Identification of an Endogenous Ligand Bound to a Native Orphan Nuclear Receptor

Orphan nuclear receptors have been instrumental in identifying novel signaling pathways and therapeutic targets. However, identification of ligands for these receptors has often been based on random compound screens or other biased approaches. As a result, it remains unclear in many cases if the reported ligands are the true endogenous ligands, – i.e., the ligand that is bound to the receptor in an unperturbed in vivo setting. Technical limitations have limited our ability to identify ligands based on this rigorous definition. The orphan receptor hepatocyte nuclear factor 4 α (HNF4α) is a key regulator of many metabolic pathways and linked to several diseases including diabetes, atherosclerosis, hemophilia and cancer. Here we utilize an affinity isolation/mass-spectrometry (AIMS) approach to demonstrate that HNF4α is selectively occupied by linoleic acid (LA, C18:2ω6) in mammalian cells and in the liver of fed mice. Receptor occupancy is dramatically reduced in the fasted state and in a receptor carrying a mutation derived from patients with Maturity Onset Diabetes of the Young 1 (MODY1). Interestingly, however, ligand occupancy does not appear to have a significant effect on HNF4α transcriptional activity, as evidenced by genome-wide expression profiling in cells derived from human colon. We also use AIMS to show that LA binding is reversible in intact cells, indicating that HNF4α could be a viable drug target. This study establishes a general method to identify true endogenous ligands for nuclear receptors (and other lipid binding proteins), independent of transcriptional function, and to track in vivo receptor occupancy under physiologically relevant conditions.


Introduction
Nuclear receptors (NRs) are ligand-dependent transcription factors that regulate the expression of genes involved in virtually all aspects of physiology and disease [1,2].The identification of ligand-receptor pairs began with the pioneering work of Jensen [3], Edelman [4] and others who injected radioactive ligands into animals and observed their binding to nuclear receptor proteins.These experiments had an inherent bias in that the probe ligand displaced binding of the true endogenous ligand, which could not be detected in these assays.The validity of the classical steroid receptor-ligand pairs are now well established, but the limitations of existing approaches leave open the possibility that additional ligands may exist for the classical steroid receptors.Indeed, such speculation was raised for estrogen receptor some time ago, as well as more recently [5,6], and for intestinal vitamin D receptor that is activated by an enterohepatic bile acid [7].
The assignment of endogenous ligands to the so-called orphan nuclear receptors is even more equivocal [2,8].Typically, orphan receptors are screened in transcription-based assays against random compound collections that include natural or synthetic molecules; in other cases candidate ligands are identified based on their structure or biological activity [9].Alternatively, compounds are added at supra-pharmacologic doses and their metabolic products are found to be ligands [10].Most recently, ligands have been reported based on fortuitous binding to heterologously produced recombinant proteins [11][12][13][14][15][16][17].Such studies have been of enormous value and have lead to the identification of many novel signaling pathways, drugs and therapeutic targets.Nonetheless, ligands have been identified for only about half of the 48 human nuclear receptors and the tally in non-human species is even lower.Furthermore, it remains unclear how many of the ligands that have been identified are the actual ligands that are bound to the receptor in vivo.For example, the first orphan receptor-ligand pair to be identified was 9-cis retinoic acid and RXR [10], yet it appears unlikely that sufficient 9-cis retinoic acid exists in vivo to serve as a true endogenous ligand [18].
HNF4a (HNF4A, NR2A1) is another orphan receptor whose endogenous ligand remains unclear [19][20][21].HNF4a is essential to early development and plays critical roles in hepatocyte differentiation [22][23][24] and in homeostasis of the adult liver, intestine, and pancreatic beta cells [25][26][27][28].In humans, mutations in the coding and promoter regions of HNF4a lead to Maturity Onset Diabetes of the Young 1 (MODY1), a heritable form of type 2 diabetes [29].Recent crystallographic studies identified a mixture of tightly bound fatty acids in the ligand binding pocket (LBP) of bacterially expressed HNF4a and HNF4c [30,31], but it remains unclear what ligands are bound when the receptor is expressed in its native physiological environment.There have also been somewhat controversial reports of fatty acyl Co-enzyme A thioesters as HNF4a ligands [32,33].
These studies highlight the need to distinguish between ligands that may bind under non-native conditions and those that are the true endogenous ligands.The most rigorous definition of a true endogenous ligand is a compound that binds the LBP in vivo in the absence of experimental probes or other perturbations.Identification of ligands by this definition requires new technical approaches.
In addition to identification of endogenous ligands for orphan (and other) nuclear receptors, new experimental tools to identify ligands are also needed to address the role of ligands in the evolution of the nuclear receptor superfamily [34][35][36].Examination of HNF4 is also instructive in this regard as it is present in the earliest metazoan organisms and is one of the most evolutionarily conserved nuclear receptors [37,38].Therefore, the question of whether HNF4 binds an endogenous ligand, the identity of that ligand and its effect on HNF4 transcriptional activity is of particular interest and may be relevant to the entire receptor superfamily.However, these issues cannot be addressed without an assay that can identify potential ligands in the absence of a presupposed function.
Here we use an affinity isolation/mass spectrometry (AIMS) approach to identify the endogenous ligand that is bound to HNF4a in mammalian cells and in mouse liver.The approach is unbiased in that it does not make any pre-assumptions as to what the ligand might be or how it might affect HNF4a function.Our results indicate that the vast majority of HNF4a is bound to a single essential fatty acid: linoleic acid (LA).Furthermore, our results show that the binding is reversible, indicating that LA is an exchangeable ligand.To our knowledge, this represents the first time an endogenous ligand has been identified by virtue of its association with a nuclear receptor in animal tissue.

