First published online 4 March 2009
doi: 10.1242/dev.030668
Development 136, 1241-1249 (2009)
Published by The Company of Biologists 2009
Caudal-like PAL-1 directly activates the bodywall muscle module regulator hlh-1 in C. elegans to initiate the embryonic muscle gene regulatory network
Haiyan Lei1,
Jun Liu2,
Tetsunari Fukushige1,
Andrew Fire3 and
Michael Krause1,*
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.
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Fig. 1. hlh-1 enhancer regions. (A) The promoter and partial
coding region of hlh-1 (up to exon II) is illustrated with regions of
interest highlighted. Shown below the gene are the bodywall muscle enhancer
regions defined by this (enh-1 to enh-4; blue boxes) and previous work (purple
boxes), with positions indicated relative to nucleotide 1 of the start codon.
Shown above the gene are the regions interrogated by qPCR after ChIP,
corresponding to newly defined muscle enhancers (red) or negative control
regions (green). The black box above enh-1 indicates the cloned genomic region
driving reporter gene expression assayed in vivo. (B) Reporter gene
constructs used to narrow down the location of enh-1. The genomic regions, as
indicated by nucleotide positions listed at the ends of each line, were cloned
and tested for bodywall muscle enhancer activity as described. Fragments that
were positive for muscle lineage expression are underlined in red; a broken
underline indicates weak or variable expression. Black underline indicates
fragments negative for muscle expression. D+C: D and C founder blastomere
embryonic muscle lineage expression; a dash indicates no expression detected.
(C) Schematic representation of bodywall muscle nuclear positions and
lineage of origin indicated by color. (D) Example of GFP observed
during embryogenesis for an enh-1-driven reporter gene showing strongest
expression in D and C lineage bodywall muscles.
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Fig. 2. Chromatin immunoprecipitation (ChIP) results identify potential in vivo
binding sites regulating hlh-1 expression. (A) The four
hlh-1 enhancer regions, and a negative control region (cntrl), were
interrogated by qPCR after a His-PAL-1 ChIP from mixed stage embryos. Of the
regions assayed, only hlh-1 enh-1 was bound by PAL-1 (red) at levels
greater than the IgG control (blue) ChIP. (B) ChIP results of
hlh-1 enhancer and control regions following overexpression of HLH-1
in mixed stage embryos and immunoprecipitation with an affinity-purified
chicken-anti-HLH-1 antibody. Enhancer regions 1, 3 and 4 show greater than
twofold enrichment (purple) compared with pre-immune serum control (blue)
ChIP. All results represent the combined data from a minimum of three
independent repetitions of chromatin preparation and ChIP.
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Fig. 3. hlh-1 enh-1 responds to both PAL-1 and HLH-1 in vivo.
(A-C) Integrated strains harboring a reporter gene (listed on the left)
with either enh-1 (A,B) or eight copies of the enh-1 P1 element (C) were
cloned upstream of the myo-2 basal promoter driving gfp
expression. On their own, these reporters resulted in GFP predominantly in
embryonic D+C bodywall muscle lineages (leftmost panels) with a decrease in
the number of GFP-positive cells upon heat shock owing to disrupted
embryogenesis. These reporter strains were genetically crossed into strains
that overproduced either PAL-1 (A,C) or HLH-1 (B) in response to heat-shock
treatment. Overproduction of either PAL-1 or HLH-1 resulted in disrupted
embryogenesis, widespread conversion of blastomeres to a bodywall muscle-like
fate and widespread activation of reporters. All embryos shown are between 5
and 6 hours of development, and are orientated (when possible) with anterior
towards the left and dorsal towards the top. Each embryo shown has a GFP (top)
and a corresponding Nomarski image (bottom). Enh-1 alone images from A are
duplicated in B. Scale bar: 10 µm.
