Supporting Information
Rozenblatt-Rosen et al. 10.1073/pnas.0812023106 SI Text mix (4304437; Applied Biosystems), an ABI Prism 7700 instru- Antibodies. The following antibodies were generated by Bethyl ment (Applied Biosystems), and the following Assays-on- Laboratories: anti-Cdc73 Ab648 (BL648, A300–170A) and Demand (Applied Biosystems): CDC73 (Hs00225998m1), Ab649 (BL649, A300–171A), anti-CPSF-160 (BL1896), anti- INTS6 (Hs00247179m1), and ACTB (4310879E), which was CPSF-100 (BL1902), anti-CPSF-73 (BL1906), anti-CPSF-30 used as an internal reference standard. For detecting INTS6 (BL1985), anti-CstF-77 (BL1894), anti-CstF-64 (BL1889), and read-through transcripts, real-time PCR quantitation was per- anti-Ints6 (BL1115). Normal rabbit IgG was obtained from formed in triplicate by using SYBR green PCR Master Mix Bethyl Laboratories. Other antibodies were obtained commer- (4309155; Applied Biosystems), and the primer pairs whose cially as follows: anti-symplekin antibodies (BD Bioscience), sequence is detailed below. GAPDH (43088313; Applied Bio- anti-RNA polymerase II antibodies (Covance), anti-histone H3 systems) was used as an internal reference standard. For deter- tri methyl K4 (Abcam), and anti-histone H3 tri methyl K36 mining transcript levels the standard curve method for relative (Upstate and Millipore). Antibodies were diluted in 5% milk/ quantitation was used. TBST according to the manufacturer’s instructions. A ReliaBlot kit (Bethyl Labratories) was used to avoid masking of protein RNA Expression Analysis. Expression levels were measured in 4 bands by the Ig heavy chain. replicates for each of the 2 CDC73 siRNAs, for a total of 8 test samples. These were invariant-set normalized together with 8 Immunopurification and Mass Spectrometry. Peptides from the control samples, so that expression levels across genes were following proteins were identified by mass spectrometry: Cdc73 comparable. Genes whose expression levels were Ͻ150 units (NP078805), Paf1 (NP061961), Leo1 (NP620147), Ctr9 (Affymetrix arbitrary units) across all samples were considered (NP055448), CPSF-160 (NP037423), CPSF-100 (NP059133), absent in test and control samples, and hence were omitted from CPSF-73 (NP057291), symplekin (NP004810), Fip1 further analysis. Student’s t tests for higher and lower mean (NP112179), CstF-77 (NP001317) CstF-64 (NP001316), expression levels in test as compared with control samples were MLL3 (NP067053), Ash2L (NP004665), Rbbp5 (NP005048), performed for each gene. Genes were considered to be up- WDR61 (NP001074025), and CPSF-30 (NP001075028). regulated if: (i) the mean expression level in the test samples was at least 2-fold that of the control samples, and (ii) the t test P Glycerol Gradient Fractionation. Cells were lysed with 1 mL of value for higher expression in the test samples was Ͻ0.0003. Nonidet P-40 lysis buffer (1), and 200 L of lysate were applied Similarly, genes were considered to be down-regulated if: (i) the to a 4.8 mL of 5–40% glycerol gradient in Nonidet P-40 lysis mean expression level in the test samples was at less than half that buffer. The gradient was centrifuged for 18 h at 4 °C in a of the control samples, and (ii) the t test P value for lower Beckman SW50 rotor at 50,000 rpm. Two hundred-microliter expression in the test samples was Ͻ0.0003. fractions were collected starting from the top of the gradient. Protein composition of the gradient fractions was analyzed by ChIPs. Quantitative ChIP assays were performed on at least 4 Western blotting of SDS/PAGE-fractionated aliquots. independent occasions; for each ChIP assay, DNA samples were quantitated in triplicate by using Power SYBR Green (Applied In Vitro Transcription-Coupled Processing Assays. The pG3CMVL3 Biosystems)-based real-time PCR. siRNA-treated HeLa cells and pCMVAdML DNA templates were constructed by inserting were treated with 1% formaldehyde and incubated for 10 min at the CMV promoter into the pG3L3-A (2) and pAdML-M3 room temperature. Glycine was added to a final concentration of plasmids, respectively. Transcription-coupled polyadenylation 0.125 M to stop the reaction. The cells were then suspended in was carried out in reaction mixtures containing bead-depleted or 0.6 mL of lysis buffer (50 mM Tris⅐Cl, pH 8.1, containing 1% Cdc73-immunodepleted nuclear extracts, 10 mM Hepes (pH Triton X-100, 0.1% deoxycholate, 150 mM NaCl, and 5 mM 7.9), 400 ng of pG3CMVL3 linearized with ApaL1, 0.5 mM each EDTA) plus protease inhibitors (leupeptin, phenylmethylsulfo- of ATP, GTP, and CTP, 15 M cold UTP, 10 Ci of [␣-32P] nyl fluoride, and aprotinin) and subjected to sonication (using a UTP, 1.5 mM MgCl2, 8 mM creatine phosphate (di-Tris), 3% Branson Sonifer 450 sonic dismembrator with a microtip at a polyvinyl alcohol, 10% glycerol, 50 mM KCl, 0.1 mM EDTA, 0.1 setting of 3). Fifteen 5-s pulses were required to shear chromatin mM DTT, and 0.25 mM PMSF. Transcription-coupled splicing to 1,000-bp fragments. The effectiveness of shearing was con- was performed