Candida albicans and identified two factors, Tye7p and Gal4p, as key regulators of glycolysis. When respiration was inhibited or oxygen was limited, a gal4tye7 C. albicans strain showed a severe growth defect when cultured on glucose, fructose or mannose as carbon sources. The gal4tye7 strain displayed attenuated virulence in both Galleria and mouse models as well, supporting the connection between pathogenicity and metabolism. Chromatin immunoprecipitation coupled with microarray analysis (ChIP-CHIP) and transcription profiling revealed that Tye7p bound the promoter sequences of the glycolytic genes and activated their expression during growth on either fermentable or non-fermentable carbon sources. Gal4p also bound the glycolytic promoter sequences and activated the genes although to a lesser extent than Tye7p. Intriguingly, binding and activation by Gal4p was carbon source-dependent and much stronger during growth on media containing fermentable sugars than on glycerol. Furthermore, Tye7p and Gal4p were responsible for the complete induction of the glycolytic genes under hypoxic growth conditions. Tye7p and Gal4p also regulated unique sets of carbohydrate metabolic genes; Tye7p bound and activated genes involved in trehalose, glycogen, and glycerol metabolism, while Gal4p regulated the pyruvate dehydrogenase complex. This suggests that Tye7p represents the key transcriptional regulator of carbohydrate metabolism in C. albicans and Gal4p provides a carbon source-dependent fine-tuning of gene expression while regulating the metabolic flux between respiration and fermentation pathways.Glycolysis is a metabolic pathway that is central to the assimilation of carbon for either respiration or fermentation and therefore is critical for the growth of all organisms. Consequently, glycolytic transcriptional regulation is important for the metabolic flexibility of pathogens in their attempts to colonize diverse niches. We investigated the transcriptional control of carbohydrate metabolism in the human fungal pathogen  Candida albicans, depends upon glycolysis for the progression of systemic disease. We investigated glycolytic transcriptional regulation in C. albicans and defined two key regulators of the pathway, Tye7p and Gal4p. We demonstrated that these factors are important for the fermentative growth of C. albicans both in vitro and in vivo and also regulate the input and output fluxes of glycolysis. The gal4tye7 strain showed attenuated virulence in a Galleria and two mouse models, potentially due to the severe growth defect in oxygen-limiting environments. Gal4p and Tye7p represent fungal specific regulators involved in the pathogenicity of the organism that may be exploited in the development of antifungal treatments. Our study describes a fungal glycolytic transcriptional circuit that is fundamentally different from that of the model yeast Saccharomyces cerevisiae, providing further evidence that the transcriptional networks in S. cerevisiae need not be generally representative of the fungal kingdom.Pathogens must be able to assimilate the carbon sources in their environment to generate sufficient energy and metabolites to survive. Since glycolysis is a central metabolic pathway, it is important for this metabolic flexibility. The most commonly isolated agent in human fungal infections,  In order to grow and thrive in a wide range of hosts, pathogens not only depend on certain virulence factors but also metabolic flexibility; therefore, they must be able to assimilate various carbon sources. Carbohydrates are the primary and preferred source of metabolic carbon for most organisms, and are used for generating energy and producing biomolecules. Most sugars are converted to glucose 6-phosphate or fructose 6-phosphate before entering the glycolytic pathway. Glycolysis is then responsible for converting these hexose phosphates into the key metabolite pyruvate while producing ATP and NADH . From thSaccharomyces cerevisiae  upstream of the glycolytic genes and Gcr2p acts as a co-activator by forming a complex with Gcr1p S. cerevisiae. Tye7p has been shown to be involved in the activation of several glycolytic genes, although not to the same extent as Gcr1p and Gcr2p GCR1 and the tye7 strain displays no growth defects under any carbon source regime Since glycolysis is a central metabolic pathway, it is strictly regulated. While there are different levels of regulation of the process, transcriptional control is common to bacteria, fungi, plants, and animals. The glycolytic enzymes are transcriptionally regulated in response to environmental conditions such as oxygen levels, carbon source and availability, and to cellular demands such as metabolite concentrations and energy needs. However, in most species the regulators of glycolytic gene expression have not been identified, so our understanding of transcriptional control of glycolysis in eukaryotes is mainly based on the non-pathogenic yeast S. cerevisiae, most organisms to do not have GCR1 or GCR2 homologs Saccharomyces-lineage exhibits a unique dependence on the fermentation pathway: these yeasts mainly ferment sugars to ethanol instead of using respiration, even under aerobic conditions S. cerevisiae up-regulates glycolysis and represses the TCA cycle in the presence of glucose allowing this aerobic fermentation behavior to occur S. cerevisiae switch to the respiratory mode. This phenomenon is known as the Crabtree effect and is due to a glucose repression circuit that is largely regulated by the transcriptional repressors Mig1p and Rgt1p, the protein kinase Snf1p, and the protein complex SCFGrr1Saccharomyces from KluyveromycesSaccharomyces-lineage and many of the genes retained from the whole-genome duplication are involved in the lifestyle adaptation to aerobic ethanol production Although the glycolytic circuit is well characterized in Saccharomyces yeasts is in contrast to that of most other eukaryotes, which are either facultative or obligate aerobes and lack the glucose repression circuit. Under aerobic conditions, Crabtree-negative cells predominately oxidize pyruvate to carbon dioxide through the TCA cycle. In the absence of oxygen, most aerobic organisms are able to utilize the fermentation pathway to some extent to continue regenerating NAD+. This difference in metabolic flux is highlighted by transcription profiles of the aerobic fungi Trichoderma reesei, Neurospora crassa, and Aspergillus oryzae, which show little or no repression of the TCA cycle in glucose rich compared to glucose poor growth conditions, and therefore do not rely as heavily on the fermentation pathway as does S. cerevisiaeThe facultative anaerobic lifestyle of Crabtree-positive Candida albicans is a facultative aerobe and thus metabolizes carbon sources in response to oxygen availability similar to that of a typical eukaryotic cell. C. albicans is responsible for various non life-threatening infections such as oral thrush and vaginitis but in extreme cases, especially in immunosuppressed individuals, it can cause potentially lethal systemic infections. In fact, Candida species are the most common isolated agent in fungal infections and the fourth leading cause of nosocomial bloodstream infections in the United States, with an attributable mortality rate of approximately 38–49% and treatment costs estimated to be $1.7 billion annually C. albicans accounts for more than half of all Candida infections The opportunistic human fungal pathogen GCR1/2 homologs, such as C. albicans, must control transcription of glycolytic genes differently than does S. cerevisiae. In this study, we characterized two fungal-specific activators of the glycolytic pathway in C. albicans, Tye7p and Gal4p. Deleting both genes resulted in severe growth defects when the mutant cells were cultured on fermentable carbon sources when respiration was inhibited or oxygen was limited, and chromatin immunoprecipitation coupled with microarray analysis (ChIP-CHIP) and transcription profiling showed these factors bind to and regulate expression of the glycolytic pathway genes. Tye7p and Gal4p are also required for complete pathogenicity as the mutant strains showed attenuated virulence. This work therefore defines the key regulatory elements controlling glycolytic gene expression in a facultative aerobic pathogen.Crabtree-negative organisms that lack C. albicans. In the well-studied yeast S. cerevisiae, Gcr1p, Gcr2p, and Tye7p are key glycolysis-specific activators. Unlike Gcr1p and Gcr2p, which are limited to Saccharomyces and closely related yeasts, Tye7-like transcription factors can be found throughout the Saccharomycotina subphylum. Therefore, while there are no homologs of Gcr1p or Gcr2p in C. albicans, there is a CaTye7p. ScTye7p and CaTye7p share 87% amino acid similarity in the DNA binding basic-helix-loop-helix (bHLH) domain but only 33% in the activation domain. A recent investigation also implicated the CaGal4p transcription regulator in the expression of genes involved in glycolysis S. cerevisiae is a well-characterized zinc cluster transcription factor that regulates galactose catabolism, it does not fulfill this role in C. albicans. ScGal4p and CaGal4p also share homology strictly in the DNA-binding domain. Due to the potential or observed involvement of these factors in aspects of carbohydrate metabolism, we tested the role of CaTye7p and CaGal4p in the control of glycolytic gene expression in C. albicans.We investigated possible transcriptional regulators of glycolytic gene expression in tye7 and gal4tye7 deletion strains to use in conjunction with our previously generated gal4 strain gal4tye7 strain with glucose media, as the doubling time was 147 min compared to that of the wild type of 123 min  are included in this group of targets. Therefore, the ChIP-CHIP data suggests that Gal4p and Tye7p are involved in the entire fermentation pathway from glucose to ethanol.The tiling array showed highly similar results but was able to identify a few additional targets including three glycolytic genes, 52 peaks  and S5. Although Gal4p and Tye7p bound many common targets, there were a significant number of individual binding events, some of which are related to carbohydrate metabolism . The cleGAL4, the binding and transcription profiles can provide different insights. Therefore, to complement the ChIP-CHIP