Ethylene Response Factor genes (ERF) which represent one of the largest gene families of plant transcription factors. How an apparently simple signalling pathway can account for the complex and widely diverse plant responses to ethylene remains yet an unanswered question. Building on the recent release of the complete tomato genome sequence, the present study aims at gaining better insight on distinctive features among ERF proteins.The phytohormone ethylene is involved in a wide range of developmental processes and in mediating plant responses to biotic and abiotic stresses. Ethylene signalling acts via a linear transduction pathway leading to the activation of Solanum lycopersicon) were isolated and shown to fall into nine distinct subclasses characterised by specific conserved motifs most of which with unknown function. In addition of being able to regulate the transcriptional activity of GCC-box containing promoters, tomato ERFs are also shown to be active on promoters lacking this canonical ethylene-responsive-element. Moreover, the data reveal that ERF affinity to the GCC-box depends on the nucleotide environment surrounding this cis-acting element. Site-directed mutagenesis revealed that the nature of the flanking nucleotides can either enhance or reduce the binding affinity, thus conferring the binding specificity of various ERFs to target promoters.A set of 28 cDNA clones encoding ERFs in the tomato (ERF genes can be clustered in two main clades given their preferential expression in reproductive or vegetative tissues. The regulation of several tomato ERF genes by both ethylene and auxin, suggests their potential contribution to the convergence mechanism between the signalling pathways of the two hormones.Based on their expression pattern, ERF tissue-specific expression combined to their responsiveness to both ethylene and auxin bring some insight on the complexity and fine regulation mechanisms involving these transcriptional mediators. All together the data support the hypothesis that ERFs are the main component enabling ethylene to regulate a wide range of physiological processes in a highly specific and coordinated manner.The data reveal that regions flanking the core GCC-box sequence are part of the discrimination mechanism by which ERFs selectively bind to their target promoters.  The gaseous plant hormone ethylene is reported to play an active role in a wide range of developmental processes, including germination, flower and leaf senescence, fruit ripening, leaf abscission, root nodulation, programmed cell death, and responses to abiotic stresses and pathogen attacks -3. Compotrans-acting factors unique to plants shown to bind specifically to GCC box cis-acting elements found in the promoter regions of ethylene-responsive genes [APETALA2[cis-regulatory element. In Arabidopsis, ERFs account for up to 53 % of the total ERF/DREB proteins [in silico using either the entire protein sequence for phylogenetic analysis [Nicotiana tabacum[Pti4, an ERF type transcription factor, is able to bind promoters lacking a GCC box cis-element [cis-elements such as VWRE (Vascular Wounding Responsive Element) [ERFs are ve genes ,6. The Eve genes . The AP2[APETALA2. The RAV[APETALA2. The ERF[APETALA2 both con[APETALA2. It has [APETALA2,12. ERF proteins . In poplproteins ,14 whereproteins . Comparaanalysis ,17, or tanalysis ,13 to ina tabacum. In addi-element , recent Element) .in silico analysis and so far the structure/function relationship has been only superficially addressed [ERFs are ubiquitous in plant kingdom and their functional implications have been studied in various plant species and in a wide range of processes including hormonal signal transduction , responsddressed .ERF gene family in the tomato is organised into 9 subclasses defined by distinct structural features. Based on functional analysis of 28 tomato ERFs and through testing their ability to activate or repress transcriptional activity of target genes, the data suggest that functional activity is conserved among ERF proteins sharing the same structural features. Moreover, data demonstrate that flanking regions of the core GCC-box sequence are part of the discrimination mechanism by which ERFs selectively bind to their target promoters. The data also show that ERF genes display tissue-specific patterns of transcript accumulation and uncover their regulation by auxin.The present study shows that the ERF cDNA clones were initially identified by TBLASTN search [http://solgenomics.net/) using a consensus sequence within the AP2/ERF domain as a query sequence. Forty nine unigenes were found from which AP2, RAV and DREB sequences were discarded based on their distinctive features regarding the number of AP2 domains, the presence of a B3-like domain and the presence of conserved amino acid residues, respectively. Using RACE-PCR extension approach, complete CDSs were obtained for 28 ERF unigenes that are representative of the main ERF sub-groups. Subsequently, building on the annotated whole tomato genome sequence recently released [in silico screening allowed the identification of up to 146 genes encoding putative AP2-containing proteins distributed into 77 ERFs, 48 DREBs, 18 AP2 and 3 RAVs  are intronless  that likely result from tandem duplication events . Half of the ERFs tested (14 out of 28) have significant effect on the synthetic promoter while only 8 ERFs are active on the native osmotin GCC-containing promoter and 4 on the native E4 lacking a conserved GCC motif.The data reveal that ERF proteins are more active on the synthetic GCC promoter than on native complex promoters. The activity of the ERFs on the GCC box Figure  indicate′-A1A2G3A4GCCGCCA11C12T13A14- 3′) or with mutated versions of this motif in gel shift assay experiments  were mutated and the binding of the ERFs was tested by EMSA and assessed by phosphoimaging. Mutation of A at position 4 (A4) into T or G dramatically decreased the affinity to the GCC box of all ERFs except that of ERF.F.5  and downstream (position 11) to the GCC box impact the binding affinity in the same way for all the ERFs tested. By contrast, substitution of G3 or C12 by another nucleotide resulted in different effect on the binding affinity depending on the ERF tested  by ethylene and 4 significantly (p<0.05) down-regulated by this hormone. Interestingly, the expression of up to 12 ERFs is also positively regulated by auxin. Among these, 4 ERF genes  are up-regulated by both hormones whereas ERF.F.5 undergo opposite regulation by auxin and ethylene. More surprisingly, 6 ERF genes  are significantly up-regulated by auxin while they are not responsive to ethylene.It has been reported that beside their regulation by ethylene, ch auxin . To tests Figure  indicatein silico screening identified 146 genes encoding putative AP2-containing proteins distributed into 77 ERFs, 48 DREBs, 18 AP2 and 3 RAVs  to ethylene responsive promoters. Upstream of ERFs, the ethylene signal propagates via a linear transduction pathway which can hardly account for the wide range of ethylene responses. In its downstream part, ethylene signalling leads to the activation of ERFs that are encoded by one of the largest multigene family of transcription factors , and theThe data indicate that all ERF proteins display a clear nuclear localization, even though three members of the family lack any predictable nuclear targeting motif suggesting that their import to the nucleus may occur by a non-conventional localization signal or they might be conveyed to this compartment via an interaction with a yet unknown nuclear localized partner.The large diversity among ERFs is well illustrated by the presence of distinctive motifs that are sufficient to discriminate between different subclasses. Recent studies already suggested a link between the structural classification of ERFs and their physiological function , yet, thcis-elements without the canonical GCC motif. While it cannot be ruled out that ERFs may induce indirectly the E4 promoter through the activation of primary target genes encoding transcriptional proteins active on the E4 promoter, our data suggest that ERFs can interact with other cis-acting elements beside the canonical GCC motif. This is in agreement with recent studies demonstrating that, in addition to the GCC box, ERFs can bind cis-acting elements such as VWRE and GCC-like [cis-acting element.Transient expression assays indicated that the activity of ERFs on synthetic GCC box promoter correlates well with their structural classification. That is, ERFs from subclass C are strong activators and those belonging to subclasses A, B and E are weak activators, whereas members of subclass F are clear repressors. By contrast, ERFs from subclasses G and H display no activity when challenged with the GCC box. Noteworthy, ERFs show weaker activity on the native complex osmotin promoter than on the synthetic promoter. This may be due to the high basal expression displayed by the native osmotin promoter in the absence of ERF effectors, which likely minimizes the induction effect observed in the presence of ERFs  the differential expression of the gene family members, (ii) the ability to negatively or positively impact transcriptional activity and, (iii) the capacity to select with specificity their target genes based on the nucleotide environment of the GCC-box. Considering the diversity of their transcriptional activity and expression patterns, Solanum lycopersicum cv MicroTom) plants were grown in climatic chamber. The conditions are the following: 14-h-day/10-h-night cycle, 25/20°C day/night temperature, 80 % hygrometry, 250 μmol.m-2.s-1 irradiance.Tomato  and the complete ERF CDS were, cloned in over-expression vector, with or without SRDX fusion in C-terminal [The full length CDS were obtained by RACE using BD SMARTterminal  (AdditioFive day-old dark-grown seedlings were treated with air or ethylene gas (50 μL/L) for 5 hours and RNA was extracted from the corresponding tissues. Auxin treatments were performed on 7 day-old light-grown seedlings by soaking (3 hours) in auxin-containing (20 μM) or auxin-free MS solution. Three independent biological repeats were performed for each experiment.RNA samples were obtained from different plant tissues: Immature Green fruit (17 days post anthesis), Mature Green fruit (1 day before Breaker), Breaker fruit, Breaker + 2 days, Breaker + 7 days, leaf, flowers, roots and stem. Real-time quantitative PCR was performed according to Pirrello et al. 2006  was used as BLAST query sequence against the tomato ITAG2.30(Sl2.40) protein database . The same database was also screened by an HMM analysis using a typical AP2/ERF domain PF00847 as query. One hundred forty six genes were identified as possibly encoding proteins containing the AP2/ERF domain (Table In order to identify Cauliflower mosaic virus to the coding region of the Green Fluorescent Protein (GFP). Reporter constructs were also generated with native promoters, E4 (S44898) and Sl-Osmotine (C08HBa0235H18.1), fused to GFP. Effectors constructs were generated by fusing the 35S promoter to the CDS of the ERF genes. For transient assays, tobacco (Nicotiana tabacum) BY-2 protoplasts were co-transformed with reporter and effector constructs [A synthetic reporter construct (4XGCC-GFP) was generated by fusing a synthetic promoter containing 4 GCC-box repeats upstream of the minimal −42 to +8 TATA box from the 35S promoter of nstructs . TransfoYFP N-terminal fusions were obtained with ERF.B1, D.1, H.1 and E.1 and used for tobacco protoplast BY-2 transfection according to Chaabouni et al. 2009 . The subhttp://www.promega.com). Primers used for amplification of ERFs were design according to manufacture recommendation and include Kozak T7 sequences. Gel retardation experiments were performed with the GCC-box probe mutated in the flanking nucleotides according to the procedure described in Tournier et al. 2003 [http://www.fujifilm.com).Proteins used for the gel shift assay were produced in vitro using a “TNT®T7 Quick kit for PCR DNA”  and the native promoters, fused to the GFP in the absence of added ERF effectors.Click here for filePrimers used for RACE amplification.Click here for filePrimers used for CDS amplification. Each pair of primers encompasses the complete CDS from the ATG to the STOP codon. Adaptors attB1 and attB2, have been added at the 5′ end of sense and antisense primers, respectively.Click here for filePrimers used for qRT-PCR. Primers designed with PRIMER EXPRESS software in sequence specific regions for each ERF. Specificity was further checked using BLAST against all tomato unigenes (Tomato unigene database).Click here for filePrimers used for the synthesis of radioactive GCCbox probes utilized in EMSA experiments.Click here for fileCoding sequences of the 28 studied ERF.Click here for file


