Richard Sterner

Affinity chromatographic purification of nucleosomes containing transcriptionally active DNA sequences

The unfolding of nucleosome cores in transcriptionally active chromatin uncovers the sulfhydryl groups of histone H3, making them accessible to SH-reagents. This has suggested that nucleosomes from active genes could be retained selectively on organomercurial/agarose columns. When nucleosomes released from rat liver nuclei by limited digestion with micrococcal nuclease were passed through an Hg affinity column, a run-off fraction of compact, beaded nucleosomes was separated from a retained nucleosome fraction. Although both contained monomer-length DNA and a full complement of core histones, histones in the retained fraction were hyperacetylated. Dot blot hybridizations showed the Hg-bound nucleosome fraction to be enriched in DNA sequences transcribed by hepatocytes (serum albumin and transferrin genes), while a brain-specific gene (preproenkephalin) was not retained, but appeared in the nucleosomes of the run-off fraction. The results are discussed in light of other evidence linking hyperacetylation of histones H3 and H4 to conformational changes at the middle of the nucleosome core.

Post-synthetic modifications of nuclear proteins. High mobility group proteins are methylated.

Abstract Calf thymus high mobility group proteins 1 and 2 (HMG-1 and HMG-2) were purified to homogeneity from a 0.35 M NaCl extract of chromatin by selective precipitation with trichloroacetic acid followed by ion exchange chromatography on CM-Sephadex. Amino acid analysis of an acid hydrolyzate of HMG-1 and HMG-2 proteins revealed the presence of a ninhydrin-positive peak identified as NG,NG-dimethylarginine. Radioactive tracer experiments confirmed the presence of methylated arginine in HMG proteins.

Protein side-chain acetylations

Publisher Summary This chapter discusses the acetylation of protein side-chains. Using preparative gel electrophoresis, it is now possible to prepare the well-defined histone substrates necessary for the studies of the kinetics and mode of action of isolated transacetylases or deacetylases. The amide linkage between the acetyl group and the ɛ-NH2 function of lysine is not stable to acid hydrolysis, and therefore standard procedures of hydrolysis and amino acid analysis cannot be used to detect ɛ-N- acetyllysine. The presence of this derivative can be determined, however, by enzymatic degradation of the modified protein and chromatographic separation of the component amino acids. The quantitation of the extent of protein acetylation may be achieved by the use of fluorodinitrobenzene (FDNB), which reacts with free amino groups to form acid-stable dinitrophenyl (DNP) derivatives. Other direct chemical procedures for the quantitation of acetyl groups in proteins include steam distillation of protein hydrolysates and the titration of the volatile acetic acid, hydrazinolysis, followed by the conversion of the acetyl hydrazide to a dinitrophenyl derivative and the extraction of acid hydrolysates with tert -butyl ethyl ether followed by gas chromatographic separation of the extracted acetic acid.

Phosphorylation and acetylation of chromatin conjugate protein A24.

Abstract An analysis was made of the phosphorylation and acetylation of chromatin protein A24, a conjugate of histone 2A and ubiquitin. 32 P-orthophosphate was incorporated into phosphoserine of histone 2A and protein A24 in Novikoff hepatoma ascites cells in culture. The ratio of 32 P incorporation and the pattern of tryptic digestion of 32 P-labeled protein A24 indicated that the histone 2A component was phosphorylated and the ubiquitin component was not. Analysis of e-N-acetyl lysine in protein A24, histone 2A and ubiquitin showed that while protein A24 and histone 2A were acetylated, ubiquitin was not. Apparently, even though it is conjugated with ubiquitin, the histone 2A portion of protein A24 has the same modifications as free histone 2A. The lack of modification of ubiquitin differs from that of high mobility group (HMG) non-histone chromatin proteins with which it is co-extracted from chromatin.

Discrete proteolytic cleavage of High Mobility Group proteins

Abstract Chromatographic fractionation on CM-Sephadex of a 0.35 M NaCl extract from calf thymus chromatin reveals the presence of a High Mobility Group (HMG) protein which comigrates electrophoretically with HMG-17. Further amino acid analysis and partial sequence determination suggest that this protein is a proteolytic degradation product of either HMG-1 or HMG-2 from which the acidic C-terminal region has been removed.