analysis, transcription profiling comparing wild type and mutant strains was performed under glucose growth conditions. The expression levels of selected carbohydrate metabolic genes is illustrated in tye7 strain confirming Tye7p's role as an activator of fermentative metabolism. Gal4p is also involved in the activation of the glycolytic/fermentation genes because the expression levels of these genes were lower in the gal4tye7 strain compared to the tye7 strain. The involvement of Gal4p in the activation of glycolytic gene expression was masked in the gal4 expression profiles, most likely because the absence of GAL4 caused an up-regulation of TYE7ChIP-CHIP is a whole-genome approach for determining binding locations of a transcription factor; however, it is insufficient to give a complete picture of a factor's biological function. As was observed with tye7 strain but not further reduced in the gal4tye7 strain indicating that Gal4p is not involved in their activation. Trehalose is a glucose disaccharide that has a role as a storage carbohydrate in yeast. Another important storage molecule in yeast is glycogen. Tye7p also bound several glycogen metabolic targets  that were subsequently down-regulated in the tye7 strain and showed no influence of Gal4p in the gal4tye7 strain. As well, Tye7p was the sole regulator of several glycerol metabolic targets . On the other hand, Gal4p activated the PDH genes with no influence from Tye7p. These genes were not down-regulated in the tye7 strain  but were reduced in the gal4tye7 strain.The ChIP-CHIP data showed that only Tye7p bound the genes involved in trehalose metabolism. Expression of the trehalose metabolic genes was down-regulated in the tye7 P-value: 1.34E-18; gal4tye7: 1.44E-15) and cellular alcohol metabolic process  being the most significantly enriched categories.The down-regulated genes were analyzed for GO enrichment. As expected, all the categories were related to carbon metabolism. Generally, the two deletion strains had similar results with glycolysis (PFK1 and PFK2) were among the top six most down-regulated genes in the tye7 strain while the gluconeogenesis-specific gene FBP1 was moderately down-regulated. Therefore, it appears that Tye7p regulates the flux between energy storage and energy production at the glucose-6-phosphate branch point  compared to the early part of the pathway, regardless of the carbon source of the growth medium CCG-3′5′-CGG  .C. albicans, this flexibility involves growth in oxygen rich environments such as the skin and oral mucosal layers and oxygen poor niches such as inner organs. Since serious systemic infections are associated with these oxygen poor conditions and the glycolytic genes are known to be up-regulated in response to hypoxia in C. albicansgal4 strain was unaffected but the tye7 and gal4tye7 strains displayed a severe growth defect . This up-regulation also explains the increase in PDH expression and is likely why some glycolytic genes were not as significantly down-regulated. Third, the expression of many more genes was altered in the hypoxia profile, but many of these could be attributed to the significant growth defect of the tye7 strain (doubling time was approximately 220 min compared to the wild type at around 110 min). We chose to just focus on the effect on metabolic gene expression as we had already established Tye7p as a metabolic regulator under normoxic conditions.To confirm that Tye7p is responsible for the induction of the glycolytic genes under hypoxic conditions we repeated the expression profile under glucose growth conditions in the presence of nitrogen instead of oxygen  difference compared to the wild type, while both the tye7 and gal4tye7 strains showed very significant  attenuated virulence  and C57BL/6J (P = 0.0007) mouse models  in the gal4tye7 infected mice compared to mice challenged with the wild type strain . These results further support the hypothesis that the reduced virulence of the double mutant strain is attributed to a growth defect due to low oxygen environments in the host, which allows the organism to survive with the infection for a longer time and increases the chance the host's immune system is able to clear the infection.Histological examination showed A/J mice  and confC. albicans and the non-pathogenic yeast S. cerevisiae. Crabtree-positive yeasts, such as S. cerevisiae, have developed a circuit that represses the respiration pathway and up-regulates the glycolytic/fermentation pathway in the presence of excess glucose even under aerobic conditions. In contrast, C. albicans is Crabtree-negative and prefers to completely oxidize carbohydrates through the respiration pathway in aerobic conditions, only relying on the fermentation pathway in the absence of oxygen. Furthermore, C. albicans up-regulates the glycolytic pathway in low oxygen conditions while S. cerevisiae does not C. albicans are similar to the majority of fungi and other eukaryotes. Therefore, it is important to extend our understanding of transcriptional control of carbohydrate metabolism beyond S. cerevisiae to other organisms.Although fungi generally have similarly designed metabolic pathways, their transcriptional regulation of these pathways can be quite different, as illustrated by the human fungal pathogen Kluyveromyces lactisS. cerevisiae and its glycolytic genes are mainly regulated by orthologs of Gcr1p and Gcr2p. It appears that K. lactis is an intermediate between S. cerevisiae and the majority of aerobic fungi that do not contain GCR1/2 homologs and therefore it is not a representative model of Crabtree-negative fungi. We characterized Tye7p and Gal4p, two transcriptional activators of the glycolytic pathway in C. albicans, which lacks GCR1/2 homologs. Deleting both factors resulted in a severe growth defect during culture on several fermentable carbon sources  when respiration was inhibited or oxygen was limited; the single mutant gal4 strain showed no growth defects while the tye7 strain displayed a slight growth defect under these conditions. All deletion strains grew at near wild type levels on a non-fermentable carbon source or with fermentable sources when the respiration pathway was not disrupted.Although the glycolytic transcriptional circuit has been studied in the yeast tye7 strain showed a down-regulation of these genes that was most significant in glucose growth conditions. Gal4p binding to the promoter sequences of the glycolytic genes was affected by the carbon source with a decrease in binding from glucose to galactose to glycerol. Gal4p's role in the activation of these genes was also carbon source-dependent with the strongest effect during growth on glucose and galactose. Furthermore, transcription profiling and qPCR confirmed that both Tye7p and Gal4p are involved in the induction of the glycolytic genes under hypoxic growth conditions.ChIP-CHIP and transcription profiling revealed that both Tye7p and Gal4p are directly involved in the activation of the entire glycolytic pathway. Tye7p bound all the glycolytic promoters in a carbon source-independent manner and the TYE7 increases the levels of trehalose and glycogen, likely a result of the severely reduced glycolytic flux due to the extremely low expression of PFK1 and PFK2. Deleting GAL4 had no effect on the storage carbohydrate levels. Therefore, Tye7p has a role in determining whether glucose is stored or utilized for energy derivation, similar to Gcr1p in S. cerevisiaeGal4p and Tye7p also regulated metabolic processes linked to the glycolytic pathway. Some of these roles are common while some are independent of one another. Tye7p bound to and activated many genes involved in trehalose and glycogen metabolism without Gal4p's involvement. As well, Tye7p strongly activated the genes encoding phosphofructokinase, which catalyzes an irreversible glycolytic-committing reaction. We showed that deleting S. cerevisiae, glucose induces PDC expression to promote fermentation while the PDH genes are unaffected and the TCA cycle is repressed A. oryzae and N. crassa, both the PDC and PDH are induced by glucose and the TCA cycle is not repressed LSC1/2), deleting GAL4 subsequently results in the down-regulation of the PDH genes and ultimately several TCA cycle genes Another equilibrium that requires regulation is the flux between fermentation and respiration. This flux depends on the competition for pyruvate between pyruvate decarboxylase (PDC) and the pyruvate dehydrogenase complex (PDH) Therefore, although both Gal4p and Tye7p are key regulators of the glycolytic pathway, each has its own distinct role. Tye7p is the central transcriptional regulator of carbohydrate metabolism that provides a strong basal level of glycolytic expression while controlling the flux into the pathway and committing the cell to glycolysis. Gal4p is a carbon source-dependent regulator that fine-tunes gene expression based on the cells' need for the fermentation pathway. It assists Tye7p by increasing glycolytic gene expression during growth on fermentable carbon sources. In the presence of fermentable carbon sources Gal4p is able to significantly enhance the energy producing part of the glycolytic pathway and promote respiration by activating the PDH to meet the increased energy needs of the cell and minimize the dependence on fermentation. This assistance is not required with non-fermentable carbon sources as the glycolytic flux is lower since gluconeogenesis is stimulated and the cells grow at a slower rate with reduced energy needs.C. albicans infections, azoles, polyenes, and echinocandins, target cell membrane and cell wall integrity; however, targeting fungal metabolism could provide for future drug development. The importance of glycolysis to C. albicans' pathogenicity has been shown as deleting CDC19 causes avirulence while a conditional mutant of FBA1 results in attenuated virulence C. albicans CDC19 has approximately 50% amino acid identity with human pyruvate kinases). However, Gal4p and Tye7p are fungal-specific regulators and therefore represent potential antifungal targets. GAL4 and TYE7 were shown to be important for the virulence in a Galleria and two mouse models. This virulence effect is likely a result of a growth defect due to the low oxygen conditions that are present in invasive infections. The ability of two-thirds of the gal4tye7 injected