Mediator coordinates PIC assembly
with recruitment of CHD1
Justin J. Lin, Lynn W. Lehmann, Giancarlo Bonora, Rupa Sridharan, Ajay A. Vashisht, Nancy Tran,
Kathrin Plath, James A. Wohlschlegel, and Michael Carey1
Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, California 90095, USA
Murine Chd1 (chromodomain helicase DNA-binding protein 1), a chromodomain-containing chromatin remod-
eling protein, is necessary for embryonic stem (ES) cell pluripotency. Chd1 binds to nucleosomes trimethylated at
histone 3 Lys 4 (H3K4me3) near the beginning of active genes but not to bivalent domains also containing
H3K27me3. To address the mechanism of this specificity, we reproduced H3K4me3- and CHD1-stimulated gene
activation in HeLa extracts. Multidimensional protein identification technology (MuDPIT) and immunoblot
analyses of purified preinitiation complexes (PICs) revealed the recruitment of CHD1 to naive chromatin but
enhancement on H3K4me3 chromatin. Studies in depleted extracts showed that the Mediator coactivator
complex, which controls PIC assembly, is also necessary for CHD1 recruitment. MuDPIT analyses of CHD1-
associated proteins support the recruitment data and reveal numerous components of the PIC, including Mediator.
In vivo, CHD1 and Mediator are recruited to an inducible gene, and genome-wide binding of the two proteins
correlates well with active gene transcription in mouse ES cells. Finally, coimmunoprecipitation of CHD1 and
Mediator from cell extracts can be ablated by shRNA knockdown of a specific Mediator subunit. Our data support
a model in which the Mediator coordinates PIC assembly along with the recruitment of CHD1. The combined
action of the PIC and H3K4me3 provides specificity in targeting CHD1 to active genes.
[Keywords: Mediator; preinitiation complex; CHD1; H3K4me3]
Supplemental material is available for this article.
Received July 26, 2011; revised version accepted September 9, 2011.
Histone H3 Lys 4 trimethylation (H3K4me3) near the
beginning of genes correlates with active transcription.
However, although most active genes contain this mod-
ification, not all H3K4me3-bearing genes are actively
transcribed (Kim et al. 2005; Ruthenburg et al. 2007).
H3K4me3 is also found in bivalent domains alongside
H3K27me3, which correlates withgene silencing. Bivalent
domains are found on promoters of important develop-
but not yet active or fully silenced (Bernstein et al. 2006).
The mechanisms by which the context-specific effects
of H3K4me3 are achieved have not been fully explored.
The coordination of transcription with histone modifi-
cations allows RNA polymerase II (Pol II) to overcome
nucleosomal barriers presented by chromatin (Li et al.
2007). Specific histone modifications recruit distinct ef-
fector proteins that alter the chromatin landscapeof active
genes, making them permissive for transcription. It is
largely unknown how the binding and function of this
chromatin machinery are coordinated with assembly of
the preinitiation complex (PIC). The PIC comprises
coactivators like the 30-subunit Mediator complex and
the 14-subunit TFIID complex, along with Pol II and the
general transcription factors (GTFs) TFIIA, TFIIB, TFIID,
TFIIE, TFIIF, and TFIIH; TFIID is both a coactivator and
a GTF (Kornberg 2005).
Mechanistic studies have established that activators
contact and recruit TFIID and Mediator, while the GTFs
and Pol II associate with these coactivators to complete
PIC assembly (Roeder 1998; Johnson and Carey 2003).
The GTFs bound at the promoter position Pol II at the
start site, melt the DNA, and facilitate the catalytic steps
of transcription initiation (Kornberg 2007). Cross-linking
studies have shown that many of the GTFs contact
promoter DNA (Lagrange et al. 1996). Moreover, Pol II,
TFIIH, and the TBP-associated factor (TAFII) subunits of
TFIID all bind both upstream of and downstream from
the start site (Martinez 2002). Additionally, proteins such
as the PAF1 complex and P-TEFb are recruited at or near
the start site to facilitate Pol II elongation. Indeed, most
genes contain a well-characterized NELF- and DSIF-
mediated Pol II pause site located 30 base pairs (bp)
downstream from the start site that must be overcome
elongation complex (SEC) (Peterlin and Price 2006; Smith
Article published online ahead of print. Article and publication date are
online at http://www.genesdev.org/cgi/doi/10.1101/gad.17554711.
2198GENES & DEVELOPMENT 25:2198–2209 ? 2011 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/11; www.genesdev.org
et al. 2011). Most human genes also contain nucleosomes
positioned near the start site (Schones et al. 2008). The
close proximity of the PIC and Pol II elongation machinery
with nucleosomes emphasizes the need to understand how
their functions are coordinated. Some chromatin factors—
like the p300 histone acetyltransferase, which stimulates
Pol II elongation, and the PAF1 complex, which controls
H3K4me3—are known to be recruited to a functional
PIC (Black et al. 2006; Wu et al. 2008; Kim et al. 2009).
However, it is unknown whether the PIC or its associated
factors control the recruitment of effector proteins that
bind to H3K4me3.
The SET1 complex is responsible for the majority of
H3K4me3 in yeast and mammalian cells (Ruthenburg
et al. 2007; Wu et al. 2008). In budding yeast, recruitment
of the SET1 complex (termed COMPASS) by PAF1, in
combination with ubiquitinylation of H2B by Rad6-Bre1,
leads to H3K4me3 (Wood et al. 2003; Kim et al. 2009).
However, in mammals and other organisms, H3K4me3 is
found at some gene promoters that have not yet generated
productive transcript (Bernstein et al. 2006). Studies have
shown that several DNA sequence-specific transcription
factors associate directly with ASH2L, a core subunit of
It is conceivable that SET1 might be recruited to a gene by
such factors under conditions in which the gene is not
The majority of H3K4me3-binding domains can be found
in effector proteins associated with chromatin modification
and remodeling (Ruthenburg et al. 2007). Chd1 (chromodo-
main helicase DNA-binding protein 1) is an H3K4me3-
associated chromatin remodeler that is required for the
expression of developmentally essential genes in mouse
embryonic stem (ES) cells (Gaspar-Maia et al. 2009).
