RUNX3 promoter, with ankylosing spondylitis (AS).To identify the functional basis for the genetic association of single nucleotide polymorphisms (SNP), upstream of the RUNX3 region. The functional effects of this SNP were tested in luciferase reporter assays. Its effects on TF binding were investigated by electrophoretic mobility gel shift assays and chromatin immunoprecipitation. RUNX3 mRNA levels were compared in primary CD8+ T cells of AS risk and protective genotypes by real-time PCR.We performed conditional analysis of genetic association data and used ENCODE data on chromatin remodelling and transcription factor (TF) binding sites to identify the primary AS-associated regulatory SNP in the RUNX3 SNP rs4648889 with AS (p<7.6×10−14) was robust to conditioning on all other SNPs in this region. We identified a 2 kb putative regulatory element, upstream of RUNX3, containing rs4648889. In reporter gene constructs, the protective rs4648889 ‘G’ allele increased luciferase activity ninefold but significantly less activity (4.3-fold) was seen with the AS risk ‘A’ allele (p≤0.01). The binding of Jurkat or CD8+ T-cell nuclear extracts to the risk allele was decreased and IRF4 recruitment was reduced. The AS-risk allele also affected H3K4Me1 histone methylation and associated with an allele-specific reduction in RUNX3 mRNA (p<0.05).The association of the RUNX3 that is modulated by rs4648889. The risk allele decreases TF binding (including IRF4) and reduces reporter activity and RUNX3 expression. These findings may have important implications for understanding the role of T cells and other immune cells in AS.We identified a regulatory region upstream of  HLA-B, ERAP1 and IL23R.RUNX3 and AS and psoriatic arthritis, another form of SpA.RUNX3 locus,RUNX3 promoter. One of these, rs4648889, lies in a putative transcription regulatory region defined by DNaseI hypersensitivity and the presence of chromatin marks correlated with active enhancers (H3K4Me1 and H3K27Ac).RUNX3 is a member of the runt domain-containing family of transcription factors (TFs), which play key roles in many developmental pathways.Runx3−/− mouse shows a complex phenotype affecting several organs, highlighting the broad spectrum of RUNX3 action.RUNX3 influences several types of immune cell that could be involved in AS, including natural killer (NK) cells and regulatory T cells,RUNX3 genotype.RUNX3 expression; the gene has two differentially expressed alternative promoters resulting in proteins that have dissimilar transcriptional activation capacity.RUNX3 promoter methylation status has also been incriminated in acute myeloid leukaemia19Ankylosing spondylitis (AS) is a common form of spondyloarthropathy (SpA) with numerous robust genetic associations, of which most are currently unexplained. Some functional protein-coding single nucleotide polymorphisms (SNPs) have been identified in genes associated with AS, such as RUNX3 are strongly associated with AS. In the recent large ImmunoChip study, the lead SNP was rs6600247 (p=1.3×10−14), which is in complete LD with rs4648889 (∼2 kb upstream of the RUNX3 promoter).RUNX3,rs4648889 on RUNX3 expression.Numerous SNPs in a ∼15 kb linkage disequilibrium (LD) block upstream of rs4648889 genotypes. Where required, DNA was extracted using the Qiagen AllPrep DNA/RNA Mini Kit .Historical typing data from the AS ImmunoChip studyGenotype data from the UK cases in the International Genetics of Ankylosing Spondylitis Consortium AS ImmunoChip study and WTCCC2 controlsrs4648889 was amplified from human genomic DNA and cloned into the TA cloning kit pCR2.1 vector , then subcloned into the pGL4.23(luc2/minP) reporter vector  at the SacI/XhoI restriction sites (primer sequences available on request). Point mutations corresponding to genetic variants (G/A) of rs4648889 were introduced using the QuikChange II XL Site-Directed Mutagenesis Kit . Luciferase reporter assay details are available online (see online supplementary methods section).The 250 bp sequence flanking Following informed consent (COREC 06/Q1606/139 and OXREC B 07/Q1605/35), venous blood samples were obtained from 19 patients with positive HLA-B27  of white British ancestry fulfilling the modified New York criteria for AS.CD8+ T cells were isolated from peripheral blood mononuclear cells using the CD8+ T-cell Isolation Kit , according to manufacturer's instructions. CD8+ T cells were then plated for 4 h in RPMI supplemented with 10% fetal bovine serum before harvesting for experiments.Nuclear extract from Jurkat cells was prepared using the Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction kit . Electrophoretic mobility gel shift assays (EMSAs) were performed with LightShift Chemiluminescent EMSA Kit  using 5 μg of nuclear extract and 0.6 ng biotin-labelled double-stranded oligonucleotides . The sequences of the synthetic single-stranded oligonucleotides used in the construction of these double-stranded oligonucleotides are listed in the online supplementary methods.Probes were prepared using a biotin 3′ end DNA labelling kit .Single-stranded biotinylated oligonucleotides were mixed and annealed at room temperature for 1 h. Unlabelled competitor probes were used in 100-fold excess.EMSAs were performed according to standard protocol (Thermo Scientific).Band intensity was quantified with ImageJ software .A detailed protocol is available online (see online supplementary methods section).4 CD8+ T cells were incubated with 1% formaldehyde for 10 min and 1.25 M glycine added for 5 min. DNA isolation was performed with DNA isolation buffer supplied by the kit. Quantitative PCR (qPCR) was performed on immune complex-associated DNA using allele-specific primers for rs4648889 , detected with SYBR Green on ABI ViiA7 PCR instruments . A compensatory factor ((log(100)/log2) was subtracted from the cycle threshold (Ct) values of the diluted input (1%) in order to calculate the Ct values of the 100% input. Calculation of relative enrichment was done as follows: signals obtained from the ChIP are divided by signals obtained from input sample (representing the amount of the chromatin used in the ChIP)=2^ (adj input-ct(IP)).Chromatin immunoprecipitation (ChIP) was performed using the Diagenode Low Cell# ChIP kit . For each ChIP, 6×10Relative occupancy was calculated as a ratio of specific signal over background: % input (specific loci)/% input (background loci).The antibodies used were: IRF4 , H3K4Me1  and IgG .RNA was isolated with TRIzol  and cDNA synthesis (for 500 ng RNA) was prepared with Superscript III from Invitrogen . A final concentration of 5 ng/μL was used in qPCR, which was performed with the ABI ViiA7 PCR instrument  using SYBR Master mix  with evaluation of dissociation curves. mRNA levels of each gene were quantified using the ΔΔCt method and normalised to β-actin. For each gene, PCR melting curves were checked to evaluate the single, specific product.The specific primers designed were RUNX3 forward: 5′-ACT CAG CAC CAC AAG CCA CT-3′ RUNX3 reverse: 5′-GTC GGA GAA TGG GTT CAG TT-3′ RUNX3 values were normalised to β-actin .Sanger sequencing in forward and reverse orientations was performed by Source Biosciences (Oxford) using the sequencing primer: 5′-GTT TCC ATT CCA CCA ACA CC-3′.RUNX3 locus, we performed conditional analysis on 4230 AS cases and 9700 matched controls, as previously described.RUNX3 SNP rs4648889.Association data for genotyped and imputed SNPs were obtained on the subset of AS cases of white British ancestry and white British controls from the recent ImmunoChip GWAS. To evaluate the presence of independent effects on genetic susceptibility at the One-way analysis of variance (ANOVA) and two-tailed Student's t test were used to determine statistical significance using the GraphPad Prism software (V.5.03) package.rs6600247 as the lead SNP at the RUNX3 locus (1.3×10−14), with rs4648889 as the next most strongly associated SNP (p=7.6×10−14). We performed conditional analysis and found no evidence of independent effects in disease susceptibility between these two SNPs, reflecting the strong LD between them  association—rs4265380 (1.7×10−7) and rs7529070 (p=1.2×10−7). Conditioning on these two SNPs independently, the strong association with the SNP rs4648889 was retained (p≤3.0×10−6 for both SNP), thereby establishing the primacy of this association with AS in this region.The previous AS ImmunoChip studyeen them . After cRUNX3 distal promoter in the region of rs4648889 (http://www.epigenomebrowser.org) and is predicted to bind several TFs. The region around rs4648889 is more likely to be functionally relevant than rs6600247, the lead SNP in the previous ImmunoChip study, considering the paucity of TF binding near the latter (RUNX3 promoter (We first identified a putative regulatory element ∼2 kb upstream of the s4648889 A. This ws4648889 A.2028 Te latter B. We thepromoter .rs4648889 (rs4648889 dimorphism on TF binding to a 50 bp DNA fragment using EMSA. Addition of nuclear extract from Jurkat cells (leukaemia T-cell line) to this DNA probe created a major protein–DNA complex (i) with a visibly weaker band for the AS-risk allele ‘A’ (∼5.4-fold±1.2 SEM less than ‘G’) than the AS-protective ‘G’ allele  to the AS-protective ‘G’ allele compared with the AS-risk ‘A’ allele  compared with the AS-risk ‘A’ allele .Sanger sequencing of one rs4648889 was also evaluated in vitro by luciferase reporter assay. The 250 bp region containing rs4648889 showed increased luciferase activity compared with the minimal promoter (minP=1) but the presence of the AS-risk allele ‘A’ reduced the enhancer activity from 9.1-fold to 4.3-fold (p≤0.01) in HEK293T cells (human embryonic kidney cell line) and from 4.0-fold to 1.9-fold (p≤0.01) in Jurkat cells  showed that RUNX3 mRNA levels, normalised against β-actin, were significantly lower in subjects with the AA than the GG genotype . No correlation was observed between RUNX3 expression and disease activity as defined either by BASDAI or CRP.Analysis of pression  in CD8+ rs4648889 is probably the primary AS-associated SNP at the RUNX3 locus and that this association can probably be best explained through its effects on gene expression. The rs4648889 AS-risk allele ‘A’ exhibits relatively low luciferase reporter activity compared with the AS-protective ‘G’ allele, less RUNX3 mRNA levels and less enrichment for H3K4Me1 histone modification of a type associated with active enhancer elements.RUNX3 expression and consequently on CD8+ T-cell development and function.RUNX3 genotype.RUNX3 enhancer. RUNX3 plays a key role in haematopoiesis and enhances CD8 T-cell development/activation and has an inhibitory effect on CD4+ T cells.RUNX3 affects NK cell development,35We have shown here that HLA-B*27 association with AS has long tempted speculation that aberrant immune responses to microbial infection, perhaps mediated through HLA-B27-restricted CD8+ cytotoxic T cells, could be involved.HLA-B*27-transgenic rats develop SpA unless reared in germ-free conditions.HLA-B*27 transgenic rats still develop SpA in the absence of CD8+ T cells.HLA-B*27, RUNX3, EOMES, TBX21, ZMIZ1, IL7 and IL7R) potentially affect lymphocyte biology.RUNX3 locus in CD8+ T cells. We have not yet extended these studies to other cell types, such as Th1, NK, dendritic or other immune cells that could also be involved in AS.RUNX3 expression could enhance CD4+ T-cell activity, which would be consistent with models of AS invoking a pathological role for CD4+ T cells.44The striking rs4648889 polymorphism. It is well known that eukaryotic transcription may be controlled by regulatory elements that are distant from their target genes; such control can be exerted (sometimes on multiple genes) through the formation of chromatin loops.RUNX3 regulatory region could influence other more distant genes relevant to AS. Establishing the full extent of the genes (and their regulation) in these pathways and their relationship to the pathogenesis of AS represents a considerable challenge. In future we will investigate TF binding and chromatin accessibility across the whole genome to explore the composition of these AS gene regulatory networks.47It is likely that most genetic associations in AS reflect relatively subtle changes in gene expression. The differences in DNA–protein complexes revealed by EMSA experiments between the AS-risk ‘A’ and AS-protective ‘G’ allele require further investigation to define the full diversity of TFs involved in these complexes and also the network of genes that might ultimately be regulated by the This work illustrates the first steps towards a more complete mechanistic understanding of just one of the many genetic associations of AS. These and related techniques have broad applications to the investigation of other similar regulatory polymorphisms that probably account for much of the genetic risk in AS. A deeper knowledge of the factors involved in these processes is likely to lead of the discovery of new therapeutic targets.


