Hnf4: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Hepatocyte nuclear factor 4

Synonyms -

Cytological map position - 29E1-29E6

Function - Transcription factor

Keyword(s) - endoderm, salivary glands, malpighian tubules, Fat storage

Symbol - Hnf4

FlyBase ID:FBgn0004914

Genetic map position - 2-

Classification - zinc finger - steroid receptor superfamily

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Laranjeira, A., Schulz, J. and Dotti, C. G. (2016). Genes related to fatty acid beta-oxidation play a role in the functional decline of the Drosophila brain with age. PLoS One 11: e0161143. PubMed ID: 27518101
Summary:
In living organisms, ageing is widely considered to be the result of a multifaceted process consisting of the progressive accumulation of damage over time, having implications both in terms of function and survival. The study of ageing presents several challenges, from the different mechanisms implicated to the great diversity of systems affected over time. The current study set out to identify genes involved in the functional decline of the brain with age and study its relevance in a tissue dependent manner using Drosophila melanogaster as a model system. The age-dependent upregulation is reported of genes involved in the metabolic process of fatty acid beta-oxidation in the nervous tissue of female wild-type flies. Downregulation of CG10814, dHNF4 and lipid mobilizing genes bmm and dAkh rescues the functional decline of the brain with age, both at the cellular and behaviour level, while over-expression worsens performance. The data proposes the occurrence of a metabolic alteration in the fly brain with age, whereby the process of beta-oxidation of fatty acids experiences a genetic gain-of-function. This event proved to be one of the main causes contributing to the functional decline of the brain with age.

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).


GENE STRUCTURE

Transcript length - 4.5 and 3.3 kb


PROTEIN STRUCTURE

Amino Acids - 666

Structural Domains

The Drosophila gene matches the mouse gene in 60 out of 66 amino acids in the zinc finger DNA binding domain, and in 140 out of 206 amino acids in the domain that specifies dimerization and ligand binding. The 12 amino acids immediately C-terminal to the zinc finger are also completely conserved. The Drosophila cDNA codes for a protein that is more than 200 amino acids longer than the rat and mouse proteins. The genomic coding structure is the same for mammalian and fly HNF4s. One exon encodes five of the eight cysteines found in the two zinc fingers; the next exon encodes the remaining three cysteines of the second zinc finger. In most steroid family members, each zinc finger (four cysteines) is encoded in a separate exon (Zhong, 1993).


Hnf4: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 13 FEB 97 

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