The EMBO Journal vol.10 no.7 pp.1853-1862, 1991
TFIID is required for in vitro transcription of the human U6 gene by RNA polymerase III
Kenneth A.Simmen, Jordi Bernues, Huw D.Parry2, Hendrik G.Stunnenberg, Anders Berkenstam, Bruno Cavallini1, Jean-Marc Egly' and lain W.Mattaj European Molecular Biology Laboratory, Meyerhofstrasse 1, Postfach 10.2209, Heidelberg, FRG and Institut de Chimie Biologique, Facult6 de M6decine, 11 Rue Humann, Strasbourg, France 2Present address: CRC Cell Cycle Genetics Group, University of Dundee, Dundee DD1 4HN, UK Communicated by I.W.Mattaj
We present evidence that transcription factor TFIID, known for its central role in transcription by RNA polymerase II, is also involved in RNA polymerase m transcription of the human U6 snRNA gene. Recombinant human TFIID, expressed either via a vaccinia virus vector in HeLa cells or in Escherichia coli, affects U6 transcription in three different in vitro assays. First, TFIID-containing fractions stimulate U6 transcription in reactions containing rate-limiting amounts of HeLa nuclear extract. Second, TFIID addition relieves transcriptional exclusion between two competing U6 templates. Third, TFUID can replace one of two heat labile fractions essential for U6 transcription. Thus, at least one basal transcription factor is involved in transcription by two different RNA polymerases. Key words: polymerase selectivity/RNA polymerase HI/transcription factor TFHD/U6 snRNA
Introduction Initiation of transcription at many eukaryotic promoters involves the association of RNA polymerase H with a group of general factors including TFIIA (STF), TFIIB (BTF3), TFHD (BTF1), TFIIE and TFIIF (RAP30/74) (for review see Saltzmann and Weinmann, 1989). Interaction of these factors with a promoter leads to the formation of a complex capable of basal level transcription. Regulation of transcription is dependent mainly on sequence-specific DNAbinding proteins (for review see Mitchell and Tjian, 1989). These act through more distant sequences to enhance or repress initiation levels from the promoter, presumably by directly or indirectly influencing the basal complex or polymerase itself (for reviews see Ptashne, 1988; Ptashne and Gann, 1990). Formation of the initiation complex is an ordered process with stable binding of TFIID to the TATA box promoter element, commonly found 25 -30 bp upstream of the transcription start site, being the first and prerequisite step for the assembly of the other general factors and RNA polymerase II into the complex (Davison et al., 1983; Reinberg et al., 1987; Buratowski et al., 1989). Yeast TFIID has been purified and the TFHD gene cloned from Saccharomyces cerevisiae (Cavallini et al., 1989; Eisenmann ( Oxford University Press
et al., 1989; Hahn et al., 1989; Horikoshi et al., 1989; Schmidt et al., 1989). Subsequently, human cDNAs for TFHD were also isolated and transcriptionally active recombinant TFIID was expressed in bacteria and HeLa cells (Peterson et al., 1990; Kao et al., 1990; Hoffmann et al., 1990). In vitro studies have shown that recombinant human TFIID can bind stably to a TATA box (Hoffmann et al., 1990; Kao et al., 1990; Peterson et al., 1990), interact directly with TFIIA and TFIIB (Peterson et al., 1990), and support basal level transcription (Hoffmann et al., 1990; Kao et al., 1990; Peterson et al., 1990). In contrast to the partially purified TFIID fraction, cloned human TFIID protein is unable to support stimulation of transcription by the activator protein Spl in reconstituted assays. This, and the discrepancy between the estimated molecular weight of native TFIID from active fractions and conceptual translation of the cloned protein led to the hypothesis that 'co-activators' exist which communicate between TFHD and regulatory proteins (Peterson et al., 1990; Ptashne and Gann, 1990; Pugh and Tjian 1990). A similar hypothesis was invoked to explain the results of in vivo transcription activation experiments (Meyer et al., 1989; Tora et al., 1989; Martin etal., 1990). A number of RNA polymerase III-transcribed eukaryotic genes have been found to have extragenic promoters exhibiting similarities to RNA polymerase H promoters (for review, see Murphy et al., 1989a). These RNA polymerase Ill genes include the U6 snRNA genes of mouse, human and Xenopus (Das et al., 1988; Kunkel and Pederson, 1988; Carbon et al., 1987) and the human 7SK gene (Murphy et al., 1987; Kleinert and Benecke, 1988). A region resembling a TATA motif in both sequence and position is present in the promoters of these genes. The sequence from -31 to -24 is CTTATAAG in the Xenopus U6 gene and TTTATATA in the human U6 gene. Deletion or mutation of the TATA element abolished polymerase HI transcription of Xenopus U6 in vivo (Mattaj et al., 1988), 7SK expression in vitro (Murphy et al., 1987) and human U6 transcription both in vitro and in vivo (Lobo and Hemandez, 1989; Kunkel and Pederson, 1989), demonstrating its functional importance. The TATA boxes of both human and Xenopus U6 genes have also been implicated as determinants of polymerase IH specificity, as their addition to the polymerase H promoter of a U2 snRNA gene converted its specificity from polymerase II to polymerase III (Mattaj et al., 1988; Lobo and Hemandez, 1989; Lobo et al., 1990). Hence, the TATA motif is important for both the efficiency and polymerase IH specificity of U6 transcription. Recently, however, it was demonstrated that the Xenopus U6 TATA element can mediate polymerase II transcription when taken out of the U6 promoter context, suggesting that polymerase III specificity of U6 transcription is not solely dependent on the TATA box (Simmen and Mattaj, 1990). Genetic studies of the requirements for pol H and pol III transcription driven 1853
K.A.Simmen et al.
