DNA Binding Activity Of Receptor Research Articles

Icariin Improves Glucocorticoid Resistance in a Murine Model of Asthma with Depression Associated with Enhancement of GR Expression and Function.

Icariin, a flavonoid glycoside isolated from Epimedium brevicornum, exerts a variety of biological activities. However, its effects on depression-induced glucocorticoid resistance in asthma and the underlying mechanisms have not been elucidated. In this study, a murine model of asthma with depression was established by exposure to ovalbumin combined with chronic unpredictable mild stress, and icariin was given orally during ovalbumin challenge and chronic unpredictable mild stress exposure. Depression-like behaviors were assessed by the open field test, forced swim test, and tail suspension test. The characteristic features of allergic asthma, including airway hyperreactivity, histopathology, inflammatory cytokine levels in bronchoalveolar lavage fluid, and immunoglobulin E and corticosterone levels in serum, were examined. Following splenocyte isolation in vitro, the inhibitory effects of corticosterone on the proliferation and cytokine secretion of splenocytes, glucocorticoid receptor DNA-binding activity, and expression of p-glucocorticoid receptor s226, glucocorticoid receptor α, and p-p38 mitogen-activated protein kinase in splenocytes were determined. We found that icariin had limited effects on depression-like behaviors, however, it markedly suppressed airway hyperresponsiveness, inflammatory infiltration in lung tissues, levels of interleukin-4, interleukin-5, and interleukin-6 in bronchoalveolar lavage fluid, and immunoglobulin E in serum. Furthermore, icariin improved the inhibitory effects of corticosterone on lipopolysaccharide-stimulated splenocytes, increased the glucocorticoid receptor expression and glucocorticoid receptor DNA-binding activity, and inhibited the phosphorylation of glucocorticoid receptors S226 and p38 mitogen-activated protein kinase. Taken together, icariin improved glucocorticoid resistance in a murine model of asthma with depression associated with enhancement of glucocorticoid receptor function and glucocorticoid receptor expression, and its effects on the glucocorticoid receptor function were related to decreased phosphorylation of glucocorticoid receptors S226 and p38 mitogen-activated protein kinase.

Modulation of androgen receptor DNA binding activity through direct interaction with the ETS transcription factor ERG

The androgen receptor (AR) is a type I nuclear hormone receptor and the primary drug target in prostate cancer due to its role as a lineage survival factor in prostate luminal epithelium. In prostate cancer, the AR cistrome is reprogrammed relative to normal prostate epithelium and particularly in cancers driven by oncogenic ETS fusion genes. The molecular basis for this change has remained elusive. Using purified proteins, we report a minimal cell-free system that demonstrates interdomain cooperativity between the ligand (LBD) and DNA binding domains (DBD) of AR, and its autoinhibition by the N terminus of AR. Furthermore, we identify ERG as a cofactor that activates AR's ability to bind DNA in both high and lower affinity contexts through direct interaction within a newly identified AR-interacting motif (AIM) in the ETS domain, independent of ERG's own DNA binding ability. Finally, we present evidence that this interaction is conserved among ETS factors whose expression is altered in prostate cancer. Our work highlights, at a biochemical level, how tumor-initiating ETS translocations result in reprogramming of the AR cistrome.

Identification and Molecular Characterization of Peroxisome Proliferator-Activated Receptor δ as a Novel Target for Covalent Modification by 15-Deoxy-Δ12,14-prostaglandin J2.

