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First published online 14 November 2007
doi: 10.1242/dev.008680
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1 Howard Hughes Medical Institute, Division of Basic Sciences, Fred Hutchinson
Cancer Research Center, Seattle, WA 98109, USA.
2 Gulbenkian PhD Programme in Biomedicine, Rua da Quinta Grande, 6, 2780-156,
Oeiras, Portugal.
3 Department of Biology, University of Washington, Seattle, WA 98195, USA.
Author for correspondence (e-mail:
jpriess{at}fhcrc.org)
Accepted 25 September 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Notch, GATA, ref-1
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Mumm and Kopan,
2000; Greenwald,
2005; Bray, 2006).
In the absence of Notch signaling, target genes can be repressed by
sequence-specific DNA binding proteins in the CSL family [for vertebrate CBF1,
Drosophila Su(H) and Caenorhabditis elegans LAG-1] in
combination with corepressors. Contact with ligand activates the transmembrane
receptor Notch, initiating processing events that eventually release the Notch
intracellular domain (NICD). NICD then translocates to the nucleus where it
binds CSL, displacing the corepressor complex and forming a ternary complex
with an essential coactivator in the mastermind-like (MAML) protein family.
The NICD-CSL-MAML ternary complex is thought to recruit chromatin-modifying
enzymes that promote target gene expression.
Notch-regulated cell fate decisions have been described in ectoderm,
mesoderm, endoderm and germline tissues. Examples include germ cell mitosis
and vulval development in C. elegans, specification of the
ectoderm/endoderm boundary in sea urchins, and binary cell fate decisions in
neural, vascular and lymphoid precursors in vertebrates
(Austin and Kimble, 1987;
Robey, 1997;
Sherwood and McClay, 2001;
Iso et al., 2003;
Greenwald, 2005). Individual
Notch targets typically are expressed in one or a few interactions, but are
not expressed in all interactions. For example, the Drosophila cut
and sim genes are Notch targets in the wing and the ventrolateral
ectoderm, respectively (Morel and
Schweisguth, 2000; Guss et
al., 2001). Similarly, the C. elegans ref-1 gene is a
direct target of multiple Notch interactions during embryogenesis, but does
not appear to be expressed in several postembryonic Notch interactions
(Neves and Priess, 2005). This
specificity suggests that CSL proteins must cooperate directly or indirectly
with tissue-restricted factors to control target gene expression. Multimerized
binding sites for CSL proteins are sufficient to promote in vitro
transcription using purified NICD, CSL and MAML proteins, and multimerized CSL
binding sites can promote Notch-dependent reporter expression in cultured
cells (Furukawa et al., 1995;
Wallberg et al., 2002).
However, multimerized CSL sites are insufficient to drive reporter expression
in Notch-activated cells in vivo (Guss et
al., 2001), and some endogenous Notch targets contain only one or
two CSL-binding sites (Yoo and Greenwald,
2005) (our unpublished results). Furthermore, fusing CSL proteins
to a strong activation domain like VP16 or a constitutively active form of
Notch (NICD) does not, in general, suffice for target gene expression
(Cooper et al., 2000;
Morel and Schweisguth, 2000).
These results together suggest that the regulation of endogenous Notch targets
involves additional combinatorial factors, and transcriptional codes that are
more complex than simply the presence of CSL-binding sites.
The promoters of some Notch-regulated genes contain conserved, paired
CSL-binding sites that are oriented head to head and spaced 15-22 bp apart
(Bailey and Posakony, 1995;
Nellesen et al., 1999;
Nam et al., 2007). Inverting
the orientation of either, or both, of the CSL paired sites significantly
impairs the expression of reporter genes
(Cave et al., 2005). Recent
structural and biochemical studies have shown that a CSL-paired site is able
to promote cooperative interactions between ternary complexes of mammalian
CSL, MAML and the NICD ankyrin domain (Nam
et al., 2007). A Drosophila CSL-paired site, called SPS
[Su(H) paired site], functions in conjunction with a binding site for
Daughterless (Da), a bHLH transcription factor that can bind directly to Su(H)
(Kramatschek and Campos-Ortega,
1994; Cooper et al.,
2000; Cave et al.,
2005). Thus, SPS + A, where A is the Da-binding site, appears to
represent a transcriptional code for Notch-bHLH synergy. Other Notch
transcriptional codes must exist, as some Notch-regulated genes with
CSL-paired sites lack predicted bHLH binding sites, and many Notch targets do
not contain CSL-paired sites (Cave et al.,
2005). For example, recent studies have provided evidence that the
C. elegans egl-43 gene is regulated by Notch signaling and the bHLH
protein HLH-2, but elements required for expression do not contain obvious
CSL-paired sites (Hwang et al.,
2007). Previous studies have shown that expression of the
Drosophila Notch targets cut and sim requires the
transcription factors Scalloped/TEF-1 and Dorsal/NF-
B, respectively,
but how these factors couple with Notch signaling is not known
(Kasai et al., 1998;
Guss et al., 2001).
