Distinct Functions Are Implicated for the GATA-4, -5, and -6 Transcription Factors in the Regulation of Intestine Epithelial Cell Differentiation

The intestinal epithelium provides an excellent model system for investigating molecular mechanisms regulating cell lineage establishment, stem cell proliferation, morphogenesis, and the specialization of cell function during terminal differentiation (see references 9 and 16 for reviews). In all vertebrates, the embryonic intestinal lumen is lined by an endoderm-derived epithelial sheet, a monolayer consisting of four principal cell types that are renewed from a proliferating stem cell population. Lineage tracing experiments (7, 39) demonstrated that the four cell types are derived from a small population of multipotential stem cells present near the villus base (the crypt). The differentiating cells migrate from the crypt toward the villus tip, where they eventually die and are extruded into the lumen. Within the epithelium, absorptive enterocytes are the predominant cell type, and they form a highly organized apical brush border. In addition, goblet cells secrete mucus through exported granules, and enteroendocrine cells secrete various hormones and growth factors. The fourth cell type, the lysozyme-producing Paneth cell, differentiates during migration toward the base of the crypt. Although cell position along the polar crypt-tip axis is clearly an important determinant, the mechanisms that regulate the decision to differentiate (and the choice of pathway) are unknown. Regulation of gut-specific differentiation programs is likely to involve lineage-restricted transcription factors that control expression of terminal-differentiation genes. Experiments using transgenic mice and cell culture transfection have analyzed the promoters of gut-specific genes in order to identify regulatory elements that mediate lineage, temporal, and spatial control (see reference 52 for a review). In a few cases, potential transcription factors have been identified that can interact with defined regulatory elements. For example, the region including nucleotides −103 to +28 of the murine intestinal fatty acid-binding protein (IFABP) gene directs proper lineage-specific expression; a repeated and conserved element in this region binds two members of the steroid hormone receptor superfamily, HNF-4 and ARP-1 (38). Other transcription factors implicated in the regulation of differentiated epithelia include HNF-1 (5), HNF-3 (8), COUP-TF (31), and Cdx-2 (48). Genetic studies with Caenorhabditis elegans and Drosophila identified several additional genes that are likely to have conserved functions in vertebrate gut development (reviewed in reference 44). GATA factors comprise a small family of transcriptional regulatory proteins defined by a highly conserved DNA-binding domain that interacts specifically with DNA cis elements containing a consensus WGATAR or related sequence. Six distinct vertebrate GATA factors have been characterized, grouped into two subfamilies based on structural and expression comparisons. The GATA-1, -2, and -3 genes each function in the hematopoietic system (35). Genetic studies defined a unique role for each in regulating the differentiation state of specific blood lineages (43). The GATA-4, -5, and -6 genes are expressed with overlapping patterns in the developing cardiovascular system and in endoderm-derived tissues including the liver, lungs, pancreas, and gut (12). Several candidate target genes for GATA-4, -5, and -6 have been identified in cardiomyocytes, and data from several experiments indicate a functional role in regulating cardiac differentiation (17, 19, 24). Disruption of GATA-4 function during embryogenesis demonstrated an additional role regulating early morphogenesis critical for normal heart development, although the relevant downstream target genes are not known (26, 32). In addition, GATA-4 is required for the development of visceral endoderm in embryonic stem cell culture (46). In contrast, the function of the GATA-4, -5, and -6 genes in the differentiating gut is not known and is largely untested. A possible role for GATA factors in regulating gut-specific gene expression was proposed based on the expression of GATA-4 and GATA-5 in the embryonic gut and the adult intestinal epithelium (2, 25, 27). Likewise, GATA-4 and GATA-6 are expressed in gastric epithelium (51), and a few likely target genes for GATA factors in the differentiated stomach have been proposed. A sequence capable of binding GATA factors is present in the promoters of the human and rat α- and β-subunit genes encoding the H+/K+-ATPase (30, 33); this proton pump is expressed in stomach parietal cells that also express GATA-4 and GATA-6 (GATA-5 was not analyzed). Similarly, a GATA-binding site is present in the promoter of the rat histidine decarboxylase gene that is expressed specifically in gastric endocrine cells containing abundant GATA-6 transcripts (10). However, these and other putative binding sites have not yet been shown to be functional regulatory elements. Furthermore, candidate targets for GATA factors in intestinal epithelium have not been identified. To address the function and conservation of GATA factors in the gut, we studied the activity of GATA-4, -5, and -6 on a putative downstream target promoter, and the expression patterns for the GATA-4, -5, and -6 genes in chick intestine, and in a human model cell culture system. The data support a hypothesis that these genes have distinct functions in progenitor and differentiated cell compartments of the intestinal epithelium. We identify the gene encoding Xenopus IFABP as a downstream target for GATA factors in differentiating intestinal enterocytes. Therefore, GATA factors are likely to have distinct functions in the regulation of the IFABP and other epithelium-specific genes.