PPARδ belongs to the peroxisome proliferator-activated receptor (PPAR) family of nuclear receptors. Upon activation by an agonist, PPARδ controls a variety of physiological processes via regulation of its target genes. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is a cyclopentenone prostaglandin that features an electrophilic, α,β-unsaturated ketone (an enone) in the cyclopentenone ring. Many of 15d-PGJ2's biological effects result from covalent interaction between C9 and the thiol group of a catalytic cysteine (Cys) in target proteins. In this study, we investigated whether 15d-PGJ2 activates PPARδ by forming a covalent adduct. Our data show that 15d-PGJ2 activates PPARδ's transcriptional activity through formation of a covalent adduct between its endocyclic enone at C9 and Cys249 in the receptor's ligand-binding domain. As expected, no adduct formation was seen following a Cys-to-Ser mutation at residue 249 (C249S) of PPARδ or with a PGD2/PGJ2 analogue that lacks the electrophilic C9. Furthermore, the PPARδ C249S mutation weakened induction of the receptor's DNA binding activity by 15d-PGJ2, which highlights the biological significance of our findings. Calculated chemical properties as well as data from molecular orbital calculations, reactive molecular dynamics simulations, and intrinsic reaction coordinate modeling also supported the selectivity of 15d-PGJ2's C9 toward PPARδ's Cys thiol. In summary, our results provide the molecular, chemical, and structural basis of 15d-PGJ2-mediated PPARδ activation, designating 15d-PGJ2 as the first covalent PPARδ ligand to be identified.

Structure activity relationship of plumbagin in BRCA1 related cancer cells

It has been shown earlier that plumbagin, a naturally occurring naphthaquinone has specific anticancer activity in BRCA1 blocked ovarian cancer cells. Plumbagin can induce estrogen dependent cell signaling and apoptosis in BRCA1 blocked ovarian cancer cells. Being a reactive oxygen species (ROS) generator and apoptosis inducing agent, plumbagin has immense potential as a promising anticancer agent. In this study we analyzed whether there would be increased anticancer activity if the positions of the functional groups on plumbagin were altered and further to analyze the detailed molecular mechanism of action of the lead molecule. Methods like MTT assay, apoptosis analysis by flow cytometry, assessment of mitochondrial membrane potential-Δψm , suppression subtractive hybridization, microarray, molecular docking and estrogen receptor-DNA binding activity by electrophoresis mobility shift assay (EMSA) were adopted for assessing the anticancer activity. Consequently we found that, plumbagin was the most potent anticancer agent when compared to structurally related compounds. The anti-cancer activities were in the order plumbagin > 1,4-naphthaquinone > juglone > lawsone > menadione. Molecular docking studies showed that plumbagin could be well docked in the receptor ligand complex of TRAIL-DR5 complexes to activate the extrinsic pathway of apoptosis. Since the antiproliferative activity of plumbagin could be reduced by inhibiting ERα, we speculated that plumbagin interferes with the binding of ERα to ERE and we confirmed this by EMSA. This study clearly indicates that plumbagin can induce multiple pathways of apoptosis and cell cycle arrest in BRCA1 blocked cells compared to unblocked cells.

A physiological role for androgen actions in the absence of androgen receptor DNA binding activity

We tested the hypothesis that androgens have physiological actions via non-DNA binding-dependent androgen receptor (AR) signaling pathways in males, using our genetically modified mice that express a mutant AR with deletion of the 2nd zinc finger of the DNA binding domain (AR ΔZF2) that cannot bind DNA. In cultured genital skin fibroblasts, the mutant AR ΔZF2 has normal ligand binding ability, phosphorylates ERK-1/2 in response to 1 min DHT treatment (blocked by the AR antagonist bicalutamide), but has reduced androgen-dependent nuclear localization compared to wildtype (WT). AR ΔZF2 males have normal baseline ERK-1/2 phosphorylation, with a 1.5-fold increase in Akt phosphorylation in AR ΔZF2 muscle vs WT. To identify physiological actions of non-DNA binding-dependent AR signaling, AR ΔZF2 males were treated for 6 weeks with dihydrotestosterone (DHT). Cortical bone growth was suppressed by DHT in AR ΔZF2 mice (6% decrease in periosteal and 7% decrease in medullary circumference vs untreated AR ΔZF2 males). In conclusion, these data suggest that non-DNA binding dependent AR actions suppress cortical bone growth, which may provide a mechanism to fine-tune the response to androgens in bone.

