12): 3697-3705. Taylor, B. L. and I. B. Zhulin (1999). "PAS domains: internal sensors of oxygen, redox potential, and light." Microbiology and molecular biology reviews : MMBR 63(2): 479-506. Tchorz, J. S., T. Suply, et al. (2012). "A modified RMCE-compatible Rosa26 locus for the expression of transgenes from exogenous promoters." PloS one 7(1): e30011. Uniacke, J., C. E. Holterman, et al. (2012). "An oxygen-regulated switch in the protein synthesis machinery." Nature 486(7401): 126-129. Whitelaw, M., I. Pongratz, et al. (1993). "Ligand-dependent recruitment of the Arnt coregulator determines DNA recognition by the dioxin receptor." Molecular and cellular biology 13(4): 2504-2514. Whitelaw, M. L., M. Gottlicher, et al. (1993). "Definition of a novel ligand binding domain of a nuclear bHLH receptor: co-localization of ligand and hsp90 binding activities within the regulable inactivation domain of the dioxin receptor." The EMBO journal 12(11): 4169- 4179. Whitelaw, M. L., J. McGuire, et al. (1995). "Heat shock protein hsp90 regulates dioxin receptor function in vivo." Proceedings of the National Academy of Sciences of the United States of America 92(10): 4437-4441. Wiesel, T. N. (1982). "Postnatal development of the visual cortex and the influence of environment." Nature 299(5884): 583-591. Wilk, R., I. Weizman, et al. (1996). "trachealess encodes a bHLH-PAS protein that is an inducer of tracheal cell fates in Drosophila." Genes & development 10(1): 93-102. Woods, S. L. and M. L. Whitelaw (2002). "Differential activities of murine single minded 1 (SIM1) and SIM2 on a hypoxic response element. Cross-talk between basic helix-loop- helix/per-Arnt-Sim homology transcription factors." The Journal of biological chemistry 277(12): 10236-10243.

Wu, H. and Y. Zhang (2011). "Mechanisms and functions of Tet protein-mediated 5- methylcytosine oxidation." Genes & development 25(23): 2436-2452. Yang, J., K. D. Kim, et al. (2008). "A novel heme-regulatory motif mediates heme-dependent degradation of the circadian factor period 2." Molecular and cellular biology 28(15): 4697-4711. Yizhar, O., L. E. Fenno, et al. (2011). "Neocortical excitation/inhibition balance in information processing and social dysfunction." Nature 477(7363): 171-178. Yun, Z., H. L. Maecker, et al. (2002). "Inhibition of PPAR gamma 2 gene expression by the HIF- 1-regulated gene DEC1/Stra13: a mechanism for regulation of adipogenesis by hypoxia." Developmental cell 2(3): 331-341. Zelzer, E., P. Wappner, et al. (1997). "The PAS domain confers target gene specificity of Drosophila bHLH/PAS proteins." Genes & development 11(16): 2079-2089. Zhang, S. J., M. Zou, et al. (2009). "Nuclear calcium signaling controls expression of a large gene pool: identification of a gene program for acquired neuroprotection induced by synaptic activity." PLoS genetics 5(8): e1000604. Zhulin, I. B., B. L. Taylor, et al. (1997). "PAS domain S-boxes in Archaea, Bacteria and sensors for oxygen and redox." Trends in biochemical sciences 22(9): 331-333.

Brunskill, E. W., D. P. Witte, et al. (1999). "Characterization of npas3, a novel basic helix-loop- helix PAS gene expressed in the developing mouse nervous system." Mechanisms of development 88(2): 237-241. Card, P. B., P. J. Erbel, et al. (2005). "Structural basis of ARNT PAS-B dimerization: use of a common beta-sheet interface for hetero- and homodimerization." Journal of molecular biology 353(3): 664-677. Chan, W. K., G. Yao, et al. (1999). "Cross-talk between the aryl hydrocarbon receptor and hypoxia inducible factor signaling pathways. Demonstration of competition and compensation." The Journal of biological chemistry 274(17): 12115-12123. Chapman-Smith, A., J. K. Lutwyche, et al. (2004). "Contribution of the Per/Arnt/Sim (PAS) domains to DNA binding by the basic helix-loop-helix PAS transcriptional regulators." The Journal of biological chemistry 279(7): 5353-5362. Chapman-Smith, A. and M. L. Whitelaw (2006). "Novel DNA binding by a basic helix-loop-helix protein. The role of the dioxin receptor PAS domain." The Journal of biological chemistry 281(18): 12535-12545. Chen, W. G., Q. Chang, et al. (2003). "Derepression of BDNF transcription involves calcium- dependent phosphorylation of MeCP2." Science 302(5646): 885-889. Coba, M. P., L. M. Valor, et al. (2008). "Kinase networks integrate profiles of N-methyl-D- aspartate receptor-mediated gene expression in hippocampus." The Journal of biological chemistry 283(49): 34101-34107. Coumailleau, P., L. Poellinger, et al. (1995). "Definition of a minimal domain of the dioxin receptor that is associated with Hsp90 and maintains wild type ligand binding affinity and specificity." The Journal of biological chemistry 270(42): 25291-25300. Coutellier, L., S. Beraki, et al. (2012). "Npas4: a neuronal transcription factor with a key role in social and cognitive functions relevant to developmental disorders." PloS one 7(9): e46604. Crews, S. T. (1998). "Control of cell lineage-specific development and transcription by bHLH- PAS proteins." Genes & development 12(5): 607-620. Crews, S. T. and C. M. Fan (1999). "Remembrance of things PAS: regulation of development by bHLH-PAS proteins." Current opinion in genetics & development 9(5): 580-587. Crews, S. T., J. B. Thomas, et al. (1988). "The Drosophila single-minded gene encodes a nuclear protein with sequence similarity to the per gene product." Cell 52(1): 143-151. Dang, E. V., J. Barbi, et al. (2011). "Control of T(H)17/T(reg) balance by hypoxia-inducible factor 1." Cell 146(5): 772-784. Davis, R. L. and H. Weintraub (1992). "Acquisition of myogenic specificity by replacement of three amino acid residues from MyoD into E12." Science 256(5059): 1027-1030. Dean, C., H. Liu, et al. (2012). "Distinct subsets of Syt-IV/BDNF vesicles are sorted to axons versus dendrites and recruited to synapses by activity." The Journal of neuroscience : the official journal of the Society for Neuroscience 32(16): 5398-5413. Dioum, E. M., J. Rutter, et al. (2002). "NPAS2: a gas-responsive transcription factor." Science 298(5602): 2385-2387. Ebert, D. H., H. W. Gabel, et al. (2013). "Activity-dependent phosphorylation of MeCP2 threonine 308 regulates interaction with NCoR." Nature 499(7458): 341-345. Ebert, D. H. and M. E. Greenberg (2013). "Activity-dependent neuronal signalling and autism spectrum disorder." Nature 493(7432): 327-337. Englund, C., P. Steneberg, et al. (2002). "Attractive and repulsive functions of Slit are mediated by different receptors in the Drosophila trachea." Development 129(21): 4941-4951. Erbel, P. J., P. B. Card, et al. (2003). "Structural basis for PAS domain heterodimerization in the basic helix--loop--helix-PAS transcription factor hypoxia-inducible factor." Proceedings of the National Academy of Sciences of the United States of America 100(26): 15504- 15509.

Farrall, A. L. and M. L. Whitelaw (2009). "The HIF1alpha-inducible pro-cell death gene BNIP3 is a novel target of SIM2s repression through cross-talk on the hypoxia response element." Oncogene 28(41): 3671-3680. Ferre-D'Amare, A. R., G. C. Prendergast, et al. (1993). "Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain." Nature 363(6424): 38-45. Flavell, S. W., T. K. Kim, et al. (2008). "Genome-wide analysis of MEF2 transcriptional program reveals synaptic target genes and neuronal activity-dependent polyadenylation site selection." Neuron 60(6): 1022-1038. Flood, W. D., R. W. Moyer, et al. (2004). "Nxf and Fbxo33: novel seizure-responsive genes in mice." The European journal of neuroscience 20(7): 1819-1826. Fukunaga, B. N., M. R. Probst, et al. (1995). "Identification of functional domains of the aryl hydrocarbon receptor." The Journal of biological chemistry 270(49): 29270-29278. Genick, U. K., G. E. Borgstahl, et al. (1997). "Structure of a protein photocycle intermediate by millisecond time-resolved crystallography." Science 275(5305): 1471-1475. Ghabrial, A., S. Luschnig, et al. (2003). "Branching morphogenesis of the Drosophila tracheal system." Annual review of cell and developmental biology 19: 623-647. Greer, P. L. and M. E. Greenberg (2008). "From synapse to nucleus: calcium-dependent gene transcription in the control of synapse development and function." Neuron 59(6): 846- 860. Gunton, J. E., R. N. Kulkarni, et al. (2005). "Loss of ARNT/HIF1beta mediates altered gene expression and pancreatic-islet dysfunction in human type 2 diabetes." Cell 122(3): 337-349. Guo, M. L., B. Xue, et al. (2012). "Upregulation of Npas4 protein expression by chronic administration of amphetamine in rat nucleus accumbens in vivo." Neuroscience letters 528(2): 210-214. Haenebalcke, L., S. Goossens, et al. (2013). "Efficient ROSA26-based conditional and/or inducible transgenesis using RMCE-compatible F1 hybrid mouse embryonic stem cells." Stem Cell Rev 9(6): 774-785. Hao, N. and M. L. Whitelaw (2013). "The emerging roles of AhR in physiology and immunity." Biochemical pharmacology 86(5): 561-570. Hao, N., M. L. Whitelaw, et al. (2011). "Identification of residues in the N-terminal PAS domains important for dimerization of Arnt and AhR." Nucleic acids research 39(9): 3695-3709. Hefti, M. H., K. J. Francoijs, et al. (2004). "The PAS fold. A redefinition of the PAS domain based upon structural prediction." European journal of biochemistry / FEBS 271(6): 1198- 1208. Henry, J. T. and S. Crosson (2011). "Ligand-binding PAS domains in a genomic, cellular, and structural context." Annual review of microbiology 65: 261-286. Hester, I., S. McKee, et al. (2007). "Transient expression of Nxf, a bHLH-PAS transactivator induced by neuronal preconditioning, confers neuroprotection in cultured cells." Brain research 1135(1): 1-11. Hirose, K., M. Morita, et al. (1996). "cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt)." Molecular and cellular biology 16(4): 1706-1713. Hoffman, E. C., H. Reyes, et al. (1991). "Cloning of a factor required for activity of the Ah (dioxin) receptor." Science 252(5008): 954-958. Hogenesch, J. B., Y. Z. Gu, et al. (1998). "The basic-helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors." Proceedings of the National Academy of Sciences of the United States of America 95(10): 5474-5479.

Holmes, J. L. and R. S. Pollenz (1997). "Determination of aryl hydrocarbon receptor nuclear translocator protein concentration and subcellular localization in hepatic and nonhepatic cell culture lines: development of quantitative Western blotting protocols for calculation of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein in total cell lysates." Molecular pharmacology 52(2): 202-211. Huang, N., Y. Chelliah, et al. (2012). "Crystal structure of the heterodimeric CLOCK:BMAL1 transcriptional activator complex." Science 337(6091): 189-194. Jiang, L. and S. T. Crews (2003). "The Drosophila dysfusion basic helix-loop-helix (bHLH)-PAS gene controls tracheal fusion and levels of the trachealess bHLH-PAS protein." Molecular and cellular biology 23(16): 5625-5637. Jiang, L. and S. T. Crews (2006). "Dysfusion transcriptional control of Drosophila tracheal migration, adhesion, and fusion." Molecular and cellular biology 26(17): 6547-6556. Jiang, L. and S. T. Crews (2007). "Transcriptional specificity of Drosophila dysfusion and the control of tracheal fusion cell gene expression." The Journal of biological chemistry 282(39): 28659-28668. Kaas, G. A., C. Zhong, et al. (2013). "TET1 controls CNS 5-methylcytosine hydroxylation, active DNA demethylation, gene transcription, and memory formation." Neuron 79(6): 1086- 1093. Kawajiri, K., Y. Kobayashi, et al. (2009). "Aryl hydrocarbon receptor suppresses intestinal carcinogenesis in ApcMin/+ mice with natural ligands." Proceedings of the National Academy of Sciences of the United States of America 106(32): 13481-13486. Kehrer, C., N. Maziashvili, et al. (2008). "Altered Excitatory-Inhibitory Balance in the NMDA- Hypofunction Model of Schizophrenia." Frontiers in molecular neuroscience 1: 6. Kewley, R. J., M. L. Whitelaw, et al. (2004). "The mammalian basic helix-loop-helix/PAS family of transcriptional regulators." The international journal of biochemistry & cell biology 36(2): 189-204. Kim, T. K., M. Hemberg, et al. (2010). "Widespread transcription at neuronal activity-regulated enhancers." Nature 465(7295): 182-187. Kimura, H., A. Weisz, et al. (2001). "Identification of hypoxia-inducible factor 1 ancillary sequence and its function in vascular endothelial growth factor gene induction by hypoxia and nitric oxide." The Journal of biological chemistry 276(3): 2292-2298. Ledent, V. and M. Vervoort (2001). "The basic helix-loop-helix protein family: comparative genomics and phylogenetic analysis." Genome research 11(5): 754-770. Leong, W. K., T. S. Klaric, et al. (2013). "Upregulation of the neuronal Per-Arnt-Sim domain protein 4 (Npas4) in the rat corticolimbic system following focal cerebral ischemia." The European journal of neuroscience 37(11): 1875-1884. Lin, Y., B. L. Bloodgood, et al. (2008). "Activity-dependent regulation of inhibitory synapse development by Npas4." Nature 455(7217): 1198-1204. Ma, P. C., M. A. Rould, et al. (1994). "Crystal structure of MyoD bHLH domain-DNA complex: perspectives on DNA recognition and implications for transcriptional activation." Cell 77(3): 451-459. Makino, Y., R. Cao, et al. (2001). "Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression." Nature 414(6863): 550-554. Makino, Y., A. Kanopka, et al. (2002). "Inhibitory PAS domain protein (IPAS) is a hypoxia- inducible splicing variant of the hypoxia-inducible factor-3alpha locus." The Journal of biological chemistry 277(36): 32405-32408. Marcheva, B., K. M. Ramsey, et al. (2010). "Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes." Nature 466(7306): 627-631. Martin, T. A., S. Jayanthi, et al. (2012). "Methamphetamine causes differential alterations in gene expression and patterns of histone acetylation/hypoacetylation in the rat nucleus accumbens." PloS one 7(3): e34236.

Marty, S., R. Wehrle, et al. (2000). "Neuronal activity and brain-derived neurotrophic factor regulate the density of inhibitory synapses in organotypic slice cultures of postnatal hippocampus." The Journal of neuroscience : the official journal of the Society for Neuroscience 20(21): 8087-8095. Massari, M. E. and C. Murre (2000). "Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms." Molecular and cellular biology 20(2): 429-440. Maya-Vetencourt, J. F., E. Tiraboschi, et al. (2012). "Experience-dependent expression of NPAS4 regulates plasticity in adult visual cortex." The Journal of physiology 590(Pt 19): 4777-4787. McIntosh, B. E., J. B. Hogenesch, et al. (2010). "Mammalian Per-Arnt-Sim proteins in environmental adaptation." Annual review of physiology 72: 625-645. Mimura, J., M. Ema, et al. (1999). "Identification of a novel mechanism of regulation of Ah (dioxin) receptor function." Genes & development 13(1): 20-25. Moglich, A., R. A. Ayers, et al. (2009). "Structure and signaling mechanism of Per-ARNT-Sim domains." Structure 17(10): 1282-1294. Moser, M., R. Knoth, et al. (2004). "LE-PAS, a novel Arnt-dependent HLH-PAS protein, is expressed in limbic tissues and transactivates the CNS midline enhancer element." Brain research. Molecular brain research 128(2): 141-149. Ohtake, F., A. Baba, et al. (2007). "Dioxin receptor is a ligand-dependent E3 ubiquitin ligase." Nature 446(7135): 562-566. Ooe, N., K. Kobayashi, et al. (2009). "Dynamic regulation of bHLH-PAS-type transcription factor NXF gene expression and neurotrophin dependent induction of the transcriptional control activity." Biochemical and biophysical research communications 378(4): 761- 765. Ooe, N., K. Motonaga, et al. (2009). "Functional characterization of basic helix-loop-helix-PAS type transcription factor NXF in vivo: putative involvement in an "on demand" neuroprotection system." The Journal of biological chemistry 284(2): 1057-1063. Ooe, N., K. Saito, et al. (2009). "Characterization of functional heterodimer partners in brain for a bHLH-PAS factor NXF." Biochimica et biophysica acta 1789(3): 192-197. Ooe, N., K. Saito, et al. (2004). "Identification of a novel basic helix-loop-helix-PAS factor, NXF, reveals a Sim2 competitive, positive regulatory role in dendritic-cytoskeleton modulator drebrin gene expression." Molecular and cellular biology 24(2): 608-616. Ooe, N., K. Saito, et al. (2007). "Characterization of Drosophila and Caenorhabditis elegans NXF-like-factors, putative homologs of mammalian NXF." Gene 400(1-2): 122-130. Partch, C. L., P. B. Card, et al. (2009). "Molecular basis of coiled coil coactivator recruitment by the aryl hydrocarbon receptor nuclear translocator (ARNT)." The Journal of biological chemistry 284(22): 15184-15192. Partch, C. L. and K. H. Gardner (2010). "Coactivator recruitment: a new role for PAS domains in transcriptional regulation by the bHLH-PAS family." Journal of cellular physiology 223(3): 553-557. Partch, C. L. and K. H. Gardner (2011). "Coactivators necessary for transcriptional output of the hypoxia inducible factor, HIF, are directly recruited by ARNT PAS-B." Proceedings of the National Academy of Sciences of the United States of America 108(19): 7739-7744. Pattabiraman, P. P., D. Tropea, et al. (2005). "Neuronal activity regulates the developmental expression and subcellular localization of cortical BDNF mRNA isoforms in vivo." Molecular and cellular neurosciences 28(3): 556-570. Pearson, J. C., J. D. Watson, et al. (2012). "Drosophila melanogaster Zelda and Single-minded collaborate to regulate an evolutionarily dynamic CNS midline cell enhancer." Developmental biology 366(2): 420-432.

Peyrefitte, S., D. Kahn, et al. (2001). "New members of the Drosophila Myc transcription factor subfamily revealed by a genome-wide examination for basic helix-loop-helix genes." Mechanisms of development 104(1-2): 99-104. Piechota, M., M. Korostynski, et al. (2010). "The dissection of transcriptional modules regulated by various drugs of abuse in the mouse striatum." Genome biology 11(5): R48. Ploski, J. E., M. S. Monsey, et al. (2011). "The neuronal PAS domain protein 4 (Npas4) is required for new and reactivated fear memories." PloS one 6(8): e23760. Pongratz, I., C. Antonsson, et al. (1998). "Role of the PAS domain in regulation of dimerization and DNA binding specificity of the dioxin receptor." Molecular and cellular biology 18(7): 4079-4088. Pongratz, I., G. G. Mason, et al. (1992). "Dual roles of the 90-kDa heat shock protein hsp90 in modulating functional activities of the dioxin receptor. Evidence that the dioxin receptor functionally belongs to a subclass of nuclear receptors which require hsp90 both for ligand binding activity and repression of intrinsic DNA binding activity." The Journal of biological chemistry 267(19): 13728-13734. Ponting, C. P. and L. Aravind (1997). "PAS: a multifunctional domain family comes to light." Current biology : CB 7(11): R674-677. Pruunsild, P., M. Sepp, et al. (2011). "Identification of cis-elements and transcription factors regulating neuronal activity-dependent transcription of human BDNF gene." The Journal of neuroscience : the official journal of the Society for Neuroscience 31(9): 3295-3308. Ramachandrappa, S., A. Raimondo, et al. (2013). "Rare variants in single-minded 1 (SIM1) are associated with severe obesity." The Journal of clinical investigation 123(7): 3042- 3050. Ramamoorthi, K., R. Fropf, et al. (2011). "Npas4 regulates a transcriptional program in CA3 required for contextual memory formation." Science 334(6063): 1669-1675. Ramocki, M. B. and H. Y. Zoghbi (2008). "Failure of neuronal homeostasis results in common neuropsychiatric phenotypes." Nature 455(7215): 912-918. Reddy, P., W. A. Zehring, et al. (1984). "Molecular analysis of the period locus in Drosophila melanogaster and identification of a transcript involved in biological rhythms." Cell 38(3): 701-710. Reisz-Porszasz, S., M. R. Probst, et al. (1994). "Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT)." Molecular and cellular biology 14(9): 6075-6086. Reppert, S. M. and D. R. Weaver (2002). "Coordination of circadian timing in mammals." Nature 418(6901): 935-941. Ribeiro, C., A. Ebner, et al. (2002). "In vivo imaging reveals different cellular functions for FGF and Dpp signaling in tracheal branching morphogenesis." Developmental cell 2(5): 677-683. Rubenstein, J. L. (2010). "Three hypotheses for developmental defects that may underlie some forms of autism spectrum disorder." Current opinion in neurology 23(2): 118-123. Rubenstein, J. L. and M. M. Merzenich (2003). "Model of autism: increased ratio of excitation/inhibition in key neural systems." Genes, brain, and behavior 2(5): 255-267. Rudenko, A., M. M. Dawlaty, et al. (2013). "Tet1 is critical for neuronal activity-regulated gene expression and memory extinction." Neuron 79(6): 1109-1122. Rutter, J., M. Reick, et al. (2001). "Regulation of clock and NPAS2 DNA binding by the redox state of NAD cofactors." Science 293(5529): 510-514. Sabatini, P. V., N. A. Krentz, et al. (2013). "Npas4 is a novel activity-regulated cytoprotective factor in pancreatic beta-cells." Diabetes 62(8): 2808-2820.

