Conditional Expression of RNA Polymerase II in Mammalian Cells

The carboxyl-terminal domain (CTD) of the large subunit of mammalian RNA polymerase II contains 52 repeats of a heptapeptide that is the target of a variety of kinases. The hyperphosphorylated CTD recruits important factors for mRNA capping, splicing, and 3′-processing. The role of the CTD for the transcription process in vivo, however, is not yet clear. We have conditionally expressed an α-amanitin-resistant large subunit with an almost entirely deleted CTD (LS*Δ5) in B-cells. These cells have a defect in global transcription of cellular genes in the presence of α-amanitin. Moreover, pol II harboring LS*Δ5 failed to transcribe up to the promoter-proximal pause sites in thehsp70A and c-fos gene promoters. The results indicate that the CTD is already required for steps that occur before promoter-proximal pausing and maturation of mRNA.

Eukaryotic mRNA synthesis is catalyzed by the multisubunit RNA polymerase II (pol II). 1 The large subunit of pol II (LS) is highly conserved among eukaryotic RNA polymerases and also shows striking homology to the large subunit of Escherichia coli RNA polymerase (1). The LS has evolved a particularly structured carboxyl-terminal domain (CTD) that is not present in other RNA polymerases (2). This CTD comprises multiple copies of a heptapeptide repeat with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The number of repeats varies from 26/27 in yeast to 52 in mouse and human cells (3). Deletion of more than half of the repeats in yeast and mouse interferes with cell viability (4 -6). Mice homozygous for a deletion of 13 repeats are smaller than wild-type littermates and have a high rate of neonatal lethality (7), suggesting that CTD is important in regulating growth during mammalian development. In cells, two forms of pol II are detectable containing either a hypophosphorylated (pol IIA) or hyperphosphorylated CTD (pol II0). Although pol IIA is consistently found in the initiation complex, pol II0 is associated with elongating complexes.
An increasing number of genes have been shown to be regulated by promoter-proximal pausing of pol II. These genes include Drosophila hsp70 and hsp26 genes, as well as the mammalian c-myc, c-fos, and immunoglobulin genes (8 -15). The passage of the paused pol II into a processive mode coincides in vivo with hyperphosphorylation of the CTD (11,16).
Recent studies suggest that the hyperphosphorylated CTD functions as a platform for the assembly of complexes that cap, splice, cleave, and polyadenylate pre-mRNA (2,17,18). Capping of mRNA occurs shortly after transcription initiation (19), preceding other mRNA processing events such as mRNA splicing and polyadenylation. The capping enzyme is not stably associated with basal transcription factors or the RNA pol II holoenzyme but is directly and specifically recruited to the hyperphosphorylated form of CTD (20 -22, 24). Similarly, several components of the splicing machinery (25,26) and related factors such as SR proteins and SR-like proteins (27)(28)(29) are recruited to pol II by the hyperphosphorylated CTD. The cleavage-polyadenylation factors CPSF and CstF specifically bind to the hyperphosphorylated CTD and copurify with pol II in a high molecular mass complex (21), suggesting that polyadenylation factors can be recruited to an RNA 3Ј-processing signal by pol II, where they dissociate from the polymerase and initiate polyadenylation. In an extension of this model, pol II is required for 3Ј-processing in vitro in the absence of transcription (30,31).
In addition to pre-mRNA maturation, hyperphosphorylation of CTD appears to play an important role in rendering pol II processive. A positive and a negative elongation factor, implicated in 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB) inhibition of transcription elongation, have been identified. DSIF (DRB sensitivity-inducing factor) is a negative elongation factor that renders elongation sensitive to DRB (32)(33)(34). It consists of p14 and p160 subunits, which have homology to the yeast Spt4-Spt5 complex (35). p160 shows homology to the bacterial elongation factor NusG (32,33) and interacts with another negative transcription factor, NELF (36). The state of CTD phosphorylation determines the negative action of DSIF on RNA elongation and provides a direct link between DSIF and the positive elongation factor P-TEFb, a CTD-specific kinase (cyclin T/cdk9), which is inhibited by DRB (37,38). Other factors, e.g. the Elongator complex, may also bind to pol II in a CTD-dependent manner (39). Taken together, a huge body of evidence suggests an important role for CTD in activation of RNA elongation and maturation of pre-mRNA in vivo.
