Expanding the paradigm for estrogen receptor binding and transcriptional activation.

Estrogen receptor (ER) binds to a spectrum of functional estrogen response elements (ERE) within the human genome, including ERE half-sites (HERE), inverted and direct repeats. This has been confounding, because ER has been reported to bind weakly, if at all, to these sites in vitro. We show that ER binds strongly to these nonconventional EREs, and the binding is enhanced by the presence of high-mobility group protein B1 (HMGB1). Collectively, these and previous findings reinforce the notion of the plasticity of strong ER/ERE interactions, consistent with their broader range of observed binding specificity. In addition, transient transfection studies using luciferase reporter gene assays show that these EREs drive luciferase activity, and HMGB1 enhances transcriptional activity. Furthermore, HMGB1 gene expression knockdown results in a precipitous drop in luciferase activity, suggesting a prominent role for HMGB1 in activation of estrogen/ER-responsive genes. Therefore, these data advocate that the minimal target site for ER is a cHERE (consensus HERE) that occurs in many different contexts and that HMGB1 enhances both the binding affinity and transcriptional activity. This challenges the current paradigm for ER binding affinity and functional activity and suggests that the paradigm requires significant reevaluation and modification. These findings also suggest a possible mechanism for a cross talk between genes regulated by ER and class II nuclear receptors.

The plasticity of estrogen receptor-DNA complexes: binding affinity and specificity of estrogen receptors to estrogen response element half-sites separated by variant spacers.

The consensus estrogen response element (cERE) contains a palindromic sequence of two 6-base pair (bp) half-sites separated by a spacer size of 3bp. This study investigates the extent to which estrogen receptors, ERalpha and ERbeta can bind target sequences not considered as conventional EREs. We determined the effect of spacer size (n=0-4) on the binding affinity and conformation of ERalpha and ERbeta in these complexes and the effect of HMGB1 on the complexation. We find (1) both receptors bind similarly and with progressively reduced affinity to cEREn, as n differs from 3; (2) however, both receptors bind as strongly to the cERE with no spacer (cERE0) as to cERE3; (3) HMGB1 enhances ER binding affinity in all complexes, resulting in strong and comparable binding affinities in all complexes examined; (4) the full-length ER binding differs strikingly from similar binding studies for the ER DNA binding domain (ERDBD), with the full-length ER dimer exhibiting strong binding affinity, enormous plasticity and retaining binding cooperativity as the spacer size varies; (5) both protease digestion profiles and monoclonal antibody binding assays indicate the conformation of the receptor in the ER/ERE complex is sensitive to the spacer size; (6) the ER/cERE0 complex appears to be singularly different than the other ER/cEREn complexes in binding and conformation. This multifaceted approach reinforces the notion of the plasticity in ER binding and leads to the hypothesis that in most cases, the minimum requirement for estrogen receptor binding is the ERE half-site, in which one or more cofactors, such as HMGB1, can cooperate to decrease ER binding specificity, while increasing its binding affinity.

TFIIA abrogates the effects of inhibition by HMGB1 but not E1A during the early stages of assembly of the transcriptional preinitiation complex.

Successful assembly of the transcriptional preinitiation complex (PIC) is prerequisite to transcriptional initiation. At each stage of PIC assembly, regulation may occur as repressors and activators compete with and influence the incorporation of general transcription factors (GTFs). Both TFIIA and HMGB1 bind individually to the TATA-binding protein (TBP) to increase the rate of binding and to stabilize TBP binding to the TATA element. The competitive binding between these two cofactors for TBP/TATA was examined to show that TFIIA binds preferentially to TBP and inhibits HMGB1 binding. TFIIA can also readily dissociate HMGB1 from the preestablished HMGB1/TBP/TATA complex. This suggests that TFIIA and HMGB1 may bind to the same or overlapping sites on TBP and/or compete for similar DNA sites that are 5' to the TATA element. In addition, EMSA studies show that adenovirus E1A(13S) oncoprotein is unable to disrupt either the preestablished TFIIA/TBP/TATA or TFIIA/TFIIB/TBP/TATA complexes, but does inhibit complex formation when all transcription factors were simultaneously added. The inhibitory effect of E1A(13S) on the assembly of the PIC is overcome when excess TBP is added back in the reaction, while addition of either excess TFIIA or TFIIB were ineffective. This shows that the main target for E1A(13S) is free TBP and emphasizes the primary competition between E1A and the TATA-element for unbound TBP. This may be the principal point, if not the only point, at which E1A can target TBP to exert its inhibitory effect. This work, coupled with previous findings in our laboratory, indicates that TFIIA is much more effective than TFIIB in reversing the inhibitory effect of HMGB1 binding in the early stages of PIC assembly, which is consistent with the in vitro transcription results.

