Binding of HMG14 non-histone protein to histones H2A, H2B, H1 and DNA in reconstituted chromatin

The interaction between calf thymus HMG14 and rat liver chromatin components has been studied via reconstitution and chemical cross-linking. Selective labeling of HMG14 with photoactivable reversible heterobifunctional reagents has allowed a clear identification of the histones interacting with it (histones H2A, H2B and H1). These results are not dependent on whether the chromatin samples used were bulk chromatin, mononucleosomes, or core particles (for H2A and H2B). In addition to histone proteins, DNA also seems to be involved in HMG14 attachment to nucleosome.     

Identification of the core-histone-binding domains of HMG1 and HMG2

High mobility group (HMG) nonhistone chromosomal proteins are a group of abundant, conservative and highly charged nuclear proteins whose physiological role in chromatin is still unknown. To gain insight into the interactions of HMG1 and HMG2 with the fundamental components of chromatin we have introduced the methodology of photochemical crosslinking. This technique has allowed us to study the interaction of HMG1 and HMG2 with the core histones, in the form of an H2A X H2B dimer and an (H3 X H4)2 tetramer, for an effective time of crosslinking of less than 1 ms and under very mild conditions. This is achieved by using flash photolysis. With this procedure we found that both HMG1 and HMG2 interact with H2A X H2B and also with (H3 X H4)2. In the second case, they seem to do this through histone H3. To obtain more information about the interactions, we split HMG1 and HMG2 into their peptides using staphylococcal proteinase. The peptides obtained, which reflect the domain distribution of these proteins, were then used along with the histone oligomers to elucidate their interactions by means of photochemical crosslinking. Results obtained indicate that the domain of HMG1 and HMG2 involved in the interaction with H2A X H2B histones is the highly acidic C-terminal, whereas the N-terminal is involved in the interactions with (H3 X H4)2 histones. In all cases, the interactions found appear appreciably strong. Along with other data published in the literature, these proteins appear to have at least one binding site per domain for the chromatin components.     

Interaction of non-histone proteins HMG1 and HMG2 with core histones in nucleosomes and core particles revealed by chemical cross-linking

Chemical cross-linking was used to study the interaction of the non-histone chromosomal proteins HMG1 and HMG2 with core histones in H1,H5-depleted nucleosomes or core particles. Cross-linking with a 'zero-length' cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and with a longer (cleavable) cross-linker dimethyl-3,3'-dithiobispropionimidate revealed an interaction of HMG1 and HMG2 with (or proximity to) core histones in both types of particles. These results indicated that the presence of the 40-50-base-pairs-long segment of the 'linker' DNA in nucleosomes was not necessary for the establishment of mutual contacts of HMG1 and HMG2 proteins with core histones. Possible implications of the interaction of HMG1 and HMG2 proteins with histones for the structure and functioning of chromatin are discussed.     

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.     

Binding of non-histone chromosomal protein HMG1 to histone H3 in nucleosomes detected by photochemical cross-linking

The interaction of non-histone chromosomal protein HMG1 with core histones in nucleosomes was studied via reconstitution and photochemical cross-linking. The results obtained indicated that photoaffinity-labeled HMG1 interacted in nucleosomes with histone H3. Similar experiments with peptides derived from HMG1 by V8 protease digestion allowed to identify N-terminal domain of HMG1 (peptide V3) as a binding region for histone H3 in nucleosomes.     

Detection by chemical cross-linking of interaction between high mobility group protein 1 and histone oligomers in free solution

Among the more abundant non-histone proteins is the high mobility group (HMG), with an unknown role in chromatin. We have investigated, by chemical cross-linking, the interaction of the protein HMG 1 with the histone dimer H2A X H2B and the histone tetramer (H3 X H4)2 in free solution. Cross-linking with dimethyl suberimidate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the cleavable cross-linker dimethyl-3,3'-dithiobispropionimidate, by two-dimensional electrophoresis reveals the existence of an interaction between HMG 1 and the histone dimer, and also between HMG 1 and the histone tetramer. In the case of the H2A X H2B dimer, the analysis of the patterns of the cross-linking products shows the presence of a trimer, (H2A X H2B) X HMG 1, and of another oligomer of higher molecular weight which also contains H2A X H2B and HMG 1. Non-histone HMG 1 has been found to interact with (H3 X H4)2, both by cross-linking kinetics and also by gel permeation chromatography, displaying a stoichiometry of one HMG 1/histone tetramer. The results have been interpreted as indicating the existence of an interaction between HMG 1 and both oligomers through two different binding sites.     

