Separation of a functional deubiquitylating module from the SAGA complex by the proteasome regulatory particle

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1 Supplementary information for Separation of a functional deubiquitylating module from the SAGA complex by the proteasome regulatory particle Sungsu Lim 1, Jaechan Kwak 1, Minhoo Kim 1 and Daeyoup Lee 1,* 1 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 335 Gwahangno, Yuseong-gu, Daejeon , Korea * daeyoup@kaist.ac.kr

2 Supplementary Figure S1. The proteasomal ATPase, Rpt2p, specifically binds to Sgf73p of the SAGA complex. (a) The proteasomal ATPase, Rpt2p, binds to the SAGA complex in vitro. Pull-down assays were performed using the indicated recombinant GST-fused subunits of the 19S RP and Spt7-TAP-tagged SAGA complexes purified from wt. The input (10%) and bound proteins were analyzed by Western blotting using an antibody against HA (Spt7p). Purified GST-fused proteins were subjected to SDS-PAGE (10%) and visualized by Coomassie blue staining. (b) Experiments were performed as described in (a), except that we used the indicated recombinant GST-fused Rpt2 mutant proteins and SAGA complexes purified from wt, sgf73δ, ubp8δ and sgf11δ cells. The input (5%) and bound proteins were analyzed by Western blotting using antibodies against CBP and MYC. (c) Pull-down assays were performed using the indicated recombinant GST-Rpt2 mutant and SAGA complexes

3 purified from wt. The input (1%) and bound proteins were analyzed by Western blotting using an antibody against CBP. Abbreviation: I, input, S, supernatants and B, beads. 2

4 Supplementary Figure S2. SAGA complexes purified from rpt2-1 show normal HAT activity. (a and b) The HAT activity of the Spt7-TAP-tag-p urified SAGA complexes shown in Figure 2a (a) and the glycerol gradient fractions of Figure 2e (b) derived from wt and rpt2-1. HAT activity is shown in CPM, and error bars represent the standard deviations obtained from three independent repeats. 3

5 Supplementary Figure S3. Specific binding between Rpt2p and Sgf73p of the SAGA 4

6 complex affects the recruitment of the SAGA complex to chromatin. The relative abundances of Spt20p, Spt8p, Ada1p, Gcn5p and Ada2p on the PMA1, ADH1 and PYK1 genes in the wt and rpt2-1 strains were measured by ChIP assays followed by q-pcr with the indicated primers. ChIP assays and q-pcr were performed on five independent extracts, and standard deviations are shown by error bars. 5

7 Supplementary Figure S4. Targeting of RNA PolII and proteasomes to promoter regions in wt and rpt2-1. (a and b) The transcription levels of RNA PolII (8WG16) (a) and the relative abundances of Rpt2p and Rpt6p (b) on the PMA1 gene in the wt and rpt2-1 strains were measured by ChIP assay followed by q-pcr using the indicated primers. ChIP assays and q- PCR were performed on three independent extracts, and standard deviations are shown by error bars. 6

8 Supplementary Figure S5. The compositions of the TREX-2 complex and SAGA-TREX-2 are unaltered in the rpt2-1 strain. (a and b) THP1-TAP-tagged TREX-2 complexes (a) and SUS1-TAP-tagged SAGA-TREX-2 (b) from wt and rpt2-1 were subjected to TAP-based purification and visualized by silver staining. Protein molecular weight standards are indicated. 7

9 Supplementary Figure S6. The enrichment of TREX-2 at the promoter region is not specific to the PMA1 gene. (a and b) Additional ChIP experiments and q-pcr were used to examine the ADH1 (a) and PYK1 (b) genes with extracts prepared from wt and rpt2-1, using antibodies against Sac3p and Thp1p. Three independent repeats were performed, and the error bars show the standard deviations. 8

10 Supplementary Figure S7. The global level of histone H2B mono-ubiquitylation in the rpt2-1 and SAC3-deleted strains is unchanged. Recovered histones from the indicated strains were analyzed by SDS-PAGE and Western blotting using an anti-yh2b antibody and an anti-h3 antibody (control). The blots shown are representative of three independent experiments. 9

11 Supplementary Figure S8. Scans of the full Western blots and Coomassie blue staining gel of each panels shown in Figure 1. 10

