Molecular Cell, Vol. 17, 683–694, March 4, 2005, Copyright ©2005 by Elsevier Inc.DOI 10.1016/j.molcel.2005.02.010
Mediator Requirement for Both
Recruitment and Postrecruitment Steps
in Transcription Initiation
Gang Wang,1Michael A. Balamotis,1
Jennitte L. Stevens,1,3Yuki Yamaguchi,2
Hiroshi Handa,2and Arnold J. Berk1,*
1Molecular Biology Institute and
Department of Microbiology, Immunology
and Molecular Genetics
University of California, Los Angeles
611 Young Drive East
Los Angeles, California 90095
2Graduate School of Bioscience and Biotechnology
Tokyo Institute of Technology
of transcriptional activation: activator-dependent as-
sembly of a preinitiation complex (Cantin et al., 2003;
Kuras and Struhl, 1999; Li et al., 1999). However, it has
been suggested that Mediator may also function at ad-
ditional steps in transcription initiation subsequent to
preinitiation complex assembly (Malik et al., 2002; My-
ers and Kornberg, 2000). For example, purified yeast
Mediator stimulates the Pol II CTD-kinase activity of
TFIIH in vitro (Kim et al., 1994), an activity that could
contribute to such a “postrecruitment” step in tran-
scription initiation. The question has also been raised
as to whether Mediator complexes participate in the
recruitment of other transcriptional coactivators such
as chromatin remodeling complexes, histone acetylase
and methylase complexes, and elongation factors
(Kingston, 1999; Taatjes et al., 2004). One of the sub-
units of yeast Mediator has histone acetylase activity
(Lorch et al., 2000), raising the possibility that once a
gene is activated, Mediator may assist in maintaining
chromatin in a hyperacetylated, open conformation.
In this study, we addressed the question of what
steps in transcription are controlled by an activator-
Mediator interaction. We focused on a specific activa-
tor-Mediator interaction through studies of recently
isolated murine embryonic stem (ES) cells lacking a
metazoan-specific Mediator subunit, MED23 (Sur2)
(Bourbon et al., 2004). Med23−/−ES cells contain largely
normal Mediator complexes, except for the absence of
the MED23 subunit, and support activation by the vast
majority of activation domains tested (Stevens et al.,
2002). However, two specific activation domains, ade-
novirus E1A conserved region 3 and the MAP kinase-
regulated activation domain of the cellular transcription
factor ELK1, are extremely defective in Med23−/−cells
(Stevens et al., 2002). These two activation domains
normally interact with Mediator complexes by binding
to the MED23 subunit, accounting for their defective
activity in Med23−/−cells (Boyer et al., 1999; Stevens et
al., 2002; Wang and Berk, 2002). For ELK1, interaction
with Mediator requires phosphorylation of the activa-
tion domain by a MAP kinase (Stevens et al., 2002).
Microarray analysis of gene expression in Med23−/−
ES cells showed that the vast majority of genes was
expressed at normal levels. However, expression of
early response genes Egr1 and Egr2 was greatly atten-
uated. These genes are normally induced by serum
growth factor activation of MAP kinase pathways and
the subsequent phosphorylation of a ternary complex
transcription factor that binds to the control regions of
Egr1 and Egr2 (Thiel and Cibelli, 2002). The ELK1 tran-
scription factor, whose activation domain is defective in
Med23−/−cells (Stevens et al., 2002), is the predominant
ternary complex factor in ES cells (M.A.B. and A.J.B.,
unpublished data), accounting for the defect in Egr1
and Egr2 transcription. This system provided us with a
powerful approach to study the mechanism of tran-
scriptional stimulation by the phospho ELK1-Mediator
interaction. We used chromatin immunoprecipitation
(ChIP) assays to compare the binding of proteins in-
volved in transcription and histone modifications at the
Mediator complexes are required for activators to
stimulate Pol II preinitiation complex assembly on an
associated promoter. We show here that for the mouse
Egr1 gene, controlled largely by MAP kinase phos-
phorylation of the ELK1 transcription factor, the
MED23 Mediator subunit that interacts with phospho-
ELK1 is also required to stimulate Pol II initiation at a
step subsequent to preinitiation complex assembly.
In Med23−/−cells, histone acetylation, methylation,
and chromatin remodeling complex association at the
Egr1 promoter were equivalent to that of wild-type
cells, yet Egr1 induction was greatly reduced. MAP
kinase activation stimulated Pol II and GTF promoter
binding. However, the difference in factor binding be-
tween wild-type and mutant cells was much less than
the difference in transcription, and Pol II remained lo-
calized to the promoter in mutant cells. These results
indicate that an interaction with MED23 stimulates ini-
tiation by promoter bound Pol II in addition to Pol II
and GTF recruitment.
Two general mechanisms contribute to transcription
regulation by eukaryotic activators. First, activators in-
teract with multiprotein coactivator complexes that
modify chromatin structure to give general transcription
factors (GTFs) and RNA polymerase II (Pol II) access to
promoter DNA (Becker and Horz, 2002; Fischle et al.,
2003; Horn and Peterson, 2002). Second, activators di-
rectly stimulate binding of Pol II and GTFs to promoter
DNA to form a preinitiation complex (Roeder, 1998).
Multisubunit Mediator complexes (Bourbon et al., 2004)
were originally identified as molecular bridges between
activators and Pol II required for activation in vitro and
in vivo (Lee and Young, 2000; Malik and Roeder, 2000;
Myers and Kornberg, 2000). Previous studies have
shown that Mediator is required for the second aspect
3Present address: Protein Sciences Department, Amgen Inc., One
Amgen Center Drive, Thousand Oaks, CA 91320.
an w30-fold induction of Egr1 RNA (27.5 ± 3.5 in three
successive, independent experiments) 30 min after the
addition of serum to wt cells. Serum induction was se-
verely attenuated in Med23−/−cells. Quantitiative RT-
PCR of Egr1 intron RNA showed the same response to
serum and to the absence of MED23 (Figure 1B, white
bars), indicating that the total RNA levels are a good
measure of the relative rates of Egr1 transcription. In
serum-treated cells, Egr1 transcription was 13.2 ± 1.6-
fold higher in wt compared to Med23−/−cells.
Serum response factor (SRF) and a ternary complex
factor (TCF) bind cooperatively to SREs to mediate
transcriptional induction in response to serum mito-
gens (Buchwalter et al., 2004; Shaw and Saxton, 2003).
There are three closely related TCFs in mammals: ELK1,
SAP1 (ELK4), and NET (SAP2/ERP/ELK3). Quantitative
RT-PCR of the mRNAs encoding these TCFs revealed
that ELK1 was expressed at more than 9-fold higher
levels than the other TCFs in the Med23−/−ES cells
(M.A.B. and A.J.B., unpublished data). Consequently,
ELK1 is likely to be the major TCF controlling Egr1 tran-
scription in these cells, and the defect in Egr1 tran-
scription is due to the inability of the ELK1 activation
domain to interact with Mediator and stimulate tran-
scription in these cells (Stevens et al., 2002).
