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Identification of Human DNA Topoisomerase I As a Cofactor For Proc. Natl. Acad. Sci. USA Vol. 90, pp. 11508-11512, December 1993 Biochemistry Identification of human DNA topoisomerase I as a cofactor for activator-dependent transcription by RNA polymerase II (transcriptional activation/repression/coactivator/acddic activator GAL4-AH) MARCUS KRETZSCHMAR*t, MICHAEL MEISTERERNSTt, AND ROBERT G. ROEDER*t *Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, NY 10021; and tLaboratorium fOr Molekulare Biologie/Genzentrum, Ludwig-Maximilians-Universitat Mflnchen, 82152 Martinsried, Federal Republic of Germany Contributed by Robert G. Roeder, September 15, 1993 ABSTRACT The transcriptional activation of eukaryotic transactivation by the acidic activator GAL4-AH. GAL4-AH class II genes by sequence-specific regulatory proteins requires contains amino acids 1-147 of the yeast activator GAL4 cofactors in addition to the general transcription factors. One (including its DNA-binding domain) fused to a short acidic cofactor (termed PC3) was purified from HeLa cells and peptide that serves as the activation domain (10-12). We have identified by sequence analysis and functional assays as human purified to near homogeneity and functionally characterized DNA topoisomerase I (EC 5.99.1.2). Under identical conditions one COF (PC3) which can both repress basal and enhance PC3 mediates both a net activation of transcription by the activator-dependent transcription. We have identified this acidic activator GAL4-AH and repression of basal transcrip- factor as DNA topoisomerase I. tion, thereby leading to a large induction oftranscription by the activator. PC3-mediated activation of transcription is depen- dent on the presence of both the GAL4-AH activation domain MATERIALS AND METHODS and the TATA-binding protein (TBP)-associated-factors Purification of PC3. As described previously (7) a HeLa (TAFs) in natural transcription factor TFIID, while repression cell nuclear extract-derived phosphocellulose 0.85 M KCl of basal transcription is observed with either TFID or the fraction was further fractionated on two successive DEAE- derived TBP alone. These results suggest novel functions, cellulose columns (DE52, Whatman), and the 0.17 M KCl apparently through distinct mechanisms, for human DNA pool ofthe second DE52 step was adjusted to 0.04 M KCl and topoisomerase I in the regulation of transcription initiation by applied to a Mono Q column (FPLC, Pharmacia). The RNA polymerase H. flowthrough was loaded without dialysis onto a heparin- Sepharose column (Pharmacia) and washed with buffer A At least three different functional classes of proteins are (identical to buffer C in ref. 8, but without Nonidet P-40) involved in the process of transcription initiation by RNA containing 40 mM KCl. PC3 was eluted with buffer A polymerase II in mammalian cells: general transcription containing 500 mM KCl and dialyzed against buffer A con- factors (GTFs), which include TFIIA, -B, -D, -E, -F, -H, and taining 100 mM KCI. For Superose-12 chromatography -G/J and RNA polymerase II itself (1); gene-specific tran- (Smart system, Pharmacia), an aliquot (250 ,g of protein) of scriptional activators (2); and another group offactors which the heparin-Sepharose fraction was concentrated to 40 ,ul by mediate the effects of activators on the general transcription centrifugation in Centricon-30 (Amicon) and loaded onto the machinery. This last group of factors includes both the column. Fractions (50 ul) were collected and dialyzed against TATA-binding protein (TBP)-associated factors (TAFs), buffer A containing 100 mM KCl. For phenyl-Superose which are tightly bound to TBP in the large TFIID complex chromatography (Smart system, Pharmacia), an aliquot (500 (3-5), and other cofactors (COFs) which can be separated ,Ag ofprotein) ofthe heparin-Sepharose fraction was adjusted chromatographically from TBP and the other GTFs (6). The to buffer B [20 mM Tris'HCl, pH 7.9 at 4°C/10% (vol/vol) molecular mechanisms by which TAFs and COFs facilitate glycerol/0.25 mM EDTA/1.5 M ammonium sulfate] and communication between activators and GTFs, and thereby loaded onto the column, which was then developed with a 1.5 enhance transcription, are still not understood, but identifi- M to 0.0 M ammonium sulfate gradient in buffer B. Fractions cation ofthe various polypeptides involved will be crucial for (100 Al) were collected and dialyzed against buffer A con- elucidation ofthis important process. Progress in understand- taining 100 mM KCI. ing the structure and function of the GTFs during formation Natural and Recombinant Transcription Factors. Recom- ofa preinitiation complex has been facilitated greatly by their binant TFIIB, TBP, and GAL4(A&94) were expressed in purification and by the cloning oftheir respective cDNAs (1). bacteria as fusion proteins with hexahistidine N-terminal tags However, while GTFs alone can lead to high basal transcrip- and purified by modifications of previously described meth- tion in vitro, activators