as above, except that 400 ng of pCMVAdML firmed by incubating a 10-L aliquot of the extract at 65 °C for linearized with Eco0109I was used as a DNA template, and 20 3 h (to reverse cross-links) and subsequently subjecting it to mM creatine phosphate was included in the reaction mixtures. electrophoresis on a 1% agarose gel. One hundred micrograms of the clarified extracts was diluted to 1 mL in lysis buffer RNA Interference. The following siGENOME duplexes (Dharma- containing protease inhibitors and then incubated with specific con) were used: CDC73 (D-015184-02, D-015184-03), CPSF-73 antiserum (a separate aliquot was taken and stored for later PCR (D-006365-01, D-006365-02), siCONTROL Lamin A/C (LMNA, analysis as 10% of the input extract). Incubations occurred D-001050-01), and siCONTROL nontargeting siRNA (LUC, overnight at 4 °C on a rocking platform, after which 45 Lof D-001210-02). For cotransfections, the indicated siRNAs were protein A-agarose slurry (Santa Cruz Biotechnology) was added, transfected along with a plasmid encoding a CDC73-insensitive and incubation was continued an additional 1–2 h. The agarose mutant or pCDNA1 vector control by using Lipofectamine 2000 was pelleted by centrifugation, and the pellets were washed (Invitrogen), according to the manufacturer’s instructions. Cells consecutively with 1 mL of lysis buffer, lysis buffer plus 500 mM were harvested 72 h after transfection. NaCl, lysis buffer plus 0.25 M LiCl, and Tris/EDTA. DNA and protein were eluted from the pellets by incubating the pellets 2 RNA Isolation and Quantitation. cDNA was diluted 1:14 and 5 L times in 0.25 mL of elution buffer (0.1 M NaHCO3 with 1% SDS was used per real-time PCR. Real-time PCR quantification was and 20 mg/mL herring sperm DNA), and protein-DNA cross- performed in triplicate, using the TaqMan universal PCR master links were reversed by incubation at 65 °C for 4 h. DNA and
Rozenblatt-Rosen et al. www.pnas.org/cgi/content/short/0812023106 1of14 protein were ethanol-precipitated overnight at Ϫ20 °C. The CACAC-3Ј; and Ext2, Fwd 5Ј-AATGCAGTGCCAAAGTTA- precipitated samples were pelleted and dissolved in proteinase K CAAAG-3Ј, Rev 5Ј-GAATCTCTGCTTATTTCACCAACA-3Ј. buffer (10 mM Tris⅐Cl, pH 7.5 with 1% SDS) and incubated with ChIP primer pairs were: INTS6,5ЈUTR, Fwd 5Ј-AGAACGGC- 1 g of proteinase K (Roche Molecular Biochemicals) for1hat GAGGCGGTGTATC-3Ј, Rev 5Ј-TTCTCAGCCCCTCTCCTCGC- 55 °C. The samples were extracted once with phenol/chloroform TACTG-3Ј; INTS6 CDS1, Fwd 5Ј-TCCATGGAACCACT- Ϫ and ethanol-precipitated overnight at 20 °C. Samples were CAAATCCAA-3Ј, Rev 5Ј-ACCTACCCAACTGCCACTCATT-3Ј; pelleted, washed with 70% ethanol, and dissolved in 100 Lof INTS6 CDS2, Fwd 5Ј-GGCAGATAGGGACCAGATCACT-3Ј, Rev Tris/EDTA. Three-microliter aliquots were used for each real- 5Ј-TTGGGCCTTCATCATTTCTCAGAT-3Ј; INTS6 3ЈUTR, Fwd time PCR to quantitate coimmunoprecipitated DNA fragments Ј Ј Ј INTS6 read-through primer pairs were: CDS, Fwd 5Ј-CAGAAAC- 5 -ATAACAACAGAGCTGCAGGAAAG-3 , Rev 5 -CCCCAT- Ј Ј Ј CACTAATGATTCGATAATAC-3Ј, Rev 5Ј-CAGTAAACTG- CACAACAGTAAACAATC-3 ; and HBG1 5 UTR, Fwd 5 - ACAC- GCTGGAGAAGATG-3Ј; Ext1, Fwd 5Ј-GGGTTATGGAAAGAT- TAATCTATTACTGCGCTG -3Ј,Rev5Ј- CCAGGATTTTT- TCAGAAGTG-3Ј, Rev 5Ј-TGTTCAGAGAATACCCAGT- GACGGA -3Ј.
1. Rozenblatt-Rosen O, et al. (2005) The parafibromin tumor suppressor protein is part of a human Paf1 complex. Mol Cell Biol 25:612–620. 2. Takagaki Y, Ryner LC, Manley JL (1988) Separation and characterization of a poly(A) polymerase and a cleavage/specificity factor required for pre-mRNA polyadenylation. Cell 52:731–742.
Rozenblatt-Rosen et al. www.pnas.org/cgi/content/short/0812023106 2of14 Fig. S1. Identification of low molecular weight Cdc73-interacting proteins and anti-Cdc73 Ab649 characterization. (a) Anti-Cdc73 Ab648 immunoprecipitates were separated on a 10% SDS/PAGE gel to identify smaller (low molecular weight) interacting proteins. (b) Characterization of anti-Cdc73 Ab649 in HeLa nuclear extracts. HeLa nuclear extracts were immunoprecipitated with anti-Cdc73 Ab648 or anti-Cdc73 Ab649 antibodies. Immunoprecipitates were resolved and then immunoblotted with anti-Cdc73 Ab649. Negative controls include normal rabbit IgG and the appropriate blocking peptides. HeLa nuclear extract is shown as input. (c) Cell lysate from HeLa cells was fractioned on a 5–40% glycerol gradient. Aliquots of the depicted fractions were used for immunoblotting with the indicated antibodies.
Rozenblatt-Rosen et al. www.pnas.org/cgi/content/short/0812023106 3of14 Fig. S2. Cdc73 specifically regulates Ints6 protein expression level. (a) U2OS cells were transfected with the indicated siRNAs and cell lysates were subjected to immunoblot analysis with the indicated antibodies. (b) HeLa cells were transfected with CDC73-1 or CPSF-73–1 siRNAs alone or with an expression construct encoding CDC73, which is no longer sensitive to CDC73-1 siRNA, or a vector control. Cell lysates were subjected to Western blot analysis with the indicated antibodies.
