Histone affinity chromatography as a tool for fractionating nonhistone chromatin proteins and studying histone-nonhistone protein interactions.

The interaction between histone and nonhistone chromatin proteins has been investigated by histone affinity chromatography, a technique that avoids precipitation of histone l nonhistone complexes. When nonhistone proteins labeled with 32P are loaded directly on histone-Sepharose columns, no binding of radioactivity to the column is observed. However, if labeled nonhistone proteins are mixed with histone-Sepharose in the presence of 4 M urea and 2 M NaCl, and the salt concentration then gradually lowered by gradient dialysis, elution of the histone-Sepharose with increasing NaCl concentration reveals the presence of several peaks of bound radioactivity which are not observed with control columns (blank Sepharose or cytochrome c linked to Sepharose). Columns of histone H2B bind more labeled nonhistones than do columns of histone Hl and the radioactive proteins are eluted at higher salt concentrations. A given peak, rechromatographed on the same histone column, is eluted at the same NaCl concentration originally required for elution. However, when rechromatographed on another type of histone column, the same peak is eluted at an NaCl concentration different from that required for the original elution. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the radioactive peaks eluted from the histone columns shows a number of highly radioactive proteins, some binding to both histone columns, and others binding preferentially to either histone H2B or Hl. These experiments therefore suggest that some nonhistone proteins, particularly the phosphorylated ones, bind to histones in a selective fashion.