Saxena, S. and P. Caroni (2011). "Selective neuronal vulnerability in neurodegenerative diseases: from stressor thresholds to degeneration." Neuron 71(1): 35-48. Scheuermann, T. H., D. R. Tomchick, et al. (2009). "Artificial ligand binding within the HIF2alpha PAS-B domain of the HIF2 transcription factor." Proceedings of the National Academy of Sciences of the United States of America 106(2): 450-455. Semenza, G. L. (2012). "Hypoxia-inducible factors in physiology and medicine." Cell 148(3): 399-408. Semenza, G. L., B. H. Jiang, et al. (1996). "Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1." The Journal of biological chemistry 271(51): 32529-32537. Shamloo, M., L. Soriano, et al. (2006). "Npas4, a novel helix-loop-helix PAS domain protein, is regulated in response to cerebral ischemia." The European journal of neuroscience 24(10): 2705-2720. Sogawa, K., R. Nakano, et al. (1995). "Possible function of Ah receptor nuclear translocator (Arnt) homodimer in transcriptional regulation." Proceedings of the National Academy of Sciences of the United States of America 92(6): 1936-1940. Spitzer, N. C. (2006). "Electrical activity in early neuronal development." Nature 444(7120): 707-712. Spitzer, N. C. (2012). "Activity-dependent neurotransmitter respecification." Nature reviews. Neuroscience 13(2): 94-106. Sudhof, T. C. (2008). "Neuroligins and neurexins link synaptic function to cognitive disease." Nature 455(7215): 903-911. Swanson, H. I., W. K. Chan, et al. (1995). "DNA binding specificities and pairing rules of the Ah receptor, ARNT, and SIM proteins." The Journal of biological chemistry 270(44): 26292-26302. Swanson, H. I. and J. H. Yang (1999). "Specificity of DNA binding of the c-Myc/Max and ARNT/ARNT dimers at the CACGTG recognition site." Nucleic acids research 27(15): 3205-3212. Sylwestrak, E. and P. Scheiffele (2013). "Neuroscience: Sculpting neuronal connectivity." Nature 503(7474): 42-43. Tanaka-Matakatsu, M., T. Uemura, et al. (1996). "Cadherin-mediated cell adhesion and cell motility in Drosophila trachea regulated by the transcription factor Escargot." Development 122(12): 3697-3705. Taylor, B. L. and I. B. Zhulin (1999). "PAS domains: internal sensors of oxygen, redox potential, and light." Microbiology and molecular biology reviews : MMBR 63(2): 479-506. Tchorz, J. S., T. Suply, et al. (2012). "A modified RMCE-compatible Rosa26 locus for the expression of transgenes from exogenous promoters." PloS one 7(1): e30011. Uniacke, J., C. E. Holterman, et al. (2012). "An oxygen-regulated switch in the protein synthesis machinery." Nature 486(7401): 126-129. Whitelaw, M., I. Pongratz, et al. (1993). "Ligand-dependent recruitment of the Arnt coregulator determines DNA recognition by the dioxin receptor." Molecular and cellular biology 13(4): 2504-2514. Whitelaw, M. L., M. Gottlicher, et al. (1993). "Definition of a novel ligand binding domain of a nuclear bHLH receptor: co-localization of ligand and hsp90 binding activities within the regulable inactivation domain of the dioxin receptor." The EMBO journal 12(11): 4169- 4179. Whitelaw, M. L., J. McGuire, et al. (1995). "Heat shock protein hsp90 regulates dioxin receptor function in vivo." Proceedings of the National Academy of Sciences of the United States of America 92(10): 4437-4441. Wiesel, T. N. (1982). "Postnatal development of the visual cortex and the influence of environment." Nature 299(5884): 583-591.

Wilk, R., I. Weizman, et al. (1996). "trachealess encodes a bHLH-PAS protein that is an inducer of tracheal cell fates in Drosophila." Genes & development 10(1): 93-102. Woods, S. L. and M. L. Whitelaw (2002). "Differential activities of murine single minded 1 (SIM1) and SIM2 on a hypoxic response element. Cross-talk between basic helix-loop- helix/per-Arnt-Sim homology transcription factors." The Journal of biological chemistry 277(12): 10236-10243. Wu, H. and Y. Zhang (2011). "Mechanisms and functions of Tet protein-mediated 5- methylcytosine oxidation." Genes & development 25(23): 2436-2452. Yang, J., K. D. Kim, et al. (2008). "A novel heme-regulatory motif mediates heme-dependent degradation of the circadian factor period 2." Molecular and cellular biology 28(15): 4697-4711. Yizhar, O., L. E. Fenno, et al. (2011). "Neocortical excitation/inhibition balance in information processing and social dysfunction." Nature 477(7363): 171-178. Yun, Z., H. L. Maecker, et al. (2002). "Inhibition of PPAR gamma 2 gene expression by the HIF- 1-regulated gene DEC1/Stra13: a mechanism for regulation of adipogenesis by hypoxia." Developmental cell 2(3): 331-341. Zelzer, E., P. Wappner, et al. (1997). "The PAS domain confers target gene specificity of Drosophila bHLH/PAS proteins." Genes & development 11(16): 2079-2089. Zhang, S. J., M. Zou, et al. (2009). "Nuclear calcium signaling controls expression of a large gene pool: identification of a gene program for acquired neuroprotection induced by synaptic activity." PLoS genetics 5(8): e1000604. Zhulin, I. B., B. L. Taylor, et al. (1997). "PAS domain S-boxes in Archaea, Bacteria and sensors for oxygen and redox." Trends in biochemical sciences 22(9): 331-333.

Bloodgood, B. L., N. Sharma, et al. (2013). "The activity-dependent transcription factor NPAS4 regulates domain-specific inhibition." Nature 503(7474): 121-125. Bonnefond, A., A. Raimondo, et al. (2013). "Loss-of-function mutations in SIM1 contribute to obesity and Prader-Willi-like features." The Journal of clinical investigation 123(7): 3037-3041. Brunskill, E. W., D. P. Witte, et al. (1999). "Characterization of npas3, a novel basic helix-loop- helix PAS gene expressed in the developing mouse nervous system." Mechanisms of development 88(2): 237-241. Card, P. B., P. J. Erbel, et al. (2005). "Structural basis of ARNT PAS-B dimerization: use of a common beta-sheet interface for hetero- and homodimerization." Journal of molecular biology 353(3): 664-677. Chan, W. K., G. Yao, et al. (1999). "Cross-talk between the aryl hydrocarbon receptor and hypoxia inducible factor signaling pathways. Demonstration of competition and compensation." The Journal of biological chemistry 274(17): 12115-12123. Chapman-Smith, A., J. K. Lutwyche, et al. (2004). "Contribution of the Per/Arnt/Sim (PAS) domains to DNA binding by the basic helix-loop-helix PAS transcriptional regulators." The Journal of biological chemistry 279(7): 5353-5362. Chapman-Smith, A. and M. L. Whitelaw (2006). "Novel DNA binding by a basic helix-loop-helix protein. The role of the dioxin receptor PAS domain." The Journal of biological chemistry 281(18): 12535-12545. Chen, W. G., Q. Chang, et al. (2003). "Derepression of BDNF transcription involves calcium- dependent phosphorylation of MeCP2." Science 302(5646): 885-889. Coba, M. P., L. M. Valor, et al. (2008). "Kinase networks integrate profiles of N-methyl-D- aspartate receptor-mediated gene expression in hippocampus." The Journal of biological chemistry 283(49): 34101-34107. Coumailleau, P., L. Poellinger, et al. (1995). "Definition of a minimal domain of the dioxin receptor that is associated with Hsp90 and maintains wild type ligand binding affinity and specificity." The Journal of biological chemistry 270(42): 25291-25300. Coutellier, L., S. Beraki, et al. (2012). "Npas4: a neuronal transcription factor with a key role in social and cognitive functions relevant to developmental disorders." PloS one 7(9): e46604. Crews, S. T. (1998). "Control of cell lineage-specific development and transcription by bHLH- PAS proteins." Genes & development 12(5): 607-620. Crews, S. T. and C. M. Fan (1999). "Remembrance of things PAS: regulation of development by bHLH-PAS proteins." Current opinion in genetics & development 9(5): 580-587. Crews, S. T., J. B. Thomas, et al. (1988). "The Drosophila single-minded gene encodes a nuclear protein with sequence similarity to the per gene product." Cell 52(1): 143-151. Dang, E. V., J. Barbi, et al. (2011). "Control of T(H)17/T(reg) balance by hypoxia-inducible factor 1." Cell 146(5): 772-784. Davis, R. L. and H. Weintraub (1992). "Acquisition of myogenic specificity by replacement of three amino acid residues from MyoD into E12." Science 256(5059): 1027-1030. Dean, C., H. Liu, et al. (2012). "Distinct subsets of Syt-IV/BDNF vesicles are sorted to axons versus dendrites and recruited to synapses by activity." The Journal of neuroscience : the official journal of the Society for Neuroscience 32(16): 5398-5413. Dioum, E. M., J. Rutter, et al. (2002). "NPAS2: a gas-responsive transcription factor." Science 298(5602): 2385-2387. Ebert, D. H., H. W. Gabel, et al. (2013). "Activity-dependent phosphorylation of MeCP2 threonine 308 regulates interaction with NCoR." Nature 499(7458): 341-345. Ebert, D. H. and M. E. Greenberg (2013). "Activity-dependent neuronal signalling and autism spectrum disorder." Nature 493(7432): 327-337.

Englund, C., P. Steneberg, et al. (2002). "Attractive and repulsive functions of Slit are mediated by different receptors in the Drosophila trachea." Development 129(21): 4941-4951. Erbel, P. J., P. B. Card, et al. (2003). "Structural basis for PAS domain heterodimerization in the basic helix--loop--helix-PAS transcription factor hypoxia-inducible factor." Proceedings of the National Academy of Sciences of the United States of America 100(26): 15504- 15509. Farrall, A. L. and M. L. Whitelaw (2009). "The HIF1alpha-inducible pro-cell death gene BNIP3 is a novel target of SIM2s repression through cross-talk on the hypoxia response element." Oncogene 28(41): 3671-3680. Ferre-D'Amare, A. R., G. C. Prendergast, et al. (1993). "Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain." Nature 363(6424): 38-45. Flavell, S. W., T. K. Kim, et al. (2008). "Genome-wide analysis of MEF2 transcriptional program reveals synaptic target genes and neuronal activity-dependent polyadenylation site selection." Neuron 60(6): 1022-1038. Flood, W. D., R. W. Moyer, et al. (2004). "Nxf and Fbxo33: novel seizure-responsive genes in mice." The European journal of neuroscience 20(7): 1819-1826. Fukunaga, B. N., M. R. Probst, et al. (1995). "Identification of functional domains of the aryl hydrocarbon receptor." The Journal of biological chemistry 270(49): 29270-29278. Genick, U. K., G. E. Borgstahl, et al. (1997). "Structure of a protein photocycle intermediate by millisecond time-resolved crystallography." Science 275(5305): 1471-1475. Ghabrial, A., S. Luschnig, et al. (2003). "Branching morphogenesis of the Drosophila tracheal system." Annual review of cell and developmental biology 19: 623-647. Greer, P. L. and M. E. Greenberg (2008). "From synapse to nucleus: calcium-dependent gene transcription in the control of synapse development and function." Neuron 59(6): 846- 860. Gunton, J. E., R. N. Kulkarni, et al. (2005). "Loss of ARNT/HIF1beta mediates altered gene expression and pancreatic-islet dysfunction in human type 2 diabetes." Cell 122(3): 337-349. Guo, M. L., B. Xue, et al. (2012). "Upregulation of Npas4 protein expression by chronic administration of amphetamine in rat nucleus accumbens in vivo." Neuroscience letters 528(2): 210-214. Haenebalcke, L., S. Goossens, et al. (2013). "Efficient ROSA26-based conditional and/or inducible transgenesis using RMCE-compatible F1 hybrid mouse embryonic stem cells." Stem Cell Rev 9(6): 774-785. Hao, N. and M. L. Whitelaw (2013). "The emerging roles of AhR in physiology and immunity." Biochemical pharmacology 86(5): 561-570. Hao, N., M. L. Whitelaw, et al. (2011). "Identification of residues in the N-terminal PAS domains important for dimerization of Arnt and AhR." Nucleic acids research 39(9): 3695-3709. Hefti, M. H., K. J. Francoijs, et al. (2004). "The PAS fold. A redefinition of the PAS domain based upon structural prediction." European journal of biochemistry / FEBS 271(6): 1198- 1208. Henry, J. T. and S. Crosson (2011). "Ligand-binding PAS domains in a genomic, cellular, and structural context." Annual review of microbiology 65: 261-286. Hester, I., S. McKee, et al. (2007). "Transient expression of Nxf, a bHLH-PAS transactivator induced by neuronal preconditioning, confers neuroprotection in cultured cells." Brain research 1135(1): 1-11. Hirose, K., M. Morita, et al. (1996). "cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt)." Molecular and cellular biology 16(4): 1706-1713.

Hoffman, E. C., H. Reyes, et al. (1991). "Cloning of a factor required for activity of the Ah (dioxin) receptor." Science 252(5008): 954-958. Hogenesch, J. B., Y. Z. Gu, et al. (1998). "The basic-helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors." Proceedings of the National Academy of Sciences of the United States of America 95(10): 5474-5479. Holmes, J. L. and R. S. Pollenz (1997). "Determination of aryl hydrocarbon receptor nuclear translocator protein concentration and subcellular localization in hepatic and nonhepatic cell culture lines: development of quantitative Western blotting protocols for calculation of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein in total cell lysates." Molecular pharmacology 52(2): 202-211. Huang, N., Y. Chelliah, et al. (2012). "Crystal structure of the heterodimeric CLOCK:BMAL1 transcriptional activator complex." Science 337(6091): 189-194. Jiang, L. and S. T. Crews (2003). "The Drosophila dysfusion basic helix-loop-helix (bHLH)-PAS gene controls tracheal fusion and levels of the trachealess bHLH-PAS protein." Molecular and cellular biology 23(16): 5625-5637. Jiang, L. and S. T. Crews (2006). "Dysfusion transcriptional control of Drosophila tracheal migration, adhesion, and fusion." Molecular and cellular biology 26(17): 6547-6556. Jiang, L. and S. T. Crews (2007). "Transcriptional specificity of Drosophila dysfusion and the control of tracheal fusion cell gene expression." The Journal of biological chemistry 282(39): 28659-28668. Kaas, G. A., C. Zhong, et al. (2013). "TET1 controls CNS 5-methylcytosine hydroxylation, active DNA demethylation, gene transcription, and memory formation." Neuron 79(6): 1086- 1093. Kawajiri, K., Y. Kobayashi, et al. (2009). "Aryl hydrocarbon receptor suppresses intestinal carcinogenesis in ApcMin/+ mice with natural ligands." Proceedings of the National Academy of Sciences of the United States of America 106(32): 13481-13486. Kehrer, C., N. Maziashvili, et al. (2008). "Altered Excitatory-Inhibitory Balance in the NMDA- Hypofunction Model of Schizophrenia." Frontiers in molecular neuroscience 1: 6. Kewley, R. J., M. L. Whitelaw, et al. (2004). "The mammalian basic helix-loop-helix/PAS family of transcriptional regulators." The international journal of biochemistry & cell biology 36(2): 189-204. Kim, T. K., M. Hemberg, et al. (2010). "Widespread transcription at neuronal activity-regulated enhancers." Nature 465(7295): 182-187. Kimura, H., A. Weisz, et al. (2001). "Identification of hypoxia-inducible factor 1 ancillary sequence and its function in vascular endothelial growth factor gene induction by hypoxia and nitric oxide." The Journal of biological chemistry 276(3): 2292-2298. Ledent, V. and M. Vervoort (2001). "The basic helix-loop-helix protein family: comparative genomics and phylogenetic analysis." Genome research 11(5): 754-770. Leong, W. K., T. S. Klaric, et al. (2013). "Upregulation of the neuronal Per-Arnt-Sim domain protein 4 (Npas4) in the rat corticolimbic system following focal cerebral ischemia." The European journal of neuroscience 37(11): 1875-1884. Lin, Y., B. L. Bloodgood, et al. (2008). "Activity-dependent regulation of inhibitory synapse development by Npas4." Nature 455(7217): 1198-1204. Ma, P. C., M. A. Rould, et al. (1994). "Crystal structure of MyoD bHLH domain-DNA complex: perspectives on DNA recognition and implications for transcriptional activation." Cell 77(3): 451-459. Makino, Y., R. Cao, et al. (2001). "Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression." Nature 414(6863): 550-554. Makino, Y., A. Kanopka, et al. (2002). "Inhibitory PAS domain protein (IPAS) is a hypoxia- inducible splicing variant of the hypoxia-inducible factor-3alpha locus." The Journal of biological chemistry 277(36): 32405-32408.

Marcheva, B., K. M. Ramsey, et al. (2010). "Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes." Nature 466(7306): 627-631. Martin, T. A., S. Jayanthi, et al. (2012). "Methamphetamine causes differential alterations in gene expression and patterns of histone acetylation/hypoacetylation in the rat nucleus accumbens." PloS one 7(3): e34236. Marty, S., R. Wehrle, et al. (2000). "Neuronal activity and brain-derived neurotrophic factor regulate the density of inhibitory synapses in organotypic slice cultures of postnatal hippocampus." The Journal of neuroscience : the official journal of the Society for Neuroscience 20(21): 8087-8095. Massari, M. E. and C. Murre (2000). "Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms." Molecular and cellular biology 20(2): 429-440. Maya-Vetencourt, J. F., E. Tiraboschi, et al. (2012). "Experience-dependent expression of NPAS4 regulates plasticity in adult visual cortex." The Journal of physiology 590(Pt 19): 4777-4787. McIntosh, B. E., J. B. Hogenesch, et al. (2010). "Mammalian Per-Arnt-Sim proteins in environmental adaptation." Annual review of physiology 72: 625-645. Mimura, J., M. Ema, et al. (1999). "Identification of a novel mechanism of regulation of Ah (dioxin) receptor function." Genes & development 13(1): 20-25. Moglich, A., R. A. Ayers, et al. (2009). "Structure and signaling mechanism of Per-ARNT-Sim domains." Structure 17(10): 1282-1294. Moser, M., R. Knoth, et al. (2004). "LE-PAS, a novel Arnt-dependent HLH-PAS protein, is expressed in limbic tissues and transactivates the CNS midline enhancer element." Brain research. Molecular brain research 128(2): 141-149. Ohtake, F., A. Baba, et al. (2007). "Dioxin receptor is a ligand-dependent E3 ubiquitin ligase." Nature 446(7135): 562-566. Ooe, N., K. Kobayashi, et al. (2009). "Dynamic regulation of bHLH-PAS-type transcription factor NXF gene expression and neurotrophin dependent induction of the transcriptional control activity." Biochemical and biophysical research communications 378(4): 761- 765. Ooe, N., K. Motonaga, et al. (2009). "Functional characterization of basic helix-loop-helix-PAS type transcription factor NXF in vivo: putative involvement in an "on demand" neuroprotection system." The Journal of biological chemistry 284(2): 1057-1063. Ooe, N., K. Saito, et al. (2009). "Characterization of functional heterodimer partners in brain for a bHLH-PAS factor NXF." Biochimica et biophysica acta 1789(3): 192-197. Ooe, N., K. Saito, et al. (2004). "Identification of a novel basic helix-loop-helix-PAS factor, NXF, reveals a Sim2 competitive, positive regulatory role in dendritic-cytoskeleton modulator drebrin gene expression." Molecular and cellular biology 24(2): 608-616. Ooe, N., K. Saito, et al. (2007). "Characterization of Drosophila and Caenorhabditis elegans NXF-like-factors, putative homologs of mammalian NXF." Gene 400(1-2): 122-130. Partch, C. L., P. B. Card, et al. (2009). "Molecular basis of coiled coil coactivator recruitment by the aryl hydrocarbon receptor nuclear translocator (ARNT)." The Journal of biological chemistry 284(22): 15184-15192. Partch, C. L. and K. H. Gardner (2010). "Coactivator recruitment: a new role for PAS domains in transcriptional regulation by the bHLH-PAS family." Journal of cellular physiology 223(3): 553-557. Partch, C. L. and K. H. Gardner (2011). "Coactivators necessary for transcriptional output of the hypoxia inducible factor, HIF, are directly recruited by ARNT PAS-B." Proceedings of the National Academy of Sciences of the United States of America 108(19): 7739-7744. Pattabiraman, P. P., D. Tropea, et al. (2005). "Neuronal activity regulates the developmental expression and subcellular localization of cortical BDNF mRNA isoforms in vivo." Molecular and cellular neurosciences 28(3): 556-570.

Pearson, J. C., J. D. Watson, et al. (2012). "Drosophila melanogaster Zelda and Single-minded collaborate to regulate an evolutionarily dynamic CNS midline cell enhancer." Developmental biology 366(2): 420-432. Peyrefitte, S., D. Kahn, et al. (2001). "New members of the Drosophila Myc transcription factor subfamily revealed by a genome-wide examination for basic helix-loop-helix genes." Mechanisms of development 104(1-2): 99-104. Piechota, M., M. Korostynski, et al. (2010). "The dissection of transcriptional modules regulated by various drugs of abuse in the mouse striatum." Genome biology 11(5): R48. Ploski, J. E., M. S. Monsey, et al. (2011). "The neuronal PAS domain protein 4 (Npas4) is required for new and reactivated fear memories." PloS one 6(8): e23760. Pongratz, I., C. Antonsson, et al. (1998). "Role of the PAS domain in regulation of dimerization and DNA binding specificity of the dioxin receptor." Molecular and cellular biology 18(7): 4079-4088. Pongratz, I., G. G. Mason, et al. (1992). "Dual roles of the 90-kDa heat shock protein hsp90 in modulating functional activities of the dioxin receptor. Evidence that the dioxin receptor functionally belongs to a subclass of nuclear receptors which require hsp90 both for ligand binding activity and repression of intrinsic DNA binding activity." The Journal of biological chemistry 267(19): 13728-13734. Ponting, C. P. and L. Aravind (1997). "PAS: a multifunctional domain family comes to light." Current biology : CB 7(11): R674-677. Pruunsild, P., M. Sepp, et al. (2011). "Identification of cis-elements and transcription factors regulating neuronal activity-dependent transcription of human BDNF gene." The Journal of neuroscience : the official journal of the Society for Neuroscience 31(9): 3295-3308. Ramachandrappa, S., A. Raimondo, et al. (2013). "Rare variants in single-minded 1 (SIM1) are associated with severe obesity." The Journal of clinical investigation 123(7): 3042- 3050. Ramamoorthi, K., R. Fropf, et al. (2011). "Npas4 regulates a transcriptional program in CA3 required for contextual memory formation." Science 334(6063): 1669-1675. Ramocki, M. B. and H. Y. Zoghbi (2008). "Failure of neuronal homeostasis results in common neuropsychiatric phenotypes." Nature 455(7215): 912-918. Reddy, P., W. A. Zehring, et al. (1984). "Molecular analysis of the period locus in Drosophila melanogaster and identification of a transcript involved in biological rhythms." Cell 38(3): 701-710. Reisz-Porszasz, S., M. R. Probst, et al. (1994). "Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT)." Molecular and cellular biology 14(9): 6075-6086. Reppert, S. M. and D. R. Weaver (2002). "Coordination of circadian timing in mammals." Nature 418(6901): 935-941. Ribeiro, C., A. Ebner, et al. (2002). "In vivo imaging reveals different cellular functions for FGF and Dpp signaling in tracheal branching morphogenesis." Developmental cell 2(5): 677-683. Rubenstein, J. L. (2010). "Three hypotheses for developmental defects that may underlie some forms of autism spectrum disorder." Current opinion in neurology 23(2): 118-123. Rubenstein, J. L. and M. M. Merzenich (2003). "Model of autism: increased ratio of excitation/inhibition in key neural systems." Genes, brain, and behavior 2(5): 255-267. Rudenko, A., M. M. Dawlaty, et al. (2013). "Tet1 is critical for neuronal activity-regulated gene expression and memory extinction." Neuron 79(6): 1109-1122. Rutter, J., M. Reick, et al. (2001). "Regulation of clock and NPAS2 DNA binding by the redox state of NAD cofactors." Science 293(5529): 510-514.

