Intra- and inter-nucleosomal interactions of the histone H4 tail revealed with a human nucleosome core particle with genetically-incorporated H4 tetra-acetylation

Abstract

Post-translational modifications (PTMs) of histones, such as lysine acetylation of the N-terminal tails, play crucial roles in controlling gene expression. Due to the difficulty in reconstituting site-specifically acetylated nucleosomes with crystallization quality, structural analyses of histone acetylation are currently performed using synthesized tail peptides. Through engineering of the genetic code, translation termination, and cell-free protein synthesis, we reconstituted human H4-mono- to tetra-acetylated nucleosome core particles (NCPs), and solved the crystal structures of the H4-K5/K8/K12/K16-tetra-acetylated NCP and unmodified NCP at 2.4 Å and 2.2 Å resolutions, respectively. The structure of the H4-tetra-acetylated NCP resembled that of the unmodified NCP, and the DNA wrapped the histone octamer as precisely as in the unmodified NCP. However, the B-factors were significantly increased for the peripheral DNAs near the N-terminal tail of the intra- or inter-nucleosomal H4. In contrast, the B-factors were negligibly affected by the H4 tetra-acetylation in histone core residues, including those composing the acidic patch, and at H4-R23, which interacts with the acidic patch of the neighboring NCP. The present study revealed that the H4 tetra-acetylation impairs NCP self-association by changing the interactions of the H4 tail with DNA, and is the first demonstration of crystallization quality NCPs reconstituted with genuine PTMs.

JQ1 affects BRD2-dependent and independent transcription regulation without disrupting H4-hyperacetylated chromatin states

“…We synthesized the full-length histone H4 protein with site-specific acetylations at K5 and K8 on a milligram scale [22], and used it as the antigen. Through screening with ELISA and Western blot, we successfully obtained two hits of hybridoma (1A9D7 and 2A7D9) that recognize the histone H4 proteins containing di-acetylation of K5acK8ac (i.e.…”

“…The full-length human H4 protein containing acetyl residue at K5 and K8 (H4K5acK8ac) was synthesized in a cell-free system essentially as previously described [22,56] and used to immunize mice. Generation, selection, and purification of antibodies were performed as previously described [24], with the following modifications.…”

Proteome-wide Structural Analysis of PTM Hotspots Reveals Regulatory Elements Predicted to Impact Biological Function and Disease

“…In several cases, regions of intrinsic structural disorder have been shown to act as regulatory elements and are often targets of modification (14–16). To account for the importance of these unresolved sites, we used measures of intrinsic disorder defined by the IUPred application (17).…”

DNA conformational transitions inferred from re-evaluation ofm|Fo| −D|Fc| electron-density maps

“…This parameter related to atomic thermal motion is expressed as a probability density function of atomic displacements from the mean position. It has been reported that tetra-acetylation of H4 histones significantly increases the B factors of wrapped DNAs in the vicinity of the tetraacetylated N-terminal tail of inter-and intra-nucleosomal H4 (Wakamori et al, 2015). From this observation and biochemical assays, it is suggested that H4 tetra-acetylation impairs the self-association of nucleosome core particles by changing the interactions of the H4 tail with DNA.…”