Chromosomal Protein HMG-17 Research Articles

Hierarchy of binding sites for chromosomal proteins HMG 1 and 2 in supercoiled deoxyribonucleic acid.

The interaction of chromosomal proteins HMG 1 and 2 with various DNA structures has been examined with plasmid pPst-0.9, which contains DNA sequences that can form the Z-DNA conformation and palindromic sequences that can form cruciform structures. Direct binding and competition experiments with 32P-labeled plasmid indicated that proteins HMG 1 and 2 preferentially bind to supercoiled form I DNA as compared to double-stranded linear DNA. The preferential binding to form I is due to the presence of single-stranded regions in this DNA. The binding of HMG 1 and 2 to the form I plasmid results in inhibition of S1 nuclease digestion in a selective manner. The B-Z junction is preferentially protected as compared to the cruciform, which in turn is more protected than other minor S1-sensitive structures present in pPst-0.9. Our results indicate that the binding of HMG 1 and 2 proteins to DNA is not random in that HMG 1 and 2 can distinguish between various S1 nuclease sensitive sites in the plasmid. The existence of a hierarchy of DNA binding sites for these proteins suggests that they can selectively affect the structure of distinct regions in the genome.

Isolation of high-mobility-group proteins HMG1 and HMG2 in non-denaturing conditions and comparison of their properties with those of acid-extracted proteins

We described a method for isolation and purification of the chromosomal proteins HMG1 and HMG2 in non-denaturing conditions which overcomes the difficulties of the published methods concerning yield and purity. The method is based on salt extraction, selective precipitation with ammonium sulfate and DEAE-cellulose chromatography. All studied properties of these proteins (formation of proteins tetramers, enhancement of micrococcal nuclease digestion of DNA and chromatin, and protection of 165-basepair DNA in chromatosome) differ significantly from the properties of HMG1 and 2 isolated under denaturing conditions.

Interaction between domains in chromosomal protein HMG-1.

Peptides corresponding to the N-terminal, central and central plus C-terminal domains of high mobility group protein HMG-1 from calf thymus have been isolated after digestion in solution with protease V8 under structuring conditions (0.35 M NaCl, pH 7.1). The effect of the interaction of these peptides with DNA on the topological properties of the nucleic acid has been studied and compared with the change in superhelicity produced by the whole protein. It appears that the region responsible for this effect is the central domain of HMG-1. The isolated N-terminal and central domains of this protein maintain their secondary and tertiary structure as observed by spectroscopic techniques. However, when the central domain is covalently linked only to the acidic C-terminal part of the molecule, its secondary and tertiary structures are lost as well as its property to alter DNA superhelicity. The results are discussed in relation to the interactions occurring between the different domains and the possible functional interactions of this protein.

Mg2+-dependent unwinding of DNA by nonhistone chromosomal protein HMG(1 + 2) from pig thymus as determined by DNA melting temperature analysis.

In the previous studies with endonucleases specific for single-stranded DNA, we have indicated that the nonhistone chromosomal protein HMG(1 + 2) prepared from pig thymus has an activity to unwind DNA partially at low protein-to-DNA weight ratios (Yoshida, M. & Shimura, K. (1984) J. Biochem. 95, 117-124). In the present work, we have pursued the unwinding reaction by HMG(1 + 2) by thermal melting temperature analysis of DNA, and by investigating the effect of Mg2+ on the reaction. The melting temperature of DNA in the presence of HMG(1 + 2) at low protein weight ratios decreased in 2 mM Tris-HCl, pH 7.8, whereas it increased at higher ratios. The depressions of melting temperature by HMG(1 + 2) at low ratios were not observed either in the system of 2 mM Tris-HCl, pH 7.8, containing EDTA or in the system containing samples treated in advance with EDTA. An addition of Mg2+ to the system reproduced the depression of melting temperature at low protein-to-DNA ratios as well as the increase at higher ratios. Analysis by Mg2+-equilibrated gel filtration revealed that HMG(1 + 2) is a Mg2+-binding protein. However, the depression of melting temperature at low protein-to-DNA ratios was not due to removal of Mg2+ from DNA by HMG(1 + 2). From these results, it is concluded that HMG(1 + 2) causes a partial DNA unwinding detectable by thermal melting temperature analysis of DNA, and that Mg2+ is necessary for the unwinding reaction.

Inhibition of transcription in somatic cells by microinjection of antibodies to chromosomal proteins.

