www.sciencemag.org/cgi/content/full/1149042/DC1

Supporting Online Material for

Demethylation of H3K27 Regulates Polycomb Recruitment and H2A Ubiquitination

Min Gyu Lee, Raffaella Villa, Patrick Trojer, Jessica Norman, Kai-Ping Yan, Danny Reinberg, Luciano Di Croce, Ramin Shiekhattar*

*To whom correspondence should be addressed. E-mail: ramin.shiekhattar@crg.es

Published 30 August 2007 on Science Express

DOI: 10.1126/science.1149042

This PDF file includes:

Materials and Methods Figs. S1 to S9 Table S1 References

Supporting Online Material

Demethylation of H3K27 regulates polycomb recruitment and H2A ubiquitination

Min Gyu Lee, Raffaella Villa, Patrick Trojer, Jessica Norman, Kai-Ping Yan, Danny Reinberg, Luciano Di Croce, Ramin Shiekhattar

Materials and Methods

Antibodies, plasmids, and cell lines

Anti-dimethyl H3K4 (07030), anti-trimethyl H3K4 (07473), anti-dimethyl H3K9

(07521), anti-trimethyl H3K9 (07442), Anti-dimethyl H3K27 (07452), anti-trimethyl H3K27

(05851), anti-dimethyl H3K36 (07369), anti-dimethyl H3K79 (07366), anti-dimethyl

H4K20 (07747), anti-ubH2A (05678), and anti-Bmi1 (05637) antibodies were purchased

from Upstate/Millipore. The anti-trimethyl H3K36 (ab9050), anti-trimethyl H3K79

(ab2621), anti-trimethyl H4K20 (ab9053), anti-H3 antibodies (ab1791), and anti-SUZ12

(ab12073) were from Abcam Ltd. Anti-Ring1A (sc-28736) and anti-ASH2L were from

Santa Cruz and Bethyl laboratories, respectively. Anti-dimethyl and monomethyl H3K27

were generous gifts from Dr. Thomas Jenuwein. Anti-FLAG antibody (F3165) was from

Sigma. Anti-UTX was generated from rabbit.

Baculoviral expression plasmid encoding FLAG-(His)6-UTX was constructed using

pFastBacHTa vector (Invitrogen) and a UTX cDNA (GenBankTM accession number

AB208795). Its catalytic mutant (FLAG-(His)6-UTX (HE/AA)) and deletion mutant (FLAG-

(His)6-∆TPR, deletion of 1-388 amino acids) were derived from FLAG-(His)6-UTX. The

UTX cDNA and its mutant (UTX (HE/AA)) were also cloned into pFLAG-CMV2 (Sigma)

vector for expression in mammalian cells and a nuclear localization signal (DPKKKRKV)

was added just before a stop codon to maximize nuclear import. Stable cell lines

2

expressing FLAG-UTX or FLAG-WDR5 were generated by cotransfecting HEK (human

embryonic kidney) 293 cells with FLAG-UTX or FLAG-WDR5 constructs and a

selectable marker for puromycin resistance.

The embryonic carcinoma cell line NTERA2 (NT2/D1) was growing in DMEM

medium supplemented with 10% FBS. Cells were treated with 1µM ATRA to induce

differentiation and harvested at 18h, 1 and 2 days.

Affinity purification

Baculoviral recombinant proteins (FLAG-(His)6-UTX, FLAG-(His)6-UTX (HE/AA),

and FLAG-(His)6-∆TPR) were purified by methods previously described for the

purification of recombinant proteins from Sf9 or Sf21 insect cells (1). In brief, insect cells

were disrupted using lysis buffer (20 mM Tris (8.0), 137 mM NaCl, 1.5 mM MgCl2, 1 mM

EDTA, 10% Glycerol, 1% triton X-100) and homogenized fifteen times using a dounce

homogenizer. Cell lysates were incubated with anti-FLAG M2 affinity resin (Sigma).

Affinity chromatography using anti-FLAG resin was performed as previously described

(2). The amounts of recombinant proteins were deduced by comparing their band

intensities with known amounts of BSA on Colloidal Blue-stained gel.

UTX-containing complex (or WDR5-containing compex) was purified from 150–

200 mg nuclear extract isolated from the stable cell lines using anti-Flag M2 affinity resin

as previously described (2). UTX-associated proteins were identified by liquid

chromatography–tandem mass spectroscopy. The amount of UTX in complexes was

determined by comparing with known amounts of BSA on silver- stained gel.

