Binding of wheat and chicken high mobility group chromosomal proteins to DNA and to wheat and chicken mononucleosomes

We have used an electrophoretic retardation assay to investigate the interactions of wheat high mobility group (HMG) proteins with DNA and with isolated trimmed mononucleosomes (complexes which contain a histone octamer and approximately 146 base pairs of DNA). In order to characterize these interactions, we have compared the binding of each of the wheat HMG proteins, HMGa, b, c, and d, with those of the low molecular weight chicken HMG proteins HMG14 and 17. These vertebrate animal HMG proteins have previously been shown to occupy two specific binding sites on animal nucleosomes and to have a greater affinity for nucleosomes than for naked DNA (Mardian, J. K. W., Paton, A. E., Bunick, G. J., and Olins, D. E. (1980) Science 209, 1534-1536; Sandeen, G., Wood, W. I., and Felsenfeld, G. (1980) Nucleic Acids Res. 8, 3757-3778). As a criterion for "specific binding," we have used the property of HMG14 and 17 binding of causing a discontinuous shift of nucleosomes to a distinct band of lower electrophoretic mobility. According to this criterion, wheat HMGb, c, and d do not bind nucleosomes specifically. These HMG proteins have approximately the same affinity for nucleosomes and naked DNA. Wheat HMGa does bind nucleosomes specifically by this criterion, but other aspects of the binding are reminiscent of histone H1-nucleosome binding. We present evidence that trimmed mononucleosomes of wheat are conformationally distinct from their animal counterparts. Despite the conformational differences, competition studies indicate that chicken and wheat mononucleosomes have essentially identical affinity for the low molecular weight animal HMG proteins.     

Effect of HMG protein 17 on the thermal stability of control and acetylated HeLa oligonucleosomes.

Many studies have implicated histone acetylation and HMG proteins 14 and 17 in the structure of active chromatin. Studies of the binding of HMG 14 and 17 to chromatin core particles have shown that there are two binding sites for HMG 14 or 17 located within 20-25 bp of the DNA ends of the core particles [13-15]. Such binding sites may result from the free DNA ends in the core particle being available for the binding of HMG 14 and 17. We have studied the effects of the binding of HMG 17 on the thermal denaturation of DNA in mono, di and trinucleosomes. In each case the binding of 1 HMG 17 molecule per nucleosome reduces the DNA premelt region by 50%, while the binding of 2 HMG 17 molecules per nucleosome abolishes the premelt region. From this it is concluded that there are two HMG 17 binding sites per nucleosome which are located between the entry and exit points to the nucleosome and the strongly complexed central DNA region. Highly acetylated mono, di and trinucleosomes have been isolated from butyrate treated HeLa S3 cells. For this series of acetylated oligonucleosomes, it has been found that there are also two HMG 17 binding sites per acetylated nucleosome.     

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.     

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.     

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.     

Interaction of maize chromatin-associated HMG proteins with mononucleosomes: role of core and linker histones

Two groups of plant chromatin-associated high mobility group (HMG) proteins, namely the HMGA family, typically containing four A/T-hook DNA-binding motifs, and the HMGB family, containing a single HMG-box DNA-binding domain, have been identified. We have examined the interaction of recombinant maize HMGA and five different HMGB proteins with mononucleosomes (containing approx. 165 bp of DNA) purified from micrococcal nuclease-digested maize chromatin. The HMGB proteins interacted with the nucleosomes independent of the presence of the linker histone H1, while the binding of HMGA in the presence of H1 differed from that observed in the absence of H1. HMGA and the HMGB proteins bound H1-containing nucleosome particles with similar affinity. The plant HMG proteins could also bind nucleosomes that were briefly treated with trypsin (removing the N-terminal domains of the core histones), suggesting that the histone N-termini are dispensable for HMG protein binding. In the presence of untreated nucleosomes and trypsinised nucleosomes, HMGB1 could be chemically crosslinked with a core histone, which indicates that the trypsin-resistant part of the histones within the nucleosome is the main interaction partner of HMGB1 rather than the histone N-termini. In conclusion, these results indicate that specific nucleosome binding of the plant HMGB proteins requires simultaneous DNA and histone contacts.     

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.     

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.     

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.     