C57BL/6J mice to clear the infection highlights the real potential of Gal4p and Tye7p as drug targets. Although the double mutant strain did show significant attenuated virulence in all three host models, it was not avirulent. This is likely attributed to the metabolic flexibility of C. albicans and its ability to use alternative carbon sources. The importance of alternative carbon metabolism to C. albicans' pathogenicity has been previously demonstrated as deleting key enzymes of the glyoxylate cycle, gluconeogenesis, and ß-oxidation pathways reduces virulence The most common types of antifungal drugs for GCR1/2 homologs and the clustering of the Gal4p motif. Species that possess GCR1/2 homologs have an enrichment of the Gal4p motif upstream of the GAL regulon genes while species lacking GCR1/2 homologs have an enrichment of the Gal4p motif in the promoter regions of the glycolytic genes. Therefore, it appears that the rewiring of Gal4p coincides with the loss/gain of GCR1/2, and that Gal4p and Tye7p likely regulate glycolysis in the Saccharomycotina species lacking GCR1/2 homologs. Intriguingly, this rewiring event also appears to coincide with changes in the galactose sensory network It appears that Gal4p and Tye7p have altered their function throughout the evolution of fungi. We analyzed other genomes in the Saccharomycotina subphylum and observed a pattern relating the presence of S. cerevisiae and closely related species, suggesting that the transcriptional networks of S. cerevisiae are not representative models of the fungal kingdom. However, there is a key distinction between these two rewiring events. While the regulatory components of the ribosomal network are different, the ultimate outcome is identical. On the other hand, carbon metabolism regulation is fundamentally different between Saccharomyces and other eukaryotes suggesting that the entire carbohydrate transcriptional network has undergone rewiring, not just the regulators. Thus, investigating the regulation of carbon metabolism in Crabtree-negative organisms is important as little can be extrapolated based on S. cerevisiae. This study thus defines the key regulatory elements of glycolytic gene expression and provides insights into the mode of transcriptional regulation of carbohydrate metabolism in a typical eukaryotic cell and a human pathogen.Along with the ribosomal transcriptional network C. albicans strains used in this study are listed in The URA3 PCR product Plasmids and oligonucleotides used in this study are listed in tye7 strain. CMM3  gal4tye7 strain. The deletion and complementation strains were created using the SAT1-flipper cassette as described tye7 disruption construct was created by cloning 500 bp flanking sequences of TYE7 into the plasmid pSFS2A. The plasmid was linearized prior to transformation. Transformants were selected on YPD plates containing 200 µg/ml of nourseothricin and confirmed by PCR. Excision was performed by incubation at 30°C for 5 hours in YP media with 2% maltose before plating on YPD plates. Confirmation of excision events was done by PCR. The process was repeated for disruption of the second allele. Revertants of tye7 and gal4 were created using the gal4tye7 strain. Complementation of tye7 was carried out by reintroducing the ORF at its native locus by replacing the upstream flanking sequence in the tye7 disruption cassette with the complete TYE7 ORF. A complementation cassette that consisted of the complete GAL4 ORF and a 500 bp downstream flanking sequence was constructed for reintroduction of GAL4 at its native locus. We selected clones that replaced the HIS1 deletion cassette and then restored histidine prototrophy by transformation with NruI-digested pGEM-HIS1 BWP17 was used for generating the 600 than CMM1. In this case, the tye7 strain was compared to BWP17 and normalized to CMM1 for graphical purposes.CMM3 and CMM1  URA3 at the RPS10 locus gal4/gal4/tye7/tye7, and gal4/GAL4/tye7/TYE7 strains were made prototrophic by targeting the URA3 marker to the RPS10 locus through StuI digestion of CIp10. Correct integration was confirmed by PCR and qPCR verified that only one copy of URA3 was integrated.As uridine auxotrophy affects virulence but can be restored by integration of 600 = 0.1 in synthetic media containing the carbon source at 2%. Cells were either grown at 30°C in flasks with shaking (aerobic conditions) or in microtiter plates without shaking (static conditions). Samples were done in triplicate and the average was used for analysis. For growth under hypoxic conditions, cells were spotted on YPD plates and incubated in an anaerobic chamber. The chamber was flushed daily with nitrogen to remove oxygen and any by-products.Serial spotting plate assays were carried out as described 600 = 2 in 40 ml of YPD, YPGal, or YPGly. Tagged ChIPs were labeled with Cy5 dye and untagged (mock) ChIPs were labeled with Cy3 dye. Microarray hybridization and washing were performed as described P-value<0.1.ChIP experiments were performed as previously described C. albicans Genome Assembly 21 MTL alpha locus https://earray.chem.agilent.com/erray/).One replicate of the ChIP-CHIP experiments for each carbon source condition was hybridized to a custom designed whole-genome tiling array for further analysis. Using the Lowess normalization of the intensity ratio of each of the 240,798 probes was done using an in-house software implementation. The signal along each chromosome was smoothed using a median filter (n = 3) followed by a Gaussian low-pass filter (σ = 150 bp). For peak localization, the smoothed signal was interpolated at 10 bp intervals using cubic-spline resampling. Peaks were reported in decreasing order of the smoothed intensities.600 = 0.8 in YPD, YPGal, or YPGly in aerated flasks (normoxia) and disrupted using acid washed glass beads. Transcriptional profiling was carried out as described P-value<0.05.RNA was extracted with the RNeasy Kit (Qiagen) as per manufacturer's instructions. Briefly, cells were grown to ODExpression profiles under hypoxic conditions were performed as described above except bottles containing YPD media were flushed with nitrogen to remove oxygen. Two biological replicates were performed and statistical analysis was done as with normoxic conditions.tye7, and gal4tye7 cultures were grown to OD600 = 0.7 in YPD in an aerated flask. The cultures were then transferred to bottles flushed with nitrogen and grown for an additional 30 min before the RNA was extracted as above and analyzed by qPCR.For qPCR analysis of glycolytic gene expression under hypoxic conditions, BWP17, 600 = 0.8 in YPD in an aerated flask. Half the culture was transferred to bottles flushed with nitrogen while the other half was left to grow in the aerated flask. At different time points the RNA was extracted and compared by qPCR.For hypoxic induction kinetic analysis, BWP17 was grown to OD2, 400 nM oligo(dT)15, 1 µM random octamers, 0.5 mM dNTPs, 200 units Superscript III reverse transcriptase; Invitrogen). The mixture was incubated for 60 min at 50°C. Aliquots were used for qPCR, which was performed using the Corbett Rotor-Gene RG3000A (Corbett Research) with SYBR Green fluorescence (Qiagen). Cycling was 10 min at 95°C followed by 40 cycles . Samples were done in triplicate and means were used for calculations. Fold changes were estimated using the comparative ΔΔCt method as described C. albicans ACT1 ORF as a reference.For qPCR, cDNA was synthesized from 5 µg of total RNA using the reverse-transcription system  or grown for 40 hours to reach stationary phase, washed twice with cold water, and resuspended in water. Cells were lysed by incubating at 95°C for 30 min and the supernatant was used for enzymatic analysis. Reactions ) were incubated at 37°C for 5 hours. Glucose amounts were assayed with the hexokinase glucose kit (Sigma) with endogenous glucose levels determined based on reactions without trehalase. A BCA protein assay (Pierce) was performed as per manufacturer's instructions. Relative trehalose levels were based on nmol trehalose per mg of cell protein. Three biological replicates were performed for each strain and condition. Glycogen levels were estimated using the iodine vapor method Trehalose levels were measured as based on previous studies 600 = 0.4 and grown for 2 hours in fresh YPD. Cells were washed twice with sterile water and resuspended in YP, YPD, YPGal, or YPGly media and grown for an additional 3 hours. The cells were then washed with TBS buffer and lysed using acid washed glass beads (same lysis buffer as ChIP-CHIP except for addition of a phosphatase inhibitor cocktail (Roche)). The protein extract was clarified by centrifugation and a BCA protein assay (Pierce) was performed as per manufacturer's instructions. Gal4p and Tye7p were separated on a 10% SDS gel and transferred to a PVDF membrane. A rabbit polyclonal antibody directed against the TAP-tag (Open Biosystems) was used (1∶2000 dilution). A mouse anti-actin monoclonal antibody  was used to probe actin as a loading control. HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (Santa Cruz) were used . The HRP signal was revealed using the Lumi-Light Western Blotting Substrate (Roche).To determine the protein expression of Gal4p and Tye7p under different carbon sources, overnight cultures in YPD were diluted to ODGalleria mellonella studies, overnight cultures were washed twice with PBS and resuspended in PBS at OD600 = 8. Larvae, in the final instar, weighing 180±10 mg were injected between the third pair prothoracic legs with 10 µl of suspension (8×105 cells). Infected larvae were incubated at 37°C in the dark with excess of a multigrain diet supplemented with glycerol and vitamins C. albicans cells were responsible for death. Kaplan-Meier survival curves were created and compared with the log-rank test (GraphPad Prism 5).For 5C. albicans in PBS. Six mice, three female and three male, were used for each experimental group except for the gal4tye7 injected A/J set where six females and six males were used. Mice were closely monitored and those showing extreme lethargy were considered moribund and were euthanized. Target organs were removed aseptically and homogenized in PBS before plating on YPD plates containing chloramphenicol (34 µg/ml). The number of yeast colonies per organ was determined, log-transformed, and compared using the Mann-Whitney test (GraphPad Prism 5). Comparative genomic hybridization (CGH) analysis was performed on all strains prior to injection to verify that no aneuploidy arose as a result of any of the genetic manipulations. All experimental procedures involving mice were approved by the Biotechnology Research Institute Animal Care Committee, which operated under the guidelines of the Canadian Council of Animal Care.Mouse studies were carried out as previously described Figure S1GAL4 and TYE7 are involved in fermentative growth with glucose, fructose or mannose as the carbon source. WT refers to strain CMM1. (A) Growth curves where strains were grown with aeration. (B) Growth curves where strains were grown without aeration.Liquid assays verify that (1.30 MB PDF)Click here for additional data file.Figure S2Overlap of binding targets for Gal4p and Tye7p under glucose, galactose, and glycerol growth conditions. Peaks common to all three carbon sources had peak intensities >2 fold in all three conditions with the tiling array data. A peak was considered to be unique to a carbon source (or sources) if the peak intensity in the condition (or conditions) was >2 fold and the peak intensities in the remaining carbon sources were <1.4 fold. This result confirms that for most targets Gal4p displays carbon-source dependent binding while Tye7p binding is more constitutive.