Supplemental Data. Antosz et al. Plant Cell (2017) /tpc SPT6/SPT6L. genomic DNA ACT2 +RT -RT +RT -RT

Transcription

1 A B C SPT6/SPT6L genomic DNA ACT2 +RT -RT Col- seedlings +RT -RT PSB-D cells Supplementl Figure 1. Expression of SPT6L nd SPT6. (Supports Figure 1.) Trnscript levels of of SPT6L (At1g6544) nd SPT6 (At1g6321) were exmined by RT-PCR (+RT) with RNA from 16 DAS Col- seedlings (A) nd PSB-D cells (B). This experiment indictes tht in line with publicly vilble microrry dt ( only SPT6L is expressed in seedlings, while the trnscripts of both SPT6 nd SPT6L re detected in PSB-D cells. As controls the ACT2 trnscript ws nlysed comprtively s well s smples without reverse trnscription (-RT) (right pnels in A,B). Moreover, the functionlity of the primer combintions ws tested in PCR rections using genomic DNA s templte (C).

2 A α-ctd-s2p α-ctd NRPB1O NRPB1A input AP input AP ELF7-GS NRPB1O NRPB1A GS-TFIIS B 1.8 Frction bound SPT6 [µm] Supplementl Figure 2. Enrichment of Ser2-phosphorylted RNAPII in TEF ffinity purifictions nd interction of SPT6L with Ser2-phosphorylted RNAPII-CTD repets. (Supports Figure 1 nd 2.) (A) Immunoblot nlysis of ffinity purifictions (AP) vs. input of ELF7-GS nd GS-TFIIS with ntibodies directed ginst Ser2-phosphorylted RNAPII-CTD (left) nd ginst non-phosphorylted RNAPII-CTD (right). The hypophosphorylted lrgest subunit NRPB1A hs slightly higher electrophoretic mobility thn the hyperphosphorylted NRPB1O (indicted by rrows). (B) MST nlysis of the interction of GST- SPT6L(Phe1218-Asp1412) with RNAPII-CTD repet peptides. A constnt mount of the GST-SPT6L protein ws incubted with incresing concentrtions of FITC-lbelled CTD repet peptides tht were either nonphosphorylted (blue), or phosphorylted t Ser2 (red) or Ser5 (green). The error brs represent the stndrd devition of three technicl experiments (independently prepred regent mixtures followed by MST mesurements). Binding ffinity (K D ) ws clculted by fitting the dt points (red line) using the K D Fit derived from the lw of mss ction ( = unbound, 1= bound) SPT6 showed n ffinity of ± 26.6 µm (R 2 =.98). For S5 nd non-phosphorylted weker binding is indicted.

3 ELF7 SPT6L RNAPII Ser2P RNAPII non-phos.5 µm.5 µm Supplementl Figure 3. ELF7 nd SPT6L co-loclise with RNAPII in euchromtin. (Supports Figure 3.) Co-loclistion nlysis of ELF7 nd SPT6L with RNAPII-Ser2P nd non-phosphorylted RNAPII within euchromtic regions of flow-sorted 8C nuclei of lef cells visulised by SIM. For comprison, in the merges the sme colour is used ech for both forms of RNAPII (green) nd both TEFs (ELF7, SPT6L; red). In the merge of the TEF signls with those of non-phosphorylted RNAPII more clerly red res (representing TEFs, but no RNAPII) re visible, while in the merge with RNAPII-S2P more prominently yellow/ornge res (representing TEFs nd RNAPII) re visible.

4 A 28 DAS B Col- tfiis ssrp1 tfiis ssrp1 28 DAS C Col- tfiis spt16 tfiis spt16 D bolting [DAS] Col- tfiis spt16 tfiis spt16 Col- tfiis spt16 tfiis spt16 E 4 b b 3 b b bolting [DAS] 2 1 Supplementl Figure 4. Anlysis of tfiis ssrp1 nd tfiis spt16 double-mutnts in comprison to the respective single-mutnts nd the Col- wild type. (Supports Figure 4.) Representtive imges of the plnts t 28 dys fter strtifiction (DAS) under long-dy conditions re shown (A,B). Freshly hrvested siliques (left) nd clered siliques (right) of tfiis spt16 nd controls re shown in (C) tfiis ssrp1 plnts re sterile, producing no seeds. The time of bolting (D,E) ws determined (n=13). Dt were nlysed by two-wy ANOVA nd error brs indicte stndrd devition clculted from the mesurements of 13 individul plnts for ech line. The letters bove the histogrm brs indicte the outcome of multi comprisons Tukey s test (p <.5).

