In vivo footprinting analysis of the hepatic control region of the human apolipoprotein E/C-I/C-IV/C-II gene locus.
Expression of both the apolipoprotein (apo)E and apoC-I genes in the liver is specified by a 319-nucleotide hepatic control region (HCR-1) that is located 15 kilobase pairs downstream of the apoE gene and 5 kilobase pairs downstream of the apoC-I gene. In vivo footprint analysis of HCR-1 in intact nuclei revealed several liver-specific protein-binding sites that were not detectable by in vitro methods. In addition to three previously identified in vitro footprints, four in vivo footprints were identified in a region of HCR-1 that is required for directing gene expression to hepatocytes. Prominent liver-specific DNase I-hypersensitive sites were associated with these footprints. Liver-specific nuclear protein binding to these sites was confirmed by oligonucleotide gel-retention assays. The in vivo analysis also identified a cluster of nuclear protein-binding sites in the Alu family repeat segment adjacent to the domain required for liver expression. Micrococcal nuclease digestion indicated the presence of a nucleosome in the central domain of HCR-1 in liver chromatin that was in phase with the nucleosome location in tissues that did not express the transgene. These results suggest that HCR-1 functions in a highly structured chromatin environment requiring a complex interaction of liver-enriched transcription factors.
Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, California 94141-9100, USA.
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Taylor
D053302:Apolipoprotein C-I; D053304:Apolipoprotein C-II; D001056:Apolipoproteins C / Q000235:genetics*; D001057:Apolipoproteins E / Q000235:genetics*; D001483:Base Sequence; D002874:Chromosome Mapping; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005809:Genes, Regulator; D006801:Humans; D008099:Liver / Q000737:chemistry*; D008969:Molecular Sequence Data
C097349:APOC4 protein, human; D053302:Apolipoprotein C-I; D053304:Apolipoprotein C-II; D001056:Apolipoproteins C; D001057:Apolipoproteins E; D003850:Deoxyribonuclease I