Histone Arginine Demethylation through Cleavage

通过切割进行组蛋白精氨酸去甲基化

• 批准号: 10693175
• 负责人: GONGYI ZHANG
• 金额: $ 42.62万
• 依托单位: NATIONAL JEWISH HEALTH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-08 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10693175
• 关键词: ATAC-seq; Acceleration; Address; Aging; Aminopeptidase; Arginine; Awareness; Binding; Biological Sciences; Breast Cancer Cell; C-terminal; Catalysis; Cell physiology; Cells; ChIP-seq; Charge; Chromosomes; Clip; Complex; Coupled; Cryoelectron Microscopy; DNA Polymerase I; Data; Defect; Development; Dioxygenases; Disease; Embryo; Embryonic Development; Endopeptidases; Enzymes; Epigenetic Process; Eukaryota; Eukaryotic Cell; Exclusion; Exopeptidase; Family; Family member; Functional disorder; Genes; Genetic Transcription; Genome; Histones; Homeostasis; Human; Immune response; In Vitro; Knock-out; Lead; Malignant Neoplasms; Methylation; Modeling; Modification; Mus; N-terminal; Nucleosomes; Peptide Hydrolases; Phosphorylation; Play; Polymerase; Process; Proliferating; Property; Protein Family; Proteins; RNA Polymerase II; Regulation; Reporting; Research Personnel; Resolution; Role; Structure; Subgroup; Surface; Tail; Time; Transcription Initiation Site; Transcriptional Regulation; Yeasts; demethylation; histone methylation; in vivo; innovation; male; melanoma; negative elongation factor; novel; recruit; transcriptome sequencing

In higher eukaryotes, RNA Polymerase II (Pol II) pausing is a critical regulation mechanism controlling development, differentiation, proliferation, immune response, and all variety of cell function. Dysfunction of the regulation will lead to developmental defects, irregular immune responses, cancers, accelerating aging, and different diseases. A major portion (over ~30%) of genes in higher eukaryotes (in human and mice, not in yeast) are regulated by Pol II pausing. The release of paused Pol II at the +1 nucleosome is thought to require phosphorylation of C-terminal domain (CTD) of Pol II, NELF, and DSIF by CDK9. However, the precise role of phosphorylation of CTD of Pol II by CDK9 in Pol II pausing regulation is not well understood, nor if other mechanisms for pause release also contribute. In this proposal, we are proposing an innovative new idea that, if correct, will be paradigm changing. That is that in addition to the known mechanisms for pause release, JMJD5 is recruited by Pol II with Ser2 phosphorylation of CTD generated by CDK9 to carry out its proteolytic function on arginine methylated histone tails to generate “Tailless Nucleosomes” at +1 from TSS for paused Pol II to overcome. The phenomenon of clipping of histone tails and high turnover rate of histone was reported more than three decades ago; however, this process is still poorly understood in part due to the lack of identified enzymes responsible for the clipping process. Despite the confirmed importance of methylation of histone arginines in transcriptional regulation, the exact function of this modification is not very well understood. At the same time, the identities of histone arginine demethylases have remained elusive, though some candidates have been assigned. We propose that arginine methylation on nucleosomes at +1 from TSS represent a marker for genes regulated by paused Pol II. Furthermore, a group of Jumonji C (JmjC) domain containing protein family could specifically clip histone tails with methylated arginines on these nucleosomes. In the past two decades, we and other researchers have revealed that the JmjC domain family members have diverse enzymatic activities. Overall, these functions are related to the JmjC/cupin-like dioxygenase domains that are the hallmark of this protein family. We now have growing evidence that a subgroup of JmjC domain family, including JMJD5, JMJD7, and possibly others, may remove histone tails with methylated arginines through novel endopeptidase and aminopeptidase activities. We claimed that there exists a third protease family in life science with both endopeptidase and exopeptidase activities. Our preliminary functional data strongly suggests that JMJD5 and JMJD7 specifically recognize methylated arginines and make cleavages in the context of histone tails. Our structural analysis of JMJD5 and JMJD7 with and without substrates revealed unique features and surface charge distribution properties of these proteins that may account for novel catalysis mechanism and specific recognition of methyl-arginine on histone tails. Knockout of JMJD5 in mice leads to early embryonic lethal. Knockouts of JMJD5 and JMJD7 lead to proliferation arrest of melanoma and breast cancer cells, as well as the dramatic increase in the overall amount of histone subunits. Preliminary ChIP-seq, ATAC- seq, MNase-seq, and RNA-seq data show drastic changes of nucleosomes profile with and without JMJD5 in a male MEF cells. We propose that cleavage of arginine methylated histone tails on nucleosomes at +1 from TSS by JMJD5, high turnover rate of histone in non-proliferating cells, phosphorylation of CTD of Pol II by CDK9, and the release of paused Pol II, are intrinsically coupled. Our lab therefore aims to address several critical questions: 1) Does the clipping of histone tails play critical roles in transcription regulation? 2) Why there exists a high turnover rate of histone in non-proliferating cells? 2) What is the exact role of histone arginine methylation? 4) Do histone arginine demethylases exist? 5) How paused RNA Polymerase II (Pol II) is regulated in higher eukaryotes? 6) Does CTD phosphorylation by CDK9 play any role in Pol II pausing regulation? 7) How does nucleosome at +1 from transcription start site (TSS) participate the regulation? To address these major questions mentioned above, we propose three specific aims: Specific aim 1: To determine if JMJD5 and JMJD7 are cognate proteases that specifically recognize histone tails with methylated arginines on nucleosomes at +1 to release paused Pol I. Specific aim 2: Determine the structural basis of the novel mechanisms of catalysis, activation regulation, and specific recognition. Specific aim 3. To elucidate the recruitment mechanism of JMJD5 by paused Pol II and changes of landscapes of nucleosomes with a and without JMJD5. Overall, our studies aim to solve a conundrum in the field of epigenetics and transcription by filling a critical gap in our understanding of general transcription regulation in higher eukaryotes.

