Chromatin immunoprecipitation of UTX (Lan et al. 2007), H3K27me3, and H3K4me2 (Rinn et

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1 Wang et al., p. 1 Supplementary Methods ChIP-chip and data analysis Chromatin immunoprecipitation of UTX (Lan et al. 27), H3K27me3, and H3K4me2 (Rinn et al. 27) were as previously described. We used affinity-purified UTX anti-serum (gift of E. Canaani) that was used successfully for UTX ChIP by multiple groups (Agger et al. 27; Issaeva et al. 27; Lan et al. 27). Genomic DNA retrieved by ChIP was hybridized to arrays tiling human promoters (Roche Nimblegen, Madison, WI) as described (Rinn et al. 27), and ChIP target genes were defined as having one or more peaks with a false discovery rate (FDR) <.2, a parameter which has been empirically shown to better match predefined positive controls (Johnson et al. 28). Systematic comparison of biological replicates of UTX ChIP-chip experiments from two different primary human fibroblast lines and independent ChIP-qPCR validations showed that the most accurate results are obtained by accepting peaks that pass the FDR filter in at least one experiment; the combined list of UTX target genes are reported thereafter. The top gene networks and pathway map were generated through the use of Ingenuity Pathways Analysis (Ingenuity Systems, Fibroblast gene expression levels were obtained by mrna hybridization to Illumina whole genome bead array per manufacturer s instruction. Enriched Gene Ontology terms were found using the software DAVID ( Array data are available at GEO ( and Stanford Microarray Database ( Human cancer data analysis Gene Module map: Gene expression compendium of 1,973 microarrays representing 22 human tumor types and diverse normal controls were as described (Segal et al. 24). Briefly, for each

2 Wang et al., p. 2 microarray, we first identified genes that were induced or repressed by at least 2-fold, and tested for their enrichment in a set of 5 genes comprised of UTX plus its genomic targets shown in Fig. 2A over that expected by chance alone (p <.5, FDR <.5, hypergeometric distribution). The end result of step one is the identification of all array that show coordinate activation or deactivation of the UTX-bound RB associated network. Second, we examined whether any clinical annotation is enriched among samples that exhibit coordinate induction or repression. Each array in the cancer compendium has been previously annotated for 283 biological or clinical attributes ( For each annotation, we compared the frequency of UTX target induction or repression among the samples versus that expected by chance alone (p <.5, FDR <.5, hypergeometric distribution). Arrays showing coordinate regulation of UTX-RB genes and their enriched clinical annotations are shown in Fig. 2C. Gene Set Enrichment Analysis: The expression level of each gene across 295 breast cancers (van de Vijver et al. 22) was compared to that of UTX and ranked based on Pearson correlation, (the gene with most similar expression to UTX being ranked number one). We tested whether genes bound by UTX protein (as measured by ChIP-chip) was more likely to show similar expression pattern with UTX itself. Specifically, the top 3% of the UTX ChIP-chip targets, sorted by ChIP/control enrichment values, was compared to the breast cancer transcriptome sorted based on similarity to UTX by GSEA (Subramanian et al. 25). Kolmogorov Smirnov scanning statistics were calculated in the observed data versus 1 permuted sets to determine the statistical significance. GSEA and gene module map provide independent and complementary methods to test the co-expression of UTX with its candidate target genes, and both methods

3 Wang et al., p. 3 Kaplan-Meier survival analysis: To determine the significance of UTX and EZH2 expression and patient outcome, the expression data of 295 breast cancer patients was mean centered, and the expression values for UTX and EZH2 were isolated. Samples with expression values below were considered to have low expression, while values above were considered to have high expression. Kaplan-Meier analysis of patient survival and metastasis-free survival were calculated with WinSTAT (R. Fitch Software). Cell culture, RNA Interference, and Plasmids Primary human fibroblasts, wild type MEFs, and TKO MEFs were cultured in 1% Fetal Bovine Serum in DMEM. TKO MEFs were as described (Sage et al. 23). Two independent sirna duplexes targeting UTX were used as previously described (Lan et al. 27). sigfp duplex has been previously described (Rinn et al. 28). sirnas targeting luciferase, RB, and HBP1 were all purchased from Thermo Scientific Dharmacon. sirnas were either transiently transfected at 5nM with Lipofectamine 2 following manufacturer s protocol (Invitrogen) or 3ug nucleofected following manufacturer s protocol specific to cell type (Lonza). For UTX overexpression, full-length UTX was cloned into MSCV-puro vector. Catalytic mutant UTX was constructed by making a single codon substitution H1126A using Quikchange II XL site directed mutagenesis kit (Stratagene). All plasmids were nucleofected at 3ug each following manufacturer s protocol specific to cell type (Lonza). UTX protein was detected by Western blotting with a rabbit polyclonal antibody (Abcam). Primer sequences for qpcr and qrt-pcr assays are listed in Supplementary Table 3. BrdU Immunofluorescence

