1. Volume 18, Issue 1, Pages 83-96 (April 2005)
PARP-1 Determines Specificity in a Retinoid Signaling Pathway via Direct Modulation of Mediator 
Rushad Pavri, Brian Lewis, Tae-Kyung Kim, F. Jeffrey Dilworth, Hediye Erdjument-Bromage, Paul Tempst, Gilbert de Murcia, Ronald Evans, Pierre Chambon, Danny Reinberg 
Molecular Cell 
Volume 18, Issue 1, Pages (April 2005)
DOI: /j.molcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1
Identification of Ligand-Dependent Activity in Nuclear Extracts
(A) Top: diagram of p(DR5)5β2G template (described in text). The region of primer hybridization is indicated (arrow), resulting in a 150 nt extension product. Bottom: in vitro transcription assay with HeLa nuclear extracts (NE). Recombinant purified RAR/RXR heterodimers were preincubated with the template, with or without 1 μM tRA, prior to NE addition and primer extension. The arrow indicates the position of the transcript.
(B) Purification scheme for ligand-dependent activity (see Supplemental Data).
(C) Immunodepletion of Mediator from 1–2 MDa Superose 6 fraction by using anti-Cdk8-coupled protein A beads. Beads were blocked with either rCdk8 or an equimolar amount of BSA prior to incubation with Superose 6 material. The Western blot shows complete depletion of Mediator components from the BSA-blocked flowthrough (FT) as compared to the rCdk8-blocked FT. Silver staining revealed that the Superose 6 material was a highly purified Mediator fraction. In the reconstituted transcription assay, the BSA-blocked FT (lacking Mediator) was unable to confer ligand dependence, whereas the rCdk8-blocked FT (containing Mediator) efficiently substituted for the 1–2 MDa fraction. Transcription assays were performed as described (Lewis et al., 2000).
(D) Titration of the Superose 6 Mediator fraction in the reconstituted transcription assay. The Mediator fraction supported ligand-dependent transcription in association with the 400 kDa Superose 6 fraction.
Molecular Cell  , 83-96DOI: ( /j.molcel )
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3. Figure 2
Identification of PARP-1 as the Sole Component of the 400 kDa Superose 6 Activity
(A) Reconstituted transcription assay with phenyl sepharose fractions (Figure 1B).
(B) Silver stain of phenyl sepharose fractions. Asterisks (*) indicate bands analyzed by mass spectrometric analysis. The arrow indicates the position of full-length PARP-1 (115 kDa). Molecular weight markers are shown on the left.
(C) Western blot of phenyl sepharose fractions used in (B) with antibodies to PARP-1. The arrow indicates position of full-length PARP-1 (115 kDa). Molecular weight markers are shown on the left.
(D) Ligand-dependent transcription can be reconstituted with recombinant PARP-1 (rPARP-1). rPARP-1 (10, 20, 50, 100, and 200 ng) efficiently substituted for native PARP-1 (phenyl sepharose material), confirming that PARP-1 was responsible for ligand dependence in the system.
(E) Diagram of PARP-1. FI and FII indicate zinc fingers I and II, respectively; NLS is the nuclear localization signal, and BRCT is the BRCA1 C terminus. The location of the active site is shown. Also shown are the various mutants and fragments used in the transcription, transfection, and IP assays.
(F) PARPABC and PARPEF were tested for their ability to confer ligand dependence. PARPABC and PARPEF (50, 100, and 200 ng in both cases) were added to the transcription reaction as shown. In contrast to the full-length protein, neither the ABC nor the catalytic domains were capable of restoring ligand dependence.
(G) PARPABC and PARPABCD were tested for ligand-dependence as in (F) above. PARPABCD, but not PARPABC, was able to efficiently restore ligand dependence, demonstrating that the BRCT domain of PARP-1 was essential for RAR-mediated transcription.
Molecular Cell  , 83-96DOI: ( /j.molcel )
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4. Figure 3 PARP-1 Functions as a Coactivator in a Physiological Context
(A) Micrococcal nuclease digestion of chromatin-assembled p(DR5)5β2G. The p(DR5)5β2G template (Figure 1A) was assembled into chromatin with S190 Drosophila embryo extract, and chromatin assembly was analyzed by micrococcal nuclease digestion.