The in vivo function of defined chromosomal proteins was examined by microinjecting purified antibody and antibody fragments into living fibroblasts. The involvement of histones and chromosomal high mobility group proteins HMG-1, 2, and 17 in transcription was visualized by studying the [3H]uridine incorporation in KD human fibroblasts after microinjection of fluoresceinated antibodies to these proteins. Nuclear uridine incorporation was not affected by microinjection of control antibodies or by the presence of immune complexes formed after microinjection of antibodies to chromosomal proteins that are not involved in transcription. In contrast, injection of anti-histone IgG, F(ab')2, or Fab and anti-HMG-17 IgG causes a significant reduction in transcription. The reduction is proportional to the amount of antibody introduced into the cell. We conclude that histones and protein HMG-17 are present on transcribed regions of the genome and that passage of RNA polymerase along the chromatin fiber is prevented by antibody binding to these proteins.

Quantitative changes of high mobility group non-histone chromosomal proteins HMG1 and HMG2 during rooster spermatogenesis

The quantitative changes of a group of non-histone chromosomal proteins identified by its solubility, electrophoretic mobility and amino acid analysis as the high mobility group proteins HMG1 and HMG2, were studied throughout rooster spermatogenesis. The ratio HMG1/HMG2 remained constant (0.66 ± 0.04) during the transition from dividing meiotic and premeiotic cells to nondividing spermatids and from transcriptionally active cells (spermatogonia, spermatocytes and early spermatids) to transcriptionally inactive late spermatids. The ratio HMG1/nucleosomal histone and HMG2/nucleosomal histone increase markedly at the end of spermiogenesis during the transition from nucleohistone to nucleoprotamine when nucleosomes are being disassembled. The high mobility group chromosomal proteins HMG1 and HMG2 were not detectable in the nuclei of rooster spermatozoa.

Phosphorylation of nuclear proteins.

Many nuclear proteins are phosphorylated: they range from enzymes to several structural proteins such as histones, non-histone chromosomal proteins and the nuclear lamins. The pattern of phosphorylation varies through the cell cycle. Although histone H1 is phosphorylated during interphase its phosphorylation increases sharply during mitosis. Histone H3, chromosomal protein HMG 14 and lamins A, B and C all show reversible phosphorylation during mitosis. Several nuclear kinases have been characterized, including one that increases during mitosis and phosphorylates H1 in vitro. Factors have been demonstrated in maturing amphibian oocytes and mitotic mammalian cells that induce chromosome condensation and breakdown of the nuclear membrane. The possibility that they are autocatalytic protein kinases is considered. The location of histone phosphorylation sites within the nucleosome is consistent with a role for phosphorylation in modulating chromatin folding.

Comparison of the structures of the chromosomal high mobility group proteins HMG1 and HMG2 prepared under conditions of neutral and acidic pH

The chromosomal proteins HMG1 and 2 have been prepared by salt extraction and phosphocelllose chromatography at neutral pH (Isackson, P.J., Debold, W.A. and Reeck, G.R. (1980) FEBS Lett. 119, 337–342) to minimize protein denaturation. The structures of these phosphocellulose-prepared high mobility group proteins have been compared with those of high mobility group proteins using the previously described acid-extraction conditions which fully denature the proteins. When compared in the same solvent conditions the acid-extracted proteins did not refold to give the same level of α-helical and tertiary folded structures as the phosphocelluose-prepared proteins, suggesting that acid treatment can cause some irreversible damage to the proteins. This findings was supported by changes in the structure observed when phosphocellulose-prepared HMG1 was neutralized after exposure to acid. Gel filtration studies reveal no difference in the size of the high mobility group proteins, phosphocellulose-prepared and acid-extracted proteins both being largely monomeric in solution. Little difference was detected in the DNA-binding properties of the two types of protein, nor was there any difference in the oxidation state of the cysteines. However, isoelectric focusing analysis revealed differences in the subfractions of HMG2 prepared by the two methods.

Demonstration of an S1-nuclease sensitive site near the human β-globin gene, and its protection by HMG 1 and 2

An S1-nuclease sensitive site exists within supercoiled plasmids containing the 5′-flanking sequences of the human β-globin gene. This site is located approximately 540 base pairs upstream from the start of the gene within a region of 52 alternating purine-pyrimidine residues which has the potential to adopt either cruciform structures or Z-form DNA. This site is protected from specific cleavage by S1-nuclease by the high-mobility-group chromosomal proteins HMG1 and 2, which may be specifically acting to protect short sequences of single-stranded DNA.

