a-non-random-mouse-model-for-pharmacological-reactivation-mecp2-on

A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all ...

University of Virginia School of Medicine

Rett syndrome (RTT) is a genetic disorder resulting from a loss-of-function mutation in one copy of the X-linked gene methyl-CpG-binding protein 2 (). Typical RTT patients are females and, due to random X chromosome inactivation (XCI), ∼50% of cells express mutant MECP2 and the other ∼50% express wild-type MECP2. Cells expressing mutant MECP2 retain a wild-type copy of MECP2 on the inactive X chromosome (Xi), the reactivation of which represents a potential therapeutic approach for RTT. Previous studies have demonstrated reactivation of Xi-linked  in cultured cells by biological or pharmacological inhibition of factors that promote XCI (called "XCI factors" or "XCIFs"). Whether XCIF inhibitors in living animals can reactivate Xi-linked  in cerebral cortical neurons, the cell type most therapeutically relevant to RTT, remains to be determined. Here, we show that pharmacological inhibitors targeting XCIFs in the PI3K/AKT and bone morphogenetic protein signaling pathways reactivate Xi-linked  in cultured mouse fibroblasts and human induced pluripotent stem cell-derived postmitotic RTT neurons. Notably, reactivation of Xi-linked  corrects characteristic defects of human RTT neurons including reduced soma size and branch points. Most importantly, we show that intracerebroventricular injection of the XCIF inhibitors reactivates Xi-linked  in cerebral cortical neurons of adult living mice. In support of these pharmacological results, we also demonstrate genetic reactivation of Xi-linked  in cerebral cortical neurons of living mice bearing a homozygous XCIF deletion. Collectively, our results further establish the feasibility of pharmacological reactivation of Xi-linked  as a therapeutic approach for RTT.

Pharmacological reactivation of inactive X-linked  in cerebral cortical neurons of living mice.

X-inactive specific transcript (Xist) is a long noncoding RNA that is essential for initiating and maintaining epigenetic silencing of one copy of the X chromosome in mammalian females. But the mechanism by which Xist localizes and spreads on the X chromosome and facilitates transcriptional silencing remains largely unknown. This limited understanding, at least in part, is due to the technical difficulties in the visualization and functional characterization of Xist. Development of a successful method for Xist tracking is a key to better understanding of the X chromosome silencing, as well as to gain insight into the regulatory role of other long noncoding RNAs. Here, we describe an alternative method for visualization of Xist lncRNA in cells using a CRISPR/Cas9-based approach. This strategy is relatively simple approach to track Xist at different stages of cell differentiation, providing mechanistic insights into the initiation, maintenance, and establishment of X inactivation.

Urszula Wasko

Department of Biochemistry and Molecular Genetics

University of Virginia School of Medicine

A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome

Urszula Wasko

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry and Molecular Genetics

University of Virginia School of Medicine

A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome

Department of Biochemistry and Molecular Genetics