by the U6 TATA element in vivo led us to speculate that there might be distinct TATA binding factors involved in transcription by the two polymerases. However, the results leading to that hypothesis were not conclusive (Simmen and Mattaj, 1990). The availability of recombinant human TFIID protein has made it possible to investigate whether TFIID is involved in U6 expression in vitro. In this report we present evidence that RNA polymerase HI transcription of human U6 in HeLa cell nuclear extracts is dependent on human TFIID. This supports earlier work which suggested that U6 forms hybrid transcription complexes with contributions from polymerase II and polymerase III factors (Bark et al., 1987; Carbon et al., 1987; Das et al., 1988; Murphy et al., 1989b; Margottin et al., 1991) and is evidence that even a basal factor can be involved in transcription by two different RNA polymerases.
Results RNA polymerase /i1 transcription of human U6 is TATA dependent A biochemical analysis of trans-acting factors involved in U6 transcription requires a faithful and efficient in vitro transcription system. The human and mouse U6 genes are transcribed well in HeLa extracts (Kunkel et al., 1986; Das et al., 1988; Reddy, 1988; Kunkel and Pederson, 1988) and we therefore used the human U6 gene for in vitro studies. Proximal promoter elements of human U6 have already been mapped both in vivo and in vitro (Kunkel and Pederson, 1988; Lobo and Hernandez, 1989). To verify that our in vitro assay was faithfully reproducing U6 transcription, a series of 10 linker-scanner mutations which altered 8 bp each across the proximal promoter between nucleotides -1 and -80 was generated. Their activity was assayed in a HeLa nuclear extract. The mutants were made in a U6 maxigene background. The maxigene (maxi) is transcribed equally with the U6 wild-type internal control (wt) (Figure 1, lane 11. Note that the U6 gene, both in vivo and in vitro, gives rise to a series of closely spaced transcripts). The activity of the mutants is displayed in lanes 1-10 where lane 1 contains the -1 to -8 mutant HLS 1, lane 2 the -9 to -16 mutant HLS 2 etc. Lane 4 shows that HLS 4, which mutates the sequence from -25 to -32 (CTTTATAT) drastically reduces transcription from the promoter. Mutants HLS 7-9 (lanes 7-9) span the proximal sequence element (PSE), a feature of all snRNA gene promoters, and are strongly deleterious. Mutants HLS 1-3, 5 and 6 all allow transcription to proceed, but at a level which is reduced compared with the wild-type gene. These results are in good agreement with previous studies of both the human and Xenopus U6 promoters (Lobo and Hemandez, 1989; Mattaj et al., 1988). With the knowledge that the in vitro transcription system was faithfully responsive to mutation, we next investigated whether the observed dependence on the TATA-like element reflected a requirement for human TFIID in RNA polymerase Ill transcription of human U6. Recombinant human TFIID protein stimulates U6 transcription in vitro Overexpression of human TFHD was effected in HeLa cells using a recombinant vaccinia virus (see Materials and methods). Following infection with either wild-type or recombinant virus, nuclear extracts were prepared from the 1854
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HeLa cells. These crude extracts did not support U6 transcription (data not shown), presumably due to virally induced alteration in host nuclear protein composition. The extracts were assayed for their ability to influence U6 transcription in three different ways. The first assay involved direct addition of the vaccinia extracts to an RNA polymerase III transcription reaction carried out in an extract similar to that used above (Figure 1), but under conditions where the rate of transcription is dependent on the amount of extract in the reaction. One might expect that if TFIID were involved in U6 transcription, a stimulation would be seen. In this experiment, U6HLS 10 (shown in Figure 1 to be transcriptionally wild-type) was co-transcribed with an internal control tRNA gene under limiting extract conditions. Weak transcription of both U6 and tRNA was seen (Figure 2A, lane 1). A T7 riboprobe (labelled RIB) was added to the extract after transcription to control for sample recovery. Addition of up to 3 Itl of extract from cells infected with wild-type vaccinia virus (wt) had no effect on transcription of U6 or tRNA (compare lanes 2-4 with lane 1). However, addition of TFIID-containing extract from cells infected with recombinant virus (vhTFIID) resulted in a clear stimulation of U6 transcription without significantly affecting tRNA activity (compare lanes 5-9 with lane 1). The stimulatory effect was maximal when 1-2 1d of vhTFIID extract was added (lanes 7 and 8). Addition of more extract resulted in a non-specific inhibition of both U6 and tRNA transcription (lane 9 and data not shown). The above experiment made use of crude nuclear extracts of vaccinia-infected cells. In order to demonstrate more clearly that the effect seen was due to TFIID itself, rather than to some secondary effect induced by the expression of TFIID in vaccinia-infected HeLa cells, we repeated the experiment making use of recombinant TFIID produced in, and partially purified from, E. coli (see Materials and methods). Control bacterial extract had no effect on either U6 or tRNA transcription (Figure 2B, lanes 1 and 6). Addition of increasing quantities of recombinant TFIIDcontaining fractions resulted, however, in a dose-dependent increase in U6 transcription (Figure 2B, lanes 2-5). A slight (maximally two-fold) stimulation of tRNA transcription was frequently observed when bacterially produced TFIID was used (Figure 2B and subsequent Figures). This was not seen with vaccinia-produced TFIID (Figure 2A) and the response was not linearly dependent on the amount of TFIID added
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Fig. 1. Linker-scanner mutants of the human U6 gene promoter. Transcription reactions in HeLa cell nuclear extract were performed as described in Materials and methods using 500 ng of each linkerscanner test plasmid (U6M) and 500 ng of U6wt as an internal standard. Lanes 1-10: HLS 1-10 with U6wt; lane 11: U6M with U6wt. The transcripts indicated are U6 maxigene (maxi) and U6 wildtype (wt), which are 112 and 106 nt long respectively. HLS 1 is mutant from positions -i to -8, HLS 2 from -9 to -16 etc.