Reconstitution of mononucleosomes: characterization of distinct particles that differ in the position of the histone core

Reconstitution of mononucleosomes from DNA and core histones was carried out to study the positioning of histone octamers on the DNA. Using random DNA molecules in the 200 to 250 bp size range we found that the reconstitution products consisted of a mixture of three different types of particles that could be separated by low ionic strength gel electrophoresis. In one particle, DNA was complexed with histones along its entire length indicating the binding of more than one histone octamer. The second particle contained only one histone core that was always associated, however, with the terminal 145 bp of the DNA regardless of its sequence which can be ascribed to a DNA end effect. Only the third particle consisted of histone octamers bound at internal positions of the DNA and is therefore the only particle suitable for investigating the influence of the DNA sequence on the positioning of the histone cores. A defined 154 bp pBR 322 restriction fragment that contains three BspRI restriction sites was also reconstituted with core histones. The accessibility of these sites to BspRI was measured in order to delineate the utility of restriction nucleases as probes for the structure of chromatin. Two sites located close to the center of the DNA were less susceptible by at least a factor of 1000 as compared to free DNA while the susceptibility of the third site in the terminal section of the DNA decreased about 50 fold after reconstitution.     

Alignment of nucleosomes along DNA and organization of spacer DNA in Drosophila chromatin

A series of mono- and dinucleosomal DNAs characterized by an about ten-base periodicity in the size were revealed in the micrococcal nuclease digests of Drosophila chromatin which have 180 +/- 5 base pair (bp) nucleosomal repeat. 20, 30, and 40 bp spacers were found to be predominant in chromatin by trimming DNA in dinucleosomes to the core position. Among several identified mononucleosomes (MN), MN170, MN180 and MN190 were isolated from different sources (the figures indicate the DNA length in bp). The presence of the 10, 20, and 30 bp long spacers was shown in these mononucleosomes by crosslinking experiments. The interaction of histone H3 with the spacer in the Drosophila MN180 particle was also shown by the crosslinking /5/. We conclude from these results that the 10 n bp long intercore DNA (n = 2, 3 and 4) is organized by histone H3, in particular, and together with the core DNA forms a continuous superhelix. Taken together, these data suggest that Drosophila chromatin consists of the regularly aligned and tightly packed MN180, as a repeating unit, containing 10 and 20 bp spacers at the ends of 180 bp DNA. Within the asymmetric and randomly oriented in chromatin MN180, the cores occupy two alternative positions spaced by 10 bp.     

HMG-D is an architecture-specific protein that preferentially binds to DNA containing the dinucleotide TG.

The high mobility group (HMG) protein HMG-D from Drosophila melanogaster is a highly abundant chromosomal protein that is closely related to the vertebrate HMG domain proteins HMG1 and HMG2. In general, chromosomal HMG domain proteins lack sequence specificity. However, using both NMR spectroscopy and standard biochemical techniques we show that binding of HMG-D to a single DNA site is sequence selective. The preferred duplex DNA binding site comprises at least 5 bp and contains the deformable dinucleotide TG embedded in A/T-rich sequences. The TG motif constitutes a common core element in the binding sites of the well-characterized sequence-specific HMG domain proteins. We show that a conserved aromatic residue in helix 1 of the HMG domain may be involved in recognition of this core sequence. In common with other HMG domain proteins HMG-D binds preferentially to DNA sites that are stably bent and underwound, therefore HMG-D can be considered an architecture-specific protein. Finally, we show that HMG-D bends DNA and may confer a superhelical DNA conformation at a natural DNA binding site in the Drosophila fushi tarazu scaffold-associated region.     

The effect of the acidic tail on the DNA-binding properties of the HMG1,2 class of proteins: insights from tail switching and tail removal

