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First published online 4 March 2009
doi: 10.1242/dev.030668
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1 National Institute of Diabetes and Digestive and Kidney Diseases, National
Institutes of Health, Bethesda, MD 20892, USA.
2 Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY
14853, USA.
3 Departments of Pathology and Genetics, Stanford School of Medicine, Stanford,
CA 94305, USA.
* Author for correspondence (e-mail: mwkrause{at}helix.nih.gov)
Accepted 9 February 2009
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Key words: C. elegans, Myogenesis, Caudal, MyoD, Embryogenesis
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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;
Charge and Rudnicki, 2004;
Tapscott, 2005). A similar,
genetically defined pathway has been shown to regulate posterior bodywall
muscle development during C. elegans embryogenesis
(Baugh and Hunter, 2006;
Fukushige et al., 2006). The
maternally supplied, Caudal-related homeobox transcription factor PAL-1
specifies the posterior somatic blastomeres called C and D
(Hunter and Kenyon, 1996;
Edgar et al., 2001). PAL-1
acts as a binary developmental switch that is toggled by nuclear levels of
POP-1/TCF that, in turn, are regulated by Wnt/MAP kinase signaling. In the
presence of high nuclear POP-1, PAL-1 drives hypodermal (skin) cell fate,
whereas in the presence of low or absent POP-1, PAL-1 directs bodywall muscle
development (Baugh et al.,
2005a; Fukushige and Krause,
2005). In the case of myogenesis, PAL-1 is required genetically to
activate key transcriptional regulators of myogenesis, including HLH-1/MRF and
UNC-120/SRF (Baugh et al.,
2005a; Baugh et al.,
2005b; Fukushige et al.,
2006; Yanai et al.,
2008).
Circumstantial evidence suggests that both HLH-1 and UNC-120 are direct
targets of PAL-1 in the posterior myogenic lineage. Both HLH-1 and UNC-120 are
detectable within posterior, PAL-1-positive muscle lineages within 60 minutes
of somatic lineage establishment
(Fukushige et al., 2006).
Similarly, expression array analysis reveals that ectopically expressed
pal-1 in early embryos results in the activation of both
hlh-1 and unc-120 within 2 hours
(Fukushige and Krause, 2005),
and that both genes are temporally downstream of PAL-1 activity in wild-type
development (Baugh et al.,
2005a; Baugh et al.,
2005b; Yanai et al.,
2008). Thus, PAL-1 sits at the top of a hierarchy of gene function
in posterior embryonic bodywall muscle development, although the molecular
details of this transcriptional cascade remain unknown.
To gain insight into the network of maternal and zygotic genes that regulate muscle module transcription factors during C. elegans development, we combined embryonic chromatin immunoprecipitation (ChIP) assays with transgene mutagenesis. Our results demonstrate that the maternal factor PAL-1 directly binds in vivo to an enhancer region within the promoter of the potent myogenic regulator hlh-1. Mutational analysis of conserved sequences within this enhancer identified two sequence elements, P1 and E1, which bind to PAL-1 and HLH-1, respectively. hlh-1 is the first direct, zygotic target of PAL-1 to be molecularly identified in vivo, providing information on PAL-1 binding site preferences. Our study also provides details of the temporal-spatial control of hlh-1, including the first direct evidence for HLH-1 positive auto-regulation. Together, these results molecularly define a network of maternal and zygotic genes that is responsible for activating and sustaining muscle development; they also suggest how extrinsic cell signals can influence the activity of key components within this network.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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); PD6817: hlh-1(cc450)/mIn1[dpy-10(e128) mIs14] II;
C-His terminal tagged hs::pal-1 (KM471); 353 bp hlh-1 enh-1
driving myo-2 basal promoter (L3136; pPD107.97) (KM480, KM481) and
the mutant derivatives P1 site mutant KM483, KM484, E1 site mutant KM485,
KM486, P1 and E1 double mutant KM487, KM488; 351 bp hlh-1 negative
control region driving myo-2 basal promoter (KM489, KM490); four copy
JKL26 with the pes-10 basal promoter (PD4743); and eight copy P1
element driving myo-2 basal promoter (KM492, KM493).
