InteractiveFly: GeneBrief

Hepatocyte nuclear factor 4 Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

A Drosophila gene, Hnf4, was selected by cross-hybridization with a probe to rat HNF4 (hepatocyte nuclear factor 4), a member of the steroid hormone receptor superfamily that plays an important role in liver-specific gene expression. Drosophila Hnf4 is transcribed maternally, and disappears soon after cellularization. It reappears zygotically, first in the posterior midgut and subsequently in the anterior midgut, salivary glands and at the growing tip of the Malpighian tubules. Although early gut development occurs normally, Drosophila mutants in Hnf4 show defects in midgut, Malpighian tubule and salivary gland development. HNF4 mRNA is distributed in a different pattern from that of Forkhead, the Drosophila homolog of the vertebrate Forkhead domain protein HNF3. Whereas forkhead is involved in early gut invagination and is expressed most strongly in the ectodermal hindgut and foregut, Drosophila Hnf4 transcription is involved in gut endoderm determination and is confined to the endodermal midgut (Zhong, 1993).

With such limited information, many questions may be asked, but few answers are currently available. Most of the unanswered questions arise from work on the vertebrate homolog of Drosophila HNF4. Recent experiments show strong parallels between Drosophila and vertebrate HNF4 biology; it is these parallels that raise the yet to be answered questions.

Xenopus HNF4 is present as a maternal protein and accumulates in growing oocytes. Xenopus HNF4 protein distributes in an animal to vegetal gradient in the embryo. This distribution remains through the 64 cell stage at which time a proportion of the protein becomes transported to the nucleus. Transcripts are absent in early embryos but become detectable in the early gastrula, when zygotic transcription has started, and accumulate during further development. Xenopus HNF4 is involved in the activation of an endodermal specific transcription factor HNF1alpha, a homeoprotein (Holewa, 1996).

Activation of HNF1alpha early in Xenopus development is via an HNF4-binding site. This HNF4 binding site is identified as an activin A responsive element in the Xenopus HNF1alpha promoter. This means that activin A signaling, apparently through Tgfß type 1 and type 2 receptors, is required for HNF4 to activate the HNF1alpha promoter. One possibility for activation of Xenopus HNF4 might be its post-transcriptional modification (Weber, 1996). The activation of HNF4 by phosphorylation makes sense as tyrosine phosphorylation of HNF4 is required for DNA binding and consequently for activation of HNF4 in cell-free systems as well as in cultured mammalian cells (Ktistaki, 1995).

The existence of maternal Xenopus HNF4 and Drosophila Hnf4 raises the question of the functions these transcription factors carry out early in development. The Xenopus protein may play a role in initiating a transcriptional hierarchy involved in the determination of endoderm. What is the role of maternally coded Drosophila Hnf4 early in development, and is there any similarity to the presumed role of the Xenopus protein? Might activation of Drosophila Hnf4 be one of the early events signaling activation of the egg following fertilization? If so, does its function depend of phosphorylation, is it a target of Decapentaplegic (a TGFß family member) signaling, and what are its targets?

Murine HNF4 is expressed in the visceral endoderm as early as 4.5 days after fertilization, during implantation and well before gastrulation (Duncan, 1994). Disruption of that gene leads to cell death in the embryonic ectoderm at 6.5 days of development, at a time when these cells normally initiate gastrulation. The result is impaired gastrulation of mouse embryos. As assessed by expression of Brachyury and HNF3ß, primitive streak formation and initial differentiation of mesoderm do occur, but are delayed by about 24 hours. This work shows that expression of murine HNF4 in the visceral endoderm is essential for embryonic ectoderm survival and normal gastrulation (Chen, 1994).

What place does zygotic Drosophila Hnf4 transcription have in the hierarchy of gene activation in the endoderm? Is it directly activated by Huckebein, or is it downstream of Serpent? Serpent is responsible for the repression of forkhead in the midgut (Reuter, 1994). Does Serpent induce Hnf4, or is Hnf4 transcription independent of Serpent? Similar questions can be asked for the murine HNF4. Where does it lie in the early developmental hierarchy of the endoderm? Resolution of these questions would go a long way to resolving the origin of vertebrate endoderm. Also, what are the interactions in mouse development that result in ectodermal cell death in murine HNF4 deficient embryos? There must be a signaling pathway between endoderm and ectoderm, but the existence of such a pathway is currently undocumented.

Drosophila HNF4 regulates lipid mobilization and β-oxidation

Drosophila HNF4 (dHNF4) is the single ancestral ortholog of a highly conserved subfamily of nuclear receptors that includes two mammalian receptors, HNFalpha and HNFgamma, and 269 members in C. elegans. dHNF4 null mutant larvae are sensitive to starvation. Starved mutant larvae consume glycogen normally but retain lipids in their midgut and fat body and have increased levels of long-chain fatty acids, suggesting that they are unable to efficiently mobilize stored fat for energy. Microarray studies support this model, indicating reduced expression of genes that control lipid catabolism and beta-oxidation. A GAL4-dHNF4;UAS-lacZ ligand sensor can be activated by starvation or exogenous long-chain fatty acids, suggesting that dHNF4 is responsive to dietary signals. Taken together, these results support a feed-forward model for dHNF4, in which fatty acids released from triglycerides activate the receptor, inducing enzymes that drive fatty acid oxidation for energy production (Palanker, 2009).

The presence of multiple HNF4 family members in mice and C. elegans complicates understanding of the physiological functions of this nuclear receptor subclass. This study characterized the single ancestral HNF4 in Drosophila, demonstrating its critical role in regulating the adaptive response to starvation. The results support a feed-forward model for dHNF4 function in which fatty acids freed from triglycerides activate the receptor, inducing the expression of enzymes that drive lipid mobilization and β-oxidation for energy production (Palanker, 2009).

Remarkably, the expression pattern of HNF4 has been conserved through evolution, from flies to mammals, with abundant dHNF4 expression in the midgut, fat body, and Malpighian tubules -- tissues that are the functional equivalents of the intestine, liver, and kidney, respectively, where mammalian HNF4 is expressed. The only exception is a lack of dHNF4 expression in the median neurosecretory cells that produce insulin-like peptides (IPCs), the functional equivalent of mammalian pancreatic β cells. dHNF4 is also expressed in the oenocytes, which have hepatocyte-like properties and contribute to lipid mobilization. The starvation sensitivity of dHNF4 mutants, however, cannot be rescued by expression of wild-type dHNF4 in the oenocytes, and starvation-induced lipid accumulation occurs normally in the oenocytes of dHNF4 mutants, leaving it unclear what role, if any, dHNF4 plays in these cells (Palanker, 2009).

Although dHNF4 mutants can survive to adulthood under ideal culture conditions, significant defects become evident upon food deprivation, including an inability to efficiently mobilize stored lipid in the midgut and fat body, increased levels of (long-chain fatty acid) LCFAs and triglycerides (TAG), and premature death. This retention of lipid is similar to the accumulation of lipids seen in the guts of nhr-49 mutant worms and the steatosis seen in liver-specific HNF4α mutant mice (Hayhurst, 2001; Van Gilst, 2005). The ability of dHNF4 mutant larvae to survive on a sugar diet indicates that glycolysis and oxidative phosphorylation are intact in these animals. Rather, starved dHNF4 mutant larvae appear to die from an inability to break down TAG and use the resulting free fatty acids for energy production via β-oxidation. Similar phenotypes are associated with defects in β-oxidation in mice and humans, which result in sensitivity to starvation, accumulation of lipid in the liver, and increased levels of free fatty acids. β-oxidation takes place in the mitochondria or peroxisomes of most organisms, with very long-chain fatty acids (VLCFAs) as the substrate for the peroxisomal pathway. Although peroxisomes are present in the gut and Malpighian tubules of Drosophila adults and VLCFAs accumulate in a VLCFA acyl-CoA synthase mutant, the existence of peroxisomes in Drosophila larvae remains unclear (Palanker, 2009).

Like mammalian HNF4α, dHNF4 mRNA is significantly upregulated in response to starvation. In addition, many genes in the β-oxidation pathway are upregulated upon starvation and display significantly reduced expression in dHNF4 mutant larvae. These include acetyl-CoA synthetase (AcCoAs), γ-butyrobetaine dioxygenases, and carnitine acyl transferases, the rate-limiting step in acyl import into mitochondria. In addition, genes that encode the four enzymatic steps of β-oxidation are all dependent on dHNF4 for their proper level of expression. Importantly, this effect is also seen in fed dHNF4 mutant larvae, where only 86 transcripts change ≥ 1.3-fold compared to wild-type, many of which encode components of the β-oxidation pathway. These include yip2, Acox57D-d, thiolase, scully, and CPTI (carnitine palmitoyltransferase) among the most highly downregulated genes in fed dHNF4 mutants (1.8- to 4-fold as determined by dChip). Thus, dHNF4 is required for both basal and starvation-induced β-oxidation gene transcription. Moreover, many of the genes in this pathway have at least one canonical binding site for an HNF4 homodimer within 1 kb of their predicted 5' end, suggesting that dHNF4 directly regulates their transcription (Palanker, 2009).