However, it is not yet clear whether the CTD of pol II is always required for transcriptional initiation in vivo. Several studies showed that pol II with a deleted CTD is transcriptionally active in vitro (Serizawa et al. (40)) and initiates and transcribes transiently transfected genes (21,41) as well as the CUP1 gene in yeast (42). Here, we show evidence that pol II with a deleted CTD is defective in global gene transcription and is unable to transcribe up to promoter-proximal pause sites on chromosomal templates.
Cell Lines and Cell Culture-All cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 g/ml streptomycin, and 2 mM L-glutamine (Life Technologies, Inc.), which in the following is referred to as growth medium. Stable cell lines were grown in the presence of 1 mg/ml G418 and 0.1 g/ml tetracycline. Raji is a Burkitt's lymphoma B-cell line carrying a t (8,14) translocation. Raji cells were stably transfected with the constructs LS*mock, LS*wt, LS*⌬31, or LS*⌬5, and polyclonal cell lines RajiLS*mock, RajiLS*wt, RajiLS*⌬31, and RajiLS*⌬5, respectively, were established after selection with G418 and tetracycline. For the expression of the various recombinant large subunits of pol II, 2 ϫ 10 7 cells were washed three times with 40 ml of RPMI 1640 medium supplemented with 1% fetal calf serum and subsequently resuspended in 20 ml of growth medium. After 24 h ␣-amanitin (2 g/ml final concentration, Roche Molecular Biochemicals) was added to the medium to inhibit the endogenous pol II. For Northern experiments the c-fos gene was activated for 1 h by 12-O-tetradecanoylphorbol-13-acetate (TPA) (100 ng/ml, Sigma) and cycloheximide (50 g/ml, Sigma). For global and c-fos nuclear run-on experiments the cells were treated for 25 min with TPA. The hsp70A gene was activated by incubating the cells in the tissue culture flasks without lids at 43°C for 2 h.
Western Blots-Lysates containing total cellular protein were analyzed by Western blotting using 6% SDS-polyacrylamide gels (45). To verify the transfer of equal amounts of proteins the membrane (Immobilon-P, Millipore) was stained with Ponceau S. The endogenous large subunit of pol II was detected by an anti-CTD-specific monoclonal mouse antibody (8WG16) (44), LS*wt, LS*⌬31, and LS*⌬5 by an anti-HA specific monoclonal rat antibody (3F10, Roche Molecular Biochemicals). Immunocomplexes were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech) using goat anti-mouse IgG or goat anti-rat IgG horseradish peroxidase conjugate (Promega) as a secondary antibody.
Northern Blots-Northern blot analysis was performed as described elsewhere (45). 10 g of total RNA was isolated using the RNeasy Midi Kit (Qiagen) and loaded per lane. Probes were generated as follows: mouse c-fos, a 506-bp polymerase chain reaction product from exon 4 using oligonucleotides 5Ј-TGCTTTGCAGACCGAGATTGC-3Ј and 5Ј-GGTAGGTGAAGACAAAGG-AAGACG-3Ј as primers; human hsp70A, a 2.4-kb EcoRI fragment from pUCHsp70A (StressGene). mRNA analysis with the ATLAS human cancer cDNA expression array was performed as described by the manufacturer (CLONTECH). Briefly, total RNA was isolated with the Atlas pure RNA isolation kit (CLONTECH) and subsequently digested with DNase I to remove any trace of DNA. By using gene-specific primers, radioactively labeled cDNAs were generated with 10 g of total RNA. ATLAS human cancer cDNA expression arrays were hybridized with 16 ϫ 10 6 dpm labeled cDNAs overnight at 68°C. Subsequently, filters were washed three times with 1% (w/v) SDS, 2ϫ SSC, and twice with 0.5% (w/v) SDS, 0.1ϫ SSC. Filters were exposed to Kodak X-Omat AR film at Ϫ80°C with intensifying screens.
