Neurotrophin-regulated gene expression is believed to play a key role in long-term changes in synaptic structure and the formation of dendritic spines. Brain-derived neurotrophic factor (BDNF) has been shown to induce increases in dendritic spine formation, and this process is thought to function in part by stimulating CREB-dependent transcriptional changes. To identify CREB-regulated genes linked to BDNF-induced synaptogenesis, we profiled transcriptional occupancy of CREB in hippocampal neurons. Interestingly, de novo motif analysis of hippocampal ChIP-Seq data identified a non-canonical CRE motif (TGGCG) that was enriched at CREB target regions and conferred CREB-responsiveness. Because cytoskeletal remodeling is an essential element of the formation of dendritic spines, within our screens we focused our attention on genes previously identified as inhibitors of RhoA GTPase. Bioinformatic analyses identified dozens of candidate CREB target genes known to regulate synaptic architecture and function. We showed that two of these, the RhoA inhibitors Par6C (Pard6A) and Rnd3 (RhoE), are BDNF-induced CREB-regulated genes. Interestingly, CREB occupied a cluster of non-canonical CRE motifs in the Rnd3 promoter region. Lastly, we show that BDNF-stimulated synaptogenesis requires the expression of Par6C and Rnd3, and that overexpression of either protein is sufficient to increase synaptogenesis. Thus, we propose that BDNF can regulate formation of functional synapses by increasing the expression of the RhoA inhibitors, Par6C and Rnd3. This study shows that genome-wide analyses of CREB target genes can facilitate the discovery of new regulators of synaptogenesis. Most excitatory synapses in the mammalian brain are found on small, actin-rich protrusions of the dendritic membrane known as dendritic spines de novo and developmental synaptogenesis Long-term changes in spine morphogenesis often depend on de novo gene expression To achieve this goal, we utilized chromatin immunoprecipitation (ChIP) and next generation sequencing to identify CREB-target sites in hippocampal neurons. Interestingly, bioinformatic analyses identified an alternate, non-canonical CRE motif that was highly enriched at CREB targeted genes, facilitated recruitment of CREB, and was sufficient for CREB-regulated transcription. Modeling of the CREB bZip-CRE crystal structure showed that this variant CRE maintained the same interactions as the canonical motif. Gene ontology analysis to select putative CREB-targets that regulate the actin cytoskeleton resulted in the identification of two CREB- and BDNF-regulated molecules known to inhibit RhoA, Par6C (Pard6a) and Rnd3 (RhoE). Both Rnd3 and Par6C have been reported to inhibit RhoA signaling via activation of p190RhoGAP, and thus play a potential role in BDNF-dependent spine formation We demonstrate that BDNF-induced CREB-dependent synaptogenesis requires the expression of the RhoA inhibitors, Rnd3 and Par6C. Moreover, analyses of CREB ChIP-Seq data identified an alternate, non-canonical, CRE motif that is occupied by CREB in the Rnd3 promoter, and is sufficient to confer CREB responsiveness. These discoveries shed insight into the processes by which CREB and neurotrophins regulate synapse formation and synaptic remodeling.http://cancan.cshl.edu/RNAi_central/main2.cgi). Serial Cloner  was used to design two complementary oligos incorporating the target sequence and a short hairpin sequence (TTCAAGAGA) surrounded by BglII and HindIII restriction sequences. The complementary oligos were annealed and cloned into either the pSUPER or the pSUPER GFP vector (Oligoengine) between the BglII and the HindII sites. Each sh-RNA was tested for effectiveness of knockdown in both HEK-293 cells and hippocampal neurons. The most effective sh-RNA was then used throughout the described studies. Exogenous knockdown by sh/si-RNAs was used based on detection limitations of endogenous Par6C and Rnd3 expression using Rnd3 and Par6C antibodies, as well as transfection efficiency limitations in cultures. Luciferase Reporter Constructs: The canonical CRE  and the novel CRE  were phosphorylated and ligated into the BglII and XhoI sites of dephosphorylated pGL3 basic (promega).The following reagents were purchased from the indicated sources: Recombinant human BDNF (Peprotech), U0126 . pCAG-ACREB Antibodies: anti-pErk , anti-Erk1/2 (mouse Santa Cruz), anti-pCREB , anti-c-Myc , anti-Rnd3 , anti-Par6 , anti-Synapsin1 , anti-VGlut1 . Primers used for ChIp-Seq analyses, and cloning primers provided in .4 cells per square centimeter) were cultured from P1–2 Sprague–Dawley rats on plates coated with poly-L-lysine  as described previously Animal use for hippocampal cultures was carried out in compliance with Washington State University IACUC approved protocols ASAF 0317-011 and ASAF 04020-003. These protocols were specifically approved by Washington State University s institutional IACUC review board. Hippocampal neurons . For transfection of 24 wells of a 24 well plate, 50 µL of Lipofectamine 2000 was added to 2.5 mL of NBA and incubated for 5 minutes. DNA plasmids for each of the treatments were mixed in separate tubes, with enough total plasmid DNA for 1 µg/well . Native media in wells were collected and kept warm, and 500 µL of warm GM was placed in the well. NBA/L2K mix was added to DNA mixtures for 20 minutes, before 100uL/well of DNA/NBA/L2K mix was added to each well for 30 minutes. After 30 minutes, media was aspirated, and 500 µL of warm native media was returned to well. In each experiment, we optimized DNA amounts to minimize toxicity and maximize transfection efficiency. Lipofectamine 2000 transfection efficiency was 0.5–5%.Organotypic hippocampal slices from P5 Sprague–Dawley rats were cultured as described previously ChIP-Seq data has been submitted to GEO and can be accessed at:.ChIP was conducted as described previously Total or nuclear RNA was isolated using the Trizol (Invitrogen) method following manufacturer’s protocol, and reverse-transcribed using MMLV (Invitrogen). Real time PCR was conducted as described previously ChIP-Seq areas of enrichment at an FDR of 0.001 were determined using a custom 500 bp sliding-window approach based on a previous algorithm De novo motif analyses utilized 500 bp regions flanking the sequence tag center of mass of the top 1000 ChIP-Seq peaks with and without the canonical CRE . The Weeder and YMF algorithms produced similar results using flags that allowed for the presence of more than one 6- mer motif on both strands ChIP-Seq data was annotated and visualized using R. Annotation was downloaded from the UCSC genome browser (mm9). Profile plots of ChIP-Seq data relative to RefSeq genes were generated in R by removing redundant RefSeq gene annotation, setting all gene lengths equal to 1, and plotting median-adjusted tag density. Statistical comparisons between ChIP-Seq data sets and genomic data were conducted in R and utilized Monte-carlo analyses and the Wilcoxon rank-sum test for significance testing. Both ChIP genomic input reads and randomized genomic extents were used to create Monte-carlo background models with equivalent results. Hippocampal neurons were transfected with mRFP-βactin±test plasmids or oligos as indicated. Expression of fluorescently tagged actin allows visualization of dendritic spine density, size and shape. Expression of low levels of βactin has no significant effect on either spine density or size or shape Transfected neurons were treated and fixed as described above. After fixation, cells were rinsed in PBS and permeabilized with 0.1% Triton X-100 detergent , followed by two rinses in PBS, and blocked with 8% bovine serum albumin  in PBS for 1 h. Cells were again rinsed with PBS, followed by a 24-h incubation period with anti-VGLUT1  or anti-synapsin1 (Synaptic Systems) diluted in 1% BSA, following the manufacturer's protocol, at 4°C. Then, cells were rinsed thrice with PBS, incubated in Alexa Fluor 488 goat-anti-mouse IgG in 1% BSA following the manufacturer's protocol (Invitrogen) for 2 h at room temperature, rinsed again with PBS, and mounted with elvanol. Imaging was performed as described above. Spines were determined to be co-localized if any portion of the spine head overlapped with VGLUT1 or Synapsin1 puncta .To analyze Par6C and Rnd3 expression, cultures were treated as specified in figure legends, and each well was lysed in 50 µL RIPA buffer (Upstate) with Phosphatase Inhibitor II and III (Sigma) and Protease Inhibitor (Roche). Samples were then dounce homogenized and spun in a microcentrifuge to pellet debris, and frozen at −80°C for storage. Samples were prepared using NuPage LDS Sample Buffer (Invitrogen) with 0.5 M DTT and heated at 70°C for 10 minutes before equal volumes were loaded into NuPage 4–12% Bis-Tris gels (Invitrogen) and run using NuPage Mops Buffer. Protein was transferred to PVDF using Tris-Glycine buffer. Blots were blocked using Aquablock (East Coast Biotech) for 1 hour, and then probed with anti-Par6C , anti-Rnd3 (Upstate), and anti-ERK ½ (Loading control)(Santa Cruz) diluted in Aquablock overnight. Blots were then washed with PBS before application of Rockland IR secondary antibodies (anti-RB 700 and anti-MS 800) for 2 hours. Finally, blots were washed with PBS again before blots were scanned using Li-COR infra-red Odyssey Scanner to image blots. Band intensities were measured using ImageJ and normalized to ERK2 band intensities.2, 3 mM CaCl2, 25 mM glucose, and 5 mM HEPES; pH was adjusted to 7.3 with KOH, and osmolality was adjusted to 310 mOsM. Cultures were allowed to equilibrate in a recording chamber mounted on an inverted microscope  for 30 min before recording. Transfected cells were visualized with fluorescence . Recording pipettes were pulled  from standard-wall borosilicate glass without filament . The pipette-to-bath d.c. resistance of patch electrodes ranged from 4.0 to 5.2 MΩ, and they were filled with an internal solution of the following composition: 25 mM CsCl, 100 mM CsCH3O3S, 10 mM phosphocreatine, 0.4 mM EGTA, 10 mM HEPES, 2 mM MgCl2, 0.4 mM Mg-ATP, and 0.04 mM Na-GTP; pH was adjusted to 7.2 with CsOH, and osmolality was adjusted to 296 to 300 mOsM. Miniature EPSCs (mEPSCs) were isolated pharmacologically by blocking GABA receptor chloride channels with picrotoxin , blocking glycine receptors with strychnine , and blocking action potential generation with tetrodotoxin . Recordings were obtained using a Multiclamp 700B amplifier . Analog signals were low-pass Bessel-filtered at 2 kHz, digitized at 10 kHz through a Digidata 1440A interface (Molecular Devices), and stored in a computer using Clampex 10.2 software (Molecular Devices). The membrane potential was held at −70 mV at room temperature (25°C) during a period of 0.5 to 2 h after removal of the culture from the incubator. Liquid junction potentials were not corrected. Data analysis was performed using Clampfit 10.2 software (Molecular Devices) and Mini-Analysis 6.0 software . The criteria for a successful recording included an electrical resistance of the seal between the outside surface of the recording pipette and the attached cell >2 GΩ and neuron input resistance >240 MΩ. A 5 min. recording time was used for analysis of the mEPSCs.Patch-clamp experiments were performed on cultured hippocampal neurons transfected with mRFP-β-actin-transfected and test plasmids ± BDNF pretreatment. Recordings were made on DIV12 to DIV14. The culture medium was exchanged by an extracellular solution containing 140 mM NaCl, 2.5 mM KCl, 1 mM MgClActivation of CREB-dependent transcription has been shown to increase synaptogenesis −11) (−10). To better examine any overlap between the two data sets, we re-mapped and re-analyzed the embryonic neuron ChIP-Seq reads with our pipeline to obtain 4,783 regions of predicted CREB enrichment (FDR of 0.001%). 