Supplementary Material to:

The homeobox protein Prox1 is a negative modulator of ERRα/PGC-1α bioenergetic functions by

Alexis Charest-Marcotte, Catherine R. Dufour, Brian J. Wilson, Annie M. Tremblay, Lillian J. Eichner, Daniel H. Arlow, Vamsi K. Mootha and Vincent Giguère

This PDF includes:

Materials and Methods

References

Figure legends for Online Supplemental Figures

Figures S1 to S7

Tables S3 to S5 Materials and Methods

Plasmids. pCMX and pCMX-ERRα were described previously (Laganière 2004). The expression vector pcDNA3/HA-hPGC-1α was provided by A. Kralli (La Jolla, CA). pcDNA3-Prox1 was subcloned from PROX1 cDNA amplified from pCMV-XL6-Prox1 (OriGene Technologies, Rockville, MD). pCMV6-Prox1 DBD mutant was designed according to two amino acid substitution mutations (N625A and R627A) as described previously (Shin, J.W. et al., 2006). pcDNA3-Prox1 NR1/2 mutant, whose LRKLL and ISQLL motifs were mutated to ARKAL and ASQAL respectively, as described previously (Qin, J. et al., 2004) was constructed by PCR site-directed mutagenesis using Pfu polymerase (Stratagene). ERRα, PGC-1α and Prox1 fragments were generated by PCR and the purified products were subcloned in pGEX2T to be bacterially expressed as GST fusion proteins. For luciferase reporters, mouse promoter sequences were obtained from the UCSC genome browser database, and cloned in pGL3 basic from C57BL/6J mouse genomic DNA using high-fidelity PCR. The Cycs and Apoc3-Apoa4 promoter constructs were described previously (Dufour 2007; Carrier 2004) and the Pdk4 (-1377 to +31) and Cs (-2492 to +61) promoter regions were cloned in pGL3 basic. The integrity of all plasmids described was verified by DNA sequencing.

Reporter Assays. HepG2 cells, cultured in DMEM/F12 (Invitrogen) supplemented with 10% fetal bovine serum were transfected using FUGENE 6 (Roche Diagnostics, Mannheim, Germany) in 12-well plates with 300 ng luciferase reporter, 100 ng pCMX- ERRα, 500 ng pcDNA3-PGC1α and 500 ng pCMV6-Prox1 wild-type or indicated mutants (or empty vector) and 1 unit of β-galactose protein (Sigma-Aldrich, G4155). Cells were harvested and assayed for luciferase activity 48 h post-transfection. Luciferase counts were normalized to β-galactose activity. Experiments were performed in duplicate and each experiment was replicated three times.

Coimmunoprecipitation, Immunoblotting and GST Pull-Down Assays. For coimmunoprecipitation studies, livers from wild-type mice were isolated, washed in PBS and homogenized in lysis buffer (sodium phosphate 20 mM, NaCl 150 mM, NP40 1%, EDTA 5 mM, PMSF 1 mM) containing protease and phosphatase inhibitor cocktails (Roche). Liver lysates were incubated at 4°C with rotation for 1 h and centrifuged at 13,000 rpm for 10 min at 4°C. Protein concentration was determined using a Bio-Rad protein assay (500-0006, Bio-Rad Laboratories, Hercules, CA) and 3 mg of lysate for each immunoprecipitation (9 mg of lysate were used for the ERRα and PGC-1α CO-IP) was pre-cleared with protein G-sepharose for 1 h at 4°C with rotation. The supernatants were collected following centrifugation at 7,000 rpm for 10 sec at 4°C and incubated overnight at 4°C with rotation with either anti-ERRα, anti-PGC-1α (rabbit polyclonal IgG, Santa Cruz Biotechnology, sc-13067), anti-Prox1 (rabbit polyclonal IgG, Proteintech Group, 51043-1-AP) or anti-HA tag (rabbit polyclonal IgG, Santa Cruz Biotechnology, sc-805). Subsequently, protein G-sepharose was added to the lysates and the samples were left to rotate at 4°C for 1 h. The immunoprecipitates were washed 5 times with lysis buffer and eluted in loading buffer and boiled for 5 min. The samples were separated on a 7.5% SDS-PAGE gel, transferred onto PVDF membranes (Amersham Biosciences) and blocked overnight at 4°C in TBS-T (Tris-buffered saline and 0.1% Tween) containing 5% skim milk. Membranes were incubated for 1 h with either anti-ERRα (1:10,000), anti-PGC-1α (1:200), anti-Prox1 (1:400) or anti-HA (1:200) antisera diluted in TBS-T containing 5% skim milk. Following 3 washes in TBS- T containing 5% skim milk, the membranes were incubated for 1 h with rabbit IgG TrueBlot (eBioscience, diluted 1:5000 in TBS-T containing 5% skim milk), washed 3 times in TBS-T and subsequently the proteins were detected using Lumi-Light Western Blotting Substrate (Roche).