The evolution of anchor DNA sequences in bacterial  and archaeal  promoters is potentially explained. The evolution of BREdown elements of archaeal promoters is potentially explained.Repeating sequences generated from RNA gene fusions/ligations dominate ancient life, indicating central importance of building structural complexity in evolving biological systems. A simple and coherent story of life on earth is told from tracking repeating motifs that generate α/β proteins, 2-double-Ψ−β-barrel (DPBB) type RNA polymerases (RNAPs), general transcription factors (GTFs), and promoters. A general rule that emerges is that biological complexity that arises through generation of repeats is often bounded by solubility and closure . Because the first DNA genomes were replicated by DNA template-dependent RNA synthesis followed by RNA template-dependent DNA synthesis via reverse transcriptase, the first DNA replication origins were initially 2-DPBB type RNAP promoters. A simplifying model for evolution of promoters/replication origins via repetition of core promoter elements is proposed. The model can explain why Pribnow boxes in bacterial transcription (i.e.,  After the advent of coding, ancient evolution of life on earth becomes a starkly simple story of replication errors or, perhaps more likely, RNA gene fusions/ligations resulting in repeating RNA sequences encoding repeating protein motifs.Eukaryotes are generally more complex than bacteria and archaea, and the tortured path to eukaryotic evolution explains increased genomic, functional and organismal complexity. At LECA , eukaryotes resulted from endosymbiosis and genetic fusion of a Lokiarchaeota phylum archaea−31TATAAAAG−24) and bacterial Pribnow boxes  (see below).In eukaryotes, genetic duplications generated RNAPs I, II and III and the carboxy terminal domain (CTD) repeat on RNAP II, which facilitated nuanced RNAP II regulation required to support complexity and multicellularity.n repeat proteins, generated from β−α−β−α repeats.n repeats to generate TIM barrels (β−α)8 and Rossmann folds (β−α)8. We conclude that ubiquitous α/β fold proteins are generated from simple (β−α)n repeats.Remarkably, glycolysis, the citric acid cycle and the glyoxylate cycle are catalyzed by ancient α/β fold proteins .4 unit, which was itself generated by earlier duplication of a (β−α)2 unit. Formation of the 8-parallel β-sheet TIM barrel gives closure to the structure. Further polymerization beyond 8 sheets breaks the barrel and can create a larger, more flexible horseshoe structure. Compared to TIM barrels, Rossmann folds are generated from (β−α)8 repeats that are rearranged into a twisted sheet (8 may have resulted from rearrangement of a TIM barrel (β−α)8. In a TIM barrel, β1−β8 curvature supports closure of the barrel. Because of necessary rearrangements, the Rossmann fold linear, twisted sheet is supported by the opposite curvature of β1−β3 and β4−β7/β8. Similarly, TOPRIM domains ∼(β−α)4-5  appear to be generated from a larger repeat such as a Rossmann-like fold. Many ATPases, GTPases and kinases are Rossmann-like folds that emerged in the RNA-protein world . Swi-Snf ATPases include 2 (β−α)6 domains . Some of these folds require rearrangements of β-sheets. Ubiquitous α/β fold proteins that account for essentially all ancient metabolism and energy transduction, therefore, were generated on a scaffold formed by simple repetition of a β−α−β−α unit.Glycolytic enzymes are of the TIM (triose phosphate isomerase) barrel fold (β−α)ed sheet  and S3.1Importantly, α/β protein folds provide both structure and solubility. The β−α−β−α unit and its larger repeats create structure through interaction of parallel β-sheets, and these folds are soluble, because β-sheets, which by themselves might form amyloid-like interactions, are each paired with an α-helix. The solubility of the β-sheet fold, therefore, appears to have been promoted by the associated helices. Early and enduring success of the α/β fold, therefore, is explained by structure, sufficient functionality and solubility. Remarkably, so far as we are aware, the active site in α/β fold proteins is always located to the C-terminal side of the β-sheets that dominate the fold. It appears that this relation may have been established ∼4 billion years ago on a ∼4.6 billion year old earth and maintained via powerful co-evolutionary forces. So far as we are aware, in a TIM barrel, a Rossmann fold or a Rossmann-like protein, no active site or allosteric site locates to the N-terminal end of the β-sheets.Multi-subunit RNAPs are of the 2-DPBB type.4) to support transcription initiationIn RNAPs, 2-DPBBs border the 2-Mg active site, and loops from the barrels bind active site Mg-I and Mg-II (Mg-A and Mg-B) . OppositHere we describe evolution of TBP (TATA-binding protein) and a primordial transcription initiation factor that gave rise to σ factors in bacteria and to TFB (Transcription Factor B) in archaea, driving divergence of bacteria and archaea.1 appears vestigial, HTH1 is not shown. HTH4 binds the “anchor” DNA -35 region of the bacterial promoter (−35TTGACA−30). This contact anchors RNAP at the promoter upstream and specifies the direction of bubble opening and downstream transcription. HTH4 is a typical HTH unit with a 8-10 amino acid H2 that makes canonical contact, via the N-terminal end of its “recognition helix” H3, to the major groove of the DNA. HTH3 is also a typical HTH that binds upstream of the initiation site where the bubble opens. HTH3 has a typical 8-10 amino acid H2. In the structure with an open bubble, the N-terminal end of HTH3 H3 does not bind in the DNA major groove, as would be expected during an initial encounter with double-stranded (ds) DNA, but no structure of HTH3 on dsDNA is currently available. Perhaps, as the bubble opens, HTH3 twists from an initial typical HTH-major groove contact. HTH2 is a highly specialized HTH-derived fold that opens the -10 region of the bacterial promoter (−12TATAATG−6). HTH2 has an elongated H2 (20 amino acids) and bulky hydrophobic residues on H3  judged inconsistent with binding to dsDNA.2 aggressively attacks dsDNA, flips out −11A of the non-template DNA strand (NDS) and then flips out −7T on the NDS to help unzip DNA to +1 for initiation on the template DNA strand (TDS).4 that binds the anchor DNA (-35) and the highly specialized HTH2 that opens the -10 region.The evolutionary model for σ combined with recent x-ray structures of initiating RNAP describes σ functions in initiation. In 43 TFEα includes a winged HTH (WHTH) motif and a Zn ribbon. The WHTH motif in TFEα is most similar to the ArsR family of WHTH transcription factors dispersed to bacteria and archaea. The WHTH motif in TFEβ is most similar to the MarR family of WHTH transcription factors dispersed to bacteria and archaea. It is clear that the WHTH motif is ancient and, because it is distributed to both bacteria and archaea, the WHTH motif may have been present at LUCA. It is unclear when TFEα/β arose in archaea . Because of the difficulty in opening B-form DNA, it is also possible that a helicase may have aided promoter and replication origin opening at LUCA, analogous (not homologous) to the eukaryotic TFIIH helicases.In addition to TBP and TFB, archaea also utilize the GTF TFEα/β.According to these simple and simplifying models, LUCA transcription and replication, on the first DNA templates, were supported by a mechanism that is very recognizable today. We posit that TBP bound multiple TATAAAAG boxes. A primordial initiation factor with 4-HTH repeats (“cyclin-like” repeats) bound to surrounding BREs (TFB-recognition elements). TFE may have been present, or TFE may have evolved separately in archaea after divergence. Also, a helicase may have facilitated promoter/replication origin opening. This model is simplifying, because it roots the tree of life at LUCA and describes the radiation of bacteria and archaea. Bacteria and archaea are posited to have diverged because they evolved to interpret, transcribe and replicate their genomes using distinct RNAP-GTF-promoter combinations. In bacteria, GTFs, RNAP and promoters became much more tightly coupled and co-dependent than in archaea, and this difference is seen comparing the degenerate HTH units of σ factors in bacteria and the more recognizable cyclin-like HTH repeats of TFB in archaea.down (TFB-recognition element downstream of TATA) (up). In the LUCA promoter/replication origin, there may have been many more than 3 repeats, but 3 repeats is sufficient to generate a model. From the proposed LUCA promoter, an archaeal promoter with a BREup, a TATAAAAG box and a BREdown can be generated via simplification, and simplification is also posited for archaeal TFB, which is posited to have lost 2-HTH repeats from a 4-HTH primordial initiation factor. Similarly, a bacterial promoter can be derived, with a −35TTGACA−30 -35 anchor region, an extended -10 region, and a Pribnow box (−12TATAATG−6) -10 region. In this model, the Pribnow box of the bacterial promoter is derived from a downstream TATAAAAG box of the LUCA promoter, explaining why Pribnow boxes, which are bound by a specialized HTH-derived domain (σ HTH2), resemble TATAAAAG boxes, which are bound by TBP (not present in bacteria) binding in the DNA minor groove. Other features of promoters can be generated by coevolution of interacting factors and promoters. For instance, alternate σ factors with different promoter recognition can be generated via co-evolutionary forces. As we have previously shown, some alternate σ factors are most similar to archaeal TFB in sequence, particularly in σ HTH4 T1-H2-T2  and TFB HTH2 T1-H2-T2 .Repeating sequences generated α/β folds, RIFT barrels, DPBBs, TBP (2 TBP-fold repeats), σ (4-HTH repeats) and TFB (2-HTH repeats). Similarly, promoters at LUCA are posited to have evolved via repetition of an AT-rich sequence. As a possible example, a hypothetical LUCA promoter is posited to be generated by alternating repeats of a TATAAAAG box and an AT-rich BREof TATA) . Three r1-HTH2 on promoter DNA.1-(HTH2/HTH1)-(HTH2/HTH1)-HTH2. Because in the model 4 of 6-HTH units are superimposed (HTH2/HTH1), 6-HTH units reduce to 4-HTH units, as are found in bacterial σ factors. What results is a promoter repeat of 3-TATAAAAG boxes and 4-BREs. Three TBP molecules bind the 3-TATAAAAG boxes and the 4-HTH repeats each bind a BRE. Because the archaeal TBP-TFB-promoter 1AIS structure was used to generate the LUCA TBP-primordial initiation factor-promoter structure, archaeal transcription is obtained from the LUCA model by simplification. To suppress unwanted transcription starts in archaeal promoters, selection against multiple TATA boxes is expected, leading to degeneration of the initial repeat structure , and a surprisingly simple and compelling model for genesis of eukaryotes is available from extensive phylogenomic studies.1YSPTSPSMore complex eukaryotic genomes allowed for duplication of genes and diversification of functions . Two-DPBB type RNAPs diversified into RNAP I, II and III, and RNAP II acquired the carboxy terminal domain (CTD), a heptapeptide  form a surprisingly dominant component of the fabric of life, as if, in early evolution, structural complexity may have been positively selected, even before active sites gained refined function and specificity. We suggest that some initial repeats must have had sufficient function to compete successfully with or to collaborate with ribozymes.n repeat proteins) have built-in structure through parallel interacting β-sheets and solubility through pairing of each β-sheet with its α-helix. As such, α/β proteins appear to have won a primordial race to structure, solubility and function. Remarkably, the β−α−β−α pattern remains discernable after almost ∼4.1 billion years of evolution on a ∼4.6 billion year old earth . This indicates, perhaps contrary to intuition, that evolution can be very conservative of core protein repeat motifs over a span of ∼4 billion years.