Nature Genetics: doi: /ng Supplementary Figure 1. ChIP-seq genome browser views of BRM occupancy at previously identified BRM targets.

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Joan Richardson
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1 Supplementary Figure 1 ChIP-seq genome browser views of BRM occupancy at previously identified BRM targets. Gene structures are shown underneath each panel.

2 Supplementary Figure 2 pref6::ref6-gfp complements ref6-1 phenotypes. Rosette leaf number was counted at bolting. Lowercase letters indicate significant differences between genetic backgrounds, as determined by the post hoc Tukey s HSD test (n = 17).

3 Supplementary Figure 3 ChIP-seq genome browser views of REF6 occupancy at previously published REF6 targets. The positions of the CTCTGYTY motifs underlying the REF6 peaks (Fig.4) are indicated by orange vertical bars. Gene structures are shown underneath each panel. The FLC locus is not targeted by REF6.

4 Supplementary Figure 4 Overlap of BRM- and REF6-occupied genes and BRM REF6 co-target genes with various epigenetic marks.

5 Supplementary Figure 5 Gene Ontology (GO) analysis of the BRM and REF6 target genes.

6 Supplementary Figure 6 REF6 expression is not reduced in brm-1. (a) qrt PCR analysis of REF6 transcript levels in brm-1 compared to wild type. (b) Immunoblot analyses showing the protein levels of REF6-GFP in wild type and brm-1. Histone 4 was used as the loading control. Numbers at the top represent relative levels of the proteins after normalization to the loading control. (c) Confocal images of root tips showing nuclear localization of GFP-tagged REF6 in brm-1 and wild-type plants. Scale bar, 20 m.

7 Supplementary Figure 7 BRM expression is not reduced in ref6-1. (a) BRM transcript levels in ref6-1 compared to wild type. (b) Immunoblot analyses showing the protein levels of BRM-GFP in wild type and brm-1. Histone 4 was used as the loading control. Numbers at the top represent relative levels of the proteins after normalization to the loading control. (c) Confocal images of root tips showing nuclear localization of GFP-tagged BRM in ref6-1 and wild-type plants. Scale bar, 20 m.

Supplementary Figure 8 REF6 physically interacts with BRM. (a) A list of peptides from SWI/SNF subunits and REF6 identified in the BRM-GFP IP MS experiments.

8 Supplementary Figure 8 REF6 physically interacts with BRM. (a) A list of peptides from SWI/SNF subunits and REF6 identified in the BRM-GFP IP MS experiments. All of these proteins were identified only in the pbrm::brm-gfp sample but not in the p35s::yfp control sample. (b) BiFC assays showing the interaction between BRM and REF6 in vivo. An unrelated nuclear protein encoded by At3G60390 was used as a negative control. (c) Immunoblot analysis showing the protein levels of the YN- or YC-tagged proteins used in the BiFC assays. YN- and YC-tagged proteins were probed with antibodies to HA and FLAG, respectively. Histone 4 was used as the loading control.

9 Supplementary Figure 9 DNA sequence from YUC3 ( bp downstream of the ATG start codon). The CTCTGYTY motifs are highlighted in red. Nucleotides in exons and the intron are shown in upper and lowercase letters, respectively.

Supplementary Figure 10 The CTCTGYTY motif contributes to the recruitment of BRM. For the full sequence of the transgene, see Supplementary Figure 9.

10 Supplementary Figure 10 The CTCTGYTY motif contributes to the recruitment of BRM. For the full sequence of the transgene, see Supplementary Figure 9. ChIP qpcr results show that binding of BRM at the transgene without the motifs (YUC3 ) is significantly less than that at the transgene containing the motifs (YUC3wt). Three independent transgenic lines were analyzed for each construct. ChIP signals are shown as percentage input. The endogenous YUC3 locus and the TA3 locus were used as the positive and negative control, respectively. Error bars, s.d. from three biological replicates. Lowercase letters indicate significant differences between genetic backgrounds, as determined by the post hoc Tukey s HSD test.

Supplementary Figure 11 Flowering time of wild-type, ref6-1, ref6-1 pref6::ref6-gfp, and ref6-1 pref6::ref6δznfs-gfp plants as determined by rosette leaf number.

