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

描述(由申请人提供)：尽管我们在理解三核苷酸重复如何导致疾病方面取得了重大进展，但其遗传不稳定的独特特性背后的机制仍未明确。在所有CAG/聚谷氨酰胺疾病中，脊髓小脑性共济失调7型(SCA7)表现出最深刻的重复扩张趋势，因此也是最戏剧性的预期。我们之前资助的建议是基于这样一个假设，即识别顺式作用元件和反式作用因子将有助于解开导致反复发生的不稳定和疾病的复杂过程。利用人类SCA7基因的13.5kb基因组片段，我们发现3‘到CAG重复序列是转基因小鼠重复不稳定所必需的。在这个3‘区内是“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，其功能源于其调节DNA结构的能力。当我们在相同的13.5kb的ataxin-7 CAG-92基因组片段和再衍生的转基因小鼠中将CTCF结合位点3‘突变为SCA7重复时，我们发现了两个惊人的观察结果：i)SCA7位点的重复不稳定需要完整的CTCF结合位点序列；ii)CTCF结合位点的突变会产生类似SCA7的神经退行性变表型。 由于后者的结果完全出乎意料，我们对这些小鼠和ataxin-7微基因内的基因组区域进行了深入的研究，并确定CTCF通过反义非编码RNA调节来自另一个启动子的ataxin-7的表达。由于ataxin-7可能是转录共激活因子发挥作用所必需的，我们的结果为CTCF的作用和反义非编码RNA表达在转录调控中的作用提供了一个新的挑衅性模型。由于CTCF结合位点已在多种三核苷酸重复疾病基因座上被发现，并已被证明在另一个基因座上调节转录，我们的发现表明重复扩大、CTCF功能、重复不稳定、表观遗传学和转录调控之间存在令人兴奋的联系。 这项建议试图通过研究转基因小鼠的过程来确定CTCF是否是调节生殖系和SCA7基因座体细胞中重复重复不稳定的反式作用因子。我们发现CTCF在促进SCA7基因上反义非编码RNA转录方面具有潜在的作用，这增加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ataxin-7的转录，从而调节STAGA复合体的共激活功能。我们将通过鉴定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基因的表达；并确定ataxin-7正义和反义非编码RNA转录本的表达模式、调控和关系。我们将确定CTCF水平是否调节STAGA复杂功能；这种调节是否依赖于ataxin-7反义非编码RNA的产生或减少；以及哪些因素影响CTCF-ataxin-7-STAGA途径。最后，我们将尝试在体内验证反义非编码RNA的功能。

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.