| Summary: | Xist (X-inactive specific transcript), a prototype long non-coding RNA, is essential for establishing X chromosome inactivation (XCI) in early embryos. However, its role in maintaining XCI in soma is unclear. When Xist was conditionally deleted in somatic cells from the inactive X (Xi∆Xist ), only rare and sporadic gene reactivation could be detected. We carried out high-resolution RNA allelotyping by padlock SNP capture on a panel of wild type and mutant mouse cell lines. We confirm that low level (~4%) gene reactivation occurs along Xi∆Xist, but the pattern is not sporadic. A gene with its upstream region enriched for SINEs (a class of retrotransposon transcribed by RNA polymerase III) is prone to be reactivated. Intriguingly, Pol III transcription from non-coding regions is largely independent of Xist-mediated transcriptional silencing. Pol III inhibition alters chromatin looping and impairs XCI suggesting a role of Pol III transcription in regulating chromatin architecture.
In the second project of my graduate research, plasmid fusion constructs of mH2A and miniSOG were established. miniSOG catalyzes polymerization of diaminobenzidine (DAB) which is osmiophilic. Therefore miniSOG can be used to label protein targets in live cells for downstream electron microscope (EM) studies. macroH2A (mH2A) is a histone variant enriched on Xi. One mH2A-miniSOG fusion construct successfully labeled Xi for fluorescent microscopy and photo-oxidation (DAB polymerization). It is ready to be used as the EM tag for future studies on the chromatin structures of the Barr body.
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