cannot stimulate transcription effi- ods (8, 13). GAL4-AH was isolated as described (14). Other ciently in systems containing highly purified GTFs (including natural GTFs (TFIIA, TFIID, TFIIE/F/H, and RNA poly- holo-TFIID) but lacking COFs (7-9). Whereas TAFs have merase II) were purified as described (7). been identified recently by affinity methods targeting the TBP In Vitro Transcription. The GAL4-AH-activated pMRG5 molecule, and several of their cDNAs subsequently have template ("GAL" in the figures) contains five GAL4 recog- been cloned (5), there have been no reports of COFs which nition sites (14) upstream of the TATA element of human have been characterized with respect to polypeptide compo- immunodeficiency virus type 1 (7), and the control pMLA53 sition. template ('MLA" in the figures) contains the adenovirus In this study we have subjected HeLa cell nuclear extracts major late core promoter (8). Transcription assays were to biochemical fractionation in an attempt to isolate, from the conducted as described (7). If not indicated otherwise the COF fraction USA (7), components which can mediate reaction mixtures included 10 ng of each template, 0.25-0.5 The publication costs of this article were defrayed in part by page charge Abbreviations: TBP, TATA-binding protein; TAF, TBP-associated payment. This article must therefore be hereby marked "advertisement" factor; COF, cofactor; GTF, general transcription factor. in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1To whom reprint requests should be addressed. 11508 Downloaded by guest on September 23, 2021 Biochemistry: Kretzschmar et al. Proc. Natl. Acad. Sci. USA 90 (1993) 11509 ,ul of TFIIA (Mono Q fraction, 2.70 mg/ml), 5 ng of recom- R.G.R., and M.M., unpublished work), whereas the others binant TFIIB (heparin-Sepharose fraction), 0.3-0.6 pl of were isolated from the crude PC1 fraction (therefore termed TFIID (DE52 fraction, 0.35 mg/ml), 1 ,ul of TFIIE/F/H PC1 pool) following the heparin-Sepharose column (see Ma- (Sephadex A-25 fraction, 0.5 mg/ml) and 0.02-0.2 ,ul ofRNA terials and Methods). One of these coactivators has been polymerase II (DE52 fraction, 0.50 mg/ml). PC3 fractions enriched and highly purified on both hydrophobic (phenyl- (concentrations indicated in the figure legends) were added Superose) and gel filtration (Superose 12) columns. The shortly (<1 min) after GAL4-AH and just before the GTFs. fractions from phenyl-Superose chromatography were ana- Preparation ofProteins for Sequence Analysis. Purified PC3 lyzed by SDS/PAGE, and one (no. 16), which was eluted at (phenyl-Superose fraction) was subjected to SDS/PAGE. 1.3 M ammonium sulfate and contained a nearly homoge- Resolved proteins were transferred to membranes, eluted, neous 100-kDa polypeptide (Fig. 1A), was found initially to and digested with endoproteinase Lys-C, and the resulting have a positive COF activity (selectively enhancing GAL4- peptides were separated and subjected to microsequence AH-dependent transcription) that was termed PC3. On Su- analysis as described (15). perose 12, PC3 was eluted with a native molecular size of about 300 kDa (data not shown). More detailed analyses of fractions 14-18 by SDS/PAGE RESULTS (Fig. 1A) and by functional assays (Fig. 1B) revealed clearly Identification of a Positive COF (PC3) as DNA Topoisom- that the 100-kDa polypeptide and the positive COF activity erase I. During purification, COF activity was assayed in a were coeluted, with a peak in fraction 16. A minor band of reconstituted transcription system containing both purified about 80 kDa also peaked in fraction 16. Because ofits perfect natural factors (TFIIA, -D, -E, -F, and -H and RNA poly- coelution with COF activity, the 100-kDa polypeptide was merase II) and purified recombinant factors (TFIIB and subjected to sequence analysis. Two internal peptide se- GAL4-AH). Initially, a previously described COF activity, quences were obtained (PVFAPPYEPLPEN and LQLF- USA (7, 8), was obtained by fractionation of HeLa nuclear MENK) and a homology search revealed complete identity extracts on phosphocellulose and DEAE-cellulose columns, with sequences (amino acids 225-237 and 551-558, respec- and further dissection of this USA fraction led to the iden- tively) in human DNA topoisomerase I, an enzyme known to tification of several distinct COF activities. One was sepa- relax negatively supercoiled DNA through single-strand rated from PC1 (positive COF 1) on the second DE52 gradient nicking, DNA unwinding, and subsequent religation. Con- and subsequently termed PC2 (M.K., Gertraud Stelzer, sistent with this observation an assay for topoisomerase I FR.NO. M LD FT 16 18 20 21 23 25 27 14 15 16 17 18 .l. .- .- It .. .. i 96- It 66- flm-. .1I B - $m I* 45 i FR.NO. 14 15 16 17 18 33 - i i GAL-_ -W 22 - .; .- . I 1 3_ A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 C FR.NO. - 1C LD 14 15 16 17 18 20 21 23 27 rT GAL4-AH D + PC3 ++ ++ a-TopoI REL -_ GAL-_ _- sUP -_- MLA-~.- 1 24 1 2 3 4 5 6 7 8 9 10 11 12 13 FIG.
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