I. Transcription Factors and Gene Regulation

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Transcription factors are trans-acting DNA-binding proteins that bind to a specific cis-acting DNA sequence within the regulatory element of a gene. Usually, control regions can be found upstream of the start site of transcription, although in some cases binding occurs within the coding region. Transcription factors bound to their cognate cis-acting DNA sequence interact with the transcriptional machinery and enable selective gene expression and regulation. Frequently, this process is governed by the binding of many different proteins to cognate DNA-binding sites, which enables combinatorial control of gene expression. An additional level of complexity is provided by protein-protein interactions between transcription factors and coactivators or corepressors. Together with the transcriptional machinery, these proteins form a multiprotein complex that enables regulated mRNA synthesis (for review see Pabo and Sauer, 1992; Giordano and Avantaggiati, 1999; Klug, 1999; Wolberger, 1999; Goodman and Smolik, 2000).

Efficient gene transcription requires a permissive chromatin environment for successful interaction between the trans-acting transcription factors of the multiprotein complex and the respective cis-acting target DNA template of the nucleosome core particle. Therefore, the modulation of chromatin structure with its effects on gene transcription represents a key mechanism for transcriptional repression, derepression, and transcriptional activation. In the following sections we briefly summarize some of the fundamental mechanisms in the formation of the multiprotein complex to provide newfound knowledge on gene transcription and liver-enriched transcription factors, and we deliberately exclude aspects of DNA repair and DNA miscoding, which have been reviewed elsewhere (Krokan et al., 2000; Thompson and Schild, 2001).

Chromatin is composed of a histone octamer, the DNA of the nucleosome core particle, and the linker DNA. The nucleosome core particle is formed by about 160 bp¹ of DNA wrapped around an octamer composed of two copies of each of the four histones H2A, H2B, H3, and H4. Within the nucleosome core particle an (H3)₂(H4)₂ tetramer, as well as an H2A-H2B dimer, could be distinguished. These histone oligomers could be recombined with DNA in vitro to generate the characteristic X-ray diffraction pattern of chromatin (Kornberg and Thomas, 1974; Kornberg and Lorch, 1999). Figure 1 shows the basic entities that form chromatin, and Fig. 2 shows a schematic transection of the nucleosome core particle based on X-ray findings by Luger et al. (1997). X-Ray and electron crystallography revealed the coiling of DNA in left-handed superhelical turns around the histones (Finch et al., 1977). Crystallographic analysis of the nucleosome showed that the histones form a left-handed protein superhelix matching that of the DNA in the nucleosome core particle (Klug et al., 1980; Arents and Moudrianakis, 1995; Kornberg and Lorch, 1999).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Schematic drawing of the basic entities that form chromatin. The histone octamer is represented as a disk, the linker DNA as a red ribbon, and the DNA of the nucleosome core particle as a black ribbon.

View larger version (61K): [in this window] [in a new window]   Fig. 2.   Depicted is a schematic transection of the nucleosome core particle based on X-ray findings of its crystal structure by Luger et al. (1997) and on findings reviewed by Bird and Wolffe (1999) and by Kornberg and Lorch (1999). Half the core particle is shown with four histone molecules and 73 DNA base pairs. Core histone fold domains for H2A, H2B, H3, and H4 are indicated by green, red, brown, and blue coloring, respectively. DNA is shown wrapped around the histone fold domains with the numbers 0 to 7 indicating turns away from the dyad axis (Dyad). CpGs accessible in the major groove to MBD proteins (MBDPs) that act as transcriptional repressors through DNA methylation are indicated (purple). The N-terminal tail of histone H4 that can be targeted by the ATP-utilizing chromatin remodeling complex NuRD (also called Mi-2 complex) is indicated by K5, K8, K12, and K16. The N-terminal helix of histone H4, which is targeted by the histone-binding protein RbAp48, a subunit of the NuRD complex, is colored yellow. The NuRD complex is able to influence transcriptional activity through ATP-dependent chromatin remodeling, chromatin deacetylation, and DNA methylation (see text for details and references). A key site of interaction between histone H4 and DNA where mutations in the protein relieve the requirement for SWI/SNF activity is shown in orange. "Lollipops" on the tails of histones H3 and H4 indicate acetylation sites.