α/β proteins ((β−α)In unexpected ways, evolution describes transcription, and transcription describes evolution. Life originated from a RNA-protein world that included 2-DPBB type RNA-template dependent RNAPs, and, therefore, RNAPs are central to the story of life on earth from: 1) the RNA-protein world to LUCA; 2) from LUCA diverging to bacteria and archaea; and 3) from endosymbiotic fusion of a Lokiarchaeota and an α-proteobacterium at LECA. Thus, a stunningly simple working model for genesis of life on earth is available from evolution of 2-DPBB type RNAPs, RNAP GTFs, RNAP promoters, RNAPs I, II and III, and the RNAP II CTD . The model describes the major branch points in evolution and provides surprising insights into eukaryote and animal complexity. The model provides a deeply conceptual understanding of life on earth.The complexity of protein structures and, therefore, of living systems can be bounded by the linked issues of: 1) solubility; and 2) closure. The repeating protein sequence folds that win tend to be soluble and to gain closure by forming pseudo-symmetric forms: i.e. dimeric repeats and tetrameric repeats. Examples of dimeric repeats: 1) double-Ψ−β-barrels; 2) 2-double-Ψ−β-barrel type RNAPs; 3) TBP (TATA-binding protein); 4) RIFT barrels; 5) TFB (Transcription Factor B); 6) TIM barrels (β−α−β−α−β−α−β−α dimer). Examples of tetrameric repeats: 1) the primordial initiation factor (4-HTH); 2) σ factor (4-HTH); 3) TIM barrels (β−α−β−α tetramer). Closure is attained by formation of barrels and/or pseudo symmetry. What this means is that there are limits to the complexity evolution will achieve, and limits are often approached by simple bounds of solubility and closure. The CTD (52 repeats in humans) is not strongly bounded by these rules because it is largely unstructured, attached to a soluble scaffold (RNAP II) and heavily modified. The length of the CTD is bounded by its functionality.


Yersinia ruckeri to survive in the aquatic systems reflects its adaptation  to the unfavorable environments. The nonspecific porins are a key factor contributing to the permeability. Here we studied the influence of the stimuli, such as temperature, osmolarity, and oxygen availability on regulation of Y. ruckeri porins. Using qRT‐PCR and SDS‐PAGE methods we found that major porins are tightly controlled by temperature. Hyperosmosis did not repress OmpF production. The limitation of oxygen availability led to decreased expression of both major porins and increased transcription of the minor porin OmpY. Regulation of the porin balance in Y. ruckeri, in spite of some similarities, diverges from that system in Escherichia coli. The changes in porin regulation can be adapted in Y. ruckeri in a species‐specific manner determined by its aquatic habitats.The capability of  Yersinia ruckeri is a Gram‐negative bacterium intermittent between planktonic and host interaction states . There are different surviving strategies developed during bacterial evolution, one of them is alteration of outer membrane permeability. General porins, the most abundant outer membrane proteins (OMPs), are responsible for the permeability of the membrane to small hydrophilic polar molecules. The balance of major OMPs, known as OmpF and OmpC, is achieved through its complex regulation often reciprocal in response to many environmental factors, such as temperature, osmotic pressure, and pH , temperature , aeration (200 rpm), and limited oxygen availability, and incubated to mid‐exponential growth phase (OD600 = 0.4–0.6). Then 1 mL of the culture was centrifuged, and the cell pellet was placed in the RNA Protect bacterial reagent  to stabilize total RNA. The rest culture was sedimented by centrifugation and 150 mg of the wet bacterial pallet was used for porin extraction.Strain μg of total RNA using the MMLV RT kit  and random primer according to the manufacturer's instruction.Total RNA was extracted from two biological replicates using the Aurum Total RNA Mini Kit  comprising a treatment with RNase‐free DNase I to eliminate DNA contamination. First‐strand cDNA synthesis was performed from 2 ompF, ompC, ompY expression was calculated as the ratio of reference to target using the ΔΔCt method, where Ct is the cycle threshold.The cDNAs were subsequently quantified by real‐time PCR amplification with primers specific to the outer membrane protein genes and 16S rDNA gene    using Student's g. The complex of peptidoglycan (PG) and OM proteins in the precipitate was treated with DNAse (Fermentas). The fraction containing PG‐associated proteins was extracted with buffer A containing 1% SDS and 0.5 mol/L NaCl. Protein fractions were analyzed by SDS‐PAGE. Gels were stained with Coomassie brilliant blue G250 in 3.5% perchloric acid solution. Original SDS‐PAGE are presented in Figures S1 and S2.Porins were isolated and purified as described previously  equipped with a high mass HMD 1 detector  in linear positive ion detection mode. A saturated solution of 3,5‐dimethoxy‐4‐hydrocinnamic (sinapinic) acid (10 mg/mL) in an acetonitrile–0.1% TFA mixture (70% acetonitrile) was used as the matrix. The sample was mixed with the matrix and spotted onto a target by the dried droplet method. 0.05 mg/mL BSA was used to calibrate the mass spectrometer.Y. ruckeri bacterial culture grown at 26°C, 300 mmol/L NaCl with aeration. BSA was analyzed as a control. MALDI‐TOF data show the presence of two proteins  similar to those it encounters in hosts or nature. Yersinia ruckeri, as all Yersinia, is a psychrophilic microorganism, it can grow and reproduce at cold temperatures from 4°C . As shown in Figure Y. ruckeri major porins are tightly controlled by temperature. In SDS‐PAGE, OmpF became predominant at low temperature (8°C), while OmpC dominated at higher temperature (37°C). However, both major porins were present in the outer membrane in almost equal amounts at the optimal growth temperature (26°C), excepting low osmolarity that resulted in considerable reduction in OmpC production.We investigated the porin expression in ompF transcription increased about four times at low temperature compared with 26°C. Increasing the temperature to 37°C promoted a 6.7‐fold reduction of ompF expression. Expression of ompC shows strong opposite effect at 8°C, it decreases up to 66 times. It should be noted that the maximum level of ompC transcription was observed at 26°C, and the further temperature increase to 37°C did not cause noticeable changes in the expression.According to the qRT‐PCR analysis, Y. ruckeri, the low temperature dramatically reduces the transcription of ompC and induces ompF. Interesting to mention that a high temperature did not significantly affect the transcriptional level of ompC and OmpC remains the prevalent protein in the outer membrane because the ompF transcription decreases. Only a very few publication concern the modulation of porins in response to changes in temperature. For Escherichia coli the porin thermoregulation observed is reciprocal increase in OmpF and decrease in OmpC at 20°C and vice versa at 37°C . However, ompC expression remained unchanged at 37°C and at 8°C both at hyperosmotic and hypo‐osmotic conditions. We suppose that no change in ompC expression under influence of different osmolarity, excluding hypo‐osmolar condition at 26°C, is due to the predominant effect of temperature. The transcriptional profile of ompC was also confirmed by SDS‐PAGE.We determined the effect of osmolarity on the transcription of ompF increased about 10‐fold at 37°C in hypo‐osmotic LB medium. The same effect was reproduced at 26°C, but the ompF transcription decreased by 2.5 times in hypo‐osmotic medium at 8°C . Interestingly, the hyperosmotic medium (300 mmol/L NaCl) did not decrease the ompF transcription and this strongly contrasts to what has been observed previously for E. coli  by normal osmolarity (150 mmol/L NaCl). The aerobic growth condition was achieved by vigorous agitation (200 rpm) in flasks. The anaerobic cultures were grown without agitation overlaid with sterile mineral oil. According to the OMP profiles in aerobic conditions  porins should be taken into account to achieve a comprehensive picture of the bacterial adaptation mechanisms. To date, our knowledge of the minor porins is very limited. Previously, we have described a new OmpY porin in ompY transcription (4.5‐fold) at 8°C and upregulates its transcription (3.4‐fold) at 26°C. Furthermore, increasing or decreasing of osmolarity led to the same effect, namely, to increased transcription (approximately fourfold) of ompY at high temperature. Lowering the temperature increased the ompY transcription by approximately threefold at 8°C (Fig. ompC transcription was documented (Fig. ompY porin gene increased insignificantly in response to anaerobiosis (Fig. ompY transcription behavior was very similar to the ompF one under most tested conditions. Because OmpY is supposed to present in small amounts in the outer membrane, the differences in the OMP profiles might be undetectable under tested conditions. It is also possible that we could not define the particular stress conditions necessary for production of considerable amounts of OmpY porin in Y. ruckeri.It is evident from qRT‐PCR data Fig. A that thiption 4.‐fold at iption 4.‐fold at ompY transcription that indicates its special role in maintaining the necessary level of porins in the bacterial cell.Thus, several stress conditions may affect the Y. ruckeri, in spite of some similarities, diverges from that system in E. coli. The changes in porin regulation can be adapted in Y. ruckeri in a species‐specific manner determined by its aquatic habitats. The study of a minor porin is of particular interest as it could provide the necessary level of porins at the special stress conditions.In summary, we report in this work that regulation of the porin balance in None declared.Figure S1. SDS‐PAGE analysis of porin samples, reflecting effects of growth temperature and osmolarity.Click here for additional data file.Figure S2. SDS‐PAGE analysis of porin samples, reflecting effects of oxygen availability.Click here for additional data file.


