PRIMER
5
Development 137, 5-13 (2010) doi:10.1242/dev.036160
Dissecting the regulatory switches of development: lessons
from enhancer evolution in Drosophila
Matthew J. Borok*, Diana A. Tran*, Margaret C. W. Ho and Robert A. Drewell†
Cis-regulatory modules are non-protein-coding regions of DNA
essential for the control of gene expression. One class of
regulatory modules is embryonic enhancers, which drive gene
expression during development as a result of transcription
factor protein binding at the enhancer sequences. Recent
comparative studies have begun to investigate the evolution of
the sequence architecture within enhancers. These analyses are
illuminating the way that developmental biologists think about
enhancers by revealing their molecular mechanism of function.
Introduction
In order to form the tissues of the embryo, the expression patterns of
genes must be tightly directed in space and time. This function is
largely controlled by cis-regulatory modules (CRMs), specific
regions of non-protein coding DNA that regulate genes on the same
chromosome. Specifically, early patterns of gene expression
regulated by CRMs are required to establish the anterior to posterior
axis of the developing embryo. The best-understood CRM is the
enhancer, a genetic switch that is bound by specific transcription
factors (TFs) and is able to drive distinct patterns of transcription of
a target gene in an orientation-independent manner (Dillon and
Sabbattini, 2000; Ptashne, 1986) (see Box 1). The binding of TFs to
enhancers recruits RNA polymerase II and the associated
transcriptional machinery to a gene’s promoter to drive the
transcription of target genes. Notably, some of the components of the
machinery, such as histone acetyltransferases and nucleosome
remodelers, have the ability to modify the chromatin environment in
order to facilitate transcription (Kim and Dean, 2003; Madisen et al.,
1998; Soutoglou and Talianidis, 2002; Utley et al., 1998).
For many years, researchers have sought to identify these modules
and their respective target genes. However, recent interest has shifted
towards a more complex analysis of individual enhancers in order to
elucidate their molecular activity. Key questions include: which TF
binding sites are responsible for functional activity; how these
binding sites are organized; and what their affinities for different TFs
are. The wealth of information that is emerging from the recent
sequencing of many different genomes has provided answers to
some of these questions, but has also brought controversy to the
field. Particularly in insects, as we discuss in this Primer,
evolutionary comparisons of enhancers across different species are
beginning to show us that slight variations in non-coding regulatory
sequences can lead to large phenotypic differences in development
(Gompel et al., 2005; Jeong et al., 2006; Prud’homme et al., 2006).
Such detailed analyses of enhancer evolution allow for a better
understanding of the organizational and structural constraints of a
CRM. In turn, the elucidation of such evolutionary constraints
Biology Department, Harvey Mudd College, 301 Platt Boulevard, Claremont, CA
91711, USA
*These authors contributed equally to this work
†
Author for correspondence ([email protected])
should reveal the nature of TF interactions with regulatory DNA
sequences, as well as what is required for the transcriptional control
of specific genes during development.
In this Primer, we briefly review our current understanding of
classical enhancer function and then delve into recent studies that
have compared enhancers in Drosophila and related insect species
to reveal how evolution acts upon mechanisms of enhancer function.
We focus on the even skipped stripe 2 enhancer (S2E) and
neurogenic ectoderm enhancers (NEEs) in particular, owing to the
lessons that these CRMs can teach us about the general principles of
transcriptional control during embryonic development.
The building blocks of gene regulation: the
functional characteristics of enhancers
Enhancers were first identified in the SV40 viral genome as regions
of non-coding DNA that are crucial for the transcription of adjacent
genes (Banerji et al., 1981; Benoist and Chambon, 1981). Functional
activity of an enhancer depends upon the binding of specific TF
proteins (activators, see Box 2) within the enhancer DNA sequence
that help to recruit RNA polymerase II and associated protein factors
to the promoter of a target gene (detailed in Box 1) (McEwan et al.,
1993; Wildeman et al., 1984). In the years after these initial
observations, enhancers were discovered for many genes in a wide
range of model organisms (Banerji et al., 1983; Shepherd et al.,
1985; Struhl, 1984). In complex eukaryotes, many of the identified
enhancers are responsible for directing spatiotemporally restricted
patterns of gene expression in the developing embryo (Choi and
Engel, 1986; Hiromi et al., 1985). Although a number of enhancers
are able to activate transcription at a given eukaryotic gene, the
regulation of key genes by a restricted set of tissue-specific
enhancers during development must be very carefully controlled
because they encode proteins that specify cellular identities in the
embryo (Caplan and Ordahl, 1978; Lewis, 1978). These crucial
developmental genes cannot, therefore, be expressed ubiquitously.
A number of emerging studies indicate that these specific promoterenhancer interactions are subject to subtle molecular control
mechanisms, including the activity of a novel class of regulatory
module – the promoter tethering element (Akbari et al., 2008;
Akbari et al., 2007; Calhoun et al., 2002; Cande et al., 2009; Fujioka
et al., 2009; Kwon et al., 2009).