We assayed the association of SRF and ELK1 with
the Egr1 control region in wt and Med23−/−cells by
ChIP (Figure 1C). Initially we used three sets of primers
to PCR amplify immunoprecipitated DNA in a single
multiplex reaction. One set of primers amplified a re-
gion from the Egr1 promoter that includes the TATA-box
and an SRE (Figure 1A). Another set amplified the 3#
end of the Egr1 coding region located w4 kb from the
promoter in this relatively small mammalian gene. As
an internal control, we included a third set of primers
for the promoter region of the constitutively expressed
translation elongation factor 2 (EF2) gene. In prelimi-
nary experiments, we found that EF2 transcription was
not significantly stimulated when serum-starved ES
cells were treated with serum during the time course of
our experiments (within 60 min after addition of serum).
Consistent with earlier work that used in vivo foot-
printing to assay the association of SRF and a TCF with
an SRE controlling Fos transcription in cultured A431
cells (Herrera et al., 1989), ChIP with anti-SRF and anti-
ELK1 showed specific binding of these two proteins to
the Egr1 promoter region to a similar extent in serum-
starved and serum-treated wt cells (Figure 1C). The
same extent of binding was also observed in Med23−/−
cells. ChIP performed by using an antibody specific for
ELK1 phosphorylated at Ser 383 by a MAP kinase re-
vealed that phosphorylation of promoter bound ELK1
in response to serum occurred to an equivalent extent
in wt and Med23−/−cells (Figure 1C). A low level of phos-
pho-ELK1 binding was observed in serum-starved cells
and may contribute to the low level of Egr1 transcrip-
tion in serum-starved cells. To map the sites of ELK1
binding more accurately, we used five sets of primers
that amplified fragments centered at –836 (fragment A),
−410 (fragment B), −62 (fragment C, the same as Egr1p
in Figure 1C), +366 (fragment D), and +674 (fragment E)
relative to the Egr1 transcription start site (Figure 1D).
ELK1 binding was greatest to fragment B in the region
of the four clustered SREs between −300 and −420 and
Table 1. Genes Reduced 2-Fold or More in Med23−/−ES Cells
Gene NameFold Decrease
Early growth response 1
Early growth response 2
Brachyury the second (T2)
Fibroblast growth factor 1
embryonic alkaline phosphatase
guanine nucleotide dissociation stimulator
for a ras-related GTPase
carbonic anhydrase II
regulator of G-protein signaling-GIPC
TNF-response element binding protein
Mouse V(preB)1 gene
polyA binding protein, testis enriched
TAPL (TAP-like ABC transporter)
growth factor inducible protein (pip92)
postsynaptic protein Cript
ribosomal protein L7
glycerol phosphate dehydrogenase 1
growth/differentiation factor 1 (GDF-1)
Fibroblast growth factor 4
G protein-coupled receptor (GPR27)
Microarray analysis of w10,000 mouse genes and ESTs revealed
the indicated annotated genes whose RNA levels were reduced
2-fold or more and their fold reduction relative to wt.
promoters and coding regions of Egr1 and Egr2 in wild-
type (wt) and Med23−/−cells. Because the phospho-
ELK1-Mediator interaction cannot occur in the absence
of MED23, steps in transcription that depend on this
interaction were revealed. The results uncovered an in-
triguing function for an activator-Mediator interaction:
enhancement of the rate of initiation by a preinitiation
Results and Discussion
SRF and ELK1 Are Bound to Egr1 SREs
before Serum Induction
Microarray analysis revealed that fewer than 1% of
genes expressed in mouse ES cells were reduced by a
factor of 2-fold or more in Med23−/−compared to wt
cells (Table 1). This and the finding that Med23−/−
mouse embryos undergo complex morphological de-
velopment before they die at about 10 days of gestation
(J.L.S. and A.J.B., unpublished data) indicate that Me-
diator lacking the MED23 subunit can support near nor-
mal expression of the vast majority of genes. Of all the
genes examined by the microarray, Egr1 was the most
severely affected by loss of MED23. Egr1 is a prototypi-
cal early response gene encoding a zinc finger tran-
scription factor. In wt ES cells it is rapidly and strongly
induced by serum growth factors through five serum
response elements (SREs) in its upstream control re-
gion (Thiel and Cibelli, 2002) (Figure 1A). Quantitiative
reverse-transcriptase-polymerase chain reaction (RT-
PCR) of total cell RNA (Figure 1B, gray bars) revealed
Postrecruitment Function of Mediator
Figure 1. Egr1 Transcriptional Control
(A) TF binding sites (Thiel and Cibelli, 2002). The line shows the promoter region amplified in ChIP.
(B) Quantitative RT-PCR showing induction of Egr1 in wt and Med23 knockout ES cells. Gray bars are total Egr1 RNA; white bars, Egr1 intron
RNA. Standard deviations are indicated, n = 3.
(C) ChIP with anti-SRF, -ELK1, and -MAP kinase-phosphorylated ELK1 (ELK1-P). In this and subsequent figures serum was removed from the
medium for 16 hr (−) or added for 30 min after 16 hr of depletion (+). Three sets of primers were used to amplify DNA from the Egr1 promoter
(Egr1p), Egr1 coding region (Egr1c), and the translation elongation factor 2 promoter region (EF2p).
(D) ChIP using primers for the indicated fragments. Scale in base pairs.
(E) Phosphorimager quantitation of the data in (D) relative to the EF2p band.
next greatest to fragment C containing the promoter
proximal SRE (Figures 1D and 1E). Similar results were
observed for phosphorylated ELK1 in serum-treated wt
and mutant cells (data not shown).
onstrating a generalized increase in H3 and H4 acetyla-
tion in response to serum in the Egr1 promoter region
specifically. Histone acetylation reached maximal levels
by 10 min after addition of serum and then declined
slowly until it approached preserum levels after 50 min
(Figure 2D). H4 acetylation in the promoter region was
unchanged by knockout of the Med23 genes (Figure
2A) and followed a similar time course (data not
shown). Scanning through the promoter region showed
that increased histone acetylation occurred primarily in
the regions of the SREs assayed by fragments B and C
(Figures 2B and 2C). It is noteworthy that despite sim-
ilar histone acetylation compared to wt cells, Egr1 tran-
scription induction was extremely defective in Med23−/−
cells (Figure 1B). We conclude that normal histone
acetylation is not sufficient for maximal Egr1 transcrip-
tion, although it may contribute to the serum stimula-
tion of the low level of Egr1 transcription observed in
ELK1 interacts with the related histone acetylases p300
and CBP (Janknecht and Nordheim, 1996; Li et al.,
2003; Nissen et al., 2001). Although the interaction does
not require ELK1 phosphorylation, MAP kinase phos-
phorylation increases associated HAT activity (Li et al.,
2003). ChIP performed by using a polyclonal antibody
raised against histone H4 peptide 2-19 acetylated at
K5, 8, 12, and 16 revealed serum-induced acetylation
of H4 in the promoter region of Egr1, but not in the
coding region of wt cells (Figure 2A). Similar results
were observed using antibodies to H4-AcK8 only; H3-
AcK9, -AcK14; and H3-AcK18 (data not shown), dem-
Figure 2. Chromatin Modification
(A) ChIP with antibody to H4 amino acids 2–19 acetylated at K5, 8, 12, and 16.
(B) Chip with this antibody using primers diagrammed at the bottom, with results quantitated in (C). Lanes are as indicated in (A). KO is
(D) The same antibody used for ChIP of chromatin crosslinked at the indicated min after addition of serum to cells depleted of serum for 16 hr.
(E) ChIP with antibody to H3 peptide with di- or trimethylated K4.
(F) ChIP with anti-BRG1 antibody.