Co-immunoprecipitation assays in HepG2 cells were done in a similar manner as described above for mouse livers with the exception that 7.8 mg of HepG2 lysate for each immunoprecipitation was used. Input represents 0.5% of the starting material used for the immunoprecipitations.

For Western blot detection to determine the siRNA knockdown efficiencies in HepG2 cells treated with either a control siRNA or an siRNA specific for ERRα or Prox1 75 µg of HepG2 lysates were separated on a 10% SDS-PAGE gel. Immunoblot detection was done using anti-ERRα (1:10,000), anti-Prox1 (1:400, Proteintech Group, 51043-1-AP) and anti-RPLP (1:2000, Proteintech Group, 11290-2-AP).

For GST Pull-Down assays, equal amounts of bacterially expressed GST and either GST- ERRα fragments, GST-PGC-1α fragments or GST-Prox1 fragments immobilized on glutathione sepharose beads were combined with 8 µl of either 35S-labeled ERRα, PGC- 1α or Prox1 proteins produced with the TNT T7 coupled reticulocyte lysate system (Promega Corp., Madison, WI) in 150 µl of GST binding buffer (20 mM Tris, pH 7.5; 100 mM KCl; 0.1 mM EDTA; 0.05% Nonidet P-40; 10% glycerol; 1 mg/ml BSA; 1 mM phenylmethylsulfonyl fluoride; protease inhibitor tablet complete mini (Roche)) for 2 h at 4°C. The beads were washed five times with cold binding buffer, and the immobilized proteins were eluted by boiling in 2x sample buffer. The eluted proteins were resolved on SDS-PAGE, and the fixed and dried gels were visualized by autoradiography.

HDAC activity assay. Three mg of mouse liver lysate was used to immunoprecipitate Prox1 or HA as control similar to that described above for coimmunoprecipitation assays. Subsequently, HDAC activity was measured using a Fluor-de-LysTM-green HDAC fluorometric activity assay kit (Enzo Life Sciences) according to the manufacturer’s instructions.