Transient Transfection Assay-A 1270-bp KpnI/PvuII fragment containing the human c-MYC promoter region was cloned upstream of the luciferase reporter gene into a Bluescript vector. Ten g of plasmid DNA was transfected into indicated cell lines by electroporation (900 microfarads, 250 V). Luciferase activity was measured after 24 h.
Nuclear Run-on Analysis-Isolation of nuclei and nuclear run-on reactions were carried out as described previously (46) with slight modifications. Briefly, cells were spun down, washed once with phosphate-buffered saline (4°C), resuspended in 10 mM Tris/HCl, pH 7.5, 10 mM MgCl 2 , 10 mM NaCl, 0.5% (v/v) Nonidet P-40 (4°C), and incubated on ice for 5 min. The nuclei were spun down at 500 ϫ g, resuspended in storage buffer (50 mM Tris/HCl, pH 8.3, 5 mM MgCl 2 , 0.1 mM EDTA/ NaOH, pH 8.0, 40% (v/v) glycerin), frozen in liquid nitrogen in portions of 100 l corresponding to 2 ϫ 10 7 nuclei, and stored at Ϫ80°C. Nuclei (100 l) were thawed on ice and incubated for 12 min at room temperature with or without 4 l of ␣-amanitin (0.1 mg/ml). Nuclei were then mixed with 100 l of reaction buffer (300 mM KCl; 5 mM MgCl 2 ; 0.5 mM of each ATP, UTP, GTP; and 100 Ci of [␣-32 P]CTP (800 Ci/mmol, 10 mCi/ml, Amersham Pharmacia Biotech); 10 mM Tris/HCl, pH 8.0, with or without 1.2% (w/v) Sarkosyl) and incubated for 15 min at 28°C. DNase I (5 l, 50 units, RNase-free, Roche Molecular Biochemicals) was added, and the incubation was continued for 12 min at room temperature. The DNase I treatment was repeated if Sarkosyl was part of the reaction buffer. After isolation of nuclear transcripts by Sephadex G-50 column filtration, labeled RNA was hybridized to DNA oligonucleotides or DNA restriction fragments (immobilized on a nylon membrane; Hybond-Nϩ; Amersham Pharmacia Biotech) at 65°C for 36 h in 5 ml of Church buffer (0.5 M sodium phosphate, pH 7.1, 7% (v/v) SDS, 0.1 mM EDTA/NaOH, pH 8.0) or to an ATLAS human 1.2 array (CLONTECH) at 68°C for 36 h in 5 ml of ExpressHyb (CLONTECH). Nylon membranes with immobilized DNA oligonucleotides or DNA restriction fragments were washed as described previously (46). ATLAS human 1.2 arrays were extensively washed in succession with 1% SDS, 2ϫ SSC; 0.5% SDS, 0.1ϫ SSC; and 1ϫ SSC, 1 mM EDTA at 45°C. Subsequently filters were treated for 15 min with 1ϫ SSC, 1 mM EDTA, 2 g/ml RNase A at 30°C and finally washed with 0.5% SDS, 0.1ϫ SSC at 45°C. Thereafter, membranes were exposed to Kodak X-Omat AR film at Ϫ80°C with intensifying screens.
DNA oligonucleotides complementary to the sense strands of human HSP70A and human c-FOS genes were synthesized according to the sequence positions described in Milner and Campbell (48)  The control oligonucleotide 7SK for pol III-specific transcription has been described previously (10).
Preparation of in Vitro Transcribed RNA by T7 RNA Polymerase-For production of uniformly labeled control RNAs, DNA fragments encompassing the c-fos region from position 67 to 533 (47) and the hsp70A region from position Ϫ65 to ϩ400 (48) were generated by polymerase chain reaction. DNA fragments were amplified with primers carrying the T7 RNA polymerase promoter for in vitro transcription by T7 RNA polymerase (Roche Molecular Biochemicals). In vitro transcription was done as recommended by the manufacturer in the presence of [␣-32 P]CTP. Full-length transcripts were isolated by polyacrylamide gel electrophoresis and used for hybridization to DNA oligonucleotides as described above.
