RNA-induced transcriptional gene silencing in Friedreich ataxia

Friedreich 共济失调中 RNA 诱导的转录基因沉默

基本信息

批准号：
8153888
负责人：
SANJAY I BIDICHANDANI
金额：
$ 23.2万
依托单位：
UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-30 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8153888
关键词：
5&apos Untranslated Regions Adopted Alleles Antisense Oligonucleotides Antisense RNA Ataxia Biological Assay Cell Line Cells Chromatin D Cells DNA Sequence Data Defect Development Elements Epigenetic Process Fibroblasts Fluorescein Friedreich Ataxia Gene Expression Gene Silencing Genes HDAC3 gene Heterochromatin Histone Deacetylase Inhibitor Human Inherited Introns Length Mammalian Cell Mediating Mediator of activation protein Methods MicroRNAs Neurons Nuclear Nucleosomes Pathogenesis Pathway interactions Patients Process Proteins RNA Recruitment Activity Repression Reverse Transcriptase Polymerase Chain Reaction Ribonuclease H Role Run-On Assays Small Interfering RNA Sorting - Cell Movement Structure Testing Transcript Transcription Initiation Site Transfection Trinucleotide Repeats Triplet Multiple Birth antigene chromatin immunoprecipitation design frataxin gene function human DICER1 protein induced pluripotent stem cell loss of function mutant novel overexpression promoter public health relevance research study stable cell line therapeutic target

项目摘要

DESCRIPTION (provided by applicant): Friedreich ataxia patients are homozygous for abnormally expanded GAA triplet-repeats in the first intron of the FXN gene. The expanded GAA triplet-repeat sequence results in a deficiency of FXN transcript which is reversed via administration of histone deacetylase inhibitors, indicating that transcriptional silencing is at least partly due to an epigenetic abnormality. Our preliminary data show that Friedreich ataxia patients have heterochromatin formation involving the critical +1 nucleosome of the FXN gene, thus offering a potential mechanism for the transcriptional silencing. We found that heterochromatin formation at the +1 nucleosome and the ensuing deficiency of FXN transcript are associated with overexpression of a novel antisense transcript, FAST1 (FXN Antisense Transcript 1), that overlaps with the +1 nucleosome, and higher levels of a novel, promoter-associated microRNA, miR-FXN1. Given that both antisense RNAs and microRNAs are able to induce heterochromatin formation, leading to transcriptional silencing in mammalian cells, we hypothesize that loss-of-function of the FXN gene in FRDA is caused by RNA-induced heterochromatin formation and transcriptional gene silencing. We will test this hypothesis by investigating the potential roles of FAST1 and miR-FXN1 in inducing heterochromatin formation and transcriptional gene silencing of the FXN gene in Friedreich ataxia. Furthermore, we will test if the epigenetic defect seen in fibroblast cell lines from patients is also detected in Friedreich ataxia neurons derived from differentiation of induced pluripotent stem cells. Implication of FAST1 and / or miR-FXN1 in the pathogenesis of Friedreich ataxia would make them rational therapeutic targets to permit specific reactivation of the FXN gene. Indeed, we will also test the feasibility of reactivation of the FXN gene in Friedreich ataxia via targeted repression of FAST1 and miR-FXN1. This proposal will enhance our understanding of the pathogenesis of Friedreich ataxia, the most common inherited ataxia, and will potentially have important implications for the development of a specific therapy. PUBLIC HEALTH RELEVANCE: Patients with Friedreich ataxia, the most common inherited ataxia, have expanded GAA repeat sequences in their FXN genes. Preliminary results indicate that this leads to altered packaging of the FXN genes, turning off gene expression, and causing a deficiency of the essential protein frataxin. Our experiments are designed to determine the precise mechanism of this altered packaging in Friedreich ataxia, and to decipher methods to reverse it and restore normal gene function.

描述（由申请人提供）：Friedreich 共济失调患者在 FXN 基因的第一个内含子中存在异常扩增的 GAA 三联体重复纯合子。扩展的 GAA 三联体重复序列导致 FXN 转录物的缺陷，通过施用组蛋白脱乙酰酶抑制剂可以逆转这种缺陷，表明转录沉默至少部分是由于表观遗传异常所致。我们的初步数据表明，Friedreich 共济失调患者具有涉及 FXN 基因关键 +1 核小体的异染色质形成，从而为转录沉默提供了潜在机制。我们发现，+1 核小体处异染色质的形成以及随之而来的 FXN 转录本的缺陷与一种新型反义转录本 FAST1（FXN 反义转录本 1）的过度表达有关，该转录本与 +1 核小体重叠，并且较高水平的新型反义转录本 FAST1（FXN 反义转录本 1）与 +1 核小体重叠。启动子相关的 microRNA，miR-FXN1。鉴于反义 RNA 和 microRNA 都能够诱导异染色质形成，从而导致哺乳动物细胞中的转录沉默，我们假设 FRDA 中 FXN 基因功能丧失是由 RNA 诱导的异染色质形成和转录基因沉默引起的。我们将通过研究 FAST1 和 miR-FXN1 在 Friedreich 共济失调中诱导异染色质形成和 FXN 基因转录基因沉默中的潜在作用来检验这一假设。此外，我们将测试患者成纤维细胞系中观察到的表观遗传缺陷是否也在诱导多能干细胞分化产生的弗里德赖希共济失调神经元中检测到。 FAST1 和/或 miR-FXN1 在 Friedreich 共济失调发病机制中的意义将使它们成为合理的治疗靶点，以允许 FXN 基因的特异性重新激活。事实上，我们还将测试通过靶向抑制 FAST1 和 miR-FXN1 在 Friedreich 共济失调中重新激活 FXN 基因的可行性。该提议将增强我们对弗里德赖希共济失调（最常见的遗传性共济失调）发病机制的理解，并可能对特定疗法的开发产生重要影响。公共健康相关性：弗里德赖希共济失调（最常见的遗传性共济失调）患者的 FXN 基因中的 GAA 重复序列有所扩展。初步结果表明，这会导致 FXN 基因包装发生改变，关闭基因表达，并导致必需蛋白 frataxin 的缺乏。我们的实验旨在确定弗里德赖希共济失调中这种包装改变的精确机制，并破译逆转它并恢复正常基因功能的方法。