Interaction of wheat high-mobility-group proteins with four-way-junction DNA and characterization of the structure and expression of HMGA gene

Plant high-mobility-group (HMG) chromosomal proteins are the most abundant and ubiquitous nonhistone proteins found in the nuclei of higher eukaryotes. There are only two families of HMG proteins, namely, HMGA and HMGB in plants. The cDNA encoding wheat HMGa protein was isolated and characterized. Wheat HMGA cDNA encodes a protein of 189 amino acid residues. At its N terminus, there is a histone H1-like structure, which is a common feature of plant HMGA proteins, followed by four AT-hook motifs. Polymerase chain reaction results show that the gene contains a single intron of 134 bp. All four AT-hook motifs are encoded by the second exon. Northern blot results show that the expression of HMGA gene is much higher in organs undergoing active cell proliferation. Gel retardation analysis show that wheat HMGa, b, c and histone H1 bind to four-way-junction DNA with high binding affinity, but affinity is dramatically reduced with increasing Mg(2+) and Na(+) ion concentration. Competition binding studies show that proteins share overlapping binding sites on four-way-junction DNA. HMGd does not bind to four-way-junction DNA.     

Linker histone interaction shows divalent character with both supercoiled and linear DNA

The interaction of linker histone H1 with both linear and superhelical double-stranded DNA has been investigated at low ionic strengths. Gel mobility retardation experiments demonstrate strikingly different behavior for the two forms of DNA. First, the experiments strongly suggest that linker histone binds to superhelical DNA in a negatively cooperative mode. In contrast, binding of linker histone to linear DNA under the conditions employed here shows no cooperativity. Second, binding of linker histone to linear DNA results in aggregation of histone-DNA complexes, even at very low levels of input histone H1. Because H1 has been shown to interact as a monomer, this aggregation is evidence of the divalent character of the linker histone, for without H1's ability to bind to two duplex strands of DNA, aggregation could not occur. Although aggregation can be made to occur with superhelical DNA, it can do so only at near-saturation levels of input histone H1. Finally, in direct competition, linker histone binds to superhelical DNA to the complete exclusion of linear DNA, indicating that the linker histone's function is related to the crossover structures that differentiate superhelical DNA from linear DNA. We develop a model that explains the observed behavior of binding of linker histone to superhelical DNA that is consistent with both the divalent character of the linker histone and the negative cooperativity by which linker histone and superhelical DNA interact.     

Molecular recognition of histone H3 by the WD40 protein WDR5

The WD40-repeat protein WDR5 is a conserved subunit of Trithorax (TRX) histone methyltransferase complexes. WDR5 has been reported to selectively bind dimethylated Lys4 (K4me2) in histone H3 to promote K4 trimethylation by TRX. To elucidate the basis of this binding specificity, we have determined the crystal structure of WDR5 bound to a histone H3 peptide bearing K4me2. The structure reveals that the N terminus of histone H3 binds as a 3(10)-helix in the central depression formed by the WD40 repeats. R2 in histone H3 is bound in the acidic channel in the protein's core, whereas K4me2 is solvent exposed and does not engage in direct interactions with WDR5. Functional studies confirm that WDR5 recognizes A1, R2 and T3 in histone H3 but has virtually identical affinities for the unmodified and mono-, di- and trimethylated forms of K4, demonstrating that it does not discriminate among different degrees of methylation of this residue.     

Histone stoichiometry and DNA circularization in archaeal nucleosomes.

Recombinant (r)HMfB (archaealhistone B fromMethanothermusfervidus) formed complexes with increasing stability with DNA molecules increasing in length from 52 to 100 bp, but not with a 39 bp molecule. By using125I-labeled rHMfB-YY (an rHMfB variant with I31Y and M35Y replacements) and32P-labeled 100 bp DNA, these complexes, designated archaeal nucleosomes, have been shown to contain an archaeal histone tetramer. Consistent with DNA bending and wrapping, addition of DNA ligase to archaeal nucleosomes assembled with 88 and 128 bp DNAs resulted in covalently-closed monomeric circular DNAs which, following histone removal, were positively supercoiled based on their electrophoretic mobilities in the presence of ethidium bromide before and after relaxation by calf thymus topoisomerase I. Ligase addition to mixtures of rHMfB with 53 or 30 bp DNA molecules also resulted in circular DNAs but these were circular dimers and trimers. These short DNA molecules apparently had to be ligated into longer linear multimers for assembly into archaeal nucleosomes and ligation into circles. rHMfB assembled into archaeal nucleosomes at lower histone to DNA ratios with the supercoiled, circular ligation product than with the original 88 bp linear version of this molecule. Archaeal histones are most similar to the globular histone fold region of eukaryal histone H4, and the results reported are consistent with archaeal nucleosomes resembling the structure formed by eukaryal histone (H3+H4)2tetramers.     