Generating transgenic lines
Promoter deletion and enhancer assays primarily used the pes-10
basal promoter vector L3135 (pPD107.94), which drives the expression of coding
regions for green fluorescent protein (GFP) fused to lacZ. Transgenic
strains were generated using 10-100 ng/µl of test plasmid and 50
µg/µl of the selectable dominant rol-6 plasmid pRF4. Fine
resolution mapping of enhancer sequences used concatenated oligonucleotides
(between one and eight copies) as simple or complex arrays (see Tables S1 and
S2 in the supplementary material) upstream of the pes-10 basal
promoter. Complex array methods used 4-8 µg/ml test plasmid, 50 µg/ml of
pRF4 (all test plasmid DNAs digested with FspI and pRF4 digested with
ScaI) and 100 µg/ml of PvuII digested N2 genomic DNA.
A full-length pal-1 cDNA was joined to a 6xHis C- or N-terminal tag (primer details can be provided on request) inserted into the hsp16.41 vector pPD49.83 KpnI/NcoI sites. Extrachromosomal transgenic lines were subsequently integrated by gamma irradiation yielding strains KM471, KM472, KM473 and KM474.
Chromatin immunoprecipitation (ChIP)
ChIP assays were performed using the Upstate ChIP kit (cat #17-295).
C-terminal 6xHis-tagged, hs::pal-1 transgenic embryos were
collected from 10 synchronized 10 cm OP50 feeding plates. After a 34°C
heat-shock for 30 minutes, embryos were incubated at room temperature for 3
hours and collected, frozen on dry ice and cracked by three freeze/thaw
cycles. Embryos were incubated in a crosslinking solution of 1.5%
formaldehyde/PBS/proteinase inhibitor buffer (Sigma, cat #P8340) for 20
minutes at 
22°C, pelleted, washed three times in cold PBS/proteinase
inhibitor buffer and used immediately or frozen at -80°C. After washing
embryos, warm nucleic SDS lysis buffer (Upstate, cat #20-163) was added, the
samples vortexed for 20 seconds and put on ice for 30 minutes to 1 hour with
several vortex treatments during incubation. Samples were sonicated on ice
(Misonix sonicator, Model #3000) using an output program of 2.5 (6 W), 30
second work/30 second stop/4 minutes to shear DNA to between 200 and 1000 bp,
as determined by gel electrophoresis. Samples were centrifuged to remove
debris. The supernatant was collected and divided into three fractions: input,
sample and IgG control. Each fraction (except input) was diluted 10-fold into
ChIP dilution buffer (Upstate, catalog number 20-153) and added to pre-cleaned
ssDNA/protein A agarose beads (Upstate, catalog number 16-157c). Sample
fractions (1500 µl) were incubated with 5 µl 6xHis antibody
(Abcam, ab9108) overnight at 4°C with constant rotation while IgG control
samples were incubated in parallel with 5 µl normal Rabbit IgG (Upstate,
catalog number 12-370). After incubation, 60 µl protein A beads were added
to each sample for 1 hour with constant rotation. Beads were washed three
times each with a low salt (Upstate, cat #20-154), followed by high salt
(Upstate, catalog number 20-155), followed by LiCl2 (Upstate,
catalog number 20-156) and finally TE buffer (Upstate, catalog number 20-157).
