Document S1. Supplemental Experimental Procedures, Five Figures

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  • Molecular Cell, Volume 39

    Supplemental Information

    The Methyltransferase Activity of Clr4Suv39h Triggers RNAi Independently of Histone H3K9 Methylation

    Erica L. Gerace, Mario Halic, and Danesh Moazed

    Supplemental Experimental Procedures

    Strain and Plasmid Construction

    To generate the Rik1-N strain, the pFA6a-N-kanMX6 (Buhler et al., 2006) served as a

    PCR template for amplification with oligos containing 80 bp of homology to the rik1+

    3UTR to produce a fragment for transformation of the ura4+-5BoxB strain. All

    transformations were performed using the lithium acetate method (Bahler et al., 1998).

    To make the chp1CD strains, the chp1+ ORF was cloned into the Pac1/Asc1 sites of the

    pFA6a-GST-natMX6 vector. The resulting plasmid (pDM1218) served as a template for

    PCR to generate a fragment comprised of 80 bp of homology to the chp1+ promoter and

    start codon, the chp1+ ORF starting with amino acid 70, adh1

    + terminator, and the nat

    R

    cassette and 80 bp of homology to the chp1+

    3UTR. This product was transformed into

    the ura4+-5BoxB, tas3-N or rik1N, chp1::ble

    R strains. To re-integrate the clr4

    + or

    clr4 H410D/C412A into the ura4+-5BoxB, tas3-N or rik1N, clr4::ble

    R, first the entire

    clr4+ locus (including promoter and terminator) was cloned from genomic DNA of a

    strain carrying a Flag-tagged clr4+ allele (SPY 1716) into the Pac1/BglII sites of the

    pFA6a-13XMyc-natMX6 vector. The H410D/C412A point mutations were generated

    using site-directed mutagenesis by overlap PCR extension (Ho et al., 1989) and the

    resulting mutated gene was cloned into the pFA6a-natMX6 vector and candidates were

    verified by sequencing. The wild-type and mutant plasmids (pDM1188 and 1191,

    respectively) were digested with Pac1 and EcoR1, and the entire digestion reaction was

    transformed into clr4 strains. To generate pREP1-3xFlag-clr4 wild-type and mutant

    plasmids, pDM1206 and 1207, clr4+ was amplified from the pDM1188 and 1191

    plasmids and inserted into the Pst1/Xma1 sites of pREP1.

  • Figure S1. CLRC subunits interact with RITS in a Dcr1 activity-dependent

    manner. Related to Figure 1.

    (A) Western blots showing that Myc-Cmc1 co-precipitates with Flag-Ago1. (B) Western

    blots showing that Flag-Clr4 co-precipitates with Myc-Ago1. (C) Western blots showing

    that the enzymatic activity of Dcr1 is required for the interaction of Tas3-TAP with Rik1-

    Myc. The decreased interaction between Tas3-TAP and Rik1-Myc in a dcr1 strain is

    not rescued by expression of Dcr1-D937A, which contains a mutation in the RNAseIII

    catalytic domain (lane 8).

  • Figure S2. Characterization of the Clr4 catalytic SET domain and histone H3K9

    mutants. Related to Figure 3.

    (A) Western blot of whole cell lysates of cells carrying a re-integrated copy of either

    wild-type 3xFlag-clr4+(SPY 1976, lane 2) or 3xFlag-clr4 H410D/C412A (SPY 1977,

    lane 3), which has two point mutations in catalytic residues of the SET domain, show that

  • the wild-type and mutant Clr4 proteins are expressed at similar levels. 3xFlag-clr4+ and

