Anti-silencing factor Epe1 associates with SAGA to regulate transcription within heterochromatin

Abstract

Heterochromatin is a highly condensed form of chromatin that silences gene transcription. Although high levels of transcriptional activities disrupt heterochromatin, transcription of repetitive DNA elements and subsequent processing of the transcripts by the RNAi machinery are required for heterochromatin assembly. In fission yeast, a JmjC domain protein, Epe1, promotes transcription of DNA repeats to facilitate heterochromatin formation, but overexpression of Epe1 leads to heterochromatin defects. However, the molecular function of Epe1 is not well understood. By screening the fission yeast deletion library, we found that heterochromatin defects associated with Epe1 overexpression are alleviated by mutations of the SAGA histone acetyltransferase complex. Overexpressed Epe1 associates with SAGA and recruits SAGA to heterochromatin regions, which leads to increased histone acetylation, transcription of repeats, and the disruption of heterochromatin. At its normal expression levels, Epe1 also associates with SAGA, albeit weakly. Such interaction regulates histone acetylation levels at heterochromatin and promotes transcription of repeats for heterochromatin assembly. Our results also suggest that increases of certain chromatin protein levels, which frequently occur in cancer cells, might strengthen relatively weak interactions to affect the epigenetic landscape.

Keywords: Epe1; SAGA; heterochromatin; histone; methylation; transcription.

Figure 1.

Overexpression of Epe1 leads to defective gene silencing. (A) Schematic diagram of the reporter genes used. Bars indicate the positions of PCR fragments used in ChIP analyses. (B, left) Tenfold serial dilution analyses of the indicated yeast strains grown on the indicated media to measure the expression of ura4⁺ reporter genes. (Right) The first two panels show ChIP analyses of H3K9me3 and Swi6 levels at repetitive DNA elements within pericentric (dh) and mating-type regions (cenH), normalized to act1⁺. The last panel shows quantitative RT–PCR (qRT–PCR) analysis of the dh and cenH transcripts, normalized to act1⁺.

Figure 2.

A genetic screen for mutations that alleviate the effects of Epe1 overexpression on heterochromatin integrity. (A) Workflow to introduce otr::ura4⁺ and nmt41-epe1⁺ into the deletion library. (B) A representative image of cells grown on medium without thiamine and containing 5-FOA. Each square represents quadruplicates of colonies of the same genotype. The box indicates the position of gcn5Δ. (C, left) Tenfold serial dilution analyses of the indicated yeast strains grown on the indicated media to measure the expression of the otr::ura4⁺ reporter gene. (Right) The first two panels show ChIP analyses of H3K9me3 and Swi6 levels at pericentric dh repeats, normalized to act1⁺. The last panel shows qRT–PCR analysis of dh transcript, normalized to act1⁺. (D) Western blot analysis to measure the levels of HA-tagged Epe1. A nonspecific band served as a loading control. (E) Coimmunoprecipitation analyses of Epe1 and Swi6. The immunoprecipitation was performed with Flag-agarose beads, and Western blot analyses were performed with Flag and Swi6 antibodies. (F) ChIP analyses of HA-Epe1 levels at pericentric dh repeats, normalized to act1⁺.

Figure 3.

The acetyltransferase activity of SAGA is required for Epe1 function. Tenfold serial dilution analyses of the indicated yeast strains grown on the indicated media to measure the expression of the otr::ura4⁺ reporter gene. (A) The effects of deletion of the SAGA acetyltransferase module. (B) The effects of deletion of the SAGA deubiquitination and SPT modules.

Figure 4.

Epe1 associates with SAGA. (A) Mass spectrometry analyses of purified protein complexes. The spectral count (left) and the sequence coverage of each protein (right) are indicated. (B) Coimmunoprecipitation analyses of Epe1 and Gcn5. The lysates were treated with benzonase before immunoprecipitation was performed with Flag-agarose beads. Western blot analyses were performed with Flag and myc antibodies. (C) ChIP analyses of Gcn5 levels at pericentric dh repeat, shown as ChIP/input normalized to the no tag control. (D) Tenfold serial dilution analyses of the indicated yeast strains grown on the indicated media to measure the expression of the otr::ura4⁺ reporter gene.

Figure 5.

SAGA regulates histone acetylation and Pol II access at heterochromatin when Epe1 is overexpressed. (A–C) ChIP analyses of the levels of H3K9ac, H3K14ac, and the Ser2 phosphorylated form (Ser2P) of Pol II at the pericentric dh repeat, shown as ChIP/input normalized to wild type.

Figure 6.

SAGA counteracts the effects of HDAC Sir2. (A) Coimmunoprecipitation analyses of Epe1-Flag and Spt7-myc. The lysates were treated with benzonase before immunoprecipitation was performed with Flag-agarose beads. Western blot analyses were performed with Flag and myc antibodies. (B,D,E,F) ChIP analyses of SAGA components, H3K9ac, H3K14ac, and Pol II Ser2P at the pericentric dh repeat, shown as ChIP/input normalized to wild type. (C) Tenfold serial dilution analyses of the indicated yeast strains grown on the indicated media to measure the expression of the imr::ura4⁺ reporter gene. (G) qRT–PCR analysis of the dh transcript, normalized to act1⁺.

Figure 7.

Model for the function of Epe1 at heterochromatin. Epe1 demethylates H3K9, competes with HDAC Clr3 for binding to Swi6, and recruits SAGA to counteract HDAC Sir2. The combined activities lead to higher levels of histone acetylation at heterochromatin, which promotes transcription of the underlying DNA repeats.