59% of these embryonic neuron ChIP-Seq peaks  were within 2 kb of a hippocampal neuron ChIP-Seq peak   suggesti−11) . A recenDe novo motif analysis of the top 500 hippocampal CREB ChIP-Seq peaks that do not contain a canonical motif identified an alternate, non-canonical, motif (TGGCG) (−11). Interestingly, ∼72% of CREB peaks were within 2 kb of a non-canonical motif and ∼69% were adjacent to the canonical CRE (TGACG) . Although some of these anomalous interactions may represent indirect binding events or lack of spatial specificity, many highly ranked loci were not adjacent to a canonical CREB CRE.  (TGGCG)  that was (TGACG) . 56% of  (TGACG) . [46]. T (TGACG)  and fore (TGACG) . Importaata sets ).We next tested whether the non-canonical motif was sufficient to drive CREB-dependent transcription. −11). Similar results were seen with both embryonic ChIP-Seq data −6) (data not shown). To test whether this motif is correlated with functional regulation of gene expression, we selected ChIP-Seq peaks adjacent to proximal promoters that contained the non-canonical, but not the canonical motif (1000 bp window). In this data the distribution of the non-canonical motif at individual genes showed tight correlation with hippocampal ChIP-Seq peaks, and with ChIP data from other studies  BDNF is known to stimulate synaptogenesis in both hippocampal and cortical neurons, and BDNF has been shown to activate CREB-dependent transcription in neurons Many Rho-family proteins play important roles in regulating the actin-cytoskeleton and neuronal morphogenesis. Specifically, the activation of Rac1-mediated signaling is thought to stimulate the formation of dendritic spines, while the activation of RhoA signaling is thought to be inhibitory to spinogenesis Organotypic hippocampal slices retain the cellular and morphological organization of the intact hippocampus and have been used extensively to study activity-dependent synaptic plasticity Par6C has been shown to stimulate spine formation in hippocampal neurons by controlling p190RhoGAP-mediated inhibition of RhoA Rnd3 is a well-established inhibitor of the RhoA signaling pathway that is believed to function by directly activating p190RhoGAP, directly inhibiting the RhoA effector ROCK, and/or inhibiting the RhoA-GEF SYX BDNF-treatment increased spine density above control levels, as did over expression of either Par6C or Rnd3 . Co-exprin vitro binding assays and reporter studies. Nevertheless, multiple epigenetic mechanisms, including DNA methylation, histone modifications, and chromatin structure, influence CREB occupancy The identification of genes directly regulated by CREB has largely relied on de novo motif analyses of ChIP-Seq peaks lacking a consensus CRE identified an alternate, non-canonical, motif (TGGCG). This motif was markedly enriched at ChIP-seq peaks (72%), and both the known and non-canonical motifs were detected in (56%) of CREB ChIP-Seq peaks. A high percentage of CREB ChIP-Seq peaks (16%) contained only the non-canonical motif. Both CREB SACO loci While the Par6C promoter contains a consensus palindromic CRE that overlaps with a CREB ChIP-Seq peak, the Rnd3 ChIP-Seq peak is not adjacent to a canonical CRE. As in earlier studies, CREB ChIP-Seq peaks were significantly enriched for consensus CRE motifs. Nevertheless, as in earlier studies, a significant fraction of peaks did not contain an adjacent CRE. These peaks could represent, noise, indirect interactions with other factors, or a non-canonical CREB-bound motif. Interestingly, Interestingly, the non-canonical “TGGCG” motif identified in this study was previously described as a cAMP responsive enhancer in the proenkephalin promoter CREB activation has been shown to stimulate the formation of dendritic spines and new synapses Although both BDNF- and CREB-dependent transcription are known to stimulate synaptogenesis, the precise mechanism and molecular changes involved are not well characterized. We show that inhibition of RhoA activity is necessary for BDNF-induced spinogenesis in hippocampal neurons. Bioinformatic analysis of CREB ChIP-seq data identified two known inhibitors of Rho, Par6C and Rnd3. We showed that Par6C expression is induced by BDNF in a CREB-dependent fashion, and that Par6C expression is necessary for BDNF-induced synaptogenesis. Similarly, Rnd3 was also transcriptionally regulated by BDNF in a CREB-dependent manner. Unexpectedly, we found that CREB bound to a non-canonical CRE in the Rnd3 promoter region. Moreover, we show here that Rnd3 expression is necessary and sufficient for synaptogenesis in hippocampal neurons. Interestingly, Rnd3 knockout mice are phenotypically similar to BDNF knockout mice, in that Rnd3 deletion leads to retarded growth, postnatal lethality, and severe motor disruption This study utilized high-throughput ChIP-Seq to identify ∼30,000 predicted CREB target sites not detected in other studies. Moreover, we have characterized and demonstrated the functionality of a non-canonical CRE motif that is enriched to approximately the same extent as the canonical motif in multiple ChIP-Sequencing data sets. Lastly, we have demonstrated how targeted analysis of the CREB ChiP-Seq data in this study can be used to identify CREB-regulated genes that may regulate a wide variety of neuronal processes.Figure S1RNAi control experiments. DIV6 cultured hippocampal neurons were transfected with mRFP-βActin ± empty vector (Control and BDNF), ± sh-Scrambled (non-specific sh-RNA construct), ± sh-Rac3 Click here for additional data file.Table S1Primers used in current study.(XLS)Click here for additional data file.Table S2Annotation of hippocampal neuron CREB ChIP-Seq data with canonical (TGACG), novel (TGGCG) CRE motifs, and closest RefSeq gene transcriptional start site . Both strands were used for motif analyses.(XLS)Click here for additional data file.Table S3Annotation of hippocampal neuron CREB ChIP-Seq data with closest embryonic forebrain CREB ChIP-Seq data Click here for additional data file.Text S1Supporting Information Legend.(DOCX)Click here for additional data file.


Escherichia coli genome have been proposed to be under the control of cAMP-bound CRP. Using the newly developed Genomic SELEX screening system of transcription factor-binding sequences, however, we have identified a total of at least 254 CRP-binding sites. Based on their location on the E. coli genome, we predict a total of at least 183 novel regulation target operons, altogether with the 195 hitherto known targets, reaching to the minimum of 378 promoters as the regulation targets of cAMP-CRP. All the promoters selected from the newly identified targets and examined by using the lacZ reporter assay were found to be under the control of CRP, indicating that the Genomic SELEX screening allowed to identify the CRP targets with high accuracy. Based on the functions of novel target genes, we conclude that CRP plays a key regulatory role in the whole processes from the selective transport of carbon sources, the glycolysis-gluconeogenesis switching to the metabolisms downstream of glycolysis, including tricarboxylic acid (TCA) cycle, pyruvate dehydrogenase (PDH) pathway and aerobic respiration. One unique regulation mode is that a single and the same CRP molecule bound within intergenic regions often regulates both of divergently transcribed operons.CRP (cAMP receptor protein), the global regulator of genes for carbon source utilization in the absence of glucose, is the best-studied prokaryotic transcription factor. A total of 195 target promoters on the  Escherichia coli  pathway, pentose-phosphate (PP) pathway and tri-carboxylic-acid (TCA) cycle Escherichia coliOCRP was the first purified cAMP-CRP is a dual regulator, acting as an activator or a repressor E. coli genome, but later improved by employing DNA tilling array for mapping of SELEX fragments on the E. coli genome As a short-cut approach for identification of the whole set of genes under the control of a given transcription factor (TF), we developed an improved “Genetic SELEX” screening system In this study, we performed a systematic search for the whole set of regulation targets of cAMP-CRP by using the Genomic SELEX screening system. Using the collection of 254 different CRP-binding sequences thus identified, a total of at least 183 promoters have been newly identified as the regulation targets of CRP. By using the LacZ reporter assay, some of these novel CRP target promoters were confirmed to be under the control of CRP in vivo. Including the hitherto identified targets, the total number of operons under the direct control of cAMP-CRP was estimated to range from minimum 378 to maximum more than 500. After analysis of the functions of newly identified regulation target operons, we propose some novel regulatory roles and the regulation modes of CRP.Escherichia coli BW25113 (W3110 lacIq rrnBT14 Insert > symbol DΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78), and crp disruptant JW5702 were the products of the Keio collection E. coli Stock Center . Cells were grown in LB medium at 37°C under aeration with constant shaking at 140 rpm. Cell growth was monitored by measuring the turbidity at 600 nm. Plasmid pRS551 was used for the reporter assay of CRP-depending promoters (see below). For maintenance of pRS551, ampicilin and kanamycin were added each at a final concentration of 50 mg/ml.E. coli BL21(DE3) transformant with the CRP expression plasmid pCRP encoding His-tagged CRP was grown in LB broth, and protein expression was induced by adding 1 mM IPTG. After 3 h of induction, cells were harvested and subjected to CRP purification. Purification of His-tagged CRP was performed according to the standard procedure E. coli K-12 W3110 genome was prepared after sonication of purified genome DNA, and cloned into a multi-copy plasmid pBR322. In each SELEX screening, the DNA mixture was regenerated by PCR. For SELEX screening, 5 pmol of the mixture of DNA fragments and 10 pmol each of His-tagged CRP were mixed in a binding buffer  and incubated for 30 min at 37°C. For the effector(+) reaction of CRP, 10 insert>symbol mµM adenosine 2′,3′-cyclic monophosphate (cAMP) (SIGMA) was added during the binding reaction and also subsequent isolation procedure of CRP-DNA complexes. The DNA-transcription factor mixture was applied to a Ni-NTA column, and