Interaction of high mobility group proteins HMG 1 and HMG 2 with nucleosomes studied by gel electrophoresis

The binding of isolated high mobility group proteins HMG (1+2) with nucleosomes was studied using gel electrophoresis. The interaction of HMG (1+2) with mononucleosomes could be detected as a new discrete electrophoretic band with a decreased mobility only after cross-linking of HMG (1+2)-nucleosome complex by formaldehyde. Approximately two molecules of the large HMG proteins were bound per nucleosomal particle of a DNA length of 185 base pairs, lacking histones H1 and H5. Using the same techniques, no binding was observed with core particles of a DNA length of 145 base pairs.     

Phosphorylation of high-mobility-group protein 14 by two specific kinases modifies its interaction with histone oligomers in free solution

Chromosomal protein HMG14 can be specifically phosphorylated by the cyclic AMP-dependent protein kinase at the N-terminus and by casein kinase 2 at the acidic C-terminus. Under the same conditions used for HMG14, HMG17 is not significantly phosphorylated by either of the two kinases. Further, we have studied the effect of phosphorylation by these kinases on the interaction of HMG14 with histone oligomers, using chemical cross-linking. Our results indicate that the phosphorylation of HMG14 by casein kinase 2 enhances its interaction with histone oligomers in free solution, whereas a minor effect was observed by phosphorylation with cyclic AMP-dependent protein kinase. In contrast, HMG17 does not interact at all with any histone oligomer in free solution under the conditions used. To gain insight into the possible effect that phosphorylation may play in vivo, the pattern of distribution among different chromatin fractions was analysed. It was found that, although phosphorylation of HMG14 by both kinases allowed reconstitution of HMG14 to chromatin, the patterns obtained showed some slight differences.     

Interactions between HMG proteins and the core sequence of DNaseI hypersensitive site 2 in the locus control region (LCR) of the human β-like globin gene cluster

HMG proteins are abundant chromosomal non-histone proteins. It has been suggested that the HMG proteins may play an important role in the structure and function of chromatin. In the present study, the binding of HMG proteins (HMG1/2 and HMG14/17) to the core DNA sequence of DNaseI hypersensitive site 2 (HS2core DNA sequence, -10681-10970 bp) in the locus control region (LCR) of the human β-like globin gene cluster has been examined by using both thein vitro nucleosome reconstitution and the gel mobility shift assays. Here we show that HMG1/2 can bind to the naked HS2core DNA sequence, however, HMG14/17 cannot. Using thein vitro nucleosome reconstitution we demonstrate that HMG14/17 can bind to the HS2core DNA sequence which is assembled into nucleosomes with the core histone octamer transferred from chicken erythrocytes. In contrast, HMG1/2 cannot bind to the nucleosomes reconstitutedin vitro with the HS2core DNA sequence. These results indicate that the binding patterns between HMG proteins and the HS2core DNA sequence which exists in different states (the naked DNA or thein vitro reconstituted nucleosomal DNA) are quite different. We speculate that HMG proteins might play a critical role in the regulation of the human β-like globin gene’s expression.     

DNA intercalators induce specific release of HMG 14, HMG 17 and other DNA-binding proteins from chicken erythrocyte chromatin.

Chicken erythrocyte nuclei were incubated with DNA intercalating agents in order to isolate from chromatin specific DNA-binding proteins whose binding specificity may be determined by DNA secondary and/or tertiary structure. The intercalating agents ethidium bromide (EtBr) and propidium iodide induce the specific release of high mobility group proteins HMG 14 and HMG 17 under low ionic strength conditions. Chloroquine (CQ) intercalation also results in the selective liberation of HMG 14 and HMG 17, but, in addition, selectively releases other nuclear proteins (including histone H1A) in a pH- and ionic strength-dependent fashion. The use of this new 'elutive intercalation' technique for the isolation and purification of 'sequence-specific' and 'helix-specific' DNA-binding proteins is suggested.     