ChIP and ChIP-on-chip. ChIP assays were performed as previously described (Dufour 2007) with modifications on adult male mouse livers. For Prox1 ChIP-on-Chip experiments, chromatin corresponding to 5 g of initial liver mass taken from a pool of 10 livers was used and pre-cleared chromatin was immunoprecipitated with 32 µg of an anti- Prox1 polyclonal antibody (Proteintech Group, 51043-1-AP) or not (no antibody control) with subsequent addition of 700 µl of a 50% slurry of salmon sperm DNA/protein A beads for 3 h at 4°C. For ERRα ChIP-on-chip experiments, chromatin corresponding to 3.3 g of initial liver mass taken from a pool of 24 livers was diluted in 2.5X ChIP dilution buffer and precleared using 500 µl of a 50% slurry of salmon sperm DNA/protein A beads for 2.5 h at 4°C. Duplicate ERRα and Prox1 ChIP-on-chip experiments were performed. Sample preparation of target enriched and no antibody control material for hybridizations to mouse extended promoter microarrays (Agilent) involves ligation- mediated PCR (LM-PCR) and Cy-dye labeling. First, 55 µl of purified non-diluted enriched and no antibody control material were independently added to a 55 µl mixture containing 11 µl NEB buffer 2 (10X), 0.5 µl BSA (10 mg/ml), 1 µl 10 mM dNTP mix, 0.2 µl T4 DNA polymerase (3U/µl) and 42.3 µl of H2O. The mixtures were incubated at 12°C for 20 min followed by addition of 12 µl of a solution containing 11.5 µl 3 M NaOAc, pH 5.2 and 0.5 µl glycogen (20 mg/ml). The samples were vortexed briefly and 120 µl of phenol/chloroform/isoamyl was added. The samples were vortexed again and centrifuged for 5 min at 13,000 rpm at RT. The upper phase was transferred to a new tube followed by addition of 2 volumes of cold ethanol. The samples were vortexed and precipitated at –80°C for 1 h. The precipitated material was centrifuged at 13,000 rpm, 4°C for 30 min, the pellets were air-dried and then resuspended with a 20 µl mixture containing 2 µl 10X T4 DNA ligase buffer, 6.5 µl annealed linkers (15 µM) (Ren 2000), 0.5 µl 0.1 M ATP, 1 µl T4 DNA ligase (400U/µl) and 10 µl H2O. The resuspended samples were incubated at 16°C overnight. The next day, a 20 µl solution containing 13.5 µl H2O, 4 µl 10X Thermopol buffer, 1.25 µl 10 mM dNTP mix and 1.25 µl of 40 uM oligo oJW102 was added. The samples were initiated to an LM-PCR program consisting of 4 min at 55°C, 3 min at 72°C, 2 min at 95°C, 15 cycles of 30 sec at 95°C, 30 sec at 60°C and 1 min at 72°C, followed by 5 min at 72°C and kept at 4°C until ready. The PCR program was paused once step 1 reached 2 min at 55°C, then a 10 µl solution comprised of 8 µl of H2O, 1 µl of 10X Thermopol buffer and 1 µl TAQ (5U/µl, Invitrogen) was added and the PCR run was continued. The LM-PCR samples were purified by QIAquick Spin kit and eluted twice with 30 µl of elution buffer provided in the kit. The purified enriched and control samples underwent several second round LM-PCRs in order to amplify enough DNA incorporated with aminoallyl-dUTPs required for subsequent labelling and hybridizations. A 35 µl solution containing 26.75 µl H2O, 4 µl 10X Thermopol buffer, 3 µl 5mM dNTP mix containing aminoallyl-dUTP (Sigma, cat# A0410) (5 mM dATP, 5 mM CTP, 5 mM GTP, 1.5 mM dTTP and 3.5 mM aminoallyl- dUTP) and 1.25 µl of 40 uM oligo oJW102 was added to 5 µl of a 5 ng/µl dilution of the amplified enriched and control samples. The samples were initiated to an LM-PCR program similar to that in the first round of amplification except that 18 cycles instead of 15 were used. The PCR program was paused once step 1 reached 2 min at 55°C, then a 10 µl solution comprised of 8 µl of H2O, 1 µl of 10X Thermopol buffer and 1 µl TAQ (5 U/µl, Invitrogen) was added and the PCR run was continued. The LM-PCR samples were purified by QIAquick Spin kit with the following exceptions. The columns were washed using a phosphate wash buffer (5 mM KPO4 pH 8.0 and 80% EtOH) and eluted twice with 35 µl of a phosphate elution buffer (4 mM KPO4 pH 8.0) not provided in the kit. Labelling of the purified samples involved coupling of Cy dyes to the aminoallyl-dUTP incorporated samples. Three tubes each containing 7.5 µg of either the enriched or control samples were dried using a SpeedVac. The samples were resuspended in 4.5 µl 0.1M Na2CO3 pH 9.0. Subsequently, 4.5 ul Cy5 (Amersham, cat# PA25001, resuspended in 73 ul DMSO) and 4.5 µl Cy3 (Amersham, cat# PA23001, resuspended in 73 µl DMSO) were added to the enriched and control samples, respectively. The samples were left at room temperature for 1.5 hr with occasional mixing every 10 min. Next, 35 µl 0.1M NaOAc pH 5.2 was added to the samples and were then purified using a QiaQuick spin kit. The samples were eluted from the columns using 52 µl elution buffer provided in the kit. The eluates from the three Cy5 enriched tubes and Cy3 control tubes were pooled together. The ∼150 µl of Cy5 and Cy3 labelled DNA samples were precipitated using 15 µL 3M NaOAc pH 5.2 and 2 volumes of EtOH at -80°C for 1 h. The samples were centrifuged at 13,000 rpm for 30 min and the DNA pellets were resuspended with 150 µl of H2O and stored at -20°C until hybridized. Samples were hybridized to microarray slides containing ~17,000 of the best-defined mouse transcripts represented as defined by RefSeq spanning from -5.5kb upstream to +2.5kb downstream of the transcriptional start sites (Agilent 244K microarray) according to the Agilent mammalian ChIP-on-chip protocol version 9.2. Following hybridization at 65°C for 40 h, the arrays were washed and scanned using a GenePix 4000B scanner and data was extracted from the images using Agilent Feature Extraction software as described in the Agilent mammalian ChIP- on-chip protocol. Data from duplicate ERRα and Prox1 ChIP-on-chips were normalized and averaged using ChIP Analytics 1.3 software. Data was processed in ChIP Analytics using the following parameters: intra-array Lowess (intensity-dependent) normalization, Whitehead Error Model v1.0 and Whitehead Per-Array Neighbourhood Model v1.0 for peak detection and evaluation. The default parameters were used to identify significant binding events as follows: 1,000 bp maximum distance for 2 probes to be considered as neighbours in a probe set, probe set p-value < 0.001 for a probe in a probe set to be considered bound, and a probe in a probe set needed to pass 1 of 2 possible filters: A) either the centre probe in a probe set had a p-value < 0.001 with at least 1 neighbouring probe with a p-value < 0.1 or B) at least 1 of the neighbouring probes in a probe set had a p-value < 0.005.

