Precise temporal control of the
eye regulatory gene Pax6 via
enhancer-binding site affinity
Yingzi Yue,1Martha L. Bulyk,1,2,3,5
and Richard L. Maas1,6
1Division of Genetics, Department of Medicine, Brigham
and Women’s Hospital and Harvard Medical School, Boston,
Massachusetts 02115, USA;2Department of Pathology, Brigham
and Women’s Hospital and Harvard Medical, School, Boston,
Massachusetts 02115, USA;3Harvard-Massachusetts Institute
of Technology Division of Health Sciences and Technology
(HST), Harvard Medical, School, Boston, Massachusetts
How transcription factors interpret the cis-regulatory
logic encoded within enhancers to mediate quantitative
changes in spatiotemporally restricted expression pat-
terns during animal development is not well understood.
Pax6 is a dosage-sensitive gene essential for eye de-
velopment. Here, we identify the Prep1 (pKnox1) tran-
scription factor as a critical dose-dependent upstream
regulator of Pax6 expression during lens formation. We
show that Prep1 activates the Pax6 lens enhancer by
binding to two phylogenetically conserved lower-affinity
DNA-binding sites. Finally, we describe a mechanism
whereby Pax6 levels are determined by transcriptional
synergy of Prep1 bound to the two sites, while timing of
enhancer activation is determined by binding site affinity.
Supplemental material is available at http://www.genesdev.org.
Received November 24, 2009; revised version accepted
March 17, 2010.
The precise spatiotemporal patterns of metazoan gene
expression are controlled by cis-regulatory modules
(CRMs), which are regions of DNA, typically a few
hundred base pairs long, that comprise binding sites for
transcriptional regulators (Yuh et al. 1998; Arnosti and
Kulkarni 2005; Davidson and Levine 2008). While current
genomic approaches have traditionally focused on identi-
fying the highest-affinity transcription factor (TF)-binding
sites, new genome-wide location analyses have demon-
strated widespread TF binding in the absence of high-
affinity sites, thus forcing a re-evaluation of the scope and
regulatory significance of lower-affinity sites (Hollenhorst
et al. 2007; Gordan et al. 2009).
The affinity of a binding site provides a mechanism to
respond to different concentrations of TFs. In develop-
ment, the ability of CRMs that contain sites of differing
affinities to interpret TF gradients has been studied in
Drosophilia melanogaster and other organisms for spatial
gradients (Driever et al. 1989; Struhl et al. 1989; Jiang and
Levine 1993; Scardigli et al. 2003; Segal et al. 2008), and
in Caenorhabditis elegans and sea urchin for temporal
gradients (Lai et al. 1988; Gaudet and Mango 2002). For
example, in C. elegans, altering PHA-4 DNA-binding
sites to higher- or lower-affinity sequences altered the
onset of pharyngeal gene expression, with higher-affinity
sites directing expression earlier in development (Gaudet
and Mango 2002). The ability to tune the timing of ex-
pression by modulating the affinity of the DNA-binding
site(s) for just one TF is conceptually appealing. However,
it remains unclear whether such a simple mechanism
could operate in the context of a complex vertebrate
Pax6 is a key regulator of metazoan eye development,
and in mammals is required in a dose-dependent fashion
for lens induction and specification (Hill et al. 1991;
Grindley et al. 1995; Ashery-Padan et al. 2000; van
Raamsdonk and Tilghman 2000; Davis-Silberman et al.
2005). Furthermore, the precise cis regulation of Pax6 is
obligatory, as alteration in either the timing or level of
Pax6 expression has profound effects on eye development
(Schedl et al. 1996; van Raamsdonk and Tilghman 2000;
Duncan et al. 2004). Several groups have molecularly
dissected regulatory elements contained within a region
of Pax6 that contains the P0 promoter and 3.9 kb of
upstream sequence (P0-3.9), and identified CRMs that
control expression in the lens (denoted as the Pax6
ectodermal enhancer or EE) and endocrine pancreas
(Williams et al. 1998; Kammandel et al. 1999; Zhang
et al. 2002, 2006). Within the EE, there is a highly
conserved minimal 104-base-pair (bp) region sufficient
to direct lens-specific expression (Williams et al. 1998),
and several TFs—including Sox2, Oct1 (Pou2f1), Meis1/2,
and Pax6 itself—have been shown to bind this region
(Zhang et al. 2002; Aota et al. 2003; Donner et al. 2007).