The effect of a high mobility group protein (HMG 17) on the structure of acetylated and control core HeLa cell chromatin

The effect of binding a high mobility group protein (HMG 17) on the stability and conformation of acetylated and control HeLa high molecular weight core chromatin (stripped of H1 and non-histone chromosomal proteins) was studied by circular dichroism and thermal-denaturation measurements. Previously it had been shown that conformational differences exist between native whole chromatin derived from butyrate-treated (acetylated) and control HeLa cells and that these conformational differences disappear by removing H1 and non-histone chromosomal proteins (Reczek, P.R., Weissman, D., Huvos, P.E. and Fasman, G.D. (1982) Biochemistry 21, 993–1002). The circular dichroism spectra and the thermal denaturation profiles of control and acetylated core chromatin were found to be similar. The circular dichroism properties of HMG 17 reconstituted highly acetylated and control core chromatin indicated the same alteration of chromatin structure at low ionic strength (1 mM sodium phosphate/0.25 mM EDTA, pH 7.0). The magnitudes of the decrease in ellipticity were proportional to the amount of HMG 17 bound and were found to be the same for both the acetylated and control core chromatin. Thermal denaturation profiles confirmed this change in structure induced by HMG 17 on control and highly acetylated core chromatin. The thermal denaturation profiles, which were resolved into three component transitions, exhibited a shifting of hyperchromicity from the lower melting transitions to the higher melting transitions, with a concomitant rise in T_m, on HMG 17 binding to both control and acetylated chromatin. The natures of the interactions of HMG 17 at higher ionic strength (50 mM NaCl/0.25 mM EDTA/1 mM sodium phosphate, pH 7.0) with acetylated and control core chromatin were slightly different, as measured by circular dichroism; however, a decrease in ellipticity was observed for both samples upon binding of HMG 17. These observations suggest that acetylation coupled with HMG 17 binding to core chromatin does not loosen chromatin structure. HMG 17 binding to control and acetylated core chromatin produces an overall stabilization and compaction of chromatin structure.     

Response of high mobility group proteins of human kidney T1 and murine L 929 cell lines to heat shock

High mobility group (HMG) proteins in human kidney T1 and murine L 929 cells have been investigated after exposure to heat shock at 41 degrees C and their influence on the organizational change of chromatin under heat shock condition has been examined. Results reveal that the two cell lines show differential response of the HMG proteins 1 & 2 and 14 & 17 to heat shock. Neither T1 nor L 929 cells show significant differences in response to heat shock with respect to the binding affinities of HMG proteins 1 & 2 or 14 & 17 to DNA, as revealed by DNase I sensitivity and chromatin reconstitution assays. Furthermore, the HMG proteins of both the non-heat shocked and the heat shocked T1 and L 929 cells can recover their chromatin activity following reconstitution. These findings suggest that although the HMG proteins might undergo some change in response to heat shock, their inherent potential of reassociation with DNA is still retained.     

Evidence for a shared structural role for HMG1 and linker histones B4 and H1 in organizing chromatin.

The high mobility group proteins 1 and 2 (HMG1/2) and histone B4 are major components of chromatin within the nuclei assembled during the incubation of Xenopus sperm chromatin in Xenopus egg extract. To investigate their potential structural and functional roles, we have cloned and expressed Xenopus HMG1 and histone B4. Purified histone B4 and HMG1 form stable complexes with nucleosomes including Xenopus 5S DNA. Both proteins associate with linker DNA and stabilize it against digestion with micrococcal nuclease, in a similar manner to histone H1. However, neither histone B4 nor HMG1 influence the DNase I or hydroxyl radical digestion of DNA within the nucleosome core. We suggest that HMG1/2 and histone B4 have a shared structural role in organizing linker DNA in the nucleosome.     

Interaction of HMG 14 and 17 with actively transcribed genes

The interaction of HMG 14 and 17 with actively transcribed genes was studied by monitoring the sensitivity of specific genes to DNAase I after reconstitution of HMG-depleted chromatin with HMG 14 and 17. Our experiments lead to the following conclusions: most actively transcribed genes become sensitized to DNAase I by HMG 14 and 17; either HMG 14 or HMG 17 can sensitize most genes to DNAase I; genes transcribed at different rates have about the same affinity for HMG 14 and 17; HMG 14 and 17 bind stoichiometrically to actively transcribed nucleosomes; and HMG 14 and 17 can restore DNAase I sensitivity to purified nucleosome core particles depleted of HMGs. This last observation suggests that during reconstitution, low levels of HMG 14 and 17 can associate with the active nucleosomes in the presence of a 10–20 fold excess of inactive nucleosomes. Consequently, we conclude that besides their association with HMGs, active nucleosomes also have at least one other unique feature that distinguishes them from bulk nucleosomes and insures proper HMG binding during reconstitution.