SUPPLEMENTAL DATA

Frizzell et al. (2009) - "Global Analysis of Transcriptional Regulation by Poly(ADP-ribose)Polymerase-1 and Poly(ADP-ribose) Glycohydrolase in MCF-7 Human Breast Cancer Cells "

Contents:page

1) Experimental Procedures ..................................................................................................... 3a) Cell proliferation analyses .............................................................................................. 3b) Cell cycle analyses......................................................................................................... 3c) Inhibitor analyses ........................................................................................................... 3

2) Oligonucleotide Sequences .................................................................................................. 5a) shRNA constructs .......................................................................................................... 5b) Site-directed mutagenesis............................................................................................... 5c) RT-qPCR ....................................................................................................................... 6d) ChIP-qPCR.................................................................................................................... 7

3) Tables Listing PARP-1- and PARG-Regulated Genes.......................................................... 9

a) Table S1: List of 154 of the most robustly regulated genes affected by PARP-1knockdown only ........................................................................................................... 9

b) Table S2: List of 167 of the most robustly regulated genes affected by PARGknockdown only ......................................................................................................... 11

c) Table S3: List of 50 of the most robustly regulated genes affected by both PARP-1and PARG knockdown ............................................................................................... 13

4) Supplemental Figures and Legends.................................................................................... 14

a) Figure S1: PARP-1 and PARG antibodies immunoprecipitate their cognateproteins from crosslinked chromatin........................................................................... 14

b) Figure S2: PARP-1 or PARG knockdown does not significantly alter MCF-7 cellproliferation or cell cycle progression......................................................................... 15

c) Figure S3: PARP-1 and PARG coordinately regulate global patterns of geneexpression in MCF-7 cells (analysis of the union of regulated genes as defined bypresent call and p-value cutoff) .................................................................................. 16

d) Figure S4: Comparison of two independent shRNA target sequences for theknockdown of PARP-1 or the knockdown of PARG................................................... 17

e) Figure S5: Stable reexpression of wild-type PARP-1 or PARG re-establishescellular PAR levels, while catalytically inactive mutants do not.................................. 19

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f) Figure S6: Treatment with PARP-1 or PARG chemical inhibitors, PJ34 orGallotannin (GT), independently confirms that some genes do not require PARP-1 or PARG enzymatic activity for proper gene expression .......................................... 20

5) References ......................................................................................................................... 22

Supplemental Materials Frizzell et al. (2009)

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1) Experimental Procedures

a) Cell proliferation analyses:For analysis of cell proliferation, stable Luc, PARP-1, and PARG knockdown cells were seededat ~2 x 104 cells per well in 6-well plates in MEM containing 5% CDCS and the additives notedin the main text. Every two days during an 8-day time course, the cells were collected bytrypsinization and counted using a hemacytometer. All experiments were conducted a minimumof three times to ensure reproducibility. Analysis of variance (ANOVA) with a p-valuethreshold of < 0.05 was used to determine the significance of differences between samples.

b) Cell cycle analyses:For FACS analysis, stable Luc, PARP-1, and PARG knockdown cells were seeded at ~5 x 105

cells per 6 cm diameter dish in MEM containing 5% CDCS and the additives noted in the maintext. After three days (at ~50 to 60% confluence), the cells were collected by trypsinization,washed twice with ice-cold PBS, and fixed with 70% ethanol for at least 1 hour at 4°C. The cellswere then washed again with ice-cold PBS and stained with a propidium iodide solution (40µg/mL propidium iodide, 0.1% Triton X-100, 200 µg/mL RNase A). The samples wereincubated at 37°C for 30 minutes and analyzed by flow cytometry at Cornell University’sBiomedical Sciences Flow Cytometry Laboratory. Briefly, DNA content was measured with aBD Biosciences LSRII (San Jose, CA). Propidium iodide was excited with a 488nm laser andemission collected through a 576/26BP filter. Gating and analysis were done using the BDBiosciences FACSDiVa software. All experiments were conducted a minimum of three times toensure reproducibility. Analysis of variance (ANOVA) with a p-value threshold of < 0.05 wasused to determine the significance of differences between samples.

c) Inhibitor analyses:

Chemical inhibitors. PJ34 was purchased from Alexis Biochemicals. Gallotannin ("GT"; a.k.a.common tannic acid) was purchased from Sigma Chemical Co. Both inhibitors were dissolvedin distilled water, pH-adjusted to approximately 7.5, and added to the cell culture medium at theconcentrations and for the times specified in the figure legends.

