Reactivation of Inactivated X-linked Genes Via Inhibition of Histone Demethylase KDM5C

项目总结/文摘

Project Summary/Abstract X-chromosome inactivation equalizes X-linked gene expression between XX female and XY male mammals. X- inactivation is separated into two distinct phases: an initiation phase and a maintenance phase. The Kalantry lab has recently found that the X-linked gene, Kdm5c, is both necessary and sufficient to cause initiation of X- inactivation in a dose-dependent manner. KDM5C is a demethylase enzyme that removes the histone H3K4me2/3 chromatin marks which are associated with active transcription. Deleting both copies of Kdm5c abrogates the initiation of X-inactivation. Deleting one copy of Kdm5c leads to deficient silencing of a subset of X-linked genes when X-inactivation initiates. The specific goal of my project is to determine the dose- dependent role of KDM5C in maintaining X-inactivation. In Aim 1 of this proposal, I will test a role for KDM5C in maintaining X-inactivation. I will generate hybrid primary cortical cells, which are in the maintenance phase of X-inactivation, with biased X-inactivation from mouse embryos with a conditional Kdm5c mutation on the active- X. By deleting one Kdm5c allele in the cortical neurons, I will test which X-linked genes require KDM5C to remain silenced through both high- and low-throughput approaches. In Aim 2 of this proposal, I will inhibit KDM5C protein by administering a validated KDM5 pharmacological inhibitor to cultured hybrid primary cortical neurons. I will test KDM5C inhibition by chromatin immunoprecipitation (ChIP) for H3K4me2/3 followed by next-generation sequencing (Seq). I will also test X-linked gene expression in the KDM5C-inhibited cortical neurons by allele- specific RNA-Seq. Through these experiments, I will learn how X-inactivation occurs and is maintained. The expected findings also promise a potential therapy for females with X-linked disorders.