After the last wash, beads were resuspended in fresh elution buffer (1% SDS,
0.1 M NaHCO3) for 30 minutes. Beads were pelleted with a brief spin
and the supernatant transferred to a new tube; this was repeated once and
elution volumes combined. The eluted volumes and the input (500 µl each)
were adjusted with 20 µl 5 M NaCl and heated at 65°C for 4 hours
followed by the addition of EDTA, Tris-HCl, proteinase K and incubation for 1
hour at 45°C. The DNA was recovered from samples using the Qiaquick PCR
Purification Kit (Qiagen) with elution off the column with 100 µl elution
buffer. Eluted DNA (0.4 µl) was used as a template in each 20 µl
quantitative PCR (qPCR) reaction.
Heat-shock experiments
Two-cell stage embryos were isolated by hand dissection and incubated at
room temperature for 25 minutes before heat-shock. Heat shock consisted of 30
minutes at 34°C with reporter gene expression assayed 4.5 hours later.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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)
(Fig. 1A). To define the
enhancer regions at higher resolution, we tested a series of additional
genomic fragments to narrow each enhancer to several hundreds of base pairs.
Each enhancer was further dissected using a series of partially overlapping
(15 bp) 54-mer oligonucleotides that were concatenated and cloned into the
pes-10 basal promoter test plasmid to generate stable transgenic
strains. Evolutionarily conserved sequences within muscle positive
concatenates were eliminated or mutated and these oligonucleotide derivatives
again tested for activity. Our analysis significantly improved the resolution
of the map of cis-acting sequence elements that function to regulate
hlh-1 expression. Three muscle enhancer elements can now be defined
within 3 kb upstream of the hlh-1 start that are centered around the
following positions relative to the translational start codon: enh-1 (-2446),
enh-2 (-1536) and enh-3 (-496) (Fig.
1A). Further details of this analysis are available in Figs S1 and
S2, and Tables S1 and S2 in the supplementary material. Most hlh-1-derived enhancers with muscle activity functioned in larval and adult bodywall muscle cells, but showed distinct preferences for certain bodywall muscle lineages in embryos. For example, enh-1 was preferentially active in the posterior C and D lineages, whereas enh-2 was active in C, D and MS muscle lineages (Fig. 1B; see Figs S1 and S2 in the supplementary material). This suggested that although there may be common sequence elements directing general bodywall muscle expression after hatching, lineage-restricted elements probably functioned during embryonic development to direct the correct spatial and temporal regulation of hlh-1 activation and maintain its expression.
PAL-1 directly binds to the hlh-1 enh-1 region
One of the important and unresolved issues for hlh-1 gene
regulation was the identity of its activator(s) during embryogenesis. In this
regard, enh-1 drew our attention because of its strong preference for
directing expression in the posterior C and D bodywall muscle lineages in
early embryogenesis (Krause et al.,
1990). Previous studies have shown that C and D blastomere
specification is dependent on the caudal-related transcription factor PAL-1
(Hunter and Kenyon, 1996).
Maternal PAL-1 protein can be first detected in the P2 and EMS blastomeres of
four-cell stage embryos and subsequently in the somatic descendents of P2 -
namely C and D. Maternal PAL-1 persists in the posterior somatic lineages
until the 100-cell stage of embryogenesis, overlapping the onset of zygotic
PAL-1 accumulation that persists in these lineages until the end of
gastrulation (350 cells) (Hunter and
Kenyon, 1996; Edgar et al.,
2001). This temporal and spatial profile of PAL-1 initially
proceeds, and is then coincident with, the expression of hlh-1 in the
C and D lineages, putting PAL-1 in the right place and time to activate this
crucial myogenic regulatory gene directly. Unfortunately, the available PAL-1
antibody failed to pull down endogenous PAL-1 protein efficiently by
immunoprecipitation (C. Hunter, personal communication) and was unsuitable for
chromatin immunoprecipitation (ChIP) studies.
