ABSTRACT: Repro9 in an allele of Mybl1 (A-Myb) transcription factor obtained in ENU screen to identify alleles causing mouse infertility. Repro9/repro9 mutant males are infertile due to meiotic arrest at pachytene stage. Mutants show wide range of abnormalities including inefficient chromosome synapsis, sex body formation and progression through meiotic cycle. Females are unaffected. To determine genes transcriptionally regulated by MYBL1 we analyzed gene expression profiles of wild type and repro9/repro9 mutant testis at 14 and 17 days postpartum. Analysis revealed many misregulated genes, in majority downregulated, at day 14 pp and even more at day 17 pp, probably due to secondary effects of meiotic arrest. Significantly misregulated genes were characterized by Gene Ontology. Comparative gene expression analysis uncovered potential targets of MYBL1 regulation that play roles in regulation of transcription, cell cycle, apoptosis, protein phosphorylation and ubiquitination, chromosome organization and others. Abstract:The transcriptional regulation of mammalian meiosis is poorly characterized, due to few genetic and ex vivo models. From a genetic screen, we identify the transcription factor MYBL1 as a male-specific master regulator of several critical meiotic processes. Spermatocytes bearing a novel separation-of-function allele (Mybl1repro9) had subtle defects in autosome synapsis in pachynema, a high incidence of unsynapsed sex chromosomes, incomplete double strand break (DSB) repair on synapsed pachytene chromosomes, and a lack of crossing-over. MYBL1 protein appears in pachynema, and its mutation caused specific alterations in expression of diverse genes, including some translated postmeiotically. These data, coupled with chromatin immunoprecipitation (ChIP-chip) experiments and bioinformatic analysis of promoters, identified direct targets of MYBL1 regulation. Meiosis in mutant females appears unaffected. These results reveal that MYBL1 is a master regulator of meiotic genes that are involved in multiple processes in spermatocytes, including chromosome synapsis, recombination, and cell cycle progression through pachynema. RNA was extracted from wt and repro9/repro9 mutant testis from 14 and 17 days old mice. This two time points were selected for sample enrichment in early/midpachytene and latepachytene/diplotene spermatocytes, respectively. Total testis RNA from wild-type (≥3) and mutant (≥3) mice was reverse-transcribed into double stranded cDNA, and biotin-labeled cRNA (GeneChip IVT labeling kit, Affymetrix, Santa Clara, CA) was hybridized to Affymetrix mouse genome 430 2.0 GeneChips containing. The raw data was processed using Affymetrix GCOS software. Two-way ANOVA analysis resolved differentially expressed (DE) genes either between genotypes and/or days using MeV(version 4.6.1) on log2-transformed expression values. Results were multiple test corrected with the Benjamini-Hochberg method to control the false discovery rate (FDR) in R. ~800 genes significant at FDR<=0.025 were selected as differentially expressed for downstream analysis.
