of 5-Hydroxymethylcytosine Marked Genes
in Embryonic Stem Cells
Ozlem Yildirim,1Ruowang Li,2Jui-Hung Hung,2Poshen B. Chen,3Xianjun Dong,2Ly-Sha Ee,3Zhiping Weng,1,2
Oliver J. Rando,1,* and Thomas G. Fazzio3,4,*
1Department of Biochemistry and Molecular Pharmacology
2Program in Bioinformatics and Integrative Biology
3Program in Gene Function and Expression
4Program in Molecular Medicine
University of Massachusetts Medical School, Worcester, MA 01605, USA
*Correspondence: email@example.com (O.J.R.), firstname.lastname@example.org (T.G.F.)
Numerous chromatin regulators are required for
embryonic stem (ES) cell self-renewal and pluripo-
tency, but few have been studied in detail. Here, we
examine the roles of several chromatin regulators
whose loss affects the pluripotent state of ES cells.
We find that Mbd3 and Brg1 antagonistically regu-
late a common set of genes by regulating promoter
nucleosome occupancy. Furthermore, both Mbd3
and Brg1 play key roles in the biology of 5-hydroxy-
methylcytosine (5hmC): Mbd3 colocalizes with Tet1
and 5hmC in vivo, Mbd3 knockdown preferentially
affects expression of 5hmC-marked genes, Mbd3
localization is Tet1-dependent, and Mbd3 preferen-
tially binds to 5hmC relative to 5-methylcytosine
in vitro. Finally, both Mbd3 and Brg1 are themselves
required for normal levels of 5hmC in vivo. Together,
our results identify an effector for 5hmC, and reveal
that control of gene expression by antagonistic chro-
matin regulators is a surprisingly common regulatory
strategy in ES cells.
scriptional regulators are essential to maintain the pluripotent
state. Several transcription factors are required for pluripotency,
including Oct4, Sox2, and Nanog, which function as ‘‘master
regulators’’ of the ES cell transcriptional network (Young, 2011).
Along with sequence-specific transcription factors, many chro-
matin regulators also play essential roles in ES cell gene regula-
tion, self-renewal,and differentiation. Several proteincomplexes
that catalyze covalent modification of histones have important
roles in ES cells, including proteins involved in histone methyla-
tion, acetylation, and ubiquitylation (Niwa, 2007; Surface et al.,
2010). Compared to most somatic cells, ES cells exhibit unusual
sition of a mark associated with active genes (H3K4me3) with
a repressive mark (H3K27me3) near the promoters of develop-
mentally regulated genes (Azuara et al., 2006; Bernstein et al.,
2006). The chromatin modifying complexes creating these
marks, MLL/SET1 Complex and Polycomb Repressive Com-
plex 2 (PRC2), respectively, are highly conserved and have
important roles in development (van Lohuizen, 1998). It has
been proposed that H3K4me3 and H3K27me3 have opposing
for future regulatory changes (Bernstein et al., 2006).
modify the spacing or subunit composition of nucleosomes,
also play important roles in ES cells (Fazzio and Panning, 2010;
Keenen and de la Serna, 2009). BAF (Brahma/Brg1 Associated
Factor) complexes are a family of ATP-dependent nucleosome
remodeling factors that share homology to yeast SWI/SNF
complex and function to both activate and silence transcription
by remodeling nucleosomes near promoters and enhancers
(Clapier and Cairns, 2009). In ES cells, a single BAF complex,
esBAF, predominates (Ho et al., 2009b). Homozygous knockout
(KO) or knockdown (KD) of any of several BAF subunits results in
defects in ES cell self-renewal and pluripotency, highlighting
their critical roles in maintaining the ES cell gene expression
pattern (Gao et al., 2008; Ho et al., 2011, 2009b; Kidder et al.,
2009; Yan et al., 2008).
Conversely, NURD (Nucleosome Remodeling and Deacety-
lase) complexes are chromatin remodeling factors that utilize
nucleosome remodeling and histone deacetylase activities
to create repressive chromatin structure (Denslow and Wade,
2007). KD or KO of the gene encoding the NURD subunit
Mbd3 in ES cells results in a defect in differentiation, as well as
altered developmental potency (Kaji et al., 2006, 2007; Zhu
et al., 2009). Mbd3 is one of four proteins named Mbd for
methyl-CpG binding domain, based on the homology of
these proteins to the methylcytosine binding domain in MeCP2
(Hendrich and Bird, 1998). However, whereas mammalian
Mbd1, Mbd2 and Mbd4 bind to cytosine-methylated substrates
in vitro, Mbd3 does not (Hendrich and Bird, 1998; Zhang et al.,
1498 Cell 147, 1498–1510, December 23, 2011 ª2011 Elsevier Inc.
1999), raising the question of what role the methyl binding
domain plays in Mbd3 biology. NURD complexes containing
either Mbd2 or Mbd3 (hereafter Mbd2/NURD and Mbd3/
NURD), or both, have been purified from mammalian cells
(Feng and Zhang, 2001; Le Guezennec et al., 2006), suggesting
these complexes may be targeted to regions of the genome with
distinct epigenetic marks.
Recently, members of the Tet family of proteins (Tet1, Tet2,
andTet3) have beenshown to carry outhydroxylation of 5-meth-
ylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC)
(Ito et al., 2010; Tahiliani et al., 2009). Knockdown of Tet1 and
Tet2 in ES cells leads to defects in differentiation (Koh et al.,
2011), while Tet1 knockdown also leads to defects in self-
renewal (Ito et al., 2010). Despite these defects in KD cells,
Tet1 KO mice are viable and fertile (Dawlaty et al., 2011).
serves as an intermediate stage in cytosine demethylation (Bhu-
tani et al., 2011), the relatively high steady-state levels of 5hmC
observed in several contexts (for example, during global ‘‘deme-
thylation’’ of paternal DNA after fertilization (Inoue and Zhang,
2011; Iqbal et al., 2011)) suggest that hydroxymethylation may
also serve a specific regulatory function, since an intermediate in
an enzymatic demethylation pathway would not be expected to
persist for hours or days. Interestingly, Mbd3 KD in ES cells has
defects similar to those of Tet1 KD ES cells, exhibiting increased
expression of some trophectoderm markers (Kaji et al., 2006;
Zhu et al., 2009). These data suggest the possibility that Mbd3
may bind hydroxymethylated regions of DNA and regulate genes
whose regulatory sequences are enriched for this modification.
Here, we investigate functional interactions among six
chromatin regulators necessary for ES cell self-renewal using
genomic expression analysis, identifying a large set of overlap-
ping targets of Mbd3 and Brg1. Genes activated by Brg1 and
repressed by Mbd3 were significantly associated with both
proteins. Mapping of Mbd3 showed that it is strongly enriched
at Polycomb target genes, and gene expression changes were
highly similar in Mbd3 KD and Suz12 KD cells, revealing that
Mbd3 plays a role in regulation of bivalent genes in ES cells.
