Mouse CHD4-NURD is required for neonatal spermatogonia survival and normal gonad development

Abstract

Testis development and sustained germ cell production in adults rely on the establishment of spermatogonia stem cells and their proper differentiation into mature gametes. Control of these processes involves not only promoting the expression of genes required for cell 2 survival and differentiation but also repressing other cell fates. This level of transcriptional control requires chromatin-remodeling complexes that restrict or promote transcription machinery. Here, we investigated the roles of the NUcleosome Remodeling and Deacetylase (NURD) complex during spermatogenesis. Our cytological and biochemical analyses revealed differential expression and composition of NURD subunits in gametocytes at different stages of testis development. Germ cell-specific deletion of the NURD catalytic component CHD4, but not CHD3, resulted in male infertility due to failed maintenance of the undifferentiated spermatogonia stem cell population. Genome-wide CHD4 localization and transcriptomic analyses revealed that CHD4 binds the promoters and regulates the expression of genes involved in spermatogonia cell survival, and differentiation. These results uncover the requirements of CHD4 in mammalian gonad development, and point to unique roles for the NURD complex with respect to other chromatin remodelers during gamete development. Significance StatementGametogenesis is a fundamental developmental program required for sustained fertility and survival of all sexually reproducing species. The developing male gamete undergoes numerous cell divisions and developmental stage transitions that are carefully monitored by epigenetic mechanisms. One prominent mechanism is directed by chromatin remodeling complexes, which modify chromatin structure and thereby control fundamental cellular processes such as gene transcription. In this work, we focused in understanding the role of CHD4 and CHD3 proteins, catalytic subunits of the NURD chromatinremodeling complex, in mouse gametogenesis. We find that CHD4 has an essential function in gametogenesis, with an absolute requirement for maintenance of and differentiation of spermatogonia populations in the developing testis. This is achieved by CHD4-mediated transcriptional regulation of genes important for spermatogonia survival, and differentiation.

A regulatory role for CHD4 in maintenance of the spermatogonial stem cell pool

“…This included the RNA-binding protein DAZL, which is thought to drive spermatogonial differentiation ( Mikedis et al., 2020 ; Schrans-Stassen et al., 2001 ), and was significantly upregulated following Chd4 knockdown (p < 0.001) ( Data S2 ). Upon mining a publicly available anti-CHD4 chromatin immunoprecipitation (ChIP) dataset produced using THY1+ undifferentiated spermatogonia ( de Castro et al., 2020 ), it was determined that 102 of the DEGs identified here are direct targets of CHD4 binding ( Data S4 ), including Dmrt1 and Gfra1 ( Figure 4 B), which are known regulators of SSC function.…”

“…The interaction between CHD4 and SALL4 is also supported by SALL4 co-IP experiments in undifferentiated spermatogonia that have been published previously ( Chan et al., 2017 ). Finally, we mined publicly available anti-SALL4 ( Lovelace et al., 2016 ) and anti-CHD4 ( de Castro et al., 2020 ) ChIP datasets from THY1+ undifferentiated spermatogonia, and identified 282 genes that were common targets of these proteins ( Data S4 ). Common gene targets included Gfra1 , which we have shown to be significantly downregulated upon Chd4 knockdown ( Figures 4 B and S4 A), as well as 18 other genes that were shown to have dysregulated expression following Chd4 knockdown in our scRNA-seq experiments ( Data S4 ).…”

“…Cumulatively, findings reported here support a circumstance in which CHD4 is an integral component of the self-renewal machinery within SSCs. Although experiments described here were primarily conducted in an in vitro setting, spermatogonial dysfunction and infertility has recently been reported following Chd4 knockout in the germline in vivo ( de Castro et al., 2020 ), suggesting that the mechanisms identified in this manuscript transcend to SSCs in the testicular microenvironment. Specifically, we propose that CHD4 acts within the NuRD nucleosome remodeling complex to activate the transcription of genes important for SSC maintenance and self-renewal.…”

“…Specifically, we demonstrated that a reduction in Chd4 expression instigates a ∼50% loss of stem cell content. In support of the notion that CHD4 expression plays a critical role in maintenance of SSCs, a recent preprint article ( de Castro et al., 2020 ) demonstrated that germline-specific Chd4 knockout mice are infertile, with a complete absence of germ cells in the adult testis. Notably, ablation of CHD4 expression in the germline from embryonic day 15.5 ( Ddx4 -Cre) did not prevent formation of the spermatogonial pool following birth.…”

The chromatin-remodeling enzyme CHD3 plays a role in embryonic viability but is dispensable for early vascular development

“…However, we found Chd3 -heterozygous mice ( Chd3 Δ/fl and Chd3 Δ/+ ) represented at expected numbers at weaning (Tables 4 and 5 ), indicating that Chd3 Δ gametes are viable and fertile. Moreover, a recent preprint reports that CHD3 plays no significant role in testis or sperm development when our Chd3 -flox allele is combined with a primordial germ cell-specific Ddx4-Cre [ 25 ]. Alternatively, CHD3 is expressed in embryonic mouse brain cortices as early as E15.5 and has been shown through shRNA electroporation studies to influence neuronal migration and laminar identity in late stages of embryonic cortical development [ 6 ].…”