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Fig. 6. Activation of Foxe3 promoter by Sip1 and Smad8. (A) Scheme of
full-length Sip1 (FL) and SBD-deleted Sip1 (SBD-) (left), and Foxe3
pro (-6.2k)-luciferase reporter construct used for transfection (right). (B)
Exogenous Sip1, either full-length or SBD-deleted form, activates the promoter
in a dose-dependent manner in transfected lens cells. Typically, 100 ng of
Sip1 expression vector causes sevenfold activation and 500 ng of the vector
elicits 20-fold activation. Using the 
EF1 expression vector, only a
marginal activation effect was observed. (C) Effect of exogenous Smads on
Sip1-dependent activation of Foxe3 promoter in transfected lens
cells. Any of the Smads, alone, has no effect on promoter activity, as
exemplified by Smad8. Of the eight Smads examined, only Smad8 augmented
Sip1-dependent activation level. This effect was absent when Sip1 lacked SBD.
(D) Activation of 3GC2-Luciferase reporter gene by expression vectors for
Smad1, Smad5 and Smad8 in transfected lens cells. (E) Effect of inhibitory
Smads on Smad8-mediated augmentation of Sip1-dependent promoter activation.
Smad6 or Smad7 individually cancelled Smad8 effect. (F) Amount-dependent
augmenting effect of exogenous Smad8 on Sip1-dependent activation of
Foxe3 promoter. Regardless of the initial activation level by
exogenous Sip1, the effect of Smad8 saturates at approximately threefold
augmentation level. (G) Effect of length of Foxe3 promoter sequence
on activation by Sip1 and augmentation by Smad8. In the scheme for the 6.2 kb
promoter sequence, CACCT sequences are indicated by dots for comparison. A
1.26 kb promoter sequence was sufficient to show Sip1-dependent activation and
Smad8-dependent augmentation. A 287 bp sequence still showed significant
response to Sip1 and Smad8, but the shortening of the promoter to 127 bp
resulted in loss of the responses. Data using 100 ng of Sip1 expression vector
is shown, but basically the same promoter-length-dependent effect was observed
using 500 ng of Sip1 expression vector as tabulated in the right panel.
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