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，但其独特的遗传不稳定性背后的机制仍然不明确。在所有CAG/多谷氨酰胺疾病中，脊髓小脑性共济失调7型（SCA7）表现出最深刻的重复扩张倾向，因此最显著的预期。我们以前资助的建议是基于这样一种假设，即确定顺式作用因素和反式作用因素将有助于揭示产生重复不稳定和疾病的复杂过程。利用来自人类SCA7基因的13.5 kb基因组片段，我们发现转基因小鼠重复序列的不稳定性需要3'到CAG重复序列。在这个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，源于其调节DNA结构的能力。当我们在相同的13.5 kb ataxin-7 CAG-92基因组片段中将CTCF结合位点3'突变为SCA7重复序列并重新衍生转基因小鼠时，我们得到了两个惊人的观察结果：i)在SCA7位点重复不稳定性需要完整的CTCF结合位点序列；ii) CTCF结合位点的突变产生sca7样神经退行性表型。