Association of Taf14 with acetylated histone H3 directs gene transcription and the DNA damage response

Abstract

The YEATS domain, found in a number of chromatin-associated proteins, has recently been shown to have the capacity to bind histone lysine acetylation. Here, we show that the YEATS domain of Taf14, a member of key transcriptional and chromatin-modifying complexes in yeast, is a selective reader of histone H3 Lys9 acetylation (H3K9ac). Structural analysis reveals that acetylated Lys9 is sandwiched in an aromatic cage formed by F62 and W81. Disruption of this binding in cells impairs gene transcription and the DNA damage response. Our findings establish a highly conserved acetyllysine reader function for the YEATS domain protein family and highlight the significance of this interaction for Taf14.

Keywords: DNA damage; Taf14; YEATS domain; gene transcription; histone acetylation.

Figure 1.

The YEATS domain of the transcriptional regulator Taf14 binds acetylated histone peptides. (A) Schematic of the YEATS proteins in S. cerevisiae. The YEATS domains in Yaf9 and Sas5 have 45% and 46% shared identity with the Taf14 YEATS domain. (B) Representative images of histone peptide microarrays of full-length Taf14. (C) A scatter plot of the relative binding intensity normalized to the most intense signal, highlighting binding of Taf14 to histone H3 acetylated peptides on duplicate arrays. (D) Western blot analyses of peptide pull-down assays with the corresponding GST-tagged Taf14 proteins and histone peptides. (E) Western blot analyses of peptide pull-down assays with the corresponding GST-tagged YEATS domains and histone peptides.

Figure 2.

Taf14 YEATS selectively binds H3K9ac. (A,B) Superimposed ¹H,¹⁵N HSQC spectra of the Taf14 YEATS domain (residues 1–132) collected upon titration with the H3K9ac_1–12 peptide (A) and unmodified H3_1–12 peptide (B). Spectra are color-coded according to the protein:peptide molar ratio. (C) Fluorescence polarization binding assays of the Taf14 YEATS domain with fluorescently labeled H3_1–20 (black), H3_1–20K9ac (blue), and H3_1–20K9,14,18ac (green) peptides. (D) Superimposed ¹H,¹⁵N HSQC spectra of the Taf14 YEATS domain collected upon titration with acetylated lysine (free amino acid).

Figure 3.

Structural basis for the interaction between Taf14 YEATS and H3K9ac. (A) The overall structure of the Taf14 YEATS domain (gray) in complex with the H3K9ac peptide (orange) depicted as cartoon. (B) Surface representation of the Taf14 YEATS domain reveals a deep binding pocket for acetylated lysine. (C) Hydrogen-bonding (blue dashes) network between the H3K9ac peptide and the Taf14 YEATS domain. Water molecules are shown as red spheres. (D) Close-up view of the K9ac-binding pocket, with the hydrogen-bonding interaction represented by blue dashes.

Figure 4.

The Taf14 YEATS domain regulates transcription and DNA damage response. (A) Schematic of the Taf14-containing complexes in S. cerevisiae with the known interacting members listed. (B) Western blot analysis of peptide pull-down assays performed with full-length Taf14 showing disruption of H3K9ac binding when the aromatic cage residue W81 is mutated. (C) Chromatin association assays with wild-type or W81A mutant Taf14 YEATS-HA expressed in wild-type BY4741 showing loss of chromatin association in the point mutant. (D) Spotting assays with BY4741 or taf14Δ strains transformed with empty vector or plasmids expressing HA-tagged full-length wild-type or taf14_W81A at the indicated temperatures. (E) Spotting assays with wild-type (BY4741) or gcn5Δ strains demonstrating that Taf14 YEATS acetyl binding becomes necessary for normal cell growth in the context of depleted histone acetylation levels. (F) Spotting assays with wild-type (BY4741) or taf14Δ strains transformed with the indicated plasmids and grown on control (SC-Ura-His) and 6-azauracil (6-AU) medium. (G) Venn diagram of differentially expressed (DE) genes identified by RNA sequencing (P < 0.01, fold change >1.5). Of the 843 DE genes identified in taf14Δ (red), 204 genes (24%) were also identified as DE between TAF14 and taf14_W81A (yellow). (H) Scatter plot of log₂-normalized gene counts in TAF14 and taf14_W81A. Of the 204 genes identified in G, 72 genes were up-regulated (orange), and 132 genes were down-regulated (blue) in taf14_W81A. (I) Gene ontology analysis of the 204 genes identified in G, showing that up-regulated genes and down-regulated genes were enriched in several molecular functions (enrichment scores shown in orange and blue, respectively) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (red). (J) Quantitative RT–PCR showing reduced transcription of several genes in both taf14Δ and taf14_W81A. In contrast, GAL7 transcript levels do not depend on Taf14 YEATS. Error bars represent standard deviations of three biological replicates. (*) P < 0.05 compared with wild type.