Sir2 is required for Clr4 to initiate centromeric heterochromatin assembly in fission yeast

Heterochromatin assembly in fission yeast depends on the Clr4 histone methyltransferase, which targets H3K9. We show that the histone deacetylase Sir2 is required for Clr4 activity at telomeres, but acts redundantly with Clr3 histone deacetylase to maintain centromeric heterochromatin. However, Sir2 is critical for Clr4 function during de novo centromeric heterochromatin assembly. We identified new targets of Sir2 and tested if their deacetylation is necessary for Clr4-mediated heterochromatin establishment. Sir2 preferentially deacetylates H4K16Ac and H3K4Ac, but mutation of these residues to mimic acetylation did not prevent Clr4-mediated heterochromatin establishment. Sir2 also deacetylates H3K9Ac and H3K14Ac. Strains bearing H3K9 or H3K14 mutations exhibit heterochromatin defects. H3K9 mutation blocks Clr4 function, but why H3K14 mutation impacts heterochromatin was not known. Here, we demonstrate that recruitment of Clr4 to centromeres is blocked by mutation of H3K14. We suggest that Sir2 deacetylates H3K14 to target Clr4 to centromeres. Further, we demonstrate that Sir2 is critical for de novo accumulation of H3K9me2 in RNAi-deficient cells. These analyses place Sir2 and H3K14 deacetylation upstream of Clr4 recruitment during heterochromatin assembly.

TAP tagging of Sir2 and sir2N247A was performed by homologous recombination across C terminal sequences of sir2 + with PCR product generated from a pFA6aTAP-NatR vector (JP1290) with homology to the sir2 ORF and 3' UTR sequences. The tagged sir2 locus was checked by sequencing, and strains were outcrossed twice prior to analysis. clr4 + reintroduction into the genome was performed using JP1326 which was digested with HpaI. Integrants were selected for growth on media lacking adenine, and single copy integration was confirmed by southern analysis.
The 3xFlag epitope was introduced between the ATG of clr4 + and the 2 nd codon by homologous recombination, in a strain where ura4 + was inserted at that site (Py1249) to generate Py1664 following selection on FOA, sequencing of the locus, and outcrossing.

