The Function And Mechanism Of Chromatin Remodeling Factor CHD2 In Spermatogenesis

The latest reported data show that from 1973 to 2018,the number of sperm in men worldwide has decreased by about 51.6%and sperm quality has also declined precipitously.According to the World Health Organization,about 15%of couples at childbearing age worldwide have infertility problems,with about 50%of them being female factors alone,30%male factors alone,and about 20%in both.Abnormal spermatogenesis is a common cause among male infertility patients.Spermatogenesis is a complex and sophisticated process of cellular differentiation that is regulated by a variety of factors,including genetic and epigenetic factors.Chromatin remodeling is an important epigenetic regulation mechanism,which plays an important role in spermatogenesis and male fertility.The abnormality or disorder of its regulation can lead to spermatogenesis disorders,sperm morphological abnormalities,azoospermia and other infertility symptoms.The CHD（chromodomain helicase/ATPase DNA binding protein）is a subfamily of the chromatin remodeling complex family that uses energy generated by hydrolysis of adenosine triphosphate（ATP）to alter the position of nucleosomes,thereby participating in the regulation of gene expression.CHD2,a member of the CHD family,has been found to be involved in the development of embryonic stem cells,nerve cells and muscle cells.In the field of reproductive research,the Rs1406714 single nucleotide polymorphism of the CHD2 gene was found to be possibly associated with non-obstructive azoospermia（NOA）in Han Chinese men.However,the relationship between CHD2 and spermatogenesis and underlying mechanism have not been investigated.Mice are commonly used model organisms in reproductive development studies and CHD2 proteins are highly homologous in humans and mice.Thus,we investigated the role and underlying mechanism of CHD2 in spermatogenesis using mice as an experimental model,and the main research results were as follows.（1）CHD2 is highly expressed in male germ cells.The expression of CHD2 m RNA and protein was analyzed by database search,RT-q PCR,immunofluorescence and other experiments.The results showed that CHD2 was highly expressed in the testis and mainly located in the nucleus of cells.CHD2 was expressed in spermatogenic cells at all stages,with the highest expression in meiotic spermatocytes.The results suggests that CHD2 may have a physiological function in testicular development or spermatogenesis.（2）Chd2 haploinsufficiency causes compromised testicular germ cell proliferation and impaired fertility.In this study,we constructed and bred Chd2 heterozygous knockout mice(Chd2^+/-)using CRISPR/Cas9 gene editing technology.By morphological observation,in vitro fertilization（IVF）experiments and litter numbers analysis,we found that the development of testicular tissue was retarded,the rate of abnormal sperm increased and fertility decreased in Chd2 haploinsufficiency mice.Flow cytometry,hematoxylin-eosin（HE）staining,immunohistochemistry（IHC）and TUNEL assay results showed that the proliferation of mouse germ cells was inhibited,but the testis structure was not significantly changed,the composition of different germ cells was not significantly changed,and germ cell apoptosis was not increased in Chd2^+/-mouse.In addition,immunofluorescence detection and other experiments revealed that Chd2^+/-mice had impaired DNA damage repair function and abnormal chromosomal synapsis during spermatogenesis.Finally,transcriptome analysis of testicular tissue based on RNA seq showed that self-renewal-associated genes such as Plzf were significantly downregulated in Chd2^+/-mice compared with wild-type mice,indicating that Chd2 haploinsufficiency inhibits spermatogonial self-renewal,resulting in smaller testes in Chd2^+/-mice.（3）CHD2 promotes spermatogonial self-renewal.After knock down of CHD2 by si RNA in spermatogonia,CCK8,EDU,clone formation,sphere-forming assay and immunofluorescence were performed.The results showed that knockdown of CHD2 inhibits proliferation and stemness of spermatogonia.Further,by RT-q PCR and Western blot,the key stemness and proliferation-related genes Oct4,Plzf,Ccnb1 and Ccnd2 were significantly downregulated after CHD2 knockdown in spermatogonia.Immunofluorescence staining and Ch IP-q PCR experiment results demonstrated that CHD2 was associated active chromatin marker H3K4me3 and CHD2 maintained H3K4me3 enrichment on promoter regions of Oct4,Ccnb1,and Ccnd2 gene.And the regulation of CHD2 on OCT4,PLZF,CCNB1 and CCND2 expression were also confirmed in vivo.（4）CHD2 promotes the m RNA stability of Oct4 and Plzf by interacting with CSTF3.LC?MS mass spectrometry and immunoprecipitation（IP）experiments confirmed that CHD2 interacted with the cleavage stimulation factor subunit 3（CSTF3）.Based on the structure of CHD2 protein,the CHD2-A expression vector containing Chromodomain,CHD2-B expression vector containing ATPase domain and CHD2-C expression vector containing C-terminal DNA-binding domain was respectively constructed,and then each recombinant vector was overexpressed in293T cells,respectively.IP experiments confirmed the interaction between CHD2-B and CSTF3.RNA immunoprecipitation（RIP）and RT-q PCR confirmed that the interaction between CHD2 and CSTF3 promoted the m RNA stability of Oct4 and Plzf.CHD2 protein recognized and bound to Oct4 and Plzf m RNA through the CHD2-A fragment.Finally,by database analysis combined with experiments,we confirmed that the binding site of CHD2 on target m RNA was ACAGG.The above results indicate that CHD2 interacts with CSTF3 to regulate target genes at RNA level.Our study is the first to reveal that CHD2 can regulate gene expression by affecting m RNA stability.（5）Chd2^+/-mice are more intolerant to cyclophosphamide-induced spermatogenic damage.Finally,we explored the significance of CHD2 mutation in the reproductive protection for cancer patients.Using the anticancer drug cyclophosphamide to construct a mouse spermatogenic injury model,it was found that compared with wild-type mice,cyclophosphamide caused more severe testes damage in Chd2^+/-mice,and Chd2^+/-mice had a higher proportion of damaged seminiferous tubules,more significant arrest of spermatogenesis,and more apoptotic germ cells.The reason may be related to the decreased self-renewal ability of spermatogonia and DNA damage repair ability caused by the deficiency of CHD2 expression in Chd2+/-mice,indicating that the spermatogenesis process in Chd2^+/-mice was more intolerant to anticancer drugs such as cyclophosphamide.In summary,for the first time,our study elucidated the relationship between CHD2 and male reproduction,and demonstrated the role of CHD2 in spermatogonial stem cell self-renewal.We confirmed the mechanism that CHD2 promotes spermatogonial self-renewal by maintaining chromatin accessibility and improving target m RNA stability.This study expands our understanding of the mechanism of chromatin remodeling factors,and provides candidate targets for the diagnosis and treatment of male infertility such as spermatogenesis disorders and the protection of spermatogenesis in cancer patients.