after washing out unbound DNA with the binding buffer containing 10 mM imidazole, DNA-protein complexes were eluted with an elution buffer containing 200 mM imidazole. DNA fragments recovered from the complexes were PCR-amplified.The genomic SELEX method was carried out as previously described E. coli DH5a. Sequencing of each clone was carried out using the T7 primer (5′-TAATACGACTCACTATAGGG-3′). For SELEX-chip (DNA tilling-array chip) analysis, PCR-amplified products of the isolated DNA-protein complexes obtained in the presence or absence of the effecter were labeled with Cy5, and mixed with the original mixture of genome DNA fragments labeled with Cy3. The fluorescent labaled DNA mixtures were hybridized to a DNA microarray consisting of 43,450 species of 60 b-long DNA probe, which are designed to cover the entire E. coli genome at 105 bp interval . The Cy5 fluorescent intensity of test sample at each probe was normalized with that of the corresponding peak of Cy3 fluorescence of the original library. The Cy5/Cy3 ratio was measured and plotted along the E. coli genome.For SELEX-clos (cloning-sequencing) analysis, PCR products were cloned into pT7 Blue-T vector (Novagen) and transformed into BamHI and EcoRI, and then ligated to pRS551. Construction of the plasmids was confirmed by DNA sequencing. Plasmids were transformed into test E. coli strains, wild-type BW25113, crp mutant JW5702. Overnight cultures in LB medium were diluted 1∶1000 into fresh medium, and cells were grown for 4 hrs to an OD600 of 0.4–0.5. The activity of β-galactosidase was measured according to the standard Miller method To measure the activity and regulation of promoters, a LacZ-reporter assay was employed using pRS551 plasmid vector E. coli, we first tried to identify the whole set of target promoters, genes and operons under the control of CRP. For this purpose, we employed ‘Genomic SELEX’ screening, in which DNA fragments carrying the recognition sequence by CRP were isolated as CRP-DNA complexes from a mixture of genome DNA fragments (28). For screening of DNA sequences with affinity to CRP, purified His-tagged CRP was mixed with a collection of E. coli genome fragments of 200–300 bp in length, and CRP-bound DNA fragments were affinity-purified. Since CRP exhibits functional inter-conversion depending on the presence or absence of specific effector cAMP In order to get insight into the genome regulation by CRP for control of carbon source utilization in malK), fucose (fucP), and mannitol (mtlA) but also amino acids and nucleosides such as alanine (cycA), serine (dsdX) and uridine (nupC).For identification of the sequences of DNA fragments bound to CRP, we first performed SELEX-clos (cloning-sequencing) analysis. A total of 160 SELEX fragments were cloned and sequenced, which were classified into 29 CRP-binding sequences, including 13 previously identified loci and 16 newly identified sites . When thyibI and mltA, both being located downstream of the cAMP-CRP binding sequence, of which the mtlA operon encoding mannitol-specific PTS permease is known to be activated by cAMP-CRP In the case of cAMP-CRP, the binding sites are mostly located in intergenic spacer regions between divergently transcribed genes. As an initial screening, we predicated that only of the divergent promoters is under the control of cAMP-CRP . For instance, the most abundant 59 clones carried the intergenic sequence between the divergently transcribed yecJ and yecR, of which one is counted as a CRP target . LikewisE. coli genome .For identification of the whole set of binding sequences by CRP, we then subjected the mixture of SELEX fragments to the newly developed DNA tilling array analysis (SELEX-chip) i genome . On the By setting the cut-off level of intensity ratio at 30, a total of 19 high-level peaks with strong affinity to cAMP-CRP were identified  while thE. coli, and a total of 195 genes or operons are listed as the regulation targets of cAMP-CRP in E. coli databases such as Regulon DB (27)  . Among t DB (27) . Except  DB (27) , group-B DB (27) .E. coli, transcription factors bound within an intergenic region between divergently organized genes or operons generally regulate transcription toward one direction. A group of CRP-binding sites are located between divergently organized genes or operons . The number of CRP targets should be more because: 1) both of the divergently transcribed genes or operons might be regulated for some of group-B and group-C cases and even for some known targets; and 2) regulation targets increase if the cut-off level of SELEX-chip is set at lower levels [note that only about half of the known CRP targets were picked up at the cut-off level herein employed].http://ai.stanford.edu/~xsliu/BioProspector/), which was successfully employed for identification of RutR box sequence The consensus recognition sequences of CRP have been proposed after sequence analysis of the hitherto identified targets , fruK (1-phosphofructokinase), yggF , ppsA (phosphoenolpyruvate synthase), talA  and tktB (transketolase II). In order to further confirm the accuracy of target prediction by the Genomic SELEX screening, we performed the lacZ promoter assay for some of the newly identified target promoters, together with some known targets, in both wild-type and crp deletion mutant. ptsG (glucose-specific PTS), dgsA , ptsH (PTS Hpr component) and glpF (glycerol facilitator), that are activated by cAMP-CRP. All the putative target genes examined were indeed found to be under the control of CRP, classified in group-A in CRP has been identified to regulate some of the enzymes for central carbon metabolism, including in vivo, either activation or repression, for the novel targets. Two promoters, talA  and fbaB , showed increased activity in the crp-disrupted mutant, indicating that these two promoters are repressed by cAMP-CRP. All other promoters tested showed decreased activity in the crp mutant, including pps (phosphoenolpyruvate synthetase), fruB (fructose-specific PTS), setB (lactose/glucose efflux system), nuoA (NADH:ubiquinone oxidoreductase), glk (glucokinase), cmtB (mannitol-specific PTS), pck (phosphoenolpyruvate carboxykinase), gpmM (phosphoglycerate mutase), atpI (ATP synthase) and pgi (glucosephosphate isomerase). For this group of promoters, cAMP-CRP is involved as an activator. The binding sites of activator CRP were mostly located upstream from the promoters while those of repressor CRP were located downstream from the target promoters  of the CRP targets in Regulon DB was, however, not identified in the Genomic SELEX screening under the conditions herewith employed, but it is noteworthy that the level of consensus sequence conservation is lower for CRP-binding sequences included in these unselected promoters see . The numE. colii.e., the respiratory electron transport pathway, including ndh (NADH dehydrogenase) and cyoABCDE (cytochrome boptype oxidase) The marked increase in the numbers of target genes under the control of CRP indicates that the regulation targets by each of a total 300 species of transcription factors in a) the genes for carbon source transporters; (b) a large number of genes coding for enzymes participating in the reversible reactions of the central carbon metabolism; (c) a small number of the genes coding for rate-controlling enzymes in the carbon metabolism; and (d) the genes for side pathways linked to the central carbon metabolism. The group-a genes encoding transporters of carbon sources are organized into more than 40 transcription units, of which more than 30 have been identified to be under the control of CRP The most important regulatory role of CRP has been believed to be the control of central metabolism E. coli contains a set of substrate-specific transport systems, which are classified on the basis of molecular composition into PTS (phosphoenolpuruvate: sugar phosphotransferase) E. colifruBKA for fructose manXYZ for mannose transport ptsG for glucose transport mtlAD for mannitol transport ptsH encoding HPr component of PTS fruAB, ptsH and pstG was confirmed to be down-regulated in the crp mutant  transporters belong to the primary transporters, which use the energy released from ATP hydrolysis to drive ions or solutes across membranes. In cAMP-CRP . LikewisfabA , gapA  and pgk (phosphoglycerate kinase) (glk (glucokinase), fruK (1-phosphofructokinase), yggF , ppsA (phosphoenolpyruvate synthase), talA  and tktB (transketolase II) are under the control of activator cAMP-CRP (in vivo reporter assay indicated that both pgi (glucosephosphate isomerase) and gpmM (phosphoglycero mutase III) promoters were activated by CRP while the fbaB  promoter was repressed by CRP  . In addicAMP-CRP . Furtherd by CRP  even thotktAB and talAB encoding the last step enzymes.The pentose phosphate (PP) pathway, also called hexose monophosphate shunt, is one of the three essential pathways, EMP (Embden-Meyerof-Parnas pathway), PP (pentose-phosphate) and TCA (tri-carboxylic-acid pathway) pathways of the central metabolism. At the entry gate, D-glucose-6P is converted to D-glucono-lactone-6P by Zwf ndh encoding NADH dehydrogenase II and cyoABCDE encoding the cytochrome bo-type oxidate, both together forming the pathway of respiratory electron transport downstream from the PDH cycle nuoABCDEFGHIJKLMN operon, NADH-ubiquinone oxidoreductase II encoded by ndh, and the cytochrome bo terminal oxidase encoded by the cyoABCDE operon. Furthermore the atpIBFHAGDC operon coding for the ATPase F1 and F0 complexes was also found to be under the control of CRP  complex, and most of the TCA cycle enzymes. Previously we identified by Genomic SELEX screening that PdhR, originally identified as the regulator of genes coding for components of PDH complex, regulates l of CRP .E. coli, two steps at the entry gate of the central metabolism for supply of fructose-1,6-bisphosphate  i.e., the conversion of phosphoenolpyruvate to pyruvate by two pyruvate kinase isozymes (PykA and PykF) Several step reactions of the central carbon metabolism are apparently irreversible, different enzymes being involved in catalysis of forward and backward reactions. These irreversible reactions, sometimes called rate-controlling reactions, are considered to be involved in switching control between glycolysis and gluconeogenesis. There are 4 steps of the irreversible reactions in hosphate , i.e., oPromoters for the genes encoding the key metabolic enzymes and the essential cell architectures are under the control of multiple regulators, each monitoring different environmental conditions or metabolic states Of the 70 transcription factors under the control of CRP, a total of 24 were newly identified in this study Click here for additional data file.Table S1Primers used for promoter assay.(XLS)Click here for additional data file.Table S2CRP-binding sequences identified by SELEX-clos.(XLS)Click here for additional data file.Table S3Regulation targets of cAMP-CRP.(XLS)Click here for additional data file.Table S4CRP-binding sequences on regulation target promoters.(XLS)Click here for additional data file.