Identification of an endogenous mammalian HNF4a ligand
To identify an endogenous ligand for HNF4a, we utilized an affinity isolation/mass-spectrometry (AIMS) approach outlined in Figure 1A.Wild type (wt) rat HNF4a2 was expressed in mammalian COS-7 cells and gas chromatography/mass spectrometry (GC/MS) was used to analyze the compounds bound to HNF4a2 immunoprecipitated (IP'd) from nuclear extracts.We found that HNF4a2 was associated with linoleic acid (LA, 9,12octadecadienoic acid, C18:2-D9,12) (Figure 1B, top panel), an essential dietary polyunsaturated fatty acid not typically found in E. coli.Quantification of the levels of LA and wt HNF4a2 protein indicated that 30-100% of the receptor is occupied by LA, depending on the experiment.Although the presence of trace amounts of other fatty acids cannot be excluded, the only peak clearly identified was that of LA.These findings suggest that LA represents the predominant fatty acid associated with HNF4a in mammalian cells.Although unlikely, a trivial explanation for these findings is that LA is not bound to HNF4a within cells, but becomes associated with the receptor after lysis.To rule out this possibility, deuterated LA ([ 2 H]LA) was added to the lysed cells and AIMS was performed as in Figure 1A.As expected, HNF4a was found associated with cellular [ 1 H]LA but not with bufferspecific [ 2 H]LA (see Supplementary Figure S1), indicating that the HNF4aNLA complex forms in cells prior to lysis.
To determine the specificity of LA binding, we asked whether LA, or other fatty acids, were bound to HNF4a proteins containing mutations in the LBP.We first mutated a key arginine residue (R226) that establishes a critical charge-charge interaction with the carboxylic acid moiety of the fatty acid [30,31].This R226M mutant did not bind LA (Figure 1B, middle), indicating that binding is specific to the LBP.Similarly, a V255M mutation that is found in patients with MODY1 [39,40] also failed to bind LA (Figure 1B, bottom).Taken together, these findings demonstrate that LA specifically binds to the HNF4a LBP within mammalian cells.
We next examined whether LA could be removed or replaced from the HNF4a LBP by manipulating the media conditions.In cells grown in lipid-depleted serum, some LA remained associated with HNF4a2, suggesting a relatively tight interaction (Figure 1C, top panel).Nonetheless, addition of exogenous LA to the lipiddepeleted media resulted in a ,3-fold increase in the amount of LA bound (peak intensity 2.2610 7 v. 8.6610 6 without LA) (Figure 1C, middle).Although we could force this increase in LA binding, we did not observe an increase in binding of a related fatty acid (palmitoleic acid, PLA, C16:1) provided under similar conditions (Figure 1C, bottom).These findings further demonstrate that HNF4a does not effectively associate with other fatty acids when expressed in mammalian cells.
To further confirm binding specificity, we sought to create an orthologous receptor-ligand pair that does not bind endogenous LA.As noted above, the guanidinium group of R226 makes direct contact with the carboxylic head group of fatty acids (Figure 1D, left) [31].We asked whether an artificial receptor-ligand pair could be established by reversing this charge interaction -i.e., by converting R226 to a negatively charged glutamate residue (R226E) whose carboxylate group is predicted by molecular modeling to interact with the amine moiety of linoleamide (LAA), a positively charged amide derivative of LA (Figure 1D, right).Indeed, AIMS analysis verified that R226E bound LAA in cells, whereas binding to LA could not be detected (Figure 1E).Taken together, these findings demonstrate the lipid binding specificity of HNF4a and suggest that LA binds HNF4a via the R226 guanidinium-carboxylate interaction.

