Executive Summary : | In therian mammals, to compensate sex-chromosome dosage between the sexes, one of the X-chromosomes in female become transcriptionally inactive. In mouse, there are two forms of X-inactivation- Imprinted and Random. At the beginning of early mouse development, paternal X is inactivated (imprinted) and subsequently, it switches to random X-inactivation, i.e., either paternal or maternal-X gets inactivated. X-inactivation is orchestrated by Xist, which exclusively expresses and coats the entire inactive-X. Xist recruits different repressive factors and modulate the 3-dimensional architecture of the X to improvise the heterochromatinization. One long-standing question is how Xist is exclusively transcribe from the inactive-X and how it is maintained through the cell divisions. It is believed that many regulatory elements, specially a series of lncRNAs (Tsix, Jpx, Ftx, Xert) regulate the Xist expression in such a way that it can only express from the inactive-X. However, genetic studies have often disproven these models. Therefore, a detailed mechanistic understanding of Xist regulation is crucial. Recently, we found a novel enhancer element (UPXist-enhancer) at the upstream region of Xist in mouse extraembryonic endoderm stem cells (XEN), which undergo imprinted X-inactivation. Moreover, we found that this enhancer actively transcribes and produces an eRNA (UPXist-eRNA). Emerging studies indicate that eRNAs play important roles in spatio-temporal gene regulation. Interestingly, a genomic deletion encompassing the locus harbouring UPXist-enhancer leads to a significant reduction in Xist expression. Based on these, we have hypothesized that UPXist-enhancer/eRNA plays an important role in the regulation of Xist and X-chromosome inactivation and could be one of the candidates in orchestrating inactive-X specific expression of Xist. Therefore, we want to characterize the role of the UPXist-enhancer/eRNA in Xist regulation and X-inactivation through the following objectives- (1) Molecular annotation of the UPXist-enhancer/eRNA- We will perform luciferase reporter assay, 5 and 3 RACE, RT-PCR and RNA-FISH to characterize UPXist-enhancer/eRNA. (2) Functional characterization of the UPXist-enhancer/eRNA- We will specifically delete the enhancer locus using CRISPR tool and assay the effect on Xist expression, X-inactivation. Additionally, to dissect the importance of UPXist-eRNA, we will perturb the transcription of UPXist eRNA through CRISPR-dCas9-KRAB interference approach without deleting the DNA locus. (3) To test if UPXist-enhancer/eRNA function is conserved in random X-inactivation in EpiSC- Our preliminary data show that UPXist-eRNA is also transcribed in epiblast stem cells (EpiSC), which undergo random X-inactivation. Therefore, we want to characterize the UPXist-enhancer/eRNA at molecular and functional level in EpiSC. Altogether, this study will provide significant insight into Xist regulation, evolution of X-inactivation and eRNA biology. |