Fig. S1. Validation of ChIP-seq binding sites by single gene ChIP-PCR Fig. S2. Transactivation potential of PPAR

Transcription

1 Fig. S1. Validation of ChIP-seq binding sites by single gene ChIP-PCR ChIP-PCR was performed on PPARγ and RXR-enriched chromatin harvested during adipocyte differentiation at day and day 6 as described in Fig. 1. Thirty out of 31 randomly selected sites displaying both PPARγ and RXR binding in ChIP-seq could be validated by single gene ChIPqPCR. Three out of three randomly selected day 6 PPARγ-only sites were found to display modest occupancy of RXR as well as PPARγ. Positive controls regions (Acox1, Fabp4 and Plin) and a negative control (Sox9 promoter, no gene control region (NC), myoglobin promoter and myoglobin exon 2) were included. Fig. S2. Transactivation potential of PPARγ:RXR binding sites identified by ChIP-seq Promoter or enhancer sequences identified as PPARγ:RXR binding sites in ChIP-seq were cloned into pgl3-basic and pgl3-promoter luciferase-reporter vectors, respectively. Constructs were transiently co-transfected with PPARγ and RXRα expression vectors in NIH-3T3 cells to test for transactivation. Four of the eight (only shown) PPARγ:RXR associated regions supported PPARγ:RXR-mediated transactivation. For comparison, PPARγ:RXR mediated transactivation of p3xppre-tk-luc where three copies of the Acox PPRE in tandem were cloned in front of a minimal thymidine kinase promoter to control luciferase expression. All results are displayed as fold activity of respective vectors without transfected PPARγ and RXR. Results are representative of three independent experiments performed in triplicate. Fig. S3. Biological replicates of PPARγ binding during adipocyte differentiation PPARγ ChIP-PCR was performed on three biological replicates and analyzed as described. Selected target sites adjacent to the following genes were investigated; Acox (chr11: ), Plin (chr7: ), Insig1 (chr: ), Angptl4 (chr17: ), Alas1 (chr9: ), Btd (chr14: ) and Cidec (chr6: ). The data is illustrated relative to day 6 (%) and representative of three biological experiments. Fig. S4. Genome wide mapping of PPARγ:RXR binding sites during adipocyte differentiation (A-D) ChIP-seq was performed on PPARγ and RXR enriched chromatin harvested during adipocyte differentiation (days -6). Target sites were detected using FindPeak peak detection (<.1 FDR). Y-axis indicates the number of annotated tags. ChIP-seq data from days, 1, 2, 3, 4, 6 viewed in the

2 UCSC browser showing PPARγ and RXR binding sites for (A) Abca1, (B) Plin and Pex11a, (C) Pnpla2 and (D) Agpat2. Fig. S6. Efficiency of PPARδ and PPARγ knockdown (A) RNA was harvested from 3T3-L1 preadipocytes (day ) exposed to the indicated shrnai expressing lentivirus and the levels of PPARγ and PPARδ mrna were determined by real-time PCR. Expression levels were normalized to expression of TFIIB. Levels in cells treated with RNAi against LacZ were set to 1. (B) Protein was harvested from 3T3-L1 preadipocytes (day 1) exposed to the indicated shrnai expressing lentivirus. Protein levels of PPARγ were determined by Western blotting using PPARγ specific antibody (sc 7196). Arrows indicate the position of PPARγ1 and -2, respectively. denotes an unspecific protein. Fig. S7. PPARγ:RXR binding and RNAPII activity PPARγ, RXR and RNAPII ChIP-seq were performed and analyzed as described. (A-F) Equalized ChIP-seq data viewed in the UCSC browser for (A) Lpin1, (B) Acsl, (C) Scd1, (D) Dgat2, (E) Taldo1, and (F) Hk2. Y-axis shows the number of annotated tags. Fig. S8. Correlation between RNAPII occupancy, pre-mrna and mrna for genes during differentiation Transcriptional activity was measured as percentage of day (vertical axis) by RNAPII occupancy, and levels of pre-mrna and mrna at day, 1, 2, 3, 4 and 6 during adipogenesis (horizontal axis, in category scale). (A-E) Profiles of two genes for each of the clusters A, B, C, D and E are shown. RNAPII occupancy was obtained from ChIP-seq analyses, and levels of pre-mrna and mrna were measured by real-time PCR using intron-exon and exon-exon spanning primers, respectively. All pre-mrna and mrna levels were normalized to TFIIB mrna and expression at day was set to. Corresponding correlation coefficients for these and additional genes can be found in Table S3.

