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.     

Binding of high-mobility-group proteins HMG 14 and HMG 17 to DNA and histone H1 as influenced by phosphorylation

We have used affinity chromatography to study the effects of phosphorylation of calf thymus high-mobility-group proteins HMG 14 and HMG 17 on their binding properties towards calf thymus single- and double-stranded DNA and histone H1. Without in vitro phosphorylation, HMG 14 and HMG17 eluted from doble-stranded DNA-columns at 200 mM NaCl. HMG 14 was released from single-stranded DNA-column at 300 mM NaCl and from H1-column at 130 mM NaCl, whereas the corresponding values for HMG 17 were 230 mM and 20 mM, respectively. Phosphorylation of HMG 14 and HMG 17 by cAMP-dependent protein kinase (A-kinase) decreased markedly their affinity (270 mM and 200 mM NaCl, respectively) for single-stranded DNA, whereas HMG 14 phosphorylated by nuclear protein kinase II (NII-kinase) eluted only slightly (290 mM NaCl) ahead of the unphosphorylated protein. HMG 14 phosphorylated by both A-kinase and NII-kinase eluted from double-stranded DNA-columns almost identically (190 mM NaCl) with the unphosphorylated protein. Interestingly, phosphorylation of HMG 14 by NII-kinase increased considerably its affinity for histone H1 and the phosphorylated protein eluted at 200 mM NaCl. Phosphorylation of HMG 14 by A-kinase did not alter its interaction towards histone H1. These results indicate that modification of HMG 14 by phosphorylation at specific sites may have profound effects on its binding properties towards DNA and histone H1, and that HMG 17 has much weaker affinity for single-stranded DNA and histone H1 than HMG 14.     

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.     

The characterisation of 1SF monomer nucleosomes from hen oviduct and the partial characterisation of a third HMG14/17-like in such nucleosomes.

Nucleosomes released from oviduct nuclei during brief micrococcal nuclease digestions are enriched in transcribed sequences (bloom K.S. and Anderson, J.N. (1978) Cell, 15, 141-150). Such nucleosomes released into this 1Sf supernatant fraction are enriched in proteins HMG14, 17 and a third lower molecular weight protein which we show in this paper to be related to HMG14 and 17. This protein, which we call HMGY, runs as a doublet on polyacrylamide gels. A similar doublet is present in smaller quantities in chicken erythrocyte nuclei. Monomer nucleosomes in the 1SF supernatant have been separated by polyacrylamide gel electrophoresis into two main bands. The slower moving band contains the three HMG proteins HMG14, 17 and Y but lacks histone H1.     

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.     

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.     

Polyamines and heparin do not appreciably influence phosphorylation of chromatin proteins HMG 14 and HMG 17 by nuclear protein kinase II

Phosphorylation of acidic substrates such as casein and phosvitin by nuclear protein kinase II is stimulated by polyamines and inhibited by heparin, which mimics an endogenous proteoglycan inhibitor. The phosphorylation in vitro of the chromatin proteins HMG 14 and HMG 17 by nuclear protein kinase II were examined in this study focusing on the modifying effects of polyamines and heparin. Both HMG proteins were phosphorylated by the enzyme, but polyamines did not appreciably influence the extent of their phosphorylation. In addition, heparin did not inhibit the kinase reaction with the HMG proteins as substrates. These results indicate that the nuclear protein kinase II does actively phosphorylate HMG 14 and HMG 17 in vitro but that in contrast to some model substrates, polyamines and heparin do not appreciably affect their phosphorylation.     

Preferential in vitro binding of high mobility group proteins 14 and 17 to nucleosomes containing active and DNase I sensitive single-copy genes

High mobility group (HMG) proteins 14 and 17 bind to mononucleosomes in vitro, but the exact nature of this binding has not been clearly established. A new method was developed to allow direct membrane transfer of DNA from HMG 14/17 bound and unbound nucleosomes, which have been separated by acrylamide gel electrophoresis. Hybridization analysis of membranes obtained by this method revealed that the HMG 14/17 bound nucleosomes of avian erythrocytes and rat hepatic tumor (HTC) cells were enriched, about 2-fold, in actively transcribed genes and also inactive but DNase I sensitive genes. Nucleosomes containing inactive, DNase I resistant genes were bound by HMG 14/17, but not preferentially. Several factors that have been reported to greatly influence the binding of HMG 14/17 to nucleosomes in vitro were tested and shown to not account for the preferential binding to DNase I sensitive chromatin. These factors include nucleosomal linker DNA length, single-stranded DNA nicks, and DNA bulk hypomethylation. An additional factor, histone acetylation, was preferentially associated with the HMG 14/17 bound chromatin fraction of avian erythrocytes, but it was not associated with the HMG 14/17 bound chromatin fraction of metabolically active HTC cells. The latter finding was true for all kinetic forms of histone acetylation.     

