(Received for publication, July 25, 1995; and in revised form, August 18, 1995)
From the
Regulation of chain elongation by RNA polymerase II can have an
important effect on gene expression (Bentley, D.(1995) Curr. Opin.
Genet. Dev. 5, 210-216; Yankulov, K., Blau, J., Purton, T.,
Roberts, S., and Bentley, D.(1994) Cell 77, 749-759);
however the mechanisms that control this step in transcription are not
well understood. The adenosine analogue
5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB) has
long been used as an inhibitor of RNA polymerase II elongation, but its
target is not known. We show that DRB is a potent inhibitor of
Cdk-activating kinase, associated with the general transcription factor
TFIIH. Two other inhibitors of this kinase, H-7 and H-8, also inhibited
transcriptional elongation. Furthermore, TFIIH kinase bound
specifically to the herpes simplex virus VP16 activation domain which
stimulates polymerase II elongation in addition to initiation
(Yankulov, K., Blau, J., Purton, T., Roberts, S., and Bentley, D.(1994) Cell 77, 749-759). Our results suggest that DRB affects
transcription by inhibiting the TFIIH-associated kinase and that this
kinase functions in the control of elongation by RNA polymerase II.
The adenosine analogue
5,6-dichloro-1--D-ribofuranosylbenzimidazole (DRB) (
)inhibits RNA polymerase II elongation; however, the
molecular basis of its effect is poorly understood. DRB inhibits the
production of long, but not short, RNAs (Chodosh et al., 1989;
Fraser et al., 1978; Tamm, 1977). It does not directly
interfere with polymerization, however, since it does not prevent
elongation in nuclear run-on reactions (Roberts and Bentley, 1992;
Tamm, 1977). DRB only inhibits transcription if it is present during or
immediately following initiation (Chodosh et al., 1989;
Kephart et al., 1992). Poor elongation appears to be an
intrinsic property of a distinct class of pol II transcription
complexes, which are resistant to DRB. We and others (Marshall and
Price, 1992; Roberts and Bentley, 1992; Bentley, 1995) have suggested
that a modification of poorly processive complexes is required to
achieve efficient elongation. DRB was postulated to antagonize a
positive transcriptional elongation factor (P-TEF) responsible for this
modification (Marshall and Price, 1992). Recently, a factor P-TEFb
required for production of long transcripts and for reconstitution of
DRB sensitivity was purified from Drosophila nuclear extract
(Marshall and Price, 1995). It is not clear, though, whether P-TEFb is
a direct target for DRB.
DRB inhibits casein kinase II (Zandomeni et al., 1986), but not protein kinase A, protein kinase C, tyrosine kinases (Meggio et al., 1990), or mitogen-activated protein kinase (Dubois et al., 1994b). Interestingly, suppression of pol II transcription by DRB in vivo coincides with reduced phosphorylation of the pol II large subunit C-terminal domain (Dubois et al., 1994a, 1994b). CTD hyperphosphorylation normally accompanies the transition from initiation to elongation (Payne et al., 1989). Therefore, reduced CTD phosphorylation in response to DRB could be an indirect effect of inhibiting transcription. Alternatively DRB could directly inhibit a CTD kinase.
A pol II CTD kinase, identical with the Cdk-activating kinase, CAK, is closely associated with the core subunits of the general transcription factor TFIIH (Feaver et al., 1994; Roy et al., 1994; Serizawa et al., 1995; Shiekhattar et al., 1995). Two distinct forms of TFIIH function in DNA repair and pol II transcription in yeast. Only the latter form is complexed with CAK (Feaver et al., 1994; Svejstrup et al., 1995). Core TFIIH subunits bind to acidic activation domains (Xiao et al., 1994), but it is not known whether CAK also associates with activators. In this paper we show that TFIIH kinase associates with an acidic activation domain and that DRB is a potent inhibitor of this kinase. We suggest a role of TFIIH-associated CAK in regulation of pol II transcriptional elongation.
