Lmo2) gene encodes a transcriptional cofactor critical for the development of hematopoietic stem cells. Several distal regulatory elements have been identified upstream of the Lmo2 gene in the human and mouse genomes that are capable of enhancing reporter gene expression in erythroid cells and may be responsible for the high level transcription of Lmo2 in the erythroid lineage. In this study we investigate how these elements regulate transcription of Lmo2 and whether or not they function cooperatively in the endogenous context. Chromosome conformation capture (3C) experiments show that chromatin-chromatin interactions exist between upstream regulatory elements and the Lmo2 promoter in erythroid cells but that these interactions are absent from kidney where Lmo2 is transcribed at twelve fold lower levels. Specifically, long range chromatin-chromatin interactions occur between the Lmo2 proximal promoter and two broad regions, 3–31 and 66–105 kb upstream of Lmo2, which we term the proximal and distal control regions for Lmo2 (pCR and dCR respectively). Each of these regions is bound by several transcription factors suggesting that multiple regulatory elements cooperate in regulating high level transcription of Lmo2 in erythroid cells. Binding of CTCF and cohesin which support chromatin loops at other loci were also found within the dCR and at the Lmo2 proximal promoter. Intergenic transcription occurs throughout the dCR in erythroid cells but not in kidney suggesting a role for these intergenic transcripts in regulating Lmo2, similar to the broad domain of intergenic transcription observed at the human β-globin locus control region. Our data supports a model in which the dCR functions through a chromatin looping mechanism to contact and enhance Lmo2 transcription specifically in erythroid cells. Furthermore, these chromatin loops are supported by the cohesin complex recruited to both CTCF and transcription factor bound regions.The Lim domain only 2 ( Lmo2 gene, have shown that Lmo2 is necessary for embryonic yolk sac erythropoiesis Lmo2 expression is maintained in erythroid cells but down regulated in the T-cell lineage Lmo2 results in the development of T-cell related diseases; indeed Lmo2 is located at a recurrent site of T-cell acute lymphoblastic leukemia (T-ALL) specific translocation Lmo2 when the gene therapy vector integrated near Lmo2Lim domain only 2 (LMO2) is a critical transcriptional regulator of hematopoiesis. Gene targeting experiments conducted to introduce null mutations in the mouse Hbb), α-globin (Hba), retinaldehyde dehydrogenase 2, c-kit and erythroid Kruppel-like factor (Eklf) Previous studies have shown that in erythroid cells LMO2 is usually present as part of a complex with the transcriptional regulators, TAL1, E47, LDB1, and GATA1 Hbb locus control region (LCR) Hbb-b1 gene Hbb LCR is in close proximity to the active Hbb genes (Hbb-b1 and Hbb-b2) whereas the intervening 50 kb of DNA sequence containing the embryonic erythroid cell expressed genes is looped out Hba, Shh, TH2, HoxB1 and olfactory receptor genes Transcription is regulated not only by the sequences immediately upstream of gene transcription start sites (TSS) but in many cases by distal regulatory elements (DRE) which can be located up or downstream of the genes they regulate. In fact genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq) analysis for several transcription factors has revealed that a significant proportion (40–60%) of transcription factor bound regions are located in the intergenic regions of the genome ≥10 kb from a gene TSS Hbb, Igf2/H19 and HoxA, however, CTCF bound regions are generally bound by CTCF in all cell types IGF2-H19 locus and at the developmentally regulated IFNG locus both cohesin and CTCF are required for maintaining higher-order chromatin conformation cis-regulatory elements not occupied by CTCF The chromatin-chromatin looping interactions that regulate cell-type specific gene expression are also present in a cell-type specific manner whereas many of the proteins present at sites of looping interactions are ubiquitously expressed. For example, CTCF participates in intra- and inter-chromosomal looping at individual gene loci including Lmo2 in erythroid cells appears to involve multiple distal regulatory elements. Three alternate upstream promoters have been identified as well as multiple conserved DRE located up to 100 kb upstream of the Lmo2 gene Lmo2 and 1 kb downstream are capable of enhancing reporter gene expression in erythroid cells and in transgenic mice suggesting that strong expression of Lmo2 in hematopoietic cells requires the combined action of upstream DRE and sequences close to the Lmo2 proximal promoter Lmo2 transcription in the context of its endogenous genomic location using the 3C technique. Our results show that multiple upstream DRE interact with the Lmo2 proximal promoter whereas intervening regions are looped away from the proximal promoter. DRE upstream of Lmo2 are bound by multiple transcription factors, p300, and associated with intergenic transcription when Lmo2 is transcribed at high levels in erythroid cells. Interaction between the DRE and the Lmo2 promoter was identified in erythroid cells but not in kidney cells suggesting a link between the looping conformation of the locus and transcriptional regulation of Lmo2. Furthermore, a CTCF and cohesin occupied region upstream of the most distal enhancer (90 DRE) also contacts the Lmo2 proximal promoter region, potentially insulating the neighboring Cell cycle associated protein 1 (Caprin1) gene from interaction with the DRE that enhance Lmo2 transcription.Transcriptional regulation of Lmo2 have been identified in the human genome and confirmed to have enhancer activity in transgenic mice Lmo2 , is located close to the Lmo2 proximal promoter and contains the distal and intermediate promoters as well as the 12 and 25 DRE. The second interaction domain located further upstream of Lmo2, which we term the distal control region (dCR), contains the 70, 75 and 90 DRE as well as a fragment upstream of the 90 DRE. These are similar, though not identical, to clusters I (−90 to −64) and II (−40 to +1) identified by Landry et al. 2009 as enriched in histone H3 acetylation in erythroid cells Next we performed a locus-wide 3C using the ey cells . These fCaprin1 and Lmo2 highlighted the 75 DRE as being bound by multiple transcription factors but did not reveal binding at the 90 and 70 DRE. By contrast our 3C data revealed a broad domain of chromatin-chromatin contacts between the 70, 75 and 90 DRE and the Lmo2 proximal promoter raising questions about the function of this dCR. We did identify additional p300 association at the 70 DRE suggesting that bound transcription factors are present which recruit p300 to this DRE , containing three transcription factor bound DRE , and a proximal interaction cluster (pCR). Furthermore, 3C experiments revealed no significant interactions between the Caprin1 TSS and the DRE suggesting these elements are specific in regulating Lmo2 transcription.Our investigation of chromatin-chromatin interactions throughout the mouse genomic region containing Lmo2 the 75 DRE contained the highest density of transcription factor association as well as p300, RAD21 and a nearby CTCF bound region. Furthermore our 3C data confirmed specific interaction of the 75 DRE with the Lmo2 proximal promoter and interaction of the Lmo2 proximal promoter with the dCR containing the 75 DRE as well as the 90 and 70 DRE. In addition, a CTCF bound region upstream of the 90 DRE is contained in the dCR. Previous studies in circulating erythroid cells of transgenic mice show that the 75 DRE has the strongest enhancer activity in erythroid cells and drives the expression of a reporter gene, cooperatively with the Lmo2 proximal promoter and +1 enhancer element Lmo2 proximal promoter/+1 DRE region to form an erythroid cell specific chromatin loop that includes other regulatory elements and a cluster of CTCF bound regions. This suggests that in the endogenous context elements throughout the dCR coordinately regulate Lmo2 transcription in erythroid cells.Using a combination of ChIP-Seq data for mature erythroid cells and HPC7 hematopoietic progenitor cells we identified multiple transcription factors bound within both the pCR and the dCR. Even considering the HPC7 ChIP-Seq data that showed more transcription factor peaks across the entire region upstream of Lmo2. Interestingly, we did not identify interactions of the 75 DRE with the fragments containing the 25 or 12 DRE. Transgenic analysis by Landry et al. 2009 of these elements did reveal that the 25 and 12 DRE conferred expression in the fetal liver whereas only the 75 DRE conferred expression in fetal liver as well as in circulating blood cells suggesting that these elements have different functional roles in regulating Lmo2 expression. The fact that we did not identify specific interactions between the 75 DRE and the fragment containing the 25 or 12 DRE suggests that the interactions of these two regions with the proximal promoter are mutually exclusive. These mutually exclusive interactions could occur in different sub-populations of cells within the anaemic spleen or the interactions could be dynamic within individual cells with the proximal promoter region alternately contacting the 25–12 region and the 70–90 region similar to the flip-flop of the Hbb LCR between the γ- and β-genes We also identified increased interaction throughout a pCR containing the 25 DRE/distal promoter and 12 DRE/intergenic promoter and the +1DRE/proximal promoter in erythroid cells compared to kidney cells, suggesting that this more proximal region also contributes to transcriptional regulation of endogenous Lmo2 proximal promoter; however the question remains as to which factors are mediating these looping interactions. LMO2 itself is one of the regulatory transcription factors bound to the upstream DRE, specifically at 75, 25 and 12, all of which