In addition to activators, another class of regulatory TFs,
repressors (see Box 2), are able to bind directly to enhancers, in this
case to prevent target gene transcription (Dearolf et al., 1989;
Stanojevic et al., 1989). Although the molecular roles of activators
and repressors in promoting or preventing the recruitment of RNA
polymerase II to gene promoters are not completely understood,
some mechanisms for the functional activity of enhancers have been
characterized. The predominant theory is that activators directly
interact with components of the basal transcription machinery via
specific protein domains, such as glutamine-rich, proline-rich and
acidic domains or hydrophobic  sheets (McEwan et al., 1993); for
a detailed review, see Kadonaga (Kadonaga, 2004). There is also
DEVELOPMENT
Summary
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Development 137 (1)
Box 1. Defining a functional enhancer cis-regulatory
module
A
Enhancer
Promoter
Gene
Enhancer
Promoter
Gene
B
C
Enhancer
Promote
r
Gene
D
Enhancer
NR
NR
HAT HAT
Promote
r
Gene
An enhancer is an orientation-independent region of non-proteincoding DNA in the genome that is associated with a promoter and
target gene (A). Transcription factor (TF) proteins (purple spheres)
bind to the enhancer (B) and interact with the transcriptional
machinery (red, green and pink spheres) (C) and eventually recruit
RNA polymerase II (gray ellipse) at the promoter of target genes to
enhance the transcription of the gene (D). The TFs bound at an
enhancer often also recruit chromatin-modifying enzymes such as
histone acetyltransferases (HAT, yellow ellipses) and nucleosome
remodeling factors (NR, orange spheres) that facilitate changes to
the chromatin environment to allow transcription to proceed.
Historically, enhancers were often initially identified in genetic
screens, as mutation of the enhancer module disrupts TF binding and
results in an associated loss of target gene expression. Later, the
development of transposon-mediated mutagenesis in Drosophila
and plants allowed more sophisticated ‘enhancer trap’ reporter gene
constructs to be used. Enhancers identified in these types of genetic
screens include those in the bithorax complex of D. melanogaster
responsible for regulating the Abdominal B gene (Celniker et al.,
1990; Karch et al., 1985) [for a detailed review on enhancer trapping
see Bellen (Bellen, 1999)]. The functional sequences of the enhancer
module are often resolved in detailed transgenic reporter gene
studies [for early resolution of the eve stripe enhancers see papers by
Goto and Harding (Goto et al., 1989; Harding et al., 1989)]. More
recently, global genome-wide approaches to identifying TF binding
sites, such as by chromatin immunoprecipitation combined with a
tiled genomic microarray (ChIP on chip) (Visel et al., 2009; Zeitlinger
et al., 2007) or bioinformatic analysis using comparative genomics
to identify genomic regions with potential cis-regulatory function
(Nobrega et al., 2003; Peterson et al., 2009), have been successful at
identifying an increasing number of enhancers.
extensive evidence that sequence-specific binding of activator TFs
to individual enhancer CRMs leads to the recruitment, via protein
interactions with co-activator enzymes, of an extensive number of
components of the RNA polymerase II transcriptional machinery,
including the Mediator complex. The Mediator complex is highly
conserved in eukaryotes from yeast to humans, although its
individual components can vary when assembled at individual
enhancers (Kadonaga, 2004). The Mediator complex facilitates
interactions between the enhancer and chromatin-modifying
enzymes, such as histone acetyltransferases and nucleosome
remodeling factors, to establish a chromatin environment that
facilitates transcription of the target gene (see Box 1). By contrast,
repressors may disrupt enhancer functional activity in one of two
ways: (1) competition, where the binding sites for repressors and
activators within an enhancer sequence overlap and, as a result,
repressor binding excludes activators; and (2) quenching, in which
repressors are able to inhibit the regulatory activity of activators
bound to nearby sites within the enhancer (Gray et al., 1994;
Kirchhamer et al., 1996; Levine and Manley, 1989; Small et al.,
1991b).
In metazoan species, enhancer CRMs are major players in the
developmental cascade that transforms the early embryo from a
mass of uniform, undifferentiated cells into a segmented and highly
organized structure of differentiated cells. In Drosophila, the
dynamic developmental specification of the embryonic body plan
and of differentiated cell fates is accomplished by a combination of
early spatiotemporal expression gradients of activator and repressor
TFs that act upon downstream embryonic enhancers. Based on the
timing of their expression during embryonic development, the genes
in this cascade represent four basic developmental TF families:
maternal, gap, pair-rule and homeotic genes (described in detail in
Box 3) (for a review, see Sauer et al., 1996). At the top of the
cascade, maternal mRNAs are deposited in the unfertilized egg cell
during oogenesis (Berleth et al., 1988; Steward et al., 1988).
Spatially restricted translation of localized maternal mRNAs in the
fertilized egg establishes TF gradients in the embryo. In turn,
maternal TFs bind at target embryonic enhancers for gap genes,
directing gap TF expression patterns in the developing embryo
(Driever and Nüsslein-Volhard, 1988; Struhl et al., 1989) (Fig. 1A).
Gap TFs further regulate downstream target genes, such as those for
pair-rule and homeotic TFs (Qian et al., 1991; Stanojevic et al.,
1989). At each step in the cascade, gene expression patterns are
controlled by specific clusters of activator and repressor binding
sites within embryonic enhancers (see Box 2). Fine-tuning of
transcription is mediated by the specific molecular properties of
individual enhancer CRMs. Whether a given TF acts as an activator
or repressor when it binds to an embryonic enhancer can be context
dependent (Ip et al., 1991; Small et al., 1996). In addition, DNA
sequences within embryonic enhancers may bind TFs with varying
affinities (Jiang et al., 1991; Struhl et al., 1989). The functional
consequence is that enhancers may require different threshold
concentrations of interacting activators and repressors in order to
regulate transcription of their target genes. The central role of
enhancers in the regulatory cascade responsible for development of
the Drosophila embryo makes in-depth analysis of their function
crucial to the field of developmental biology.
A CRM paradigm: even skipped stripe 2 enhancer
Probably the best-characterized embryonic CRM is the even skipped
(eve) S2E in Drosophila melanogaster (see Box 2). eve is a pair-rule
gene that is expressed under the control of various neighboring CRMs
in seven distinct stripes in the developing embryo (Macdonald et al.,
1986) (Fig. 1B-D). The pattern of eve expression that S2E directs is
entirely derived from the combination of transcription factor binding
sites (TFBSs, see Box 2) embedded within its DNA sequence (Small
et al., 1991b) (Fig. 1). Pioneering studies in the laboratory of Michael
Levine twenty years ago discovered that twelve different binding sites
for Kruppel (KR), Giant (GT), Bicoid (BCD) and Hunchback (HB)
DEVELOPMENT
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Development 137 (1)
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7
A
B
Hunchback
Kruppel
Giant
1 kb
eve
St 3+7
St 2
St 4+6
St 1+5
C
D
eve stripe 2
Fig. 1. Transcription factor gradients activate even skipped
expression. (A)The localization patterns of four major transcription
factors (TFs) in the early Drosophila embryo are shown. Embryos are
oriented anterior to the left and dorsal is up. The Bicoid (blue) and
Hunchback (purple) TFs are activators of the even skipped (eve) stripe 2
enhancer, whereas the Kruppel (red) and Giant (green) TFs are
repressors. Both activators are broadly expressed in the anterior half of
the embryo, but repressor expression is more restricted. (B)Map of the
eve genomic locus, including the embryonic enhancers (orange)
responsible for eve (black) expression. Each enhancer drives eve
expression in one or two developing parasegments of the embryo.
Together, the enhancers drive expression in seven stripes (St 1-7) in
odd-numbered parasegments. (C)The minimal eve stripe 2 enhancer (St
2) of D. melanogaster contains twelve transcription factor binding sites.
The locations of color-coded binding sites for the activators and
repressors shown in A are indicated. Two clusters of binding sites (black
bars) at each end of the minimal stripe 2 module are thought to be
particularly important for functional activity. (D)Sharp borders of eve
stripe 2-directed expression are established by high concentrations of
Giant (green) at the anterior boundary and Kruppel (red) at the
posterior boundary.
TFs are located in the minimal S2E, a 480 bp region of the CRM that
is sufficient to drive eve stripe 2 expression (Fig. 1C) (Goto et al.,
1989; Small et al., 1991b; Stanojevic et al., 1989). Notably, eight of
the twelve TFBSs form two tight clusters at each end of the minimal
S2E. Each cluster has closely spaced activator binding sites, each of
which strongly overlaps with a repressor binding site (Fig. 1C). These
observations suggested two important ideas regarding the molecular
architecture of an enhancer: (1) clustering of TFBSs is crucial to
enhancer function; and (2) the overlap between activator and repressor
TFBSs enables the characteristically sharp boundaries between stripes
of gene expression that are driven by many early embryonic enhancers
(Fig. 1D).
Analysis of the TF gradients present in the developing Drosophila
embryo reveals how S2E directs eve expression with such sharp
boundaries. Activation of S2E-driven expression of eve occurs
through the binding of the HB and BCD activators (see Box 3),
Box 2. Glossary of specialized terms
Activator. A transcription factor that, when bound within a
particular enhancer, is able to recruit the transcriptional machinery
to upregulate transcription of a target gene under control of the
enhancer.
Cluster. A group of closely spaced transcription factor binding sites
thought to be the hub of enhancer activity. Clusters usually include
binding sites for both activators and repressors.
Eve stripe 2 enhancer (S2E). An extensively studied embryonic
enhancer of the even skipped gene.
Neurogenic ectodermal enhancer (NEE). A specific type of
enhancer that directs gene expression in the neurogenic ectoderm.
Spatial and temporal expression of NEE-driven genes depends upon
the activators Dorsal and Twist, and the repressor Snail.
Orthologous gene. A gene derived from the gene of a common
ancestor.
PATSER. A pattern search program that identifies potential
transcription factor binding sites in a given DNA sequence using a
position-weighted matrix.
Position-weighted matrix (PWM). Generated from a compilation
of experimentally verified binding site sequences for a specific
transcription factor (TF), it is a mathematical matrix that indicates the
probability of finding a specific nucleotide at each position in the
binding site.
Repressor. A TF that, when bound within a particular enhancer,
blocks transcription of the target gene.
Transcription factor binding site (TFBS). A sequence of DNA
known to be bound by a specific TF protein that influences
transcription of nearby target genes.
