ADP-ribosylation of chromosomal proteins and mouse mammary tumor virus gene expression. Glucocorticoids rapidly decrease endogenous ADP-ribosylation of nonhistone high mobility group 14 and 17 proteins

The relationship between endogenous ADP-ribosylation of chromosomal proteins and glucocorticoid-regulated mouse mammary tumor virus gene expression was investigated in cultured mouse mammary tumor cells. It was observed that glucocorticoids quickly decreased endogenous (ADP-ribose)n on the nonhistone high mobility group (HMG) 14 and 17 proteins. The half-time for this loss was 8 and 17 min, respectively, for the two proteins. (ADP-ribose)n on HMG 1 and 2 and on histone H1 was less susceptible to hydrolysis during glucocorticoid treatment. The rapid loss of (ADP-ribose)n from HMG 14 and 17 occurred in the same time frame as the induction of mouse mammary tumor virus RNA synthesis by glucocorticoids in these cells (Young, H. A., Shih, T. Y., Scolnick, E. M., and Parks, W. P. (1977) J. Virol. 21, 139-149). 3-Amino-benzamide, a specific inhibitor of (ADP-ribose)n synthetase, increased mouse mammary tumor virus RNA levels with an accompanying decrease in endogenous ADP-ribosylation of HMG 14 and 17. These results show that a decrease in endogenous ADP-ribosylation of HMG 14 and 17 is a consequence of glucocorticoid action and suggest that loss of (ADP-ribose)n from these proteins may be an important event in mouse mammary tumor virus gene expression.     

Analysis of age-associated alteration in the synthesis of HMG nonhistone proteins of the rat liver

HMG proteins were extracted with 5% PCA or 0.35 M NaCl from whole tissue, nuclei or chromatin of the liver of young (19 weeks) and old (118 weeks) male rats. They were resolved on acetic acid-urea polyacrylamide gel. The electrophoretic patterns of the major HMG proteins 1, 2, 14 and 17 of both ages are similar. The in vitro synthesis of HMG 1 and 2 decreases, but that of HMG 14 and 17 increases considerably in the liver of old rats. The synthesis of different HMG proteins is modulated differentially by spermine, butyrate, dexamethasone and 3-aminobenzamide in the liver of young and old rats. These findings suggest that HMG proteins contribute to alterations in the organization of chromatin and expression of genes during aging.     

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%.     

Developmental changes in the expression of high mobility group chromosomal proteins

The high mobility group (HMG) chromosomal proteins may modulate the structure of distinct regions in chromatin, thereby affecting processes such as development and differentiation. Here we report that the levels of the HMG chromosomal proteins and their mRNAs change significantly during erythropoiesis. Erythroid cells from 5-day chicken embryos contain 2.5-10 times more HMG mRNAs than cells from 14-day embryos, whereas circulating cells from adult animals are devoid of HMG and most other mRNAs. Nuclear run-off experiments and Northern analysis of RNA from various developmental stages and from Percoll-fractionated cells indicate that the genes are transcribed in early cells of either the primitive or definitive erythroid lineage. The rate of synthesis of the various HMGs changes during erythropoiesis; in erythroid cells from 7-day embryos the ratio of HMG-14b or HMG-17 to HMG-14a is, respectively, 8 and 10 times lower than in 9-day erythroids. HMG-14a, the major chicken HMG-14 species, is synthesized mainly in primitive cells, while HMG-14b is preferentially synthesized in definitive cells. Thus, the change from primitive to definitive erythroid lineage during embryogenesis is accompanied by a change in the expression of HMG chromosomal proteins. Conceivably, these changes may affect the structure of certain regions in chromatin; however, it is not presently clear whether the switch in HMG protein gene expression is a consequence or a prerequisite for proper differentiation.     

