From the Laboratoire de Génétique Moléculaire, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris Cedex 05, France
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ABSTRACT |
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Actinomycin D and Several cyclin-dependent kinases (CDK) have been shown to
phosphorylate the CTD and regulate transcription. CDK7, and its partner, cyclin H, are subunits of the general transcription factor, TFIIH, a component of the preinitiation complex (14, 15); CDK8 and its
partner cyclin C belong to the RNAP II holoenzyme (16, 17);
CDK9/PITALRE, and its partners, cyclins T1 and T2, are subunits of the
transcription elongation factor P-TEFb (18). 5,6-Dichloro-1- Involvement of the CDK7 and CDK9/PITALRE kinases in transcription is
probably best documented for the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter. Transcriptional activation
of the HIV LTR at the level of elongation is a key step for the viral
replication cycle and has been extensively analyzed (reviewed in Refs.
22 and 23). Like other retroviruses, the HIV can integrate in the
cellular genome and remain silent for an indefinite period (24). In the
latently infected cells, the basal transcription directed by the HIV
LTR is inefficient as most of the transcription initiation events abort
approximately 60-80 nucleotides (including the TAR RNA) downstream of
the transcription initiation site (25). A great variety of stimulations
switch the latent-infected cells to cells producing viral proteins
including Tat and viral particles. The Tat protein binds the TAR RNA
and activates the transcription directed by the LTR promoter (reviewed in Refs. 26 and 27). Tat binds to a number of components of the basal
transcriptional machinery such as TATA-binding protein or the RNAP II
holoenzyme (28, 29). Phosphorylation of the CTD assisted by the viral
protein Tat is essential in establishing an efficient transcription of
the entire viral genome (reviewed in Ref. 30). The Tat protein first
facilitates the phosphorylation of the CTD by CDK7. In a second step,
Tat recruits the CDK9/PITALRE CTD kinase (21, 31).
Therefore, to investigate whether an enhanced CTD phosphorylation
influences the efficiency of actinomycin D and Plasmids--
The luciferase cDNA was placed under the
control of the Rous sarcoma virus (RSV) LTR, pRSVLuc, (32) or the HIV-1
ARV-2 LTR between nucleotides Cells and Transfection--
Murine Ltk
In transient transfection assays, cells were transfected by the
standard calcium phosphate procedure and left to grow for 24 h
before addition of the various drugs; semi-confluent growing cells,
8 × 105 cells per well of a 24-well tissue culture
plate, were transfected with 0.25 µg of luciferase expression vector
supplemented with 2 µg of DNA with plasmid pSP64 (Promega) as a
carrier. Actinomycin D, Luciferase Assay--
Cells were washed twice in ice-chilled
phosphate-buffered saline and lysed on ice in buffer A (25 mM Tris-H3PO4, pH 7.8, 10 mM MgCl2, 1% Triton X-100, 1 mM
2-mercaptoethanol 1 mM EDTA, and 15% glycerol). Luciferase
activity was determined using a Berthold luminometer and mixing 150 µl of cell lysate to 100 µl of buffer A containing 1.2 mM ATP and 0.33 mM luciferin and measuring the light emission for 10 s after mixing.
RNA Extraction and Northern Blots--
Total cellular RNAs were
isolated by the guanidinium thiocyanate method. 15 µg of total RNA
were denatured 15 min at 65 °C in 6% formaldehyde, run on a 1.2%
agarose, 6.3% formaldehyde gel. Equal loading of each lane was checked
by ethidium bromide staining. The gels were blotted onto Hybond N nylon
membrane (Amersham Pharmacia Biotech) that were UV cross-linked,
prehybridized at 65 °C for 1 h in 0.5 M
Na2HPO4, 1% bovine serum albumin, 1 mM EDTA, and 7% SDS and hybridized overnight at 65 °C
to radiolabeled probes in the prehybridization solution. The membranes
were washed twice at room temperature in 2× NaCl/citrate buffer (SSC),
0.1% SDS for 1 h and twice at 42 °C in 0.2× SSC, 0.1% SDS
for 1 h. Membranes were autoradiographed. Quantification was
performed with a FUJI BAS-1 PhosphorImager. Before reprobing, the
membranes were boiled for 10 min in 0.1% SDS to strip off the
hybridized probe. DNA probes for luciferase from pRSVL (32),
cytoplasmic actin from pAL41 (38), mouse 18 S ribosomal RNA from pMSE2
(39), WAF-1 (40), and HSC73 from pRC62 (41) were labeled by random priming.
Primer Extension and DNA Sequencing--
The Alu5 luciferase
antisense primer, 5'-TCTTTATGTT TTTGGCGTCT TCCAT, was end-labeled with
T4 kinase. 20 µg of total RNA were denatured 10 min at 75 °C and
annealed to the primer overnight at 42 °C in 20 µl 10 mM Pipes, pH 6.4, and 400 mM NaCl, overlaid with 20 µl of mineral oil. Nucleic acids were precipitated in ethanol
and redissolved in 20 µl of 50 mM Tris-HCl, pH 8.2, 6 mM MgCl2, 10 mM dithiothreitol, 100 µM of the four dNTPs, 0.1 unit/µl RNasin (Promega), and
1 unit/µl SuperScript II RNase H Single-strand DNA Probes and Run-on Assays--
The primers
GCCCTCAGATGCTGCATATA and CGGTCCATCCTCTAGAGGAT were used to generate the
5' probes (177 base pairs from
Run-on assays were performed following established procedures (42).
2 × 107 nuclei were allowed to transcribe in
vitro for 20 min at 30 °C in the presence of
[ Western Blots--
The cells were dissolved in 1× Laemmli
buffer, and the samples were heated for 5 min at 95 °C before
loading on sodium dodecyl sulfate-5% polyacrylamide gels. The RNAP II
largest subunit was detected with the POL 3/3 antibody that recognizes
an epitope located outside the CTD (43). This monoclonal antibody was
visualized with an anti-mouse IgG horseradish peroxidase conjugate
(Promega) and chemiluminescence (Pierce).
To establish whether the increase in luciferase synthesis might be
related to a general interference with transcription, we investigated
the response to actinomycin D, another transcriptional inhibitor acting
through a different mechanism. The luciferase activity increased up to
130-fold in muHL6b cells treated with an optimal actinomycin D
concentration around 0.3 µg/ml and up to 45-fold in the huHL6 cells,
culminating for 0.2 µg/ml of actinomycin D (Fig. 1B). Due
to a higher actinomycin toxicity, the increase in luciferase activity
dropped more rapidly than with the muHL6b cells. This increase was
exponential between 0 and 20 h of treatment (Fig. 1C),
but to be observed, the cells had to remain exposed to the drug until
lysis; and when actinomycin D was removed after 6 h, no
significant increase was observed 18 h later (data not shown).