    3xFlag-clr4 H410D/C412A were re-integrated at the endogenous clr4+ locus. The

    membrane stained with PonceauS prior to blotting serves as a loading control. (B) The

    re-integration of wild-type 3xFlag-clr4+ rescues silencing of a clr4 strain. In contrast,

    the integration of the catalytically inactive 3xFlag-clr4 H410D/C412A, does not rescue

    silencing of a clr4 strain. (C) Chromatin immunoprecipitation experiments showing that

    clr4 H410D/C412A does not support H3K9 methylation. Wild-type 3xFlag-clr4+

    or

    3xFlag-clr4 H410D/C412A were introduced into clr4 cells. While the integration of

    clr4+ restores H3K9diMe at the dg repeats of the otr in clr4 cells, the re-integration of

    clr4 H410D/C412A does not result in any detectable H3K9 methylation. (D) Detection

    of Argonaute-associated centromeric small RNAs by splinted ligation in wild-type and

    the indicated mutant cells. Change in copy number of histone H3 has little or no effect

    on siRNA levels. For analysis of histone H3K9 mutations, a strain in which two out of

    the three copies of H3 were deleted was used (Mellone et al., 2003). The single-copy H3

    strain (H3-SC, lane 2, SPY 1575) has similar dg and dh siRNA levels as a wild-type

    strain containing all three copies of H3 (lane 1). The H3K9 mutant background strains,

    H3K9A (SPY 1576), H3K9R (SPY 1577) are isogenic to (SPY 1575). (E) Quantitative

    RT-PCR for centromeric dg transcripts in wild-type, clr4, the single-copy H3 (H3-SC),

    H3K9A, and H3K9R cells. In the H3K9 mutant cells, dg transcripts are derepressed to

    levels that are the same (H3K9R) or below (H3K9A) that observed in clr4 cells. Fold

    increase is calculated relative to the euchromatic tdh1 transcript. The error bars represent

    standard deviations for three independent experiments.

  • Figure S3. Rik1-N is functional and Pol II occupancy at the ura4+-5BoxB locus is

    not affected by Rik1 tethering. Related to Figure 4.

    (A) ChIP experiment showing that H3K9 methylation levels are similar in wild-type

    rik1+ (untagged) and rik1-N cells but are reduced in dcr1 cells. (B) ChIP experiment

    showing that Rik1-N silencing was not accompanied by a change in RNA pol II

    occupancy at the ura4+-5BoxB locus. Tas3-N served as a control and, consistent with

    previous results (Buhler et al., 2006), also showed no change in RNA pol II occupancy.

    PCR amplifications were performed in the presence of dCTP-32

    P with oligos mb

    263/264, mb21/mb134 and mb90/mb91 for cen dh, ura4+ and act1

    +, respectively (Buhler

    et al., 2006). Gels were imaged by phosphor imager screen and quantified using the

    Quantity One software (BioRad). Fold enrichments were calculated by normalizing to

    act1+ and relative to the dcr1 strain (A) and the untagged strain (B), which was given

    the value of 1.0. No ab, no antibody control.

  • Figure S4. Genetic requirements for silencing induced by Rik1- N versus Tas3-

    N. Related to Figure 5.

    (A) Silencing of ura4+-5BoxB induced by Tas3-N requires stc1

    + as shown by lack of

    growth on 5-FOA medium (top panels). In contrast, silencing by Rik1- N can still occur

    in stc1 cells as indicated by growth on 5-FOA medium (bottom panels). Rik1 can

    therefore mediate RNAi-dependent silencing in the absence of stc1+. (B) Western blots

    of co-immunoprecipitation experiments showing that the interaction of Rik1-Myc with

    Tas3-TAP is diminished in dcr1 and clr4 cells, but the interaction is only weakly

    affected by the deletion of stc1+(compare lanes 7 to 9). (C) Silencing of ura4

    +-5BoxB

    induced by Tas3-N requires CLRC subunits, as indicated by loss of growth on 5-FOA

    medium in rik1, cmc1 and cmc2 strains. (D) Western blot showing that Chp1 and

    Chp1CD proteins are expressed at similar levels. Non-specific band labeled with an

    asterisk serves as a loading control. Blot was probed with Chp1 (Abcam, 18191).

  • Figure S5. Rik1C disrupts centromeric silencing. Related to Figure 6.