Plasmid construction
All PCR-generated cloned fragments were confirmed by sequence analysis. Constructs for expression of GST fusion proteins were generated by amplification of sir2 cDNA sequences with primers bearing EcoR1 restriction sites, and cloned into EcoR1 digested pGEX-GK (Guan and Dixon, 1991).
Plasmids for integration of wild type or N247A mutant Sir2 linked to his3 + (JP1267 and 1312) were generated by PCR amplification of genomic sir2 + with primers containing Not1 and Pst1 restriction sites, and cloned into Not1/ Pst1 digested JP1142, which is pRO319 (Adams et al., 2005) that has been converted to an integration vector by AatII digestion to remove ARS sequences. Mutagenesis of sir2N247A was performed by PCR with a mutagenic primer incorporating a 5' Sph1 site, and with the 3' Pst1 primer. The N247A mutation was incorporated into JP1312 by digestion of JP1267 with Pst1 and Sph1 and replacement with the mutant PCR product.
The sir2 expression construct JP1613 was generated by PCR of Sir2 cDNA with primers bearing SalI and BamHI sites, which did not include the stop codon. The PCR fragment was cloned into XhoI and BglII sites of JP1611, which is a C terminal 3xV5 tagging vector in a pREP81 backbone. JP1611 was generated by release of the 3xV5 tag from pSLF972 (kind gift from Susan Forsburg) through XhoI, SacI digestion and recloning of the 3xV5 tag into XhoI, SacI digested JP802 which is a derivative of pREP81.
The plasmid used for reintegration of clr4 + linked to ade6 + at single copy into the genomic locus following linearization with HpaI (JP1326) was derived from JP1084 (Partridge et al., 2007). The his3 + marker was released by FseI digestion and replaced with ade6 + from pRO317 (Adams et al., 2005).
Plasmids for episomal expression of genomic 3xFlag-clr4 + linked to his3 + (JP1636) and leu1 + (JP2100) were generated by PCR amplification of the 3xFlag-clr4 + locus from Py1664 with primers incorporating SacII and SalI restriction sites (used previously to amplify clr4 + , Partridge et al 2007), and were cloned into SacII/ SalI sites of JP1049 (pR0319-his3 + (Adams et al., 2005) or JP1050 (pRO320-leu1 + (Adams et al., 2005). Mutagenesis of JP1636 was performed by in phusion PCR to generate H410K mutant of genomic 3xFlag-clr4 + (JP2111).Plasmids for episomal expression of genomic clr4 + (JP1078 and empty vector JP1045) have been described previously (Partridge et al., 2007) Fluorogenic peptide deacetylation assay A 25 L deacetylase reaction was assembled comprising 0.8 M GST, GST-Sir2, or GST-Sir2N247A, 5mM Fluor-de-lys green substrate, 8mM NAD + and 1mM histone peptide where applicable in a reaction buffer of 50 mM Tris pH 8.0, 100 mM NaCl, and 1 mM DTT. Enzymatic reactions were performed for 3 h while dark at ambient temperature (22º C). Termination of the enzymatic reaction was achieved by addition of 25 l quenching and developing mixture comprising 0.25 L 2 mM trichostatin A (10 M final in quenched reaction mixture), 0.5 L 50 mM Nicotinamide (0.5 mM), and 1.25 L trypsin-based developer concentrate to the 25 L enzymatic reaction mixture. Endpoint fluorescence was measured 15 min after addition of developer using a Synergy HT BioTek Scanner (485 nm excitation/520 nm emission). A standard curve was subsequently constructed by serial dilution of the deacetylated fluorogenic substrate, and was used to equate experimentally observed fluorescence units with molar quantities of substrate deacetylated. 32 P NAD + hydrolysis assay for Sir2 deacetylase activity 0.2 µM affinity-purified GST, GST-Sir2, and GST-Sir2N247A were coincubated overnight at ambient temperature (22ºC) with 2.5 µCi 32 P NAD + (ARC 0141, American Radiolabeled Chemicals Inc.; specific activity 800 Ci/mmol) in the presence of 0.1 µg/uL calf thymus histones (Sigma Life Sciences) or 0.5 mM acetylated or unacetylated histone H3 1-19 or H4 1-19 N-terminal peptides (Hartwell Center, St Jude Children's Research Hospital), in a 10 µL reaction mixture buffered with 50 mM Tris pH 8.0, 100 mM NaCl, 1 mM DTT. Following the coupled 32 P NAD + hydrolysis/substrate deacetylation reaction, products were diluted 50-fold, and 3 µL of this mixture was resolved by reverse-phase TLC (LKSD silica gel 60 A, Whatman Inc.; mobile phase of 80% EtOH, 20% 2.5 M ammonium acetate). The relative mobility of hydrolyzed radioligands was assessed by autoradiography, and the extent of 32 P NAD + hydrolysis product evolution was determined by quantitative densitometry (UN-SCAN-IT gel™ Version 6.1 gel analysis and graph digitizing software, Silk Scientific Inc.). Figure S1. De-novo silencing defects in sir2 deficient cells correspond with loss of centromere function. A. Increased incidence of lagging chromosomes in late anaphase upon clr4 + reintroduction in sir2 clr4 cells. Yeast strains were fixed, stained with anti-tubulin antibodies, and DAPI. The percentage of cells with visibly lagging chromosomes was determined among late anaphase cells. Double-blind experiments were conducted in duplicate, with N≥200 for each strain background indicated. Figure S2. De-novo silencing defects in sir2 clr4 to clr4 + cells are not due to defective clr4 transcription, and can be complemented by re-expression of sir2 + in clr4 + reintegrant backgrounds. A. clr4 + transcript levels are not decreased following reintegration of clr4 + into sir2clr4backgrounds. Quantitative real time PCR of clr4 + transcripts relative to the euchromatic control, adh1 + , to monitor effects of reintegration of clr4 + into various mutant backgrounds. Data represent SEM of 2 biological replicate experiments.B. Re-expression of sir2 + in clr4 + reintegrant strains complements the heterochromatic silencing defect. Yeast strains bearing the centromeric ura4 + reporter were transformed with vectors expressing the V5 tag alone, or Sir2-V5. Serial dilution spotting assays were performed on media lacking leucine (to maintain selection for plasmids), and selective for growth of ura4 expressing (-ura) or ura4 silenced cells (+FOA). C. Expression of Sir2-V5 was confirmed by western analysis. Western analysis of extracts prepared from cells described in (A), using antibodies that recognize the V5 tag, or tubulin as a loading control. Figure S3. De-novo silencing defects in sir2 clr4 to clr4 + cells are epigenetically heritable. Where indicated, yeast strains bearing the ura4 + centromeric transgene were restruck on nonselective medium, cultured for a time period corresponding to approximately 100 cell doublings, and assayed for growth by serial dilution spotting assays on nonselective (complete) medium and medium selective against uracil auxotrophy (+FOA). Figure S4. GST-Sir2, but not GST-Sir2N247A or GST alone, promotes deacetylation of acetylated peptide substrates in vitro, and H4K16Ac is slightly increased in vivo in sir2D cells. GST-Sir2 promotes the evolution of higher mobility 32 P NAD + hydrolysis products upon coincubation with radiolabeled cofactor, exclusively in the presence of acetylated peptide substrates. Representative autoradiographs following TLC separation of reaction products after coincubating A. GST alone, B. GST-Sir2, or C. GST-Sir2N247A with the substrates indicated in the presence of 32 P NAD + , hydrolysis of which is required for coupled substrate deacetylation by Sir2 and its homologs. Relative evolution of higher mobility 32 P NAD + hydrolysis products was determined by quantitative densitometry, as presented in Figure 5E. D. Western analysis of H4K16Ac in WT and sir2 cells, using a H4K16G mutant strain as control for antibody specificity. Relative levels of the immunoreactive species indicated were quantified by densitometry. Experimental data presents the average of 2 experimental replicates from each of 2 distinct biological samples. Figure S5. Centromeric transcript accumulation in H3K4A and H3K4Q mutants; H3K4Q mutation does not abolish de-novo centromeric silencing. H3K4Q mutation produces significantly elevated centromeric dh transcript accumulation (A.), while accumulation of centromeric dg transcripts is comparatively attenuated (B.). mRNA transcripts were evaluated by qRT-PCR amplification of cDNA and normalized to transcript levels of the act1 + euchromatic control. Data presents the average of 2 experimental replicates. C. H3K4Q mutation does not abolish de-novo centromeric silencing. mRNA transcripts were evaluated by qRT-PCR as in panels A and B, but with 3 distinct biological replicates for the K4Q clr4 to clr4 + reintroduction strain only. Figure S6. Heterochromatin establishment at centromeres appears defective in H3K14A mutant cells. Quantitative PCR assessment of dh transcripts relative to adh1 + following reintegration of clr4 + into the clr4genomic locus of H3K14A mutant cells. clr4 + reintegration into single copy H3/H4 clr4 cells resulted in suppression of centromeric transcription, whereas transcripts remained high following reintegration of clr4 + into clr4H3K14A mutant cells. Note that transcript levels are also strongly elevated in H3K14A mutant cells under maintenance conditions. Figure S7. Episomal genomic Flag-clr4 + expression compensates for clr4 function at centromeres and centromeric heterochromatin assembly is defective in histone H3 mutant backgrounds. A. Plasmid based Flag-clr4 + overexpression overcomes centromeric silencing defects in clr4 mutants. Serial dilutions of the indicated strains each bearing the centromeric ura4 + transgene were assayed for growth on medium lacking histidine (-His), as well as medium lacking histidine and uracil (-His -Ura), and medium lacking histidine and containing 5-fluoro-orotic acid (-His +FOA). B. Swi6 does not associate with centromeres in H3K14A mutant background. ChIP for Swi6 reveals a similar loss of recruitment of Swi6 to centromeric repeats (dh) in H3K14A and clr4 backgrounds. C. Expression of Flag-Clr4 is not reduced by H3K14R mutation. Protein extracts were made from single copy H3/H4 strains expressing WT or H3K14R mutant that were transformed with episomal Flag-Clr4. Western analysis revealed that levels of Flag-Clr4 were slightly upregulated in H3K14R cells compared with wild type, when normalized to the tubulin loading control. D. Anti-Flag ChIP reveals a defect in Flag-Clr4 recruitment to centromeres in H3K14R mutant strains. Anti-Flag ChIP was performed on WT or H3K14R histone mutant strains transformed with Flag or Flag-Clr4 expression vectors, and analyzed by quantitative real time for enrichment of centromeric dh and euchromatic adh1 + sequences. Figure S8. Catalytic mutant clr4H410K is stably expressed but is not enriched at centromeres. A. Flag-clr4H410K is expressed as a stable protein. Western blot against-epitope tagged Flag-Clr4 and Flag-clr4H410K provides evidence for specific detection of an immunoreactive species of the expected molecular mass. Denaturing protein extracts were prepared from equivalent cell inputs, resolved by SDS-PAGE and probed using antibodies against the Flag epitope. This blot was stripped and reprobed with a tubulin specific antibody, providing a loading control. Plasmid based overexpression of Flag-Clr4 and Flag-clr4H410K were driven by the genomic clr4 promoter. B. Flag-clr4H410K is not enriched at centromeres. Q-PCR analysis of ChIP for Flag-Clr4 or Flag-clr4H410 enrichment (in otherwise clr4 cells) at centromeric dh sequences compared with adh1 + euchromatic control. Data averaged from 3 experimental replicates, with SEM shown.

