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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2006.00147.x |
Transcription Factors Ets2 and
Sp1 Act Synergistically with Histone Acetyltransferase p
Hai-Jing SUN1,2,
Xin XU1, Xiu-Li WANG1, Liang WEI1, Fen LI1,
Jun LU1*, and Bai-Qu HUANG1
1 Institute of Genetics and
Cytology,
Received:
November 17, 2005
Accepted:
December 23, 2005
This work
was supported by the grants from the National Natural Science Foundation of
author: Tel, 86-431-5099768; Fax, 86-431-5681833; E-mail, [email protected] or
[email protected]
Abstract There has been considerable interest
in researching the regulatory mechanisms that control the synthesis of
interleukin (IL)-12, which plays a central role in the differentiation of
T-helper-1 cells. In this study, we performed a series of transient
transfection experiments designed to elucidate the functional relationship
between the IL-12 promoter-specific transcription factors (Ets2 and Sp1) and
histone acetylation modification in IL-12 regulation mediated by p300 and
various histone deacetylases (HDACs). Results presented in this report
demonstrated that the transcription factors Ets2 and Sp1 acted synergistically
with p300 to activate the human IL-12 promoter. The histone acetyltransferase
(HAT) activity of p300 was required for this synergic effect, because the
adenovirus E
Key words Ets2; Sp1; histone acetyltransferase;
p300; interleukin-12
Interleukin (IL)-12 is a
disulfide-bonded heterodimeric lymphokine that acts as a growth factor for
activated T cells, independent of IL-2, and synergizes with suboptimal
concentrations of IL-2 to induce lymphokine-activated killer cells [1]. IL-12
is produced by macrophages and dendritic cells and is required for
cell-mediated immunity against intracellular microbes. IL-12 is a heterodimeric
molecule (p70) consisting of a heavy chain (p40) and a light chain (p35). IL-12
p40 is the determining subunit in IL-12 function, and the
regulation of its expression in antigen-presenting cells is a critical event
in the pathogenesis of infectious and inflammatory diseases [2].
Regulation of p40 is
fulfilled primarily at the transcription level and is likely to be controlled
by regulatory elements in the promoter region that can influence the
transcriptional activity of the gene. The promoter of the IL-12 p40 gene
contains multiple response elements that act as protein binding sites. For
instance, NF-kB and Ets2
are the two most important transcription factors in IL-12 expression
regulation, which bind to the –114/–104 and –212/–207 regions [3,4]. It has been reported that C/EBPb increases IL-12 gene transcription by
interacting with the –80/–74 region of the promoter [5]. Moreover, Sp1 and
AP-1 were found to contribute equally to the regulation of IL-12
expression [6,7].
It is now clear that
chromatin remodeling, an epigenetic process that modulates the structure and
function of chromatin, participates in the activation/suppression of gene expression.
The reversible acetylation/deacetylation modification of core histone tails,
accomplished by histone acetyltransferases (HATs) and histone deacetylases
(HDACs), is a major mechanism in chromatin remodeling [8]. p300, a protein that
is associated with the adenovirus E
counteracting enzymes of HATs, catalyzing the deacetylation of histones. So
far, eleven human HDAC proteins, all of which share a highly conserved
catalytic domain, have been identified [21]. There has been evidence that HDACs
regulate gene transcription, not only by deacetylating core histones, but also
by deacetylating various transcription factors [22–24].
We have previously
reported that p300 was able to increase endogenous IL-12 p40 mRNA
expression, and that the transcription factor C/EBP can be acetylated by p300
[16,17]. In this study, we further investigated the effects of two other IL-12
gene transcription factors (Ets2 and Sp1) on p300-mediated activation of
the IL-12 promoter. Our results indicated that Ets2 and Sp1 worked
synergistically with p300 to activate the IL-12 gene. Also, E
Materials and Methods
Reagents
Cell culture reagents
were provided by Gibco-BRL, and cell culture plastic ware was from
Cell lines and cell
culture
293T embryonic kidney
cells were cultured in Dulbecco Eagle’s minimum essential medium (Gibco
Laboratories,
Plasmids
A 950-bp fragment of
IL-12 p40 promoter amplified by polymerase chain reaction (PCR) from the human
genomic DNA was cloned into the luciferase reporter construct, pREP4, at the BglII
site. Ets2 expression vectors were gifts from Dr. Jürgen DITTER (University
Tübingen,
Transient transfection
and luciferase assay
293T cells were
transfected using a standard calcium phosphate method using 2.5´105 cells with 1 mg of DNA, unless indicated otherwise. Cells were
harvested 24 h after transfection. Cell lysates were collected and luciferase
activity was measured with a Turner design TD-20/20 luminometer in the
Dual-luciferase assay system (Promega,
Results
Several transcription
factors acted synergistically with p300 to activate the human IL-12 p40 promoter
The IL-12 p40 promoter contains
binding sites for a number of transcription factors, such as NF-kB, Ets2, C/EBP, Sp1 and AP-1, and these
transcription factors have been found to be able to activate the IL-12 p40
promoter [3–7]. A
schematic presentation of the IL-12 p40 promoter structure and the binding
sites for relevant transcription factors is shown in Fig. 1. All these
transcription factors interact with p300 and use CBP/p300 as a coactivator [25–29]. It is highly likely that these transcription
factors recruit p300 on the IL-12 p40 promoter to promote IL-12
expression. In a previous report we showed that both C/EBP and c-Rel could
function together with p300 to activate IL-12 p40 expression [16,17]. To
investigate if Ets2 and Sp1 have a similar synergistic action with p300, we
performed co-transfection and luciferase IL-12 p40 promoter reporter assay
experiments with constructs of Ets2, Sp1 and p300. As shown in Fig. 2,
Ets2 and Sp1 increased IL-12 promoter-driven luciferase activity by 36.6-fold
and 2.3-fold, respectively. When the cells were co-transfected with Ets2/Sp1
and p300wt, the activity of the IL-12 reporter gene was significantly
increased by 62.9-fold and 9.9-fold, respectively (Fig. 2). These
results demonstrated that both Ets2 and Sp1 had similar effects to C/EBP and
c-Rel in activating the IL-12 promoter in cooperation with p300, indicating
that as a coactivator, p300 was involved in the transcription factor-mediated
transactivation of the IL-12 promoter.
