The Combinatorial Role of Architectural Proteins in Insulator Function and Dynamics during the Ecdysone Response in D. melanogaster Open Access
Van Bortle, Kevin Thomas (2014)
Abstract
Recently developed genomic strategies for assaying chromosome architecture have significantly improved our ability to investigate the nature of genome organization and the players involved. In particular, recent high-throughput chromosome conformation capture studies provide evidence that eukaryotic genomes are organized into tissue-invariant, sub-megabase sized structures called Topologically Associating Domains (TADs). The conserved nature of TADs across diverse cell types suggests a pre-defined, bottom-up pattern of chromosome organization, yet how these structures are established and maintained during development remain important questions. The borders of TADs are highly enriched for architectural proteins, previously characterized for their role in insulator function. However, a majority of architectural protein binding sites (APBSs) localize within topological domains, suggesting sites associated with TAD borders represent a functionally different subclass of these regulatory elements. By mapping the genome-wide target sites for several Drosophila architectural proteins, including previously uncharacterized profiles for Pol III transcription factor TFIIIC and SMC-containing condensin complexes, I uncovered an extensive pattern of colocalization in which architectural proteins establish dense, high occupancy clusters at the borders of topological domains. Enhancer-blocking activity and TAD border strength scale with the occupancy level of APBSs, suggesting co-binding by multiple architectural proteins underlies the functional potential of these loci. Parallel analyses in mouse and human stem cells further suggest that clustering of architectural proteins is a general feature of genome organization, and that conserved APBSs may underlie the tissue-invariant nature of TADs. These results provide a novel, integrative model for understanding the role of architectural proteins in insulator function and topological domain organization. Profiling of architectural protein binding dynamics in response to signaling events, including 20-hydroxyecdysone (20HE) and TGF-β, further reveals a role for these proteins in defining the transcriptional response to extracellular stimuli. Finally, I present evidence that transcriptomic and proteomic codon usage is altered in response to 20HE in a manner that reflects the differentiation status of the cell. tRNA abundance predicts preferential codon incorporation in proteins, and increasing and decreasing tRNA isoacceptors lead to increased and decreased codon usage, respectively, in proteins, together suggesting tRNA levels play an important role in regulating the translational output of a cell.
Table of Contents
Table of Contents
CHAPTER 1: INTRODUCTION .......................................................................................1
Interphase Chromosome Organization............................................................................... 3
Differentiation, Replication, and Genome Stability............................................................... 3
Genomic Strategies........................................................................................................ 4
Mediators of Nuclear Organization..................................................................................... 6
Chromatin Insulators...................................................................................................... 7
Composition and Evolution............................................................................................... 7
Insulator Protein CTCF.................................................................................................... 8
Insulators in D. melanogaster......................................................................................... 10
tDNAs and TFIIIC.......................................................................................................... 11
Distribution and Chromatin Structure................................................................................ 12
Long-range Interactions................................................................................................. 14
Role in Nuclear Organization........................................................................................... 18
Polycomb..................................................................................................................... 20
Composition and Evolution.............................................................................................. 20
Distribution and Chromatin Structure................................................................................ 22
Long-range Interactions................................................................................................. 23
Role in Nuclear Organization........................................................................................... 24
Transcription Factories.................................................................................................. 27
Composition and Evolution............................................................................................. 27
Enhancer-Promoter Interactions...................................................................................... 29
Perspectives................................................................................................................ 33
Remaining questions concerning the role of insulators in chromosome
organization................................................................................................................. 37
CHAPTER 2: DROSOPHILA CTCF TANDEMLY ALIGNS WITH OTHER INSULATOR PROTEINS AT THE BORDERS OF H3K27ME3 DOMAINS
Abstract....................................................................................................................... 39
Introduction.................................................................................................................. 40
Results......................................................................................................................... 43
dCTCF sites align with Drosophila specific insulator proteins Su(Hw) and
BEAF-32....................................................................................................................... 43
dCTCF sites are enriched for multiple DNA motifs................................................................ 47
dCTCF recruits unique Mod(mdg4) isoform(s) ..................................................................... 50
Aligned dCTCF sites are enriched for CP190, Mod(mdg4) isoforms, and
additional co-factors ..................................................................................................... 52
Aligned dCTCF sites commonly flank the borders of H3K27me3 domains................................. 55
Insulator knockdown results in H3K27me3 loss within repressed domains............................... 56
The even-skipped gene provides a model for dCTCF alignment at H3K27me3
domain borders............................................................................................................ 62