showed increased interaction with the Lmo2 promoter suggesting an important role for the LMO2 complex in chromatin looping. In support of this, a recent study found LDB1 (a member of the LMO2 complex) at regions of chromatin interaction with the LDB1 bound Hbb-b1 promoter Hbb-b1 and the LCR Lmo2 locus. Cohesin (RAD21) is bound within both the dCR and pCR, specifically at the 75, 25 DRE as well as at the proximal promoter suggesting cohesin bound at the upstream DRE supports erythroid cell specific looping interactions with the Lmo2 proximal promoter.We identified erythroid-cell specific chromatin loops between a dCR and the Lmo2 upstream region, all of which occur within erythroid cell specific interacting domains, though the majority of the CTCF sites were not specific to erythroid cells. This is similar to the findings at the Hbb locus where CTCF bound regions, invariant between cell types, formed cell type specific chromatin loops Caprin1 from interacting with the DRE that enhance Lmo2 transcription in erythroid cells. As overexpression of Caprin1 causes inhibition of cell division it is critical to prevent its aberrant upregulation in rapidly cycling erythroid cells Lmo2 locus being supported by cohesin recruited both to CTCF bound regions  as well as at transcription factor and p300 bound enhancers not associated with CTCF .Cohesin is also recruited to CTCF occupied regions Lmo2 dCR is more reminiscent of the human Hbb LCR Lmo2 dCR may function to facilitate the physical interaction of the dCR with the Lmo2 proximal promoter through recruitment of both regions to a shared transcription factory. This co-localization at a shared transcription compartment would then allow transcription factors recruited to the dCR to influence the basal transcriptional machinery recruited to the Lmo2 proximal promoter thereby enhancing Lmo2 transcription.Our analysis indicates that a large portion of the dCR is transcribed at moderate levels in erythroid cells but not in kidney. Whereas short eRNAs have been identified at enhancers in neuronal cells the broad domain of intergenic transcription we observed throughout the Lmo2-Caprin1 locus adopts a tissue-specific conformation in erythroid cells. This tissue specific organization of the locus brings several DRE into proximity with the Lmo2 proximal promoter while excluding the Caprin1 TSS. A proximal control region, immediately upstream of the Lmo2 proximal promoter, contains the 12 and 25 DRE as well as the distal and intermediate promoters and is more closely associated with the Lmo2 proximal promoter in erythroid cells compared to kidney. Furthermore, a distal control region, covering 39 kb and containing three DRE , forms a strong interaction with the Lmo2 proximal promoter and shares many features with the well characterized Hbb LCR. A CTCF bound region upstream of the 90 DRE flanks the dCR and may function as an insulator preventing the interaction of Caprin1 with the erythroid cell specific Lmo2 enhancers.In conclusion, we found that the mouse All experiments were approved by the University Animal Care Committee (UACC) at the University of Toronto and the Bioscience Local Animal Care Committee (LACC).8) from the spleen of mice 5 days after phenylhydrazine treatment. This treatment induces haemolytic anaemia, as a result of which the spleen becomes the major site of red blood cell production Adult globin expressing mature erythroid cells were isolated from C57/Blk6 mice in large numbers (>1×10Lmo2-Caprin1 locus (RP23-76D2) with the Alpha Aortic Actin 2 BAC clone (RP23-2N15) followed by digestion with HindIII. The digested DNA was then ligated, and purified using phenol/chloroform extraction and ethanol precipitation. HindIII restriction enzyme digestion efficiency was confirmed to be between 85 and 95% efficient at several genomic fragments in anaemic spleen and kidney cells  was subsequently used for quantification. PCR products of the ligated fragments were quantified using real-time quantitative PCR (qPCR) on the Bio-Rad CFX-384 cycler. All data points were generated from an average of three-five independent 3C experiments with the qPCR performed in triplicate. Standard curves were generated by 5 fold serial dilution of the 3C control template and run in parallel with 3C experimental samples. The primers used in qPCR are listed in Gapdh or Epn1 were used for normalization of expression levels. The primers used for real-time RT-qPCR are listed in RNA from anaemic spleen and kidney was isolated using TRIzol, according to the manufacturer`s instructions (Invitrogen). The iScript First strand synthesis cDNA kit from Bio-Rad was used for preparation of random-hexamer primed cDNA. Amplification in qPCR was performed on the Bio-Rad CFX-384 cycler using the standard curve method. The reaction mixture contained 2X Bio-Rad iTaq SYBR green mastermix, 0.3 pM of each primer, 1uL cDNA (10 times diluted from a 20 uL of reverse transcription reaction). The conditions for qPCR were as follows: 94°C for 3 min followed by 40 cycles at 94°C for 30 s, 62°C for 30 s. Expression levels of The 3C data were analyzed by two-way ANOVA using Sigma Plot12. Post tests (Holm-Sidak method) were performed to assess significant differences between anaemic spleen and kidney samples at specific genomic locations.ChIP-Seq raw data for GATA1, KLF1, LDB1, TAL1, and MTGR1 Figure S1The Lmo2/Caprin1 region on mouse chromosome 2. Primers used in chromosome conformation capture (3C) and HindIII restriction sites are shown across the Lmo2/Caprin1 region of mouse chromosome 2. Promoters and distal regulatory elements (DRE) are depicted in red and black respectively. Anchor fragments used in the Caprin1, 75 DRE and Lmo2 3C experiments are marked with an asterisk (*). Distal promoter (pP), proximal promoter (pP).(PDF)Click here for additional data file.Figure S2Distal regulatory elements upstream of Lmo2 overlap transcription factor bound regions in HPC7 hematopoietic progenitor cells. The mouse Lmo2-Caprin1 region. Distal regulatory element (DRE) homology regions are indicated by black boxes joined by a line to delineate the human enhancer construct used in the generation of transgenic mice. Coloured boxes represent peaks identified from transcription factor ChIP-Seq data from HPC7 hematopoietic progenitor cells obtained from Wilson et al. 2010. Proximal promoter (pP), distal promoter (dP).(PDF)Click here for additional data file.Figure S3The Caprin1 TSS does not interact with distal regulatory elements upstream of Lmo2. Quantitative chromosome conformation capture (3C) was performed to detect chromatin-chromatin interactions between the Caprin1 TSS and distal regulatory elements (DRE). The profile of interactions identified in anaemic spleen (red) and kidney (blue) is displayed. Black box indicates the anchor fragment at Caprin1 and alternating intensities of grey boxes indicate the fragments investigated for interactions. Data points are an average of three independent biological replicates. Error bars depict the SEM, no significant differences were identified throughout this region.(PDF)Click here for additional data file.Figure S4CTCF bound upstream of Lmo2 in different cell types. The mouse Lmo2 upstream region on chromosome 2 is depicted with chromosome coordinates shown at the top. HindIII restriction sites are indicated by blue lines. The two Lmo2 promoters are indicated by red boxes. Distal regulatory element (DRE) homology regions are indicated by black boxes joined by a line to delineate the human enhancer construct used in the generation of transgenic mice. Mouse ENCODE ChIP-Seq data from B Ren (Ludwig Inst. for Cancer Research) and M Snyder (Stanford University) for CTCF in different cell types are shown below the DRE. Proximal promoter (pP), distal promoter (dP), murine erythroleukemia cells (MEL differentiated with 2% DMSO), bone marrow (BM), embryonic stem cells (ES-Bruce4), mouse embryonic fibroblasts (MEF).(PDF)Click here for additional data file.Figure S5Restriction digestion efficiency in chromosome conformation capture. Restriction digestion efficiency was between 85 and 95% at several HindIII restriction sites. Lmo2 proximal promoter (pP), Distal regulatory element (DRE), Caprin1 promoter (Caprin1P). “U” denotes a restriction fragment upstream of the indicated element.(PDF)Click here for additional data file.Table S1Coordinates of distal regulatory elements located upstream of the Lmo2 promoter in the mouse genome. Distal regulatory elements are named acording to their distance upstream of the annotated Lmo2 transcription start site overlapping the proximal promoter. Coordinates are given for homology regions identified by BLAT. All fragments were mapped in NCBI m37 mouse assembly (mm9).(PDF)Click here for additional data file.Table S2Coordinates of the Lmo2 proximal and distal promoters in the mouse genome. The coordintes of proximal promoters and distal promoters for the Lmo2 gene in the mouse genome are listed in the table. Coordinates are given for homology regions identified by BLAT. All fragments were mapped in NCBI m37 mouse assembly (mm9).(PDF)Click here for additional data file.Table S3Chromatin immunoprecipitation sequencing data. Transcription factor binding sites have been obtained from three different cell types; differentiated murine erythroleukemia cells (MEL), hematopoietic progenitor cells (HPC7), and GIE-ER4 a GATA1-null erythroblast cell line in which GATA1 activity was restored. CTCF, DNaseI hypersensitivity, p300 and RAD21 data have been obtained from the mouse ENCODE project, sources listed .(PDF)Click here for additional data file.Table S4Relative transcript abundance in intergenic regions upstream of Lmo2.(PDF)Click here for additional data file.Table S5Primers. Specific primers are listed for the chromosome conformation capture (3C) and RT-qPCR analyses. Left primer (L), right primer (R), primers used to test HindIII restriction digestion efficiency are marked as REX.(PDF)Click here for additional data file.