Rozenblatt-Rosen et al. www.pnas.org/cgi/content/short/0812023106 4of14 Fig. S3. Cdc73 knockdown results in significant reduction in histone H3 lysine 36 trimethylation across the INTS6 locus. ChIPs were carried out in CDC73 or LUC siRNA-treated cells with the depicted antibodies, and precipitated chromatin was used for PCR amplification with the following primer sets: 5ЈUTR (a), CDS1 (b), CDS2 (c), and 3ЈUTR (d). Error bars indicate SEM for 4 independent experiments.
Rozenblatt-Rosen et al. www.pnas.org/cgi/content/short/0812023106 5of14 Fig. S4. Cdc73 is required for cross-linking of 3Ј mRNA processing factors to the INTS6 locus. ChIPs were carried out in CDC73 or LUC siRNA-treated cells with the depicted antibodies, and precipitated chromatin was used for PCR amplification with the following primer sets: INTS6 CDS1 (a), INTS6 CDS2 (b), and INTS6 3Ј UTR (c). Error bars indicate SEM for 4 independent experiments.
Rozenblatt-Rosen et al. www.pnas.org/cgi/content/short/0812023106 6of14 Fig. S5. Cdc73 and 3Ј processing factors do not cross-link to the hemoglobin, gamma A (HBG1) locus. ChIPs were carried out in CDC73 or LUC siRNA-treated cells with the following antibodies: no antibody control, lanes 2 and 8; Cdc73, lanes 3 and 9; Leo1, lanes 4 and 10; CPSF-30, lanes 4 and 11; CPSF-73, lanes 5 and 12; CstF-64, lanes 6 and 13, and CstF-77, lanes 7 and 14. Precipitated chromatin from a representative ChIP was used for semiquantitative PCR amplification with a5ЈUTR HBG1 primer set for 32 cycles, and the products were separated on a 2% agarose gel.
Rozenblatt-Rosen et al. www.pnas.org/cgi/content/short/0812023106 7of14 Table S1. Genes whose expression is decreased upon depletion of Cdc73 Ratio Con/ Probe Gene Gene CDC73 Rank set symbol Gene name Accession no. Entrez ID P value siRNA
1 218578at CDC73 Cell division cycle 73 NM024529 79577 0.0002683 8.71 2 235283at INTS6 Integrator complex subunit 6 AA262171 26512 2.08E-08 5.85 3 230251at C6orf176 Chromosome 6 open reading frame 176 AA041523 90632 1.21E-05 5.65 4 235196at CDC73 Cell division cycle 73 AA447464 79577 0.0001877 5.12 5 218819at INTS6 Integrator complex subunit 6 NM012141 26512 4.18E-05 4.39 6 215071sat HIST1H2AC Histone cluster 1, H2ac AL353759 8334 0.0002255 4.14 7 229828at CDC73 Cell division cycle 73 AL044007 79577 3.65E-05 4.05 8 218303xat KRCC1 Lysine-rich coiled-coil 1 NM016618 51315 3.60E-10 3.88 9 233329sat KRCC1 Lysine-rich coiled-coil 1 AK025986 51315 8.53E-05 3.76 10 217967sat FAM129A Family with sequence similarity 129, member A AF288391 116496 0.0002924 3.57 11 236535at SMC6 Structural maintenance of chromosomes 6 AW069285 79677 1.92E-05 3.40 12 223681sat INADL InaD-like (Drosophila) AB044807 10207 0.0002141 3.39 13 38892at KIAA0240 KIAA0240 D87077 23506 8.38E-07 3.39 14 222239sat INTS6 Integrator complex subunit 6 AL117626 26512 9.01E-08 3.33 15 228544sat CSRP2BP CSRP2 binding protein AA780252 57325 1.01E-05 3.21 16 1558801at — —- AK055769 — 4.42E-05 3.19 17 222908at FAM38B Family with sequence similarity 38, member B AW269818 63895 0.0001254 3.19 18 216399sat SCAPER S phase cyclin A-associated protein in the ER AK025663 49855 1.38E-05 3.17 19 1558826at C20orf174 chromosome 20 open reading frame 174 AL713683 128611 2.26E-05 3.16 20 228575at IL20RB Interleukin 20 receptor beta AL578102 152028 9.42E-05 3.14 21 1558827aat C20orf174 Chromosome 20 open reading frame 174 AL713683 128611 0.0002223 3.11 22 228734at UBE2V2 Ubiquitin-conjugating enzyme E2 variant 2 BF447286 7336 0.0001005 3.09 23 238576at — — AA169515 — 2.52E-05 3.07 24 218519at SLC35A5 Solute carrier family 35, member A5 NM017945 55032 4.32E-06 3.01 25 202847at PCK2 Phosphoenolpyruvate carboxykinase 2 NM004563 5106 2.65E-05 3.00 (mitochondrial) 26 221050sat GTPBP2 GTP binding protein 2 NM019096 54676 0.0001417 2.94 27 219966xat BANP BTG3 associated nuclear protein NM017869 54971 0.0002329 2.94 28 225575at LIFR Leukemia inhibitory factor receptor alpha AI680541 3977 9.51E-07 2.93 29 233186sat BANP BTG3 associated nuclear protein AK001039 54971 0.0003287 2.91 30 226364at HIP1 Huntingtin interacting protein 1 AU145049 3092 0.0002698 2.88 31 228928xat BANP BTG3 associated nuclear protein AL157429 54971 0.0002237 2.87 32 225571at LIFR Leukemia inhibitory factor receptor alpha AA701657 3977 4.86E-07 2.87 33 229257at — KIAA1856 protein AI625045 84629 7.25E-08 2.85 34 229173at — KIAA1715 