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Previous Article | Next Article 
Journal of Virology, December 2000, p. 11394-11397, Vol. 74, No. 23
0022-538X/00/$04.00+0
Ternary Complex Factors and Cofactors Are Essential for
Human T-Cell Leukemia Virus Type 1 Tax Transactivation of the
Serum Response Element
Maureen
Shuh
and
David
Derse*
Basic Research Laboratory, Division of Basic
Sciences, National Cancer Institute, Frederick, Maryland
21702-12011
Received 13 June 2000/Accepted 15 September 2000
 |
ABSTRACT |
The human T-cell leukemia virus type 1 Tax protein activates the
expression of cellular immediate early genes controlled by the serum
response element (SRE), which contains both the serum response factor
(SRF) binding element (CArG box) and the ternary complex factor (TCF)
binding element (Ets box). We show that TCF binding is necessary for
Tax activation of the SRE and that Tax directly interacts with TCFs in
vitro. In addition, Tax interactions with CREB binding protein (CBP)
and p300- and CBP-associated factor were found to be essential for Tax
activation of SRF-mediated transcription.
| |
TEXT |
Human T-cell leukemia virus type 1 (HTLV-1) encodes a 40-kDa trans-regulatory protein, Tax,
that was shown to activate c-fos transcription by
interacting directly with the serum response factor (SRF)
(5-7), which binds the central CC(A/T)6GG
(CArG) sequence of the serum response element (SRE). Once SRF occupies the CArG element, the ternary complex factor (TCF) establishes protein
interaction with SRF and binds DNA at the upstream GGA(A/T) site. Sap-1
or Sap-1a, Sap-2 or Sap-1b, and Elk-1, which are members of the Ets
binding protein family, have been identified as TCFs (3, 4, 11,
12, 16, 18). It is known that signal-induced activation of
c-fos expression requires SRF, TCF, and the CREB binding
protein (CBP) (13, 14, 19). We have asked whether other
cellular factors, in addition to SRF, are necessary for Tax-mediated
activation of the SRE.
As an initial test of the role of TCFs in Tax-mediated induction of
SRF-directed transcriptional activity, we compared the abilities of Tax
proteins to activate reporter gene expression controlled by promoters
containing either a full-length enhancer with a CArG box and a
5'-end-proximal TCF binding site (pSRE-Luc) or an enhancer with only
the CArG box (pCArG-Luc) (Fig. 1A).
Jurkat cells were transfected with each reporter plasmid in combination with a Tax expression plasmid (pRS-HTax1C) or an empty vector (pBS-KS-RSPA). Tax activated luciferase expression from pSRE-Luc by
almost 70-fold (Fig. 1B). In contrast, pCArG-Luc, which lacks a TCF
binding site, was not activated by Tax (Fig. 1B). For comparison, cotransfections were also performed with pHTLV-Luc, which contains the
HTLV-1 promoter, or pNF-
B-Luc, which contains NF-B binding sites.
Tax activated luciferase expression directed by pHTLV-Luc and
NF-B-Luc by approximately 25-fold and 70-fold, respectively (Fig.
1B). Thus, the TCF binding site is essential for Tax activation of the
SRE.
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FIG. 1.
Cotransfection of luciferase reporter plasmids and Tax
in Jurkat cells. (A) The transcription factor recognition sequences of
the four luciferase reporter plasmids are highlighted. The plasmid
pSRE-Luc contains five copies of the SRE element, which includes the
CArG box and TCF binding site, pCArG-Luc contains five copies of the
CArG box, pHTLV-Luc contains the HTLV-1 LTR with its three CRE sites,
and pNF-B-Luc contains five copies of the NF-B DNA binding site.
(B) Luciferase assay in which 2 µg of pRS-HTax1C or pBS-RSPA
(nonspecific Rous sarcoma virus promoter-containing plasmid) was
cotransfected with 2 µg of the indicated reporter plasmids in 2 × 106 Jurkat T cells using Superfect (Qiagen) in a
six-well plate. The reporter plasmids are shown on the y
axis, and the Tax activation levels normalized for background
luciferase expression are presented on the x axis. All
experiments were performed at least three times, and the error bars
represent the standard deviations.
|
|
A similar pattern of Tax transactivation of reporter plasmids was
observed in transfections of human 293 cells with the difference that
pSRE-Luc was activated only fivefold by Tax (Fig.
2A). Ectopic expression of Elk-1
increased Tax activation of pSRE-Luc approximately 16-fold; however,
Elk-1 expression alone had no effect on pCArG-Luc activity (Fig. 2A).
Elk-1 protein was detected in 293 cells which had been transfected with
an Elk-1 expression plasmid but not in untransfected cells (Fig. 2B).
