Role Of Sumoylation In The Epigenetic Regulation And Tumor Biology

Post translational modification is one of the important regulatory mechanisms of protein function in cell,and it has always been the hot topic in the basic research area.Sumoylation is an important post translational modification.Sumoylation pathway includes a series of cascade enzymatic reactions,adding SUMO proteins to the lysine residue of the substrates through the E1/E2/E3 ligases.This modification could regulate protein subcellular localization,protein-protein interaction,and so on.Here,we carried out two parts of studies with sumoylation as the main line,to explore the regulation of sumoylation on protein function and its roles in cancer development.Part Ⅰ: The regulatory mechanism of SUMO1 modification of histone H3K36 methyltransferase SETD2SETD2/HYPB,knew as the orthologous gene of the yeast set2,was discovered and separated from the CD34+ hematopoietic stem/progenitor cells by our group,and was identified as the histone H3K36 methyltransferase.It plays an important role in many biological processes,including the transcriptional initiation,alternative splicing,homologous recombination,mismatch repair,chromatin remodeling and so on.The aberration of SETD2 was discovered in a variety of cancers,suggesting that SETD2 may act as a tumor suppressor.Regulatory mechanism studies of SETD2 were relatively fewer.Recent studies indicate that SETD2 is regulated by the microRNA and long non coding RNA.Our previous studies indicated that sumoylation pathway may be involved in regulation of SETD2 function.In this study,by using SUMO consensus motif prediction,gene cloning and in vitro expression,co-immunoprecipitation,cell transfection and western blot analysis,we investigated whether SETD2 is modified by sumoylation,and whether this modification can affect SETD2 function.Co-immunoprecipitation assays showed that SETD2 could interact with UBE2I(Ubc9),the unique E2 ligase in sumoylation pathway.Meanwhile,it could also interact with most members of the PIAS protein family,which are the important E3 ligases in the SUMOylation pathway.After revealing the interaction between SETD2 and main ligases of the sumoylation pathway,we did find that SETD2 could be modified by SUMO1 in vitro.Subsequently,lysine 1594 of SET enzymatic domain was identified as the SUMO1 modification site of SETD2 by site directed mutagenesis approach.Furthermore,our co-immunoprecipitation result showed that SETD2 could also interact with SENP1,the main desumoylation enzyme of the SUMO1 modified substrates.More interesting,histone H3K36me3 level could be regulated by enforced overexpression of SENP1,and this regulation relies on the desumoylation activity of SENP1.Taken together,these data suggest that sumoylation pathway is involved in the regulation of histone H3K36 trimethytransferase activity of SETD2.Part Ⅱ: Function and mechanism study of SUMO1 modification of MAFB in Colorectal cancerMAFB is a member of the Maf(avian musculoaponeurotic fibrosarcoma oncogene homolog)transcriptional factor protein family,which contains bzip(Basic Leucine Zipper)domain and plays important roles in regulating the growth and differentiation of the organs and the tissues.In some tumors,it has been found that MAFB acts as a tumor promoting factor in many cancers.In this study,we analyzed the available online data of tumors TCGA database using the online tool cbioportal,and found that the expression level of MAFB appears to be significantly high in the majority of the tumors,including colorectal cancer(CRC),as compared with normal tissue.This result was further confirmed by gene expression level detection and immunohistochemistry analysis of clinical CRC samples.Moreover,our data showed that MAFB expression level was associated with the malignant degree of CRC.Next,we knocked down MAFB in CRC cell lines with shRNA,and found S/G2 M phase cells were significantly reduced as compared with that in control group.Further analysis showed that mRNA levels of a series of cell cycle associated factors were dysregulated,including CDK6,CDKN3,CUL1,CUL3,and etc.Moreover,dual luciferase reporter gene assay revealed that MAFB directly regulated the transcription of CDK6.Meanwhile,mechanical investigation verified that Homo sapiens MAFB could be sumo-modified,this was consistent with its mouse orthologue MafB.Different from mouse MafB,Homo sapiens MAFB could only be modified by SUMO1 at lysine32.And the dual luciferase assay demonstrated that this modification was critical for MAFB regulated CDK6 transcription.In line with this,lysine32 mutated MAFB could not rescue the abnormal phenotype in MAFB knocked down CRC cells.Therefore,these data suggest that MAFB may promote CRC tumor cell proliferation via regulating CDK6 transcription,and this regulation relies on the SUMO1 modification of MAFB.