Genome wide chromatin occupancy of <i>mrhl</i> RNA and its role in gene regulation in mouse spermatogonial cells

<i>Mrhl</i> RNA is a nuclear lncRNA encoded in the mouse genome and negatively regulates Wnt signaling in spermatogonial cells through p68/Ddx5 RNA helicase. <i>Mrhl</i> RNA is present in the chromatin fraction of mouse spermatogonial Gc1-Spg cells and genome wide chromatin occupancy of <i>mrhl</i> RNA by ChOP (Chromatin oligo affinity precipitation) technique identified 1370 statistically significant genomic loci. Among these, genes at 37 genomic loci also showed altered expression pattern upon <i>mrhl</i> RNA down regulation which are referred to as GRPAM (Genes Regulated by Physical Association of <i>Mrhl</i> RNA). p68 interacted with <i>mrhl</i> RNA in chromatin at these GRPAM loci. p68 silencing drastically reduced <i>mrhl</i> RNA occupancy at 27 GRPAM loci and also perturbed the expression of GRPAM suggesting a role for p68 mediated <i>mrhl</i> RNA occupancy in regulating GRPAM expression. Wnt3a ligand treatment of Gc1-Spg cells down regulated <i>mrhl</i> RNA expression and also perturbed expression of these 27 GRPAM genes that included genes regulating Wnt signaling pathway and spermatogenesis, one of them being Sox8, a developmentally important transcription factor. We also identified interacting proteins of <i>mrhl</i> RNA associated chromatin fraction which included Pc4, a chromatin organizer protein and hnRNP A/B and hnRNP A2/B1 which have been shown to be associated with lincRNA-Cox2 function in gene regulation. Our findings in the Gc1-Spg cell line also correlate with the results from analysis of mouse testicular tissue which further highlights the <i>in vivo</i> physiological significance of <i>mrhl</i> RNA in the context of gene regulation during mammalian spermatogenesis.