Med23−/−cells (Figure 1B). ChIP performed by using an
anti-H3 antibody that binds total H3 and H3.3 indepen-
dently of covalent modifications showed that H3 bind-
ing in the Egr1 coding region was reduced in wt, but
not Med23−/−cells after serum addition to an extent
similar to that for the anti-acH4 signal in Figure 2A (see
Figure S1 in the Supplemental Data available with this
article online). This result is consistent with the de-
crease in nucleosome density in the coding regions of
highly transcribed yeast genes that has been noted re-
cently (Lee et al., 2004).
Trimethylation of H3K4 has been associated with
actively transcribed chromatin in yeast (Ng et al., 2003;
Santos-Rosa et al., 2002). Recent work indicates that
di- and trimethylated H3K4 are enriched specifically in
the promoter regions of transcribed genes in mamma-
lian cells (Liang et al., 2004). ChIP performed by using
antibodies specific for di- and trimethylated H3K4 re-
vealed that dimethyl H3K4 is modestly enriched in the
promoter region versus the coding region of Egr1 in wt
ES cells, whereas trimethylated H3K4 is greatly en-
riched in the promoter region (Figure 2E). High H3K4
methylation was apparent in serum-starved cells when
Egr1 transcription was low and was not significantly
stimulated by addition of serum. The level of H3K4
methylation was similar to the level observed in the pro-
moter region of the actively transcribed EF2 gene. Sig-
nificantly, the extent of H3K4 methylation was not af-
fected greatly by knockout of the Med23 genes.
We assayed association of chromatin remodeling
complexes with the Egr1 gene by ChIP by using anti-
body to BRG1, the ATPase subunit of both SWI/SNF-
and RSC-type chromatin remodeling complexes ex-
pressed in mouse ES cells (LeGouy et al., 1998; Wang,
2003). BRG1 binding to both the Egr1 promoter and
coding regions in both serum-starved and serum-
treated cells was above the background level of bind-
ing observed for the promoter of the IL12b gene that is
Postrecruitment Function of Mediator
not transcribed in ES cells (Figure 2F). BRG1 binding
was modestly enriched in the promoter compared to
the coding region of Egr1 and was comparable to the
level of binding to the EF2 promoter. A slight increase
in binding was observed in wt cells in response to se-
rum, but in general BRG1 binding to Egr1 chromatin
was comparable when comparing wt and Med23−/−
cells. Based on these results, it is unlikely that differ-
ences in the binding of SWI/SNF- or RSC-type chro-
matin remodeling complexes account for the very
large difference in Egr1 transcription between wt and
(Figure 3D). Also consistent with the transcription as-
says, Pol II binding to the coding region was greatly
diminished in Med23−/−cells. Surprisingly, a significant
signal was observed for Pol II binding to the Egr1 pro-
moter in both wt and mutant cells before addition of
serum when the rate of Egr1 transcription was very low
(Figure 1B). These ChIP signals for Pol II binding in se-
rum-starved cells were well above background binding
to nonimmune rabbit IgG (Figure 3D). Moreover, pro-
moter regions were not nonspecifically precipitated by
this anti-Pol II antibody because the promoter of the
nontranscribed IL12b gene was not brought down in
the same immunoprecipitation (Figure 3E). Pol II bind-
ing to the Egr1 promoter region increased significantly
in response to serum in wt cells and less so in Med23−/−
cells (Figure 3D).
To accurately measure the influence of serum treat-
ment on Pol II binding to the Egr1-promoter region,
ChIPed DNA from four successive, independent experi-
ments was analyzed by quantitative PCR (Figure 3F).
To determine if the increase in Egr1 transcription in re-
sponse to serum is exclusively the result of enhanced
Pol II binding to the promoter, these values were com-
pared to the quantitative analysis of Egr1 transcription
(Figure 1B). Pol II binding to the Egr1 promoter in wt
cells increased by a factor of 5.8 ± 1.5 in response to
serum. However, Egr1 transcription in serum-treated wt
cells increased by a factor of 27.5 ± 3.5. These results
indicate that the rate of initiation by Pol II molecules
bound to the Egr1 promoter in serum-treated wt cells
must be w4–5 times the rate in serum-starved wt cells
to account for the discrepancy between the w6-fold
increase in Pol II binding to the Egr1 -promoter region
and the w30-fold increase in the overall rate of Egr1
transcription. Comparing serum-induced wt cells to se-
rum-starved Med23−/−cells, 6.6 ± 1.6-fold more Pol II
bound to the Egr1 promoter (Figure 3F) resulted in a
107 ± 24-fold increase in Egr1 transcription (Figure 1B).
Clearly, the rate of Egr1 transcription is not regulated
solely through enhanced binding of Pol II to the pro-
The influence of MED23 on Egr1 transcription is most
clearly shown by comparing the serum-treated wt and
mutant cells, as histone acetylation, methylation, and
chromatin remodeling complex association at the Egr1
promoter region were equivalent after serum treatment
(Figure 2). Consequently, no apparent significant differ-
ences in chromatin structure influence the comparison.
Quantitative PCR of ChIPed promoter DNA (Figure 3F)
showed that Pol II binding was 2.7 ± 1.1-fold higher in
wt compared to Med23−/−cells. Thus, interactions
made by this Mediator subunit result in an w3-fold
increase in Pol II binding to the Egr1 promoter. How-
ever, this w3-fold difference in promoter binding was
considerably smaller than the difference in the rate of
Egr1 transcription in wt versus Med23−/−serum-treated
cells, which was 13.2 ± 1.6-fold (Figure 1B). Thus, while
MED23 increased Pol II recruitment to the Egr1 pro-
moter, the magnitude of this increase, w3-fold, could
not account fully for the increase in Egr1 transcription,
w13-fold. To account for this difference, we conclude
that the presence of MED23 also resulted in an w4- to
5-fold increase in the rate of transcription initiation by
Pol II molecules bound to the Egr1 promoter.
MED23-Dependent Increase in Mediator Binding
in Response to Serum
Mediator binding was assayed by ChIP using antibod-
ies to two Mediator subunits, MED1(TRAP220) and
MED17(CRSP77), in a single immunoprecipitation reac-
tion. As predicted from in vitro binding studies with the
ELK1 activation domain (Cantin et al., 2003; Stevens et
al., 2002), ChIP showed that serum and the subsequent
phosphorylation of ELK1 greatly stimulated Mediator
binding in vivo to the Egr1 control region in wt cells,
and this stimulation was greatly reduced in Med23−/−
cells (Figure 3A). It is difficult to determine from this
data whether or not a low level of Mediator was associ-
ated with the Egr1 promoter before addition of serum
due to the fact that the ChIP signal at the promoter was
only slightly higher than what is presumably a back-
ground signal for the Egr1 coding region, and PCR am-
plification of the promoter region from input DNA ampli-
fied simultaneously showed a similar increase over the
coding region signal (Figure 3A, input). However, it is
clear that Mediator binding increased greatly in re-
sponse to serum in wt cells. More accurate mapping of
Mediator binding sites was accomplished by PCR by
using primers for fragments A–E (Figure 3B). Binding
was greatest to fragment C spanning the transcription
start site and the single promoter proximal SRE and
was next highest to fragment B close to the clustered
SREs at approximately −400 (Figures 3B and 3C).
