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• W2012075768 abstract "The TFIID complex is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFs) and is the only component of the general RNA polymerase II (RNAP II) transcription machinery with intrinsic sequence-specific DNA-binding activity. Binding of transcription factor (TF) IID to the core promoter region of protein-coding genes is a key event in RNAP II transcription activation and is the first and rate-limiting step of transcription initiation complex assembly. Intense research efforts in the past have established that TFIID promoter-binding activity as well as the function of TFIID-promoter complexes is tightly regulated through dynamic TFIID interactions with positive- and negative-acting transcription regulatory proteins. However, very little is known about the role of post-translational modifications in the regulation of TFIID. Here we show that the human TFIID subunits hsTAF5 and hsTAF12 are modified by the small ubiquitin-related modifier SUMO-1 in vitro and in human cells. We identify Lys-14 in hsTAF5 and Lys-19 in hsTAF12 as the primary SUMO-1 acceptor sites and show that SUMO conjugation has no detectable effect on nuclear import or intranuclear distribution of hsTAF5 and hsTAF12. Finally, we demonstrate that purified human TFIID complex can be SUMO-1-modified in vitro at both hsTAF5 and hsTAF12. We find that SUMO-1 conjugation at hsTAF5 interferes with binding of TFIID to promoter DNA, whereas modification of hsTAF12 has no detectable effect on TFIID promoter-binding activity. Our observations suggest that reversible SUMO modification at hsTAF5 contributes to the dynamic regulation of TFIID promoter-binding activity in human cells. The TFIID complex is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFs) and is the only component of the general RNA polymerase II (RNAP II) transcription machinery with intrinsic sequence-specific DNA-binding activity. Binding of transcription factor (TF) IID to the core promoter region of protein-coding genes is a key event in RNAP II transcription activation and is the first and rate-limiting step of transcription initiation complex assembly. Intense research efforts in the past have established that TFIID promoter-binding activity as well as the function of TFIID-promoter complexes is tightly regulated through dynamic TFIID interactions with positive- and negative-acting transcription regulatory proteins. However, very little is known about the role of post-translational modifications in the regulation of TFIID. Here we show that the human TFIID subunits hsTAF5 and hsTAF12 are modified by the small ubiquitin-related modifier SUMO-1 in vitro and in human cells. We identify Lys-14 in hsTAF5 and Lys-19 in hsTAF12 as the primary SUMO-1 acceptor sites and show that SUMO conjugation has no detectable effect on nuclear import or intranuclear distribution of hsTAF5 and hsTAF12. Finally, we demonstrate that purified human TFIID complex can be SUMO-1-modified in vitro at both hsTAF5 and hsTAF12. We find that SUMO-1 conjugation at hsTAF5 interferes with binding of TFIID to promoter DNA, whereas modification of hsTAF12 has no detectable effect on TFIID promoter-binding activity. Our observations suggest that reversible SUMO modification at hsTAF5 contributes to the dynamic regulation of TFIID promoter-binding activity in human cells. To initiate mRNA synthesis, RNA polymerase II (RNAP II) 1The abbreviations used are: RNAP II, RNA polymerase II; HSV-1, herpes simplex virus 1; PIC, preinitiation complex; TBP, TATA-binding protein; TAF, TBP-associated factor; TF, transcription factor; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; HA, hemagglutinin; (HA)3, triple-HA epitope tag; aa, amino acid(s); NEM, N-ethylmaleimide; Ni-NTA, nickel-nitrilotriacetic acid; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; f:TFIID, FLAG:epitope-tagged TFIID complex; ATPγS, adenosine 5′-O-(thiotriphosphate).1The abbreviations