The human genome consists of 2.91 billion base pairs, which would be theoretically about 1.8 m long if stretched out as one long chain (Venter et al., 2001). DNA is organized into chromatin to achieve the required high level of compaction to pack this DNA into a nucleus with a diameter less than 6 nm (Lewin, 1994). The orderly packaging of DNA in the nucleus plays an important role in the functional aspects of gene regulation. Only a small percentage of chromatin is made available to transcription factors and the transcriptional machinery, whereas the remainder of the genome is in a state that is essentially inaccessible to the RNA polymerases. ATP-dependent chromatin remodeling as well as chromatin modifications by acetylation of lysines, DNA methylation, phosphorylation of serines and threonines, and ubiquitination of lysines play key roles in altering chromatin higher order structure and function (Bradbury, 1992; Shilatifard, 1998; Giordano and Avantaggiati, 1999; Spencer and Davie, 1999; Stein et al., 1999). Acetylations and phosphorylations markedly affect the charge densities of well defined, very basic N- and C-terminal domains of histones (for details see also Fig. 2), whereas ubiquitination adds a bulky globular protein, ubiquitin, to lysines in the C-terminal tails of H2A and H2B (for review see Bradbury, 1992; Bird and Wolffe, 1999; Kornberg and Lorch, 1999).

New findings on ATP-dependent chromatin remodeling as well as chromatin modifications by covalent acetylation, phosphorylation, ubiquitination, and DNA methylation demonstrate the importance of these alterations for the regulation of many genes, although their precise role in liver gene expression remains largely unknown. The following sections provide an overview of recent data and hopefully stimulate further investigations on the role of chromatin remodeling and chromatin modifications in liver function, liver regeneration, and liver development. Additionally, the concepts of epigenetics and position-effect variegation (PEV), and their possible impact on gene transcription and expression, are discussed, because these concepts are of fundamental importance in gene transcription but are widely neglected in molecular investigations of liver-specific gene expression.

1. ATP-Utilizing Chromatin Remodeling Complexes: Switch/Sucrose Nonfermenting and Relatives. The activation of a gene requires accessibility for transcription factors, activators, coactivators, and transcription machinery to the various regulatory regions. Several ATP-consuming chromatin remodeling complexes have been identified that enable gene activation by altering the stable structure of nucleosomes (for review see Devine et al., 1999; Muchardt and Yaniv, 1999; Wade and Wolffe, 1999; Tyler and Kadonga, 1999; Sudarsanam and Winston, 2000).

2. Chromatin Modification: Reversible Acetylation of Histone Lysines. Expressed genes are located in highly acetylated chromatin. The acetylation status of nucleosomes is regulated by a group of enzymes, histone acetyltransferases (HATs) and HDACs. Examples of acetylation sites of histones H3 and H4 are shown in Fig. 2. Both groups of enzymes contain numerous family members, most of which have been highly conserved during evolution. The noncatalytic components of these complexes can either target the catalytic unit to specific sites of the genome or regulate its enzymatic specificity. DNA methylation and histone acetylation have also been linked together, whereby methylation is used to direct gene repression through a histone deacetylase complex (Gray et al., 1999) (see also Fig. 3).

View larger version (42K): [in this window] [in a new window]   Fig. 3.   This schematic model on increasing levels of transcriptional repression induced by DNA methylation and MeCP2 binding followed by corepressor and deacetylase binding with subsequent deacetylation and chromatin compaction was adapted with modifications from Jones and Laird (1999). Nucleosome core particles are shown as gray disks with DNA wrapped around as black ribbon. Acetylation, methylation, MeCP2 binding, corepressor and deacetylase binding are represented by red, green, blue, gray, and dark blue spheres, respectively.

3. Chromatin Modification: Reversible Phosphorylation of Histone Serines and Threonines. Histone H1 and H3 phosphorylations correlate with the process of chromosome condensation. The subunits of histone H1 kinase have now been shown to be cyclins and the p34CDC2 kinase product of the cell cycle control gene CDC2. It is probable that all of the processes that control chromosome structure and function relationships are also involved in the control of the cell cycle (Bradbury, 1992; Spencer and Davie, 1999).