The high-mobility group (HMG) proteins HMG1, HMG2 and HMG2a are relatively abundant vertebrate DNA-binding and bending proteins that bind with structure specificity, rather than sequence specificity, and appear to play an architectural role in the assembly of nucleoprotein complexes. They have two homologous "HMG-box" DNA-binding domains (which show about 80 % homology) connected by a short basic linker to an acidic carboxy-terminal tail that differs in length between HMG1 and 2. To gain insights into the role of the acidic tail, we examined the DNA-binding properties of HMG1, HMG2b and HMG2a from chicken erythrocytes (corresponding to HMG1, HMG2 and HMG2a in other vertebrates). HMG1, with the longest acidic tail, is less effective than HMG2a and 2b (at a given molar input ratio) in supercoiling relaxed, closed circular DNA, in inducing ligase-mediated circularisation of an 88 bp DNA fragment, and in binding to four-way DNA junctions in a gel-shift assay. Removal of the acidic tail increases the affinity of the HMG boxes for DNA and largely abolishes the differences between the three species. Switching the acidic tail of HMG1 for that of HMG2a or 2b gives hybrid proteins with essentially the same DNA-binding properties as HMG2a, 2b. The length (and possibly sequence) of the acidic tail thus appears to be the dominant factor in mediating the differences in properties between HMG1, 2a and 2b and finely tunes the rather similar DNA-binding properties of the tandem HMG boxes, presumably to fulfill different cellular roles. The tail is essential for structure-selective DNA-binding of the HMG boxes to DNA minicircles in the presence of equimolar linear DNA, and has little effect on the affinity for this already highly distorted DNA ligand, in contrast to binding to linear and four-way junction DNA.     

Release of nucleosomes from nuclei by bleomycin-induced DNA strand scission

Mononucleosomes were released from both isolated mammalian (hog thyroid) and protozoan (Tetrahymena) nuclei by the bleomycin-induced DNA-strand breaking reaction. Trout sperm nuclei, on the other hand, were protected from the bleomycin-mediated DNA degradation. The mononucleosomes released from the bleomycin-treated nuclei contained the core histones H2A, H2B, H3, and H4; while HMG1 and HMG2 proteins, in addition to the core histones, were detected in the mononucleosomes obtained by micrococcal nuclease digestion of nuclei. HMGs, but not H1 histone, were dissociated into the supernatant by cleavage of chromatin DNA with bleomycin, whereas both HMGs and H1 were found in that fraction by digestion of nuclei with micrococcal nuclease. HMG1 and HMG2 were exclusively dissociated from chromatin with 1 mM bleomycin under the solvent condition where the DNA strand-breaking activity of the drug is repressed. These observations suggest the possibility that bleomycin preferentially binds to linker DNA regions not occupied by H1 histone in chromatin and exclusively dissociates HMG proteins and breaks the DNA strand. The results of the effects on bleomycin-induced DNA cleavage of nuclei of various drugs including polyamines, chelating agents, intercalating antibiotics such as mitomycin C or adriamycin, and radical scavengers are also presented.     

Structure of nucleosomes and organization of internucleosomal DNA in chromatin

We have compared the mononucleosomal pattern produced by micrococcal nuclease digestion of condensed and unfolded chromatin and chromatin in nuclei from various sources with the repeat length varying from 165 to 240 base-pairs (bp). Upon digestion of isolated H1-containing chromatin of every tested type in a low ionic strength solution (unfolded chromatin), a standard series of mononucleosomes (MN) was formed: the core particle, MN145, and H1-containing, MN165, MN175, MN185, MN195, MN205 and MN215 (the indexes give an approximate length of the nucleosomal DNA that differs in these particles by an integral number of 10 bp). In addition to the pattern of unfolded chromatin, digestion of whole nuclei or condensed chromatin (high ionic strength of Ca2+) gave rise to nuclei-specific, H1-lacking MN155. Digestion of H1-lacking chromatin produced only MN145, MN155 and MN165 particles, indicating that the histone octamer can organize up to 165 bp of nucleosomal DNA. Although digestion of isolated sea urchin sperm chromatin (repeat length of about 240 bp) at a low ionic strength gave a typical "unfolded chromatin pattern", digests of spermal nuclei contained primarily MN145, MN155, MN235 and MN245 particles. A linear arrangement of histones along DNA (primary organization) of the core particle was found to be preserved in the mononucleosomes, with the spacer DNA length from 10 to 90 bp on one (in MN155) or both sides of core DNA being a multiple of about 10 bp. In MN235, the core particle occupies preferentially a central position with the length of the spacer DNA on both sides of the core DNA being usually about 30 + 60 or 40 + 50 bp. Histone H1 is localized at the ends of these particles, i.e. close to the centre of the spacer DNA. The finding that globular part of histones H3 and sea urchin sperm H2B can covalently bind to spacer DNA suggests their involvement in the organization of chromatin superstructure. Our data indicate that decondensation of chromatin is accompanied by rearrangement of histone H1 on the spacer DNA sites adjacent to the core particle and thus support a solenoid model for the chromatin superstructure in nuclei in which the core DNA together with the spacer DNA form a continuous superhelix.