In vitro, Saccharomyces cerevisiae ISWI and CHD remodelers require ∼30–85 bp of extranucleosomal DNA to reposition nucleosomes, but linker DNA in S. cerevisiae averages <20 bp. To address this discrepancy between in vitro and in vivo observations, we have mapped the genomic distributions of the yeast Isw1, Isw2, and Chd1 remodelers at base-pair resolution on native chromatin. Although these remodelers act in gene bodies, we find that they are also highly enriched at nucleosome-depleted regions (NDRs), where they bind to extended regions of DNA adjacent to particular transcription factors. Surprisingly, catalytically inactive remodelers show similar binding patterns. We find that remodeler occupancy at NDRs and gene bodies is associated with nucleosome turnover and transcriptional elongation rate, suggesting that remodelers act on regions of transient nucleosome unwrapping or depletion within gene bodies subsequent to transcriptional elongation.ATP-dependent nucleosome remodelers influence genetic processes by altering nucleosome occupancy, positioning, and composition.  In vitro, yeast ISWI and CHD remodelers require 30–85 bp of extranucleosomal DNA in order to efficiently remodel chromatin, but in vivo, yeast linker DNA is, on average, <20 bp. By mapping yeast Isw1, Isw2, and Chd1 on native chromatin, we find that these remodelers bind to extended regions of linker DNA adjacent to transcription factor binding sites within nucleosome depleted regions. Remodeler binding is associated with nucleosome turnover and transcription rate, suggesting that ISWI and CHD remodelers help to reestablish proper chromatin structure following transcriptional elongation.Eukaryotic genomes are compacted into chromatin, which restricts access to DNA. In order for cells to transcribe, replicate, and repair DNA, chromatin structure must be altered. Eukaryotes have evolved chromatin remodeling enzymes that use energy derived from ATP hydrolysis to modulate chromatin structure.  Nucleosome remodelers use the energy of ATP hydrolysis to alter histone-DNA contacts, slide nucleosomes, and exchange or remove histones and entire nucleosomes. They are ubiquitous throughout eukaryotic evolution in vitroin vivoin vitro as a dimeric sensor of linker length, with one ATPase molecule contacting extranucleosomal DNA on each side of a nucleosome, and the efficiency of its nucleosome repositioning activity is decreased with decreasing linker length in vitroS. cerevisiae is 165 bp S. pombe, where the Chd1-like remodelers hrp1 and hrp3 act within gene bodies Yeast Isw1, Isw2, and Chd1 remodeler complexes bind ∼30–85 bp of extranucleosomal DNA, which is required for efficient nucleosome repositioning To address this paradox, we mapped the genomic distributions of yeast Isw1, Isw2, and Chd1 on native chromatin. We show that yeast ISWI and CHD remodelers are highly enriched at nucleosome-depleted regions (NDRs) flanking transcription units, where they bind to extended stretches of linker DNA flanking transcription factor binding sites (TFBSs). We find that remodeler binding is positively associated with nucleosome turnover and transcriptional elongation rate, suggesting that ISWI and CHD remodelers first associate with naked DNA within NDRs and subsequently relocate to gene bodies following nucleosome disruption by RNA Polymerase II transit, upon which there is ample linker DNA to promote their efficient remodeling activity.S. cerevisiae Isw1, Isw2, and Chd1. N-ChIP does not rely on formaldehyde fixation, which crosslinks primary amines such as those in lysine-rich histones We performed immunoprecipitation of uncrosslinked (native) chromatin digested with micrococcal nuclease (MNase) combined with high-throughput paired-end sequencing (N-ChIP-seq) for P = 0.009) and 2.26–2.76  times greater than that seen in input samples . We also detected strong enrichment of Isw2 but little or no binding of Isw1 or Chd1 at the A+T rich yeast recombination enhancer , further arguing against nonspecific association of remodelers with free DNA during our experiments.The sharp patterns of phased nucleosomes on either side of binding sites for Abf1, Reb1 and other TFs is consistent with their known role in creating NDRs that lead to transcriptional activation Our Isw2 N-ChIP-seq results are somewhat different from those generated using ChIP-exo. We found Isw2 to be enriched upstream of ORF 5′ ends to a distance of nearly 1 kb in some cases , whereasin vitro preference for substantial extranucleosomal DNA and its action positioning nucleosomes adjacent to NDRs in vitro association with significant amounts of linker DNA Our results demonstrate that yeast ISWI and CHD remodelers associate not only with gene bodies, where their effects are seen, but also with regions of extended linker DNA flanking TFBSs within NDRs. In the case of Isw2, robust binding to linker DNA within NDRs is consistent both with its This model predicts that Chd1 and Isw1 association with gene bodies depends on transcription rate. As such, we would not expect to observe an association between remodeler binding and steady-state expression levels, as infrequently transcribed genes may yield mRNAs with long half-lives and vice versa What is the relationship of ISWI and CHD remodelers to transcription? Previous work has demonstrated that simultaneous deletion of Isw1, Isw2, and Chd1 in yeast does not alter promoter nucleosome occupancy or positioning Drosophila ISWI is similarly abundant It is estimated that there is sufficient Isw2 for a molecule be bound every 2–5 kb of DNA, with a similar abundance for Chd1 and twice that amount for Isw1 . Additionally, high salt is required for remodeler extraction from cells, indicating that they are bound to chromatin 600 = 0.6–0.8. Mutation of Chd1 K407 was carried out as follows. Primers F- GGTACCCTTGGGAGAATGCCACAGAT -3′5′-, containing a KpnI site (underlined) and R- ATCGATCTTCGTCAACGGCCATAAAT -3′5′-, containing a ClaI site (underlined) were used to amplify nt 961–1543 of CHD1. The amplified fragment was TOPO-cloned into pCR2.1. pCR2.1-CHD1-961–1543 was digested with KpnI and ClaI and the CHD1 fragment was ligated into pRS406 and mutagenized with the QuikChange kit (Agilent). Mutation was confirmed by sequencing and introduced a strain harboring a Chd1-3xFLAG allele via the pop-in/pop-out method Yeast cells were grown in YPD and harvested at OD2. Chromatin was digested with MNase for 2.5 or 10 min and digestion was stopped by the addition of EDTA to 10 mM. MNase-treated nuclei were then passed 4 times through a 20-gauge needle and 4 times through a 26-gauge needle Nuclei were isolated from yeast cells and stored at −80°C in 5 ml sorbitol-PIPES-calcium (SPC) buffer as described th volumes of 5 M NaCl and 0.5 mM EDTA were added to IP and input samples and RNA was digested with 1 µg RNase A for 10 min at 37°C. SDS was added to IP and input samples to a concentration of 0.5% and protein was digested with 80 µg Proteinase K for 20 min at 65°C. 20 µg glycogen was added to each IP and input sample followed by extraction with 1 volume phenol/chloroform/isoamyl alcohol. DNA was precipitated with 2 volumes 100% ethanol for 30 min at −80°C and washed twice with 500 µl 100% ethanol. Precipitated DNA was resuspended in 20 µl 0.1X TE buffer, pH 8.0 and quantified by PicoGreen 100 µl of each chromatin sample was saved as input. 100 µl of FLAG M2 magnetic beads  were used for all remodeler-FLAG immunoprecipitations (IPs). Beads were washed 3 times with PBS/0.5% BSA and added to the chromatin samples. IPs were incubated overnight at 4°C with end-over-end rotation. Beads were washed 3 times with IP buffer  supplemented with 10 µg/ml LPC and 1 mM PMSF and resuspended in 400 µl IP buffer. 1/50X-ChIP was performed as above with the following modifications. In addition to washing in IP buffer, IPs were washed twice with TSE I  and twice with TSE II . Elution and crosslink reversal was performed overnight at 65°C in elution buffer . Following elution, 1 volume of TE buffer pH 8.0 was added to each sample to dilute the SDS to 0.5% for RNase A and proteinase K treatment as above.Sequencing libraries were prepared according to our previously described modification of the standard Illumina protocol http://www.novocraft.com) and processed as described http://www.gnuplot.info). The V-plot scale represents the percentage of fragments used in constructing each V-plot contained in each bin (pixel) of the plot .Paired-end sequencing data were processed and aligned to the V64/SacCer3 genome build with Novoalign  N-ChIP-seq signal for each base pair ±1 kb of the midpoint of each Isw2 K215R-bound region, site of Isw2 remodeling, Isw2 ChIP-exo site, and random site was determined as above and averaged to generate aggregate plots.Isw2 K215R ChIP-chip binding sites, sites of chromatin remodeling by Isw2, and random sites were obtained from Whitehouse et al. For DNA bendability analysis, 29 100 bp regions bound by Isw2 without Isw1 and/or Chd1 and 29 100 bp regions bound by Isw1 and/or Chd1 without Isw2 were obtained. Regions were analyzed using the bend.it server 2(IP/input) for each base pair ±1 kb of each ORF 5′ or 3′ coordinate was determined using a custom Perl script 2(IP/input) N-ChIP-seq signal within each NDR.The SGD verified ORFs list was used to determine transcription unit length and ORF 5′ and 3′ end coordinates. For heatmaps, the logSequencing data generated in this publication have been deposited with GEO (GSE39331).Figure S1Additional Isw1, Isw2, and Chd1 N-ChIP-seq profiles. Signal tracks of Isw1, Isw2, and Chd1 binding in representative regions of the genome. Counts/bp is indicated on the Y-axis.(TIF)Click here for additional data file.Figure S2V-plots of wild-type Isw1, Isw2 and Chd1 ChIP and input data at Abf1, Cbf1, Mbp1, and Reb1 sites. Note the discrete lower size limit of flanking nucleosomes at ∼147 bp in 10′ MNase-treated samples, indicating that these nucleosomes are tightly wrapped.(TIF)Click here for additional data file.Figure S3V-plots of wild-type and catalytically inactive Isw1, Isw2, and Chd1 ChIP and input data at ChIP-exo-defined Reb1 binding sites. Binding sites are derived from the data of Rhee and Pugh (TIF)Click here for additional data file.Figure S4Loss of remodeler catalytic activity does not alter remodeler fragment size distributions. Size distributions of mapped paired-end 2.5′ MNase-digested wild-type and K215R Isw2 and wild-type and K407R Chd1 ChIP and input fragments.(TIF)Click here for additional data file.Figure S5V-plots of catalytically inactive Isw1, Isw2 and Chd1 ChIP and input data at Abf1, Cbf1, Mbp1, and Reb1 sites.(TIF)Click here for additional data file.Figure S62(Isw1 IP/input) native and crosslinked 2.5′ MNase signal ±1 kb of verified ORF 5′ ends ranked descending by gene expression level.Isw1 association with 5′ NDRs is captured by X-ChIP-seq. Heatmaps of log(TIF)Click here for additional data file.Table S1RAD5 mutation is repaired Yeast strains used in this study. All strains are derived from W1588-4C, which is isogenic to W303-1A except that a weak (DOC)Click here for additional data file.Table S2Numbers of mapped pairs and supernucleosomal/nucleosomal AUC ratios for sequenced samples. ChIP/input is the ChIP supernucleosomal/nucleosomal AUC ratio divided by the input supernucleosomal/nucleosomal AUC ratio.(DOC)Click here for additional data file.


