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：
8325145
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/8325145
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共济失调患者对于FXN基因的第一个内含子中异常扩展的GAA三胞胎重复均具有纯合。扩展的GAA三重态重复序列导致FXN转录本的缺乏，该转录物通过组蛋白脱乙酰基酶抑制剂而反转，表明转录沉默至少部分是由于表观遗传异常引起的。我们的初步数据表明，Friedreich共济失调患者的形成异染色质，涉及FXN基因的临界+1核小体，因此为转录沉默提供了潜在的机制。我们发现，在+1核小体处的异染色质形成，随之而来的FXN转录本的缺乏与新型反义转录本的过表达相关，FAST1（FXN抗义转录本1）与+1核小体和较高水平的新型启动子与启动子相关的microRNA，miR miR miR-fxn1，更高水平与+1核小体重叠。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的转录沉默，我们假设FRDA中FXN基因的功能丧失是由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在诱导FXN基因在Friedreich ataxia中的异染色质形成和转录基因沉默中的潜在作用来检验这一假设。此外，我们将测试是否在诱导多能干细胞的分化中得出的弗里德里希共济失调神经元中还检测到来自患者的成纤维细胞细胞系中的表观遗传缺陷。 Fast1和 /或miR-FXN1在弗里德里希共济失调的发病机理中的影响将使它们成为合理的治疗靶标，以允许FXN基因的特定重新激活。实际上，我们还将通过靶向抑制FAST1和miR-FXN1来测试FXN基因在Friedreich共济失调中重新激活的可行性。该提案将增强我们对弗里德里希共济失调的发病机理的理解，弗里德里希共济失调是最常见的共济失调，并有可能对特定疗法的发展具有重要意义。公共卫生相关性：最常见的遗传性共济失调的弗里德里希共济失调患者在其FXN基因中扩大了GAA重复序列。初步结果表明，这导致FXN基因的包装改变，关闭基因表达并导致必需蛋白质Frataxin的缺乏。我们的实验旨在确定Friedreich共济失调中这种改变包装的精确机制，并破译方法逆转并恢复正常基因功能。