Southern Blotting-DNA purification, digestion with suitable restriction enzymes, and Southern blotting were performed by standard methods (45). The following probe was used in Fig. 1B, a 1.2-kb BamHI-HindIII fragment of pMC1neo Poly(A) (Stratagene). The description of the plasmids used in Fig. 4 for nuclear run-on analysis is as follows: S471-1, a pUC12 vector containing a 1.5-kb SacI fragment of c-myc, was digested with SacI and gave rise to fragments 1 (pUC12) and 4 (c-myc) (49). BT34-8, a SP65 vector containing a 1.2-kb EcoRI fragment ( B ) of the Ig -heavy chain, was digested with EcoRI and gave rise to fragments 2 (SP65) and 5 ( B ) (49). BT20-21, an SP65 vector containing a 0.9-kb EcoRI/SacI fragment ( A ) of the Ig -heavy chain, was digested with EcoRI/SacI and gave rise to fragments 3 (SP65) and 6 ( A ) (49).

Conditional Expression of the Large Subunit of pol II-
The gene for the ␣-amanitin-resistant, large subunit of pol II (LS*wt) and CTD deletion mutants thereof (⌬31 consisting of only 31 repeats; ⌬5 of 5 repeats) were cloned under the control of a tetracycline (Tc)-regulated promoter into an episomal, Epstein-Barr virus-derived vector (LS*mock) (Fig. 1A). Plasmids were stably transfected into the Burkitt lymphoma cell line, Raji, and polyclonal cell lines resistant to neomycin were isolated (RajiLS*mock, RajiLS*wt, RajiLS*⌬31, and RajiLS*⌬5). The number of episomes in the cell lines varied from 40 to 70 copies/cell (Fig. 1B).
Tc-regulated expression of the LS was checked for each cell line in the presence and absence of ␣-amanitin. Expression of the recombinant LS was observed as early as 6 h after removal of Tc (Fig. 2, B-D) and was maximal after 24 h. At this time, ␣-amanitin was added. The complete inhibition of transcriptional activity of the endogenous pol II was achieved after an additional 24 h (51, data not shown) and was accompanied by a substantial degradation of the endogenous LS ( Fig. 2A). LS*wt and LS*⌬31 were well expressed at day 2 and day 3 in the presence of ␣-amanitin (Fig. 2, B and C). In contrast, expression of LS*⌬5 was high at day 2 but declined at day 3 in the presence of ␣-amanitin (Fig. 2D), suggesting that LS*⌬5 cannot support its own expression. Sequence analysis revealed no additional mutations in LS*⌬5 except the deletion of CTD (data not shown).
The CTD-less subunit has previously been reported to display transcriptional activity on transiently transfected reporter gene constructs (21,41). Its transcriptional activity in Ra-jiLS*⌬5 cells was tested after transient transfection of plasmid DNA carrying a c-myc promoter cloned upstream of the luciferase gene. Cells expressing LS*⌬5 (ϪTc) showed an ϳ20-fold higher luciferase activity as cells non-expressing LS*⌬5 (ϩTc) (Fig. 2E). In a similar experiment, a CMV promoter-driven luciferase construct showed a 46-fold higher luciferase activity in cells expressing LS*⌬5 (data not shown). The transfection data indicate that the CTD-less large subunit is assembled into a transcriptionally active complex in RajiLS*⌬5 cells.
Viability of Cells Expressing CTD Deletion Mutants-Truncation of the CTD has been reported to affect viability of cells to various extents (4). Raji cells expressing LS*wt with a full size CTD proliferated in the presence of ␣-amanitin. At the beginning, the cells run through a crises with reduced viability when they were grown for the first time in the presence of ␣-amanitin (Fig. 3A). After a period of 3 weeks, however, cells proliferated at a similiar rate as RajiLS*mock cells in the absence of ␣-amanitin (data not shown). Repression of LS*wt after 45 days by addition of Tc resulted in growth inhibition of cells, indicating that LS*wt was still required for growth (Fig. 3A).