We show that Mediator is still present at the promoter when the Pol II machinery is recruited in the absence of an activator, in this case through a direct fusion between a basal transcription factor and a heterologous DNA binding protein bound to the promoter. Moreover, transcription resulting from activator-independent recruitment of the Pol II machinery is impaired by inactivation of the essential Mediator subunit Med17 due to the loss of Pol II from the promoter. Our results strongly support that Mediator is an integral component of the minimal machinery essential in vivo for stable Pol II association with the promoter.Mediator is a prominent multisubunit coactivator that functions as a bridge between gene-specific activators and the basal RNA polymerase (Pol) II initiation machinery. Here, we study the poorly documented role of Mediator in basal, or activator-independent, transcription  Mediator is a large multisubunit complex, conserved throughout eukaryotes, that plays an essential role in transcription of protein-encoding genes. It was first discovered in yeast due to its ability to support activator-driven transcription on naked DNA templates in a cell-free system reconstituted with purified RNA polymerase II (Pol II) and general transcription factors , Transcription Factor (TF) IIB, TFIIF, TFIIE and TFIIH) . Mediatoet al.  on the chromosome and plasmids RY2844  or RY2882  in the W303-1A genetic background  were replaced by TRP1. This strain was crossed with Y84 and appropriate segregants combining met4Δ::TRP1 and MED14-TAP-klTRP1 were selected by tetrad analysis and confirmed by polymerase chain reaction (PCR).Genotypes are given in e et al.  upstreamy et al. . Y822 ant et al. . Y959 anYNB medium contains 0.7% yeast nitrogen base, 0.5% ammonium sulfate and 2% glucose. Complete Supplement Mixture (CSM) medium contains, in addition, a CSM amino acid drop-out mixture.HIS3, CEN) was generated from pRS314-TFIIB-RFX  (a gift from Michel Strubin) by subcloning an Xba1-Xho1 fragment containing the promoter region of TBP followed by the ORFs of TFIIB and the human regulatory factor X (RFX) protein. A short linker coding for the nuclear localization signal (NLS) of SV40 and the HA epitope from influenza virus is found in between TFIIB and RFX. YCp91-LexA  contains the entire LexA coding sequence (residues 1–202) under the control of the ADH1 promoter, followed by SV40 NLS, an HA epitope and the CYC8 terminator  contains the SPT15 coding sequence and terminator in place of the HA epitope  was constructed by cloning into YCplac33-GFP (a gift from Dieter Kressler) a PCR fragment spanning nucleotides −1 to −400 of MET17 and containing at position −200 the sequence CAGTTGCCTAGCAACTACATATGGTCACC (refer-red to as X box) including the RFX binding site found in the polyomavirus enhancer (underlined). The xhis3 fragment in YCplac33-xhis3-GFP originates from a LexAop-his3 construct containing a LexA operator inserted into a derivative of his3 lacking the Tc element and a functional Gcn4 binding site  were constructed by cloning into YCplac33-GFP PCR fragments spanning nucleotides −1 to −392 of PHO5 or nucleotides −1 to −500 of GAL1, and containing the X box inserted at position −167 and −200, respectively. YCplac33-LexAop-MET17-GFP was constructed by cloning into YCplac33-GFP a fragment, engineered by PCR, containing the sequence CTACTGTATGTACATACAGTAGTTTGTT (LexA operator underlined) inserted at −200 in a MET17 fragment spanning from −1 to −400 (relative to ATG).pRS313-TFIIB-RFX (rminator . YCp91-L epitope . pRS315-650 = 1. TAP-tagged proteins were immunoprecipitated by incubating the same amount of crosslinked chromatin with 30 µl of rabbit Immunoglobulin G (IgG)-agarose (Sigma) for 4 h at 4°C. DNA was quantified by real-time PCR using the LightCycler 480 instrument (Roche), and SYBR Premix Ex TaqTM (Takara). Sequences of primers are given in Supplementary Table S2. A typical run included duplicates of each IP and input DNA, and serial dilutions of one input DNA to create a standard curve and determine the efficiency of the amplification. Data were analysed with the LightCycler 480 software using the ‘second derivative maximum’ method for quantification. The level of occupancy at a specific DNA locus is calculated as the percentage of DNA present in the immunoprecipitate relative to the total input. Relative occupancy was obtained by normalizing to occupancy at the IME2 locus.Cell fixation, chromatin preparation and immunoprecipitation were performed essentially as described previously . In Figuet al.  and the suspension was mixed with 400 µl of cold phenol saturated with 0.1 M citrate buffer pH 4.3 (Sigma). The mixture was incubated for 8 min at 65°C with agitation, quickly frozen in liquid nitrogen, incubated again at 65°C with agitation for 4 min and centrifuged at room temperature for 10 min, 12 000 rpm. The aqueous phase was extracted once with 1 vol acidic phenol/chloroform (1:1), once with 1 vol chloroform and was precipitated with 0.1 vol 3 M LiCl and 2.5 vol absolute ethanol. After centrifugation, the RNA pellet was washed with absolute ethanol, air-dried and resuspended in water.Total RNA was extracted with hot acidic phenol following a protocol derived from Schmitt et al. . BrieflySupplementary Table S2.Reverse transcription (RT)-quantitative PCR was conducted following a two-step procedure using the RevertAid H Minus M-MuLV reverse transcriptase (Thermo Scientific) and random hexamers for priming, and the SYBR Green I Master mix and LightCycler 480 system (Roche) for real-time PCR. Sequence of primers is given in 32P-5′-labelled gene-specific oligonucleotides and 20 nmol of each dNTP was heated for 15 min at 65°C and cooled down on ice. Ten microliters containing 200 units of SuperScript II Reverse Transcriptase (Invitrogen) in 2× first-strand buffer and 0.02 M DTT was added, and the mixture was incubated at 42°C for 30 min. Reaction was stopped by adding 10 µl containing 0.9 M LiCl, 40 µg of glycogen and 10 µg of RNase. After 5 min at room temperature, the DNA was ethanol-precipitated, centrifuged, air-dried, dissolved in 6 µl of formamide-containing loading buffer and electrophoresed in a 6% denaturing polyacrylamide gel. Sequence of primers is given in Supplementary Table S3.Primer extension was performed as follows. Ten microliters containing 10 µg of RNA, 1 pmol of Protein extracts were prepared using mechanical breakage with glass beads as described in Dunn and Wobbe . One micin vivo, we took advantage of molecular tools allowing artificial recruitment of the Pol II machinery to a test promoter in the absence of classical activator protein in yeast  fused to the coding sequence of the GFP was introduced, with a plasmid bearing TFIIB or TFIIB-RFX, into a strain containing the chromosomal TFIIB allele under control of the glucose-repressed GAL1 promoter to shut down expression of the endogenous TFIIB. As expected, the xMET17 promoter remained inactive in cells expressing TFIIB in the presence of 0.5 mM methionine, and attaching TFIIB to RFX led to a strong increase in xMET17-GFP transcription . Therefore, artificial tethering of TFIIB to MET17 results in efficient and accurate transcription initiation in the absence of the activator.To assess the role of Mediator in basal transcription east see A. We firin yeast ,35. The hut down ,37. MET1thionine . A plasmcription B. We concription C. MoreovxMET17 promoter when transcription is driven by TFIIB-RFX, we carried out chromatin immunoprecipitation (ChIP) experiments using cells containing TAP-tagged versions of Med5 and Med14, two Mediator subunits belonging to the middle and tail modules, respectively. The results for Med14-TAP and Med5-TAP showed a 3- to 4-fold increase in occupancy at the xMET17 promoter in the strains expressing TFIIB-RFX compared with the strains expressing TFIIB   . Therefot xMET17 A, and tht levels B nor GFPt levels C. As alrt levels , transcrof genes , which sin vivo .We next used a mutation in the Med17 subunit located at the Mediator head module, knowing that this module makes multiple contacts with Pol II in the structural models ,41. Med1in vivo . We used at 37°C . Mutant  at 28°C . In contkin28-ts3 mutation already known to cause a global shutdown of mRNA synthesis at 37°C . However, occupancy was decreased by 3- to 4-fold in the mutant cells compared with the wild-type cells at 37°C. Therefore, inactivation of Med17 and Kin28 has the same effect on transcriptional activation of xMET17-RFX by TFIIB-RFX.For comparison, ChIP experiments were also performed with strains carrying a temperature-sensitive mutation in the kinase subunit of TFIIH. We used the  at 37°C ,30. The HIS3, PHO5 or GAL1 promoters  for all three genes . Moreover, as in the case of xMET17-GFP, inactivation of Med17 resulted in a decrease in transcription activation of the three promoters by TFIIB-RFX . For instance, Pol II occupancy at xhis3-GFP and xPHO5-GFP was decreased by 4- and 6-fold, respectively, in the med17-138 mutant compared with the wild-type cells after 60 min at 37°C  selection. We analysed TSS positions at ple TSSs . The resple TSSs . TherefoLexAop-MET17-GFP test reporter containing one LexA operator inserted 69 bp upstream of the TATA box of MET17 (see schematic in LexAop-MET17 in the TAP-tagged strain expressing LexA-TBP compared with the TAP-tagged strain expressing LexA alone (med17-138 mutant and the isogenic wild-type strain. As shown in LexAopMET17-GFP at 28°C. In contrast, occupancy was decreased 9-fold in the mutant compared with the wild type cells after 60 min at 37°C. Therefore, transcription activation by tethering TBP to the promoter is also accompanied by recruitment of Mediator and is also sensitive to inactivation of Med17.Finally, we asked whether Mediator is also required when the Pol II machinery is recruited to DNA by tethering TBP instead of TFIIB. To this end, we used a LexA-TBP hybrid containing TBP fused to the full-length bacterial repressor LexA , and a Lin vivo independently of the presence of an activator, solely via its ability to interact with Pol II and the GTFs. Mediator has been known for a long time to form a biochemically stable complex with Pol II (in vivo has also been reported recently (in vivo in the absence of interaction with an activator. This scarcity of data is certainly a consequence of the transient nature of the interaction between Mediator and Pol II at promoters, which occurs only during transcription initiation and must be broken to allow Pol II escape (Conventional and genome-wide ChIP analyses in yeast have clearly demonstrated that Mediator is crosslinked by formaldehyde preferentially to enhancers rather than to core promoters where Pol II binds; moreover, based on time-course