However, although H3K4me3 is present across a large
subset of genes, occupancy of mouse Chd1 correlates
primarily with active gene promoters that display enrich-
ment of Pol II. Human CHD1 has been reported to deposit
the histone variant H3.3 in vivo (Konev et al. 2007). The
CHD1 complex has been shown to associate with the
PAF1 elongation complex in Saccharomyces cerevisiae
(Warner et al. 2007). However, in S. cerevisiae, while
CHD1 is capable of remodeling promoter nucleosomes,
it does not exhibit preferential binding to H3K4me3
chromatin (Ehrensberger and Kornberg 2011). Thus, in
higher eukaryotes, CHD1 is a model H3K4me3 effector
used in developmental decisions and elsewhere; its en-
hancement of gene expression is linked to both transcrip-
tion and H3K4me3. Since in vitro studies have demon-
strated that the CHD1 double chromodomains bind
H3K4me3 nucleosomes and tail peptides with seemingly
et al. 2010), an important question is why Chd1 is found
only at actively transcribed H3K4me3 genes and not at
To investigate the biochemical mechanisms by which
H3K4me3 effectors function in a transcription- and
chromatin-specific context, we used multidimensional
protein identification technology (MuDPIT) proteomics
in conjunction with immunoblotting to identify factors
found to be enriched in the context of a PIC assembled
on chromatin in vitro. Proteomic techniques have been
previously used to successfully analyze PICs on nonchro-
matin templates in yeast (Ranish et al. 2003). Surprisingly,
we found that binding of CHD1 was stimulated by the
activator on unmodified chromatin templates but was
enhanced on methylated templates. CHD1 was observed
to also participate in higher-order protein complex in-
teractions with components of the transcription machin-
ery located at or downstream from the start site and was
a prime candidate for further mechanistic investigation.
Using the immobilized chromatin template assay, we
combined PIC-stimulated recruitment with functional
transcription assays. Our studies show that the specific
recruitment of CHD1 to an active gene is achieved by
linking it to assembly of an active transcription complex
via the action of Mediator.
H3K4me3 stimulates transcription in vitro
To study the mechanism by which H3K4me3 contributes
to active gene transcription, we used the methods de-
veloped by Shokat and colleagues (Simon et al. 2007) to
ical analysis. In this method, Lys 4 of histone H3 was
mutated to a cysteine to generate H3K4C (Fig. 1A).
Recombinant H3K4C was reacted with an ethylamine
substrate [(2-bromoethyl) trimethylammonium bromide]
to generate the methyl-lysine analog (MLA) of H3K4me3.
The MLA was validated by immunoblotting with an
antibody to H3K4me3 (Fig. 1A) and quantitated by electro-
spray ionization mass spectrometry, which revealed that
66%ofH3 was modified (datanot shown).Asshowninthe
Figure 1B schematic, unmodified naive or synthetically
methylated H3was assembledintooctamersandtheninto
chromatin on biotinylated DNA templates containing a
of chromatin template were first normalized based on the
extent of chromatin assembly as shown in Figure 1B.
Subsequently, in vitro transcription was performed in
HeLa nuclear extracts on equivalent amounts of chro-
matin. The data revealed stimulation by the activator
GAL4-VP16 on the H3K4me3 versus the naive unmod-
ified templates (Fig. 1C). Multiple repeats were quanti-
tated, graphed, and subjected to statistical analysis to
reveal that the stimulation was indeed reproducible and
significant (Fig. 1D). This result demonstrated an ability
to recreate, in a cell-free system, the transcription stimu-
lation imparted by H3K4me3 and offered the possibility
of generating mechanistic insights into its function. We
addressed the molecular basis for this stimulation by first
analyzing the composition of the PICs formed via immo-
bilized template assays.
Activator-mediated recruitment of CHD1
The immobilized template assay is a powerful method for
understanding the mechanism of gene activation in vitro
because it permits a comparison of transcriptional activity
CHD1 is recruited to the PIC
GENES & DEVELOPMENT2199
with the composition of the PICs. In a typical experiment,
biotinylated chromatin templates are attached to para-
magnetic streptavidin-coated beads and incubated either
in nuclear extract or with pure proteins. After washing the
beads,theproteinscaptured bythe templateare eluted and
analyzed by immunoblotting or MuDPIT.
Supplemental Figure 1A shows a typical time-course
experiment on naive chromatin in which we determined
the optimal conditions for chromatin binding of various
GTFs, coactivators (Mediator and TAFs), and complexes
associated with H3K4me3, including PAF1 and SET1.
Immunoblotting for each subunit of every transcription
factor is nearly impossible given the large number of
polypeptides constituting the PIC. We therefore chose
specific subunits to represent various factors within the
PIC. The activator recruited the Mediator with the fastest
kinetics as previously reported (Black et al. 2006). Impor-
tantly, we observed activator-stimulated recruitment of
the PAF1 and SET1 complexes, although they bound
more slowly than Mediator. The coordinated recruit-
ment of PAF1, SET1, and the PIC was expected based on
Shilatifard’s previous studies in yeast (Wu et al. 2008;
Kim et al. 2009), with the caveat that PAF1 and SET1
bound in the absence of transcription, whereas cotran-
scriptional phosphorylation of Ser 5 in the Pol II
C-terminal domain (CTD) is necessary for PAF1 binding
in yeast. Immunoblotting revealed that no detectable Ser 5
phosphorylated Pol II was observed bound to the immo-
bilized template in the absence of ATP (Supplemental
Fig. 1B; data not shown). This result was consistent with
numerous studies showing that phosphorylated Pol II
does not join the PIC (Drapkin et al. 1994).
