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: 7760578
• 负责人: ALBERT R LA SPADA
• 金额: $ 36.71万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 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 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7760578
• 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如何引起疾病的理解取得了重大进展，但其遗传不稳定性独特特性的基础机制仍未定义。在所有CAG/聚谷氨酰胺疾病中，脊髓脑性共济失调7型（SCA7）表现出最深刻的重复膨胀趋势，因此是最引人注目的预期。我们以前资助的建议基于以下假设：鉴定顺式作用元素和跨性因素将有助于揭示产生重复不稳定和疾病的复杂过程。使用来自人类SCA7基因的13.5 Kb基因组片段，我们发现转基因小鼠重复不稳定性需要3'至CAG重复序列。在这个3'区域内是“ CCCTC结合因子”（CTCF）的结合位点，该蛋白具有多种功能，其源于其调节DNA结构的能力。当我们在相同13.5 kb ataxin-7 CAG-92基因组片段和重新衍生的转基因小鼠中突变CTCF结合位点3'与SCA7重复重复时，我们做出了两个引人注目的观察结果：i）完整的CTCF结合位点序列需要在SCA7 Locus重复不稳定。 ii）CTCF结合位点的突变产生SCA7样神经退行性表型。 由于后者的结果是完全出乎意料的，因此我们深入研究了ataxin-7 Mini-Gene内的这些小鼠和基因组区域，并确定CTCF通过反义非编码RNA从替代启动子中调节ataxin-7的表达。由于ataxin-7可能是转录共激活因子的功能所需的，因此我们的结果表明了一个挑衅性的CTCF作用的新型模型以及反义非编码RNA在转录调控中的作用。由于在多种三核苷酸重复疾病基因座上发现了CTCF结合位点，并已被证明可以调节另一个基因座的转录，因此我们的发现表明重复扩张，CTCF功能，重复不稳定，表观遗传学和转录调节之间存在令人兴奋的联系。 该提案旨在通过研究转基因小鼠的过程来确定CTCF是否是调节SCA7基因座中种系和体细胞中重复不稳定性的跨作用因子。我们发现CTCF在SCA7基因座上促进反义非编码RNA转录中的潜在作用增加了一种有趣的可能性，即CTCF结合调节ataxin-7转录，从而通过促进反义非编码RNA的表达来调节Ataxin-7转录，从而调节Staga复杂的共激活因子功能。我们将通过表征Ataxin-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ataxin-7基因表达；并确定Ataxin-7感官和反义非编码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复合函数；如果这种调节依赖于ataxin-7反义非编码RNA的产生或减少；哪些因素会影响CTCF-ataxin-7- Staga途径。最后，我们将尝试验证体内反义非编码RNA的功能。