An intronic locus control region plays an essential role in the establishment of an autonomous hepatic chromatin domain for the human vitamin D-binding protein gene.
The human vitamin D-binding protein (hDBP) gene exists in a cluster of four liver-expressed genes. A minimal hDBP transgene, containing a defined set of liver-specific DNase I hypersensitive sites (HSs), is robustly expressed in mouse liver in a copy-number-dependent manner. Here we evaluate these HSs for function. Deletion of HSI, located 5' to the promoter (kb -2.1) had no significant effect on hDBP expression. In contrast, deletion of HSIV and HSV from intron 1 repressed hDBP expression and eliminated copy number dependency without a loss of liver specificity. Chromatin immunoprecipitation analysis revealed peaks of histone H3 and H4 acetylation coincident with HSIV in the intact hDBP locus. This region contains a conserved array of binding sites for the liver-enriched transcription factor C/EBP. In vitro studies revealed selective binding of C/EBPalpha to HSIV. In vivo occupancy of C/EBPalpha at HSIV was demonstrated in hepatic chromatin, and depletion of C/EBPalpha in a hepatic cell line decreased hDBP expression. A nonredundant role for C/EBPalpha was confirmed in vivo by demonstrating a reduction of hDBP expression in C/EBPalpha-null mice. Parallel studies revealed in vivo occupancy of the liver-enriched factor HNF1alpha at HSIII (at kb 0.13) within the hDBP promoter. These data demonstrate a critical role for elements within intron 1 in the establishment of an autonomous and productive hDBP chromatin locus and suggest that this function is dependent upon C/EBPalpha. Cooperative interactions between these intronic complexes and liver-restricted complexes within the target promoter are likely to underlie the consistency and liver specificity of the hDBP activation.
Hiroki
Tomoko
T
Department of Medicine, University of Pennsylvania, 415 Curie Boulevard, Philadelphia, PA 19104, USA.
Liebhaber
Stephen A
SA
Cooke
Nancy E
NE
D000107:Acetylation; D000818:Animals; D001483:Base Sequence; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D003850:Deoxyribonuclease I / Q000378:metabolism; D005786:Gene Expression Regulation; D051538:Hepatocyte Nuclear Factor 1-alpha / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D007438:Introns / Q000235:genetics*; D008099:Liver / Q000378:metabolism*; D019901:Locus Control Region / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D017384:Sequence Deletion; D014809:Vitamin D-Binding Protein / Q000235:genetics*