Ligand-dependent dynamics of retinoic acid receptor binding during early neurogenesis.

Abstract

Among its many roles in development, retinoic acid determines the anterior-posterior identity of differentiating motor neurons by activating retinoic acid receptor (RAR)-mediated transcription. RAR is thought to bind the genome constitutively, and only induce transcription in the presence of the retinoid ligand. However, little is known about where RAR binds to the genome or how it selects target sites.
We tested the constitutive RAR binding model using the retinoic acid-driven differentiation of mouse embryonic stem cells into differentiated motor neurons. We find that retinoic acid treatment results in widespread changes in RAR genomic binding, including novel binding to genes directly responsible for anterior-posterior specification, as well as the subsequent recruitment of the basal polymerase machinery. Finally, we discovered that the binding of transcription factors at the embryonic stem cell stage can accurately predict where in the genome RAR binds after initial differentiation.
We have characterized a ligand-dependent shift in RAR genomic occupancy at the initiation of neurogenesis. Our data also suggest that enhancers active in pluripotent embryonic stem cells may be preselecting regions that will be activated by RAR during neuronal differentiation.

Full text

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RESEARCH Open Access
Ligand-dependent dynamics of retinoic acid
receptor binding during early neurogenesis
Shaun Mahony1†, Esteban O Mazzoni2†, Scott McCuine3, Richard A Young3, Hynek Wichterle2, David K Gifford1*
Abstract
Background: Among its many roles in development, retinoic acid determines the anterior-posterior identity of
differentiating motor neurons by activating retinoic acid receptor (RAR)-mediated transcription. RAR is thought to
bind the genome constitutively, and only induce transcription in the presence of the retinoid ligand. However,
little is known about where RAR binds to the genome or how it selects target sites.
Results: We tested the constitutive RAR binding model using the retinoic acid-driven differentiation of mouse
embryonic stem cells into differentiated motor neurons. We find that retinoic acid treatment results in widespread
changes in RAR genomic binding, including novel binding to genes directly responsible for anterior-posterior
specification, as well as the subsequent recruitment of the basal polymerase machinery. Finally, we discovered that
the binding of transcription factors at the embryonic stem cell stage can accurately predict where in the genome
RAR binds after initial differentiation.
Conclusions: We have characterized a ligand-dependent shift in RAR genomic occupancy at the initiation of
neurogenesis. Our data also suggest that enhancers active in pluripotent embryonic stem cells may be preselecting
regions that will be activated by RAR during neuronal differentiation.
Background
Cellular competence, fate determination, and differentia-
tion are influenced by the external signals cells receive.
While these external signals can take the form of steroid
hormones, protein growth factors, or other molecules,
their presence is typically communicated by signal-
responsive transcription factors (TFs). The effect of a
signal on gene expression, and ultimately on cell fate,
depends on where such TFs bind to the genome. There-
fore, understanding how signal-responsive TFs are inte-
grated into a dynamic cellular context will further our
knowledge of the mechanisms guiding the acquisition of
specific cellular identities.
In the developing neural tube, retinoid signaling initi-
ates neural differentiation [1], specifies caudal hindbrain
and rostral cervical spinal identity [2,3], and controls
patterning and differentiation of spinal motor neurons
and interneurons [4-6]. Retinoic acid (RA) is the most
commonly used neuralizing agent during in vitro
embryonic stem (ES) cell differentiation since exposure
to it results in a rapid transition from pluripotent
embryoid bodies to committed neuronal precursors. The
response to RA during neuronal development is
mediated by the action of retinoic acid receptor iso-
forms (collectively abbreviated here as RARs). It has
been proposed that RARs are constitutively bound to
target sites in the absence of retinoids [7], recruiting co-
repressors such as Ncor1 and Ncor2 [8]. In the presence
of the retinoid ligand, RAR (often heterodimerized with
RXR) recruits co-activators (Ncoa1 and Ncoa2), p300,
and core components of the transcriptional machinery
[7]. However, the proposed independence of RAR bind-
ing from the presence of the ligand has only been con-
firmed at a small number of sites.
While some characterization of RAR genomic binding
has recently been carried out in mouse ES and human
breast cancer cell lines [9-11], it is unknown which
genes are targeted by RAR during neurogenesis, and
how RAR binding targets are selected. Chromatin acces-
sibility and protein cooperativity may both play roles in
restricting the cohort of bound locations under a given
* Correspondence: gifford@mit.edu
† Contributed equally
1Computer Science and Artificial Intelligence Laboratory, Massachusetts
Institute of Technology, 32 Vassar Street, Cambridge, MA 02139, USA
Full list of author information is available at the end of the article
Mahony et al. Genome Biology 2011, 12:R2
http://genomebiology.com/2011/12/1/R2
© 2011 Mahony et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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set of cellular conditions. For example, in human breast
cancer cell lines, RAR binding is highly coincident with
the binding of estrogen receptor (ER)a, FoxA1, and
Gata3 [10,11], and FoxA1 is required for RAR recruit-
ment [10]. Recent work has demonstrated that TF bind-
ing also correlates with nucleosome-free regions [12],
certain histone modifications [13-17], and the occupancy
of other regulatory proteins [18,19] in the same cellular
conditions. It is not known how these relationships
extend through developmental time at individual enhan-
cers. Enhancers may be entirely developmental stage-
specific, in which case the sites bound by a regulator in
one developmental stage should not be coincident with
the sites bound by a subsequent stage-specific TF. Alter-
natively, enhancers may be reused across developmental
time, and the occupancy patterns of regulatory proteins
or epigenetic markers may anticipate the future binding
of newly activated TFs during differentiation [20,21].
Determining the dynamics of RAR binding during early
neuronal development may therefore yield insight into
the precise temporal response of cells to retinoid signal-
ing and how enhancers are organized to facilitate this
response.
In this study, we examine the genome-wide binding of
RARs during RA induced differentiation of ES cells into
spinal motor neurons [22]. Retinoid signaling initiates
the transition from pluripotency to neurogenesis in this
model system, and provides rostro-caudal information
to developing motor neurons. By profiling the binding
of active RAR isoforms in both the presence and
absence of retinoid signaling, we observe that only a
small subset of sites are constitutively bound. An addi-
tional set of sites is bound only in the presence of RA,
and the existence of this set provides a convenient
opportunity to examine how pre-RA occupied and post-
RA occupied sites correlate with the relatively well-char-
acterized regulatory network in mouse ES cells. We find
that binding information for ES cell TFs and other regu-
latory proteins accurately predicts both constitutive and
exclusively post-RA RAR binding. The binding of core
ES cell regulators is highly correlated with pre-RA
bound RAR sites, slightly less correlated with post-RA
bound RAR sites, and much less correlated with the
binding of other TFs in further differentiated tissues,
arguing that the active regulatory network may be one
of the most important determinants of TF binding.
Results
RAR ChIP-seq profiles direct genomic interactions during
early differentiation
Using a pan-RAR antibody, we profiled the genome-
wide occupancy of RAR isoforms in differentiating
embryoid bodies after 8 hours of exposure to RA, find-
ing significant ChIP-seq enrichment at 1,924 sites.
We also profiled RAR occupancy in the same develop-
mental stage but in the absence of retinoid signaling,
finding 1,822 sites of significant enrichment. A number
of previously characterized retinoic acid response ele-
ments (RAREs) were observed to be bound in both con-
ditions, including RAREs at Rarb, Hoxa1, and Cyp26a1
(Figure 1) [23]. A recent promoter-focused ChIP-chip
study of RAR in mouse embryonic stem cells [9] sug-
gested that few RAR binding sites contained ‘direct-
repeat’ hormone response elements. In contrast, we find
that high-similarity hormone response element motifs
occur at RAR ChIP-enriched sites at a higher rate than
that observed in published ChIP-seq studies of other
nuclear hormone receptors such as ERa, Esrrb, and
Nr5a2 [10,24-26] (Additional file 1). The most frequent
motifs at our enriched sites are the direct-repeat motifs
with spacers of 5 bp or 2 bp (DR5 and DR2, respectively;
Additional file 1), which RAR is known to preferentially
bind [23,27]. The binding events with the highest ChIP-
enrichment are more likely to contain high-similarity
matches to the DR5 and DR2 motifs (Additional file 2),
suggesting that many of the most enriched sites represent
direct RAR-DNA binding events.
RAR binding shifts in response to RA exposure
In contradiction to the model of RAR constitutively
binding to its targets [7], only 507 of the predicted RAR
binding events are significantly enriched both in the pre-
sence and absence of retinoid exposure, where signifi-
cant enrichment is defined by our binding event
detection methodology (see Materials and methods).
Figure 1 presents a clustergram of all sites bound before
or after RA exposure, and is arranged according to the
pattern of enrichment across both conditions. As the
figure indicates, we need to be cautious when determin-
ing if a site is bound exclusively in one condition. For
instance, some sites display similar enrichment levels
across both conditions, but this enrichment level is only
deemed significant in one condition (that is, it falls
below the significance threshold in the other condition).
After further analysis, we define a set of 638 sites that
are bound exclusively in the presence of retinoid signal-
ing, as they are not significantly enriched in the absence
of RA exposure (compared with control), and their
levels of ChIP-seq enrichment are significantly different
in the presence and absence of RA (see Materials and
methods). Conversely, at least 539 sites are bound only
in the absence of retinoid exposure.
