AmphiVent1 and AmphiVent2 promoters appear to be correctly regulated in the context of a vertebrate embryo. Finally, we show that AmphiVent1 is able to directly repress promoters of AmphiGoosecoid and AmphiChordin genes. Repression of genes encoding dorsal-specific signaling molecule Chordin and transcription factor Goosecoid by Xenopus and zebrafish Vent genes represents a key regulatory interaction during vertebrate axis formation. Our data indicate high evolutionary conservation of a core Bmp-triggered gene regulatory network for dorsoventral patterning in chordates and suggest that co-option of the canonical Wnt signaling pathway for dorsoventral patterning in vertebrates represents one of the innovations through which an increased morphological complexity of vertebrate embryo is achieved.Formation of a dorsoventral axis is a key event in the early development of most animal embryos. It is well established that bone morphogenetic proteins (Bmps) and Wnts are key mediators of dorsoventral patterning in vertebrates. In the cephalochordate amphioxus, genes encoding Bmps and transcription factors downstream of Bmp signaling such as Vent are expressed in patterns reminiscent of those of their vertebrate orthologues. However, the key question is whether the conservation of expression patterns of network constituents implies conservation of functional network interactions, and if so, how an increased functional complexity can evolve. Using heterologous systems, namely by reporter gene assays in mammalian cell lines and by transgenesis in medaka fish, we have compared the gene regulatory network implicated in dorsoventral patterning of the basal chordate amphioxus and vertebrates. We found that Bmp but not canonical Wnt signaling regulates promoters of genes encoding homeodomain proteins AmphiVent1 and AmphiVent2. Furthermore,  Vent and VoxGoosecoid, Chordin, Nodal are expressed in early chordate embryo Goosecoid promoter in Xenopus embryo AmphiVent1 and AmphiChordinAmphiVent1 is expressed throughout the mesendoderm AmphiVent1 and vertebrate Vent genes during early development, their dorsal expression is similar as exemplified by downregulation at the dorsal lip of the blastopore and neural plate Establishment of a dorsoventral (DV) axis is a key event in early development of any bilaterian animal embryo. The crucial step in DV axis formation is specification of the dorsal and ventral mesoderm. In vertebrates, the establishment of the organizer involves activation of several genes Xvent-2 promoter via Smad1 in XenopusXenopus and zebrafish. In the zebrafish embryo Wnt8 directly activates Vent and Vox genes through β-catenin Xenopus Xvent-1 and Xvent-2 genes contain conserved Lef/Tcf binding sites in the promoter. Xwnt-8 protein can activate Xvent-1 promoter and the activation depends on the functional Lef/Tcf binding site Xvent-2 promoter revealed that mutation of the Lef/Tcf binding site decreases expression of the reporter gene Multiple transcriptional inputs are likely required for the correct regulation of Vent genes. Among those Bmp-mediated activation of vertebrate Vent genes is well documented. Bmp2 and Bmp4 activate AmphiVent1 gene regulation, functional properties of Vent proteins, and identification of direct targets of AmphiVent1 transcription factor. Using luciferase reporter assays in P19 murine embryonal cells we have demonstrated Bmp-mediated activation of the AmphiVent1 5′genomic non-coding regions (putative promoter) via Smad1/Smad4 proteins. Similar to vertebrate Xvent-2B gene promoter, AmphiVent1 promoter responsiveness to Bmp signaling is co-stimulated by zinc finger transcription factor OAZ. Furthermore, reporter plasmids where expression of GFP is controlled by Xenopus Xvent-2B and amphioxus AmphiVent1 promoters show a highly similar expression pattern in transgenic medaka embryos. We found that AmphiVent1 protein acts as a transcriptional repressor with the repression domain located at its N-terminus that appears to interact with groucho family co-repressor Grg4. As in the case of its vertebrate orthologues, AmphiVent1 protein can suppress the activity of amphioxus Chordin and Goosecoid gene promoters. Our data thus provide evidence for a remarkable conservation of Bmp-triggered gene regulatory network mediating DV patterning in vertebrates and basal chordates. On the other hand, our data suggest an increased complexity of DV pattern regulation in vertebrates. The canonical Wnt signaling regulatory input for ventral-specific gene expression appears to be lacking in cephalochordates (this study) In this study we have investigated the role of AmphiVent1 homeodomain protein in the molecular events responsible for DV patterning in amphioxus. We have specifically focused on three main areas: the role of Bmp and canonical Wnt signaling in AmphiVent1AmphiVent2http://genome.jgi-psf.org/Brafl1/Brafl1.home.html). Both of them encode the Q50 homeodomain protein with the Vent-specific T47 substitution Xenopus laevis. Higher number of Vent genes in Xenopus laevis may be caused by a recent duplication of its genome as only two Vent genes are found in another frog Xenopus tropicalis (http://www.ensembl.org). Only a single Vent gene is found in the genome of humans and chimpanzee. Interestingly, a functional copy of a Vent gene has been lost from the mouse genome; only a fragment of the Vent-type homeodomain in the mouse genome can be identified . Since AmphiVent2 has not been previously characterized at all we performed an expression analysis using real-time quantitative RT-PCR. As shown in AmphiVent2 displays similar but not identical temporal and quantitative regulation of mRNA expression as compared to AmphiVent1.Two Vent-like genes, XVent-2B gene is sufficient for Bmp-mediated regulation AmphiVent1 (−1230/+20) and AmphiVent2 (−912/+22) genes putatively containing promoters and generated luciferase reporter gene constructs. We next tested their activity in P19 cells in the absence and presence of exogenous Bmp (heterologous human BMP2 was used in this study unless indicated otherwise). The promoter of Xenopus Xvent-2B gene known to be activated by Bmp signaling in the embryo and in P19 cells VENTX2 gene was not inducible by BMP2. However, it is very likely that the 5′genomic non-coding region of VENTX2 used in our study did not contain a complete promoter and so Bmp-responsive elements might have been missing. Applying different doses of BMP2 (from 12 ng/ml up to 400 ng/ml) resulted in rather similar promoter inductions  promoter (5′genomic non-coding region) of ductions . Similarductions . In the  activin  that areAmphiVent1 promoter regulation. We have chosen AmphiVent1 since the corresponding gene has previously been characterized and represents an important marker of ventral mesoderm in amphioxus AmphiVent1 gene promoter is mediated by the members of the Smad group of proteins. Co-transfection of common partner human Smad4 with receptor-activated human Smad1 into P19 cells resulted in activation of the AmphiVent1 promoter  were cloned upstream of the luciferase reporter gene . Only when all six SBE's were mutated, the Bmp responsiveness of AmphiVent1 reporter gene was completely lost (AmphiVent1 is able to function as an autonomous Bmp response element (BRE). Such BRE activity was previously ascribed to a specific region of Xenopus Xvent-2B promoter AmphiVent1 promoter, only the cluster of proximal SBE's can function as an autonomous BRE when fused to a heterologous promoter   protein, which associates with Smad1 in response to BMP2, allowing selective recognition of the BRE in nd Smad4 . The humt 4-fold . Cotranst 4-fold , suggestivation) . These ronstruct . The mosXvent-1B and Xvent-2B genes via binding of the Lef/Tcf/β-catenin complex to their promoters AmphiVent1 and AmphiVent2 genes, we first searched for Lef/Tcf binding motifs within their promoters. We found putative Lef/Tcf elements (5′-CTTTGTT-3′) in both AmphiVent1 (position −596/−590) and AmphiVent2 (position −450/−446) promoters  (VENTX2 (−248/+65) does not contain any Lef/Tcf binding sequence (data not shown). To examine whether promoters of AmphiVent1, AmphiVent2, Xvent-1B and Xvent-2B genes and 5′genomic non-coding region of VENTX2 gene are responsive to canonical Wnt signaling, we cotransfected their reporter genes into 293T cells together with N-terminally truncated β-catenin (β-cateninΔN). It is well established that β-cateninΔN is a constitutively active form of β-catenin , which is able to interact with endogenous LEF/TCF transcription factors, thus mimicking activation of canonical (Wnt/β-catenin) signaling. Mouse Sp5 promoter is known to be responsive to canonical Wnt signaling and was used as a positive control Xvent-1B and Xvent-2B promoters was significantly stimulated by cotransfection of human β-cateninΔN  resulted in a modest repression of the reporter gene suggesting an indirect type of regulation. These results confirm previously published data showing direct regulation of the two Xenopus vent genes by canonical Wnt signaling AmphiVent1 and AmphiVent2 genes are not directly regulated by canonical Wnt signaling. However, our conclusions were based on rather limited 5′genomic non-coding regions that might be sufficient for Bmp-responsivness but not necessarily for responsiveness to canonical Wnt signaling. To provide more definitive answer about the possible role of Wnt/β-catenin signaling in the regulation of AmphiVent1 and AmphiVent2 expression we pharmacologically manipulated Wnt pathway in vivo in the developing amphioxus embryos. To activate the canonical Wnt signaling, we used 6-Bromoindirubin-3′-oxime (BIO), a potent and less toxic inhibitor of glycogen synthase kinase-3β (GSK-3β) as compared to lithium (Li+)+ administration; Axin2 is a direct target of Wnt/β-catenin signaling whose expression is induced by activated Wnt signaling and acts therefore in a negative feedback loop Axin2 is currently the most reliable and frequently used natural readout of Wnt/β-catenin signaling in vivo (http://www.stanford.edu/group/nusselab/cgi-bin/wnt/reporters). As shown in AmphiAxin expression and downregulation of AmphiFoxQ2 expression. In contrast, however, expression of AmphiVent1 and AmphiVent2 has not been significantly upregulated in the presence of activated Wnt/β-catenin signaling.It was shown that Wnt/β-catenin signaling directly regulates romoters . Promotectively) . In contateninΔN . ConversateninΔN . Next weXenopus and zebrafish, amphioxus does not use this pathway for specification of the ventral fate. In summary, our data argue that, in contrast to the situation with Bmp signaling, participation of the Wnt/β-catenin signaling pathway in regulation of Vent genes is not conserved among chordates.Available evidence thus suggests that although canonical Wnt signaling plays a prominent role in the early establishment of ventral mesoderm in AmphiVent1 and AmphiVent2 promoters in silico using Family Relations software  we identified highly conserved sequence motifs that are 80% similar within a sliding 20 bp window (AmphiVent2 gene whereas the promoter of AmphiVent1 contains a single nucleotide change (GcAAC) in the FoxD