AID Targeting Mechanisms for IgH Switch Recombination and Somatic Hypermutation

描述（由申请人提供）：我们计划研究IgH类别转换重组（CSR）和IG可变区（VDJ）外显子体细胞超突变（SHM），重点是阐明将激活诱导的胞苷脱氨酶（AID）（CSR和SHM的起始物）导向特定靶点的机制。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。我们建议阐明的机制，DSB的产生和定向加入CSR过程中。IgH开关（S）区是AIDS启动的DSB的靶标，其在两个不同的S区之间连接以实现CSR，并且由具有不寻常特征的重复序列组成。我们开发了一个强大的新的V（D）J乘客等位基因检测系统，使我们能够表明，S区进行SHM和DSB时，插入在CSR激活的B细胞和GC B细胞中的V（D）J外显子的位置，并进行，第一次，在核心S区的SHM模式的深入分析。我们建议使用乘客等位基因系统进行系统的分析，小鼠和人类的S区图案，以及那些从其他物种，以确定艾滋病靶向图案，并阐明如何定向特异性连接的S区DSBs是强制执行，以促进生产，删除CSR。在生发中心（GC）B细胞的SHM期间，V（D）J外显子通过与激活CSR的B细胞中的S区不同的机制被AID活性靶向。在V（D）J外显子内，互补决定区（CDR）是主要的AID靶标，即使它们没有富集已知的AID热点基序。我们建议使用乘客等位基因系统来剖析CDR中的潜在AID靶向元件，并测试以下假设：在抗体亲和力成熟过程中，通过获得AID热点基序来促进抗HIV广泛中和抗体可变区中的高水平SHM、缺失和插入。我们还将检验GC B细胞中VH（D）JH外显子的会聚转录靶向SHM的AID以及IgH 3'调节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，并含有可疑的艾滋病启动的致癌易位。我们现在建议通过我们新开发的高通量易位测序方法鉴定小鼠GC B细胞中的AIDS依赖性DSB/易位脱靶，并通过另一种高通量方法Gro-Seq表征其转录特征。我们还将在我们的乘客等位基因系统中测试先前定义的和新鉴定的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优先的AID靶点，如果是的话，阐明它们内部的潜在靶基序和转录作用。

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.