spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

doi: 10.1242/10.1242/dev.00348

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Solano, P. J.
Right arrow Articles by Maschat, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Solano, P. J.
Right arrow Articles by Maschat, F.

Genome-wide identification of in vivo Drosophila Engrailed-binding DNA fragments and related target genes

Pascal Jean Solano1,*, Bruno Mugat1,*, David Martin2, Franck Girard1, Jean-Marc Huibant1, Conchita Ferraz1, Bernard Jacq2, Jacques Demaille1 and Florence Maschat1,{dagger}

1 Institut de Génétique Humaine (UPR 1142). 141 rue de la Cardonille, 34396 Montpellier, France
2 Laboratoire de Génétique et Physiologie du Développement (UMR 6545), IBDM, Parc Scientifique de Luminy, 13288 Marseille, Cedex 9, France



View larger version (53K):

[in a new window]
  		Fig. 5. Engrailed is a direct repressor of frizzled 2 expression. (A) fz2 expression was analyzed by in situ hybridization (in blue) in wild-type (WT) and engrailed mutant (en-) genetic backgrounds. Embryos were double stained with anti-Engrailed antibody (in brown). (B) Schematic representation of the (1A4-GFP) transgene, where a trimer of 170 bp 1A4 fragment has been cloned upstream to hsp70 minimal promoter and GFP reporter gene. (C) GFP expression is shown in different genetic backgrounds: (1A4-GFP) corresponds to the normal expression of the transgene (1, 3, 5, 7, 9); (1A4-GFP/VP16-En) corresponds to GFP expression in the presence of the activator form of Engrailed, driven either by (hs-Gal4) (2, 4, 6, 8) or by (en-Gal4) (10). GFP expression was analyzed in either late L3 larval tissues (1-8) or in embryos (9, 10): (1, 2) hindgut (d, dorsal; v, ventral); (3, 4) midgut; (5, 6) salivary gland; (7, 8) wing imaginal disc; (9, 10) embryo. White arrowheads show the position of stripes. Bracket in 2 indicates GFP expression in both dorsal and ventral compartments. (D) GFP expression of late third instar larvae is shown in the hindgut (GFP) and can be compared with endogenous fz2 expression, detected by an anti-Frizzled 2 antibody in red (anti-Fz2) or with engrailed expression, detected by polyclonal anti-Engrailed antibody in red (anti-En). Merged images are shown.

 


View larger version (40K):

[in a new window]
  		Fig. 2. (A) Identification of Engrailed-binding consensus sequence. The YAATYANB consensus was deduced from sequence analysis of 107 selected clones, as defined in the Material and Methods. For each position, the ratio of A, C G or T is indicated. (B) Gel shift assay was performed on a pentamer of the motif CAATTAGC, the sequence of which is shown below the gel. Labeled DNA fragment was analyzed in the absence (-) or in the presence of different amounts of Engrailed protein: 1=2x10-10 M; 2=3x10-10 M; 3=5x10-10 M; 4=10-9 M. Competition experiments were performed in the presence of 5x10-10 M En protein (+) and in the presence of 300-fold excess of different DNAs: D2, polyhomeotic D2 fragment, corresponding to a specific Engrailed-binding fragment (Serrano et al., 1995Go); C, double strand monomer `CAATTAGC'; N, polyhomeotic N fragment that does not bind Engrailed specifically in vitro (Serrano et al., 1995Go); Cm, double strand mutated monomer `CAGCCGGC'. Supershift assays were performed in the presence of 5x10-10 M En protein (+) and of 4F11 monoclonal anti-Engrailed antibody. F indicates free DNA. The asterisks indicate the Engrailed protein-DNA complexes.

 


View larger version (49K):

[in a new window]
  		Fig. 1. (A) Strategy of Engrailed chromatin immunoprecipitation, cloning procedure and design of the probes used on Southern blot to test the specificity of the procedure. The En probe corresponds to the chromatin preparation used to construct the library. (B) Southern blots performed on 14 inserts isolated from the library. Clone inserts, visualized with ethidium bromide (EtBr), have been transferred onto nitrocellulose. Blots have been hybridized with EN probe and with Background probe, as indicated and described in A.

 


View larger version (54K):

[in a new window]
  		Fig. 3. Specific Engrailed binding to the isolated genomic DNA fragments. The ability of Engrailed to bind the immunoprecipitated fragments was tested in vitro by gel shift assays, as shown here for the 1A4, 2H10, 1B12 and 2C5 fragments. In each case, the sequence of the fragment is shown, as well as a map of the surrounding transcription units. The fragments are around 100 bp in size, surrounding the consensus Engrailed-binding motifs (highlighted in bold in the + strand, and underlined in the opposite strand). Labeled DNA fragments were analyzed in the absence (-) or in the presence of different amounts of Engrailed protein: 1=3x10-10 M; 2=5x10-10 M; 3=10-9 M. Competition experiments were performed in the presence of 10-9 M En protein (+), and in the presence of a 300-fold excess of different DNAs: D2, polyhomeotic D2 fragment; A, the cold fragment itself; N, polyhomeotic N fragment. Supershift assays were performed in the presence of 4F11 monoclonal anti-Engrailed antibody, and in the presence (+) or in the absence (-) of 10-9 M En protein. F indicates free DNA. The asterisks mark the Engrailed protein-DNA complexes.

 


View larger version (52K):

[in a new window]
  		Fig. 4. Engrailed misexpression induces changes in the expression of potential Engrailed target genes. In situ hybridization using anti-sense RNA probes were carried out on late third instar wing imaginal discs in different genetic backgrounds: wild-type (WT) (D,G,J,M,P); MS1096 X UAS-VP16-En (E,H,K,N,Q); MS1096X UAS-En (F,I,L,O,R). Overexpression of (VP16-En), driven by MS1096-Gal4, was detected in the wing pouch, using anti-HA antibody (B), when compared with wild type (A). Overexpression of the normal form of Engrailed was detected with polyclonal anti-En antibody (C). Note that (VP16-En) overexpression leads to a posterior distortion of the disc, whereas Engrailed (En) overexpression leads to an anterior distortion. (D-F) In situ hybridization with ß3-tubulin probe. (G-I) In situ hybridization with frizzled 2 probe. (J-L) In situ hybridization with hibris probe. (M-O) In situ hybridization with branchless probe. Normal expression of bnl is indicated by an arrowhead. Arrows indicate the ectopic expression of bnl in the wing pouch. (P-R) In situ hybridization with frazzled probe. Discs are oriented anterior leftwards, ventral upwards.

 


View larger version (93K):

[in a new window]
  		Fig. 6. Engrailed directly binds 1A4 fragment on polytene chromosomes. (A) In situ hybridization on polytene chromosomes. Localization of the transgene on 2L chromosome, in position 25C (shown by the arrow), using 1A4-GFP biotinylated DNA probe, detected by FITC anti-biotin antibody. Banding of the chromosome is shown by DAPI staining. (B1,B2,C1,C2) Immunodetection of Engrailed-binding sites on 2L polytene chromosome, in tramtrack heterozygous background [ttk804/+], identified by anti-Engrailed antibody (in red). (B1) Engrailed-binding sites of [1A4-GFP/+; ttk804/+] salivary glands. (B2) Same as B1 with DAPI staining. (C1) Engrailed-binding sites of [ttk804/+] salivary glands. (C2) Same as C1 with DAPI staining. The white arrow shows an additional Engrailed binding site at the insertion point of the transgene and the bracket indicates two sites close to it.

 




© The Company of Biologists Ltd 2003