motif . AmphiFoxD formed a specific complex with the probe, which corresponded to the AmphiVent2 promoter region  probe. Binding site specificity was confirmed by EMSA in the presence of increasing amounts of non-specific (unrelated) double-stranded oligonucleotide or AmphiFoxD binding site derived from AmphiVent2 promoter region. As shown in AmphiVent2, P19 cells were transfected with luciferase reporters containing AmphiVent1 or AmphiVent2 promoters in the presence or absence of an expression plasmid encoding AmphiFoxD. AmphiFoxD can significantly repress AmphiVent2-luc but not AmphiVent1-luc promoter construct , AmphiVent2 (0.9 kb) and Xenopus Xvent-2B (0.3 kb) promoters are activated in medaka embryos. Corresponding EGFP reporter constructs p817-AmphiVent1, p817-AmphiVent2 and p817-Xvent-2B were injected into medaka embryos at the single cell stage and their transient expression was monitored during early embryogenesis. At early gastrula stage AmphiVent1 and AmphiChordin (−1354/+118) promoter regions. Cotransfections of AmphiGoosecoid-luc and AmphiChordin-luc plasmids with an expression vector carrying the AmphiVent1 coding sequence resulted in downregulation of reporter genes 2.8-fold and 6.8-fold, respectively  was generated and designated AmphiGoosecoid(mut)-luc  generated a transcription factor with properties comparable to wild-type AmphiVent1  and AmphiVent1(N51Q), respectively, that contain point mutations in the DNA-binding helix of the homeodomain. Based on previous report AmphiGoosecoid and AmphiChordin reporter genes (AmphiGoosecoid and AmphiChordin genes.One of the most prominent functions of vertebrate Vent transcription factors is repression of organizer-specific genes such as ectively . Paired-ectively . In caseectively . Althougectively . It was mut)-luc . BindingphiVent1 . It is wer genes  despite er genes , inset. Orthologous genes encoding components of Bmp signaling and transcription factors downstream of Bmp signaling (such as Vent and Goosecoid) are expressed in highly similar patterns in vertebrates and a cephalochordate amphioxus in vitro using heterologous cell lines and heterologous proteins or heterologous animal model system (medaka) which may induce a bias in the results and influence our interpretation. In addition, since no data on transgenic amphioxus are available for AmphiVent1 or AmphiVent2 promoter constructs used in this study, we do not know whether we have isolated sufficiently large promoter fragments that can account for the entire expression domain of these two genes.Here, we performed a detailed molecular analysis of gene regulatory network underlying DV patterning in a basal chordate amphioxus. Although we believe that the data presented in this study are relevant for amphioxus it must be emphasized that most of the experiments were performed AmphiVent1 and AmphiVent2) Xenopus, respectively. A single Vent gene (VENTX) is present in the genome of humans and chimpanzee. In amphibians and teleosts mutational analysis of Vent genes suggested their prominent role in DV patterning Vent, Vox, Ved and Xenopus Xvent-1/Xvent1b and Xvent-2/Xvent2b genes are homeodomain-containing repressor proteins, which at early stages of development suppress organizer-specific genes Goosecoid and Chordin Xenopus Goosecoid promoter region contains the Xvent-2 binding site and is directly repressed by Xvent-2 Goosecoid and Chordin gene promoters by the AmphiVent1 protein. However, it appears that in mammals Vent genes have completely abandoned their prominent function within the Bmp-Chordin signaling network VENTX gene that is located on mouse chromosome 7 between Utf1 and Adam8. This region precisely corresponds to a syntenic region where human orthologue VENTX is situated on human chromosome 10. Moreover, the functional role of VENTX is not clear. We have shown that in contrast to AmphiVent1/2 and Xvent1/2 promoters, the promoter of VENTX is not Bmp-inducible. Expression of the VENTX gene has only been detected in immature bone marrow, indicating that the gene may be involved in the maintenance of hematopoietic stem cells in the adult VENTX in the clinical behavior of hematopoietic malignancies Xenopus Vent proteins was confirmed by microinjection of VENTX mRNA that resulted in ventralized zebrafish embryo Xenopus Xvent-2 seems to be an activator, whereas human VENTX protein functions as a suppressor of canonical Wnt signaling. Gao et al. proposed that Xvent-2 interacts with Tcf/Lef directly through its homeodomain and activates gene expression by the N-terminal transactivation domain Genetic and molecular studies have identified a remarkably conserved function of the Bmp-Chordin signaling network in animal DV patterning Xenopus embryo. Thus, Vent genes have likely been recruited as one of the key players in this complicated system of molecular asymmetry.In conclusion, genome analyses, structure-function and developmental studies point to a dynamic evolution of vent genes. The loss of a presumed ancestral role of Vent genes in chordate DV patterning is particularly intriguing. One plausible explanation of such an impressive damnation of Vent genes in mammals lies probably in marked evolutionary changes in early development of chordates. Sanders and co-workers Goosecoid, BAMBI, Tsg display extreme similarity between amphioxus and vertebrate embryos at early stage of development Xenopus and zebrafish) embryo Bmp signaling is mediated by Vent genes Xvent-2 promoter has revealed BRE in its proximal region Xvent-2 contains Smad and OAZ binding sites, which are known mediators of Bmp4 signaling. Indeed, it was shown that OAZ is able to form a complex with Smad1 and Smad4 upon Bmp stimulation Xvent-2 gene promoter leads to promoter activation. Our data support a concept of a highly conserved role of Bmp regulation in the establishment of D/V axis in chordates. Bmp signaling proteins activate amphioxus AmphiVent1 and AmphiVent2 genes. Detailed analysis of the regulation of AmphiVent1 promoter region has revealed that the activation is mediated by Smad1/Smad4 proteins. Furthermore, zinc finger protein OAZ seems to be involved in the activation of AmphiVent1 in a similar way as shown previously for the Xenopus Xvent-2 gene. There is a notable difference in the promoter structure between cephalochordates and amphibian genes with regard to positioning and significance of individual SMAD binding motifs. BRE of the Xenopus Xvent-2 gene is constrained within a short promoter region between nucleotides −243 to −191 Xvent-2 promoter. In contrast, we have identified six putative Smad binding elements (SBE) in the Bmp inducible region of AmphiVent1 gene promoter between nucleotides −1230 to +20. Deletion and mutational analysis has revealed that none of these SBE's plays a dominant role in the activation of the gene by Bmp signaling since point mutations of individual SBE's did not lead to the complete loss of Bmp inducibility. Bmp-mediated activation was abolished only in the case of simultaneous mutation of all six SBE's within the −1230 to +20 promoter region of AmphiVent1. Therefore, the significance of six SBE's within the AmphiVent1 promoter region appears to be equally balanced. Seemingly this fact is not even influenced by the presence of OAZ binding site in the vicinity of one of the SBE's. It is of note that the human VENTX gene promoter contains a putative SBE, which however does not provide any detectable Bmp inducibility AmphiVent1 promoter with regard to Bmp responsiveness through multiplication of SBE's. In contrast, the evolution of vertebrate Vent gene regulation proceeded by a distinct mechanism. The apparent robustness of Bmp-mediated regulation of Xenopus Vent genes was achieved by co-option of Vent binding sites into their promoters. These sites provide a positive autoregulatory loop  controlling chordate DV patterning  of early Xenopus Xvent-2 and Xvent-1 genes as well as zebrafish Vent and VoxXvent-1 and Xvent-2 by canonical Wnt signaling , is not responsive to canonical Wnt signaling. Taking into account an apparent loss of the mouse Vent gene, the available data point to Vent-independent formation of mesoderm in mammals.Loss-of-function experiments indicate an important role of canonical Wnt signaling in the regulation of Vent genes and specification of vertebrate ventral mesoderm Xenopus and zebrafish Vent genes. In addition to stimulation triggered by Wnt8, β-catenin participates in the suppression of vertebrate Vent genes in dorsal lip at early stages of development. The downregulation is achieved by β-catenin-dependent activation of dorsal-lip-specific transcription factors such as goosecoid, which in turn represses Vent gene expression AmphiGoosecoid and AmphiVent1 genes in early amphioxus embryo, which makes the above-mentioned mechanism plausible in amphioxus as well It seems that Wnt/β-catenin signaling plays a dual role in the regulation of Wnt8 and AmphiVent1 overlap in early amphioxus embryo AmphiVent1 and Wnt8 are co-expressed dorsolaterally in regions of invaginated mesendoderm, which constitute the prospective paraxial mesoderm AmphiVent1 and AmphiVent2 gene promoters by in silico analysis, our experimental data in vitro (reporter assays) and in vivo  do not indicate that the promoters are regulated by canonical Wnt signaling, which is congruent with results obtained by Li+ treatment that has not impact on DV patterning in amphioxus-We were interested in whether the Wnt-mediated regulation of Vent genes is an evolutionarily conserved feature between cephalochordates and ‘lower’ vertebrates. It was shown previously that the expression patterns of It is interesting to note that canonical Wnt signaling is broadly used for the establishment of anteroposterior (A/P) axis in diverse deutorostomes  and protostomes Summarized, the available experimental evidence indicates that the canonical Wnt signaling pathway does not play a role in the establishment of ventral fate in cephalochordate amphioxus . This isIn conclusion, our data show that there is a remarkably conserved gene regulatory network in which Bmp signaling induces transcriptional repressor Vent, which in turn represses genes encoding transcription factor Goosecoid and signaling molecule Chordin. We propose that this gene regulatory network was a key module recruited in early chordate evolution for establishment of DV patterning. This network remains in use in the present-day chordates such as amphioxus, fish and amphibians.in vivo experiments were performed after approval by the Animal Care Committee of the Institute of Molecular Genetics (study ID#36/2007) and in compliance with national and institutional guidelines (ID#12135/2010-17210).Housing of animals and Xenopus Xvent-2 (X98849), Xenopus Xvent-1B (AJ131094), Xenopus Xvent-1 (X92851), amphioxus AmphiVent2 , amphioxus AmphiVent1 (AAK58840), Hs six2 (AF332196). Promoter alignments in http://family.caltech.edu) For phylogenetic analysis, we aligned sequences and generated neighbor-joining trees with bootstrap with ClustalX and the GeneDoc program. To construct a Vent tree, we used the following organisms (with accession numbers): Human VENTX2 (AF068006), Pan