Study On The Function And Regulation Mechanism Of H3K4me2 In The Formation Of Chicken SSCs

Spermatogonial stem cells(SSCs)are a kind of adult stem cells unique to male animals that can transmit the parental genetic information to their offspring.They have the ability to self-renew and differentiate and can replace embryonic stem cells(ESCs),For treatment and genetic modification,and does not involve ethical issues and immune rejection issues.To this end,scientists at home and abroad are committed to inducing the formation of a large number of functional SSCs in vitro,and in the treatment of infertility in clinical medicine.However,with the in-depth study of the mechanism of SSCs,it has been found that the presence of in vitro induction efficiency is low,the number of acquired cells is small and the quality is poor,and in vitro induction of differentiation and culture system instability and other issues.This has led to great restrictions on the application of SSCs.Therefore,it is particularly important to systematically study the regulatory mechanisms that affect the occurrence of SSCs.ChIP-Seq technology was used to detect the level of histone H3K4 methylation in ESCs,PGCs,and SSCs during the differentiation of chicken male germ cells.It was determined that histone H3K4me2 was involved in the formation of chicken SSCs.There is a differential distribution,suggesting that it plays an important regulatory role in the formation of SSCs,but its specific mechanism of action is not known.In order to systematically study the specific regulation of H3K4me2 on SSCs.In this study,combined with a feeder-free RA culture inducing system and chicken embryonic vascular injection system established in the laboratory,the RNA interference technique was used to knock down the pre-selected histone methylation enzyme MLL2 and demethylase LSD 1.Expression,thereby altering the level of histone H3K4me2 methylation during in vivo and in vitro SSCs formation,exploring the role of histone H3K4me2 in the regulation of chicken SSCs,and on this basis searching for histone H3K4me2 by RNA-seq and CHIP-qPCR The relationship between methylation and related target genes,elaborates the regulation of histone H3K4me2 methylation and gene interactions on the formation of chicken SSCs,provides a theoretical basis for clarifying the mechanism of germ cell development and differentiation,and provides a fundamental basis for the treatment of male infertility.Solutions,while providing reference materials for improving poultry productivity in production practices.The results of this study are as follows:(1)The time-dependent expression and localization of histone methylation-modifying enzymes and H3K4me2 in different tissues and cells of chickens Screening of specific methyltransferases(ASH2L,MLL2,MLL5)and demethylases(LSD1,KDM5A,KDM5B)that determine H3K4me2 levels during male germ cell development,RT-qPCR detection of each group of proteins The expression and distribution of the modified enzyme in different cells and tissues of chicken showed that ASH2L(23.03±0.02),MLL2(467±0.016),AMLL5(9.46±0.046)and LSD1(846.81±0.024)were the highest in chicken testis.However,the expression of KDM5A(140.06±0.024)and KDM5B(13.47±0.516)was highest in ovarian tissues and lower in testis.At the same time,these histone-modifying enzymes ASH2L(2.79±0.080),MLL2(1.05±0.004),LSD1(4.03±0.082),KDM5A(3.88±0.008),and KDM5B(2.51±0.081)expressed the highest in chicken PGCs,while MLL5(The highest expression level was 5.24±0.080)in chicken SSCs.Western blot showed higher levels of histone H3K4me2 methylation in PGC and SSC cells compared to ESC cells.There was no differential expression of histone H3K4me2 methylation levels in adult heart,liver,spleen,lung,kidney,testis,and ovarian tissue.(2)Screening of histone H3K4me2 methylation-modifying enzyme during the regulation of chicken SSCs formation According to NCBI database provided ASH2L,MLL2,MLL5,LSD1,,KDM5A and KDM5B CDS sequences,three shRNA target sites were designed,and the sequencing results showed that each lentivirus interfering vector was successfully constructed;successfully constructed transfected interfering plasmids were transfected.The RNA was extracted from chicken DF1 cells with good growth.The best interference sequences of each vector were ASH2L-shRNA2,MLL2-shRNA3,MLL5-shRNA3,L SD1-shRNA1,KDM5A-shRNAl and KDMSB-shRNAl by RT-qPCR.Achieve 88%,65%,79%,68%,60%,and 67%;Western Blot results further show that only MLL2-shRNA3 can significantly reduce the level of histone H3K4me2 methylation,while LSD1-shRNAl can significantly increase histone H3K4me2 Methylation levels.Therefore,the histone H3K4 methylase MLL2 and demethylase LSD1 were identified as key candidate enzymes for regulating H3K4me2 in the formation of chicken SSCs.