Estrogen receptor regulates MyoD gene expression by preventing AP-1-mediated repression

Cell growth and differentiation are opposite events in the myogenic lineage. Growth factors block the muscle differentiation program by inducing the expression of transcription factors that negatively regulate the expression of muscle regulatory genes like MyoD. In contrast, extracellular clues that induce cell cycle arrest promote MyoD expression and muscle differentiation. Thus, the regulation of MyoD expression is critical for muscle differentiation. Here we show that estrogen induces MyoD expression in mouse skeletal muscle in vivo and in dividing myoblasts in vitro by relieving the MyoD promoter from AP-1 negative regulation through a mechanism involving estrogen receptor/AP-1 protein–protein interactions but independent of the estrogen receptor DNA binding activity.

DNA Binding Activity of the Aryl Hydrocarbon Receptor Is Sensitive to Redox Changes in Intact Cells

The potential involvement of vicinal dithiols in the transformation of the aryl hydrocarbon (Ah) receptor from its ligand binding to DNA binding form in Hepa-1 cells was explored through the use of diamide and phenylarsine oxide (PAO), which have been shown to specifically form a stable ring complex with vicinal sulfhydryl groups in selected proteins. Pretreatment with diamide and PAO rapidly prevented the inducer-dependent formation of the Ah receptor/xenobiotic response element complex detected by electrophoretic mobility shift assays and suppressed Ah receptor-mediated transcription. Diamide and PAO also inhibited DNA binding activity of the nuclear Ah receptor subsequent to its translocation to the nucleus but to a lesser extent than that observed with pretreatment conditions. The Ah receptor exhibited much higher sensitivity to cellular redox changes than Sp1, a transcription factor previously shown to be very sensitive to redox regulation. Diamide added to nuclear extracts inhibited Ah receptor DNA binding more than when it was added in intact cells. In contrast, Ah receptor DNA binding activity was more sensitive to PAO when it was added to intact cells than when it was added to nuclear extracts. Finally, dithiol 2,3-dimercaptopropanol was over 100 times more effective than monothiol 2-mercaptoethanol in reversing the PAO-dependent inhibition of Ah receptor DNA binding activity. This suggests that vicinal sulhydryl residues may be involved in DNA binding of the Ah receptor.

Suppression ofCYP1A1Transcription by H2O2Is Mediated by Xenobiotic-Response Element

We have previously demonstrated that H2O2downregulatesCYP1A1andCYP1A2transcription in isolated rat hepatocytes (C. W. Barker,et al., 1994,J. Biol. Chem.269, 3985–3990). In the present study, induction of chloramphenicol acetyltransferase (CAT) expression driven by 3.1 kb of ratCYP1A1upstream regulatory sequences was suppressed by 56% in Hepa-1 cells treated with H2O2. Similarly, H2O2inhibited CAT expression from vectors containing two copies of either xenobiotic-response element (XRE) 1 or XRE2. H2O2did not inhibit basal CAT expression in cells that were not treated with the inducer β-napthoflavone. Electrophoretic mobility shift assays demonstrated that the suppression of XRE-dependent transcription by H2O2was not due to changes in nuclear aryl hydrocarbon (Ah) receptor DNA binding activity. Several types of experiments indicated that modulation of XRE enhancer strength by various means could modify H2O2-dependent suppression of CAT expression. Conditions that increased the transactivation potential of the Ah receptor (increase in XRE copy number or shortening of the distance between XREs and the minimalCYP1A1promoter) attenuated the action of H2O2, while conditions that reduced XRE-mediated transactivation potential (decrease in XRE copy number, increase of the distance between the XRE and the promoter, or reduction of the number of bound Ah receptors by lowering the concentration of inducer) potentiated the inhibitory action of H2O2.

Down-regulation of nuclear aryl hydrocarbon receptor DNA-binding and transactivation functions: requirement for a labile or inducible factor.