FT gene, which encodes a flowering signal in plants. These modifications dampen FT expression at dusk to prevent precocious flowering.The seasonal cue day length regulates the timing of the floral transition in plants through periodic histone modifications of the  Arabidopsis, FT expression is rhythmically activated by the output of the photoperiod pathway CONSTANS (CO), specifically at the end of long days. How FT expression is modulated at an adequate level in response to the long-day cue to set a proper flowering time remains unknown. Here, we report a periodic histone deacetylation mechanism for the photoperiodic modulation of FT expression. We have identified a plant-unique core structural component of an Arabidopsis histone deacetylase (HDAC) complex. In long days, this component accumulates at dusk, and is recruited by a MADS-domain transcription factor to the FT locus specifically at the end of the day, leading to periodic histone deacetylation of FT chromatin at dusk. Furthermore, we found that at the end of long days CO activity not only activates FT expression but also enables HDAC-activity recruitment to FT chromatin to dampen the level of FT expression, and so prevent precocious flowering in response to the inductive long-day cue. These results collectively reveal a periodic histone deacetylation mechanism for the day-length control of flowering time in higher plants.The developmental transition from a vegetative to a reproductive phase  is timed by the seasonal cue day length or photoperiod in many plant species. Through the photoperiod pathway, inductive day lengths trigger the production of a systemic flowering signal, florigen, to provoke the floral transition. FLOWERING LOCUS T (FT), widely conserved in angiosperms, is a major component of the mobile florigen. In the long-day plant  Arabidopsis, FT mRNA expression is rhythmically activated by the gene CONSTANS, which is the output of the photoperiod pathway, specifically at the end of long days. In this study, we aimed to address how the level of FT expression is modulated in response to the long-day cue to set the appropriate flowering time. We show that on long days a transcription factor recruits histone deacetylase activity to remove acetyl marks from histones at the FT gene specifically at dusk, thereby dampening FT mRNA expression upon its transcriptional activation by CONSTANS. This sets the correct level of FT expression at the end of long days, and thus prevents precocious flowering in response to the long-day cue.The timing of the developmental transition from a vegetative to a reproductive phase is critically important for reproductive success in flowering plants. Plants synchronize the timing of their floral transition with the changing seasons to flower at a suitable time. The change in day length, or photoperiod, is a key seasonal cue, especially at high latitudes. It is through the photoperiod pathway that day lengths trigger the production of a systemic flowering signal, florigen, to induce the transition from vegetative to reproductive growth and flowering. The FT protein is a major component of the mobile florigen signal. In the model flowering plant  The timing of the developmental transition from a vegetative to a reproductive phase is critical for reproductive success in flowering plants. Plants synchronize their timings of floral transition with changing seasons to flower at a suitable time. The change in day length or photoperiod is a key seasonal cue, especially at high latitudes, and is perceived in leaves, leading to the production of florigen in leaf vasculature Arabidopsis thalianaArabidopsis, a facultative long-day plant that flowers rapidly under inductive long days, the output of the photoperiod pathway CONSTANS (CO) activates FT expression in leaf veins CO mRNA expression in the late afternoon in long days, which coincides with light exposure resulting in CO protein accumulation towards the day's end, because light stabilizes the CO protein FT expression activation in the phloem companion cells specifically at the end of long days, resulting in rhythmic FT expression FT expression is repressed. Upon its production in dusk, the FT protein moves from phloem to SAM to induce flowering. FT and its function as florigen are conserved in the flowering plants so far examined; the level of FT expression at the end of long days in Arabidopsis plays a primary determining role for when a plant to flower FT expression specifically at dusk remain to be identified.FLOWERING LOCUS T (FT), first identified in Histone acetylation and de-acetylation can regulate chromatin structure and gene expression. Histone acetyltransferases (HATs) add acetyl groups to core histone tails and typically function to promote transcription of target loci, whereas histone deacetylases (HDACs) remove acetyl groups and are often linked with transcriptional repression Arabidopsis, functional disruption of HATs such as GCN5 and AtHAC1, or HDACs including HDA6 and HDA19, leads to pleiotropic abnormalities in growth and development AtHAC1 or HDA6 function causes upregulation of a potent floral repressor FLOWERING LOCUS C (FLC), resulting in a delay in flowering Histone acetylation is involved in plant gene regulation. In Arabidopsis, there are six Sin3 homologs including SNL1–SNL6 (SNL for SIN3-LIKE), and four close RPD3 homologs including HDA19, HDA9, HDA7, and HDA6SAP18 homolog in Arabidopsis, AtSAP18, that has been shown to play a role in salt stress response and floral organ formation Arabidopsis genome does not encode an apparent homolog of the yeast SAP30; hence, whether there are Sin3-RPD3 like co-repressor complexes for histone deacetylation in Arabidopsis remains to be addressed.HDACs such as the reduced potassium dependency-3 (RPD3) type are often found in multiprotein co-repressor complexes, among which the yeast Sin3-HDAC complex has been well characterized Arabidopsis there are two functional relatives of the yeast SAP30, named as SAP30 FUNCTION-RELATED 1 (AFR1) and SAP30 FUNCTION-RELATED 2 (AFR2), acting as part of HDAC complexes (AFR1-HDAC or AFR2-HDAC) to modulate the acetylation level of FT chromatin upon FT activation at the end of long days (LDs) (16-h light/8-h dark). Both AFR1 and AFR2 proteins accumulate at the end of LDs, and bind to FT chromatin at dusk, but not in the middle of the day. Moreover, we found that the MADS-domain transcription factor AGAMOUS LIKE 18 (AGL18) directly interacts with AFR1 and AFR2 and recruits these proteins and presumably AFR1/AFR2-HDAC to FT chromatin specifically at the end of LDs leading to histone deacetylation upon FT activation. The output of the photoperiod pathway CO at the end of LDs not only activates FT expression, but also enables the recruitment of AFRs to the FT locus to dampen FT expression and set it at an adequate level, preventing precocious flowering in response to the day length cue in Arabidopsis.Here we report that in Arabidopsis exists, we first examined whether Arabidopsis has any functional relatives of ScSAP30 because it lacks of an apparent homolog of the full-length ScSAP30. In the C-terminal region of ScSAP30 there is a domain important for its function: the 30-amino acid Sin3-binding region (SBR) Arabidopsis protein database and found that there are nine proteins containing a region with a similarity to SBR, among which AT1G75060 and AT1G19330 are the top two hits  assays in which non-fluorescent N-terminal and C-terminal fragments of enhanced yellow fluorescent protein (EYFP) were fused to the full-length AT1G75060, AT1G19330, AtSAP18, or HDA19 proteins. When AT1G75060-cEYFP and nEYFP-AtSAP18 were simultaneously expressed in onion epidermal cells, in the nuclei fluorescence was observed , demonstAFR1 and AFR2, we identified their loss-of-function mutants, and each carries a transfer DNA (T-DNA) insertion. In afr1-1 and afr1-2, a T-DNA is inserted into the first intron of AFR1, resulting in a great reduction, but not elimination of AFR1 expression  Col, as revealed by the total number of leaves formed prior to flower formation from the primary SAM of an examined line, the developmental standard for Arabidopsis flowering-time measurement , and found it flowered earlier than either single mutant in both LDs and short days , because CO is highly expressed to activate FT expression at this time point. CO expression remained unchanged in the afr1 afr2 double mutant compared to WT  methytransferase CLF and the PKDM7B H3 lysine-4 demethylase , are required for continuous FT repression along day/night cycles AFR1 and AFR2 could repress FT expression in other times of LDs beside ZT16. Upon loss of AFR1 and AFR2 function, FT expression in LDs was not affected from ZT0 through ZT14, but upregulated only from ZT14 to ZT20 , were fused to the β-GLUCURONIDASE (GUS) gene in frame and the fusions were introduced into Arabidopsis by transformation. Histochemical staining revealed that both AFR1 and AFR2 were strongly expressed in leaf vasculature in addition to root tips and shoot apices . Upon their expression in Arabidopsis roots, these fusion proteins were specifically localized in the nuclei  assays were performed using the AFR1:HA-expressing seedlings. We found that at the end of LDs (ZT16), AFR1 bound to the proximal FT promoter region, but not in the first exon or the first intron (FT locus in the middle of the day (ZT8)  . Moreovet at ZT8  and S10BFT chromatin at the day's end, may cause periodic histone deacetylation in LDs. To test this, we examined steady-state levels of histone H3 acetylation of FT chromatin in WT and afr1 afr2 seedlings (and rosette leaves) at ZT8 and ZT16. Loss of AFR1 and AFR2 function led to an increase of steady-state level of acetylated H3 in the proximal FT promoter region, but not in the first exon or the first intron at ZT16, whereas at ZT8 there was no noticeable change in all three regions examined , AGL18, and AGAMOUS LIKE 15 (AGL15), are involved in FT repression in the vasculature to inhibit flowering in vivo association of AGL18 with AFR1, we conducted co-immunoprecipitation experiments using the seedlings expressing AFR1:HA and a functional AGL18:FLAG  in both afr1 afr2 and agl15 agl18 seedlings increased to about 2-fold of the WT level, and that in the quadruple, FT levels were slightly higher than either double mutant  was crossed to agl15 agl18, and the seedlings of afr1 AFR1:HA and agl15 agl18 afr1 AFR1:HA harvested at ZT16 were subjected to ChIP assays with anti-HA. We found that loss of AGL15 and AGL18 function nearly eliminated AFR1 binding to FT chromatin at the end of LDs  to co and created co afr1 AFR1:HA. Next, we carried out ChIP assays with the AFR1:HA-expressing seedlings harvested at ZT16, and found that CO, indeed, is required for AFR1 binding to FT chromatin at ZT16  to the FT locus at the end of LDs to modulate FT expression. Our findings collectively uncover a temporal chromatin mechanism, namely periodic histone deacetylation, for the day-length regulation of flowering time in higher plants.In this study, we have revealed that the plant-unique AFR1 and AFR2 mediate periodic histone deacetylation on the rhythmically expressed florigen gene FT locus, accompanying rhythmic CO-mediated FT activation at the end of LDs, AFR1 and AFR2 (presumably AFR-HDAC) are recruited to the actively transcribed FT chromatin for histone deacetylation. Given that disruption of AFR1 and AFR2 function leads to an increase of steady-state level of acetylation on FT chromatin at the end of LDs, there must be a HAT (or HATs) to add acetyl groups, suggesting that constant cycles of acetylation and de-acetylation occur at the FT locus upon its activation by CO at dusk. AFR1/AFR2-HDAC is expected to act antagonistically to a HAT to regulate FT expression. Of note, the HAT(s) has not been identified yet.The histone acetylation mark added by HATs can be rapidly removed by HDACs. Both HATs and HDACs have been found to associate with actively transcribed genes in yeast and human, and are often synchronously recruited to certain target loci, leading