Abstract
Studies using transgenic mice indicate that expression of the human apolipoprotein (apo) A-I gene in the liver and small intestine is controlled by spatially distinct cis-acting DNA elements; hepatic expression is controlled by a domain defined by nucleotides -256 to -1, while small intestinal expression requires elements positioned 9 kilobases 3' to the gene, between nucleotides -1300 and -200 of the convergently transcribed apoC-III gene. In this report we have mapped this enhancer to a 260-base pair (bp) region of the apoC-III promoter spanning nucleotides -780 to -520. The elements contained within this 260-bp apoC-III domain are sufficient to direct a pattern of expression in villus-associated enterocytes distributed along the duodenal-to-ileal axis that resembles that of mouse and human apoA-I. However, the elements produce inappropriate activation of apoA-I expression in proliferating and nonproliferating crypt epithelial cells, and in subpopulations of cholecystokinin- and serotonin-producing enteroendocrine cells. Cis-acting suppressors of these inappropriate patterns of expression are located outside of nucleotides -1300 to -200 of the human apoC-III gene. DNase I protection and gel mobility gel shift assays identified two 21-bp sequences, nucleotides -745 to -725 and -700 to -680 of human apoC-III, which bind nuclear proteins present in a human enterocyte-like cell line (Caco-2). These sequences are conserved in the orthologous mouse apoC-III gene. The 260-bp apoC-III element is the first intestinal enhancer that has been identified in an in vivo system and should provide insights about how cell lineage-specific, differentiation-dependent, and cephalocaudal patterns of gene expression are established and maintained in the perpetually renewing gut epithelium. In addition, novel intestinal transcription factors may bind to the enhancer and regulate its activity.
Highlights
Enterocytes, goblet cells, and enteroendocrine cells all differentiate as they migrate in vertical coherent bands from each crypt up an adjacent villus to an apical extrusion zone located near the villus tip [19, 20]
Different fragments from the 1.1-kb apoC-III fragment, previously shown to be necessary for intestinal expression [14], were placed upstream of nucleotides -256 to +22 of the human apoA-1 gene linked to a chloramphenicol acetyltransferase (CAT) reporter, and the resulting recombinant plasmids were used for transient transfections of an established human enterocyte-like cell line (Caco-2), a nonintestinal epithelial cell line (HeLa), and a hepatocyte-like cell line (HepG2)
The positive effects produced by nucleotides -780 to -690 are cell-specific; no increase in CAT activity was detectable in HepG2 or HeLa cells over that produced by apoA_I- 256 to +22CAT alone
Summary
Laboratory of Biochemical Genetics and Metabolism, RockefellerUniversity, 1230 York Ave., New York, NY 10021. The determinants of intestinal expression appear to reside in a 1.1-kb fragment that extends from nucleotides -200 to -1300 of the adjacent, convergently transcribed human apoC-III gene [14]. The intestine's four principal epithelial cell lineages, enterocytic, goblet, enteroendocrine, and Paneth cell, are all derived from multipotent stem cells located near the base of each crypt [15,16,17]. We have used transgenic mice and cultured cell lines to map further the intestinal enhancer contained in the human apoA-I/C-III/A-IV gene cluster, to define its effects on gene expression along the crypt-villus and duodenal-colonic axes, and to characterize nuclear proteins that may modulate its activity
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