부산대학교 도서관

The lncRNA Xist is the master regulator of X chromosome inactivation in female mammals. Its mono-allelic expression triggers a cascade of well characterised events leading to the stable silencing of the X chromosome. However, how Xist coordinates these events is still unclear. We developed a system to dissect Xist function by making a library of deletions of Xist cDNA. The cDNAs were then stably transfected into a male ES cell line under an inducible promoter. The parent cell line was derived from an interspecific cross between different mouse strains, allowing subsequent allele-specific analysis based on a previously described set of strain-specific SNPs. This approach allowed full and partial loss of silencing activity to be discriminated by isolating nascent transcripts upon prolonged Xist expression, and using them to make libraries for high-throughput sequencing. It was therefore possible to map regions of Xist required for specific functions, notably gene silencing and recruitment of Polycomb repressor proteins. Using this approach, the B-repeats were identified as the segment of the RNA crucial for de novo recruitment of both PRC1 and PRC2 Polycomb complexes, whereas the A-repeats were confirmed to be essential for Xist-mediated early silencing of genes. To complement these analyses, an in vitro assembly system was set-up to study the direct interaction between key regions of Xist RNA and defined nuclear proteins at the biochemical/proteomic level, obtaining a comprehensive list of functional candidates. Interestingly, the nuclear matrix protein hnRNP-K was found among the factors significantly enriched over the segment of Xist previously identified as essential for Polycomb recruitment. We thus investigated the functional role of hnRNP-K in mediating the interaction between Xist and Polycomb with a number of in vivo and in vitro assays, and found that hnRNP-K is required to mediate contact between PRC1 and Xist through its KI domain.