The 19S proteasome is directly involved in the regulation of heterochromatin spreading in fission yeast

Abstract

Cumulative evidence suggests that non-proteolytic functions of the proteasome are involved in transcriptional regulation, mRNA export, and ubiquitin-dependent histone modification and thereby modulate the intracellular levels of regulatory proteins implicated in controlling key cellular functions. To date, the non-proteolytic roles of the proteasome have been mainly investigated in euchromatin; their effects on heterochromatin are largely unknown. Here, using fission yeast as a model, we randomly mutagenized the subunits of the 19S proteasome subcomplex and sought to uncover a direct role of the proteasome in heterochromatin regulation. We identified a mutant allele, rpt4-1, that disrupts a non-proteolytic function of the proteasome, also known as a non-proteolytic allele. Experiments performed using rpt4-1 cells revealed that the proteasome is involved in the regulation of heterochromatin spreading to prevent its uncontrolled invasion into neighboring euchromatin regions. Intriguingly, the phenotype of the non-proteolytic rpt4-1 mutant resembled that of epe1Δ cells, which lack the Epe1 protein that counteracts heterochromatin spreading. Both mutants exhibited variegated gene-silencing phenotypes across yeast colonies, spreading of heterochromatin, bypassing of the requirement for RNAi in heterochromatin formation at the outer repeat region (otr), and up-regulation of RNA polymerase II. Further analysis revealed Mst2, another factor that antagonizes heterochromatin spreading, may function redundantly with Rpt4. These observations suggest that the 19S proteasome may be involved in modulating the activities of Epe1 and Mst2. In conclusion, our findings indicate that the proteasome appears to have a heterochromatin-regulating function that is independent of its canonical function in proteolysis.

Keywords: 19S RP; Epe1; RNA interference (RNAi); RNAi; chromatin regulation; epigenetics; heterochromatin; heterochromatin spreading; non-proteolytic function; proteasome.

Figure 1.

Proteasome is involved in the regulation of heterochromatin. A, schematic diagram of the 26S proteasome, 19S lid and base, and the six-ATPase ring. Candidate subunits for random mutagenesis are highlighted in red. B, centromere distribution profiles for the 19S RP and 20S CP of the proteasome. Schematic representations of the three S. pombe centromeres are shown (top), along with H3K9me2 levels at the pericentromeric regions of wild-type and clr4Δ cells (middle) and ChIP-seq analysis of proteasome subcomplexes in Rpn1-FLAG cells (for the 19S RP, red) and Pre1-FLAG cells (for the 20S RP, blue) (bottom). C, 5-fold serial dilutions of a selection of screened proteasome mutants were spotted onto the indicated plates, in the order of increasing level of heterochromatin de-repression.

Figure 2.

Regulation of heterochromatin by proteasome is non-proteolytic. A, poly-ubiquitylated proteins do not accumulate in rpt4-1 cells. Wild-type, rpt4-1, mts2-1, and rpt3-1 cells were collected, and whole-cell extracts were subjected to Western blotting with FK2 antibodies against poly-ubiquitin. Rpt2 was used as a loading control. B, Rum1-FLAG proteins do not accumulate in rpt4-1 cells. Wild-type, rpt4-1, and mts3-1 cells were grown to log phase in YES at 30 °C and then shifted to 37 °C. The cells were collected at the indicated times, and whole-cell extracts were subjected to Western blotting with anti-FLAG antibodies. Rpt2 was used as a loading control. C, Rpn1-TAP-tagged 26S proteasomes were purified and visualized using silver staining. Protein molecular weight standards are indicated. Asterisk denotes the FLAG-tagged Rpn2 subunit in the rpt4-1 proteasome. D, schematic representation of the rpt4-1 mutation (D249V). Domains present in Rpt4p are shown (top) and the partial amino acid sequences of Rpt4 from eight species are aligned. The mutation site is highlighted in pink.

Figure 3.

rpt4-1 cells show variegated silencing at pericentromeric regions. A and B, schematic diagrams of the otr1::ade6+ and otr1::ura4+ reporters (top). rpt4-1 causes variegated silencing at the centromere otr. 5-Fold serial dilutions of single rpt4-1 colonies harboring ade6+ (A) or ura+ (B) at the otr were spotted onto the indicated plates (bottom). C, rpt4-1 epe1Δ cells retain the variegated silencing at the centromere. 5-Fold serial dilutions of single rpt4-1 epe1Δ colonies harboring ade6+ at the otr were spotted onto the indicated plates. D, rpt4-1 causes chromosome segregation defects. 5-Fold serial dilutions of a population (top) or single colonies (bottom) of rpt4-1 cells harboring ade6+ at the otr were spotted onto low-adenine (low ade) medium or medium containing 10 μg/ml TBZ. N/S, non-selective.

Figure 4.

Rpt4 regulates heterochromatin spreading. A, schematic diagram showing the position of the IRC1L::ura4+ insertion at centromere 1. Shading denotes the heterochromatin regions. B, rpt4-1 silences the ura4+ reporter outside the IRC1L. The indicated strains were spotted onto medium supplemented with or without FOA (top), and FOA-resistant colonies were selected and spotted onto medium supplemented with or without FOA (bottom). C and D, 5-fold serial dilutions of the indicated strains were spotted onto medium supplemented with or without FOA.

Figure 5.

rpt4-1 cells form heterochromatin in the absence of active RNAi. A, 5-fold serial dilutions of the indicated strains harboring ade6+ at the otr were spotted onto medium supplemented with or without adenine (Ade). B, 5-fold serial dilutions of the indicated strains were spotted onto YES media. The last column has been enlarged to enable a clear comparison of the cell growth of the indicated strains. C, ChIP-qPCR analysis of H3K9me2 levels at dg relative to act1+. D, ChIP-qPCR analysis of Swi6 levels at dg relative to act1+. Immunoprecipitated DNA was recovered using the Chelex-100 resin (Bio-Rad) and quantified by quantitative PCR using the primers listed in supplemental Table S2.

Figure 6.

rpt4-1 reduces transcription at pericentromeric heterochromatin in the absence of RNAi. A, ChIP-qPCR analysis of pol II levels at dg relative to act1+. B, quantitative RT-PCR analysis of dg transcript levels in the indicated strains, normalized to those of act1+. C, sRNA-seq analysis of the indicated strains across centromere 3.

Figure 7.

Model for the proteolytic and non-proteolytic roles of the proteasome in heterochromatin. In wild-type cells, the proteasome actively degrades the poly-ubiquitylated Epe1 in the repeat regions, while regulating Epe1 and/or Mst2 to facilitate boundary maintenance mechanisms. The effect of boundary maintenance is sufficient to antagonize heterochromatin spreading. In rpt4-1 cells, the mutant proteasome does not affect the degradation of the poly-ubiquitylated Epe1 but exhibits impaired regulation of Epe1 and/or Mst2. The effect of boundary maintenance is not sufficient to counteract heterochromatin spreading, and heterochromatin spreads beyond the natural border.