A four-and-a-half LIM FHL2 acts as a coactivator for the Wilms' tumor suppressor WT1 during gonadal differentiation

Inaugural-Dissertation zur Erlangung der Doktorwürde der Fakultät für Biologie der Albert-Ludwigs-Universität zu Freiburg im Breisgau

Vorgelegt von Xiaojuan Du aus Peking, V.R.China Mai 2001 Dekan: Prof. Dr. Hans Kleinig Leiter der Arbeit: Prof. Dr. Roland Schüle Referent: Prof. Dr. Driever Korreferent: PD. Dr. Matthias Hammerschmidt

Tag der Bekanntgabe des Prüfungsergebnisses: 5.7.2001 To my husband Baocai Xing and my son Chen Xing Acknowledgement

I would like to thank Prof. Dr. Roland Schüle for offering me such an excellent opportunity to come to and work in Germany. I could not finish my work without his supervision. when I got good results with my experiments, his smiles gave me confidence. He gave me good advice and encouragement when I am frustrated. I not only learnt the scientific knowledge from him but also obtained the capability of facing the frustration and solving the problem under his guidance. I owe all I have learnt and achieved to him. I appreciate the opportunity to come here so that I could meet many nice German people. I thank Prof. Hausmann and PD. Dr. Scherer for the arrangement of my "Oberseminars". I would like to thank PD. Dr. Christoph Englert and Dagmar Wilhelm for the coorperation. I learnt whole mount in situ hybridization technique from them. I am grateful to Prof. Edgar Wagner and Jürgen Eich. They offered me the chance to do my assistant courses in their nice lab. I enjoyed the work with them very much. I appreciate the Wednesday's seminars in the Schüle lab. From Prof. Schüle and my colleagues, I learnt how to present my data and to plan the next step for my project. I am grateful to Dr. Judith Müller for fruitful discussion and criticism of my project and thesis. I deeply appreciate the discussion and criticism of my thesis from Thomas Günter and Philippe Ulsemer. I am very grateful to Philip Hublitz. I thank him for all his support during my Ph.D. work. He always presents his warm friendship and kind help to me. I will remember him forever. I want to give my thanks to Dominica Willmann. She is a nice colleague and a good friend. I thank her for her support and the computer work. I got a lot support from her when I am frustrated. I enjoyed our excursions in the famous black forest very much. I am grateful to Natercia Conceicao. She was a good company when I missed my family. I thank my pretty nice colleague Silke Winkler for her nice suggestions to my thesis. I am grateful to all the members in Schüle's group especially our technicians Luka Mercep, Sandra Vomstein, Simone Erhardt and Awuku Osei. I can not proceed my work without them. I thank Luka Mercep for his tolerance to share the working bench with me. His humorous jokes made me happy. I am grateful to Manuela Kelter. She was my first friend in Germany. I appreciate her concerns about me and my family. I appreciate the friendship from my Chinese colleague Dongya Huang. She is a very good friend. When I missed my family, I could always talk to her in my mother language. I am very lucky to make a good Chinese friend overseas. She is always there when I need her. I will keep my nice experience in beautiful Freiburg in my memory forever. Summary

An essential step during sex determination is the maintenance of the Müllerian duct in females which gives rise to oviduct, uterus and the upper part of vagina and its regression in males caused by the expression of Müllerian inhibiting substance (MIS). The Wilms' tumor suppressor (WT1) and the orphan nuclear (SF-1) bind cooperatively to the of MIS and activate its transcription. In the ovary, on the other hand, the orphan DAX-1 binds to SF-1. The binding inhibits WT1/SF-1 transactivation and thereby suppresses induction of MIS expression. In addition, WT1 itself is also responsible for directly upregulating DAX-1 transcription. In summary, little is known about the molecular mechanisms that regulates transcription of the MIS gene. Importantly, so far no bona fide coactivators for WT1 have been described. Recently, a four-and-a-half LIM protein family was identified that can function as transcriptional coactivators. FHL2, a member of this family, is strongly expressed in heart and is also found in ovary and prostate. In the work presented here, the expression pattern and the physiological role of FHL2 during gonadal differentiation are analyzed. Whole mount in situ hybridizations of mouse embryos show strong spatio-temporal expression of FHL2 in both male and female gonads. FHL2 expression overlaps with that of WT1 during gonadal differentiation. During testis differentiation FHL2 is coexpressed with WT1, SF-1 and MIS, whereas in ovarian differentiation FHL2 is coexpressed with WT1 and DAX-1. Pull-down assays reveal that FHL2 interacts specifically with WT1 in vitro, but fails to do so with other transcription factors involved in gonadal differentiation such as SF-1, SOX9 or . The WT1 interaction domains in FHL2 are mapped to the N-terminal part LIM domains LIM0-2. The FHL2 interaction domain in WT1 is mapped to amino acids 182 to 298. FHL2 interacts with WT1 in vivo in a yeast two- hybrid experiment and in coimmunoprecipitation in mammalian cells. In this study, the physiological importance of this interaction is demonstrated in transient transfection assays. FHL2 potentiates the synergism of WT1 and SF-1 and superactivates MIS . This potentiation of transactivation by FHL2 is specific for the WT1 (-KTS) isoform and needs the ligand binding domain of SF-1. Moreover, FHL2 has the potential to coactivate WT1 in the context of the DAX-1 promoter. FHL2 does not bind directly to DNA but is recruited to the DAX-1 promoter via binding to the WT1. Taken together, the data show that FHL2 is a novel transcriptional coactivator of WT1. The ability to functionally interact with WT1 and modulate DAX-1 and MIS expression allows FHL2 to act in the molecular fine-tuning of that control gonadal differentiation. PUBLICATIONS: (articles in Chinese)

Du X et al., " Detection of Coxsackie B3 viral genome in the organ sections of infected mice by in situ hybridization" Chinese Journal of Epidemiology 1990; 11:70.

Chen H, Du X et al., "SRY gene detection by DNA amplification for sex discrimination" Medical Study Communication 1991; 20(7):28.

Du X et al., "In situ determination of viral RNA in the myocardial biopsy sample of a patient with myocarditis" Chinese Journal of Epidemiology 1992; 13:311.

Du X et al., "Distribution of viral RNA in the organs of infected mice" Viral myocarditis in children, Hei Long Jiang Science and Technology Press, 1993:34.

Chen H, Du X et al., "Isolation, cloning and sequencing of the SRY gene from a Chinese individual" Chinese Biochemical Journal 1993; 9(2):219.

Du X et al., "Genetic diagnosis of 46,XY true hermaphroditism" Chinese Journal of Pediatric Surgery, 1994; 15(5):262.

Chen H, Xiong J, Du X et al., " A study on the detection of SRY gene in 8 cases of sexual abnormality" Chinese Journal of Medical Genetics 1995; 12(1):26.

Chen H, Du X et al., "An improvement for direct sequencing double-stranded PCR product" Chinese Journal of Medical Genetics 1995; 12(5): 292.

Chen H, Du X et al., "Gene analysis in 46,XY true hermaphroditism and preliminary study of its mechanism" Chinese Journal of Medical Genetics 1996; 3(5): 289.

Du X et al., "Mutation of the RET proto-oncogene in Hirschsprung's disease" Chinese Journal of Medical Genetics, 1997; 14(3):146.

Du X et al., "Expression of human SRY gene and the DNA-binding property of the product Chinese Journal of Medical Genetics, 1998; 15(5):290. Index - I-

1. Introduction...... 1 1.1 Regulation of transcription ...... 1 1.2 Transcription factors...... 3 1.3 Transcriptional coregulators ...... 7 1.3.1 Coactivators in transcriptional regulation by nuclear receptors (NRs) ...... 7 1.3.2 Corepressors complexes in transcriptional regulation by NRs ...... 9 1.3.3 Cell and promoter-specific coactivators...... 9 1.4 Four-and-half LIM (FHL) can act as tissue-specific coactivators...... 10 1.4.1 The structural characteristics of the FHL family ...... 10 1.4.2 Expression pattern and functional activities of FHL family members...... 11 1.4.3 Properties of FHL2...... 11 1.5 Gene regulation during sex differentiation ...... 12 1.5.1 Embryonic differentiation of gonads ...... 12 1.5.2 Gene regulation during sexual differentiation...... 13 1.6 The aim of this study ...... 22

2. Materials and methods...... 23

2.1 Materials...... 23 2.1.1 Plasmids ...... 23 2.1.2 Enzymes ...... 24 2.1.3 Oligonucleotides...... 24 2.1.4 Antibodies...... 25 2.1.5 Bacteria and cultural media for E.coli...... 25 2.1.6 Cells and culture media ...... 26 2.1.7 Radioactive materials ...... 26 2.1.8 Kits ...... 26 2.1.9 Electrophoresis buffers and solutions ...... 26 2.1.10 Solutions for Western-blot ...... 27 2.1.11 Solutions for nuclear extract from culture cells...... 27 2.1.12 Buffers used in transient transfections ...... 27 2.1.13 Chemicals ...... 27 2.1.14 Devices...... 28

2.2 Methods ...... 29 2.2.1 Methods for DNA...... 29 Index - II-

2.2.1.1 Agarose gel electrophoresis...... 29 2.2.1.2 Recovery of DNA fragment from agarose gels ...... 29 2.2.1.3 DNA restriction digestion...... 29 2.2.1.4 Modification of DNA ends ...... 29 2.2.1.5 DNA ligation ...... 30 2.2.1.6 Preparation and transformation of competent E. coli ...... 30 2.2.1.7 Analytical isolation of plasmid DNA from E. coli...... 30 2.2.1.8 Polymerase chain reaction (PCR)...... 31 2.2.1.9 DNA sequencing...... 31 2.2.1.10 Preparative isolation of plasmid DNA from E. coli...... 31 2.2.1.11 Annealing of oligonucleotides...... 31 2.2.1.12 Radiolabeling of double stranded oligonucleotides...... 32

2.2.2 Methods dealing with protein...... 32 2.2.2.1 SDS-polyacrylamide-gel-electrophoresis ...... 32 2.2.2.2 Western-blotting and immuno-detection ...... 33 2.2.2.3 Immunoprecipitation ...... 33 2.2.2.4 Electrophoretic mobility shift assay (EMSA)...... 33 2.2.2.5 Coupled in vitro transcription/translation ...... 34 2.2.2.6 Protein expression in E. coli ...... 34 2.2.2.7 Glutathione-S-transferase (GST) pulldown...... 34 2.2.2.8 Protein measurement ...... 35

2.2.3 Cell culture and transient transfection ...... 35 2.2.3.1 Permanent cell culture ...... 35 2.2.3.2 Transient transfection using the calcium phosphate method ...... 35 2.2.3.3 Measurement of luciferase activity...... 36 2.2.3.4 Nuclear extract from culture cells...... 36

2.2.4 Work with embryos ...... 36 2.2.4.1 Preparation of embryos for in situ hybridization (ISH) ...... 36 2.2.4.2 Labeling of the cRNA probe...... 36 2.2.4.3 Whole mount in situ hybridization ...... 37

2.2.5 Yeast two hybrid assay...... 37 Index - III-

3. Results...... 38

3.1 Expression of FHL2 during sex development ...... 38 3.1.1 FHL2 is co-expressed with SF-1, WT1 and MIS in testis...... 39 3.1.2 FHL2 is co-expressed with WT1 and DAX-1 in ovary ...... 40

3.2 Direct interaction between FHL2 and WT1...... 42 3.2.1 FHL2 interacts with WT1 in vitro...... 42 3.2.2 Mapping of the WT1 interaction domains in FHL2...... 42 3.2.3 Mapping of the FHL2 interaction domain in WT1 ...... 43 3.2.4 FHL2 interacts with WT1 in vivo...... 44

3.3 FHL2 regulates genes during gonadal differentiation ...... 46 3.3.1 FHL2 upregulates the male specific gene MIS via WT1/SF-1...... 46 3.3.2 Transcriptional upregulation of the 2×MIS-RE1 reporter gene by FHL2 is specific for the WT1(-KTS) forms ...... 49 3.3.3 FHL2 regulation of the 2×MIS-RE1-TATA-luciferase reporter gene is specifically dependent on SF-1 ...... 50 3.3.4 The SF-1-LBD is required for the transcriptional upregulation of the MIS promoter by FHL2...... 50 3.3.5 FHL2 upregulates the female specific gene, DAX-1 ...... 52

3.4 FHL2 and WT1 form a complex on the DAX-1 promoter...... 53

4. Discussion...... 56

4.1 FHL2 is co-expressed with WT1 during gonadal differentiation...... 56 4.2 FHL2 interacts with WT1 in vitro and in vivo ...... 57 4.3 FHL2 is a coactivator in the transcriptional activation of the MIS gene ...... 59 4.4 FHL2 acts as a coactivator in the transcriptional upregulation of the DAX-1 gene by WT1 ...... 63 4.5 FHL2 fine-tunes gene regulation during gonadal differentiation...... 64

5. References...... 65 Abbreviation

ACTR Activator of the thyroid and AF Activation function ATCC American type culture collection bp Basepair BSA Bovine serum albumin CBP CREB-binding protein CRE cAMP-response element CREB cAMP-response element binding protein CTE C-terminal extension DMEM Dulbecco's modified Eagle medium EDTA Ethylene diaminetetracetic acid ERα/β α or β DAX-1 Dosage sensitive sex reversal-adrenal hypoplasia congenia critical region on the X , gene 1 DBD DNA-binding domain DR Direct repeat ( of DNA-consensus motifs) DTT Dithiothreitol FCS Fetal calf serum FHL Four and a half LIM domains GCNF GR GST Glutathione-S-transferase h hours HDAC Histone deacetylase HRE Hormone responsive element HRP Horse radish peroxydase IPTG Isopropyl-β-D-thiogalactocid kDa Kilodalton LBD Ligand binding domain min Minutes MIS Müllerian ducts inhibiting substance N-CoR Nuclear receptor co-repressor NTD N-terminal domain PAGE Polyacrylamide-gel electrophoresis p/CAF p300/CBP-associated factor pol II DNA-dependent RNA polymerase II PVDF Polyvinyldifluorid RAR Retinoic acid receptor RNAPII RNA-polymerase II RT Room temperature RXR SDS Sodium dodecyl sulfate SF-1 Steroidogenic factor-1 SHR steroid SMRT Silencing mediator for RXR and TR SP1 Stimulating protein-1 SRC-1 Steroid receptor coactivator-1 TAF TBP-associated factor TBP TATA-box binding protein TEMED N,N,N',N',-Tetramethylethylendiamine TFIIA, B, D, E, F, H, J Transcription factors A, A, D, E, F, H, J for pol II. TIFI-2 Transcriptional intermediary factor-2 TR VDR WT1 Wilms' tumor suppressor Introduction - 1 -

1. Introduction

1.1 Regulation of transcription

Gene expression in eukaryotic cells Multicellular organisms are composed of hundreds of different cell types. All the cells contain the same set of genes. The phenotypic differences that distinguish the various kinds of cells in a higher eukaryote are largely due to differences in the expression of genes that code for proteins. Thus, gene regulation controls the function of different cells during differentiation and development. Gene expression in eukaryotes in response to environmental and developmental stimuli is a complex phenomenon involving the coordinated silencing and activation of genes by the action of transcription factors and coregulators.

Transcription in eukaryotic cells The initiation of mRNA transcription is a key issue in the regulation of gene expression. In karyotes, mRNA synthesis is catalysed by RNA polymerase II (pol II) which consists of multiple subunits. All subunits assemble to form a holoenzyme complex prior to binding to the promoter (Orphanides et al., 1996). The promoter is defined as the region upstream of the protein-coding sequence and contains a variety of binding sites (response elements) for regulatory factors involved in the initiation of transcription. For pol II, some elements and factors involved in transcription are common: they are found in a variety of promoters and are used constitutively; others are gene-specific and they are signal-regulated. The elements occur in different combinations in individual promoters. A pol II dependent promoter consists of a startpoint and some short sequences located immediately upstream of the startpoint. Pol II forms an initiation complex surrounding the startpoint. Typically, a sequence called TATA box is found at -35, CAAT boxes at -75, and GC boxes at -90. The TATA box is the crucial positioning component of the core promoter, it is recognized by the TATA-box binding protein (TBP). The CAAT box plays a role in determining the efficiency of the promoter and is recognized by a variety of factors, such as CCAAT/enhancer-binding protein (C/EBP) and CCAAT-transcription factor (CTF). The GC box contains the sequence GGGCGG and multiple copies are often present in promoters. The GC box is recognized by transcription factors of the SP1 family. All promoters probably require one or more of these DNA elements in order to function efficiently. But in some cases the same DNA element can be recognized by different proteins and a particular protein can recognize more than one type of DNA element. Introduction - 2 -

Enhancers are another type of DNA elements that are able to stimulate transcription in either orientation if located either upstream or downstream of the startpoint. Enhancer elements are targets for tissue-specific and/or temporal regulation. Enhancer-binding proteins interact with promoter bound proteins to stimulate transcription initiation. In summary, the initiation of eukaryotic mRNA transcription at a promoter involves a large number of transcription factors that bind to a variety of DNA elements. The factors which act in conjunction with pol II can be divided into three general groups: 1) The general factors, which are required for the initiation of RNA synthesis in all promoters. They join the pol II to form a complex surrounding the startpoint and determine the site of RNA initiation. The general factors together with pol II constitute the basal transcription apparatus. 2) The upstream factors, which are DNA-binding proteins that recognize specific short consensus elements located upstream of the startpoint. The activity of these factors is not regulated. 3) The inducible factors which have a regulatory role. They are synthesized or activated at specific times or in specific tissues, and are therefore responsible for the control of tissue-specific and developmental transcription patterns. Fig.1-1 depicts the basic transcription machinery.

