In Vivo Analysis of Mouse H1 Histone Function

描述(由申请人提供)：这个项目的目标是促进我们对H1连接器组蛋白功能的了解，并了解这个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起关键作用，从而影响基因表达以及其他需要DNA的过程。我们关于H1组蛋白功能的大部分知识都来自于体外实验。我们的方法是分析H1组蛋白在小鼠体内的功能。小鼠(和其他哺乳动物)的H1组蛋白由至少8个亚型组成，这些亚型在它们的初级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、潜在的染色质功能调节水平。我们研究连接子组蛋白功能的策略一直是通过基因打靶产生和鉴定一个或多个H1基因失活的小鼠。我们已经产生了一个庞大的小鼠品系，包括6只单一的H1缺失小鼠和几个复合缺失品系和培养的细胞系。我们使用这些突变体来表明，与低等生物不同，H1组蛋白对哺乳动物的发育是必不可少的。一些突变体已经分析了它们对基因表达的影响。结果表明，个体亚型的丧失以及H1总量的减少会导致特定基因表达的变化。我们现在建议使用我们独特的一组小鼠品系来：(1)研究个体H1亚型和H1化学计量比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缺失细胞和小鼠中特定基因附近的染色质结构；以及(2)研究H1连接体组蛋白在体内染色质的结构、组成和翻译后修饰中的作用。我们还建议开发极低含量或完全缺乏H1组蛋白的新细胞系。我们还建议通过基因替换策略在小鼠身上进行特定H1亚型功能的体内测试。有证据表明，H1组蛋白是视网膜母细胞瘤蛋白调节的细胞周期蛋白依赖的蛋白的下游靶点。因此，在正常和恶性细胞中，H1组蛋白可能是细胞周期调节与染色质结构、基因表达和其他基因组活动之间的关键信息转导。

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.