used are: RNAP II, RNA polymerase II; HSV-1, herpes simplex virus 1; PIC, preinitiation complex; TBP, TATA-binding protein; TAF, TBP-associated factor; TF, transcription factor; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; HA, hemagglutinin; (HA)3, triple-HA epitope tag; aa, amino acid(s); NEM, N-ethylmaleimide; Ni-NTA, nickel-nitrilotriacetic acid; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; f:TFIID, FLAG:epitope-tagged TFIID complex; ATPγS, adenosine 5′-O-(thiotriphosphate). must assemble in an ordered fashion with a set of general transcription factors, TFIIA, -IIB, -IID, -IIE, -IIF, and -IIH, to form a so-called preinitiation complex (PIC) at the core promoter region of protein-coding genes (1Roeder R.G. Trends Biochem. Sci. 1996; 21: 327-335Abstract Full Text PDF PubMed Scopus (718) Google Scholar, 2Lee T.I. Young R.A. Annu. Rev. Genet. 2000; 34: 77-137Crossref PubMed Scopus (625) Google Scholar, 3Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (844) Google Scholar). The initial, and rate-limiting, step for PIC assembly is binding of TFIID, a multiprotein complex composed of the TATA-binding protein TBP and at least 13 TBP-associated factors (TAFs), to core promoter sequence elements (1Roeder R.G. Trends Biochem. Sci. 1996; 21: 327-335Abstract Full Text PDF PubMed Scopus (718) Google Scholar, 2Lee T.I. Young R.A. Annu. Rev. Genet. 2000; 34: 77-137Crossref PubMed Scopus (625) Google Scholar, 3Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (844) Google Scholar, 4Gangloff Y. Romier C. Thuault S. Werten S. Davidson I. Trends Biochem. Sci. 2001; 26: 250-257Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 5Tora L. Genes Dev. 2002; 16: 673-675Crossref PubMed Scopus (192) Google Scholar). In addition to sequence-specific DNA binding, the TFIID complex provides several enzymatic activities that all reside in its largest subunit TAF1 (6Wassarman D.A. Sauer F. J. Cell Sci. 2001; 114: 2895-2902Crossref PubMed Google Scholar). These include histone acetyltransferase activity (7Mizzen C.A. Yang X.J. Kokubo T. Brownell J.E. Bannister A.J. Owen-Hughes T. Workman J. Wang L. Berger S.L. Kouzarides T. Nakatani Y. Allis C.D. Cell. 1996; 87: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar), protein kinase activity (8Dikstein R. Ruppert S. Tjian R. Cell. 1996; 84: 781-790Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), and ubiquitin-activating/-conjugating activity (9Pham A.-D. Sauer F. Science. 2000; 289: 2357-2360Crossref PubMed Scopus (203) Google Scholar). How exactly TFIID enzymatic activities contribute to RNAP II transcription is still unknown. In accordance with its crucial role in PIC assembly, TFIID is considered one of the key targets of transcription regulation pathways. Biochemical and genetic studies have shown that TFIID binding to promoters as well as the stability and functionality of TFIID-promoter complexes are subject to regulation by gene-specific activators and repressors (10Hoffmann A. Oelgeschläger T. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8928-8935Crossref PubMed Scopus (76) Google Scholar, 11Roeder R.G. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 201-218Crossref PubMed Scopus (141) Google Scholar) and by an array of ubiquitous regulators of TFIID (TBP) activity, including NC2 (Dr1/DRAP1), Mot1/BTAF1, and the NOT complex (11Roeder R.G. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 201-218Crossref PubMed Scopus (141) Google Scholar, 12Pugh B.F. Gene (Amst.). 2000; 255: 1-14Crossref PubMed Scopus (154) Google Scholar, 13Lee T.I. Young R.A. Genes Dev. 1998; 12: 1398-1408Crossref PubMed Scopus (155) Google Scholar, 14Pereira L.A. Klejman M.P. Timmers H.T. Gene (Amst.). 