The effects of dHNF4 are not, however, restricted to lipid oxidation. Three predicted lipase transcripts, lip3, CG6295, and CG2772, are significantly reduced in starved dHNF4 mutants compared to starved controls, suggesting a direct role for the receptor in the release of LCFAs from TAG. Similarly, the CGI-58 homolog encoded by CG1882, which activates lipolysis in mammals, is upregulated in starved dHNF4 mutants, whereas adipokinetic hormone receptor (AKHR) is downregulated, consistent with its normal role in mobilizing stored lipids analogous to mammalian β-adrenergic signaling. No effect, however, is seen on expression of brummer, which encodes adipose triglyceride lipase, suggesting that dHNF4 does not have a direct impact on brummer-mediated lipolysis. A VLCFA-CoA ligase gene (bubblegum) and LCFA-CoA ligase gene (CG6178) are also affected in starved dHNF4 mutants. These enzymes activate free fatty acids for either catabolic or anabolic processes. Effects were also seen on lipid synthesis, with downregulation of a predicted fatty acid synthase (CG3523), a predicted acetyl-CoA carboxylase (CG11198), and two predicted glycerol-3-phosphate acyltransferases (GPATs) (CG3209 and CG17608) in starved dHNF4 mutants. Two predicted stearoyl-CoA desaturase genes, CG15531 and desat1, which catalyze the production of monounsaturated fatty acids, are also downregulated in the mutant. A similar result has been reported for NHR-49, where a stearoyl-CoA desaturase gene is a key functional target of the receptor. The glyoxylate pathway is also affected in nhr-49 mutants under both fed and starved conditions. This pathway is analogous to mammalian ketogenesis, in which fatty acid β-oxidation products are used for energy production. Genes for the central enzymes in this pathway, malate synthase and isocitrate lyase, have not yet been identified in D. melanogaster. However, CG12208, which is predicted to contribute to glyoxylate catabolism, is downregulated in both fed and starved dHNF4 mutants. Taken together, these effects on transcription define a central role for dHNF4 in balancing lipid anabolic and catabolic pathways in response to dietary conditions (Palanker, 2009).

Although genome-wide ChIP suggests that HNF4α is a promiscuous regulator of many actively transcribed genes in pancreatic islets and hepatocytes, relatively few targets have been identified in functional studies. These include multiple apolipoprotein genes, NTCP, and L-FABP, indicating roles in very low-density lipoprotein secretion, high-density lipoprotein synthesis, and bile acid homeostasis. Only a few β-oxidation genes have been studied in HNF4α mutant mice, with contradictory results. HNF4α can bind to the promoters of liver CPT-I and MCAD (which encodes an acyl-CoA dehydrogenase), and CPT-I mRNA levels are reduced in HNF4α mutant livers. In contrast, other studies show that CPT-I, CPT-II, and MCAD transcripts are elevated in HNF4α mutant livers. Given the current results, it would be interesting to resolve these contradictory observations and more broadly examine other steps in the β-oxidation pathway in HNF4α mutant mice (Palanker, 2009).

Although dHNF4 is widely expressed in the larval midgut, its activity appears to be spatially restricted, primarily to a band of cells at the anterior end of the midgut that lie at the base of the gastric caeca. This is the primary site for nutrient absorption into the circulatory system, suggesting that dHNF4 is responsive to dietary signals. In the fat body, the dHNF4 ligand sensor is inactive during most of larval development, when fat storage is favored over fat breakdown, but becomes activated at the end of larval growth, when lipolysis and autophagy are initiated in the fat body. Similarly, a significant increase in GAL4-dHNF4 activation is seen in the fat body of starved larvae, concurrent with the increase in fatty acid β-oxidation that is required to survive these conditions. Consistent with this model, the dHNF4 LBD can be activated by ectopic bmm expression or exogenous LCFAs in cultured larval organs. The most effective LCFAs, palmitic acid (C16:0) and oleic acid (C18:1), are relatively abundant in Drosophila larvae and are the primary constituents of triglycerides that are hydrolyzed upon starvation, making them logical signaling intermediates. In addition, the dHNF4 LBD copurifies with palmitic acid, indicating that fatty acid binding has been conserved through evolution. Two different point mutations that change conserved amino acids, each of which is predicted to disrupt dHNF4 fatty acid binding, block the ability of the LBD to respond to starvation or exogenous fatty acids, suggesting that direct fatty acid binding is essential for dHNF4 transcriptional activity (Palanker, 2009).

Taken together, these studies support a feed-forward model in which dHNF4 functions as a sensor for free fatty acid levels in the larva, driving their catabolism through β-oxidation. Upon nutrient deprivation, TAG is hydrolyzed into free LCFAs. As has been shown in mammalian cells, it is likely that these cytosolic LCFAs can translocate into the nucleus, activating dHNF4. This, in turn, leads to the transcriptional induction of key genes involved in TAG breakdown, acyl-CoA production, acyl import into mitochondria, and β-oxidation. The net result of this response is consumption of the dHNF4-activating signal (LCFAs), maintaining energy homeostasis through ATP production. This feed-forward model is consistent with the phenotypes of dHNF4 mutants, which are starvation sensitive and accumulate TAG and LCFAs (Palanker, 2009).

Crystal structure studies of mammalian HNF4α have shown that the fatty acid-bound form of the LBD can assume both open and closed positions, with helix 12 either extended or held against the body of the LBD, whereas the HNF4γ LBD appears to be locked in the open configuration. The current data, however, indicate that the Drosophila HNF4 LBD is responsive to the nutritional status of the animal and can be activated by exogenous LCFAs. Although it is possible that fatty acids act as exchangeable ligands for dHNF4 in vivo, the current model is also consistent with a role for fatty acid binding in constitutive dHNF4 transcriptional activation. dHNF4 mRNA is significantly induced upon starvation. The resultant newly synthesized protein could act as a sensor for free fatty acids, binding those molecules and thereby forming active dHNF4 transcription complexes. In contrast, dHNF4 protein that is synthesized during stages with less metabolic demand would have access to lower free fatty acid levels and thus remain less active. This model is consistent with ligand sensor studies, in which newly synthesized GAL4-dHNF4 LBD is inactive in the presence of abundant nutrients and is highly active upon food deprivation. This work sets the stage for biochemical studies that address the mechanisms of dHNF4 activation in vivo and whether protein turnover, posttranslational modification, and/or differential cofactor binding modulate the activation of dHNF4 by LCFAs. It also raises the interesting possibility that vertebrate HNF4 may function as a fatty acid sensor (Palanker, 2009).

The feed-forward model for dHNF4 function is reminiscent of that ascribed to a mammalian nuclear receptor, PPARα. PPARα binds LCFAs and directly regulates genes that are orthologous to dHNF4 targets, including many genes in the β-oxidation pathway. PPARα mutant mice are defective in the adaptive response to starvation and display increased plasma free fatty acids and fatty liver with enlarged hepatocyte lipid droplets -- phenotypes shared with dHNF4 mutants. A similar conclusion has been reached in a characterization of C. elegans nhr-49 mutants. There are no orthologs of the PPAR nuclear receptor subclass in lower organisms, including Drosophila and worms. This is in spite of the fact that PPARα functions (the ability to sense nutrient deprivation and mobilize stored forms of energy to maintain homeostasis) are essential for animal survival. Indeed, even unicellular fungi have an analogous pathway, where the Oaf1/Pip2 transcription factor heterodimer binds the fatty acid oleate and regulates peroxisomal β-oxidation. It is proposed that the ancestral function of HNF4 has been adopted by PPARα during the course of evolution. These studies also indicate additional functions for dHNF4 beyond lipid mobilization that provide possible directions for future research. In addition, phenotypic characterization of dHNF4 mutants under different environmental and dietary conditions may provide new insights into dHNF4 activities and raise new implications for the regulation and function of its vertebrate counterparts (Palanker, 2009).

Sir2 acts through Hepatocyte Nuclear Factor 4 to maintain insulin signaling and metabolic homeostasis in Drosophila

SIRT1 is a member of the sirtuin family of NAD+-dependent deacetylases, which couple cellular metabolism to systemic physiology. This study shows that loss of the Drosophila SIRT1 homolog sir2 leads to the age-progressive onset of hyperglycemia, obesity, glucose intolerance, and insulin resistance. Tissue-specific functional studies show that Sir2 is both necessary and sufficient in the fat body to maintain glucose homeostasis and peripheral insulin sensitivity. This study reveals a major overlap with genes regulated by the nuclear receptor Hepatocyte Nuclear Factor 4 (HNF4). Drosophila HNF4 mutants display diabetic phenotypes similar to those of sir2 mutants, and protein levels for dHNF4 are reduced in sir2 mutant animals. Sir2 exerts these effects by deacetylating and stabilizing dHNF4 through protein interactions. Increasing dHNF4 expression in sir2 mutants is sufficient to rescue their insulin signaling defects, defining this nuclear receptor as an important downstream effector of Sir2 signaling. This study provides a genetic model for functional studies of phenotypes related to type 2 diabetes and establishes HNF4 as a critical downstream target by which Sir2 maintains metabolic health (Palu, 2016).

This study shows that sir2 mutants display a range of metabolic defects that parallel those seen in mouse Sirt1 mutants, including hyperglycemia, lipid accumulation, insulin resistance, and glucose intolerance. These results suggest that the fundamental metabolic functions of Sirt1 have been conserved through evolution and that further studies in Drosophila can be used to provide insights into its mammalian counterpart. An additional parallel with Sirt1 is seen in tissue-specific studies, where sir2 function is shown to be necessary and sufficient in the fat body to maintain insulin signaling and suppress hyperglycemia and obesity, analogous to the role of Sirt1 in the liver and white adipose. These results are also consistent with published studies of insulin sensitivity in Drosophila, which have shown that the fat body is the critical tissue that maintains glucose and lipid homeostasis through its ability to respond properly to insulin signaling (Palu, 2016).

These studies also define the dHNF4 nuclear receptor as a major target for Sir2 regulation. Consistent with this, dHNF4 mutants display a range of phenotypes that resemble those of sir2 mutants, including hyperglycemia, obesity, and glucose intolerance. As expected, these defects are more severe in dHNF4 loss-of-function mutants, consistent with sir2 mutants only resulting in a partial loss of dHNF4 protein. Sir2 interacts with dHNF4 and appears to stabilize this protein through deacetylation. This is an established mechanism for regulating protein stability, either through changes in target protein conformation that allow ubiquitin ligases to bind prior to proteasomal degradation, or through alternate pathways. Further studies, however, are required to determine if this is a direct protein-protein interaction or part of a higher order complex (Palu, 2016).