Furthermore, we found that Mbd3 binding strongly overlaps
with Tet1 binding profiles, that Mbd3-bound genes are enriched
for DNA marked with 5hmC, and that KD of Tet1 abrogated
Mbd3 binding to its genomic targets. Mbd3 binds to DNA
harboring 5hmC, but not 5mC, in vitro, suggesting that Mbd30s
requirement for Tet1 to bind chromatin in vivo is mediated by
direct binding to 5hmC. Finally, we find that Mbd3 and Brg1
are required for normal bulk levels of 5hmC in ES cells. Together,
these data identify a novel effector function for 5hmC in vivo,
indicating that it is not simply a demethylation intermediate,
and further identify a positive feedback loop in which Mbd3 is
both dependent upon 5hmC for chromatin binding and is neces-
sary for normal levels of 5hmC within the genome.
Brg1 and Mbd3 Have Opposing Effects on Expression
of Shared Target Genes
To better understand chromatin regulation of the ES cell
transcriptional regulatory network, we sought to identify the
transcriptional targets and functional relationships among six
chromatin regulators with important roles in ES cell self-renewal
and pluripotency: Tip60, p400, Suz12 (involved in H3K27 meth-
ylation), Ash2l (involved in H3K4 methylation), Mbd3 and Brg1.
We measured genome-wide mRNA changes upon knockdown
(KD) of each factor alone or in all pairwise combinations (to iden-
S1 available online). Consistent with previous data (Fazzio et al.,
2008b), KD of Tip60 or p400, alone or in combinations with other
factors, exhibited similar changes in gene expression (Figure 1A
and Figure S1A), while Brg1 and Mbd3 KD profiles were poorly
correlated with these gene expression effects. Principal compo-
nent analysis (PCA) confirmed that KD of Tip60 or p400 elicited
the strongest changes in gene expression (Figure 1B).
Surprisingly, Brg1 KD and Mbd3 KD affected the same prin-
cipal component as one another but in opposite directions,
of a common set of targets. Indeed, the overlap of genes
misregulated upon Brg1 or Mbd3 KD was highly significant
(p < 2.2e-16). As genes that were similarly up- or downregulated
in both Mbd3 KD and Brg1 KD cells were also nonspecifically
affected by other KDs (Figure S1B), we therefore focused
on the large group of genes upregulated upon Mbd3 KD and
downregulated upon Brg1 KD (Figure 1C). We validated several
of these common Brg1/Mbd3 targets by quantitative RT-PCR
(RTqPCR) (Figure S1C), and found that expression of these
genes was also altered in independent Mbd3 or Brg1 KDs using
nonoverlapping esiRNAs (Figure S1D) and in KDs of additional
subunits of either the Mbd3/NURD or BAF complexes (Fig-
ure S1E). We next searched for epistatic effects between
Mbd3 and Brg1. Double KD of Brg1 and Mbd3 resulted in a
more wild-type mRNA profile than either single KD, indicating
that these proteins play antagonistic roles at their common
target genes (Figures 1C and Figure S1C).
What features do Mbd3/Brg1 target genes share in common?
In normal ES cells, Mbd3/Brg1 target genes are expressed
at moderate to high levels (Figure 1D). Shared targets between
these complexes were enriched for a number of functional
categories, several of which relate to cellular adhesion and
signaling (Table S2). Among the common targets of Brg1 and
Mbd3, we noted genes encoding signaling molecules with
important functions in ES cell self-renewal or differentiation,
including Wnt3a and Tgfb1 (Figure S1C). These findings suggest
that Mbd3/NURD and BAF complexes function in opposition to
fine-tune the expression of a set of genes required for ES cell
viability or self-renewal.
Mbd3 Binds Just Downstream of the TSS
Are the joint Brg1/Mbd3-regulated genes direct targets of these
complexes? While Brg1 has been mapped genome-wide in ES
cells (Ho et al., 2009a), Mbd3 localization is currently unknown.
We therefore carried out ChIP-Seq for Mbd3 in murine ES cells,
finding that Mbd3 localized largely to promoters (Figures 2A–2C,
Figure S2,and Figures S3Aand S3B),with only weak localization
to enhancers (Figures S4A and S4B). Genes upregulated upon
Mbd3 KD were associated with somewhat higher levels of
Mbd3 than were unaffected genes (Figure S3C), although, as is
commonly observed with chromatin regulators (Fan et al.,
Cell 147, 1498–1510, December 23, 2011 ª2011 Elsevier Inc. 1499
Figure 1. Antagonistic Effects of Brg1 and Mbd3 on Gene Expression in mES Cells
(A) Gene expression data for single and double knockdowns of Brg1, Mbd3, Ash2l, Suz12, p400, and Tip60. Heatmap shows pairwise Pearson correlation
coefficients for the 21 datasets. Four major clusters emerge, roughly corresponding to Brg1, Mbd3, Ash2l/Suz12, and Tip60/p400.
(B) Principal component analysis. Genes significantly misregulated (adjusted p value < 0.01) in any data set from (A) were subjected to principal component
analysis. Shown are individual data sets plotted along three most prominent principal components, which account for 87% of the total variance in gene
(C) Mbd3 and Brg1 antagonistically regulate a common set of genes. Unsupervised clustering of genes misregulated in both the Mbd3 KD and Brg1 KD datasets
(adjusted p value < 0.05). Clustering was performed on data sets containing either Mbd3 KD or Brg1 KD (or both), as well as the Tip60, p400, Suz12, and
Ash2l single KD datasets for contrast.
(D) Genes regulated by both Mbd3 and Brg1 tend to be moderately to highly expressed. Shown are mRNA abundance distributions (expressed as log2
of microarray probe intensity) for all genes, and for genes regulated by either or both Mbd3 and Brg1.
See also Figure S1, Table S1, and Table S2.
1500 Cell 147, 1498–1510, December 23, 2011 ª2011 Elsevier Inc.
2005; Jiang et al., 2011; Rando and Chang, 2009; Wu et al.,
2011b), only a small subset of Mbd3-bound loci are transcrip-
tionally affected by Mbd3 loss (Figure 2A).
Comparing Mbd3 maps to prior maps of Brg1 binding
(Ho et al., 2009a), we found significant overlap between the
binding sites of the two factors (Figure 2B and Figure S4C).
Figure 2. Genome-wide Localization of Mbd3
(A) Mbd3 KD effects on gene expression. Genes are sorted by change in mRNA abundance in Mbd3 KD, shown here in Log2.
(B) Mbd3 was mapped across the genome in ES cells by ChIP-Seq. Left panel: Mbd3 mapping data for 4 kb surrounding the transcriptional start sites (TSS) of
17,992 genes for which Mbd3KD gene expression data were available, with heatmap yellow saturating at 20 ppm normalized abundance. Right panel: published
data for Brg1 (Ho et al., 2009a). In both panels, genes are sorted as in (A).
(C) Mbd3 binds downstream of Brg1. Averaged data for all genes in (B) are shown relative to the TSS.
repressed by Mbd3 and activated by Brg1.
