IDO1 gene is highly inducible by IFN-γ and TNF-α through interaction with cis-acting regulatory elements of the promoter region. Accordingly, functional polymorphisms in the IDO1 promoter could partly explain the interindividual variability in IDO expression that has been previously documented.Indoleamine 2,3-dioxygenase (IDO) catalyzes the first and rate-limiting step of the kynurenine pathway that is an important component of immunomodulatory and neuromodulatory processes. The IDO1 promoter, which correlates significantly with serum tryptophan concentration, controlled partially by IDO activity, in female subjects, but not in males. Although this VNTR does not appear to affect basal or cytokine-induced promoter activity in gene reporter assays, it contains novel cis-acting elements. Three putative LEF-1 binding sites, one being located within the VNTR repeat motif, were predicted in silico and confirmed by chromatin immunoprecipitation. Overexpression of LEF-1 in luciferase assays confirmed an interaction between LEF-1 and the predicted transcription factor binding sites, and modification of the LEF-1 core sequence within the VNTR repeat motif, by site-directed mutagenesis, resulted in an increase in promoter activity.A PCR-sequencing strategy, applied to DNA samples from healthy Caucasians, allowed us to identify a VNTR polymorphism in the IDO1 promoter revealed a cis-acting element interacting with the most downstream factor of the Wnt signaling pathway, suggesting novel mechanisms of regulation of IDO1 expression. These data offer new insights, and suggest further studies, into the role of IDO in various pathological conditions, particularly in cancer where IDO and the Wnt pathway are strongly dysregulated.The identification of a VNTR in the  The heme enzyme indoleamine 2,3-dioxygenase  catalyzes the initial and rate-limiting step of the kynurenine pathway that converts 95% of the essential amino acid tryptophan (Trp) to kynurenine derivatives. IDO is constitutively expressed in many cells and tissues and is also a cytokine-inducible enzyme, with IFN-gamma (IFN-γ) being the most potent known transcriptional inducer of IDO. The overexpression of IDO has a dual consequence: depleting Trp in local microenvironments and increasing the concentration of downstream metabolites of the kynurenine pathway.via IDO overexpression has also been shown to be involved in the immune escape of tumor cells which is a crucial feature of cancer progression IDO activation is of great interest in the field of immunology as a consequence of Trp starvation, as well as the generation of immunomodulatory kynurenine metabolites. It results in the inhibition of growth of various pathogens and, thus, participates in antimicrobial host defence mechanisms Furthermore, as Trp is also involved in neurological functions as a precursor in the synthesis of serotonin, a neurotransmitter that modulates mood, cognition and several neuroendocrine rhythms In vitro studies have shown that the response of IDO to IFN-γ stimulation varies greatly between various human cell lines Although the role of IDO and its induction by cytokines are now well recognized, not many studies have focused on the mechanisms of variability of IDO activity, which could have numerous relevant clinical implications. IDO1) which were shown in vitro to be responsible for impaired functional activity and could therefore result in reduced IDO activity in the general population IDO1 were screened for nucleotide variations. As the promoter of IDO1 carries numerous well-identified responsive element sequences that are involved in the regulation of IDO expression IDO1 gene. The screening of the IDO1 promoter in healthy Caucasian subjects allowed us to identify a Variable Number of Tandem Repeats (VNTR) polymorphism. A significant association of this VNTR with decreased circulating tryptophan concentrations was observed in healthy female subjects. The contribution of the VNTR polymorphism to the variability of IDO expression was further studied by functional analyses. Using in vitro and in silico approaches, we revealed the existence of previously unknown cis-acting regulatory elements that could participate in transcriptional regulation and, therefore, in interindividual variability of IDO1 expression.The currently known causes of variation in IDO activity remain sparse. Physiological states such as ageing or pregnancy, as well as pathological conditions such as infection, inflammation or tumor proliferation, have been demonstrated to modify IDO expression and/or activity, but they all mainly reflect the various mechanisms involved in the regulation of IDO expression IDO1, a PCR-sequencing strategy was developed and applied to a 1576 bp-sequence upstream of the ATG initiation codon. Sequencing was applied to PCR products amplified from genomic DNA samples of 41 unrelated healthy individuals of Caucasian origin. A VNTR polymorphism was identified in both heterozygous and homozygous states. This VNTR polymorphism comprises a 24-bp tandem repeat motif located approximately 1.3 kb upstream of the ATG initiation codon  and 47 females . As serum samples were collected after an overnight fast, the effects of diet on circulating Trp levels can be considered as negligible. First, the complete sequencing of the 1.6-kb promoter region of IDO1 excluded the presence of any other polymorphism in the 94 tested samples, and none of the samples carried the functional coding variants, i.e. the 9-bp deletion and/or the rs35099072 SNP. Mean (± S.D.) values of serum Trp (µM), serum Kyn (µM) and Kyn/Trp ratio (µmol/mmol), in all tested subjects, as well as in males and females separately, are reported according to VNTR genotypes in versus 66.1±15.6 µM; p = 0.0006). In all subjects, no statistically significant association was observed between the VNTR genotype and serum Trp, serum Kyn or Kyn/Trp ratio. However, a lower Trp concentration was observed for females with a *V2/*V2 genotype compared to those with a *V1/*V1 (p = 0.076) or a *V1/*V2 (p = 0.028) genotype. Furthermore, the Trp concentration in females with a *V2/*V2 genotype was significantly lower than that in males with a *V1/*V1 (p = 0.008), *V1/*V2 (p = 0.01) and *V2/*V2 (p = 0.0007) genotype.In order to study the possible association between the VNTR genotype and IDO activity, tryptophan and kynurenine concentrations were measured in sera from 94 subjects of the Haguenau cohort, comprising 47 males . Stimulation in the presence of both IFN-γ and TNF-α resulted in a 250- and 277-fold increase in *V1 and *V2 luciferase activity, respectively . These data confirm the induction of IDO1 expression via a transcriptional mechanism through cytokines stimulation. Note, however, no statistical difference was observed between *V1 and *V2 luciferase activities in any of the above experimental conditions.A gene reporter assay was developed in order to assess the impact of the VNTR on the transcriptional activity of the IDO1 promoter, the 1.6-kb promoter region of IDO1 was analysed using MatInspector prediction software. Three critical putative binding sites for the LEF-1/TCF transcription factor were identified in the *V1 allele, one site being located within the 24-bp motif of the VNTR, as shown in *V2 allele, which carries two repeat motifs of the VNTR, exhibits four potential TFBSs for LEF-1/TCF in the 1.6-kb promoter region.To assess whether the VNTR polymorphism could have an influence on upstream regulatory factors of the *V1 allele, and the successful chromatin immunoprecipitation was checked via the GAPDH positive control. Three specific primer pairs were designed for PCR amplification of LEF-1 genomic sequences from immunoprecipitates.The results shown in IDO1 promoter, which confirms the LEF-1/TCF binding sites predicted by MatInspector.ChIP assays were used to confirm the presence of potential binding sites for LEF-1/TCF predicted by MatInspector. The assays were conducted with extracts of HeLa cells, that were genotyped as homozygotes for the *V2 allele relative to *V1, gene reporter assays were conducted with the overexpression of an activated form of LEF-1. In this assay, activated LEF-1 is expressed due to the VP16-TCF expression vector possessing a virtual DNA-binding domain for TCF/LEF and a VP16 activation domain, in place of a β-catenin-binding domain IDO1 promoter activity for both *V1 and *V2 alleles. However, under the experimental conditions, no significant difference in promoter activity was observed between the two VNTR alleles.To evaluate the functional significance of the additional TFBS for LEF-1/TCF in the IDO1, the *V1 allele was modified by mutating the core sequence of the binding site localized in the 24-bp motif of the VNTR by site-directed mutagenesis, such that the MatInspector software could no longer identify a binding site at this location. Thus, a third pGL4 construct containing the mutated core sequence of the LEF-1/TCF binding site (pGL4-V1Mut) was used in gene reporter assays, as described previously .To investigate the influence of the TFBS for LEF-1/TCF on the transcriptional activity of the 1.6-kb promoter region of eviously . As showAdditional gene reporter assays conducted with the overexpression of an activated form of LEF-1 showed no statistical difference in the promoter activity of pGL4-V1Mut compared to pGL4-V1 and pGL4-V2, under basal conditions . FurtherIDO1 gene would be of particular interest in association studies between IDO and various pathological conditions. Several variations in the IDO1 genomic sequence have already been documented in sequence databases, but their functional consequences have not yet been studied et al. IDO1 gene. Two of these, a missense mutation  and a 9-bp deletion in exon 7, result, in vitro, in significantly reduced protein expression and, consequently, in almost complete loss of enzyme activity IDO1 polymorphisms, since each was identified in only one African-American subject, and, accordingly, can be expected to have a minor phenotypic effect in the general population. In addition, Nishizawa et al. IDO1 genomic variations as a potential determinant of pre-eclampsia. The sequencing of placental genomic DNA from pre-eclamptic and normotensive pregnant women revealed three new rare IDO1 variants, consisting of one silent mutation, one missense mutation (p.Arg193Gly) and a 4-bp deletion in the proximal 5′-UTR. However, no functional analyses were performed for the two coding SNPs, and the 4-bp deletion did not affect gene expression of IDO1 in in vitro experiments Variability in IDO activity can result in significant imbalances in the serotonin/kynurenine pathways which could have clinical immunological and neuropsychiatric implications. As inherited variants of coding and non-coding sequences of a gene are recognized as an important cause of interindividual variations in protein expression and/or activity, the identification of functional genetic polymorphisms in the cis-acting elements spanning through the promoter region and transcription factors, variations in the sequence of cis-acting elements could have an effect on gene expression IDO1, the promoter region has been well characterized and contains several cis-acting response elements that are involved in the up-regulation of the gene by cytokines, such as IFN-γ, the most potent inducer of IDO activity, as well as TNF-α IDO1 promoter activity, with a synergistic activation by IFN-γ and TNF-α IDO1 promoter region, consisting of a 24-bp repeat motif located 1.3-kb upstream of the ATG initiation codon. It was identified by a PCR-sequencing strategy applied to 41 DNA samples and allowed us to characterize two different alleles, named *V1 and *V2, that carry one or two repeats, respectively, the *V1 allele corresponding to the reference sequence listed in Genbank (accession number NC_000008.10). This polymorphism appears to be common in Caucasians, the frequency of the *V1 allele being 46–48% and that of the *V2 variant being 52–54% in our study. No additional VNTR allele with more than two motif repeats was identified.As promoter regions control gene expression by interactions between *V2/*V2 genotype displayed a significant and a trend toward lower serum Trp concentration compared to females with a *V1/*V2 and *V1/*V1 genotype, respectively. IDO is known to be induced by soluble hormones, such as human chorionic gonadotropin, prolactin and estrogens, supporting the hypothesis of a hormonal control of IDO expression et al. To assess the impact of the VNTR polymorphism on IDO activity, 47 males and 47 females from a cohort of 300 healthy Caucasian subjects were selected based on their genotype, and their sera were analysed to determine Trp and Kyn concentrations. We first observed a significant lower serum Trp