The binding interaction of HMG-1 with the TATA-binding protein/TATA complex.

High mobility protein-1 (HMG-1) has been shown to regulate transcription by RNA polymerase II. In the context that it acts as a transcriptional repressor, it binds to the TATA-binding protein (TBP) to form the HMG-1/TBP/TATA complex, which is proposed to inhibit the assembly of the preinitiation complex. By using electrophoretic mobility shift assays, we show that the acidic C-terminal domain of HMG-1 and the N terminus of human TBP are the domains that are essential for the formation of a stable HMG-1/TBP/TATA complex. HMG-1 binding increases the affinity of TBP for the TATA element by 20-fold, which is reflected in a significant stimulation of the rate of TBP binding, with little effect on the dissociation rate constant. In support of the binding target of HMG-1 being the N terminus of hTBP, the N-terminal polypeptide of human TBP competes with and inhibits HMG-1/TBP/TATA complex formation. Deletion of segments of the N terminus of human TBP was used to map the region(s) where HMG-1 binds. These findings indicate that interaction of HMG-1 with the Q-tract (amino acids 55-95) in hTBP is primarily responsible for stable complex formation. In addition, HMG-1 and the monoclonal antibody, 1C2, specific to the Q-tract, compete for the same site. Furthermore, calf thymus HMG-1 forms a stable complex with the TBP/TATA complex that contains TBP from either human or Drosophila but not yeast. This is again consistent with the importance of the Q-tract for this stable interaction and shows that the interaction extends over many species but does not include yeast TBP.

Influence of HMG-1 and adenovirus oncoprotein E1A on early stages of transcriptional preinitiation complex assembly.

The TATA-binding protein (TBP) in the TFIID complex binds specifically to the TATA-box to initiate the stepwise assembly of the preinitiation complex (PIC) for RNA polymerase II transcription. Transcriptional activators and repressors compete with general transcription factors at each step to influence the course of the assembly. To investigate this process, the TBP.TATA complex was titrated with HMG-1 and the interaction monitored by electrophoretic mobility shift assays. The titration produced a ternary HMG-1.TBP. TATA complex, which exhibits increased mobility relative to the TBP. TATA complex. The addition of increasing levels of TFIIB to this complex results in the formation of the TFIIB.TBP.TATA complex. However, in the reverse titration, with very high mole ratios of HMG-1 present, TFIIB is not dissociated off and a complex is formed that contains all factors. The simultaneous addition of E1A to a mixture of TBP and TATA; or HMG-1, TBP, and TATA; or TFIIB, TBP, and TATA inhibits complex formation. On the other hand, E1A added to the pre-established complexes shows a significantly reduced capability to disrupt the complex. In add-back experiments with all complexes, increased levels of TBP re-established the complexes, indicating that the primary target for E1A in all complexes is TBP.

Reexamination of the high mobility group-1 protein for self-association and characterization of hydrodynamic properties.