Here we decode a cis-regulatory logic whereby DNA-
binding site affinities of a novel EE regulator, Prep1, are
used to control the timing of Pax6 activation in the
Results and Discussion
Prep1 (also known as Pknox1) is a homeobox TF in the
TALE superfamily that was identified previously along
with other closely related Meis TFs as a regulator of the
Pax6 pancreatic enhancer (Zhang et al. 2006). Prep1
homozygous-null mouse embryos (Prep1?/?) do not sur-
vive to midgestation, while Prep1 hypomorphs, where
RNA expression is reduced to ;2% of wild-type levels
due to a retroviral insertion in first intron of Prep1
(Prep1i/i), show highly variable phenotypes (Ferretti
et al. 2006). We found that Prep1 trans-heterozygotes
(Prep1i/?) generally survive until embryonic days 12.5–
14.5 (E12.5–E14.5), and exhibit distinct and highly pene-
trant phenotypes (Fig. 1A; Supplemental Table 1). Strik-
ingly, Prep1i/?mutants lack an external eye, with an
arrest in eye development by E10.5 (Fig. 1A), the same
[Keywords: Transcription factor; protein-binding microarray; DNA-
binding site affinity; eye development; mathematical modeling; gene
4These authors contributed equally to this work.
5E-MAIL firstname.lastname@example.org; FAX (617) 525-4705.
6E-MAIL email@example.com; FAX (617) 525-4751.
Article published online ahead of print. Article and publication date are
online at http://www.genesdev.org/cgi/doi/10.1101/gad.1890410.
980GENES & DEVELOPMENT 24:980–985 ? 2010 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/10; www.genesdev.org
time point at which eye development arrests in Pax6
tissue, and marker analyses revealed that neither lens
epithelial cells (Foxe3+), nor lens fiber cells (g-crystallin+),
nor lens precursor cells (Pax6+) formed (Fig. 1B). Foxe3 is
a dose-dependent Pax6 target gene in the presumptive
lens epithelium (Fig. 1C) and a definitive marker of lens
specification (Blixt et al. 2007). Prep1i/?mutant mice do
not initiate Foxe3 expression (Fig. 1C), and while Pax6 is
initially expressed in the presumptive lens epithelium of
Prep1i/?mutants, its expression is subsequently lost
specifically in the lens preplacode, which fails to thicken
or invaginate at E10.5 to form the lens placode (Fig. 1C).
Conversely, Prep1 is coexpressed with Pax6 in the pre-
sumptive lens epithelium, and does not depend on Pax6
for its expression (Supplemental Figs. 1, 2). These data
identify Prep1 as a novel regulator of lens induction, and
also as a putative regulator of Pax6 expression.
Pax6 regulation during lens development is complex,
with multiple enhancers controlling the earliest phase of
expression in lens preplacodal epithelium, and the Pax6
EE having a more significant role later during lens in-
duction (Williams et al. 1998; Dimanlig et al. 2001). We
evaluated whether Pax6 regulatory elements might be
regulated directly by Prep1 by crossing a P0-3.9 Pax6
reporter transgene (Fig. 2C; Rowan et al. 2008) into the
Prep1i/?mutant background. Strikingly, the activity of
this GFP reporter in the eye was reduced dramatically in
E10.0 Prep1i/?mutants, while pancreatic enhancer activ-
ity was unaltered (Fig. 2A). The expression of other TFs
that reside upstream of the Pax6 EE was retained in
Prep1i/?mutants, ruling out indirect regulation via these
factors (Supplemental Fig. 3).
We sought to determine whether the P0-3.9 region con-
tained potential Prep1-binding sites. To search the P0-3.9
region for potential Prep1-binding sites, we used protein-
binding microarray (PBM) data on Prep1 (Berger et al.
2008), which provide comprehensive and quantitative in
vitro assessment of binding to all possible 8-bp DNA
sequences. Along with a previously identified binding
site in the Pax6 P0-3.9 region that is required for Pax6
pancreatic enhancer activity (denoted P1) (Zhang et al.