In the present study, we used the ref-1 gene to study
tissue-specific, Notch-dependent gene expression. The ref-1 gene is a
direct target of many, though not all, Notch-mediated cell interactions in
embryogenesis, and in addition is expressed in several cells independently of
Notch signaling (Neves and Priess,
2005; Ross et al.,
2005; Lanjuin et al.,
2006). Expression of ref-1 in some cells involves DNA
sequences located over 8 kb upstream of the start codon
(Ross et al., 2005), in
contrast to most C. elegans genes that have very small promoter
regions (Okkema and Krause,
2005). These and other results suggest that ref-1 has a
complex promoter, with separable, dispersed elements controlling expression in
different cell types (Neves and Priess,
2005; Ross et al.,
2005; Lanjuin et al.,
2006). Here, we present the first characterization of a
Notch-dependent enhancer element from the ref-1 promoter. The
enhancer is highly conserved in other Caenorhabditis species, and
confers Notch-specific expression in a cell interaction in the mesoderm, and
in two interactions in the endoderm. Notch-dependent expression in the
endoderm, but not mesoderm, requires predicted binding sites for GATA
transcription factors. We show that ELT-2/GATA is necessary for one of the two
endodermal interactions, and is sufficient for ectopic, Notch-dependent
expression in multiple cell types. Endoderm expression does not require
CSL-paired sites, but instead involves CSL sites closely flanked by predicted
GATA-binding sites. We demonstrate that LAG-1/CSL can interact directly with
ELT-2/GATA in vitro, providing a possible basis for Notch-GATA synergy.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The following
extrachromosomal arrays were created for this study. zuEx132,
[ref-1600bp::gfp(pKG55)]; zuEx133,
[ref-1600bp(WCATAR)::gfp(pKG58)]; zuEx141,
[ref-1600bp(WCATAR;1-GATA)::gfp]; zuEx150,
[ref-1600bp(WCATAR;2-GATA)::gfp]; zuEx139,
[ref-1600bp(WCATAR;3-GATA)::gfp]; zuEx144,
[ref-1600bp(WCATAR;4-GATA)::gfp]; zuEx151,
[ref-1600bp(RAGGCAA;1-CSL)::gfp]; zuEx148,
[ref-1600bp(RAGGCAA;2-CSL)::gfp]; zuEx154,
[ref-1600bp(RAGGCAA;3-CSL)::gfp]; zuEx149,
[ref-1600bp(RAGGCAA;4-CSL)::gfp]; zuEx142,
[enhA::gfp(pAN20)]; zuEx162, [C.b.
enhA::gfp]; zuEx178, [C.r.
enhA::gfp]; zuEx191,
[enhA(RAGGCAA)::gfp(pAN29)]; zuEx172,
[enhA(1-CSLinverted)::gfp]; zuEx177,
[enhA(2-CSLinverted)::gfp]; zuEx171,
[enhA(3-CSLinverted)::gfp]; zuEx179,
[enhA(3-CSL and 3-GATA interchanged)::gfp]; zuIs173
(pKG82). wIs88 (hs-med-1) was provided by Morris Maduro
(Maduro et al., 2001) and
caIs7 (hs-elt-2) was provided by Jim McGhee
(Fukushige et al., 1998).
Transgenics
Standard techniques were used to manipulate DNA. All enhancer elements were
fused to a pes-10 minimal promoter to drive expression of a
GFP::HIS2B fusion protein (pAP10)
(Gaudet and Mango, 2002).
Constructs were injected at 40 ng/µl with 100 ng/µl rol-6 DNA.
At least two independent lines were analyzed for each transgene, and at least
20 embryos were examined per line. Promoter/enhancer mutagenesis and inversion
of binding sites was performed using the QuikChange site-directed mutagenesis
kit (Stratagene, La Jolla, CA). Predicted CSL sites were inverted by reversing
individual RTGGGAA or TTCCCAY sequences. The inverted 1-CSL and
2-CSL sequences would conform with the YRTGRGAA consensus CSL-binding
site described previously (Yoo et al.,
2004).
RNAi
RNAi against embryonically expressed genes were typically performed by
injection. Briefly, young hermaphrodites were injected with dsRNA produced by
in vitro transcription (0.5-2 µg/µl) and the progeny analyzed after 24
hours. For elt-2 RNAi, injected animals were allowed to develop for
24 hours at 22°C, then an additional 12 hours at 15°C before scoring
progeny. RNAi targeting maternally expressed genes was performed by feeding
(Timmons and Fire, 1998).
Ectopic expression of ELT-2/GATA
For ectopic expression during AB interactions, two- to eight-cell embryos
were heat-shocked at 33°C for 30 minutes then allowed to recover at
20°C for 1-1.5 hours. For ectopic expression during the pm8 interaction,
E16 stage embryos were heat-shocked for 30 minutes at 33°C, allowed to
recover for 2-3 hours, and then analyzed.
GST pulldowns
GST pulldowns were performed essentially as described
(Poortinga et al., 1998).
Briefly, full-length ELT-2 was expressed from the T7 promoter in the pCITE
vector using the Promega TnT in vitro expression kit (Promega, Madison, WI)
with 0.01 mM ZnCl2 and labeled with 35S. In vitro
translated proteins were first pre-cleared by incubation with GST alone on
glutathione-Sepharose beads, then incubated with GST, GST-LAG-1
(Kovall and Hendrickson, 2004)
or GST-Cad ICD proteins. Beads were washed four times with PBS + 0.5% Nonidet
P40 and potential protein-protein complexes were resolved by SDS-PAGE and
visualized by autoradiography. GST-Cad ICD is a GST fusion to the C-terminus
(200 aa) of the intracellular domain of Drosophila DE-cadherin, and
was kindly provided by Susan Parkhurst (Fred Hutchinson Cancer Research
Center, Seattle, WA).
DNA binding
Electrophoretic mobility shift assays (EMSA) were performed essentially as
described (Stroeher et al.,
1994). The 32P-labeled probe used for ELT-2 EMSA was
from the pho-1 gene: catcgagtagccaACTGATAAaagacattactacaa. ELT-2 was
synthesized in vitro from the T7 promoter in the pCITE 4a vector (pAN47) using
the Promega TnT in vitro expression kit (Promega, Madison, WI, USA) and
labeled with 35S. The 32P-labeled probe used for GST-LAG
EMSA was:
gaattctcgcgactCGTGGGAAaatgggcggaagggcacCGTGGGAAaatagttccaggaattc.
This sequence has been used previously for EMSA with LAG-1
(Hwang et al., 2007), and the
underlined region has been used for EMSA with CBF1/CSL
(Zimber-Strobl et al., 1994).
GST-LAG-1(192-663) has been described previously
(Kovall and Hendrickson,
2004). EMSA reactions used 25 ng GST-LAG-1 or 3 µl ELT-2 TnT
lysate and 0.1 µg of poly(dI-dC) per reaction. Probes were at 0.02 µM
[LAG-1 EMSA] and 0.1 µM [ELT-2 EMSA]. Binding reactions were incubated for
30 minutes at 22°C.
Genomic sequence analysis
DNA searches were performed using Genome enhancer
(www.genomeenhancer.org).
SPS searches used a 36 bp window to find both RTGGGAA and TTCCCAY sequences
located within 3 kb of predicted genes. A 50 bp window was used to search for
clusters of two TTCCAY and TGATAR sequences.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Hermann et al., 2000).
