Identification of nonhistone chromatin proteins in chromatin subunits.

Abstract

Rat liver chromatin was digested by micrococcal nuclease. More than 80% of the enzyme-digested chromatin could be recovered after centrifugation. Treatment with sodium deoxycholate and Triton X-100 at concentrations of 0.5% in the final chromatin suspension gave a higher recovery. Chromatin subunits were fractionated on a 5-30% linear sucrose density gradient. Approximately 35% of the chromatin subunits could be recovered from the gradient. Chromatin subunits and their DNA fragments were identified by gel electrophoresis and ultracentrifugation. The presence of nonhistone chromatin proteins (NHCP) in chromatin subunits was demonstrated by the following criteria: (i) Quantitative analysis showed that the mass ratio of histone to NHCP, in the presence or absence of detergents, was 1:0.25 or 1:0.1, respectively. (ii) After the removal of acid-soluble protein from the subunits, it was found that most of the phenol-soluble NHCP were similar to total chromatin NHCP. However, four major fractions of these phenol-soluble NHCP were found to be enriched in the subunits as identified by two-dimensional polyacrylamide gel electrophoresis. (iKi) Experiments using an exchange of isotope-labeled and nonlabeled chromatin showed that NHCP were tightly bound to the chromatin subunits. In 1969, Murray (1) reported that native or partially dehistonized chromatin could be digested by deoxyribonuclease I and a region of chromatin protected by proteins could be isolated. Clark and Felsenfeld (2) used chemical probes to measure the accessibility of the DNA in chromatin and reported that about half of the DNA could be titrated with divalent cations, polylysine, or histones and could be subjected to staphylococcal nuclease or deoxyribonuclease I digestion. Others also used nucleases as probes to study the interaction of DNA and histones, as well as to isolate an active and inactive chromatin fraction (3,4). Hewish and Burgoyne (5) demonstrated that a uniform size of chromatin DNA could be obtained by Ca++-Mg++-dependent endonuclease digestion. The electron microscopic observations made by Olins and Olins (6,7) and Woodcock et al. (8,9) indicate that chromatin structure resembles beads on a string, greatly substantiating the biochemical investigations (1-5).Recently the nonhistone chromatin proteins (NHCP) were absent from the llS particles. This report deals with the identification of NHCP in rat liver chromatin subunits. We have found that these subunits are specifically enriched in at least four major fractions of NHCP.MATERIALS AND METHODS All chemicals and organic solvents used in these studies were of reagent grade.Male albino Wistar rats (200 g) were used. Food was removed the night before experiments.[3H]leucine (250 AOi) was given to each rat intraperitoneally (i.p.) for pulse-labeling, and the liver was then removed for the isolation of nuclei. Preparation of Nuclei and Chromatin. Five grams of liver were homogenized in 5 volumes of Medium A (0.25 M sucrose-10 mM Tris-HUl, pH 8.0-3 mM MgCl2-0.1 mM phenylmethylsulfonylf...

Nuclear matrix DNA from chicken erythrocytes contains beta-globin gene sequences.

“…Thus it is reasonable to suggest that the nucleotide sequence alone is insufficient for genomic activation of tissue-specific transcription. Although gene-specific nonhistone proteins have not yet been found in chromatin obtained from specialized tissues, results supporting that possibility have been reported (8)(9)(10)(11)(12)(13).…”

“…Thus it is reasonable to suggest that the nucleotide sequence alone is insufficient for genomic activation of tissue-specific transcription. Although gene-specific nonhistone proteins have not yet been found in chromatin obtained from specialized tissues, results supporting that possibility have been reported (8)(9)(10)(11)(12)(13).Of the total nuclear proteins, the insoluble nonhistones are significant in their content of tissue-specific proteins (14), in the structure of the nuclear matrix (15), and in their association with transcriptionally active genes (16). Recently, studies by Pettijohn's group also suggested that an insoluble nonhistone protein of high molecular weight appears to be specific to cells undergoing active proliferation (17).…”

Monoclonal antibodies to a phosphoprotein from chromatin of rat liver

“…In a previous study we described the production of a polyclonal antibody to a rat liver nuclear phosphoprotein (B2: Mr 68000, pI6.5-8.2) (Zhao & Liew, 1982). This non-histone chromosomal structure and subsequently demonstrated to be preferentially associated with actively transcribed nucleosomes (Liew & Chan, 1976;Chan & Liew, 1977, 1979Liew et al, 1984a). In the present report we carried this immunological approach one step further with the production of several monoclonal antibodies to this phosphoprotein.…”

“…In recent years the highly heterogeneous nonhistone chromosomal proteins, several of which are associated with the core particles of nucleosomes (Liew & Chan, 1976;Defer et al, 1978;Zachau et al, 1982;Klug, 1983;Klug et al, 1983), have come under close scrutiny as potential regulators of gene expression (see reviews by Liew, 1979;Philips et al, 1980;Cartwright et al, 1982;Liew et al, 1984b). For example, HMG 14 and 17 appear to be potential regulators due to their ability to confer sensitivity to deoxyribonuclease in certain selective transcribable regions of the genome (see reviews by Weisbrod & Weintraub, 1979;Johns, 1982).…”

A chromosomal phosphoprotein is preferentially released by mild micrococcal-nuclease digestion

“…Two groups of nonhistone chromosomal proteins, which co-sediment with the chromatin monomers generated by digestion with micrococcal nuclease and isolated by sucrose-density-gradient centrifugation, have been identified in our laboratory. One group of nonhistone chromosomal proteins is highly phosphorylated, with Mr 68000 and pI6.5-8.2, and is associated with the monomers (Liew & Chan, 1976;Chan & Liew, 1977, 1979aZhao & Liew, 1982), and the second group of proteins is found associated with the heterogeneous ribonucleoprotein particles (Suria & Liew, 1979).…”