MCELROY-DISSERTATION-2016.Pdf (6.703Mb)

Balancing transcriptional activity in Drosophila through protein- protein interactions on chromatin The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation McElroy, Kyle A. 2016. Balancing transcriptional activity in Drosophila through protein-protein interactions on chromatin. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493492 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Balancing transcriptional activity in Drosophila through protein-protein interactions on chromatin A dissertation presented by Kyle McElroy to The Department of Molecular and Cellular Biology in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Biology Harvard University Cambridge, Massachusetts April 2016 ©2016 Kyle Andrew McElroy All rights reserved. ii Dissertation Advisor: Professor Mitzi I Kuroda Kyle McElroy Balancing transcriptional activity in Drosophila through protein-protein interactions on chromatin Abstract Chromatin plays a vital role in the implementation of gene expression programs. Several disparate groups of regulatory proteins alter chromatin state through post-translational modification of histone proteins, nucleosome remodeling, and higher order chromatin structure in order to affect gene expression. Several of these key groups, such as the Male-Specific Lethal complex and Polycomb Group have been well characterized in Drosophila. Yet aspects of their biology at the molecular level, such as the means by which they are faithfully targeted to regulated loci throughout the genome and the molecular mechanisms they employ to alter transcriptional state, still remain unexplained. In this dissertation I explore how identifying protein-protein interactions on chromatin reveals insights into these unanswered questions critical to chromatin biology. My results highlight the importance of balancing active and repressive chromatin states for the proper maintenance of gene expression. The Male-Specific Lethal complex is the dosage compensation complex in Drosophila, which upregulates gene expression on the male X chromosome approximately two-fold. The MSL complex catalyzes an acetyl mark which may create a uniquely permissive chromatin state to promote transcriptional elongation. A proteomic screen for MSL-interacting proteins identified UpSET, the Drosophila homolog of yeast SET3 and mammalian MLL5. Interestingly, SET3 and UpSET have been characterized to assemble into histone deacetylase complexes. I employed genetic, genomic, and proteomic techniques to assess whether UpSET plays a role in dosage compensation. UpSET appears to play a role in limiting the level of activation of the MSL complex. Surprisingly, UpSET appears to play a more important role in the maintenance of heterochromatin. iii The Polycomb Group is comprised of a well characterized set of developmental repressors. The PcG assembles into several multiprotein complexes to maintain the repressed state. The PcG is opposed by a group of activators known as the Trithorax group. Although the PcG and TrxG often appear to be recruited to the same genomic elements in different tissues, whether they might interact directly was not known. In a collaboration with Dr. Hyuckjoon Kang, I characterized the TrxG protein Female sterile (1) homeotic and found that it interacts specifically with PRC1. The data support a model that bivalency, a poised state observed in mammalian stem cells, may be critical, perhaps transiently, in the developing Drosophila embryo. The mechanism of coordination amongst the various PcG complexes on chromatin is not well understood. We also identified the Sex comb on midleg protein, a known member of the PcG, as a potential physical bridge between PRC1 and PRC2. In these sets of experiments, I have characterized instances of crosstalk between activating and repressing regulators which are critical for the proper maintenance of chromatin state. Perturbations of these interactions may lead to an imbalance of regulators on chromatin and aberrant transcriptional activity. These findings highlight the need for tuning gene expression state and suggest chromatin- based mechanisms by which this can be accomplished. iv Table of Contents Abstract……………………………………………………………………………………………………………………………………………….…iii Table of Contents………………………………………………………………………………………………………………………………….…v List of Figures…………………………………………………………………………………………………………………………………………vii List of Tables……………………………………………………………………………………………………………………………………………x Acknowledgements…………………………………………………………………………………………………………………………………xi Chapter 1. Introduction…………………………………………….………………………………………………………………..………1 On the importance of chromatin…..