Genomic Plasticity in the Human U2 snRNA Cluster

染色体易位和畸变在癌症中很常见，但对染色体脆性的分子机制知之甚少。我们正在使用人类RNU 2基因座（编码U2 snRNA）作为模型系统，以了解中期染色体脆性的原因。编码U2 snRNA（mRNA剪接装置的关键组分）的基因被组织为一个基本上完美的串联阵列，包含5至>22个拷贝的人染色体17 q21上的6.1 kb重复单元。U2串联重复序列的规律性，关于U2 snRNA转录的丰富功能数据，以及诱导RNU 2脆性的各种条件，都使RNU 2成为研究基因功能和中期染色质凝聚之间关系的良好系统。最重要的是，U2基因是唯一的实验系统，其中可以通过将克隆的脆性位点（即串联重复的U2基因或其突变体）插入新的染色体基因座来从头产生新的脆性位点。我们之前已经表明：活性U2 snRNA转录是RNU 2中期脆性所必需的;所有诱导脆性的条件也诱导p53; CS B（Cockayne综合征组B蛋白）（一种转录偶联修复（TCR）所需的假定转录延伸因子）的缺失导致组成性RNU 2脆性;并且结合CS B的p53的C末端结构域（CTD）本身可以诱导脆性。这些数据表明，激活的p53通过调节CSB功能诱导U2基因的脆性，并且CSB是U2转录或修复受损U2基因所需的。我们提出了一个多方面的实验方法来测试，修改，或放弃这个工作模型。具体地说， 我们将检测非洲爪蟾卵母细胞（Aim 1）和人类体细胞（Aim 2）中活跃转录U2 snRNA基因的染色质结构;我们将检测CSB在U2转录（Aim 3）和DNA修复（Aim 4）中的功能，沿着XPG、BRCA 1和BRCA 2;我们将检测p53对CSB功能的调节（Aim 5）;我们将通过微阵列技术来研究CSB活性的丧失会影响哪些细胞mRNA（目的6）。我们的研究将有助于阐明细胞周期转录调控、染色体高阶结构、肿瘤染色体不稳定的分子机制以及Cockayne综合征的病因。

Chromosome translocations and aberrations are common in cancer, but the molecular mechanisms responsible for chromosome fragility are poorly understood. We are using the human RNU2 locus (encoding U2 snRNA) as a model system to understand the causes of metaphase chromosome fragility. The genes encoding U2 snRNA, a key component of the mRNA splicing apparatus, are organized as a single essentially perfect tandem array containing 5 to >22 copies of a 6.1 kb repeat unit on human chromosome 17q21. The regularity of the U2 tandem repeat, the wealth of functional data regarding U2 snRNA transcription, and the variety of conditions that induce RNU2 fragility, all make RNU2 a good system for studying the relationship between gene function and metaphase chromatin condensation. Most importantly, the U2 genes are the only experimental system in which a new fragile site can be created de novo by inserting a cloned fragile site (i.e. tandemly repeated U2 genes or mutants thereof) into a new chromosomal locus. We have shown previously that: active U2 snRNA transcription is required for RNU2 metaphase fragility; all conditions that induce fragility also induce p53; loss of CSB (Cockayne Syndrome Group B protein), a putative transcription elongation factor required for transcription-coupled repair (TCR), causes constitutive RNU2 fragility; and the C-terminal domain (CTD) of p53, which binds CSB, can itself induce fragility. The data suggest that activated p53 induces fragility of the U2 genes by modulating CSB function, and that CSB is required either for U2 transcription, or for repair of damaged U2 genes. We propose a multifaceted experimental approach to test, modify, or discard this working model. Specifically, we will examine the chromatin structure of actively transcribing U2 snRNA genes in Xenopus oocytes (Aim 1) and in human somatic cells (Aim 2); we will assay the function of CSB in U2 transcription (Aim 3) and in DNA repair (Aim 4) along with XPG, BRCA1, and BRCA2; we will assay modulation of CSB function by p53 (Aim 5); and we will ask by microarray technology what cellular mRNAs are affected by loss of CSB activity (Aim 6). Our studies could shed light on cell cycle control of transcription, higher order chromosome structure, the molecular mechanisms of chromosome instability in cancer, and the causes of Cockayne Syndrome.