We engineered N- and C-terminal 6xHis-tagged versions of PAL-1 driven
by a heat shock promoter and made stable integrated transgenic strains to
assay by ChIP if PAL-1 was directly binding to hlh-1 enhancer
sequences in vivo. To ensure that these 6xHis-tagged versions of PAL-1
retained wild type activity, we tested each in a biological assay. We employed
an in vivo blastomere cell fate conversion assay rather than rescuing the
pal-1 mutant, as the rescue experiments would be complicated by a
need for the transgene to be expressed both maternally and zygotically. We
have shown previously that ectopic expression of either endogenous or
transgenic pal-1 in early embryos is sufficient to convert almost all
blastomeres to a body wall muscle-like fate when POP-1 levels are depleted by
RNAi (Fukushige and Krause,
2005). We found that both the N- and C-terminal 6xHis-tagged
PAL-1 proteins were indistinguishable from wild-type PAL-1 in this assay (see
Fig. S3 in the supplementary material), demonstrating that both fusion
proteins retained biological activity in vivo. We chose to focus on the
C-terminal tagged transgene for all subsequent experiments. We monitored by
western blot analysis the level of C-terminal 6xHis-tagged PAL-1 protein
(His-PAL-1) accumulation following heat shock induction and found that under
optimal conditions, heat-shock induced His-PAL-1 accumulation was robust with
maximum levels of protein detected 3 hours after induction (see Fig. S4 in the
supplementary material).
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To gain additional insight into hlh-1 regulation and to control
for ChIP assay specificity, we also looked for occupancy at any of these
hlh-1 genomic regions by HLH-1 itself, given that previous studies
have suggested that HLH-1 positively regulates its own expression
(Krause et al., 1994). Our
attempts to ChIP endogenous levels of HLH-1 from wild-type embryos or strains
carrying a high copy number hlh-1 promoter transgene failed to
generate a signal above background. Consequently, we generated integrated
transgenic lines expressing a full-length hlh-1 cDNA under the
control of the heat-shock promoter, induced expression, prepared chromatin as
described above and ChIPed with an affinity-purified chicken anti-HLH-1
antibody. Western blot analysis demonstrated that heat-shock-induced HLH-1
protein levels from the transgene accumulated to between 20- and 100-fold
higher levels than endogenous HLH-1 (data not shown). ChIP data demonstrated
in vivo HLH-1 occupancy of enh-1, enh-3 and enh-4 regions of the
hlh-1 gene, suggesting one or more of these sites may mediate its
positive auto regulation (Fig.
2B). The clear distinction between positive enhancer elements from
PAL-1 and HLH-1 ChIP experiments, coupled with the lack of binding to our
negative control regions, suggested the antibodies used for ChIP had
specificity. As both experimental approaches relied on overexpression of the
transcription factor prior to ChIP, probably resulting in unnatural binding
conditions, the positive results were validated by extensive analysis of
functional cis-acting elements.
Both PAL-1 and HLH-1 alone can activate transcription through enh-1
We focused on the role of enh-1 in hlh-1 gene regulation because
this region demonstrated binding by both PAL-1 and HLH-1 in our ChIP assays.
We tested whether this region alone was sufficient to drive reporter
expression in response to either endogenous factors or ectopic PAL-1 and HLH-1
by cloning a 353 bp genomic fragment upstream of the myo-2 basal
promoter driving gfp expression
(Okkema et al., 1993).
Multiple independent integrated enh-1::myo-2::gfp::lacZ transgenes
resulted in GFP in early embryonic C- and D-derived muscle in otherwise
wild-type animals (Fig. 3). Two
independent enh-1::myo-2::gfp::lacZ lines were crossed into animals
carrying integrated version of the heat-shock promoter driving either PAL-1-or
HLH-1-encoding cDNAs. In the absence of heat shock induction of either of
these two factors, the enh-1::myo-2::gfp::lacZ reporter genes
retained the early embryonic muscle expression pattern that was seen when
assayed on their own. By contrast, heat shock induction of either PAL-1
(Fig. 3A) or HLH-1
(Fig. 3B) in the presence of
the reporter resulted in widespread and strong GFP in 92% (n=176) and
94% (n=165) of the embryos, respectively. Heat-shock treatment of
these enh-1 reporters in an otherwise wild-type background resulted in 53%
(n=77) GFP-positive embryos, with a slight decrease in number of
positive cells per embryo owing to heat shock disruption of normal development
(Fig. 3A,B). As a control for
enh-1 specificity to respond to these induced factors, we also tested a
negative control region within the hlh-1 promoter
(Fig. 1A) in this assay; this
region showed no response to either PAL-1 or HLH-1 (data not shown).
