Molecular Basis of Immunoglobulin Heavy Chain Switch

描述（由申请方提供）：抗体（免疫球蛋白，IG）类别转换导致B淋巴细胞从产生IgM转为产生IgG、伊加或IgE，从而提高抗体清除体内病原体和细菌毒素的能力。类别转换通过染色体内DNA重组事件发生，必须仔细控制以避免与其他染色体的异常重组（易位）。然而，癌基因和IgH位点之间确实发生易位，这可导致B细胞淋巴瘤。在类别转换期间，激活诱导的胞苷脱氨酶（AID）在IG重链基因座（IgH）中的开关（S）区域启动DNA双链断裂（DSB）的形成，这是类别转换所必需的。我的第一个目标是确定AID如何使IG S区的dC脱氨基，形成dU，导致DSB。我们已经表明，艾滋病诱导的dC脱氨基导致DNA单链断裂（SSB）通过碱基切除修复途径。这些SSB随后如何转换为DSB尚不清楚。我们已经报道了另一种DNA修复途径，错配修复（MMR）对这一步很重要，我们将研究它的作用。我们将通过确定S区域中AID诱导的dU的频率和位点，AID靶标的频率和位置如何影响转换频率，以及MMR蛋白是否可能被AID本身募集到S区域来研究SSB如何转化为DSB。在目标2中，我们将在本基金的当前期限内继续研究我们的发现，即AID可以在活化的B细胞中IgH位点以外的位点引发DSB。我们将确定是什么使这些其他位点成为AID的靶点，这些DSB是否导致染色体断裂、缺失和易位，以及MMR和其他已知参与CSR的DNA修复蛋白（例如ATM、H2 AX和53 BP 1）是否参与制造或预防这些DSB。 公共卫生相关性：大多数人成熟B细胞淋巴瘤的特征是相互染色体易位，通常涉及免疫球蛋白（IG）基因。长期以来的假设，这些易位是由错误的抗体类别转换，一个正常的过程，是一个有效的体液免疫反应，是必不可少的，只是最近得到验证。抗体类别转换是一个需要将DNA断裂引入到IG基因中的过程，进入称为转换区的特殊区域，然后将这些DNA断裂末端之一与同一染色体上同一IG基因座中的另一个DNA断裂末端连接。诱导DNA断裂的机制以及如何调节该过程以防止将DNA断裂连接到另一条染色体，这可能导致癌基因的激活，从而导致B细胞淋巴瘤尚不清楚。这里提出的实验将确定启动类别转换和染色体易位的酶AID的活性是如何调节的，以及它如何选择其靶点。我们已经证明，细胞中一种特殊的DNA修复途径，称为错配修复，这是修复DNA合成过程中错误所必需的，是类别转换过程中大多数DNA断裂形成所必需的。我们试图进一步了解它在将艾滋病诱导的病变转化为DNA断裂中的作用。错配修复是消除突变和微卫星不稳定性，从而防止细胞转化和恶性肿瘤的重要修复系统。这一通路增加DNA断裂的假设似乎违反直觉，但直接来自我们对这一通路在类别转换过程中的作用的理解。 在小鼠成熟B细胞浆细胞瘤中，c-myc癌基因和IG位点之间的染色体易位需要AID。此外，许多起源于生殖中心B细胞的人B细胞淋巴瘤显示涉及IG转换区的染色体易位，表明涉及类别转换过程。由于与强IgH增强子并列，IgH易位通常大大增加癌基因表达，导致细胞转化和恶性肿瘤。然而，靶向IG基因座以外的其他基因座的AID是否导致在这些位点形成DNA断裂尚不清楚。通过使用全基因组搜索方法，我们已经在基因组的其他几个位点发现了艾滋病依赖性DNA断裂。我们将检查这些其他位点是否与IG位点的染色体易位有关。我们将调查是什么使这些其他网站的目标艾滋病，并为DNA断裂的形成。因此，我们的研究也将阐明国际援助如何选择其目标这一重要问题。

DESCRIPTION (provided by applicant): Antibody (immunoglobulin, Ig) class switch causes B lymphocytes to switch from producing IgM to producing IgG, IgA or IgE, which improves the ability of the antibody to remove pathogens and bacterial toxins from the body. Class switching occurs by an intrachromosomal DNA recombination event that must be carefully controlled in order to avoid aberrant recombination with other chromosomes (translocations). However, translocations do occur between oncogenes and the IgH locus, and this can lead to B cell lymphomas. During class switching, activation-induced cytidine deaminase (AID) initiates the formation of DNA double strand breaks (DSBs) at switch (S) regions in the Ig heavy chain gene locus (IgH), which are necessary for class switching. My first Aim is to determine how deamination of dC's in Ig S regions by AID, forming dU's, results in DSBs. We have shown that AID-induced deamination of dC leads to DNA single-strand breaks (SSBs) via the base excision repair pathway. How these SSBs are then converted to DSBs is less clear. We have reported that another DNA repair pathway, mismatch repair (MMR) is important for this step, and we will investigate its role. We will investigate how SSBs are converted to DSBs by determining the frequency and sites of AID- induced dU's in S regions, how the frequency and positions of AID targets affects frequency of switching, and whether MMR proteins might be recruited to S regions by AID itself. In Aim 2 we will follow up on our finding during the current term of this grant that AID can instigate DSBs at sites other