(B) Transcription assay by using chromatin templates. S190-assembled chromatin was used in the reconstituted transcription system, and reactions were performed as before. PARP-1, Mediator, p300, and Swi/Snf were added where indicated. PARP-1 (native and recombinant) and Mediator established ligand dependence on chromatin templates only when p300 and Swi/Snf were present in the system (compare lanes 5 and 6 to lanes 15 and 16). This assay thus shows a coactivator role for PARP-1 in RAR-mediated, ligand-dependent transcription.
(C) PARPABCD, but not PARPABC or PARPEF, efficiently coactivated RAR-mediated ligand-dependent transcription in a chromatin context, underscoring the requirement of the BRCT domain for PARP-1 function.
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5. Figure 4 RA-Inducible Expression of RARβ Is Impaired in PARP-1−/− MEFs
(A) PARP-1+/+ and PARP-1−/− MEFs were treated with tRA for 15 hr, followed by RNA extraction and RT-PCR. The top panel represents an RARβ2-specific amplicon. Conserved sequences in RARβ, RARα, and RARγ were amplified as shown in subsequent panels. RA-inducible expression of all RARβ isoforms was abolished in PARP-1−/− MEFs, whereas RARα and RARγ expression profiles were similar in wt and knockout MEFs.
(B) PARP-1−/− cells were transfected with constructs expressing PARP-1 wt (PARPFL), DNA binding mutant (PARPDBD), and catalytic mutant (PARPcat) proteins. After transfection for 48 hr, cells were induced with tRA for 15 hr followed by RNA extraction and RT-PCR as before. RA-inducible expression of RARβ2 was efficiently restored either by wt PARP-1 (PARPFL) or by the catalytically inactive PARP-1 mutant (PARPcat), but not by the DNA binding mutant (PARPDBD).
(C) PARP-1−/− cells were transfected with constructs expressing PARPABC, PARPABCD, and PARPEF. Experiments were performed as in (B) above. PARPABCD, but not PARPABC or PARPEF, was able to completely restore RA-dependent expression of RAR.
Molecular Cell  , 83-96DOI: ( /j.molcel )
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6. Figure 5 PARP-1 Binds Directly to RAR and TR
(A) Immunoprecipitation (IP) assay for RAR and PARP-1 interaction. Native PARP-1 (phenyl sepharose fraction) and f-RAR were incubated together in equimolar amounts with anti-FLAG agarose beads in the presence or absence of 1 μM tRA. The beads were then washed, and the bound material was eluted and analyzed by SDS-PAGE and Western blot by using antibodies as indicated.
(B) Interaction between PARP-1 and RAR via reciprocal IP. The experiment was performed as in (A) with the exception that anti-FLAG beads were replaced with anti-PARP-1-coupled protein A beads. The RAR-PARP-1 interaction was independent of, but stimulated upon, ligand binding.
(C) and (D) Mapping of the RAR-PARP-1 interaction via anti-FLAG IPs. Experiments were performed as in (A). RAR bound domain AB (DBD), ABC, and ABCD, but not domain D or the catalytic domain.
(E) PARP-1 interacts with TR. Equimolar amounts of f-TRα and native PARP-1 were incubated with anti-FLAG beads in the absence or presence of 0.1 μM T3, and the IP was performed as in (A). Antibodies to TRα were used for the Western blot. RAR-TR interaction, although independent of ligand, was markedly stimulated upon ligand addition.
(F) RAR interacts with Mediator. The active Superose 6 Mediator material (1–2 MDa) was incubated with f-RARα in the presence or absence of 1 μM tRA, and IP was performed as above.
(G) PARP-1 binds Mediator via the BRCT domain (PARPD). Left: His-PARPD was mixed with purified Mediator and incubated with Ni-NTA beads. Bound material was eluted with Imidazole and analyzed as above. Right: reciprocal IP for Mediator-PARPD interaction by using antibodies to Cdk8.
