Alteration in the nucleosome and chromatin structures upon interaction with platinum coordination complexes

The interaction of various platinum coordination complexes with nucleosomes and chromatin has been investigated by ultraviolet absorption spectrophotometry, circular and electric linear dichroism, and thermal denaturation, at low binding ratios (r < 0.1–0.2). The general trend of the changes in these physicochemical properties is similar to that observed for the DNA-platinum complexes, which indicates that the same binding sites are involved in the platinum interaction with DNA and with its nucleoprotein complex. The cis-bidentate ligands, cis-dichlorodiammine, diaminocyclohexane and ethylenediamine platinum(II), showed a distinct behavior, with a more important destabilization of the DNA structure in the nucleoprotein than the trans-bidentate ligand, trans-dichlorodiammine-Pt(II), and monodentate ligand, diethylenetriamine-Pt(II). The drastic decrease of the negative electric dichroism in the 260 nm absorption band of the bases, observed with the five ligands, indicates a profound alteration of the DNA arrangement in chromatin and nucleosomes, attributed to a condensation of its superhelical structure. Some differences with previous observations on DNA complexes with the same platinum compounds indicate the possible formation of protein-DNA crosslinks in chromatin and nucleosomes. These could have some importance for the biological effects.     

The differential effects produced by cis- and trans-DDP on DNA in calf thymus nucleosomes

Calf thymus nucleosomes containing H1 were treated with dichlorodiammineplatinum (DDP) at low binding ratios (r = 0.05–0.15). Change in the electrophoretic mobility of the extracted nucleosomal DNA was observed following treatment with cis-DDP and little change with trans-DDP. There was a decrease in the electrophoretic mobility of the nucleosomal DNA as well as obliteration of the nucleosomal repeat distance. The fluorescence intensity of terbium binding to the extracted DNA showed minimal change following drug treatment. However, the thermal melting behavior of the nucleosomal DNA was altered to a greater extent following cis-DDP treatment at 280 rather than 260 nm and a destabilization of the DNA helix was observed. These data suggest that in the whole nucleosome, cis-DDP produces greater structural effects on the packaged DNA than trans-DDP, although similar amounts of drug are bound with both isomers.     

Validation of Cis and Trans Modes in Multistep Phosphotransfer Signaling of Bacterial Tripartite Sensor Kinases by Using Phos-Tag SDS-PAGE

Tripartite sensor kinases (TSKs) have three phosphorylation sites on His, Asp, and His residues, which are conserved in a histidine kinase (HK) domain, a receiver domain, and a histidine-containing phosphotransmitter (HPt) domain, respectively. By means of a three-step phosphorelay, TSKs convey a phosphoryl group from the γ-phosphate group of ATP to the first His residue in the HK domain, then to the Asp residue in the receiver domain, and finally to the second His residue in the HPt domain. Although TSKs generally form homodimers, it was unknown whether the mode of phosphorylation in each step was intramolecular (cis) or intermolecular (trans). To examine this mode, we performed in vitro complementation analyses using Ala-substituted mutants of the ATP-binding region and three phosphorylation sites of recombinant ArcB, EvgS, and BarA TSKs derived from Escherichia coli. Phosphorylation profiles of these kinases, determined by using Phos-tag SDS-PAGE, showed that the sequential modes of the three-step phosphoryl-transfer reactions of ArcB, EvgS, and BarA are all different: cis-trans-trans, cis-cis-cis, and trans-trans-trans, respectively. The inclusion of a trans mode is consistent with the need to form a homodimer; the fact that all the steps for EvgS have cis modes is particularly interesting. Phos-tag SDS-PAGE therefore provides a simple method for identifying the unique and specific phosphotransfer mode for a given kinase, without taking complicated intracellular elements into consideration.     

The Differences Between Cis- and Trans-Gene Inactivation Caused by Heterochromatin in Drosophila

Position-effect variegation (PEV) is the epigenetic disruption of gene expression near the de novo–formed euchromatin-heterochromatin border. Heterochromatic cis-inactivation may be accompanied by the trans-inactivation of genes on a normal homologous chromosome in trans-heterozygous combination with a PEV-inducing rearrangement. We characterize a new genetic system, inversion In(2)A4, demonstrating cis-acting PEV as well as trans-inactivation of the reporter transgenes on the homologous nonrearranged chromosome. The cis-effect of heterochromatin in the inversion results not only in repression but also in activation of genes, and it varies at different developmental stages. While cis-actions affect only a few juxtaposed genes, trans-inactivation is observed in a 500-kb region and demonstrates а nonuniform pattern of repression with intermingled regions where no transgene repression occurs. There is no repression around the histone gene cluster and in some other euchromatic sites. trans-Inactivation is accompanied by dragging of euchromatic regions into the heterochromatic compartment, but the histone gene cluster, located in the middle of the trans-inactivated region, was shown to be evicted from the heterochromatin. We demonstrate that trans-inactivation is followed by de novo HP1a accumulation in the affected transgene; trans-inactivation is specifically favored by the chromatin remodeler SAYP and prevented by Argonaute AGO2.     