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Fig. 4. Identification of hlh-1 enh-1 subelements involved in embryonic
bodywall muscle expression. (A) A series of partially overlapping
oligonucleotides from the hlh-1 enh-1 region were tested in
transgenic strains for their ability to drive embryonic expression in the
posterior PAL-1-dependent D+C lineages. Positive fragments are underlined in
red, with broken underlines indicating weak or variable expression. Black
underlines indicate fragments negative for expression. Scoring: no expression
is indicated by a dash (-); body wall muscle (bwm) expression in <10% of
strains (+/-), 10-25% of strains (+), 26%-50% of strains (++), >51% of
strains (+++); n.d. is not tested. (B) Best case representation of
expression for D+C-positive reporter genes (JKL28 shown). Normarski image at
top with corresponding GFP pattern below. (C) An alignment of sequences
upstream of the hlh-1 homologs in C. remanei and C.
briggsae to the C. elegans enh-1 region; dashes indicate spaces,
Ns within remanei sequence reflect a break in contigs used for
alignment. Four substantial blocks of conserved sequence are identified, as
shown in the boxes. The positions for the putative PAL-1-binding site (P1) and
HLH-1-binding site (E1) are indicated relative to enh-1 derivatives shown in
A. Black lines below the sequence mark the extent of oligonucleotides used in
enhancer assays as detailed in A; brown lines underscore the sequence
multimerized for the eight copy P1 element.
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Fig. 5. PAL-1 binding. (A) Electromobility gel shifts were used to
determine whether bacterially produced PAL-1 protein could specifically bind
the P1 site of hlh-1 enh-1. A biotin-labeled 20-mer oligonucleotide
(60 fmol) centered on the wild-type P1 site sequence was annealed to a
100-fold excess of a non-labeled complementary oligonucleotide and run on the
gel alone (lane 1) or after incubation with PAL-1 protein (lanes 2-10); free
probe is indicated by a black arrow and PAL-1 bound probe is indicated by a
white arrow. PAL-1 binding (lane 2) was competed with increasing amounts of
either the wild-type probe (lanes 3-6) or a mutated sequence that eliminated
the putative PAL-1-binding site (lanes 7-10). The mutant probe failed to
compete for binding and did not bind PAL-1 on its own (lane 11). (B)
Promoter regions upstream of hlh-1, unc-120 and hnd-1 that
contained sequence elements resembling the core PAL-1-binding site (ATTTATG)
from hlh-1 enh-1 were interrogated by qPCR after overexpression of
6xHis tagged PAL-1 and ChIP. Only hlh-1 enh-1 and the region
upstream of unc-120 were reproducibly identified as being bound to
PAL-1, as assayed by ChIP.
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Fig. 6. PAL-1 and HLH-1 function through the P1 and E1 sites of hlh-1
enh-1, respectively. The hlh-1 enh-1 reporter gene constructs are
diagrammed with the sequences of the P1 and E1 sites detailed. Wild-type (top)
and mutant enh-1 sequences in which one or both of the sites have been altered
were tested as integrated transgenes for their ability to respond to
overproduction of either PAL-1 or HLH-1 in embryos. A region of the
hlh-1 promoter lacking muscle enhancer activity served as a negative
control (see Fig. 1). The
percentage of embryos with gfp reporter gene expression at 4.5 hours
after heat-shock treatment is shown. Note that P1 is required for response to
PAL-1, E-1 is required for response to HLH-1, and elimination of both P1 and
E1 significantly drops the responsiveness of the reporter gene to either
factor. Names of two independent strains tested for each integrated reporter
gene construct are listed on the left.
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Fig. 7. A model of PAL-1 activation of the muscle module in posterior C and D
lineages of the embryo. PAL-1 binds to enh-1 and activates hlh-1
expression (and possibly unc-120) in C and D lineage descendents
destined to become bodywall muscle. HLH-1 functions as a positive
auto-regulatory factor for its own expression, acting through multiple
enhancer elements, and also activates unc-120. Together, HLH-1 and
UNC-120 activate downstream muscle-specific genes. In C lineage descendent
blastomeres that are not fated to become muscle, an unidentified repressor
blocks PAL-1 activation through enh-1, ensuring that hlh-1 expression
and its positive auto-regulation remains switched off.
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© The Company of Biologists Ltd 2009
