AID Targeting Mechanisms for IgH Switch Recombination and Somatic Hypermutation

描述（由申请人提供）：我们拟研究IgH类开关重组（CSR）和Ig可变区（VDJ）外显子体细胞超突变（SHM），重点阐明激活诱导胞苷脱氨酶（AID） （CSR和SHM的启动物）到达特定靶点的机制。基于各种证据，我们假设特定的序列基序和独特的转录特征将AID靶向特定的基因组位点。基于支持的初步数据，我们建议用强大的新方法来检验这一假设。我们建议阐明CSR过程中DSB的产生和定向连接的机制。IgH switch (S)区域是艾滋病启动的dsb的靶标，它们连接在两个不同的S区域之间以实现CSR，由具有不寻常特征的重复序列组成。我们开发了一种强大的新型V (D) J乘客等位基因检测系统，该系统使我们能够在csr激活的B细胞和GC B细胞中插入V (D) J外显子时，S区域发生SHM和dsb，并首次对核心S区域的SHM模式进行深入分析。我们建议使用乘客等位基因系统对小鼠和人类以及其他物种的S区基序进行系统分析，以确定AID靶向基序，并阐明如何强制S区dsb的定向特异性连接以促进多产的、缺失的CSR。在生发中心（GC） B细胞SHM过程中，AID活性通过不同的机制靶向V (D) J外显子，而不是通过CSR激活的B细胞中的S区域。在V(D)J外显子中，互补决定区（cdr）是主要的AID靶点，尽管它们并不富含已知的AID热点基序。我们建议使用客运等位基因系统来解剖cdr中潜在的AID靶向元件，并验证在抗体亲和成熟过程中获得AID热点基序可以促进抗hiv广泛中和抗体可变区域的高水平SHMs、缺失和插入的假设。我们还将验证GC B细胞中VH(D)JH外显子的聚合转录靶向SHM的AID以及IgH 3'调控区的元件调节这种转录的假设。许多人B细胞淋巴瘤是GC起源的，并有疑似艾滋病引发的致癌易位。我们现在建议通过我们新开发的高通量易位测序方法来鉴定小鼠GC B细胞中aids依赖性DSB/易位脱靶，并通过另一种高通量方法Gro-Seq来表征它们的转录特征。我们还将在我们的乘客等位基因系统中测试先前定义和新发现的AID非ig基因靶标（“脱靶”）的活性，以阐明靶向基序和转录在使其成为AID靶标中的作用。最后，我们将扩展我们的乘客等位基因分析，以测试易位的人类B细胞癌基因，以测试它们是否确实是AID的优先靶标，如果是的话，阐明它们内部潜在的靶基序和转录的作用。

DESCRIPTION (provided by applicant): We propose to study IgH class switch recombination (CSR) and Ig variable region (VDJ) exon somatic hypermutation (SHM) with a focus on elucidating mechanisms that direct Activation Induced Cytidine Deaminase (AID), the initiator of CSR and SHM, to specific targets. Based on various lines of evidence, we hypothesize that particular sequence motifs and unique transcription features target AID to particular genomic sites. Based on supporting preliminary data, we propose to test this hypothesis with powerful new approaches. We propose to elucidate mechanisms of DSB generation and directional joining during CSR. IgH switch (S) regions are targets of AID-initiated DSBs that are joined between two different S regions to achieve CSR and are composed of repetitive sequences with unusual characteristics. We developed a powerful new V (D) J passenger allele assay system that allowed us to show that S regions undergo both SHMs and DSBs when inserted in place of a V (D) J exon in CSR-activated B cells and in GC B cells and to perform, for the first time, in depth analysis of SHM patterns of core S regions. We propose to use the passenger allele system to perform a systematic analysis of mouse and human S region motifs, as well as those from other species to identify AID targeting motifs and to elucidate how orientation-specific joining of S region DSBs is enforced to promote productive, deletional CSR. V (D) J exons are targeted by AID activity during SHM in germinal center (GC) B cells through different mechanisms than S regions in B cells activated for CSR. Within V(D)J exons, complementarity determining region (CDRs) are prime AID targets even though they are not enriched for known AID hotspot motifs. We propose to use the passenger allele system to dissect potential AID targeting elements in CDRs and test the hypothesis that high levels of SHMs, deletions and insertions in anti-HIV broadly neutralizing antibody variable regions are promoted by acquisition of AID hotspots motifs during the antibody affinity maturation process. We also will test the hypothesis that convergent transcription of VH(D)JH exons in GC B cells targets AID for SHM and that that elements of the IgH 3' regulatory region regulate such transcription. Many human B cell lymphomas are of GC origin and harbor suspected AID-initiated oncogenic translocations. We now propose to identify AID-dependent DSB/translocation off-targets in mouse GC B cells by our newly developed high throughput translocation sequencing method and to characterize their transcriptional signatures by Gro-Seq, another high throughput approach. We also will test activity of previously defined and newly identified AID non-Ig gene targets ("off-targets") in our passenger allele system to elucidate targeting motifs and roles of transcription in making them AID targets. Finally, we will extend our passenger allele analyses to test translocated human B cell oncogenes to test whether they are, indeed, preferential AID targets and, if so, to elucidate potential target motifs within them and roles for transcription.