Intriguingly, some of the shift in RAR binding sites
may be explained by a ligand-dependent shift in RAR’s
binding preference. Sites bound only in the absence of
RA contain more direct repeat motifs with 0-bp or 1-bp
spacers than sites bound only in the presence of RA
(Additional files 3 and 4). Prior studies have shown that
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such motif configurations can be bound by RAR [28,29].
On the other hand, sites bound exclusively in the pre-
sence of RA contain more DR5 motifs. These direct
repeat motifs are amongst the set of sequence features
that have the most significant difference in occurrence
frequency between RAR sites bound exclusively in the
presence or absence of retinoid signaling (Additional file
5). However, only approximately 14% of exclusively pre-
RA sites contain high similarity matches to the DR0 or
DR1 motifs, while only 13% of exclusively post-RA sites
contain high similarity DR5 motifs. Therefore, a poten-
tial shift in RAR’s direct binding preference offers only a
partial explanation for the observed condition-exclusive
binding patterns.
By comparing the relative occurrence of all known TF
binding motifs in each condition-exclusive set, we also
find that exclusively post-RA sites contain significantly
more E-box and ETS-family motifs than exclusively pre-
RA sites (Additional file 5). Exclusively post-RA sites also
contain more instances of a palindromic motif with con-
sensus sequence ‘TCTCGCGAGA’. It is not known which
proteins may interact with this motif, although the motif is
over-represented in mammalian promoter regions [30],
and has recently been characterized as a regulatory
sequence [31]. The observation of these over-represented
secondary motifs suggests that some of the exclusively
post-RA binding sites may occur due to ligand-dependent
interactions between RAR and cofactors, or some may
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Figure 1 RAR binding shifts in response to RA exposure. (a) The plots in the two leftmost columns show enrichment over all 1,822 pre-RA
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region. (b) Examples of constitutive and ligand-specific RAR binding at four loci (Rqcd1, Cyp26a1, Hoxa1, Hoxb4/Hoxb5).
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potentially represent indirect binding events caused by
enhancer-promoter looping. Most of the motifs with sig-
nificantly higher relative frequency in the exclusively pre-
RA sites are related to DR0 or DR1 patterns.
A compact retinoid response is directly mediated by RAR
In order to determine which RAR binding sites are asso-
ciated with transcriptional regulation, we characterized
the early transcriptional response to retinoid signaling.
Despite the dramatic consequences initiated by RA expo-
sure, microarray-based gene expression analysis reveals
that only 96 genes are differentially expressed given 8
hours of RA exposure (more than two-fold change, P <
0.01; Additional file 6). Of these, 81 genes are up-regu-
lated. The most prevalent theme in the expression
response is the acquisition of rostro-caudal identity; 12
anterior Hox genes are significantly up-regulated, along
with the Hox co-factors Meis1, Meis2, Pbx2, and other
positioning genes such as Tshz1 and Cdx1. While RARb
is up-regulated, the response also attenuates retinoid sig-
naling via the induction of retinoid metabolism genes
(Cyp26a1, Dhrs3, Rbp1) and a repressor of RAR, Nrip1
[32]. Thirty-five significantly up-regulated genes are
within 20 kbp of a post-RA RAR binding event, including
many of the most differentially expressed genes (Figure 2;
Additional file 6). Exclusively post-RA RAR targets are
no less associated with differential expression than the
constitutively bound targets; while 20 significantly up-
regulated genes are nearby constitutively bound RAR
sites, 15 up-regulated genes are only bound after RA.
RAR binding is associated with RNA polymerase II
initiation
The set of RAR binding sites near differentially
expressed genes represents a small proportion of the
total complement of post-RA RAR binding sites. It is
likely that many other RAR binding sites play regulatory
roles during the retinoid response that are not apparent
from microarray-based differential expression analysis.
We used ChIP-seq to characterize RNA polymerase II
(Pol2) initiation (as signified by Pol2 CTD serine 5
phosphorylation, Pol2-S5P [33-35]) and elongation (as
signified by Pol2 CTD serine 2 phosphorylation, Pol2-
S2P [33-35]) after 8 hours of RA exposure. We identi-
fied 3,409 significant Pol2 initiation events, of which 424
were within 5 kbp of post-RA RAR binding events. Of
these RAR-associated Pol2-S5P events, 402 (95%) are
within 1 kbp of the transcription start sites, or within
the gene body, of 269 known genes and non-coding
RNAs. Significant enrichment of Pol2-S2P is observed
within or at the 3’ end of 214 genes (80%) bound by
RAR and Pol2-S5P, demonstrating that many of these
genes are actively transcribed post-RA (for example, see
Figure 3). Therefore, the correlation between RAR
binding and Pol2 initiation and elongation suggests that
RAR may play a wider role in driving and maintaining
transcription beyond that observed from microarray-
based differential expression analysis. We again find no
evidence that exclusively post-RA RAR binding sites are
less associated with Pol2 initiation than constitutively
bound sites; both sets of sites are coincident with Pol2-
S5P events at similar rates.