There are two major classes of Mediator complexes,
those containing a cyclin-dependent kinase-cyclin mod-
ule and those lacking the module (Holstege et al., 1998;
Naar et al., 2002; Samuelsen et al., 2003; Sato et al.,
2004; Wang et al., 2001). Recent studies have indicated
that Mediator complexes containing the CDK-cyclin
module have reduced association with Pol II (Naar et
al., 2002; Samuelsen et al., 2003; Sato et al., 2004) and
are unable to stimulate transcription in response to ac-
tivators in vitro (Taatjes et al., 2002). However, ChIP per-
formed by using anti-CDK8 antibody produced very
similar results to ChIP with anti-MED1 and anti-MED17
(Figure S2), indicating that a CDK8-containing form of
Mediator interacts with activation domains bound to
control regions in vivo, just as it does in vitro, and may
contribute to transcriptional activation (Cantin et al.,
2003; Stevens et al., 2002).
Pol II Binding to the Egr1 Gene
As expected from the transcription results (Figure 1B),
ChIP revealed a very large increase in Pol II binding to
the Egr1- coding region in response to serum in wt cells
Figure 3. Mediator and Pol II Binding
(A) Mediator crosslinked to chromatin was immunoprecipitated with a mixture of anti-MED1 and anti-MED17.
(B) ChIP performed by using primers for the indicated fragments with quantiation shown in (C).
(D) ChIP performed by using control IgG and antibody against a peptide from the amino-terminal region of the large Pol II subunit (α-Pol II
here and in subsequent figures).
(E) ChIP with control IgG and with primers for the promoter region of IL12b.
(F) Quantitative PCR of Egr1 promoter region ChIP using α-Pol II with standard deviations indicated, n = 4.
(G) ChIP with primers indicated in (B), quantitated in (H) relative to the EF2p band for the +serum samples.
Mapping of Pol II binding throughout the Egr1 pro-
moter region by multiplex PCR with primer sets A–E
(Figure 3B) provided additional data in support of the
conclusion that an interaction with MED23 increases
the rate of Pol II initiation at the Egr1 promoter. For
serum-treated Med23−/−cells, Pol II binding was con-
centrated at the promoter (Figures 3G and 3H). In con-
trast, for wt cells plus serum, high levels of Pol II also
were observed in the promoter proximal coding region
fragments D and E. These results indicate that the rate
at which each promoter-bound Pol II molecule initiates
transcription at the Egr1 promoter is greater in wt com-
pared to Med23−/−cells since a much larger proportion
of the Pol II molecules associated with the promoter
region transcribed into the coding region. If the increase
in transcription in wt compared to Med23−/−cells was
due only to an increase in Pol II binding to the promoter,
we would expect the distribution of Pol II in Med23−/−
cells plus serum to be similar to that for wt, with the
amount of Pol II in each region being decreased. Be-
Postrecruitment Function of Mediator
cause this was not the case, the difference in the distri-
bution of Pol II through the promoter proximal region
is a second argument that an interaction with MED23
increases the rate of transcription initiation for each
molecule of Pol II at the Egr1 promoter. This is probably
a consequence of the ELK1-Mediator interaction ob-
served in vitro that, like Egr1 induction, is dependent on
both MED23 and MAP kinase phosphorylation of ELK1
(Stevens et al., 2002). To gain further insight into the
transcription step stimulated by this activator-Mediator
interaction, we analyzed GTF binding, Pol II CTD phos-
phorylation, and elongation factor binding by ChIP.
these factors were stably associated with this small
fraction of Egr1 promoters. Instead, it seems more
likely that in uninduced serum-starved cells there is a
dynamic equilibrium between binding and dissociation
of Pol II, TFIIB, E, and H (and presumably TFIIF) from
TFIID-TFIIA complexes on Egr1 promoters (Figure 4E,
top). As a result, Pol II, TFIIB, E, and H would be bound
to only a fraction of promoters at the moment of formal-
dehyde crosslinking, accounting for the observed data.
It is possible that Mediator complexes also are revers-
ibly associated with the small fraction of Egr1 promot-
ers bound by Pol II and GTFs in serum-starved cells.
However, because a high background of signal in ChIP
assays with anti-Mediator antibodies prevented us
from determining the level of Mediator binding in se-
rum-starved cells (Figure 3A), we could not reach a reli-
able conclusion on this point.
Serum treatment clearly increased Mediator binding
to the Egr1 promoter in wt cells, for which the ChIP
signal was well above background (Figure 3A). The re-
sulting additional interactions between phospho-ELK1
and the MED23 Mediator subunit may stabilize the pre-
initiation complex, resulting in the observed w3-fold
increase in binding of Pol II, TFIIB, E, and H in response
to serum (Figure 4E). This is similar to the stimulation
of preinitiation complex assembly resulting from the
phospho-ELK1-Mediator interaction observed in vitro
on a synthetic promoter, with binding sites for only the
single Gal4-ELK1 fusion protein studied (Cantin et al.,
2003). In the case of the more complex endogenous
Egr1 control region, other transcription factors likely
promote the assembly of the preinitiation complexes
observed before the addition of serum. Sp1 was sug-
gested to have this function at promoters rapidly in-
duced by NF-κB (Ainbinder et al., 2002).
Association of General Transcription Factors
ChIP assays of general transcription factor (GTF) bind-
ing in wt cells revealed that each one assayed associ-
ated with the Egr1 promoter both in the absence of se-
rum, when the transcription rate was low, and after
addition of serum, when transcription occurred at a
high rate in wt cells (Figure 4). However, there were in-
teresting differences in the level of binding of the dif-
ferent GTFs. TBP and TFIIA binding increased only
modestly at best in response to serum (Figures 4A and
4B). In contrast, TFIIB, E, and H behaved like Pol II.
Although binding was clearly detected at the Egr1 pro-
moter in the absence of serum, it increased signifi-
cantly in response to serum (Figures 4C and 4D). In
Med23−/−cells, GTF binding was similar to wt cells in
the absence of serum, but the stimulation of TFIIB, E,
and H binding by serum was reduced compared to wt
cells. Scanning through the promoter region showed
that, for all the GTFs assayed, binding was centered
over the transcription start site (Figures 4B and 4D, and
data not shown). ChIP performed by using antibody to
TAF1 showed similar results to those for TBP (data not
shown), indicating that TFIID complexes were associ-
ated with the promoter. We were not able to obtain reli-
able ChIP results using antibodies prepared against
TFIIF. Quantitative PCR of ChIPed DNA showed that
TFIIH binding to the Egr1 promoter was 3.3 ± 0.9-fold
higher in wt versus Med23−/−serum-treated cells. This
is similar to the fold increase observed for Pol II, which
was 2.7 ± 1.1. Because TFIIH is the last GTF to associ-
ate with a promoter during preinitiation complex as-
sembly (Roeder, 1998) and quantitative Egr1 promoter
binding assays showed a similar increase in Pol II and
TFIIH binding in wt compared to Med23−/−cells, we
conclude that MED23 stimulated preinitiation complex
assembly w3-fold in serum-treated cells.
The observation that TFIID and A binding in wt cells
did not increase significantly with serum, whereas bind-
ing of TFIIB, E, H, and Pol II did, suggests that in the
absence of serum, more Egr1 promoters were bound
by TFIID and TFIIA than by TFIIB, E, H, and Pol II. The
maximum ChIP signal possible corresponds to protein
binding to the two Egr1 promoters in every diploid cell.
