Two Modes of Transcriptional Activation
at Native Promoters by NF-jB p65
Dominic van Essen1, Bettina Engist1, Gioacchino Natoli2, Simona Saccani1*
1 Max Planck Institute for Immunobiology, Freiburg, Germany, 2 Department of Experimental Oncology, European Institute of Oncology (IEO), IFOM-IEO Campus, Milan, Italy
The NF-jB family of transcription factors is crucial for the expression of multiple genes involved in cell survival,
proliferation, differentiation, and inflammation. The molecular basis by which NF-jB activates endogenous promoters
is largely unknown, but it seems likely that it should include the means to tailor transcriptional output to match the
wide functional range of its target genes. To dissect NF-jB–driven transcription at native promoters, we disrupted the
interaction between NF-jB p65 and the Mediator complex. We found that expression of many endogenous NF-jB
target genes depends on direct contact between p65 and Mediator, and that this occurs through the Trap-80 subunit
and the TA1 and TA2 regions of p65. Unexpectedly, however, a subset of p65-dependent genes are transcribed
normally even when the interaction of p65 with Mediator is abolished. Moreover, a mutant form of p65 lacking all
transcription activation domains previously identified in vitro can still activate such promoters in vivo. We found that
without p65, native NF-jB target promoters cannot be bound by secondary transcription factors. Artificial recruitment
of a secondary transcription factor was able to restore transcription of an otherwise NF-jB–dependent target gene in
the absence of p65, showing that the control of promoter occupancy constitutes a second, independent mode of
transcriptional activation by p65. This mode enables a subset of promoters to utilize a wide choice of transcription
factors, with the potential to regulate their expression accordingly, whilst remaining dependent for their activation on
Citation: van Essen D, Engist B, Natoli G, Saccani S (2009) Two modes of transcriptional activation at native promoters by NF-jB p65. PLoS Biol 7(3): e1000073. doi:10.1371/
The goal of understanding transcriptional activation
encompasses the description of an unbroken chain of events
leading from the binding of a transcription factor to its
natural target promoters in an intact cell, until the initiation
of mRNA synthesis by RNA polymerase II (pol-II). In the case
of the NF-jB family of transcription factors, this is a
challenging task, since the tremendous functional diversity
of its target genes makes it difficult to imagine a single
activation mechanism able to satisfy the needs of all of them.
Transcription factors belonging to the NF-jB family are
found in metazoan organisms ranging from insects to
mammals, and are essential in regulating the activation of
hundreds of genes in response to various extracellular
stimuli and developmental cues . In most vertebrate cell
types, NF-jB exists as a combination of five related proteins:
p65, c-Rel, RelB, p50, and p52. They share a structurally
conserved Rel homology region at their amino terminus,
which is responsible for dimerization, interaction with
inhibitory IjB proteins, nuclear entry, and binding to their
specific DNA target sequences (known as jB sites). In
unstimulated cells, dimers of NF-jB are held in the
cytoplasm through the binding of inhibitory proteins (IjBs
or p100), but upon stimulation they are released to enter the
nucleus. There they are capable of binding with high affinity
to their target sequences, found both in gene promoters and
in enhancer regions . In contrast to our detailed under-
standing of the signalling events that control the level of NF-
jB present in the nucleus, little is known about the
mechanisms of transcriptional activation by the various
dimer species whilst bound to endogenous target genes. It is
particularly unclear whether promoter binding by a given
NF-jB dimer always triggers the same fixed response,
leading to an identical transcriptional output at all genes,
or, as seems more reasonable, different genes should
somehow be able to fine tune their transcription levels after
binding and activation by NF-jB. However, the transcrip-
tional activation domain of p65 has been extensively studied
in vitro and on artificial reporter plasmids, and the data
from these systems provide a foundation on which one can
try to build an understanding of its function on natural
During the last two decades, experiments using reconsti-
tuted cell-free systems have succeeded in defining the
minimal apparatus needed to drive activated transcription.
An essential set of general transcription factors (GTFs) is
sufficient to direct the binding of, and initiation of basal
transcription by pol-II at the core regions of most promoters
. In order to respond to transcriptional activators such as
NF-jB, though, additional elements are required, foremost
Academic Editor: Jonathan D Ashwell, National Cancer Institute, United States of
Received November 11, 2008; Accepted February 17, 2009; Published March 31,
Copyright: ? 2009 van Essen et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author
and source are credited.
Abbreviations: BiFC, bimolecular fluorescence complementation; ChIP, chromatin
immunoprecipitation; DC, dendritic cell; GTF, general transcription factor; LPS,
lipopolysaccharide; pol-II, RNA polymerase II; shRNA, small hairpin RNA; TAD,
transcriptional activation domain; TFIIB, transcription factor IIB; TNF, tumor necrosis
factor; TSS, transcriptional start site
* To whom correspondence should be addressed. E-mail: email@example.com.
PLoS Biology | www.plosbiology.orgMarch 2009 | Volume 7 | Issue 3 | e10000730549
P PL Lo oS S BIOLOGY
amongst which is the Mediator complex. This is a large, multi-
subunit complex, which was independently identified by
several laboratories through its ability to bind to various
transcriptional activation domains (including that of p65), or
by its necessity as a co-factor for transcriptional activation in
vitro by other transcription factors (reviewed by Malik and
Roeder ). Using highly purified components in vitro, the
combination of pol-II, GTFs, and the Mediator complex is
sufficient to drive transcriptional activation by NF-jB .
Conversely, depletion of Mediator from total nuclear extracts
using antibodies abolishes all in vitro transcription by pol-II,
including the response to activators such as p65 [6,7].
The capacity of p65 to activate transcription has also been
the subject of numerous studies using synthetic reporter
plasmids in transfected cell lines. In this context, the carboxy
terminus of p65 (like those of c-Rel and RelB) is able to drive
transcription in isolation when fused to a heterologous
DNA-binding domain, leading to its definition as a tran-
scriptional activation domain (TAD [8,9]). Since the Medi-
ator complex has been shown to interact with the TAD of
p65 , a straightforward model would be that direct
binding to Mediator constitutes the initial, essential step in
p65-driven transcription; however, to our knowledge this has
never been tested in vivo at native promoters. Part of the
reason for this may be that the Mediator complex is essential
for viability, and thus it is not readily amenable to loss-of-
function-based experiments in intact cells. In order to test
the requirement for this interaction at native NF-jB target
promoters, we sought to disrupt the contact between p65
and Mediator by eliminating a single Mediator subunit in
We found that contact with Mediator is indeed essential for
p65 to drive the expression of many NF-jB target genes.
Unexpectedly, though, many others were still expressed
normally even when this contact was disrupted. Further
experiments revealed that p65 has a second, independent
mode of transcriptional activation, which acts by regulating
promoter occupancy by secondary transcription factors.
Removal of Trap-80 Disrupts the Interaction of p65 with
We wanted to identify a subunit of the Mediator complex
that directly contacts p65, and whose removal would abolish
the interaction of p65 with the remaining complex. As a cue,
we noted that the Drosophila NF-jB homologue Dif has been
shown to interact with Med17 (amongst other Mediator
subunits ). Although the TAD of p65 shows no obvious
sequence homology with that of Dif, we speculated that it
might nonetheless contact the corresponding mammalian
Mediator subunit, Trap-80. Using the yeast two-hybrid
system, we were able to detect an interaction between the
amino-terminus of Trap-80 and the far carboxy-terminus of
p65 (Figure S1A). Since none of the known components of
the Mediator complex are well conserved between yeast and
mammals at the primary amino acid sequence level , this
strongly suggests that the interaction of p65 with Trap-80 is
direct; however, at this point we could not exclude that it may
be bridged or stabilized by interaction with some endogenous
An over-expressed, tagged form of Trap-80 could be co-
immunoprecipitated with p65 from nuclear extracts of
transfected HEK-293 cells (Figure S1B), confirming that the
two proteins can associate into a complex together. To
establish whether they occupy adjacent positions within the
complex, we used the bimolecular fluorescence complemen-
tation (BiFC) approach . We co-expressed fusion proteins
of p65 joined, via a short peptide linker, to an amino-
terminal fragment of the fluorescent protein Venus, and of
Trap-80 similarly joined to a complementary fragment from
the Venus carboxy-terminus. Neither of these fragments is
itself fluorescent, but if brought sufficiently close by an
interaction between their respective fusion partners, they
form a bimolecular fluorescent entity (the maximum permis-
sible distance separating the tethered ends is limited by the
peptide linkers (16 amino acids, or around 60 A˚each), and
has been empirically estimated at around 100 A˚—roughly
comparable to the diameter of the Rel homology region of
p65 ). Fusions of Venus fragments to the carboxy-
terminus of p65, or to the amino-terminus of Trap-80 were
nonfluorescent when expressed alone, but, in close agree-
ment with the yeast two-hybrid experiments, cells co-
expressing both together were fluorescent, indicating that
the two proteins are juxtaposed in vivo (Figure S1C).
Together, these data suggested that Trap-80 forms part of
the contact surface of the Mediator complex through which it
interacts with p65. Although this interaction may include
regions of contact with other Mediator subunits, we
considered that Trap-80 was a good candidate as a subunit
whose removal might destabilize binding by p65. Therefore,
we attempted to disrupt the p65-Mediator interaction by
generating cell lines in which Trap-80 expression was stably
knocked-down by RNA interference. At the outset, this
seemed a risky approach, since in other systems Trap-80 has
been shown to be essential for cell viability. Yeast with a null
mutation of the homologous Srb4 gene are nonviable, and in
cells carrying a temperature-sensitive allele, most mRNA
synthesis ceases at the restrictive temperature [15,16]. Like-
wise, dTrap-80 is needed for both basal and activated
transcription in Drosophila SL2 cells , and Boube et al.