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(Figure 2B, lanes 2-5). This effect has not been investigated further. Hence, the TFIID fractions were able to stimulate RNA polymerase III transcription. However, this ability was not universal, but promoter-specific, since U6 and tRNA responded differently. Analogy with RNA polymerase II A
A role for TFIID in U6 gene transcription
transcription suggests that the presence of an essential TATA element in the U6 promoter might be the basis for this specificity. In order to show that the TATA element was necessary to observe the effect of TFHD we compared the result of addition of bacterially-produced TFIID to HLS 10 and HLS 4 templates. HLS 4 is mutant in the TATA-like B coo
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Fig. 2. Recombinant human TFIID stimulates human U6 transcription in vitro in a TATA dependent manner. (A) Vaccinia-expressed TFIID stimulates human U6 transcription in vitro. All transcription reactions were performed as described in Materials and methods using a limiting amount of extract (3 Al; 30 yg protein) with 500 ng of U6HLS 10 as test template, and 7.5 ng of a tRNA gene, Mcetl (Ciliberto et al., 1982), as internal standard for RNA polymerase Ill transcription. Reactions were supplemented with the vaccinia wild-type or TFIID recombinant HeLa nuclear extracts as follows; lane 1: no addition; lanes 2-4: adding 0.25, 1 and 3 til of vaccinia wild-type extract (wt; 9 AgIAl protein); lanes 5-9: adding 0.25, 0.5, 1, 2 and 3 yd of vaccinia human TFHD extract (vhTFIID; 7 yg/dl protein). Alpha-amanitin was present at 2 jig/ml. The transcripts indicated are U6 maxigene (U6M) and Mcetl (tRNA). Markers in lane M are end-labelled fragments of Hpall-cut pBR322 DNA. RIB is a T7-produced riboprobe added to the reactions after transcription to control for RNA recovery. (B) Bacterially expressed human TFIID stimulates human U6 transcription in vitro. All transcriptions were performed exactly as in (A) using a limiting amount of extract (3 Ad; 30 Ag protein). Reactions were supplemented with bacterial TFIID (bhTFIID) as follows; lane 1: no addition; lanes 2-5: adding 0.125, 0.25, 0.5 and 1 ALl of bhTFIID. The reaction in lane 6 was supplemented with 2 y1 of control bacterial extract. Expression and partial purification of human TFIID and preparation of control extracts from Ecoli are described in Materials and methods. U6 maxigene (U6M) and tRNA transcripts (tRNA) are indicated. (C) Recombinant TFIID cannot stimulate transcription of a U6 TATA mutant. In vitro transcription reactions were carried out as above, using nuclear extract (4 Al; 40 jig protein) with 500 ng of U6 test templates as indicated, and 7.5 ng of Mcetl as internal standard for RNA polymerase III transcription. Alpha-amanitin was present at 2 /tg/ml. Reactions in lanes 2 and 4 were supplemented with 0.5 Al bhTFIID fraction. U6 maxigene (U6M) and tRNA transcripts (tRNA) are indicated. 1855
K.A.Simmen et al.
