Hansenula polymorpha are able to assimilate nitrate and use it as nitrogen source. The genes necessary for nitrate assimilation are organised in this organism as a cluster comprising those encoding nitrate reductase (YNR1), nitrite reductase (YNI1), a high affinity transporter (YNT1), as well as the two pathway specific Zn(II)2Cys2 transcriptional activators . Yna1p and Yna2p mediate induction of the system and here we show that their functions are interdependent. Yna1p activates YNA2 as well as its own (YNA1) transcription thus forming a nitrate-dependent autoactivation loop. Using a split-YFP approach we demonstrate here that Yna1p and Yna2p form a heterodimer independently of the inducer and despite both Yna1p and Yna2p can occupy the target promoter as mono- or homodimer individually, these proteins are transcriptionally incompetent. Subsequently, the transcription factors target genes containing a conserved DNA motif (termed nitrate-UAS) determined in this work by in vitro and in vivo protein-DNA interaction studies. These events lead to a rearrangement of the chromatin landscape on the target promoters and are associated with the onset of transcription of these target genes. In contrast to other fungi and plants, in which nuclear accumulation of the pathway-specific transcription factors only occur in the presence of nitrate, Yna1p and Yna2p are constitutively nuclear in H. polymorpha. Yna2p is needed for this nuclear accumulation and Yna1p is incapable of strictly positioning in the nucleus without Yna2p. In vivo DNA footprinting and ChIP analyses revealed that the permanently nuclear Yna1p/Yna2p heterodimer only binds to the nitrate-UAS when the inducer is present. The nitrate-dependent up-regulation of one partner protein in the heterodimeric complex is functionally similar to the nitrate-dependent activation of nuclear accumulation in other systems.A few yeasts, including  Hansenula polymorpha (syn. Pichia angusta) is able to use nitrate as sole nitrogen source  and DH5α [fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17] were used for routine plasmid preparation.The H. polymorpha strains were grown at 37°C in rich medium (YPD) containing 2% glucose, 1% yeast extract and 2% peptone, or in a minimal medium (MM) containing 2% glucose, 0.2% yeast nitrogen base lacking of both amino acids and ammonium sulphate , plus 0.5% ammonium sulphate (corresponding to 75mM NH4+) or 50 mM of proline, glutamine or glutamate. When necessary, 1.8% agar or 0.006% leucine were added. The standard growth conditions for northern blots and MNase digestion were as follows: cells were grown in 250 ml flasks with 50 ml medium in an orbital incubator at 37°C and 200 rpm. Pre-culturing in YPD for 15 hours, the cells were washed with sterile water and inoculated (106 cells/ml) on minimal medium containing 50 mM proline as nitrogen source. After 16 hours culture the cells were again washed with sterile water and then transferred to a MM containing one of the following nitrogen sources: 50 mM NaNO3, 50 mM NH4Cl, 50 mM L-glutamine, 50 mM Na-glutamate, 50 mM L-proline, 50 mM NH4NO3, 50 mM NaNO3 + 50 mM L-glutamine, 50 mM NaNO3 + 50 mM Na-glutamate, 50 mM NaNO3 + 50 mM L-proline. After the optimal repressing conditions were established, four standard conditions were used: 50 mM L-proline ; 50 mM NaNO3 ; 50mM L-glutamine ; 50 mM L-glutamine + 50 mM NaNO3 . When not otherwise stated, cultures were further incubated for 2 hours and then harvested.All in vivo footprinting of H. polymorpha were as follows: cells were grown in 500 ml flasks with 100 ml of MM containing 50 mM proline as nitrogen source at 37°C and 180 rpm. After pre-culturing for 15 hours, cells were washed with sterile water and inoculated (106 cells/ml) in MM containing one of the following nitrogen sources: 50 mM L-proline  or 50 mM NaNO3 . Cultures were further incubated for 2 hours. Guanines and adenines methylation of genomic DNA in whole and living cells was performed by adding to each flask 10 ml of a solution containing 9,925 ml of 20 mM MES buffer pH 6.2 and 75 μl of pure dimethylsulphate . Cells were incubated for 2 minutes at 37°C and 180 rpm. Methylation was stopped by adding TLE-β stop buffer . Cells were then harvested for genomic DNA extraction.The growth conditions for H. polymorpha strains L1-GFP-1 and L1-GFP-M2 harbouring the chimeric constructs YNA1(p)::YNA1::GFP and MOX(p)::YNA2::GFP respectively, were as follows: cells were pre-cultivated in 100 ml flasks with 20 ml of MM supplemented by 50 mM proline as nitrogen source, at 37°C and 180 rpm. Cells were washed with sterile water and resuspended in 20 ml of MM w/o nitrogen source. 3 ml of each sample was aliquotted in a 15 ml-tube containing MM plus 50 mM of nitrogen source related to the physiological condition under investigation. Cells were then incubated at 37°C, 180 rpm, at different times (from 20 minutes to 2 hours) and observed by confocal microscopy.Growth conditions for H. polymorpha strain AL-YFP18, harbouring the chimeric cassettes YNA1(p)::YNA1::YFPB::LEU2::YNA1(t) and YNA2(p)::YNA2::YFPC::PUR7::YNA2(t), was pre-grown in 100 ml flasks containing 20 ml of MM supplemented by 50 mM Glutamine as sole nitrogen source. Cells were washed with sterile water and resuspended in 20 ml of MM w/o nitrogen source. 3 ml of each sample was aliquotted in 15 ml-tube containing MM plus 50 mM of nitrogen source related to the physiological condition under investigation. Cells were incubated at 37°C, 180 rpm for 2 hours and observed by confocal microscopy.The yna116632 and L1-yna2Δ12274–12595 harbouring the YNA1(p)::YNA1::GFP and MOX(p)::YNA2::GFP constructs, respectively, were grown at 37°C, 180 rpm, on MM containing ammonium as nitrogen source and glucose as carbon source. After 18 h, cells were harvested, washed and cultivated for additional 2 hours on MM containing nitrate (I) or glutamine (R). For strains harbouring the construct MOX(p)::YNA2::GFP, methanol was used as carbon source and inducer.For chromatin immunoprecipitation (ChIP) assays with the GFP antibody, strains L1-YNA1 amplified using primers YNA1ef and YNA1dr and inserted in EcoRI of EBOX37p6. pA2 contains the YNA2 gene, amplified using primer YNA2df and YNA2dr cloned in BamHI of EBOX42p1. EBOX37p6 and the EBOX42p1 are two H. polymorpha replicative plasmids . The pYNA1LEU-I plasmid, containing the LD-I cassette  using primers leu2cf and leu2cr and cloned into the EcoRV restriction site of this intermediate construct (EcoRV is in the YNA1 gene), the latter having been previously linearized with EcoRV and then dephosphorylated. The LEU2 containing PCR fragment was cut with SmaI and then ligated with the plasmid. The construction was confirmed by restriction analyses and sequencing . The pYNA2LEU plasmid, containing the D2 cassette , dephosphorylated and finally ligated with a SmaI/SmaI-end 2122 bp DNA fragment containing the Candida albicans LEU2 gene obtained as described above. The construction was confirmed by restriction analyses and sequencing.Plasmid pA1 contains cassette , derivescassette , derives(1–185) and Yna2p(1–180) truncated forms in E. coli, YNA1(1–555) and YNA2(1–540) were cloned by Gateway Cloning Technology, following manufacturer instructions. Yna1p(1–185) and Yna2p(1–180) contain putative nuclear localisation signals, activation domains and Zn(II)2Cys6 binding domains. In addition, Yna1p(1–185) harbours a putative nuclear export signal (NES), which is not predicted for Yna2p. YNA1(1–555) and YNA2(1–540) were amplified from plasmids by using primers GATYNA1_fw, GATYNA1tr_rv and GATYNA2_fw, GATYNA2tr_rv respectively. pDEST17 destination vector was used as final expression vector in E. coli BL21DE3 strain. Gene expression is driven by E. coli T7 promoter and mediated by T7 RNA polymerase.For the expression of Yna1pScLEU2, MOX(p)::eGFP-Tamo derived from pHIPX4, ATP-end-labeled YNT1 oligonucleotide ] and specific competitors. ATP-labelled amplification products were separated on a sequencing gel which was dried and exposed to intensifying screens. To investigate the H. polymorpha YNT1 promoter, Primer1_sense, Primer2_sense, Primer3_sense were used  coated with Ab290 Abcam anti-GFP antibody. In the next step, each mixture was washed and the Yna::GFP-DNA complexes eluted using TES buffer at 65°C. Samples were treated with Proteinase K for 1,5 h and DNA isolated by phenol-chloroform extraction and 2-propanol precipitation. Each pellet was eluted in 30 μl of Tris-EDTA solution. Samples were analysed by quantitative PCR, using different concentrations of genomic DNA ranging from 10 ng to 10 pg as serial dilutions to generate a standard curve. The sequence of the primers YT_Ch_fw and YT_Ch_rv used to amplify a region of 114 bp containing the Yna1/Yna2 binding site are indicated in H. polymorpha cells were as indicated above. 