Fig.1-1 A model of the basic transcription machinery. General transcription factors recruit pol II to the TATA-box of a promoter to initiate the transcription. Inducible transcription factors bind to the upstream DNA elements such as CAAT and GC-box to regulate transcription. Inducible transcription factors, e.g. nuclear hormone receptors, bind to enhancers to regulate transcription. Cofactors can upregulate or downregulate transcriptions by interacting with the transcription factors. Introduction - 3 -

The general transcription factors (GTFs) have been named TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH and TFIIJ (TF stands for transcription factor and II for genes that are transcribed by pol II) (Zawel and Reinberg, 1993). All GTFs are multiprotein complexes. The GTFs can assemble sequentially onto promoter DNA to form stable nucleoprotein complexes to recruit pol II and the whole complex is termed preinitiation complex (PIC). In its most general form, the conventional model for ordered transcription initiation by pol II is characterized by a distinct series of events: (1) Recognition of core promoter elements by TFIID, (2) recognition of the TFIID-promoter complex by TFIIB, (3) recruitment of a TFIIF/pol II complex, (4) binding of TFIIE and TFIIH to complete the PIC, (5) promoter melting and formation of an "open" initiation complex, (6) synthesis of the first phosphodiester bond of the nascent mRNA transcript, (7) release of pol II contacts with the promoter, and (8) elongation of the RNA transcript. TFIIA can join the complex at any stage after TFIID binding and stabilizes the initiation complex (Orphanides et al., 1996). The largest subunit in pol II contains a carboxy-terminal domain (CTD) which is heavily phosphorylated during the transition from initiation to elongation. The TFIIH contains a kinase specific for the CTD and the kinase activity can be stimulated by TFIIE. Many of the proteins in the PIC are the targets of transcriptional regulators. The assembly of the GTFs is subject to regulation by activator and repressor proteins: activator can recruit GTFs to a promoter thereby accelerating the assembly process, whereas repressor proteins can inhibit transcription by blocking the assembly of GTFs.

1.2 Transcription factors Except RNA polymerase, any protein that is needed for the initiation of transcription is defined as a transcription factor. Transcription factors bound at upstream elements influence the initiation of transcription by contacting other factors in the basal apparatus. Transcription factors need to bind their specific DNA elements (response elements) located in enhancers or promoters of their target genes in order to regulate transcription.

Nuclear receptors are transcription factors In humans, more than 24 classes of special transcription factors, so-called nuclear receptors (NRs) have been identified. NRs exert diverse roles in the regulation of growth, development, and homeostasis (Beato et al., 1995; Kastner et al., 1995; Mangelsdorf and Evans, 1995; Sadovsky and Crawford, 1998). Based on their importance in biology and medicine, NR represent one of the most intensively studied and best-understood classes of transcription factors at the molecular level (Glass and Rosenfeld, 2000). The NR superfamily includes: 1) receptors for steriod hormones, such as estrogens (ER) and glucocorticoid (GR); 2) receptors for nonsteroidal ligands, such as thyroid hormones (TR) and retinoic acid (RAR); 3) receptors that bind diverse products of the lipid metabolism, such as fatty acids and prostaglandins; 4) so-called orphan nuclear receptors for which regulatory ligands have not been identified (Mangelsdorf and Evans, 1995). Introduction - 4 -

Structural characteristics of nuclear receptors Analysis of the structure of nuclear hormone receptors showed that the modular structural motifs within nuclear receptors provide functional regions responsible for their activities. The domain structure of NRs typically consists of five regions A/B, C, D, E and F (Fig.1-2). The amino-terminal A/B contains the ligand-independent activation domain (AF-1). The A/B domain is usually not well conserved among nuclear receptors. Region C and E contain the conserved DNA-binding domain (DBD) and the ligand-binding domain (LBD). The LBD is connected to the DBD by a short flexible hinge domain (domain D). Domain F contains ligand-dependent activation domain (AF-2). The DBD is the most conserved domain in NR superfamily. The DBD contains two modules followed by a carboxy-terminal extension (CTE). The so-called P-box located in the first zinc finger directly contacts DNA and discriminates between different DNA-binding sites (Luisi et al., 1991; Schwabe et al., 1993). The second zinc finger is involved in homodimerization through the so-called D-box (Glass, 1994). The CTE effectively extends the DNA-binding surface of some nuclear receptors. For example, SF-1 binds stably as a monomer by using the CTE to extend the DNA-binding surface (Ueda et al., 1992). The LBD consists of 12 α-helixes (H1-H12) and participates in nuclear receptor hetero- or homodimerization (Bourguet et al., 1995; Perlmann et al., 1996), transcription repression and ligand-induced transcriptional activation (Baniahmad et al., 1995; Danielian et al., 1992; Horlein et al., 1995; Leng et al., 1995). Conformational changes are induced within the LBD by ligand binding. One obvious difference between the unliganded (apo form) and the liganded (holo form) LBD structures is a positional reorientation of H12. H12 is indispensible for the transcriptional activation function of the LBD and contains the so-called AF-2 core motif. H12 protrudes from the LBD core in the apo conformation whereas it folds back towards the LBD core between H3, H4 and H5 in its holo conformation (Danielian et al., 1992; Barettino et al., 1994; Durand et al., 1994; Vivat et al., 1997). The ligand-induced conformational changes most likely result in the formation of novel surfaces in the holo-LBD which, in turn, allow direct protein-protein interactions with cofactors (Chambon, 1996; Glass et al., 1997; Greschik et al., 1999). Thus, the AF-2 is responsible for the interactions of NR with coactivators (Mangelsdorf and Evans, 1995). In fact, domain D and E serve as the interface for a multitude of cooperating proteins transducing activating or repressing transcriptional signals. Introduction - 5 -

Fig.1-2. Schematic structure of nuclear receptors. The structure of NRs may be divided into five regions based on structure and function similarities (denoted A/B, C, D, E and F). Region C and E contain the conserved DBD and LBD. The DBD domain contains two zinc finger modules followed by a carboxy-terminal extension (CTE). The P-box of the first zinc finger motif is responsible for distinguishing the DNA half site. The D-box of the second zinc finger motif is involved in specific homodimerization. The CTE helix extends the DNA-binding surface of some NRs such as SF-1. The LBD consists of 12 α-helixes (H1-H12) and participates in nuclear receptor hetero- or homodimerization, transcription repression and ligand-induced transcriptional activation. The C-terminal activation function domain (AF-2, domain F) is involved in the ligand-dependent interactions of nuclear receptor with coactivators. (modified after Beato et al., 1995).

The transcriptional activities of nuclear receptors Nuclear receptors can activate or repress target genes by binding directly to DNA response elements as either homo- or heterodimers. Alternatively, NR can interact with other classes of transcription factors. Members of the NR family regulate transcription by several mechanisms (Fig.1-3). NRs activate transcription by following mechanisms: 1) The prototypic activity of NRs is ligand-dependent activation of transcription upon binding to specific hormone-response elements (HREs) in target genes (Mangelsdorf and Evans, 1995; Stunnenberg, 1993). 2) NRs also contribute to gene activation by acting as coactivators for other transcription factors, e.g. in the case of the glucocorticoid receptor for certain STAT-5-responsive genes (Doppler et al., 2001; Wyszomierski and Rosen, 2001). 3) A number of orphan NRs, such as CARβ and RORβ, Introduction - 6 - constitutively activate transcription (Harish et al., 2001; Greiner et al., 2000). Several mechanisms of transcriptional inhibition by NR have also been established: 1). A subset of NRs that heterodimerize with the retinoid X receptor, including the thyroid hormone receptor (TR) and retinoid acid receptor (RAR), are capable of actively repressing target genes upon binding to HREs in the absence of ligand (Fondell et al., 1993). 2). Several NRs, exemplified by the glucocorticoid receptor are capable of inhibiting the activities of other classes of transcription factors, such as AP-1, in a ligand-dependent manner. This effect does not require DNA binding by the NR and is refered to as transrepression (Schüle et al., 1991).

Fig.1-3. Transcriptional activities by NRs. Members of the NR family can both activate and inhibit gene expression. (A) The prototypic ligand- dependent activation of transcription by NRs upon binding to specific HREs in target genes. (C) NRs activate transcription by acting as coactivators for other transcription factors. (B) NRs heterodimerized with RXR, including TR and RAR, actively repress target genes upon binding to HREs in the absence of ligand. (D) Several NRs, inhibit transactivation of transcription factors, e.g. GR acts on AP-1, in a ligand- dependent manner (adapted from Glass and Rosenfeld, 2000). Introduction - 7 -

1.3 Transcriptional coregulators Coregulators do not bind DNA directly but are recruited to promoters by physical interaction with DNA-bound transcription factors. Coregulators play essential roles in the action of these transcription factors. Transcription coregulators can be subdivided into coactivators, which mediate gene activation (Anzick et al., 1997; Chen et al., 1997; Hong et al., 1997; Torchia et al., 1997; Voegel et al., 1998), and corepressors, which mediate gene silencing (Heinzel et al., 1997; Nagy et al., 1997; Lavinsky et al., 1998; Kao et al., 1998; Burke and Baniahmad, 2000).

1.3.1 Coactivators in transcriptional regulation by nuclear receptors (NRs) The transcription activation by NRs involves multiple cofactors that act both in a sequential and combinatorial manner to reorganize chromatin templates (Pollard and Peterson, 1998) and to modify and recruit basal factors and pol II. A combination of genetic, biochemical, and functional data suggests that several factors, including the BRG (SWI/SNF) complex, CBP/p300, p160 factors, P/CAF, and the TRIP/DRIP/ARC complexes, discussed below, are likely to be critical regulators for NR-regulated genes (Glass and Rosenfeld, 2000)(Fig.1-4).

Fig.1-4 Coactivator and corepressor complexes in NR controled transcription. Introduction - 8 -

Coactivator complexes include SWI/SNF, CBP/SRC-1/p/CAF and TRAP/DRIP/ARC. The SWI/SNF complex possesses ATP-dependent chromatin remodeling activities. The CBP/p/CAF complexes possesses histone acetyltransferase activities. These complexes may act in concert to relieve chromatin- mediated repression, with the TRAP/DRIP/ARC complex functioning to recruit core transcription factors. Corepressor complexes include the SIN3/HDAC complex, which has been proposed to be recruited via the NR corepressors NCoR or SMRT. This complex possesses histone deacetylase activity and is thought to reverse actions of histone acetyltransferase-containing complexes (adapted from Class and Rosenfeld, 2000).

Chromatinized transcription units are repressed compared to naked DNA. Thus, a critical aspect of gene activation involves nucleosomal remodeling (Wu, 1997; Guschin and Wolffe, 1999). Two general classes of chromatin remodeling complexes seem to play critical roles in transcriptional activation by NRs; ATP-dependent nucleosome remodeling complexes, and complexes containing histone acetyltransferase activity (HAT) (Kingston and Narlikar, 1999). The hyperacetylated regions of the genome are more actively transcribed than hypoacetylated regions (Pazin and Kadonaga, 1997). Specific recruitment of a complex with HAT activity to a promoter may play a crucial role in overcoming repressive effects of chromatin structure on transcription (Pazin and Kadonaga, 1997; Struhl and Moqtaderi, 1998; Wade et al., 1998). Thus, the transcription activation might involve the exchange of complexes containing histone deacetylase (HDAC) function with those containing HAT activity (Glass and Rosenfeld, 2000). The yeast SWI/SNF complex (Peterson, 1996; Workman and Kingston, 1998) modulates chromatin structure in an ATP-dependent manner and facilitates the binding of sequence-specific transcription factors to nucleosomal DNA (Owen-Hughes and Workman, 1996). BRG1 and hBrm are identified as homologs of SWI2/SNF2 in mammals (Khavari et al., 1993; Muchardt and Yaniv, 1993; Dingwall et al., 1995; Tsukiyama and Wu, 1995). CBP (CREB-binding protein) and p300 (McKenna et al., 1999; Torchia et al., 1997) serve as essential coactivators for many classes of sequence-specific transcription factors and possess HAT activity (Ogryzko et al., 1996). P/CAF (p300/CBP-associated factor) is an essential component of multisubunit coactivator complexes and acetylate histones in the context of nucleosomes (Grant et al., 1998; Utley et al., 1998). p160 factors were identified as coactivators that interact with NRs in a ligand-dependent manner (Cavailles et al., 1994; Halachmi et al., 1994) and also associate with CBP (Kamei et al., 1996; Yao et al., 1996). Three related genes were identified that encode the p160 factors, refered to as SRC-1/NcoA-1 (steroid receptor coactivator-1/nuclear receptor coactivator-1)/p160, TIF-2 (transcriptional intermediary factor-2))/GRIP-2/NcoA-2 (Hong et al., 1997; Torchia et al., 1997; Voegel et al., 1998), and p/CIP/AIB-1/ACTR (activator of the thyroid and RA receptor)/RAC/TRAM-1 (Anzick et al., 1997; Chen et al., 1997; Torchia et al., 1997). The Introduction - 9 - carboxyl terminus of SRC-1 (Spencer et al., 1997), TIF-2 (Hong et al., 1997) and p/CIP (Chen et al., 1997) contain HAT activity. TR-associated protein (TRAP) complex (Fondell et al., 1996a) and a very similar complex of vitamin D receptor (VDR) interacting proteins (DRIPs) were isolated (Rachez et al., 1998) and shown to potentiate transactivation function of TR and VDR, respectively, in vitro (Fondell et al., 1996a; Fondell et al., 1999; Rachez et al., 1999). Recent studies have shown that the TRAP/DRIP/ARC (activator-recruited cofactor) complex function as mediators for transcription activation by recruiting core transcription factors (Yuan et al., 1998; Treuter et al., 1999; Malik and Roeder, 2000).

1.3.2 Corepressors complexes in transcriptional regulation by NRs N-CoR (nuclear receptor corepressor) (Horlein et al., 1995; Kurokawa et al., 1995) and SMRT (silencing mediator of retinoid and thyroid hormone receptor) (Chen and Evans, 1995) were identified as NR corepressors. Both factors can also act as corepressors for other transcription factors, suggesting that they play a more general roles in the regulation of gene expression (Heinzel et al., 1997; Xu et al., 1998). N-CoR and SMRT both contain transferrable repression domians and they associate with mSin3a. The mSin3 proteins are components of corepressor complexs that also contain histone deacetylases (HDAC1/HDAC2 in mammals) (Vidal et al., 1991; Horlein et al., 1995; Taunton et al., 1996; Alland et al., 1997; Hassig et al., 1997a; Heinzel et al., 1997; Kadosh and Struhl, 1997; Laherty et al., 1997; Nagy et al., 1997; Zhang et al., 1997). Recently, distinct corepressor domains of N-CoR have been demonstrated to directly interact with different histone deacetylases, including HDAC4, HDAC5, and HDAC7, implying a redundant or combinatorial deacetylase-dependent code of repression (Downes et al., 2000; Huang et al., 2000).

1.3.3 Cell and promoter-specific coactivators Most of the coactivator complexes not only interact with members of the NR family, but also with other sequence-specific DNA-binding transcription factors (Xu et al., 1999). However, in vivo, only distinct complexes may be required for selective and tissue-specific gene activation (Xu et al., 1999). This raises the question how target gene selection, and tissue or development- specific gene activation is achieved in the organism. Resent studies suggest that cell-specific coactivators may also play critical roles in gene-specific transcriptional activation. For example, peroxisome proliferator-activated receptor γ (PPARγ) can activate transcription of the uncoupling protein 1 (UCP-1) gene in brown fat. The PPARγ coactivator (PGC-1) was identified as a cell- specific coactivator which is expressed exclusively in brown fat and skeletal muscle and is Introduction - 10 - markedly upregulated by exposure to cold (Puigserver et al., 1998). A number of coactivators have been identified that exhibit relative preferences for a subset of NRs. For example, ARA70 ( coactivator 70) is reported to enhance androgen receptor function in transfected cell lines (Yeh and Chang, 1996; Miyamoto et al., 1998). Importantly, the four-and-a- half LIM (FHL) proteins, discussed below, were identified as a family of tissue-specific coactivators (Taniguchi et al., 1998; Fimia et al., 1999; Muller et al., 2000; Scholl et al., 2000).

1.4 Four-and-half LIM (FHL) proteins can act as tissue-specific coactivators

The LIM domain (abbreviated from the first three proteins where the motif was found, Lin-11, Isl-1 and Mec-3) has been found in a variety of proteins (Freyd et al., 1990; Karlsson et al., 1990; Way and Chalfie, 1988). LIM domains mediate protein-protein interaction (Arber and Caroni, 1996; Schmeichel and Beckerle, 1994b) and are characterized by a highly conserved double zinc finger motif, Cys-X2-Cys-X16-23-His-X2-Cys/His-X2-Cys-X2-Cys-X16-23-Cys-X2-Cys/His/Asp, where the Cys and His residues co-ordinate the binding of two Zn2+ ions (Dawid et al., 1998; Michelsen et al., 1993). LIM proteins are involved in processes such as cell identity, differentiation, and growth control (Fujii et al., 1994; Sanchez-Garcia and Rabbitts, 1994). So far three classes of LIM proteins were identified: 1) LIM-HD proteins contain LIM domains and a homeodomain; 2) LIM-K proteins contain LIM domains and a protein kinase domain; 3) LIM- only proteins consist of LIM domains only (Jain et al., 1996; Chan et al., 1998).

