THE DOUBLESEX TRANSCRIPTION FACTOR: STRUCTURAL AND FUNCTIONAL STUDIES OF A SEX- DETERMING FACTOR by JAMES ROBERT BAYRER Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. Michael A. Weiss Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY January, 2006 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents List of Tables iii List of Figures iv Acknowledgements vii List of Abbreviations viii Abstract xi Chapter I 1 "Introduction, review of the literature, and statement of purpose" Chapter II 60 "Expression, crystallization and preliminary X-Ray and NMR characterization of the Drosophila transcription factor Doublesex" Chapter III 83 "Dimerization of Doublesex is mediated by a cryptic UBA Domain: Implications for sex- specific gene regulation" Chapter IV 126 "Sex-specific gene regulation: Intersexual Drosophila development due to misfolding of a novel UBA domain" Chapter V 163 "Hydrogen exchange reveals a stabile dimeric core of Doublesex CTD" Chapter VI 190 "Summary and future directions" Appendix I 226 "Residue environments" i Appendix II 254 "Three helix domains" Appendix III 271 "Doublesex CTD sequence homologs" Appendix IV 272 "UBA Domains and electrostatics" Appendix V 275 “Expression and purification of intact Doublesex” Appendix VI 277 "Expression and purification of intact Intersex" Appendix VII 281 "Characterization of the putative Intersex-binding groove" Appendix VIII 292 "Yeast One-hybrid control studies" Appendix IX 296 "Residual energy in dimeric systems" Appendix X 297 "Salt bridge and hydrogen bond interactions in the Doublesex CTD" Appendix XI 301 "Dimerization-coupled folding of a sex-specific UBA domain in a transcription factor" Bibliography 315 ii List of Tables Table I-1 DNA binding and oligomerization properties of Doublesex 37 Table II-1 X-ray data-collection and analysis statistics 71 Table II-2 Triple-resonance experiments 72 Table III-1 X-ray data collection and refinement statistics 98 Table III-2 Model-building and refinement statistics 99 Table III-3 Yeast two-hybrid analyses 100 Table V-1 Hydrogen exchange parameters for native CTDF-p 179 Table V-2 Hydrogen exchange parameters for G398A CTDF-p 180 iii List of Figures Figure I-1 Sex determining hierarchy in Drosophila melanogaster 38 Figure I-2 DSXF homologs 40 Figure I-3 Larval segmentation and imaginal disc location 42 Figure I-4 Segmental origins and dimorphic features of the genital disc 44 Figure I-5 Dsx alternative mRNA splicing and protein organization 46 Figure I-6 Regulation of sxl transcripts 48 Figure I-7 Organization of the yp fbe region and co-regulator binding sites 50 Figure I-8 DM GMSA of the dsxA binding site 52 Figure I-9 DM domain (residues 41-81) solution structure 54 Figure I-10 Courtship ritual of Drosophila melanogaster 56 Figure I-11 Common α-helical dimerization motifs 58 Figure II-1 Crystals of CTDF-p 73 Figure II-2 X-ray diffraction pattern of native CTDF-p 75 Figure II-3 Harker section from anomalous scattering Patterson map 77 Figure II-4 1D 1H NMR spectra of native CTDF and 398A CTDF-p 79 Figure II-5 1H-15N HSQC spectra of CTDF and CTDF-p 81 Figure III-1 Sexual differentiation cascade in D. melanogaster and domain organization of DSX 100 Figure III-2 Sequence alignment of DSX homologs in insect alleles 102 Figure III-3 Comparison of CTDF-p secondary structure with that predicted previously 104 Figure III-4 Structure of CTDF-p 106 iv Figure III-5 Unusual structure and packing of bent helix α2 108 Figure III-6 Alignment of CTDF-p and Classical UBA Domains 110 Figure III-7 Stereo diagrams salt bridge interactions in CTDF-p 112 Figure III-8 Stereo view of protomeric UBA mini-core 114 Figure III-9 Hydrophobic core and key dimer interface residues 116 Figure III-10 Dimerization interface and potential Ub-binding surface 118 Figure III-11 Surface representation of CTDF-p dimer color-coded according to extent of sequence conservation among insect dsx alleles 120 Figure III-12 Ribbon model depicting the environments ofL373, M377, and I395 122 Figure III-13 Ribbon stereo comparison of CUE and DSX CTDF-p dimerization motifs 124 Figure IV-1 Sexual differentiation cascade in D. melanogaster and domain organization of DSX 139 Figure IV-2 Structure of CTDF-p 141 Figure IV-3 Biochemical and Cell-Based Studies of CTDF 143 Figure IV-4 Serial Dilution of CTDF-p 145 Figure IV-5 Schematic of yeast one-hybrid system 147 Figure IV-6 CD Studies 149 Figure IV-7 Comparison of of 1H-15N HSQC spectra of CTDF-p and CTDF 151 Figure IV-8 The distal portion of the female-specific tail of DSXF is flexible in solution 153 Figure IV-9 NMR Studies of native CTDF-p and G398A substitution 155 v Figure IV-10 Sequence Conservation of female and male CTD