Tcrb) locus. These include transcriptional promoters and enhancers, which interact with each other in conformational space to form a promoter-enhancer holocomplex. The holocomplex subsequently recruits the SWI/SNF chromatin remodeling complex, leading to locus activation. However, the factors that mediate conformation and epigenetic changes required for efficient Tcrb gene assembly are unknown. We hypothesized that active transcription may stabilize the holocomplex. In order to test this, we chemically induced promoter-proximal-pausing in proT cells. We discovered that in the absence of elongating RNA polymerase II, the promoter-enhancer holocomplex is disrupted without any robust changes in transcription factor binding, structural protein recruitment or histone acetylation across the locus. Ongoing work seeks to decipher whether the transcriptional machinery is required at all for holocomplex stability. Meanwhile, in order to dissect the spatial and epigenetic mechanisms that lead to Tcrb activation, our lab has generated mutated versions of Tcrb cis-elements in a proT cell line model using Zinc Finger Nucleases. Preliminary studies on these mutants have shown that the holocomplex formation requires the enhancer (or enhancer bound elements) and also CTCF sites flanking the locus. Ongoing studies seek to identify which CTCF sites are necessary and sufficient for holocomplex stability. Using these and additional mutants, we are testing the role played by cis-elements in maintaining the chromatin and looping of Tcrb, which are essential for recombination.The adaptive immune system endows mammals with a sophisticated mechanism to survive in a world filled with pathogens. This dynamic defense is generated by V(D)J recombination, which enables lymphocytes to uniquely interact with an enormous range of epitopes on foreign antigens. V(D)J recombination is a set of sequentially controlled DNA cleavage and repair events, which brings variable (V), diversity (D) and joining (J) gene segments together to form functional antigen receptor genes. However, the recombination process must be stringently regulated to prevent formation of chromosomal translocations, which can lead to tumors. The process of V(D)J recombination is regulated at the level of tissue, stage and allele specificity by genetic and epigenetic mechanisms. Our lab has characterized several genetic elements that regulate chromatin accessibility and recombination at the T cell receptor beta (


Drosophila melanogaster CBP (nejire) ChIP–seq peaks with regions bound by 40 different transcription factors in early embryos, and we found high co-occupancy with the Rel-family protein Dorsal. Dorsal is required for CBP occupancy in the embryo, but only at regions where few other factors are present. CBP peaks in mutant embryos lacking nuclear Dorsal are best correlated with TGF-ß/Dpp-signaling and Smad-protein binding. Differences in CBP occupancy in mutant embryos reflect gene expression changes genome-wide, but CBP also occupies some non-expressed genes. The presence of CBP at silent genes does not result in histone acetylation. We find that Polycomb-repressed H3K27me3 chromatin does not preclude CBP binding, but restricts histone acetylation at CBP-bound genomic sites. We conclude that CBP occupancy in Drosophila embryos preferentially overlaps factors controlling dorso-ventral patterning and that CBP binds silent genes without causing histone hyperacetylation.CBP and the related p300 protein are widely used transcriptional co-activators in metazoans that interact with multiple transcription factors. Whether CBP/p300 occupies the genome equally with all factors or preferentially binds together with some factors is not known. We therefore compared  Drosophila embryos and find that master regulators of dorsal-ventral patterning, the transcription factors Dorsal and Medea, target CBP to the genome. CBP occupies mainly active genes in embryos, where histones become acetylated. Surprisingly, the presence of CBP at silent genes does not result in histone acetylation. We find that repressive chromatin prevents histone acetylation by CBP. Our results demonstrate that CBP preferentially associates with some gene regulatory networks and that CBP binds silent genes without causing histone acetylation. These data have implications for prediction of transcriptional regulatory sequences and for understanding gene regulation by one of the most widely used co-activators in animal cells.Development of an embryo into different cell types relies on regulation of gene expression, whereby genes are coordinately turned on or off. CBP and the related p300 protein are central regulators of gene expression in animal cells. These co-activator proteins facilitate gene activation by a multitude of transcription factors, possibly through their histone acetyltransferase activity. Consequently, loss of CBP/p300 gene function disrupts development and is lethal in mice, worms, and flies. How CBP/p300 is targeted to regulatory DNA sequences is not understood. Here, we have compared genome occupancy of CBP with 40 different transcription factors in  Here, we use the early Drosophila melanogaster embryo to compare the genomic distribution of p300/CBP with 40 transcription factors involved in embryonic patterning and cell differentiation.CREB-binding protein (CBP) and its paralog p300 are widely used transcriptional co-regulators with histone acetyltransferase (HAT) activity  is cell-lethal, whereas the hypomorphic 1nej allele reduces CBP expression approximately two-fold, and causes embryonic patterning phenotypes 1nej embryos tld gene activation 10BToll, results in high Dorsal concentration in all embryonic nuclei, generating embryos consisting entirely of presumptive mesoderm. In embryos derived from rm9/rm10Toll mutant mothers twist (twi) and snail (sna) with low-affinity bindning sites are turned on in ventral, presumptive mesodermal cells where Dorsal concentration is highest. When Dorsal sites are positioned in proximity to AT-rich binding sites, Dorsal is converted to a repressor that recruits the co-repressor Groucho and thereby prevents expression of dorsal ectoderm targets such as zerknüllt (zen), tld, and dpp in lateral and ventral parts of the embryo Embryonic dorsal-ventral patterning is controlled by an intra-nuclear concentration gradient of Dorsal, a Rel-family transcription factor related to NF-κB. Over 50 Dorsal target genes are known, constituting one of the best understood gene regulatory networks in animal development . The CBP serum is affinity-purified and CBP-specific, and quantitatively similar levels of CBP occupancy is found at several loci with another CBP antibody .We compared the published CBP binding data in antibody . To calc7gd embryos. These CBP peaks were compared to the occupancy of the previously mapped 40 transcription factors We identified 3013 high-confidence peaks in wild-type and 1939 CBP peaks in 7gd mutant embryos. As shown in 7gd mutant embryos than CBP peaks in wild-type. By contrast, many other factors overlap the 7gd CBP peaks better than wild-type peaks. This indicates that CBP occupies regions bound by multiple factors to a larger extent in 7gd than in wild-type embryos (see below). In 7gd embryos that lack Dorsal in the nucleus, CBP continues to associate with 84% of Dorsal-binding regions, suggesting that other factors maintain CBP binding at these places in the absence of Dorsal. However, CBP occupancy at the top 300 Dorsal-binding regions is significantly lower in 7gd embryos compared to wild-type (paired T-test p = 5.22×10−9), showing that CBP occupies many Dorsal targets in 7gd embryos less strongly. We therefore extracted the regions where the CBP-peaks were completely lost in 7gd embryos, and compared them to all of the regions bound by the 40 factors in wild-type (not only the top 300 regions). High-confidence CBP peaks in wild-type with a CBP enrichment of less than 0.5  in 7gd were considered to be completely lost. It was found that 76% and 67% of the peaks that are lost in 7gd embryos overlap Dorsal or GAF respectively, but that no other factor overlaps these CBP-peaks to more than 22% , a transducer of Dpp-signaling  in Race and pannier in 7gd mutants they become expressed throughout the entire circumference of the embryo. Thus, Dpp-signaling occurs in the entire embryo in 7gd mutants, thereby providing a genetic background in which binding of CBP to Dpp-target genes can be visualized.To identify Dorsal-independent transcription factor sites that are co-occupied by CBP, we next compared the CBP peaks in ignaling . In gd7 s absent . This reush is hardly detectable in wild-type embryos, but highly evident in 7gd embryos. Similar results were obtained for other Dpp-target genes, including Race, tail-up, GATAc, and pannier . Our results indicate that CBP becomes recruited to Dpp-target genes upon signaling, consistent with previous observations that Dpp-signaling is impaired in CBP mutant embryos As illustrated in  pannier . As expe pannier . We conf7gd, those where CBP binding increases at least 2-fold in 7gd versus wild-type (7gd Up), those where CBP binding decreases at least 2-fold in 7gd versus wild-type (7gd Down), and those that are completely lost in 7gd. We first looked at how many other factors that occupy these CBP-bound regions, measured in their HOTness. High occupancy target (HOT) regions are defined as genomic sites binding at least one of the 40 transcription factors Drosophila, C. elegans, and human genomes, and are associated with open chromatin, but their function is not understood 7gd have a high HOTness, whereas unchanged, down and lost in 7gd are decreasingly HOT. This is also illustrated in 7gd versus wild-type is plotted against HOTness. Remarkably, there is an almost perfect correlation between the difference in CBP occupancy in 7gd versus wild-type embryos and mean HOTness. Thus, in 7gd embryos, CBP is present at regions where many factors are bound, but decreases or is lost at regions bound by only few factors. However, the highest mean CBP occupancy in wild-type is found at regions where CBP binding decreases in 7gd embryos  strongly overlap H3K4me1, a histone modification associated with enhancer sequences, as well as the “active” histone marks H3K18ac and H3K27ac, but are depleted of H3K27me3-repressed chromatin  and H3K4me3 (active promoters), histone acetylation, but also with H3K27me3 (Polycomb-repressed chromatin)  . Unchangromatin) . Like gd7gd embryos overlap mainly Dorsal and GAF , CBP binding is selectively reduced at regions where Dorsal, but few other factors bind. Interestingly, strong CBP-peaks that are decreased in 7gd embryos (7gd Down) overlap more with the inactive chromatin mark H3K27me3 than with the active histone marks H3K18ac, H3K27ac, and H3K4me3 .Regions where CBP binding is decreased in  and GAF . Especia H3K4me3 . The regequences . Regions7gd overlap virtually none of the top 300 sites for the 40 transcription factors  and dorsal ectoderm-targets (up-regulated in pipe and 7gd) as defined in 7gd mutant embryos. Taken together, our results show that differences in CBP occupancy correlate well with changes in gene expression genome-wide.Dorsal-regulated genes have previously been identified by comparing the difference in gene expression in  embryos . All genecreased . We then7gd, rm9/rm10Toll, and 10BToll mutant embryos by ChIP-qPCR. In these mutant backgrounds the entire embryo is converted to dorsal ectoderm (7gd), neuroectoderm (rm9/rm10Toll), or mesoderm (10BToll). We normalized the binding of Dorsal, CBP, and histone modifications to two intergenic regions, selected based on the absence of protein binding and histone modifications, and plotted the fold enrichment relative these intergenic sites. The histone modifications