to dynamic cycles of acetylation and deacetylation FT promoter may result from transient binding of HATs and AFR1/AFR2-HDAC to FT chromatin. Consistent with the transient or unstable AFR1-HDAC binding, our ChIP analysis shows that AFR1 is moderately enriched at the FT promoter , the Gcn5 HAT and several HDACs are synchronously recruited to ARG1 chromatin, and these opposing activities result in no noticeable changes of steady-state acetylation levels at the ARG1 locus, as measured by ChIP Interestingly, the steady-state acetylation levels of d of LDs , althougFT locus at the end of LDs. AGL18 is well expressed from ZT8 to ZT16 (unpublished data), but the AGL18 protein binds to FT chromatin  only at ZT16. This indicates that the chromatin state of the FT locus may play a role for the AGL18 binding to it; for instance, the state may determine whether a cis-element such as a CArG motif, recognized typically by a MADS domain, in FT promoter is accessible to AGL18.In this study we have revealed that the MADS-domain transcription factor AGL18 and presumably AGL15 act to recruit AFR1/AFR2-HDAC to the CO is required for, but not directly involved in the recruitment of AFR1 to FT chromatin at dusk. The CO protein directly binds to FT proximal promoter and also associates with NY-F transcriptional factors that bind to FT distal promoter, to promote FT expression specifically at the end of LDs FT proximal promoter and/or consequent transcriptional activation of FT expression enable AGL18 recruiting AFRs to the FT locus. It is likely that CO activity may cause a change in FT chromatin state so that the cis-element(s) such as a CArG motif or its variant in the FT promoter become accessible by AGL18. In short, in the photoperiodic flowering regulation CO plays a dual role: activating FT expression and enabling AFR-HDAC-mediated FT downregulation to set a proper level of FT expression at the end of LDs.We found that the output of the photoperiod pathway FT proximal promoter. CO activity not only enables AGL18-AFR-HDAC binding to FT chromatin to dampen FT expression, but may also facilitate dynamic recruitment of a HAT to the FT locus to promote its expression given the dynamic nature of histone acetylation and deacetylation on FT chromatin at dusk  is largely conserved, and the CO-FT regulatory module underlying the photoperiodic flowering-time control in the dicotyledonous LD plant Arabidopsis, also plays a central role for flowering-time control in the monocotyledonous short-day plant rice FT expression control in Arabidopsis may modulate the expression of FT homologs in other plant species and so confer an optimal flowering time in response to photoperiodic changes.co, ft, fve, agl15-3, and agl18-1 were described previously afr1-1 , afr1-2 , afr2-1 , and afr2-2 (CS842844) were obtained from the Arabidopsis Biological Resource Center and directly used in this study. Plants were grown in long days (16-h light/8-h dark) or short days (8-h light/16-h dark) under cool white fluorescent light at 22°C The pGADT7 and/or pGBKT7 vectors, which were subsequently introduced into the yeast strain AH109 according to the manufacturer's instructions (Clontech). Yeast cells were spotted on selective media lacking of leucine, tryptophan, and histidine.The Matchmaker GAL4 Two-Hybrid System 3 (Clontech) was adapted for this assay. Full-length coding sequences for AFR1, AFR2, AtSAP18, HDA9, HDA19, and SNL2 were cloned into the pSAT1A-nEYFP-N1/pSAT1-nEYFPC1 vectors and/or for a C-terminal EYFP fragment in the pSAT1AcEYFP-N1/pSAT1-cEYFP-C1-B vectors The full-length coding sequences for AFR1, AFR2, AtSAP18, HDA19, AGL15, and AGL18 were fused in frame with either the coding sequence for an N-terminal EYFP fragment in the UBQ10 (its expression is not influenced by day-night cycles) UBQ10 is calculated as 2−ΔCt [ΔCT = CT (gene of interest) − CT (UBQ10)]. The primer pair for CO amplification has been described previously AFR1, AFR2, FT, and UBQ10 amplifications are specified in Total RNAs were extracted from aerial parts of 10- to 11-d-old seedlings grown in long days (at various time points) or 21-d-old seedling grown in short day using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions. cDNAs were reverse transcribed from the total RNAs. Real-time quantitative PCR (qRT-PCR) was performed on an ABI Prism 7900HT sequence detection system using a SYBR green PCR master mix; PCR was conducted as follows: 50°C (2 min), 95°C (10 min), and 40 cycles of 95°C (15 s) and 60°C (60 s). Each sample was quantified at least in triplicate, and the constitutively expressed AFR1-GUS and AFR2-GUS, genomic fragments of 3.0-kb AFR1 (1.0-kb 5′ promoter plus 2.0-kb genomic coding sequence) and 3.9-kb AFR2 (1.2-kb 5′ promoter plus 2.7-kb genomic coding sequence) were inserted upstream of GUS in the pMDC162 vector AFR1 and AFR2 coding sequences (except the stop codons) were inserted between the 35S promoter and GFP in the pMDC85-GFP vector AFR1 and AFR2 are in frame with GFP).To construct pAFR1-AFR1:HA plasmid, a 3.0-kb AFR1 genomic fragment (1.0-kb 5′ promoter plus the 2.0-kb full-length genomic coding sequence except the stop codon) was first fused with a 3xHA tag, and cloned into the pHGW vector pAFR2-AFR2:FLAG construction, a 3.9-kb AFR2 genomic fragment (1.2-kb 5′ promoter plus the 2.7-kb full-length genomic coding sequence except the stop codon) was first fused with a 3xFLAG tag, and cloned into pHGW. To construct p35S-AGL18:FLAG, the full-length AGL18 coding sequence without the stop codon (772 bp) was first fused with a 3xFLAG tag, and the AGL18:FLAG fusion was subsequently placed downstream of the 35S promoter in the pB2GW7 vector To generate the Co-immunoprecipitation experiments were carried out as previously described FT and the constitutively expressed TUB2 and TUB8 fragments on an ABI Prism 7900HT sequence detection system using a SYBR Green PCR master mix. The primers used are specified in ChIP experiments were carried out as described previously with minor modifications Figure S1Alignments of AFR1 and AFR2 relatives. Numbers refer to amino acid residues. Identical residues among these proteins are shaded black, whereas similar residues are shaded gray. (A) Alignment of the Arabidopsis AFR1 (AtAFR1) and AFR2 (AtAFR2) with the yeast Sap30 (ScSap30). The conserved residues in the putative SBRs are indicated by asterisks (*). (B) Alignment of AtAFR1 with its homologs from other plants including Populus trichocarpa (PtSFR), Picea sitchensis (PsSFR), and Physcomitrella patens (PpSFR). The GenBank accession numbers for PtSFR, PsSFR, and PpSFR are XP_002302473.1, ADE76839.1, and XP_001766227.1, respectively.(EPS)Click here for additional data file.Figure S2Direct interactions of AT1G75060 (AFR1) and AT1G19330 (AFR2) with SNL2 and HDA9 proteins in yeast. The indicated full-length proteins were fused with the GAL4-BD or AD domains. Yeast cells harboring the fusion proteins, BD and/or AD (as indicated), were grown on the selective synthetic defined media lacking of Trp (W), Leu (L), and His (H).  Interactions of SNL2 with AFR1 and AFR2 in yeast.  Interactions of HDA9 with AFR1 and AFR2 in yeast.(TIF)Click here for additional data file.Figure S3Analysis of AFR1 and AFR2 expression in afr1 and afr2 seedlings by semiquantitative RT-PCR. The constitutively expressed TUB2 served as a control. (A) Analysis of the full-length AFR1 and AFR2 transcripts. (B) Analysis of 5′- or 3′-truncated AFR2 transcripts in afr2 mutants.(EPS)Click here for additional data file.Figure S4Rescue of afr1 and afr2 mutants by pAFR1-AFR1:HA and pAFR2-AFR2:FLAG, respectively. Plants were grown in short days, and total leaf number for each line (9–11 plants per line) was scored. Bars indicate SD. (A) Flowering times of afr1-1 and a transgenic line of pAFR1-AFR1:HA in afr1-1 . (B) Flowering times of afr2-1 and a transgenic line of pAFR2-AFR2:FLAG in afr2-1 .(EPS)Click here for additional data file.Figure S5Analysis of flowering time and leaf initiation rate of afr1 afr2 in LDs. 12 plants were scored for each line; bars for SD. (A) Days to flowering of Col and afr1 afr2. (B) Total leaf numbers at flowering of Col and afr1 afr2. (C) Leaf initiation rates of Col and afr1 afr2 plants.(EPS)Click here for additional data file.Figure S6Relative AFR1 and AFR2 transcript levels in Col and co seedlings at the end of LDs, quantified by qRT-PCR. The transcript levels were normalized first to the endogenous control UBQ10, and relative fold changes to Col are presented. Bars indicate SD of triplicate measurements.(EPS)Click here for additional data file.Figure S7AFR1 and AFR2 moderately repress FT expression in short days. (A) Flowering times of the indicated lines grown in short days. Total leaf number for each line (8–14 plants per line) was scored. Error bars indicate SD. (B) FT mRNA levels in Col and afr1 afr2 seedlings over a 24-h short-day cycle. Total RNAs were extracted from 21-d old seedlings every 4 h and quantified by qRT-PCR. Bars indicate SD of three measurements.(EPS)Click here for additional data file.Figure S8Biological repeats of FT, AFR1, and AFR2 expression analyses. (A) A biological repeat of the FT expression analysis presented in AFR1 and AFR2 expression analysis presented in (EPS)Click here for additional data file.Figure S9Relative levels of AFR1:HA and AFR2:FLAG proteins over a 24-h LD cycle. The proteins in respective 10- to 11-d-old seedlings were analyzed by western blotting. The intensities of protein bands were quantified by the ImageJ 1.44j program, and relative levels to ZT0 are presented. Note that the time of the day has a significant effect on the levels of both AFR1 and AFR2 proteins in the seedlings, as revealed by single-factor ANOVA tests . (A) Relative AFR1:HA levels over a 24-h LD cycle. Average values of three biological repeats are presented; bars for SD. (B) Relative AFR2:FLAG levels over a 24-h LD cycle. Average values of four biological repeats are presented; bars for SD.(EPS)Click here for additional data file.Figure S10Biological repeats of the ChIP assays on AFR1 and AFR2 binding to FT chromatin. (A) A biological repeat of the ChIP analysis presented in (EPS)Click here for additional data file.Figure S11ChIP analysis of the histone H3 acetylation state of FT chromatin in Col and afr1 afr2 seedlings grown in LDs. Amounts of the immunoprecipitated genomic DNA was quantified and normalized first to the constitutively expressed TUB2. Subsequently, the fold enrichments were calculated by dividing the levels of acetylated H3 in afr1 afr2 (at ZT8 or ZT16) or the WT at ZT16, by the level in the WT at ZT8. Shown are the means and SD of two ChIP experiments.(EPS)Click here for additional data file.Figure S12Direct interactions of AGL18 with AFR2 and AGL15 with AFR1. (A) BiFC analysis of the interaction of AGL18 with AFR2 in onion epidermal cells. Yellowish-green signals indicate the binding of AGL18 with AFR2 in the nuclei (indicated by the blue fluorescence from DAPI). Bar = 20 µm. (B) AFR1 interacts with AGL15, but not FLC, in yeast cells. The full-length AFR1, and full-length FLC and the 208-aa AGL15 (without MADS domain) were fused with the GAL4-AD and BD domains, respectively. Yeast cells were grown on the selective synthetic defined media lacking of W, L, and H or lacking of W and L. Of note, FLC directly interacts with SVP and binds to FT chromatin to repress FT expression (TIF)Click here for additional data file.Figure S13Characterization of the p35S-AGL18:FLAG line and a biological repeat of ChIP analysis of AGL18 binding to FT chromatin. (A) Flowering times of the p35S-AGL18:FLAG line  grown in LDs. Overexpression of AGL18 causes moderate late flowering t test . Bars indicate SD. (B) A biological repeat of ChIP analysis of AGL18 binding to FT chromatin presented in (EPS)Click here for additional data file.Figure S14Analysis of AGL15 and AGL18 expression in Col and agl15 agl18 seedlings by RT-PCR. The constitutively expressed TUB2 served as a control.(EPS)Click here for additional data file.Figure S15Examination of CO interaction with AFR1, AFR2, or AGL18 by the yeast-two-hybrid assay. The full-length CO was fused with GAL4-AD, whereas the full-length AFR1, AFR2, and AGL18 were fused with GAL4-BD. Yeast cells were grown on the selective synthetic defined media lacking of W, L, and H or lacking of W and L.(TIF)Click here for additional data file.Table S1List of primers used in this study.(DOCX)Click here for additional data file.