4. Chromatin Modification: Reversible Ubiquitination of Histone Lysines. Ubiquitin-dependent proteolytic pathways are largely responsible for selective protein turnover in the cytosol of eukaryotes. Although ubiquitinated histones are present in substantial levels in vertebrate cells, the roles they play in specific biological processes and the cellular factors that regulate this modification are not well characterized. Ubiquitinated H2B (uH2B) has been identified in the yeast S. cerevisiae, and mutation of the conserved ubiquitination site could confer defects in mitotic cell growth and meiosis. uH2B was not detected in rad6 mutants, which are defective for the ubiquitin-conjugating enzyme Ubc2, thus identifying Rad6 as the major cellular activity that ubiquitinates H2B in yeast (Robzyk et al., 2000).

5. Chromatin Modification: Reversible DNA Methylation. Cytosine residues in the sequence 5'CpG (cytosine-guanine) are often postsynthetically methylated in animal genomes. The methyl-CpG-binding proteins MeCP1 and MeCP2 interact specifically with methylated DNA and mediate transcriptional repression. MeCP2 is an abundant nuclear protein that is essential for mouse embryogenesis (Nan et al., 1998). MeCP2 binds tightly to chromosomes in a methylation-dependent manner. It contains a transcriptional-repression domain that can function at a distance in vitro and in vivo. A region of MeCP2 that localizes with the transcriptional-repression domain associates with a corepressor complex containing the transcriptional repressor mSin3A and histone deacetylases (see Fig. 3). Transcriptional repression in vivo is relieved by the deacetylase inhibitor trichostatin A, indicating that deacetylation of histones (and/or of other proteins) is an essential component of this repression mechanism. Two global mechanisms of gene regulation, DNA methylation and histone deacetylation, can be linked by MeCP2 (Nan et al., 1998).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Shown is a model adapted with modifications from Bird and Wolffe (1999) on different levels of transcriptional activity due to activated, repressed, and derepressed basal DNA transcription with quantitative estimates. It was proposed that the formation of chromatin between a DNA template and a histone octamer leads to a repressive effect on basal transcriptional activity when compared with transcription from a naked DNA template. Further repression results from additional methylation of DNA. It was assumed that DNA methylation may expand the range of transcriptional regulation significantly beyond that which could be achieved by chromatin modification alone.

The inheritance of information during cell replication on the basis of gene expression levels is known as epigenetics, as opposed to genetics, which refers to information inherited on the basis of gene sequence. Enzymatic methylation of the C-5 position of cytosine residues can effect epigenetic inheritance by altering the expression of genes and by transmission of DNA methylation patterns through cell division (Bird and Wolffe, 1999; Jones and Laird, 1999; Wolffe and Matzke, 1999). Epigenetic control of gene expression can be considered from the standpoint of normal development, which requires stable repression of genes not required in specific cell types (Wolffe and Matzke, 1999). Interactions between repeated DNA sequences can trigger the formation and the transmission of inactive genetic states and DNA modifications. Methylation induced by DNA repeats can template chromatin modifications and transcriptional repression by MeCP2 binding to methylated CpG with subsequent recruitment of histone deacetylase (Nan et al., 1998; Jones and Laird, 1999) (see also Fig. 3).

The chromosomes of most higher eukaryotes consist of distinct regions that are cytologically distinguishable owing to differences in condensation. In a typical chromosome, heterochromatin differs from euchromatin in sequence composition, function, and cytological appearance and is predominantly located in the pericentric region. The DNA of heterochromatin consists almost entirely of repetitive sequences and encodes relatively few genes. In Drosophila, genes juxtaposed to heterochromatin are frequently inactivated, a phenomenon known as PEV. Inactivation is believed to result from the spreading of the heterochromatin state along the chromosome (Dorer and Henikoff 1994, 1997). The extent of PEV spreading may vary from cell to cell, producing mosaic expression of nearby genes. In contrast with the growing understanding of transacting factors, little is known of cis-acting requirements for heterochromatin formation and PEV. Experiments with Drosophila using a mini-white reporter gene, a commonly used eye color marker in Drosophila P transposons, and PEV to explore the requirements for heterochromatin formation revealed that variegated expression of mini-white occurs when it is present in repeat arrays. Variegation was particularly strong for repeated transposons at a euchromatic site near heterochromatin, but also resulted from repeats at a site distant from heterochromatin (Dorer and Henikoff, 1994). Inactivation strengthened with increasing copy number, a phenomenon that can also be observed for the transgene in numerous transgenic animals and plants (Dorer and Henikoff, 1997; Garrick et al., 1998). Experiments using the lox/Cre system of site-specific recombination to generate transgenic mouse lines showed that the reduction in copy number results in a methylation at the transgene locus (Garrick et al., 1998).

