Regulation Of The Expression Of SARS-CoV-2 Target Genes In Testis

Since COVID-19 broke out in late December 2019,the way of human production and lifestyle has been profoundly changed.As of February 27,2021,the virus had infected over113 million people worldwide,and the number of deaths had totaled more than 2.5 million.COVID-19 is pneumonia caused by SARS-CoV-2 coronavirus infection.The last two decades of the 21 st century have seen emerging zoonotic coronavirus(CoV)diseases,including severe acute respiratory syndrome(SARS)(Holmes 2003),Middle East respiratory syndrome(MERS)(Graham et al.,2013).SARS-CoV-2 shares 80% nucleotide identity to SARS-CoV.Both viruses can use ACE2 and TMPRSS2 as receptors to invade host cells.It is now known that the testicular cells of the infected SARS were seriously damaged,Ig G was highly expressed in the Sertoli cells and germ cells of the patients.However,the potential effects of SARS-CoV-2 on testicular tissue remain to be explored.Infectious and inflammatory conditions in the reproductive system are among major aetiological factors.Various microorganisms including,bacterial,virus and protozoa,can impair male fertility.Microbial pathogens directly interact with sperm may exert harmful effect including,sperm death,decreasing sperm motility.At present,some studies have suggested that SARS-COV-2 may infect testis and affect spermatogenesis.This raise a question that what kind of cell cluster can be infected by SARS-CoV-2? What is the expression level of receptor in different cells? How is the abnormal expression and regulation of genes affected by virus invading host cells realized? These questions are of great value for understanding the mechanism of SARS-CoV-2 and deserve further exploration.The SARSCoV-2 target gene in this study consists of the genes corresponding to 332 proteins interacting with the SARS-CoV-2 virus genome in host cells and the SARS-CoV-2 receptor gene.In this study,we make fully use of testis scRNA-seq datasets to explore receptor expression levels within each cell cluster are accurately quantitated.Besides,the ACE2 positive rates among divergent ages are compared.Furthermore,the binding proteins of SARS-CoV-2 proteins were analyzed by multi-omics in different cell types during spermatogenesis.In the last,interactive modifications for specific cell types were analyzed.Specific process and results are as follows.1.Analysis the ACE2 expression and distribution patternThrough analysis scRNA-seq datasets,these two datasets can be cluster into 12 cell types consisting of 4 somatic cells and 8 spermatogenic cell types.Sertoli cells,Leydig cells as well as SSC have a higher ACE2 positive proportion.In the comparison of men with different ages,the highest ACE2 positive rate was found in men aged 30,the relatively low ACE2 positive rate was found in men aged 20 and the lowest in men aged 60,suggesting that the ACE2 expression level may be closely related to age.2.Multi-omics regulation of viral target proteins during spermatogenesisFirst,we clustered different cell types in testicular tissue according to their expression status.Results show that compared with somatic cells,each cell in the cluster related to spermatogenesis correlation.the correlation in each cell cluster is higher.In general,the expression level of target genes involved in spermatogenesis is relatively high when compared to somatic cells.Target genes are mainly involving in regulation of cellular protein translation,cell division as well as cell proliferation.Second,the histone modifications in pachytene spermatocytes and round spermatid stages were fully analyzed.Active histone modification such as H3K4me1,H3K27 ac as well as H3K4 me,has higher modification intensity at gene promoters.Some genes are regulated by corresponding histone modification.Third,we analyzed chromatin open region in target cells.It is obvious that there are obvious open regions of chromatin at the target gene promoters of spermatogonia.In spermatogonia,the open chromatin regions at promoters contain a variety of transcription factor binding sites include: GATA1,LEF1,MYC,NRF1,SP1 and TCF7.There are three transcription factors including ZNF263,HOXD10 and FOXA1,which bind into promoters at sperm.Forth,the further analysis was made from the perspective of DNA methylation.The methylation level at gene promoters has divided the genes into three categories: high methylation genes,middle methylation genes and low methylation genes.The modification levels of H3K4me1 and DNA methylation showed a seesaw dynamic pattern.In other words,the promoter with high DNA methylation has low H3K4me1 modification level.The promoter with low DNA methylation has high H3K4ME1 modification and vice versa.Last,we found that viral target genes also have unique miRNA regulation characteristics.The results showed that multiple miRNAs and target genes formed a tight regulatory network.It could be seen that multiple miRNAs formed regulatory relationships with their target genes.For instance,miR-34 c could induce apoptosis of primary spermatocytes and male germ stem cells and let-7a overexpression might be a sign of spermatogenesis failure.3.Synergistic regulation of viral original protein genesWe first focused on pachytene spermatocytes and round spermatid,results showed that target genes of SARS-CoV-2 have higher modification level of active histones.There were247 and 191 genes with two histone modifications,including H3K4me1 and H3K27 ac,at the same time.the modification levels of these two modifications were changing dynamically.Based on protein interaction networks,there are some specific nodes that are very important for SARS-CoV-2 infection among these selected nodes with a degree greater than 3,such as LARP1,BSG,EIF4 H,RAE1,PRKACA.In addition,through protein interaction network,related genes with low methylation and high active histone modification levels have greater connectivity and antiviral effects.