11 Supplementary Figure 11 Flowering time of wild-type, ref6-1, ref6-1 pref6::ref6-gfp, and ref6-1 pref6::ref6δznfs-gfp plants as determined by rosette leaf number. Error bars, s.d. from 17 plants. Lowercase letters indicate significant differences between genetic backgrounds, as determined by the post hoc Tukey s HSD test.

12 Supplementary Figure 12 Phenotypes of Col, ref6-1, brm-1, and brm-1 ref6-1 plants. Top, 4-week-old plants. Bottom, rosette leaves from each genetic background as indicated. Scale bar, 1 cm.

Supplementary Figure 13 BRM and REF6 directly co-activate a set of genes. (a) Venn diagrams showing a statistically significant overlap between genes upregulated in brm-1 and ref6-1.

13 Supplementary Figure 13 BRM and REF6 directly co-activate a set of genes. (a) Venn diagrams showing a statistically significant overlap between genes upregulated in brm-1 and ref6-1. (b) Venn diagrams showing statistically significant overlaps between BRM REF6 co-target genes and genes reduced in brm-1 or ref6-1. (c) Venn diagrams showing the lack of statistically significant overlaps between BRM REF6 co-bound genes and genes induced in brm-1, ref6-1, or brm-1 ref6-1.

14 Supplementary Figure 14 BRM is not required for the ability of REF6 to remove trimethyl groups from H3K27me3. Top, mean density of H3K27me3 at REF6-occupied genes in wild type (WT), brm-1, ref6-1, and brm-1 ref6-1. The average H3K27me3 signal within 2-kb genomic regions flanking the center of REF6 peaks is shown. Bottom, ChIP-seq genome browser views of REF6 occupancy at selected loci in wild type and brm-1. Gene structures are shown underneath each panel.

15 Supplementary Figure 15 Correlation analyses of biological replicates of ChIP-seq data. The x axis represents normalized signal intensity from the first replicate in log 2 scale. The y axis represents the log 2 value of normalized signal intensity from the second replicate. The correlation analyses show highly positive correlations between the biological repeats, suggesting that the binding profiles are generally similar for the two ChIP-seq replicates.

16 Supplementary Table 1. Oligonucleotides Used in This Study Primers used for ChIP-qPCR analyses AT1G76590-F: 5 -TCGATTCTTTCCGTTTCTGCTCTC-3 AT1G76590-R: 5 -TCTCTTCCATGCTTCCCTCTCAAA-3 SNRK2.8-F: 5 -TGGAGAGGTACGAAATAGTGAAGG -3 SNRK2.8-R: 5 -TTGGCCTCGCTCGATGAACTT -3 AT2G40320-F: 5 -CCCGACCACTGTATGAGGAATG-3 AT2G40320-R: 5 -GCTGCCACCGCCAAAACT-3 AT3G51910-F: 5 -CCTCGTCATTTCAAACACAGCA-3 AT3G51910-R: 5 -TCGCGGTAATTTTCCAGCAAC-3 CYP707A3-F: 5 -GGGATGGAACTCAACTCAACACTT-3 CYP707A3-R: 5 -CAGAGAAAATAAACAGGGGACACT-3 AT5G55090-F: 5 -ACTTCTTCGCCGTCAAATCC-3 AT5G55090-R: 5 -CTAATCAACGGCTCCGGTAACTTC -3 CYP707A1-F: 5 -AAGGTTACAATTCGATGCCAGTGA-3 CYP707A1-R: 5 -TGTTCGTCGGTTAGCTCTTCTTTG-3 YUC3-F: 5 -TGGCTCGCGGACAAGACTA -3 YUC3-R: 5 -TTTATGATTCCGGGGACGATTTT-3 AT5G51670-F: 5 -GGCCAGCCTCCGTTCTTT-3 AT5G51670-R: 5 -ATATCGCCATTTCTTCCATCTCT-3 TA3-F: 5 -GATTCTTACTGTAAAGAACATGGCATTGAGAGA-3 TA3-R: 5 -TCCAAATTTCCTGAGGTGCTTGTAACC-3 SVP-F: 5 -CAACGGCGAGACAAGTGA-3 SVP-R: 5 -GAGAGAAAGAAAGACCTGGAGC-3 Primers used for ChIP-qPCR analyses in Figure 4b AT1G04610-ChIP-F1: GGGGACAAGTTTGTACAAAAAAGCAGG AT1G ChIP-R1: TAGGGGCTATAAAGATTTGAACTTTTGG Primers for generating ProREF6:REF6-GFP vector REF6-P-attB-F: 5 - GGGGACAAGTTTGTACAAAAAAGCAGGCTTC CACCTAAAAACAGAGCTGGCTCTC-3 REF6-attB-R: 5 - GGGGACCACTTTGTACAAGAAAGCTGGGTC CCTTTTGTTGGTCTTCTTAACCGAA-3 Primers for generating ProREF6:REF6-ΔZnFs-GFP vector REF6-P-attB-F: 5 - GGGGACAAGTTTGTACAAAAAAGCAGGCTTC CACCTAAAAACAGAGCTGGCTCTC-3 REF6-attB-R3: 5 -GGGGACCACTTTGTACAAGAAAGCTGGGTC TCGTTTGGCTGTTGACCTGG-3 Primers for CTCTGTTT motif deletion YUC3-wt-F: 5 - GGGGACAAGTTTGTACAAAAAAGCAGGCTTCTCGTTCGTAGCGCTgtaagtac-3 YUC3-wt -R: 5 -GGGGACCACTTTGTACAAGAAAGCTGGGTC AGTATAGTCTTGTCCGCGAGCC-3 YUC3-Δ-F: 5 -taggggctataaagatttgaac-3 YUC3-Δ-R: 5 -cagtatctaatagtctgtgttt-3 1

