HNF4

EVOLUTIONARY HOMOLOGS

A comparative tree of DNA-binding domain amino acid sequences reveals the evolutionary affinities of Drosophila nuclear receptor proteins. Knirps shows no close affinities to other nuclear receptor proteins. Drosophila Ecdysone receptor sequence is most similar to murine RIP14. Tailless has a close affinity to murine Tlx. Drosophila E78 and E75 fall in the same subclass as Rat Reverb alpha and beta, and C. elegans "CNR-14." Drosophila HR3 is in the same subclass as C. elegans "CNR-3." Drosophila HNF-4 is most closely related in sequence to Rat HNF-4. Drosophila Ftz-F1 and Mus ELP show sequence similarity to each other. Drosophila Seven up is closely related to Human COUP-TF. Drosophila Ultraspiracle is in the same subfamily as Human RXRalpha, Human RXRbeta, and Murine RXRgamma. The latter two groups, containing Ultraspiracle and Seven up, show a distant affinity to each other. Four other subfamilies show no close Drosophila affinities. These are: 1) C. elegans rhr-2, 2) Human RARalpha, beta and gamma, 3) Human thyroid hormone receptor alpha and beta, and 4) Human growth hormone receptor, glucocorticoid receptor, and progesterone receptor (Sluder, 1997).

Each ovariole of the pharate adult silkworm, Bombyx mori, contains approximatedly 120 developing follicles arranged in a linear array, with each follicle along the length of the developing ovariole representing a specific (and unique stage of follicle development; developing follicles differ from their adjacent neighbors by approximately 2 hours in the developmental program. The most advanced follicles enter vitellogenesis as early as the spinning stage, while choriogenesis is initiated by the first (terminal) follicles 5 days after larval-pupal ecdysis. Two silkmoth nuclear receptor isoforms, BmHNF-4a and BmHNF-4b, that are related to the mammalian orphan receptor HNF-4, were characterized. Their characterization reveals that they differ from each other only in their 5' UTR and N-terminus of the predicted polypeptides. In ovarian tissue, the two receptors are expressed as a delayed response to 20-hydroxy-ecdysone and their expression increases during vitellogenesis. BmHNF-4 mRNA is localized in the cytoplasm of follicular cells and a binding activity that recognizes a mammalian HNF-4 response element is present in follicular cell nuclear extracts. BmHNF-4 mRNA is also present in the oocyte, the unfertilized egg and the early embryo, thus displaying a behavior reminiscent of maternal mRNA. Both mRNA isoforms are found in the embryo following fertilization and their abundance is modulated during ensuing embryogenesis. In contrast to the rather limited distribution of HNF-4 in mammalian tissues, in the silkmoth BmHNF-4 is expressed in most larval and pharate adult tissues. During embryogenesis BmHNF-4a is mainly present in the fat body, gut and Malpighian tubules, while BmHNF-4b, detectable in all tissues except for the silkgland, is predominantly expressed in the testis. The dual pattern of expression of the BnHNF-4 gene during oogenesis (accumulation of maternal mRNA in the oocyte fraction, and mRNA and DNA binding protein present in the nuclei of follicular cells) bears a striking resemblance to the expression pattern of the GAGA factor during Drosophila oogenesis. These results challenge the notion that the HNF-4 class of orphan receptors encompasses members that are only expressed in liver, kidney and intestine or in the functional analgs of insects (fat body, Malpighian tubules and gut) BmHNF-4 is expressed in the most larval and pharate adult tissues of the silkmoth, but shows tissue-specific accumulation of different isoforms (Swevers, 1998).

Xenopus HNF1alpha, activated shortly after zygotic transcription starts, is expressed in liver, kidney, stomavh and gut. Mutational analysis of the HNF1alpha promoter shows that HNF1 and HNF4 binding sites are essential for proper embryonic regulation. Since by injecting HNF4 mRNA into fertilized eggs the endogenous HNF1alpha gene is activated ectopically and HNF4 is present as a maternal protein within an animal to vegetal gradient in the embryo, it is assumed that HNF4 initiates a transcriptional hierarchy involved in determination of different cell fates (Holewa, 1996)

A second Xenopus HNF4 gene has been identified which is more distantly related to mammalian HNF4 than the previously isolated gene. This new gene is named HNF4ß , to distinguish it from the known HNF4 gene, which is now called HNF4alpha. There are two splice variants of HNF4ß with additional exons that seem to affect messenger RNA stablity. HNF4ß is a functional transcription factor acting in a sequence specific manner on HNF4 binding sites known for HNF4alpha, but in comparison, it appears to have a lower DNA binding activity than the alpha isoform and is a weaker transactivator. The two factors differ with respect to their tissue distribution in adult frogs: whereas HNF4alpha and HNF4ß are both expressed in liver and kidney, HNF4ß is also expressed in the stomach, intestine, lung, ovary and testis. Both factors are maternal proteins and present at constant levels throughout embryogenesis. Whereas HNF4alpha is expressed early during oogenesis and is absent in the egg, HNF4ß is first detected in the latest stage of oogenesis, and transcripts are present in the egg and early cleavage stages. Furthermore, zygotic HNF4alpha transcripts appear in early gastrula and accumulate during further embryogenesis, whereas HNF4ß mRNA transiently appears during gastrulation before it accumulates again at the tail bud stage. All of these distinct characteristics of the newly identified HNF4 protein imply that the alpha and ß isoforms have different functions in development and in adult tissues (Holewa, 1997).

