Investigating the roles of H2A.Z in transcriptional regulation and nucleosomal organization through its relationships with other chromatin proteins Öffentlichkeit
Torres, Erica (Spring 2018)
Abstract
The well-organized chromatin structure of DNA packaged into nucleosomes can determine how genetic information encoded by DNA is used in different conditions. Factors contributing to DNA accessibility include the activity of chromatin remodeling complexes and exchanging histones for variant histones within nucleosomes. The histone variant H2A.Z acts to promote or repress transcription by either stabilizing or destabilizing nucleosome structure, but the underlying mechanisms are unclear. Therefore, much work is needed to understand the context that makes H2A.Z incorporation into chromatin necessary for transcriptional regulation.
Previous work in Arabidopsis thaliana showed that the repressive function of the BRM ATPase of the SWI2/SNF2 complex makes the transcriptional activating function of H2A.Z necessary for transcription of the developmental switch gene Flowering Locus C (FLC). Thus, I performed chromatin and transcriptional profiling in Arabidopsis plants to evaluate the genetic interaction between BRM and H2A.Z at target genes. I found 8 classes of genes involved in transcriptional regulation and responses to stimuli that establish that H2A.Z and BRM directly regulate transcription of genes either redundantly or with opposing roles. Profiling genomic nucleosomal changes resulting from loss of BRM and/or H2A.Z showed that H2A.Z is associated with varying nucleosome dynamics, and BRM tends to destabilize or reposition nucleosomes flanking nucleosome-depleted regions.
To identify additional factors opposing H2A.Z-mediated transcription at the FLC locus, I conducted a forward genetic suppressor screen. I identified mutants depleted of H2A.Z-containing nucleosomes that still showed FLC transcription. By mapping suppressor mutations from the screen, I identified 9 candidate H2A.Z antagonists. Further work to distinguish whether the candidate mutations identified can suppress H2A.Z-nucleosome depletion phenotypes has potential to expand our understanding of what repressive factors make H2A.Z necessary for transcription.
In summary, I comprehensively profiled how H2A.Z and BRM interact to contribute to transcriptional regulation and nucleosomal organization and identified additional factors that may necessitate the role of H2A.Z for transcriptional regulation. These findings expand our view of how H2A.Z interacts with other factors in a complex chromatin context to regulate genomic processes, which have biological implications for development and how organisms respond to their environment.
Table of Contents
Table of Contents
CHAPTER1: INTRODUCTION - 1
Chromatin structure and regulation - 2
SWR1 and H2A.Z - 5
SWI2/SNF2 and BRM - 9
SWR1 and SWI2/SNF functions overlap in the genome -12
Scope of the dissertation - 12
Figure 1.1. Many factors influence nucleosome position and stability. - 15
Figure 1.2. Experimental models - 16
Literature Cited -17
CHAPTER 2: THE HISTONE VARIANT H2A.Z AND CHROMATIN REMODELER BRAHMA ACT COORDINATELY AND ANTAGONISTICALLY TO REGULATE TRANSCRIPTION AND NUCLEOSOME DYNAMICS - 26
Abstract - 26
Introduction - 27
Results - 29
Discussion - 51
Materials and Methods - 60
Acknowledgements - 66
Figure 2.1. H2A.Z and BRM regulate transcription through various cooperative and antagonistic relationships. - 67
Figure 2.2. Gene Ontology (GO) analysis summary of BRM and ARP6/H2A.Z regulated genes. - 70
Figure 2.3. H2A.Z levels in chromatin are independent of BRM and dependent on ARP6. - 72
Figure 2.4. BRM is flanked by two well-positioned nucleosomes that are disrupted by transcription. - 74
Figure 2.5 Nucleosome patterns surrounding BRM at DE BRM target genes show distinct occupancy patterns. - 76
Figure 2.6. BRM contributes to nucleosome stability and positioning differentially at nucleosome-depleted regions and flanking areas. - 77
Figure 2.7. The arp6 mutant genome contains large genomic deletions. - 79
Figure 2.8. H2A.Z contributes to a range of nucleosome changes. - 80
Figure 2.9. Quantifying H2A.Z contributions to nucleosome occupancy, positioning, and fuzziness changes in arp6 mutants. - 81
Figure 2.10. BRM destabilizes nucleosomes where BRM and H2A.Z overlap. - 83
Figure 2.11. BRM and H2A.Z destabilize the +1 nucleosome at DE targets. - 84
Figure 2.12. Nucleosome patterns at coordinately and antagonistically H2A.Z and BRM regulated gene sets. - 86
Figure 2.13 BRM and H2A.Z overlap with PIF4 peaks but do not affect the surrounding chromatin environment. - 88
Figure 2.14 BRM and H2A.Z overlap with PIF5 peaks but do not affect the surrounding chromatin environment. - 90
Figure 2.15. BRM and H2A.Z can contribute to nucleosome stability at FRS9 binding sites. - 92
Table 2.1. Summary of MEME-TOMTOM output showing transcription factors (TFs) that potentially associate with our 8 classes of DE genes - 94
Literature Cited - 96
CHAPTER 3: A GENETIC SUPPRESSOR SCREEN TO IDENTIFY H2A.Z ANTAGONISTS - 120
Abstract - 120
Introduction - 121
Results - 123
Discussion -130
Materials and Methods - 135
Acknowledgements - 143
Figure 3.1. Forward genetic suppressor screen study design - 144
Figure 3.2. Identifying eoa2 mutants and performing mapping backcrosses - 146
Figure 3.3. Identifying 9 candidate causal eoa2 mutations - 148
Figure 3.4. Phenotyping complementation transformations is inconclusive in identifying the causal eoa2 mutation. -150
Figure 3.5. FLC levels were not rescued to arp6-like levels in eoa2 complementation lines. - 151
Figure 3.6. FLC shows tissue specific expression levels. - 153
Figure 3.7. ARP6 and BLISTER do not physically interact. - 154
Table 3.1. Top causal candidate eoa2 mutations - 155
Table 3.2. Primers used for InFusion cloning of the candidate genes into the pCAMBIA Agrobacterium transformation vector - 156
Table 3.3. Transformant qPCR methods and materials summary - 157
Table 3.4. Primers to confirm that Agrobacterium and Arabidopsis transformants have the correct insert - 159
Literature Cited -160
CHAPTER 4: DISCUSSION – IMPLICATIONS AND FUTURE DIRECTIONS - 165
Figure 4.1. Hypocotyl elongation phenotypes may indicate a physiological link between ARP6 and BRM function. - 175
Figure 4.2. BRM and H2A.Z may contribute to nuclear localization of light-responsive genes together with PIF4 for their transcriptional activation. - 177
Table 4.1. Disease resistance genes potentially impacted by deletions in arp6 mutants. - 178
Table 4.2. Genes with light responsive nuclear re-positioning are targeted by PIF4, H2A.Z, and BRM. - 179
Literature Cited - 180
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