concentration in females compared to males, as reported previously in other studies IDO1 promoter activity IDO1, and that both IFN-γ and TNF-α induce promoter activity, a greater activation being obtained with IFN-γ and a synergy being observed with a combination of the two cytokines. However, no significant difference in promoter activity was seen between *V1 and *V2 alleles under any experimental condition. Based on these results, the absence of any difference in promoter activity between the two alleles, even after synergistic induction, may not have been unexpected, since the VNTR polymorphism does not modify any critical response element, such as ISRE or GAS.In our experiments involving HeLa cells, a cell line which was previously used to study the mechanism of IFN-γ action on cis-acting elements in the promoter region of IDO1 in order to investigate whether the VNTR polymorphism could affect interactions between putative binding sites and transcription factors. In addition to the response elements for IFN-γ and TNF-α previously described, the in silico analysis predicted that the LEF-1/TCF transcription factor could interact with the promoter region of IDO1 at three different sites, with one putative consensus sequence being located in the 24-bp motif of the VNTR. As TFBS prediction programs cannot assert the existence of a TFBS or infer the functionality of a site IDO1 promoter was confirmed by specific PCR amplifications using chromatin immunoprecipitation of HeLa cellular extracts. To investigate the functionality of the potential sites, additional gene reporter assays were performed using an overexpression of a functional LEF-1. As a member of the TCF/LEF family of transcription factors, LEF-1 is one of the major nuclear transducers involved in Wnt signaling. However, it has no transcriptional activation potential in isolation and requires binding with β-catenin, the β-catenin activation domain and TCF/LEF DNA-binding domain forming a bipartite transcriptional activator of target genes β-catenin TCF/LEF interaction *V1 and *V2 alleles was found to be significantly increased relative to the negative control expression vector, thus confirming the existence of specific and functional binding sites for LEF-1 on the studied 1.6-kb promoter region of IDO1. This result confirms the reliability of MatInspector prediction of potential binding sites for this transcription factor. However, no difference in promoter activity was observed between *V1 and *V2 alleles, thus suggesting the insertion of the 24-bp motif, that creates one additional copy of a LEF-1 response element, may not interact with LEF-1, and, furthermore, that the other three LEF-1 sites most likely contribute to the global increase of IDO1 promoter activity following overexpression of LEF-1. Interestingly, following IFN-γ and/or TNF-α stimulation, promoter activities of *V1 and *V2 alleles were similarly increased, but to a lesser extent than those observed in the first experiments without LEF-1 overexpression, relative to the promoterless pGL4 vector. These results suggest the interaction between LEF-1 response elements and VP16-TCF product alters the IDO1 promoter responsiveness to cytokines. It is noteworthy that Robinson et al.IDO1 promoter gave rise to a significant increase of transcription activation by IFN-γ, compared to that observed with the undeleted promoter. Accordingly, the authors suggested that regulatory elements within this region could provide additional mechanisms of IDO1 regulation. As two of the LEF-1 sites we characterized are located between the two ISRE sites, they could then be hypothesized to correspond to those suggested additional cis-acting elements in the IDO1 promoter.A bioinformatic analysis was undertaken to predict potential IDO1 promoter activity relative to the activity of the *V1 and *V2 alleles, suggesting that the LEF-1 site within the VNTR has a repressive effect on IDO1 promoter activity. This result could be explained by the fact that LEF-1 can also interact with proteins of the Groucho family, proteins that are known to repress transcriptional activation by β-catenin-TCF complexes The modification, by site-directed mutagenesis, of the LEF-1 binding site located within the 24-bp motif of the VNTR, appeared to increase IDO1 gene. Although this VNTR appears to have no major consequence on basal or cytokine-induced transcriptional activity in vitro, it seems to participate in Trp homeostasis in female subjects. Furthermore, the identification of this polymorphism has uncovered previously unknown cis-acting elements for the LEF-1 transcription factor. Variations in the number of these response elements could alter IDO1 expression, particularly in pathological conditions involving the Wnt signaling pathway.In conclusion, we have characterized a previously unknown VNTR polymorphism in the promoter region of the IDO1 promoter.41 anonymous DNA samples, previously obtained from unrelated healthy Caucasian volunteers and declared to the Commission Nationale de l′Informatique et des Libertés for research studies on genetic polymorphism identification (SPFCO/cellule de bioéthique/n° DC-2008-642), were used to screen the genomic sequence of the et al. A further 300 serum and DNA samples from young healthy adults, prepared as previously described by Bouatia-Naji A written informed consent was obtained from all participants.IDO1 gene from each of the initial 41 DNA samples was sequenced in order to screen for genetic polymorphisms. Accordingly, a DNA fragment covering the 1576 bp-sequence upstream of the ATG initiation codon was amplified from each DNA sample using Phusion™ High-Fidelity DNA polymerase , according to the manufacturer's instructions. Each fragment was directly sequenced using the BigDye® Terminator v3.1 cycle sequencing kit  and an automated 3130XL genetic analyzer (Applied Biosystems). All primers were designed according to the nucleotide sequence of the IDO1 gene published in GenBank (accession number NT_007995) and are listed in The promoter region of the IDO1 promoter region and applied to the 300 DNA samples from the Haguenau cohort. A short PCR fragment encompassing the VNTR region was amplified with a pair of specific primers  and a 232-bp fragment  allowed the characterization of the VNTR genotype of each sample.A rapid and simple genotyping assay was developed to genotype the VNTR polymorphism that was identified within the  primers . The PCRIDO1 genotyping assays were performed on 94 out of the 300 DNA samples from the Haguenau cohort to screen the 9-bp deletion and the rs35099072 SNP, previously characterized as functional IDO1 variants by Arefayene et al.Additional www.Genomatix.de) was used to search the IDO1 gene for potential Transcription Factor Binding Sites (TFBSs) in the 1.6-kb promoter region. A restrictive threshold of 0.85 and the matrix library v7.0 were used.The prediction software MatInspector  and subsequently cloned into the pGL4.11 [Luc2P] vector  which contains the firefly luciferase cDNA. After cloning, both recombinant vectors pGL4-V1 and pGL4-V2 were sequence-verified to avoid constructs containing sequence mismatches and correct orientation confirmed.Genomic DNA samples carrying a homozygous VNTR genotype, i.e. promoter . These pThe LEF-1/TCF consensus sequence identified in the repeat motif of the VNTR by MatInspector was mutated using the QuickChange II Site Directed Mutagenesis Kit , according to the manufacturer's recommendations. This mutagenesis step was applied to the pGL4-V1 construct with complementary primers designed to create a 2-bp change in the LEF-1/TCF core sequence. The corresponding plasmid was named pGL4-V1Mut and was used for gene reporter assays. The sequences of the mutated primers are shown in 2 at 37°C. The day before transfection, 4.104 cells were freshly plated on 24-well plates in 0.5 mL of complete media. Twenty-four hours after plating, HeLa cells were transfected with 1.25 µL of lipofectamine LTX (Invitrogen).The HeLa human cervical carcinoma cell line  was maintained and cultured in DMEM supplemented with 1% of a combined solution of 100 U/mL penicillin and 100 µg/mL streptomycin and 10% FBS (Invitrogen). The cells were grown in 5% COrenilla luciferase, used to normalize transfection efficiency. Four hours after transfection, the medium was removed and replaced with fresh complete medium with or without IFN-γ (10 ng/mL), TNF-α (5 ng/mL), or a combination of both cytokines . Luciferase activities were measured 24 h after cytokine induction.In a first set of transfection experiments, HeLa cells were transfected with 250 ng of the insertless pGL4.11-basic vector or one of the recombinant vectors, pGL4-V1, pGL4-V2 and pGL4-V1Mut, and 12.5 ng of the control reporter PGL4.74 plasmid (Promega) that contains the In a second set of transfections, 125 ng of the pGL4.11 basic vector or one of the promoter constructs was co-transfected with 12.5 ng of PGL4.74 and 125 ng of the expression vector VP16-TCF; pcDNA 3.3 was used as a negative expression control. As previously, a cytokine induction was performed 4 h after transfection and luciferase activities were measured 24 h later.firefly luciferase activity (fLA) and the renilla luciferase activity (rLA) were measured sequentially with a Glomax luminometer (Promega). The relative luciferase activity (RLA) was calculated as follows: RLA  =  fLA/rLA. The transfection and reporter gene assays were performed in triplicates and repeated in three independent experiments. Results are expressed as relative firefly luciferase units normalized to renilla luciferase. Data are expressed as mean ± SEM.The luciferase activities were measured using the Dual-Glo Luciferase Reporter Assay System (Promega) following the manufacturer's recommendations. The 6 HeLa cells were cross-linked for 10 min at 37°C in 1% formaldehyde. Cross-linking was stopped by adding 0.125 M of glycine for 5 min at room temperature. Cells were then washed twice with cold PBS 1X, re-suspended in 300 µL of SDS lysis buffer containing protease inhibitors and pelleted for 5 min at 2000 g at 4°C. Cross-linked DNA was sheared by sonication to yield DNA fragments between 200 and 500 bp in size and then diluted 10-fold with ChIP dilution buffer. At this step, 20 µL of the sample was used as input material to quantify the amount of DNA present in each sample before immunoprecipitation. Samples were pre-cleared for 4 h with 75 µL of protein A-agarose/salmon sperm DNA (50% slurry) and immunoprecipitated overnight at 4°C with rotation with 10 µg of an antibody to acetylated histone H3 (Millipore) for positive control experiments, 20 µg of an anti-LEF-1 antibody , or without antibody as a negative control.. The following day, 60 µL of protein A-agarose/salmon sperm DNA (50% slurry) was added to the sample for 4 h at 4°C with agitation to collect the antibody/histone complex. Beads were then collected by gentle centrifugation and washed according to the Upstate protocol. The histone complexes were eluted from the beads with 500 µL elution buffer  at room temperature for 30 min with rotation. Cross-links were reversed by adding 20 µL of NaCl 5M to the eluates and heating at 65°C overnight. The sample was then treated with 10 µL of 0.5 M EDTA, 20 µL of 1 M Tris-HCl, pH 6.5, and 2 µL of 10 mg/mL Proteinase K for 2 h at 45°C. DNA was recovered by phenol/chloroform extraction and ethanol precipitation. PCR used to isolate each of the 3 potential TFBSs for LEF-1, as well as the GAPDH promoter sequence used as a positive control, were performed with specific primers listed in ChIP analysis was performed according to the Upstate Biotechnology  protocol with modifications. 3.10*V1/*V1, *V1/*V2 and *V2/*V2 genotype, respectively, were determined by HPLC/MS-MS using a method described by Schefold et al.Trp and kynurenine (Kyn) concentrations in serum samples from 47 male  and 47 female  healthy subjects of the Haguenau cohort, that comprise three groups of 31, 31 and 32 subjects with a 2 testing. The luciferase assay data were tested using a non-parametric Mann-Whitney test (two-tailed). The above statistical calculations were run with the SPSS software version 15.0 . Serum Trp, serum Kyn and Kyn/Trp ratio were each compared with genotype frequencies in all subjects, as well as in males and females separately, using a non-parametric Mann-Whitney test (two-tailed) with use of GraphPad Prism version 5.02 for Windows. Findings were considered statistically significant at p value<0.05.Observed genotype frequencies of the VNTR polymorphism were examined for deviation from the Hardy-Weinberg equilibrium by χTable S1Mean (± S.E.M) luciferase activity values observed with pGL4-basic, pGL4-V1, pGL4-V2 and pGL4-V1mut constructs in HeLa cells.(DOC)Click here for additional data file.


