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.