Proliferation and viability of RajiLS*⌬31 cells was significantly reduced by ␣-amanitin. The viability of cells steadily decreased in the presence of ␣-amanitin (Fig. 3A), whereas the total number of living cells remained relatively constant at the beginning (Fig. 3B). Thus, the cells could still proliferate for a limited time in the presence of ␣-amanitin. RajiLS*mock and RajiLS*⌬5 cells died between day 4 and day 8 after addition of ␣-amanitin. In conclusion, expression of LS*wt from a heterologous promoter could fully restore viability and proliferation in the presence of ␣-amanitin, LS*⌬5 failed, and LS*⌬31 had an intermediate phenotype. Expression of the VP16 fusion protein (TcTA) had no apparent effect on proliferation of RajiLS*mock cells (data not shown).
Dominant-negative Phenotype of LS*⌬5-We next tested whether expression of LS*⌬31 and LS*⌬5 had a dominantnegative effect on transcription of the endogenous pol II. For this purpose, nuclear run-on experiments were performed with nuclei of cells expressing the endogenous LS together with LS*wt, LS*⌬31, or LS*⌬5 (Fig. 4). The transcription rate for two genes was determined. One DNA fragment covering c-myc exon 2 (Fig. 4A, fragment 4) and two fragments corresponding to the Ig heavy chain gene locus (fragments 5 and 6) were separated by agarose gel electrophoresis and blotted onto a nylon membrane. Fragments 1-3 correspond to vector sequences of pBR322 derivatives. Run-on RNA of untransfected Raji cells does not give rise to vector-specific hybridization signals (Ref. 49 and data not shown), whereas run-on RNA derived from RajiLS*mock cells produced clear signals (Fig.  4C). These signals are derived from RNA transcribed from sequences of the episomal vector backbone in transfected cells. The vector-specific signals further increased in RajiLS*wt cells (Fig. 4D) consistent with the observation that LS*wt was strongly expressed at day 1 after induction (Fig. 2B). In contrast, expression of LS*⌬5 almost completely repressed the transcriptional run-on activity for episomal sequences in Raji cells (Fig. 4F). Expression of LS*⌬31 showed an intermediate effect (Fig. 4E). Expression of LS*wt, LS*⌬31, and LS*⌬5 did not affect the transcription rates for the c-myc and Ig genes (Fig. 4, D-F). Taken together, LS*⌬5 showed a strong dominant-negative effect on transcription of vector sequences, whereas it showed no effect on transcription of two chromosomal genes.
Treatment of Raji Cells with ␣-Amanitin Does Not Affect Steady-state mRNA Levels in Raji Cells-Deletion of CTD has been shown to affect mRNA 5Ј-capping and splicing and 3Јprocessing of the primary transcript but appears to have little effect on the production of pre-mRNA (21,22). This is consistent with the observation that transcription by pol II in in vitro We used DNA microarrays to analyze the extent to which LS*⌬5 is able to restore gene expression in Raji cells. For this purpose, expression of LS*wt and LS*⌬5 was induced in Raji cells according to the scheme (Fig. 5A). Total RNA was isolated 24 h after addition of ␣-amanitin. The levels of steady-state RNAs were analyzed for 588 genes using Atlas human cancer cDNA expression arrays (CLONTECH). RajiLS*mock and RajiLS*wt cells revealed no significant difference in steadystate mRNA levels after treatment with ␣-amanitin for 24 h (Fig. 5B). This was surprising since pol II activity is entirely blocked 24 h after addition of ␣-amanitin. The result raises the question whether inhibition of pol II transcription in RajiLS*mock cells does lead to a stabilization of mRNA. Thus, the analysis of steady-state mRNA turned out to be unsuitable to study the transcriptional activity of LS*⌬5.
