(a) Analysis method of gene set enrichment pattern18. For each sample (array) genes over- and under- expressed relative to the mean across samples are scored. The fraction of these differentially expressed genes that belong to each of the tested gene sets is then calculated, and its significance over random is estimated, producing a P value. In the second step of the analysis, the over-representation of particular sample groups among the samples enriched for each gene set is assessed. (b) Gene set enrichments in ES cells compared to other cell types. Columns represent individual samples (sample annotations on bottom), sample group names are indicated above. Rows represent individual gene sets (names indicated on left). Red – gene-set enrichment for overexpression, green - gene-set enrichment for underexpression, black – no significant enrichment. (c) Enrichment pattern across sample groups. Numbers indicate P-values for gene set enrichment significance within sample group, in negative log, e.g., 4 symbolizes P=10^-4.

(a) Gene set enrichments in the breast compendium tumors stratified by ER status, tumor size (T Size) or intrinsic subtype. Stratification for the latter parameter was done by employing an expression-profile based classification method35,48. S – small, L – large, Lum – luminal. (b) Samples were divided into six groups representing different combinations of tumor grade and ER-status, and enrichment of gene sets was tested across these groups (left). A similar analysis was performed for grade and tumor size (right). (c) Enrichments in tumors stratified by lymph-node metastasis absence (LN-) or presence (LN+), distant metastasis (Met), or disease-induced mortality (Death). (d) Kaplan-Meier analyses of overall survival in patients included in three of the five studies analyzed. Patients showing both overexpression of the ES exp1 set and underexpression of the PRC2 targets set were labeled as ES (red), those showing the reversed pattern were labeled as Non-ES (blue), and the remainder were labeled as No signature (No sig, pink). P values indicate significance of survival difference between the ES patient group and all other patients.

(a) Gene set enrichment pattern across 157 normal brain and glioma samples41 of various subtypes (bottom). Gene sets subtracted for proliferation genes are indicated as noprol. ODG – oligodendroglioma, AC- astrocytoma, GBM – glioblastoma multiforme. (b) Gene set enrichment pattern across normal bladder samples and grade 2 and 3 transitional cell carcinomas (TCC)42. Invasive TCCs represent a tumor stage more advanced than superficial tumors.

(a) Enrichment pattern of indicated gene sets (rows) across 1,211 breast cancer samples included in 6 profiling studies (columns). Red/green – significantly over- or underexpressed gene sets. Shown are 1,089 tumors for which both ER status and grade annotations were available. Brown bars (bottom) indicate individual tumor annotations for grade, ER status, and tumor size (T Sz), where available. S – tumor smaller than 2cm across (pathological T1), L – tumor larger than 2cm (pathological T2 or T3). (b) Gene set enrichments in the breast compendium tumors stratified by tumor grade.

(a) Enrichment patterns in individual samples of ES cells and other cell types27 of the indicated ES gene sets (as in Fig. 1), and of three Proliferation gene sets: Proliferation Function - genes functionally associated with cell proliferation (compiled from several GO categories), Cycling Genes – genes showing cell-cycle stage-specific expression40, Proliferation Cluster – defined in tumor expression data35. Bar indicates difference in enrichment pattern between ES gene sets and proliferation gene sets in cultured tumor cell lines. (b) Gene set enrichment patterns across grade, ER-status and intrinsic subtypes after subtraction of the Proliferation Function genes from all gene sets. Subtraction of the two other proliferation sets is shown in Supplementary Fig. 2. (c) Enrichment pattern across grade of different subsets of the ES exp1 gene set, based on their cellular function (GO).

(a) Expression pattern of 59 genes encoding ES-associated transcription regulators (rows) across the breast cancer compendium samples (columns), sorted by grade and ER status (indicated in bottom). 12 additional genes in the ES TFs set were not represented in most of the arrays used are therefore not shown. Red/green – two-fold or higher over- or underexpression, respectively. White – missing data. (b) Core set of 9 closely correlated ES transcription regulators, as determined by the pvclust method (Supplementary Figure 4b). (c) Gene set enrichment in the breast cancer compendium samples of the 68 ES-associated transcription regulators (ES TFs), the Core 9 gene subset (Core 9), and top ranking 100 genes in the nearest neighbor expression correlation analysis (NN top 100) – see panel f. Shown are enrichments in individual tumors and in tumors stratified by grade, ER status and intrinsic subtype. Only samples showing enrichment for at least one set are presented. (d) Analysis as in C in glioma samples. NB – normal brain, other annotations as in Figure 5a. (e) Analysis as in C in bladder carcinoma samples. NB – normal bladder, other annotations as in Figure 5b. (f) 1,700 transcription regulators in the human genome were ranked according to the similarity of their expression pattern in the breast cancer compendium to the expression of the Core 9 gene cluster (nearest neighbor analysis). Shown are the top-ranking 100 genes, in rank order (top down).