A Novel Mechanism for LEF1 Promoter Regulation in Colon Cancer

结肠癌中 LEF1 启动子调控的新机制

• 批准号: 8718884
• 负责人: Andria Denmon
• 金额: $ 5.15万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2016-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8718884
• 关键词: APC gene; Affect; Affinity Chromatography; African American; American; American Cancer Society; Binding; Binding Sites; Biochemical; Biological Assay; Cancer Etiology; Cell Nucleus; Cessation of life; Colon Carcinoma; Colorectal Cancer; Complex; Custom; DNA; DNA Binding; DNA Sequencing Facility; Diagnosis; Disease; Dominant-Negative Mutation; Down-Regulation; Elements; Ethnic Origin; Family; Functional RNA; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Immunoprecipitation; Incidence; Individual; Intestines; Introns; Knowledge; Lead; Length; Link; Malignant Neoplasms; Mass Spectrum Analysis; Messenger RNA; Methods; Mutation; Nucleotides; Play; Process; Protein Isoforms; Proteins; RNA; RNA Binding; RNA Polymerase II; Recruitment Activity; Regulation; Repression; Response Elements; Role; Signal Transduction; Site; TCF Transcription Factor; TCF7L2 gene; Technology; Testing; Transcript; Transcription Repressor/Corepressor; Transfection; United States; Work; X Inactivation; YY1 Transcription Factor; Yin-Yang; cancer initiation; carcinogenesis; cell transformation; design; gene repression; improved; in vitro Assay; lymphoid enhancer-binding factor 1; member; mortality; novel; novel therapeutic intervention; promoter; public health relevance; research study; therapeutic target; transcription factor; tumor progression

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.

描述（由申请人提供）：在美国，结肠癌是癌症相关死亡的第三个主要原因，非裔美国人的发病率和死亡率比任何其他种族都高。今年，美国癌症协会估计，将被诊断出102,480名美国人患有结肠癌，其中50,830人将屈服于该疾病。迄今为止，大肠癌是与过度活跃，异常的WNT信号相关的最臭名昭著的癌症。大多数病例来自腺瘤性息肉大肠杆菌基因中的灭活突变，该基因编码B-catenin销毁复合物的关键成分。稳定的，细胞质，B-蛋白链蛋白的增加导致该调节剂的涌入核与淋巴样增强剂因子/T细胞因子（LEF/TCF）一起工作，以激活Wnt靶基因的转录，例如CMYC和LEF1。 LEF1基因座包含两个启动子。启动子1（P1）是WNT靶标的，它会产生全长淋巴增强子1（FL-LEF-1），一种与B-catenin：启动子2（P2）一起使用的同工型，可产生主导的LEF-1（DNLEF-1），这是一种缺乏B-catenin结合域的同种型，因此可以降低B-catenin结合域，因此可以下降。重要的是，仅在结肠癌中表达了靶向WNT的Fl-Lef-1同工型。 DNLEF-1产生的P2启动子被涉及上游阻遏元件和多功能蛋白质Yin Yang 1（yy1）的未知机制积极沉默。由于DNLEF-1的重新引入可以通过与Wnt靶基因结合以下调来反对Wnt信号传导，因此阐明其转录抑制的机制很重要，并确定如何在癌症中重新表达该调节剂。提出了以下目的和方法：1）确定YY1是否使用RNA依赖机制沉默P2。提出了RNA免疫沉淀测定和体外生化方法。 2）定义上游阻遏物元件的核苷酸上下文和相关蛋白质。用于抑制的瞬态转染测定法将用于定义元素内的核心序列。 DNA亲和色谱和质谱法将鉴定与这些关键序列相互作用的蛋白质3）检验以下假设：来自上游P1的转录会影响下游P2的转录活性。 P1转录将使用自定义设计的故事压迫器调制。定义P2抑制的机制将导致对多启动基因基因座的表达更好地理解，从而突出癌症和其他疾病的广泛影响和新的治疗方法的潜力。