The Mechanism Of Heat Shock Transcription Factor 5 In Spermatogenesis

Part I Dissecting the structure and function of HSF5 proteinObjective To analyze the sequence homology of HSF5(Heat shock transcription factor 5,HSF5)between human and mouse,to analyze the structure and function of HSF5 protein,and to verify its expression and localization during spermatogenesis.Methods The HSF5 protein domain was analyzed,and the full-length HSF5 protein(1625aa)and truncated body protein(11-228aa 418-577aa)were purified according to the structural domain characteristics of HSF5 protein.Gel migration experiment(EMSA),membrane interference experiment(BLI)and magnetic tweezer binding experiment were used to explore the binding effect of HSF5 protein to single and double stranded DNA.HSF5 antibody was prepared by immunizing rabbit and mouse with HSF5 purified antigen.The expression of HSF5 protein in different tissues of wild-type mice was verified by qPCR,Westen blot and frozen section of testicular tissue,and the localization of HSF5 in human and mouse testicular tissues was verified.Results(1)Phylogenetic tree analysis showed that HSF5 protein was highly conserved between species,and HSF5 localization in human testicular tissue was the same as that in mice.(2)The results of qPCR and Western blot showed that HSF5 was highly expressed in mouse testicular tissue,and showed a trend of elevated expression during spermatogenesis.(3)Purified mouse antibody and rabbit antibody confirmed that HSF5 was distributed in the nucleus of spermatocytes in a haze and was specifically localized to the XY body of spermatocytes in pachytene stage.The strong signal on the XY body of spermatocytes in pachytene stage started from mid-pachytene stage.It weakened slightly in Late pachytene stage and disappeared in Diplotene stage,but its diffuse localization in the nucleus persisted until the round sperm stage.(4)HSF5 DNA binding domain is located at the position of amino acid 11-228aa.HSF5 protein exposed to DNA binding domain has single-stranded DNA binding function and can specifically bind spermatocytes chromatin.Conclusions HSF5 protein is highly conserved and consistent in localization between humans and mice.It is a testis-specific chromatin-binding protein with single-stranded DNA binding function.It is located at the stage from pachytene spermatocytes to round spermatids.In the nucleus and highly enriched in the XY body of pachytene spermatocytes.Part Ⅱ Analysis of the causes of spermatogenesis dysfunction in Hsf5 knockout miceObjective Analyze the specific stage of spermatogenesis arrest in Hsf5 knockout mice and the chromosomal behavior involved in HSF5 protein,and explore the causes of spermatogenesis disorder in Hsf5 knockout mice.Methods Chromosome spreading and frozen sections of testicular tissue were used to explore the specific stage of spermatogenesis arrest in Hsf5-/-and the chromosomal behavior involved in HSF5.RNA Seq and Cut&Tag experiments were used to further verify the cause of spermatogenesis disorder in Hsf5 knockout mice.Results(1)Hsf5-/-mice results in meiosis arrest of spermatocyte in Mid-pachytene stage.(2)The XY chromosome agglutination defect of pachytene spermatocytes in Hsf5/-mice showed an elongated form that could not agglutinate.(3)Meiotic chromosome silencing(MSCI)failed in Hsf5-/-mice,and RNA POLII was abnormally activated.(4)RNA Seq analysis results showed that compared with wild-type mice,the transcription of a large number of reproduction-related genes on spermatocytes of Hsf5-/-mice was decreased and accompanied by a variety of abnormal alternative splicing,but the expression of genes on X and Y chromosomes was significantly up-regulated.qPCR results confirmed that the gene expression on XY chromosome of Hsf5-/-mice testicular tissue was significantly higher than that of wild-type mice.(5)Cut&Tag analysis showed that H3K4me3,a marker of activated chromatin,showed a peak on the X and Y chromosomes of Hsf5-/-mice spermatocytes,and it was noted that the chromatin packaging on XY chromosomes was loose.(6)Frozen sections of testicular tissue and chromosome spreading results also confirmed that H3K4me3,an activated chromatin marker,was highly signaled in Hsf5-/-spermatocytes,while H3K9me3,an inhibitory chromatin marker,was low signaled.