Histone Arginine Demethylation through Cleavage

通过切割进行组蛋白精氨酸去甲基化

基本信息

批准号：
10256759
负责人：
GONGYI ZHANG
金额：
$ 43.36万
依托单位：
NATIONAL JEWISH HEALTH
依托单位国家：
美国
项目类别：
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/10256759
关键词：
ATAC-seq 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 DNA Polymerase II DNA-Directed RNA Polymerase Data Defect Development Dioxygenases Disease Embryo Embryonic Development Endopeptidases Enzymes Epigenetic Process Eukaryota Eukaryotic Cell 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 Process Property Protein Family Proteins Regulation Reporting Research Personnel Resolution Role Structure Subgroup Surface Tail Time Transcription Initiation Site Transcriptional Regulation Yeasts base demethylation histone methylation in vivo innovation male malignant breast neoplasm melanoma negative elongation factor novel recruit transcriptome sequencing

项目摘要

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) 暂停是控制发育、分化、增殖、免疫反应和各种细胞功能障碍。调节将导致发育缺陷、不规则的免疫反应、癌症、加速衰老和不同的疾病。高等真核生物（人类和小鼠，而非酵母）中的大部分基因（超过 30%）是 Pol II 暂停的调节被认为需要在 +1 核小体处释放暂停的 Pol II。 CDK9 对 Pol II、NELF 和 DSIF 的 C 末端结构域 (CTD) 进行磷酸化。 Pol II 暂停调节中 CDK9 对 Pol II CTD 的磷酸化尚不清楚，也不清楚其他情况暂停释放机制也有所贡献。在这项提案中，我们提出了一个创新的想法：如果正确的话，将会改变范式。也就是说，除了已知的暂停释放机制之外， JMJD5 被 Pol II 招募，并通过 CDK9 产生的 CTD Ser2 磷酸化来进行蛋白水解精氨酸甲基化组蛋白尾部的功能，在 TSS 的 +1 处生成“无尾核小体”，用于暂停 Pol II 需要克服。组蛋白尾部剪切和组蛋白高周转率的现象被报道超过三十年前；然而，由于缺乏已识别的酶，这一过程仍然知之甚少。尽管已证实组蛋白精氨酸甲基化的重要性。转录调控，但这种修饰的确切功能尚不清楚。组蛋白精氨酸去甲基化酶的身份仍然难以捉摸，尽管一些候选者已被证实我们认为 TSS 的+1 处核小体上的精氨酸甲基化代表基因的标记。此外，一组包含 Jumonji C (JmjC) 结构域的蛋白家族可以受暂停的 Pol II 调节。在这些核小体上用甲基化精氨酸特异性地夹住组蛋白尾部。在过去的二十年里，我们和其他研究人员揭示了 JmjC 域家族成员总体而言，这些功能与 JmjC/cupin 样双加氧酶相关。结构域是该蛋白质家族的标志，我们现在有越来越多的证据表明 JmjC 的一个亚组。结构域家族，包括 JMJD5、JMJD7 和可能的其他家族，可能会去除甲基化的组蛋白尾部我们声称存在第三种通过新型内肽酶和氨肽酶活性产生的精氨酸。生命科学中的蛋白酶家族具有肽链内切酶和肽外切酶活性。数据强烈表明 JMJD5 和 JMJD7 特异性识别甲基化精氨酸并进行切割我们对有底物和无底物的 JMJD5 和 JMJD7 进行结构分析。这些蛋白质的独特特征和表面电荷分布特性可能解释新的催化作用组蛋白尾部甲基精氨酸的机制和特异性识别 JMJD5 小鼠中导致早期。 JMJD5 和 JMJD7 的敲除会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。 seq、MNase-seq 和 RNA-seq 数据显示，在有和没有 JMJD5 的情况下，核小体谱发生了巨大变化。我们建议在 TSS 的 +1 处切割核小体上的精氨酸甲基化组蛋白尾部。 JMJD5、非增殖细胞中组蛋白的高周转率、CDK9 对 Pol II 的 CTD 进行磷酸化，以及暂停的 Pol II 的释放本质上是耦合的。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 2）为什么非增殖细胞中组蛋白周转率较高 2) 组蛋白精氨酸甲基化的确切作用是什么？ 4) 组蛋白精氨酸去甲基化酶是否存在？暂停的 RNA 聚合酶 II (Pol II) 在高等真核生物中是如何调节的？ 6) CTD 磷酸化是否受以下因素影响？ CDK9 在 Pol II 暂停调节中发挥任何作用？ 7) 转录起始位点 +1 处的核小体如何发挥作用？（TSS）参与监管？针对上述主要问题，我们提出三个具体目标：具体目标1：确定 JMJD5 和 JMJD7 是否是特异性识别组蛋白尾部的同源蛋白酶核小体上 +1 处的甲基化精氨酸释放暂停的 Pol I。具体目标 2：确定催化、激活调节和特异性识别新机制的结构基础。具体目标 3. 通过暂停 Pol II 和变化来阐明 JMJD5 的招募机制有和没有 JMJD5 的核小体景观。总的来说，我们的研究旨在通过填补关键空白来解决表观遗传学和转录领域的难题帮助我们理解高等真核生物的一般转录调控。