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Fig. 3. U6 template exclusion is relieved by recombinant TFIID. (A) Schematic outline of template exclusion assay. (B) Competition between two U6 templates in vitro is relieved upon addition of vaccinia-expressed TFIID and phosphocellulose fractions capable of tRNA transcription. Lanes 1-3 show transcription from each template individually (150 ng of either U6 template, 7.5 ng of tRNA) following pre-incubation as depicted in Figure 3A, with addition of NTPs only at to. Lanes 4-11 were pre-incubated with U6wt before addition of U6M and tRNA templates at to immediately followed by phosphocellulose (PC) fractions as indicated (3.5 I1 each fraction). Vaccinia wild-type (wt) or vaccinia recombinant TFIID (vhTFIID) extracts (1 Al) were added at to as shown in lanes 8-11. Transcripts indicated are from Mcetl (tRNA), U6 wild-type (U6) and U6 maxigene (U6M). The arrow indicates a non-specific transcript derived from the U6 plasmid vectors. Markers in lane M are end-labelled fragments of HpaII-cut pBR322 DNA. Protein concentrations are given either in Figure 2 or in Materials and methods. (C) Competition between two U6 templates in vitro is relieved upon addition of bacterially-expressed TFIID and phosphocellulose fractions capable of tRNA transcription. Transcription reactions and pre-incubations were as in Figure 3B, with a-amanitin present at 2 /4g/ml. Lanes 4-9 were pre-incubated with U6wt before addition of U6M and tRNA templates at to in conjunction with phosphocellulose (PC) fractions as indicated (3.5 ytl each fraction). Bacterial recombinant TFIID (bhTFIID) (0.5 M11) was added at to in lanes 8-9.
region of the U6 promoter. No transcription of HLS 4 was seen, even after addition of exogenous TFIID (Figure 2C, lanes 3 and 4). This is consistent with, but does not prove, that the effect of TFIID is mediated by binding to the TATA element. Recombinant TFIID can relieve exclusion between two U6 templates in vitro The above experiment suggested that TFIID was involved in U6 transcription. Since we lose U6 transcription activity on fractionation (unpublished data), it was impossible to test the involvement of TFIID directly by its addition to highly purified fractions of HeLa cell extract. Therefore, use was made of other methods. The second assay used to study TFIID involvement in U6 transcription was template exclusion. Such template exclusion assays have proved useful in defining steps in the assembly of various pre-initiation
1856
complexes (Davison et al., 1983; Lassar and Roeder, 1983; Fire et al., 1984; Van Dyke et al., 1989). The aim was to better define the proteins whose stable sequestration by a pre-incubated U6 template results in exclusion of a second U6 template. The experimental protocol is outlined schematically in Figure 3A. Components were added at t_20 and to in descending order as indicated. The ability of factor-containing fractions to relieve exclusion was thus tested by adding them immediately after the second template and ribonucleotide triphosphates at to. In Figure 3B the activity of two U6 templates and a tRNA template are shown in the absence of any second template (lanes 1-3). For unknown reasons, the pre-incubation step of the template exclusion protocol results in the production of an additional transcript (marked with an arrow). This transcript is derived from the U6 vector sequences (data not shown) and is a convenient control for sample recovery. A
A role for TFIID in U6 gene transcription
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Fig. 4. Recovery of U6 transcription in a heat treated nuclear extract requires TFIID and a phosphocellulose fraction. (A) Vaccinia-expressed human TFIID can restore RNA polymerase II transcription of the Adenovirus major late promoter in a heat treated HeLa nuclear extract. The nuclear extracts derived from vaccinia-infected cells and phosphocellulose fractions of a HeLa nuclear extract were assayed for TFIID function as determined by their ability to complement an extract depleted of TFIID by heating to 47°C for 15 min (Nakajima et al., 1988). G-less reactions with the adenovirus major late promoter were performed as described in Materials and methods. Reactions containing heat treated extract (1 tl; 10 jig protein) were complemented as follows; lane 1: no addition; lane 2: wt extract (1 pl); lane 3: vhTFIID extract (1 A1); lanes 4 and 5: PC0.35M and PCO.45M fractions (4 Al) respectively. Protein concentrations are as in Figures 2 and 3. The transcript indicated as AdMI is the correctly initiated 190 nt Gless product of AdM1404[180] transcription. Markers in lane M are end-labelled fragments of HpaII-cut pBR322 DNA. (B) tRNA transcription is heat labile and not dependent on TFIID. RNA polymerase III transcription reactions were as described in Materials and methods with 500 ng Mcetl template and 4 Al heat treated nuclear extract (HTNE) with a-amanitin present at 2 Ag/mI. Reactions were complemented with 4 p1 in total of the PC fractions (lanes 2-6) and 1 Atl bhTFIID fraction (lanes 5-6) as indicated. Lanes 7-9 show the ability of the PC fractions alone to support tRNA transcription. Protein concentrations are as in Figures 2 and 3. The tRNA transcripts are bracketed. (C) U6 transcription requires two heat labile components, TFIID and a phosphocellulose fraction. Transcription reactions were as in (B) but with 1 Mg U6M template. Reactions were complemented with PC fractions (4 M1 in total) and wt (lanes 5-8) or vhTFIID (lanes 9-11) nuclear extract (1 Al) as indicated. Lane 12 shows a test of the ability of a mixture of the two PC fractions to support U6 transcription in the absence of heat treated extract. Lane 13 is a control U6M transcription carried out in non-heated HeLa nuclear extract. U6 maxigene transcripts (U6M) are indicated. Markers in lane M are end-labelled fragments of HpaII-cut pBR322 DNA. (D) U6 transcription requires two heat labile components, TFIID and a phosphocellulose fraction. Transcription reactions were as in 4C. Reactions were complemented with PC fractions (4 A1 in total) and 1 A1 of bhTFIID fraction (lanes 4-5) as indicated. In lane 6 bacterial control extract (2 M1) was added. 1857
K.A.Simmen et al.