5 μl of each culture were spotted on a cover slide. For Yna1p::GFP and Yna2p::GFP subcellular fluorescence detection, a Leica TCS SP2 confocal microscope was used . For YFP  and DAPI  detection an Olympus FV1000 microscope was used.Growth conditions for H. polymorpha requires the presence of nitrate and, at the same time, the absence or low levels of reduced nitrogen sources [YNA2 gene are strongly repressed by glutamine, whereas addition of ammonium does not result in complete repression. The least repressive effect is exerted by proline as the steady state mRNA levels on proline plus nitrate are from 40% to 85% of those obtained upon incubation on nitrate alone. This indicates that nitrogen catabolite repression (NCR) depends on the metabolic rate by which a given metabolite is converted to the possibly direct effector for NCR, glutamine. Induction of the structural nitrate utilisation genes  has a considerable lag-time of around 40 minutes whereas the regulatory gene YNA1 is already transcribed after 20 minutes of transfer to nitrate  were unable to utilise nitrate or nitrite as nitrogen sources due to the lack of expression of assimilatory and transporter genes  this is only true for nitrate as sole nitrogen source, when other nitrogen sources were employed , the absence of YNA2 led to a defect in YNA1 expression. This clearly indicates that Yna2p is required for full expression of its partner activator in this genetic background.As expected from previous work, both er genes . Each mugene see  Figs. As-workers  found thYNA1 and YNA2 genes were fused to GFP. YNA1-GFP expression was driven by its native promoter, whereas YNA2-GFP was under the control of the methanol-inducible promoter (MOX(p) from methanol oxidase gene). Heterologous YNA2-GFP expression by the MOX promoter was chosen because Yna2p-GFP should also be studied in YNA1Δ backgrounds and, as determined above, the YNA2 gene is almost not expressed in a strain lacking Yna1p  revealed that both Yna1p::GFP and Yna2p::GFP are located predominantly in the nucleus under all physiological conditions tested, namely non-inducing , inducing , inducing-repressing  or repressing  conditions. YNA1 mRNA is barely detectable under these repressing conditions (YNA1Δ strain (L1-yna1I6632), Yna2p::GFP also locates constitutively and predominantly to the nucleus , Yna1p::GFP has lost its capacity to accumulate in the nucleus and uniformly distributes throughout the cell under all tested physiological conditions  as well as of the structural genes necessary for nitrate assimilation.This corroborates our previous results that nuclear accumulation of Yna1p depends on the presence of Yna2p which putatively regulates the function of Yna1p subcellular localization signals. In this context, Yna2p may either mask the Yna1p NES or be involved in exposing the NLS of Yna1p. A clear signal was only detected under inducing (I) and non-inducing conditions (NI) but was absent under repressing conditions . This result could be due to the small amount of Yna1p::YFP, that is expected to be present in cells grown on glutamine although the Yna1p::GFP signal was readily detected on this N-source compare . Alternain silico analysis of the structural and regulatory gene promoters , and probe 2 (-200 to -235) extends more distally into the promoter region. The truncated forms Yna1p(1–180) and Yna2p(1–185) containing the predicted Zn(II)2Cys6 DNA-binding domain, a putative dimerization domain and the predicted NLS  or even abrogated (probe 2) the formation of the complex. This indicates that the HIS-Yna1p: probe 2 complexes are less stable than complexes formed with probe 1 containing a longer AT-rich stretch in the sequence towards the YNT1 ORF. For HIS-Yna2p the scenario was even clearer, as this protein does not form a detectable complex with probe 2 lacking the 3′AT-rich sequences. These data indicate that in vitro both proteins are able to independently form stable and specific complexes with DNA when both the conserved 5′CGGAGA3′ motif as well as the 3′AT-rich sequences are available.Thus, we suspected that these sequences may mediate nitrate induction by providing recognition sites for the Yna1p and Yna2p proteins. On the basis of these nucleotide alignments, we first performed band shift experiments with recombinant forms of Yna1 and Yna2. We synthesized double-stranded, partially overlapping oligonucleotides representing the region -180 to -235 of the g assays . Both prpurified . The purr region . The forYNT1 promoter are targeted in vivo and under which conditions putative recognition sequences interact with the activators. To this end, we performed in vivo methylation protection footprinting experiments to single out the purines protected by a DNA-binding protein under a given physiological condition [YNT1 promoter. The pattern obtained after ligation-mediated PCR is shown in YNA1Δ and YNA2Δ strains incubated on nitrate (inducing conditions), the protected region is not present and this suggests that in vivo both proteins in the heterodimer are required to form this protection pattern. Due to this large protection window, we were not able to determine exactly which guanines and adenines are participating in the complex-DNA interaction. Besides the protected bases, we also noted the appearance of at least two hypersensitive adenines (indicated by circled dots) in the autoradiograph which were not present in the in vitro methylation control (indicated by V) in a region corresponding to the central part of probe 1 and the end of probe 2. It should be remarked that these hypersensitive bands always specifically appeared in both mutant strains when they were shifted to inducing conditions (nitrate). Generally, the appearance of such residues displaying hypersensitivity towards the methylating agent dimethylsulphate (DMS) is a good indication of local DNA structure distortions generated by binding events. The same principle would apply when Yna1p is binding alone. Note that YNA1 is only expressed to a detectable level in a YNA2Δ strain under nitrate induction  assays. To this end we performed the ChIP analysis on H. polymorpha cells grown under inducing  and repressing  conditions using the GFP-tagged versions of the activators as tag for IP . The presence of the fusion proteins in the different backgrounds was analysed by qPCR targeting 114 nucleotides of the YNT1 promoter region which contains the Yna1p/Yna2p 5′-CGGAGAATA-3′ binding motif. ChIP results are shown in YNT1 promoter strongly only under inducing conditions (I), but can barely be detected under repressing conditions  was tested in the ChIP assay. Also in this case, significantly more Yna2 protein is found associated with the YNT1 promoter region under induced conditions compared to repressed conditions. But a striking feature of Yna2p binding was observed in the strain lacking Yna1p: in this case a roughly 10-fold higher amount of Yna2::GFP is precipitated compared to the amount detected in the wild type background. Strikingly, this hyper accumulation of Yna2::GFP is dependent on the presence of nitrate and does not occur on the repressing nitrogen source (glutamine). These data suggest that also Yna2p –similar to Yna1p –can bind as monomer or homodimer and needs the inducer for this activity. Obviously, in the absence of the heterodimer forming Yna1p, Yna2::GFP is present at higher amounts indicating that Yna1p balances the heterodimer formation with Yna2p under inducing conditions. The fact that Yna2 expression is driven by the strong MOX promoter cannot solely account for the hyper-accumulation of the GFP fusion protein as the amount of Yna2::GFP present at the YNT1 promoter in the wild type background (putative heterodimer) is only around twice the amount of Yna1::GFP in the same heterodimeric complex (wild type background). In contrast, when Yna1p::GFP is lacking, the amount of Yna2p::GFP present as homodimer or monomer is roughly 10-fold higher than when Yna2p::GFP occupies the promoter in the heterodimeric Yna1p/Yna2p form.The strains used for this analysis expressed the fusion proteins either in the wild type background  or in the mutant lacking the heterodimer partner protein . Each of these nucleosomes is flanked by MNase sensitive sites located at positions +85, -81, -275, -449, -655. A very strong MNase hypersensitive site  between the MNase cutting sites corresponding to the end of nucleosomes -1 (position -275) and +1 (position + 85). Taken together, both these findings indicate that a nucleosome-free region may be extant between these nucleosomes potentially providing a docking platform for wide-domain and/or pathway-specific transcription factors. Upon nitrate induction a striking nucleosome rearrangement becomes detectable in which all nucleosomes in the transcribed region (+1–+6) as well two promoter nucleosomes (-1 and -2) lose their position. Chromatin restructuring in transcribed regions is known to be a consequence of transcription and thus it is not surprising that, when the gene is induced, nucleosomes starting from the transcriptional start point (nucleosome -1) and downstream lose their positioning. In many cases nucleosomes upstream of the transcriptional start point are remodelled by specific transcription factors and this process is independent from transcription [H. polymorpha, we performed MNase assays in YNA1Δ and YNA2Δ strains and probed again the YNT1 region for nucleosomal positioning. As seen in YNT1 ORF and consistent with lack of gene transcription in both strains under inducing conditions. But also the upstream nucleosomes -1 and -2 do not rearrange in the mutants: this indicates that both regulators are involved in remodelling the chromatin in the promoter of this target gene. We also tested for chromatin structure changes for another gene belonging to the pathway. To this end, we targeted the nitrite reductase gene YNI1 and analysed the MNase pattern obtained from cultures grown under conditions where the gene is silent  or activated (inducing conditions). This analysis is presented in YNT1 pattern. Nucleosomes are strictly positioned under non-expressing conditions and lose