Loss of chromosomal high mobility group proteins HMG1 and HMG2 when mouse neuroblastoma and Friend erythroleukemia cells become committed to differentiation.

Chromosomal high mobility group (HMG) proteins HMG1 and HMG2 from mouse neuroblastoma cells and Friend erythroleukemic cells were analyzed by acetic acid/urea/polyacrylamide gel electrophoresis. Compared to rapidly growing cells, levels of HMG1 and HMG2 were decreased in mouse neuroblastoma cells that had been induced to differentiate by serum deprivation. This comparison revealed a reciprocal relationship between these HMG proteins and H10, a histone known to be in higher concentrations in nondividing cells. When cell growth was inhibited by means of density inhibition, however, HMG1 and -2 levels were not affected in either HeLa or mouse neuroblastoma cells, even though H10 did not accumulate. This observation establishes that HMG1 and -2 contents are not correlated with mitotic rate per se. Treatment of mouse neuroblastoma by sodium butyrate, which stops cell division without commitment to differentiation, had no effect on the level of HMG1 and -2. However, the level was decreased by dibutyryl cyclic AMP and dimethyl sulfoxide treatments, which, like serum deprivation, induced irreversible morphological differentiation in the neuroblastoma cells. Moreover, induction of differentiation (hemoglobin synthesis) in Friend erythroleukemic cells by dimethyl sulfoxide showed a decrease in the contents of HMG1 and -2. These observations suggest that preferential loss of HMG1 and -2 in mouse neuroblastoma and Friend erythroleukemia cells may be related to commitment of these cells to differentiation.

MRNA of chromosomal proteins HMG-1 and HMG-2 are polyadenylated

Total RNA from rat thymus was isolated and fractionated into poly A − and poly A + RNA. The translation products directed by the poly A − and poly A + RNA were immunoprecipitated with affinity pure antibodies to chromosomal protein HMG-1. Examination of the immunoprecipitates reveals that the m-RNA for chromosomal proteins HMG-1 and HMG-2 is polyadenylated. Thus, the m-RNA for these chromosomal proteins differ from those for the nucleosomal histones and resemble those coding for the bulk of the cellular proteins.

Immunochemical detection of chromosomal protein HMG-17 in chromatin subunits.

Chromosomal protein HMG-17, purified from calf thymus, has been used to elicit specific antibodies in rabbits. Specific serological reaction between the antigen and the antisera is demonstrated by solid-phase radioimmunoassay and by competitive inhibition assays. The antisera did not cross-react with histones or other chromosomal HMG proteins. The antisera bound specifically to chromatin subunits isolated from HeLa cells, demonstrating that it may be used to study the in situ organization of this chromosomal protein. Chromatin purified from HeLa nuclei was digested with micrococcal nuclease, and the resulting mono- and oligonucleosomes were fractionated on a sucrose gradient. Analyses of the content of chromosomal proteins HMG-1, HMG-17, and H4 in different size nucleosomal particles, by the solid-phase radioimmunoassay, reveal that the distribution of HMG-17 was the same as that of H4, but different from that of HMG-1.

Circular dichroism, thermal denaturation, and deoxyribonuclease I digestion studies of nucleosomes highly enriched in high mobility group proteins HMG 1 and HMG 2.

Salt-soluble (S) nucleosomes that contain near equimolar high mobility group nonhistone chromosomal proteins HMG 1 and HMG 2 and lack histone H1 were isolated from mouse myeloma nuclei. Comparisons of the sedimentation, near-UV circular dichroism, thermal denaturation, and pattern of DNase I digestion of S nucleosomes with these properties of nucleosome cores or "typical" nucleosomes containing H1 did not detect significant differences. These results indicate that HMG 1 and 2 do not affect the conformation and stability of nucleosomes or nucleosomal DNA and are consistent with the proposal that major functions of HMG 1 and 2 are to replace H1 and maintain the (micro) solubility and accessibility of local chromatin regions. In contrast to these similarities, the initial rate of DNase I digestion of S nucleosomes was approximately 3 times that of chromatin depleted in S nucleosomes. This is consistent with a relation of S nucleosomes to transcription and suggests that subtle factors (not necessarily HMG 1 and 2) determine DNase I susceptibility.

