TFIIB-related factor 2 (Brf2) is a member of the family of TFIIB-like core transcription factors. Brf2 recruits RNA polymerase (Pol) III to type III gene-external promoters, including the U6 spliceosomal RNA and selenocysteine tRNA genes. Found only in vertebrates, Brf2 has been linked to tumorigenesis but the underlying mechanisms remain elusive. We have solved crystal structures of a human Brf2-TBP complex bound to natural promoters, obtaining a detailed view of the molecular interactions occurring at Brf2-dependent Pol III promoters and highlighting the general structural and functional conservation of human Pol II and Pol III pre-initiation complexes. Surprisingly, our structural and functional studies unravel a Brf2 redox-sensing module capable of specifically regulating Pol III transcriptional output in living cells. Furthermore, we establish Brf2 as a central redox-sensing transcription factor involved in the oxidative stress pathway and provide a mechanistic model for Brf2 genetic activation in lung and breast cancer.  •Architectural conservation of TFIIB and TFIIB-related factors•Brf2 is a redox-sensing RNA polymerase III core transcription factor•Brf2 regulates cellular responses to oxidative stress•Brf2 amplification enables cancer cells to evade oxidative stress-induced apoptosis Direct redox-sensing by the RNA polymerase III core transcription factor Brf2 couples cellular responses to oxidative stress and regulation of transcriptional output, contributing to the ability of cancer cells to evade death induced by reactive oxygen species. In the eukaryotic nucleus, RNA polymerase (Pol) III transcribes genes encoding essential RNAs, including tRNAs and the 5S rRNA. The accurate recruitment of Pol III to its target genes and the formation of a transcriptionally active pre-initiation complex (PIC) occur through the association of Pol III with several specific transcription factors but TFIIIB is the key factor required for this process . TFIIIB c, a complex binding to the PSE  upstream of the transcriptional start site and a proximal sequence element (PSE) located further upstream . The TAT the PSE . Activel the PSE  and enco the PSE .Brf2 and Brf1 are part of a family of TFIIB-like transcription factors that share structural and functional features with the archetypal Pol II transcription factor TFIIB . These fPol III transcription is tightly regulated during the cell-cycle and its upregulation has been linked to tumorigenesis . RecentlWe obtained X-ray crystallographic structures of Brf2-TBP complexes bound to the U6 snRNA , RPPH1, and TRNAU1 promoters at resolutions of 1.9 Å, 2.2 Å, and 2.7 Å, respectively . In all The overall architecture of the Brf2-TBP/DNA complex  is reminWhether associated with Brf2 or TFIIB, TBP interacts with the TATA box in an undistinguishable manner, generating a virtually identical local bend in the DNA. However, as a result of specific Brf2/DNA interactions, the path of the DNA backbone deviates at the TATA flanking regions B. Modeli+3 and G′+4   at position +4, is notably enriched at Brf2-dependent promoters  A. The siromoters A. We invoters  at the vast majority of Brf2-dependent promoters, since a base-specific hydrogen bond is only possible with the amine group of a pyrimidine nucleobase C or purine nucleobase A at that position. Indeed, substitution of C−4 with a G or a T residue, or exchange of both adjacent pyrimidines with purines, resulted in reduced binding  folds into a TBP “anchor domain,” which structurally resembles Brf1 homology region II and binds TBP on its convex surface similarly to other TBP-associated factors  Figure B. RemarkA Brf2-specific short structured element (residues 357–363), the “molecular pin,” encompasses a conserved LPPC motif and lies at the ternary interface between the Brf2 C-terminal cyclin repeat, TBP, and the DNA C. This hA recurring theme in redox signaling by transcription factors is the presence of a reactive cysteine thiol that can cycle through reduced and oxidized states, “sensing” the redox environment of the cell . We notia of the cysteine residues and thus resulting in a sulfur atom that is more susceptible to oxidation , a potent inducer of oxidative stress. Remarkably, all the Brf2-dependent transcripts tested were severely reduced upon treatment, while leucine tRNA precursors (p-tRNA), a Brf1-dependent transcript, remained unchanged A. We theSeCys tRNA is essential for the synthesis of selenoproteins, the vast majority of which are involved in ROS detoxification and in the maintenance of cellular redox homeostasis . To testConversely, to test the functional consequences of decreasing Brf2 activity during oxidative stress in cancer cells, we reduced the levels of Brf2 via small interfering RNA (siRNA) in A549 