Molecular Cell  , 83-96DOI: ( /j.molcel )
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7. Figure 6
ChIP Assays
(A) Top: diagram of the endogenous mouse RARβ2 (mRARβ2) promoter. Positions of primers for amplification of promoter and coding regions are indicated with arrows. TRE is tetradecanoyl phorbol ester-like response element, CRE is cAMP response element, Inr is initiator. Bottom: P19 cells were treated with tRA for 1 hr followed by ChIP analysis. Input material was incubated with a panel of antibodies as shown. PARP-1 was constitutively promoter bound as was RAR. The Mediator subunits Med6 and Med10 were also constitutively engaged on the promoter, whereas the Cdk8 subunit was lost upon ligand addition. The presence of Pol II, TBP, and TFIIB under uninduced and induced states indicated that the preinitiation complex was assembled at this promoter even in the transcriptionally repressed state. However, TFIIH (ERCC3), which is required for transcription initiation, was recruited upon RA-induction.
(B) Multiplex PCR by using primers specific to the coding region (exon 3) of the mRARβ2 gene along with the promoter-specific primers used in (A). The products are of different sizes and can be separated by electrophoresis. PARP-1 was absent in the coding region as was RAR, whereas Pol II was detected upon RA treatment.
(C) ChIP on the Brachyury T promoter. P19 cells were induced with 1% DMSO for 24 hr followed by ChIP analysis. PARP-1 and RAR were constitutively absent on this promoter.
(D) ChIP on non-RAR regulated promoters, β-Actin and GAPDH in P19 cells. Neither RAR nor PARP-1 was localized to these promoters, showing that PARP-1 recruitment to RAR-regulated promoters was specific.
(E) ChIP on the dio1 promoter by using antibodies as indicated. Experiments were performed as above except that the cells were induced with T3 for 1.5 hr. PARP-1 was constitutively localized to this.
(F) ChIP analysis comparing RARβ2 promoter occupancy in PARP-1+/+ and PARP-1−/− MEFs. Experiments were performed as in (A) above. Significantly, chromatin remodeling upon RA induction was likely unaffected in the absence of PARP-1, as evidenced by the similar profiles of NCoR and p300 occupancy in both PARP-1+/+ and PARP-1−/− cells. However, in the absence of PARP-1, Mediator did not attain its active conformation upon RA induction as evidenced by the retention of Cdk8 after RA treatment. Consequently, TFIIH (ERCC3) was not recruited, and the gene was silent.
(G) Presence of CRSP bypasses the requirement of PARP-1 for ligand-dependent transcription in a physiological context. Transcriptions were performed on chromatin templates as described for Figure 3. Highly purified CRSP (see silver stain) was added alone or with PARP-1. CRSP was able to confer ligand-dependence by itself (lanes 3 and 4), and addition of PARP-1 had no effect on ligand dependence (lanes 5 and 6), indicating that in the presence of active Mediator (CRSP) PARP-1 was not essential for ligand dependence. In contrast, the inactive Cdk8-containing Mediator (Med) required PARP-1 for ligand dependence (compare lanes 1 and 2 with lanes 7 and 8).
Molecular Cell  , 83-96DOI: ( /j.molcel )
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8. Figure 7 PARP-1 Functions at a Step Prior to TFIID and Mediator
(A) The order-of-addition experiments were performed as shown in the scheme. The normal transcription reaction was slightly modified, including pre or postincubation steps as shown.
(B) and (C) Preincubation of PARP-1 alone or with TFIID and/or Mediator confers ligand dependence ([C], compare lanes 3–6 with lanes 11 and 12). However, preincubation of TFIID, or Mediator, or both abolished ligand dependence ([C], compare lanes 7–10 with lanes 13 and 14). PARP-1 did not confer ligand dependence if added after PIC formation ([B], compare lanes 5 and 6 with lanes 9 and 10). Additionally, the system was TAF dependent ([B], lanes 9 and 10) and TFIIH addition after PIC formation did not affect ligand dependence ([B], lanes 7 and 8). See text for details.
Molecular Cell  , 83-96DOI: ( /j.molcel )
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9. Figure 8
Mechanistic Model for the Role of PARP-1 in RAR-Mediated Transcription
The model depicts the role of PARP-1 as a gene-specific coactivator responsible for the ligand-dependent activation of Mediator in RAR-dependent transcription. The model has been simplified for clarity. See text for a detailed description.
Molecular Cell  , 83-96DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions