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PgmNr 113: Identification of post-translational regulators of MeCP2 levels as potential therapeutic targets for MECP2 duplication syndrome.

M. Zaghlula 1,2; J.-Y. Kim 2,3; L. Nitschke 2,4; H.H. Jeong 2,3; C.E. Alcott 2,5,6; J.-P. Revelli 2; Z. Liu 2,3; S.J. Elledge 7,8; H.Y. Zoghbi 1,2,3,4,5,6,9

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1) Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, TX.; 2) Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, TX.; 3) Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX.; 4) Integrative Program in Molecular and Biomedical Sciences, Baylor College of Medicine, Houston, TX.; 5) Program in Developmental Biology, Baylor College of Medicine, Houston, TX.; 6) Medical Scientist Training Program, Baylor College of Medicine, Houston, TX.; 7) Department of Genetics, Harvard Medical School, Boston, MA.; 8) Howard Hughes Medical Institute, Harvard Medical School, Boston, MA.; 9) Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX.

Advances in clinical sequencing continue to highlight the involvement of dosage-sensitive genes in the pathogenesis of neurological disorders. Maintenance of the levels of these proteins within a narrow range is crucial for proper brain function. Methyl-CpG-binding protein 2, MECP2, is one such gene: loss-of-function mutations in MECP2 cause Rett syndrome (RTT) while duplications spanning the MECP2 locus cause MECP2 Duplication syndrome (MDS). Both disorders are severe and progressive; current treatments are restricted to symptom management. Importantly, normalization of MeCP2 abundance has been shown to rescue disease phenotypes in mouse models of both disorders. As specific post-translational modifications of proteins can be determinants of protein stability, we set out to identify post-translational regulators of MeCP2 that can be targeted therapeutically.
To this end, we performed cell-based siRNA and CRISPR screens in a reporter system that allows us to monitor changes in MeCP2 levels. From these screens we selected two promising candidates, the atypical kinase RIOK1 and the ubiquitin protease USP1, to perform mechanistic and genetic interaction studies.
First, we confirmed that shRNA-mediated knockdown of RIOK1 decreases endogenous levels of MeCP2 in HEK293T cells and show nuclear interaction of these proteins in cells. Next, we developed a loss-of-function mouse model of Riok1 and found that MeCP2 levels are reduced in heterozygous knockout mice by 15%. As this effect is relatively modest, we hypothesized that one of its orthologs, RIOK2, might play a compensatory role. As we found that knockdown of RIOK2 reduces MeCP2 levels in cells, we are now following up with in vivo to test further hypotheses about the genetic relationship between the RIO Kinases and MeCP2.
Our studies with the deubiquitinating enzyme USP1 revealed that genetic as well as pharmacological targeting of this screen candidate are able to reduce MeCP2 stability robustly in cells. We also found that knockdown of Usp1 in the mouse brain lowers MeCP2 levels by 30%. Ongoing studies aim to elucidate whether USP1 directly targets MeCP2 for ubiquitin proteolysis and whether the strong reduction in MeCP2 protein stability in vivo is sufficient to ameliorate disease phenotypes in a mouse model of MECP2 Duplication Syndrome.
Overall, this screening approach is a powerful and translationally invaluable path to identify drug targets in the treatment of many dosage-sensitive disorders.