Transcription, Chromatin and DNA repair

免疫球蛋白(Ig)基因在B细胞发育过程中经历了三种遗传修饰，即V(D)J重组、体细胞超突变和类别转换。要发生这些反应，RAGS和AID酶必须能够进入重组和超突变位点。重组酶RAG1和RAG2在祖细胞B和T细胞中表达，促进V(D)J重组。另一方面，胞苷脱氨酶AID通过在Ig基因上将胞苷转化为尿嘧啶来调节体细胞的超突变和类别转换。 在V(D)J、转换和超突变过程中的可及性似乎是由基因转录提供的，可能是通过染色质重塑的方式，使Ig基因暴露于酶活性。然而，虽然可及性模型为特定靶向提供了理论基础，但尚不清楚在存在活跃转录的情况下，RAG和AID下游的DNA损伤如何处理。 去年，我们发表的数据显示，在ATM酶的引导下，一种特定的DNA修复途径下调了DNA损伤部位的转录。这一发现是使用rRNA基因作为一个系统进行的，主要是因为i)这些基因在细胞中高度丰富(每个基因组+400个拷贝)，ii)在一个被称为核仁的高度定义的微环境中在细胞核中分割，以及iii)它们的转录完全由DNA聚合酶I驱动。这三个特征使rRNA基因成为解决细胞动力学复杂问题的理想系统。然而，由于细胞中的大多数基因(包括免疫球蛋白基因)是由聚合酶II转录的，我们发表的结果是否揭示了B淋巴细胞重组或高突变的机制仍不清楚。为了将我们的研究扩展到聚合酶II转录，我们现在已经产生了在特定基因组位置携带大量聚合酶II基因拷贝(阵列)的转基因小鼠。此外，我们还开发了另一种表达荧光标记的聚合酶II的转基因菌株。从产生的几个转基因创始人中，我们目前正在选择一种最好地提供体内聚合酶II转录可视化手段的单株。一旦这只小鼠被选中，我们将分离小鼠胚胎成纤维细胞，开始我们的polII共聚焦显微镜研究。 正如前面提到的，DNA重组和超突变部位的酶可及性也是由染色质重塑提供的。核小体是由一个组蛋白八聚体包裹147bp的DNA形成的，它代表了DNA在细胞核中紧凑的第一级，也是DNA修复的障碍。为了中和染色质凝聚和促进修复，细胞进化出染色质重塑复合体和专门的修复酶，这些酶共价修饰核心组蛋白并破坏DNA-染色质结构。进化上保守的复合体NuA4已被证明通过组蛋白乙酰转移酶Tip60和依赖于ATP的染色质重构体P400参与DNA修复。关于这个复合体在修复中的作用，人们知道的大部分都是通过间接的方式揭示的，因为它的基因消融会导致死亡，至少在哺乳动物中是这样。因此，为了确定NuA4复合体是否在Ig基因重组和超突变中发挥作用，我们建立了Tip60酶在B细胞或T细胞中唯一缺失的小鼠模型。我们的初步研究表明，在无特定病原体的条件下，这些动物基本上是正常的，但T和B细胞发育不足。我们目前正在使用共聚焦显微镜、流式细胞术、分子生物学技术和深度测序来分析Tip60-/-细胞的表型。这些研究将帮助我们了解染色质解压是否在抗体分子的构建中发挥作用。

Immunoglobulin (Ig) genes undergo three genetic modifications during B cell development, namely V(D)J recombination, somatic hypermutation, and class switching. For these reactions to occur, RAGs and the AID enzymes must gain access to recombination and hypermutation sites. The recombinases RAG1 and RAG2 are expressed in progenitor B and T cells and promote V(D)J recombination. The cytidine deaminase AID on the other hand mediates both somatic hypermutation and class switching by converting cytidines into uracils at Ig genes. Accessibility during V(D)J, switching, and hypermutation appears to be provided by gene transcription, presumably by way of chromatin remodeling, which exposes Ig genes to enzymatic activity. However, while the accessibility model provides a rationale to specific targeting, it is unclear how DNA lesions downstream of RAG and AID can be processed in the presence of active transcription. Last year we published data showing that a particular DNA repair pathway, led by the ATM enzyme, downregulates transcription at sites of DNA damage. This discovery was made using rRNA genes as a system, mainly because i) these genes are highly abundant in the cell (+400 copies per genome), ii) are compartmentalized in the cell nucleus in a highly defined microenvironment known as the nucleolus, and iii) their transcription is exclusively driven by DNA polymerase I. These three features make of rRNA genes an ideal system to address complex questions on cellular dynamics. However, because most genes in the cell (including immunoglobulin genes) are transcribed by polymerase II, it is still unclear whether our published results shed light to the mechanism of recombination or hypermutation in B lymphocytes. To extend our studies to polymerase II transcription we have now generated transgenic mice carrying at a particular genomic site a large number of copies (an array) of polymerase II genes. In addition, we have also developed another transgenic strain expressing a fluorescently labeled polymerase II. Out of several transgenic founders produced, we are currently in the process of selecting a single line that will best provide a means to visualize polymerase II transcription in vivo. Once this mouse is selected we will then isolate mouse embryonic fibroblasts to begin our polII confocal microscopy studies. As previously mentioned, enzyme accessibility to sites of DNA recombination and hypermutation is also provided by chromatin remodeling. Nucleosomes, formed by the wrapping of 147 bp of DNA around a histone octamer, represent the first level of DNA compaction in the nucleus and an obstacle to DNA repair. To counteract chromatin condensation and facilitate repair, cells have evolved chromatin remodeling complexes and specialized repair enzymes that covalently modify core histones and disrupt DNA-chromatin structures. The evolutionarily conserved complex NuA4 has been shown to be involved in DNA repair via the histone acetyltransferase Tip60 and the ATP-dependent chromatin remodeler p400. Most of what is known about this complex's role in repair was revealed by indirect means because its genetic ablation leads to lethality, at least in mammals. Thus to determine whether the NuA4 complex plays a role in Ig gene recombination and hypermutation we have generated mouse models where the Tip60 enzyme is exclusively deleted in B or T cells. Our preliminary studies indicate that under specific pathogen free conditions these animals are largely normal but have deficiencies in T and B cell development. We are currently using confocal microscopy, flow cytometry, molecular biology techniques, and deep sequencing to dissect the phenotype of Tip60-/- cells. These studies will help us understand whether chromatin decompaction plays a role in the building of the antibody molecule.