troglodytes Vent (XP_521666), zebrafish Vox (AF255045), zebrafish Vent (AF255044), Xenopus Xvent-2B (AJ131095), ATCATGAATGAATAACAATGACGTTG; reverse 616B, GTTGTCGCGTGTTCGTCACTGGA; AmphiVent2: forward 822A, TGAAATTTGTTCGCTTACAGTGTA; reverse 822C, GGTCGACGATTGACAGCAGTG; AmphiGoosecoid: forward 918A, CAATGGGCAGGTTGATAATCCACT; reverse 918B, CGTGACTGTTTCCGCTGCTTTGTC; AmphiChordin: forward 839A, CAGACAACGTCAAAAGACAGTCAA; reverse 839B, TTCAGAGAATGTTTGCGTCGTCAA; Xvent-2b: forward 542A, GAGAGGCTTCCCAATAGCTA; reverse 542B, CTGTATTAGTCCTTGTGTTC; VENTX2: forward 818A, CATCGCCTCTCCATTAACCAG; reverse 818C, GCCAAAGCTGGAGAGGCGCAT. PCR products were cloned into pGL3-basic vector and sequenced. AmphiVent1 promoter 5′ deletion constructs were obtained by PCR using corresponding oligonucleotides. Site-directed mutagenesis of promoter constructs was performed using QuikChange kit (Stratagene). Smad binding sites within amphiVent1 promoter were mutagenized converting the wild-type core of SBE sequence AGAC into AcAt. The wild-type P3 homeodomain binding site ATTATTGTAAT in amphiGoosecoid promoter was mutagenized into cTTcTTGTccT. Tcf/Lef binding sites in AmphiVent1 and AmphiVent2 promoters were mutagenized converting the wild-type CTTTGTT into aggTGTT. Tcf/Lef binding sites in Xvent-1B and Xvent-2B promoters were mutagenized converting the wild-type CTTTGAT into aggTGAT. All constructs were verified by sequencing. The 5xGal4E1b and 3xP3HD-luc constructs were described previously The amphioxus amphiVent1, amphiVent2, amphiGoosecoid, amphiChordin, xenopus XVent2b and human VENTX2 promoters were amplified by PCR using a corresponding genomic DNA as a template. The oligonucleotides for PCR were as follows (5′-3′): AmphiVent1: forward 616A, 293T, MDA-MB468 and P19 cells were purchased from ATCC. 293T and MDA-MB468 cells were cultured in Dulbecco's modified Eagle's medium (SIGMA) supplemented with 10% Fetal bovine serum (PAA laboratories), 2 mM L-glutamine, 100 units/ml penicillin, and 0.1 mg/ml streptomycin (SIGMA). P19 cells were cultured in Dulbecco's modified Eagle's medium (SIGMA) supplemented with 5% Fetal bovine serum (PAA laboratories), 2 mM L-glutamine, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. Cells were passaged every three days and maintained at 37°C in an atmosphere of humidified air with 5% CO2. Cells were plated in 24-well plates 24 hours prior to transfection. Each well was transfected with 100 ng of the reporter gene and 50 ng of the expression vector (when applicable) using Fugene 6 (Roche) according to the manufacturer's protocol. The total amount of DNA transfected per well was 300 ng and was adjusted with pUC18 when necessary. A β-Galactosidase expression plasmid was cotransfected to normalize the transfection efficiency. In some experiments, 24 hours after transfection recombinant human BMP2, BMP4, BMP7, TGFβ or Activin B  was added.Unless indicated otherwise, Bmp signaling was stimulated by the addition of human BMP2 at a final concentration of 20 ng/ml. Alternatively, Bmp pathway stimulation was elicited by transfection of 50 ng of the caAlk2 expression vector. Two days after transfection, the cells were lysed in 100 ul of 1× passive lysis buffer (Promega). Luciferase reporter assays were performed using Luciferase Reporter assay kit (Promega). β-Galactosidase was detected with Galacto-Star system (Applied Biosystems). All transfection experiments were performed at least three times and a representative experiment is shown. In each experiment triplicate assays were performed; graph values represent the average of triplicates +/− standard deviation. Statistical significance was determined using Student t-test in Microsoft Excel.CTTAAGTAAACAAACA; FoxD1 BS 2, AGGCCGTAAACAAACA; FoxD1 BS 1(K.O.), CTTAAGTACCCAAACA; FoxD2 BS, CTTAAGTAAACAATGG; FoxC1 BS, CCAAAGTAAATAAACA; FoxC1 BS (K.O.), CCAAAGTAAATTAACA; amphiFoxD BS V1, TCACAGCAAACAATTA; amphiFoxD BS V2, TCAATGTAAACAATAG. The following double-stranded oligonucleotides were used in EMSA with amphiVent1-HD and Pax6-HD : amphiGoosecoid promoter WT, GCATGCTAAATTATTGTAATGAATGCGCA; amphiGoosecoid promoter mut, GCATGCTAAcTTcTTGTccTGAATGCGCA; P1/2, TCGACTGAGTCTAATTGAGCGTCT; P1, TCGACCCTAATGATTACCCTCGA; P2, TCGACCCTAATCGATTACCCTCGA; P3, TCGACCCTAATGCGATTACCCTCGA; P4, TCGACCCTAATGCGCATTACCCTCGA. Double-stranded oligonucleotides containing indicated homeodomain and FoxD binding sites were radioactively labeled at the 5′ends with γ32PdATP using polynucleotide kinase (Boehringer Manheim) and purified on microspin columns (Amersham Biosciences). 32P-labeled oligonucleotides were incubated with bacterially-purified 6xHis-tagged or GST- tagged homeodomain proteins in binding buffer  for 15 minutes at RT. In some experiments increasing amounts of unlabelled double-stranded oligonucleotides were added to binding reaction to test for specificity. Samples were analysed by 6 % polyacrylamide gel electrophoresis and autoradiography.The following double-stranded oligonucleotides were used in EMSA with amphiFoxD : FoxD1 BS 1, 35S-labeled Grg4 was prepared by TNT Quick Coupled Transcription/Translation Systems according to the manufacturer's protocol (Promega). GST fusion expression plasmids were transformed into BL21 CodonPlus (DE3)-RIPL cells (Stratagene). A single colony from the transformation was cultured in 2 ml LB medium containing 50 µg/ml of chloramphenicol and 30 ng/ml of kanamycin overnight at 37°C. The cultures were transferred to 100 ml of LB without antibiotics. The expression of the fusion construct was induced by adding IPTG to a final concentration of 2 mM for 2 hours. The cells were harvested by centrifugation and resuspended in 5 ml of NETN buffer . Lysozyme was added to a final concentration of 0.1 mg/ml. The lysates were incubated on ice for 20 min, sonicated and centrifuged to remove the cell debris. The supernatant was incubated with 200 µl of glutathione-Sepharose beads slurry (BD Bioscience) for 1 hour at 4°C. The beads were washed three times by 5 ml of binding buffer  containing 0.1% NP40. GST fusion proteins bound to the beads were analyzed by SDS-PAGE. Beads containing normalized amounts of fusion proteins were blocked by binding buffer containing 0.05% of NP40 and 5 mg/ml of BSA for 2 hours at 4°C and resuspended in 150 µl of binding buffer containing 0.05% NP40, 1 mg/ml BSA and 100 µg/ml Ethidium Bromide. The beads were incubated overnight at 4°C with 3 µl of 35S-labeled Grg4. The beads were washed three times with 500 µl of binding buffer containing 0.05% NP40 and boiled with SDS sample buffer. The amount of Grg4 was detected by autoradiography.I-SceI meganuclease transgenesis in medaka fish was performed as previously described I-SceI-plasmids  and 0.25U/µl I-SceI meganuclease in 0.5 × I-SceI buffer /0.5 × Yamamoto's embryo rearing medium. Final concentration of injected plasmids was 10 ng/µl of AmphiVent1-GFP and 15 ng/µl of XVent-2B-GFP, respectively. The expression of the transgene was detected as early as at an early gastrula stage (St.13) and onward B. floridae were collected into RNA later (Ambion). Some embryos treated with DMSO (control) or 3 µM Wnt signaling activator 6-Bromoindirubin-3′-oxime  http://frodo.wi.mit.edu/. The housekeeping gene encoding ribosomal protein L32, AmphiRPL32, was used as internal control gene to standardize the quality of different cDNA preparations. Primer sequences were as follows: AmphiRPL32 (117 bp product): forward 1093A, GGCTTCAAGAAATTCCTCGTT; reverse 1093B, GATGAGTTTCCTCTTGCGTGA; AmphiVent1 (204 bp product): forward 1094A, ACGTCTGACGAGGAGGAAGA; reverse 1094B, GTACTTCTGCAGGCGGAAAC; AmphiVent2 (219 bp product): forward 1096A, GACGAGGAGATCGACGTTGT; reverse 1094B, GTACTTCTGCAGGCGGAAAC; AmphiAxin (148 bp product): forward 1098A, TCATGTGCTACCCTCCATTTC; reverse 1098B, TCATCCAGTCGTTCCTCATTC; AmphiFoxQ2 (255 bp product): forward 1103A, TCTACCAGTGGATCATGGACAA; reverse 1103B, CGTACTGCATGTAGGGATGCT.Developing embryos of Figure S1Activation of Amphioxus and Xenopus Vent gene promoters by Bmp2, BMP4 and BMP7. (A) P19 cells were transfected with luciferase reporters containing AmphiVent1, AmphiVent2, Xvent-2B and VENTX2 5′genomic non-coding regions in the absence (open bars) and presence (black bars) of increasing amounts of exogenous human BMP2. Numbers indicate final concentration of BMP2 in the cell culture medium (in ng/ml). (B) P19 cells were transfected with luciferase reporters containing AmphiVent1, AmphiVent2 and Xvent-2B 5′genomic non-coding regions in the absence of BMP ligand (open bars), or in the presence of either BMP4  or BMP7 . (C) P19 cells were transfected with luciferase reporter containing AmphiVent1 5′genomic non-coding region in the absence of ligands (open bar) or presence of human BMP2 (50 ng/ml), human TGF β (20 ng/ml) and human Activin B (10 ng/ml), respectively. **P<0.01, ***P<0.001.(0.38 MB TIF)Click here for additional data file.Figure S2Wnt3A activates Xenopus Xvent-2B but not AmphiVent1 and AmphiVent2 promoters. Indicated luciferase reporter plasmids were transfected into 293T cells in the absence or presence of Wnt3A conditioned medium. Please, note that fold induction of individual reporter genes was normalized to activation of the promoter-less construct pGL3-basic. **P<0.01, ***P<0.001.(0.10 MB TIF)Click here for additional data file.Figure S3Transient expression of EGFP in medaka embryos injected with p817-AmphiVent2. (A-A′) EGFP expression driven by AmphiVent2 promoter at early gastrula stage. (B-C′) EGFP expression patterns in mid-gastrula stage medaka embryos; dorsal (B-B′) and lateral (C-C′) views show EGFP fluorescence in the blastoderm around the most dorsal region of embryonic shield (Sh). Dashed line indicates the borders of the blastoderm. White arrowheads depict the most dorsal embryonic shield of the medaka embryo, where Chordin and Goosecoid are expressed. GR-germ ring, Sh-embryonic shield.(4.01 MB TIF)Click here for additional data file.Figure S4Modulation of TCF/LEF-mediated transcription by Vent proteins. (A) Schematic diagram of TCF/LEF reporter gene pTOPFLASH. (B) TCF/LEF reporter gene pTOPFLASH was cotransfected into 293T cells with CMV-based expression plasmids encoding LEF1, stabilized version of β-catenin (β-cateninΔ) and the indicated Vent protein. Please note that transfection of plasmid encoding AmphiVent1 fusion with strong transcriptional activator VP16 does not lead to detectable activation of pTOPFLASH above vector control. *P<0.05, **P<0.01, ***P<0.001.(0.09 MB TIF)Click here for additional data file.Figure S5Xvent-2B but not AmphiVent1 or AmphiVent2 positively autoregulates its own expression. (A-C) 293T cells were transfected with (A) luciferase reporter containing Xvent-2B promoter in the absence or presence of an expression vector encoding Xvent-2B, (B) luciferase reporters containing AmphiVent1 and AmphiVent2 in the absence or presence of an expression vector encoding AmphiVent1, (C) luciferase reporters containing AmphiVent1 and AmphiVent2 in the absence or presence of an expression vector encoding AmphiVent2. Please note, that only Xvent-2B significantly activates its own promoter. *P<0.05, **P<0.01.(0.15 MB TIF)Click here for additional data file.