(E) Mbd3 and Brg1 physically associate. Western blots for Brg1 following immunoprecipitation with the indicated antibodies.
(F) Brg1 is required for Mbd3 localization. Mbd3 was mapped genome-wide in Brg1 KD cells, and data for all genes are averaged and plotted as in (C).
See also Figure S2, Figure S3, and Figure S4.
Cell 147, 1498–1510, December 23, 2011 ª2011 Elsevier Inc. 1501
We validated Brg1 and Mbd3 binding at several common target
promoters in ES cells by qPCR (Figure S3B). Interestingly, Mbd3
binding typically occurred ?100-200 bp downstream of the
average peak of Brg1 (Figure 2C), suggesting that these com-
plexes bind in an oriented fashion with respect to the direction
of transcription (see below). Furthermore, both Mbd3 and Brg1
were significantly associated with their antagonistically-regu-
lated target genes (Figure 2D and Figure S4D).
How are Brg1 and Mbd3 recruited to common promoters? We
first asked whether Brg1 and Mbd3 physically interact in vivo
using IP-westerns. Immunoprecipitation using anti-Mbd3 anti-
body, but not control IgG, coprecipitated Brg1 (Figure 2E), indi-
cating that these proteins interact in ES cells, as observed in
K562 cells (Mahajan et al., 2005) and suggested by mass spec-
trometry of esBAF (Ho et al., 2009b). To further probe the func-
tional relationship between Brg1 and Mbd3, we mapped Mbd3
genome-wide in Brg1 KD ES cells (Figure 2F), finding that
Mbd3 localization was lost upon Brg1 KD. Together, these
results indicate that Brg1 and Mbd3 directly associate with and
regulate a common set of target genes in ES cells.
Mbd3 Binding Is Enriched at Bivalent Genes
We next compared Mbd3 binding to previously published ChIP-
Seq datasets in murine ES cells. Figure 3A shows the difference
in Mbd3 binding levels between genes bound by a particular
factor and those unbound, as defined in (Kim et al., 2010).
Mbd3 binding was substantially higher at targets of the Poly-
comb proteins Ezh1, Suz12, Phc1, and Eed, as well as the
histone modifications associated with most Polycomb targets
in ES cells: H3K27me3 and H3K4me3 (Figures 3A–3C). Further-
more, similar to Polycomb localization, Mbd3 localization was
strongest at CpG-rich promoters and weakest at CpG-poor
promoters (Figure 3D). Finally, Mbd3 was localized to all four
Hox clusters, as previously observed for several PRC1 and
PRC2 subunits (Boyer et al., 2006; Figure S3A).
These data suggest that Mbd3 may function with Polycomb
and/or MLL/SET1 complexes to regulate gene expression. Inter-
estingly, we noted that the gene expression profile of Mbd3 KD
ES cells strongly correlated with that of Suz12 KD ES cells,
and to a lesser extent with that of Ash2l KD ES cells (Figure 1A),
indicating that both H3K4me3 and H3K27me3 affect many
‘‘bivalent’’ genes similarly, and that Mbd3 may also regulate
these genes. Indeed, most genes significantly up- or downregu-
lated upon Mbd3 KD were changed in the same direction upon
KD of either Suz12 or Ash2l (Figures 3E and 3F), although the
effects of Suz12 or Ash2l on gene expression were generally
less severe. Thus, Mbd3 is directly associated with a significant
fraction of Polycomb-bound genes, and contributes to their
To attempt to understand why only a subset of Mbd3-bound
genes were affected transcriptionally by Mbd3 knockdown, we
carried out a multivariate regression analysis (see Experimental
Procedures). Linear combinations of genome-wide binding data
for 38 factors or modifications were optimized to best explain
the effects of Mbd3 KD on expression of Mbd3/Brg1 common
targets (Figures S5A and S5B). The full model exhibited a high
correlation (R = 0.40) between predicted and measured effects
of Mbd3 KD on gene expression. The strongest predictors of
Mbd3 effects on gene expression included Tet1 and 5-hydroxy-
methylcytosine localization (see below). Beyond these, strong
predictors of Mbd3 function included H3K27me3 (individual
R = 0.29), Mbd3 (R = 0.13), and transcription factors such as
Esrrb, E2F1, and Klf4, whereas core pluripotency factors such
as Oct4, Sox2, and Nanog contributed little (Figure S5B and
data not shown). These data are consistent with proteomics
studies showing that Esrrb physically associates with the BAF
and Mbd3/NURD complexes (van den Berg et al., 2010), and
suggest that genes regulated by this transcription factor are
particularly sensitive to Mbd3 function.
Opposing Chromatin Remodeling Functions of Mbd3
and Brg1 Regulate Recruitment of RNA Polymerase II
How do Mbd3 and Brg1 control gene expression at their
common target genes? NURD complexes contain both a Swi2/
Snf2-related ATPase (Mi-2) and a pair of histone deacetylase
subunits (Hdac1 and Hdac2). We therefore analyzed nucleo-
some occupancy (measured by H3 abundance) and H4 acetyla-
tion at Mbd3/Brg1 targets by ChIP-qPCR. Genes repressed by
Mbd3 exhibited decreased H3 occupancy and increased H4
acetylation upon Mbd3 KD (Figures 4A and 4B), suggesting
that the Mbd3 complex represses target genes by deacetylating
and stabilizing nucleosomes at promoters. Conversely, Brg1
knockdown resulted in variable effects on H4 acetylation, but
consistent increases in H3 occupancy (Figures 4A and 4B).
Thus, we find that Brg1 and Mbd3 antagonistically control nucle-
osome occupancy at target genes, with Brg1-mediated nucleo-
some loss associated with gene activation, and competing
nucleosome stabilization and deacetylation by Mbd3 associated
with gene repression.
How do these changes in chromatin architecture affect
transcription? Many genes in eukaryotes are regulated by tran-
scriptional pausing (Guertin et al., 2010). Given the localization
of Mbd3 downstream of the TSS (Figure 2C), we considered
the hypothesis that Mbd3 could play a role in enforcing a tran-
scriptional pause. We therefore carried out whole-genome
RNA Polymerase II (Pol II) mapping in EGFP KD, Mbd3 KD,
and Brg1 KD ES cells. Overall, genome-wide Pol II localization
was similar in all three KDs (Figure 4C), exhibiting in each case
the expected promoter-proximal peaks corresponding to bidi-
rectional transcription (Core et al., 2008; Seila et al., 2008). We
vated by Brg1 (Figure 4D). If Mbd3 functioned solely to arrest Pol
II,thenmRNA increases inMbd3 KDshould bereflected in a loss
of 50Pol II and an increase in downstream Pol II. Instead, we find
that the 50peak of Pol II increases at these target genes in the
Mbd3 KD, and decreases in the Brg1 KD, mirroring the effects
of each KD on mRNA levels. Since the global Pol II profiles are
nearly identical for all three KDs (Figure 4C), these data are
consistent with a primary role for Mbd3 and Brg1 in regulating
recruitment of Pol II specifically to target promoters, although
an additional role in regulating Pol II pausing cannot be ruled out.
