Paralog-dependent Specialization of Paf1C subunit, Ctr9, for Sex Chromosome Gene Regulation and Male Germline Differentiation in Drosophila

Abstract

Testis-specific gene regulatory mechanisms govern the differentiation of germ cells into mature sperms. However, the molecular underpinnings are not fully elucidated. Here, we show the subunits forming Polymerase Associated Factor 1 Complex (Paf1C), a transcription regulator conserved across eukaryotes, have their individual paralogs predominantly expressed in Drosophila testes. One of these, namely, Ctr9 paralog enriched in testes (Ctr9t) was found to play a critical and nonredundant role in post-meiotic spermatid differentiation and male fertility in D. melanogaster. A proximity proteome analysis provides evidence that Ctr9t forms complexes with other paralog members. We show endogenous Ctr9t is expressed in germ cells at spermatocyte stages, accumulating in a distinct compartment within the nucleolus. There, Ctr9t co-localizes with Spermatocyte arrest (Sa), a testis-specific paralog of TATA-binding protein (TBP)-associated factor 8 (TAF8). We further demonstrate that ctr9t function is crucial for maintaining Sa in the nucleolus, but not vice versa. Transcriptome profiling reveals that Ctr9t acts as an activator for the set of male fertility genes on Y chromosome, but it also acts as a global repressor of X chromosome genes. Collectively, our results shed light on the nucleolus-associated, paralog-dependent regulation of gene expression from sex chromosomes, which ensures the terminal differentiation of male germ cells. Author summaryA wide repertoire of genes takes a coordinated action during sperm formation. However, gene expression during spermatogenesis is controlled by heretofore unelucidated mechanisms. In this study, we show Drosophila expresses testis-specific paralogs of the individual subunits forming a conserved transcription regulator called Paf1C. We generated a loss of function allele of one of the paralog members which we named Ctr9t, and demonstrate that Ctr9t function is crucial for post-meiotic spermatid differentiation and male fertility. Therefore, the paralog has established a nonredundant role for spermatogenesis. Using newly generated antibodies, we show Ctr9t protein is expressed in germ cells at spermatocyte stages and, intriguingly, enriched in the nucleolus. There, Ctr9t interacts with Spermatocyte arrest (Sa), a key transcription factor essential for meiotic and post-meiotic differentiation processes. By further performing whole testis transcriptome analyses, we reveal that Ctr9t acts as a regulator of gene expression from sex chromosomes. Importantly, the activation of male fertility genes located on Y chromosome relies on Ctr9t function, which can explain the defects observed in ctr9t mutant germ cells. Altogether, we propose that Ctr9t acts as a key player in the spermatocyte nucleolus where it controls the expression of sex chromosome genes and ensures functional sperm formation.