Role of a versatile peptide motif in controlling Hox nuclear export and autophagy in theDrosophilafat body

Abstract

Hox proteins are major regulators of embryonic development, acting in the nucleus to regulate the expression of their numerous downstream target genes. By analyzing deleted forms of the Drosophila Hox protein Ultrabithorax (Ubx), we revealed the presence of an unconventional Nuclear Export Signal (NES) that overlaps with the highly conserved hexapeptide (HX) motif. This short linear motif was originally described as mediating the interaction with the PBC proteins, a generic and crucial class of Hox transcriptional cofactors in development and cancer. Here we show that the HX motif is involved in the interaction with the major CRM1/Embargoed exportin protein. This novel role was found in several Drosophila and human Hox proteins. We provide evidence that HX-dependent Hox nuclear export is tightly regulated in the Drosophila fat body to control the onset of autophagy. Our results underline the high molecular versatility of a unique short peptide motif for controlling context-dependent activity of Hox proteins at both transcriptional and non-transcriptional levels.consensus distribution of hydrophobic residues (indicated below the graph). NetNES predicts a putative NES with a good confidence score in the region upstream of the HX (magenta peak). E. BiFC between wild type or HX-mutated VC-Scr fusion protein and VN-Emb, as indicated. Fusion proteins are expressed with the Antennapedia (Antp)-Gal4 driver. Graphs on the right show the statistical quantification of BiFC signal in the nucleus (N) and cytoplasm (C) of epidermal cells of stage 10 live embryos. The HX mutation affects the Scr-Emb interaction only in the cytoplasm. F. Genetic interaction between Scr and Emb. The co-expression of cold HA-Emb triggers Scr in the cytoplasm except if the HX motif is mutated. Quantification is performed as described in the Figure 2. Expanded View 5. Analysis of Drosophila and human Hox protein sequences withNetNES. The sequence of putative NESs is provided when the prediction score was good enough (magenta peak above the red line threshold). No NES was predicted in Antp and AbdB. NES were predicted in the N-terminal region of Dfd and Scr, and in the homeodomain (HD) of AbdA, HOXB4, HOXA5, HOXA7 and HOXA9. Expanded View 6. Analysis of Drosophila and human Hox protein sequences withLocNES. The sequence of putative NESs is provided when the prediction score was good enough. NESs were predicted only in Antp, AbdA and AbdB. Of note, one of the NES predicted in AbdB contains the derived HX motif (corresponding to a single W residue: bolded and red). Expanded View 7. Conservation of the non-canonical NES covering the HX motif in Dfd (A) and Scr (B) proteins from invertebrate and vertebrate species. Dm: Drosophila melanogaster. Am: Apis melifera. Bm: Bombyx mori. Tc: Tribolium castaneaum. Csa: Cupiennus salei. Mm: Mus musculus. Dr: Danio rerio. Nv: Nasonia vitripennis. Red arrowheads highlight hydrophobic residues of the putative NESs. Expanded View 8. The HX motif of human HOXA5 is part of a NES mediating CRM1dependent export in live H...