Knockdownof Tet1Impairs Mbd3Recruitment toTarget
Despite being named Methyl Binding Domain based on
its homology to the methyl binding domain from MeCP2,
1502 Cell 147, 1498–1510, December 23, 2011 ª2011 Elsevier Inc.
Figure 3. Mbd3 Directly Regulates Polycomb Target Genes
(A) Mbd3 binding at Polycomb target genes. For 34 mapped factors with thresholded binding defined in (Kim et al., 2010), Mbd3 binding levels (mean ChIP signal
for 1 kb centered on the +200 position) were calculated for bound and unbound subsets of genes. Genes annotated as unbound by all 34 factors were removed
from this analysis. Factors are sorted according to the relative Mbd3 binding at factor targets relative to nontargets.
(B) Mbd3 binding at three ES cell ‘‘modules.’’ Average Mbd3 binding for all genes, and for the three modules defined in (Kim et al., 2010), is plotted relative to
(C) Mbd3 binding at Polycomb targets and nontargets. For various Polycomb-related marks, averaged Mbd3 profile is shown for bound and unbound genes.
(D) Mbd3 binds preferentially to high-CpG promoters. Mbd3 binding data are averaged for high, intermediate, and low CpG (HCP, ICP, and LCP) promoters, as
defined in (Weber et al., 2007).
(E) Mbd3 KD affects Polycomb targets. Clustered mRNA data for KD of Suz12, Ash2l, Mbd3, or Brg1, and assorted double knockdowns. Genes significantly
misregulated in any of the included datasets are shown.
(F) Scatterplot of Mbd3 KD gene expression versus Ash2l KD and Suz12 KD. Only genes showing significant misregulation in Mbd3 KD are shown.
See also Figure S3 and Figure S5.
Cell 147, 1498–1510, December 23, 2011 ª2011 Elsevier Inc. 1503
mammalian Mbd3 does not appear to associate with cytosine-
methylated DNA (Hendrich and Bird, 1998; Zhang et al., 1999).
Interestingly, we noted that one prominent phenotype of Mbd3
null or KDES cells—upregulation of genesinvolved in trophecto-
derm differentiation—shares similarities to that of ES cells
5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC).
We therefore speculated that Mbd3 might be regulated by
5hmC, rather than 5mC. To test this hypothesis, we compared
Mbd3 localization to maps of Tet1 in murine ES cells (Wu et al.,
2011b). Similar to Tet1 localization, Mbd3 was found at CpG-
rich promoters, and correlated with Polycomb localization
patterns (Figures 3C and 3D). High levels of 5hmC accompany
Mbd3-associated genes in ES cells (Wu et al., 2011a; Figure 5A),
and Mbd3 and Tet1 localization patterns exhibited strong over-
lap (Figure 5B). Furthermore, genes repressed by Mbd3 were
associated with significantly higher levels of Tet1 and 5hmC
than genes unaffected by Mbd3 KD (Figures S5B and S5C).
We therefore asked whether cytosine hydroxymethyla-
tion functions in Mbd3 localization. Since, in ES cells, Tet1 KD
significantly decreases hydroxymethylation levels, we carried
out genome-wide mapping of Mbd3 in Tet1 KD ES cells.
Strikingly, Mbd3 was completely delocalized in Tet1 KD cells,
5B–5E). Thus, hydroxymethylation, or some other aspect of Tet1
function, is necessary for Mbd3 association with target genes.
Our results suggest that hydroxymethylation is not solely an
intermediate state in a cytosine demethylation pathway, but
that it also plays a distinct role in gene regulation via Mbd3
recruitment. As Mbd3 does not specifically bind to 5mC
in vitro, we considered the hypothesis that it might instead
bind to 5hmC. To test this, we purified Mbd3/NURD complex
from ES cells (Figures 6A and Figures S6A and S6B), finding
that this complex contained low levels of Tet1 (Figure 6B), iden-
tifying a direct physical link between Tet1 and Mbd3.
We carried out electrophoretic mobility shift assays (EMSAs)
using probes with unmodified cytosine (C), 5mC, or 5hmC. As
shown in Figure 6C, Mbd3/NURD complex had little effect on
the methylated probe, but strongly shifted the 5hmC probe,
whereas an untagged purification did not shift any of the probes
Figure 4. Mbd3 and Brg1 Regulate Chromatin Structure and Transcription Initiation at Target Genes
(A) H3 occupancy at Mbd3/Brg1 targets. H3 occupancy is plotted for Mbd3 KD and Brg1 KD relative to control KD. Shown are mean ± SEM.
(B) H4 acetylation at Mbd3/Brg1 targets. As in (A), for H4 acetylation. Data are normalized to nucleosome occupancy (H3 ChIP). Shown are mean ± SEM.
(C) Genome-wide RNA Polymerase II (Pol II) mapping. Average TSS-aligned profiles of Pol II occupancy is shown for all genes for control, Brg1 KD, and Mbd3
(D) Pol II levels at the 50end correlate with mRNA abundance changes in Brg1 and Mbd3 KDs. As in (C), but only for genes repressed by Mbd3 and activated
1504 Cell 147, 1498–1510, December 23, 2011 ª2011 Elsevier Inc.
Figure 5. Mbd3 Associates with Hydroxymethylated Regions of the Genome
(A) Mbd3-bound genes are associated with high levels of hydroxymethylation. Hydroxymethylation data from (Wu et al., 2011a) are averaged for genes with the
indicated levels of Mbd3 binding.
(B) Mbd3 colocalizes with Tet1. Left panel: Tet1 (Wu et al., 2011b) mapping data are shown for all named genes, sorted by Tet1 binding levels. Middle and right
panels: Mbd3 localization is shown for control and Tet1 KD ES cells.
(C) Mbd3 binding data for control and Tet1 KD are plotted as in Figures 2C and 2D.
(D) qPCR validation of Mbd3 binding at 6 selected target loci in GFP or Tet1 KD. Shown are mean ± SEM.
(E) Knockdowns of Tet1, Brg1, and Mbd3 do not affect protein levels of the other remaining factors. Knockdowns of the various factors were assayed by western
blot, as indicated.
See also Figure S5.
Cell 147, 1498–1510, December 23, 2011 ª2011 Elsevier Inc. 1505
(Figure S6C). Interestingly, Mbd3/NURD also shifted the unmod-
ified ‘‘C’’ probe, but the shifted band was broader than the
uniform shift of the 5hmC probe. These data suggest that
Mbd3/NURD complex employs distinct modes of binding to
unmodified and hydroxymethylated DNA.
To determine whether Mbd3/NURD’s specificity for 5hmC
over 5mC was due to Mbd3, we tested recombinant Mbd3 for
binding to the same probes. Consistent with the results obtained
using the full complex, recombinant Mbd3 exhibited strongest
binding to hydroxymethylated DNA probes, with comparable
but slightly reduced binding to unmodified probe, and dramati-
cally lower binding to methylated probe (Figure 6D and Fig-
ure S6D). As a control, recombinant methyl binding domain
from Mbd1 specifically shifted the methylated probe (Figure 6E),
as expected (Hendrich and Bird, 1998; Ohki et al., 2001).