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TFIID is required for in vitro transcription of the human U6 gene by RNA polymerase III
Kenneth A.Simmen, Jordi Bernues, Huw D.Parry2, Hendrik G.Stunnenberg, Anders Berkenstam, Bruno Cavallini1, Jean-Marc Egly' and lain W.Mattaj European Molecular Biology Laboratory, Meyerhofstrasse 1, Postfach 10.2209, Heidelberg, FRG and Institut de Chimie Biologique, Facult6 de M6decine, 11 Rue Humann, Strasbourg, France 2Present address: CRC Cell Cycle Genetics Group, University of Dundee, Dundee DD1 4HN, UK Communicated by I.W.Mattaj
We present evidence that transcription factor TFIID, known for its central role in transcription by RNA polymerase II, is also involved in RNA polymerase m transcription of the human U6 snRNA gene. Recombinant human TFIID, expressed either via a vaccinia virus vector in HeLa cells or in Escherichia coli, affects U6 transcription in three different in vitro assays. First, TFIID-containing fractions stimulate U6 transcription in reactions containing rate-limiting amounts of HeLa nuclear extract. Second, TFIID addition relieves transcriptional exclusion between two competing U6 templates. Third, TFUID can replace one of two heat labile fractions essential for U6 transcription. Thus, at least one basal transcription factor is involved in transcription by two different RNA polymerases. Key words: polymerase selectivity/RNA polymerase HI/transcription factor TFHD/U6 snRNA
Introduction Initiation of transcription at many eukaryotic promoters involves the association of RNA polymerase H with a group of general factors including TFIIA (STF), TFIIB (BTF3), TFHD (BTF1), TFIIE and TFIIF (RAP30/74) (for review see Saltzmann and Weinmann, 1989). Interaction of these factors with a promoter leads to the formation of a complex capable of basal level transcription. Regulation of transcription is dependent mainly on sequence-specific DNAbinding proteins (for review see Mitchell and Tjian, 1989). These act through more distant sequences to enhance or repress initiation levels from the promoter, presumably by directly or indirectly influencing the basal complex or polymerase itself (for reviews see Ptashne, 1988; Ptashne and Gann, 1990). Formation of the initiation complex is an ordered process with stable binding of TFIID to the TATA box promoter element, commonly found 25 -30 bp upstream of the transcription start site, being the first and prerequisite step for the assembly of the other general factors and RNA polymerase II into the complex (Davison et al., 1983; Reinberg et al., 1987; Buratowski et al., 1989). Yeast TFIID has been purified and the TFHD gene cloned from Saccharomyces cerevisiae (Cavallini et al., 1989; Eisenmann ( Oxford University Press
et al., 1989; Hahn et al., 1989; Horikoshi et al., 1989; Schmidt et al., 1989). Subsequently, human cDNAs for TFHD were also isolated and transcriptionally active recombinant TFIID was expressed in bacteria and HeLa cells (Peterson et al., 1990; Kao et al., 1990; Hoffmann et al., 1990). In vitro studies have shown that recombinant human TFIID can bind stably to a TATA box (Hoffmann et al., 1990; Kao et al., 1990; Peterson et al., 1990), interact directly with TFIIA and TFIIB (Peterson et al., 1990), and support basal level transcription (Hoffmann et al., 1990; Kao et al., 1990; Peterson et al., 1990). In contrast to the partially purified TFIID fraction, cloned human TFIID protein is unable to support stimulation of transcription by the activator protein Spl in reconstituted assays. This, and the discrepancy between the estimated molecular weight of native TFIID from active fractions and conceptual translation of the cloned protein led to the hypothesis that 'co-activators' exist which communicate between TFHD and regulatory proteins (Peterson et al., 1990; Ptashne and Gann, 1990; Pugh and Tjian 1990). A similar hypothesis was invoked to explain the results of in vivo transcription activation experiments (Meyer et al., 1989; Tora et al., 1989; Martin etal., 1990). A number of RNA polymerase III-transcribed eukaryotic genes have been found to have extragenic promoters exhibiting similarities to RNA polymerase H promoters (for review, see Murphy et al., 1989a). These RNA polymerase Ill genes include the U6 snRNA genes of mouse, human and Xenopus (Das et al., 1988; Kunkel and Pederson, 1988; Carbon et al., 1987) and the human 7SK gene (Murphy et al., 1987; Kleinert and Benecke, 1988). A region resembling a TATA motif in both sequence and position is present in the promoters of these genes. The sequence from -31 to -24 is CTTATAAG in the Xenopus U6 gene and TTTATATA in the human U6 gene. Deletion or mutation of the TATA element abolished polymerase HI transcription of Xenopus U6 in vivo (Mattaj et al., 1988), 7SK expression in vitro (Murphy et al., 1987) and human U6 transcription both in vitro and in vivo (Lobo and Hemandez, 1989; Kunkel and Pederson, 1989), demonstrating its functional importance. The TATA boxes of both human and Xenopus U6 genes have also been implicated as determinants of polymerase IH specificity, as their addition to the polymerase H promoter of a U2 snRNA gene converted its specificity from polymerase II to polymerase III (Mattaj et al., 1988; Lobo and Hemandez, 1989; Lobo et al., 1990). Hence, the TATA motif is important for both the efficiency and polymerase IH specificity of U6 transcription. Recently, however, it was demonstrated that the Xenopus U6 TATA element can mediate polymerase II transcription when taken out of the U6 promoter context, suggesting that polymerase III specificity of U6 transcription is not solely dependent on the TATA box (Simmen and Mattaj, 1990). Genetic studies of the requirements for pol H and pol III transcription driven 1853
K.A.Simmen et al.