Although two papers have shown that mammalian Sirt1 can control HNF4A transcriptional activity through a protein complex, only one gene was identified as a downstream target of this regulation, PEPCK, leaving it unclear if this activity is of functional significance. The current study suggests that this regulatory connection is far more extensive. The observation that one third of the genes down-regulated in sir2 mutants are also down-regulated in dHNF4 mutants (including pepck), and most of the genes up-regulated in sir2 mutants are up-regulated in dHNF4 mutants, establishes this nuclear receptor as a major downstream target for Sir2 regulation. It will be interesting to determine if the extent of this regulatory connection has been conserved through evolution (Palu, 2016).

Despite this regulatory control, the over-expression of an HNF4 transgene was only able to partially restore the insulin signaling response and not the defects in carbohydrate homeostasis in sir2 mutants. This lack of complete rescue is not surprising, given that the Sirt1 family targets a large number of transcription factors, histones, and enzymes, providing multiple additional pathways for metabolic regulation. Moreover, the activity or target recognition of dHNF4 may be altered when it is hyperacetylated, in which case merely over-expressing this factor would not fully restore normal function. Future studies can examine more direct targets, both previously characterized and uncharacterized, for their functions in suppressing diabetes downstream of Sir2-dependent regulation (Palu, 2016).

Finally, sir2 mutants represent a new genetic model for studying the age-dependent onset of phenotypes related to type 2 diabetes. Newly-eclosed sir2 mutant adults are relatively healthy, with elevated levels of free glucose and glycogen but otherwise normal metabolic functions. Their health, however, progressively worsens with age, with two-week-old sir2 mutants displaying lipid accumulation, fasting hyperglycemia, and reduced insulin signaling accompanied by insulin resistance. This is followed by the onset of glucose intolerance by three weeks of age. Previous studies of type 2 diabetes in Drosophila have relied on dietary models using wild-type animals that are subjected to a high sugar diet. Although this is a valuable approach to better define the critical role of diet in diabetes onset, it is also clear that the likelihood of developing type 2 diabetes increases with age. The discovery that sir2 mutants display this pathophysiology provides an opportunity to exploit the power of Drosophila genetics to better define the mechanisms that lead to the stepwise onset of metabolic dysfunction associated with diabetes (Palu, 2016).

Drosophila HNF4 directs a switch in lipid metabolism that supports the transition to adulthood

Animals must adjust their metabolism as they progress through development in order to meet the needs of each stage in the life cycle. This study shows that the dHNF4 nuclear receptor acts at the onset of Drosophila adulthood to direct an essential switch in lipid metabolism. Lipid stores are consumed shortly after metamorphosis but contribute little to energy metabolism. Rather, dHNF4 directs their conversion to very long chain fatty acids and hydrocarbons, which waterproof the animal to preserve fluid homeostasis. Similarly, HNF4alpha is required in mouse hepatocytes for the expression of fatty acid elongases that contribute to a waterproof epidermis, suggesting that this pathway is conserved through evolution. This developmental switch in Drosophila lipid metabolism promotes lifespan and desiccation resistance in adults and suppresses hallmarks of diabetes, including elevated glucose levels and intolerance to dietary sugars. These studies establish dHNF4 as a regulator of the adult metabolic state (Storelli, 2018).

Drosophila breeds and feeds on rotting fruits, which are ephemeral ecosystems. Thus, while the Drosophila larva lives inside a semi-liquid nutritive substrate and feeds extensively to support its rapid increase in mass, the mature adult fly emerges into an unpredictable environment where it uses flight for dispersal and the colonization of more favorable niches. Similar to all insects, the large surface-to-volume ratio of Drosophila adults makes them vulnerable to dehydration. In addition, the metabolic demands of flight require a high rate of gas exchange via the respiratory system, increasing water loss. This study shows that the successful transition from a stationary and protected pupa to a motile adult that can survive in a dry environment is dependent on the dHNF4 nuclear receptor, which acts in the oenocytes to direct a transcriptional program that supports the rapid production of very long-chain fatty acids (VLCFAs) and cuticular hydrocarbons. This activity is consistent with earlier studies, which have demonstrated an important role for oenocytes in VLCFA/hydrocarbon synthesis and desiccation resistance, and places dHNF4 as a central transcriptional regulator for oenocyte function. This study shows, however, that whole-body and hemolymph TAG levels drop to low levels between emergence and day 3 of adulthood, on either a sugar-free diet or a rich 15% sugar diet. The lipids contained in larval adipose cells thus appear to be consumed independently of nutritional input, suggesting that they contribute to non-energetic functions. Moreover, control adults can survive for weeks in the absence of stored lipids, further indicating that they provide a relatively minor source of energy at this stage in development (Storelli, 2018).

It is proposed that lipids in the newly emerged adult are shunted toward a purpose that is more profound than energy production at this stage in development -- the formation of a waterproof cuticle -- and that this occurs in a dHNF4-dependent manner. Stored lipid levels are unaffected in newly emerged dHNF4 mutant adults relative to controls. Lipids, however, accumulate rapidly after this stage in oenocytes, in the presence or absence of nutrients, representing a developmental role for dHNF4 in maintaining lipid homeostasis in this cell type. The timing of lipid accumulation in the oenocytes of starved and fed dHNF4 mutants coincides with the timing of lipid loss and larval adipose cell death. Genetic studies demonstrate that dHNF4 is specifically required in oenocytes to suppress developmental steatosis. This appears to be due to its role in inducing the expression of genes involved in VLCFA elongation. Oenocyte-specific silencing of KAR or ACC recapitulates the developmental steatotic phenotype, while silencing genes that encode downstream or upstream steps in the hydrocarbon biosynthetic pathway has no effect on the lipid levels in oenocytes. Further studies are needed to determine the molecular mechanisms that link VLCFA elongation with oenocyte lipid homeostasis. Finally, adults normally emerge with low hydrocarbon levels that increase dramatically over 3 days, even in the absence of food, in agreement with the hypothesis that the larval adipose cells provide the primary source of precursors for hydrocarbon production. These results are consistent with previous reports, which have shown that dietary manipulation during larval life affects the blend of hydrocarbons in young adult. Consistent with this, oenocyte-specific dHNF4 RNAi in mature adults was shown to lead to a similar degree of lipid accumulation as that seen upon inducing RNAi at emergence. Further studies are required to address this continuing role for dHNF4 in mature adults. In addition, the mechanisms that partition stored lipids toward VLCFA/hydrocarbon production or energy production remain to be determined. Finally, the key functions described for dHNF4 in oenocytes are shared between males and females, indicating that they are not sex specific (Storelli, 2018).

Studies of starvation resistance indicate that newly emerged Drosophila adults can tolerate the absence of nutrients for at least a week. During the non-feeding period of metamorphosis, pupae consume about half of their glycogen and TAG stores and most of the trehalose gathered during the larval stages. In spite of this depletion of energy reserves, sufficient nutrients persist to allow the newly emerged adult to survive and disperse in their new environment. Under hydrated conditions, control lines emerge with the ability to survive from 1 to 3 weeks in the absence of nutrients. It is suggested that these conditions provide insufficient humidity to accurately distinguish the effects of dehydration from the effects of nutrient depletion. Without moisture or food, Drosophila die in less than a day, with a median lifespan of 13 hr. Thus, as is well known in mammals, sufficient hydration is critical for assessing the ability of animals to properly mobilize stored nutrients for survival during starvation (Storelli, 2018).

Changes in metabolism must accompany each stage in development in order to allow normal progression through the life cycle. Although little is known about how development and metabolism are coupled, nuclear receptors appear to play a central role in this process. Consistent with this, a previous study of dHNF4 showed that it is highly up-regulated at the end of metamorphosis as the fly begins its adult life, coordinately inducing genes involved in glucose homeostasis and oxidative phosphorylation (Barry, 2016). Functional studies showed that dHNF4 acts at this stage in the insulin-producing cells to maintain glucose-stimulated insulin secretion and acts in the fat body to promote glucose clearance. This study expands upon these activities for dHNF4 at the onset of adulthood to include an essential role in supporting VLCFA and hydrocarbon production in the oenocytes to allow adult survival and dispersion (Storelli, 2018).

Key genes involved in VLCFA/hydrocarbon production are induced at the onset of adulthood in control adults and reduced in expression in dHNF4 mutants, including KAR, Cpr, Cyp4g1, and several genes encoding predicted elongases. In addition, dHNF4 transcriptional activity can be activated by LCFAs and VLCFAs, which appear to act as ligands for this nuclear receptor. This is consistent with studies of mammalian HNF4α, which have identified LCFAs as ligands that can trigger the conformational changes required for coactivator recruitment. Thus, the free fatty acids generated by lipolysis from the larval adipose cells could act as ligands for dHNF4 in oenocytes, driving the transcription of genes in the VLCFA/hydrocarbon pathway. The resulting VLCFAs might further activate dHNF4, providing a feed-forward loop to enhance VLCFA/hydrocarbon production. These levels of regulation could ensure that free fatty acids are rapidly and efficiently converted into VLCFAs and hydrocarbons to provide an effective waterproof barrier for the young adult (Storelli, 2018).

Simple carbohydrates are required for optimal Drosophila longevity, yet dHNF4 and VLCFA/hydrocarbon biosynthesis are required in oenocytes to provide tolerance to these nutrients. In addition, increased humidity is sufficient to rescue sugar toxicity and hyperglycemia in dHNF4 mutants, suggesting that defects in fluid homeostasis are contributing to these phenotypes. Defects in fluid homeostasis could occur in dHNF4 mutants because of impaired waterproofing of the cuticle or the trachea. Parvy and colleagues demonstrated that, besides providing precursors for hydrocarbon synthesis, VLCFA metabolism in oenocytes is required to remotely waterproof the respiratory system in larvae. Notably, disrupting VLCFA production in oenocytes via ACC, KAR, or FASN^CG17374 RNAi in this cell type results in defects in tracheal waterproofing and larval lethality. Lethality between larval and pupal stages was observed when these RNAi constructs were driven with a constitutive oenocyte-specific GAL4 driver. However, defects in tracheal air filling or lethality are not seen in dHNF4 mutant larvae or in animals with constitutive dHNF4 RNAi expression in oenocytes, suggesting that there are no significant effects on tracheal waterproofing in these animals. In addition, altering cuticular hydrocarbon production while leaving VLCFA synthesis intact (by silencing Cyp4g1 in oenocytes) induces sugar toxicity and defects in glucose homeostasis. Thus it is postulated that reduced hydrocarbon production by oenocytes and altered cuticular waterproofing play central roles in the physiological defects observed in dHNF4 mutants (Storelli, 2018).