Verification of PARP-1 and PARG inhibitor activity. To verify the inhibitory activities of PJ34and GT, we monitored autoPARylation of PARP-1 by using an immunoprecipitation-Westernblotting protocol. Briefly, parental MCF-7 cells were seeded at ~4 x 105 cells per 10 cmdiameter plate and grown for at least 3 days in MEM containing 5% CDCS and the additivesnoted above. At ~80% confluence, the cells were treated with PJ34 (1 µM) or GT (100 µM) for6 hrs immediately prior to collection of the cells for analysis. The cells were then rinsed withice-cold PBS, collected into ice-cold PBS (containing PJ34 or GT where appropriate), andpelleted by centrifugation. The cell pellets were resuspended in 300 mL of lysis buffer [25 mMTris•HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 0.1 mM EDTA, 0.1% NP-40, 1 mM DTT, anda protease inhibitor cocktail (Roche Molecular Biochemicals)]. The samples were mixed at 4°Cfor 30 minutes and centrifuged at 12,000 rpm for 20 min at 4°C in a microcentrifuge. Theresulting supernatants were used for immunoprecipitation of PARP-1, which was performed for2 hrs at 4°C using the PARP-1 polyclonal antibody described in the main text. Immune

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complexes were collected by the addition of 40 µL of a 50% protein A-agarose slurry with anadditional 2 hr incubation at 4°C. The agarose beads were washed three times for 5 min each inwash buffer [25 mM Tris•HCl (pH 7.5), 300 mM NaCl, 10% glycerol, 0.1 mM EDTA, 0.1%NP-40, and 1 mM DTT]. Bound proteins were eluted by boiling in SDS loading solution andanalyzed by Western blotting for PARP-1 and PAR.

Effect of PARP-1 and PARG inhibition on gene expression. Parental MCF-7 cells were treatedwith PJ34 (1 µM) or GT (100 µM) for 6 hrs immediately prior to collection of the cells foranalysis. The cells were seeded at ~1.5 x 105 cells per well in 6-well plates and grown for 3 daysin MEM containing 5% CDCS and the additives noted in the main text. Total RNA was isolatedusing Trizol Reagent (Invitrogen), reverse transcribed, and subjected to real-time quantitativePCR using gene specific primers. All target gene transcripts were normalized to the β-actintranscript. All experiments were conducted a minimum of three times with independent RNAisolations to ensure reproducibility. Analysis of variance (ANOVA) with a p-value threshold of< 0.05 was used to determine the significance of differences between samples.

Supplemental Materials Frizzell et al. (2009)

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2) Oligonucleotide Sequences

a) shRNA constructs:

Target Sequence SourceLuc 5’ - gatatgggctgaatacaaa - 3’ Reynolds et al (1)hPARP-1 #1 5’ - gggcaagcacagtgtcaaa - 3’ Ju et al and Shah et al (2,3)hPARP-1 #2 5’ - acacctctctactatataa - 3’ Dharmacon siDESIGN® Center*

hPARG #1 5’ - caataccactcctgaaaca - 3’ Dharmacon siDESIGN® Center*hPARG #2 5’ - agagaccgctgaccattca - 3’ Dharmacon siDESIGN® Center*

*Sequences were chosen based on priority score according to criteria described at the website.

b) Site-directed mutagenesis:

• Primers for site-directed mutagenesis to generate an RNAi-resistant hPARP-1 cDNA:

1. Recognition site #1 (Wt and CatMut)5’ - caggttacccaagggcaaacatagcgttaaaggtttgggcaaaac - 3’ and5’ - gttttgcccaaacctttaacgctatgtttgcccttgggtaacctg - 3’

Specific changes relative to the first nucleotide of the first codon:g2835a, c2838t, t2841c, c2844t

2. Recognition site #2 (Wt)5’ - ctggtgtgaatgacacgtcgctgctgtataacgagtacattgtc - 3’ and5’ - gacaatgtactcgttatacagcagcgacgtgtcattcacaccag - 3’

Specific changes relative to the first nucleotide of the first codon:c2946g, t2949g, a2952g, a2955g

3. Recognition site #2 (CatMut)5’ - ctggtgtgaatgacacgtcgctgctgtataacaagtacattgtc - 3’ and5’ - gacaatgtacttgttatacagcagcgacgtgtcattcacaccag - 3’

Specific changes relative to the first nucleotide of the first codon:c2946g, t2949g, a2952g, a2955g

• Primers for site-directed mutagenesis to generate an RNAi-resistant rPARG cDNA:

1. Recognition site #1 (Wt and CatMut)5’ - tttgcacccagccaataccgcttctcaagcagaagatgaacc - 3’ and5’ - ggttcatcttctgcttgagaagcggtattggctgggtgcaaa - 3’

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Specific changes relative to the first nucleotide of the first codon:a1827g, g1833c, a1836g

2. Recognition site #2 (Wt and CatMut) - Not alteredDifferences between the human-based PARG shRNA#2 sequence and the rat

PARG cDNA abrogated the need to alter the #2 recognition site

c) RT-qPCR:

Gene Name Primer Sequenceβ-ACTIN forward 5’-AGCTACGAGCTGCCTGAC-3’β-ACTIN reverse 5’-AAGGTAGTTTCGTGGATGC-3’DNAJC12 forward 5’-GAATGTCACCCAGACAAGC-3’DNAJC12 reverse 5’-GAATGGCATCGACATCTG-3’GAPDH forward 5’-CCCAACCACCTGCTGCTTTAACCTG-3’GAPDH reverse 5’-TGGCTTTGGAGTTGGAGATTTTTGG-3’GDF15 forward 5’-CTACAATCCCATGGTGCTCA-3’GDF15 reverse 5’-TATGCAGTGGCAGTCTTTGG-3’ITPR1 forward 5’- TGCCTCCACAATTCTACG-3’ITPR1 reverse 5’- TGAATGTCCCACAGTTGC-3’LGALS3BP forward 5’-AATGTCACCATGAGTGTGG-3’LGALS3BP reverse 5’-ACTGACGACAGGGTGATG-3’MTR forward 5’-ACAACAGCCTATGTCCTCTG-3’MTR reverse 5’-CCATCATAGAAGGCGTTTC-3’NAT1 forward 5’-CTTCACCCTCACCCATAGGA-3’NAT1 reverse 5’-TTTGGGCACAAGCTTTCTCT-3’NELL2 forward 5’-TGAAGGGAACCACCTACC-3’NELL2 reverse 5’-ATTTGCCATCCACATACG-3’NFAT5 forward 5’-ACCTCTTCCAGCCCTACCAT-3’NFAT5 reverse 5’-CCTCTTCGGTGTTGATGGAT-3’NVL forward 5’-ACGAAGAATTGTAGCCCAAC-3’NVL reverse 5’-CGAGTCTGGTCGATTAGTAGC-3’PARP-1 forward 5’-GTGTGGGAAGACCAAAGGAA-3’PARP-1 reverse 5’-TTCAAGAGCTCCCATGTTCA-3’PARG forward 5’-GACGCAATCTCTTCCACACA-3’PARG reverse 5’-TGAGTCAGGATGGAGGGAGT-3’PFDN1 forward 5’-TGCCTTCTCCCATACATTCC-3’PFDN1 reverse 5’-CAGGATTATGGCGTCCATCT-3’PHF3 forward 5’- AATTCCACACCCTCTTGTG-3’PHF3 reverse 5’- TGCTGTCGCTTCAGTTTC-3’PLA2G2A forward 5’- GATCCAGGGAGCATTCAC-3’PLA2G2A reverse 5’- TGTTTGTTCTGCACTCCTG-3’PVALB forward 5’-CTGAACGCTGAGGACATC-3’PVALB reverse 5’-TTCACATCATCCGCACTC-3’

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RAPGEF4 forward 5’-ATGGAAACGGGCTCTAAC-3’RAPGEF4 reverse 5’-AGCAGGACGAAGAGGAAC-3’SEMA4G forward 5’-TGGGGGTCTTGTTAGTCTGG-3’SEMA4G reverse 5’-GTGAGGATGCTGAGGAGGAG-3’SOCS2 forward 5’-ACACGTCAGCACCATCTCTG-3’SOCS2 reverse 5’-TGGCACCGGTACATTTGTTA-3’TMOD3 forward 5’-GGAAGTAGTAATGGTGTTGACC-3’TMOD3 reverse 5’-GCTCATCAAATACCGGAAG-3’

d) ChIP-qPCR:

Gene Name Primer SequenceDNAJC12 promoter forward 5’-GCTATGTGGAACATGCTGCT-3’DNAJC12 promoter reverse 5’-GTCCTTCTTCCCTCGGAAAC-3’GDF15 -500 forward 5’-ACACATCAAGGTTGCCCTTC-3’GDF15 -500 reverse 5’-TGGTGAAAAACAAAGGAAGCA-3’GDF15 0 forward (promoter) 5’-CTCAGATGCTCCTGGTGTTG-3’GDF15 0 reverse (promoter) 5’-CTCGGAATCTGGAGTCTTCG-3’GDF15 1500 forward 5’-TTTGACTGCCAGAAGAAAAGC-3’GDF15 1500 reverse 5’-AGGCAGCCTGAGATTCCAAC-3’ITPR1 promoter forward 5’-ACTGAGGTCGCGGTTTGTAT-3’ITPR1 promoter reverse 5’-AAGGAGCCGTGTTGTGACTT-3’LGALS3BP -500 forward (promoter) 5’-GGGCACCCCTCTCTCTACAC-3’LGALS3BP -500 reverse (promoter) 5’-TGATTGTTGCTGGACTCAGG-3’LGALS3BP 0 forward 5’-GGGGCATTTCAGAGATGAGA-3’LGALS3BP 0 reverse 5’-GTTTGGGGTAGAGGCACAAA-3’LGALS3BP 500 forward 5’-ACAGAAACCCCAGCATCATC-3’LGALS3BP 500 reverse 5’-CTCTGCACTCCTGTCCTTCC-3’LGALS3BP 1000 forward 5’- CTCAGTGAGGCAATCAGCAG-3’LGALS3BP 1000 reverse 5’-CAAGGCTCATCCAGAACCAT-3’LGALS3BP 1500 forward 5’-TCCACCCTCTCTGTGCTCTT-3’LGALS3BP 1500 reverse 5’-GACAGTGCCATGCAACCTT-3’LGALS3BP 2000 forward 5’-GACTGGTCCTTTGACCCAGA-3’LGALS3BP 2000 reverse 5’-CCAATCCCGGAAGACATCTA-3’NAT1 promoter forward 5’-CCGGCTGAAATAACCTGGTA-3’NAT1 promoter reverse 5’-TATGTGCCAGCCACACTTTC-3’NELL2 promoter forward 5’-TCCCCGGAGGAGCAGTCT-3’NELL2 promoter reverse 5’-CGCCCGAACCTGTTGTAAAG-3’NVL promoter forward 5’-TGCAACCAAACGGATCAATA-3’NVL promoter reverse 5’-TGAATTAAGTATTAGATTTCCCACTCA-3’PVALB -1000 forward (promoter) 5’-GCTCCCCTATCTGCACACTC-3’PVALB -1000 reverse (promoter) 5’-CAAAGGCTGTTTGGAAGCTC-3’PVALB 0 forward 5’-CTGCTGCATCCCTCTATCCT-3’PVALB 0 reverse 5’-CTCACTTCCCGACAGGACTT-3’RAPGEF4 promoter forward 5’-GTAACTCCCGACGACAGCTC-3’

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RAPGEF4 promoter reverse 5’-CTGTCACAGCCTGGAAACAA-3’SEMA4G promoter forward 5’-AAACGGACCTCAGAAAACCA-3’SEMA4G promoter reverse 5’-CCATGGTGAGAGGGAGTTGT-3’SOCS2 promoter forward 5’-TTCAAGCTTTCGAGCAGTGA-3’SOCS2 promoter reverse 5’-CCCTTAACAATCACGGGAAA-3’

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3) Tables Listing PARP-1- and PARG-Regulated Genes

Table S1. List of 154 of the most robustly regulated genes affected by PARP-1 knockdownonly.

Gene Log2 FC Gene Log2 FC Gene Log2 FC

ADAMTSL3 -0.711ADCK2 0.519ALDH5A1 0.519AMY1A -0.695ANKRD17 -0.536AQP3 0.919ARL4D -0.693ATP2A3 0.651AVEN 0.522BCAT1 0.564BCORL1 0.511C10orf110 -0.940C10orf56 0.837C10orf84 0.732C11orf32 0.561C11orf9 -0.514C18orf25 0.800C2orf34 0.628C8orf1 -0.575CALML5 0.921CAMK2N1 -0.515CAV1 -0.744CCDC14 -1.631CDC42BPB 0.504CDC6 0.554CEACAM6 1.069CENPI -0.610CHKB -0.580CPM 0.656CREB3L1 0.672CRISPLD2 -0.650DBR1 0.576DDX19A 0.633DEGS1 -0.595DKFZp667G2110 -0.570DKFZP686A01247 -0.516DNAJC12 -0.656DST 0.503

DUSP2 -0.755DUSP5 0.654DYNC2LI1 0.641DZIP3 -0.667EIF2C4 -1.404ELF5 0.584ENAH -0.588ETS2 0.540EXTL2 -0.568F2RL1 0.602FADS1 0.572FAM117A -0.587FARP1 0.517FHL1 0.604FHL2 0.872FLJ23172 0.804FZD3 0.501FZD7 -0.880GDF15 1.036GNAS -0.780GP1BA 0.695GPR30 -0.519GPR64 -0.554GUSBP1 -0.568HADHA -0.561HHEX 0.501HIST1H2AM 0.618HMGA2 -0.787HTR2C -0.656HYAL2 0.594ID1 -0.606ID2 -0.536ID3 -0.660INHBB 0.617IQSEC1 0.584KIAA0040 0.601KIAA0367 0.868KIAA0683 0.532

KIAA0776 -0.583KLF4 0.574KPNA5 -0.822LAMC1 -0.531LOC92249 -0.617LRRFIP2 0.533LTBP1 0.508MANEA 0.671MAP3K5 -0.718MAP3K9 0.515MARS -0.617MCAM -0.618MED6 0.622MORC4 0.507MUC5AC 0.661MZF1 -0.663NAV3 -0.555NCKAP1 -0.598NEK4 0.552NETO2 -0.642NFAT5 -0.808NIPSNAP3B -0.827NMB -0.550NR2F2 0.528NRF1 0.588NVL -0.736ODF2 0.597OSBPL1A -0.549PADI2 0.698PAH -0.758PALMD -0.662PARP1 -2.711PARP12 0.775PCGF1 -0.753PDLIM7 0.740PER3 -0.574PFDN1 -1.027PFTK1 -0.501