HNF4a ligand binding is reversible in mammalian cells
Previous studies suggested that a variety of fatty acids bind in a non exchangeable fashion to bacterially expressed HNF4a and HNF4c [30,31].The inability to exchange with free fatty acids would exclude the type of ''on/off'' regulatory switch that is characteristic of ligand-modulated nuclear receptors.If so, the fatty acid might serve more like a co-factor than a hormone [41].However, these conclusions were based on in vitro studies using HNF4 expressed in bacterial environments where protein folding, redox state and lipid milieu differ dramatically from the native mammalian environment.Thus, we utilized the AIMS technique to determine the ability of bound LA to freely exchange within mammalian cells.On-rate experiments were performed by adding [ 2 H]LA to cycloheximide-treated cells (Figure 2, left).In the absence of new HNF4a2 synthesis, we found that within 5 hours more than half the endogenous [ 1 H]LA that bound to existing HNF4a2 was replaced with exogenous [ 2 H]LA.Conversely, offrate experiments showed that pre-bound [ 2 H]LA was replaced with exogenously added [ 1 H]LA within a similar time frame (Figure 2, right).These results indicate that LA is an exchangeable ligand that binds native HNF4a in a reversible fashion.

The HNF4a ligand binding pocket is unoccupied in the livers of fasted animals
We next sought to determine what ligands may bind HNF4a in vivo under relevant physiological conditions.Livers were removed from young adult male C57BL/6 mice that were either fed a standard diet (fed), fasted for 24 hrs (24-hr fast), or fasted for 24 hrs followed by refeeding (re-fed).Hepatic HNF4a was then IP'd from isolated nuclei and quantified by immunoblot (IB) analysis (Figure 3A and 3B).GC/MS analysis of the isolated protein demonstrated that HNF4a bound LA in both the fed and re-fed state but not after a 24-hr fast (Figure 3C).No other fatty acid was found bound to HNF4a even when dietary LA was depleted by fasting, further confirming a preference of HNF4a for LA.Gel shift analysis showed that HNF4a protein from livers of fasted animals binds DNA well, indicating that the LA-free HNF4a retains DNA-binding function (Supplementary Figure S2).The decrease in HNF4a-bound LA was surprising since the total free fatty acid pool increases in fasting livers [42].However, LA did not increase in 24-hr fasted livers; if anything, LA levels decreased by ,27% in fasted vs. fed or re-fed livers (Figure 3D).This is consistent with the dietary requirement for LA and may at least partially explain the decrease in HNF4a occupancy in fasted mice (Figure 3C).These findings demonstrate that LA is bound to native HNF4a in vivo, and that binding is a physiological marker of the fed, but not the fasted, state.