Briefly, the entire endoderm in C. elegans is derived from a single
embryonic blastomere called E; successive stages of the endodermal primordium
are called E2, E4, etc., corresponding to the number of E descendants
(Fig. 1A). All E4 cells express
the receptor LIN-12/Notch, but only the left two E4 cells contact
ligand-expressing cells outside the primordium. The E4-Notch interaction
induces ref-1 expression in the left side of the E4 and E8 primodium,
where REF-1 functions to downregulate LIN-12/Notch expression. Because
LIN-12/Notch persists only in the right primordial cells, a subsequent
interaction at the E16 stage induces ref-1 expression on the right
side of the primordium (Neves and Priess,
2005; Hermann et al.,
2000) (Fig. 1A).
Thus, ref-1 expression switches from the left E4 and E8 cells to the
right E16 cells (Fig. 1A). The
E4 and E16 interactions cause a handed rotation in the positions of anterior
endodermal cells, resulting in an asymmetrical twist in the developing
intestine (Hermann et al.,
2000).
|
). In
the present study, DNA fragments from this region were fused to a GFP reporter
containing a heterologous minimal promoter, and transgenes were injected into
wild-type worms to test for endoderm expression
(Fig. 2A). These experiments
identified a 153 bp element (called enhA;
Fig. 2B) that was sufficient to
recapitulate the normal pattern of ref-1 expression for both Notch
interactions in the endoderm (Fig.
1B,C and Fig. 2A;
Table 1); smaller fragments
showed no expression (Fig.
2A).
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Part of the enhA element overlaps a sequence we noted
previously as being highly conserved in promoters of ref-1
orthologues in Caenorhabditis briggsae and Caenorhabditis
remanei - strains that are thought to have diverged from C.
elegans by about 100 million years
(Fig. 2C)
(Neves and Priess, 2005).
Recent genomic sequence from a fourth nematode species, Caenorhabditis
brenneri (Washington University Genome Sequencing Center) includes a
predicted orthologue of ref-1 with a similar highly conserved
upstream sequence (Fig. 2C).
The conserved region occurs at varying distances from the predicted initiator
ATG (from about -300 bp in C. elegans to -1400 bp in C.
briggsae) and in both orientations with respect to coding regions (data
not shown). We constructed GFP transgenes using the conserved regions from
C. briggsae or C. remanei, and found that each transgene was
expressed in C. elegans in the Notch-activated E8, E16 and pm8 cells
(Fig. 3C,D; data not shown).
Each of the conserved sequences contains multiple RTGGGAA sequences that are
predicted LAG-1/CSL-binding sites (labeled 1-CSL to 4-CSL in
Fig. 2B,C)
(Christensen et al., 1996). A
region from the C. elegans enhA sequence that includes
2-CSL and 3-CSL (bold line in
Fig. 2B) was tested for in
vitro binding of LAG-1/CSL (Fig.
4A). In these experiments, wild-type or mutated
enhA DNA was used to compete binding between LAG-1 and a
labeled probe containing two CSL sites that has been described previously and
which binds LAG-1 in vitro (Fig.
4A) (Hwang et al.,
2007). The wild-type sequence from enhA
competed effectively for LAG-1 binding; competition was not observed when both
2-CSL and 3-CSL were mutated from RTGGGAA to RAGGCAA, and
LAG-1 appeared to bind both sites comparably
(Fig. 4A). A GFP transgene was
constructed where all four of the predicted CSL-binding sites in
enhA were mutated from RTGGGAA to RAGGCAA. This transgene
was not expressed in the Notch-activated E4, E8, E16 or pm8 cells
(Fig. 1D;
Table 2), but was expressed in
several Notch-independent cells (data not shown). For these several reasons we
conclude that enhA is a Notch-regulated enhancer for
endodermal and mesodermal gene expression.
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). However, the enhA-related sequences
from all four Caenorhabditis species share two motifs in addition to
CSL sites (Fig. 2C). The first,
CTCAAG, is a core binding motif for some NK-type homeodomain transcription
factors such as mammalian TTF1 (thyroid transcription factor) and
Drosophila Tinman (Guazzi et al.,
1990; Kelly et al.,
1996; Gajewski et al.,
1997; Li et al.,
1998). The extended CTCAAGTGC sequence in enhA
is nearly identical to a repeated CTCAAGTGG binding site for Tinman in the
D-mef2 gene (Gajewski et al.,
1997). We found that mutating the CTCAAGTGC sequence to CTGTGCTGC
or CTCAAGTTC abolished or markedly reduced expression in pm8
(Fig. 3E,I;
Table 2). The C.
elegans genome encodes four NK-type homeobox transcription factors:
CEH-22, CEH-24, CEH-27 and CEH-28. At least one of these factors (CEH-24) is
expressed in pm8, but does not appear essential for at least some aspects of
pm8 differentiation (Harfe and Fire,
1998). ceh-24 is a direct target of Notch signaling in
pm8, but we do not yet know whether CEH-24 has a redundant role in regulating
ref-1 expression in pm8 (our unpublished results).
The enhA element also contains multiple, conserved
WGATAR sequences that are predicted binding sites for GATA transcription
factors (for a review, see Patient and
McGhee, 2002). Mutating all of the WGATAR sequences in
enhA to WCATAR did not prevent expression in pm8
(Fig. 3F, and
Table 2), but abolished
expression in both sets of Notch-regulated endodermal cells
(Fig. 1E and
Table 2). Mutating only the
fourth site (4-GATA) or the non-conserved first site
(1-GATA) had little if any effect on expression levels in the
endodermal cells (Table 2).
However, mutating either 2-GATA or 3-GATA markedly reduced
endoderm expression (Table 2
and data not shown). These results together suggest that
enhA contains distinct sequences that collaborate with
CSL-binding sites to drive Notch-dependent expression in either mesodermal or
endodermal cells.
ELT-2/GATA regulates Notch-dependent expression in the endoderm
The finding that WGATAR sequences in enhA are crucial
for endoderm expression suggests that GATA transcription factors cooperate
with Notch signaling in the endoderm. Genetic and molecular studies have shown
that the specification of the E blastomere and the differentiation of
intestinal cells are controlled by a regulatory cascade of multiple GATA
transcription factors (for reviews, see
Maduro and Rothman, 2002;
McGhee, 2007). The GATA
factors have overlapping patterns of expression, beginning in the parent of
the E blastomere (MED-1, MED-2), in the E blastomere (END-1, END-3), in the
daughters of the E blastomere (ELT-2, ELT-7) or in their descendants (ELT-4).