Caenorhabditis elegans HNF4α-related nuclear hormone receptor NHR-62 is required for metabolic and physiologic responses associated with DR-induced longevity. nhr-62 mediates the longevity of eat-2 mutants, a genetic mimetic of dietary restriction, and blunts the longevity response of DR induced by bacterial food dilution at low nutrient levels. Metabolic changes associated with DR, including decreased Oil Red O staining, decreased triglyceride levels, and increased autophagy are partly reversed by mutation of nhr-62. Additionally, the DR fatty acid profile is altered in nhr-62 mutants. Expression profiles reveal that several hundred genes induced by DR depend on the activity of NHR-62, including a putative lipase required for the DR response. This study provides critical evidence of nuclear hormone receptor regulation of the DR longevity response, suggesting hormonal and metabolic control of life span.Dietary restriction (DR) extends lifespan in a wide variety of species, yet the underlying mechanisms are not well understood. Here we show that the  Caenorhabditis elegans, genes implicated in the TOR pathway, autophagy, protein synthesis and energy homeostasis have been shown to modulate the dietary restriction response; however their mechanism of action is still unclear. In this work, we find that the C. elegans nuclear hormone receptor, nhr-62, is required for longevity in multiple DR regimens, providing the first evidence of a nuclear receptor required for DR-induced longevity. Additionally, nhr-62 is required for physiologic changes associated with DR, including increased autophagy and decreased levels of triglycerides, possibly through lipolysis. Moreover, nhr-62 is responsible for regulating hundreds of genes under DR, as measured by qPCR and RNA-seq. Importantly, this work is the first to report transcriptome analysis of DR in C. elegans and the first to provide functional evidence that nuclear receptors are key regulators of the DR longevity response, which imply hormonal and metabolic control of longevity, possibly through alterations in fat metabolism, lipolysis, and autophagy.Dietary restriction extends the life span of diverse species across taxa, yet the underlying mechanisms are poorly understood. In humans there are clear health benefits associated with DR such as improved serum cholesterol and lipid levels. In  Genetic and environmental factors can cause profound changes in organism lifespan. Genetic alterations that stimulate robust longevity across taxa include reduced insulin/IGF and TOR signaling, reduced mitochondrial function, and reduced signaling from germline stem cells Caenorhabditis elegans several different DR regimens can induce longevity. The two most widely used are dilution of bacterial food and the genetic DR mimetic eat-2. Dilution of bacterial food in liquid culture (BDR) was first demonstrated by Klass in 1977 to extend C. elegans life span, and variations of this method have been shown to enhance longevity by 20–100% ad libitum until adulthood, and then are shifted to liquid culture containing a dilution of bacterial food. The eat-2 mutation affects the function of a pharyngeal acetylcholine receptor subunit, which reduces pharyngeal pumping rate and subsequent food intake throughout the life of the animal, and extends life span by 15–40% C. elegans adults include intermittent feeding (IF), in which worms are fed every two days, dietary deprivation (DD), in which adult worms are completely removed from food, and solid DR (sDR) where bacteria is diluted on solid agar plates In C. elegans, a few key transcriptional regulators of the DR response have started to emerge. PHA-4 is a FOXA homolog required for eat-2 and BDR induced longevity From genetic studies in nhr-62 as required for DR-induced metabolic and longevity responses.In an effort to identify regulators of DR-induced longevity, we specifically focused on nuclear hormone receptors (NHRs). Nuclear hormone receptors are transcription factors that respond to fat-soluble hormones, such as steroids and fatty acids, to directly regulate gene transcription. They are broadly implicated in the regulation of development, metabolism and homeostasis, and are well poised to coordinate events throughout the body in response to hormonal or nutritional signals eat-2 mutation (a genetic DR mimetic), and screened for a loss of eat-2-induced longevity. We screened 246 of the 284 C. elegans NHRs in a genetic background more sensitive to RNAi . Knockdown of most NHR genes had no effect on eat-2 longevity  yet largely maintained DR-induced life span extension  consisting of DNA- and ligand-binding domains , and a short isoform B (353 AA) consisting of only the LBD , which removes a 658 bp region, including part of exon one, and results in an immediate stop codon, thus removing the DBD of the receptor. Although nhr-62(tm1818) is likely a strong loss-of-function allele, it may not be null because the predicted B isoform containing the LBD only is intact. We introduced this mutation into eat-2(ad465) animals and measured the lifespan of the double mutant. Similar to RNAi, nhr-62(tm1818) significantly suppressed the lifespan of eat-2(ad465) animals. Importantly, the nhr-62(tm1818) mutant had little or no effect on wild-type (N2), suggesting that nhr-62 does not suppress eat-2 longevity through general sickness (nhr-62(tm1818) consistently suppressed eat-2(ad465) lifespan, suppression was not always complete, suggesting other activities still contribute to the response.To validate our RNAi results, we obtained the deletion allele, sickness . Though nhr-62 in DR-induced longevity, we generated an extra chromosomal line, dhEx627, expressing wild-type nhr-62, and introduced it into eat-2;nhr-62 mutant animals to measure whether complementing nhr-62 function was sufficient to restore longevity. As expected, the dhEx627 array restored longevity to eat-2;nhr-62 double mutants  without affecting pharyngeal pumping rate  animals fed daf-2 (IR) or cco-1 (cytochrome c oxidase) RNAi. As expected, daf-2 RNAi and cco-1 RNAi robustly increased the life span of wild-type animals. Moreover, RNAi knockdown similarly increased longevity in the nhr-62(tm1818) background  signaling or reduced mitochondrial function have both been shown to extend life span ckground . These rnhr-62(tm1818) is also required for DR-induced longevity by BDR, we measured the lifespan of wild-type and nhr-62(tm1818) worms fed bacteria at ten different concentrations. We started with our ad libitum bacterial concentration of 2.5×109 CFU/ml followed by nine subsequent two-fold serial dilutions. At the level of DR associated with the longest lifespan , wild-type worms exhibited a 188% increase in median lifespan compared to ad libitum conditions (nhr-62(tm1818) worms exhibited a 40% increase in median lifespan compared to ad libitum conditions, identical to the wild-type DR response curve at these high nutrient conditions. However, at the optimal DR condition, the DR response curve for nhr-62(tm1818) mutants diverged from the wild-type curve; these animals were unable to respond to DR as effectively, with only a partial increase in the median lifespan at all subsequent concentrations . As previously described, transgene expression was observed from embryo to adult in the pharynx and intestine. We also observed clear expression in various neurons including sensory neurons, motor neurons, hermaphrodite specific neuron, and pharyngeal neurons (data not shown). A similar pattern of expression was seen in the eat-2(ad465) background. A full-length integrated low copy nhr-62::gfp expressing strain from the TransgeneOme Project eat-2 backgrounds. Again no obvious change in nhr-62::gfp intensity or localization was observed in the eat-2 background, nor were changes seen in nhr-62 mRNA levels as measured by qPCR. Notably several tissues that express nhr-62::gfp, such as the intestine, ASI sensory neurons, and hypodermis, are major endocrine tissues that coordinate metabolic states and have been implicated in aging We obtained a transgenic strain from the Hope laboratory containing 2 kb of the ad libitum conditions. Since nhr-62 shares homology to HNF4α-like NHRs known to function in fat metabolism such as nhr-49 and nhr-80nhr-62 might be necessary for proper fat regulation under DR. To test this hypothesis, we treated wild-type, nhr-62(tm1818), eat-2(ad465), and eat-2;nhr-62 animals with the lysochrome dye Oil Red O, which stains neutral triglycerides and lipids  nhr-62 regulates fatty acid composition under DR, we quantified individual fats by gas chromatography. eat-2;nhr-62 double mutants exhibited increased levels of saturated fatty acids compared to wild-type. Moreover, eat-2;nhr-62 double mutants showed a significant reduction of various monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) compared to eat-2 mutants alone , a nucleophilic elbow, and other features typical of lipases  mutants. We found that let-363 knockdown induced longevity in both wild-type and nhr-62 mutants  kinase. Down-regulation of  mutants . These rnhr-62 promotes DR longevity and associated physiological processes, we speculated that it could modulate the overall transcriptional change upon DR. To test this hypothesis, we performed RNA-seq on wild-type, nhr-62(tm1818), eat-2(ad465), and eat-2;nhr-62 animals. This approach not only enabled us to identify genes regulated under DR, but also which genes showed nhr-62 dependence. Our criteria for differential gene regulation included a greater than 1.5-fold change in expression compared to wild-type, a false discovery rate less than 0.05, and greater than 10 reads when normalized to the base mean. For each sample we typically obtained 25–37 million unambiguous reads, and could assign these to approximately 15,000 genes. Heat maps of the mean Euclidean distance between samples showed that nhr-62 was most similar to wild-type, while eat-2 was most different, with eat-2;nhr-62 in between  back to wild-type levels , phosphorus metabolism , nucleosome assembly and organization,  protein, amino acid, histidine, and heterocyclic amine metabolism  and short chain dehydrogenase metabolism   ,S7. OtheC. elegans we identified the HNF4α homolog, nhr-62, as an important mediator of the DR response. Several lines of evidence are consistent with the notion that nhr-62 promotes aspects of DR-induced metabolism and longevity. RNAi knockdown or mutation of nhr-62 specifically suppressed eat-2 DR-induced longevity, but not that of reduced insulin/IGF receptor (daf-2) or mitochondrial function (cco-1) arguing for a DR specific function. Importantly, nhr-62 mutation prevented animals from fully responding to the longevity-inducing effects of bacterial food dilution (BDR), thereby making it one of only a handful of DR-regulating genes that clearly modulates multiple forms of DR. By comparing eat-2 to eat-2;nhr-62 mutants we found a reversal of Oil Red O staining intensity, triglyceride levels, and autophagy induction, without seeing a difference in body size or pumping rate. Additionally, we found that nhr-62 mutation decreased the mono- and polyunsaturated fatty acid content of dietary restricted worms. Finally, we found a reversal of the DR-induced transcriptional regulation of a predicted lipase required for DR, as well as a plethora of genes through RNA-seq analysis. These results point to a model in which a dietary restricted state promotes activation of nhr-62, the lipase C40H1.8, and numerous other genes to modulate DR-induced longevity, possibly through fat metabolism, autophagy, and other processes  