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Fig. 4. Snail2 binds specifically to E boxes surrounding the ATG codon of
Cad6B. (A) Chick Cad6B is encoded by 12 exons on
chromosome 2. Sequence analysis identified six E boxes with the motif CAGGTA
clustered upstream and downstream of the ATG located in exon 2 of
Cad6B. Primers and probes for chromatin immunoprecipitation (ChIP)
and QPCR assays were designed to anneal to the region between E boxes within
each clustered E-box pair. For luciferase experiments, the region of sequence
spanning each clustered pair of E boxes that was cloned into the luciferase
reporter vector is indicated. (B) ChIP experiments identify the
preferential association of Snail2 with E boxes in the premigratory neural
crest region of the chick trunk and midbrain, with E1 and E2>E3 and
E4>EA and EB. At each axial level, the experiment was repeated at least
three times, and the results presented are representative of a typical
experiment. No association of Snail2 to distal sequences lacking E-box motifs
in exon 11 of the Cad6B coding region was observed, and no E-box
motifs were amplified by QPCR performed on samples immunoprecipitated with
control antibodies (GFP, IgG) or with no antibodies added. (C)
Electrophoretic mobility shift assay (EMSA) identifying the specific
interaction of Snail2 with E box 1 and E box 2 in the Cad6B
regulatory region (see also Fig. S3 in the supplementary material).
Double-stranded 33P-end-labeled E-box oligonucleotide probes were
incubated with control or Snail2 nuclear extract in the presence or absence of
various competitor DNA oligonucleotide probes. P, no protein; C, 10 µg
control nuclear extract. In the remaining lanes, 10 µg Snail2 nuclear
extract was added to each binding reaction. S, no competitors added; WT and
MUT, 10-fold and 100-fold molar excess of either unlabeled wild-type or
unlabeled mutant E-box probes added, respectively. Retarded Snail2-E-box
complexes are identified by arrows, and unbound probes are indicated by
arrowheads. Two bands corresponding to E-box complexes were observed in the
presence of the Snail2 nuclear extract. These bands were competed by the
addition of wild-type E-box oligonucleotides, but to a much reduced efficiency
with mutated E-box oligonucleotides. (D) Luciferase assays demonstrate
that Snail2 represses luciferase expression from wild-type
Cad6B E box-luciferase reporters but not from reporters with mutated
E boxes. E1* and E2*, E1 and E2 double mutant;
E3* and E4*, E3 and E4 double mutant. Results are
reported as fold repression (relative to empty vector), and are an average of
at least three independent QPCR experiments.
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