AID Targeting Mechanisms for IgH Switch Recombination and Somatic Hypermutation

描述(申请人提供)：我们建议研究IgH类开关重组(CSR)和免疫球蛋白可变区(VDJ)外显子体细胞超突变(SHM)，重点在于阐明将CSR和SHM的启动子--激活诱导的胞苷脱氨酶(AID)定向到特定靶点的机制。根据各种证据，我们假设特定的序列基序和独特的转录特征针对特定的基因组位置。在支持初步数据的基础上，我们建议用强有力的新方法来检验这一假说。我们建议阐明CSR过程中DSB的产生和定向连接的机制。IgH开关(S)区是艾滋病引发的双链断裂的靶点，连接在两个不同的S区域之间以实现CSR，并且由具有不同特征的重复序列组成。我们开发了一个功能强大的新的V(D)J乘客等位基因分析系统，使我们能够证明当插入到CSR激活的B细胞和GC B细胞中的V(D)J外显子时，S区域同时经历了SHMS和DSB，并首次对S核心区域的SHM模式进行了深入分析。我们建议利用乘客等位基因系统对小鼠和人类S区域的基序以及其他物种的基序进行系统的分析，以识别艾滋病靶向基序，并阐明S区域DSB如何实施定向特异性连接以促进生产性的、缺失性的CSR。在生发中心(GC)B细胞中，V(D)J外显子通过不同于被CSR激活的B细胞中的S区域的机制，在SHM过程中被AID活性靶向。在V(D)J外显子中，互补决定区(CDR)是主要的AID靶点，尽管它们不富含已知的AID热点基序。我们建议使用乘客等位基因系统来剖析CDR中潜在的AID靶向元件，并检验这样一种假设，即在抗体亲和力成熟过程中，获得AID热点基序会促进高水平的SHM、抗HIV广中和抗体可变区的缺失和插入。我们还将检验这样的假设，即GC B细胞中VH(D)JH外显子的融合转录针对SHM的AID，以及IgH 3‘调节区的元件调节这种转录。许多人类B细胞淋巴瘤起源于GC，并存在疑似艾滋病引发的致癌易位。我们现在建议用我们新开发的高通量易位测序方法来鉴定小鼠GC B细胞中依赖AID的DSB/易位非靶标，并用另一种高通量方法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的和新发现的AID非免疫球蛋白基因靶标的活性，以阐明靶向基序和转录在使其成为AID靶标中的作用。最后，我们将把我们的乘客等位基因分析扩展到测试易位的人类B细胞癌基因，以测试它们是否确实是优先的AID靶标，如果是，则阐明它们内部的潜在靶标基序和转录作用。

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.