Inner nuclear membrane protein LBR preferentially interacts with DNA secondary structures and nucleosomal linker

The lamin B receptor (LBR) is an integral protein of inner nuclear membrane whose nucleoplasmic amino-terminal domain contributes to the attachment of the membrane to chromatin. Here we analyzed the interactions of a recombinant GST protein containing the amino-terminal domain of the protein with in vitro reconstituted nucleosomes and short DNA fragments. Data show that the LBR amino-terminal domain (AT) binds linker DNA but does not interact with the nucleosome core. Titration and competition studies revealed that the interaction between LBR AT and DNA is saturable, of high affinity (K(D) approximately 4 nM), independent of DNA sequence, and enhanced by DNA curvature and supercoiling. In this respect, LBR amino-terminal domain binding to nucleosomes is similar to that of histone H1 and non histone proteins HMG1/2 which both bind preferentially to linker DNA and present a significant affinity for DNA secondary structures.     

A thermodynamic model for Nap1-histone interactions

The yeast nucleosome assembly protein 1 (yNap1) plays a role in chromatin maintenance by facilitating histone exchange as well as nucleosome assembly and disassembly. It has been suggested that yNap1 carries out these functions by regulating the concentration of free histones. Therefore, a quantitative understanding of yNap1-histone interactions also provides information on the thermodynamics of chromatin. We have developed quantitative methods to study the affinity of yNap1 for histones. We show that yNap1 binds H2A/H2B and H3/H4 histone complexes with low nm affinity, and that each yNap1 dimer binds two histone fold dimers. The yNap1 tails contribute synergistically to histone binding while the histone tails have a slightly repressive effect on binding. The (H3/H4)(2) tetramer binds DNA with higher affinity than it binds yNap1.     

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.     

Structural investigations of DNA-histone complexes. A spin label study.

We have prepared two acridine spin labels, 6-chloro-9-[4-(2,2,6,6-tetramethyl-1-piperidinyloxy)amino]-2-methoxyacridine (I) and 9-[4-(2,2,6,6-tetramethyl-1-piperidinyloxy)amino]-acridine (II) and have used them to study the binding of lysine-rich histone (H1) to DNA using electron spin resonance (ESR). ESR spectra of I in the presence of DNA, polydA-polydT and polydG-polydC were characteristic of highly immobilized radicals with maximum hyperfine splitting (2T11) of 59G, 62.5G and 59G respectively. However, the 2T11 values for II in the same systems were 55.5G, 55.5G and 62.5G respectively. Addition of H1 at a low P/D released ionically bound I and II from DNA. In the presence of 0.1 M NaCl, which prevents ionic binding, H1 still caused a significant release of bound II but not I from DNA. At a high P/D (with or without NaCl) H1 caused no displacement of either I or II. Our findings suggest that H1 does not affect the intercalating sites and probably binds to one of the grooves of DNA, most probably the major groove, and specifically in the A-T-rich regions.     

HP1a Recruitment to Promoters Is Independent of H3K9 Methylation in Drosophila melanogaster

Heterochromatin protein 1 (HP1) proteins, recognized readers of the heterochromatin mark methylation of histone H3 lysine 9 (H3K9me), are important regulators of heterochromatin-mediated gene silencing and chromosome structure. In Drosophila melanogaster three histone lysine methyl transferases (HKMTs) are associated with the methylation of H3K9: Su(var)3-9, Setdb1, and G9a. To probe the dependence of HP1a binding on H3K9me, its dependence on these three HKMTs, and the division of labor between the HKMTs, we have examined correlations between HP1a binding and H3K9me patterns in wild type and null mutants of these HKMTs. We show here that Su(var)3-9 controls H3K9me-dependent binding of HP1a in pericentromeric regions, while Setdb1 controls it in cytological region 2L:31 and (together with POF) in chromosome 4. HP1a binds to the promoters and within bodies of active genes in these three regions. More importantly, however, HP1a binding at promoters of active genes is independent of H3K9me and POF. Rather, it is associated with heterochromatin protein 2 (HP2) and open chromatin. Our results support a hypothesis in which HP1a nucleates with high affinity independently of H3K9me in promoters of active genes and then spreads via H3K9 methylation and transient looping contacts with those H3K9me target sites.     

