In Vivo Analysis of Mouse H1 Histone Function

描述（由申请人提供）：该项目的长期目标是提高我们对H1连接组蛋白功能的认识，并了解该染色质蛋白家族中存在的多样性的功能意义。在大多数真核细胞中，H1连接蛋白几乎与核小体核心颗粒一样丰富。因此，H1组蛋白在染色质纤维的结构中起着关键作用。H1组蛋白影响基因表达以及许多其他需要接触DNA的过程。我们对H1功能的了解大多来自体外实验。我们的方法是分析H1组蛋白在小鼠体内和胚胎干细胞（ES）中的功能。小鼠（和其他哺乳动物）H1组蛋白由至少8种非等位变异或亚型组成，它们的初级序列和在发育和组织分化过程中的表达差异很大。这些亚型提供了额外水平的染色质功能调节。我们研究连接体组蛋白功能的策略是生成和表征一个或多个H1基因被基因靶向灭活的小鼠和胚胎干细胞系。我们已经产生了大量的小鼠菌株，包括单一null突变体（8种亚型中的6种已灭活）和复合null菌株。我们最具信息量的菌株之一有3个H1基因同时失活（三重敲除，TKO）。这种突变表明，与不太复杂的真核生物不同，H1组蛋白对哺乳动物的发育至关重要。分析了部分突变株和TKO ES细胞对基因表达的影响。结果表明，与体外实验得出的结论相反，H1不是转录的全局抑制因子。重要的是，我们发现H1参与促进印迹基因位点的DNA甲基化。我们现在建议使用我们独特的小鼠品系和胚胎干细胞系：(1)了解H1控制DNA甲基化的机制。我们还建议在全基因组范围内鉴定H1影响DNA甲基化的基因组位点。(2)评估H1翻译后修饰和H1亚型多样性在H1调节基因表达和DNA甲基化能力中的作用。(3)开发新的小鼠突变株和ES细胞系，特别是条件三重H1 null，并利用它们研究H1在出生后发育、细胞分化和组织特异性基因表达中的作用。我们的研究表明，H1组蛋白密切参与控制基因表达、DNA甲基化、印迹和其他表观遗传修饰。这些过程是正常发育所必需的，它们的异常与癌症和其他人类疾病有关。我们认为我们有一个独特的机会来帮助理解真核生物染色体的这个主要组成部分的许多重要功能。

DESCRIPTION (provided by applicant): The long-term goals of this project are to advance our knowledge about the functions of H1 linker histones and to understand the functional significance of the diversity present in this family of chromatin proteins. In most eukaryotic cells H1 linker histones are nearly as abundant as nucleosome core particles. Therefore, H1 histones play a key role in the structure of the chromatin fiber. H1 histones affect gene expression as well as many other processes requiring access to DNA. Much of our knowledge about the functions of H1 has been derived from in vitro experiments. Our approach is to analyze the functions of H1 histones in vivo in mice and in embryonic stem (ES) cells. The mouse (and other mammalian) H1 histones consist of at least 8 nonallelic variants or subtypes that differ considerably in their primary sequences and in their expression during development and tissue differentiation. These subtypes offer an additional level of regulation of chromatin function. Our strategy for studying the functions of linker histones has been to generate and characterize mice and ES cell lines in which one or more H1 genes have been inactivated by gene targeting. We have generated a large collection of mouse strains consisting of both single null mutants (6 of the 8 subtypes have been inactivated) and compound null strains. One of our most informative strains has 3 H1 genes inactivated simultaneously (triple knock-out, TKO). This mutant demonstrated that, unlike in less complex eukaryotes, H1 histones are essential for mammalian development. Some of the mutant strains and TKO ES cells have been analyzed for effects on gene expression. The results showed that, contrary to conclusions derived from in vitro experiments, H1 is not a global repressor of transcription. Importantly, we discovered that H1 is involved in promoting DNA methylation at imprinted gene loci. We now propose to use our unique set of mouse strains and ES cell lines to: (1) Understand the mechanism(s) by which H1 controls DNA methylation. We also propose to identify on a genome-wide scale the sites in the genome at which H1 affects DNA methylation. (2) Assess the role of H1 posttranslational modifications and H1 subtype diversity in the ability of H1 to regulate gene expression and DNA methylation. (3) Develop new mutant mouse strains and ES cell lines, specifically a conditional triple H1 null, and use them to study the role of H1 in postnatal development, cell differentiation and tissue-specific gene expression. Our studies suggest that H1 histones are intimately involved in controlling gene expression, DNA methylation, imprinting and other epigenetic modifications. These processes are required for normal development and abnormalities in them are associated with cancer and other human diseases. We think we have a unique opportunity to help understand some of the numerous important functions of this major component of eukaryotic chromosomes.