DNA binding protein controls plant transcription when oxygen is at a premium - During hypoxia, the plant transcription factor HRA1 counterbalances the upregulation of anaerobic gene expression triggered by a stabilized plant ethylene responsive factor. Arabidopsis thaliana ERF-VII factor RAP2.12 is maladaptive. This suggests that transcriptional activation under hypoxia that leads to anaerobic metabolism may need to be fine-tuned. However, it is presently unknown whether a counterbalance of RAP2.12 exists. Genome-wide transcriptome analyses identified an uncharacterized trihelix transcription factor gene, which we named HYPOXIA RESPONSE ATTENUATOR1 (HRA1), as highly up-regulated by hypoxia. HRA1 counteracts the induction of core low oxygen-responsive genes and transcriptional activation of hypoxia-responsive promoters by RAP2.12. By yeast-two-hybrid assays and chromatin immunoprecipitation we demonstrated that HRA1 interacts with the RAP2.12 protein but with only a few genomic DNA regions from hypoxia-regulated genes, indicating that HRA1 modulates RAP2.12 through protein–protein interaction. Comparison of the low oxygen response of tissues characterized by different levels of metabolic hypoxia  revealed that the antagonistic interplay between RAP2.12 and HRA1 enables a flexible response to fluctuating hypoxia and is of importance to stress survival. In Arabidopsis, an effective low oxygen-sensing response requires RAP2.12 stabilization followed by HRA1 induction to modulate the extent of the anaerobic response by negative feedback regulation of RAP2.12. This mechanism is crucial for plant survival under suboptimal oxygenation conditions. The discovery of the feedback loop regulating the oxygen-sensing mechanism in plants opens new perspectives for breeding flood-resistant crops.Transcriptional activation in response to hypoxia in plants is orchestrated by ethylene-responsive factor group VII (ERF-VII) transcription factors, which are stable during hypoxia but destabilized during normoxia through their targeting to the N-end rule pathway of selective proteolysis. Whereas the conditionally expressed ERF-VII genes enable effective flooding survival strategies in rice, the constitutive accumulation of N-end-rule–insensitive versions of the  HRA1 is transcriptionally activated upon ERF-VII RAP2.12 stabilization and encodes a trihelix DNA binding protein that functionally interacts with RAP2.12 to curtail its activity. In addition to its negative regulation of RAP2.12, HRA1 negatively regulates activation of its own promoter. This RAP2.12-HRA1 control unit allows plants to modulate the extent of the response to hypoxia, including anaerobic enzyme production, to levels that improve endurance of the stress. Our results emphasize the importance of a strategy that can counterbalance energy-inefficient survival responses.Respiratory metabolism in land plants requires oxygen availability to be able to generate ATP, which is essential for biosynthetic processes. Cellular hypoxia can be triggered as a consequence of environmental events (mainly floods), anatomical constraints (low tissue permeability to gases), or elevated cellular respiration, and it is unfavorable to growth due to the resultant decline in ATP. The adaptation of plants to fluctuating oxygen levels inside tissues requires the dynamic regulation of mechanisms that ensure cell viability and ultimately organism survival, but only a few molecular components of this homeostatic network are known. Direct hypoxia-sensing entails the posttranslational stabilization of a subgroup of plant ethylene-responsive factor (ERF) transcription factors, which coordinate the expression of hypoxia-inducible genes. Turnover of these ERFs is determined by an oxygen-dependent pathway of proteasomal degradation. Here, we demonstrate that the hypoxia-inducible transcription factor gene  In higher plants, respiratory metabolism requires molecular oxygen as the terminal electron acceptor to generate ATP. Limited oxygen availability (hypoxia) can occur in plant cells due to floods, frosts, and excessive respiration, requiring physiological acclimation to constraints in ATP availability for growth and development Arabidopsis thaliana, the presence of the ERF-VII factor RAP2.12 in the nucleus is inversely correlated to cellular oxygen levels, due to an oxygen-dependent branch of the N-end rule pathway of targeted protein degradation Low oxygen responses are coordinately regulated in plants by ethylene-responsive factor group VII (ERF-VII) transcription factors (TFs), primary activators of anaerobic gene expression At3g10040), which we named HRA1 (HYPOXIA RESPONSE ATTENUATOR 1). Here, we present evidence that HRA1 encodes a transcriptional repressor that attenuates the anaerobic response induced by ERF-VIIs in a tissue-specific manner. We show that HRA1 imposes an additional level of negative regulation on RAP2.12, besides the ERF-VII's oxygen-dependent instability. RAP2.12 transcriptionally activates HRA1, which in turn binds RAP2.12 and restrains its function. Additionally, HRA1 interacts with its own promoter, limiting its activation by RAP2.12 through a negative feedback mechanism. Thus, transcriptional activation by RAP2.12 is controlled under normoxia by its N-end rule susceptibility and under oxygen deficiency by HRA1. The spatial and temporal regulation of both factors appears to be a key to modulation of transcriptional activity and survival of transient hypoxia.Among the genes that are induced by oxygen deficiency, those encoding known or putative TFs deserve special attention as candidate modulators of transcription under hypoxia. When hypoxia-responsive genes are compared across different plant species, a few TFs in addition to ERF-VIIs are consistently up-regulated by oxygen deprivation A. thaliana Columbia-0 genome encodes 30 genes belonging to the plant-specific family of trihelix TFs At3g10040 (HRA1) is the only trihelix family member up-regulated by oxygen deprivation  and wild type Arabidopsis seedlings (HRA1 significantly reduced the up-regulation of 30 out of the 49 (61%) core hypoxia-responsive genes ADH1 and PDC1. The inhibition of hypoxic transcript accumulation by HRA1 overexpression contrasted to the constitutive up-regulation of the hypoxia-responsive genes observed in 35S:HA:RAP2.12 transgenic seedlings, which constitutively accumulate RAP2.12 due to masking of its N-terminus from the N-end rule machinery HRA1 expression  reporter fusion demonstrated that the HRA1 protein localized to the cell nucleus , consisteedlings . Overexppression , suggesteedlings . It was HRA1 expression affect plant performance under submergence-induced hypoxia. We compared two independent 35S:HRA1:FLAG transgenic genotypes (OE-HRA1#1 and #2) (hra1-1 and hra1-2)  OE-HRA1#1 plants   and two  hra1-2)  and S5 wte stage  and, in 1 plants . Underwa1 plants . This wahra1-1 mutant, and generally unable to recover during postsubmergence. Contrastingly, the fully expanded and mature leaves of OE-HRA1#1 plants were more sensitive to dark submergence than the wild type, but the shoot apical meristem performance was less damaged  reporter. As RrLuc activity was unaffected by concurrent transfection of the 35S:HRA1 effector, we conclude that HRA1 expression does not affect RAP2.12 stability, at least in isolated leaf protoplasts  reporter confirmed that basal HRA1 promoter activity, detectable under normal growth conditions, was restricted to the shoot apical region and leaf vasculature in aboveground tissues   using seedlings deprived of oxygen for 2 h, in the same manner as the transcriptome analysis. To identify chromatin bound by HRA1-FLAG, ciations , correspciations . We then1g01040) . The sma1 itself  were con1 plants . This le1–123) was sufficient for interaction   and the eraction , while ieraction . The inttoplasts .in planta; We also tested HRA1 interaction with the other four Arabidopsis ERF-VII factors in the yeast-two-hybrid system . Interesluciferase (PpLuc) reporter fusion to measure the activity of the RAP2.12-responsive PDC1 promoter (−911 to −1 relative to the start codon) in transiently transfected Arabidopsis mesophyll protoplasts. We confirmed that 14–35835S:RAP2.12 effector plasmid DNA enhanced the luciferase activity of promPDC1:PpLuc  was transactivated in a dosage-dependent manner by RAP2.12 and repressed by HRA1 itself in mesophyll protoplasts . The absence of candidate transcriptional repressors among HRA1 targets , along with HRA1's ability to restrain anaerobic promoter activation even after ablation of its DNA binding domain , either at the transcriptional or post-translational level of regulation  was used as the wild type ecotype. hra1-1  and hra1-2  T-DNA mutants were obtained from the European Arabidopsis Stock Center (uNASC) and the Arabidopsis Biological Resource Center, respectively. Mutants were genotyped using standard nonquantitative PCR on genomic DNA, using primers listed in −2 s−1 light intensity). Experiments in sterile conditions were performed with 4-d-old seedlings grown in liquid MS medium  under continuous shaking conditions, or with 2- and 3-wk-old plants grown vertically on solid MS medium . For the DNA microarray, chromatin immunopurification, and ADH assay experiments, sterilized seeds were grown for 7 d on solid MS medium in vertical orientation in a growth chamber  under a long day cycle , at 23°C, before treatments Seeds were sown in a moist mixture of soil∶perlite∶sand mixture 3∶1∶1, stratified at 4°C in the dark for 48 h and germinated at 23°C day/18°C night under a neutral day cycle  or old leaves (10th to 12th leaves) from five plants. Three biological replicates were used, and the experiment was repeated two times with comparable results. Gene expression data are mean ± s.d.50), standard error, and the 95% confidence interval were determined using the 9-d recovery time point exactly as described previously In an independent submergence survival assessment system Additional abiotic stress treatments were carried out on liquid-grown 4-d-old seedlings. The following conditions were used: 2 h at 4°C (cold stress); pinching of the seedlings with 10 consecutive pin pricks, 1 h before sampling ; 3 h in the presence of 150 mM sodium chloride ; 3 h in the presence of 5 mM hydrogen peroxide (oxidative stress); 3 h in the presence of 100 mM mannitol (osmotic stress); 3-h-long desiccation under laminar air flux (dehydration stress); 90 min at 38°C (heat stress). Control plants were maintained at 23°C with continuous shaking.Coding and upstream regulatory sequences were amplified from appropriate Arabidopsis cDNA or genomic DNA templates using Phusion High Fidelity DNA-polymerase (New England Biolabs). Fusion sequences were generated by overlapping PCR. Whenever the GATEWAY cloning system (Life Technologies) was exploited, sequences were cloned into pENTR/D-TOPO and the resulting entry vectors were recombined into destination vectors using the LR reaction mix II (Life Technologies). A list of plasmid constructs generated in this study and primers used for cloning can be found in HRA1 in the OE-HRA1#1 and OE-HRA1#2 transgenics, named 35S-HRA1-FLAG, was prepared by cloning the full-length HRA1 cDNA with gwHRA1_5′UTR_Fw and gwHRA1_3′UTR_Rv primers and recombination into the p35S:GATA-HF vector HRA1 cDNA linked to a C-terminal FLAG tag [NH2-Gly7-FLAG(AspTyrLysAsp4Lys)Gly3-His6-COOH]. A further 35S-HRA1 overexpression construct, used to obtain a third transgenic lacking any C-terminal epitope tag (OE-HRA1#3), was produced by amplification of HRA1 coding sequence with gwHRA1_Fw and gwHRA1_Rv primers and subsequent cloning in the pK7WG2 vector A construct for overexpression of 35S:RAP2.12:RrLuc construct exploited for RAP2.12:RrLuc DNA sequence into p2GW7; this sequence, in turn, was produced by overlapping PCR after separate amplification of the RAP2.12 full CDS, from a cDNA template, and Renilla reniformis luciferase CDS, from the 35S:RrLuc plasmid  35S:HRA1:YFPn and 14–358:YFPc35S:RAP2.1235S:YFPn and 35S:YFPc vectors were co-transfected into protoplasts. For each construct, 10 µg plasmid DNA was used. Fluorescence was observed with a Nikon ViCo microscope using filters for YFP , TRITC , and DAPI . Micrographs are representative of three independent experiments.−135S:RrLuc plasmid DNA promoter:PpLuc, 3 µg transformation−1) harboring the Photinus pyralis luciferase gene were co-transfected into protoplasts, along with the specified effector plasmid(s) encoding TFs . pAVA 393 35S:GFP construct, when needed. Samples were subsequently processed with the Dual-Luciferase Reporter Assay System (Promega), and luciferase activity was quantified with a Lumat LB 9507 luminometer (Bechtold Technologies). Each experiment was repeated three times and a representative replicate was shown. Relative luciferase intensity values (PpLuc/RrLuc) are presented as mean ± s.d. of four independent transfections.In promoter transactivation assays performed with protoplasts, 3 µg transformation35S:RAP2.12:RrLuc plasmid DNA was supplemented with increasing amounts of the 35S:HRA1 effector construct and transfected into a mesophyll protoplast suspension. In this case, 35S:PpLuc was used for normalization. Relative Renilla luciferase intensity values (RrLuc/PpLuc) are presented as mean ± s.d. of four independent transfections.RAP2.12:RrLuc protein stability from 35S:HRA1:GFP-His6-FLAG translational fusion construct (named 35S-HRA1-GFP), was generated by subcloning of a HRA1:GFP-His6 construct, obtained by recombining the full-length HRA1 cDNA in the pEarleyGate103 vector −1 4′, 6-diamidino-2-phenylindole (DAPI) for 10 min, washed in water for 10 min under vacuum, and observed with a Leica SP2  confocal microscope at the Microscopy Core Facility, Institute for Integrative Genome Biology, University of California, Riverside. GFP was viewed by excitation at 488 nm and emission at 500–600 nm. DAPI stained nuclei were visualized with a UV laser by excitation at 350 nm and emission at 399–600 nm.For HRA1 localization in plant tissue, a Histochemical GUS staining was carried out according to Ubiquitin10 (At4g05320) or β-TUB2 (At5g62690) as reference genes and relative expression values were calculated using the comparative Ct method HRA1-derived transcripts: Those named “sgHRA1_Endo” and “sgHRA1_Tot” were exploited to measure HRA1 mRNA levels in RNA extraction, removal of genomic DNA, cDNA synthesis, and RT-qPCR analyses were performed as described previously OE-HRA1#1, and OE-HRA1#2—were profiled under the two conditions. CEL files of two OE lines were processed as biological replicates along with two Col-0 replicates, using R and Bioconductor package. The NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) accession number for the generated dataset is GSE50679. The microarray dataset generated by http://www.ncbi.nlm.nih.gov/geo/) accession no. GSE29187. CEL files of aerobic-treated 5-wk-old rosette tissues of Col-0 and a transgenic overexpressing N-terminally HA-tagged RAP2.12 (RAP) line were processed together with the HRA1 microarray dataset . After computing the absent and present calls using the Affymetrix MAS 5.0 algorithm p<0.01 , in at least one comparison for each Affymetrix probe set. The DEGs were further analyzed using fuzzy k-means clustering with FANNY function. Clustering results were visualized using the Multi Expression Viewer (MEV) software (http://www.tm4.org/mev/) A. thaliana from http://geneontology.org (downloaded 17 Jan 2012).Total RNA was extracted from frozen tissue using the RNeasy Plant Kit  and quantified with a NanoDrop 1000 spectrophotometer . RNA quality was checked using the Agilent 2100 Bioanalyzer , and biotin-labeled cRNA was prepared with the GeneChip IVT Labeling Kit . Hybridizations against the Arabidopsis ATH1 Genome Array were performed by the Institute for Integrative Genome Biology (IIGB) Genomics Core Facility, University of California, Riverside. Transcriptomes of each of the three genotypes—namely, Col-0, g for 20 min at 4°C to obtain a nuclear pellet that was washed five times with 5 ml of M2 buffer  Triton X-100, 0.5 tablet Complete Protease Inhibitor Cocktail per 25 ml). The final wash was performed with 5 ml M3 buffer . The nuclear pellet was resuspended in 1 ml sonication buffer  and sonicated using a Bioruptor UCD-200  on ice, following the manufacturer's instruction. The sample was centrifuged twice at 15600 g for 10 min at 4°C and the supernatant was used for chromatin immunoprecipitation (ChIP) with 20 µl of EZview Red ANTI-FLAG® M2 Affinity Gel  following the manufacturer's instruction with IP buffer  Triton X-100, 0.05% (w/v) SDS). After the samples were reverse-crosslinked,, the DNA was purified using the Qiagen PCR purification kit. Library construction involving end repair, A-tailing, and ligation to an adapter was conducted using the End-It DNA End-Repair Kit , Klenow fragment  and Fast-Link DNA Ligation Kit (Epicentre). Two custom barcodes of four nucleotides were used for multiplexing (Col-0 [5′-GTAT-3′] and OE-HRA1#1 [5′-ACGT-3′]). Samples were submitted to the IIGB Genomics Core Facility, University of California, Riverside, for single-end sequencing with 100 cycles using the Illumina Hiseq2000 platform. Raw data in fastq file format were imported into R using the ShortRead package A. thaliana genome TAIR10 version (http://www.arabidopsis.org) using Bowtie (ver. 0.12.7) ChIP-seq libraries were prepared using the protocol of Soluble protein samples from total tissue extracts were separated by SDS-PAGE on 10% polyacrylamide Bis-Tris NuPAGE midigels (Life Technologies) and then transferred onto a polyvinylidene difluoride membrane by means of the Trans-Blot Turbo System (Bio-Rad). Detection of the HRP-conjugated secondary antibody  was performed with the LiteAblot Turbo Extra-Sensitive Chemiluminescent Substrate (EuroClone). Antibodies against PDC (product code AS10 691) and ADH (product code AS10 685) were purchased from Agrisera and antisera against FLAG (A8592) from Sigma-Aldrich. Equal loading of total protein samples was checked by amido black staining, as described in ADH-specific activity was measured as described previously Soluble carbohydrates analyzed in Saccharomyces cerevisiae strain MaV203 was transformed with the different combinations of bait (obtained after recombination of the inserts into pDEST32), prey (obtained after recombination of the inserts into pDEST22), and control vectors. Empty pDEST32 and pDEST22 were used as negative controls. Yeast transformation was performed according to the LiAc/SS carrier DNA/PEG method The ProQuest Two-Hybrid System (Life Technologies) was used. t test or one-way ANOVA, coupled with Tukey's posttest, for general comparisons, or Dunnet's posttest, for multiple comparisons with a reference sample, where appropriate. Mean values that were significantly different (p<0.05) from each other are marked with lower case letters or asterisks inside the figures. The statistical evaluation of the submergence survival, DNA microarray, and ChIP-seq experiments is described under the respective subsections.Significant variations between genotypes or treatments were evaluated statistically by Student's Full protein coding sequences of plant trihelix proteins were obtained from GenBank and aligned using ClustalW Figure S1HRA1 is the only low oxygen-responsive trihelix-coding gene in Arabidopsis. A comprehensive selection of low oxygen-related Genevestigator A. thaliana (63 experiments) was queried for the expression of all the trihelix genes encoded in the genome  and then filtered on At3g10040 (HRA1), according to the following criteria: |Fold change|≥2, p<0.05.(TIF)Click here for additional data file.Figure S2Oryza sativa) in a yeast-two-hybrid assay and bimolecular fluorescence complementation SH4-like2 (At1g31310) was identified as a putative target of HRA1 by ChIP-seq analysis (HRA1 is a member of the plant trihelix TF family. Sequences of the conserved trihelix domain (69 amino acids in GT-1) of Arabidopsis, rice, and poplar trihelix protein family members were used for phylogenetic tree reconstruction as described previously analysis . Low oxy(TIF)Click here for additional data file.Figure S3HRA1 is an early hypoxia-responsive gene. (A) HRA1 mRNA is kept constant at medium to low levels across Arabidopsis tissues at various plant developmental stages. Average gene expression data were retrieved from the Genevestigator webtool HRA1 mRNA accumulation is not sustained along the progression of the stress, unlike the case of the typical hypoxia marker gene ADH1. Data are mean ± s.d. (n = 3) from RT-qPCR analyses. Aerial and root tissues were collected from 3-wk-old plants grown on solid MS medium. Aerobic shoot samples were used as the reference. (C) The hypoxia-induced HRA1 mRNA is translated ubiquitously in the plant. The cartoons depict the absolute signal values of HRA1 transcript in translatomes (polysome-associated mRNA populations) isolated from different leaf and root cell types www.efp.ucr.edu.