Percharde et al. 1
Ncoa3 functions as essential Esrrb coactivator to sustain embryonic stem cell self-renewal and reprogramming
Michelle Percharde, Fabrice Lavial, Jia-Hui Ng, Vibhor Kumar, Rute A Tomaz, Nadine Martin, Jia-Chi Yeo, Jesús Gil, Shyam Prabhakar, Huck-Hui Ng, Malcolm G Parker and Véronique Azuara
List of Supplemental Material
1. Figure S1 (related to Figure 1) 2. Figure S2 (related to Figure 2) 3. Figure S3 (related to Figure 3) 4. Figure S4 (related to Figure 4) 5. Figure S5 (related to Figure 5) 6. Figure S6 (related to Figure 6) 7. Figure S7 (related to Figure 6) 8. Figure S8 (related to Figure 6) 9. Table S1 (related to Figure 6) 10. Table S2 (related to Figure 6) 11. Table S3 (related to Figure 6) 12. Table S4 (related to Figure 6) 13. Table S5 (related to Figure 6) 14. Table S6 (list of shRNA and primer sequences used in this study)
Note: Tables S1-S4 contain significantly enriched GO terms for Ncoa3 chIP-seq peaks (FDR q-value<0.05). Table S5 shows significantly altered targets upon Ncoa3 knockdown as identified by microarray (fold-change >1.5). These are available online as separate spreadsheets
Supplemental Materials and Methods
Supplemental References
Percharde et al. 2
Figure S1. Analysis of the Esrrb LBD requirement in ESCs. (A) Diagram indicating Flag-tagged wild-type (Wt) and Esrrb deletion mutants lacking either the
LBD (ΔLBD) or AF-1 (ΔAF-1) domain. (B) Expression of Flag-tagged wild-type (Wt)
Esrrb or deletion mutants in ESCs. (C) Luciferase assays for Esrrb-bound targets.
ESCs were transfected with the indicated ERRE-containing reporter plus either control (shScrambled) or shRNA to endogenous Esrrb (shEsrrb), and with or without
RNAi-resistant Esrrb rescue constructs as depicted in (A). (D) Indicated morphology and Alkaline phosphatase (AP) staining for colonies scored in self-renewal assays, categorized as undifferentiated (>85% AP-positive), mixed (15-85% AP-positive) or differentiated (<15% AP-positive). (E) Structural basis for diethylstilbestrol (DES)- induced ERR inactivation. Crystal structure of the ERRγ LBD complexed with DES
(green), superimposed upon the apoLBD (yellow). DES binding induces a rotational shift in helix 11 (H11), leading to steric clashing of F435 (corresponding to F310 in
Esrrb) with H12 in the agonist position. Adapted with permission from (Greschik et al.
2004). (F) The activity of luciferase reporters from (C) was measured in ESCs following 24 hours treatment with increasing doses of DES (from 3-12.5µM). No significant cell toxicity was noticed within this range of DES concentrations. (G-H) AP staining of ESCs following treatment with DES for 4 days. Colonies were quantified as in Figure S1D. Data are mean +/- SEM of three independent experiments.
Statistical significance of the difference between numbers of undifferentiated colonies in samples compared to ethanol-treated (0µM DES) ESCs was calculated using
Student’s t-test; ** denotes P-values <0.01.
Figure S2. Analysis of PRC and Ncoa3 as putative Esrrb coactivators in ESCs.
(A) Expression profiling of members of the PGC-1 coactivator family in ESCs, as assessed by qRT-PCR and normalised to two housekeeping genes. Expression levels in brown adipose (IMBAT) cells with and without CL-316,243, a β3-AR ligand and inducer of PGC-1α expression (Debevec et al. 2007), are shown as controls. (B) Percharde et al. 3 shRNA-mediated depletion of PRC in ESCs 48 hours following transfection. Data for
(A-B) are mean +/- SEM of three independent biological replicates. ** denotes P- values <0.01 as calculated using Student’s t-test. (C) Activity of Enhancer-Luc reporters in ESCs following cotransfection with shScrambled (control), shEsrrb or shPRC. (D-E) AP staining and quantification of ESCs plated at low density then transfected with shScrambled, shPRC or shEsrrb and selected with Puromycin for 5 days. Data are mean +/- SEM of three independent experiments. Student’s t-test of the difference in undifferentiated colonies in shPRC or shEsrrb compared to shScrambled is shown; *** P-values <0.001. (F) Co-immunoprecipitation (Co-IP) of ectopically expressed Flag-Esrrb Wt or point-mutant forms and Ncoa3 in COS-1 cells. (G) Interaction between endogenous Esrrb and Ncoa3 proteins in ESC extracts, as assessed by Co-IP and western blot. At least two experiments were performed with similar results. (H) Depletion of Ncoa3 protein 48 hours after transfection with siControl or siNcoa3 oligos. Tubulin is shown as a loading control.
(I) Luciferase assays assessing the ability of Wt Esrrb to rescue reporter activity with or without simultaneous Ncoa3 knockdown. Three experiments were performed with similar results.