Deletion of CTD Affects Global Gene Transcription-Deletion of the CTD has no general effect on the transcriptional activity of transiently transfected reporter constructs (21,22,41). To address the question of the transcriptional activity of LS*⌬5 on chromosomal genes more directly, we performed run-on exper-iments with nuclei of RajiLS*mock, RajiLS*wt, and RajiLS*⌬5 cells in the presence and absence of ␣-amanitin. Run-on RNAs were hybridized to 1176 gene and 15 control probes spotted on to a nylon filter (Atlas 1.2 DNA array, CLONTECH) (Fig. 6,  A-D). With this technique 48% of the gene probes generated a positive and reproducible transcription signal for RajiLS*mock cells in the absence of ␣-amanitin (Fig. 6A, data not shown). Almost all signals disappeared, if the run-on reaction was performed in the presence of ␣-amanitin (Fig. 6B, data not shown). RajiLS*wt cells gave a very similar pattern of transcriptional active genes in the presence of ␣-amanitin as RajiLS*mock cells in the absence ␣-amanitin (Fig. 5C, data not shown). It should be noted that cells expressing RajiLS*wt produced reproducibly higher transcription signals for a small number of gene probes (data not shown). This suggests that this polymerase may have lost a negative control. RajiLS*⌬5 cells almost entirely failed to produce specific transcription signals in the presence of ␣-amanitin. The few observed signals that were reproducibly detectable could be divided into two classes. To class 1 belong the gene probes that also produced signals in RajiLS*mock cells in the presence of ␣-amanitin (Fig.  5, spot 12/l). Signals for spots of class 2 are observed in Ra-jiLS*⌬5 cells but not in RajiLS*mock cells (spots at position 2/g and 2/l). The intensity of the signals for these spots is, however, only 5-10% of the signal intensity observed in RajiLS*wt cells, indicating that LS*⌬5 can transcribe these genes only at low rate. We also tested the pol II-specific run-on activity of RajiLS*⌬31 cells in the absence and presence of ␣-amanitin. The results were very similar to the results obtained with RajiLS*wt cells and gave no indication for genes that were significantly altered in the transcription rate (data not shown).
The almost complete disappearance of pol II-specific transcription signals for probes on filters B and D in ␣-amanitintreated cells is not due to an inefficient labeling of RNAs in the run-on reaction. Nuclei incorporated still a high amount of label, indicating that pol I and pol III transcription were not affected by ␣-amanitin in the nuclei of RajiLS*mock and Ra-jiLS*⌬5 cells (Fig. 6E). In conclusion, deletion of the CTD affects global run-on activity of pol II. We next asked, whether deletion of CTD affected initiation and/or elongation of RNA from promoter-proximal pause sites.
Deletion of CTD Affects Initiation of pol II-We have shown previously that transiently expressed LS*wt but not LS*⌬5 is able to induce expression of the hsp70A and c-fos genes in 293 cells after appropriate stimuli (51). We confirmed this observation for Raji cells. LS*wt and LS*⌬31 induced transcription of the hsp70A gene after heat shock and c-fos transcription after phorbol ester activation, whereas LS*⌬5 could not (Fig. 7, A  and B). hsp70A and c-fos belong to the class of genes that are regulated by promoter-proximal pausing of pol II (12,14,52). In the uninduced stage, pol II pauses in a region approximately 20 -40 bp downstream of the initiation site. Upon activation of the gene, the CTD of the paused pol II presumably becomes hyperphosphorylated, rendering pol II processive for transcription and recruiting mRNA maturation factors. The hsp70A and c-fos genes were used to determine if pol II with a deleted CTD is still able to initiate and to transcribe up to the respective promoter-proximal pause site. The distribution of pol II in the promoter-proximal region of the hsp70A and c-fos genes was studied in nuclear run-on experiments. To determine the position from which pol II continues transcription in isolated nuclei, labeled run-on RNAs were hybridized to a set of antisense oligonucleotides, each 50 nucleotides long, spanning the respective promoter region.
