X-linked microRNA genes escape Meiotic Sex Chromosome Inactivation

Date

2014

Authors

Sosa, Enrique

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Abstract

MicroRNAs (miRNAs) are small, non-coding RNAs known to regulate gene expression at the post-transcriptional level, and potentially at the transcriptional level as well. Studies with gene knockouts have demonstrated that miRNAs are critically required for normal spermatogenesis and male fertility in mammals. It was previously shown that ~20% of testicular miRNAs map to the X-chromosome and that ~40% of these display testis-specific or testis-preferential expression. Surprisingly, real-time qPCR further revealed an increase in the expression of a majority of the X-linked miRNA transcripts in spermatocytes at a time when all 364 X-linked messenger RNA-encoding genes studied to date decline in expression due to Meiotic Sex Chromosome Inactivation (MSCI). Many of these same X-linked miRNA genes also appear to escape a secondary repressive effect induced by Post Meiotic Sex Chromatin (PMSC) in spermatids. We hypothesized that elevated levels of X-linked miRNA transcripts in primary spermatocytes and round spermatids are due to ongoing, active transcription, indicating transcriptional escape of these genes from the repressive effects of MSCI and/or PMSC, respectively. To test this hypothesis, we used immunofluorescence staining, DNA-fluorescence in situ hybridization (DNA-FISH) and RNA-FISH to examine nascent transcription in spermatogenic cells from testes of adult mice. Our data confirm three patterns of expression of X-linked miRNA genes during spermatogenesis - Type I, suppressed by MSCI and PMSC; Type II, escaping MSCI but not PMSC; and Type III, escaping both MSCI and PMSC, respectively, and that escape of these genes from MSCI or PMSC occurs by ongoing de novo transcription of these genes in primary spermatocytes and/or round spermatids, respectively. Further, three-dimensional analyses obtained by reconstructing confocal images of spermatocyte nuclei indicate Type II and III X-linked miRNA genes that escape MSCI undergo de novo transcription at the periphery or completely outside of the heterochromatic XY body, while Type I X-linked miRNA genes and all X-linked mRNA genes are silenced by virtue of their location within the boundaries of the XY body. Analysis of available genome-wide ChIP-Seq data for specific histone modifications found associated with X-linked miRNA genes in spermatocytes and spermatids suggest that Type III X-linked miRNA genes retain chromatin modifications characteristic of active genes in spermatocytes, and analysis of the location of miRNA genes on the mouse X chromosome suggest that most X-linked miRNA genes evolved more recently than most X-linked mRNA genes. Taken together, these results support the notion that many X-linked miRNAs participate in functions that are critically required for spermatogenesis and that they have therefore evolved the capability to escape MSCI and/or PMSC to facilitate their ongoing expression in these cells.

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Keywords

Meiotic Sex Chromosome Inactivation, microRNAs, nascent transcription, spermatogenesis

Citation

Department

Integrative Biology