Understanding of diabetes in humans provides possible models to explain the physiological mechanisms that link dHNF4 activity in oenocytes to fluid and carbohydrate homeostasis. In mammals, excess blood glucose cannot be effectively reabsorbed by the kidney and is excreted with urine. One possibility is that dHNF4 mutants provide a sensitized genetic context for the development of a hyperosmolar hyperglycemic state in Drosophila. Increased transepidermal water loss could reduce hemolymph volume, contributing to the development of hyperglycemia on a sugar-containing diet. This hypothesis could explain the elevated glucose levels observed in dHNF4 mutants and the normalization of glucose levels in high humidity. Further studies are required to test this possibility and dissect the mechanisms that couple dHNF4 function in oenocytes to systemic carbohydrate homeostasis (Storelli, 2018).

A number of studies have focused on characterizing the signaling pathways that govern glucose sensing and metabolism in Drosophila. These studies have provided evidence for glucose-induced cellular damage via oxidative stress or the formation of advanced glycation end-products. In spite of these efforts, however, more remains to be learned about the mechanisms underlying dietary sugar toxicity. This work suggests unexpected roles for oenocyte lipid metabolism and hydration status in regulating glucose homeostasis and suppressing sugar toxicity in Drosophila (Storelli, 2018).

Interestingly, the role of VLCFAs in reducing trans-epidermal water loss is conserved through evolution. Similarly, mouse mutants for Elovl1 and Elovl4 are born normally but die shortly thereafter from acute dehydration, similar to dHNF4 mutant adults. This lethality is accompanied by reduced epithelial barrier function, defects in the lamellar structure of newborn skin, reduced levels of VLCFAs, and the absence of key acylceramides that contribute to skin hydrophobicity. Genetic studies of the gene encoding ELOVL3, which elongates C20 fatty acids to C22 and C24, revealed similar functions. Elovl3 mutant mice have disrupted skin and hair morphology resulting in increased trans-epidermal water loss. Taken together, these results indicate that VLCFA production is critical for proper lipid metabolism and survival after birth in mouse, revealing a perinatal transition that parallels the role for VLCFAs and hydrocarbons in newly emerged Drosophila adults. Interestingly, these studies also link Elovl gene expression to HNF4α function in mice. Elovl3, Elovl5, and KAR are all expressed at reduced levels in HNF4α mutant hepatocytes, indicating that this regulatory link is conserved through evolution. Further studies are required to determine if HNF4α exerts a similar role in mammalian skin and if its activity is required for epithelial barrier function and desiccation resistance (Storelli, 2018).

Molecular basis for autosomal-dominant renal Fanconi Syndrome caused by HNF4A

HNF4A is a nuclear hormone receptor that binds DNA as an obligate homodimer. While all known human heterozygous mutations are associated with the autosomal-dominant diabetes form MODY1, one particular mutation (p.R85W) in the DNA-binding domain (DBD) causes additional renal Fanconi syndrome (FRTS). This study finds that expression of the conserved fly ortholog HNF4 harboring the FRTS mutation in Drosophila nephrocytes caused nuclear depletion and cytosolic aggregation of a wild-type HNF4 reporter protein. While the nuclear depletion led to mitochondrial defects and lipid droplet accumulation, the cytosolic aggregates triggered the expansion of the endoplasmic reticulum (ER), autophagy, and eventually cell death. The latter effects could be fully rescued by preventing nuclear export through interfering with serine phosphorylation in the DBD. These data describe a genomic and a non-genomic mechanism for FRTS in HNF4A-associated MODY1 with important implications for the renal proximal tubule and the regulation of other nuclear hormone receptors (Marchesin, 2019).

HNF4A is specifically expressed in the kidney in proximal tubules and is required for the terminal differentiation of proximal tubular cells. However, out of all of the known mutations, only the R85W mutation causes FRTS. Using fly nephrocytes as a model for proximal tubules, this study shows that both dHNF4 KD and overexpression of the FRTS mutation (R167W in flies) causes mitochondrial alterations and LD accumulation. By using a dHNF4-GFP as a reporter protein, this study shows that the FRTS mutation provokes the nuclear export of the wild-type protomer in a dominant-negative manner, possibly explaining the similarity to the KD. The nuclear export is additionally associated with cytoplasmic aggregate formation, increased autophagy, severe ER morphology changes, and eventually cell death. All of these phenotypes are not seen in the KD, suggesting that they are the result of the aggregate formation. However, they could be observed when expressing wild-type dHNF4 at high levels, suggesting dominant-negative effects in this condition as well. For both dHNF4R167W/FRTS and dHNF4highOE, these effects could be rescued by the increase in nuclear levels through dephosphorylation at S169 (Marchesin, 2019).

Crucial for the understanding of the investigated mutations are structural studies of the HNF4A homodimer/DNA complex, in which the homodimer forms multiple domain-domain junctions and a convergence zone or 'nerve center,' in which the two LBDs, the upstream positioned DBD, and the hinge region meet. S87 maps precisely to this center, leading to unfavorable charge repulsion upon phosphorylation and disengagement of the quaternary structure needed for DNA binding. In another nuclear receptor, constitutive androstane receptor (CAR), the corresponding phosphorylation leads to the formation of inactive homodimers, providing support for this model. As R85 is in close contact with the DNA bases and backbone of the dipartite DR1 element in both upstream and downstream protomers of the homodimer, the lack of DNA binding in the R85W mutant may promote phosphorylation-dependent conversion of active to inactive homodimers and, finally, nuclear export of both mutant and wild-type protomers (Marchesin, 2019).

By contrast, the effects of the MODY mutation R171W resembled those of the phosphorylation-deficient S169A mutant, which is supported by two phosphorylation prediction algorithms, showing that R171W/MODY (or R89W/MODY) but not R167W/FRTS (or R85W/FRTS) reduces the probability for phosphorylation at S169 (or S87). In addition, the available HNF4A structure shows that R89 is closer to the S87 convergence zone compared to R85, which may explain that at the R171 (or R89) position, the substitution with the bulky tryptophan could interfere with the S169 (or the mammalian S87) phosphorylation site. Unlike for R167W (or R85W), the reduced DNA binding of R171W (or R89W) may therefore not result in phosphorylation-dependent dominant-negative effects, which is consistent with the haploinsufficiency that has been proposed for most MODY1 mutations. The reverse conclusion would be that haploinsufficiency alone is not enough to cause FRTS and that dominant-negative effects are required in addition (Marchesin, 2019).

As a consequence of the nuclear export due to the dominant-negative mechanism, this study uncovered cytotoxic effects that were not seen in the dHNF4 KD and hence are most likely linked with the aggregation of potentially misfolded proteins in the cytoplasm. Co-labeling with the nuclear envelope marker lamin D shows that the aggregate formation already commences during nuclear export. It can therefore be assumed that the small nuclear pore size causes an accumulation of dHNF4 beneath the nuclear envelope, which in turn favors aggregate formation. Although the order of cytosolic events is not entirely clear, it is speculated that the aggregates first pose a challenge for the clearance capacity of the proteasome, which is the normal site of HNF4A degradation. As a result, the ER may help by translocating chaperones into the cytoplasm to stimulate autophagy, as has been described for BiP and PDI. Consequently, the ability of the ER to address its own misfolded membrane-bound or luminal proteins could be weakened, thereby causing ER stress. Previously, cytoplasmic aggregates due to dominant-negative mutations or polyQ expansions have been observed in two other nuclear receptors, the thyroid receptor and the androgen receptor, respectively. In the latter case, the cytoplasmic aggregates were also associated with ER stress (Marchesin, 2019).

While dHNF4 has been shown to act as a regulator of mitochondria gene expression in the fly, the role of mammalian HNF4A in regulating mitochondrial function has so far been poorly investigated. Using the iREC system, this study showed that the R85W mutation affects mitochondria by decreasing the transcription of genes for mitochondrial structure and function. This was accompanied by a reduction in β-oxidation, leading to LD accumulation. As the HNF4A R85W also showed lower mRNA levels, it was not possible to assess the contribution of any dominant-negative effects in this model. Also, it was not possible to directly measure any effects of HNF4A R85W on DNA binding as was done in the COS-7 cell system. Nevertheless, both the nephrocyte and iREC results suggest that the effect on mitochondria could be one of the reasons why HNF4A R85W affects the fatty acid-consuming proximal tubules. In this manner, HNF4A-associated FRTS would be related to mitochondriopathies with isolated Fanconi syndrome or Fanconi syndrome as renal manifestation in more widespread disease. Given that HNF4A may be regulated by free fatty acids, it would be interesting to explore how extensively HNF4A is controlled by the reabsorption of exogenous fatty acids in proximal tubules (or nephrocytes) and whether fatty acid re-esterification and storage in LDs counteracts HNF4A activation (Marchesin, 2019).

In conclusion, these findings establish HNF4A as a master regulator of lipid metabolism in nephrocytes with high relevance for proximal tubular cells. This study further provides insight into the molecular basis of HNF4A-associated FRTS that has remained enigmatic since the identification of the first patient with the R85W mutation. The results suggest a rationale for treatments aimed at preventing the nuclear export of HNF4A in such patients. As serine phosphorylation in the DBD has been shown to be conserved in many nuclear hormone receptors, blocking this phosphorylation as a means to increase nuclear activity should also have more general implications (Marchesin, 2019).