To more thoroughly interrogate and compare the com-
ponents of PICs formed on naive and H3K4me3 chroma-
tin, we scaled up the immobilized template reactions and
performed MuDPIT analysis. We compared two primary
criteria in our analysis—the proteins whose binding
appeared to be stimulated by activator, and those that
were further enriched by H3K4 methylation. Our goal was
to identify H3K4me3-enriched factors that interact with
the PIC and participate in transcription initiation on
chromatin. PIC-related complexes detected in the MudPIT
data are summarized as a table in Figure 2A. (The anno-
tated data set with accompanying graphs representing
peptide spectra focused on transcription/chromatin
proteins are shown in Supplemental Table 1 and Supple-
mental Fig. 2A–D.) Relative enrichment of specific PIC
components was calculated by average NSAF (normalized
spectral abundance factor) for all subunits of a complex
and ranked by color. A gradient of red to yellow to light
blue represents proteins ranked in the 90th to 20th percen-
tiles, while values shown in solid blue represent proteins
ranking below the 20th percentile. The most enriched
factor was Mediator. The Mediator is a direct target
of VP16 (Uhlmann et al. 2007), and its recruitment by
activator represented a validation of the overall approach
(Fig. 2A). Indeed, for most of the Mediator subunits, the
MuDPIT data indicated that no binding occurred in the
absence of activator. Among other transcription factors,
the number of peptides decreased substantially, but Pol II,
the known or suspected H3K4me3 effector proteins, CHD1
binding was strongly stimulated by activator on naive
templates. CHD1 was further enriched on methylated tem-
plates in the absence of activator. In addition, we observed
activator-stimulated enrichment of the PAF complex. The
PAF1 complex is necessary for recruitment of the SET1
complex COMPASS inyeast (Kroganetal.2003;Wood etal.
2003). CHD1 has been reported to associate with PAF1 in
HeLa extracts (Sims et al. 2007; Warner et al. 2007).
Despite the fact that our approach detected activator-
stimulated recruitment of proteins like Mediator, the
spectral counting-based quantitation in MuDPIT is not
ideal for detecting small differences in abundance as
compared with other mass spectrometry techniques like
SILAC (Bartke et al. 2010). We therefore used immuno-
blotting to validate candidates chosen for further mecha-
nistic analysis. Figure 2B shows a recruitment time course
of CHD1 versus the Mediator and TFIID on both naive
and H3K4me3 templates. Quantitation of the blots from
nuclear extract. (A) Schematic of the trimethyl-lysine analog
synthesis at histone 3 Cys 4. Below the schematic is a Western
blot showing the specific detection of H3K4me3 MLA by
antibody (Abcam anti-K4me3). (B) Schematic of the immobi-
lized chromatin template and chromatin normalization. The
extent of chromatin assembly was monitored by EMSA; equiv-
alent amounts of chromatin were used in all experiments. (C)
Schematic and autoradiograph of in vitro transcription as
detected by primer extension on unmodified (Naive) and syn-
thetic H3K4me3 chromatin templates in the presence and
absence of the activator GAL4-VP16 and acetyl-CoA (AcCoA)
using HeLa nuclear extract. (AcCoA was necessary for optimal
levels of transcription.) (D) Signal quantitation and statistical
analysis of the stimulation by H3K4me3. Three repeats of the
transcription experiment were quantitated using Imagequant
TL software, graphed, and subjected to a Student’s t-test as
a measure of statistical significance between naive unmodified
and H3K4me3 chromatin.
H3K4me3 stimulates in vitro transcription from
Lin et al.
2200 GENES & DEVELOPMENT
multiple experiments revealed that the amount of Media-
tor remained roughly constant between naive versus
H3K4me3 chromatin (Fig. 2C). In contrast, the activator
stimulated a significant twofold to threefold increase
with CHD1 on the H3K4me3 chromatin, with the effect
being most evident at the 60-min time point. Moreover,
CHD1 bound with a slightly higher affinity to the
H3K4me3 versus naive templates in the absence of acti-
vator, although not to the extent observed in the MuDPIT
analysis (Fig. 2B,C).The data suggestthat the activator and
distinguish between the effect of chromatin and the effect
of activator on the recruitment of CHD1, we compared its
binding on chromatin and naked DNA templates. The
data in Supplemental 2E show a significant increase in
CHD1 binding in response to activator even on naked
DNA templates. These data imply that the recruitment of
CHD1 might be linked directly to PIC assembly.
The role of Mediator in recruitment of CHD1
Biochemical analysis of PICs assembled in vitro revealed
a dual requirement for the coactivators TFIID and Medi-
ator to achieve efficient binding of the GTFs and Pol II
(Johnson and Carey 2003). To assess the role of the
coactivators in recruitment of CHD1, we prepared nuclear
extracts depleted of either TFIID or Mediator. Our immu-
nodepletion protocol removed ;90% or more of the
Mediator and TFIID but did not significantly affect the
levels of CHD1, other H3K4me3-related proteins, or Pol II
(Fig. 3A; Supplemental Fig. 3A). We found that activator-
dependent CHD1recruitmentwas significantly reduced in
immobilized template assays performed in the Mediator-
depleted extracts on both chromatin and naked DNA
(Fig. 3B,C). Unlike Mediator, immunodepletion of TFIID
had little effect on recruitment of CHD1 (Supplemental
Fig. 3B). Our data suggest that the Mediator is required
for recruitment of CHD1. Importantly, the Mediator
dependence of CHD1 recruitment observed on naked
DNA reinforces the idea that chromatin need not be
present for CHD1 recruitment to PICs. The results
suggest that Mediator coordinates the recruitment of a
key chromatin remodeling enzyme with PIC assembly.
These data reinforce the findings in Figure 2 and provide
a basis for how Chd1 is preferentially localized to active
genes in mouse ES cells.
Mediator stimulates CHD1 function
We took two approaches to determine the role of Mediator
in recruiting CHD1. First, Mediator was immunoaffinity-
purified from HeLa cell lines expressing Flag-tagged Med
29 and used to supplement our Mediator-depleted extract
in add-back experiments to rescue CHD1 recruitment.
Second, we expressed and purified Flag-CHD1 from the
baculovirus system and used MuDPITanalysis to identify
other factors that were consistently associated with
CHD1, albeit in low abundance, in solution in HeLa
extracts. This is a standard proteomic approach for iden-
tifying candidate proteins that might interact in the
context of a PIC. The caveats in such analyses are that
associated factors are usually present in substoichiomet-
ric amounts in solution, and the technology does not
distinguish between direct and indirect associations.
