A Novel Mechanism for LEF1 Promoter Regulation in Colon Cancer

描述(申请人提供)：在美国，结肠癌是癌症相关死亡的第三大原因，非洲裔美国人的发病率和死亡率高于任何其他种族。今年，美国癌症协会估计，将有102,480名美国人被诊断出患有结肠癌，其中50,830人将死于这种疾病。到目前为止，结直肠癌是最臭名昭著的癌症，与过度活跃、异常的Wnt信号有关。大多数病例源于腺瘤性息肉病结肠基因的失活突变，该基因编码b-连环蛋白破坏复合体的关键成分。稳定的细胞质b-catenin的增加导致这种调节因子流入细胞核，在那里它与淋巴增强因子/T细胞因子(Lef/TCFs)一起激活Wnt靶基因的转录，如cMYC和LEF1。LEF1基因包含两个启动子。启动子1(P1)是Wnt的靶标，它产生全长淋巴增强因子-1(FL-Lef-1)，这是一种与b-连环蛋白起作用的异构体：启动子2(P2)产生主导的负LEF-1(dnLEF-1)，这是一种缺乏b-连环蛋白结合域的异构体，因此可以下调Wnt靶基因。重要的是，只有Wnt靶向的FL-Lef-1亚型在结肠癌中表达。产生dnLEF-1的P2启动子被一种未知的机制主动沉默，该机制涉及上游阻遏元件和多功能蛋白阴阳1(YY1)。由于重新引入dnLEF-1可以通过与Wnt靶基因结合来下调Wnt信号，因此阐明其转录抑制的机制并确定该调控因子如何在癌症中重新表达具有重要意义。提出了以下目标和方法：1)确定YY1是否使用RNA依赖机制来沉默P2。提出了RNA免疫沉淀法和体外生化方法。2)确定上游阻遏元件的核苷酸背景和相关蛋白质。用于抑制的瞬时转染法将用于确定该元件内的核心序列。DNA亲和层析和质谱学将鉴定与这些关键序列相互作用的蛋白质3)检验上游P1的转录影响下游P2转录活性的假设。P1转录将使用定制设计的TALE抑制因子进行调节。明确P2抑制的机制将有助于更好地理解多启动子基因座的表达，从而突出潜在的广泛影响和新的治疗方法在癌症和其他疾病。

DESCRIPTION (provided by applicant): In the United States, colon cancer is the third leading cause of cancer-related deaths with African-Americans having a higher incidence and mortality rate than any other ethnicity. This year, the American Cancer Society estimates that 102,480 Americans will be diagnosed with colon cancer and of those cases, 50,830 individuals will succumb to the disease. To date, colorectal cancer is the most notorious cancer linked to overactive, aberrant Wnt signaling. Most cases derive from inactivating mutations in the adenomatous polyposis coli gene, which encodes a key component of the b-catenin Destruction Complex. The increase in stable, cytoplasmic, b- catenin results in an influx of this regulator int the nucleus where it works with Lymphoid Enhancer Factor/T Cell Factors (LEF/TCFs) to activate transcription of Wnt target genes, such as CMYC and LEF1. The LEF1 locus contains two promoters. Promoter 1 (P1), which is a Wnt target, produces full-length Lymphoid Enhancer Factor-1 (FL-LEF-1), an isoform that works with b-catenin: promoter 2 (P2) produces dominate negative LEF-1 (dnLEF-1), an isoform that lacks the b-catenin binding domain, and can therefore down-regulate Wnt target genes. Importantly, only the Wnt-targeted FL-LEF-1 isoform is expressed in colon cancer. The dnLEF-1 producing P2 promoter is actively silenced by an unknown mechanism involving an upstream repressor element and the multifunctional protein, Yin Yang 1 (YY1). Since re-introduction of dnLEF-1 can oppose Wnt signaling by binding to Wnt target genes for downregulation, it is important to elucidate the mechanism of its transcriptional repression and determine how this regulator can be re-expressed in cancer. The following aims and methods are proposed: 1) Determine whether YY1 uses an RNA-dependent mechanism to silence P2. RNA immunoprecipitation assays and in vitro biochemical approaches are proposed. 2) Define the nucleotide context and associated proteins of the upstream repressor element. A transient transfection assay for repression will be used to define the core sequences within the element. DNA affinity chromatography and mass spectrometry will identify proteins interacting with these key sequences 3) Test the hypothesis that transcription from upstream P1 affects transcriptional activity of downstream P2. P1 transcription will be modulated using a custom designed TALE-repressor. Defining the mechanism of P2 repression will lead to a better understanding of expression from multi-promoter gene loci, and thus highlight the potential for broad impact and new therapeutic approaches in cancer and other diseases.