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

基本信息

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
项目类别：
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

项目摘要

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染色体失活均衡XX女性和XY雄性哺乳动物之间的X连锁基因表达。 X- 灭活分为两个不同的阶段：一个起始阶段和一个维护阶段。 Kalantry实验室最近发现，X连锁基因KDM5C既需要且足以引起X-的启动以剂量依赖性的方式灭活。 KDM5C是一种去除组蛋白的脱甲基酶与主动转录相关的H3K4ME2/3染色质标记。删除KDM5C的两个副本废除X灭活的启动。删除一份KDM5C的副本导致一部分的沉默不足 X灭活启动时X连锁基因。我项目的具体目标是确定剂量 - KDM5C在维持X灭活中的依赖作用。在该提案的目标1中，我将测试KDM5C的角色维持X灭活。我将生成混合原代皮质细胞，该细胞处于 X灭活，在活性 - 有条件的KDM5C突变的小鼠胚胎中偏置X灭活 X.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保持通过高通量和低通量方法沉默。在该提案的目标2中，我将抑制KDM5C蛋白通过将经过验证的KDM5药理学抑制剂用于培养的杂交原发性皮质神经元。我会通过染色质免疫沉淀（CHIP）测试KDM5C对H3K4ME2/3的抑制测序（SEQ）。我还将在等位基因的KDM5C抑制皮质神经元中测试X连锁基因表达特定的RNA-seq。通过这些实验，我将了解X灭活是如何发生和维持的。这预期的发现还有助于对患有X连锁疾病的女性进行潜在的疗法。