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A series of eight partially overlapping (15 bp) 54-mer oligonucleotides that spanned the enh-1 region were tested as concatenates for their ability to drive embryonic expression in the C and D bodywall muscle lineages (Fig. 4A; see Tables S1 and S2 in the supplementary material). Three of these (JKL26, JKL28 and JKL30) showed strong expression in embryonic C and D lineages. As a test for HLH-1 dependence of these oligonucleotide concatenates, we put those that showed weak (JKL22 and JKL24) or strong (JKL26, JKL28 and JKL30) activity into a balanced hlh-1(cc450)/mIn1(mIs14) mutant background and assayed expression in heterozygous and homozygous hlh-1-null mutants. JKL22 failed to express in any animals, suggesting the original expression in D+C lineages was a false positive result. JKL24 and JKL26 were active in both heterozygous and homozygous hlh-1 mutants, demonstrating that neither region was dependent on wild-type HLH-1 activity to enhance muscle expression. By contrast, JKL30 enhanced muscle expression in heterozygous hlh-1 mutant animals, but was not expressed in hlh-1(cc450) homozygous mutants, demonstrating that this construct was dependent on HLH-1. We were unable to generate lines by injecting JKL28 reporters into the hlh-1 balanced mutant background, so we tested this construct by crossing two independent reporters into the mutant background. Like JKL30, JKL28 required HLH-1 activity for expression. We also tested the ability of JKL26 (strain PD4743) to respond to PAL-1 by knocking down maternal and early embryonic pal-1 expression by RNAi injection into parental hermaphrodites. Whereas 64% (n=76) of embryos from untreated hermaphrodites showed D+C expression of the reporter gene, only 9% (n=96) of embryos from pal-1 RNAi-treated hermaphrodites had GFP-positive cells (see Fig. S5 in the supplementary material), demonstrating that PAL-1 is required for efficient JKL26 bodywall muscle expression in D+C lineages.
A phylogenetic comparison of genomic regions represented by reporter
constructs JKL26 through JKL30 revealed four blocks of conserved sequence in
the alignment of sequences from three species
(Fig. 4C). Two of the conserved
blocks (P1 and Block 3) contained sequences that are identical to known
Caudal-binding sites [TTTATG (Dearolf et
al., 1989; Maurer et al.,
2007)], the Drosophila homolog of PAL-1 (TESS:
http://www.cbil.upenn.edu/cgi-bin/tess/tess).
Block 2 had a single TCF/LEF-like binding site [MAMAG
(Travis et al., 1991;
Waterman et al., 1991)], the
vertebrate homolog of POP-1 that functions in concert with PAL-1 in the C and
D lineages. This sequence is also similar to a consensus binding site sequence
derived from a limited number of known C. elegans POP-1 sites
(Arata et al., 2006;
Korswagen et al., 2000;
Lam et al., 2006;
Maduro et al., 2005;
Shetty et al., 2005). Finally,
the E1 site (CAACTG) is a bHLH factor binding E-box sequence
(Blackwell and Weintraub, 1990;
Kophengnavong et al., 2000)
and we have previously shown that HLH-1 can bind canonical E-box sequences in
vitro (Krause et al.,
1997).
