ABSTRACT
Epigenetics refers to heritable patterns of gene expression that do not depend on alterations of the genomic DNA sequence. Nickel compounds have demonstrated carcinogenicity without any associated mutagenesis, suggesting that its mechanism of carcinogenesis is epigenetic in nature. One such potential mechanism is the heterochromatinization of chromatin within a region of the genome containing a gene sequence, inhibiting any further molecular interactions with that underlying gene sequence and effectively inactivating that gene. We report here the observation, by atomic force microscopy and circular dichroism spectropolarimetry, that nickel ion (Ni(2+)) condenses chromatin to a greater extent than the natural divalent cation of the cell, magnesium ion (Mg(2+)). In addition, we use a model experimental system that incorporates a transgene, the bacterial xanthine guanine phosphoribosyl transferase gene (gpt), differentially near, and far from, a heterochromatic region of the genome, in two cell lines, the Chinese hamster V79-derived G12 and G10 cells, respectively, to demonstrate by a DNase I protection assay that nickel treatement protects the gpt gene sequence from DNase I exonuclease digestion in the G12 cells, but not in the G10 cells. We conclude that condensation of chromatin by nickel is a potential mechanism of nickel-mediated gene regulation.
Subject(s)
Carcinogens/toxicity , Heterochromatin/drug effects , Nickel/toxicity , Animals , Cell Line , Cobalt/pharmacology , Cricetinae , Cricetulus , Deoxyribonuclease I/metabolism , Gene Silencing/drug effects , Heterochromatin/genetics , Heterochromatin/metabolism , Magnesium/pharmacology , Nucleosomes/drug effects , Nucleosomes/metabolismABSTRACT
The retinoid X receptor (RXR) is a ligand-activated transcription factor that plays an important role in growth and development and the maintenance of cellular homeostasis. A thermodynamic ultraviolet circular dichroism, tryptophan fluorescence and ligand binding activity with guanidine as a chemical denaturant are consistent with a two step mechanism. The dimeric LBD equilibrates with a monomeric intermediate (DeltaG(0)(H(2)O) equal to 8.3 kcal/mol) that is in equilibrium with the unfolded state (DeltaG(0)(H(2)O) equal to 2.8 kcal/mol). The intermediate was characterized by analytical ultracentrifugation, spectroscopy, and collisional fluorescence quenching, which imply that the monomeric intermediate maintains a high degree, but not all, of native secondary structure. Although intrinsic fluorescence from native and intermediate suggests little change in tryptophan environments, fluorescence intensities from fluorescein reporter groups differ significantly between the two structures. Analysis of the collisional quenching results imply that the intermediate is characterized by tryptophans with increased accessibility to small solutes and less overall compactness than the native protein.
Subject(s)
Retinoid X Receptors/chemistry , Retinoid X Receptors/metabolism , Acrylamide/pharmacology , Alitretinoin , Circular Dichroism , Dose-Response Relationship, Drug , Fluorescein/metabolism , Fluorescent Dyes/metabolism , Humans , Ligands , Nitrates/pharmacology , Protein Denaturation/drug effects , Protein Folding , Protein Multimerization , Protein Structure, Quaternary , Protein Structure, Tertiary , Thermodynamics , Tretinoin/metabolism , Tryptophan , UltracentrifugationABSTRACT
A new method for analyzing three-state protein unfolding equilibria is described that overcomes the difficulties created by direct effects of denaturants on circular dichroism (CD) and fluorescence spectra of the intermediate state. The procedure begins with a singular value analysis of the data matrix to determine the number of contributing species and perturbations. This result is used to choose a fitting model and remove all spectra from the fitting equation. Because the fitting model is a product of a matrix function which is nonlinear in the thermodynamic parameters and a matrix that is linear in the parameters that specify component spectra, the problem is solved with a variable projection algorithm. Advantages of this procedure are perturbation spectra do not have to be estimated before fitting, arbitrary assumptions about magnitudes of parameters that describe the intermediate state are not required, and multiple experiments involving different spectroscopic techniques can be simultaneously analyzed. Two tests of this method were performed: First, simulated three-state data were analyzed, and the original and recovered thermodynamic parameters agreed within one standard error, whereas recovered and original component spectra agreed within 0.5%. Second, guanidine-induced unfolding titrations of the human retinoid-X-receptor ligand-binding domain were analyzed according to a three-state model. The standard unfolding free energy changes in the absence of guanidine and the guanidine concentrations at zero free-energy change for both transitions were determined from a joint analysis of fluorescence and CD spectra. Realistic spectra of the three protein states were also obtained.
