Document S1. Supplemental Experimental Procedures, Five Figures ...

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-λN strain, the pFA6a-λN-kanMX6 (Buhler et al., 2006) served as a

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

3’UTR 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 chp1ΔCD 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+

3’UTR. This product was transformed into

the ura4+-5BoxB, tas3-λN or rik1λN, chp1Δ::ble

R strains. To re-integrate the clr4

+ or

clr4 H410D/C412A into the ura4+-5BoxB, tas3-λN or rik1λN, clr4Δ::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 clr4Δ 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 dcr1Δ 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 clr4Δ strain. In contrast,

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

silencing of a clr4Δ 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 clr4Δ cells. While the integration of

clr4+ restores H3K9diMe at the dg repeats of the otr in clr4Δ 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, clr4Δ, 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 clr4Δ cells. Fold

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

standard deviations for three independent experiments.

Figure S3. Rik1-λN 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-λN cells but are reduced in dcr1∆ cells. (B) ChIP experiment

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

occupancy at the ura4+-5BoxB locus. Tas3-λN 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-α32

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 dcr1Δ 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- λN versus Tas3-

λN. Related to Figure 5.

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

+ as shown by lack of

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

in stc1Δ 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 dcr1Δ and clr4Δ 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-λN requires CLRC subunits, as indicated by loss of growth on 5-FOA

medium in rik1Δ, cmc1Δ and cmc2Δ strains. (D) Western blot showing that Chp1 and

Chp1ΔCD proteins are expressed at similar levels. Non-specific band labeled with an

asterisk serves as a loading control. Blot was probed with αChp1 (Abcam, 18191).

Figure S5. Rik1ΔC disrupts centromeric silencing. Related to Figure 6.

(A) Derepression of the ura4+ reporter at the imr locus was observed for the rik1ΔC-TAP

strain, to the same level observed for rik1Δ 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+ dcr1Δ::TAP-kanR

6

SPY 399 h+ leu1-32 ura4-D18 ade6-216M imr1R(Nco1)::ura4+ clr4 Δ::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+ rik1Δ::kanR

3

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

3


tas3+ ::N-kanR rik1::natR 1


tas3+ ::N-kanR cmc1::natR 1


tas3+ ::N-kanR cmc2::natR 1

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

rik1706-1040::TAP-hphR 1


rik1+ ::N-kanR 1


rik1706-1040+ ::N-kanR 1


rik1+ ::N-kanR dcr1::natR 1


rik1+ ::N-kanR cmc2::natR 1


rik1+ ::N-kanR cmc1::natR 1


rik1+ ::N-kanR ago1::natR 1


rik1+ ::N-kanR hrr1::natR 1


rik1+ ::N-kanR clr4::natR 1


rik1+ ::N-kanR swi6::natR 1


rik1+ ::N-kanR sir2::natR 1


rik1+ ::N-kanR tas3::natR 1


rik1+ ::N-kanR chp1::natR 1


rik1+ ::N-kanR cid12::natR 1


rik1+ ::N-kanR rdp1::natR 1


rik1+ ::N-kanR chp1::bleR 1


rik1+ ::N-kanR chp12-69::natR 1

SPY 1575 ura4-D18 ade6-210 otr1R(Sph1)::ade6+ h3.1/h4.1Δ::his3 h3.3/h4.3Δ::arg3

4

SPY 1576 ura4-D18 ade6-210 otr1R(Sph1)::ade6+ h3.1/h4.1Δ::his3 4

h3.3/h4.3Δ::arg3 h3.2-K9A/h4.2 SPY 1577 ura4-D18 ade6-210 otr1R(Sph1)::ade6+ h3.1/h4.1Δ::his3

h3.3/h4.3Δ::arg3 h3.2-K9R/h4.2 4


rik1+ ::N-kanR dcr1::natR leu1ΔbleR 1


rik1+ ::N-kanR ago1::natR leu1ΔbleR 1


tas3+ ::N-kanR chp1::bleR 1

SPY 1716 h+ leu1-32 ura4-D18 ade6-210 otr1R(Sph1)::ade6+ 3xFlag-clr4

1

SPY 1780 h+ leu1-32 ura4-D18 ade6-216M natR::ago1p-3xFlag-ago1 3xMyc-cmc1

1

SPY 1830 h+ leu1-32 ura4-D18 ade6-216M imr1R(Nco1)::ura4+ rik1+::13xMyc-hphR

1

SPY 1833 h+ leu1-32 ura4-D18 ade6-216M imr1R(Nco1)::ura4+ rik1+-13xMyc-hphR tas3+::TAP-kanR

1

SPY 1834 h+ leu1-32 ura4-D18 ade6-216M imr1R(Nco1)::ura4+ rik1+::13xMyc-hphR tas3+-TAP::kanR clr4Δ::natR

1


rik1+ ::N-kanR leu1ΔbleR 1

SPY 1896 h+ leu1-32 ura4-D18 ade6-216M imr1R(Nco1)::ura4+ rik1+::13xMyc-hphR tas3+::TAP-kanR dcr1Δ::natR

