Targeting of somatic hypermutation and gene conversion

描述(申请人提供)：体细胞过度突变(SM)和基因转换(GC)是负责免疫球蛋白(Ig)可变区组装后多样化的突变反应。它们在B细胞发育和有效的体液免疫中起着关键作用。SM和GC在力学上非常相似，都有转录和激活诱导脱氨酶(AID)的需要，很可能是共同的启动DNA损伤。在我们对SM和GC的理解中，一个根本的鸿沟涉及到它们针对Ig基因的机制。这个问题具有重要的临床意义，因为SM的错误定位与B细胞恶性肿瘤的癌基因突变和染色体易位有关。我们的长期目标是确定以SM和GC为靶点的Ig基因的分子机制。这一提议的基础是两个中心假设：第一，SM和GC的靶向依赖于位于Ig基因座的特定顺式作用DNA序列(靶向元件)；第二，这些元件的靶向功能是有限的或有限制的，因此相邻的基因不会受到影响。这项提案的具体目标是确定这些靶向元件，并确定它们作用的染色体区域的边界。我们选择鸡B细胞系DT40中的免疫球蛋白轻链基因座(IGL)作为我们的实验系统是因为以下几个原因：1)这个基因座非常紧凑，可以同时进行SM和GC；2)DT40允许通过同源重组快速而简便地操纵基因组；以及3)我们已经创建了新的DT40细胞系，其中正常的IGL启动子已经被一个强大的、增强子无关的启动子(来自人延长因子1a基因)所取代。靶基因座在高水平转录，并经过有效的GC，使我们能够区分IGL基因座中DNA元件的潜在靶向活性和它们的转录调节活性。使用这些新的DT40细胞系，我们将系统地删除部分IGL基因座，以确定靶向元件。此外，我们还将在野生型IGL基因座的不同位置插入突变盒，以确定突变活性区域的边界。这些实验的结果应该会为理解蛋白质因子和DNA序列合作靶向SM和GC到Ig基因的机制奠定重要进展。

DESCRIPTION (provided by applicant): Somatic hypermutation (SM) and gene conversion (GC) are mutagenic reactions responsible for the post-assembly diversification of immunoglobulin (Ig) variable regions. They play critical roles in B cell development and effective humoral immunity. SM and GC are mechanistically very similar, sharing a requirement for transcription and the activation induced deaminase (AID) and likely a common initiating DNA lesion. A fundamental gap in our understanding of SM and GC concerns the mechanism by which they are targeted to Ig genes. This issue has important clinical implications because mistargeting of SM has been linked to oncogene mutations and chromosomal translocations in B cell malignancies. Our long term goal is to determine the molecular mechanism that targets SM and GC to Ig genes. Two central hypotheses form the foundation for this proposal: first, that targeting of SM and GC relies on specific cis-acting DNA sequences (targeting elements) that reside in the Ig loci and second, that the targeting function of these elements is circumscribed, or bounded, so that adjacent genes are not affected. The specific aims of this proposal are to identify these targeting elements and to determine the boundaries of the chromosomal domain over which they act. We have chosen the Ig light chain locus (IgL) in the chicken B cell line DT40 as our experimental system for several reasons: 1) this locus is very compact and can undergo both SM and GC; 2) DT40 allows rapid and facile manipulation of the genome by homologous recombination; and, 3) we have created novel DT40 cell lines in which the normal IgL promoter has been replaced by a strong, enhancer-independent promoter (from the human elongation factor 1a gene). The targeted locus is transcribed at high levels and undergoes efficient GC, allowing us to distinguish the potential targeting activity of DNA elements in the IgL locus from their transcriptional regulatory activity. Using these new DT40 cell lines, we will systematically delete portions of the IgL locus to identify targeting elements. In addition, we will insert a mutational cassette at various positions in the wild type IgL locus to identify the boundaries of the mutationally active domain. The results of these experiments should set the stage for significant progress toward an understanding of the mechanism by which protein factors and DNA sequences collaborate to target SM and GC to Ig genes.