PARP-1 Attenuates Smad-Mediated Transcription Peter Lönn, Lars P. van der Heide, Markus Dahl, Ulf Hellman, Carl-Henrik Heldin, Aristidis Moustakas  Molecular Cell  Volume 40, Issue 4, Pages 521-532 (November 2010) DOI: 10.1016/j.molcel.2010.10.029 Copyright © 2010 Elsevier Inc. Terms and Conditions

Molecular Cell 2010 40, 521-532DOI: (10.1016/j.molcel.2010.10.029) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 PARP-1 Forms Complexes with Smad4 (A–C) Pull-down assays with GST and GST-Smad4 (S4) proteins (A) and endogenous PARP-1 from 293T cells (B) or with 200 ng of recombinant human PARP-1 (C). A nonspecific protein is marked (∗) on the Coomassie-stained gel. Input (5%) 293T total cell lysate (TCL) was immunoblotted (IB). (D) GST-Smad4 (S4) forms complexes with a DNA-binding mutant of Myc-PARP-1 expressed in 293T cells. The protein complex was unaffected by the presence of 50 μg/ml EtBr. The input (5% of TCL) shows the expression levels of Myc-PARP-1 mutant C21, 125G. Recombinant proteins are shown after Coomassie staining. (E and F) Pull-down assays with GST-Smad4 (S4) MH1, MH2, linker+MH2 (L+MH2) proteins (E), and Myc-PARP-1 expressed in 293T cells (F). Input (5% of TCL) was immunoblotted. (G) Immunofluorescence microscopy of endogenous Smad4 and PARP-1 prior to or after stimulation of HaCaT cells with 2.5 ng/ml TGF-β for 1 hr. Merged pictures and nuclear (DAPI) staining are also shown (scale bar, 10 μm). (H and I) CoIP of endogenous PARP-1 with endogenous Smad4 (H) and of endogenous Smad4 with endogenous PARP-1, Smad2, and Smad3 (I) with or without TGF-β stimulation (5 ng/ml, 0.5 hr) in HaCaT cells. A nonspecific protein is marked (∗) in (H). Molecular size markers are shown (A and E). See also Figure S1. Molecular Cell 2010 40, 521-532DOI: (10.1016/j.molcel.2010.10.029) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Smad2, Smad3, and Smad4 Interact with PARP-1 (A) Pull-down assay with the indicated GST fusion proteins and Myc-PARP-1 from 293T cells. Inputs of the cellular (5% of TCL) and recombinant proteins are shown. (B) GST-Smad3 (S3) forms complexes with a DNA-binding mutant of Myc-PARP-1 expressed in 293T cells. The protein complex was unaffected by the presence of 50 μg/ml EtBr. The input (5% of TCL) shows the expression levels of Myc-PARP-1 mutant C21, 125G. Recombinant proteins are shown after Coomassie staining. (C) Pull-down assay with the indicated GST fusion proteins and Myc-PARP-1 from 293T cells with or without cotransfected Flag-Smad2 or Flag-Smad3. Cellular (5% of TCL) and recombinant protein inputs, a nonspecific protein (∗), and molecular size markers are shown. (D) CoIP of endogenous Smad2 and Smad3 with PARP-1 from nuclear HaCaT extracts with or without TGF-β stimulation (5 ng/ml, 0.5 hr). PARP-1 immunoblot of total nuclear cell lysates (NCL) serves as loading control. (E) CoIP of stoichiometric complexes of Flag-Smad2/Smad3/Smad4 with endogenous PARP-1 from 293T cells after TGF-β stimulation (2.5 ng/ml) for the indicated times. (F) Diagram of GST-fused human PARP-1 with its domains, DNA-binding domain (DBD), nuclear localization signal (NLS), BRCA1 C-terminus (BRCT)/automodification domain (AMD), and catalytic domain (CD). Fragments A–F with their amino acid coordinates are listed. Purified GST-PARP-1 fragments are shown after Coomassie staining. (G–J) GST pull-down assays demonstrate specific binding of Myc-Smad4 to the D fragment of GST-PARP-1 (G) and of 6 × Myc-Smad3 to the A, B, and D fragments of GST-PARP-1 (I). The 293T input (5% TCL) shows transfected levels of Myc-Smad4 and 6 × Myc-Smad3. GST pull-down assays in the presence of EtBr demonstrate direct protein-protein interactions in the absence of contaminating DNA (H and J). The assays were performed as in (G) and (I) in the absence or presence of 50 μg/ml EtBr. In (H) and (J), a − indicates pull-down with GST protein alone. Molecular Cell 2010 40, 521-532DOI: (10.1016/j.molcel.2010.10.029) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 PARP-1 PARylates Smad3 and Smad4 (A and B) PARylation of endogenous Smads. Immunoprecipitation of endogenous Smad2/Smad3 from HaCaT cells stimulated with or without 5 ng/ml TGF-β for 30 min. Anti-PAR immunoblot indicates the PARylated Smad species. Immunoblots with anti-Smad2/3 and anti-PARP-1 antibodies