The in vitro reconstitution of nucleosome and its binding patterns with HMG1／2 and HMG14／17 proteins

Using atomic force microscopy (AFM), the dynamic process of the in vitro nucleosome reconstitution followed by slow dilution from high salt to low salt was visualized. Data showed that the histone octamers were dissociatedfrom DNA at 1M NaC1. When the salt concentration was slowly reduced to 650 mM and 300 mM, the core histones bound to the naked DNA gradually. Once the salt concentration was reduced to 50 mM the classic “beads-on-a-string“ structure was clearly visualized. Furthermore, using the technique of the in vitro reconstitution of nucleosome,the mono- and di- nucleosomes were assembled in vitro with both HS2core (-10681 to -10970 bp) and NCR2 (-372to -194 bp) DNA sequences in the 5‘flanking sequence of human b-globin gene. Data revealed that HMG 1/2 and HMG 14/17 proteins binding to both DNA sequences are changeable following the assembly and disassembly of nucleosomes. We suggest that the changeable binding patterns of HMG 14/17 and HMG1/2 proteins with these regulatory elements may be critical in the process of nucleosome assembly, recruitment of chromatin-modifying activities, and the regulation of human b-globin gene 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.     

Isolation of oligonucleosomes from active chromatin using HMG17-specific monoclonal antibodies.

We report the preparation of HMG17-containing oligonucleosomes from chicken embryos and from liver and oviduct of laying hens. Monoclonal antibodies against HMG17 were used for their isolation. An unusual size distribution with respect to their repeat number was observed. The oligonucleosomes of repeat number up to N6 were highly enriched for DNA of the vitellogenin II gene (liver) and for DNA of the ovalbumin and lysozyme genes (oviduct).     

Histone H1 and HMG 14/17 are deposited nonrandomly in the nucleus.

We have studied the assembly of histone H1 and the high mobility group nonhistones 14/17 by isopycnic analysis after crosslinking density labeled MSB cell nuclei or chromatin. Carbodiimide crosslinking produces dense poly-H1 and hybrid density H1-H2A histone dimers, indicating that new H1 is deposited nonrandomly, albeit nonconservatively relative to new core histones. Core histone-HMG crosslinking with succinimidyl propionate yields dense HMG 14 in uniformly dense particles and new HMG 17 crosslinked to both dense and light protein, implying that HMG 14 and 17 each deposit nonrandomly; but differently with respect to new core octamers. Propionimidate crosslinking yields dense H1-HMG 17 dimers, suggesting that the interactions of new 14/17 with H1 (new HMG 14-old H1, new HMG 17-new H1) are reciprocal to their interactions with the core histones.     

Properties of P-form DNA as revealed by circular dichroism

Investigations of DNA using CD spectroscopy show that the P-form is available in a wide variety of methanol–ethanol mixtures when the water content is low. Increasing the temperature or the ethanol content of a 95% methanol solution causes DNA to undergo a cooperative transition to the P-form. However, this transition cannot be reversed on cooling, or on adding methanol. Thus P-form DNA appears to be stable at high methanol concentrations, but it is usually not observed because the DNA is trapped by a kinetic barrier. P-form DNA will instantaneously assume the native B-form on addition of water, confirming earlier reports that P-form DNA is not strand separated [E. Kay (1976) Biochemistry 15 , 5241]. CD spectra extended to 190 nm show that there is no base–base interaction in the P-form. However, the P-form is extremely stable to heat denaturation in solvents which promote hydrogen bonding between the base pairs. A number of models that can account for the properties of P-form DNA are discussed.     

Thermal denaturation and fluorescence study of nucleosomes containing non-histone chromosomal protein HMG2

Interaction of calf thymus non-histone chromosomal protein HMG2 with H1,H5-depleted nucleosomes from chicken erythrocytes was studied by means of thermal denaturation and an N-(3-pyrene)maleimide fluorescence probe. Under low ionic conditions (2 mM Tris buffer plus EDTA) addition of 1-2 molecules of HMG2 per nucleosome markedly stabilized the segment of the linker DNA against thermal denaturation. Under approximately physiological ionic conditions (0.1 M NaCl) addition of two HMG2 molecules per nucleosome, labeled by N-(3-pyrene)maleimide at the sulfhydryl groups of Cys-110 of histones H3, resulted in a decrease of the pyrene excimer fluorescence corresponding to the slight movement of the sulfhydryl groups of the two histone H3 molecules apart.