To test the functional significance of these conserved sequence elements, a series of 13 oligonucleotides that removed or mutated bases that were within conserved blocks were tested for muscle enhancer activity as concatenates cloned upstream of the pes-10 basal promoter (see Tables S1 and S2 in the supplementary material). The results demonstrated that both the P1 and E1 sites were crucial for bodywall muscle enhancer activity in the embryonic C and D lineages. Some mutations within conserved Block 2 and 3 sequences retained muscle enhancer activity, including weak activity for an oligonucleotide lacking the Caudal-like binding site within Block 3. We concluded that site P1 was responsible for much of the muscle enhancer activity of JKL26, possibly serving as a direct PAL-1-binding site. E1 appeared to be an HLH-1 binding site that was responsible for JKL28 and JKL30 muscle enhancer activity, consistent with our hlh-1(cc450) results above.
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Several POP-1-binding sites have been previously identified in C.
elegans (Arata et al.,
2006; Korswagen et al.,
2000; Lam et al.,
2006; Maduro et al.,
2005; Shetty et al.,
2005). The conserved sequences within Block 2 of enh-1 match the
eight-base POP-1 consensus (C/T)TTTG(A/T)(A/T)(A/G/C) with the exception of
position 7 [Block 2 has a C, rather than (A/T) in this position]. Using a
previously studied, truncated POP-1 bacterial expression clone kindly provided
by D. Eisenmann, we assayed Block 2 by EMSA. Although we could easily
demonstrate binding of POP-1 to control oligonucleotides that matched the
consensus perfectly (CTTTGATC), POP-1 failed to bind a probe centered on the
Block 2 site (TTTTGACG) (data not shown; see Table S1 in the supplementary
material). Changing the seventh position C to match the consensus resulted in
strong binding by POP-1 in vitro. Thus, we were unable to provide any positive
evidence for Block 2 serving as a binding site for POP-1, suggesting one or
more additional factors are needed to bind this sequence, either on their own
or in combination with POP-1.
The putative PAL-1 and HLH-1 binding sites of enh-1 function in vivo
To test the ability of the P1 and E1 elements of enh-1 to respond to PAL-1
and HLH-1 in vivo, we made a series of site-directed mutations in P1, E1 or
both sites within the enh1::myo-2::gfp::lacZ transgene and tested the
ability of chromosomal integrants of each mutant version to respond to heat
shock-induced PAL-1 or HLH-1. Mutation of the P1 site almost completely
abolished the response of the reporter to PAL-1, but did not affect its
response to HLH-1 (Fig. 6).
Conversely, mutation of E1 eliminated the response of the reporter gene to
HLH-1, but not its ability to respond to PAL-1. The E1 site mutation did
diminish PAL-1 responsiveness of enh-1 (92.4% to 74.4%), presumably because
this alteration eliminates the positive auto-regulatory HLH-1 feedback loop on
this transgene. Finally, mutation of both the P1 and E1 sites of enh-1
severely reduced the responsiveness of the reporter to either PAL-1 or HLH-1.
As a control, we tested each of these mutant reporters alone after heat shock
and found very little expression in the absence of the PAL-1 or HLH-1 encoding
transgenes; 4% (n=75) for the P1 mutant, 4% (n=78) for E1
mutant, and 3% (n=76) for the P1, E1 double mutant. These results
demonstrate that the P1 and E1 sites are both necessary and sufficient for
robust enh-1 responsiveness in vivo to PAL-1 and HLH-1, respectively.
PAL-1 binding site specificity
To determine whether additional sequence information was responsible for
PAL-1 specificity for the P1 site, we focused on the potential contribution of
flanking sequences that extended from the core. Each of the three
transcription factors (HLH-1, UNC-120 and HND-1) that define the regulatory
muscle module (Baugh et al.,
2005a; Baugh et al.,
2005b; Fukushige et al.,
2006) have one or more perfect matches to the P1 core sequence
(TTTATG). Two such sequences exist in the enh-1 region of hlh-1 (P1
and Block 3), there is one matching sequence in the enh-2 region of
hlh-1, one matching sequence at -2405 from the ATG of
unc-120, and one matching sequence at -3589 from the ATG of
hnd-1. We interrogated the ability of heat-shock-induced PAL-1 to
bind to each of these sites in vivo by ChIP-qPCR. Only two sites were positive
for PAL-1 binding, hlh-1 enh-1 and the site upstream of
unc-120 (Fig. 5B),
suggesting that hlh-1 and unc-120, but not hnd-1,
are direct targets of PAL-1.