by the U6 TATA element in vivo led us to speculate that there might be distinct TATA binding factors involved in transcription by the two polymerases. However, the results leading to that hypothesis were not conclusive (Simmen and Mattaj, 1990). The availability of recombinant human TFIID protein has made it possible to investigate whether TFIID is involved in U6 expression in vitro. In this report we present evidence that RNA polymerase HI transcription of human U6 in HeLa cell nuclear extracts is dependent on human TFIID. This supports earlier work which suggested that U6 forms hybrid transcription complexes with contributions from polymerase II and polymerase III factors (Bark et al., 1987; Carbon et al., 1987; Das et al., 1988; Murphy et al., 1989b; Margottin et al., 1991) and is evidence that even a basal factor can be involved in transcription by two different RNA polymerases.
Results RNA polymerase /i1 transcription of human U6 is TATA dependent A biochemical analysis of trans-acting factors involved in U6 transcription requires a faithful and efficient in vitro transcription system. The human and mouse U6 genes are transcribed well in HeLa extracts (Kunkel et al., 1986; Das et al., 1988; Reddy, 1988; Kunkel and Pederson, 1988) and we therefore used the human U6 gene for in vitro studies. Proximal promoter elements of human U6 have already been mapped both in vivo and in vitro (Kunkel and Pederson, 1988; Lobo and Hernandez, 1989). To verify that our in vitro assay was faithfully reproducing U6 transcription, a series of 10 linker-scanner mutations which altered 8 bp each across the proximal promoter between nucleotides -1 and -80 was generated. Their activity was assayed in a HeLa nuclear extract. The mutants were made in a U6 maxigene background. The maxigene (maxi) is transcribed equally with the U6 wild-type internal control (wt) (Figure 1, lane 11. Note that the U6 gene, both in vivo and in vitro, gives rise to a series of closely spaced transcripts). The activity of the mutants is displayed in lanes 1-10 where lane 1 contains the -1 to -8 mutant HLS 1, lane 2 the -9 to -16 mutant HLS 2 etc. Lane 4 shows that HLS 4, which mutates the sequence from -25 to -32 (CTTTATAT) drastically reduces transcription from the promoter. Mutants HLS 7-9 (lanes 7-9) span the proximal sequence element (PSE), a feature of all snRNA gene promoters, and are strongly deleterious. Mutants HLS 1-3, 5 and 6 all allow transcription to proceed, but at a level which is reduced compared with the wild-type gene. These results are in good agreement with previous studies of both the human and Xenopus U6 promoters (Lobo and Hemandez, 1989; Mattaj et al., 1988). With the knowledge that the in vitro transcription system was faithfully responsive to mutation, we next investigated whether the observed dependence on the TATA-like element reflected a requirement for human TFIID in RNA polymerase Ill transcription of human U6. Recombinant human TFIID protein stimulates U6 transcription in vitro Overexpression of human TFHD was effected in HeLa cells using a recombinant vaccinia virus (see Materials and methods). Following infection with either wild-type or recombinant virus, nuclear extracts were prepared from the 1854
.t=X_S-*.w
HeLa cells. These crude extracts did not support U6 transcription (data not shown), presumably due to virally induced alteration in host nuclear protein composition. The extracts were assayed for their ability to influence U6 transcription in three different ways. The first assay involved direct addition of the vaccinia extracts to an RNA polymerase III transcription reaction carried out in an extract similar to that used above (Figure 1), but under conditions where the rate of transcription is dependent on the amount of extract in the reaction. One might expect that if TFIID were involved in U6 transcription, a stimulation would be seen. In this experiment, U6HLS 10 (shown in Figure 1 to be transcriptionally wild-type) was co-transcribed with an internal control tRNA gene under limiting extract conditions. Weak transcription of both U6 and tRNA was seen (Figure 2A, lane 1). A T7 riboprobe (labelled RIB) was added to the extract after transcription to control for sample recovery. Addition of up to 3 Itl of extract from cells infected with wild-type vaccinia virus (wt) had no effect on transcription of U6 or tRNA (compare lanes 2-4 with lane 1). However, addition of TFIID-containing extract from cells infected with recombinant virus (vhTFIID) resulted in a clear stimulation of U6 transcription without significantly affecting tRNA activity (compare lanes 5-9 with lane 1). The stimulatory effect was maximal when 1-2 1d of vhTFIID extract was added (lanes 7 and 8). Addition of more extract resulted in a non-specific inhibition of both U6 and tRNA transcription (lane 9 and data not shown). The above experiment made use of crude nuclear extracts of vaccinia-infected cells. In order to demonstrate more clearly that the effect seen was due to TFIID itself, rather than to some secondary effect induced by the expression of TFIID in vaccinia-infected HeLa cells, we repeated the experiment making use of recombinant TFIID produced in, and partially purified from, E. coli (see Materials and methods). Control bacterial extract had no effect on either U6 or tRNA transcription (Figure 2B, lanes 1 and 6). Addition of increasing quantities of recombinant TFIIDcontaining fractions resulted, however, in a dose-dependent increase in U6 transcription (Figure 2B, lanes 2-5). A slight (maximally two-fold) stimulation of tRNA transcription was frequently observed when bacterially produced TFIID was used (Figure 2B and subsequent Figures). This was not seen with vaccinia-produced TFIID (Figure 2A) and the response was not linearly dependent on the amount of TFIID added
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Fig. 1. Linker-scanner mutants of the human U6 gene promoter. Transcription reactions in HeLa cell nuclear extract were performed as described in Materials and methods using 500 ng of each linkerscanner test plasmid (U6M) and 500 ng of U6wt as an internal standard. Lanes 1-10: HLS 1-10 with U6wt; lane 11: U6M with U6wt. The transcripts indicated are U6 maxigene (maxi) and U6 wildtype (wt), which are 112 and 106 nt long respectively. HLS 1 is mutant from positions -i to -8, HLS 2 from -9 to -16 etc.