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PGK1 -0.700PIK3R1 -0.654PLA2G2A -0.803PLEKHB1 0.595PSEN2 -0.675PTGER3 -0.536PTPN3 0.504RAI16 0.594RAP2C 0.652RBM21 0.541RHBDD3 0.539RHOBTB3 -0.511RRAGD 0.662SCAMP1 -0.536

SCN1A -1.036SEMA4D -0.517SEPP1 -0.524SLC16A2 -0.985SLC29A1 0.536SLC2A10 0.554SMOX 0.571SORD 0.542SSBP2 -0.520SSH1 -0.567SYCP2 -1.048TBCA -0.812TBCE 0.511TBX10 0.627

TNRC9 0.901TPK1 0.602TRPC1 -0.733UBN1 -0.645UTP18 -0.661UTRN -0.577VGLL1 0.903WASL 0.521YIPF4 0.541ZNF133 -0.519ZNF35 -0.524ZNF571 -0.550

List of genes regulated by PARP-1 only (from Fig. 3A), which include genes passing the presentcall, p-value, and fold change criteria noted in the text. The degree of change upon PARP-1knockdown is listed as log2 fold change relative to the Luc control.

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Table S2. List of 167 of the most robustly regulated genes affected by PARG knockdownonly.

Gene Log2 FC Gene Log2 FC Gene Log2 FC

ABCA5 -0.517ABCC4 0.504ABCD1 0.684ACOT2 0.703ACOX1 -0.812AGPAT3 0.524AMDHD2 -0.660AMPH -0.779AOX1 0.703AP4E1 0.772APH1B -0.755ARL4C -0.615ARMC6 0.525ARPC4 0.722ATP11A -0.528ATP11B 0.660AZIN1 -0.618BBS1 -0.557BCAR3 0.502BFSP1 -0.519BHLHB2 -0.903BRAF -0.818C14orf45 -0.617C20orf117 -0.773C5orf13 0.831C9orf156 0.594CARD14 -0.528CASP2 0.624CDC14B -0.844CDKN2C -0.509CES2 -0.707CLIC4 -0.580CLTA -0.702CNN2 0.664COL11A2 0.668COQ7 -0.553CRISP3 0.551CRLF1 -0.584CROT 0.519DEPDC5 0.611DICER1 -0.627

dJ222E13.2 0.521DLEU2 -0.762DTNA -0.511EEF1D -0.720EFEMP1 0.834EHF -0.547EMCN -0.625ENPEP -0.515EZH1 -0.681FAM116B -0.884FBXO22 -0.973FBXW7 0.603FGFR1 0.719FLJ12151 -0.583FLJ21820 -0.561FLJ22662 0.602GALNT12 0.609GDAP1 -0.559GEM -0.722GLE1L 0.586GNE 0.871GPC5 0.553GSPT1 0.712GTF2F1 0.614GULP1 0.654HLA-A -0.510HNRPD -0.552H-plk 0.680HSF1 0.583IFT122 -0.872INSIG1 0.571IREB2 0.528ITFG2 0.653KCNJ5 0.796KIAA0562 0.593KIAA2010 -0.810KIF14 -0.552KLF3 0.663KRT86 0.540KYNU 0.566LIG3 0.530

LOC441296 -0.559LOC93349 0.567LRRC40 -0.954LTBP3 0.695MAP4 -0.521MARCKS -0.655MAX -0.815MBNL2 0.608MCFD2 0.577MED6 -1.098MIER2 -0.523MLF1 -0.578MNS1 -0.682MTSS1 -0.684MUM1 1.199MYO9B 0.551NAT1 -0.857NCAM2 -0.957NEK1 -0.793NEK7 -0.920NFATC1 0.542NFYB 0.509NUP62CL -0.865OLR1 0.573OPN3 0.651OR7E37P -0.738P18SRP 0.591PARD3 0.545PARG -1.514PCP4 0.502PDCD4 0.523PHF3 -0.744PHLPPL -0.658PLD3 -0.652PPAP2B 0.598PPFIBP2 -0.754PPP1R15A -0.778PPP1R9A -0.630PRO0149 0.750PRO1843 -0.537RAB28 0.529

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RASL11B -0.584RBM7 0.503RET -0.599RPL23 -0.957RRM2 -0.545RXRB 0.657SAV1 0.647SF3B3 0.604SIPA1L1 -0.612SKIP -0.562SLC6A14 1.362SNRPN 0.935SOCS2 -0.974SPATA2 0.702SRGAP3 -0.768