cells challenged with t-BHP. A549 are epithelial human lung adenocarcinoma cells displaying high Brf2 expression and a generally increased resistance to t-BHP treatment when compared to MRC5 fibroblasts A and 7D.d Pol II core promoter element , which have been involved in open complex formation and/or its stabilization  was co-expressed with a TBP-core construct (169–339) and used for structural determination. Full-length Brf2  was cloned into pSBET and used for biochemical assays. The Quickchange Site-Directed Mutagenesis kit (Agilent Technologies) was used to generate the Brf2 point mutants.2, 2 mM DTT) in hanging drop plates. Following harvesting and cryo-cooling, diffraction data were collected at the Diamond Light Source (UK) and ESRF (France) synchrotrons. The structure was solved by molecular replacement, using TFIIB-TBP/DNA (Protein Data Bank: 1C9B) as the search model.Complexes were assembled at a final concentration of 60 μM and crystals were grown by mixing 1 μl each of complexes and crystallization solution .Pull-Down experiments were performed by incubation of bait and prey proteins in binding buffer and loaded onto a His SpinTrap columns . Following washing and elution, samples were analyzed by SDS-PAGE.Full-length Brf2 samples were digested with trypsin and infused into an LTQ Velos Orbitrap mass spectrometer (Thermo Fisher Scientific) to characterize cysteine oxidation states.Binding of 5′-Alexa488-labeled oligonucleotides were monitored at different Brf2-TBP concentrations by fluorescence anisotropy at 25°C on a POLARstar Omega plate reader (BMG Labtech).Immunopurification from cells lysed in RIPA buffer were carried out using a chip-grade Brf2 antibody  covalently coupled to epoxy-magnetic beads (Life Technologies), according to the manufacturer’s protocol.Cells were transfected with 1.5 μg of pCDNA3.1 (empty vector control), Brf2WT-pCDNA3.1, Brf2C361A-pCDNA3.1, or Brf2C361D-pCDNA3.1 DNA in a 6-well plate format, with Lipofectamine 2000 according to manufacturer’s instructions. In For Brf2 knockdown, cells were transfected with siGENOME Human Brf2 siRNA  in Total RNA was extracted from treated cells with TRIzol reagent (Life Technologies) according to the manufacturer’s instructions. SeCys p-tRNA was quantified using the relative standard curve method and the 5S rRNA as an endogenous control. SeCys m-tRNA was monitored using a previously published protocol  with minCell viability and apoptosis were assessed by flow cytometry using annexin V and propidium iodide staining.c-binding-deficient Brf2 mutants and performed EMSAs in presence of SNAPc. A.V. designed and supervised research and wrote the manuscript with contributions from all authors.J.G. carried out purification of protein/nucleic acid complexes, performed biochemical experiments, including EMSAs, fluorescence polarization and IPs, prepared Brf2-TBP/DNA crystals, collected crystallographic data, and solved the Brf2-TBP/DNA structures. K.S. originally cloned Brf2, carried out cellular work, and performed qRT-PCR and FACS analysis. J.G. and K.S. generated Brf2 mutants. N.G. preliminary expressed and purified Brf2. M.W. and A.J.T. performed mass spectrometry analysis. P.C., O.D., and N.H. generated SNAP


MaDEAR1 expression was repressed by ethylene, with reduced levels of histone H3 and H4 acetylation at its regulatory regions during fruit ripening. In addition, MaDEAR1 promoter activity was also suppressed in response to ethylene treatment. More importantly, MaDEAR1 directly binds to the DRE/CRT motifs in promoters of several cell wall-modifying genes including MaEXP1/3, MaPG1, MaXTH10, MaPL3, and MaPME3 associated with fruit softening during ripening and represses their activities. These data suggest that MaDEAR1 acts as a transcriptional repressor of cell wall-modifying genes, and may be negatively involved in ethylene-mediated ripening of banana fruit. Our findings provide new insights into the involvement of DREB TFs in the regulation of fruit ripening.Ethylene plays an essential role in many biological processes including fruit ripening via modulation of ethylene signaling pathway. Ethylene Response Factors (ERFs) are key transcription factors (TFs) involved in ethylene perception and are divided into AP2, RAV, ERF, and DREB sub-families. Although a number of studies have implicated the involvement of DREB sub-family genes in stress responses, little is known about their roles in fruit ripening. In this study, we identified a DREB TF with a EAR motif, designated as MaDEAR1, which is a nucleus-localized transcriptional repressor. Expression analysis indicated that  Arabidopsis, which reveals a linear transduction pathway with the transduction of ethylene signal from receptors to dedicated transcription factors (TFs)  . The lasrs (TFs) .Arabidopsis cDNA  were induced by drought, salt and heat , ERF and dehydration-responsive element-binding protein (DREB), according to the number and similarity of the AP2/ERF domains . DREB TFsis cDNA . Extensiand heat . By contpression , whereassalinity . Except salinity . These Esalinity , RAP2.1 salinity , and GhDsalinity . DespiteMaACS1 and MaACO1 promoters to suppress their activities whereas MaERF9 activates MaACO1 promoter activity , polygalacturonase (MaPG1), xyloglucan endotransglycosylase/hydrolase (MaXTH10), pectate lyase (MaPL3), and pectin methylesterase (MaPME3). Our results suggest that MaDEAR1 may be acting as a negative regulator of cell wall-modifying genes, unraveling new information on EAR motif-containing DREB TFs in relation to fruit ripening.In this study, we identified a DREB TF with EAR motif, designated as MaDEAR1, which is a nucleus-localized transcriptional repressor. MaDEAR1 was ethylene- and ripening-inhibited, with reduced levels of histone H3 and H4 acetylation at its regulatory regions during fruit ripening. More importantly, MaDEAR1 binds to and represses promoters of several cell wall-modifying genes associated with fruit softening, including expansins  fruit at the 70–80% plump stage were obtained from a local commercial plantation near Guangzhou, southern China. Harvested fruit were separated into fingers, and fruit of uniform weight, shape, and maturity with no visual defects were selected, rinsed in tap water, and then air-dried before treatments were applied. The post-harvest treatments include a control , ethylene-induced ripening , and 1-methylcyclopropene (1-MCP)-delayed ripening , as described previously by Pre-climacteric banana (Nicotiana benthamiana) plants were grown under a 16-h light (25°C) and 8-h dark (22°C) photoperiod. Four- to six-week-old tobacco plants were used for transient assays.Tobacco bright yellow 2 (BY-2) suspension cells were cultured and prepared as described by DREB gene containing an EAR motif, with complete start and stop codons, termed MaDEAR1 , was identified and selected from banana whole-genome sequence. This segment was cloned and sequenced. Alignments were carried out on ClustalX (version 1.83) and GeneDoc software, and a phylogenetic tree was constructed using the Neighbor–Joining method in the MEGA5 program.Frozen banana fruit pulp were ground in liquid nitrogen using a mortar and pestle. Total RNA was extracted using the hot borate method described by Supplementary Table S1. qRT-PCR was carried out on a Bio-Rad CFX96 Real-Time PCR System using the SYBR®Green PCR Supermix Kit (Bio-Rad Laboratories) following the manufacturer’s instructions. MaRPS2  was selected as a reference gene according to our previous study on the selection of reliable reference genes under different experimental conditions , then the fusion construct and positive control GFP vector were electroporated into the Agrobacterium tumefaciens strain GV3101 using Gene PulserXcellTM Electroporation Systems . The primers for construct development are listed in Supplementary Table S1. The Agrobacterium harboring MaDEAR1-GFP or the positive control was inoculated for 16 h at 28°C. Cells were pelleted, resuspended at OD600 = 0.1 in infiltration buffer , incubated for 4 h at room temperature, then was infiltrated into the abaxial side of 4- to 6-week-old tobacco leaves using a 1-mL needleless syringe as described previously by The coding sequences of MaDEAR1, and genes including MaEXP1/3, MaPG1, MaXTH10, MaPL3, and MaPME3 involved in cell wall loosening of banana fruit associated with softening (Supplementary Table S1). After sequencing, conserved cis-element motifs of promoters were predicted using Plant-CARE1 databases.Genomic DNA was extracted from banana leaves using the DNeasy Plant Mini Kit (Qiagen). The promoters of oftening , were isSupplementary Table S1. The PCR product was inserted into the pGreenII 0800-LUC double reporter vector (KpnI and NcoI sites to fuse it with the Firefly luciferase (LUC) reporter gene (MaDEAR1 pro-LUC). A Renilla luciferase (REN) under the control of the 35S promoter at the same vector was used as an internal control. The construct CaMV35S-REN/MaDEAR1 pro-LUC (∼20 μg) was transformed into tobacco BY-2 protoplasts (∼2 × 104) by polyethylene glycol (PEG) methods as described previously . The suspensions were transferred to microfuge tubes and centrifuged at 12,000 g for 10 min. The pellets were suspended in 1.7 M sucrose, 10 mM Tris-HCl (pH 8.0), 2 mM MgCl2, 0.15% Triton X-100, 5 mM β-mercaptoethanol, 0.1 mM PMSF, and protease inhibitors and centrifuged through a layer of the same buffer in microfuge tubes. The chromatin extracts were lysed in a buffer containing 50 mM Tris-HCl, pH 8, 10 mM EDTA, 1% SDS, and