Thus, moderate actinomycin D or Activation by Actinomycin D Is a Characteristic of the HIV LTR in
Transient Transfection Assays--
The increased expression of the
luciferase gene in stably transfected cells might relate to a
positional effect in the region of plasmid DNA insertion. Indeed, Tat
transactivation of the HIV-1 LTR has been reported to differ for
integrated versus unintegrated vectors (44). Therefore, HeLa
cells were transiently transfected with the pHIVLucA41 plasmid. The
luciferase activity in the lysates was strongly enhanced when the cells
were exposed 24 h to actinomycin D after transfection (Fig.
2). The strongest effects were obtained around 0.1 µg/ml drug, and higher concentrations led to a drop in
activation. The optimal drug concentration was lower than with the
related huHL6 cells as the calcium phosphate treatment exacerbated an
extensive cell death. The transient transfection assays allowed us to
compare different promoters. Therefore, HeLa cells were transfected
with plasmids associating the luciferase cDNA to other promoters
such as the Rous sarcoma virus LTR (RSV), the cytomegalovirus early
promoter (CMV), or the human HSP70 promoter (there was no need to
stress the cells to observe a relatively high basal level of
HSP70-driven luciferase expression). In the two latter cases, the
luciferase activity in the lysates decreased with increasing concentrations of actinomycin D (Fig. 2). However, the Rous sarcoma virus LTR resisted inhibition up to 0.1 µg/ml actinomycin D.
These results, which were also obtained upon transient transfection of
NIH 3T3 cells (not shown), suggest that the effect of actinomycin D is
a characteristic of the HIV LTR promoter.
Transcriptional Inhibitors Enhance HIV LTR-driven mRNA
Accumulation--
To establish that the increase in luciferase
activity reflected an increase in the corresponding mRNA, total
RNAs were isolated from muHL6b cells incubated with actinomycin D for
24 h and were analyzed by Northern blot. The luciferase probe
detected a major RNA species between the 18 S and the 28 S rRNAs and
a minor species below the 18 S RNA (Fig.
3A). The size of the major
species (2.9 ± 0.2 kilobases) was consistent with the 2.62 kilobase pairs separating the expected transcription initiation from
the termination site on the pHIVLucA41 plasmid and likely corresponded
to a full-length luciferase mRNA as the luciferase cDNA coding
sequence spans over 1650 bases (32). In muHL6b cells treated for
24 h with actinomycin D, both RNA species markedly increased in a
dose-dependent manner (Fig. 3A, left). The
highest increase was observed with cells treated with 0.2 µg/ml
actinomycin D. At higher concentrations (2 µg/ml), the signal
corresponding to luciferase mRNA remained as in the controls and
decreased at 20 µg/ml (not shown). The increase in luciferase
mRNA was observable after 6 h of exposure to 0.2 µg/ml
actinomycin D (Fig. 3A, right). When the muHL6b cells were
exposed to
To evaluate the general transcriptional inhibition, the above-mentioned
Northern blots were rehybridized with cellular genes probes. In RNAs
prepared from muHL6b cells, the actin and the 70-kDa heat shock cognate
(HSC73) probes each detected a single mRNA species with the
expected sizes. When the cells were exposed to actinomycin D at
concentrations above 0.02 µg/ml, both the actin and HSC73 mRNA
abundance decreased indicating that these genes were inhibited (Fig.
3A). The decrease was more pronounced for HSC73 due to the
shorter half-life of the corresponding mRNA in murine cells. Both
signals were distinctly stronger with 0.2 µg/ml than with 2 µg/ml
actinomycin D indicating that for this concentration, which was optimal
for luciferase induction, the transcriptional inhibition was not
complete. When the muHL6b cells were exposed to
When the RNAs were prepared from actinomycin-treated huHL6 cells, a
strong increase in luciferase mRNA was also observed, but the minor
species was less abundant than in muHL6b cells (Fig. 3C).
Very low concentrations of actinomycin D (0.006 µg/ml) have been
reported to promote an increase in p21WAF-1 mRNA in MRC5 human
fibroblasts (45). However, no reliable changes in p21WAF-1 signals were
found in huHL6 cells exposed to actinomycin D in the 0.02 to 0.1 µg/ml range. Primer extension using RNAs prepared from actinomycin
D-treated huHL6 cells demonstrated that under these conditions, the
luciferase mRNAs were correctly initiated at the usual +1 position
(Fig. 4).
Thus, treatments that determine an enhanced luciferase mRNA
accumulation lead to a neat decrease in housekeeping gene mRNAs indicating a general transcriptional arrest. The increase in luciferase mRNA abundance was more pronounced with actinomycin D than with Increased Transcription Driven by the HIV LTR in Nuclei Isolated
from Cells Treated with Actinomycin D--
To establish that the
accumulation of luciferase mRNA was due to an enhanced
transcription of the corresponding gene, run-on assays were performed
with nuclei prepared from huHL6 cells untreated or exposed to
actinomycin D. The actinomycin D treatment determined a 13-fold
increase in the luciferase gene signal (Fig.
5A). In contrast, the actin
and HSC73 gene signals remained unaffected, and the
ribosomal gene signal decreased more than 100 times as expected from
the known high susceptibility of class I gene transcription (5). To
demonstrate that the luciferase gene transcription in actinomycin
D-treated cells was attributable to RNAP II transcription, the run-ons
were also performed in the presence of
To discriminate between an enhanced initiation of transcription and an
enhanced elongation of transcription, the run-ons were repeated using
short probes corresponding to either the 5' end (5'-AS) or the middle
(M-AS) of the luciferase gene. When RNAs were obtained from nuclei of
untreated cells, 5'-AS probe hybridized much stronger than the M-AS
probe (Fig. 5B). When RNAs were obtained from nuclei of
actinomycin-treated cells, the 5'-AS signal decreased about 2-fold. But
in contrast, the M-AS signal increased 18-fold. This result indicates
that, in cells treated with actinomycin D, the transcription of the
luciferase gene driven by the HIV-1 LTR is enhanced at the level of elongation.
HIV LTR Activation by Actinomycin D Does Not Involve the TAR
Sequence--
To dissect the promoter elements required for
actinomycin stimulation, HeLa cells were transiently transfected with
different plasmids containing fragments of the HIV LTR wild type or
mutated and exposed or not to actinomycin D for 24 h before lysis.