Rhabdomyosarcoma (RMS) and Ewing sarcoma (ES) are among the most common pediatric sarcomas (Arndt et al.,  These translocations lead to expression of characteristic fusion proteins that consist of the DNA-binding domain of PAX3 or PAX7 and the transactivation domain of the forkhead transcription factor FOXO1 on chromosome 13. Ewing sarcoma (ES) on the other hand is characterized by a translocation involving the ubiquitously expressed, putative RNA-binding EWSR1 gene and the friend leukemia integration 1 (FLI1) transcription factor, forming the EWS/FLI1 fusion protein as the most common fusion event.Alveolar RMS (ARMS) harbors reciprocal chromosomal translocations between chromosome 2 (or chromosome 1) and chromosome 13, yielding The genetic basis of these diseases is therefore thought to be transcriptional deregulation of normal developmental processes. Hence, improving current treatment approaches will require likely a more detailed understanding of the biology of these oncogenic fusion proteins as well as of the underlying biology of the tumor cells. Importantly, however, available mouse models for ARMS using PAX3/FOXO1 as oncogenic driver as well as (so far unsuccessful) attempts to generate a mouse model for ES based on expression of EWS/FLI1 have unraveled the importance of largely unknown secondary hits which are required for tumorigenesis. Identification of such secondary hits likely requires large-scale next-generation sequencing of which several projects are now getting under way. One critical challenge in this area, however, is the availability of sufficient and high-quality tumor and normal tissue specimen.Since transcription factors (TF) are thought to be undruggable based on their overall structure and the complexity of eukaryotic transcription, novel treatment approaches likely have to be indirect. One such indirect possibility might lie in the epigenetic control of gene expression which seems to play a major role also in sarcomas, as discussed during the conference. Additional areas that are beginning to emerge are the detailed characterization of the role played by embryonal signaling pathways such as Wnt, Notch, and Hedgehog (Hh) in tumorigenesis of sarcomas.in vitro system that allows transformation of primary satellite cells by specific oncogenes such as RAS together with deletion of CDKN2A, thereby creating a defined RMS tumorigenicity model.Different levels of gene regulation by EWS/FLI1 were discussed by H. Kovar  who also investigated the role of the Notch pathway in ES. O. Delattre  described their attempts to generate a mouse model for ES and also described identification of an additional novel translocation, BCOR-CCNB3. The next two talks under this topic looked at specific aspects in rhabdomyosarcoma (RMS), namely the biological role of CB1, a well-known downstream target gene. Interestingly, inhibition of CB1 was found to abrogate lung metastasis in a xenograft mouse model . Finally, S. Hettmer  reported on a promising in vitro and in vivo.S. Lessnik  discussed a novel target gene repressed by EWS/FLI1, namely lysyl oxidase (LOX) that inhibits the transformed phenotype of ES cells. For transcriptional repression, association of EWS/FLI1 with histone deacetylases and the NuRD complex were identified as necessary elements. As a component of the Wnt signaling pathway, DKK2 was reported by G. Richter  to be highly overexpressed in ES and critical for malignant cell outgrowth Apart from histone acetylation, also histone methylation is an important epigenetic mechanism contributing to genetic heterogeneity. E. Lawlor  described studies investigating the role of the polycomb repressor complex in response to EWS/FLI1 expression. Finally, activity of the fusion TF might also be regulated by protein–protein interactions. One interesting co-factor to follow up in the future might be E2F .T. Triche  discussed the prognostic significance of non-coding RNA identified by a human exon array. In ES, ErbB4 might play a role in metastasis , while cul4, an E3-ubiquitin ligase, might have a general role in ES . In RMS, novel pathways that were subject of discussion included the role of pro-protein convertases which are involved in processing of important therapeutic targets such as IGF1R . Several novel target genes of PAX3/FOXO1 were characterized by different groups such as carnitine palmitoyltransferase and pleiotrophin , JARID2 , and p-cadherin . Genome-wide ChIP-seq experiments could also help to identify novel critical pathways. Such experiments were presented by L. Cao  for PAX3/FOXO1. Among the important receptor targets that he identified as direct targets of the fusion protein were FGFR4, c-MET, IGFR1R, and TRAIL receptor 2. Interestingly, about 25% of the identified binding sites seem to lie in the first intron and some even further downstream within genes. Another important new field for sarcoma biology is the role of miRNAs in the tumorigenic process. Some miRNA termed the myomiRs are important for myogenic differentiation and hence RMS biology. These include miR206 and miR1 which were discussed by S. Subramanian  and C. Ponzetto .in vitro culture system of isolated single muscle fibers that contained satellite cells in their natural niche. Ectopic expression of PAX3/FOXO1 was able to induce expression of myogenin but not myoD. However, it remained unclear whether the satellite cells become fully transformed. D. Loeb  then looked at another interesting gene in the context of ES, namely WT1. This transcription factor might play an important role for angiogenesis in this tumor.Despite its relevance for tumor biology, characterization of cancer stem cells in sarcoma is still in its infancy. Attempts to define a subpopulation of ES cells by side-population analysis were discussed by M. Hotfilder . Evasion of apoptosis, one of the hallmarks of human cancers, can also be the cause of treatment resistance. Indeed, sensitivity of sarcoma cells to treatment with TRAIL was reported by two researchers . Along similar lines, blockade of survivin might enhance sensitivity to therapeutic T cells . P. Zammit  described an K-RAS was discussed by D. Langenau . This model allows in vivo imaging of developing tumors. While not all cells in a tumor might have tumor-propagating potential, these cells clearly can enter the vasculature and play a critical role in metastasis, at least in zebrafish.An important tool not only to advance our understanding of tumor biology but also to prioritize drugs for pre-clinical development is the availability of genetically engineered animal models. While no mouse model is available up to now for ES that is driven by the EWS/FLI1 oncogene, C. Keller  described mice expressing PAX3/FOXO1 that will form ARMS in collaboration with p53 deletion. Interestingly, he used different driver genes along the myogenic lineage to initiate expression of the fusion protein and found the most penetrating model to be with myf6. From this data he concluded that the cell of origin for ARMS is a fetal myoblast. In contrast to ARMS, several mouse models are available that give rise to ERMS, the RMS subtype without expression of the fusion protein. H. Hahn  reported on recent findings in the Ptch heterozygous mouse model that leads to activation of the Hh pathway and ERMS. An interesting zebrafish model utilizing activated J. Toretsky  discussed interaction of EWS/FLI1 with RNA helicase A which could be utilized as therapeutic target and the improvements that were made in developing a specific small therapeutic molecule. Another approach was described by B. Schäfer  who investigated post-translational modifications of PAX3/FOXO1. Several phosphorylation sites were identified and a candidate upstream kinase (PLK1) was identified that might regulate PAX3/FOXO1 activity. The same phosphorylation sites were also investigated biochemically in much more detail by A. Hollenbach . Finally, the role of gene amplification in the pathogenesis of RMS was addressed by F. Barr .Several important signaling pathways were highlighted in this session. These included a CRKL/Yes axis , targeting of c-Met by a small-molecule inhibitor  and the influence of the Notch pathway on invasion of RMS cells . The possibility of targeting both tumor and stromal cells by inhibiting the PDGFR pathway was brought up by M. Ehman . A new and exciting area of research is represented by the newly discovered ALK mutations that occur in RMS and which can be targeted by small-molecule inhibitors .The last two sessions of the conference closed the circle to discuss potentially novel treatment options. The first talk in this session was given by P. Houghton  who summarized the efforts made by the Pediatric Pre-clinical Testing Program (PPTP) to identify important novel drugs for sarcoma treatment. Interestingly, response rates for known drugs in the xenograft models are around 30%, while it was only 9% for experimental drugs. For sarcomas, one important class of drugs might be topoisomerase I inhibitors. Furthermore, inhibition of the IGF1R resulted in suppression of sarcoma growth. Since the PPTP program works with animal models, it will be crucial to develop strategies for clinical translation. J. Chisholm  described the structure used in Europe to achieve this goal, and which is called ITCC (innovative treatments for children with cancer). Several trials are now implemented under this umbrella. One of the targets that are currently being developed at the Royal Marsden was discussed in the final talk by S. Gatz , namely FGFR, which was found mutated in some ARMS and overexpressed in most of them.Moving further to the clinics, U. Dirksen  summarized the current state of the art on diagnosis, treatment and prognostic factors for ES in Europe. E. Koscielniak  gave an overview on the role of molecular signatures in soft tissue sarcoma and their impact on therapy stratification of RMS. S. Stegmaier  discussed the prognostic relevance of fusion transcript markers in soft tissue sarcoma. Some discrepancy in the field concerns the clinical outcome of different PAX/FOXO1 fusions, as different outcomes depending on distinct PAX/FOXO1 fusion products have been found in the US, but not in the European CWS trials. Finally, a novel fusion transcription factor, EWSR1/NFAT2c was described in ES .This conference was the first specialized international meeting that brought together leading biologists and clinicians involved in early clinical trials to discuss recent advances in translational cancer research on two groups of sarcomas, namely RMS and ES, in the pediatric and young adult population. Since fusion TF are often the only genetic abnormality present at high frequency in these tumors, they are hypothesized to be one of the tumor-initiating events and might represent important therapeutic targets. Therefore, a large effort is still directed at identification of specific, either activated or repressed target genes. Several speakers discussed novel techniques such as next-generation sequencing and whole genome ChIP-seq to identify such target genes including mechanisms of transcriptional regulation. Several new targets genes that have now been individually characterized were highlighted as novel therapeutic targets at the conference. Another important area in this field is the development of genetic animal models to further advance our understanding of the biology of the diseases and to pre-clinically evaluate novel targeted compounds. A specific session addressed the progress in this field spanning from mouse to zebrafish models. Especially for ES, the lack of an appropriate mouse model still represents a major bottleneck.In two forward-looking sessions, future research directions were discussed. Among them were methods to unravel the still unknown cell of origin for these sarcomas. Novel insights into this aspect might be provided by future collaborations with developmental biologists. Additional identified areas included the search for novel genetic alterations, the role of the micro- and macroenvironment, the significance of tumor heterogeneity and pharmacogenomics. Regarding novel therapeutic targets, more emphasis might be put on understanding the biology of the prevalent genetic aberrations found in these tumors, namely the fusion TF themselves. However, important downstream target genes include some well-known oncogenes against which direct inhibitors are available and therefore development of therapies using these targets might be more straightforward. Hopefully, a next conference planned 2 years from now will further prioritize the most promising therapeutic avenues.