Wang et al., p. 1 

Supplementary Methods

ChIP-chip and data analysis

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

al. 2007) 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. 2007;

Issaeva et al. 2007; Lan et al. 2007). Genomic DNA retrieved by ChIP was hybridized to arrays

tiling human promoters (Roche Nimblegen, Madison, WI) as described (Rinn et al. 2007), and

ChIP target genes were defined as having one or more peaks with a false discovery rate (FDR) <

0.20, a parameter which has been empirically shown to better match predefined positive controls

(Johnson et al. 2008). 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, www.ingenuity.com). 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

(http://david.abcc.ncifcrf.gov/). Array data are available at GEO

(http://www.ncbi.nlm.nih.gov/geo/) and Stanford Microarray Database (http://smd.stanford.edu/)

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. 2004). Briefly, for each

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 50 genes comprised of UTX plus its genomic targets shown in

Fig. 2A over that expected by chance alone (p < 0.05, FDR < 0.05, 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 (http://robotics.stanford.edu/~erans/cancer/experiment_attributes.html). For

each annotation, we compared the frequency of UTX target induction or repression among the

samples versus that expected by chance alone (p < 0.05, FDR < 0.05, 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. 2002) 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 30% 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. 2005). Kolmogorov–Smirnov

scanning statistics were calculated in the observed data versus 1000 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

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 0

were considered to have ‘low’ expression, while values above 0 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 10% Fetal Bovine

Serum in DMEM. TKO MEFs were as described (Sage et al. 2003). Two independent siRNA

duplexes targeting UTX were used as previously described (Lan et al. 2007). siGFP duplex has

been previously described (Rinn et al. 2008). siRNAs targeting luciferase, RB, and HBP1 were

all purchased from Thermo Scientific Dharmacon. siRNAs were either transiently transfected at

50nM with Lipofectamine 2000 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

Wang et al., p. 4 

BrdU staining was performed as previously described (Liu et al. 2007) with the following

modifications: TKO MEFs were incubated with BrdU for one hour. Since wild type MEFs and

TKO MEFs had nucleofection efficiencies < 90%, 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 10% 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 100μg/ml ampicillin. The bacterium was used to inoculate six-well plates

that contained NGM agar, 1 mM IPTG, and 25 μg/ml carbenicillin. About 10 synchronized L3

worms for each strain were placed in the first well, transfered every 24 hours for two days, and

maintained at 20°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(n4075)/nT1[qIs51]. Strains were maintained as described in Brenner (Brenner 1974).

Wang et al., p. 5 

References

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Brenner, S. 1974. The genetics of Caenorhabditis elegans. Genetics 77(1): 71-94. 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. 2004. Gene expression signature of fibroblast serum response predicts human cancer progression: Similarities between tumors and wounds. PLoS Biology 2(2): 206-214.

Issaeva, I., Zonis, Y., Rozovskaia, T., Orlovsky, K., Croce, C.M., Nakamura, T., Mazo, A., Eisenbach, L., and Canaani, E. 2007. Knockdown of ALR (MLL2) reveals ALR target genes and leads to alterations in cell adhesion and growth. Mol Cell Biol 27(5): 1889-1903.

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. 2008. Systematic evaluation of variability in ChIP-chip experiments using predefined DNA targets. Genome Res 18(3): 393-403.

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. 2007. A histone H3 lysine 27 demethylase regulates animal posterior development. Nature 449(7163): 689-694.

Liu, H., Adler, A.S., Segal, E., and Chang, H.Y. 2007. 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. 2007. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129(7): 1311-1323.

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Sage, J., Miller, A.L., Perez-Mancera, P.A., Wysocki, J.M., and Jacks, T. 2003. Acute mutation of retinoblastoma gene function is sufficient for cell cycle re-entry. Nature 424(6945): 223-228.

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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. 2002. A gene-expression signature as a predictor of survival in breast cancer. N Engl J Med 347(25): 1999-2009.

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 <0.05.

Supplementary Table 3. Primer sequences for qPCR and qRT-PCR assays.

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 ~700 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) 100 have ‘high’ UTX expression and 57 have ‘low’ UTX expression.

For patients with high UTX expression (B), 60 have ‘high’ EZH2 expression and 100 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. 2004). While a few genes (such as CCND3, CENPF,

PCNA, shown in red) are induced by serum stimulation, most genes, including UTX-dependent

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.

2.500

0.000

5.090

0.000

H3K4me2

H3K27me3

log2

(rat

io)

log2

(rat

io)

3.000

0.000

3.000

0.000

UTX_2

UTX_1

log2

(rat

io)

log2

(rat

io)

Wang et al., Supplementary Fig.1

2.500

0.000

5.330

0.000

H3K4me2

H3K27me3

log2

(rat

io)

log2

(rat

io)

3.000

0.000

3.000

0.000

UTX_2

UTX_1lo

g2 (r

atio

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g2 (r

atio

)

A BC

hIP

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t

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0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20

Prob

abilit

y of

Sur

viva

l

Survival (years)

Tumors with Low EZH2

Low UTX

High UTX

p = 0.001

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20

Prob

abilit

y of

Sur

viva

l

Survival (years)

Tumors with High EZH2

Low UTX

High UTX

p = 0.240

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20

Prob

abilit

y of

Sur

viva

l

Survival (years)

Tumors with Low UTX

High EZH2

Low EZH2

p = 0.24

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20

Prob

abilit

y of

Sur

viva

l

Survival (years)

Tumors with High UTX

High EZH2

Low EZH2

p = 3x10-7

BA

DC

Wang et al., Supplementary Fig. 2

0

10

20

30

40

50

60

70

80

90

RB

FibroblastESC

RB network genes Olfactory Receptors

49

0

65

81

Num

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ound

by

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Wang et al., Supplementary Fig. 3

B

A

0.25 0.5 1 2.0 4.0

-2 -1 0 1 2

0 hr

0.25

hr

0.5

hr1

hr1.

5 hr

2 hr

3 hr

4 hr

6 hr

8 hr

10 h

r12

hr

16 h

r20

hr

24 h

r36

hr

-RBBP6-RBBP4

-RBBP5

-HBP1

CENPFCDC2--

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-

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