REGULATION

Protein Interactions

When Drosophila and rat HNF4 mRNAs are co-translated and tested in a gel-shift assay, the translation products form an intermediate gel-shift band indicative of heterodimer fomation. This is further evidence of the close relationship between these two proteins (Zhong, 1993).

A modified tandem affinity purification strategy identifies cofactors of the Drosophila nuclear receptor dHNF4

With the completion of numerous genome projects, new high-throughput methods are required to ascribe gene function and interactions. A method proven successful in yeast for protein interaction studies is tandem affinity purification (TAP) of native protein complexes followed by MS. TAP, using Protein A and CBP tags, is not generally suitable for the purification and identification of proteins from tissues. A head-to-head comparison of tags shows that two others, FLAG and His, provide protein yields from Drosophila tissues that are an order of magnitude higher than Protein A and CBP. FLAG-His purification works sufficiently well so that two cofactors of the Drosophila nuclear receptor protein dHNF4 could be purified from whole animals. These proteins, Hsc70 and Hsp83, are important chaperones and cofactors of other nuclear receptor proteins. However, this is the first time that they have been shown to interact with a non-steroid binding nuclear receptor. The two proteins increase the ability of dHNF4 to bind DNA in vitro and to function in vivo. The tags and approaches developed here will help facilitate the routine purification of proteins from complex cells, tissues and whole organisms (Yang, 2006).

Previous studies of the Drosophila Ecdysone nuclear receptor and its heterodimer partner Ultraspiracle (EcR/USP) have shown that they require Hsc70 and Hsp83 for full DNA binding activity. To see if these two chaperones act similarly to increase the DNA-binding activity of dHNF4, electrophoretic gel mobility shift assays were performed in the presence of rabbit reticulocyte lysate (RT) or purified Hsc70 and Hsp83 proteins. Reticulocyte lysate is a rich source of Hsp90 and Hsp70, mammalian Hsp83 and Hsc70 homologues, respectively. The mobility shift assays show that reticulocyte lysate and both purified proteins enhance the binding of dHNF4 to its cognate DNA element. Interestingly, the enhancing activity of the reticulocyte lysate was higher than for either Hsc70 or Hsp83 on their own. Saturation of dHNF4 DNA binding activity enhancement was reached with Hsc70 or Hsp83 amounts of about 1.2 mg per reaction. When both chaperones were combined, however, dHNF4 DNA-binding activity could be further enhanced, suggesting that the two proteins act in a complementary or synergistic fashion (Yang, 2006).

As with EcR, dHNF4 associates specifically with the Hsp83 and Hsc70 chaperone proteins, and that these play an important role in DNA binding. Interestingly, this is the first non-steroid binding nuclear receptor shown to bind these proteins. Although the natural ligand for dHNF4 is unknown, mammalian HNF4 protein expressed in bacteria has been shown to bind small fatty acids related to and including palmitate within its ligand binding domain. This is the case for dHNF4 expressed in bacteria or Sf9 cells. Chaperones may be required to stabilize dHNF4 prior to fatty acid binding, or to facilitate exchange with some other native ligand. Additional or alternative roles are the modulation of intra- or inter-molecular interactions that control DNA binding and/or transcriptional activity. Mammalian HNF4 proteins play pivotal roles in lipid metabolism and associated diseases such as diabetes and heart disease. Hence, the interactions described in this study, and their molecular and physiological effects, will be important subjects for further study (Yang, 2006).

DEVELOPMENTAL BIOLOGY

Embryonic

Hnf4 is expressed in developing Drosophila embryos in midgut, fat bodies and Malpighian tubules, a strikingly similar pattern to its limited expression in the adult intestine, liver and kidney of the mouse homolog.

Maternal mRNA is deposited in the egg by nurse cells. The highest level of Drosophila HNF4 mRNA is found in stage 1 and stage 2 embryos where the mRNA shows uniform ectodermal distributed. During cleavage stages, the mRNA is retained at the peripheral regions of the syncytial blastoderm. Just before cellularization, the only detectable stain in the syncytial blastoderm is terminal, with the posterior end being stained more strongly than the anterior. From 2 to 6 hours after fertilization, there is no detectable Drosophila HNF4 mRNA. The mRNA then reappears between 6 and 8 hours, initially in the endodermal cells corresponding to the invaginating posterior midgut primordium and later in the anterior midgut primordium. The stain grows more intense, definitely conforming to the distribution of the dividing endodermal cells in the midgut. The cells of the foregut and hindgut contain little or no HNF4 messenger RNA.

Still later (stage 14/15) a variety of tissues contain HNF4 mRNA. These include fat bodies, Malpighian tubules, salivary glands and one cluster of cells, found on either side of each of the embryonic abdominal segments; the nature of these cells is unknown but may be related to the peripheral nervous system or endocrine gland. Staining in the Malpighian tubules is confined to the distal part of each tubule. This distal region contains dividing cells that are responsible for the elongation of the Malpighian tubules. At the end of stage 15, when the fused midgut has contracted to form four loops, the most heavily stained region in the midgut is observed in the midgut caeca and in the first and fourth loops from which gut primordia (nests of imaginal cells rather than imaginal discs) arise in larvae (Zhong, 1993).

Conflicting with the results given just above is another paper, reporting that there is no indication that Drosophila Hnf4 is expressed in the fat body, and that it is not involved in the development of this tissue (Hoshizaki, 1994).

Comparison of Drosophila Hnf4 distribution with that of Forkhead, shows that Hnf4 transcription is confined to the midgut, while Forkhead is present in foregut and hindgut. Both proteins are present in the salivary gland and the Malpighian tubules (Zhong, 1993).

EFFECTS OF MUTATION

A Drosophila mutant that has a chromosome deletion spanning the Hnf4 locus fails to develop tissues where Hnf4 is expressed during late embryogenesis. Early midgut, Malpighian tubule and salivary gland development appear to be normal. However, after stage 10, when the HNF4 mRNA reappears in wild type embryos, there are clearly visible defects in midgut, Malpighian tubule and salivary gland development in the mutants. Both the anterior and posterior midgut fail to further invaginate, the malpighian tubules fail to grow and the salivary gland invagination is arrested. At around stage 16, the endodermal part of the midgut is clearly missing, the Malpighian tubules are not formed, and the salivary glands do not invaginate properly and are reduced significantly in size (Zhong, 1993).

The Drosophila HNF4 nuclear receptor promotes glucose-stimulated insulin secretion and mitochondrial function in adults

Although mutations in HNF4A were identified as the cause of Maturity Onset Diabetes of the Young 1 (MODY1) two decades ago, the mechanisms by which this nuclear receptor regulates glucose homeostasis remain unclear. This study reports that loss of Drosophila HNF4 recapitulates hallmark symptoms of MODY1, including adult-onset hyperglycemia, glucose intolerance and impaired glucose-stimulated insulin secretion (GSIS). These defects are linked to a role for dHNF4 in promoting mitochondrial function as well as the expression of Hex-C, a homolog of the MODY2 gene Glucokinase. dHNF4 is required in the fat body and insulin-producing cells to maintain glucose homeostasis by supporting a developmental switch toward oxidative phosphorylation and GSIS at the transition to adulthood. These findings establish an animal model for MODY1 and define a developmental reprogramming of metabolism to support the energetic needs of the mature animal (Barry, 2016).

The association of MODY subtypes with mutations in specific genes provides a framework for understanding the monogenic heritability of this disorder as well as the roles of the corresponding pathways in systemic glucose homeostasis. This paper investigated the long-known association between HNF4A mutations and MODY1 by characterizing a whole-animal mutant that recapitulates the key symptoms associated with this disorder. Drosophila HNF4 is shown to be required for both GSIS and glucose clearance in adults, acting in distinct tissues and multiple pathways to maintain glucose homeostasis. Evidence is provided that dHNF4 promotes mitochondrial OXPHOS by regulating nuclear and mitochondrial gene expression. Finally, the expression of dHNF4 and its target genes is shown to be dramatically induced at the onset of adulthood, contributing to a developmental switch toward GSIS and oxidative metabolism at this stage in development. These results provide insights into the molecular basis of MODY1, expand understanding of the close coupling between development and metabolism, and establish the adult stage of Drosophila as an accurate context for genetic studies of GSIS, glucose clearance, and diabetes (Barry, 2016).

Drosophila HNF4 mutants display late-onset hyperglycemia accompanied by sensitivity to dietary carbohydrates, glucose intolerance, and defects in GSIS - hallmarks of MODY1. These defects arise from roles for dHNF4 in multiple tissues, including a requirement in the IPCs for GSIS and a role in the fat body for glucose clearance. The regulation of GSIS by dHNF4 is consistent with the long-known central contribution of pancreatic β-cells to the pathophysiology of MODY1 (Fajans and Bell, 2011). Similarly, several MODY-associated genes, including GCK, HNF1A and HNF1B, are important for maintaining normal hepatic function. These distinct tissue-specific contributions to glycemic control may explain why single-tissue Hnf4A mutants in mice do not fully recapitulate MODY1 phenotypes and predict that a combined deficiency for the receptor in both the liver and pancreatic β-cells of adults would produce a more accurate model of this disorder (Barry, 2016).

