In Vivo Analysis of Mouse H1 Histone Function

描述（由申请人提供）：该项目的目标是提高我们对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。H1连接体组蛋白在染色质结构中发挥关键作用，从而影响基因表达以及其他需要进入DNA的过程。我们对H1组蛋白功能的了解大多来自体外实验。我们的方法是在小鼠体内分析H1组蛋白的功能。小鼠（和其他哺乳动物）H1组蛋白由至少8个亚型组成，它们的主要序列和在发育和组织分化过程中的表达都有很大的不同。这些亚型提供了一个额外的，潜在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一个或多个H1基因被基因靶向灭活的小鼠并对其进行表征。我们已经产生了大量的小鼠菌株，包括6只H1阴性小鼠和几个复合阴性小鼠和培养细胞系。我们利用这些突变体表明，与低等生物不同，H1组蛋白对哺乳动物的发育至关重要。已经分析了一些突变体对基因表达的影响。结果表明，单个亚型的缺失以及H1总量的减少导致了特定基因表达的变化。我们现在建议使用我们独特的小鼠品系：(1)研究个体H1亚型和H1化学计量影响体内基因转录和调控的机制。我们将比较野生型和突变型h1 -贫细胞和小鼠中特定基因附近的染色质结构；(2)研究H1连接体组蛋白在体内染色质结构、组成和翻译后修饰中的作用。我们还建议开发含有极少量或完全缺乏H1组蛋白的新细胞系。我们还建议通过基因替代策略在小鼠中进行特异性H1亚型功能的体内测试。有证据表明，H1组蛋白是由视网膜母细胞瘤蛋白调节的周期蛋白依赖性激酶的下游靶点。因此，在正常和恶性细胞中，H1组蛋白可能是细胞周期调节因子与染色质结构、基因表达和基因组其他活动之间的关键信息转导器。

DESCRIPTION (provided by applicant): The 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. H1 linker histones play a key role in the structure of chromatin and thereby affect gene expression as well as other processes requiring access to DNA. Most of our knowledge about the functions of H1 histones has been derived from in vitro experiments. Our approach is to analyze the functions of H1 histones in vivo in mice. The mouse (and other mammalian) H1 histones consist of at least 8 subtypes that differ considerably, both in their primary sequences and in their expression during development and tissue differentiation. These subtypes offer an additional, potential level of regulation of chromatin function. Our strategy for studying the function of linker histones has been to generate and characterize mice in which one or more H1 genes has been inactivated by gene targeting. We have generated a large repertoire of mouse strains consisting of 6 single H1 null mice and several compound null strains and cultured cell lines. We have used these mutants to show that, unlike in lower organisms, H1 histones are essential for mammalian development. Some of the mutants have been analyzed for their effects on gene expression. The results show that loss of individual subtypes as well as reduction in total amount of H1 causes changes in expression of specific genes. We now propose to use our unique set of mouse strains to: (1) study the mechanisms by which individual H1 subtypes and H1 stoichiometry affect gene transcription and regulation in vivo. We will compare the chromatin structure in the vicinity of specific genes in wild-type and mutant H1-depleted cells and mice; and (2) study the role of H1 linker histones in the structure, composition and post-translational modifications of chromatin in vivo. We also propose to develop new cell lines with extremely low amounts or completely lacking H1 histones. We also propose to perform an in vivo test of specific H1 subtype function by a gene replacement strategy in mice. There is evidence that H1 histones are downstream targets of cyclin-dependent kinases regulated by the retinoblastoma protein. Thus H1 histones may be key transducers of information between cell cycle regulators and chromatin structure, gene expression and other activities of the genome in normal and malignant cells.