Figure S9. Sir2 deletion does not reduce Clr4 levels or expression from the endogenous clr4 + locus. A. Sir2 deletion does not reduce detectable levels of Flag-Clr4 when expression is
driven from the endogenous clr4 + locus. Anti-Flag immunoprecipitated samples were prepared from cell lysates normalized to contain equal amounts of total protein, resolved by SDS-PAGE, and probed by western blot against the Flag epitope. Crude lysates were also resolved by SDS-PAGE and probed by western blot against tubulin, to provide a loading control for sample input to the immunoprecipitation experiment. Relative levels of the immunoreactive species indicated were subsequently quantified by densitometry. Experimental data presents the average of 3 experimental replicates from each of 2 distinct biological samples. B. Sir2 deletion does not reduce steady state levels of Flag-clr4 + mRNA transcripts when expression is driven from the endogenous clr4 + locus. qRT-PCR amplification of random primed cDNA from yeast strains of the genotype indicated was performed, and clr4 + transcript levels were normalized to transcript levels of the adh1 + control. Experimental data presents the average of 2 experimental replicates. C. Sir2 deletion does not impact clr4 + transcript levels when it is expressed from genomic plasmid. qRT-PCR analysis of random primed cDNA from indicated yeast strains. clr4 + transcript levels were normalized to adh1 + , and expression of clr4 + from genomic clr4 + vector in clr4 cells was set to 100. Data represents average and SEM for 2 experimental replicates, with replicate experiments performed on 2 independent transformants for the triple deletion strain (n=4).

Table S1
Strain Genotype Figure   Py