Epe1 recruits BET family bromodomain protein Bdf2 to establish heterochromatin boundaries

Abstract

Heterochromatin spreading leads to the silencing of genes within its path, and boundary elements have evolved to constrain such spreading. In fission yeast, heterochromatin at centromeres I and III is flanked by inverted repeats termed IRCs, which are required for proper boundary functions. However, the mechanisms by which IRCs prevent heterochromatin spreading are unknown. Here, we identified Bdf2, which is homologous to the mammalian bromodomain and extraterminal (BET) family double bromodomain proteins involved in diverse types of cancers, as a factor required for proper boundary function at IRCs. Bdf2 is enriched at IRCs through its interaction with the boundary protein Epe1. The bromodomains of Bdf2 recognize acetylated histone H4 tails and antagonize Sir2-mediated deacetylation of histone H4K16. Furthermore, abolishing H4K16 acetylation (H4K16ac) with an H4K16R mutation promotes heterochromatin spreading, and mimicking H4K16ac by an H4K16Q mutation blocks heterochromatin spreading at IRCs. Our results thus illustrate a mechanism of establishing chromosome boundaries at specific sites through the recruitment of a factor that protects euchromatic histone modifications. They also reveal a previously unappreciated function of H4K16ac in cooperation with H3K9 methylation to regulate heterochromatin spreading.

Keywords: BET; H4K16; acetylation; boundary; heterochromatin.

Figure 1.

Bdf2 is required for boundary function. (A,C,G) Schematic diagrams of the IRC1R∷ura4⁺, L5-IRC1R-ura4⁺, and IRC3L∷ura4⁺ reporters. (B,D,H) Tenfold serial dilution analyses of the indicated yeast strains were grown on the indicated medium to measure the spreading of heterochromatin into the ura4⁺ reporter. (E,I) ChIP analysis of H3K9me2 and Swi6 levels at the ura4⁺ reporters, normalized to act1. The data are averages of three experiments, and error bars represent standard deviation. (F) qRT–PCR analysis of emc5 transcript levels, normalized to act1. Data presented are averages of three experiments, and error bars represent standard deviation.

Figure 2.

Bdf2 is enriched at IRCs. (A,C,E) ChIP–chip analysis of Bdf2-Flag and H3K9me2 levels across centromeres I, II, and III. The data are averages of two microarrays. Black arrows in the diagrams indicate the positions of IRCs. Bars indicate tRNA genes. Note that centromere II does not contain IRCs. (B,D,F) ChIP-qPCR analysis of Bdf2-Flag levels at boundary regions, normalized to act1. The positions of amplified regions used for qPCR quantification are indicated. The data are averages of three experiments, and error bars represent standard deviation.

Figure 3.

Epe1 recruits Bdf2 to IRCs. (A) Multidimensional protein identification technology (MudPIT) mass spectrometry analyses of purified Bdf2-Flag and Epe1-Flag complexes. The number of Bdf2 and Epe1 peptides identified and the percentage of each protein that these peptides cover are indicated. (B,G) Cell extracts from the indicated strains were incubated with Flag-agarose beads to isolate Epe1-Flag. Bound proteins were resolved by SDS-PAGE, and Western blot analyses were performed with Myc and Flag antibodies. (C,I) Serial dilution analyses were performed to measure heterochromatin spreading outside of IRC1R∷ura4⁺. (D,H) ChIP analysis of Bdf2-Flag and Epe1-Flag levels at IRCs, normalized to act1. The data are averages of three experiments, and error bars represent standard deviation. (E) Diagram of Bdf2 constructs used in yeast two-hybrid analysis. (F) Yeast two-hybrid analysis of Bdf2 with Epe1. Bdf2 was fused with the Gal4 DNA-binding domain, and Epe1 was fused with an activation domain. Interaction between Bdf2 and Epe1 resulted in the activation of a HIS3 reporter gene, allowing cells to grow in the absence of histidine.

Figure 4.

