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M8 motif; SILAC; Quantitative proteomics
Abstract:
The motif TMTCGCGANR (M being C or A, R being A or G, and N any nucleotide) called M8 was discovered as a putative cis-regulatory element present in 368 human gene promoters. Of these, 236 (64%) are conserved within promoter sequences of four related organisms: human, mouse, rat and dog. However, transcription factors (TFs) interacting with the M8 motif has not yet been described. We previously reported the use of quantitative proteomics coupled to one-step DNA affinity purification as a means of screening for TFs associated with given functional DNA elements. The procedure is performed in-vitro employing SILAC-labeled nuclear extracts and making use of a well-characterized cis-regulatory motifs. Building on that, in this study we have combined our method with statistical analysis to filter out false positive hits from the one-step DNA affinity pull-down experiments. This resulted in the identification of zinc finger BED domain-containing protein 1 (ZBED1), alpha globin transcription factor CP2 (TFCP2), upstream binding protein 1 (UBP) and transcription factor CP2 like 1(TFCP2L1), as specific M8 interacting factors. We validated our screen demonstrating the in vivo binding of alpha globin transcription factor TFCP2 to selected genes harboring M8-containing promoters using ChIP (chromatin immuno-precipitation) assays. This not only implicates a functional role of the above proteins in regulating M8 motif containing genes, but also suggests the potential use of our approach to decipher protein-DNA interactions occurring in living cells.
