The interaction of high mobility proteins HMG14 and 17 with nucleosomes.

The interaction of the high mobility group proteins, HMG14 and HMG17, with nucleosome core particles has been studied. The results show that two molecules of HMG14/17 can be bound tightly but reversibly to each core particle and that their affinity for core particles is greater than their affinity for histone-free DNA of core size. Thermal denaturation and nuclease digestion studies suggest that major sites of interaction are located near the ends of the nucleosome core DNA. When nucleosome preparations from chicken erythrocyte nuclei stripped of HMG proteins are partially titrated with HMG14/17, the nucleosome-HMG complex fraction is enriched in beta-globin gene sequences.     

Primary organization of nucleosomes. Interaction of non-histone high mobility group proteins 14 and 17 with nucleosomes, as revealed by DNA-protein crosslinking and immunoaffinity isolation

The binding sites for histones and high mobility group proteins (HMG) 14 and 17 have been located on DNA in the nucleosomal cores and H1/H5-containing nucleosomes. The nucleosomes were specifically associated with two molecules of the non-histone proteins HMG 14 and/or HMG 17 when followed by DNA-protein crosslinking and immunoaffinity isolation of the crosslinked HMG-DNA complexes. HMGs 14 and 17 were shown to be crosslinked in a similar manner to each core DNA strand at four sites: to both 3' and 5' DNA ends and also at distances of about 25 and 125 nucleotides from the 5' termini of the DNA. These sites are designated as HMG(143), (0), (25) and (125). The site HMG(125) is located at the place where no significant histone-DNA crosslinking was observed. The HMG(125) and HMG(25) sites lie opposite one another on the complementary DNA strands across the minor DNA groove and are placed, similarly to histones, on the inner side of the DNA superhelix in the nucleosome. The crosslinking of HMG 17 to the 3' ends of the DNA is much weaker than that of HMG 14. These data indicate that each of two molecules of HMG 14 and/or HMG 17 is bound to the double-stranded core DNA at two discrete sites: to the 3' and 5' ends of the DNA and at a distance of 20 to 25 base-pairs from each DNA terminus inside the nucleosome on a histone-free DNA region. Binding of HMG 14 or 17 does not induce any detectable rearrangement of histones on DNA and both HMGs seem to choose the same sites for attachment in nucleosomal cores and H1/H5-containing nucleosomes.     

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 b-like globin gene cluster has been examined by using both the in 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 the in 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 reconstituted in 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 the in 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.     

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.     

Nonhistone nuclear high mobility group proteins 14 and 17 stabilize nucleosome core particles

Nucleosome core particles form well defined complexes with the nuclear nonhistone proteins HMG 14 or 17. The binding of HMG 14 or 17 to nucleosomes results in greater stability of the nucleosomal DNA as shown by circular dichroism and thermal denaturation. Under appropriate conditions the binding is cooperative, and cooperativity is ionic strength dependent. The specificity and cooperative transitions of high mobility group (HMG) binding are preserved in 1 M urea. Specificity is lost in 4 M urea. Thermal denaturation and circular dichroism show a dramatic reversal of the effects of urea on nucleosomes when HMG 14 or 17 is bound, indicating stabilization of the nucleosome by HMG proteins. Complexes formed between reconstructed nucleosomes containing purified inner histones plus poly(dA-dT) and HMG 14 or 17 demonstrate that the HMG binding site requires only DNA and histones. Electron microscopy reveals no major structural alterations in the nucleosome upon binding of HMG 14 or 17. Cross-linking the nucleosome extensively with formaldehyde under cooperative HMG binding conditions does not prevent the ionic strength-dependent shift to noncooperative binding. This suggests mechanisms other than internal nucleosome conformational changes may be involved in cooperative HMG binding.     

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.     

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.     

Nucleosome core binding region of chromosomal protein HMG-17 acts as an independent functional domain.

Chromosomal proteins HMG-14 and HMG-17 have a modular structure. Here we examine whether the putative nucleosome-binding domain in these proteins can function as an independent module. Mobility shift assays with recombinant HMG-17 indicate that synthetic molecules can be used to analyze the interaction of this protein with the nucleosome core. Peptides corresponding to various regions of the protein have been synthesized and their interaction with nucleosome cores analyzed by mobility shift, thermal denaturation and DNase I digestion. A 30 amino acid long peptide, corresponding to the putative nucleosome-binding domain of HMG-17, specifically shifts the mobility of cores as compared to free DNA, elevates the tm of both the premelt and main melt of the cores and protects from DNase I digestion the same nucleosomal DNA sites as the intact protein. The binding of both the peptide and the intact protein is lost upon digestion of the histone tails by trypsin. The nucleosomal binding sites of the peptide appear identical to those of the intact protein. Thus, a region of the protein can acts as an independent functional domain. This supports the notion that HMG-14 and HMG-17 are modular proteins. This finding is relevant to the understanding of the function and evolution of HMG-14/-17, the only nucleosome core particle binding proteins known to date.     