2003; 315: 1-13Crossref PubMed Scopus (43) Google Scholar). In addition, TFIID subunits are subject to post-transcriptional modifications. Earliest studies demonstrated that several subunits of TFIID are phosphorylated during mitosis and that TFIID isolated from mitotic cells fails to respond to transcription activators in vitro (15Segil N. Guermah M. Hoffmann A. Roeder R.G. Heintz N. Genes Dev. 1996; 10: 2389-2400Crossref PubMed Scopus (160) Google Scholar). Results of more recent studies demonstrated gene-specific effects upon hsTAF10 methylation by the SET9 protein methyltransferase (16Kouskouti A. Scheer E. Staub A. Tora L. Talianidis I. Mol. Cell. 2004; 14: 175-182Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) and that the C-terminal domain of TAF1 is a substrate for the protein kinase CKII (17Sawa C. Nedea E. Krogan N. Wada T. Handa H. Greenblatt J. Buratowski S. Mol. Cell. Biol. 2004; 24: 4734-4742Crossref PubMed Scopus (30) Google Scholar). Exactly how and to what extent these TAF modifications modulate TFIID functions is not known. In recent years, regulation of transcription factor activity through modification with SUMO proteins has attracted considerable attention (18Seeler J.S. Dejean A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 690-699Crossref PubMed Scopus (575) Google Scholar, 19Verger A. Perdomo J. Crossley M. EMBO Rep. 2003; 4: 137-142Crossref PubMed Scopus (369) Google Scholar, 20Gill G. Genes Dev. 2004; 18: 2046-2059Crossref PubMed Scopus (618) Google Scholar, 21Gill G. Curr. Opin. Genet. Dev. 2003; 13: 108-113Crossref PubMed Scopus (193) Google Scholar). SUMO proteins are small ubiquitin-related modifiers that are conjugated to target proteins through an enzymatic pathway, which is similar to ubiquitylation, but which involves a distinct set of enzymes (22Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1376) Google Scholar, 23Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (652) Google Scholar). Like ubiquitin, SUMO proteins are expressed as inactive precursors that have to be processed by SUMO-specific proteases to expose a C-terminal double-glycine motif required for conjugation. The processed form of SUMO is activated in an ATP-dependent reaction in which a thioester bond is formed between its C-terminal glycine residue and a cysteine residue in the activating E1 enzyme (SAE1/2). Following transfer to a conjugating E2 enzyme (Ubc9), SUMO is covalently attached to a target protein lysine residue via isopeptide bond formation. Whereas SUMO E1 and E2 enzymes are sufficient to modify target proteins in vitro (23Melchior F. Annu. Rev. Cell Dev. Biol. 2000; 16: 591-626Crossref PubMed Scopus (652) Google Scholar), several SUMO E3 ligases have been described that enhance transfer of SUMO from the E2 enzyme to specific substrates (22Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1376) Google Scholar). Importantly, SUMO modification is a reversible and dynamic process, and SUMO conjugates can be removed from substrate proteins by SUMO-specific proteases (22Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1376) Google Scholar). SUMO modification occurs in many cases, but not exclusively, within a consensus motif ΨKXE, where Ψ is a large hydrophobic amino acid residue and X is any amino acid residue (22Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1376) Google Scholar, 24Rodriguez M.S. Dargemont C. Hay R.T. J. Biol. Chem. 2001; 276: 12654-12659Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar). Here we investigated SUMO-1 modification of several TFIID subunits containing the ΨKXE motif. We show that hsTAF5 and hsTAF12 are SUMO-1-modified in human cells. We further show that recombinant hsTAF1, hsTAF5, hsTAF12, and hsTBP can be SUMO-1-modified using a minimal SUMO conjugation system composed of recombinant E1 and E2 enzymes and SUMO-1. TAF5 and TAF12 can also be efficiently sumoylated in purified TFIID complex, whereas SUMO acceptor sites in TAF1 and TBP appear to become inaccessible upon assembly into TFIID. We identified the principal SUMO-1 acceptor sites in hsTAF5 and hsTAF12 and show that SUMO modification does not affect nuclear import or the global nuclear distribution of hsTAF5 and hsTAF12 in human cells. Finally, we present results of in vitro DNA binding experiments showing that SUMO modification of purified human TFIID at hsTAF5 inhibits TFIID DNA-binding activity. Cell Culture—HeLa cells were grown in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum. HeLa-6His:Myc: SUMO-1 cells (25Bailey D. O'Hare P. J. Gen. Virol. 