(TIF)Click here for additional data file.Figure S4OE-HRA1#1 and OE-HRA1#2 7-d-old seedlings (35S:HRA1:FLAG genotype), in aerobic conditions, after 2 h hypoxia and after subsequent recovery in normal atmosphere (2 h). (B) Schematic diagram of the HRA1 open reading frame . Triangles mark the positions of the two T-DNAs present in the insertional mutants hra1-1 (red triangle) and hra1-2 (orange triangle). Occurrence of tandem T-DNA repetitions could be inferred after sequencing of their flanking regions (see Tot and HRA1Endo RT-qPCR amplicons shown in HRA1 transcripts level in the two hra1 mutants and the wild type. The mRNA was isolated from 7-d-old seedlings exposed to control or hypoxic conditions (2 h hypoxia in the dark), and 30 cycles of amplification were performed, using primer set1 and 2 (see Molecular features of HRA1 overexpressing and mutant lines used in this study. (A) Western blot showing the accumulation of the HRA1-FLAG protein in nd 2 see . The ana(TIF)Click here for additional data file.Figure S5hra1-1 and hra1-2 mutant alleles. For the amplification of either hra1-1 or hra1-2 genomic DNA, the LBb1 primer, annealing on the left T-DNA border, was used in combination with an upstream (gwHRA1_580_Fw) or downstream (gwHRA1_Rv) primer Click here for additional data file.Figure S6hra1 mutant, and HRA1 overexpressing plants at the 10-leaf rosette stage . Slight differences in tolerance were detected between plants treated at this developmental age and older plants (compare to results HRA1 overexpression in air but not as a response to submergence. Petiole lengths were recorded at the 10-leaf rosette stage (L0) and after 3 d of treatments , and petiole elongation rates were calculated as (L3−L0)/3. Data are mean ± s.d. (n = 10).Altered HRA1 levels reduce plant ability to survive submergence. (A) Representative pictures (upper part) and median lethal time  of wild type, (TIF)Click here for additional data file.Figure S7n = 3). Data are mean ± s.d. One-way ANOVA on single time points was performed, and no statistically significant differences from the wild type were found.Soluble sugar contents in soil-grown plants at the late vegetative rosette stage (3.90) of development. The amount of soluble sugars  was assessed, at the beginning  and the end of the photoperiod , in the aerial tissues of plants of the same age as those evaluated in (TIF)Click here for additional data file.Figure S8HRA1 and ADH1 expression over midterm submergence. HRA1 responds to submergence-induced hypoxia and peaked within the first 6 h, in the wild type. ADH1 was measured as a downstream target of the treatment and equally produced a peak of expression within the first 6 h of treatment, although the reciprocal position of ADH1 and HRA1 expression maxima cannot be concluded from the present analysis. Data are mean ± s.d. (n = 3) of relative mRNA levels, normalized by setting as 1 the wild type expression value in air (time = 0) for both the old and the young leaves. Note the higher steady-state level mRNA of both hypoxia markers in the young leaves, either under aerated conditions and over the stress. Asterisks mark statistically significant differences, in the overexpressors (blue asterisks) or in the mutant (red asterisks), from the wild type mean expression value after separate one-way ANOVA for each time point (p<0.05). Differences in ADH1 expression in the hra1-1 mutant, which are present at 2 and 6 h submergence in the young leaves only and disappear towards the end of the treatment, are supposed to be correlated with the time span of HRA1 activity.Patterns of (TIF)Click here for additional data file.Figure S9hra1-1 and hra1-2 plants display a similar molecular phenotype. Comparison of ADH1, PDC1, and HRA1 mRNA levels in young leaf samples from 4-wk-old air-grown plants indicates that basal expression of hypoxia markers is higher than the wild type in either hra1-1  of relative mRNA levels, normalized by setting the wild type expression value as 1. Asterisks mark statistically significant differences from the wild type after one-way ANOVA .Young leaves of see also  or the i(TIF)Click here for additional data file.Figure S10OE-HRA1#1, and hra1-1 leaf samples using specific polyclonal antibodies, as indicated by the arrows. The same membrane was cut in two parts for separate hybridization with the different antisera, and each portion was eventually exposed for the convenient time for protein detection. We loaded 50 µg total proteins in each well, and equal loading of the samples was visualized through amido black staining of the PVDF membrane Images of the Western blot membranes magnified in (TIF)Click here for additional data file.Figure S11HRA1 promoter activity in 4-wk-old plants, visualized by GUS-reporter staining. During normal growth (“Control”), the promoter was active in the apical meristem zone, which includes the emerging leaves, and in leaf veins. After 12 h of submergence, the activity was markedly enhanced in the same tissues but also expanded to mesophyll and epidermal tissues. Scale bar, 1 cm.Profile of (TIF)Click here for additional data file.Figure S12OE-HRA1 plants are more strongly affected by carbon starvation and senesce earlier than the other genotypes. Plants were grown as in Excessive HRA1 accumulation is detrimental for survival to mild (2 d) or prolonged (9 d) continuous darkness. Mature leaves from (TIF)Click here for additional data file.Figure S13HRA1 overexpressing plants. (A) Representative pictures of wild type, HRA1 mutant, and overexpressing plants, grown at the end of the vegetative phase  in the overexpressing plants. No obvious differences from the wild type were recorded in the hra1-1 or hra1-2 mutants. (B) Rosette growth parameters and total leaf anthocyanin content in OE-HRA1 soil-grown plants at a stage corresponding to stage 3.90 in the wild type n = 4 for rosette FW and anthocyanin content; n = 6 for rosette size and leaf number). *p<0.05 statistically significant means from the wild type, after one-way ANOVA. (C) Above, phenotype of plants progressing through the reproductive phase  was assessed both in terms of days from germination and number of leaves to floral induction and consistently showed a delay in the transgenic plants if compared with the wild type, independent of the photoperiodic flowering pathway . Mean ± s.d. (n = 12), *p<0.05 statistically significant means from the wild type, after one-way ANOVA. Data describing plant yield (bottom right) are mean ± s.d. (n = 4). *p<0.05 statistically significant differences from the wild type after one-way ANOVA.Phenotypical characterization of (TIF)Click here for additional data file.Figure S14Identification of HRA1 binding sites by chromatin immunopurification and fragment sequencing (ChIP-seq). (A) Pie chart displaying the percentage of HRA1 binding peaks that map to defined domains of annotated genes. Upstream, peak maps 5′ of the predicted transcription start site; downstream, peak maps 3′ of the transcriptional unit of the gene; inside, peak maps within the annotated mRNA; overlapStart, peak overlaps with the predicted transcriptional start site of the gene; overlapEnd, peak overlaps with the end of the gene; includeFeature, peak includes the entire gene. (B) Venn diagram comparison of the 1,295 DEGs, identified by five comparisons in DNA microarray analysis, and the 146 putative targets of HRA1 binding, identified by ChIP-Seq. The overlap was seven genes. (C) Heatmap summarizing the expression values of the seven genes identified in both the DEG and ChIP-Seq datasets , as extr(TIF)Click here for additional data file.Figure S15194–431), lacking the predicted trihelix DNA binding domain, is able to interact with RAP2.12 (RAP2.121–177) in the yeast-two-hybrid assay and also when used as the prey construct.HRA1 interacts with RAP2.12. (A) Schematic view of the domains present on HRA1 and RAP2.12 proteins. (B) Yeast-two-hybrid between an N-terminal RAP2.12 fragment (bait) and HRA1 (prey), showing that amino acids 1–123 of RAP2.12, corresponding to a variable region preceding the ERF DNA binding domain (TIF)Click here for additional data file.Figure S16in vivo. (A) Survival of protoplasts incubated for 18 h was evaluated by calculating the proportion of alive, fluorescein diacetate-stained, cells n = 8), and *p<0.05 indicates statistically significant difference from the control transformation . Protoplasts were transfected with 3 µg individual plasmid DNA in every transformation. (B) Phenotype of soil-grown plants overexpressing HRA1 alone or HRA1 and the stable RAP2.1214–358 version at the same time.Interaction of HRA1 with RAP2.12 has an impact (TIF)Click here for additional data file.Table S1HRA1 according to the PlantTFDB database.List of low oxygen-inducible trihelix coding genes in four mono- and dicotyledonous plant species. In bold are genes belonging to the same orthologous group as (DOCX)Click here for additional data file.Table S2OE-HRA1 lines.DNA microarray analysis of (XLSX)Click here for additional data file.Table S3OE-HRA1#1. The p values indicate significance of fold changes based on Poisson distribution.Chromatin immunopurification and fragment sequencing analysis with (XLSX)Click here for additional data file.Table S4List of ChIP-PCR primers.(DOCX)Click here for additional data file.Table S5HRA1, endogenous HRA1 and ADH1 transcript levels in wild type, and OE-HRA1 and hra1 seedlings in normoxia or after 2 h hypoxia. Fold change values were calculated relatively to the wild type control. Data are means ± s.d. (n = 4). *p adj. <0.05 and **p adj. <0.01 were calculated separately for control and hypoxic conditions, after one-way-ANOVA.Comparison of total (DOCX)Click here for additional data file.Table S6Referenced list of the plasmid constructs produced in this study.(DOCX)Click here for additional data file.Table S7Oligonucleotide primers used for gene cloning and RT-PCR screening.(DOCX)Click here for additional data file.Table S8Oligonucleotide primers used for qPCR analyses.(DOCX)Click here for additional data file.