Figure 2:
DRB specifically inhibits TFIIH-associated
kinase. A, phosphorylation of pol II by VP16-associated kinase
is stimulated by TFIIE and inhibited by DRB. Kinase reactions contained
5 µg/ml calf thymus pol II, 0.8 µg/ml recombinant TFIIE, and
1.0 (left panel) or 1.5 µg/ml fraction 5 from the VP16
affinity column (right panel). B, kinase reactions
with three recombinant substrates using VP16-associated kinase
(fraction 5, Fig. 1) or highly purified TFIIH (HAP fraction
(Gerard et al., 1991)). Autoradiograms of phosphorylated
GST-CTD, TFIIE p56, and TFIIF rap74 are shown. C, CAK,
immunoprecipitated from Xenopus egg extract (Poon et
al., 1994), is inhibited by DRB. Substrates were GST-CTD
() and GST-Cdk2(K33R), a kinase deficient mutant of Cdk2
(
). D, dose response curves for DRB (
), H-7
(
), and H-8 (
) inhibition of pol II phosphorylation by
VP16-fraction 5 as in A. E, DRB specifically inhibits
the kinase, but not the ATPase, activity of TFIIH. GST-CTD
phosphorylation by VP16-fraction 5 (
) or pure TFIIH (
)
data are from B. ATPase activity in the presence of DNA was
determined for pure TFIIH (
), VP16-fraction 5 (
) and HeLa
nuclear extract (NE) (⊞). Total autophosphorylation
activity of nuclear extract (NE KINASE,
) was
determined using 1.5 µg of HeLa nuclear extract under standard
kinase assay conditions without added
substrate.
Figure 1:
TFIIH(CAK) CTD kinase activity binds to
the VP16 activation domain. HeLa nuclear extract was fractionated on
equivalent GST-VP16 or control GST affinity columns. The columns were
loaded in 50 mM KCl and eluted in steps of increasing KCl
concentration as indicated by arrows. L, load; FT, flow-through. CTD KINASE, 0.3 µl of each
fraction was assayed in kinase reactions with GST-CTD substrate. No CTD
kinase activity was detected in fractions 2-8 from the control
GST column (data not shown). Western blot analysis of the GST and
GST-VP16 affinity column fractions: 5 µl of fraction FT, 15 µl
of fractions 1 and 2, and 30 µl of fractions 3-8,
respectively, were loaded on SDS gels, blotted, and reacted with
monoclonal antibodies against the 70-kDa subunit of KU,
p34 and the p62 and p40
subunits
of TFIIH.
The protein kinase assay of immunoprecipitated CAK from Xenopus eggs was described previously (Poon et al., 1994). Kinase reactions were with 50 µg/ml of GST-CTD or GST-Cdk2(K33R), respectively.
We previously observed that an excess of the herpes simplex
virus VP16 activation domain in trans (``squelching'') (Gill
and Ptashne, 1988) specifically inhibited production of long
transcripts in Xenopus oocytes presumably by titrating a
factor(s) required for efficient elongation (Yankulov et al.,
1994). The similar effects of squelching and DRB on transcriptional
elongation prompted us to look for a DRB target by VP16 affinity
chromatography. Because DRB inhibits pol II CTD phosphorylation in
vivo (Dubois et al., 1994a), we looked for a CTD kinase
activity that bound to the VP16 activation domain and was inhibited by
DRB. HeLa nuclear extract was fractionated on GST-VP16 affinity resin,
and fractions were assayed for phosphorylation of a GST-CTD substrate.
We detected a CTD kinase activity which bound to the GST-VP16 column (Fig. 1) but not to control GST or mutant GST-VP16 (GST-SW6
(Walker et al., 1993)) columns (data not shown). The CTD
kinase co-eluted with two subunits of TFIIH: p62 and
p40, the catalytic subunit of CAK. (There was less than
expected kinase activity in fraction 6, possibly due to an inhibitor or
a phosphatase). Two other CTD kinases, p34
(Cisek and
Corden, 1989) and DNA-dependent protein kinase monitored by detection
of the p70 KU subunit (Peterson et al., 1992), were not
retained on the VP16 column (Fig. 1). The CTD kinase activity in
VP16-fraction 5 was immunodepleted by anti-p62 antibodies (
)indicating that it was associated with TFIIH. Xenopus TFIIH from oocyte extracts was also specifically retained on a
GST-VP16 column. (
)
The VP16-associated GST-CTD kinase was
assayed for activity on a physiological substrate, purified calf thymus
pol II. Phosphorylation of pol II large subunit by VP16-fraction 5 was
stimulated by DNA and recombinant TFIIE in accordance with the
properties of TFIIH (Lu et al., 1992). Importantly, pol II
phosphorylation under these conditions was inhibited by DRB with an
IC of about 25 µM (Fig. 2, A and D).