N29672 80856 8.22E-05 2.84 35 231202at ALDH1L2 Aldehyde dehydrogenase 1 family, member L2 AI654224 160428 1.49E-06 2.83 36 225937at FP6778 — BF002121 — 2.97E-10 2.80 37 222103at ATF1 Activating transcription factor 1 AI434345 466 3.18E-07 2.80 38 224649xat — — AI765014 — 3.99E-05 2.79 39 220404at GPR97 G protein-coupled receptor 97 NM014076 222487 4.58E-07 2.79 40 243309at — — AA398658 145788 6.60E-06 2.78 41 229145at C10orf104 — AA541762 119504 0.0001071 2.77 42 209741xat SCAPER S phase cyclin A-associated protein in the ER AF119814 49855 9.50E-06 2.77 43 228543at CSRP2BP CSRP2 binding protein AA780252 57325 1.96E-05 2.77 44 232362at CCDC18 Coiled-coil domain containing 18 AL139421 343099 9.66E-06 2.73 45 239173at INADL InaD-like (Drosophila) AI697184 10207 0.000143 2.73 46 226760at — — BF666325 203411 0.0001927 2.69 47 202106at GOLGA3 Golgi autoantigen, golgin subfamily a, 3 NM005895 2802 0.0002169 2.68 48 226019at OMA1 OMA1 homolog, zinc metallopeptidase AI927931 115209 6.22E-05 2.67 49 238681at GDPD1 Glycerophosphodiester phosphodiesterase domain R46180 284161 4.06E-06 2.67 containing 1 50 202293at STAG1 Stromal antigen 1 AW168948 10274 5.04E-05 2.66 51 225935at FP6778 — AI350995 — 0.0001191 2.65 52 226899at UNC5B unc-5 homolog B AK022859 219699 0.0002915 2.65 53 235109at ZBED3 zinc finger, BED-type containing 3 AI887983 84327 9.58E-05 2.63 54 230660at SERTAD4 SERTA domain containing 4 AU146709 56256 0.0002307 2.63 55 205194at PSPH phosphoserine phosphatase NM004577 5723 1.21E-05 2.62 56 225477sat MRPS25 Mitochondrial ribosomal protein S25 AL138444 64432 0.0001011 2.61 57 203563at AFAP1 Actin filament associated protein 1 NM021638 60312 1.79E-09 2.59 58 210410sat MSH5 MutS homolog 5 AF034759 4439 4.62E-06 2.59 59 226886at — — AF131846 — 1.69E-05 2.56
Rozenblatt-Rosen et al. www.pnas.org/cgi/content/short/0812023106 8of14 Ratio Con/ Probe Gene Gene CDC73 Rank set symbol Gene name Accession no. Entrez ID P value siRNA
60 1564786at — — BC043578 338667 2.43E-05 2.55 61 203513at SPG11 Spastic paraplegia 11 NM025137 80208 6.37E-07 2.55 62 225603sat — — BE962119 286144 0.0002307 2.55 63 232293at LCORL Ligand dependent nuclear receptor corepressor-like AL133031 254251 0.0002874 2.54 64 219502at NEIL3 Nei endonuclease VIII-like 3 NM018248 55247 0.0001251 2.53 65 229674at SERTAD4 SERTA domain containing 4 AL035414 56256 3.28E-05 2.52 66 225442at DDR2 Discoidin domain receptor family AI799915 4921 3.25E-05 2.52 67 225497at ATE1 Arginyltransferase 1 AL589591 11101 0.0002547 2.51 68 214681at GK glycerol kinase AI830490 2710 4.40E-05 2.51 69 231953at BPTF Bromodomain PHD finger transcription factor AK027184 2186 1.09E-06 2.51 70 225798at JAZF1 JAZF zinc finger 1 AL047908 221895 1.72E-05 2.49 71 227628at — — AL571557 493869 0.0003066 2.49 72 212486sat FYN FYN oncogene related to SRC N20923 2534 8.31E-05 2.49 73 220088at C5AR1 Complement component 5a receptor 1 NM001736 728 0.000206 2.48 74 218628at CCDC53 Coiled-coil domain containing 53 NM016053 51019 0.0001216 2.48 75 210621sat RASA1 RAS p21 protein activator M23612 5921 4.86E-05 2.48 76 225994at CPSF2 Cleavage and polyadenylation specific factor 2 AK023583 53981 4.68E-06 2.47 77 1560066at — — BC015907 — 1.72E-05 2.46 78 225332at KRTAP4–7 Keratin associated protein 4–7 BF674064 85287 0.0001016 2.46 79 227465at — — AL514301 23383 4.76E-06 2.46 80 208051sat PAIP1 Poly(A) binding protein interacting protein 1 NM006451 10605 1.12E-05 2.46 81 209064xat PAIP1 Poly(A) binding protein interacting protein 1 AL136920 10605 1.66E-05 2.46 82 225128at KDELC2 KDEL (Lys-Asp-Glu-Leu) containing 2 AL548941 143888 2.44E-05 2.44 83 204531sat BRCA1 Breast cancer 1, early onset NM007295 672 3.19E-06 2.44 84 218783at — — AL133049 — 0.0001532 2.44 85 225885at EEA1 Early endosome antigen 1 AI336848 8411 0.0001022 2.42 86 226267at JDP2 Jun dimerization protein 2 AA716425 122953 5.18E-05 2.42 87 225837at C12orf32 Chromosome 12 open reading frame 32 AL577977 83695 3.45E-05 2.41 88 219602sat FAM38B Family with sequence similarity 38, member B NM022068 63895 0.000148 2.41 89 203879at PIK3CD Phosphoinositide-3-kinase, catalytic, delta U86453 5293 9.20E-06 2.40 polypeptide 90 235545at DEPDC1 DEP domain containing 1 AI810054 55635 0.0001867 2.39 91 229431at RFXAP Regulatory factor X-associated protein AI742868 5994 6.44E-06 2.39 92 235704at — — AI307251 — 6.20E-06 2.39 93 227375at ANKRD13C Ankyrin