Together, these data confirm the hypothesis that Mbd3 and
Mbd3 complexes do not bind to DNA harboring 5mC, but can
bind 5hmC-marked DNA in a manner qualitatively distinct from
that of DNA containing unmodified cytosine.
Given the physical interaction between Mbd3 and Tet1, we next
asked if Mbd3 affected 5hmC in vivo. Compared to control cells,
Tet1 KD ES cells exhibited reduced 5hmC levels by dot blotting
and thin layer chromatography (Figures 7A–7C), as previously
reported (Koh et al., 2011; Wu et al., 2011a; Ficz et al., 2011).
To our surprise, both Mbd3 KD and Brg1 KD ES cells also
exhibited strong reductions in global 5hmC levels as assayed
by dot blotting (Figure 7A and Figure S7). This effect of Mbd3
KD on bulk 5hmC levels was independently quantified by thin
layer chromatography (Figures 7B and 7C). To extend these
results to in vivo targets of Mbd3, we measured the enrichment
of 5hmC (Song et al., 2011) at several Mbd3 target genes. We
observed similar losses of 5hmC at six loci in Tet1 KD and in
Mbd3 KD cells (Figure 7D). Finally, we compared 5hmC staining
of individual cells by immunofluorescence in control cells to cells
depleted of Tet1, Mbd3, or Brg1. Consistent with the above
results, we observed noticeably reduced 5hmC levels in most
Tet1, Mbd3 and Brg1 KD cells compared to control (Figure 7E).
Therefore, we conclude that Mbd3 and Brg1 function globally
to establish or maintain normal levels of 5hmC in ES cells.
Theepigenetic controlof stemcell pluripotency and self-renewal
has been subject to intense scrutiny in recent years. Extensive
attention has been given to specific coactivators/corepressors
such as Polycomb Repressive Complexes, whereas other im-
portant chromatin regulators have received less attention.
Here, we focused on Brg1 and Mbd3, components of coactiva-
tor and corepressor complexes, the mechanisms by which they
antagonistically regulate a group of common target genes, and
their role in the biology of 5hmC.
Antagonistic Control of Gene Expression by BAF
and Mbd3/NURD Complexes
Several hundred genes were antagonistically regulated by
Brg1 and Mbd3, showing increased expression in Mbd3KD,
decreased expression in Brg1KD, and wild-type-like expression
the ‘‘bivalent’’ chromatin architecture seen in ES cells, in which
the activation-associated histone mark H3K4me3 and the
repression-associated H3K27me3 mark co-occur at a large
number of ‘‘master regulator’’ genes involved in cell fate deci-
sions (Bernstein et al., 2006). However, reduction of H3K4me3
Figure 6. Mbd3 Directly Binds to Hydroxymethylated DNA In Vitro
(A) Silver stain showing tandem affinity purification from untagged or Mbd3-
6His-3XFLAG ES cells.
(B) Western Blot of purifications described in (A) for indicated proteins. Mta1 is
a component of the Mbd3/NURD complex. Preliminary mass spec results also
identified most other known NURD subunits in this purification (data not
(C) EMSA assay using Mbd3/NURD complex and DNA probes containing
unmodified cytosine (C), methylated cytosine (5mC) or hydroxymethylated
(D and E) EMSA assay using recombinant mouse Mbd3 (D) or recombinant
Mbd1 methyl-binding domain (E), and various modified probes as in (C).
See also Figure S6.
1506 Cell 147, 1498–1510, December 23, 2011 ª2011 Elsevier Inc.
in ES cells by KD of Ash2l (Fazzio et al., 2008b) or Dpy-30 (Jiang
et al., 2011) does not cause self-renewal defects, nor does
H3K4me3 appear to be important for expression of most genes
in ES cells (Jiang et al., 2011; Table S1). Indeed, our results with
Ash2l and Suz12 knockdowns show that reduction of either
H3K4me3 or H3K27me3 results, ‘‘paradoxically,’’ in similar
effects on gene expression (Figures 3E and 3F). Conversely,
we show here that Mbd3 and Brg1 exhibit opposing functional
effects on RNA Polymerase II recruitment and gene expression
at several hundred genes. Together, these data suggest that
the juxtaposition of opposing histone modifications or chromatin
regulators near the regulatory sequences of shared target genes
is a common regulatory strategy in ES cells.
Mbd3/NURD Plays a Central Role
in 5-Hydroxymethylcytosine Biology
The inability of Mbd3 to bind specifically to 5mC (Hendrich and
Bird, 1998; Zhang et al., 1999) raises the question of what
Figure 7. Mbd3 Is Required for Global Hydroxymethylation In Vivo
(A) Dot blots of 5hmC. Top panel shows positive (5hmC) and negative (5mC) controls for antibody specificity. Bottom panels showdilution series of genomic DNA
isolated from the indicated knockdown ES cells. Mbd3 and Brg1 KDs have similar effects to Tet1KD on bulk 5hmC levels.
(B) Thin layer chromatography of radioactively end-labeled bases from MspI-digested genomic DNA (Ficz et al., 2011) from the indicated knockdowns.
(C) Quantitation of 5hmC levels (normalized to levels of T) measured as in (B) for 5 independent replicate experiments. Columns show mean ± SEM.
(D) Tet1 and Mbd3 knockdowns havesimilareffects on hydroxymethylation of target gene promoters. Hydroxymethylated DNA was isolated by capture of biotin-
glucosylated 5hmC-containing DNA fragments (Song et al., 2011), and fold enrichment over input was assessed by qPCR at the indicated loci and expressed as
fold change relative to 5hmC levels in control (EGFP) KD cells. Data represent mean ± SEM.
(E) Immunofluorescence imaging of Mbd3 and 5hmC. Immunofluorescence images are pseudocolored blue (DAPI), green (Mbd3), and red (5hmC) for the
indicated KDs – top panel shows 5hmC data only, bottom panel shows all 3 colors.
See also Figure S7.
Cell 147, 1498–1510, December 23, 2011 ª2011 Elsevier Inc. 1507
function the ‘‘methyl binding domain’’ of this protein serves. We
ylation of 5mC to form 5hmC, exhibit phenotypes similar to
those of Mbd3 KD ES cells (Kaji et al., 2006; Koh et al., 2011;
Zhu et al., 2009). Intriguingly, one of the few sequence differ-
ences in the methyl binding domain between human/mouse
Mbd3 and remaining Mbd family members is a substitution
of a phenylalanine for a tyrosine (numbered Y34/F34 in human
Mbd1/Mbd3). This sequence change is largely responsible for
Mbd30s lack of 5mC binding in vitro (Saito and Ishikawa, 2002).