Highly purified human TFIIH (HAP fraction,
Gerard et al., 1991) and the VP16-associated kinase were
compared for sensitivity to DRB using three recombinant substrates:
GST-CTD, TFIIE, and TFIIF. Both kinases phosphorylated the p56, but not
the p34, subunit of TFIIE and the rap74, but not the rap30 subunit of
TFIIF, ()in agreement with the reported substrate
specificity of TFIIH (Ohkuma and Roeder, 1994). The two kinase
preparations were inhibited by DRB with IC
values between
10 and 50 µM for all three recombinant substrates (Fig. 2B) in agreement with the results for calf thymus
pol II. Furthermore, CAK prepared by immunoprecipitation from Xenopus egg extract with anti-p40
anti-peptide
antibody (Poon et al., 1994) phosphorylated both
GST-Cdk2(K33R) and GST-CTD in a DRB-sensitive manner which closely
resembled TFIIH (Fig. 2C).
The previously identified
inhibitors of TFIIH kinase, H-7 and H-8 (Serizawa et al.,
1993b), also inhibited phosphorylation of GST-CTD (not shown) and pol
II by the VP16-associated kinase, but at substantially higher
concentrations than DRB (Fig. 2D). In contrast, the
DNA-dependent ATPase activity of highly purified TFIIH was not
inhibited even at 1 mM DRB (Fig. 2E and data
not shown), nor did DRB inhibit the overall ATPase activity or total
autophosphorylation activity of crude HeLa nuclear extract (Fig. 2E). DRB at 1 mM also had no effect on
the 5`-3` and 3`-5` helicase activities of purified TFIIH. ()It was previously demonstrated that H-8 had no effect on
TFIIH ATPase activity (Serizawa et al., 1993b).
To address
whether inhibition of elongation is a general property of TFIIH kinase
inhibitors, we investigated the effects of H-7 and H-8 on pol II
transcription in HeLa nuclear extract (Fig. 3). Transcription
reactions were performed with a template containing five GAL4 binding
sites fused upstream of the HIV2 TATA box and TAR sequences
(pGal-HIV2 CAT). Transcription was activated by recombinant
GAL4-AH or GAL4-VP16. Read-through (RT) and prematurely terminated (TM)
transcripts were quantified by PhosphorImager analysis of RNase
protection assays (Fig. 3A). Addition of DRB, H-7, or
H-8 reduced the elongation efficiency (RT/RT + TM) from 70 to 23%
in the presence of GAL4-AH (Fig. 3A). Similar results
were obtained when transcription was activated by GAL4-VP16 (Fig. 3B). The dose-response curves in Fig. 3B show that whereas DRB significantly inhibited
elongation at 10 µM, H-7 and H-8 were only effective in
excess of 50 µM. H-7 and H-8 also inhibited elongation
through the mouse c-myc gene in HeLa extract. (
)A
similar inhibitory effect (DRB > H-7, H-8) on elongation of
transcripts from the mouse c-myc, HIV2 LTR, and
Gal
-E1b promoters (Yankulov et al., 1994) was
observed in injected Xenopus oocytes. (
)
Figure 3:
DRB, H-7, and H-8 inhibit transcriptional
elongation by pol II in vitro. A, RNase protection of
transcripts from the pGal-HIV2 CAT and the pol III control
pSPVA plasmids. In vitro transcription in HeLa nuclear extract
was activated with GAL4-AH at 5 µg/ml which stimulated
transcription 15-fold relative to a control without activator (not
shown). DRB was added at 75 µM and H-7 and H-8 at 200
µM. Read-through (RT) and terminated (TM) bands which were quantified by PhosphorImager are marked.