The interaction of platinum complexes with nucleosomes investigated with fluorescent probes

The fluorescent probes, N-(3-pyrene)maleimide, which is specific for histone H3, and terbium (Tb³⁺), which is specific for guanine single-stranded residues in DNA, are used to investigate the interaction of platinum complexes (cis- and trans-dichlorodiammineplatinum(II)) with rat liver and calf thymus nucleosomes. At low concentrations of the drug, lower than most of those reported previously in studies investigating the interaction of the drugs with isolated DNA, N-(3-pyrene)maleimide studies show that profound modifications occur near or in the cysteinyl binding site of histone H3. H3 dimer formation appears to be the cause of the change induced by trans-DDP; however, the effects observed with the cis-isomer do not seem to be correlated with dimer formation. At short incubation times, Tb³⁺ fluorescence shows small changes in DNA conformation, but they are slight when compared to the effect observed with proteins at the same length of incubation. SDS-polyacrylamide gels indicate some changes in protein composition, and agarose gels display a decrease in ethidium bromide staining of the cis-treated DNA. The results suggest that the protein portion, predominantly histone H3, as well as DNA are targets for the platinum derivatives in the nucleosome.     

Synthesis and receptor binding in trans-CD ring-fused A-CD estrogens: Comparison with the cis-fused isomers

Ligands which selectively activate only one of the estrogen receptors, ERα or ERβ, are current pharmaceutical targets. Previously, we have reported on substituted cis A-CD ligands in which the B-ring of the steroidal structure has been removed and cis refers the stereochemistry of the CD ring junction as compared to trans in estradiol. These compounds often showed good potency and selectivity for ERβ. Here we report the synthesis and binding affinities for a similar series of trans A-CD ligands, and compare them to the cis-series. Counterintuitively, trans A-CD ligands, which are structurally more closely related to the natural ligand estradiol, show weaker binding and less β-selectivity than their cis-counterparts.     

Stereochemical aspects of the metabolism of the isomeric methylcyclohexanols and methylcyclohexanones

1. The seven isomeric optically inactive forms of methylcyclohexanol (i.e. 1-, and cis- and trans-2-, -3- and -4-) are excreted by rabbits mainly as glucuronides of the thermodynamically more stable forms of the alcohols. The eighth isomer, cyclohexylmethanol, however, undergoes aromatization in vivo, giving rise to benzoic acid and hippuric acid. The (±)-2-, (±-3- and 4-methylcyclohexanones are reduced in the rabbit and excreted mainly as the glucuronides of the thermodynamically more stable forms of the corresponding methylcyclohexanols. 2. Racemic cis- and trans-2-methylcyclohexanol and 2-methylcyclohexanone are all excreted as conjugated trans-2-methylcyclohexanol. However, when the (±)-cis-alcohol or the (±)-ketone is fed, (+)-trans-2-methylcyclohexanol is excreted, whereas when the (±)-trans-alcohol is fed it is excreted as the (±)-trans-alcohol. 3. Racemic cis- and trans-3-methylcyclohexanol and 3-methylcyclohexanone are all excreted as conjugated racemic cis-3-methylcyclohexanol. cis- and trans-4-Methylcyclohexanol and 4-methylcyclohexanone are all excreted as conjugated trans-4-methylcyclohexanol. 4. The metabolic differences between the various methylcyclohexanols are explicable in terms of their conformations and of Vennesland's (1958) hypothesis of the role of NADH in dehydrogenation reactions.     