We performed the same transactivation assay with expression of Sap-1
instead of Elk-1, and Sap-1 increased Tax activation of pSRE-Luc but
not of pCArG (data not shown). Therefore, Tax activation of the SRE in
293 cells may be augmented by ectopic expression of a TCF in this cell
line.
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FIG. 2.
Cotransfection of Tax with luciferase reporter plasmids
in 293 cells. (A) Luciferase assay in which 0.25 µg of pRS-HTax1C was
cotransfected with 0.25 µg of the indicated reporter plasmids in
5 × 104 293 cells with FuGene6 reagent (Roche) in
24-well plates. In experiments using Elk-1, either the Elk-1 expression
plasmid (pElk-1-RSPA) or pBS-RSPA was transfected at 0.25 µg per
well. The reporter plasmids are shown on the y axis, and
activation levels by Tax are represented on the x axis. The
experiment was repeated at least three times, and the error bars
represent the standard deviations. (B) Expression level of Elk-1 in 293 cells as shown by Western blotting. Lysates are labeled at the top.
Lanes 1 and 4 were loaded with mock-transfected lysates, lanes 2 and 5 were loaded with lysates from cells transfected with pElk-1-RSPA, and
lanes 3 and 6 were loaded with lysates from cells transfected with both
Elk-1 and Tax expression plasmids. Lysates were analyzed on 4 to
12% Bis-Tris gels (Novex), probed with a 1:1,000 dilution of rabbit
anti-Elk-1 ( Elk-1) (New England Biolabs) and rabbit anti-Tax
antibodies, and visualized by chemiluminescence (New England
Biolabs).
|
|
We next asked whether Tax directly interacts with either Sap-1a or
Elk-1 proteins in vitro. Glutathione S-transferase
(GST)-Sap-1, GST-Elk-1, and GST were immobilized on
glutathione-Sepharose beads and incubated with in vitro-translated
35S-labeled Tax using the GST pull-down protocol previously
described (21). The 35S-labeled Tax was retained
on GST-Elk-1 and GST-Sap-1 beads but did not bind GST control beads
(compare Fig. 3A, lanes 1 and 4, with
Fig. 3B, lane 1). As a positive control, 35S-labeled SRF
was incubated with GST-Elk-1 and GST-Sap1 and was retained on both
matrices (Fig. 3A, lanes 2 and 5) but did not bind to GST (Fig. 3B,
lane 4). A 50-kDa SRF polypeptide, which was synthesized in vitro from
an alternative AUG start site located at nucleotide 312 of the cDNA
(17) also bound the immobilized TCFs (Fig. 3A, lanes 2 and
5, lower band). In a reciprocal experiment, 35S-labeled
SRF, Elk-1, and Sap-1 were incubated with GST-Tax, and all three
proteins were retained on the GST-Tax matrix (Fig. 3A, lanes 8 to 10).
The 35S-labeled input proteins for all GST pull-down assays
are shown in Fig. 3C. Our results show that Tax and TCFs directly
interact in vitro and suggest that Tax activation of the SRE in vivo
may depend on Tax-TCF and Tax-SRF interactions.
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FIG. 3.
GST pull-down assays reveal Tax-TCF interactions. (A)
Two-microgram quantities of GST-Elk-1, GST-Sap-1 (both GST-TCF
constructs were the gifts of Andrew Sharrocks, University of
Manchester), and GST-Tax (a gift from Susan Marriott, Baylor
University), which were bound to a 50% glutathione-Sepharose slurry,
used to pull down the indicated 35S-labeled proteins. Lanes
denoted by "None" contain the GST matrix alone; no lysate was
incubated with the matrix. Bound proteins were analyzed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and
autoradiographed. (B) Results of the GST pull-down assay using the
negative-control GST matrix are shown. (C) Full-length SRF, Elk-1,
Sap-1a, and Tax were translated in vitro in a rabbit reticulocyte
lysate system (Promega TNT system). Ten microliters (10% of total
lysate) of input protein was used to indicate the amount of
35S-labeled lysate that was loaded onto the GST-bound
matrix.
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|
Mutant Tax proteins with specific transactivation phenotypes provide
important tools for determining Tax function, particularly in the
characterization of protein-protein interaction domains. To identify
other components necessary for Tax activation of the SRE, we
cotransfected Jurkat cells with a series of mutant Tax proteins and
pSRE-Luc. Tax-M47, which contains two amino acid changes (L319R and
L320S), cannot activate viral gene expression from the HTLV-1 long
terminal repeat (LTR) due to the inability of Tax-M47 to interact with
the p300- and CBP-associated factor (PCAF) (15, 22). No
activation of pSRE-Luc was seen with Tax-M47 (Fig.