Cold induced MYB 1 (CMYB1). Tissue-specific gene expression analysis revealed that CMYB1 was highly expressed in rice stems and nodes. qRT-PCR assay indicated that CMYB1 was dramatically induced by cold stress (>100-folds) and induced by exogenous ABA and osmotic stress. Interestingly, CMYB1 showed rhythmic expression profile in rice leaves at different developmental stages. Subcellular localization assay suggested that CMYB1-GFP (green fluorescent protein) fusion protein was localized in the nuclei. Moreover, CMYB1 exhibited the transcriptional activation activity when transiently expressed in rice protoplast cells. Taken together, CMYB1 probably functions as a transcriptional activator in mediating stress and rhythm responsive gene expression in rice.Through analysis of cold-induced transcriptome, a novel gene encoding a putative MYB transcription factor was isolated and designated  Environmental stresses such as cold, drought, and salinity adversely affect plant growth and crop productivity. To cope with such stress, plants initiate a number of molecular, cellular, and physiological changes to respond and adapt to various stresses. The transcriptional gene regulation of stress-related genes is crucial in these responses and adaptations , 2. The  Arabidopsis and rice. A number of MYB genes have been found to be induced by abiotic stress and their genetic engineering may improve stress tolerance in plants. AtMYB60, a R2R3-MYB gene of Arabidopsis, was the first identified transcription factor involved in the regulation of stomata movements [ AtMYB60 was specifically expressed in guard cells, and its expression was negatively modulated by drought. A null mutation in AtMYB60 resulted in the constitutive reduction of stomata opening and decreased wilting under water stress conditions. OsMYB3R-2 is a MYB transcription factor induced by cold, drought, and salt stresses in rice. The Arabidopsis and rice transgenic plants overexpressing OsMYB3R-2 showed increased cold tolerance [ OsMYB3R-2 functions as a MYB transcription factor targeting OsCycB1;1, which was involved in the G2/M phase transition at low temperature. The transcript level of OsCPT1, a putative member of DREB1/CBF pathway, was also enhanced by OsMYB3R-2 [ OsMYB2 was functionally studied. Expression of OsMYB2 was upregulated by salt, cold, and dehydration stresses. Overexpression of OsMYB2 increased salt, cold, and dehydration tolerance. The enhanced proline and soluble sugar accumulations and activities of antioxidant enzymes, including peroxidase, superoxide dismutase, and catalase, may underlie the enhanced stress tolerance [ Arabidopsis and rice, several MYB genes from other plants, such as apple, wheat, and tobacco, also showed the roles in improving abiotic stress tolerance [MYB transcription factors are present widely in eukaryotes . MYB proovements . AtMYB60olerance , 19. Furolerance . Besidesolerance –23. CMYB1 was identified through transcriptome analysis in rice under cold stress. Gene expression analysis suggested that CMYB1 was largely induced upon cold stress and also induced by osmotic and drought stresses. Interestingly, CMYB1 showed the significant rhythmic expression profile, implying that CMYB1 might play an important role in cold tolerance through a circadian rhythm regulation manner.In the present study, a novel MYB transcription factor geneOryza sativa L. subsp. japonica) was used in this experiment. The rice seeds were sterilized in 0.3% NaClO for 15 minutes, germinated, and cultured in Yoshida's nutrition solution in the growth chamber with a light cycle of 16 h light at 28°C and 8 h dark at 22°C until three-leaf stage. Some seedlings were transferred to the field for further growing; then the stem, node, leaf blade, leaf sheath, immature panicle, mature panicle, and anther were harvested at proper time.Rice cultivar Jiucaiqing , and 0.1 mM ABA, respectively, or cultured in the nutrition solution under the condition of 4°C. The seedlings were sampled at different time points after each treatment, immediately frozen in liquid nitrogen, and stored at −80°C until RNA was isolated. For dehydration treatment, the seedlings were taken off from solution and kept at a natural dehydration situation for 12 h and then put back in the nutrition solution for recovery.μg of purified total RNA using the reverse transcription system  according to the manufacturer's protocol.The total RNA was extracted using the Trizol reagent  according to the manufacturer's protocol. The remaining genomic DNA in the RNA samples was removed by treating with DNase I  at 37°C for 15 min. The first-strand cDNA was synthesized with 2  CMYB1 was amplified from cDNA of three-leaf rice seedling by PCR using the primers as follows: forward primer: 5′-TGGGAGGAGTTCGGTTTT-3′; reverse primer: 5′-CCTCTTCTTCCCGCCTTA-3′. The PCR product was purified and cloned into pEASY-T vector  and sequenced. Plasmid was isolated from single clone, which was confirmed with correct sequencing result, and used as template for next construction of expression proteins.The coding sequence of CMYB1 primers  were used for quantitative real-time PCR analysis. A constitutively expressed gene 18S rRNA was used as an internal control [ 18S rRNA were as follows: forward primer: 5′-ATGGTGGTGACGGGTGAC-3′; reverse primer: 5′-CAGACACTAAAGCGCCCGGTA-3′.The control . The pri CMYB1::GFP fusion protein, the CMYB1 fragment was amplified using the primers as follows: forward primer: 5′-GCTCTAGAAATGGAGATGGCCTGTT-3′; reverse primer: 5′-GTTCCCGGGTGTCACAAGCAC-3′, where the underlines indicate the restriction digestion site of Xba I and Sma I, respectively. Then this fragment was cloned into the Xba I and Sma I sites of plant transient expression vector pA7 [ CMYB1::GFP fusion protein under the control of CaMV35S promoter. 35S::GFP fusion protein was used as control. Both of the fusion proteins were then transferred into onion epidermal cells by particle bombardment and the cells were examined with confocal laser scanning microscopy .To constructctor pA7  to const CMYB1 was determined in rice protoplast by dual luciferase reporter assay system . To construct reporter plasmids, we used the pGL3-Basic vector, in which two random cis-elements, 5×GAL4 and 3×DRE, and a minimal TATA promoter [ CMYB1 effector plasmid was constructed by inserting DNA fragments containing CMYB1 coding region and GAL4-DNA binding domain sequence of pGBKT7 vector  into the Spe I and Xba I sites of pA7. Similarly, 35S::DBD and 35S::DBD-ZFP179 plasmids [ cis-element was constructed [The transcriptional activity assay ofpromoter  were cloplasmids  were constructed . The ren CMYB1.Based on the cold-responsive transcriptome analysis in rice (our unpublished data), an EST probe (probe ID: Os.8149.1.S1_at) encoding a putative MYB family transcription factor (Gene locus name: LOC_Os02g46030.1) was identified. The predicted protein product comprised 491 amino acids with a conversed MYB domain at the N-terminal and was designatedCMYB1 belongs to the R1-type subfamily of MYB proteins and was homologous to many other MYB transcription factors containing single MYB domain , it was found that expression of CMYB1 was significantly subjected to circadian rhythm regulation. The similar results were obtained in rice leaves during different developmental stages, including vegetative, reproductive, and ripening stages  and RVE2, were also involved in circadian rhythm. Although inactivation of RVE1 does not affect circadian rhythmicity, it was speculated that RVE1 was a clock output affecting plant development. It was also suggested that RVE2 was possibly part of a regulatory feedback loop that controls a subset of the circadian outputs. As CMYB1 expression was also strictly circadian rhythm regulated, this branch of MYB family might be involved in circadian rhythmicity, especially functioning in the regulation of circadian outputs regulating plant development and adaptation to environment changes.Through investigation ofg stages . It seem CMYB1 was analyzed through the MatInspector program (http://www.genomatix.de/). The result showed that the promoter sequence of CMYB1 contained many putative plant hormone-related cis-acting elements, such as ABA-responsive element (ABRE), auxin response element (AuxRE), brassinosteroid (BR) response element (BRRE), and ethylene response element factors (EREF), indicating that CMYB1 might be a central crosstalk of various plant hormones regulating plant growth and development. The presence of ABRE in the promoter region and the fact that CMYB1 was induced by exogenous ABA treatment suggested that CMYB1 probably functions in the ABA-dependent pathway of stress response in rice. Interestingly, we also found a circadian-related cis-element existing in the promoter region  35S promoter in upstream of luciferase. The effector plasmids were composed of two parts. One was 35S::DREB1A expression protein that could bind to the DRE fragments. Another one was 35S promoter fused to the GAL4 DBD (DNA binding domain) or GAL4DBD-CMYB1 cDNA. 35S promoter fused to the GAL4DBD-ZFP179 cDNA was used as positive control (CMYB1 displayed significant induction in the expression of the luciferase reporter gene, which was about 3-fold to the one harboring DREB1A and GAL4DBD proteins. This result indicated that CMYB1 had transcriptional activity in rice protoplast and functioned as a transcriptional activator. Based on the subcellular localization and transcription activation activity, it could be confirmed that CMYB1 functions as a transcription activator in rice cells.The transcriptional activity of control . As show CMYB1 which was dramatically induced by cold stress. CMYB1 is a nuclear protein exhibiting transcriptional activation activity, implying that CMYB1 functions as a transcription activator in rice cells. Interestingly, we found that expression of CMYB1 was significantly regulated by circadian rhythm. As CMYB1 was largely induced upon cold stress, it is hypothesized that CMYB1 probably promotes cold-responsive gene expression to respond to low temperature in the night. With temperature increases in the day, expression of CMYB1 gradually decreases. However, this hypothesis needs further experimental validation by use of CMYB1 null mutant or transgenic approach.In this paper, we reported a novel MYB transcription factor gene


