Epigenetic regulation of the canonical histone genes during development Restricted; Files Only
O'Haren, Tommy (Spring 2026)
Abstract
To ensure that the embryo can package exponentially increasing amounts of DNA, replication-dependent histones are some of the earliest transcribed genes from the zygotic genome. Zygotic histone gene expression coincides with the emergence of many epigenetic processes that control transcription from the previously quiescent genome. However, how these mechanisms identify and regulate the uniquely stored and processed histone genes is not known. In Drosophila melanogaster, pioneering transcription factors Zelda and CLAMP collaborate across the genome to regulate zygotic genome activation and target early activated genes. We hypothesized that Zelda helps identify histone genes for early embryonic expression and helps assist CLAMP in their activation. Yet, we found that depletion of zelda in the early embryo does not affect histone mRNA levels or prevent the recruitment of Histone Locus Body (HLB) factors. We also uncovered that known histone regulating factor Mxc, which does not bind DNA, localizes to the centromeres in embryos devoid of canonical histone gene sequence, suggesting protein-protein interactions or likely phase separation drives recruitment. Finally, as the histone genes are present in multiple, clustered copies within many metazoan genomes called Histone Loci, it was unclear how cells regulate expression from neighboring, nearly identical arrays of genes. Using long-read sequencing techniques like DiMeLo-seq and Fiber-seq and analyzing single molecule data, we discovered that histone gene expression likely varies from cell to cell as different subsections of the histone gene cluster are enriched for active epigenetic marks and RNA Polymerase II across chromatin fibers. In addition, we discovered that in the early embryo, typically only one histone gene shows increased accessibility compared to the others within a histone gene array, suggesting there is histone gene specific regulation across the locus.
Table of Contents
Chapter I – Introduction and Background
1.0 Overview and Motivation………………………………………………………………… 1
1.1 Molecular Biology of Early Embryonic Development……………………………………2
1.1.1 Metazoan Embryogenesis is Conserved……………………………………………. 2
1.1.2 Reprogramming of the Germline Epigenetic Landscape……………………………3
1.1.3 Maternal Loading of Products into the Embryo……………………………………. 4
1.1.4 The Maternal to Zygotic Transition…………………………………………………6
1.1.5 Epigenetic Features Emerge During Zygotic Genome Activation…………………. 6
1.2 Replication-Dependent Histone Biology…………………………………………………. 9
1.2.1 The Nucleosome, the basic unit of chromatin……………………………………… 9
1.2.2 Histone Variants……………………………………………………………………10
1.2.3 Replication-Dependent Histone Gene Organization……………………………….11
1.2.4 Replication-Dependent Histone mRNA Biosynthesis…………………………….. 12
1.2.5 The Histone Locus Body in Early Drosophila Development……………………... 14
1.2.6 Histone Dysregulation in Disease………………………………………………….15
1.2.7 Gaps in Knowledge………………………………………………………………...17
1.3 Long-read Sequencing as a Tool for the Histone Locus…………………………………18
1.3.1 Justification………………………………………………………………………...18
1.3.2 Next Generation Sequencing Techniques…………………………………………. 18
1.3.3 Long Read Sequencing Techniques……………………………………………….. 20
Chapter II – Zelda is dispensable for Drosophila melanogaster histone gene regulation……….24
2.0 Abstract………………………………………………………………………………….. 24
2.1 Introduction………………………………………………………………………………25
2.2 Results……………………………………………………………………………………27
2.2.1 Zelda localizes to TAGteam sites in the histone gene array early during
embryogenesis……………………………………………………………………………27
2.2.2 Zelda reduction in the embryo has a slight effect on HLB size……………………28
2.2.3 Zelda reduction in the embryo has little effect on histone transcript levels………. 31
2.2.4 Zelda and CLAMP do not affect reciprocal localization to the histone genes……..32
2.2.5 Histone sequences lacking TAGteam sites still recruit HLB factors in vivo and in
vitro………………………………………………………………………………………32
2.3 Discussion………………………………………………………………………………..35
2.4 Supplemental Figures…………………………………………………………………….36
Chapter III – Drosophila HLB factor Mxc localizes localizes to other repetitive regions in
embryos lacking endogenous histone genes…………………………………………………43
3.0 Abstract………………………………………………………………………………….. 43
3.1 Introduction………………………………………………………………………………44
3.2 Results……………………………………………………………………………………46
3.2.1 Mxc continues to form puncta in Drosophila embryos lacking the histone genes... 46
3.2.2 Combining a fluorescent embryo sorting system and DamID enriches for embryos
lacking the histone genes and transient DNA-protein interactions………………………49
3.2.3 Mxc localizes to the centromeres in absence of the endogenous histone locus……54
3.3 Discussion………………………………………………………………………………..58
3.4 Supplemental Figures…………………………………………………………………….63
Chapter IV – Single molecule profiling of the accessibility and epigenetic landscape of the
Drosophila Histone Locus…………………………………………………………………... 65
4.0 Abstract………………………………………………………………………………….. 65
4.1 Introduction………………………………………………………………………………66
4.2 Results……………………………………………………………………………………69
4.2.1 Differences in cis elements in the H3-H4 promoter allow long-read sequencing to
map to the Drosophila histone locus……………………………………………………..69
4.2.2 The 5kb histone array acts a regulatory unit of the histone locus………………….71
4.2.3 Subsections of arrays within the Histone Locus are differentially bound by
regulatory machinery across nuclei……………………………………………………... 75
4.2.4 Footprinting analysis reveals specific promoter and gene accessibility patterns
within arrays……………………………………………………………………………...78
4.3 Discussion………………………………………………………………………………..85
Chapter V – Conclusions and Future Directions………………………………………………... 88
Chapter VI – Materials and Methods…………………………………………………………….92
6.1 Drosophila strains………………………………………………………………………..92
6.1.1 Chapter II strains………………………………...………………………………..92
6.1.2 Chapter III strains…………………………………………………………………92
6.1.3 Chapter IV strains…………………………………………………………………93
6.2 Cloning and transgenesis……………………………………………………………....... 93
6.3 ChIP-seq data analysis and visualization……………………………………………….. 94
6.4 Quantitative real-time PCR……………………………………………………………....94
6.5 Embryo immunofluorescence…………………………………………………………... 95
6.6 Quantitative microscopy………………………………………………………………... 96
6.7 Polytene chromosome FISH and immunofluorescence……………………………….....96
6.8 EMSAs and probes…………………………………………………………………....… 97
6.9 Embryo sorting and collection………………………………………………………….. 98
6.10 DamID experiments…………………………………………………………………….99
6.11 DamID-seq sequencing and data analysis……………………………………………... 99
6.12 DiMeLo-seq experiments……………………………………………………………...102
6.13 DiMeLo-seq sequencing and data analysis……………………………………………104
6.14 Fiber-seq experiments………………………………………………………………....105
6.15 Fiber-HMM analysis…………………………………………………………………..106
Chapter VII – References……………………………………………………………………….107
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File download under embargo until 27 May 2028 | 2026-03-18 16:44:08 -0400 | File download under embargo until 27 May 2028 |
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