Demethylation assay

The histone demethylase assay was performed as previously described (3). In

brief, peptides (0.1 µg) or bulk histones (4 µg, Sigma) were mixed with the indicated

3

amounts of recombinant proteins or complex in a histone demethylase (JHDM) assay

buffer (50 mM HEPES (pH 8.0), 100 µΜ (NH4)2(SO4)2, 1 mM α-ketoglutarate, 2 mM

ascorbate, 5% glycerol, and 0.2 mM PMSF) in a final volume of 10 µl and incubated at

37°C for 5 - 7 h (unless specifically indicated). The reaction was stopped by adding

SDS-PAGE sample buffer, subjected to SDS-PAGE, followed by Western blot analysis

as previously described (3). Specific antibodies were used to monitor levels of individual

methyl mark.

Histone methyltransferase assay

Reconstituted nucleosomes were incubated with UTX-containing complex (or

WDR5-containing compex) as previously described (4).

Mass spectrometric analysis

Demethylation reaction mixtures were desalted with a C18 ZipTip (Millipore) using a

modified version of an earlier procedure(5). In brief, the ZipTips were equilibrated by

washing with 50% acetonitrile/0.1% trifluoroacetic acid (TFA), followed by washing with

0.1% TFA. The reaction mixture was then applied to ZipTips. The ZipTips were

extensively washed with 0.1% TFA and eluted with 70% acetonitrile/0.1% TFA. The

eluates were analyzed by a MALDI-TOF mass spectrometer at The Wistar proteomics

facility.

For mass spectrometric sequencing, excised bands containing UTX-associated

proteins were processed as previously described (6). MS/MS spectra of the peptides

were interpreted using a program (Proteomics Sequest Browser) developed in the

Harvard Microchemistry Facility.

4

RNA interference, quantitative RT-PCR, and chromatin immunoprecipitation.

RNA interference was performed using small interfering RNAs (siRNA) purchased from

Dharmacon’s siGENOME collection. Small interfering RNA against luciferase (sense

strand, 5’-AACGUACGCGGAAUACUUCGA-3’) was used as a control. In brief, cells are

transfected with siRNAs against luciferase or UTX using Lipofectamine 2000 (Invitrogen)

according to the manufacturer’s instructions. Forty to forty-eight hours later, a second

transfection was performed. Cells were incubated for 90–96 h and harvested about 6 d

after the initial transfection. For chromatin immunoprecipitation, cells were treated with

1% HCHO. For quantitative RT-PCR, total RNA was isolated using the Qiagen RNeasy

kit and reverse-transcribed using Invitrogen’s First Strand Synthesis kit, followed by

double digestion with RNase H and RNase A. Each sample was amplified with

Finnzymes DyNAmo HS SYBR Green qPCR kit using the Opticon2 (MJ Research) and

was analyzed to quantify mRNA levels of both GAPDH and HOX genes using Opticon2

software. Messenger RNA levels of HOX clusters were normalized to GAPDH levels.

The relative mRNA level represents the fold change over the control. Chromatin

immunopurification assay was performed as previously described (7). Chromatin

immunoprecipitates for proteins and methyl marks were amplified by quantitative PCR,

normalized to input, and calculated as % of input. Relative occupancy represents the

fold change in % of input over the control (e.g., siUTX / siLuciferase), and enrichment

levels indicate the fold change over IgG control. The PCR primer and siRNA sequences

are available upon request. Data are presented as the mean ± S.E.M. Where indicated,

statistical p values were determined using a student’s t-test.

B

AHuman UTX (1401aa)

Human UTY (1347aa)

Human JMJD3 (1682aa)

D. melanogaster CG5640 (1136aa)

TPR JmjC

83%

56%

59%

*

r.UTX

r.UTX(H

E/AA)

Mar

ker

250

150

98

64

50

36

22

kDa

Colloidal Blue

C

0 1/4 1 4Incubation (h) 0 1/4 1 4

r.UTX (400 ng) r.UTX (800 ng)