17 Primers for EMSA assays YUC3-wt: 5 - CCCTAAAACAGAGCATCCTCTGTTTCTGTTACTCTGTTTTCTAAAACAGAGCAGTA-3 YUC3-m1: 5 - CCCTAAAAttttGCATCCTCTGTTTCTGTTACTCTGTTTTCTAAAAttttGCAGTA-3 YUC3-m2: 5 - CCCTAAAAttttGCATCCaaaaTTTCTGTTACTCTGTTTTCTAAAACAGAGCAGTA-3 YUC3-m3: 5 - CCCTAAAAttttGCATCCTCTGTTTCTGTTACaaaaTTTTCTAAAACAGAGCAGTA-3 YUC3-m4: 5 - CCCTAAAAttttGCATCCTCTGTTTCTGTTACaaaaTTTTCTAAAAttttGCAGTA-3 YUC3-m5: 5 - CCCTAAAAttttGCATCCaaaaTTTCTGTTACaaaaTTTTCTAAAAttttGCAGTA-3 Primers for BiFC assays REF6-attB-F: 5 -GGGGACAAGTTTGTACAAAAAAGCAGGCTTC ATGGCGGTTTCAGAGCAGAGT -3 REF6-attB-R: 5 -GGGGACCACTTTGTACAAGAAAGCTGGGTC CCTTTTGTTGGTCTTCTTAACCGAA-3 BRM-attB-F: 5 - GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCAATCTGGAGGCAGTGGC-3 BRM-attB-R: 5 -GGGGACCACTTTGTACAAGAAAGCTGGGTC TAAATGGCTAGGCCGTCTTTTACCAG-3 Gene-specific primers pairs for qrt-pcr AT1G76590-F: 5 -TCGATTCTTTCCGTTTCTGCTCTC-3 AT1G76590-R: 5 -TCTCTTCCATGCTTCCCTCTCAAA-3 SNRK2.8-F: 5 -TGGAGAGGTACGAAATAGTGAAGG-3 SNRK2.8-R: 5 -TTGGCCTCGCTCGATGAACTT-3 CYP707A1-F: 5 -CCAAACTCCCACTCCCTCCG-3 CYP707A1-R: 5 -CGAACTTAGCAGCCTCTGGACTC-3 YUC3-F: 5 -TGGCTCGCGGACAAGACTA-3 YUC3-R: 5 -TTTATGATTCCGGGGACGATTTT-3 AT5G51670-F: 5 -GGCCAGCCTCCGTTCTTT-3 AT5G51670-R: 5 -ATATCGCCATTTCTTCCATCTCT-3 AT3G51910-F: 5 -AAGCGTCATGGCTTGGTGAA-3 AT3G51910-R: 5 -CCTCGTCATTTCAAACACAGCA-3 CYP707A3- F: 5 -CTTGTCCAGGCAATGAGTTAG-3 CYP707A3-R: 5 -CCAAGGCAATAGGCAATCC-3 AT5G55090-F: 5 -TGTGTTGGATCAACGGTTTT-3 AT5G55090-R: 5 -GATCACCCGGTAAAGAATCG-3 AT2G40320-F: 5 -CCCGACCACTGTATGAGGAATG-3 AT2G40320-R: 5 -GCTGCCACCGCCAAAACT-3 BRM-F: 5 -TTTAGAAAGGAAAAGGATTAGGC-3 BRM-R: 5 -GCCGTTCGCATAACCTCA-3 REF6-F: 5 -TGAGTCAGAAGATAATGGCG-3 REF6-R: 5 -CTATTCGTTTGGCTGTTGAC-3 GAPDH-F: 5 -CTTGGAAGGAGCTAGGAATTGACA-3 GAPDH-R: 5 -ATGTGTTTCCCTGCACCTTCTC-3 2