For PGC-1α ChIP assays, mouse livers were first homogenized using a polytron homogenizer in cold PBS. The cell pellet was then resuspended in PBS containing 2 mM EGS (ethylene glycol-bis [succinimidylsuccinate], cat# 21565, Thermo Scientific) containing protease and phosphatase inhibitor cocktails (Roche) and left to rotate at room temperature for 25 min to crosslink protein-protein interactions. The cells were washed twice with cold PBS and then resuspended with PBS containing 1% formaldehyde and left to rotate for 12 min to crosslink protein-DNA interactions. The cells were washed twice with cold PBS and ChIP assays were performed in a similar manner to as described above with the following modifications. Liver chromatin corresponding to 2.25 g from a pool of 6 livers was pre-cleared and immunoprecipitated with 40 µg of a polyclonal PGC-1α antibody (sc-13067, Santa Cruz Biotechnology) or not (no antibody control) with subsequent addition of 320 µl of a 50% slurry of salmon sperm DNA/protein A beads for 3 h at 4°C.

Serial ChIP. Re-chip experiments with anti-ERRα or anti-Prox1 antibodies were performed as previously described (Dufour 2007). Quantification of ERRα and Prox1 promoter occupancy was done by calculating ERRα and Prox1 enrichment relative to the initial 10% input sample and normalized against two amplified regions using the control primers, located approximately 4kb upstream of the ERRα and 49 kb upstream of the Prox1 transcriptional start site (Table S4).

Quantitative Real-Time PCR. To assess the enrichment of ERRα and Prox1 at specific promoters identified from the ChIP-on-chip experiments, quantitative PCR (qPCR) was performed using the same ChIP material used for hybridizations as described previously (Dufour 2007). Enrichment of DNA fragments was normalized against two amplified regions using the control primers, located approximately 4kb upstream of the ERRα and 49 kb upstream of the Prox1 transcriptional start site. Specific mouse primers designed and used for ChIP-qPCR analysis are shown in Table S4.

Ingenuity Pathway Analysis of Target Genes. Analysis of the ChIP-on-chip target genes for significant biological pathways and networks were done using Ingenuity Pathways Analysis software (Ingenuity® Systems, www.ingenuity.com). Canonical pathways analysis identified significant pathways from the Ingenuity’s Pathways Analysis library of canonical pathways. Fisher’s exact test was used to calculate a p-value determining the probability that the association between the genes in the dataset and the canonical pathway is explained by chance alone. Networks of genes were algorithmically generated based on their connectivity by overlaying the target genes onto a global molecular network developed from information contained in the Ingenuity Pathways Knowledge Base.

Computational Motif Discovery. Creating datasets: 1 kb of sequence centered on the probe with the highest signal for each bound region of each factor was used. Only bound intervals with P < 10^-5, and only those that are clearly associated with a single TSS (i.e. no bidirectional promoters) were included, to avoid situations where the same genomic sequence gets represented twice. Genes were separated into four categories: bound by ERRα only, bound by Prox1 only, and bound by both ERRα and Prox1 within 1000bp of each other, and not bound (not appearing anywhere in either the ERRα or the Prox1 ChIP-chip results; P > 10^-3). Ambiguous cases were excluded from the analysis, like genes bound by both ERRα and Prox1 but with the binding sites farther than 1000 bp apart. For each of the three bound sets, ERRα only, Prox1 only, and both ERRα and Prox1, matched control (not bound) regions were created. The control regions are carefully matched so that they have the same distribution of distance from the TSS of the nearest gene and the same representation of promoter types (HCP, ICP, and LCP) as the bound regions. There are 5 matched control sequences for each bound sequence.

Finding enriched known motifs: known enriched motifs were searched using MOTIFCLASS software from the CREAD package (3) to test all of the positional weight matrix (PWMs) in TRANSFAC 11.1 for enrichment in the bound sets compared to their matched controls. MOTIFCLASS attempts to find a score cutoff for the PWM that maximizes its enrichment in the bound set compared to the background set, and then computes a P value for the enrichment using a permutation test. All motifs at P < 0.05 corrected for multiple testing are reported as enriched.

De novo motif discovery : All 6-, 7-, 8-, and 9-mer DNA sequences for enrichment in bound regions compared to matched controls were tested, as well as the same k-mer motifs allowing instances to have 1 mismatch, using a feature of motifADE that computes P values for enrichment using the hypergeometric cumulative distribution (4). siRNA and Quantitative Reverse Transcription PCR. HepG2 cells were cultured in DMEM (Invitrogen, cat# 10569-044) supplemented with 10% FBS and pen/strep and maintained at 70% confluency. HepG2 cells were transfected with either control siRNA (5’-CUUCCUCUCUUUCUCUCCCUUGUGA-3’) or On-Target Smartpool control from Dharmacon or specific siRNAs against ERRα (5’- AGAGGAGUAUGUUCUACUAAAGGCC-3’ or On-Target Smartpool from Dharmacon or Prox1 (5’-GUCAAUAAACUGUCCUGGGUCUAGCUC-3’ or On-Target Smartpool from Dharmacon using HiPerfect reagent (according to manufacturer instructions) for 72 h. For quantitative reverse transcription PCR, cDNA was prepared from total RNA isolated from the HepG2 siRNA knockdown samples. cDNA was obtained from 4 ug of total RNA by reverse transcription with Oligo(dT) primer, dNTPs, 5X 1st strand buffer, DTT, RNase inhibitor, and Superscript II RNase H Reverse Transcriptase. cDNA was amplified using specific primers (Table S5) along with the SYBR PCR Master Mix (Qiagen) and a LightCycler instrument (Roche). Relative fold expression levels of the analyzed genes were normalized to HPRT1 levels. Primer efficiences were used in the calculations.

