Regulation of X-chromosome inactivation and imprinting by non-coding elements

PROJECT SUMMARY Interactions between long noncoding RNAs and epigenetic complexes are now increasingly recognized as important regulators of developmental decisions. A classic example is X-chromosome inactivation (XCI), an epigenetic process by which one X-chromosome becomes inactivated in the early embryo. XCI can take place randomly or in an imprinted manner on the paternal X. In the mouse, random XCI is observed in the embryo proper (soma), and imprinted XCI in the placenta. Both require the ‘X-inactivation center’ (Xic), an X-linked region that harbors several essential noncoding genes. A key gene is Xist, which produces a noncoding RNA that ‘paints’ the X-chromosome and recruits silencing factors in cis. Our work has shown that, during random XCI, Xist is regulated both positively and negatively by Xic factors, with Jpx in the positive pathway and Tsix/Xite in the negative pathway. We have also shown that homologous X- chromosome pairing coordinates which X chromosome will be inactivated. In this funding cycle, we have further advanced understanding of the Xic’s role in X-chromosome counting, allelic choice, and imprinting. For the counting mechanism, Jpx RNA — an Xist activator — serves as an X-linked “numerator” by titrating the autosomal “denominator”, CTCF. This titration accounts for the X-to-autosome (X:A) ratio and creates a permissive state for Xist induction. We also learned that X-X pairing requires a new long noncoding RNA called “PAR-TERRA” produced from the X-linked pseudoautosomal region (PAR). Interestingly, all pairing interactions take place within a “tetrad” of two Xic’s and two PAR’s. X-X pairing is then resolved by mRNA decapping enzyme, DCP1A. For imprinted XCI, we have generated an ex vivo cellular model to study XP imprinting. We have demonstrated that a 200-kb Xic region is sufficient to direct Xist imprinting and that Xist imprinting requires an unpaired state in the male germ line that is akin to meiotic sex chromosome inactivation (MSCI). Our research has generated new opportunities and many additional mechanistic questions. In the next funding cycle, we aim to (i) investigate mechanisms underlying X-X pairing and breaking of X-chromosome symmetry, (ii) determine how Jpx-CTCF titration regulates the initiation of XCI, and (iii) elucidate the mechanisms of imprinted XCI. The proposed work will require five years to complete and is expected to train three postdoctoral fellows for academic careers in the fields of epigenetics and RNA biology.