In the structure of Mbd1 bound to methylated DNA, Y34 is
located immediately adjacent to the 5-methyl group of 5mC
(Ohki et al., 2001; Figure S6E). Thus, loss of the hydroxyl group
(Y34F) at this position in Mbd3 could almost perfectly compen-
sate for the additional hydroxyl group in 5hmC relative to 5mC,
thus allowing direct binding of Mbd3 to this modification in a
manner structurally analogous to the binding of remaining Mbd
members to 5mC.
Consistent with this hypothesis, we found that Mbd3 localiza-
tion patterns are similar to Tet1 localization patterns, and Mbd3
is enriched at genes with high levels of hydroxymethylation (Wu
et al., 2011b). Like Tet1, Mbd3 is associated largely with CpG-
rich promoters bound by Polycomb, and is required for normal
expression of many of these targets. Mbd3 localization requires
Tet1, suggesting that hydroxymethylation plays a role in Mbd3
recruitment in vivo. Finally, we found that Mbd3 preferentially
binds in vitro to 5hmC-containing probes relative to 5mC-con-
taining probes. Thus, our data are most consistent with a model
in which Tet1-catalyzed hydroxymethylation serves to recruit
Mbd3/NURD complex, and thus Mbd3/NURD may be an
effector that mediates some of the effects of hydroxymethylation
on gene expression.
Interestingly, we found that purified Mbd3/NURD also bound
to unmodified probe in vitro, but that this binding was qualita-
tively distinct from binding to the 5hmC probe: the shifted
band was more discrete and of higher mobility when the probe
was hydroxymethylated. One potential explanation for this could
be the binding location of the complex on the probe, as probe
bound at either end should have higher mobility than internally
bound probe. At present it is unclear to us why Tet1KD, which
results in incomplete loss of 5hmC in vivo, almost completely
abolishes Mbd3 localization. Given the increase in 5mC ob-
served in Tet1KD cells at loci that lose 5hmC (Ficz et al., 2011;
Wu et al., 2011a), and our observation that 5mC inhibits Mbd3
with 5hmC might be unfavorable for Mbd3 binding. Future
studies will dissect the details of how Mbd3/NURD differentially
interacts with unmodified and hydroxymethylated DNA, and with
hemi-modified or symmetrically modified DNA.
Finally, we found that KD of either Mbd3 or Brg1 results in
reduction of bulk levels of 5hmC in vivo. This could occur via
several possible mechanisms: as two examples, Mbd3 could
bind to a region of hydroxymethylated DNA and recruit Tet
enzymes to hydroxylate adjacent methylcytosines, or Mbd3
could bind to hydroxymethylated loci and protect them from
further steps in a demethylation pathway. Either way, the exten-
sive interdependency between these factors – Tet1 is necessary
for Mbd3 localization, Mbd3 is necessary for cytosine hydroxy-
methylation – is reminiscent of other codependencies in chro-
matin pathways. For instance, Polycomb is required for H2A.Z
incorporation at promoters of developmental genes in ES cells,
and H2A.Z is in turn required for Polycomb binding (Creyghton
et al., 2008). Dissecting the detailed mechanistic basis for such
interdependencies is a challenging goal for studies on chromatin
Together, these data describe the first downstream effector
that regulates expression of 5hmC-marked genes, suggesting
that 5hmC plays a role in gene regulation beyond serving simply
as an intermediate in a demethylation pathway. It will be of great
interest in future studies to determine whether Mbd3 also plays
a role in 5hmC biology in other contexts such as early embryos
(Inoue and Zhang, 2011; Iqbal et al., 2011), imprinting (Reese
et al., 2007), or neurons (Kriaucionis and Heintz, 2009). Finally,
our discovery of an interdependent regulatory network consist-
ingof5hmCand twoantagonisticchromatin regulatorssuggests
that control of gene expression by opposing chromatin regula-
tors is a common regulatory strategy in pluripotent ES cells.
RNA interference using endoribonuclease III digested siRNAs (esiRNAs) was
performed as described previously (Fazzio et al., 2008b), using E14 mouse
ES cells. All KDs were performed for 48 hr to achieve effective KD, but
avoid some indirect effects of prolonged loss of chromatin regulators. Stable
Mbd3 KD lineswere made by infection of ES cells withlentiviralshRNA vectors
from the TRC library (Open Biosystems).
Array hybridizations were performed at the Sandler Asthma Basic Research
(SABRE) Center Functional Genomics Core Facility as described previously
(Fazzio et al., 2008a). For each single and double KD, a linear model was fit
to the comparison to estimate the mean log2 (fold change) in 2 biological
replicates and to calculate a moderated t-statistic, B statistic, false discovery
rate and p value for each probe. Adjusted p values were produced as decribed
previously (Holm, 1979). All procedures were carried out using functions in the
R package limma (Gentleman et al., 2004; Smyth, 2004) or made4 (Culhane
et al., 2005) in R/Bioconductor. Enrichment of Gene Ontology terms was
performed with DAVID 6.7 (Dennis et al., 2003). mRNA data are available at
GEO, accession GSE31008.
Chromatin immunoprecipitation and deep sequencing library construction
were performed using minor modifications of established protocols (Barski
et al., 2009; Lee et al., 2006; Extended Experimental Procedures). Data are
available at GEO, accession GSE31690.
Multivariate Linear Regression Model
Multivariate linear regression was used to predict factors that regulate
Mbd3/Brg1-target genes. 38 ChIP-seq datasets were used to analyze 40
100 bp bins surrounding the TSS of each gene that changed oppositely
upon Mbd3 KD and Brg1 KD, with another bin representing the gene body.
Binding of each of the 38 factors was compared to gene expression changes
(see Extended Experimental Procedures for details).
An ES cell line with a C-terminal 6-Histidine-3X FLAG tag placed just upstream
of the Mbd3 stop codon was constructed as described in the Supplementary
Experimental Procedures. Mbd3/NURD complex was purified from ?4 3 108
of these cells by sequential affinity purification steps using FLAG-M2 Agarose
followed by TALON Agarose beads.
1508 Cell 147, 1498–1510, December 23, 2011 ª2011 Elsevier Inc.
EMSA assays were performed on 5% polyacrylamide 0.53 TBE gels.
Biotinylated probes corresponding to +529–+628 of the Tgfb1 gene were
made by PCR using Phusion polymerase and NTPs containing either unmod-
ified, methylated, or hydroxymethylated dCTP. The PCR primer sequences for
probe construction were: biotin-TGCCTCTTGAGTCCCTCGCATC and AGTG
GGTGTTCTTAAATAGGGGAGCT. Binding reactions were performed in 13
Binding Buffer (LightShift Chemiluminescent EMSA Kit; Thermo Scientific)
with 100 mM KCl, 8% glycerol, 0.02% NP-40, 5 mM MgCl2, 0.85 mg BSA,
30 ng yeast genomic DNA, 0.5 ng probe, 1 mM ATP, and with or without 1 ml
purified Mbd3/NURD or recombinant Mbd3, as indicated. After electropho-
resis, samples were transferred to charged Nylon membrane and probed
with streptavidin-HRP according to the instructions in the EMSA kit.