1.4.1 The structural characteristics of the FHL family FHL stands for four-and-a-half LIM protein. The FHL family includes FHL1 (SLIM1) (Lee et al., 1998; Taniguchi et al., 1998), FHL2 (SLIM3, DRAL) (Genini et al., 1997; Chan et al., 1998), FHL3 (SLIM2) (Morgan and Madgwick, 1996), FHL4 (Morgan and Madgwick, 1999) and ACT (activator of CREM in testis) (Fimia et al., 2000). All members of the FHL family contain four and half LIM domains and share high homology throughout their amino acid sequences. All these proteins range in size from 31 to 35 kD. Fig.1-5 depicts the domain structure of FHL2. Introduction - 11 -

Fig.1-5. Domain structure of the FHL2 protein. FHL2 contains a half LIM domain at the N-terminus and four LIM domains at the C-terminus. The amino acids sequence of the zinc motif is: Cys-X2-Cys-X16-23-His-X2-Cys/His-X2-Cys-X2-Cys-X16-23-Cys-X2- Cys/His/Asp

1.4.2 Expression pattern and functional activities of FHL family members

FHL2 (also called DRAL for downregulated in rhabdomyosarcoma LIM protein or SLIM3) was the first FHL protein identified due to its differential expression in normal human myoblasts versus the malignant counterparts, rhabdomyosarcoma cells. FHL2 expression is detected predominantly in heart (Genini et al., 1997; Muller et al., 2000). Both, FHL1 and FHL3 are mainly expressed in skeletal muscle (Greene et al., 1999; Morgan et al., 1995). FHL4 is highly expressed in testis and ACT expression is found exclusively in testis (Morgan and Madgwick, 1999; Fimia et al., 1999). The functional analysis revealed that FHL family members act as tissue-specific coactivators (Scholl et al., 2000). FHL1 was found to interact with the mammalian homologue of the Drosophila transcription factor suppressor of hairless (Taniguchi et al., 1998). ACT was identified as a coactivator for CREM (cAMP-responsive element modulator). It binds to CREM and stimulates the transcriptional activity of CREM in yeast and mammalian cells (Fimia et al., 1999). FHL2, discussed below, was identified as a coactivator for the antrogen receptor (Müller et al., 2000). Hence, the available evidence suggests that these five LIM-only proteins might share a similar function in the modulation of tissue-specific or developmentally regulated gene transcription (Scholl et al., 2000).

1.4.3 Properties of FHL2 The FHL2 gene is located on chromosome 2q12-q13 (Chan et al., 1998) and encodes a protein of 279 amino acids (Fig.1-5). Each LIM domain of FHL2 is separated by eight amino-acid residues (Chan et al., 1998). Expression studies revealed that FHL2 is expressed in human skeletal muscle (Morgan and Madgwick, 1996), however, the highest level of FHL2 expression is detected in Introduction - 12 - heart (Chan et al., 1998; Muller et al., 2000) and ovary (Scholl et al., 2000). In our previous study, FHL2 was identified as the first LIM-only coactivator of the androgen receptor (AR) with a unique tissue-specific expression pattern (Müller et al., 2000). In the adult, FHL2 was found expressed in the myocardium of the heart and in the epithelial cells of the prostate, where it colocalizes with the AR in the nucleus (Müller et al., 2000). FHL2 contains a strong, autonomous transactivation function and binds specifically to the AR in vitro and in vivo. In an agonist- and AF-2-dependent manner FHL2 selectively increases the transcriptional activity of AR, but not that of any other nuclear receptor (Müller et al., 2000). In addition, the transcription of the prostate-specific AR target gene probasin is coactivated by FHL2. In more recent studies, FHL2 was found to interact with the polypyrimidine tract binding protein-associated splicing factor (Dye and Patton, 2001), indicating a possible role for LIM domains in the localization of splicing factors to subnuclear speckles. Another report showed that FHL2 was also found to bind to hCDC47 which participates in the regulation of mammalian DNA replication in S phase (Chan et al., 2000). FHL2 may act as a molecular adaptor to form a multicomplex with hCDC47 in the nucleus, thus it may play an important role in the specification or maintenance of the terminal differentiated phenotype of heart muscle cells (Chan et al., 2000). In the same study, FHL2 was found expressed in colorectal adenocarcinoma SW480 and HeLa cell, suggesting that FHL2 protein may associate with cancer development. FHL2 displays high homology with ACT and FHL3, and all these three proteins have intrinsic activation function (Fimia et al., 2000). FHL2 was found to interact with FHL3 (Fimia et al., 2000; Li et al., 2001) and both proteins are expressed in ovary (Fimia et al., 2000). Both FHL2 and FHL3 were shown to interact with CREB (cAMP-response element binding protein). ACT was identified as an activator of CREM in testis, and a weak but significant interaction was also observed between FHL2 or FHL3 and CREM (Fimia et al., 2000). Taken together, these data suggest that FHL members may share similar and redundant functions in transcriptional activation.

1.5 Gene regulation during sex differentiation

1.5.1 Embryonic differentiation of gonads In mammals, the gonads are derived from the urogenital ridge which has to go through a bipotential-gonad stage before the sex determination is made. The bipotential gonad can differentiate into testis or ovary depending on whether SRY (Sex determining Region on the Y chromsome) is present or not. In the absence of SRY, gonad differentiation follows the ovary pathway and the Müllerian ducts will develop into female internal genitalia (oviduct, uterus and the upper end of vagina). Conversely, SRY induces testis formation and the Wolffian ducts develop into male internal genitalia (epididymus, vas deferens and seminal vesicle) (Fig.1-6), whereas the Müllerian ducts regresses. Introduction - 13 -

Fig.1-6. Representation of the development of gonads and their ducts in mammals. Both the Wolffian and Müllerian ducts are present at the bipotential-gonad stage. In male, the Wolffian ducts normally form the epididymus, vas deferens and seminal vesicle. In female, Müllerian ducts will develop into oviduct, uterus and the upper end of vagina (adapted from Developmental Biology VI by Scott F. Gilbert).

1.5.2 Gene regulation during sexual differentiation During gonadal differentiation, a cascade of genes including nuclear receptors and other transcription factors are active and crosstalk to each other. This complex regulation pattern is time- and dosage-sensitive. Wilms' tumor suppressor protein (WT1) and the orphan nuclear receptor steroidogenic factor 1 (SF-1) are essential transcription factors during gonadal differentiation, since mice with gene disruption for either SF-1 or WT1 lack gonads (Kreidberg et al., 1993; Luo et al., 1994). An essential step during male sexual differentiation is the regression of the Müllerian ducts caused by the expression of Müllerian inhibiting substance (MIS). MIS is produced by testicular cords shortly after tetis differentiation is triggered by SRY. SF-1 was found to bind to the promoter of the MIS gene and activate its transcription (Shen et al., 1994). WT1 interacts and synergizes with SF-1 to upregulate the expression of MIS in male (Nachtigal et al., 1998). In females, on the other hand, downregulation of MIS allows the development of the Müllerian ducts giving rise to oviduct, uterus and upper end of vagina. In the ovary, the orphan Introduction - 14 - nuclear receptor DAX-1 will bind to SF-1, inhibiting transactivation by SF-1 and thereby suppressing the induction of MIS gene expression. In addition, DAX-1 was identified as a direct WT1 target gene. DAX-1 expression is upregulated by WT1 (Kim et al., 1999) (Fig.1-7).

Fig.1-7 Schematic presentation of gonadal differentiation. The bipotential-gonad follows the female pathway regulated by the action of the DAX-1 gene and the male pathway by the SRY gene in conjunction with autosomal genes such as SOX9. In female, the Müllerian ducts differentiate into the female genitalia. The testes produce MIS which causes the Müllerian ducts to regress. WT1 and SF-1 interact and synergize with each other to upregulate the transcription of the MIS gene. DAX-1 interacts with SF-1 and disrupts the synergy between WT1 and SF-1 to suppress the upregulation of MIS.

The role of the orphan nuclear receptor SF-1 in sex development

SF-1 is expressed in both male and female mouse embryos from the very early stages of gonadogenesis when the urogenital ridge forms (Ikeda et al., 1994). SF-1 null mice lack gonads (Luo et al., 1994; Sadovsky et al., 1995), demonstrating that SF-1 is a critical factor for gonadal development. With the onset of testicular differentiation, the level of SF-1 transcripts increases in testes. More strikingly, SF-1 transcripts in ovaries decrease coincident with sexual differentiation, which suggests that persistent SF-1 expression may impair ovarian differentiation. SF-1 is a member of the orphan nuclear receptor family capable of activating a variety of genes involved in steroid biosynthesis (Ikeda et al., 1993; Ikeda et al., 1994; Lala et al., 1992; Shen et al., 1994). The promoters of all these genes contain the consensus SF-1 response element TCAAGGTCA (Wilson et al., 1993). SF-1 binds DNA as a monomer. SF-1 is a typical member Introduction - 15 - of the NR superfamily. However, SF-1 lacks a typical AF-1 domain in the NTD (Mangelsdorf and Evans, 1995; Ito et al., 1998). SF-1 is a major regulator of the development and function of the hypothalamic-pituitary- adrenal-gonadal axes. SF-1 regulates gene transcription through its interaction with nuclear receptor coactivators such as steroid receptor coactivator-1 (SRC-1) and CBP/p300 (Ito et al., 1998; Monte et al., 1998) as well as by direct interactions with other transcription factors such as DAX-1, Egr-1, SOX9, WT1 and CREB (De Santa Barbara et al., 1998; Halvorson et al., 1998; Ito et al., 2000; Ito et al., 1997; Nachtigal et al., 1998). The interaction between SF-1 and SRC-1 requires both the activation function-2 (AF-2) and the proximal interactive domain (PID) which positioned between residues 226-230 (ELILQ) of SF-1 (Crawford et al., 1998; Crawford et al., 1997). This interaction potentiates the activity of SF-1. To interact with DAX-1, SF-1 requires a carboxy-terminal repressive domain (R domain, amino acids 437 to 447) and the PID (Fig.1-8). SF-1 activity is repressed by the interaction with DAX-1 (Crawford et al., 1998). Thus, the PID is required for SF-1 to interact with both activator and repressor molecules (Crawford et al., 1998).

Fig.1-8. Schematic diagram of SF-1. SF-1 binds extended consensus sequence by its DBD. The proximal interaction domain (PID) is required for the interaction of SF-1 with DAX-1 and SRC-1. The repressive domain (R domain) and the PID are required for the interactions of SF-1 with DAX-1 (adapted from Crawford et al., 1998).

No high affinity ligand for SF-1 has been identified so far (Christenson et al., 1998; Mellon and Bair, 1998) though some oxysterols were found to stimulate SF-1-dependent transcription (Lala et al., 1997). As mentioned above, SF-1 binds directly to the MIS promoter and upregulates the expression of the MIS gene (Shen et al., 1994; Giuili et al., 1997). Recently, several transcription factors, including WT1, SOX9 and GATA-4, have been found to bind to and synergize with SF-1 in the upregulation of the MIS gene (Nachtigal et al., 1998; De Santa Barbara et al., 1998; Tremblay and Viger, 2001a). However, the cofactors involved in this regulation mechanism have not been described. Introduction - 16 -

DAX-1 is involved in ovarian development

Sex reversal occurs in males who have a duplicated short arm of the human X chromsome and thus carry two copies of a 160-kb Xp locus. This phenomenon is termed dosage sensitive sex reversal (DDS) (Bardoni et al., 1994). Mutations or deletions of the same region of Xp in 46 XY males cause X-linked adrenal hypoplasia congenita (AHC), a rare life-threating disorder of adrenal gland development. Positional cloning of the AHC gene led to the isolation of a gene, designated DAX-1 (dosage-sensitive sex-reversal, AHC, on the X-chromosome, gene 1.) (Muscatelli et al., 1994; Zanaria et al., 1994) (Fig.1-9).

Fig.1-9. Two copies of the DAX-1 locus reverse a XY individual to a female. In XY normal individual, SRY and one copy of DAX-1 produce testes. In XX normal individual, one active copy of DAX-1 produces ovaries. The XY individuals with two active copies of the DAX-1 locus develop poorly formed gonad. Since neither MIS nor testosterone is produced, the phenotype is female. (adapted from Gilbert, Developmental Biology).

DAX-1 encodes an atypical member of the nuclear hormone receptor family that retains the conserved ligand binding domain but lacks the typical zinc finger DNA-binding motif. The DAX-1 protein can be divided in two portions: 1) The N-terminal domain, which defines a novel DNA- binding domain that consists of a 3.5 times repeat motif of 66-67 amino acid (Fig.1-10), which is unique among transcription factors. 2) The C-terminal part, with characteristics of a nuclear hormone receptor dimerization and ligand-binding domain. Introduction - 17 -

Fig.1-10. Schematic structure of DAX-1. DAX-1 is an unusual nuclear receptor. It contains a 3.5 times repeat of a 66-67 amino acids domain that functions as a DNA binding domain (DBD). In the C-terminus a nuclear receptor dimerization and ligand- binding domain is found. (adapted from Zanaria et al., 1994).

The expression pattern of DAX-1 and SF-1 overlap in many tissues, including the gonads, adrenal cortex and pituitary gonadotropes (Ikeda et al., 1996; Swain et al., 1996). DAX-1 can form heterodimers with SF-1, which blocks the synergism of SF-1/WT1 (Nachtigal et al., 1998) and SF-1/GATA-4 (Tremblay and Viger, 2001a) and supresses SF-1 activation of its target gene MIS (Ito et al., 1997). DAX-1 can also act as an adaptor molecule capable of recruiting the nuclear receptor corepressor N-CoR to SF-1 (Crawford et al., 1998) (Fig 1-11).

Fig.1-11. Model of the interaction of SF-1 and DAX-1. SF-1 is known to regulate the transcription of a variety of genes in the adrenal and reproductive axis. Transcription factors such as CBP and SRC-1 have been shown to enhance SF-1-dependent transcription. DAX-1 does not contain a typical DNA-binding domain, but it has been shown to interact with hairpin loops in the promoters of some target genes (e.g. steroidogenic acute regulatory gene). DAX-1 represses transcription probably by recruiting N-CoR. DAX-1 binds to SF-1 and strongly inhibits SF-1-dependent transcription. GTFs, general transcription factors. (adapted from Yu et al., 1998). Introduction - 18 -

Although DAX-1 does not contain a typical zinc-finger DNA-binding domain, DAX-1 binds to DNA hairpin structures and acts as a repressor of StAR (steroinogenic acute regulatory protein) gene expression. The C-terminal domain of DAX-1 appears to be involved in transcription control (Ito et al., 1997; Lalli et al., 1997). The majority of mutations in DAX-1 are nonsense or frameshift mutations resulting in truncation of the carboxy-terminus of the DAX-1 protein. AHC mutations of DAX-1 do not recruit N-coR to SF-1, thus decreasing their ability to repress SF-1 activity (Crawford et al., 1998). Thus, DAX-1 may function as a potential mediator of ovarian development by suppressing the activation function of SF-1.

WT1 regulates gene transcription involved in sexual differentiation

The Wilms' tumor suppressor gene, WT1 was initially identified through its association with Wilms' tumorsÐembryonic kidney tumors that arise from abnormal proliferation of the metanephric blastema (Coppes et al., 1993; Cowell et al., 1991; Haber et al., 1990; Huff and Saunders, 1993; Little et al., 1992; Tadokoro et al., 1992). A subset of Wilms' tumors (10%-15%) contain mutant WT1. WT1 is located at human chromosome 11p13 (Haber et al., 1992). The human WT1 gene spans approximately 50 kb and includes 10 exons, which generate a 3kb mRNA (Call et al., 1990; Gessler et al., 1990). Sequence analysis of WT1 identified a polypeptide composed of two function domains: a proline-glutamine-rich amino terminus and four zinc fingers at the carboxy terminus (Call et al., 1990). The last three carboxy-terminal zinc fingers of WT1 have 64% identity to those of the early growth response gene-1 (EGR-1) (Call et al., 1990; Gessler et al., 1990). Consistent with a possible role as a transcription factor, WT1 interacts with specific DNA sequences located upstream of a large number of genes, including genes that encode growth factors and their receptors (Menke et al., 1998). WT1 can function as either a transcription activator or a repressor depending on the cell type and promoter context. The WT1 mRNA is alternatively spliced, reflecting the presence or absence of two exons (Haber et al., 1991). Exon 5, encodes a 17 amino acid segment in the amino terminus of the protein; exon 9 encodes the three amino acids lysine, threonine and serine (KTS). The KTS domain is located between the third and fourth zinc fingers. The alternatve splicing gives rise to four WT1 isoforms: WT1(+/+) refers to WT1(+exon5/+KTS), WT1(-/+) to WT1(-exon5/+KTS), WT1 (+/-) to WT1(+exon5/-KTS) and WT1(-/-) to (-exon5/-KTS) (Fig.1-12). Introduction - 19 -

Fig.1-12 leads to four different WT1 isoforms. Splice 1 results in the presence or absence of exon 5 which codes for 17 amino acids. Alternative splice 2 differs in three amino acids (KTS) between zinc fingers three and four. The two alternative splicing sites give rise to four WT1 isoforms.