were additionally normalized to histone H3 levels . This is consistent with the genome-wide correlation of reduced CBP occupancy with lower gene expression and lack of Dorsal protein . This shows that the amount of CBP bound to a genomic region is not the only determinant of histone acetylation levels. In 10BToll embryos, twi expression is turned on in the entire embryo, and CBP as well as histone acetylations are present in high amounts .In the mesoderm, Dorsal targets such as e embryo . Less CB protein . Interessna promoter is not decreased in 7gd mutant embryos compared to wild-type . We therefore measured histone lysine methylation at Dorsal-target genes, a modification mutually exclusive to lysine acetylation. Interestingly, we observed high amounts of H3K27me3 in 7gd and rm9/rm10Toll embryos , which is likely explained by the higher amounts of H3K27me3 in rm9/rm10Toll as compared to 10BToll embryos . We note that H3K18ac levels in mutant embryos correlate with changes in CBP amount to a better extent than other histone acetylations, indicating that H3K18 may be a major in vivo target for CBP's HAT activity.Interestingly, the high levels of H3K27me3 over the tld and zen are repressed by Dorsal, and therefore more highly expressed in 7gd mutants that lack nuclear Dorsal, but completely repressed in rm9/rm10Toll and 10BToll mutants, except at the embryonic poles  and acetylation of histones increases . This is consistent with the genome-wide correlation of CBP occupancy and gene expression , there is equivalent amounts of histone acetylation in 10BToll embryos . Taken together, our analysis of Dorsal-target genes in different tissues shows that CBP can bind to these genes when they are silenced, but that this does not result in high levels of histone acetylation.The dorsal ectoderm targets ic poles . In gd7 pression . In both embryos . This maIn conclusion, our results suggest, 1) that CBP binding does not always correlate with gene expression or Dorsal binding, 2) that H3K18 acetylation levels closely follow CBP-binding, 3) that changes in histone acetylation can occur without a corresponding change in CBP binding, 4) that CBP binding is not prevented by the presence of H3K27me3 (Polycomb)-repressed chromatin, 5) but that H3K27me3-chromatin may restrict histone acetylation by CBP and other HATs.Drosophila embryo are associated with CBP to a larger extent than e.g. anterior-posterior patterning.CBP and the related p300 protein are widely used transcriptional co-activators in metazoans that interact with transcription factors of all major families Drosophila embryos to previously mapped transcription factors 7gd mutants), and found that CBP peaks that overlap regions where Dorsal, but few other factors bind in wild-type are selectively reduced in 7gd mutant embryos. Instead, strong CBP-bound regions in 7gd mutants overlap best with regions bound by the Smad protein Medea, a mediator of Dpp-signaling. We and others have previously shown that signaling by the TGF-ß molecule Dpp is exceptionally sensitive to a small decline in the level of CBP in Drosophila embryos By comparison of CBP-bound regions in 2–4 hour old in vivo. However, many of the Bicoid peaks are found in HOT regions that bind several transcription factors. Therefore, it may not be Bicoid that targets CBP to these sites. Furthermore, although the shape of the Bicoid gradient is slightly changed in embryos from the CBP hypomorph 1nej, activation of Bicoid-target genes is not compromised by the decrease in CBP levels in 1nej embryos hb, otd, kni, and eve , Paired, Caudal, and Bicoid . Previou and eve . Thus, atterning .7gd embryos are lacking strong GAF binding, despite their high HOTness  gene, which is co-occupied by Dorsal and CBP but where CBP binding is reduced more than two-fold in 7gd embryos  compared to Dorsal target gene regions where CBP binding is not changed . What maintains CBP binding on these genes in the absence of Dorsal is not clear. Presumably, other factors recruit CBP to these sites in the absence of Dorsal, but we have not found a common factor for the regions where CBP binding is unchanged. We note, however, that GAGA-factor (GAF) associates with many of the CBP-binding regions in wild-type embryos, but much less with CBP-binding regions in 7gd embryos. It is possible that GAF contributes to the recruitment of CBP to chromatin.As summarized in  embryos . Althoug embryos , there idpp, zen, and tld, are activated in all cells of 7gd mutant embryos , the twi promoter contains less histone acetylation than in the dorsal ectoderm (7gd embryos) although H3K27me3 levels are reduced and CBP binding not decreased compared to dorsal ectoderm  2540–3190 fusion protein was used to immunize rabbits and the resulting serum affinity-purified as described in S2 cells . The rabS2 cells . A guineDrosophila embryos were collected on grape-juice plates, dechorionated, and used to prepare chromatin extracts as described below or fixed for in situ hybridization. Wild-type embryos were collected from 1118w flies, and embryos where Dorsal fails to enter nuclei were collected from 7gd homozygous mothers. Embryos with uniformly high levels of Dorsal in all nuclei were collected from 10BToll heterozygous females obtained directly from the balanced stock (10BToll/TM3 Sb Ser/OR60). rm9/Tollrm10Toll trans-heterozygous females were used to collect embryos with intermediate levels of Dorsal in all nuclei.Two- to four-hour old Whole-mount RNA in situ hybridization using digoxigenin-labeled probes was performed as described previously 2, 1% NP40, and proteinase inhibitors (Roche)). Lysate was shaken at 4°C and centrifuged. Pre-clearing and immunoprecipitation with the CBP antibody was done as previously described Two to four hour old embryos were dechorionated and crushed in a Dounce homogeniser in Lysis buffer  for 15 minutes at room temperature. The reaction was stopped with 0.225 M glycine and nuclei washed 3 times in Buffer A1 and once in Lysis buffer . Nuclei were resuspended in Lysis buffer with 0.1% SDS and 0.5% N-lauroylsarcosine and sonicated in a Bioruptor (Diagenode). Chromatin extract was centrifuged to remove debris and diluted in an equal amount of lysis buffer, followed by snap freezing in liquid nitrogen and stored at −80°C.Two to four hour embryos were dechorionated, crushed in a Dounce homogeniser, and cross-linked with 1.8% formaldehyde in Buffer A1  blocked with 1 mg/ml BSA (Sigma Aldrich) were mixed with indicated antibodies. A bead-antibody complex was formed at 4°C for at least 4 hours. Beads with bound antibody were captured on magnet, and beads were resuspended in chromatin extract corresponding to 30–40 µl of embryos followed by incubation at 4°C over night. Beads were washed 5 minutes each with sonication buffer , WashA , WashB  and TE. Beads were transferred in TE to new tubes and resuspended in Elution buffer . Cross-linking was reversed at 68°C for at least 4 hours and Proteinase K treated followed by DNA purification with phenol-chloroform extraction and ethanol precipitation. The DNA was resuspended in 200 µl 0.1×TE.The ChIP material was analysed either by qPCR or sent to the Uppsala Genome Center for SOLiD (TM) ChIP-Seq Library preparation (Rev date 18 March 2010), size selection (100–150 bp+adapters 90 bp ≈250 bp), and sequencing using SOLiD4 50 bp fragment run. Approximately 10 ChIPs for each genotype were pooled and used for the ChIP-seq libraries.Mi-2 locus or to two intergenic sites with background levels of binding. For histone antibodies, values were further normalized to total amount of histone H3.For ChIP-qPCR, duplicates with 2 µl DNA each were used for analysis by qPCR, using 300 nM primers , and iQ Drosophila melanogaster reference sequence (release 5) using the classical mapping in Applied Biosystems Bioscope software v1.2.1. The number of uniquely mapping reads was 21,667,438 (wild-type input), 17,131,635 (wild type CBP), 25,683,953 (7gd input) and 18,325,130 (7gd CBP). Average read-count per nucleotide was calculated for IP and input samples. For regions where the IP sample had at least the average read-count, a ratio of IP-input  was calculated. If the read count in the input was below the average read-count (in the input sample) it was set to the average. All ratio values were then adjusted by reducing each value with the average read-count in IP minus the average read-count in input. This linear adjustment was to normalize for differences in sequencing depth of IP and input. Then the ratio value at an interval of 35 bp was extracted across the genome and median smoothed using a window size of 350 bp. Windows with fewer than 5 data points were discarded. The dynamic range of CBP in wild type was −2.0 to +5.1 and CBP in gd7 −1.8 to +5.1.Reads were aligned against the 7gd were extracted, corresponding to a cut-off of 1.9 in wild type and 1.9 in 7gd. Bound regions were then defined as regions of at least 200 bp and a region was extended as long as there was a value within 200 bp of the previous value. The value of each detected region was set to the average of the highest five consecutive ratio values. The center of the peak was set to the middle position of the five highest consecutive ratio values. When comparing CBP in 7gd to CBP in wild type we did not normalize the two data sets to each other since the dynamic range and the 5% cut-off was more or less identical.To calculate peaks and bound regions, the 5% highest ratio values in both wild-type and When the overlap of bound regions of dataset X was to be compared to bound regions of other datasets first all regions of dataset X was used. Overlaps of at least one nucleotide were scored. Then an increasing cut-off for the binding values of data set X was applied. We used 20 cut-offs from the lowest to the highest binding value of dataset X in steps of maximum value minus minimum value then divided by 20. We plotted percent overlap using an increasing cut-off until about five percent of the binding regions of dataset X remained. In plots where only three cut-offs are shown, the cut-offs are; 1) no cut-off , 2) an average of cut-offs 1 and 3, 3) the highest cut-off where only the ∼5% most enriched regions remained.7gd embryos were calculated (as described above) within each CBP bound region from WT embryos. In regions for which the 7gd peak values were below 0.5  CBP was considered lost. Next, a ratio of 7gd and WT peak values was calculated. Regions with less than a two-fold difference (−1 to 1 in log2 ratio) were considered unchanged. Regions with more than two-fold higher 7gd peaks were defined as going up and regions with more than two-fold higher WT peaks where defined as going down. When CBP peak values in WT embryos were determined within CBP bound regions in 7gd embryos we found only two regions where no CBP could be detected in WT. We therefore did not define a class of regions unique for 7gd embryos.CBP peak values in All statistics was performed using Statistica 10.0 (Statsoft). All reported T-tests are two-tailed without assuming equal variance. When multiple histone acetylations were compared between genotypes, all replicates from each acetylation were treated as one sample.The ChIP-seq data is deposited in GEO under accession number GSE34221.Figure S1Drosophila S2 cells. Primers with 5′T7 RNA polymerase-binding sites  and lysed for 30 minutes at room temperature. Centrifugation at 13000 rpm for 5 min followed, and the supernatant was saved. The protein concentration was measured by the BCA protein assay kit (Thermo scientific). SDS-PAGE sample buffer was added, the samples denatured at 95°C for 5 min and centrifuged at 13000 rpm for 5 min before loading on 7.5% SDS-PAGE gels. The gel was either Coomassie-stained (B) or transferred to PVDF membrane (Bio Rad laboratories) at 30 V over night (A). The membrane was blocked in PBS containing 5% non-fat dry milk and incubated with the affinity-purified rabbit anti-dCBP serum (1∶200 in PBS containing 1% BSA) over night. The membrane was washed with PBS three times and incubated with HRP-coupled anti-rabbit antibody  for one hour followed by ECL detection , and exposure to a Luminiscent Image Analyzer . Expression of the loading controls that we used to re-probe the membrane with was affected by the CBP RNAi treatment. We therefore compared total protein concentration between samples on Coomassie-stained gels. Arrow in A) points to full-length CBP, the other bands represent degradation products since they are also reduced in strength by CBP RNAi treatment. C) Comparison of ChIP signals obtained with the rabbit anti-dCBP aa 2540–3190 serum with ChIP signals from a guinea-pig anti-dCBP aa 1–178 serum The affinity-purifed rabbit serum is CBP specific. A–B) Western blot and Coomassie-staining of CBP RNAi-treated ng sites  were use(PDF)Click here for additional data file.Figure S27gd mutant embryos. A) In situ hybridization of Dpp target genes Race and pnr in wild-type (1118w) and 7gd derived embryos. Two to four hour old embryos were hybridized with digoxigenin-labeled probes and are oriented with anterior to the left, and dorsal up. Note the expanded expression of the Dpp target genes in 7gd mutant embryos. B) ChIP-qPCR of CBP recruitment to Dpp target genes Race and pnr. Values are presented as fold over CBP binding at Mi-2 whose expression and CBP binding is unaffected by the levels of Dorsal. As a negative control, the average of background CBP binding at two intergenic loci that do not bind CBP is included (IG). C–F) CBP and Medea occupancy overlap at the Dpp-target gene loci Race (Ance) (C), pnr (D), GATAc (grn) (E), and tup (F). CBP ChIP-seq peaks  in wild-type (wt) and 7gd mutant embryos, as well as Medea ChIP-chip peaks in wt are shown. Occupancy is plotted as log2-fold enrichment over input.CBP occupies Dpp-target genes in (PDF)Click here for additional data file.Figure S3eve (A), hb (B), kni (C), and otd (oc) (D). CBP ChIP-seq peaks  in wild-type (wt) and 7gd mutant embryos, as well as Bicoid ChIP-chip peaks in wt are shown. Occupancy is plotted as log2-fold enrichment over input.CBP and Bicoid do not co-occupy Bicoid-target genes. A–D) CBP and Bicoid occupancy does not overlap at the Bicoid-target gene loci (PDF)Click here for additional data file.Figure S47gd embryos that lack nuclear Dorsal, CBP occupancy is reduced at some Dorsal-target genes (A–C), but relatively unaffected at other Dorsal-target genes (D–F) compared to wild-type. ChIP-seq peaks for CBP in wild-type (wt) and 7gd mutant embryos (raw data without cut-off), as well as Dorsal ChIP-chip peaks in wild-type are shown for the Mef2 (A), htl (B), brk (C), neur (D), ind (E), and ths (E) loci. Occupancy is plotted as log2-fold enrichment over input.CBP occupancy at Dorsal-target genes. In (PDF)Click here for additional data file.Table S1Overlap between previously published CBP ChIP-seq peaks in 0–4 hour embryos (XLSX)Click here for additional data file.Table S27gd embryos and the 300 most strongly occupied regions for 40 sequence specific transcription factors 7gd compared to wt (7gd Up), similar levels of CBP occupancy (7gd Unchanged), at least 2-fold less occupancy in 7gd (7gd Down), and wt CBP peaks with no or background levels of binding in 7gd (7gd Lost).Overlap between CBP ChIP-seq peaks from 2 to 4 hour old wt or (XLSX)Click here for additional data file.Table S37gd embryos and all or the 300 most strongly occupied regions for 40 sequence specific transcription factors Overlap between CBP ChIP-seq raw data from 2 to 4 hour old wt or (XLSX)Click here for additional data file.Table S47gd embryos.Overlap between the 300 most strongly occupied regions for 40 sequence specific transcription factors (XLSX)Click here for additional data file.Table S57gd embryos and all regions for 40 sequence specific transcription factors 7gd compared to wt (7gd Up), similar levels of CBP occupancy (7gd Unchanged), at least 2-fold less occupancy in 7gd (7gd Down), and wt CBP peaks with no or background levels of binding in 7gd (7gd Lost).Overlap between CBP ChIP-seq peaks from 2 to 4 hour old wt or (XLSX)Click here for additional data file.Table S67gd, rm9/rm10Toll, and 10BToll embryos. qPCR values were compared to input, and then normalized to two intergenic sites with background levels of binding. For histone antibodies, values were further normalized to total amount of histone H3. Values represent the average and standard deviation of the indicated number of independent ChIP experiments.Binding of CBP, Dorsal, and indicated histone modifications at Dorsal target genes, expressed as fold over background. ChIP was performed on 2 to 4 hour old wt, (XLSX)Click here for additional data file.Table S77gd embryos.Peak position, peak value, and distance to closest TSS for CBP ChIP-seq peaks from 2 to 4 hour old wt or (XLSX)Click here for additional data file.Table S8Primer sequences.(PDF)Click here for additional data file.


The complexity and diversity of eukaryotic organisms are a feat of nature's engineering. Pulling the strings of such an intricate machinery requires an even more masterful and crafty approach. Only the number and type of responses that they generate exceed the staggering proportions of environmental signals perceived and processed by eukaryotes. Hence, at first glance, the cell's sparse stockpile of controlling factors does not seem remotely adequate to carry out this response. The question as to how eukaryotes sense and respond to environmental cues has no single answer. It is an amalgamation, an interplay between several processes, pathways, and factors—a combinatorial control. A short description of some of the most important elements that operate this entire conglomerate is given in this paper. The orchestration of various biological processes with high fidelity requires a precise control over temporal and spatial expression of genes. The regulation of gene expression in eukaryotes can occur at various steps, namely, transcription, m-RNA splicing, translation, and Posttranslational modifications.Transcriptional control can be achieved at any of the various stages—initiation, elongation, and termination. Initiation is marked by the assembly of the preinitiation complex at the promoter of the corresponding gene. The Preinitiation complex, comprised of RNA polymerase II and its auxiliary components  that bound to the core promoter, can only initiate basal levels of transcription . Proper Some regulators act by increasing (enhancers) or decreasing (repressors) the rates of transcription by stabilizing or destabilizing the interactions of the polymerase with the DNA or the auxiliary components, respectively. Others act by modifying chromatin structure. Chromatin structure greatly affects transcription in eukaryotes as it determines the accessibility of different regions of DNA to the incoming binding factors. Some regions are tightly coiled and are lesser accessible to the transcriptional machinery, while others are loosely coiled and easily accessible. A class of regulators called chromatin modifiers (CM) affects the rate of transcription initiation by increasing or decreasing accessibility of various regions. CMs function by two mechanisms—chromatin remodelling or by facilitating histone tail modifications. Both of these processes are ATP intensive and involve either increasing or decreasing interactions between the parent DNA and the histone cores . Another class of molecules called coregulators binds to regulators and affects transcription by either stabilizing or destabilizing interactions between regulators and the Basal-transcription machinery. Long noncoding RNA molecules constitute a special class of coregulators because of their proteinaceous nature. The latter is also the reason why the study of these molecules has been of great interest in recent times. Some of the findings will be presented in a later section.Transcriptional elongation involves the actual process of m-RNA synthesis from parent DNA strand. It has acquired great interest in the recent times because of its possible role as a checkpoint for control of gene regulation. Transcriptional pausing is a very common feature in elongation. Pausing occurs due to interactions between the RNA polymerase molecule and the m-RNA molecule, which may assume secondary structures such as hairpin loops and lead to consequent transcriptional attenuation. The latter may lead to premature termination and may consequently result in alternate products, thereby, affecting gene expression. Certain in vitro simulation studies have shown that pausing of the polymerase is briefly followed by microbursts in m-RNA yield, which was explained by the authors as a consequence of collision of two RNA polymerase molecules .The primary m-RNA product may be subject to alternate splicing or preliminary RNA editing. Both of these processes may yield different translational precursors and, consequently, different protein products. The mature m-RNA transcript is finally translated into the corresponding protein product. Control at the level of translation is achieved by numerous mechanisms. One mechanism that has been extensively studied involves the role of small non-coding RNA molecules. These RNA molecules are majorly of two types—small interfering RNA (siRNA) and micro-RNA (miRNA). Both siRNA and miRNA molecules inhibit translation by two predominant mechanisms. siRNA molecules lead to the recruitment of RNA-induced silencing complexes (RISCs), which cut up the target mRNA molecules. On the other hand, miRNA molecules lead to the recruitment of microRNA-induced silencing complex (MISC), which physically blocks ribosomal translocation during translation. The role of small non-coding RNAs has frequently been noticed in the process of cell differentiation .The final control checkpoint comes at the level of posttranslational modifications (PTMs). These affect the structure and consequently the activity, stability, and functioning of the protein. PTMs are hence critical to the cellular role of proteins. PTM in histones such as methylation, acetylation and ubiquitination at specific residues critically affects the conformational states of chromatin in the cell by altering the stability of histone-DNA interactions .Although numerous checkpoints exist, complex controls of gene expression are noticed almost extensively at the level of transcriptional initiation. Transcriptional Initiation controls are extremely diverse and complex and display extreme variability in terms of the number of types of underlying molecular signals. The reasons supporting the latter are presented below.The number of genes in an organism by far outnumber the numbers of transcription factors. For example, the human genome has about 20,000–25,000 genes. Each of these genes has multiple, unique temporal, and spatial expression patterns. Yet the number of DNA binding transcription factors is around 1850 ..18].The posttranslational states of transcription factors affect the functionality of the transcription factors. Scientific literature evidences of various posttranslational events such as acetylation, phosphorylation, and methylation and their effects on the functions of different classes of TFs have been recorded. The High-Motility Group Box (HMGB) family of proteins posees a good example of the same.The HMGB class of proteins has significant roles in DNA maintenance and recognition processes. They are seen to possess DNA binding and DNA bending functions. The post-translational modifications affect these functions of the HMGB proteins. In bovine HMGB1 proteins, acetylation of Lys 3 increased the former's affinity for