sequences 157 Figure IV-11 Surface representation of CTDF-p dimer color-coded according to extent of sequence conservation among insect dsx alleles 159 Figure IV-12 Environment of G398 and G398D mutant side chain in hypothetical isolated CTDF-p protomer 161 Figure V-1 Thermal denaturation of native CTDF-p monitored by CD 180 Figure V-2 Protection factors for slowly exchanging amides for native and 398A substitution 182 Figure V-3 Stereo diagram of Group I residues 184 Figure V-4 Stereo diagram of Group II and III residues 186 F Figure V-5 CD-detected guanidine hydrochloride titration of native CTD -p in D2O at 30 °C 188 Figure VI-1 Schematic of putative DSX binding sites in promoter regions of suspected DSX-regulated genes 212 Figure VI-2 Crystal contacts suggest potential tetramer surfaces 214 Figure VI-3 Fluorescence studies of the CTDF-p and ubiquitin 216 Figure VI-4 NMR footprinting studies of the CTDF-p and Ub 218 Figure VI-5 Reverse footprint of 15N-Ub with unlabeled CTDF-p 220 Figure VI-6 Model of di-Ub binding a UBA monomer 222 Figure VI-7 Organization of a sex-specific transcription complex 224 vi Acknowledgements I would first like to thank my committee for their support and assistance throughout my studies at Case: Tony Berdis, Mike Weiss, John Mieyal, George Dubyak, and Peter Harte. Within the Weiss Lab, Nelson Phillips imparted invaluable hands-on education and support. Dr. Wan and Narendra Narayana were great company on our long synchrotron trips and always willing to assist with crystallography. Dr. Li has wonderful, unbridled support and enthusiasm for science and for all students. Rupi Singh has provided great personal and professional support, and given me confidence for my next phase of training. Dr. Hua and especially Yanwu Yang have been very kind in help with set-up and interpretation of our NMR data. I especially want to thank Wei Zhang, my partner-in-crime and friend during this thesis work. I am very grateful to my advisor, Mike Weiss, who has served as my mentor in personal and professional growth. His scientific rigor and expectations serve as a constant challenge to better myself, and have instilled a spirit of independence and continual learning that will serve me throughout the rest of my career. Finally, I would like to thank my wife and my family for their love and support throughout my training. I’ve had some great days and more than a few truly lousy ones at the bench, but their constant love and encouragement kept me going. Thank you! vii List of Abbreviations CCD, charge coupled device CD, circular dichroism CTD, C-terminal domain CUE, coupling of ubiquitin to ER degredation DSX, Doublesex fbe, fat body enhancer FRU, Fruitless HPLC, high-performance liquid chromatography HSQC, heteronuclear single-quantum coherence HX, hydrogen exchange IPTG, isopropyl-β-D-thiogalactoside IX, intersex, Ub, ubiquitin NMR, Nuclear Magnetic Resonance SAD, single wavelength anomalous dispersion/diffraction SEC, size-exclusion chromatography SeMet, selenomethionine SXL, Sex Lethal TRA, Transformer Ub, ubiquitin UBA, ubiquitin-associated domain Y1H, yeast one-hybrid Y2H, yeast two-hybrid yp, yolk protein viii THE DOUBLESEX TRANSCRIPTION FACTOR: STRUCTURAL AND FUNCTIONAL STUDIES OF A SEX-DETERMINING FACTOR Abstract by JAMES ROBERT BAYRER Doublesex (DSX) is a transcription factor responsible for the regulation of sexual differentiation in Drosophila. Alternate splicing gives rise to male- and female-specific isoforms. A potent modulator of the yolk protein gene (yp), the male isoform (DSXM) represses transcription of yp whereas the female isoform (DSXF) is an activator. DSX contains two recognized domains, an N-terminal DNA-binding domain (shared between isoforms) and a C-terminal domain (CTD) that is responsible for oligomerization and presumably transcriptional regulation. The CTDs contain sex-specific C-terminal sequences with opposite gene-regulatory properties. We have solved the crystal structure of the core CTD dimerization domain to a resolution of 1.6 Å using single-wavelength anomalous dispersion (SAD) phasing. The crystal structure reveals a novel dimeric arrangement of ubiquitin-associated (UBA) folds. To our knowledge this is its first report in a transcription factor, and the first structure of a dimeric UBA domain. Dimerization is mediated by a non-canonical hydrophobic interface extrinsic to the putative Ub-binding surface. The unexpected observation of a UBA fold in DSX extends the repertoire of α-helical dimerization elements in transcription factors. ix Intersexual development of XX Drosophila (karyotypic females) is associated with mutation G398D, encoded in female-specific exon 4. G398 lies within CTD.