Histone Arginine Demethylation through Cleavage

通过切割进行组蛋白精氨酸去甲基化

• 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
• 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

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(PolII)的暂停是一种关键的调控机制 发育、分化、增殖、免疫反应等各种细胞功能。脑部功能障碍 监管将导致发育缺陷、免疫反应不规律、癌症、加速衰老和 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暂停调整。核小体+1处暂停的POL II的释放被认为需要 CDK9对POL II、NELF和DSIF C末端结构域(CTD)的磷酸化然而，它的确切作用是 在POL II中，CDK9对POL II中CTD的磷酸化还不是很清楚，也不是很清楚 暂停释放的机制也起到了作用。在这项提案中，我们提出了一个创新的新想法， 如果是正确的，将是范式的改变。也就是说，除了用于暂停释放的已知机制之外， JMJD5通过CDK9产生的CTD的Ser2磷酸化被POL II招募来进行其蛋白降解 TSS对精氨酸甲基化的组蛋白尾巴产生+1无尾核小体的作用 波尔II需要克服。 组蛋白尾部被剪断、组蛋白转化率高的现象已有较多报道。 然而，三十年前，人们对这一过程仍然知之甚少，部分原因是缺乏已确定的酶 负责剪裁过程。尽管已证实组蛋白精氨酸甲基化在 转录调控，这种修饰的确切功能还不是很清楚。同时， 组蛋白精氨酸脱甲基酶的特性仍然难以捉摸，尽管一些候选基因已经 已分配。我们认为TSS核小体+1处的精氨酸甲基化是基因的一个标志 受暂停的Pol II调节。此外，一组含有Jumonji C(JmjC)结构域的蛋白家族可以 具体地说，在这些核小体上用甲基化精氨酸剪切组蛋白尾巴。 在过去的二十年里，我们和其他研究人员揭示了JmjC结构域的家族成员 具有不同的酶活性。总体而言，这些功能与JmjC/Cupin样双加氧酶有关 这些结构域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JMJD5、JMJD7和其他可能的家族，可能会去掉甲基化的组蛋白尾巴 精氨酸通过新的内肽酶和氨基肽酶活性。我们声称存在第三个 具有内肽酶和外肽酶活性的生命科学中的蛋白水解酶家族。我们的初步职能部门 数据有力地表明，JMJD5和JMJD7特异性地识别甲基化的精氨酸并进行切割 在组蛋白尾巴的背景下。我们对有底物和无底物的JMJD5和JMJD7的结构分析揭示了 这些蛋白质的独特特征和表面电荷分布特性可能是新型催化作用的原因 甲基精氨酸在组蛋白尾巴上的作用机理和特异性识别。小鼠JMJD5基因敲除导致早期 胚胎致死。JMJD5和JMJD7基因敲除导致黑色素瘤和乳腺癌的增殖受阻 细胞，以及组蛋白亚基总量的急剧增加。初步芯片-SEQ，ATAC- SEQ、MNase-seq和RNA-seq数据显示，在有和没有JMJD5的情况下， 雄性MEF细胞。我们认为TSS中精氨酸甲基化的组蛋白尾巴在核小体上的+1处被切割 通过JMJD5，非增殖细胞中组蛋白的高周转率，CDK9对Pol II的CTD的磷酸化，以及 释放暂停的Pol II，本质上是耦合的。 因此，我们的实验室致力于解决几个关键问题：1)组蛋白尾巴的剪裁是否起作用 转录调控中的关键作用？2)为什么组蛋白在非增殖期有很高的转换率 细胞？2)组蛋白精氨酸甲基化的确切作用是什么？4)组蛋白精氨酸去甲基酶存在吗？5) 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的磷酸化是通过 CDK9在POL II暂停调控中起什么作用？7)核小体如何从转录起始点+1开始 (TSS)参与监管？ 针对上述主要问题，我们提出了三个具体目标：具体目标1： 确定JMJD5和JMJD7是否是专门识别组蛋白尾巴的同源蛋白酶 核小体+1上的甲基化精氨酸释放暂停的Pol I特定目标2：确定 催化、激活调节和特异性识别等新机制的结构基础。 具体目的3.阐明停顿的Pol II对JMJD5的招募机制和细胞周期的变化 有无JMJD5的核小体景观。 总体而言，我们的研究旨在通过填补一个关键空白来解决表观遗传学和转录领域的一个难题 在我们对高等真核生物一般转录调控的理解中。