Identification And Mechanism Studies Of Functional SNPs Associated With Leukemia Risk

Background and ObjectCancers have become the leading threat to human health worldwide.Among them,the incidence and mortality of leukemia both rank in the top ten on a global scale.Leukemia ranks first among pediatric cancers,accounting for about 28%of newly diagnosed childhood cancers and seriously threatening the health of adults and adolescents.With the use of targeted drugs,the 5-year survival rate of leukemia patients have been improved greatly,but timely detection and diagnosis of leukemia is still an urgent needs.It is necessary to find more effective molecular biomarkers for the prevention and early diagnosis of leukemia.Some natural molecular markers,such as single nucleotide polymorphisms(SNPs),have important potential in the cancer prediction,diagnosis and treatment.Genome wide association studies(GWAS)have identified hundreds of SNPs associated with leukemia risk.However,analysis of the functional results of SNPs has shown that up to 90%of the genetic variation in disease susceptibility are located in the non-coding regions,which makes it a particularly challenge to elucidate their function and mechanisms in the occurrence and development of diseases.However,most of the genetic variations in disease susceptibility are located in the non-coding regions,which makes it a particularly challenge to elucidate their function and mechanisms in the occurrence and development of diseases.In addition,gene delivery efficiency is also a limiting factor for vitro functional studies of leukemia-associated suspension cell lines.Therefore,the biological regulatory functions and pathogenic mechanisms of most SNPs associated with leukemia risk are still not clear.This research aims to identify gene regulatory SNPs associated with leukemia risk using a highly efficient paralleled report gene analysis system and delves into the herein mechanism leading to disease susceptibility.The results provide detailed functional annotation of these functional leukemia risk loci and help provide solid evidence and support for future clinical application as possible biomarkers in the diagnosis and treatment of leukemia.In addition,to solve the problem of low transfection efficiency of leukemia suspension cell lines,we developed a transfection-positive cell sorting system based on a membrane-anchoring affinity tag to facilitate gene function studies in related cell lines in vitro.Results1.Parallel Reporter Gene Analysis Identifies Multiple Functional Leukemia Risk SNPsWe performed DiR-seq analysis of 226 risk-related GWAS SNPs in the leukemia cell line K-562 and obtained 85 functional SNPs with altered reporter activity.Among the most significant 55 regulatory SNPs,16 SNPs had reduced transcriptional activity compared with the reference allele,and 39 SNPs had increased transcriptional activity compared with the reference allele.Then,we performed a combinational analysis with genomic and epigenomic data to evaluate the chromatin openness(FAIRE-Seq),accessibility(ATAC-seq and DNase-seq),and histone modification status(H3K4mel,H3K4me3,and H3K27ac ChIP-seq)of the SNP regions to further identify functional SNPs in active chromatin region.Moreover,we confirmed the chromatin opening status surrounding the SNPs by FAIRE-qPCR in K-562 cells.Finally,we chose rs1388941 and rs6858698 as the preferred functional SNP candidates to study the function and mechanism.2.Roles of Regulatory SNP rs1388941 in Leukemia RiskFor functional SNP rs1388941,we confirmed that the risk G allele could significantly up-regulate the enhancer activity in DiR-qPCR analysis and dualluciferase reporter activity analysis in K-562 cells.Multiomics analysis based on ChIPseq,DNase-Seq,FAIRE-Seq,and ATAC-seq,as well as qPCR and Sanger sequencing analysis of ChIP-DNA and FAIRE-DNA,showed that the mutation significantly remodeled chromatin structure and modification state.More importantly,the G allele has higher chromatin activity than the A allele.Computational prediction revealed that the risk G allele makes this SNP locus region a stronger typical FLI1 transcription factor binding site.We identified CSGALNACT1 as the target gene of rs 1388941 through eQTL analysis in the GTEx database and further confirmed this using CRISPRa and CRISPRi experiments in leukemia cells.In addition,we found that in the CRISPR/Cas9 genomeedited monoclonal cells with the rs 1388941 region being deleted,the chromatin openness of this region was significantly reduced,and the expression of CSGALNACT1 was significantly down-regulated.CCK-8 assay showed that cell proliferation was impeded upon the SNP knockout.Moreover,we found that the overexpression of the CSGALNACT1 gene increased the STAT5 activity,which subsequently upregulated the CyclinD1 expression and promoted cell proliferation.In this section,we demonstrate a possible mechanism for rs 1388941 in leading to leukemia predisposition,by which the risk G allele enhances FLI1 binding and drives specific upregulation of downstream gene CSGALNACT1,which thereby promotes the expression of cyclin and accelerates cell cycle progression.3.Roles of Regulatory SNP rs6858698 in Leukemia RiskFor the leukemia risk rs6858698 locus,we further confirmed that the enhancer activity of the risk C allele was significantly higher than that of the reference G allele by reporter gene analysis assay.Meanwhile,our ChIP-qPCR and FAIRE-qPCR confirmed the epigenomic analysis,indicating that rs6858698 is located in an open active chromatin region and the enhancer region of leukemia cell lines.Through further ATAC-seq footprint analysis in Jurkat cells and JASPAR database prediction,we found that the C to G mutation of rs6858698 created a conserved E2F1 binding motif.Further ChIP analysis confirmed that E2F1 was able to bind to this locus.In addition,we identified CAMK2D as the target gene of rs6858698 using CRISPRa and CRISPRi assay.Gene expression analysis of clinical samples from TCGA and GTEx databases showed that the expression level of CAMK2D in leukemia samples was significantly