Function of Chromatin Features in Cellular Programming

染色质特征在细胞编程中的作用

• 批准号: 10206199
• 负责人: Patrick J. Murphy
• 金额: $ 38.5万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10206199
• 关键词: Binding; Biological Process; Biology; Chromatin; DNA Binding; DNA Sequence; DNA-Binding Proteins; Defect; Development; Developmental Biology; Disease; Elements; Embryo; Embryonic Development; Enhancers; Epigenetic Process; Failure; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genetic Transcription; Genome; Germ Cells; Histone H2A; Histones; Link; Location; Malignant Neoplasms; Modeling; Nucleosomes; Outcome; Pattern; Polymerase; Role; Series; Testing; Transcriptional Activation; Variant; Zebrafish; carcinogenesis; cell type; developmental disease; genetic manipulation; genome-wide; in vivo; next generation; prevent; programs; stem cell function; stem cells; transcription factor; zebrafish development

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.

项目总结/摘要 维持稳定的基因转录模式对于细胞编程至关重要。同样，有序切换 从一种转录模式到另一种转录模式，称为重编程，对于发育以及 许多其他生物过程。失调的重编程可能会产生灾难性的后果， 从发育性疾病到癌症。值得注意的是，表观遗传异常， 分化和不适当的细胞编程与致癌作用有着错综复杂的联系。我们的目标是 定义H2A.Z（组蛋白H2 A的变体形式）在体内调节细胞编程中的功能。我们将 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，以功能性地测试H2A.Z模式如何调节细胞的几个方面， 重编程，包括转录激活。基因转录的调控通过两个主要的途径进行， 组成部分：增强子，主要的顺式调节DNA序列组成部分，和转录因子，主要的 反式调节DNA结合蛋白组分。然而，由于与体内植入相关的技术挑战， 研究中，一些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。比如什么 调节基因组中成千上万个增强子中细胞类型特异性增强子的激活？ 同样地，在数百万个小的DNA结合基序中，转录因子如何能够选择性地结合在一个小的DNA结合基序上？ 位置的离散子集？一个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， 增强剂。我们最近定义了专门的“占位”核小体在发育中的功能， 将配子重新编程为斑马鱼的干细胞。占位核小体使适当的全基因组 表观遗传标记的模式化并促进合子基因组的激活。我们确定少校 占位核小体的功能单位，组蛋白变体H2A.Z，定位于增强子区域 在斑马鱼发育的后续阶段，当分化和细胞类型特化发生时。 这使我们假设H2A.Z模式的重组是控制发育的主要因素， 细胞类型规格。为了验证这一假设，我们将从基因上操纵H2A.Z定位的调节因子 在斑马鱼胚胎中，然后评估全基因组对增强子活性的影响，以及细胞类型特异性 转录因子结合该项目的成功完成将明确H2A.Z在控制基因 表达模式和细胞编程。我们建议这个更广泛的概念，其中的变化 表观遗传标记是细胞重编程的基础，可能是生物学中的一般原则，与 发育生物学、干细胞功能和致癌作用。