The role of gene enhancer elements in colon cancer

项目(摘要/摘要！ 结直肠癌(CRC)每年与超过50,000名美国成年人的死亡有关。 在之前的获奖期，我们表明除了基因突变，疾病 进展伴随着基因增强子元件的表观遗传变化 基因时断时续。我们称这些变异为增强子基因座，或VEL。值得注意的是，结肠肿瘤 显示了一种重复激活的增强剂的共同模式 所有的病人样本。这些重复激活的增强子构成了CRC的签名。在……里面 在这个相互竞争的续订申请中，我们将调查三个非互斥的假设 以揭示在CRC中形成这些签名VEL的机制。特定目标1测试 假设VEL是规范CRC突变的直接结果 癌基因和肿瘤抑制因子。这一假设将通过H3K27ac芯片序列进行验证 人肠道类器官中每个已知结直肠癌的增强子表观基因组分析 驱动基因通过CRISPR/Cas9顺序突变，以重述腺瘤- Vogelgram预测的癌症序列。《特定目标2》验证了这一假设 转录因子驱动签名细胞的形成。这一假设将得到检验。 通过敲除和过度表达结合到签名膜上的转录因子 在结直肠癌细胞系和肠道器官模型中，随后分析结直肠癌的染色质 签名的维尔斯。特异靶3验证了增强子的体细胞indel突变假说 元素驱动着VEL的形成。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-产生插入突变，然后 增强子活性的功能分析。最后，我们提出了第四个目标，以评估 VEL是结直肠癌致瘤性所必需的。CRISPR-基于CAS9的战略将用于 破坏结直肠癌细胞系中的标志性Vel，然后量化它们在小鼠体内的生长 相对于未经编辑的对照细胞的异种移植。我们希望获得对以下方面的基本见解 表观遗传增强子失调是结直肠癌发生的根本原因，可能导致 为患者提供靶向治疗的基金会。 好了！

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. !