Abstract
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.
Highlights
The human apolipoprotein1 E gene spans 3.6 kb [1,2,3] and is located at the 5Ј end of a 45-kb cluster of apolipoprotein genes on chromosome 19, all of which have the same transcriptional orientation [4]
Bound nuclear factors protect the underlying nucleotides from being digested; these sites are identified by subsequent ligation-mediated polymerase chain reaction (LMPCR), and the exact location of protected sites is determined by analyzing the sequence of guanine nucleotides in amplified control DNA (Fig. 2, lane G)
A cluster of five footprints was detected in the Alu family sequence adjacent to hepatic control region (HCR)-1 (Fig. 2, E and F), these footprints tended to be less distinct than those located within the liver-specific HCR-1 domain
Summary
The human apolipoprotein (apo) E gene spans 3.6 kb [1,2,3] and is located at the 5Ј end of a 45-kb cluster of apolipoprotein genes on chromosome 19, all of which have the same transcriptional orientation [4]. Simonet et al [16, 17] demonstrated that expression of the apoE and apoC-I genes in the liver requires the presence of a distal downstream tissue-specific enhancer. The presence of a previously characterized enhancer element, which lacks tissue specificity, in the promoter of the apoE gene [20, 21] was required for transcriptional activation These results suggested that interaction of a unique hepatocyte-specific combination of distal elements in the HCR with a nonspecific activator sequence in the promoter directed the expression of the apoE/C-I/C-II locus in the liver
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