A proposed model of RAR functionality suggests that
it acts as a transcriptional repressor in the absence of
RA signaling, and becomes an activator after ligand
binding [7]. To assess the dynamics of RAR’s interac-
tions with Pol2, we compare the post-RA Pol2 ChIP-seq
profiles with Pol2-S2P and Pol2-S5P ChIP-seq data from
the pluripotent state [36]. Of the 424 RAR-associated
Pol2-S5P events characterized post-RA, the majority
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Gene Functions
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RA metabolism
RA signaling
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Rarb
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(liganded)
RAR
(unliganded)
Retinoic
Acid
-RA+RA
Figure 2 Direct binding of RAR mediates the initial response to
RA during early neurogenesis. Genes with more than five-fold
differential expression after 8 hours of RA exposure are listed. RAR
binds to many of the up-regulated genes, with binding more likely
for greater degrees of up-regulation. Red arrows indicate post-RA
RAR binding within 20 kbp of the gene. Black dashed lines indicate
pre-RA RAR binding within 20 kbp. Three functional groups of
genes are indicated by coloring the gene names. Information for all
more than two-fold differentially expressed genes is tabulated in
Additional file 2.
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(390) are also enriched for Pol2-S5P in the pluripotent
state. The pre-RA pattern of RAR binding does not
seem to affect the behavior of Pol2 at these sites; both
constitutive and exclusively post-RA RAR binding sites
are coincident with constitutive Pol2 initiation events at
similar rates. From the 214 RAR-bound genes that dis-
played enrichment for both initiating and elongating Pol2
after RA exposure, 54 (25%) also display evidence of Pol2
elongation in the pluripotent state. Genome-wide, we
find a set of only 27 significant Pol2-S5P initiation events
that are bound by Pol2 after RA exposure but show no
evidence of enrichment in pluripotent cells. Only 11 of
these events are near RAR binding events. Surprisingly,
this compact set of RAR targets for which Pol2 is not
poised in pluripotent cells includes Hoxa1, Cyp26a1,
RARb, and Stra8 (for example, see Figure 3). Therefore,
these critical RA-responsive genes are constitutively
bound by RAR, but Pol2 is only recruited to their promo-
ters after RA exposure.
In summary, our examination of potential interactions
between RAR and Pol2 before and after retinoid expo-
sure adds complexity to the proposed model of RAR
functionality. Only a small set of important retinoid
targets fit the simple model of RAR recruiting Pol2 to
the transcription start site only after RA exposure. Many
more RAR target genes already have poised Pol2 before
retinoid signaling, regardless of whether RAR is consti-
tutively bound. A further set of bound genes is already
being actively transcribed before RA exposure.
RAR binding is associated with ES cell regulatory state
DNA-binding preference alone is not sufficient to
explain the specificity of RAR’s post-RA genomic occu-
pancy. At least 150,000 high-similarity matches to the
DR2 and DR5 motifs do not display significant RAR
binding either before or after RA exposure. One possibi-
lity is that RAR bound sites are distinguished by their
chromatin structure profiles and the occupancy of other
regulatory proteins in the surrounding genomic region.
To assess the regulatory state of RAR binding sites, we
compare constitutively bound sites (by definition occu-
pied both post-RA and in the preceding pluripotent
state) to published ChIP-seq data in mouse ES cells,
including data for multiple TFs, co-factors, histone
modifications, and chromatin modifying proteins
[24,37-41].
Pol2-S2P (+RA)
Pol2-S5P (+RA)
Pol2-S5P (ES)
RAR (+RA)
RAR (-RA)
Pol2-S2P (+RA)
Pol2-S5P (+RA)
Pol2-S5P (ES)
RAR (+RA)
RAR (-RA)
50
50
50
100
100
100
50
50
50
50
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0
0
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5
4
8
4
3 : 6 r h
c
0
0
0
5
7
8
4
3 : 6 r h
c
Figure 3 Constitutive RAR binding without ES cell-poised Pol2 at Stra8 and Rarb. RAR is constitutively bound at these targets, but no
enrichment of poised/initiating polymerase (Pol2-S5P) is observed in ES cells at these loci. Within 8 hours of retinoid exposure, the initiating and
elongating forms of Pol2 are recruited to these genes.
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