These GATA factors are expressed in all endodermal cells, in contrast to the
left-right asymmetric, Notch-dependent expression of ref-1. We were
unable to assign a role to any of the GATA factors in the first endodermal
Notch interaction using RNAi or mutant analysis (data not shown); this
analysis was complicated by the requirement for END-1 and END-3 in endoderm
specification, such that many affected embryos lacked endoderm
(Maduro et al., 2005).
However, we found that ELT-2/GATA was involved in the second (E16) endodermal
interaction: most embryos lacked E16 expression of the integrated
ref-1(1.8kb)::GFP transgene (24/32 embryos) and of the
non-integrated enhA::GFP transgene
(Table 1 and
Fig. 1G, bottom panel) after
depletion of ELT-2 by RNAi. Moreover, many of the elt-2(RNAi) embryos
showed intestinal morphogenesis defects similar to those described previously
in ref-1 or lin-12/Notch mutants [34% of embryos
(n=82) lacked intestinal twist] (see also
Hermann et al., 2000;
Neves and Priess 2005).
Previous studies have analyzed the regulation of the pho-1 gene by
ELT-2/GATA (Hawkins and McGhee,
1995). All endodermal cells express pho-1, and expression
is not known to involve Notch signaling. Endodermal expression requires a
pho-1 promoter element that contains three WGATAR sequences, and
which lacks predicted CSL-binding sites
(Fukushige et al., 2003). The
GATA site most critical for pho-1 expression in the endoderm has the
extended sequence ACTGATAA, and DNA from the pho-1 promoter
containing this sequence binds ELT-2 in vitro
(Fig. 4B)
(Hawkins and McGhee, 1995).
The 3-GATA site in enhA has the identical
sequence - ACTGATAA - and our above results showed that 3-GATA and
2-GATA are critical for endodermal expression of
enhA::GFP. To test whether ELT-2 could bind
2-GATA and/or 3-GATA, we used enhA DNA
(bold line in Fig. 2B) as
competitor for labeled pho-1 DNA
(Fig. 4B). These results
demonstrate that ELT-2 can bind both 2-GATA and 3-GATA in
vitro, and that binding to 3-GATA is reproducibly stronger than to
2-GATA (Fig. 4B).
These in vitro and in vivo experiments suggest that ELT-2/GATA cooperates with
Notch signaling to regulate ref-1 expression in the E16 endodermal
cells.
ELT-2/GATA and enhA are sufficient to drive Notch-regulated expression in non-endodermal cells
enhA promotes Notch-dependent transgene expression in
endodermal (E8, E16) and mesodermal (pm8) cells, yet
enhA::GFP is not expressed in numerous Notch interactions
that occur either earlier or later in embryogenesis, or occur in ectodermal
cells [see Priess (Priess,
2005) for a review of Notch interactions in the embryo]. At the
4-cell stage, for example, a daughter of the AB blastomere that is an
ectodermal precursor undergoes a Notch interaction, and at the 12-cell stage a
different set of AB descendants undergo a distinct Notch interaction. Notch
targets in the ref-1 family, such as hlh-26, are expressed
in both sets of Notch-activated AB descendants (numbered 1 and 2 in
Fig. 5A), but not in other AB
descendants (arrow in Fig. 5A);
the enhA::GFP transgene is not expressed in any
of these AB descendants (Table
1). To test the hypothesis that enhA plus
ELT-2/GATA is sufficient for Notch-specific expression, we used heat shock to
misexpress ELT-2 during early embryogenesis. The enhA
transgene was expressed strongly in all of the AB descendants that are
activated by Notch signaling at the 4-cell and 12-cell stages, but was not
expressed in any of the other AB descendants, or in any other cells in the
embryo (Fig. 5C,D). To confirm
that the predicted GATA-binding sites in enhA were
important for ectopic Notch-specific expression, the heat shock experiment was
repeated using a transgene with all four WGATAR sequences mutated to WCATAR.
This modified transgene was not expressed in any of the early embryonic cells
following heat-shock expression of ELT-2/GATA
(Fig. 5E,F).
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), yet we observed enhA::GFP
expression only in the Notch-activated AB descendants following heat shock.
The canonical model for Notch signaling provides a possible explanation for
the restricted expression of enhA::GFP; CSL proteins are
complexed with co-repressors on Notch target genes, preventing expression in
the absence of Notch signaling (see Introduction). Consistent with this view,
LAG-1/CSL is present in all of the early embryonic nuclei
(Christensen et al., 1996;
Hermann et al., 2000). If
LAG-1/CSL-mediated repression prevents expression of
enhA::GFP in cells that do not receive Notch signals,
depleting LAG-1/CSL should allow expression in all cells with heat-shock
ELT-2/GATA. Instead, we found that no cells expressed
enhA::GFP in lag-1(RNAi) embryos following heat
shock (0/54 embryos scored between the 28- and 44-cell stages). To address the
possibility that depleting LAG-1/CSL by RNAi indirectly prevented gene
expression, we performed a complementary experiment by mutating all of the
predicted LAG-1/CSL binding sites in the enhA::GFP
transgene and analyzing expression in an otherwise wild-type background.
Heat-shock ELT-2/GATA failed to induce expression of the mutated transgene,
despite the fact that the transgene retained multiple WGATAR sequences, and
the transgene was present as a multi-copy array
(Fig. 5G,H). Together, these
results suggest that Notch signaling in AB descendants does not simply
alleviate repressive functions of LAG-1/CSL, thereby allowing ELT-2/GATA to
independently activate gene expression. Instead, these results argue that
Notch signaling and ELT-2/GATA must synergize to promote gene expression;
neither is sufficient in the absence of the other.
Notch-GATA synergy does not require a CSL-paired site
The first and second predicted LAG-1/CSL binding sites (1-CSL and
2-CSL) in enhA are conserved with the same
inverted orientation and approximate spacing in three of the
Caenorhabditis species (Fig.