This core set exhibits evolutionarily conserved binding regions and is enriched in functional responses to hypoxia. Computational prediction of enriched transcription factor binding sites identified sequence motifs corresponding to several stress-responsive transcription factors, such as activator protein 1 (AP1), cAMP response element-binding (CREB), or CCAAT-enhancer binding protein (CEBP). Experimental validations on HIF-regulated promoters suggest a functional role of the identified motifs in modulating HIF-mediated transcription. Accordingly, transcriptional targets of these factors are over-represented in a sorted list of hypoxia-regulated genes. Altogether, our results implicate cooperativity among stress-responsive transcription factors in fine-tuning the HIF transcriptional response.The transcriptional response driven by Hypoxia-inducible factor (HIF) is central to the adaptation to oxygen restriction. Despite recent characterization of genome-wide HIF DNA binding locations and hypoxia-regulated transcripts in different cell types, the molecular bases of HIF target selection remain unresolved. Herein, we combined multi-level experimental data and computational predictions to identify sequence motifs that may contribute to HIF target selectivity. We obtained a core set of  Oxygen is essential for the survival of all eukaryotic cells, and metazoans are heavily dependent on this element to meet their large metabolic demands. At the cellular level, 90% of oxygen is consumed in oxidative phosphorylation. Consistent with a central role of oxygen in aerobic metabolism, all metazoan cells respond to an imbalance between demand and supply of oxygen (hypoxia) by activating a gene expression program aimed at restoring oxygen supply and reducing its consumption. The cellular response to hypoxia is mainly controlled by the evolutionarily conserved Hypoxia Inducible Factor (HIF) family of basic helix-loop-helix transcription factors. HIFs are heterodimers of a beta subunit , and an alpha subunit (HIFα) A large number of studies focusing on single genes have identified individual HIF targets that, collectively, account for the functional responses to hypoxia, mainly metabolic adaptation and induction of angiogenesis ET-1 under hypoxia PAI-1 induction by hypoxia has been linked to cooperative promoter activation by CEBPα, HIF1α and EGR-1 LDHA promoter) or identical sites (BNIP3) Understanding the mechanisms that explain HIFs transactivation selectivity is of paramount importance to expand our knowledge on transcriptional regulation and to improve the sensitivity and specificity of genome-wide efforts to characterize the HIF transcriptional response. DNA accessibility of transcription factor binding sites (TFBSs) can clearly contribute to binding selectivity Gene expression profiling indicates that the expression of thousands of genes changes with hypoxia, with vast cell-type differences in the specific genes being regulated http://www.ncbi.nlm.nih.gov/geo/) Gene profiling experiments of hypoxic cell cultures were downloaded from the Gene Expression Omnibus database .HeLa cells were maintained in Dulbecco’s modified Eagle medium supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin and 5% (v/v) fetal bovine serum. Cells were grown at 37°C and 5% CO7 HeLa cells were subjected to hypoxia (1% oxygen) for six hours or left untreated . Following treatments, cells were crosslinked with 1% formaldehyde for 12 min at 4°C. Cross-linking was stopped by the addition of glycine . Cell lysis was achieved by scraping in 1 ml of lysis buffer . Cell lysates were incubated on ice for 10 min and then sonicated to shear DNA to fragments between 200 and 1500 bp. Only experiments that showed homogeneous sonication across all samples (from normoxia and hypoxia treatments) were continued. 50 µl of each sample was stored (input), while 100 µl were diluted in 1 ml of immunoprecipitation buffer . Lysates were precleared with 200 µg of a Salmon Sperm DNA/Protein A agarose 50% slurry  for 1 h at 4°C; and then immunoprecipitated twice, initially with whole rabbit serum for 6 h (IgG control) and then overnight at 4°C with a polyclonal anti-HIF1 alpha antiserum . Immunocomplexes were recovered by addition of 400 µg of Salmon Sperm DNA/Protein A agarose 50% slurry, and then sequentially washed in Low Salt Wash Buffer , High Salt Wash Buffer , LiCl buffer , and twice in TE buffer . Elution of protein-bound DNA was performed twice with 1% SDS 0.1 M NaHCO3. Eluates were pooled, and crosslinking was reversed by the addition of 200 mM NaCl  and overnight incubation at 65°C. Protein and RNA were removed by the addition of proteinase K (30 µg/sample) and RNAse (200 mg/µl) for 2 hours at 42°C, and immunoprecipitated DNA was purified by phenol-chloroform extraction and ethanol precipitation. Successful ChIP was assayed by standard PCR using two primer pairs, targeting the functional EGLN3 HRE and a control region in the same locusChromatin immunoprecipitation was performed as previously described The ChIP-chip method was carried out as previously described Amplified DNA from normoxic and hypoxic chromatin immunoprecipitation samples was labelled with Cy3 and Cy5 fluorescent dyes, respectively, and hybridized to microarrays following guidelines from the microarray manufacturer . Hybridized microarray slides were scanned in an Agilent DNA microarray scanner  at 5 µm resolution, and acquired microarray images were quantified with GenePix 6.0 software . A total of six hybridizations were conducted, corresponding to four biological replicates. The two technical replicates were dye-swap experiments, where normoxic samples were labelled with Cy5 and hypoxic samples with Cy3.A custom alternative promoter microarray was used for ChIP-chip hybridizations The R/Bioconductor packages “Ringo” and “limma” were used to analyze ChIP-chip readouts The microarray experiments described above have been deposited in ArrayExpress under accession number E-MEXP-3499.http://frodo.wi.mit.edu/primer3/). All measurements were carried out in triplicate. Threshold cycle (Ct) values for each sample were interpolated in a standard curve of input DNA dilutions to obtain % of input absolute values. Enrichment of HIF1 alpha binding to target loci was calculated as the ratio of the amounts of target sequence (measured as % of input) detected in hypoxic vs noxmoxic ChIP samples (% of input hypoxia/% of input normoxia). For validation of ChIP-chip candidates, three negative control regions  following the manufacturer’s recommendations. Primers targeting candidate ChIP-enriched regions were designed with Primer Express software v2.0 (Applied Biosystems) and Primer3 (Custom scripts written in Perl were used to indentify evolutionarily conserved HIF binding sites (HBSs) within ChIP-chip regions and to select HBSs that showed evolutionary conservation. Conservation of the HIF binding consensus RCGTG in four mammalian species, including mouse, was required for the evolutionary conservation filter. HBSs were further selected to map to genes robustly induced by hypoxia, as indicated by the results of a meta-analysis of gene expression profiling experiments in hypoxic cell cultures loci that contained conserved RCGTG motifs in their non-coding sequences were first selected. For these genes, each of their probes was examined, and only genes for which all of their associated probes exhibited a mean fold value within 0.25 standard deviations of the global mean in each of the 19 datasets employed in the meta-analysis were considered as not induced by hypoxia. The selected coordinates of conserved RCGTG motifs mapping to these loci were extended as previously described for the set of core HIF binding sites. Genomic regions from this collection were further selected to match the frequency of genomic locations (relative to the TSS) found in the core HBR set. Briefly, Perl scripts were used to annotate core HBRs as promoter, 5′UTR, intronic or 3′UTR genomic locations and to choose, from the whole collection, a random sample according to the proportions of genomic locations found in the core HBR set. Similarly as with the set of core HIF binding regions, multiple sequence alignments corresponding to the selected control regions were retrieved.To obtain a set of background (control) genomic regions, custom perl scripts were used to exploit the microarray meta-analysis results for the identification of genes harbouring conserved RCGTG motifs but that are unlikely to be modulated by hypoxia. To this end, gene In silico transcription factor binding site predictions were carried out employing custom scripts written in Perl. Position-weight matrices (PWMs) collections were downloaded from the public databases JASPAR (2010 release) and Transfac (7.0 version) PWM conversion:The weight of base b in position i is calculated as:The corrected probability is obtained from the raw matrix by adding a pseudocount:Information content calculation:Subsequently, log-odd matrices were used to screen core HIF binding regions and genomic-matched background regions for the presence of putative TFBSs or other sequence motifs. Perl scripts were employed to split each sequence into overlapping fragments of length equal to that of the PWM under analysis. For each fragment, a score value was calculated by summing up the log-odd frequencies obtained by substitution of nucleotides found in the fragment in the corresponding position of the PWM. The contribution of each base to the score was weighted by the information content of its position in the matrix, as detailed below.i is the information content of position i and Wb,i the log-odd weight of base b in position i.Score calculation:Finally, the resulting score was normalized by subtraction of the minimum score and division by the score range, and compared with a threshold value. Fragments showing a score above the threshold were considered as putative TFBSs, and the evolutionary conservation of nucleotides in each motif was evaluated for matrix positions with information content over 60%. Putative TFBSs showing evolutionary conservation in four mammalian species (including mouse) and whose score was over the threshold value were recorded as present (respresented as 1). Otherwise, they were considered absent from the analyzed sequence (represented as 0).For each PWM, the three strategies proposed in MATCH versus the background collection. In particular, we considered significant PWMs with a p value lower than 0.05. No correction for multiple comparisons was applied to these p values.Fisher’s exact test was used to identify PWMs showing significant enrichment in the set of core HIF binding regions Additionally, the Weka machine learning workbench CA9 promoter region (hg19 coordinates chr9∶35673508–35673956), which was subcloned into the pGL3-Basic plasmid (Invitrogen). The Human GYS1 reporter construct (hg19 chr19∶49496421–49496978) has been previously described LDHA promoter construct (mm9 chr7∶54101027–54101258) and the derived HRE and CREB binding site mutations were a kind gift from Peter Ratcliffe Human genomic DNA extracted from HeLa cells was used as template for PCR amplification of the Reporter assays were performed using the human cervical-carcinoma cell line HeLa. Cells were seeded on six-well plates (2.5·105 cells/well) 6 h prior to transfection. Per well, a 4.5 µg DNA mixture containing 1.5 µg of the indicated reporter construct or empty plasmid and 0.25 µg of a plasmid encoding for Renilla (sea pansy) luciferase under the control of a null promoter  was used for transfection using the calcium phosphate method. 