Function of HNF4a ligand binding
For classical receptor-ligand pairs, ligand binding induces a conformational change that results in co-regulator recruitment and subsequent transcriptional modulation.Therefore, many previous attempts at ligand identification for orphan receptors utilized transcriptional activation and/or coactivator recruitment as the primary screen.However, unlike AIMS, these approaches are biased in that they can only identify ligands that induce transcriptional activity.Nonetheless, once we had identified LA in AIMS as binding HNF4a, it seemed reasonable to determine whether LA could transcriptionally activate HNF4a2 in transfected CV-1 cells exposed to lipid-depleted medium.No positive effect of LA was observed on full length HNF4a2 with or without expression of PGC1a (data not shown), a key coactivator that mediates HNF4a activity in the liver [43].Since lack of apparent LA-responsiveness could reflect the high constitutive activity mediated by the ligand-independent AF-1 domain [44], we next examined N-terminally deleted (DN) HNF4a2 constructs that lack the AF-1.LA also had no effect on DN-HNF4a2 in the presence (Figure 4A) or absence (data not shown) of PGC1a.Similarly, significant LA-responsiveness was not observed on other wellcharacterized HNF4a responsive promoters (ApoB, PEPCK, G6Pase, data not shown).These findings provide further support for the utility of the AIMS approach since LA cannot be identified as an HNF4a ligand by commonly used transcriptional screening strategies.
The apparent lack of LA responsiveness could reflect a constitutive activity that is inherent to ligand-free HNF4a.To explore this possibility, we examined the transcriptional activity of the DN derivatives of the two non-LA binding HNF4a mutants (DN-V255M and DN-R226M) identified in Figure 1B.Since both mutants retained significant constitutive activity in the presence of PGC1a (Figure 4: V255M, 100% activity; R226M, 63%), we conclude that unoccupied HNF4a can activate transcription.Similar experiments with HNF4a R226E and its orthologous ligand LAA were not interpretable as R226E does not bind DNA well (data not shown).Nonetheless, the HNF4a mutants that were tested demonstrate that LA-free HNF4a, which is characteristic of the fasted state, retains significant transcriptional activity (Figure 4 and data not shown).
Despite the clear ligand-independent transactivation observed in the transfection assays on select target genes, it remained possible that ligand binding could modulate HNF4a activity on other target genes that we did not examine.It has been well established for other NR-ligand pairs that ligands can have different effects on different promoters [9,[45][46][47].Furthermore, we hypothesized that LA might have a more pronounced effect on endogenous targets with the appropriate chromatin structure.Therefore, we used a genome-wide approach to ask whether there  are any endogenous HNF4a target genes that are affected by exogenously added LA.We first verified by GC/MS that we could sufficiently deplete tissue culture cells and HNF4a of endogenous LA by incubating the cells in serum free media for 60 hr (Supplementary Figure S3).Then we used a recombinant adenovirus system to ectopically express HNF4a in a human colon cancer cell line, HCT116, which does not express endogenous HNF4a protein (Supplementary Figure S4); since HNF4a is normally expressed in the colon, this cell line should provide the appropriate environment to examine the effect of HNF4a on endogenous target genes.The adenovirus system also allowed for a prolonged incubation in serum free media in order to maximally deplete the cells of LA, while still being able to express HNF4a (Supplementary Figure S4).
We performed expression profiling using Affymetrix whole human genome Gene Chips on infected cells incubated in the absence or presence of 30 mM LA for 60 hrs.The results show that whereas the majority of the genes (72%) were not affected by the addition of LA in the absence of HNF4a (Figure 5, left pie chart), among the 168 genes that were constitutively activated 3-fold or more by HNF4a expression, ,73% were down regulated 20% or more by the addition of LA.Interestingly, only two HNF4a target genes were up regulated 20% or more by LA (Figure 5) (see Supplementary Table S1 for a list of HNF4a targets affected by LA).We next examined the effect of LA on several individual HNF4a target genes by quantitative real time PCR (qRT-PCR) and again observed a modest decrease in target gene expression, verifying the array results (Figure 6B).However, we also noticed that there was a modest but consistent decrease in the amount of HNF4a protein in the samples containing LA (Figure 6A).Therefore, we normalized each individual qRT-PCR result to its appropriate HNF4a protein level and found that the decrease by LA was no longer significant (Figure 6C).These results confirm that ligand-free HNF4a has considerable constitutive transactivation activity and that the presence of LA does not enhance that activity.If anything, the presence of LA seemed to modestly repress the constitutive activity of HNF4a, although that effect may be mediated by an alteration in the HNF4a protein level.