100

Sabatini, P. V., N. A. Krentz, et al. (2013). "Npas4 is a novel activity-regulated cytoprotective factor in pancreatic beta-cells." Diabetes 62(8): 2808-2820. Saxena, S. and P. Caroni (2011). "Selective neuronal vulnerability in neurodegenerative diseases: from stressor thresholds to degeneration." Neuron 71(1): 35-48. Scheuermann, T. H., D. R. Tomchick, et al. (2009). "Artificial ligand binding within the HIF2alpha PAS-B domain of the HIF2 transcription factor." Proceedings of the National Academy of Sciences of the United States of America 106(2): 450-455. Semenza, G. L. (2012). "Hypoxia-inducible factors in physiology and medicine." Cell 148(3): 399-408. Semenza, G. L., B. H. Jiang, et al. (1996). "Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1." The Journal of biological chemistry 271(51): 32529-32537. Shamloo, M., L. Soriano, et al. (2006). "Npas4, a novel helix-loop-helix PAS domain protein, is regulated in response to cerebral ischemia." The European journal of neuroscience 24(10): 2705-2720. Sogawa, K., R. Nakano, et al. (1995). "Possible function of Ah receptor nuclear translocator (Arnt) homodimer in transcriptional regulation." Proceedings of the National Academy of Sciences of the United States of America 92(6): 1936-1940. Spitzer, N. C. (2006). "Electrical activity in early neuronal development." Nature 444(7120): 707-712. Spitzer, N. C. (2012). "Activity-dependent neurotransmitter respecification." Nature reviews. Neuroscience 13(2): 94-106. Sudhof, T. C. (2008). "Neuroligins and neurexins link synaptic function to cognitive disease." Nature 455(7215): 903-911. Swanson, H. I., W. K. Chan, et al. (1995). "DNA binding specificities and pairing rules of the Ah receptor, ARNT, and SIM proteins." The Journal of biological chemistry 270(44): 26292-26302. Swanson, H. I. and J. H. Yang (1999). "Specificity of DNA binding of the c-Myc/Max and ARNT/ARNT dimers at the CACGTG recognition site." Nucleic acids research 27(15): 3205-3212. Sylwestrak, E. and P. Scheiffele (2013). "Neuroscience: Sculpting neuronal connectivity." Nature 503(7474): 42-43. Tanaka-Matakatsu, M., T. Uemura, et al. (1996). "Cadherin-mediated cell adhesion and cell motility in Drosophila trachea regulated by the transcription factor Escargot." Development 122(12): 3697-3705. Taylor, B. L. and I. B. Zhulin (1999). "PAS domains: internal sensors of oxygen, redox potential, and light." Microbiology and molecular biology reviews : MMBR 63(2): 479-506. Uniacke, J., C. E. Holterman, et al. (2012). "An oxygen-regulated switch in the protein synthesis machinery." Nature 486(7401): 126-129. Whitelaw, M., I. Pongratz, et al. (1993). "Ligand-dependent recruitment of the Arnt coregulator determines DNA recognition by the dioxin receptor." Molecular and cellular biology 13(4): 2504-2514. Whitelaw, M. L., M. Gottlicher, et al. (1993). "Definition of a novel ligand binding domain of a nuclear bHLH receptor: co-localization of ligand and hsp90 binding activities within the regulable inactivation domain of the dioxin receptor." The EMBO journal 12(11): 4169- 4179. Whitelaw, M. L., J. McGuire, et al. (1995). "Heat shock protein hsp90 regulates dioxin receptor function in vivo." Proceedings of the National Academy of Sciences of the United States of America 92(10): 4437-4441. Wiesel, T. N. (1982). "Postnatal development of the visual cortex and the influence of environment." Nature 299(5884): 583-591.

101

102

Appendix 1

bHLH–PAS proteins in cancer

David C. Bersten, Adrienne E. Sullivan, Daniel J. Peet and Murray L. Whitelaw

School of Molecular and Biomedical Science (Biochemistry), and Australian Research

Council Special Research Centre for the Molecular Genetics of Development,

The University of Adelaide, Adelaide, South Australia, Australia

To whom correspondence should be addressed: A/Prof. Murray Whitelaw, School of

Molecular and Biomedical Science (Biochemistry), The University of Adelaide, North Tce,

Adelaide, SA 5005, Australia Email: [email protected]

Nat Rev Cancer. 2013 Dec;13(12):827-41.

103

Bersten, D.C., Sullivan, A.E., Peet, D.J. & Whitelaw, M.L. (2013). bHLH–PAS proteins in cancer. Nature Reviews Cancer, v. 13 (12), pp. 827-841

NOTE:

This publication is included after page 103 in the print copy

of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1038/nrc3621

Appendix 2

Regulation of the Neuronal Transcription Factor NPAS4 by REST

and microRNAs.

David C. Bersten, Josephine A. Wright, Peter J. McCarthy and Murray L. Whitelaw

School of Molecular and Biomedical Science (Biochemistry) and Australian Research

Council Special Research Centre for the Molecular Genetics of Development,

The University of Adelaide, Adelaide, South Australia, Australia

To whom correspondence should be addressed:

David C Bersten

School of Molecular and Biomedical Science (Biochemistry),

The University of Adelaide, North Tce, Adelaide, SA 5005, Australia

Email: [email protected]

Biochim Biophys Acta. 2014 Jan;1839(1):13-24.

104

Bersten, D.C., Wright, J.A., McCarthy, P.J. & Whitelaw, M.L. (2014) Regulation of the neuronal transcription factor NPAS4 by REST and microRNAs. Biochimica et Biophysica Acta, v. 1839 (1), pp. 13-24

NOTE:

This publication is included after page 104 in the print copy

of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1016/j.bbagrm.2013.11.004

Appendix 3

Human Variants in the Neuronal basic Helix-Loop-Helix /Per-

Arnt-Sim (bHLH/PAS) Transcription Factor Complex

NPAS4/ARNT2 Disrupt Function.

David C. Bersten1, John B. Bruning1, Daniel J. Peet1 and Murray L. Whitelaw1

1School of Molecular and Biomedical Science (Biochemistry), and Australian Research

Council Special Research Centre for the Molecular Genetics of Development,

The University of Adelaide, Adelaide, South Australia, Australia

To whom correspondence should be addressed: A/Prof. Murray Whitelaw, School of

Molecular and Biomedical Science (Biochemistry), The University of Adelaide, North Tce,

Adelaide, SA 5005, Australia Email: [email protected]

PLoS One. 2014 Jan 17;9(1)

105

Human Variants in the Neuronal Basic Helix-Loop-Helix/ Per-Arnt-Sim (bHLH/PAS) Transcription Factor Complex NPAS4/ARNT2 Disrupt Function

David C. Bersten, John B. Bruning, Daniel J. Peet, Murray L. Whitelaw* School of Molecular and Biomedical Science (Biochemistry), and Australian Research Council Special Research Centre for the Molecular Genetics of Development, The University of Adelaide, Adelaide, South Australia, Australia

Abstract Neuronal Per-Arnt-Sim homology (PAS) Factor 4 (NPAS4) is a neuronal activity-dependent transcription factor which heterodimerises with ARNT2 to regulate genes involved in inhibitory synapse formation. NPAS4 functions to maintain excitatory/inhibitory balance in neurons, while mouse models have shown it to play roles in memory formation, social interaction and neurodegeneration. NPAS4 has therefore been implicated in a number of neuropsychiatric or neurodegenerative diseases which are underpinned by defects in excitatory/inhibitory balance. Here we have explored a broad set of non-synonymous human variants in NPAS4 and ARNT2 for disruption of NPAS4 function. We found two variants in NPAS4 (F147S and E257K) and two variants in ARNT2 (R46W and R107H) which significantly reduced transcriptional activity of the heterodimer on a luciferase reporter gene. Furthermore, we found that NPAS4.F147S was unable to activate expression of the NPAS4 target gene BDNF due to reduced dimerisation with ARNT2. Homology modelling predicts F147 in NPAS4 to lie at the dimer interface, where it appears to directly contribute to protein/protein interaction. We also found that reduced transcriptional activation by ARNT2 R46W was due to disruption of nuclear localisation. These results provide insight into the mechanisms of NPAS4/ARNT dimerisation and transcriptional activation and have potential implications for cognitive phenotypic variation and diseases such as autism, schizophrenia and dementia.

Citation: Bersten DC, Bruning JB, Peet DJ, Whitelaw ML (2014) Human Variants in the Neuronal Basic Helix-Loop-Helix/Per-Arnt-Sim (bHLH/PAS) Transcription Factor Complex NPAS4/ARNT2 Disrupt Function. PLoS ONE 9(1): e85768. doi:10.1371/journal.pone.0085768 Editor: Masaru Katoh, National Cancer Center, Japan Received September 11, 2013; Accepted December 6, 2013; Published January 17, 2014 Copyright: ß 2014 Bersten et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by the Australian Research Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]

Introduction expression of NPAS4 [10,12,13]. In response, NPAS4 activates a battery of genes to increase the number of inhibitory synapses, Basic Helix-Loop-Helix Per-Arnt-Sim homology (bHLH-PAS) maintaining homeostasis of neuron activity [10,14]. Not surpris- proteins are signal regulated and/or tissue specific dimeric ingly, NPAS4 null mice are hyperactive, prone to seizures, and transcription factors involved in a diverse array of physiological display several defects in social anxiety and cognitive impairments and pathological functions [1–3]. They mediate processes such as similar to those observed in autism and schizophrenia [10,15]. the cellular response to hypoxia (Hypoxia Inducible Factors NPAS4 null mice also have a much reduced life span due to (HIF1a/HIF2a)) [2], the maintenance of circadian rhythms extensive neurodegeneration, thought to be caused by glutamate (Circadian Locomotor Output Cycles Kaput (CLOCK)) [4], the neurotoxicity [16]. More recently, conditional deletion of NPAS4 clearance of environmental pollutants (Aryl hydrocarbon Receptor in the CA3 region of the hippocampus in adult mice has shown it (AhR)/Dioxin Receptor (DR)) [1], and appetite control (Single is required for contextual memory formation [11]. minded 1 (Sim1)) [5,6]. The above bHLH-PAS transcription Neuropsychiatric disorders encompass a diverse array of factors must heterodimerise with an obligate nuclear partner phenotypes and while a strong genetic component has been protein, Aryl hydrocarbon Receptor Nuclear Translator (ARNT/ suggested, the precise genes involved have been difficult to identify ARNT2) or Brain and Muscle ARNT-Like (BMAL1/BMAL2), to [15,17,18]. In addition, modifier or susceptibility genes are activate or repress gene expression[1]. Dimerisation is predomi- hypothesised to be required in concert with other mutations to nantly mediated through the conserved N-terminal bHLH and drive aberrant neurological phenotypes [19,20]. Recent reports PAS repeat domains (PASA and PASB) to allow binding to from large scale whole genome and exome sequencing projects asymmetric E-BOX-like elements in regulatory regions of target have highlighted the contribution that rare, de novo variants make genes [7–9]. to disease [19,21–23]. Indeed, the large spectrum of phenotypes Neuronal PAS factor 4 (NPAS4) is a bHLH-PAS transcription that underlie neuropsychiatric disease may reflect a multivaried factor whose expression and activity is tightly coupled with nature of deleterious, rare genetic variants associated with disease. neuronal activity [10,11]. Ischemia, seizure, neuronal depolarisa- Many neuropsychiatric diseases, including schizophrenia and tion, and models of learning all rapidly and transiently increase

PLOS ONE | www.plosone.org 1 January 2014 | Volume 9 | Issue 1 | e85768 NPAS4 and ARNT2 Variants Disrupt Activity autism, are thought to have related defects which disrupt the exon I expression analysis. NPAS4 expression was induced with balance between neuronal excitation and inhibition [24–27]. 1 mg/ml doxycyline for 24 hrs. There is strong evidence from loss of function mutations associated with these diseases that synapse dysfunction may play a key role in Transient transfections and luciferase assays disrupting this homeostatic balance [17,28,29]. In addition, HEK293T cells in 24 well plates were transiently transfected glutamergic hyperexcitation, which may also arise from disruption with a DNA cocktail containing 200 ng firefly luciferase reporter of the excitatory/inhibitory balance or other neuronal stressors, plasmid pML-6xCME-Luc or empty pML-Luc control [38], 50 ng has been linked to the progression of neurodegenerative disorders each of ARNT1 or ARNT2 expression plasmid, pEF-IRESpuro- [30,31]. hSIM1-2myc, pEF-IRESpuro-hSIM2-2myc [39,40] or pEFBOS- Given NPAS4 is activated following neuronal activity to control hNPAS4-mycFlag expression vectors and 200 pg of phRL-CMV the balance of excitatory and inhibitory synapses, and that defects Renilla luciferase reporter plasmid (Promega) using Fugene6 in memory, social anxiety, age related neurodegeneration, (Roche) according to the manufacturers’ instructions. Plasmid hyperactivity and seizures are observed in NPAS4 null mice, concentrations were normalised using empty expression plasmids. NPAS4 has been implicated in a number of neuropsychiatric and After 24 or 48 hrs, relative luciferase activities for each bHLH/ neurodegenerative diseases [15,16,29,32]. Here we sought to test PAS variant were assayed using a DLR kit (Promega) and whether human variants in NPAS4 or ARNT2 might compromise normalized to relative WT activity. For immunoprecipitations, transcriptional function. We screened several variants listed in 1 mg of pCI-eGFP, 2 mg of ARNT2 and 2 mg of NPAS4 public databases and found examples where single amino acid expression plasmids were cotransfected into 56105 293T cells changes in NPAS4 or ARNT2 resulted in mild or dramatic loss of using Fugene6 (Roche). function. We elucidated mechanisms of disrupted heterodimerisation or impaired nuclear localisation to attenuate activity of the RNA extraction, cDNA synthesis and quantitative real NPAS4/ARNT2 complex. This study outlines a strategy to time PCR highlight and prioritise non-synonymous variants in human HEK293-TREX cells were lysed in 500 ml trizol (Invitrogen) transcription factors which might then be screened in patient and RNA isolated. 2 mg of RNA was used in each reverse cohorts to investigate their possible contribution to disease. transcription reaction (Superscript III, Invitrogen) and cDNA was diluted 10 fold in 1X Tris-EDTA (TE) pH 8.0.for real time PCR. Materials and Methods Real time PCR was performed in triplicate using Fast SYBR Green Master Mix (Applied Biosystems) on a StepOne Plus Real- Cloning and Cell Culture time PCR system (Applied Biosystems) using primers specific to pCMV-hNPAS4-mycFlag was purchased from Origene (Rock- human BDNF exon I [41], NPAS4 [10] and RNA polymerase 2A ville,USA). NPAS4-mycFlag was subsequently subcloned into [42] and spanning an intron where possible. BDNF gene induction pENTR1a (Invitrogen) using SmaI/EcoRV. pEF-IRESpuro- by each NPAS4 variant was normalised to RNA polymerase 2A. hSIM1-2Myc and pEF-IRESneo-hARNT1, pEF-IRESneo- Melt curves of PCR products were analysed to confirm a single hARNT2, pEF-IRESpuro-hARNT1-3xFlag, pEF-IRESneo- amplicon and real time PCR results were analysed and ‘QGene’ hARNT2-3xFlag have been described previously [33]. pEFBOS- analysis software [43]. Gtwy and pcDNA-FRT/TO-Gtwy were created by inserting KpnI/EcoRV GtwyA from pLV410 [34] into either pEF-BOS or Immunoblotting and Immunoprecipitations pcDNA-FRT/TO. NPAS4, ARNT2 and SIM1 variants were For immunoprecipitations, cells were washed twice in PBS and cloned using overlap extension PCR or Gibson Isothermal lysed in 20 mM HEPES, pH 8.0, 420 mM NaCl, 0.5% Igepal, assembly essentially as described [35,36] using Phusion proof 25% glycerol, 0.2 mM EDTA, 1.5 mM MgCl2, 1 mM DTT and reading polymerase (New England Biolabs). Primers were protease inhibitors (Sigma). 100 mg of protein whole cell extract designed to incorporate the indicated human variants (Table S1) was diluted to 1 mg/ml in immunoprecipitation (IP) buffer and the mutations were confirmed by sequencing. NPAS4 variants (20 mM HEPES, pH 8.0, 150 mM NaCl, 150 mM KCl, 0.1% were either subcloned into pEFBOS-Gtwy or pcDNA5-FRT/TO- glycerol, 1 mM EDTA, 1 mM DTT and protease inhibitors) and Gtwy plasmids using LR recombination (Invitrogen). pcDNA3.2- incubated with 50 mL of BSA blocked Flag M2 resin (Sigma). The d2nucEGFP was created by first subcloning d2eGFP from resin was washed twice with 1 mL of IP wash buffer (250 mM pd2eGFP-T4 N1 (Clonetech) into pENTR1a using EcoRI/NotI, NaCl, 20 mM HEPES pH 8.0, 0.1% Igepal, and 1 mM EDTA) then D2nucEGFP was digested from pNSEN [37] with AgeI/ and the bound material boiled in 20 ml of SDS sample buffer. HindIII and inserted into pENTR1a-d2eGFP. pcDNA3.2-DEST Input sample (10%) and immunoprecipitations were then run on (Invitrogen) was then recombined with pENTR1a-d2nucEGFP by 7.5%SDS-PAGE gels and transferred to nitrocellulose. Lysates LR recombination. from reporter gene assays were separated on 7.5% SDS-PAGE gel Human Embryonic Kidney HEK293T (ATCC, CCL-3216), and transferred to nitrocellulose. Proteins were detected using the HEK293-TREX (Invitrogen) and Neuro2A (ATCC, CCL-131) anti-ARNT2 (Santa Cruz), anti-FLAG (Sigma), anti-Myc (4A6, cells were maintained in DMEM medium supplemented with 10% Upstate) and anti-a-Tubulin antibodies (MCA78G, Serotec). FCS, 1 mM L-glutamine, 100 units/ml penicillin, and 100 mg/ml Primary antibodies were detected using horseradish peroxidise- streptomycin. HEK293-TREX stable cell lines were generated conjugated secondary antibodies and visualised using chemilumi- 5 according to manufacturer instructions (Invitrogen). Briefly 6610 nescence. Quantification of ARNT2 co-immunoprecipition band HEK293TREX cells were transfected with 2 mg of pOG44 and intensity was estimated using ImageLab software (BioRad). 200 ng of pcDNA5-FRT/TO-hNPAS4-mycFlag plasmid using Fugene6 (Roche). 48 hrs after transfection the cells were expanded Immunofluorescence and selected in 200 mg/ml Hygromycin B (Invitrogen) to generate pEF-IRESpuro-hARNT2-3xFlag or pEF-IRESpuro- stable cell lines. At least two independently derived WT NPAS4 hARNT2.R46W-3xFlag (50 ng) were cotransfected with 200 ng cell lines and one cell line for each variant were used for BDNF of pcDNA3.2-d2nucEGFP into HEK293T cells plated on Poly-D-

PLOS ONE | www.plosone.org 2 January 2014 | Volume 9 | Issue 1 | e85768 NPAS4 and ARNT2 Variants Disrupt Activity

Lysine (70–150 KDa; Sigma) coated coverslips using Fugene6 transcription with ARNT1 or ARNT2 [53,54]. To explore this (Roche). After 24 hrs, Cells were fixed 4% Paraformaldehyde further we then tested the ability of a subset of NPAS4 variants, (PFA) for 20 mins, washed in PBS and permeablised using 2% including F147S, to activate the reporter gene in HEK293T cells Triton-X-100/PBS for 10 mins. The fixed cells were then blocked in combination with either ARNT1 or ARNT2 as the dimerisation using horse serum, and ARNT2 detected using anti-Flag antibody partner (Fig. 1C). NPAS4 was able to strongly activate the reporter (1:1000; Sigma) and visualised using a TxRed-conjugated second- when dimerising with either ARNT1 or ARNT2, although the ary antibody. Coverslips were mounted onto slides using Prolong F147S variant remained inactive irrespective of coexpression with Gold mounting medium containing DAPI (Invitrogen) and images ARNT1 or ARNT2 (P,0.0001, Fig. 1C). The loss of function taken using a Ziess deconvolution microscope. Images were then NPAS4.F147S protein was expressed at similar levels and size false coloured and overlayed using ImageJ imaging software [44]. (,100 KDa) to that of WT NPAS4 or other variants by western blot analysis (Fig. 1D). Given that NPAS4 is a neuronal Homology modeling transcription factor, we were interested in establishing whether Homology models were created in the ICM-Pro program suite there were neuron specific effects not observed in HEK293T cells. [45] using the homology add-on [46,47]. Individual subunits of the Using Neuro2A (N2A) cells we found that WT NPAS4 was active heterodimer were first created separately using the sequence for with either ARNT1 or ARNT2, but the F147S variant again failed human NPAS4 (Uniprot number Q8IUM7) and for human Arnt2 to activate the reporter gene (P,0.0001, Fig. 1E). We did not (Uniprot number Q9HBZ2). A search for homologous structures observe any consistent changes in any of the other variants or in in the Protein Data Bank was performed and the Clock-BMAL the overall protein expression of these variants in N2A cells (Fig. E structure was the structure with the highest homology (PDB:4F3L) and F). [48]. The NPAS4 homology model was created using the To further characterise the mechanism of the loss of function CLOCK protein as a three-dimensional template and the ARNT2 NPAS4.F147S variant we generated HEK293-TREX inducible homology model was created using BMAL as the three- cell lines where NPAS4 or NPAS4 variant expression can be dimensional molecular template. After creation of individual induced from a defined locus upon treatment with doxycycline. subunit models, they were both subjected to regularization and This allows for equivalent expression of NPAS4 variants, model refinement within ICM-Pro (energy minimization, optimi- facilitating direct comparison of activities. NPAS4 has been zation of geometry, and easing of clashing side-chains). Both previously shown to be a critical activator of Brain Derived subunits were then docked together using the CLOCK-BMAL Neurotrophic Factor (BDNF) following neuronal depolarisation structure to guide the docking. Further model refinement and [10,41]. Furthermore, defective BDNF expression and function is regularization was then performed to ensure the integrity of the also an important contributor to neurological disease [55,56]. We dimer interface. Finally, many loops were identified and subjected therefore next investigated whether the NPAS4.F147S and a to loop modeling to improve clashing at the dimer interface using subset of variants spanning the C-terminus could induce BDNF the ICM-Pro loop modeling utility [49]. Figures were created exon I expression using 293TREX cells. Overexpression of WT using PyMOL (The PyMOL Molecular Graphics System, Version NPAS4 and the C-terminal variants were able to strongly induce 1.2r3pre, Schro¨dinger, LLC.). the expression of BDNF exon I, however, there was a ,90% reduction in the ability of F147S to induce BDNF exon I Statistical Analysis expression (Fig. 2A). Similar reduction in the ability of Statistical significance was performed using GraphPad Prism 5 NPAS4.F147S to activate BDNF exon I expression was found in program for Windows (GraphPad Software Inc.) and evaluated by transient transfection experiments where ARNT2 was coexpressed the Student’s t test or ANOVA with the level of significance set at (Fig. S1). We did not observe any significant differences in the p,0.05. ANOVA statistical analysis was performed on log ability of any other NPAS4 variants to activate BDNF I expression transformed data with Tukey’s post hoc analysis. Data are (Fig. 2A). expressed as mean 6SEM unless otherwise indicated. The NPAS4.F147S variation was located towards the end of the PASA domain in a region where amino acids important for Results dimerisation have been previously identified (Fig.S2A and Fig. 1A) [33,57]. We were therefore interested to test whether the F to S To investigate whether variants in NPAS4 would disrupt conversion could disrupt dimerisation between NPAS4 and function we examined protein coding variants from the 1000 ARNT2. We used co-immunoprecipitation to show that genomes project, the NHLBI exome sequencing project and the NPAS4.F147S failed to form a heterodimer with ARNT2 NCBI SNP database [50–52]. Initially we examined 13 non- (Fig. 2B). Other NPAS4 variants, which showed near wild type synonymous NPAS4 variants, which represented all reported activities on the reporter gene (Fig 1C), showed similar ARNT2 mismatch variants at the time we initiated this study, for their co-immunoprecipitation to wild type NPAS4. Homology model- ability to activate a bHLH-PAS responsive luciferase reporter gene ling of NPAS4 and ARNT2 using the crystal structure for containing six repeats of a bHLH-PAS core binding element CLOCK and BMAL as a template revealed F147 to lie on the (pML-6xCME-Luc, [33]). The single nucleotide variants were surface (Fig. 3A) and position at the proposed dimerisation spread throughout the protein sequence and spanned the C- interface between NPAS4 and ARNT2 (Fig. 3B) [48]. The terminal and PAS regions (Fig. 1A). Coexpression of wild type phenylalanine residue appears to be in close proximity to several (WT) NPAS4 and ARNT2, but not ARNT2 alone, strongly hydrophobic residues in ARNT2 and may make contacts within activated the reporter gene (Fig. 1B). The overwhelming majority this interface to promote dimerisation (Fig. 3A). Substitution of the of variants were able to activate the reporter to a similar extent as large, hydrophobic phenylalanine with a small, polar serine may WT NPAS4, however, variant F147S completely ablated the therefore explain the loss in dimerisation. We hypothesised that ability of NPAS4 to activate the reporter (Fig. 1B). substitution of F147 with an alanine may partially disrupt function Although NPAS4 expression most strongly overlaps with by removing the larger phenylalanine residue, but maintaining ARNT2 within the brain, in biochemical experiments NPAS4 hydrophobicity. We therefore tested this mutant and found appears to show little bias between binding and activating NPAS4.F147A significantly (P,0.001) decreased reporter gene