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pax6 and Irx3 undergoes both decompaction and displacement towards the nuclear centre coincident with transcriptional onset. Conversely, down-regulation of Fgf8 as neural differentiation commences correlates with a more peripheral nuclear position of this locus. During normal neural differentiation, fibroblast growth factor (FGF) signalling is repressed by retinoic acid, and this vitamin A derivative is further required for transcription of neural genes. We show here that exposure to retinoic acid or inhibition of FGF signalling promotes precocious decompaction and central nuclear positioning of differentiation gene loci. Using the Raldh2 mutant as a model for retinoid deficiency, we further find that such changes in higher order chromatin organisation are dependent on retinoid signalling. In this retinoid deficient condition, FGF signalling persists ectopically in the elongating body, and importantly, we find that inhibiting FGF receptor (FGFR) signalling in Raldh2−/− embryos does not rescue differentiation gene transcription, but does elicit both chromatin decompaction and nuclear position change. These findings demonstrate that regulation of higher order chromatin organisation during differentiation in the embryo can be uncoupled from the machinery that promotes transcription and, for the first time, identify FGF as an extrinsic signal that can direct chromatin compaction and nuclear organisation of gene loci.Changes in higher order chromatin organisation have been linked to transcriptional regulation; however, little is known about how such organisation alters during embryonic development or how it is regulated by extrinsic signals. Here we analyse changes in chromatin organisation as neural differentiation progresses, exploiting the clear spatial separation of the temporal events of differentiation along the elongating body axis of the mouse embryo. Combining fluorescence in situ hybridisation with super-resolution structured illumination microscopy, we show that chromatin around key differentiation gene loci  Changes in the position of genes within the nucleus and in their local organisation frequently correlate with whether or not genes are turned on. However, little is known about how such nuclear organisation is controlled and whether this can be separated from the mechanisms that promote transcription. We show here that central nuclear position and chromatin de-compaction correlate with onset of expression at key neural differentiation gene loci in the mouse embryo. Conversely, the locus of a gene that is down-regulated as neural differentiation commences exhibits a shift towards the nuclear periphery as this takes place. Importantly, we show that signalling through the fibroblast growth factor (FGF) pathway regulates changes at this level of nuclear organisation. FGF represses differentiation gene transcription and keeps differentiation gene loci compact and at the nuclear periphery. By blocking FGF signalling in a retinoid deficient embryo in which differentiation genes are not expressed, we further show that control of nuclear organisation by FGF is not just a consequence of gene transcription. These findings are the first to demonstrate that such higher order nuclear organisation is regulated in the developing embryo, that this takes place downstream of FGF signaling, and can be uncoupled from the machinery of gene transcription. A further critical level of regulation is provided by so-called higher order chromatin organisation, which includes changes in local chromatin compaction and nuclear position of gene loci. Such changes have been documented in Sox1, Sox3 and Pax6Raldh2 which encodes retinaldehyde dehydrogenase 2 - an RA synthesising enzyme - in the presomitic mesoderm and RA receptor beta (RARb) in the forming neuroepithelium as well as promoting expression of Cyp26a1, encoding a RA catabolising enzyme reviewed in During vertebrate embryonic development, induction of the future brain is followed by the progressive generation of neural tissue as the body axis elongates and this provides a unique opportunity to investigate steps leading to the onset of neural differentiation. New neural tissue arises from the stem zone/caudal lateral epiblast (adjacent to the primitive streak), which includes resident axial stem cells Fgf8 morphants, and Ph2α acts downstream of FGF signalling, which is necessary, although not sufficient for Ph2α expression Fgf8 or for PcG genes  also share a common gastrulation failure phenotype, with some reported proliferation defects The underlying molecular mechanisms through which this opposing signalling switch controls differentiation onset in the embryonic body axis are not well understood, but might involve changes in gene expression determined by altered chromatin structure around target gene loci. One way in which chromatin compaction is locally regulated is by the action of polycomb repression complexes (PRC); and there is some evidence implicating FGF signalling in the regulation of polycomb complex component genes. Polycomb group (PcG) proteins are key regulators of cell growth and differentiation genes 3′ Hoxb1 transgene to the 5′ end of the Hoxd cluster elicited such chromatin changes in a cellular context in which Hoxb1 is not transcribed Retinoic acid can signal directly to chromatin via liganded retinoic acid receptor – retinoid X receptor (RAR-RXRs) heterodimers and their sequence specific binding to retinoic acid response elements (RAREs) and this is known to attenuate binding of PRC2 components and to decrease H3K27me3 enrichment at these sites A further important manifestation of higher order chromatin organisation that frequently correlates with transcription is the position of a locus with respect to the nuclear periphery, which can be a repressive environment. Although recent studies have shown that artificial tethering to the nuclear periphery need not necessarily lead to gene silencing Pax6 and Irx3 as differentiation takes place and compared this with the Fgf8 locus, which is transcriptionally downregulated as cells leave the stem zone (Pax6) we analysed chromatin organisation around loci in the Raldh2−/− mutant embryo, which is unable to synthesize RA in the elongating embryonic body axis Raldh2 mutants we dissected the consequences of FGF loss in a context in which many differentiation genes fail to be transcribed. Our data demonstrate, for the first time, that FGF signalling acts upstream of mechanisms that direct higher-order chromatin organisation around differentiation gene loci and further reveal that such mechanisms can be uncoupled from the machinery that mediates transcription of such genes.To assess changes in higher-order chromatin organisation during the progressive generation of neural tissue in the elongating body axis of the mouse embryo, we used fluorescence in situ hybridisation (FISH) combined with super-resolution structured illumination microscopy (SIM). We analysed changes in higher-order chromatin organisation at the loci of exemplar neural progenitor genes tem zone . As retiPax6 transcription in the E8.5 mouse embryo involves a change in chromatin compaction, fosmid probes separated by 65 kb and specific for sequences flanking Pax6 (Pax6 transcription) and in the neural tube, where Pax6 is now transcribed  (Hba-a1), which is not transcribed in the embryo at this stage 2d values for each data set, as this is the value that scales linearly with genomic separation 2d values for stem zone and preneural tube (p>0.05), but a clear increase in inter-probe distances across Pax6 in neural tube in comparison with measurements in either stem zone or preneural tube nuclei  were not significantly different between stem zone, neural tube and somite data sets  and those that will later come to express it , and that it specifically decompacts coincident with onset of Pax6 transcription in the neural tube. Analysis of genome-wide histone modification data sets in mouse ES cells and derived neural progenitors Pax6 locus is subject to H3K27me3 enrichment in ES cells and is relieved of this mark upon neural differentiation, suggesting that this locus is a target of polycomb mediated repression (To determine if the onset of ing Pax6  were useing Pax6 . Images sitions) . Fosmids p<0.05, , S3. Addral tube . In cont p>0.05, . This copression .Fgf8, which is expressed in the stem zone and downregulated as neural differentiation commences. Fgf8 is marked by H3K27me3 and H3K4me3 in mES cells and upon neural differentiation H3K4me3 is lost and H3K27me3 retained Fgf8 is a smaller gene than Pax6 and FISH signals from fosmids flanking this locus (100 kb separation) were barely resolved in any tissue assessed  Pax6 locus was carried out on E8.5 Raldh2−/− embryos. There was no difference in chromatin compaction (d2) between stem zone and neural tube  Pax6. The repression of Spry2  in stem zone and somites than in neural tube, this difference was abolished in PD173074-treated embryos. In these conditions the chromatin across Pax6 appears to decompact in stem zone and the somites to the level normally seen in the neural tube . Together these data indicate that FGF signalling acts upstream of mechanisms that regulate chromatin compaction and nuclear position at Pax6.Analysis of chromatin compaction across the  by FISH  revealed by FISH  and cont p<0.05; . This inites see . BlockinIrx3. Like Pax6, onset of Irx3 transcription takes place in the neural tube of the elongating body and is initially broadly expressed across the dorso-ventral axis Irx3 expression domain (n = 0/5 DMSO treated and n = 4/6 PD173074 treated embryos)  . Also lills Irx3  FISH analls Irx3 . Further somites , S7B. ThPax6 expression in the absence of retinoid signalling, Raldh2 mutant embryos, and their wild type littermates produced from heterozygous Raldh2+/− crosses, were cultured with PD173074 or DMSO. Strikingly, blocking FGFR signalling did not rescue Pax6 expression in the neural tube of Raldh2−/− embryos  that directs nuclear position of the Fgf8 locus.Although we could not use FISH to measure chromatin compaction at the Fgf8 locus is influenced by FGF signalling itself, as transcription of Fgf genes can be maintained by positive auto-regulatory feedback loops e.g. Raldh2−/− mutants (Fgf8 locus in the stem zone (where this gene is expressed) in comparison with wildtype and Raldh2−/− DMSO controls. Strikingly, in the Raldh2 −/− mutant neural tube blocking FGFR signalling also rescued the failure to shift to the nuclear periphery observed in untreated mutants (Fgf8 transcripts are still detected in PD173074 exposed embryos (Fgf8 mRNA Fgf8 transcripts were detected in the stem zone of PD173074 treated embryos by whole mount in situ hybridisation (Fgf8 transcription is lost). Overall, these findings demonstrate that FGFR signalling regulates nuclear position of the Fgf8 locus and that it is responsible for the persistent central location of this gene in the retinoid deficient neural tube.It is further possible that the location of the  mutants . In both mutants , S5. Fgf embryos  and thisdisation , indicatFgf8 locus. These data demonstrate that FGF can constrain differentiation via multiple mechanisms that control higher-order chromatin organisation.This study reveals changes in higher-order chromatin organisation during neural differentiation in the mouse embryo and demonstrates, for the first time, that this level of organisation is regulated by key signalling pathways that direct differentiation , has associated H3K27me3 in ES cells  in which chromatin decompaction (observed around Pax6 and Irx3) took place without a concomitant shift towards the nuclear centre. This suggests that de-compaction may be contingent upon a more central nuclear position.Importantly, although nuclear position of the Pax6 nor Irx3 loci, nor the whole Fgf8 region is a LAD in either ES cells or ES-derived neural progenitors  generally have low levels of transcription. Genome-wide