Enhanced myocardial RNA synthesis in spontaneously hypertensive rats possible role of high-mobility group non-histone proteins

Cardiac hypertrophy in spontaneously hypertensive rats is associated with increased nuclear RNA polymerase activity. In order to explore mechanisms facilitating the interaction of the enzyme with its endogenous template, we compared the structure of nuclear chromatin from myocytes of 20-week-old spontaneously hypertensive rats and normotensive Wistar-Kyoto controls. Enhanced RNA synthesis in hypertensive rats was accompanied by increased susceptibility to digestion by deoxyribonuclease I. Nick translation of nuclei also resulted in higher nucleotide incorporation in hypertensive rats. Salt-extraction abolished the differences in deoxyribonuclease I sensitivity between the two animal groups. Reconstitution with either 0.35 M NaCl-extract or high mobility group (HMG) non-histone proteins restored digestion susceptibility but did not equalize SHR and WKY cells. SDS-polyacrylamide gel electrophoresis of 0.35 M NaCl-extracts and supernatants from deoxyribonuclease I digestion revealed the presence of HMG proteins which were preferentially released in hypertensive rats. There was a small but statistically significant increase in nuclear HMG protein content in hypertensive rats (0.12 +/- 0.02 mg/mg DNA vs. 0.09 +/- 0.02 mg/mg DNA in Wistar-Kyotos, P less than 0.05) but no difference in their electrophoretic appearance. These results indicate that chromatin structure is altered in the hypertrophied myocardium with resultant increase in deoxyribonuclease I susceptibility. This increase appears to be partly dependent on the high-mobility group non-histone proteins.     

Alterations in amounts and covalent modifications of low-molecular-weight chromosomal proteins in Chinese hamster ovary cells during polyamine depletion

The effect of polyamine depletion on phosphorylation and ADP-ribosylation of low-M_r chromosomal proteins was studied in intact, mutant Chinese hamster ovary cells (CHO-P22) devoid of ornithine decarboxylase activity. When starved of polyamines for 6 days, severe polyamine deficiency develops and the cells gradually stop growing. The rate of DNA synthesis was retarded to 16% of the control value and to 29% in density-inhibited cells. The synthesis of high-mobility-group (HMG) proteins was decreased by 65% in polyamine-depleted cells and by 40% in density-inhibited cells. The synthesis of core histones was decreased by 40% both in polyamine-depleted and density-inhibited cells. In polyamine-depleted cells the molar ratio of the higher-M_r HMG proteins (HMG 1 + 2) to the lower-M_r HMG proteins (HMG 14 + P) was about one-half of that found in cells grown in the presence of putrescine or in density-inhibited cells. In contrast to HMG proteins, no major differences were found in the content of core histones in these cell populations. In the perchloric acid-soluble fraction of nuclear proteins, ³²P was incorporated mainly into histone H1, HMG P and a protein migrating more slowly than HMG 1 (protein P1). Specific changes in the ³²P-labeling and migration of a number of protein bands, including histone H1, was observed in polyamine-depleted cells as compared to cells grown in the presence of putrescine or to density-inhibited cells. ADP-ribosylation experiments using [³H]adenosine showed a different pattern of label distribution; the higher-M_r HMG proteins from polyamine-depleted cells contained about one-half the amount of label found in the proteins from control cells. The lower-M_r HMG proteins and histone H1 were the preferentially labeled proteins in polyamine-depleted cells. Labeling of core histones with [³²P]orthophosphate or [³H]adenosine did not differ markedly in the two cell populations. The results obtained using intact polyamine auxotrophic cells indicated that polyamine depletion is connected with more severe alterations in amounts and covalent modifications (phosphorylation and ADP-ribosylation) of HMG chromosomal proteins and histone H1 than core histones.     

The Saccharomyces cerevisiae ACP2 gene encodes an essential HMG1-like protein.