We found that adding back Mediator, purified under
high-stringency conditions, to Mediator-depleted extract
PIC. (A) MuDPITanalyses were performed on PICs
assembled on chromatin arrays: a chart of PIC
components, H3K4me3 effector proteins, and
other factors recruited. Average enrichment in
the presence of GAL4-VP16 and further enrich-
ment on H3K4me3 chromatin are shown as a heat
map from average NSAFe5 values for the factors in
the complex. Average NSAFe5 values for each
complex are ranked by percentile in a three-color
gradient from high (red) to medium (yellow) to low
(blue) as shown. (B) Time courses of PIC recruit-
ment in HeLa extracts were compared between
Naive and H3K4me3 templates by immunoblot-
ting to validate enrichment of select factors as
determined by the MuDPIT screen. (C) Quantita-
tion and statistical analysis of Western blot signals
for CHD1 and Mediator enriched on H3K4me3
versus Naive chromatin. Signals were normalized
to VP16. Student’s t-test was used to calculate the
statistical significance of the differences at the
60-min time point. Assays were performed in
H3K4me3 effectors are recruited to the
CHD1 is recruited to the PIC
GENES & DEVELOPMENT2201
was able to rescue CHD1 recruitment to the immobilized
template on naked DNA (Fig. 4A). Quantitation of
triplicate repeats, normalized to VP16 activator binding,
revealed that restoration of CHD1 recruitment in Medi-
ator-depleted extract is reproducible and significant. One
prediction of the hypothesis that CHD1 somehow at-
taches to the PIC is that CHD1 should interact with PIC
components. To test this hypothesis, recombinant CHD1
was incubated with HeLa nuclear extracts in 100 mM
KCl using lower and higher DNase/heparin conditions
(see the Materials and Methods) and then repurified at
high salt (300 mM NaCl) to examine interacting proteins.
MuDPIT analysis of proteins bound to CHD1 revealed
subunits from the Mediator and other PIC components
present in the lower heparin conditions (Fig. 4B; Supple-
mental Table 2A). These components included the SEC,
TFIID, TFIIH, INO80, and the SET1 and PAF1 complexes.
TFIIH, PAF1, and FACT complexes were still detected
at the higher heparin conditions (Fig. 4B; Supplemental
Table 2B) and may possibly help bridge the CHD1–
Mediator interaction. Nonetheless, the observation that
CHD1 pull-down experiments mainly detect components
of the PIC and associated chromatin remodeling factors
adds additional strength to the idea that CHD1 is indeed
a component of the PIC bound at a promoter.
CHD1 is necessary for efficient transcription in vitro
We next focused on the function of CHD1 at a promoter.
To establishthatrecombinantCHD1from the baculovirus
system is functional, we performed a chromatin remodel-
ing assay. In the presence of ATP, CHD1 is active in the
in vitro remodeling assay, as illustrated by the shift in
mobility of the nucleosome on a gel upon CHD1 treat-
ment (Fig. 5A). Additionally, the remodeling is signifi-
cantly more pronounced on H3K4me3 chromatin. This
result agrees with the generally accepted view that one
role of H3K4me3 is to enhance CHD1 remodeling of
nucleosomes (Petesch and Lis 2008).
To determine whether CHD1 contributes to transcrip-
tional activation on H3K4me3 chromatin, we prepared
extracts depleted of the protein using a native CHD1
antibody. Using this method, we depleted CHD1 from the
HeLa extracts by >90% (Fig. 5B). The in vitro transcrip-
tion experiment in Figure 5C shows that depletion of
CHD1 decreases transcriptional activation, whereas ad-
dition of recombinant CHD1 restores the normal level
of activated transcription. The effect is specific to the
CHD1-depleted extracts, as additionofCHD1 tothe mock-
depleted extracts has no additional effect on transcription
and even inhibits at high concentrations (Supplemental
Fig. 4). Note that CHD1 depletion reduces but does not
abolish transcription activation on the H3K4me3 chroma-
tin template. This result could indicate that CHD1 is not
absolutely required for, but does strongly contribute to,
H3K4me3 chromatin transcription in our system. Collec-
tively, the data in Figure 5, A–C, establish that CHD1 is
active in remodeling and necessary for efficient activated
One possibility is that CHD1 recruitment to the PIC
through the Mediator facilitates Pol II initiation on
chromatin. If so, this could be a rate-limiting step that
would be overcome by prebinding the Mediator and
CHD1 to the template in the presence of ATP prior to
adding HeLa extract and nucleotides. First, we tested
binding of purified CHD1 and Mediator to chromatin.
Figure 5D quantitates an immobilized template assay
showing that purified CHD1 binds better in the presence
of Mediator. Experiments on both naive unmodified and
of CHD1 is further enriched on the H3K4me3 chromatin
(Fig. 5D). We cannot say that this is a direct interaction, as
neither protein is completely pure. Next, we tested the
effect of prebinding Mediator and CHD1 on transcription
of H3K4me3 templates. The data in Figure 5E establish
that preincubation of Mediator and CHD1 with the
chromatin template stimulates transcription in an ATP-
dependent manner. Preincubation of Mediator and CHD1
in the absence of ATP failed to elicit a stimulatory effect
within the short 10-min time frame of the in vitro
transcription experiment. This result implies that the
ATP-dependent activity shown in Figure 5A is necessary
for transcription. We conclude that CHD1 enhances
PIC. (A) Immunoblot of Mediator subunits and control proteins
in Mediator-depleted (DMediator) and mock-depleted (Mock)
nuclear extract loaded in threefold steps. (B) Immunoblot (left
panel) and quantitation (right panel) comparing CHD1 recruit-
ment time courses in an immobilized template assay (IT) on
H3K4me3 chromatin in Mock versus DMediator extracts. West-
ern blot signal was quantified using LiCOR imaging software,
and the statistical significance between Mock- and Mediator-
depleted extracts was calculated using Student’s t-test. Signals
were normalized to VP16. (C) CHD1 recruitment in Mock-
versus Mediator-depleted extracts repeated using naked DNA
Mediator plays a key role in CHD1 recruitment to the
Lin et al.