(0.53 MB PDF)Click here for additional data file.Figure S3Both Gal4p and Tye7p are induced by glucose but Tye7p has a higher constitutive expression. Protein expression levels of Gal4p and Tye7p under different carbon sources are presented. YP media with no additional carbon source was included to establish the basal level of expression. Actin was used as the loading control.(0.24 MB PDF)Click here for additional data file.Figure S4HXK2 (A), CDC19 (B), and TDH3 (C) were determined in tye7 and gal4tye7 strains relative to the wild type (BWP17) under normoxic growth conditions. ACT1 was used as the reference. TDH3 was included since its expression was unchanged according to the transcription profiles despite being bound by Gal4p and Tye7p Click here for additional data file.Figure S5GAL4 and TYE7 are important for hypoxic growth at both 30°C and 37°C. (A) Strains were serially diluted on YPD plates and incubated in an anaerobic jar at 37°C for 2 days. WT refers to strain CMM1. (B) Strains were serially diluted on two separate YPD plates and incubated in an anaerobic jar at 30°C for 4 days. One representative dilution is shown. The WT for the top row is CMM1 and the WT for the bottom row is BWP17. One copy of either GAL4 or TYE7 is able to restore the growth defect of the double mutant strain although the GAL4 revertant does not grow at wild type levels since TYE7 is still deleted.(1.67 MB PDF)Click here for additional data file.Figure S6GAL4 and TYE7 are initially induced by hypoxia. The expression levels of GAL4, TYE7, and CDC19 were measured in BWP17 by qPCR at different time points following a shift from normoxic to hypoxic growth conditions. ACT1 was used as the reference.(0.57 MB PDF)Click here for additional data file.Table S1Doubling times under fermentative growth conditions.(0.02 MB XLS)Click here for additional data file.Table S2Gal4p ChIP-CHIP targets with glucose as the carbon source.(0.03 MB XLS)Click here for additional data file.Table S3Tye7p ChIP-CHIP targets with glucose as the carbon source.(0.05 MB XLS)Click here for additional data file.Table S4Top Gal4p YPD binding peaks with tiling array.(0.02 MB XLS)Click here for additional data file.Table S5Top Tye7p YPD binding peaks with tiling array.(0.02 MB XLS)Click here for additional data file.Table S6ChIP-CHIP binding enrichments of selected metabolic genes under different carbon sources.(0.03 MB XLS)Click here for additional data file.Table S7tye7 and gal4tye7 strains under different carbon sources and oxygen levels.Expression levels of selected metabolic genes for (0.03 MB XLS)Click here for additional data file.Table S8tye7 strain in glucose growth conditions.Genes down-regulated in (0.03 MB XLS)Click here for additional data file.Table S9gal4tye7 strain in glucose growth conditions.Genes down-regulated in (0.03 MB XLS)Click here for additional data file.Table S10tye7 strain in galactose growth conditions.Genes down-regulated in (0.02 MB XLS)Click here for additional data file.Table S11gal4tye7 strain in galactose growth conditions.Genes down-regulated in (0.04 MB XLS)Click here for additional data file.Table S12tye7 strain in glycerol growth conditions.Genes down-regulated in (0.03 MB XLS)Click here for additional data file.Table S13gal4tye7 strain in glycerol growth conditions.Genes down-regulated in (0.03 MB XLS)Click here for additional data file.Table S14tye7 strain in glucose hypoxic growth conditions.Genes down-regulated in (0.07 MB XLS)Click here for additional data file.Table S15tye7 strain in glucose hypoxic growth conditions.Genes up-regulated in (0.05 MB XLS)Click here for additional data file.Table S16Genes down-regulated in BWP17 strain in glucose hypoxic growth conditions.(0.04 MB XLS)Click here for additional data file.Table S17Genes up-regulated in BWP17 strain in glucose hypoxic growth conditions.(0.05 MB XLS)Click here for additional data file.Table S18Strains used in this study.(0.03 MB XLS)Click here for additional data file.Table S19Plasmids used in this study.(0.02 MB XLS)Click here for additional data file.Table S20Primers used in this study.(0.02 MB XLS)Click here for additional data file.Dataset S12 (fold enrichment) with negative values indicating enrichment for Gal4p or Tye7p binding. Significant binding events are highlighted in green.ChIP-CHIP normalized tiling array data. Columns correspond to (L to R) Tye7p YPD, YPGal, YPGly; Gal4p YPD, YPGal, YPGly. The start (>) and end (|) of each ORF is indicated. Values given for each probe are log(3.52 MB ZIP)Click here for additional data file.


Drosophila melanogaster. The male X chromosome is targeted by the male-specific lethal complex believed to mediate the 2-fold up-regulation of the X-linked genes, and the highly heterochromatic fourth chromosome is specifically targeted by the Painting of Fourth (POF) protein, which, together with heterochromatin protein 1 (HP1), modulates the expression level of genes on the fourth chromosome. Here we use chromatin immunoprecipitation and tiling microarray analysis to map POF and HP1 on the fourth chromosome in S2 cells and salivary glands at high resolution. The enrichment profiles were complemented by transcript profiles to examine the link between binding and transcripts. The results show that POF specifically binds to genes, with a strong preference for exons, and the HP1 binding profile is a mirror image of POF, although HP1 displays an additional “peak” in the promoter regions of bound genes. HP1 binding within genes is much higher than the basal HP1 enrichment on Chromosome 4. Our results suggest a balancing mechanism for the regulation of the fourth chromosome where POF and HP1 competitively bind at increasing levels with increased transcriptional activity. In addition, our results contradict transposable elements as a major nucleation site for HP1 on the fourth chromosome.Two specific chromosome-targeting and gene regulatory systems are present in  Drosophila this mechanism is achieved by a 2-fold up-regulation of the single male X chromosome. Flies also possess an additional chromosome-wide regulatory system that regulates the transcriptional output from genes on the fourth chromosome. In this case the protein Painting of Fourth (POF), together with heterochromatin protein 1 (HP1) bind to the fourth chromosome and fine tune gene expression. By using a high resolution map of POF and HP1 binding, we can show that they bind to the same sequences on the fourth chromosome. We also demonstrate that POF and HP1 bind to active genes with preferences for exon sequences. In gene regulatory mechanisms, including chromosome-wide gene regulation, a simple on/off switch is often not enough. Our findings support the presence of a balancing mechanism in which the dual recruitment of a repressing and a stimulating factor makes the transcription efficiency more stable and less sensitive to fluctuations.Species where males and females have a different number of sex chromosomes have to equalize the transcriptional output from the genes located on the X chromosome. In  Drosophila the small fourth chromosome is also considered to be highly heterochromatic , and 1 μl of 20 mg/ml proteinase K were added before an additional incubation at 45 °C for 1 h. The DNA was recovered by phenol/chloroform extraction followed by ethanol precipitation. The immunoprecipitated DNA was then dissolved in 20 μl water.For our ChIP experiments we used Schneider's ribed by . For salThe immunoprecipitated DNA and corresponding amounts of input DNA were amplified using ligation-mediated PCR. The linkers used and the ligation procedure were as described . The ampD. melanogaster genomic release 4. Repeated regions were removed from the design using a repeat masker. Three pseudogenes  are present on the fourth chromosome. Because of high sequence identity between these three genes they were not included in further analysis. Thus, 89 genes on the fourth chromosome were included in the analysis. The array production, probe labeling, and hybridization were conducted by Nimblegen Systems Inc. (http://www.nimblegen.com). In total, 15 ChIP experiments and four cDNA samples for transcript profiling were hybridized. The complete dataset is available at http://www.ncbi.nlm.nih.gov/geo/. The raw enrichment data (ChIP/input) were very consistent between the three ChIP replicates and between consecutive probes . All values below these cutoffs were set to zero. A relative gene transcription level was set as the mean enrichment of the 50% highest probes within exons of each gene . To calc+RNA was isolated using DynabeadsOligo (dT)25 . Two biological replicates were used of both salivary glands (300 pairs per replicate) and S2 cells. The cells or tissue were frozen at −70 °C and homogenized in 0.1 M TRIS-HCl (pH 8.0), 0.5 M LiCl, 10 mM EDTA, 1% SDS, and 5 mM DTT. Dynal's recommendations were then followed. The poly(A)+RNA was converted to cDNA using an ImPromII first strand synthesis kit (Promega) according to the supplier's recommendations. The cDNA was then used as a template for forming double-stranded cDNA using a Superscript double stranded cDNA synthesis kit (Invitrogen). The double-stranded cDNA was purified by phenol-chloroform extraction followed by passage through S200HR columns , and 4 μg of the double-stranded cDNA from each sample was sent to Nimblegen for labeling and hybridization using the same array design as for the ChIP-chip.For transcript profiling experiments, poly(A)P element insertions on the fourth chromosome, we used the data from Sun et al. [n = 17) or nonvariegated (n = 6). The highest relative gene expression value and relative gene HP1 enrichment levels  within 1 kb (±500 bp) were determined for each insertion, then mean expression and HP1 enrichment levels were calculated.To determine expression (transcript levels) and HP1 enrichment close to n et al.  and theiFigure S1(A) POF enrichment before and after ligation-mediated PCR as determined by real-time PCR.