In RajiLS*mock, RajiLS*wt, and RajiLS*⌬5 cells, a strong transcription signal is produced for hsp70A on oligonucleotide C, which corresponds to the previously described paused pol II site at the hsp70A promoter (Fig. 7C, lanes 1, 5, and 9). Upon heat shock, processivity of pol II is induced, and transcription signals on oligonucleotides D to I strongly increase (lanes 2, 6, and 10). For the c-fos gene, similar results were obtained. In the uninduced situation, promoter-proximal pausing of pol II was observed in cells expressing LS*wt in the presence of ␣-amanitin (Fig. 7D, lane 7) but not in RajiLS*mock and RajiLS*⌬5 cells (lanes 3 and 11, respectively). As expected, c-fos was inducible by TPA only in RajiLS*wt cells but not in RajiLS*⌬5 cells (lanes 8 and 12, respectively). From these results we conclude that pol II with a deleted CTD is unable to initiate and transcribe to the respective pause site proximal of the hsp70A and c-fos promoters. DISCUSSION We have established human B-cell lines conditionally expressing the large subunit of pol II to study the effect of CTD deletions on transcription initiation and promoter-proximal pausing. Long term cultures could be established in the presence of ␣-amanitin from Raji cells expressing LS*wt but not from cells expressing LS*⌬31 and LS*⌬5. Raji cells expressing LS*wt initially run through a crisis but thereafter proliferated quite normally in the presence of ␣-amanitin, with a doubling time of 30 h. Thus, constitutive expression of LS*wt from a heterologous promoter does not conflict with proliferation and long term survival of Raji cells. Currently we do not know the reason for the initial crisis. A possible reason for the crisis could be the point mutation in LS*wt that confers ␣-amanitin resistance. Cells expressing this mutant may have an altered gene expression pattern and require an adaptation phase for growth. In contrast, expression of LS*⌬31, even though it initially prolonged survival, and LS*⌬5 failed to replace the endogenous LS in regard to long term survival. This is in line with an earlier report that ratL6 myoblasts expressing LS*⌬31 showed a limited growth in a colony assay, whereas LS*⌬5 failed to support growth (4).
In this study we were particularly interested in the question how deletion of the CTD affects global gene expression in Raji cells. LS*⌬5 has been shown to be defective in enhancer-driven expression of transiently transfected genes (41). The mutant has also been reported to be unable to facilitate pre-mRNA maturation in transient transfection experiments (21). However, LS*⌬5 is able to transcribe transiently transfected CMV promoter and SP1-driven promoter constructs (41). These results could be confirmed for the CMV promoter and in addition for the c-myc promoter in RajiLS*⌬5 cells. pol II with a deleted CTD has also been reported to initiate at the Drosophila hsp70 gene promoter and to transcribe to the promoter-proximal pause site in vitro (53). Thus, it was not clear whether LS*⌬5 has a general defect in initiation and/or elongation.
A first approach to compare steady-state mRNA levels in cells expressing LS*wt and LS*⌬5 turned out to be unsuccessful, because treatment with ␣-amanitin for 24 h did not lead to significant changes in steady-state mRNA levels. This unexpected result suggested that inhibition of pol II transcription by ␣-amanitin may lead to a global stabilization of mRNA in Raji cells. Stabilization of mRNA by protein synthesis inhibitors, like cycloheximide, is a well known phenomenon that most likely results from the inhibition of the synthesis of factors required for mRNA degradation. Stabilization of mRNA by transcription inhibitors has been reported for the transferrin receptor gene (54) and multidrug resistance gene mdr1 (55) but is not yet known to be a general phenomenon. This observation certainly deserves further analysis. Here, this phenomenon made a comparison of mRNAs levels in cells expressing LS*wt and LS*⌬5 impossible.