Discussion
Orphan nuclear receptors are central regulators of a wide range of physiological and pathological events.Over the past ,20 years, a number of orphan receptors have been ''de-orphanized,'' yet some of those ligand assignments may not represent the actual ligand that is bound to the receptor in vivo -i.e., the true endogenous ligand (TEL).Identification of synthetic or nonendogenous ligands remains critical as those compounds serve as important experimental tools and provide critical mechanistic insights into the function of nuclear receptors.Nonetheless, a complete understanding of receptor-regulated networks requires knowledge of the TELs as well as how the TELs are generated and what their affect may be on receptor activity.Existing experimental strategies are not specifically designed to identify TELs and are often biased by an implicit assumption that the TEL is contained within a particular compound library or biological extract.Our work establishes the AIMS approach as a viable strategy to identify TELs based purely on their association with a receptor in vivo: no pre-assumptions are required as to the nature of the ligand.AIMS is likely to be applicable to virtually all NRs as the only requirement is an antibody, or other means to selectively isolate the receptor.Perhaps the only limitation to this approach is the ability to obtain sufficient amounts of native receptor to ensure that the cognate ligand is within the detection limits of the assay.
In addition to identifying endogenous ligands, the AIMS approach has the unique advantage of being a functionindependent assay.Many of the existing screening technologies are biased by the assumption that the ligand functions as a transcriptional modulator -this excludes the possibility of identifying NR ligands that selectively modulate non-genomic NR functions such as kinase activation, proteasomal degradation and intracellular trafficking, etc. [48][49][50][51].Indeed, the existence of some TELs may have been overlooked because they would not have been detected by transcription-or coactivator-based screens.HNF4a is a case in point: here we use the AIMS approach to demonstrate that LA is the endogenous ligand bound to rat, mouse and human HNF4a (Figures 1,2,3 and S3) and that addition of LA fails to alter the activation by the receptor (Figures 4,5,6).

HNF4a is transcriptionally active in the absence of ligand
We also show here that during fasting native HNF4a exists in an LA-free state (Figure 3), and that the ligand-free HNF4a-PGC1a complex is transcriptionally active (Figure 4).The ligandindependent transcriptional activity that is characteristic of HNF4a in the fasted state is consistent with the physiology of the fasted liver.Indeed, hepatic gluconeogenesis is stimulated by fasting-induced expression and recruitment of PGC1a to gluco- neogenic HNF4a target genes [43].Ligand-independent activity is also consistent with structural studies that show that the position of the HNF4a AF-2 is altered primarily by binding of co-factor peptides [52].Thus, our finding of transcriptional activity for ligand-free HNF4a is consistent with the known structural and physiological activities of this receptor.

HNF4-LA, a primitive receptor-ligand pair?
The origin of NRs and their ligands is a hotly debated topic (see [34] and references therein), and one that can benefit from experimental approaches such as the AIMS assay.There are basically two camps, one that proposes that the ancestral NR was unliganded (e.g., [53]) and the other that proposes that NRs always had ligands (often referred to as the ligand exploitation hypothesis) (e.g., [54]).Since HNF4 is one of the few NRs found in primitive metazoans such as sponge and coral reef [37,38], it could be viewed as an ancient receptor and possibly even one of the founders of the superfamily.As such, it is tempting to speculate on the significance of our findings on the evolution of receptor-ligand pairs.There is evolutionary logic to the notion that a relatively simple lipid (such as LA) would have been available to ancient organisms and could thus serve as a primordial ligand [55].Binding of more complex ligands (e.g., steroids, retinoids, etc.) may have been a later development associated with more recently evolved receptor proteins.If that is the case, and if the more ancient forms of HNF4 also bound LA, then the results presented here could impact the discussion of the origins of ligand-dependent transcription.One possibility is that the modern HNF4aNLA pair accurately reflects the primitive receptor-ligand pair, which would suggest that ligand binding activity was acquired in advance of the ability to modulate transcription.It would also suggest that subsequent receptor-ligand pairs acquired the AF-2-mediated conformational switch associated with transcriptional activation later on during evolution.If so, HNF4 could represent a ''missinglink'' in the evolutionary pathway from a non ligand-regulated transcription factor to a transcription factor that can both bind and respond to ligand.The alternative hypothesis is that ligand binding and ligand-regulated transcription appeared at the same time for HNF4 but ligand-regulated transcription was subsequently lost at some point during the evolution to modern mammalian HNF4a.Additional studies are clearly required to appropriately address these important questions; the application of the AIMS assay to ancient NRs should prove useful in this regard.
Is there a non transcriptional function for the HNF4a ligand?
It remains possible that the HNF4 ligand used to have, and/or continues to have, an as yet-to-be determined function that is distinct from transcriptional modulation.The physiological linkage of receptor occupancy to the fasting/fed state raises the possibility that the ligand contributes to a non-genomic mechanism that affects this physiological transition.Potential non-genomic activities that have been linked to other nuclear receptors include kinase activation, proteasomal degradation and intracellular trafficking, etc. [48][49][50][51].The identification of LA as an endogenous HNF4a ligand will open up new avenues of research that explore the potential for LA to modulate non-transcriptional activities.This demonstrates the power and utility of the AIMS approach that identifies ligands based on binding rather than preconceived notions of function.