The expression of any gene is accomplished primarily through the interaction of protein transcription factors with characteristic nucleotide sequences located in the control regions of the gene, which are most commonly located near to, or upstream from, the actual coding region. The binding of a set of such factors, or regulatory proteins, acts as a molecular switch for the activation of the RNA polymerase II (RNA pol II) and other components of the transcriptional machinery, which are common to all genes. The supply of a particular combination of such transcription factors ensures that a gene is switched on in the right cell or tissue and at the right time (Duncan et al., 1998; Klug, 1999).

Transcription initiation by RNA pol II requires interaction between cis-acting promoter elements and trans-acting factors. The eukaryotic promoter consists of core elements, which include the TATA and CAAT box and other DNA sequences that define transcription start sites, and regulatory elements, which either enhance or repress transcription in a gene-specific manner. The core promoter is the site for assembly of the transcription preinitiation complex, which includes RNA pol II and the general transcription factors TBP, transcription factor IIB (TFIIB), TFIIE, TFIIF, and TFIIH (for review see Roeder, 1996; Hampsey, 1998; Shilatifard, 1998).

Regulatory elements bind gene-specific factors, which affect the rate of transcription by interacting, either directly or indirectly, with components of the general transcriptional machinery. A third class of transcription factors, termed coactivators, is not required for basal transcription in vitro but often mediates activation by a broad spectrum of activators. Accordingly, coactivators are neither gene-specific nor general transcription factors, although gene-specific coactivators have been described in metazoan systems including humans. Transcriptional repressors include both gene-specific and general factors. Similar to coactivators, general transcriptional repressors affect the expression of a broad spectrum of genes yet do not repress all genes. General repressors either act through the core transcriptional machinery or are histone-related and presumably affect chromatin function, thus preventing RNA transcription ( Chang and Jaehning, 1997; Hampsey, 1998; Yamaguchi et al., 1998).

Figure 5 depicts a schematic model on the formation of the multiprotein complex within the promoter region of a gene. In this model acetylated chromatin is made accessible for transcription factors (DNA-binding transactivators) in the control region (promoter regions and enhancer-binding sites) of the respective gene by ATP-dependent chromatin remodeling complexes. After transcription factor binding, the RNA polymerase II, general initiation factors, and mediators bind at the promoter region. Then RNA polymerase II elongation factors bind additionally to the multiprotein complex to enable mRNA transcription. In this process extensive protein-protein interactions occur that enable the fine tuning of an orchestrated regulation of gene transcription (for review see Roeder, 1996; Hampsey, 1998; Shilatifard, 1998; Wolberger, 1999).

View larger version (41K): [in this window] [in a new window]   Fig. 5.   Formation of the multiprotein complex at the promoter of a gene. The depicted model shows acetylated chromatin (acetylation = red spheres) that is selectively remodeled by ATP-dependent chromatin remodeling complexes (blue sphere and black sphere) and thus allowing the binding of DNA-binding transactivators close to the promoter of the gene. Then the RNA polymerase II, general initiation factors, and mediators bind at the promoter followed by the binding of RNA polymerase II elongation factors to the multiprotein complex, finally leading to mRNA transcription. The proteins that form the multiprotein complex are represented by spheres in different shapes and colors.

	II. Classification of Liver-Enriched Transcription Factors

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Transcription factors achieve recognition of the DNA-binding site through protein-DNA and protein-protein interactions via discrete substructures or protein domains that serve binding to DNA. The DNA-binding motifs of transcription factors contain characteristic amino acid sequences and form characteristic three-dimensional structures that allow the classification of different types of transcription factors. The three-dimensional structure of these DNA-binding motifs leads to DNA sequence-specific DNA binding through the formation of hydrogen bonds and Van der Waals contacts (Pabo and Sauer 1992; Klug, 1999).