2002; 83: 2951-2964Crossref PubMed Scopus (66) Google Scholar) were kindly provided by Peter O'Hare (Marie Curie Research Institute, Oxted, UK) and were grown in the presence of 2 μg/ml puromycin to select for 6His:Myc:SUMO-1 expression. Antibodies—Rabbit polyclonal antibodies for hsTAF5, hsTAF6, hsTAF9, hsTAF12, and hsTBP were a kind gift of R. G. Roeder (The Rockefeller University, New York, NY). Rabbit polyclonal antibody raised against the N-terminal part of VP22 (AGV31) has been described previously (26Elliott G. O'Hare P. Cell. 1997; 88: 223-233Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar). Mouse monoclonal antibodies for the HA:epitope tag (F7), hsTAF1 and hsSUMO-1, as well as rabbit polyclonal antibody for the 6His:epitope tag were purchased from Santa Cruz Biotechnology. Plasmids—pTOG5TdT(–41TATA/+33) contains five binding sites for the yeast activator GAL4 in front of a murine TdT core promoter variant with a consensus TATA box inserted 30 bp upstream of the transcription start site and was obtained by inserting the HindIII/BamHI fragment from pG5TdT(–41TATA/+33) (27Martinez E. Zhou Q. L'Etoile N.D. Oelgeschläger T. Berk A.J. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11864-11868Crossref PubMed Scopus (74) Google Scholar) into pGEM7Zf(+) (Promega) at XbaI and BamHI sites. pGEM7Zf(+)-(HA)3:hsTAF1, -(HA)3:hsTAF4, -(HA)3:hsTAF5, and -(HA)3:hsTAF12 were used for protein expression by coupled in vitro transcription/translation and were constructed by inserting the cDNA for a triple-HA epitope tag ((HA)3; amino acid sequence M(GYPYDVPDYAV)3GH) fused to the N terminus of hsTAF1, hsTAF4, hsTAF5, or hsTAF12 (28Hoffmann A. Roeder R.G. J. Biol. Chem. 1996; 271: 18194-18202Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 29Tao Y. Guermah M. Martinez E. Oelgeschläger T. Hasegawa S. Takada R. Yamamoto T. Horikoshi M. Roeder R.G. J. Biol. Chem. 1997; 272: 6714-6721Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 30Hisatake K. Hasegawa S. Takada R. Nakatani Y. Horikoshi M. Roeder R.G. Nature. 1993; 362: 179-181Crossref PubMed Scopus (149) Google Scholar), respectively, into pGEM7Zf(+) at EcoRI and BamHI sites. pcDNA5-FRT (Invitrogen) derivatives pcDNA5-(HA)3:hsTAF5 and pcDNA5-(HA)3:hsTAF12 were constructed accordingly and were used to express (HA)3-tagged hsTAF5 and hsTAF12 in mammalian cells. cDNAs for (HA)3:hsTAF5K14R and (HA)3:hsTAF12K19R were obtained by PCR-based site-directed mutagenesis and inserted into pcDNA5-FRT vectors. Plasmids pcDNA5-(HA)3:SUMO1-hsTAF5(Δ1–13) and pcDNA5-(HA)3:SUMO1-hsTAF12(Δ1–18) were used to express in human cells (HA)3:epitope-tagged versions of the mature form of hsSUMO-1 (aa 1–97) fused to the N terminus of either hsTAF5 lacking the first 13 amino acids (Δ1–13) or hsTAF12 lacking the first 18 amino acids (Δ1–18), respectively. To prevent cleavage of the SUMO-1 moiety from the fusion proteins by SUMO-specific proteases, residue Gly-97 in the C-terminal double-glycine motif of the mature form of SUMO-1 was mutated to alanine (G97A). pET11d-6His:Myc:hsSUMO-1-(1–97) was used for expression of 6His:Myc:epitope-tagged mature form of SUMO-1 (aa 1–97) in Escherichia coli. To construct pET11d-6His:Myc:hsSUMO-1-(1–97), the cDNA for 6His:Myc:SUMO-1 (aa 1–97) was amplified from pcDNA3-Myc-SUMO-1 (25Bailey D. O'Hare P. J. Gen. Virol. 