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					                                                                SIGuide.doc Nature 2004-12-27836F


A sumoylation-dependent pathway mediating transrepression of inflammatory response

genes by PPAR

Pascual et. al



Supplementary figures

Figure S1. Efficacy of siRNAs directed against NCoR, HDAC3, HDAC7, PIAS1 and Ubc9.

Efficiency of knockdown was evaluated out in primary macrophages transfected with the indicated

specific and control siRNAs. Protein levels were determined by Western blotting using specific

antibodies.



Figure S2 NCoR and HDAC3 are required for PPAR-mediated transrepression. a, RAW264.7

cells were transfected with the indicated siRNAs and PPAR expression and iNOS-luciferase reporter

plasmids. Cells were treated with LPS and/or Ro as indicated and assayed for luciferase activity 16

hours later. b, Primary macrophages were pre-treated with 10 nM Trichostatin A (TSA) for 1 h

followed by treatment with 1 M Ro for 2h. Cells were stimulated with LPS for 6h and total RNA

was analyzed by Northern Blotting analysis. c, siRNAs directed against HDAC3, but not HDAC7

abolish transrepression by Ro. RAW264.7 cells were transiently transfected with the HDAC3 and

HDAC7 siRNAs and PPAR expression and iNOS-luciferase reporter plasmids. Cells were treated

with LPS and/or Ro as in panel a prior to assay for luciferase activity.



Figure S3 PPAR transrepression does not require sequence specific DNA binding and can be

extended to other LPS-dependent promoters. a, RAW264.7 cells were transfected with flag-tagged

wild type PPAR or flag-tagged PPARCA126/EA127. Ligand-dependent recruitment of tagged PPAR

proteins to the endogenous iNOS and CD36 promoter was determined by ChIP assay using anti-Flag


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                                                                SIGuide.doc Nature 2004-12-27836F

antibody. b, Expression data derived from microarray analysis of primary macrophages treated with

LPS in the presence or absence of the PPAR-specific agonist GW7845. c, ChIP analysis for NCoR

and PPAR on the indicated promoters was carried out using primary macrophages as in Figure 1f.

Figure S4 PIAS1 interacts with PPAR and is required for ligand-dependent inhibition of iNOS

gene activation. a, Schematic diagram of WT-PIAS1 and PIAS1-N isolated from a yeast two-hybrid

screen. NRbox1,2,3, motifs mediating nuclear receptor interaction; SAP, scaffold attachment

protein, first identified as chromatin interaction domain; RING, domain essential for E3 ligase

activity. b, Yeast two-hybrid assay demonstrating interactions between PPAR and PIAS1.

Gal4DBD-T7 and Gal4AD-p53 serve as a positive control. c, The N-terminus of PIAS1 is a

dominant inhibitor of transrepression of the iNOS promoter by PPAR. RAW264.7 cells were co-

transfected with the iNOS-luciferase reporter, PPAR and PIAS1-N expression plasmids as indicated.

36 h post-transfection, cells were treated with Ro and LPS for 6 h prior to assay of luciferase activity.



Figure S5 Sumoylation is required for PPAR transrepression and enhances ligand dependent

association with NCoR. a, RAW264.7 cells were transfected with the iNOS reporter, PPAR

expression plasmid and Ubc9-siRNA. 36 h post-transfection cells were pretreated with Ro for 1 h

and LPS for 6 h prior to assay of luciferase activity. b, Macrophages were transfected with

mammalian two-hybrid reporter, VP-16 PPARWT and either GalDBD-NCoR IDC (C-terminal

nuclear receptor interaction motif) or GalDBD-NCoR IDC (NCoR a.a. 1-2277 without IDC).

Control or PIAS1 siRNA was co-transfected and cells were cultured with 0.01M TSA prior to

treatment with 1M Ro. Cells were assayed for luciferase activity 16 hours after Ro treatment. c,

Macrophages were transfected with mammalian two-hybrid reporter, VP-16 PPARWT and either

GalDBD-HDAC3 or GalDBD. Control or Ubc9 siRNA was co-transfected and cells were cultured




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with 0.01M TSA prior to treatment with 1M Ro. Cells were assayed for luciferase activity 16

hours after Ro treatment.




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