Broad phosphorylation mediated by testis-specific serine/threonine kinases contributes to spermiogenesis and male fertility.

Genetic studies elucidate a link between testis-specific serine/threonine kinases (TSSKs) and male infertility in mammals, but the underlying mechanisms are unclear. Here, we identify a TSSK homolog in Drosophila, CG14305 (termed dTSSK), whose mutation impairs the histone-to-protamine transition during spermiogenesis and causes multiple phenotypic defects in nuclear shaping, DNA condensation, and flagellar organization in spermatids. Genetic analysis demonstrates that kinase catalytic activity of dTSSK, which is functionally conserved with human TSSKs, is essential for male fertility. Phosphoproteomics identify 828 phosphopeptides/449 proteins as potential substrates of dTSSK enriched primarily in microtubule-based processes, flagellar organization and mobility, and spermatid differentiation and development, suggesting that dTSSK phosphorylates various proteins to orchestrate postmeiotic spermiogenesis. Among them, the two substrates, protamine-like protein Mst77F/Ser9 and transition protein Mst33A/Ser237, are biochemically validated to be phosphorylated by dTSSK in vitro, and are genetically demonstrated to be involved in spermiogenesis in vivo. Collectively, our findings demonstrate that broad phosphorylation mediated by TSSKs plays an indispensable role in spermiogenesis.

Cis- and trans-regulation by histone H4 basic patch R17/R19 in metazoan development.

The histone H4 basic patch is critical for chromatin structure and regulation of the chromatin machinery. However, the biological roles of these positively charged residues and the mechanisms by which they regulate gene expression remain unclear. In this study, we used histone mutagenesis to investigate the physiological function and downstream regulatory genes of H4 residues R17 and R19 in Drosophila. We found all histone mutations including R17A/E/H and R19A/E/H (R17 and R19 of H4 are substituted by A, E and H respectively) result in a range of growth defects and abnormalities in chromosomal high-order structures, whereas R17E mutation is embryonic lethal. RNA-seq demonstrates that downregulated genes in both R17A and R19A show significant overlap and are enriched in development-related pathways. In addition, Western and cytological analyses showed that the R17A mutation resulted in a significant reduction in H4K16 acetylation and male offspring, implying that the R17 may be involved in male dosage compensation mechanisms. R19 mutation on the other hand strongly affect Gpp (Dot1 homologue in flies)-mediated H3K79 methylation, possibly through histone crosstalk. Together these results provide insights into the differential impacts of positive charges of H4 basic patch R17/R19 on regulation of gene transcription during developmental processes.

Transcription-coupled donor DNA expression increases homologous recombination for efficient genome editing.

Genomes can be edited by homologous recombination stimulated by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated peptide 9]-induced DNA double-strand breaks. However, this approach is inefficient for inserting or deleting long fragments in mammalian cells. Here, we describe a simple genome-editing method, termed transcription-coupled Cas9-mediated editing (TEd), that can achieve higher efficiencies than canonical Cas9-mediated editing (CEd) in deleting genomic fragments, inserting/replacing large DNA fragments and introducing point mutations into mammalian cell lines. We also found that the transcription on DNA templates is crucial for the promotion of homology-directed repair, and that tethering transcripts from TEd donors to targeted sites further improves editing efficiency. The superior efficiency of TEd for the insertion and deletion of long DNA fragments expands the applications of CRISPR for editing mammalian genomes.