Set1-catalyzed H3K4 Trimethylation Regulates Histone Gene Expression And Cell Aging

In eukaryotic cells,the basic structural unit of chromatin is the nucleosome,which is formed by the core histones H3,H4,H2 A and H2 B and 147 bp of core DNA.To maintain normal chromatin structure and function,in DNA replication,cells not only need to synthesize large amounts of histones,but also limit most histone synthesis to S phase.Insufficient histone levels may trigger cell cycle arrest in Saccharomyces cerevisiae and disrupt the S phase of mammals.Decreased or absent expression of core histones during DNA replication disrupts chromatin structure,delays S phase and leads to mitotic arrest.Overexpression or excessive accumulation of histones outside the S phase is also toxic to cells.In summary,misregulation of histone gene expression leads to defects in cell cycle progression,genomic stability,DNA damage response and transcriptional regulation.Therefore,it is important to study the regulatory factors and regulatory mechanism of histone gene expression.Although individual histone modifications have been reported to regulate histone gene expression,the effect of most histone modifications on histone expression is unclear.In this study,we identified the amino acid residues and histone post-translational modifications that regulate histone gene expression by screening histone H3/H4 mutant libraries and elucidated the underlying mechanisms.The following are our research: 1.Screen histone H3/H4 mutant library.The histone H3/H4 mutant library was screened by Western blot to look for regulatory factors that could change histone levels.The results showed that the histone levels were reduced in 15 point mutations,and the histone levels were increased in 5 point mutations.2.Study the effect of Set1-catalyzed H3K4me3 on intracellular histone expression.Among the above 20 mutants,we found that mutation of H3K4 significantly reduced histone levels.H3K4 can be methylated and methylated H3K4 is directly related to transcription,so we focused on the regulation of H3K4 methylation on histone expression.In Saccharomyces cerevisiae,Set1 is the only enzyme that catalyzes H3K4 methylation.Loss of SET1 not only dimishes H3K4me3,but also reduces histone expression.At the same time,knocking out SPP1,which is the subunit of Set1 complex and specifically regulates H3K4me3,or mutating the histone sites that affects H3K4me3(H3R2A,H3T6 A,H3K14A/Q),reduce intracellular histone histones.These results demonstrate that Set1 complex catalyzed H3K4me3 is essential for maintaining normal intracellular histone potein levels.Further,we find that Set-catalyzed H3K4me3 positively regulates histone gene transcription by q RT-PCR.3.Investigete the mechanism by which Set1-catalyzed H3K4me3 regulates histone gene expression.In the histone gene pair HHT1-HHF1,HHT2-HHF2,HTA1-HTB1,there is a negative regulatory region(NEG),which is bound the HIR/Asf1/Rtt106 complex,and Rtt106 inhibits histone genes expression.We explored whether the mutation of H3K4 and the deletion of SET1 had an effect on the binding of Rtt106 at the histone locus by chromatin immunoprecipitation(Ch IP).These results showed that Rtt106 reduced at NEG regions but significantly increased in the mutant,indicating that the Set1-catalyzed H3K4me3 prevents Rtt106 spread to the histone gene coding region.Further,we examined the binding ability of Rtt106 to histones in wild-type strains and set1? mutants by immunoprecipitation(in vivo and in vitro Co-IP),and found that knockdown of SET1 significantly reduced the binding of Rtt106 to histones.Next,we examined the interaction between Rtt106 and H3K4me3 recombinant histones or H3K4me3 octamers.The results showed that H3K4me3 directly inhibited Rtt106 bind to histones.These data demonstrate that Set1-catalyzed H3K4me3 restricts Rtt106 spread from the NEG region of three HIR-dependent loci to the coding region of the gene by hindering the binding of Rtt106 to histones.4.Set1-catalyzed H3K4me3 regulates chronological life span.There is a tight correlation between histone gene expression and cell chronological life span.Set1-catalyzed H3K4me3 regulates histone gene expression,so we analyze the effection of histone gene expression on chronological life span by aging curve assay,and found that Set1-catalyzed H3K4me3 maintains nomal cell chronological life span by promoting histone gene expression.Our research not only reveal the new mechanism of histone expression regulation from the perspective of histone modifications,but also provide new clues for histone expression regulating cell aging,and provide a good target for the study of anti-aging drugs.