STK3 0.507STX6 -0.646TAF1 -0.793TBC1D5 -0.729TFF1 -0.576TFF3 -0.869TGFB2 0.694TMC5 0.640TMCO3 0.824TMSL8 -1.295TNFSF13 -0.627TPM4 0.799TPR 0.554TTC12 -0.615TTC30A -0.803

TWIST1 -0.586URG4 -1.281USP24 0.583VAPA -0.528VAV3 -0.738VCX -0.950VCX2 -0.555WSB2 -0.661WTAP -0.512YWHAZ -0.926ZIC1 0.708ZNF165 0.506ZNF202 0.614ZNF606 -0.731

List of genes regulated by PARG only (from Fig. 3A), which include genes passing the presentcall, p-value, and fold change criteria noted in the text. The degree of change upon PARGknockdown is listed as log2 fold change relative to the Luc control.

Supplemental Materials Frizzell et al. (2009)

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Table S3. List of 50 of the most robustly regulated genes affected by both PARP-1 andPARG knockdown.

Gene PARP-1 PARG Gene PARP-1 PARG

ALDH1A3 1.41 0.99ALOX15 0.70 0.69ANKRD12 -0.51 -0.54ATXN10 1.14 1.26C10orf97 0.59 0.66C14orf78 0.65 0.60CALM1 0.78 0.92CCNA2 0.50 0.56CENTD1 -0.87 -0.58CTDSPL 0.70 0.91CYB561 0.68 0.78EPS15 0.66 0.79FGFR2 0.65 0.53FLJ11151 1.24 1.25GRSF1 0.52 0.72HGD 0.66 0.80ITPR1 -0.73 -0.98KCNK5 0.60 0.94KIAA0999 0.65 0.73LGALS3BP 0.79 0.62LYRM1 0.65 0.55MBOAT2 0.60 0.62MSMB 0.76 0.62MTR 0.60 0.82MYB -0.63 -0.86

NELL2 -0.54 -1.14NR4A2 0.53 0.53OGFR 0.87 0.79PAQR6 -0.78 -0.74PCCA 0.52 0.61PDLIM5 0.73 0.65PGM3 0.66 0.75PPP2R1B 0.56 0.61PRODH 0.89 0.82PRUNE 0.50 0.53PVALB -0.64 -0.57QPRT 0.85 0.86RIPK2 0.69 0.94RPL23AP7 0.75 1.02SERINC3 0.63 0.60SGK3 0.89 0.66SLC35A3 0.65 0.94ST3GAL5 -0.67 -0.57TARP -1.03 -1.44TBC1D4 0.83 0.69TGOLN2 0.58 0.57TMOD3 0.91 1.12TROVE2 -0.85 -0.89UBE2B 0.51 0.52ZMYM6 0.70 0.60

List of genes regulated by both PARP-1 and PARG (from Fig. 3A), which include genes passingthe present call, p-value, and fold change criteria noted in the text. The degree of change uponPARP-1 or PARG knockdown is listed as log2 fold change relative to the Luc control.

Supplemental Materials Frizzell et al. (2009)

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4) Supplemental Figures and Legends

Figure S1. PARP-1 and PARG antibodies immunoprecipitate their cognate proteins fromcrosslinked chromatin.(A) Top, Crosslinked and sheared chromatin from MCF-7 cells was subjected to ChIP-Westernanalyses for PARP-1 and PARG, demonstrating the ability of PARP-1 and PARG antibodies toimmunoprecipitate their cognate proteins. In, Input; NA, no antibody control; IP,immunoprecipitate. Bottom, A higher resolution Western blot demonstrates the ability of thePARG antibody to specifically enrich for two PARG isoforms, denoted by asterisks, relative tothe input material during ChIP. Isoforms not enriched during ChIP are denoted by pluses.(B) and (C) ChIP-qPCR analyses demonstrate a reduction in PARP-1 ChIP signal uponknockdown of PARP-1 (B) and, likewise, a reduction in PARG ChIP signal upon knockdown ofPARG (C), indicating that the antibodies specifically recognize their cognate proteins.

PARP-1 PARG

In NA IP In NA IPA

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Figure S2. PARP-1 or PARG knockdown does not significantly alter MCF-7 cellproliferation or cell cycle progression.(A) PARP-1 or PARG knockdown does not significantly affect the proliferation of MCF-7 cells.The specified knockdown cell lines were seeded and counted every two days over an 8-dayperiod. Data is shown as the mean ± SEM for four independent experiments. The significantdifferences between the samples as determined by ANOVA with a p-value threshold of < 0.05,are indicated by an asterisk (i.e. day 8). All expression and ChIP experiments were conducted 3to 4 days post plating, as indicated, where growth differences were determined to beinsignificant. Stock cells were maintained as subconfluent cultures and passaged at the pointindicated.(B) PARP-1 or PARG knockdown does not significantly affect cell cycle progression. Thespecified knockdown cell lines were subjected to FACS analysis to determine percent of cells ineach cell cycle phase. Data is shown as the mean ± SEM for four independent experiments. Thedifferences between the samples are not significantly different as determined by ANOVA with ap-value threshold of < 0.05.