PLoS Biology | www.plosbiology.orgMarch 2009 | Volume 7 | Issue 3 | e1000073 0550
Two Modes of Activation by p65
Transcriptional activation by the NF-jB family of transcription
factors is crucial for the expression of multiple genes involved in
cell survival, proliferation, differentiation, and inflammation. The
activation domain of the p65 subunit of NF-jB has been extensively
studied in vitro and on artificial reporter plasmids, but the molecular
basis by which it drives expression of natural target genes in vivo is
still not well understood. Moreover, it is unclear how any single
activation mechanism could allow different target genes to fine tune
their timing and expression according to their biological require-
ments. To address this, we experimentally blocked the interaction of
p65 with the Mediator complex—a key factor for transcription by
most, if not all, activators. While this prevented expression of many
NF-jB–dependent genes, others were unaffected, revealing that p65
is able to drive their expression by an independent mode, which
does not depend on direct contact with Mediator. Further experi-
ments indicated that p65 accomplishes this by controlling the
recruitment of other, secondary transcription factors to its target
promoters. This may enable NF-jB to retain overall control over
activation of its target genes, but at the same time allow secondary
transcription factors to specify appropriate expression levels
according to the cell-type and stimulus.
 have shown in the Drosophila epidermis that mutation of
dTrap-80 is lethal for cells.
Strikingly, then, we were able to generate clonal lines of
mouse 3T3 fibroblasts in which Trap-80 mRNA expression was
reduced by .90% compared to wild-type levels (Figure 1A),
and Trap-80 protein levels were no longer detectable by
western blotting (Figure 1B). These Trap-80–deficient fibro-
blasts proliferated equivalently to control cells and appeared
morphologically normal (Figure S2), could be grown in
culture for at least 12 wk, and expanded by at least 1020-
fold (;30 passages; unpublished data). Moreover, microarray
analysis indicated that the expression levels of .96% of
transcripts were changed by less than 1.5-fold in Trap-80–
deficient cells (see Figure 2B later).
We used the Trap-80–deficient cells to determine whether
Trap-80 is indeed essential for binding of p65 to the Mediator
complex. To this end, we tested whether p65 could be co-
precipitated with an alternative Mediator subunit, Trap-95,
from nuclear extracts of Trap-80–deficient fibroblasts. We
used streptavidin beads to pull-down the Mediator complex
from cells expressing a biotin-tagged allele of Trap-95. In
cells containing Trap-80, p65 was pulled-down with the
Mediator complex, reconfirming their in vivo interaction
(Figure 1C). However, no p65 was pulled-down from Trap-80
knock-down cells, indicating that the Trap-80 subunit is
required for the interaction between p65 and Mediator in
Therefore, Trap-80–deficient cells represent an experi-
mental system with which we could test the importance of the
interaction with the Mediator complex for transcriptional
activation in vivo by p65.
Trap-80–Dependent and –Independent NF-jB Target
We examined the expression of endogenous NF-jB target
genes in Trap-80–deficient cells, in response to stimulation
with the cytokine tumour necrosis factor-a (TNF-a). 3T3
fibroblasts are a particularly useful model system in which to
study activation by p65, since in these cells most NF-jB–
driven transcription relies on this subunit [18,19]. We
predicted that if transcriptional activation of endogenous
genes by p65 depends on its interaction with Mediator, as
implied by in vitro studies [6,10], then they should not be
expressed in Trap-80–deficient cells. In agreement with this,
we found that expression of the Ip-10 and Il-6 genes was
abolished in cells lacking Trap-80 (Figure 2A). Two inde-
pendent small hairpin RNAs (shRNAs) targeting Trap-80 gave
the same result, and expression could be restored by
reconstitution of Trap-80 knock-down cells with an shRNA-
resistant form of Trap-80, ruling out the possibility that the
block in expression was caused by an off-target effect of the
shRNAs (Figure S4).
Unexpectedly, however, two other NF-jB target genes, Mip-
2 and Nfkbia, were unaffected by the absence of Trap-80, and
Figure 1. Removal of Trap-80 Disrupts the Interaction of p65 with Mediator
(A) Knock-down of Trap-80 mRNA by RNA interference. Expression level of Trap-80 mRNA in 3T3 fibroblasts expressing an shRNA targeting Trap-80,
shown as a percentage of the level in control cells (expressing an irrelevant shRNA, hereafter referred to as wild type). mRNA levels were measured by
quantitative PCR and normalized with respect to Tbp. Error bars indicate standard errors; the results presented here are representative of more than ten
(B) Knock-down of Trap-80 protein levels. Nuclear extracts were prepared from wild-type (wt) or Trap-80 knock-down (kd) fibroblasts, and endogenous
Trap-80 (upper panel) or PCNA (as a loading control, lower panel) was detected by western blotting.
(C) Co-precipitation of p65 and Trap-95 depends on the presence of Trap-80. Wild-type and Trap-80 knock-down fibroblasts were simultaneously
transduced with retroviruses expressing biotin-tagged Trap-95 and the BirA biotin ligase, and nuclear extracts were prepared after stimulation with
TNF-a. The Mediator complex, containing biotinylated Trap-95, was pulled down using streptavidin beads (biot P.D.), and any associated p65 was
detected by western blotting. Pull-down using in vivo biotinylation is our method of choice for analysis of proteins bound to the endogenous Mediator
complex, due to the extremely high affinity of the streptavidin-biotin interaction. The levels of biotin-tagged Trap-95 were similar in both cell types, as
measured by the level of co-expressed Tomato fluorescent protein (Figure S3). Dotted lines indicate where nonrelevant lanes have been cropped from
PLoS Biology | www.plosbiology.org March 2009 | Volume 7 | Issue 3 | e10000730551
Two Modes of Activation by p65
were expressed in cells containing either shRNA at the same
level as in control cells (Figure 2A). To verify that tran-
scription of these genes was indeed dependent on p65, we
analysed their expression in fibroblasts derived from p65-
knockout mice. In agreement with earlier published results
[18,19], production of Mip-2, Nfkbia, and Ip-10 mRNA was
completely abolished, and Il-6 mRNA levels were strongly
reduced (Figure S5). Thus, in 3T3 fibroblasts, p65-dependent
genes can be subdivided, depending on whether they require
the interaction of p65 with the Mediator complex for their
expression (Trap-80–dependent; exemplified by Ip-10 and Il-
6), or instead can be expressed even when this interaction is
disrupted (Trap-80–independent; exemplified by Mip-2 and
To examine the generality of this grouping, we performed a
microarray analysis of the levels of 29,000 transcripts in wild-
type and Trap-80 knock-down cells, before and after
stimulation of NF-jB activity using TNF-a. Genes induced
by TNF-a are dominated by known NF-jB targets, and their
promoters are significantly enriched for NF-jB binding
motifs (Tables S1 and S2). Amongst these TNF-a–induced
genes, Trap-80–dependent genes are strongly enriched (22%,
compared with ,3% of non-TNF-a–induced genes; Figure
2B)—supporting the importance of the interaction with
Mediator for p65-driven transcription. On the other hand,
when considering all Trap-80–dependent genes, although
TNF-a–induced genes are significantly over-represented
(10%, compared with ,0.2% of Trap-80–independent genes;
Figure S6), the majority are unaffected by TNF-a treatment,
indicating that NF-jB is not alone in its functional require-
ment for Trap-80. A subset of both Trap-80–dependent and
Trap-80–independent NF-jB target genes were validated by
quantitative reverse transcription (RT)-PCR (Figure S7), and
the results closely correlated with those of the microarray (r¼
0.85). We chose to focus on Ip-10, Il-6, Mip-2, and Nfkbia for
further study, since these displayed clear-cut dependencies
Interaction of p65 with Mediator Drives Recruitment of
We initially considered the mechanism of activation of
Trap-80–dependent genes. First, we performed chromatin
immunoprecipitation (ChIP) using antibodies against p65, to
establish whether disrupting its interaction with Mediator
could somehow inhibit p65 from binding to some of its target
promoters. We found that p65 was efficiently recruited to the
promoters of the Trap-80–dependent genes Ip-10 and Il-6
upon TNF-a stimulation, and its level of binding was only
slightly reduced in Trap-80–deficient compared to wild-type
cells (Figure 3A). Moreover, the level of p65 binding to the
Trap-80–independent Nfkbia promoter was also slightly
reduced to a similar extent, arguing that this is not sufficient
to explain the failure in Ip-10 and Il-6 transcription. Binding
to the Mip-2 promoter was completely unaffected.
Next, we did ChIP with antibodies against pol-II to
investigate whether its recruitment to promoters was a
consequence of the p65-Mediator interaction. Indeed, asso-
ciation of pol-II with the promoters of Ip-10 and Il-6 was
completely prevented in Trap-80–deficient cells (Figure 3B).
In contrast, it was strongly recruited to the Mip-2 promoter
both with and without Trap-80. Pol-II was also recruited to
the Nfkbia promoter in the absence of Trap-80, but at a
reduced level, mirroring the lower level of p65 binding noted
We also examined the recruitment of the general tran-
scription factor IIB (TFIIB) to promoters, in the presence and
Figure 2. Trap-80–Dependent and –Independent Genes
(A) Expression of Mip-2, Nfkbia, Ip-10, and Il-6 mRNA in subconfluent cultures of wild-type and Trap-80 knock-down fibroblasts, after stimulation with
TNF-a. mRNA levels are expressed relative to unstimulated, wild-type cells; the results presented here are representative of more than ten experiments.
(B) Trap-80–dependence of TNF-a–induced versus noninduced genes. Cumulative percentage of transcripts whose expression in Trap-80 knock-down
cells exceeds the indicated level in wild-type cells, expressed both as the D log signal of the microarray probes (upper scale) and the calculated relative
expression level using a standard curve generated by quantitative PCR of a subset of 36 genes (lower scale). The red line represents the top 100 TNF-a–
induced transcripts; the black line represents 5,000 transcripts whose levels were unchanged by TNF-a stimulation. Table S1 lists the top 50 Trap-80–
dependent and –independent TNF-a–induced genes.