Hepatocyte nuclear factor 4 (HNF-4) defines a new subgroup of nuclear receptors that exist in solution and bind DNA exclusively as homodimers. The putative ligand binding domain (LBD) of HNF-4 is responsible for dimerization in solution and prevents heterodimerization with other receptors. The role of the LBD in DNA binding by HNF-4 was investigated by using electrophoretic mobility shift analysis. A comparison of constructs containing either the DNA binding domain (DBD) alone or the DBD plus the LBD of HNF-4 shows that dimerization via the DBD is sufficient to provide nearly the full DNA binding affinity of the full-length HNF-4. In contrast, dimerization via the DBD is not sufficient to produce a stable protein-DNA complex, whereas dimerization via the LBD increases the half-life of the complex by at least 100-fold. Circular permutation analysis shows that full-length HNF-4 bends DNA by approximately 80 degrees while the DBD bends DNA by only 24 degrees. Nonetheless, analysis of other constructs indicates that the increase in stability afforded by the LBD could be explained only partially by an increased ability to bend DNA. In comparison, coimmunoprecipitation studies show that dimerization via the LBD produces a protein-protein complex that is much more stable than the corresponding protein-DNA complex. These results have lead to a model in which dimerization via the LBD stabilizes the receptor on DNA by converting an energetically favorable two-step dissociation event into an energetically unfavorable single-step event (Jiang, 1997).

At E4.0 the inner cell mass of the mouse blastocyst consists of a core of embryonic ectoderm cells surrounded by an outer layer of primitive (extraembryonic) endoderm, which subsequently gives rise to both visceral endoderm and parietal endoderm. Shortly after blastocyst implantation, the solid mass of ectoderm cells is converted by a process known as cavitation into a pseudostratified columnar epithelium surrounding a central cavity. The trophectoderm-derived extraembryonic ectoderm undergoes a similar cavitation process at a slightly later stage. Cavitation, this type of morphogenetic conversion, also occurs during the formation of other hollow (tubular) structures that arise from solid primordia, such as the ducts of various exocrine glands. In early postimplantation mouse development, cavitation prepares the embryo for gastrulation, the process by which the three germ layers are formed. Two cell lines, which form embryoid bodies that do (PSA1) or do not (S2) cavitate, have been used as an in vitro model system for studying the mechanism of cavitation in the early embryo. Evidence is provided that cavitation is the result of both programmed cell death and selective cell survival, and that the process depends on signals from visceral endoderm. Bmp2 and Bmp4 are expressed in PSA1 embryoid bodies and embryos at the stages when visceral endoderm differentiation and cavitation are occurring, and blocking BMP signaling via expression of a transgene encoding a dominant negative mutant form of BMP receptor IB inhibits expression of the visceral endoderm marker, Hnf4, and prevents cavitation in PSA1 embryoid bodies. Furthermore, addition of BMP protein to cultures of S2 embryoid bodies induces expression of Hnf4 and other visceral endoderm markers and also cavitation. Taken together, these data indicate that BMP signaling is both capable of promoting, and required for differentiation of, visceral endoderm and cavitation of embryoid bodies. Based on these and other data, a model is proposed for the role of BMP signaling during peri-implantation stages of mouse embryo development. It is suggested that BMP4 produced in the ectoderm acts on the primitive endoderm to promote visceral endoderm differentiation. BMP2 produced in the endoderm may also play a role in the process. BMP4 produced in the ectoderm and/or BMP2 produced in the visceral endoderm act(s) on the ectoderm [perhaps in conjunction with other, visceral endoderm-derived signals(s)] to promote programmed cell death of the inner cells and the differentiation of the outer layer of columnar cells (Coucouvanis, 1999).

The human homolog of the rat and mouse HNF4 splice variant HNF4 alpha 2 have been isolated, as well as a previously unknown splice variant of this protein, which is here called HNF alpha 4. In addition, novel HNF4 subtype (HNF4 gamma) derived from a different gene has been cloned. The genes encoding HNF 4 alpha and HNF4 gamma are located on human chromosomes 20 and 8, respectively. HNF4 gamma is expressed in the kidney, pancreas, small intestine, testis, and colon but not in the liver, while HNF4 alpha RNA is found in all of these tissues. HNF4 gamma is significantly less active than HNF4 alpha 2, and the novel HNF4 alpha splice variant HNF4 alpha 4 has no detectable transactivation potential. Therefore, the differential expression of distinct HNF4 proteins may play a key role in the differential transcriptional regulation of HNF4-dependent genes (Drewes, 1996).

HNF4 binds fatty acids

HNF4 alpha is an orphan member of the nuclear receptor family with prominent functions in liver, gut, kidney and pancreatic beta cells. The x-ray crystal structure of the HNF4 alpha ligand binding domain, which adopts a canonical fold, has been solved. Two conformational states are present within each homodimer: an open form with alpha helix 12 (alpha 12) extended and collinear with alpha 10 and a closed form with alpha 12 folded against the body of the domain. Although the protein was crystallized without added ligands, the ligand binding pockets of both closed and open forms contain fatty acids. The carboxylic acid headgroup of the fatty acid ion pairs with the guanidinium group of Arg(226) at one end of the ligand binding pocket, while the aliphatic chain fills a long, narrow channel that is lined with hydrophobic residues. These findings suggest that fatty acids are endogenous ligands for HNF4 alpha and establish a framework for understanding how HNF4 alpha activity is enhanced by ligand binding and diminished by MODY1 mutations (Dhe-Paganon, 2002).