As the HIV LTR transactivation by the viral Tat protein has been extensively investigated, for standardization purposes, the same plasmids were cotransfected with a Tat expression vector in a parallel
experiment. The luciferase synthesis driven by the CMV promoter
(pCMVLuc) was inhibited by both actinomycin and Tat and was used as a
reference (Table I, top part).
In contrast, both actinomycin and Tat enhanced the luciferase synthesis
driven by the HIV-1 LTR fragments of various lengths and isolates
(plasmids pLTRWTLuc, pLTRXLuc, pLTRBLuc, pLTR476Luc, and pHIVLucA41)
(Table I, top part). The actinomycin D treatment increased
more than 10-fold the amount of luciferase, whereas Tat coexpression
led to several 100-fold increases. In this set, the length of the LTR
sequences upstream from the TATA box or trimming off the 3' end
nucleotides down to +46 minimally affected the Tat response and did not
influence the actinomycin response. In contrast, deletion of the
nucleotides downstream +32 (relative to the initiation site), in
pLTRB DNA Sequences Upstream and Downstream from the TATA Box Confer HIV
LTR Sensitivity to Actinomycin D--
To dissect further the DNA
sequence elements involved in actinomycin D activation, we took
advantage of a 10-base pair homology between the HIV and CMV TATA boxes
in the core promoter elements (Fig. 6).
The HI/MV promoter carried the HIV sequences upstream from the TATA
box, and the TATA box fused to the CMV sequences downstream from it.
The pHI/MVLuc plasmid lacked the TAR DNA sequence and was not enhanced
in a Tat cotransfection assay as expected (Table I, middle).
However, its actinomycin inducibility was close to that of
pLTRB
The CM/IV chimeric promoter associated the CMV sequences upstream from
the TATA box and the TATA box was fused to the HIV sequences downstream
from it. The pCM/IVLuc contained the TAR sequence and was Tat-inducible
although less efficiently than the parental HIV LTR (Table I,
middle). The latter result confirmed a previous observation
establishing that the TAR sequences remain functional within
heterologous promoters (49). The pCM/IVLuc was actinomycin-inducible
but with a lower efficiency than the parental pHIVLucA41 confirming
that sequences downstream from the TATA box, overlapping the TAR
element, contribute to the response.
The NF
In conclusion, the actinomycin stimulation of the HIV promoter requires
the integrity of the HIV TATA box and Sp1 sites but is independent of
the NF Activation of the HIV-LTR Is Sensitive to CDK9/PITALRE Protein
Kinase Inhibitors--
Protein kinase inhibitors such as DRB block the
Tat transactivation (50). DRB inhibits various protein kinases
including CDK7 (20) and CDK9/PITALRE (18). Tat transactivation of the HIV LTR has been shown to rely on the recruitment of the CDK7 and
CDK9/PITALRE kinases onto the transcription complex (21, 31, 51). The
efficiency of kinase inhibitors in blocking the Tat activation matches
their capacity to inhibit CDK9/PITALRE in vitro (37).
Therefore, to evaluate the contribution of these kinases in actinomycin
stimulation of the HIV LTR, huHL6 cells were exposed during 24 h
to a combination of actinomycin D and various kinase inhibitors.
Concentrations of TRB and T276339 in the micromolar range led to a
dramatic decrease in actinomycin-induced luciferase synthesis (Fig.
7A). H7 and DRB were a little
less efficient, followed by T172298. The other compounds tested such as
T172299 or T525636 and T163693 (not shown) had no significant effects
when applied at concentrations below 20 µM. In a control experiment, TRB and T276339 were found to be the strongest inhibitors of Tat transactivation followed by DRB and H7, T172298 being the weakest as previously reported (Fig. 7B). The other
compounds tested, T172299, T525636, and T163693 (not shown), had no
significant effects when applied at concentrations below 20 µM.
Thus, the capacities to inhibit Tat and actinomycin D transactivations
are correlated. TRB, T276339, T172298, DRB, and H7 are strong
inhibitors of CDK9/PITALRE, and out of this set, only T172298, DRB, and
H7 inhibit CDK7 (37). Hence, CDK9/PITALRE is likely to be required for
actinomycin stimulation as previously reported for the Tat-activated
transcription of the HIV LTR (21, 31).
The Average CTD Phosphorylation Is Enhanced in Cells Exposed to
Actinomycin D--
CDK9/PITALRE phosphorylates the CTD of RNAP II
largest subunit (21), and actinomycin D enhances the average
phosphorylation of the CTD in HeLa cells (7, 8). Indeed, the proportion in hyperphosphorylated (IIo) form of the CTD increased markedly in
huHL6 cells exposed during 8 h to actinomycin D concentrations as
low as 0.06 µg/ml (Fig. 8A).
Identical effects were observed with murine cells (not shown).
To strengthen the link between CTD phosphorylation and HIV LTR
activation, cells were treated with
Thus, actinomycin D and Inhibitors of CDK9/PITALRE Promote CTD Dephosphorylation in
Mammalian Cells--
DRB promotes a bulk dephosphorylation of the CTD
(7). To identify which kinase was essential to the average CTD
phosphorylation steady state in mammalian cells, the effect of the
CDK9/PITALRE inhibitors was investigated in HeLa cells. Five, of the
eight inhibitors tested, promoted average CTD dephosphorylation within 1 h of treatment (Fig.
9A). TRB and T276339 were the
most efficient (their effect was detectable above 6 µM)
closely followed by DRB and H7, and T172298 was the least efficient.
T172299, T163693, and T525636 had no detectable effects at 50 µM (not shown). Although higher kinase inhibitor
concentrations were required to observe average CTD dephosphorylation
than to inhibit HIV LTR expression, both effects were correlated (Table
II). The inhibition of Tat-activated HIV
LTR expression is correlated with the capacity to inhibit CDK9/PITALRE
in vitro (37), which suggests that this kinase provides an
essential contribution to the average CTD phosphorylation in mammalian
cells.
Addition of DRB prior to actinomycin D prevents the bulk CTD
phosphorylation (8). Therefore, we evaluated the capacity of the
various inhibitors of CDK9/PITALRE to prevent the CTD
hyperphosphorylation induced by actinomycin D. In HeLa cells, the
stronger inhibitors, TRB or T276339 at concentrations as low as 10 µM, were found to prevent the average CTD phosphorylation
promoted by actinomycin D (Fig. 9B). DRB and H7 required
higher concentrations (40 µM), and even higher
concentrations of T172298 (60 µM) had to be used to
observe any effect. The other compounds, T172299, T163693, and T525636
were inefficient up to 60 µM (not shown).
Thus, the capacity to inhibit the CTD phosphorylation promoted by
actinomycin D correlates with the transcription inhibition efficiency.
