Transcriptional regulation in endoderm development: characterization of an enhancer controlling Hnf3g expression by transgenesis and targeted mutagenesis

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Abstract
The hepatic nuclear factor 3γ ( Hnf3g ) is a member of the winged helix gene family of transcription factors and is thought to be involved in anterior–posterior regionalization of the primitive gut. In this study, cis ‐regulatory elements essential for the expression of Hnf3g in vivo have been characterized. To this end, a 170 kb yeast artificial chromosome (YAC) carrying the entire Hnf3g locus was isolated and modified with a lacZ reporter gene. The two mouse lines carrying the unfragmented Hnf3g – lacZ YAC showed tissue‐specific, copy number‐dependent and position‐independent expression, proving that 170 kb of the Hnf3g locus contain all elements important in the regulation of Hnf3g . Cis ‐regulatory elements necessary for expression of Hnf3g were identified in a three‐step procedure. First, DNase I hypersensitive site mapping was used to delineate important chromatin regions around the gene required for tissue‐specific activation of Hnf3g . Second, plasmid‐derived transgenes and gene targeting of the endogenous Hnf3g gene locus were used to demonstrate that the 3′‐flanking region of the gene is necessary and sufficient to direct reporter gene expression in liver, pancreas, stomach and small intestine. Third, a binding site for HNF‐1α and β, factors expressed in organs derived from the endoderm such as liver, gut and pancreas, was identified in this 3′‐enhancer and shown to be crucial for enhancer function in vitro . Based on its expression pattern we inferred that HNF‐1β is a likely candidate for directly activating Hnf3g gene expression during development.