Because the TBP and TFIIA signals did not increase
significantly in response to serum, TFIID and TFIIA may
be bound to all or nearly all Egr1 promoters before se-
rum addition. In contrast, only a fraction of Egr1 pro-
moters had associated TFIIB, E, H, and Pol II in serum-
starved cells since the ChIP signal for these factors
increased after serum addition. It seems unlikely that
Salt Sensitivity of Pol II Binding to the Egr1
The observation that preinitiation complexes are as-
sembled on the uninduced Egr1 promoter before addi-
tion of serum raised the question of whether these
polymerases are associated with preinitiation com-
plexes that have not initiated transcription, or polymer-
ase molecules that initiated transcription and paused,
as observed at uninduced heatshock promoters origi-
nally in Drosophila (Lis, 1998) and also in mammalian
cells (Corey et al., 2003). To address this question, we
determined whether Pol II associated with the Egr1 pro-
moter in the absence of serum is stable to treatment
with high salt. In contrast to the polymerase in preinitia-
tion complexes, elongating multisubunit RNA polymer-
ases bound to template with associated nascent tran-
scripts (ternary complexes) remain stably associated
with template DNA in >0.5 M monovalent cation
(Rhodes and Chamberlin, 1974; Roe et al., 1984). For
mammalian RNA polymerase II, synthesis of only two
phosphodiester bonds generates a complex stable to
0.35 M KCl, and synthesis of ten phosphodiester bonds
yields a complex stable to 0.6 M KCl (Cai and Luse,
1987). Structural studies of an RNA polymerase II elon-
gation complex suggest that this increase in salt sta-
bility results from closing of the moveable clamp do-
main of the large subunit over downstream DNA (Gnatt
et al., 2001).
Figure 4. ChIP of GTFs
(A) ChIP using antibodies to TBP and TFIIA.
(B) Anti-TBP ChIP using the indicated primers, quantitation at right.
(C) ChIP using antibodies to TFIIB, TFIIE, and TFIIH.
(D) Anti-TFIIE ChIP using the primers shown in (B), quantitation at right.
(E) Model for GTF binding to the Egr1 promoter. See text for discussion.
Consistent with these in vitro results on the salt sta-
bility of Pol II template association, a recent study in
yeast showed that increasing the salt concentration of
media to 0.4 M NaCl led to a rapid loss of Pol II and
GTFs from the promoter regions of most genes without
dissociation of elongating Pol II from coding regions,
as assayed by ChIP (Proft and Struhl, 2004). In our ex-
periments, we added NaCl to 0.5 M to media of wt cells
that were serum starved or treated with serum for 30
min. Formaldehyde was subsequently added to cross-
link chromatin, and the remaining steps of the ChIP
analysis were performed by using antibody to the large
Postrecruitment Function of Mediator
coding region, in both serum-starved and serum-treated
cells. In contrast to the EF2 promoter, Pol II binding
to the Hsp70A1 promoter was not decreased by salt
treatment. This is consistent with previous observa-
tions indicating that in non-heat-shocked cells, Pol II
has initiated and paused near the Hsp70A1 promoter
(Corey et al., 2003).
When the same anti-Pol II immunoprecipitates were
analyzed by PCR by using primers for the Egr1 pro-
moter and coding regions (Figures 5A and 5C), the
analysis showed that salt treatment did not reduce Pol
II binding to the Egr1 coding region in serum-treated
cells, or to the EF2 coding region in serum-starved and
serum-treated cells. However, Pol II binding to the Egr1
promoter region, as well as the EF2 promoter region
analyzed in the same reactions, clearly decreased with
salt treatment, in sharp contrast to the Hsp70A1 pro-
moter. We conclude that Pol II associated with the Egr1
promoter region in serum-starved cells has not initiated
and then paused at an early point in elongation, as is
the case for Pol II at the Hsp70A1 promoter. (It was
necessary to use a different set of primers for the EF2
coding region in the multiplex PCRs of Figures 5B and
5D than in C because the primer pair for EF2 coding
region used in C interfered with amplification of the
Hsp70A1 promoter region. The alternative amplified
product from the EF2 coding region in Figure 5C was
smaller than the amplicon from the EF2 promoter re-
gion.) Similar salt sensitivity of Pol II binding to the Egr1
promoter region was observed in serum-starved and
treated Med23−/−cells, as expected.
Wt ES cells also were either maintained at 37°C or
incubated at 43°C for 5 min to induce the heat shock
response (Figure 5D). As observed earlier, the Pol II
ChIP signal for the Hsp70A1 gene from cells at 37°C
was not affected by high salt, whereas the signal from
the EF2 promoter in the same immunoprecipitated DNA
was decreased. After a shift to 43°C for 5 min, Pol II
increased greatly in the Hsp70A1 coding region as well
as in the promoter region (Figure 5D, 43°C). The signal
for elongating polymerase in the Hsp70A1 coding re-
gion as well as the signals for elongating Pol II in the
EF2 coding region at 37°C and at 43°C were not de-
creased by high-salt treatment. However, Pol II in the
promoter region of the heat-shock-induced Hsp70A1
gene was decreased by high salt. This result suggests
that much of the increased Pol II at the Hsp70A1 gene
in response to heat shock is associated with preinitia-
tion complexes rather than paused, elongating poly-
merases as observed in nonshocked cells. Pol II bind-
ing to the induced Egr1 promoter in serum-treated cells
(Figure 5C) was also salt sensitive, like Pol II at the EF2
promoter under all conditions. These results suggest
that there is a substantial pause after Pol II promoter
binding before the polymerase initiates and enters the
salt-stable elongation conformation. This pause may be
due to abortive initiation (Goodrich and Tjian, 1994; Tin-
tut et al., 1995) or another slow step in promoter escape
and the transition to a stable elongation complex. Such
a pause may account for the higher level of Pol II ob-
served in the promoter regions compared to the coding
regions of the transcribed genes in most of these
Figure 5. Salt Stability of Pol II Binding
(A) Primers used.
(B) Wt ES cells were depleted of serum (−) or treated with serum
for 30 min after depletion (+). NaCl was added to the medium to a
final concentration of 0.5 M (+) or not added (−), and formaldehyde
was added subsequently to crosslink chromatin. The standard
ChIP procedure with anti-Pol II and the indicated primers was ap-
plied to all samples.
(C) ChIP as in (B) with the indicated primers.
(D) Wt ES cells were incubated continuously at 37°C or shifted to
43°C for 5 min. NaCl was added to the medium to 0.5 M as indi-
cated, formaldehyde was added subsequently and the ChIP proto-
col was performed with the indicated primers.
subunit of Pol II. As a control, the promoter region of
the Hsp70A1 gene (Corey et al., 2003) was analyzed in
multiplex PCRs by using primers for the promoter and
coding regions of the EF2 gene (Figures 5A and 5B). As
observed in S. cerevisiae (Proft and Struhl, 2004), salt
treatment resulted in a substantial decrease in Pol II
binding to the EF2 promoter region, but not to the EF2
tion by the anti-Ser5-P H14 monoclonal antibody is not
an accurate measure of the number of CTD repeats
phosphorylated on Ser5. Consequently, stimulation of
CTD phosphorylation by the phospho-ELK1-MED23 in-
teraction may have been missed by this assay.