Figure S3. Crosstalk between Ncoa3, Esrrb, Oct4 and Nanog in ESCs. (A)
Western blot for the indicated proteins in ZHBTc4 ESCs (Niwa et al. 2000) treated with 1µg/mL Doxyclicline (Dox) for 24 hours to delete Oct4. (B) ChIP assays in
ZHBTc4 cells after 24 hours with or without Dox treatment, expressed relative to input. Enrichment in control cells for each individual experiment is set at 100%. Data are mean +/- SD of at least two independent experiments. Dotted lines indicate background enrichment by control IgGs. (C) Western blot in RCNβHB ESCs
(Chambers et al. 2007), treated with 1µM Tamoxifen (OHT) for 24 hours to delete
Nanog. (D) ChIP in RCNβHB ESCs performed after 24 hours with or without OHT treatment. Data are mean +/- SD of at least three independent experiments and are Percharde et al. 4 displayed as in (B). (E-H) Co-IP assays to determine whether Ncoa3 interacts with
Oct4 and Nanog. ESC lysates were immunoprecipitated with (E) anti-Oct4 or (F) anti-
Ncoa3 antibodies, and immunoblotted for Esrrb (top panels) and either Ncoa3 or
Oct4 respectively (bottom panels). Immunoblotting for Esrrb is shown as a positive control for these Co-IPs. (G) ESCs were immunoprecipitated with anti-Nanog antibodies, and immunoblotted for Oct4 or Ncoa3; immunoblotting for Oct4 in the same experiment is used as a positive control. (H) Cell lysates from COS-1 cells transiently cotransfected with either Ncoa3 and Flag-Esrrb (top) or Ncoa3 and Nanog
(bottom) expression vectors were immunoprecipitated using anti-Ncoa3 antibodies
(BDBiosciences, mouse monoclonal #611105), and immunoblotted for Flag-Esrrb
(positive control) or Nanog.
Figure S4. Ectopic expression of Ncoa3 enhances ESC self-renewal. (A) Self- renewal assay of stable ESC lines expressing CAG-Control, CAG-Ncoa3, or CAG-
Nanog vectors. Pooled clones once generated were re-plated at clonal density, and then maintained in absence or presence of LIF as indicated for 5 days. Cells were fixed, stained for AP and counted as in Figure S1D. Data are mean +/- SEM of three independent experiments. Statistical significance of the difference in undifferentiated colonies compared to ESC CAG-Control was calculated using Student’s t-test; * P- values <0.05, ** P-values <0.01. Nanog-overexpressing ESCs have been previously shown to support LIF-independent self-renewal (Chambers et al. 2003; Mitsui et al.
2003) and were used as a positive control. (B) Quantitative expression analysis of
ESC-related genes in stable ESC lines. Data are mean +/- SEM of three independent experiments. (C) Expression profiles of Ncoa3, Esrrb, and two early differentiation markers (Fgf5 and Gata6) in ESC CAG-Control or ESC CAG-Ncoa3 cells during embryoid body (EB)-mediated differentiation.
Figure S5. Ncoa3 is required for somatic cell reprogramming. (A) Microarray expression data (Mikkelsen et al. 2008) showing upregulation of indicated Percharde et al. 5 endogenous factors during reprogramming. (B) Immunofluorescence staining for indicated proteins and GFP in iPSC clone 1. Scale bars, 50µm. (C) Expression of
Ncoa1, Ncoa2 and Ncoa3 in MEFs infected with shScrambled or shNcoa3 #2 measured 3 days after lentiviral transduction, as assessed by qRT-PCR and normalised to two housekeeping genes. Note that the expression of Ncoa3 is over
10-fold lower than in ESCs as shown in Fig. 2A. Data are mean +/- SEM of three experiments. Student’s t-test; * P-values <0.05. ** P-values <0.01. (D) Schematic depicting Ncoa3 knockdown and reprogramming experiments performed in Oct4GFP and p53-/- MEFs. (E) Quantification of BrdU-positive cells in shScrambled or shNcoa3 infected MEFs following a 16-hour pulse-labelling period, performed 3 days after lentiviral infection. Micrographs show representative BrdU-staining (green) and DAPI
(blue) in both cell types. At least 350 cells were counted per sample; scale bars,
25µm. (F) Quantification of nuclei staining positive for cleaved Caspase-3, a marker of apoptosis, in MEFs 3 days after infection with lentiviruses. Data are expressed as numbers of apoptotic cells per 500 cells counted. Micrograph shows example staining for Caspase-3 (red) overlaid with DAPI (blue) upon Staurosporine treatment; scale bar, 50µm. Results for (E-F) are mean +/- SEM of three independent infections.
Figure S6. Ncoa3 ChIP-seq in ESCs. (A) Genome browser screenshots showing binding of Esrrb, Nanog, Ncoa3, Oct4 and Sox2 to the indicated genomic loci. (B)
GREAT Gene Ontology analysis of significantly enriched phenotypes associated with
Ncoa3-Esrrb co-bound targets.
