Functions of Mammalian H1 Linker Histones in Gene Regulation and Chromatin Activity

哺乳动物 H1 连接组蛋白在基因调控和染色质活性中的功能

• 批准号: 10703445
• 负责人: ARTHUR I SKOULTCHI
• 金额: $ 50.67万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-12 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10703445
• 关键词: ATAC-seq; Address; Affect; Biochemical; Biology; C-terminal; Cell Cycle; Cell Proliferation; ChIP-seq; Chromatin; Chromatin Fiber; Chromosomes; DNA; DNA Polymerase II; Deposition; Development; Drosophila genus; ES Cell Line; Embryonic and Fetal Development; Engineering; Essential Genes; Euchromatin; G1 Phase; Gene Expression Regulation; Gene Family; Genes; Genetic Transcription; Goals; Heterochromatin; Hi-C; Histone H3; Histones; In Vitro; Kinetics; Knowledge; Lysine; Malignant Neoplasms; Mammalian Chromosomes; Messenger RNA; Methods; Methylation; Mitosis; Modeling; Mus; Nucleosomes; Outcome; Phase Transition; Play; Post-Translational Protein Processing; Protein Family; Proteins; RNA; RNA Splicing; Role; S phase; Salivary Glands; Structural Protein; Structure; Testing; Transcription Elongation; Variant; design; embryonic stem cell; epigenetic regulation; human disease; in vivo; insight; mutant; organizational structure; promoter; stem cell survival; transcriptome sequencing

The goals of this proposal are to elucidate the functions of mammalian H1 linker histones in gene regulation and chromatin activity during the cell cycle and to reveal functional differences amongst the 11 variants that constitute the mammalian H1 gene family. Along with the four core histones (H2A, H2B, H3 and H4) that assemble DNA into nucleosomes, the H1 linker histones play key roles in organizing the structure of the chromatin fiber. In comparison to the progress in understanding the roles of core histones, our knowledge about the functions of linker histones in chromatin biology is quite limited. Much of our knowledge about H1’s roles is derived from in vitro studies, whereas our understanding of its functions in vivo remains very incomplete. We propose to help fill this gap by elucidating the functions, mechanisms of action and variant-specific activities of mammalian H1 linker histones in gene regulation and chromatin activity in vivo. Our strategy for studying the in vivo functions of mammalian H1’s has been to generate and characterize mouse embryonic stem cells and mice that are partially depleted of H1 by inactivation of several H1 genes. By analyzing these H1-depleted models, we showed that H1 is essential for mammalian development, plays unexpected roles in epigenetic regulation, and is very important for the integrity and silencing of heterochromatin. Furthermore, our studies with Drosophila H1 showed that it is required for late replication of heterochromatin during endoreplication of salivary gland polytene chromosomes. Recently, we also discovered that, in addition to their important functions in heterochromatin, mammalian H1’s also play important roles in euchromatin where they inhibit deposition of active gene body chromatin marks (methylation of H3K79, H3K36 and H3K4) and affect Pol II transcription elongation and alternative mRNA splicing outcomes. To gain a deeper understanding of the mechanisms by which mammalian H1’s participate in gene regulation and chromatin activities, we propose to pursue the following specific aims: (1) Understand the functions of H1 in RNA Pol II transcription, mRNA splicing and gene body histone H3 lysine methylation; (2) Determine the roles of H1 in late S phase replication timing of heterochromatin and its reorganization during the mitosis to G1 phase transition; (3) Identify functional differences amongst H1 variants and the contributions of their N- and C-terminal domains and post-translational modifications. The successful completion of this project will provide important insights into the ways in which this major constituent of chromatin regulates the structure and activity of mammalian chromosomes.

本研究的目的是阐明哺乳动物H1接头组蛋白在基因调控中的功能 和染色质活性，并揭示11种变异体之间的功能差异， 构成哺乳动物H1基因家族。沿着的是四种核心组蛋白（H2 A、H2 B、H3和H4）， 在将DNA组装成核小体的过程中，H1接头组蛋白在组织核小体的结构中起着关键作用。 染色质纤维与理解核心组蛋白作用的进展相比，我们对 连接组蛋白在染色质生物学中的功能是相当有限的。我们对H1角色的大部分了解 来自体外研究，而我们对它在体内的功能的理解仍然非常不完整。我们 建议通过阐明以下方面的功能、作用机制和变体特异性活动来帮助填补这一空白： 哺乳动物H1接头组蛋白在体内基因调控和染色质活性中的作用。 我们研究哺乳动物H1的体内功能的策略是产生和表征 小鼠胚胎干细胞和通过几种H1基因失活而部分耗尽H1的小鼠。通过 通过分析这些H1缺失的模型，我们发现H1对哺乳动物的发育至关重要， 在表观遗传调控中具有意想不到的作用，并且对于异染色质的完整性和沉默非常重要。 此外，我们对果蝇H1的研究表明，它是异染色质后期复制所必需的， 在唾液腺多线染色体的内复制过程中。最近我们还发现， 除了在异染色质中的重要功能外，哺乳动物H1在常染色质中也发挥着重要作用， 它们抑制活性基因体染色质标记（H3 K79、H3 K36和H3 K4的甲基化）的沉积并影响 Pol II转录延长和可变mRNA剪接结果。 深入了解哺乳动物H1参与基因调控的机制 和染色质的活动，我们建议追求以下具体目标：（1）了解H1的功能， RNA Pol II转录、mRNA剪接和基因体组蛋白H3赖氨酸甲基化;（2）确定基因体组蛋白H3赖氨酸甲基化在RNA Pol II转录和mRNA剪接中的作用 异染色质的复制时机及其在有丝分裂到G1期过程中的重组 （3）识别H1变体之间的功能差异及其N-和C-末端的贡献 结构域和翻译后修饰。 该项目的成功完成将提供重要的见解的方式，这一主要 染色质的组成部分调节哺乳动物染色体的结构和活性。