ASH2L In The MLL Gene Rearrangement Of Acute Leukemia On The Regulation Mechanism Of HOXC8 Gene Histone Modification

PartIThe study of HOXC8 gene expression in MLL rearranged acute leukemiaObjective:HOXC8 genes are frequently dysregulated in human leukemia with the gene rearrangement between mixed lineage leukaemia(MLL)and partner genes.To explore the possible mechanism by which the histone methylases of ASH2L(absent-small-homeotic-2-like)protein regulate the target genes,we measured the level of HOXC8 gene expression in two MLL rearranged acute leukemia cell lines(RS4:11 and THP-1 cells).Methods:The mRNA levels of HOXC8 genes were quantified using real-time quantitative reverse transcription PCR)(qRT-PCR)with SYBR Green by CFX connect RT-PCR System from RS4:11 ? THP-1 cell lines.The relative expression level of mRNA was analyzed by 2(-ACT)method.Results:The over-expression and good stability of MLL target genes HOXC8 were found in two MLL-rearranged acute leukemia cell lines,especially in RS4:11 cells.Conclusion:MLL regulates a number of genes,including HOXC8.High-level expression of HOXC8 in our results provide detailed experimental data for the follow-up theoretical study of methylation of histones,and the potential role of HOXC8 in MLL-rearranged acute leukemia will be investigate in part 2 and part 3.Part IIH3K4me3 modification on HOXC8 promoter treated by ASH2L-siRNA in MLL-rearranged acute leukemiaObjective:MLL1 fusion protein alone exhibits poor histone lysine methyltransferases(HKMTS)activity on catalyzing histone H3 Lys4 tri-methylation(H3K4me3)in MLL rearranged acute leukemia.To explore the HKMT effect of another regulatory protein within complex proteins associated with Setl(COMPASS)-ASH2L,we analyzed H3K4me3 modification on HOXC8 promoter under the action of ASH2L regulation.Methods:After RNAi knockdown of ASH2L,chromatin immunoprecipitation(ChIP)-real-time PCR(RT-PCR)and western blot were used to detect the expression of specific regions of the HOXC8 promoter,RBBP5,WDR5,MLL gene and BRTF gene in two MLL rearranged acute leukemia cell lines(RS4:11 and THP-1 cells).Results:The HOXC8 gene and protein level were significantly down-regulated upon treatment with ASH2L-siRNA(specific regions of the HOXC8 promoter located Ok and 3kb(-3.0 kb)upstream of the transcriptional start site in RSH:11 cells,and-3.OK,-2.OK upstream of the transcriptional start site,+1.4K downstream of the transcriptional start site in THP-1 cells).The expression level of BRTF,RBBP5,WDR5 and MLL gene located at different transcriptional start site of HOXC8 prometer were significantly down-regulated in RSH:11 cell line(P<0.05).Also,BPTF and RBBP5 gene were down-regulated of HOXC8 prometer in THP-1 cell line(P<0.05).Conclusion:Based on the experiment results,we suggest a new histone modification concept about the ASH2L protein in MLL rearranged acute leukemia,which can not carry out methyltransferase activity independently.Here protein-protein interactions of ASH2L with other COMPASS members and chromatin remodeling factor-BRTF,are essential for its activating function on HOXC8 gene transcription.PartIIIInteraction mechanism of ASH2L and other regulatory proteins in MLL rearranged acute leukemiaObjective:Through the previous study of Part 1 and Part 2,we found ASH2L was significantly associated with histone methylase of HOXC8 gene in MLL rearranged acute leukemia.The aim of this study was to investigate the innteraction mechanism of histone methylase about the ASH2L and other regulatory proteins in COMPASS.Methods:Based on the epigenome libarary,the effective cells,epigenome genes and small interference RNA about MLL-C,WDR5,RBBP5,BPTF were screened.The investigation of the potential relationship between ASH2L and other regulate genes in MLL1 core complex were detected by RT-PCR and western blot test.Results:Apart from ASH2L,WDR5,RBBP5,MLL1 and BPTF proteins have regulation effect on the expression level of HOXC8 gene.The WDR5 protein level was significantly down-regulated upon treatment with ASH2L-siRNA in RS4:11 cells(P<0.05).Although the MLL1 and RBBP5 protein levels also have downward trend,but there were no statistical significance(P>0.05).Conclusion:Because wild type MLL1 gene only maintained a weak direct interaction with target genes in MLL rearranged acute leukemia,WDR5 gene is critical for the MLL1 core complex to regulates the expression of target genes.We suspected that full activation of WDR5 requires RBBP5-ASH2L heterodimer within MLL family core complexes that can orderly interact with MLL1 and amplifying histone methylation effect of HOXC8 gene H3K4me3.