PLOS ONE | www.plosone.org 3 January 2014 | Volume 9 | Issue 1 | e85768 NPAS4 and ARNT2 Variants Disrupt Activity

Figure 1. Functional Analysis of human NPAS4 Variants. A, Schematic of domains within NPAS4 showing positions of analysed non- synonymous variants. B, A screen of NPAS4-mycFlag variants using NPAS4/ARNT2 activation of a reporter gene (6xCME-Luc) in HEK293T cells transfected with NPAS4-MycFlag and ARNT2 expression vectors. Values represent average percentage activity of WT NPAS4-mycFlag 6SD of two

PLOS ONE | www.plosone.org 4 January 2014 | Volume 9 | Issue 1 | e85768 NPAS4 and ARNT2 Variants Disrupt Activity

independent experiments. C and E, NPAS4-MycFlag variant activation of the 6xCME-Luc reporter gene upon co-expression of ARNT1 or ARNT2 as the dimerisation partner in HEK293T cells (C) or Neuro2A cells (E). Data are mean percentage activity of WT NPAS4-mycFlag 6SEM of at least 3 independent experiments. Statistical significance was calculated using an ANOVA compared to WT NPAS4-mycFlag. Cell lysates from reporter assays in C and E were separated by SDS-PAGE and NPAS4-mycFlag protein detected by immunoblotting using a-Flag antibodies, with a-tubulin was used as a loading control. Representative western blots of NPAS4-mycFlag variants are shown for HEK293T cells (D) and Neuro2A cells (F). ****p,0.0001. doi:10.1371/journal.pone.0085768.g001 activity to approximately 65% of wt, supporting our hypothesis of clustered LoF mutations (Figure S2B). As these variants (Fig. 4A). Furthermore, mutation of the corresponding phenylal- replaced well conserved residues and had drastically altered side anine residue to alanine in related bHLH/PAS proteins SIM1 chain chemistry, we reasoned they might alter protein activities. (SIM1.F160A) and SIM2 (SIM2.F160A) also significantly (P, Using luciferase reporter gene assays we found that the NPAS4 0.001) reduced reporter gene activities to approximately 60% and E257K variant significantly (p,0.05) reduced activity to ,70% 40%, respectively, suggesting this mechanism of dimerisation may when compared to WT (Fig. 5A). The reduced reporter activity be shared among bHLH-PAS transcription factors (Fig. 4B). observed with the NPAS4.E257K mutation did not appear to be NPAS4.F147S and NPAS4.F147A also showed significantly (P, due a reduction in dimerisation with ARNT2 (Fig. 4C and 4D) or 0.0001 and P,0.001, respectively) reduced dimerization with protein expression (Fig. 5B). SIM1 G254E and G254R both endogenous ARNT2 in co-immunoprecipition experiments com- reduced luciferase reporter activity, however only SIM1.G254R pared to WT NPAS4 (Fig. 4C and 4D). Dimerisation with reached statistical significance (G245E, p.0.05; G254R, p,0.01). ARNT2 appeared to be greater with NPAS4.F147A than The SIM1.G254R variant reduced reporter activity to ,60% of NPAS4.F147S, consistent with reporter gene data. Alignment of WT, which may in part be due to a reduction in SIM1 protein the human bHLH-PAS transcription factors revealed that the expression (Fig 5B). phenylalanine at this position was conserved among all the bHLH- Since our data revealed certain variants in NPAS4 to disrupt the PAS transcription factors, suggesting that this may represent an transcriptional output from NPAS4, we then predicted that important amino acid for a conserved mode of dimerisation variants in ARNT2 might indirectly disrupt NPAS4 dependent (Figure S2A). transcriptional output. We therefore examined single nucleotide Recently we have found a number of Loss of Function (LoF), variants in ARNT2, concentrating on those that invoked dramatic non-synonymous variants of SIM1 in a cohort of children changes in side chain chemistry within functional N-terminal displaying early onset, morbid obesity[58,59]. A number of these domains where LoF variants were likely to occur. We selected and LoF SIM1 variants were clustered around PASA and PASB tested four variants in ARNT2, R46W, R107H, R402Q and domains. In addition, a number of residues within a homologous W410R, for their ability to activate the 6xCME-Luciferase region of PASB have been shown to be important for dimerisation reporter gene in combination with NPAS4. Expression of NPAS4 of CLOCK and BMAL [48]. New variants from recent alone was unable to activate the reporter, however when ARNT2 sequencing projects became available during this study and we was coexpressed we observed strong activation (Fig. 5C). Both identified one additional variant in NPAS4 (E257K) and two ARNT2.R46W and ARNT2.R107H significantly (P,0.01 and variants in SIM1 (G254E and G254R) which lay within this region P,0.05, respectively) reduced activation of the reporter to 55%

Figure 2. NPAS4 Variant F147S reduces dimerisation with ARNT2 and fails to activate BDNF expression. A, 293TREX cells containing site specific, stable integration of WT NPAS4-mycFlag, NPAS4-mycFlag variants or an empty vector were induced with 1 mg/ml doxycycline for 24 hrs and Brain Derived Neurotrophic Factor (BDNF) Exon I mRNA expression measured by quantitative real-time PCR and normalised to RNA Polymerase 2A. Data are mean 6SEM of at least 4 independent experiments; WT is an average of two independently derived cell lines and least 4 independent experiments. Statistical significance is calculated using an ANOVA compared to WT NPAS4-mycFlag, ****p,0.0001. B, Immunoblotting of whole cell extracts and a-Flag coimmunoprecipitates from HEK293T cells transiently transfected with NPAS4-MycFlag and ARNT2 expression constructs. a-Flag Abs used to detect NPAS4-MycFlag and a-ARNT2 Abs used to detect overexpressed ARNT2. Data are representative of three independent experiments. doi:10.1371/journal.pone.0085768.g002

PLOS ONE | www.plosone.org 5 January 2014 | Volume 9 | Issue 1 | e85768 NPAS4 and ARNT2 Variants Disrupt Activity

Figure 3. Homology model highlighting residue Phe147 of NPAS4. NPAS4 is colored green and ARNT2 is colored yellow. A) The NPAS4 subunit is depicted as a surface representation. The location of Phe147 is shown by red color. B) The NPAS4-ARNT2 heterodimer is depicted as a ribbons diagram. Phe147 is shown at the interface with side chain shown as sticks colored red, together with amino acids within 3.5 angstroms. Phe 147 is part of a hydrophobic pocket formed with ARNT2 residues (Leu 133, Leu 141) and NPAS4 residues (Leu 146, Trp 182). doi:10.1371/journal.pone.0085768.g003 and 65% compared to WT ARNT2, respectively. No significant Discussion differences were observed with ARNT2.R402Q or ARNT2.W410R (Fig. 5C). Expression of these ARNT2 variants Protein coding variants are estimated to be abundantly was comparable to WT ARNT2 (Fig. 5D). Previously, it has been generated in healthy individuals, but can also be important shown that constitutively nuclear localisation of ARNT1 is contributors to disease [51,61]. The notion of de novo or rare controlled by a small N-terminal bi-partite nuclear localisation variant generation as drivers of complex diseases such as autism is sequence (NLS) [60]. Alignment of ARNT1 and ARNT2 revealed being increasingly appreciated [21,23]. Although large scale that ARNT2 also shared this NLS and that R46W is within a set of sequencing projects have produced bioinformatic estimations for basic residues that comprise the NLS (Fig. 6A). We hypothesised the contribution of natural protein coding variation to protein that the LoF exhibited by the ARNT2.R46W variant may be due dysfunction, this has not been addressed experimentally [61]. to loss of nuclear localisation and therefore performed immuno- Intellectual disability, autism and schizophrenia have been linked cytochemistry on Flag tagged WT or ARNT2.R46W expressed in to mutations in synaptic proteins, some of which have been shown HEK293T cells (Fig. 6B). As expected, WT ARNT2 was to underpin defects in synapse structure, function or homeostatic constitutively nuclear in almost all transfected cells, whereas balance in animal models [28,62–64]. It has therefore been ARNT2.R46W was cytoplasmic in the majority of transfected cells hypothesised that alterations to activity-dependent neuronal (Fig. 6B). We conclude that weak reporter gene activity of variant signalling may disrupt neuronal homeostasis as a common R46W is therefore due to deficient nuclear import. The mechanism in neuropsychiatric disease [17,29]. ARNT2.R107H variant mildly impaired activity. R107 lies within NPAS4 is a transcription factor critical for maintaining a proposed helix of the bHLH domain of ARNT2 and mapping homeostatic balance of excitation/inhibition within neurons and this amino acid on the homology model of ARNT2 predicts that has been implicated in several neurological diseases [11,15,29,32]. R107 is also surface exposed, positioned at the interface between Mice lacking NPAS4 exhibit many hallmarks of both neuropsy- NPAS4 and ARNT2. The R to H transition may slightly weaken chiatric and neurodegenerative diseases, but as yet this transcrip- dimerisation between NPAS4 and ARNT2 although this was not tion factor has not been linked to any human disorder. While this evident from co-immunoprecipition experiments with NPAS4 may seem surprising, many of these diseases are multifaceted, do (data not shown). not manifest until later in life and may be dependent upon In summary, we have analysed a series of natural human additional environmental or genetic susceptibility factors. variants in NPAS4 and ARNT2 and discovered a subset which The rapid expansion in genome sequencing data has recently compromise transcriptional activation. LoF variants are clustered provided a wealth of human protein coding variants with little or within domains with known roles in dimerisation and nuclear no associated phenotypic information. Here we tested the effects of localisation. Taken together, this data suggests that while many several human protein coding variants in NPAS4 and ARNT2 on single amino acid variants in the NPAS4 and ARNT2 transcrip- activity of the NPAS4/ARNT2 transcription factor complex. We tional complex may not compromise activity, distinct sites in the initially screened all NPAS4 non-synonymous coding variants N-terminal functional domains are sensitive to alteration and available in the dbSNP database at the outset of the study, in the warrant further investigation into possible links with neurological context of an NPAS4/ARNT2 heterodimer. We found disorders. NPAS4.F147S to have a near complete loss of function on both

PLOS ONE | www.plosone.org 6 January 2014 | Volume 9 | Issue 1 | e85768 NPAS4 and ARNT2 Variants Disrupt Activity

Figure 4. NPAS4.F147A, SIM1.F160A, and SIM2.F160A show reduced activities on a reporter gene. A, Comparison of WT NPAS4-mycFlag and NPAS4.F147A-mycFlag activities on reporter gene 6xCME-Luc in HEK293T cells transfected with NPAS4-MycFlag and ARNT2 expression vectors. B, Comparison of WT SIM-2myc proteins with SIM1.F160A-2myc and SIM2.F160A-2myc variants on reporter gene 6xCME-Luc in HEK 293T cells cells transfected with SIM1-2Myc, SIM2-2Myc and ARNT2 expression vectors. C. Immunoblotting of whole cell extracts and a-Flag coimmunoprecipitates from HEK293T cells transiently transfected with NPAS4-MycFlag. a-Flag Abs used to detect NPAS4-MycFlag and a-ARNT2 Abs used to detect endogenous ARNT2. D. ARNT2 coimmunopreciption band intensity quantitation of 3 independent a-Flag coimmunoprecipitation experiments. Data are mean relative luciferase activities 6SEM of 3 experiments. Statistical significance is calculated using an unpaired two tailed students t-test (A and B) or ANOVA (D) compared to WT.***p,0.001, ****p,0.0001. doi:10.1371/journal.pone.0085768.g004 a reporter gene and the endogenous BDNF target gene, which was reduce induction of the reporter gene and/or BDNF. We note that a consequence of disrupted dimerisation with ARNT2. In contrast, the majority of these variants (8/13) lie in a region spanning 300 the other twelve NPAS4 variants we screened failed to significantly amino acids following PASB (Fig 1A), which is predicted to be

PLOS ONE | www.plosone.org 7 January 2014 | Volume 9 | Issue 1 | e85768 NPAS4 and ARNT2 Variants Disrupt Activity

Figure 5. Partial loss of activity variants found in NPAS4, SIM1 and ARNT2. A, Variants of NPAS4-mycFlag, and SIM1-2myc were coexpressed with ARNT2 and assessed for activation of reporter gene 6xCME-Luc in HEK293T cells. Data are presented as average percentage activities (6SEM of 3 independent experiments) of the relevant WT heterodimers which have been normalised 100%. B, Western blot of reporter assay lysates from A to verify expression of hSIM1-2myc and hNPAS4-mycFlag (using a-Myc antibodies), hARNT2 (a-ARNT2 antibodies) and tubulin (a- tubulin antibodies). C, NPAS4-mycFlag activation of reporter gene 6xCME-Luc in combination with the indicated ARNT2-3xFlag variants in HEK293T cells. Data are presented as average percentage activities (6SEM of 3 independent experiments) of WT NPAS4-mycFlag/ARNT2-3xFlag heterodimer which has been normalised to 100%. D, Western blot of reporter assay lysates from C to detect expressed hNPAS4-MycFlag and hARNT2-3xFlag with a-flag antibodies, or tubulin using a-tubulin antibodies. Statistical significance was calculated using an ANOVA comparing relative luciferase activities to WT, * p,0.05, **p,0.01. doi:10.1371/journal.pone.0085768.g005 largely unstructured and seemingly tolerant for variation. As SNP We recently found that rare variants within the bHLH-PAS databases expanded, we tested variants in NPAS4 and ARNT2 transcription factor SIM1 were present in severely obese children which we predicted might be prone to altered activity due to or adults with hyperphagic Prada-Willi Like syndrome [58,59]. position in key domains and distinct change in amino acid side While non-synonymous variants with partial loss of function were chain chemistry. This led to the discovery that variant E257K in found throughout SIM1, those with the most severe effects on NPAS4 had a mild attenuation of activity (,70% of WT), an activity were clustered within N-terminal domains important for observation recapitulated with similarly located variants (G254E, dimerisation and DNA binding. Furthermore, using a reverse G254R) within the related bHLH-PAS SIM1 transcription factor bacterial two-hybrid system, we have previously defined several (Fig 5A). In addition, we successfully predicted and verified the amino acids within the PASA domain of bHLH-PAS heterodimers R46W variant in ARNT2, which lies within a nuclear localisation that are important for dimerisation [33]. These tend to be sequence, to have significantly weaker activity than wild type due clustered within a conserved b-sheet towards the end of PASA, to attenuated nuclear uptake. where the NPAS4 variant F147 resides (Figure S2A). Using homology modelling of NPAS4 and ARNT2 based on the

PLOS ONE | www.plosone.org 8 January 2014 | Volume 9 | Issue 1 | e85768 NPAS4 and ARNT2 Variants Disrupt Activity

Figure 6. Variant R46W in ARNT2 disrupts nuclear localisation. A, Alignment of the N-terminal basic residues of ARNT1 nuclear localisation sequence with WT ARNT2 and ARNT2.R46W. B, Immunofluorescence of HEK293T cells transfected with either WT ARNT2-3xFlag or ARNT2.R46W- 3xFlag expression vectors using a-Flag antibodies (Red) and nuclei stained with DAPI (Blue). doi:10.1371/journal.pone.0085768.g006

CLOCK/BMAL heterodimer crystal structure we confirmed that encompassing NPAS4 has been found to be associated with F147 likely lies at the NPAS4/ARNT2 PASA dimer interface. bipolar disorder [20]. Effects of the variants we tested includes Furthermore, maintenance of hydrophobicity by substitution with near complete loss of function of the NPAS4/ARNT2 heterodi- alanine at this position only partially disrupted reporter activation mer (NPAS4.F147S) and partial loss of function (NPAS4.E257K, and dimerisation (Fig. 4A, Fig. 4C, and Fig. 4D), suggesting that ARNT2.R46W and ARNT2.R107H). While we cannot assess the the presence of a large hydrophobic residue is required for optimal impact of these variants on phenotype, experiments using NPAS4 dimerisation. Sequence alignments of human bHLH-PAS tran- deficient mice suggest affected phenotypes may encompass low scription factors also revealed that the F147 residue is highly severity mild memory deficits and social interaction deficits, conserved at the corresponding position in other bHLH-PAS through to more severe conditions of epilepsy, schizophrenia or transcription factors (Figure S2A). Consistent with this notion, age related neurodegeneration [10,11,15,16]. Our results now replacing this phenylalanine with alanine in SIM1 and SIM2 also seem to warrant targeted sequencing of NPAS4 in cohorts of led to partial loss of function of the SIM/ARNT2 heterodimers neuropsychiatric and/or dementia patients. (Fig 4B). Within the CLOCK/BMAL structure the A’a helix of Finally, the number of new variants identified from ongoing CLOCK PASA make strong interactions with the b-sheet faces of sequencing projects has significantly expanded since the initiation BMAL, and conversely the BMAL A’a helix makes contacts with of this study. The methods outlined here provide a rapid and the b-sheet face of CLOCK to mediate dimerisation [48]. This efficient way of testing for loss of function or weak activity helps explain the lack of NPAS4/ARNT2 interaction for variations in bHLH-PAS transcription factors and can be used to NPAS4.F147S, and also why a screen for PAS A mutations in examine other disease related bHLH-PAS transcription factors ARNT which inhibit AhR/ARNT dimerisation recovered mod- such as SIM1 or HIF1a, as additional variants from new ifications within the surface exposed b-sheet face of ARNT [33]. sequencing projects becomes available. Taken together this supports the notion of F147 being a key residue for dimerisation among bHLH-PAS transcription factors Supporting Information and outlines the importance of this region for intermolecular interaction and heterodimerisation between PASA domains Figure S1 NPAS4.F147S has reduced ability to induce [33,48]. BDNF exon I mRNA expression in HEK293T cells. This study of human NPAS4 and ARNT2 non-synonymous HEK293T cells transiently transfected with NPAS4-MycFlag, variants is the first screen that we are aware of that assess the ARNT2, or control expression vectors. Brain Derived Neuro- specific activities of natural transcription factor variants. While trophic Factor (BDNF) exon I mRNA expression measured by protein coding variants in NPAS4 have yet to be associated with a quantitative real-time PCR and normalised to RNA Polymerase human disorder, the region surrounding NPAS4 has been found to 2A. Data are mean 6SEM of 3 independent experiments. be deleted in a boy with intellectual disability [65], and a region

PLOS ONE | www.plosone.org 9 January 2014 | Volume 9 | Issue 1 | e85768 NPAS4 and ARNT2 Variants Disrupt Activity

Statistical significance is calculated using an ANOVA compared to Table S1 Oligonucleotides used for cloning bHLH-PAS WT NPAS4-mycFlag. ** p,0.01, ***p,0.001. variants. (TIF) (DOCX) Figure S2 Conservation of the PAS domains of bHLH- PAS transcription factors using multiple sequence Acknowledgments alignments. Multiple sequence alignments of selected regions We thank Prof J. Pelletier (McGill University) for the pML-6xCME-Luc of A) basic helix loop helix (bHLH) and PASA regions or B) the vector and the 3rd year University of Adelaide Molecular and Structural PASB regions of human bHLH-PAS transcription factors. Biology Class of 2012 for their help with cloning and analysis of expression Conserved residues of selected variants (ARNT2 R107, plasmids. NPAS4.F147, NPAS4.G208, NPAS4.E257 and SIM1 G254) in this study are highlighted in red, activity deficient variants from Author Contributions CLOCK/BMAL structure [1], ARNT/AhR [2,3] or SIM1 [4,5] Conceived and designed the experiments: DCB MLW. Performed the are in magenta. experiments: DCB JBB. Analyzed the data: DCB JBB DJP MLW. Wrote (DOCX) the paper: DCB MLW.

