3 - Measurement of Steroid Receptor Binding to DNA

Abstract

Steroid receptors form a large super family of ligand-dependent DNA binding proteins that function in the modulation of gene transcription. Such control of gene expression appears to be the primary mechanism by which steroid receptors play a role in cell growth, development, and differentiation in eukaryotic organisms. Receptor modulation of transcription occurs through binding to specific DNA elements, called steroid response elements; such binding leads to either the activation or repression of RNA synthesis at nearby transcriptional start-sites. Over the last decade, there has been a virtual explosion of information on the structure/function relationships for a variety of steroid by cleaving the DNA with piperidine and running the resulting fragments on a sequencing gel. There should be a series of strong bands corresponding to cleavage at methylated guanine residue; weak bands corresponding to cleavage at methylated adenine residues may also be present. An over methylated probe shows skewing of bands toward the bottom of the gel with little or no intact probe near the top; a shorter incubation time with dimethyl sulfate (DMS) should be used. An under methylated probe remains uncleaved; in such cases, longer methylation times are necessary. The concentration of DNA binding proteins of interest can be a limiting factor in methylation interference, since more receptor/DNA complex is required than for other procedures. This is because the complexed DNA must be recovered from one gel, processed, and run on a second gel. The use of purified proteins is advisable, where possible.