maps of Lamin B1 association in mouse ES cells show that many neural differentiation genes are located in LADs genitors  suggestiRaldh2 mutant, where many differentiation genes fail to be transcribed, restores chromatin decompaction and a more central nuclear position of the Pax6 locus suggests that RA acts first to inhibit FGFR signalling and that FGF is upstream of molecular mechanism(s) that direct higher order chromatin organisation at differentiation gene loci. This result is further supported in wildtype embryos in which FGFR signalling is blocked, as here Pax6 transcription extends caudally, being precociously expressed in the preneural tube, but we detect Pax6 decompaction and its more central nuclear position in stem zone cells, which have yet to express Pax6. Importantly, these experiments uncouple regulation of higher order chromatin organisation from gene expression itself and indicate that these large-scale chromatin changes take place as an initial step in the differentiation process. Although we assess these changes by detailed investigation around two exemplar neural differentiation genes, FGF signalling in this context represses expression of many such genes, including Sox1 and Sox3  and prevents the onset of ventral patterning and neuron production in the newly generated spinal cord Our discovery that blocking FGFR signalling in the Pax6 and Irx3 loci in somites, where this gene is never expressed. These somites will have formed (1 somite every 2 hours) during the 7 h period of exposure to FGFR inhibitor, at the start of which these cells would have been experiencing FGF signalling in the presomitic mesoderm. The reorganisation of Pax6 and Irx3 loci in this context may thus reflect the finding that high level FGF signalling is required for mesoderm induction, while reduction elicits neural differentiation, as observed in Fgfr1 mutant mice, reviewed in Intriguingly, blocking FGFR signalling also led to decompaction and a more central nuclear position of Hba-a1 locus and inter-probe distance for the region of the Fgf8 locus remain unchanged in all tissues examined. The Fgf8 locus does, however, alter its nuclear position in response to changes in FGFR signalling. When FGFR signalling is blocked in either wildtype or Raldh2 mutant embryos the Fgf8 locus remains close to the nuclear periphery in all tissues examined, including the stem zone where this gene is normally expressed and in the Raldh2 −/− mutant, where this inhibition of FGF signalling rescues the ectopic centralised location of Fgf8 locus. Although location at the nuclear periphery generally correlates with gene repression, we do detect some intronic Fgf8 transcripts in PD173074 treated embryos, indicating that in the timeframe of this experiment the peripheralisation of the Fgf8 locus does not simply correlate with loss of transcription. This may reflect an initial heterogeneous response to the loss of FGF signalling across the stem zone cell population, however, active transcription and peripheral locus position it is not incompatible with transcription Pax6 and Irx3 compaction and peripheral location, and that promote a central position of Fgf8 within the nucleus. This shows that in this context FGF signalling influences multiple distinct molecular mechanisms, which regulate chromatin compaction and promote movement towards or away from the nuclear centre in a locus specific manner.Importantly, we show that blocking FGFR signalling does not lead to global chromatin reorganisation, as inter-probe distances and the fractional radius for control Pax6Attenuation of FGF signalling in human embryonic stem (hES) cells and mouse epiblast stem cells leads to loss of self-renewal in situ hybridization (ISH) or for FISH as described below. Heterozygous Raldh2 mutant CD1 mice Raldh2−/−, Raldh2+/− and wildtype embryos. These were either dissected, genotyped as described previously 2, 20% O2. These were then dissected, genotyped, fixed and processed for FISH. For treatment with retinoic acid wild type CD1 E8-8.5 embryos were dissected to give explants pairs of the caudal embryo  washed and stored in dH20 prior to pre-hybridisation steps and denaturation as previously described ot1 DNA (Invitrogen) and 5 µg sonicated salmon sperm DNA (sssDNA) were used per coverslip, denatured and hybridised to coverslips Mouse embryos stored in 100% MeOH were cleared in xylene, embedded in wax, sectioned at 7 microns and dried down on thin TESPA-coated 50×22 #1.5 coverslips (Scientific Laboratory Supplies Ltd) suitable for OMX microscopy. The protocol for FISH on mouse tissue sections was then adapted from x and y, using predetermined shifts which were measured using a target lens and 100-nm Tetraspeck fluorescent beads (Invitrogen) in the Softworx alignment tool (Applied Precision).Samples were imaged on a Deltavision 3D OMX Structured Illumination Microscope (Applied Precision) using a protocol adapted after For analysis of chromatin compaction and nuclear position, measurements were made in images of >50 nuclei per region in each of 3 different embryos per condition.Stem zone was defined as epiblast cells adjacent and just caudal to the node (∼5 sections per embryo), preneural tube as neuroepithelium rostral to the node underlain by notochord and presomitic mesoderm, neural tube as neuroepithelium flanked by 2 or 3 most recently formed somites, and these adjacent somites were also used to represent somitic tissue. As nuclei in tissues are not as spherical as in cultured cells it was not possible to apply standard nuclear segmentation tools to define nuclear position. Instead sections in which a fosmid signal and nuclear edge were in sharp focus were used to measure the shortest distance from the probe centre to the periphery. The broadest distance across the nucleus was also measured as an indication of nuclear diameter and this was halved and data presented as a proportion the nuclear radius . Super resolution images were uploaded into an OMERO server (Open Microscopy Environment) and ROIs containing hybridisation signals for both dig and biotin-labelled probes were identified by manual inspection in OMERO-insight. ROIs typically extended over several z-sections to accommodate the whole volume of the signals. These ROIs were analysed by a custom script developed in MATLAB . This script first segments the objects defined by each probe from the background using Otsu thresholding and then calculates the xyz coordinates the centroid in each object. The centroids of these two objects and the distance between them, d (µm), were then output to a spread-sheet. The inter-probe distance was then squared because in interphase nuclei the mean physical distance squared between two points is linearly related to the known genomic distance rd and 1st inter-quartiles and the middle line represents the median. Non-parametric Mann-Whitney U test was used for analyses as data were not normally distributed. For comparison between explant pairs, a paired-sample Wilcoxon signed-rank test was used , Irx3 (B), Fgf8 (C) and Hba-a1 (D) in mouse embryonic stem (ES) cells and ES cell derived neural progenitors (NP) Click here for additional data file.Figure S2Fgf8 locus. (A) Fosmids flanking the Fgf8 locus mapped to the mm9 assembly of the mouse genome; (B) Examples of FISH images in DAPI-stained nuclei for the Fgf8 -flanking probe pairs in stem zone, neural tube, and somite; (C) Box-plot of inter-probe distances (µm2) for Fgf8 flanking probes in each tissue, indicating no difference in distances despite transcriptional down regulation of Fgf8 in the neural tube and somites; (D) Boxplot showing that blocking FGFR signalling also did not alter inter-probe distances between fosmids flanking the Fgf8 locus (images in Assessment of chromatin compaction at the (TIF)Click here for additional data file.Figure S3Fgf8 and its neighbouring genes NPM3 and Mgea5/OGA. Localisation of mRNAs for Fgf8, NPM3 and Mgea5/OGA in the caudal regions of the mouse embryos at  E8-8.25 and at E8.5 . Asterisk indicates position of the node. All three genes are expressed in the stem zone at E8-8.25, at E8.5 NPM3 and Mgea5/OGA transcripts are also detected more rostrally, but are still downregulated in the neural tube. (D) Table of primers used for NPM3, Mgea5 and Irx3 (Expression patterns of and Irx3  in situ (TIF)Click here for additional data file.Figure S4Spry2, and does not alter chromatin compaction or nuclear position across the control Hba-a1 locus. (A) Expression of Spry2 in embryos following exposure to vehicle control DMSO or the FGFR antagonist PD173074. (B) Box-plot of inter-probe distances (µm2) for Hba-a1 flanking probes in each tissue assessed in DMSO and PD173074 treated embryos, showing that FGFR signalling has no effect on chromatin compaction around the Hba-a1 locus in the stem zone (p = 0.55) or neural tube (p = 0.08). (C) Graph of data distribution for fractional radius measurements in DMSO and PD173074 treated tissues, showing that the Hba-a1 locus does not change nuclear position in the stem zone (p = 0.23) or neural tube (p = 0.83) after treatment with DMSO or FGFRI.Exposure to FGFR inhibitor PD173074 represses FGFR target (TIF)Click here for additional data file.Figure S5Fgf8 locus compaction and nuclear position in wildtype and in conditions lacking retinoid, FGFR, and both RA and FGFR signalling. Exemplar images of fosmid pairs across the Fgf8 locus in nuclei from each tissue and condition assessed. Compaction of the genomic region around the Fgf8 locus does not alter with Fgf8 transcriptional activity, including in conditions in which retinoid or FGFR signalling or both are attenuated. However, nuclear position of the Fgf8 locus is regulated by retinoid and FGF signalling Click here for additional data file.Figure S6Fgf8 mRNA following inhibition of FGFR signalling. Intronic Fgf8 mRNA was detected in the stem zone of (A) wildtype (n = 4/4) and (B) PD173074 treated embryos (n = 3/4). Scale bar = 100 microns.Detection of intronic (TIF)Click here for additional data file.Figure S7Pax6 and Irx3 loci relocate to the centre of the nucleus in somite tissue treated with FGFR inhibitor. (A) Graph of data distribution for Pax6 fractional radius measurements for somites in wild-type, Raldh2−/−, DMSO and PD173074 treated wild-type and PD173074 treated mutant tissues. These data show that FGF signalling is required for the proper localisation of the Pax6 locus close to the nuclear periphery in somites. (B) Graph of data distribution for fractional radius measurements in DMSO and PD173074 treated tissues, showing that the Irx3 locus displays a more central localisation within the nucleus when FGF signalling is blocked (p<0.05).(TIF)Click here for additional data file.Figure S8Pax6, Irx3, Fgf8 and control Hba-a1 loci. Genomic regions around (A) Pax6, (B) Irx3 (C) Fgf8 and (D) Hba-a1 are not associated with Lamin B1 binding in ES cells or neural progenitors Click here for additional data file.Figure S9Fgf8, Irx3 and Pax6 flanking fosmids was carried out using a standard PCR protocol (B) to amplify regions at the 5′ and 3′ends of fosmids obtained from the WIBR-1 Mouse Fosmid Library (Whitehead Institute/MIT Center for Genomic Research) ; Primer sequences and PCR product sizes are shown in A.PCR verification of fosmid sequences. Verification of (TIF)Click here for additional data file.Table S1http://jun2007.archive.ensembl.org/Mus_musculus/index.html). Fosmids highlighted with an asterisk were previously used in Names and co-ordinates and sizes of fosmids used. All fosmid names are from Ensembl (r 45) (DOC)Click here for additional data file.Table S2Pax6, Irx3, Hba-a1 and Fgf8 loci in the stem zone, pre-neural tube (except Hba-a1), neural tube and somites. P-values are from Mann-Whitney analysis.Squared inter-probe distances of fosmids surrounding the (DOC)Click here for additional data file.Table S3Pax6, Irx3, Fgf8 and Hba-a1 chromatin compaction.Statistical significance between stem zone and neural tube in three embryos analysed for (DOC)Click here for additional data file.Table S4Pax6 chromatin compaction and nuclear localisation.P-values from the paired-sample Wilcoxon signed-rank test showing statistical differences between individual embryo explant pairs analysed for (DOC)Click here for additional data file.


