title: Functional synthetic Antennapedia genes and the dual roles of YPWM motif and linker size in transcriptional activation and repression.
abstract: Segmental identity along the anteroposterior axis of bilateral animals is specified by Hox genes. These genes encode transcription factors, harboring the conserved homeodomain and, generally, a YPWM motif, which binds Hox cofactors and increases Hox transcriptional specificity in vivo. Here we derive synthetic Drosophila Antennapedia genes, consisting only of the YPWM motif and homeodomain, and investigate their functional role throughout development. Synthetic peptides and full-length Antennapedia proteins cause head-to-thorax transformations in the embryo, as well as antenna-to-tarsus and eye-to-wing transformations in the adult, thus converting the entire head to a mesothorax. This conversion is achieved by repression of genes required for head and antennal development and ectopic activation of genes promoting thoracic and tarsal fates, respectively. Synthetic Antennapedia peptides bind DNA specifically and interact with Extradenticle and Bric-à-brac interacting protein 2 cofactors in vitro and ex vivo. Substitution of the YPWM motif by alanines abolishes Antennapedia homeotic function, whereas substitution of YPWM by the WRPW repressor motif, which binds the transcriptional corepressor Groucho, allows all proteins to act as repressors only. Finally, naturally occurring variations in the size of the linker between the homeodomain and YPWM motif enhance Antennapedia repressive or activating efficiency, emphasizing the importance of linker size, rather than sequence, for specificity. Our results clearly show that synthetic Antennapedia genes are functional in vivo and therefore provide powerful tools for synthetic biology. Moreover, the YPWM motif is necessary--whereas the entire N terminus of the protein is dispensable--for Antennapedia homeotic function, indicating its dual role in transcriptional activation and repression by recruiting either coactivators or corepressors.
authors.affiliation: Department of Cell Biology, Biozentrum, University of Basel, 4056 Basel, Switzerland.
authors.forename: Dimitrios K
authors.initials: DK
authors.lastname: Papadopoulos
authors.forename: Diana
authors.initials: D
authors.lastname: Reséndez-Pérez
authors.forename: Diana L
authors.initials: DL
authors.lastname: Cárdenas-Chávez
authors.forename: Karina
authors.initials: K
authors.lastname: Villanueva-Segura
authors.forename: Ricardo
authors.initials: R
authors.lastname: Canales-del-Castillo
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Felix
authors.forename: Raphael
authors.initials: R
authors.lastname: Fünfschilling
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Gehring
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics*; D000818:Animals; D051943:Antennapedia Homeodomain Protein / Q000235:genetics* / Q000378:metabolism; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D019521:Body Patterning / Q000235:genetics*; D004330:Drosophila / Q000196:embryology* / Q000235:genetics; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000235:genetics*; D018014:Gene Transfer Techniques; D005813:Genes, Synthetic / Q000235:genetics; D018398:Homeodomain Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D010957:Plasmids / Q000235:genetics; D012097:Repressor Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D051943:Antennapedia Homeodomain Protein; D051792:Basic Helix-Loop-Helix Proteins; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C497974:Antp protein, Drosophila; C459600:BAB2 protein, Drosophila; C083405:exd protein, Drosophila; C091023:gro protein, Drosophila