Discussion................................................................................................................... 65
Experimental Procedures................................................................................................ 71
Acknowledgements........................................................................................................ 76
CHAPTER 3: INSULATOR FUNCTION AND TOPOLOGICAL DOMAIN BORDER STRENGTH SCALE WITH ARCHITECTURAL PROTEIN OCCUPANCY
Abstract....................................................................................................................... 78
Introduction.................................................................................................................. 79
Results........................................................................................................................ 82
Characterization and genome-wide mapping of TFIIIC in D. melanogaster............................... 82
Relationship to SMC-containing cohesin and condensin complexes.......................................... 85
TFIIIC clusters with CTCF and other architectural proteins.................................................... 89
Clustering of architectural proteins scales with TAD border strength....................................... 91
High occupancy APBSs associate with robust enhancer blocking activity.................................. 95
High occupancy APBSs are characterized by DNase I hypersensitivity through-
out Drosophila development............................................................................................ 99
Mammalian TFIIIC and CTCF cluster at TAD borders.......................................................... 100
Discussion.................................................................................................................. 106
Experimental Procedures.............................................................................................. 112
Acknowledgements...................................................................................................... 124
CHAPTER 4: CTCF-DEPENDENT CO-LOCALIZATION OF CANONICAL SMAD SIGNALING FACTORS AT ARCHITECTURAL PROTEIN BINDING SITES
Abstract..................................................................................................................... 126
Introduction................................................................................................................ 127
Results....................................................................................................................... 130
Genome-wide mapping of Drosophila Smad proteins........................................................... 130
SMM modules overlap Drosophila CTCF and other architectural proteins................................. 134
CTCF-dependent co-localization of Smad proteins at APBSs.................................................. 137
dCTCF-dependent Smad binding occurs at low occupancy APBSs within
topological domains...................................................................................................... 140
dCTCF binding remains constant in response to DPP-induced signaling
events........................................................................................................................ 145
Discussion................................................................................................................... 149
Experimental Procedures................................................................................................ 152
Acknowledgements........................................................................................................ 155
CHAPTER 5: DYNAMIC RECRUITMENT OF REGULATORY FACTORS TO ARCHITECTURAL PROTEIN BINDING SITES DURING STEROID-HORMONE SIGNALING EVENTS
Abstract...................................................................................................................... 157
Introduction................................................................................................................. 158
Results........................................................................................................................ 159
Ecdysone treatment leads to changes in the genome-wide distribution of
architectural proteins..................................................................................................... 159
Discussion.................................................................................................................... 163
Experimental Procedures................................................................................................ 165
Acknowledgements........................................................................................................ 167
CHAPTER 6: DYNAMIC CODON USAGE AND tRNA ABUNDANCE IN RESPONSE TO ECDYSONE INDUCED DIFFERENTIATION
Abstract...................................................................................................................... 169
Introduction................................................................................................................. 170
Results........................................................................................................................ 173
Ecdysone induces differentiation of Drosophila Kc167 Plasmatocyte cells
into macrophages.......................................................................................................... 173
Transcriptome and quantitative proteomics after 48h Ecdysone treatment............................... 175
Codon usage after 48hrs 20HE mirrors preferential codon incorporation in
differentiation genes...................................................................................................... 177
Small RNA profiling identifies changes in tRNA and miRNA abundance.................................... 179
tRNA abundance predicts preferential codon incorporation in polypeptides............................... 181
Upregulation of selenocysteine tRNA and an experimental system to determine
whether increasing tRNASeC regulates selenoprotein synthesis.............................................. 186
Discussion................................................................................................................... 189
Experimental Procedures................................................................................................ 191
Acknowledgements........................................................................................................ 193
CHAPTER 7: DISCUSSION ........................................................................................... 194
Building a new model for insulator function and chromosome domain organization................... 195
tRNAs: a likely key player in cellular identity and differentiation........................................... 202
CURRICULUM VITAE................................................................................................... 207
LITERATURE CITED .................................................................................................... 211
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