This correlation links CTD phosphorylation to the actinomycin-promoted
HIV LTR stimulation and strongly suggests that CDK9/PITALRE is involved
in both processes.
In this study we show that treatment of mammalian cells with
actinomycin D and The Accumulation of HIV-driven mRNAs Is Due to an Enhanced
Elongation of Transcription--
The accumulation of HIV-driven
luciferase mRNAs in actinomycin D-treated cells is attributable to
an enhanced transcription at the level of elongation since run-on
transcription assays indicate a higher polymerase density in the midst
of the luciferase gene in nuclei isolated from actinomycin D-treated
cells, whereas the RNAP II density decreases at the 5' end of the
luciferase gene. There are several reports on class II mRNA
accumulation in cells treated with inhibitors of transcription. For
instance, treatment of cells with low concentrations of actinomycin D
(0.007 µg/ml) trigger a p53-dependent accumulation of
several mRNAs including p21WAF-1, cyclin G1, and cyclin
G2 mRNAs in human fibroblasts (45, 52). DNA-damaging agents and
Transcriptional Activation of the HIV LTR Does Not Involve the TAR
nor the NF
Actinomycin D and topoisomerase inhibitors activate the NF Involvement of the CDK9/PITALRE Kinase in HIV LTR Transcriptional
Activation--
The transcription from the HIV LTR has been
extensively investigated. To summarize, in the absence of Tat, the HIV
LTR directs the synthesis of a significant basal level of transcripts
but most of them are short, transcription aborts after the TAR element. The TATA-binding protein would bind first to the promoter and recruit
the RNAP II (70). Tat would interact with the TAR RNA hairpin and
recruit histone acetylases on the LTR promoter (71-73). But an
increased histone acetylation affects HIV LTR transcription only when
integrated into the genome (65, 73); it is unlikely, therefore, to
contribute to actinomycin D stimulation. More significant to our
purpose, Tat mediates the recruitment of CDK7 (29) and CDK9/PITALRE
(21, 31, 37) which phosphorylate the CTD and promote an efficient
transcription elongation throughout the entire viral genome.
Phosphorylation of the CTD by the CDK9/PITALRE of the positive
transcription elongation factor, P-TEFb, might be the key step in
switching the competence of RNAP II to proceed transcription beyond the
TAR region. As the actinomycin-activated (this work) and the
Tat-activated (37, 50) transcription are very sensitive to CDK9/PITALRE
inhibitors in correlation with their inhibition efficiencies in
vitro, this kinase is likely to contribute to both the Tat and the
actinomycin D stimulation of the HIV LTR. In agreement with this
hypothesis, actinomycin D (this work) and Tat both stimulate
transcription at the elongation level.
The recruitment of P-TEFb by Tat involves a direct interaction between
Tat and cyclin T1 but, in contrast to the human cyclin T1, the murine
cyclin T1 is defective in binding to Tat (74, 75). Indeed, Tat
transactivation of the HIV LTR is defective in rodent cells. However,
actinomycin D is as efficient to stimulate the HIV LTR transcription in
both human and murine cells. The actinomycin D treatment may bypass a
requirement in Tat/cyclin T1 interaction to recruit P-TEFb.
Involvement of the CDK9/PITALRE Kinase in CTD Phosphorylation in
Vivo--
Here we report on a collection of compounds that promote CTD
dephosphorylation in vivo with an efficiency correlating
with their capacity to inhibit CDK9/PITALRE in vitro.
Phosphorylation of the CTD is a dynamic process involving permanent
phosphorylation and dephosphorylation reactions. Inhibiting
CDK9/PITALRE would prevent CTD phosphorylation and result in the
accumulation of dephosphorylated CTD. CTD dephosphorylation
systematically required higher inhibitor concentrations than
transcription impairment. However, it should be kept in mind that RNAP
II exists under various structures, core enzyme and holoenzymes (76).
As the holoenzymes are organized around the CTD, their reactivities
toward CTD kinases and CTD phosphatases are expected to differ from one
holoenzyme to the other. A specific class of holoenzymes (with a
characteristic kinase inhibitor sensitivity) is likely to be involved
in the HIV LTR transcription. In contrast, the CTD dephosphorylation detected by Western blot averages the behavior of all classes of RNAP
II molecules. Thus, the correlations between the in vitro and in vivo inhibitor effects strongly suggest that
CDK9/PITALRE is an essential contributor to CTD phosphorylation
in vivo.
Inactivation of KIN28, the yeast CDK7 homologue results in a rapid
dephosphorylation of the CTD in yeast cells (77). However, the yeast
CDK9/PITALRE homolog has not yet been identified and might also be the
KIN28 gene product. In mammalian cells, both CDK7 and CDK9/PITALRE are
likely to be essential to ensure CTD phosphorylation in
vivo. Such a double requirement has been described for HIV
promoter activation (30). Phosphorylation of the CTD is enhanced in
cells exposed to actinomycin D and
An enhanced CTD kinase activity should promote an enhanced average CTD
phosphorylation. Activation of peripheral blood lymphocytes and
differentiation of promonocytic cell lines is accompanied with an
enhanced CDK9/PITALRE activity that is proposed to contribute to the
high levels of HIV transcription (78). Alternatively, blocking CTD
dephosphorylation should promote the accumulation of phosphorylated
RNAP II, actinomycin D may thus block the RNAP II dephosphorylation.
However, neither actinomycin D nor Inappropriate CTD Phosphorylation May Contribute to Inhibition of
Transcription--
Actinomycin D and
During severe stress, a block in CTD phosphate exchange due to CTD
kinase and CTD phosphatase inactivations is likely to contribute to a
general shut-off of transcription except for the heat shock genes
(80-82). As the Tat protein remains bound to the elongating transcriptional complex (83) and to the RNAP II holoenzyme (29), it is
likely to "freeze" the RNAP II CTD in a phosphorylated state in the
vicinity of a transcriptionally active HIV LTR. Indeed, the Tat protein
has evolved not only to recruit CTD kinases (30) but also to inhibit a
phosphatase that specifically dephosphorylates the CTD (84). The Tat
protein represses several cellular genes at the transcriptional level
(72), and it may be part of the HIV viral strategy to escape the
transcriptional initiation block caused by a "frozen" CTD phosphorylation.