Extracellular Flux (XF) Analysis. HepG2 cells cultured in DMEM (Invitrogen, cat# 10569-044) supplemented with 10% FBS and pen/strep and maintained at 70% confluency were transfected with either control siRNA or siRNA against ERRα or Prox1 and left for 48 h. The cells were then trypsinized, counted and seeded at 30,000 cells/well in XF24-well plates (Seahorse Bioscience) and grown at 37˚C/5% CO2 for 20-24 h. The plates were then washed and incubated for 1 h in a 37˚C incubator under air in 675 µl of assay medium (Seahorse Bioscience, supplemented with 25mM glucose, 2mM Glutamax, 1mM Na pyruvate, pH adjusted to 7.4). For each plate, 7 wells were seeded with HepG2 cells treated with siRNA against either ERRα or Prox1 and 6 wells were seeded with HepG2 cells treated with control siRNA. Concentrated stocks of oligomycin (1 mM, Sigma cat# O4876), FCCP (300 µM, Sigma cat# C2920) and rotenone (1 mM, Sigma cat# R8875) were first prepared in DMSO and diluted 1/100 in assay medium. 75 µl of the test compounds were loaded into the appropriate reagent delivery ports of the measuring sensor cartridge, which was then loaded with an XF24 plate containing a calibration solution of known pH and oxygen concentration. Following a 30 min calibration of the biosensors the plate containing the calibration solution was exchanged for the cell culture plate. Each run consisted of 3 cycles of 2 min sample mixing and 2 min waiting followed by 4 baseline measurement cycles of 3 min sample mixing, 2 min waiting and 3 min measurement of oxygen consumption (OCR) and extracellular acidification (ECAR) rate. Test compounds were added sequentially after baseline readings with 2 measurement cycles following each injection. The OCR and ECAR rates were calculated for each well as a percentage of the average of the third and fourth measurement point of the basal rate just prior to the addition of oligomycin. The rates determined for each well were normalized to cell number determined at the end of the experiment using a haemocytometer. The OCR and ECAR rates at each time point were averaged from 6-7 replicate wells. Experiments were performed in triplicate and data are shown as mean rates +/- SEM.

References

Carrier, J.C., Deblois, G., Champigny, C., Levy, E., Giguere, V. 2004. Estrogen-related receptor α (ERRα) is a transcriptional regulator of apolipoprotein A-IV and controls lipid handling in the intestine. J Biol Chem 279:52052-52058.

Dufour, C.R., Wilson, B.J., Huss, J.M., Kelly, D.P., Alaynick, W.A., Downes, M., Evans, R.M., Blanchette, M., Giguere, V. 2007. Genome-wide orchestration of cardiac functions by the orphan nuclear receptors ERRα and γ. Cell Metab 5:345–356.

Laganière, J., Tremblay, G.B., Dufour, C.R., Giroux, S., Rousseau, F., Giguère, V. 2004. A polymorphic autoregulatory hormone response element in the human estrogen related receptor α (ERRα) promoter dictates PGC-1α control of ERRα expression. J Biol Chem 279:18504-18510.

Qin, J., Gao, D.M., Jiang, Q.F., Zhou, Q., Kong, Y.Y., Wang, Y., Xie, Y.H. 2004. Prospero-related homeobox (Prox1) is a corepressor of human liver receptor homolog-1 and suppresses the transcription of the cholestérol 7-alpha-hydroxylase gene. Mol Endocrinol 18:2424-2439.

Ren, B. et al. (2000). Genome-wide location and function of DNA binding proteins. Science 290:2306-2309. Shin JW, Min M, Larrieu-Lahargue F, Canron X, Kunstfeld R, Nguyen L, Henderson JE, Bikfalvi A, Detmar M, Hong YK. (2006) Prox1 promotes lineage-specific expression of fibroblast growth factor (FGF) receptor-3 in lymphatic endothelium: a role for FGF signaling in lymphangiogenesis. Mol.. Biol. Cell 17:576-584.

Figure legends for Supplemental Figures

Figure S1. Prox1 interacts with and influences the transcriptional activity of ERRα and PGC-1α. Effects of Prox1 on the transcriptional activity of ERRα and PGC-1α. The Cycs and Apoc3-Apoa4 promoters were cloned upstream of the luciferase reporter gene and cotransfected in HepG2 cells with empty vector (-), ERRα, PGC-1α, or a combination of both expression vectors in the presence or absence of Prox1.

