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

Abstract

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/EBPα to HSIV. In vivo occupancy of C/EBPα at HSIV was demonstrated in hepatic chromatin, and depletion of C/EBPα in a hepatic cell line decreased hDBP expression. A nonredundant role for C/EBPα was confirmed in vivo by demonstrating a reduction of hDBP expression in C/EBPα-null mice. Parallel studies revealed in vivo occupancy of the liver-enriched factor HNF1α 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/EBPα. 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.

We thank Jean Richa and the Transgenic and Chimeric Mouse Core of the University of Pennsylvania and the Center for Molecular Studies in Digestive and Liver Diseases (NIH P30 DK50306) for the generation of transgenic lines and Chris Laing in our laboratory who developed the ELISA for mouse serum DBP. We are also grateful to Philip Phuc Le, Klaus Kaestner, and Linda Greenbaum for liver RNA samples from the C/EBPα- and C/EBPβ-null mice.

This study was supported by grant NIH R01 GM32035 (N.E.C. and S.A.L.).