A General Inhibition of Transcription May Contribute to Stress
Activation of the HIV LTR--
The actinomycin D and the -amanitin are commonly used
to inhibit transcription. Unexpectedly, however, the transcription of
the human immunodeficiency virus (HIV-1) long terminal repeats (LTR) is shown to be activated at the level of elongation, in human and murine
cells exposed to these drugs, whereas the Rous sarcoma virus LTR, the
human cytomegalovirus immediate early gene (CMV), and the HSP70
promoters are repressed. Activation of the HIV LTR is independent of
the NF
B and TAR sequences and coincides with an enhanced average
phosphorylation of the C-terminal domain (CTD) from the largest subunit
of RNA polymerase II. Both the HIV-1 LTR activation and the bulk CTD
phosphorylation enhancement are prevented by several CTD kinase
inhibitors, including
5,6-dichloro-1-
-D-ribofuranosylbenzimidazole. The
efficacies of the various compounds to block CTD phosphorylation and
transcription in vivo correlate with their capacities to
inhibit the CDK9/PITALRE kinase in vitro. Hence, the
positive transcription elongation factor, P-TEFb, is likely to
contribute to the average CTD phosphorylation in vivo and
to the activation of the HIV-1 LTR induced by actinomycin D.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Amanitin and actinomycin D are commonly used inhibitors of
transcription.
-Amanitin binds to the largest subunits of RNA polymerase II (RNAP II)1 (1,
2) and RNAP III (3), with RNAP II being the most sensitive. As a
consequence, the incorporation of new ribonucleotides into the nascent
RNA chains is blocked (4). Actinomycin D is generally thought to
intercalate into DNA thereby preventing the progression of RNA
polymerases, with RNAP I being the most sensitive (5, 6). In previous
work, we have shown that the average phosphorylation of RNAP II
C-terminal domain (CTD) increases in cells exposed to actinomycin D (7,
8). The activity of RNAP II is regulated by multisite phosphorylation
on the CTD (9). The underphosphorylated CTD mediates multiple
protein-protein interactions involved in the assembly of a
preinitiation complex. The subsequent phosphorylation of the CTD occurs
along the initiation of transcription and contributes to disrupt some
of the interactions that lead to the assembly of the preinitiation
complex on promoters. Phosphorylation of RNAP II at this step is
required to elongate transcription and mediates the recruitment of
various enzymatic complexes involved in processing of the primary
transcript (10-12). In contrast, phosphorylation of the CTD prior to
the formation of the preinitiation complex represses the expression of
specific genes (13). Hence, the increase in average phosphorylation of the CTD promoted by actinomycin D raises the possibility that different
genes may have different susceptibilities to this drug.
-D-ribofuranosylbenzimidazole (DRB) is
another widely used transcriptional inhibitor (19) that inhibits CDK7 (20) and CDK9/PITALRE (21). The average CTD phosphorylation is
decreased in cells exposed to DRB (7), suggesting that these kinases
might contribute to global CTD phosphorylation in vivo.
-amanitin on
transcription of identified genes, we followed their effect on the
expression of a reporter gene driven by the HIV-1 LTR or by the human
cytomegalovirus (CMV) immediate early promoters. Unexpectedly, both
drugs were found to promote an enhanced reporter expression when the
corresponding cDNA was placed under the control of the HIV-1 LTR
promoter. This stimulation is shown to be at the level of elongation of
transcription and is suggested to be linked to an enhanced average CTD
phosphorylation which may involve the CDK9/PITALRE CTD kinase.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
167 and +46, pHIVLucA41 (33). Plasmid
pLTRWTLuc contained the HIV-1 ARV-2 LTR wild type sequences from
644
to +83, plasmid pLTR
kBLuc derived from pLTRWTLuc, and the NF
B
sites A and B were deleted and replaced by a BclI linker
(34). Plasmid pLTR476Luc contained the luciferase cDNA controlled
by the HIV-1 ARV-2 LTR wild type sequences from
177 to +83; in the
plasmid pLTR361
Sp1Luc derived from pLTR476Luc, the three Sp1 sites
(
75 to
50) have been replaced by the sequence 5'-ATATCGTGGC
CTGTGTAGTC CGTGCC. Plasmids pLTRXLuc, pLTRBLuc, and pLTRB
TARLuc
contained the luciferase cDNA controlled by the HIV-1 Bru LTR
between, respectively, nucleotides
644 to +83,
489 to +83, and
489 to +32 (35). Plasmid pHSPLuc contains the luciferase cDNA
under the control of the human HSP70 promoter (36), and plasmids
pCMVTat and pCMVLuc contain the human cytomegalovirus (CMV) immediate
early promoter followed by the cDNAs coding for either the Tat
protein or luciferase (35). To generate plasmid pCM/IVLuc, the CMV
sequences between
324 and
18 (relative to the transcription
initiation site) were amplified by PCR from plasmid pCMVLuc using the
primers 5'-GCGATCTCGA GCGTCAATGACG GTAAATG and 5'-GCATGCAGCT GCTTATATAG
ACCTCC. The amplified fragment was digested with XhoI and
PvuII and inserted between the unique XhoI and
PvuII sites of pHIVLucA41. To generate plasmid pHI/MVLuc,
the pCMVLuc sequences between
18 and +738 were amplified by PCR using
the primers 5'-GAATATCAGC TGCTCGTTTA GTGAACCGTC AG and 5'-CTGGCATGCG
AGAATCTGAC GCAGGCAGTT. The amplified fragment was digested with
PvuII and BstEII and inserted between the unique
PvuII and BstEII sites of pHIVLucA41. To generate
pmTATALuc, the pHIVLucA41 sequences were amplified by PCR using the
primers 5'-GCAAAAAGCA GCTGCTTGTC TGCAGCATCT GAG and 5'-AACCTGATAT
CCCCTCGAGG TCACGT that replaced the TATA box sequence by GACA. The
amplified fragment was digested with XhoI and
PvuII and inserted between the unique XhoI and
PvuII sites of pHIVLucA41. All these plasmids were
controlled by sequencing the 3' ends of the promoter fragments.
, NIH 3T3, and human
HeLa cells (MRL2 strain) were cultured in Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) supplemented with 10% fetal calf
serum. The muHL6b were derived from Ltk
cells after cotransfection
with pHIVLucA41 and pAGO, plasmid carrying the herpes thymidine kinase
gene. The huHL6 cells were derived from HeLa cells cotransfected with
pHIVLucA41 and pRSVtkneo, a plasmid carrying the neomycin resistance
gene. Clones were subcloned and selected, respectively, in HAT or G418 medium for luciferase expression.
-amanitin, and DRB were purchased from
Sigma. TRB, T276339, H7, T172298, T525636, T172299, and T163693 were
kindly provided by Dr. Osvaldo Flores as 10 mM stock
solutions in dimethyl sulfoxide (37).
reverse transcriptase
(Life Technologies, Inc.). Primer extension was performed at 42 °C
for 60 min using the Alu5 luciferase primer. Nucleic acids were
precipitated in ethanol, redissolved in formamide loading buffer, and
run on a 10% denaturing polyacrylamide-urea gel. The dried gels were
exposed for autoradiography. DNA sequences were obtained using the Alu5
primer, plasmid pHIVLucA41, and a T7 Sequencing kit (Amersham Pharmacia Biotech).
