Sunday, June 1, 2025
6/1/2025
Developmental regulation of histone genes by pioneer factors and three-dimensional architecture - PROJECT SUMMARY: Maternally deposited factors control the earliest stages of animal development. When maternal factors are depleted, the transcriptional responsibility shifts to the zygote. This process, known as zygotic genome activation (ZGA), is conserved across animals and is necessary for accurate gene expression and cellular differentiation. Maternal histone proteins are especially critical during early embryogenesis as their levels regulate the timing of cellular/nuclear divisions: depletion of histones leads to cell cycle arrest, while histone overexpression causes asynchronous cell division. Expression of the zygotic histones genes is activated early, prior to ZGA. The histone genes are targeted by a suite of unique regulatory factors collectively called the Histone Locus Body (HLB). However, it is unclear how the HLB components identify the locus as none directly interact with DNA. Recent data suggest that zygotic histone genes are targeted by specialized transcription factor called pioneer factors, which recognize nucleosome-obstructed binding sites. Pioneer factors facilitate overall ZGA by recruiting chromatin remodeling complexes in preparation for other transcription factors. Pioneer factors are therefore excellent candidates that may target the silent zygotic histone locus and prepare it fore HLB formation and histone gene expression. My preliminary data suggest that the pioneer factor Zelda, the “master regulator” of ZGA, occupies the Drosophila histone locus prior to zygotic histone gene expression. A histone gene array lacking Zelda binding sites is less efficient at recruiting HLB factors needed for histone gene expression compared to a wild type array. During ZGA, Zelda cooperates with another pioneering factor CLAMP. We previously discovered that CLAMP participates in zygotic HLB formation and histone gene expression. In this proposal, I propose to leverage powerful tools in Drosophila melanogaster to investigate the contribution of pioneer factors to histone gene regulation during ZGA. I hypothesize that Zelda and CLAMP collaborate to prepare the zygotic histone locus for HLB formation and organization. In Aim 1, I will determine the role of Zelda in zygotic HLB formation by leveraging a transgenic histone array lacking Zelda binding sites, artificially tethering Zelda to the transgene at different developmental time points around ZGA, and assaying HLB formation by microscopy. In Aim 2A, I will define the relationship of Zelda and CLAMP at the zygotic histone locus by performing pioneer factor CUT&RUN at histone loci in the presence and absence of the other pioneer factor. In Aim 2B, I will probe the role of pioneer factors in histone locus organization through DamID and long-read sequencing. The long-term goal of this project is to define the contribution of pioneer factors to regulation of the indispensable histone genes. The need for precise zygotic histone regulation is broadly conserved, and insights from Drosophila will advance our understanding of mechanisms that contribute to early human development.
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