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

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 both steady-state and nascent 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 impairs differentiation to mature spermatids. Moreover, our work reveals unanticipated roles for Pol II pausing in the regulation of meiosis during spermatogenesis, with the presence of paused Pol II influencing double strand break formation by Spo11, and disruption of Spo11 expression in germ cells lacking NELF. Overall design: Investigation of single cell RNA-seq on whole testis in Control (N=4), NELF-B cKO (N=2), and TDP-43 cKO (N=3) C57BL/6 mice.