This study used metabolomics, RNA-seq, and ChIP-seq to provide initial insights into the molecular mechanisms by which dHNF4 exerts its effects on systemic metabolism. These studies revealed several downstream pathways, each of which is associated with maintaining homeostasis and, when disrupted, can contribute to diabetes. These include genes identified in previous study of dHNF4 in larvae that act in lipid metabolism and fatty acid β-oxidation, analogous to the role of Hnf4A in the mouse liver to maintain normal levels of stored and circulating lipids (Hayhurst, 2001; Palanker, 2009). Extensive studies have linked defects in lipid metabolism with impaired β-cell function and peripheral glucose uptake and clearance, suggesting that these pathways contribute to the diabetic phenotypes of dHNF4 mutants. An example of this is pudgy, which is expressed at reduced levels in dHNF4 mutants and encodes an acyl-CoA synthetase that is required for fatty acid oxidation (Xu, 2012). Interestingly, pudgy mutants have elevated triglycerides, reduced glycogen, and increased circulating sugars, similar to dHNF4 mutants, suggesting that this gene is a critical downstream target of the receptor. It is important to note, however, that the metabolomic, RNA-seq, and ChIP-seq studies were conducted on extracts from whole animals rather than individual tissues. As a result, some of the findings may reflect compensatory responses between tissues, and some tissue-specific changes in gene expression or metabolite levels may not be detected by the current approach. Further studies using samples from dissected tissues would likely provide a more complete understanding of the mechanisms by which dHNF4 maintains systemic physiology (Barry, 2016).

Notably, the Drosophila GCK homolog encoded by Hex-C is expressed at reduced levels in dHNF4 mutants. The central role of GCK in glucose sensing by pancreatic β-cells as well as glucose clearance by the liver places it as an important regulator of systemic glycemic control. Functional data supports these associations by showing that Hex-C is required in the fat body for proper circulating glucose levels, analogous to the role of GCK in mammalian liver. Unlike mice lacking GCK in the β-cells, no effect is seen on glucose homeostasis when Hex-C is targeted by RNAi in the IPCs. This is possibly due to the presence of a second GCK homolog in Drosophila, Hex-A, which could act alone or redundantly with Hex-C to mediate glucose sensing by the IPCs. In mammals, GCK expression is differentially regulated between hepatocytes and β-cells through the use of two distinct promoters, and studies in rats have demonstrated a direct role for HNF4A in promoting GCK expression in the liver. These findings suggest that this relationship has been conserved through evolution. In addition, the association between GCK mutations and MODY2 raise the interesting possibility that defects in liver GCK activity may contribute to the pathophysiology of both MODY1 and MODY2 (Barry, 2016).

Interestingly, gene ontology analysis indicates that the up-regulated genes in dHNF4 mutants correspond to the innate immune response pathways in Drosophila. This response parallels that seen in mice lacking Hnf4A function in enterocytes, which display intestinal inflammation accompanied by increased sensitivity to DSS-induced colitis and increased permeability of the intestinal epithelium, similar to humans with inflammatory bowel disease. Disruption of Hnf4A expression in Caco-2 cells using shRNA resulted in changes in the expression of genes that act in oxidative stress responses, detoxification pathways, and inflammatory responses, similar to the effect seen in dHNF4 mutants. Moreover, mutations in human HNF4A are associated with chronic intestinal inflammation, irritable bowel disease, ulcerative colitis, and Crohn's disease, suggesting that these functions are conserved through evolution. Taken together, these results support the hypothesis that dHNF4 plays an important role in suppressing an inflammatory response in the intestine. Future studies are required to test this hypothesis in Drosophila. In addition, further work is required to better define the regulatory functions of HNF4 that are shared between Drosophila and mammals. Although the current work suggests that key activities for this receptor have been conserved in flies and mammals, corresponding to the roles of HNF4 in the IPCs (β-cells) for GSIS, fat body (liver) for lipid metabolism and glucose clearance, and intestine to suppress inflammation, there are likely to be divergent roles as well. One example of this is the embryonic lethality of Hnf4A mutant mice, which is clearly distinct from the early adult lethality reported here for dHNF4 mutants. Further studies are required to dissect the degree to which the regulatory functions of this receptor have been conserved through evolution (Barry, 2016).

It is also important to note that mammalian Hnf4A plays a role in hepatocyte differentiation and proliferation in addition to its roles in metabolism. This raises the possibility that early developmental roles for dHNF4 could impact the phenotypes reported in this study in adults. Indeed, all of the current studies involve zygotic dHNF4 null mutants that lack function throughout development. In an effort to address this possibility and distinguish developmental from adult-specific functions, a conditional dHNF4 mutant allele is currently being constructed using CRISPR/Cas9 technology. Future studies using this mutation should allow conducting a detailed phenotypic analysis of this receptor at different stages of Drosophila development (Barry, 2016).

It is also interesting to speculate that the current functional studies of dHNF4 uncover more widespread roles for MODY-associated genes in glycemic control, in addition to the link with MODY2 described in this study. HNF1A and HNF1B, which are associated with MODY3 and MODY5, respectively, act together with HNF4A in an autoregulatory circuit in an overlapping set of tissues, with HNF4A proposed to be the most upstream regulator of this circuit. The observation that Drosophila do not have identifiable homologs for HNF1A and HNF1B raises the interesting possibility that dHNF4 alone replaces this autoregulatory circuit in more primitive organisms. The related phenotype of these disorders is further emphasized by cases of MODY3 that are caused by mutation of an HNF4A binding site within the HNF1A promoter. Consistent with this link, MODY1, MODY3 and MODY5 display similar features of disease complication and progression, and studies of HNF1A and HNF4A in INS-1 cells have implicated roles for these transcription factors in promoting mitochondrial metabolism in β-cells. In line with this, mitochondrial diabetes is clearly age progressive, as are MODY1, 3, and 5, but not MODY2, which represents a more mild form of this disorder. Furthermore, the severity and progression of MODY3 is significantly enhanced when patients carry an additional mutation in either HNF4A or mtDNA. Overall, these observations are consistent with the well-established multifactorial nature of diabetes, with multiple distinct metabolic insults contributing to disease onset (Barry, 2016).

RNA-seq analysis supports a role for dHNF4 in coordinating mitochondrial and nuclear gene expression. This is represented by the reduced expression of transcripts encoded by the mitochondrial genome, along with effects on nuclear-encoded genes that act in mitochondria. In addition, ChIP-seq revealed that several of the nuclear-encoded genes are direct targets of the receptor. Mitochondrial defects have well-established links to diabetes-onset, with mutations in mtDNA causing maternally-inherited diabetes and mitochondrial OXPHOS playing a central role in both GSIS and peripheral glucose clearance. Consistent with this, functional studies indicate that dHNF4 is required to maintain normal mitochondrial function and that defects in this process contribute to the diabetic phenotypes in dHNF4 mutants (Barry, 2016).

It is important to note that the number of direct targets for dHNF4 in the nucleus is difficult to predict with the current dataset. A relatively low signal-to-noise ratio in ChIP-seq experiment allowed identification of only 37 nuclear-encoded genes as high confidence targets by fitting the criteria of proximal dHNF4 binding along with reduced expression in dHNF4 mutants. Future ChIP-seq studies will allow expansion of this dataset to gain a more comprehensive understanding of the scope of the dHNF4 regulatory circuit and may also reveal tissue-restricted targets that are more difficult to detect. Nonetheless, almost all of the genes identified as direct targets for dHNF4 regulation correspond to genes involved in mitochondrial metabolism, including the TCA cycle, OXPHOS, and lipid catabolism, demonstrating that this receptor has a direct impact on these critical downstream pathways that influence glucose homeostasis (Barry, 2016).

An unexpected and significant discovery in these studies is that dHNF4 is required for mitochondrial gene expression and function. Several lines of evidence support the model that dHNF4 exerts this effect through direct regulation of mitochondrial transcription, although a number of additional experiments are required to draw firm conclusions on this regulatory connection. First, most of the 13 protein-coding genes in mtDNA are underexpressed in dHNF4 mutants. RNA-seq studies have been conducted of Drosophila nuclear transcription factor mutants and similar effects on mitochondrial gene expression have not been reported previously. Second, dHNF4 protein is abundantly bound to the control region of the mitochondrial genome, representing the fifth strongest enrichment peak in the ChIP-seq dataset. Although the promoters in Drosophila mtDNA have not yet been identified, the site bound by dHNF4 corresponds to a predicted promoter region for Drosophila mitochondrial transcription and coincides with the location of the major divergent promoters in human mtDNA. It is unlikely that the abundance of mtDNA relative to nuclear DNA had an effect on the ChIP-seq peak calling because the MACS2 platform used for this analysis accounts for local differences in read depth across the genome (including the abundance of mtDNA). In addition, although the D-loop in mtDNA has been proposed to contribute to possible false-positive ChIP-seq peaks in mammalian studies, the D-loop structure is not present in Drosophila mtDNA. Nonetheless, additional experiments are required before it can be concluded that this apparent binding is of regulatory significance for mitochondrial function. Third, the effects on mitochondrial gene expression do not appear to be due to reduced mitochondrial number in dHNF4 mutants. This is consistent with the normal expression of mt:Cyt-b in dHNF4 mutants, which has a predicted upstream promoter that drives expression of the mt:Cyt-b and mt:ND6 operon (although mt:ND6 RNA could not be detected in northern blot studies). Fourth, immunostaining for dHNF4 shows cytoplasmic protein that overlaps with the mitochondrial marker ATP5A, in addition to its expected nuclear localization. Some of the cytoplasmic staining, however, clearly fails to overlap with the mitochondrial marker, making it difficult to draw firm conclusions from this experiment. Multiple efforts to expand on this question biochemically with subcellular fractionation studies have been complicated by abundant background proteins that co-migrate with the receptor in mitochondrial extracts. New reagents are currently being developed to detect the relatively low levels of endogenous dHNF4 protein in mitochondria, including use of the CRISPR/Cas9 system for the addition of specific epitope tags to the endogenous dHNF4 locus. Finally, multiple hallmarks of mitochondrial dysfunction were observed, including elevated pyruvate and lactate, specific alterations in TCA cycle metabolites, reduced mitochondrial membrane potential, reduced levels of ATP, and fragmented mitochondrial morphology. These phenotypes are consistent with the reduced expression of key genes involved in mitochondrial OXPHOS, and studies showing that decreased mitochondrial membrane potential and ATP production are commonly associated with mitochondrial fragmentation (Barry, 2016).