(B) POF enrichments in a randomly chosen 10-kb region  from the three biological replicates of S2 cells. The effect of the seven-probe median smoothing process is shown below the raw data from the replicates.(C) The enrichment profile in the 216–227-kb genomic region from Johansson et al.  comparedy-axis represents % of input as determined by real-time PCR.(D) The two HP1 antibodies (C1A9 and PRB291C) give comparable enrichment profiles. PRB291C was used in the ChIP-chip experiments. The (134 KB PDF)Click here for additional data file.Figure S2The gene binding values for POF and HP1 were individually calculated from each of the three replicates of POF in S2 Cells (PS2), POF in Salivary Glands (PSG), HP1 in S2 Cells (HS2), and HP1 in Salivary Glands (HSG). Pairs of replicates were then compared. Binding values for: POF to genes in S2 cells are plotted in (A–C); POF to genes in salivary glands in (D–F), HP1 to genes in S2 cells in (G–I), and HP1 to genes in salivary glands in (J–L). The replicates used for further analysis are indicated by asterisks.(106 KB PDF)Click here for additional data file.Figure S3Relative transcription values for Chromosome 4 genes in S2 cells and salivary glands were individually calculated from data acquired from each of the two replicates of S2 cells (TP_S2) and salivary glands (TP_SG). Pairs of replicates were then compared, using correlation plots. The replicates used for further analysis are indicated by asterisks.(42 KB PDF)Click here for additional data file.Figure S4The plot shows normalized log2 differences in transcript levels and POF binding, respectively, between S2 cells and salivary glands.(63 KB PDF)Click here for additional data file.Table S1(29 KB XLS)Click here for additional data file.http://www.ncbi.nlm.nih.gov/geo) (accession number GSE8301).The complete dataset mentioned in this paper is available from the National Center for Biotechnology Information (NCBI) (


Bdnf. In a genome-wide chromatin immunoprecipitation analysis (ChIP-Seq), we identified 176 sites of CaRF-specific binding (peaks) in neuronal genomic DNA. 128 of these peaks are within 10kB of an annotated gene, and 60 are within 1kB of an annotated transcriptional start site. At least 138 of the CaRF peaks contain a common 10-bp motif with strong statistical similarity to the cCaRE, and we provide evidence predicting that CaRF can bind independently to at least 64.5% of these motifs in vitro. Analysis of this set of putative CaRF targets suggests the enrichment of genes that regulate intracellular signaling cascades. Finally we demonstrate that expression of a subset of these target genes is altered in the cortex of Carf knockout (KO) mice. Together these data strongly support the characterization of CaRF as a unique transcription factor and provide the first insight into the program of CaRF-regulated transcription in neurons.Calcium-Response Factor (CaRF) was first identified as a transcription factor based on its affinity for a neuronal-selective calcium-response element (CaRE1) in the gene encoding Brain-Derived Neurotrophic Factor (BDNF). However, because CaRF shares no homology with other transcription factors, its properties and gene targets have remained unknown. Here we show that the DNA binding domain of CaRF has been highly conserved across evolution and that CaRF binds DNA directly in a sequence-specific manner in the absence of other eukaryotic cofactors. Using a binding site selection screen we identify a high-affinity consensus CaRF response element (cCaRE) that shares significant homology with the CaRE1 element of  The function of any given transcription factor is determined in large part by its DNA binding specificity, which defines its potential target genes. Over 1000 gene products are annotated as transcription factors in the mammalian genome, the vast majority of which belong to large families classified by the homology of their DNA binding domains  Some of the most important recent insights into transcription factor biology have come from the application of technologies that capture the full complement of transcription factor binding sites across the genome MECP2, FOXP2, CBP, and GTF2I/GTF2IRD1, are associated with cognitive impairment in humans Transcription factors are essential for accommodating intracellular states to extracellular stimuli. In the nervous system, transcription factors play an important role in coordinating neuronal responses following changes in synaptic activity - a key stimulus for shaping brain development, driving synaptic plasticity, and promoting survival of mature neurons Bdnf promoter IV Bdnf none of CaRF's target genes are known. Surprisingly, CaRF shares no sequence similarity with any known family of transcription factors, and it has no homologs in the mammalian genome. Given this evidence that CaRF is a unique transcription factor, we set out to characterize CaRF's DNA binding properties and identify its potential target genes that might in turn suggest biological functions for CaRF beyond Bdnf regulation.Calcium-Response Factor (CaRF) is a novel nuclear protein first identified as a binding protein for a calcium-response element (CaRE1) in in vitro binding site selection and genome-scale in vivo chromatin immunoprecipitation followed by sequencing (ChIP-Seq) as tools to identify and characterize high affinity CaRF-binding DNA sequences. These data reveal a large set of putative CaRF target genes in mouse cortical neurons, providing the first insight into the CaRF regulon.Here we show that the DNA binding domain of CaRF has been highly conserved over evolution, and we use both Carf exon 8 KO mice were generated by homologous recombination EcoRI and XbaI sites. The construct was transformed into Top10 E. coli and expression of CaRF was induced for 3 hours with 0.1mM IPTG. Bacteria were lysed by sonication and CaRF was purified over a ProBond nickel resin column. After washing to remove unbound protein, binding was competed with 50mM imidazole to remove nonspecific proteins and then CaRF was eluted with 250mM imidazole. Fractions containing CaRF as determined by Western analysis were pooled and concentrated to a final concentration of 100ng/uL.Full-length mouse CaRF was subcloned in the bacterial expression vector pThioHisA  at the in vitro transcribed and translated (TNT) protein  or bacterially expressed CaRF as described above. 2µL TNT or purified CaRF protein was incubated with 50fmol 32P nick labeled  or polynucleotide kinase  end-labeled annealed oligonucleotide probes prior to separation on 6% non-denaturing acrylamide gels. In competition assays, unlabeled probes were added to nuclear extracts in 100-fold molar excess (unless otherwise noted) to the TNT protein for 30 min. prior to addition of the radiolabeled probe. Gels were dried and visualized by phosphorimager . Mobility-retarded bands were quantified relative to probe intensity in the same lane using the ImageQuant image analysis program . Oligonucleotide probes are listed in Table S1.EMSAs were performed as described Table S1. Full length human CaRF (hCaRF) was tagged at its N-terminus with the FLAG-epitope (GACTACAAGGACGATGACGATAAA) and used for in vitro TNT as above. For the binding selection, random 16mer nucleotide sequences were synthesized  within a 66bp oligo, then made double-stranded by a single round of PCR using primers against the flanking sequences. Oligos were incubated with TNT hCaRF or a control TNT master mix and immunoprecipitated with the M2 anti-FLAG epitope antibody . Coimmunoprecipitated oligos were purified and amplified by PCR, then used for an additional three rounds of selection. A subset of the final samples were radiolabeled and tested by EMSA for their ability to bind hCaRF. The remaining samples were ligated into pBSK , the inserts were sequenced and 62 were aligned using ClustalW  http://weblogo.berkeley.edu/) PCR-assisted binding site selection was performed essentially as described Carf+/+ (WT) or Carf−/− (KO) pups were plated at a density of 10 million cells/10cm dish. 50 million neurons of each genotype were used for ChIP. Because the Carf mice are on a mixed C57BL6/129SvJ background, WT and KO siblings were crossed (WTxWT and KOxKO) to generate the pups for this experiment in order to minimize genetic background variations. At 4DIV, cultured neurons were treated overnight with tetrodotoxin , then on DIV5 protein-DNA complexes were processed for ChIP. 4µL purified anti-CaRF antibody (#4510)Carf promoter, striatum was dissected from brains of Carf WT or KO mice and snap frozen in liquid nitrogen. 