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Fig. 6. Shn proteins contribute to Bmp signaling by functioning as scaffolding
factors. Shn proteins provide a framework that integrates Smads,
co-activators/co-repressors and other transcription factors. The large size of
Shn proteins may provide the flexibility to recognize different partners and
to act through a variety of cis-elements. On genes such as Xvent2, Id3,
brk, bam and gsb, that contain the GRCKNC(N5)GTCTG consensus,
Smad1/Mad and Smad4/Med bind to GRCKNC and GTCT sites, probably as a
heterotrimeric complex (Gao et al.,
2005 ) (not represented in this figure). Shn/Shn1 interaction with
the MH2 domains of Smads could stabilize the complex and provide docking sites
for cell/tissue-specific co-repressors, as in A, or co-activators, as
in B. Shn binding in A and B is highly sensitive to the spacing between
the Smad sites indicating steric constraints
(Gao et al., 2005;
Pyrowolakis et al., 2004).
(C) Shn promotes activation of Ubx in the Drosophila
midgut through a promoter element that contains sites for Mad and an
NF B-like site directly bound by Shn. In this context, there is no
apparent requirement for Med binding to DNA. (D) In contrast, the mouse
PPAR enhancer that is activated by Shn2 requires sites for Smad4 and
C/EBP , but does not contain Smad1 motifs
(Jin et al., 2006). The
sensitivity of these enhancers to alterations in spacing between binding sites
has not been tested.
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T) at position 4. Point mutations and
deletions in the Xvent2-BRE variants are marked in green. (B)
Mutant BREs were tested for their response to Bmp signaling in
Xenopus animal cap assays. Xvent2-BRE-luciferase reporters
(top) were microinjected into two- to four-cell stage embryos (bottom), animal
cap explants were dissected at stage 8-9 and cultured until siblings reached
early gastrula stages, then processed for luciferase assays. (C, left)
Mutant Xvent2-BREs respond identically in Xenopus and
Drosophila. Both the wild-type BRE and Xvent2-sub2, which
contains two transversions within the five-nucleotide spacer (see A), are
stimulated 
10- to 11-fold in response to CABR, compared with reporter
alone. By contrast, Xvent2-del2 bearing a two-nucleotide spacer
deletion (see A) fails to respond. In the experiment shown, 100 pg of CABR
mRNA/embryo was used to stimulate expression; however, the
Xvent2-del2 reporter fails to respond even at 2 ng. All results are
presented as fold activation relative to wild type in the absence of CABR. We
consistently observed higher luciferase counts for Xvent2-sub2
relative to wild type. (C, right) Wild type and Xvent2-sub2 respond
to endogenous Bmp signaling, whereas the Xvent2-del2 mutant does not.
Transgenic embryos containing BRE multimers driving GFP are at tailbud stage
31/32. In situ hybridization shows that wild type and Xvent2-sub2
mutants direct expression in a pattern similar to the endogenous
Xvent2 mRNA and Xvent2 transgenes described previously.
Transgenic Xvent2-del2 embryos had variable expression patterns that
are likely to reflect position effects due to random integration sites.
