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

项目总结/摘要 X染色体失活使XX雌性和XY雄性哺乳动物之间的X连锁基因表达相等。X-的 灭活分为两个不同的阶段：起始阶段和维持阶段。Kalantry实验室 最近发现，X连锁基因Kdm 5c是引起X染色体启动所必需的，也是足够的。 以剂量依赖性方式灭活。KDM 5C是一种去甲基化酶， H3 K4 me 2/3染色质标记，其与活跃转录相关。删除Kdm 5c的两个副本 消除X-失活的起始。缺失Kdm 5c的一个拷贝导致Kdm 5c的一个子集的缺陷沉默。 当X染色体失活启动时，X染色体连锁基因。我的项目的具体目标是确定剂量- KDM 5C在维持X-失活中的依赖性作用。在本提案的目标1中，我将测试KDM 5C的角色 维持X失活。我将产生混合的初级皮层细胞，它们处于维持阶段， X-失活，来自小鼠胚胎的偏向X-失活，在活性- X.通过删除皮层神经元中的一个KDM 5c等位基因，我将测试哪些X连锁基因需要保留KDM 5c 通过高通量和低通量方法沉默。在本提案的目标2中，我将抑制KDM 5C蛋白 通过向培养的杂交原代皮层神经元施用经验证的KDM 5药理学抑制剂。我会 通过染色质免疫沉淀（ChIP）检测H3 K4 me 2/3对KDM 5C的抑制作用，然后进行下一代 测序（Seq）。我还将通过等位基因测试KDM 5C抑制的皮质神经元中的X连锁基因表达- 特异性RNA测序通过这些实验，我将了解X-失活是如何发生和维持的。的 预期的发现也预示着X连锁疾病女性的潜在治疗。

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.