Writing and Interpreting the Chromatin Enhancer Code in Myeloid Cells

描述(由申请人提供)：后生动物生物中基因表达的时间和空间控制定义了细胞异质性、细胞类型特定的表型特化和细胞谱系的稳定性。这项建议致力于描述在不同的哺乳动物细胞系中建立选择性和可遗传的调控染色质景观和基因表达程序的机制。具体地说，利用新的哺乳动物模型系统以及已建立的模型和技术，我的目标是确定指定转录因子的细胞谱系如何影响特定谱系特异的调控染色质特征，并与之协作，例如在引导细胞分化中掺入组蛋白变体H3.3和H3K4(H3K4me1)的单甲基化。转录因子PU.1与C/EBP1和AP1一起指导髓系细胞的分化，并在空间和时间上明确地与带有H3K4me1和其他特征的“增强子组蛋白密码”修饰的谱系特异性增强子联系在一起。使用生化方法，我建议研究组蛋白修饰酶的活性和包含在髓系特定转录因子复合体中的辅因子特性。转录因子可以直接招募染色质机制到谱系特异性增强子，或者，这些因子可以以染色质模板依赖的方式顺序招募，并且这些可能的机制对于建立和繁殖谱系特异性染色质景观的相对贡献将被评估。此外，利用成熟的造血细胞分化模型和表型分析，结合新的小鼠模型，将在体内评估H3.3在细胞分化和谱系维持方面的功能需求。此外，我将使用基因组分析技术来表征H3.3对相关染色质特性的离散功能影响，如可及性、增强子转录和组蛋白的翻译后修饰。这些研究将解决染色质生物学和表观遗传学领域中的一个悬而未决的问题，即指定转录因子的谱系如何谱系特定的染色质景观，定义发育潜力，对次生因素的反应，以及可遗传的转录程序。增加对决定细胞命运的机制的了解将增强我们对这些表观遗传机制如何影响人类健康、发育过程和肿瘤发生的理解。 与公共卫生相关：真核生物的特征是数千种分化的细胞类型，它们都来自单一基因组的“编程”。这些研究旨在揭示染色质特征的调节机制，该机制控制细胞类型特定基因表达程序的建立和可遗传维持。这些途径的改变可能导致发育缺陷和肿瘤发生，因此，更好地理解转录因子和染色质特征如何共同控制细胞分化和身份将对人类健康产生重要影响。

DESCRIPTION (provided by applicant): Temporal and spatial control of gene expression in metazoan organisms defines cellular heterogeneity, cell type-specific phenotypic specialization, and cell lineage stability. This proposal pursues the description of mechanisms for the establishment of selective and heritable regulatory chromatin landscapes and gene expression programs in distinct mammalian cell lineages. Specifically, utilizing a combination of novel mammalian model systems and established models and techniques I aim to determine how cell lineage specifying transcription factors affect and collaborate with characteristic lineage specifi regulatory chromatin features, such as incorporation of the histone variant H3.3 and monomethylation of H3K4 (H3K4me1), in directing cellular differentiation. The transcription factor PU.1 directs myeloid cell differentiation together with C/EBP1 and AP1, and is clearly linked, spatiotemporally, to lineage specific enhancers decorated with H3K4me1 and other characteristic "enhancer histone code" modifications. Using biochemical approaches, I propose to investigate the histone modifying enzyme activity and cofactor identity contained within myeloid lineage specifying transcription factor complexes. Transcription factors may directly recruit chromatin machinery to lineage specific enhancers, or alternatively, these factors may be sequentially recruited in a chromatin template dependent manner and the relative contributions of these possible mechanisms for establishing and propagating lineage specific chromatin landscapes will be assessed. Additionally, using well- established models of hematopoietic cell differentiation and phenotypic analysis combined with novel mouse models, the functional requirements for H3.3 in cellular differentiation and lineage maintenance will be assessed in vivo. Additionally, I will employ genomic analytical techniques for characterization of discrete functional effects of H3.3 on associated chromatin characteristics such as accessibility, enhancer transcription, and post-translational modification of histones. These studies will address an outstanding question in the field of chromatin biology and epigenetics, namely, how lineage specifying transcription factors template lineage specific chromatin landscapes defining developmental potential, responsiveness to secondary factors, and heritable transcription programs. Increased knowledge of mechanisms of cell fate determination will enhance our understanding of how these epigenetic mechanisms impact human health, during development and in tumorogenesis. PUBLIC HEALTH RELEVANCE: Eukaryotic life is characterized by thousands of differentiated cell types which all derive their "programming" from a single genome. These studies aim to reveal mechanisms for regulation of chromatin characteristics that control the establishment and heritable maintenance of cell type-specific gene expression programs. Alterations in these pathways can result in developmental defects and tumorigenesis, and therefore, an improved understanding of how transcription factors and chromatin features together control cellular differentiation and identity will have an important impact on human health.