RESUMO
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.
Assuntos
Proteínas de Transporte , Proteínas do Citoesqueleto , Proteínas de Homeodomínio/genética , Proteínas do Tecido Nervoso , Colágenos não Fibrilares , Análise de Sequência de DNA/métodos , Fatores de Transcrição , Animais , Autoantígenos/biossíntese , Autoantígenos/genética , Encéfalo/embriologia , Encéfalo/metabolismo , Núcleo Celular/metabolismo , Colágeno/biossíntese , Colágeno/genética , Citoplasma/metabolismo , Distonina , Eletroporação , Embrião de Mamíferos/citologia , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Camundongos , Modelos Genéticos , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/embriologia , Medula Espinal/metabolismo , Células-Tronco/citologia , Colágeno Tipo XVIIRESUMO
In a recent gene-trap screen, we identified the gene coding for Epidermal Bullous Pemphigoid Antigen 1 (BPAG1) as a putative transcriptional target of Engrailed and of other homeoproteins with a glutamine in position 50 of their homeodomain. We now show that the nuclear addressing of the homeodomains of Engrailed (EnHD) and Antennapedia (AntpHD) upregulates BPAG1e transcription in immortalized human keratinocytes (GMA24FIA) expressing En1. This upregulation is not observed with AntpHD-Q50A, a variant of AntpHD in which a single mutation abolishes its high-affinity binding to target DNA, thus strongly suggesting that BPAG1e upregulation homeodomains reflects their specific recognition of homeoprotein-binding sites in the BPAG1e locus. This is further confirmed by DNase I footprinting and electrophoretic mobility shift assays that reveal, within the cloned BPAG1e promoter, several sites of direct interaction with EnHD and Engrailed. Co-transfection experiments in GMA24FIA human keratinocytes, COS-7 simian fibroblasts, and CHP-100 human neuroepithelial cells show that Engrailed, Hoxa-5, and Hoxc-8 regulate BPAG1e promoter activity and that this regulation is context-dependent. Finally, using a mouse line with LacZ inserted within the En1 locus, we identify the keratinocytes of the ventral paws, including the epithelial cells of the eccrine tubules, as a strong site of En1 expression throughout adulthood. We therefore propose that BPAG1e, a 230 kDa keratin-binding protein expressed in keratinocytes and participating in the maintenance of hemidesmosomes at the dermis-epidermis border, is directly regulated by homeoprotein transcription factors.