Resurrection of HNF4a as a drug target?
Previous studies suggested that a wide variety of fatty acids are associated with bacterially expressed HNF4 [30,31], though LA was not among these.In retrospect, this is undoubtedly due to the fact that E. coli do not have LA [56].Although HNF4aNLA interactions were not examined in those studies, the authors mention that the fatty acids they did observe bound to HNF4 could not be removed without denaturing the protein.This in turn led to the notion that synthetic HNF4a ligands would have little utility as they would not be able to compete with a non exchangeable endogenous ligand.Hence, HNF4a, despite its many links to human disease, was no longer deemed a suitable drug target.In sharp contrast, we show here that when HNF4a is expressed in its native environment, it specifically and reversibly associates with one predominant fatty acid: LA.We also show that under certain physiological conditions, the LBP of HNF4a is unoccupied indicating that ligand binding is specifically linked to physiological status.These findings suggest that HNF4a may indeed be a tractable target for small molecule drug discovery.The predicted function of such synthetic ligands, however, will benefit from an understanding of the functional role of the endogenous ligand.

Materials and Methods
37 hr after transfection, fresh media containing 30 mM [ 2 H]LA was added and the cells were incubated at 37uC for 5 hr to allow for exchange with pre-bound, non-labeled endogenous LA.Cells were then treated with 50 mM cycloheximide for 1.5 hr.To remove the [ 2 H]LA, the media was replaced with fresh media containing 30 mM non-labeled LA and 50 mM cycloheximide.Nuclear extracts were prepared after 0, 1, 2, 3 and 5 hr and analyzed as described above for the on-rate experiments.

Mouse liver extraction for LA measurement
To determine the amount of LA in whole liver tissue of fed, fasted, and 24 hr fasted animals, mouse liver tissue (n = 6 per condition, ,20 mg per liver) was homogenized in PBS, then extracted with 1.1 ml of ethyl acetate.PBS containing various concentrations of an LA standard were extracted simultaneously and used to establish a standard curve.Heptadecanoic acid (20 nmole, Sigma) was added to all samples as an internal control for extraction efficiency.

Identification of candidate LA-responsive HNF4a target genes
Human colorectal carcinoma HCT116 cells (#CCL-247, ATCC) maintained in McCoy's 5A (Hyclone) media supplemented with 10% Tet System Approved fetal bovine serum (Clontech) and penicillin/streptomycin at 37uC and 5% CO 2 were grown to 50-60% confluency at which point the media was changed to McCoy's 5A, penicillin/streptomycin plus 0.15% fatty acid-free BSA (EMD Biosciences) plus vehicle (0.1% DMSO) or 30 mM linoleic acid (LA) (Sigma).The cells were then immediately coinfected with 2.5 MOI each of the Adeno-X Tet-On virus (Clontech) and the recombinant adenovirus expressing VSVtagged rat HNF4a1 under control [58].(HNF4a1 differs from HNF4a2 by 10 amino acids in the F domain; both activate transcription well although HNF4a2 is somewhat more responsive to co-activators than HNF4a1 [59].The DNA binding domain of rat and human (and mouse) HNF4a is 100% identical and hence these species are anticipated to have very similar if not identical DNA binding specificity (see Supplementary Figure S5).At 12 hr post infection, 0.5 mg/ml doxycycline (Sigma) was added to induce expression of HNF4a and the cells were maintained under serumfree conditions with vehicle or 30 mM LA for an additional 48 hr (60 hr total in serum-free conditions with vehicle or LA).All cells received the same amount of doxycycline and Tet-On virus, but only those cells also infected with the Adeno.ratHNF4a1.VSV expressed HNF4a protein.RNA isolated from one-half of three biological replicate plates using the RNeasy Mini kit (Qiagen) was pooled and hybridized to the GeneChip Human Genome U133 plus 2.0 (Affymetrix) as per the manufacturer's protocol in the Genomics Core in the UCR Institute for Integrated Genome Biology.Each pooled RNA sample was analyzed on two separate arrays and the results were averaged (8 arrays total for the four different conditions -+/2LA +/2HNF4).Whole cell extracts harvested from the other half of the plate [60] were used to verify HNF4a protein levels by IB analysis.

