An induction gene trap for identifying a homeoprotein-regulated locus.
An important issue in developmental biology is the identification of homeoprotein target genes. We have developed a strategy based on the internalization and nuclear addressing of exogenous homeodomains, using an engrailed homeodomain (EnHD) to screen an embryonic stem (ES) cell gene trap library. Eight integrated gene trap loci responded to EnHD. One is within the bullous pemphigoid antigen 1 (BPAG1) locus, in a region that interrupts two neural isoforms. By combining in vivo electroporation with organotypic cultures, we show that an already identified BPAG1 enhancer/promoter is differentially regulated by homeoproteins Hoxc-8 and Engrailed in the embryonic spinal cord and mesencephalon. This strategy can therefore be used for identifying and mutating homeoprotein targets. Because homeodomain third helices can internalize proteins, peptides, phosphopeptides, and antisense oligonucleotides, this strategy should be applicable to other intracellular targets for characterizing genetic networks involved in a large number of physiopathological states.
Mainguy
G
G
CNRS, UMR 8542, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris Cedex 05 France.
Montesinos
M L
ML
Lesaffre
B
B
Zevnik
B
B
Karasawa
M
M
Kothary
R
R
Wurst
W
W
Prochiantz
A
A
Volovitch
M
M
D000818:Animals; D001324:Autoantigens / Q000096:biosynthesis / Q000235:genetics; D001921:Brain / Q000196:embryology / Q000378:metabolism; D002352:Carrier Proteins; D002467:Cell Nucleus / Q000378:metabolism; D003094:Collagen / Q000096:biosynthesis / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D018274:Electroporation; D004622:Embryo, Mammalian / Q000166:cytology; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008957:Models, Genetic; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013116:Spinal Cord / Q000196:embryology / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology; D014157:Transcription Factors; D000097805:Collagen Type XVII
D001324:Autoantigens; D002352:Carrier Proteins; D003094:Collagen; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D020033:Protein Isoforms; D012333:RNA, Messenger; D014157:Transcription Factors; D000097805:Collagen Type XVII; C095667:Dst protein, mouse; C489816:Hoxc8 protein, mouse; C106185:engrailed homeobox proteins