Assays for interaction of transcription factor with nucleosome

This chapter describes various methods for the analysis of binding of transcription factors to nucleosomal DNA-binding sites; these methods are essentially the same as those used routinely to study the protein–DNA interaction in the absence of histones. Special considerations for using nucleosomal DNA as a template for factor binding include (1) ascertaining whether the reconstituted nucleosome is homogeneous in terms of translational and rotational positioning, (2) the ways in which the histone–DNA contacts and nucleosome structure are affected by the factor binding, and (3) the extent to which the detection of the ternary complex is disturbed by the nucleosomal or factor-free DNA complex. The electrophoretic mobility shift assay is a sensitive and fast method and usually is the first method of choice for any protein–DNA-binding study. Potential shortcomings are the aggregation and instability of a bound complex during electrophoresis; sometimes the instability of a nucleosome may seriously hamper the evaluation of results. Dimethyl sulfate (DMS) methylation protection analysis is a powerful method for studying the binding of a transcription factor to its nucleosome template; this is especially useful when many nonspecific interactions occur or when histone–DNA interactions make the evaluation of a specific protein–nucleosome interaction by DNase I footprinting difficult.