Figure S7. DNA sequence motifs represented at Ncoa3 ChIP-seq peaks. DNA sequences found by MEME with associated E-values, generated from the top 500 sites (100bp wide) of Ncoa3 ChIP-seq peaks. The four top significant motifs (E- values <0.01) are shown. MEME provides an E-value of the found motif, which is a product of P-values. See http://www.sdsc.edu/~tbailey/MEME-protocol- draft2/protocols.html. Percharde et al. 6
Figure S8. Genome-wide analysis of Ncoa3 targets in ESCs. (A) Validation of
Ncoa3 microarray. cDNA generated from the same samples used for the Ncoa3 knockdown microarray was analysed via qRT-PCR for expression changes to the indicated genes. Candidates from class Ia and Ib Ncoa3-bound targets were selected based on their identification in Ncoa3 class Ia or Ib GO analysis, and significant downregulation (P<0.05) upon Esrrb knockdown, as determined by microarray (Feng et al. 2009). Data were normalised to two housekeeping genes and expressed as mean +/- SEM of three biological replicates. Significant expression changes were identified by Student’s t-test, * P-values <0.05, ** P-values <0.01. (B) Spatial heatmap for Class III Ncoa3-bound genes, grouped into two subclusters according to the absence (IIIa) or the presence (IIIb) of H3K4me1. No Polycomb occupancy was detected at either subcluster, represented by Suz12 and Ezh12. The nearest genes within 100Kbp of Ncoa3 peaks in each subcluster were then analysed for significant fold-change (>1.5) upon Ncoa3 knockdown. The proportion of those genes that are either upregulated (red) or downregulated (green) for IIIa and IIIb are displayed as a pie chart. (C) Western blot showing Ncoa3 knockdown by siNcoa3 in lysates used for
Co-IP experiments. (D-E) Co-IP of Esrrb immunoblotted for (D) Esrrb and (E) Oct4 in
ESCs treated for 48 hours with siControl or siNcoa3 oligos. At least two experiments were performed with similar results.
Percharde_GENESDEV/2012/195545_FigS1. A B
1 99 203 433aa Wt Esrrb anti-Flag 48 kDa ΔLBD Esrrb 38 kDa AF-1 Esrrb Δ 25 kDa
C Esrrb Klf4 Nanog Sox2 activity(%) Luciferase Enhancer-Luc shScrambled shEsrrb Wt Esrrb rescue ΔLBD Esrrb rescue ΔAF-1 Esrrb rescue D E F 100 Esrrb H12 ERRb-LUC Klf4-LUCKlf4 80 Nanog-LUCNanog Sox2-LUCSox2 60 undifferentiated undifferentiated activity(%)
40 mixed
Luciferase 20
0 0 2 4 6 8 10 12 14 apoLBD LBD.DES
differentiated differentiated [DES] /µM G H Ethanol 6.25 µM DES 120 ** ** ** 100 undifferentiated mixed 80 differentiated 60 12.5 µM DES - LIF Colonies(%) 40
20
0 0 6.25 12.5 -LIF 1 2 3 4 [DES] /µM Percharde_GENESDEV/2012/195545_FigS2. A B C 0.4 Esrrb Klf4 Sox2 ESC PRC
1.2 IMBAT 120 120 120 0.3 IMBAT + CL 1 100 100 100 0.8 80 80 80 0.2 0.6 60 60 60 ** 40 Expression 0.4 40 40 0.1 Lucactivity(%) 20 20 20 0.2 Relativeexpression 0 Enh-Luc 0 0 0 0 + + + + + + + + + PGC-1α PGC-1β PRC shScrambled + - - + - - + - - Pgc-1a Pgc-1b PRC shEsrrb - + - - + - - + - shPRC - - + - - shRNA + - - + Scrambled PRC shRNA PRCshRNA PRC shRNA PRCshRNA E PRCshRNA
D ERRbshRNA ERRb shRNA ERRbshRNA 120%120 ERRbshRNA Scrambled shRNA ScrambledshRNA Scrambled shRNA ScrambledshRNA *** 100%100 undifferentiated shScrambled shPRC shEsrrb 80%80 mixed differentiated 60%60 40%40 Colonies(%) 20%20 0%0
F G IP: IP: IB: Inputs anti-Flag anti-Ncoa3 IB: anti-Ncoa3 160 kDa 160 kDa
IP: Inputs anti-Ncoa3 IP: IB: anti-Flag IB: anti-Esrrb
48 kDa 48 kDa
