Drosophila Brakeless Interacts with Atrophin and Is Required for Tailless-Mediated Transcriptional Repression in Early Embryos
Figure 5
Bks Interacts with Tll and Atrophin
(A) Binding of Bks to Tll in vitro. Left panel shows that in vitro–translated Tll interacts with bacterially produced GST-BksA, but not with GST alone. In the right panel, in vitro–translated Bks-B binds weakly to a GST-Tll fusion protein lacking the DNA binding domain (GST-Tll 101–452), and more strongly with GST-full-length Tll.
(B–E) Genetic interaction of bks with tll mutants. Cellularizing embryos hybridized with a kni probe are oriented with anterior to the left and dorsal up. The kni pattern in wild-type (wt) embryos (B) and embryos from bks278 heterozygous mothers (C) are indistinguishable. In tll1 homozygous embryos (D), the posterior kni domain expands slightly towards the posterior. In tll1 embryos derived from bks278 heterozygous females (E), there is a further expansion of the kni pattern (see arrow).
(F) Bks interacts with the C-terminus of Atrophin. Amino acids (aa) 1,324–1,966 of Atrophin binds the ligand binding domain of Tll, as well as GST-BksA. Truncation of the conserved Bks D2 region (GST-Bks 1–780) does not disrupt binding, but a weaker, independent interaction is found with the D2 domain together with the zinc finger (GST-Bks 834–1,151).
(G) Bks and Tll can be co-immunoprecipitated with Atrophin from Drosophila S2 cells. A stable cell line expressing V5-tagged Bks-B was generated and transiently transfected with FLAG-tagged Tll. Immunoprecipitations with V5, Atrophin, and FLAG antibodies were performed from these cells and compared to normal S2 cells lacking tagged Bks and Tll. The leftmost panel shows a short exposure of a membrane immunoblotted with the V5 antibody, demonstrating the presence of Bks-V5 in transfected cells. The middle panel shows a longer exposure of the same membrane, where Bks-V5 is co-immunoprecipitated with endogenous Atrophin. In the right panel, FLAG-Tll is detected both in the Atrophin and FLAG immunoprecipitates. Arrowheads point to Bks-V5 and Tll-FLAG.
(H) The human Bks homolog ZNF608 (aa 1–600) interacts with aa 600-1191 from human Atrophin-1, showing that the Bks-Atrophin interaction is evolutionarily conserved.