repeat domain 13C AA152232 81573 5.27E-05 2.38 94 223598at RAD23B RAD23 homolog B AF262027 5887 1.14E-06 2.38 95 225048at PHF10 PHD finger protein 10 AI818048 55274 3.88E-06 2.38 96 204886at — — AL043646 — 7.05E-05 2.37 97 233208xat CPSF2 Cleavage and polyadenylation specific factor 2 AA583986 53981 9.39E-05 2.37 98 242245at SYDE2 Synapse defective 1 N90719 84144 3.65E-05 2.36 99 223062sat PSAT1 Phosphoserine aminotransferase 1 BC004863 29968 1.77E-05 2.35 100 202557at STCH Stress 70 protein chaperone AI718418 6782 3.30E-05 2.35 101 203956at MORC2 MORC family CW-type zinc finger 2 NM014941 22880 1.55E-06 2.34 102 219109at SPAG16 Sperm associated antigen 16 NM024532 79582 2.29E-06 2.34 103 202215sat NFYC Nuclear transcription factor Y, gamma NM014223 4802 5.22E-07 2.34 104 224665at C10orf104 Chromosome 10 open reading frame 104 BE874771 119504 2.50E-06 2.33 105 215136sat EXOSC8 Exosome component 8 AL050353 11340 5.28E-07 2.33 106 229492at VANGL1 Vang-like 1 R85437 81839 8.99E-05 2.32 107 226020sat DAB1 Disabled homolog 1 AI927931 1600 7.70E-05 2.32 108 225931sat RNF213 Ring finger protein 213 AI954660 57674 3.46E-05 2.32 109 227561at DDR2 Discoidin domain receptor family, member 2 W73819 4921 0.0002083 2.32 110 1553743at FAM119A Family with sequence similarity 119, member A NM145280 151194 0.0001034 2.32 111 223275at PRMT6 Protein arginine methyltransferase 6 BC002729 55170 1.89E-06 2.32 112 219581at TSEN2 tRNA splicing endonuclease 2 homolog NM025265 80746 4.30E-06 2.32 113 225120at PURB Purine-rich element binding protein B N25931 5814 5.11E-06 2.32 114 1558233sat ATF1 Activating transcription factor 1 CA337502 466 1.76E-07 2.31 115 225600at — — AW303300 286144 4.66E-06 2.31 116 224664at C10orf104 Chromosome 10 open reading frame 104 BE962336 119504 2.74E-05 2.31 117 227290at — — AU157881 — 4.69E-06 2.30 118 224248xat NIP30 NEFA-interacting nuclear protein NIP30 AF271785 80011 1.18E-05 2.30 119 226725at — — AI435399 — 1.54E-05 2.30
Rozenblatt-Rosen et al. www.pnas.org/cgi/content/short/0812023106 9of14 Ratio Con/ Probe Gene Gene CDC73 Rank set symbol Gene name Accession no. Entrez ID P value siRNA
120 218316at TIMM9 Translocase of inner mitochondrial membrane 9 NM012460 26520 8.10E-06 2.29 homolog 121 222844sat SRR Serine racemase AF169974 63826 2.16E-07 2.29 122 227069at FP6778 — AA806989 — 0.0001032 2.29 123 227531at — — AI816849 — 1.15E-05 2.29 124 1553972aat CBS Cystathionine-beta-synthase BC007257 875 3.85E-06 2.29 125 241937sat WDR4 WD repeat domain 4 AA577678 10785 9.18E-06 2.28 126 1554078sat DNAJA3 DnaJ (Hsp40) homolog, subfamily A, member 3 BC032100 9093 6.01E-05 2.28 127 222798at PTER Phosphotriesterase related BF112019 9317 0.0003339 2.27 128 227565at — — BE501881 — 0.0001452 2.27 129 229551xat ZNF367 Zinc finger protein 367 N62196 195828 0.0001279 2.27 130 222360at DPH5 DPH5 homolog AI291720 51611 1.05E-06 2.27 131 212906at GRAMD1B GRAM domain containing 1B BE044440 57476 6.80E-07 2.27 132 219291at DTWD1 DTW domain containing 1 NM020234 56986 2.95E-05 2.26 133 209285sat C3orf63 Chromosome 3 open reading frame 63 N38985 23272 0.0001448 2.26 134 1554447at — — BC029480 — 1.10E-06 2.26 135 204162at NDC80 NDC80 homolog, kinetochore complex component NM006101 10403 5.01E-06 2.25 136 230569at — — AA868380 57587 0.0002902 2.25 137 222890at CCDC113 Coiled-coil domain containing 113 BG054922 29070 0.0002072 2.24 138 204962sat CENPA Centromere protein A NM001809 1058 4.91E-08 2.24 139 235848xat — — N35250 — 0.0001248 2.24 140 223477sat — — AF061733 — 6.84E-07 2.23 141 224694at ANTXR1 Anthrax toxin receptor 1 AF279145 84168 9.60E-07 2.23 142 202259sat — —NM014887 10443 7.11E-07 2.23 143 205632sat PIP5K1B Phosphatidylinositol-4-phosphate 5-kinase, type I, NM003558 8395 5.87E-06 2.23 beta 144 225599sat — — AW303300 286144 8.54E-05 2.22 145 220206at ZMYM1 Zinc finger, MYM-type 1 NM024772 79830 0.0001715 2.22 146 226519sat AGXT2L2 Alanine-glyoxylate aminotransferase 2-like 2 AL561859 85007 7.88E-05 2.22 147 226308at NY-SAR-48 Sarcoma antigen NY-SAR-48 AA099118 93323 2.70E-05 2.21 148 228402at ZBED3 Zinc finger, BED-type containing 3 AI679968 84327 0.0001578 2.21 149 226355at WDR51A WD repeat domain 51A AW001089 25886 3.88E-05 2.21 150 229983at TIGD2 Tigger transposable element derived 2 AI610112 166815 9.48E-07 2.20 151 218967sat PTER Phosphotriesterase related NM030664 9317 6.68E-05 2.20 152 218911at YEATS4 