The phosphorylation of high mobility group proteins 14 and 17 and their distribution in chromatin

The phosphorylation of the high mobility group (HMG) proteins has been investigated in mouse Ehrlich ascites, L1210 and P388 leukemia cells, human colon carcinoma cells (HT-29), and Chinese hamster ovary cells. HMG 14 and 17, but not HMB 1 and 2, were phosphorylated in the nuclei of all cell lines with a serine being the site of modification for both proteins in Ehrlich ascites cells. Phosphorylation of HMG 14 and 17 was greatly reduced in cultured cells at plateau phase in comparison to log phase cells, suggesting that modification of HMG 14 and 17 is growth-associated. However, phosphorylation was not linked to DNA synthesis, since incorporation of 32P did not vary through G1 and S phase in synchronized Chinese hamster ovary cells. Treatment of HT-29 or Ehrlich ascites cells with sodium butyrate reduced HMG phosphorylation by 30 and 70%, respectively. The distribution of the phosphorylated HMG proteins in chromatin was examined using micrococcal nuclease and DNase I. 32P-HMG 14 and 17 were preferentially associated with micrococcal nuclease-sensitive regions as demonstrated by the release of a substantial fraction of the phosphorylated forms of these proteins under conditions which solubilized less than 3% of the DNA. Short digestions with DNase I did not show a marked release of 32P-HMG 14 or 17.     

Evidence for the location of high mobility group protein T in the internucleosomal linker regions of trout testis chromatin.

Antibodies against the trout testis non"histone chromosomal protein, high mobility group protein T (HMG-T), have been elicited in goats. The antiserum was shown to be specific for HMG-T and did not cross-react with histone 1 or with the other two trout testis HMG proteins, H6 and ubiquitin. Purified anti-HMG-T IgG was used to determine the location of HMG-T within chromatin subunits separated on sucrose gradients. Binding of fluorescent labeled anti-HMG-T to these subunits clearly supports the notion that this protein is associated not with the nucleosome core but rather with the internucleosomal linker regions, and previously suggested (Levy W., B., Wong, N.C.W., and Dixon, G. H. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2810-2814).     

Distribution of high mobility group and other acid-soluble proteins in fractionated chromatin

Chromatin was fractionated by digestion with deoxyribonuclease II and precipitation with MgCl₂. The Mg²⁺-soluble fraction, known to be enriched in transcribed DNA sequences, was enriched also in high mobility group proteins 1 and 2 and contained almost all other acid-soluble nonhistone proteins.     

The release of high mobility group protein H6 and protamine gene sequences upon selective DNase I degradation of trout testis chromatin.

Limited digestion of trout testis nuclei with DNase I selectively degrades the protamine genes. Concomitant with the degradation of transcribed DNA sequences a series of chromosomal proteins are released; among these, the major species corresponds to the high mobility group protein H6. The amounts of H6 released from chromatin by limited DNase I action and that in the residual nuclear pellet have been determined. A very high proportion of H6 is associated with DNase I sensitive chromatin regions.     

The intracellular distribution and function of the high mobility group chromosomal proteins

This brief review provides a framework for discussing current approaches being used to determine the cellular localization and function of the high mobility group chromosomal (HMG) proteins. The four main constituents of this group (HMG 1, 2, 14, 17) are present in all four eukaryotic kingdoms, have a relatively well conserved primary sequence and contain several functional domains which enable them to interact with DNA, histones and other components of the genome. The evolutionary conservation in the primary and tertiary structure as well as the observed correlations between cell phenotype and quantitative changes in protein levels and in post-synthesis modifications suggests that these proteins are components obligatory for proper cellular function. Proteins HMG 1, 2 are DNA-binding proteins which can distinguish between various types of single-stranded regions of the genome. Proteins HMG 14, 17 may be involved in maintaining specific chromatin regions in particular conformations. The data available presently suggests that these proteins are important structural elements of chromatin and chromosomes.     

Synthesis of high mobility group proteins in regenerating rat liver.

Incorporation of [3H]lysine into the non-histone chromosomal proteins HMG1, HMG2, and HMG17 and into each of the five major classes of histones was measured in rat liver at various times after partial hepatectomy. Histone synthesis was closely coupled temporally to that of DNA, although a small amount of histone was shown to be produced before DNA replication began. In contrast, the incorporation curves for the high mobility group (HMG) proteins showed little correlation with that for DNA. At 4 h after partial hepatectomy, protein synthesis had virtually ceased. Thereafter, the rates of synthesis of the HMG proteins rose steadily so that by 12 h, well before the onset of DNA replication they had reached about two-thirds of the maximum rates attained during the first cell division cycle. Histones had only reached about one-sixth of their maximum rates at this time. The lack of coupling betweeen the synthesis of the HMG proteins and DNA was confirmed by experiments with inhibitors of DNA replication. Reduction of DNA synthesis to less than 10% of the uninhibited rate had little or no effect on incorporation into the HMG proteins, whereas, under similar conditions, the rate of synthesis of histones was reduced by more than 50%.     

An improved large scale fractionation of high mobility group non-histone chromatin proteins.

1. Methodology is presented for the large scale preparation and fractionation of high mobility group proteins from calf thymus chromatin. The total high mobility group protein from approx. 1 kg calf thymus tissue can be separated into five fractions by CM-Sephadex C25 ion-exchange chromatography. High mobility group proteins 1 and 2 comprise two fo the fractions. From a third fraction two more chromatin proteins, protein 3 and 17, can be isolated by trichloroacetic acid precipitation and CM-cellulose chromatography at pH 5.5. 2. The four proteins thus purified are lysine-rich proteins. Proteins 1 and 2 are additionally characterised by their high contents of acidic amino acids, as described previously (Goodwin, G. H. and Johns, E. W. (1973) Eur. J. Biochem. 40, 215-219). Proteins 3 and 17, having lower contents of acidic amino acids, are basic proteins similar to the histones. All four proteins exhibit single N-terminal amino acids; glycine is the N-terminal group of proteins 1, 2 and 3; protein 17 has a proline N-terminal amino acid. The proteins are not highly phosphorylated nor are they associated with appreciable quantities of nucleic acid.     

