title: Long-range downstream enhancers are essential for Pax6 expression.
abstract: Pax6 is a developmental control gene with an essential role in development of the eye, brain and pancreas. Pax6, as many other developmental regulators, depends on a substantial number of cis-regulatory elements in addition to its promoters for correct spatiotemporal and quantitative expression. Here we report on our analysis of a set of mice transgenic for a modified yeast artificial chromosome carrying the human PAX6 locus. In this 420 kb YAC a tauGFP-IRES-Neomycin reporter cassette has been inserted into the PAX6 translational start site in exon 4. The YAC has been further engineered to insert LoxP sites flanking a 35 kb long, distant downstream regulatory region (DRR) containing previously described DNaseI hypersensitive sites, to allow direct comparison between the presence or absence of this region in the same genomic context. Five independent transgenic lines were obtained that vary in the extent of downstream PAX6 locus that has integrated. Analysis of transgenic embryos carrying full-length and truncated versions of the YAC indicates the location and putative function of several novel tissue-specific enhancers. Absence of these distal regulatory elements abolishes expression in specific tissues despite the presence of more proximal enhancers with overlapping specificity, strongly suggesting interaction between these control elements. Using plasmid-based reporter transgenic analysis we provide detailed characterization of one of these enhancers in isolation. Furthermore, we show that overexpression of a short PAX6 isoform derived from an internal promoter in a multicopy YAC transgenic line results in a microphthalmia phenotype. Finally, direct comparison of a single-copy line with the floxed DRR before and after Cre-mediated deletion demonstrates unequivocally the essential role of these long-range control elements for PAX6 expression.
authors.affiliation: MRC Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, UK. dirkjan@hgu.mrc.ac.uk
authors.forename: Dirk A
authors.initials: DA
authors.lastname: Kleinjan
authors.forename: Anne
authors.initials: A
authors.lastname: Seawright
authors.forename: Sebastien
authors.initials: S
authors.lastname: Mella
authors.forename: Catherine B
authors.initials: CB
authors.lastname: Carr
authors.forename: David A
authors.initials: DA
authors.lastname: Tyas
authors.forename: T Ian
authors.initials: TI
authors.lastname: Simpson
authors.forename: John O
authors.initials: JO
authors.lastname: Mason
authors.forename: David J
authors.initials: DJ
authors.lastname: Price
authors.forename: Veronica
authors.initials: V
authors.lastname: van Heyningen
mesh_terms: D000818:Animals; D002531:Cerebellum / Q000196:embryology / Q000378:metabolism; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics; D004742:Enhancer Elements, Genetic; D005136:Eye Proteins / Q000096:biosynthesis* / Q000235:genetics; D018628:Gene Dosage; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D018398:Homeodomain Proteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008850:Microphthalmos / Q000196:embryology / Q000378:metabolism; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics; D050436:Regulatory Elements, Transcriptional; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D005136:Eye Proteins; D018398:Homeodomain Proteins; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D020033:Protein Isoforms; D012097:Repressor Proteins; D049452:Green Fluorescent Proteins