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(Figure 2B, lanes 2-5). This effect has not been investigated further. Hence, the TFIID fractions were able to stimulate RNA polymerase III transcription. However, this ability was not universal, but promoter-specific, since U6 and tRNA responded differently. Analogy with RNA polymerase II A
A role for TFIID in U6 gene transcription
transcription suggests that the presence of an essential TATA element in the U6 promoter might be the basis for this specificity. In order to show that the TATA element was necessary to observe the effect of TFHD we compared the result of addition of bacterially-produced TFIID to HLS 10 and HLS 4 templates. HLS 4 is mutant in the TATA-like B coo
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Fig. 2. Recombinant human TFIID stimulates human U6 transcription in vitro in a TATA dependent manner. (A) Vaccinia-expressed TFIID stimulates human U6 transcription in vitro. All transcription reactions were performed as described in Materials and methods using a limiting amount of extract (3 Al; 30 yg protein) with 500 ng of U6HLS 10 as test template, and 7.5 ng of a tRNA gene, Mcetl (Ciliberto et al., 1982), as internal standard for RNA polymerase Ill transcription. Reactions were supplemented with the vaccinia wild-type or TFIID recombinant HeLa nuclear extracts as follows; lane 1: no addition; lanes 2-4: adding 0.25, 1 and 3 til of vaccinia wild-type extract (wt; 9 AgIAl protein); lanes 5-9: adding 0.25, 0.5, 1, 2 and 3 yd of vaccinia human TFHD extract (vhTFIID; 7 yg/dl protein). Alpha-amanitin was present at 2 jig/ml. The transcripts indicated are U6 maxigene (U6M) and Mcetl (tRNA). Markers in lane M are end-labelled fragments of Hpall-cut pBR322 DNA. RIB is a T7-produced riboprobe added to the reactions after transcription to control for RNA recovery. (B) Bacterially expressed human TFIID stimulates human U6 transcription in vitro. All transcriptions were performed exactly as in (A) using a limiting amount of extract (3 Ad; 30 Ag protein). Reactions were supplemented with bacterial TFIID (bhTFIID) as follows; lane 1: no addition; lanes 2-5: adding 0.125, 0.25, 0.5 and 1 ALl of bhTFIID. The reaction in lane 6 was supplemented with 2 y1 of control bacterial extract. Expression and partial purification of human TFIID and preparation of control extracts from Ecoli are described in Materials and methods. U6 maxigene (U6M) and tRNA transcripts (tRNA) are indicated. (C) Recombinant TFIID cannot stimulate transcription of a U6 TATA mutant. In vitro transcription reactions were carried out as above, using nuclear extract (4 Al; 40 jig protein) with 500 ng of U6 test templates as indicated, and 7.5 ng of Mcetl as internal standard for RNA polymerase III transcription. Alpha-amanitin was present at 2 /tg/ml. Reactions in lanes 2 and 4 were supplemented with 0.5 Al bhTFIID fraction. U6 maxigene (U6M) and tRNA transcripts (tRNA) are indicated. 1855
K.A.Simmen et al.