CTD Ser2 phosphorylation at the Egr1 promoter in-
creased in response to serum in both wt and Med23−/−
cells (Figure 6C). Consequently, it seems unlikely that
the Med23−/−cell defect in initiation resulted from a de-
fect in CTD phosphorylation at Ser2. After serum addi-
tion and the resulting stimulation of transcription, Ser2
phosphorylation was much higher at the 3# end of the
Egr1 and EF2 genes than in the promoter region, as has
been observed in yeast (Cho et al., 2001; Komarnitsky
et al., 2000). Surprisingly, the signal for Ser2 phosphor-
ylation (Figure 6C) relative to total Pol II in the Egr1 cod-
ing region (Figure 6A) was reproducibly higher in
Med23−/−compared to wt cells. This might be ex-
plained by more extensive Ser2 phosphorylation of Pol
II in the mutant cells resulting in more efficient immuno-
precipitation by the anti- Ser2-P antibody. Perhaps this
was a consequence of the slower rate of initiation at
the Egr1 promoter in the mutant cells, allowing a longer
period for association with P-TEFb.
Elongation factors DSIF (Spt4-Spt5) and NELF func-
tion to inhibit promoter proximal elongation, and this
inhibition is relieved by the kinase activity of P-TEFb
(CDK9-Cyclin T) (Renner et al., 2001; Yamaguchi et al.,
1999). Elongation factors NELF, DSIF, and P-TEFb were
each readily detected at the Egr1 promoter before addi-
tion of serum in both wt and Med23−/−cells (Figures
6D–6F). They each increased in response to serum to
an extent similar to that observed for Pol II, as expected
for factors that bind to the polymerase. The ChIP sig-
nals for these elongation factors paralleled the signals
for Pol II (Figure 6A). Consequently, the amount of these
factors associated per Pol II molecule bound to the
Egr1 promoter was the same in wt and Med23−/−cells
plus or minus serum. As in Drosophila (Andrulis et al.,
2000; Kaplan et al., 2000; Wu et al., 2003), NELF was
confined to the promoter region whereas DSIF and
P-TEFb traveled with elongating polymerase into the
The Egr1 control region is organized into a chromatin
structure that is poised to respond rapidly to MAP ki-
nase signaling. Kinetic analysis of Pol II binding indi-
cated that transcription initiation is stimulated within
one minute of serum addition to wt cells (Figure S3).
Figure 6. CTD Phosphorylation and Elongation Factor Binding
(A–F) are ChIPs of the same preparation of crosslinked chromatin
performed with the indicated antibodies. In (F), IP was performed
first with anti-Pol II, the immunoprecipitate was eluted and precipi-
tated a second time with anti-CDK9.
CTD Phosphorylation and Association
of Elongation Factors
The results presented above indicate that the MED23
Mediator subunit is required for a high rate of Pol II
initiation at the Egr1 promoter in response to serum.
Because phosphorylation of the Pol II CTD and the
binding of elongation factors have been implicated in
controlling transcription initiation and elongation at
other promoters (Ainbinder et al., 2004; Andrulis et al.,
2000; Kaplan et al., 2000; Renner et al., 2001; Shim et
al., 2002; Wu et al., 2003), we analyzed these processes
at the Egr1 promoter. Even though the rate of Egr1 tran-
scription initiation in serum-starved cells was very low,
especially in Med23−/−cells, phosphorylation of CTD
Ser5 at the promoter, as measured by ChIP with the
anit-Ser5-P H14 monoclonal antibody (Patturajan et al.,
1998), was evident in both wt and Med23−/−cells (Fig-
ure 6B). An increase in phospho-Ser5 paralleled the
increase in total Pol II after addition of serum to both
wt and Med23−/−cells (compare Figures 6A and 6B).
Consequently, although S. cerevisiae Mediator stimu-
lates TFIIH phosphorylation of the CTD in vitro (Kim et
al., 1994), these ChIP experiments did not reveal a
requirement for the MED23 subunit for Ser5 phosphory-
lation at the Egr1 promoter in mouse cells. It is interest-
ing that MED23 does not appear to be conserved be-
tween S. cerevisiae and metazoans (Bourbon et al.,
2004). It seems likely that stimulation of TFIIH phos-
phorylation of Ser5 requires other, conserved Mediator
subunits. However, it is possible that immunoprecipita-
Previous studies of the mechanism of Mediator func-
tion in activator-dependent transcription in vivo re-
vealed that Mediator is required for an activator to stim-
ulate binding of Pol II and GTFs to a target promoter—
the recruitment steps of transcription initiation (Kuras
and Struhl, 1999; Li et al., 1999). This is consistent with
a requirement for Mediator (Ranish et al., 1999) and
specific activation domain-Mediator interactions (Can-
tin et al., 2003) to stimulate pre-initiation complex as-
sembly in vitro. The results presented here demonstrate
that an activator-Mediator interaction can also stimu-
late a postrecruitment step in transcription initiation
Postrecruitment Function of Mediator
and Carol Eng for technical assistance. This work was supported
by NIH grant CA25235.
in vivo. The phospho-ELK1-MED23 interaction in wt
cells stimulated recruitment of preinitiation complexes
to the Egr1 promoter by a factor of w3 compared to
Med23−/−cells where this interaction cannot occur (Fig-
ures 3F and 4C). However, the rate of Egr1 transcription
was w13-fold higher in wt compared to Med23−/−cells
(Figure 1B), indicating that Pol II bound to the Egr1 pro-
moter initiated transcription approximately four to five
times faster in wt compared to Med23−/−cells. This
increase in the rate of Pol II initiation was also apparent
from the increased association of Pol II with the pro-
moter proximal coding region relative to the promoter
in wt compared to Med23−/−cells (Figures 3G and 3H).
In contrast to Pol II initiation, the stimulation of his-
tone acetylation in the Egr1 promoter region in re-
sponse to serum occurred normally in Med23−/−cells
(Figures 2A–2C). This result indicates that recruitment
and activation of histone acetylases by phospho-ELK1
(Janknecht and Nordheim, 1996; Li et al., 2003; Nissen
et al., 2001) are independent of Mediator recruitment
and that histone acetylation alone is not sufficient to
activate transcription fully. These results suggest that
recruitment of HAT complexes and Mediator are inde-
pendent processes, which are both required to activate
transcription. TFIID and A binding to the Egr1 promoter
also were not dependent on the phospho-ELK1-Media-
tor interaction. The binding of these core preinitiation
complex factors is probably directed by transcription
factors other than ELK1 that bind to the Egr1 control
region (Figure 1A).
The ability of Mediator to influence both Pol II binding
and the rate of initiation by promoter bound Pol II is
similar to the ability of the E. coli CAP activator to stim-
ulate transcription from class II promoters by both
increasing the binding of RNA polymerase to promoter
DNA and stimulating the rate of isomerization of the
RNA polymerase-promoter closed complex to an open
complex (Busby and Ebright, 1999). The MED23-depen-
dent stimulation of initiation by promoter bound Pol II
at the Egr1 promoter may also involve facilitation of
promoter melting. Further studies will be required to re-
solve this issue. However, it seems likely that stimula-
tion of the rate of initiation by promoter bound preinitia-
tion complexes through activator-Mediator interactions
contributes to activation of genes in addition to Egr1.
This activity of Mediator may be particularly important
in the activation of other rapidly induced genes with
preinitiation complexes assembled on their promoters
before induction, such as genes that are rapidly in-
duced by TNF-α via NF-κB (Ainbinder et al., 2002).
