Lysine Crotonylation:identification Of Transferase,function And The Study Of Crosstalk With H3K4 Methylation

Histone posttranslational modifications are critically important for epigenetic regulation within which lysine acetylation is best known for its positive role in transcriptional regulation.Recent studies indicate that in addition to acetylation,lysine residues in histones are subjected to various types of fatty acylations including propionylation,butyrylation and crotonylation.Lysine crotonylation,initially identified on core histones by mass spectrometry,has been shown to involve in spermatogenesis and active gene expression in inactive X chromosome.However,prior to our study the enzymes responsible for histone lysine crotonylation and the function of lysine crotonylation remain poorly defined.In this study we focus our effort on the identification and functional characterization of histone lysine crotonyltransferases(HCT).By using immunofluorescence staining,we screened for HCT activity among known histone acetyltransferases(HAT).We found that among the known HATs,CBP/p300 and MOF but not others possess HCT activity.Moreover,in order to investigate the evolutionary conservatism of lysine crotonylation,we carried out sequence evolution analysis and experimental verification.We provide evidence that Esal,the yeast homolog of mammalian MOF,is responsible for bulk histone crotonylation in yeast.The finding that CBP/p300 and MOF catalyze both histone acetylation and crotonylation makes the functional studies of histone crotonylation technically difficult.To address the function of histone crotonylation,we resorted to make novel CBP and p300 mutants with defective HAT but active HCT activity.Toward this direction,we have generated a p300 I1395G and CBP I1432G mutants with deficient HAT but competent HCT activity.Importantly,through the luciferase reporter assay,we found that these HAT-deficient CBP/p300 mutants are capable of enhancing transcriptional activation by various transcription factors as effectively as wild-type CBP/p300.Furthermore,we demonstrate these CBP/p300 mutants can substitute the endogenous CBP/p300 to enhance transcriptional activation in cell,which correlates with enhanced promoter crotonylation and recruitment of DPF2,a selective reader for crotonylated histones.Together we have identified CBP,p300 and MOF as HCTs and provide evidence that MOF is an evolutionarily conserved HCT.More importantly,we provide first cellular evidence that CBP/p300 can facilitate transcriptional activation through histone acylation other than acetylation,thus supporting an emerging role for the non-acetylation type of histone acylation in transcription and possibly other chromatin-based processes.In the second section we aimed for identification of histone fatty acylation reader proteins.We found that WDR5 protein specifically bound to acetylated or crotonylated H3 polypeptides.WDR5 is an essential subunit of H3K4 methyltransferase MLL/SET complexes,suggesting that WDR5 may act as a bridge molecule for the crosstalk between acetylation/crotonylation and methylation.We found that increasing the levels of cellular histone acetylation/crotonylation by inhibition of deacetylase activity led to increased levels of H3K4 methylation,whereas knockdown of CBP/p300 or inhibition of their enzyme activity by C646 compound resulted in reduced levels of H3K4 methylation.These evidences indicate that intracellular acetylation/crotonylation were necessary for the maintenance of H3K4 methylation.Therefore,our study will provide novel insights into the molecular mechanism by which WDR5 modulates the crosstalk between histone acetylation/crotonylation and methylation and the functional significance of this crosstalk.