A Rik1-associated, cullin-dependent E3 ubiquitin ligase is essential for heterochromatin formation

Abstract

Heterochromatin is critical for proper centromere and telomere function, and it plays a key role in the transcriptional silencing of specific genomic loci. In fission yeast, the Rik1 protein functions with the Clr4 histone methyltransferase at an early step in heterochromatin formation. Here, we use mass spectrometry and tandem affinity purification of a Rik1-TAP fusion protein to identify Rik1-associated proteins. These studies identify two novel proteins, Raf1 and Raf2, which we find are required for H3-K9 methylation and for transcriptional silencing within centromeric heterochromatin. We also find that subunits of a cullin-dependent E3 ubiquitin ligase are associated with Rik1 and Clr4, and Rik1-TAP preparations exhibit robust E3 ubiquitin ligase activity. Furthermore, expression of a dominant-negative allele of the Pcu4 cullin subunit disrupts regulation of K4 methylation within heterochromatin. These studies provide evidence for a novel Rik1-associated E3 ubiquitin ligase that is required for heterochromatin formation.

Figure 1.

The Rik1 TAP prep contains a high-molecular-weight E3 ligase activity. (A) SDS PAGE of the Rik1-TAP preparation stained by silver. (B) A sample of the TAP eluate from strain CYP11 (Rik1-TAP) was preincubated on ice with or without human UbcH5b (an E2 enzyme), ATP, radiolabeled ubiquitin, and histone H2B as indicated. Following prebinding, the reactions were initiated with an E1 enzyme, followed by transfer to 30°C. Reaction products were separated by 12% SDS PAGE and conjugates were detected by autoradiography. The CYP11 TAP preparation displayed robust polyubiquitylation activity (as indicated by the ladder of bands ascending from monomeric ubiquitin) that was dependent on the addition of exogenous E1 and E2 enzymes. The reduction of laddering seen in the absence of exogenous H2B suggests that H2B does serve as an in vitro substrate. The components included in each lane are indicated in the panel legend above each lane. (C) A sample of the TAP eluate from strain CYP54 (Raf2-TAP) was assayed for polyubiquitylation activity as described for B. (D) A sample of the TAP eluate from strain CYP52 (Clr4-TAP) was assayed for polyubiquitylation activity as described for B.

Figure 2.

Clr4 is a stable component of the Rik1 E3-containing complex. (A) A schematic of the purification of the material used in B. A Rik1-TAP Clr4–13myc strain (CYP56) was first purified by the TAP strategy and then submitted to additional Superose 6 gel filtration. (B) Concentrated fractions, prepared as in A, were analyzed by Western blot with an anti-9E10 monoclonal antibody (odd fractions only) and by the E3 assay as described above. Both polyubiquitylation activity and Clr4-myc immunoreactivity elute in a high molecular fraction, confirming that the Clr4 protein present in the Rik1-TAP preparation is directly associated with the E3 ligase activity.

Figure 3.

The raf gene deletions exhibit centromere-specific silencing defects. (A) A schematic diagram of the centromeric and pericentromeric regions of S. pombe chromosome 1. The relative positions of the ura4⁺ reporter integrants are shown with their corresponding strain designations. The strongest points of repression in cenI correspond to the otr (CYP27), followed by the imr (CYP26) and central core (CYP25) in hierarchical order. Strain CYP28 contains the ura4⁺ reporter at a euchromatic location. Note that strain CYP27 also contains a swi6 deletion, which partially derepresses this strain and sensitizes this background to the effects of other silencing factor mutations. (B) Overnight cultures of raf1Δ and raf2Δ strains in the indicated reporter backgrounds (illustrated in A) were compared with clr4Δ and rik1Δ control strains. Cells were diluted and spotted onto nonselective supplemented EMMG (N/S sections), EMMG lacking only uracil (–Ura sections), or supplemented EMMG containing 1 g/L 5-fluoroorotic acid (FOA sections). Cells were spotted in serial fourfold dilutions.

Figure 4.

Deletion of the raf genes results in TBZ hypersensitivity. Overnight cultures of raf1Δ and raf2Δ strains in the CYP28 background were compared with clr4Δ, rik1Δ, and wild-type CYP28. Cells were diluted and spotted onto nonselective supplemented EMMG (left panel; “N/S”) or EMMG media containing 10 μg/mL Thiabendazole (right panel; “+TBZ”). Cells were spotted in serial fourfold dilutions.

Figure 5.

The raf1 and raf2 genes contribute to the regulation of histone H3 methylation. (A). Chromatin immunoprecipitation analyses were performed with the indicated strains that each harbor the imrL::ura4⁺ pericentric reporter (CYP26 background). Immunoprecipitations used antisera against histone H3 dimethyl-Lys 4. Primer sets that amplify the imrL::ura4⁺ reporter and a reference set that amplifies the fbp1⁺ euchromatic control locus were multiplexed in PCR reactions. (WCE) Whole-cell extract before immunoprecipitation; (IP) immunoprecipitated DNA. Data were quantified by PhosphorImager and appear below their corresponding lanes expressed as a ratio of ratios using the fbp1⁺ control for normalization. (B) Chromatin immunoprecipitations were performed with antisera against H3 dimethyl-Lys 9. DNA was multiplexed and quantified as described for A.

Figure 6.

A Pcu4 dominant-negative allele impairs heterochromatin formation. (A) Strains CYP26 (containing a ura4⁺ transgene inserted into imrL of cenI) or CYP28 (a euchromatic insertion) were transformed with an episomal copy of either wild-type pcu4⁺ (WT lanes) or the pcu4-K680R dominant-negative allele (D/N lanes) expressed from a low-expression nmt1 promoter. Transformants were first plated onto –Leu –Ura solid media to select for derepression of the ura4⁺ transgene and cultured overnight in –Leu –Ura liquid media, prior to dilution into EMM –Leu. Cells were grown 10 generations in –Leu media to allow for heterochromatin reformation and silencing of the ura4⁺ cassette and plated in fourfold dilutions onto EMMG –Leu (“nonselective”; N/S section) or EMMG –Leu –Ura (–Ura section) to assess silencing of the transgene. (B) Transformants of CYP26 and CYP28, grown as described in A, were subjected to chromatin immunoprecipitation as described in Materials and Methods. Primer sets that amplify the imrL::ura4⁺ reporter and a reference set that amplifies the fbp1⁺ euchromatic control locus were multiplexed in PCR reactions. (WCE) Whole-cell extract; (K4Me) immunoprecipitated with antisera against histone H3 dimethyl-K4; (K9Me) immunoprecipitated with antisera against histone H3 dimethyl-K9.