H I Esrrb Klf4 Nanog Sox2 140 140 140 140 120 120 120 120 100 100 100 100 Ncoa3 80 80 80 80 60 60 60 60 Tubulin 40 40 40 40 Lucactivity(%) 20 20 20 20 0 0 0 0 Enhancer-Luc +1 +2 3+ 4+ +1 +2 +3 +4 +1 +2 3+ 4+ + + + + shScrambled + - - - + - - - + - - - +1 -2 -3 4- shEsrrb - + + + - + + + - + + + - + + + Wt Esrrb rescue - - + + - - + + - - + + - - + + siControl + + + - + + + - + + + - + + + - siNcoa3 - - - + - - - + - - - + - - - + Percharde_GENESDEV/2012/195545_FigS3. A B [Doxycycline (Dox)] Esrrb Klf4 Nanog Sox2 Inter. - + 120 120 120 120 700 Esrrb Ncoa3 100 100 100 100 600 Ncoa3 500 Esrrb 80 80 80 80 400 60 60 60 60 Nanog 300 Enrichment 40 40 40 40 200 Oct4 20 20 20 20 100 0 0 0 0 0 Tubulin [Dox] - + - + - + - + - + - + - + - + - + - + [-Dox] [-Dox] [-Dox] [-Dox] [-Dox] [+Dox] [+Dox] [+Dox] [+Dox] [+Dox] C D IP:EsrrbIP: Ncoa3 IP:EsrrbIP: Ncoa3 IP:EsrrbIP: Ncoa3 IP:EsrrbIP: Ncoa3 IP:EsrrbIP: Ncoa3 [Tamoxifen (OHT)] - + Esrrb Klf4 Nanog Sox2 Inter. Ncoa3 140 140 140 140 700 120 120 120 120 600 Esrrb Esrrb 100 100 100 100 500 Ncoa3 80 80 80 80 400 Nanog 60 60 60 60 300
Enrichment 40 40 40 40 200 Oct4 20 20 20 20 100 Tubulin 0 0 0 0 0 [OHT] - + - + - + - + - + - + - + - + - + - + IP: [-4OHT]Ncoa3 IP: [-4OHT]Ncoa3 IP: [-4OHT]Ncoa3 IP: [-4OHT]Ncoa3 IP: [-4OHT]Ncoa3
[+4OHT] [+4OHT] [+4OHT] [+4OHT] [+4OHT] E F IP:Esrrb[-4OHT] IP:Esrrb[-4OHT] IP:Esrrb[-4OHT] IP:Esrrb[-4OHT] IP:Esrrb[-4OHT] IP: IP: IB: anti-Esrrb IB: anti-Esrrb
IB:
G H IP: IP: IB: anti-Flag IB: anti-Oct4
48 kDa
IB: anti-Ncoa3 anti-Nanog
37 kDa Percharde_GENESDEV/2012/195545_FigS4.
A [- LIF] [+ LIF] 120 120 * ** 100 100 undifferentiated mixed 80 80 differentiated 60 60
40 40 Colonies(%)
20 20
0 0
B 36 ESC CAG-Control 34 ESC CAG-Ncoa3 32 ESC CAG-Nanog 30 28 266 245 224 203 Relative expression 2 Ncoa3 Nanog Esrrb Klf4 Pou5f1 Rex1 Sox2 1 0 Ncoa3 Nanog Esrrb Klf4 Pou5f1 Rex1 Sox2
C 16 Ncoa3 4 Esrrb 14 12 3 ESC CAG-Control 10 ESC CAG-Ncoa3 8 2 6 4 1 Relative expression Relative expression 2 0 0 0 2 4 6 0 2 4 6
Fgf5 Gata6 350 15 300 250 10 200 150 100 5 50
Relative expression 0 Relative expression 0 0 2 4 6 0 2 4 6 -5 Percharde_GENESDEV/2012/195545_FigS5.
A B Nanog GFP Esrrb Nanog MEF DOX d4 400 6000 d8 300 d12 4000 d16 200 MCV6 Par al iPS Oct4 GFP 2000 B-iPSC GFP 100 MCV8.1 iPSC Probeintensity ESC 0 0
Esrrb GFP
Ncoa1 Ncoa2 Ncoa3 800 800 800 600 600 600 400 400 400 Ncoa3 GFP
Probeintensity 200 200 200 0 0 0
C D 0.025 shScrambled shNcoa3 OSKM plate harvest 0.02 seed shRNA Oct4GFP infection infection onto iPS 0.015 MEFs feeders 0.01 Expression 0.005 * ** -3.25 -3 0 +2 +12-14 0 Time (days) Ncoa1Ncoa1 Ncoa2 Ncoa3 Ncoa3
E F
shScrambled 100 40 35 80 30 60 25 shNcoa3 20
-positive cells -positive 40 15
Apoptotic cells Apoptotic 10 BrdU 20
% 5
0 0 s c h o s h a N S cr shNcoa3 10-100nM shScrambled Staurosporine Percharde_GENESDEV/2012/195545_FigS6. A
Esrrb Klf4
Nanog Sox2
B
embryonic lethality embryonic growth retardation abnormal embryonic growth/weight/body size abnormal embryonic tissue morphology Embryonic development Fertility reduced male fertility reduced fertility Metabolism/proliferation Organogenesis abnormal metabolism abnormal physiological response to xenobiotics decreased cell proliferation
thin myocardial wall abnormal intestinal goblet cells abnormal cardiac muscle morphology microcephaly abnormal head morphology abnormal heart morphology embryonic lethality during organogenesis
1.E-03 1.E-05 1.E-07 1.E-09 1.E-11 1.E-13 Binomial P-value Percharde_GENESDEV/2012/195545_FigS7.