The TM bands have an average of 1.55 fewer labeled residues than the RT
band. %RT/RT + TM values are given below each lane. nd,
not determined.
-Amanitin (
A) (5 µg/ml) was
added with DRB in lane 3. B, DRB is a more potent
inhibitor of elongation than H-7 and H-8. pGal
-HIV2 CAT
transcription was activated with GAL4-VP16 (5 µg/ml) in the
presence of various concentrations of inhibitor as shown below each bar. RNase protection assays were quantified by PhosphorImager
as in A.
In this paper we show that H-7, H-8, and DRB inhibit both TFIIH (CAK) kinase and pol II elongation. The dose-response curves for inhibition of transcription and kinase activity were similar for each compound, and DRB was always more effective than H-7 and H-8. It is particularly significant that TFIIH kinase is highly sensitive to DRB, which has long been known to interfere with transcript elongation (Fraser et al., 1978; Tamm, 1977), although its mechanism of action has remained obscure. Casein kinase II is quite specifically inhibited by DRB (Zandomeni et al., 1986), but it is unlikely to be involved in the effect on elongation, because it is unaffected by H-7 and H-8 at the concentrations we used (Hidaka and Kobayashi, 1992). Conversely protein kinase A and protein kinase C, which are inhibited at low concentrations of H-7 and H-8, are unaffected by DRB (Meggio et al., 1990). We suggest that the relevant target for these inhibitors of pol II elongation is the kinase associated with the general transcription factor TFIIH.
H-8 had no effect on basal transcription in a highly purified reconstituted system (Serizawa et al., 1993a), but inhibited transcription in crude yeast cell extract (Li and Kornberg, 1994). We show that in crude HeLa nuclear extract H-7, H-8, and DRB inhibit pol II elongation (Fig. 3). In Drosophila extract P-ETFb is required for production of long transcripts and for sensitivity to DRB (Marshall and Price, 1995). Sensitivity of pol II transcription to inhibitors of TFIIH kinase may therefore require additional elongation factors such as P-ETFb, which are not present in highly purified systems. It is not clear whether these factors are substrates for TFIIH kinase. Our results suggest a role for TFIIH kinase in stimulation of pol II elongation, but they do not exclude additional functions in regulating initiation or promoter clearance.
A role of TFIIH kinase in stimulating elongation is consistent with our observation that this kinase binds to the VP16 activation domain which is a potent stimulator of pol II elongation (Yankulov et al., 1994). This activation domain was previously shown to make direct contacts with subunits of the core TFIIH (Xiao et al., 1994). It is not known, however, whether TFIIH kinase activity is directly stimulated by acidic activators.
TFIIH kinase phosphorylates pol II CTD, TATA-binding protein, and the large subunits of TFIIF and TFIIE in vitro (Ohkuma and Roeder, 1994; Fig. 2B). The relative importance of these phosphorylations for transcriptional elongation is not established. The modification of a known elongation factor, TFIIF, could stimulate processivity by stabilizing its interaction with pol II which is quite labile (Price et al., 1989). A role for the CTD in elongation is suggested by the observation that pol II is hypophosphorylated when it enters the preinitiation complex and when it pauses shortly after initiation, whereas the actively elongating form is hyperphosphorylated (Lu et al., 1991; O'Brien et al., 1994; Payne et al., 1989; Weeks et al., 1993). The timing of CTD phosphorylation therefore appears to coincide with the time when DRB is effective, during or immediately following initiation (Cisek and Corden, 1989; Kephart et al., 1992). Furthermore, DRB inhibits CTD phosphorylation in vivo (Dubois et al., 1994a, 1994b). These data are consistent with the idea that CTD phosphorylation by TFIIH is the DRB-sensitive modification (Marshall and Price, 1992; Roberts and Bentley, 1992; Bentley, 1995), which stimulates elongation by pol II.