The role of the Xist 5’ m6A region and RBM15 in X chromosome inactivation

Background: X chromosome inactivation in mammals is regulated by the non-coding (nc) RNA, Xist, which represses the chromosome from which it is transcribed. High levels of the N6-methyladenosine (m6A) RNA modification occur within Xist exon I, close to the 5’ end of the transcript, and also further 3’, in Xist exon VII. The m6A modification is catalysed by the METTL3/14 complex that is directed to specific targets, including Xist, by the RNA binding protein RBM15/15B. m6A modification of Xist RNA has been reported to be important for Xist–mediated gene silencing. Methods: We use CRISPR/Cas9 mediated mutagenesis to delete sequences around the 5’ m6A region in interspecific XX mouse embryonic stem cells (mESCs). Following induction of Xist RNA expression, we assay chromosome silencing using allelic RNA-seq and Xist m6A distribution using m6A-seq. Additionally, we use Xist RNA FISH to analyse the effect of deleting the 5’ m6A region on the function of the endogenous Xist promoter. We purify epitope tagged RBM15 from mESCs, and then apply MS/MS analysis to define the RBM15 interactome. Results: We show that a deletion encompassing the entire Xist 5’ m6A region results in a modest reduction in Xist-mediated silencing, and that the 5’ m6A region overlaps essential DNA elements required for activation of the endogenous Xist promoter. Deletion of the Xist A-repeat, to which RBM15 binds, entirely abolishes deposition of m6A in the Xist 5’ m6A region without affecting the modification in exon VII. We show that in mESCs, RBM15 interacts with the m6A complex, the SETD1B histone modifying complex, and several proteins linked to RNA metabolism. Conclusions: Our findings support that RBM15 binding to the Xist A-repeat recruits the m6A complex to the 5’ Xist m6A region and that this region plays a role in Xist-mediated chromosome silencing.

XIST-Promoter Demethylation as Tissue Biomarker for Testicular Germ Cell Tumors and Spermatogenesis Quality

Background: The event of X chromosome inactivation induced by XIST, which is physiologically observed in females, is retained in testicular germ cell tumors (TGCTs), as a result of a supernumerary X chromosome constitution. X chromosome inactivation also occurs in male germline, specifically during spermatogenesis. We aimed to analyze the promoter methylation status of XIST in a series of TGCT tissues, representative cell lines, and testicular parenchyma. Methods: Two independent cohorts were included, comprising a total of 413 TGCT samples, four (T)GCT cell lines, and 86 testicular parenchyma samples. The relative amount of methylated and demethylated XIST promoter fragments was assessed by quantitative methylation-specific PCR (qMSP) and more sensitive high-resolution melting (HRM) methylation analyses. Results: Seminomas showed a lower amount of methylated XIST fragments as compared to non-seminomas or normal testis (p < 0.0001), allowing for a good discrimination among these groups (area under the curve 0.83 and 0.81, respectively). Seminomas showed a significantly higher content of demethylated XIST as compared to non-seminomas. The percentage of demethylated XIST fragment in cell lines reflected their chromosomal constitution (number of extra X chromosomes). A novel and strong positive correlation between the Johnsen’s score and XIST demethylation was identified (r = 0.75, p < 0.0001). Conclusions: The X chromosome inactivation event and demethylated XIST promoter are promising biomarkers for TGCTs and for assessing spermatogenesis quality.

The Human Xist Gene Promoter Prevents Silencing of an Integrated Reporter Gene.

Epigenetic silencing and position dependent expression are long-standing problems which continue to limit the development of gene replacement therapy. As a strategy to overcome this problem we have tested the ability of the human XIST (X inactivation-specific transcript) gene promoter to overcome epigenetic silencing. The XIST gene is one of a relatively small number of genes which are expressed from the inactive X chromosome. The product of this gene is an untranslated structural RNA which coats the X chromosome destined for inactivation prior to H3 Lys 9 hypoacetylation, H3 Lys 9 methylation, CpG island methylation and the subsequent silencing of most of the genes on the chromosome. Continued expression of the XIST gene in this highly repressive environment is required to maintain the chromosome in an inactive state. The region of the proximal promoter of the XIST gene on the inactive X chromosome has been shown to retain an active chromatin structure. Based on these findings we hypothesized that the XIST gene promoter would be able to resist the epigenetic changes which lead to transgene silencing. To test this idea, we subcloned a minimal XIST promoter upstream of an enhanced GFP reporter gene in a pUC-based plasmid which also contained a neomycin resistance gene. The same plasmid, with a CMV promoter, served as a control vector. The plasmids were electroporated into mouse erythroleukemia (MEL) cells and then grown in media containing G418. The MEL cell line was chosen because genes transferred into these cells are frequently silenced and because it is often used as a first screen for vectors with potential for use in therapeutic gene transfer to erythroid cells. Individual colonies were selected and G418 removed. After expansion of the clones, flow cytometry was used to determine the percentage of cells in each clonal population which were expressing GFP as determined by comparison to the untransduced MEL cell line. Silencing typically involves a gradual decrease in the proportion of cells expressing the integrated transgene. Statistical analyses of results were performed using the t-test. 16 XIST and 13 CMV clones were available for analysis at the start of the experiment (time 0). 11/13 CMV clones and 12/16 XIST clones initially expressed GFP. Of the clones which were expressing GFP, the average percentages of positive cells were higher for those with the XIST promoter (63% vs. 41%, p= 0.015). Expression was reanalyzed after 6 weeks of culture in the absence of G418 selection. At this time point, the average percentage of GFP expressing cells was much higher for the XIST clones (57% vs. 19%, p=0.00008) and when analyzed for silencing, XIST clones were expressing at an average of 90% of their time 0 levels vs. 46% for the CMV clones (p=.0008). These results indicate that the XIST promoter is resistant to silencing in our model system and is a candidate for further development and mechanistic studies.