The WT1(-KTS) forms bind to GC-rich response element (EGR core consensus) with high affinity. WT1 (+KTS) forms don't bind to the EGR core consensus, but rather bind to a DNA-sequence that contains EGR core consensus and additional flanking sequence. (Cook et al., 1996; Little et al., 1996). WT1 (+KTS) forms preferentially associate with splicing factors (Larsson et al., 1995) and are incorporated into spliceosome in vitro (Davies et al., 1998). Thus, it appears that the two major splicing variants of WT1 may participate in different aspects of gene expression, namely, the WT1 (-KTS) forms participate in transcription and the WT1 (+KTS) forms in RNA processing. The WT1(+exon5) forms, unlike the KTS insertion, are only found in mammalian WT1 (Kent et al., 1995). The WT1 (+exon5) forms have been shown to effect both, cell proliferation and apoptosis (Englert et al., 1995; Johnstone et al., 1996; Menke et al., 1997; Murata et al., 1997; Mayo et al., 1999). Recent study (Richard et al., 2001) identified the exon 5 encoded 17 AA insertion of WT1 as a regulated transcriptional activation domain, that functionally interacts with the prostate apoptosis response factor par4. Since the WT1 (+exon5) forms were found to be overexpressed in some Wilms' tumor and also in leukemia (Pritchard-Jones and King- Underwood, 1997; Pritchard-Jones and Renshaw, 1997; Iben and Royer-Pokora, 1999), WT1 may also have an oncogenic role in leukemia in addition to its defined function as a tumor suppressor. Thus, it seems that WT1 can function both as a repressor and as an activator of transcription through the interaction of separate WT1 domains with various cofactors (Wang et al., 1993). The domain structure of WT1 is depicted in Fig.1-13. Introduction - 20 -

Fig.1-13. Domain structure of the WT1 protein. WT1 protein is alternatively spliced. Exon 5 encodes a 17 amino acids domain and exon 9 codes for the three amino acids, KTS between zinc finger III and IV. The WT1 self-association domain and the repression domain are mapped to amino acid 2-126 and 84-180, respectively. The activation domain of WT1 is mapped to amino acid 180-250.

Besides mediating tumor suppression and tumorgenesis in Wilms' tumor and leukemia, WT1 plays essential roles in the development of the urogenital system. WT1 starts to be expressed from the early stage of the urogenital ridge and persists during kidney and gonad development (Pritchard-Jones et al., 1990). Mice with a disrupted WT1 gene lack kidneys and gonads (Kreidberg et al., 1993). As a result of gonadal degeneration, the internal and external genitalia develop along the female pathway. These data suggest that WT1 regulates target genes that are essential for gonadogenesis. In vitro studies have shown that WT1 and SF-1 form heterodimers (Nachtigal et al., 1998). The functional significance of this interaction is supported by the finding that cotransfection of SF-1 with the WT1(-KTS) form augments SF-1-dependent transcriptional activation of the MIS promoter. Interestingly, the WT1(+KTS) form is able to induce the SF-1 promoter, which suggests that the WT1(+KTS) form may act in an autoregulatory loop to regulate SF-1 expression (Parker et al., 1999). This suggests a role of the WT1 (+KTS) form in testicular development. WT1 apparently is not essential for SF-1 expression, as WT1 knockout mice still have detectable SF-1 transcripts in the degenerating gonads (Parker et al., 1999). WT1 may regulate SF-1 activity by two mechanisms: facilitating SF-1-dependent activation of downstream genes such as MIS and increasing the levels of SF-1 (Parker et al., 1999). In the developing murine gonadal ridge, WT1 expression precedes expression of DAX-1 (Kim et al., 1999). The spatial distribution profiles of both proteins in the developing gonad Introduction - 21 - overlap. Sequence comparison of the human and murine DAX-1 promoters revealed two potential WT1 binding sites (desiganated WB1 and WB2). Footprinting analysis, transient transfections, promoter mutagenesis, and mobility shift assays suggest that WT1 binds to these two binding sites and regulates DAX-1 gene expression (Kim et al., 1999). In summary, WT1 functions as a transcription factor that upregulates the transcription of the MIS gene through a synergistic interaction with SF-1. In addition, WT1 upregulates the transcription of the DAX-1 gene during gonadal differentiation. Introduction - 22 -

1.6 The aim of this study An essential step during sex determination is the maintenance of the Müllerian ducts in females giving rise to oviduct, uterus and the upper end of vagina and its regression in males caused by the expression of Müllerian inhibiting substance (MIS). Therefore, the regulation of MIS expression is an important event in sexual differentiation. In the testis, the nuclear receptor steroidogenic factor 1 (SF-1) binds to a specific response element in the MIS promoter and activates the transcription of the MIS gene. Wilms' tumor suppressor protein (WT1) interacts and synergizes with SF-1 to upregulate the transcription of the MIS gene. In the ovary, on the other hand, the nuclear orphan receptor DAX-1 binds to SF-1 and functions as a repressor, disrupting the synergism of WT1/SF-1. This leads to the suppression of MIS gene expression. In addition, WT1 was defined as an upstream regulator of the DAX-1 gene. WT1 induces DAX-1 gene expression. This led to the assumption that protein-protein interactions might regulate the spatio- temporal fine-tuning of WT1 action. However, no WT1 coactivators important in the transcriptional upregulation of the MIS gene have been described. Recently, the members of a four-and-a-half LIM (FHL) family were identified as transcriptional coactivators. FHL2, a member of this family is expressed in heart, ovary and prostate. The FHL family members share a high homology in amino acids sequence and structure. In addition to the tissue-specific expression pattern, several FHL members are expressed in gonads. FHL2 and FHL3 were found in ovary. FHL4 was shown to be mainly expressed in testis and ACT (activator of CREM in testis) was found exclusively expressed in testis. Taken together, these data suggest that FHL proteins may be involved in gonadal differentiation. Therefore, I aimed to investigate the expression pattern of FHL2 during the development of embryonic gonads in mouse. I compare the FHL2 expression pattern with that of WT1 during gonadal differentiation. The aim of this study was to analyze the potential interaction between FHL2 and WT1 in vitro and in vivo and to demonstrate the physiological relevance of this interaction. Materials and methods - 23 -

2. Materials and methods

2.1 Materials

2.1.1 Plasmids pCMX: Eukaryotic expression plasmid, containing the cytomegalovirus (CMV) promoter and the T7 promoter (Umesono et al., 1991). pCMX-FHL2: Eukaryotic expression plasmid for FHL2 in pCMX (Müller et al., 2000). pCMX-mSF-1: Eukaryotic expression plasmid for mouse orphan nuclear receptor SF-1 in pCMX (Greschik et al., 1999) pCMX-mSF-1-GCNF: Eukaryotic expression plasmid for the mouse SF-1 mutant which consists of SF-1 DNA-binding domain and GCNF ligand-binding domain (Greschik et al., 1999). pCMX-PL2-K.ATG: Eukaryotic expression plasmid, containing a Kozak sequence followed by an ATG in front of a multi-cloning-site in pCMX. Constructed by Eric Metzger (Schüle lab). pCMX-PL2-K.ATG-mGCNF: Eukaryotic expression plasmid for mouse GCNF in pCMX- PL2-K.ATG (Greschik et al., 1999). pCMX-PL2-K.ATG-WT1-mutants: Eukaryotic expression plasmids for WT1 mutant proteins in pCMX-PL2-K.ATG. pCMX-PL2-K.ATG-3×Flag-WT1(-/-): Eukaryotic expression plasmid for WT1(-/-) fusion protein tagged with three repeats of the Flag-epitope at N-terminus in pCMX-PL2-K.ATG. pCMX-Flag: Eukaryotic expression plasmid. A Flag epitope is inserted in front of a multi- cloning-site in pCMX. pCMX-Flag-FHL2: Eukaryotic expression plasmid for Flag-FHL2 in pCMX (Müller et al., 2000). pGL2-basic: Reporter plasmid (Promega). pGL2-DAX-1 promoter: Contains DAX-1 promoter in pGL2-basic (Kim et al., 1999). pTATA-LUC: Reporter plasmid, containing the β-globin-TATA box sequence in pGL2-basic (Altshmied and Duschl, 1997). p2×MIS-RE1-TATA-LUC: Reporter plasmid, containing 2 repeats of the MIS-RE1(SF-1- responsive element of the MIS promoter) in pTATA-LUC. pBSK (pBluescript SK): Vector plasmid for in vitro transcription driven by T7 or T3 promoter (Stratagene). pBSK-WT1: Plasmid for in vitro transcription of WT1 cRNA probe (Herzer et al., 1999). pBSK-FHL2: Plasmid for in vitro transcription of full-length mouse FHL2 cRNA probe. Constructed by Judith Müller (Schüle lab). Materials and methods - 24 - pGBKT 7, pACT2: Library vectors for the GAL4DBD/AD two-hybrid-system (Clontech). pGBKT 7-WT1: Yeast expression plasmid for full-length WT1 (-/-) fused to the GAL4 DBD. Constructed by Philip Hublitz (Schüle lab). pGBKT7-WT1(182-298): Yeast expression plasmid for the WT1 (-/-) deletion mutant WT1 (182-298) fused to GAL4-DBD. Constructed by Philip Hublitz (Schüle lab). pACT2-FHL2: Yeast expression plasmid for FHL2 fused to Gal-AD (Müller et al., 2000). pGEX-4T1: Prokaryotic GST gene fusion vector (Pharmacia). pGEX-4T1-FHL2: Prokaryotic expression plasmid for GST-FHL2 fusion protein in pGEX- 4T1. Constructed by Holger Greschik (Schüle lab). pGEX-4T1-FHL2LIM0-1, pGEX4T1-FHL2LIM0-2, pGEX4T1-FHL2LIM3-4, pGEX4T1-FHL2LIM1-2, pGEX4T1-FHL2LIM0, pGEX4T1-FHL2LIM1, and pGEX4T1-FHL2LIM2: Prokaryotic expression plasmids for the corresponding GST-FHL2 mutant fusion proteins in pGEX-4T1. All constructed by Holger Greschik (Schüle lab).

2.1.2 Enzymes Restriction enzymes New England BioLAbs (NEB)/Takara. Klenow fragment NEB CIP NEB. T4 DNA ligase NEB. VentTM DNA polymerase NEB. T7 RNA polymerase Promega. T3 RNA polymerase Promega. RNase A Boehringer Mannheim. RNase Inhibitor (RNasin) Promega. Proteinase K Boehringer/Roche

2.1.3 Oligonucleotides Oligonucleotides were ordered from Interactiva (Ulm).

ATG-FLAG-x2: 5'-AATTCATGGACTACAAGCATGACGACGGTATGGACTACAAGCATGACGACGGTG-3' (Sense) 5'-AATTCACCGTCGTCATGCTTGTAGTCCATACCGTCGTCATGCTTGTAGTCCATG-3' (Antisense)

MIS-RE: 5'-GATCCCACTGTCCCCCAAGGTCAA-3' (Sense) 5'-GATCTTGACCTTGGGGGACAGTGG-3' (Antisense) Materials and methods - 25 -

MIS-M1: 5'-GATCCCACTGTCCCCAAAGGTCAA-3' (Sense) 5'-GATCTTGACCTTTGGGGACAGTGG-3' (Antisense)

WB1 5'-GATCCGCGCGTCCCCCTCCACAA-3' (Sense) 5'-GATCTTGTGGAGGGGGACGCGCG-3' (Antisense)

WB2 5'-GATCCCAAGAGGAGGAGGCGGACCGA-3' (Sense) 5'-GATCTCGGTCCGCCTCCTCCTCTTGG-3' (Antisense)

WB1+WB2 5'-GATCCGCGCGTCCCCCTCTGCGCCCTTGTCCAAGAGGAGGAGGCGGACCGA-3' (Sense) 5'-GATCTCGGTCCGCCTCCTCCTCTTGGACAAGGGCGCAGAGGGGGACGCGCG-3' (Antisense)

WT1 298-449: 5'-CGGAATTCGCATCTGAGACCAGTG-3' (Sense) 5'-GAAGATCTTCAAAGCGCCAGCTGG-3' (Antisense)

WT1 182-298: 5'-CGGAATTCGGCCAGCAGGGCTC-3' (Sense) GAAGATCTTGCCGACCGTACAAGAGTCG (Antisense)

2.1.4 Antibodies Mouse α-Flag-antibody (M2): monoclonal antibody, 4.4 µg/µl. (Sigma-Aldrich, USA). Mouse α-Flag-antibody (M5): monoclonal antibody, 3.4 µg/µl. (Sigma-Aldrich, USA). Mouse α-FHL2-antibody (11-134): monoclonal antibody, 1.13 µg/µl (Müller et al., 2000). Rabbit α-WT1-antibody (C-19): affinity-purified rabbit polyclonal antibody, IgG, 200 µg/ml (Santa Cruz, USA). Anti-XpressTM antibody: 2 µg/µl. (Invitrogen, Netherlands).

2.1.5 Bacteria and cultural media for E.coli E. coli DH5α: supE44 ∆lacU169(φ80lacZ∆M15) hsdR17 recA1 endA1 gyrA96 thi- 1relA1 (Hanahan, 1983) E. coli NM554: recA13 araD139 ∆(ara-leu)7696 ∆(lacZ)l7A galU galK hsdR rpsL (Strr) mcrA mcrB (Raleigh et al., 1988). E. coli BL21 (DE3): hsdS gal(λcIts857 ind1 sam7 nin5 lacUV5-T7 gene 1) (Studier et al., 1990 & 1991). Materials and methods - 26 -

LB medium powder and LB-agar were purchased from Gibco-BRL company (Eggenstein). LB-Amp-medium: LB medium containing 100 µg/ml ampicillin.

2.1.6 Cells and culture media 293: human embryonic kidney cell line (ATCC# CRL 1573) cultured in DMEM with 10% FCS. COS-7: African green monkey kidney cell line, SV40 transformed (ATCC# CRL 1651) cultured in DMEM with 10% FCS. The Dulbecco's Modified Eagle Medium (DMEM) is supplemented with 2 mM glutamine, 100 IU/ml penicillin and 100 mg/ml streptomycin.

PBS: 137 mM NaCl; 2.7 mM KCl; 8 mM Na2HPO4; 1.8 mM KH2PO4 Trypsin EDTA: 0.25% Trypsin, 0.04% EDTA in PBS buffer.

Dulbecco's Modified Eagle Medium (DMEM) Bio Whittaker (Belgium) Fetal calf serum (FCS) Gibco BRL Penicillin/streptomycin (100×) Gibco BRL. Glutamine (200 mM) Gibco BRL. Trypsin/EDTA Gibco BRL.

2.1.7 Radioactive materials All radioactive materials were purchased from Pharmacia/Amersham. [α-32P] dATP, 10 mCi/ml; [α-33P] ddNTPs, 450 µCi/ml; L-[35S]-Methinion, 10 mCi/ml.

2.1.8 Kits UltraCleanTM 15 DNA Purification Kit MO BIO. ECLTM Western-Detection Kit: Pharmacia/Amersham. Plasmid Maxi Kit: Qiagen. TNTTM Coupled Reticulocyte Lysate: Promega.

2.1.9 Electrophoresis buffers and solutions TAE buffer (50×): 2 M Tris/HCl; 5.7% (v/v) acetic acid; 50 mM EDTA; pH 8.0. TBE buffer (5×): 440 mM Tris; 440 mM boric acid; 10 mM EDTA; pH 8.4. DNA loading buffer (10×): 50% (w/v) saccrose; 50 mM EDTA (pH 8.0); 0.5% (v/v) SDS; 0.06% (w/v) bromide phenol blue; 0.06% (w/v) xylencyanol. SDS loading buffer (5×): 250 mM Tris-HCl; 500 mM DTT; 10% (w/v) SDS; 0.5% (w/v) Materials and methods - 27 -

bromide phenol blue; 50% glycerol (v/v); pH 6.8. SDS-PAGE buffer (10×): 250 mM Tris-HCl; 2.5 M glycin; 1% (w/v) SDS; pH 8.3.

Coomassie bright blue staining buffer: 2.5 g Coomassse R250; 45.5% (v/v) CH3OH; 9.2% (v/v) acetic acid. Coomassie bright blue destaining buffer: 25% (v/v) isopropanol; 10% (v/v) acetic acid.

2.1.10 Solutions for Western-blot

Anode buffer 1: 300 mM Tris; 20% (v/v) CH3OH; pH 10.4

Anode buffer 2: 25 mM Tris; 20% (v/v) CH3OH.

Cathode buffer: 25 mM Tris; 40 mM 6-amino phexanoic acid; 20% (v/v) CH3OH; pH 9.4.

2.1.11 Solutions for nuclear extract from culture cells

Buffer A: 10 mM HEPES-KOH, pH 7.9; 1.5 mM MgCl2; 10 mM KCl; 0.5 mM DTT; 0.2 mM Pefablock T buffer: 50 mM Tris-HCl, pH 7.4; 250 mM NaCl; 20% glycerol; 2 mM DTT; 1% Triton X- 100; 1 mM Pefablock; 0.2 mM NaVO4; 5 mM β-glycerophophate.

2.1.12 Buffers used in transient transfections

Luciferase-assay-buffer: 200 mM Tricine; 1.07 mM(MgCO3).4Mg(OH)2; 0.1 M MgSO4; 10 mM EDTA, pH 8.0; 33 mM DTT; 0.5 M ATP; 270 mM acetyl- coenzyme A; 470 mM lightfly Luciferin. 2×BES: 50 mM N,N-bis-[2 hydroxyethyl]-2-aminoethansulfic acid; 280 mM NaCl; 1.5 mM

Na2HPO4.2H2O; pH 6.95.

2.1.13 Chemicals All basic chemicals were purchased from Fluka, Roth or Sigma. The chemicals are analytical grade (p.a.).