42 to +136), whereas the primers
CAGCTATTCTGATTACACCCG and ATTCGCCTCTCTGATTAACG were used for the M
probes (127 base pairs from +1111 to +1238). These primers were used to
amplify DNA fragments by symmetric PCR (30 cycles) using pHIVLucA41Luc
as a starting template. The single-strand antisense probes were
amplified from these DNA fragments by asymmetric PCR (30 cycles) using
only one of the primers.
-32P]UTP, non-radioactive ATP, CTP, and GTP with or
without
-amanitin (0.1 µg/ml), and the resulting RNAs were
isolated. These RNAs were hybridized to Hybond-N+ membranes (Amersham
Pharmacia Biotech) on which either linearized and denatured plasmids (5 µg) or single-strand probes (1 µg) had been slotted. Hybridizations
were at 65 °C in Church buffer (0.5 M sodium phosphate,
7% SDS, 1 mM EDTA, 1% serum albumin). Dehybridization
washes were done at 65 °C in 0.2× SSC, 0.5% SDS.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Amanitin and Actinomycin D Activate the HIV LTR in Stably
Transfected Cells--
To investigate the dose effect of
-amanitin
on the expression of a reporter gene under the control of a defined
promoter, firefly luciferase activity was followed in lysates from
muHL6b cells exposed to varying amounts of the drug for 24 h. This
clonal cell line was derived from murine Ltk
cells stably transfected with a plasmid, pHIVLucA41, in which the luciferase cDNA had been placed under the control of an HIV LTR. Unexpectedly, increasing amounts of luciferase activity were found in lysates from muHL6b cells
exposed during 24 h to
-amanitin up to 30 µg/ml (Fig.
1A). The highest stimulation
(~28-fold) was achieved at 10 µg/ml, a rather elevated
concentration. To extend these observations to other cell systems, we
stably transfected HeLa cells with pHIVLucA41 and isolated the huHL6
clonal human cell line.
-Amanitin also stimulated the luciferase
expression in huHL6 cells but with much lower efficiency than in the
murine cells (only 3.5-fold).
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Fig. 1.
Actinomycin D and
-amanitin activate HIV LTR-driven luciferase
expression in stably transfected cell lines. Increase in
luciferase activity in lysates from muHL6b (
) and huHL6 (
) cells
that have been incubated with various concentrations of
-amanitin
(A) and actinomycin D (B) for 24 h.
C, time course variation of luciferase activity in muHL6b
cells incubated with 0.2 µg/ml actinomycin D. An exponential curve
fitting is drawn. The luciferase activity in control cells was taken as
the unity.
-amanitin concentrations enhance
luciferase synthesis in murine and human cell lines stably transfected
with a plasmid associating the HIV-1 LTR to the luciferase cDNA.
But no stimulation was observed when actinomycin D (0.2 µg/ml) and
-amanitin (10 µg/ml) were added simultaneously (data not shown).
This finding as well as the bell-shaped dose-response curves suggests
two opposing effects: an activation and an inhibition. At high drug
concentration, the latter overcomes.
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Fig. 2.
Actinomycin D activation is a characteristic
of the HIV LTR in cell transient transfection assays. HeLa cells
were transiently transfected with plasmids containing the luciferase
gene under the control of different plasmids as follows: pHIVLucA41
( ), pCMVLuc (
), pRSVLuc (
), and pHSPLuc (
). Actinomycin D
was added to the cells 24 h after transfection, and the cells were
lysed 48 h after transfection. The luciferase activity in control
cells was taken as the unity.
-amanitin, the 2.9 kilobase pairs of luciferase mRNA
abundance also increased with time (Fig. 3B).
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Fig. 3.
Northern blot analysis of RNAs isolated from
cells treated with actinomycin D and
-amanitin. A, RNAs from muHL6b
cells incubated 24 h with varying amounts of actinomycin D
(left panel). RNA extracted at different time points from
muHL6b cells incubated with 0.2 µg/ml actinomycin D (right
panel). B, RNAs from huHL6 cells were incubated 24 h with varying amounts of actinomycin D. C, RNA from muHL6b
cells or incubated with 10 µg/ml
-amanitin for 0, 16, and 20 h. The RNAs were blotted on nitrocellulose membranes after agarose gel
electrophoresis. The membranes were dehybridized and rehybridized
sequentially with radiolabeled probes. The position of the ribosomal
RNAs detected by ethidium bromide staining is indicated.
-amanitin, the HSC73
mRNA levels also decreased (Fig. 3B).
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Fig. 4.
Mapping of the transcriptional start site of
the luciferase mRNAs. RNAs isolated from actinomycin D-treated
(0.1 µg/ml, 18 h) huHL6 cells were annealed to the Alu5
luciferase antisense primer and extended with reverse transcriptase.
The reverse transcripts (RT) were run on a
polyacrylamide-urea denaturing gel along with the products of plasmid
pHIVLucA41 sequencing using the same primer.
-amanitin, as anticipated by luciferase activity determinations.
-amanitin in the assay (0.1 µg/ml). Addition of
-amanitin to nuclei prepared from control
untreated cells did not affect the 18 S rRNA signal as expected since
RNAP I is insensitive to this compound; however, it completely wiped
off the luciferase and the actin signals. The luciferase and actin
signals as well as the remaining 18 S signal were also suppressed by
addition of
-amanitin to nuclei from actinomycin
D-treated cells. First, it should be emphasized that this
finding does not conflict the data provided in Fig. 1A as
in vitro
-amanitin poisoning of RNAP II is likely to be fast and complete, whereas in vivo it is a slow, incomplete
process controlled by the penetration of the drug (46, 47). Second, an
involvement of RNAP II in ribosomal DNA transcription has been shown to
occur in yeast cells that lack RNAP I activity (48).
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Fig. 5.
