Study On The Mechanism Of Distal Regulatory Elements Upstream Of Human C-myb Gene

The transcription factor c-Myb is a very important regulator in the process of hematopoiesis and plays an important role in cell proliferation,differentiation and apoptosis.The expression level of c-myb in hematopoietic progenitor cells is very high,and its expression will decrease or not be expressed as cells differentiate.Recent reports of abnormal expression of c-myb have caused various cancers,such as leukemia,breast cancer,and colon cancer,which have attracted people's attention.Although there have been many reports on the regulation of c-myb expression,since c-myb lacks a typical promoter and multiple factors are involved,there are still many questions to be resolved about the detailed transcriptional mechanism of c-myb.A number of studies have demonstrated that distal regulatory elements play a very important role in the regulation of c-myb expression,but the detailed regulatory mechanisms are still unclear.Pre-laboratory data indicate that in mouse myeloid leukemia M1 cells,there are interactions between the DNA regulatory elements and the c-myb promoter in the 25 kb,50 kb,and 70 kb upstream regions of the c-myb promoter,through DNA-loop space.The structure is close to the promoter region of c-myb gene,activates gene expression and induces tumorigenesis,and finds that transcription factors Hoxa9,PU.1,etc.can regulate c-myb transcription by binding these distal elements.Leukemia provides new ideas,so we want to know more about whether there are similar remote regulatory elements in human leukemia and how these regulatory elements regulate c-myb transcription.Subsequently,we used the circular chromosome conformation capture(4C)method in the human leukemia cell line K562,and used the promoter of the human chromosome 6 c-myb gene as a bait to find the upstream 34 k,88k and promoter promoters.Mutual interaction sites confirmed that these two upstream distal regulatory elements may regulate the expression of the c-myb gene.The results of the dual fluorescence reporter system further indicated the enhancer activity of-34 k and-88 k,and we found the enrichment of the apparently modified H3K27 ac and the binding of the transcription factors GATA1 and TAL1 in these regions,so we suspect that these transcription factors may Participate in the transcriptional regulation of c-myb.Based on the above research basis,we would like to know more about how these remote regulatory elements and apparent modifications and transcription factors regulate the c-myb gene.In this study,we continue to use the human erythroleukemia cell line K562 as a model to study the apparent modification of the c-myb gene-34 k,-88 k on human chromosome 6 and the effect of transcription factors on gene transcriptional regulation.Hemin induced differentiation of K562 cells by chromatin immunoprecipitation(ChIP-qPCR)and detected the enrichment of histone H3K27 ac and transcription factors GATA1 and TAL1 in the-34 k and-88 k regions,and we constructed Targeting gRNAs upstream of c-myb 34 k,88k,using the CRISPR/Cas9 system to carry the corresponding epigenetic modification enzymes to modify the remote regulatory region,to detect the effect of activation and inhibitory epigenetic modification on c-myb expression Finally,we co-transfected dCas9-p300 and enhancer gRNA into K562 cells to monitor the expression of c-myb after hemin induced cell differentiation.The results are as follows:1)After hemoglobin(hemin)induced differentiation of K562 cells for 24 hours,the histone modification H3K27 ac enrichment of the c-myb gene distal regulatory region-34 k,-88 k decreased,and the enrichment of transcription factors GATA1 and TAL1 decreased.2)Successfully constructed a gRNA targeting the c-myb gene promoter,-34 k,-88 k,-53 k on chromosome 6 of human erythroleukemia cell line K562(promoter is a positive control,-53 k is a negative control),and sequencing The results indicated that all gRNAs were successfully cloned into the pSPgRNA empty vector plasmid without mutation.3)ChIP-qPCR results showed that dCas9-p300 successfully targets the-34 k,-88 k regions and increases the H3K27 ac levels in these regions.4)dCas9-p300 targets the c-myb gene-34 k region and increases H3K27 ac in this region and binds to transcription factors GATA1 and TAL1 to activate c-myb gene expression;dCas9-p300 targets c-myb gene-88 k region This region H3K27 ac binds to the transcription factor GATA1 to activate the expression of the c-myb gene.5)ChIP-qPCR results showed that dCas9-DNMT3 A successfully targetd to the-34 k region and increased the level of DNA methylation in this region.6)dCas9-DNMT3 A targets c-myb gene-34 k region and increases DNA methylation level inhibits c-myb gene expression7)dCas9-p300 can reduce the differentiation of k562 cells to a certain extent after targeting the distal enhancerAll of the above results indicate that in human erythroid leukemia K562,the enrichment of histone H3K27 ac and transcription factors GATA1,TAL1,etc.is decreased after hemin induces cell differentiation.In the upstream of the c-myb gene,the-34 k region is used as an enhancer.When the acetyltransferase dCas9-p300 targets the region to increase the histone H3K27 ac,the binding of the transcription factors GATA1 and TAL1 activates the expression of the c-myb gene.The methyltransferase dCas9-DNMT3 A targets this region to increase DNA methylation and inhibit the expression of c-myb gene.The-88 k region acts as an enhancer,and the acetyltransferase dCas9-p300 targets the region to increase histone H3K27 ac,and binds to the transcription factor GATA1 to activate c-myb gene expression.This study demonstrates that the apparent modification of these remote regulatory elements can affect the expression of c-myb gene,while dCas9-p300 can reduce the differentiation of k562 cells to a certain extent after targeting the distal enhancer,so these regulatory sites can As a drug target,it provides a new breakthrough for further research on leukemia.