mutants. One potential explanation for this observation is that by mutating nhr-62, the optimum DR threshold has been shifted to a new concentration. However, nhr-62(tm1818) mutants do not exhibit a globally shifted BDR response curve since the highest three food concentrations give median life spans that align precisely with wild-type. At further food dilution, nhr-62(tm1818) mutation significantly suppressed life span extension compared to wild-type, which results in a parallel response but with lower median life spans compared to wild-type. These results are consistent with a blunted response to DR in the lower nutrient range. In contrast to a “master regulator” of DR, which might completely abrogate life span extension at all food concentrations, this type of response curve could be indicative of a gene that plays a role in modulating or fine tuning the BDR longevity response at specific nutrient levels. It is also possible that nhr-62 mutation disrupts metabolism in a non-specific manner that is incompatible with a proper DR response. What argues against this is that nhr-62 mutation on its own had very little effect on gene expression or physiology under ad libitum conditions, suggesting nhr-62 is engaged in a regulatory role primarily under DR states.It was unexpected to see a partial BDR response by nhr-62 exhibits several phenotypes suggesting it functions in mobilizing fat to partition energetic and biosynthetic demands under nutrient limitation. Notably nhr-62 mutation prevents some of the reduction of TAG and Oil Red O staining stores seen under DR longevity. Several dynamic changes in fatty acid metabolism have been observed upon DR shifts in other species. In flies there is a shift toward increasing fatty acid synthesis and breakdown, and inhibiting this suppresses DR longevity nhr-62 alters fatty acid composition in the eat-2 background, increasing the amount of the saturated fatty acid palmitate, and reducing the amount of various MUFAs and PUFAs, including C18:1n7, C20:3n6, C20:4n6, C20:4n3, and C20:5n3 fatty acids. It is possible that these changes could contribute towards the suppression of life span. Interestingly, a handful of these fatty acids have been recently implicated in longevity. The omega-6 fatty acid, DGLA (C20:3n6) stimulates autophagy and extends C. elegans life span lipl-4, which stimulates autophagy C40H1.8 lipase could stimulate autophagy, since RNAi against this gene diminished DR longevity and GFP::LGG-1 punctual formation. Alternately, lipolysis could liberate fatty acids that serve a signaling role, binding to NHR-62 or related receptors. Future studies should further clarify the complex relationships between fatty acid composition, lipolysis, autophagy and longevity in the DR response.eat-2 to wild-type. Amongst enriched categories are genes involved in phosphorus metabolism, unsaturated fatty acid metabolism, eicosanoid and ceramide metabolism, lipid modification and transport, amino acid, amine and chitin metabolism, and neuropeptide signaling. Notably mutation of nhr-62 reversed the regulation of about 600 of these genes, suggesting a key role in mediating the DR response. These genes are candidates for nhr-62 targets, and possibly important effectors of the DR response , lipid binding , fatty acid remodeling (acyl coA thiolases F57F4.1), β-oxidation  and fatty acid elongation . Presumably some of these genes may mediate the physiologic changes relevant to DR. Indeed, vitellogenin knockdown has been previously shown to extend life By RNA-seq analysis we identified significant changes in the expression of approximately 3,000 genes comparing response . Among tlet-363/TOR RNAi induced longevity in the nhr-62 mutant background. We also found that the autophagy gene lgg-1 was up-regulated 1.5-fold, in a manner dependent on nhr-62. Consistently, we observed that autophagic vesicles visualized with GFP::LGG-1 were increased under DR in an nhr-62 dependent manner. Previous studies have shown that lgg-1 and other autophagy genes are required for DR and various longevity pathways Dietary restriction is associated with reduced TOR signaling, reduced protein synthesis, and increased autophagy inf-1, whose down-regulation has been shown to extend life span nhr-62 dependence. It is possible that these minor transcriptional changes collectively recapitulate the physiologic effect of DR. Alternately, modest regulation of a critical enzyme or key regulator in the pathway might be sufficient. nhr-62 might also regulate unidentified post-transcriptional modifiers of these processes, especially given the many kinases and phosphatases affected  and fungal induced proteins, which are speculated to be involved in innate immunity. In the future it will be important to understand how the diverse transcriptional output of nhr-62 relates to longevity and to identify direct targets by ChIP-seq.Reduced protein translation extends life span and may be one of the important outputs of the DR response affected ,S7. Manynhr-62. The C. elegans genome has undergone a radical expansion of the HNF4 family of nuclear receptors, making an assignment of homologous function for nhr-62 challenging. One such HNF4-like nematode receptor, nhr-49, has been proposed as PPARα-like because it is involved in the starvation response, turns on genes implicated in β-oxidation, fatty acid desaturation, binding and transport. However, in our hands, nhr-49 knockdown did not abrogate the DR response. Conceivably, nhr-62 functions similar to the PPARs as well, since these nuclear receptors are known lipid sensors that regulate metabolism under different nutritional states. Corton et al originally observed a substantial overlap in the expression profile of DR and PPAR nuclear receptor regulation in mice nhr-62 functions like HNF4, which regulates apolipoprotein levels and lipid and glucose homeostasis. Notably, both PPARs and HNF4 nuclear receptors can bind to fatty acids, which may be regulating their activity nhr-62 is involved in fat metabolism raises the possibility it too is regulated by fatty acid like ligands.It is currently unclear which mammalian homolog is most functionally analogous to C. elegans transcription factor SKN-1/NF-E2 regulates DR-induced longevity from the pair of ASI sensory neurons Interestingly, the E. coli (OP50) at 20°C unless otherwise noted. Standard procedures for culturing and maintaining strains were used eat-2(ad465), nhr-62(tm1818), nre-1(hd20);lin-15b(hd126), UL1385 mvEx5591(Y67A6A.2::gfp + unc-119), eat-2(ad465);mvEx5591(Y67A6A.2::gfp + unc-119), OP403 wgIs403(nhr-62::TY1 EGFP 3× FLAG (91H02);unc-119(+)); unc-119(tm4063), QU1 izEx1(plgg-1::gfp::lgg-1 + rol-6), eat-2(ad465); izEx1(plgg-1::gfp::lgg-1 + rol-6), eat-2(ad465);nhr-62(tm1818) dhEx627(pmyo-3::cfp + fosmid WRM065CF04). The dhEx627 extra-chromosomal rescue strain was generated by injecting DNA from worm fosmid WRM065CF04 (10 ng/µl), a co-injectable marker (pmyo-3::cfp at 20 ng/µl) and salmon sperm DNA at 70 ng/µl. Stable arrays were selected for and maintained based on expression of the co-injected marker. For imaging of nhr-62::gfp, day 1 adult worms were visualized using a Zeiss Axioskop2 Plus microscope and representative images were processed with ImageJ All strains were grown and maintained on NGM agar seeded with egg laying defective), in which unlaid eggs hatch inside the mothers, were censored from the aging experiment. The number of aging experiments performed, mean, median, and maximum lifespans, log-rank analysis, number of worms used in each experiment and worms censored were recorded (nre-1(hd20);lin-15b(hd126) was used. Eggs were pipetted at a target density of 15 eggs per well into 12 well cell culture plates with RNAi NGM seeded with RNAi clone of interest. At L4, or day 0 of adult, 15 µl of FUdR (5-Fluoro-2′-deoxyuridine) (50 mM) was pipetted into each well to inhibit progeny production. On day 7 of adulthood, the number of worms alive was determined for each well. On day 15 of adulthood, the number of worms alive was again determined and the percent surviving was calculated. For RNAi experiments, worms were maintained on NGM plates with 20 µg/ml carbenicillin, 10 µg/ml tetracycline, and 1 mM IPTG. Worms were transferred to fresh RNAi plates every other day. For BDR longevity experiments, lifespans were recorded as previously described 2HPO4, 6 g KH2PO4, 1 ml cholesterol at 5 mg/mL in ethanol, and MilliQ H2O to 1 L) with carbenicillin (50 µg/ml), kanamycin (10 µg/ml), and tetracycline (1 µg/ml) added to inhibit bacteria replication. 90 worms were then moved into defined bacteria concentrations and scored every 3–4 days for survival at a density of 15 worms per well. On every score day worms were transferred to freshly made bacteria condition. FUdR was added to the BDR liquid media for the first 14 days of adulthood to inhibit progeny production. Bacteria concentration was determined through serial dilution, plating, and counting of colony forming units (CFUs). The number of aging experiments performed, mean, median, and maximum lifespans, log-rank analysis, number of worms used in each experiment and worms censored were recorded  and the Pierce BCA Protein Assay Kit (Thermo Scientific) and analyzed on a Biotek Power Wave XS plate reader according to manufacturer's instructions. Three technical replicates were preformed for every biological replicate. Standards were also done in triplicate. Statistics were done using GraphPad Prism software.2SO4 (Sigma) in methanol (Biosolve) was added to each sample and incubated at 80°C for 20 minutes with shaking. 0.75 ml of water  and 350 µl of hexane (Sigma) were next added and the sample was incubated with shaking for 10 min to extract FAMEs into the hexane layer. The hexane layer was removed, concentrated by evaporation, and used directly for analysis. FAME analysis was carried out using an Agilent 7890A GC equipped with a flame ionization detector (FID)  and a DB-23 column  using helium as a carrier gas at a flow rate of 3 ml/min. 1 µl per sample was injected in pulsed-splitless mode. The initial oven temperature was set to 80°C, held for 1 minute; increased to 170°C at 6.5°C/min; then increased to 215°C at 2.75°C/min. FAMEs were identified and compared against known reference standards for quantification.Day 1 adults animals were collected in 100 µl M9 buffer, washed 2-times, and run through 10-freeze/thaw cycles before being sonicated to lyse the cuticle and tissue. Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific) according to manufacturer's instructions. 50 µl was removed to a 1.5 ml gas chromatography (GC) vial for derivatization. To each vial, 200 ng each of methyl C11:0, C13:0, and C23:0  were added as internal reference standards. To convert fatty acids into their fatty acid methyl ester (FAME) derivatives, 0.5 ml of 2.5% Hama-1 and cdc-42 was used as control. 