2002; 83: 2951-2964Crossref PubMed Scopus (66) Google Scholar) and inserted into pET11d (Novagen) using NcoI and ClaI restriction sites. Bacterial expression vectors for 6His: TBP and 6His:TAF12, 20-kDa and 15-kDa variants, were obtained from Robert G. Roeder (The Rockefeller University, New York, NY). Infection of HeLa and HeLa-6His:Myc:SUMO-1 Cells with HSV-1— HeLa or HeLa-6His:Myc:SUMO-1 cells were seeded in 6-well plates at 3 × 105 cells/well and infected the following day with wild-type HSV-1 strain 17syn+ in Dulbecco's modified Eagle's medium containing 2% fetal bovine serum at a multiplicity of infection of 100 plaque-forming units per cell. The virus inoculum was allowed to adsorb to cells for 1 h at 37 °C, then replaced with fresh medium, and the infected cells were further incubated at 37 °C. Cells were lysed in SDS loading buffer containing 25 mmN-ethylmaleimide (NEM) 17 h post infection and analyzed by SDS-PAGE and immunoblotting. Cold Shock Treatment of Hela-6His:Myc:SUMO-1 Cells—6His:Myc: SUMO-1 cells were seeded in 6-well plates at 3 × 105 cells/well. The following day, cells were subjected to 4 °C for 10 min. After further incubation at 37 °C for 5 h cells were lysed in SDS loading buffer containing 25 mm NEM and analyzed by SDS-PAGE and immunoblotting. Transient Transfection—Transient transfections were performed using GeneJuice transfection reagent (Novagen) according to the manufacturer's protocol. Typically, transfections were performed in 10-cm tissue culture plates with 2 × 106 cells and 5 μg of plasmid DNA. Enrichment of SUMO-1-conjugated Proteins from Human Cell Lysates— Cells were lysed by 10-min incubation at 85 °C in 1 ml of preheated radioimmune precipitation assay lysis buffer (50 mm Tris·HCl, pH 7.5, 300 mm NaCl, 0.1% SDS, 1% sodium deoxycholate, 0.5% Triton X-100, 1 mm dithiothreitol) supplemented with 1 mm phenylmethylsulfonyl fluoride (Sigma), 0.2% protease inhibitor mix P-8340 (Sigma), and 25 mm NEM. After centrifugation for 20 min at 10,000 × g and 4 °C, cleared lysates were incubated for 6 h at 4°C either with 100 μl (50% slurry in lysis buffer) of Ni-NTA-agarose resin (Qiagen) to enrich proteins conjugated to 6His:Myc:SUMO-1, or with 100 μl (50% slurry in lysis buffer) of anti-FLAG antibody resin (M2-agarose, Sigma) to enrich transiently expressed FLAG:epitope-tagged proteins. After binding, the resins were rinsed with 1 ml of radioimmune precipitation assay lysis buffer containing 150 mm NaCl, 25 mm NEM, and, in the case of Ni-NTA resin, 10 mm imidazole, and eluted with 100 μl of radioimmune precipitation assay lysis buffer supplemented with either 250 mm imidazole (Ni-NTA resin) or 0.5 mg/ml FLAG peptide (M2-agarose, Sigma). 25-μl aliquots of the eluates were analyzed by SDS-PAGE and immunoblotting. SDS-PAGE and Immunoblotting—Proteins were separated by SDS-PAGE using pre-cast NuPAGE SDS gels (Invitrogen) and transferred to nitrocellulose membranes (Schleicher and Schuell) using an XCell II™ blot module (Invitrogen). Membranes were washed in H2O for 10 min, stained with Aurodye Forte (Amersham Biosciences) according to the manufacturer's instructions, blocked with 5% dried nonfat milk in TBS-T20 (10 mm Tris·Cl, pH 8.0, 25 mm NaCl, 0.1% Tween 20), and incubated with primary antibody in blocking buffer. Membranes were washed three times for 10 min in TBS-T20 and incubated for 1 h with the appropriate horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences) diluted in blocking buffer. Membranes were washed again three times for 10 min in TBS-T20 and detected with enhanced chemiluminescence reagent (ECL, Amersham Biosciences). Immunocytochemistry—HeLa cells were grown on coverslips and fixed using 4% paraformaldehyde in PBS (0.43 mm Na2HPO4, 0.14 mm KH2PO4, 13.7 mm NaCl, 0.27 mm KCl) for 10 min, washed three times for 5 min in PBS, permeabilized with 0.5% Triton X-100 in PBS for 15 min, washed again three times for 5 min in PBS, and blocked with 10% fetal bovine serum in PBS for 1 h. Coverslips were then incubated for 1 h with primary antibody (1:1000 dilution in blocking buffer), washed three times for 5 min with PBS, and further incubated for 1 h with fluorescein-conjugated secondary antibody (Vector Laboratories, 1:1000 dilution in blocking buffer). Coverslips were washed