Purification of transcription cofactor complex CRSP

Transcription of protein coding genes in metazoans involves the concerted a ction of enhancer binding proteins and the RNA polymerase II apparatus. The cross talk between these two classes of transcription factors is mediated by an elaborate set of cofactor complexes, For the activation of transcript ion by the promoter specificity protein 1 (Spl), TATA binding protein-assoc iated factors in the TFIID complex originally were identified as necessary coactivators, but the identity of additional cofactors required for activat ed transcription was unknown. Recently, we have reported the isolation and properties of a cofactor complex, CRSP (cofactor required for Sp1), which f unctions in conjunction with the TATA binding protein-associated factors to promote efficient activation of transcription by Sp1, CRSP contains unique subunits as well as polypeptides that are shared with other cofactor compl exes, Here, we report a detailed purification protocol for the isolation of CRSP from human HeLa cells, Our purification strategy takes advantage of t he ability of CRSP to bind Ni2+-nitrilotriacetic acid-agarose resin as well as other conventional chromatographic resins, We also describe a streamlin ed purification protocol that allows a more rapid and efficient means to is olate active CRSP.