2C). The orientation and spacing are similar to CSL-paired sites
that are found in some Notch-regulated genes in both Drosophila and
vertebrates (see Introduction). We therefore asked whether 1-CSL or
2-CSL were essential for Notch-regulated expression. Mutating either
sequence from RTGGGAA to RAGGCAA markedly reduced pm8 expression
(Table 2). However, inverting
1-CSL or 2-CSL did not noticeably affect expression in pm8.
Thus, 1-CSL and 2-CSL are important for pm8 expression in
combination with a possible NK site, but do not appear to function as a
CSL-paired site. Mutating or inverting 1-CSL did not noticeably
affect the level of endodermal expression. By contrast, mutating either
2-CSL or 3-CSL markedly decreased endodermal expression
(Table 2). Combined with our
results on mutating individual GATA sites (above), these results suggest that
sites 2-CSL, 2-GATA, 3-CSL and 3-GATA provide the major
input to Notch-regulated endodermal expression
(Fig. 2B).
Although in this study we have not examined in detail the possible spacing
or orientation requirements for the sites, we found that inverting
2-CSL or 3-CSL markedly reduced endoderm expression, as did
interchanging the positions of 3-CSL and 3-GATA
(Table 2). A potential function
of oriented CSL- and GATA-binding sites would be to facilitate physical
interactions between LAG-1/CSL and ELT-2, or proteins associated with these
transcription factors. To test whether LAG-1 could bind ELT-2 in vitro, GST
fusion proteins were generated with full-length LAG-1 (residues 1-674) or with
the LAG-1 core (amino acids 192-663); the core is conserved among CSL
orthologues and has been used for crystal studies of LAG-1-DNA complexes
(Kovall and Hendrickson, 2004;
Wilson and Kovall, 2006). We
found that both LAG-1 fusion proteins bound full-length ELT-2 (aa 1-434), with
much stronger binding by the LAG-1 core
(Fig. 6). In similar
experiments, neither full-length nor core LAG-1 showed significant binding to
the N-terminal half of ELT-2 (aa 1-226), but both reproducibly bound a
C-terminal region (aa 227-404) that contains the ELT-2 DNA-binding domain
(Fig. 6).
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Guss et al.,
2001). In addition to CSL sites, enhA contains
highly conserved WGATAR and CTCAAG sequences. WGATAR is a predicted binding
site for GATA transcription factors, and CTCAAG sequences can function as
bindings sites for members of the NK family of homeodomain proteins
(Gajewski et al., 1997). The
fact that these sites have remained in close proximity in all four
Caenorhabditis species, despite considerable variation in the
position and orientation of the enhancer, suggests that they have a functional
relationship. Vertebrate GATA-4 and Nkx2-5 function synergistically in
activating expression of cardiac genes such as atrial natriuretic factor
(ANF); the ANF promoter has conserved binding sites for both factors, and
Nkx2-5 and GATA-4 proteins interact in vitro and in vivo
(Durocher et al., 1997). In
C. elegans, the mesodermal cell pm8 expresses both the NK
transcription factor CEH-24 (Harfe and
Fire, 1998), and the GATA factors ELT-5 and ELT-6 (our unpublished
results) (Koh and Rothman,
2001). However, our studies suggest that the CTCAAG and WGATAR
sequences in enhA have at least partially separate roles
with respect to Notch interactions in the mesoderm and endoderm, respectively.
For example, mutations in all of the WGATAR sequences abolish Notch-dependent
expression in the endoderm, but have no apparent effect on Notch-dependent
expression in the mesoderm. It remains possible that GATA and NK proteins
function together in Notch independent events in embryonic or postembryonic
development, or in physiological contexts not addressed in our study.
GATA factors and Notch interactions in the endoderm
The predicted GATA-binding sites in enhA are essential
for two Notch interactions that occur in the endoderm, and we present evidence
that ELT-2/GATA acts in concert with Notch signaling in the second
interaction. The finding that heat shock ELT-2/GATA is sufficient to activate
reporter expression in diverse Notch interactions argues that there are no
additional endoderm-specific factors that are essential for Notch-regulated
expression. Indeed, the combination of inducible ELT-2/GATA with the
enhA::GFP reporter might be a useful tool to identify new
Notch interactions in C. elegans or in other animals. There are six
GATA factors, including ELT-2, that are expressed before or during the first
endodermal Notch interaction (Maduro and
Rothman, 2002). We consider the atypical GATA factors MED-1 and
MED-2 to be unlikely candidates for combinatorial factors in the first
interaction, because their predicted binding sites are not present in
enhA, and heat shock MED-1 is unable to drive ectopic
enhA transgene expression in any of several
Notch-activated cells (our unpublished results). Thus, ELT-2 and/or the
remaining GATA factors are candidates for functionally redundant combinatorial
factors in the first endodermal Notch interaction.
Notch interactions in the endoderm are required to generate a morphological
twist in the intestine, but Notch mutants have an otherwise
well-differentiated intestine (Hermann et
al., 2000). By contrast, ELT-2/GATA appears to regulate the
expression of numerous intestine-specific genes in both embryonic and
postembryonic development (Pauli et al.,
2006; McGhee et al.,
2007). Analysis of the ELT-2 target pho-1 has identified
a 79 bp element that is important for endoderm expression; this element
contains three WGATAR sequences, but no predicted CSL-binding sites
(Fukushige et al., 2005) (our
unpublished observations). Multi-copy arrays of transgenes with the
pho-1 element can drive endoderm-specific expression, but only if the
element is multimerized in each transgene to contain between 12 and 24 WGATAR
sequences. In contrast, enhA transgenes with CSL-binding
sites showed strong reporter expression with only three (C. briggsae)
or four (C. elegans) WGATAR sequences. Heat-shock-induced expression
of ELT-2/GATA was capable of driving enhA::GFP expression
either before or after ELT-2-dependent expression of ref-1 normally
occurs in the endoderm. However, in all cases, expression was restricted to
Notch-activated cells. Similarly, heat shock ELT-2/GATA was unable to drive
transgene expression when the CSL-binding sites were mutated in
enhA, or when LAG-1/CSL activity was depleted by RNAi.
From these results we conclude that ELT-2/GATA is insufficient to drive
expression enhA::GFP in the absence of Notch signaling,
and that this insufficiency is not caused by CSL-mediated repression.
