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Fig. 7. Repression of Hb9 by homeodomain transcription factors. (A)
Plasmids encoding progenitor factors, Nkx2.2, Nkx6.1, Pax6 and Irx3, were
co-transfected with the 2.5 kb distal+TK:luciferase reporter into 293 cells
and luciferase activity was used to monitor the fold repression relative to
vector-only transfections. (B) Hb9 and EnR-Hb9 (Hb9 homeodomain linked to eh1
engrailed repressor domain) repressed the synergistic activation of M250 by
Isl1/Lhx3/NeuroM in transfected P19 cells. By contrast, Hb9-HD and Hb9-HD
fused VP16 activation domain (VP16-Hb9) did not. (C,D) Gel retardation
analysis reveals that Hb9 binds to the M50 and M100 (not shown) portions of
its enhancer. Antibodies against Hb9 supershifted or disrupted the DNA:protein
complex, whereas IgG control serum had no effect on DNA binding. Mutation of
the ATTA sequences within M50 (ATTAm) disrupted Hb9 binding. The protein
complex formed by Isl1/Lhx3 relies on the same ATTA elements for binding
(Lee and Pfaff, 2003),
suggesting Hb9 may compete with the LIM factors for access to the
enhancer.
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