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The expression of the eight copy P1 reporter gene alone in hypodermis and
bodywall muscle cells derived from the C blastomere was different from all
other enh-1 fragments tested. We have shown previously that hypodermal and
bodywall muscle fates in this lineage are specified by PAL-1 acting in concert
with different levels of nuclear POP-1 (Fukushige et al., 2005). Comparing the
sequence of the eight copy P1 oligonucleotide to a closely related enh-1
subfragment that was bodywall muscle specific (JKL26) suggested the putative
TCF/POP-1 binding site in Block 2 might play a role in bodywall muscle
specificity (see Fig. 4C). As
noted above, we have been unable to demonstrate in vitro that POP-1 alone
binds to Block 2 sequences, therefore, we tested the role of POP-1 in
mediating the differences in expression seen with JKL26 and eight copy P1 in
vivo using RNAi. Heat-shock induction of PAL-1 in embryos harboring the eight
copy P1 reporter and depleted of POP-1 by RNAi resulted in a similar magnitude
of effect on reporter gene expression as it did in embryos with wild-type
levels of POP-1; there was an increase in both the number of GFP positive
embryos (6-fold) and number of GFP positive cells per embryo (threefold).
Although the disruption of embryogenesis due to POP-1 depletion prevents a
direct comparison of numbers, this result suggests the absence of POP-1 has
little effect on the activity of the eight copy P1 reporter. Similarly, RNAi
of lit-1, which blocks POP-1 nuclear export, resulted in an
2.5-fold increase in the number of GFP-positive cells per embryo when
comparing heat shock-induced pal-1 expression to the non-heat shocked
controls. We concluded that the eight copy P1 element reporter responds to
PAL-1 independently of the nuclear levels of POP-1, consistent with the
expression of this reporter gene in both hypodermal and bodywall muscles
derived from the C lineage.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Fukushige et al., 2006). We
have limited evidence that unc-120 may also be a direct PAL-1 target
gene in the posterior muscle lineage. The molecular details for posterior
bodywall muscle can now be modeled (Fig.
7). PAL-1 activation of hlh-1 (and perhaps
unc-120) initiates a self-sustaining positive-feedback loop through
HLH-1 that results in an irreversible commitment of these cells to the
bodywall muscle cell fate. Positive auto-regulation of HLH-1 is robust because
of the presence of multiple HLH-1-binding sites in its own enhancer regions.
We find no evidence that the third factor in the previously described muscle
module, HND-1 (Baugh et al.,
2005a; Baugh et al.,
2005b; Fukushige et al.,
2006), is directly controlled by PAL-1 (see
Fig. 5). It is possible that
HND-1 is controlled independently of PAL-1 to provide a transient and
redundant function to HLH-1 in these lineages. Indeed, HND-1 is expressed in
multiple embryonic lineages and has multiple roles during development in
C. elegans and other organisms
(Cserjesi et al., 1995;
Firulli, 2003;
Mathies et al., 2003;
Srivastava et al., 1995).
Therefore, the role of HND-1 is distinct from the main transcriptional
activators HLH-1 and UNC-120 that persist through post-embryonic development
and that represent the core transcriptional engine driving myogenesis and
post-embryonic muscle cell growth in our model.