Previous studies of the 25 kDa high mobility group-1 (HMG-1) protein have generated conflicting results regarding whether HMG-1 exists as a monomer or is capable of oligomerizing to (functional) tetramers. To resolve this question, sedimentation velocity analysis yielded a s20,w value of 2.59S, which is consistent with a monomeric protein. Equilibrium sedimentation data were obtained for three HMG-1 concentrations at two rotor speeds. The six sets of data were fit to both an ideal single component and monomer-dimer equilibrium model, with essentially identical fits produced for both models, with the latter indicating a low extent (7%) of dimerization. Reaction of HMG-1 with glutaraldehyde produced a small population of oligomers consistent with a low level of dimers. This supported the monomer-dimer equilibrium model. Surprisingly, gel permeation chromatography yielded an apparent molecular mass of approx. 55 kDa for both HMG-1 and HMG-2. This finding is considered anomalous and presumably due to the high negative charge density in the C terminus of HMG-1. The sedimentation data also permit one to model HMG-1 as a hydrated prolate ellipsoid with a major axis/minor axis ratio of 2. 79. The collective evidence from the sedimentation and chemical cross-linking studies strongly supports a moderately asymmetric monomer in solution and unequivocally eliminates the possibility of a highly extended shape for HMG-1 or the existence of any extensive oligomerization.

High mobility group protein, HMG-1, contains insignificant glycosyl modification.

High mobility group protein-1 (HMG-1) is a ubiquitous, highly conserved, and abundant nuclear protein. Recent findings suggest that HMG-1 may serve as a DNA chaperone protein and play a role in the regulation of transcription. There is a mounting interest in elucidating the mechanism by which HMG-1 protein takes part in these activities. HMG-1 has been reported to undergo an extensive array of posttranslational modifications, including glycosylation. We extend the earlier findings on the glycosylation of HMG-1 by quantitating the amount of carbohydrate on HMG-1 from calf thymus and chicken erythrocytes isolated by 2 different purification procedures. In addition, 2 different developmental stages (embryonic and adult) were examined in the chicken erythrocytes. The glycosyl composition was quantitated using the Dionex HPAE-PAD II system. Furthermore, the presence of O-linked GlcNAc on HMG-1 was determined by the enzymatic incorporation of 3H-galactose into HMG-1 protein. Contrary to earlier reports, less than 0.5 mol of total monosaccharides (Fuc, Man, GalNH2, GlcNH2, Gal) were detected per mole of HMG-1 protein, regardless of the source of the protein or the method of isolation. In addition, less than 0.002 mol of O-linked GlcNAc per mole of HMG-1 protein was detected. Thus, insignificant amount of glycosylation was found on HMG-1 protein. Because O-linked GlcNAc modification of proteins is believed to be a reversible posttranslational event, more definitive studies will need to be conducted before ruling out that the function of HMG-1 protein is not regulated by glycosylation.

cis-diamminedichloroplatinum (II) modified chromatin and nucleosomal core particle.

The influence of cis-diamminedichloroplatinum (II) (cis-DDP) binding to chromatin in chicken erythrocyte nuclei and the nucleosomal core particle is investigated. The cis-DDP modifications alter DNA-protein interactions associated with the higher order structure of chromatin to significantly inhibit the rate of micrococcal nuclease digestion and alter the digestion profile. However, cis-DDP modification of core particle has little effect on the digestion rate and the relative distribution of DNA fragments produced by microccocal nuclease digestion. Analysis of the monomer DNA fragments derived from the digestion of modified nuclei suggests that cis-DDP binding does not significantly disrupt the DNA structure within the core particle, with its major influence being on the internucleosomal DNA. Together these findings suggest that cis-DDP may preferentially bind to the internucleosomal region and/or that the formation of the intrastrand cross-link involving adjacent guanines exhibits a preference for the linker region. Sucrose gradient profiles of the modified nucleoprotein complexes further confirm that the digestion profile for micrococcal nuclease is altered by cis-DDP binding and that the greatest changes occur at the initial stages of digestion. The covalent cross-links within bulk chromatin fix a sub-population of subnucleosomal and nucleosomal products, which are released only after reversal by NaCN treatment. Coupled with our previous findings, it appears that this cis-DDP mediated cross-linking network is primarily associated with protein-protein crosslinks of the low mobility group (LMG) proteins.

cis-Diamminedichloroplatinum (II) modified chromatin and nucleosomal core particle probed with DNase I.