4 Wang et al., p. 4 BrdU staining was performed as previously described (Liu et al. 27) with the following modifications: TKO MEFs were incubated with BrdU for one hour. Since wild type MEFs and TKO MEFs had nucleofection efficiencies < 9%, a YFP plasmid was co-nucleofected for these cell lines and only nucleofected YFP+ cells were scored. Serial Cell Count Cells were re-plated at 1% confluence into a 96-well plate one day after plasmid or sirna insertion. Number of viable cells was quantified every 12 hours in triplicate using MTT cell proliferation kit (Roche). C. elegans RNAi Bacterial culture for the UTX-1 RNAi feeding clone was grown for ~7 hours at 37 C in LB medium containing 1μg/ml ampicillin. The bacterium was used to inoculate six-well plates that contained NGM agar, 1 mm IPTG, and 25 μg/ml carbenicillin. About 1 synchronized L3 worms for each strain were placed in the first well, transfered every 24 hours for two days, and maintained at 2 C. The F1 progeny from the last transfer were scored for the Muv phenotype using DIC microscopy. Strains used in this study were: N2, lin-15a(n767), lin-8(n111), lin- 38(n751), lin-56(n2728), lin-35(n745), lin-53(n833), lin-15b(n744), and mys- 1(n475)/nT1[qIs51]. Strains were maintained as described in Brenner (Brenner 1974).

5 Wang et al., p. 5 References Agger, K., Cloos, P.A., Christensen, J., Pasini, D., Rose, S., Rappsilber, J., Issaeva, I., Canaani, E., Salcini, A.E., and Helin, K. 27. UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development. Nature 449(7163): Brenner, S The genetics of Caenorhabditis elegans. Genetics 77(1): Chang, H.Y., Sneddon, J.B., Alizadeh, A.A., Sood, R., West, R.B., Montgomery, K., Chi, J.T., van de Rijn, M., Botstein, D., and Brown, P.O. 24. Gene expression signature of fibroblast serum response predicts human cancer progression: Similarities between tumors and wounds. PLoS Biology 2(2): Issaeva, I., Zonis, Y., Rozovskaia, T., Orlovsky, K., Croce, C.M., Nakamura, T., Mazo, A., Eisenbach, L., and Canaani, E. 27. Knockdown of ALR (MLL2) reveals ALR target genes and leads to alterations in cell adhesion and growth. Mol Cell Biol 27(5): Johnson, D.S., Li, W., Gordon, D.B., Bhattacharjee, A., Curry, B., Ghosh, J., Brizuela, L., Carroll, J.S., Brown, M., Flicek, P. et al. 28. Systematic evaluation of variability in ChIP-chip experiments using predefined DNA targets. Genome Res 18(3): Lan, F., Bayliss, P.E., Rinn, J.L., Whetstine, J.R., Wang, J.K., Chen, S., Iwase, S., Alpatov, R., Issaeva, I., Canaani, E. et al. 27. A histone H3 lysine 27 demethylase regulates animal posterior development. Nature 449(7163): Liu, H., Adler, A.S., Segal, E., and Chang, H.Y. 27. A transcriptional program mediating entry into cellular quiescence. PLoS Genet 3(6): e91. Rinn, J.L., Kertesz, M., Wang, J.K., Squazzo, S.L., Xu, X., Brugmann, S.A., Goodnough, L.H., Helms, J.A., Farnham, P.J., Segal, E. et al. 27. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129(7): Rinn, J.L., Wang, J.K., Allen, N., Brugmann, S.A., Mikels, A.J., Liu, H., Ridky, T.W., Stadler, H.S., Nusse, R., Helms, J.A. et al. 28. A dermal HOX transcriptional program regulates site-specific epidermal fate. Genes Dev 22(3): Sage, J., Miller, A.L., Perez-Mancera, P.A., Wysocki, J.M., and Jacks, T. 23. Acute mutation of retinoblastoma gene function is sufficient for cell cycle re-entry. Nature 424(6945): Segal, E., Friedman, N., Koller, D., and Regev, A. 24. A module map showing conditional activity of expression modules in cancer. Nat Genet 36(1): Subramanian, A., Tamayo, P., Mootha, V.K., Mukherjee, S., Ebert, B.L., Gillette, M.A., Paulovich, A., Pomeroy, S.L., Golub, T.R., Lander, E.S. et al. 25. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 12(43): van de Vijver, M.J., He, Y.D., van't Veer, L.J., Dai, H., Hart, A.A., Voskuil, D.W., Schreiber, G.J., Peterse, J.L., Roberts, C., Marton, M.J. et al. 22. A gene-expression signature as a predictor of survival in breast cancer. N Engl J Med 347(25):