Figure S2. Schematic representation of ERRα, Prox1 and ERRα/Prox1 ChIP-on-chip targets associated with biological canonical pathways determined by Ingenuity Pathway Analysis software (Ingenuity Systems, www.ingenuity.com, P < 0.05). ERRα and Prox1 are enriched at promoters of genes involved in common and distinct pathways and can regulate a distinct subset of genes in the same biological process.

Figure S3. Biological processes associated with genes with segments within their extended promoter bound by ERRα, Prox1 or ERRα/Prox1. (A) Pie charts representing the major cellular functions associated with ERRα, Prox1 or ERRα/Prox1 common targets enriched in mouse liver. (B) Comparison between the functionally enriched biological processes associated with the ERRα, Prox1 or ERRα/Prox1 common targets. The biological processes associated with genes with promoter regions that are recognized by ERRα, Prox1 or ERRα/Prox1 were evaluated using Gene Ontology. Target genes specific for ERRα or ERRα/Prox1 were enriched for processes related OXPHOS, carbohydrate and lipid metabolism.

Figure S4. Endogenous Prox1 interacts with ERRα and PGC-1α in HepG2 cells. Lysates from HepG2 cells were subjected to immunoprecipitation with either PGC-1α, ERRα, Prox1 or a non-specific IgG antibody (C) and then subjected to immunoblot analysis with the indicated antibody.

Figure S5. Divergent regulation of genetic programs by ERRα and Prox1. Real-time qRT-PCR was performed on RNA isolated from HepG2 cells treated with control siRNA or siRNA against ERRα (red) or Prox1 (green). The expression of specific and common ERRα and Prox1 target genes was determined and the data is shown as relative fold expression levels compared to control siRNA and normalized to HPRT1 levels. Data represent mean ± s.d of triplicate independent experiments. *P < 0.05.

Figure S6. Divergent regulation of mitochondrial functions by ERRα and Prox1. Cellular oxygen consumption (A) and (B) extracellular acidification rates were measured in intact HepG2 cells treated with either control siRNA or an siRNA Dharmacon On-Target Smartpools against ERRα or Prox1. Rates determined following sequential addition of oligomycin, FCCP and rotenone were taken from an average of 2 measurement readings and are expressed as a percentage of the baseline rates. *P < 0.05. (C) Western blot analysis of ERRα on lysates prepared from the HepG2 knockdown samples is shown with the respective antibodies as indicated. Detection of RPLP was used a control. (D) Western blot analysis of Prox1 on lysates prepared from the HepG2 knockdown samples is shown with the respective antibodies as indicated. Detection of RPLP was used a control.

Figure S7. Lack of HDAC activity associated with immunoprecipitated Prox1 in mouse liver. Prox1 was immunoprecipitated as in Fig. 1A. Control (HA) and Prox1 bead-bound immunoprecipitates were subjected to HDAC activity assay with Fluor-de-Lys kit according to the manufacturer’s instructions.

Supplementary Table 3. Transcription factor enriched binding sites (P < 0.05, Fold ≥ 1.5) in ERRα, ERRα/Prox1 or Prox1 target genes

Factor ERRα ERRα/Prox1 Prox1

SF1 5.4 2.9 NA ERR 5.2 2.8 NA RORα1 2.9 2.2 NA v-ErbA 2.0 1.6 NA COUPTF 1.8 1.5 1.6 HNF4 1.8 2.2 3.1 GCNF 1.8 2.0 NA COUPTF 1.8 1.6 NA COUPTF 1.7 2.2 1.7 PPARG 1.7 1.8 1.5 PPAR 1.6 1.8 1.7 CREB 1.6 NA NA ATF2:c-Jun 1.6 NA NA NF-1 NA 1.7 NA STAT NA 1.6 NA PPARα:RXRα NA 1.5 NA C/EBPβ NA 1.8 2.2 C/EBP NA 2.0 1.8 HNF4α1 NA 2.8 1.7 HLF NA NA 1.7 HNF1 NA NA 1.6 HNF4α NA 2.2 1.6 CDP (Cux1) NA NA 1.5 C/EBPδ NA NA 1.5

Table S4

Mouse primers used for ChIP quantitative PCR analysis.