The top motifs found by meme with E-value in top 500 sites (100 bp wide) of Ncoa3. Meme provides E-value of the found motif, which is product of P-values. See http://www.sdsc.edu/~tbailey/MEME-protocol-draft2/protocols.html
Oct-Sox
E-value = 1.5e-006 Esrrb
E-value = 5.7e-004
motif3
E-value = 1.1e-003 motif4
E-value = 2.1e-003
motif5 :
E-value = 1.2e+004
Percharde_GENESDEV/2012/195545_FigS8. A
2 Class Ia shScrambled !!!!! shNcoa3 !!!!!!! 1.5 1 * * 0.5 ** * Relativeexpression ** ** 0 ** Ncoa3 Epha4 Fancc Gadd45a Hsf2 Inhbb Prdm14 Smarcad1 Tgif1
2 Class Ib 1.5
1 * ** * ** 0.5 ** * * * * ** Relativeexpression 0 Esrrb Ccnd3 Klf2 Klf4 Klf5 Oct4 Prdm5 Sall3 Sox2 Tbx3 Tcfap2c Trim24 Zfp42 Zfp57 B
Class IIIa Ezh2 Ezh2 Ncoa3 H3K27ac H3K4me1 Suz12 20% 10% Ncoa3 80% -regulated
IIIa Upregulated Class IIIb Downregulated
4.7% Ncoa3 100% -regulated
IIIb !!!!!!!!!!!!!! "#$%&'(!)*'!+%,-.%+/'&+!01&!-.2++!333!2&'!244'4!516!/1!7&'8#1%+!-.%+/'$9!:1/#-'!/*2/!7&'8#1%+! -.%+/'$!;/1!4'/'&<#5'!-.2++!32!3,!=!33!254!333!>!62+!415'!%+#5$!?*3@A+'B!+#$52.!10!)"+!C+&&,=!D-/E=! F1GH=!:251$=!:&I2H9!!?.2++333!#+!516!4#8#4'4!#5!/61!+%,-.%+/'&+9!J#4/*!10!6#5416!#+!HK,!2&1%54! -'5/'&!;/1/2.!E!L,7>9!)*'!71&/#15!10!$'5'+!;5'2&'+/!6#/*!MNN!L,7>!6*#-*!2.+1!+*16!+#$5#0#-25/!01.4C D ! E -*25$'!15!:-12O!K51-KA4165!#+!2+!+%-*!;! -.2++!MA!MO9IP=! anti-Esrrb -.2++HAMM9I!P l anti-Oct4 !-.2++!O2AMNP!;QNP!%7&'$%.2/'4>= IB: Ncoa3 42 kDa !-.2++O,AE9RP!;!2..!%7&'$%.2/'4> 48 kDa siContro siControl siControl
)*'!7&1,.' 48 kDa 42 kDa siNcoa3 siNcoa3 Percharde et al. 15
Supplemental Table S6. shRNA, primer and probe sequences
shRNA name shRNA sequence / reference
pLKO.1 shScrambled CAACAAGATGAAGAGCACCAA
pLKO.1 shNcoa3 #1 CGGCAGGCACTTGAAATGAAA
pLKO.1 shNcoa3 #2 GCCGATTACAGTGCCACTTTA
pLKO.1 shPRC GAGAGCTGCTAGTGTGGAAA
pSUPER.shScrambled GACGTTAGCAATCGAGCTC
pSUPER.puro-shGFP (Loh et al. 2006)
pSUPER.puro-shEsrrb (Loh et al. 2006)
qPCR primer Primer sequence (5’3’) name
Actin Fw: CGAGGCCCAGAGCAAGAG Rv: CGTCCCAGTTGGTAACAATGC
B2m Fw: TGGTGCTTGTCTCACTGACC Rv: TATGTTCGGCTTCCCATTCT
Ccnd3 Fw: ACCTTTGCGATGTATCCTCC Rv: TTCGATCTGTTCCTGGCAGG
Cdx2 Fw: TCAACCTCGCCACAACCTTCCC Rv: TGGCTCAGCCTGGGATTGCT
Esrrb Fw: GCACCTGGGCTCTAGTTGC Rv: TACAGTCCTCGTAGCTCTTGC
Epha4 Fw: AGATGTGATCAAAGCCATCG Rv: TCTAACAAGGCTGTGTTTGG
Fancc Fw: TGAGAGTCCCAGTGGAGACC Rv: GTGATCTGTACAAGGTCTGG
Fgf5 Fw: TGTGTCTCAGGGGATTGTAGG Rv: AGCTGTTTTCTTGGAATCTCTC
Gadd45a Fw: AAGTGCTCAGCAAGGCTCGG Rv: CACGGATGAGGGTGAAATGG
Gata3 Fw: AGTCGAGGCCCAAGGCACGA Rv: GCTGCCGACAGCCTTCGCTT
Gata6 Fw: GAGCTGGTGCTACCAAGAGG Rv: TGCAAAAGCCCATCTCTTCT
Inhbb Fw: GCGTCTCCGAGATCATCAGC Rv: CTTGACCCGTACCTTCCTCC
Hsf2 Fw: TCAACATGTGTCAGAAGAGG Rv: GGCACAACTTCTGACGATGC
Klf2 Fw: CTTGCAGCTACACCAACTGC Rv: TCCTTCCCAGTTGCAATGAT
Klf4 Fw: GTGCCCCGACTAACCGTTG Rv: GTCGTTGAACTCCTCGGTCT
Klf5 Fw: GGATCTGGAGAAGCGACGTA Rv: TGAGTCCTCAGGTGAGCTTTT
L19 Fw: TGATCTGCTGACGGAGTTG Rv: GGAAAAGAAGGTCTGGTTGGA
