X chromosome inactivation (XCI) is the most important silencing event undergone by female mammalian cells. Virtually all known epigenetic silencing mechanisms come into play during this process, which makes it a great model system to study them. Among the actors involved, some are trans-acting, such as the polycomb repressive complexes (PRC) 1 and 2, and some are cis-acting like the long non-coding RNA Xist that coats the inactive X chromosome. To find new trans epigenetic modifiers, we perform high-throughput screens in female Xmas (X-linked marker active silent) embryonic stem cells undergoing X inactivation. These cells possess a different reporter gene (GFP or mCherry) in the Hprt allele of the X chromosome that becomes silenced with the rest of the chromosome, thus allowing the monitoring of XCI by FACS. Target genes are then knocked-down by shRNAs to see if this interferes with X gene silencing.
On the other hand, cis-acting effectors include structural elements like the Dxz4 macrosatellite repeat, which marks the boundary between the two inactive X’s mega domains. Other repetitive elements such as tandem or interspersed repeats also seem to have roles in the three-dimensional conformation of the inactive X. One of such is the locus of the repetitive long non-coding RNA Firre. Although the transcript RNA has been reported to have different roles, the genomic locus has a seems to be involved in setting the inactive X’s characteristic conformation. This locus has been showed to co-localize with Dxz4 only in the inactive X and the nuclear periphery.
During my PhD, I plan on using screening techniques to discover new trans-acting epigenetic modifiers, and to study cis-acting repetitive elements by using an array of different genomic techniques.