higher than that in normal tissues.Notably,high expression of the CAMK2D gene in leukemia patients was significantly associated with lower overall survival,suggesting that CAMK2D is associated with poor prognosis in leukemia.Further analysis in K562 cells showed that the overexpression of CAMK2D upregulated the STAT5 and NFκB activities and affected the expression of downstream target genes,including Bcl-2,PIM-1,CAT,GLRX,and c-FOS.In brief,our results systematically demonstrate that the risk C allele of rs6858698 affects several downstream signaling pathways and increases disease risk by upregulating the expression of CAMK2D mediated by E2F1.4.A Transfection-positive Cell Sorting System Based on A Membrane-anchoring Affinity TagWhen conducting in vitro pathogenic function studies of acquired disease-related genes,gene delivery efficiency becomes an important limiting factor in the functional study of leukemia-related genes.To solve the problem of low transfection efficiency in hard-to-transfect cells such as leukemia cells and to facilitate functional studies of related genes in these cells,we developed an affinity cell sorting system that can efficiently enrich transfection-positive cells.In this system,membrane-localized EGFP fluorescent protein with affinity tag Twin-Strep-Tag(TST)was used as the plasmid marker.The fluorescent affinity sorting tag could be expressed in transfected positive cells and displayed on the cell surface.Based on the high affinity between affinity tag and ligand,efficient separation of transfected positive cells could be achieved by ligand-coupled magnetic beads.In addition,the fluorescent proteins in the sorting tags make it easy to analyze the positive cells by fluorescence microscopy and flow cytometry.We compared the ability of six membrane localization signal sequences to target sorting tags to cell membranes in Lenti-X 293T cells,including three glycosylated phosphatidylinositol(GPI)variants,TST-EGFP-GPIBY55,TST-EGFP-GPIDAF,and TST-EGFP-GPICEAM7 and three transmembrane domain(TMD)variants,TST-EGFPTMITB3,TST-EGFP-TMITA5,and TST-EGFP-TMITAV.The laser scanning confocal microscope analysis showed that all six variants of the sorting tag could be expressed at high levels in Lenti-X 293T cells and displayed on the cell membrane.Moreover,in K-562,Lenti-X 293T,and 22Rv1 cell lines,affinity cell sorting using Strep-Tatin coupled magnetic beads could achieve efficient enrichment of the transfected positive cells.Among them,GPI membrane-anchored type sorting tags showed better performance than TMD-type tags.The enriched K-562 cells resulted in a final positive cell percentage of up to 95.0%.Meanwhile,the enrichment folds determined with EGFP RNA level of the three GPI-type sorting tags were over ten times in K-562 cells.Then we constructed vectors for gene overexpression,shRNA knockdown,and CRISPR/Cas9 genome editing experiments using the TST-EGFP-GPIBY55 sorting tag and applied them in gene function studies.In the gene overexpression assay,the gene overexpression fold dramatically increased from 10 times to 58 times in the cells enriched by affinity sorting.In the shRNA knockdown assay,the knockdown efficiency increased from 12%to 53%in the sorted cells.In addition,sorted cells from CRISPR/Cas9 genome editing experiments showed more significant gene editing,with an increase in indel percentage from 20%to 79%.In conclusion,our novel affinity cell sorting system can greatly increase the proportion of transfection-positive cells and effectively promote the study of gene function.ConclusionsThis project used an efficient parallel reporter analysis system,combined with high-throughput sequencing data of whole-genome chromatin activity analysis,and identified 55 leukemia risk-related SNPs with potential gene regulatory activity.The mechanism of disease susceptibility caused by two functional risk SNPs,rs1388941 and rs6858698,was further explored.We elucidated the underlying biological functions and mechanisms of genetic variation in functional leukemia risk through a series of molecular and cellular experiments.We demonstrate a possible mechanism for rs 1388941 in leading to leukemia predisposition,by which the risk G allele enhances transcription factor FLI1 binding promoted chromatin opening in the locus region and drives specific upregulation of downstream gene CSGALNACT1,which thereby promotes the expression of cyclin and accelerates cell proliferation.In addition,our results systematically demonstrate that the reference G allele of rs6858698 SNP mutates into the risk C allele to form a conserved E2F1 binding motif,which drives the upregulation of CAMK2D mediated by enhancing E2F1 chromatin binding.Up-regulated CAMK2D can significantly improve the activity of STAT5 and NF-κB signaling pathways,thereby affecting the expression of many downstream target genes,and participating in the physiological processes of cell proliferation,apoptosis,cell diferentiation,and oxidative stress,thus affecting the occurrence and development of leukemia.Our results have important implications for understanding the mechanism of genetic susceptibility to cancer,which not only reveal the detailed gene function annotation for functional leukemia risk variants but also provide potential biomarkers for the diagnosis and treatment of leukemia.At the same time,it provides strong evidence support for its application in clinical therapy.In addition,to overcome the problem of the low transfection efficiency in leukemia suspension cell lines,we developed a novel affinity cell sorting system based on a membrane-anchoring affinity tag to enrich transfected positive cells.This enabled efficient enrichment of transfected positive cells in shRNA gene knockdown and CRISPR/Cas9 genome editing monoclonal assays performed in leukemia cell lines,which greatly improves the efficiency of gene function study.The one-step affinity cell sorting system can simply and efficiently increase the proportion of transfected positive cells and effectively promote the study of gene function in cell lines that were difficult to transfect.