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.
Highlights
Gene regulation at the level of transcription requires the presence of a promoter, sometimes in conjunction with additional cis-regulatory elements such as enhancers and repressors
Strong GFP expression was seen in the pancreas of older transgenic mice (> 3months). This result was surprising as a recent report described the presence of pancreatic expression in transgenic mice carrying a Pax6 GFP reporter BAC containing a smaller portion of the Pax6 locus than our yeast artificial chromosomes (YAC) transgenic lines (Kim and Lauderdale, 2006)
Multispecies sequence comparisons of the Pax6 locus have revealed a large number of evolutionarily conserved regions (ECRs), not all of which have been characterized to date
Summary
Gene regulation at the level of transcription requires the presence of a promoter, sometimes in conjunction with additional cis-regulatory elements such as enhancers and repressors. Developmental control genes form a particular class of genes with respect to their transcriptional regulation, as they often display highly complex spatiotemporal as well as quantitative expression patterns. Their expression needs to be induced or switched off at the right time and place in the embryo. A full characterization of the cis-regulatory elements required for Pax transcription in different tissues will be essential for studying the molecular mechanisms controlling its expression, and greatly enhance the identification of the trans-acting factors binding to these elements, leading to a better definition of the regulatory networks that specify eye, brain and pancreas development
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