Our evidence demonstrates, both in vivo and in vitro, that PAL-1 can bind
the sequence ATTTATGAC. We have begun to analyze the distribution of this
sequence throughout the genome of C. elegans to determine whether
other possible PAL-1 target genes can be identified. Previous studies of
potential direct or indirect downstream targets of PAL-1 in both hypodermal
and bodywall muscle cells identified and validated 21 genes
(Baugh et al., 2005a). Six of
those genes, including hlh-1 and unc-120, have a single
perfect match to this 9 bp consensus sequence within the 4 kb upstream of the
translational start codon; the other positive genes in this group are
spp-10, R02D3.1, cwn-1 and pal-1 itself. By
comparison, a search of 71 randomly selected genes had no sequences matching
this consensus within the 4 kb putative promoter regions. Restricting the
consensus sequence to 8 bp (ATTTATGA) hits nine of the previously identified
21 genes compared with 15/71 random genes; restricting to 7 bp (ATTTATG) hits
14 of the 21 genes compared with 25/71 random genes. We conclude that our
results provide improved insight into potential direct PAL-1 target genes and
specific sites that mediate PAL-1 binding, but that additional binding site
specificity is probably provided by chromatin context and/or interacting
proteins. This underscores the difficulty in assigning functional significance
to genome-scale assays for transcription factor binding sites and highlights
the need for experimental validation.
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;
Fukushige et al., 2006). An
intriguing possibility is that the conserved sequence that resembles a C.
elegans POP-1 binding site in the enh-1 region of hlh-1 (Block
2) mediates part of this switch. Located next to the PAL-1-binding site,
occupancy of this site by POP-1 in hypodermal lineages that have relatively
high levels of nuclear POP-1 could interfere with PAL-1 binding, thus
repressing hlh-1 in these non-muscle lineages
(Fig. 7). Conversely, the
relatively low levels of POP-1 that are present in muscle cell precursors
could allow PAL-1 to bind to enh-1 and activate hlh-1, as well as be
permissive for HLH-1-positive auto-regulation that also occurs through enh-1.
Consistent with this notion, our reporter gene studies demonstrate a role of
POP-1 in determining enh-1-driven expression in response to PAL-1. However,
our inability to demonstrate in vitro binding of POP-1 to Block 2 sequences
suggests that another factor may mediate this response. A test of five
additional genes (hmg-1.2, hmg-3, hmg-4, hmg-12 and sem-2;
see Fig. S6 in the supplementary material) encoding HMG-containing factors
present in early embryogenesis failed to reveal an essential role for these
POP-1-related factors in myogenesis. So, although our results reaffirm the
crucial role for POP-1 in regulating gene expression, it leaves unanswered the
molecular basis for its function. Clearly, understanding the molecular
mechanisms underlying POP-1 function during embryogenesis will go a long way
to revealing the logic of cell fate specification. From a methodological perspective, our results demonstrate the feasibility of using ChIP in C. elegans to determine transcription factor binding sites in vivo. The lack of homogeneity among cells within the developing C. elegans embryo makes ChIP of cell type-specific transcription factors challenging. Indeed, we were unable to generate a ChIP signal from endogenous levels of HLH-1 when we used multiple antibodies and strains that were transgenic for a multicopy hlh-1 promoter-driven reporter gene that included the upstream enhancers that we have demonstrated here bind HLH-1 in vivo. This is a theme that will be common for many of the existing reagents for specific DNA-binding proteins. We have shown that these limitations can be overcome by either epitope-tagging and/or overexpressing the factor of interest. Although overexpression from heat-shock promoters far exceeds physiological levels when measurable, the technique remains a viable option. We were able to identify DNA-binding sites in vivo using ChIP after overexpression of either PAL-1 or HLH-1 and validate these sites by extensive mutational analysis, demonstrating that at least a subset of sites identified this way are biologically relevant. Thus, the use of epitope-tagged factors in transgenic animals offers the possibility to use ChIP to decipher DNA-binding elements in the C. elegans embryo and in larvae.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/8/1241/DC1
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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