CCCTC-binding factor (CTCF) in trinucleotide repeat instability and disease

三核苷酸重复不稳定性和疾病中的 CCCTC 结合因子 (CTCF)

• 批准号: 7570106
• 负责人: ALBERT R LA SPADA
• 金额: $ 35.74万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7570106
• 关键词: Activator Appliances; Affect; Binding; Binding Sites; CAG repeat; CCCTC-binding factor; Cell Differentiation process; Code; Complementary DNA; Complex; DNA Structure; Development; Disease; Elements; Employee Strikes; Epigenetic Process; Excision; Exons; Functional RNA; Funding; Gene Expression; Genes; Genetic; Genetic Transcription; Genomics; Germ Cells; Germ Lines; Human; Methylation; Modeling; Mus; Mutate; Mutation; Pathway interactions; Pattern; Polyadenylation; Process; Production; Property; Proteins; RNA; RNA Interference; RNA methylation; Regulation; Research Personnel; Role; SCA7 protein; Signal Transduction; Somatic Cell; Spinocerebellar Ataxias; Testing; Trans-Activators; Transcript; Transcriptional Regulation; Transgenic Mice; Trinucleotide Repeats; Type 7 Spinocerebellar Ataxia; base; chromatin immunoprecipitation; cis acting element; in vivo; mouse CCCTC-binding factor; mutant; neurodegenerative phenotype; novel; polyglutamine; premature; programs; promoter; recombinase; stem

DESCRIPTION (provided by applicant): Despite significant advances in our understanding of how trinucleotide repeats cause disease, mechanisms underlying their unique property of genetic instability remain ill-defined. Of all CAG/polyglutamine diseases, spinocerebellar ataxia type 7 (SCA7) displays the most profound repeat expansion tendency and hence the most dramatic anticipation. Our previously funded proposal was based upon the hypothesis that the identification of cis-acting elements and trans-acting factors would help to unravel the complex processes that produce repeat instability and disease. Using a 13.5 kb genomic fragment from the human SCA7 gene, we found that sequences 3' to the CAG repeat are required for repeat instability in transgenic mice. Within this 3' region is a binding site for the "CCCTC-binding factor" (CTCF), a protein with a variety of functions that stem from its ability to modulate DNA structure. When we mutated the CTCF binding site 3' to the SCA7 repeat in the same 13.5 kb ataxin-7 CAG-92 genomic fragment and re-derived transgenic mice, we made two striking observations: i) an intact CTCF binding site sequence is required for repeat instability at the SCA7 locus; and ii) mutation of the CTCF binding site yields a SCA7-like neurodegenerative phenotype. As the latter result was completely unexpected, we have intensively studied these mice and the genomic region within the ataxin-7 mini-gene, and have determined that CTCF regulates expression of ataxin-7 from an alternative promoter through an antisense non-coding RNA. As ataxin-7 may be required for the function of a transcriptional co-activator, our results suggest a provocative novel model for CTCF action and for the role of antisense non-coding RNA expression in transcription regulation. As CTCF binding sites have been found at a variety of trinucleotide repeat disease loci and have been shown to regulate transcription at one other locus, our findings suggest an exciting connection between repeat expansion, CTCF function, repeat instability, epigenetics, and transcription regulation. This proposal seeks to determine if CTCF is the trans-acting factor regulating repeat instability in the germ line and in somatic cells at the SCA7 locus by studying the process in transgenic mice. Our discovery of a potential role for CTCF in the promotion of antisense non-coding RNA transcription at the SCA7 locus raises the intriguing possibility that CTCF binding regulates ataxin-7 transcription and thus STAGA complex co-activator function by promoting expression of an antisense non-coding RNA. We will test if CTCF regulates ataxin-7 gene expression through an antisense non-coding RNA by characterizing an ataxin-7 alternative promoter and antisense non-coding RNA; and determining the expression pattern, regulation, and relationship of ataxin-7 sense and antisense non-coding RNA transcripts. We will determine if CTCF levels modulate STAGA complex function; if such modulation relies on production or reduction of the ataxin-7 antisense non-coding RNA; and what factors affect the CTCF - ataxin-7- STAGA pathway. Finally, we will attempt to validate the function of the antisense non-coding RNA in vivo.

描述（由申请人提供）：尽管我们对三核苷酸重复序列如何引起疾病的理解取得了重大进展，但其遗传不稳定性的独特性质的机制仍然不明确。在所有CAG/多聚谷氨酰胺疾病中，脊髓小脑共济失调7型（SCA 7）显示出最深刻的重复扩增趋势，因此最引人注目的预期。我们以前资助的提案是基于这样的假设，即顺式作用元件和反式作用因子的鉴定将有助于解开产生重复不稳定和疾病的复杂过程。使用来自人SCA 7基因的13.5kb基因组片段，我们发现CAG重复序列3'端的序列是转基因小鼠中重复序列不稳定性所必需的。在该3'区域内是“CCCTC结合因子”（CTCF）的结合位点，CTCF是一种具有多种功能的蛋白质，这些功能源于其调节DNA结构的能力。当我们在相同的13.5kb共济失调蛋白-7 CAG-92基因组片段和再衍生的转基因小鼠中突变SCA 7重复序列的3'端CTCF结合位点时，我们得到了两个惊人的观察结果：i）完整的CTCF结合位点序列是SCA 7基因座处重复序列不稳定性所必需的;和ii）CTCF结合位点的突变产生SCA 7样神经退行性表型。 由于后一结果完全出乎意料，我们深入研究了这些小鼠和共济失调蛋白-7微型基因内的基因组区域，并确定CTCF通过反义非编码RNA从替代启动子调节共济失调蛋白-7的表达。由于共济失调蛋白-7可能是转录共激活因子的功能所必需的，我们的研究结果表明了CTCF作用和反义非编码RNA表达在转录调控中的作用的一种挑衅性的新模型。由于CTCF结合位点已被发现在各种三核苷酸重复疾病基因座，并已被证明在一个其他基因座的转录调节，我们的研究结果表明重复扩增，CTCF功能，重复不稳定性，表观遗传学和转录调控之间的一个令人兴奋的连接。 该建议旨在通过研究转基因小鼠中的过程来确定CTCF是否是调控生殖系和体细胞中SCA 7位点的重复不稳定性的反式作用因子。我们发现CTCF在SCA 7位点促进反义非编码RNA转录中的潜在作用，这提出了一种有趣的可能性，即CTCF结合通过促进反义非编码RNA的表达来调节共济失调蛋白-7转录，从而调节STAGA复合物共激活因子功能。我们将通过表征共济失调蛋白-7替代启动子和反义非编码RNA来测试CTCF是否通过反义非编码RNA调节共济失调蛋白-7基因表达;并确定共济失调蛋白-7正义和反义非编码RNA转录本的表达模式、调节和关系。我们将确定CTCF水平是否调节STAGA复合体功能;这种调节是否依赖于共济失调蛋白-7反义非编码RNA的产生或减少;以及哪些因素影响CTCF -共济失调蛋白-7- STAGA通路。最后，我们将尝试在体内验证反义非编码RNA的功能。