Chromatin and nucleosomal core particles were modified with cis-diamminedichloroplatinum (II) and the nucleoprotein complexes then digested with DNase I. Limited digestion of the modified chromatin containing cis-Pt(NH3)2Cl2 mediated cross-links involving the non-histone chromosomal proteins (Scovell et al. (1987) Biochem. Biophys. Res. Commun. 142, 826-835) does not release the low mobility proteins and excises only about 20% of the high mobility proteins 1, 2, and E. This supports previous findings that the low mobility proteins are involved primarily in protein-protein cross-links. In addition, the covalent cross-links between DNA and the high mobility proteins 1, 2, and E are relatively inaccessible to DNase I, in marked contrast to their accessibility to micrococcal nuclease. Furthermore, gels of the denatured DNA fragments obtained from digestion of both modified chromatin and nucleosomal core particle reveal virtually no difference in the 10n base repeat pattern, indicating no detectable change in the DNA-protein interactions upon DNA modification. This suggests that the predominant modification produced on core particle DNA, whether contained within higher order chromatin structure or in the core particle itself, is one which does not significantly alter the helical twist of the DNA within these nucleoprotein assemblies.

cis-Diamminedichloroplatinum(II) selectively cross-links high mobility group proteins 1 and 2 to DNA in micrococcal nuclease accessible regions of chromatin.

The reaction of cis-diamminedichloroplatinum(II) with chicken erythrocyte nuclei produces covalent cross-linking of HMG proteins 1, 2 and E to DNA, in addition to cross-links amongst LMG proteins. This is supported by and consistent with the observations that all cross-links are chemically reversed by NaCN treatment, while only the cross-links involving the HMG proteins 1,2 and E are eliminated after a limited digestion with micrococcal nuclease. Having identified the subset of proteins selectively cross-linked to DNA by the bifunctional cis-(NH3)2PtCl2, a tentative model is proposed for the interactions between DNA and HMG proteins 1 and 2 in bulk chromatin. In addition, possible modes of action for this anti-neoplastic drug are suggested in light of these findings.

Unwinding of supercoiled DNA by cis- and trans-diamminedichloroplatinum(II): influence of the torsional strain on DNA unwinding.

The effective unwinding angle, phi, for cis-diamminedichloroplatinum(II) (cis-DDP) and trans-DDP was determined by utilizing high resolution gel electrophoresis and supercoiled phi X174 RF DNA as a substrate. The effective unwinding angle was calculated by equating the reduction in mobility of the DDP-modified DNA to the removal of a number of superhelical turns. The value of the effective unwinding angle for both DDP isomers was greatest at the low levels of DDP bound and decreased with increasing amounts of unwinding agent. The cis-isomer is a better unwinding agent than is the trans-isomer, being nearly twice as effective in unwinding the supercoiled DNA at the DDP levels investigated. A comparison of the magnitude of phi below rb values of 0.005 and those at high levels of binding reveals that the extent of torsional strain in the supercoiled DNA influences the magnitude of the unwinding of the DNA by these complexes. When this method is used in the analysis of the unwinding angle for a covalently bound species on supercoiled DNA, it may provide a more reliable estimate of the magnitude of phi at high degrees of supercoiling and at low levels of modification.

S1 nuclease sensitivity to cis- and trans-diamminedichloroplatinum(II) modified DNAS: influence of (G+C) content and nucleotide sequence.