The first DNA-binding motif identified in X-ray crystallographic studies is the helix-turn-helix (HTH) motif (Wintjens and Rooman, 1996). POU domain transcription factors have two separate helix-turn-helix DNA-binding subdomains, the POU homeodomain (POUhd) and the POU-specific domain (POUs). Each subdomain recognizes a specific subsite of 4 or 5 bp in the octamer recognition sequence (Van Leeuwen et al., 1997). The POU domain family of transcription factors was defined after the observation that the products of three mammalian genes, Pit-1, Oct-1, and Oct-2, and the protein encoded by the Caenorhabditis elegans gene unc-86, shared a region of homology, known as the POU domain (Schonemann et al., 1998).

Molecular characterization of the genes whose sequence alterations cause impressive phenotypes in the fruit fly, Drosophila melanogaster, has led to the identification of the human homeobox genes, also referred to as the HOX genes and defined as "master genes" for their crucial role in embryogenesis (McGinnis and Krumlauf, 1992). They all share a homeobox region, known as a 180-bp highly conserved sequence encoding a 60-amino acid DNA-binding domain, also called the "homeodomain", conferring to the resulting proteins the ability to act as transcription factors (Gehring et al., 1994; Chariot et al., 1999). The 39 human HOX genes are organized in four distinct clusters (loci A, B, C, and D) and can be aligned on the basis of homology within the homeobox to define paralogs (Acampora et al., 1989; Scott, 1992). Besides a critical involvement in cell phenotype determination along the anterior-posterior axis during embryonic development, the HOX genes also play a key role in differentiation and tumoral development (Chariot et al., 1999; Cillo et al., 1999; Morata and Sanchez-Herrero, 1999).

The liver-enriched transcription factor hepatocyte nuclear factor-1 (HNF-1) contains a variant homeodomain and shares homeodomain, as well as short acidic and basic sequences, with the POU family of transcriptional activators (Baumhueter et al., 1990). HNF-1 is composed of HNF-1 or HNF-1 homo- or heterodimers (Song et al., 1998).

The hepatocyte nuclear factor-3 (HNF-3)/fork head (fkh) family contains a large number of transcription factors and folds into a winged helix motif. Despite having almost invariable amino acid sequences in their principal DNA-binding helices, HNF-3/fkh proteins show a wide diversity of sequence-specific binding. Previous studies of chimeric HNF-3/fkh proteins demonstrated that the binding specificity is primarily influenced by a region directly adjacent to the binding helix (Marsden et al., 1998; Jin et al., 1999). In NMR and X-ray crystallographic studies it is found that in comparison with HNF-3, the HNF-3/fork head (fkh) family member Genesis contains an extra small helix directly prior to the N terminus of the primary DNA contact helix. Due to the insertion of this helix, a shorter and slightly repositioned primary DNA contact helix is observed, which is believed to lead to the DNA-binding specificity differences among various family members (Marsden et al., 1998).

The liver-enriched transcription factor hepatocyte nuclear factor-4 (HNF-4) belongs to the group of zinc finger proteins and is frequently seen as a member of the nuclear receptor superfamily with unknown ligand (Taraviras et al., 1994). Zinc-fingers are small DNA-binding peptide motifs. These motifs can be used as modular building blocks for the construction of larger protein domains that recognize and bind to specific DNA sequences (Klug, 1999). Steroids and thyroid hormones, as well as vitamin D, retinoids, and some nutrient metabolites (fatty acids, prostaglandins, farnesol metabolites) act through binding to members of the zinc-finger containing superfamily of nuclear hormone receptors. These receptor proteins bind directly to specific DNA recognition sequences (hormone response elements) in the promoter region of target genes to facilitate transcription. The formation of several sets of heterodimers among family members as well as cross-talk with other signaling systems results in an intricate regulatory network with distinct particularities for each receptor type (Meier, 1997).

HNF-6 is a liver-enriched transcription factor that contains a single-cut domain and a novel type of homeodomain. Comparative trees of mammalian, Drosophila, and C. elegans proteins showed that HNF-6 defines a new class of homeodomain proteins called onecut class. It could be demonstrated that C. elegans proteins of this class bind to HNF-6 DNA targets. Thus, depending on their sequence, these targets determine for HNF-6 at least two modes of DNA binding, which hinge on the homeodomain and on the linker that separates it from the cut domain, and two modes of transcriptional stimulation, which hinge on the homeodomain (Lannoy et al., 1998).

