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Supporting Information 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 (Hs00225998࿝m1), Ab649 (BL649, A300–171A), anti-CPSF-160 (BL1896), anti- INTS6 (Hs00247179࿝m1), 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 (NP࿝078805), Paf1 (NP࿝061961), Leo1 (NP࿝620147), Ctr9 (Affymetrix arbitrary units) across all samples were considered (NP࿝055448), CPSF-160 (NP࿝037423), CPSF-100 (NP࿝059133), absent in test and control samples, and hence were omitted from CPSF-73 (NP࿝057291), symplekin (NP࿝004810), Fip1 further analysis. Student’s t tests for higher and lower mean (NP࿝112179), CstF-77 (NP࿝001317) CstF-64 (NP࿝001316), expression levels in test as compared with control samples were MLL3 (NP࿝067053), Ash2L (NP࿝004665), Rbbp5 (NP࿝005048), performed for each gene. Genes were considered to be up- WDR61 (NP࿝001074025), and CPSF-30 (NP࿝001075028). 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.
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