(7)Frozen sections of testis tissue of Hsf5 knockout mice showed that the retrotransposon LINE1 abnormally activated high signal,while there was no abnormal activation signal of LINE 1 in the testis tissue of wild-type mice.Conclusions The meiotic arrest of Hsf5 knockout mice is at the mid-pachytene stage,and the main causes of spermatogenesis defects are the failure of meiotic sex chromosome silencing(MSCI)and the abnormal activation of retrotransposons.Part Ⅲ Exploring the specific mechanism of HSF5Objective To find the interaction protein and regulatory pathway of HSF5,and analyze the specific mechanism of HSF5 in meiotic chromosome silencing and retrotransposon inhibition.Methods The differential proteins of HSF5-IP products in testicular tissue of wild-type and Hsf5 knockout mice were identified by immunoprecipitation mass spectrometry(IPMS).The IP-MS and RNA Seq data were comprehensively analyzed to screen the key differential genes and proteins jointly annotated by transcriptome and proteome of wildtype and Hsf5 knockout mice,and the screened interaction proteins were verified by COIP.In wild-type and Hsf5 knockout mice,qPCR,Western blot,chromosome spreading and frozen sections of testicular tissue were used to further analyze the expression and localization of the screened proteins,their complexes and their downstream proteins.The expression and localization of HSF5 were reverse verified in the knockout mice of the verified interaction protein to determine the upstream and downstream relationship between them.The reasons of retrotransposon activation and whether the piRNA pathway is affected by Hsf5 gene knockout were explored by experimental methods such as sulfite sequencing,unit site methylation detection(BSP)and small RNA sequencing.Results(1)A variety of meiotic related proteins were identified by IP-MS,SCML2 and MDC1 were mainly associated with XY body formation and MSCI.Co-IP verified that HSF5 interacted with SCML2 and MDC1.Hsf5 knockout resulted in approximately 60%SCML2 could not be located in XY body of pachytene spermatocyte,and its complex USP7 and downstream protein BRG1 were both abnormally located,while Hsf5 was normally located in Scml2-/-mice.(2)MDC1 could be localized to XY body in spermatocytes of Hsf5-/-mice,but the localization of MDC1 was elongated with the change of XY body morphology,while the localization of Hsf5 disappeared completely in spermatocytes of Mdc1-/-mice.(3)The comprehensive analysis of RNA Seq and IPMS showed that the transcription and translation levels of PIWIL1(PIWIL1)were significant differences in Hsf5 knockout mice.The transcription level of PIWIL1 in testicular spermatocytes of wild-type mice was 10 times higher than that of Hsf5-/-mice.The expression of HSF5 protein in IP samples of wild-type testicular tissue was 2,043,961,while it was 0 in the the testicular tissue IP sample of Hsf5-/-mice.The results of qPCR and Western blot showed that the expression of PIWIL1(Piwill)decreased significantly in the testis of Hsf5-/-mice.The results of frozen sections of testicular tissue showed that compared with the wild type,the localization of PIWIL1 in frozen sections of testicular tissue of Hsf5-/-mice was significantly weakened.(4)The methylation level of Line 1 DNA in testicular tissue of Hsf5-/-mice decreased significantly.(5)The production of mature piRNA(pachytene piRNA)decreased in Hsf5-/-mice.In Hsf5-/mice,the piRNA cluster and expression of piRNA decreased significantly,what’ more,the mRNA level corresponding to the decreased cluster also decreased significantly.Go analysis showed that the genes affected by Hsf5-/-were related to germ cell development and spermatogenesis.Conclusions The function of HSF5 is located upstream of SCML2 and downstream of MDC1.It causes meiotic chromosome silencing failure by affecting the function of SCML2.It causes the inhibition of retrotransposon at the transcriptional and post transcriptional levels by affecting the shear of PIWIL1,the reprint of piRNA and the methylation of Line1.The normal establishment of MSCI can protect XY chromosome from the attack of retrotransposon,The abnormally established MSCI enhanced the activation of transposons to attack the genome of germ cells,and finally led to the complete stagnation of germ cell development..