PLoS Biology | www.plosbiology.org March 2009 | Volume 7 | Issue 3 | e10000730552
Two Modes of Activation by p65
absence of Trap-80. TFIIB is an essential component of the
pre-initiation complex, shown in vitro to be required for the
recruitment of pol-II . Consistent with this, TFIIB
appeared at the Ip-10 and Mip-2 promoters concomitantly
with pol-II in wild-type fibroblasts (Figure 3C), although at
later time points the relative levels of promoter-associated
TFIIB declined. In Trap-80–deficient cells, the recruitment of
TFIIB to the Mip-2 promoter was unimpaired (and even
slightly augmented; Figure 3C). However, TFIIB levels at the
Trap-80–dependent Ip-10 promoter were severely reduced,
foretelling the failure of this promoter to recruit pol-II.
Since Trap-80 seemed not to be required for the recruit-
ment of pol-II or TFIIB to Trap-80–independent promoters,
we wondered whether a Mediator complex containing Trap-
80 associates with these promoters at all. To check this, we
used antibodies against Trap-80 to examine its presence at
promoters by ChIP. As expected, we detected Trap-80 at the
promoters for the Trap-80–dependent Ip-10 and Il-6 genes
(Figure 3D). We also found Trap-80, though, at the Mip-2 and
Nfkbia promoters, despite the fact that these genes can still be
expressed normally in cells where Trap-80 levels have been
knocked-down. This suggests that a Mediator complex which
ordinarily contains Trap-80 is involved in transcriptional
activation at all of these promoters, but that the Trap-80
subunit is functionally essential only at some of them.
However, one caveat to this interpretation is that the
apparent difference in Trap-80 dependency between the
two classes of promoters might be only quantitative, and the
seemingly Trap-80–independent Mip-2 and Nfkbia promoters
might actually manage to bind to the low level of residual
Trap-80 remaining in the knock-down cells. To deal with this
concern, we also checked for the presence of Trap-80 at these
promoters in Trap-80–deficient cells. Trap-80 was undetect-
able at any promoters in Trap-80 knock-down cells, including
Figure 3. Interaction of p65 with Mediator Drives Recruitment of pol-II
(A) Trap-80 knock-down does not prevent p65 recruitment to its target promoters.
(B) Trap-80 knock-down blocks pol-II recruitment to Trap-80 dependent promoters.
(C) Trap-80 knock-down blocks TFIIB recruitment to Trap-80 dependent promoters.
(D) Presence of Trap-80 at promoter regions.
(E) Trap-80 knock-down blocks Med-26 recruitment to Trap-80 dependent promoters.
ChIP using antibodies against p65 (A), pol-II (B), TFIIB (C), Trap-80 (D), and Med-26 (E), after stimulation with TNF-a. Bound DNA was analyzed using
primers and probes specific for promoter regions. The amounts are presented as the percentage recovered out of the total input DNA (percent input).
All measurements were performed in duplicate; the results presented here are representative of two to five experiments.
PLoS Biology | www.plosbiology.org March 2009 | Volume 7 | Issue 3 | e10000730553
Two Modes of Activation by p65
those of Mip-2 and Nfkbia—confirming that it is truly
dispensable for the expression of these genes.
In Trap-80–deficient cells, the Mediator complex is
‘‘invisible’’ when using antibodies against Trap-80. We there-
fore used antibodies against another component, Med-26, to
assess the involvement of the Mediator complex when Trap-
80 is missing. After stimulation of Trap-80–deficient cells we
could still find roughly normal levels of Med-26 at the Mip-2
promoter, confirming that its transcription involves the
Trap-80–independent participation of Mediator, and also
serving as a control that in these cells the Mediator complex is
not drastically disrupted (Figure 3E). In these cells, however,
Med-26 was undetectable at the Trap-80–dependent Ip-10
promoter. This supports the notion that the inability of p65
to interact with the Mediator complex in Trap-80–deficient
cells underlies their failure to transcribe Trap-80–dependent
Interestingly, we noticed that Trap-80 was present on
promoters at above-background levels in resting wild-type
cells, preceding the stimulus-induced promoter-binding by
NF-jB (Figure 3D; compare with Figure 3A). This was
particularly apparent at the promoters for Nfkbia and Ip-10,
and in parallel experiments in which HA-Trap-80 was stably
over-expressed by around 10–1003, we could also detect HA-
Trap-80 at the Mip-2 and Il-6 promoters before stimulation
(Figure S8). In contrast, we detected Med-26 at the Ip-10 and
Mip-2 promoters only after transcription was induced by
stimulating wild-type cells with TNF-a (Figure 3E). The Med-
26 subunit is associated with an active subcomplex of
Mediator that is able to bind pol-II, and which accounts for
its transcriptional cofactor activity in vitro [21–24]. Our
results indicate that while some Mediator seems to be
preloaded on promoters in vivo, as has recently been
described in yeast , contact with p65 is required for the
establishment of an active, Med-26-containing complex at
target promoters upon stimulation.
Taken together, our data indicate that one mechanism of
transcriptional activation by p65 depends on its direct
interaction with Mediator, and that this is essential for
expression of a subset of its target genes in vivo. Without
Trap-80, p65 binding to the promoters of these genes is not
prevented, but once bound it is unable to interact with the
Mediator complex, and thereby drive the recruitment of pol-
II and the initiation of transcription.
Artificial Contact with Mediator Can Bypass the
Requirement for Trap-80
Three predictions arise from this model: first, the binding
sites for p65 should be situated close to the transcriptional
start sites of Trap-80–dependent promoters. We analysed the
TNF-a–induced genes revealed by the microarray, and could
identify conserved (between mouse and human) NF-jB
binding motifs with high confidence in 85% of Trap-80–
dependent promoters. At .92% of these, the promoter-
proximal site lies within 800 bp of the transcriptional start
site (see later), consistent with a direct role for p65 in
interacting with Mediator to recruit pol-II.
Second, one should be able to bypass the need for Trap-80
by artificially recruiting an alternative transcriptional activa-
tion domain capable of interacting with a different Mediator
subunit, to Trap-80–dependent promoters (schematically
depicted in Figure 4). To attempt this, we chose to use the
well-studied transcriptional activation domain of the herpes
simplex virus VP16 protein. Transcriptional activation by the
VP16 TAD depends on its direct interaction with the Med25
subunit of the Mediator complex, which can occur through
either of two subregions (H1 and H2 ). Also, the only
proteins it can pull-down from total nuclear extracts are
Mediator components , implying that additional, un-
wanted interactions with other nuclear constituents are weak
or nonexistent. To effect recruitment to NF-jB target
promoters, we used the Rel homology region of p65 (p65
DBD, encompassing both its DNA-binding domain and also
the region required for regulation by IjBa). When over-
expressed in wild-type cells, the p65 DBD is able to out-
compete full-length p65 for binding to jB sites in promoters,
and acts as a dominant-negative allele (Figure S9). We
generated retroviruses encoding fusion proteins between
the p65 DBD and the H1 region of the VP16 TAD, since the
H2 region has been shown to make nonessential contacts with
Trap-80 [4,26]. After stimulation with TNF-a, Trap-80–
deficient fibroblasts transduced with a control virus encoding
full-length p65 still showed severely impaired Ip-10 expres-
sion compared to wild-type fibroblasts (although the over-
expression of p65 did slightly increase Ip-10 levels above those
seen in untransduced cells; Figure 4). Expression of the p65
DBD fused to the H1 region of VP16, however, fully restored
Ip-10 expression in the absence of Trap-80, to levels that even
exceeded those seen in wild-type fibroblasts (Figure 4). Thus,
when contact between p65 and Mediator is prevented by the
absence of Trap-80, artificial contact with a different
Mediator subunit is sufficient to rescue expression of a
Trap-80–dependent NF-jB target gene.
Mutations of p65 That Prevent Interaction with Trap-80
The third prediction is that it should be possible to mimic
the absence of Trap-80 at NF-jB–dependent promoters by
introducing mutations into p65 that disrupt its interaction
with Trap-80. Two transcriptional activation regions have
previously been identified within the carboxy-terminus of
p65 (TA1 and TA2 [8,9]). We generated mutant forms of p65
in which either or both of these regions were deleted, and
assayed their in vivo interaction with Trap-80 using BiFC. As
a negative control we used the p65 DBD, which lacks the
entire carboxy-terminus. All mutants were expressed at
comparable levels, as detected by western blotting (unpub-
lished data), and interacted to similar extents with full length
p65 (Figure 5B). However, deletion of either TA1 or TA2
alone each diminished the interaction with Trap-80, and
deletion of both together (p65DTA1&2) completely reduced
it to background levels (Figure 5A). We next tested the ability
of each mutant to rescue NF-jB target gene expression in
TNF-a–stimulated p65-knockout fibroblasts. Transduction
with viruses encoding full-length p65, or p65 with deletions
of either TA1 or TA2 alone, restored transcription of both
the Ip-10 and Mip-2 genes to levels that equalled or even
exceeded those in wild-type cells (Figure S10). Notably
though, the p65 mutant lacking both TA1 and TA2 was
completely unable to drive transcription of the Trap-80–
dependent Ip-10 gene, but it could still activate expression of
the Trap-80–independent Mip-2 gene to wild-type levels
(Figure 5C). Thus, p65 can activate transcription of Trap-
80–dependent and –independent genes using separable
regions within its carboxy-terminus. These findings can be
PLoS Biology | www.plosbiology.orgMarch 2009 | Volume 7 | Issue 3 | e1000073 0554
Two Modes of Activation by p65
explained by an inability of the p65DTA1&2 mutant to
interact with Mediator. However, since it could also be
argued that deletion of a substantial domain from p65 may
have other, additional consequences for the protein’s
function, we sought to identify more subtle mutations in
which interaction with Trap-80 was still disrupted. We used
the p65 mutant lacking TA2 as a template, since this protein
drives transcription of Ip-10 and Mip-2 normally, but depends
on TA1 for its interaction with Trap-80 (Figures 5A and S10).