3mH3K27

2mH3K27

H3

Figure S1. UTX specifically demethylates tri- and dimethyl H3K27. (A) Diagrammatic representation of the UTX family members. UTX shows significant sequence identities with human UTY (83%, 1172/1393 residues), human JMJD3 (56%, 384/687 residues) and D. melanogaster CG5640 (59%, 490/827 residues). (B) Colloidal Blue staining of recombinant (r.) UTX and UTX (HE/AA) proteins isolated from Sf9 insect cells. In UT X mutant, histidine and glutamate (amino acid positions 1146 and 1148, respectively) residues were mutated to alanine. These residues are predicted as Fe (II) binding sites and conserved in other JmJC domains. Asterisk indicates a nonspecific polypeptide. (C) Concentration- and time-dependent demethylation of methylated H3K27 by recombinant UTX. Histones were mixed with recombinant UTX (400 or 800 ng) and analyzed at various time points (0, 1/4, 1, 4h).

A

C

B%

inte

nsity

100

80

60

40

20

0

2895 2913 29222904 2931 2940

% in

tens

ity

1mH3K27+UTX

100

80

60

40

20

0

1mH3K27

2970

3mH3K27

2925 2943 29522934 2961

% in

tens

ity

3mH3K27+UTX

100

80

60

40

20

0

% in

tens

ity

100

80

60

40

20

0

Me

2910 2928 29372919 2946 2955

% in

tens

ity%

inte

nsity

100

80

60

40

20

0

2mH3K27+UTX

100

80

60

40

20

0

2mH3K27

Me

Figure S2. UTX removes methyl groups from tri- and dimethyl H3K27 peptides.Synthetic 3mH3K27 (A), 2mH3K27 (B) and 1mH3K27 (C) peptides were incubated with orwithout UTX, and analyzed by a MALDI-TOF mass spectrometry. “Me” indicates a loss ofmethyl group.

CB

A

3mH3K27

2mH3K27

3mH3K9

3mH4K20

FLAG-protein

Transfection

UTXUTX(H

E/AA)

Moc

k

r.∆TPR

− + + ++r.U

TX

3mH3K27

H3*

250

150

98

64

50

36

22

kDa r.∆TPR

Mar

ker

Colloidal Blue

Figure S3. In vivo demethylation of methylated H3K27 by UTX and the effect of TPR domain on UTX activity. (A) Analysis of global levels of histone methyl marks following UTX overexpression. HeLa cells were transfected with expression plasmids encoding a ß -galactosidase (Mock), UTX, or a UTX mutant (UTX-HE/AA). Equal amounts of whole cell lysates were subjected to SDS–PAGE followed by Western blot analysis using antibodies against various methyl marks. (B) Colloidal Blue staining of recombinant (r.) ∆TPR isolated from Sf21 insect cells. (C) Comparison of demethylase activities of recombinant UTX and its deletion mutant (r.∆TPR)). “+” represents ~ 800 ng of recombinant proteins. For data in (C), histones were mixed with recombinant proteins. Reaction mixtures were subjected to SDS-PAGE, followed by Western blot analysis.

A

B

mR

NA

leve

ls o

f UT

X

siLuciferase siUTX

0

0.5

1

1.5

UTX

Actin

siUTX

siLus

ifera

se

Figure S4. Knockdown of UTX by siRNA. (A) Analysis of UTX protein levels following siRNA treatment. HEK293 cells were transfected with siRNA against UTX and harvested. Whole cell extracts were subjected to SDS–PAGE followed by Western blot analysis using antibodies against UTX or actin. (B) Analysis of UTX mRNA levels by quantitative RT-PCR (qRT-PCR) after treatment of HEK293 cells with siRNAs against luciferase or UTX

A

B

kDa

250

150

98

64

50

36

Blot with anti-UTX

NEr.U

TX

UTX

DAPI

anti-UTX

Figure S5. Characterization of anti-UTX antibody and nuclear localization of UTX. (A) Analysis of anti-UTX antibody by Western blotting using nuclear extracts (NE) and r.UTX. (B) Immunostaining showing nuclear localization of endogenous UTX in HeLa cells. HeLa cells were fixed, and stained with anti-UTX antibody. Nuclei were counterstained with DAPI.