GENE PRIMER Apoc3-Apoa4 forward 5’-CACACTGACCTCCACCTGTGATC-3’ reverse 5’-GATTACTAACGATAGGTCCAGAGGG-3’

Actc1 forward 5’-CAGGAAGAACCTTGCTGGCTCTCC-3’ reverse 5’-GCTTCCCCAGGGCACAGAGCTTTG-3’

Aldoa forward 5’-CCAAGATTTCCCGACTGAGGTGG-3’ reverse 5’-CCCTTTTCTGTGCTTCTGATGGC-3’

Crsp3 forward 5’-CTCCAAACAGGTCGCAGTTCC-3’ reverse 5’-GGTGCTTACCCGTTACCAGCC -3’

Cs forward 5’-TGTTAAGACGATTCTAGAGCCCGA-3’ reverse 5’-GGTCCAAATATACCTGCATGAGAACAG -3’

Cycs forward 5’-CTAGCATGGTAGGATGTGCCCAGAG-3’ reverse 5’-CACGCACGTCCACGCCTTAC -3’

ERRα control forward 5’-TTGGCATTGATATTGGGGGTGGGAGCAACT-3’ reverse 5’-GACTTCTTACTTTGACGCTTTCCTCCATCG-3’

Fabp1 (ERR) forward 5’-CTGAATGGAGGAGACAGAATGG-3’ reverse 5’-CATCACCCACCTTTCCCTAGAG-3’

Fabp1 (PROX1) forward 5’-GTCCCTCTGCTTCCCTTCCAG-3’ reverse 5’-GAAGTTGCATTGGAATTTTGTTGG-3’

Fgfr3 forward 5’-CCTTCCTTGTCTCCCCCCAAC -3’ reverse 5’-GAGTTCCAGGTCCCAAGTAAAAC-3’

Foxa2 forward 5’-GCCTCGGTGTTTCAAGGTTACTTTTC-3’ reverse 5’-GATCCTCCTGAAGTCATCCCACAAG-3’

G6pc forward 5’-GCCTCCCCCATAGATTGGTTGGTCGG-3’ reverse 5’-CCTCCCCCTCCTGAGTATTGGCATTAC-3’

Gabpa forward 5’-CACCCCGTCTTTTCTGCTTTTCGAGTC-3’ reverse 5’-CTTCTGGGTTTCTTCACGAGGAGAGAG-3’

Igf2 forward 5’-CCCAATGGATGATGAAATCTAAATTGCAG-3’ reverse 5’-GTTTTATGGTCCCCAAAGCACACTG-3’

Mtch2 (ERR) forward 5’-CAGTTCTTCCAGTTGTTAGTCCG-3’ reverse 5’-GTCTGGGCGTGGGCTCCTC-3’

Mtch2 (PROX1) forward 5’-GAATTGACAGTATTTCGCAGTACACG-3’ reverse 5’-CCCCCTCTCTGAACTCACTGAAAGTG-3’ mTor forward 5’-CCCTGTAGTGTAGCGTTTGAAAGCC -3’ reverse 5’-CCAGCACTGAGGGAGAGGCAAG-3’

Nek8 forward 5’-GGGGAAGTACCTCGCCTGTTTTGG-3’ reverse 5’-CCAGCCTGTGCCCTTGGAAGATAG -3’

Nrip1 forward 5’-CAGGGCAACTCACTTGGAAGTGG-3’ reverse 5’-CACAGGTTTAGCAAGTGGGCG-3’

Pck1 forward 5’-CAAATGTTGTGTAAGGACTCACTATGG-3’ reverse 5’-GGCTCGCCTCTGACGTAAGGGG-3’

Pdk4 forward 5’-GGATAGATCCCAGGTCGCTAGG-3’ reverse 5’-GGCTACTGTAAAAGTCCCGCTCTG-3’

Pparg (ERR) forward 5’-CCAGTTCAGGATTGAGTTCACTGTCTAC-3’ reverse 5’-GAATAGCGACCACTTTCATGGCTTATAG-3’

Pparg (PROX1) forward 5’-GGGCTCCAATTTTTTCATTGTGAC-3’ reverse 5’-CCACACAGGTAACAAAATACATTTCTAAGG-3’

Ppargc1a forward 5’-CCAGCCAGACAACCACCCACGG-3’ reverse 5’-GGCTTTGGCAGATCGGATTTGC -3’

Ppargc1b forward 5’-GCTGAAGGTCAGGAGTGCAGAC-3’ reverse 5’-CTTCCAGTCTTTATCCAGCCC-3’

Prkab2 (ERR) forward 5’-GATCCAGCTCCTCTCCTATCC-3’ reverse 5’-GTGAGTTCCAGGACAGCCAGG-3’

Prkab2 (PROX1) forward 5’-CTGATCGATGATCGTGTGCTGATTCACC-3’ reverse 5’-ACATGAGCTAGTCAGTCCATTTGGGC-3’

Prkaca forward 5’-CTCACTCTGACCTTGACCCACAGG-3’ reverse 5’-GGGGAGTTTTGCCACAGCTAAGTG-3’

Prox1 forward 5’-GAGTTATCTTATCTTCTTCAGTCCAC -3’ reverse 5’-GACTGGCTGATCACACCCAC-3’ Prox1 control forward 5’-CCAAGCACAAATATCTAATCACCCTTTC-3’ reverse 5’-CTTCTTGATAGGTTTATGGGTTGGGC-3’

Saa3 forward 5’-CGGGTCATCCAGAAACAAGCTG-3’ reverse 5’-CAGAGAACGTGCTGTGCTGTATTTAGAC-3’