By initially substituting blocks of seven amino acids within
TA1 (e.g., TA1 mut528–534 and TA1 mut535–541), and
subsequently by mutating adjacent pairs of amino acids, we
were able to identify a p65 mutant in which only two amino
acid changes result in the abolition of the interaction with
Trap-80 (TA1 DF539AA; Figure 5D). This mutant can still
activate transcription of the Trap-80–independent Mip-2
gene, but is inactive at the Trap-80–dependent Ip-10
promoter (Figure 5F).
Thus, using two independent approaches—knock-down of
Trap-80 and targeted mutation of the p65 carboxy-termi-
nus—we find that contact with the Mediator complex
through the Trap-80 subunit is responsible for transcrip-
tional activation by p65 at a subset of its target genes in vivo.
p65 Controls the Recruitment of Secondary Transcription
The observation that expression of many endogenous
target genes (including Mip-2 and Nfkbia) is unimpaired in
Trap-80–deficient fibroblasts, though, indicates that p65 can
utilize a second mode of transcriptional activation at these
promoters, which does not depend on either of the tran-
scriptional activation domains identified in earlier in vitro
studies. To try to uncover features that might explain their
different requirements for activation by p65, we compared
the promoters of Trap-80–dependent and –independent
TNF-a–induced genes identified by our microarray analysis.
We could identify conserved jB motifs in a similar fraction of
Trap-80–dependent and –independent TNF-a–induced genes
(85% versus 92%, respectively), and the consensus sequence
did not obviously differ between the two (Figure S11).
However, for a substantial fraction of Trap-80–independent
promoters, the most proximal predicted jB site was .1 kb
from the transcriptional start site (29%; Figure 6A). This is
significantly different from the Trap-80–dependent genes,
and suggests p65 may not be directly involved in assembly of
the pre-initiation complex at these promoters. With this in
mind, we investigated whether the two classes of promoters
could be distinguished by the presence or absence of binding
motifs for other transcription factors. Although we were
unable to find any clear-cut motifs that could unambiguously
discriminate between Trap-80–dependent and –independent
promoters, there were clear differences in the ‘‘signatures’’ of
transcription factor binding sites associated with the two
promoter classes (Figure S12 and Table S2). Trap-80–
independent promoters were highly enriched for the pres-
ence of GC-box motifs (the binding site for Sp1 and related
transcription factors) compared with total mouse promoters,
although this enrichment did not reach statistical significance
Figure 4. Artificial Contact with Mediator Rescues Expression of Trap-80–Dependent Genes
The experimental rationale is depicted schematically on the left.
On the right, Trap-80 knock-down fibroblasts were transduced with a retrovirus driving expression of full-length p65 (blue triangles), or of a fusion of
the p65 DBD with the H1 region of VP16 (p65DBD-VP16-H1; red triangles), and expression of Ip-10 mRNA was compared to wild-type (wt) and
untransduced Trap-80 knock-down cells. The result is representative of five experiments. Similar fusions using both the H1 and H2 regions of VP16, or
fusions to full-length p65, gave the same results (not shown).
PLoS Biology | www.plosbiology.org March 2009 | Volume 7 | Issue 3 | e10000730555
Two Modes of Activation by p65
when compared with Trap-80–dependent promoters. On the
other hand, Trap-80–dependent promoters were themselves
strongly enriched for the presence of a TATA-box, and for
binding sites for the Ap-1 and HSF families of transcription
factors. All promoters induced by TNF-a contained statisti-
cally elevated levels of NF-jB-binding and E-box motifs when
compared with total mouse promoters.
These findings prompted us to investigate the co-occu-
pancy of endogenous promoters by other transcription
factors alongside p65. The promoters for Mip-2 and Nfkbia,
as well as that of Ip-10, contain putative binding sites for Ap-
1, ATF/CREB, and Sp1, in addition to NF-jB. We performed
ChIP using antibodies against c-Jun and Jun-D (which form
part of Ap-1), ATF-3, and Sp1. All of these transcription
factors were recruited to both the Trap-80–independent Mip-
2 and Nfkbia promoters, and the Trap-80–dependent Ip-10
promoter, upon stimulation of fibroblasts with TNF-a (Figure
6B). Remarkably, in every case, binding to these promoters
was totally abolished in p65-knockout fibroblasts. This effect
was specific for NF-jB–dependent genes, since Sp1 remained
bound to the promoters of control, housekeeping genes in
both wild-type and p65-knockout cells (Figure S13 ). Thus,
at native NF-jB target promoters, the initial recruitment of
p65 is required for the subsequent binding of other,
secondary transcription factors.
To explore whether this phenomenon also occurs in
another cell type, we used lipopolysaccharide (LPS)-stimu-
lated primary dendritic cells (DCs), derived in vitro using cells
from wild-type and p65-knockout mice. Unlike the situation
in fibroblasts, many NF-jB target genes are expressed in DCs
in the absence of p65; however, the Vcam-1 and Ip-10 genes
are still largely p65-dependent (Figure S14A). The promoters
for both of these genes contain binding sites for Ap-1, and in
wild-type DCs both are able to recruit c-Jun upon LPS
Figure 5. Mutations of p65 That Prevent Interaction with Trap-80
(A) Deletion of TA1 and TA2 prevents the interaction of p65 with Trap-80.
(B) All p65 deletion mutants tested can still homodimerize.
(A, B, D, and E) depict fluorescence intensities of HEK-293 cells after co-transfection with vectors expressing the indicated mutants of p65 fused to Venus
fragment 1 (V1), and either V2-Trap-80 (A and D) or p65-V2 (B and E). Intensities are expressed as a percentage of the level in cells co-expressing p65-V1
and V2-Trap-80 (shown in [A]). The residual ;30% fluorescence seen with the p65 DBD in (A) is similar to the level seen in cells co-expressing V1 and V2
fused to control, noninteracting proteins. Error bars indicate standard errors of independent transfections. The results presented here are representative
of two to four experiments.
(C) Deletion of TA1 and TA2 prevents expression of Ip-10 but does not affect expression of Mip-2. p65-knockout fibroblasts were transduced with
retroviruses driving expression of the p65 DBD (blue triangles) or of p65DTA1&2 (red triangles), and expression of Ip-10 and Mip-2 was compared to
wild-type and untransfected p65-knockout cells.
(D) Mutations in TA1 that prevent the interaction of p65DTA2 with Trap-80.
(E) All p65 mutants tested can still homodimerize.
(F) Mutants that do not interact with Trap-80 cannot drive expression of Ip-10, but expression of Mip-2 is unaffected. Expression of Mip-2 (left) and Ip-10
(right) mRNA in p65-knockout fibroblasts transduced with retroviruses expressing the indicated p65 mutants, after stimulation for 1 h with TNF-a.
PLoS Biology | www.plosbiology.org March 2009 | Volume 7 | Issue 3 | e10000730556
Two Modes of Activation by p65
stimulation (Figure S14B). As we had observed in fibroblasts,
though, binding to both promoters was prevented in p65-
An Artificially Recruited Secondary Transcription Factor
Drives Transcription in the Absence of p65
The above results indicate that one mechanism by which
p65 could drive transcriptional activation at Trap-80–
independent promoters would be by controlling the recruit-
ment of secondary transcription factors whose activities do
not require Trap-80 (as illustrated in Figure 7). If this
explanation is correct, we should be able to rescue expression
of Trap-80–independent genes in p65-knockout fibroblasts
by bringing one of the relevant transcription factors to their
promoters. We decided to attempt this using the transcrip-
tional activation domain from Sp1. Sp1 is recruited in a p65-
dependent fashion to NF-jB target promoters (Figure 6B).
The binding site for Sp1 is frequently found in Trap-80–
independent promoters (GC-box; Figure S12), and it has been
implicated in the expression of several NF-jB–regulated
genes (e.g., Mcp-1 ). Moreover, while the Sp1 TAD requires
Mediator for its activity , it does not directly interact with
the Mediator complex, so we reasoned that it was unlikely to
show a particular dependency on the Trap-80 subunit.
Using a similar strategy to that used earlier (Figure 4), we
generated retroviruses encoding a fusion protein between the
p65 DBD and the Sp1 TAD, and used these to infect p65-
knockout fibroblasts. Expression of the Trap-80–independent
Mip-2 gene was completely restored to wild-type levels in
infected cells (Figure 7). This demonstrates that recruitment
of Sp1, an event that is normally controlled by p65, is
sufficient to drive transcription even in an experimental
setting in which p65 itself is absent. Therefore, the ability of
p65 to control the binding of secondary transcription factors
such as Sp1 to target gene promoters constitutes a second,
indirect, mode of transcriptional activation, independent
from its direct interaction with Mediator via Trap-80.
Although Sp1 binding sites are enriched at the promoters
of Trap-80–independent genes, there also exist instances at
those of Trap-80–dependent genes (e.g., Ip-10, Figure 6B). In
such cases, binding of Sp1 to the promoter (along with other
transcription factors) is not sufficient to drive transcription
in Trap-80–deficient cells. In line with this, artificial recruit-
ment of the Sp1 TAD to the Trap-80–dependent Ip-10
promoter failed to restore its expression in p65-knockout
cells (Figures 7 and S15). A difference between the Trap-80–
independent and Trap-80–dependent genes, then, corre-
sponds to the ability of secondary transcription factors
(exemplified here by Sp1) to drive their transcription
following p65-dependent recruitment.