2202 GENES & DEVELOPMENT
transcription in a Mediator- and ATP-dependent man-
ner (Esnault et al. 2008; Boeing et al. 2010).
Genomic Chd1 binding overlaps with Mediator in vivo
Our hypothesis is that CHD1 binding to the PIC is
controlled by the Mediator, possibly through bridging
factors. CHD1 can then act on the H3K4me3 nucleosomes
that are found at gene promoters. This hypothesis predicts
that CHD1 occupancy would correlate with Mediator
predominantly at active genes. To address this issue, we
first examined the recruitment of CHD1 along with Medi-
ator in a U2OS cell line bearing a doxycycline-inducible
TetR-VP16-activated reporter gene (Fig. 6A). CHD1 re-
cruitment closely follows Mediator recruitment in a time
course of doxycyclineinduction at the promoter, but is not
observed at the 39 end of the reporter gene at the time
points tested (Supplemental Fig. 5A). These data suggest
that Mediator and CHD1 join the VP16-stimulated PIC as
it is assembled in vivo.
We next examined two previously published genome-
wide data sets in mouse ES cells (Gaspar-Maia et al. 2009;
Sridharan et al. 2009; Kagey et al. 2010). We analyzed
binding eventsfor the Med1 Mediatorsubunitonthe same
promoter regions that were analyzed on promoter arrays
for Chd1 and H3K4me3. Previous studies have reported
that Chd1, which is required for ES cell self-renewal,
localizes to only 12% of the promoter regions of bivalent
genes and is highly enriched at active H3K4me3 genes.
We found a significant genome-wide overlap for Chd1,
Med1, and H3K4me3 (Fig. 6B; Supplemental Fig. 5B,C).
As shown previously, Chd1 localizes predominantly to
promoters occupied by H3K4me3. This correlation is
even stronger when bivalent promoters are excluded from
the pool of H3K4me3-positive promoters (Supplemental
Fig. 5B). Similarly, Med1 occupancy is significantly re-
duced across bivalent promoters (Supplemental Fig. 5C).
These data imply that the mechanism to ‘‘poise/prime’’
bivalent genes for future activation is not PIC-dependent.
Although not all Med1-bound, H3K4me3-occupied genes
are Chd1 targets, both Med1 and H3K4me3 are found at
the vast majority of Chd1-bound promoters. Binding of
Med1 and Chd1, along with H3K4me3, is indicative of
highly active transcription as shown in Figure 6C (see
also Supplemental Fig. 5D). We suggest, based on the
data above, that the Mediator coordinates H3K4me3-
stimulated initiation of transcription by Chd1.
Med1 is required for coimmunoprecipitation
of Mediator and CHD1
One problem with studying cross-talk between Mediator
and CHD1 in cells is that indiscriminate disruption of the
Mediator is likely to have widespread indirect conse-
quences. It was therefore necessary to identify specific
Mediator subunits that might be involved in the re-
cruitment. Using in vitro transcription and translation,
we synthesized individual Mediator subunits and studied
their affinity for Flag-CHD1 bound to the Flag antibody
resin (Supplemental Fig. 6). Inthis typeof analysis, binding
does not necessarily indicate a direct interaction because
transcription factors are known to be present in reticulo-
cyte lysates and the baculovirus-synthesized CHD1 may
contain contaminants from insect cells. Nevertheless, this
using purified complexes. (A) Immunoblot analysis
of CHD1 recruitment to naked DNA templates in
highly purified Mediator 6 activator. Mediator was
normalized to levels found in the HeLa nuclear
extract used in Figures 2 and 3. The top panel is
a representative immunoblot of the Mediator com-
plementation experiment, while the bottom panels
are graphs analyzing triplicate experiments. Quanti-
tation was performed using LiCOR imaging software.
Signals were normalized to VP16. (B) Chart of PIC-
related complexes found associated with recombinant
Flag-tagged human CHD1 incubated with HeLa nu-
clear extract at higher and lower heparin conditions.
Average NSAFe5 values for each complex are ranked
by percentile in a three-color gradient from high (red)
to mid (yellow) to low (blue) as shown. Note that the
cutoff values differ from the chart in Figure 2.
Characterization of CHD1 recruitment
CHD1 is recruited to the PIC
GENES & DEVELOPMENT2203
approach represents the most logical way to determine
which Mediator subunit is involved in recruitment of
CHD1. Our assay identified Med1 as the subunit with
the highest affinity among the 29 subunits tested. This
result, however, does not preclude the possibility that
other Mediator subunits may also be involved.
Med1 was then knocked down in 293Tcells along with
a control subunit, Med23, using lentiviruses encoding
shRNAs targeting the two subunits. Both Med1 and
Med23 are known to be targeted by specific activators
in vivo (Yuan et al. 1998; Boyer et al. 1999). Mediators
lacking Med1 and Med23 have been isolated (Ito et al.
2000; Stevens et al. 2002). Thus, disruption of Med1 or
Med23 is unlikely to have a strong impact on Mediator
structure. We then performed coimmunoprecipitation
experiments with CHD1 as bait. We chose Med6, Med7,
and Med14 as subunits indicative of the three known
structural modules of Mediator (Conaway et al. 2005). The
data in Figure 7 show that CHD1 coimmunoprecipitated
with Mediator subunits in extracts from cells treated with
the virus alone and with the Med23 shRNA knockdown
virus but not in the Med1 shRNA knockdown. The data
suggest that select disruption of the Mediator can abolish
its ability to coimmunoprecipitate with CHD1.
Our data support the concept that activator-stimulated
PICassembly isphysicallyand mechanistically linked with
the subsequent effects of H3K4me3. The use of MuDPIT
and immunoblotting to interrogate the PICs formed on
chromatin, coupled with the purification and analysis
of CHD1, allowed us to determine that the Mediator
ation. (A) Autoradiograph of nucleosome remodeling assay
demonstrating that recombinant CHD1 is enzymatically active
on Naive and H3K4me3
templates. (B) Western blot of CHD1-depleted nuclear extract
and a silver-stained gel of purified recombinant Flag-CHD1
expressed in baculovirus. (C) Autoradiograph of in vitro tran-
scription primer extension assays using CHD1-depleted nuclear
extracts as shown. Transcription was compared between CHD1-
depleted (DCHD1) and mock-depleted nuclear extracts and
rescued with the addition of purified CHD1 as shown. Signals
were quantitated using Imagequant TL and normalized to
Mock-treated + activator. (D) Graph showing CHD1 recruit-
ment to H3K4me3 versus Naive immobilized chromatin tem-
plates with purified Mediator and activator. Trace levels of
CHD1 were detected by immunoblotting in the purified Medi-
ator alone. CHD1 recruitment in each lane was normalized to
recruited Mediator. (E) Levels of transcription were measured in
a rate-limiting assay performed as illustrated by the schematic.