Mixl1 Fw: GACAGACCATGTACCCAGAC Rv: GCTTCAAACACCTAGCTTCAG Percharde et al. 16
Nanog Fw: CTTACAAGGGTCTGCTACTGA Rv: TCTGCTTCCTGGCAAGGACC
Ncoa1 Fw: AGATGGAAACCAGGTCAGTAAGA Rv: TCCAGAGTGATCGAATCTGCC
Ncoa2 Fw: ATCTCGAAAATTGCTGACCCTG Rv: TGGCAACACAAAGAGACGCA
Ncoa3 Fw: TCTCAGCGACATCGACAACTT Rv: GCCTGTAGTAAAAGCGGTCCTA
Oct4 Fw: CGTGGAGACTTTGCAGCCTG Rv: GCTTGGCAAACTGTTCTAGCTCCT
PGC-1α Fw: CCCTGCCATTGTTAAGACC Rv: TGCTGCTGTTCCTGTTTTC
PGC-1β Fw: CCACAGCCCACTTCCAGAGA Rv: CTTGCCAAGAGAGTCGCTTTGT
Pou5f1 Fw: CGTGGAGACTTTGCAGCCTG Rv: GCTTGGCAAACTGTTCTAGCTCCT
Prdm5 Fw: GCATGTAAAGCCTTGTTCCG Rv: GTGTGTCTTCTTATGACTCC
Prdm14 Fw: GCATCCTGGTTCCCACAGAG Rv: CTGCAGAACACGCCAAAGTG
PRC Fw: GGCCCAGCACAGGAGTAGTC Rv: CAAGAAAGGAGGCGAGAGAA
Sall3 Fw: AGAAGCCCTTTGGCTGCACC Rv: ATCTCAGAGAACTTGAGAGC
Smarcad1 Fw: CTCTTGTCAACTAAAGCTGG Rv: CTCTTCAATAGTTCCTTGGC
Sox2 Fw: AAAACCACCAATCCCATCCA Rv: CGAAGCGCCTAACGTACCAC
T Fw: GCTTCAAGGAGCTAACTAACGAG Rv: CCAGCAAGAAAGAGTACATGGC
Tbx3 Fw: GAACCTACCTGTTCCCGGAAA Rv: CAATGCCCAATGTCTCGAAAAC
Tcfap2c Fw: GGCTGACTATTTAACGAGACC Rv: CAGTTTTGTATGTTCGGCTCC
Tcfcp2l1 Fw: GAGTCTTGTTTTATCCTCAGC Rv: ACAGCAGCTGACAGTGGTGC
Tgif1 Fw: CCCTCAGAGCAAGAGAAAGC Rv: GGAAATCGTGAACTGATTTGG
Trim24 Fw: AAGCCTGAAGACTTTGTAGC Rv: ACAGTCTTCTGCTTCATGCC
Zfp42 Fw: CTCCTAGCCGCCTAGATTTCCA Rv: CGTGTCCCAGCTCTTAGTCCATT
Zfp57 Fw: GTCAGAAACCTTCAAGAACC Rv: CTGGTGATTAAAGAGGAAGG
ChIP primer name Primer sequence (5’3’)
Esrrb-ERRE Fw: ATAGAACCCACAACGTGACC Rv: AGTGGTCAAAGCCACTCTGC
Klf4-ERRE Fw: TAGATAGCTAAAGCAGGGGG Rv: TGTCTTTGCAACTGGCCTCC
Nanog-ERRE Fw: TCGCCAGGGTCTGGAGGTGC Rv: TCCCACCTGCAGGGTCCACC
Sox2-ERRE Fw: ACTTCTCAACCTCACTGACC Rv: TGGAGTTTGGGAAATTCGACC
Intergenic-Chr.8 Fw: AAGGGGCCTCTGCTTAAAA Rv: AGAGCTCCATGGCAGGTAGA
Percharde et al. 17
DNA Probe Sequence (ERREs are underlined with mutated bases in lowercase; Oct-Sox element is in bold)
Esrrb-Wt CCTGCAGCATCCCTGCTTCAAGGTCAACTGAAAAGCCTGGGCTCCAGCAG
Esrrb-mut CCTGCAGCATCCCTGCTTCAAaaTCAACTGAAAAGCCTGGGCTCCAGCAG
Klf4-Wt TGCTTTTGACCTTGGAGGCCCCAGGGCACTGTTATTTTCC
Klf4-mut TGCTTTTGAttTTGGAGGCCCCAaaGCACTGTTATTTTCC
Nanog-Wt CCAGTCTGGGTCACCTTACAGCTTCTTTTGCATTACAATGTCCATG
Nanog-mut CCAGTCTGaacCACCTTACAGCTTCTTTTGCATTACAATGTCCATG
Sox2-Wt CCACCACTTGCAAGGGCAGGCAGAGGTCGAATTTCCCAAA
Sox2-mut CCACCACTTGCAAaaGCAGGCAGAaaTCGAATTTCCCAAA
Supplemental Material and Methods
Cell Culture. Mouse E14Tg2A ESCs were cultured on 0.1% gelatin-coated plates and maintained in GMEM supplemented with 10% fetal calf serum (FCS), MEM non- essential amino acids, beta-mercaptoethanol, L-glutamine, sodium pyruvate, sodium bicarbonate, penicillin/streptomycin and LIF. Mouse ZHBTc4 (Niwa et al. 2000) and
RCNβHB (Chambers et al. 2007) ESCs have been previously described. iPSCs were generated and maintained in DMEM supplemented with 15% KnockOut serum replacer (GIBCO), beta-mercaptoethanol, L-glutamine, MEM non-essential amino acids, penicillin-streptomycin and LIF. MEF, COS-1 and 293T cells were cultured in