Two-dimensional similarity plots. A graphical method that
identifies regions of similarity between two sequences.
which are present at high concentrations in the anterior of the
embryo (Fig. 1A). Binding of the KR and GT repressors (see Box
3), which are present in distinct non-overlapping patterns in the
anterior half of the embryo, prevents activation of S2E (Small et al.,
1991b) and thus delineates the boundaries of the second stripe of eve
expression (Fig. 1D). As a result, in the presumptive second
abdominal segment (the spatial interval in the embryo where HB and
BCD activators are present and KR and GT repressors are absent),
HB and BCD are able to occupy activator binding sites in S2E
(Small et al., 1991a) (Fig. 1C). With strong activation and no
repression, S2E only drives eve expression in a narrow 2- to 3-cellwide region of the developing embryo (Macdonald et al., 1986) (Fig.
1D). By integrating the study of the spatial distribution of TFs in the
embryo, the binding of these TFs at specific enhancers and the
resulting patterns of target gene expression, we begin to see how an
embryonic enhancer is analogous to a central processing unit. The
enhancer must be capable of receiving input signals from TFs and
of outputting a response in the form of directing a very specific
spatiotemporal pattern of target gene expression.
Evolution of the eve stripe 2 embryonic enhancer
Recent evolutionary analyses of TFBSs within enhancer sequences
and their relative rate of evolutionary turnover are beginning to
provide a useful perspective on the molecular mechanisms of
enhancer function. Early comparative studies in the Kreitman
laboratory concentrated on evolutionary analysis of the sequence of
S2E across several Drosophila species, including D. melanogaster,
D. yakuba, D. erecta and D. pseudoobscura (see Fig. 2A for
phylogenetic relationship). Their studies revealed that all of the
Drosophila species investigated possess an orthologous early
embryonic enhancer (see Box 2) capable of driving eve expression
DEVELOPMENT
Bicoid
PRIMER
Development 137 (1)
Box 3. Key players in anterioposterior patterning in
early Drosophila development
MAT
Time
Gap
Pair-rule
Segment polarity
Homeotic
Early in Drosophila embryonic development, the initial anterior-toposterior morphogen gradient comprises maternally deposited (MAT)
mRNAs in the unfertilized egg, including bicoid and hunchback.
Translation produces the MAT transcription factors (TFs), which can
activate or repress spatiotemporally restricted patterns of expression
of gap genes, such as Kruppel, knirps and giant. In turn, these gap
TFs regulate downstream expression of pair-rule genes, such as eve,
in stripes in the developing Drosophila embryo. Pair-rule TFs regulate
segment polarity genes further downstream in the cascade. In
addition, gap and pair-rule TFs both regulate the expression of the
homeotic genes, which ultimately define segmental identity.
Maternal genes
Bicoid (BCD). The unfertilized egg contains a broad, maternally
deposited distribution of bicoid mRNA, which is transported to the
anterior pole of the developing embryo prior to translation. After
translation, BCD protein diffuses from a high concentration at the
anterior end across the embryo and acts as an activator of
hunchback.
Hunchback (HB). The hunchback gene is activated by BCD in the
anterior half of the embryo. Maternally deposited hunchback mRNAs
also help to produce this protein in the embryo. HB protein functions
as a regulator of other gap genes and as a threshold level-dependent
activator or repressor at different enhancers of the pair-rule genes,
including even skipped (eve).
Gap genes
Kruppel (KR). The Kruppel gene is repressed by high levels of HB at
the anterior end of the embryo, leading to its localization in the
middle of the embryo, where HB levels begin to diminish. KR protein
functions as a repressor of downstream target genes, including eve.
Knirps (KNI). The knirps gene is repressed by intermediate levels of
HB, and thus KNI protein is present in a peak posterior to that of KR.
KNI functions as a repressor of eve.
Giant (GT). The giant gene is repressed by low levels of HB, and is
therefore only expressed in one anterior and one posterior region of
the embryo, where HB is not present. GT protein functions as a
repressor of eve.
Pair-rule genes
Even skipped (EVE). The eve gene is expressed in seven stripes in
the developing embryo. eve enhancers are activated by BCD and HB,
and repressed by HB, KR, KNI and GT. EVE protein establishes
segmentation in the embryo.
in a similar spatiotemporal stripe 2 pattern when tested in transgenic
D. melanogaster (Ludwig et al., 1998). This functional conservation
partially extends to chimeric enhancers created by juxtaposing two
halves of the S2E, each half taken from a different Drosophila
species, which are capable of directing a similar pattern of gene
expression in transgenic D. melanogaster (Ludwig et al., 2000).
However, the expression directed by the chimeric enhancers is not
identical to the activity of the endogenous eve S2E. In some embryos
carrying the chimeric enhancers, reporter gene expression undergoes
a posterior shift or expansion (Ludwig et al., 2000), suggesting that
nuanced enhancer architecture might be encoding important
functional information. Bioinformatic analysis revealed that the
TFBSs contained within the S2E are not well-conserved between
orthologs (Ludwig and Kreitman, 1995; Ludwig et al., 1998). There
is, in fact, significant turnover of TFBSs between the Drosophila
S2E orthologs studied: of the seventeen known binding sites
analyzed in the 798 bp full-length S2E from D. melanogaster, only
three are completely conserved at the sequence level in the other
three Drosophila species studied (Ludwig et al., 1998) (Fig. 2B). In
addition, none of the sixteen binding sites that were surveyed from
the full-length D. melanogaster S2E are conserved in all thirteen
sequenced Drosophila species (Ludwig et al., 2000). Detailed
sequence alignment revealed that binding sites for BCD, HB, KR
and GT identified in D. melanogaster are subject to extensive
nucleotide substitutions in other Drosophila species (Ludwig et al.,
2000; Ludwig et al., 1998). The key question therefore becomes:
how is the functional activity of the enhancer preserved at all,
despite the evolutionary turnover of TFBSs within the CRM?
Some resolution to this intriguing issue is being provided by
exciting new studies that have expanded the evolutionary scope of
S2E analysis. These studies analyze evolutionarily divergent species
outside of the Drosophila genus (Fig. 2). Drosophila species
generally have small, compacted genomes with relatively uniformly
conserved non-coding DNA. In comparison, species of the true fruit
flies (Tephritidae family, Sepsid species) have genomes that are
between 4- and 6-times larger than that of D. melanogaster and
contain blocks of conserved non-coding sequence flanked by
regions that are poorly conserved (Peterson et al., 2009). The
increased evolutionary divergence between these species, combined
with the differences in overall genome structure, enable a more
accurate measure of TFBS turnover and of the functional
significance of sequence conservation.
Recent studies have compared the eve regulatory locus in
Drosophila with that of Sepsid species, which diverged
approximately 100 million years ago (Hare et al., 2008a) (Fig. 2A).
Nevertheless, eve is expressed in the same seven transverse stripes
in Sepsid embryos as in Drosophila embryos (Hare et al., 2008a). In
addition, many of the TFs upstream in the developmental cascade,
including HB, GT and KR, are also expressed in conserved
embryonic patterns in the Sepsid species Themira minor. Despite the
fact that eve regulatory regions are found to be only minimally
conserved between these groups, Sepsid eve enhancers identified
through clusters of HB, Caudal (CAD), Knirps (KNI), KR and BCD
binding sites are able to drive conserved gene expression patterns in
transgenic D. melanogaster.
Functional conservation of the Sepsid and Drosophila S2E
orthologs despite relatively low sequence conservation indicates that
some other shared molecular property is responsible for their
activity. The identity of this common molecular mechanism is
currently the focus of active research and debate. Bioinformatic
analysis in the Eisen laboratory demonstrates that there has been
large-scale reorganization of the TFBSs in the eve enhancers from
different Drosophila and Sepsid species (Fig. 2B), indicating that
the spatial organization of the binding sites within enhancer regions
may not be crucial (Hare et al., 2008a). However, detailed analysis
of the architecture of S2E orthologs reveals the existence of 20-30
bp blocks of highly conserved sequence enriched in pairs of
neighboring or overlapping TFBSs. This suggests that the relative
position of binding sites to one another might be more important
than their overall spatial arrangement within an enhancer (Hare et
al., 2008a) (Fig. 2B).
DEVELOPMENT
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Development 137 (1)
PRIMER
A
9
B
eve stripe 2 enhancer binding sites
D. melanogaster
D. simulans
D. yakuba
D. erecta
D. pseudoobscura
D. virilis
Sepsis cynipsea
?
125
100
75
50
25
Million years ago
Themira minor
0
BCD HB GT KR SLP1
Fig. 2. Phylogenetic relationship and eve stripe 2 enhancer architecture of Drosophila and Sepsid species. (A)The Drosophila (green box)
diverged from the Sepsid family (blue box) approximately 100 million years ago (Mya). The distantly related Drosophila species, D. melanogaster and
D. virilis, diverged 60 Mya (Tamura et al., 2004). (B)The organization of a subset of bioinformatically predicted binding sites for the transcription
factors Bicoid (BCD, blue), Hunchback (HB, purple), Giant (GT, green), Kruppel (KR, red) and Sloppy paired 1 (SLP1, yellow) within the minimal eve
stripe 2 enhancer [summarized from Hare (Hare et al., 2008a)] are shown for eight species: D. melanogaster, D. simulans, D. yakuba, D. erecta,
D. pseudoobscura, D. virilis, Sepsis cynipsea and Themira minor.
S2E has been built, which could support the idea that this CRM is a
loose cluster of TFBSs with as yet undefined organizational
requirements. Further analysis of S2E will continue to shed light on
the molecular mechanisms of enhancer regulation. In the meantime,
studies of additional Drosophila CRMs may be beginning to
elucidate this issue.
Molecular motifs in neurogenic ectodermal
enhancer architecture
The functional significance of TFBSs has also been investigated in
NEEs (see Box 2), which are crucial for dorsoventral patterning of
the neurogenic ectoderm in the developing Drosophila embryo
(Erives and Levine, 2004). The gene expression patterns directed by
this class of CRMs are mediated largely by three TFs: the activators
Dorsal (DL) and Twist (TWI) and the repressor Snail (SNA)
(reviewed by Rusch and Levine, 1996). The genomes of D.
melanogaster, D. pseudoobscura and D. virilis were analyzed for
the presence of a consensus NEE motif comprising several
sequences, including closely spaced binding sites for DL and TWI
and a SNA binding site that overlapped with the TWI binding site
(Crocker et al., 2008b). Using their consensus motif, the authors
identified five NEEs in D. melanogaster, five in D. virilis and four
in D. pseudoobscura. In agreement with the findings of Hare et al.
for the eve gene (Hare et al., 2008a), orthologous NEE-driven genes
from different Drosophila species show nearly identical embryonic
expression patterns. However, when the bioinformatically identified
NEEs were tested in transgenic D. melanogaster, they produced
different patterns of expression. Although the predicted NEEs
directed expression in a band on the ventral side of the embryo
extending along the entire anterioposterior axis (as is normal), the
number of nuclei in the band was not consistent. As the
computational criteria by which the NEEs were identified depended
on homology to a consensus sequence, the orthologous NEEs were
very similar in their organization of DL, TWI and SNA TFBSs.
These results raise a fundamental question: how are the endogenous
NEE-driven patterns of gene expression the same in all species,
whereas the identified NEE orthologs direct different patterns of
reporter gene transcription in transgenic D. melanogaster?
A crucial difference between NEE orthologs that appears to
regulate the distinct functional activity of these enhancers from
different Drosophila species is the spacing between DL and TWI
DEVELOPMENT
Several crucial issues were raised following the conclusions
drawn from the initial studies of the orthologous insect S2Es (Hare
et al., 2008a). A primary concern is that the S2E sequences in
Drosophila and Themira are not as diverged as originally indicated,
and therefore the lack of sequence homology does not indicate a lack
of conserved TF organization. Using two-dimensional similarity
plots (see Box 2), Crocker and Erives aligned the S2Es from
Drosophila melanogaster and Themira putris and found extensive
homology between a series of specific 14-41 bp stretches of
sequence. The order of these sequence blocks is conserved across
the entire length of the enhancer, suggesting that their position
relative to each other is crucial. As a result, these authors argue that
these sequences harbor binding sites for KR, BCD and GT and thus
that the TFBS architecture is largely unchanged between these insect
families (Crocker and Erives, 2008a). In response, Hare et al. note
that the plots only align half of the minimal S2E region; by contrast,
the other half of S2E exhibits little or no conservation despite its
necessity for proper functional activity of the S2E module (Hare et
al., 2008b). Furthermore, computational studies using PATSER [a
bioinformatics tool that can be used to scan a given DNA sequence
with a position-weighted matrix (PWM, see Box 2) representing a
consensus binding site for a specific transcription factor] to predict
TFBSs within S2E orthologs reveals that an extensive genomic
reorganization of binding sites has occurred between Drosophila and
Sepsid families. Indeed, 24 of the TFBSs analyzed in this study are
not conserved between Drosophila and Sepsids, despite the fact that
many of these sites bind conserved proteins known to regulate eve
expression through S2E, such as HB, BCD, GT, KR and an
additional pair-rule repressor TF, Sloppy paired 1 (SLP1) (Andrioli
et al., 2002; Hare et al., 2008b). The studies by Hare et al. suggest
that TFBSs are the essential molecular components within enhancer
regions that, even when extensively reorganized, can still function
to produce the same patterns of expression (Hare et al., 2008a; Hare
et al., 2008b).
Overall, there exist caveats to the use of sequence alignments,
including similarity plots, which can overemphasize weak
homology across short stretches of sequence that are sometimes near
the threshold for statistical significance. Although sequence analysis
and alignment approaches can be useful in the identification of
potential regions of conservation, the biological relevance of such
alignments is not easily deciphered. To date, no functional synthetic
A
PRIMER
Development 137 (1)
C
Drosophila melanogaster
+2 bp
B
D
Drosophila virilis
Fig. 3. Changing regulatory environments can drive
compensatory changes in enhancer organization. In (A) D.
melanogaster and (B) D. virilis, the Dorsal (orange) and Twist (gray)
activators cooperatively bind to neurogenic ectoderm enhancer (NEE)
sequences (green) to upregulate the transcription of target genes
(arrow). This interaction is dependent upon compatibility between the
transcription factors and their respective binding sites. In the ~60
million years of evolution separating D. virilis and D. melanogaster
(Tamura et al., 2004), protein sequences have diverged only slightly, but
enough to affect the DNA-binding domains of the proteins. As a result,
in the vnd NEEs of (C) D. melanogaster and (D) D. virilis, the spacing
between a conserved Dorsal binding site (orange boxes) and a
conserved Twist binding site (gray boxes) is different. The intervening
sequence between the binding sites is only 37.5% conserved, but there
is also a deletion of two nucleotides (white boxes) in the D. virilis NEE.