H3K4me3 chromatin templates were preincubated with activa-
tor, Mediator, and CHD1 as indicated. After 30 min, unbound
protein was removed, and buffer with or without ATP was
added. After a further 30-min incubation, the buffer was re-
moved and beads were suspended in transcription buffer con-
taining HeLa nuclear extract and nucleotides. After 10 min, the
transcription reaction was subjected to primer extension anal-
ysis to measure mRNA. Signals were quantitated using Image-
quant TL and normalized to the signal in lane 6.
Characterization of CHD1 during transcription initi-
32P-labeled 601 mononucleosomal
analysis of doxycycline-induced enrichment of VP16, Med1-
containing Mediator, CHD1, and Pol II on a stably integrated
doxycycline-inducible Luciferase reporter in U2OS cells during
a time course. (B) Venn diagram showing distribution and overlap
of genes for Chd1 and Med1 binding and H3K4me3 (excluding
H3K27me3) occupancy across the mouse ES cell genome.
P-values are as follows: Chd1-Med1, P-value = 1.42 3 10?216;
Chd1-H3K4me3 (excluding H3K27me3), P-value < 9.34 3 10?322;
Med1-H3K4me3 (excluding H3K27me3), P-value < 1.05 3 10?321.
(C) Box plot of gene expression levels of Chd1-positive, Med1-
positive, and H3K4me3 (excluding H3K27me3)-positive genes
and all other genes, including singly and doubly bound as well
as unbound genes. Chd1-, Med1-, and H3K4me3-cobound genes
show an overall higher level of transcription. For triple-positive
versus all other genes: D = 0.4276, P-value < 2.2 3 10?16.
Chd1 and Mediator binding correlate in vivo. (A) ChIP
Lin et al.
2204GENES & DEVELOPMENT
coordinates assembly of the PIC and recruitment of CHD1.
Our data provide a basis for the specificity of CHD1 for
Our major finding was that CHD1 binding was largely
coupled to activator-mediated PIC assembly but was also
weakly stimulated by H3K4me3 alone. Indeed, the PIC
and H3K4me3 together caused the most efficient recruit-
ment of CHD1. Previous studies have shown that CHD1
binds to H3K4me3, an observation highlighted by recent
proteomic analyses (Sims et al. 2005; Bartke et al. 2010;
Vermeulen et al. 2010). However, this event alone seems
to contribute only a small amount of CHD1 binding to an
active gene. Indeed, recent studies have demonstrated
that in Drosophila, the CHD1 chromodomains, which
recognize H3K4me3, are not required for its colocalization
to active genes (Morettini et al. 2011). Moreover, Kornberg
and colleagues (Ehrensberger and Kornberg 2011) have
recently found that S. cerevisiae CHD1, whose chromo-
domains do not recognize H3K4me3, selectively removes
nucleosomes at the promoter in vitro and in vivo in an
activator-dependent manner. In mammalian cells, the
close correlation between localization of Mediator, Chd1,
and H3K4me3 and active transcription in mouse ES cells
reinforces the notion that the major mechanism for Chd1
localization is PIC assembly. Dual interactions between
the transcriptional machinery and the SET1-catalyzed
H3K4me3 modification reinforce the specificity of CHD1.
Among our findings was that the SET1 complex is also
recruited to the PIC (as shown in Supplemental Fig. 1A).
It has been reported that some activators interact with
subunits of the SET1 complex and that some promoters of
inactive genes are bound by activators. CHD1, however,
is found only at genes that are actively transcribed. In-
active genes are typically di- or trimethylated at H3K9 or
H3K27. Hence, bivalent domains might be a natural by-
product of PIC absence. In such cases, where full PIC
assembly might be limited by an incomplete complement
of activators, CHD1 occupancy may be prevented by both
the absence of Mediator and the presence of repressive
complexes like PRC1 or HP1 that bind the modifications
that correlate with inactive genes.
MuDPITanalysis of proteins bound to CHD1 led to the
discovery that it associates with several proteins normally
found near or immediately downstream from the start
site, including the PAF1 complex, INO80, P-TEFb, NELF,
TAFIIs, TFIIH, and Mediator, among others. P-TEFb re-
leases the stalled Pol II found 30–50 bp downstream from
many genes by phosphorylating DSIF and NELF (Peterlin
and Price 2006). Similarly, numerous TAFIIsubunits and
the TFIIH XPB subunit cross-link immediately down-
stream from the start site in vitro (Dvir et al. 2001). These
data, along with the observation that many genes contain
a nucleosome positioned near the start site (Schones et al.
2008), reinforce the notion that CHD1 action is function-
ally coupled to regulatory events associated with the pro-
INO80 is unclear, but studies have implicated this ATP
remodeling complex in promoter-dependent transcription
(Cai et al. 2007). We also found small amounts of PAF1
associated with CHD1 as previously observed by others,
suggesting the possibility that the two factors interact
directly (Sims et al. 2007; Warner et al. 2007).
Analysis of higher-stringency CHD1 pull-downs sug-
gested that among the interacting proteins, TFIIH has
a high affinity (Fig. 4B). TFIIH has previously been shown
to bind Mediator in work done by others (Esnault et al.
2008; Boeing et al. 2010). This suggests that TFIIH may
contribute to the docking of CHD1 with Mediator. Indeed,
our unpublished immobilized template data support a
Mediator–TFIIH–CHD1 interaction. However, it remains
a possibility that this may be enabled by factors copurify-
ing with Mediator or with CHD1 from the insect cell
expression system. These issues notwithstanding, our data
suggest that Mediator controls CHD1 recruitment.