DMEM supplemented with 10% FBS, glutamine and penicillin/streptomycin. PlatE cells were as 293T, supplemented with 1µg/ml Puromycin and 10µg/ml Blasticidin
(Sigma). Where used, diethylstilbestrol (DES, Sigma) or vehicle (ethanol) control was added to the culture medium at the indicated concentration.
Differentiation experiments. For embryoid body (EB)-mediated differentiation,
ESCs were trypsinized to single-cell suspension and 1x105 cells plated per well in
ESC media minus LIF on ultra low attachment 6-well plates (Corning). For retinoid- Percharde et al. 18 mediated differentiation, cells were plated in normal ESC culture conditions, and next day media replaced with ESC media minus LIF containing 1µM all-trans retinoic acid
(Sigma), and cultured for the indicated number of days.
RNAi knockdown. pLKO.1 shRNA vectors targeting Ncoa3, or a scrambled control, were purchased from Sigma and used in ESC and iPSC assays. OnTarget Plus siNcoa3 pool and siControl (Dharmacon) were used in luciferase assays. pSuper.shEsrrb has been described previously (Loh et al. 2006). See Table S6 for shRNA sequences. For Ncoa3 knockdown in ESCs, E14 were plated in 6-well plates and 5 hours later were transfected with 1µg linear pLKO.1 vectors using
Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol. 1µg/ml
Puromycin (Sigma) was added 24 hours post transfection and cells harvested after
96 hours Puromycin selection. For Ncoa3 knockdown in MEFs, lentiviruses harbouring pLKO.1 vectors were produced in 293T cells, concentrated, and viral titer calculated according to GFP fluorescence using pLKO.3G (Addgene). MEFs were seeded in 12-well plates and after 5 hours infections performed. Cells were incubated with viruses for 24 hours before replacement with normal MEF media.
Self-renewal assays. To generate cell lines overexpressing indicated constructs,
E14 ESCs were transfected with the relevant linearized vectors using standard
Lipofectamine 2000 protocols (Invitrogen). 24 hours following transfection,
Puromycin (1µg/ml, Sigma; pPyCAG-IP) or Zeocin (300µg/ml, Invitrogen; pCDNA4/TO) was added. Cells were maintained under selection for 8-9 days, then colonies pooled and cells re-plated at clonal density. The next day, media was exchanged for media minus LIF where indicated and cells maintained for 5 days before fixation and staining for AP using standard protocols (Sigma).
RNA extraction, reverse transcription and real-time quantitative PCR. RNA was extracted using the RNeasy mini kit and treated on-column with DNase I (Qiagen), Percharde et al. 19 then used to generate cDNA according to the Superscript II RT-PCR Kit (Invitrogen).
Gene expression analysis was performed via quantitative PCR using SYBR Green
Master Mix (Sigma) on an ABI StepOne or 7900HT Real-Time PCR machine.
Expression levels were normalized to two housekeeping genes (Actin, L19 or B2m).
See Supplemental Table S6 for a full list of primer sequences used.
