SUPPRESSOR AND ACTIVATOR FUNCTIONS MEDIATED BY A REPEATED HEPTAD SEQUENCE IN THE LIVER FATTY-ACID-BINDING PROTEIN GENE (FABPL) - EFFECTS ONRENAL, SMALL-INTESTINAL, AND COLONIC EPITHELIAL-CELL GENE-EXPRESSION IN TRANSGENIC MICE

A 35-nucleotide sequence in the liver fatty acid-binding protein gene (Fabpl) has been identified that interacts with nuclear proteins prese nt in adult mouse liver, kidney, stomach, small intestine, and colon, The binding site consists of a direct heptad repeat (TTCTGNNTT) separa ted by five nucleotides, Both heptads are required for formation of st able complexes with nuclear proteins in gel mobility shift assays, The in vivo functions mediated by the repeats were determined by comparin g the expression of four Fabpl/human growth hormone fusion genes in mu ltiple 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, Tr ansgene expression was defined by RNA blot hybridizations and by light and electron microscopic immunohistochemistry. The heptad repeat func tions to suppress expression in tubular epithelial cells of the proxim al nephron, in hepatocytes, in the mucus-producing pit cells of the ga stric epithelium, and in absorptive enterocytes located in the proxima l small intestine, There is a gradient of escape from enterocytic supp ression as one moves from the proximal to distal small intestine, This escape progresses to involve successively less differentiated cells l ocated closer and closer to the stem cell zone in crypts of Lieberkuhn . The heptad repeat activates gene expression in the colonic epitheliu m so that all proliferating and nonproliferating cells in colonic cryp ts distributed from the cecum to the rectum support transgene expressi on, The heptad has no obvious sequence similarities to known transcrip tion factor binding sites, suggesting that mediators of its in vivo ac tivities are likely to be novel, One candidate factor is a 90-kDa prot ein identified in Southwestern blots, The 90-kDa protein also binds to an element in the matrix metalloproteinase-2 gene that functions as a n enhancer in renal cells, shares sequence homology with the heptad, a nd generates similar-sized complexes in gel mobility shift assays as t he Fabpl repeat. The heptad repeat represents a target for identifying transcription factors that regulate gene expression between gut and r enal epithelia and that also regulate the differentiation program of t he intestine's principal epithelial lineage as a function of its locat ion along the duodenal colonic axis, Finally, the Fabpl regulatory ele ments described in this report should be useful for delivering a varie ty of gene products throughout the colonic epithelium of transgenic mi ce.