3–5 independent samples were pooled, homogenized, and processed for ChIP as above. Immunoprecipitations were performed at 4°C overnight with 5–10µg of each specific antibody and assayed by real-time PCR using SYBR green detection. Antibodies used in this study include purified normal mouse polyclonal IgG  and anti-RNA polymerase II, clone CTD4H8 which recognizes the largest subunit of RNA polymerase II . For input samples, 25µl (6–7% of the amount used for IP with specific antibody) was added to 75µl of elution buffer, NaCl was added to 200mM final concentration, and samples were reverse crosslinked at 65°C overnight. All ChIP pulldowns are displayed as % of input for each sample (WT or KO). Data presented are the result of two independent experiments, and statistical significance was evaluated by a Student's two-tailed unpaired t-test.Chromatin Immunoprecipitation (ChIP) was performed as described http://www.appliedbiosystems.com. The purified adaptor-ligated ChIP DNA fragments were subject to 6% native-PAGE for In-Gel PCR reaction. A gel slice containing 175∼200bp adaptor-ligated ChIP DNA fragments was cut and shredded. Then to add bar codes, PCR Platinum Supermix , 50pmol of PCR primers containing bar codes, 0.5µl Taq DNA polymerase (NEB), and 0.15µl Pfu Turbo DNA polymerase (Stratagene) were added into the shredded gel slice. The adaptor-ligated ChIP DNA fragments were amplified by 15 cycles of In-Gel PCR reactions. After the PCR reaction, gel pieces were filtered out by 0.45µm filter spin column and the amplified ChIP-Seq library was purified by MinElute PCR purification kit (Qiagen). The library was purified by one more round of 6% PAGE. A gel slice containing 200∼250bp PCR products was cut and shredded, and the amplified library was extracted out of the gel by passive elution in elution buffer (1.5M ammonium acetate in 1× TE). Gel pieces were filtered out by filter spin column and both ChIP-Seq libraries were purified by Qiaquick PCR purification kit (Qiagen). Samples were affixed to a slide and sequenced on a SOLiD sequencer version 2. After filtering with 3bp mismatches allowed in 35bp reads, just over 5 million uniquely mapped reads  were obtained for each library (WT and KO). The resulting sequences were formatted for alignment in the UCSC genome browser.DNA fragments coimmunoprecipitated with CaRF were repaired using the End-It DNA End Repair Kit , purified using the MinElute Reaction Cleanup Kit  and eluted in 20µl EB buffer. The resulting DNA fragments were ligated to adaptors for the SOLiD sequencer  for 20 minutes at RT using the Quick Ligase Kit (NEB), followed by purification using the MinElute Reaction Cleanup Kit. Sequences of adaptor and barcodes can be found at Carf WT neurons relative to the negative control ChIP samples from Carf KO neurons. First we determined the false detection ratio by using a sliding window with a width of 240bp for every 10bp in the mouse genome. Repetitive regions are excluded from our analysis since it is impossible to assign 35bp reads uniquely to such regions R and λN, respectively. It follows that D is a Skellam distribution dI is the modified Bessel function of the first kind of order d. The construction of the null distribution from Eq. (1) takes unequal numbers of reads in the two samples into account by shifting the mode of the distribution. To determine the number of reads required for a 240bp window to be significant, we use the local false detection rate (locFDR) framework D, f(D), can be written as the mixture f(D) = 0f0p(D)+1f1p(D), where 0f is the null density, 1f is the density of windows corresponding to true peaks and 0p+1p = 1 with p0d) is related to the more familiar FDR E is the expectation with respect to the mixture density f. For a fixed threshold fp = 0.01 and empirically estimated λR = 0.15273 and λN = 0.21607, we find that the critical difference is d0 = 6 fragments. Inserting the empirical distribution and the Skellam null distribution from Eq. (1), we find that the FDR is ∼1×10−7.To determine CaRF-specific binding sites, we developed an algorithm to identify the sequences specifically enriched in the ChIP samples from Regions of the genome determined to be specifically bound by CaRF were downloaded using the GALAXY Carf heterozygote (HET×HET) cross were removed, the cortex was dissected and rapidly frozen on liquid nitrogen, then the thawed tissue was used for RNA harvesting. For evaluation of Carf mRNA expression, brains from single P0 pups from a Carf HET×HET cross were used for neuron culture as described above. Tail biopsies were clipped during dissection and genotyped prior to sample harvesting to identify WT and KO pups. On day 5 in culture, RNA was harvested, and cDNA was synthesized as above. All primers used in this analysis are listed in Table S1. To measure Carf mRNA we used primers against exons 11–12, distal to the Carf exon 8 deletion. Samples were normalized to Gapdh as a control for sample handling. Data shown for Carf are the result of measurements in 8 individual pups of each genotype. Data for other Carf target genes are the result of measurements in 4–6 individual pups of each genotype. For quantitation of chromatin immunoprecipitation of RNA polymerase II on the Carf gene, the primers listed in Table S1 were used to amplify a 173bp region flanking the ChIP peaks in Carf exon 1. Statistical significance was determined by a Student's unpaired 2-tailed t-test, and p<0.05 was considered significant.For evaluation of CaRF target gene expression, brains from single P0 pups from a Mus musculus genome reveals no significant homology of this protein to any other gene product. However we find gene products with significant similarity to CaRF at the amino acid level in 37 different species (Table S2), including 35 vertebrates and the deuterostome Branchiostoma floridaeDrosophila melanogaster or Caenorhabitas elegans genomes, a conserved CaRF ortholog is present in the genome of the cnidarian starlet sea anemone Nematostella vectensis, which diverged from the vertebrate lineage over 700 million years ago Showing that a protein domain is conserved over evolution is one way of suggesting its importance for function of the protein. Sequence analysis of CaRF against build 37 of the E. coli binds Bdnf CaRE1 by EMSA and is competed with the same specificity as FLAG-tagged CaRF synthesized using a eukaryotic in vitro transcription and translation system  were aligned, revealing a 12bp consensus CaRF binding motif (cCaRE) with the sequence 5′- YSANAACGAGGC - 3′ . Mapping these fragments to the reference genome gives a profile of the DNA regions that are enriched in the pulldown, however the challenge is to determine which of the enriched regions (“peaks”) are statistically significant at a given threshold and which correspond to the genomic background. Thus a peak is defined as region that contains significantly more reads from an experimental pulldown than from a negative control. In this case, for our negative control we performed ChIP with the same anti-CaRF antiserum from neuronal cultures made from Carf KO mice, which are null for CaRF protein. Independent libraries were constructed from the genomic DNA coimmunoprecipitated from the WT and KO cells, and genomic regions specifically bound by CaRF (CaRF ChIP peaks) were identified using the statistical algorithm described in detail in the Given this evidence that CaRF is a direct sequence-specific DNA binding protein, we wanted to find endogenous gene targets of CaRF. To identify CaRF binding sites genome-wide, we performed chromatin immunoprecipitation with an anti-CaRF antiserum from cultured Table S4). 128 of the 176 peaks (73%) are within 10kB of an annotated gene, and 60/128 (47%) of these are within 1kB of the annotated transcriptional start site (TSS). Graphing the position of each of these peaks relative to the TSS shows an enrichment in the 200bp just 5′ to the TSS . The motif extracted from this set of sequences (chCaRE) has the sequence 5′-RRARYGAGGC-3′,  (5′-YSANAACGAGGC-3′) and the CaRE1 (5′-CTATTTCGAGGC-3′) CaRF binding sequences  found in a CaRF ChIP peak from the promoter of the Camk2n1 gene , we estimated the likelihood that CaRF would bind by EMSA to each individual chCaRE motif. Strikingly, 45% of the peaks (62/138) contain a chCaRE sequence that we predict would bind with high affinity to CaRF, while an additional 19.5% (27/138) have sequences that we predict would bind with lower affinity. Only 35.5% of the chCaRE sequences (49/138) have sequences that we predict would show no significant affinity for CaRF by EMSA. Whether CaRF may bind to other sequence motifs in these ChIP peaks or whether CaRF may bind these variant chCaRE motifs in collaboration with a binding partner remains unknown. Nonetheless, taken together these data provide strong evidence to suggest that the ChIP peaks represent sites of high affinity CaRF binding to neuronal genomic DNA in vivo.By comparing these sequence requirements at each position across the camCaRE to the sequences of the individual chCaRE sites identified in each of the ChIP peaks (http://gostat.wehi.edu.au). This analysis revealed two statistically significant overrepresented GO categories  terms for the genes on this list was conducted using GOstat Carf mRNA is still transcribed. Quantitative PCR of Carf using primers distal to the exon 8-deleted region reveal that Carf mRNA expression is significantly elevated in the absence of CaRF protein . In an independent set of experiments, we identified a highly similar motif (the 10bp chCaRE) when we aligned the fragments of neuronal genomic DNA that specifically coimmunoprecipitated with CaRF by ChIP. The chCaRE motif was found in 78% of the 176 CaRF ChIP peaks, and by testing the requirement for each degenerate base across the chCaRE motif we provide evidence that CaRF is likely to show affinity for at least 64.5% of the variant chCaRE motifs in vitro. These data raise the question of whether CaRF also directly associates with the other 35.5% of the ChIP peaks that contain base changes in key positions within the chCaRE motif, as well as those that lack a sequence with any similarity to the cCaRE.We took two complementary approaches to identifying CaRF binding sites. First we conducted a PCR-assisted binding site selection screen Transcription factors vary in their requirement for a consensus motif to guide their selection of genomic binding sites Epha3 and Epha6 are regionally expressed in the developing brain, where they play important roles in neural patterning Caly  and Cacng2 (Stargazin) contribute to the regulation of AMPA-type glutamate receptor trafficking to synapses Carf KO mice show abnormalities in several tests of learning and memory Carf knockout mice, and whether any the gene products identified in this study contribute to the memory phenotypes in these mice, will be interesting questions to explore.GOstat analysis of the genes that neighbor the CaRF ChIP peaks revealed overrepresentation of gene products involved in signaling pathways, suggesting the possibility that CaRF-dependent transcription may modulate intracellular states in