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.

Public Health Relevance

The cleavage of histone tails, arginine demethylation of histone tails, high turnover rate of histone in non-proliferating cells, RNA Polymerase II (Pol II) pausing regulation in higher eukaryotes, roles of nucleosomes at +1 in regulating Pol II pausing, and the role of the phosphorylation of carboxyl terminal domain of Pol II by CDK9 in pausing regulation, remain poorly understood and controversial. We found that a group of Jumonji C domain containing proteins, which are essential for embryonic development and highly expressed in numerous cancers, could specifically remove histone tails with methylation on arginine residues to generate ?Tailless Nucleosomes?, and propose that cleavage of histone tails, arginine demethylation of histone tails, high turnover rate of histone in non-proliferating cells, and Pol II release from pausing, are intrinsically coupled. !

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function B Study Section (MSFB)
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Carter, Anthony D
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National Jewish Health
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