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Fig. 1. Zn-finger domains of Sens mediate DNA binding, repression and
activation. (A) Schematic of the ac-luc reporter used in
the S2 cell transcription assay. E1, E2 and E3 represent the E-boxes, and S
represents the S-box in the 470 bp ac proximal enhancer. (B)
Alignment of the fly (D. m) Sens Zn-finger (ZF) domains with the
corresponding Zn-finger domains of Homo sapiens (H.s) Gfi1
and Caenorhabditis elegans (C.e) PAG-3 shows that Zn-finger
domains of GPS proteins and the linker regions that connect them are highly
conserved. Stars represent the cysteines in the C2H2 structure. Squares
represent the amino acids that are predicted to contact DNA in C2H2-type
Zn-finger domains. Circles represent the amino acids in linkers that have the
potential to be phosphorylated. Blue boxes denote divergent amino acids.
(C) EMSA assay using a previously characterized probe (R21, see
Materials and methods) and Sens with different types of Zn-finger mutations.
Sens loses its ability to bind to DNA if the cysteines in Zn finger 1, 2 or 3
are mutated. The amino acids that were predicted to contact DNA in Zn fingers
2 and 3 but not in Zn finger 1 seem to be crucial for DNA binding. Zn finger 4
seems to be dispensable for DNA binding. (D) Activation assays in S2
cells show that all Zn fingers are involved in the synergism with proneural
proteins to upregulate the expression of the ac-luc reporter. Sens
fails to synergize with proneural proteins when either Zn finger 2 or 3 is
mutated, but exhibits some synergism upon mutating Zn finger 1 or 4.
(E,F) Repression assays on the ac-luc reporter using
wild-type and Zn-finger mutant Sens expression constructs. Sens loses its
ability to repress the ac-luc reporter at low levels if Zn finger 1,
2 or 3, but not 4, is mutated.
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