This raises the question of why promoter-bound Sp1
Figure 6. p65 Is Required for Promoter Occupancy by Secondary Transcription Factors
(A) Positions of promoter-proximal NF-jB binding motifs relative to the transcriptional start site. Cumulative percentage of promoters in which the
closest conserved NF-jB binding motif lies within the indicated distance to the TSS. Promoters analysed are those of the top 30 Trap-80–dependent and
30 Trap-80–independent genes, from amongst the 200 most induced by TNF-a.
(B) Recruitment of p65 and secondary transcription factors to the Mip-2, Nfkbia, and Ip-10 promoters in wild-type and p65-knockout fibroblasts. ChIP
using antibodies against p65, Sp1, c-Jun, JunD, or ATF-3, after stimulation with TNF-a. The results presented here are representative of two to three
PLoS Biology | www.plosbiology.org March 2009 | Volume 7 | Issue 3 | e10000730557
Two Modes of Activation by p65
cannot drive transcription of Trap-80–dependent genes, such
as Ip-10. Transcriptional activation by Sp1 in vitro depends
on its direct interaction, through TAFII110, with a TFIID
complex containing TAFII250 . However, not all tran-
scriptionally active genes in human cells are found in
association with TAFII250, nor in yeast cells with its
homologue TAFII145 [31–33]. We therefore examined TA-
FII250 occupancy at the Ip-10 and Mip-2 promoters. We found
that TAFII250 is recruited to the endogenous Mip-2 promoter
upon stimulation with TNF-a, but no such recruitment was
seen at the promoter for Ip-10 (Figure S16). Thus, the
differential responsiveness of these two promoters to bound
Sp1 can be explained by their respective abilities to recruit a
TFIID complex containing TAFII250; this, in turn, accounts
for the ability of p65 to activate transcription of Mip-2, but
not Ip-10, in the absence of Trap-80. Differential TAF usage
by promoters may represent a widely used additional level of
control over the activity of bound transcription factors. It has
been shown in both yeast and mammals that promoters differ
in their requirement for a TFIID complex containing
TAFII250/145 [15,34,35]. Although the correlation is not
absolute, one predictive factor for TAF-independence is the
presence of a TATA-box, and it is worth noting that this
motif is enriched in Trap-80–dependent promoters (Figure
S12 and Table S2). However, just as there does not appear to
be any single transcription factor binding motif that
unequivocally separates the two classes of promoter, the
association of Trap-80–independent promoters with TA-
FII250 presence is not perfect, and there exist some Trap-80–
dependent genes whose human counterparts are bound by
TAFII250 (e.g., Adm and Cebpb ), and some Trap-80–
independent promoters which contain a TATA-box (e.g.,
Ccl7). Thus, while Sp1 serves as a successful example in the
case of the Mip-2 promoter, we certainly do not suggest that
all Trap-80–independent transcription is mediated by the
same secondary transcription factor.
Rather, our data indicate that each NF-jB–dependent
promoter contains a combination of sites for the binding of
various transcription factors, any of which could drive
transcription if present and active in that promoter context.
Importantly, though, in fibroblasts and DCs this binding is
subject to overall upstream control by p65, and it is likely that
this reflects a general mechanism used by NF-jB–dependent
promoters in other cell types.
When the studies described here were initiated, numerous
in vitro data were known about transcription by NF-jB, but
the actual mechanism of transcription downstream of p65
binding to endogenous genes in vivo was unclear. Since the
Mediator complex was known to play an important role in
most, if not all, transcription by pol-II, we set out to disrupt
its interaction with p65, as a means to dissect p65-driven
transcription. We found that expression of some NF-jB
target genes depends on direct contact between p65 and
Mediator, which occurs through the Trap-80 subunit and the
TA1 and TA2 regions of p65. This contact is needed for the
Figure 7. An Artificially Recruited Secondary Transcription Factor Drives Transcription in the Absence of p65
On the left is a cartoon illustrating our model for the events occurring at Trap-80–independent promoters. In wild-type cells, initial p65 binding brings
about an undefined change to target promoters, which allows the binding of various secondary transcription factors. Once bound, these can drive the
recruitment of pol-II, and so enable transcription. In p65-knockout cells, secondary transcription factors cannot bind and no transcription takes place. If
binding of a secondary transcription factor is artificially restored (in this case Sp1), transcription is rescued at promoters that can recruit the relevant co-
factors (in this case TAFII250).
On the right, p65-knockout cells were transduced with a retrovirus expressing a fusion of the p65 DBD with the Sp1 TAD (red triangles, p65DBD-Sp1),
and expression of Mip-2 and Ip-10 mRNA was compared to untransduced wild-type and p65-knockout fibroblasts. The result is representative of three
PLoS Biology | www.plosbiology.org March 2009 | Volume 7 | Issue 3 | e10000730558
Two Modes of Activation by p65
establishment of an active, Med-26–containing Mediator
complex at promoters, recruitment of TFIIB and pol-II, and
thereby the initiation of transcription. While this result is not
surprising, it does provide important confirmation that
events hitherto only described in minimalist in vitro experi-
ments are necessary, and sufficient, for the expression of
some genes in their natural context in vivo.
The finding that 3T3 fibroblasts remain viable even after
depletion of Trap-80 to ,10% of normal levels is remarkable,
considering that its yeast homologue Srb4 is required for
most, if not all, transcription by pol-II . Srb4 is essential
for the integrity of the yeast Mediator complex, which,
without it, dissociates at the boundary between the structur-
ally conserved head and middle modules [36,37]. In mamma-
lian cells, like in yeast, the Mediator complex is critical for
pol-II–driven transcription: its addition is required for the in
vitro activity of various transcriptional activators [10,29], and
its depletion from mammalian nuclear extracts abolishes all
transcription by pol-II [7,38]. Hence, the survival of fibro-
blasts after Trap-80 has been knocked-down implies that
some Mediator activity must still remain. One possibility is
that the amount of cellular Mediator is not normally limiting
for the expression of essential genes, and that the residual
5%–10% level of Trap-80 in knock-down cells suffices for
these. However, we find that the expression of .96% of
transcripts changes by less than 1.5-fold in Trap-80–deficient
cells (Figure 2B). This finding argues that, instead, Trap-80–
deficient cells contain Trap-80–less, but otherwise functional,
Mediator complexes. Proteomic analyses have so far identi-
fied Trap-80 to be part of a common core of subunits, shared
by all Mediator species [23,39]. It seems likely, then, that
Trap-80 does not play the same essential structural role as
yeast Srb4, and that Mediator complexes that normally
incorporate Trap-80 are still able to at least partially
assemble when this subunit is missing. This interpretation is
supported by our finding that the Mediator complex,
revealed by the Med-26 subunit, is recruited to the Mip-2
promoter even in Trap-80–deficient cells (Figure 3E).
We have shown that the interaction of p65 with Mediator
through Trap-80 is sufficient to drive transcription. However,
the discovery that a subset of p65-dependent genes are
transcribed normally even when the interaction of p65 with
Mediator is abolished was completely unanticipated. More-
over, a mutant form of p65 that not only cannot interact with
Trap-80, but that also lacks both previously identified tran-
scriptional activation domains, can still activate the Trap-80–
independent Mip-2 gene in vivo (p65DTA1&2, Figure 5C). This
finding prompted us to examine more closely the events that
occur at promoters upon engagement of NF-jB. Remarkably,
we found that without the binding of p65, NF-jB target
promoters cannot be bound by many other transcription
factors. Thus, it appears that a p65-containing NF-jB dimer
binds to target promoters as a lone, ‘‘pioneer’’ transcription
factor, and controls their subsequent co-occupancy by
secondary transcription factors (illustrated in Figure 7).
One model for this, which we do not favour, could be that
secondary transcription factors bind to promoters via direct,
co-operative interactions with p65. Such a scenario has been
previously shown in the context of particular promoters
containing juxtaposed binding sites (e.g., HIV1-LTR ,
Ifnb1 ), but this arrangement is not a general feature of
NF-jB target promoters. Moreover, it seems unlikely that
pairwise interactions with p65 could account for the binding
of multiple transcription factors to each of many different
promoters (and at non-NF-jB target promoters, co-operative
binding with p65 is clearly not required; see Figure S13).
A more plausible possibility is that p65 controls promoter
accessibility by inducing local alterations to chromatin. In
macrophages, NF-jB–driven activation is accompanied by
nucleosome remodeling at target gene promoters .
However, we could detect no differences in promoter
accessibility to micrococcal nuclease digestion after stimula-
tion of wild-type and p65-knockout fibroblasts (unpublished
data). Alternatively, p65 binding may bring about changes to
histone modifications, several of which have been described
to be associated with the expression of NF-jB target genes in
different systems (e.g., lysine acetylation [43,44] and methyl-
ation , serine phosphorylation [46,47]). Further experi-
ments are required to determine whether these could
account for the control over secondary transcription-factor
In p65-knockout cells, artificial recruitment of a secondary
transcription factor is sufficient to restore gene expression
(Figure 7), indicating that regardless of the mechanism, the
regulation of promoter occupancy constitutes a second,
independent mode of transcriptional activation by p65.
What, though, could be the benefit of having a second
mode of transcriptional activation? After all, in real,
nonexperimentally manipulated cells, an intact Mediator
complex containing Trap-80 is always present. We can
envisage two situations in which the ability of p65 to control
recruitment of secondary transcription factors to a promoter
could be important. First, if the only means by which p65
could activate transcription was through its direct binding to
Mediator, then the transcriptional output at every NF-
jB?dependent promoter should be the same, and upon its
release from promoters, transcription would necessarily halt.
There are numerous mechanisms that control the longevity of
promoter-bound p65, including nuclear export by resynthe-
sized IjB molecules , ubiquitination and proteasomal
degradation , and replacement by other NF-jB dimer
species . Considering the tremendous diversity of NF-jB
target genes, though, it seems inconceivable that the optimal
biological window and level of expression for all of them can
be identical (and experimentally this is not the case; compare,
for example, expression of Mip-2 and Il-6 in Figure 2A). By
endowing p65 with the ability to license promoters for the
binding of secondary transcription factors, there is a means
to customize expression levels, and prolong transcription
after the departure of p65. From the point of view of a
promoter, this would be an attractive solution, since NF-jB–
dependence can be retained at the same time as tailoring the
expression pattern by selecting binding sites for appropriate
secondary transcription factors.