Multi-cell biochemical assays and single cell fluorescence measurements revealed that the elongation rate of Polymerase II (PolII) in eukaryotes varies largely across different cell types and genes. However, there is not yet a consensus whether intrinsic factors such as the position, local mobility or the engagement by an active molecular mechanism of a genetic locus could be the determinants of the observed heterogeneity. Here by employing high-speed 3D fluorescence nanoimaging techniques we resolve and track at the single cell level multiple, distinct regions of mRNA synthesis within the model system of a large transgene array. We demonstrate that these regions are active transcription sites that release mRNA molecules in the nucleoplasm. Using fluctuation spectroscopy and the phasor analysis approach we were able to extract the local PolII elongation rate at each site as a function of time. We measured a four-fold variation in the average elongation between identical copies of the same gene measured simultaneously within the same cell, demonstrating a correlation between local transcription kinetics and the movement of the transcription site. Together these observations demonstrate that local factors, such as chromatin local mobility and the microenvironment of the transcription site, are an important source of transcription kinetics variability. A growing evidence points to a significant variance characterizing the transcription processHowever, conclusive evidence able to identify whether factors such as genomic position, local mobility and microenvironment, are indeed the primary determinant of the observed variability in the kinetics of transcription, in particular elongation, is missing. In particular, the question whether increased local mobility of individual chromatin regions correlates to their transcriptional output is still the subject of active researchpetals surround the denser chromatin array.The reasons for the large variability affecting PolII transcription have been so far difficult to isolate, even in single cell experiments. To do so, it would be necessary to first isolate those extrinsic factors, such as cell to cell variability, that contribute a large part of this variance. This would allow assessing the role of the dynamic, spatial properties of the active site in regulating transcription kinetics. We propose here an original application of a fast 3D nanoimaging method, namely 3D orbital particle tracking, towards this goal. We sought to exploit the observation of a spatial structure in the organization of fluorescently labeled nascent mRNAs within the model system represented by the transgene array of U2OS 263 cells, a cell line allowing simultaneous labeling of both a gene and its transcriptAlthough transgene arrays are artificial systems, they have been highly valuable in the past to study the dynamics of large scale chromatin domains101112We developed a way to visualize the spatial and fluorescence trajectory of each of the petals at high resolution over space and time by means of a fluorescence nanoimaging method based on 3D orbital tracking in a 2-photon laser-scanning microscope. 3D orbital tracking is ideally suited to capture fluctuations in the fluorescence signal arising from the enzymatic activity of PolII within the reference system of the active fluorescently labeled array at ms temporal resolution. In combination with phasor analysis, a fit-free method that allows us to visualize and to compare PolII elongation rates within different petals, we measure a fourfold variation in the average elongation rates across identical copies of the same gene measured simultaneously within the same cell.Furthermore, the position of individual fluorescent particles within the orbit can be determined with nm-accuracy, depending exclusively on the measured signal to noise ratio17We found that the mRNA fluorescence signal, following induction in U2OS 263 cells, concentrates in multiple well-defined regions surrounding the denser core of the labeled transgene array , of apprIn order to capture the rapid dynamic processes occurring in each petal and to decouple them from the overall motion of the gene array we employed 3D orbital tracking to follow the labeled transgene array at millisecond temporal resolution and with nm precision in a 2-photon laser scanning microscope Each petal can be individually resolved by the microscope PSF, and its angular trajectory over time tracked continuously by fitting the carpet with a Gaussian function line by lineWe found that fluorescence trajectories are not temporally cross correlated (data not shown) among petals surrounding the same chromatin locus, supporting the notion that each petal on the surface of the denser chromatin array acts as an independent locus. These observations argue in favor of the possibility of measuring the transcriptional activity of distinct petals within the transgene as an interesting model of transcription from tandemly repeated genesd. A cross correlation signal indicates that molecules that leave one point can reach the other. The delay in such spatial cross-correlation reflects the average physical length of the path taken by a molecule. Starting from the fluorescence intensity carpet collected along the trajectory of the scanning beam, a new carpet can be therefore constructed: the intensity values in each column is indicative of diffusion or flow from that point to a point at a distance d along the orbit in the direction followed by the scanning beam22We validated this hypothesis by performing tracking-Pair Correlation Analysis, a fluorescence fluctuations microscopy method that provides information on the degree of molecular diffusion or flow between two points along the path of a fast scanning laser beamd = 12 pixels distance between the columns of the intensity carpet. A strong spatial cross-correlation is observed between the petals and the distal extremities of the orbit lobes, confirming that most of the mRNA molecules that reach further into the nucleoplasm originate from the transcribing petals. Barriers to diffusion, in the form of dark pCF carpet columns, are instead observed for displacements towards or away from the center of the array, confirming that mRNA synthesis occurs on the surface and that the chromatin density within the array impedes any mRNA diffusion or flow. Finally, mRNA nucleoplasmic diffusion is detected at the distal extremities of the trefoil lobe. Interestingly, a significant difference can be observed in the molecular flow of mRNA molecules that originate from each of the three petals. As illustrated in We use fluctuation correlation spectroscopy in combination with the phasor analysis to extract transcription kinetics, and in particular PolII elongation kinetics within the petals. As illustrated in This observation is in qualitative agreement with recent observations of the dynamics of fluorescently labeled mRNA on a single transcription site26To extract elongation kinetics we adopt here a novel strategy, based on the calculation of the phasor of the ACF. Phasor analysis, i.e. the representation of the harmonic components of a signal as a point on the unit circle in the complex plane, was successfully applied to study lifetime data2830Using this method, as is schematically illustrated in et alThe parameters for the simulations were taken from Darzacq N = 100) initiating, elongating and being released from an individual gene  displays a quite distinct average elongation value, pointing to the fact that PolII elongation on the same gene displays a large variability across different cellsALL in The phasor approach allows prompt comparison of elongation velocities across different cells, across different petals within the same cell, and even between temporally distinct portions of the same petal. E) PolII elongation rate can change by more than a factor of four between the slowest and fastest petal, while the variance of elongation rates measured within a petal can span over an order of magnitude.Since an individual array gives rise to multiple petals, the tracking-phasor analysis allows comparing the transcription kinetics of multiple, identical genetic loci within the same cell and at the same time. Furthermore, our approach allows measuring the temporal evolution of PolII elongation within an individual petal over time. Since phasor analysis divides the carpet in periods, corresponding to a distinct time, it is possible to observe the evolution of PolII elongation rate along a specific petal. The capability to perform observations on a single locus as a function of time with high spatial and temporal resolution prompted us to address the longstanding question whether a relationship exists between transcriptional activity and the physical movements of the inducted genemax measured from the saturation value of the MSD is a common indicator of chromatin mobilityIn the polar coordinates reference system centered on the transgene array, the motion of the petals is predominantly angular, as can be observed in y = αAt were performed for each MSD curve in each of the two regimes (0–5 s) and (10–100 s). In the first regime the exponent α was found to vary between a minimum of 0.31 and a maximum of 1.43 and in the second regime between a minimum of 0.09 and a maximum of 0.46.Power Law fits, in the form Simulations of chromatin dynamicsThe MSD analysis allows establishing a direct correlation between the overall mobility of individual petals and the elongation rate measured using the phasor analysis.max as the extrapolation of the power law behavior observed in the 10–100 s regime at t = 512 s. Although strictly speaking this is not a radius of constraint, since no constrained motion is ultimately observed for most petals, Rmax nonetheless provides an indication of the spatial range that can be explored by the petals on a timescale of approximately 10 minutes. The average Rmax = (5/2 * MSD)0.5 extrapolated from the intermediate regime to τ = 512 s) is about 210 nm, and the petals that display a lower overall mobility  appear to be elongating at a lower rate than the more mobile ones, as illustrated by the scatter plot reported in R = 0.52, p-value = 0.008).For most of the petals no obvious saturation of the MSD curve was observed at longer times, the most likely explanation being slow rotations of the transgene array itself on a timescale longer than a few hundred seconds. We therefore arbitrarily define Rmax analysis provides an indication for limiting long time scales, the cross-correlation indicates that directional movements occur with a specific delay with respect to maxima of transcriptional activity. This result can be reconciled only with the existence of a molecular mechanism driving the displacement of the active transcription site, possibly in a spring and ratchet mechanismFurther light can be shed on whether an active mechanism is indeed responsible for the motion of the transcribing petals. By cross-correlating the fluorescence intensity of each petal at each time to its angular displacement we found that changes in transcript output correlate to movements of the active transcription site. The diagrams in This is consistent with the drastic reduction observed in the amplitude of the angular fluctuations upon energy starvation of the cells achieved using Sodium Azide and 2-DeoxyGlucose