The Study Of Histone Crotonylation Biological Function And Decrotonylases And Large-scale Identification Of Lysine Crotonylation

Histone crotonylation is a new covalent type of modification which is a dynamic process analogus to histone acetylation regulated by HCTs(Histone crotonyltransferases)and HDCRs(Histone decrotonylases).Previous studies state that CBP/p300 have the HCT enzymatic activity.But the HDCRs are still needs to be identified and the biological functions of histone crotonylation are largely unknown.First,we have examined and identified the major cellular enzyme for histone decrotonylation.Base on the ball-and-stick models of a crotonyl group and an acetyl group,We used immunofluorescence staining assays to screen the 18 members of HDAC(Histone deacetylase)family,our results demonstrate that among histone deacetylase SIRT1 and class ? HDACs are active for histone decrotonylation and class? HDACs are the major histone decrotonylases.Importantly,we have generated a novel HDAC1(AGG-VRPP)mutant which maintains HDCR but loss HDAC activity.Our luciferase reportor assay results provide first evidence that histone decrotonylation without deacetylation is sufficient to repress transcription.RNA-seq data and ChIP-qPCR data suggest that decrotonylation without deacetylation is able to repress transcription as broadly as both decrotonylation and deacetylation.Compared with the results of HDAC1 wild-type and HDAC1(AGG-VRPP)mutant,we can conclude that histone crotonylation is not functionally redundant as histone acetylation and is likely to be essential for transcription.Second,we identified the substrates of lysine crotonylation by LC-MS/MS and studied the biological function of lysine crotonylation.Since we want to identify as many crotonylated proteins as possible,we treated HeLa cells with 20mM sodium crotonate(NACR)which can dramatically increase protein crotonylation level in cells.The total proteins from HeLa cells were digested to different peptides by trypsin,and then enriched by pan-lysine crotonylation antibody for crotonylated peptides.Our results show that 366 new crotonylated proteins and 550 new crotonylated sites were identified by LC-MS/MS.Then we analyse the crotonylation proteomics profile.GO analysis results indicate that these proteins are involved in different important biological processes,including RNA processing,mRNA metabolic process,chromosome organization,DNA metabolic process,DNA repair,ribosome biogenesis,DNA packaging and chromatin assembly process.Quantitative MS results show that crotonylation modification can repress the DNA replication related proteins such as MCM 4,MCM 5,MCM 6,MCM 7 and PCNA to binding to chromatin and regulate the cell cycle,Comparing acetylome with crotonylome,the results suggest that some proteins can be both crotonylated and acetylated.This implies that these two types of lysine modifications may have different cellular functions.