Drosophila, cis-regulatory regions termed PcG response elements (PREs) act as nucleation sites for PcG proteins to create large repressive PcG domains that are marked by trimethylation of lysine 27 on histone H3 (H3K27me3). In addition to an action in cis, PREs can interact over long distances, thereby enhancing PcG dependent silencing. How PcG domains are established, which factors limit their propagation in cis, and how long range interactions of PREs in trans affect the chromatin structure is largely unknown.Polycomb group (PcG) proteins dynamically define cellular identities through the epigenetic repression of key developmental genes. In Drosophila genome generates an artificial PcG domain and we analyze its organization by quantitative ChIP and ChIP-on-chip experiments. Intriguingly, a boundary element and known insulator proteins do not necessarily interfere with spreading of H3K27me3. Instead, domain borders correlate with the presence of promoter regions bound by RNA Polymerase II and active chromatin marks. In contrast, genes that are silent during early fly development get included within the PcG domain and this incorporation interferes with gene activation at later developmental stages. Moreover, trans-interaction of the transgenic PRE with its homologous endogenous PRE results in increased PcG binding, correlating with reinforced silencing of genes within the domain borders.We demonstrate that the insertion of a PRE-containing transgene in the Our results suggest that higher-order organization of PcG-bound chromatin can stabilize gene silencing within PcG domains. Further we propose that multi-protein complexes associated with active promoters are able to define the limits of PcG domains. Future work aimed to pinpoint the factors providing this barrier function will be required to understand the precise molecular mechanism by which active promoter regions can act as boundaries to stop spreading of H3K27me3. Polycomb-group proteins (PcG) proteins were initially discovered in flies for their role in maintaining the repressed state of homeotic (HOX) genes in appropriate spatial domains, after the segmentation gene products have disappeared in early embryonic development. In addition to their function as cellular memory system, PcG factors dynamically regulate cell fate decisions and control cell proliferation Biochemical studies showed that PcG proteins exist in distinct large multiprotein complexes Drosophila, PcG complexes are recruited to chromatin via regulatory DNA elements called PcG response elements (PREs) In Functionally it has been proposed that spreading of the repressive histone mark H3K27me3 is blocked by insulator proteins. Binding sites for the insulator protein Su(Hw) have been shown to interfere with the spreading of the H3K27me3 mark Importantly, the action of PREs to form large repressive H3K27me3 domains is not restricted to the linear chromatin fiber: PREs can interact at long distances Using a previously established transgenic fly line Fab-7 is a well-defined chromosomal element that contains a PRE and is involved in the regulation of the homeotic gene Abdominal-B (Abd-B) within the homeotic bithorax complex (BX-C) Fab-7 region, upstream of a mini-white reporter and inserted at the scalloped (sd) locus of the X chromosome (referred to as the Fab-X line) has been previously shown to ectopically recruit PcG proteins mini-white eye color reporter gene and of the endogenous sd gene downstream of Fab-7Fab-7 containing transgene is inserted 1.6 kb upstream of the scalloped-RD (sd-RD) promoter (Fab-7 containing transgene at the sd gene locus results in spreading of H3K27me3 downstream of the transgenic PRE into flanking genomic regions that are not significantly methylated in wild type (WT) embryos , where its levels significantly decrease close to background levels. Intriguingly, spreading is unidirectional, since no increased H3K27me3 levels were found upstream of the transgene insertion site .Next we asked why spreading is unidirectional and what prevents the coating of a larger region by the H3K27me3 mark. We considered two parameters: chromatin boundaries or insulators, and the presence of active chromatin components. The proximal end of the 3.6 kb ion site . Since wsd locus in wild type Dosophila embryos R85 allele, a null mutant for GAF To examine the effect of endogenous insulator proteins in blocking spreading of the H3K27me3 mark at the transgenic gene locus, we compared the genomic location of the domain borders of the ectopic PcG domain with the previously published distribution of six insulator proteins at the sd gene locus. Immediately upstream of the transgene insertion site is the promoter of the gene PGRP-LE that is highly tri-methylated at H3K4, both at the embryonic stage  is tri-methylated on H3K4 and is adjacent to the Fab-7-mediated H3K27me3 domain boundary  embryos showed that RNA Pol II and H3K4me3 levels correlated strongly at the examined promoter regions . High leDrosophila development (sd-RE promoter could be detected in early embryogenesis (2–4 hours after egg laying). Expression levels are slightly decreased at later developmental stages but stay elevated throughout fly development. In contrast, no significant transcription can be detected from the sd-RD and CG8509 gene loci in early embryonic development and these genes become moderately activated at later developmental stages. Surprisingly, a similar expression pattern was observed for the PGRP-LE gene, whose promoter region is bound by RNA Pol II and marked by H3K4me3 in embryos, but is not detectably transcribed in embryonic development. Together, these results show that promoter regions poised for gene activation by the presence of the RNA Pol II complex, and H3K4me3 demarcate the artificial PcG domain, indicating a potential role of active chromatin components in delimiting these repressive H3K27me3 domains without a need of active transcription per se.We next performed qRT-PCR experiments to examine expression of genes at the sd gene locus during elopment . We detetrans interactions of the transgenic Fab-7 copy with the endogenous Fab-7 element at the BX-C Fab-7 mediated pairing was shown to be dependent on sequence homology since deletion of the endogenous Fab-7 copy  results in loss of pairing of the BX-C with the transgenic Fab-7 copy and reduced silencing of the reporter gene present in the construct as well as of the sd gene. We have previously shown that PcG-mediated repression is enhanced by sd gene locus and the endogenous PcG domain at the BX-C has an impact on PcG binding. Therefore, we compared levels of PcG proteins and of H3K27me3 at the Fab-7 PRE and at flanking genomic regions of the sd gene locus in the presence or absence of Fab-7 interaction with the BX-C. qChIP experiments in Drosophila embryos revealed that the PcG proteins Polycomb (PC) and Polyhomeotic (PH) are detected at the transgenic Fab-7 element in the absence of the endogenous copy in embryos  we detected significantly increased levels of PcG proteins at the Fab-7 PRE  or absence  of Fab-7 pairing during Drosophila development , which can explain the milder scalloped phenotype observed in Fab-X, Fab-71 flies Fab-7 cannot efficiently silence genes far away from its integration site. In contrast, long-range interaction of the artificial PcG domain at the sd gene locus with the BX-C can decrease transcription significantly. Notably, no major changes in expression levels of more distal genes (located up to 100 kb away from the transgene insertion site) could be detected at the embryonic or adult stage . However, at the adult stage we observed a 2-fold decrease in RNA Pol II levels at the sd-RD and CG8509 promoter regions in Fab-X versus wild type (WT) flies  flies . These rIn this study we analyzed the mechanisms impinging on the formation, stability and silencing ability of an ectopically generated PcG domain. Our data show that PcG proteins bind specifically to the transgenic PRE. Importantly, chromatin surrounding the PRE-containing transgene becomes tri-methylated on H3K27, forming a repressive H3K27me3 domain of over ten kb in size. We found that H3K27me3 can spread over boundary elements and silent gene promoters, whereas promoter regions of genes that are either expressed or poised for activation demarcate the PcG domain. We propose that protein complexes associated with active promoter regions are major determinants in blocking H3K27me3 spreading. Furthermore, chromatin analysis of the transgenic Fab-X system showed that long-range interactions of Fab-7 elements increase the efficiency of PcG binding, resulting in enhanced gene silencing and decreased RNA Pol II affinity to promoter regions. To our knowledge this is the first report demonstrating the effect of long-range interactions of PREs on chromatin structure.Fab-7 PRE at genomic loci that usually have low PcG-binding affinity is sufficient for recruitment of PcG proteins and for marking of the transgene as well as the surrounding chromosome region with H3K27me3. Is this mark deposited by spreading of PcG complexes or by PRE-bound complexes looping out to reach neighboring chromatin regions? Interestingly, we did neither observe homogeneous levels of H3K27me3, nor did we see a gradual decrease of H3K27me3 levels from the PRE outwards. Instead, peaks and valleys appear in the profile. This argues against a linear spreading of PcG proteins along the chromatin fiber. A more likely scenario is that PcG complexes bound at PREs may loop out and contact downstream chromatin regions. Indeed, we have shown recently that a transgenic PRE can contact a downstream promoter via chromatin looping The insertion of the sd gene locus in wild type flies  We found that H3K27me3 can spread from a transgenic PRE over gene promoter regions where H3K4me3 and RNA Pol II are absent, whereas promoters marked by RNA Pol II and H3K4me3 demarcate the borders of the PcG domain. Likewise, many endogenous PcG domains are sharply bordered by gene promoter regions associated with active histone marks sd gene locus is the chromatin regulator GAF. GAF has been shown to counteract heterochromatin spreading by directing histone H3 replacement by the H3.3 variant Drosophila) family members sd promoter region that is tri-methylated at H3K4 and interfere with the spreading of the H3K27me3 mark (data not shown). Therefore, insulator proteins, transcription factors and cofactors, the TrxG machinery and RNA polymerase II are candidate to delimit PcG domains. Whether they block spreading collectively or whether one of the proteins suffices to fulfill this function remains to be studied.What is the molecular mechanism at the basis of the block? One possibility is that the active mark H3K4me3 directly interferes with the deposition of a methyl mark on H3K27. H3K4me3 has been shown to inhibit methyltransferase activity catalyzed by PRC2 activity in an allosteric fashion when nucleosomes are methylated on lysine 4 in a symmetric manner Fab-7 PRE can associate in the nucleus to enhance PcG-mediated silencing sd gene locus on the X chromosome is stronger when the endogenous Fab-7 element is present at the BX-C in chromosome 3, showing that long-distance interactions between these loci can reinforce silencing by stabilizing the association of PcG proteins with chromatin. However, a single Fab-7 transgene is sufficient for the recruitment of PcG proteins and its associated histone mark H3K27me3 even in the absence of the endogenous copy, although binding levels of PcG proteins are significantly lower and H3K27me3 is just above background. Strongly increased H3K27me3 levels at the Fab-7-containing transgenic locus in the Fab-X line may be due to targeting of the transgene to a large PcG body and subsequent communication with the BX-C locus PcG proteins have a speckled distribution within the nucleus cis might be essential for efficient recruitment of PcG proteins. In line with this hypothesis, ChIP-chip analysis of PcG proteins in Drosophila embryos Fab-7 sequence at the BX-C is not bound by PcG proteins