Transcription, Chromatin and DNA repair

免疫球蛋白（IG）基因在B细胞发育过程中经历三种遗传修饰，即V（D）J重组、体细胞超突变和类转换。为了使这些反应发生，RAG和AID酶必须能够进入重组和超突变位点。重组酶RAG 1和RAG 2在祖细胞B和T细胞中表达并促进V（D）J重组。另一方面，胞苷脱氨酶AID通过在IG基因处将胞苷转化为尿嘧啶来介导体细胞超突变和类别转换。 在V（D）J、转换和超突变过程中的可接近性似乎是由基因转录提供的，推测是通过染色质重塑的方式，其使IG基因暴露于酶活性。然而，尽管可及性模型为特异性靶向提供了理论基础，但目前尚不清楚RAG和AID下游的DNA损伤如何在存在活性转录的情况下进行处理。 去年我们发表的数据显示，由ATM酶领导的一种特定的DNA修复途径下调了DNA损伤位点的转录。这一发现是使用rRNA基因作为一个系统，主要是因为i）这些基因在细胞中非常丰富（每个基因组+400个拷贝），ii）在细胞核中被划分在一个高度定义的微环境中，称为核仁，iii）它们的转录完全由DNA聚合酶I驱动。这三个特征使得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，因此我们发表的结果是否揭示了B淋巴细胞中的重组或超突变机制仍不清楚。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点携带大量聚合酶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聚合酶II的转基因菌株。在产生的几个转基因创始人，我们目前正在选择一个单一的线，将最好地提供一种手段，可视化聚合酶II在体内转录的过程。一旦选择了这只小鼠，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。 如前所述，酶对DNA重组和超突变位点的可及性也由染色质重塑提供。核小体是由147 bp的DNA包裹在组蛋白八聚体周围形成的，代表了细胞核中DNA压缩的第一个层次，也是DNA修复的障碍。为了抵消染色质凝聚并促进修复，细胞已经进化出染色质重塑复合物和专门的修复酶，其共价修饰核心组蛋白并破坏DNA-染色质结构。进化上保守的复合物NuA 4已被证明通过组蛋白乙酰转移酶Tip 60和ATP依赖性染色质重塑蛋白p400参与DNA修复。关于这种复合物在修复中的作用的大部分已知信息都是通过间接手段揭示的，因为它的基因切除会导致致命性，至少在哺乳动物中是这样。因此，为了确定NuA 4复合物是否在IG基因重组和超突变中起作用，我们产生了小鼠模型，其中Tip 60酶仅在B或T细胞中缺失。我们的初步研究表明，在特定的病原体自由的条件下，这些动物基本上是正常的，但有缺陷的T和B细胞的发展。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剖析Tip 60-/-细胞的表型。这些研究将帮助我们了解染色质解压缩是否在抗体分子的构建中起作用。

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.