YEATS domain containing 4 NM006530 8089 8.31E-07 2.19 153 218684at LRRC8D Leucine rich repeat containing 8 family, member D NM018103 55144 3.85E-05 2.19 154 223700at MND1 Meiotic nuclear divisions 1 homolog AY028916 84057 2.13E-06 2.19 155 239808at PITPNC1 Phosphatidylinositol transfer protein, cytoplasmic 1 AI084489 26207 4.69E-06 2.19 156 200053at SPAG7 Sperm associated antigen 7 NM004890 9552 1.86E-06 2.19 157 227771at LIFR Leukemia inhibitory factor receptor alpha AW592684 3977 3.33E-05 2.18 158 213220at — — AV706096 92482 6.36E-05 2.18 159 227967at TUBGCP5 Tubulin, gamma complex associated protein 5 N47680 114791 1.73E-05 2.18 160 228813at HDAC4 Histone deacetylase 4 AW206037 9759 0.0003241 2.18 161 213924at MPPE1 Metallophosphoesterase 1 BF476502 65258 9.74E-05 2.18 162 226472at PPIL4 Peptidylprolyl isomerase (cyclophilin)-like 4 BF515889 85313 4.90E-07 2.17 163 222819at CTPS2 CTP synthase II AK025654 56474 7.53E-05 2.17 164 218370sat S100PBP S100P binding protein NM022753 64766 0.0001708 2.16 165 221703at BRIP1 BRCA1 interacting protein C-terminal helicase 1 AF360549 83990 6.10E-07 2.16 166 203493sat CEP57 Centrosomal protein 57kDa AL525206 9702 2.51E-05 2.16 167 218330sat NAV2 Neuron navigator 2 NM018162 89797 0.0001473 2.16 168 202660at ITPR2 Inositol 1,4,5-triphosphate receptor, type 2 AA834576 3709 0.000272 2.16 169 225987at STEAP4 STEAP family member 4 AA650281 79689 7.46E-06 2.16 170 204653at TFAP2A Transcription factor AP-2 alpha BF343007 7020 1.49E-05 2.15 171 206101at ECM2 Extracellular matrix protein 2 NM001393 1842 0.0003057 2.15 172 203285sat HS2ST1 Heparan sulfate 2-O-sulfotransferase 1 NM012262 9653 0.0001728 2.15 173 227094at DHTKD1 Dehydrogenase E1 and transketolase domain AI934407 55526 2.34E-05 2.15 containing 1 174 231851at RAVER2 Ribonucleoprotein, PTB-binding 2 AL359613 55225 7.19E-05 2.14 175 228512at PTCD3 Pentatricopeptide repeat domain 3 AW138833 55037 5.13E-06 2.13 176 205963sat DNAJA3 DnaJ (Hsp40) homolog, subfamily A, member 3 NM005147 9093 0.0002725 2.13 177 225808at — — AA883486 124512 3.03E-05 2.13 178 200991sat SNX17 Sorting nexin 17 NM014748 9784 0.0002325 2.13
Rozenblatt-Rosen et al. www.pnas.org/cgi/content/short/0812023106 10 of 14 Ratio Con/ Probe Gene Gene CDC73 Rank set symbol Gene name Accession no. Entrez ID P value siRNA
179 209096at UBE2V2 Ubiquitin-conjugating enzyme E2 variant 2 U62136 7336 5.17E-06 2.12 180 235609at BRIP1 BRCA1 interacting protein C-terminal helicase 1 BF056791 83990 0.0001061 2.12 181 202528at GALE UDP-galactose-4-epimerase NM000403 2582 2.45E-06 2.12 182 225193at KIAA1967 KIAA1967 BC003172 57805 7.37E-05 2.12 183 225856at — — BF512028 — 0.0001056 2.12 184 236665at CCDC18 Coiled-coil domain containing 18 BF056459 343099 2.88E-07 2.12 185 218412sat GTF2IRD1 GTF2I repeat domain containing 1 NM016328 9569 4.06E-05 2.12 186 204340at TMEM187 Transmembrane protein 187 NM003492 8269 4.92E-05 2.11 187 242835sat — — AW772084 — 6.81E-05 2.11 188 209661at KIFC3 Kinesin family member C3 BC001211 3801 8.84E-06 2.11 189 212908at DNAJC16 DnaJ (Hsp40) homolog, subfamily C, member 16 AK022530 23341 2.71E-06 2.11 190 226563at SMAD2 SMAD family member 2 BF793454 4087 0.0002413 2.10 191 209916at DHTKD1 Dehydrogenase E1 and transketolase domain BC002477 55526 2.77E-05 2.10 containing 1 192 218016sat POLR3E Polymerase (RNA) III (DNA directed) polypeptide E NM018119 55718 2.14E-06 2.09 193 225012at HDLBP High density lipoprotein binding protein BE378479 3069 9.91E-05 2.09 194 218919at ZFAND1 Zinc finger, AN1-type domain 1 NM024699 79752 8.60E-06 2.08 195 230054at PRRT1 Proline-rich transmembrane protein 1 AW134492 80863 0.0001155 2.08 196 204361sat SKAP2 Src kinase associated phosphoprotein 2 AB014486 8935 1.99E-05 2.08 197 213077at YTHDC2 YTH domain containing 2 AL049305 64848 8.99E-05 2.08 198 223476sat C12orf65 Chromosome 12 open reading frame 65 AW007238 91574 0.0002582 2.08 199 223438sat PPARA Peroxisome proliferator-activated receptor alpha BC004162 5465 0.0001936 2.08 200 219336sat ASCC1 Activating signal cointegrator 1 complex subunit 1 NM015947 51008 6.45E-06 2.07 201 226762at PURB Purine-rich element binding protein B AV709094 5814 0.0002622 2.07 202 222589at NLK Nemo-like kinase AI967933 51701 0.0002063 2.06 203 218705sat SNX24 Sorting nexin 24 NM014035 28966 2.82E-05 2.06 204 229333at — — BE466139 — 0.0001238 2.06 205 213007at FANCI Fanconi