NE

f-L3

MB

TL1

f-W

DR

5

IP: anti-FLAG

UTX

L3MBTL1

WDR5

Blot: anti-UTX

Blot: anti-FLAG

f-WDR5Nuclear Extract

P11

0.1 M 0.3 M 0.5 M 1.0 M

DE52

0.1 M 0.35 M

Anti-FLAG (M2) agarose

BA

H3

H3

H2A/H2BH4

Inpu

t

15 17 19 21 23 25 27 29 31 33 35 37

UTX

ASH2L

RBBP5

WDR5

15 17 19 21 23 25 27 29 31 33 35 37 39

CBB staining

f-WDR5: Superose 6

[ H]-Fluorography3

1mH3K4

2mH3K4

3mH3K4

Ponceau-Red

Ponceau-Red

Ponceau-Red

Nat.Nuc.

_

f-L3

MB

TL1

f-W

DR

5

Rec. Nuc.

HKMT assayC D

Western

Figure S6. UTX is present in WDR5-containing complex. (A) Schematic representation for multiple steps used to purify WDR5 protein complexes. Nuclear extracts (NE) were purified from a HEK293 stable cell line expressing FLAG-tagged WDR5 (f-WDR). (B) Analysis of WDR5 protein complexes by Western blotting using anti-UTX antibody. Nuclear extracts and f-L3MBTL1 eluate were used as i nput and a negative control, respectively. (C) Western blot analysis of histone lysine methyltransferase (HKMT) assay using antibodies against various methyl marks. Nucleosomes were reconstituted using recombinant histones and DNA. Reconstituted (Rec.) nucleosomes (Nuc.) were used as substrates and mixed with either f-L3MBTL1 or f-WDR5 eluates. Native (Nat.) nucleosomes (Nuc.) were used as a positive control for western blot analysis. (D) Western blot analysis and HKMT assay of WDR5-containing complex fractionated by Superose 6 gel filtration.

0

1

2

SUZ12 ASH2L 3mH3K4 2mH3K4

Promoter occupancy

siLuciferasesiUTX

qChIP

Rel

ativ

e oc

cupa

ncy

at r

egio

n a

of H

OX

A13

0

1

2

SUZ12 ASH2L 3mH3K4 2mH3K4

siLuciferasesiUTX

qChIP

Rel

ativ

e oc

cupa

ncy

at r

egio

n a

of H

OX

C4

A

B

Figure S7. UTX knockdown does not affect promoter occupancy of PRC2, ASH2L and methylated H3K4. (A and B) Analysis of promoter occupancy of SUZ12 (a PRC2 subunit), ASH2L (a subunit of MLL complex), tri- and dimethyl H3K4 at the region a of HOXA13 (A) and region a of HOXC4 (B) genes. The regions a of HOXA13 and HOXC4 are indicated in Fig. 2, A and B, respectively. For data in [(A) and (B)], HEK 293 cells were treated with siRNAs against luciferase or UTX, and then used for ChIP assay. The representative values of % input for HOXA13 and HOXC4 are as follows: SUZ12 (0.01, 0.07), ASH2L (0.02, 0.06), 3mH3K4 (0.11, 1.19), and 2mH3K4 (4.48, 13.4), respectively.

Re

lativ

eo

ccu

pa

ncy

of

UT

X

0 18 240

1.5

3

7.5

6

4.5

910.5

1213.5

A

Re

lativ

eo

ccu

pa

ncy

of

SU

Z1

2

0 18 24

B

Re

lativ

eo

ccu

pa

ncy

of

3m

H3

K2

7

0 18 24

C

Re

lativ

eo

ccu

pa

ncy

of

3m

H3

K4

0 18 240

1

2

5

4

3

6789

D

RA (hours)