The synergy between
transcription factors and p300 was acetylase-dependent
To determine whether or
not the synergy was acetylase-dependent, we performed co-transfection studies
using constructs of p300wt and p300DHAT. As shown
in Fig. 3, transcription factors acted synergistically with p300wt to
activate the human IL-12 promoter. When Ets2 and Sp1 were co-transfected with
p300DHAT, this synergistic
effect disappeared [Fig. 3(A,B)]. The induction fold of reporter gene in
cells co-transfected with p300wt was significantly different (P<0.01)
from that in cells co-transfected with p300DHAT [Fig. 3(A), column 4 vs. 5; Fig. 3(B), column 4 vs. 5]. This result indicated that the synergy between
transcription factors and p300 was acetylase-dependent.
Adenovirus E
To further confirm the
function of p300 acetylase activity on Ets2- and Sp1-activited gene expression,
we carried out co-transfection with a vector expressing wild type E
The synergy of Ets2/Sp1
and coactivator p300 can be counteracted by HDACs
Because acetylation and
deacetylation are reversible modifications catalyzed by HATs and HDACs, we
asked if HDACs are able to counteract the synergistic effects of p300 on the
IL-12 promoter. To test this, we performed transient expression assays by
co-transfecting Ets2, Sp1, p300 and six human HDACs (1–6). As shown in Fig. 5, certain HDACs strongly
inhibited the synergistic effects between p300 and Ets2/Sp1. Specifically,
HDAC5 exerted little effect on Ets2/p300 synergy [Fig. 5(A), lane 15],
whereas HDAC4 had a significant inhibitory effect on Sp1/p300 synergy [Fig.
5(B), lane 15]. Conversely, p300 was able to rectify the inhibitory ability
of HDACs, to a variable extent [Fig. 5(A), lanes 11–16; Fig.
5(B), lanes 11–16]. Data presented in Fig. 5 clearly
demonstrated that HDACs repressed the expression of IL-12 promoter
reporter activated by Ets2 and Sp1 [Fig. 5(A), lanes 5–10; Fig.
5(B), lanes 5–10].
Discussion
It has been known that,
as a HAT, p300 is also a “factor acetyltransferase” (FAT). Besides histones, many transcription
factors have been reported to be substrates of p300 acetylase. The acetylase
activity of p300 plays an important role in its action on transcription
factors. Acetylation of these factors results in alterations of their DNA
binding and transcription activation properties, as well as their interactions
with other proteins [29,33]. In this report, we showed that the HAT activity of
p300 is necessary for the synergy between transcription factors and p300 (Fig.
2). We speculate that the transcription factor Ets2 may be acetylated by
p300 when activating IL-12. It is noteworthy from our results that
although the p300 HAT activity was important for the synergistic effect between
p300 and Sp1, this effect was not as prominent as that for Ets2 (Fig. 4).
We thought that the acetylase activity of p300 was not always necessary for its
action on transcription factors. Suzuki et al. reported that the p300
function was acetylase-independent although the HAT domain played a role in
activating Sp1 [34]. Clearly, further studies are needed to fully address this
issue.
HDACs may achieve the
inhibition by deacetylating histones or transcription factors. Human HDACs fall
into three classes. Class I HDACs (HDAC1, 2, 3 and 8) are orthologous of yeast
RPD3; class II comprising HDAC4, 5, 6, 7, 9 and 10, are yeast HDA1 orthologous [21]; the yeast
silencing protein SIR2 homologues (SIRTs) are classified as class III HDACs.
The co-transfection results presented in this report revealed that different
HDACs exhibited a variable extent of inhibitory effects on transcription
factor/p300 synergy in activating the IL-12 promoter. One distinct feature of
class II HDACs is their ability to shuttle between the nucleus and the
cytoplasm. It is therefore expected that different HDACs have diverse functions
in vivo, resulting in different inhibitory abilities.
It was reported that
Ets2 had a physical interaction with p300, and p300 was indispensable for Ets2
when it activated the human stromelysin promoter [26]. Integrating the results presented in Fig. 3(A),
Fig. 4(A) and Fig. 5(A), we speculate that Ets2 is regulated by
reversible acetylation when it activates IL-12 and acetylated Ets2 is
needed for its function. All these results support our view that reversible
acetylation is involved in the activation of the IL-12 promoter.
Apart from its HAT
activity, the p300 protein may act as a bridging factor to connect
sequence-specific transcription factors to the basal transcription machinery.
Experimental data from our previous study and this study revealed that the
Ets2, Sp1, C/EBP and NF-kB binding
sites at the IL-12 p40 promoter were involved in p300-mediated activation of
the gene. As a bridging factor, p300 may play a role in the assembly of all
these transcription factors to form a transcription complex on the IL-12
promoter. Our previous co-immuoprecipitation data support this assumption, as
they showed that transcription factor C/EBP had a physical interaction with
p300 [17].
To conclude,
data arising from this study indicate that histone acetyltransferase p300
synergistically upregulates IL-12 expression with the transcription factors Ets2
and Sp1. This synergy can be inhibited by E
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