Aryl hydrocarbons (AHs) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene activate the sequence-specific DNA-binding activity of the AH receptor. In the rat hepatocyte-derived cell line LCS7, DNA-binding activity peaked after 30 min and was then down-regulated, reaching negligible levels by 2 h. Down-regulation could be blocked, and DNA-binding activity maintained at maximum for many hours by inhibiting protein or RNA synthesis, implying that down-regulation is a mediated process requiring a labile or inducible protein. CYP1A1 transcription and in vivo DNA-protein interactions at xenobiotic response elements were down-regulated in parallel with DNA-binding activity in nuclear extracts, and these changes could also be blocked by inhibitors of protein synthesis. The correlation between AH receptor DNA-binding activity, intensity of in vivo footprints at xenobiotic response elements, and CYP1A1 transcription rate implies that down-regulation of AH receptor DNA-binding activity is important in regulating CYP1A1 transcription and that receptor is required continuously to maintain transcription. This correlation extends to the murine hepatoma cell line Hepa-1c1c7, in which slower kinetics of activation and down-regulation of CYP1A1 transcription paralleled slower activation and down-regulation of AH receptor DNA-binding activity. The difference in kinetics between cell lines also implies that AH receptor DNA-binding activity is modulated by a mechanism that may be influenced by cell-specific regulatory pathways. The above observations in conjunction with mixing experiments and comparisons of cytoplasmic and nuclear extracts indicate that down-regulation of AH receptor DNA-binding activity is probably due either to degradation or to conversion of the receptor to form that is inactive in both DNA binding and transactivation.

Down-Regulation of Nuclear Aryl Hydrocarbon Receptor DNA-Binding and Transactivation Functions: Requirement for a Labile or Inducible Factor

Aryl hydrocarbons (AHs) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene activate the sequence-specific DNA-binding activity of the AH receptor. In the rat hepatocyte-derived cell line LCS7, DNA-binding activity peaked after 30 min and was then down-regulated, reaching negligible levels by 2 h. Down-regulation could be blocked, and DNA-binding activity maintained at maximum for many hours by inhibiting protein or RNA synthesis, implying that down-regulation is a mediated process requiring a labile or inducible protein. CYP1A1 transcription and in vivo DNA-protein interactions at xenobiotic response elements were down-regulated in parallel with DNA-binding activity in nuclear extracts, and these changes could also be blocked by inhibitors of protein synthesis. The correlation between AH receptor DNA-binding activity, intensity of in vivo footprints at xenobiotic response elements, and CYP1A1 transcription rate implies that down-regulation of AH receptor DNA-binding activity is important in regulating CYP1A1 transcription and that receptor is required continuously to maintain transcription. This correlation extends to the murine hepatoma cell line Hepa-1c1c7, in which slower kinetics of activation and down-regulation of CYP1A1 transcription paralleled slower activation and down-regulation of AH receptor DNA-binding activity. The difference in kinetics between cell lines also implies that AH receptor DNA-binding activity is modulated by a mechanism that may be influenced by cell-specific regulatory pathways. The above observations in conjunction with mixing experiments and comparisons of cytoplasmic and nuclear extracts indicate that down-regulation of AH receptor DNA-binding activity is probably due either to degradation or to conversion of the receptor to form that is inactive in both DNA binding and transactivation.

Differential roles of heat shock protein 70 in the in vitro nuclear import of glucocorticoid receptor and simian virus 40 large tumor antigen.