2-fold serial dilutions of genomic DNA were denatured in 0.4 M NaOH/10mM
EDTA at 95?C for 10 min, then added to an equal volume of cold 2M ammo-
nium acetate (pH 7.0). Denatured DNA samples were spotted onto nitrocellu-
Membrane was blocked with 5% nonfat milk for 1 hr and incubated with rabbit
anti-5hmC, detected by HRP-conjugated secondary antibody and enhanced
Cells were resuspended in PBS, cytospun onto glass slides, fixed in 4%
paraformaldehyde for 10 min at room temperature, washed twice with PBS,
permeabilized in 2% Triton X-100-PBS for 10 min, and blocked with 5%
FBS and 0.1% Triton X-100 in PBS for 15 min at room temperature. Cells
were incubated with primary antibody diluted 1:500 with 5% FBS and 0.1%
Triton X-100 in PBS for 30 min at room temperature, washed 3 3 5 min with
PBS, and incubated for 30 min at room temperature with secondary antibody
diluted 1:200 with 5% FBS and 0.1% Triton X-100 in PBS. DAPI was added in
mounting medium (Vectashield, H1500).
Supplemental Information includes Extended Experimental Procedures,
two tables, and seven figures and can be found with this article online at
We thank the Rando and Fazzio labs for critical reading of the manuscript;
J. Benanti and P. Kaufman for technical advice; C. Li for assistance with
TLC; D. Carone for assistance with IF; and J.M. Bishop, in whose lab some
of these experiments were performed. We thank C. He and C. Song for the
generous gift of reagents for isolation of 5hmC. T.G.F. is supported in part
by grant CA140854 from NCIand is aPew Scholar intheBiomedical Sciences.
andLeilaMathers Charitable Foundation. Z.W.issupportedby NSFgrant DBI-
decision to publish, or preparation of the manuscript. O.Y., T.G.F., and O.J.R.
designed all experiments. O.Y. and T.G.F. carried out mapping experiments.
O.Y. performed 5hmC quantitation, T.G.F. carried out gene expression and
gel-shift experiments, and L.E. and P.B.C. performed experiments in Figures
4A and 4B. T.G.F. analyzed gene expression data. O.J.R., X.D., J.H.H., R.L.,
and Z.W. analyzed localization data. O.J.R., O.Y., and T.G.F. wrote the paper.
Received: April 22, 2011
Revised: October 12, 2011
Accepted: November 23, 2011
Published: December 22, 2011
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EXTENDED EXPERIMENTAL PROCEDURES
80% Confluent ES cells were crosslinked by addition of formaldehyde (Sigma) to a final concentration of 1% and incubated at room
temperature(RT) for10min.Crosslinkingwas quenched with125mMGlycine.Cells werewashed twicewithice coldPBScontaining
PMSF, collected in PBS, and pelleted at 1,000 g for 5 min, at 4?C. Cell pellets were either flash frozen in liquid nitrogen and stored in
?80?C, or processed immediately. Pellets were resuspended in 270 ml SDS-Lysis Buffer (1% SDS, 10 mM EDTA and 50 mM Tris-Cl,
[pH 8.1]) including protease inhibitor complex (Sigma) and PMSF (Sigma). Samples were sonicated in a Bioruptor (UCD-200) at high
setting 3 times for 15 min of 30 s on/30 s off cycles, followed by a 14,000 rpm spin at 4?C for 1 hr. Supernatants were transferred to
a new tube and pellets were resuspended in 150 ml of lysis buffer and processed in a Bioruptor using the previous settings for two
more 15 min cycles. Supernatants were collected after a 13,000 rpm spin at 4?C for 10 min and combined with supernatants fromthe
previous centrifugation. Chromatin was quantified by measuring A260. 50 mg of protein A magnetic beads (NEB) were washed twice
with 600 ml of 0.1 M sodium phosphate (NaK) buffer (pH 8.0) and antibody was coupled in 200 ml NaK buffer for 30min at RT. Antibody
coupled beads were blocked with 5 mg/ml BSA-PBS solution for 1 hr at 4?C. 70 mg of chromatin for each immunoprecipitaton was
diluted 1:10 in IP-Blocking buffer (5 mg/ml BSA and 5 mg/ml yeast tRNA in 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM
Tris-Cl (pH 8.1), 167 mM NaCl), combined with antibody coupled magnetic beads and incubated at 4?C overnight (up to 16 hr).
X-100); 3 times with NaCl buffer (0.5 M NaCl in RIPA); 3 times with LiCl buffer (0.3 M LiCl, 0.5% NP40, 0.5% Na-Deoxycholate in
RIPA); twice with 0.2% Triton X-100 TE buffer and once with TE buffer (pH 8.0).
Washed beads were resuspended in 300 ml TE and incubated at 65?C overnight in after adding the following (final concentrations):
SDS (3%), proteinaseK (1 mg/ml) (Sigma) and glycogen (0.5 mg/ml) (Ambion). Eluted material was transferred to a new tube and the
beads were resuspended in 150 ml 0.5M NaCl-TE solution and incubated at 65?C for one hour. Eluted materials were combined and
PCI (Phenol-Chloroform-Isoamylalcohol) extracted using phase-lock tubes (Eppendorf). Ethanol precipitated ChIP DNA was treated
with RNase (QIAGEN) for 2 hr and with CIP for an hour at 37?C (NEB calf alkaline phosphatase - 0.25U/ml in 1x NEB Buffer 3). Reac-
tions were then cleaned up with QIAGEN MiniElute spin columns.
The following antibodies were used in this study for ChIP, IP, western blotting, dot blotting and meDIP: Mbd3, Abcam (ab3755) and
Bethyl (A302-528A); Brg1, Bethyl (A300-813A); Pol II, Santa Cruz (sc-899X); Ac-H4, Millipore (06-866); H3, Abcam (ab1791); Tet1,
Millipore (09-872); 5hmC, Active Motif (39791); 5mC, Eurogentec (BI-MECY-0500); Mta1, Bethyl (A300-280A); and beta-actin, Sigma
Generation of Stable Mbd3 Knockdown Lines
80%–90% confluent 293T cells were transfected with 15 mg of shRNA containing vector, 20 mg of pCMVD8.9 and 3 mg of pVSV-G
using Lipofectamine 2000 in OPTI-MEM. The viral supernatant was collected at 48 hr and 72 hr and filtered with low protein binding
a 37?C water bath. After pelleting the cellular debris with centrifugation, the supernatant was filtered and combined with the filtered
growth medium. Virus was concentrated with ultracentrifugation at 4?C, 27,000 rpm for 2 hr in an SW28 rotor. The viral pellet was
resuspended in 15 ml HBS and ultra filtered with Ultracell-100k filters (Amicon). ES cells were infected in 2 ml ES medium containing
6 mg/ml Polybrene in a 6-well-plate with 100ul of the concentrated virus. The next day, cells were replated on 10 cm dishes and anti-
biotic selection started 24 hr after the infection. Resistant colonies were picked, expanded and tested for knockdown efficiency via
RTqPCR and western blotting.