The most critical WGATAR sequence in the promoter of the ELT-2 target
pho-1 has the extended sequence ACTGATAA; this same sequence is
critical for expression of the ELT-2 target ges-1, and ELT-2 binds
this sequence in vitro (Egan et al.,
1995; Hawkins and McGhee,
1995; Fukushige et al.,
2005). We showed here that an ACTGATAA sequence (3-GATA)
is one of the two most important WGATAR sequences required for endodermal
expression of enhA::GFP, and that ELT-2 binds this
sequence in vitro. However, the second critical GATA site in
enhA is TTTGATAA (2-GATA), and is completely
conserved in each of the four Caenorhabditis species. This sequence
differs from 19 experimentally confirmed WGATAR sequences important for
ELT-2-dependent intestine-specific expression in C. elegans, and is
very rare among potential GATA-binding sites found in a survey of over 70
intestine-specific or -enriched genes that are likely to be regulated by ELT-2
(McGhee et al., 2007).
Moreover, our in vitro competition experiments suggest that ELT-2 has a lower
affinity for the 2-GATA site than for the 3-GATA site. A
possible explanation for the conservation of 2-GATA is that it is a
preferred binding site for GATA factors other than ELT-2 that presumably
regulate expression in the first endodermal Notch interaction. If so,
transgenes containing only 2-GATA might be expected to show higher
levels of expression after the first endodermal interaction than transgenes
with only 3-GATA. We did not observe this difference in expression;
however, both transgenes were assayed as multi-copy arrays that may have
masked such differences. An alternative possibility for the conservation of
2-GATA is that presence of the NICD-CSL-MAML ternary complex
facilitates binding of ELT-2 to the site, thus allowing gene expression to be
dependent on Notch signaling. We have shown that the C-terminal half of ELT-2,
that contains the DNA-binding domain, can bind LAG-1/CSL in vitro.
Interestingly, core LAG-1 shows much stronger binding to ELT-2 in vitro than
does full-length LAG-1, raising the possibility that in vivo binding to ELT-2
might be modulated by protein interactions that alter the conformation of
LAG-1. In future experiments, it will be important to determine whether CSL
sites or components of the ternary complex impact the recruitment of
ELT-2/GATA to 2-GATA or 3-GATA in vivo.
Studies in several systems have shown that GATA factors often act in
combination with other types of transcription factors. For example, vertebrate
GATA3 recruits Smad3 to regulate target gene expression during T-cell
development, and GATA4 recruits STAT proteins in the developing heart
(Blokzijl et al., 2002;
Wang et al., 2005).
Drosophila studies have shown synergy between Rel/NF-B type
transcription factors and the GATA factor Serpent for immunity gene expression
(Senger et al., 2004). The
structures of the N- and C-terminal domains of LAG-1/CSL are remarkably
similar to the N- and C-terminal Rel-homology regions of Rel/NF-B
proteins (Kovall and Hendrickson,
2004). It will be of interest to determine whether Serpent/GATA
binds Rel/NF-B proteins directly, and whether these interactions have
structural parallels with ELT-2/GATA binding to LAG-1/CSL.
Towards a Notch-GATA transcriptional code
RTGGGAA and RTGAGAA sequences that are potential binding sites for CSL
proteins occur frequently in the C. elegans genome (>80,000
copies), and are often found near genes that are unlikely to be Notch targets,
such as genes encoding metabolic enzymes (Genome Enhancer,
http://genomeenhancer.org/;
our unpublished observations). What sequence motifs determine whether a gene
will respond to Notch signaling? CSL-paired sites provide a paradigm for a
Notch transcriptional code by facilitating cooperative binding of CSL ternary
complexes (Nam et al., 2007)
(see Introduction). Cooperative binding involves residues in the Notch ANK
domain that are conserved in vertebrates and Drosophila, but that are
not present in the ANK domains of the C. elegans Notch proteins GLP-1
or LIN-12 (Nam et al., 2007).
In computer searches, we identified nine C. elegans genes that
contained a potential CSL paired site within 3 kb of the predicted initiator
ATG (see Materials and methods). However, none of the promoters of the
orthologous C. briggsae genes appeared to contain potential CSL
paired sites, with the exception of the enhA element from
ref-1. Because our results show that the `paired' orientation of CSL
sites in enhA is not essential for Notch-dependent
expression, it is unlikely that CSL-paired sites represent an important
transcriptional code for Notch regulation in C. elegans.
enhA appears to promote Notch-specific expression in
the endoderm primarily through two GATA sites (2-GATA and
3-GATA) and two CSL sites (2-CSL and 3-CSL); point
mutations in any one of these sites, or changing the orientation of either CSL
site, markedly reduces or eliminates enhA::GFP
expression. The spacing and orientations of the 2-GATA, 3-CSL and
3-GATA sites have been completely conserved in all four
Caenorhabditis species. In contrast, sequences outside the enhancer
are highly divergent, and the position and orientation of the enhancer varies
considerably with respect to the initiator ATG of the various ref-1
orthologues. These observations argue against the possibility that the
multiple sites have independent and equivalent functions, and simply
contribute quantitatively to increase gene expression. Because ELT-2/GATA and
LAG-1/CSL can interact in vitro, it is possible that some of the oriented
sites in enhA facilitate a similar interaction between
these proteins in vivo. It is also possible that the sites orient CSL and GATA
proteins to create unique binding surfaces for coactivators, analogous to the
recruitment of the coactivator CBP by multiple proteins that bind the
mammalian IFNβ enhancer (Merika et
al., 1998).