at the adult stage, although the other PREs of the locus stay efficiently bound. This dynamic PcG behavior, which was previously observed for other PREs A previous report demonstrated physical interaction of multiple PREs at the BX-C locus cis-regulation imposed on the surrounding DNA by individual PREs, global locus regulation relying on higher-order chromatin folding with the contribution of multiple PREs from the same locus and trans-regulation involving three-dimensional contacts among PREs located at large distances might contribute to impart to each PcG target locus its specific chromatin state leading to its precise regulatory output.In summary, 1118 line (referred to as wild type) was obtained from R. Paro . Transgenic lines used in this study have been previously described in Fab-71 lines the Fab-7 containing transgene is inserted at position 15698606 of chromosome X, 1.648 kb downstream of the PGRP-LE gene and 1.670 kb upstream of the sd-RD transcript (flybase release FB2008_07). For all experiments including the Fab-X and Fab-X, Fab-71 lines, flies were grown at 28,5°C since PcG-mediated silencing was stronger at higher temperatures.Flies were raised in standard corn meal yeast extract medium. The Oregon-R wChIP of whole embryos or homozygous adult flies was essentially performed as previously described Rp49 was used in the PCR experiments.For quantitative ChIP, after immunoprecipitation and DNA purification, enrichment of specific DNA fragments was analyzed by real-time PCR, using the Roche Light Cycler equipment and accessories as described in For ChIP-on-chip assays precipitated DNA was amplified by ligation mediated PCR (LM PCR). To verify the linear response of the LM PCR, amplified DNA was tested by Southern blot analysis using genomic DNA fragments containing known PcG binding sites Drosophila melanogaster genome sequence (Flybase release 4.3) with an average oligonucleotide length of 50 bp and an average inter-oligo spacing of 130 bp from Nimblegen Systems Inc. (GEO accession: GPL3353) were used to hybridize embryo ChIP samples (H3K4me3 and H3K27me3) and adult ChIP sample (H3K4me3). Adult ChIP sample (H3K27me3) was hybridized on a 385K genome tiling array provided by NimbleGen (Design Name: DMEL ChIP Set3) with an oligonucleotide length of 50 bp and a median probe spacing of 97 bp. Specific (Cy5) and Mock IP (Cy3) signal intensities as quantified and provided by Nimblegen were converted to log2 ratio and normalized using loess normalization http://cav-ouranos.igh.cnrs.fr/data/NIMBLEGEN_DATA_Schuttengruber_Cavalli.rarA 385K tiling microarray representing the euchromatic, non-repetitive regions of A single replicate was performed of WT and Fab-X lines in embryos and adults. In these cases, spreading of repressive histone marks from the PRE-containing transgene and formation of the artificial PcG domains was confirmed by ChIP experiments analyzed by quantitative PCR see . Thus inDrosophila embryos or adult flies was done using Trizol Reagent according to the manufacturer’s instructions (Sigma). RT PCR was performed using Superscript III First Strand Synthesis Kit from Invitrogen following the manufacturer’s instructions. Reverse transcription was primed using hexamer primers. qPCR analysis was done as described for ChIP experiments. The copy number for each investigated gene was normalized to a housekeeping gene (Rp49). To detect nascent transcripts, at least one primer of the amplicon is located within an intron of the investigated gene. Primer sequences are listed in RNA isolation of whole Genome wide H3K27me3 domain data were derived from Figure S1Chromatin state at the mini-white gene of the Fab-7 containing transgene. (A and B) ChIP-on-chip analysis of the mini white gene from embryos (A) or adult flies (B) in the indicated fly lines. Although the graph displays the white gene as it is annotated in the endogenous genome, the fly lines used in this study carry the 1118w mutation, which deletes almost all DNA sequences at the white locus. Thus, the ChIP on chip signal corresponding to white comes from the transgene. Fold changes between the specific IP and mock IP are plotted on the Y axis. On the X axis, genomic coordinates and white gene are indicated. P indicates the white promoter. CDS marks the white coding region. I-1 indicates the first intron of the endogenous white gene. Note that in the mini-white gene of the transgene the first large intron is deleted and therefore no significant ChIP-on-chip signal is detected. Notably, the white gene is not expressed during early development and therefore does not interfere with the spreading of the H3K27me3 mark. The weak H3K4me3 signals associated with the white promoter region might represent a subset of cells, where the mini-white gene escapes PRE-mediated silencing and gets activated, accounting for the variegated eye phenotype observed in this fly line.(PDF)Click here for additional data file.Figure S2Distribution of chromatin insulator binding sites at the sd gene locus. Annotated genes at the sd gene locus (X chromosome position 15628000 to 15668000) are shown on the top. Orange asterisk marks the transgene insertion site. Below, binding sites for the indicated insulator protein are shown (PDF)Click here for additional data file.Figure S3Chromatin state of the Fab-7 PRE and the sd gene locus as a function of Fab-7 long-range interactions at the adult stage. (A) ChIP analysis on female adult flies. Fly genotypes and antibodies used for IP are indicated at the top of the panels. Immunoprecipitated DNA was analysed by semi quantitative PCR with primers amplifying the Fab-7 element (Fab-7), the white promoter region (mini-white) or the Rp49 gene (Rp49). Note that Fab-7 primers amplify both the endogenous Fab-7 sequence and the transgenic element. (B) ChIP analysis on female adult flies using H3K27me3 or H3K4me3 antibodies. Immunoprecipitated DNA was analysed by semi quantitative PCR using primers amplifying genomic regions at the sd gene locus or the Rp49 gene. Position of PCR fragments is indicated at the bottom of the figure. Note that the “PRE 5′end” amplicon only amplifies the transgenic Fab-7 element.(PDF)Click here for additional data file.Figure S4Chromatin state of the bithorax complex (BX-C). (A) ChIP-on-chip analysis of the BX-C in female adult flies of the indicated fly lines using PC, PH and H3K27me3 antibodies. Fold changes between specific IP the and mock IP are plotted on the Y axis. On the X axis, genomic coordinates and Hox genes are indicated. The Fab-7 element is indicated by the blue bar. The bx, bxd and mcp PREs are represented by blue asterisks. Note that in WT flies PRC1 components (PC and PH) are not significantly bound at the Fab-7 sequence, while PC and to a very weak extent PH proteins are bound to the transgenic Fab-7 copy in the Fab-X, Fab-71 line. In contrast, strong binding of both PRC1 components to Fab-7 element is observed in the Fab-X line. H3K27me3 levels are lower at the transgenic Fab-7 copy compared to the endogenous sequences, but do not synergize as a function of Fab-7 pairing. (B) ChIP-on-chip analysis of BX-C locus in 4–12 hour old embryos of the indicated fly lines using PC, PH and H3K27me3 antibodies. Fold changes between specific IP the and mock IP are plotted on the Y axis. On the X axis, genomic coordinates and Hox genes are indicated. The Fab-7 element is indicated by the blue bar. The bx, bxd and mcp PREs are represented by blue asterisks.(PDF)Click here for additional data file.Figure S5Gene expression at the sd gene locus after insertion of the Fab-7 containing transgene. (A) Map of the sd gene locus on chromosome X. Orange asterisk indicates the transgene insertion site. (B–C) RT PCR analysis of genes up and downstream the transgene insertion site in embryos (B) or adult flies (C). RNA was extracted from 4–12 hours old embryos or from female adult flies. RNA levels were normalized to the housekeeping gene Rp49.(PDF)Click here for additional data file.Figure S6The number of PREs within a PcG domain correlates with the intensity of H3K27me3. (A) An example of a domain with lower H3K27me3 intensity (few PH peaks). (B) An example of a domain with higher H3K27me3 intensity (large number of PH peaks). The plots show the ratio (fold change) of specific IP versus mock IP along parts of chromosome 3L and 2R. Significantly bound regions  are indicated in red. (C) Correlation between number of PH peaks and the average intensity of H3K27me3 domains. For calculating the average intensity per domain the area covered by a domain was divided by the size of the domain in bps. Pearson’s correlation co-efficient (CC) was calculated using the R package.(PDF)Click here for additional data file.Table S1Sequences of primers used in the study.(PDF)Click here for additional data file.


We provide evidence for expression of these regulators very early in salmonid development and in tissues outside of the hypothalamic-pituitary-adrenal/gonadal (HPAG) axis. Although the function of these factors in sexual differentiation have been defined, their roles in early development before sexual fate decisions and in tissues beyond the brain or gonad are essentially unknown.The products of dax1 and dax2, foxl2b and mis were isolated and the regulatory regions that control their expression were characterized. Transposon integrations are implicated in the shaping of the dax and foxl2 loci. Splice variants for cyp19b1 and mis in both embryonic and adult tissues were detected and characterized. We found that cyp19b1 transcripts are generated that contain 5'-untranslated regions of different lengths due to cryptic splicing of the 3'-end of intron 1. We also demonstrate that salmon mis transcripts can encode prodomain products that present different C-termini and terminate before translation of the MIS hormone. Regulatory differences in the expression of two distinct aromatases cyp19a and cyp19b1 are exerted, despite transcription of their transactivators  occurring much earlier during embryonic development.Bacterial artificial chromosomes containing salmon dax, foxl2, mis and other differentiation factors that indicate that they have functions that are more general and not restricted to steroidogenesis and gonadogenesis. Spliced cyp19b1 and mis transcripts are generated that may provide regulatory controls for tissue- or development-specific activities. Selection of cyp19b1 transcripts may be regulated by DAX-1, FOXL2 and SF-1 complexes that bind motifs in intron 1, or by signals within exon 2 that recruit splicing factors, or both. The potential translation of proteins bearing only the N-terminal MIS prodomain may modulate the functions of other TGF β family members in different tissues. The expression patterns of dax1 early in salmon embryogenesis implicate its role as a lineage determination factor. Other roles for these factors during embryogenesis and outside the HPAG axis are discussed.We report the embryonic and extragonadal expression of  Sex determination and early gonadal development depends upon specific embryonic gene programs that activate differentiation of the bipotential primodium. These processes hinge upon the expression of both sex-linked and autosomal genes that promote or antagonize male or female cell fate pathways. The products of some genes are essential for female (WNT-4 (Wingless-related MMTV integration site-4) and follistatin)  and maledax1, foxl2 and sf1 each play critical roles in the transcriptional regulation of aromatase (cyp19) genes ; BAC 105M10 containing dax2 [GenBank:HM159470]; BAC 217E24 containing foxl2a ortholog (pseudogene) [GenBank:HM159471]; BAC 261D01 containing foxl2b diverged paralog [GenBank:HM159472], and BAC 19C14 containing mis [GenBank:HM159473].