References 1. McIntosh BE, Hogenesch JB, Bradfield CA (2010) Mammalian Per-Arnt-Sim 22. Chahrour MH, Yu TW, Lim ET, Ataman B, Coulter ME, et al. (2012) Whole- proteins in environmental adaptation. Annual review of physiology 72: 625–645. exome sequencing and homozygosity analysis implicate depolarization-regulated 2. Semenza GL (2012) Hypoxia-inducible factors in physiology and medicine. Cell neuronal genes in autism. PLoS genetics 8: e1002635. 148: 399–408. 23. Neale BM, Kou Y, Liu L, Ma’ayan A, Samocha KE, et al. (2012) Patterns and 3. Furness SG, Lees MJ, Whitelaw ML (2007) The dioxin (aryl hydrocarbon) rates of exonic de novo mutations in autism spectrum disorders. Nature 485: receptor as a model for adaptive responses of bHLH/PAS transcription factors. 242–245. FEBS letters 581: 3616–3625. 24. Yizhar O, Fenno LE, Prigge M, Schneider F, Davidson TJ, et al. (2011) 4. Reppert SM, Weaver DR (2002) Coordination of circadian timing in mammals. Neocortical excitation/inhibition balance in information processing and social Nature 418: 935–941. dysfunction. Nature 477: 171–178. 5. Tolson KP, Gemelli T, Gautron L, Elmquist JK, Zinn AR, et al. (2010) 25. Rubenstein JL (2010) Three hypotheses for developmental defects that may Postnatal Sim1 deficiency causes hyperphagic obesity and reduced Mc4r and underlie some forms of autism spectrum disorder. Current opinion in neurology oxytocin expression. The Journal of neuroscience: the official journal of the 23: 118–123. Society for Neuroscience 30: 3803–3812. 26. Rubenstein JL, Merzenich MM (2003) Model of autism: increased ratio of 6. Michaud JL, Boucher F, Melnyk A, Gauthier F, Goshu E, et al. (2001) Sim1 excitation/inhibition in key neural systems. Genes, brain, and behavior 2: 255– haploinsufficiency causes hyperphagia, obesity and reduction of the paraven- 267. tricular nucleus of the hypothalamus. Human molecular genetics 10: 1465– 27. Kehrer C, Maziashvili N, Dugladze T, Gloveli T (2008) Altered Excitatory- 1473. Inhibitory Balance in the NMDA-Hypofunction Model of Schizophrenia. 7. Probst MR, Fan CM, Tessier-Lavigne M, Hankinson O (1997) Two murine Frontiers in molecular neuroscience 1: 6. homologs of the Drosophila single-minded protein that interact with the mouse 28. Sudhof TC (2008) Neuroligins and neurexins link synaptic function to cognitive aryl hydrocarbon receptor nuclear translocator protein. The Journal of disease. Nature 455: 903–911. biological chemistry 272: 4451–4457. 29. Ebert DH, Greenberg ME (2013) Activity-dependent neuronal signalling and 8. Lindebro MC, Poellinger L, Whitelaw ML (1995) Protein-protein interaction via autism spectrum disorder. Nature 493: 327–337. PAS domains: role of the PAS domain in positive and negative regulation of the 30. Saxena S, Caroni P (2011) Selective neuronal vulnerability in neurodegenerative bHLH/PAS dioxin receptor-Arnt transcription factor complex. The EMBO diseases: from stressor thresholds to degeneration. Neuron 71: 35–48. journal 14: 3528–3539. 31. Bezprozvanny I, Mattson MP (2008) Neuronal calcium mishandling and the 9. Chapman-Smith A, Whitelaw ML (2006) Novel DNA binding by a basic helix- pathogenesis of Alzheimer’s disease. Trends in neurosciences 31: 454–463. loop-helix protein. The role of the dioxin receptor PAS domain. The Journal of 32. Morrow EM, Yoo SY, Flavell SW, Kim TK, Lin Y, et al. (2008) Identifying biological chemistry 281: 12535–12545. autism loci and genes by tracing recent shared ancestry. Science 321: 218–223. 10. Lin Y, Bloodgood BL, Hauser JL, Lapan AD, Koon AC, et al. (2008) Activity- 33. Hao N, Whitelaw ML, Shearwin KE, Dodd IB, Chapman-Smith A (2011) dependent regulation of inhibitory synapse development by Npas4. Nature 455: Identification of residues in the N-terminal PAS domains important for 1198–1204. dimerization of Arnt and AhR. Nucleic acids research 39: 3695–3709. 11. Ramamoorthi K, Fropf R, Belfort GM, Fitzmaurice HL, McKinney RM, et al. (2011) Npas4 regulates a transcriptional program in CA3 required for contextual 34. Skalamera D, Ranall MV, Wilson BM, Leo P, Purdon AS, et al. (2011) A high- memory formation. Science 334: 1669–1675. throughput platform for lentiviral overexpression screening of the human 12. Shamloo M, Soriano L, von Schack D, Rickhag M, Chin DJ, et al. (2006) ORFeome. PloS one 6: e20057. Npas4, a novel helix-loop-helix PAS domain protein, is regulated in response to 35. Gibson DG (2011) Enzymatic assembly of overlapping DNA fragments. cerebral ischemia. The European journal of neuroscience 24: 2705–2720. Methods in enzymology 498: 349–361. 13. Flood WD, Moyer RW, Tsykin A, Sutherland GR, Koblar SA (2004) Nxf and 36. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, 3rd, et al. (2009) Fbxo33: novel seizure-responsive genes in mice. The European journal of Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature neuroscience 20: 1819–1826. methods 6: 343–345. 14. Kim TK, Hemberg M, Gray JM, Costa AM, Bear DM, et al. (2010) Widespread 37. Yoo AS, Staahl BT, Chen L, Crabtree GR (2009) MicroRNA-mediated transcription at neuronal activity-regulated enhancers. Nature 465: 182–187. switching of chromatin-remodelling complexes in neural development. Nature 15. Coutellier L, Beraki S, Ardestani PM, Saw NL, Shamloo M (2012) Npas4: a 460: 642–646. neuronal transcription factor with a key role in social and cognitive functions 38. Moffett P, Pelletier J (2000) Different transcriptional properties of mSim-1 and relevant to developmental disorders. PloS one 7: e46604. mSim-2. FEBS letters 466: 80–86. 16. Ooe N, Motonaga K, Kobayashi K, Saito K, Kaneko H (2009) Functional 39. Farrall AL, Whitelaw ML (2009) The HIF1alpha-inducible pro-cell death gene characterization of basic helix-loop-helix-PAS type transcription factor NXF in BNIP3 is a novel target of SIM2s repression through cross-talk on the hypoxia vivo: putative involvement in an ‘‘on demand’’ neuroprotection system. The response element. Oncogene 28: 3671–3680. Journal of biological chemistry 284: 1057–1063. 40. Woods SL, Whitelaw ML (2002) Differential activities of murine single minded 1 17. Ramocki MB, Zoghbi HY (2008) Failure of neuronal homeostasis results in (SIM1) and SIM2 on a hypoxic response element. Cross-talk between basic common neuropsychiatric phenotypes. Nature 455: 912–918. helix-loop-helix/per-Arnt-Sim homology transcription factors. The Journal of 18. Burmeister M, McInnis MG, Zollner S (2008) Psychiatric genetics: progress biological chemistry 277: 10236–10243. amid controversy. Nature reviews Genetics 9: 527–540. 41. Pruunsild P, Sepp M, Orav E, Koppel I, Timmusk T (2011) Identification of cis- 19. O’Roak BJ, Deriziotis P, Lee C, Vives L, Schwartz JJ, et al. (2011) Exome elements and transcription factors regulating neuronal activity-dependent sequencing in sporadic autism spectrum disorders identifies severe de novo transcription of human BDNF gene. The Journal of neuroscience: the official mutations. Nature genetics 43: 585–589. journal of the Society for Neuroscience 31: 3295–3308. 20. Psychiatric GCBDWG (2011) Large-scale genome-wide association analysis of 42. Olechnowicz SW, Fedele AO, Peet DJ (2012) Hypoxic induction of the regulator bipolar disorder identifies a new susceptibility locus near ODZ4. Nature genetics of G-protein signalling 4 gene is mediated by the hypoxia-inducible factor 43: 977–983. pathway. PloS one 7: e44564. 21. Sanders SJ, Murtha MT, Gupta AR, Murdoch JD, Raubeson MJ, et al. (2012) 43. Muller PY, Janovjak H, Miserez AR, Dobbie Z (2002) Processing of gene De novo mutations revealed by whole-exome sequencing are strongly associated expression data generated by quantitative real-time RT-PCR. BioTechniques with autism. Nature 485: 237–241. 32: 1372–1374, 1376, 1378–1379.

PLOS ONE | www.plosone.org 10 January 2014 | Volume 9 | Issue 1 | e85768 NPAS4 and ARNT2 Variants Disrupt Activity

44. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 55. Chang Q, Khare G, Dani V, Nelson S, Jaenisch R (2006) The disease years of image analysis. Nature methods 9: 671–675. progression of Mecp2 mutant mice is affected by the level of BDNF expression. 45. Abagyan RA, Totrov M.M., and Kuznetsov D.A. (1994) ICM: A New Method Neuron 49: 341–348. For Protein Modeling and Design: Applications To Docking and Structure 56. Egan MF, Kojima M, Callicott JH, Goldberg TE, Kolachana BS, et al. (2003) Prediction From The Distorted Native Conformation. J Comp Chem 15: 488– The BDNF val66met polymorphism affects activity-dependent secretion of 506. BDNF and human memory and hippocampal function. Cell 112: 257–269. 46. Cardozo T, Totrov M, Abagyan R (1995) Homology modeling by the ICM 57. Sun W, Zhang J, Hankinson O (1997) A mutation in the aryl hydrocarbon method. Proteins 23: 403–414. receptor (AHR) in a cultured mammalian cell line identifies a novel region of 47. Abagyan R, Batalov S, Cardozo T, Totrov M, Webber J, et al. (1997) Homology AHR that affects DNA binding. The Journal of biological chemistry 272: modeling with internal coordinate mechanics: deformation zone mapping and 31845–31854. improvements of models via conformational search. Proteins Suppl 1: 29–37. 58. Bonnefond A, Raimondo A, Stutzmann F, Ghoussaini M, Ramachandrappa S, 48. Huang N, Chelliah Y, Shan Y, Taylor CA, Yoo SH, et al. (2012) Crystal et al. (2013) Loss-of-function mutations in SIM1 contribute to obesity and structure of the heterodimeric CLOCK:BMAL1 transcriptional activator Prader-Willi-like features. The Journal of clinical investigation 123: 3037–3041. complex. Science 337: 189–194. 59. Ramachandrappa S, Raimondo A, Cali AM, Keogh JM, Henning E, et al. (2013) Rare variants in single-minded 1 (SIM1) are associated with severe 49. Arnautova YA, Abagyan RA, Totrov M (2011) Development of a new physics- obesity. The Journal of clinical investigation 123: 3042–3050. based internal coordinate mechanics force field and its application to protein 60. Eguchi H, Ikuta T, Tachibana T, Yoneda Y, Kawajiri K (1997) A nuclear loop modeling. Proteins 79: 477–498. localization signal of human aryl hydrocarbon receptor nuclear translocator/ 50. Fu W, O’Connor TD, Jun G, Kang HM, Abecasis G, et al. (2012) Analysis of hypoxia-inducible factor 1beta is a novel bipartite type recognized by the two 6,515 exomes reveals the recent origin of most human protein-coding variants. components of nuclear pore-targeting complex. The Journal of biological Nature. chemistry 272: 17640–17647. 51. Genomes Project C, Abecasis GR, Altshuler D, Auton A, Brooks LD, et al. 61. MacArthur DG, Balasubramanian S, Frankish A, Huang N, Morris J, et al. (2010) A map of human genome variation from population-scale sequencing. (2012) A systematic survey of loss-of-function variants in human protein-coding Nature 467: 1061–1073. genes. Science 335: 823–828. 52. Abecasis GR, Auton A, Brooks LD, DePristo MA, Durbin RM, et al. (2012) An 62. Won H, Lee HR, Gee HY, Mah W, Kim JI, et al. (2012) Autistic-like social integrated map of genetic variation from 1,092 human genomes. Nature 491: behaviour in Shank2-mutant mice improved by restoring NMDA receptor 56–65. function. Nature 486: 261–265. 53. Ooe N, Saito K, Mikami N, Nakatuka I, Kaneko H (2004) Identification of a 63. Auerbach BD, Osterweil EK, Bear MF (2011) Mutations causing syndromic novel basic helix-loop-helix-PAS factor, NXF, reveals a Sim2 competitive, autism define an axis of synaptic pathophysiology. Nature 480: 63–68. positive regulatory role in dendritic-cytoskeleton modulator drebrin gene 64. Clement JP, Aceti M, Creson TK, Ozkan ED, Shi Y, et al. (2012) Pathogenic expression. Molecular and cellular biology 24: 608–616. SYNGAP1 mutations impair cognitive development by disrupting maturation of 54. Ooe N, Saito K, Kaneko H (2009) Characterization of functional heterodimer dendritic spine synapses. Cell 151: 709–723. partners in brain for a bHLH-PAS factor NXF. Biochimica et biophysica acta 65. Floor K, Baroy T, Misceo D, Kanavin OJ, Fannemel M, et al. (2012) A 1 Mb de 1789: 192–197. novo deletion within 11q13.1q13.2 in a boy with mild intellectual disability and minor dysmorphic features. European journal of medical genetics 55: 695–699.

PLOS ONE | www.plosone.org 11 January 2014 | Volume 9 | Issue 1 | e85768

bHLH PAS A

hNPAS1 PLPGAISSQLDKASIVRLSVTYLR 96 SLVQERSFFVRMKSTLTKRG 259 LGHTLPPAPLAELPLHGHMI 305 hNPAS3 PLPAAITSQLDKASIIRLTISYLK 102 DNTLERSFFIRMKSTLTKRG 275 VAHALPPPTINEVRIDCHMF 333 hSIM1 PLPSAITSQLDKASIIRLTTSYLK 51 EYEIERSFFLRMKCVLAKRN 172 VGHSLPPSAVTEIKLHSNMF 232 hSIM2 PLPSAITSQLDKASIIRLTTSYLK 51 EYEIERSFFLRMKCVLAKRN 172 VGQSLPPSAITEIKLYSNMF 232 hHIF1a PLPHNVSSHLDKASVMRLTISYLR 68 EQNTQRSFFLRMKCTLTSRG 179 ICEPIPHPSNIEIPLDSKTF 242 hHIF2a PLPHSVSSHLDKASIMRLAISFLR 65 DMSTERDFFMRMKCTVTNRG 178 MCEPIQHPSHMDIPLDSKTF 244 hARNT1 PTCSALARKPDKLTILRMAVSHMK 140 CMGSRRSFICRMRCGSSSVD 275 QVTSSPNCTDMSNVCQPTEF 363 hARNT2 PTCSALARKPDKLTILRMAVSHMK 114 CMGSRRSFICRMRCGNAPLD 249 QVTSSPVCMDMNGMSVPTEF 336 hBMAL1 PTCNAMSRKLDKLTVLRMAVQHMK 80 CSGARRSFFCRMKCNRPSVK 216 HSHVVPQPVNGEIRVKSMEY 287 hBMAL2 PQCNPMARKLDKLTVLRMAVQHLR 121 YSGSRRSFFCRIKSCKISVK 257 QPYIVPQ-NSGEINVKPTEF 334 hCLOCK PGN---ARKMDKSTVLQKSIDFLR 82 KSKNQLEFCCHMLRGTIDPK 190 ATPQFIKEMCTVEEPN-EEF 276 hNPAS2 PGN---TRKMDKTTVLEKVIGFLQ 55 KSDSDLEFYCHLLRGSLNPK 206 ATPQFLKEMCIVDEPL-EEF 251 hNPAS4 PLAEADKVRLSYLHIMSLACIYTR 51 ALDTDRLFRCRFNTSKSLRR 159 RPRPGPGPGPGPASLFLAMF 217 hAhR PFPQDVINKLDKLSVLRLSVSYLR 78 SPLMERCFICRLRCLLDNSS 232 IATPLQPPSILEIRTKNFIF 287 * : . :: . : * ::

Loop Helix Gβ Linker Aβ

PAS B

hNPAS1 GLTILACESRVSDHMDLGPSELVGR-SCYQFVHGQDATRIRQSHVDLLDKG--QVMTGYY 368 hNPAS3 DLNIIYCENRISDYMDLTPVDIVGK-RCYHFIHAEDVEGIRHSHLDLLNKG--QCVTKYY 396 hSIM1 DMKLIFLDSRVAELTGYEPQDLIEK-TLYHHVHGCDTFHLRCAHHLLLVKG--QVTTKYY 295 hSIM2 DLKLIFLDSRVTEVTGYEPQDLIEK-TLYHHVHGCDVFHLRYAHHLLLVKG--QVTTKYY 295 hHIF1a DMKFSYCDERITELMGYEPEELLGR-SIYEYYHALDSDHLTKTHHDMFTKG--QVTTGQY 305 hHIF2a DMKFTYCDDRITELIGYHPEELLGR-SAYEFYHALDSENMTKSHQNLCTKG-—QVVSGQY 307 hARNT1 EGIFTFVDHRCVATVGYQPQELLGK-NIVEFCHPEDQQLLRDSFQQVVKLKG-QVLSVMF 427 hARNT2 DGIITFVDPRCISVIGYQPQDLLGK-DILEFCHPEDQSHLRESFQQVVKLKG-QVLSVMY 401 hBMAL1 DGKFVFVDQRATAILAYLPQELLGT-SCYEYFHQDDIGHLAECHRQVLQTRE-KITTNCY 403 hBMAL2 NGKFVYVDQRATAILGYLPQELLGT-SCYEYFHQDDHNNLTDKHKAVLQSKE-KILTDSY 350 hCLOCK EWKFLFLDHRAPPIIGYLPFEVLGT-SGYDYYHVDDLENLAKCHEHLMQYG--KGKSCYY 339 hNPAS2 EWKFLFLDHRAPPIIGYLPFEVLGT-SGYDYYHIDDLELLARCHQHLMQFG--KGKSCCY 314 hNPAS4 DLALLDISESVLIYLGFERSELLCK-SWYGLLHPEDLAHASAQHYRLLAESGDIQAEMVV 282 hAhR DFTPIGCDAKGRIVLGYTEAELCTRGSGYQFIHAADMLYCAESHIRMIKTG--ESGMIVF 351 . :: * * . :.

Bβ Cα Dα Eα Fα Gβ

1. Huang N, Chelliah Y, Shan Y, Taylor CA, Yoo SH, et al. (2012) Crystal structure of the heterodimeric CLOCK:BMAL1 transcriptional activator complex. Science 337: 189- 194. 2. Hao N, Whitelaw ML, Shearwin KE, Dodd IB, Chapman-Smith A (2011) Identification of residues in the N-terminal PAS domains important for dimerization of Arnt and AhR. Nucleic acids research 39: 3695-3709. 3. Sun W, Zhang J, Hankinson O (1997) A mutation in the aryl hydrocarbon receptor (AHR) in a cultured mammalian cell line identifies a novel region of AHR that affects DNA binding. The Journal of biological chemistry 272: 31845-31854. 4. Bonnefond A, Raimondo A, Stutzmann F, Ghoussaini M, Ramachandrappa S, et al. (2013) Loss-of-function mutations in SIM1 contribute to obesity and Prader-Willi-like features. The Journal of clinical investigation 123: 3037-3041. 5. Ramachandrappa S, Raimondo A, Cali AM, Keogh JM, Henning E, et al. (2013) Rare variants in single-minded 1 (SIM1) are associated with severe obesity. The Journal of clinical investigation 123: 3042-3050.

Primer&Name& Sequence& ARNT2 F 5’ GGCGCAGACGGAACAAGATG 3’ ARNT2 R 5’ CCTTGGGAGTAGATCTGCTG 3’ hARNT2 R46W F 5' CCTGCCCGTGGAGGAAAGTGGAGATCTGGAATGGACTTCGATGATGAAGA 3' hARNT2 R46W R 5' TCTTCATCATCGAAGTCCATTCCAGATCTCCACTTTCCTCCACGGGCAGG 3' hARNT2 R107H F 5' CCAGACAAGCTCACCATCCTCCATATGGCCGTCTCGCACATGAAG 3' hARNT2 R107H R 5' CTTCATGTGCGAGACGGCCATATGGAGGATGGTGAGCTTGTCTGG 3' hARNT2 R402Q F 5' CCAAGTCCTGTCGGTCATGTATCAATTTCGAACCAAGAACCGGGAG 3' hARNT2 R402Q R 5' CTCCCGGTTCTTGGTTCGAAATTGATACATGACCGACAGGACTTGG 3' hARNT2 W410R F 5' CGCACCAAGAACCGGGAGCGCATGCTGATCCGCACCAGCAGCT 3' hARNT2 W410R R 5' AGCTGCTGGTGCGGATCAGCATGCGCTCCCGGTTCTTGGTGCG 3' hNPAS4 F147A F 5' TGCCCTGGACACTGATCGGCTAGCCCGCTGCCGCTTCAAC 3' hNPAS4 F147A R 5' GTTGAAGCGGCAGCGGGCTAGCCGATCAGTGTCCAGGGCA 3' hNPAS4 F147S F 5' CTGGACACTGATCGCCTCTCCCGCTGCAGATTCAACAC 3' hNPAS4 F147S R 5' GTGTTGAATCTGCAGCGGGAGAGGCGATCAGTGTCCAG 3' hNPAS4 G208C F 5' CCTGGCCCTGGCCCTTGCCCTGCTAGCCTCTTCC 3' hNPAS4 G208C R 5' GGAAGAGGCTAGCAGGGCAAGGGCCAGGGCCAGG 3' hNPAS4 E257K F 5' GGTATGGACTGCTGCACCCCAAAGATCTGGCCCACGCTTCTGC 3' hNPAS4 E257K R 5' GCAGAAGCGTGGGCCAGATCTTTGGGGTGCAGCAGTCCATACC 3' hNPAS4 W293R F 5' CAAGACTGGAGGCTGGGCGCGCATTTACTGCCTGTTATACTCAGAAGGTC 3' hNPAS4 W293R R 5' GACCTTCTGAGTATAACAGGCAGTAAATGCGCGCCCAGCCTCCAGTCTTG 3' hNPAS4 C296R F 5' AGGCTGGGCATGGATTTACGGTCTACTATACTCAGAAGGTCCAGAGGGA 3' hNPAS4 C296R R 5' TCCCTCTGGACCTTCTGAGTATAGTAGACCGTAAATCCATGCCCAGCCT 3' hNPAS4 M317L F 5' CCCAATCAGTGACCTCGAGGCCTGGAGCCTCCGC 3' hNPAS4 M317L R 5' GCGGAGGCTCCAGGCCTCGAGGTCACTGATTGGG 3' hNPAS4 P344A F 5’ ATGCTGGCAAGCTTCCCTGAAAACATTCTTTCCCAG 3’ hNPAS4 P344A R 5’ CAGGGAAGCTTGCCAGCATGGTCGGAGTGCC 3’ hNPAS4 M359I F 5’ AAGAGTGCTCGAGCATTAACCCACTCTTCACCGC 3’ hNPAS4 M359I R 5’ GGTTAATGCTCGAGCACTCTTCCTGGGAAAGAATGTTTTC 3’ hNPAS4 P472S F 5’ GATCAGTTAACGTCCAGCAGTGCAACCTTCCCAG 3’ hNPAS4 P472S R 5’ GCACTGCTGGACGTTAACTGATCAGAGAAGGTCGCAGTG 3’ hNPAS4 Q500K F 5' CCTCGGTCAGATCTTATGAAGACAAGTTGACTCCCTGCACCTCC 3' hNPAS4 Q500K R 5' GGAGGTGCAGGGAGTCAACTTGTCTTCATAAGATCTGACCGAGG 3' hNPAS4 T587M F 5’ GACTGCATGCTGCTAGCCCTAGCCCAGCTCCG 3’ hNPAS4 T587M R 5’ TAGGGCTAGCAGCATGCAGTCCCCATTACCAGGGC 3’ hNPAS4 N702D F 5’ CCTGATGACATGTTTCTAGAAGAGACGCCCGTGGAAG 3’ hNPAS4 N702D R 5’ CTCTTCTAGAAACATGTCATCAGGGTCAGCCAGGAAGT 3’ hNPAS4 D750Y F 5’ CTGTCCCCCGAGTACCACAGCTTCCTGGAGGACC 3’ hNPAS4 D750Y R 5’ GCTGTGGTACTCGGGGGACAGGTTGTTGCAAGGC 3’ hNPAS4 T777I F 5' CCCCTATGATGGGTTTATTGATGAGCTCCATCAACTCCAGAGC 3' hNPAS4 T777I R 5' GCTCTGGAGTTGATGGAGCTCATCAATAAACCCATCATAGGG 3' hNPAS4 AscI F 5' CGATCGCCATGTACCGCTC 3' hNPAS4 F1 5' GCTCGTTTAGTGAACCGTCAG 3' hNPAS4 R1 5' GCCTTGCAGTGGGCTAGTTAG 3' hNPAS4 F2 5' CCCATTTGCCCACCCCATCC 3' hNPAS4 R2 5' CTAGTCCGCCAGTGCCAGC 3' hNPAS4 F3 5' GAGGCCTCTCCAGTCAAGCAG 3' hNPAS4 R3 5' AGGGGTCACAGGGATGCCACCCG 3' hNPAS4 bglI F 5' GAGGAGATCTGCCGCCGC 3' EF Pst I F 5’ TTTTTTTCTTCCATTTCAGGTGTCGTGA 3’ SIM1 PstI R 5’ GAAGGCTGGTTTGGAGGCTG 3’ hSIM1 G254E F 5' TCCAGGGTGGCGGAGCTCACGGAGTACGAACCTCAGGACCTGA 3' hSIM1 G254E R 5' TCAGGTCCTGAGGTTCGTACTCCGTGAGCTCCGCCACCCTGGA 3' hSIM1 G254R F 5' TCCAGGGTGGCGGAGCTCACGCGTTACGAACCTCAGGACCTGA 3' hSIM1 G254R R 5' TCAGGTCCTGAGGTTCGTAACGCGTGAGCTCCGCCACCCTGGA 3' hSIM1 F160A F 5' CCCTACCACTCTCACTTCGTGCAGGAGTATGAGATCGAGCGCTCCGCCTTCCTACGTATGAAGTGCGTCTTGG 3' hSIM1 F160A R 5' ACCTTGTAGCCGCCACAGGTGAGGCCGGCGTTACGCTTGGCCAAGACGCACTTCATACGTAGGAAGGCGGAGC 3' hSIM2L PvuII F 5' TGCCAAGCTGCTCCCGCTG 3' hSIM2L PvuII R 5' GGGCCAGCACGCGGTGG 3' hSIM2L F160A F 5' GAGTATGAGATAGAGAGATCTGCCTTTCTTCGAATGAAATGTGTCTTGG 3' hSIM2L F160A R 5' ATTTCATTCGAAGAAAGGCAGATCTCTCTATCTCATACTCTTGGAGCAGG 3'

