Long noncoding RNAs (lncRNAs) are important regulators of various biological processes, including spermatogenesis. Our previous studies have revealed the regulatory loop of mrhl RNA and Wnt signaling, where mrhl RNA negatively regulates Wnt signaling and gets downregulated upon Wnt signaling activation. This downregulation of mrhl RNA is important for the meiotic progression of spermatogonial cells. In our present study, we identified the transcription factor Sox8 as the regulatory link between mrhl RNA expression, Wnt signaling activation, and meiotic progression. In contrast to reports from other groups, we report the expression of Sox8 in germ cells and describe the molecular mechanism of Sox8 regulation by mrhl RNA during differentiation of spermatogonial cells. Binding of mrhl RNA to the Sox8 promoter is accompanied by the assembly of other regulatory factors involving Myc-Max-Mad transcription factors, corepressor Sin3a, and coactivator Pcaf. In the context of Wnt signaling, Sox8 directly regulates the expression of premeiotic and meiotic markers. Prolonged Wnt signaling activation in spermatogonial cells leads to changes in global chromatin architecture and a decrease in levels of stem cell markers.KEYWORDS Myc-Mad-Max, Sin3A, Sox8, Wnt signaling, mrhl RNA T he complexity of the eukaryotic genome has been attributed to its multifaceted regulatory networks. Over recent years, significant advancements in high-throughput analyses of the transcriptome have demonstrated the abundance of transcripts that do not code for proteins (noncoding RNAs [ncRNAs]). These ncRNAs are broadly classified as small and long noncoding RNAs. The small ncRNAs, such as microRNA (miRNA) and small interfering RNA (siRNA), play important roles in transcriptional and posttranscriptional gene regulation, while Piwi-associated RNAs (piRNAs) are involved in transposon regulation (1, 2). Another class is that of the long noncoding RNAs (lncRNAs), which are of various sizes between 200 bp and several kilobases (3). The role of lncRNAs in a plethora of functions, for example, dosage compensation (Xist and roX) (4, 5), genomic imprinting (Air and Kcnq1ot1) (6, 7), pluripotency (Evx1as and Hoxb5/6as) (8), cell differentiation and development (Fendrr, Bvht, Miat, Hotair, etc.) (9), nuclear architecture (NEAT1) (10), chromosome segregation (Concr) (11), immune response (lincRNA-EPS, EGOT) (12, 13), etc., have been characterized. lncRNAs bring about such copious functions by employment of diverse mechanisms such as translational inhibition (lincRNA-p21) (14), mRNA degradation (1/2-sbs RNAs) (15), RNA decoys (Gas5) (16)
Sox8 is a developmentally important transcription factor that plays an important role in sex maintenance and fertility of adult mice. In the B-type spermatogonial cells, Sox8 is regulated by the long noncoding RNAs (lncRNA) Mrhl in a p68-dependant manner under the control of the Wnt signaling pathway. The downregulation of Mrhl leads to the meiotic commitment of the spermatogonial cells in a Sox8 -dependant manner.
Sox8 is a developmentally important transcription factor that plays an important role in sex maintenance and fertility of adult mice. In the B-type spermatogonial cells, Sox8 is regulated by the lncRNA Mrhl in a p68-dependant manner under the control of the Wnt signalling pathway. The downregulation of Mrhl leads to the meiotic commitment of the spermatogonial cells in a Sox8-dependant manner. While the molecular players involved in the regulation of transcription at the Sox8 promoter have been worked out, our current study points to the involvement of the architectural proteins, CTCF and cohesin, in mediating a chromatin loop. This loop brings the Sox8 promoter in contact with a silencer element present within the gene body in the presence of lncRNA Mrhl concomitant with transcriptional repression. Further, lncRNA Mrhl interacts with the Sox8 locus through the formation of a DNA:DNA:RNA triplex which is necessary for the recruitment of PRC2 to the locus. The downregulation of lncRNA Mrhl results in the promoter-silencer loop giving way to a promoter-enhancer loop. This active transcription associated chromatin loop is mediated by YY1 and brings the promoter in contact with the enhancer present downstream of the gene.
Proliferation and meiotic division of spermatogonial stem cells are highly regulated biological processes that occur during spermatogenesis. In addition to protein coding genes, mammalian genome encodes thousands of lncRNAs which are spatio-temporally expressed and play key role(s) in cellular differentiation and development. Mrhl lncRNA is one such mono-exonic polyadenylated non-coding RNA encoded within the 15th intron of the mouse Phkb gene. Mrhl lncRNA is expressed in mouse testis amongst other tissues. The RNA is nuclear localized and predominantly bound to chromatin in GC-1 spg cells (derived from B type spermatogonia). It regulates multiple genes belonging to different biological processes including Wnt signaling. Wnt activation of GC-1 spg cells downregulates Mrhl lncRNA expression, in turn activating the expression of meiotic marker genes and downregulating stem cells markers. We have mapped the genomic loci bound by Mrhl lncRNA and identified 37 genes to be regulated by its physical association. Sox8 gene, one among these, is regulated through its interaction at its promoter through RNA: DNA: DNA triplex structure and chromatin looping mediated by the architectural proteins CTCF and YY1. Here we summarize our major findings on this novel lncRNA starting from its discovery to biological function(s) particularly during meiotic commitment/initiation in mouse spermatogenesis.
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