[GenBank:BAC clone sequence data were annotated using BLAST homolog searches and EMBOSS programs package . AlignmeBAC DNA was labeled with Spectrum Orange using a nick translation kit (Abbott Molecular). Human placental DNA (2 μg) and Cot-1 DNA  or Cot-1 DNA  were added to the probe mixture for blocking. Hybridizations were carried out at 37°C overnight and post-hybridization washes were as recommended by the manufacturer (Abbott Molecular) with minor modifications . AntibodThe chromosome preparations were obtained from blood of the Norwegian strain of Atlantic salmon (2N = 58) by methods described previously for salmonid fishes . Brieflymis and cyp19 promoters [To identify the location of potential transcription factor binding elements in the promoters of our GOI, we preliminarily used MatInspector (Genomatix Software GmbH). Our designation of OCT3/4 consensus binding motifs 5'-ACACGCAT-3' or 5'-ATTTGCAT-3' are based on work done in ES cells ,71. The romoters . We use romoters . Sequencromoters ,53-55,74ARE: androgen response element; BMP: bone morphogenetic protein; CRE: cyclic AMP response element; EGR-1: early growth response-1; ERE: estrogen response element; OCT3/4: octamer binding factor 3/4; PPARE: peroxisome proliferator-activated receptor element; RXR/RAR: retinoid X receptor/retinoic acid receptor; SMAD: mothers against decapentaplegic; SP-1: stimulatory protein-1; TGFβ: transforming growth factor β; WNT: wingless-related MMTV integration site.KRVS designed and coordinated the study, performed GOI-specific PCRs of early development stage cDNAs, promoter analysis and drafted the manuscript. MY performed GOI-specific PCRs of adult tissue cDNAs and semi-quantitative analysis. RY performed BAC sequence data analysis and annotations. JdB performed transposable element analysis. LR performed hybridizations to isolate and identify GOI-containing BACs. SS, AR and EBR performed BAC clone preparation and sequencing. RBP performed FISH on BAC DNA. WSD and BFK obtained funding and contributed to experimental design and analysis. All authors read and approved the final manuscript.Graphical presentation of number of mortalities found throughout early development. Day represents post-fertilization age.Click here for filecDNA expression profiles for ten genes of interest in twelve different adult tissues. cDNA expression profiles for ten genes of interest (GOI) in twelve different tissues: 1: kidney, 2: muscle, 3: skin, 4: gut, 5: gill, 6: spleen, 7: brain, 8: heart, 9: testis, 10: liver, 11: eye and 12: pyloric caecum. Semi-quantitative levels of expression were calculated for each GOI as signal intensity relative to ubiquitin levels for three individual fish. Mean values (bars) of three individuals plus standard error are shown.Click here for filemis transcriptsAlignment of partial . Sequence alignment of mis cDNAs and amino acid residues based on sequence upstream from the C-terminal hormone portion of salmon MIS. A. The stop codon for mis (629 bp) is highlighted in red. The open reading frame of mis (396 bp) cDNA is in-frame for encoding the bioactive hormone portion of MIS and does not contain a stop codon. Two potential protease recognition motifs (RGQR AND RATR) for translated MIS (396 bp) are boxed. B. The stop codons for mis (629 bp) and mis (436 bp) are highlighted in red. Putative cleavage RLRR recognition sites are boxed.Click here for filedax1 geneProximal promoter sequences of the Atlantic salmon . The potential binding elements of various transcription factors, TATA boxes, exon 1 and initiator methionine codon are labeled.Click here for filedax2 geneProximal promoter sequences of the Atlantic salmon . The potential binding elements of various transcription factors, TATA boxes, exon 1 and initiator methionine codon are labeled.Click here for filemis geneProximal promoter sequences of the Atlantic salmon . The potential binding elements of various transcription factors, TATA boxes, exon 1 and initiator methionine codon are labeled.Click here for filefoxl2b geneProximal promoter sequences of the Atlantic salmon . The potential binding elements of various transcription factors, TATA boxes, exon 1 and initiator methionine codon are labeled.Click here for filefoxl2a geneProximal promoter sequences of the Atlantic salmon pseudogenic . Potential TATA boxes and the 5'-end portion of a region of sequence similarity shared with rainbow trout foxl2 genes are labeled.Click here for filein situ hybridization of dax1-containing BACFluorescent .Click here for filein situ hybridization of dax2-containing BACFluorescent .Click here for filein situ hybridization of mis-containing BACFluorescent .Click here for filePrimer oligonucleotide sequences used in RT-PCRs.Click here for file


We summarize recent findings linking inflammatory hypoxia to chromatin modifications, in particular to repressive histone signatures. We focus on the role of Hypoxia-Induced Factor-1 in promoting the activity of specific histone demethylases thus deeply modifying chromatin configuration. The consequences of these changes are depicted in terms of gene expression and cellular phenotypes. We finally integrate available data to introduce novel speculations on the relationship between inflammation, histones, and DNA function and integrity. Epigenetic control is a motor of gene regulation. In a broad sense, it can be viewed as a “bridge” between genotype and phenotype, as being capable of changing the function of a given locus without modification of the underlying sequence. This occurs because both DNA and histones can be chemically modified in the cell without changing the base composition, and these modifications are read by the transcriptional apparatus as a on/off signal. DNA can be methylated, when needed, mainly on promoter cytosines, whereas histones can be modified on lysines, and less frequently on arginines, by methylation and other modifications, creating a template for heritable changes in gene expression, the epigenome. In evolutionary terms, the evolving epigenome is probably increasing the variability and adaptation of species, as recently shown for Schistosoma . MoreoveMore than 40 years ago, Vincent Allfrey proposed that histone modifications could contribute significantly to the regulation of gene expression  but onlyHistone methylation was once considered enzymatically irreversible until a histone lysine demethylase (LSD1) was identified . Subsequ2 to long distances and wide areas via an intricate network of vessels, culminating in capillaries. Low oxygenation (hypoxia) occurs when cells are at a distance greater then roughly 200 nm from these capillaries, while, within this distance, oxygen can freely diffuse from cell to cell [2 consuption by the pathogen or first-aid immune cells, or transient ischemia due to vasoconstriction or compression by local edema; ischemia may be a more relevant factor in subacute or chronic inflammations, due for instance to vessel clogging by incoming cellular infiltrate; moreover, some anatomical districts may be more prone to undergo inflammatory hypoxia [In multicellular organisms, oxygen is supplied essentially by blood circulation, which brings molecular O to cell . Hypoxia hypoxia .α, which binds to and activates regulatory regions of target genes [β, and this interaction is controlled by oxygen levels. When oxygen is abundant, there is little HIF-1α, it is destroyed under the direction of the von Hippel-Lindau (VHL) protein—and what is left cannot bind HIF-1β. At low oxygen levels, these restraints are lifted [Hypoxia, including that induced in inflammatory processes, induces Hypoxia-Inducible Factor (HIF-1)et genes . To do se lifted .HIF-1 is pleiotropic: targets include factors involved in metabolism and angiogenesis, like the inducible form of nitric oxide (NO) synthase (iNOS), vascular endothelial growth factor (VEGF), glucose transporter-1, and several glycolytic enzymes. Accordingly, studies utilizing knock-out mice have shown that HIF-1 deletion is lethal during midterm of embryonic development resulting in major defects particularly in the vascular system . Inflammation is usually self-contained both in temporal and structural terms, but one complicating aspect is that a vicious circuit  may poteIn addition to the above-mentioned HIF's direct transactivation of target genes, recent work pointed to HIF-1 as being capable of “indirect”, albeit dramatic modification of chromatin structure, particularly in terms of histone code configuration.What is the link between HIF-1 and histone modifications? First, in recent years, evidence has pointed towards HIF-1 capability of HDAC recruitment and regulation , 21. TheBefore speculating on the consequences of JMJD activation in inflammatory hypoxia, we should recollect what is known on the putative role of H3K9 modification in cell physiology.Currently, H3K9 methylation has been linked to the determination and building of repressive heterochromatin; for instance, it is established that H3K9 di- and trimethylation creates the binding site for HP1, a conserved heterochromatin protein that mediates gene silencing, heterochromatin compaction, and late replication . H3K9 diIn Drosophila, it has been shown that HP1 is required for the normal organization of the nucleolus and heterochromatic DNA repeats, and that H3K9 methylation protects these repeats from being excised from the genome by the enzyme Ligase 4 . OverallOn a speculative basis, the removal of H3K9 mark and the lifting of this protection in inflammation could expose the cell to the occurrence of cancer initiating events . Thus, besides the long recognized role of (chronic) inflammation as instrumental to cancer progression , 30, newImportantly, the notions discussed in the present paper indicate one possible (epigenetic) mechanism whereby the following may occur HIF-1 mediated induction of histone demethylases, removal of H3K9 methyl marks, loosening of chromatin compaction, exposure of DNA to instability, or abnormal transcription of otherwise silent genes.The perspective linking inflammation to cancer initiation is obviously a worrisome one, and it is likely that safeguard mechanisms, such as p53 activation, would be called often for extra work to protect tissues from dangerous karyotypes arising .α gene in cells treated by the proinflammatory fatty acid palmitate [Which transcriptome scenario—hence cellular phenotype—should be expect within inflammatory hypoxic areas? In a simplified (and speculative) view, together with the large panel of targets known to be affected by HIF-1 as a transactivator, we could witness a whole set of genes being rapidly derepressed as a result of increased JMJD function, with unpredictable consequences on cell life. Moreover, in a genomic “topographical” sense, the extent of this derepression could be significant if long distance spreading of the newly acquired modification would take place . Given talmitate . Alteratalmitate . Likely,Cellular consequences could than range from minor to significant, depending on the cell maturation level, the time frame of activation, and the relevance of some target genes. In an inflammatory context, metaplasia of differentiated cells has been postulated as one possible outcome . HypoxiaThe discovery of a relationship between environmental clues and H3 lysine demethylases sheds new light on the intricacies of gene expression and advances our understanding on the epigenetics of inflammation. κBhigh transcriptional activity is due to enzymatic erasing of H3K9 methyl marks from its promoter [κBactivity) in shaping our evolving epigenome [We should here stress that, although in this paper we have focused mainly on the reduction of oxygen tension, it is clear that, in inflammation, a whole array of molecular events could concur to alter the histone code, with important consequences on cell life. For instance, it was found that, in hyperglycemic memory,NFpromoter , 44. Thipigenome , 46.Moreover, recent findings suggest that even trans-generational differences can be due to environmental clues  acting in the temporal windows of gametogenesis and embryo implantation, during which massive epigenetic reprogramming takes place . The impTogether, these findings have broad implications for how we envisage the mechanisms underlying epigenetics and the every day physiology and offer us a mechanistic glimpse into the exciting world of dynamic chromatin.


