Elucidation of Initiation and Maintenance Mechanisms of X Chromosome Inactivation
X chromosome inactivation is a program of gene silencing on one of two female mammalian X chromosomes to equalize X-linked gene expression to XY male counterparts. This developmentally-regulated chromatin change is initiated on either the maternal or paternal X chromosome early in embryonic development and, once established, is maintained on the chosen chromosome for the lifetime of the female. The onset of X chromosome inactivation is regulated by the long noncoding transcript Xist and an open question is the field is how embryonic developmental cues trigger expression of Xist and onset of X chromosome inactivation. The correlation of pluripotency with repression of Xist in the mouse system has led to a model where pluripotency transcription factors repress X chromosome inactivation by binding to a region within the first intron of Xist gene. Thus differentiation would release the repression of Xist. We rigorously tested this intron1 hypothesis in a transgenic mouse model and refute that intron1 binding is responsible for the developmental regulation of X chromosome inactivation.
A second set of studies focused on the maintenance phase of X chromosome inactivation with the goal of discovering novel chromatin factors that contribute to the remarkable stability of gene silencing on the entire X chromosome. We took an unbiased screening approach, designing a high throughput assay with primary mouse cells bearing reporters on the inactive X, and screened genome-wide siRNA and chemical libraries. We report that knockdown of chromatin-associated protein Atf7ip or its previously characterized interactors reactivates silenced genes of the inactive X chromosome. From chemical screening, we found that the compound Resveratrol can lead to reactivation of silenced genes as part of a novel drug combination. We show evidence that Resveratrol inhibits the dNTP biosynthetic enzyme ribonucleotide reductase in this context. This finding has spurred a collaboration investigating Resveratrol as part of a rationale drug combination for cancer therapy. In summary, these studies demonstrate that X chromosome inactivation is powerful and flexible model for the interrogation of mammalian chromatin regulation mechanisms with relevance for disease therapy.