12 Supplementary Figure S9. Scans of the full Western blots of each panels shown in Figure 2. 11

13 Supplementary Table S1. Saccharomyces cerevisiae strains used Yeast strain Genotype Reference yss501 MATa, can1-100, ade2-1, his3-11, -15, leu2-3, -112, trp1-1, ura3-1 Ref 14 yss106 MATa, can1-100, ade2-1, his3-11, -15, leu2-3, -112, trp1-1, ura3-1 RPT2ΔRKKRK yss503 sgf73δ::natnt2 yss506 sus1δ::trp1 yss401 SAC3- GFP::HIS5 yss403 SAC3- GFP::HIS5 RPT2ΔRKKRK yss405 SAC3- GFP::HIS5, sgf73δ::natnt2 yss411 SAC3- GFP::HIS5, sus1δ::trp1 yss420 THP1- GFP:: natnt2 yss421 THP1- GFP:: natnt2, RPT2ΔRKKRK yss422 THP1- GFP:: natnt2, sus1δ::trp1 yss423 NUP1- GFP:: natnt2 yss424 NUP1- GFP:: natnt2, RPT2ΔRKKRK yss425 NUP1- GFP:: natnt2, sus1δ::trp1 yss426 MEX67- GFP:: natnt2 yss427 MEX67- GFP:: natnt2, RPT2ΔRKKRK yss428 MEX67- GFP:: natnt2, sus1δ::trp1 yss429 MTR2- GFP:: natnt2 yss430 MTR2- GFP:: natnt2, RPT2ΔRKKRK yss431 MTR2- GFP:: natnt2, sus1δ::trp1 yss513 sac3δ::kanmx6 yss509 MATa ura3 52, lys2 801, ade2 101, trp1-δ63, his3-δ200, leu2-δ1, wt Ref 38 yss510 MAT-alpha ura3-52, leu2δ1, cim3-1 Ref 38 yss310 SPT7- TAP::TRP1 SPT7- yss313 TAP::TRP1, 3FLAG-SGF73::kanMX6, UBP8-, GCN5-3HA::HIS3 yss314 RPT2ΔRKKRK SPT7-TAP::TRP1, 3FLAG-SGF73::kanMX6, UBP8-13MYC::natNT2, GCN5-3HA::HIS3 yss340 ADA1- yss341 12

14 yss342 yss343 yss344 yss345 yss346 yss347 yss348 yss349 yss350 yss351 yss352 yss353 yss354 yss355 yss315 yss316 yss320 yss321 yss322 yss323 RPT2ΔRKKRK ADA1- SPT8- RPT2ΔRKKRK SPT8- SPT20- RPT2ΔRKKRK SPT20- ADA2- RPT2ΔRKKRK ADA2- GCN5- RPT2ΔRKKRK GCN5- UBP8- RPT2ΔRKKRK UBP8- SGF73- RPT2ΔRKKRK SGF73- SGF11- RPT2ΔRKKRK SGF11- SGF73- TAP::HIS3, GCN5-13MYC::TRP1, UBP8-3HA::kanMX6 RPT2ΔRKKRK SGF73-TAP::HIS3, GCN5-13MYC::TRP1, UBP8-3HA::kanMX6 RPN1- TAP::HIS3 RPT2ΔRKKRK RPN1-TAP:: natnt2 RPN11- TAP::HIS3 RPT2ΔRKKRK RPN11-TAP:: natnt2 13

15 Supplementary Table S2. The q-pcr primers used for ChIP analyses PYK1_#1 F PYK1_#1 R PYK1_#2 F PYK1_#2 F PYK1_#3 F PYK1_#3 F ADH1_PRO F ADH1_PRO R ADH1_ORF1 F ADH1_ORF1 R ADH1_ORF2 F ADH1_ORF2 R PMA1_A F PMA1_A R PMA1_B F PMA1_B R PMA1_C F PMA1_C R INT 5_F INT 5_R Q-PCR primer GCTACGTAAATGTGTTCCGCAC GGTACCTAGCATCATATGGGAAGG GTCCGTATGAACTTCTCTCACG GTGGGATTGGGTAGTCAACATC CACCGAAACCGTCGCTGCCTCCGCT TACGGGCTTCAACATCATCAGTCCA GTTTCCGGGTGTACAATATGG CTATTGTATATCTCCCCTCCGC CTGGTTACACCCACGACGGTTCTT GCAGACTTCAAAGCCTTGTAGACG CGGTAACAGAGCTGACACCAGAGA ACGTATCTACCAACGATTTGACCC GAAACGGAGAAACATAAACAGG GTCTCGAGGCCTGGAAGTGC CGACGACGAAGACAGTGATAACG ATTGAATTGGACCGACGAAAAACATAAC AAGTCGTCCCAGGTGATATTTTGCA AACGAAAGTGTTGTCACCGGTAGC GGCTGTCAGAATATGGGGCCGTAGTA CACCCCGAAGCTGCTTTCACAATAC 14