………………………………………………………………………………………......3 Chromatin-based modulation of gene expression….…..………………………………………………………………4 Transcription and Chromatin….…..……………………………………………………………………………………………..5 Chromatin state and the histone code….…..……………………………………………………………………………..10 The Male-specific Lethal Complex in Drosophila……..……………………………………………………………….12 The Polycomb Group…..……………………………………………………………………………………………………………17 The Trithorax Group……..………………………………………………………………………………………………………….22 Constitutive Heterochromatin and HP1……………………………………………………………………………………26 Summary and Overview of the Dissertation……………………………………………………………………………..30 References……………………………………………………………………………………………………………………………….32 Chapter 2. Differential effects of the loss of UpSET in heterochromatin and the dosage-compensated X chromosome in Drosophila…………………………………………………….…………………………………………………………40 Abstract……………………………………………………………………………………………………………………………………42 Introduction……………………………………………………………………………………………………………………………..43 Results……………………………………………………………………………………………………………………………………..45 Discussion………………………………………………………………………………………………………………………………..74 Materials and Methods……………………………………………………………………………………………………………79 References……………………………………………………………………………………………………………………………….86 Chapter 3. Characterization of PRC1 interactors reveals functional link to PRC2 or TrxG proteins………90 Abstract…………………………………………………………………………………………………………………………………….92 Introduction………………………………………………………………………………………………………………………………93 Results……………………………………………………………………………………………………………………………………...97 Discussion……………………………………………………………………………………………………………………………….129 Materials and Methods…………………………………………………………………………………………………………..133 References…………………………………………………………………………………………………………………………..….138 Chapter 4. Summary and Perspectives………………………………………………………………………………………….…141 References……………………………………………………………………………………………………………………………..149 Appendix 1. Follow-up Studies on the kinase Jil-1……………………………………………………………………………151 Abstract………………………………………………………………………………………………………………………………….153 Introduction……………………………………………………………………………………………………………………………154 v Results and Discussion………………………………………………………………………………………………………….158 Materials and Methods…………………………………………………………………………………………………………182 References……………………………………………………………………………………………………………………………184 Supplementary Information……………………………………………………………………………………………………………187 vi List of Figures Chapter 1. Introduction…………………………………………………………………………………………………………………………..1 Figure 1-1: The classical view of euchromatin vs heterochromatin…………………………………………….6 Figure 1-2: The Male-Specific Lethal Complex is the Dosage Compensation Complex for Drosophila……………………………………………………………………………………………………………………13 Figure 1-3: The PcG forms multiprotein complexes that maintain gene repression………………….19 Figure 1-4: The TrxG forms multiprotein complexes that maintain gene activation…………….……24 Figure 1-5: Heterochromatin formation is mediated by Su(var)3-9 and HP1……………………….……28 Chapter 2. Differential effects of the loss of UpSET in heterochromatin and the dosage-compensated X chromosome in Drosophila…………………………………………………………………………………………………………………..40 Figure 2-1: upSET is an essential gene in Drosophila………………………………………………………………..46 Figure 2-2: UpSET-BioTAP localizes to active TSS in embryos…………………………………………………..49 Figure 2-3: UpSET-BioTAP ChIP peaks are enriched for active TSS and transcriptional elongation chromatin signatures…………………………………………………………………………………………………51 Figure 2-4: UpSET-BioTAP ChIP-seq data correlates most strongly with modEncode ChIP-chip datasets for PolII……………………………………………………………………………………………………….53 Figure 2-5: UpSET-BioTAP shows differential enrichment by chromosome arm…………………….55 Figure 2-6: Generating upSET mutant S2 lines……………………………………………………………………….58 Figure 2-7: upSET mutant cells have broader H4K16ac peaks on the X chromosome…………….61 Figure 2-8: Analysis of histone post-translational modifications from bulk histones in upSET mutant cells………………………………………………………………………………………………………………64 Figure 2-9: H3K9me2 depletion following upSET mutation…………………………………………………….67 vii Figure 2-10: Nascent-RNA-seq reveals misregulation of transcription in upSET mutants……….70 Figure 2-11: Increased transcription of heterochromatin and X-linked genes in upSET mutants…………………………………………………………………………………………………………………….72 Figure 2-12: UpSET: a protein with diverse repressive responsibilities…………………………………..75 Chapter 3. Characterization of PRC1 interactors reveals functional link to PRC2 or TrxG proteins…………90 Figure 3-1: Pc and E(z) BioTAP suggest PRC1 and PRC2 are largely distinct and reveal interesting novel interactions…………………………………………………………………………………………………….….94

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