Immunochemical studies of high mobility group non-histone chromatin proteins HMG 1 and HMG 2

Sera were raised to non-histone chromatin proteins HMG 1 and HMG 2. Immunoperoxidase staining localised these proteins on chromosomes during mitosis and indicated a cell cycle-related variation in these proteins during interphase. Some species differences in HMG 1 and HMG 2 were also observed.     

Preferential affinity of high molecular weight high mobility group non-histone chromatin proteins for single-stranded DNA.

We have subjected proteins dissociated from chicken erythrocyte or calf thymus chromatin by 0.35 M NaCl to sequential chromatography on columns containing immobilized double-stranded DNA and single-stranded DNA. At 0.2 M NaCl, 1 mM Tris . Cl (pH 7.5), the high molecular weight, high mobility group proteins (HMG-1, HMG-2, and HMG-E), were not retained by double-stranded DNA columns, but were retained by single-stranded DNA columns. Thus, in that solvent, those proteins exhibit selective affinity for single-stranded DNA. This suggests that the functions of the high molecular weight, high mobility group proteins might involve destabilizing the DNA double helix by virtue of their preferential affinity for single-stranded DNA.     

Monoclonal antibodies as probes for the complexity, phylogeny, and chromatin distribution of high mobility group chromosomal proteins 1 and 2

Monoclonal antibodies were prepared against the high mobility group (HMG) proteins 1, 2a, and 2b from hen erythrocyte chromatin. One antibody that recognized multiple sites along HMG-1, -2a, and -2b reacted strongly with HMG proteins from all vertebrates tested. In contrast, five antibodies that detected unique epitopes on chicken HMG-1 and -2a recognized antigenic sites that exhibited restricted phylogenic distributions. The differential reactivity of these antibodies on vertebrate proteins was in agreement with traditional taxonomy in that the avian HMGs were most closely related to those from reptiles and less related to those from mammals, amphibians, bonyfish, and especially the jawless fish. Mononucleosomes generated by mild digestion of erythrocyte chromatin with micrococcal nuclease were highly enriched in HMG-2a. One antigenic determinant located within the N-terminal domain of HMG-2a was freely accessible to its antibody when the protein was bound to these mononucleosomes. In contrast, two antibodies that recognized determinants in the central region of HMG-2a exhibited little chromatin binding activity. The masking of the central domain by DNA binding was presumably not responsible for these results because all three determinants were available for antibody binding when HMG-2a was bound to DNA in vitro. Therefore, the central region of HMG-2a may be masked from antibody binding by protein-protein interactions in chromatin.     

Studies on the association of the high mobility group non-histone chromatin proteins with isolated nucleosomes.

Nucleosomes have been isolated from rabbit thymus by sucrose gradient centrifugation, and their high mobility group (HMG) protein content analysed by electrophoresis on polyacrylamide gels. The results suggest that proteins HMG 14 and HMG 17 are associated with the core particle of the nucleosome, and that there are two or more sub-populations of both HMG 1 and HMG 2 molecules. One sub-population appears to be fairly tightly bound to the nucleosome, while another is rapidly released from the chromatin by digestion with micrococcal nuclease. The latter fraction may participate in a higher order folding of the nucleosomes.     

Evidence for a quantitative tissue-specific distribution of high mobility group chromosomal proteins

The quantitative tissue specificity of the high mobility group (HMG) chromosomal proteins was investigated. Perchloric acid (PCA) extracts of four different chicken tissues and erythrocytes contained three proteins which comigrated on NaDodSO4-polyacrylamide gels with the HMG's 1,2, and E from erythrocyte nuclei. These three HMG's from embryonic skeletal muscle and erythrocytes also comigrated on two-dimensional gels, employing an acid-urea system in the first dimension and an NaDodSO4 system in the second. Interpretation of the two-dimensional gels suggested that the two low molecular weight proteins of this triplet arose from the HMG 2 band of the acid-urea gels. These have been designated HMG 2A and HMG 2B. Three proteins of similar molecular weights were also found in the PCA extracts of calf thymus. They were arranged in a similar but not identical pattern on two-dimensional gels. Thus, these three HMG's appear to be neither tissue nor species specific. In addition, the 2.0% PCA extracts of all chicken tissues examined contain a 38 000-dalton (38K) nuclear protein which coisolates with the HMG's. These four proteins are found in different relative amounts in each of the four chicken tissues and erythrocytes. They are found in the same relative amounts, however, in embryonic skeletal muscles from different chicken strains with widely different highly repetitive sequence content, suggesting that none of these individual proteins is selectively localized to constitutive heterochromatin. The quantitative tissue specificity of the HMG's and the 38K protein, however, suggests that they may participate in regulating cell-specific gene expression.     

Selective distribution of histone H1 variants and high mobility group proteins in chromosomes

Recent results on the differential distribution of sequence variants of histone H1, of proteins of the HMG 1/2 family, and of HMGI in polytene chromosomes are reviewed. Several organisms are known to contain two different HMG 1/2 proteins. InChironomus, one of them is restricted to decondensed puffs and may have a specific function. One of the H1 variants ofChironomusis found only in a minority of chromosome bands and differs from the other H1 proteins of the organism by genomic organization and by an inserted structural motif that is also present in single H1 variants of other organisms.