Gene expression is controlled by proximal promoters and distal regulatory elements such as enhancers. While the activity of some promoters can be invariant across tissues, enhancers tend to be highly tissue-specific.in vivo using the hydrodynamic tail vein injection assay. Fifty-eight percent of the predictions yielded significant enhancer activity in the mouse liver, whereas a control set of five sequences was completely negative.We compiled sets of tissue-specific promoters based on gene expression profiles of 79 human tissues and cell types. Putative transcription factor binding sites within each set of sequences were used to train a support vector machine classifier capable of distinguishing tissue-specific promoters from control sequences. We obtained reliable classifiers for 92% of the tissues, with an area under the receiver operating characteristic curve between 60%  and 98% (for heart promoters). We next used these classifiers to identify tissue-specific enhancers, scanning distal non-coding sequences in the loci of the 200 most highly and lowly expressed genes. Thirty percent of reliable classifiers produced consistent enhancer predictions, with significantly higher densities in the loci of the most highly expressed compared to lowly expressed genes. Liver enhancer predictions were assessed de novo prediction of enhancers.We conclude that promoters of tissue-specific genes often contain unambiguous tissue-specific signatures that can be learned and used for the  A fundamental question in biology is how cells and tissues differentiate and maintain their identity from essentially the same genome. Wide variation in spatial, temporal and condition-dependent expression patterns of more than 20,000 genes in the human genome  is requicis-regulatory sequences, such as enhancers, are specifically bound by TFs and are central players in the control of transcription in multicellular eukaryotes. The regulation of promoters by distal enhancers involves DNA looping or scanning and/or higher-order conformation changes in chromatin  vector (Promega) using the Gateway system (Life Technologies). Sequence and orientation of the insert were re-verified by Sanger sequencing, and approximately 200 μg of endotoxin-free plasmid DNA was isolated using the EndoFree Plasmid Midi prep (Qiagen).Sequences selected for ApoE liver enhancer  was injected along with 2 μg of pGL4.74[hRluc/TK] vector to correct for injection efficiency, into at least three CD1 mice  using the TransIT EE hydrodynamic gene delivery system  according to the manufacturer’s protocol. Negative (empty pGL4.23[luc2]) and positive  conAUC: Area under the receiver operating characteristic curve; bp: Base pair; CNE: Conserved non-coding element; DHS: DNase I hypersensitive site; NK: Natural killer; PWM: Position-weight matrix; SVM: Support vector machine; TF: Transcription factor; TSS: Transcription start site; UTR: Untranslated region.The authors declare that they have no competing interests.IO conceived the work and supervised the analysis. LT carried out the analysis. RPS and MJK conducted the mouse experiments. NA supervised the experiments. The manuscript was written by LT and IO, with contributions from the other authors. All authors read and approved the final manuscript.Figures S1 to 15 and Supplementary notes.Click here for fileA table listing the motif ranks for the promoter-based models.Click here for fileA table summarizing the performance of the promoter-based models.Click here for fileA summary of results obtained with the hydrodynamic tail vein injection assay.Click here for fileA list of transcription factors known to be relevant for liver function.Click here for file


Drosophila muscleblind was previously shown to be expressed in embryonic somatic and visceral muscle subtypes, and in the central nervous system, and to depend on Mef2 for transcriptional activation. Genomic approaches have pointed out candidate gene promoters and tissue-specific enhancers, but experimental confirmation of their regulatory roles was lacking. In our study, luciferase reporter assays in S2 cells confirmed that regions P1 (515 bp) and P2 (573 bp), involving the beginning of exon 1 and exon 2, respectively, were able to initiate RNA transcription. Similarly, transgenic Drosophila embryos carrying enhancer reporter constructs supported the existence of two regulatory regions which control embryonic expression of muscleblind in the central nerve cord  and somatic  musculature . Both NE and ME were able to boost expression from the Hsp70 heterologous promoter. In S2 cell assays most of the ME enhancer activation could be further narrowed down to a 1200 bp subregion (ME.3), which contains predicted binding sites for the Mef2 transcription factor. The present study constitutes the first characterization of muscleblind enhancers and will contribute to a deeper understanding of the transcriptional regulation of the gene.The phylogenetically conserved family of Muscleblind proteins are RNA-binding factors involved in a variety of gene expression processes including alternative splicing regulation, RNA stability and subcellular localization, and miRNA biogenesis, which typically contribute to cell-type specific differentiation. In humans, sequestration of Muscleblind-like proteins MBNL1 and MBNL2 has been implicated in degenerative disorders, particularly expansion diseases such as myotonic dystrophy type 1 and 2.  Drosophila and associated to the development of the embryonic peripheral nervous system musclebIind transcripts themselves generate at least fourteen transcript isoforms. Most of them share common 5′ sequences but differ at the 3′-ends, encoding proteins of different lengths and carboxyl termini. The muscleblind transcriptional unit is large and has a complex organization with ten exons distributed over about thirty times more than the average gene length in DrosophilaMuscleblind proteins were initially identified in Drosophila, which has a single gene, three Muscleblind-like homologs  exist in humans and mice Myocyte enhancer factor 2D (Mef2D) In contrast to Drosophila Muscleblind shows tissue-specific expression during development. In eye-antennal imaginal discs Muscleblind is required for the formation of photoreceptor rhabdomeres, identifying muscleblind as a general factor required for terminal differentiation of adult ommatidia muscleblind caused defects on muscle attachments to the epidermis and disrupted Z-band formation in muscle sarcomeres muscleblind in the myoblast fusion process through a splice-independent regulation of muscle protein 20 (Mp20), a gene that promotes myoblast fusion Drosophila Myocyte enhancer factor 2 (Mef2) activates muscleblind in embryos, placing this gene downstream of Mef2 function in the myogenic differentiation program in flies muscleblind chaste mutation has revealed that the gene is not only required during embryo development but also in adult brain, where it is necessary for the normal development of neural circuitry that regulate female sexual receptivity Dystrophia Myotonica Protein Kinase (DMPK) gene CCCH-type zinc finger nucleic acid binding protein (CNBP) Muscleblind-like proteins are critically involved in many pathogenesis pathways, but most notably in myotonic dystrophies type 1 and type 2 . To test this hypothesis we defined two regions as potential promoters of muscleblind. P1 ranged from −180 to +335 (515 bp long) while P2 spanned from −243 to +343 (586 bp long)  involving the f exon 2 , which sbp long) A, definDrosophila promoter elements C,D). Furthermore, these motifs were phylogenetically conserved among Drosophila species in the Multiz Alignments & phastCons Scores provided by the UCSC, supporting the relevance of these non-coding regions (data not shown). To test the functional relevance of the putative promoters we generated reporter constructs in which P1 and P2 drove expression of Firefly luciferase. In addition, we also tested the activity of shorter versions, contained in the longer ones, of 220 bp long  and 397 bp long , respectively, in an attempt to define minimal promoters. Dual luciferase reporter assays in Drosophila S2 cells transfected with the resulting constructs revealed that P1 was able to boost luciferase readings more than 100 fold higher relative to the promoter-less control. This was 2.5 fold the transcription measured for P1.1, 12 fold higher than the luciferase activity driven by P2 and 7 times the activity measured for P2.1 , was used as the reference DNA to align orthologous sequences from 12 Drosophilids. Using the bioinformatics tool rVista, an intronic sequence showing above 90% identity in a 100 bp window between Drosophila melanogaster and D.mojavensis was selected. This sequence, refered to as H region, was 872 bp long and was located in intron 2 (cis-regulatory modules (CRMs) M1, M2, M3 and ML  promoter and is specifically designed to avoid chromatin configuration effects (“position effects”) by flanking the eGFP reporter cDNA with two copies of insulator sequences from the gypsy transposon ;To test the regulatory potential of the highly conserved (H) and the Mef2-bound  genomic regions involving the Hsp70 promoter, M2 drove robust eGFP expression in the somatic musculature of late embryos, approximately starting from stage 13. Notably, no eGFP expression was detected in other muscle derivatives or tissues where endogenous muscleblind is normally detected, particularly the CNS  and “NE” (from nervous system enhancer). Importantly, both ME and NE were capable of activating a heterologous promoter, a typical ability of transcriptional enhancer elements.We have previously shown that Muscleblind is localized in the nuclei of embryonic pharyngeal, visceral and somatic muscles, in the larval photoreceptor system, and in repeated clusters of cells within the central nervous system  the CNS I. As co the CNS D–F, thu the CNS . First s the CNS G. In sumuscleblind promoters, we used luciferase reporter assays in Drosophila S2 cells. We used S2 cells because this is a well characterized cell line whereas muscle or neuron-specific cell lines were not immediately available. Both enhancer regions were cloned upstream to each of the putative promoters P1, P1.1, P2 and P2.1 in their forward , thus confirming that the enhancer regions do not have any transcriptional activity by themselves. When promoters were in the forward orientation we observed that the transcription originated from P1 and P1.1 was strongly enhanced by ME, whereas NE had no effect on P1.1, or even decreased transcription when in combination with P1 , although both ME and NE still managed to significantly potentiate transcription from P1 and P1.1. Thus, promoter activity is orientation-dependent, as reversed promoters expressed significantly less luciferase reporter, and the activity of the ME and NE enhancers on P1 and P2 suggests enhancer-promoter communication specificity.To test the ability of ME and NE genomic regions to enhance transcription from the putative  forward  and reve with P1 B. Simil with P1 C. As coME function was further analyzed to narrow down sequences necessary for enhancer activity. This involved testing three smaller regions, approximately 1 kb each, here referred to as ME.1, ME.2 and ME.3 according to their relative position in the original region A. TheseDrosophila and human MBNL1 promoter sequences was patently non-existent. However, analysis of available cDNA sequences and ESTs in the MBNL1 locus suggested that human MBNL1 might also use TSS located in exon 1 and in exon 2  or spinocerebellar ataxia 8 (SCA8) muscleblind genes both in Drosophila and in vertebrates.cis-regulation of muscleblind, we analyzed a genomic DNA fragment harboring the muscleblind locus and its upstream region, looking for CRMs that regulate basal initiation of transcription (promoters) and tissue-specific expression (enhancers). In silico and in vivo studies identified two putative promoters, P1 in exon 1 and P2 in exon 2, and two intronic tissue-specific regulatory elements, a region of 3340 bp which drives specific expression in somatic muscle (ME) and a region of 830 bp which drives expression in central nervous system (NE). Both enhancers had been selected because of their enrichment in Mef2 binding sites according to ChIP-on-chip data muscleblind in the embryo muscleblind, which also includes expression in visceral and pharyngeal musculature, the Bolwiǵs organ  and the imaginal discs As a means of dissecting the Hsp70 promoter in transgenic embryos. However, in luciferase S2 assays, ME preferentially activated the P1 promoter while NE showed preference for P2, although the enhancer activity of NE was smaller than that of ME and transcription arising from P2 was on average one tenth of that from P1  was used to subclone into the pGL3-Basic vector (Promega) previously linearized with the same enzymes. ME, ME.1, ME.2, ME.3  and NE were obtained from Drosophila genomic DNA by high fidelity PCR and were cloned into KpnI/SacI digested pGL3-Basic vectors already including different promoter regions. To generate transgenic flies, M1, M2, M3, H and ML fragments were PCR amplified from genomic DNA and were cloned into the BglII and XbaI sites of the Drosophila expression vector pH-Stinger PstI, BglII, XhoI (5′) and HindIII, PstI (3′) adapter sites, were cloned into the pUC57 vector and subsequently transferred to the PstI site of the pH-Stinger vector. To generate ME-Hsa-P1 and ME-Hsa-P2 constructs, M2 was amplified with specific oligos containing BglII/XhoI adapters, digested, and cloned into the corresponding sites of the pH-Stinger vector already containing the candidate human promoters. All constructs were confirmed by sequencing. Description of used primers is in P1 and P2, and their shorter versions P1.1 and P2.1, promoter regions were synthesized by GenScript with Drosophila melanogaster Schneider 2 cells (S2) were cultured at 27°C in growing media containing 90% Schneideŕs insect media (Gibco), 10% heat inactivated fetal bovine serum (FBS), 100 units/ml of penicillin and 100 mg/ml of streptomycin. 