2006), we identified several lower-scoring putative bind-
ing sites throughout the region, including a pair of sites in
the EE (denoted L1, L2) that are separated from each other
by 34 bp (Fig. 2B,C). The high-scoring P1 sequence was
a perfect match to the Prep1 DNA-binding site motif
(Berger et al. 2008), while L1 and L2 each had a single
phylogenetically conserved mismatch (L1: TTGTCA;
L2: CTGTCT) from the optimal core 6-bp sequence 59-
CTGTCA-39 (Fig. 2B,C). The single base mismatches in
the L1 and L2 sites are consistent with the PBM data,
indicating that L1 and L2 are lower-affinity Prep1-binding
Motivated by the PBM data, we used surface plasmon
resonance (SPR) to quantitatively measure the binding
affinity of Prep1 to genomic sequences encompassing the
L1, L2, and P1 sites (Majka and Speck 2007). P1 is a high-
affinity Prep1-binding site, with a dissociation constant
(Kd) of 146 3.4 nM (mean 6 SD), whereas L1 and L2 are of
approximately sixfold to sevenfold lower affinity, with
Kd values of 106 6 4.3 and 92 6 16 nM, respectively
(Fig. 3A,B). These relative affinity differences agree well
with estimates derived independently from the PBM data
(Fig. 3B). Searching for Prep1-binding sites in multiple
aligned genomes, we found two lower-scoring sites, spaced
34 bp apart, aligning to L1 and L2 (Fig. 2C; Supplemental
Fig. 4) throughout the vertebrate lineage, suggesting con-
servation of affinity and function.
The L1 site was previously mutated and shown to be
essential for EE activity (Zhang et al. 2002); hence, we
sought to test the functional significance of the L2 site for
EE regulation. Pax6 P0-3.9 reporters containing an abla-
tion of the L2 site resulted in loss of reporter activity in
the developing lens (Fig. 3C). Additional reporter analyses
indicated that L1 and L2 function nonredundantly as
critical Pax6 regulators (Fig. 3C). These results suggested
three possible models: (1) Prep1 binds cooperatively to
the two sites, but when either site is mutated, it is in-
sufficiently bound to activate detectable levels of expres-
sion. (2) Prep1 binds independently to each site, with
Whole-mount control or Prep1i/?mutants at E10.5, E12.5, and
E14.5. The inset shows high magnification of eye region (boxed).
Arrows show the absence of eyes. (B) Sections through E12.5 control
or Prep1i/?mutant eyes stained with hematoxylin and eosin to show
histology, or stained with antibodies to Foxe3, g-crystallin, or Pax6
as indicated. (C) Sections through E9.5 or E10.5 control or Prep1i/?
mutant embryos stained with antibodies for Pax6 or Foxe3. Arrows
indicate presumptive lens ectoderm, and arrowheads indicate non-
lens head ectoderm. Bars: B,C, 100 mm. (l) Lens; (r) retina.
Genetic requirement for Prep1 in lens induction. (A)
Affinity-dependent Pax6 cis regulation
GENES & DEVELOPMENT 981
DNA-bound Prep1 proteins acting synergistically to ac-
tivate detectable levels of transcripts. (3) Individual DNA-
bound Prep1 proteins cannot activate expression, and
binding to both sites is required to form a larger, multi-
component complex required for Pax6 expression.
We used SPR to measure the binding of full-length
Prep1 to a DNA probe containing the portion of the EE
spanning the L1 and L2 sites in the event that regions
outside the DNA-binding domain may mediate coopera-
tive protein interactions. We did not detect any cooper-
ative binding (Supplemental Fig. 5), arguing against model
1. Model 2 proposes synergistically acting lower-affinity
sites. We reasoned that our mutant reporter constructs
(DL1L2 and L1DL2) might be rescued by substituting the
remaining, native lower-affinity site in each of the
constructs with a high-affinity site resembling the P1
site (DL1L2* and L1*DL2). Strikingly, both constructs
rescued EE reporter activity, but only to a modest extent
and only in a minority of transgenic embryos, while
expression from an L1L2 to L2L1 swap or from the
L1*L2* double-mutant construct was not significantly
different from that of L1L2 (Fig. 4A). These results
demonstrate several important features of the EE ele-
ment: (1) Prep1 occupancy at either site under native
conditions is below saturation. (2) Prep1 bound to either
L1 or L2 sites can provide the necessary requirements for
activation (thereby ruling out model 3). (3) Prep1 mole-
cules bound to L1 and L2 act synergistically and without
a preferred order relative to each other to achieve wild-
type EE expression levels.
