Nuances of context-dependent transcription factor function Restricted; Files Only
Hodkinson, Lauren (Spring 2024)
Abstract
Despite binding similar cis elements, transcription factors often perform context-dependent functions at different genomic loci. Furthermore, transcription factors can be involved in large scale coordinated gene events where hundreds of genes need to be targeted and regulated with strict temporal or special requirements. How transcription factors integrate cis sequence and genomic context to perform their context-dependent functions is still poorly understood. One example of a context-dependent transcription factor is involved in coordinated gene regulation the Drosophila protein Chromatin-Linked Adapter for MSL Proteins (CLAMP), which targets similar GA-rich cis elements on the X-chromosome and at the histone locus but recruits very different, locus-specific transcription factors to each of these contexts. We utilized several different techniques to interrogate CLAMP function at the histone locus as a context-dependent transcription factor. First, we focused on understanding how differences in the CLAMP-binding GA-repeat element within the in the promoter of H3 and H4 (H3/H4p) may impact the overall regulation of histone genes. We found that the H3/H4p GA-repeat is variable across the ~100 histone gene arrays however this sequence variation, and subsequent factor targeting, likely does not confer differential expression of the histone genes. Next, we investigated how CLAMP function at the histone genes is impacted by the identity of its cis binding elements. Leveraging a powerful transgenic histone array system, we discovered that X-linked CLAMP sequences do not functionally substitute for GA-repeats in the histone gene array. Our results suggest that transcription factors incorporate cis sequences and flanking sequence to govern their regulatory function at target loci. Finally, we explored what additional DNA factors may be regulating histone gene expression. Through our undergraduate driven in silico screen, we identified 9 novel histone locus regulatory factor candidates that warrant future wet lab studies to interrogate how they may influence histone biogenesis. Combined, these findings broaden our understanding of the nuanced mechanisms of coordinated histone gene regulation.
Table of Contents
Table of Contents
Chapter 1 – Introduction............................................................................................................ 19
1.1 Overview................................................................................................................................. 20
1.2 Coordinated gene expression occurs on different scales in the nucleus................................. 21
1.2.1 Coordinating zygotic genome activation.............................................................................. 23
1.2.2 Coordinating dosage compensation..................................................................................... 25
1.2.3 Coordinated expression of the replication dependent histone genes.................................... 27
1.3 The gene clustering and genomic organization facilitate coordinated gene expression......... 29
1.3.1 Nuclear bodies and three-dimensional genome architecture enable more efficient gene regulation 29
1.3.2 Organizing genes into clusters or repetitive arrays can facilitate coordinated gene expression 31
1.3.3 Combined genomic organizational strategies...................................................................... 32
1.4 Several properties can influence transcription factor targeting and function......................... 33
1.4.1 Cis element sequence........................................................................................................... 35
1.4.2 Cofactors.............................................................................................................................. 36
1.5 Context-dependent transcription factors................................................................................. 38
1.5.1 CLAMP as a context-dependent transcription factor........................................................... 39
1.5.2 CLAMP function at the histone locus is a model for context-dependent transcription factor function 40
1.6 Goals and major findings........................................................................................................ 41
1.7 Summary................................................................................................................................. 43
1.8 References............................................................................................................................... 45
Chapter 2 - Cis element length variability does not confer differential transcription factor occupancy at the histone locus 55
2.1 Abstract................................................................................................................................... 56
2.2 Introduction............................................................................................................................. 57
2.3 Results..................................................................................................................................... 60
2.3.1 The GA-repeat is variable in length across the histone gene arrays.................................... 60
2.3.2 CLAMP, GAF, and Psq all target the GA-repeats in the H3/H4p....................................... 62
2.3.3 GA-binding factors do not show preference for GA-repeat length at the histone locus...... 64
2.4 Discussion............................................................................................................................... 69
2.5 Conclusions............................................................................................................................. 72
2.6 Methods................................................................................................................................... 73
2.7 Acknowledgments................................................................................................................... 77
2.8 Supplemental Figures.............................................................................................................. 78
2.9 References............................................................................................................................... 80
Chapter 3 Sequence reliance of a Drosophila context-dependent transcription factor........ 87
3.1 Abstract................................................................................................................................... 88
3.2 Introduction............................................................................................................................. 89
3.3 Materials and Methods............................................................................................................ 92
3.4 Results and Discussion............................................................................................................ 97
3.4.1 CLAMP targets GA-rich cis elements at different loci........................................................ 97
3.4.2 CLAMP requires GA-rich sequences to bind in vitro and in vivo....................................... 99
3.4.3 The GA-repeats must reside in the H3/H4p for Mxc recruitment in vivo.......................... 103
3.4.4 CLAMP binding sequences from different loci do not functionally substitute in the context of the histone array in chromosomal females 106
3.4.5 X-linked sequences in the context of the histone gene array attract MSL2 in chromosomal males 110
3.5 Acknowledgements............................................................................................................... 114
3.6 Supplemental Figures............................................................................................................ 115
3.7 References............................................................................................................................. 116
Chapter 4 - MSL2 targets histone genes in Drosophila virilis............................................... 122
4.1 Abstract................................................................................................................................. 123