4 biological replicates containing 300–400 worms each and four technical replicates were tested for each experiment. qPCR primers sequences are in Synchronized worms were collected into TRIzol (Invitrogen) when reaching adulthood but bearing no embryos. Total RNA and cDNA was prepared using RNeasy Mini kit (QIAGEN) and iScript cDNA Synthesis Kit (Bio-Rad) respectively. qRT-PCR was performed with Power SYBR Green master mix (Applied Biosystems) on a ViiA7 384 Real-Time PCR System (Applied Biosystems). A combination of nhr-62 RNAi experiments, 3 biological replicates with 31 animals (345 seam cells) of GFP::LGG-1 (L4440), 26 animals (225 seams cells) of GFP::LGG-1 (nhr-62i), 62 animals (773 seams cells) of eat-2 GFP::LGG-1 (L4440), and 58 animals (598 seam cells) of eat-2 GFP::LGG-1 (nhr-62i) were scored. For C40H1.8 RNAi experiments, 3 biological replicates with 50 animals (323 seam cells) of GFP::LGG-1 (L4440), 43 animals (382 seams cells) of GFP::LGG-1 (C40H1.8i), 100 animals (933 seams cells) of eat-2 GFP::LGG-1 (L4440), and 99 animals (896 seam cells) of eat-2 GFP::LGG-1 (C40H1.8i) were scored.Counting of puncta formation was done as previously described eat-2, nhr-62;eat-2, nhr-62, N2, giving a total of 12 samples. Sequencing reads were clipped to remove adapter sequence using fastx_clipper (http://hannonlab.cshl.edu/fastx_toolkit/), clipped reads were aligned to the C. elegans reference genome (WBcel215.67) using tophat version 1.3  with option −g1 to ensure unique mapping and option −G to pass the Caenorhabditis_elegans.WBcel215.67.gtf. The number of reads mapping to each Ensemble gene was counted with htseq-count (http://www-huber.embl.de/users/anders/HTSeq/doc/count.html#count). Statistical analysis and drawing of plots was performed in R (http://www.r-project.org/) using the bioconductor package Deseq  and R function gplots (http://cran.r-project.org/web/packages/gplots/index.html). Scatterplots of the RNA-seq data were drawn to illustrate differential expression between samples. A heatmap showing Euclidean distances between samples  was plotted in R. For each data comparison a list of “significant differentially expressed genes” was compiled from the Deseq-result as follows: FDR<0.05, mean normalized count >10 and absolute Foldchange (FC) >1.5. Furthermore a list of genes, which are up or down regulated by eat-2, compared to the control (eat-2 vs N2), but show that they are rescued in the nhr-62;eat-2 phenotype  was compiled and referred to as Rescued-Genes. Further analysis of “significant differentially expressed genes” and “Rescued-Genes” was performed as follows:Worms of indicated genotypes were synchronized by two rounds of bleaching and approximately 300 worms for each genotype were handpicked into TRIzol (Invitrogen) when they just reached adulthood without carrying embryos. Total RNA was prepared with RNeasy Mini Kit (QIAGEN). cDNA library was subsequently constructed by TruSeq RNA Sample Preparation Kit (Illumina). Illumina sequencing (100 bp single end) was performed on 3 biological replicates for each of the 4 treatments A Venn diagram was plotted in R for “significant differentially expressed genes” to show the overlap of the significant differentially expressed genes.Annotation and Gene-enrichment analysis of “significant differentially expressed genes” and “Rescued-Genes” was performed with David ReviGO analysis Figure S1nhr-49 by RNAi does not abolish longevity of eat-2(ad465) mutant animals (log-rank p<0.001). (B) Percentage of animals alive on day 15 fed bacteria expressing different RNAi constructs. nhr-49 RNAi resulted in shortevity. Knockdown of the nhr-62 paralogs nhr-21, nhr-4, nhr-34, and nhr-100 by RNAi does not suppress eat-2;nre-1;lin-15b longevity. (C) Median lifespan of wild-type (N2) or eat-2(ad465) worms fed nhr-62, nhr-4, or nhr-21 RNAi.Related NHRs do not mediate DR longevity response. (A) Knockdown of (TIF)Click here for additional data file.Figure S2nhr-62 overexpression. (A) Pumping rates of day 1 wild-type (N2) and wild-type(dhEx627) worms are not different. (B) Body length of day 1 adult wild-type(dhEx627) are shorter than wild-type (N2) worms. (C) Oil Red O staining of wild-type (N2) and wild-type(dhEx627). Oil Red O intensity in wild-type(dhEx627) is slightly decreased relative to wild-type (N2). ***p<0.001 by unpaired t-test. Mean (Center Line) ± SD (Box) with bars representing an outlier coefficient of 1.5.Physiologic traits of (TIF)Click here for additional data file.Figure S3nhr-62 mutation. (A) Pumping rates of day 1 adult eat-2(ad465) and eat-2;nhr-62 mutants are reduced relative to wild-type (N2) and nhr-62(tm1818) mutants (combined data from 2 independent experiments). (B) Relative body length of day 1 adult eat-2(ad465) and eat-2;nhr-62 mutants are smaller than ad libitum controls. (C) Day 1 adult eat-2(ad465) and eat-2;nhr-62 worms have reduced total progeny compared to ad libitum controls. nhr-62(tm1818) mutants have a modest but significant decrease in total hatched progeny compared to wild-type (N2) (combined data from 3 independent experiments). *p<0.05, **p<0.01, ***p<0.001 by Single Factor ANOVA with Tukey test. Mean (Center Line) ± SD (Box) with bars representing an outlier coefficient of 1.5.Physiologic traits of (TIF)Click here for additional data file.Figure S4fat-2, a Δ12-desaturase involved in PUFA synthesis. Levels of fat-2 mRNA were significantly up-regulated under DR as measured by qPCR. Mutation of nhr-62 partially suppressed this up-regulation, though this did not reach significance (p = n.s.). *p<0.05 by single factor ANOVA with Tukey test. Mean±SEM.Expression levels of (TIF)Click here for additional data file.Figure S5C40H1.8 partially suppresses DR-induced longevity.  C40H1.8 RNAi slightly reduces the lifespan of eat-2;nre-1;lin-15b worms relative to gfp RNAi fed controls (2 independent experiments). (C) Percent change in median lifespan of eat-2;nre-1;lin-15b relative to nre-1;lin-15b animals fed either C40H1.8 RNAi or gfp RNAi calculated from the experiments shown in A and B.(TIF)Click here for additional data file.Figure S6C40H1.8 and homologs. Level of (A) C40H1.7 mRNA (B) C40H1.8 mRNA and (C) C40H1.9 mRNA in wild-type (N2) worms fed C40H1.8 RNAi relative to wild-type (N2) worms fed gfp RNAi. *p<0.05 by student's unpaired t-test. Mean±SEM.qPCR of (TIF)Click here for additional data file.Figure S7C40H1.8. Sequence alignment of C40H1.8 to lipase class 3 proteins in various species. Catalytic triad (CT), nucleophilic elbow (NE), dashes represent breaks in the amino acid sequence. Accession numbers are C40H1.8:NP_001021213.1, Atg15p:EEU05398.1, Os11g0299300:ABA92846.1, C40H1.9:NP_001021214.2, diacyclglycerol lipase D. melanogaster:ACF37118.1, diacyclglycerol lipase M. musculus:NP_659164.2, and diacyclglycerol lipase H. sapiens:NP_631918.3.Sequence alignment of predicted lipase (TIF)Click here for additional data file.Figure S8Analysis of RNA-seq data. (A) Heatmap showing Euclidean distances between RNA-seq samples . (B) Venn diagram shows the number of distinct and overlapping regulated genes from RNA-seq. (C–N) qPCR validation of RNA-seq candidates. *p<0.05, **p<0.01, ***p<0.001 by single factor ANOVA with Tukey test. Mean±SEM.(TIF)Click here for additional data file.Table S1Plate Aging.(XLSX)Click here for additional data file.Table S2BDR Aging.(XLSX)Click here for additional data file.Table S3Genes tested in DR lipase screen.(XLSX)Click here for additional data file.Table S4eat-2 versus N2.Genes regulated in (XLSX)Click here for additional data file.Table S5eat-2 versus N2.Gene enrichment DAVID analysis of genes regulated in (XLSX)Click here for additional data file.Table S6nhr-62 rescued genes.(XLSX)Click here for additional data file.Table S7Gene enrichment DAVID analysis of rescued genes.(XLSX)Click here for additional data file.Table S8qPCR primers.(XLSX)Click here for additional data file.


Arabidopsis Phytochrome (phy)-Interacting bHLH Factors  are critically necessary to maintaining this developmental state and that light activation of phy induces a switch to photomorphogenic development by inducing rapid degradation of the PIFs. Here, using integrated ChIP–seq and RNA–seq analyses, we have identified genes that are direct targets of PIF3 transcriptional regulation, exerted by sequence-specific binding to G-box (CACGTG) or PBE-box (CACATG) motifs in the target promoters genome-wide. In addition, expression analysis of selected genes in this set, in all triple pif-mutant combinations, provides evidence that the PIF quartet members collaborate to generate an expression pattern that is the product of a mosaic of differential transcriptional responsiveness of individual genes to the different PIFs and of differential regulatory activity of individual PIFs toward the different genes. Together with prior evidence that all four PIFs can bind to G-boxes, the data suggest that this collective activity may be exerted via shared occupancy of binding sites in target promoters.Dark-grown seedlings exhibit skotomorphogenic development. Genetic and molecular evidence indicates that a quartet of  Arabidopsis bHLH family), discriminate between target genes. However, the specific question of whether, and to what extent, closely related sub-family members, with potential overlapping functional redundancy (like the quartet of Phytochrome (phy)-Interacting bHLH transcription Factors  studied here), share regulation of target genes through shared binding to promoter-localized consensus motifs does not appear to have been widely investigated. Here, using ChIP–seq analysis, we have identified genes that bind PIF3 to conserved, sequence-specific sites in their promoters; and, using RNA–seq, we have identified those genes displaying altered expression in various pif mutants. Integration of these data identifies those genes that are likely direct targets of transcriptional regulation by PIF3. Our data suggest that the PIF quartet members share directly in transcriptional activation of numerous target genes, potentially via redundant promoter occupancy, in a manner that varies quantitatively from gene to gene. This finding suggests that these PIFs function collectively as a signaling hub, selectively partitioning common upstream signals from light-activated phys at the transcriptional-network interface.An important issue in understanding mechanisms of eukaryotic transcriptional regulation is how members of large transcription-factor families, with conserved DNA–binding domains (such as the 162-member  A key component of the successful colonization of land by terrestrial flowering plants was the evolution of a developmental strategy termed skotomorphogenesis (etiolated growth). This strategy enabled post-germinative seedlings emerging from buried seed to grow heterotrophically, on seed reserves, rapidly upwards through the subterranean darkness to the soil surface. Coupled with this was the evolution of a photosensory mechanism to trigger a switch to autotrophic, photomorphogenic (deetiolated) development upon emergence into sunlight.pif mutant (pifq), lacking PIF-family members PIF1, PIF3, PIF4 and PIF5 (termed the PIF quartet), displays morphogenic development in total darkness that strongly phenocopies that of normal light-grown seedlings pif-mutant combinations progressively exhibit increasingly photomorphogenic phenotypes in darkness, it appears that the PIF quartet members act with partially additive or overlapping redundancy to drive the skotomorphogenic pathway Genetic evidence indicates that a small subfamily of basic helix-loop-helix (bHLH) transcription factors, termed PIFs (for Phytochrome (phy)-Interacting Factors) are centrally critical to the promotion of such skotomorphogenic development in dark-grown seedlings The phy family of sensory photoreceptors  has a central role in inducing the switch from skotomorphogenic to photomorphogenic development (deetiolation) in response to initial exposure of dark-grown seedlings to light pifq mutant in the dark are normally altered by prolonged light in fully deetiolated wild-type (WT) seedlings pif1pif3pif4pif5 mutants A limited number of transcriptome analyses, using Affymetrix ATH1 microarrays, aimed at identifying genes regulated by the phy-PIF signaling pathway during deetiolation have been reported Arabidopsis, using either ChIP-chip