Function of Chromatin Features in Cellular Programming

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PROJECT SUMMARY / ABSTRACT Maintaining stable gene transcription patterns is critical for cellular programming. Likewise, orderly switching from one transcription pattern to another, termed reprogramming, is necessary for development, as well as for numerous other biological processes. Dysregulated reprogramming can have catastrophic consequences, with outcomes ranging from developmental disease to cancer. Notably, epigenetic abnormalities, failure to differentiate, and inappropriate cellular programming are intricately linked to carcinogenesis. Our objective is to define the function of H2A.Z (a variant form of histone H2A) in regulating cellular programming in vivo. We will utilize the zebrafish model for early embryonic development, combined with a series of next-generation genome- wide sequencing approaches, to functionally test how H2A.Z patterns regulate several aspects of cellular reprogramming, including transcriptional activation. Regulation of gene transcription occurs through two primary components: enhancers, the major cis-regulatory DNA sequence component, and transcription factors, the major trans-regulatory DNA-binding protein component. However, due to technical challenges associated with in vivo studies, several critical unknowns limit our understanding of how these elements function. For example, what regulates activation of cell-type-specific enhancers among the many thousands of enhancers in the genome? Likewise, among millions of small DNA-binding motifs, how are transcription factors able to selectively bind at a discrete subset of locations? One interesting possibility is that interplay between transcription factors, polymerase machinery, and epigenetic marks, regulates the binding of transcription factors, and the activity of enhancers. We recently defined the function of specialized ‘Placeholder’ nucleosomes in developmental reprogramming of gametes to stems cells in zebrafish. Placeholder nucleosomes enable proper genome-wide pattering of epigenetic marks and facilitate activation of the zygotic genome. We determined that the major functional unit of Placeholder nucleosomes, the histone variant H2A.Z, becomes localized to enhancer regions during the subsequent stages of zebrafish development, when differentiation and cell-type specification occurs. This led us to hypothesize that reorganization of H2A.Z patterns is a major factor in controlling developmental cell-type specification. To test this hypothesis, we will genetically manipulate the regulators of H2A.Z localization in zebrafish embryos, and then assess genome-wide impacts on enhancer activity, and cell-type-specific transcription factor binding. Successful completion of this project will define the role of H2A.Z in controlling gene expression patterns and in cellular programming. We propose that this broader concept, where changes in epigenetic marks underlie cellular reprogramming, might be a general principle in biology, with relevance to developmental biology, stem cell function, and carcinogenesis.