The specificity of STAT1 transcription factor was determined quantitatively by measuring the relative binding affinity to hundreds of variants of the gonsensus site. The binding specities of eight mutants of STAT1 were also investigated. The mutants showed either overall reduced binding specificity or altered specificity accross the binding site. The effect of CpG methylaton within the binding site was also investigated, which demonstrated verry little effect on binding. A previously described method, Spec-Seq, was used to determine the binding specificititys of STAT1 and the mutants. Libreries of DNAs were enzymatically methylated and the same method was used for studying their binding affinities in comparison with the non-methylated counterparts. The proteins and DNAs were incubated for 30 min in in suitable buffer and the bound and unbound bands were resolved in 12% polyacrylamide gels. the DNAs were purified from the bound and unbound bands and sequenced.
Overall design
18 independent Spec-Seq experiments were designed for each of the STAT1 and its mutants. four other Spec-Seq experiments were performed for the comperative analysis of the effect of CpG methylation across the binding site. One of these libreries were enzymatically methylated using CpG-methyltransferase (NEB).
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