Agarose (Gibco) IPTG (Roth) Amplify solution (Amersham) Long RangerTM (FMC Bioproducts) BSA (Sigma-Aldrich) Luciferase reagent (Promega) DNA marker -1Kb ladder(Gibco) Skimmed milk powder (Fluka) DNA marker -123 bp ladder(Gibco) Nonidet P40 (Fluka) DNA marker V Nucleotide triphosphate (Boehringer/Roche) EDTA (Serva) Poly(dIdC) (Boehringer/Roche) Ethanol (Roth) Protein marker-prestained (Gibco) Ethidium bromide (Roth) SDS (Serva) GammaBind G Sepharose (Pharmacia) TALON Metal Affinity Resin (Clontech) Materials and methods - 28 -

Gel SlickTM (FMC Bioproducts) TEMED (Serva) Glutathione-Sepharose 4B (Pharmacia) Triton X-100 (Fluka) HEPES (Roth) Tween-20 (Fluka)

2.1.14 Devices Bacteria incubator (Heraeus) Balance (Sartorius, LP6200S) Bohemian wheel (Grünewald GmbH & Co KG) Camera AxioCam (Zeiss) Centrifuges: Eppendorf table centrifuge 5415C and 5417C (Eppendorf-Nethele-Hinz GmbH) Megafuge 3.0R (Kendro) J2HS-centrifuge (Beckmann) L7 Ultracentrifuge (Beckmann) EMSA apparatus (Hoefer) Geiger Counter LB122 (Beckmann) Gel documentation (Eagle Eye) (Stratagene) Gel electrophoresis chamber (BioRad) Gel dryer (BioRad) Heating block (Techne Driblock) LS 6500 Scitilation Counter (Beckmann) Luminometer ML 3000 (Dynatech/EG&G Berthold) Magnetic stirrer (IKA-Labortechnik)

Microscope MZ95 (Leica) Parafilm (American National Can) PCR machine 9600 (Perkin Elmer) pH meter (Knick) Photometer (Curix 60) (AGFA) Photometer (MR7000) (Dynatech) Rotors JA10, JA20, TI70 (Beckmann) Scintilation cocktail (Beckmann) Vortex (Heidolph) Waterbath (Haake) Western-blot apparatus (Fröbel) Whatman 3 MM paper (Schleicher and Schuell) X-ray film (Fuji and Kodak) X-ray film cassette (Cawo) Materials and methods - 29 -

2.2 Methods

2.2.1 Methods for DNA

2.2.1.1 Agarose gel electrophoresis DNA fragments were seperated in 0.8-3.3% agarose gels according to the size of the DNA fragments to be analyzed. Either TAE or TBE electrophoresis buffers were used. For regular sized DNA, the 1Kb DNA ladder (Gibco) was used as a standard. For smaller DNA fragments, the 123bp ladder (Gibco) and marker V (Boehringer) were used. The electrophoresis were carried out at 8 V/cm gel length. The gel was stained in ethidium bromide and visualized under UV-light at 345 nm (Eagle Eye II, Stratagene).

2.2.1.2 Recovery of DNA fragment from agarose gels The gel slice containing the desired DNA fragment was cut out under long wave UV-light. DNA was purified according to the manufacturer's recommendation (Ultra CleanTM). The DNA was eluted in water or TE.

2.2.1.3 DNA restriction digestion DNA was incubated with 4-12 units of enzyme per µg DNA in the corresponding buffer for 1 h at 37¡C. The restriction reaction was then analyzed by agarose gel electrophoresis.

2.2.1.4 Modification of DNA ends Plasmid DNA was dephospharylated at 5' ends to avoid vector religation. Linearized plasmids were incubated with 5 units of calf intestinal alkaline phosphatase (CIP) per nmol free 5'- DNA-ends for 1h at 37¡C. Then the DNA is purified with the UltraCleanTM 15 DNA Purification Kit. The large fragment of DNA polymerase I (Klenow fragment) was used to generate blunted DNA. Klenow fragment has 5'-3' polymerase function which can fill the 5' overhang. Klenow fragment was used in any of the restriction reaction buffers. After restriction digestion, 1 unit Klenow fragment per µg DNA and 0.23 mM of each dNTPs were added and incubated at 25¡C for 15 min and the reaction was stopped by adding EDTA to 10 mM and heating at 75¡C for 10 min. For cloning of double stranded oligonucleotides, T4 polynucleotide kinase was used to catalyze phosphate transfer from ATP to the 5'- hydroxyterminus of polynucleotides. The standard kinase reaction was as following: double stranded polynucleotides 1 µg 10× T4 polynucleotide kinase buffer 2 µl 10 mM ATP 2 µl T4 polynucleotide kinase (10 U/µl) 1 µl µ ddH2O to 20 l Materials and methods - 30 -

The reaction was incubated at 37¡C for 30 min. The enzyme was inactivated by incubation at 65¡C for 15 min.

2.2.1.5 DNA ligation T4 DNA ligase was used to join two DNA fragments. 5'-terminal phosphate groups and ATP are needed for T4 DNA ligase reaction. In the ligation reaction, the total DNA amount varies from 0.1-1 µg and the molar ratio of vector DNA to insert DNA was around 1:3. The reaction was incubated with 150 weiss units T4 ligase in ligation buffer for 1-2 hrs at RT.

2.2.1.6 Preparation and transformation of competent E. coli To prepare competent E. coli cells, 500 ml LB mudium was inoculated with 25 ml of a overnight E. coli culture and incubated at 37¡C until a OD600= 0.5 was reached. The bacteria were cooled on ice and then centrifuged at 2000 rpm (JA-10 rotor, Beckman) for 10 min at

4¡C. The pellet was resuspended in 30 ml TFB I-buffer (30 mM KOAc, 50 mM MnCl2, 100 mM RbCl2, 10 mM CaCl2,, 15% (v/v) glycerol, pH 5.8) and then incubated on ice for 10 min. After centrifugation at 4000 rpm for 10 min at 4¡C, the pellet was resuspended in 4 ml TFB µ II-buffer (10 mM MOPS, 75 mM CaCl2,, 10 mM RbCl2,, 15% (v/v) glycerol, pH 7.0). 50 l aliquots were stored at -80¡C. For transformation, 10 µl ligation mix or 100 ng plasmid DNA were incubated with the competent cells on ice for 20 min. Following a heat shock at 42¡C for 2 min, the competent cells were cooled on ice for 1 min and transferred to LB-Amp medium or spread on LB-Amp plates with the respective selection marker.

2.2.1.7 Analytical isolation of plasmid DNA from E. coli E. coli cultures were grown in 3 ml LB medium containing 100 µg ampicillin under constant shaking overnight at 37¡C. The next day, 1 ml E. col cultures were transferred to an eppendorf tube and centrifuged at 14000 rpm for 1 min. After resuspending in 50 µl buffer P1, the bacteria were lysed by the addition of 100 µl buffer P2. Then 75 µl buffer P3 was added to precipitate proteins and attached genomic DNA. The mixture was incubated on ice for 10 min. After centrifugation at 14000 rpm for 10 min, 200 µl of the supernatant was mixed with 300 µl 6 M guanidiumthiocyanate and 400 µl isobutanol. After centrifugation at 14000 rpm for 10 min, the pellet was washed with 70% ethanol, air dried and dissolved in 30 µl TE buffer.

Buffer P1: 50 mM Tris-HCl, pH 8.0; 10 mM EDTA; 10 µg/µl RNase. Buffer P2: 200 mM NaOH; 10% (w/v) SDS. Buffer P3: 3 M KOAc, pH 5.5. TE buffer: 10 mM Tris-HCl, pH 8.0; 1 mM EDTA. Materials and methods - 31 -

2.2.1.8 Polymerase chain reaction (PCR)

A standard PCR reaction contains 100 ng DNA, 0.1 mM dNTP, 15 mM MgCl2, 10 pmol primers, 2 U VentTM polymerase in a final volumed 100 µl. The annealing temperature depends on the primers' annealing temperatures. The typical cycle mode of a PCR reaction is: a) 1 cycle with: 5 min denaturation at 94¡C b) 30 cycles each with: 30 sec denaturation at 94¡C 30 sec annealing at 50-65¡C 1 min per kb DNA extension at 72¡C c) 1 cycle with: 10 min final extension at 72¡C d) 4¡C to coll down.

2.2.1.9 DNA sequencing  DNA sequencing was carried out with ABI PRISM BigDyeTM Terminator Cycle Sequencing Ready Reaction Kits (PE Biosystems). The sequencing reaction was set up according to the manufacturer's recommendation (PE Biosystems) as following: plasmid DNA 500 ng primers 3.5 pmol BigDye Mix 4 µl total volume 20 µl The BigDye Mix contains the terminators labeled with fluorescent dyes. The sequencing reaction was performed with 25 PCR cycles. The following PCR program was used for each cycle: 30 sec denaturation at 96¡C 15 sec annealing at 50¡C 4 min extension at 60¡C  Analysis of the sequences was carried out with ABI PRISM 3100 Genetic Analyzer System (PE Biosystems).

2.2.1.10 Preparative isolation of plasmid DNA from E. coli Large scale preparation of plasmid DNA was performed with the Qiagen Plasmid Maxi Kit. 150 ml LB medium which contains 100 µg /ml ampicillin was inoculated with 50 µl of E coli suspension and cultured at 37¡C overnight. The plasmid DNA isolation was performed according to the manufacturer's instruction. The plasmid DNA was dissolved in 300-400 µl TE buffer. The concentration of the plasmid DNA was measured by reading the absorption at µ 260 nm. 1 A260 unit = 50 g/ml double stranded DNA.

2.2.1.11 Annealing of oligonucleotides In a typical annealing reaction, two single stranded complementary oligonucleotides were incubated in a buffer containing 10-20 mM MgCl2 at 95¡C for 10 min and then cooled down Materials and methods - 32 - at RT within 10 min. The double stranded oligonucleotides were precipitated by addition of 1/10 volume 3 M NaOAc, 2.5 volumes ethanol, and cooled at -80¡C for 5-30 min, followed by centrifugation at 15000 rpm for 30 min at 4¡C. The pellet was washed with 70% ethanol and air dried, then dissolved in TE buffer. The concentration of double stranded µ oligonucleotides was estimated at A260 (1 A260 correspons to 40 g/ml).

2.2.1.12 Radiolabeling of double stranded oligonucleotides The double stranded oligonucleotides with 5' overhang ends are radiolabeled with Klenow fragment. A typical labeling reaction is composed of: double stranded oligonucleotides 100 ng 10x Klenow buffer 2 µl dCTP, dGTP, dTTP (5 mM each) 1 µl α-32P-dATP (10 mCi/ml) 3 µl Klenow fragment (5 U/µl) 1 µl µ ddH2O to 20 l The reaction is incubated at 30¡C for 30 min, 1 µl of dNTP (2 mM of each dCTP, dGTP, dTTP and dATP) was added and incubated at RT for 5 min. The reaction was stopped by µ µ addition of 1 l 0.5 M EDTA. Then 30 l ddH2O was added to the reaction and the unincorporated free nucleotides were eliminated by passing through a Microspin column (Microspin S-200 HR; Pharmacia). The incorporated α-32P-dATP was measured in a scintillation counter (LS6500, Beckman).

2.2.2 Methods dealing with protein

2.2.2.1 SDS-polyacrylamide-gel-electrophoresis Proteins are dissociated into their individual polypeptide subunits by a strongly anionic detergent, sodium dodecyl sulfate (SDS). The denatured polypeptides bind SDS to get a uniform negative charge and therefore migrate through a polyacrylamide-gel according to their sizes. Before loading on the SDS-polyacrylamide gel, the proteins were denatured at 95¡C with SDS-loading buffer for 5 min. The discontinous buffer system was used in SDS- PAGE. The sample and the stacking gel contain Tris-glycine (pH 6.8), the upper and lower buffer reservoirs contain Tris-glycine (pH 8.3), and the resolving gel contains Tris-HCl (pH 8.8). All the components in the system contain 0.1% SDS. After SDS-PAGE, the non- radioactive labeled proteins were stained with Commassie bright blue. For radiolabeled proteins, the gel was rinsed in fixer solution (25% isopropanol and 10% acetic acid) followed by incubation with amplifier (Amersham) for 15 min. The gel was dried before visualizing by autoradiography. Materials and methods - 33 -

2.2.2.2 Western-blotting and immuno-detection To transfer the proteins from SDS-polyacrylamide gels onto the blotting membrane (PVDF), the following procedure was used. Anode: 2 pieces of Whatman 3 MM-filter paper soaked in anode buffer 1 4 pieces of Whatman 3 MM-filter paper soaked in anode buffer 2 PVDF-membrane (Hybond Immobilon, pre-rinsed in methanol) (Millipore) SDS- polyacrylamide-gel Cathode: 6 pieces of Whatman 3 MM-filter paper soaked in cathode buffer The transfer was conducted for 1.5 h with 1 mA per cm2 of gel size. The transfer was controled by Ponceau S staining. The membrane then was incubated in blocking buffer, 5% (w/v) skimmed milk powder in PBST (0.5% Tween-20 in PBS) buffer for 1h at RT. After blocking, the membrane was hybridized with primary antibody for 1 h in blocking buffer at RT. The concentration of the antibodies was chosen as needed (from 1-10 µg/ml). After washing 3-5 times in PBST buffer, the membrane was incubated with HRP-conjugated secondary antibody (Sigma) for 1 h at RT. The membrane was incubated in the detection solution of the ECL-Kit (Amersham, Pharmacia) before exposed to a X-ray film.

2.2.2.3 Immunoprecipitation Coimmunoprecipitation was performed with nuclear extract of transfected 293 cells. 4 µg of antibody was used for an immunoprecipitation with 200 µg of nuclear extract. The antibody was immobilized to Gamma-bind G sepharose beads and incubated with the precleared nuclear extract in binding buffer for 3 hrs at 4¡C. After three washes with washing buffer each for 5 min, the immunoprecipitated proteins were boiled in SDS sample buffer for 5 min and subjected to SDS-PAGE followed by Western-blot and immuno-detection.

Binding buffer: 20 mM Tris, pH 7.5; 2 mM DTT; 20% glycerol; 100 mM NaCl Washing buffer: 20 mM Tris-HCl (pH 8.0), 250 mM NaCl, 0.5% NP-40 and 0.5 mM Pefablock.

2.2.2.4 Electrophoretic mobility shift assay (EMSA) Proteins that bind specifically to DNA fragment retard the mobility of the DNA fragment during native electrophoresis, resulting in discrete bands corresponding to the individual protein-DNA complexes. The WB1+WB2 fragment which contains two WT1-binding sites were labeled with 32P by Klenow reaction. Nuclear extracts of transiently transfected 293 cells were incubated with the 32P-labeled WB1+WB2 oligonucleotide in the binding buffer for 30 mins at RT. The EMSA reaction was saperated in a 4.5% native polyacrylamide gel in 0.5×TBE. The electrophoresis was conducted in 0.5×TBE at 150 V at 4¡C. After the end of the run, the gel was dried before exposed to X-ray film. Materials and methods - 34 -

µ µ Binding buffer: 50 mM KCl, 1 mM MgCl2, 1 M ZnSO4, 10 mM Tris, pH 8.0, 1 g dIdC, 30 µg BSA and 4% glycerol.

2.2.2.5 Coupled in vitro transcription/translation WT1 and mutants thereof were in vitro translated in TNT coupled reticulocyte lysate (Promega). The TNT coupled reticulocyte lysate contains all the factors that are needed for the transcription from plasmid DNA to mRNA, and the translation from mRNA to protein. The in vitro transcription/translation reaction was carried out in a total volume of 50 µl, which contains 25 µl reticulocyte tysate, 2 µl transcription/translation buffer, 1 µl amino acid mixture, 1 µl T7-RNA-polymerase, 1 µl RNase-inhibitor and 1 µg plasmid DNA (i.e. pCMX- PL2-K.ATG-WT1). The reaction was incubated at 30 ¡C for 1.5 h and then stored at -20¡C. Proteins were labeled with 35S-methionine. The radiolabeled proteins were separated by SDS- polyacrylamide-gel-electrophoresis. The gel was dried and visualized by autoradiography.

2.2.2.6 Protein expression in E. coli E. coli BL21 DE3plys GOLD were transformed with pGEX expression plasmids and incubated in LB medium containing 100 µg/ml ampicillin and 34 µg/ml chloramphenicol overnight at 37¡C. The next day, the E. coli cultures were diluted 1:7 and incubated at 37¡C until the OD600=0.6-0.7 was reached. After induction with 1 mM IPTG for 5 h at RT, the bacteria were harvested by centrifugation at 6000 rpm for 10 mins at 4¡C. The pellet was resuspended in 30 ml of lysis buffer. To lyse the E. coli, the cells were frozen in liquid nitrogen and thawed at 37¡C for 3 times followed by ultrasonication with a Bandalin sonifying device. The debris of the bacteria were spined down by a 30 min centrifugation at 16000 rpm (JA20-rotor, J2-HS centrifuge, Beckman) at 4¡C. The supernatant was supplemented with 10% glycerol and 1 ml aliquots were stored at Ð80¡C.

Lysis buffer: 50 mM Tris/HCl (pH 8.0), 250 mM NaCl, 10% (v/v) Triton X-100, 1 mM DTT and 0.5 mM PefablockTM

2.2.2.7 Glutathione-S-transferase (GST) pulldown Glutathione-S-transferase (GST)-pulldown is a useful method to analyze protein-protein interaction. GST fusion proteins were expressed in E.coli and whole cell extracts were prepared as described. The E.coli expressed GST fusion protein was then immobilized on Glutathione-sepharose 4B (Pharmacia) and incubated with 35S-labeled in vitro translated protein in the binding buffer for 60 min at 4¡C. After extensive washing, bound proteins were seperated by SDS-PAGE and visualized by autoradiography. Ten percent of the in vitro translated proteins were loaded as input control.

Binding buffer: 20 mM HEPES (pH 7.7), 150-300 mM KCl, 0.1 mM EDTA, 25 mM MgCl2, Materials and methods - 35 -

10 mM DTT, 0.15% (v/v) NP-40.