Many transcription factors bind DNA to form dimeric (2:1) protein-DNA complexes. Examples include basic region leucine zipper (bZIP) proteins and basic region helix-loop-helix zipper (bHLHZIP) proteins. These two families of transcription factors follow an assembly pathway in which two protein monomers bind DNA sequentially and form their dimerization interface while bound to DNA (Kohler et al., 1999). Dimerization of these transcription factors stabilizes the protein-DNA complexes and can lead either to homodimers with the same transcription factor or to heterodimers with other members of the same family of transcription factors (Horiuchi et al., 1997).

The bZIP family of proteins is one of the largest and most conserved groups of eukaryotic transcription factors/repressors (Niu et al., 1999). These transcription factors use an atypically simple motif for DNA recognition called the basic region, yet family members discriminate differentially between target sites that differ only in half-site spacing. Two such sites are the cAMP-response element (CRE) and the AP-1 target site (Metallo and Schepartz, 1994). The DNA-binding motif of transcription factors belonging to the bZIP family is bipartite, consisting of a dimerization interface termed "leucine zipper" and a DNA contact surface termed the "basic region". Specificity of DNA binding has been shown to be imparted by the basic region (Agre et al., 1989).

The CCAAT/enhancer-binding proteins (C/EBP, C/EBP, C/EBP, and C/EBP) form a subfamily of bZIP transcription factors that display sequence homology within the bZIP domain. The conserved basic region in this subfamily contains two motifs that exhibit significant homology to the bipartite nuclear localization signal promoting nuclear transport of a bZIP transcription factor (Williams et al., 1997).

Further important members of the bZIP family of transcription factors are c-jun, c-fos (AP-1), and CREB. The molecular chaperone bZIP enhancing factor (BEF) has been shown to increase DNA binding of transcription factors that contain a basic region leucine zipper (bZIP) DNA-binding domain. BEF stimulates DNA binding by recognizing the unfolded leucine zipper and promoting the folding of bZIP monomers to dimers. Antisense experiments indicate that BEF is required for efficient transcriptional activation by bZIP proteins in vivo (Virbasius et al., 1999).

	III. Molecular Regulation of Liver Function

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The transcription rate of genes encoding liver-specific proteins is distinctly higher in hepatocytes as compared with other cell types (Powell et al., 1984). The transcription of several hepatic genes is activated during liver development and later modulated depending on extracellular stimulation (Schmid and Schulz, 1990; Cascio and Zaret, 1991; Shiojori et al., 1991). Experiments using a cDNA library from mouse liver poly(A)+ RNA that was then differentially screened with poly(A)+ RNA from liver and nonliver cells provided strong evidence that the predominant control of liver-specific gene expression resides at the level of transcription (Derman et al., 1981; Aran et al., 1995). Clones proven to be liver-specific were picked and used as templates for hybridization with radioactive RNA newly transcribed in vitro in nuclei isolated from liver and nonliver tissues. The hybridization signals obtained with RNA synthesized with liver nuclei were at least 10 times more intense than those obtained with nuclei from other tissues. Because the cDNA clones represented an unbiased population of transcripts, the findings led to the conclusion that liver-specific gene expression is primarily a consequence of transcriptional regulation (Derman et al., 1981).

Transient transfection assays in which the introduced gene does not integrate into the genome have been instrumental in identifying the regulatory sequences in DNA that confer liver-specific gene expression. Analyses performed on a wide variety of genes that code for entirely different proteins show shared regulatory sequences. Moreover, characterization of the regulatory sequences of a number of genes has shown that each gene contains a combination of some or all of the liver-specific shared motifs (Benvenisty and Reshef, 1991; Aran et al., 1995). It is this combination of cis-regulatory elements rather than a single element that appears to be required for liver-specific gene expression. Finally, these shared motifs bind distinct cognate liver-enriched transcription factors and have aided in isolating and characterizing these factors (for review see De Simone and Cortese, 1991; Lai and Darnell, 1991; Aran et al., 1995).