Reaction of cis and trans isomers of platinum(II) diamminedichloride with purine,adenine and its derivatives in dilute solutions

Reactions of the cytostatic and antitumour agent, cis-platinum(II) diamminedichloride, and its trans isomer with purine, adenine and its N-9 derivatives were followed spectrophotometrically in dilute aqueous solutions. While the cis isomer formed with all compounds studied complexes with the metal-to-ligand ratios ML, ML₂ and M₂L, it was found that the trans isomer did not form the complexes ML₂ with adenosine and AMP. The values of dissociation constants, reaction rate constants and activation energies of the reactions of the cis and trans isomers with purine, adenine and its derivatives were in most cases comparable. Lower stability was exhibited only by the complexes of trans-platinum(II) diamminedichloride (trans-Pt(II)) with AMP as well as by its complexes M₂L with adenine and adenosine. The ability of cis-platinum(II) diamminedichloride (cis-Pt(II)) to react with two adenine residues can explain the greater tendency of the cis isomer to form intrastrand cross-links in nucleic acids as compared with the trans isomer.     

Geometric specificity of porcine liver carboxylesterase and its application for the production of cis-arylacrylic esters

Porcine liver carboxylesterase (carboxylic-ester hydrolase, EC 3.1.1.1) hydrolyses trans isomers of three different methyl 3-arylacrylates approximately one order of magnitude faster than the corresponding cis isomers. This phenomenon can be used for preparative production of cis esters from their trans counterparts as exemplified by methyl cinnamate. A solution of commercial, predominantly trans methyl cinnamate was irradiated by ultraviolet light and the resultant mixture of trans and cis esters was passed through a column packed with immobilized esterase. The effluent contained mainly trans cinnamic acid and cis methyl cinnamate. The latter was then extracted with methylene chloride, and the cis ester was isolated by evaporating the solvent. By esterifying the co-produced trans acid, the process can be made continuous.     

Plug-and-Play Pairing via Defined Divalent Streptavidins

Streptavidin is one of the most important hubs for molecular biology, either multimerizing biomolecules, bridging one molecule to another, or anchoring to a biotinylated surface/nanoparticle. Streptavidin has the advantage of rapid ultra-stable binding to biotin. However, the ability of streptavidin to bind four biotinylated molecules in a heterogeneous manner is often limiting. Here, we present an efficient approach to isolate streptavidin tetramers with two biotin-binding sites in a precise arrangement, cis or trans. We genetically modified specific subunits with negatively charged tags, refolded a mixture of monomers, and used ion-exchange chromatography to resolve tetramers according to the number and orientation of tags. We solved the crystal structures of cis-divalent streptavidin to 1.4 Å resolution and trans-divalent streptavidin to 1.6 Å resolution, validating the isolation strategy and explaining the behavior of the Dead streptavidin variant. cis- and trans-divalent streptavidins retained tetravalent streptavidin's high thermostability and low off-rate. These defined divalent streptavidins enabled us to uncover how streptavidin binding depends on the nature of the biotin ligand. Biotinylated DNA showed strong negative cooperativity of binding to cis-divalent but not trans-divalent streptavidin. A small biotinylated protein bound readily to cis and trans binding sites. We also solved the structure of trans-divalent streptavidin bound to biotin-4-fluorescein, showing how one ligand obstructs binding to an adjacent biotin-binding site. Using a hexaglutamate tag proved a more powerful way to isolate monovalent streptavidin, for ultra-stable labeling without undesired clustering. These forms of streptavidin allow this key hub to be used with a new level of precision, for homogeneous molecular assembly.     

Genetic Control of Individual Differences in Gene-Specific Methylation in Human Brain

We have observed extensive interindividual differences in DNA methylation of 8590 CpG sites of 6229 genes in 153 human adult cerebellum samples, enriched in CpG island “shores” and at further distances from CpG islands. To search for genetic factors that regulate this variation, we performed a genome-wide association study (GWAS) mapping of methylation quantitative trait loci (mQTLs) for the 8590 testable CpG sites. cis association refers to correlation of methylation with SNPs within 1 Mb of a CpG site. 736 CpG sites showed phenotype-wide significant cis association with 2878 SNPs (after permutation correction for all tested markers and methylation phenotypes). In trans analysis of methylation, which tests for distant regulation effects, associations of 12 CpG sites and 38 SNPs remained significant after phenotype-wide correction. To examine the functional effects of mQTLs, we analyzed 85 genes that were with genetically regulated methylation we observed and for which we had quality gene expression data. Ten genes showed SNP-methylation-expression three-way associations—the same SNP simultaneously showed significant association with both DNA methylation and gene expression, while DNA methylation was significantly correlated with gene expression. Thus, we demonstrated that DNA methylation is frequently a heritable continuous quantitatively variable trait in human brain. Unlike allele-specific methylation, genetic polymorphisms mark both cis- and trans-regulatory genetic sites at measurable distances from their CpG sites. Some of the genetically regulated DNA methylation is directly connected with genetically regulated gene expression variation.