Analysis of Array Data
All arrays were analyzed using GC Robust Multi-array Average (GCRMA) background adjustment and quantile normalization on probe-level data sets with R software (http://www.bioconductor.org).When comparing two categories (i.e., +/2LA or +/2HNF4) we included only those probe sets for which at least one treatment group had 100% Present (P) or Marginal (M) calls (determined by the MAS5 algorithm), as has been previously described [61].
Quantitative Real-Time PCR Quantitative real-time PCR (qRT-PCR) analysis was used to verify the relative expression of select genes, with preference given to either known HNF4a targets [21], or genes that contained promoters previously indicated in the literature to bind HNF4a in human cells [22,62].Genomic DNA was removed by DNAse I treatment at 37uC for 1 hr and the RNA was reverse transcribed using random hexamers and SuperScript III (Invitrogen).The resulting cDNA was diluted and analyzed using a MyiQ singlecolor real-time PCR detection system (Bio-Rad) and SYBR Green.Gene-specific primers (see Supplementary Table S2) were validated over four orders of magnitude and analyzed with the iQ5 Optical System Software (Bio-Rad); primer pairs were deemed valid if an input log plot amount versus C T generated an efficiency of 100%610% and a correlation coefficient of R 2 = 0.95060.05.Each biological triplicate was analyzed in technical triplicate.The relative expression level of the genes was evaluated using the Pfaffl method [63], normalizing to cyclophilin A (PPIA) expression.

Supporting Information
Supporting Materials and Methods S1 Reagents, plasmids, ectopic expression of HNF4a proteins in COS-7 cells, preparation of mouse liver nuclear extracts, immunoblot (IB) analysis, reporter gene assay, molecular modeling are described in detail.Found at: doi:10.1371/journal.pone.0005609.s001(0.09 MB DOC) Figure S5 Alignment of human, mouse and rat HNF4a2 amino acid sequence.Alignment of the amino acid sequence of these receptors shows the high degree of similarity, especially in the DBD and LBD, including the residues that contact that the ligand.Found at: doi:10.1371/journal.pone.0005609.s006(0.12 MB PDF) Table S1 Effect of linoleic acid on HNF4a target genes (Affymetrix array results).Found at: doi:10.1371/journal.pone.0005609.s007(0.08 MB PDF) Table S2 Primers used for qRT-PCR to verify HNF4a target genes.Found at: doi:10.1371/journal.pone.0005609.s008(0.06 MB PDF)

Figure 1 .
Figure 1.Identification of linoleic acid (LA) as the endogenous ligand for mammalian-expressed HNF4a2.(A) Affinity isolation/massspectrometry (AIMS): rat HNF4a2 was purified by immunoprecipitation (IP) from crude nuclear extracts of transfected COS-7 cells using an HNF4a specific antibody (HNF4a Ab).The amount of HNF4a2 protein recovered was determined by immunoblot (IB) analysis and bound ligands bound were identified by GC/MS.(B) GC/MS chromatograms (10 to 12 min) comparing compounds extracted from IP'd material from mock-transfected (black) to HNF4a2-transfected (red) (wt and LBP mutants R226M and V255M) COS-7 cells grown in 10% bovine calf serum (Full Serum).Insets: HNF4a IB showing input (In, 2% of total), material not bound by the Protein A Sepharose (unbound, UB, 2%), and IP'd material (IP, 4%).(C) Same as in (B) except lipid-depleted serum (Stripped Serum) was used and vehicle (DMSO), linoleic acid (LA, 30 mM) or palmitoleic acid (PLA, 30 mM) were added to the media as indicated.Arrow, position of LA and PLA peak as determined by standards.(D) Predicted model of HNF4a2 (WT) with bound LA

Figure 2 .
Figure 2. Binding of LA to HNF4a2 is exchangeable in mammalian cells.The on-and off-rate of LA binding to rat HNF4a2 in mammalian cells was determined by incubating COS-7 cells transfected with HNF4a2 wt with [ 2 H]LA (30 mM) for 1 to 5 hr after (on-rate) or before (off-rate) a 1.5hr treatment with cycloheximide (50 mM, CHX).[ 1 H]LA (open) and [ 2 H]LA (filled) bound to HNF4a2 was determined as in Figure 1 and graphed as a percent of the total bound LA normalized to the amount of HNF4a2 protein in the immunoprecipitate.Absolute amounts of LA and HNF4a2 protein are noted above each bar.doi:10.1371/journal.pone.0005609.g002

