The role of gene enhancer elements in colon cancer

项目（摘要/摘要！ 结直肠癌 (CRC) 每年导致超过 50,000 名美国成年人死亡。 在上一期颁奖期间，我们展示了除了基因突变之外，疾病 进展伴随着基因增强子元件的表观遗传变化，这些变化 基因的开启和关闭。我们将这些变异增强子基因座（VariantEnhancer Loci，VEL）称为变体增强子基因座。值得注意的是，结肠肿瘤 来自不同个体的增强子显示出反复激活的共同模式 跨患者样本。这些反复激活的增强子构成了 CRC 的特征。在 在这个竞争性续订申请中，我们将研究三个非互斥的假设 揭示这些签名 VEL 在 CRC 中形成的机制。具体目标 1 测试 VEL 是典型 CRC 突变的直接结果的假设 癌基因和肿瘤抑制基因。该假设将通过 H3K27ac ChIP-seq 进行检验 对人类肠道类器官中的增强子表观基因组进行分析，其中每个已知的 CRC 驱动基因通过 CRISPR/Cas9 连续突变以重现腺瘤- Vogelgram 预测的癌序列。具体目标 2 检验以下假设： 转录因子驱动特征 VEL 的形成。这个假设将被检验 通过与特征 VEL 结合的转录因子的敲低和过度表达 在 CRC 细胞系和肠道类器官模型中进行研究，然后对 CRC 的染色质进行分析 签名 VEL。具体目标 3 检验增强子中体细胞插入缺失突变的假设 元素驱动 VEL 形成。 CRISPR-Cas9基因组编辑策略将用于纠正 或者在 CRC 细胞系中引入候选增强子创建插入缺失突变，然后 增强子活性的功能分析。最后，我们提出第四个目标来评估是否 VEL 是 CRC 致瘤性所必需的。基于 CRISPR-Cas9 的策略将用于 破坏 CRC 细胞系中的特征 VEL，然后量化其在小鼠体内的生长 异种移植物相对于未经编辑的对照细胞。我们期望获得基本见解 表观遗传增强子失调是结直肠癌肿瘤发生的根本原因，可能奠定 为患者的靶向治疗奠定了基础。 ！

PROJECT(SUMMARY/ABSTRACT! Colorectal cancer (CRC) is associated with the deaths of over 50,000 adult Americans annually. In the previous award period, we showed that in addition to genetic mutation, disease progression is accompanied by epigenetic changes at gene enhancer elements that switch genes on and off. We term these Variant Enhancer Loci, or VELs. Remarkably, colon tumors from different individuals show a common pattern of enhancers that are recurrently activated across patient samples. These recurrently activated enhancers constitute a signature of CRC. In this competing renewal application, we will investigate three non-mutually exclusive hypotheses to uncover the mechanism by which these signature VELs form in CRC. Specific Aim 1 tests the hypothesis that the VELs are a direct consequence of mutations in canonical CRC oncogenes and tumor suppressors. This hypothesis will be tested through H3K27ac ChIP-seq analysis of the enhancer epigenome in human intestinal organoids in which each of known CRC driver genes were sequentially mutated via CRISPR/Cas9 to recapitulate the adenoma- carcinoma sequence predicted by the Vogelgram. Specific Aim 2 tests the hypothesis that transcription factors drive formation of the signature VELs. This hypothesis will be tested through knockdown and overexpression of transcription factors that bind to the signature VELs in CRC cell lines and intestinal organoid models, followed by analysis of chromatin at CRC signature VELs. Specific Aim 3 tests the hypothesis that somatic indel mutations in enhancer elements drive VEL formation. CRISPR-Cas9 genome editing strategies will be used to correct or introduce candidate enhancer-creating indel mutations in CRC cell lines, followed by functional analysis of enhancer activity. Lastly, we propose a fourth Aim to assess whether VELs are required for tumorigenicity of CRC. CRISPR-Cas9-based strategies will be used to disrupt signature VELs in CRC cell lines, followed by quantification of their growth in mouse xenografts relative to unedited control cells. We expect to gain fundamental insights to epigenetic enhancer dysregulation as a root cause of CRC tumorigenesis that could lay the foundation for targeted therapies for patients. !