Nuclear import of glucocorticoid receptors (GRs) was analyzed in vitro with digitonin-permeabilized cells (S. A. Adam, R. Sterne-Marr, and L. Gerace, J. Cell Biol. 111:807-816, 1990). Indirect immunofluorescence methods were used to monitor the transport of GRs from rat hepatoma and fibroblast cell cytosol into HeLa nuclei. In vitro nuclear import of GRs was shown to be hormone dependent and to require ATP and incubation at ambient temperatures (i.e., 30 degrees C). Hormone-dependent dissociation of GR-bound proteins, such as the 90-kDa heat shock protein, hsp90, is part of an activation process that is obligatory for the expression of the receptor's DNA-binding activity. Inhibition of in vitro GR activation by Na2MoO4 blocked hormone-dependent nuclear import, demonstrating that receptor activation is required for nuclear import. The addition to GR-containing cytosol of antiserum directed against the cytosolic 70-kDa heat shock protein, hsp70, while effective in blocking the nuclear import of simian virus 40 large tumor antigen (SV40 TAg), did not affect hormone-dependent nuclear import of endogenous, full-length GRs or an exogenously added truncated GR protein (i.e., XGR556) that lacks a hormone-binding domain but possesses a constitutively active nuclear localization signal sequence (NLS). Depletion of hsp70 from HeLa cell cytosol did not affect the nuclear import of exogenously added XGR556 but led to inhibition of SV40 TAg nuclear import. Thus, two closely related NLSs, one contained within GRs and the other contained within SV40 TAg, are distinguished by their differential requirements for hsp70 in vitro.

Action of ?pure? antiestrogens in inhibiting estrogen receptor action

The mechanism of action of the pure antiestrogens ICI 164384 and ICI 182780 has been investigated. Both antagonists are steroidal antiestrogens with 7 alpha-alkylamide side-chains. The antiestrogens reduce the cellular content of the estrogen receptor by reducing the half-life of the protein. A potential mechanism for this effect is suggested by the observation that the DNA binding activity of receptors which have been over-expressed in cells was inhibited in vitro. The inhibitory activity of analogues of ICI 164384 with different side chain lengths correlates with their ability to function as pure antiestrogens in vivo. Since the estrogen binding site overlaps with residues involved in dimerisation, the antiestrogens are likely to bind to a similar site and may therefore with receptor dimerisation in the hormone binding domain by means of the 7 alpha side-chain. We propose that the increased turnover of the receptor in the presence of ICI 164384 and ICI 182380 is a consequence of impaired dimerisation of the proteins.

Expression of androgen receptor in insect cells. Purification of the receptor and renaturation of its steroid- and DNA-binding functions.

A full-length rat androgen receptor cDNA was used to produce a recombinant baculovirus (AcrAR) by homologous recombination. Spodoptera frugiperda (Sf9) cells infected with this virus expressed a 110-kDa polypeptide that amounted up to about one-third of total cell protein. Studies with AR antibodies confirmed that this protein was indeed rAR. Only a minor portion of the recombinant AR was soluble in buffers without ionic detergents, but its complete solubilization was achieved in 6 M guanidine HCl (GdnHCl). Electron microscopy of cell pellets revealed that AR was localized to electron-dense cytoplasmic aggregates. The soluble cytosolic receptor was biologically active, in that it bound [3H]mibolerone with high affinity and specificity and interacted with an androgen-responsive element. The functions of the GdnHCl-solubilized AR were partially restored by a 20-50-fold dilution. The solubilized receptor was purified to an apparent homogeneity in a single step by gel filtration on a Sephacryl S-400 column in the presence of 6 M GdnHCl. The homogeneous AR protein could be renatured to bind [3H]mibolerone, interact specifically with a DNA element, and be recognized by receptor antibodies. Receptor-DNA interaction was stabilized by an antibody directed against the N-terminal part and abolished by an antibody against the hinge region of the receptor Zn2+ ions were essential for the purified receptor to refold into a specific DNA-binding form during the renaturation, with the optimal ZnCl2 concentration being 50-100 microM depending on the buffer conditions. Cd2+ ions were also capable of restoring the receptor's DNA-binding activity and did so at concentrations 10-fold lower than those of the Zn2+ ions.

Effects of antioestrogens on the DNA binding activity of oestrogen receptors in vitro.