indicate cellular protein levels. Proteasomal inhibition with 25 μM MG-132 does not significantly alter the degree of Smad PARylation (A). Combined silencing of PARP-1 with siRNA and 3-AB abolishes the PARylated endogenous Smad species (B). (C) PARP-1 directly PARylates Smad3 and Smad4, but not Smad2. Recombinant PARP-1 incorporates 32P-β-NAD into PAR-chains on the indicated GST fusion proteins, as analyzed after GST pull-down. Recombinant protein input levels stained with Coomassie and molecular size markers are shown. (D) C-terminally phosphorylated Smad3 (p-Smad3/p-S3) is a better PARylation substrate than unphosphorylated Smad3. Autoradiography based on incorporation of 32P-β-NAD into PAR-chains on the indicated GST and His-tag fusion proteins by recombinant PARP-1. GST-Smads alone or after formation of complexes with the indicated His-tagged Smads were introduced to the in vitro PARylation reaction. Input levels of recombinant proteins are shown after Coomassie brilliant blue staining. Note the difference in size of the GST-fused versus the 6× His-tagged Smads. Molecular size markers are shown. (E) Smad3 PARylation is inhibited by 3-AB. Autoradiography of 32P-PAR chains incorporated into GST-Smad3 by PARP-1 in the absence or presence of 1 mM 3-AB. (F) PARP-1 PARylates the Smad3 MH1 domain. Autoradiography of 32P-PAR chains incorporated into full-length (FL) and deletion (Δ) mutants of GST-Smad3 by PARP-1. (G) Exon 3 (TID) of Smad2 interferes with PARylation of Smad2. Autoradiography of 32P-PAR chains incorporated into full-length (FL), insertion (+), and deletion (Δ) mutants of GST-Smad2 or GST-Smad3 by PARP-1. Molecular size markers are shown. See also Figures S2 and S3. Molecular Cell 2010 40, 521-532DOI: (10.1016/j.molcel.2010.10.029) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 PARP-1 Regulates the Residence of Smad Complexes on DNA (A) TGF-β induces a nuclear Smad3/Smad4/PARP-1 complex that binds to SBE DNA and gradually dissociates with time. Biotinylated 4 × CAGA oligonucleotide affinity precipitates (DNAp) endogenous PARP-1, Smad3, and Smad4 from HaCaT cells stimulated with 5 ng/ml TGF-β for the indicated time points. Total protein levels are shown along with α-tubulin (α-Tub) that serves as loading control. (B) Endogenous PARP-1 silencing prolongs the residence of the Smad3/Smad4 complex on SBE DNA. DNAp experiment performed as in (A), except that HaCaT cells were transfected with control or PARP-1-specific siRNA prior to stimulation with TGF-β. (C) DNAp of the endogenous Smad3/Smad4 complex from HaCaT cells after stimulation with 5 ng/ml TGF-β for the indicated time periods in the presence of vehicle (DMSO) or 5 mM 3-AB. Total cell lysates show the expression levels of the proteins. Note the enhanced signal of SBE-bound Smad4, p-Smad3, and Smad3 at the 3 hr time point. (D and E) ChIP assay for endogenous phospho-Smad3 (p-Smad3) from HaCaT cells indicates enhanced recruitment of this protein to the proximal PAI-1 (D) or SnoN (E) enhancers after stimulation with TGF-β for 1 hr and further increases after PARP-1 depletion. Control chIPs with rabbit preimmune IgG are also shown. The chIP input levels are shown in Figures S4B and S4C. Mean values from triplicate quantitative PCR determinations and SD are shown. Stars indicate statistical significance, p < 0.05. Each assay was repeated at least two or three times. See also Figure S4. Molecular Cell 2010 40, 521-532DOI: (10.1016/j.molcel.2010.10.029) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 PARP-1 Dissociates Smad Complexes from DNA (A) Binding of Smad3 to CAGA SBE DNA enhances Smad3 PARylation by PARP-1. Autoradiography of 32P-PAR chains incorporated into the protein complex of GST-Smad4 and His-Smad3 by PARP-1. The indicated DNA oligonucleotides were added to the reaction, and the PARylated proteins were analyzed without prior pull-down. DNA oligonucleotide sequences are shown in the Supplemental Experimental Procedures. GST protein input levels and molecular size markers are shown. (B) PARP-1-dependent catalysis of β-NAD dissociates the Smad3/Smad4 complex from the CAGA SBE. Endogenous Smad3/Smad4 complex was affinity purified from HaCaT cell extracts using the CAGA SBE (DNAp), recombinant PARP-1 with buffer and cofactors was added to the DNA-bound protein complex (PAR), and finally a second affinity purification (DNAp) was performed. In addition to Smad3 and Smad4, phospho-Smad3 (p-Smad3) in the complex is shown. (C) Dissociation of phospho-Smad3 from DNA is eliminated after inhibiting PARP-1 with 3-AB. Experiment performed as in (B), except that 1 mM 3-AB was added in the PARylation reaction. (D) Endogenous PARP-1 depletion enhances CAGA12 SBE promoter activity induced by 5 ng/ml TGF-β for 16 hr in HaCaT cells. Conditions were similar to Figure 4B, except that the cells were transfected with siRNAs and with the promoter plasmids using DuoFect. Mean values from triplicate determinations normalized to the β-galactosidase measurements are plotted with SD. Each assay was repeated at least two or three times. (E) Inhibition of endogenous PARP-1 by 3-AB enhances CAGA12 SBE promoter activity induced by 5 ng/ml TGF-β for 16 hr in HepG2 cells, in the absence or presence of 5 mM 3-AB added 0.5 hr prior to TGF-β. Mean values from triplicate determinations normalized to the β-galactosidase measurements are plotted with SD. Each assay was repeated at least two or three times. (F) Wild-type and DNA-binding deficient PARP-1 repress TGF-β-induced CAGA12 SBE promoter activity. NMuMG cells were transfected with the indicated wild-type and mutant PARP-1 constructs together with the CAGA12 SBE promoter construct prior to stimulation with 5 ng/ml TGF-β for 6 hr. Mean values from triplicate determinations normalized to the GFP measurements are plotted with SD. Each assay was repeated at least two or three times. Wild-type and C21, 125G mutant PARP-1 immunoblot in the same lysate shows expression levels of the transfected proteins. A nonspecific protein is marked (∗) just above the specific PARP-1 protein bands. See also Figure S5. Molecular Cell 2010 40, 521-532DOI: (10.1016/j.molcel.2010.10.029) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 PARP-1 Regulates the Transcriptional Output of TGF-β Signaling (A) Efficiency of PARP-1 silencing in HaCaT cells. Quantitative RT-PCR analysis of endogenous PARP-1 mRNA levels after transfection of control or specific siRNA, and corresponding PARP-1 immunoblot and α-tubulin loading control. The graph shows mean values with SD, derived from triplicate samples, normalized to the housekeeping GAPDH mRNA and further normalized to the levels of the control condition, which is set to ten. The experiment was repeated at least two or three times. (B–D) Endogenous PARP-1 knockdown enhances the induction of Smad7 (B), SnoN (C), or PAI-1 (D) mRNA by 5 ng/ml TGF-β for the indicated time points. Quantitative RT-PCR analysis was performed as in (A). The graph shows mean values with SD, derived from triplicate samples, normalized to the housekeeping GAPDH mRNA and further normalized to the level of the control condition, which is set to one. Each assay was repeated at least two or three times. Stars indicate statistical significance, p < 0.05. (E) Endogenous PARP-1 depletion enhances PAI-1 protein induction by TGF-β. Immunoblot of endogenous PAI-1 protein levels in cells transfected with control or specific siRNA. The efficiency of PARP-1 silencing is shown along with controls of TGF-β stimulation (phospho-Smad2/3) and loading controls (Smad2/3). See also Figure S6. Molecular Cell 2010 40, 521-532DOI: (10.1016/j.molcel.2010.10.029) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 7 PARP-1 Regulates TGF-β Signaling and EMT (A) Endogenous PARP-1 depletion in NMuMG cells enhances Pai-1 or Gadd45γ mRNA induction by 1 ng/ml TGF-β for 1 hr, whereas knockdown of endogenous Smad4 suppresses the enhancement of both basal and TGF-β-stimulated mRNA levels. Quantitative RT-PCR was performed as in Figure 6A. Mean values, derived from triplicate measurements and SD are shown. Each assay was repeated at least two or three times. (B) Phase-contrast microscopy of NMuMG cells indicates EMT into parallel bundles of spindle-like cells after PARP-1 silencing. Cells were stimulated with TGF-β for 24 hr (scale bar, 10 μm). (C and D) Immunofluorescence analysis of endogenous fibronectin (C) and ZO-1 (D) levels in NMuMG cells treated exactly as in (B) (scale bar, 10 μm). See also Figure S7. Molecular Cell 2010 40, 521-532DOI: (10.1016/j.molcel.2010.10.029) Copyright © 2010 Elsevier Inc. Terms and Conditions