Although unexpected, the proposal that dHNF4 may directly regulate mitochondrial gene expression is not unprecedented. A number of nuclear transcription factors have been localized to mitochondria, including ATFS-1, MEF2D, CREB, p53, STAT3, along with several nuclear receptors, including the estrogen receptor, glucocorticoid receptor, and the p43 isoform of the thyroid hormone receptor. The significance of these observations, however, remains largely unclear, with few studies demonstrating regulatory functions within mitochondria. In addition, these factors lack a canonical mitochondrial localization signal at their amino-terminus, leaving it unclear how they achieve their subcellular distribution. In contrast, one of the five mRNA isoforms encoded by dHNF4, dHNF4-B, encodes a predicted mitochondrial localization signal in its 5'-specific exon, providing a molecular mechanism to explain the targeting of this nuclear receptor to this organelle. Efforts are currently underway to conduct a detailed functional analysis of dHNF4-B by using the CRISPR/Cas9 system to delete its unique 5' exon, as well as establishing transgenic lines that express a tagged version of dHNF4-B under UAS control. Future studies using these reagents, along with available dHNF4 mutants, should allow dissection of the nuclear and mitochondrial functions of this nuclear receptor and their respective contributions to systemic physiology (Barry, 2016).

Finally, it is interesting to speculate whether the role for dHNF4 in mitochondria is conserved in mammals. A few papers have described the regulation of nuclear-encoded mitochondrial genes by HNF4A. In addition, several studies have detected cytoplasmic Hnf4A by immunohistochemistry in tissue sections, including in postnatal pancreatic islets and hepatocytes. Moreover, the regulation of nuclear/cytoplasmic shuttling of HNF4A has been studied in cultured cells. The evolutionary conservation of the physiological functions of HNF4A, from flies to mammals, combined with these prior studies, argue that more effort should be directed at defining the subcellular distribution of HNF4A protein and its potential roles within mitochondria. Taken together with these studies in Drosophila, this work could provide new directions for understanding HNF4 function and MODY1 (Barry, 2016).

Physiological studies by George Newport in 1836 noted that holometabolous insects reduce their respiration during metamorphosis leading to a characteristic 'U-shaped curve' in oxygen consumption. Subsequent classical experiments in Lepidoptera, Bombyx, Rhodnius and Calliphora showed that this reduction in mitochondrial respiration during metamorphosis and dramatic rise in early adults is seen in multiple insect species, including Drosophila. Consistent with this, the activity of oxidative enzyme systems and the levels of ATP also follow a 'U-shaped curve' during development as the animal transitions from a non-feeding pupa to a motile and reproductively active adult fly. Although first described over 150 years ago, the regulation of this developmental increase in mitochondrial activity has remained undefined. This study shows that this temporal switch is dependent, at least in part, on the dHNF4 nuclear receptor. The levels of dHNF4 expression increase dramatically at the onset of adulthood, accompanied by the expression of downstream genes that act in glucose homeostasis and mitochondrial OXPHOS. This coordinate transcriptional switch is reduced in dHNF4 mutants, indicating that the receptor plays a key role in this transition. Importantly, the timing of this program correlates with the onset of dHNF4 mutant phenotypes in young adults, including sugar-dependent lethality, hyperglycemia, and defects in glucose-stimulated insulin secretion, indicating that the upregulation of dHNF4 expression in adults is of functional significance. It should also be noted, however, that dHNF4 target genes are still induced at the onset of adulthood in dHNF4 mutants, albeit at lower levels, indicating that other regulators contribute to this switch in metabolic state. Nonetheless, the timing of the induction of dHNF4 and its target genes in early adults, and its role in promoting OXPHOS, suggest that this receptor contributes to the end of the 'U-shaped curve' and directs a systemic transcriptional switch that establishes an optimized metabolic state to support the energetic demands of adult life (Barry, 2016).

Interestingly, a similar metabolic transition towards OXPHOS was recently described in Drosophila neuroblast differentiation, mediated by another nuclear receptor, EcR. Although this occurs during early stages of pupal development, prior to the dHNF4-mediated transition at the onset of adulthood, the genes involved in this switch show a high degree of overlap with dHNF4 target genes that act in mitochondria, including ETFB, components of Complex IV, pyruvate carboxylase, and members of the α-ketoglutarate dehydrogenase complex. This raises the possibility that dHNF4 may contribute to this change in neuroblast metabolic state and play a more general role in supporting tissue differentiation by promoting OXPHOS (Barry, 2016).

Only one other developmentally coordinated switch in systemic metabolic state has been reported in Drosophila and, intriguingly, it is also regulated by a nuclear receptor. Drosophila Estrogen-Related Receptor (dERR) acts in mid-embryogenesis to directly induce genes that function in biosynthetic pathways related to the Warburg effect, by which cancer cells use glucose to support rapid proliferation (Tennessen, 2011; Tennessen, 2014b). This switch toward aerobic glycolysis favors lactate production and flux through biosynthetic pathways over mitochondrial OXPHOS, supporting the ~200-fold increase in mass that occurs during larval development. Taken together with the current work on dHNF4, these studies define a role for nuclear receptors in directing temporal switches in metabolic state that meet the changing physiological needs of different stages in development. Further studies should allow better definition of these regulatory pathways as well as determine how broadly nuclear receptors exert this role in coupling developmental progression with systemic metabolism (Barry, 2016).

Although little is known about the links between development and metabolism, it is likely that coordinated switches in metabolic state are not unique to Drosophila, but rather occur in all higher organisms in order to meet the distinct metabolic needs of an animal as it progresses through its life cycle. Indeed, a developmental switch towards OXPHOS in coordination with the cessation of growth and differentiation appears to be a conserved feature of animal development. Moreover, as has been shown for cardiac hypertrophy, a failure to coordinate metabolic state with developmental context can have an important influence on human disease (Barry, 2016).

In addition to promoting a transition toward systemic oxidative metabolism in adult flies, dHNF4 also contributes to a switch in IPC physiology that supports GSIS. dHNF4 is not expressed in larval IPCs, but is specifically induced in these cells at adulthood. Similarly, the fly homologs of the mammalian ATP-sensitive potassium channel subunits, Sur1 and Kir6, which link OXPHOS and ATP production to GSIS, are not expressed in the larval IPCs but are expressed during the adult stage. They also appear to be active at this stage as cultured IPCs from adult flies undergo calcium influx and membrane depolarization upon exposure to glucose or the anti-diabetic sulfonylurea drug glibenclamide. In addition, reduction of the mitochondrial membrane potential in adult IPCs by ectopic expression of an uncoupling protein is sufficient to reduce IPC calcium influx, elevate whole-animal glucose levels, and reduce peripheral insulin signaling. This switch in IPC physiology is paralleled by a change in the nutritional signals that trigger DILP release. Amino acids, and not glucose, stimulate DILP2 secretion by larval IPCs. Rather, glucose is sensed by the corpora cardiaca in larvae, a distinct organ that secretes adipokinetic hormone, which acts like glucagon to maintain carbohydrate homeostasis during larval stages. Interestingly, this can have an indirect effect on the larval IPCs, triggering DILP3 secretion in response to dietary carbohydrates (Kim, 2015). Adult IPCs, however, are responsive to glucose for DILP2 release (Park, 2014). In addition, dHNF4 mutants on a normal diet maintain euglycemia during larval and early pupal stages, but display hyperglycemia at the onset of adulthood, paralleling their lethal phase on a normal diet. Taken together, these observations support the model that the IPCs change their physiological state during the larval-to-adult transition and that dHNF4 contributes to this transition toward glucose-stimulated insulin secretion. The observation that glucose is a major circulating sugar in adults, but not larvae, combined with its ability to stimulate DILP2 secretion from adult IPCs, establishes this stage as an experimental context for genetic studies of glucose homeostasis, GSIS, and diabetes. Functional characterization of these pathways in adult Drosophila will allow he power of model organism genetics to be harnessed to better understand the regulation of glucose homeostasis and the factors that contribute to diabetes (Barry, 2016).

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).

HNF4A is essential for specification of hepatic progenitors from human pluripotent stem cells

The availability of pluripotent stem cells offers the possibility of using such cells to model hepatic disease and development. With this in mind, a protocol was extablished that facilitates the differentiation of both human embryonic stem cells and induced pluripotent stem cells into cells that share many characteristics with hepatocytes. The use of highly defined culture conditions and the avoidance of feeder cells or embryoid bodies allowed synchronous and reproducible differentiation to occur. The differentiation towards a hepatocyte-like fate appeared to recapitulate many of the developmental stages normally associated with the formation of hepatocytes in vivo. In the current study, the feasibility of using human pluripotent stem cells to probe the molecular mechanisms underlying human hepatocyte differentiation was addressed. This study demonstrates the following: (1) that human embryonic stem cells express a number of mRNAs that characterize each stage in the differentiation process, (2) that gene expression can be efficiently depleted throughout the differentiation time course using shRNAs expressed from lentiviruses and (3) that the nuclear hormone receptor HNF4A is essential for specification of human hepatic progenitor cells by establishing the expression of the network of transcription factors that controls the onset of hepatocyte cell fate (DeLaForest, 2011).

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).