2.2.2.8 Protein measurement To determine protein concentration, a standard curve with the 1-10 µg BSA was generated. The absorption at 595 nm was read with a Spectro-photometer (Molecular Devices GmBH). The protein concentration was obtained by comparing absorption values of the protein samples at 595 nm with that of the BSA-standard-curve.

2.2.3 Cell culture and transient transfection

2.2.3.1 Permanent cell culture The cells are cultured under sterilized condition. Cells were split and subcultured at a ratio of 1:10 when reaching 80% confluency. To split the culture, the cells were washed with PBS and incubated with Trypsin-EDTA for 3-5 min. Trypsin-EDTA was inactivated by the addition of 5 ml fresh medium. Cells were counted in a Neubauer chamber and resuspended in fresh medium. The cells were kept at 37¡C with 5% CO2 humified atmosphere. To store cells in liquid nitrogen, cells were resuspended in the same medium supplemented with 20% FCS and 10% DMSO. 1.5 ml aliquots were frozen in liquid nitrogen.

2.2.3.2 Transient transfection using the calcium phosphate method Transient transfection assays were carried out in 24- or 12-well plate. 293 cells were seeded at a density of 0.5×105 cells per well and COS-7 cells at a density of 0.25×105 cells per well (24- well plate). Both, 293 and COS-7 cells were transfected with expression and reporter plasmids using a standard calcium phosphate precipitation method. A standard transfection mixture ready to use for two wells in a 24-well plate experiment was composed as following: Reporter plasmid 500 ng expression plasmids 20-1000 ng carrier DNA (PUC18) to 8 µg µ CaCl2 solution 20 l µ ddH2O to 80 l 80 µl 2×BES was added drop by drop under constant mixing. The mixture was left at RT for 15 min to form the precipitate. 78 µl of the precipitate was added to each well. After 8 hours of transfection, cells were washed with PBS and cultured for an additional 18 hours. For transfections in 10 cm dishes, a standard transfection mixture for two wells was composed as following: carrier DNA (PUC18) 20 µg expression plasmids 20 µg µ CaCl2 solution 224 l µ ddH2O to 924 l Materials and methods - 36 -

924 µl 2×BES was added as discribed above. After 20 min at RT, 920 µl of the precipitate was added to each dish.

2.2.3.3 Measurement of luciferase activity The transfected cells were washed 2 times with PBS and lysed by the addition of 50 µl reporter-lysis buffer (Promega). After 10 min incubation at RT, the cell lysates were transfered into eppendorf tubes and the insoluble materials were pelleted by centrifugation at 14000 rpm for 3 min. 10 µl of the supernatants were transfered into 96-well-plates and the luciferase activity was measured in a luminometer (Berthold). For measurement of luciferase activity, 40 µl of luciferase assay buffer was used. The protein concentration for each well was measured with 3 µl of the cell lysates. The relative luciferase activity of each sample was standarderized to the protein concentration of the sample and defined as activity/mg protein.

2.2.3.4 Nuclear extract from culture cells Aspirate the medium and wash the cells 2 times with PBS. 1 ml cold PBS was added to a 10 cm dish and the cells were scrabed and transferred into an eppendorf tube. After centrifugation for 10 seconds, the cells were resuspended in 400 µl buffer A and incubated on ice for 10 min. After 10 seconds vortexing, the nuclei were pelleted by centrifugation at 14000 rpm for 10 min. The supernatant was removed and the pellet (nuclei) was resuspended in 400 µl T buffer and incubated on ice for 10 min. After centrifugation at 14000 rpm for 10 min at 4¡C, the supernatant (nuclear extract) was saved and the protein concentration was measured.

2.2.4 Work with embryos

2.2.4.1 Preparation of embryos for in situ hybridization (ISH) Mouse embryos from E11.5 to E18.5 were dissected in cold PBS and fixed in 4% paraformaldehyde overnight. The embryos were washed in PBS-T (PBS with 0.1% Tween- 20), then dehydrated with a gradient methanol series (25%, 50%, 75%, and 100% in PBS-T) for 10 min each, and stored at -20¡C.

2.2.4.2 Labeling of the cRNA probe cRNA probes were labeled with digoxigenin. All steps were performed with RNAse-free solutions to avoid RNAse contamination. The plasmids were linearized, purified with the UltraClean method and resuspended in a suitable volume of nuclease free water. The transcription reaction was: DNA 1 µg 10× transcription buffer 2 µl labeling mix 2 µl RNAase inhibitor 1 µl Materials and methods - 37 -

RNA polymerase (Sp6, T3 or T7) 1 µl µ ddH2O to 20 l The reaction was incubated for 2 hrs at 37 ¡C. The unincorporated free nucleotides were removed by precipitating the RNA probe: Add 2.5 µl 4 M LiCl, 75 µl ethanol to the transcription reaction, incubate on ice for 1 hour, centrifuge at 15000 rpm for 30 mins at 4¡C. The pellet was washed with 70% ethanol, air dried, resuspended in 50µl DEPC-water and stored at -80¡C.

2.2.4.3 Whole mount in situ hybridization First, the embryos were rehydrated in a decreasing methanol series (90%, 80%, 75%, 50%, 25%) for 10 min each and stopped in PBS-T. The embryos were then digested with 4.5 µg/ml proteinase K for 10 mins at RT. The digestion was stopped by washing in freshly prepared 2 mg/ml glycine in PBS-T. The embryos were refixed in 4% paraformadehyde-0.2% glutaraldehyde for 15 min. After prehybridization at 65¡C for 3 h, the embryos were hybridized with digoxygenin-UTP (DIG) labeled probes at 70¡C overnight. The unhybridized probes were removed by washing in prehybridization solution for 5 min at 70¡C followed by a wash with 2×SSC (pH 4.5), 2×SSC (pH 7.0) and maleic acid buffer (100 mM maleic acid, 150 mM NaCl, pH 7.5). The embryos were preblocked with 10% goat serum in Boehringer Blocking Reagent at 4¡C for 2 h before incubated with alkaline-phosphatase (AP)-conjugated anti-DIG antibody at 4 ¡C overnight. After 5 washes with 0.1% BSA in PBS-T, the embryos were incubated with AP1 buffer (100 mM Tris pH 9.5, 100 mM NaCl, 50 mM MgCl2) for 10 mins at RT. The staining was carried out in AP substrate precipitating BM purple

(Boehring/Roche). The staining was observed under a microscope (Leica, MZ95) and reactions were stopped by washing with PBS. Photographs were taken with a AxioCam camera (Zeiss).

2.2.5 Yeast two hybrid assay Yeast two hybrid studies were performed as recommended by the manufacturer (Clontech Matchmaker Two-Hybrid system). Briefly, HF7c cells were transformed using the LiAc method (Guthrie and Fink) with pGBKT7, pGBKT7-WT1, or Gal4-DBD-WT1-IAD (carrying trp-auxotrophy markers) in concert with either pACT2 or pACT2-FHL (carrying leucine auxotrophy markers) and plated on selection media. Single yeast colonies were resuspended in TE and parallel-plated on double dropout plates (-leu, trp) to assess co- transformation efficacy and on triple dropout plates (-leu, trp, his), respectively, to assay the interaction of bait and prey. Double dropout plates were incubated 3 days, triple dropout plates were incubated for 7 days at 30¡C and then photographed. The experimant was performed with the assistance of Philip Hublitz. Results - 38 -

3. Results

3.1 Expression of FHL2 during sex development In adult, FHL2 is expressed strongly in heart and also in ovaries and prostate (Müller et al., 2000). To investigate the expression pattern of FHL2 in embryonic development, whole mount in situ hybridization is performed with full-length FHL2 cRNA probe. Mouse embryos are prepared from E12.5 until E18.5. FHL2 transcripts are found in the developing gonads in addition to heart (data not shown). The in situ hybridization with FHL2 antisense cRNA reveals expression in both testis and ovary during a period from E13.5 to E16.5, whereas no staining is obtained with sense probe. The FHL2 transcript fades to background level at E17.5. Fig.3-1a shows the expression of FHL2 in testis. Fig.3-1b shows FHL2 expression in ovary.

Fig.3-1a Expression of FHL2 in mouse embryonic testis. In situ hybridization of gonads from mouse embryos. The FHL2 antisense probe shows expression in testis from E13.5 until E16.5. The corresponding sense control probe shows no signal. The original magnification is 6 fold. Results - 39 -

Fig.3-1b Expression of FHL2 in mouse embryonic ovaries. In situ hybridization of gonads from mouse embryos. The FHL2 antisense probe shows expression in the ovaries from E13.5 until E16.5. The corresponding sense control probe shows no signal. The original magnification is 6 fold.

3.1.1 FHL2 is co-expressed with SF-1, WT1 and MIS in testis In this study, whole mount in situ hybridizations show FHL2 expression in mouse testis during a period between E13.5 and E16.5, which is a critical stage for testis differentiation. During testis differentiation, the MIS gene plays an important role and is first expressed from E12.5 and persists throughout testis differentiation (Behringer et al., 1994). SF-1 and WT1 have been indicated in the regulation of MIS expression (Shen et al., 1994; Nachtigal et al., 1998). SF-1 is expressed from E9.5 and persists throughout the male gonadal differentiation (Luo et al., 1994). WT1 expression (Kreidberg et al., 1993) overlaps with that of SF-1 and MIS. To demonstrate co-expression of WT1 and FHL2 during testis development, a series of in situ hybridization with a WT1 probe was performed. Fig.3-2 compares the expression of FHL2 and WT1 in the mouse testis at E14.5. Results - 40 -

Fig.3-2 Comparison of the expression pattern of WT1 and FHL2 in mouse testis at E14.5. In situ hybridizations are performed with WT1 and FHL2 cRNA probes. The antisense probes show intensive signals (upper panel) whereas the sense control probes show no expression.

Taken together, FHL2 shows an ovlapping expression pattern with SF-1, WT1 and MIS in testis during the period between E13.5 and E16.5. Fig.3-3 illustrates the overlapping expression pattern of these genes in testis.

Fig.3-3 Overview of genes expressed in testis differentiation. The green squares represent expression. Empty squares represent no expression. The expression of SF- 1, WT1 and MIS are summarized from published data (Luo et al., 1994; Kreidberg et al., 1993; Behringer et al., 1994). FHL2 expression is detected from E13.5 until E16.5 in the present study. FHL2 is co-expressed with SF-1, WT1, and MIS in testis during the period between E13.5 and E16.5.

3.1.2 FHL2 is co-expressed with WT1 and DAX-1 in ovary In the mouse ovary, FHL2 is expressed from E13.5 until E16.5. No transcripts are detectable by E17.5. In ovarian differentiation, DAX-1 is an important transcription factor involved in ovarian gene regulation. DAX-1 transcripts were detected in the genital ridge at E10.5 in both sexes. RNA transcripts decrease dramatically in the male gonad at E12.5, but are maintained in the female gonad (Swain et al., 1996). WT1 was defined as an upstream Results - 41 - regulator gene of DAX-1. WT1 is found in the urogenital ridge of male and female mice at E9 and the expression lasts till late stages of embryonic development (Kreidberg et al., 1993). It is well noted that SF-1 is expressed in the urogenital ridges of both sexes at E9 and the transcripts decline dramatically to undetectable level in the ovary from E13.5 to E16.5 (Luo et al., 1994). The disappearance of SF-1 coincidents with the expression of FHL2 from E13.5 to E16.5 in ovary. Taken together, FHL2, WT1 and DAX-1 are co-expressed in the ovary during the period between E13.5 and E16.5 (Fig.3-4).

Fig.3-4. Overview of genes expressed during ovarian differentiation. The green squares represent expression. Empty squares represent no expression. The expression of SF- 1, WT1, and DAX-1 are summarized from published data (Luo et al., 1994; Kreidberg et al., 1993; Swain et al., 1996). FHL2 expression is detected from E13.5 until E16.5 in the present study. FHL2 is co-expressed with WT1 and DAX-1 in ovary during the period between E13.5 and E16.5.

In situ hybridization with a WT1 probe is also performed to validate the co- expression of WT1 and FHL2 in ovaries. Fig.3-5 shows the in situ hybridization performed with mouse ovaries at E14.5.

Fig.3-5 Comparison of WT1 and FHL2 expression in mouse ovaries at E14.5. In situ hybridizations are performed with WT1 and FHL2 cRNA probes from mouse ovaries at E14.5. The antisense probes show intensive expression (upper panel) whereas the sense control probes show no signal. Results - 42 -

3.2 Direct interaction between FHL2 and WT1

3.2.1 FHL2 interacts with WT1 in vitro Since FHL2 is co-expressed with WT1 in the developing gonads during a well defined period, pull-down assays were performed to further investigate if the two proteins physically interact. GST-FHL2 fusion protein is immobilized on Glutathione Sepharose beads and incubated with 35S-methionine labeled in vitro translated WT1 protein. Fig.3-6 demonstrates that WT1 binds specifically to GST-FHL2 proteins but fails to bind GST. To address whether FHL2 binds to other transcription factors involed in gonadal differentiation, pull-down experiments are performed with GST-FHL2 fusion proteins and 35S-methionine labeled in vitro translated SF-1, SOX9 and p53. GST-FHL2 fails to bind any of these proteins, thus demonstrating specificity of the association between FHL2 and WT1.

Fig.3-6 FHL2 interacts with WT1 in vitro. GST-pull-down assays are performed with 35S-methionine labeled in vitro translated WT1, SF-1, SOX9 or p53 and E.coli expressed GST-FHL2 protein or GST alone. 10% of the in vitro translated proteins used in pull-downs are loaded in the first lane of each panel as input control.

3.2.2 Mapping of the WT1 interaction domains in FHL2 To map the region of FHL2 involved in WT1 association, FHL2 deletion mutants are generated (Fig.3-7). Deletion of the N-terminal two and half LIM domains results in a construct (GST-FHL2-LIM3-4) that has lost the ability to bind WT1. In contrast the N- terminal LIM domains (GST-FHL2-LIM0-2) interacts with WT1 as stong as full-length GST-FHL2. However, neither the N-terminal half LIM domain LIM0 (GST-FHL2-LIM0), nor the single LIM domain LIM1 (GST-FHL2-LIM1) or LIM2 (GST-FHL2-LIM2) alone are able to interact with WT1, while the mutants GST-FHL2-LIM0-1 or GST-FHL2-LIM1-2 interact with WT1 to a lesser extend (Fig.3-7). Therefore, LIM1 either needs the N-terminal Results - 43 - half LIM domain LIM0 or the adjacent LIM domain LIM2 to form an interaction platform for the association with WT1.

Fig 3-7. Mapping of the WT1 interaction domain in FHL2. Schematic presentation of the FHL2 deletion mutants and pull-down analysis with in vitro translated WT1. GST-FHL2-LIM0-2 interacts with WT1 as strong as full-length GST-FHL2. 10% of the in vitro translated WT1 protein is loaded at the first lane as input control.

3.2.3 Mapping of the FHL2 interaction domain in WT1 To determine the FHL2 interaction domain in WT1, truncated WT1 proteins were generated by deleting both the N- and C-termini. The four zinc finger domain of WT1(298-429) fails to interact with FHL2, while the corresponding N-terminal part of WT1(1-298) still binds to GST-FHL2. Further deletions generating the mutant WT1(1-182) abolished the interaction with FHL2. Thus, the interaction domain is mapped to amino acids 182-298 in WT1. Neither full-length WT1 protein nor the WT1 deletion mutant proteins bind to GST protein. Results - 44 -

Fig.3-8. Mapping of the FHL2 interaction domain in WT1. Schematic structure of the WT1 deletion mutants. GST pull-down experiments were performed with 35S-labeled in vitro translated WT1 proteins and GST-FHL2 fusion protein or GST alone. The interaction domain is mapped to WT1(182-298). 10% of the in vitro translated proteins are loaded in the first lane of each pull-down panel as input control.

3.2.4 FHL2 interacts with WT1 in vivo To investigate whether FHL2 and WT1 interact with each other in vivo, co- immunoprecipitations are performed. 293 cells are cotransfected with WT1 and either Flag- FHL2 or untagged-FHL2 as control. Nuclear extracts are prepared and the expression of both proteins is controled by Western-blotting (Fig.3-9).

Fig.3-9 Expression of WT1 and FHL2 proteins in transfected 293 cells. The Western-blot is performed with the α-WT1 antibody C19 (upper part) and the α-FHL2 antibody 11-134 (lower part). The expression plasmids used for transfections are shown on the top. The specific bands representing WT1, Flag-FHL2 and FHL2 are indicated with arrow heads. Results - 45 -

Immunoprecipitations are performed with the monoclonal α-Flag antibody M5 immobilized with GammaBind sepharose beads. The immuno-complexes are fractioned by 10% SDS polyacrylamide gel electrophoresis. Western-blots are decorated with the monoclonal α-Flag antibody M2 and the α-WT1 antibody C-19. The Western blot analysis show that WT1 is coimmunoprecipitated with Flag-FHL2, demonstrating that FHL2 associates with WT1 in vivo. No WT1 protein is detected in the control immunoprecipitation from nuclear extracts containing WT1 and untagged FHL2. This control demonstrates the specificity of the association between Flag-FHL2 and WT1 in vivo (Fig.3-10). The M5 antibody also detects an unspecific signal in the Western-blot.

Fig.3-10 WT1 coimmunoprecipitates with Flag-FHL2. Nuclear extracts from 293 cells transfected with WT1 and either FHL2 or Flag-FHL2 are immunoprecipitated with the monoclonal α-Flag antibody M5. Western blots are either decorated with the α-WT1 antibody (upper panel) or with the α-Flag antibody (lower panel). 10% of the extract used for immunoprecipitation is loaded as input control in lanes 2 and 4. The IPs are loaded in lanes 3 and 5. No signal is obtained from the nuclear extract containing WT1 and untagged FHL2 protein (lane 3). M5 antibody shows an unspecific signal in the Western-blot (lane 1).

