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. 2011 Jul 19;108(29):11959-64.
doi: 10.1073/pnas.1108686108. Epub 2011 Jun 28.

Functional synthetic Antennapedia genes and the dual roles of YPWM motif and linker size in transcriptional activation and repression

Affiliations

Functional synthetic Antennapedia genes and the dual roles of YPWM motif and linker size in transcriptional activation and repression

Dimitrios K Papadopoulos et al. Proc Natl Acad Sci U S A. .

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Full-length and synthetic Antp genes cause homeotic transformations and head-involution defects in the embryo. (Upper) UAS Antp constructs (full-length and synthetic) generated for the functional analysis of the YPWM motif and the N terminus. The short linker (SL) contains the sequence RSQF and the long linker (LL) contains the sequence RSQFGKCQ. The C terminus of synthetic peptides lacks the terminal 14 residues and contains only the sequence TKGEPGS. All construct numbers are used for reference in the subsequent figures. (Lower) Comparative analysis of head-involution defects generated by constitutive expression of full-length and synthetic Antp genes, using nullo-Gal4. Only proteins bearing a YPWM motif (Center and Right column) could induce transformation of head to mesothorax (T2). The size of thoracic segments is indicated by lines. The numbers 1–3 indicate segment identity: (1) prothoracic, (2) mesothoracic, or (3) metathoracic. Red arrows show the prothoracic beards, and blue asterisks indicate pronounced head-involution defects, where present. Note the mild head defects in the synthetic AAAAAntp line, shown by the abnormal development of the mouth hook.
Fig. 2.
Fig. 2.
YPWM-dependent repression and activation of Antp target genes in the embryo mediated by full-length and synthetic Antp genes. (A) Repression of Scr in the labial (lb, solid brackets) and prothoracic (T1, dashed brackets) segments by full-length and synthetic Antp constructs. Ectopic Antp is depicted in green and Scr in red. For each line a close-up of the region of repression is shown, and below it an overview of an entire embryo is shown. Note the overlay of both gene products in the AAAA-substituted lines, indicated by the yellow color. Staining in the WT embryo is for the endogenous proteins. (B) Ectopic activation of Tsh in the embryonic head segments by full-length and synthetic Antp genes. Tsh is shown in red and ectopic Antp in green. The upper image for each construct depicts Tsh, and the lower image shows its overlay with ectopic Antp. The domain of ectopic Tsh expression is outlined by dashed lines. Staining in the WT embryo is for the endogenous genes. All UAS lines were induced using a ptc-Gal4 driver.
Fig. 3.
Fig. 3.
Tarsal transformations caused by full-length proteins and synthetic Antp peptides. (A) The presence of the YPWM motif in Antp full-length and synthetic peptides greatly enhances the transformation, but its absence in synthetic peptides does not completely abolish it. Note the leg bristles (black arrows) that indicate weak transformation of the third antennal segment (A3) to a tarsus and the presence of apical/preapical bristles (red arrowheads), indicating mesothoracic leg (T2) identity. Curved lines show antennal segments. 2, second antennal segment; 3, third antennal segment; Ar, arista; transformed segments are identified in red type; tarsal claws are indicated by blue asterisks. (B) Eye-to-wing transformations induced in a toyD2 mutant background. The synthetic genes behave comparably to the full-length Antp73b allele. W, wings; L, legs.
Fig. 4.
Fig. 4.
Repression of genes required for antennal development by ectopic expression of full-length and synthetic Antp genes. (A) Repression of Salm in the antennal portion of the eye-antennal disc mediated by full-length and synthetic Antp genes. All proteins featuring a functional YPWM motif repress Salm. The constructs with the AAAA substitution do not repress Salm. (B) Repression of dan in the antennal disc by full-length and synthetic Antp genes. All lines behave as in A.
Fig. 5.
Fig. 5.
Ectopic activation of leg-specific genes by full-length and synthetic Antp genes in the antennal disc. Ectopic activation of grn in the antennal disc by synthetic and full-length Antp constructs. Arrows show the ectopic expression of β-galactosidase upon Antp gain of function in the antennal portion of the disc, as represented schematically. A Dll-Gal4 driver has been used throughout.

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