REFERENCES

Search PubMed for articles about Drosophila Hnf4

Barry, W. E. and Thummel, C. S. (2016). The Drosophila HNF4 nuclear receptor promotes glucose-stimulated insulin secretion and mitochondrial function in adults. Elife 5. PubMed ID: 27185732

Briancon, N., et al. (2004). Expression of the alpha7 isoform of hepatocyte nuclear factor (HNF) 4 is activated by HNF6/OC-2 and HNF1 and repressed by HNF4alpha1 in the liver. J. Biol. Chem. 279(32): 33398-408. 15159395

Chen, W. S., et al. (1994). Disruption of the HNF-4 gene, expressed in visceral endoderm, leads to cell death in embryonic ectoderm and impaired gastrulation of mouse embryos. Genes Dev. 8: 2466-77. PubMed Citation: 7958910

Clotman, F., et al. (2002). The onecut transcription factor HNF6 is required for normal development of the biliary tract. Development 129(8): 1819-28. 11934848

Coucouvanis, E. and Martin, G. R. (1999). BMP signaling plays a role in visceral endoderm differentiation and cavitation in the early mouse embryo. Development 126(3): 535-546. PubMed Citation: 9876182

DeLaForest, A., et al. (2011). HNF4A is essential for specification of hepatic progenitors from human pluripotent stem cells. Development 138(19): 4143-53. PubMed Citation: 21852396

Dhe-Paganon, S., Duda, K., Iwamoto, M., Chi, Y. I. and Shoelson, S. E. (2002). Crystal structure of the HNF4 alpha ligand binding domain in complex with endogenous fatty acid ligand. J. Biol. Chem. 277(41): 37973-6. 12193589

Drewes, T., et al. (1996). Human Hepatocyte nuclear factor 4 isoforms are encoded by distinct and differentially expressed genes. Mol. Cell. Biol. 16: 925-931. PubMed Citation: 8622695

Duncan, S. A., et al. (1994). Expression of transcription factor HNF-4 in the extraembryonic endoderm, gut, and nephrogenic tissue of the developing mouse embryo: HNF-4 is a marker for primary endoderm in the implanting blastocyst. Proc. Natl. Acad. Sci. 91: 7598-7602. PubMed Citation: 8052626

Duncan, S. A., Nagy, A. and Chan, W. (1997). Murine gastrulation requires HNF-4 regulated gene expression in the visceral endoderm: tetraploid rescue of Hnf-4-/- embryos. Development 124: 279-287. PubMed Citation: 9053305

Duncan, S. A., et al. (1998). Regulation of a transcription factor network required for differentiation and metabolism. Science 281: 692-695. PubMed Citation: 9685261

Fajans, S. S. and Bell, G. I. (2011). MODY: history, genetics, pathophysiology, and clinical decision making. Diabetes Care 34: 1878-1884. PubMed ID: 21788644

Galson, D. L., et al. (1995). The orphan receptor hepatic nuclear factor 4 functions as a transcriptional activator for tissue-specific and hypoxia-specific erythropoietin gene expression and is antagonized by EAR3/COUP-TF1. Mol. Cell. Biol. 15(4): 2135-44. 7891708

Gupta, R. K., et al. (2007). Expansion of adult β-cell mass in response to increased metabolic demand is dependent on HNF-4α. Genes Dev. 21: 756-769. Medline abstract: 17403778

Hatzis, P. and Talianidis, I. (2003). Dynamics of enhancer-promoter communication during differentiation-induced gene activation. Mol. Cell 10: 1467-1477. 12504020

Hayhurst, G. P., Lee, Y. H., Lambert, G., Ward, J. M. and Gonzalez, F. J. (2001). Hepatocyte nuclear factor 4alpha (nuclear receptor 2A1) is essential for maintenance of hepatic gene expression and lipid homeostasis. Mol Cell Biol 21: 1393-1403. PubMed ID: 11158324

Holewa, B., Pogge, E., et al. (1996). Transriptional hierarchy in Xenopus embryogenesis: HNF4 a maternal factor involved in the developmental activation of the gene encoding the tissue specific transcription factor HNF1alpha (LFB1). Mech. Dev. 54: 45-57. PubMed Citation: 8808405

Holewa, B., et al. (1997). HNF4ß, a new gene of the HNF4 family with distinct activation and expression profiles in oogenesis and embryogenesis of Xenopus laevis. Mol. Cell. Biol. 17: 687-694. PubMed Citation: 9001222

Hong, S. K., et al. (2002). Isolation and expression of a novel neuron-specific onecut homeobox gene in zebrafish. Mech. Dev. 112(1-2): 199-202. 11850195

Hoshizaki, D. K., et al. (1994). Embryonic fat-cell lineage in Drosophila melanogaster. Development 120: 2489-99. PubMed Citation: 7956826

Jiang, G., Lee, U. and Sladek, F. M. (1997). Proposed mechanism for the stabilization of nuclear receptor DNA binding via protein dimerization. Mol. Cell. Biol. 17(11): 6546-6554

Kim, J. and Neufeld, T. P. (2015). Dietary sugar promotes systemic TOR activation in Drosophila through AKH-dependent selective secretion of Dilp3. Nat Commun 6: 6846. PubMed ID: 25882208

Ktistaki, E., et al. (1995). Recruitment of hepatocyte nuclear factor 4 into specific intranuclear compartments depends on tyrosine phosphorylation that affects its DNA-binding and transactivation potential. Proc. Natl. Acad. Sci. 92: 9876-9880

Kuo, C. J., et al. (1992). A transcriptional hierarchy involved in mammalian cell-type specification. Nature 355: 457-61

Makita, T., et al. (2001). A developmental transition in definitive erythropoiesis: erythropoietin expression is sequentially regulated by retinoic acid receptors and HNF4. Genes Dev. 15: 889-901. 11297512

Marchesin, V., Perez-Marti, A., Le Meur, G., Pichler, R., Grand, K., Klootwijk, E. D., Kesselheim, A., Kleta, R., Lienkamp, S. and Simons, M. (2019). Molecular basis for autosomal-dominant renal Fanconi Syndrome caused by HNF4A. Cell Rep 29(13): 4407-4421. PubMed ID: 31875549

Miquerol, L., et al. (1994). Expression of the L-type pyruvate kinase gene and the hepatocyte nuclear factor 4 transcription factor in exocrine and endocrine pancreas. J. Biol. Chem. 269: 8944-51.

Miura, N. and Tanaka, K. (1993). Analysis of the rat hepatocyte nuclear factor (HNF) 1 gene promoter: synergistic activation by HNF4 and HNF1 proteins. Nucleic Acids Res 21: 3731-6

Morrisey, E. E., et al. (1998). GATA6 regulates HNF4 and is required for differentiation of visceral endoderm in the mouse embryo. Genes Dev. 12(22): 3579-90

Nagy, P., Bisgaard, H. C. and Thorgeirsson, S. S. (1994). Expression of hepatic transcription factors during liver development and oval cell differentiation. J Cell Biol 126: 223-233

Palanker, L., Tennessen, J. M., Lam, G. and Thummel, C. S. (2009). Drosophila HNF4 regulates lipid mobilization and beta-oxidation. Cell Metab 9: 228-239. PubMed ID: 19254568

Palu, R.A. and Thummel, C.S. (2016). Sir2 acts through Hepatocyte Nuclear Factor 4 to maintain insulin signaling and metabolic homeostasis in Drosophila. PLoS Genet 12: e1005978. PubMed ID: 27058248

Parvy, J. P., Napal, L., Rubin, T., Poidevin, M., Perrin, L., Wicker-Thomas, C. and Montagne, J. (2012). Drosophila melanogaster Acetyl-CoA-carboxylase sustains a fatty acid-dependent remote signal to waterproof the respiratory system. PLoS Genet 8(8): e1002925. PubMed ID: 22956916

Reuter, R. (1994). The gene serpent has homeotic properties and specifies endoderm versus ectoderm with the Drosophila gut. Development 120(5): 1123-1135

Sluder, A. E., Lindbloom, T. and Ruvkun, G. (1997). The Caenorhabditis elegans orphan nuclear hormone receptor gene rhr-2 functions in early embryonic development. Dev. Biol. 184: 303-319

Spath, G. F. and Weiss, M. C. (1998). Hepatocyte nuclear factor 4 provokes expression of epithelial marker genes, acting as a morphogen in dedifferentiated hepatoma cells. J. Cell Biol. 140(4): 935-946

Storelli, G., Nam, H. J., Simcox, J., Villanueva, C. J. and Thummel, C. S. (2018). Drosophila HNF4 directs a switch in lipid metabolism that supports the transition to adulthood. Dev Cell 48(2):200-214. PubMed ID: 30554999

Swevers, L. and Iatrou, K. (1998). The orphan receptor BmHNF-4 of the silkmoth bombyx mori: ovarian and zygotic expression of two mRNA isoforms encoding polypeptides with different activating domains. Mech. Dev. 72(1-2): 3-13

Torres-Padilla, M. E., Fougere-Deschatrette, C. and Weiss, M. C. (2001). Expression of HNF4alpha isoforms in mouse liver development is regulated by sequential promoter usage and constitutive 3' end splicing. Mech. Dev. 109(2): 183-93. 11731232

Van Gilst, M. R. et al. (2005). Nuclear hormone receptor NHR-49 controls fat consumption and fatty acid composition in C. elegans. PLoS Biol. 3: e53. PubMed Citation: 15719061

Viollet, B., Kahn. A. and Raymondjean, M. (1997). Protein kinase A-dependent phosphorylation modulates DNA-binding activity of hepatocyte nuclear factor 4. Mol. Cell. Biol. 17: 4208-4219

Weber, H., et al. (1996). Mesoderm and endoderm differentiation in animal cap explants: identification of the HNF4-binding site as an activin A responsive element in the Xenopus HNF1alpha promoter. Development 122: 1975-1984

Wisely, G. B., et al. (2002). Hepatocyte nuclear factor 4 is a transcription factor that constitutively binds fatty acids. Structure 10(9): 1225-34. 12220494

Yang, P., Sampson, H. M. and Krause, H. M. (2006), A modified tandem affinity purification strategy identifies cofactors of the Drosophila nuclear receptor dHNF4. Proteomics 6(3): 927-35. 16400689

Zhong, W, Sladek, F. M. and Darnell, J. E. (1993). The expression pattern of a Drosophila homolog to the mouse transcription factor HNF-4 suggests a determinative role in gut formation. EMBO J. 12: 537-44

Xu, X., Gopalacharyulu, P., Seppanen-Laakso, T., Ruskeepaa, A. L., Aye, C. C., Carson, B. P., Mora, S., Oresic, M. and Teleman, A. A. (2012). Insulin signaling regulates fatty acid catabolism at the level of CoA activation. PLoS Genet 8: e1002478. PubMed ID: 22275878