Enhancer of split gene complex [E(spl)-C]. The E(spl)-C contains 12 genes that inhibit neural development. In a cell line derived from the central nervous system, cohesin and the PRC1 PcG protein complex bind and repress E (spl)-C transcription, but the repression mechanisms are unknown. The genes in the E(spl)-C are directly activated by the Notch receptor. Here we show that depletion of cohesin or PRC1 increases binding of the Notch intracellular fragment to genes in the E(spl)-C, correlating with increased transcription. The increased transcription likely reflects both direct effects of cohesin and PRC1 on RNA polymerase activity at the E(spl)-C, and increased expression of Notch ligands. By chromosome conformation capture we find that the E(spl)-C is organized into a self-interactive architectural domain that is co-extensive with the region that binds cohesin and PcG complexes. The self-interactive architecture is formed independently of cohesin or PcG proteins. We posit that the E(spl)-C architecture dictates where cohesin and PcG complexes bind and act when they are recruited by as yet unidentified factors, thereby controlling the E(spl)-C as a coordinated domain.The cohesin protein complex functionally interacts with Polycomb group (PcG) silencing proteins to control expression of several key developmental genes, such as the Drosophila  The cohesin protein complex, named for its role in sister chromatid cohesion and chromosome segregation, plays multiple dosage-sensitive roles in gene transcription . Mild dii.e., NIPBL) cohesin loading factor bind to virtually all extragenic enhancers and many active promoters, and decreases in cohesin dosage reduce enhancer-promoter looping interactions  just downstream of the transcription start site (Cohesin directly facilitates binding of the PRC1 Polycomb group (PcG) complex to active genes, where PRC1 inhibits premature entry of underphosphorylated Pol II into elongation (invected and engrailed gene complex has the cohesin-H3K27me3 state in ML-DmBG3 (BG3) cells derived from Drosophila central nervous system but not in Sg4 cells or wing discs. In Sg4 cells and anterior wing disc, the invected-engrailed gene complex has a PcG-silenced state with H3K27me3 and PRC1 but no cohesin, and in anterior wing disc, it is transcriptionally active and binds cohesin and PRC1 without H3K27me3 -C] in BG3 cells. This complex contains twelve short genes, many of which encode helix-loop-helix (HLH) DNA binding proteins that repress neural fate, and which are transcriptionally activated by the Notch receptor. In BG3 cells, the entire 50-kb complex binds cohesin and has the H3K27me3 histone modification made by the PRC2 complex -chip and ChIP-quantitative polymerase chain reaction (qPCR) were performed as previously described -qPCR as previously described -C in BG3 cells .RNAi-mediated depletion of cohesin, kollerin, or PRC1 subunits increases RNA produced by the active E(spl)-C genes by 5- to 100-fold in BG3 cells, depending on the gene and the extent of depletion . RNA turSerrate Notch ligand gene expression that occur upon cohesin or PRC1 depletion -C genes before cohesin or PRC1 depletion, indicating that the genes are poised for increased transcription . The incHLHmβ and HLHm3 genes, which show the highest levels of paused Pol II by PRO-seq, before and after cohesin and PRC1 depletion. NICD binding increased from undetectable to 3- and 1.5-fold enrichment at the HLHmβ and HLHm3 Su(H) binding sites in the E(spl)-C when BG3 cells were depleted for the Rad21 cohesin subunit, and similar increases occurred when the Ph subunit of PRC1 was depleted  binding was detected at these sites in untreated cells, and the levels were not significantly altered by cohesin, kollerin, or PRC1 depletion (Activation of Notch releases the Notch intracellular fragment (NICD) that translocates to the nucleus and interacts with the Suppressor of Hairless [Su(H)] protein bound upstream of each E(spl)-C gene. We used ChIP-qPCR to measure the levels of Su(H) and NICD at known Su(H) binding sites upstream of the depleted . Signifiepletion .HLHmβ and HLHm3, in addition to higher levels of paused Pol II, show regions of lower H3K27me3 and H2Aub histone marks than other E(spl)-C genes . Close examination reveals significant H2Aub levels over the bodies of both of these genes, and H3K27me3 and Pc over HLHm3 . By ChIP-qPCR, these genes show a 7- to 10-fold enrichment of H3K27me3 at their promoters . These observations, and the fact that cohesin and PRC1 depletion alters expression of HLHmβ and HLHm3 in the same manner as it alters expression of the other E(spl)-C genes, argues that they are under the same type of regulation by cohesin and PRC1. The lowest levels of PcG histone marks in both cases are between the promoter-paused Pol II and the upstream Su(H)-binding sites, suggesting that greater Pol II and Su(H) occupancy may create a region of reduced nucleosome density .We examined NICD occupancy of Su(H) binding sites upstream of other E(spl)-C genes, but it was not detectable even after cohesin or Ph depletion. The difficulty in detecting NICD at the E(spl)-C genes arises from several factors. NICD is usually present at low levels, does not directly bind DNA, and NICD bound to genes is subject to ubiquitination and degradation . The genAlthough we did not detect NICD at any of the E(spl)-C genes examined in control cells, the basal levels of E(spl)-C transcription are Notch-dependent. Overnight treatment of BG3 cells with DAPT, a γ secretase inhibitor that blocks Notch receptor processing, reduces E(spl)-C transcripts . The incHLHmβ and HLHm3 increased to 10-fold and 6-fold enrichment -C. For example, cohesin and PRC1 could form a structure that restricts access of NICD to the entire gene complex, or cohesin and PRC1 bound to the E(spl)-C might also facilitate NICD degradation.The aforementioned experiments argue that when Notch activation is low, depletion of cohesin or PRC1 increases E(spl)-C transcription by increasing the amount of NICD bound to Su(H) upstream of the individual E(spl)-C genes. This could reflect increased Figure S1B).We considered the possibility that depletion of cohesin or PRC1 might have similar direct effects on E(spl)-C transcription if their binding is codependent but found that this is not the case. Cohesin interacts directly with PRC1 . There iFigure S2). The transcript increases upon Rad21 and Pc codepletion were either similar to the increases seen by depletion of one or the other, or slightly greater. This argues that cohesin and PRC1 together target the same step in E(spl)-C transcription, so that loss of one is equivalent to functional loss of both.Simultaneous depletion of Rad21 and Pc did not give a synergistic increase in E(spl)-C transcripts, even with submaximal depletions of both -C. Two anchors  are outside of the complex on either side, two  are at the inside ends, and one (c) is in the center of the complex (We used chromosome conformation capture (3C) analysis ( complex . The int complex . In contTo test whether cohesin or PRC1 proteins facilitate the extensive looping interactions within the E(spl)-C, we performed 3C analysis after depletion of Rad21 or Pc. Although these depletions greatly increased transcription, there were only minor quantitative changes in the interaction of anchor d with immediately flanking regions  bind cohesin and Pol II in Sg4 cells and show low levels of expression that is not altered by cohesin depletion -C in Sg4 cells, in which there is no H3K27me3 in the E(spl)-C .An early origin of DNA replication is centered over the E(spl)-C in BG3 and other cell lines (We considered the idea that the E(spl)-C 3C structure may be formed by insulator proteins, which show less variation in binding between different cell types. However, inspection of genomic insulator ChIP data (The Chromator (Chro) chromodomain protein binds at the telomere-proximal (right) end of the E(spl)-C in BG3 cells, and several kilobases upstream of the centromere-proximal end . We thusHLHm3 gene in the middle of the complex, even though the Chro-Pzg/Z4 binding sites are located outside the complex. The location of the Chro binding sites and the changes in E(spl)-C expression upon Chro or Pzg/Z4 depletion suggested that Chro and Pzg/Z4 might affect the E(spl)-C architecture, but depletion of Chro or Pzg/Z4 did not measurably alter the interactions measured by 3C .Depletion of Chro or Pzg/Z4 in BG3 cells increased expression of multiple genes in the E(spl)-C several-fold . This inFigure S5, A and B). We were unable to detect Chro protein by Western blot, and Chro RNAi treatment slightly increased Chro RNA transcripts . Intriguingly, however, Chro dsRNA decreased Pzg/Z4 RNA to a similar extent as Pzg/Z4 RNAi treatment , suggesting that Chro RNAi likely decreases the levels of both proteins.Pzg/Z4 RNAi treatment decreased Pzg/Z4 protein and RNA in a dosage and time-dependent manner -C expression . Although Rad21 RNA transcripts increased, the Rad21 protein levels were not appreciably altered . We found, however, that the levels of the Delta and Serrate Notch ligand gene RNAs increased 2- to fourfold upon Chro or Pzg/Z4 RNAi treatment, suggesting that increased Notch receptor activation may contribute to increased E(spl)-C expression . Chro binds to the Delta gene promoter region, but not to Serrate in BG3 cells -C expression indirectly by altering cohesin or Notch activity. Chro and Pzg/Z4 depletion increased vice versa -C by performing Rad21 ChIP after Pzg/Z4 depletion. We found, however, that Pzg/Z4 depletion did not alter cohesin levels at the HLHmβ and HLHm3 promoter regions . We posit, therefore, that the effect of Chro and Pzg/Z4 on E(spl)-C expression is most likely caused by increased expression of Notch ligands.We also considered the possibility the Chro-Pzg/Z4 complex might control E(spl)-C transcription by influencing cohesin binding based on immunostaining of salivary gland polytene chromosomes and genomic ChIP patterns. Chro, Pzg/Z4, and cohesin all bind preferentially to polytene interband regions, which represent less compacted, transcriptionally active chromatin, as revealed by DAPI staining . In mostce versa . This alIn these studies we investigated the regulation of the E(spl)-C complex by cohesin, PRC1, and the Chro-Z4/Pzg protein complex in BG3 cells, in which the E(spl)-C has a rare restrained state with a cohesin-H3K27me3 overlap. We find that the E(spl)-C has a highly self-interactive structure that is unexpectedly independent of these protein complexes and the level of gene expression. Depletion of any of these three protein complexes, however, significantly increases E(spl)-C transcription. As discussed herein, the effects of these three protein complexes on E(spl)-C expression likely reflect changes in expression of Notch ligands, and in the cases of cohesin and PRC1, potentially direct effects on activator and Pol II activity at the E(spl)-C genes .Enhancer of split complexes with similar structures are conserved in insects and crustaceans over 420 million years -C has a structure in which all positions within the complex interact with each other at a high frequency, but not with flanking regions. Surprisingly, we found that this architecture is independent of cohesin, the PcG complexes, the Chro-Pzg/Z4 complex, transcription, and stage of the cell cycle. We thus do not know the factors that establish this striking architecture, which defines the E(spl)-C as a structurally independent domain. We also do not yet know the factors that control recruitment of cohesin and PcG complexes to the locus. We speculate, however, that this architecture coordinates transcriptional control of the entire E(spl)-C, based on the finding that in BG3 cells, cohesin, PRC1, and the H2Aub and H3K27me3 histone modifications made by the PRC1 and PRC2 complexes are co-extensive within this architectural domain. Although no known insulators or boundary elements flank the E(spl)-C, and depletion of the CP190 protein required for activity of all known Drosophila insulators does not alter E(spl)-C expression, it is likely that the unknown factors that form this structure limit the spread of these protein complexes and histone marks. The E(spl)-C architectural domain may be evolutionarily significant, because Notch-regulated on years .spl-1N rough eye and bristle phenotypes. These phenotypes are sensitive to mutations in the E(spl)-C and cohesin genes in a highly dosage-sensitive manner, and modest changes in the E(spl)-C architecture or recruitment of cohesin or PcG proteins may have similar effects (Possible clues to the identities of the factors that control the E(spl)-C architecture and/or the recruitment of cohesin and PcG complexes may arise in genetic screens for factors that alter E(spl)-C sensitive phenotypes, such as the Protocadherin beta (Pchdb) gene complex is down-regulated in the embryonic fibroblasts and brains of mice heterozygous mutant for the Nipbl cohesin loading factor, and brains of mice that are homozygous mutant for the SA1 cohesin subunit  complex, helping determine which genes in the complex are active -C genes.Previous studies showed that depletion of cohesin or PRC1 increases expression of the and gene . This liBecause cohesin and PRC1, unlike the Chro-Pzg/Z4 complex, bind directly to the E(spl)-C, it is also possible that they also directly control association of NICD with the Su(H) protein bound upstream of the active genes. For example, they could potentially interact with NICD or the Su(H) complex, and interfere with NICD association, or somehow facilitate ubiquitination and degradation of NICD. The lack of an effect of cohesin or PRC1 depletion on NICD association with E(spl)-C genes after EDTA treatment does not rule out this possibility, because under these conditions, the amount of NICD is no longer limiting.NIPBL and cohesin subunit genes -C transcription independently of any effects on Notch ligand expression or NICD association with the E(spl)-C genes. This is because both bind throughout the complex, and the PRC1-generated H2Aub repressive histone mark is co-extensive with the E(spl)-C architectural domain. Importantly, all genes in BG3 cells that show rare extended overlap of cohesin and the PRC2-generated H3K27me3 modification, such as the ctivated ,b. It isAt all genes examined that are strongly repressed by cohesin, cohesin restricts the transition of paused RNA Pol II into elongation, irrespective of whether or not they have the H3K27me3 mark (