    (A) Derepression of the ura4+ reporter at the imr locus was observed for the rik1C-TAP

    strain, to the same level observed for rik1 cells, indicated by lack of growth on 5-FOA

    medium.

  • Supplemental Tables

    Table S1. The list of oligonucleotides used in this study.

    Name Sequence

    EG211 5-CGCTTATTTAGAAGTGGCGCGC-3 EG212 5-CGATATCATCATTGTTGGTCGTGGAG-3 EG220 5-GAAGTACCCCATTGAGCACGG-3 EG221 5-CAATTTCACGTTCGGCGGTAG-3 pMO283 5-GTCGAGGATTTCGACCAGGATATG-3 pMO284 5-AGCTCCATAGACTCCACGACCAAC-3 DM554 5-AATGACAATTCCCCACTAGCC-3 DM555 5-ACTTCAGCTAGGATTCACCTGG-3 DM558a 5-GAAAACACATCGTTGTCTTCAGAG-3 DM559a 5-CGTCTTGTAGCTGCATGTGAA-3 matM_F 5-GTCTACTGAACGTACTCCGAGAC-3 matM_R 5-GCTGGTACTTATAACCAGGGTACATT-3 110a.tdh1_F 5-CCAAGCCTACCAACTACGA-3 110a.tdh1_R 5-AGAGACGAGCTTGACGAA-3 110b.dgB_F 5-CGACCACCCTGACTTGTTCT-3 110b.dgB_R 5-GGGTTCCAAGACTCGTCAAA-3 110e.dhE_F 5-GCCCATTCATCAAACGAGTC-3 110e.dhE_R 5-GATTCGGCACCTTTGTCATT-3 mb21 5-TACATAACTATGTCCCCTGGTATCGGC-3 mb90 5-CAACCCTCAGCTTTGGGTCTTG-3 mb91 5-TCCTTTTGCATACGATCGGCAATAC-3 mb134 5-TTAATGCTGAGAAAGTCTTTGCTGATATGC-3 mb263 5-TGAATCGTGTCACTCAACCC-3 mb264 5-CGAAACTTTCAGATCTCGCC-3

  • Table S2. The list of strains used in this study.

    Strain Genotype Source

    SPY 28 h+ leu1-32 ura4-D18 ade6-216M imr1R(Nco1)::ura4+ 7

    SPY 44 h+ leu1-32 ura4-D18 ade6-216M imr1R(Nco1)::ura4+

    rik1::TAP-kanR 3

    SPY 72 h- 8

    SPY 86 h+ leu1-32 ura4-D18 ade6-216M imr1R(Nco1)::ura4+

    dcr1::TAP-kanR 6

    SPY 399 h+ leu1-32 ura4-D18 ade6-216M imr1R(Nco1)::ura4+ clr4

    ::natR 2

    SPY 440 h- ura4+::5BoxB/hphR 2

    SPY 452 h- ura4+::5BoxB-hphR tas3+ ::N-kanR 2

    SPY 797 h+ otr1R(Sph1)::ura4+ ura4DS/E leu1-32 ade6-M210 natR-

    ago1p-3xFlag::ago1+ 5

    SPY 822 h+ leu1-32 ura4-D18 ade6-216M imr1R(Nco1)::ura4+

    rik1::kanR 3

    SPY 825 h+ leu1-32 ura4-D18 ade6-216M imr1R(Nco1)::ura4+ clr4

    ::kanR 3

    SPY 993 h- ura4+::5BoxB-hphR tas3+ ::N-kanR rik1::natR 1

    SPY 995 h- ura4+::5BoxB-hphR tas3+ ::N-kanR cmc1::natR 1

    SPY 996 h- ura4+::5BoxB-hphR tas3+ ::N-kanR cmc2::natR 1

    SPY 1103 h+ leu1-32 ura4-D18 ade6-216M imr1R(Nco1)::ura4+

    rik1706-1040::TAP-hphR 1

    SPY 1124 h- ura4+::5BoxB-hphR rik1+ ::N-kanR 1

    SPY 1218 h- ura4+