Plasmids. Esrrb, Klf4 and Sox2 luciferase reporters have been described previously
(Feng et al. 2009), and Nanog-Luc (Rodda et al. 2005). Nanog-Luc and pSG5.Flag- mEsrrb were kindly provided by P. Robson and V. Cavailles, respectively. Esrrb deletion mutants were generated by substituting full length Esrrb for truncated versions (99-433aa; ΔAF-1 or 1-203aa: ΔLBD) inserted into BamHI and XbaI sites downstream of the Flag-tag. Esrrb point-mutants (C120G/C123G: DBD-mutant,
K259A: AF2-mut1, L424A/F425A: AF2-mut2) were generated using the QuikChange
XL Mutagenesis kit (Stratagene). For stable overexpression in ESCs, cDNAs encoding Esrrb or Ncoa3 were cloned into XhoI and PacI sites in the pPyCAGIP vector (Chambers et al. 2003), and into KpnI/NotI sites in pCDNA4/TO (Ncoa3). Oct4 and Sox2 were subcloned from pMXs into BstXI sites in pCDNA4/TO. Constructs were verified by sequencing and western blot.
Western blotting. For whole cell extracts, cells were scraped in ice-cold RIPA buffer, subjected to two freeze-thaw cycles then incubated with rotation for 20 min,
4°C. Protein concentration was determined using the BCA assay kit (Pierce) and 20-
30µg total protein was prepared in SDS buffer and separated on 4-12% pre-cast
NuPAGE Bis-Tris gels (Invitrogen) then transferred to PVDF membranes (GE
Healthcare). Membranes were probed with primary antibodies in 5% milk-TBST, followed by the appropriate HRP-conjugated secondary antibodies (Dako).
Immunofluorescence. Cells were seeded on gelatin-coated glass coverslips and fixed for immunofluorescence after the indicated number of days with 4% Percharde et al. 20 paraformaldehyde, 10 min. Slides were washed in PBS, blocked and permeabilized at room temperature before incubating overnight at 4°C with the relevant primary antibodies. Next day, secondary antibodies conjugated to Alexa-fluor 488nm, 568nm or 633nm (Invitrogen) were added and incubated for 1 hour at room temperature.
Coverslips were washed and mounted in Vectorshield containing DAPI and slides visualised using a Leica SP5 confocal microscope.
BrdU and Caspase-3 staining. BrdU incorporation and staining was carried out as previously described (Banito et al. 2009), 3 days after lentiviral infection. MEFs were fixed in 4% PFA and staining for cleaved Caspase-3 carried out as for other immunofluorescence experiments. Uninfected MEFs treated with 10-100nM staurosporine for 16 hours to induce apoptosis were stained for Caspase-3 alongside shRNA-transduced cells as a positive control.
ChIP-seq data processing. ChIP-seq was performed on chromatin prepared from mouse ESCs, using an Illumina Genome Analyzer II. The sequenced tags of the
Ncoa3 ChIP and control samples were aligned to mouse genome (version mm8).
Peak detection was performed using MACS with options (P-value=1.0E-5, gsize=1.87E9, mfold=10,30). The peaks for Oct4, Sox2, Esrrb and CTCF were taken from (Chen et al. 2008). Peaks for Nr5a2 were taken from (Heng et al., 2010). In order to compare peak overlaps of Esrrb, Oct4, Sox2 and CTCF within 200bp, binomial tests were used to assess the significance of the observed intersections.
Significantly represented DNA motifs found by MEME were generated from the top
500 sites (100bp) from Ncoa3 ChIP-seq peaks.
Spatial heatmap. For clustering multiple ChIP-seq signals at given bed file, each
ChIP-seq data was first arranged in form of tag-count in 200bp bins. Here the bed file contains centres of Ncoa3 peaks. Log of the tag-count signal was used to make the heatmap plot and clustering. For each ChIP-seq data, the maximum value within 2kb Percharde et al. 21 around each given position in the bed file was taken in one column of a 2- dimensional (n x k) matrix, where n is the number of positions in bed file and k is the number of ChIP-seq libraries. This 2D matrix was provided as input for the K-means clustering program in Matlab to generate classes for each position. After achieving classes, one 2D matrix (n x 2w) was for made for each tag-count data taking values with in w bins around each position as a row of the matrix. Each 2D matrix corresponding to a ChIP-seq library was then sub-plotted using the imagesc function in Matlab.
Gene ontology. For finding the enrichment of terms for genes near selected peaks,
GREAT (McLean et al. 2010) was used such that the whole genome was considered as background (binomial mode). Only the terms with binomial Q-value better than
0.05 were selected from output. Top results are reported here such that multiple redundant terms with same genes are represented by only one term with highest binomial P-value.
Microarray. RNA was obtained from E14 ESCs transfected with shScrambled or shNcoa3 #2 and harvested 3 days after Puromycin selection. Three biological replicates per knockdown condition were isolated. Reverse transcription, amplification, and cRNA biotinylation were performed using Illumina TotalPrep RNA
Amplification Kit according to the manufacturer's instructions. Biotinylated cRNA was then hybridized to an Illumina Mouse WG-6 v2.0 expression beadchip, and analysed as previously described (Feng et al. 2009). Results obtained were rank invariant normalised, and the correlation between Ncoa3 knockdown and Esrrb knockdown (3 days after Puromycin selection) was calculated using previously published microarray data (Feng et al. 2009).
Percharde et al. 22
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