UV-damaged DNA , while tOne method of control of gene expression is by regulating of availability of TFs. In the HMGB1 proteins, it has so been found that the post-translational state of these proteins affects their cellular location . Such coChromatin structure often prevents interaction of the transcription machinery with the promoter of certain genes and hence acts as a repressor. In the presence of tissue-specific cofactors, some transcription factors have the ability to modify chromatin structure and thus allow tissue-specific expression of certain genes. This involves ATP-dependant noncovalent modification of local chromatin structure and addition or removal of covalent groups from histone tails.MyoD is a member of the myogenic basic helix-loop-helix family of regulatory factors (MRFs) and is essential for the induction of muscle-specific genes like myogenin, which leads to myogenesis during embryogenesis. This factor is indispensible for commitment to the skeletal muscle lineage , 24. Myo It was found that MyoD-mediated activation of muscle-specific genes is heavily dependent on the SWI/SNF enzyme which is an ATP-dependant chromatin remodelling complex , 26. On This example elucidates that acetylation pattern; chromatin structure and its remodelling in part dictate tissue-specific behaviour of transcription factors.The activity of transcription factors and hence the different patterns of gene expression in different tissues are controlled by another class of molecules, known as the coregulators. These molecules greatly increase the variety of responses that can be elicited by a limited repertoire of transcription factors. Coregulators do not typically have DNA binding activity, rather they exhibit protein-protein interaction and bind to the DNA bound transcription factors. Each type of cell has a specific set of co-regulators and this plays a major role in determining the response to a particular signal. A transcription factor bounded to a specific DNA sequence can act as either an activator or a repressor depending upon the coregulator associated with it. NcoR have histone deacetylase activity and play a role in gene silencing. Another class of co-regulators binds to transcription factors and is involved in recruiting RNA polymerase and the core transcription machinery. This includes members of the TRAP/DRIP/Mediator/ARC family.Coregulators can perform various functions and can be broadly classified on that basis. A class of coregulators can modify histones making certain genes accessible (coactivators) or inaccessible (corepressors) to the transcription machinery. For example, p300 and CBP are powerful histone acetyltransferases (HATs) and hence they act as coactivators, whereas corepressors like Finally, a third class of co-regulators has ATP-dependent DNA unwinding activity and is essential for the transcription of certain genes. The SWI/SNF complex belongs to this group .In many cases, TFs do not bind to the DNA even though the REs of TFs are freely available for binding. The reason for this is that the population of TFs present exists in conformational states that do not favour DNA binding. In such cases, co-regulators bind to the TFs prior to the DNA binding event. This shifts the populations to conformational states that are stabilized by binding with a specific RE sequence  29]..29].NMR studies of p53 binding to Apoptosis stimulating protein of p53 (ASPP) are a testament to this mechanism . While pCo-regulators have the ability to recruit secondary co-regulators that further diversify the function of a particular transcription factor. For example, the co-activator CBP can bind to transcription factors as well as recruit coactivators like SRC-1 and pCAF, which have similar HAT activity. In addition to serving as a primary coactivator, CBP can act as a secondary co-activator to PCG-1 that binds to another transcription factor. Such varied and complex interplay of molecules having flexible nature gives cells the option of forming innumerable biological programs using a limited arsenal of transcription factors  23]..23].Some DBTFs have binding sites on the promoters of a variety of genes that they induce in different tissues. Along with this, they also induce certain tissue-specific genes in some cells and not in the others. This discretion can be, in part, attributed to the presence of certain co-regulators in some tissue as against the others. This phenomenon is observed in the following cases.Expression of immunoglobulin (Ig) genes requires octamer binding proteins (Oct) to bind to the promoters of Ig genes. Oct-1 is expressed in many cells; however, Oct-2 is mostly restricted to B cells. Therefore, it was hypothesized that Oct-2 is the factor that enables B cells to produce Ig molecules. However, Oct-2 alone was unable to perform this function in cell free systems and in transfected cells. Biochemical purification of Oct-1 and 2 revealed a co-activator, OCA-B which has chromatin modifying enzymatic activity . It recrα-SM actin and calponin [Serum response factor (SRF) binds to the promoters of many muscle genes and induces their expression. SRF also has binding site in the promoters of many nonmuscle-specific genes. SRF is also found in the cells and tissues other than muscle. Therefore, there must be a molecule that is associated with SRF, which imparts muscle-specific function. From in silico data, it was evident that myocardin is one such molecule as it can interact with SRF via an SAP domain and it is found only in smooth and cardiac muscle cells . Experimcalponin . Myocardcalponin , 39. FroLargely, transcription is controlled at the co-regulator level especially through co-activators. Even the co-regulators themselves can be a target for developmental or physiological signals. The combinatorial control of gene expression cannot be fully appreciated without considering the co-regulators. Transcription factors and their associated co-regulators form complexes that control transcription. By the different permutations of co-regulators, even a small number of DBTFs binding to a limited number of cis regulatory elements can form a system, which can execute the innumerable gene expression profiles that are necessary for sustaining eukaryotic organisms. Furthermore, the biological system gains additional control of gene expression by modifying the co-regulators via different cell signalling pathways, which allow the regulators to target different subsets of co-regulators at different genes or in different cell types.Most of the genome consists of non-coding DNA regions. Recent research is indicative of the significance of the long non-coding RNA (lnc-RNA), which is the byproducts of transcription of these regions. Lnc-RNA molecules are 200 nt or greater in length and are seen to play an important role in transcriptional control of gene regulation. They are poorly conserved through the course of evolution, unlike other conserved RNA regulators like micro RNAs, siRNAs, and so forth that function by means of base-pairing mechanisms. There is no concrete mechanism to explain the mechanism of control exercised in the case of lnc-RNAs, but four distinct mechanisms have been observed  . The latter physically associates with PRC2 complexes at the FLC locus and mediates the chromatin modifications that lead to transcriptional repression. The PRC2 complexes are important in carrying out the epigenetic changes observed [Such mechanisms are observed in plants also. For example, the vernalization-mediated epigenetic control of the Flowering Locus C (FLC) in observed .In other instances, lnc-RNAs have been found to impersonate target DNA and hence interfere in the assembly of the regulatory transcriptional machinery. Such a mechanism has mostly been associated with transcriptional repression by means of misleading the effector molecules . An examIn many cases, lnc-RNAs may act as guides and aid in directing the effector molecules, which may be cis-regulators, transacting proteins, or TFs, to their respective binding sites . These aXIC is the recruitment of polycomb repressive complex 2 (PRC2) which is achieved through PRC2 binding to RepA, which is an lnc-RNA transcribed from the 5′end of the Xist gene [RepA guides PRC2 to its target site.  Such a mechanism is prevalent in X-chromosome inactivation (XIC). An important step in ist gene ; that isSometimes, lnc-RNAs might act as scaffolds or recruitment sites of multiple proteins that are to be targeted to a particular gene. By doing so, they aid in bringing together multiple proteins in both time and space .The HOTAIR lnc-RNA is one such example. This stretch of lnc-RNA contains two sites—a 300 nt stretch in its 5′end and a 700 nt stretch towards its 3′end. The former is the binding site for PRC2 proteins, while the latter forms the binding site for LSD1/CoREST/REST complex . Both of The HOTAIR molecule is unique in that it can both act as a scaffold as described above or as a guide that aids in targeting of PRC2 to transregions of the genome. Such integrated approaches have also been found in case of other lnc-RNA molecules, whose functions are implicated in other cellular events . This laThe cell performs an amazing feat interpretation constantly varying external signals and keeping with an ever-changing pattern of gene expression. It does so with an intricate network of mediators and signalling molecules; those are integrated together by using an equally complex system of signal transduction. The plethora of internal signalling required for gene expression is created by the cells by cleverly utilising a comparatively limited reservoir of molecules. The principle of combinatorial control of gene expression that is addressed in this paper sheds light on the mechanisms used by the biological systems to achieve tight regulation of the entire process. The cells use the limited amount of resources available at their disposal, in the most innovative and economic ways to achieve the same. It points to a situation where the cells utilize a handful of molecules in various permutations and combinations to accomplish the startling task of constantly switching on genes and switching them off according to their needs. The controls of gene expression show complex and diverse patterns and are subject to various constraints such as factor availability. It is also noticed that although one tries to map the entire process by a linear series of events, the various steps are very closely knit together (reviewed in ). In sucThe cis regulatory modules, regulators, and coregulators are at the foundations of these mechanisms. Emerging discoveries regarding the roles of lnc-RNAs point to newer biochemical pathways and emphasise the notion of combinatorial control. These studies point to a new era, which might look to alter gene expression profiles by increasing the concentrations of these RNA products within the cell. Each of these elements shows a variety and flexibility of function depending upon their conformational states. Additionally, the situation is made more interesting by availability of various regulators and coregulators, which aids in the further selection of the combination required. The use of regulators and co-effectors with less than 100% binding efficiencies in systems, whose performance is optimal for satisfying the cell's needs, is a testament to the efficiency of the latter.The use of such complex signalling cascades also gives the cell greater control over the process as it now contains more than one regulatory step. Such a high degree of control is indispensable for the cell. The holistic understanding of these mechanisms will aid in improving the techniques used in r-DNA technology, developmental biology, and so forth. Such improvement will aid in the development of applications that benefit humanity.