We have investigated the effect of a series of steroidal oestrogen antagonists, related to ICI 164,384, on the DNA binding activity of mouse oestrogen receptors expressed in insect cells. The analogues possess different side chains at the 7-position of the B ring in the steroid. Inhibition was observed when the length of the side-chain was 15-16 carbon atoms but not 10 or 20 carbon atoms and only when the 7 alpha isomer was used. The DNA binding activity of receptors expressed in COS-1 cells was also inhibited after extended periods of incubation with antioestrogens but not that of the human receptor in breast cancer cell-extracts. We have proposed that ICI 164,384 might disrupt receptor dimerisation, and therefore the variation in its ability to inhibit DNA binding activity may reflect differences in dimer stability. Since the DNA binding activity of in vitro translated receptors was inhibited when they were translated in the presence of the antioestrogen we suggest that ICI 164,384 might prevent the formation of receptor dimers without necessarily being able to disrupt preformed dimers.

Redox manipulation of DNA binding activity and BuGR epitope reactivity of the glucocorticoid receptor

Treatment of the transformed glucocorticoid receptor with hydrogen peroxide promotes the formation of disulfide bonds and inhibits the ability of the receptor to bind to DNA (Tienrungroj, W., Meshinchi, S., Sanchez, E. R., Pratt, S. E., Grippo, J. F., Holmgren, A., and Pratt, W. B. (1987) J. Biol. Chem. 262, 6992-7000). It has not been determined whether the inhibition of DNA binding activity is due to disulfide bonds formed within the DNA binding domain or between the DNA binding domain and another region of the receptor. In this paper, we examined the ability of hydrogen peroxide to inactivate the DNA binding activity of the mouse glucocorticoid receptor. We show that inhibition of DNA binding activity caused by hydrogen peroxide can be accounted for entirely by the formation of disulfide bonds between cysteine residues lying within the 15-kDa tryptic fragment containing the DNA binding domain of the receptor. Reversal of the peroxide-induced inactivation of DNA binding activity requires both zinc and a thiol-disulfide exchange reagent, such as dithiothreitol. Peroxide also eliminates recognition of the intact receptor and the 15-kDa tryptic fragment by the BuGR monoclonal antibody, and the reactivity of the BuGR epitope is restored by reduction without a requirement for zinc. Pretreatment of the receptor with methyl methanethiosulfonate inhibits much of the peroxide-mediated inactivation of the BuGR epitope but pretreatment with N-ethylmaleimide does not. Similarly, DNA binding activity of the receptor is inhibited by methyl methanethiosulfonate but not by N-ethylmaleimide. These results are consistent with the proposal that peroxide promotes the formation of disulfide bonds between thiols that lie spatially close to one another in the 15-kDa tryptic fragment, resulting in rapid elimination of zinc. Restoration of the zinc finger structure restores DNA-binding activity but restoration of the BuGR epitope requires only reduction without restoration of the zinc fingers.

Aryl hydrocarbon (Ah) receptor DNA-binding activity. Sequence specificity and Zn2+ requirement.

The aryl hydrocarbon (Ah) receptor, also called the xenobiotic or TCDD receptor, mediates transcriptional activation of the cytochrome P-450c (CYP1A1) gene by interacting with Ah or xenobiotic response elements. This paper presents evidence that a metal ion, probably Zn2+, is an essential cofactor for the Ah receptor. This paper also maps in detail the interactions between the Ah response element XRE1 and the Ah receptor from the rat hepatocyte-derived cell line LCS7. Interactions were mapped by three methods, 1) methylation interference footprinting, 2) mobility shift competition experiments, using a series of synthetic oligonucleotides with systematic alterations in the Ah response element core sequence, and 3) orthophenanthroline/Cu+ footprinting. These findings suggest the following consensus sequence for DNA recognition by the Ah receptor: CNA/TNA/TCACGCA/TA/T. The chelators 1,10-phenanthroline and oxalic acid inhibited the sequence-specific DNA-binding activity of the AH receptor in a concentration dependent manner, suggesting that the DNA-binding activity of the receptor requires divalent metal ions. Inhibition was due to metal-chelation, since: 1) inhibition was almost completely prevented by the presence of Zn2+, or other divalent metal ions having high affinity for the chelators used, while metal ions with low affinity did not protect; 2) the DNA-binding activity of the receptor could be restored by dialysis to remove 1,10-phenanthroline, but only in the presence of Zn2+, while dialysis in the absence of metal ions reversed inhibition by the nonchelating isomer 4,7-phenanthroline. The involvement of a divalent cation in receptor function, possibly bound via sulfhydryls, was also suggested by the finding that Cd2+ and Co2+ inhibited DNA-binding activity. Once bound to the XRE1 DNA sequence, the receptor could not be inhibited by 1,10-phenanthroline, suggesting that the essential metal ion must become inaccessible to chelation when the receptor binds DNA. The Zn2+ requirement of the Ah receptor is similar to that of the estrogen and the glucocorticoid receptors and is consistent with the hypothesis that the Ah receptor is a member of the steroid and thyroid hormone receptor superfamily.