neurons. Interestingly, the known functions of these gene products suggest possible mechanisms through which CaRF could contribute to experience-dependent synaptic development and plasticity. For example the Eph family receptor tyrosine kinases Carf WT and KO neurons then developed a statistical method to identify only those genomic regions significantly enriched in the pulldown from the WT cells. This method of analysis yielded a relatively small number of peaks compared with other genome-scale ChIP studies Bdnf gene, which could indicate either that CaRF was not bound to Bdnf CaRE1 under the conditions used in this study or that this interaction was undetectable for technical reasons. In addition, despite the evidence that the transcriptional activity of CaRF can be acutely enhanced by the activation of L-type voltage gated calcium channels in neurons Table S4) and several large published sets of neuronal activity-dependent genes Despite the evidence for the enrichment of functional pathways among the putative CaRF targets, it remains possible that our list of genomic CaRF binding sites is incomplete. To ensure the specificity of ChIP using the anti-CaRF antibody, we sequenced ChIP libraries made from both Carf KO mice, demonstrating that CaRF expression is important for their proper regulation. It is interesting that whereas in heterologous overexpression assays CaRF acts as a CaRE1-dependent transcriptional activator of a Bdnf promoter IV-luciferase reporter Carf KO mice. Although loss of CaRF may indirectly elevate gene expression, because the genes we have assayed here directly neighbor sites of CaRF binding in vivo, these data raise the possibility that CaRF acts as a direct repressor of a subset of its target genes. The functions of many transcriptional regulators are highly context dependent, and can be influenced by cell type Bdnf promoter IV reporter plasmid Carf transcripts, we have found that the Carf locus also encodes a C-terminally truncated Carf variant through the use of an alternative polyadenylation site in the intron just following the exons that encode the DNA binding domain Carf KO mouse with the identification of genomic binding sites of CaRF presented here we expect these data will provide a crucial starting point for a detailed study of the molecular mechanisms that regulate CaRF-dependent transcription in neurons.Nonetheless, we have demonstrated that at least a subset of the putative CaRF target genes show altered expression in the brains of Table S1Oligonucleotides used in this study.(0.07 MB DOC)Click here for additional data file.Table S2http://www.ensembl.org) and XM, XR, and NR sequences are from NCBI (http://www.ncbi.nlm.nih.gov/entrez).CaRF orthologs. CaRF orthologs were identified by BLAST search for translated nucleotide sequences with high similarity to Mus musculus CaRF. Orthologs from 37 species were identified, and accession numbers are listed below. ENS sequences are from Ensembl ((0.04 MB DOC)Click here for additional data file.Table S3Identification of a high affinity CaRF binding motif by PCR-assisted site selection. Oligos selected after four rounds of coimmunoprecipitation with CaRF were cloned in the vector pBluescript and sequenced. 62 sequences were aligned and the best fitted 16bp motif is shown for each clone. Capital letters indicate bases within the random 16mer sequence and lower case letters indicate the flanking sequences.(0.04 MB DOC)Click here for additional data file.Table S4http://genome.ucsc.edu). The nearest annotated gene within 10kB of the ChIP peak is listed under Gene Symbol. The Motif column shows the sequence of the common 10bp motif found by PRIORITY analysis of the ChIP peak sequences. The sequences not included in the analysis are marked “too short”, and sequences analyzed that did not contain a conserved motif are marked “none.” Finally based on in vitro analysis of CaRF's tolerance for base pair changes across the motif as shown in Genomic locations of CaRF ChIP peaks and identification of a conserved in vivo CaRF binding motif. Peak files derived from the CaRF ChIP were loaded into the UCSC browser to analyze genomic location. Location denotes the chromosomal position of each peak. The peak start and end numbers are in reference to build 37 of the Mus musculus genome Click here for additional data file.


Cspg2, Igfbp5, Mab21l2, Nrf2f, Olfm3, Spag5, Spock1, Spon1 and Tgfb2, was confirmed by quantitative RT-PCR, with five of these genes: Cspg2, Mab21l2, Olfm3, Spag5 and Tgfb2, identified as candidate direct Pax6 target genes by quantitative chromatin immunoprecipitation (qChIP). In Mab21l2 and Tgfb2 promoter regions, twelve putative individual Pax6-binding sites were tested by electrophoretic mobility shift assays (EMSAs) with recombinant Pax6 proteins. This led to the identification of two and three sites in the respective Mab21l2 and Tgfb2 promoter regions identified by qChIPs. Collectively, the present studies represent an integrative genome-wide approach to identify downstream networks controlled by Pax6 that control mouse lens and forebrain development.Lineage-specific DNA-binding transcription factors regulate development by activating and repressing particular set of genes required for the acquisition of a specific cell type. Pax6 is a paired domain and homeodomain-containing transcription factor essential for development of central nervous, olfactory and visual systems, as well as endocrine pancreas. Haploinsufficiency of Pax6 results in perturbed lens development and homeostasis. Loss-of-function of Pax6 is incompatible with lens lineage formation and results in abnormal telencephalic development. Using DNA microarrays, we have identified 559 genes expressed differentially between 1-day old mouse Pax6 heterozygous and wild type lenses. Of these, 178 (31.8%) were similarly increased and decreased in Pax6 homozygous embryonic telencephalon . In contrast, 381 (68.2%) genes were differently regulated between the lens and embryonic telencephalon. Differential expression of nine genes implicated in lens development and homeostasis:  Embryonic organ development is contingent on complex coordinated interactions of multiple transcription factors that regulate the expression of selected downstream target genes. The Paired (Pax) family of genes has been shown to control development of many organs, such as brain, ear, eye, kidney, muscle, pancreas and thyroid During early stages of visual system formation, Pax6 is required for the establishment of lens progenitor cells −/− embryonic dorsal (cortex) and ventral  telencephalon, E12 and E15, identified a number of novel Pax6-regulated genes in situ hybridization analysis of hundreds of co-expressed genes in midgestation mouse embryo (E14.5) resulted in the prediction of 30 genes regulated by Pax6 in the embryonic cortex, with ∼ one half considered as putative direct target genes Drosophila, DNA microarray expression studies identified batteries of genes regulated by eyeless (ey), a fly homologue of Pax6 Two recent high throughput studies, focused on the function of Pax6 in mouse telecephalon, have shown a number of novel genes whose expression is controlled by Pax6. A DNA microarray study of Pax6In this study, we have analyzed differential gene expression in newborn mouse Pax6 heterozygous and wild type lens and compared this to expression profiles in embryonic forebrain from Pax6 homozygous embryos +/− to wild type mice  from a total number of over 22,000 mouse genes represented on the array. The lens transcriptome was represented by 7,009 (∼32%) genes with a median of RMA-normalized raw signal intensities above 100, and 1,844 (∼8%) genes with signal intensities between 50–100, respectively. The boxplot shown in +/− lens originates most likely from variable phenotypes of individual mutant lenses +/− lenses measured using both the microarrays and by qRT-PCR is shown in To assess differential gene expression in newborn mouse Pax6 heterozygous lenses compared to wild type lenses, we performed DNA microarray hybridizations using the Affymetrix Mouse Genome 430 2.0 Arrays. Three biological replicate experiments were performed and analyzed as described in Cspg2, Igfbp5, Olfm3, Spock1, Spon1 and Tgfb2 (see The 559 genes were organized according to Gene Ontology (GO) categories of biological process, molecular function and cellular compartment . This angfb2 see .Mab21l2, NeuroD1, Nfil3, and Tgfb2. The expression of Mab21l2 Mab21l2, a gene up-regulated in Pax6+/− lenses, to Nr2f2 and Spag5, to the genes listed in A subcategory “Eye development” within the GO Biological process  yielded Analysis of differentially expressed genes using the KEGG Pathway classification Differential expression of nine genes, Cspg2, Igfbp5, Mab21l2, Nrf2f, Olfm3, Spag5, Spock1, Spon1 and Tgfb2 , in Pax6+/− lens.To further validate the microarray results Sey/Sey embryonic telencephalon Both lens and brain are of the ectodermal germ layer origin. Specific roles of Pax6 were established in the development of both tissues As lens cells and cortical neurons represent distinct cell types, it is not surprising that the majority of 381 (68.2%) Pax6-differentially expressed genes in lens show “opposite” changes in their expression in Pax6 homozygous telencephalon Cspg2, Igfbp5, Mab21l2, Nrf2f, Olfm3, Spag5, Sparc1, Spon1 and Tgfb2 genes  contains both direct and indirect Pax6 target genes. In a separate report , we assessed whether b2 genes  are Pax6gfb2 see  confirmeDrosophila paired “consensus” Direct binding of Pax6 in Mab21l2 and Tgfb2 genes in lens chromatin suggests two novel regulatory mechanisms for embryonic eye development (see Discussion). Therefore, we wanted to localize the putative Pax6-binding sites within the regions occupied by Pax6 in lens chromatin. Using the 20 base pair Pax6 PD “consensus” sequence Gata1, HNF4α, MyoD, Mitf, Nr2e3, Nrl, Oct4, Pdx1, PU.1, Pax5/BSAP, Pax6, Runx1, Runx2 and Sox9, in various cell types. Here, we examined the function of Pax6 in mouse lens development by identifying batteries of genes differentially expressed in Pax6 heterozygous lens; Pax6 homozygous embryos (Sey/Sey) do not form any lens −/− mouse embryo A major question in developmental biology is to elucidate the function of lineage-specific DNA-binding transcription factors encoded by “stage-selector” genes, such as Sey/Sey