Xylella fastidiosa (Xf) infection, we investigated expression and regulatory mechanisms of the cytochrome P450 monooxygenase CYP736B gene in both disease resistant and susceptible grapevines.Plant cytochrome P450 monooxygenases (CYP) mediate synthesis and metabolism of many physiologically important primary and secondary compounds that are related to plant defense against a range of pathogenic microbes and insects. To determine if cytochrome P450 monooxygenases are involved in defense response to CYP736B gene was composed of two exons and one intron with GT as a donor site and AG as an acceptor site. CYP736B transcript was up-regulated in PD-resistant plants and down-regulated in PD-susceptible plants 6 weeks after Xf inoculation. However, CYP736B expression was very low in stem tissues at all evaluated time points. 5'RACE and 3'RACE sequence analyses revealed that there were three candidate transcription start sites (TSS) in the upstream region and three candidate polyadenylation (PolyA) sites in the downstream region of CYP736B. Usage frequencies of each transcription initiation site and each polyadenylation site varied depending on plant genotype, developmental stage, tissue, and treatment. These results demonstrate that expression of CYP736B is regulated developmentally and in response to Xf infection at both transcriptional and post-transcriptional levels. Multiple transcription start and polyadenylation sites contribute to regulation of CYP736B expression.Cloning of genomic DNA and cDNA revealed that the CYP736B gene is involved in defense response at a specific stage of Xf infection in grapevines; multiple transcription initiation and polyadenylation sites exist for CYP736B in grapevine; and coordinative and selective use of transcription initiation and polyadenylation sites play an important role in regulation of CYP736B expression during growth, development and response to Xf infection.This report provides evidence that the cytochrome P450 monooxygenase  Arabidopsis and function as part of the highly sophisticated network of plant defense reactions . The current study identified a cluster of three cytochrome P450 monooxygenase genes  that are organized as tandem repeats and flanked upstream with a RE-LTR sequence on chromosome 7. This organization is likely the result of gene duplication [CYP736B was expressed at very low levels in stem tissues but at higher levels in leaf tissues of both PD-resistant and -susceptible grapevines with or without Xf infection. While the level of CYP736B expression was quite different between leaf and stem tissues, patterns of gene expression in leaf and stem tissues were similar in both PD- resistant and susceptible grapevines. Differential expression in response to Xf infection between genotypes suggested that CYP736B may be involved in the host response to Xf infection. It is known that the relative density of Xf populations in stem tissues are much lower in PD-resistant compared to -susceptible grapevines [Xf infection in PD- resistant grapevines. In fact, a wide range of cytochrome P450 monooxygenases mediate biosynthesis of lignins, terpenes, alkaloids, and a variety of other secondary compounds. Various biochemical pathways are involved in this process: the DIMBOA biosynthesis pathway that is initiated in response to wounding and naphthalic anhydride treatment [Cytochrome P450 monooxygenase genes encode for a superfamily of enzymes in plants that seem to be involved in response to abiotic and biotic stresses ,15. Althlication -18. We fapevines . This phreatment , and thereatment . Cytochrreatment ,11,21. HCYP736B changed differently, depending on growth stage and Xf infection status. Induced expression levels of CYP736B were correlated positively with resistance and negatively with susceptibility in both stem and leaf tissues 6 weeks post-inoculation. These results support the conclusion that CYP736B is involved in the defense response against Xf infection in grapevines. We further demonstrated that expression of CYP736B was post-transcriptionally regulated via pre-mRNA splicing. The very low frequency of unspliced CYP736B transcripts in leaf tissues of PD-resistant grapevines and the very high frequency of unspliced CYP736B transcripts in leaf tissues of PD- susceptible grapevines  may reveal an important aspect of the grapevine Xf interaction; correctly spliced CYP736B transcripts would be functional in PD-resistant grapevines, whereas unspliced CYP736B transcripts would not be functional in PD- susceptible grapevines. Differences in CYP736B expression levels between stems and leaves of grapevines would suggest that the levels of monooxygenase biosynthesis also vary. It seems that CYP736B expression involves dynamic regulatory mechanisms at both transcription initiation and the post-transcription modification levels via a developmental-dependent splicing pathway in PD- resistant grapevines. This result provides the first evidence that CYP736B is involved in the defense response at a specific stage of pathogenesis in grapevines.In this study, transcription and pre-mRNA splicing patterns of Arabidopsis [Arabidopsis revealed that most cytochrome P450 promoters contain the recognition sites MYB and MYC, an ACGT-core sequence, and TGA and W-boxes for WRKY transcription factors [CYP736B transcripts, whereas two CAAT boxes and a G-box were located at the TSS1-containing region, and one W-box was located at the TSS2-containing region. Previous studies indicated that TATA boxes are the most common regulatory elements found in promoters of eukaryotic genes because they are associated with basal transcription initiation by RNA polymerase II, especially with cis-acting elements that enhance or repress transcription [Arabidopsis respectively [cis-elements located within each CYP736B transcript are involved in physical interaction of the transcript with corresponding transcription factors, variation in frequencies of each transcription initiation site could reflect differences in CYP736B regulatory mechanisms triggered by Xf. If so, selective use of transcription initiation sites would play an important role in regulation of CYP736B expression in response to Xf infection.Transcriptional initiation of mRNAs is preceded by formation of a pre-initiation complex in eukaryotic cells. Diverse transcriptional initiation sites have been discovered by large scale mapping of mRNA start sites in the human genome . Use of bidopsis . At pres factors . Upon idcription . I-box  tail by poly(A) polymerase immediately after transcription ,34. The in vitro .CYP736B transcripts in both PD-resistant and -susceptible grapevines. There were 1 to 3 major polyadenylation sites with A or U as the terminal acceptor for polyadenylation. This result suggests that A and U are targets by poly(A) polymerase to synthesize poly(A) tails of CYP736B transcripts. Although there are examples of polyadenylation sites located between the poly(A) signal and a potential U-rich downstream element, a poly(A) signal could determine the optimal cleavage sites for CYP736B pre-mRNA polyadenylation. One possibility could be that the poly(A) polymerase-centered polyadenylation complex functions within the branched 3'UTR structure of CYP736B transcripts with some degree of flexibility after co-activation by poly(A) signals in grapevines. Given that total usage frequency of poly(A)2 and poly(A)3 sites changed differently between PD- resistant and susceptible grapevines after Xf inoculation, it is possible that bacterial virulence factors are involved directly or indirectly in modulation of CYP736B transcript polyadenylation in grapevines.The research presented here revealed multiple poly(A) signals and multiple polyadenylation sites at the 3' UTR of CYP736B splicing. The combination of different transcription initiation sites and different 3' polyadenylation sites determined efficiency of CYP736B splicing in both PD- resistant and susceptible grapevines. For instance, transcription initiation from the TSS1-containing region always produced unspliced CYP736B RNAs in either PD- resistant or susceptible leaf tissues 6 weeks after Xf inoculation. As distance from the TSS-containing region to the start codon ATG decreased, frequency of spliced CYP736B RNA increased. Similarly, transcription termination at the poly(A)1 region of the 3'UTR always produced the highest frequency of spliced CYP736B RNAs. More importantly, frequency of correct CYP736B pre-mRNA splicing was correlated with both transcription initiation and termination sites distant to translation start and stop codons. The closer the distance from transcription initiation site to translation start codon and the distance from poly(A) site to translation stop codon, the higher the frequency of correctly spliced CYP736B transcripts. These relationships seem to be determined by changes in free energy within both 5'UTR and 3'UTR sequence-confined 2D structure constraints. Thus, our study revealed that both transcription initiation and termination have significant effects not only on relative abundance of CYP736B transcripts, but also on pre-mRNA splicing. Grapevine genotype/Xf interactions and their effect on CYP736B expression could be regulated by selective usage of transcription initiation and polyadenylation sites. The detailed regulatory mechanism of how Xf infection and grapevine/bacterial interactions affect selection of transcription initiation sites, polyadenylation sites, and pre-mRNA splicing remains to be understood. This report provides evidence that the cytochrome P450 monooxygenase CYP736B gene is involved in defense response at a specific stage of Xf infection in grapevines; multiple transcription initiation and polyadenylation sites exist for CYP736B in grapevine; and coordinated and selective use of transcription initiation and polyadenylation sites play an important role in regulation of CYP736B expression during growth, development and response to Xf infection.Transcription initiation, capping, cleavage/polyadenylation and pre-mRNA splicing are complex processes and tightly coupled to RNA polymerase II transcription. Transcription by RNA polymerase II and pre-mRNA processing are coordinated within the nucleus . In geneV. rupestris 'A. de Serres × V. arizonica b42-26) × F8909-17 (V. rupestris 'A. de Serres' × V. arizonica/candicans b43-17)) [Xf (1 × 108 cfu/ml) as treatment groups or inoculated with 10 μl of Xf-free PW3 liquid media as control groups on the stem 10 cm above the pot surface following the procedure of Krivanek et al [The PD-resistant 9621-67 and -susceptible 9621-94 lines were selected from the 9621 population (D8909-15 (b43-17)) . A total [Xf 1 × 8 cfu/ml Xf, respectively. Collected tissues were immediately placed in liquid nitrogen and stored at - 80°C until use. Symptoms were recorded as they appeared over a 12 week period on a separate set of 3 inoculated and 3 non-inoculated plants for each genotype grown under the same conditions.Leaf and stem tissues of three plants per treatment were collected at week 1, 6 and 10 after inoculation with or without AM475392.1, CAAP02000243.1, CAAP02005006.1) using the BLAST http://www.ncbi.nlm.nih.gov/ and FGENESH http://www.softberry.com/berry.phtml programs. A pair of primers, P450F1 and P450R1 . RT-PCR analysis of gene expression was completed in a 25 μl of reaction that consisted of 1× reaction buffer (with 1.5 mM MgCl2), 2 mM dNTPs, 100 ng of cDNA template, 40 ng of primers, and 0.2 μl of Ampli Taq Gold DNA polymerase (5U/μl) . The RT-PCR program consisted of preheating at 94°C for 5 min, 30 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 3 min followed by a 72°C extension for 10 min. RT-PCR products were analyzed on a 1.2% of agarose gel in 1× TBE buffer with ethidium bromide staining. Real-time quantitative PCR analysis of gene expression was completed in a 25 μl of reaction that consisted of 1× SYBR Green PCR Master Mix , 100 ng of template cDNA and 0.3 μM of each primers SYBR-P450F1 and SYBR-P450R1 . First strand cDNA was synthesized using oligo(dT)The Northern Max Formaldehyde-Based System for Northern Blots  was used in this study according to the manufacturer's instruction. A full-length cDNA of grape cytochrome P450 monooxygenase gene CYP736B was labeled with biotin-dUTP at room temperature and used as probe for hybridization at 42°C for 18 hrs. Hybridization signals were detected using the BrightStar BioDetect Nonisotopic Detection Kit .Determination of 5' upstream transcription start regions and 3' poly(A) termination signal regions were done with the 5'RACE and 3'RACE Kits . The two gene specific primers used for upstream 5'RACE cDNA cloning were P450-5'RACE GSP2 and P450-5'RACE GSP3 Table . The two® 3:1 Agarose gel , transferred to a Nylon membrane, and detected by the BrightStar BioDetect Nonisotopic Detection Kit .Two gene specific primers, P450-5'RACE GSP3 for 5' primer extension analysis and P450-3'RACE GSP2 for 3'primer extension analysis, were designed to determine upstream transcriptional initiation regions and downstream transcriptional termination regions of grape P450B transcripts, respectively Table . Total R2), 2 mM dNTPs, 100 ng of cDNA template, 40 ng of primers, and 0.125 μl of TAKARA Taq polymerase (1U/μl) in a 25 μl reaction. The PCR program consisted of preheating at 98°C for 10 sec, 30 cycles of 98°C for 10 sec, 55°C for 30 sec, and 72°C for 5 min, and post-PCR extension at 72°C for 10 min. The relative abundance of each specific 5'UTR and 3'UTR primer-defined P450 transcript was measured using Real-time Quantitative PCR as described above.To determine relationships among different transcription initiation sites, pre-mRNA splicing, and polyadenylation signals, a modified 5'UTR-defined long 3'RACE method was designed in combination of the representative specific 5'UTR primers with the standard 3'RACE technology . These specific 5'UTR primers were -300UF, -140UF, and -60UF , and cloned into pGEM-T easy vector . Cloned DNA fragments were sequenced with M13F, M13R and a pair of internal P450 primers, P450F3 and P450R3  and 95% (p < 0.05). The Student T-test was used to analyze significance between treatments for gene expression at the same confidence levels of 99% (p < 0.01) and 95% (p < 0.05).Gene expression experiments in this study were repeated three times with three plants of each genotype at each time of sample collection. The statistic significance of experimental variation was calculated using ANOVA at confidence levels of 99% (DC, HL, YT and AW designed and conducted research experiments. DC was involved most expression analyses and the manuscript preparation. HL, AW, EC and DS were involved in organization of the work, writing and editing of the manuscript. HL was PI and initiator of the project. All authors took part in drafting, reviewing and approval of the final manuscript.