We employed a biophysical model (Bintu et al. 2005) of
the Pax6 EE to investigate whether a model based on
transcriptional synergy between Prep1 bound indepen-
dently to the L1 and L2 sites could explain the behavior of
our reporter constructs and provide further testable pre-
dictions. Expression was modeled as equilibrium binding
of Prep1, with DNA-bound Prep1 recruiting a required
cofactor (e.g., TFIID) or RNA polymerase II. The model
was parameterized directly by using our SPR-determined
binding affinities, and indirectly by requiring consistency
with relative expression levels from our reporter con-
structs. In the absence of Prep1-binding cooperativity, we
find that synergistic activation is required byPrep1 bound
at L1 and L2 in order to achieve agreement with the ex-
perimental reporter expression data (Fig. 4B,C). The levels
of Prep1 protein that are consistent with the relative
reporter expression data lie in a narrow range (Fig. 4C,
shaded region) that is consistent with published nuclear
TF concentrations (Gregor et al. 2007; Giorgetti et al.
2010). For this range of Prep1 concentrations, the model
predicts that the EE should be highly sensitive to Prep1
levels. This prediction is consistent with the dramatically
reduced EE activity observed in Prep1i/?mutant mice
In light of the model, we re-examined why both Prep1-
binding sites are highly conserved as lower-affinity sites,
given that substitution of both sites for high-affinity
versions (L1*L2*) did not alter reporter activity at
E10.5. We found that, at concentrations of Prep1 below
those modeled for E10.5, the predicted difference in
expression between the L1*L2* and L1L2 constructs is
enhanced (Supplemental Figs. 6, 7). Prep1 mRNA and
protein levels are known to increase during early mouse
development (Ferretti et al. 1999), as does EE activity
(Williams et al. 1998). We predicted, therefore, that the
L1*L2* construct should be expressed at higher levels
than L1L2 at stages preceding E10.5, when Prep1 levels
are lower (see Supplemental Figs. 1, 2). We tested this
prediction, and indeed found that L1*L2* directed in-
appropriately high reporter activity at E9.5 (Fig. 4D). Our
interpretation, therefore, is that Prep1-binding site affin-
ity provides a mechanism for controlling the timing of
Pax6 expression in lens development. Additionally, we
detected specific regions of expanded reporter activity in
visualization of E10 control or Prep1i/?mutant eyes expressing the P0-3.9-GFPCre transgene, or section through the pancreas of the same
embryo stained with antibodies for Pdx1 and GFP. Dotted line marks the optic vesicle. (B) Logo of the Prep1 DNA-binding site motif (Berger et al.
2008), with a box showing the CTGTCA core sequence. (C) Schematic of the Pax6 P0-3.9 genomic region used in the transgenic mouse line. P0
box represents the location of the Pax6 P0 promoter. Shown in the University of California at Santa Cruz Genome Browser view are PBM scores
(enrichment score, $0.37) from a sliding 8-bp window. Hits in the minimal essential region of the EE (cyan) or Pancreas enhancer (magenta) are
labeled L1, L2, and P1; sequences are shown boxed below. Red nucleotides indicate divergence from the boxed CTGTCA core sequence.
Prep1 is required for Pax6 EE activation and binds multiple highly conserved sites upstream of Pax6. (A) Whole-mount GFP
Rowan et al.
982 GENES & DEVELOPMENT
L1*L2* transgenic embryos (Fig. 4; Supplemental Fig. 8),
suggesting additional selective pressure against high-
affinity Prep1 sites in the EE. These results provide
a mechanistic link between Prep1-binding site affinity
and the timing of Pax6 expression during eye develop-
Deciphering the cis-regulatory logic of enhancers gov-
erning the expression of developmental genes requires
the description of coordinate action of multiple DNA-
bound TFs. To what extent the role of individual sites can
be dissected within the context of larger, complex verte-
brate enhancers remains an open question. Here we
describe a mammalian enhancer using binding site affin-
ity of a single TF, Prep1, to regulate the temporal control
of gene expression. Specifically, mutating the lower-
affinity, native L1 and L2 Prep1 sites to higher-affinity
sites resulted in high-level EE activity at an earlier
developmental time point (Fig. 4D).
Coordination of the timing and level of Pax6expression
is of particular importance because of the exquisite
sensitivity of eye development to even subtle changes
in Pax6 levels; both Pax6 heterozygotes (mouse) and
PAX6 hypomorphs (human) exhibit developmental phe-
notypes (Hill et al. 1991; Glaser et al. 1994; Schedl et al.
1996; van Raamsdonk and Tilghman 2000; Sansom et al.
