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

Abstract

NaCl, and the salt concen- tration 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 con- trol columns (blank Sepharose or cytochrome c linked to Sepharose). Columns of histone H2B bind more la- beled nonhistones than do columns of histone Hl and the radioactive proteins are eluted at higher salt con- centrations. A given peak, rechromatographed on the same histone column, is eluted at the same NaCl con- centration originally required for elution. However, when rechromatographed on another type of histone column, the same peak is eluted at an NaCl concentra- tion different from that required for the original elu- tion. Sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis 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. Both histone and nonhistone chromatin proteins are thought to be involved in regulating chromatin structure and function. The histones function mainly by packing DNA into compact structures known as v bodies or nucleosomes (l-3). This complexing of DNA with histones generally renders the DNA inefficient as a template for RNA synthesis (4). The