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Fig. 3. U6 template exclusion is relieved by recombinant TFIID. (A) Schematic outline of template exclusion assay. (B) Competition between two U6 templates in vitro is relieved upon addition of vaccinia-expressed TFIID and phosphocellulose fractions capable of tRNA transcription. Lanes 1-3 show transcription from each template individually (150 ng of either U6 template, 7.5 ng of tRNA) following pre-incubation as depicted in Figure 3A, with addition of NTPs only at to. Lanes 4-11 were pre-incubated with U6wt before addition of U6M and tRNA templates at to immediately followed by phosphocellulose (PC) fractions as indicated (3.5 I1 each fraction). Vaccinia wild-type (wt) or vaccinia recombinant TFIID (vhTFIID) extracts (1 Al) were added at to as shown in lanes 8-11. Transcripts indicated are from Mcetl (tRNA), U6 wild-type (U6) and U6 maxigene (U6M). The arrow indicates a non-specific transcript derived from the U6 plasmid vectors. Markers in lane M are end-labelled fragments of HpaII-cut pBR322 DNA. Protein concentrations are given either in Figure 2 or in Materials and methods. (C) Competition between two U6 templates in vitro is relieved upon addition of bacterially-expressed TFIID and phosphocellulose fractions capable of tRNA transcription. Transcription reactions and pre-incubations were as in Figure 3B, with a-amanitin present at 2 /4g/ml. Lanes 4-9 were pre-incubated with U6wt before addition of U6M and tRNA templates at to in conjunction with phosphocellulose (PC) fractions as indicated (3.5 ytl each fraction). Bacterial recombinant TFIID (bhTFIID) (0.5 M11) was added at to in lanes 8-9.
region of the U6 promoter. No transcription of HLS 4 was seen, even after addition of exogenous TFIID (Figure 2C, lanes 3 and 4). This is consistent with, but does not prove, that the effect of TFIID is mediated by binding to the TATA element. Recombinant TFIID can relieve exclusion between two U6 templates in vitro The above experiment suggested that TFIID was involved in U6 transcription. Since we lose U6 transcription activity on fractionation (unpublished data), it was impossible to test the involvement of TFIID directly by its addition to highly purified fractions of HeLa cell extract. Therefore, use was made of other methods. The second assay used to study TFIID involvement in U6 transcription was template exclusion. Such template exclusion assays have proved useful in defining steps in the assembly of various pre-initiation
1856
complexes (Davison et al., 1983; Lassar and Roeder, 1983; Fire et al., 1984; Van Dyke et al., 1989). The aim was to better define the proteins whose stable sequestration by a pre-incubated U6 template results in exclusion of a second U6 template. The experimental protocol is outlined schematically in Figure 3A. Components were added at t_20 and to in descending order as indicated. The ability of factor-containing fractions to relieve exclusion was thus tested by adding them immediately after the second template and ribonucleotide triphosphates at to. In Figure 3B the activity of two U6 templates and a tRNA template are shown in the absence of any second template (lanes 1-3). For unknown reasons, the pre-incubation step of the template exclusion protocol results in the production of an additional transcript (marked with an arrow). This transcript is derived from the U6 vector sequences (data not shown) and is a convenient control for sample recovery. A
A role for TFIID in U6 gene transcription
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Fig. 4. Recovery of U6 transcription in a heat treated nuclear extract requires TFIID and a phosphocellulose fraction. (A) Vaccinia-expressed human TFIID can restore RNA polymerase II transcription of the Adenovirus major late promoter in a heat treated HeLa nuclear extract. The nuclear extracts derived from vaccinia-infected cells and phosphocellulose fractions of a HeLa nuclear extract were assayed for TFIID function as determined by their ability to complement an extract depleted of TFIID by heating to 47°C for 15 min (Nakajima et al., 1988). G-less reactions with the adenovirus major late promoter were performed as described in Materials and methods. Reactions containing heat treated extract (1 tl; 10 jig protein) were complemented as follows; lane 1: no addition; lane 2: wt extract (1 pl); lane 3: vhTFIID extract (1 A1); lanes 4 and 5: PC0.35M and PCO.45M fractions (4 Al) respectively. Protein concentrations are as in Figures 2 and 3. The transcript indicated as AdMI is the correctly initiated 190 nt Gless product of AdM1404[180] transcription. Markers in lane M are end-labelled fragments of HpaII-cut pBR322 DNA. (B) tRNA transcription is heat labile and not dependent on TFIID. RNA polymerase III transcription reactions were as described in Materials and methods with 500 ng Mcetl template and 4 Al heat treated nuclear extract (HTNE) with a-amanitin present at 2 Ag/mI. Reactions were complemented with 4 p1 in total of the PC fractions (lanes 2-6) and 1 Atl bhTFIID fraction (lanes 5-6) as indicated. Lanes 7-9 show the ability of the PC fractions alone to support tRNA transcription. Protein concentrations are as in Figures 2 and 3. The tRNA transcripts are bracketed. (C) U6 transcription requires two heat labile components, TFIID and a phosphocellulose fraction. Transcription reactions were as in (B) but with 1 Mg U6M template. Reactions were complemented with PC fractions (4 M1 in total) and wt (lanes 5-8) or vhTFIID (lanes 9-11) nuclear extract (1 Al) as indicated. Lane 12 shows a test of the ability of a mixture of the two PC fractions to support U6 transcription in the absence of heat treated extract. Lane 13 is a control U6M transcription carried out in non-heated HeLa nuclear extract. U6 maxigene transcripts (U6M) are indicated. Markers in lane M are end-labelled fragments of HpaII-cut pBR322 DNA. (D) U6 transcription requires two heat labile components, TFIID and a phosphocellulose fraction. Transcription reactions were as in 4C. Reactions were complemented with PC fractions (4 A1 in total) and 1 A1 of bhTFIID fraction (lanes 4-5) as indicated. In lane 6 bacterial control extract (2 M1) was added. 1857
K.A.Simmen et al.
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