Shc1 forward 5’- CAGCTTCTGTTTCCTCTCTATCCC-3’ reverse 5’- CACTCTCCCAGCCCAATACAAAC-3’

Sirt7 forward 5’-GCACTTTAAGGGAGAAGGCATTAGCAG -3’ reverse 5’-GACTGCATAGGGAGTTCTAGGTCAGCC-3’

Sod2 forward 5’-CCGTCCTCCCCTCCGCTGAT-3’ reverse 5’-CGCTGCTCTCCTCAGAACACG-3’

Supplementary Table 5

Human primers used for qRT-PCR. GENE PRIMER

Aco2 forward 5’-GATCCACGAGACCAACCTGAAGAA-3’ reverse 5’-CCTTCATTCTGTTGAGGGCACTGC-3’

Aldob forward 5’-CAAGGCTGCAAACAAGGAGGC-3’ reverse 5’-GAAGAACCCGTGTGAACATACTGTCC-3’

Aldoc forward 5’-GCCTGTCCCATCAAGTATACCCCAG-3’ reverse 5’-GATGCCTCTTCTTCGCTCTGACC-3’

Atp5g3 forward 5’-CCAATTTCTGCATCAGTGTTATCTCGAC-3’ reverse 5’-GCTGATTGCACTGGTCTGAAACTCC-3’

Atp5l forward 5’-CTGCTGTGACTTACTCGAAGCCTC-3’ reverse 5’-GACATAAAACCACATCAACACCTCAGTG-3’

Cox7a2 forward 5’-GTCAGATTGGGCAGAGGACGATAAG-3’ reverse 5’-GTCACTCCTGCTTCTTGGGAAATG-3’

Cs forward 5’-CAACTCAGGACGGGTTGTTCCAGG-3’ reverse 5’- GTAGTAATTCATCTCCGTCATGCC-3’

Cycs forward 5’-CGTGTCCTTGGACTTAGAGAGTGGG-3’ reverse 5’-GGCTGTGTAAGAGTATCCAGGG-3’

Dlst forward 5’-GTTCAGAACATCGGGAGAAAATGA-3’ reverse 5’-GTTCCTGCAAGGCAAAGGCTGAGG-3’

G6PC forward 5’-GTGGTTGGGATTCTGGGCTGTG-3’ reverse 5’-GATGCTGTGGATGTGGCTGAAAG-3’

Gapdh forward 5’-CCCACTCCTCCACCTTTGACGC-3’ reverse 5’-GGGTCTACATGGCAACTGTGAGG-3’

Gpi1 forward 5’-GTGGGAGGACGCTACTCGCTGTG-3’ reverse 5’-GTCGTGCGGAAGTGCTGGTC-3’

Hprt1 forward 5’-CTAATCATTATGCTGAGGATTTGG-3’ reverse 5’-CTATTCAGTGCTTTGATGTAATCCAGC-3’

Idh3b forward 5’-GATGTGCTTGTGATGCCCAATCTC-3’ reverse 5’-GTGATACTCAAGATTAAGATGCCG-3’ Ldha forward 5’-GACCTACGTGGCTTGGAAGATAAGTGGT-3’ reverse 5’-AATCTGGGTGCAGAGTCTTCAGAGAGAC-3’

Ndufs1 forward 5’-GGTGGAAGCAATTCGGAAGAACC-3’ reverse 5’-CTGGGAGAATAACATCAGCTATGGGAG-3’

Ogdh forward 5’-AGATCATCCGTCGGCTGGAGATGG-3’ reverse 5’-CTTCTCAGAGGACCACTTCCGCTG-3’

Pck1 forward 5’-CTGGGAACACAAACTTGCTGG-3’ reverse 5’-GCTTCCCTGGACAACTTTGGC-3’

Pdk1 forward 5’-CGCACAATACTTCCAAGGAGACCTG-3’ reverse 5’-CAGCCTCGTGGTTGGTGTTGTAATG-3’

Pfkp forward 5’-CGCTACTGTGTCCAACAATGTGC-3’ reverse 5’-CGAAAATGTATGCGGCATCAGCTC-3’

Pgm2 forward 5’-CCATTAGGGACCTTACAACTGGC-3’ reverse 5’-CACAGCTCTGCATAGTACTTGATTTTGG-3’

Suclg1 forward 5’-GTGAAATTGGTGGTAATGCAGAAGAGAA-3’ reverse 5’-TACTGACCACAACTCCTGCACTCTGAAG-3’

Suclg2 forward 5’-GCTGATCCTAAGGTTGAAGCCATC-3’ reverse 5’-TTGGCTGCATCCTCCAGGTCAATG-3’

Sdhd forward 5’-CCACCATTCTGGCTCCAAGGCTGC-3’ reverse 5’-CAGCTTTCTGCAAGGCATCCCCAT-3’