RNA polymerase II pausing is essential during spermatogenesis for appropriate gene expression and completion of meiosis.

Male germ cell development requires precise regulation of gene activity in a cell-type and stage-specific manner, with perturbations in gene expression during spermatogenesis associated with infertility. Here, we use steady-state, nascent and single-cell RNA sequencing strategies to comprehensively characterize gene expression across male germ cell populations, to dissect the mechanisms of gene control and provide new insights towards therapy. We discover a requirement for pausing of RNA Polymerase II (Pol II) at the earliest stages of sperm differentiation to establish the landscape of gene activity across development. Accordingly, genetic knockout of the Pol II pause-inducing factor NELF in immature germ cells blocks differentiation to spermatids. Further, we uncover unanticipated roles for Pol II pausing in the regulation of meiosis during spermatogenesis, with the presence of paused Pol II associated with double-strand break (DSB) formation, and disruption of meiotic gene expression and DSB repair in germ cells lacking NELF.