在高等真核生物中，RNA聚合酶II（Pol II）的停顿是一种重要的调控机制， 发育、分化、增殖、免疫应答和各种细胞功能。功能障碍 调节将导致发育缺陷，不规则的免疫反应，癌症，加速衰老， 不同的疾病。 高等真核生物（人类和小鼠，而不是酵母）中的大部分基因（超过30%）是 由Pol II暂停调节。在+1核小体处暂停的Pol II的释放被认为需要 通过CDK 9对Pol II、NELF和DSIF的C-末端结构域（CTD）的磷酸化。然而， 在Pol II暂停调节中，CDK 9对Pol II CTD的磷酸化还没有很好的理解，如果其他的 用于暂停释放的机制也有贡献。在这份提案中，我们提出了一个创新的新想法， 如果正确的话，将是范式的改变。即除了用于暂停释放的已知机制之外， JMJD 5被Pol II募集，其中CTD的Ser 2磷酸化由CDK 9产生，以进行其蛋白水解。 在精氨酸甲基化组蛋白尾部上的功能，以在TSS的+1处产生“无尾核小体”，用于暂停 二是克服。 组蛋白尾部剪切和组蛋白高更新率现象的报道超过 然而，由于缺乏确定的酶，这一过程仍然知之甚少 负责剪辑过程。尽管组蛋白甲基化的重要性已被证实， 虽然这种修饰与转录调控密切相关，但这种修饰的确切功能还不是很清楚。与此同时， 组蛋白精氨酸脱甲基酶的身份仍然难以捉摸，尽管一些候选者已经被发现。 分配。我们认为TSS +1处核小体上的精氨酸甲基化是基因的标记， 由暂停的Pol II调节。此外，一组含有Jumonji C（JmjC）结构域的蛋白质家族可 特异性地用这些核小体上的甲基化精氨酸剪切组蛋白尾部。 在过去的二十年里，我们和其他研究人员已经发现，JmjC结构域家族成员 具有多种酶活性。总体而言，这些功能与JmjC/cupin样双加氧酶有关 这是这个蛋白质家族的标志。我们现在有越来越多的证据表明JmjC的一个亚群 结构域家族，包括JMJD 5、JMJD 7和可能的其他结构域家族，可以去除具有甲基化的组蛋白尾部， 通过新的内肽酶和氨肽酶的活性来合成氨基糖苷。我们声称存在第三个 蛋白酶家族在生命科学中具有内肽酶和外肽酶活性。我们的初步功能 数据有力地表明，JMJD 5和JMJD 7特异性地识别甲基化的丝氨酸并进行裂解 in the context上下文of histone组蛋白tails尾.我们对JMJD 5和JMJD 7在有和没有底物的情况下的结构分析显示， 这些蛋白质的独特特征和表面电荷分布特性可以解释新的催化作用 甲基精氨酸对组蛋白尾部的特异性识别。小鼠中JMJD 5的敲除导致早期 胚胎致死JMJD 5和JMJD 7的敲除导致黑色素瘤和乳腺癌的增殖停滞 细胞，以及组蛋白亚基总量的急剧增加。初步ChIP-seq，ATAC- seq、MNase-seq和RNA-seq数据显示，在有和没有JMJD 5的情况下， 雄性MEF细胞。我们提出核小体上的精氨酸甲基化组蛋白尾部在距离TSS +1处发生切割 非增殖细胞中组蛋白的高周转率，Pol II的CTD被CDK 9磷酸化，以及 暂停的Pol II的释放是内在耦合的。 因此，我们的实验室旨在解决几个关键问题：1）组蛋白尾部的剪切是否起作用？ 在转录调控中的重要作用2)为什么非增殖细胞中存在高的组蛋白周转率 细胞？2)组蛋白精氨酸甲基化的确切作用是什么？4)组蛋白精氨酸脱甲基酶存在吗？第五章） RNA聚合酶II（Pol II）在高等真核生物中是如何被调控的？6)CTD磷酸化是否通过 CDK 9在Pol II暂停调节中起作用吗？7)位于转录起始位点+1的核小体 (TSS)参与监管？ 为了解决上述主要问题，我们提出三个具体目标：具体目标1： 为了确定JMJD 5和JMJD 7是否是特异性识别组蛋白尾部的同源蛋白酶， 在+1处，核小体上的甲基化精氨酸释放暂停的Pol I。具体目标2：确定 催化、活化调节和特异性识别的新机制的结构基础。 具体目标3。为了阐明JMJD 5通过暂停Pol II的募集机制， 具有和不具有JMJD 5的核小体的景观。 总的来说，我们的研究旨在通过填补一个关键的空白来解决表观遗传学和转录领域的难题。 在我们对高等真核生物一般转录调控的理解中。