GATA factors appear to have conserved roles in endoderm development in many
animals, and Notch signaling is involved in the development of several
endoderm-derived tissues (Contakos et al.,
2005; Fre et al.,
2005; Matsuda et al.,
2005; Morrisey et al.,
1998; Reiter et al.,
1999; Reuter,
1994; van Es et al.,
2005; Zhu et al.,
1997). Thus, we consider it likely that examples of Notch-GATA
synergy will occur in other animals. Our computer searches of genomic DNA from
the four sequenced Caenorhabditis species do not reveal genes other
than ref-1 that have orthologues with the identical number, spacing,
and orientation, of the CSL and GATA sites found in enhA
(J. Rasmussen, A.N. and J.R.P., unpublished results). It is possible that all
of these sequence features are crucial for Notch-GATA synergy, but that some
of the orthologous genes are not regulated by Notch signaling. Alternatively,
a subset of sites in enhA might promote a core, conserved
interaction between CSL and GATA factors, with additional sites optimizing the
transcriptional response. Within about 25 minutes after first contacting a
signaling cell, a Notch-expressing embryonic cell in C. elegans can
process the Notch receptor, activate transcription of a ref-1::GFP
reporter, translate the mRNA, then produce and fold GFP into a visibly
fluorescent protein (Neves and Priess,
2005). This is a remarkably short interval, and allows
Notch-mediated cell fate decision to occur within a typical embryonic cell
cycle lasting 30 minutes or less. Postembryonic cell cycles can extend to
several hours in C. elegans and other animals, and thus might allow
enhancers regulated by Notch and GATA more time to assemble
transcriptionally-competent protein complexes.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Artavanis-Tsakonas, S., Rand, M. D. and Lake, R. J.
(1999). Notch signaling: cell fate control and signal integration
in development. Science
284,770
-776.
Austin, J. and Kimble, J. (1987). glp-1 is required in the germ line for regulation of the decision between mitosis and meiosis in C. elegans. Cell 51,589 -599.[CrossRef][Medline]
Bailey, A. M. and Posakony, J. W. (1995).
Suppressor of hairless directly activates transcription of enhancer of split
complex genes in response to Notch receptor activity. Genes
Dev. 9,2609
-2622.
Blokzijl, A., ten Dijke, P. and Ibanez, C. F. (2002). Physical and functional interaction between GATA-3 and Smad3 allows TGF-beta regulation of GATA target genes. Curr. Biol. 12,35 -45.[CrossRef][Medline]
Bray, S. J. (2006). Notch signalling: a simple pathway becomes complex. Nat. Rev. Mol. Cell Biol. 7, 678-689.[CrossRef][Medline]
Brenner, S. (1974). The genetics of
Caenorhabditis elegans. Genetics
77, 71-94.
Cave, J. W., Loh, F., Surpris, J. W., Xia, L. and Caudy, M. A. (2005). A DNA transcription code for cell-specific gene activation by Notch signaling. Curr. Biol. 15, 94-104.[CrossRef][Medline]
Christensen, S., Kodoyianni, V., Bosenberg, M., Friedman, L. and Kimble, J. (1996). lag-1, a gene required for lin-12 and glp-1 signaling in Caenorhabditis elegans, is homologous to human CBF1 and Drosophila Su(H). Development 122,1373 -1383.[Abstract]
Contakos, S. P., Gaydos, C. M., Pfeil, E. C. and McLaughlin, K. A. (2005). Subdividing the embryo: a role for Notch signaling during germ layer patterning in Xenopus laevis. Dev. Biol. 288,294 -307.[CrossRef][Medline]
Cooper, M. T., Tyler, D. M., Furriols, M., Chalkiadaki, A., Delidakis, C. and Bray, S. (2000). Spatially restricted factors cooperate with Notch in the regulation of Enhancer of split genes. Dev. Biol. 221,390 -403.[CrossRef][Medline]
Durocher, D., Charron, F., Warren, R., Schwartz, R. J. and Nemer, M. (1997). The cardiac transcription factors Nkx2-5 and GATA-4 are mutual cofactors. EMBO J. 16,5687 -5696.[CrossRef][Medline]
Egan, C. R., Chung, M. A., Allen, F. L., Heschl, M. F., Van Buskirk, C. L. and McGhee, J. D. (1995). A gut-to-tail switch in embryonic expression of the Caenorhabditis elegans ges-1 gene centers on two GATA sequences. Dev. Biol. 170,397 -419.[CrossRef][Medline]
Fre, S., Huyghe, M., Mourikis, P., Robine, S., Louvard, D. and Artavanis-Tsakonas, S. (2005). Notch signals control the fate of immature progenitor cell in the intestine. Nature 435,964 -968.[CrossRef][Medline]
Fukushige, T., Hawkins, M. G. and McGhee, J. D. (1998). The GATA-factor elt-2 is essential for formation of the Caenorhabditis elegans intestine. Dev. Biol. 198,286 -302.[Medline]
Fukushige, T., Goszczynski, B., Tian, H. and McGhee, J. D.
(2003). The evolutionary duplication and probable demise of an
endodermal GATA factor in Caenorhabditis elegans.
Genetics 165,575
-588.
Fukushige, T., Goszczynski, B., Yan, J. and McGhee, J. D. (2005). Transcriptional control and patterning of the pho-1 gene, an essential acid phosphatase expressed in the C. elegans intestine. Dev. Biol. 279,446 -461.[CrossRef][Medline]
Furukawa, T., Kobayakawa, Y., Tamura, K., Kimura, K., Kawaichi, M., Tanimura, T. and Honjo, T. (1995). Suppressor of hairless, the Drosophila homologue of RBP-J kappa, transactivates the neurogenic gene E(spl)m8. Jpn. J. Genet. 70,505 -524.[CrossRef][Medline]
Gajewski, K., Kim, Y., Lee, Y. M., Olson, E. N. and Schulz, R. A. (1997). D-mef2 is a target for Tinman activation during Drosophila heart development. EMBO J. 16,515 -522.[CrossRef][Medline]
Gaudet, J. and Mango, S. E. (2002). Regulation
of organogenesis by the Caenorhabditis elegans FoxA protein PHA-4.
Science 295,821
-825.
Greenwald, I. (2005). LIN-12/Notch signaling in C. elegans. In Wormbook (ed. The C. elegans Research Community), Wormbook, doi/10.1895/wormbook.1.10.1., http://www.wormbook.org.
Guazzi, S., Price, M., De Felice, M., Damante, G., Mattei, M. G. and Di Lauro, R. (1990). Thyroid nuclear factor 1 (TTF-1) contains a homeodomain and displays a novel DNA binding specificity. EMBO J. 9,3631 -3639.[Medline]
Guss, K. A., Nelson, C. E., Hudson, A., Kraus, M. E. and
Carroll, S. B. (2001). Control of a genetic regulatory
network by a selector gene. Science
292,1164
-1167.