5 Col- Col- spt16 spt16 tfiis tfiis ssrp1 ssrp1 elf7 elf7 tfiis spt16 tfiis spt16 tfiis elf7 tfiis elf7 tfiis ssrp1 tfiis ssrp1 Supplementl Figure 5. Lef vein ptterning of Col-, single- nd double mutnts. (Supports Figure 4.) Vein pttern of clered leves of the indicted plnt lines. Representtive first leves (left) nd second leves (right) of 21 d old plnts re shown. All imges re of the sme mgnifiction except for tfiis elf7. Size brs indicte 1 mm.

6 A 28 DAS Col- tfiis elf7 tfiis elf7 B bolting [DAS] b b C leves t bolting b c d Supplementl Figure 6. Anlysis of tfiis elf7 double-mutnts in comprison to the respective single-mutnts nd the Col- wild type. (Supports Figure 4.) Representtive imges of the plnts t 28 dys fter strtifiction (DAS) under long-dy conditions re shown (A). The time of bolting in DAS (B) nd the number of leves t bolting (C) were determined (n=13). Dt were nlysed by two-wy ANOVA nd error brs indicte stndrd devition clculted from the mesurements of 13 individul plnts for ech line. The letters bove the histogrm brs indicte the outcome of multi comprisons Tukey s test (p <.5).

7 Col- spt16-1 elf7-2 spt16-1 elf7-2 Supplementl Figure 7. Phenotype of spt16 elf7 double-mutnt plnts in comprison to the respective single-mutnts nd Col-. (Supports Figure 4.) Representtive imge plnts t 28 dys fter strtifiction (DAS) under long-dy conditions. For the cross of the ssrp1 nd elf7 mutnts, no homozygous double-mutnts were obtined. The proportion of 18.4% non-germinting seeds observed with the segregting popultion (ELF7 +/- SSRP1 -/-, n = 49) indictes tht this double-mutnt combintion is lethl.

8 * CstF77 CstF64 GS GS GS * * CBB stin Supplementl Figure 8. Isoltion of the CstF complex. (Supports Figure 1 nd 2.) Elutes of ffinity purifictions from cells expressing CstF77-GS, CstF64-GS or unfused GS fter SDS- PAGE nd Coomssie-stining of the gel. The unfused GS-tg nd GS-fusion proteins re indicted by sterisks. The migrtion positions of moleculr weight mrker (in kd) re indicted on the left.

9 A bolting [DAS] b c d B reltive FLC expression ,d b,d c d,,b Supplementl Figure 9. Erly bolting phenotype of cdc73 cstf64 double-mutnt plnts. (Supports Figure 5.) (A) Bolting time in DAS of the indicted genotypes (n=15) under LD conditions. (B) FLC trnscript levels of the different genotypes quntified by qrt-pcr from three biologicl replictes (independently pooled collections of tissues) nd t lest two technicl replictes ech. Dt were nlysed by two-wy ANOVA nd error brs indicte stndrd devition. The letters bove the histogrm brs indicte the outcome of multi comprisons Tukey s test (p <.5), nd for the brs (in B) tht re indicted with more thn one letter bove the br, the brs shring the sme letter re not significntly different.

10 Col cdc73 elf7 spt16 ssrp1 SPT4-R3 tfiis Supplementl Figure 1. 35S pre-rrna levels in mutnts deficient in different TEFs. (Supports Figure 6.) Quntifiction of RNA gel blot nlyses of totl RNA isolted from Col-, the indicted T-DNA insertion mutnts nd SPT4-R3, which is plnt line expressing nti-sense RNA directed ginst SPT4 tht contins strongly reduced mounts of SPT4-1 nd SPT4-2 trnscripts (Dürr et l., 214). The blots were hybridised with rdio-lbelled probes detecting the 35S pre-rrna (ITS1) nd UBQ5. The dt depicted in the histogrm represent the mount of the 35S pre-rrna signl reltive to the UBQ5 signl (quntified using phosphorimger) nd they correspond to men vlues of three experiments. Error brs represent stndrd devition nd nlysis of the dt by one-wy ANOVA reveled tht the dtsets re not significntly different from tht of Col-.