The primary structures of non-histone chromosomal proteins HMG 1 and 2

FEBS LettersVolume 122, Issue 2 p. 264-270 Full-length articleFree Access The primary structures of non-histone chromosomal proteins HMG 1 and 2 John M. Walker, John M. Walker Chester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, Fulham Road, London, SW3 6JB, England Present address: School of Biological Sciences, Hatfield Polytechnic, Hatfield, Herts, England Search for more papers by this authorKeith Gooderham, Keith Gooderham Chester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, Fulham Road, London, SW3 6JB, England Present Address: MRC Clinical and Population Cytogenetics Unit, Western General Hospital, Crewe Road, Edinburgh, Scotland Search for more papers by this authorJeremy R.B. Hastings, Jeremy R.B. Hastings Chester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, Fulham Road, London, SW3 6JB, EnglandSearch for more papers by this authorElaine Mayes, Elaine Mayes Chester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, Fulham Road, London, SW3 6JB, EnglandSearch for more papers by this authorErnest W. Johns, Ernest W. Johns Chester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, Fulham Road, London, SW3 6JB, EnglandSearch for more papers by this author John M. Walker, John M. Walker Chester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, Fulham Road, London, SW3 6JB, England Present address: School of Biological Sciences, Hatfield Polytechnic, Hatfield, Herts, England Search for more papers by this authorKeith Gooderham, Keith Gooderham Chester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, Fulham Road, London, SW3 6JB, England Present Address: MRC Clinical and Population Cytogenetics Unit, Western General Hospital, Crewe Road, Edinburgh, Scotland Search for more papers by this authorJeremy R.B. Hastings, Jeremy R.B. Hastings Chester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, Fulham Road, London, SW3 6JB, EnglandSearch for more papers by this authorElaine Mayes, Elaine Mayes Chester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, Fulham Road, London, SW3 6JB, EnglandSearch for more papers by this authorErnest W. Johns, Ernest W. Johns Chester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, Fulham Road, London, SW3 6JB, EnglandSearch for more papers by this author First published: December 29, 1980 https://doi.org/10.1016/0014-5793(80)80453-4Citations: 75 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat References 1 J.D. McGhee, G. Felsenfeld, Ann. Rev. Biochem., 49, (1980), 1115– 1156. 2 J.M. Walker, G.H. Goodwin, B.J. Smith, E.W. Johns, E.H. Stotz A. Neuberger Comprehensive Biochemistry 19B, (1980), Elsevier/North-Holland Amsterdam, New York 3 A. Rabbani, G.H. Goodwin, E.W. Johns, Biochem. Biophys. Res. Commun., 81, (1978), 351– 358. 4 R. Sterner, L.C. Boffa, G. Vidali, J. Biol. Chem., 253, (1978), 3830– 3836. 5 D.C. Watson, E.H. Peters, G.H. Dixon, Eur. J. Biochem., 74, (1977), 53– 60. 6 Rabbani, A., Goodwin, G. H., Walker, J. M., Brown, E. and Johns, E. W. (1980) 109, 294–298. 7 S. Spiker, J.K.W. Mardian, I. Isenberg, Biochem. Biophys. Res. Commun., 82, (1978), 129– 135. 8 C.R. Alfageme, G.T. Rudkin, L.H. Cohen, Proc. Natl. Acad. Sci. USA, 73, (1976), 2038– 2042. 9 G.H. Goodwin, R.H. Nicolas, E.W. Johns, Biochim. Biophys. Acta, 405, (1975), 280– 291. 10 G.H. Goodwin, A. Rabbani, R.H. Nicolas, E.W. Johns, FEBS Lett., 80, (1977), 413– 416. 11 C. Sanders, PhD Thesis, London University, (1975), 12 G.H. Goodwin, L. Woodhead, E.W. Johns, FEBS Lett., 73, (1977), 85– 88. 13 J.M. Walker, J.R.B. Hastings, E.W. Johns, Eur. J. Biochem., 76, (1977), 461– 468. 14 J.M. Walker, G.H. Goodwin, E.W. Johns, FEBS Lett., 100, (1979), 394– 398. 15 W.B. Levy, G.H. Dixon, Nucl. Acids. Res., 5, (1978), 4155– 4163. 16 S. Weisbrod, H. Weintraub, Proc. Natl. Acad. Sci. USA, 760, (1979), 630– 634. 17 J.M. Walker, B.P. Parker, E.W. Johns, Int. J. Pept. Prot. Res., 12, (1978), 269– 276. 18 J.M. Walker, K. Gooderham, E.W. Johns, Biochem. J., 181, (1979), 659– 665. 19 M.W. Hunkapiller, L.E. Hood, Biochemistry, 17, (1978), 2124– 2129. 20 J.M. Walker, J.R.B. Hastings, E.W. Johns, Nature, 271, (1978), 281– 21 J.M. Walker, G.H. Goodwin, E.W. Johns, P. Wietzes, W. Gaastra, Int. J. Pept. Prot. Res., 9, (1977), 220– 223. 22 J.M. Kinkade, R.D. Cole, J. Biol. Chem., 241, (1966), 5790– 5797. 23 J.M. Kinkade, R.D. Cole, J. Biol. Chem., 241, (1966), 5798– 5805. 24 J.M. Walker, G.H. Goodwin, E.W. Johns, Eur. J. Biochem., 62, (1976), 461– 469. 25 R. Sterner, G. Vidali, V. Allfrey, J. Biol. Chem., 254, (1979), 11577– 11583. Citing Literature Volume122, Issue2December 29, 1980Pages 264-270 ReferencesRelatedInformation