Harfe, B. D. and Fire, A. (1998). Muscle and nerve-specific regulation of a novel NK-2 class homeodomain factor in Caenorhabditis elegans. Development 125,421 -429.[Abstract]
Hawkins, M. G. and McGhee, J. D. (1995).
elt-2, a second GATA factor from the nematode Caenorhabditis elegans.
J. Biol. Chem. 270,14666
-14671.
Hermann, G. J., Leung, B. and Priess, J. R. (2000). Left-right asymmetry in C. elegans intestine organogenesis involves a LIN-12/Notch signaling pathway. Development 127,3429 -3440.[Abstract]
Hwang, B. J., Meruelo, A. D. and Sternberg, P. W.
(2007). C. elegans EVI1 proteo-oncogene, EGL-43, is necessary for
Notch-mediated cell fate specification and regulates cell invasion.
Development 134,669
-679.
Iso, T., Kedes, L. and Hamamori, Y. (2003). HES and HERP families: multiple effectors of the Notch signaling pathway. J. Cell. Physiol. 194,237 -255.[CrossRef][Medline]
Kasai, Y., Stahl, S. and Crews, S. (1998). Specification of the Drosophila CNS midline cell lineage: direct control of single-minded transcription by dorsal/ventral patterning genes. Gene Expr. 7,171 -189.[Medline]
Kelly, S. E., Bachurski, C. J., Burhans, M. S. and Glasser, S.
W. (1996). Transcription of the lung-specific surfactant
protein C gene is mediated by thyroid transcription factor 1. J.
Biol. Chem. 271,6881
-6888.
Koh, K. and Rothman, J. H. (2001). ELT-5 and
ELT-6 are required continuosly to regulate epidermal seam cell differentiation
and cell fusion in C. elegans. Development
128,2867
-2880.
Kovall, R. A. and Hendrickson, W. A. (2004). Crystal structure of the nuclear effector of Notch signaling, CSL, bound to DNA. EMBO J. 23,3441 -3451.[CrossRef][Medline]
Kramatschek, B. and Campos-Ortega, J. A. (1994). Neuroectodermal transcription of the Drosophila neurogenic genes E(spl) and HLH-m5 is regulated by proneural genes. Development 120,815 -826.[Abstract]
Lanjuin, A., Claggett, J., Shibuya, M., Hunter, C. P. and Sengupta, P. (2006). Regulation of neuronal lineage decisions by the HES-related bHLH protein REF-1. Dev. Biol. 290,139 -151.[CrossRef][Medline]
Li, J., Gao, E. and Mendelson, C. R. (1998).
Cyclic AMP-responsive expression of the surfactant protein-A gene is mediated
by increased DNA binding and transcriptional activity of thyroid transcription
factor-1. J. Biol. Chem.
273,4592
-4600.
Maduro, M. F. and Rothman, J. H. (2002). Making worm guts: the gene regulatory network of the Caenorhabditis elegans endoderm. Dev. Biol. 246,68 -85.[CrossRef][Medline]
Maduro, M. F., Meneghini, M. D., Bowerman, B., Broitman-Maduro, G. and Rothman, J. H. (2001). Restriction of mesendoderm to a single blastomere by the combined action of SKN-1 and a GSK-3beta homolog is mediated by MED-1 and -2 in C. elegans. Mol. Cell 7, 475-485.[CrossRef][Medline]
Maduro, M. F., Hill, R. J., Heid, P. J., Newman-Smith, E. D., Zhu, J., Priess, J. R. and Rothman, J. H. (2005). Genetic redundancy in endoderm specification within the genus Caenorhabditis. Dev. Biol. 284,509 -522.[Medline]
Matsuda, Y., Wakamatsu, Y., Kohyama, J., Okano, H., Fukuda, K.
and Yasugi, S. (2005). Notch signaling functions as a binary
switch for the determination of glandular and luminal fates of endodermal
epithelium during chicken stomach development.
Development 132,2783
-2793.
McGhee, J. D. (2007). The C. elegans intestine. In Wormbook (ed. The C. elegans Research Community), WormBook, doi/10.1895/wormbook.1.133.1, http://www.wormbook.org.
McGhee, J. D., Sleumer, M. C., Bilenky, M., Wong, K., McKay, S. J., Goszczynski, B., Tian, H., Krich, N. D., Khattra, J., Holt, R. A. et al. (2007). The ELT-2 GATA-factor and the global regulation of transcription in the C. elegans intestine. Dev. Biol. 302,627 -645.[CrossRef][Medline]
Merika, M., Williams, A. J., Chen, G., Collins, T. and Thanos, D. (1998). Recruitment of CBP/p300 by the IFN beta enhanceosome is required for synergistic activation of transcription. Mol. Cell 1,277 -287.[CrossRef][Medline]
Morel, V. and Schweisguth, F. (2000).
Repression by suppressor of hairless and activation by Notch are required to
define a single row of single-minded expressing cells in the
Drosophila embryo. Genes Dev.
14,377
-388.
Morrisey, E. E., Tang, Z., Sigrist, K., Lu, MM., Jiang, F., Ip,
H. S. and Parmacek, M. S. (1998). GATA6 regulates HNF4 and is
required for differentiation of visceral endoderm in the mouse embryo.
Genes Dev. 12,3579
-3590.
Mumm, J. S. and Kopan, R. (2000). Notch signaling: from the outside in. Dev. Biol. 228,151 -165.[CrossRef][Medline]
Nam, Y., Sliz, P., Pear, W. S., Aster, J. C. and Blacklow, S.
C. (2007). Cooperative assembly of higher-order Notch
complexes functions as a switch to induce transcription. Proc.
Natl. Acad. Sci. USA 104,2103
-2108.
Nellesen, D. T., Lai, E. C. and Posakony, J. W. (1999). Discrete enhancer elements mediate selective responsiveness of enhancer of split complex genes to common transcriptional activators. Dev. Biol. 213, 33-53.[CrossRef][Medline]
Neves, A. and Priess, J. R. (2005). The REF-1 family of bHLH transcription factors pattern C. elegans embryos through Notch-dependent and Notch-independent pathways. Dev. Cell 8,867 -879.[CrossRef]