16 h after transfection, cells where washed, replated in 24-well plates, and incubated in normoxia, in the presence of DMOG  or in hypoxia for an additional 16 hours. After treatments, cells were lysed and the firefly and Renilla luciferase activities of the lysate were determined using a dual-luciferase system . The firefly luciferase activity was normalized to that of Renilla luciferase. Each experimental condition was assayed in duplicate. Hypoxia or DMOG fold induction values for each experiment were analyzed by repeated measures ANOVA with a Dunnet posthoc correction, comparing values of the wild-type promoter construct to each of the introduced mutations.http://www.broadinstitute.org/gsea/index.jsp). We employed a ranked list of core hypoxia-regulated genes derived from a meta-analysis of 16 hypoxia gene expression experiments Gene-set enrichment analysis (GSEA) was carried out as previously described  for six hours. Amplified samples from normoxic and hypoxic cells were competitively hybridized to a proximal promoter microarray that tiles a subset of human 7000 genes Next, we compared HIF1α ChIP-chip predictions in the four cell lines by analyzing the overlap of reported binding locations . The majbona fide HBSs to computationally identify enriched TFBSs that could be indicative of transcription factor cooperation. To focus on binding locations for which there is evidence of transcriptional modulation of nearby genes in hypoxia, we sought to combine the core set of HIF1 binding locations with HIF transactivation data. To this end, we employed our previous microarray meta-analysis study in silico sequence analyses, we focused on HIF binding sites whose sequence showed evolutionary conservation in mammalian species  databases and custom scripts based on the MATCH algorithm As an independent assessment of enriched motifs that is less dependent on the composition of the core set, we compared the results of the previous analysis with a variable selection approach implemented in the Weka machine learning software bona fide HIF binding regions.We next asked whether the TFs associated to the enriched TFBSs may share any common characteristics. Gene annotation enrichment analysis  of thesein silico, we next set out to validate some of these predictions experimentally. To this end, we selected hits for enriched TFBSs focusing on: 1) HREs located close to the TSSs of genes, to be able to study these promoter regions in in cellulo reporter assays, and 2) TFBS predictions located close to the Hypoxia Response Element (HRE). According to these criteria, we evaluated a CREB binding site prediction in the LDHA promoter  and upon treatment with the prolyl hydroxylase inhibitor DMOG (500 µM). As expected, mutation of the HRE in all the studied promoters completely abrogated induction by either hypoxia or DMOG treatment . ImportaHRE core . This reHRE core . Finally or DMOG , reachinbona fide HIF binding regions play a functional role in the transactivation by hypoxia or DMOG treatment of HIF-responsive promoters. Moreover, our data also suggests that diverse stress-responsive transcription factors, probably through modulation of basal transcription or recruitment of common cofactors, contribute to the specification of HIF target selectivity.Collectively, these results indicate that at least some of the TFBSs computationally predicted as enriched in a core set of http://www.broadinstitute.org/gsea/index.jsp). This collection comprises over 3000 gene sets from various sources including experimentally derived lists of targets for specific transcription factors. Thus, this analysis identifies sets of functionally related genes, such as those co-regulated in response to specific genetic and chemical perturbations, that are significantly enriched in the top positions of a list of genes induced by hypoxia.The results in the previous section were restricted to a limited set of validated promoters. However, if the involved transcription factors are of general relevance to HIF mediated transcription, some of their targets would be expected to be common with HIFs. To judge the potential generality of the involvement of CEBPs, CREB and AP1 in modulation induction of HIF transcriptional targets, we employed a gene-set enrichment analysis (GSEA) As expected, GSEA analysis revealed a statistically significant enrichment of well-characterized HIF targets in this sorted list . These gThe complete elucidation of the molecular principles governing the translation of genomic information to gene regulation remains a central question in biology. In particular, understanding the mechanisms dictating target selection by HIF transcription factors is of fundamental importance to truly dissect the genes directly modulated by HIFs, and therefore to completely characterize the transcriptional response to hypoxia that these factors orchestrate, and its interactions with other transcriptional pathways. Several mechanisms have been proposed to contribute to selective DNA binding and gene regulation by transcription factors with largely generic DNA binding domains Chromatin accessibility has been shown to play an important role in dictating transcription factor binding BNIP3HIF1ADNA methylation of a HIF binding site was originally shown to block HIF1α binding to the 3′ erythropoietin enhancer bona fide HIF binding regions  of transcription factor targets in a sorted list of genes regulated by hypoxia In conclusion, the data presented herein demonstrates that integration of high-throughput chromatin immunoprecipitation and gene expression data is a successful approach to select high-quality core HIF binding regions, and provides experimental proof of principle for the biological relevance of enriched transcription factor binding sites other than the HIF binding consensus in HIF-mediated transcription. Specifically, our results suggest that diverse stress-responsive transcription factors, in particular CEBPs, contribute to fine-tuning of the HIF-mediated transcriptional response.Figure S1Construction of a background set of control sequences resembling core HIF binding regions.(PDF)Click here for additional data file.Table S1HRE, AP1, CREB, CEBPB binding sites and control mutations introduced in LDHA, CA9 and GYS1 promoter constructs.(XLSX)Click here for additional data file.Table S2Correlation between normoxic basal expression and hypoxia inducibility (known HIF targets).(XLSX)Click here for additional data file.Table S3Candidate HIF1alpha-binding regions identified by ChIP-chip analysis in HeLa cells.(XLSX)Click here for additional data file.Table S4Distribution of conserved HBSs in ChIP-chip results across different cell types.(XLSX)Click here for additional data file.Table S5Integrated set of core HIF binding regions.(XLSX)Click here for additional data file.Table S6Enriched gene annotation clusters in core HIF binding regions.(XLSX)Click here for additional data file.Table S7Enriched gene annotation terms in transcription factors associated to sequence motifs overrepresented in core HIF binding regions.(XLSX)Click here for additional data file.


Drosophila it is not generally observed in mammalian cells. However, a number of regions have recently been shown to come into close proximity with their homologous allele, and it has been proposed that pairing might be involved in the establishment or maintenance of monoallelic expression. Here, we investigate the pairing properties of various imprinted and non-imprinted regions in mouse tissues and ES cells. We find by allele-specific 4C-Seq and DNA FISH that the Kcnq1 imprinted region displays frequent pairing but that this is not dependent on monoallelic expression. We demonstrate that pairing involves larger chromosomal regions and that the two chromosome territories come close together. Frequent pairing is not associated with imprinted status or DNA repair, but is influenced by chromosomal location and transcription. We propose that homologous pairing is not exclusive to specialised regions or specific functional events, and speculate that it provides the cell with the opportunity of trans-allelic effects on gene regulation.Although somatic homologous pairing is common in  Often, co-regulated genes are found in the same transcription factory, bringing together various regions from different chromosomes Drosophila. It underlies the phenomenon of transvection which refers to changes in gene activity through interaction of regulatory elements of one allele with its homologue Tight spatial and temporal regulation of gene expression requires several interleaved layers of control. Apart from protein factor binding to trans to regulate gene expression.However, not all examples of homologous pairing in somatic cells involve monoallelically expressed regions. Renal oncocytoma cells show pairing of the q arms of chromosome 19 as a chromosomal abnormality associated with misregulation of gene expression Kcnq1 cluster is a large imprinted region located on distal mouse chromosome 7. Imprinted protein-coding genes of this cluster are expressed from the maternal allele, while a long non-coding RNA expressed from the paternal allele covers the locus to create a repressive compartment Kcnq1 imprinted region for interactions with its homologous allele. We adapted the linear 4C technique Mus spretus (SD7) results in a subset of 3C restriction fragments being informative for their allelic origin  we find pairing events in around 4% of foetal liver nuclei, twice as often as for a control probe covering a region around the myc gene  ‘TouKcnq1 imprinted region, and further away along chromosome 7 . In ES cells  are a common phenomenon which constantly jeopardises genomic integrity, and homologous recombination (HR) is a major repair pathway rescuing these lesions. In both yeast and mammals, sister chromatids are preferred partners for HR but DSBs can also be efficiently repaired between homologous chromosomes Kcnq1 locus, the paternal allele is transcriptionally silenced while a number of protein coding genes are expressed from the maternal allele. It has been shown by RNA immuno FISH that the active maternal allele colocalises with regions of high RNA polymerase II (RNA PolII) concentration while the paternal allele does not It has previously been shown that transcription can reposition genes and mediate preferential co-associations between chromosomal regions We have demonstrated for the first time by allele specific 4C-Seq and by extensive DNA FISH analysis that many loci pair with their homologous allele. Pairing is not limited to regions of mono-allelic expression, involves larger chromosomal regions and brings the two homologous chromosomes into close proximity. While pairing events did not coincide with DNA repair, they took place at sites of ongoing transcription.Tsix on one allele, thereby creating a window of opportunity for mono-allelic expression of Xisttrans-allelic cross talk to ensure that one but only one allele gets inactivated. However, for imprinted regions the situation is different. Here, each allele comes in pre-marked and there is no immediate requirement for communication between homologous alleles. Nevertheless, short interallelic distances were observed in late S phase for the Prader-Willi/Angelman imprinted region in human Kcnq1 and adjacent Igf2/H19 clusters in the mouse, but not for a number of other imprinted clusters. Pairing at distal chromosome 7 was not limited to the imprinted region, and in fact loss of imprinting did not change pairing frequency. Thus, we conclude that homologous pairing is not a general feature of mono-allelically expressed regions. However, this does not preclude an involvement of pairing and trans-allelic effects on the regulation of imprinted regions. In fact, it was shown that introducing a third copy of human chromosome 15 disrupted pairing and affected gene expression at the Prader-Willi/Angelman region Kcnq1 domain which is silenced by a coating RNA, homologous pairing might be involved in the escape of imprinting of several interspersed biallelically expressed genes, especially as we find that pairing is associated with transcription.Homologous pairing has been implicated in the establishment of mono-allelic silencing of the X chromosome. Indeed, during a period of high chromatin mobility, the two X inactivation centres ‘kiss’ which is followed by transient downregulation of Homologous pairing has also been speculated to be linked with DNA repair. The genome is constantly challenged by double strand breaks (DSB) brought about by internal or external chemical insults or the collapse of stalled replication forks , which provides SNPs to distinguish between parental alleles. ES cell genotypes were either C57BL6/JOlaHsd or C57BL6 x SD7. The hybrid ES cell line C57BL6×SD7 carries allele specific information and was newly derived using a modification of a protocol previously described All experimental procedures were conducted under licences by the Home Office (UK) in accordance with the Animals (Scientific Procedures) Act 1986. We used C57BL6/JOlaHsd or SD7 as wild-type strains. SD7 contains the distal region of cis and trans interactions. The linear 4C-Seq protocol was adapted from 3C was performed as described DNA FISH probes were directly labelled essentially as described 0t1 DNA, 10 µg salmon sperm DNA in 50% formamide/10% dextran sulphate/1xSSC) were applied to cells using cover slips sealed on with rubber cement. Samples were denatured at 78°C for 2 min and incubated at 37°C over night. For chromosome painting, directly labelled ready-to-use XMP XCyting mouse chromosome paints (Metasystems) were mixed with precipitated FISH probes. After probe hybridisation, slides were washed in 50% formamide/2×SSC , 0.2×SSC , 2×SSC , 2×SSC , PBS . For immunostaining, slides were blocked in 3% BSA, 0.05% azide, 0.1% Tween-20 in PBS for 30 min, and incubated for 1 h with primary antibody in blocking solution  software. All acquired images were post-analysed by eye to identify pairing events. For each biological sample the frequency of pairing events was determined from two sets of 300 imaged nuclei, scored by two different people in a sample blind way. A FISH signal was counted as ‘paired’ if i) the spots were so close together that the MetaCyte software would only recognise one signal but two spots were discernible by eye, ii) the MetaCyte signal annotation was in the middle of the two spots and iii) there were no other signals visible in the nucleus. Since the z-planes of the image stacks are 0.5 µm apart, this was considered the maximal resolution of the analysis. Paired signals are therefore less than 0.5 µm apart.For DNA immuno FISH, pairing events were identified using automated image capture followed by manual acquisition of image stacks using ISIS software . For chromosome painting, nuclei were imaged on an Olympus IX81 confocal microscope (FV1000) using a 60×1.35 NA plan apochromat lens and Olympus fluoview software (version 3.0). Deconvolution of captured image stacks was performed with Huygens Professional software . Imaris software  was used for image analysis and 3D modelling.A computational model was developed in R to simulate the position of two FISH signals inside the nucleus which display preferential radial positions. The constraint on radial distribution is attained by setting up two exclusion limits for each allele in the nucleus: a central exclusion limit (minimum distance from the nuclear centre) and a peripheric exclusion limit (maximum distance from the centre). For each simulated signal, the limits are randomly chosen based on a normal distribution whose mean and standard deviation are input by the user. By adjusting the input variables the radial distribution of simulated signals is matched to the radial distribution of the respective measured FISH signals. As an output, the model displays the distance between two simulated FISH signals.ES cells were fixed in 4% PFA in PBS for 10 min. Dye Cycle Violet staining was performed according to instructions of the manufacturer . Briefly, fixed cells were washed in 0.1 M Tris-Cl (pH 7.4) and then incubated with 10 µM Dye Cycle Violet in 0.1 M Tris-Cl for 30 min at 37°C. Samples were FACS sorted on a BD FACS Aria3 using violet 405 nm excitation with a 450/40 nm bandpass filter. Cell fractions were attached to slides using a cytospin . DNA FISH was performed as normal.Figure S1Radial distributions of DNA FISH signals and simulated counterparts. Radial distances for KvDMR (A) and myc (B) DNA FISH signals from E13.5 liver nuclei were determined and plotted as a histogram . Radial distances >1 can occur if the nucleus is not a perfect sphere. For both probes, FISH signals show a highly non-random radial distribution and thus cannot be compared to a simulated data set in which signals are placed in a sphere at random. Therefore, locations of signals were simulated to reflect the radial distribution of the respective probe , but are otherwise random. The distance between pairs of simulated signals was then calculated and is plotted in C) Histogram of interallelic distances of KvDMR and myc signals, measured and simulated. Interallelic distances for 600 nuclei were grouped into four equal bins (bin width  = 0.7r). Bin centres are indicated . The histogram displays the same data that is shown as Tukey box-whisker plots in (TIF)Click here for additional data file.Figure S2Pairing of the KvDMR region in ES cells. A) Pairing frequency of the KvDMR and myc regions in ES cells. Each dot represents one sample from one cell passage, and for each sample 300 nuclei were counted in four technical replicates. Differences were assessed by unpaired t-test, ns: not significant, *: p<0.05, ***: p<0.001. KvDMR signals show higher pairing frequency than myc signals. B) Distances between homologous alleles in E13.5 liver represented as Tukey box-whisker plots (n = 600). Differences were assessed by one-way ANOVA with Bonferroni’s multiple comparison post test. Simulated: a group of spots displaying the same radial distributions as KvDMR and myc FISH signals, respectively, were placed into a sphere at random and their interallelic distances determined. While interallelic distances between myc signals show a distribution expected from their radial positions (no difference between sample and simulated), KvDMR signals are significantly closer together than expected . Also, KvDMR signals are generally closer together than myc signals.(TIF)Click here for additional data file.Figure S3Pairing frequencies of imprinted regions in ES cells. Each dot represents the mean of three to four samples taken from different passages with the pairing frequency determined for each sample in four technical replicates of 300 nuclei. Whiskers represent standard deviation. Background shading indicates high pairing frequency , medium  and low pairing frequency .(TIF)Click here for additional data file.Figure S4Paired KvDMR FISH signals do not colocalise with markers for DNA double strand breaks or repair by homologous recombination. 3D representations of image stacks from E13.5 liver cells. Red: KvDMR DNA FISH signals, green: immunostaining for markers of DNA double strand breaks (γH2AX (A), p53bp1 (B)), or markers for homologous recombination (Rad51 (C), Rad52 (D)), blue: DAPI counterstain. Numbers for analysed pairing events are indicated. No overlap of immuno and FISH signals was observed.(TIF)Click here for additional data file.Figure S5Pairing is not specific to a certain cell cycle stage. A) Histogram of PFA fixed ES cells stained with DyeCycle Violet showing DNA content distribution with peaks for G1 and G2 phase cells. The high proportion of S-phase cells is typical for ES cells. B) Pairing frequencies for subpopulations of cells FACS sorted for cell cycle stages. Red dots: KvDMR signals, green squares: myc signals. Each dot represents the mean of three samples taken from different passages with the pairing frequency determined for each sample in two to four technical replicates of 300 nuclei.(TIF)Click here for additional data file.Figure S6Pairing frequency is correlated with expression. A) RNA Seq reads (ES cell dataset from B) The percentage of DNA FISH probe covered by genic sequence was determined using the Ensembl annotation and plotted against the pairing frequency in ES cells. No significant Pearson correlation is observed .(TIF)Click here for additional data file.Table S1BACs labelled to create FISH probes.(DOC)Click here for additional data file.Table S2Antibodies.(DOC)Click here for additional data file.File S1Derivation and characterisation of the ES cell line B6xSD7.(DOC)Click here for additional data file.File S2Details of the linear 4C-Seq analysis.(DOC)Click here for additional data file.Movie S1Regional pairing brings chromosome domains close together.(MOV)Click here for additional data file.


Bacillus subtilis, transcription of the tryptophan biosynthetic operon is regulated by an attenuation mechanism. When intracellular tryptophan levels are high, the TRAP protein binds to the 5′ leader region of the nascent trp mRNA and induces transcription termination prior to the structural genes. In limiting tryptophan, TRAP does not bind and the operon is transcribed. Two competing RNA secondary structures termed the antiterminator and terminator (attenuator) can form in the leader region RNA. In prior attenuation models, the only role of TRAP binding was to alter the RNA secondary structure to allow formation of the attenuator, which has been thought function as an intrinsic transcription terminator. However, recent studies have shown that the attenuator is not an effective intrinsic terminator. From these studies it was not clear whether TRAP functions independently or requires the presence of the attenuator RNA structure. Hence we have further examined the role of the attenuator RNA in TRAP-mediated transcription termination. TRAP was found to cause efficient transcription termination in the trp leader region in vivo when the attenuator was mutated or deleted. However, TRAP failed to induce transcription termination at these mutant attenuators in a minimal in vitro transcription system with B. subtilis RNA polymerase. Further studies using this system showed that NusA as well as the timing of TRAP binding to RNA play a role in the observed differences in vivo and in vitro.In  This binding prevents formation of the antiterminator structure, which allows formation of the attenuator and the trp genes are not expressed. Conversely, when tryptophan is limiting, TRAP does not bind RNA, and the antiterminator structure forms, which allows transcription of the trp genes  and BG4233 (argC4 ΔmtrB) were used as hosts for transformation and integration of gene fusions. BG4233 contains a deletion in mtrB (from codons 7–62), which encodes TRAP B. subtilis was transformed by natural competence E. coli BL21(DE3)  and was purified as described previously B. subtilis NusA was expressed in E. coli strain M15(pREP4) using the plasmid pQE30-NusA, which was a gift from Tina Henkin, encoding a N-terminal His-tagged B. subtilis NusA protein and was purified using Qiagen Ni-NTA beads as per manufacturer's instructions.TRAP was expressed in trp attenuator were created using QuikChange Site-Directed Mutagenesis (Stratagene) using pUCtrpL plasmid as the template trpL contains a 730-bp EcoRI-HindIII fragment including the B. subtilis trp promoter and leader sequence and the start of trpE (−411 to +318 relative to the start of transcription). The resulting plasmids with mutant leader sequence were used as templates for PCR to generate transcriptional fusions with lacZ. PCR was used to introduce EcoRI and BamHI restriction endonuclease sites at the ends of a DNA fragment containing the trp promoter, leader region, and the first 45 codons of trpE followed by a stop codon. Transcriptional fusions with lacZ were made by digesting the PCR product with EcoRI and BamHI, and ligating the DNA into similarly digested pDH32 PstI, transformed into B. subtilis BG2087 and BG4233, and integrated into the amyE locus by homologous recombination, and the −amyE phenotype was confirmed on starch plates Mutations in the B. subtilis strains containing lacZ reporter fusions were grown in 0.2% acid-hydrolyzed casein, 1X MOPS solution pH 7 , 1.3 mM K2HPO4, 10 µM FeCl3, 5 µg/ml arginine, and 5 µg/ml chloramphenicol in the presence or absence of 50 µg/ml L-tryptophan at 37°C to an absorbance between 0.4 and 0.6 at 600 nm. Cells (1.5 ml) were pelleted, washed with TE buffer pH 8, and resuspended in 1.5 ml of Z-buffer . Cells were lysed by addition of lysozyme to 0.1 mg/ml with incubation at 37°C for 5 minutes, followed