Nucleosome cores have two specific binding sites for nonhistone chromosomal proteins HMG 14 and HMG 17.

The binding of HMG 14 (or 17) to nucleosome cores produces two major additional bands on nondenaturing polyacrylamide gels. Bound HMG 14 alters the relative densities of the end-labeled DNA fragment distribution produced by deoxyribonuclease I digestion of reconstructed poly(deoxyadenylate-deoxythymidylate) nucleosome cores. These results indicate nucleosome cores have two specific binding sites for HMG 14 (or 17).

Chromatin subunits elicit species-specific antibodies against nucleoprotein antigenic determinants.

Nucleosomes composed of 195 base pairs of DNA associated with histones H2A, H2B, H3, and H4 purified from chicken erythrocyte nuclei were used to elicit antibodies in rabbits. Specific serological reaction between the antisera and the nucleosomes is demonstrated by immunodiffusion, immunofluorescence, microcomplement fixation, solid-phase radioimmunoassay, immunosedimentation, and polyacrylamide gel electrophoresis of 5'-32P end-labeled nucleosomes. The antisera did not react with DNA extracted from these nucleosomes, core histones, or the cross-linked histone octamer from chicken erythocytes, calf thymus total histones, or chromosomal proteins HMG-1 or HMG-17. Nucleosome antigenicity was not affected by redigestion with micrococcal nuclease. Digestion with DNase I brought about 50% loss of reactivity while digestion with trypsin or proteinase K resulted in total loss of activity. The antisera reacted strongly with trimer, dimer, and monomer nucleosomes as well as with the core particle (145 base pairs of DNA) and subnucleosome (greater than 145 base pairs) obtained from chicken. It reacted less well with nucleosomes obtained from HeLa cells and was almost totally devoid of activity against chromatin particles obtained from rat liver or wheat germ. Experiments employing the technique of transferring proteins from a polyacrylamide gel to diazobenzyloxymethyl paper and visualization of antigens by autoradiography excluded the possibility that the serum contains antibodies against tissue-specific antigens which are found in small amounts but are very immunogenic. It is concluded that most of the anitbodies in the sera are directed against nucleoprotein antigenic determinants composed of the N-terminal portion of the histones and segments of DNA. Antibody binding is dependent on contact between the histone and DNA segments and is independent of the integrity of the entire nucleosome. Thus, certain histone DNA contacts remain intact even though the structure of the nucleosome has been disrupted.

Quantitative analysis of non-histone chromosomal proteins HMG 14 and HMG 17 by polyacrylamide gel electrophoresis

Quantitative analysis of non-histone chromosomal proteins HMG 14 and HMG 17 by polyacrylamide gel electrophoresis

The isolation of three new high mobility group nuclear proteins

In addition to the four high mobility group non-histone chromosomal proteins HMG 1, 2, 14 and 17 and histone H1, perchloric acid extracts of nuclei contain a number of other smaller low molecular weight proteins. Three of these proteins (HMG 18, 19A, and 19B) have been purified and characterized. Protein HMG 18 has high lysine and alanine contents, resembling histone H1. Proteins HMG 19A and 19B have high contents of basic and acidic amino acids and resemble HMG proteins 1, 2, 14 and 17. N-terminal sequence analyses of the proteins show that they are not degradation products of histones or the other HMG proteins. However, there are sequence similarities between HMG 18 and histone H5, and between HMG 19B and HMG 17, supporting the view that the HMG proteins and the lysine-rich histones are functionally related.

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%.

The primary structure of the nucleosome-associated chromosomal protein HMG 14

The primary structure of the nucleosome-associated chromosomal protein HMG 14