Impact of a Uty gene trap on the regulation of genes and the abundance of corresponding transcripts in adult mouse somatic (non-reproductive) cells (RNA-Seq)

In addition to sperm-related genes, the male-specific chromosome Y (chrY) contains a class of ubiquitously expressed and evolutionary conserved dosage-sensitive regulator genes, which include the neighboring Uty, Ddx3y and (in mice) Eif2s3y genes. However, the impact of targeted mutations of these genes on the functions of adult non-reproductive somatic cells is still unknown due to lack of experimental evidence. We thus compared adult male mice carrying a gene trap within their Uty gene (UtyGT) to wild-type (WT) isogenic controls, and performed deep sequencing of RNA and genome-wide profiling of chromatin in extracts from either cardiac tissue, cardiomyocyte-specific nuclei and purified cardiomyocytes. The impact of UtyGT on gene transcription was mostly limited to its effects on genes surrounding the locus of insertion, i.e. Uty, Ddx3y, long non-coding RNAs (lncRNAs) contained within their introns, Eif2s3y, as well as on the autosomal Malat1 lncRNA. Notwithstanding, UtyGT also caused coordinate changes in the abundance of hundreds of mRNA transcripts related to particular cell functions, including RNA processing and translation. The results altogether indicated that tightly co-regulated chrY genes had nonetheless more widespread effects on the autosomal transcriptome in adult somatic cells, most likely due to mechanisms other than just transcriptional regulation of protein-coding genes. Adult cardiomyocytes were obtained from ventricles from either UtyGt(XS0378)Wtsi (which carries a gene trap within the 4th intron of the Uty gene) or their wild-type counterparts from the isogenic strain C57BL/6.Y<129P2> adult mice. The hearts were digested by retrograde perfusion of a solution of collagenase type 2 (Worthington, Lakewood, NJ) in calcium-free solutions, followed by gravity sedimentation within 5% bovine serum albumin (BSA) solutions supplemented in a step-wise fashion with increasing CaCl2 concentrations. After isolation, the cells were plated on laminin-coated dishes, and used 4 hours after plating for RNA isolation. For each strain, the procedure was repeated three times to obtain triplicate samples for the RNA-Seq analysis.