Appendix 4

Inducible and Reversible Lentiviral and Recombination Mediated Cassette Exchange (RMCE) Systems for Controlling Gene Expression

1,2David C. Bersten, 1,2Adrienne E. Sullivan, 1,2Dian Li, 1,2Veronica Bhakti, 3,4Stephen J. Bent and 1,2Murray L. Whitelaw

1School of Molecular and Biomedical Science (Biochemistry), 2Australian Research Council Special Research Centre for the Molecular Genetics of Development, 3School of Molecular and Biomedical Science (Genetics), and 4Robinson Research Institute

The University of Adelaide, Adelaide, South Australia, Australia

To whom correspondence should be addressed:

A/Prof. Murray Whitelaw; David Bersten

School of Molecular and Biomedical Science (Biochemistry),

The University of Adelaide, North Tce, Adelaide, SA 5005, Australia

Email: [email protected]; [email protected]

Manuscript

106

15/5/2014 Inducible and Reversible Lentiviral and Recombination Mediated Cassette Exchange (RMCE) Systems for Controlling Gene Expression

1,2David C. Bersten, 1,2Adrienne E. Sullivan, 1,2Dian Li, 1,2Veronica Bhakti, 3,4Stephen J. Bent and 1,2Murray L. Whitelaw

1School of Molecular and Biomedical Science (Biochemistry), 2Institute of Molecular Pathology, 3School of Molecular and Biomedical Science (Genetics), and 4Robinson Research Institute

The University of Adelaide, Adelaide, South Australia, Australia

To whom correspondence should be addressed:

A/Prof. Murray Whitelaw; David Bersten

School of Molecular and Biomedical Science (Biochemistry),

The University of Adelaide, North Tce, Adelaide, SA 5005, Australia

Email: [email protected]; [email protected]

Running Title; Inducible and reversible platforms for controlling gene expression

Abstract

Manipulation of gene expression to invoke loss of function (LoF) or gain of function (GoF) phenotypes is important for interrogating complex biological questions both in vitro and in vivo. Doxycycline (Dox)-inducible gene expression systems are commonly used although success is often limited by high background and insufficient sensitivity to Dox. Here we develop broadly applicable platforms for reliable, tightly controlled and reversible Dox- inducible systems for lentiviral mediated generation of cell lines or FLP Recombination- Mediated Cassette Exchange (RMCE) into the Collagen 1a1 (Col1a1) locus (FLP-In Col1a1) in mouse embryonic stem cells. We significantly improve the flexibility, usefulness and robustness of the Dox-inducible system by using Tetracycline (Tet) activator (Tet-On) variants which are more sensitive to Dox, have no background activity and are expressed from single Gateway-compatible constructs. We demonstrate the usefulness of these platforms in ectopic gene expression or gene knockdown in multiple cell lines, primary neurons and in FLP-In Col1a1 mouse embryonic stem cells. We also improve the flexibility of RMCE Dox-inducible systems by generating constructs that allow for tissue or cell type-specific Dox-inducible expression and generate a shRNA selection algorithm which can effectively predict potent shRNA sequences able to knockdown gene expression from single integrant constructs. These platforms provide flexible, reliable and broadly applicable inducible expression systems for studying gene function.

Significance Statement

Inducible expression systems are commonly used to investigate disease relevant gene function. However, significant limitations in current Doxycycline (Dox) inducible ectopic or knockdown gene expression systems often remain, including high background expression in the absence of Dox, low sensitivity to Dox, labour intensive screening of shRNA sequences for knockdown and complex, unreliable establishment of inducible expression systems. We describe simple, modular, and all-inclusive Dox-inducible expression systems delivered through lentiviral infection or Recombination Mediated Cassette Exchange (RMCE), which overcome many previous limitations. We demonstrate improvements in background expression, sensitivity, and shRNA screening, which allow for efficient generation of reliable gain or loss of gene expression systems.

Introduction

Control of gene expression in mammalian cells and in animal models is of significant importance to basic and applied biological research. The ability to temporally control gene expression in a cell or tissue specific manner enables complex biological questions to be addressed with relevance to disease. While transgenesis or conditional gene knockouts have enabled advances, they are generally limited to permanent and complete gene deletion/ectopic expression and are time consuming, expensive and technically challenging. Doxycycline (Dox)-inducible expression systems have been used widely for ectopic expression and knockdown of genes both in culture cells and in vivo, however significant challenges concerning the reliability of expression in cell lines or animals and significant background expression have limited their use. Recently, site-specific insertion of inducible microRNA-30 context (miR30c) based shRNA cassettes in embryonic stem cells have enabled rapid generation of mice with inducible gene knockdown (1,2). However, these systems employ Tet activators driven from different genomic sites, do not provide expression in all tissues, and lack a flexible platform to gain tissue-specific expression. Impaired Dox-inducible expression in some tissues may be due to lower promoter activity in these tissues, decreased accessibility of the transcriptional machinery to the loci expressing either the Tet-activator (Tet-ON) or the Tet- responsive factor in certain tissues, or limited delivery of Dox to these tissues. In addition, the algorithms used to predict potent miR30c flanked shRNA sequences able to knockdown gene expression from singly integrated constructs are yet to be fully developed, thus time-consuming testing of multiple sequences for each target gene of interest is still required.

Here we generate flexible and reliable Dox-inducible expression systems which can be rapidly employed for GoF and LoF, overcoming difficulties previously encountered with Dox- inducible expression systems. We designed and generated Gateway-compatible lentiviral or Recombination Mediated Cassette Exchange (RMCE) vectors, which contain all the components necessary to gain strong Dox-inducible expression from a near-zero background. We developed a modular platform, which can easily be adjusted to gain tissue-specific expression, and use alternative Tetracycline activators to increase the sensitivity to Dox, which should overcome poor response in some tissues due to limited delivery of Dox. We also designed and tested a miR30c shRNA prediction tool, which enables effective knockdown of

2 target genes from singly integrated Dox-inducible cassettes. These platforms provide a broadly applicable Tet-On system and enable improved methods for rapid generation of cell lines and mice in order to study gain and loss of gene function.

Material & Methods

Generation of Lentiviral and RMCE Constructs pCMV-Tet-On variant 16 (V16; V9I, F67S, R171K, F86Y, A209T)(3) was kindly provided by A.T. Das, pCMV-Tet-On(4), and Tet-On 3G (V10; F67S, R171K, F86Y, A209T)(3) purchased from Clonetech. Optimised Tet-responsive promoter pTRE3G and pTRE3G-luciferase were also purchased from Clonetech(5). Tet-On variants were subcloned into pEFIRES-puro using BamHI/EcoRI. pLVTPT plamids were constructed by initially creating pTRE-3G-gtwyA by inserting GtwyA from pEFIRESpuro-eYFP-GtwyA (A gift from S. Furness, Monash University) into pTRE-3G using EcoRV. pTRE-3G-gtwyA-IRES-eGFP was created by digesting GtwyA-IRES-eGFP from pCEMM-NTAP-GtwyA-IRES-eGFP (6) with NheI and inserting it into the NheI site of pTRE-3G. pLV-TRE3G-GtwyA was then created by inserting XhoI/NheI TRE-3G-gtwyA into SalI/NheI digested pLV410 (A gift from S. Barry, CHRI, Adelaide) and then MluI PGK-eGFP from pLVx-eGFP-shRNA (A gift from K. Jensen, University of Adelaide) was inserted into MluI digested pLV-TRE3G-GtwyA to create pLV- TRE3G-GtwyA-PGK-eGFP. We then created pLVTPT by inserting EcoRI/NotI Tet-on3G from pEFIRES-puro-Tet-On 3G into EcoRI/NotI digested pENTR1a, eGFP from pLV-TRE3G- GtwyA-PGK-eGFP was the removed with XhoI/XbaI and replaced with SalI/SpeI Tet-On 3G from pENTR1a-Tet-On 3G. pLV-TRE3G-IRES-eGFP was created by removing TRE3G- gtwyA-IRES-eGFP with XhoI/NheI from pTRE-3G-gtwyA-IRES-eGFP and inserted into SalI/NheI digested pLV410. pLVTPTIG was then constructed by first creating pLV-TRE3G- Tet-on3G -IRES-eGFP by LR recombination between pENTR1a-Tet-On 3G and pLV-TRE3G- IRES-eGFP, then SalI/NheI TRE3G-Tet-On-3G-IRES-eGFP was inserted into pLVTPT using SalI/XhoI to create LVTPTIG. pLVTPTIP was created by digesting pLVTPT with NheI and inserting XhoI digested IRES-Puro PCR product generated from pEFIRES-puro using Phusion polymerase and EF F 5’GGGTGGAGACTGAAGTTAGGC 3’ IRES-Puro XbaI R 5’TGAGATCTCTAGATCGTGCGCTCCTTTCGGTCG3’.

FLP-Inducer was created by first removing IRES-Puro from pEFIRES-puro-Tet-On 3G by digestion and relegation with EcoRV/BsaBI. Isothermal assembly was then used to insert TRE- 3G-GtwyA-SV40pA and PGK-ATG-FRT-2xSV40pA PCR products generated from PGK- ATG-FRT (Addgene #20734) and pTRE3G-GtwyA using TRE3G F 5’AAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTC 3’, TRE3G R 5’GATGTGCGCTCTGCCCACTGACGGGCACCGGAGCCTCACGAAGCTGGCGCGCCTC TAGAGCCGCAGACATGAT 3’, PGK ATG F 5’ctggccttttgctcacatggctcgacagatcccccaattcaCgacctcgaaattctaccg 3’, PGK ATG R 5’ GAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTagtggtaaactcgatccagac 3’, into HindIII digested pEF-Tet-On-3G. LoxP-STOP-LoxP was generated by nested PCR using the following primers LSL F AflII 5’gatctagtcttaagATAACTTCGTATAGCATACATTATACGAAGTTATgatatctcgatcccccgggtc

3 3’,LSL R XcmI 5’actagatcCCAGTCTAGACATGGATAACTTCGTATAATGTATGCTATACGAAGTTATaa gcggccatcgaattcgg 3’, LSL F Iso 5’ttctctccacaggtgtccactcccagttcaattacagctcttaagATAACTTCGTATAGCATACATT3’, LSL R Iso 5’ATTCCAGAGCAGAGTTTATGACTTTGCTCTTGTCCAGTCTAGACATGGATAACTTC G 3’ and inserted into AflII/XcmI FLP-Inducer plasmids by isothermal assembly. Human Synapsin I promoter (hSynI) was amplified from pLenti-Synapsin-hChR2(H134R)-EYFP- WPRE (Addgene #20945) using hSyn Iso F 5’AACTCATCAATGTATCTTATCATGTCTGCGGCTCTAGAGGtaagtgtctagactgcagagggc 3’ and hSyn Iso R 5’ gctagcctatagtgagtcgtattaagtactctagccttaagtttctcgactgcgctctcagg 3’, and human Glial Fibrillary Acidic Protein (GFAP) promoter was amplified from human genomic DNA using GFAP Iso F 5’ AACTCATCAATGTATCTTATCATGTCTGCGGCTCTAGAGGcccacctccctctctgtgc 3’, and GFAP Iso R 5’GCTAGCCTATAGTGAGTCGTATTAAGTACTCTAGCCTTAACCTGCTCTGGCTCTGC TCG 3’and inserted into AscI/AflII digested FLP-Inducer by isothermal assembly. Finally the CAG promoter was inserted into FLP-Inducer by first removing CAG from pCAG-ASIP using XhoI/BamHI and inserting it into SalI/BamHI digested pEYFP-ACN (Addgene #20344). We then removed the CAG promoter using AscI/AflII and ligated it into AscI/AflII digested pFLP- Inducer.

pENTR1a-dsRed-m30c was constructed by first cloning dsRed-Express (Clonetech) into pENTR1a (Invitrogen) with SalI/NotI, then the miR30 based context was cloned into NotI/XbaI sites of pENTR1a-dsRed using the following primers miR30 5’Arm 5’cgtaaGCGGCCGCGTCGACTAGGGATAACAGGGTAATTGTTTGAATGAGGCTTCAG TACTTTACAGAATCGTTGCCTGCACATCTTGGAAACACTTGCTGGG 3, miR30 mid Arm 5’CTTGGAAACACTTGCTGGGATTACTTCTTCAGGTTAACCCAACAGAAGGCTCGAG CAACCAGATATCGAATTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACT 3’, miR30 3’Arm 5’GGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTT TTACAAAGCTGAATTAAAATGGTATAAATTAAATCACTTTCTAGAcgtaa 3’. pENTR1a-eYFP-m30c and pENTR1a-dnucTomato-m30c were subsequently constructed by replacing the dsRed with KpnI/NotI eYFP or SmaI/NotI dnucTomato from peYFP-ACN and pNESN (A gift from A. Yoo and G.R. Crabtree, Stanford University), respectively.

miR30 shRNA sequences were then inserted into pENTR1a-dsRed-m30c by digesting the plasmid with XhoI/EcoRV and inserting PCR generated ~190bp products using 97mer template shRNA primers (Ultramer IDTdna, Supplementary Table 1) and M30c Iso F 5’CTTGCTGGGATTACTTCTTCAGGTTAACCCAACAGAAGGCTCGAGaaggtatatTGCTG TTGACAGTGAGCG3’ and M30c Iso R 5’AACAAGATAATTGCTCCTAAAGTAGCCCCTTGAATTCGATTCCGAGGCAGTAGGC A3’ amplification primers using isothermal assembly as described (7). Multiplex cloning of 10 or more shRNA clones was achieved by mixing 190bp generated shRNA cloning templates and assembly into XhoI/EcoRV digested pENTR1a-miR30c plasmids. Individual shRNA clones were then identified by sequencing. Using this technique cloning efficiency was close to 100% 4 and 50-70% of these could be confirmed by sequencing. Fluorescent miR30-shRNA or Flag tagged NPAS4, SIM2s and SIM2l cDNAs were recombined into pFLP-Inducer or pLVTPT vectors by LR recombination.

Lentiviral production, Infections and cell line generation

Lentivirus was produced by transient transfection in HEK293T cells using calcium phosphate precipitation, and for primary neuron infections was concentrated (0.5-1x109 TU/ml) and viral titre estimated as described (8). Stable cell lines were produced by infecting cells with virus plus 8g/ml polybrene at <20% infection rate and subsequently selected in puromycin (200- 1000 ng/ml). Primary mouse embryonic day 16 cortical neurons were infected with for overnight and washed 3 times in Neurobasal/ 2% B27/1% Penicillin-Streptomycin/1% Glutamine media.

Cell culture, Doxycycline inductions and transfections

HEK293T and MEF’s were cultured in DMEM (Gibco #12430), 10%FBS, 1% GlutaMAX (Gibco), 1% Penicillin/Streptomycin (Invitrogen), while LNCaP, DU145, MDA-MB-231, MDA-MB453, MCF7 were cultured in RPMI 1640 (Gibco #11835) with the same additives. Mouse embryonic stem cells were maintained on irradiated primary MEF’s in DMEM (Gibco #11995), 15%FBS, 1% L-Glutamine, 1% Penicillin/Streptomycin, 55M -Mecaptoethanol (Gibco #21985) and 1:10,000 ESGRO (LIF; Millipore). Transfections were carried out according to manufacturer instructions using Fugene6 (Roche) unless otherwise indicated. Expression in cell lines and reporter experiments used induction with 1g/ml Doxycyline (Sigma) unless otherwise indicated. Immortalised Maged1 knockout MEFs were a kind gift from Prof. Olivier de Backer, University of Namur, Belgium.

Luciferase Reporter Gene Assays

~1x105 HEK293T cells were transfected with 50ng of pEFIRES-puro-Tet-On/Tet-On-V16/Tet- On-3G, 200pg pCMV-RL and 200ng of pTRE3G-Luc with Fugene6 (Roche). Cells were lysed 48hrs after transfection and firefly and renilla luciferase were measured on a GloMax96 Microplate Luminometer using the Dual Luciferase Assay System (Promega).

Col1a1 targeting and screening of mouse embryonic stem cells

C57B6/Sv129 hybrid mouse embryonic stem cells were used to target the Col1a1 locus with pCol1A-frt-hygro-pA (Addgene # 20730) by homologous recombination essentially as described (9,10). Embryonic stem cell genomic DNA was digested with EcoRI and an XcmI fragment from pCol1A-frt-hygro-pA was used as a southern probe to identify correctly targeted colonies, which were termed mES-Col1a1-FLP-In cells

Recombination mediated cassette exchange (RMCE) in mES-Col1a1-FLP-In cells.

0.8-1.5 x 107 mES-Col1a1-FLP-In cells grown on MEFs were electroporated twice using a Biorad x-cell gene pulse electroporator set at 400V and 125 µF with 25µg of pPGK-FLPo-bpA (Addgene #13793) and 50µg of pFLP-Inducer plasmid and plated on irradiated hygromycin

5 resistant MEFs (Millipore). 36-48hrs post electroporation cells were incubated with 140 µg/ml Hygromycin containing media and colonies picked typically after 6-8 days of selection. Using this technique we were able to generate ~30 hygromycin resistant colonies per electroporation, all of which were able to induce expression in the presence of Dox.

CRE mediated excision of LoxP-STOP-LoxP from mES-FLP-Inducer-LSL cells

1 x 107 mES-FLP-Inducer-LSL-dsRed-m30c-shP53(814) cells were electroporated using a Biorad x-cell gene pulse electroporator set at 250V and 500 µF with 40µg of pCAG-GS-Cre plasmid. 2 days after electroporation the cells were treated with 2 µg/ml Dox for 24hrs or left untreated and cells were imaged using fluorescence microscopy. Genomic DNA was extracted before and after electroporation with the CRE expressing plasmid for genotyping.

Cell Florescence and FACS Analysis

Cell fluorescence was visualised using an inverted microscope (Zeiss) and images manipulated and overlayed using ImageJ software. FACS analysis was performed on a BD Canto flow cytometer and data analysed using FlowJo software.

RNA extraction, cDNA synthesis, and Real-time PCR

Cells were lysed in Trizol (Invitrogen) and RNA was isolated, purified and reverse transcribed using superscript III (Invitrogen) and cDNA was diluted for real time PCR. Real time PCR was performed in triplicate using primers specific to BDNF exon I F 5’ TTGAAGCTTTGCGGATATTGCG 3’, BDNF exon I R 5’ AAGTTGCCTTGTCCGTGGAC 3’ NPAS4(11), hSIM2L F 5' AGCAGCTCGTCTCCAGCTAA 3', hSIM2L R 5' GTGTCCTCGCCGAACCTG 3', hSIM2s (12), human RNA polymerase 2A (Pol2a)(13) or GAPDH(11) and spanned an intron where possible. The expression level was normalised to RNA polymerase 2A and presented as fold induction over parent cell line. Melt curves of PCR products were used to confirm a single amplicon.

Western Blot

Lysates were prepared in 20mM HEPES pH 8.0, 420mM NaCl, 0.5% Igepal, 25% Glycerol,

0.2mM EDTA, 1.5mM MgCl2 1mM DTT and protease inhibitors, separated on 7.5-10% SDS- PAGE gel and transferred to nitrocellulose. Proteins were detected using anti-FLAG (M2, Sigma), anti-ARNT1 (Abcam, Ab2), anti-P53 (Ab-1, Calbiochem) anti-ARNT2 (Santa Cruz, M-165) and anti--Tubulin antibodies (MCA78G, Serotec). Primary antibodies were detected using horseradish peroxidise-conjugated secondary antibodies and visualised using chemiluminescence. shRNA design algorithm

Our approach for predicting the efficacy of candidate shRNAs was based on an implementation of a support vector machine (SVM) using the statistical programming language R and the e1071 module. The bases of the candidate siRNA guide strand sequences were encoded using the method described by Sciabola et al, 2012 (14), and flanking regions corresponding to 10 bases on the 5’ end of the guide sequence (3’ in the mRNA sequence) and 16 bases on the 3’ end of the guide sequence (5’ in the mRNA sequence) were included in the SVM data table. Additional data incorporated into the data sets included the siRNA scoring output from mir- scan (15), the posterior probability output from contrafold (16), and the output from sfold’s 6 sirna module(17). The source code for the shR-Thing algorithm is available at https://code.google.com/p/shr-thing/

Generation of a miR30 shRNA selection algorithm.

The training data set comprised the reported knockdown efficacy data sets from Fellmann et al, 2011 (18) and Tan et al, 2012 (19), using the ProdEn score reported by Fellmann, and a transformed score with similar properties to Fellmann’s ProdEn score, generated by averaging the log2-transformed hybridization and sequencing values from the sort 6 round, taking 2 to the power of that (removing the log transformation), multiplying by 4, and subtracting 10. This process was empirically determined to yield a similar distribution of scores from the Tan data set as Fellmann’s ProdEn score.

The SVM was trained using the Fellmann and Tan data sets using eps regression and a radial kernel. The SVM was tuned, but shown not to be greatly sensitive to the cost and gamma parameters, which were set at 2.25 and 0.1, respectively. The oligo sequences that received the highest scores from the SVM run were checked manually to exclude homopolymers greater than length 4, and the hairpin sequence was generated following Dow et al, 2012 (1) .

Statistical Analysis

Data was graphed and statistical analysis performed using GraphPad Prism 5 (GraphPad Software Inc.). Statistical differences were evaluated by the Student’s t-test with the level of significance set at p <0.05.

Ethics

To generate primary mouse neuron cultures, pregnant mice were CO2 euthanatized and embryonic cortices were dissected with approval from the University of Adelaide animal ethics committee (Ethics approval # 0000009506).