this positioning upon gene induction. Remarkably, the nucleosome-free-region seems to be more extended in this gene than in YNT1. Nevertheless, a nucleosome-free region still extends here between nucleosomes -1 and + 1. As in YNT1, in YNI1 all nucleosomal rearrangements are strictly dependent on the presence of both transcriptions factors (When the gene is silent under non-inducing (NI) and inducing-repressing (IR) conditions, three positioned nucleosomes are detectable in the promoter region  no effect of the YNA2 mutation on YNA1 expression was reported, our experiments clearly reveal that, in the absence of Yna2p, YNA1 expression is almost completely abolished under all conditions with the exception that it is weakly expressed on nitrate as sole nitrogen source. Considering that the YNA1 gene promoter carries a potential binding site for the activators which may operate as heterodimer, this observation might be consistent with the proposed auto-regulatory function of the Yna1p-Yna2p heterodimer for the expression of the YNA1 gene. Interestingly, YNA2 is constitutively and strongly expressed independently of nitrate . This indicates that YNA2 gene expression is not regulated by the Yna1-Yna2 heterodimer. Instead, Yna1p could bind as homo-dimer to a different sequence or may indirectly influence the YNA2 expression, a situation that has been detected in our ChIP analysis of Yna1::GFP in the YNA2 mutant  but our microscopic studies clearly showed nuclear positioning of Yna1p::GFP under all conditions, including glutamine. This would mean that some other mechanism keeps Yna1p::GFP in the nucleus. Alternatively, Yna1p::YFP levels expressed from its own promoter might be insufficient for detection by fluorescence microscopy. This in fact seems to be the most likely explanation because YNA1 is expressed to a very low level on glutamine 2Cys6 type  are also constitutively nuclear and bound to cognate UAS (Upstream Activation Sequences), but this does not automatically lead to target gene activation. Only upon dissociation of a repressing cofactor (e.g. GAL80p from GAL4p) the activation domain is exposed and target genes are expressed [A. nidulans, the proline activator PrnA has also a permanent nuclear localisation, but needs the inducer to bind to its DNA target sites in vivo [H. polymorpha the Yna1p-Yna2p heterodimer shows constitutive nuclear localization. This implies that the presence of nitrate elicits heterodimer DNA-binding and/or a conformational activation of the DNA-bound proteins.To our surprise, this heterodimer is constitutively located to the nucleus and does not need the inducer to trigger nuclear accumulation. This is in contrast to the best studied fungal model system the cell . Similarnduction . It shouxpressed . Only a  in vivo . Neverth in vivo . In H. pYna2p is crucial for the nuclear import or retention of Yna1p. It is possible that the NLS of Yna1p is weakly active in the absence of Yna2p. Alternatively or additionally, Yna2p may mask the Yna1p-NES (“AKLTTLLNEMDDLRLD” ), thus px-CCG Zn(II)2Cys6 protein target site [5′-CGGAGAATA-3′  [H. polymorpha UAS for the nitrate gene expression and the transcriptional activators Yna1p and Yna2p bind this site preferentially as a heterodimer.We identified a common motif in the promoters of the Yna1p-Yna2p target genes. This nucleotide motif does not conform to the typical CCG-nget site ,56 but hGAATA-3′ , similar= A,C,T) . In concin vivo footprinting results from the YNT1 promoter region suggest, however, that this form of the transcription factor complex does not bind DNA in the absence of nitrate but needs the inducer to specifically interact with their recognition sequences in target genes. Our chromatin-immunoprecipitation studies confirmed these results by demonstrating quantitatively that in vivo both Yna1p and Yna2p only bind to the YNT1 promoter when the inducer is present. Thus, although both proteins are present in the nucleus under all conditions, they still need the inducer to bind to their target promoters. At the moment it is not clear which biochemical changes occur upon nitrate induction that transform the inactive transcription factors to an active heterodimeric complex. A mechanism how a fungal nitrate regulator can respond to the presence of the inducer was recently discovered in A. nidulans [H. polymorpha, is unknown.We have also shown in this study that Yna1p and Yna2p physically interact and that this interaction is crucial for bringing the heterodimer to the nucleus or for keeping it there. Our nidulans . In thisH. polymorpha nitrate metabolism. Our data show the existence of hierarchies of repressing compounds, glutamine being the strongest repressing metabolite, and proline being a neutral, or nearly-neutral, nitrogen source. It has been proposed that the existence of such hierarchies is generally related to the conversion rate at which a given nutritional nitrogen compound is transformed into the effector molecules which respond to and signal the “nitrogen state” of the cell [This work also elucidates new aspects in the regulation of the the cell .YNA1. This latter sequence arrangement results in a nitrate-responsive positive auto-regulation loop at the level of transcription factor gene expression and a balanced stoichiometry in the Yna1p/Yna2p heterodimer in which Yna2p is expressed to high levels constitutively and Yna1p is induced to high levels when the inducer and thus the potential substrate for the assimilation process becomes available. Although both proteins can bind individually, the balanced stoichiometry seems to be important for optimal function of the activator complex. To ensure optimal binding, binding sites of the regulators in the investigated target gene promoters appear to lie preferentially in a nucleosome-free region that presumably allows efficient DNA recognition without nucleosomal barriers. Ultimately these binding events lead to a drastic chromatin rearrangement, followed by the transcriptional activation process. The latter is likely mediated through the recruitment of remodelling complexes, presumably containing histone acetyl-transferases and/or ATP-dependent remodelling factors along with the pre-initiation complex.Taken the results of this study together we propose that the “CGGAGA” DNA motif present in the nitrate-responsive gene promoters serve as upstream-activating sequence (UAS) that mediate nitrate induction by serving as binding site for a functional Yna1p/Yna2p heterodimer. Formation of the heterodimer itself does not need the inducer, but stable binding of the heterodimeric activator complex to target DNA and transcriptional competence requires nitrate either as effector molecule or as signalling compound. However, the activator complex seems to be sensitive to glutamine repression because neither interaction nor transcriptional activity is detected under these conditions. UAS are conserved in promoters of all known genes involved in nitrate assimilation, including in the promoter of S1 FigH. polymorpha L1 cells, pre-grown in proline, were incubated in nitrate for the indicated time period. Numbers below the individual lanes represent relative expression values calculated from densitometric measurements of the autoradiograph, normalized to the loading control signal (actin gene HpACT).(TIF)Click here for additional data file.S2 FigA. Schematic representation of the YNA1 locus and adjacent regions and of the LD-I cassette used to generate the yna1I6632::LEU2 allele . Probe-Y1, used for Southern analyses is also indicated (panel C). B. Growth tests show that L1- yna1I6632 is unable to used nitrate as sole nitrogen source for growth. L1+p37 (Leu+) is the wt strain with a LEU2 containing vector (EBOX37p6); yna1Δ is the L1I6632-yna1 mutant; yna1Δ+pA1 is the same mutant with a YNA1 containing plasmid; yna1Δ−pA1 is a plasmid-less derivative of the previous strain. C. Southern blot showing the I6632yna1 null allele. Genomic DNA, digested with EcoRV, was hybridized with probe-Y1 (panel A). Lane 1, wt (L1); lane 2, null mutant (L1- yna1I6632)(TIF)Click here for additional data file.S3 FigA. Schematic representation of the YNA2 locus and adjacent regions and of the strategy used to generate the yna212274–12595Δ:: LEU2 allele . Probe-Y2, used for Southern analyses is also indicated (panel C). B. Growth tests showi that L1- yna212274–12595Δ is unable to use nitrate as sole nitrogen source for growth. L1+p42 (Leu+) is the wild type with a LEU2 containing vector (EBOX42p1); yna2Δ is the L1- yna2Δ12274–12595 mutant; yna2Δ+pA2 is the same mutant with a YNA2 containing plasmid; yna2Δ−pA2 is a plasmid- less derivative of the previous strain. C. Southern blot showing the yna2Δ12274–12595 null allele. Genomic DNA, digested with PstI, was hybridized with probe-Y2 (panel A). Lane 1, wt (L1); lane 2, null mutant (L1- yna212274–12595Δ)(TIF)Click here for additional data file.S4 FigMOX promoter (right panel) was analyzed by confocal microscopy in the wild type (L1) strain and in strains lacking YNA1 (L1-yna1I6632) or YNA2 (L1-yna2Δ12274–12595) under non-inducing , inducing-repressing  or repressing  conditions. Below the recorded fluorescent images the corresponding DIC image is shown. Scale bars refer to 2 μm.Yna1p expressed as a GFP fusion from its own promoter (left panel) and Yna2p-GFP expressed from the (TIF)Click here for additional data file.S5 FigA.nidulans.The sequences aligned comprise the DNA binding domain (DBD) showing the six conserved