The sensitivity of S1 nuclease to cis- and trans-(NH3)2PtCl2 modified DNAs is examined as a function of the level of cis- and trans-(NH3)2PtCl2 bound, the % (G+C) content in DNA from different sources and the sequence dependence in poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC). The extent of DNA digested increases with increasing levels of either isomer and is inversely influenced by the % (G+C) content of the DNA. However, the difference in the extent of digestion between the cis-and trans-(NH3)2PtCl2 modified DNAs at equivalent levels of bound isomer follows the order, calf-thymus greater than M. lysodeikticus greater than poly(dG-dC).poly(dG-dC). While there is virtually no difference in the digestion profiles for poly(dG-dC).poly(dG-dC) modified with the two isomers, there is a striking difference in the extent of digestion between cis- and trans-(NH3)2PtCl2 modified poly(dG).poly(dC). These results are discussed in light of the possible modes of binding for cis-(NH3)2PtCl2, previously reported findings on modified DNA and possible implications for modifications in cellular chromatin.

Laser raman spectroscopy of a complementary base pair in the Hoogsteen configuration.

Cocrystallization of m9Ade and m1Thy from water yielded crystals of te Hoogsteen base pair, m9Ade x m1Thy, as demonstrated by X-ray diffraction data. The Raman spectrum of the Hoogsteen base pair in the crystalline state was obtained and compared to the crystals of the individual monomers. Frequency changes between the heterobase dimer and both m9Ade and m1Thy are indicative of the base pairing interaction. Utilizing previously reported vibrational analyses for m9Ade, the frequency changes are clearly associated with modes involving the internal coordinates most sensitive to a Hoogsteen hydrogen bonding interaction. The spectrum of an equimolar mixture of e9Ade and ch1Ura in CHCl3 was also compared to the spectrum of the separate monomers in solution. The numerous frequency shifts and band intensity changes observed in the mixture from that of the individual monomers suggest a cyclic dimer is formed although the nature of the base pairing interaction is not clear.

The interaction of aquated cis-[(NH3)2Pt(II)]with nucleic acid constituents. II. Modified nucleosides.

The binding interaction of aquated cis-(NH3)2Pt(II) with 1-methylguanosine (1-MeGuo), 7-methylguanosine (7-MeGuo), 1-methyladenosine (1-MeAdo+) and protonated adenosine has been studied using UV difference spectroscopy. The magnitude of the binding constants for the 1 : 1 interaction of cis-(NH3)2Pt(II) with 1-MeDuo and 1-MeAdo are log K = 3.5 and 3.7, respectively. These data are presented and compared to other cis-(NH3)2Pt(II)-nucleoside interactions.

Raman spectral studies of poly(1-methylinosinic acid).

The synthesis and characterization of poly(1-methylinosinic acid) are described. Laser Raman spectra of poly (mII) were obtained as a function of temperature in D2O solution. Thermal melting profiles derived from the intensity variations of the 712, 795, 814, 986, 1333, 1509, 1550 and 1680 cm-1 bands indicate a cooperative melting temperature of 9 +/- 1 degree C. The low temperature form of poly(mII) exhibits a carbonyl frequency at 1710 cm-I which is decreased to 1680 cm-I upon melting. The Raman hypochromism in the bands reported are equal to or much larger than any reported for other nucleic acids. The data are consistent with the low temperature form of poly(mII) being an ordered single stranded unit with a high degree of basestacking. The melting profiles obtained from the uv and cd spectra are consistent with and support the Raman data. This single stranded RNA exhibits an uncharacteristic behavior in that it melts cooperatively.

Raman pH profiles for nucleic acid constituents I. Cytidine and uridine ribonucleosides.

Raman spectra of aqueous solutions of uridine and cytidine have been recorded as a function of pH with the band intensities and vibrational frequencies monitored to determine bands which may be considered as diagnostic of the concentration of the various species. Quantitative band intensity measurements indicate that not all pH-sensitive bands can be considered as diagnostic of the pK value for the acid form of the nucleoside, and for the percent species in solution. Although the accuracy of the Raman band intensity method is inherently less than that of the titrimetric or visible-ultraviolet spectrophotometric methods, the pK values and percent species agree well with those obtained from these methods. The utility of the results obtained from the pH profiles for cytidine is discussed in terms of the effect of acidification on the structural and conformational characteristics of polycytidylic acid in solution.