The 2.7 Å X-ray crystal structure of the HNF4gamma ligand binding domain (LBD) revealed the presence of a fatty acid within the pocket, with the AF2 helix in a conformation characteristic of a transcriptionally active nuclear receptor. GC/MS and NMR analysis of chloroform/methanol extracts from purified HNF4alpha and HNF4gamma LBDs has identified mixtures of saturated and cis-monounsaturated C14-18 fatty acids. The purified HNF4 LBDs interact with nuclear receptor coactivators, and both HNF4 subtypes show high constitutive activity in transient transfection assays, that is reduced by mutations designed to interfere with fatty acid binding. The endogenous fatty acids do not readily exchange with radiolabeled palmitic acid, and all attempts to displace them without denaturing the protein failed. These results suggest that the HNF4s may be transcription factors that are constitutively bound to fatty acids (Wisely, 2002).

Role for HNF-4 in gastrulation

Immediately prior to gastrulation the murine embryo consists of an outer layer of visceral endoderm (VE) and an inner layer of ectoderm. Differentiation and migration of the ectoderm then occurs to produce the three germ layers (ectoderm, embryonic endoderm and mesoderm) from which the fetus is derived. An indication that the VE might have a critical role in this process emerged from studies of Hnf-4-/- mouse embryos which fail to undergo normal gastrulation. Since expression of the transcription factor HNF-4 is restricted to the VE during this phase of development, it is proposed that HNF-4-regulated gene expression in the VE creates an environment capable of supporting gastrulation. Using Hnf-4-/- embryonic stem cells it can be demonstrated that HNF-4 is a key regulator of tissue-specific gene expression in the VE, required for normal expression of secreted factors including alphafetoprotein, apolipoproteins, transthyretin, retinol binding protein, and transferrin. Furthermore, specific complementation of Hnf-4-/- embryos with tetraploid-derived Hnf-4+/+ VE rescues their early developmental arrest, showing conclusively that a functional VE is mandatory for gastrulation (Duncan, 1997).

Expression of vertebrate HNF4

Expression of HNF4, a transcription factor in the steroid hormone receptor superfamily, is detected only in the visceral endoderm of mouse embryos during gastrulation and is expressed in certain embryonic tissues from 8.5 days of gestation. To examine the role of HNF4 during embryonic development, the gene was disrupted in embryonic stem cells. The homozygous loss of functional HNF4 protein is an embryonic lethal. Cell death is evident in the embryonic ectoderm at 6.5 days, when these cells normally initiate gastrulation. As assessed by expression of Brachyury and HNF3 beta, primitive streak formation and initial differentiation of mesoderm do occur, but with a delay of approximately 24 h. Development of embryonic structures is severely impaired. These results demonstrate that the expression of HNF4 in the visceral endoderm is essential for embryonic ectoderm survival and normal gastrulation (Chen, 1994).

The expression of HNF4 mRNA in postimplantation mouse embryos has been analyzed by in situ hybridization. Expression is found in the primary endoderm at embryonic day 4.5 and is restricted to the columnar visceral endoderm cells of the yolk sac from day 5.5 to day 8.5. HNF4 mRNA is first detected in the liver diverticulum and the hindgut of these embryonic tissues at day 8.5. Later, HNF4 transcripts are observed in the mesonephric tubules, pancreas, stomach, and intestine and, still later, in the metanephric tubules of the developing kidney. This expression pattern suggests that HNF4 has a role in the earliest stages of murine postimplantation development as well as in organogenesis (Duncan, 1994).

The oval cells are thought to be the progeny of a liver stem cell compartment. Strong evidence now exists indicating that these cells can participate in liver regeneration by differentiating into different hepatic lineages. To better understand the regulation of this process the expression of liver-enriched transcriptional factors (HNF1 alpha and HNF1 beta, HNF3 alpha, HNF3 beta, and HNF3 gamma, HNF4, C/EBP, C/EBP beta, and DBP) has been studied in an experimental model of oval cell proliferation and differentiation . The expression of these factors has been compared to that observed during late stages of hepatic ontogenesis. The steady-state mRNA levels of four "liver-enriched" transcriptional factors (HNF1 alpha, HNF3 alpha, HNF4, and C/EBP beta) gradually decreases during the late period of embryonic liver development, while three factors (HNF1 beta, HNF3 beta, and DBP) increase. In the normal adult rat liver the expression of all the transcription factors are restricted to the hepatocytes. However, during early stages of oval cell proliferation both small and large bile ducts start to express HNF1 alpha and HNF1 beta, HNF3 gamma, C/EBP, and DBP, but not HNF4. At the later stages all of these factors are also highly expressed in the proliferating oval cells. Expression of HNF4 is first observed when the oval cells differentiate morphologically and functionally into hepatocytes and form basophilic foci. At that time the expression of some of the other factors is also further increased. It is suggested that the upregulation of the "establishment" factors (HNF1 and -3) may be an important step in oval cell activation. The high levels of these factors in the oval cells and embryonic hepatoblasts further substantiates the similarity between the two cell compartments. Furthermore, the data suggest that HNF4 may be responsible for the final commitment of a small portion of the oval cells to differentiate into hepatocytes that form the basophilic foci and eventually regenerate the liver parenchyma (Nagy, 1994).