experiments and mutant studies, it was also shown that Mediator association with promoters is independent of Pol II, GTFs and core promoter sequences ,20,21,47h Pol II ,48, and h Pol II ,49. The recently  but, to I escape , whereasI escape .med17-138 demonstrate a role of Mediator in assembly of the basal Pol II initiation machinery at promoters even in the absence of signals transmitted by activators. Importantly, this conclusion was reached using two GTFs tethered to four different test promoters through two different DNA binding proteins. Strikingly, inactivation of Med17 has a more pronounced effect on Pol II recruitment to opMET17 via LexA-TBP than on Pol II recruitment to xMET17 via TFIIB-RFX (compare in vivo. This view is in line with a previous in vitro study showing that the requirement of human Mediator for basal transcription in nuclear extracts can be alleviated by increasing the TFIIB amount (med17-138 mutant at a restrictive temperature is also certainly owing to incomplete inactivation of Med17. Indeed, it was observed that the MED6-101 dominant mutation could suppress the temperature-sensitive phenotype of med17-138 but not the lethality associated with the med17-null allele, indicating that the Med17-138 protein retains some function even at a non-permissive temperature (ACT1 than at IME2 in med17-138 after 60 min at a non-permissive temperature (Our results with  compare  and 7. TB amount . Part ofperature . This experature  and 7.xMET17 promoter by TFIIB-RFX, even though this module is required for activation of MET genes by Met4 (in vivo confirmation to the finding that Mediator head alone is sufficient to stimulate basal transcription in vitro (In contrast with Med17, we have found that inactivation of subunits of the tail module does not affect activation of the  by Met4 . In agrein vitro ,54.Structural studies showed that the yeast Mediator complex adopts two different conformations when free or when associated with Pol II: the free complex presents a compact conformation with the middle and tail modules folded on each other, whereas the complex associated with Pol II presents an extended conformation, with Pol II binding in a pocket generated when Mediator changes conformation . It is nMediator is often presented as a relay conveying regulatory signals from gene-specific activators bound at distal regulatory elements to the basal Pol II machinery located at the core promoter. The present study shows that the yeast Mediator functions not only through targeted recruitment by gene-specific factors, but also in an untargeted manner, strongly supporting a model in which Mediator is an integral part of the basic Pol II initiation machinery and can exert a function independently of regulatory signals conveyed by activators. Considering the functional and structural conservation of Mediator ,56, thisSupplementary Data are available at NAR Online.Centre National de la Recherche Scientifique, and grants from Agence Nationale pour la Recherche [ANR-06-JCJC-0045 and ANR-08-BLAN-0229]; and Association pour la Recherche sur le Cancer [subvention 4901]. Funding for open access charge: Centre National de la Recherche Scientifique.Conflict of interest statement. None declared.


The gaseous plant hormone ethylene regulates a multitude of growth and developmental processes. How the numerous growth control pathways are coordinated by the ethylene transcriptional response remains elusive. We characterized the dynamic ethylene transcriptional response by identifying targets of the master regulator of the ethylene signaling pathway, ETHYLENE INSENSITIVE3 (EIN3), using chromatin immunoprecipitation sequencing and transcript sequencing during a timecourse of ethylene treatment. Ethylene-induced transcription occurs in temporal waves regulated by EIN3, suggesting distinct layers of transcriptional control. EIN3 binding was found to modulate a multitude of downstream transcriptional cascades, including a major feedback regulatory circuitry of the ethylene signaling pathway, as well as integrating numerous connections between most of the hormone mediated growth response pathways. These findings provide direct evidence linking each of the major plant growth and development networks in novel ways.DOI:http://dx.doi.org/10.7554/eLife.00675.001 All multicellular organisms, including plants, produce hormones—chemical messengers that are released in one part of an organism but act in another. The binding of hormones to receptor proteins on the surface of target cells activates signal transduction cascades, leading ultimately to changes in the transcription and translation of genes.Ethylene is a gaseous plant hormone that acts at trace levels to stimulate or regulate a variety of processes, including the regulation of plant growth, the ripening of fruit and the shedding of leaves. Plants also produce ethylene in response to wounding, pathogen attack or exposure to environmental stresses, such as extreme temperatures or drought. Although the effects of ethylene on plants are well documented, much less is known about how its functions are controlled and coordinated at the molecular level.Arabidopsis thaliana. Ethylene is known to exert some of its effects via a protein called EIN3, which is a transcription factor that acts as the master regulator of the ethylene signaling pathway. To identify the targets of EIN3, Chang et al. exposed plants to ethylene and then used a technique called ChIP-Seq to identify those regions of the DNA that EIN3 binds to. At the same time, they used genome-wide mRNA sequencing to determine which genes showed altered transcription.Here, Chang et al. reveal how ethylene alters the transcription of DNA into messenger DNA (mRNA) in the plant model organism, Over the course of 24 hr, ethylene induced four distinct waves of transcription, suggesting that discrete layers of transcriptional control are present. EIN3 binding also controlled a multitude of downstream transcriptional cascades, including a major negative feedback loop. Surprisingly, many of the genes that showed altered expression in response to EIN3 binding were also influenced by hormones other than ethylene.In addition to extending our knowledge of the role of EIN3 in coordinating the effects of ethylene, the work of Chang et al. reveals the extensive connectivity between pathways regulated by distinct hormones in plants. The results may also make it easier to identify key players involved in hormone signaling pathways in other plant species.DOI:http://dx.doi.org/10.7554/eLife.00675.002 ETHYLENE RESPONSE FACTOR1 [ERF1]), it is challenging to distinguish immediate early targets from those further downstream. To understand the dynamics of the EIN3-mediated ethylene transcriptional response, we performed a genome-wide study of the ethylene-induced EIN3 protein-DNA interactions using chromatin immunoprecipitation followed by sequencing (ChIP-Seq) and simultaneously determined the repertoire of target genes that are transcriptionally regulated by ethylene (mRNA-Seq). Tracing the transcriptional cascade, we asked if EIN3-mediated genes contribute to a component of the ethylene transcriptional response. For a select number of EIN3 targets that are putative transcriptional regulators, DNA-binding motifs were identified using protein binding microarrays (PBM) and the enrichment for these motifs in the promoters of ethylene response genes was determined.Despite the importance of the plant hormone ethylene, we lack a comprehensive understanding of how its linear signaling pathway mediates many different morphological responses. The dynamic ethylene physiological response, a rapid growth inhibition independent of the master transcriptional regulator ETHYLENE INSENSITIVE3 (EIN3), followed by an EIN3-dependent sustained growth inhibition, calls for a temporal study of ethylene transcriptional regulation . EIN3 haArabidopsis genome associated with 1314 genes (−87) (EIN3-BINDING F-BOX PROTEIN 2 (EBF2), is shown in EBF2 promoter region (We performed ChIP-Seq using a native antibody that recognizes EIN3  as well 14 genes . We refees (−87) , and de es (−87) . We detees (−87)  (Solano es (−87) . One exar region .10.7554/The majority of studies that exist in the literature have shown that EIN3 acts as an activator, and we observed this activation at the genome-wide level . We founNumerous studies have reported that transcription factor binding does not necessarily coincide with changes in transcription , especia−10) (Projection of the dynamic EIN3 binding (ChIP-Seq) onto the transcriptional ethylene response (mRNA-Seq) using the Dynamic Regulatory Events Miner (DREM)  revealed−10) . These w−10) . Genes t−10) .−10)  . The expA recurring theme throughout this study is that the key players in the ethylene transcriptional response regulated by EIN3 are involved in plant hormone response pathways, and we anticipate a dense network of interconnections between the coregulated hormone pathways because hormones operate in concert, synergistically/antagonistically regulating growth and development. Although hormone pathway interconnections have been previously described by many groups , here weGiven that EIN3, the master regulator of the ethylene transcriptional response, acts at the culmination of the ethylene signal transduction pathway and is the transcriptional initiator of the ethylene response, one would expect a large number of downstream effectors to coordinate the transcriptional cascade and feedback regulators to maintain the circuitry in a homeostatic state as opposed to a feed-forward runaway response. Analysis of the ethylene-regulated EIN3 targets reveals a number of sites of ethylene signaling modulation of which the majority are negative regulators, supporting the idea that EIN3 is at the end of a signal transduction pathway, and that this regulatory logic dictates a negative feedback loop for homeostatic adaptable systems. More specifically, several negative regulators of the ethylene signaling pathway  were tar−96)  , inset (−96) . Many ofbidopsis . Hormonebidopsis . Many debidopsis  . The fibidopsis .RAP2.6L, ERF1. EIN3 also targets four JAZ repressors, two of which are transcriptionally regulated by ethylene . In general, ethylene and jasmonate are known to function synergistically and in the presence of jasmonate, JAZ1 proteins bound to EIN3 are degraded, relieving the EIN3 transcriptional activation  and transcription (PIF3). The regulatory logic of EIN3 binding results in an up-regulation of the gibberellin response; GID receptors target DELLA repressors for degradation, which releases PIF3 from repression, resulting in the activation of the gibberellin transcriptional response. Additional support for feed-forward transcription is provided by over-representation of the PIF3 motif in the promoter sequences of the ethylene transcriptional response genes . HormoneEthylene and auxin co-regulation occurs at both the level of transport and transcriptional response, as EIN3 modulates a regulator of auxin efflux (PID) and its upstream activator (PBP1), and at least seven auxin response proteins . EIN3 al−5)  . Future HOOKLESS1 (HLS1) and HLS1-LIKE HOMOLOG2 (HLH2), and to a lesser extent HLH1  DNA-binding protein  and, in fact, we found that the Physcomitrella patens (moss) EIN3 protein binds a very similar motif sequence to that of the Arabidopsis thaliana EIN3  was the parent strain for these experiments. Genotypes used for this study include wild-type Col-0, and mutants ein3-1 (ein3-1/eil1-1 (hls1-1 (hls1)  , hlh1, hh2, hlh3 .