or ChIP-seq analysis of factor binding sites, coupled predominantly with Affymetrix ATH1 microarrays (representing about 80% of the protein-coding genes in the genome) for expression analysis The advent of ChIP-chip and ChIP-seq technology has provided the opportunity to identify genes that contain binding sites for transcription factors of interest, on a genome-wide scale in vivo to four selected promoters using ChIP-PCR analysis pif-mutant lines, we have defined the genes regulated by PIF3, genome-wide, in dark-grown seedlings. By merging these datasets, we have identified those genes whose expression is, at least partially, directly regulated by promoter-bound PIF3. In addition, by profiling the expression of a selected subset of these direct PIF3-targets in multiple additional pif-mutant combinations, we have addressed the question of whether PIF1, 3, 4 and 5 display qualitative and/or quantitative functional divergence in regulating shared target genes.Despite these advances, the use of the ATH1 microarray for expression analysis in many of these studies means that important expression changes in genes not present on this array might have been missed. In addition, the question of whether, and to what extent, closely related transcription-factor family members, such as the PIF quartet, with apparently shared DNA-target-sequence specificity, contribute toward the transcriptional regulation of common target genes does not appear to have been addressed in many existing studies of eukaryotic systems pif3-3 null-mutant seedlings were used for ChIP-seq analysis. DNA prepared from MYC-antibody-generated immunoprecipitates from four independent biological replicates of each genotype was subjected to high-throughput sequencing. Statistically-significant binding peaks were defined by comparing the parallel P3M and WT ChIP samples within each replicate using the MACS algorithm Two-day-old dark-grown wild-type (WT) and MYC-epitope-tagged-PIF3 (P3M)-expressing, This analysis identified 1064 reproducible peaks which form our “high-confidence” set of PIF3-binding sites . These sde novo motif discovery on the +/−100 bp regions surrounding the 1064 “high-confidence” PIF3 binding-peak summits described above. Two E-box (CANNTG) variants were identified as statistically overrepresented motifs within these PIF3-binding regions (CACGTG (‘G-box’) variant is well-established as a preferred PIF-binding motif CACATG variant is previously undescribed as a PIF3-binding motif, although PIF1 Using the MEME program  regions . The CACcis-elements.A broader analysis shows that 64% of the G-box and 30% of the PBE-box motifs present in the 2 kb windows surrounding the PIF3-binding summits cluster within the designated 200-bp binding sites . SimilarPIL1, PHYB, and RGA1) or PBE-box-  containing probes generated from various genomic PIF3-binding sites identified in the ChIP-seq analysis. in vitro binding-assay data indicate that the coincidence of PIF3-binding sites with the G-box or PBE-box motif in the ChIP-seq assay likely results from their direct interaction in vivo, and that the PBE-box is indeed another sequence-specific PIF-binding, E-box variant genome wide. Because all of the PIF3-binding sites tested by ChIP-qPCR in in vivo-binding of PIF3 to these motifs.To examine the potential direct interaction of PIF3 with the newly-identified PBE-box compared to that of the G-box, we performed DNA-Protein-Interaction (DPI)-ELISA ATHB-2 probe, which contains one G-box and one PBE-box, provides an interesting insight. Neither the competitor mutated in both motifs . Initially, from the 1064 binding sites defined above, we identified 709 sites that are both intergenic and G- and/or PBE-box-coincident . For thePIL1 and ATHB-2, as examples. The chromosomal region surrounding the PIL1 gene shows a single PIF3-binding peak that is coincident with three G-box motifs located in the PIL1 promoter region. ATHB-2 is somewhat unusual in that it displays five specific PIF3-binding peaks in its extensive 5′-upstream region, each coincident with 1 to 3 G-box motifs  and pif1pif3pif4pif5 (pifq) Arabidopsis seedlings. Genes displaying Statistically-Significant Two-Fold (SSTF) expression changes in the three mutant genotypes compared to the WT and each other were identified as being regulated by the relevant mutated PIF(s)  , S7, S8.gene-set . Similargene-set . CompariThe data indicate that 254 (74%) PIF3-regulated genes are also redundantly transcriptionally regulated by one or more of the other three PIF-quartet proteins. Conversely, 1740 (86%) PIF-quartet-regulated genes show no significant PIF3 dependence , whereasPIL1 and ATHB2 in pifq mutant, demonstrating the robust dependence of full expression on the presence of the PIF-quartet. Comparison of the expression peaks for the pif145 and pifq mutants also suggests that PIF3 acts in the absence of the other three quartet members to promote a moderate increase in transcript levels. Overall, the combined PIF-regulated expression-patterns and promoter-located PIF3-binding sites displayed by these two genes render them likely direct-targets of transcriptional regulation by PIF3 and one or more other quartet members in promoting skotomorphogenic development.To identify the genes that both physically bind PIF3 in their promoters, in a G-box- or PBE-box-coincident manner, and display PIF-regulated expression (defined here as “direct-target genes”), we merged our ChIP-seq and RNA-seq data. This permitted gene-by-gene visualization of the PIF3-binding peaks and PIF-dependent transcription, genome-wide, as shown for pif genotype. The quantitatively robust responsiveness of the PIF3-bound genes to the presence of PIF3 in the pif145 mutant background is evident from these data (Class Z-associated bar graphs). This robust PIF3-responsiveness was validated using RT-qPCR for selected members of the 19 PIF3-induced, Z-Class gene-set, having a range of quantitative dependence on this bHLH factor  shows that there is a reciprocal continuum in the magnitude of the relative contributions of PIF3 and the PIF1/4/5-trio to the collective activity of the PIF quartet in transcriptionally activating these genes , PIF3-promoted expression increases in the pif145 mutant compared to the pifq mutant by RT-qPCR. This suggests that a subset of Y-Class genes may represent additional bona fide autonomously-PIF3-regulated genes that are below the resolution of the SSTF criteria we imposed on our RNA-seq analysis. We have therefore designated these 17 as Class YZ1.5 genes, having moderate (>1.5-fold), but statistically significant, regulation by PIF3  that display promoter-located, PIF3-binding sites, but lack evidence of SSTF-level PIF3 regulation in our RNA-seq analysis . Neverthse genes . To more by PIF3 . The evi by PIF3 , it is ppifq mutant and wild type  are also PIF1/4/5-induced  that, in dark-grown seedlings, exhibit a PIF-quartet-dependent expression pattern, that is rapidly reversed (within 1 h) upon initial exposure to phy-activating R light PSY and KAI2) exhibit derepression here in the dark-grown pifq mutant. No individual PIF3 contribution to this repression was detectable here. Collectively, these data indicate that PIF3 acts predominantly, if not exclusively, to activate the expression of direct-target genes in dark-grown seedlings. Conversely, the 94% (108/115) of light-induced Class 7 genes that do not display G- or PBE-box coincident PIF3-binding peaks, might suggest that one or more of the proposed direct targets of the PIF quartet   quartet  can act Recently, we defined a small core set of 14 Class 7 PIF-quartet-regulated genes  that display rapid, reciprocal, transcriptional responsiveness to light and vegetative shade in dark-grown and light-grown seedlings, respectively pif triple mutants compared to the pifq mutant and WT  , with those previously identified as being rapidly (within 1 h) repressed by initial exposure of dark-grown seedlings to red light PIL1) . The eviPIL1) , or indiPIL1)  by PIF1,The predicted or established functional diversity of the PIF direct-target genes identified here  suggestsArabidopsis bHLH family A central issue in understanding mechanisms of eukaryotic transcriptional regulation is how members of large transcription-factor families, with conserved DNA-binding domains  transgenic line pif3pif1pif4pif5 (pif145) pif1pif3pif4 (pif134), pif1pif3pif5 (pif135), pif3pif4pif5 (pif345), and pif1pif3pif4pif5 (pifq) The Colombia-0 ecotype of Stratified seeds were irradiated with WL at 21°C for 3 h to induce germination, followed by a FR pulse for 15 min to suppress pseudo dark effects Arabidopsis 2-d-old dark-grown whole seedlings as described Arabidopsis seedlings grown under the same conditions were used as the negative control following the same assay procedure.The ChIP assay was performed using about 2 g of www.illumina.com) with some modifications. Four ChIP samples from technical replicates of each biological replicate were pooled together and concentrated to increase the starting amount of DNA. The end repair of DNA fragments was performed using End-It DNA End-Repair Kit (Epicentre). The A-tailing was added to the end-repaired DNA fragments using Klenow Fragment (NEB), and then Illumina's PE adapters were ligated by T4 DNA Ligase (Promega) at 16°C overnight. The adapter-ligated DNA fragments in the 200–300 bp size-range were selected by the gel purification, and then were amplified using Phusion High-Fidelity DNA Polymerase (NEB) with the Illumina PE PCR primer set. The library was purified using an Agencourt AMPure XP system (Beckman Coulter Genomics), and then validated by Bioanalyzer 2000 (Agilent).The ChIP-seq library was constructed according to Illumina's instructions  by employing modified parameters .The statistical identification of PIF3-binding peaks was performed separately for each biological replicate using MACS Four independent biological replicates of ChIP-seq data were collected, and replicate-specific binding peaks, identified in at least one other replicate, were defined as reproducible, if the distance between the summits of each replicate were less than 100 bp. For each reproducible peak, a mean summit position was assigned as the average position of the individual replicate-specific summits, and the PIF3-binding sites were defined as the 201 bp windows centered at each reproducible mean-summit position.De novo PIF3-binding motif discovery was performed on the 201-bp defined binding sites using MEME Definition of the closest neighboring genes to each binding peak was approached by scanning the regions within +/−5 kb centered at each peak summit, using CisGenome 25 (Invitrogen), and fragmented using Fragmentation Reagents (Ambion) at 70°C for 2.5 min in 20 µl of reaction. The polyA-tailed 3′-end fragments were captured by another run of mRNA purification as described above, and then treated by Antarctic Phosphatase (NEB) and T4 Polynucleotide Kinase (NEB) at 37°C for 1 h and 2 h, respectively. The sample was purified using RNeasy MinElute Cleanup Kit (Qiagen) according to Illumina's protocol. The eluted mRNA fragments were ligated with 2.5 µM of Illumina's SRA 5′ adaptor by T4 RNA Ligase 1 (NEB) at 20°C for 4 h. The 3′ cDNA adapter derived from Illumina's v1.5 sRNA 3′ adapter was conjugated with the anchored oligo (dT)20 primer, and introduced through reverse transcription using the SuperScript III First-Strand Synthesis System (Invitrogen). The first-strand cDNA was purified using the Agencourt AMPure XP system, and then amplified by PCR reaction using Phusion High-Fidelity DNA Polymerase with