Supporting Online Material for - Science · 9/28/2007 · Min Gyu Lee, Raffaella Villa, Patrick...
Transcript of Supporting Online Material for - Science · 9/28/2007 · Min Gyu Lee, Raffaella Villa, Patrick...
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: [email protected]
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
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1.25
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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
60
150
120
90
180210240270
J
0
10
20
50
40
30
60708090
HO
XA
2re
lativ
em
RN
Ale
vel
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12141618
HO
XA
3re
lativ
em
RN
Ale
vel
L
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15
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9
18212427
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XB
1re
lativ
em
RN
Ale
vel
M
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18212427
HO
XB
2re
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em
RN
Ale
vel
N
0
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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
4
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10
8
6
RA - +
RA - +0
2
4
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10
8
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
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