cysteines, the putative nuclear localization signals (NLS1 and NLS2) and the dimerization domain characterized by the presence of regularily spaced leucines and confirmed so far to be functionally required for in vitro DNA binding of NirA in (TIF)Click here for additional data file.S6 FigI, input; W, protein composition from the column first wash; 1, first elution fraction; 2, second elution fraction; 3, third elution fraction; 4, fourth elution fraction; 5, fifth elution fraction. Fractions 3 are used for band shift assays shown in (TIF)Click here for additional data file.S7 FigA. Location of the analyzed region in the context of the nitrate utilization cluster. The EcoRI-EcoRV fragment used as probe is also shown. B. Representative autoradiograms showing the MNase digestion patterns of the analyzed region. Relevant MNAse hypersensitive sites are indicated. IR, induced-repressed conditions (nitrate + glutamine); R, repressed conditions (glutamine); NI, non-induced conditions (proline); I, induced conditions (nitrate); n-DNA, naked DNA. Two MNase concentrations were used for each condition, as represented by the horizontal triangles above the lanes . See C. Schematic representation of the chromatin organization of the YNI1 promoter region in the physiological conditions examined. Ovals indicate positioned nucleosomes, vertical arrows indicate MNase cutting sites, their thickness being roughly proportional to site sensitivity. YNI1 translational start is indicated by an horizontal arrow at position +1, as well as the YNT1 end . Putative GATA recognition sites are indicated by grey triangles at positions -123 and -162.(TIF)Click here for additional data file.S8 FigYNI1 gene and chromatin structure in the promoter established in the wild type strain and in the regulatory mutants. All growth conditions were as in as in yna1I6632, and Δ2 L1- yna212274–12595Δ. The RNAs were prepared from the same cultures used for chromatin analysis. A. Northern blots. B. MNase digests. The chromatin organization of the wild type (L1 strain) is schematized to the left of each panel, that of the mutants to the right of each panel. Arrows, translational start. Grey triangles, nucleosome free regions (nfr). Dots, hypersensitive sites present in L1- yna212274–12595Δ (Δ2) under R and RI conditions.Expression of the (TIF)Click here for additional data file.S9 FigH. polymorpha L1, L1- yna1I6632 (Δ1) and L1- yna212274–12595Δ (Δ2), grown under I conditions were examined. Results for the L1 strain at 120 min are also shown to allow comparisons to the standard conditions. Total RNAs were prepared from the same cultures used for chromatin analysis (A) Northern blots (B) MNase digestion pattern and nucleosomal organization patterns. The chromatin organization is schematized to the left of each of the 20 min panels. For the L1, 120 min panel, to the left, chromatin structure under induced (I) conditions, to the right chromatin structure under non induced (NI) condition. Dots indicate two MNase sensitive sites present in the wt after 20 min induction (positions -618 and -509) which are absent after 2 hours induction.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


CTCF knockdown also causes a termination defect on the U2 snRNA genes (U2), by affecting recruitment of negative elongation factor (NELF). In addition, CTCF is required for recruitment of positive elongation factor b (P-TEFb), which phosphorylates NELF, DSIF, and Ser2 of the RNAP II CTD to activate elongation of transcription of c-myc and recognition of the snRNA gene-specific 3’ box RNA processing signal. These findings implicate CTCF in a complex network of protein:protein/protein:DNA interactions and assign a key role to CTCF in controlling RNAP II transcription through the elongation checkpoint of the protein-coding c-myc and the termination site of the non-coding U2, by regulating the recruitment and/or activity of key players in these processes.CTCF is a versatile transcription factor with well-established roles in chromatin organization and insulator function. Recent findings also implicate CTCF in the control of elongation by RNA polymerase (RNAP) II. Here we show that CTCF knockdown abrogates RNAP II pausing at the early elongation checkpoint of  BRD4Bromodomain Containing 4ChIPChromatin immunoprecipitationCTCFCCCTC binding factorCDK9cyclin-dependent protein kinase 9CKIIcasein kinase IICTDRNAP II carboxyl-terminal domainDRB5,6-dichlorobenzimidazone-1-β-D-ribofuranosideDSIFDRB-sensitivity-inducing factorGRO-seqglobal run on sequencingGSTglutathione S-transferaseDKknockdownKSHVKaposi's sarcoma-associated herpes virusIPTGIsopropyl β-D-1-thiogalactopyranosideLANALatency-Associated Nuclear AntigenNELFnegative elongation factorNFkBNuclear Factor Kappa BP-TEFbpositive elongation factor bPBSPhosphate-buffered salineqPCRquantitative real-time PCRqRT-PCRquantitative reverse-transcriptase PCRRNAP IIRNA polymerase IIsnRNAsmall nuclear RNASTX4Syntaxin 4TSStranscription start sitesiRNAsmall interfering RNAU2RAU2read-aroundU2RTU2 read-throughWBP5WW Domain Binding Protein 5.CCCTC binding factor (CTCF) is a highly conserved and ubiquitously-expressed transcription factor, which regulates gene expression and organizes chromatin structure.c-myc, pax-6, and chicken lysozyme genes.CTCF binding sites are important elements of insulators, which block communication between adjacent regulatory elements and prevent spreading of heterochromatin.in vivo and this phosphorylation can be recapitulated in vitro by casein kinase II (CKII).CTCF is a 727 amino-acid protein with a central zinc-finger DNA binding domain.in vitro transcription system.U2.Approximately 20% of CTCF sites are located within 2 kb of transcription start sites (TSS), suggesting a role for CTCF in the regulation of transcription at the 5′ end of genes.c-myc, which occurs within 100 bp of the TSS, and on termination of transcription of U2. Our results indicate that CTCF knockdown causes an increase in RNAP II transcription through both the early elongation checkpoint of c-myc and the normal transcription termination site of U2. CTCF is required for the efficient recruitment or retention of NELF and DSIF at sites of RNAP II stalling/termination. The association of these factors correlates with repression of c-myc and efficient termination of transcription of U2. CTCF also enhances P-TEFb recruitment, which is required for RNAP II release from the elongation checkpoint on protein-coding genes and for efficient 3’ box-dependent processing of snRNA gene transcripts. In contrast, NELF and DSIF are not required for CTCF binding, indicating that CTCF recruitment initiates a cascade of interactions that lead either to RNAP II stalling followed by the transition to productive elongation (c-myc) or to termination of transcription (U2). Our data therefore highlights a new function of CTCF as a regulator of RNAP II stalling at the elongation checkpoint of c-myc and at the termination site of U2. In addition, the interactions between CTCF and NELF, DSIF and P-TEFb we describe provide a molecular mechanism for the effect of CTCF on transcription elongation.To investigate the role of CTCF in RNAP II stalling/termination of transcription, we have analyzed the effect of CTCF knockdown on RNAP II stalling at the early elongation checkpoint of L-glutamine at 37ºC and 5% CO2. siRNAs targeting CTCF, the Nelf-E subunit of NELF and the Spt5 subunit of DSIF  were transfected using Lipofectamine 2000  according to the manufacturer's instructions.HeLa cells were grown in DMEM medium supplemented with 10% fetal calf serum, 100 U/mL penicillin, 100 ug/ml streptomycin and 2 mM HeLa nuclear extracts were produced as described.Western blot analysis was performed as previously describedHeLa cells were transfected using Lipofectamine 2000  before being subjected to ChIP analysis following the protocol from the Farnham LabU2. The 3’end of the probes corresponds to positions −130 (probe PSE), +48 (probe R1), +208 (probe R2), +288 (probe R3), +368 (probe R4), +448 (probe R5), +528 (probe R6), +608 (probe R7), relative to the site of transcription initiation. The 3’end of the 80-nucleotide 5S RNA probe corresponding to position +32, relative to the site of transcription initiation was used as a control for the level of transcription. Hybridization signals were quantified by phosphorimager, corrected for the background level (PSE) and normalized to probe 1.Nuclear run-on analysis was carried out as described 6 control or CTCF KD HeLa cells using TRIzol®  according to the manufacturer's instruction. As 7SK RNA levels relative to total RNA were equivalent in control and knockdown samples, these were used for normalization. cDNA was amplified by qRT-PCR using QuantiTect SYBR Green PCR . Sequences of primer pairs are given in Supplementary data, RNA was extracted from 6 × 10RNase protection was carried out as described previously.7 infected Sf9 cells were lysed in high salt buffer  by sonication. After centrifugation, the cleared lysate was incubated for 2h at 4°C on a rotating platform with 2 mL Ni-NTA agarose beads , previously equilibrated in high salt buffer. Beads were then washed twice with TN buffer  containing 10 mM imidazole and 20 mM imidazole and once with NE buffer . Beads were then incubated 4 times with 1 mL NE buffer containing 250 mM imidazole to elute his-CTCF. FLAG-Nelf-E containing NELF complex was purified as described.To generate the pFastBac-hisCTCF vector, human CTCF cDNA  was amplified by PCR using