The compatibility of netropsin and actinomycin binding to natural deoxyribonucleic acid.

The simultaneous binding of netropsin and actinomycin to four natural DNAs was studied to determine the influence of one ligand on the binding of the other. Actinomycin binds specifically to GC sites, whereas netropsin binds specifically to AT sites. Spectral titrations, thermal denaturation, and analytical buoyant density centrifugation were employed to measure the binding interference of these drugs. The binding of actinomycin to DNA was decreased by the presence of netropsin. Increasing the GC content of the DNA resulted in a decreased effect of netropsin on actinomycin binding. Quantitative analysis of the binding parameters indicated that netropsin and actinomycin can bind in close proximity along the DNA chain. Supercoiled DNA gave the same result as linear DNA. These results imply that DNA can absorb alterations in conformation within a short distance.     

Modulation of action of wheat seedling endonucleases WEN1 and WEN2 by histones

Wheat core histones and various subfractions of histone H1 modulate differently the action of endonucleases WEN1 and WEN2 from wheat seedlings. The character of this modulation depends on the nature of the histone and the methylation status of the substrate DNA. The modulation of enzyme action occurs at different stages of processive DNA hydrolysis and is accompanied by changes in the site specificity of the enzyme action. It seems that endonuclease WEN1 prefers to bind with protein-free DNA stretches in histone H1-DNA complex. The endonuclease WEN1 does not compete with histone H1/6 for DNA binding sites, but it does compete with histone H1/1, probably for binding with methylated sites of DNA. Unlike histone H1, the core histone H2b binds with endonuclease WEN1 and significantly increases its action. This is associated with changes in the site specificity of the enzyme action that is manifested by a significant increase in the amount of low molecular weight oligonucleotides and mononucleotides produced as a result of hydrolysis of DNA fragments with 120–140-bp length. The WEN2 endonuclease binds with histone-DNA complexes only through histones. The action of WEN2 is increased or decreased depending on the nature of the histone. Histone H1/1 stimulated the exonuclease activity of WEN2. It is supposed that endonucleases WEN1 and WEN2, in addition to the catalytic domain, should have a regulatory domain that is involved in binding of histones. As histone H1 is mainly located in the linker chromatin areas, it is suggested that WEN2 should attack DNA just in the chromatin linker zones. As differentiated from WEN2, DNA hydrolysis with endonuclease WEN1 is increased in the presence of core histones and, in particular, of H2b. Endonuclease WEN1 initially attacks different DNA sites in chromatin than WEN2. Endonuclease WEN2 activity can be increased or diminished depending on presence of histone H1 subfractions. It seems that just different fractions of the histone H1 are responsible for regulation of the stepwise DNA degradation by endonuclease WEN2 during apoptosis. Modulation of the action of the endonucleases by histones can play a significant role in the epigenetic regulation of various genetic processes and functional activity of genes.     

Attenuation of DNA-protein interactions associated with intrinsic, sequence-dependent DNA curvature.

Inherently curved DNA segments, associated with short runs of adenines, have been identified in many gene regulatory regions, yet their physiological significance remains unknown. The observations reported in this study indicate that intrinsically bent nucleic acid fragments are characterized by substantially attenuated affinities toward DNA-binding proteins involved in structural functions, such as H1 histone and protamine, as well as toward various DNA-modifying enzymes including ligases and exo- and endonucleases. Two mechanisms might be responsible for the altered binding properties. According to the first mechanism, the attenuated binding affinities and the bending represent two independent consequences of the unique structural parameters exhibited by A-tracts. Indeed, analysis of the degradation products obtained upon exposure of the curved sequences to various chemical nucleases points toward the narrowing of the DNA minor groove, a conformational modulation known to characterize A-tracts and to run along the axially-bent motifs, as a potential determinant of the observed binding attenuation. Alternatively, the conformational constraints which result from the stable bending might act to modulate the strength of DNA-protein interactions. Although the factor directly responsible for the altered binding affinities revealed by the bent sequences cannot as yet be conclusively resolved, it is proposed that a reiteration of this specific factor, being either an A-tract or a bend, in phase with the DNA helical repeat acts to amplify the modulation of the binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of DNA binding by the ADR1 zinc finger transcription factor as determined by SPR