again three times with PBS and mounted on a slide using Vectashield mounting media (Vector Laboratories). Cells were visualized using an Axiovert S100TV confocal microscope (Zeiss). Images were processed using Metamorph version 4.1.4 (Universal Imaging Corp.) and Photoshop version 7 (Adobe) software. Expression and Purification of Proteins—Protein expression by coupled in vitro transcription/translation was carried out for 90 min at 30 °C in 25, μl reactions containing 0.5 μg of the relevant expression vectors, 20 μl of rabbit reticulocyte lysate (Promega), and either 40 μm cold methionine (Promega) or 15 μCi of 35S-labeled methionine (Amersham Biosciences, 1000 Ci/mmol). Recombinant hsSAE1/2, hsUbc9, 6His:TBP, and the 20-kDa and 15-kDa variants of hsTAF12 were expressed and purified as detailed previously (28Hoffmann A. Roeder R.G. J. Biol. Chem. 1996; 271: 18194-18202Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 31Oelgeschläger T. Tao Y. Kang Y.K. Roeder R.G. Mol. Cell. 1998; 1: 925-931Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 32Desterro J.M. Rodriguez M.S. Kemp G.D. Hay R.T. J. Biol. Chem. 1999; 274: 10618-10624Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 33Tatham M.H. Jaffray E. Vaughan O.A. Desterro J.M.P. Botting C.H. Naismith J.H. Hay R.T. J. Biol. Chem. 2001; 276: 35368-35374Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar). The 6His:Myc: epitope-tagged mature form of human SUMO-1 (6His:Myc:SUMO-1 (aa 1–97)) was expressed in E. coli BL21codon+ (Stratagene) and purified from bacterial lysates on Ni-NTA resin (Qiagen) according to the manufacturer's protocol. FLAG:epitope-tagged TFIID complex (f:TFIID) was purified by immunoaffinity chromatography from the HeLa 3–10 P11 (Whatman) 0.85 m KCl/DEAE-Sepharose FF (Amersham Biosciences) 0.3 m KCl TFIID fraction as described before (34Chiang C.-M. Ge H. Wang Z. Hoffmann A. Roeder R.G. EMBO J. 1993; 12: 2749-2762Crossref PubMed Scopus (171) Google Scholar). In Vitro SUMO-1 Conjugation Assay—SUMO-1 conjugation assays were performed either in buffer containing an ATP-regenerating system (50 mm Tris·HCl, pH 7.6, 5 mm MgCl2, 10 mm creatine phosphate, 3.5 units·ml–1 creatine kinase, 0.6 unit·ml–1 inorganic pyrophosphatase, 2 mm ATP) or in TMDA buffer (50 mm Tris·HCl, pH 7.5, 5 mm MgCl2, 1 mm dithiothreitol, 5 mm ATP). 20-μl reactions contained 100 ng of purified recombinant hsSAE1/2 (E1), 400 ng of purified recombinant hsUbc9 (E2), 1 μg of purified recombinant human 6His:Myc: SUMO-1-(1–97), and either 100 ng of purified recombinant protein substrate or 5 μl of coupled in vitro transcription/translation reactions. Reactions were incubated at 30 °C or 37 °C for 1–2 h as indicated. Preparation of Immobilized Promoter DNA Templates—762-bp linear biotinylated G5TdT(–41TATA/+33) promoter DNA fragments were amplified by PCR from plasmid pTOG5TdT(–41TATA/+33) using a 5′-biotinylated reverse primer. PCR products were purified from agarose gels and immobilized on M-280 streptavidin-coated magnetic beads (Dynal) according to the manufacturer's instructions. TFIID DNA Binding Assay—TFIID DNA binding was performed at 30 °C for 1 h in transcription buffer (20 mm HEPES·KOH, pH 8.4, 5 mm MgCl2, 0.05% Igepal CA-630 (Sigma), 5 mm dithiothreitol) containing 0.1 mg/ml FLAG peptide (Sigma). 50-μl binding reactions contained 10 ng of highly purified recombinant TBP or 200 ng of immunoaffinity-purified FLAG:epitope-tagged TFIID (f:TFIID), and 500 fmol of immobilized promoter template. f:TFIID-promoter complexes were separated from unbound proteins in a magnetic particle separator, washed with 200 μl of transcription buffer without FLAG peptide, and eluted from immobilized template with SDS loading buffer. Proteins present in the combined unbound and wash fractions were quantitatively recovered by binding to 16 μl of StrataClean suspension (Stratagene) for 30 min at 4 °C and elution with SDS loading buffer. In vitro SUMO-1 conjugation of f:TFIID prior to or