Systematic identification of transcription factor binding sites

转录因子结合位点的系统鉴定

• 批准号: 7964016
• 负责人: Minoru Ko
• 金额: $ 20.31万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7964016
• 关键词: Aging; Antibodies; Binding; Binding Sites; CDX2 gene; Cells; Chromatin; Classification; Complex; Consensus; Consensus Sequence; DNA; DNA Sequence; ES Cell Line; Embryo; Gene Targeting; Genes; Genomics; Goals; Hour; Laboratories; Maintenance; Methods; Mus; Open Reading Frames; Organ; Play; Procedures; Replacement Therapy; Reporting; Role; Sampling; Stem cells; Testing; Tetracyclines; Undifferentiated; Work; cell bank; embryonic stem cell; genome-wide; overexpression; pluripotency; promoter; scale up; self-renewal; transcription factor

We have optimized ChIP conditions using FLAG-antibody and have also set up the analysis pipeline for ChIP-DNA sequencing procedure. We have used the ES cell line, where the Cdx2 transcription factor can be overexpressed in the absence of tetracycline for 48 hours. We have then isolated CDX2-chromatin complexes using FLAG-antibody and sequenced these DNA fragments. Compared to control samples, we have identified several thousand mouse genomic loci where these sequence tags are clustered. The consensus CDX2-binding sequence motifs deduced from these binding sites matched previously reported consensus sequences. The results indicate that our method works successfully. Interestingly, ChIP-Seq revealed that CDX2 binds to promoters of up-regulated target genes. By contrast, genes down-regulated by CDX2 did not show CDX2 binding, but were enriched with binding sites for POU5F1, SOX2, and NANOG. Because the genome-wide binding sites can be analyzed for any TF tagged with Flag, our method can be scaled up to a large number of TFs. We are currently testing the potential to scale up the procedure for the analysis of 60 TFs.

我们使用FLAG抗体优化了ChIP条件，并建立了ChIP-DNA测序程序的分析管道。我们已经使用了ES细胞系，其中Cdx 2转录因子可以在四环素不存在下过表达48小时。然后，我们使用FLAG抗体分离了CDX 2-染色质复合物，并对这些DNA片段进行了测序。与对照样品相比，我们已经确定了几千个小鼠基因组位点，这些序列标签聚集在一起。从这些结合位点推导出的CDX 2结合序列基序与先前报道的一致序列相匹配。结果表明，我们的方法是成功的。有趣的是，ChIP-Seq揭示了CDX 2与上调靶基因的启动子结合。相比之下，被CDX 2下调的基因不显示CDX 2结合，但富含POU 5 F1、SOX 2和NANOG的结合位点。 由于可以分析任何标记有Flag的TF的全基因组结合位点，因此我们的方法可以扩大到大量TF。我们目前正在测试扩大60个TF分析程序的可能性。