the following primers (GCGGCCGGATCCGAAGGTGATGCAGTCGAAGCCATTG and GCGGCCGA ATTCTCACCGGTCCATCATGCTGAGGATC), digested by BamHI and EcoRI endonucleases and inserted between BamHI and EcoRI of the pFastBac HT vector . The pFastBac-hisCTCF vector was used to produce recombinant hisCTCF protein using a Bac-to-Bac® Baculovirus expression system according to the manufacturer's instructions (Life Technologies). His-CTCF purification was performed as follow. Briefly, 3 × 10During his-CTCF purification (see cloning and protein purification), 50 uL of Ni-NTA agarose beads bound by his-CTCF were kept after the washing steps and incubated with 250 uL of HeLa nuclear extract for 1h at 4°C on a rotating platform. Beads were washed 8 times with RIPA buffer , 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, Complete™ protease inhibitor cocktail ), boiled in Laemmli buffer and analyzed by western blot.For the FLAG-pull-down assay, 500 ng of his-CTCF and 500 ng of FLAG peptide, FLAG-NELF or FLAG-DSIF were incubated in 200 uL buffer  for 1 h at 4°C on a shaker. Anti-FLAG® M2 affinity gel , previously saturated in bovine serum albumin (BSA), were added to the mixture and incubated for 1h at 4°C on a shaker. Beads were washed 4 times with buffer and samples were eluted 3 times using the FLAG peptide. Eluted samples were boiled in Laemmli buffer and analyzed by western blot. For the GST pull-down assay, 500 ng of GST, GST-Nelf-A or GST-Nelf-E diluted in 100 uL of NP40 buffer  was added to 20 uL of glutathione sepharose 4B  for 1h at 4°C on a shaker. Beads were washed 4 times with NP40 buffer and resuspended in 50 uL of NE buffer . His-CTCF (500 ng) was added to the mixture for 1 h at 4°C on a shaker. Beads were washed 5 times with 50 uL of NE buffer, boiled in Laemmli buffer, and analyzed by Western blot.The published datasets for Nelf-E, Spt5, and input reads in HeLa-S3 were obtained fromDe novo motif discovery on the 1044 CTCF peaks close to a TSS was performed with the MEME suite MEME-ChIPAll sequences were mapped using Bowtie2D) of CTCF was used to assess its function in expression of the proto-oncogene c-myc, as a model of a protein-coding gene where CTCF binds at an early elongation checkpoint and represses expression.D on transcription of U2 as CTCF binds at the point where RNAP II terminates in a NELF-dependent manner, approximately 800 bp downstream from the transcription start site (TSS).D was effective as determined by RNA and Western blot analysis , suggesting that any effect of CTCF KD on transcription of c-myc and U2 is not due to drastic rearrangements of nucleosomes.Our observation that CTCF binds at sites of Nelf-E association in both protein-coding and snRNA genes D increases the level of RNAP II along the transcription unit of c-myc , suggesting that CTCF is either causing RNAP II to stall at the checkpoint or repressing initiation. The resolution achieved by ChIP (approximately 200 bp) does not allow differentiation between RNAP II at the TSS and the early elongation checkpoint. In agreement with these results, qRT-PCR analysis of mature (mRNA) and nascent (pre-mRNA) transcripts after CTCF KD using primer pairs specific for exonic or intronic sequences shows that levels of both pre-mRNA and mRNA downstream from the checkpoint increase . The level of c-Myc protein also appears to increase slightly upon CTCF KD . These results are in line with a role for CTCF as a repressor of transcription of c-myc.CTCF KD also causes the RNAP II level to increase toward the 3’ end of the U2 transcription unit . Loss of CTCF causes a defect in termination of transcription very similar to that seen when NELF is knocked down.STX4 and WBP5 genes where no CTCF binding sites are located within the transcription unit .c-myc and terminate transcription of U2.CTCF KD on the association of the Nelf-A subunit of NELF and the Spt5 subunit of DSIF with c-myc and U2 was determined by ChIP (S4). On c-myc, the level of Nelf-A and Spt5 drops after CTCF KD , despite an increase in the cellular level of Spt5 . As NELF and DSIF are recruited to the genes through their interaction with RNAP II, we have also calculated the ratio of Nelf-A and Spt5 to RNAP II. The drop in the ratio of Nelf-A and Spt5 to RNAP II after CTCF KD is even more striking . The level of both proteins peaks in the promoter-proximal region of c-myc in control conditions and drop after CTCF KD . As CDK9 phosphorylates Ser2 of RNAP II CTDD. ChIP analysis indicates that phosphorylation of Ser2 is lower across the promoter-proximal region of c-myc, while phosphorylation of Ser5 is much less affected . CTCF therefore plays a role in recruitment of P-TEFb to this region of c-myc. CTD Ser2 phosphorylation may be dispensable for c-myc transcription in the absence of CTCF because a checkpoint is not established.We next investigated the effect of CTCF KD, both recruitment of CDK9 and cyclin T1 to U2 and phosphorylation of CTD Ser2 are impaired (S4B). Phosphorylation of CTD Ser5 is instead largely unaffected . We have shown that CDK9 is recruited to U2 and is required for recognition of the snRNA gene-specific 3’ box.U2 is impaired upon CTCF KD, 3’ box-directed RNA processing may also be affected. RNase protection of U2 transcripts detects 3 RNAs, corresponding to pre-U2 snRNA (pre-U2), the read-through product that has escaped 3’ box-directed processing (U2RT), and read around RNA that is produced by RNAP II that has escaped transcription termination and read into the next tandem U2 (U2RA).D. Thus, CTCF also helps to recruit P-TEFb to U2 for efficient recognition of the 3’ box recognition.Upon CTCF Kc-myc and U2, we have investigated whether CTCF interacts with these factors. Recombinant his-tagged CTCF produced using a baculovirus system was incubated with nuclear extract and the interaction partners were pulled down using nickel beads and analyzed by Western blot , confirming the interaction between these factors.As CTCF is required for NELF, DSIF, and P-TEFb recruitment to c-myc and U2. The next question was whether NELF can in turn regulate CTCF binding. We have investigated the effect of NELF KD on CTCF and RNAP II association with c-myc and U2. In line with the finding that loss of one subunit of NELF affects stability of the complex,D of the Nelf-E subunit efficiently reduces the level of Nelf-A detected by ChIP , arguing for feedback regulation between these factors.NELF KU2 transcription unit to terminate transcription.In conclusion, CTCF helps to recruit and/or stabilize NELF at the end of the D on CTCF, RNAP II and NELF association with c-myc and U2. The knockdown efficiency has been tested by ChIP  and Western blot , the elongation checkpoint is not established and RNAP II transcribes c-myc without pausing at an early elongation checkpoint.On U2, the level of RNAP II decreases along the transcription unit upon DSIF KD .Conversely, on c-myc to first stall transcription while recruitment of P-TEFb promotes the transition to productive elongation. On U2, instead, recr-uitment of NELF and P-TEFb by CTCF helps to couple termination of transcription to RNA 3’ end formation ,CTCF also helps recruitment of P-TEFb to the elongation checkpoint of D causes an increase in RNAP II on c-myc, an increase in nascent and steady state transcripts and an increase in levels of the c-myc protein, CTCF is acting as a repressor. In addition, our results suggest that production of mature c-myc mRNA is not strictly dependent on CTD Ser2 phosphorylation at the early elongation checkpoint. GRO-seq analysis indicates that, when CTCF is not knocked down, 2 CDK9 inhibitors, DRB and KM05382, inhibit transcription of c-myc,As CTCF Kin vitro, highlighting that interactions between CTCF and these factors in vivo underlies the effect of CTCF on transcription elongation. The C-terminus of CTCF can be phosphorylated in vitro by nuclear extracts and casein kinase II (CKII).c-myc early elongation checkpoint. Furthermore, CTCF-CTCF interactions are known to be involved in the formation of gene loops.D would perturb these loops and allow RNAP II to proceed.Our results indicate that CTCF can interact directly with DSIF and NELF and either directly or indirectly with P-TEFb . Recruitment of P-TEFb by CTCF will result in phosphorylation of NELF, which then leaves the elongation complex, phosphorylation of DSIF, which is converted into a transcriptional activator and phosphorylation of Ser2 of the RNAP II CTD.Our findings suggest a model where a network of interactions between CTCF, NELF, DSIF, and RNAP II underlies an early elongation checkpoint on some protein-coding genes U2, which activates 3’ box-directed processing of snRNA gene transcripts.D may contribute to the termination defect through the effect on 3’ box recognition.D causes a similar defect without affecting RNA 3’ end formation.D this mark could also contribute to efficient termination of transcription.CTCF also recruits P-TEFb to