Hepatocyte nuclear factors (HNFs) are a heterogeneous class of evolutionarily conserved transcription factors that are required for cellular differentiation and metabolism. Mutations in HNF-1alpha and HNF-4alpha genes impair insulin secretion and cause type 2 diabetes. Regulation of HNF-4/HNF-1 expression by HNF-3alpha and HNF-3beta was studied in embryoid bodies in which one or both HNF-3alpha or HNF-3beta alleles were inactivated. HNF-3beta positively regulates the expression of HNF-4alpha/HNF-1alpha and their downstream targets, implicating a role in diabetes. HNF-3beta is also necessary for expression of HNF-3alpha. In contrast, HNF-3alpha acts as a negative regulator of HNF-4alpha/HNF-1alpha, demonstrating that HNF-3alpha and HNF-3beta have antagonistic transcriptional regulatory functions in vivo. HNF-3alpha does not appear to act as a classic biochemical repressor but rather exerts its negative effect by competing for HNF-3 binding sites with the more efficient activator HNF-3beta. In addition, the HNF-3alpha/HNF-3beta ratio is modulated by the presence of insulin, providing evidence that the HNF network may have important roles in mediating the action of insulin (Duncan, 1998).

Hepatocyte nuclear factor 4alpha (HNF4alpha) is essential for the establishment and maintenance of liver-specific gene expression. The HNF4alpha gene codes for several isoforms whose developmental and physiological relevance has not yet been explored. HNF4alpha1 and HNF4alpha7 originate from different promoters, while alternative splicing in 3' leads to HNF4alpha2 and HNF4alpha8, respectively. HNF4alpha7/alpha8 are abundantly expressed in embryonic liver and fetal-like hepatoma cells. HNF4alpha1/alpha2 transcripts are up-regulated at birth and represented the only isoforms in adult-like hepatoma cells. In line with its expression profile, HNF4alpha7 activates more avidly than HNF4alpha1 reporter plasmids for genes that are expressed early. The expression patterns of both isoforms together with the differences observed in their transcriptional activities provide elements accounting for fine-tuning of the activity of HNF4alpha. The sequential expression of HNF4alpha7/alpha8 and HNF4alpha1/alpha2 during mouse liver development is the only modification in liver-enriched transcription factors thus far recorded, which parallels the transition from the fetal to the adult hepatic phenotype (Torres-Padilla, 2001).

Targets of vertebrate HNF4

The gene encoding the tissue-specific transcription factor HNF1alpha (LFB1) in Xenopus embryos is transcriptionally activated shortly after mid-blastula transition. The HNF1alpha protein is localized in the nuclei of the liver, gall bladder, gut and pronephros of the developing larvae. In animal cap explants treated with activin A together with retinoic acid, HNF1alpha is induced in pronephric tubules and epithelial gut cells, i.e. in mesodermal as well as in endodermal tissues. HNF1alpha can also be induced by activin A, but not by retinoic acid alone. To define the promoter element responding to the activin A signal, various HNF1alpha promoter luciferase constructs were injected into fertilized eggs the isolated animal caps were cultured in the presence of activin A. From the activity profiles of the promoter mutants used, the HNF4-binding site is identified as an activin-A-responsive element. Since HNF4 is a maternal protein in Xenopus and localized in an animal-to-vegetal gradient in the cleaving embryo, it is speculated that the activin A signal emanating from the vegetal pole cooperates with the maternal transcription factor HNF4 to define the embryonic regions expressing HNF1alpha (Weber, 1996).

Homeoprotein hepatocyte nuclear factor-1 alpha (HNF1 alpha), and HNF4 are regulated coordinately or in a hierarchy by a higher-order locus, independent of other hepatic transactivators. HNF4 was implicated as an essential positive regulator of HNF1 alpha, as deletion of an HNF4 binding site in the HNF1 alpha promoter abolished promoter activity, and HNF4 potently transactivates the HNF1 alpha promoter in cotransfection assays. Moreover, genetic complementation of dedifferentiated hepatomas with HNF4 complementary DNA rescues expression of endogenous HNF1 alpha messenger RNA and DNA-binding activity. These studies therefore define an HNF4 - HNF1 alpha transcriptional hierarchy operative in differentiated hepatocytes, but selectively inhibited by an extinguishing locus and somatic mutations that antagonize the liver phenotype (Kuo, 1992).

The L-pyruvate kinase (L-PK) gene is a target of HNF1 and HNF4. L-PK is slightly active in normal and tumoral endocrine pancreatic tissues while, in vivo, this gene is not transcribed in the exocrine pancreas. Nevertheless, the L-PK gene is re-expressed at a very low level in cultured cells derived from an exocrine pancreas carcinoma. The L-PK gene is activated early in the development of endodermal tissues (for example, the yolk sac and primitive intestine); it remains transcribed in fetal pancreas. In the adult, L-PK gene expression is restricted to some endocrine cells. HNF1 and HNF4 are the main tissue-restricted transcription factors involved in tissue-specific expression of the L-PK gene. HNF1 concentration is similar in liver and all pancreatic cells. HNF4 concentration is high in liver, much lower in the islets of Langerhans, endocrine pancreatic tumors, and cultured insulinoma cells, and is scarcely detectable in adult exocrine pancreas. This distribution of HNF4 parallels the expression of the L-PK gene. In vivo footprinting experiments show that the HNF1 binding site is similarly occupied in both adult liver and pancreas, where this gene is found to be practically inactive. However, in the latter tissue, the HNF4 binding site is differently occupied, with respect to the liver. Since the chromatin structure remains open around the L-PK promoter in pancreas, the L-PK gene can probably be re-expressed under certain circumstances, for instance in cancerous pancreatic cells (Miquerol, 1994).