−1 for 0, 0.25, 0.5, 1, 4, 12, and 24 hr.Three-day-old etiolated seedling tissue was used for these experiments unless otherwise noted. Seeds were sterilized and sown on Murashige and Skoog  media pH5.7, containing 1% sucrose and 1.8% agar. After stratification for 3 days in the dark at 4°C, exposure to light for 2–4 hr to induce germination, seeds were dark-grown in hydrocarbon free air at 24°C for 3 days. Etiolated seedlings were subsequently treated with ethylene gas at 10 μl lEtiolated seedlings were collected in the dark, immersed in 1% formaldehyde solution, and cross-linked under vacuum for 15 min. A final concentration of 125 mM glycine was used to quench the formaldehyde for 5 min under vacuum. Cross-linking under vacuum resulted in translucent etiolated seedling tissue. Tissue was liquid nitrogen ground and extraction of chromatin was performed as described in .Chromatin immunoprecipitation (ChIP) was performed as described in  with modAffinity-purified rabbit polyclonal antibodies capable of detecting the C-terminus of EIN3 were used in immunoprecipitation reactions. Details regarding the generation of EIN3 antibodies were previously described . Prior tein3-1 mutant).Quantitative PCR revealed that relative ChIP enrichment for the promoter of ERF1 performed with the Dynabeads M-280 Sheep anti-Rabbit IgG was higher in comparison to Dynabeads Protein A . Thus, DChIP was performed in chromatin derived from wild-type Col-0 three-day-old etiolated seedlings treated with 0, 0.25, 0.5, 1, 4, 12, and 24 hr of ethylene. Two independent biological replicates were used in two replicates experiments for timepoints, 0, 0.5, 1, 4 hr ethylene gas treatment. Single replicates exist for 0.25, 12, 24 hr of ethylene gas treatment.Total RNA was extracted from liquid nitrogen ground etiolated seedlings using the Qiagen RNeasy Plant Mini Kit with Qiashredder columns , with DNaseI  treatment prior to RNA precipitation in sodium acetate and ethanol. Concentrations of RNA were determined using the ND-1000 spectrometer . Experiments were performed in three biological replicates for timepoints, 0, 0.25, 0.5, 1, 4, 12, 24 hr ethylene gas treatment.Resulting ChIP DNA from two pooled ChIP reactions above was used to generate a sequencing library as per the Illumina ChIP-Seq manufacturer’s instructions. The Illumina Genome Analyzer II  was used to sequence the single-read ChIP-Seq libraries as per manufacturer’s instructions, for 36–43 bps . Raw seqAt least 80 μg total RNA was subject to polyA selection using the Poly(A)Purist MAG Kit . PolyA RNA was subsequently concentrated by ammonium acetate ethanol precipitation and concentrations were determined using the Qubit fluorometer  and the Quant-iT RNA Assay Kit . 50–100 ng of polyA RNA was used in a strand-specific library preparation as per the SOLiD Total RNA-Seq Kit protocol  and AMPure XP beads  were used for purification of cDNA and amplified DNA. Samples were barcoded for multiplexing using the SOLiD RNA Barcoding Kit  as per manufacturer’s instructions; final size selection was performed using AMPure XP beads instead of the PAGE purification recommended in the protocol. Size selected libraries were then purified using the MinElute Gel Extraction Kit . Resulting concentrations of libraries were detecting using the Qubit fluorometer and Quant-iT dsDNA High-Sensitivity Assay Kit . RNA libraries were sequenced for 50 bps on the SOLiD4 platform  .ftp://ftp.arabidopsis.org/). The analysis variable for the ELAND alignment program was set to eland_extended, as read length was greater than 32 bases . Resulting aligned unique single copy reads were used in ChIP-Seq peak analysis  EIN3 ChIP samples as controls. Three software packages: spp   .EIN3 binding profiles of previously determined targets are shown in www.ebi.ac.uk/Tools/msa/clustalw2/). Consensus motif representation of the three EIN3 binding sites in the promoter of EBF2 is shown in Motif identification was performed with the matrix screening software Patser  and the Arabidopsis thaliana  was performed using STAMP . Details of the design and use of universal PBMs has been described elsewhere . Here, wThe creation of a position frequency matrix (PFM) from a PBM experiment is non-trivial. For each TF, we therefore evaluated a panel of three algorithms and chose the PFM with the highest performance. For each TF, we ran each algorithm individually on both PBM experiments (HK and ME array designs). The resulting PFMs were then used to score the probe sequences of the opposite array, and these predictions were evaluated based on their Pearson correlation with the actual intensities across all probes. Based on these evaluations, a final PFM was chosen for each TF from the six possible PFMs .We chose three algorithms based on their high performance on an independent PBM dataset (data not shown). Two of the methods, BEEML-PBM , and FeaThe presence of these motifs in the promoter region (−1000bp) of genes that were transcriptionally induced/repressed by ethylene was evaluated to find candidate transcription factors that may be involved in regulating the secondary ethylene transcriptional response. The matrix screening software Patser  was usedftp://ftp.arabidopsis.org/). Two perfect matches per location were allowed. Exonic expression was determined (RPKM) using mRNA-Seq reads mapping in exons in the direction of transcription. Genes were denoted as expressed if they contained RPKM values greater than one for at least one biological replicate in one timepoint. Differentially expressed genes were then called , and log2 normalized with respect to the 0 hr ethylene gas treatment control , non-ethylene-regulated (EIN3-NR), and transcription not detected in etiolated seedlings (EIN3-ND). The heatmap  revealedThe kinetics of transcription was determined for all genes that were transcriptionally regulated by ethylene, and EIN3-R, and reflects the previous growth inhibition study kinetics . The eth−10) were observed. There was a variable amount of noise and percentage of hormone-related genes in each wave of transcription (−10) was used to identify groups of genes with a significant percentage (>15%) of EIN3 candidate targets.Four temporally distinct (Hypergeometric p<0.001) EIN3-modulated waves of transcription  included high throughput data (ChIP-chip and ChIP-Seq) for several transcription factors including AGL15, HY5, GL3, AtbHLH15, WRKY53, GL1, E2F, and SEP3 as well as literature curated data  in addition to other datasets including relevant ethylene microrarray studies in etiolated seedlings , we used the annotation in the Arabidopsis Hormone Database   exist in Arabidopsis genome. The protein sequences of the HLHs are homologous to the full-length protein . Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see eLife. Your article has been favorably reviewed by Detlef Weigel (eLife Deputy Editor) and an outside peer reviewer. Both discussed their comments and the following reflects their combined assessment of your work.Thank you for sending your work entitled “Temporal transcriptional response to ethylene gas drives growth hormone cross-regulation in Arabidopsis” for consideration at The manuscript by Chang et al. greatly advances our understanding of how the effects of the plant hormone ethylene are mediated at the molecular level. The authors present a coordinated analysis of transcriptional changes and genome-wide binding of the key transcription factor EIN3 (which accumulates upon ethylene treatment and which is both necessary and sufficient for most ethylene responses) during a time course after addition of ethylene. There are very few reports in the literature tracking genome-wide transcription factor binding after a stimulus, and the integration with gene expression measurements greatly increases the inferences that can be drawn from the genome-wide DNA binding studies. This tour de force will set a new bar for the analysis of transcription factor networks.Using ChIP-Seq and RNA-Seq, the authors analyzed young Arabidopsis seedlings for EIN3 binding and transcriptional modulation after addition of ethylene. Important findings are that there are several discrete waves of transcriptional responses; that a negative feedback loop represses ethylene receptor expression; and that much of the previously known cross-talk between hormone pathways involves EIN3 binding to genes regulated by several hormones. The interaction with other hormone pathways is nicely demonstrated with the knockout of a small family of closely related EIN3 targets, which reproduces aspects of phenotypes seen in mutants deficient in the response to another hormone, auxin.The authors also introduce a clever shortcut for understanding the regulatory logic of the later waves of ethylene triggered gene expression. They first nominate candidates for transcription factors that themselves are transcriptionally responsive to EIN3 and that mediate these later responses. The authors then use microarrays to identify sequences that 12 such candidate factors can bind to in vitro. These motifs are then confirmed to be overrepresented in the promoters of the late EIN3 targets.The only major criticism is that the authors should think a bit harder about their nomenclature. It seems that “targets” refer to any genomic sequence that is bound by EIN3 (or any gene that is within a certain distance from an EIN3 binding site). More commonly, “target” is reserved for bona fide repressed or activated genes. For example, the sentence “many EIN3 targets were not transcriptionally ethylene-regulated (EIN3-NR) (67%), while a subset of the EIN3 targets was transcriptionally ethylene-regulated” should be rephrased as “only about 30% of EIN3 binding sites were associated with transcriptional changes; at least two third were not”.Minor comments:1) The first wave of transcriptional regulation does not look very much like a set of coordinated genes. There are many genes that appear to move in the opposite