Fgfr1 gene at the top of major ontogenic pathways that enable gastrulation, tissue development and organogenesis. Using genome-wide sequencing and loss and gain of function experiments the present investigation reveals a mechanism that underlies global and direct gene regulation by the nuclear form of FGFR1, ensuring that pluripotent Embryonic Stem Cells differentiate into Neuronal Cells in response to Retinoic Acid. Nuclear FGFR1, both alone and with its partner nuclear receptors RXR and Nur77, targets thousands of active genes and controls the expression of pluripotency, homeobox, neuronal and mesodermal genes. Nuclear FGFR1 targets genes in developmental pathways represented by Wnt/β-catenin, CREB, BMP, the cell cycle and cancer-related TP53 pathway, neuroectodermal and mesodermal programing networks, axonal growth and synaptic plasticity pathways. Nuclear FGFR1 targets the consensus sequences of transcription factors known to engage CREB-binding protein, a common coregulator of transcription and established binding partner of nuclear FGFR1. This investigation reveals the role of nuclear FGFR1 as a global genomic programmer of cell, neural and muscle development.Genetic studies have placed the  Fgfr1 gene interfere with gastrulation, as well as with development of the neural plate and neural crest, central nervous system, and somites by affecting the expression of diverse genes [Fgfr1 at the top of the developmental hierarchy.Development of a multicellular organism from a single cell is regulated by myriads of TFs and requires the coordinated regulation of multi-gene programs. The "Integrative Nuclear Fibroblast Growth Factor Receptor-1 (FGFR1) Signaling" (INFS) pathway has been shown to mediate cellular development and differentiation programs activated by numerous signals . At the se genes –6 and mise genes , 8 that C. elegans [The  elegans  FGF orth elegans –13. Indi elegans . For exa elegans . The acc elegans .The INFS mechanism is involved primarily in developmental transitions, most commonly the switches to differentiation and post-mitotic development , 10. TraFgfr1, Fgf-2 and Th genes, and synergistically activate isolated RA- and Nur-responsive elements [Within mouse Embryonic Stem Cells (ESCs), core networks of interconnected TFs control the ability of these cells to maintain the pluripotent state or differentiate into lineages of all three germ layers , 26. Retelements , 28, 35.How can a single nuclear protein program the development of ESCs—a process that involves the coordinated regulation of thousands of genes that are located on different chromosomes and contain diverse regulatory elements? Given that nFGFR1 binds to and activates CBP , a histoTo identify the chromatin-binding patterns of nFGFR1, RXR and Nur77, we performed ChIP-seq on extracts from pluripotent ESCs maintained in the presence of leukemia inhibitory factor (LIF), essential for maintaining stem cell pluripotency, and from LIF-free monolayers treated with 1 μM RA for 2 days to induce Neuronal Cell (NC) differentiation. In both ESCs and NCs, the peaks of nFGFR1, RXR and Nur77 binding were heterogeneously distributed across the chromosomes Fig —S1C Fig.To determine whether DNA nFGFR1 targeting of any part of the genome is dependent on co-targeting with RXR and/or Nur77 , 28, we To identify all mRNA genes expressed in both ESCs and NCs, we performed RNA-seq. In total, 14,443 expressed genes were detected, of which 1,834 were up-regulated and 1,477 down-regulated (fold change (FC) ≥-/+2.0 and p-value <0.035) during RA-induced neuronal differentiation .Combined analyses of the ChIP-seq and RNA-seq data sets revealed that >85% of the sites within the proximal promoter (-1kb to +1kb TSS) that were bound by nFGFR1, RXR, Nur77, individually or in combination, were associated with expressed genes . Thus, anFGFR1 targeted not only mRNA-encoding genes, but also expressed miRNA genes. Using miRNA-seq we identified 534 miRNAs that were expressed and 24 that were differentially regulated. In ESCs, nFGFR1 bound to the proximal promoters of only two expressed miRNAs. However, during RA-induced NC differentiation this number increased to 14 ; among tOne established marker of gene activation is histone variant H3.3 , which iWe next sought to identify biological pathways and networks in which the differentially expressed genes whose promoters are targeted by nFGFR1 are involved. As a first step, we utilized Ingenuity Pathway Analysis (IPA), an established tool for analyzing comprehensive genomic data.Tp53), checkpoint kinase 1/2, Dkk1 and Camk2d , anand Irx3 , the Rarreceptor  and the [Cyp26a1 , 49)  domain is deleted (FGFR1(SP-/NLS) (TK-)). This exclusively nuclear protein prevents endogenous nFGFR1 from binding to targeted gene promoters [To determine whether nFGFR1 influences the expression of pluripotency genes, we modulated nFGFR1 function and measured levels of the candidate mRNA targets by real-time quantitative PCR (RT-qPCR). Since both the deletion and siRNA-mediated inhibition of the romoters  and the romoters , 42, 54.Ctcf, Suz12, Klf4, Sox2, E2f1, and Stat3) or not bound (Oct4 and Nanog) by endogenous nFGFR1. In ESCs, FGFR1(SP-/NLS)(TK-) led to a significant increase in the basal expression of all pluripotency genes examined (TK-) on genes that were down-regulated during NC differentiation and either bound . This exclusively nuclear protein binds to CREs within promoters , activatHox genes encode homeo-domain TFs, which are present in all metazoans and ensure both that the general body plan is established and that the central nervous system is organized [Hox clusters are activated sequentially, according to chromosomal position (3’ to 5’) [The rganized . In resp’ to 5’) .Hoxd1 and Hoxd13 genes, in NCs it bound at Hoxa1, 2 and 4, as determined by both ChIP-seq experiments , which includes Hoxa1, critical for activating downstream Hoxa and Hoxb genes [Hoxa1, 4 and 7 mRNAs , FGFR1(SP-/NLS) enhanced the expression of the Hoxa1 and Hoxa2 mRNAs  significantly augmented the RA-induced up-regulation of the Cyp26a1 mRNA , Lfng, Nkd1, Nrarp, Mesp2 and Porcn  construct, which encodes a constitutively active nFGFR1, and a luciferase reporter construct containing motifs for the binding of pluripotency TFs, including SMAD (SMAD binding element (SBE1-4)) and MYC (E-Box). Transfection of FGFR1(SP-/NLS) markedly reduced SMAD3-dependent transcriptional activity in NCs , indicatSmad1 gene and SBE-4, as well as of the Myc genes and E-BOX, support the concept of dual-level transcription control by nFGFR1, i.e., (i) targeting a TF-encoding gene, and (ii) control the trans-acting function of that TFs.nFGFR1 targeting of both the The present study establishes nFGFR1 as a global factor that controls the genome by binding the promoters of thousands of genes, and consensus DNA sequences of diverse TF families. As such, it identifies a previously unknown, multifaceted form of control of major ontogenic pathways and gene networks.nFGFR1 targets genes on all mouse chromosomes in a nonrandom manner, and has a distribution similar to those of two established nFGFR1 partner TFs: RXR and Nur77. Like the binding sites of RXR and Nur77, those of nFGFR1 were present throughout the genome, but most highly concentrated within regulatory regions. For example, upstream and bidirectional promoters and 5’ UTRs were frequently bound in both ESCs and NCs, and >85% of all nFGFR1 peaks were found within the proximal promoters (-1kb to +1kb TSS) of expressed genes . These din vivo [One feature that distinguishes the interactions of nFGFR1 with the genome from those of RXR and Nur77 is the several-fold increase in the number of promoter sites occupied by nFGFR1 only in genes that are differentially regulated in the course of RA-triggered differentiation . Also, tin vivo .The roles of nFGFR1, together and independently of RXR and Nur77, are further illustrated by the overlapping lists of recognition motifs in the target DNAs of these proteins . nFGFR1 Klf4, Sox2, Stat3, E2f1, Esrrb and Suz12, Smad1, Zfx, Tcfcp2l1, and Ctcf genes during RA-induced differentiation appears to be mediated by the recruitment of nFGFR1 to the proximal promoter, and the disassociation of RXR and Nur77 from many of the same sites (TK-) is consistent with the previously demonstrated blockade of RA-induced NC differentiation  how does blocking nFGFR1 disrupt cellular development and the associated gene activities that are initiated by RA and other ontogenic signals; and (3) how does the constitutively active nFGFR1 protein program the differentiation of stem and progenitor cells in vitro and in vivo?Genetic experiments have positioned FGFR1 at the top of the developmental hierarchy. The present investigation begins to answer important questions that have arisen from these findings: (1) how does disruption of a single gene, This study reveals the basic components of an emerging paradigm for master ontogenic networks involved in cell pluripotency, tissue and organ development, and axis specification being controlled by nFGFR1 . In assonFGFR1 has been proposed to act as a gate-opening factor in the feed-forward-and-gate module for control of CBP. Feed-forward loops are common in biological networks, serving as pulse generators, response-delaying circuits, signal-to-noise enhancers and signal integrators. nFGFR1-controlled feed-forward-and-gate loops are positioned at several strategic nodes that may increase the efficiency and reproducibility of ontogenic pathways. The overall importance of such mechanisms in animal development is supported by the evolutionary emergence of nuclear FGFs  and FGFRAmerican Type Culture Collection) were propagated as previously described [Cultures of undifferentiated mouse ESCs , in which the SP is replaced with a canonical NLS, normally provided by the FGF-2 ligand, is a constitutively active nuclear protein which binds to CBP and nFGFR1 regulated gene promoters , 62, 63.in vivo , and actin vivo , 28, 35.in vivo , 62, 63.in vivo , 54. PlaThe nuclear presence of FGFR1 was demonstrated in several