than the IgH locus in activated B cells. We will determine what makes these other sites targets for AID, and if these DSBs lead to chromosome breaks, deletions and translocations, and whether MMR and other DNA repair proteins known to be involved in CSR, for example, ATM, H2AX, and 53BP1 are involved in making or preventing these DSBs. PUBLIC HEALTH RELEVANCE: The majority of human mature B-cell lymphomas are characterized by reciprocal chromosomal translocations that often involve the immunoglobulin (Ig) genes. The longstanding assumption that these translocations are generated by erroneous antibody class switching, a normal process that is essential for an effective humoral immune response, was verified only recently. Antibody class switching is a process that requires the introduction of DNA breaks into the Ig genes, into special regions called switch regions, and then joining one of these DNA break ends with another DNA break end in the same Ig locus, on the same chromosome. The mechanism of induction of DNA breaks and also how this process is regulated to prevent joining the DNA breaks to another chromosome, which can result in activation of oncogenes and thus B cell lymphomas not understood. The experiments proposed here will determine how the activity of the enzyme AID, which initiates class switching and also chromosomal translocations, is regulated and how it chooses its targets. We have shown that a specific DNA repair pathway in cells, called mismatch repair, which is essential for repairing mistakes during DNA synthesis, is required for formation of most of the DNA breaks during class switching. We seek to further understand its role in converting AID-induced lesions to DNA breaks. Mismatch repair is an essential repair system for eliminating mutations and microsatellite instability, thereby protecting against cellular transformation and malignancy. The hypothesis that this pathway increases DNA breaks seems counterintuitive, but arises directly from our understanding of the role of this pathway during class switching. AID is required for chromosomal translocations between the c-myc oncogene and the Ig locus in mature B-cell plasmacytomas in mouse. Also, many human B-cell lymphomas that originate from germinal center B cells show chromosomal translocations involving the Ig switch regions, suggesting the involvement of class switch processes. IgH translocations often greatly increase oncogene expression due to juxtaposition to the strong IgH enhancers, leading to cellular transformation and malignancy. Whether AID targeting to other loci besides the Ig loci leads to formation of DNA breaks at these sites is, however, unknown. By the use of a genome- wide search method, we have found AID-dependent DNA breaks at several other sites in the genome. We will examine whether these other sites are involved in chromosomal translocations with the Ig locus. We will investigate what makes these other sites targets for AID, and for DNA break formation. Thus our studies will also shed light on the important question of how AID chooses its targets.