Second, jB sites are not always located in promoters close
to the transcriptional start sites, and in some cases can be
several kilobases away (Figure 6A; examples include Mcp-1
and JunB). At such a distance, looping of the intervening DNA
would be required to bring bound p65 into the proximity of
core promoter elements, and this may not allow a sufficiently
stable interaction with Mediator to enable nucleation of the
pre-initiation complex. However, the local presence of p65 is
nevertheless adequate to regulate the recruitment of secon-
dary transcription factors. In turn, these newly arrived
PLoS Biology | www.plosbiology.orgMarch 2009 | Volume 7 | Issue 3 | e1000073 0559
Two Modes of Activation by p65
transcription factors can stably bind to the promoter, and
themselves interact with components of the pre-initiation
complex to drive transcription. In this model, the Trap-80–
independent mode of activation by p65 is critical to permit it
to operate at enhancers.
A corollary of activation by p65 in this way is that the activity
of a given target promoter, although entirely NF-jB–depend-
ent, will depend on the availability of suitable secondary
transcription factors. Since this is determined by both cell-type
and stimulus, this mode of activation is likely to be essential to
allow genes controlled by NF-jB to attain an appropriate
pattern of expression in different biological contexts.
Materials and Methods
Plasmids. For expression in yeast, fragments from the N- or C
termini of p65 (NT: amino acids [aa] 1–305; CT1: aa 306–549, CT2:
431–549) were cloned into pAct2. Full-length Trap-80, and amino or
carboxy terminal fragments (NT: aa 1–335, CT: aa 336–649) were
cloned into pGBT9. For expression in HEK-293 cells, the coding
sequences for full-length mouse p65 and Trap-80 were cloned in
pCDNA3. Trap-80 was tagged at the N terminus with the HA epitope
MYPYDVPDYA. For BiFC, p65 and mutants thereof were fused to
Venus fragment 1 (V1: aa 1–158) or fragment 2 (V2: aa 159–239) using
the linker sequence SRGSGGGGSGGGGSSG, and Trap-80 was fused
to V2. p65 mutations are as follows: DTA1 is truncated at aa 519;
DTA2 lacks aa 441–474; mut528–534 and mut535–541 each substitute
7 aa for AAASAAA; DF539AA substitutes aa 539 and 540 for AA
(numbers refer to aa positions in full-length p65). Trap-80 was
knocked-down using hairpins directed against the sequences AGA-
GATGGTCGGGTAATCA or GACATTGGTGATCTTGGCA (in the
Trap-80 CDS), cloned into pSuper-Retro-Puro. The shRNA-resistant
Trap-80 contains two silent point mutations (underlined): AGA-
GACGGTCGGGTCATCA, and was cloned in pMY-IRES-GFP. For
generation of biotin-tagged Trap-95, the Escherichia coli BirA coding
sequence was cloned into pMY-IRES-Bsd (conferring resistance to
blasticidin), and full-length Trap-95 was tagged at the C terminus
with the peptide GLNDIFEAQKIEWH, and cloned in pMY-IRES-
Tomato (expressing red fluorescent Tomato protein). For expression
in fibroblasts, HA-Trap-80, full-length p65 and mutants thereof, and
the p65 DBD (aa 1–305), either alone or fused to VP16-H1 (aa 411–
456) or Sp1 TAD (aa 92–551), were all cloned in pMY-IRES-GFP.
Antibodies. Polyclonal antibodies against HA, p65, pol-II Rbp1
subunit, Trap-80, Med-26, TFIIB, ATF3, c-Jun, Jun-D, Sp1, and c-Rel
were from Santa Cruz, that against TAFII250 was from Abcam.
Monoclonal anti-p65 (N terminus) was from Santa Cruz. Monoclonal
anti-HA is produced by the hybridoma 12CA5.
Yeast 2 hybrid. Y153 yeast were grown at 30 8C in YAPD medium
and transformed using lithium acetate. Transformants were selected
by growth on YNB plates without tryptophan or leucine, and
additionally lacking histidine and containing 25 mM 3-amino triazole
to select for interactions between hybrid proteins. Expression of LacZ
was screened by transfer of colonies to nitrocellulose, lysis, and
incubation at 37 8C with X-Gal.
Cell culture. HEK-293 and Ecotropic-Phoenix cells were were
transfected using CaPO4. BiFC fluorescence intensities in transfected
cells were measured by flow cytometry. 3T3 cells were infected with
retroviral supernatants from Ecotropic-Phoenix packaging cells.
Retroviral gene expression was monitored using flow cytometry to
measure co-expressed fluorescent proteins. Where necessary, cells
were sorted to obtain equivalent expression levels. Primary DCs were
generated from foetal liver progenitor cells by culture for 8–10 d with
GM-CSF (4% supernatant from transfected X63 cells). Cells were
stimulated with 5 ng ml?1mouse TNF-a or with 100 ng ml?1LPS.
Immunoprecipitations and pull-downs. Nuclei were isolated by cell
lysis in L1 buffer (50 mM Tris, 2 mM EDTA, 0.1% NP-40, 10%
Glycerol [pH8]) and nuclear proteins were extracted using L1 þ250
mM NaCl for 10 min. After centrifugation, the salt in the supernatant
was diluted to 100 mM. For immunoprecipitations, extracts were
incubated overnight with 2 lg antibody followed by 30 min with 5 ll
protein A- or protein G-sepharose, per 100 lg total protein. For pull-
down of in vivo biotinylated Trap-95, extracts were incubated with 2
ll streptavidin-M280 magnetic beads (Dynal) per 100 lg total protein.
Bound material was washed in L1 þ150 mM KCl and analysed by
Microarray analysis. Total RNA was prepared with RNeasy
(Qiagen) from three independent samples per group, and used to
prepare labelled ss-cDNA for hybridization on Affymetrix Mouse
Gene 1.0 ST microarrays. The data have been deposited at the
National Center for Biotechnology Information (NCBI) Gene
Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo), with acces-
sion number GSE12697. Only transcripts whose microarray D log
signals were reproducible between groups for all samples (r/? x , 0.5
and p , 0.1) were considered for analysis. A set of 36 transcripts were
measured by RT-PCR from the same samples, and used to generate a
standard curve (r ¼ 0.84) to quantify the microarray probe signals.
NF-jB binding motifs conserved between mouse and human were
identified in the region ?9,000 to þ1,000 bp relative to the mouse
transcriptional start site (TSS) using Consite (matrices MA0061, 0101,
and 0107, conservation .70%, TF score .85% ). The fraction of
promoters for which the most proximal jB site was .1 kb (kilobase
pair) from the TSS were compared using the Fisher’s exact test. Over-
represented motifs in sets of promoters were identified using
ChIP and PCR. ChIP was performed as described , using
primers which amplify promoter regions within 300 bp upstream of
the TSS (binding sites for the transcription factors studied all lie
within 6500 bp of the TSS at the promoters analysed). All PCR was
performed using quantitative real-time analysis with gene-specific
fluorescent probes. Primer sequences are available on request.
Figure S1. p65 Interacts with Trap-80
(A) Yeast two-hybrid experiments. Yeast cells were sequentially
transformed with plasmids encoding the Gal4 activation domain
fused to either the amino terminus of p65 (p65 NT) or two nested
carboxy terminal fragments (p65CT1/CT2), followed by the Gal4
DNA-binding domain fused to either full length, or amino- or
carboxy-terminal Trap-80 (Trap-80 NT and Trap-80 CT). The blue
colour of colony lifts when Trap-80 is combined with p65 CT2, or
when Trap-80 NT is combined with p65 CT1 or CT2 is indicative of
an in vivo interaction between the two hybrid proteins. The failure of
p65 CT1 to drive LacZ expression when co-transformed with full-
length Trap-80 is probably due to its weak expression: western
blotting of yeast cell extracts using antibodies specific for the
carboxy-terminus of p65 revealed only very low amounts of the
shorter p65 fragment (CT2), and undetectable levels of the longer
fragment (CT1, not shown). Yeast co-transformants containing all
combinations of full-length Trap-80 or Trap-80 NT, combined with
p65 CT1 or CT2, grew on medium lacking histidine (not shown).
(B) Co-immunoprecipitation of p65 and Trap-80. HEK-293 cells were
co-transfected with expression vectors for p65 and a haemagglutinin
(HA) epitope-tagged allele of Trap-80, and nuclear extracts were
prepared after stimulation with TNF-a to induce nuclear entry of NF-
jB. p65 was detected by western blotting after immunoprecipitation
using an anti-HA antibody, or without antibody (left panel). In the
reciprocal experiment, tagged Trap-80 was detected with anti-HA
after immunoprecipitation using anti-p65 (right panel). The level of
p65 in total nuclear extract is shown as ‘‘input’’; HA-Trap-80 was not
easily detectable in nuclear extracts before immunoprecipitation.
(C) BiFC mediated by in vivo interaction between p65 and Trap-80.
HEK-293 cells were transfected with vectors expressing Venus
fragment 2 fused to the N terminus of Trap-80 (V2-Trap-80; i, iii,
iv), Trap-80–V2 (v), or p65-V2 (vi), either alone or together with p65-
V1 (i, ii, vi), p65DBD-V1 (iv), or V1-p65 (v). Fluorescence from either
p65-V1 or V2-Trap-80 was undetectable when transfected alone (ii,
iii). (iv) and (v) are controls for the specificity of the interaction based
on the yeast two-hybrid results (above), wherein the interacting
carboxy-terminus of p65 is deleted (iv), or the two proteins are fused
at the noninteracting termini (v). The residual ’30% fluorescence
seen in these cases is similar to the level seen in cells co-expressing V1
and V2 fused to control, noninteracting proteins. The strong self-
association of p65 into homodimers serves as a positive control (vi).
The fluorescence intensity of transfected cells is expressed as a
percentage of the level in cells co-expressing p65-V1 and V2-Trap-80
(i). Error bars indicate standard errors of independent transfections.
The results presented here are representative of three experiments.
Found at doi:10.1371/journal.pbio.1000073.sg001 (3.97 MB TIF).