48 h before transfection, 106 log-phase cells were transferred onto 24-well plates (300 μl per well). 4 μl of Cellfectin reagent (Invitrogen) in 200 μl of serum and antibiotic-free medium were used to co-transfect 450 ng of the pGL3 reporter plasmid of interest and 25 ng of Renilla luciferase. A GFP expressing vector served as transfection efficiency control. Cells were incubated 16 h with transfection mix, then media was replaced by Schneideŕs complete medium and the culture was additionally maintained 24 h at 27°C. Luciferase expression was monitorized using the Dual-Luciferase Reporter Assay System (Promega). Briefly, this involved adding 100 μl of lysis buffer per well, shaking for 15 min and transferring the lysate to a white 96-well plate with 40 μl of luciferase substrate. After 10 s of luminescence detection, Stop&Glo buffer was added and luminescence measurement was repeated. Luminescence readings used an EnVision plate reader (PerkinElmer). In cell culture luciferase reporter assays, all graphs show the average of three independent experiments with three technical replicates each. P-values were obtained using a two-tailed, non-paired t-test (α = 0.05). Welch’s correction was applied when variances were significantly different.Embryos were fixed for 20 min using 4% paraformaldehyde in PBS, devitelinized with a heptane:methanol 1∶1 mixture, and blocked with 0.1% Triton-X-100 in PBS (PBT) with 1% BSA for 15 min and later with 2% BSA in PBT for 30 min. Subsequently they were incubated with rabbit anti-GFP (1∶200 Torrey Pines Biolabs) antibody diluted in blocking solution containing 1% donkey serum for 2 h at room temperature. After washes embryos were incubated with an anti-rabbit-FITC  secondary antibody for 45 min. Muscleblind detection used sheep anti-Muscleblind 1∶500  for 2 h Drosophila genome project strain 1; Gr22b1 Gr22d1 cn1 CG33964R4.2 bw1 sp1; LysC1 MstProx1 GstD51 Rh61y . 1w1118y flies were also from Bloomington IN. All constructs were injected into 1118w embryos by BestGene, typically resulting in 2–6 independent transgenic lines.Genomic DNA was extracted from the Drosophila BDGP R5/dm3 and human GRCh37releases.Predictions of transcription factor binding sites used JASPAR


In budding yeast, the Sir2, Sir3 and Sir4 proteins form SIR complexes, required for the assembly of silent heterochromatin domains, and which mediate transcription silencing at the telomeres as well as at silent mating type loci. In this study, under fluorescence microscopy, we found most Sir3-GFP expressions in the logarithmic phase cells appeared as multiple punctations as expected. However, some differences in the distribution of fluorescent signals were detected in the diauxic~early stationary phase cells. To clarify these, we then used ChIP on chip assays to investigate the genome-wide localization of Sir3. In general, Sir3 binds to all 32 telomere proximal regions, the silent mating type loci and also binds to the rDNA region. However, the genome-wide localization patterns of Sir3 are different between these two distinct growth phases. We also confirmed that Sir3 binds to a recently identified secondary binding site, PAU genes, and further identified 349 Sir3-associated cluster regions. These results provide additional support in roles for Sir3 in the modulation of gene expression during physical conditions such as diauxic~early stationary phase growing. Moreover, they imply that Sir3 may be not only involved in the formation of conventional silent heterochromatin, but also able to associate with some other chromatin regions involved in epigenetic regulation. In the eukaryotic nucleus, genomic DNA is packaged into chromatin. The nucleosome is the basic unit of chromatin organization and consists of around 146 base pairs of DNA wrapped twice around an octamer core of histones  –9.Saccharomyces cerevisiae, is a eukaryotic microorganism. Its well-known silent heterochromatic DNA domains are silent mating type loci (HML and HMR), telomeric DNA regions [in vitro. In addition, in the presence of purified yeast nucleosome arrays in an in vitro system, the three Sir proteins require coupling to AAR synthesis for the formation of SIR-nucleosome filaments [The budding yeast,  regions , 10 and  regions –12. The  regions –16. SIR  regions –20. Sir2 regions –12. O-ac regions –25, appe regions . It has  regions  and to i regions  in vitrilaments . Based oilaments , 30–31. in vitro[Sir3 contains a bromo-adjacent homology (BAH) domain in its N-terminal region (amino acids 11 to 196)  and an Ain vitro. Two othin vitro. The 464in vitro–42.in vitro, and preferentially interacts with nucleosomes’ hypomethylated H3K79 and hypoacetylated H4K16 [It has been reported that Sir3 binds to the tail and core regions of the nucleosome ed H4K16 , 43. Theed H4K16 . The Sired H4K16 –29, 45. ed H4K16 .Sir proteins are located at the periphery of the nucleus in 6-10 punctate foci by fluorescence detection/trace methods . It is bWhen yeast cells are inoculated in a rich liquid culture such as YEPD medium, they metabolize glucose in the medium and produce ethanol. When glucose becomes limiting, cells switch to rapid growth with the fermentation of glucose at logarithmic phase to slowly grow with the aerobic utilization of ethanol and undergo a diauxic shift. After a transit diauxic shift, cells grow slowly in the postdiauxic phase as an early stage of the stationary phase. When the ethanol is totally consumed, cells stop growing, exit the proliferating stage and enter the quiescent state as the late stationary phase, a true growth arrest state, i.e., the growth period when cell numbers are no longer increasing. Many gene expressions change during the postdiauxic phase and stationary phase .In this study, we mainly focused on two distinct growth phases: the logarithmic and diauxic~early stationary phases. First, we carefully examined the localization of Sir3 by a current popularly use of fluorescence microscopy. Then, we used a genome-wide approach to analyze changes in Sir3 spreading to map the genome-wide localization patterns of Sir3 via the chromatin immunoprecipitation (ChIP) on chip assay. We found that the genome-wide localization patterns of Sir3 were different from those at these two distinct growth stages, and further analyzed our newly identified Sir3-associated cluster regions in diauxic~early stationary phase cells.The yeast strains used for this study were W303, DMY748  and DMY3993  that were kindly gifts from Dr. Danesh Moazed. These wild type Sir3, Sir3-GFP and Sir3-Myc fusion genes are located at the original chromosomal Sir3 gene locus. Table S1 provids primers for semi-quantitative PCR of ChIP DNA. The assay is expressed as fold enrichment over input and is normalized to an internal control (Actin).Fluorescence images were obtained by a 100X Plan-Apochromat 1.4 numerical aperture lens and an electron-multiplying CCD camera  on an inverted Olympus IX71 wild field fluorescence microscope with a DeltaVision Core system (Applied Precision). Serial focal sections of the z-axis with a spacing of 0.2 µm were collected. Deconvolution and maximal projection of the images were performed by Softworx software (Applied Precision). The analysis of the fluorescence images were performed by UVP BioSpectrum imaging system (UVP) associated software and ZEN (Carl Zeiss) packaged software.The preparation method of EM samples were mainly followed the manufacturer's instructions (Leica Microsystems) and modified, see previous descriptions –56. BrieS. scerevisiae whole genome tiling array chip (NimbleGen), until the step of scanning the chip, and data analysis using the NimbleScan program and SignalMap software. Data had been deposited in GEO (accession #GSE39437).The assays were as described previously  with somIn this study, first, we monitored the growth curves of the yeast strain with a wild type Sir3, Sir3-GFP and Sir3-Myc fusion gene on chromosomes at the authentic Sir3 location, respectively. We observed all three strains appeared to have similar growth curve patterns in a standard culture of YEPD medium . It was suggested that fusion Sir3 gene with GFP or Myc tag should not affect the normal growth of yeast. According to the growth curves, we then focued and chose to harvest cells of all strains grown either on an OD valued around 3 of the logarithmic phase (Log) or on an OD valued around 10 of diauxic~early stationary phase (Sta) for all further experiments.In Sir3-GFP strain grown to the logarithmic phase, under fluorescence microscopy, we found that Sir3 distribution appeared mainly as multiple discrete punctations , S1B thaBecause overexpression of Sir3 causes it to spread for a longer distance along the telomeres , we specTo investigate the detailed genome-wide localization pattern of Sir3, we applied the ChIP assays in combination with a NimblGen high-resolution whole genome tiling array chip (ChIP on chip). Genome-wide localization of the Sir proteins has been previously reported using other microarray chips –58 and CHML locus. In contrast, the Sir3 association domain at HMR was clearly separated from its telomeric association domain  genes have been identified as secondary recruitment sites for Sir3  and in tInterestingly, at most newly discovered localizations of Sir3, Sir3 were not only distributed at promoter regions but also spread on the coding regions of genes. Therefore, to classify these newly discovered localizations,, we aligned the newly identified genes by their transcription start sites (TSS) and plotted the association patterns of Sir3 binding clusters using the parameters of enrichment ratio against the distance from the TSS. Five cluster pattern sets were generated by K-means clustering analysis with K = 5 . The cluster 1 set was shown to have a peak of Sir3 binding at promoters (around 2kb upstream of the TSS), whereas in the cluster 2 set, the peak extended through the TSS and exhibited as a plateau. The cluster sets 3 and 5 contained one major and one minor peak. The major peak of cluster 3 appeared at upstream of TSS, but that of cluster 5 was at downstream of TSS. In addition, both the minor peaks of these two clusters were at around 2.5kb and 3kb downstream of TSS, respectively. In addition, in cluster 4 set, a broad peak was shown at around 1~2kb downstream of TSS. However, it remains to be clarified whether these 5 cluster sets individually correlate with distinct biological function/process and whether these associations of Sir3 within the coding region of genes represent a distinct functional classification and whether Sir3 plays a critical role in the modulation of gene expression in these regions.TSA1, GAD1, SOD1, SGF29 and HST1. Nevertheless, through an ontological analysis, these genes’ products were found to be diverse with roles encompassing many biological functions, such as DNA/RNA binding, protein binding, transcriptional regulation, transferase activity and signal transduction  are older than cells usually used in most of bioassays. They are naturally undergoing older processes. As aging is one of the cellular functions that Sir2 is involved in, it is possible that the initial stage of aging related function of Sir3 may be directly related to its interactions with Sir2.Overall, the genome-wide Sir3 association pattern in diauxic~early stationary phase cells has opened a new aspect in the field of epigenetics and transcriptional regulation and might provide some initial clues into the mechanisms connected with responses to environmental change and aging process.Click here for additional data file.