Even small changes in transcription factor protein domains between
different species can drive the architectural reorganization of DNA
binding sites. In this case, a decrease in the spacing between sites
preserves enhancer functional activity.
activator binding sites. For example, in the D. melanogaster ventral
nervous system defective (vnd) NEE, the DL and TWI TFBSs are
separated by 10 bp (Fig. 3A,C). In the orthologous D. virilis vnd
NEE, the distance between the sites is shortened to 8 bp (Crocker et
al., 2008b) (Fig. 3B,D). Could this relatively minor modulation of
TFBS architecture have a pronounced influence on functional
activity? In several experiments, Crocker and colleagues found that
adding or deleting just a few base pairs between the two TFBSs in
an NEE from one species to mimic the spacing in a second species
(or another NEE from the same species) is in fact enough to recreate
the pattern of gene expression directed by the second NEE (Crocker
et al., 2008a; Crocker et al., 2008b). What could be directing these
subtle modifications in CRM architecture? Previous studies suggest
that slight differences in the trans environment of different species
can drive evolutionary changes at the sequence level in CRMs
(Gasch et al., 2004; Ronshaugen et al., 2002). A close investigation
of DL and TWI protein sequences has revealed that several changes
have occurred between the species, including one mutation in D.
melanogaster DL that is known to affect DL-TWI gene activation
(Jia et al., 2002). Therefore, the way in which these two proteins
interact with their DNA binding sites, and thus with each other
indirectly, has probably slightly changed between D. virilis and D.
melanogaster (Fig. 3). These modifications at the protein level in
turn lead to the co-evolution of the regulatory sequences bound by
these TFs (Fig. 3). The pattern of expression of NEE-driven genes
is crucial for the development of the fly body plan, so we can assume
that any change in expression of NEE-target genes would probably
be strongly selected against in evolution. Thus, as the trans
environment changes, natural selection also demands compensatory
changes in the cis-regulatory sequences. In the case of the NEEs, cisevolution appears to act by modulating the spacing of binding sites
to ensure that functional activity of the CRM is maintained in a given
species (Fig. 3). Accordingly, when NEEs are tested in a different
species the trans environment will be different and thus the NEEs
will not respond to the upstream TFs with the same transcriptional
outputs.
Is transcription factor binding site organization
crucial for enhancer function?
The current debate over the functional significance of TFBS
organization in CRMs highlights a key mystery in developmental
biology: how exactly does an enhancer function at the molecular
level? Without a doubt, the TF binding sites within an enhancer are
crucially important. However, studies of the eve stripe 2 regulatory
module suggest that binding site turnover occurs frequently in
evolution, resulting in changes to the overall number and
architecture of TFBSs within orthologous enhancers. Surprisingly,
these pronounced differences appear to have no significant effect on
the regulation of target gene transcription, suggesting that enhancers
are under selective pressure to be functionally robust to evolutionary
changes at the sequence level. Our own studies on early embryonic
enhancers from the homeotic genes in Drosophila (Ho et al., 2009)
have corroborated these discoveries. In general, enhancers from
different species maintain their conserved functional activity despite
significant modulation of their sequence architecture during
evolution.
Can we use these observations of enhancer activity to develop
a clearer idea of the molecular function of CRMs? Recent studies
do in fact lend support to the information display/billboard model
of enhancer function (Fig. 4A). The information display/billboard
model suggests that an enhancer functions not as a single large
processor, but as a series of autonomous signals, each of which
can have an effect on target gene transcription (Arnosti et al.,
1996; Kulkarni and Arnosti, 2003). In this model, as long as there
is never a net decrease in activator and repressor TFBSs, the
enhancer can continue to function normally. The only requirement
is for the presence of the correct combination of TFBSs
somewhere in the enhancer sequence (Fig. 4A). Could this loose
requirement be all that is needed to establish a precise response to
the complex binding of multiple TFs at a CRM? Hare and
colleagues concede that certain binding site clusters may not be
so flexible (Hare et al., 2008a). Functionally linked sites, such as
the tight clusters at either end of the minimal S2E, may not be
subject to an equivalent rate of evolutionary turnover as more
independent sites within the CRM. This is certainly the case with
the NEEs, where even slight changes in the spacing between
binding sites can produce large differences in target gene
expression in the same trans environment (Crocker et al., 2008b).
The most parsimonious explanation for these conflicting
discoveries is that individual CRMs may in fact be very
idiosyncratic. Enhancers may function differently depending
upon the TFs by which they are bound and the genes they
regulate. This might reflect the unique properties of specific TFs.
