Targeting of somatic hypermutation and gene conversion

描述（由申请人提供）：体细胞超突变（SM）和基因转化（GC）是导致免疫球蛋白（Ig）可变区组装后多样化的致突变反应。它们在B细胞发育和有效的体液免疫中起关键作用。SM和GC在机制上非常相似，对转录和激活诱导脱氨酶（AID）有相同的要求，并且可能有共同的起始DNA损伤。我们对SM和GC的理解存在一个根本性的差距，即它们针对Ig基因的机制。这个问题具有重要的临床意义，因为SM的错误靶向与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SM和GC靶向Ig基因的分子机制。两个中心假设构成了这一建议的基础：第一，SM和GC的靶向依赖于位于Ig位点的特定顺式作用DNA序列（靶向元件）；第二，这些元件的靶向功能是受限的，因此邻近基因不受影响。本建议的具体目的是确定这些靶向元件，并确定其作用的染色体结构域的边界。我们选择鸡B细胞系DT40中的IgL轻链基因座作为实验系统，主要有以下几个原因：1)该基因座结构紧凑，可以经历SM和GC；2) DT40允许通过同源重组快速简便地操作基因组；3)我们创造了新的DT40细胞系，其中正常的IgL启动子被一个强大的、与增强子无关的启动子（来自人类延伸因子1a基因）所取代。目标基因座的转录水平很高，并经历了高效的GC，使我们能够区分IgL基因座中DNA元件的潜在靶向活性及其转录调控活性。使用这些新的DT40细胞系，我们将系统地删除IgL位点的部分以识别靶向元件。此外，我们将在野生型IgL基因座的不同位置插入一个突变盒，以确定突变活性域的边界。这些实验的结果应该为理解蛋白质因子和DNA序列协同作用将SM和GC靶向Ig基因的机制奠定重要的基础。

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genome by homologous recombination; and, 3) we have created novel DT40 cell lines in which the normal IgL promoter has been replaced by a strong, enhancer-independent promoter (from the human elongation factor 1a gene).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.