4.2 Introduction........................................................................................................................... 124
4.3 Methods................................................................................................................................. 128
4.4 Results................................................................................................................................... 133
4.4.1 Sequence differences between D. virilis histone loci......................................................... 133
4.4.2 Both D. virilis histone loci are targeted by Mxc and CLAMP........................................... 134
4.4.3 MSL2 targets the major D. virilis histone locus................................................................. 136
4.4.4 MSL2 does not directly interact with histone array sequence........................................... 140
4.4.5 CLAMP does not require the GA-rich elements to interact with the virilis H3/H4 promoter in vitro 142
4.4.6 The D. virilis H3/H4 promoter does not promote Mxc recruitment in D. melanogaster... 144
4.5 Discussion............................................................................................................................. 146
4.6 Acknowledgments................................................................................................................. 152
4.7 Supplemental Figures and Tables......................................................................................... 153
4.8 References............................................................................................................................. 164
Chapter 5 - A bioinformatics screen reveals Hox and chromatin remodeling factors at the Drosophila histone locus 174
5.1 Abstract................................................................................................................................. 175
5.2 Introduction........................................................................................................................... 176
5.3 Methods................................................................................................................................. 179
5.3.1 GEO Datasets..................................................................................................................... 179
5.3.2 Bioinformatic Analysis and Data Visualization................................................................. 181
5.3.3 Criteria for Positive Candidates vs. Negative Candidate................................................... 182
5.4 Results................................................................................................................................... 183
5.4.1 Validating the bioinformatics pipeline by mapping TATA-associated factors to the histone gene array 183
5.4.2 Candidate DNA-binding factors that did not pass the bioinformatics screen.................... 186
5.4.3 Candidates that passed the bioinformatics screen.............................................................. 192
5.4.4 Hox factors localize to the Drosophila histone gene array when overexpressed in cell culture 194
5.4.5 Power and limitations of the screen................................................................................... 197
5.5 Discussion............................................................................................................................. 199
5.6 Conclusions........................................................................................................................... 205
5.7 Acknowledgments and Authors' contributions..................................................................... 206
5.8 References............................................................................................................................. 207
Chapter 6 - A cost-free CURE: Using bioinformatics to identify DNA-binding factors at a specific genomic locus 220
6.1 Abstract................................................................................................................................. 221
6.2 Introduction........................................................................................................................... 222
6.2.1 Intended audience............................................................................................................... 224
6.2.2 Learning time..................................................................................................................... 225
6.2.3 Prerequisite student knowledge.......................................................................................... 225
6.2.4 Learning objectives............................................................................................................ 225
6.3 Procedures............................................................................................................................. 226
6.3.1 Materials............................................................................................................................. 226
6.3.2 Student instruction.............................................................................................................. 228
6.3.3 Faculty Instruction.............................................................................................................. 228
6.3.3.1 Background..................................................................................................................... 228
6.3.3.2 Tutorials........................................................................................................................... 230
6.3.3.3 Work days........................................................................................................................ 231
6.3.3.4 Poster session.................................................................................................................. 232
6.3.3.5 Notes and recommendations........................................................................................... 233
6.4 Discussion............................................................................................................................. 237
6.4.1 Field testing........................................................................................................................ 237
6.4.2 Evidence of student learning.............................................................................................. 238
6.4.3 Possible modifications........................................................................................................ 241
6.5 Summary............................................................................................................................... 242
6.6 Acknowledgments................................................................................................................. 243
6.7 References............................................................................................................................. 244
Chapter 7 – Discussion.............................................................................................................. 253
7.1 Overview: Coordinated gene regulation is a heavy burden.................................................. 254
7.2 The Drosophila melanogaster histone locus is inherently absurd......................................... 254
7.3 The impact of cis element length on transcription factor recruitment.................................. 257
7.4 A combination of cues can influence transcription factor function...................................... 260
7.5 Regulatory mechanisms of histone gene expression vary between species.......................... 263
7.6 The HLB is a wild unknown entity....................................................................................... 266
7.7 Histone gene regulation is way more complicated than you think....................................... 268
7.8 The future of studying coordinated histone gene regulation: closing remarks..................... 272
7.9 References............................................................................................................................. 274
Appendix A – Supplemental Data of Chapter 5..................................................................... 279
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