fashion at different time points. This would appear to go beyond the “noise” that the authors use to describe it. Either this should be cleaned up in the sense that the genes that are most coordinated are kept and the others are removed, or the authors need to better explain their rationale for keeping this cluster.2) Results: over-representation of DNA binding motifs for the 12 candidate factors in EIN3 targets: we suspect it matters which wave is examined; please comment.3) Results: is the binding of EIN3 to promoters of HLS paralogs associated with overall sequence conservation of the promoters?ein3-1 eil1-1 double mutant background.4) In addition, it would be nice to know if transcript levels of the other family members decreased in an 5) Similarly, one would want to know if other auxin related functions like root gravitropism or bud emergence were impaired in the quadruple mutant and if any of its phenotypes could be rescued by exogenous addition of auxin and/or ethylene. The only major criticism is that the authors should think a bit harder about their nomenclature. It seems that “targets” refer to any genomic sequence that is bound by EIN3 (or any gene that is within a certain distance from an EIN3 binding site). More commonly, “target” is reserved for bona fide repressed or activated genes. For example, the sentence “many EIN3 targets were not transcriptionally ethylene-regulated (EIN3-NR) (67%), while a subset of the EIN3 targets was transcriptionally ethylene-regulated” should be rephrased as “only about 30% of EIN3 binding sites were associated with transcriptional changes; at least two third were not”.We have modified our nomenclature to reflect the commonly used meaning of “target”. Putative EIN3 targets identified by ChIP-Seq are now referred to as “EIN3 candidate targets”. The subset of EIN3 candidate targets that are ethylene-regulated are referred to as “EIN3 targets”.Minor comments:1) The first wave of transcriptional regulation does not look very much like a set of coordinated genes. There are many genes that appear to move in the opposite fashion at different time points. This would appear to go beyond the “noise” that the authors use to describe it. Either this should be cleaned up in the sense that the genes that are most coordinated are kept and the others are removed, or the authors need to better explain their rationale for keeping this cluster.Although genes in the first wave have lower expression levels and higher noise, EIN3 is still significantly associated with genes in this wave . The gene labels in the expression matrix were permuted 1000 times while the TF-gene interaction table remained as the original. None of the 1000 permutations resulted in EIN3 associated with the first wave bifurcation, reassuring that the enrichment of EIN3 with the first path is strong.-10, pathway hypergeometric test) would not be associated with this set of genes.It is likely that if these genes were not part of a coordinated set of genes , EIN3 regulation (p<102) Results: over-representation of DNA binding motifs for the 12 candidate factors in EIN3 targets: we suspect it matters which wave is examined; please comment.Three of the twelve candidate factors are in two of the waves of transcription that are regulated by EIN3. The nine others occur in other waves of transcription. Note that the two genes that are repressed by ethylene do not contain over-representation of DNA binding motifs in the promoters of ethylene response genes. It is unknown why the DNA binding motif of At3g24120 is not over-represented in ethylene response genes. Further experimentation (ChIP-Seq with antibodies generated to these transcriptions factors) is required to understand these results.3) Results: is the binding of EIN3 to promoters of HLS paralogs associated with overall sequence conservation of the promoters?Using ClustalW, we aligned the promoters of HLS1, HLH1, HLH2, HLH3 (3000bp upstream) as well as the EIN3 binding sites in these genes . The bin4) In addition, it would be nice to know if transcript levels of the other family members decreased in an ein3-1 eil1-1 double mutant background.ein3-1 eil1-1 mutant background with the wildtype Col-0 cannot be quantified. We know from earlier studies  that HLS1 expression is somewhat restricted and constitutive in many tissues. It is possible the homologs of HLS1 play specific and unique roles in other cell types/tissues.In general expression of HLH1/2/3 are low  in etiolated seedlings ; the dif5) Similarly, one would want to know if other auxin related functions like root gravitropism or bud emergence were impaired in the quadruple mutant and if any of its phenotypes could be rescued by exogenous addition of auxin and/or ethylene.arf2 mutants were reported to have increased leaf longevity , and as mentioned previously, it appears the quadruple mutant exhibits the converse phenotype, accelerated development. However, the regulatory logic of ARF2 as a repressor of the auxin response is not simple, if it were, the HLS/HLH quadruple mutant resulting in an increase of ARF2 would therefore result in enhanced repression of the auxin response. However, using Biomaps, genes up-regulated in the quadruple mutant were significantly enriched for “response to auxin” function (unpublished observation). Finally, it is currently not possible to apply exogenous auxin with the expectation of proper delivery of the hormone and complementation of the many phenotypes observed. Regarding ethylene, the quadruple mutant is partially insensitive to ethylene and thus cannot be rescued by the hormone.We did not specifically observe and/or quantify root gravitropism phenotypes in the quadruple mutant. We observed an increased number of rosette leaves in the quadruple mutant (n=21), in comparison to wild-type plants (n=12). This phenotype suggests an enhanced not impaired axillary bud emergence, which is expected because auxin inhibits axillary bud emergence in wildtype plants. Concurrent mutations in HLS1 and its homologs resulted in various floral phenotypes. Mutants flowered earlier (2 weeks long day/3 weeks short day) in comparison to the wild type (4 weeks long day/8 weeks short day). Additionally, the quadruple mutant plants lacked apical dominance, producing numerous inflorescences that were sterile. The increased number of rosette leaves and the early flowering phenotype indicate that the quadruple mutants have an accelerated development rate than that of wild type. Interestingly, we found that ARF2 protein accumulates in the quadruple mutant.


SOX2 is a key gene implicated in maintaining the stemness of embryonic and adult stem cells. SOX2 appears to re-activate in several human cancers including glioblastoma multiforme (GBM), however, the detailed response program of SOX2 in GBM has not yet been defined.SOX2 gene in LN229 GBM cells reduces cell proliferation and colony formation. We then comprehensively characterize the SOX2 response program by an integrated analysis using several advanced genomic technologies including ChIP-seq, microarray profiling, and microRNA sequencing. Using ChIP-seq technology, we identified 4883 SOX2 binding regions in the GBM cancer genome. SOX2 binding regions contain the consensus sequence wwTGnwTw that occurred 3931 instances in 2312 SOX2 binding regions. Microarray analysis identified 489 genes whose expression altered in response to SOX2 knockdown. Interesting findings include that SOX2 regulates the expression of SOX family proteins SOX1 and SOX18, and that SOX2 down regulates BEX1 (brain expressed X-linked 1) and BEX2 (brain expressed X-linked 2), two genes with tumor suppressor activity in GBM. Using next generation sequencing, we identified 105 precursor microRNAs (corresponding to 95 mature miRNAs) regulated by SOX2, including down regulation of miR-143, -145, -253-5p and miR-452. We also show that miR-145 and SOX2 form a double negative feedback loop in GBM cells, potentially creating a bistable system in GBM cells.We show that knockdown of the SOX2 response program in LN229 GBM cells. The insights gained from our integrated analysis further our understanding of the potential actions of SOX2 in carcinogenesis and serves as a useful resource for the research community.We present an integrated dataset of ChIP-seq, expression microarrays and microRNA sequencing representing the  The SOX (SRY-like HMG box) gene family represents a family of transcriptional factors characterized by the presence of a homologous sequence called the HMG (high mobility group) box. The HMG box is a DNA binding domain that is highly conserved throughout eukaryotic species. So far, twenty SOX genes have been identified in humans and mice and they can be divided into 10 subgroups on the basis of sequence similarity and genomic organization . SOX genSOX2, one of the key members of the SOX family gene, is highly expressed in embryonic stem cells  and UTF1 (undifferentiated embryonic cell transcription factor 1)  to identify binding consensus sequences enriched in the SOX2 binding regions that we identified. We found a consensus sequence wwTGnwTw with a very high log-likelihood score of 13920.71. The output matrix for this consensus sequence is shown in Additional File SOX2 binding regions  and scanned all TF motif matrices (PWM databases) using the human transcription factor subset of the Transfac professional 7.0. Matched matrices with likelihood (LR) ratios of 500 or higher were tabulated and frequencies calculated , POU1F1 (POU class 1 homeobox 1), TEF1 , AREB6  and GR .We were curious whether known TFs could bind to the POU1F1 is the POU class 1 homeobox 1. OCT family TFs also contain POU domains. These suggest that SOX2 and many POU domain proteins may act together to control gene expression. SOX2 and OCT family TFs such as OCT1  and OCT3/4  are well known to work synergistically in embryonic stem cells . The mouse sox2 consensus motif in the mouse ES cells found by Chen et al. has the sequence 5'-CATTGTT-3' [We applied ChIP-seq technology to identify global TTGTT-3' . The simSOX2 consensus is consistent with previous in vitro studies showing that the HMG domain of SOX proteins binds to the minor groove of DNA through AT rich sequences with a heptamer motif WWCAAAG (W = A or T) [SOX2 specific binding sequences. Before the development of ChIP-chip or ChIP-Seq technologies, Mertin et al. determined the DNA-binding properties of SOX9 using random oligonucleotide selection assay [SOX9 binding. The HMG domain in SOX family proteins forms an L-shaped module composed of three helices that binds to DNA in the minor groove. SOX proteins are categorized into Groups A-G based on their sequence homology [SOX2 belongs to Group A  and SOX9 belongs to group E [SOX2 and SOX9 only