A short region between -118 and -8 is crucial for cell type-specific expression of the HNF1 gene in the hepatoma cell line (HepG2 cells). This region contains two positive cis-elements: site A, to which the transcription factor HNF4 protein can bind, and site B, to which the HNF1 protein can bind. Mutational analyses of these sites and cotransfection assays suggested that the HNF4 protein and HNF1 protein can transactivate the HNF1 gene (Miura, 1993).

Stable expression of an epitope-tagged cDNA of the hepatocyte-enriched transcription factor, hepatocyte nuclear factor (HNF)4, in dedifferentiated rat hepatoma H5 cells is sufficient to provoke reexpression of a set of hepatocyte marker genes. The effects of HNF4 expression extend to the reestablishment of differentiated epithelial cell morphology and simple epithelial polarity. The acquisition of epithelial morphology occurs in two steps. First, expression of HNF4 results in reexpression of cytokeratin proteins and partial reestablishment of E-cadherin production. Only the transfectants are competent to respond to the synthetic glucocorticoid dexamethasone, which induces the second step of morphogenesis, including formation of the junctional complex and expression of a polarized cell phenotype. Cell fusion experiments revealed that the transfectant cells, which show only partial restoration of E-cadherin expression, produce an extinguisher that is capable of acting in trans to downregulate the E-cadherin gene of well-differentiated hepatoma cells. Bypass of this repression by stable expression of E-cadherin in H5 cells is sufficient to establish some epithelial cell characteristics, implying that the morphogenic potential of HNF4 in hepatic cells acts via activation of the E-cadherin gene. Thus, HNF4 seems to integrate the genetic programs of liver-specific gene expression and epithelial morphogenesis (Spath, 1998).

Phosphorylation of HNF4

HNF4 is a phosphoprotein. Phosphorylation at tyrosine residue(s) is important for its DNA-binding activity and consequently, for its transactivation potential, both in cell-free systems and in cultured cells. Tyrosine phosphorylation does not affect the transport of HNF-4 from the cytoplasm to the nucleus but has a dramatic effect on its subnuclear localization. HNF4 is concentrated in distinct nuclear compartments, as evidenced by in situ immunofluorescence and electron microscopy. This compartmentalization disappears when tyrosine phosphorylation is inhibited by genistein. The correlation between the intranuclear distribution of HNF4 and its ability to activate endogenous target genes demonstrates a phosphorylation signal-dependent pathway in the regulation of transcription factor activity (Ktistaki, 1995).

Hepatocyte nuclear factor 4 (HNF4), a liver-enriched transcription factor of the nuclear receptor superfamily, is critical for liver development and liver-specific gene expression. Its DNA-binding activity is modulated posttranslationally by phosphorylation. In vivo, HNF4 DNA-binding activity is reduced by fasting and by inducers of intracellular cyclic AMP (cAMP) accumulation. A consensus protein kinase A (PKA) phosphorylation site located within the A box of its DNA-binding domain has been identified, and its role in phosphorylation-dependent inhibition of HNF4 DNA-binding activity has been investigated. Mutants of HNF4 in which two potentially phosphorylatable serines have been replaced by either neutral or charged amino acids are able to bind DNA in vitro with affinity similar to that of the wild-type protein. However, phosphorylation by PKA strongly represses the binding affinity of the wild-type factor but not that of HNF4 mutants. Accordingly, in transfection assays, expression vectors for the mutated HNF4 proteins activate transcription more efficiently than do those from the wild-type protein, when cotransfected with the PKA catalytic subunit expression vector (See Drosophila PKA). Therefore, HNF4 is a direct target of PKA that might be involved in the transcriptional inhibition of liver genes by cAMP inducers (Viollet, 1997).

Transcriptional regulation of HNF4

GATA6 belongs to a family of zinc finger transcription factors that play important roles in transducing nuclear events that regulate cellular differentiation and embryonic morphogenesis in vertebrate species. To examine the function of GATA6 during embryonic development, gene targeting was used to generate GATA6-deficient [GATA6(-/-)] ES cells and mice harboring a null mutation in GATA6. Differentiated embryoid bodies derived from GATA6(-/-) ES cells lack a covering layer of visceral endoderm and severely attenuate, or fail to express, genes encoding early and late endodermal markers, including HNF4, GATA4, alpha-fetoprotein (AFP), and HNF3beta. Homozygous GATA6(-/-) mice die between embryonic day (E) 6.5 and E7. 5 and exhibit a specific defect in endoderm differentiation, including severely down-regulated expression of GATA4 and the absence of HNF4 gene expression. Moreover, widespread programmed cell death was observed within the embryonic ectoderm of GATA6-deficient embryos, a finding also observed in HNF4-deficient embryos. Consistent with these data, forced expression of GATA6 activates the HNF4 promoter in nonendodermal cells. Finally, to examine the function of GATA6 during later embryonic development, GATA6(-/-)-C57BL/6 chimeric mice were generated. lacZ-tagged GATA6(-/-) ES cells contribute to all embryonic tissues with the exception of the endodermally derived bronchial epithelium. Taken together, these data suggest a model in which GATA6 lies upstream of HNF4 in a transcriptional cascade that regulates differentiation of the visceral endoderm. In addition, these data demonstrate that GATA6 is required for establishment of the endodermally derived bronchial epithelium (Morrisey, 1998).