The bromodomains of Bdf2 are required for boundary function. (A) Schematic diagram of Bdf2 protein. The positions of mutations are indicated. (B) Peptide pull-down assays of the indicated recombinant proteins with biotinylated H4 tail (2–24) unmodified and tetra-acetylated at the K5, K8, K12, and K16 (Ac-H4) peptides as well as H3 tail (1–21) unmodified and diacetylated at the K9 and K14 (Ac-H3) peptides. (C) Serial dilution analysis to measure heterochromatin spreading outside of IRC1R∷ura4⁺. (D,E) ChIP analysis of H3K9me2 and Swi6 levels at IRC1R∷ura4⁺ and Bdf2-Flag levels at IRC1R and IRC3L, normalized to act1. The data are averages of three experiments, and error bars represent standard deviation. (F) Western blot analysis of Bdf2-Flag levels. (G) Schematic diagram of the L5-gbs-ura4⁺ reporter. (H) Serial dilution analysis to measure heterochromatin spreading to ura4⁺. All strains used contain epe1Δ to rule out the possibility that Bdf2 recruits Epe1 to establish heterochromatin boundaries. (I) ChIP analysis of H3K9me2 and Swi6 levels at L5-gbs-ura4⁺ and Bdf2-GBD levels at 3xgbs, normalized to act1. The data are averages of three experiments, and error bars represent standard deviation. All strains used contain epe1Δ.

Figure 5.

Bdf2 counteracts Sir2-mediated histone deacetylation. (A,C) Serial dilution analysis to measure heterochromatin spreading outside of IRC1R∷ura4⁺. (B,D,E) ChIP analysis of H3K9me2 and Swi6 levels at IRC1R∷ura4⁺ and H4K16ac levels at IRC1, normalized to act1. The data are averages of three experiments, and error bars represent standard deviation. (F, left) Coomassie-stained gel of recombinant Sir2. (Right) HDAC assays were performed with recombinant Sir2, a tetra-acetylated histone H4 peptide, and recombinant Bdf2. The production of nicotinamide was measured via the generation of nicotinic acid and free ammonia by nicotinamidase.

Figure 6.

H4K16ac modulates heterochromatin boundary function. (A–C) ChIP–chip analysis of H4K16ac levels across centromeres I, II, and III. The Bdf2-Flag profile is also shown for comparison. (D) Serial dilution analysis to measure heterochromatin spreading outside of IRC1R∷ura4⁺. All strains used are in an h4.1Δ/h4.3Δ background. (E,F) ChIP analysis of H3K9me2 and Swi6 levels at IRC1R∷ura4⁺ and Bdf2 levels at IRC1, normalized to act1. The data are averages of three experiments, and error bars represent standard deviation. All strains used are in an h4.1Δ/h4.3Δ background.

Figure 7.

(A) Serial dilution analysis to measure heterochromatin spreading outside of IRC1R∷ura4⁺. All strains used are in an h4.1Δ/h4.3Δ background. (B–D) ChIP analysis of H3K9me2 and Swi6 levels at IRC1R∷ura4⁺ and H4K16ac and Bdf2 levels at IRC1, normalized to act1. The data are averages of three experiments, and error bars represent standard deviation. (E) A model by which H4K16ac and Bdf2 regulate heterochromatin spreading and boundary function. Heterochromatin spreading is mediated by cycles of Clr4-mediated H3K9me and the recruitment of Swi6. Sir2 mediates the deacetylation of H3K9, which is required for creating the substrate for Clr4, as well as the deacetylation of H4K16, which facilitates nucleosome compaction to bring the adjacent nucleosome closer to Clr4. Bdf2 is recruited to IRCs through Epe1, which is highly enriched at IRCs. Mst1 mediates H4K16ac at IRCs, which further stabilizes binding of Bdf2 to IRCs through its bromodomains. Bdf2 protects H4 tails from deacetylation by Sir2, preventing further heterochromatin spreading.