To confirm in vivo interaction between FHL2 and WT1, a yeast two hybrid analysis was performed. Full-length WT1 and the FHL2-interaction domain in WT1(182-298) (see Fig.3-8) are fused to the Gal4-DNA-binding domain (DBD) to generate the bait proteins. FHL2 is fused to the Gal4 activation domain to generate the prey protein (pACT2-FHL2). The two hybrid assay shows that both, the full-length WT1 and the FHL2 interaction domain in WT1(182-298) associate with FHL2 in yeast (Fig.3-11), thus confirming the in vivo interaction between FHL2 and WT1. Results - 46 -

Fig.3-11. WT1 and FHL2 interact in vivo in yeast. WT1 and the FHL2-interaction domain in WT1 (aa 182-298) fused to the Gal4-DBD interact with hFHL2-Gal4-AD in a yeast two hybrid analysis. Yeast cells are plated on -Trp/-Leu media to confirm transformation efficiency and on triple dropout media in the presence of 5 mM 3-Aminotriazole (3- AT) to demonstrate the in vivo interaction. p53/T (T=SV40 large T antigen) is used as a positive control.

3.3 FHL2 regulates genes during gonadal differentiation

3.3.1 FHL2 upregulates the male specific gene MIS via WT1/SF-1 The sex-determining cascade is thought of as a combinatorial network rather than a linear pathway (Koopman, 1999). Several transcription factors such as SF-1, WT1, DAX-1, and SOX9 are involved in the transcriptional regulation of the MIS gene. However, the tissue- specific coactivators that link these factors and the transcription machinery are missing. In this study, it is demonstrated that FHL2 and WT1 show an overlapping expression pattern and that the two proteins interact specifically with each other in vitro and in vivo. Therefore, the next step is to investigate the functional consequence of the physical protein-protein interaction between FHL2 and WT1 at the level of transcriptional regulation. In testis, FHL2 is co-expressed with SF-1, WT1 and MIS. It was demonstrated that SF-1 upregulates the transcription of the MIS gene by binding directly to a conserved upstream regulatory element, MIS-RE1 (Shen et al., 1994). Also, WT1 interacts with SF-1 and synergistically upregulates transcription of the MIS gene (Nachtigal et al., 1998). To determine whether FHL2 has a regulatory function in MIS transcription, a TATA-luciferase- reporter plasmid containing a duplicate MIS-RE1 elements (2×MIS-RE1) is generated and tested in transient transfections in 293 and COS-7 cells. To confirm the WT1/SF-1 synergy on the MIS-RE1, the 2×MIS-RE1-TATA-luciferase reporter is cotransfected with SF-1 and WT1(-/-). The 2×MIS-RE1-TATA-luciferase reporter is activated by SF1 and a synergy Results - 47 - between WT1 (-/-) and SF-1 is observed in both cell lines. Fig.3-12 shows the transfection data in 293 cells.

Fig.3-12 WT1 activates MIS-RE1 dependent reporters by synergizing with SF-1. The 2×MIS-RE1-TATA-luciferase reporter is cotransfected with expression plasmids for SF-1 and WT(-/-) in 293 cells. Luciferase activity is reported as relative light units (RLU). The experiments are repeated at least 5 times.

The 2×MIS-RE1-TATA-luciferase reporter is cotransfected with SF-1, WT1(-/-) and increasing amount of FHL2. With low dose of WT1(-/-) and SF-1, for example, 40ng WT1(- /-) and 50ng SF-1, a further superactivation is obtained when FHL2 is titrated in the range from 50 to 150 ng. The highest fold activation is 7.9 (Fig.3-10). In contrast, FHL2 is neither able to activate the 2×MIS-RE1-TATA-luciferase reporter alone nor in combination with WT1 or SF-1 alone. These results show that the coactivation of the 2×MIS-RE1-TATA- luciferase reporter by FHL2 requires both WT1(-/-) and SF-1. The same results are obtained in both 293 and COS-7 cell lines. Fig.3-13 shows the result in COS-7 cells. Results - 48 -

Fig 3-13. FHL2 coactivates MIS-RE1 reporters via WT1/SF-1. Cos-7 cells are cotransfected with the 2×MIS-RE1-TATA-luciferase reporter and the expression plasmids for SF-1, WT1(-/-) and FHL2. The luciferase activity is shown as relative light units (RLU). FHL2 transactivates the reporter only when cotransfected with WT1(-/-) and SF-1. The experiments are repeated at least five times.

It is notable that at higher concentrations of expres WT1 and SF-1, WT1(-/-) synergizes with SF-1 to potently activate the MIS-RE1 reporter. Under these conditions, FHL2 is unable to further superactivate (Fig.3-14). The same results are observed in both, 293 and COS-7 cell lines. Fig.3-14 shows the result in 293 cells. The data suggest that FHL2 acts as a coactivator of WT1, thereby fine-tuning MIS expression. This fine-tuning seems to be important for the transcriptional activation of MIS gene in testis especially when WT1 and SF-1 amounts are limited. Results - 49 -

Fig.3-14 FHL2 coactivates MIS-RE1 reporters with limited amounts of SF-1 and WT1(-/-). 293 cells are transiently transfected with the 2×MIS-RE1-TATA-luciferase reporter and the expression plasmids coding for SF-1, WT1(-/-) and FHL2. The luciferase activity is reported as relative light units (RLU). FHL2 transactivates 2×MIS-RE-TATA-luciferase reporter only when cotransfected with limited amounts of plasmids coding for WT1 and SF-1. The experiments are repeated at least five times.

3.3.2 Transcriptional upregulation of the 2×MIS-RE1 reporter gene by FHL2 is specific for the WT1(-KTS) forms It has been demonstrated that the WT1(-KTS) forms are involved in transcription regulation, whereas WT1(+KTS) forms associate with splicesome and participate in splicing events (Larsson et al., 1995). To investigate whether WT1(+KTS) is involved in the transcriptional upregulation of MIS-RE1 dependent reporters by FHL2, transfections are performed with plasmids coding for WT1(+KTS), SF-1 and FHL2. The result is shown in Fig.3-15. As expected, neither synergism of SF-1 with WT1 (+KTS) nor superactivation by FHL2 is observed in the transfection.

Fig.3-15. FHL2 coactivation of the 2×MIS-RE1 reporter gene is specific for the WT1(-KTS) form. 293 cells are transfected with the 2×MIS-RE1-TATA-luciferase reporter and expression plasmids for FHL2, SF-1, and WT1(-/-) or WT1(-/+). The luciferase activity is reported as relative light units (RLU). FHL2 transactivates 2×MIS-RE1-TATA-luciferase reporter when cotransfected with SF-1 and WT1(-/-). When cotransfected with SF-1 and WT1(-/+), FHL2 does not show any transactivation. The experiments are repeated at least five times. Results - 50 -

3.3.3 FHL2 regulation of the 2×MIS-RE1-TATA-luciferase reporter gene is specifically dependent on SF-1 SF-1 regulates MIS expression by binding to the MIS-RE1. The MIS-RE1 contains an extended half site, CCAAGGTCA. The orphan nuclear receptor germ cell nuclear factor (GCNF) binds to a similar extended half-site TCAAGGTCA, (Greschik et al., 1999). To investigate whether FHL2 transactivation of the MIS dependent reporter is specific for SF-1, transient transfections are performed with the 2×MIS-RE1-TATA-luciferase reporter and expression plasmids coding for FHL2, WT1 and GCNF. The result is shown in Fig.3-16. FHL2 fails to transactivate the reporter in the presence of WT1 and GCNF. The results demonstrate that FHL2 dependent transactivation specifically requires a WT1/SF-1 complex.

Fig.3-16 FHL2 transactivation is specific for SF-1. 293 cells are transiently transfected with the 2×MIS-RE1-TATA-luciferase reporter and expression plasmids coding for FHL2, WT1(-/-), SF-1 or GCNF. The luciferase activity is reported as relative light units (RLU). The experiments are repeated at least five times.

3.3.4 The SF-1-LBD is required for the transcriptional upregulation of the MIS promoter by FHL2 It was suggested that either a ligand or a cofactor is required for SF-1 to upregulate MIS gene expression (Shen et al., 1994). However, no high affinity ligand for SF-1 has been identified so far (Christenson et al., 1998; Mellon and Bair, 1998) though some oxysterols were shown to stimulate SF-1-dependent transcription (Lala et al., 1997). It was indicated that WT1 associates with the DBD of SF-1 and this association results in a synergistic activation (Nachtigal et al., 1998). The data presented here show that FHL2 interacts directly with WT1 and that the interaction results in a further superactivation of MIS-RE1 dependent reporters Results - 51 - in the presence of SF-1. To investigate whether the LBD of SF-1 is required in this context, two SF-1 mutants are generated. SF-1∆LBD which lacks the LBD and SF-1(DBD)- GCNF(LBD) in which the SF-1-LBD is replaced by the GCNF-LBD. Transient transfections are performed with expression plasmids for FHL2, WT1(-/-), and either SF-1∆LBD or SF- 1(DBD)-GCNF(LBD). The result is shown in Fig.3-17. FHL2 transactivates the 2×MIS- RE1-TATA-luciferase reporter in the presence of WT1(-/-) and wild type SF-1. However, when cotransfected with expression plasmids for WT1 and either SF-1∆LBD or SF-1(DBD)- GCNF(LBD), FHL2 fails to superactivate the 2×MIS-RE1-TATA-luciferase reporter. These results indicate that the SF-1-LBD is required for transactivation by FHL2 though, as shown in Fig. 3-6, no direct contact between FHL2 and SF-1 is detected. Results - 52 -

Fig.3-17. The SF-1-LBD is required for the superactivation by FHL2. (a) 293 cells are transiently transfected with the 2×MIS-RE1-TATA-luciferase reporter and expression plasmids for FHL2, WT1(-/-), SF-1 or SF-1(∆LBD). (b) 293 cells are transiently transfected with the 2×MIS-RE1-TATA-luciferase reporter and expression plasmids for FHL2, WT1(-/-), SF-1 or SF-1(DBD)-GCNF(LBD). The luciferase activity is reported as relative light units. FHL2 transactivates the reporter in a dosage-dependent manner when cotransfected with plasmids coding for WT1(-/-) and SF-1 proteins. The experiments are repeated at least five times.

3.3.5 FHL2 upregulates the female specific gene, DAX-1 FHL2 is co-expressed with WT1 and DAX-1 in the ovary during a period from E13.5 to E16.5. DAX-1 was demonstrated to be a direct target of WT1 (Kim et al., 1999). To determine the regulatory consequence of the physical interaction between FHL2 and WT1 on the DAX-1 promoter, transient transfections are carried out in 293 and COS-7 cells. To confirm the WT1 mediated upregulation of the DAX-1 promoter, a DAX-1-promoter- luciferase reporter is cotransfected with expression plasmid coding for WT1(-/-). WT1(-/-) alone transactivates the DAX-1 promoter in a dosage-dependent manner (Fig 3-18). Then, the DAX-1-promoter-luciferase reporter is cotransfected with expression plasmids for FHL2 and WT1(-/-). Fig.3-18 shows that FHL2 potentiates the activation of WT1(-/-) on the DAX- 1 promoter in a dosage dependent manner.

Fig.3-18. FHL2 potentiates WT1 activation on the DAX-1 promoter. 293 cells are transiently transfected with the DAX-1-promoter-luciferase reporter, and expression plasmids for WT1(-/-) and FHL2. The luciferase activity is reported as relative light units (RLU). The experiments are repeated at least five times. Results - 53 -

3.4 FHL2 and WT1 form a complex on the DAX-1 promoter. WT1 regulates DAX-1 by binding to the two WT1-binding sites (WB1+WB2) in the DAX-1 promoter (Kim et al., 1999) (Fig.3-19).

Fig.3-19. Alignment of the murine (mDAX-1) and human (hDAX-1) promoter sequence. The dots represent regions which are not conserved. The characteristic TATAA box and a putative SF-1-binding site are boxed. Two potential WT1 recognition elements are underlined. The nucleotide numbering is related to the adenosine residue of the ATG initiation codon, which is numbered as +1 (from Kim et al., 1999).

The transfection experiments show that FHL2 can potentiate the activation of the DAX-1 promoter by WT1(-/-). To determine whether this potentiation is due to the formation of a FHL2-WT1 complex on WB1+WB2, EMSAs are performed. Nuclear extracts from 293 cells transiently transfected with either pCMX-3×Flag-WT1(-/-) and FHL2 or WT1 and Flag- FHL2 were used for EMSAs. Expression in the transfected 293 cells is controled by Western-blotting (Fig.3-20). Results - 54 -

Fig.3-20 The expression of FHL2 and WT1 in transfected 293 cells. The Western-blots are performed with nuclear extracts from 293 cells after transfections. The expression plasmids used are indicated on the top. The Western-blots are decorated with α-WT1 antibodies (upper part) and αFHL2 antibodies (lower part), respectively. The specific bands representing WT1 and FHL2 proteins are indicated by arrow heads.

The EMSAs (Fig.3-21) show that the nuclear extracts containing 3×Flag-WT1(-/-) and FHL2 form a DNA-protein complex with the 32P-labeled oligonucleotide WB1+WB2 (lane 1). The formation of the DNA-protein complex is specific since it is competed by an 100-fold excess of unlabeled WB1+WB2 oligonucleotide (Lane 2) but not by addition of an unrelated competitor (lane 3). Addition of monoclonal anti-Flag antibodies (M5) causes a retardation in mobility, resulting in a supershift complex (lane 4). No supershifted complex is observed by the addition of normal mouse serum (lane 5), verifying the presence of Flag- tagged WT1 in the complex. Conversely, DNA-protein complexes form by incubating nuclear extracts containing Flag-FHL2 and WT1(-/-) with the radiolabeled WB1+WB2 probe. The DNA-protein complex (lane 6) is specifically disrupted by a 100-fold molar excess of unlabeled WB1+WB2 oligonucleotide (lane 7), whereas the unrelated competitor fails to do so (lane 8). Addition of the monoclonal anti-Flag antibody M5 to the complex causes an retardated supershift complex (lane 9). No mobility change is observed by addition of unrelated control-serum (lane 10), thus indicating the presence of FHL2 in the DNA- protein complex. Mobility shift assays with nuclear extracts containing both, untagged WT1 and FHL2 show the same specific DNA-protein complex (lane 11) that is competed with an excess of unlabeled probe (lane 12). Since no Flag tagged protein is present, addition of monoclonal M5 antibody fails to retard the migration of the complex (lane 14). Results - 55 -

Fig.3-21. FHL2 and WT1 form a complex on DNA. EMSAs are performed with 32P-labeled WT1-binding sites WB1 and WB2. The nuclear extracts containing 3×Flag-WT1 and FHL2 or WT1 and Flag-FHL2 form a stable DNA-protein complex (lanes 1-5 and 6-10, respectively). The presence of 3×Flag-WT1 and Flag-FHL2 in the complex is demonstrated by a supershift using α-Flag monoclonal antibody M5 (lane 4, 9 and 14), respectively. Competition experiments are performed with a 100-fold molar excess of either unlabeled WB1+WB2 (lanes 2, 7 and 12) or unrelated oligonucleotides (lanes 3, 8 and 13). Nuclear extract containing both, untagged WT1 and FHL2 is used as a control for the M5 supershift (lanes 11-15). Discussion - 56 -

4. Discussion

4.1 FHL2 is co-expressed with WT1 during gonadal differentiation In this study, the expression of FHL2 and its functional role in developing gonads is analyzed. Whole mount in situ hybridization data show that FHL2 starts to be expressed in both, testes and ovaries from E13.5 until E16.5. WT1 transcripts are also detected in both gonads during the same period. FHL2 and WT1 transcripts disappear from the gonads at E17.5. Thus, FHL2 and WT1 show an overlapping expression pattern during a period between E13.5 and E16.5 in testis and ovary. During gonadal differentiation, a cascade of transcription factors show a spatio-temporal expression pattern consistant with their regulatory function. For example, testis differentiaton is initiated by expression of the SRY gene during a short period between E10.5 and E12.5 (Koopman et al., 1990). Once the testicular cords form and produce MIS at E12.5, SRY transcription is turned off (Koopman et al., 1990). The spatio-temporal expression pattern of FHL2 implicates a function in transcriptional regulation of genes involed in gonadal differentiation. In the mouse, MIS starts to be expressed at E12.5 and persists throughout testis differentiation to assure that the Müllerian ducts do not develop into female genitalia. Therefore, the adequate expression of MIS is critical for proper male gonadal differentiation. SF-1 and WT1 have been shown to interact with each other and synergistically upregulate transcription of the MIS gene in Sertoli cells (Nachtigal et al., 1998; Shen et al., 1994). SF-1 is expressed in the urogenital ridges of both sexes at E9 and ceases by E12.5 in females. In males, however, SF-1 expression persists in Sertoli cells (Luo et al., 1994). WT1 is also expressed in the urogenital ridge of male and female mice at E9 and lasts until late stages of embryonic development in both sexes (Kreidberg et al., 1993). However, no coactivators involed in the transcriptional regulation of the MIS gene by WT1 have been described. Therefore, FHL2 is the first coactivator whose spatio-temporal expression pattern overlaps with that of SF1, WT1 and MIS in testis differentiation. In the female, the expression of SF-1 is turned off from E13.5 to E16.5 and reappears at late embryonic stage (E18.5) in the mouse (Ikeda et al., 1994). WT1 expression persists in the ovary until late embryonic stages. DAX-1 is expressed at E10.5 in the genital ridge of both sexes. DAX-1 is strongly downregulated at E12.5 in the testis but is maintained in the ovary. Taken together, FHL2 expression overlaps with the expression of WT1 and DAX-1 in ovarian differentiation, suggesting an additional regulatory interaction in the ovary. Since the transcripts of FHL2 disappear at E17.5 (data not shown) and reappear in adult ovary (Fimia et al., 2000), this may suggest additional physiological function in adult ovary. Discussion - 57 -

4.2 FHL2 interacts with WT1 in vitro and in vivo The physical interaction of FHL2 and WT1 is revealed by the in vitro GST pulldown experiments. The data show that FHL2 interacts specifically with WT1 but not with SF-1, SOX9, or p53. The in vivo interaction between FHL2 and WT1 is demonstrated in a yeast two hybrid assay and is further confirmed by coimmunoprecipitation. Therefore, FHL2 is identified as a novel WT1-binding protein. The WT1 interaction domain in FHL2 is mapped to the LIM domains LIM0-2. In the FHL2 deletion mutants FHL2 LIM0-1 and FHL2 LIM1- 2 interaction is decreased but not abolish, while the N-terminal half LIM domain LIM0, the single LIM domains LIM1 or LIM2 have lost the ability to interact with WT1. These results demonstrate that LIM1 either needs the N-terminal half LIM domain LIM0 or the LIM2 domain to form a plateform that suffices the interaction with WT1. Since LIM domains are thought to function as protein interaction modules, FHL proteins interact with numerous proteins by different combination of the LIM domains. LIM domains fold independently and are held together by a linker region (Dawid, 1998; Konrat et al., 1998). A single LIM domain, or even a single zinc finger module of a LIM domain, can function as a protein-binding interface so that a single LIM domain could be functionally bipartite (Schmeichel and Beckerle, 1994a). Thus, FHL proteins can be involved in many different interactions due to its modular structure. Recent studies have identified several FHL2 interacting proteins such as androgen receptor (AR), hCDC47 or FHL3 (Müller et al., 2000; Chan et al., 2000; Fimia et al., 2000). It was reported that FHL2 interacts with AR or hCDC47 through different domains. The interaction with AR requires both the N- and C- terminal domains of FHL2, whereas the interaction with hCDC47 involves the LIM domains 2 and 3 together with the first half LIM domain. The LIM domain 2 of FHL3 was shown to be required for the interaction with FHL2 (Li et al., 2001). Taken together, the data suggest that FHL2 could be involved in the organization or the regulation of very diverse multiprotein complexes. Table 3-1 summarizes the interaction partners of FHL2.

