Regulated histone methyltransferase and demethylase complexes in the control of genes by nuclear receptors.

Liganded nuclear receptors (NRs) are DNA-binding transcription factors that control the transcription of target genes. Such NRs exert their transcriptional functions via ligand binding-induced interactions with a number of coregulator complexes to reorganize chromatin state. Intensive investigation of NR coregulator complexes has revealed that, besides histone acetylation, histone methylation is critical for ligand-dependent transcriptional controls by NRs. Our recent biochemical screening for NR coregulator complexes showed that the enzymatic activities of these histone methylation/demethylation complexes are under the control of posttranslational modifications (PTMs) of their catalytic subunit. Characterization of such regulated complexes has established the concept that transcriptional coregulator complexes sense and decode cellular signals at the molecular level. In this symposium review, we will illustrate our recent findings regarding PTM-based regulation of NR transcriptional control and discuss how these findings are applicable to the diverse roles of NR coregulators in interpreting regulatory signals into proper gene regulation.

Binding affinity of GM3 lactone for influenza virus.

We investigated the interaction of GM3 lactone with influenza virus. The specific bindings of influenza virus and its hemagglutinin to GM3 lactone-containing mixed monolayers were studied by using a quartz-crystal microbalance. It has been known that gangliosides as receptors for influenza virus are also substrates for virus neuraminidase. GM3 lactone, however, was found to bind to influenza virus hemagglutinin, but not to be substrate for virus neuraminidase.

Quantitative measurements of the interaction between monosialoganglioside monolayers and wheat germ agglutinin (WGA) by a quartz-crystal microbalance.

Monosialogangliosides (GM1, GM2, GM3 and GM4) were reconstituted in lipid monolayers at the air-water interface. The binding amounts and the initial binding rates of wheat germ agglutinin (WGA) to the monosialoganglioside monolayers were quantitatively studied by use of a quartz-crystal microbalance (QCM). A QCM was horizontally attached to the monolayer from the air phase, and the binding behavior (mass increase) was followed by the frequency decrease of the QCM. WGA binding affinities for the ganglioside monolayers were influenced by hydrophilic head groups of lipid matrices, densities of gangliosides, and sequences of oligosaccharide in gangliosides. Binding of WGA to the gangliosides reconstituted in a phosphatidylcholine (sphingomyelin and distearoylphosphatidylcholine) matrix was strongly suppressed, but not in a neutral glycolipids (GlcCer, GalCer, and LacCer), dipalmitoylphosphatidylethanolamine, and dipalmitoylphosphatidylethanolamine matrix. WGA showed high affinity for monolayers containing 20 mol% gangliosides, but only low affinity for 100% ganglioside monolayers. WGA preferably binds to gangliosides in the following sequence: GM3 > GM4 >> GM2 = GM1. No affinities of WGA for GM2 and GM1 were observed. The combined techniques of monolayer and QCM have the advantages of investigating recognition properties of gangliosides.

Kinetic measurements of DNA hybridization on an oligonucleotide-immobilized 27-MHz quartz crystal microbalance.

A highly sensitive 27-MHz quartz-crystal microbalance, on which a 10-30-mer oligonucleotide was immobilized as a probe molecule, was employed to detect hybridization of complementary oligonucleotides in aqueous solution. From frequency decreases (mass increases due to the hybridization) with passage of time, kinetic parameters such as association constants (K(a)) and binding and dissociation rate constants (k(1) and k(-1)) could be obtained, as well as binding (hybridization) amount at the nanogram level (delta m). Kinetic studies were carried out by changing various parameters: (i) the immobilization method of a probe oligonucleotide on Au electrode, (ii) number of mismatching bases in sequences of target oligonucleotides, (iii) length of both probe and target oligonucleotides, (iv) hybridization temperature, and (v) ionic strength in solution. The obtained results were compared with those obtained by a surface plasmon resonance method using a BIAcore system.