Mechanisms of Human Lymphoid Chromosomal Translocation

 描述(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，其中包含1,800个来自癌症患者的肿瘤染色体易位，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脆弱区，它们占了人类B细胞淋巴瘤的大部分。虽然易位可能跨越癌基因附近的10-100kb区域，但我们已经确定的脆弱区长度只有25-600bp，并且比附近的DNA更容易发生DNA断裂。确定这些脆弱区的分子和结构基础是这项提案的主要重点。我们已经确定，这些脆弱区中的所有CG和WGCW位点都是AID(激活诱导脱氨酶)的作用位点，AID通过将C转化为U或将甲基C转化为T而产生G：U碱基对错配，从而导致碱基对错配转化为双链DNA断裂(DSB)。我们已经确定的7个人类脆弱区没有一个位于启动子或启动子附近，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原因导致这些25-600bp的脆弱区达到导致DSB的单链DNA状态。这项研究中研究的所有易位都发生在人类前B细胞分化过程中。我们的分析有三个人类前B细胞来源，其中两个是原代细胞。目标1使用四种平行的方法来定义相对于邻近DNA的7个脆弱区的单链DNA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法识别单链DNA区域。我们将确定7个脆弱区之间单链DNA特征的位置、长度和程度的相关性，以及7个脆弱区之间的共同序列和特征(DNA重复、蛋白质结合基序、DNA结构基序)。在Aim 1C中，我们映射了人类前B细胞中的所有非编码RNA，以确定非编码RNA是否在7个脆弱区的边界处启动，从而产生拓扑紧张。在Aim 1D中，我们测试脆弱区中称为R环的改变的DNA结构(Yu&Lieber，2003)。对于目标2，我们已经证明了当移到其他核位置时，即使是在非淋巴系人类细胞中，这些区域也是脆弱的。AIM 2中的新的初步数据表明，在酿酒酵母极其敏感和定量的遗传分析中，这些区带是脆弱的。我们证明了bcl2MBR只有在转录通过它发生并且以一种拓扑依赖的方式发生时才是脆弱的。Aim 2A-C测试其他6个脆弱区的转录和拓扑依赖的脆性，以及复制起点邻近的影响。目的2D-E诱变bcl2MBR脆性区以确定其最小特征及其对激活Artemis的敏感性。这些研究与所有细胞和肿瘤中的脆弱区广泛相关。

 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.