The idea that the Mediator serves as a platform for other
proteins and conveys signals to the general transcription
machinery was originally proposed by Young (Chao et al.
1996). The concept that Mediator coordinates events at
chromatin is emerging and makes sense given that the
machineries coevolved in almost all eukaryotes. In addi-
tion to Shilatifard’s work on COMPASS (Krogan et al.
2003; Wu et al. 2008), a previous study in our laboratory
(Black et al. 2006) linked p300-mediated acetylation with
PIC assembly. We alsoobservedinour MuDPITanalysis of
PICs the enrichment of other chromatin factors—such as
GCN5, NuA4, and other CHD family members—in an
activator-stimulated fashion. Most importantly, Mediator
has been linked directly to recruitment of Pol II SEC via
the Med26 subunit (Takahashi et al. 2011). It would be
interesting if this event was linked to the action of CHD1
at the start site. Boyer and colleagues (Ding et al. 2008)
have shown that the connectivity also extends to silencing
factors, as G9a links directly with the Cdk8 module of the
Our current view of the protein–protein interactions
identified by proteomic and mechanistic studies is that
CHD1 is recruited only to active genes by its interaction
with the PIC via the Mediator. Activators are known to
interact directly with Mediator and TFIID. The VP16
activation domain docks with Mediator via the MED25
subunit (Yang et al. 2004; Uhlmann et al. 2007) and with
TFIID via TAF9 (Goodrich et al. 1993). VP16 is known to
recruit both complexes to DNA in vitro and in vivo (Berk
vivo. Immunoblots showing specific shRNA knockdown of
Med1 and Med23 subunits of Mediator in 293Tcells (left panels)
and Med1-dependent coimmunoprecipitation of Mediator by
targeted immunoprecipitation of CHD1 (right panel). Subunits
representing the head (Med6), middle (Med7), and tail (Med14)
modules of Mediator are blotted.
CHD1 associates with Med1-containing Mediator in
CHD1 is recruited to the PIC
GENES & DEVELOPMENT2205
et al. 1998). TFIID and Mediator also interact to form
a coactivator complex (Johnson and Carey 2003), which is
necessary in vitro for binding of the GTFs and Pol II. Pol II
binds tightly to the active form of Mediator in a some-
what mutually exclusive manner with the Cdk8 module
(Paoletti et al. 2006).
In conclusion, our data suggest that the Mediator not
only serves as a docking platform for activator-stimulated
PIC assembly of the GTFs, but coordinates the recruit-
ment of CHD1 during active transcription as well. As the
Mediator signifies active genes, the specificity of CHD1
recruitment can best be described as cooperative protein–
protein interactions between the PIC, CHD1, and
Materials and methods
Methyl-lysine histone octamer preparation
Lys 4 of histone H3 was mutated to a cysteine by site-directed
mutagenesis of Xenopus H3.1 bearing a C110A mutation,
expressed and purified from Escherichia coli inclusion bodies,
and subjected to chemical alkylation by (2-bromoethyl) trime-
thylammonium bromide (Simon et al. 2007) before assembly
into histone octamers (Luger et al. 1997).
A 602-bp biotinylated PCR fragment that directly encompasses
G5E4T (Johnson and Carey 2003) was assembled into chromatin
by salt dilution as described previously (Steger et al. 1997) and
was validated by EMSA in native PAGE. Chromatin was immo-
bilized on M280 streptavidin beads (Dynal) in chromatin-binding
buffer (20 mM HEPES at pH 8.0, 150 mM KCl, 10% glycerol, 4
mM MgCl2, 1 mM DTT, 200 mg/mL BSA).
Immobilized template recruitment assay
The 40-mL immobilized template recruitment assays contained
80 mg of HeLa nuclear extract, and 50 ng of chromatin or naked
DNA template in immobilized template binding buffer (120
mM KCl, 10 mM HEPES at pH 8.0, 5% glycerol, 0.2 mg/mL BSA,
0.05% NP-40). After the indicated time periods, the beads were
washed three times in immobilized template buffer. Captured
PICs were incubated with 500 mM ATP in immobilized tem-
plate binding buffer where indicated in Supplemental Figure 1B
for Pol II phosphorylation assays. Bound protein was eluted in
10 mL of 23 Laemmli buffer, fractionated by SDS-PAGE, and
immunoblotted. For proteomic analysis, the 1-h time point was
scaled up and subjected to MuDPIT. Antibodies used in immu-
noblotting included MED23 (BD Pharmingen), Pol II CTD
8WG16 (QED Bioscience), TFIIB (Tantin et al. 1996), WDR5
(Upstate Biotechnology), ASH2L, RBBP5, PAF1, and CHD1
(Bethyl Laboratories). All other antibodies were purchased from
Santa Cruz Biotechnologies.
Extract and protein preparation
HeLa nuclear extract (Dignam et al. 1983) and GAL4-VP16 were
prepared as previously described. Immunodepletion of TFIID
from 1 mL of HeLa nuclear extract was performed using 200 mg
each of antibodies against TAF4, TAF1, and TAF3 (Santa Cruz
Biotechnologies). Mediator was depleted from 1 mL of extract
CHD1 was depleted with an antibody against CHD1 (Bethyl).
Antibodies were cross-linked to protein A and G paramagnetic
beads (Invitrogen) using 20 mM dimethylpimelimidate in 0.1 M
sodium borate buffer (pH 9) and washed extensively with 50 mM
glycine (pH 2.5). The cross-linked beads were equilibrated in buffer
D (20 mM HEPES at pH 7.9, 0.1 mM EDTA, 20% glycerol, 0.1 M
KCl) and incubated with HeLa nuclear extract in buffer D for 4 h
at 4°C. The supernatant was isolated and used for immobilized
template analysis as described above.
Recombinant Flag-tagged human CHD1was purified from SF9
cells using a baculovirus overexpression system (Invitrogen).