Received: September 24, 2004
Revised: January 2, 2005
Accepted: February 2, 2005
Published: March 3, 2005
Ainbinder, E., Revach, M., Wolstein, O., Moshonov, S., Diamant, N.,
and Dikstein, R. (2002). Mechanism of rapid transcriptional induc-
tion of tumor necrosis factor alpha-responsive genes by NF-kap-
paB. Mol. Cell. Biol. 22, 6354–6362.
Ainbinder, E., Amir-Zilberstein, L., Yamaguchi, Y., Handa, H., and
Dikstein, R. (2004). Elongation inhibition by DRB sensitivity-induc-
ing factor is regulated by the A20 promoter via a novel negative
element and NF-kappaB. Mol. Cell. Biol. 24, 2444–2454.
Andrulis, E.D., Guzman, E., Doring, P., Werner, J., and Lis, J.T.
(2000). High-resolution localization of Drosophila Spt5 and Spt6 at
heat shock genes in vivo: roles in promoter proximal pausing and
transcription elongation. Genes Dev. 14, 2635–2649.
Becker, P.B., and Horz, W. (2002). ATP-dependent nucleosome re-
modeling. Annu. Rev. Biochem. 71, 247–273.
Bourbon, H.M., Aguilera, A., Ansari, A.Z., Asturias, F.J., Berk, A.J.,
Bjorklund, S., Blackwell, T.K., Borggrefe, T., Carey, M., Carlson, M.,
et al. (2004). A unified nomenclature for protein subunits of media-
tor complexes linking transcriptional regulators to RNA polymerase
II. Mol. Cell 14, 553–557.
Boyer, T.G., Martin, M.E., Lees, E., Ricciardi, R.P., and Berk, A.J.
(1999). Mammalian Srb/Mediator complex is targeted by adenovi-
rus E1A protein. Nature 399, 276–279.
Buchwalter, G., Gross, C., and Wasylyk, B. (2004). Ets ternary com-
plex transcription factors. Gene 324, 1–14.
Busby, S., and Ebright, R.H. (1999). Transcription activation by ca-
tabolite activator protein (CAP). J. Mol. Biol. 293, 199–213.
Cai, H., and Luse, D.S. (1987). Transcription initiation by RNA poly-
merase II in vitro. Properties of preinitiation, initiation, and elonga-
tion complexes. J. Biol. Chem. 262, 298–304.
Cantin, G.T., Stevens, J.L., and Berk, A.J. (2003). Activation do-
main-mediator interactions promote transcription preinitiation
complex assembly on promoter DNA. Proc. Natl. Acad. Sci. USA
Cho, E.J., Kobor, M.S., Kim, M., Greenblatt, J., and Buratowski, S.
(2001). Opposing effects of Ctk1 kinase and Fcp1 phosphatase at
Ser 2 of the RNA polymerase II C-terminal domain. Genes Dev. 15,
Corey, L.L., Weirich, C.S., Benjamin, I.J., and Kingston, R.E. (2003).
Localized recruitment of a chromatin-remodeling activity by an ac-
tivator in vivo drives transcriptional elongation. Genes Dev. 17,
Fischle, W., Wang, Y., and Allis, C.D. (2003). Histone and chromatin
cross-talk. Curr. Opin. Cell Biol. 15, 172–183.
Gnatt, A.L., Cramer, P., Fu, J., Bushnell, D.A., and Kornberg, R.D.
(2001). Structural basis of transcription: an RNA polymerase II elon-
gation complex at 3.3 A resolution. Science 292, 1876–1882.
Goodrich, J.A., and Tjian, R. (1994). Transcription factors IIE and
IIH and ATP hydrolysis direct promoter clearance by RNA polymer-
ase II. Cell 77, 145–156.
Herrera, R.E., Shaw, P.E., and Nordheim, A. (1989). Occupation of
the c-fos serum response element in vivo by a multi-protein com-
plex is unaltered by growth factor induction. Nature 340, 68–70.
Holstege, F.C., Jennings, E.G., Wyrick, J.J., Lee, T.I., Hengartner,
C.J., Green, M.R., Golub, T.R., Lander, E.S., and Young, R.A. (1998).
Dissecting the regulatory circuitry of a eukaryotic genome. Cell 95,
Horn, P.J., and Peterson, C.L. (2002). Molecular biology. Chromatin
higher order folding–wrapping up transcription. Science 297,
Supplemental Data including three figures, one table, and Supple-
mental Experimental Procedures are available online with this arti-
cle at http://www.molecule.org/cgi/content/full/17/5/683/DC1/.
We thank Michael Carey, Albert Courey, Michael Grunstein, Siavash
Kurdistani, and Stephen Smale for helpful suggestions and com-
ments; Weidong Wang (National Institutes of Health) for antibody
to BRG1; Michael Grunstein for antibodies to acetylated histones;
Molecular Cell Download full-text
Janknecht, R., and Nordheim, A. (1996). MAP kinase-dependent
transcriptional coactivation by Elk-1 and its cofactor CBP. Bio-
chem. Biophys. Res. Commun. 228, 831–837.
Kaplan, C.D., Morris, J.R., Wu, C., and Winston, F. (2000). Spt5 and
spt6 are associated with active transcription and have characteris-
tics of general elongation factors in D. melanogaster. Genes Dev.
Kim, Y.J., Bjorklund, S., Li, Y., Sayre, M.H., and Kornberg, R.D.
(1994). A multiprotein mediator of transcriptional activation and its
interaction with the C-terminal repeat domain of RNA polymerase
II. Cell 77, 599–608.
Kingston, R.E. (1999). A shared but complex bridge. Nature 399,
Komarnitsky, P., Cho, E.J., and Buratowski, S. (2000). Different
phosphorylated forms of RNA polymerase II and associated mRNA
processing factors during transcription. Genes Dev. 14, 2452–2460.
Kuras, L., and Struhl, K. (1999). Binding of TBP to promoters in vivo
is stimulated by activators and requires Pol II holoenzyme. Nature
Lee, C.K., Shibata, Y., Rao, B., Strahl, B.D., and Lieb, J.D. (2004).
Evidence for nucleosome depletion at active regulatory regions ge-
nome-wide. Nat. Genet. 36, 900–905.
Lee, T.I., and Young, R.A. (2000). Transcription of eukaryotic pro-
tein-coding genes. Annu. Rev. Genet. 34, 77–137.
LeGouy, E., Thompson, E.M., Muchardt, C., and Renard, J.P. (1998).
Differential preimplantation regulation of two mouse homologues
of the yeast SWI2 protein. Dev. Dyn. 212, 38–48.
Li, Q.J., Yang, S.H., Maeda, Y., Sladek, F.M., Sharrocks, A.D., and
Martins-Green, M. (2003). MAP kinase phosphorylation-dependent
activation of Elk-1 leads to activation of the co-activator p300.
EMBO J. 22, 281–291.
Li, X.Y., Virbasius, A., Zhu, X., and Green, M.R. (1999). Enhance-
ment of TBP binding by activators and general transcription
factors. Nature 399, 605–609.
Liang, G., Lin, J.C., Wei, V., Yoo, C., Cheng, J.C., Nguyen, C.T.,
Weisenberger, D.J., Egger, G., Takai, D., Gonzales, F.A., and Jones,
P.A. (2004). Distinct localization of histone H3 acetylation and H3–
K4 methylation to the transcription start sites in the human ge-
nome. Proc. Natl. Acad. Sci. USA 101, 7357–7362.