Isolation of Hydroxymethylated DNA
HydroxymethylatedDNAwasisolated aspreviouslydescribed (Songetal.,2011).Briefly, purifiedgenomicDNA,sonicatedintoshort
fragments (200–500 bp), was incubated with b-glucosyltransferase (b-GT) and UDP-6-N3-Glucose, which resulted in N3-glucosy-
magnetic beads and eluted in 100 mM DTT for 2 hr at room temperature. Isolated DNA was cleaned up using Micro Bio-Spin 6 spin
columns (Bio-Rad) followed by MinElute reaction clean up columns (QIAGEN) and subjected to qPCR.
Deep Sequencing Library Construction
ChIP material was size selected (100–800 bp in size) on a 2% TAE agarose gel using the MiniElute columns QiaQuick (QIAGEN). Gel
purified DNA fragments were blunt ended and phosphorylated with the EPICENTRE End-it-Repair kit (13 buffer, 0.25 mM dNTPs,
1 mM ATP, 1 ml/50 ml reaction of Enzyme mix) for 1hr at RT and cleaned up with QIAGEN MiniElute spin columns. Adenosine nucle-
torswerethenligatedusingthe EPICENTRE Fast-Linkligationkit: 11.5mlAtailed DNAelutedfromaMinElute columnwasmixedwith
1.5 ml 103 ligation buffer, 0.75 ml 10 mM ATP, 0.5 ml Illumina DNA adaptors and 1 ml ligase. The reaction was incubated for 1hr at RT
andsubsequentlysupplemented with7.5 mlwater,1ml103buffer,0.5ml10mMATPand 1mlligase, andincubatedovernightat16?C.
Cell 147, 1498–1510, December 23, 2011 ª2011 Elsevier Inc. S1
Theligation reactionwas cleaned up with MiniElutecolumns (with anadditional washstepto eliminate all theexcess adaptors) and
the adaptor ligated fragments were amplified by PCR as follows:
0.75 ml of each Illumina genomic DNA sequencing primers, 6 ml 103 Pfx buffer 1.8 ml 10 mM dNTPs, 1.2 ml 50 mM MgSO4 and 1 ml
Pfx DNA polymerase (Invitrogen) were added to 30 ml DNA template in a 100 ml reaction. The cycling parameters were: 1. 94?C 20; 2.
94?C 1500; 3. 65?C 10; 4. 68?C 3000; 5. repeat from step 2, 16 times; 6. 68?C 50. The PCR product was size selected (250–450 bp) from
a 2% TAE agarose gel using QiaQuick columns (QIAGEN). Gel purified fragments were finally precipitated with Sodium acetate and
ethanol and pellets were resuspended (25 nM final concentration) in TE buffer and subjected to SOLEXA single-read sequencing at
the UMass Medical School deep sequencing core facility.
Mbd3 datasets were collected for two independent ChIP samples using independent antibodies (obtained from Bethyl and
Abcam). Datasets were well-correlated (Figure S2), although the Bethyl library exhibited higher genome-wide background than
the Abcam library and so was not used for most analyses. Nonetheless, all major biological conclusions were reproduced with either
Multivariate Adaptive Regression Splines (MARS) Model
MARS models were used to predict the log2 expression fold change upon Mbd3 knockdown using 38 ChIP-seq datasets (log2 of
histone modification and transcription factor binding; called features). Each gene was divided into 41 bins, 40 100-bp bins for its
promoter region ([?2k, +2k] around TSS) and one bin for the remaining gene body (from +2k to the transcriptional termination
site), and the bin that led to the highest correlation coefficient was chosen as representative signal for that gene. Feature selection
was performed and the final model includes 12 features. A MARS model isjudged by Person’s correlation coefficient (R) between the
criteria: percentage of models that contain the feature after feature selection, and generalized cross validation (GCV) (Friedman,
1991). We constructed the model with the 568 transcripts on which knocking down Brg1 and Mbd3 had opposite effects (Brg1 up-
regulated and Mbd3 downregulated, or Brg1 downregulated and Mbd3 upregulated) that were significant in both knockdown data-
sets (adjusted p value < 0.01).
A targeting construct containing one homology arm extending from ?2.5 kb upstream of the Mbd3 transcriptional termination site
stream was constructed. Sequence encoding 36 amino acids (SGRGSHHHHHHAGMDYKDHDGDYKDHDIDYKDDDDK) that include
a 6-Histidine and a triple FLAG tag was inserted just upstream of the normal Mbd3 stop codon. A LoxP-Hygromycin-LoxP selection
cassette was introduced at the upstream end of the second homology arm and a diptheria toxin A (DTA) counterselection cassette
just downstream of the second homology arm (Figure S6A). 25 mg of the resulting plasmid was linearized with Asc I and electropo-
rated into E14 ES cells. Clones were screened by PCR, followed by western blotting to ensure correct integration (Figure S6B).
The lysate was clarified twice by centrifugation and incubated for three hours in 200 ml of FLAG M2 Agarose bead slurry (Sigma) that
had previously been equilibrated in Lysis buffer. Beads were washed five times and Mbd3/NURD complex was eluted five times with
0.5 mg/ml 33 FLAG peptide (Sigma) in Lysis Buffer, with 15 min incubations prior to recovery of each elution. Pooled FLAG elutions
were incubated with 100 ml of pre-equilibrated TALON Agarose beads for 2 hr, washed five times with Lysis buffer, and eluted five
times in Lysis buffer + 250 mM imidazole (10 min incubations per elution). Pooled eluates from the TALON beads were dialyzed over-
night in Buffer H (25 mM HEPES-KOH [pH 7.6], 0.1 mM EDTA, 0.5 mM EGTA, 2 mM MgCl2, 20% glycerol, 0.02% NP40) with 100 mM
KCl, aliquoted, flash-frozen in liquid nitrogen and stored at ?80?C.
Purification of Recombinant Mbd3 or Mbd1-MBD
The longest isoform of Mbd3 (Mbd3a) and the first 75 amino acids (comprising the methylcytosine binding domain) of Mbd1 were
PCR amplified from mouse ES cell cDNA with Phusion DNA polymerase, cloned into pCR2.1, sequence verified, subcloned into
pQE80L in frame with an N-terminal six histidine tag encoded within the vector and transformed into BL21 pLysS cells. 200 ml
cultures were grown to early log phase, induced with 0.5 mM IPTG for 3.5 hr, washed with PBS and frozen. Frozen pellets were re-
suspended in lysis buffer (see Mbd3/NURD purification above), sonicated several times and centrifuged twice to clarify lysates.
Lysates were incubated with 200 ml of TALON resin for 2 hr, washed 6 times with 1 ml lysis buffer and eluted 5 times in lysis buffer
containing 250 mM imidazole. Purified proteins were dialyzed overnight in Buffer H (see above), aliquoted and flash frozen in liquid
S2 Cell 147, 1498–1510, December 23, 2011 ª2011 Elsevier Inc.