Yet treatment of such NB cell lines with retinoic acid results in growth control and induction of differentiation. This indicates that the signaling pathways that regulate cell growth and differentiation are not functionally lost but dysregulated. Agents such as retinoic acid normalize the signaling pathways and impose growth control and induction of differentiation. Recent studies in embryonic stem cells indicate that polycomb repressor complex proteins (PRC1 and PRC2) play a major role in regulating stem cell lineage specification and coordinating the shift from a transcriptome that supports self-renewal or growth to one that specifies lineage and controls growth. We have shown that in NB, the PRC2 complex is elevated in undifferentiated NB tumors and functions to suppress a number of tumor suppressor genes. This study will review the role of MYC genes in regulating the epigenome in normal development and explore how this role may be altered during tumorigenesis.It is well known that Neuroblastoma (NB) patients whose tumors have an undifferentiated histology and a transcriptome enriched in cell cycle genes have a worse prognosis. This contrasts with the good prognoses of patients whose tumors have histologic evidence of differentiation and a transcriptome enriched in differentiation genes. Tumor cell lines from poor prognosis, high-risk patients contain a number of genetic alterations, including amplification of MYCN, 1pLOH, and unbalanced 11q or gains of Chr 17 and 7, and exhibit uncontrolled growth and an undifferentiated phenotype in  Ever since MYCN was discovered to be the commonly amplified sequence in neuroblastoma (NB) tumors some 30 years ago, the mechanisms by which it contributes to NB tumorigenesis and strategies to target it have been intensively investigated . The finFunctionally MYC family genes  have been implicated in nearly every biologic process evaluated. Evidence indicates that MYC and MYCN are functionally interchangeable with MYCN having a more restricted spatial and temporal role during development . In thisFigure 1). Based on studies using naked DNA, two prevailing views emerged as to how MYC affects transcription: (1) a direct mechanism in which MYC/MAX dimers upon binding to E-boxes recruit higher order chromatin complexes that activate or repress transcription or (2) an indirect mechanism in which MYC/MAX dimers compete with other bHLH-Zip transcription factors for E-box binding to alter target gene activity. In searching for a common MYC target signature investigators have proposed numerous target signatures in different cellular contexts, but none have proven true in all cellular contexts (MYC is a member of a basic helix-loop-helix transcription-leucine zipper (bHLH-Zip) factor family whose members dimerize with MAX and bind to specific DNA sequences called E-boxes . Nucleosomes are then joined by a stretch of linker DNA (20 bp). When DNA is tightly complexed to histones, transcription is silenced. Activation of gene transcription requires loosening of DNA–histone interactions to enable access by transcription factors, the melting of the DNA that enables access of the basal transcription machinery including RNA polymerase II (Pol II) and the sliding of the nucleosomes to enable transcriptional elongation. The accessibility of DNA to DNA binding transcription factors is dynamically regulated by post-translational modifications to histone “tails” such as methylation (methyltransferases and demethylases), acetylation (acetyltransferases and deacetylases), phosphorylation (kinases and phosphatases), ADP-ribosylation, and ubiquitination .The necessity to compact some 2 m worth of DNA into the 10–20 μm nucleus of a cell has resulted in the evolution of a dynamic packaging system in eukaryotes that allows for the regulated sequestration or exposure of stretches of DNA. DNA (about 146 bp) is wrapped twice around an octamer of histone proteins  and stabilized by histone H1, forming the nucleosome, the basic unit of chromatin .When MYC function was first being described over 20 years ago, it was noted that MYC preferentially binds to “open” nucleosomes and DNase sensitive loci that we now know characterize euchromatin . EuchromSaccharomyces cerevisiae as members of the SAGA and NuA4 HAT complexes, respectively, and have been shown to have similar function in mammals . Concordantly, in embryonic stem cells (ESCs) MYC has been shown to elicit its transcriptional induction through the mammalian NuA4 complex and in neural stem cells MYCN and GCN5 have similar transcriptional signatures . Although TBP has no HAT activity, its binding to MYC suggests MYC is able to recruit Pol II machinery to activate transcription . Therefore, through its binding partners MYC is able to regulate the chromatin landscape to exert its function.Further work revealed that, via TRRAP, MYC was able to recruit histone acetyltransferases (HATs), GCN5 and Tip60 to its targets. GCN5 and Tip60 were first identified in gnatures . Both GCgnatures . In addiFigure 2D). GCN5 and Tip60 function to promote transcription by their acetyltransferase activity, but it is not limited to histones. It has also been demonstrated that MYC is a substrate of acetyltransferase activity. GCN5, Tip60, and CBP/p300 all acetylate MYC . As MYC’s bHLH domain is required for its transactivation function and binding to INI1, INI1 appears indispensable for proper MYC function. Furthermore, intact SWI/SNF complexes are also needed for MYC function. BRG1 is a functional component of the SWI/SNF complex and its ATPase-defective mutant abrogates histone remodeling activity and blocks transcription of MYC targets  and functioning to amplify transcription of actively transcribed genes  suppressible MYCN SHEP NB model system and ChIP hybridizations to gene promoters (ChIP-Chip) revealed that MYCN binding associates with euchromatic histone marks H3K4me and H3K9Ac . The cored genes . An inteed genes . This imde novo factors to bind to DNA to initiate its transcriptional function , only OSK can bind to distal enhancer regions at an early time during reprogramming. Subsequently MYC is recruited to the TSS to stabilize chromatin binding and promote transcription. Furthermore, OSK proteins are all able to bind to one side of the DNA helix, suggesting they are able to bind to heterochromatic regions. MYC does not bind to heterochromatic regions. MYC is predominantly found on euchromatic regions indicating it requires a prior transcriptional stimuli or function .de novo transcription, the question arises as to how MYC can promote the induction of transcription initiated by reprogramming factors. Regulation of gene transcription in complex chromatin models is still evolving and occurs in at least five steps: (1) initiation, (2) promoter pausing, (3) mRNA capping, (4) promoter escape, and (5) transcript elongation. A clue to MYC function during initiation lies with MYC binding to TBP , p-Ser5-Pol II. Genome-wide analyses of Pol II binding demonstrated that p-Ser5-Pol II commonly resides at promoters or is paused 20–40 bases downstream of the promoters of many non-expressed genes. The capping machinery is recruited to the CTD of Pol II and results in the addition of a methylated guanine at the 5′-triphosphate of the nascent mRNA strand. MYC also binds to TFIIH and the increased p-Ser5-Pol II increases capping associated methylation. Capping is required for the recruitment of the translation machinery and mRNA binding to ribosomes. The acetylated lysine binding bromodomain containing protein BRD4, recruits P-TEFb complex which phosphorylates serine 2 on the CTD region of Pol II (p-Ser2-Pol II) causing elongation of mRNA transcripts and promoter escape  results in cells with a relative nuclear size twice that of the parental or control-transfected (8B) cell lines which contain a single copy of MYCN and do not express MYCN mRNA . Decreased euchromatic regions have also been identified in tumor models when MYC is knocked down . In NB, the combined treatment of RA and Interferon-gamma (INF-γ) has been shown to dramatically decrease cell growth and induce differentiation , via SIN3 a transcriptional corepressor, to target loci to induce transcriptional silencing . Thus thntiation . It was ntiation . The preAnother model of MYCN-mediated transcriptional repression invokes the association of MYC binding to the MIZ and SP1 transcriptional activators resulting in the recruitment of HDACs. This has been demonstrated for two genes associated with good prognosis and differentiation in NB, NTRK1(TrkA), and p75(NGFR). Upon induction of MYCN, there is increased HDAC1 and decreased acetylated H3 binding over the TSS of these promoters. The functional presence of MYCN was found to attenuate their promoter activity . One canMost recently MYC has been found to transcriptionally amplify epigenetic modifiers with transcriptional repressive activities. For example, in ESCs MYC stimulates all components of the polycomb repressive complex 2 (PRC2), including embryonic ectoderm development (Eed), suppressor of zeste 12 (Suz12), and histone methyltransferase enhancer of zeste homolog 2 (EZH2), in ESCs . PRC2 fuFurthermore, in NB cell lines and tumor samples, MYCN stimulates B cell-specific Moloney murine leukemia virus integration site (BMI1) expression. BMI1 is a member of the PRC1 complex whose targets are often hypermethylated suggesting epigenetic silencing . As partGiven MYCN’s role in stimulating expansion of neural progenitors, it is not unexpected that differentiation genes like NTRK1 or p75(NGFR) are repressed by MYCN. A global survey by ChIP-Chip of ESCs for MYC and Miz-1 binding sites indicated that almost 30% of Miz-1-regulated genes could be repressed by MYC. These included homeobox genes, developmental proteins and genes involved in regulation of apoptosis. In NB tumor samples, within the MYCN gene signature set, 30% of the down-regulated genes in the set are neuronal tissue-specific genes while only 2% of such genes are up-regulated . This isSince MYC has been implicated in oncogenic transformation, researchers and clinicians have sought to target MYC and its oncogenic functions. A common mode of function of therapeutic interest has been MYC’s chromatin remodeling and transcriptional activities. It was rationalized that since MYC binds and recruits coactivators containing the bromodomain and extraterminal (BET) members of human bromodomain proteins , which associate with acetylated histones, it could be functionally targeted . This inFigure 4; Figure 4A) or a small molecule inhibitor of EZH2, 3-Deazaneplanocin A  led to decreased growth and induction of neurites. Mechanistically this was also accompanied by induction of cell death  which was due to increased levels of activated caspase 3/7 activity  since the apoptotic effects of DZNep could be reversed by a caspase 3/7 inhibitor . The finding that DZNep inhibited the growth of NB tumor xenografts is promising . However, all mice still succumbed to tumors suggesting that combination therapies may be required to more efficaciously block tumor growth.As MYC and MYCN have been shown to induce EZH2 expression and activity  it is reMYC’s dysregulation of epigenetic writers, readers, or erasers thus provides a novel avenue that can be therapeutically developed since many of these proteins have targetable enzyme activities .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Core promoter elements are thought to be the docking site for transcription factors binding, and thereby essential for transcription initiation. The role of core promoter elements has been studied for decades in reporter systems. the central model claim that each cis sequence has an epigenetic role in navigating transcription initiation. We use here the MHC class I gene as model to study transcription in a transgenic mouse.MHC class I genes are ubiquitously expressed and subject to both tissue-specific and hormonal regulation. The core promoter contains four conventional elements: CCAAT, a TATAA-like element, an Sp1 binding site and a canonical Inr. The in vivo function of these elements was determined by mutating each individually within the context of the native gene.Surprisingly, none of the elements was essential for transcription since no single mutation eliminated transcription. Indeed, mutation of any one of the elements resulted in increased promoter activity, indicating that these elements function as transcriptional modulators. Further, each of the elements was found to have a distinct function, contributing uniquely to tissue-specificity, hormonal responses or both.The core promoter elements do not affect start site selection, demonstrating that they do not invoke a cryptic promoter. However, they do modulate relative start site usage. The patterns of chromatin modification reflect the expression status of the different promoters. In tissues where the different promoters constructs support active transcription, histone H3K4 trimethylation is high and H3K9 trimethylation is low. Conversely, H3K9 trimethylation is high and H3K4 trimethylation low across the gene in tissues where the promoter constructs are less active. The wild type promoter is activated by interferon, while the Inr and Sp1 mutants repress transcription in response to interferon treatment.Finally, the CAAT element was found to have dual function, both as a transcriptional regulator and as a barrier element. The barrier function correlated with the binding of C/EBP and CTCF to the wild type CAAT element and variegated expression across generations when the CAAT element was mutated.Remarkably, these results demonstrate that none of the elements homologous to canonical core promoter elements are necessary for promoter activity. However, they do contribute to the fine tuning of the tissue specific patterns of expression, extracellular signaling, overall promoter activity and chromatin modifications.


