Functional Analysis Of Accelerated Evolved DNase ? Hypersensitive Sites In Brain-related Gene Regulatory Regions Of Primates

The differences in brain between humans and non-human primates are largely unknown,especially the human-specific social and cognitive behaviors as well as some mental disorders.However,the complex drivers at the genome level behind these differences remain unclear.It is the main goal of brain evolution research to reveal the relationship between gene mutations and trait variation between primates.It has been hypothesized that variation in the regulatory elements of a large number of non-coding regions is one of the important factors that contribute to the phenotypic differences between humans and other non-human primates.Therefore,evolutionary and functional analysis of potential brain-related gene regulatory regions contributes to a better understanding to the adaptive evolution of the human brain,which is the main purpose of this research.The DNase I hypersensitive site(DHS)on the whole genome is conjugated to a variety of active regulatory elements,including promoters,enhancers,silencers,insulators and gene locus control regions.The current mature DHS-seq technology enables high-throughput DHS data production and continuous improvement of DHS maps.Among them,hundreds of different cell lines have been published in the ENCODE program and the Human Epigenome Project,and DHS data of different tissues have been widely used in the study of regulatory regions.In recent years,studies of the evolution of multiple DHSs between primates have demonstrated that these regions play an important role in gene regulation and thus affect human-specific traits.In addition,in the previous study,our group combined with DHSs to carry out evolutionary analysis and functional analysis of primate between major histocompatibility complex(MHC)and TRIM(Tripartite motif)gene family regulatory regions,further confirming the applicability of DHSs in the study of primate evolution.In this study,we used bioinformatics methods,combined with DHS data,to perform functional analysis of accelerated evolutionary regulatory elements in primate brain-related gene regulatory regions,speculating that these accelerated evolutionary DHSs are in the evolution of primate brains.Role and meaning.The CRISPR/Cas9 system,Western Blotting and qPCR(real-time quantitative PCR)were used to verify the functionalization of accelerated evolution DHS.The results are as follows:(1)In this study,we identified 2397 brain-related accelerated DHSs(brain-accelerated DHSs)based on previous analysis in our group,and revealed the data through genome annotation files and genomic regulatory regions.The distribution characteristics of brainaceDHSs on the genome: most of the brain-aceDHSs are enriched within 10 kb of the upstream and downstream of the transcription start site,and are mainly distributed in the intron and intergenic regions,only a few of which are located in the gene.The coding region is in the promoter.Among them,75 brain aceDHS overlaps with promoter region,303 overlaps with enhancer,and 1121 overlaps with region with transcriptional element characteristics.(2)According to the published Hi-C database,544 target genes potentially regulated by brain-aceDHSs were identified to be enriched in the biological functions of cell proliferation and cycle,and closely related to brain and neural development.In addition,some brainaceDHSs target genes play a role in Down's syndrome and brain diseases.According to the existing human,chimpanzee and rhesus monkey brain tissue expression data,it was found that some target genes(some target genes do not have expression data)showed a difference in expression patterns between humans and non-human primates.(3)Through a multiple sequence alignment and transcription factor binding site database,the alignment indicated that there are 61 potential human-specific transcription factor binding sites in brain-aceDHS,including CTCF,FOXH1 and FOXQ1.In addition,based on GWAS SNP,Hi-C and brain tissue eQTL SNP data,16 GWAS SNPs and 82 eQTL SNPs were located in brain-aceDHS,which regulate genes involved in brain development or other human diseases.(4)In these brain-aceDHSs,we used CRISPR-Cas9 knockout technology,western blotting and qPCR techniques confirmed that knockdown of brain-aceDHS(chr12: 131621602-131623539)significantly decreased the expression of its target gene GPR133,indicating The enhancer function of brain-aceDHS.GPR133 gene is associated with glioblastoma,further demonstrating the association of SNPs in brain-aceDHSs with brain diseases.In summary,the analysis of the variation of brain-related gene regulatory regions in this study provides important clues for a better understanding of the genetic basis of human brain phenotypic differences in adaptive evolution.