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.
| Sharov, Alexei A; Nishiyama, Akira; Piao, Yulan et al. (2011) Responsiveness of genes to manipulation of transcription factors in ES cells is associated with histone modifications and tissue specificity. BMC Genomics 12:102 |
| Sharov, Alexei A; Piao, Yulan; Ko, Minoru S H (2010) Gene expression profiling of mouse embryos with microarrays. Methods Enzymol 477:511-41 |
| Nishiyama, Akira; Xin, Li; Sharov, Alexei A et al. (2009) Uncovering early response of gene regulatory networks in ESCs by systematic induction of transcription factors. Cell Stem Cell 5:420-33 |
