|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
|
Fig. 3. Multiple Tbx6 binding is required for Mesp2 activation. (A)
The sequence of the mouse Mesp2 enhancer region that contains four
presumptive Tbx6 binding sites (Sites B, D, F and G). Sites A and C are
presumptive RBPJ-
binding sites
(Yasuhiko et al., 2006). The
Tbx6 consensus binding site was originally reported by White and Chapman
(White and Chapman, 2005).
(B) Site G, but not Site F, binds to Tbx6 in an electromobility shift
assay (EMSA). Site G produced a bandshift indicating a single bound Tbx6
molecule (lane 16), whereas Site B produced two bands (lane 1). Mutated
oligonucleotide probes mB1, mD and mG produced no shifted bands (lanes 5, 11
and 20, respectively). The mB2 probe showed a single shifted band, implying
the loss of one Tbx6 binding site in Site B (lane 6). (C,D)
Luciferase reporter assays were conducted using several mutated enhancer
elements. Luciferase activity was measured after transfection of reporter
constructs along with an empty vector (None), Tbx6 expression vector (+Tbx6),
or both Tbx6 and Notch intracellular domain expression vectors (+Tbx6 +NICD).
Each reporter construct is presented schematically to the left of each graph.
Black oval, mutated Tbx6 binding site; white oval, wild-type Tbx6 binding
site; arrow, transcription start site. The number of wild-type Tbx6 binding
sites is also indicated (number of Tbx6 binding: C, 4 to 2; D, 2 to 0). To the
left are representative images of lacZ staining in transgenic embryos
with P2PSME-lacZ reporters bearing the indicated enhancers. The
number of lacZ-positive/transgene-positive embryos is indicated. The
results of a consecutive series of reporter assays, as described in C, are
shown in D, but on a different scale owing to the steep declines in activity.
The P2EmDG lane represents the same data in both C and D. Each luciferase
assay was performed in triplicate in at least three independent experiments.
Error bars represent s.d.
|
