HNF-4 (hepatocyte nuclear factor 4) is a protein enriched in liver extracts that binds to sites required for the transcription of the genes for transthyretin (TTR), the carrier protein in the serum for vitamin A and thyroid hormone, and for apolipoprotein CIII (apoCIII), a major constituent of chylomicrons and very low-density lipoproteins (VLDL). Synthetic oligonucleotides derived from amino acid sequence of affinity-purified HNF-4 protein (54 kD) were used in the polymerase chain reaction (PCR) to isolate a cDNA clone encoding the protein. Cell type specificity is based on differential gene expression, which is in turn determined, at least in part, by the particular set of transcription factors present and active in a given cell at a given time. Many such factors have been identified and characterized, particularly in the liver where there is a wide range of transcriptionally controlled genes (McKnight and Palmiter 1979;Derman et al. 1981). Some transcription factors, such as AP-I and Sp-1, seem to be present in all cells at all times, but other factors have a more limited distribution. Whether there is a discernible logic that explains the distribution of the many factors has yet to be determined. Two aspects of this problem are particularly important. First, is the distribution of factors in different tissues controlled at the level of transcription? If so, then a cascade of transcriptional regulation that ultimately results in cell specificity is indicated. Second, is any particular factor central to the accomplishment of a particular metabolic or physiologic goal? Such a goal might be suggested by a factor acting on an interrelated set of genes.

Although transcriptional hierarchies have been extensively studied in invertebrates, their involvement in mammalian cell-type specification is poorly understood. Here we report a hepatocyte transcriptional cascade suggested by the expression patterns of hepatic transcription factors in dedifferentiated hepatomas and hepatocyte: fibroblast hybrids in which the liver phenotype was extinguished. These results indicated that the homeoprotein hepatocyte nuclear factor-1 alpha (HNF-1 alpha), and HNF-4, a member of the steroid hormone receptor superfamily, were regulated coordinately or in a hierarchy by a higher-order locus, independently of other hepatic transactivators. HNF-4 was implicated as an essential positive regulator of HNF-1 alpha, as deletion of an HNF-4 binding site in the HNF-1 alpha promoter abolished promoter activity, and HNF-4 potently transactivated the HNF-1 alpha promoter in cotransfection assays. Moreover, genetic complementation of dedifferentiated hepatomas with HNF-4 complementary DNA rescued expression of endogenous HNF-1 alpha messenger RNA and DNA-binding activity. Our studies therefore define an HNF-4----HNF-1 alpha (4----1 alpha) transcriptional hierarchy operative in differentiated hepatocytes but selectively inhibited by an extinguishing locus and somatic mutations which antagonize the liver phenotype.

The core promoter of eukaryotic genes is the minimal DNA region that recruits the basal transcription machinery to direct efficient and accurate transcription initiation. The fraction of human and yeast genes that contain specific core promoter elements such as the TATA box and the initiator (INR) remains unclear and core promoter motifs specific for TATA-less genes remain to be identified. Here, we present genome-scale computational analyses indicating that ∼76% of human core promoters lack TATA-like elements, have a high GC content, and are enriched in Sp1 binding sites. We further identify two motifs -M3 (SCGGAAGY) and M22 (TGCGCANK) -that occur preferentially in human TATA-less core promoters. About 24% of human genes have a TATA-like element and their promoters are generally AT-rich; however, only ∼10% of these TATA-containing promoters have the canonical TATA box (TATAWAWR). In contrast, ∼46% of human core promoters contain the consensus INR (YYANWYY) and ∼30% are INR-containing TATA-less genes. Significantly, ∼46% of human promoters lack both TATA-like and consensus INR elements. Surprisingly, mammalian-type INR sequences are present -and tend to cluster -in the transcription start site (TSS) region of ∼40% of yeast core promoters and the frequency of specific core promoter types appears to be conserved in yeast and human genomes. Gene Ontology analyses reveal that TATA-less genes in humans, as in yeast, are frequently involved in basic "housekeeping" processes, while TATAcontaining genes are more often highly regulated, such as by biotic or stress stimuli. These results reveal unexpected similarities in the occurrence of specific core promoter types and in their associated biological processes in yeast and humans and point to novel vertebrate-specific DNA motifs that might play a selective role in TATA-independent transcription.

Epithelial formation is a central facet of organogenesis that relies on intercellular junction assembly to create functionally distinct apical and basal cell surfaces. How this process is regulated during embryonic development remains obscure. Previous studies using conditional knockout mice have shown that loss of hepatocyte nuclear factor 4␣ (HNF4␣) blocks the epithelial transformation of the fetal liver, suggesting that HNF4␣ is a central regulator of epithelial morphogenesis. Although HNF4␣-null hepatocytes do not express E-cadherin (also called CDH1), we show here that E-cadherin is dispensable for liver development, implying that HNF4␣ regulates additional aspects of epithelial formation. Microarray and molecular analyses reveal that HNF4␣ regulates the developmental expression of a myriad of proteins required for cell junction assembly and adhesion. Our findings define a fundamental mechanism through which generation of tissue epithelia during development is coordinated with the onset of organ function.cell junctions ͉ organogenesis ͉ transcription