Run-on transcription from huHL6 nuclei and
mapping of the transcription initiation site. A, nuclei
were isolated from huHL6 cells that had been exposed or not to 0.1 µg/ml actinomycin D (AcD) during 18 h. The nuclei were allowed
to transcribe in vitro in the presence of XTPs with
(+ -AM) or without (
) 0.1 µg/ml
-amanitin. In
vitro transcribed RNAs were hybridized to linearized plasmids
carrying the luciferase (LUC), actin (ACT),
70-kDa heat shock cognate protein (HSC), and ribosomal RNA
(18 S) sequences. DNA from plasmid pSP64 (SP6)
was used as a negative control. B, nuclei were isolated from
huHL6 cells that had been exposed (actinomycin D, AcD) or
not (
) to 0.12 µg/ml actinomycin D during 8 h. In
vitro transcribed RNAs were hybridized to single-strand antisense
DNA probes. The 5'-AS probe extends from
42 to +136 relative to the
transcription initiation site (30 transcribed uridines), whereas
the M-AS probe extends from +1111 to +1238 (30 uridines), in the
midst of the luciferase gene. The radioactivity hybridizing to each
probe was quantified with a PhosphorImager.
TARLuc, resulted in a dramatic decrease in Tat inducibility (as
expected since this deletion impaired the formation of the TAR RNA
hairpin) and led to a 2-fold decrease in actinomycin D inducibility.
Thus, the 14 nucleotides at the 3' end of the TAR element (from +32 to
+46) that are essential for the Tat response provided a limited
contribution to the actinomycin response.
Molecular dissection of the DNA sequences involved in actinomycin D
stimulation of transiently transfected plasmids
29 and
19 in the HIV LTR and CMV
promoters include the TATA box and are identical (Fig. 6). 24 hours
after transfection, half of the wells with cells transfected with the
luciferase expression vectors alone were exposed to actinomycin D (0.12 µg/ml). 48 hours after transfection, the cells were assayed for
luciferase expression. The actinomycin and Tat stimulations are
relative to the untreated cells transfected with the vectors alone
(taken as unity). The complete experiment was repeated three times
independently, all plasmids were analyzed each time. The stimulations
are given as the mean values obtained in the three experiments with
groups of four wells. The luciferase activities provided in parentheses
in relative light units (RLU) (mean of four wells) have all been
obtained in the same experiment with untreated cells transfected with
the expression vectors alone.
TARLuc indicating that the HIV sequences between
19 and +32
were not involved.
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Fig. 6.
Alignment of the CMV and HIV core
promoters. Alignment of the CMV (87) and HIV-1 LTR (88) promoter
sequences from 167 to +47 relative to the transcription initiation
sites. The identical 10-base pair sequence blocks are in
bold. The transcription initiation sites, the NF
B- and
Sp1-binding sites, as well as the TATA box sequences are indicated. The
PvuII site in the HIV-1 LTR was used to engineer the pCM/IV
and pHI/MV chimeric promoters.
B Binding Sequences of the HIV LTR Are Not Required for
Actinomycin D Stimulation--
Two NF
B-binding sequences, a cluster
of three Sp1 elements, and the TATA box are regulatory elements that
have been identified within the HIV LTR (22, 23). When the TATA box
sequence (plasmid pHIVLucA41) was replaced by GACA (plasmid mTATA), the
basal level of luciferase expression was around half that of its
parental pHIVLucA41; the Tat stimulation, however, was abolished, and
the actinomycin D stimulation was depressed (Table I, lower
part). Disruption of the three Sp1 sites in plasmid
pLTR361
Sp1Luc minimally affected the basal level of expression
compared with the corresponding wild type plasmid pLTR476Luc but led to
a decreased actinomycin D inducibility, thereby suggesting the
involvement of the Sp1-binding sites. The Tat stimulation dropped
10-fold with the Sp1-less plasmid. In plasmid pLTR
BLuc, both the
A and B NF
B sequences have been deleted from the corresponding wild
type plasmid pLTRWTLuc. Luciferase synthesis was induced by actinomycin
D to a similar extent in cells transfected by either plasmid.
Involvement of NF
B in the actinomycin D-induced HIV-1 activation is
therefore very limited.
B-binding sites.
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Fig. 7.
CDK9/PITALRE kinase inhibitors prevent
actinomycin- and Tat-induced HIV LTR activation. A,
huHL6 cells were exposed 24 h to actinomycin D (0.2 µg/ml) in
the presence of varying concentrations of kinase inhibitors.
B, HeLa cells were cotransfected with the pHIVLucA41 and
pCMVTat plasmids. 24 h after transfection, they were exposed for
the following 24 h to indicated concentrations of protein kinase
inhibitors as follows: TRB ( ), T276339 (
), H7 (
), DRB (
),
T172298 (
), and T172299 (
). Compounds T525636 and T163693 behaved
like T172299; they had no significant effect up to 20 µM;
the corresponding data are therefore not represented. The level of
luciferase expression detected in cells treated with actinomycin but in
the absence of kinase inhibitor was taken as the unity. Exponential
curve fittings are drawn; at high kinase inhibitor concentration, the
luciferase levels narrow to zero in the case of actinomycin stimulation
but remain above 0.13 in the case of Tat activation as in the latter
case, the reporter protein and the corresponding mRNA accumulate
before the addition of the drugs.
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Fig. 8.
Increased average phosphorylation of the CTD
in cells exposed to actinomycin D and
-amanitin. A, huHL6 cells were
exposed to actinomycin D during 8 h at the indicated
concentrations (µg/ml). B, muHL6b and huHL6 cells were
exposed to 10 µg/ml of
-amanitin during 3, 6 or 9 h. Whole
cell lysates were analyzed by Western blot with the POL 3/3 antibody.
The hyperphosphorylated (IIo) and underphosphorylated (IIa) subunits of
the largest RNAP II subunit are indicated.
-amanitin, and the lysates were
analyzed by Western blot. In the muHL6b cells, both the intensities of
the IIa and IIo forms decreased with time of treatment (Fig. 8B). Indeed,
-amanitin had been shown to promote the
degradation of the largest subunit of RNAP II in murine cells (47).
Careful quantification of gel scans indicated that the IIa band
intensity reproducibly decreased more rapidly than the IIo band,
resulting in close to a 4-fold increase in the IIo/IIa ratio after
9 h of treatment. The increase in the IIo/IIa ratio was less than
2-fold in the HeLa-derived huHL6 cells exposed to
-amanitin during
the same period. This result strengthens the link between CTD
phosphorylation imbalance and HIV LTR transcription as luciferase
synthesis was much less inducible by
-amanitin in the huHL6 cells
than in the muHL6b cells (Fig. 1C).
-amanitin treatment promoted an increase in
the proportion of RNAP II phosphorylated on its CTD in correlation with
HIV LTR induction.
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Fig. 9.
The increased average phosphorylation of the
CTD in cells exposed to actinomycin D is prevented by inhibitors of
CDK9/PITALRE. A, HeLa cells were exposed to kinase
inhibitors for 1 h at the indicated concentrations
(µM). B, HeLa cells were preexposed for 1 h to the kinase inhibitors (10 or 40 µM), then
actinomycin D (0.5 µg/ml) was added (+) or not ( ) during 1 additional hour. Whole cell lysates were electrophoresed like in Fig.