Analysis of the binding of high mobility group protein 17 to the nucleosome core particle by 1H NMR spectroscopy

The binding of high mobility group (HMG) protein 17 to the nucleosome core particle has been studied in D2O solution using 1H NMR at 500 MHz. Spectra were obtained for purified HMG 17, purified nucleosome core particles, and the reconstituted HMG 17-nucleosome core particle complex at 0.1, 0.2, 0.3, and 0.4 M NaCl. Subtraction of the core particle spectra from spectra of the core particle reconstituted with HMG 17 demonstrated those regions of HMG 17 which interact with the nucleosome at different ionic strengths; the resonance peaks of interacting groups are broadened due to their restricted mobility. At 0.1 M NaCl, the mobility of all the amino acid side chains of HMG 17 was restricted, indicating complete binding of HMG 17 to the much larger nucleosome core particle. At 0.2 M NaCl most of the amino acids were free with the exception of arginine and proline which are confined to or predominant in the basic central region of HMG 17. These amino acids were completely free only at 0.4 M NaCl. We conclude that the entire HMG 17 molecule interacts with the nucleosome core particle at physiological ionic strength. The acidic COOH-terminal region of HMG 17 is released from interaction with the core histones at an NaCl concentration between 0.1 and 0.2 M and so binds weakly at physiological ionic strength. The basic central region binds more strongly to the core particle DNA, being completely released only at much higher ionic strength, between 0.3 and 0.4 M NaCl.     

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.     

High mobility group protein 17 cross-links primarily to histone H2A in the reconstituted HMG 17-nucleosome core particle complex

The "neighbor relationship" of lamb thymus high mobility group (HMG) protein 17 to native HeLa nucleosome core particle histones in the reconstituted complex has been studied. 125I-Labeled HMG 17 was cross-linked to core histones using the protein-protein cross-linking reagent 2-iminothiolane. Specific cross-linked products were separated on a two-dimensional Triton-acid-urea/sodium dodecyl sulfate-gel system, located by autoradiography, excised, and quantified. Disulfide bonds in the cross-links were then cleaved, and the protein constituents were identified by sodium dodecyl sulfate-gel electrophoresis. HMG 17 cross-linked primarily to histone H2A while lower levels of cross-linking occurred between HMG 17 and the other histones. In contrast, cross-linking between 2 HMG 17 molecules bound on the same nucleosome core particle was relatively rare. We have concluded that H2A comprises part of the HMG 17 binding site. Less contact occurs between HMG 17 and the other core histones, and there is little contact possible between the 2 bound HMG 17 molecules. These results are in agreement with the current model for the structure of the nucleosome and the proposed binding sites for HMG 17.     

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.     

Conformation of the HMG17-nucleosome complex

The nucleosome core binds more than two molecules of HMG17 at low ionic strength (8.9 mM Tris-HCl/8.9 mM boric acid/0.25 mM Na2EDTA, pH 8.3). Circular dichroism of the complexes showed only minor conformational changes of the nucleosome core DNA on binding of HMG17, with no detectable change in the histone secondary structure. The fluorescence of N-(3-pyrene) maleimide bound to -SH groups at Cys-110 of H3 histones in the core particle suggested that the structure of the histone octamer assembly changed little upon binding of HMG17 to the nucleosome. These observations support the idea that even a high level of HMG17 binding, e.g., four HMGs per nucleosome, alone, does not open up the core particle.     

Conformation of the HMG 14 nucleosome core complex from flow birefringence.

Flow birefringence and extinction angles have been measured for HMG 14 complexes with nucleosome core particles from chicken erythrocytes under cooperative "tight" binding conditions, and for the uncomplexed core particles used in the preparations. Results are interpreted using optical models for the observed DNA anisotropy, and are compared to recent small angle neutron scattering results. (19) The studies effectively rule out highly distorted DNA conformations and configurations in which DNA ends are unwound and extended. It is concluded that the most likely conformation of the complex is one in which the DNA superhelix is radially increased, either uniformly or bilaterally, with the DNA ends remaining tightly bound to the particle. This conformation does not require large changes in spatial relationships between the DNA ends compared to the uncomplexed core as would accompany, for example, significant unwinding of the ends. However, it may lead to more subtle but possibly highly significant differences in the angles at which the DNA exits the core particle.     