1

SPY 1901 h+ otr1R(Sph1)::ura4+ ura4DS/E leu1-32 ade6-M210 natR-ago1p-3xFlag::ago1+ rik1+::TAP-kanR

1


tas3+ ::N-kanR clr4::bleR 1

SPY 1910 h+ leu1-32 ura4-D18 ade6-216M imr1R(Nco1)::ura4+ rik1+::13xMyc-hphR rdp1+::TAP-kanR

1


rik1+ ::N-kanR 3xFlag-clr4-

clr4ter::natR 1


tas3+ ::N-kanR 3xFlag-clr4

H410D/C412A-clr4ter::natR 1


tas3+ ::N-kanR chp12-69::natR 1


tas3+ ::N-kanR 3xFlag-clr4-

clr4ter::natR 1


tas3+ ::N-kanR 3xFlag-clr4

H410D/C412A-clr4ter::natR 1

SPY 1947 natR::ago1p-3xmyc-ago1 3xFlag-clr4 1

SPY 1976 h+ leu1-32 ura4-D18 ade6-216M imr1R(Nco1)::ura4+ 3xFlag-clr4-clr4ter::natR

1

SPY 1977 h+ leu1-32 ura4-D18 ade6-216M imr1R(Nco1)::ura4+ 3xFlag-clr4 H410D/C412A-clr4ter::natR

1

SPY 2005 h+ otr1R(Sph1)::ura4+ ura4DS/E leu1-32 ade6-M210 natR-ago1p-3xFlag::ago1+ rik1+::TAP-kanR dcr1Δ::hphR

1


tas3+ ::N-kanR stc1Δ::natR 1


rik1+ ::N-kanR stc1Δ::natR 1

SPY 2031 h+ leu1-32 ura4-D18 ade6-216M imr1R(Nco1)::ura4+ rik1+-13xMyc-hphR tas3+::TAP-kanR stc1Δ::natR

1

1= this study, 2= (Buhler et al., 2006), 3= (Hong et al., 2005), 4= (Mellone et al., 2003)

5=(Buker et al., 2007), 6=(Motamedi et al., 2004), 7= Dr. Shiv Grewal, 8= Dr. Charles

Hoffman

Table S3. The list of plasmids used in this study.

Name Construct Source

pDM 817 pREP-3xFlag-ago1 2 pDM 829 pREP-1 4 pDM 831 pREP-3xFlag-ago1 D580A 2 pDM 914 pREPNFlag-dcr1 3 pDM 915 pREPNFlag-dcr1 D937A 3 pDM 1188 pFA6a-clr4p-3xFlag-clr4-clr4ter-natMX6 1 pDM 1191 clr4p-3xFlag-clr4 H410D/C412A-clr4ter-natMX6 1 pDM 1206 pREP-clr4p-3xFlag-clr4-clr4ter 1 pDM 1207 pREP-clr4p-3xFlag-clr4 H410D/C412A-clr4ter 1 pDM 1218 pFA6a-chp1-natMX6 1

1= this study, 2=(Buker et al., 2007), 3=(Colmenares et al., 2007), 4= Dr. Kathy Gould

Supplemental References

Bahler, J., Wu, J.Q., Longtine, M.S., Shah, N.G., McKenzie, A., 3rd, Steever, A.B.,

Wach, A., Philippsen, P., and Pringle, J.R. (1998). Heterologous modules for efficient

and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14, 943-

951.

Buhler, M., Verdel, A., and Moazed, D. (2006). Tethering RITS to a nascent transcript

initiates RNAi- and heterochromatin-dependent gene silencing. Cell 125, 873-886.

Buker, S.M., Iida, T., Buhler, M., Villen, J., Gygi, S.P., Nakayama, J., and Moazed, D.

(2007). Two different Argonaute complexes are required for siRNA generation and

heterochromatin assembly in fission yeast. Nat Struct Mol Biol 14, 200-207.

Colmenares, S.U., Buker, S.M., Buhler, M., Dlakic, M., and Moazed, D. (2007).

Coupling of double-stranded RNA synthesis and siRNA generation in fission yeast

RNAi. Mol Cell 27, 449-461.

Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K., and Pease, L.R. (1989). Site-directed

mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51-59.

Hong, E.J., Villen, J., Gerace, E.L., Gygi, S.P., and Moazed, D. (2005). A cullin E3

ubiquitin ligase complex associates with Rik1 and the Clr4 histone H3-K9

methyltransferase and is required for RNAi-mediated heterochromatin formation. RNA

Biol 2, 106-111.

Mellone, B.G., Ball, L., Suka, N., Grunstein, M.R., Partridge, J.F., and Allshire, R.C.

(2003). Centromere silencing and function in fission yeast is governed by the amino

terminus of histone H3. Curr Biol 13, 1748-1757.

Motamedi, M.R., Verdel, A., Colmenares, S.U., Gerber, S.A., Gygi, S.P., and Moazed, D.

(2004). Two RNAi complexes, RITS and RDRC, physically interact and localize to

noncoding centromeric RNAs. Cell 119, 789-802.

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