11 Supplementl Tble 1. Degree of coloclistion of ELF7 nd SPT6L with RNAPII (phosphorylted t Ser2 or non-phosphorylted) No. nuclei % No. nuclei % Voxel intensities RNAPII non-phos RNAPII non-phos ELF SPT6L RNAPII Ser2P 13 1 RNAPII Ser2P 13 1 Co-loclistion RNAPII non-phos coloclising to ELF RNAPII non-phos coloclising to SPT6L ELF7 coloclising to RNAPII non-phos SPT6L coloclizing to RNAPII non-phos RNAPII non-phos coloclising to RNAPII Ser2P RNAPII non-phos coloclising to RNAPII Ser2P RNAPII Ser2-P coloclising to RNAPII non-phos RNAPII Ser2P coloclising to RNAPII non-phos ELF7 coloclising to RNAPII Ser2P SPT6L coloclising to RNAPII Ser2P RNAPII Ser2P coloclising to ELF RNAPII Ser2P coloclising to SPT6L Voxel intensities clculted by Imris 8.

12 Supplementl Tble 2. Overview of phenotypes observed for single- nd double mutnts defective in vrious TEFs. genotype Col- tfiis ssrp1 spt16 elf7 tfiis trit 1 ssrp1 tfiis spt16 tfiis elf7 ssrp1 elf7 spt16 elf7 vibility - rosette ø inflorescence No n.d. bolting [DAS] n.d. bolting [leves] n.d. n.d. + - n.d. lef veins n.d. seed set Quntittive differences between the different genotypes regrding the trits nlysed in this report re summrised here. 2 The bolting time [DAS] reltive to Col- (+++) is reduced in some genotypes (++). 3 The lef number t bolting is incresed (++++) or reduced (++,+) reltive to Col- (+++) in some genotypes.