2009). Lens development, particularly when the EE be-
comes active (E8.75), is highly sensitive to Pax6 concen-
tration, and a threshold model for Pax6 function has been
proposed (van Raamsdonk and Tilghman 2000). The
lower affinities of L1 and L2 may have been evolution-
arily selected to be most responsive to the developmental
concentrations of Prep1 present when increasing levels of
Pax6 expression are required to coordinate lens morpho-
genesis. Other enhancers have been shown to contribute
(A) SPR dose response curves for GST-Prep1 on L1, L2, or P1. (B) Kd
values of Prep1 for L1-, L2-, and P1-binding sites and fold change
in affinity relative to the P1 site (numbers >1 indicate decreased
affinity) as determined by SPR and PBM data. (C) Whole-mount view
of b-galactosidase-stained E10.5 embryos expressing wild-type P0-
3.9-L1L2 or P0-3.9-L1DL2 transgenes. The arrow points to the lens,
while the arrowhead points to the pancreas. A summary is shown for
Pax6 reporters lacking either L1 or L2 sites (21) or genetically
deficient for Prep1 as assayed at E10.5. Only nucleotides that diverge
from the wild-type EE sequence are shown. Lightly shaded L1 or L2
boxes indicate lower-affinity sites; white boxes indicate ablated
binding sites. Binding site ablations for L1 and L2 are genetically
indicated with D for deletion, although they are specific point
mutations that have been tested by PBM and biochemical analyses
to have no detectable Prep1 binding. Activity indicates relative
b-galactosidase (name in blue) or GFP (name in green) reporter
activity in the lens.
L1 and L2 are essential lower-affinity Prep1-binding sites.
Summary and examples of Pax6 reporter constructs with mutations
in L1 or L2 sites that create high-affinity binding sites (asterisks and
black boxes). The ratio indicates the number of transgenic embryos
with lens b-galactosidase staining as a fraction of all transgenic
embryos. Activity indicates relative b-galactosidase reporter activity
in the lens. (B) Modeling of EE activation versus Prep1 concentration
(log2 scale) for L1L2 modeled with synergy (black solid line) or
without synergy (black dashed line), single-site mutations (DL1L2,
L1DL2, gray dashed line), or high-affinity site mutations (DL1L2*,
L1*DL2, gray solid line; L1*L2*, blue solid line). (C) Ratio of
activation levels for reporter constructs modeled in B to facilitate
the comparison of model predictions and the ratio of relative
reporter levels. Single high-affinity site mutations (L1*DL2) versus
wild-type (L1L2) reporter modeled with synergy (black line) or
single-site ablation (L1DL2, gray line); double high-affinity site
mutation (L1*L2*) versus wild-type (L1L2) reporter modeled with
synergy (red line). We conservatively estimate the ratio differences
between L1*DL2 and L1DL2 to be at least twofold, between L1L2
and L1*DL2 to be at least fourfold, and between L1*L2* and L1L2 to
be at most 1.3-fold. These estimates are based on b-galactosidase
staining intensities and histochemical development times. The
shaded area defines the predicted range of physiological concentra-
tion for Prep1. (D) b-Galactosidase staining of an eye from an E9.5
L1*L2* transgenic embryo was more intense than from wild-type
(P0-3.9-L1L2) embryos of increasingly older ages up to E10.25.
L1 and L2 interact synergistically to activate the EE. (A)
Affinity-dependent Pax6 cis regulation
GENES & DEVELOPMENT983
to Pax6 lens expression both earlier and later than the EE
(e.g., SIMO element) (Kleinjan et al. 2001), and may
function coordinately with the EE to regulate Pax6. It is
possible that Prep1 may regulate Pax6 expression via
some of these other enhancers and possibly the P0
promoter (see Fig. 2C) in addition to the EE to account
for the severe lens phenotype we observed in Prep1i/?
The quantitative measurement and modeling of bind-
ing site affinities has led to the development and testing
of biophysical models of transcription (Wang et al. 1999;
Bintu et al. 2005; Kim and O’Shea 2008; Segal et al. 2008;
Gertz et al. 2009; Kim et al. 2009). Here we demonstrate
that a biophysical model, parameterized with biochemi-
cal and in vivo reporter analyses, can describe the activity
of the Pax6 EE as a function of Prep1 levels and binding
site affinities. Additionally, our model provides a mecha-
nism for decoupling the timing and levels of gene expres-
sion, whereby the affinities of the L1 and L2 sites for
Prep1 dictate when Pax6 is expressed, while the level of
activation is modulated by the synergistic activity of
Prep1 molecules noncooperatively bound to the two
sites. The strict maintenance of the presence, affinity,
and spacing of the L1 and L2 sites throughout the
vertebrate lineage suggests evolutionary conservation of
this regulatory mechanism.
