Mechanism of Chromatin Organization and Dynamics in Development

描述（申请人提供）：表观遗传谱的建立和维持在发育、生理和疾病的基因表达调控中起着重要作用。我们的建议侧重于了解启动子和增强子组蛋白标记如何影响体内核小体的定位，以及它们如何反过来受到转录的影响。模式生物斑马鱼是一种独特的脊椎动物系统来解决这些问题。在母系-合子过渡（MZT）之前，早期斑马鱼胚胎的基因组没有转录，也没有被H3K4me3或H3K27me3等组蛋白标记所占据。因此，该系统允许研究从非转录基因组到转录基因组的转变。此外，可以收集大量的阶段同步胚胎进行基因组学实验。我们建议使用RNA-seq， Pol II和组蛋白标记ChIP-seq，以及核小体-seq来回答斑马鱼MZT期间转录开始前，期间和之后的问题：(1)在斑马鱼早期胚胎发育中哪些基因是母系负载的，哪些是合子表达的？(2)当不同的组蛋白标记在不同的启动子和增强子上建立时，启动子和增强子标记是如何相关的，它们与母体负载的转录因子和合子表达的转录因子的存在有什么关系？(3)内在DNA序列、组蛋白标记建立、Pol II结合、转录起始和延伸对体内核小体定位的影响是什么？总的来说，该项目将为基因调控与胚胎发育中表观遗传谱的建立和维持之间的相互关系提供见解。

DESCRIPTION (provided by applicant): The establishment and maintenance of epigenetic profiles play an important role in the regulation of gene expression in development, physiology, and diseases. Our proposal focuses on understanding how promoter and enhancer histone marks influence nucleosome positioning in vivo, and how in turn they are influenced by transcription. The model organism zebrafish is a unique vertebrate system to address these questions. Before the maternal-zygotic transition (MZT), the genome of early zebrafish embryos is not transcribed and is not occupied by histone marks such as H3K4me3 or H3K27me3. This system therefore allows the study of the transition from a non-transcribed to a transcribed genome. Moreover, large numbers of stage-synchronized embryos can be collected for genomics experiments. We propose to use RNA-seq, Pol II and histone mark ChIP-seq, and nucleosome-seq before, during, and after the onset of transcription during zebrafish MZT to answer the following questions: (1) which genes are maternally loaded versus zygotically expressed in zebrafish early embryonic development? (2) when are different histone marks established at different promoters and enhancers, how are promoter and enhancer marks related and how are they related to the presence of maternally loaded versus zygotically expressed transcription factors? (3) What is the effect of intrinsic DNA sequence, histone mark establishment, Pol II binding, transcription initiation and elongation on nucleosome positioning in vivo? Collectively, the project will provide insights into the interrelationship between gene regulation and the establishment and maintenance of epigenetic profiles in embryonic development. PUBLIC HEALTH RELEVANCE: Epigenetic regulation plays a central role in human development, physiology, and disease. However, little is known about when, where and how epigenetic profiles are established and maintained in early embryonic development in vertebrate systems. We propose to use zebrafish embryos during early development to understand two important aspects of epigenetic regulation - the relative timing and coordination pattern of histone mark establishment, and the positioning of nucleosomes during the onset of transcription. Insights from these experiments will not only help understand gene regulation and epigenetic establishment of pluripotency in early embryonic development, but will also inform studies on directed cell differentiation, reprogramming, and regenerative medicine.