4A). Tax with the G148V mutation
(Tax-G148V), which is analogous to Tax-M22 (T130S and L131A), can
activate the CREB and SRF transcriptional pathways but cannot activate
NF-B due to a defect in associating with IKK
(2, 8, 9, 22,
23). Tax-G148V retained almost 50% of wild-type Tax activity on
pSRE-Luc (Fig. 4A). Both Tax-H3S and Tax-K88A are incapable of
activating the HTLV-1 promoter, but they differ in their functional
defects: Tax-H3S is unable to interact with the bZIP domain of CREB
(1), whereas Tax-K88A is unable to bind CBP (10).
Tax-H3S activated the SRE enhancer by almost 70% in Jurkat cells
compared to the level of enhancement by wild-type Tax, whereas
Tax-K88A was unable to activate pSRE-Luc (5% of the wild-type level of
Tax) (Fig. 4A). To make certain that the defect in SRE activation was
not due to a defect in TCF binding, we performed a GST pull-down assay
with Tax-M47 and Tax-K88A. Compared to wild-type Tax (Fig. 4B, lane 2),
Tax-M47 (Fig. 4B, lane 3) and Tax-K88A (Fig. 4B, lane 4) were not
impaired in their ability to bind Elk-1. However, the inability of
Tax-M47 and Tax-K88A to interact with PCAF and CBP, respectively, was
detrimental to Tax transactivation of pSRE-Luc. None of the proteins
bound the GST-only matrix (Fig. 4B, lanes 6 to 8), and the input
proteins (Fig. 4C, lanes 1 to 3) are shown for comparison of the
amounts of protein retained on GST-Elk-1 beads. The data show that
both the CBP and PCAF binding domains of Tax are required for Tax
activation of transcription from the SRE enhancer and that these Tax
motifs are not important for Elk-1 binding in vitro.
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FIG. 4.
Functional analysis of SRE transactivation phenotypes of
mutant Tax proteins. (A) Jurkat cells were cotransfected with pSRE-Luc
and plasmids bearing genes encoding the indicated mutant Tax proteins
as described for Fig. 1. The levels of transactivation are reported
relative to the levels of activation of Tax by the wild type. (B)
Results of a GST pull-down assay to determine whether Tax-M47 and -K88A
retain their ability to interact with GST-Elk-1 are shown. The GST
matrix is shown at the top of each gel, and the 35S-labeled
proteins are shown immediately above each lane. The experiments were
performed as described for Fig. 3. (C) The 35S-labeled
proteins Tax, Tax-M47, and Tax-K88A are shown to indicate the amounts
of proteins loaded onto the matrices.
|
|
We have presented the results of functional experiments showing that
Tax activation of SRF-dependent transcription is dependent on TCF
binding to the SRE. Furthermore, transient transfections with mutant
Tax proteins revealed that CBP and PCAF are essential for Tax
activation of SRF- and TCF-dependent transcription. We also
demonstrated that Tax interacts directly with TCFs in vitro, which
suggests that Tax activation of the SRE requires contacts with TCFs as
well as SRF. We are currently analyzing the composition and assembly of
complexes formed on the SRE in the presence of Tax using nuclear
extracts and recombinant proteins.
The relative importance of various cellular transcription factor
pathways to HTLV-1-mediated T-cell transformation has been examined
using the mutant proteins Tax-M47 and -M22 (20, 22). The
former is defective for activation of the CREB and SRF pathways, while
the latter is unable to activate NF-B. In the past, the effects of
these mutations have been interpreted in the context of a
CREB-versus-NF-B dichotomy. However, neither mutation can discriminate between the CREB and SRF pathways and no mutant Tax proteins that specifically target SRF or TCF interactions have been
identified. At present only Tax-H3S, which is specifically defective
for CREB interactions, is informative in this regard and is now being
tested in T-cell transformation assays.
| |
ACKNOWLEDGMENTS |
We thank Andrew Sharrocks for his generous gifts of plasmids pAS535
and pAS600 and Susan Marriott for her gift of plasmid pGEX-Tax. We also
thank A. Sharrocks, S. Marriott, and Ralf Janknecht for helpful
discussions and advice and Huey-Jane Liao for critically reading the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Basic Research
Laboratory, Division of Basic Sciences, National Cancer Institute,
Frederick, MD 21702-1201. Phone: (301) 846-5611. Fax: (301) 846-6863. E-mail: derse{at}mail.ncifcrf.gov.
Present address: Department of Biological Sciences, Loyola
University New Orleans, New Orleans, LA 70118.
| |
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Journal of Virology, December 2000, p. 11394-11397, Vol. 74, No. 23
0022-538X/00/$04.00+0
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