Integrative Analysis Reveals Functional Roles Of Epigenetic Regulation In Mediating Alternative Splicing In Blood Cancer

Gene expression regulation is not only including the transcription level of regulation,but also contrains the post-transcriptional processing of pre-mRNA,which decides the fate of cells.Alternative splicing is a pre-mRNA process mainly controlled by the posttranscriptional regulation.With the coming of next generation sequencing technology,how to get the biological characteristics from large-scale biological data and public omics data resources and discover the relationship between alternative splicing and the diseases has become the frontier problem in post-genomic era.Accumulating evidence suggests alternative splicing is a co-transcriptionally splicing process which not only controlled by RNA-binding splicing factors,but also mediated by epigenetic regulators,such as chromatin structure,nucleosome density,and histone modification.Aberrant alternative splicing plays an key role in abnormal cell differentiation and physiological disorder,prompts various diseases,including cancers.This study mainly introduces epigenetic modification H3K79me2 enrichment in alternative splicing sites in multiple tissues of human normal and disease cells.The main works can be described as following.First of all,using the high-throughput sequencing technologies and existing open data resources,we created ChIP-seq of H3K79me2 and RNA-seq dataset of 34 kinds of different tissue of human disease and normal cells.Utilizing the computer simulation technology,H3K79me2 enrichment peak analysis was carried out on the dataset of Ch IP-seq and alternative splicing events analysis of RNA-seq.According to the calculated ψ-value,created five kinds of alternative splicing models respectively.We integrated alternative splicing events derived from RNA-seq with ChIP-seq of H3K79me2 across 34 different normal and cancer cell types,and found the higher enrichment of H3K79me2 in two AS types,skipping exon(SE)and alternative 3’ splicing site(A3SS).We did hierarchy clustering according to the distribution and characteristics of ψ-value,and preliminary found cancer cells,including the cells of malignant blood cancer,have significant difference of normal cells.We present H3K79me2 may cause cancer by regulation of alternative splicing events.Secondly,We used the SOM clustering for the dimension reduction as a feature extraction associated with exon skipping sites.By applying self-organizing map(SOM)clustering,we unveiled two clusters mainly comprised of blood cancer cell types with a strong correlation of H3K79me2 and exon skipping event,one is mainly for myeloid leukemia cell lines,especially MLL-r acute myeloid leukemia,the other including most types of lymphocytic leukemia cell lines.Our findings reveal the reason of higher enrichment of H3K79me2 in MLL-r acute myeloid leukemia cells from epigenetic level.The exon skipping event is the improtant link between epigenetic modification H3K79me2 and MLL-r acute myeloid leukemia.Next,in order to explain H3K79me2 regulatory mechanism of alternative splicing,we did the GO and KEGG passway analysis of a set of genes from exon skipping event sites and non skipping event sites.Remarkably,the expression of transcripts associated with exon skipping were not significantly different from those without exon skipping,indicating the involvement of H3K79me2 on splicing has little impact on full mRNA transcription.Interestingly,cancer pathways,transcriptional misregulation,spliceosome,AML and CML were among top significantly enriched pathways in the overlapped genes of two SOM clusters of leukemia.Then we searched splicing factors or RBP motifs in the sequences spanning around skipping sites.Interestingly,we found that two enriched motifs,SRSF2 and U2AF2,were previously reported to be highly involved in AML progression through aberrant splicing regulation.Finally,To functionally characterize the role of H3K79me2 or DOT1 L in mediating exon skipping sites in leukemia cells,we conducted several functional assays in five selected a lymphoblastoid cell line GM12878.DOT1 L knockdown by siDOT1 L clearly reduced the DOT1 L protein level in all three cell lines.we found that the exon skipped sites were able to switch to exon inclusion in both DOT1 L knockdown AML cell lines,suggesting H3K79me2 is involved in the exon skipping process.The results show the regulatory role of H3K79me2 in the process of exon skipping and pathogenesis of leukemia.Our results reveal the novel epigenetic regulatory and functional roles of H3K79me2 in mediating co-transcriptional splicing process and the regulation function in the development of leukemia.It is shows the regulating relationship of epigenetic modification,exons skipping alternative splicing events,splicing factors and malignant leukemia.The regulatory main line is epigenetic regulation – molecular biology mechanism–disease development.