HOXA2 promoter

HOXB3 promoter

Re

lativ

eo

ccu

pa

ncy

of

UT

X

0 18 24

E

RA (hours)0

1.5

3

7.5

6

4.5

910.5

1213.5

Re

lativ

eo

ccu

pa

ncy

of

SU

Z1

2

0 18 24

F

Re

lativ

eo

ccu

pa

ncy

of

3m

H3

K2

70 18 24

G

Re

lativ

eo

ccu

pa

ncy

of

3m

H3

K4

0 18 24

H

0

0.25

0.5

1.25

1

0.75

1.51.75

22.25

0

0.5

1

2.5

2

1.5

33.5

44.5

0

0.25

0.5

1.25

1

0.75

1.51.75

22.25

0

0.25

0.5

1.25

1

0.75

1.51.75

22.25

0

0.25

0.5

1.25

1

0.75

1.51.75

22.25

HO

XA

1re

lativ

em

RN

Ale

vel

0 24RA (hours) 0 24 0 24

I

0

30

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120

90

180210240270

J

0

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60708090

HO

XA

2re

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12141618

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3re

lativ

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9

18212427

HO

XB

1re

lativ

em

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Ale

vel

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9

18212427

HO

XB

2re

lativ

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N

0

1

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4

3

6789

HO

XB

2re

lativ

em

RN

Ale

vel

0 24RA (hours) 0 24 0 24

Figure S8. UTX activates HOX genes during RA-induced differentiation of NT2/D1 cells. (Ato H) UTX occupancy [(A) and (E)], SUZ12 occupancy [(B) and (F)], trimethyl H3K27 levels [(C)and (G)], trimethyl H3K4 levels [(D) and (H)] at HOXA2 and HOXB3 genes were analyzed byquantitative chromatin immunoprecipitation (qChIP) assay. The relative occupancy represents thefold change in % of input over control (see “Materials and Methods”). The values of % input set as1 in panels A-H are as follows: A, 0.0007%; B, 0.033%; C, 0.76%; D, 0.69%; E, 0.0036%; F,0.12%; G, 0.62%; H, 1.28%. (I to N) Analysis of mRNA levels of HOXA1 (I), HOXA2 (J), HOXA3(K), HOXB1 (L), HOXB2 (M), HOXB3 (N) following RA treatment. Data are presented as themean ± SEM of three independent experiments.

0

2

4

12

10

8

6

Enr

ichm

entl

evel

sof

UT

X

OCT4 promoter

Rel

ativ

em

RN

Ale

vel

0

10

20

50

40

30

RA (days) 0 1 2

60

Enr

ichm

entl

evel

sof

3mH

3K27

0

2

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6

RA - +

RA - +0

2

4

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6

Enr

ichm

entl

evel

sof

UT

X

HOXA13 promoter

Rel

ativ

em

RN

Ale

vel

0

0.5

1

2.5

2

1.5

RA (days) 0 1 2

3

RA - +

RA - +

A B

C D

E F

IgGanti-UTX

IgGanti-UTX

IgGanti-3mH3K27

Enr

ichm

entl

evel

sof

3mH

3K27

0

75

150

450

375

300

225

IgGanti-3mH3K27

Figure S9. UTX and trimethyl H3K27 are not involved in the repression of OCT4 and HOXA13during RA-induced differentiation of NT2/D1 cells. (A to D) UTX occupancy [(A) and (B)] andtrimethyl H3K27 levels [(C) and (D)] at OCT4 (left panels) and HOXA13 (right panels) genes wereanalyzed by quantitative chromatin immunoprecipitation (qChIP) assay following RA treatment. Rab-bit IgG was used as a control for ChIP assay. (E and F) Analysis of mRNA levels of OCT4 (E) andHOXA13 (F) following RA treatment. Data are presented as the mean ± SEM of three independentexperiments.

14

Table S1. Summary of mass spectrometric identification of UTX-associated proteins

Protein name Protein GenBank

Accession #

Number of

unique peptides

Total number of

peptides

MLL2, isoform a EAW58034.1 5 10

MLL2, isoform b EAW58035.1 50 137

MLL3, isoform1 NP_067053.1 7 13

NCOA6 NP_054790 7 22

Nedd4-binding protein2

(NEDD-BP2)

AAI26467.1 15 43

UTX CAI40508.1 41 192

PAX-interacting protein 1

(PTIP)

NP_031375.3 21 82

KIAA1558 BAB13384 7 15

Zinc finger 281(ZnF281) NP_036614 19 87

KIAA2032 XP_509637.2 9 44

ASH2L BAA35127.1 3 4

RBBP5 NP_005048 9 30

WDR5 NP_060058 9 57

15

References

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2. R. I. Gregory, T. P. Chendrimada, R. Shiekhattar, Methods Mol. Biol. 342, 33 (2006).

3. M. G. Lee, J. Norman, A. Shilatifard, R. Shiekhattar, Cell 128, 877 (2007).

4. K. Nishioka, D. Reinberg, Methods 31, 49 (2003).

5. Y. Shi et al., Cell 119, 941 (2004).

6. D. A. Bochar et al., Cell 102, 257 (2000).

7. M. A. Hakimi et al., Proc. Natl. Acad. Sci. U S A 99, 7420 (2002).