Six families of liver-enriched transcription factors have been characterized so far: HNF-1, HNF-3, HNF-4, HNF-6, C/EBP, and D-binding protein (DBP). The analysis of the tissue distribution of these factors and the determination of their hierarchical relations have led to the hypothesis that the cooperation of liver-enriched transcription factors with the ubiquitous transactivating factors is necessary, and possibly even sufficient, for the maintenance of liver-specific gene transcription (Hayashi et al., 1999).

HNFs are a heterogeneous class of evolutionarily conserved transcription factors that contain several families of liver-enriched transcription factors that are required for cellular differentiation and metabolism (Duncan et al., 1998). The liver-enriched transcription factor family containing the C/EBPs was formerly called HNF-2 and will be reviewed separately along with the D-binding protein (DBP).

	IV. Hepatocyte Nuclear Factors

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The HNF-1, HNF-3, HNF-4, and HNF-6 families of transcription factors contain several members. It should be noted that liver-enriched transcription factors are not exclusively expressed in the liver. For example, HNF-1, -3, -3, -3, -4, and -6 are also expressed in pancreatic -cells (Vaisse et al., 1997). HNF-1 and HNF-4 play there a critical role in normal pancreatic -cell function. Mutations in these liver-enriched transcription factors result in two forms of maturity-onset diabetes of the young (MODY), MODY3 and MODY1, respectively (Yamagata et al., 1996; Vaisse et al., 1997; Chevre et al., 1998). There are many more examples of relevant extrahepatic functions of liver-enriched transcription factors, but it is beyond the scope of this review to provide a complete summary of those extrahepatic functions.

HNF-1 is a transcriptional regulator composed of HNF-1 and HNF-1 hetero- and homodimers. These homeoproteins share identical DNA-binding domains but have different transcriptional activation properties (Kuo et al., 1991; Song et al., 1998).

The HNF-1 gene was assigned by somatic cell hybrids and recombinant inbred strain mapping to mouse chromosome 5 near Bcd-1 and to human chromosome 12 region q22-qter, revealing a different chromosomal region for these two species (Kuo et al., 1990). The HNF-1 gene was assigned to human chromosome 17 and murine chromosome 11. These chromosomal localizations differ from that of the HNF-1 gene, indicating that both genes are not clustered on the genome (Bach et al., 1991).

HNF-1 is one of the most important transactivators of liver-specific albumin transcription (Maire et al., 1989). HNF-1 acts as an accessory factor to enhance the inhibitory action of insulin on mouse glucose-6-phosphatase gene transcription (Streeper et al., 1998). HNF-1 is also an accessory factor required for activation of glucose-6-phosphatase gene transcription by glucocorticoids (Lin et al., 1998). Several lines of evidence point to a direct transactivation of the mouse ferrochelatase promoter by HNF-1 in the liver (Muppala et al., 2000).

Plasma lipoprotein(a) concentrations are highly heritable and predominantly determined by the liver-specific apolipoprotein(a) [apo(a)] gene. Elevated levels of lipoprotein(a) in the plasma are a risk factor for coronary artery disease and stroke. Positive regulation of transcription of the apo(a) gene is dependent on the binding of HNF-1 to a regulatory element located downstream of the mRNA start site (Wade et al., 1994).

HNF-1 is able to repress the transcription of liver-specific genes as demonstrated for the sucrose-isomaltase gene. Glucose represses transcription of this gene in cooperation with three HNF-1-binding sites in the sucrose-isomaltase promoter. Mutagenesis of the HNF-1-binding sites showed that the two distal HNF-1-binding sites are crucial for the glucose regulation of the sucrose-isomaltase gene (Rodolosse et al., 1998).

A number of genes that are predominantly expressed in the liver are positively regulated by HNF-1 interacting with the respective cis-acting HNF-1-binding elements in the promoters of these genes (see also Table 1).

View this table: [in this window] [in a new window]   TABLE 1 Shown are examples of liver-specific genes that contain a regulatory element with a HNF-1 binding site. The species of the investigated gene with its regulatory sequence as well as the respective references are indicated. The positions of the HNF-1 binding sites have preferably been taken from published DNase I footprinting studies, if available. The next preference is for chemical modifications, and the last for gel retardation assays. In case of different positional information for both DNA strands, the more upstream position has been taken for the 5'-border and the more downstream position for the 3'-border of the site. If not stated otherwise, the position numbers generally refer to the transcription start site (t.s.s.). Occasionally they may refer to the translation start codon stated as ATG or to a defined restriction site. When the authors emphasized a specific motif within the published regulatory sequence, it is written in capitals whereas the rest of the sequence is written in lowercase letters.  indicates a continuing sequence in the next line.