Illumina's sRNA PCR primer set. The size of purified library DNA was validated by Bioanalyzer 2000.Total RNA was extracted from 2-d-old dark-grown seedlings using QIAshredder column and RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions. The sequencing library construction was adapted from 3′-end RNA-seq protocol Libraries from the 1st biological replicate were assayed by 36-cycle single-end sequencing on the Illumina GAIIx platform, while libraries from the 2nd and 3rd biological replicates were assayed by 50-cycle single-end sequencing on the HiSeq2000 platform. For consistency, only the 5′-end 36-nt trimmed reads from the 2nd and 3rd replicates, as well as the full-length 36-nt reads from the 1st replicate, were aligned to the TAIR9 representative transcriptome using Bowtie with zero mismatches allowed. Only reads mapping uniquely to the 3′-end 500-bp region of the coding strand were counted for gene expression. Differentially expressed genes were identified using the edgeR package E. coli as described previously The recombinant protein GST-PIF3-Flag and the GST control were purified from The DPI-ELISA assays were performed as described The EMSA assays were performed as described RT-qPCR was performed as described PIL1 promoter region (pPIL1) upstream of the ATG was amplified by PCR using the pPIL1-F1/R1 primer set, and then the XhoI/EcoRI fragment was cloned into the pBluescript II SK (pBSK) vector (Stratagene) to produce pBSK-pPIL1. The G-box mutations were introduced by two-step PCR amplification , and the XhoI/MfeI fragment from the pPIL1-F1/R5 primer set was cloned to replace the unmutated fragment of pBSK-pPIL1 to produce pBSK-mpPIL1. The HindIII/BamHI fragment containing the omega-LUC+-rbcS terminator from the pENTR/D-TOPO\arGIp::LUC+ construct was cloned into pBSK-pPIL1 and pBSK-mpPIL1, respectively, to produce pBSK-pPIL1:LUC and pBSK-mpPIL1:LUC. The CDS of Renilla Luciferase (RLUC) was amplified by PCR using the Rluc-F1/R1 primer set, and then the NcoI/PmlI fragment was cloned into the pCAMBIA1302 binary vector to produce pC1302-35S:RLUC. The PstI/SacI fragments from pBSK-pPIL1:LUC and pBSK-mpPIL1:LUC were then sub-cloned into pC1302-35S:RLUC to produce pC1302-pPIL1:LUC-35S:RLUC (pPIL1:LUC) and pC1302-mpPIL1:LUC-35S:RLUC (mpPIL1:LUC), respectively.The 1.8 kb PIF3, PIF4 and PIF5 were amplified from Arabidopsis (Col-0 ecotype) genomic DNA by PCR using the TOPO-PIF3p-LP1/RP1, TOPO-PIF4p-LP1/RP1 and TOPO-PIF5p-LP1/RP1 primer sets, respectively. The PCR products were cloned into the pENTR/D-TOPO vector (Invitrogen) to produce the pPIF3, pPIF4 and pPIF5 entry clones. For the PIF1 promoter, the first 2 kb fragment upstream of the ATG was amplified by PCR using the TOPO-PIF1p-LP3/RP1 primer set, and then was cloned into the pENTR/D-TOPO vector to produce the intermediate entry clone. The second fragment of 2–4 kb upstream of ATG was amplified using the NotI-PIF1p-LP/XcmI-PIF1p-RP primer set, and then the NotI/XcmI fragment of the PCR product was subcloned into the intermediate entry clone to produce the pPIF1 entry clone. All four entry clones were subcloned into the gateway compatible pGWB3 binary vector pPIF:GUS constructs.The 2.5–3.0 kb promoter regions upstream of the ATG of Arabidopsis plants as described The constructs were transformed into LUC was represented by its enzyme activity compared to the RLUC internal control.The 2-d dark-grown seedlings of independent transgenic lines were ground in liquid nitrogen, and total protein was extracted in LUC extraction buffer  at 3× w/v) as described 3Fe(III)(CN)6, 0.5 mM K4Fe(II)(CN)6, 1 mM EDTA, 1% Triton X-100, 1 mg/ml X-gluc). Data of biological triplicates were collected from two independent transgenic lines, and representative images are shown for each transgene.Histochemical GUS staining assays were performed on 2-d-old dark-grown seedlings as described ChIP-seq and RNA-seq data reported in this study have been deposited in the Gene Expression Omnibus database under the accession number GSE39217.Figure S1ChIP-qPCR validation of PIF3-binding sites. (A–C) PIF3-binding sites containing the G-box (A), PBE-box (B), or both motifs (C) were validated using independent ChIP-qPCR tests. Sites are named according to adjacent genes. The relative enrichment level is presented as the percentage of input-control DNA that is immunoprecipitated for the P3M and WT samples. Data are presented as the mean of biological triplicates +/− SEM.(TIF)Click here for additional data file.Figure S2in vitro binding to the G-box and PBE-box motifs. EMSA assays show that the recombinant GST-PIF3 protein (Lane 3), but not GST by itself (Lane 2), specifically binds to the wild-type (WT) biotinylated G-box and PBE-box probes. For probes containing single G-box or PBE-box motif , the cold competitor probes mutated at the motif  did not exhibit competitive effects similar to the WT competitor (Lane 4). For probes containing two G-box motifs (PIL1b and RGA1), the mut probes at either one motif (Land 6 and 7) showed competitive effects similar to the WT probes (Lane 4), whereas the mut probe at both motifs (Lane 5) did not show efficient competition. The ATHB-2 probe contains one G-box and one PBE-box motif separated by 5 bp. The mut probe at only the G-box (Lane 6) displayed no competition, as did the probe mutated at both G-box and PBE-box motifs (Lane 5), while the mut probe at only PBE-box motif (Lane 7) showed a competitive effect similar to the WT probe (Lane 4). 1 both G-boxes mutated, 2 only 1st G-Box mutated, 3 only 2nd G-Box mutated, 4 G-box and PBE-box mutated, 5 only 1st PBE-box mutated.EMSA assays of PIF3 (TIF)Click here for additional data file.Figure S3pif145 mutant compared to that in the pifq mutant (pif145/pifq), and the genes are arrayed according to this ratio. We assayed the expression patterns of 12 (A) and 20 (B) genes in each class using RT-qPCR, and the expression relative to WT was normalized to the PP2AA3 internal control, for the pif3, pif145 and pifq mutants. The data are presented as the mean of biological triplicates +/− SEM in the vertical bar-graph boxes, compared to the RNA-seq data for these genes. Genes highlighted-in-red exhibited statistically-significant differences between pif145 and pifq in the RT-qPCR tests, by Student's t-test (P<0.05) (indicated by red star above the pif145 bar), while genes highlighted-in-blue did not. Data from RNA-seq are shown as the fold-change determined by the edgeR package in each pif mutant compared to WT. The internal percentage values in each box represent the relative contribution of PIF3 to the total PIF-quartet-induced transcription for each tested gene, as shown in RT-qPCR validation of PIF3- and PIF-quartet-regulated genes. (A and B) The orange horizontal bar graphs show the relative PIF3-induced expression of 19 Z-Class (A) and 88 Y-Class (B) PIF-induced genes as determined by RNA-seq analysis. The relative PIF3-induced expression value was calculated as the ratio of the level in the (TIF)Click here for additional data file.Figure S4pifq and WT. The separate activities of PIF3 and the combined PIF1/4/5-trio, are defined as the differences in expression between pif145 and pifq, and between pif145 and WT, respectively. The relative contribution is then defined as the percentage of the collective PIF-quartet regulation contributed by the separate activities of PIF3 and the combined PIF1/4/5-trio, respectively.Relative contribution of PIF3 and the PIF1/4/5-trio to the collective regulation of the PIF quartet. The relative contribution of PIF3 and the PIF1/4/5-trio to the collective regulation of the PIF quartet is plotted for the 107 PIF3-bound, PIF-induced genes (Classes Y and Z). The degree of collective regulation by the PIF quartet is defined as the difference in expression between (TIF)Click here for additional data file.Figure S5Functional categorization of PIF3-bound, PIF-regulated genes. (A) The total PIF3-bound, PIF-regulated genes identified by integrating our ChIP-seq and RNA-seq analyses  were assigned to functional categories, color-coded as shown. This assignment was based on the Gene Ontology annotations for biological and/or molecular function in the TAIR database. The percentage of the total annotated genes within each category was calculated after excluding the genes annotated as having unknown function. (B) PIF-induced and PIF-repressed genes in Classes X, Y and Z were assigned separately to the functional categories as described in (A). The first numbers in parentheses represent the genes with functional annotation, and the second numbers represent the total genes in each class.(TIF)Click here for additional data file.Figure S6TCP15 (A), IAA29 (B), ATHB52 (C), CKX5 (D), XTR7 (E), SNRK2.5 (F), AT5G02580 (G), and SDR (H), respectively. The ChIP and RNA tracks show the pile-up distribution of the combined raw reads from four biological replicates of ChIP-seq data and three replicates of RNA-seq data, respectively. P3M- and WT-ChIP: DNA immunoprecipitated from PIF3-Myc-expressing and from wild-type seedlings, respectively. WT-, pif3-, pif145- and pifq-RNA: RNA from genotypes used for expression analysis. Binding Site: 201 bp defined as the PIF3-binding site. Summit, predicted PIF3-binding center defined from the binding-peak maximum. G- and PBE-box: Vertical lines indicate motif positions.Correlated PIF3-binding and PIF-regulation of eight M-class genes. (A–H) Visualization of ChIP-seq and RNA-seq data in the genomic regions encompassing (TIF)Click here for additional data file.Figure S7Comparison of potential direct targets of PIF3, PIF4 and/or PIF5. (A) Venn diagram shows overlap of genes associated with PIF3, PIF4 and/or PIF5 binding sites. (B) Venn diagram shows PIF-quartet-regulated genes bound by PIF3, PIF4 and/or PIF5.(TIF)Click here for additional data file.Table S1List of PIF3-binding peaks from four ChIP-seq replicates. (A) List of PIF3-binding peaks from the 1st replicate. (B) List of PIF3-binding peaks from the 2nd replicate. (C) List of PIF3-binding peaks from the 3rd replicate. (D) List of PIF3-binding peaks from the 4th replicate.(XLS)Click here for additional data file.Table S2List of reproducible PIF3-binding sites.(XLS)Click here for additional data file.Table S3List of PIF3-bound genes.(XLS)Click here for additional data file.Table S4pifq/WT comparison (PIF-quartet-regulated genes).List of SSTF genes from (XLS)Click here for additional data file.Table S5pif3/WT comparison (PIF3-regulated genes (loss-of-function)).List of SSTF genes from (XLS)Click here for additional data file.Table S6pif145/WT comparison (PIF1/4/5-trio-regulated genes (loss-of-function)).List of SSTF genes from (XLS)Click here for additional data file.Table S7pifq/pif145 comparison (PIF3-regulated genes (gain-of-function)).List of SSTF genes from (XLS)Click here for additional data file.Table S8pifq/pif3 comparison (PIF1/4/5-trio-regulated genes (gain-of-function)).List of SSTF genes from (XLS)Click here for additional data file.Table S9Composite list of PIF3-regulated genes.(XLS)Click here for additional data file.Table S10Composite list of PIF1/4/5-trio-regulated genes.(XLS)Click here for additional data file.Table S11List of PIF3-, PIF4 and/or PIF5-bound, PIF-regulated  genes.(XLS)Click here for additional data file.Table S12List of rapidly-light-regulated W-class genes.(XLS)Click here for additional data file.Table S13in vitro binding probes.List of oligonucleotides used in this study. (A) List of ChIP-qPCR primers. (B) List of RT-qPCR primers. (C) List of cloning primers. (D) List of (XLS)Click here for additional data file.