(3)IH3K4me2 regulates the formation of chicken SSCs The function of H3K4me2 in the formation of SSCs was studied in vivo and in vitro.The results showed that in vitro,RT-qPCR results showed that shRNA-MLL2 significantly down-regulated the expression of MLL2 in chicken ESCs(0.34±0.01 vs 1.00±0.02),shRNA-LSDl significantly down-regulated the expression of LSD1(0.42±0.03 vs 1.00±0.02);Western Blot results showed that the level of H3K4me2 methylation in shRNA-MLL2 group was decreased in ESCs,and the level of H3K4me2 methylation in shRNA-LSD1 interference group was increased;the morphology of each group of cells induced to the 12th day was observed.It was found that RA induced group and shRNA-LSD1 group could form Spermatogonial stem cells-like(SSCs-like)cells when they were induced to 12 days,and clustered into grape clusters,while RA+ shRNA-MLL2 group could not Spermatogonial stem cells were induced;RT-qPCR results showed that the expression of Cvh,Blimp-1,Stra8,LIN28a,integrin a6 and integrin ?1 in the shRNA-LSD1 group was up-regulated compared to the RA-induced group.However,in the RA+shRNA-MLL2 group,expression was consistently low;cytochemical immunoassay results showed that Cvh+ and C-kit+ in the RA+shRNA-LSD1 group were comparable to the RA-induced group(16.14%±0.42)at 4 days of induction.Cvh+ and C-kit+ double positive cells increased significantly(26.03%± 0.47)In the RA+shRNA-MLL2 group,Cvh+ and C-kit+double positive cells decreased significantly(8.98%±0.25).Also on the 12th day after induction,compared with the RA-induced group(6.15%±0.43),the integrin ?6+ and integrin ?1+double-positive cells in the RA+shRNA-LSD1 group were significantly increased(9.31%±0.47),whereas in the RA+shRNA-MLL2 The number of integrin a6+ and integrin ?1+ double positive cells in the group was significantly decreased(5.32%±0.25);flow cytometric analysis showedthat after induction to 4d,Cvh(8.6±0.3%)and C-kit(7.2±0.1%)in the LSD1 interference group.The proportion of positive cells was significantly higher than that of RA group,while the proportion of Cvh(2.2±0.1%)and C-kit(1.5±0.1%)positive cells in the MLL2 interference group was significantly lower than that in the RA group.Similarly,the proportion of integrin a6(14.9±0.2%)and integrin ?1(15.5±0.2%)positive cells in the LSD1 interference group was significantly higher than that in the RA group after 12 days of induction,whereas integrin a6 in the MLL2 interference group(10.5±0.2%).The proportion of positive cells with integrin ?1(5.6±0.2%)was significantly lower than that of RA group.In vivo,fluorescence microscopy revealed that Lentiviral interfering vectors could express green fluorescent protein in chick embryos for 5.5 days,while the blank group showed no green fluorescence;RT-qPCR results showed that the lentivirus interfering vector shRNA-MLL2 in chicken embryos And shRNA-LSD1 can significantly interfere with the mRNA levels of MLL2 and LSD1,and the interference efficiency can reach 68%and 76%respectively.Western blot results showed that the level of H3K4me2 methylation decreased after interference with MLL2,while the level of H3K4me2 methylation increased after interference with LSD1.RT-qPCR results showed that compared with the control group,Dazl,Stra8,integrin a6,integrin ?1,and other reproductive marker genes were significantly downregulated to 0.37±0.05,0.43±0.02 in the testes incubated up to 18 days,0.19±0.03 and 0.78±0.06,while the shRNA-LSD1 injection group also significantly increased the reproductive-related genes such as Stra8,integrin a6,and integrin ?1 to 1.1 ±0.03,1.51±0.01,and 1.69±0.06.Flow cytometry results showed that:There was no significant difference between the Blank group(26.3%±0.1;26%±0.2)and the shRNA-NC group(9.4%±0.1;9.7%±0.1)in hatching chicken embryos at 18 days,while shRNA-MLL2 group Number of medium SSCs(The percentage of SSCs in shRNA-LSD1 group(31.3%±0.4;12.4%±0.3)was significantly higher than that of Blank group.And shRNA-NC group(P<0.01);Western blot results showed that compared with the control group,intergrina6 and intergrin(31 protein expression in the LSD1 interference group was higher than the control group,while the interleukin6 and intergrin?1 protein expression in the MLL2 interference group.Lower than the control group.(4)H3K4me2 regulates the apparent mechanism of differentiation of chicken SSCs Illumina Solexa transcriptome sequencing technique was used to sequence transcriptomes of SSCs in different treatment groups.The results showed that there were 17431 differentially expressed genes in shRNA-MLL2 and Blank groups,of which 8473 genes were up-regulated and 8958 genes were expressed relative to the Blank group.Down-regulation;There were 17334 differentially expressed genes in the shRNA-LSD1 and Blank groups,of which 9086 genes were up-regulated and 8248 genes were down-regulated relative to the Blank group.GO analysis results showed that the histone methylation enzyme MLL2 and the demethylase LSD1 have catalytic activity and are associated with the binding of proteins and DNA in SSCs,and are also closely related to biological processes such as cell differentiation,reproductive process,and spermatogenesis;and KEGG Pathway further demonstrated that MLL2 and LSD1 can affect the expression of multiple genes in TGF-?,Wnt,and MAPK pathways closely related to the formation of spermatogonial stem cells;CHIP-qPCR results indicate that MLL2 and LSD1 can interact with DDX4,BMP4,DAZL,and Blimp-1.Promoter binding of the reproductive marker gene,such as LIN28a,affects the distribution level of H3K4me2 in the binding region and regulates gene expression.After knocking down MLL2,H3K4me2 levels of the DDX4,BMP4,DAZL,Blimp-1,and LIN28a gene promoters were reduced,and gene expression was inhibited,thereby inhibiting the formation of spermatogonial stem cells.After knocking down LSD1,the H3K4me2 levels of these gene promoters are increased,gene expression is activated,and the formation of spermatogonial stem cells is promoted.