The order of recruitment of factors to the HNF-4alpha regulatory regions was followed upon the initial activation of the gene during enterocyte differentiation. An initially independent assembly of regulatory complexes at the proximal promoter and the upstream enhancer regions was followed by the tracking of the entire DNA-protein complex formed on the enhancer along the intervening DNA until it encountered the proximal promoter. This movement correlates with a unidirectional spreading of histone hyperacetylation. Transcription initiation coincides with the formation of a stable enhancer-promoter complex and remodeling of the nucleosome situated at the transcription start site. The results provide experimental evidence for the involvement of a dynamic process culminating in enhancer-promoter communication during long-distance gene activation (Hatzis, 2003).

At the very beginning of the differentiation program (time 0), both the enhancer and the promoter are already occupied by the cognate DNA binding proteins. At this stage at least three basal transcription factors, TFIIA, TFIIB, and TBP, are also detectable at the proximal promoter. This complex may be viewed as a signature structure that creates a so-called poised or committed state to mark the gene for subsequent events. The nucleosomal organization of the HNF-4 regulatory regions also indicates that the gene is in a transcriptionally competent state from the beginning of the program. Unlike in nonexpressing cell lines, where positioning of nucleosomes is random, in CaCo-2 cells the proximal promoter is occupied by an array of positioned nucleosomes, while at the enhancer area two positioned nucleosomes are followed by a nucleosome-free region. In addition, the H3 component of the nucleosomes at the proximal promoter is methylated at the lysine 4 residue, a modification that has been proposed to correspond to an epigenetic mark for active chromatin. Another interesting feature of this state is that the binding site of HNF-3, a so-called pioneer factor that can disrupt higher order chromatin structure, is located at the nucleosome-free region of the HNF-4alpha enhancer. Since HNF-3-mediated disruption of internucleosomal interactions can affect the length of linker DNA, the formation of a nucleosome-free region at the enhancer may well be the result of HNF-3 action at an earlier stage of differentiation. Consistent with the above is the fact that the CaCo-2 cell culture model mimics only terminal enterocyte differentiation, since the line originates from enterocytes that have already passed through early developmental decisions determining lineage commitment (Hatzis, 2003).

In the second temporally separable phase, a selective recruitment of histone acetyltransferases (CBP and P/CAF) and the Brg-1 chromatin remodeling protein to the enhancer (20 hr time point) was observed. This coincided with the first histone H3 and H4 hyperacetylation signals confined to the enhancer region. At the same time, other components of TFIID, TFIIH, the mediator component TRAP-220, and RNA pol-II were recruited to the proximal promoter. Since in the previous step TAF1 and TAF10 were absent from the promoter, it is speculated that the TBP detected at time 0 is not part of the classical TFIID complex. Whether the TFIID detected at the 20 hr time point is generated by a progressive assembly of TAFs onto promoter-bound TBP or by an exchange of a TAF-less or nonclassical TFIID by a TAF-containing TFIID is not known. The key characteristic of this early stage is that all the major components of the general transcription machinery as well as CTD serine 5-phosphorylated RNA pol-II are stably assembled at the promoter, without initiating transcription. This indicates that recruitment of pol-II to the transcription start site is not sufficient for transcription, and other factors or events are also required for its escape from the promoter. In this regard it is important to note that at this period there was no indication for potential enhancer-promoter synergy, suggesting that the assemblies of the complexes of different compositions at the two regions are independent of each other (Hatzis, 2003).

During the ensuing time period (40-80 hr), the immunoprecipitates of all enhancer-associated factors (HNF-1alpha, C/EBPalpha, HNF-3ß, CBP, P/CAF, and Brg-1) contained DNA fragments corresponding to the regions between the enhancer and the promoter. Since at the same time these factors are also found to be associated with the enhancer region, this finding points to the formation of a binary crosslinked DNA complex composed of the enhancer and intermediary region DNA, bridged by the factors associated with them. Importantly, none of the factors recruited to the proximal promoter are detected in the intervening regions at any time during the differentiation program. The potential scenario that multiple molecules of the enhancer-recruited proteins first associate with the enhancer and then, at least part of them, may escape the enhancer DNA and scan freely toward the promoter, while others remain associated with the enhancer, is rather unlikely considering the sequence-specific DNA binding properties of HNF-3ß and C/EBPalpha, which can associate selectively with the enhancer region. Furthermore, in the next step (80-110 hr) the immunoprecipitates of all of the promoter-recruited factors contain enhancer DNA fragments, and immunoprecipitates of all of the enhancer-binding factors contain promoter DNA segments. In other words, if the detection of these two distant DNA sequences in the immunoprecipitates of factors that are recruited to one or the other region were to be interpreted as the result of either independent recruitment or free diffusion from one region to the other, then one would have to assume that general transcription factors or RNA pol-II would suddenly be recruited to a far-upstream location at the time of transcription initiation, a possibility which is hard to conceptualize. The reduction and subsequent disappearance of the ChIP signals of the enhancer-associated factors from the intervening regions at the times of active transcription (80-110 hr) is also inconsistent with the continuous escape of DNA binding factors from the enhancer. The possibility of direct recruitment of RNA pol-II followed by a long-range transfer to the promoter or the activation of a cryptic promoter at the upstream region could also be ruled out, since recruitment of RNA pol-II together with other general transcription factors to the proximal promoter could be observed long before enhancer-promoter complex formation and since no transcript corresponding to upstream sequences could be detected at any time during the differentiation program. Therefore, the direct evidence provided by the continuous ChIP signal observed with the enhancer DNA, together with the above-mentioned considerations, corroborate the claim that the signals detected at the intervening regions correspond to a complex containing enhancer DNA. These observations thus indicate that the entire DNA-protein complex forms on the enhancer tracks along the intervening region toward the promoter, a process that is in agreement with a recently proposed 'facilitated tracking' hypothesis. This model assumes that the enhancer-bound complex tracks via small steps along the chromatin until it encounters the cognate promoter, at which stage a stable looped structure is formed. Important components of the tracking complex are the histone acetyltransferases CBP and P/CAF. These proteins may modify the chromatin as they move along the DNA. The unidirectional spreading of H3 and H4 hyperacetylation from the HNF-4alpha enhancer toward the promoter in a temporally identical manner to CBP and P/CAF occupancy demonstrates that this is indeed the case. Cooperation between histone acetyltransferases and ATP-dependent chromatin remodeling complexes has been proposed to be important for gene activation. The presence of Brg-1, a catalytic subunit of the human SWI/SNF complex, in the tracking complex provides important clues with respect to the dynamics of the process. The coordinated action of the acetylases and Brg-1 should lead to the acetylation of the histone tails of the neighboring nucleosome, which in turn would create a new interaction surface for the bromodomains of Brg-1, CBP, and P/CAF. This would facilitate the propagation of the complex to the next nucleosome, thus creating sequential signals for a stepwise process, powered by the ATP-ase activity of Brg-1. An important characteristic of the HNF-4alpha enhancer tracking is its unidirectional path. Although the results of this work do not provide an answer for the question of how this one-way course is controlled, it is speculated that sequences upstream of the HNF-4alpha enhancer may act as insulators that block the movement of the enhancer complex toward the opposite direction (Hatzis, 2003).

