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

通过抑制组蛋白脱甲基酶 KDM5C 重新激活失活的 X 连锁基因

• 批准号: 10471168
• 负责人: Megan Brianne Trotter
• 金额: $ 3.54万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10471168
• 关键词: Alleles; Animal Model; Bioinformatics; Biological Models; Cells; Chromatin; Computer software; Data; Data Set; Dependence; Disease; Doctor of Philosophy; Dose; Embryo; Enzymes; Epigenetic Process; Female; Fluorescent in Situ Hybridization; Gene Expression; Gene Mutation; Gene Silencing; Genes; Genetic Diseases; Genetic Transcription; Goals; Growth; Histones; Hybrids; Knowledge; Learning; Link; LoxP-flanked allele; Maintenance; Mammals; Memory; Molecular and Cellular Biology; Mouse Strains; Mus; Mutate; Mutation; Neurodevelopmental Disorder; Neurons; Parents; Partner in relationship; Pharmacology; Phase; Proteins; RNA; Regulation; Rett Syndrome; Role; Scientist; Sex Differences; Single Nucleotide Polymorphism; Somatic Cell; Tamoxifen; Testing; Therapeutic; Transcriptional Regulation; Transgenes; Work; X Chromosome; X Inactivation; autosome; career; chromatin immunoprecipitation; daughter cell; epigenetic silencing; experimental study; girls; histone demethylase; inhibitor; male; multidisciplinary; next generation sequencing; pyrosequencing; therapy development; transcriptome sequencing

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.

项目摘要/摘要 X染色体失活使X连锁基因在XX雌性和XY雄性哺乳动物中的表达相等。X- 失活分为两个不同的阶段：启动阶段和维护阶段。卡兰特里实验室 最近发现X连锁基因Kdm5c是引起X-DNA合成的必要条件和充分条件。 以剂量依赖的方式灭活。KDM5C是一种去甲基酶，可以去除组蛋白 与激活转录相关的H3K4me2/3染色质标记。删除Kdm5c的两个副本 取消X-失活的启动。删除Kdm5c的一个副本会导致对Kdm5c子集的缺陷沉默 当X失活开始时，X连锁的基因。我的项目的具体目标是确定剂量- KDM5C在维持X-失活中的依赖作用。在本提案的目标1中，我将测试KDM5C的角色 在维持X-失活方面。我将产生混合原代皮质细胞，它们处于维持阶段 X失活，带有偏向的X失活，来自Kdm5c有条件突变的小鼠胚胎。 通过删除皮质神经元中的一个Kdm5c等位基因，我将测试哪些X连锁基因需要KDM5C保持不变 通过高吞吐量和低吞吐量方法都保持沉默。在本提案的目标2中，我将抑制KDM5C蛋白 通过给予培养的混合原代皮质神经元有效的KDM5药理抑制剂。这就做 用染色质免疫沉淀(CHIP)检测KDM5C对H3K4me2/3的抑制作用，然后是下一代 测序(SEQ)。我还将测试KDM5C抑制的皮质神经元中X连锁基因的表达-等位基因- 特异的RNA序列。通过这些实验，我将了解X失活是如何发生和维持的。这个 预期的发现也为患有X连锁障碍的女性提供了一种潜在的治疗方法。