Results

Reduced background expression and improved sensitivity to Doxycycline using the 3rd generation Tet-activator, Tet-On3G

Significant improvements have recently been made to the Tet-On inducible expression systems, including the molecular evolution of high sensitivity, low background variants of the Tet-activator and low background Tet- responsive promoters (3,5). Before incorporating these components into lentiviral or RMCE expression systems, we confirmed these improvements to the Tet-activator using luciferase reporter assays containing a minimal CMV promoter and seven optimised Tet response element (TRE3G) repeats (5). We observed strong Dox-inducible expression from all Tet-On variants (Fig. 1a), however significant background “leaky” expression was observed in the absence of Dox when either the wildtype (WT) Tet-On or the Tet-On V16 variants were employed as Tet-activators in HEK293T cells (Fig 1b). Activation of the TRE promoter in the absence of Dox was negligible when the Tet-On 3G variant was used (Fig 1b). The inability to gain Dox-inducible expression in some tissues, as described by several groups using the Tet-activator system, has been suggested to be due to low Dox concentration in these tissues. This issue may be overcome by using more sensitive Tet- activator variants (2,20,21). We therefore tested the sensitivity of Tet-On 3G to Dox, and

7 observed that the Tet-On 3G variant was active at Dox concentrations as low as 1ng/ml, revealing dramatically increased sensitivity to Dox compared to the Tet-On (Fig 1c).

A flexible lentiviral platform for Dox-inducible shRNA and cDNA expression in primary and immortalised cells

Next we sought to incorporate the optimised Tet-On 3G variant and TRE3G promoter into flexible platforms that would enable inducible expression or knockdown in readily generated stable cell lines or transduced primary and post-mitotic cells. We initially observed that generation of stable cell lines using a two plasmid system, where the Tet-activator and the TRE promoter-driven response gene were delivered on separate plasmids, was not only time consuming but resulted in cell lines displaying heterogeneous Dox-inducibility (data not shown). We reasoned that creation of constructs where all the components were present in cis would expedite cell line generation and allow homogenous inducibility in all cells. We placed the TRE3G promoter upstream of a Gateway A exchange cassette, followed by the constitutive Phosphoglycerate Kinase-1 promoter (PGKp) to drive expression of the Tet-On 3G and either the EGFP or Puromycin resistance gene linked by an internal ribosome entry site (IRES) (termed LVTPTIG and LVTPTIP, respectively; Fig 1e). To allow for broad applications, such as use in primary or post-mitotic cell types and rapid generation of stable cell lines, which may prove refractory to transfection, we utilised a third-generation lentiviral platform to deliver the expression cassette (Fig 1e). We also generated several Gateway donor plasmids containing dsRED, nucTomato, or EYFP with a downstream miR30c cassette, for producing shRNA sequences that are formatted for processing to siRNA to achieve gene knockdown (Fig 1d).

These lentiviral constructs were efficient in producing high virus titre and transducing a number of different mouse and human cell lines (Fig. 2, Fig. 3 and supplementary Figure 1). Initially, we tested these constructs in HEK293T cells stably transduced with a construct containing dsRed downstream of the TRE3G promoter (Fig. 2a). In the absence of Dox, dsRed was undetectable, while EGFP driven from the PGK promoter was expressed in transduced cells (Fig. 2b). After 24hrs of Dox treatment dsRed was robustly expressed throughout the EGFP-expressing population, indicating that the lentiviral constructs can produce complete populations of expressing cells (Fig. 2b). As previously reported, the Dox-induced expression also increased expression from the downstream PGK promoter as apparent by an increase in EGFP signal (Fig. 2b). This is presumably due to transcriptional read-through from the binding and activation of Tet-activator and the lack of polyadenylation sequences in lentivector constructs (22,23) (Fig. 2b). This allows for positive feed-forward expression of the Tet-On 3G, strengthening induction from the Tet-responsive TRE3G promoter.

To investigate the inducible expression of these constructs further, we used the LVTPTIP lentivector to generate stable HEK293T cell lines harbouring the cDNA for dsRed fused with miR30c shRNA cassettes targeting either luciferase (shGL3) or the basic Helix Loop Helix /Per-Arnt-Sim homology domain (bHLH/PAS) transcription factor neuronal PAS domain protein 4 (NPAS4) (sh2182 and sh275134). We transduced the HEK293T cells such that only 20-30% of cells were infected, limiting the number of integrants per cell to approximately 1 (24). The inducibility of the dsRed-shGL3 cell line was assessed using FACS analysis. In the absence of Dox, dsRed fluorescence was completely absent from all cells and indistinguishable from parental HEK293T cells (Fig. 2c). After 24hrs of Dox treatment most of the cells became

8 RFP positive, and by 48hrs the vast majority of cells strongly expressed RFP (Fig. 2c). To assess whether Dox-inducible expression from constructs integrated at low copy number in cell lines was sufficient to knockdown gene expression, we transiently overexpressed Flag-tagged NPAS4 in stable HEK293T cell lines and induced shRNA expression for 72hrs with Dox (Fig. 2d). We observed potent knockdown of NPAS4 in sh275143-expressing cells (high effect siRNA) but not in control shGL3 or sh2182-expressing cells (low effect siRNA) (Fig. 2d). We were also able to generate very high titre (0.5-1x109 TU/ml) using this lentivector platform, which enabled its use in primary cell transduction experiments. We cultured primary mouse embryonic day 16 post mitotic cortical neurons in vitro and successfully transduced them with LVTPT-dsRed-shGL3 lentivirus, gaining strong Dox-inducible expression in the vast majority of neurons (Fig. 2e). This suggests our platform will be useful for primary cell or in vivo inducible LoF and GoF experiments.

In addition to achieving successful shRNA production, we generated several different cell lines bearing inducible cDNA expression. Robust, inducible expression has been observed in 8 different cell types with a range of cDNAs. For example, Melanoma Antigen Family D isoform 1b (MAGED1b) was reintroduced into Maged1 knockout mouse embryonic fibroblasts (MEFs) using LVTPTIP lentivector transduction and puromycin selection. We observed strong expression within 4hrs of Dox treatment from a background free of detectable expression in the absence of Dox (Fig. 3a). In addition, the LVTPTIP lentivector provided inducible expression of both the short and long isoforms of the bHLH/PAS transcription factor Single Minded 2 (SIM2s or SIM2l) in prostate and breast cancer cell lines (Fig. 3b and Supplementary Figure 1). These lines consistently showed strong Dox-inducible expression within 4hrs of treatment with minimal or no background expression in the absence of Dox (Fig. 3b and Supplementary Figure 1). The lack of any detectable expression in the absence of Dox is of particular importance when working with genes detrimental to cell survival or linked to growth and differentiation. We therefore analysed the expression of SIM2 mRNA by quantitative real time PCR in stable LNCaP prostate cancer cell lines to determine whether there was any background expression that was undetectable by western blot. Importantly, we observed no significant (p=0.18 SIM2s, p=0.35 SIM2l) background expression in the absence of Dox when compared to parental cell lines (Fig. 3c), indicating that in general these platforms could be used to investigate ectopic expression of genes which function in apoptosis, or shRNA production which targets genes essential for cell survival, growth or differentiation.

Generation of FLP-In Col1a1 targeted mouse embryonic stem cells

In addition to lentiviral-mediated gene delivery to primary neurons and generation of stable cell lines, we were also interested in applying this technology to generate inducible expression from a defined locus in mouse embryonic stem (mES) cells. These modified mES cells can be used to study function of genes during in vitro differentiation protocols and in generating mouse models with inducible gain and loss of gene function. Following methods employed by the Jaenisch lab (10) we used a region 3’ of the coding sequence in the Col1a1 locus for targeted homologous recombination to insert a FLP/FRT based RMCE (Recombination Mediated Cassette Exchange) construct into hybrid S129/C57BL6 mES cells. RMCE allows rapid and site-specific exchange of single Dox-inducible cassettes into the Col1a1 locus and has been used to generate transgenic animals with ubiquitous Dox-inducible expression (10,25). The Col1a1 targeting construct contained a neomycin selection cassette 9 flanked by FRT sites, followed by a hygromycin cassette lacking a promoter or start codon as used by Jaenisch and colleagues (10,25) (Fig. 4a). We generated two independently derived mES cell lines by homologous recombination (F1 and F12 mES cell lines) and screened for correctly targeted single integrants by southern blot (Fig. 4a and 4b).

Inducible and reversible shRNA and cDNA expression from the Col1a1 locus using an optimised Flp-In/Tet-Activator system- pFLP-Inducer

Next we generated a platform to facilitate highly efficient FLP-mediated insertion of Dox-inducible cassettes into the Col1a1 locus from a single construct. These constructs contain a PGK promoter followed by an ATG translation start codon, a lone FRT site and two SV40 polyadenylation (PolyA) sequences to stop transcription from the endogenous Col1a1 gene (Fig. 1e and 4a). Downstream of this is the TRE3G promoter, Gateway A cloning cassette, an SV40 PolyA sequence and a strong constitutive promoter (Human elongation factor-1 alpha (EF1 or CMV early enhancer/chicken beta actin (CAG)) driving the expression of the Tet- On3G activator followed by an SV40 polyA sequence. We engineered specific AscI and AflII restriction sites to flank the promoter driving the Tet-On3G to allow for efficient promoter exchange, and an XcmI site before the Tet-On3G start codon to allow insertion of a LoxP- flanked SV40 polyA (LSL) sequence to stop transcription (Fig. 1e). We have created a number of different Gateway-compatible Flp-In cassettes with either strong constitutive promoters followed by LSL (EF1 and CAG) or tissue-specific promoters (human Glial Fibrillary Acidic Protein (hGFAP), astrocytes; human Synapsin I (hSynI), neurons; modified minimal rat Probasin promoter (ARR2Pb), prostate), to allow for use in tissue/cell-specific Dox-inducible applications (Fig. 1e and Supplementary Figure 2, and Supplementary Figure 3). Electroporation of the Flp-In construct along with the codon optimised FLP (FLPo)-expressing vector (26) generally resulted in highly efficient (near 100%) generation of approximately 30 hygromycin-resistant, Dox-inducible colonies when using RMCE S129/C57BL6 cells.

We have generated more than 30 separate dsRed miR30c shRNA-expressing mES lines using the Flp-Inducer constructs and consistently observed strong Dox-inducible expression in all hygromycin-resistant lines generated (Fig. 4c and 4d). Using FACS analysis and fluorescence microscopy we showed that background fluorescence was indistinguishable from that of the parental mES RMCE line and induction of dsRed expression was largely uniform and reversible (Fig. 4c, 4d and 4e; supplementary figure 4). We also tested the ability of these FLP-Inducer mES cell lines to efficiently knockdown gene expression when producing shRNA from a single site in mES cells by using a well-defined potent shRNA against p53(814)(18). Within 2 days of Dox addition we observed an almost complete knockdown of p53 protein, which was not observed using a non-specific shRNA (shGL3). Knockdown of p53 protein was maintained in the presence of Dox for 6 days and reversed after 4-6 days of Dox withdrawal (Fig. 4f), demonstrating the efficacy and reversibility of this system.

Optimisation of shRNA prediction for loss of gene function experiments

We noted that the success of LoF experiments using this system largely relies on the use of potent and specific miR30c shRNAs. shRNAs produced ectopically from a single copy expression cassette that achieve high knockdown efficiency are rare, and as yet such sequences 10 are difficult to reliably predict using current siRNA design algorithms. Recently, a number of high throughput experiments have been performed to identify potent shRNA sequences which, when embedded with the miR30-based context, successfully produce functional siRNAs (18,19). We utilised this data, which assesses the potency of ~60,000 miR30c shRNA sequences, and added variables to account for entropy and mRNA fold data in order to develop an updated siRNA selection algorithm to identify potent miR30c shRNA sequences.

We tested the ability of this shRNA selection algorithm to effectively predict efficient knockdown from singly integrated constructs using our mES FLP-Inducer system. We choose to target two genes, Arnt and Arnt2, which express bHLH/PAS transcription factors in embryonic stem cells (27) and can be readily detected by western immunoblot. We cloned the top 10 shRNA sequences predicted by our algorithm against either Arnt or Arnt2 using a multiplex cloning technique (see methods) and generated mES-FLP-Inducer cell lines for each construct. We then tested each shRNA for knockdown of ARNT or ARNT2 protein following 48 hrs of Dox treatment and observed ~60% (12/20) of shRNA constructs could effectively knockdown gene expression (Fig. 5a and Fig 5b).

Inducible expression of tagged proteins is of particular use in studying transcription factor occupancy of target sites in the genome and transcription factor function in protein complexes. However, excessive overexpression and issues with temporal control or background expression can lead to false positive discovery in such experiments. We therefore created FLP-Inducer cell lines containing Flag tagged NPAS4 to assess background and inducible expression levels of a prototypical cDNA in this system. We observed rapid induction of NPAS4-3xFlag protein expression following 6hrs of Dox addition in the mES cells (Fig 6a). Npas4 mRNA and protein expression is highly restricted to the brain and upregulated following neuronal depolarisation (28,29). To assess the level of expression compared to endogenously expressed NPAS4 we compared mRNA of Col1a1-FLP-Inducer mES cells to mouse cortical neurons cultured in vitro and depolarised with 55mM KCl for 1 hr. After 6hrs of Dox treatment NPAS4 mRNA levels were similar to the endogenous levels in KCl depolarised neurons (Fig 6b). Importantly, we also observed no statistical difference between expression of NPAS4 in parental mES cells or untreated mES-FLP Inducer-NPAS4-3xFlag cells (Fig 6b) indicating a distinct lack of background “leaky” expression compared to previous Tet-On systems expressing cDNAs from the Col1a1 locus (30,31). Ectopic NPAS4 expression in mES-FLP-Inducer–NPAS4-3xFlag cells was also able to activate the downstream target gene Bdnf after 6hrs of Dox induction, indicating that short periods of Dox induction could potentially be used to identify primary target genes of transcription factors (Fig 6C). Many tissues have poor permeability or delivery of Dox and are therefore exposed to lower Dox concentrations, thus strong activation of the Tet- On protein at low Dox concentrations is important for inducible expression in some tissues. Using the FLP-Inducer-NPAS4-3xFlag mES cells we found robust protein expression following 24hrs of treatment with Dox at concentrations as low as 10ng/ml (Fig 6D).

11 Discussion

Here we have engineered optimised, broadly applicable platforms for generation of Dox-inducible expression in immortalised cell lines, primary neurons and use with RMCE at a specific site in embryonic stem cells. Using a 3rd generation Tet-activator system we have created lentiviral constructs capable of generating near homogenous Dox-inducible cell lines free of background expression and capable of strong and reversible ectopic expression or gene knockdown. We have also engineered a flexible Flp-In RMCE system for generation of site- specific Dox-inducible ectopic expression or gene knockdown in mouse embryonic stem cells. While similar lentiviral platforms have been described previously (22,32,33), our platform utilises the Tet-On3G variant (3) which is demonstrably more sensitive to Dox and has lower background expression than other Tet-On variants. We also used the optimised TRE-3G Tet- responsive promoter, which exhibits lower background expression (5). We demonstrate that in the absence of Dox, expression of cDNAs from our lentiviral platform in stable cell lines is largely indistinguishable from uninfected parent cell lines, demonstrating absence of background leaky expression that often plagues these systems. Importantly, we describe an shRNA prediction tool that can effectively predict high potency shRNA target sequences when imbedded in the miR30 context, and we show that more than half of the sequences tested had the ability to knockdown gene expression from a single copy, Dox-inducible cassette in embryonic stem mES cells. This prediction tool may significantly reduce the number of shRNA sequences needed to be tested to identify potent miR30c shRNA sequences for use in vitro and in vivo. In addition, application of this prediction tool to genome wide knockdown experiments may improve the identification of positive hits in screening experiments. Recently, through a series of optimisation experiments, miR30c based shRNA backbone and loop sequences have been further modified to improve processing and target gene knockdown independent of siRNA target sequence (34). While we have not yet examined the effect of these modifications with our shRNA selection algorithm we anticipate that this may further improve the efficiency of miR30 shRNA mediated gene knockdown.

Vector expression systems that utilise viral delivery are prone to stochastic gene silencing through epigenetic mechanisms, proviral integration-specific promoter interference, or genomic deletion due to selective pressure or genomic instability. These mechanisms result in heterogeneous expression in cell populations and have significantly hindered the use of lentiviral-based systems in studying genes essential for cell survival or proliferation. Here we have developed a lentiviral platform which overcomes many of these challenges by using a single vector expression system that contains all the components necessary for Dox-inducible expression, linked to either fluorescent or antibiotic-selectable markers, and absent of any background leaky expression. This allows for uniform Dox-inducible cell populations to be selected, which facilitates the investigation of LoF and GoF of genes essential for cell differentiation, proliferation or survival.

The FLP-Inducer system described here also provides a flexible platform, which allows for rapid and reversible Dox-inducible expression from a single defined locus. RMCE delivery of these constructs to the Col1a1 locus in mES cells at efficiencies approaching 100% allows for facile generation of Dox-inducible cell lines. One of the key advantages of this system is

12 that it can be readily adapted for use in targeting alternative genomic loci, which may be advantageous in certain tissues, or in creating multi-allelic Dox-inducible mice or generating site-specific FLP-In cell lines using FRT/FLP-mediated recombination. The fact that the Tet- activator and Tet-responsive factor(s) are expressed from a single locus simplifies the system and reduces potential positional effects, which may lead to silencing of transgene expression. The use of this high sensitivity, low background Tet-On 3G system also allows for reduced doxycycline levels to be used in vivo or in vitro to gain strong Dox-inducible expression, minimising potential doxycycline off-target effects and/or toxicity. Furthermore, the activity of the Tet-On3G should be sufficient to gain Dox-inducible expression even with limited doxycycline delivery in brain and some other peripheral tissues (2,21).

We have engineered our constructs to be Gateway-compatible, allowing the use of existing publicly available Gateway-compatible mouse and human ORF libraries (http://www.orfeomecollaboration.org) (35,36). This broadens the potential use of this system, as it requires minimal molecular biology experience to generate GoF or LoF constructs. Importantly, the current promoters in these RMCE constructs can easily be exchanged for tissue- or cell type-specific promoters. Alternatively, the use of strong constitutive promoters (CAG and EF1) followed by LSL can be used in conjunction with tissue-specific CRE driver mice to gain tissue- or cell type-specific expression. There are now large numbers of CRE, tamoxifin inducible CRE Knock-In, or BAC-CRE driver lines publically available to gain specific tissue, regional or cell type specific expression (http://www.neuroscienceblueprint.nih.gov; http://www.gensat.org/cre.jsp; http://cre.jax.org; http://www.creportal.org/(37-39)). We believe this added flexibility may address some of the problems encountered with previous Tet-On systems and broaden the potential application of this technology. The emergence of technologies allowing for efficient site specific pronuclear transgene delivery by integrase (40), CRISPR(41) or TALEN(42) technologies may also expedite the generation of Dox-inducible animal models and inbred mouse strains commonly used in immunology and neuroscience research. These systems significantly advance those used in mouse genetics to allow complex biological questions to be addressed in vivo in a similar fashion to that of less complex model organisms.

Funding

This research was supported by the Australian Research Council.

Acknowledgements

We would like to thank Dr. A.S. Yoo (Washington University), Prof. G.R. Crabtree (Stanford University), S. Furness (Monash University) and Dr. A.T. Das (University of Amsterdam) for providing reagents/DNA clones.

13 1. Dow, L.E., Premsrirut, P.K., Zuber, J., Fellmann, C., McJunkin, K., Miething, C., Park, Y., Dickins, R.A., Hannon, G.J. and Lowe, S.W. (2012) A pipeline for the generation of shRNA transgenic mice. Nat Protoc, 7, 374-393. 2. Premsrirut, P.K., Dow, L.E., Kim, S.Y., Camiolo, M., Malone, C.D., Miething, C., Scuoppo, C., Zuber, J., Dickins, R.A., Kogan, S.C. et al. (2011) A rapid and scalable system for studying gene function in mice using conditional RNA interference. Cell, 145, 145-158. 3. Zhou, X., Vink, M., Klaver, B., Berkhout, B. and Das, A.T. (2006) Optimization of the Tet- On system for regulated gene expression through viral evolution. Gene Ther, 13, 1382- 1390. 4. Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W. and Bujard, H. (1995) Transcriptional activation by tetracyclines in mammalian cells. Science, 268, 1766- 1769. 5. Loew, R., Heinz, N., Hampf, M., Bujard, H. and Gossen, M. (2010) Improved Tet- responsive promoters with minimized background expression. BMC Biotechnol, 10, 81. 6. Burckstummer, T., Bennett, K.L., Preradovic, A., Schutze, G., Hantschel, O., Superti- Furga, G. and Bauch, A. (2006) An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells. Nat Methods, 3, 1013-1019. 7. Gibson, D.G., Young, L., Chuang, R.Y., Venter, J.C., Hutchison, C.A., 3rd and Smith, H.O. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods, 6, 343-345. 8. Tiscornia, G., Singer, O. and Verma, I.M. (2006) Production and purification of lentiviral vectors. Nat Protoc, 1, 241-245. 9. Wu, S., Ying, G., Wu, Q. and Capecchi, M.R. (2008) A protocol for constructing gene targeting vectors: generating knockout mice for the cadherin family and beyond. Nat Protoc, 3, 1056-1076. 10. Beard, C., Hochedlinger, K., Plath, K., Wutz, A. and Jaenisch, R. (2006) Efficient method to generate single-copy transgenic mice by site-specific integration in embryonic stem cells. Genesis, 44, 23-28. 11. Bersten, D.C., Wright, J.A., McCarthy, P.J. and Whitelaw, M.L. (2014) Regulation of the neuronal transcription factor NPAS4 by REST and microRNAs. Biochim Biophys Acta, 1839, 13-24. 12. Farrall, A.L. and Whitelaw, M.L. (2009) The HIF1alpha-inducible pro-cell death gene BNIP3 is a novel target of SIM2s repression through cross-talk on the hypoxia response element. Oncogene, 28, 3671-3680. 13. Bersten, D.C., Bruning, J.B., Peet, D.J. and Whitelaw, M.L. (2014) Human Variants in the Neuronal Basic Helix-Loop-Helix/Per-Arnt-Sim (bHLH/PAS) Transcription Factor Complex NPAS4/ARNT2 Disrupt Function. PLoS One, 9, e85768. 14. Sciabola, S., Cao, Q., Orozco, M., Faustino, I. and Stanton, R.V. (2013) Improved nucleic acid descriptors for siRNA efficacy prediction. Nucleic Acids Res, 41, 1383-1394. 15. Matveeva, O.V., Nazipova, N.N., Ogurtsov, A.Y. and Shabalina, S.A. (2012) Optimized models for design of efficient miR30-based shRNAs. Front Genet, 3, 163. 16. Do, C.B., Woods, D.A. and Batzoglou, S. (2006) CONTRAfold: RNA secondary structure prediction without physics-based models. Bioinformatics, 22, e90-98. 17. Ding, Y., Chan, C.Y. and Lawrence, C.E. (2004) Sfold web server for statistical folding and rational design of nucleic acids. Nucleic Acids Res, 32, W135-141. 18. Fellmann, C., Zuber, J., McJunkin, K., Chang, K., Malone, C.D., Dickins, R.A., Xu, Q., Hengartner, M.O., Elledge, S.J., Hannon, G.J. et al. (2011) Functional identification of optimized RNAi triggers using a massively parallel sensor assay. Mol Cell, 41, 733-746. 19. Tan, X., Lu, Z.J., Gao, G., Xu, Q., Hu, L., Fellmann, C., Li, M.Z., Qu, H., Lowe, S.W., Hannon, G.J. et al. (2012) Tiling genomes of pathogenic viruses identifies potent