13 Supplementl Tble 3. Oligonucleotide primers used in this study nd construction of plsmids use plsmid restr. site AAGGATCCATGGCGGCTGCGGCTTTTGGGCA Insertion of CDKC2 CDS in pcmbi23-gs pcmbi 23- CDKC2-GS BmHI GCGGAGCTCCGGTTGCCATCCATATTGTTGGT Insertion of CDKC2 CDS in pcmbi23-gs pcmbi 23- CDKC2-GS ScI TATAGTCGACATGGATACGAGGTTTCCGTTC Insertion of NRPB1 CDS in pcmbi23-gs pcmbi 23- NRPB1-GS SlI CGCGTCTAGAAGGGTTGCCTTTATCATCCTTAC Insertion of NRPB1 CDS in pcmbi23-gs pcmbi 23- NRPB1-GS XbI CGGAGCTCCCTGTGCTTGCGTTTAGATGGT Insertion of SPT16 CDS in pcmbi23-gs pcmbi 23- SPT16-GS ScI GCTCTAGAATGGATCCATTATCAGT Insertion of CDC73 CDS in pcmbi 23-GS pcmbi 23-CDC73-GS XbI TCCCCCGGGCCAGAATGCGACCG Insertion of CDC73 CDS in pcmbi 23-GS pcmbi 23-CDC73-GS SmI GCTCTAGAATGGCTTCATCATCA Insertion of CSTF64 CDS in pcmbi 23-GS pcmbi 23-CSTF64-GS XbI TCCCCCGGGCCTGAAGGCTGCATCAT Insertion of CSTF64 CDS in pcmbi 23-GS pcmbi 23-CSTF64-GS SmI GCTCTAGAATGGCTGATAAGTAC Insertion of CSTF77 CDS in pcmbi 23-GS pcmbi 23-CSTF77-GS XbI TCCCCCGGGCCGCCAGTGCTACCAGA Insertion of CSTF77 CDS in pcmbi 23-GS pcmbi 23-CSTF77-GS SmI GCTCTAGAATGGCGTCGTACCG Insertion of ELF7 CDS in pcmbi 23-GS pcmbi 23-ELF7-GS XbI TCCCCCGGGCCTTCAGAATAATC Insertion of ELF7 CDS in pcmbi 23-GS pcmbi 23-ELF7-GS SmI GCTCTAGAATGGCAGACTCTCGGAATGGTAAT Insertion of SPT16 CDS in pcmbi23-gs pcmbi 23-SPT16-GS XbI AATTGCGGCCGCATATGGCGGACGGCCACTCC Insertion of SSRP1 CDS in pcmbi23-gs pcmbi 23-SSRP1-GS NotI CGGAGCTCGTTACTATCGGAATCGTTTCCTGAGT Insertion of SSRP1 CDS in pcmbi23-gs pcmbi 23-SSRP1-GS ScI TTGGGCCCAACAATGGGAAGCGCACCAGCTCAGATTC Insertion of SPT4-2 CDS in pcmbi23-gs pcmbi23-spt4-2-gs SmI TTGAGCTCTCACATGCGTTTCGGCAGAACAT Insertion of SPT4-2 CDS in pcmbi23-gs pcmbi23-spt4-2-gs ScI AATTCTCGAGTTTGTGAAAAGCCCATCAAACTTTGG Insertion of TFIIS promoter in pgreenii179-gs pgreenii179:tfiisp::gs-tfiis XhoI AACTCGAGACGTTCCGACAATCCCTAGCTCA Insertion of TFIIS promoter in pgreenii179-gs pgreenii179:tfiisp::gs-tfiis XhoI CGGGATCCATGGAGAGTGATTTGATTGATTTG Insertion of TFIIS CDS in pgreenii179-gs pgreenii179:tfiisp::gs-tfiis BmHI AAGAATTCCTCAACAGAACTTCCAGTGGTTG Insertion of TFIIS CDS in pgreenii179-gs pgreenii179:tfiisp::gs-tfiis EcoRI CGGGATCCCAAAGTCTCGGATGATT Insertion of SPT6 CDS ( ) in pgex5x-1 pgex5x-1-spt6_prtil BmHI ACGCGTCGACTCAGTCATCAATATGCCT Insertion of SPT6 CDS ( ) in pgex5x-1 pgex5x-1-spt6_prtil SlI CGGGATCCGATTCTGAATTCTTTGGC Insertion of ELF7 CDS (41-589) in pqe9 pqe9-elf7_prtil BmHI CCCAAGCTTTCATTCAGAATAATCATC Insertion of ELF7 CDS (41-589) in pqe9 pqe9-elf7_prtil HindIII AAGGATCCCTATCACCAGCTAGCCCCTAC Insertion of NRPB1 CDS ( ) in pqe9 pqe9-nrpb1_prtil BmHI ATATAAGCTTTCAAGGGTTGCCTTTATCATCC Insertion of NRPB1 CDS ( ) in pqe9 pqe9-nrpb1_prtil HindIII CGGGATCCGTATTAGATTCTGGGTTA Insertion of SPT6 CDS ( ) in pqe9 pqe9-spt6_prtil BmHI ACGCGTCGACCTACATCGGTACCTTGGCAGC Insertion of SPT6 CDS ( ) in pqe9 pqe9-spt6_prtil SlI ATGGATCCATGGAGAGTGATTTGATTGATT Insertion of TFIIS CDS in pqe9 pqe9-tfiis BmHI ATATCTGCAGTCAACAGAACTTCCAGTGGT Insertion of TFIIS CDS in pqe9 pqe9-tfiis PstI AGCCAAGAAGACCGAACTCA qrt-pcr FLC TTTGTCCAGCAGGTGACATC qrt-pcr FLC GTTGTAACAAGATGGATGCCA qrt-pcr EF1