The high-mobility-group (HMG) proteins, a group of nonhistone chromatin-associated proteins, have been extensively characterized in higher eucaryotic cells. To test the biological function of an HMG protein, we have cloned and mutagenized a gene encoding an HMG-like protein from the yeast Saccharomyces cerevisiae. A yeast genomic DNA library was screened with an oligonucleotide designed to hybridize to any yeast gene containing an amino acid sequence conserved in several higher eucaryotic HMG proteins. DNA sequencing and Northern (RNA) blot analysis revealed that one gene, called ACP2 (acidic protein 2), synthesizes a poly(A)+ RNA in S. cerevisiae which encodes a 27,000-molecular-weight protein whose amino acid sequence is homologous to those of calf HMG1 and HMG2 and trout HMGT proteins. Standard procedures were used to construct a diploid yeast strain in which one copy of the ACP2 gene was mutated by replacement with the URA3 gene. When this diploid was sporulated and dissected, only half of the spores were viable. About half of the nonviable spores proceeded through two or three cell divisions and then stopped dividing; the rest did not germinate at all. None of the viable spores contained the mutant ACP2 gene, thus proving that the protein encoded by ACP2 is required for cell viability. The results presented here demonstrate that an HMG-like protein has an essential physiological function.     

Characterization of high mobility group protein levels during spermatogenesis in the rat

The distribution, quantitation, and synthesis of high mobility group (HMG) proteins during spermatogenesis in the rat have been determined. HMG1, -2, -14, and -17 were isolated from rat testes by Bio-Rex 70 chromatography combined with preparative gel electrophoresis. Amino acid analysis revealed that each rat testis HMG protein was similar to its calf thymus analogue. Tryptic peptide maps of somatic and testis HMG2 showed no differences and, therefore, failed to detect an HMG2 variant. Testis levels of HMG proteins, relative to DNA content, were equivalent to other tissues for HMG1 (13 micrograms/mg of DNA), HMG14 (3 micrograms/mg of DNA), and HMG17 (5 micrograms/mg of DNA). The testis was distinguished in that it contained a substantially higher level of HMG2 than any other rat tissue (32 micrograms/mg of DNA). HMG protein levels were determined from purified or enriched populations of testis cells representing the major stages of spermatogenesis; spermatogonia and early primary spermatocytes, pachytene spermatocytes, early spermatids, and late spermatids; and testicular somatic cells. High levels of HMG2 in the testis were due to pachytene spermatocytes and early spermatids (56 +/- 4 and 47 +/- 6 micrograms/mg of DNA, respectively). Mixtures of spermatogonia and early primary spermatocytes showed lower levels of HMG2 (12 +/- 3 micrograms/mg of DNA) similar to proliferating somatic tissues, whereas late spermatids had no detectable HMG proteins. The somatic cells of the testis, including isolated populations of Sertoli and Leydig cells, showed very low levels of HMG2 (2 micrograms/mg of DNA), similar to those in nonproliferating somatic tissues. HMG proteins were synthesized in spermatogonia and primary spermatocytes, but not in spermatids. Rat testis HMG2 exhibited two bands on acid-urea gels. A "slow" form comigrated with somatic cell HMG2, while the other "fast" band migrated ahead of the somatic form and appeared to be testis-specific. The "fast" form of HMG2 accounted for the large increase of HMG2 levels in rat testes. These results show that the very high level of HMG2 in testis is not associated with proliferative activity as previously hypothesized.     

Studies on the high-mobility-group non-histone proteins from hen oviduct.

Nuclear high-mobility-group (HMG) proteins were isolated from hen oviduct. These were proteins HMG-1, -2, -3, -14 and -17, which are equivalent to the classification of calf thymus HMG proteins. Hen oviduct proteins HMG-1 and -2 were individually isolated by HCIO4.extraction and CM-Sephadex chromatographic separation. Their mol.wts. were determined as 28 000 and 27 000, respectively. The proteins have a high content of acidic and basic amino acids. The association of proteins HMG-1 and -2 with the genome of hen oviduct nuclei was probed by a limited digestion with nucleases. Hen oviduct nuclei were incubated with deoxyribonuclease I or micrococcal nuclease until 10% of the DNA was digested. The nuclear suspension was centrifuged and the contents of proteins HMG-1 and -2 in the supernatant and sediment fractions were analysed by polyacrylamide-gel electrophoresis. HMG proteins were found to be preferentially released by micrococcal-nuclease digestion rather than by deoxyribonuclease I.     

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.