Volume 19, Issue 6, Pages 817-828 (September 2005) SUMO Modification Is Involved in the Maintenance of Heterochromatin Stability in Fission Yeast  Jin A. Shin, Eun Shik Choi, Hyun Soo Kim, Jenny C.Y. Ho, Felicity Z. Watts, Sang Dai Park, Yeun Kyu Jang  Molecular Cell  Volume 19, Issue 6, Pages 817-828 (September 2005) DOI: 10.1016/j.molcel.2005.08.021 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 SUMO Is Required for Heterochromatic Silencing at the mat3 Locus and the Centromere (A) Deletion of pmt3+ slightly reduces silencing of the ura4+ gene inserted at the mat3 locus. Wild-type (wt) or Δpmt3 cells were spotted onto selective uracil-free (−Ura), counter-selective 5′-fluoroorotic acid (FOA), and nonselective (N/S) plates (top). The data show that the effect of Δpmt3 on silencing is not gene or locus specific. Silencing of ade6+ inserted at the mat3 locus or outer repeat of centromere 1 (otr1) is also reduced by Δpmt3 deletion (bottom). Strains used are: wt (PG9), Δpmt3 (SPS207) in mat3::ura4+; wt (Hu514), Δpmt3 (SPS203) in mat3::ade6+; and wt (Hu50), Δpmt3 (SPS204) in otr1R::ade6+. Δpmt3 cells have lower viability than wt by about 10-fold. Thus, to adjust the number of Δpmt3 cells to that of wt, Δpmt3 cells were spotted ten times more than wt and then were incubated for 2 days longer at 30°C. (B) Colony color assay confirming silencing defects in Δpmt3. The wt and Δpmt3 cells were plated onto low-adenine medium. After a 5 day incubation, the colony color was compared. (C) RT-PCR analysis of ade6+ from the same strains as (A) and (B). The relative fold ratio shown beneath each lane was calculated by dividing the level of heterochromatic ade6+ with that of euchromatic lys1+. (D) Δpmt3 does not alter H3 K9 methylation but significantly increases H3 K4 methylation of the ade6+ reporter gene inserted at the silent mat3. H3 K9 and K14 acetylations are also increased, although the effects are milder. The levels of H3 K9 and K4 methylation and H3 K9 and K14 acetylation in wt and Δpmt3 deletion derivative were determined by ChIP analysis. Relative fold enrichment shown beneath each lane was calculated by dividing the ratio of the ade6+ loci/leu1+ PCR products in the ChIP sample with that in the whole-cell extract (WCE) sample. (E) Deletion of pmt3+ causes significant increase in H3 Lys4 methylation at nontranscribable loci within the K region. ChIP analysis was performed by using the same sample as (D) for four different loci (K1–K4) within the silent K region between mat2 and mat3. Molecular Cell 2005 19, 817-828DOI: (10.1016/j.molcel.2005.08.021) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Hus5 Interacts with Heterochromatin (A) Tagging and expression of Hus5 in wt or Δclr4 cells. Anti-HA immunoprecipitates of WCEs (2 mg) prepared from the indicated strains were subject to Western blot analysis with anti-HA antibody. (B) Hus5 localizes to the mat3 locus in a heterochromatin-dependent manner. The levels of Hus5-HA at mat3::ade6+ locus were determined by ChIP analysis with an anti-HA antibody. Relative fold enrichment shown beneath each lane was calculated by dividing the ratio of the ade6+/leu1+ PCR products in the ChIP sample with that in the WCE sample. Fold enrichment with negative control (wt cells with no tag) was not determined (ND). (C) Yeast two-hybrid analysis showing physical interactions between Hus5 and Swi6 (top) or Clr4 (bottom). Yeast PJ694A cells (see Experimental Procedures) were transformed with a vector expressing either the Gal4-activation domain (AD) or the Gal4-AD fused to Hus5 and a vector expressing either the Gal4-DNA binding domain (BD) or the Gal4-BD fused to Swi6 (top) or Clr4 (bottom). Transformants were grown on nonselective (control), adenine-free (−Ade), or histidine-free (−His) medium containing 5 mM 3-aminiotriazole (3-AT) at 30°C for 3–5 days before being photographed. (D) Hus5 interacts with Swi6 and Clr4 in vivo. Extracts prepared from cells either overproducing HA-Swi6, Myc-Hus5, or both were incubated with anti-HA antibody. The immunoprecipitated fractions were analyzed by Western blotting with the anti-Myc antibody. The expression of HA-Swi6 or Myc-Hus5 in each sample was confirmed by Western blot analysis with anti-HA or an anti-Myc antibodies (top). In addition, extracts prepared from cells either overproducing HA-Clr4, Myc-Hus5, or both were incubated with an anti-Myc antibody. The immunoprecipitated fractions were analyzed by Western blotting with the anti-HA antibody. The expression of HA-Clr4 or Myc-Hus5 in each sample was confirmed by Western blot analysis with anti-HA or anti-Myc (bottom). Molecular Cell 2005 19, 817-828DOI: (10.1016/j.molcel.2005.08.021) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Hus5 Binds to Heterochromatin in a Clr4 and Swi6-Dependent Manner (A) A diagram of the mat locus and the locations of primers used (1–33) are shown at the top. IR-L and IR-R represent the left and right boundary of heterochromatin, respectively. ChIP analysis with an anti-HA antibody was performed with wt or Δclr4 cells expressing Hus5-HA to measure the Hus5-HA levels throughout the mat locus. Relative fold enrichment of Hus5-HA at each mat locus was calculated by dividing the ratio of each locus/leu1+ PCR product in the ChIP sample with that in the WCE. The quantified ratio is plotted in alignment with the