Regulation of the Stability of the Histone H2A–H2B Dimer by H2A Tyr57 Phosphorylation

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

Diazirine Photocrosslinking Recruits Activated FTO Demethylase Complexes for Specific <i>N</i>⁶‑methyladenosine Recognition

<i>N</i>⁶-methyladenosine (m⁶A) is a prevalent modification of RNAs. m⁶A exists in mRNA and plays an important role in RNA biological pathways and in RNA epigenetic regulation. We applied diazirine photocrosslinking to the event of m⁶A recognition mediated by the fat mass and obesity associated (FTO) demethylase. A highly photoreactive diazirine adjacent to m⁶A on the RNA successfully recruited activated FTO complexes with an m⁶A preference. The process of recognition of m⁶A via FTO using diazirine photocrosslinking was controlled by the α-ketoglutarate (α-KG) cosubstrate and the Fe­(II) cofactor, which are involved in m⁶A oxidative demethylation. In addition, FTO bound to ssRNAs prior to the m⁶A recognition process. Diazirine photocrosslinking contributes to increasing the chances of capturing activated FTO complexes with specific m⁶A recognition and provides new insights into the dynamic FTO oxidative demethylation process

Hybridization-Sensitive Fluorescent Oligonucleotide Probe Conjugated with a Bulky Module for Compartment-Specific mRNA Monitoring in a Living Cell

Live-cell RNA imaging at specific intracellular locations is technically limited because of the diffusive nature of small oligonucleotide probes. The bulky fluorescent light-up probes that possess streptavidin or gold nanoparticles at the end of oligonucleotides were designed and synthesized. The bulky probes allowed nucleus- and cytoplasm-selective monitoring of endogenous mRNAs through nuclear and cytoplasmic microinjection, respectively. Simultaneous use of bulky and unbulky probes conjugated with different fluorescent dyes enabled dual color imaging of mRNAs present in nucleus and cytoplasm. Furthermore, we observed that the fluorescence near the cell edge in a living HeLa cell traveled over time in coordination with the dynamic formation and deformation of the pseudopodial protrusions after lipofection of the bulky probes

Base-Resolution Analysis of 5‑Hydroxymethylcytosine by One-Pot Bisulfite-Free Chemical Conversion with Peroxotungstate

5-Hydroxymethylcytosine (^hmC) is an essential intermediate in the active DNA demethylation pathway. Here we report a new base-resolution method for measuring ^hmC by combining peroxotungstate-mediated oxidation and sequencing analysis. We reveal that an oxidized product of ^hmC, trihydroxylated thymine (^thT), tolerated the incorporation of dATP as a substrate in the process of DNA polymerase elongation. By comparing the results of Sanger sequencing before and after the oxidation, we observed that ^hmC sites on single-stranded DNAs could be discriminated from unmethylated cytosines. We found that a thermal cycle condition during peroxotungstate treatment enhanced the oxidation reaction of ^hmC in double-stranded DNA. Furthermore, Illumina sequencing analysis of ^hmC-containing synthetic genome fragments enabled us to identify simultaneously the positions of ^hmC in base resolution. This bisulfite-free simple ^hmC detection technique could facilitate the acquisition of epigenomic information