Table.3-1 FHL2 interaction partners Interaction partners Interacting LIM domains in References FHL2 Androgen receptor N- and C-terminal domains Müller et al., 2000 hCDC47 LIM0 and LIM2-3 Chan et al., 2000 CREM/CREB Unknown Fimia et al., 2000 FHL3 Unknown Li et al., 2001 PSF Unknown Dye and Patton, 2001 WT1 LIM0-2 present study Discussion - 58 -

In this study, the FHL2 interaction domain in WT1 is mapped to WT1 amino acids 182-298. WT1 is a zinc-finger containing transcription factor that functions both, as activator and repressor depending on the cell type and promoter architecture. The activation domain in WT1 was mapped to residues 181-250 (Wang et al., 1993; Wang et al., 1995), the repressor domain was mapped to residues 85-124 (Madden et al., 1993; Wang et al., 1993). WT1 can also self-associate and form homodimers via a dimerization domain which is located in amino acids 1-182 (Reddy et al., 1995). Recent studies have shown that WT1 interacts with a number of proteins. The zinc fingers of WT1 have been shown to interact with several proteins such as the tumor suppressor p53 (Maheswaran et al., 1993) or the prostate apoptosis factor par4 (Johnstone et al., 1996). Several proteins have been identified that also interact with the N-terminus of WT1, including Ciao 1, a novel member of the WD40 protein family (Johnstone et al., 1998) and the chaperone Hsp70 (Maheswaran et al., 1998). Taken together, the present study shows that FHL2 is the first coactivator that interacts with the WT1 activation domain. Table 3-2 summarizes the WT1 interacting proteins.

Table. 3-2 Summary of WT1 interacting proteins and the interaction domains in WT1 Partner proteins Site of expression Interaction References domain in WT1 Splicing machinery Ubiquitous Unknown Larsson et al., 1992 components p53 Ubiquitously induced Zinc fingers Maheswaran et al., in response to DNA 1 and 2 1993 damage WT1 Developing N-terminus, Reddy et al., 1995; urogenital system amino acids Englert et al., 1995; 1-45 and 157-253 Holmes et al., 1997 UBC9 Ubiquitous Zinc fingers Wang et al., 1996 par-4 Ubiquitous; Zinc fingers Johnstone et al., 1996 high in postnatal testis and ovary Ciao1 Ubiquitous N-terminus Johnstone et al., 1998 Hsp70 Many sites, N-terminus Maheswaran et al., including podocates 1998 SF-1 Sertoli cells N-terminus Nachtigal et al., 1998 WTAP Ubiquitous Unknown Little et al., 2000 CBP/p300 Ubiquitous First two zinc Wang et al., 2001 fingers FHL2 Developing testis and amino acids Present study ovary 182-298 Discussion - 59 -

4.3 FHL2 is a coactivator in the transcriptional activation of the MIS gene The functional consequence of the physical interaction between FHL2 and WT1 is demonstrated in transient transfections. The results show that FHL2 superactivates the induction of MIS-RE1 dependent reporter genes by WT1/SF-1 in a dose-dependent manner. To address whether the superactivation by FHL2 is dependent on SF-1, the orphan nuclear receptor GCNF was tested. GCNF was shown to bind to the same binding site as SF-1 (Greschik et al., 1999). However, GCNF can not replace SF-1 in cotransfection experiments, demonstrating that the superactivation by FHL2 is dependent on the presence of SF-1. Recent data suggested that a ligand or coactivator is required for SF-1 function. No high affinity ligand for SF-1 has been identified so far (Christenson et al., 1998; Mellon and Bair, 1998) though some oxysterols were shown to stimulate SF-1-dependent transcription (Lala et al., 1997). To determin the necessity of the SF-1-LBD in the superactivation by FHL2, transfection is carried out with the SF-1 mutant SF-1∆LBD. The transfection data show that FHL2 does not show any superactivation when coexpressed with WT1 and SF-1∆LBD. Thus the SF-1-LBD is required for the transcriptional coactivation of the MIS gene by FHL2. Since the association of FHL2 and SF-1 is ruled out by GST pull-down experiment, the exact mechanism for this SF-1 LBD dependency is not known at present. A postulated regulation mechanism is illustrated in Fig.4-1. Discussion - 60 -

Fig.4-1 The upper panel compares the sequences of the MIS proximal promoter regions from human, mouse, rat, and bovine (from De santa barbara et al., 1998). The characteristic TATA box, the SF-1-binding site (SF-1-BS), the SOX-binding site (SOX-BS), and a putative GATA site (GATA) that are conserved among the different species are indicated. Numbers below the sequences correspond to the human gene. The lower panel shows a schematic presentation illustrating the putative transcriptional regulation of the MIS promoter. The MIS-RE1 sequence is presented below. SF-1 binds to the extended consensus half site, CCAAGGTCA on the MIS-RE1. The direct interaction between WT1 and SF-1 needs the DNA-binding domain (DBD) of SF-1. WT1 synergizes with SF-1 by binding to the sequence CCCCCAAGGTCA (Nachtigal et al., 1998). DAX-1 antagonizes the synergy of WT1 and SF-1 by interacting with SF-1 (Nachtigal et al., 1998). SF-1 activates transcription by associating with coactivators such as SRC-1 and CBP (Ito et al., 1998; Monte et al., 1998). FHL2 interacts physically with WT1.

SRY is not required for the regulation of the MIS gene During sex development, one central event of the male sex-determination pathway is the active regression of Müllerian ducts in the male, due to the production of MIS in Sertoli cells. Since XY mice that lack MIS develop as pseudohermaphrodities (Behringer et al., 1994), and addition of exogenous MIS results in partial sex reversion in fetal XX rats (Vigier et al., 1987), the regulation of MIS expression is of major importance in gonadal differentiation. Molecular studies have been performed by several groups to reconstruct the pathway of primary male sex determination which is initiated by SRY and terminated by MIS secretion. Since SRY initiates the determination of Sertoli cells, and determined Sertoli cell express MIS, one attractive hypothesis was that the SRY gene product, a high mobility group box containing transcription factor directly controls MIS expression (Haqq Discussion - 61 - et al., 1993 and 1994; Harley and Goodfellow, 1994). However, the time lag between SRY and MIS expression (Hacker et al., 1995) and the absence of any transactivation domain in the SRY protein (Dubin and Ostrer, 1994; Poulat et al., 1997) have led many investigators to refuse this hypothesis (Shen et al., 1994; Greenfield and Koopman, 1996; Giuili et al., 1997). It has been suggested that other regulatory genes that are located downstream of SRY have to fulfil this role. Deletion analysis of the MIS promoter has led to the identification of a 180-bp segment required for correct MIS expression in primary Sertoli cells (Shen et al., 1994). Analysis of this region in humans, bovines, mice and rats (Shen et al., 1994) indicate that it contains at least two highly conserved sequence elements and a characteristic TATA box (see Fig.4-1). The proximal element which includes an extended consensus half site, is known to interact with SF-1. The distal element was shown to interact with SOX9.

Transcriptional upregulation of the MIS gene by SF-1 in concert with SOX9, GATA- 4 and WT1 The existence of a SOX-binding element in the 180-bp minimal MIS promoter led to the identification of SOX9 as a regulator of MIS gene expression (De Santa Barbara et al., 1998). SOX9 was initially identified by positional cloning as a gene that associates with the skeletal malformation syndrome campomelic dysplasia, in which two-thirds of XY individuals show sex reversal (Foster et al., 1994; Wagner et al., 1994). SOX9 was shown to be expressed concomitant with or shortly after SRY, and its upregulation proceded the onset of MIS expression in mice. One characteristics of SOX proteins is the ability to bend DNA by angles of 60-85¡ (Giese et al., 1992). The closely spaced SOX- and SF-1-binding sites in the proximal MIS promoter (Fig.4-1), along with the ability of SOX proteins to bend DNA, raised the possibility that SOX9 and SF-1 interact with each other. It has been demonstrated that SOX9 interacts with SF-1 in vitro and in vivo. The strongest interaction occurs between the N-terminal region of SOX9 and the C-terminal region of SF-1. It was demonstrated that SOX9 and SF-1 activate MIS expression additively. The potentiation of SF-1 activity by SOX9 requires both the SOX9 transactivation domain and the SF-1 ligand-binding domain (De Santa Barbara et al., 1998). In addition, SF-1 is able to interact with various other transcription factors such as DAX-1, WT1 and GATA-4 (De Santa Barbara et al., 1998; de Santa Barbara et al., 2000; Halvorson et al., 1998; Ito et al., 2000; Ito et al., 1997; Nachtigal et al., 1998). SF-1 associates with WT1 through the DBD of SF-1 and the association results in an synergistic activation of the MIS gene. It is known that SF-1 regulates gene transcription through its interactions with nuclear receptor coactivators such as steoid receptor coactivator-1 (SRC- 1) and CBP/p300 (Ito et al., 1998; Monte et al., 1998). DAX-1 antagonizes the synergistic activation of SF-1 with WT1 by interacting directly with the SF-1 ligand-binding domain (Nachtigal et al., 1998). DAX-1 was reported to act as a repressor for SF-1 mediated Discussion - 62 - transcriptional activation by the following three mechanisms (Fig.4-2): 1) DAX-1 forms heterodimer with SF-1 (Ito et al., 1997). 2) DAX-1 recruits N-CoR to SF-1-responsive promoters (Crawford et al., 1998). 3) DAX-1 interacts with hairpin loops in SF-1- responsive promoters to interfere with SF-1-dependent transcriptional activation (Parker et al., 1999).

Fig.4-2. DAX-1 mediated repression of SF-1-responsive promoters (from Parker et al., 1999).

In conclusion, proper spatio-temporal expression of the MIS gene requires the concerted action of several transcription factors that include SF-1, WT1, SOX9, GATA-4 and DAX-1.

Transcription coactivation of the MIS gene by FHL2 This study shows that SF-1 and WT1 synergize to activate a 2×MIS-RE1-TATA-luciferase reporter (Fig.3-9). The data further reveal that FHL2 potentiates the synergistic activation of 2×MIS-RE1-TATA-luciferase reporter by WT1/SF-1 only at limiting amounts of SF-1 and WT1 (Fig.3-11). At high concentrations of SF-1 and WT1 proteins, FHL2 fails to superactivate the 2×MIS-RE1-TATA-luciferase reporter. This data suggest that FHL2 functions as a coactivator in the upregulation of the MIS gene especially when the protein concentrations of SF-1 and WT1 are not sufficient for maintaining adequate transcription levels of the MIS gene. Therefore, FHL2 may function as a WT1 coactivator in the fine- tuning of gonadal gene regulation. Interestingly, the FHL family members FHL3 and ACT are also expressed in the ovary. Furthermore, the three proteins interact with CREB (Fimia et al., 2000). Their structural and functional similarities may allow them to play a Discussion - 63 - compensatory role in gonadal differentiation if the function of one member is impaired. This may explain why FHL2 null mice do not show any phenotype (Chu et al., 2000). Most coactivators constitute HAT activity. However, no HAT activity for FHL members has been described so far. In pull-down assays, FHL2 interacts directly with HAT activity containing coactivator such as TIF2 (our unpublished data). Thus, FHL2 may recruit TIF2 and the TIF2 associated HAT activity in a specific FHL2/TIF2 conatining coactivator complex necessary for the regulation of the MIS gene during gonadal development (Fig.4-1).

4.4 FHL2 acts as a coactivator in the transcriptional upregulation of the DAX-1 gene by WT1 The data of this study show that FHL2 potentiates the activation of the DAX-1 promoter by WT1. This potentiation is SF-1 independent. This result is consistant with the co- expression pattern of FHL2 with WT1 and DAX-1 in the developing ovaries. It has been proposed that DAX-1 is required for ovarian, but not testicular, development since mice with a XY karyotype and a Dax-1 deletion develop as males (Bardoni et al., 1994). In the mouse, DAX-1 expression starts at the same time as Sry expression in the genital ridge of both, XX and XY embryos. As testis development proceeds DAX-1 is rapidly downregulated, whereas it persists throughout ovary development. Analysis of the DAX-1 promoter reveals two WT1-binding sites WB1 and WB2 (Kim et al., 1999) WT1 and DAX-1 show an overlapping expression pattern during gonadal differentiation and WT1 expression precedes that of DAX-1. Furthermore, WT1 can activate the DAX-1 promoter by binding to two WT1 responsive elements, thus demonstrating that DAX-1 is an immediate target of WT1 (Kim et al., 1999). It was also shown that DAX-1 and SF-1 transcripts colocalize during mouse embryonic development in the urogenital ridge. The slightly earlier onset of SF-1 expression and its ability to bind specifically to a conserved sequence in the DAX-1 5'-flanking region suggested that SF-1 may also activate DAX-1 expression. However, Dax-1 expression persists in the gonad and hypothalamus of SF-1 disrupted mice, establishing that SF-1 is not required for Dax-1 gene expression. This study shows that FHL2 and WT1 exert an overlapping expression pattern in the developing ovary. Furthermore, the two proteins interact with each other physically. The functional consequence of this interaction is demonstrated by a potent coactivation of WT1 dependent genes such as DAX-1. Taken together, the data suggest that FHL2 functions as a coactivator of WT1 during ovarian differentiation. Discussion - 64 -

4.5 FHL2 fine-tunes gene regulation during gonadal differentiation. Here, I present FHL2 as a coactivator of WT1 that upregulates MIS and DAX-1 gene expression in both male and female sexual differentiation (Fig.4-3).

Fig.4-3 Schematic representation of the transcriptional regulation of gene products involved in gonadal differentiation. MIS is a crucial factor to maintain proper male sexual differentiation. SF-1 activate transcription by binding to the MIS promoter. Alternatively spliced WT1 forms play different roles. A constant ratio between WT1(+KTS) and WT1(-KTS) is needed to maintain its function. WT1 (-KTS) forms interact and synergize with SF-1 to upregulate transcription of the MIS gene where as WT1(+KTS) forms participate in the post transcriptional gene regulation. DAX-1 is required in female sexual development and antagonizes the transcriptional activation of the MIS gene by SF-1. FHL2 functions as a coactivator to superactivate the transcriptional upregulation of both, the MIS gene in testis and the DAX-1 gene in ovary.

As discussed above, in the male, SF-1 is a main regulator for the transcriptioal upregulation of the MIS gene. WT1 is an important synergistic partner of SF1 in this process. FHL2 functions as a WT1 coactivator to superactivate MIS gene expression. In the female, on the other hand, DAX-1 functions as a suppressor of SF-1 to repress the transcription of the MIS gene. WT1 upregulates DAX-1 expression by directly binding to the DAX-1 promoter (Kim et al., 1999). In the present study, I show that FHL2 interacts specifically with WT1 and that this interaction potentiates the transcriptional activation of the DAX-1 gene. In summary, my data demonstrate that FHL2 acts in both sexes as a WT1 coactivator in the fine-tuning of developmental regulated gonadal genes. References - 65 -

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I hereby declare that this is an original work performed by myself and this work has not been submitted elsewhere as a thesis.

Xiaojuan Du