after DNA binding was carried out in 50-μl reactions as described under “In Vitro SUMO-1 Conjugation Assay.” hsTAF5 and hsTAF12 Are Modified by SUMO-1 in Human Cells—Protein sequence analysis revealed that several human TFIID subunits, including hsTBP, hsTAF1, hsTAF2, hsTAF4, hsTAF5, and hsTAF12, contain the sumoylation target consensus sequence ΨKXE (22Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1376) Google Scholar, 24Rodriguez M.S. Dargemont C. Hay R.T. J. Biol. Chem. 2001; 276: 12654-12659Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar) (Fig. 1). We therefore wanted to investigate whether sumoylation of TFIID subunits occurs in human cells. Detection of SUMO-1 conjugates is often difficult, because only a small percentage of cellular SUMO-1 target proteins is modified at any given time (22Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1376) Google Scholar). For this reason, SUMO conjugation is generally investigated in cells that overexpress SUMO proteins, and, as a consequence, contain elevated levels of SUMO-1 conjugates. Detection of SUMO targets is further facilitated by expression of epitope-tagged versions of SUMO proteins, allowing for enrichment of SUMO conjugates from cell lysates by affinity chromatography methods. To investigate SUMO-1 modification of human TFIID subunits, we made use of a human cell line that constitutively expresses elevated levels of 6His:Myc:epitope-tagged human SUMO-1 protein (25Bailey D. O'Hare P. J. Gen. Virol. 2002; 83: 2951-2964Crossref PubMed Scopus (66) Google Scholar). Cells were lysed in the presence of SDS and N-ethylmaleimide (NEM) to inhibit SUMO-specific proteases, and cell lysates were passed over nickel-charged agarose resin (Ni-NTA-agarose, Qiagen) to enrich proteins conjugated to 6His:Myc:SUMO-1. Analysis of Ni-NTA-bound fractions by immunoblotting using TAF-specific antibodies revealed immunoreactive bands with an apparent molecular weight expected for mono-sumoylated forms of hsTAF5 and hsTAF12 present in HeLa-6His:Myc:SUMO-1 cell lysates, but not in lysates of HeLa control cells (Fig. 2A, compare lanes 2 and 3). SUMO-1-modified hsTAF5, but not SUMO-1-modified hsTAF12, could also be detected in whole cell lysates of HeLa-6His:Myc:SUMO-1 cells (Fig. 2B and data not shown). Unfortunately, we were unable to obtain evidence for SUMO-1 modification of other TFIID subunits with antibodies available to us. Previous studies reported that stress conditions such as heat shock, serum starvation, and infection by viruses affect SUMO-1 conjugation in human cells (25Bailey D. O'Hare P. J. Gen. Virol. 2002; 83: 2951-2964Crossref PubMed Scopus (66) Google Scholar, 35Hong Y. Rogers R. Matunis M.J. Mayhew C.N. Goodson M. Park-Sarge O.K. Sarge K.D. J. Biol. Chem. 2001; 276: 40263-40267Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 36Matsuzaki K. Minami T. Tojo M. Honda Y. Uchimura Y. Saitoh H. Yasuda H. Nagahiro S. Saya H. Nakao M. Biochem. Biophys. Res. Commun. 2003; 306: 32-38Crossref PubMed Scopus (43) Google Scholar). To investigate whether sumoylation of TFIID subunits in HeLa-6His:Myc: SUMO-1 is regulated, we examined the sumoylation status of endogenous hsTAF5 protein in response to different stress conditions, including heat shock, cold shock, serum starvation, and virus infection. Heat treatment (1 h at 42 °C) and serum starvation (24 h at 37 °C) had no detectable effect on hsTAF5 sumoylation levels (data not shown). In contrast, cold shock (10 min at 4 °C) and infection with herpes simplex virus 1 (HSV-1) resulted in a dramatic loss of intracellular SUMO-1 conjugates and a corresponding loss of SUMO-1-modified hsTAF5 (Fig. 2B). Importantly, neither cold shock nor HSV-1 infection led to a detectable decrease in the absolute levels of cellular hsTAF5 protein. Thus, the observed loss of SUMO-1-modified hsTAF5 in response to stress can be attributed to either down-regulation of intracellular SUMO-1-activating/-conjugating activity and/or