The liver-specific enhancer of the serum albumin gene contains an essential segment, designated eH, which binds the hepatocyte nuclear factor 3a (HNF3a) and ubiquitous nuclear factor 1/CCAAT transcription factor (NF1/CTF) proteins in tight apposition. We previously showed that activation of transcription by the eH site was correlated with an increase in intracellular HNF3ae levels during the in vitro differentiation of the hepatic cell line H2.35. We now show that transfection of an HNF3oa cDNA expression vector into dedifferentiated H2.35 cells is sufficient to induce transcription from the eH site. Mutational analysis of the enhancer demonstrates that NF1/CTF cooperates with HNF3a to induce enhancer activity. However, when the eH site is removed from the context of the enhancer, NF1/CTF can inhibit transcriptional activation by HNF3a. We conclude that the ternary complex of HNF3a, NF1/CTF, and the eH site forms a novel, composite regulatory element that is sensitive to the local DNA sequence environment and suggest that the transcriptional stimulatory activity of NF1/CTF depends on its higher-order interactions with other proteins during hepatocyte differentiation. Cellular differentiation is governed by the binding of transcription factors to DNA in specific combinations, often leading to the formation of large arrays of protein-DNA complexes. Interactions between different transcription factors bound to adjacent sites can lead to striking changes in their respective activities. For example, the yeast protein MCM1 (PRTF) activates a-cell-specific promoters when bound adjacent to the al transcription factor, but MCM1 represses a-cell-specific promoters when bound next to the a2 protein (1,29). In this report, we study the interactions between two liver transcription factors when they are tightly juxtaposed at a site on DNA and explore how the genetic context of the proteins' DNA binding sites governs their interactions during hepatic differentiation.Serum albumin gene transcription is an excellent marker for liver differentiation in mammals because it is activated early during liver development (8,49,51,53) and increases to a rate 1,000-fold greater in the liver than in other tissues (33,44). Tissue specificity is controlled in part by an enhancer element that lies 10 kb upstream of the transcription start site and functions selectively in the liver of transgenic mice (43). The albumin enhancer contains three sites, designated eE, eG, and eH, that are essential for enhancer activity in various hepatocyte-derived cell lines, such as H2.35 cells (24,25,34). The eE site binds liver-enriched C/EBP-related proteins (6, 11, 56), and the eG site binds a family of transcription factors, hepatocyte nuclear factor 3a (HNF3a), HNF3,, and HNF3y, expressed in liver and lung (31). The HNF3 family contains a conserved DNA binding domain found in the developmentally important Drosophila fork head protein (54). The eH site was defined by a large DNase I footprint with nuclear extracts from mouse liver and H2.35 cells (34,58); ...

The extracellular matrix (ECM) promotes the differentiation of many cell types, and ECM remodeling in the liver has been implicated in embryonic development, tissue injury, and oncogenesis. Integrins are heterodimeric ECM receptors that play critical roles in transducing the composition of the ECM in the cell environment. We previously showed that mouse H2.35 cells, a conditionally transformed, liver-derived cell line, assume a more differentiated hepatocyte morphology and enhanced liver-specific gene expression when the cells are cultured on gelatinous ECM substrata. Here we show that H2.35 cells express relatively high levels of ␣ 3 ␤ 1 -integrins, similar to that previously shown for immature hepatocytes, transformed hepatocytes, and biliary cells. However, the cell morphological responses that depend on ␣ 3 ␤ 1 -integrin have not been defined. We found that transfecting H2.35 cells with antisense RNA construct directed to ␣ 3 -subunit messenger RNA perturbs the initial cell attachment to laminin and collagen, and strongly inhibits cell morphological, proliferative, and gene expression responses to a collagen gel substratum. In situ hybridization to mouse embryo tissues demonstrates the presence of ␣ 3 -subunit messenger RNAs in newly formed hepatocytes. We suggest that ␣ 3 ␤ 1 -integrins are important for immature and transformed hepatocytes to respond morphologically to the extracellular matrix. (HEPATOLOGY 1998;28:1095-1104.)Tissue-specific gene expression, morphogenesis, and cell migration are promoted by interactions between cells and the surrounding extracellular matrix (ECM). For example, early hepatocyte differentiation begins as foregut endodermal cells enter the new collagenous environment of the surrounding mesenchyme, 1 and the role of the ECM in adult hepatocyte differentiation is well established. [2][3][4][5][6][7][8] Other examples of ECMpromoted differentiation include the differentiation of keratinocytes, 9 gastrulation in Pleurodeles embryos, 10 and the epithelial conversion of kidney mesenchyme 11 (for review, see Adams and Watt 12 ). The ECM is present in all animals from the earliest stages of development and is composed of a mixture of proteins and proteoglycans such as collagens, laminin, and fibronectin. An important set of membrane receptors for ECM molecules is the integrin family of proteins. [13][14][15] Integrins are heterodimers of ␣ and ␤ subunits, with each subunit containing an N-terminal extracellular domain, a transmembrane domain, and a C-terminal intracellular domain. Integrins activate common as well as subgroupspecific intracellular signaling pathways in response to the ECM. 16,17 Membrane proximal events triggered by integrin ligation include the activation of the focal adhesion kinase 18 by the ␤ subunit and the recruitment of adapter protein Shc by certain ␣ subunits within heterodimers. 19 More ␣ subunits are known than ␤ subunits, and the particular combination of ␣ and ␤ partners determines the ligand specificity for ECM molecules. An ECM protein can be recognized...

The liver-specific enhancer of the serum albumin gene contains an essential segment, designated eH, which binds the hepatocyte nuclear factor 3 alpha (HNF3 alpha) and ubiquitous nuclear factor 1/CCAAT transcription factor (NF1/CTF) proteins in tight apposition. We previously showed that activation of transcription by the eH site was correlated with an increase in intracellular HNF3 alpha levels during the in vitro differentiation of the hepatic cell line H2.35. We now show that transfection of an HNF3 alpha cDNA expression vector into dedifferentiated H2.35 cells is sufficient to induce transcription from the eH site. Mutational analysis of the enhancer demonstrates that NF1/CTF cooperates with HNF3 alpha to induce enhancer activity. However, when the eH site is removed from the context of the enhancer, NF1/CTF can inhibit transcriptional activation by HNF3 alpha. We conclude that the ternary complex of HNF3 alpha, NF1/CTF, and the eH site forms a novel, composite regulatory element that is sensitive to the local DNA sequence environment and suggest that the transcriptional stimulatory activity of NF1/CTF depends on its higher-order interactions with other proteins during hepatocyte differentiation.