At the proximal promoter, ChIP signals of enhancer-recruited factors (HNF-1alpha, C/EBPalpha, HNF-3ß, CBP, P/CAF, and Brg-1) were first observed at 60 hr of the differentiation program, with an increased intensity at 80 and 110 hr. Concurrently, immunoprecipitates of proximal promoter-associated proteins (HNF-6, TFIIA, TFIIB, TBP, TAF1, TAF10, TFIIH, TRAP-220, and pol-II) contained DNA fragments corresponding to the enhancer region. In the experimental system employed, the simultaneous presence of the two DNA fragments in immunoprecipitates of this variety of factors demonstrates that the two regions come into close proximity to form a higher order complex by looping out the intervening DNA. This stable enhancer-promoter complex formation coincides with promoter hyperacetylation, phosphorylation of pol-II at the serine 2 position of its carboxy-terminal domain, remodeling of the nucleosome located at the transcription start site and finally with the release of pol-II from the promoter (Hatzis, 2003).

In summary, these results on HNF-4alpha enhancer-mediated activation demonstrate a dynamic mechanism, which accounts for many features of long-distance gene regulation that have been described for other genes. The ability to dissect the process to at least four temporally separable steps, all of which could be influenced by physiological signals, further emphasizes the complexity of the pathways that have evolved to regulate differential gene expression (Hatzis, 2003).

The hepatocyte nuclear factor (HNF) 4alpha gene possesses two promoters, proximal P1 and distal P2, whose use results in HNF4alpha1 and HNF4alpha7 transcripts, respectively. Both isoforms are expressed in the embryonic liver, whereas HNF4alpha1 is almost exclusively in the adult liver. A 516-bp fragment, encompassing a DNase I-hypersensitive site associated with P2 activity that is still retained in adult liver, contains functional HNF1 and HNF6 binding sites and confers full promoter activity in transient transfections. A critical role of the Onecut factors in P2 regulation has been demonstrated using site-directed mutagenesis and embryos doubly deficient for HNF6 and OC-2 that show reduced hepatic HNF4alpha7 transcript levels. Transient transgenesis shows that a 4-kb promoter region is sufficient to drive expression of a reporter gene in the stomach, intestine, and pancreas, but not the liver, for which additional activating sequences may be required. Quantitative PCR analysis has revealed that throughout liver development HNF4alpha7 transcripts are lower than those of HNF4alpha1. HNF4alpha1 represses P2 activity in transfection assays and as deduced from an increase in P2-derived transcript levels in recombinant mice in which HNF4alpha1 has been deleted and replaced by HNF4alpha7. It is concluded that although HNF6/OC-2 and perhaps HNF1 activate the P2 promoter in the embryo, increasing HNF4alpha1 expression throughout development causes a switch to essentially exclusive P1 promoter activity in the adult liver (Briancon, 2004).

