Mechanisms of Human Lymphoid Chromosomal Translocation

 描述（由申请人提供）：染色体易位是许多癌症的关键起始点，但人类易位的分子机制尚不清楚。我们有一个独特的数据库，包含1,800个来自癌症患者的肿瘤性染色体易位，这些易位连接已经测序。我们利用这些来自人类B细胞淋巴瘤的数据来定义这些患者易位发生的DNA区域的独特之处。我们已经确定了7个明确的DNA脆性区，占大多数的人类B细胞淋巴瘤。尽管易位可能跨越癌基因附近的10-100 kb区域，但我们已经鉴定的脆性区的长度仅为25-600 bp，并且比附近的DNA更容易受到DNA断裂的影响100-1000倍。确定这些脆弱区的分子和结构基础是这项建议的主要重点。我们已经确定，这些脆性区内的所有CG和WGCW位点都是AID（活化诱导脱氨酶）的作用位点，AID通过将C转化为U或甲基-C转化为T而产生G：U碱基对错配。由此产生的碱基对错配可以转化为双链DNA断裂（DSB）。我们已经确定的7个人类脆性区中没有一个位于启动子中或附近，而启动子是小鼠模型中发生易位的地方。重要的是，AID需要单链DNA（ssDNA）作为底物。因此，我们的主要目标之一是确定是什么导致这些25-600 bp的脆弱区达到导致DSB的ssDNA状态。在该提议中研究的所有易位都发生在人前B细胞分化期间。我们有三种来源的人前B细胞用于我们的分析，其中两种是原代细胞。目的1使用四种平行的方法来定义7个脆性区相对于附近DNA的ssDNA特征。在目标1A和B中，我们使用亚硫酸氢盐和高锰酸盐化学探针鉴定ssDNA的区域。我们将确定ssDNA特征的位置、长度和程度在7个脆性区之间的相关性，以及7个脆性区之间共有的序列和特征（DNA重复序列、蛋白质结合基序、DNA结构基序）。在Aim 1C中，我们绘制了人类前B细胞中的所有非编码RNA，以确定非编码RNA是否在7个脆性区的边界处启动以产生拓扑张力。在Aim 1D中，我们测试了脆弱区中被称为R环的改变的DNA结构（Yu & Lieber，2003）。对于目标2，我们已经表明，当移动到其他核位置时，即使在非淋巴样人类细胞中，这些区域也是脆弱的。目标2中的新的初步数据表明，这些区域在S.啤酒。我们表明，bcl-2 MBR是脆弱的，只有当转录发生通过它和拓扑依赖的方式。目的2A-C测试其他6个脆性区的转录和拓扑依赖性脆性，以及复制起点邻近的影响。目的2D-E诱变bcl-2膜生物反应器（MBR）脆性带，以确定其最小特征及其对活化卤虫的敏感性。这些研究与所有细胞和肿瘤中的脆性区广泛相关。

 DESCRIPTION (provided by applicant): Chromosomal translocations are the key inception point of many cancers and yet the molecular mechanism of translocations in humans is unclear. We have a unique database of 1,800 neoplastic chromosomal translocations from cancer patients for which the translocation junctions have been sequenced. We exploit these data from human B-cell lymphomas to define what is distinctive about the DNA regions where these patient translocations occurred. We have identified 7 well-defined DNA fragile zones that account for the majority of human B cell lymphomas. Although the translocations can potentially span 10-100 kb regions near oncogenes, the fragile zones we have identified are only 25-600 bp in length and are 100-1000-fold more susceptible to DNA breakage than nearby DNA. Determining the molecular and structural basis of these fragile zones is the major focus of this proposal. We have determined that all of the CG and WGCW sites within these fragile zones are sites of action of AID (activation-induced deaminase) which generates G:U base-pair mismatches by converting either C to U or methyl-C to T. The resulting base-pair mismatches can then be converted to double-strand DNA breaks (DSBs). None of the 7 human fragile zones we have identified are located in or near promoters, which is where translocations occur in murine models. Importantly, AID requires single-stranded DNA (ssDNA) as a substrate. Thus, one of our major goals is to determine what causes these 25-600 bp fragile zones to achieve a ssDNA state that leads to DSBs. All of the translocations studied in this proposal occurred during human pre-B cell differentiation. We have three sources of human pre-B cells for our analyses and two of these are primary cells. Aim 1 uses four parallel approaches to define the ssDNA character of the 7 fragile zones relative to nearby DNA. In Aim 1A & B, we identify regions of ssDNA using bisulfite and permanganate chemical probing. We will determine how well the location, length and degree of ssDNA character is correlated among the 7 fragile zones and what sequences and features (DNA repeats, protein binding motifs, DNA structural motifs) are in common among the 7 fragile zones. In Aim 1C we map all noncoding RNAs in human pre-B cells to determine whether noncoding RNAs are initiated at the boundaries of the 7 fragile zones to create topological tension. In Aim 1D, we test for altered DNA structures called R-loops in the fragile zones (Yu & Lieber, 2003). For Aim 2, we had already shown that the zones are fragile when moved to other nuclear locations, even in non-lymphoid human cells. New preliminary data in Aim 2 demonstrates that these zones are fragile in an extremely sensitive and quantitative genetic assay in S. cerevisiae. We show that the bcl-2 MBR is fragile only when transcription occurs through it and in a topologically-dependent manner. Aim 2A-C tests the other 6 fragile zones for transcription- and topologically-dependent fragility, and the effect of replication origin proximity. Aim 2D-E mutagenizes the bcl-2 MBR fragile zone to determine the minimal features and its sensitivity to activated Artemis. These studies are broadly relevant to fragile zones in all cells and tumors.