Function of Chromatin Features in Cellular Programming

项目摘要/摘要 保持稳定的基因转录模式对细胞编程至关重要。同样，有序的切换 从一种转录模式到另一种转录模式，称为重新编程，对于发育以及对 还有许多其他的生物过程。失控的重新编程可能会产生灾难性的后果， 结果从发育疾病到癌症不一而足。值得注意的是，表观遗传异常，未能 分化和不适当的细胞编程与癌症的发生有着错综复杂的联系。我们的目标是 确定H_2A.Z(组蛋白H_2A的变种形式)在调节体内细胞编程中的功能。我们会 利用斑马鱼模型进行早期胚胎发育，结合一系列下一代基因组-- 广泛的测序方法，从功能上测试H2A.Z模式如何调控细胞的几个方面 重新编程，包括转录激活。基因转录的调节主要通过两种途径进行 成分：主要的顺式调节DNA序列成分增强子和主要的转录因子 反式调节DNA结合蛋白成分。然而，由于与体内相关的技术挑战， 研究表明，有几个关键的未知因素限制了我们对这些元素如何发挥作用的理解。例如，什么 调控基因组中数千个增强子中特定细胞类型增强子的激活？ 同样，在数百万个小的dna结合基序中，转录因子是如何选择性地结合在 位置的离散子集？一种有趣的可能性是转录因子之间的相互作用， 聚合酶机制和表观遗传标记，调节转录因子的结合，以及转录因子的活性 增强剂。我们最近定义了特殊的占位符核小体在发育中的功能 斑马鱼中配子到干细胞的重新编程。占位符核小体支持正确的全基因组 表观遗传标记的图案化和促进合子基因组的激活。我们断定少校 占位符核小体的功能单位，组蛋白变异体H2A.Z，变得局限于增强子区 在斑马鱼发育的后续阶段，当分化和细胞类型指定发生时。 这导致我们假设H_2A.Z模式的重组是控制发育的主要因素 单元类型规范。为了验证这一假设，我们将从基因上操纵H2A.Z本地化的调节因子 在斑马鱼胚胎中，然后评估全基因组对增强子活性的影响，以及特定细胞类型 转录因子结合。该项目的成功完成将确定H2A.Z在控制基因中的作用 表达模式和细胞编程。我们建议，这个更广泛的概念，其中变化 表观遗传标记是细胞重新编程的基础，可能是生物学中的一个一般原则，与 发育生物学、干细胞功能和癌症发生。

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.