Transcriptional regulation in endoderm development: characterization of an enhancer controlling Hnf3g expression by transgenesis and targeted mutagenesis.
The hepatic nuclear factor 3gamma (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-1alpha and beta, 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-1beta is a likely candidate for directly activating Hnf3g gene expression during development.
Hiemisch
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Molecular Biology of the Cell I Division, German Cancer Research Center, Heidelberg.
Schütz
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Kaestner
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KH
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D018244:Chromosomes, Artificial, Yeast; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004707:Endoderm / Q000502:physiology*; D004742:Enhancer Elements, Genetic; D005753:Gastric Mucosa / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D018390:Gene Targeting; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D051543:Hepatocyte Nuclear Factor 3-gamma; D007421:Intestine, Small / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000235:genetics*; D010179:Pancreas / Q000378:metabolism; D051381:Rats; D017384:Sequence Deletion; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C496506:Foxa3 protein, mouse; C496507:Foxa3 protein, rat; D051538:Hepatocyte Nuclear Factor 1-alpha; C496488:Hnf1a protein, mouse; C496489:Hnf1a protein, rat; C496496:Hnf1b protein, mouse; D009687:Nuclear Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051543:Hepatocyte Nuclear Factor 3-gamma; D051539:Hepatocyte Nuclear Factor 1-beta; D003850:Deoxyribonuclease I