anemia, complementation group I W74442 55215 0.0003217 2.06 206 236641at KIF14 Kinesin family member 14 AW183154 9928 8.83E-05 2.06 207 202294at STAG1 Stromal antigen 1 AI126490 10274 0.0002133 2.06 208 209531at GSTZ1 Glutathione transferase zeta 1 BC001453 2954 1.91E-05 2.06 209 228461at SH3MD4 SH3 multiple domains 4 AL566989 344558 4.52E-05 2.05 210 227527at MLL2 Myeloid/lymphoid or mixed-lineage leukemia 2 AI394529 8085 9.67E-05 2.05 211 225357sat INOC1 INO80 complex homolog 1 AI659419 54617 9.50E-05 2.05 212 217127at CTH Cystathionase (cystathionine gamma-lyase) AL354872 1491 6.44E-05 2.05 213 222672at LYRM4 LYR motif containing 4 AA708831 57128 7.58E-05 2.05 214 217367sat ZHX3 Zinc fingers and homeoboxes 3 AB007855 23051 0.0002525 2.05 215 202710at BET1 Blocked early in transport 1 homolog BC000899 10282 0.0001199 2.05 216 225310at RBMX RNA binding motif protein, X-linked AI928344 27316 0.00011 2.05 217 213704at RABGGTB Rab geranylgeranyltransferase, beta subunit AA129753 5876 0.0001485 2.04 218 209862sat CEP57 Centrosomal protein 57kDa BC001233 9702 0.0001212 2.04 219 228737at TOX2 TOX high mobility group box family member 2 AA211909 84969 0.0001553 2.04 220 225087at C16orf63 Chromosome 16 open reading frame 63 AA524072 123811 0.000103 2.04 221 226881at GRPEL2 GrpE-like 2, mitochondrial BF185904 134266 0.0001832 2.04 222 221685sat CCDC99 Coiled-coil domain containing 99 AF269167 54908 2.03E-07 2.04 223 212023sat MKI67 Antigen identified by monoclonal antibody Ki-67 AU147044 4288 5.53E-05 2.03 224 203710at ITPR1 Inositol 1,4,5-triphosphate receptor, type 1 NM002222 3708 1.56E-05 2.03 225 227955sat — — BE670307 — 8.47E-06 2.03 226 230748at — — AI873273 440459 3.52E-05 2.03 227 209268at VPS45 Vacuolar protein sorting 45 homolog AF165513 11311 1.22E-06 2.03 228 1558044sat — — BF692729 — 0.000105 2.03 229 226259at EXOC6 Exocyst complex component 6 AL137438 54536 0.000279 2.03 230 210813sat XRCC4 X-ray repair complementing defective repair in BC005259 7518 4.38E-05 2.03 Chinese hamster cells 4 231 224060sat DPH5 DPH5 homolog AF157319 51611 4.04E-06 2.03 232 220603sat MCTP2 Multiple C2 domains, transmembrane 2 NM018349 55784 1.87E-05 2.02
233 228385at DDX59 DEAD (Asp-Glu-Ala-Asp) box polypeptide 59 AI831738 83479 1.60E-05 2.02 234 228590at PTCD3 Pentatricopeptide repeat domain 3 AA045257 55037 0.0001407 2.02 235 204279at PSMB9 Proteasome (prosome, macropain) subunit, beta type, NM002800 5698 1.44E-06 2.02 9 236 205356at USP13 Ubiquitin specific peptidase 13 NM003940 8975 6.72E-05 2.02
Rozenblatt-Rosen et al. www.pnas.org/cgi/content/short/0812023106 11 of 14 Ratio Con/ Probe Gene Gene CDC73 Rank set symbol Gene name Accession no. Entrez ID P value siRNA
237 205053at PRIM1 Primase, DNA, polypeptide 1 NM000946 5557 3.72E-05 2.02 238 224830at NUDT21 nudix AL520677 11051 9.08E-06 2.02 239 220187at STEAP4 STEAP family member 4 NM024636 79689 1.84E-06 2.01 240 1553984sat DTYMK Deoxythymidylate kinase AF258562 1841 0.0003488 2.01 241 217895at PTCD3 Pentatricopeptide repeat domain 3 NM017952 55037 0.0001058 2.01 242 227871at CHM Choroideremia AA878377 158677 2.89E-06 2.01 243 212295sat SLC7A1 Solute carrier family 7 AA148507 6541 1.41E-05 2.01 244 228299at KCTD20 Potassium channel tetramerisation domain AV707142 222658 0.0001453 2.00 containing 20 245 223406xat NIP30 NEFA-interacting nuclear protein AF212249 80011 3.34E-05 2.00 246 221865at C9orf91 Chromosome 9 open reading frame 91 BF969986 203197 5.46E-06 2.00 247 206833sat ACYP2 Acylphosphatase 2, muscle type NM001108 98 0.0002535 2.00 248 218766sat WARS2 Tryptophanyl tRNA synthetase 2 NM015836 10352 0.0001177 2.00
Rozenblatt-Rosen et al. www.pnas.org/cgi/content/short/0812023106 12 of 14 Table S2. Genes whose expression is increased upon depletion of Cdc73 Ratio Con/ Probe Gene Gene CDC73 Rank set symbol Gene name Accession no. Entrez ID P value siRNA
