Acetylation at Lys56 or Lys122 of H3 decreases transcription barrier at SHL(0) and facilitates RNAPII progression.

(A) Secondary structure of histone H3, showing the acetylated residues (yellow). (B) SHLs, transcription direction, and Lys56, Lys64, and Lys122 are shown on the nucleosome structure (PDB ID: 7K61). Histones H2A, H2B, H3, H4, and DNA are colored pale yellow, pale red, pale blue, pale green, and gray, respectively. Lys56, Lys64, and Lys122 are shown in blue, purple, and orange, respectively. (C) Schematic representation of the transcription assay. (D) Transcription assays using free DNA, and unmodified, H3K56ac, H3K64ac, H3K122ac, and H3K4/9/14/18/23/27ac nucleosome templates. RNA transcripts were analyzed by denaturing PAGE and detected by DY647 fluorescence. (E-H) Quantification of transcripts corresponding to (E) run-off, (F) SHL(-5), (G) SHL(-1), and (H) SHL(0) positions. Amounts of transcripts were normalized to those of the unmodified nucleosome template. Bar graphs show the means of three independent experiments, and dots indicate the individual values. Error bars represent the standard deviation.

Cryo-EM data collection, processing, refinement, and validation statistics

Cryo-EM analysis of the H3K56ac nucleosome reveals weakened density at the entry/exit DNA region.

(A) Cryo-EM density maps of the unmodified, H3K56ac, H3K64ac, and H3K122ac nucleosomes. H2A, H2B, H4, and DNA are colored yellow, red, green, and gray, respectively. H3 in the unmodified, H3K56ac, H3K64ac, and H3K122ac nucleosomes is colored cyan, blue, dark blue, and purple, respectively. The density map corresponding to Ac-Lys56 is shown in magenta. (B) Schematic representation of the micrococcal nuclease digestion assay. (C) Micrococcal nuclease digestion assays of the unmodified, H3K56ac, H3K64ac, and H3K122ac nucleosomes. Deproteinized DNA was analyzed by non-denaturing PAGE and stained with ethidium bromide. (D) Quantification of the micrococcal nuclease assay. Band intensities indicated by asterisks in (C) were normalized with those of the undigested DNA. Bar graphs show the means of three independent experiments, and dots indicate the individual values. Error bars represent the standard deviation.

Acetylation at Lys122 of H3 reorients the side chain away from the nucleosomal DNA.

(A-C) Close-up views of the regions surrounding the acetylated residues in H3K56ac (A), H3K122ac (B), and H3K64ac (C) nucleosomes. H2A, H2B, H4, and DNA are colored yellow, red, green, and gray, respectively. H3 in the unmodified, H3K56ac, H3K64ac, and H3K122ac nucleosomes is colored cyan, blue, dark blue, and purple, respectively. Acetylated lysine residues and their corresponding unmodified lysine residues are shown in magenta. The distances between the nitrogen atom at the zeta position of the lysine or acetyl-lysine residue and the nearest oxygen atom of the DNA backbone are shown in (B).

Acetylation at Lys56 and Lys122 of H3 likely promotes transcription through distinct yet complementary mechanisms.

The direct contacts between the distal DNA end of the nucleosome and RNAPII, as well as the histone-DNA interactions near the dyad axis, likely contribute to RNAPII pausing at the SHL(0) position. The acetylation at Lys56 of H3 enhances the flexibility of the DNA ends of the nucleosome, which may alleviate steric clashes between the DNA and RNAPII and facilitate RNAPII passage. In contrast, the acetylation at Lys122 of H3 weakens histone-DNA interactions near the dyad axis, thereby reducing RNAPII pausing and enhancing transcription at the SHL(0) region.