The ADR1 protein recognizes a six base-pair consensus DNA sequence using two zinc fingers and an adjacent accessory motif. Kinetic measurements were performed on the DNA-binding domain of ADR1 using surface plasmon resonance. Binding by ADR1 was characterized to two known native binding sequences from the ADH2 and CTA1 promoter regions, which differ in two of the six consensus positions. In addition, non-specific binding by ADR1 to a random DNA sequence was measured. ADR1 binds the native sites with nanomolar affinities. Remarkably, ADR1 binds non-specific DNA with affinities only approximately tenfold lower than the native sequences. The specific and non-specific binding affinities are conferred mainly by differences in the association phase of DNA binding. The association rate for the complex is strongly influenced by the proximal accessory region, while the dissociation reaction and specificity of binding are controlled by the two zinc fingers. Binding kinetics of two ADR1 mutants was also examined. ADR1 containing an R91K mutation in the accessory region bound with similar affinity to wild-type, but with slightly less sequence specificity. The R91K mutation was observed to increase binding affinity to a suboptimal sequence by decreasing the complex dissociation rate. L146H, a change-of-specificity mutation at the +3 position of the second zinc finger, bound its preferred sequence with a slightly higher affinity than wild-type. The L146H mutant indicates that beneficial protein-DNA contacts provide similar levels of stabilization to the complex, whether they are hydrogen-bonding or van der Waals interactions.     

Interaction of Hoechst 33258 with repeating synthetic DNA polymers and natural DNA

Fluorescence, circular dichroism and sedimentation through cesium chloride gradient techniques were performed to study the physical properties of the binding of the bisbenzimidazole dye Hoechst 33258 (H33258) to natural DNAs and synthetic polynucleotides of defined repeating units. These studies show that Hoechst 33258 exhibits at least two modes of interaction with duplex DNA: (1) a strong base pair specific mode which requires at least 4 consecutive AT base pairs and (2) a weaker mode of binding which is significantly reduced in the presence of high salt (0.4 M NaCl) and exhibits no apparent base specificity. The H33258 binding was found to be sensitive to the substitutions in the minor groove elements of a series of synthetic polynucleotides supporting the model of H33258 binding in the minor groove of the DNA with AT rich sequences. Similar mode of binding was predicted in natural DNAs by methylation of dye-DNA complexes. Footprint analysis of the complex of dye to a pBR322 fragment also supports that a minimum of 4 consecutive AT base pairs are required for H33258 binding to DNA.     

The Drosophila nuclear lamina protein YA binds to DNA and histone H2B with four domains

Dramatic changes occur in nuclear organization and function during the critical developmental transition from meiosis to mitosis. The Drosophila nuclear lamina protein YA binds to chromatin and is uniquely required for this transition. In this study, we dissected YA's binding to chromatin. We found that YA can bind to chromatin directly and specifically. It binds to DNA but not RNA, with a preference for double-stranded DNA (linear or supercoiled) over single-stranded DNA. It also binds to histone H2B. YA's binding to DNA and histone H2B is mediated by four domains distributed along the length of the YA molecule. A model for YA function at the end of Drosophila female meiosis is proposed.     

Physical association of eukaryotic initiation factor 4G (eIF4G) with eIF4A strongly enhances binding of eIF4G to the internal ribosomal entry site of encephalomyocarditis virus and is required for internal initiation of translation

Mammalian eukaryotic initiation factor 4GI (eIF4GI) may be divided into three similarly sized regions. The central region (amino acids [aa] 613 to 1090) binds eIF3, eIF4A, and the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) and mediates initiation on this RNA. We identified the regions of eIF4GI that are responsible for its specific interaction with the IRES and that are required to mediate 48S complex formation on the IRES in vitro. Mutational analysis demarcated the IRES binding fragment of eIF4GI (aa 746 to 949) and indicated that it does not resemble an RNA recognition motif (RRM)-like domain. An additional amino-terminal sequence (aa 722 to 746) was required for binding eIF4A and for 48S complex formation. eIF4GI bound the EMCV IRES and beta-globin mRNA with similar affinities, but association with eIF4A increased its affinity for the EMCV IRES (but not beta-globin RNA) by 2 orders of magnitude. On the other hand, eIF4GI mutants with defects in binding eIF4A were defective in mediating 48S complex formation even if they bound the IRES normally. These data indicate that the eIF4G-eIF4A complex, rather than eIF4G alone, is required for specific high-affinity binding to the EMCV IRES and for internal ribosomal entry on this RNA.