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.     

Isolation and characterization of acetylated histones H3 and H4 and their assembly into nucleosomes

Nucleosome and chromatin structure/function relationships of histone acetylations are not understood. To address these questions we have developed chromatographic procedures that separate subtypes of H3 and the acetylated states of histone H3 and H4 in exceptionally pure forms. The sites of acetylation of the intermediately acetylated states of H3 have been determined and show a specific pattern of acetylation. An unexpected finding was the identification of a fifth site of acetylation in H3 at lysine 27. Nucleosome particles with fully acetylated H3 and H4 have been assembled on the Lytechinus variegatus 5 S rRNA DNA phasing sequence and characterized. These defined acetylated H3 and H4 particles migrate more slowly in polyacrylamide nucleoprotein particle gels than the control particles indicating a subtle effect of acetylation in nucleosome structure. However, DNA footprinting of these particles using DNase I show only small changes when compared to control particles over the core particle DNA length. It is shown further that H3 cysteines in the particle containing fully acetylated H3 and H4 were not accessible to iodoacetamide indicating that protein factors additional to H3 and H4 acetylation are required to make H3 cysteines accessible to the label. These findings are consistent with the proposal that histones H3, H4 acetylations exert their major effects outside of the core particle 146-base pair DNA, either on the DNA segment entering and leaving the nucleosome or possibly on the internucleosome interactions that involve the amino-terminal domains of the core histones in organization and stability of higher order chromatin structures.     

Binding of C-reactive protein to chromatin and nucleosome core particles. A possible physiological role of C-reactive protein

By using a variety of biochemical techniques, chromatin and chromatin fragments have been identified as probable physiological ligands for C-reactive protein. Studies using 14C-labeled C-reactive protein show that binding to chromatin is saturable with a Kd = 8 X 10(-7) M, a value indicating that the affinity of C-reactive protein for chromatin is at least four times its affinity for phosphorylcholine. At saturation, there is approximately one C-reactive protein-binding site for every 160 base pairs of DNA in chromatin. The interaction of C-reactive protein with chicken erythrocyte nucleosome core particle has been studied. Fifty per cent inhibition of the binding of C-reactive protein to phosphorylcholine is obtained at a core particle concentration of 1.25 X 10(-9) M, indicating that the affinity of C-reactive protein for one of the sites on core particles is at least 2400 times greater than the affinity of C-reactive protein for phosphorylcholine. The possibility that C-reactive protein may act as a scavenger for chromatin fragments released from damaged cells is discussed.     

A nonuniform distribution of excision repair synthesis in nucleosome core DNA

We have investigated the distribution in nucleosome core DNA of nucleotides incorporated by excision repair synthesis occurring immediately after UV irradiation in human cells. We show that the differences previously observed for whole nuclei between the DNase I digestion profiles of repaired DNA (following its refolding into a nucleosome structure) and bulk DNA are obtained for isolated nucleosome core particles. Analysis of the differences obtained indicates that they could reflect a significant difference in the level of repair-incorporated nucleotides at different sites within the core DNA region. To test this possibility directly, we have used exonuclease III digestion of very homogeneous sized core particle DNA to "map" the distribution of repair synthesis in these regions. Our results indicate that in a significant fraction of the nucleosomes the 5' and 3' ends of the core DNA are markedly enhanced in repair-incorporated nucleotides relative to the central region of the core particle. A best fit analysis indicates that a good approximation of the data is obtained for a distribution where the core DNA is uniformly labeled from the 5' end to position 62 and from position 114 to the 3' end, with the 52-base central region being devoid of repair-incorporated nucleotides. This distribution accounts for all of the quantitative differences observed previously between repaired DNA and bulk DNA following the rapid phase of nucleosome rearrangement when it is assumed that linker DNA and the core DNA ends are repaired with equal efficiency and the nucleosome structure of newly repaired DNA is identical with that of bulk chromatin. Furthermore, the 52-base central region that is devoid of repair synthesis contains the lowest frequency cutting sites for DNase I in vitro, as well as the only "internal" locations where two (rather than one) histones interact with a 10-base segment of each DNA strand.