14 GGACAAATGGGATTTTGTCAG qrt-pcr EF1 GTGGAAGAGCTGCCGTTGAATT qrt-pcr SPT6 GCACAATCCTCCCTTCACCAAT qrt-pcr SPT6 GTTGTTGGGGACCTGGGAAG qrt-pcr SPT6L ATCCATCCCATGTCCGACATC qrt-pcr SPT6L GCTGGAATCCACGAGACAAC qrt-pcr Actin 2 AAGCCTTTGATCTTGAGAGCTT qrt-pcr Actin 2 GGTAACATTGTGCTCAGTGGTGG ChIP qpcr, Actin2 GGTGCAACGACCTTAATCTTCAT ChIP qpcr, Actin2 AGATGGTTTCGGGGAGGAAAATCAC ChIP qpcr, At3g226 GCTTCTCTTTCGACAGGCCAGAG ChIP qpcr, At3g226 GGATGCGATCATACCAGCACTAATG ChIP qpcr, 18S rdna -1 GAGGGATGCAACACGAGGACTTC ChIP qpcr, 18S rdna-1 CGGGGGCATTCGTATTTCATAGTCA ChIP qpcr, 18S rdna-2 GACTAGGACGGTATCTGATCGTCTTC ChIP qpcr, 18S rdna-2 TAGGGTTCTTAGTTGATCTTGTATTGAGCTC ChIP qpcr, TA3 TTTGCTCTCAAACTCTCAATTGAAGTTT ChIP qpcr, TA3 TTTCGAGACGGCGACACCAATG ChIP qpcr, U6-1 GAGAGTAGCTGATCCTAAATGCTATC ChIP qpcr, U6-1 AAACGATGCGTTGGGATAGGTC ChIP qpcr, TA2 ATACTCTCCACTTCCCGTTTTTCTTTTTA ChIP qpcr, TA2 ACTGGCTCATCGTTCTCCATT ChIP qpcr, 7SL-1 ATTTGTTTTGATTGCTCGGTTT ChIP qpcr, 7SL-1 ACTTTCCATTCGGAGTTTTTGT ChIP qpcr, U6-26 GCCTGCTTCTCTTCTTTCAGATT ChIP qpcr, U6-26 CGA CCC TCC CCT AAA TCA CTC CA ChIP qpcr, 3'ETS ACT TGG CGA GGT CCG GAA TCT TAC ChIP qpcr, 3'ETS GAAGGCGAAGATCCAAGACAAGGAA ChIP qpcr, UBQ5 GGAGGACGAGATGAAGCGTCGA ChIP qpcr, UBQ5 ATCGACGACGAATACGAAAG Expression of CSTF64 (SALK_88877) CTAAGTTGTTCCTCGGTCGC Expression of CSTF64 (SALK_88877) TCGTTTCCTGAAGACGGACC Expression of CSTF77 (SALK_1482) CAAAGCTCTCTTGGCCAAAG Expression of CSTF77 (SALK_1482) GCTCTAGAATGGATCCATTATCAGT Expression of CDC73 (SALK_8357) TTCGTTCTGCTTCTGACTCTG Expression of CDC73 (SALK_8357) ATCGACGACGAATACGAAAG Genotyping, T-DNA insertion cstf64-3 (SALK_88877) CTAAGTTGTTCCTCGGTCGC Genotyping, T-DNA insertion cstf64-3 (SALK_88877) TCGTTTCCTGAAGACGGACC Genotyping, T-DNA insertion cstf77-2 (SALK_1482) CAAAGCTCTCTTGGCCAAAG Genotyping, T-DNA insertion cstf77-2 (SALK_1482)

15 GCTCTAGAATGGATCCATTATCAGT TTCGTTCTGCTTCTGACTCTG ATCCTCTGGAATGTTGATAGT TTTCCTCTTGTCACTTGCCAT ATTTTGCCGATTTCGGAAC CCCTCATCTTACGCGTATCAGA AATTAAGCTTAGTTACTATCGGAATCGTTTCCT CTATCTCTGCATTGCCTCTTAGC TACTTGTCTAACGCAGCGAAATC GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC TTGGACCCTTCAATTCGTGATG CCTGGCCCTTTTCTTCCTCA GTTGCCCGTCTCACTGGTGA TCGATACCTGTCCAAAACAG AGACTTCAGTTCGCAGC CTCACACGAATAAGGTACAAAGTTC GAAGGCGAAGATCCAAGACAAGGAA GGAGGACGAGATGAAGCGTCGA Genotyping, T-DNA insertion cdc73-2 (SALK_8357) Genotyping, T-DNA insertion cdc73-2 (SALK_8357) Genotyping, T-DNA insertion tfiis-1 (SALK_56755) Genotyping, T-DNA insertion tfiis-1 (SALK_56755) Genotyping, T-DNA insertion SALK LBb1.3 (tfiis-1) Genotyping, T-DNA insertion ssrp1-2 (SALK_1283) Genotyping, T-DNA insertion ssrp1-2 (SALK_1283) Genotyping, T-DNA insertion spt16-1 (SAIL_392_G6) Genotyping, T-DNA insertion spt16-1 (SAIL_392_G6) Genotyping, T-DNA insertion SAIL LB (SPT16) Genotyping, T-DNA insertion elf7-2/-3(salk_4665/_19433) Genotyping, T-DNA insertion elf7-2/-3(salk_4665/_19433) Genotyping, T-DNA insertion SALK LBb1.3 (elf7-2, elf7-3, ssrp1-2) RNA gel blot probe for pre-rrna ITS1 RNA gel blot probe for pre-rrna ITS1 RNA gel blot probe FLC ntisense RNA gel blot probe for UBQ5 RNA gel blot probe for UBQ5