JMJD5 Proteolytically Processes Monomethylated Histone H3 N-tail

The histone H3 N-terminal region is a flexible tail which bears residues, with their side chains, that can be methylated, acetylated, and phosphorylated by different nuclear enzymes. Through these side-chain posttranslational modifications, the H3 N-tail contacts with DNA and histones to regulate chromatin folding and gene expression. Specific enzymatic activities have been identified to reverse all the side-chain modifications of these N-tail residues. H3 N-tail modifications also include cleavage of its peptide bond by amide hydrolysis. Emerging evidence reveals that H3 N-tail cleavage differing from H3 protein turnover is also regulated by enzymatic activities. Recently, histone N-terminal region proteolytic cleavage has been shown to be required during cell differentiation in both mammals and yeast to alter gene expression. H3 N-tail cleavage sweeps away N-tail activities along the removed section and creates a new but shorter N-tail. Inasmuch, it serves as one of the posttranslational modifications in response to environmental changes, Cell stress conditions such as nutritional depletion affect H3 N-tail activity by triggering the above modifications including proteolytic cleavage. Nevertheless, the nuclear enzyme responsible for H3 N-tail cleavage remains unknown.Histone H3 N-terminal region (N-tail) in chromatin compaction is under multiple levels of posttranslational modification regulation including methylation, acetylation, phosphorylation as well as proteolytic cleavage. The mechanism underlying H3 N-tail proteolytic cleavage however, remains largely elusive. Here we report that JMJD5, the sole Jumonji C (JmjC) domain containing factor, is responsible for H3 N-tail proteolytic cleavage. The protein's secondary structural alignment revealed that JMJD5 and Cathepsin L (LI and L2) shared the same type of structural orientation. For both JMJD5 and Cathepsin L, their N-helical regions represent the regulatory domains and their C-terminal enzymatic regions extend into a-sheet conformation similar to 5 trypsin-1, which was reported to cleave histone tails at the carboxyl side of lysine or arginine residue, i.e., "K-X", or "R-X" motifs where X can be any residues. Then, In vitro H3 peptide digestion revealed that JMJD5 exclusively cleaves mMK H3-peptides, while little or no cleavage effect of JMJD5 on dimethyl-lysine (dMK), trimethyl-lysine (tMK), or unmethyl-lysine (uMK) H3-peptides was observed. At last, in order to conclude the structural characteristics of JMJD5, we constructed the N-terminal domain (1-270), the JmjC domain (271-416), and JMJD5 full length (1-416) in glutathione S-transferase (GST) recombinant form. Intriguingly, the C-terminal JmjC domain rather than the N-terminal helical domain retained the major H3 binding activity. A strong JmjC domain-JmjC domain interaction was detected which suggests that JMJD5 preferentially undergo a cis-homodimerization between two monomers in the C-terminal JmjC domain, the JMJD5 N-terminal helical domain alone functioned dominant negatively on H3 N-tail cleavage in HeLa cells for over expression of this domain dramatically reduced basal H3 degradation level in HeLa cells. At the same time, we detected some of those mono methylation dependent genes expression in A549 cells, JMJD5 overexpression suppressed those genes including STAT1 and STAT4 with mMK4-H3 or mMK9-H3 in their promoters for expression. In contrast, JMJD5-ND, the dominant negative form, failed to inhibit mMK4/mMK9-dependent gene regulation.Intriguingly, when cells stimulated with MNNG which commomly used in our lab, MMR related genes were up regulated togather with JMJD5 gene dramaticly. Overexpress or SiRNA interfere JMJD5 expression could shorten or extend the MNNG induced cell cycle arrest. JMJD5 was reported as an histone demethylase before, phenomenons above suggested that JMJD5 may induced by MNNG to remove the inhibitive methylation marker (dMK36) on the transcribed region of those MMR genes in order to promote their transcription.Above all, our data suggest that JMJD5 regulates H3 N-tail-dependent gene expression in mammalian cells via mMK9 cleavage which sweeps away en masse modifications along the tail.