have about 61% amino acid sequence identity in the HMG domain. The sequence variations may explain the similar AT rich properties yet different consensus in their binding regions for SOX2 and SOX9. Additional functional binding assays including mutagenesis and footprinting analysis will be needed to confirm the binding activities and specificities. Further experimentation is therefore warranted.The AT rich sequence we identified for  A or T) ,36. Theron assay  and theyhomology . SOX2 be group E . The ami group E . A sequeSOX2 encompass important GO categories: 196 out of 792 genes (about 25%) were found to have signal transducer activity (GO:0004871), 101 of 410 belong to transmembrane receptor genes (about 25%) (GO: 0004888), and 92 of 365 are kinase genes (about 25%) (GO:0016301). Signal transducer, receptor and kinase genes are important genes that play an essential role in cellular functions and therefore it is not surprising that SOX2 is an essential gene that plays important roles in development and in carcinogenesis.It was a surprise to find that about one quarter of genes regulated by BEX1 (brain expressed X-linked 1) and BEX2 (brain expressed X-linked 2) were up regulated after SOX2 knockdown  proliferation by measuring BrdU incorporation and by MTT assay, cell migration using Boyden chamber assay, and capillary tube formation in vitro [SOX18 promoted capillary tube formation of HUVECs in vitro, whereas expression of dominant-negative SOX18 impaired tube formation of HUVECs [SOX18 is a potential target for antiangiogenic therapy of human cancers. The role of SOX18 in GBM has not been studied. Taking together, SOX2 could act through SOX1 and SOX18, and thus play roles in both maintaining stem cell properties of glioma cells and forming tumor vasculature in gliomas, which are two major obstacles preventing us from treating these tumors effectively.We found that 18 Table . SOX1 plntiation  and is antiation . Bylund proteins . Howeverormation . Young ein vitro . They shf HUVECs . TherefoSOX2 knockdown  are altered in response to wn Table . We show cancers , and miR cancers  and blad cancers . The relSOX2 and miR-145 form a double-negative regulation loop in GBM cells  and maintained in DMEM with 10% fetal bovine serum. Cell proliferation was analyzed using the MTT assay kits  according to the manufacturer's protocol. For Soft agar colony formation assay, cells were trypsinized and counted. 10,000 cells were seeded in six-well plates. After 2 weeks of growth, colonies with a diameter greater than 4 mm were counted. Experiments were performed in quadruplicates .6 LN229 cells were used for chromatin immunoprecipition (ChIP) assay, carried out according to the manufacturer's instructions . Antibodies used for ChIP included SOX2  and IgG . For ChIP, SOX2 antibody was tested for its specificity and specific band was found  (http://www.rajajothi.com/sissrs/) [SOX2 ChIP-seq as positive and IgG control ChIP-seq data as negative input.About 3 × 10d Figure . ChIP as earlier . Fragmen earlier . For thisissrs/)  with defSOX2 binding regions is always recorded on the positive strands, for genes mapped to the positive strand, the distance is the end position of the SOX2 binding region minus the TSS start position; for genes mapped to the negative strand, the distance is the TSS start position minus the SOX2 binding region start position.To calculate the distance to the TSS start site, annotations from the UCSC (hg18) were used. We also took into consideration the direction of the strand when we calculated the distance to TSS. As the ® Green (Invitrogen) dye detection method on ABI PRISM 7900 HT Sequence Detection System under default conditions: 95°C for 10 min, and 35 cycles of 95°C for 15 s and 55°C for 1 min. Comparative Ct method was used for quantification of the transcripts.We selected a list of binding peaks for validation using quantitative real-time PCR. The primers were listed in the Additional File SOX2 targets in GBM cells versus the 817 unique SOX2 targets in human ES cells, we GSEA (http://www.broadinstitute.org/gsea/index.jsp). The parameters used were: 1000 permutations using the C5 gene sets (GO gene sets), the diff_of_classes algorithm as metric for ranking genes, weighted enrichment statistic, minimum gene set size of 3. Other parameters were set as default.High-Throughput GoMiner  was usedSOX2 binding regions identified by ChIP-seq were extended to 100 bp 5' and 3' and the sequences were retrieved in FASTA format. The sequences were first subjected to the RepeatMask program (http://www.repeatmasker.org/) to mask all human repeats. We used the MotifSampler to find over-expressed motifs in the SOX2 binding regions with the default parameters. The over represented sequences were used as input for the Weblog program (http://weblogo.berkeley.edu/) [http://bioinformatics.psb.ugent.be/webtools/plantcare/html/Motif_Sampler.html). Human upstream sequences from EPD (The Eukaryotic Promoter Database) (epd_homo_sapiens_499_chromgenes_non_split_3.bg) were downloaded from the motif scanner web site and used as the background model. The human subset of the Transfac professional 7.0 PWM matrices was used. Matched TF matrices with likelihood ratios (LR) of 500 or higher were tabulated and their frequencies calculated.To identify novel motifs, ey.edu/)  to displSOX2 SiRNAs (Ambion Inc) were used for transient knockdown of SOX2. The SiRNA sequences are: s13295, sense sequence AGUGGAAACUUUUGUCGGATT and antisense sequence, UCCGACAAAAGUUUCCACUCG; S13296, sense sequence ACCAGCGCAUGGACAGUUATT and anti-sense sequence UAACUGUCCAUGCGCUGGUTC. LN229 cells were seeded into six well plates, cultured overnight and transfected with SOX2 SiRNAs at a final concentration of 100 nM using TransIT-OT1  according to the manufacturer's instructions. At 72 hours after tansfection, cells were harvested for western blot analysis and for microarray analysis.SOX2 Knockdown or mock knockdown) were used for analysis. Two biological replicates were performed. The normalized data for the replicates were averaged for each gene. To identify differentially expressed genes, a two fold cutoff value was used.The Applied Biosystems' microarray platform was used using the standard array hybridization protocol as we described previously . The ABISOX2 binding regions identified by ChIP-seq (Table S2) was annotated with the genome annotation of hg18 for their associated genes using the nearest gene within 50 kb of the TSS (transcription start site) or TES (transcription end site). As the published human ChIP-Chip data in human embryonic stem cells [SOX2 binding regions coordinates (Table S3 of the Boyer's paper) into the hg18 coordinates using the tool liftover (http://genome.ucsc.edu/cgi-bin/hgLiftOver). We re-annotated the human ES SOX2 binding regions with hg18 annotations using the chromosomal coordinates with the same criteria as we used for the GBM SOX2 ChIP-seq data. There were 1,075 human ES SOX2 binding regions that could be annotated with nearby genes. To identify overlapping genes between SOX2 ChIP-seq and the microarray data, we used the gene symbols from the HUGO gene nomenclature committee (http://www.genenames.org/) to compare the two lists. To compare the human ChIP-seq data with the sox2 targets that were identified in mouse ES cells [http://www.ncbi.nlm.nih.gov/homologene) to identify the human homologues for the mouse sox2 targets and then used the human homologues to compare with the human SOX2 ChIP-seq data.The human genome build hg18 and its associated annotations were used for all analysis. The em cells  was annoES cells , we usedRNA was isolated from cells using mirVana™ miRNA Isolation Kit  according to the manufacturer's instructions. Sequencing library preparation was carried out according to Illumina mirna sample preparation protocol. Small RNA samples were sequenced using GA2 sequencer (Illumina)For data analyses, Solexa adapters were first trimmed from raw sequences using custom Perl scripts, and the trimmed sequences were then aligned to known human miRNAs precursors (miRBase release 14) using miRExpress. The -t Differentially expressed miRNAs were identified by statistics analysis using edgeR package from bioconductor. The top® Green (Invitrogen) dye detection method on ABI PRISM 7900 HT Sequence Detection System under default conditions: 95°C for 30 s, and 40 cycles of 95°C for 15 s and 60°C for 20 s. Comparative Ct method was used for quantification of the transcripts.We selected a list of miRNAs for validation using quantitative real-time PCR. The primers were available upon request. Three replicates were run. Real-time PCR was performed using the SYBRmicroRNA-145 precursor mimics was obtained from Ribobio company (Guanzhou China). A scrambled precursor with no homology to the human genome was used as controls. LN229 cells were transfected with the precursor mimics by lipofectamine 2000 (Invitrogen).SOX2 knockdown and control data from this study have been submitted to Gene Expression Omnibus (GEO) under accession No.GSE23839. The SOX2 ChIP-seq data from this study have been submitted to Gene Expression Omnibus (GEO) under accession No. GSE23795.The array data for ChIP-seq: Chromatin immunoprecipitation-Sequencing; SRY: sex determining region Y; ShRNA: small hairpin RNA.BL and XF designed this study. XF, JY, LL performed the experiments. WY performed data analysis. SZ, LH, DRG, GF help to design the study and wrote the paper. All authors read and approved the final manuscript.SOX2 binding regions in GBM cells identified by ChIP-seq.Click here for fileSOX2 Chip_seq datasets from mouse ES cellsCommon genes from the two .Click here for fileOver represented GO terms in 3162 unique SOX2 targets in GBM cells.Click here for fileOver represented GO terms in 817 unique SOX2 targets in human ES cells.Click here for fileSOX2 targets that are related to regulation of cell differentiation defined by GO term GO:0045595 (REGULATION OF CELL DIFFERENTIATION).Click here for fileet al.'s paper SOX2 targets that are related to stemness defined by 378 genes overexpressed in human embryonic stem cells according to 5 or more out of 20 profiling studies in the Table S1 of Ben-Porath ['s paper .Click here for fileSOX2 consensus matrixThe .Click here for fileSOX2 matrixThe frequency of the matches to the .Click here for fileSOX2 binding regionsFrequencies of known TFs binding sites co-occurred in the .Click here for fileSOX2-Knockdown cells and control cellsDifferentially expressed genes between .Click here for fileSOX2 binding and changed in gene expression.Click here for fileSOX2 regulated miRNAs identified by Illumina's next generation sequencing.Click here for filePrimers for Q-RT-PCR for confirmation of ChIP-Seq data.Click here for file