Figure S2. Trap-80 Knock-Down Fibroblasts
(A) Proliferation of Trap-80–deficient fibroblasts. Parallel cultures of
fibroblasts expressing either the irrelevant shRNA, or an shRNA
targeting Trap-80, were grown for 1 wk and the increase in cell
PLoS Biology | www.plosbiology.org March 2009 | Volume 7 | Issue 3 | e10000730560
Two Modes of Activation by p65
number was determined. Proliferation is expressed as the percentage
increase compared to the control culture; error bars indicate
(C) Morphological appearance of Trap-80–deficient fibroblasts. Phase
contrast images (original magnification 1003) taken of exponentially
growing cultures of wild-type or Trap-80 knock-down fibroblasts.
Found at doi:10.1371/journal.pbio.1000073.sg002 (2.57 MB TIF).
Figure S3. Expression Levels of Trap-95-Biotin
Intensity of Tomato fluorescence, expressed via an IRES sequence
from a bi-cistronic mRNA also expressing Trap-95-biotin, in
transduced wild-type (top) and Trap-80 knock-down (bottom)
fibroblasts. The mean fluorescence level of the population is
indicated. Trap-95-biotin itself co-migrates with one of two naturally
biotinylated carboxylase proteins in mammalian cells, precluding
direct detection on western blots.
Found at doi:10.1371/journal.pbio.1000073.sg003 (717 KB TIF).
Figure S4. Controls for the Specificity of Trap-80 Knock-Down
(A) Two independent hairpins targeting Trap-80 were used to
generate clonal lines of 3T3 fibroblasts. A comparable number of
clones was generated using each of the shRNAs targeting Trap-80,
and using an irrelevant, scrambled shRNA. The degree by which Trap-
80 mRNA was knocked-down varied from one clone to another, in the
range of ’70%–95% reduction of wild-type levels (not shown). The
residual levels of Trap-80 mRNA are shown for a single clone
expressing each shRNA, which were chosen for further study. mRNA
levels are expressed as a percentage of the level in control cells. Trap-
80 shRNA number 1 corresponds to the shRNA used in the
experiments described in the main text. Error bars indicate standard
errors; the results presented here are representative of more than ten
(B) Expression of Mip-2, Nfkbia, Ip-10, and Il-6 mRNA in fibroblasts
expressing either of the two shRNAs targeting Trap-80, after
stimulation with TNF-a.
(C) Reconstitution of Trap-80 in knock-down cells. Fibroblasts
expressing an shRNA targeting Trap-80 were transduced with a
retrovirus driving expression of an shRNA-resistant form of Trap-80
(red triangles; total Trap-80 mRNA levels were around 1003 greater
than those of wild-type cells [not shown]), and mRNA was measured
as in (B).
Found at doi:10.1371/journal.pbio.1000073.sg004 (3.97 MB TIF).
Figure S5. Expression of Both Trap-80–Dependent and Trap-80–
Independent NF-jB Target Genes Requires p65
mRNA expression of Mip-2, Nfkbia, Ip-10, and Il-6, in normal and p65-
knockout fibroblasts stimulated with TNF-a. The results presented
here are representative of more than ten experiments.
Found at doi:10.1371/journal.pbio.1000073.sg005 (1.69 MB TIF).
Figure S6. TNF-a-Induction of Trap-80–Dependent Versus Trap-80–
Cumulative percentage of transcripts whose expression after 1 h
TNF-a stimulation exceeds that in unstimulated fibroblasts by the
indicated level, expressed both as the D log signal of the microarray
probes (upper scale) and the calculated fold induction using a
standard curve generated by real-time PCR of a subset of 36 genes
(lower scale). The red line represents the top 100 Trap-80–dependent
transcripts; the black line represents 5000 Trap-80–independent
Found at doi:10.1371/journal.pbio.1000073.sg006 (1.29 MB TIF).
Figure S7. Trap-80–Dependent and –Independent Genes
mRNA expression of a subset of Trap-80–dependent (A) and Trap-
80–independent (B) genes, in wild-type and Trap-80 knock-down
Found at doi:10.1371/journal.pbio.1000073.sg007 (509 KB TIF).
Figure S8. Presence of HA-Trap-80 at Promoter Regions
ChIP using antibodies against the HA epitope, from fibroblasts
retrovirally over-expressing HA-Trap-80, or from control untrans-
Found at doi:10.1371/journal.pbio.1000073.sg008 (1.88 MB TIF).
Figure S9. p65 DBD Alone Acts as a Dominant Negative Allele
Wild-type 3T3 fibroblasts were transduced with a retrovirus driving
expression of the p65 DBD (red triangles), and expression of Mip-2
and Ip-10 mRNA was compared to wild-type and p65 knockout
fibroblasts. The result is representative of three experiments.
Found at doi:10.1371/journal.pbio.1000073.sg009 (943 KB TIF).
Figure S10. Transcriptional Activity of p65 Deletion Mutants
p65 knockout fibroblasts transduced with retroviruses expressing the
indicated p65 mutants were stimulated with TNF-a for 1 h, and their
expression of Mip-2 (left) and Ip-10 (right) mRNA was compared to
that of wild-type cells. Note that the level of expression of p65
mutants in transduced cells exceeds that of endogenous p65 in wild-
type cells by several-fold (not shown), which may account for the
heightened expression of Mip-2. The results presented here are
representative of three experiments.
Found at doi:10.1371/journal.pbio.1000073.sg010 (1.61 MB TIF).
Figure S11. Consensus NF-jB Binding Motifs in Trap-80–Dependent
and –Independent Promoters
Logos of position weight matrices of NF-jB binding motifs conserved
between mouse and human in Trap-80–dependent (top; n¼36) and –
independent (bottom; n ¼ 58) TNF-a–induced genes. Promoters
analysed are those of the top 30 Trap-80–dependent and 30 Trap-80–
independent genes, from amongst the 200 most induced by TNF-a.
Found at doi:10.1371/journal.pbio.1000073.sg011 (677 KB TIF).
Figure S12. Transcription Factor Binding Sites Associated with Trap-
80–Dependent and –Independent Promoters
Over-represented motifs detected in promoters of TNF-a–induced
genes. See Table S2 for matrices and p-values. No motifs were
statistically over-represented in Trap-80–independent promoters
compared with Trap-80–dependent promoters.
Found at doi:10.1371/journal.pbio.1000073.sg012 (1.08 MB TIF).
Figure S13. p65-Independent Binding of Sp1 to the Promoters of
ChIP using antibodies against Sp1 in wild-type and p65-knockout
fibroblasts. Primers were chosen to amplify the promoter regions of
MeCP2 (A) or Tk (B), and adjacent control regions located upstream
or downstream at the indicated positions. Error bars indicate the
standard errors of independent immunoprecipitations (from both
unstimulated and TNF-a–stimulated cells).
Found at doi:10.1371/journal.pbio.1000073.sg013 (1.79 MB TIF).
Figure S14. p65-Dependent Recruitment of c-Jun to Promoters in
(A) p65-dependent gene transcription in dendritic cells. mRNA
expression of Vcam-1 and Ip-10 in normal and p65-knockout DCs
stimulated with LPS.
(B) ChIP using antibodies against c-Jun.
Found at doi:10.1371/journal.pbio.1000073.sg014 (2.51 MB TIF).
Figure S15. p65 DBD-Sp1 Binds to Promoters
p65 knockout 3T3 fibroblasts were transduced with a retrovirus
driving expression of the p65 DBD-Sp1, and ChIP was performed
using antibodies against the p65 N terminus.
Found at doi:10.1371/journal.pbio.1000073.sg015 (969 KB TIF).
Figure S16. The Mip-2, But Not the Ip-10 Promoter Recruits TAFII250
ChIP using antibodies against TAFII250 in wild-type fibroblasts.
Found at doi:10.1371/journal.pbio.1000073.sg016 (815 KB TIF).
Table S1. Top 50 Trap-80–Dependent and Trap-80–Independent
Found at doi:10.1371/journal.pbio.1000073.st001 (177 KB DOC).
Table S2. Over-Represented Motifs in TNF-a–induced Gene Pro-
Found at doi:10.1371/journal.pbio.1000073.st002 (113 KB DOC).
We would like to thank A. Beg for the p65-deficient 3T3s, S. Michnick
for plasmids encoding Venus fragments, G. Nolan for the Ecotrophic
Phoenix cells, T. Kitamura for the pMY-IRES-GFP plasmid, and A.
Wu ¨rch and J. Wersing for cell sorting. We are grateful to the lab of T.
Borggrefe for help with yeast experiments, to G. Mittler for advice
PLoS Biology | www.plosbiology.org March 2009 | Volume 7 | Issue 3 | e10000730561
Two Modes of Activation by p65
and discussions, and to R. Grosschedl for critical reading of the Download full-text
Author contributions. DvE, GN, and SS conceived and designed the
experiments. DvE, BE, GN, and SS performed the experiments. DvE
and SS analyzed the data. DvE wrote the paper.
Funding. This work was partially supported by Marie Curie grant
Competing interests. The authors have declared that no competing
1. Hoffmann A, Baltimore D (2006) Circuitry of nuclear factor kappaB
signaling. Immunol Rev 210: 171–186.
2. Natoli G, Saccani S, Bosisio D, Marazzi I (2005) Interactions of NF-kappaB
with chromatin: the art of being at the right place at the right time. Nat
Immunol 6: 439–445.
3. Orphanides G, Lagrange T, Reinberg D (1996) The general transcription
factors of RNA polymerase II. Genes Dev 10: 2657–2683.
4. Malik S, Roeder RG (2005) Dynamic regulation of pol II transcription by
the mammalian Mediator complex. Trends Biochem Sci 30: 256–263.
5.Guermah M, Tao Y, Roeder RG (2001) Positive and negative TAF(II)
functions that suggest a dynamic TFIID structure and elicit synergy with
traps in activator-induced transcription. Mol Cell Biol 21: 6882–6894.
6. Acevedo ML, Kraus WL (2003) Mediator and p300/CBP-steroid receptor
coactivator complexes have distinct roles, but function synergistically,
during estrogen receptor alpha-dependent transcription with chromatin
templates. Mol Cell Biol 23: 335–348.