Different TFs will use distinct molecular interactions to bind to
DNA and the mechanisms by which they promote transcription
will also vary. Innovative work from the Carroll laboratory on
CRMs that regulate yellow gene expression in wing pigmentation
in different Drosophila species has demonstrated that enhancers
with novel regulatory activity can in fact be generated by
modifications to TF binding (Gompel et al., 2005) (reviewed by
Prud’homme et al., 2007). In a recent study investigating the
evolution of Dorsal target enhancers in Drosophila, the mosquito
Anopheles gambiae and the flour beetle Tribolium castaneum,
putative orthologous enhancers were found to lack sequence
conservation. However, what does appear to be under
DEVELOPMENT
10
Development 137 (1)
PRIMER
A Information display/billboard
Enhancer
11
B Enhanceosome model
Enhancer
OFF
Enhancer
Enhancer
Enhancer
ON
Enhancer
Enhancer
Enhancer
Partially ON
Enhancer
Fig. 4. Information display/billboard and enhanceosome models of cis-regulatory module function. (A)In the information display/billboard
model, the transcriptional machinery (gray shape) samples the regulatory landscape found at an enhancer module (blue box) (Kulkarni and Arnosti,
2003). If the basal transcriptional machinery encounters only repressors (orange hexagons), the target gene will not be upregulated. The binding of
only activators (purple ellipses) at the regulatory module will result in strong target gene expression. A combination of signals from specific DNA
sites being bound by repressors and activators will lead to an intermediate (or more probably spatiotemporally restricted) level of target gene
activity. An information display enhancer is tolerant of evolutionary binding site turnover because the transcriptional machinery samples only
discrete regions of the enhancer and the output signal will not necessarily differ if transcription factor binding sites are located in different regions
within the enhancer sequence. (B)In an enhanceosome, the enhancer can only function when a large regulatory protein complex has assembled
(purple, blue, green and pink ellipses). If a repressor (orange hexagon) occludes binding sites, or if one of the components of the complex is absent,
the enhancer will not drive target gene expression. An enhanceosome will not tolerate binding site turnover, as the protein complex is extremely
stereospecific.
do not appear to have significant binding site turnover, these CRMs
are able to tolerate slight shifts in TFBS position within the module.
Indeed, it seems that evolution may have favored these shifts in
response to a changing trans environment between different species.
In the case of the NEEs, the binding site architecture of a CRM is
able to co-evolve with a changing regulatory environment,
demonstrating that molecular mechanisms of enhancer function can
be plastic. These recent studies demonstrate that evolution can
impact enhancers via a number of distinct mechanisms including,
but not limited to, reorganization of the total number, binding
affinity and spatial arrangement of TFBSs. These discoveries have
increased our understanding of the precise molecular mechanisms
of enhancer-mediated transcriptional control for a number of key
genes during development.
Conclusions
Future studies now need to ask precisely what it is within the
enhancer sequence, or the genomic context, or the TFs themselves,
that contributes to the differences in functional activity between
enhancers. These questions will be answered largely through a
combination of detailed bioinformatic analyses of genomic
sequences and functional tests across related eukaryotic species. As
our knowledge of TF binding activity during embryogenesis
increases, computational tools, including position-weight matrices,
can be used to find binding sites that are conserved between species
and to compare their distribution in genomic DNA sequences. In
vivo functional studies should follow such investigations, guided by
rapid cell-based assays to screen potential sequences. Although
these types of experiments have been performed on a few enhancers,
the next step is to apply the same techniques to the ever-growing
array of other known enhancers. In addition, expanding our current
catalog of known enhancer CRM orthologs to other insects and
mammals/vertebrates would greatly contribute to our understanding
DEVELOPMENT
evolutionary constraint is the position of these enhancers relative
to their target gene. As a result, as these enhancers are modified
over evolutionary time, they become capable of directing
divergent patterns of gene expression (Cande et al., 2009).
In support of the idea of idiosyncratic CRMs, there are
examples of enhancers that do not appear to fit well with the
information display/billboard model. In vertebrates, the number
of well-characterized enhancers is low and few examples support
the information display model perfectly. For example,
mammalian sequences with multiple binding sites for the GATA1
TF will continue to function as an enhancer if only a single
binding site is intact. However, a very particular site must remain
intact: eliminating two of the three GATA1 sites in an enhancer
may not have any effect on its function, but the single loss of the
third crucial site can completely abolish enhancer activity in mice
(Cheng et al., 2008). This example stresses the functional
importance of a subset of possible binding sites, although the
issue of overall TFBS architecture at mammalian enhancers
remains largely unexplored.
Perhaps the best-studied counter-example to the information
display/billboard model in vertebrates is the interferon  (IFNB1)
enhanceosome. The IFNB1 gene is upregulated in mammals in
response to viral infection and the enhancer that directs this
expression requires a very specific set of TFs to carry out this
regulatory activity (Thanos and Maniatis, 1995). Every TF
component is required for the enhanceosome to successfully form
and to mediate the upregulation of the IFNB1 gene (Panne et al.,
2007) (Fig. 4B). As a result of this highly structured enhancer-TF
complex, there has been virtually no evolutionary binding site
turnover at this CRM throughout 100 million years of mammalian
evolution. The architectural constraints on the Drosophila NEEs
may be somewhere between the two extremes presented by the
IFNB1 enhanceosome and the S2E orthologs. Although the NEEs
PRIMER
of the evolution of CRM function. Only by exploring the evolution
of a wide range of different enhancers can we hope to understand
exactly how these CRMs function and direct the precise patterns of
gene expression that give rise to the elegant complexity of
embryonic development.
Acknowledgements
Research in our laboratory is supported by funding to R.A.D. from the National
Science Foundation and National Institutes of Health and a Howard Hughes
Medical Institute Undergraduate Science Education Program grant to the
Biology department at Harvey Mudd College. M.J.B. was supported by an
Engman award. D.A.T. was supported as an Arnold and Mabel Beckman
Foundation Scholar. M.C.W.H. received support from the Merck-American
Association for the Advancement of Science (AAAS) Undergraduate Science
Research Program. Deposited in PMC for release after 12 months.
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