as illustrated in Here we have shown that we can follow continuously for up to hours and at an unprecedented ms temporal resolution both the transcriptional activity and spatial trajectory of identical active genes, simultaneously within the same cell. This is made possible by the peculiar, and previously underappreciated arrangement of active transcription sites in U2OS 263 cells: a corolla of active transcription regions surrounding the compact chromatin array.Furthermore, our experimental strategy provides a time range going from minutes, which is the timescale of gene bursting, to seconds where we can detect the addition of individual coat protein on mRNA molecules following PolII elongationInstead, we analyzed here the continuous time series using an original approach based on the use of the phasors of the intensity ACF function to fingerprint PolII elongation kinetics. The rates of elongation that we have obtained are in excellent agreement with elongation rates recently measured using biochemical assays. We also confirmed the previously reported large variability in elongation speeds, but this time in the same cell and at loci that are in close proximity (~1 μm). In particular, we observed, as reported in et alet alWe then asked the question whether there is any correlation between the observed variability of transcription kinetics and the local chromatin mobility, as reflected by the petal angular trajectories. Although investigations on the dynamics of fluorescently labeled loci date back to the 1997 work by Sedat and coworkersIn summary, we introduced a method that allowed monitoring at high spatial and temporal resolution both the kinetics and the physical displacement of multiple transcription sites simultaneously within a model cell line. As illustrated in Future experiments should compare the transcription kinetics of tandem repeats, which we explored here, to those of individual endogenous loci. In particular, genome editing techniques such as CRISPR/Cas9 will enable not only to fluorescently tag endogenous lociFurther experiments will be also necessary to elucidate the molecular link between transcription and the movements of the active loci, in order to determine whether these movements are originated by the direct action of PolII or by downstream steps in the transcription chain, such as splicing and mRNA 3′ end processing. The current implementation of orbital tracking that allowed us monitoring transcription kinetics in vivo, is amenable to be extended to other high temporal resolution studies of chromatin related processes that involve the in-vivo activity of an enzyme, such as monitoring binding and unbinding rates of transcription factors and other chromatin binding proteins, such as those involved in DNA damage response.Microscopy experiments were performed on a Zeiss Axiovert 135 microscope frame, modified for orbital particle tracking as discussed in the x, y, z position using the angular and axial modulation values. Dwell time per pixel was 256 μs, yielding a total time per orbit of 16.384 ms. Output powers below 2 mW at the sample plane were employed to minimize phototoxic effects and photo-damage to the living cells.During 3D particle tracking the laser beam has to be driven in a closed orbit around the particle. The voltage applied to the scanners was such as to yield a pixel size of 65 nm. A typical radius for the circular orbit was set at 10 pixels. The half-distance between the upper and lower orbit was set at 15 pixels, and we typically performed four orbits above and four orbits below the particle before calculating its U2OS 263 cells (a kind gift of Dr. Xavier Darzacq) were cultured in low glucose DMEM (Life Technologies) supplemented with 10% Tet Approved FBS (ClonTech) and 1% Pen/Strep. Cells were imaged under Leibovitz medium (Life Technologies) supplemented with 10% Tet approved FBS. Cells were transfected using X-treme gene solution (Roche), using an X-treme gene to DNA ratio of 8 μl: 2 μg according to the manufacturer's instructions and after testing for optimal transfection conditions. Cells were imaged 12 h to 48 h after transfection. Transcription was induced perfusing the cell wells with 10 μg/ml Doxycycline (Sigma) solution. pTet-On vector was purchased from ClonTech, MS2-EGFP plasmid was obtained from Addgene (plasmid 27121) and mCherry-Lac Repressor was also obtained from Addgene (plasmid 18985). ActinomycinD (Sigma) was used at a concentration of 10 μg/ml. Transfection was also performed using a Gibco Cell Porator Electroporation system, according to the manufacturer's instructions and with the following parameters , obtaining similar results to chemical transfection.F(t) a point on the unit circle in the complex plane is calculated using the S and G coordinates, according to Equation 1Δt of our measurement and by the time bin used to calculate the ACF, typically 256 s yielding N = 1024. The angle φ of each phasor component is given by: The phasor analysis of autocorrelation curves as the one displayed in 2829E.G. and P.A. designed the experiments. P.A. performed the experiments. P.A. and E.G. analyzed the data. P.A. and E.G. wrote the manuscript.Supplementary Materials and DiscussionSupplementary Movie 1


Co-ordinated with the entire pathway of aliphatic glucosinolate biosynthesis, distinct patterns of epigenetic marks are associated with the recruitment of the methionine chain-elongation pathway from leucine synthesis. Aliphatic glucosinolates (GLSs) are derived from chain-elongated methionine produced by an iterative three-step process, known to be evolutionarily recruited from leucine biosynthesis. The divergence of homologous genes between two pathways is mainly linked to the alterations in biochemical features. In this study, it was discovered that a distinct pattern of histone modifications is associated with and/or contributes to the divergence of the two pathways. In general, genes involved in leucine biosynthesis were robustly associated with H3k4me2 and H3K4me3. In contrast, despite the considerable abundances of H3K4me2 observed in some of genes involved in methionine chain elongation, H3K4me3 was completely missing. This H3K4m3-depleted pattern had no effect on gene transcription, whereas it seemingly co-evolved with the entire pathway of aliphatic GLS biosynthesis. The results reveal a novel association of the epigenetic marks with plant secondary metabolism, and may help to understand the recruitment of the methionine chain-elongation pathway from leucine biosynthesis. Brassica vegetables , once collectively referred to as mustard oil glucosides, have long been part of human life via conferring distinct flavour and aroma in getables . In the Arabidopsis  works in methionine chain elongation, while IPMDH2 and IPMDH3 are involved in leucine biosynthesis Heynh., ecotype Colombia (Col-0), was used in all the experiments. Seeds were surface-sterilized and germinated on half-strength Murashige and Skoog (1/2 MS) agar medium containing 1% sucrose, and grown in a growth chamber with a photoperiod of 16h light at 22 °C and 8h dark at 20 °C for 7 d. Seedlings were transferred to soil and grown under the same conditions.2, 1mM phenylmethylsulphonyl fluoride (PMSF), 5mM β-mercaptoethanol, 1 tablet of complete Protease Inhibitor Cocktail ] for 20min at 4 °C with gentle shaking. The suspension was filtered into a new 50ml conical tube through two layers of Miracloth , placed in a plastic funnel, and centrifuged at 4000rpm for 20min at 4 °C. The pellet was resuspended in 1ml of buffer B  and centrifuged at 14 000rpm for 10min. The pellet was resuspended in 650 μl of buffer C , placed on top of 650 μl of a buffer C cushion, and centrifuged at 14 000rpm for 1h at 4 °C. The nuclear pellet was resuspended in 500 μl of buffer D  to release chromatin.ChIP was performed as described previously, with minor modifications (–1 complete Protease Inhibitor Cocktail) and pre-cleared with Dynabeads Protein A (Invitrogen). The pre-cleared chromatin (10 μl) was saved as the chromatin input control sample. The remaining chromatin was incubated with 10 μl of antibodies  overnight at 4° C, and then captured by Dynabeads Protein A. After incubation, beads were washed five times in the following buffers: (i) low salt wash buffer ; (ii) high salt wash buffer ; (iii) LiCl buffer ; and (iv) TE buffer, twice . Chromatin was eluted from beads with 200 μl of elution buffer , treated with RNase for 2h at 3 7°C, deproteinized with proteinase K for 2h at 45 °C, and de-cross-linked at 65 °C for 8h. DNA was purified using MinElute PCR Purification Columns (Qiagen).Chromatin was sonicated into fragments with a length of 200–300bp and centrifuged at 14 000rpm for 5min at 4 °C. The supernatant was diluted 10-fold with dilution buffer . PCRs were performed in a 7500 Real-Time PCR machine (Applied Biosystems) according to the manufacturer’s instructions. To calculate the ChIP enrichment, input chromatin samples were defined as 100, and each ChIP sample was compared with its corresponding input. Data are presented as percentages of input DNA. All data were averages of three independent experiments. Primer sequences used for qPCR are listed in ACTIN7 (AT5G09810) was used as an internal standard to normalize the relative expression levels. Gene-specific primers were designed and are listed in Supplementary Table S2 at JXB online. All data are shown as the average of three independent experiments.Total RNA was extracted with TRIzol (Invitrogen Inc.) from the same 3-week-old rosette leaf samples that were used for the ChIP experiments. RNA was reverse transcribed with SuperScript III reverse transcriptase (Invitrogen). cDNA was analysed by qPCR. Arabidopsis genome (TAIR10) as previously described (The ChIP-Seq data sets for various histone modifications were obtained from the NCBI Short Read Archive (SRA) and aligned to the Arabidopsis data sets (Supplementary Table S1 at JXB online). The intensity of histone marks was counted using a 50bp sliding window around the TSS (± 1kb). As shown in Post-translational modifications of histone play crucial roles in maintaining normal transcription levels and patterns by directly or indirectly shaping the structural properties of the chromatin. To explore the distinct association with certain types of histone modification at the pathway level, occurrences of six chromatin marks representing the active chromatin state  and the repressed chromatin state  were examined and the results were compiled into two sets, with the first focusing on methionine chain elongation and the second on leucine biosynthesis. Data on histone modifications were obtained from publically available in silico analysis, the occurrences of two marks at each individual gene locus were experimentally checked by performing ChIP coupled with qPCR (ChIP–qPCR). As shown in To validate the patterns of the H3K4me2 and H3K4me3 marks revealed by Arabidopsis has shown that both marks are predominantly absent from pericentromeric heterochromatin regions  cells of CBCAT3 is functional in both pathways while BCAT4 is specialized in the methionine-chain elongation pathway  in representative secondary and primary metabolism pathways.Table S1. List of genome-wide histone modification data sets used in the study.Table S2. Primer sequences used in the study.Table S3. List of genes used in the analysis of Fig. S1.