7. Mittler G, Kremmer E, Timmers HT, Meisterernst M (2001) Novel critical
role of a human Mediator complex for basal RNA polymerase II
transcription. EMBO Rep 2: 808–813.
8. Ballard DW, Dixon EP, Peffer NJ, Bogerd H, Doerre S, et al. (1992) The 65-
kDa subunit of human NF-kappa B functions as a potent transcriptional
activator and a target for v-Rel-mediated repression. Proc Natl Acad Sci U
S A 89: 1875–1879.
9.Schmitz ML, Baeuerle PA (1991) The p65 subunit is responsible for the strong
transcription activating potential of NF-kappa B. Embo J 10: 3805–3817.
10. Naar AM, Beaurang PA, Zhou S, Abraham S, Solomon W, et al. (1999)
Composite co-activator ARC mediates chromatin-directed transcriptional
activation. Nature 398: 828–832.
of Mediator are coactivators of lipopolysaccharide- and heat-shock-induced
transcriptional activators. Proc Natl Acad Sci U S A 101: 12153–12158.
12. Boube M, Joulia L, Cribbs DL, Bourbon HM (2002) Evidence for a mediator
of RNA polymerase II transcriptional regulation conserved from yeast to
man. Cell 110: 143–151.
13. Hu CD, Chinenov Y, Kerppola TK (2002) Visualization of interactions
among bZIP and Rel family proteins in living cells using bimolecular
fluorescence complementation. Mol Cell 9: 789–798.
14. Chen FE, Huang DB, Chen YQ, Ghosh G (1998) Crystal structure of p50/p65
heterodimer of transcription factor NF-kappaB bound to DNA. Nature
15. Holstege FC, Jennings EG, Wyrick JJ, Lee TI, Hengartner CJ, et al. (1998)
Dissecting the regulatory circuitry of a eukaryotic genome. Cell 95: 717–728.
16. Thompson CM, Young RA (1995) General requirement for RNA polymer-
ase II holoenzymes in vivo. Proc Natl Acad Sci U S A 92: 4587–4590.
17. Boube M, Faucher C, Joulia L, Cribbs DL, Bourbon HM (2000) Drosophila
homologs of transcriptional mediator complex subunits are required for
adult cell and segment identity specification. Genes Dev 14: 2906–2917.
18. Hoffmann A, Leung TH, Baltimore D (2003) Genetic analysis of NF-kappaB/
19. Ouaaz F, Li M, Beg AA (1999) A critical role for the RelA subunit of nuclear
factor kappaB in regulation of multiple immune-response genes and in
Fas-induced cell death. J Exp Med 189: 999–1004.
a postrecruitment role for the TATA box and TFIIB. Genes Dev 13: 49–63.
21. Malik S, Gu W, Wu W, Qin J, Roeder RG (2000) The USA-derived
transcriptional coactivator PC2 is a submodule of TRAP/SMCC and acts
synergistically with other PCs. Mol Cell 5: 753–760.
22. Naar AM, Taatjes DJ, Zhai W, Nogales E, Tjian R (2002) Human CRSP
interacts with RNA polymerase II CTD and adopts a specific CTD-bound
conformation. Genes Dev 16: 1339–1344.
23. Sato S, Tomomori-Sato C, Parmely TJ, Florens L, Zybailov B, et al. (2004) A
set of consensus mammalian mediator subunits identified by multidimen-
sional protein identification technology. Mol Cell 14: 685–691.
24. Taatjes DJ, Naar AM, Andel F 3rd, Nogales E, Tjian R (2002) Structure,
function, and activator-induced conformations of the CRSP coactivator.
Science 295: 1058–1062.
25. Andrau JC, van de Pasch L, Lijnzaad P, Bijma T, Koerkamp MG, et al. (2006)
Genome-wide location of the coactivator mediator: Binding without
activation and transient Cdk8 interaction on DNA. Mol Cell 22: 179–192.
26. Mittler G, Stuhler T, Santolin L, Uhlmann T, Kremmer E, et al. (2003) A
novel docking site on Mediator is critical for activation by VP16 in
mammalian cells. Embo J 22: 6494–6504.
27. Marin M, Karis A, Visser P, Grosveld F, Philipsen S (1997) Transcription
factor Sp1 is essential for early embryonic development but dispensable for
cell growth and differentiation. Cell 89: 619–628.
28. Ping D, Boekhoudt G, Zhang F, Morris A, Philipsen S, et al. (2000) Sp1
binding is critical for promoter assembly and activation of the MCP-1 gene
by tumor necrosis factor. J Biol Chem 275: 1708–1714.
29. Ryu S, Zhou S, Ladurner AG, Tjian R (1999) The transcriptional cofactor
complex CRSP is required for activity of the enhancer-binding protein Sp1.
Nature 397: 446–450.
30. Chen JL, Attardi LD, Verrijzer CP, Yokomori K, Tjian R (1994) Assembly of
recombinant TFIID reveals differential coactivator requirements for
distinct transcriptional activators. Cell 79: 93–105.
31. Kim TH, Barrera LO, Zheng M, Qu C, Singer MA, et al. (2005) A high-
resolution map of active promoters in the human genome. Nature 436:
32. Kuras L, Kosa P, Mencia M, Struhl K (2000) TAF-Containing and TAF-
independent forms of transcriptionally active TBP in vivo. Science 288:
33. Li XY, Bhaumik SR, Green MR (2000) Distinct classes of yeast promoters
revealed by differential TAF recruitment. Science 288: 1242–1244.
34. Walker SS, Shen WC, Reese JC, Apone LM, Green MR (1997) Yeast
TAF(II)145 required for transcription of G1/S cyclin genes and regulated by
the cellular growth state. Cell 90: 607–614.
35. Wang EH, Tjian R (1994) Promoter-selective transcriptional defect in cell
cycle mutant ts13 rescued by hTAFII250. Science 263: 811–814.
36. Linder T, Zhu X, Baraznenok V, Gustafsson CM (2006) The classical srb4–
138 mutant allele causes dissociation of yeast Mediator. Biochem Biophys
Res Commun 349: 948–953.
37. Takagi Y, Calero G, Komori H, Brown JA, Ehrensberger AH, et al. (2006)
Head module control of mediator interactions. Mol Cell 23: 355–364.
38. Baek HJ, Malik S, Qin J, Roeder RG (2002) Requirement of TRAP/mediator
for both activator-independent and activator-dependent transcription in
conjunction with TFIID-associated TAF(II)s. Mol Cell Biol 22: 2842–2852.
39. Paoletti AC, Parmely TJ, Tomomori-Sato C, Sato S, Zhu D, et al. (2006)
Quantitative proteomic analysis of distinct mammalian Mediator com-
plexes using normalized spectral abundance factors. Proc Natl Acad Sci U S
A 103: 18928–18933.
40. Perkins ND, Agranoff AB, Pascal E, Nabel GJ (1994) An interaction between
the DNA-binding domains of RelA(p65) and Sp1 mediates human
immunodeficiency virus gene activation. Mol Cell Biol 14: 6570–6583.
41. Thanos D, Maniatis T (1995) Virus induction of human IFN beta gene
expression requires the assembly of an enhanceosome. Cell 83: 1091–1100.
42. Ramirez-Carrozzi VR, Nazarian AA, Li CC, Gore SL, Sridharan R, et al.
(2006) Selective and antagonistic functions of SWI/SNF and Mi-2beta
nucleosome remodeling complexes during an inflammatory response.
Genes Dev 20: 282–296.
43. Boekhoudt GH, Guo Z, Beresford GW, Boss JM (2003) Communication
between NF-kappa B and Sp1 controls histone acetylation within the
proximal promoter of the monocyte chemoattractant protein 1 gene. J
Immunol 170: 4139–4147.
44. Dong J, Jimi E, Zhong H, Hayden MS, Ghosh S (2008) Repression of gene
expression by unphosphorylated NF-kappaB p65 through epigenetic
mechanisms. Genes Dev 22: 1159–1173.
45. Saccani S, Natoli G (2002) Dynamic changes in histone H3 Lys 9
methylation occurring at tightly regulated inducible inflammatory genes.
Genes Dev 16: 2219–2224.
46. Anest V, Hanson JL, Cogswell PC, Steinbrecher KA, Strahl BD, et al. (2003)
A nucleosomal function for IkappaB kinase-alpha in NF-kappaB-depend-
ent gene expression. Nature 423: 659–663.
47. Yamamoto Y, Verma UN, Prajapati S, Kwak YT, Gaynor RB (2003) Histone
H3 phosphorylation by IKK-alpha is critical for cytokine-induced gene
expression. Nature 423: 655–659.
48. Arenzana-Seisdedos F, Thompson J, Rodriguez MS, Bachelerie F, Thomas
D, et al. (1995) Inducible nuclear expression of newly synthesized I kappa B
alpha negatively regulates DNA-binding and transcriptional activities of
NF-kappa B. Mol Cell Biol 15: 2689–2696.
49. Saccani S, Marazzi I, Beg AA, Natoli G (2004) Degradation of promoter-
bound p65/RelA is essential for the prompt termination of the nuclear
factor kappaB response. J Exp Med 200: 107–113.
50. Saccani S, Pantano S, Natoli G (2003) Modulation of NF-kappaB activity by
exchange of dimers. Mol Cell 11: 1563–1574.
51. Sandelin A, Wasserman WW, Lenhard B (2004) ConSite: web-based
prediction of regulatory elements using cross-species comparison. Nucleic
Acids Res 32: W249–W252.
52. Thijs G, Lescot M, Marchal K, Rombauts S, De Moor B, et al. (2001) A
higher-order background model improves the detection of promoter
regulatory elements by Gibbs sampling. Bioinformatics 17: 1113–1122.
53. Saccani S, Pantano S, Natoli G (2001) Two waves of nuclear factor kappaB
recruitment to target promoters. J Exp Med 193: 1351–1359.
PLoS Biology | www.plosbiology.org March 2009 | Volume 7 | Issue 3 | e10000730562
Two Modes of Activation by p65