18 Supplementary Table 2. Summary of mapped reads for ChIP-seq Library Total reads a Total mapped reads b (% of total) Unique mapped reads c (% of total mapped) BRM-GFP-Input 30,126,285 23,336,191 (77%) 17,450,223 (75%) BRM-GFP-IP-bio1 22,356,677 14,817,442 (66%) 12,056,062 (81%) BRM-GFP-IP-bio2 40,532,890 11,695,919 (29%) 9,550,750 (82%) ref6 BRM-GFP-Input 14,129,455 9,541,114 (68%) 7,443,958 (78%) ref6 BRM-GFP-IP-bio1 33,942,989 27,361,598 (81%) 22,327,671 (82%) ref6 BRM-GFP-IP-bio2 35,807,662 27,475,903 (77%) 21,968,071 (80%) REF6-GFP-Input 26,981,831 20,862,733 (77%) 15,649,890 (75%) REF6-GFP-IP-bio1 13,647,711 7,855,852 (58%) 6,403,041(82%) REF6-GFP-IP-bio2 31,177,000 20,519,440 (66%) 15,936,302 (78%) brm REF6-GFP-Input 15,829,582 12,058,982 (76%) 8,972,179 (74%) brm REF6-GFP-IP-bio1 31,843,533 22,467,663 (71%) 17,774,115 (79%) brm REF6-GFP-IP-bio2 34,442, ,009 (2.88%) 653,660 (66%) WT-Input 8,017,975 7,584,026 (94.59%) 3,576,620 (47% WT-H3K27me3-bio1 8,164,349 7,252,632 (88.83%) 3,622,886 (50%) WT-H3K27me3-bio2 6,082,955 4,943,531 (81.27%) 3,011,501 (61%) brm-input 8,358,050 7,718,081 (92.34%) 3,601,583 (47%) brm-h3k27me3-bio1 7,063,378 5,843,984 (82.74%) 3,578,111 (61%) brm-h3k27me3-bio1 6,901,326 5,128,340 (74.31%) 2,654,092 (52%) ref6-input 7,940,179 7,399,281 (93.19%) 3,880,118 (52%) ref6-h3k27me3-bio1 6,586,343 5,624,399 (85.39%) 2,470,708 (44%) ref6-h3k27me3-bio1 7,634,641 2,003,253 (26.24%) 1,475,339 (74%) brm ref6-input 6,836,530 6,217,561 (90.95%) 3,431,813 (55%) brm ref6-h3k27me3-bio1 6,258,129 5,454,647 (87.16%) 2,919,059 (54%) brm ref6-h3k27me3-bio1 5,065,437 4,188,949 (82.70%) 1,773,043 (42%) a Number of sequence reads before data analyses; b Number of reads that aligned to TAIR10 genome sequences; c Number of unique mapped reads.