mouse embryos Using the Pax6 lens haploinsufficiency experimental model, we identified 559 genes differentially expressed between wild type and Pax6 heterozygous newborn mouse lens. We then took advantage of similar analysis in telencephalon microdissected into the cortex and ganglionic eminence (GE) in in vitro assays, we identified at least three Pax6-binding sites  suggest this enzyme as Pax6 regulated gene In addition, our data identified perturbed expression of a number of genes that participate in a large number of signal transduction pathways see . Pax6 exMab21l2 locus  obtained from 6-week old lenses in situ hybridization studies using an atlas of ∼1,000 spatial gene expression patterns of the midgestation mouse embryo in situ hybridization studies A summary of multiple strategies to identify top candidate genes regulated by Pax6 is shown in One of the major limitations of genome-wide studies of embryonic development is the limited availability of adequate biological materials. Tissue and organ development depend on formation of multiple cell progenitors of different embryonic origins and their mutual interactions in a complex three-dimensional landscape mediated by short-, mid- and long-range signaling lacZ/+ mice 5′-CCGGCCGCTTGGGTGGAG- 3′, antisense 5′-CGG TCCGCCACACCCAGC- 3′. After enucleation, the eyes were fixed for 24 h in 4% paraformaldehyde, washed extensively in phosphate buffered saline (PBS), incubated in ascending  concentrations of sucrose/PBS for 8 h to overnight at 4°C and shock frozen in tissue freezing medium . Sections of 10–12 µm were cut at −30°C, washed three times in PBS (5 min each), and blocked with 2% BSA in PBS (45 min at room temperature). The primary Pax6 antibody  was diluted (1∶50) in blocking solution and incubated at 4°C overnight. After three washes in PBS (5 min each), the secondary antibody, diluted in blocking solution, was applied for 1 h . After three washes with PBS, counterstaining was performed with DAPI and the sections were embedded with fluorescent mounting medium .Eyes were obtained from Pax6lacZ/+) 1-day old lenses (2 lenses from the same mouse per sample) using the RNeasy MiniElute Kit  according to manufacturer's instructions. RNA quality was determined with an Agilent 2100 Bioanalyzer and cDNAs were then generated with the Ovation™ Biotin RNA amplification and Labeling System  using 50 ng of RNA according to the manufacturer's protocol. Three biological replicates were subsequently hybridized on Mouse Genome 430 2.0 Arrays . Animal husbandry and experiments were conducted in accordance with the approved protocol of the Albert Einstein College of Medicine Animal Institute Committee and the ARVO Statement for the use of animals in Ophthalmic and Vision Research.Total RNA was isolated from individual wild type (NMRI background) and Pax6 heterozygous (Pax6+/−) P1 lens were identified using triplicate sets of Robust multichip average (RMA)-normalized Affymetrix CEL files Genes/mRNAs differentially regulated between wild-type and heterozygous (Pax6+/− lenses were determined using qRT-PCR (see 20) primers (Invitrogen) and Superscript ™ III Reverse Transcriptase (Invitrogen), according to the manufacturer's protocol. The cDNA was diluted 10 times and qRT-PCR was conducted using an Applied Biosystems  7900HT fast Real-Time PCR system with Power SYBR® Green PCR master mix (ABI). qRT-PCR was conducted with the primers shown in Relative expression levels of ten genes encoding Cspg2, Igfbp5, Mab21l2, Nr2f2, Olfm3, Pax6, Spag5, Spock1, Spon1 and Tgfb2 in WT and Pax62O using the QIAquick Spin Gel Purification Kit (Qiagen).For the ChIP-on-chip studies, the “standard” assays using chromatin prepared from 20 lenses The PCR primers were designed using Primer3 and their specificity was checked using BLAST. Default parameters were used , and PCR products were limited to 80–100 bp. The primers are given in t values of series dilution of input samples  were used to generate standard curves. Immunoprecipitation data were referred to the standard curve and normalized to relative input units. Every sample was tested in triplicate per individual 384-well microplate and repeated as three independent biological experiments.Raw data were calculated and analyzed using SDS2.1 software (ABI). CCWTSANTKMNYANNTTCACG, http://mobyle.pasteur.fr/cgi-bin/MobylePortal/portal.py?formfuzznuc). Second, to identify those Pax6-binding sites that contain the homeodomain binding sequence (underlined), the PHO Drosophila paired protein consensus sequence ATTAGTCACGCTTGA-3′5′-CAATTATTNNNNCWTGANNG-3′5′-MN, P6PHD , incubated with 0.5–1 ng of the 5′-end labeled oligonucleotide in the presence of 2 µg of poly[d(I−C)]  at room temperature for 10 mins. The optimal amounts of proteins were determined using the P6CON probe as we described elsewhere Eleven double stranded oligonucleotide probes  were labFigure S1Immunofluorescence detection of Pax6 in lens epithelium. Panels (A–C) are newborn Pax6 WT lenses, panels (D–F) are Pax6 heterozygous lenses. White arrows in (F) demonstrate cells expressing higher levels of Pax6 in the epithelium than those cells marked by the red arrows. Abbreviations: epithelium; e, fiber cells; f. Scale bar = 20 µm.(5.47 MB TIF)Click here for additional data file.Figure S2+/− lens by qRT-PCR. Relative expression levels of Serpinb6b, Rdm1, Ctsh, Zw10, Stmn2, Cdh11, Aldh1a3, Kif22 and Wdhd1 transcripts in wild type  and Pax6+/−  lenses were determined using qRT-PCR as described in Verification of microarray results of up-regulated transcripts in Pax6(0.62 MB TIF)Click here for additional data file.Figure S3+/−  lenses were determined using qRT-PCR as described in Verification of microarray results of down-regulated transcripts by qRT-PCR. Relative expression levels of Acvr1b, Dnase2b, Sultx1, Rock1, Camk1d and Gaa transcripts in wild type  and Pax6(0.96 MB TIF)Click here for additional data file.Figure S4Genes regulated by Pax6 in lens and one region of embryonic telencephalon. A representative list of 43 genes regulated by Pax6 in lens. 19 of these genes shown here  were also differentially expressed in a single region of the developing telencephalon. These genes were grouped into six categories: Chromatin regulation, Mitosis, and Signaling .(2.02 MB TIF)Click here for additional data file.Figure S5Identification of Pax6-binding in regulatory regions of Cspg2/Vcan in lens chromatin by ChIP-on-chip. The upper part shows chromosomal localization, direction of transcription and evolutionar conservation of the genomic regions from eight species as displayed by the UC Santa Cruz Genome Browser. Integrate ChIP signal (input) is shown from three (one) biological replicates, respectively.(6.94 MB TIF)Click here for additional data file.Figure S6Identification of Pax6-binding in regulatory regions of Mab21l2 in lens chromatin by ChIP-on-chip. The upper part shows chromosomal localization, direction of transcription and evolutionar conservation of the genomic regions from eight species as displayed by the UC Santa Cruz Genome Browser. Integrate ChIP signal (input) is shown from three (one) biological replicates, respectively.(7.54 MB DOC)Click here for additional data file.Figure S7Identification of Pax6-binding in regulatory regions of Olfm3 in lens chromatin by ChIP-on-chip. The upper part shows chromosomal localization, direction of transcription and evolutionar conservation of the genomic regions from eight species as displayed by the UC Santa Cruz Genome Browser. Integrate ChIP signal (input) is shown from three (one) biological replicates, respectively.(6.48 MB TIF)Click here for additional data file.Figure S8Identification of Pax6-binding in regulatory regions of Spag5 in lens chromatin by ChIP-on-chip. The upper part shows chromosomal localization, direction of transcription and evolutionar conservation of the genomic regions from eight species as displayed by the UC Santa Cruz Genome Browser. Integrate ChIP signal (input) is shown from three (one) biological replicates, respectively.(6.24 MB TIF)Click here for additional data file.Figure S9Identification of Pax6-binding in regulatory regions of Tgfb2 in lens chromatin by ChIP-on-chip. The upper part shows chromosomal localization, direction of transcription and evolutionar conservation of the genomic regions from eight species as displayed by the UC Santa Cruz Genome Browser. Integrate ChIP signal (input) is shown from three (one) biological replicates, respectively.(6.20 MB TIF)Click here for additional data file.Figure S10A list of putative Pax6-binding sites in Mab21l2 and Tgfb2 loci. A) P6CON, PHO and P6PHD “consensus” sequences. B) Alignment with twelve predicted Pax6-binding sites (site 1 to 12). These sites are grouped as “active” and “inactive” sites. Conserved nucleotide (upper case letters), non-conserved nucleotides (lower case letters). Total number of missmatches (n) between the examined site and the “consensus” sequence and orientation (ori) of the respective site in the promoter  is also given.(0.69 MB TIF)Click here for additional data file.Table S1+/− lens is shown. Although a number of transcripts show relatively small fold changes, they were included as they both passed the statistical criteria, and, may represent genes whose expression is severely deregulated in a hypothetical Pax6−/− lens.A “559” master gene list of differentially expressed transcripts in Pax6 heterozygous lens. The transcripts are organized alphabetically into two group, upregulated (yellow) and downregulated (blue), respectively. Average fold change (FC) in Pax6(0.10 MB XLS)Click here for additional data file.Table S2Primers for qRT-PCR.(0.05 MB DOC)Click here for additional data file.Table S3Primers used for Pax6 analysis in lens chromatin by qChIP.(0.04 MB DOC)Click here for additional data file.Table S4A list of putative Pax6-binding sites in Mab21l2 and Tgfb2 loci. A) P6CON, PHO and P6PHD “consensus” sequences. B) Alignment with twelve predicted Pax6-binding sites (site 1 to 12). These sites are grouped as “active” and “inactive” sites. Conserved nucleotide (upper case letters), non-conserved nucleotides (lower case letters). Total number of missmatches (n) between the examined site and the “consensus” sequence and orientation (ori) of the respective site in the promoter  is also given.(0.03 MB DOC)Click here for additional data file.