RESUMEN
In the regulation processes of gene expression, genomic DNA and nuclear proteins, including histone proteins, cooperate with each other, leading to the distinctive functions of eukaryotic cells such as pluripotency and differentiation. Chemical modification of histone proteins and DNA has been revealed as one of the major driving forces in the complicated epigenetic regulation system. However, understanding of the precise molecular mechanisms is still limited. To address this issue, researchers have proposed both biological and chemical strategies for the preparation and detection of modified proteins and nucleic acids. In this review, we focus on chemical methods around the field of epigenetics. Chemical protein synthesis has enabled the preparation of site-specifically modified histones and their successful application to various inâ vitro assays, which have emphasized the significance of posttranslational modifications of interest. We also review the modification-specific chemical reactions against synthetic and genomic DNA, which enabled discrimination of several modified bases at single-base resolution.
Asunto(s)
ADN/metabolismo , Epigenómica , Histonas/metabolismo , 5-Metilcitosina/química , 5-Metilcitosina/metabolismo , ADN/química , Metilación de ADN , Histonas/química , Humanos , Procesamiento Proteico-Postraduccional , Técnicas de Síntesis en Fase SólidaRESUMEN
Histone H2A and H2B form a H2A-H2B heterodimer, which is a fundamental unit of nucleosome assembly and disassembly. Several posttranslational modifications change the interface between the H2A-H2B dimer and the H3-H4 tetramer and regulate nucleosome stability. However, posttranslational modifications associated with the interface between H2A and H2B have not been discussed. In this paper, it is shown that Tyr57 phosphorylation in H2A strongly influences H2A-H2B dimerization. Tyr57-phosphorylated H2A was chemically synthesized and utilized to reconstitute the H2A-H2B dimer and nucleosome as well as canonical H2A. Thermal shift assays showed that phosphorylation destabilized the dimer and facilitated dissociation of H2A and H2B from the nucleosome structure. The proximity between H2A Tyr57 and the H2B αC helix is assumed to lead the destabilization. The DNA accessibility of the nucleosome was estimated by using micrococcal nuclease. The phosphorylated nucleosome did not change DNA accessibility compared to that of the canonical nucleosome. It is demonstrated that phosphorylation at Tyr57 changes the H2A-H2B dimer interaction but does not interfere with histone-DNA interactions. This work on the destabilization of the H2A-H2B dimer by Tyr57 phosphorylation is a promising step in elucidating control mechanisms of dynamic behavior of H2A and H2B through posttranslational modifications.
Asunto(s)
Histonas/química , ADN/química , Dimerización , Modelos Moleculares , Fosforilación , Conformación Proteica , Pliegue de Proteína , Estabilidad ProteicaRESUMEN
A new fluorescent nucleotide with desmethyl thiazole orange dyes, D'(505), has been developed for expansion of the function of fluorescent probes for live-cell RNA imaging. The nucleoside unit of D'(505) for DNA autosynthesis was soluble in organic solvents, which made the preparation of nucleoside units and the reactions in the cycles of DNA synthesis more efficient. The dyes of D'(505)-containing oligodeoxynucleotide were protonated below pH 7 and the oligodeoxynucleotide exhibited hybridization-sensitive fluorescence emission through the control of excitonic interactions of the dyes of D'(505). The simplified procedure and effective hybridization-sensitive fluorescence emission produced multicolored hybridization-sensitive fluorescent probes, which were useful for live-cell RNA imaging. The acceptor-bleaching method gave us information on RNA in a specific cell among many living cells.
Asunto(s)
Benzotiazoles/metabolismo , Colorantes Fluorescentes/metabolismo , Imagen Molecular/métodos , Hibridación de Ácido Nucleico/métodos , Sondas de Ácido Nucleico/metabolismo , Oligonucleótidos/metabolismo , Quinolinas/metabolismo , ARN/metabolismo , Nucleótidos de Timina/metabolismo , Benzotiazoles/química , Supervivencia Celular , Dicroismo Circular , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Células HeLa , Humanos , Espacio Intracelular/metabolismo , Sondas de Ácido Nucleico/química , Oligonucleótidos/síntesis química , Oligonucleótidos/química , Fotoblanqueo , Protones , Quinolinas/química , Coloración y Etiquetado , Nucleótidos de Timina/químicaRESUMEN
The chemical synthetic route to histone H2A is described. An H2A-H2B dimer, histone octamer, and nucleosome were reconstituted with the synthetic H2A. Fluorescein-labeled H2A and multiply modified H2A, which has three different posttranslational modifications, were also synthesized, and applied to live-cell imaging and in vitro nucleosome stability assays, respectively.