Vast numbers of lower-affinity TF-binding sites are
present in vertebrate genomes, but the functions of such
sites remain unclear. Here we identified a functional role
for lower-affinity TF-binding sites in establishing the
temporal control of gene expression in the developing
lower-affinity sites is a signature of CRMs that interpret
temporal and/or spatial gradients of their upstream activa-
tors. Genomic searches for regulatory regions containing
such conserved lower-affinity TF-binding sites (Jaeger et al.
2010) may identify other delicately calibrated enhancers
controlling key developmental genes.
Materials and methods
Full descriptions of the Materials and Methods are available in the
P0-3.9-GFPCre mice were described previously, and were maintained as
heterozygotes on an FVB background (Rowan et al. 2008). Pax6Sey-Neu
mice were maintained as heterozygotes on a C3H background. Prep1i(gift
from Dr. Francesco Blasi) and Prep1?(gift from Dr. Neal Copeland) mice
were maintained as heterozygotes on a C57BL/6 background (Ferretti
et al. 2006). Transgenic constructs were based on the pLNGLKS reporter
plasmid containing the 526-bp Pax6 EE directing lacZ expression (Zhang
etal. 2002),except for the P0-3.9-L1DL2-lacZreporter,whichwas basedon
the P0-3.9-lacZ reporter line (Zhang et al. 2006). Site-directed mutations
were constructed using the QuikChange Lightning Site-Directed Muta-
genesis Kit (Stratagene) according to the manufacturer’s directions.
For antibody staining, primary antibodies used were mouse anti-Pax6
(1:10; Development Studies Hybridoma Bank, The University of Iowa),
rabbit anti-Pax6 (1:1000; Covance Research Products), rabbit anti-Foxe3
(1:1000; gift from Dr. Peter Carlsson), goat anti-g-crystallin (1:1000; Santa
Cruz Biotechnologies), goat anti-Pdx1 (1:5000; gift from Dr. Chris Wright),
guinea pig anti-Prep1 (1:200) (Zhang et al. 2006), guinea pig anti-Six3
(1:500; Abcam), rabbit anti-Sox2 (1:1000; Chemicon), and goat anti-Meis1/2
(1:250; Santa Cruz Biotechnologies). Secondary antibodies were generated
in donkey versus the appropriate species, and were directly conjugated
with Cy3 (Jackson Immunologicals) or Alexa Fluor 488 (Molecular
Protein expression and SPR
GST-Prep1 homeodomain and full-length ORFs (Berger et al. 2008) were
expressed in Escherichia coli. Tagged protein was purified using GSTrap
FF affinity columns (GE Healthcare) on an AKTA prime plus FPLC
(GE Healthcare). Concentration of purified protein was determined
by Coomassie Bradford assay. SPR was performed on a Biacore 3000.
Biotinylated oligos were immobilized onto a Sensor Chip SA (Biacore).
Serial concentrations of protein samples were diluted in running buffer
and applied to the Sensor Chip at 25 mL/min, using the KINJECT option,
250-mL sample (1000-sec dissociation phase). Binding rate constants
and equilibrium Kd values were determined using Scrubber2 software
Computational analysis and modeling
The P0-3.9 region was searched for Prep1 DNA-binding site sequences
using custom Perl scripts (available on request). Eight-base-pair sequences
were scored according to their PBM enrichment score, a statistical
measure that ranges from ?0.5 to +0.5 and indicates the binding
preference of the protein, assayed in a universal PBM experiment, for
a particular 8-mer as compared with all other 8-mers (Berger et al. 2008).
Prep1 PBM data are available via the UniPROBE database (Newburger and
An equilibrium thermodynamic model, described recently by Bintu
et al. (2005), was used to model transcriptional activation by the EE.
Activation curves were modeled and visualized in Matlab. A full de-
scription of the model and parameterization method is in the Supplemen-
We thank Francesco Blasi, Steve Gisselbrecht, Craig Kaplan, Daniel
O’Connell, and Rolf Stottmann for critical comments on the manuscript,
and members of the Maas and Bulyk laboratories for helpful discussion.
We are especially grateful to Neal Copeland and Francesco Blasi for gifts of
Prep1 mutant mice. This work was supported by a fellowship from the
Canadian Institutes of Health Research to S.R, NIH grant R01 HG003985
to M.L.B., and NIH grant R01 EY10123 to R.L.M.
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Affinity-dependent Pax6 cis regulation
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