The JmjC domain protein Epe1 prevents unregulated assembly and disassembly of heterochromatin

Abstract

Heterochromatin normally has prescribed chromosomal positions and must not encroach on adjacent regions. We demonstrate that the fission yeast protein Epe1 stabilises silent chromatin, preventing the oscillation of heterochromatin domains. Epe1 loss leads to two contrasting phenotypes: alleviation of silencing within heterochromatin and expansion of silent chromatin into neighbouring euchromatin. Thus, we propose that Epe1 regulates heterochromatin assembly and disassembly, thereby affecting heterochromatin integrity, centromere function and chromosome segregation fidelity. Epe1 regulates the extent of heterochromatin domains at the level of chromatin, not via the RNAi pathway. Analysis of an ectopically silenced site suggests that heterochromatin oscillation occurs in the absence of heterochromatin boundaries. Epe1 requires predicted iron- and 2-oxyglutarate (2-OG)-binding residues for in vivo function, indicating that it is probably a 2-OG/Fe(II)-dependent dioxygenase. We suggest that, rather than being a histone demethylase, Epe1 may be a protein hydroxylase that affects the stability of a heterochromatin protein, or protein-protein interaction, to regulate the extent of heterochromatin domains. Thus, Epe1 ensures that heterochromatin is restricted to the domains to which it is targeted by RNAi.

Figure 1

Epe1 restrains heterochromatin to its normal domain. (A) Location of ura4⁺ markers at the extremities of cen1. Outside of cen1 markers were inserted at the HpaI (site 1) and XhoI (site 2) sites. At the extremities of the otr, markers were inserted in opposite orientations at the BglII site (sites 3 and 4). (B) epe1Δ causes expansion of centromeric heterochromatin. epe1Δ cells with the ura4⁺ marker inserted at the indicated site were spotted onto the indicated media. (C) H3K9me2 ChIP analysis of wild-type (WT) and epe1Δ strains containing the indicated ura4⁺ insertion compared with the uraDS/E mini-gene.

Figure 2

Epe1 is required for normal centromeric heterochromatin integrity. (A) A ura4⁺ marker was inserted at the NcoI site of the imr and an ade6⁺ marker was inserted in the SphI site in the otr. (B) Loss of Epe1 causes variegation of silencing at the centromere otr. Single colonies of epe1Δ with ade6⁺ at the SphI site in the otr were spotted onto media containing a low level of adenine. (C) Loss of Epe1 causes loss of silencing at the centromere imr. Cells containing ura4⁺ marker inserted in the NcoI site of the imr were preselected on either media lacking uracil or containing FOA. Colonies were then spotted onto the indicated plates. (D) Loss of Epe1 causes chromosome segregation defects. Single colonies of epe1Δ mutants with ade6⁺ at the otr were spotted onto media containing low levels of adenine and media containing 15 μg/ml TBZ. (E) epe1Δ cells exhibit lagging chromosomes (indicated by arrow). The number of anaphase cells with lagging chromosomes was assessed in white and red/pink otr1:ade6⁺ epe1Δ cells as well as wild-type (WT) and swi6Δ.

Figure 3

Loss of Epe1 causes heterochromatin to oscillate. Strains were constructed with an ade6⁺ marker inserted at the SphI site on the right otr of cen1 and a ura4⁺ marker at the XhoI site on the same side of the centromere (A) or on the opposite side of the centromere (B). Single colonies were picked from wild-type (WT) and epe1Δ strains and spotted onto the indicated plates. Single red or white colonies were replated onto the indicated media, 500 of the resulting colonies were classified according to their colour. The numbers indicated are representative of several experiments.

Figure 4

Heterochromatin oscillates independently of boundaries and the RNAi pathway. (A) Loss of Epe1 causes disruption of silencing at the ade6:L5-ura4 ectopic silencer; a fragment of the otr of cen3 (L5) is inserted at the euchromatic ade6⁺ locus adjacent to a ura4⁺ gene. Unselected colonies were plated onto indicated media. (B) Loss of Epe1 allows spreading of heterochromatin in the absence of boundary elements. White colonies containing the ade6:L5-ura4 ectopic silencer were replated and the colour of the resulting colonies was assessed. The numbers indicated are representative of several experiments. (C) Loss of Epe1 alleviates silencing of a ura4⁺ marker genes inserted 150 bp distal to mat3 (mat3-M(EcoRV):ura4⁺). Unselected single colonies were plated onto indicated media.

Figure 5

Loss of Epe1 allows heterochromatin to expand without active RNAi. epe1Δ can partially rescue the phenotypes of rpb2-m203 (A) and dcr1Δ (B) mutants, which have defective RNAi. (C) ChIP analysis of levels of H3K9me2 on the centromeric outer repeats (dg region) was compared to that of the euchromatic fbp1⁺ gene. (D) epe1Δ causes expansion of telomeric heterochromatin. Strains were used which contain a minichromosome that has an ade6⁺ gene next to a telomere. Red wild-type (WT) and epe1Δ colonies were picked and spotted or plated onto media containing limited adenine, the colour of the resulting colonies was assessed. The numbers presented are representative of several experiments.

Figure 6

epe1Δ mutants display low levels of siRNAs derived from centromeric transcripts. (A) Centromeric transcripts levels are reduced in an epe1Δdcr1Δ compared with a dcr1Δ mutant. The transcripts were detected using a centromere-specific probe. As a loading control, levels of larger ribosomal RNAs were visualised by EtBr staining. (B) RT–PCR to analyse levels of centromeric transcript in cells containing a ura4⁺ marker in the centromere (cen1-imr1L(NcoI):ura4⁺) grown in the absence of uracil and in media containing FOA. (C) Levels of siRNAs are reduced in an epe1Δ mutant. siRNAs were detected using a probe specific for the dh repeats. As a loading control, the blot was also hybridised with a probe specific for a snoRNA.

Figure 7

Epe1 requires iron- and 2-OG-binding residues for activity. (A) Multiple alignments of JmjC domain proteins. The Saccharomyces cerevisiae (sc) JHD1, Homo sapiens (hs) JHDM1A and S. pombe (sp) Epe1 proteins are shown. Predicted Fe(II)-binding residues are highlighted in red and the predicted 2-OG-binding residues are highlighted in green. The conserved Fe(II)- and 2-OG-binding residues, H297 and K314, of Epe1 are indicated by ♦. (B) Strains containing the ura4⁺ or ade6⁺ marker inserted at the SphI site of the otr. (C, D) Plasmids containing Epe1 or Epe1 point mutants were overexpressed from an nmt41 promoter in strains with ade6⁺ (C) or ura4⁺ (D) at the SphI site of otr1. Overexpression of Epe1 and epe1H297A and K314A point mutants cause disruption of centromeric silencing. (E) Western blot of extracts from cells overexpressing Epe1 or Epe1 point mutants from an nmt41 promoter in a wild-type background. (F) Genomic epe1H297A and K314A mutants exhibit variegation of centromeric silencing. epe1H297A and K314A point mutants with otr1R(SphI):ade6⁺ were spotted onto media containing low adenine. (G) Genomic epe1H297A and K314A mutants of Epe1 cause spreading of centromeric heterochromatin. epe1H297A and K314A point mutants with ura4⁺ at the XhoI site adjacent to cen1 were spotted onto the indicated media.

Figure 8

Model for the function of Epe1. Epe1 prevents the oscillation of silent chromatin. In dcr1Δ cells, residual pockets of H3K9me2 may act as nucleation sites from which, in the absence of Epe1, heterochromatin can spread.