map of the mat locus. (B) A diagram of centromere 1 and locations of primers used are shown on top. Primer pairs designated as cnt1 and imr-1 are located within the central core region, which is known to be nonheterochromatic. Primer pairs designated imr-2, otr-1, and otr-2 are located within centromeric heterochromatin. The primer pair designated as cen1-R is located at a euchromatic region outside of the centromere. ChIP analysis with the anti-HA antibody was performed with wt or Δclr4 cells to measure Hus5-HA levels throughout centromeric regions. Molecular Cell 2005 19, 817-828DOI: (10.1016/j.molcel.2005.08.021) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Swi6 and Clr4 Can Be Sumoylated In Vivo (A) Modification of Swi6 in vivo. Left, a slow-migrating band of Swi6 was only detected in Δulp1 cells expressing the mature form of Pmt3 (Pmt3-GG, lane 3), but not in wt (Hu514, lane 1) or Δulp1 cells containing empty vector (lane 2). Right, immunoprecipitation (IP) and Western blot (WB) analyses identify the slow-migrating bands as SUMO conjugated forms of Swi6. Sumoylated Swi6 was identified by IP using the anti-HA antibody (which recognizes HA-tagged Swi6) followed by Western blot using the anti-Myc antibody. IP using an anti-GST antibody was performed as a negative control. (B) SUMO modification of Clr4 in vivo. Sumoylation of Clr4 was shown by Western blot and IP as described in (A). (C) Diagram showing the SUMO consensus motif (ΨKxE) in Swi6 (top). Other domains of Swi6 are also shown (CD, chromodomain; IVR, intervening region; and CSD, chromoshadow domain). K103R or E105R indicates the mutated form of potential SUMO conjugation residue Lys103 to arginine or consensus residue E105 to arginine via site-directed mutagenesis (bottom). The sumoylation of each form was analyzed by Western blotting with the anti-HA antibody. (D) SUMO consensus motifs (ΨKxE) in Clr4 (top). Other domains are also shown (CD, chromodomain; SET, set domain; hatched box, pre-SET domain; and gray box, post-SET domain). K109R or K160R indicates the mutated form of potential SUMO conjugation residue Lys109 or Lys160 to arginine, respectively. DM represents mutation at both residues (K109R and K160R) (lower). Sumoylated Clr4 was determined by IP using the HA antibody, followed by Western blot using anti-HA or anti-Myc. Molecular Cell 2005 19, 817-828DOI: (10.1016/j.molcel.2005.08.021) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Defective Sumoylation of Swi6 or Chp2 Impairs Heterochromatic Silencing (A) Sequence alignment of Swi6 and Chp2 SUMO consensus motifs with several chromodomain proteins. Conserved amino acids are marked by black or gray boxes. An asterisk indicates the Lys103 residue, and a circle indicates the Glu105 residue. (B) Mutation of Lys103 to Arg (K103R) or Glu105 to Arg (E105R) impairs silencing at mat3::ade6+. Wt, Δswi6, swi6-K103R, swi6-E105R, Δchp2, chp2-K198R, chp2-E200R, or swi6-K103R chp2-K198R (DM) cells were spotted onto selective adenine-free (−Ade) and nonselective (N/S) plates. (C) Colony color assays confirmed silencing defects in the above mutant strains. Wt or mutant cells indicated were plated onto low-adenine medium, and colony colors were assessed after a 5 day incubation at 30°C. (D) Effect of swi6-K103R, swi6-E105R, chp2-K198R, chp2-E200R, or swi6-K103R chp2-K198R (DM) on H3 K9 methylation and H3 K4 methylation of mat3::ade6+. The levels of H3 K9 and K4 methylation were determined by ChIP analysis, as described in Figure 1D. (E) A graph showing the relative level of H3 K4 methylation in wt or mutant cells, as indicated. Three independent experiments were performed, and the data are presented as the mean of triplicates ± SD. (F) Colony color assay showing silencing defects in Δpmt3, swi6-K103R, or Δpmt3 swi6-K103R. Cells were plated onto low-adenine medium, and the colony colors were compared after a 5 day incubation. Molecular Cell 2005 19, 817-828DOI: (10.1016/j.molcel.2005.08.021) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Defective Sumoylation of Swi6 Impairs Its Binding to Heterochromatic Regions (A) Western blot analysis of GST-Swi6 and GST-Swi6-K103R to confirm equal expression. Extracts prepared from the indicated strains were subject to Western blot analysis with an anti-GST antibody (top). Total proteins were shown by staining with Ponceau red to confirm equal loading (bottom). (B) Mutation of Lys103 of Swi6 to Arg (K103R) compromised its binding to the ade6+ reporter inserted near the silent mat3 locus. ChIP analysis with an anti-GST antibody was performed with wt or swi6-K103R cells in which GST-Swi6 (wt) or GST-Swi6-K103R (swi6-K103R) is expressed under the nmt41 promoter to measure GST-Swi6 or GST-Swi6-K103R levels at ade6+ or leu1+. Wt cells bearing swi6+ not tagged with GST (no tag) were used as a negative control. Relative levels of GST-Swi6 or GST-Swi6-K103R binding to ade6+ are shown beneath each lane as the mean of triplicates ± SD. (C) Mutation of Lys103 of Swi6 to Arg (K103R) compromised its binding to the silent mat locus. Chromatin immunoprecipitated DNAs from (B) were amplified by primer sets located throughout mat locus (1–33). Relative fold enrichment was calculated as described in Figure 3A. Molecular Cell 2005 19, 817-828DOI: (10.1016/j.molcel.2005.08.021) Copyright © 2005 Elsevier Inc. Terms and Conditions