Figure 3 .
Figure 3. Native hepatic HNF4a binds endogenous LA in fed but not fasted mice.(A) Design: As in Figure 1A except with livers from male C57BL/6 mice fed a standard diet (Fed), fasted for 24 hr (24hr Fast) or fasted for 24 hr and re-fed for 24 hr (Re-fed).(B) HNF4a from hepatic nuclei was IP'd in the absence (2Ab) or presence (+Ab) of the HNF4a-specific antibody and quantified by IB.HNF4a resolves as a doublet in this gel system.In, 2% of total input; UB, 2% of unbound material; IP, 2% of IP'd material.(C) GC/MS chromatograms (9 to 11 min) comparing compounds extracted from IP'd material from fed, fasted and re-fed animals with (red) or without (black) HNF4a Ab.Arrow, LA peak.(D) Quantification of the amount of LA in the liver of fed, 24-hr fasted and re-fed mice using GC/MS.Shown are average

Figure 5 .
Figure 5. Genome-wide expression profiling of HNF4a in the presence and absence of LA.HCT116 cells infected with a tetracyclineinducible recombinant adenovirus expressing rat HNF4a1 (Adeno.ratHNF4a1.VSV) were incubated for 60 hr in serum free media in the absence (DMSO) or presence of 30 mM LA.The experiment, consisting of four experimental conditions (2HNF4 2LA, 2HNF4 +LA, +HNF4 2LA, +HNF4 +LA), was performed in biological triplicate.All samples had doxycycline and the Tet-On virus; samples without HNF4a (2) and with HNF4a (+) differed only in infection with Adeno.ratHNF4a1.VSV.Pie charts, results of Affymetrix expression profiling.Effect of LA ($20% change) on all genes in the absence of HNF4a (left); effect of LA ($20% change) on 168 genes up regulated $3-fold by HNF4a (right).doi:10.1371/journal.pone.0005609.g005

Figure 6 .
Figure 6.Effect of LA on HNF4a protein levels and target gene expression.Experimental design as described in Figure 5. (A) One of five representative IBs performed on whole cell extracts of adenovirus-infected HCT116 cells (left) and average quantification of all five blots normalized to Coomassie stain (right).(B,C) qRT-PCR results normalized to cyclophilin A (PPIA) mRNA (B) or to PPIA mRNA and HNF4a protein levels (C) of select HNF4a targets.The average qRT-PCR result from technical triplicates of each biological sample was normalized to the appropriate HNF4a protein level.Error bars represent standard deviation of the biological plus technical replicates.Statistics: two-tailed t-test, +HNF42LA vs. +HNF4+LA.*, p,0.05.doi:10.1371/journal.pone.0005609.g006

Figure S1 Figure S2
Figure S1 Binding of LA to HNF4a2 occurs within cells.Deuterated LA was added to nuclear extracts before IP and subsequent GC/MS.The ratio of [ 1 H]LA to [ 2 H]LA shows that the LA that is bound to HNF4a2 is derived from endogenous [ 1 H]LA.Found at: doi:10.1371/journal.pone.0005609.s002(0.07 MB PDF) Figure S2 HNF4a from fasted mouse liver binds DNA.EMSA of nuclear extracts from the livers of fed and 24-hr fasted mice showing that HNF4a from fasted animals binds DNA, indicating that the apoHNF4a is functional.Found at: doi:10.1371/journal.pone.0005609.s003(1.25 MB PDF) Figure S3 Validation of experimental system for LA candidate gene identification I. GC/MS of endogenous HNF4a IP'd from human tissue culture cells (Hep3B) incubated in media without serum +/2 LA for 60 hr showing that LA can be depleted from the cells and HNF4a.Found at: doi:10.1371/journal.pone.0005609.s004(0.23 MB PDF) Figure S4 Validation of experimental system for LA candidate gene identification II.IB analysis of extracts from human colon cancer cells HCT116 infected with recombinant adenovirus expressing HNF4a showing a lack of expression of endogenous HNF4a and a robust expression of the recombinant HNF4a.Found at: doi:10.1371/journal.pone.0005609.s005(6.90 MB PDF)