Briefly, cells were resuspended in 0.3 M buffer F (0.3 M NaCl,
20% glycerol, 20 mM HEPES at pH 7.9, 4 mM MgCl2, 0.2%
Triton X-100, 0.1% NP-40) and sonicated. Lysates were then
treated with DNase I (1 U/mL) and heparin (12.5 mg/mL) and
cleared by centrifugation at 30,000g. The resulting lysate was
bound to M2 anti-Flag resin (Sigma), washed extensively in 0.5 M
buffer F, and eluted using 33 Flag peptide (0.25 mg/mL; Sigma).
Mediator was purified from HeLa cells expressing Flag-tagged
human Intersex (Med29) as described previously (Sato et al.
2003). The HeLa Intersex cell line was a gift from Joan and Ron
MuDPIT analysis of immobilized templates
and purified proteins
For MuDPIT analysis, the equivalent of 300 immobilized tem-
plate reactions were pooled together. Samples were eluted in 50
mM Tris (pH 8.0) and 6 M urea. For CHD1, ;250 mg of Flag-
CHD1 was immobilized on Flag antibody beads and incubated
with 0.45 mL of HeLa nuclear extract in buffer containing 100
mMKCl in thepresence of either 1 U/mLDNase and 12.5 mg/mL
heparin (low stringency) or 75 mg/mL heparin and 10 U/mL
DNase I in the presence of 2 mM CaCl2(high stringency). Bound
proteins were washed in 0.3 M buffer F, and F-CHD1 and
interacting proteins were then eluted using a 33 Flag peptide
(0.25 mg/mL; Sigma). Protein samples were precipitated in 20%
TCA, washed with cold acetone, and digested with trypsin. The
digested peptide samples were then fractionated with sequential
cation exchange and reverse-phase chromatography and eluted
directly into aLTQ-Orbitrap mass spectrometer(Thermo Fisher).
MS/MS spectra were collected as described (Law et al. 2010). Data
analysis was performed with the SEQUEST and DTASelect2
algorithms and filtered with at least two peptides per protein
and a peptide-level false-positive rate of <5% as estimated by
a decoy database strategy (Law et al. 2010). NSAF values were
calculated as described in Law et al. (2010). Proteins were
assigned into complexes using the online resource CORUM
(Ruepp et al. 2008) and Mediator submodules as published
previously (Bourbon 2008).
In vitro transcription assays
The 40-mL standard reactions in Figure 1 contained 50 ng of
linear chromatin template, nuclear extract, and GAL4-VP16 as
described previously (Black et al. 2006). After 60 min, RNA was
harvested and analyzed by primer extension as described pre-
viously. Transcription of immobilized chromatin templates in
Figure 5 was carried out on 50 ng of linear H3K4me3 chromatin
templates, which were bound with saturating levels of GAL4-
VP16, followed by removal of excess activator. Templates were
then bound with saturating amounts of Mediator and CHD1, as
determined by immobilized template assays, and incubated for
30 min at 30°C in the presence or absence of ATP, after which
unbound protein was removed. Eighty micrograms of HeLa extract
and NTPs were added, and transcriptionwas allowed to proceed for
Lin et al.
2206GENES & DEVELOPMENT
10 min under standard conditions as described previously (Black
et al. 2006).
Cross-correlation of genomic chromatin immunoprecipitation
(ChIP) data sets
ChIP–chip data sets from experiments performed on mouse ES
cells were used to determine whether gene promoters were
enriched for Chd1 (Gaspar-Maia et al. 2009) and H3K4me3 or
H3K27me3 (Sridharan et al. 2009). ChIP-seq data for Med1-
occupied genes were from Supplemental Table 5 of the study by
Young and colleagues (Kagey et al. 2010). The ChIP–chip exper-
iments were performed on Agilent promoter arrays with probes
coveringtheregion?5.5 kbto +2.5 kbrelative tothe transcription
start site. Hence, only those Med1-enriched regions that had
significant peaks within this 8-kb region, based on the location of
enriched regions from Supplemental Table 4 inKagey et al. (2010),
were used for furtheroverlap analysis. The overlap of genes whose
promoters were enriched for each feature (Med1, Chd1, and
H3K4me3 without H3K27me3) was determined pairwise, and
the significance of the overlap was evaluated using the hyper-
geometric test. Box plots were used to visualize the distribution
co-occupancy by all three features as compared with those for gene
sets with different combinations of the three features in their
promoter regions or their complete absence. Gene expression
levels were obtained from Supplemental Table 2 in Gaspar-
Maia et al. (2009). A two-sample Kolmogorov-Smirnov test was
used to compare the distributions of expression values between
the two sets of genes.
shRNA knockdown of Mediator subunits
293T cells were grown to 30% confluency in 10-cm dishes and
infected with lentiviruses expressing shRNAs targeting MED23
(TTGTGAGTGTCATCAGCAGCC) and MED1 (GTCATGGA
GAAGAGGGTTGTG). After 96 h, cell lysates were prepared
and immunoblotted to determine the extent of MED1 and
MED23 knockdown using the MED6 antibody as a control.
The lysates were subjected to immunoprecipitation with a
CHD1 antibody (Bethyl Laboratories). The immunoprecipitates
were blotted with antibodies to CHD1, MED6, MED7, and
MED14 (Santa Cruz Biotechnologies) to determine the amount
ChIP from the U2OS cells, various times after doxycyline
treatment, was performed using antibodies to CHD1, MED1,
VP16, and Ser 5 Pol II (Abcam) as previously described (Black
et al. 2006).
We thank Joan and Ron Conaway for gifts of TFIIH and the Flag-
Med29 cell line, and Matteo Pellegrini for advice on statistical
analyses. This work was supported by NIH grants R01-GM74701
(to M.C.) and R01-GM089778 (to J.A.W.); funds from the Jonsson
Cancer Center at UCLA (to J.A.W.); the USPHS National Re-
search Service Award GM07104 (to G.B.); the Jonsson Cancer
Center Foundation at UCLA (to R.S.); the NIH Director’s Young
Innovator Award DP2OD001686 (to K.P.); a CIRM Young In-
vestigator Award RN1-00564 (to K.P.); and the Eli and Edythe
Broad Center of Regenerative Medicine and Stem Cell Research
at UCLA (to K.P.).
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CHD1 is recruited to the PIC
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