Single-Cell RNA-seq Uncovers Dynamic Processes Orchestrated by RNA-Binding Protein DDX43 in Chromatin Remodeling during Spermiogenesis

Abstract Mammalian spermatogenesis shows prominent chromatin and transcriptomic switches with delicate morphological and functional alterations 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. In addition, the loss of its ATP hydrolysis activity by a missense mutation in the ATPase motif replicates the infertility phenotype in global Ddx43 knockout mice. Single-cell RNA sequencing (scRNA-seq) 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 (eCLIP-seq) further identify Elfn2 as DDX43-targeted hub gene. Elfn2 knockdown in testis leads to a phenotype similar to Ddx43 knockout or Ddx43 ATPase-dead knock-in. These findings illustrate an essential role for DDX43 in post-meiotic chromatin remodeling and highlight the single-cell-based strategy for a refined dissection of cell-state-specific regulation of germline development.