Sulfhydryl group content of chicken progesterone receptor: effect of oxidation on DNA binding activity.

DNA binding activity of chicken progesterone receptor B form (PRB) and A form (PRA) has been examined. This activity is strongly dependent upon the presence of thiols in the buffer. Stability studies showed that PRB was more sensitive to oxidation than was PRA. Receptor preparations were fractionated by DNA-cellulose chromatography to DNA-positive and DNA-negative subpopulations, and sulfhydryl groups were quantified on immunopurified receptor by labeling with [3H]-N-ethylmaleimide. Labeling of DNA-negative receptors with [3H]-N-ethylmaleimide showed 21-23 sulfhydryl groups on either PRA or PRB form when the proteins were reduced and denatured. A similar number was seen without reduction if denatured DNA-positive receptor species were tested. In contrast, the DNA-negative PRB had only 10-12 sulfhydryl groups detectable without reduction. A similar number (12-13 sulfhydryl groups) was found for PRA species that lost DNA binding activity after exposure to a nonreducing environment in vitro. We concluded that the naturally occurring receptor forms unable to bind to DNA, as well as receptor forms that have lost DNA binding activity due to exposure to a nonreducing environment in vitro, contain 10-12 oxidized cysteine residues, likely present as disulfide bonds. Since we were unable to reduce the disulfide bonds when the native DNA-negative receptor proteins were treated with dithiothreitol (DTT), we speculate that irreversible loss of DNA binding activity of receptor in vitro is due to oxidation of cysteine residues that are not accessible to DTT in the native state.

Modification of deoxyribonucleic acid-thyroid hormone receptor interaction by histones.

The effects of histones on receptor-DNA interaction were examined using an in vitro DNA-cellulose-binding assay of [125I]T3 receptor. H1 histones bound to DNA-cellulose strongly inhibited binding of receptor to DNA-cellulose. DNA-cellulose column chromatography showed that 80% of T3-binding activity attached to DNA-cellulose could be eluted using 1 mg H1 histone/ml at low ionic strength. The potent inhibitory activity of H1 histones on receptor-DNA binding was reversed by removal of H1 histones from DNA-cellulose or by removal of H1 histones from receptor. The interaction was specific for the DNA-binding activity of receptor, since H1 histones inhibit neither T3-binding activity nor core histone-binding activity of receptor. In contrast, low concentrations of core histones enhanced binding of T3 receptor to DNA-cellulose. This effect was also seen when DNA-cellulose was treated with core histones and then deprived of free core histones. The enhancement was reversed by removal of core histones bound to DNA-cellulose. A tryptic fragment of the receptor, which lost DNA-binding activity but retained hormone and core histone-binding activities, was capable of binding to DNA-cellulose in the presence of core histones. These data suggest that enhanced binding of receptor to DNA-cellulose by core histones is mediated through the core histone-binding activity of receptor. A heat-labile protein factor in nuclear extracts (possibly receptor itself) also enhanced receptor binding to DNA-cellulose. Our data suggest important roles of histones in the organization of nuclear thyroid hormone receptors in chromatin. Since H1 histone participates in condensation of chromatin and is enriched in transcriptionally inactive chromatin, inhibition of DNA-binding activity of receptor by H1 histone bound to DNA could explain the preferential binding of nuclear thyroid hormone receptor to transcriptionally active chromatin.