Structural insight into the recognition of acetylated histone H3K56ac mediated by the bromodomain of CREB‐binding protein

Abstract

Coordinates of the CBP bromodomain in complex with H3K56ac as described in this article have been deposited in the PDB with accession number 5GH9.

TRIM66 reads unmodified H3R2K4 and H3K56ac to respond to DNA damage in embryonic stem cells

“…2c, d). Based on previous researches reporting the acetyl-lysine binding partners of Bromodomain 3–12 , we chose a series of related peptides derived from H3 and H4, to test their interactions with TRIM66-WT 965-1160 by ITC experiments. No obvious bindings were observed between TRIM66-WT 965-1160 with these acetylated peptides, except for the H3 48-57 K56ac peptide, which bound to TRIM66-WT 965-1160 with a K D of 109 μM (Fig.…”

“…The construct of full-length H4 inserted into a pET3a plasmid was expressed in E. coli BL21 (DE3). The expression of full-length histone H3K56ac was performed in BL21 (DE3) cells transformed with pBK-AxcKRS3 and pET22b encoding H3 with amber codons at the K56 site 12 . The DNA fragment of full-length TRIM66 was synthesized by General Biosystems and ligated into fugw vector (Addgene).…”

“…Histone H4 was induced at A600 = 0.8 using 0.5 mM IPTG and the cells were incubated at 37 °C for 6 h. Histone H3K56ac was supplemented with 20 mM nicotinamide, 10 mM acetyl-lysine (Kac), and 0.2% arabinose, and incubated for another 30 min before induction with 0.5 mM IPTG 12 . The purification procedure of histone was shown in Supplementary Methods.…”

“…The recombination methods of the H3-H4 tetramer and the H3-H4-ASF1a complex in vitro were shown in Supplementary Methods 12,53 . About 50 μg protein, GST, GST-TRIM66-WT 965-1160 , or its mutant (D986A, N1123A, and D986A/N1123A) were bound to glutathione-sepharose beads (GE Healthcare) in a buffer (20 mM Tris, 200 mM NaCl, and 1 mM DTT) at pH 7.5 and incubated at 4 °C for 4 h. The glutathione-sepharose beads were pelleted and washed thrice with washing buffer.…”

“…Both PHD finger and Bromodomain are evolutionarily and structurally conserved modules. The majority of canonical single PHD fingers are reported to read the N-terminal tail of histone H3, mainly the methylation status of H3K4 (H3K4me2/3 or H3K4me0) 2 , whereas the Bromodomains are able to read the acetylated lysine, especially that on histone H3 and H4 3–12 . In recent years, the structure and function of the PHD Bromodomains of TRIM24, TRIM28, and TRIM33 have been studied extensively 5,6,13 .…”