Serum colloid osmotic pressure is believed to control hepatic output of plasma proteins. Many plasma proteins that are secreted from the liver, including albumin, have a HNF-1-binding site in their promoter. The activity of HNF-1 in highly differentiated hepatoma cells was shown to be modulated by a fluctuation in the level of oncotically active macromolecules like dextran or albumin in the surrounding cell culture medium. Higher oncotic pressures lead to a decrease in HNF-1 mRNA levels (Pietrangelo and Shafritz, 1994).

1. Dimerization Cofactor of Hepatocyte Nuclear Factor-1 and Liver-Specific Gene Expression. Interestingly, HNF-1, but not HNF-1, is expressed in the liver. Under physiologic conditions as well as in transfection experiments with HNF-1 and HNF-1, stable homodimer formation can be found in the liver, whereas in other organs, heterodimers also are detected. From these data it was assumed that the extent of heterodimerization may be regulated in a tissue-specific manner. Furthermore, it could be shown that exclusive expression of HNF-1 is associated with repression of a subset of hepatocyte-specific genes in the dedifferentiated hepatocyte cell line C2, in differentiated F9 cells, in somatic hybrids between hepatocytes and fibroblasts, and in the lung (Mendel et al., 1991a).

The mammalian HNF-3/fkh family consists of at least 30 distinct members and is expressed in a variety of different cellular lineages (Qian and Costa, 1995). The HNF-3 gene subfamily is composed of three proteins (, , and ) that mediate hepatocyte-enriched transcription of numerous genes whose expression is necessary for organ function (Samadani and Costa, 1996). All three transcription factors share strong homology in the winged-helix/fork head DNA-binding domain (region I) that overlaps with the nuclear localization signal (Qian and Costa, 1995). HNF-3, -, and - are able to recognize the same DNA sequence (Samadani and Costa, 1996; Pani et al., 1992a,b). They also possess two similar stretches of amino acids at the carboxyl terminus (regions II and III) and a fourth segment of homology at the amino terminus (region IV) (Pani et al., 1992a,b).

The HNF-3 proteins demonstrate homology with the Drosophila homeotic gene fork head in regions I, II, and III, suggesting that HNF-3 may be its mammalian homolog (Pani et al., 1992a). Experiments using site-directed mutagenesis within regions II and III (amino acids 361-458) of HNF-3 demonstrated their importance for transactivation. In cotransfection assays with expression vectors that produced different truncated HNF-3 proteins, amino-terminal sequences defined by conserved region IV also contributed to transactivation, but region IV activity required the participation of the region II-III domain (Pani et al., 1992a).

HNF-3 and HNF-3 regulate gene expression in endoderm-derived hepatocytes, and intestinal, pancreatic, and bronchiolar epithelium (Rausa et al., 1997; Clevidence et al., 1998). HNF-3 may also play an important role in development and maintenance of urogenital tract epithelial cells (Clevidence et al., 1998; Kopachik et al., 1998). HNF-3 and HNF-3 are members of a large family of developmentally regulated transcription factors that participate in embryonic pattern formation (Rausa et al., 1997; Clevidence et al., 1998).

Stimulation of HNF-3 gene transcription upon retinoic acid-induced differentiation of mouse F9 embryonal carcinoma cells can give rise to three distinct differentiated cell types; visceral endoderm, parietal endoderm, and primitive endoderm, which indicates that HNF-3 may play an important role in differentiation during primitive endoderm formation, an extremely early event during murine embryogenesis (Jacob et al., 1994).

A number of liver-specific genes that are predominantly expressed in the liver are positively regulated by HNF-3, -, or - through interaction with the respective cis-acting HNF-3-binding elements in the promoters of these genes (see also Table 2). In contrast, HNF-3 bound to the HNF-3-binding site of the human aldolase B promoter completely antagonizes transactivation of the liver-specific aldolase B gene by HNF-1 and DBP (Gregori et al., 1993).