Arabidopsis) and known to alleviate the stress caused by restricted water availability. As its target genes are still unknown despite the original cloning of Asr1 cDNA 20 years ago, we examined the tomato genome for specific loci interacting in vivo with this conspicuous protein.Identifying the target genes of transcription factors is important for unraveling regulatory networks in all types of organisms. Our interest was precisely to uncover the spectrum of loci regulated by a widespread plant transcription factor involved in physiological adaptation to drought, a type of stress that plants have encountered since the colonization of land habitats 400 MYA. The regulator under study, named ASR1, is exclusive to the plant kingdom  on leaves from stressed tomato plants, using a high-quality anti-ASR1 antibody. In this way, we unraveled a novel repertoire of target genes, some of which are clearly involved in the response to drought stress. Many of the ASR1-enriched genomic loci we found encode enzymes involved in cell wall synthesis and remodeling as well as channels implicated in water and solute flux, such as aquaporins. In addition, we were able to determine a robust consensus ASR1-binding DNA motif.The finding of cell wall synthesis and aquaporin genes as targets of ASR1 is consistent with their suggested role in the physiological adaptation of plants to water loss. The results gain insight into the environmental stress-sensing pathways leading to plant tolerance of drought. Plant species in arid zones are constantly exposed to drought stress . ToleranArabidopsis) ASR proteins  are considered to be a subgroup of the LEA superfamily [Solanum genus [Vitis vinifera (grape) [Arabidopsis thaliana[The widespread   and ii)  thaliana. Given t thaliana,16, we h thaliana.in vivo binding sites of DNA-associated proteins, including TFs. As it proved to be useful to map global binding sites precisely for any nuclear protein of interest believed to associate with chromatin, ChIP-seq has emerged as a powerful tool in eukaryotes, particularly in mammals, including humans [Therefore, we were convinced that it was worthwhile pursuing the challenge to identify the direct target genes of ASR1, our regulator of interest, which is thought to control the downstream network necessary for cellular adjustment to water loss. We believed that knowledge on this particular “targetome” would generate valuable mechanistic insights into the genetic program leading to such a physiological adaptation. To achieve this goal, we carried out ChIP-sequencing (ChIP-seq), a strategy that combines chromatin immunoprecipitation (ChIP) with massively parallel (throughput) DNA sequencing to identify the g humans ,19, and g humans -20. In tAfter shearing DNA through sonication of lysed nuclei, we determined the average size of the resulting DNA fragments by means of gel electrophoresis. They were approximately 400 bp Figure A, a suitAfter the ASR1 protein was successful purified .We performed ChIP, using stressed tomato leaves as the starting tissue and purified anti-ASR1 antibody for the IP assay. The recovered DNA was subjected to high throughput sequencing on Illumina Hiseq 2000 equipment. To identify immuno-enriched regions, we made use of the Macs software program   expected by chance using the 33.2% GC content in the tomato genome [Additionally, we used the software program "consensusCounter"  from both transgenic lines were significantly lower (p < 0.01) in comparison with WT plants  were decreased only in one Asr1-silenced line  after a 6-hr water stress treatment . Silenced plants showed a marked decrease (p < 0.0001) in Asr1 mRNA in comparison with WT plants Figure A. When nThese results, obtained with representatives of cell wall- and aquaporin-associated genes, indicate that the binding of ASR1 to its target genes is truly productive and results in gene regulation upon the induction of water stress, confirming previous hints of ASR1 being a direct transcriptional activator ,13,28.The identified consensus motif sequence Figure A was conFor our genome-wide analysis, we carefully followed the current guidelines for ChIP-seq  and asseASR1 showed a binding preference (70%) for gene regions, which is expected for a TF. Considering that tomato genes (including their introns) represent only approximately 13% of the whole genome (even considering non-protein-coding RNA genes) , this pra priori, for example, those encoding cell wall proteins and aquaporins (AQP), which are related to the physiological response to limited water accessibility [A comprehensive analysis of the target genes was made difficult by the diversity of functional categories  encountered. For this reason, we explored over-represented genes whose molecular or cellular functions seemed to be more influenced by ASR1 sibility .Arabidopsis gene has been reported to be up-regulated during drought stress [Solyc10g054820.1 were found lower in ASR1-silenced plants compared to WT ones, thus validating the results of ASR1 binding at a functional level. In agreement with an expected delay between TF binding and effective transcription due to chromatin remodeling [Aquaporin genes belong to the Major Intrinsic Protein (MIP) gene family. In plants, the number of AQP genes present in a single species is rather high, with more than 30 AQP genes frequently found in each genome . Plant At stress . Moreovemodeling , such diThe plant cell wall provides structural support during development and represents the first line of defense against biotic and abiotic stressors, including drought. In recent years, evidence has accumulated for a dedicated maintenance mechanism for plant cell integrity under diverse biotic and abiotic stress; however, the underlying mechanism remains to be elucidated. Thus, the discovery of cell wall-related genes as targets of ASR1 makes sense in light of the complex network of polymers essential for maintaining turgor pressure. Water loss in this matrix results in a severe disruption of cell wall integrity, which can be irreversible . In addiSolyc08g082650.2.1, which is annotated as a cellulose synthase-like (CSL) protein. The three closest genes related to Solyc08g082650.2.1 in Arabidopsis thaliana are the paralogous cellulose synthase-like G (CSLG) proteins 2  and 3 , which contain a relatively high degree of identity (48-50%) in the region between aa 26–205 (p range = 5 - 6e-43). By searching the GENEVESTIGATOR platform (http://www.genevestigator.com), we found that AtCSLG2/G3 appear to be active during leaf senescence and that osmotic or water stress stimulates their expression up to 50-fold.In particular, we found that ASR1 binds directly to several cell wall-related genes. One such gene is Arabidopsis thaliana is the plasmodesmata callose-binding protein 3 (AtPDCB3) gene, with a high degree of identity . AtPDCB3 exhibits specific callose-binding activity in vitro and localizes to plasmodesmata [Arabidopsis and tomato.Another cell wall-associated gene that emerged as a direct target of ASR1 is Solyc03g115200.2.1, which is annotated as Glucan endo-1,3-β-glucosidase 1. The closest ortholog in odesmata . Depositodesmata ,50. InteAnother work seeking targets of tomato ASR1 was able to identify ABI4, a gene involved in seed germination . Howeverin vivo ASR-binding DNA motif with little variation: (A/T)(A/G)GCCCA, almost identical to the one very recently found for ASR5 in rice subjected to Aluminium stress [Arabidopsis[Arabidopsis, which would be consistent with the known connection between adaptation to abiotic stress and oxidative respiration through mitochondrial electron transport in plants [Our results also revealed a consensus m stress  and to tm stress  , which has already been performed [At this point, it is worth mentioning the scope of ChIP in general. This procedure, due to its crosslinking step, is also able to detect proteins indirectly binding to DNA, for example by forming a DNA-interacting complex. Nevertheless, even in this second scenario, the information gained by ChIP is useful. The way to discriminate between direct and indirect binding is to perform erformed ,57.In summary, we have uncovered a novel repertoire of target genes of the TF ASR1, some of which are clearly involved in the response and physiological adaptation of plants to water stress. These findings will hopefully enable us to gain additional insight into both the early environmental stress-sensing molecular events triggered by ABA and the late physiological adjustments that finally confer tolerance to drought.Solanum lycopersicum) seeds were germinated on blotting paper for 7 days and then transferred to pots containing soil mix in a growth chamber under a photoperiod of 16 hr light/8 hr darkness at 26°C. Plants were used 4 weeks later.Commercial tomato . For expression analysis, the procedure was the same except that we applied a 6-hr water stress treatment, as a 3-hr period was insufficient to yield significant changes in expression.Solanum lycopersicum) L. cv Moneymaker seeds were obtained from Meyer Beck (Berlin), and the plants were handled as previously described [Agrobacterium tumefaciens into tomato cotyledons. Emerging shoots were excised and selected on Murashige and Skoog media containing kanamycin (100 mg/l). When the plants developed roots, they were transferred to soil in the greenhouse for subsequent selection. The initial screening for the 39 lines was based on a diminished expression displayed in Northern blots. For the expression assays, we used five WT tomato plants and 10 Asr1-silenced plants (five from line 5 and five from line 17).Tomato (escribed . The 348escribed  between This strategy combines chromatin immunoprecipitation (ChIP) with high throughput DNA sequencing to identify the binding sites of nuclear proteins connected non-covalently to DNA. A key success factor is the generation of a high-quality antibody against the purified protein of interest. In this work, ChIP-seq was chosen to identify the targets of ASR1 starting out with the purification of ASR1, against which antibodies were produced.Escherichia coli BL21 strain, which has a lac-driven T7 RNA polymerase gene. For testing induction, low-scale cultures were started and Asr1 was indirectly induced by IPTG at different times. As a negative control, a culture of plasmid-free E. coli BL21 was used. Crude protein extracts were run in 15% PAGE, transferred to a nitrocellulose membrane and stained with Coomassie Blue.Expression of recombinant ASR1 was achieved using a plasmid  that contains the tomato Asr1 cDNA driven by the prokaryote promoter sequence recognized by the RNA polymerase of bacteriophage T7 (construct T7-ASR1). The recombinant plasmid was introduced into the 2+column  using its natural histidine-rich tract as a tag. The ASR1 protein appeared to be pure, with no detectable contaminant proteins, after elution through a non-linear continuous gradient of imidazol  and inoculated into rabbits. Bleedings were performed for titrations of antisera.Total immunoglobulins from antisera were precipitated with ammonium sulfate, dialyzed and purified with a “Hi-trap protein G” column (Pharmacia Biotech). Their presence was monitored through absorbance at 280 nm. Immunoglobulin-rich fractions