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PARP-1Luc PARG

GrowthRange for

Experiments

PARP-1 shRNA

Luc shRNA

PARG shRNA

+

–

–

–

+

–

–

–

+

Differencessignificant at

p < 0.05

*

*

*

*

Stock CellsPassaged

Supplemental Materials Frizzell et al. (2009)

page 16

Figure S3. PARP-1 and PARG coordinately regulate global patterns of gene expression inMCF-7 cells.(A) Venn diagram of PARP-1- and PARG-regulated genes in MCF-7 cells as defined by shRNA-mediated knockdown and expression microarrays. Genes passing both present call and p-value <0.05 criteria for at least one factor represent the union, whereas genes regulated by both factorsrepresent the commonly regulated genes or intersection (see also main Fig. 2).(B) Heatmap showing the expression profiles of the union of regulated genes. The genes areranked in the heatmap by log2 fold change in the PARP-1 knockdown cell line (see color scale).(C) Correlation analysis of the union of regulated genes from (A). The Spearman correlationcoefficient (c.c.) and p-value are indicated.

Union(1875 genes)

PA

RG

(L

og

2 F

old

)

PARP-1 (Log2 Fold)

-2.0

-1.0

0

1.0

2.0

-2 -1 0 1 2

c.c. = 0.740p < 2.2 x 10-16

-0.5

-0.5 0

Union(1875 Genes)

Gen

es

B

PARP-1 PARG

shRNAA

Union

C

PARP-1 PARG

737 485 653

Intersection

Classification by p-value

p-value< 0.05

Log2

FoldChange

-0.5

0.5

0

Supplemental Materials Frizzell et al. (2009)

page 17

Figure S4. Comparison of two independent shRNA target sequences for the knockdown ofPARP-1 or the knockdown of PARG. [See the next page](A) Knockdown of PARP-1 or PARG by two independent shRNA target sequences. Whole celllysates were collected from Luc, PARP-1, and PARG stable shRNA-mediated single knockdowncell lines. Two independent shRNA sequences (#1 and #2) targeting PARP-1 (top) or PARG(bottom) were analyzed by Western blotting for their ability to knockdown their cognate proteinsrelative to the Luc control.(B) RT-qPCR analysis confirms the knockdown PARP-1 and PARG mRNA in the singleknockdown cell lines described in (A). Total RNA was isolated from Luc, PARP-1, and PARGsingle knockdown cells, reverse transcribed, and subjected to RT-qPCR using gene-specificprimers to PARP-1 and PARG. Each bar is the mean + SEM for three independent RNAisolations.(C) Comparable effects on gene expression for single and double PARP-1 knockdown. Luc andPARP-1 single and double knockdown MCF-7 cells were seeded and grown under the sameconditions described in the main text. Total RNA was isolated, reverse transcribed, andsubjected to RT-qPCR using gene-specific primers. The effect of single knockdown (PARP-1shRNA #1 or PARP-1 shRNA#2) and double knockdown were compared for the PARP-1 targetgenes identified in Fig. 3D (left). Each bar represents the mean + SEM from three or moreindependent determinations. A correlation analysis comparing the effects of PARP-1 shRNA #1and PARP-1 shRNA #2 at 40 target genes indicates that both shRNAs produce similar effects ontarget gene expression. The Spearman correlation coefficient (c.c.) and p-value are indicated(right).(D) Comparable effects on gene expression for single and double PARG knockdown.Experiments similar to those described for PARP-1 in (C) were performed for PARG. See (C)for descriptions.

Supplemental Materials Frizzell et al. (2009)

page 18

Figure S4 (continued) - see the legend on the previous page

0

0.2

0.4

0.6

0.8

1.0

1.2

Rel

ativ

e E

xpre

ssio

n L

evel

PARP-1 mRNA

Luc

PARP-1 #1

PARP-1 #2

0

0.2

0.4

0.6

0.8

1.2

1.0

Rel

ativ

e E

xpre

ssio

n L

evel

PARG mRNA

Luc

PARG #1

PARG #2

#2#1

PARP-1Luc

αPARP-1

#2#1

PARGLuc

αPARG

0

0.5

1.0

1.5

2.0

2.5

3.0

0 0.5 1.0 1.5 2.0 2.5 3.0

PARP-1 shRNA #1

PA

RP

-1 s

hR

NA

#2

c.c. = 0.589p < 7.06 x 10-5

0

0.5

1.0

1.5

2.0

2.5

0 0.5 1.0 1.5 2.0 2.5

PARG shRNA #1

PA

RG

sh

RN

A #

2

c.c. = 0.685p < 1.31 x 10-6

BA

C

Rel

ativ

e E

xpre

ssio

n

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

GD

F15

TMO

D3

LGA

LS3B

PN

FAT5

RA

PGEF

4

MTR

PHF3

SEM

A4G

PARP-1 shRNA #1

PARP-1 shRNA #2

PARP-1 shRNA #1 + #2

D

Rel

ativ

e E

xpre

ssio

n

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

GD

F15

TMO

D3

LGA

LS3B

PN

FAT5

RA

PGEF

4

MTR

PHF3

SEM

A4G

PARG shRNA #1

PARG shRNA #1 + #2

PARG shRNA #2

Supplemental Materials Frizzell et al. (2009)