laboratories in non-transformed cells , 65, canESC or NC monolayers on 100mm plates were cross-linked with 1% formaldehyde  for 10 min while shaking at room temperature and rinsed three times with cold phosphate-buffered saline. Cells from five plates were pooled and harvested in phosphate-buffered saline with protease inhibitors by 10 min centrifugation  at 4°C. ChIP was performed with 112 μg of chromatin and 3 μg of FGFR1, RXR or Nur77 antibodies using the Invitrogen MAGnify and Millipore kits, according to manufacturer’s instructions with slight modifications. Genomic DNA was precipitated with ethanol, treated with RNase A and proteinase K, and purified using the Qiagen PCR purification kit. For ChIP-seq, chromatin was further prepared using the Tru-seq ChIP Sample Preparation Kit and purified library DNA was captured on an Illumina flowcell for cluster generation and sequenced on an Illumina Hisequation 2000, following the manufacturer's protocols. In a pilot ChiP-seq experiment and in independent ChiP assays . One tenth of the synthesized cDNA was used as the template for real time-quantitative PCR. Twenty-five μl real time PCR reactions were performed on the BioRad MyiQ Cycler with iQ SYBR Green Supermix (Bio-Rad). RT-qPCR using the amplification cycles: Initial denaturation for 8:30 min at 95°C, followed by 35x cycle 2 . Melt curve data collection was enabled by decreasing the set point temperature after cycle 2 by 0.58°C. The specificity of amplicons was confirmed by generating the melt curve profile of all amplified products. Gene expression was quantified as described in . All oliCells were transfected with DNA using Lipofectamine 2000 and luciferase activity was measured using the Promega Luciferase Assay System on a BioTek Plate Reader, as described previously , 28, 42.qPCR was used to determine the relative amount of specific loci in IP, Input, and IgG (Preimmune) samples. qPCR was performed using iQ SYBR Green Supermix (Bio-Rad) on a Bio-Rad iCycler, as described above. Two and half μl of ChIP, IgG and 10% total input DNA was used in duplicate reactions of at least two independent experiments. Data are expressed as % Input. All oligonucleotide sequences for ChIP-qPCR can be found in For ChIP-seq, raw FASTQ reads were aligned to the mouse genome build mm10 using Bowtie 1.0 software with the following command: bowtie—m 1—p—q—S. All peaks we called were from SAM files using Model-based Analysis of ChIP-Seq (MACS) version 2.0 default settings  with the following command: macs2 callpeak—p—t—f—g—n—B. MACS2 uses a dynamic Poisson distribution to calculate peaks.RNA-seq was analyzed using the Tuxedo pipeline . Raw FAShttp://cistrome.org/ap/) and UCSC PhastCons conservation scores were used to detemermine the average conservation score profiles around the peak centers. For Galaxy/Cistrome, genome coordinates from all peak sets were first converted as BED files to the mm9 genome assembly using the LiftOver tool from the UCSC Table browser.As functional genomic regions are often evolutionarily conserved between different species, we compared the evolutionary conservation of our ChIP-seq peaks with flanking non-peak regions as an indicator of good data quality and correct data preprocessing. ChIP-seq peaks were input as BED files to the 'Conservation Plot' tool in the Galaxy/Cistrome platform (http://genome.ucsc.edu/) using the Galaxy web based platform (https://main.g2.bx.psu.edu/). Overlapping peak sets for FGFR1, RXR and Nur77 were generated using the concatenate, cluster and subtract tools from Galaxy. To find FGFR1+RXR+Nur77 binding sites we first identified FGFR1 peaks that directly overlapped either RXR or Nur77 by a minimum of -1 base pair (bp) and clustered these into single peak sets. Intervals from the above generated FGFR1+RXR and FGFR1+Nur77 peak sets, which further overlapped each other by a minimum of-1bp, were again clustered into the single FGFR1+RXR+Nur77 peak set. To identify genomic sites in which FGFR1 was bound with RXR, but not with Nur77, we subtracted the FGFR1+RXR+Nur77 peaks from the FGFR1+RXR peak set by a minimum of 1bp using the subtraction tool. The same was done for Nur77 by subtracting the FGFR1+RXR+Nur77 peak set from the FGFR1+Nur77 peak set. To identify the genomic sites in which FGFR1 was bound alone we further subtracted all FGFR1 peaks in either ESCs or NCs that overlapped either RXR and/or Nur77 by a minimum of 1bp in their respective sample. The same method was also used for RXR and Nur77.ChIP-seq tracks were generated from MACS2 output files and uploaded to the UCSC genome browser  main program in the Galaxy/Cistrome platform.To further relate binding to gene expression, we chose to focus on binding sites found within the proximal promoter to ensure the target promoter is associated with the designated expressed gene. We identified FGFR1, RXR and/or Nur77 peaks described above, which directly overlapped the proximal promoter by a minimum of 20bp using the join tool in Galaxy. Gene names for each promoter-binding site were obtained using the ref tool in UCSC. We next combined the total expressed and non-expressed UCSC genes from our RNA-seq data with the UCSC proximal promoter binding sites for each factor using the “Vlookup” function in Microsoft excel. We further filtered these promoter sites into non-expressed, expressed and differentially regulated genes for downstream analysis.https://www.analysis.ingenuity.com) web based software. Peaks bound to genes that were significant by RNA-seq (≥1.18 FC) but not by our cut-off (≥2.0) were also uploaded for network building but were not considered differentially regulated. All peak sets were analyzed using Core Analysis with mouse as the organism and all tissues and cell lines under a stringent filter. IPA uses a repository of biological interactions and functional annotations to build relationships  and sources major NCBI databases , microRNA-mRNA target databases, GWAS databases, and KEGG.We imported the differentially regulated genes that met the log ratio cut-off-/+ 2.0 and p-value ≤0.05 bound by each factor into Ingenuity Pathway Analysis , down-regulated (green), and unchanged molecules (gray) are designated in each Canonical Pathway and the top 8 pathways are shown for each peak set. The top disease and biological function p-values are also calculated using the right-tailed Fisher’s exact test and the top five in each category are listed for each peak set.To identify motifs, FASTA data from each of the original peak sets was extracted using the Fetch Sequences tool in the Galaxy platform and uploaded to MEME-ChIP motif software. MEME-ChIP was run on all the factors using the following parameters: meme-chip-noecho-oc. -index-name index.html-time 300-db db/JASPAR_CORE_2014_vertebrates.meme-db db/uniprobe_mouse.meme-meme-mod anr-meme-minw 6-meme-maxw 30-meme-nmotifs 5-dreme-e 0.05-centrimo-score 5-centrimo-ethresh 10 Galaxy213-_DNA_.fasta. JASPER and UNIPROBE were the chosen candidate databases. Motifs with a p-value ≤002 were selected for further analysis. For FGFR1, we focused on motifs that were present in both pluripotent and/or both RA replicates.S1 FigA) nFGFR1, (B) RXR and (C) Nur77 was highest on chromosomes 8 and 11 and lowest on chromosomes X and Y. Average Phastcon scores for (D) nFGFR1, (E) RXR and (F) Nur77 in ESCs and NCs display a high level of evolutionary conservation compared to non-flanking regions. As functional regions of DNA are often conserved between species, this further demonstrates the importance of nFGFR1 as a genomic regulator, and serves as an indicator of good data quality and correct data preprocessing. (G) UCSC genome browser views of nFGFR1, RXR and Nur77 binding within Fgfr1, Fgf-2, Rarα and Rarβgenes. (TIF)Figures show length of individual chromosomes as % of the entire mouse genome and % of peaks found on individual chromosomes. In both ESCs and NCs, the relative distribution of peaks for ((TIF)Click here for additional data file.S2 FigNearly 90% of proximal promoter (-1kb to +1kb TSS) peaks (identified by ChIP-seq) are associated with active genes (mRNA detected by RNA-seq). (TIFF)(TIF)Click here for additional data file.S3 Fig(A) Genomic distribution of H3.3. ChIP-seq for H3.3 was performed after 48h RA-induced NC differentiation, as described. We identified a total of 10,634 H3.3 sites that were primarily localized within the proximal promoter, gene body and intergenic regions (B) H3.3 peak enrichment within promoter and genic regions. H3.3 peaks were enriched 8-fold within the upstream proximal promoter (-1kb), 23-26-fold within the bidirectional promoter and 5’UTR, and 5-fold within coding exons. No such enrichment was found in the downstream promoter (+1kb), the 3’UTR or introns. (C) Colocalization of nFGFR1 with RXR and Nur77 coincides with H3.3 in RA-induced NCs. Within the genome, only 5% of individual nFGFR1, RXR and Nur77 binding sites overlapped with H3.3. When FGFR1 was bound with either factor separately this overlap increased to approximately 25%, and when nFGFR1 was bound with both factors combined this further increased to 63%. (D) H3.3 incorporates into promoters targeted by nFGFR1. Within the proximal promoter of expressed genes, H3.3 was incorporated into sites containing nFGFR1 to markedly greater degree (>81%), than sites containing only RXR, Nur77 or H3.3, but not nFGFR1 (15%). (E) Within the proximal promoter of differentially regulated genes, a similar preferential incorporation of H3.3 was observed