14 antiviral shRNAs and reveals a role for secondary structure in shRNA efficacy. Proc Natl Acad Sci U S A, 109, 869-874. 20. Herold, M.J., van den Brandt, J., Seibler, J. and Reichardt, H.M. (2008) Inducible and reversible gene silencing by stable integration of an shRNA-encoding lentivirus in transgenic rats. Proc Natl Acad Sci U S A, 105, 18507-18512. 21. Kleibeuker, W., Zhou, X., Centlivre, M., Legrand, N., Page, M., Almond, N., Berkhout, B. and Das, A.T. (2009) A sensitive cell-based assay to measure the doxycycline concentration in biological samples. Hum Gene Ther, 20, 524-530. 22. Shin, K.J., Wall, E.A., Zavzavadjian, J.R., Santat, L.A., Liu, J., Hwang, J.I., Rebres, R., Roach, T., Seaman, W., Simon, M.I. et al. (2006) A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression. Proc Natl Acad Sci U S A, 103, 13759-13764. 23. Zuber, J., McJunkin, K., Fellmann, C., Dow, L.E., Taylor, M.J., Hannon, G.J. and Lowe, S.W. (2011) Toolkit for evaluating genes required for proliferation and survival using tetracycline-regulated RNAi. Nat Biotechnol, 29, 79-83. 24. Stegmeier, F., Hu, G., Rickles, R.J., Hannon, G.J. and Elledge, S.J. (2005) A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells. Proc Natl Acad Sci U S A, 102, 13212-13217. 25. Hochedlinger, K., Yamada, Y., Beard, C. and Jaenisch, R. (2005) Ectopic expression of Oct-4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues. Cell, 121, 465-477. 26. Raymond, C.S. and Soriano, P. (2007) High-efficiency FLP and PhiC31 site-specific recombination in mammalian cells. PLoS One, 2, e162. 27. Hao, N., Bhakti, V.L., Peet, D.J. and Whitelaw, M.L. (2013) Reciprocal regulation of the basic helix-loop-helix/Per-Arnt-Sim partner proteins, Arnt and Arnt2, during neuronal differentiation. Nucleic Acids Res, 41, 5626-5638. 28. Lin, Y., Bloodgood, B.L., Hauser, J.L., Lapan, A.D., Koon, A.C., Kim, T.K., Hu, L.S., Malik, A.N. and Greenberg, M.E. (2008) Activity-dependent regulation of inhibitory synapse development by Npas4. Nature, 455, 1198-1204. 29. Moser, M., Knoth, R., Bode, C. and Patterson, C. (2004) LE-PAS, a novel Arnt- dependent HLH-PAS protein, is expressed in limbic tissues and transactivates the CNS midline enhancer element. Brain Res Mol Brain Res, 128, 141-149. 30. Haenebalcke, L., Goossens, S., Dierickx, P., Bartunkova, S., D'Hont, J., Haigh, K., Hochepied, T., Wirth, D., Nagy, A. and Haigh, J.J. (2013) The ROSA26-iPSC mouse: a conditional, inducible, and exchangeable resource for studying cellular (De)differentiation. Cell Rep, 3, 335-341. 31. Carey, B.W., Markoulaki, S., Beard, C., Hanna, J. and Jaenisch, R. (2010) Single-gene transgenic mouse strains for reprogramming adult somatic cells. Nat Methods, 7, 56- 59. 32. Meerbrey, K.L., Hu, G., Kessler, J.D., Roarty, K., Li, M.Z., Fang, J.E., Herschkowitz, J.I., Burrows, A.E., Ciccia, A., Sun, T. et al. (2011) The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo. Proc Natl Acad Sci U S A, 108, 3665- 3670. 33. Brown, C.Y., Sadlon, T., Gargett, T., Melville, E., Zhang, R., Drabsch, Y., Ling, M., Strathdee, C.A., Gonda, T.J. and Barry, S.C. (2010) Robust, reversible gene knockdown using a single lentiviral short hairpin RNA vector. Hum Gene Ther, 21, 1005-1017. 34. Fellmann, C., Hoffmann, T., Sridhar, V., Hopfgartner, B., Muhar, M., Roth, M., Lai, D.Y., Barbosa, I.A., Kwon, J.S., Guan, Y. et al. (2013) An optimized microRNA backbone for effective single-copy RNAi. Cell Rep, 5, 1704-1713.

15 35. Yang, X., Boehm, J.S., Salehi-Ashtiani, K., Hao, T., Shen, Y., Lubonja, R., Thomas, S.R., Alkan, O., Bhimdi, T., Green, T.M. et al. (2011) A public genome-scale lentiviral expression library of human ORFs. Nat Methods, 8, 659-661. 36. Goshima, N., Kawamura, Y., Fukumoto, A., Miura, A., Honma, R., Satoh, R., Wakamatsu, A., Yamamoto, J., Kimura, K., Nishikawa, T. et al. (2008) Human protein factory for converting the transcriptome into an in vitro-expressed proteome. Nat Methods, 5, 1011-1017. 37. Gong, S., Doughty, M., Harbaugh, C.R., Cummins, A., Hatten, M.E., Heintz, N. and Gerfen, C.R. (2007) Targeting Cre recombinase to specific neuron populations with bacterial artificial chromosome constructs. J Neurosci, 27, 9817-9823. 38. Taniguchi, H., He, M., Wu, P., Kim, S., Paik, R., Sugino, K., Kvitsiani, D., Fu, Y., Lu, J., Lin, Y. et al. (2011) A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex. Neuron, 71, 995-1013. 39. Eppig, J.T., Blake, J.A., Bult, C.J., Kadin, J.A. and Richardson, J.E. (2012) The Mouse Genome Database (MGD): comprehensive resource for genetics and genomics of the laboratory mouse. Nucleic Acids Res, 40, D881-886. 40. Tasic, B., Hippenmeyer, S., Wang, C., Gamboa, M., Zong, H., Chen-Tsai, Y. and Luo, L. (2011) Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proc Natl Acad Sci U S A, 108, 7902-7907. 41. Yang, H., Wang, H., Shivalila, C.S., Cheng, A.W., Shi, L. and Jaenisch, R. (2013) One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering. Cell, 154, 1370-1379. 42. Wefers, B., Meyer, M., Ortiz, O., Hrabe de Angelis, M., Hansen, J., Wurst, W. and Kuhn, R. (2013) Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides. Proc Natl Acad Sci U S A, 110, 3782-3787.

16 Figure legends

Figure 1. Construction of sensitive, low background Dox-inducible lentiviral and Flp-In systems. (a) Tetracycline-responsive luciferase reporter gene assays in HEK293T cells using Tet-On variants with (black) or without (grey) 1g/ml doxycycline for 24 hrs. (b) Expanded background luciferase activities in the absence of doxycycline from (a). Data are mean relative luciferase activities ± SEM of 3 independent experiments. (c) The sensitivity of Tet-On or Tet- On3G to doxycycline using a tetracycline-responsive luciferase reporter gene in HEK293T cells in the presence (black) or absence (grey) of indicated concentrations of doxycycline. Data show fold inductions of mean relative luciferase for doxycycline treated versus non-treated cells, ± SEM of 3 independent experiments. (d) Schematic of vectors for fluorescent miR30c donor plasmids containing dsRed, dnucTomato, or eYFP. (e) Schematic of vectors for Lentiviral (LVTPTIG and LVTPTIP) and FLP-Inducer (Col1a1-FLP-Inducer and Col1a1-FLP-Inducer- LSL) doxycycline-inducible destination plasmids.

Figure 2. Lentiviral LVTPT system allows for inducible cDNA and shRNA expression in stable cell lines and primary neurons. (a) Vector diagram of constitutively expressed (Tet- On3G and eGFP) and doxycycline inducible (dsRed (cDNA), Tet-On3G and eGFP) components of the LVTPT system (b) dsRed (left), eGFP (middle), or merged (right) fluorescence images of HEK293T cells infected with LVTPTIG-dsRed virus in the absence (lower panel) or presence (upper panel) of 1g/ml Doxycycline for 24hrs (c) FACS analysis of dsRed expressing cells in uninfected (orange) or HEK293T cells stably infected with LVTPTIP- dsRed-shGL3 and selected with puromycin in the absence (red) or presence of 1g/ml Doxycycline for 24 (green) or 48 (blue) hrs. (d) LVTPTIP elicits effective RNAi in HEK293T cells. HEK293T cells stably infected with LVTPTIP-dsRED-shGL3 (negative control), LVTPTIP-dsRED-shNPAS4 2182 (impotent shRNA), or LVTPTIP-dsRED-shNPAS4 275134 (potent shRNA) were transfected with expression vector pEFBOS-NPAS4-3xFlag and treated with (+) or without (-) 1g/ml doxycycline for 72 hrs. Whole cell extracts were analysed for NPAS4 (anti-Flag) and tubulin levels by immunoblot. (e) dsRed fluorescence images of embryonic day 16 mouse cortical neurons cultured in vitro and infected with concentrated LVTPT-dsRed-shGL3 virus and treated with (right panel) or without (left panel) 1g/ml Doxycycline for 24 hr.

Figure 3. Efficient generation of doxycyline inducible stable cell lines free of background expression. (a) Knock out Maged1 Mouse Embryonic Fibroblasts (MEFs) were stably transduced with LVTPTIP-3xFlag-MAGED1b and treated with 1g/ml Doxycycline for 4, 8 or 24hrs. Whole cell extracts were analysed for MAGED1b (anti-Flag) and tubulin by immunoblot. (b) LNCaP prostate cancer cells were stably transduced with LVTPTIP-SIM2s- 3xFlag or LVTPTIP-SIM2l-3xFlag and treated with 1g/ml Doxycycline for 4, 8 or 24hrs. Whole cell extracts were analysed for SIM2s or SIM2l (anti-Flag) and tubulin by immunoblot. (c) mRNA of SIM2s and SIM2l from LNCaP prostate cancer cells from (b) was measured by real time PCR and normalised to RNA polymerase subunit 2A (Pol2A). Data are mean normalised mRNA fold induction over parent cell line ± SEM of 3 independent experiments. Statistical significance was determined using an unpaired student t-test. n.s. (not significant).

Figure 4. Inducible and reversible expression and knockdown from the Col1a1 locus using the FLP-Inducer system. (a) Schematic representation of Col1a1-FLP-In targeting strategy. Electroporation of pFLP-Inducer-dsRed-shGL3 and pFLPo recombinase into mouse embryonic stem (mES) cells pre-targeted with Col1a1 FRT hygromycin (–ATG) (promoter) enables gain of hygromycin resistance and site specific integration of the FLP-Inducer construct. (b) Southern Blot of two mouse embryonic stem cell lines containing Col1a1-PGK-FRT-neo-FRT (–ATG) Hygro targeted to the Col1a1 locus and two G418 resistant WT lines, using a 5’ internal probe. (c) dsRed microscopy of a Col1a1-FLP-Inducer- dsRed-shGL3 hygromycin resistant mES cell line in the presence (right panel) or absence (left panel) of 1g/ml Doxycycline for 24hrs. (d) FACS analysis of parent mES Col1a1 FRT hygromycin (–ATG) (promoterless) cells (Orange) or mES Col1a1-FLP-Inducer- dsRed-shGL3 cells (blue and red) in the presence (red) or absence (blue and orange) of 1g/ml Doxycycline for 24hrs. (e) Median dsRed fluorescence in FACS analysis in (d). (f) shRNA mediated knockdown of P53 from the Col1a1-FLP-Inducer mES cells. Col1a1-FLP-Inducer-dsRed-shGL3 or Col1a1-FLP-Inducer- dsRed-shP53 were incorporated into the Col1a1 locus in mES cells by recombination mediated cassette exchange (RMCE) and shRNA induced using 1g/ml Doxycycline, then allowed to recover by Doxycycline removal for indicated periods. P53 and tubulin protein expression were determined by immunoblot.

Figure 5. Efficient prediction of shRNA sequences for knockdown of ARNT1 or ARNT2 in mES_FLP-Inducer cells. Induced shRNA knockdown of (a) ARNT1 or (b) ARNT2 in mES-FLP-Inducer-nucTomato-m30c-shRNA cells treated with 1g/ml Dox for 48hrs (+) versus untreated (-). Western blotting was used to detect ARNT1, ARNT2, and tubulin using - ARNT1, -ARNT2, or -tubulin antibodies, respectively.

Figure 6. Inducible, background free expression of a cDNA from the Col1a1 locus (a) Col1a1-FLP-Inducer-mNPAS4-xFlag mouse embryonic stem (mES) cells were cultured in the absence or presence of 1g/ml Dox for 6 hrs and whole cell extracts analysed for NPAS4- 3xFlag by immunoblotting with anti-Flag antibodies. n.s. represents a nonspecific band (b) NPAS4 mRNA was measured using real time PCR of Col1a1-FLP parental cells, Col1a1-FLP- Inducer-mNPAS4-xFlag cultured in the absence (Ctrl) or presence of 1g/ml Dox for 6 hrs, and mouse embryonic day 16 (E16) cortical neurons cultured in vitro and pre-treated with 2M Tetrodotoxin (TTX) and 100M D (-)-2-amino-5-phosphopentanoic acid (AP-5) (TTX/AP5) channel blockers for 24 hrs prior to 55mM KCl depolarisation treatment for 1hr. Data are mean ± SD of 3 independent experiments. Statistical analysis was calculated using two-tailed students t-test of 3 independent experiments. not significant (n.s.) (c) BDNF exon I mRNA was measured using real time PCR of Col1a1-FLP parental cells and Col1a1-FLP-Inducer- mNPAS4-xFlag cultured in the absence or presence of 1g/ml Dox for 6 hrs (d) Col1a1-FLP parental cells (mES) or Col1a1-FLP-Inducer-mNPAS4-xFlag cells were cultured in the absence or presence of indicated concentrations of Dox for 24 hrs and whole cell extracts analysed for NPAS4-3xFlag by immunoblotting with anti-Flag antibodies. n.s. represents a nonspecific band.

22 Supplementary Data

Supplementary Figure 1. Generation of multiple SIM2 Dox-inducible breast and prostate cancer cell lines using LVTPTIP lentivectors. (a,b,c) SIM2s-3xFlag or SIM2l-3xFlag containing LVTPTIP lentiviruses were infected into (a) prostate DU145 or (b) breast MDA- MB231, MCF7, MDAMB-453 cancer cell lines and selected with puromycin. SIM2 expression was induced with 1g/ml DOX (a) and (b) or 100ng/ml (c) for indicated time. SIM2s, SIM2l (-Flag) and tubulin protein levels were detected using western blot.

Supplementary Figure 2. Schematic of Dox-Inducible lentivectors and FLP-Inducer vectors. All FLP-Inducer plasmids were constructed with a 5’ PGK promoter ATG and single FRT recombination site followed two SV40 polyadenylation sequences, the TRE3G promoter which has minimised background expression(4), Gateway recombination cassette (ccdB death gene and Chloramphenicol (Cm) antibiotic flanked byAttR1 and AttR2 LR recombination sites) and a SV40 polyadenylation sequence. 3’ of this lay various promoters which drive the expression of the Tet-On3G which is followed by an SV40 polyadenylation sequence. FLP- Inducer-EF-LSL contains a constitutive EF1 promoter followed by a LoxP flanked SV40 polyadenylation sequence; FLP-Inducer-CAG and FLP-Inducer-CAG-LSL contain a cytomegalovirus (CMV) intermediate enhancer chicken -Actin promoter (FLP-Inducer-CAG- LSL also contains a LoxP flanked SV40 polyadenylation sequence following the CAG promoter); FLP-Inducer-hGFAP contains a human Glial fibrillary acidic protein (GFAP) promoter; FLP-Inducer-hSynI contains a human Synapsin I promoter and FLP-Inducer- ARR2pb contains a Probasin promoter (pb) with two Androgen Receptor response elements enhancers (ARR2)(43).

Supplementary Figure 3. Inducible and reversible expression of dsRed in Col1a1-FLP- Inducer embryonic stem cells. FACS analysis of dsRed expression in Col1a1-FLP-Inducer-EF (dsRed-shGL3) embryonic stem cells in (a) “On” 1g/ml Doxycycline for 0 (red), 4 (blue), 8 (orange), 24 (light green), 48 (dark green) hrs (b) “On” 1g/ml Doxycycline for 0 (blue), 48 (light green) hrs and “Off” Doxycycline for 24 (red) or 48 (orange) hrs (c) “On” 1g/ml Doxycycline for 0 (blue) or 6 (orange) days and “Off” Doxycycline for 6 (red) days. (d) dsRed fluorescence in Col1a1-FLP-Inducer-EF(dsRed-shGL3) embryonic stem cells “On” 1g/ml Doxycycline for 0 (blue) or 6 (orange) days and “Off” Doxycycline for 6 (red) days. Data are average median dsRed fluorescence ± SD of 3 independent experiments. (d) needs to be altered to match figure

Supplementary Figure 4. Cre mediated excision of loxP STOP LoxP enables inducible expression in embryonic stem cells. (a) Schematic diagram of CRE mediated excision of a LoxP flanked STOP sequence which then enables Dox inducible expression. (b) bright field (left) or fluorescence (right) of mES-Col1a1-FLP-Inducer-LSL-dsRed-shP53 (814) cells treated with 1g/ml Dox for 24hrs (bottom 2 panels) or left untreated (top). The bottom panel cells have been electroporated with a CRE expressing plasmid prior to treatment with Dox. (c) RT- PCR on genomic DNA extracted from mES-Col1a1-FLP-Inducer-LSL-dsRed-shP53 cells electroporated (+) or not electroporated (-) with a CRE expressing plasmid.

26 miR30&shRNA&template Sequence shGL3&(1309)& TGCTGTTGACAGTGAGCGCCCGCCTGAAGTCTCTGATTAATAGTGAAGCCACAGATGTATTAATCAGAGACTTCAGGCGGTTGCCTACTGCCTCGGA P53&shRNA&814 TGCTGTTGACAGTGAGCGCCCACTACAAGTACATGTGTAATAGTGAAGCCACAGATGTATTACACATGTACTTGTAGTGGATGCCTACTGCCTCGGA NPAS4&shRNA&275134& TGCTGTTGACAGTGAGCGCCGCCGAGATTCGGAACCTCAATAGTGAAGCCACAGATGTATTGAGGTTCCGAATCTCGGCGTTGCCTACTGCCTCGGA NPAS4&shRNA&2182& TGCTGTTGACAGTGAGCGCGGTTGACCCTGATAATTTATTTAGTGAAGCCACAGATGTAAATAAATTATCAGGGTCAACCATGCCTACTGCCTCGGA mARNT1&(4033) TGCTGTTGACAGTGAGCGCGGAGGCTATTTTTTTATTCTATAGTGAAGCCACAGATGTATAGAATAAAAAAATAGCCTCCTTGCCTACTGCCTCGGA mARNT1&(3284) TGCTGTTGACAGTGAGCGCCCCAGCCAAAATGGAACTTTATAGTGAAGCCACAGATGTATAAAGTTCCATTTTGGCTGGGTTGCCTACTGCCTCGGA mARNT1&(3751) TGCTGTTGACAGTGAGCGCCAGGGTCACTTGATAATTATATAGTGAAGCCACAGATGTATATAATTATCAAGTGACCCTGTTGCCTACTGCCTCGGA mARNT1&(415) TGCTGTTGACAGTGAGCGACCAGGGAAAATCATAGTGAAATAGTGAAGCCACAGATGTATTTCACTATGATTTTCCCTGGCTGCCTACTGCCTCGGA mARNT1&(282) TGCTGTTGACAGTGAGCGCGGGCTGGATTTTGATGATGAATAGTGAAGCCACAGATGTATTCATCATCAAAATCCAGCCCTTGCCTACTGCCTCGGA mARNT1&(414) TGCTGTTGACAGTGAGCGCGCCAGGGAAAATCATAGTGAATAGTGAAGCCACAGATGTATTCACTATGATTTTCCCTGGCATGCCTACTGCCTCGGA mARNT1&(522) TGCTGTTGACAGTGAGCGCCCAGACAAGCTAACCATCTTATAGTGAAGCCACAGATGTATAAGATGGTTAGCTTGTCTGGTTGCCTACTGCCTCGGA mARNT1&(1498) TGCTGTTGACAGTGAGCGCCAGATGAAATTGAGTATATTATAGTGAAGCCACAGATGTATAATATACTCAATTTCATCTGATGCCTACTGCCTCGGA mARNT1&(3235) TGCTGTTGACAGTGAGCGCTGCAGCCAAAGAGTATTTAAATAGTGAAGCCACAGATGTATTTAAATACTCTTTGGCTGCAATGCCTACTGCCTCGGA mARNT1&(300) TGCTGTTGACAGTGAGCGCGAAGTAGAAGTGAACACTAAATAGTGAAGCCACAGATGTATTTAGTGTTCACTTCTACTTCATGCCTACTGCCTCGGA mARNT2&(2221) TGCTGTTGACAGTGAGCGCCGCAGGGGACTGGCAACTATATAGTGAAGCCACAGATGTATATAGTTGCCAGTCCCCTGCGTTGCCTACTGCCTCGGA mARNT2&(5565) TGCTGTTGACAGTGAGCGCGGGCAACATAATGAAACTAATTAGTGAAGCCACAGATGTAATTAGTTTCATTATGTTGCCCATGCCTACTGCCTCGGA mARNT2&(377) TGCTGTTGACAGTGAGCGAGCGGAACAAGATGACTCAATATAGTGAAGCCACAGATGTATATTGAGTCATCTTGTTCCGCCTGCCTACTGCCTCGGA mARNT2&(4696) TGCTGTTGACAGTGAGCGCCAATGCCATTATCCTGCAAAATAGTGAAGCCACAGATGTATTTTGCAGGATAATGGCATTGATGCCTACTGCCTCGGA mARNT2&(4612) TGCTGTTGACAGTGAGCGAGGGGGAGAAGTCAGAAATGAATAGTGAAGCCACAGATGTATTCATTTCTGACTTCTCCCCCCTGCCTACTGCCTCGGA mARNT2&(4329) TGCTGTTGACAGTGAGCGCGGACAGGATTCTTGCTTGCTATAGTGAAGCCACAGATGTATAGCAAGCAAGAATCCTGTCCTTGCCTACTGCCTCGGA mARNT2&(3708) TGCTGTTGACAGTGAGCGCCCCTGTGTCAGTCAGATTTAATAGTGAAGCCACAGATGTATTAAATCTGACTGACACAGGGATGCCTACTGCCTCGGA mARNT2&(3186) TGCTGTTGACAGTGAGCGAGGTTAGGATACCTCGACTCTATAGTGAAGCCACAGATGTATAGAGTCGAGGTATCCTAACCCTGCCTACTGCCTCGGA mARNT2&(318) TGCTGTTGACAGTGAGCGCGATGGTGAAGGTCCCAGTAAATAGTGAAGCCACAGATGTATTTACTGGGACCTTCACCATCTTGCCTACTGCCTCGGA mARNT2&(4611) TGCTGTTGACAGTGAGCGCGGGGGGAGAAGTCAGAAATGATAGTGAAGCCACAGATGTATCATTTCTGACTTCTCCCCCCATGCCTACTGCCTCGGA

107