page 19

Figure S5. Stable reexpression of wild-type PARP-1 or PARG re-establishes cellular PARlevels, while catalytically inactive mutants do not.(A) and (B) RNAi-resistant wild-type (Wt) or catalytically inactive (CatMut) PARP-1 (A) orPARG (B) were stably expressed in their respective MCF-7 knockdown cells, as shown in Fig. 7.Whole cell lysates from each cell line were isolated and assayed for total cellular PAR levels byWestern blot.

Empty

Luc PARP-1shRNA:

Add-back:

Wt

CatMut

Empty

A

PAR

Empty

Luc PARGshRNA:

Add-back:

Wt

CatMut

Empty

B

PAR*

Supplemental Materials Frizzell et al. (2009)

page 20

Figure S6. Treatment with PARP-1 or PARG chemical inhibitors, PJ34 or Gallotannin(GT), independently confirms that some genes do not require PARP-1 or PARG enzymaticactivity for proper gene expression. [See next page](A) PJ34 and GT inhibit and enhance the autoPARylation of PARP-1, respectively, withoutaltering total PARP-1 or PARG levels. Parental MCF-7 cells were seeded and grown to ~80%confluence and subsequently treated with PJ34 (1 µM) or GT (100 µM) for 6 hrs immediatelyprior to collection of the cells. (Left) Whole cell extracts were monitored for PARP-1 andPARG by Western blotting under the treatment conditions noted. U, Untreated; PJ, PJ34-treated;GT, gallotannin-treated. (Right) PARP-1 was immunoprecipitated from whole cell extracts(bottom) and analyzed for autoPARylation by Western blotting under the treatment conditionsnoted using a PAR-specific antibody (top-bracket). Oligo(ADP-ribosyl)ated PARP-1 andpoly(ADP-ribosyl)ated are indicated.(B) and (C) Total RNA was isolated from PJ34- or GT-treated cells, reverse transcribed, andsubjected to real-time qPCR using gene-specific primers. Each bar represents the mean + SEMfrom three or more independent determinations. Bars that do not share at least one lower caseletter marking (a, b, c, or d) with each graph are statistically different, as determined by analysisof variance (ANOVA) with a p-value threshold of < 0.05. (B) PJ34 has no effect on LGALS3BPor NELL2 gene expression, independently confirming that PARP-1 enzymatic activity is notrequired to regulate these genes. The knockdown/add-back data is shown for comparison. (C)GT has no effect on LGALS3BP gene expression, independently confirming that PARGenzymatic activity is not required to regulate this gene. The effect of GT on NELL2 is shown asa control. The knockdown/add-back data is shown for comparison.

Supplemental Materials Frizzell et al. (2009)

page 21

Figure S6 (continued) - see the legend on the previous page

A

PARP-1

PARG

U GTPJ U GTPJ

PAR

PARP-1

Poly

Oligo

0

0.5

1.0

1.5

2.5

Rel

ativ

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xpre

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n

LGALS3BP

Luc PARP-1

Empty

Empty W

t

CatMut

PJ34

2.0

aa,c

a,c

b

c

B

Add-back:

shRNA:

NELL2

0

0.2

0.4

0.6

1.0

1.4

1.6

Rel

ativ

e E

xpre

ssio

n

Luc PARP-1

Empty

Empty W

t

CatMut

PJ34

1.2

0.8

a

a

a

b

a

0

0.5

1.0

1.5

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Rel

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Luc PARG

Empty

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t

CatMut

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a,c

c

NELL2

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1.2

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Empty W

t

CatMut

GT

0.8

a

bb,d

c,d

c

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Add-back:

shRNA:

Supplemental Materials Frizzell et al. (2009)

page 22

5) References

1. Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W. S., and Khvorova, A. (2004)Nat Biotechnol 22(3), 326-330

2. Ju, B. G., Solum, D., Song, E. J., Lee, K. J., Rose, D. W., Glass, C. K., and Rosenfeld, M.G. (2004) Cell 119(6), 815-829

3. Shah, R. G., Ghodgaonkar, M. M., Affar el, B., and Shah, G. M. (2005) Biochem BiophysRes Commun 331(1), 167-174