were collected and concentrated by Vivaspin columns .The purified antibodies were diluted in PBS to conduct an affinity chromatography protocol using ASR1 covalently bound to a cyanogen-activated Sepharose solid matrix . Dot blots and subsequent Ponceau Red staining were performed to visualize the eluted fractions that contained the protein material  of the antibody, we first performed a non-crosslinking ChIP procedure using fruit as the starting tissue. The protocol was stopped at the crosslinking reversal step. Precipitated proteins were eluted with 0.1 M glycine at a pH of 2.4. The sample was then transferred into a tube containing enough 1 M Tris-base to neutralize the low pH and loaded onto an SDS-PAGE gel for subsequent Western blot to detect the immunoprecipitated ASR1. Because the same primary anti-ASR1 antibody was used for both IP and Western blot, for the latter we used a secondary anti-native rabbit immunoglobulin TrueBlot antibody  to avoid the detection of immunoglobulins that had been run (denatured) on the gel and whose quality was tested in this assay.Following post-stress leaf chromatin immunoprecipitation, the DNA was purified using “AMPXP” beads according to the manufacturer’s protocol (Ambion). The DNA was eluted in 10 μl MilliQ water followed by two repeat purifications. The DNA fragments were refilled to get blunt ends using the Klenow fragment of DNA polymerase. Fragmented ends were phosphorylated by T4 kinase and ligated to different double-stranded adaptors by concentrated DNA ligase. The adaptors had 5 nucleotides in their 3′ ends as “bar codes” for sample identification in multiplex runs. The DNA was re-purified with shorter incubation times to favor the loss of adaptor primer dimers shorter than 200 bp that were generated during ligation. PCR reactions were performed using 0.5 μl of dimer-free adaptor samples, adaptor-specific primers and high-fidelity thermostable DNA polymerase  with low cycle numbers (between 12 and 18) until a product concentration of 5 ng/μl was reached. Cycling conditions were as follows: 1 starting denaturation cycle of 30 sec at 98°C, 12–18 cycles of 10 sec at 98°C (denaturing), 30 sec at 65°C , 30 sec at 72°C (elongation) and a final elongation step for 5 min at 72°C. Amplified products with each bar code were combined and re-purified with beads and resuspended. The concentration of each sample for sequencing was 2 ng/μl.DNA samples from the library were sequenced on a HiSeq Illumina® machine  at BTI, Cornell University campus, Ithaca, NY, USA).http://www.ncbi.nlm.nih.gov/sra/?term=SRX257002.The raw sequencing data have been uploaded at the following NCBI website, publicly available: http://cancan.cshl.edu/labmembers/gordon/fastx_toolkit/). Sequences were aligned with those from the tomato genome (version SL2.40) available on the webpage of the Genome Project [http://solgenomics.net/organism/Solanum_lycopersicum/genome) using the software program Bowtie [The reads were processed into separate sequences according to bar codes and trimmed by using the fastx toolbox software  in a final volume of 25 μl. A DNA Engine Opticon (MJ Research Inc.) thermocycler was used with annealing temperatures set to achieve 90% amplification efficiency . Denaturation curves were calculated and the amplified DNA was run through an agarose gel to ensure the existence of a single product. The conditions were as follows: 1 cycle of 5 min at 94°C , 35 cycles of 30 sec at 94°C (denaturation), 30 sec at 59/64°C  and 30 sec at 72°C (elongation).Solyc03g115200.2 gene (3–200), we used 58.6°C and for the Solyc10g054820.1 gene (10–820), we used 62.4°C. qRT-PCR was validated by a standard and melting curve. Primers are listed in Additional file Solyc03g115200.2 (3–200) and Solyc10g054820.1 (10–820) were normalized to Ubi3 or EF-1 levels. Statistical analysis was performed with the GraphPad software program using a one-way ANOVA statistical test with a 95% confidence level.For RNA extraction, we used the TriReagent kit (MRC Inc.) with 300 mg of previously mortar-ground leaves as a starting material and 1.5 ml of TriReagent solution according to the manufacturer’s protocol. All RNA samples were quantified using a Nanodrop 2000 (Thermo Scientific) spectrophotometer. To eliminate contamination of the samples by residual DNA, 10 μg of each RNA sample were treated with 12.5 U DNAseI (Invitrogen). Reverse transcription was achieved using 2 μl of DNAseI-treated RNA, 50 U MMLV-RT (Promega) and oligo-dT (50 pmoles) in a 25 μl final volume for 1 hr at 42°C. To prevent RNA degradation, 10 U of RNAseOUT (Invitrogen) was added. Following reverse transcription, qPCR was carried out using 5 μl of a 1/10 dilution of the cDNA samples (obtained as described above) per PCR reaction. Reactions were performed in a DNA Engine Opticon (MJ Research Inc.) thermocycler. We used 0.625 U of Taq DNA Polymerase (Invitrogen), 3 mM magnesium chloride, 2 mM of dNTPs mixture (Fermentas) and 0.2 μM of each primer (IDT Inc.) in a final volume of 25 μl. We used Sybr Green® (Roche) as the fluorophore. Reactions were conducted under the following cycling conditions: 2 min of denaturation at 94°C, 40 cycles of 30 sec of denaturation at 94°C, 30 sec of annealing and 30 sec of elongation at 72°C. A melting curve was generated between 65 and 95°C with readings at every 0.5°C. For Asr1, Ubi3 and EF-1 quantitation we used 67°C as the annealing temperature. For the The Data set 1 contains a table with the list of peaks found with the Macs software. Data set 2 shows a list of all the genes with peaks near them. Data set 3 shows a list of the gene categories, the over-represented functions and the over-represented groups obtained after running Mapman software. Data set 4 shows the count of reads for ASR1-binding sequences along all the peaks found by the Macs software. The Additional file The scripts and in-house softwares developed for this paper are included in the Additional file MYA: Million years ago; LEA: Late embryogenesis abundant; ABA: Abscisic acid; ASR: ABA/ Stress/Ripening ; ChIP: Chromatin immunoprecipitation; DNA: Deoxyribonucleic acid; cDNA: Complementary DNA; TF: Transcription factor; ROC: Receiver operating characteristic; MIP: Major intrinsic proteins; AQP: Aquaporins; WT: Wild type.All the authors declare that they have no competing interests.MMR and RMG  performed all the experimental work on Asr1 purification, antibody making, ChIP for deep sequencing as well as subsequent ChIP-qPCR and qRT-PCR for validation of individual loci and transcripts; JJG and SZ provided the equipment and setups for library making and deep sequencing (SZ made the libraries); TD carried out the initial bioinformatic analyses; PGT developed in-house softwares for in-depth bioinformatic interpretation of the data; FC and PGD generated the ASR1-silenced plants used for functional validation; KA interpreted the results regarding aquaporin genes; JME interpreted the results regarding cell wall-related genes, JDSS made valuable suggestions throughout the work; NDI introduced the theoretical frame, coordinated the project and drafted the manuscript. All authors read and approved the final manuscript.2+ affinity column chromatography. Figure S2. Purification of the anti-ASR1 antiboby. Figure S3. Testing another housekeeping gene for normalization of transcript levels. Table S1. Primers used for ChlP validation. Table S2. Primers used for expressions analysis (qRT-PCR). List 1. Software parameters used in this work.Purification of ASR1 by a NiClick here for fileData set 1. Table with the list of peaks found with the Macs software.Click here for fileTomatoProgramCode zip file. In-house softwares developed for this paper.Click here for fileData set 2. List of all the genes with peaks in or near them.Click here for fileData Set 3. List of the gene categories, the over-represented functions and the over-represented groups obtained after running the Mapman software.Click here for fileData set 4. Count of reads for ASR1-binding sequences along all the peaks found by the Macs software.Click here for file


Sox gene function in vertebrates is confounded by functional compensation between three closely related family members. In Drosophila, only two group B Sox genes, Dichaete and SoxN, have been shown to function during embryonic CNS development, providing a simpler system for understanding the functions of this important class of regulators.Group B Sox domain transcription factors play conserved roles in the specification and development of the nervous system in higher metazoans. However, we know comparatively little about how these transcription factors regulate gene expression, and the analysis of Drosophila genome. We show that Dichaete plays key roles in CNS development, regulating aspects of the temporal transcription factor sequence that confer neuroblast identity. Dichaete also shows a complex interaction with Prospero in the pathway controlling the switch from stem cell self-renewal to neural differentiation. Dichaete potentially regulates many more genes in the Drosophila genome and was found to be associated with over 2000 mapped regulatory elements.Using a combination of transcriptional profiling and genome-wide binding analysis we conservatively identify over 1000 high confidence direct Dichaete target genes in the Our analysis suggests that Dichaete acts as a transcriptional hub, controlling multiple regulatory pathways during CNS development. These include a set of core CNS expressed genes that are also bound by the related Sox2 gene during mammalian CNS development. Furthermore, we identify Dichaete as one of the transcription factors involved in the neural stem cell transcriptional network, with evidence supporting the view that Dichaete is involved in controlling the temporal series of divisions regulating neuroblast identity. Sox1, 2 and 3, which primarily act as transcriptional activators, and the group B2 genes Sox14 and 21, which act mainly as transcriptional repressors and appear to antagonise group B1 functions  and from this study, abstracted from Additional file Click here for fileGene Ontology Biological Process enrichment analysis of Dichaete target genes. Click here for fileCooccur Analysis of TF overlaps. Headers: tf1, transcription factor 1 dataset; tf2, transcription factor 2 dataset; overlapped_in_tf1, number of TF1 binding interval overlapping TF2; n_tf1, number of binding intervals in TF1 dataset; percent_in_tf1, fraction of TF1 dataset overlapping with TF2; overlapped_in_tf2, number of TF2 binding intervals overlapping with TF1; n_tf2, number of TF2 binding intervals in dataset; percent_in_tf2, fraction of TF2 intervals overlapping TF1; nsamples, number of resampling runs; pvalue, raw p value, padjust; adjusted p value; Gene 1, TF 1 gene name; Gene 2, TF2 gene name. Click here for fileGenes bound by Dichaete and Prospero. Click here for fileDrosophila orthologues of mouse Sox2 bound genes in the Dichaete core binding set. Click here for fileDrosophila orthologues of mouse Sox2 bound genes in the Dichaete target gene set. Click here for fileDichaete Core binding intervals in. bed format. Click here for fileTable of transcription factor binding data used in overlap analysis. Click here for file