1 1556346at COTL1 Coactosin-like 1 AJ227860 23406 1.53E-06 13.9 2 223908at HDAC8 Histone deacetylase 8 AF212246 55869 2.54E-05 7.72 3 214963at NUP160 Nucleoporin 160kDa AK026236 23279 1.01E-07 5.87 4 240769at METAP2 Methionyl aminopeptidase 2 AI733395 10988 0.000248 5.42 5 223666at SNX5 Sorting nexin 5 BC002724 27131 2.03E-05 4.90 6 201008sat TXNIP Thioredoxin interacting protein AA812232 10628 0.00016 4.34 7 202912at ADM Adrenomedullin NM001124 133 2.97E-07 4.32 8 238464at ANKRD36 Ankyrin repeat domain 36 AA463221 375248 4.66E-07 3.91 9 208926at NEU1 Sialidase 1 (lysosomal sialidase) U84246 4758 6.60E-05 3.83 10 201009sat TXNIP Thioredoxin interacting protein AI439556 10628 0.000119 3.70 11 201236sat BTG2 BTG family, member 2 NM006763 7832 5.93E-05 3.60 12 214960at API5 Apoptosis inhibitor 5 AF229253 8539 3.68E-07 3.58 13 228181at SLC30A1 Solute carrier family 30 AI553933 7779 1.22E-07 3.49 14 1555225at C1orf43 Chromosome 1 open reading frame 43 BC008306 25912 8.74E-07 3.46 15 224469sat INF2 Inverted formin, FH2 and WH2 domain containing BC006173 84800 1.03E-06 3.44 16 209006sat C1orf63 Chromosome 1 open reading frame 63 AF247168 57035 1.19E-08 3.36 17 231078at — — H69701 — 0.000259 3.34 18 218012at TSPYL2 TSPY-like 2 NM022117 64061 0.000156 3.32 19 38037at HBEGF Heparin-binding EGF-like growth factor M60278 1839 2.60E-06 3.32 20 207813sat FDXR Ferredoxin reductase NM004110 2232 7.06E-05 3.19 21 203821at HBEGF Heparin-binding EGF-like growth factor NM001945 1839 5.47E-05 3.18 22 243560at — — BF886735 — 9.92E-07 3.07 23 221577xat GDF15 Growth differentiation factor 15 AF003934 9518 9.63E-06 3.06 24 209007sat C1orf63 Chromosome 1 open reading frame 63 AF267856 57035 5.83E-05 2.92 25 242269at — — AW771314 440556 6.57E-05 2.85 26 1556361sat ANKRD13C Ankyrin repeat domain 13C BC016937 81573 0.000107 2.83 27 217202sat GLUL Glutamate-ammonia ligase U08626 2752 0.000225 2.80 28 236718at — — AI278445 — 0.000236 2.75 29 229106at DYNLL2 Dynein, light chain, LC8-type 2 AA401429 140735 3.53E-07 2.72 30 232216at YME1L1 YME1-like 1 AA828049 10730 0.000247 2.67 31 219689at SEMA3G Sema domain, immunoglobulin domain NM020163 56920 0.000165 2.67 32 203068at KLHL21 Kelch-like 21 NM014851 9903 4.27E-05 2.57 33 216841sat SOD2 Superoxide dismutase 2, mitochondrial X15132 6648 0.000134 2.52 34 242939at TFDP1 Transcription factor Dp-1 AI950069 7027 3.72E-06 2.51 35 200648sat GLUL Glutamate-ammonia ligase NM002065 2752 7.03E-05 2.50 36 204958at PLK3 Polo-like kinase 3 NM004073 1263 3.58E-05 2.46 37 204141at TUBB2A Tubulin, beta 2A NM001069 7280 2.57E-05 2.40 38 217682at C16orf72 Chromosome 16 open reading frame 72 AW503390 29035 3.62E-05 2.35 39 223394at SERTAD1 SERTA domain containing 1 BC002670 29950 0.0001 2.35 40 200644at MARCKS-like 1 NM023009 65108 9.07E-06 2.33 41 205960at PDK4 Pyruvate dehydrogenase kinase, isozyme 4 NM002612 5166 0.000294 2.32 42 1554014at CHD2 Chromodomain helicase DNA binding protein 2 BC007347 1106 0.00017 2.32 43 202022at ALDOC Aldolase C, fructose-bisphosphate NM005165 230 5.23E-06 2.30 44 1554719at NDUFA10 NADH dehydrogenase (ubiquinone) 1 alpha BC031332 4705 2.67E-06 2.27 subcomplex, 10 45 208290sat EIF5 Eukaryotic translation initiation factor 5 NM001969 1983 0.000208 2.26 46 209260at SFN Stratifin BC000329 2810 7.57E-05 2.25 47 214962sat NUP160 Nucleoporin 160kDa AK026236 23279 2.63E-09 2.25 48 202284sat CDKN1A Cyclin-dependent kinase inhibitor 1A NM000389 1026 8.37E-06 2.24 49 208711sat CCND1 Cyclin D1 BC000076 595 5.44E-05 2.23 50 201565sat ID2 Inhibitor of DNA binding 2 NM002166 3398 0.000228 2.23 51 204379sat FGFR3 Fibroblast growth factor receptor 3 NM000142 2261 4.74E-06 2.22 52 209189at FOS v-fos FBJ murine osteosarcoma viral oncogene homolog BC004490 2353 2.31E-06 2.21 53 233999sat TTC26 Tetratricopeptide repeat domain 26 AL137393 79989 6.37E-08 2.21 54 229566at — — AA149250 440449 1.85E-05 2.20 55 221496sat TOB2 Transducer of ERBB2, 2 D64109 10766 0.000134 2.20 56 208922sat NXF1 Nuclear RNA export factor 1 BC004904 10482 1.99E-07 2.19 57 203159at GLS Glutaminase NM014905 2744 1.41E-05 2.12 58 237464at — — AI241501 387254 9.67E-05 2.12 59 226858at CSNK1E Casein kinase 1, epsilon T51255 1454 1.87E-06 2.09 60 33323rat SFN Stratifin X57348 2810 2.21E-06 2.09
Rozenblatt-Rosen et al. www.pnas.org/cgi/content/short/0812023106 13 of 14 Ratio Con/ Probe Gene Gene CDC73 Rank set symbol Gene name Accession no. Entrez ID P value siRNA
61 236712at — — AI668706 — 0.000251 2.08 62 239615at — — AI768445 — 3.90E-05 2.08 63 225844at POLE4 Polymerase (DNA-directed), epsilon 4 (p12 subunit) NM019896 56655 7.07E-08 2.07 64 218325sat DIDO1 Death inducer-obliterator 1 NM022105 11083 1.00E-05 2.05 65 1556545at — — AK056941 — 0.000145 2.03 66 33322iat SFN Stratifin X57348 2810 1.65E-06 2.02 67 204269at PIM2 Pim-2 oncogene NM006875 11040 0.000148 2.02
Rozenblatt-Rosen et al. www.pnas.org/cgi/content/short/0812023106 14 of 14