Lis, J. (1998). Promoter-associated pausing in promoter architec-
ture and postinitiation transcriptional regulation. Cold Spring Harb.
Symp. Quant. Biol. 63, 347–356.
Lorch, Y., Beve, J., Gustafsson, C.M., Myers, L.C., and Kornberg,
R.D. (2000). Mediator-nucleosome interaction. Mol. Cell 6, 197–201.
Malik, S., and Roeder, R.G. (2000). Transcriptional regulation
through Mediator-like coactivators in yeast and metazoan cells.
Trends Biochem. Sci. 25, 277–283.
Malik, S., Wallberg, A.E., Kang, Y.K., and Roeder, R.G. (2002).
TRAP/SMCC/mediator-dependent transcriptional activation from
DNA and chromatin templates by orphan nuclear receptor hepato-
cyte nuclear factor 4. Mol. Cell. Biol. 22, 5626–5637.
Myers, L.C., and Kornberg, R.D. (2000). Mediator of transcriptional
regulation. Annu. Rev. Biochem. 69, 729–749.
Naar, A.M., Taatjes, D.J., Zhai, W., Nogales, E., and Tjian, R. (2002).
Human CRSP interacts with RNA polymerase II CTD and adopts a
specific CTD-bound conformation. Genes Dev. 16, 1339–1344.
Ng, H.H., Robert, F., Young, R.A., and Struhl, K. (2003). Targeted
recruitment of Set1 histone methylase by elongating Pol II provides
a localized mark and memory of recent transcriptional activity. Mol.
Cell 11, 709–719.
Nissen, L.J., Gelly, J.C., and Hipskind, R.A. (2001). Induction-inde-
pendent recruitment of CREB-binding protein to the c-fos serum
response element through interactions between the bromodomain
and Elk-1. J. Biol. Chem. 276, 5213–5221.
Patturajan, M., Schulte, R.J., Sefton, B.M., Berezney, R., Vincent,
M., Bensaude, O., Warren, S.L., and Corden, J.L. (1998). Growth-
related changes in phosphorylation of yeast RNA polymerase II. J.
Biol. Chem. 273, 4689–4694.
Proft, M., and Struhl, K. (2004). MAP kinase-mediated stress relief
that precedes and regulates the timing of transcriptional induction.
Cell 118, 351–361.
Ranish, J.A., Yudkovsky, N., and Hahn, S. (1999). Intermediates in
formation and activity of the RNA polymerase II preinitiation com-
plex: holoenzyme recruitment and a postrecruitment role for the
TATA box and TFIIB. Genes Dev. 13, 49–63.
Renner, D.B., Yamaguchi, Y., Wada, T., Handa, H., and Price, D.H.
(2001). A highly purified RNA polymerase II elongation control sys-
tem. J. Biol. Chem. 276, 42601–42609.
Rhodes, G., and Chamberlin, M.J. (1974). Ribonucleic acid chain
elongation by Escherichia coli ribonucleic acid polymerase. I. Isola-
tion of ternary complexes and the kinetics of elongation. J. Biol.
Chem. 249, 6675–6683.
Roe, J.H., Burgess, R.R., and Record, M.T., Jr. (1984). Kinetics and
mechanism of the interaction of Escherichia coli RNA polymerase
with the lambda PR promoter. J. Mol. Biol. 176, 495–522.
Roeder, R.G. (1998). Role of general and gene-specific cofactors in
the regulation of eukaryotic transcription. Cold Spring Harb. Symp.
Quant. Biol. 63, 201–218.
Samuelsen, C.O., Baraznenok, V., Khorosjutina, O., Spahr, H., Kie-
selbach, T., Holmberg, S., and Gustafsson, C.M. (2003). TRAP230/
ARC240 and TRAP240/ARC250 Mediator subunits are functionally
conserved through evolution. Proc. Natl. Acad. Sci. USA 100,
Santos-Rosa, H., Schneider, R., Bannister, A.J., Sherriff, J., Bern-
stein, B.E., Emre, N.C., Schreiber, S.L., Mellor, J., and Kouzarides,
T. (2002). Active genes are tri-methylated at K4 of histone H3. Na-
ture 419, 407–411.
Sato, S., Tomomori-Sato, C., Parmely, T.J., Florens, L., Zybailov, B.,
Swanson, S.K., Banks, C.A., Jin, J., Cai, Y., Washburn, M.P., et al.
(2004). A set of consensus mammalian mediator subunits identified
by multidimensional protein identification technology. Mol. Cell 14,
Shaw, P.E., and Saxton, J. (2003). Ternary complex factors: prime
nuclear targets for mitogen-activated protein kinases. Int. J. Bio-
chem. Cell Biol. 35, 1210–1226.
Shim, E.Y., Walker, A.K., Shi, Y., and Blackwell, T.K. (2002). CDK-9/
cyclin T (P-TEFb) is required in two postinitiation pathways for tran-
scription in the C. elegans embryo. Genes Dev. 16, 2135–2146.
Stevens, J.L., Cantin, G.T., Wang, G., Shevchenko, A., and Berk,
A.J. (2002). Transcription control by E1A and MAP kinase pathway
via Sur2 mediator subunit. Science 296, 755–758.
Taatjes, D.J., Naar, A.M., Andel, F., 3rd, Nogales, E., and Tjian, R.
(2002). Structure, function, and activator-induced conformations of
the CRSP coactivator. Science 295, 1058–1062.
Taatjes, D.J., Marr, M.T., and Tjian, R. (2004). Regulatory diversity
among metazoan co-activator complexes. Nat. Rev. Mol. Cell Biol.
Thiel, G., and Cibelli, G. (2002). Regulation of life and death by the
zinc finger transcription factor Egr-1. J. Cell. Physiol. 193, 287–292.
Tintut, Y., Wang, J.T., and Gralla, J.D. (1995). Abortive cycling and
the release of polymerase for elongation at the sigma 54-depen-
dent glnAp2 promoter. J. Biol. Chem. 270, 24392–24398.
Wang, G., and Berk, A.J. (2002). In vivo association of adenovirus
large E1A protein with the human mediator complex in adenovirus-
infected and -transformed cells. J. Virol. 76, 9186–9193.
Wang, G., Cantin, G.T., Stevens, J.L., and Berk, A.J. (2001). Charac-
terization of mediator complexes from HeLa cell nuclear extract.
Mol. Cell. Biol. 21, 4604–4613.
Wang, W. (2003). The SWI/SNF family of ATP-dependent chromatin
remodelers: similar mechanisms for diverse functions. Curr. Top.
Microbiol. Immunol. 274, 143–169.
Wu, C.H., Yamaguchi, Y., Benjamin, L.R., Horvat-Gordon, M., Wash-
insky, J., Enerly, E., Larsson, J., Lambertsson, A., Handa, H., and
Gilmour, D. (2003). NELF and DSIF cause promoter proximal paus-
ing on the hsp70 promoter in Drosophila. Genes Dev. 17, 1402–
Yamaguchi, Y., Takagi, T., Wada, T., Yano, K., Furuya, A., Sugimoto,
S., Hasegawa, J., and Handa, H. (1999). NELF, a multisubunit com-
plex containing RD, cooperates with DSIF to repress RNA polymer-
ase II elongation. Cell 97, 41–51.