Suppressor and Activator Functions Mediated by a Repeated Heptad Sequence in the Liver Fatty Acid-binding Protein Gene (Fabpl): EFFECTS ON RENAL, SMALL INTESTINAL, AND COLONIC EPITHELIAL CELL GENE EXPRESSION IN TRANSGENIC MICE

Abstract

A 35-nucleotide sequence in the liver fatty acid-binding protein gene (Fabpl) has been identified that interacts with nuclear proteins present in adult mouse liver, kidney, stomach, small intestine, and colon. The binding site consists of a direct heptad repeat (TTCTGNNTT) separated by five nucleotides. Both heptads are required for formation of stable complexes with nuclear proteins in gel mobility shift assays. The in vivo functions mediated by the repeats were determined by comparing the expression of four Fabpl/human growth hormone fusion genes in multiple pedigrees of adult transgenic mice. The transgenes contained (i) nucleotides -596 to +21 of Fabpl linked to the human growth hormone reporter, (ii) 4 additional copies of the 35-base pair element placed at nucleotide -596 of Fabpl, (iii) 4 additional copies of the sequence placed just upstream of its endogenous site at nucleotide -132, and (iv) a sequence identical to (iii) but with all heptad repeats mutated within each of the 4 additional copies of the 35-base pair element. Transgene expression was defined by RNA blot hybridizations and by light and electron microscopic immunohistochemistry. The heptad repeat functions to suppress expression in tubular epithelial cells of the proximal nephron, in hepatocytes, in the mucus-producing pit cells of the gastric epithelium, and in absorptive enterocytes located in the proximal small intestine. There is a gradient of escape from enterocytic suppression as one moves from the proximal to distal small intestine. This escape progresses to involve successively less differentiated cells located closer and closer to the stem cell zone in crypts of Lieberkühn. The heptad repeat activates gene expression in the colonic epithelium so that all proliferating and nonproliferating cells in colonic crypts distributed from the cecum to the rectum support transgene expression. The heptad has no obvious sequence similarities to known transcription factor binding sites, suggesting that mediators of its in vivo activities are likely to be novel. One candidate factor is a 90-kDa protein identified in Southwestern blots. The 90-kDa protein also binds to an element in the matrix metalloproteinase-2 gene that functions as an enhancer in renal cells, shares sequence homology with the heptad, and generates similar-sized complexes in gel mobility shift assays as the Fabpl repeat. The heptad repeat represents a target for identifying transcription factors that regulate gene expression between gut and renal epithelia and that also regulate the differentiation program of the intestine's principal epithelial lineage as a function of its location along the duodenal-colonic axis. Finally, the Fabpl regulatory elements described in this report should be useful for delivering a variety of gene products throughout the colonic epithelium of transgenic mice.