3 Fig. S9. Knock-down of PPARγ inhibits induction of novel PPARγ target genes early in adipogenesis 3T3-L1 cells were infected with lentivirus expressing shrna against LacZ or PPARγ as described in Fig. 4. RNA was harvested at day and day 2 of adipogenesis and the levels of known and novel putative PPARγ target genes involved in glycerolipid metabolism (Lipe, Fabp4, Acsl1, Agpat2, Gpat3 and Gpd1), glucose metabolism (Hk2, Taldo and Rpia) and regulation of transcription (Srebf1c, Cebpa, and PPARγ), respectively, were determined by real-time PCR. Expression levels were normalized to expression of TFIIB. Expression levels at day in LacZ shrnai expressing cells were set to 1. denotes mrnas significantly induced from day to day 2 (P <, in Student s t-test) Fig. S. Rosiglitazone induces mrna expression of novel putative PPARγ target genes 3T3-L1-adipocytes were treated with the PPARγ specific agonist rosiglitazone (BRL463) or vehicle (DMSO) for 12 h. RNA was harvested, and the levels of known and novel putative PPARγ target genes involved in glycerolipid metabolism (Fabp4, Acsl1, Agpat2, Gpat3 and Gpd1), glucose metabolism (Hk2, Taldo and Rpia) and regulation of transcription (Insig1), respectively, were determined by real time PCR. Expression levels were normalized to expression of TFIIB and mrna levels in vehicle treated cells were set to 1. denotes significantly induced mrnas (P <, in Student s t-test)

4 Figure S1 3,2 RXR RXR Recoveries in % 2,4 1,6,8 Sox9 No gene Myogp Myogex2 Acox1 Plin Fabp4 Aqpat2 Irak2ChIP2 Alas1 Pdk4ChIP1 Chr12confirm Chr13confirm Chr16confirm Obfc2a Slc16a Sfrs3 Samd12 ADAM12 SCD1 Insig1 Prok C7Rik Acsl Pex13 Btd Ltc4s Slc1a Pdk4ChIP3DR1 MoGAT AcslDR2 AcslDR1 Mitch2 ZNF236 Cidec GS2 LPL CD36 Decr Mageb18 Eif1a 2, PPARγ PPARγ Recoveries in % 1, 1,, Negative control regions Sox9 No gene Myogp Myogex2 Acox1 Known PPARγ:RXR binding sites Plin Fabp4 Aqpat2 Irak2ChIP2 Alas1 Pdk4ChIP1 Chr12confirm Chr13confirm Chr16confirm Obfc2a Slc16a Sfrs3 Samd12 ADAM12 SCD1 Insig1 Prok C7Rik Acsl Pex13 Btd Ltc4s Slc1a Pdk4ChIP3DR1 Novel PPARγ:RXR Binding sites MoGAT AcslDR2 AcslDR1 Mitch2 ZNF236 Cidec GS2 LPL CD36 Decr Mageb18 PPARγ "only" binding sites Eif1a

5 Figure S2 Reporter only 2ng mpparγ2 + mrxrα ng mpparγ2 + mrxrα 8 Rel. LUC xPPRE Pdk4 CD36 Lpl Mtch2 Rik493146C7 Enhancers Promoters

6 Figure S3 14 PPARγ ChIP Relative recovery ACO Plin Insig1 Angptl4 Alas1 Btd Cidec 2

7 Figure S4A PPARγ RXR Chr Abca1

8 Figure S4B PPARγ RXR Chr Plin Pex 11a

9 Figure S4C PPARγ RXR Chr Pnpla

10 Figure S4D PPARγ RXR Chr Agpat

11 Figur S

12 A Relative mrna levels Figure S PPARγ PPARδ B PPARγ2 PPARγ1 RNAi: LacZ PPARγ PPARδ PPARγ+PPARδ

13 Figure S7A PPARγ RXR RNAPII Chr Lpin

14 Figure S7B PPARγ RXR RNAPII Chr Acsl

15 Figure S7C PPARγ RXR RNAPII Chr Scd

16 Figure S7D PPARγ RXR RNAPII Chr Dgat

17 Figure S7E PPARγ RXR RNAPII Chr Taldo

18 Figure S7F PPARγ RXR RNAPII Chr Hk2

19 Axis Title Figure S8 A B Cdkn2a Actb Chart Title Akt Tuba1a RNAPII pre-mrna mrna C D Rps Lpl Eif Pparg E Pgk1 6 4 Rpia

20 Figure S9 A LacZ RNAi D LacZ RNAi D2 Pparγ RNAi D Pparγ RNAi D2 Lipe Fabp4 Acsl1 Agpat2 Gpat3 Gpd1 B 8 7 LacZ RNAi D LacZ RNAi D Pparγ RNAi D Pparγ RNAi D2 1 Hk2 Rpia Taldo Srebf1c Cebpa Pparγ

21 Figure S DMSO 1µM Rosiglitazone 1. Fabp4 Acsl1 Agpat2 Gpat3 Lipe Gpd1 Hk2 Rpia Taldo Insig 1