by addition of Triton X-100 to a final concentration of 0.1%. Cell lysate (0.1 ml) was added to 0.9 ml of Z-buffer and assayed for β-galactosidase activity B. subtilis cells were grown in LB overnight at 37°C and then diluted 1/20 into 100 ml of LB. The cells were grown at 37°C to an A600 of 0.6, harvested, and resuspended in a hot phenol mixture containing equal volumes of RNA extraction buffer , chloroform, and phenol. The mixture was incubated at 75°C for 10 minutes with shaking and the aqueous phase was collected following centrifugation. RNA was then precipitated by adding 3 volumes of ethanol, followed by incubation at −20°C for 1 hour trp leader region. RNA made from this template is complementary to +1 to +376 of the trp leader region, and contains 15 bases of non-complementary sequence at each end. The DNA templates were transcribed in vitro using T7 RNAP as described previously 32P-labeled antisense probe in hybridization buffer, and assays were performed as per manufacturer's instructions (RPA III™ Ambion). Reaction products were separated on 8% denaturing polyacrylamide gels, dried, and exposed to phosphorstorage screens. Size markers were created by T7 RNAP transcription of two DNA templates containing substitutions in the U-stretch of the trp attenuator. PCR was used to place a T7 promoter upstream of the template with an XbaI site from +133–138, or a PstI site from +134–139 relative to the start of transcription. The 137 nt marker was generated by transcription of the XbaI cleaved template. Similarly transcription of the No Binding Site template cut with PstI yielded a 134 nt RNA marker PCR was used to generate a DNA template with a T7 promoter positioned to transcribe antisense RNA to the in vitro transcription attenuation assays were created by PCR amplification of pUCtrpL plasmids using the M13 Forward and M13 Reverse primers. PCR products were purified on 1% agarose gels using the QIAGEN MinElute Gel Extraction Kit. Transcription reactions contained 50 µg/ml B. subtilis σA RNA polymerase (RNAP), 20 nM DNA template, 20 mM Tris-HCl (pH 8.0), 4 mM MgCl2, 0.1 mM EDTA, 4 mM spermidine, 5 mM DTT, 1 mM tryptophan, 500 µM NTPs (unless otherwise noted in the figures), 1 µCi [α-32P] UTP 3000 Ci/mmol DNA templates for trp leader DNA in pUCtrpL to generate the plasmid pUCtrpLEco116. Since the Mismatch Mutant alters the ability to place the EcoRI restriction site at +116, an alternative analogous C125G Mismatch mutation was examined in this assay.For block-and-release transcription assays, a cleavage-defective mutant E111Q EcoRI (EcoRI*) protein was added to the DNA template prior to initiating the transcription reaction. EcoRI* bound to the DNA blocks the transcription elongation complex (TEC) such that the active site of RNAP is stalled 12–13 nts upstream of the first G of the EcoRI site trp leader region was previously found to contain a cryptic promoter that allows transcription to initiate at +37 in vitrotrpLEco116 plasmid as template and the M13 reverse primer together with a +37trpL primer (5′ CAGCTTGACAAATACACAAGAGTGTGTTATAATGCAATTAGAATG 3′) that binds from +31 to +42 in the trp leader region and converts the cryptic promoter into a consensus promoter (−35 TTGACA and −10 TATAAT separated by 17 bases). This +37trpL template directs transcription to initiate at +37, and promotes more efficient transcription initiation at low NTPs in vitro transcription reactions were initiated in the absence of CTP with 50 µg/ml B. subtilis σA RNA polymerase (RNAP), 20 mM DNA template, 20 mM Tris-HCl (pH 8.0), 4 mM MgCl2, 0.1 mM EDTA, 4 mM spermidine, 5 mM DTT, 8 µM ATP and GTP, 2 µM UTP, and 1 µCi [α-32P] UTP 3000 Ci/mmol, and were incubated at 37°C for 10 minutes The B. subtilis trp attenuator is unusual among characterized bacterial transcription terminators. While it contains features in common with intrinsic terminators, including a stem-loop structure followed by a U-stretch AG TRAP binding site  GC content as compared to canonical intrinsic terminators The base-paired stem of the in vivo using transcriptional trp operon lacZ fusions. Each fusion contains the trp promoter and regulatory leader region (either WT or mutant), the first 40 codons of trpE and then a stop codon, followed by the entire coding segment of lacZ  was compared to that in the strain containing TRAP grown in the presence of tryptophan.TRAP-mediated termination at each of these mutant attenuators was assessed  of lacZ . Our tratrp attenuator was regulated approximately 18-fold in response to exogenous tryptophan in BG2087 , as compared to the WT attenuator (BG4233 WT: 286U). This observation suggests that a small amount of termination occurs at the WT attenuator in the absence of TRAP. In the presence of TRAP and exogenous tryptophan, TRAP induced termination at this mutant attenuator so as to regulate transcription 11-fold  the A+U-rich segment immediately following the TRAP binding site was replaced with a 14 base segment composed of Gs and Cs. In the second template (No Stem/No U GC2), the entire A+U-rich region after the TRAP binding site was replaced with a 28 residue GC segment. Replacing 14 residues of the A+U-rich segment proximal to the TRAP binding with a GC segment substantially reduced the ability of TRAP to induce termination in the leader region  probe is complementary to +1 to +141 of transcripts from the US mutant leader region from the Z fusion . If TRAPZ fusion . When th shorter . When thanscript . The ideobserved .trp mRNAs with the Disrupted Stem (DS) mutant attenuator  or the Disrupted stem (DS) mutant attenuator terminate at approximately the same location as transcripts that terminate at the WT attenuator.Similar results were obtained with a probe designed to hybridize to tenuator . When thobserved  lane 10.subtilis . TogethetrpE are observed. These observations support the conclusion that the changes in β-galactosidase activity observed when cells containing these fusions are grown in the presence of tryptophan reflect increased transcription termination in the leader region.In lanes 6 and 10 strong bands are seen corresponding to transcripts terminating at the US and DS mutant attenuators whereas little or no fully protected probes resulting from transcripts that read through the leader region into trp attenuators was also examined in vitro using an attenuation assay described previously trp promoter and leader region, including the WT or mutant attenuator, as well as the first 45 codons of the trpE gene , as well as those that read through the attenuator and continue to the end of the template (RT:320 nt) (trp attenuator.The ability of TRAP to induce transcription termination at three mutant rpE gene . Transcr:320 nt) . In the :320 nt) . Additio:320 nt) . These oin vivo results . Adding 0.5 M KCl dissociates EcoRI* from the DNA, releasing the blocked TECs to continue transcribing the template DNA.Finally, to ensure sufficient time for TRAP to bind the nascent transcript before RNAP progresses beyond the attenuator, we blocked the transcription elongation complex (TEC) with EcoRI* bound to the DNA in vitro transcription assays that is identical to that described in trp leader region For these studies, we constructed a template for trp attenuator yields two major transcripts that are similar to those seen previously with the WT template  mutant, or within the attenuator containing a single mismatch in the stem (MM) is, at least in part, related to the timing of TRAP binding relative to the position of the transcribing RNAP. However, none of the approaches used to provide additional time for TRAP to bind to the nascent transcript before RNAP has extended beyond the attenuator region, resulted in increased termination attenuator with the disrupted stem (DS). In contrast, TRAP induced termination at this DS attenuator in vivo such that transcription of lacZ was reduced 15 fold  is deleted lacZ mRNA levels were measured directly to assess transcriptional regulation independent of changes in translation. They found that TRAP mediated approximately 9-fold reduction in mRNA levels from a similar fusion in which the trp attenuator  or U-stretch (US) abolished TRAP-mediated termination in a simple in vitro transcription attenuation assay using B. subtilis RNAP  attenuator. In contrast, TRAP induces significant termination at the DS attenuator in vivo . In contrast, the other predicted RNA structures in the trp leader region, including the 5′ stem-loop, the antiterminator and the ribosome binding site sequestering structure, have been extensively mapped trp leader regions from several other species of bacteria that contain a gene encoding TRAP and contain a predicted TRAP binding site upstream of trpE, also contain predicted attenuators that appear unlikely to function as intrinsic terminators. These attenuators also display low GC content in their base-paired stems and interruptions in their U-stretches. Moreover, three species including B. megaterium, B. pseudoformis, and B. halodurans, have predicted attenuators with multiple mismatches in their predicted hairpin stems , and contains three mismatches near the base of the predicted stem B. subtilis trp operon, likely play a minor role in TRAP-mediated control of transcription. The leader regions from these bacilli also contain A+U-rich segments both upstream and downstream of the predicted attenuator similar to that in the B. subitlis trp leader, which we have shown is important for TRAP-mediated termination. Hence, it seems likely that the mechanism of TRAP-mediated attenuation in these species is similarly to that we have described for B. subtilis.In addition to finding that the attenuator is not essential for TRAP-mediated transcription termination in stems . In parttrp attenuator, forward translocation may not occur efficiently in vivo that TRAP-mediated transcription termination within the leader region does not require the attenuator, but does require an A+U-rich region following the TRAP binding site, which may serve to pause RNAP. Our revised model for transcription termination within the trp leader region suggests that TRAP functions not only to alter the RNA secondary structure but also to directly induce RNAP to terminate transcription, possibly by directly interacting with RNAP. Consistent with this model, we have recently isolated a TRAP (E60K) mutant that binds tryptophan and RNA with similar properties as the WT protein but shows 3-fold less termination in the trp leader region in cells grown in the presence of excess tryptophan. Hence Glu60 may interact with RNA polymerase to induce termination.In the forward translocation model of intrinsic termination, the U-stretch following the hairpin stem acts to pause RNAP, and formation of the hairpin stem induces RNAP to move forward along the DNA et al., the majority of mechanistic studies of intrinsic transcription termination are based on a small set of model terminators mostly from E. coli and closely related bacteria Mycobacterium, Helicobacter, Treponema, Synechocystis, Mycoplasma, and Borrelia do not appear to contain canonical terminator structures downstream of their genes B. subtilis trp attenuator, the E. coli trpA and asnU genes contain features that resemble intrinsic terminators at the end of these genes but do not induce termination. The E. coli asnU gene is followed by a GC rich hairpin stem and a U-stretch, however termination of transcription of this gene is Rho-mediated E. coli trp operon contains an apparent intrinsic terminator after trpA. However, termination is inefficient at this site and occurs further downstream by Rho-dependent termination As recently pointed out by Peters