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Fig. 3. Regulation of the Six2 promoter by Six proteins.
(A,C) E11.5 and E12.5 wild-type mouse embryos show Six2
promoter (-893 to +37)-driven lacZ expression in the maxilla (m),
first branchial arch (arrows), second branchial arch (arrowhead) and limbs
(l), recapitulating Six2 endogenous expression. (B,D)
E11.5 and E12.5 Six2 mutant embryos show unchanged staining in these
embryonic areas in the absence of Six2. (E) Nucleotide sequence of the
proximal Six2 promoter, with Six-binding sites (highlighted in gray)
and the sequence of Hoxa2 binding sites (red) indicated. The identification of
Six-binding sites is based for site 1 on the Six-binding consensus and
footprinting analysis (Spitz et al.,
1998) (N.B. and E.K., unpublished) and for site 2 on the
Six-binding consensus, in vitro binding and functional analysis
(Spitz et al., 1998;
Brodbeck et al., 2004) (N.B.
and E.K., unpublished). Numbers indicate nucleotide positions relative to the
transcriptional start site (+1). (F) Unchanged Six2 binding in the
presence of Hoxa2. Six2 was translated in rabbit reticulocytes in vitro and
incubated with a labeled Six2 promoter fragment (-181 to -48).
Increasing amounts of pcDNA3-Hoxa2 programmed reticulocytes were added to the
binding reaction mix, keeping the total amount of extract constant in each
binding mix by adding unprogrammed reticulocytes. Arrow, the Six2-probe
complex; arrowhead, the Hoxa2-probe complex. No ternary complex was observed;
however, addition of Hoxa2 did not perturb Six2 binding to the probe.
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