CTD Ser-2 phosphorylation increases under nitrogen starvation, and the stress-responsive MAP kinase pathway, mediated by Wis1 MAPKK and Sty1 MAPK, is critical for this stress response. Sty1 phosphorylates Lsk1, the catalytic subunit of CTDK-I. Furthermore, a feedback loop stemming from activated Mei2 to Win1 and Wis4 MAPKKKs operates in this pathway and eventually enhances CTD Ser-2 phosphorylation and ste11 transcription. Hence, in addition to starting meiosis, Mei2 functions to reinforce the commitment to it, once cells have entered this process. This study also demonstrates clearly that the stress-responsive MAP kinase pathway can modulates gene expression through phosphorylation of Pol II CTD.The RRM-type RNA-binding protein Mei2 is a master regulator of meiosis in fission yeast, in which it stabilizes meiosis-specific mRNAs by blocking their destruction. Artificial activation of Mei2 can provoke the entire meiotic process, and it is suspected that Mei2 may do more than the stabilization of meiosis-specific mRNAs. In our current study using a new screening system, we show that Mei2 genetically interacts with subunits of CTDK-I, which phosphorylates serine-2 residues on the C-terminal domain of RNA polymerase II (Pol II CTD). Phosphorylation of CTD Ser-2 is essential to enable the robust transcription of  ste11 gene is transcribed robustly only when serine-2 residues on the C-terminal domain (CTD Ser-2) of RNA polymerase II are phosphorylated. We show that the stress-responsive MAP kinase cascade transmits the environmental signal to stimulate CTD Ser-2 phosphorylation. Sty1 MAP kinase appears to phosphorylate and activate the catalytic subunit of CTDK-I, which in turn phosphorylates CTD Ser-2. We demonstrate further that Mei2, expression of which depends on Ste11, can activate the MAP kinase cascade, forming a feedback loop. Thus, we clarify here three important issues in cellular development: the physiological role of CTD Ser-2 phosphorylation, the molecular function of the stress-responsive MAP kinase pathway, and the presence of positive feedback that reinforces the commitment to meiosis.Hundreds of genes are newly expressed during meiosis, a process to form gametes, and the control of meiosis-specific gene expression is not simple. The master regulator of meiosis in fission yeast, Mei2, blocks an RNA destruction system that selectively degrades meiosis-specific mRNAs, highlighting the importance of post-transcriptional control in meiotic gene expression. Here we present another example of unforeseen regulation for meiosis. Ste11 is a key transcription factor responsible for the early meiotic gene expression in fission yeast. The  The cell cycle programs for mitosis and meiosis appear to be strictly segregated from each other, although they are likely to have molecular mechanisms in common. Analyses in lower eukaryotes have shown that factors required exclusively for meiosis, generated through the transcriptional activation of meiosis-specific genes, are largely responsible for the segregation of these two processes ste11, which encodes a key transcription factor that regulates the mei2 gene. Pol II CTD serves as a binding scaffold for a variety of nuclear factors, and its phosphorylation status has been implicated in regulation of an ever-increasing number of functions necessary to execute complex transcriptional processes ste11 transcription, but is not absolutely necessary for the mitotic program. Essentially the same conclusions have been reached independently by others, through global gene expression analysis To identify possible upstream or downstream effectors of Mei2, we devised a new screening system and found that a subunit of CTDK-I, which is a CDK-like kinase complex that phosphorylates the C-terminal repeat domain of the largest subunit of RNA polymerase II (Pol II CTD) ste11 via a feedback loop comprising the stress-responsive MAP kinase cascade and the phosphorylation of Pol II CTD by CTDK-I.Taken together, the results of our present study demonstrate a new regulatory paradigm for meiosis by Mei2 in fission yeast, i.e., that this master meiotic regulator ensures the commitment to meiosis by strengthening the transcription of mei2-L-SATA allele driven by the authentic mei2 promoter. This allele contains a combination of two mutations, mei2-L and mei2-SATA. The former mutation confers temperature-sensitivity to the Mei2 protein (our unpublished results), whereas the latter activates this gene constitutively, overriding the inhibitory phosphorylation by Pat1 kinase http://old.genedb.org/genedb/pombe/) to encode a homologue of the γ subunit of RNA polymerase II C-terminal domain kinase I (CTDK-I). CTDK-I belongs to the CDK family, but in addition to the catalytic subunit α and the cyclin-like regulatory subunit β conserved among these family members, it contains a third γ subunit lsk1 and lsc1 genes encode the α and β subunits of the CDK proteins, respectively lsg1.The haploid fission yeast strain JV312 harbors the Saccharomyces cerevisiae CTDK-I γ (CTK3) was found not to be high  Ste11 regulates the transcription of many genes essential for sexual development, including mei2ste11 has been shown to suppress ectopic meiosis induced by the pat1 mutation and restore vegetative growth, by blocking the expression of mei2ste11Δ cells show G1 arrest retardation under conditions of nitrogen starvation, even more extensively than lsg1Δ, lsk1Δ or lsc1Δ cells, while mei2Δ cells are not so much affected  of RNA polymerase II rpb1 alleles (reported by J. Karagiannis and kindly provided to us), namely rpb1-12×CTD and rpb1-12×S2ACTD. The former allele produces Rpb1 carrying a CTD that consists of 12 copies of the authentic heptad repeat (YSPTSPS), whereas the latter produces Rpb1 with 12 copies of a mutant heptad repeat in which Ser-2 is substituted by alanine (YAPTSPS) rpb1-12×S2ACTD allele were impaired severely in terms of conjugation and sporulation . These results indicate clearly that activation of Mei2 can stimulate expression of ste11 through phosphorylation of PolII CTD.To evaluate physiological significance of the feedback, we examined whether activation of Mei2 would result in enhancement of t manner . Deletioild-type  or mei2Δste11 during meiosis under physiological conditions. To do so, we used the mei2-FA allele, which produces inactive Mei2 protein ste11 and mei2 in wild-type and mei2-FA cells subjected to nitrogen starvation. As shown in ste11 mRNA, normalized by ribosomal RNA, and that of mei2 mRNA also, were higher in wild-type cells than in mei2-FA cells, and the difference became greater in later stages. This suggests that activated Mei2 protein in wild-type cells indeed enhances ste11 expression via feedback.We finally evaluated the contribution of the feedback regulation to the expression of Taken together, we propose that fission yeast possess a regulatory circuit, as depicted in ste11 gene, which encodes a transcription activator essential for the expression of mei2 and other genes crucial for sexual development. However, this loss of function does not significantly affect the gene expression required for vegetative growth. In an independent study, Hermand and colleagues have performed genome-wide mapping of three kinds of CTD kinases and also of serine 2- and 5-phosphorylated Pol II in fission yeast to investigate the link between CTD phosphorylation and specific cellular events ste11 gene, and remarked that the phosphorylation of CTD Ser-2 plays a highly specialized role in gene regulation in fission yeast, unlike in other organisms, and is virtually confined to the regulation of a single key gene controlling sexual differentiation. Our study fully supports this notion. While a subsequent study ste11 transcrition can be compensated partially by loss of Ser-7 phosphorylation, the nature of such extreme specification and its evolution is an intriguing enigma.In our present study, we have demonstrated that a genetic interaction exists between the subunits of CTDK-I, a protein kinase complex that phosphorylates RNA polymerase II CTD, and the master meiotic regulator in fission yeast, Mei2. Furthermore, our analyses indicate that a loss of CTDK-I function impairs the transcription of the ste11 has been known for some time ste11 expression by the Sty1 MAPK cascade. It has been shown that when phosphorylated and activated by Wis1 MAPKK, the Sty1 protein migrates to the nucleus and resides on the promoter regions of stress-responsive genes S. cerevisiae Hog1 in vitro. While the phosphorylation of Lsk1 in vivo remains to be confirmed, it appears to be conceivable that Sty1 may also be recruited to the ste11 promoter and phosphorylate CTDK-I staying there, which in turn phosphorylates CTD and licenses RNA polymerase II to transcribe the gene. In this regard, it is noteworthy that hsp9, which encodes a small heat-shock protein hsp9 and other CSRE genes upon osmotic stress in an Atf1-dependent manner, but does not necessarily phosphorylate Atf1 as a substrate ste11 and hsp9 may be similarly regulated by the Sty1 – CTDK-I – CTD phosphorylation system. However, conventional Chip analyses have not provided convincing evidence for the association of Sty1 with the ste11 promoter, and we are conducting further experiments to scrutinize this possible scheme.Our present data have further shown that the stress-responsive MAP kinase pathway is crucial for the activation of CTDK-I under conditions of nitrogen starvation. The requirement for Sty1 MAPK and its target Atf1 for the expression of ste11 through the Sty1 – CTDK-I – CTD phosphorylation system. From our findings we can outline a framework of the molecular mechanisms that differentiate the mitotic and meiotic programs in fission yeast as in The results of our present analyses demonstrate unambiguously that a feedback-regulatory system operates in fission yeast during the meiotic cell cycle. In this feedback loop, the active form of Mei2 can eventually stimulate the stress-responsive MAPKKKs and enhance the transcription of S. pombe strains used in this study are listed in S. pombe experiments were as described previously S. pombe was performed using the lithium acetate method The ura4+ cassette used for insertion mutagenesis was amplified by PCR using the primers N18AGCTTAGCTACAAATCCCACTGGCT and N18TGTGATATTGACGAACTTTTTGAC (N18: 18b random DNA sequence). The PCR products were then introduced into JV312 (mei2-L-SATA ura4-D18) cells, and transformants were plated onto SD lacking uracil and incubated at 25°C. Colonies were selected, and the site of ura4+ integration was determined via the sequencing of inverse PCR products The Samples were prepared for flow cytometry essentially as described previously lsg1Δ) cells were grown to mid-log phase in MM medium and shifted to MM-N medium. The cells were collected 2.5 h after the shift, and total RNA was extracted as described previously JY450 (wild-type) and JT659 , or Sty1KD-3Pk (NJ767) were subjected to nitrogen starvation for 1 h. Extracts were prepared, protein immunoprecipitated, and the immuno-complexes tested for kinase activity as described Figure S1S. pombe Lsg1 with its S. cerevisiae orthologue CTK3. A ClustalW alignment of Lsg1 and CTK3 is shown.Comparison of (TIF)Click here for additional data file.Figure S2lsg1Δ mutant in sexual development. (A) FACS analysis of DNA content in cells subjected to nitrogen starvation. Cells of diploid strains JY362 (wild-type), JT665 (lsg1Δ), JZ403 (ste11Δ) and JY776 (mei2Δ) cultured in liquid MM medium to mid-log phase were shifted to MM-N medium. Aliquots were taken at indicated time and the DNA content per cell was determined by FACS analysis. (B) Northern blot analysis of ste11 and rbp1 in JY450 (wild-type) and JT659 (lsg1Δ) cells. The cultures were grown to the mid-log phase, shifted to nitrogen-free medium, and sampled at the indicated intervals. Total RNA (10 µg) from each sample was resolved by gel electrophoresis and subjected to northern blot analysis to detect ste11 and rbp1 transcripts. rRNAs stained with ethidium bromide are shown as a loading control. (C) Recovery of mating and subsequent sporulation by ste11 overexpression in the CTDK-I deletion mutants. Cells of the homothallic haploid strains JY450 (wild-type), JZ396 (ste11Δ), JT659 (lsg1Δ), JT660 (lsk1Δ), and JT661 (lsc1Δ), harboring either the pREP41-ste11 or control pREP41 vectors, were examined for mating and subsequent sporulation after incubation on SSA plates at 30°C for three days.Deficiency of the (TIF)Click here for additional data file.Figure S3ste11 is a major target of CTDK-I. (A) Comparison of the global gene expression profiles between the wild-type (JY450) and lsg1Δ (JT659) fission yeast strains. Cells of each strain were grown to the mid-log phase in liquid MM and shifted to nitrogen-free MM-N. The cells were harvested 2.5 h after this shift. RNA was prepared from each sample and analyzed on a DNA microarray covering 4,997 genes. (B) Relative changes in gene expression levels caused by loss of lsg1 function. The ste11 gene and genes encoding its upstream regulators on the cAMP and stress-responsive MAPK pathways were examined. The values are presented using binary logarithms.Expression of (TIF)Click here for additional data file.Figure S4lsg1-gfp (JT670), lsk1-gfp (JT671), or lsc1-gfp (JT672) were grown to the mid-log phase and shifted to nitrogen-free medium. Cells were sampled at indicated time, and subjected to immunoblotting with antibody specific for GFP. α-tubulin is shown as a loading control. (B) A strain expressing fcp1-gfp (JT673) was analyzed as in (A).Quantification of subunits of CTDK-I and a phosphatase Fcp1 in cells subjected to nitrogen starvation. (A) Strains expressing either (TIF)Click here for additional data file.Figure S5pcr1Δ (JX25), and rst2Δ (JX231) cells subjected to nitrogen starvation were examined for CTD Ser-2 phosphorylation by immunoblotting with antibody specific for the Ser-2 phosphorylated form of the CTD (H5). α-tubulin is shown as a loading control.Pcr1 and Rst2 do not contribute to CTD Ser-2 phosphorylation significantly. Wild-type (JY450), (TIF)Click here for additional data file.Table S1lsg1-deletion mutant. Genes regulated by Ste11 are marked in blue, and hsp9 is marked in yellow.Genes downregulated in the (XLS)Click here for additional data file.Table S2S. pombe strains used in this study.(DOC)Click here for additional data file.