Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697, USA.
Nucleic Acids Res. 2010 Oct;38(19):6375-88. doi: 10.1093/nar/gkq492. Epub 2010 Jun 4.
Lymphoid enhancer factor 1 (LEF-1) mediates Wnt signaling via recruitment of β-catenin to target genes. The LEF1 gene is aberrantly transcribed in colon cancers because promoter 1 (P1) is a Wnt target gene and is activated by TCF-β-catenin complexes. A second promoter in intron 2 (P2) produces dominant negative LEF-1 isoforms (dnLEF-1), but P2 is silent because it is repressed by an upstream distal repressor element. In this study we identify Yin Yang 1 (YY1) transcription factor as the P2-specific factor necessary for repression. Site-directed mutagenesis and EMSA were used to identify a YY1-binding site at +25 in P2, and chromatin immunoprecipitation assays detected YY1 binding to endogenous LEF1 P2. Mutation of this site relieves P2 repression in transient transfections, and knockdown of endogenous YY1 relieves repression of integrated P2 reporter constructs and decreases the H3K9me3 epigenetic marks. YY1 is responsible for repressor specificity because introduction of a single YY1-binding site into the P1 promoter makes it sensitive to the distal repressor. We also show that induced expression of dnLEF-1 in colon cancer cells slows their rate of proliferation. We propose that YY1 plays an important role in preventing dnLEF-1 expression and growth inhibition in colon cancer.
淋巴增强因子 1（LEF-1）通过募集β-连环蛋白到靶基因来介导 Wnt 信号通路。LEF1 基因在结肠癌中异常转录，因为启动子 1（P1）是 Wnt 的靶基因，并被 TCF-β-连环蛋白复合物激活。第二个位于内含子 2 中的启动子（P2）产生显性负 LEF-1 异构体（dnLEF-1），但 P2 是沉默的，因为它被上游远端抑制元件抑制。在这项研究中，我们确定 Yin Yang 1（YY1）转录因子是 P2 特异性必需的抑制因子。通过定点突变和 EMSA 鉴定了 P2 中+25 处的 YY1 结合位点，染色质免疫沉淀检测到 YY1 与内源性 LEF1 P2 的结合。该位点的突变可在瞬时转染中解除 P2 的抑制，内源性 YY1 的敲低可解除整合的 P2 报告基因构建体的抑制，并减少 H3K9me3 表观遗传标记。YY1 负责抑制因子的特异性，因为将单个 YY1 结合位点引入 P1 启动子时，它对远端抑制因子变得敏感。我们还表明，诱导结肠癌细胞中 dnLEF-1 的表达会降低其增殖速度。我们提出，YY1 在防止 dnLEF-1 表达和抑制结肠癌生长中发挥重要作用。
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