title: Hoxb1 enhancer and control of rhombomere 4 expression: complex interplay between PREP1-PBX1-HOXB1 binding sites.
abstract: The Hoxb1 autoregulatory enhancer directs segmental expression in vertebrate hindbrain. Three conserved repeats (R1, R2, and R3) in the enhancer have been described as Pbx-Hoxb1 (PH) binding sites, and one Pbx-Meinox (PM) binding site has also been characterized. We have investigated the importance and relative roles of PH and PM binding sites with respect to protein interactions and in vivo regulatory activity. We have identified a new PM site (PM2) and found that it cooperates with the R3 PH site to form ternary Prep1-Pbx1-Hoxb1 complexes. In vivo, the combination of the R3 and PM2 sites is sufficient to mediate transgenic reporter activity in the developing chick hindbrain. In both chicken and mouse transgenic embryos, mutations of the PM1 and PM2 sites reveal that they cooperate to modulate in vivo regulatory activity of the Hoxb1 enhancer. Furthermore, we have shown that the R2 motif functions as a strong PM site, with a high binding affinity for Prep1-Pbx1 dimers, and renamed this site R2/PM3. In vitro R2/PM3, when combined with the PM1 and R3 motifs, inhibits ternary complex formation mediated by these elements and in vivo reduces and restricts reporter expression in transgenic embryos. These inhibitory effects appear to be a consequence of the high PM binding activity of the R2/PM3 site. Taken together, our results demonstrate that the activity of the Hoxb1 autoregulatory enhancer depends upon multiple Prep1-Pbx1 (PM1, PM2, and PM3) and Pbx1-Hoxb1 (R1 and R3) binding sites that cooperate to modulate and spatially restrict the expression of Hoxb1 in r4 rhombomere.
authors.affiliation: Molecular Genetics Unit, Department of Molecular Biology and Functional Genomics, Istituto Scientifico H. San Raffaele, Università Vita Salute San Raffaele, via Olgettina 58, 20132 Milan, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Ferretti
authors.forename: Francisco
authors.initials: F
authors.lastname: Cambronero
authors.forename: Stefan
authors.initials: S
authors.lastname: Tümpel
authors.forename: Elena
authors.initials: E
authors.lastname: Longobardi
authors.forename: Leanne M
authors.initials: LM
authors.lastname: Wiedemann
authors.forename: Francesco
authors.initials: F
authors.lastname: Blasi
authors.forename: Robb
authors.initials: R
authors.lastname: Krumlauf
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000196:embryology / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002642:Chick Embryo; D004331:Drosophila melanogaster; D018274:Electroporation; D004622:Embryo, Mammalian / Q000378:metabolism; D004625:Embryo, Nonmammalian; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D009841:Oligonucleotides / Q000737:chemistry; D000075062:Pre-B-Cell Leukemia Transcription Factor 1; D011485:Protein Binding; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism*; D019076:Transgenes
chemical_list: C101121:HOXB1 homeodomain protein; D018398:Homeodomain Proteins; D009841:Oligonucleotides; C481797:Pbx1 protein, mouse; C491412:Pknox1 protein, mouse; D000075062:Pre-B-Cell Leukemia Transcription Factor 1; D014157:Transcription Factors