up-regulation of intracellular SUMO isopeptidase activity. In summary, these observations demonstrate that hsTAF5 and hsTAF12 are modified by SUMO-1 in human cells and that TAF SUMO-1 conjugation observed in HeLa-6His:Myc: SUMO-1 cells is a regulated event. Recombinant hsTBP, hsTAF1, hsTAF5, and hsTAF12 Are Substrates for SUMO-1 Modification in Vitro—Next, we analyzed SUMO-1 modification of single recombinant TFIID subunits in vitro. Highly purified bacterially expressed recombinant hsTAF12 or hsTBP, or recombinant hsTAF1, hsTAF4, or hsTAF5 expressed by coupled in vitro transcription/translation, were incubated with a minimal SUMO-1 conjugation system composed of purified recombinant human E1 (SAE1/2) and E2 (Ubc9) enzymes, and purified mature SUMO-1 (aa 1–97) (32Desterro J.M. Rodriguez M.S. Kemp G.D. Hay R.T. J. Biol. Chem. 1999; 274: 10618-10624Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 33Tatham M.H. Jaffray E. Vaughan O.A. Desterro J.M.P. Botting C.H. Naismith J.H. Hay R.T. J. Biol. Chem. 2001; 276: 35368-35374Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar). Immunoblot analysis of reaction products revealed SUMO-1 modification of hsTBP, hsTAF1, hsTAF5, and the 20-kDa variant of hsTAF12 (Fig. 3, A–D). SUMO-1 modification was not detected with hsTAF4 (Fig. 3D), which contains a SUMO consensus target site (Fig. 1), and with the 15-kDa variant of hsTAF12 (Fig. 3A), which lacks the N terminus of full-length TAF12 (28Hoffmann A. Roeder R.G. J. Biol. Chem. 1996; 271: 18194-18202Abstract Full Text Full Text PDF PubMed Scopu" @default.
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• W2012075768 title "SUMO-1 Modification of Human Transcription Factor (TF) IID Complex Subunits" @default.
• W2012075768 cites W1744223828 @default.
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• W2012075768 cites W1829776195 @default.
• W2012075768 cites W1938987188 @default.
• W2012075768 cites W1971650764 @default.
• W2012075768 cites W1974551833 @default.
• W2012075768 cites W1976251488 @default.
• W2012075768 cites W1976822357 @default.
• W2012075768 cites W1987934394 @default.
• W2012075768 cites W1990115520 @default.
• W2012075768 cites W1991189921 @default.
• W2012075768 cites W1993602761 @default.
• W2012075768 cites W2002905985 @default.
• W2012075768 cites W2006098987 @default.
• W2012075768 cites W2011991138 @default.
• W2012075768 cites W2013241678 @default.
• W2012075768 cites W2013338348 @default.
• W2012075768 cites W2019006831 @default.
• W2012075768 cites W2028585544 @default.
• W2012075768 cites W2029189334 @default.
• W2012075768 cites W2040119344 @default.
• W2012075768 cites W2044799375 @default.
• W2012075768 cites W2050895547 @default.
• W2012075768 cites W2051166632 @default.
• W2012075768 cites W2053060343 @default.
• W2012075768 cites W2060817283 @default.
• W2012075768 cites W2061777334 @default.
• W2012075768 cites W2061857365 @default.
• W2012075768 cites W2075570601 @default.
• W2012075768 cites W2076532555 @default.
• W2012075768 cites W2085299836 @default.
• W2012075768 cites W2085515309 @default.
• W2012075768 cites W2086778920 @default.
• W2012075768 cites W2094209629 @default.
• W2012075768 cites W2105214322 @default.
• W2012075768 cites W2108625845 @default.
• W2012075768 cites W2108958484 @default.
• W2012075768 cites W2112441454 @default.
• W2012075768 cites W2115428749 @default.
• W2012075768 cites W2122044343 @default.
• W2012075768 cites W2129098267 @default.
• W2012075768 cites W2131428601 @default.
• W2012075768 cites W2140235682 @default.
• W2012075768 cites W2143883087 @default.
• W2012075768 cites W2145138555 @default.
• W2012075768 cites W2150084185 @default.
• W2012075768 cites W2162240068 @default.
• W2012075768 cites W2164154631 @default.
• W2012075768 cites W2167331352 @default.
• W2012075768 cites W2332271182 @default.
• W2012075768 cites W2413273374 @default.
• W2012075768 cites W4232123327 @default.
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