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.

在较高的真核生物中，RNA聚合酶II（POL II）暂停是控制的关键调节机制发育，分化，增殖，免疫反应和各种细胞功能。功能障碍调节将导致发育缺陷，不规则免疫反应，癌症，加速衰老，并不同的疾病。较高的真核生物（在人和小鼠中，而不是酵母中）的主要基因（超过30％）的主要部分是由pol II暂停。 +1核小体处暂停的Pol II的释放被认为需要 CDK9的POL II，NELF和DSIF的C末端结构域（CTD）的磷酸化。但是，精确的角色在POL II暂停调节中，CDK9对POL II的CTD的磷酸化尚不清楚，也不是其他暂停释放的机制也有助于。在此提案中，我们提出了一个创新的新想法，如果正确，将是更改范式的。也就是说，除了已知的暂停释放机制外， JMJD5由POL II募集，由CDK9产生的CTD的Ser2磷酸化募集以进行蛋白水解在精氨酸甲基化的组蛋白尾部上的功能，从TSS中产生+1的“无型核小体”，以暂停 pol II要克服。据报道，Hisstone Tails剪裁的现象和Hisstone的高离职率的据报道超过三十年前；但是，由于缺乏确定的酶，该过程仍然很少理解负责剪辑过程。尽管证实了组蛋白精氨酸甲基化的重要性转录调节，这种修饰的确切功能不是很好地理解。同时，组蛋白精氨酸脱甲基酶的身份仍然难以捉摸，尽管有些候选者是分配。我们提出，TSS的+1的核小体上的精氨酸甲基化代表了基因的标记受暂停的pol II监管。此外，一组含有蛋白质家族的Jumonji C（JMJC）域可以在这些核渗透器上特异性夹子组蛋白尾部用甲基化精氨酸。在过去的二十年中，我们和其他研究人员透露JMJC域名家庭成员具有潜水员的酶活性。总体而言，这些功能与JMJC/Cupin样二氧酶有关这是该蛋白质家族的标志的域。现在，我们有越来越多的证据表明JMJC子组域家族，包括JMJD5，JMJD7以及其他可能的域家族，可以用甲基化去除组蛋白尾巴精氨酸通过新型内肽酶和障碍酶活性。我们声称存在三分之一蛋白酶在生命科学中具有内肽酶和毒素酶活性。我们的初步功能数据强烈表明JMJD5和JMJD7专门识别甲基化精氨酸并进行裂解在组蛋白尾部。我们对带有和没有底物的JMJD5和JMJD7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能解释了新型催化组蛋白尾巴上甲基精氨酸的机理和特异性识别。小鼠的JMJD5敲除通往早期胚胎致死。 JMJD5和JMJD7的敲除导致黑色素瘤和乳腺癌的增殖细胞，以及组蛋白亚基的整体量的急剧增加。初步的芯片序列，atac-- Seq，Mnase-Seq和RNA-Seq数据显示了有或没有JMJD5的核小体轮廓的急剧变化雄性MEF细胞。我们提出了在+1的核小体上的精氨酸甲基化组蛋白尾巴的切割由JMJD5，非增生细胞中组蛋白的高周转率，CDK9的POL II的CTD磷酸化，并且暂停的pol II的释放是本质上耦合的。因此，我们的实验室的目的是解决几个关键问题：1）剪裁Hisstone Tails会发挥作用在转录调控中的关键作用？ 2）为什么在不加销的情况下存在较高的Hisstone的离职率细胞？ 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是否存在？ 5）如何在较高的真核生物中调节暂停的RNA聚合酶II（POL II）？ 6）通过 CDK9在Pol II暂停法规中起任何作用？ 7）从转录起始位点+1的核小体如何（TSS）参与法规？为了解决上述这些主要问题，我们提出了三个具体目的：特定目的1：确定JMJD5和JMJD7是否是同源蛋白酶，专门识别具有的组蛋白尾巴 +1的核小体上的甲基化精氨酸以释放暂停的polI。特定目标2：确定催化，激活调节和特定识别的新型机制的结构基础。特定目的3。阐明暂停的Pol II的JMJD5的募集机制和变化具有A和没有JMJD5的核小体的景观。总体而言，我们的研究旨在通过填补临界差距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中一般转录调节的理解中。