6 Wang et al., p. 6 Supplementary Table 1. Genome-wide location analysis of UTX occupancy in primary human fibroblasts. UTX bound 2459 sites on promoters, defining 1945 unique genes. Columns A-J indicate the chromosomal location, enrichment, false discovery rate, and gene name of promoter sites significantly bound by UTX. A notable subset of UTX-occupied genes is associated with an RB network (column K). Most of UTX-occupied promoters are also occupied by H3K4me2 somewhere along the same promoter (column L); a minority of these promoters exhibit occupancy of H3K27me3 (column M). Supplementary Table 2. Gene Ontology terms of UTX target genes enriched for H3K4me2, H3K27me3, both, or neither histone modifications. UTX target genes with univalent H3K4me2 identify broad functional categories, such as cellular biogenesis and cell cycle. UTX target genes are relatively depleted of co-occupancy with H3K27me3 based on chance alone (see text), and this category includes known H3K27me3 targets, such as HOX genes. In contrast, UTX targets with bivalent H3K27me3 and H3K4me2 are highly enriched for protocadherin genes, leading to striking enrichment of GO terms homophilic cell adhesion and cell-cell adhesion. UTX target genes with neither H3K27me3 or H3K4me2 are strongly enriched for olfactory receptors, leading to striking enrichment of GO terms sensor perception of smell and related terms. We note that while the HOX genes are frequently occupied by UTX in fibroblasts, approximately half of the HOX genes show univalent H3K4me2 while other show H3K27me3. Thus, HOX genes do not emerge as a strong category in this analysis. All GO term enrichments shown have Benjamini Hochberg FDR <.5. Supplementary Table 3. Primer sequences for qpcr and qrt-pcr assays.

7 Wang et al., p. 7 Supplementary Figure 1. (A) UTX occupancy and histone modifications on an olfactory receptor gene cluster across ~2 Mb of human chromosome 11. (B) UTX occupancy and histone modifications on a protocadherin gene cluster across ~7 Kb of human chromosome 5. UTX_1 and UTX_2 are two independent replicates of ChIP-chip experiments done in human fibroblasts from lung and foot, respectively, showing very similar patterns of UTX occupancy. Supplementary Figure 2. Conditional relationship between UTX and EZH2 expression and breast cancer patient outcome. Kaplan-Meier analyses of primary human breast cancer patients stratified by high or low UTX and EZH2 expression are shown. (A, C) UTX level is a significant predictor of survival in tumors with low or normal EZH2 level than in those with high EZH2 level. (B, D) Conversely, EZH2 level is a significant predictor of survival in patients with normal or high UTX levels, but not in patients with low UTX level. Patient numbers: for patients with low EZH2 expression (A) 1 have high UTX expression and 57 have low UTX expression. For patients with high UTX expression (B), 6 have high EZH2 expression and 1 have low EZH2 expression. For patients with high EZH2 expression (C), 69 have high UTX expression and 69 have low UTX expression. For patients with low UTX expression (D), 78 have high EZH2 expression and 57 have low EZH2 expression. Supplementary Figure 3. (A) Little modulation of genes in the UTX-bound RB associated network upon serum stimulation of fibroblasts. Each column is a sample after serum stimulation for the indicated times; each row is a gene. Scale for heat map is shown below. The full dataset was previously reported (Chang et al. 24). While a few genes (such as CCND3, CENPF, PCNA, shown in red) are induced by serum stimulation, most genes, including UTX-dependent

8 Wang et al., p. 8 RBBPs such as HBP1 and RBBP6, are not induced by serum. (B) Modulation of UTX occupancy of RB network genes in a cell-type specific manner. UTX binds the RB network in fibroblasts but not in mouse embryonic fibroblasts (ESCs); in contrast, UTX binds olfactory receptor genes in both cell types.

9 Wang et al., Supplementary Fig.1 A 3. UTX_1 B 3. UTX_1 ChIP/input UTX_2 H3K27me3 ChIP/input UTX_2 H3K27me H3K4me2 5.9 H3K4me2

10 Wang et al., Supplementary Fig. 2 A 1.2 Tumors with Low EZH2 B 1.2 Tumors with High UTX Probability of Survival 1 High UTX Low UTX.2 p = Survival (years) Probability of Survival 1 Low EZH High EZH2.2 p = 3x Survival (years) C 1.2 Tumors with High EZH2 D 1.2 Tumors with Low UTX Probability of Survival Low UTX.4 High UTX.2 p = Survival (years) Probability of Survival 1.8 Low EZH2.6.4 High EZH2.2 p = Survival (years)

11 Wang et al., Supplementary Fig. 3 A hr.25 hr.5 hr 1 hr 1.5 hr 2 hr 3 hr 4 hr 6 hr 8 hr 1 hr 12 hr 16 hr 2 hr 24 hr 36 hr - -ID2 -CCND3 -RBBP5 -CDC2 CENPF PCNA -RBBP6 -RBBP4 -HBP1 -JARID1B -KAT2B B Number of genes bound by UTX RB network genes Olfactory Receptors Fibroblast ESC