Mechanisms of Transcriptional Regulation by MTG16 in the Intestine

项目摘要 炎症性肠病（IBD），包括溃疡性结肠炎和克罗恩病，影响着150万人， 美国的IBD患者中结肠炎相关癌（CAC）或结直肠癌的发病率是 几乎是普通人群的六倍。因此，IBD的发病率和死亡率相当高， 高了解IBD和CAC发病机制背后的分子机制可能会改善 筛查和治疗。像其他由慢性炎症驱动的癌症一样，CAC的特征是遗传性的。 肠细胞中的突变和异常基因表达程序。富含亮氨酸重复序列的G蛋白- 偶联受体5（LGR 5）是增加肠干细胞中WNT信号传导的膜受体， 赋予自我更新和增殖。此外，LGR 5+干细胞被认为是肿瘤的起源细胞。 肠恶性肿瘤;因此，Lgr 5的转录受到严格调控。然而，转录的机制 LGR 5的调节在很大程度上是未知的。 16号染色体上的髓样易位基因（MTG 16）是最初发现的转录共阻遏物， 作为治疗相关急性髓细胞白血病的易位融合伴侣。Mtg 16-/-小鼠表现出异常， 基线肠道表型和增加的结肠炎和CAC的化学模型的易感性。初步 使用荧光素酶报告基因分析和染色质免疫沉淀（ChIP）的数据表明，MTG 16抑制 通过占据Lgr 5的内含子1表达Lgr 5。使用酵母双杂交筛选和蛋白质复合物 通过免疫沉淀（Co-IP），我们鉴定了延伸因子MLL 1、AFF 4和DOT 1 L作为潜在的MTG 16 互动伙伴MTG 16中某些Nervy同源区（NHR）的突变或缺失被消除 酵母双杂交生长试验中的相互作用和葡聚糖硫酸钠诱导的结肠炎的严重程度增加 in vivo.综上所述，我们假设MTG 16中的特定NHR与转录和延伸因子结合， 调节Lgr 5的表达，影响肠上皮的稳态、损伤和肿瘤发生。 这一假设将通过两个重点，机制和假设驱动的目标进行测试。具体目标1： 肠中Lgr 5的内源性MTG 16阻遏复合物的组分将使用Co- IP和连续ChIP在年轻成年小鼠结肠（YAMC）细胞中。突变型MTG 16的慢病毒过表达 在先前的CRISPR产生的MTG 16缺陷YAMC系中，将用于确定NHR依赖性， 复合物的形成和Lgr 5的抑制。在特定目标2中，新的MTG 16 NHR突变小鼠将 用于检测NHR对肠上皮稳态、损伤和肿瘤发生的依赖性。重要的是， 阐明MTG 16在肠上皮生物学中的作用将进一步加深我们对干细胞生物学的理解， 可能阐明靶向促增殖和致瘤途径的新机制。

PROJECT SUMMARY Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn’s disease, affects 1.5 million people in the United States. The incidence of colitis-associated cancer (CAC), or colorectal cancer in patients with IBD, is nearly six-fold higher than that of the general population. Therefore, the morbidity and mortality of IBD is quite high. Understanding the molecular mechanisms behind IBD and CAC pathogenesis may lead to improved screening and therapeutics. Like other cancers driven by chronic inflammation, CAC is characterized by genetic mutations and aberrant gene expression programs in intestinal cells. Leucine-rich repeat-containing G protein- coupled receptor 5 (LGR5) is a membrane receptor that increases WNT signaling in intestinal stem cells, conferring self-renewal and proliferation. Furthermore, LGR5+ stem cells are thought to be the cells-of-origin of intestinal malignancy; thus, transcription of Lgr5 is tightly regulated. However, mechanisms of transcriptional regulation of Lgr5 are largely unknown. Myeloid translocation gene on chromosome 16 (MTG16) is a transcriptional co-repressor originally discovered as a translocation fusion partner in therapy-related acute myeloid leukemia. Mtg16-/- mice exhibit aberrancies in baseline intestinal phenotypes and increased susceptibility to chemical models of colitis and CAC. Preliminary data using a luciferase reporter assay and chromatin immunoprecipitation (ChIP) indicate that MTG16 represses Lgr5 expression through occupancy of intron 1 of Lgr5. Using a yeast two-hybrid screen and protein complex immunoprecipitation (Co-IP), we identified the elongation factors MLL1, AFF4, and DOT1L as potential MTG16 interacting partners. Mutation or deletion of certain Nervy homology regions (NHRs) in MTG16 abolished interaction in yeast two-hybrid growth assays and increased the severity of dextran sodium sulfate-induced colitis in vivo. Taken together, we hypothesize that specific NHRs in MTG16 bind to transcription and elongation factors to regulate Lgr5 expression, impacting homeostasis, injury, and tumorigenesis in the intestinal epithelium. This hypothesis will be tested via two focused, mechanistic, and hypothesis-driven Aims. In Specific Aim 1, the components of the endogenous MTG16 repression complex of Lgr5 in the intestine will be elucidated using Co- IP and sequential ChIP in in young adult mouse colon (YAMC) cells. Lentiviral overexpression of mutant MTG16 in a previously CRISPR-generated MTG16-deficient YAMC line will be used to determine NHR dependency of complex formation and Lgr5 repression in intestinal cells. In Specific Aim 2, novel MTG16 NHR mutant mice will be used to test for NHR dependency on intestinal epithelial homeostasis, injury, and tumorigenesis. Importantly, clarifying the role of MTG16 in intestinal epithelial biology will further our understanding of stem cell biology and may elucidate novel mechanisms for targeting pro-proliferative and tumorigenic pathways.