8.
Kinase inhibitors block Tat- and actinomycin-dependent
transcription and promote CTD dephosphorylation
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amanitin leads to a strong enhancement of transcription directed by HIV LTR promoters. This enhancement is
observed both in stably transfected cells and during transient transfection assay. To observe a stimulation of RNAP II transcription in cells exposed to inhibitors of transcription is unexpected and
entirely novel. This effect is a characteristic of the HIV LTR and is
observed with moderate drug concentrations, which nevertheless lead to
a marked inhibition of class II gene expression. At high drug
concentrations, the inhibiting effects are predominant.
-amanitin have independently been reported to block the degradation
and therefore to promote the accumulation of the p53 protein which is
involved in p21WAF-1 promoter activation (53, 54).
Actinomycin D and
-amanitin have both been shown to promote an
increase in tumor necrosis factor-
mRNA (55). tumor necrosis
factor-
activates the HIV-1 LTR through a p53-responsive element
(56). However, the p53-mediated cellular responses to DNA damage are
disrupted in HeLa cells (57), and no reliable changes in
p21WAF-1 mRNA levels are detected in actinomycin
D-treated HeLa-derived huHL6 cells. Therefore, the accumulation of
luciferase mRNAs described in the present work is unlikely to
follow a p53-dependent mechanism. The mRNAs previously
known to accumulate in actinomycin and amanitin-treated cells are
unstable, and increasing their half-lives may lead to their
accumulation. To our knowledge, a unique study reports that RNAP I
transcription initiation rate increases on rRNA genes in cells exposed
to concentrations of actinomycin D slightly lower than those used in
the present study (58). Hence, this is the first report that elongation
of RNAP II transcription can be stimulated in cells exposed to
actinomycin D or
-amanitin.
B-binding Sequences--
The transcriptional activation
of the HIV LTR by actinomycin D is shown to be TAR-independent. The HIV
LTR sequence between
19 and +32 overlapped the inducer of short
transcripts element (25) and high affinity binding sites for LBP-1
(59). These factors are unlikely to be involved in the actinomycin
response as the inducibility of the pHI/MVLuc chimera (HIV sequences
167 to
19) and pLTRB
TAR (HIV sequences
488 to +32) were
similar (Table I). However, mutagenesis of either the TATA box or the Sp1-binding sites depresses the actinomycin mutagenesis-induced HIV LTR
transcription and, as previously reported, the Tat-stimulated transcription as well (reviewed in Refs. 22 and 23).
B factor
(60), and many cases of NF
B-dependent activations of the
HIV LTR have been reported. However, stimulation of the HIV LTR by Tat
is NF
B-independent in the J-Jhan lymphoblastoid cells (61).
Furthermore, an NF
B-independent activation of the HIV LTR has also
been reported after treatment with phorbol ester (62, 63), UV
irradiation (64), histone deacetylase inhibitors (65), protein kinase
inhibitors (such as 2-aminopurine) (66), okadaic acid treatment (67),
heat shock (68), and peroxovanadium compounds (69). Similarly, the
NF
B-binding sequences are not required for actinomycin stimulation.
-amanitin. Involvement of
CDK9/PITALRE is suggested as this enhancement does not occur when its
inhibitors are present.
-amanitin treatment did affect
the activities of either the CTD phosphatase or CDK9/PITALRE in HeLa
cell lysates (data not shown). Although the involvement of other CTD
kinases cannot be excluded, it should be considered that an increased
reactivity of RNAP II toward CDK9/PITALRE could lead to an enhanced
average CTD phosphorylation. For instance, moderate concentrations of
actinomycin D and
-amanitin, such as those used in this study,
promote major chromatin rearrangements (79) that may increase the
accessibility of the CTD to kinases or decrease its accessibility to phosphatases.
-amanitin have been described
to act through distinct mechanisms. We now propose that the enhanced
proportion in phosphorylated CTD promoted by actinomycin D and
-amanitin influences their capacities to inhibit transcription
in vivo. Indeed, phosphorylation of the CTD prior to the
formation of the preinitiation complex of transcription represses
initiation of transcription on specific genes (13).
-amanitin
conditions that stimulate the HIV LTR are the same as those that
promote the average CTD phosphorylation. Therefore, the bulk CTD
phosphorylation promoted by these drugs may bypass the requirement in
the viral protein Tat to recruit kinases to phosphorylate the CTD on
the HIV LTR. Osmotic shock (64), UV irradiation (64), DNA damage (85), heat shock (68, 86), okadaic acid treatment (67), and peroxovanadium compounds (69) have been shown to activate the HIV LTR transcription. Interestingly enough, osmotic shock, peroxovanadate, UV irradiation (data not shown), okadaic acid, and heat shock (8, 80) all promote CTD
phosphorylation in vivo. Most of these stresses partially inhibit the global transcriptional activity. Hence, an enhanced average
phosphorylation of CTD may result in HIV LTR activation in a very large
number of situations when class II gene transcription is impaired.
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ACKNOWLEDGEMENTS |
---|
We are indebted to Drs. Ekkehard Bautz, Osvaldo Flores, Uriel Hazan, Ula Hibner, Nicole Israël, Olivier Jean-Jean, Ian Maxwell, Rick Morimoto, and David Price for the generous gifts of antibodies and plasmids.
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FOOTNOTES |
---|
* This work was supported by grants from the Association pour la Recherche sur le Cancer, the Ligue Nationale Contre le Cancer, and the Directorate General from the European Communities, Human Capital and Mobility contract CHRX-CT93-0260.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to this work.
§ Present address: Universitá degli Studi di Roma, "La Sapienza," Istituto di Parassitologia, Piazzale A. Moro, 5, 00185 Roma, Italy.
¶ To whom correspondence should be addressed. Tel.: 331-4432-3410; Fax: 331-4432-3941; E-mail: bensaude{at}biologie.ens.fr.
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ABBREVIATIONS |
---|
The abbreviations used are:
RNAP, RNA polymerase
II;
HIV-1, human immunodeficiency virus type 1;
LTR, long terminal
repeats;
CTD, C-terminal domain;
RSV, Rous sarcoma virus;
CMV, cytomegalovirus;
DRB, 5,6-dichloro-1--D-ribofuranosylbenzimidazole;
CDK, cyclin-dependent kinases;
PCR, polymerase chain reaction;
Pipes, 1,4-piperazinediethanesulfonic acid.
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REFERENCES |
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