within sites containing nFGFR1 greater than sites containing only RXR, Nur77 or H3.3. (TIFF)(TIF)Click here for additional data file.S4 Fig(B), (C) and (D); a pink border represents genes or groups of genes bound by nFGFR1, in which the degree of gene upregulation (red) and downregulation (green) is denoted by the color intensity. A rainbow color represents a group of genes that contains members that are both up- and down-regulated. Grey symbols represent genes bound by FGFR1 but were not differentially regulated according to our cut-off. A double border denotes a group or complex of functionally related genes within the pathway. A complete interpretation of network shapes and interactions can be found in Material and Methods. All p-values were calculated using the right-tailed Fisher’s exact test. (A) Genome browser views of nFGFR1, RXR and Nur77 binding within the proximal promoter of Tp53, Chek2, Dkk1 and Camk2d genes in ESCs and NCs. (B) “CREB signaling in neurons” pathway based on nFGFR1 promoter binding in pluripotent ESCs. The top differentially regulated genes include CamkII, Adenylate cyclase (AC), Phospholipase C (PLC), and G-protein β(Gβ). (C) “Axonal guidance signaling canonical pathway” based on nFGFR1 promoter binding in NCs. nFGFR1 targets promoters of the ephrins, ephrin receptors, integrins and BMP7 genes. (D) “Wnt/B-catenin pathway” based on nFGFR1 promoter binding in NC. The top differentially regulated genes include Rar, Sox, c-Jun, Wnt, Dkk1 and Frizzled. (TIFF)All pathways are based on proximal promoter (-1kb to +1kb TSS) binding of designated factor to differentially expressed genes . For canonical pathways displayed in (TIF)Click here for additional data file.S5 ig(D) and (F); a pink border represents genes or groups of genes bound by nFGFR1, in which the degree of gene upregulation (red) and downregulation (green) is denoted by the color intensity. A rainbow color represents a group of genes that contains members that are both up- and down-regulated. Grey symbols represent genes bound by FGFR1 but were not differentially regulated according to our cut-off. A double border denotes a group or complex of functionally related genes within the pathway. A complete interpretation of network shapes and interactions can be found in Material and Methods. All p-values were calculated using the right-tailed Fisher’s exact test. (A) List of top canonical pathways based on differentially regulated genes bound by FGFR1 and RXR in pluripotent ESCs. The top 8 significant pathways are shown. (B) Top canonical pathways are based on differentially regulated genes bound by FGFR1 and Nu77 in ESCs. The top 8 significant pathways are shown. (C) Top canonical pathways based on differentially regulated genes bound by nFGFR1 and RXR in NC. The top 8 significant pathways are shown. (D) “Role of Nanog in mammalian ESC pluripotency” pathway based on nFGFR1 and RXR binding in NCs. The top differentially regulated genes bound by nFGFR1-RXR include Wnt, Frizzled, Bmp, and Smads. (E) Top canonical pathways based on differentially regulated genes bound by nFGFR1 and Nur77 in NCs. The top 8 significant pathways are shown. (F) “Dopamine DARPP32 Feedback in cAMP signaling” pathway based on nFGFR1 and Nur77 binding in RA-induced NCs. The top differentially regulated genes include Dopamine receptors 2/3/4, Protein kinase C (PKC), Phospholipase C (PLC), and Potassium inwardly-rectifying channel (KCNJ). (G) Top canonical pathways based on differentially regulated genes bound by nFGFR1, RXR and Nur77 in ESCs. The top 8 significant pathways are shown. (H) Top canonical pathways based on differentially regulated genes bound by FGFR1, RXR and Nur77 in RA-induced NCs. The top 8 significant pathways are shown. (TIFF)All pathways are based on proximal promoter (-1kb to +1kb TSS) binding of designated factor to differentially expressed genes . For canonical pathways displayed in (TIF)Click here for additional data file.S6 Fig(A) Average Phastcon scores for nFGFR1 from second ChIP-seq replicate after 48h RA-induced NC differentiation. (B) Genomic distribution of nFGFR1 within the proximal promoter, distal promoter, genic and intergenic regions. We identified a total of 14,082 genomic peaks of which 11,223 were localized within the proximal promoter. (C) nFGFR1 peak enrichment within promoter and genic regions. nFGFR1 peaks were enriched over 30-fold in the upstream proximal promoter (-1kb), over 116- fold within the bidirectional promoter and 94-fold within the 5’UTR. No enrichment was observed in the downstream promoter, 3’UTR or introns. (TIFF)(TIF)Click here for additional data file.S7 Fig(A) UCSC genome browser views of nFGFR1, RXR and Nur77 binding to promoters of core pluripotent genes  in pluripotent ESCs and RA-differentiated NCs. (B) Independent ChIP assay demonstrating nFGFR1 binding within the proximal promoter of the Suz12 gene (C) Dominant negative FGFR1(TK-) disrupts the expression of core pluripotent genes in the presence of LIF. mRNA expression levels were measured using RT-qPCR with extracts from ESCs transfected with either β-gal (control) or FGFR1(TK-) and subsequently maintained in the presence of +LIF or +RA for 48 hours. In the presence of LIF, blocking both membrane bound and nFGFR1 increased the levels of all genes examined. However, its effect on the RA-induced downregulation of nearly all genes was markedly diminished as compared to FGFR1 (SP-/NLS)(TK-). P value <0.05 * different from β-gal+LIF; + different from β-gal+RA. (TIFF)(TIF)Click here for additional data file.S8 Fig(A) UCSC genome browser views of nFGFR1, RXR, and Nur77 binding within the Hoxa-Hoxd gene cluster. (B) Independent ChIP assays demonstrating nFGFR1 binding within the proximal promoter of the hoxa1, hoxa2 and hoxa3 genes. (C) UCSC genome browser views of nFGFR1, RXR and Nur77 binding within the proximal promoter of Cyp26a1. (D) Independent ChIP assays demonstrating nFGFR1 binding within the proximal promoter of the Cyp26a1 gene. (E) FGFR1(SP-/NLS) augments the RA-induced expression of Cyp26a1. mRNA expression levels were measured using RT-qPCR with extracts from ESCs transfected with either β-gal (control) or FGFR1(SP-/NLS) and subsequently maintained in the presence of +LIF or +RA for 48 hours. In the presence of RA, transfection of FGFR1(SP-/NLS) significantly augmented the expression of Cyp26a1. P value <0.05 * different from β-gal+LIF; + different from β-gal+RA. (TIFF)(TIF)Click here for additional data file.S9 Fig(A) UCSC Genome browser views of nFGFR1, RXR, and Nur77 binding to within the promoter of Notch1, Perlecan (Hspg2), Dusp6, Lfng, Nkd1, Nrarp, Pornc and Mesp2. (B) Independent ChIP assay showing nFGFR1 binding within the Mesp2 gene body (C) FGFR1(SP-/NLS)(TK-) disrupts the expression of Mesp2 and antagonizes or blocks the RA-induced repression of Notch1 and Hspg2 mRNA expression. mRNA expression levels were measured using extracts from ESCs transfected with either β-gal (control) or FGFR1(SP-/NLS)(TK) and subsequently maintained in the presence of +LIF or +RA for 48 hours. P value <0.05 * different from β-gal+LIF; + different from β-gal+RA. (TIFF)(TIF)Click here for additional data file.S10 Fig(A) UCSC genome browser views of nFGFR1, RXR and Nur77 binding within the promoter of Pax3, Id3, Cdx1 and Irx3. (B) FGFR1(SP-/NLS) (TK-) antogonizes or blocks the RA-induced activation of Pax3, Id3, Cdx1 and Irx3. mRNA expression levels were mesured using extracts from ESCs transfected with either β-gal (control) or FGFR1(SP-/NLS) (TK-) and subsequently maintained in the presence of +LIF or +RA for 48 hours. P value <0.05 * different from β-gal+LIF; + different from β-gal+RA. (TIFF)(TIF)Click here for additional data file.S11 Fig(A) All motif analyses were carried out using MEME-ChIP software. The over-represented DNA sequences reveal nFGFR1 binding to over 30 motifs representing consensus sequences of diverse TFs, all of which are known to interact with nFGFR1 binding partner and transcriptional coactivator CBP. Consistent with partial overlap of nFGFR1 peaks with RXR and/or Nur77, we found a number of the consensus sequences in which (i) nFGFR1 shares with RXR and/or Nur77 , (ii) consensus sequences targeted by nFGFR1 alone  and (iii) consensus sequences targeted by RXR and/or Nur77 but not shared with nFGFR1 . In cases of TFs like CTCF, Klf4, SOX2, STAT3, and TP-53, nFGFR1 interacts both with their cognate DNA sequences as well as genes that encode these TFs. This implies a dual-level of regulation in which nFGFR1 controls generation of TFs as well as their downstream functions. (B) FGFR1(SP-/NLS) inhibits SBE-4 activation in the presence of Smad3. mESCs were transfected with the SBE-4-luc construct and either β-gal, FGFR1 (SP-/NLS), SMAD3 or SMAD3+FGFR1(SP-/NLS), and subsequently maintained in the presence of +LIF or +RA for 24 hours. Luciferase activity was normalized to β-gal. In the presence of RA, nuclear active FGFR1(SP-/NLS) significantly decreased the SMAD3-dependent activation of SBE4-luc. (C) FGFR1 (SP-/NLS) augments E-BOX activation in the presence of LIF and RA. mESCs were transfected with the E-BOX-luc construct and either β-gal or FGFR1(SP-/NLS), and subsequently maintained in the presence of +LIF or +RA for 24 hours. Luciferase activity was normalized to β-gal. Transfection of FGFR1 (SP-/NLS) significantly enhanced E-BOX dependent transcriptional activity in the presence of either LIF of RA treatment. (TIFF)(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.