HNF4 and erythropoiesis

The erythropoietin (Epo) gene is regulated by hypoxia-inducible cis-acting elements in the promoter and in a 3' enhancer, both of which contain consensus hexanucleotide hormone receptor response elements that are important for function. A group of 11 orphan nuclear receptors, transcribed and translated in vitro, were screened by the electrophoretic mobility shift assay. Of these, hepatic nuclear factor 4 (HNF-4), TR2-11, ROR alpha 1, and EAR3/COUP-TF1 were shown to bind specifically to the response elements in the Epo promoter and enhancer and, except for ROR alpha 1, form DNA-protein complexes that have mobilities similar to those observed in nuclear extracts of the Epo-producing cell line Hep3B. Moreover, both anti-HNF-4 and anti-COUP antibodies are able to supershift complexes in Hep3B nuclear extracts. Like Epo, HNF-4 is expressed in kidney, liver, and Hep3B cells but not in HeLa cells. Transfection of a plasmid expressing HNF-4 into HeLa cells enables an eightfold increase in the hypoxic induction of a luciferase reporter construct that contains the minimal Epo enhancer and Epo promoter, provided that the nuclear hormone receptor consensus DNA elements in both the promoter and the enhancer are intact. The augmentation by HNF-4 in HeLa cells can be abrogated by cotransfection with HNF-4 delta C, which retains the DNA binding domain of HNF-4 but lacks the C-terminal activation domain. Moreover, the hypoxia-induced expression of the endogenous Epo gene is significantly inhibited in Hep3B cells stably transfected with HNF-4 delta C. In contrast, cotransfection of EAR3/COUP-TF1 and the Epo reporter either with HNF-4 into HeLa cells or alone into Hep3B cells suppresses the hypoxia induction of the Epo reporter. These electrophoretic mobility shift assay and functional experiments indicate that HNF-4 plays a critical positive role in the tissue-specific and hypoxia-inducible expression of the Epo gene, whereas the COUP family has a negative modulatory role (Galson, 1995).

The cytokine erythropoietin (Epo) promotes erythropoietic progenitor cell proliferation and is required for erythropoietic differentiation. The primary physiological regulator of Epo expression in late embryos and in postnatal stages is oxygen tension. A mostly unknown hypoxia sensing mechanism results in the activity of the transcription factor HIF1 (hypoxia-inducible factor 1), which binds to a defined sequence in the 3' enhancer of the Epo gene and initiates Epo expression. In the fetal liver, Epo is expressed primarily by hepatocytes, a property which is conserved in hepatocellular carcinoma cell lines such as Hep3B and HepG2, in which Epo expression is induced in response to hypoxia. Adjacent to the HIF1-binding site in the mouse Epo 3' enhancer is the sequence TGACCTCTTGACCC, which is known as a DR2 element because of the direct repeat of the hexameric sequence TGACC(C/T) spaced by two nucleotides. The Epo enhancer DR2 element substantially augments hypoxic induction of Epo gene reporter constructs in transfected Hep3B cells, but is not itself responsible for responding to hypoxia. HNF4 is currently believed to be the primary factor that is responsible for Epo gene regulation through the DR2 element. HNF4 is expressed in the fetal liver and postnatal kidney, the two major sites of Epo expression, and introduction of an HNF4 expression construct in transfected HeLa cells (which do not normally express HNF4) confers hypoxic inducibility to an Epo reporter gene. HNF4 appears to function synergistically with HIF1 on the Epo enhancer by direct protein-protein interaction and through the recruitment of transcriptional coactivators (Makita, 2001 and references therein).

The Epo gene is a direct transcriptional target gene of retinoic acid signaling during early erythropoiesis (prior to embryonic day E12.5) in the fetal liver. Mouse embryos lacking the retinoic acid receptor gene RXRalpha have a morphological and histological phenotype that is comparable with embryos in which the Epo gene itself has been mutated, and flow cytometric analysis indicates that RXRalpha-deficient embryos are deficient in erythroid differentiation. Epo mRNA levels are reduced substantially in the fetal livers of RXRalpha^-/- embryos at E10.25 and E11.25, and genetic analysis shows that the RXRalpha and Epo genes are coupled in the same pathway. The Epo gene is shown to be retinoic acid inducible in embryos, and the Epo gene enhancer contains a DR2 sequence that represents a retinoic acid receptor-binding site and a retinoic acid receptor transcriptional response element. However, unlike Epo-deficient embryos that die from anemia, the erythropoietic deficiency in RXRalpha^-/- embryos is transient; Epo mRNA is expressed at normal levels by E12.5, and erythropoiesis and liver morphology are normal by E14.5. HNF4, like RXRalpha a member of the nuclear receptor family, is abundantly expressed in fetal liver hepatocytes, and is competitive with retinoic acid receptors for occupancy of the Epo gene enhancer DR2 element. It is proposed that Epo expression is regulated during the E9.5-E11.5 phase of fetal liver erythropoiesis by RXRalpha and retinoic acid, and that expression then becomes dominated by HNF4 activity from E11.5 onward. This transition may be responsible for switching regulation of Epo expression from retinoic acid control to hypoxic control, as is found throughout the remainder of life (Makita, 2001).

Expansion of adult β-cell mass in response to increased metabolic demand is dependent on HNF-4α

The failure to expand functional pancreatic β-cell mass in response to increased metabolic demand is a hallmark of type 2 diabetes. Lineage tracing studies indicate that replication of existing β-cells is the principle mechanism for β-cell expansion in adult mice. This study demonstrates that the proliferative response of β-cells is dependent on the orphan nuclear receptor hepatocyte nuclear factor-4α (HNF-4α), the gene that is mutated in Maturity-Onset Diabetes of the Young 1 (MODY1). Computational analysis of microarray expression profiles from isolated islets of mice lacking HNF-4α in pancreatic β-cells reveals that HNF-4α regulates selected genes in the β-cell, many of which are involved in proliferation. Using a physiological model of β-cell expansion, it is shown that HNF-4α is required for β-cell replication and the activation of the Ras/ERK signaling cascade in islets. This phenotype correlates with the down-regulation of suppression of tumorigenicity 5 (ST5) in HNF-4α mutants, which is identified as a novel regulator of ERK phosphorylation in β-cells and a direct transcriptional target of HNF-4α in vivo. Together, these results indicate that HNF-4α is essential for the physiological expansion of adult β-cell mass in response to increased metabolic demand (Gupta, 2007).

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