Mammalian spermatogenesis shows prominent chromatin and transcriptomic switches in germ cells, but it is unclear how such dynamics are controlled. Here we identify RNA helicase DDX43 as an essential regulator of the chromatin remodeling process during spermiogenesis. Testis-specific Ddx43 knockout mice show male infertility with defective histone-to-protamine replacement and post-meiotic chromatin condensation defects. The loss of its ATP hydrolysis activity by a missense mutation replicates the infertility phenotype in global Ddx43 knockout mice. Single-cell RNA sequencing analyses of germ cells depleted of Ddx43 or expressing the Ddx43 ATPase-dead mutant reveals that DDX43 regulates dynamic RNA regulatory processes that underlie spermatid chromatin remodeling and differentiation. Transcriptomic profiling focusing on early-stage spermatids combined with enhanced crosslinking immunoprecipitation and sequencing further identifies Elfn2 as DDX43-targeted hub gene. These findings illustrate an essential role for DDX43 in spermiogenesis and highlight the single-cell-based strategy to dissect cell-state-specific regulation of male germline development.

HMG protein Tox4 is a regulator of PP1 phosphatases with unknown function in development. Here we show that Tox4 conditional knockout in mice reduces thymic cellularity, partially blocks T cell development, and decreases ratio of CD8 to CD4 through decreasing proliferation and increasing apoptosis of CD8 cells. In addition, single-cell RNA-seq discovered that Tox4 loss also impairs proliferation of the fast-proliferating double positive (DP) blast population within DP cells in part due to downregulation of genes critical for proliferation, notably Cdk1. Moreover, genes with high and low expression level are more dependent on Tox4 than genes with medium expression level. Mechanistically, Tox4 may facilitate transcriptional reinitiation and restrict elongation in a dephosphorylation-dependent manner, a mechanism that is conserved between mouse and human. These results provide insights into the role of TOX4 in development and establish it as an evolutionarily conserved regulator of transcriptional elongation and reinitiation.