As a consequence we measured the transcription rate of genes in nuclear run-on experiments. More than 500 transcriptionally active genes in phorbol ester-stimulated Raji cells were analyzed. These genes were also found to be transcribed in cells expressing LS*wt but were repressed or strongly reduced in transcription in cells expressing LS*⌬5, indicating that LS*⌬5 has a severe and general defect in transcription in vivo. We did not detect a single gene whose transcription was not severely affected. Notably, the low but significant transcription signals that were still detectable for a few genes in RajiLS*⌬5 but not in RajiLS*mock cells in the presence of ␣-amanitin indicate that the mutant LS*⌬5 is not transcriptionally dead. This is in agreement with the transient transfection experiments by us and others (21,22,41). The ability of LS*⌬5 to transcribe from some promoters in transfection may rely on the fact that transiently transfected DNA does not establish proper chromatin and may be easily accessible for the transcriptional machinery. Genes packaged in regular chromatin may be less accessible to the transcriptional machinery, particularly if the CTD is deleted. In conclusion, a minimal size of the CTD appears to be required for the transcription of all mammalian genes. However, we cannot rule out that very few of the ϳ100,000 estimated genes do not require the function of CTD for its transcription. For example, the CUP1 gene in yeast has been reported to be transcribed quite efficiently by pol II with a deleted CTD (42).
What could be the reason that pol II without a CTD is unable to transcribe chromatin-packaged genes? In addition to binding of pre-mRNA maturation factors, CTD may permit recruitment of factors to the transcriptional machinery, either directly or indirectly, that allows transcription of chromatin-packaged DNA templates. Such a factor could be the Elongator complex, for example, which harbors HAT activity and binds to pol II FIG. 6. Transcriptional activity of LS*⌬5 in global nuclear run-on experiment. RajiLS*mock, RajiLS*wt, and RajiLS*⌬5 cells were transferred for 24 h in Tc-free growth medium and subsequently cultivated in the absence (A) or presence of ␣-amanitin (␣-am.) (B-D) for additional 24 h. After treating the cells for 20 min with TPA, nuclei were isolated, and nuclear run-on reactions were carried out in the presence of Sarkosyl. Nuclei of ␣-amanitin-treated cells were also treated with ␣-amanitin in the run-on reaction as described under "Materials and Methods." Nuclear run-on RNAs were hybridized to ATLAS human 1.2 arrays (CLONTECH). Filters were washed and exposed to Kodak XAR-5 films. Signals were quantified by using a PhosphorImager system. A representative part of the filter (quadrant A) is shown. The total incorporation of radioactivity from [␣-32 P]CTP was determined from four independent nuclear run-on reactions after isolation of labeled nuclear RNA and normalized to incorporated radioactivity from RajiLS*mock nuclei not treated with ␣-amanitin (E).
only if the CTD is hyperphosphorylated (39). Alternatively, the phosphorylated CTD may be required to release inhibitory factors from pol II, e.g. DSIF, which interfere with elongation (32,33,36). Since phosphorylation of the CTD is assumed to be a critical step in activation of promoter-proximal paused pol II, we asked if the transition from a paused to an elongating pol II is affected if CTD is deleted. At the hsp70A and the c-fos promoters, pol II with a deleted CTD was unable to produce a transcription signal in nuclear run-ons, neither in uninduced nor induced cells. The run-on reactions have been carried out in the presence of Sarkosyl, which has been described to release nucleosomes from DNA (23) and activate paused pol II (8). If pol II with a deleted CTD would be present at the pause site, Sarkosyl should induce its transcriptional activity in nuclear run-on experiments. Therefore, the hsp70A and c-fos genes in Raji cells do not harbor a pol II with a LS*⌬5 at promoterproximal pause sites. Thus, LS*⌬5 has a defect in initiation or in the very early steps of RNA elongation prior to the polymerase undergoing its conformational change required for elongation. We found evidence that LS*⌬5 may have, dependent on the promoter, a defect at both levels. If the endogenous LS and LS*⌬5 were coexpressed in the absence of ␣-amanitin, LS*⌬5 showed a dominant-negative effect on transcription of the episomal construct. This suggests that LS*⌬5 can still somehow interact with or bind to the promoter but is unable to elongate. As a consequence the promoter is blocked for binding of endogenous pol II. Interestingly, this dominant-negative effect of LS*⌬5 was not seen for the c-myc, Ig , c-fos, and hsp70A genes. For these genes, CTD may already be required for binding of pol II to the promoter. pol II with a deleted CTD cannot compete in binding. From these data we conclude that the CTD not only fulfills important tasks in RNA elongation and maturation of pre-mRNA but also in initiation and/or early elongation.