| Background18-trisomy syndrome(18-trisomy syndrome)is an autosomal aneuploidy disease with abnormal organs and systems due to the presence of extra human chromosome 18,and its clinical manifestations are abnormal organs and systems.The availability of chromatin is critical to the regulation of gene expression in specific cellular and biological processes,and the gene regulation related to clinical phenotype development and development of trisomy 18 syndrome has long been poorly understood.Due to the heterogeneity of the cell population,each cell presents a different epigenomic landscape.In this study,sc-ATAC-seq(single-cell assay for transposase accessible chromatin,sc-ATAC-seq)technology was used to address cellular heterogeneity while studying epigenetically regulated transcription factors and their potential functions in trisomy 18 syndrome.ObjectiveThe open region of cord blood mononuclear cells in trisomy 18 syndrome was analyzed at the single-cell level to obtain the map of cord blood mononuclear cells,the enrichment degree of various cell-level transcription factors was analyzed to obtain the cell-level differential transcription factors,the cell populations that play important functions in the disease were obtained,and the key transcription regulatory factors were found.To predict the target genes and analyze the genes related to trisomy 18 syndrome,deepen the understanding of the disease mechanism of trisomy 18 syndrome,and provide a basis for the research on the mechanism of the occurrence and development of trisomy 18 syndrome.MethodsIn this study,sc-ATAC-Seq was used to analyze transcription factors in open chromatin regions of umbilical cord blood mononuclear cells in patients with trisomy 18 syndrome and controls.After dimensional-reduction cluster analysis of the original data,cell identification was carried out.Cell population types were identified using genes specifically expressed by cell populations.Transcription factors specifically expressed by cell populations were analyzed,and the differential transcription factors of each cell population in the disease group and the control group were analyzed.After verification,the target genes that meet the expectations of the experiment were predicted,and the function of the target genes was analyzed.The genes and pathways involved in the control of key transcription factors in trisomy 18 syndrome were identified.Results(1)A total of 11,611 cells were captured from the single-cell ATAC sequencing library of the disease group and the control group,including 5,296 cells in the ES group and 6,315 cells in the NC group.Seven main groups of immune cells were identified: B cells,CD4+T cells,CD8+T cells,dendritic cells,monocytes,natural killer cells,T cells,and CD4+T cells.Compared with NC group,the proportion of B cells,CD4+T cells and T cells in ES group significantly changed.Twentyseven transcription factors specifically expressed in each cell population were obtained.(2)Fourteen differentially expressed transcription factors were screened,of which 12 were down-regulated and 2 were up-regulated.Analysis of 14 transcription factors in the cell population determined that the top three important cell populations in the disease group were the B cell population,monocyte population,and T cell population.The four common differential transcription factors of B cell group,monocyte group and T cell group are TEAD1,ESRRA,PBX1 and TEAD2,respectively.In addition,the two transcription factors of TEAD4 and Twister2 are analyzed by literature,and these six transcription factors with low expression in the disease group compared with the control group are finally selected for verification.Four transcription factors were verified by q PCR: TEAD1,TEAD2,TEAD4 and Twist2.(3)Four validation results were analyzed to conform to the expected motif characteristics of transcription factors(TEAD1,TEAD2,TEAD4,Twist2),and the motif characteristics of the TEAD family were similar.By analyzing the verified target genes of TEAD2 and TEAD4 in the database,the top 100 significant target genes were obtained.Among them,the top 15 significant transcription factor TEAD2 target genes are: LSP1,LY6G6 D,LOC643733,SPOCK1,ALOX15,DGK,STAB2,COL17A1,ADAMTS1,PKHD1L1,SH3RF2,LPAR6,DPP6,NRCAM,Na ALADL2;Function analysis of target gene GO Among the top 10 results of biological processes,4 functions were related to the nervous system,and 4 KEGG pathways of the top 10 target genes were related to heart disease.The top 100 most significant targets of another transcription factor,TEAD4,and the top 15 most significant targets are: LAMA2,PAWR,SAG,LOC100240735,LIN7 A,MUC17,CHRFAM7 A,GDNF,DPYSL5,ABCA9,ATP6V1G2,NPC1L1,MYOF,CACNB4,MAP7D2;Among the top 10 results of GO biological process(BP)functional analysis,6 GO functions were related to development,and 1 of the 4 pathways in KEGG enrichment analysis was related to nervous system.The relevant data of the two transcription factors TEAD1 and Twist2 have not been included in the Knock TF database for the time being.Through direct literature review,the functions of these two transcription factors and the potential roles of participating pathways in the disease group are summarized.Conclusion(1)The experimental results of this study suggest that the immune system of trisomy 18 syndrome is abnormal.Compared with the NC group,the proportion of B cells and CD4+T cells in the ES group is significantly increased,the proportion of T cells is significantly decreased,the proportion of NK cells and DC cells is slightly decreased,and the proportion of monocytes and CD8+T cells is not significantly changed.(2)The transcription factors specifically expressed by 27 cell groups of 6 types(T cell group,B cell group,monocyte group,CD8+T cell group,CD4+T cell group,NK cell group)were potential cell population marker molecules,which could provide reference for cell population identification by subsequent single cell sequencing.(3)The top three important cell populations are B cell population,monocyte population and T cell population.(4)Four important transcription factors were identified,including TEAD1 and TEAD2,which are related to heart and cardiovascular development and may regulate the heart development of trisomy 18 syndrome.The GO enrichment of TEAD2 target genes is related to the nervous system,and the down-regulation of TEAD2 may be involved in the abnormal development of the nervous system in trisomy 18 syndrome.TEAD4 is related to growth and development,and the down-regulation of this transcription factor may be involved in the developmental abnormalities of trisomy 18 syndrome.Twist2 is a skeletal abnormal transcription factor that may contribute to Trisomy 18 syndrome. |