Here, we have addressed the mechanisms underlying the transcriptional hyper-reactivity of junb. Using transfection and pharmacological assays to complement chromatin immunoprecipitation analyses addressing the localization of histones, polymerase II, negative elongation factor (NELF)-, DRB sensitivity-inducing factor (DSIF)- and Positive Transcription Factor b complexes, we demonstrate that junb is a RNA Pol II-paused gene where Pol II is loaded in the transcription start site domain but poorly active. Moreover, High salt-Recovered Sequence, chromosome conformation capture (3C)- and gene transfer experiments show that (i) junb is organized in a nuclear chromatin loop bringing into close spatial proximity the upstream promoter region and the downstream enhancer and (ii) this configuration permits immediate Pol II release on the junb body on binding of LPS-activated NF-κB to the enhancer. Thus, our work unveils a novel topological framework underlying fast junb transcriptional response in DCs. Moreover, it also points to a novel layer of complexity in the modes of action of NF-κB.The  Dendritic cells (DCs) are professional antigen-presenting cells, which are key actors in the induction of adaptive and memory immunities as well as in tolerance to self-antigens . After primer extension, amplified 3 C products were quantified by qPCR as previously described  and reverse . In all, 25 μl of this PCR product were digested with Dde I and ligated using T4 DNA ligase (Invitrogen) before a second digestion with Eco RI. Finally, this reaction was diluted into a solution of DC2.4 genomic DNA digested with DdeI and EcoRI (rather than in H2O) to obtain a final DNA concentration similar to that of the 3C reactions (12.5 ng/µl). These dilutions were used to generate standard curves for qPCR quantifications. The 3C-qPCR primer sequences are given in The 3C is a proximity assay  in whiche et al.  and modi  that was either wild-type (p-junb-Luc-κB) or mutated (p-junb-Luc-κBmut) on the κB sites that are essential for induction by NF-κB ] most often mark regulatory regions of transcription-competent/active genes, revealing recruitment sites of transcription factors/cofactors/machineries (junb to be a paused gene (i.e. loaded with Pol II at the TSS) in DCs (see later in the text). However, it was interesting to note the presence of an NFR containing the E domain in non-stimulated DC2.4 cells (i.e. with no NF-κB/p65 bound) and not just under conditions of stimulation by LPS (i.e. in the presence of bound NF-κB/p65) .As a first step to our transcriptional studies, we investigated histone distribution on the ells, H3 A and H4 hineries  but are junb gene body before stimulation, which was consistent with basal transcriptional activity, with a stronger accumulation at the beginning of the gene. On stimulation by LPS, H3K9Ac and H3K4Me3, both increased on the junb coding region, which were two observations consistent with increased transcriptional activity. Interestingly, H3K9Me3 , consistently with its putative repressive role. Taken with the analysis of the other histone marks, this was suggestive of a scenario whereby, on LPS stimulation, the junb gene was subjected to histone modifications with antagonistic effects to ensure only transient expression.Next, we studied histone marks associated with either active  or inactive (H3K9Me3) transcription ,59. H3K9 H3K9Me3 D was prejunb transcriptional regulation are confined to two regions in DCs: (i) the E region and (ii) the TSS + ∼1000 bp upstream of it. Moreover, the changes in both transcription-active and -inactive histone marks were suggestive of subtle dynamic chromatin changes on the junb locus.Thus, nucleosome distribution was consistent with the idea that the main determinants of junb locus in the −1350/+4500 region. In non-stimulated DC2.4 cells, ChIPs reproducibly revealed two similar peaks of Pol II accumulation with hardly any detectable polymerase in between  the transcription peak is transient (most probably in the few-minutes range) and (iii) cells cannot be perfectly synchronized.We then analyzed Pol II distribution on the  between A. One co between , though  between A. Not fijunb transcription was maximal after LPS stimulation (junb (see later in the text). It is worth noting that, similarly to phospho-Ser5, phospho-Ser2 appeared as two peaks centered on the TSS and E domains when detectable. It was however intriguing to find it associated with junb TSS, as this modification is usually not found at the beginning of genes but rather on their body and/or at their end  phosphorylation occurring on serines 2 and 5 of its 52 heptad repeats, as they are known to regulate Pol II activity ,61. In kmulation C, possibheir end ,61.junb by LPS , which also phosphorylates NELF and DSIF ,60–62. In by LPS C with a n by LPS . Moreoveunb mRNA D and Jununb mRNA E on stimjunb is a ‘paused’ gene in non-stimulated DCs with accumulation of NELF, DSIF and Pol II principally phosphorylated on CTD Ser5 in the TSS region, consistent with basal transcription and (ii) pausing is relieved on transcriptional activation, which is associated with more efficient recruitment of CDK9, presumably phosphorylating CTD Ser2 (and maybe other substrates), as described in liver cells stimulated by IL-6 .Thus, taken together, our data suggest that (i)  by IL-6 . The finjunb gene, we first assessed the possibility of a complex transcriptional pattern at this locus. Sensitive RT-qPCR assays using first random hexanucleotides for priming reverse transcription and then specific oligonucleotides for amplification of small amplicons, did not support the idea of sense transcription downstream of E and antisense transcription from E towards the TSS. They did not support sustained antisense transcription in the proximal upstream region of the TSS either (not shown). We however do not exclude that minor transcription of small labile RNAs could occur at junb, as has been observed on certain genes  possibly cross-linking chromatin elements distantly located on the DNA fiber but spatially close. The detection of Pol II and CDK9 ChIP signals in the E region  and contains the upstream junb NFR. The other one is located downstream of the junb transcribed region (between +1694 and +3960 relative to the TSS) and contains the E region. Unfortunately, the lack of other suitable restriction enzyme sites (i.e. accessible to their cognate enzymes in the HRS assay) did not allow us to narrow down with more precision the limits of these two HRSs (not shown). It is worth noting that we did not detect any significant change in junb HRSs between unstimulated and LPS-stimulated DC2.4 cells.Work by two of us previously showed that the genomic regions at the basis of chromatin loops can often be evidenced using the so-called HRS assay. This assay allows trapping of such sequences into deoxyribonucleoproteic complexes that precipitate on high-salt treatment of cell nuclei ,47. Theyjunb gene is flanked by two HRSs that are suggestive of two attachment sites of this locus to proteinacious intranuclear structures. Although not directly demonstrating that these two domains interact physically in vivo, these data are consistent with the idea of a loop organization of the junb locus.In conclusion, independently of LPS stimulation, the junb locus can be organized in a chromatin loop bringing together the TSS and E domains in DC2.4 cells, we resorted to the 3C assay, a technique that was designed to assess spatial proximity of DNA elements in their native chromatin context  as an anchor to test spatial proximity with other Dde I sites scattered on the junb locus  reporter gene whose size is approximately that of junb and (ii) the E domain was cloned either downstream of luc or just upstream of the promoter  less basal transcription in the case of E-mP-luc as compared with Emut-mP-luc in the experiments presented in κB than in that of p-junb-Luc-κBmut in the experiments presented in Second, the fragments −206 to +2237  of the p-junb-Luc-junb promoter and E domains leads to stronger LPS responsiveness. This is consistent with the idea that the loop configuration of the junb locus bringing about these two domains provides increased transcriptional reactivity.Thus, forced proximity of the junb is an immediate early gene in LPS-stimulated BMDCs and that its protein product, JunB, contributes to BMDC activation via stimulation of inflammatory cytokine genes  that is most probably attached to a nuclear structure that remains to be defined. This brings the TSS and the E domains into close spatial proximity, which, most probably, provides a favorable topology for fast transcriptional activation upon binding of NF-κB to the E enhancer (see later in the text). Moreover, this loop is relaxed after induction. HRS- and 3C assay resolutions rely on the position of available restriction sites in the analyzed regions. Owing to this technical constraint, which is particularly limiting when dealing with short loci, we could unfortunately not establish whether the 2 NFRs identified by ChIP on each side of the junb gene (one including the TSS upstream region and the other the E domain) reside close to, or overlap partially with the two domains forming the stem of the chromatin loop. Whatever the answer to this issue, our data are consistent with the idea that most, if not all, major determinants of junb transcription control in DCs may reside within the chromatin loop  were already present on the junb gene body in non-stimulated DCs and increased on junb transcriptional activation. Interestingly also, an inactive transcription-associated histone mark, H3K9Me3, which persisted for at least 2 h post-stimulation (not shown), was observed on junb at the peak of transcriptional activity. Though paradoxical at first glance, this was consistent with the fact that junb gene activation by LPS is transient and suggested that both transcription activation and termination programs must be activated coordinately to avoid improperly high and/or protracted expression of this gene and, consequently, abnormal immune responses.We also report that contexts ,27, thisjunb locus is organized in a chromatin loop in non-stimulated DCs raises a number of questions. At the present stage of investigations, we do not exclude that the reasons for, and the roles of, this loop configuration are multiple and non-exclusive. A first possibility would be that looping and attachment to a nuclear structure would insulate junb from its closest neighbor genes, PRDDX2 and HOOK2 , to avoid transcriptional cross-interferences and to ensure separate and differentiated expression, as has been suggested for other genes  in ChIP experiments. Whether this relaxation participates actively to transcription termination by preventing Pol II recycling from the end of junb to the TSS constitutes an interesting possibility to explore. Another one to consider is, however, that fast reactivation of junb would simply be useless in terms of JunB protein accumulation after DCs have already been activated once. Supporting this idea, JunB protein, most probably due to stabilization mechanisms, continues to be present at high and stable levels till cell death occurring by 24–48 h post-LPS stimulation, i.e. long after junb mRNA levels have returned to basal level  and the TSS domain in various genes. Such transcription factors include Klf1, GATA-1, -2 and -3, STAT6, ERα, AR, FXR, Lef1 as well as NF-κB see ,76]. Mor. Mor76)]lsewhere ,60–62.junb chromatin loop is formed during ontogeny and how it is maintained in DCs is still an open issue. Proteins such as CTCF, SATB1 and cohesin A have recently been shown to be instrumental for chromatin loop formation/maintenance and/or attachment to the nuclear matrix in specific studies [see (junb locus in DCs (not shown). Moreover, although searches in the USCS genomic database showed histone distribution (including the 2 NFRs) on the junb locus similar to that in DCs, no such proteins could be found on, or close to, junb (not shown) in other cell contexts, suggesting that their implication in chromatin loops is not universal. As junb is not silent but subjected to basal transcription in non-stimulated DCs, it is important to consider that transcription factors/cofactors other than NF-κB might be key for chromatin loop configuration of the junb locus, as some of them have been shown to be essential for induction (see earlier in the text) or maintenance [see ; fellowships were from Ligue Nationale contre le Cancer (to T.G.) and Ministry of Higher Education and Research (MESR) and from the Ligue contre le Cancer (to F.C. and G.M.T.). Funding for open access charge: Laboratory funds.MP’s laboratory is an Conflict of interest statement. None declared.