Lin28A and Lin28B are homologous RNA-binding proteins that participate in the development of primordial germ cells. The mechanisms underlying expression and regulation of Lin28A have been well documented, but such information for Lin28B is limited. In this study, a fragment of the Lin28B promoter was cloned, the pEGFP-pLin28B vector was constructed. DF-1 chicken fibroblasts were transfected and the expression of green fluorescent protein (GFP) was measured. Furtherly, Lin28B promoter of different lengths fragments was cloned using the chromosome-walking method and the fragments were ligated into the PGL3-Basic vector, and transfected into DF-1 cells. Results of dual-luciferase reporter assay showed that the core of the Lin28B promoter was included in the sequence from –1 431 to –1 034 bp. The binding sites of the transcription factor TCF7L2 was showed within this sequence by bioinformatics analysis. The promoter activity of Lin28B was downregulated (P<0.05) when the TCF7L2 binding site was mutated. Further experiments suggested that Lin28B promoter activity responded to the activation or inhibition of Wnt signaling. Results of chromatin immunoprecipitation and quantitative PCR showed that β-catenin-TCF7L2 may be enriched in the Lin28B promoter core area. In vivo and in vitro activation or inhibition of Wnt signaling significantly up- or down-regulated (P<0.05) Lin28B expression. H3K4me2 enriched in the promoter of Lin28B, which affected the regulation of Wnt signaling to Lin28B. In conclusion, our results showed that H3K4me2 and Wnt5a/β-catenin/TCF7L2 were the positive regulators of Lin28B expression. Findings of this study may lay a theoretical foundation for illuminating the mechanism underlying Lin28B expression.

Lin28A和Lin28B是参与原始生殖细胞发育的同源RNA结合蛋白。Lin28A的表达和调控的基本机制已得到充分证明，但是Lin28B的此类信息有限。在这项研究中，克隆了Lin28B启动子的片段，构建了pEGFP-p Lin28B载体。转染DF-1鸡成纤维细胞，并测量绿色荧光蛋白（GFP）的表达。此外，Lin28B使用染色体行走方法克隆了不同长度的启动子，并将这些片段连接到PGL3-Basic载体中，并转染到DF-1细胞中。双荧光素酶报告基因分析的结果表明，Lin28B启动子的核心包含在–1 431至–1 034 bp的序列中。通过生物信息学分析在该序列内显示了转录因子TCF7L2的结合位点。当TCF7L2结合位点发生突变时，Lin28B的启动子活性被下调（P <0.05）。进一步的实验表明，Lin28B启动子活性响应Wnt信号的激活或抑制。染色质免疫沉淀和定量PCR结果表明，β-catenin-TCF7L2可能在Lin28B启动子核心区域富集。体内和体外对Wnt信号的激活或抑制显着上调或下调（P <0.05）Lin28B表达。H3K4me2富含Lin28B的启动子，这影响了Wnt信号向Lin28B的调控。总之，我们的结果表明，H3K4me2和Wnt5a /β-catenin/ TCF7L2是Lin28B的正调控因子表达。这项研究的发现可能为阐明Lin28B表达基础的机制奠定理论基础。