protease inhibitors, and were sheared to an average length of 500 bp by sonication at 4°C using the Sonics VCX800 apparatus followed by centrifugation. The supernatants were diluted 10-fold with 1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.0), and 167 mM NaCl. A 1 mL aliquot of the dilution was immunoprecipitated with specific antibodies of anti-acetyl-histone H3 (anti-H3ac) and anti-acetyl-histone H4 (anti-H4ac) (Millipore), while immunoglobulin G (IgG) was used as a negative control. ChIP assays were repeated with three biological replicates. The DNA cross-linked to immunoprecipitated proteins and input DNA were detected by qRT-PCR. MaACT2 (GSMUA_Achr9G03170_001) was used as internal control since its histone acetylation level is stable during ripening (-ΔCt (=2-[Ct(IP)-Ct(Input)]). The primers used for ChIP-qPCR analysis are listed in Supplementary Table S1.Chromatin immunoprecipitation (ChIP) was performed as described earlier . Unripe ripening . The perMaDEAR1 was cloned into pGEX-4T-1 (Amersham Biosciences) to fuse in frame with GST and expressed in BM Rosetta (DE3) by induction with 1 mM of isopropyl-β-D-thiogalactopyramoside (IPTG) for 6 h at 30°C. The recombinant protein was purified with Glutathione Sepharose 4B . The EMSA was performed using the EMSA kit (Thermo) according to the manufacturer’s instructions. The probes containing the DRE/CRT (core sequence for A/GCCGAC) element (2 × 10-6 μmol) derived from MaEXP1/3, MaPG1, MaXTH10, MaPL3, and MaPME3 promoters were labeled with biotin using DNA 3′ End Biotinylation Kit (Thermo). The same unlabeled DNA fragment with 2 × 10-5 μmol, 2 × 10-4 μmol, or 2 × 10-3 μmol, respectively, was used as a competitor. After cross-linking, the membrane was detected by the chemiluminescence method according to the manufacturer’s protocol on a ChemiDocTM MP Imaging System (Bio-Rad). The primers used in protein expression and EMSA assays are listed in Supplementary Table S1.Supplementary Table S1. For transcriptional activity analysis of MaDEAR1, the coding sequence of MaDEAR1 was inserted into the constructed pBD vector driven by the 35S promoter as the effector, and the double reporter vector includes a native GAL4-LUC, and an internal control REN driven by 35S promoter, which was modified based on pGreenII 0800-LUC reporter vector  in figures. Least significant difference (LSD) at the 5% level was estimated using DPS software .DREB gene containing an EAR motif, designated as MaDEAR1 (M. acuminata DREB and EAR motif protein 1) , which was found down-regulated during fruit ripening, did attract our attention. MaDEAR1 encodes a protein of 184 amino acids, with calculated molecular weight of 20.24 kDa and pI value of 10.10. Analysis of deduced amino acid sequence of MaDEAR1 revealed a typical AP2/ERF domain of 58 amino acids with the conserved valine (V) and glutamic acid (E) at the 14th and 19th positions, respectively , which are considered to be essential sites for the binding of DREBs to the DRE cis-elements , suggesting that the MaDEAR1 might function as a transcriptional repressor. Phylogenetic analyses showed that DREB proteins can be classified into six sub-groups (A1–A6), in which MaDEAR1 together with AtRAP2.1, GmDREB2, GhDBP1, and MsDREB5 belong to A-5 sub-group . Collectively, these data suggest that MaDEAR1 is an A-5 sub-group member of the DREB family.Based on gene annotation, bioinformatics and RNA sequencing analyses , one fulelements . In addiin vivo, we fused the MaDEAR1 coding region in-frame with the N-terminal side of green fluorescent protein (GFP) under the control of CaMV 35S promoter. The MaDEAR1-GFP and GFP control plasmids were transiently expressed in tobacco leaves by Agrobacterium infiltration. While control GFP accumulated in both nucleus and cytoplasm, the MaDEAR1-GFP fusion protein was clearly localized in the nucleus . This suggests that MaDEAR1, like other reported DREB proteins, is a nuclear protein which is a typical feature of TFs.To examine the sub-cellular localization of MaDEAR1 in vivo, a dual luciferase assay was performed. The dual-luciferase reporter harbors five copies of the GAL4 DNA-binding element and CaMV 35S fused to the firefly luciferase (LUC) reporter, whereas a renilla luciferase (REN) reporter under the control of the 35S promoter was used as an internal control. Full-length MaDEAR1 was fused with GAL4 DNA-binding domain (GAL4-BD) as the effector, and the empty GAL4-BD (pBD-empty) was used as a negative control . As shown in Figure 2C, compared with the pBD-empty control, pBD-MaDEAR1 significantly repressed the expression of the LUC reporter, with approximately fivefold less LUC/REN value than the control. To further confirm whether the conserved EAR motif (DLNQAP) was important for the MaDEAR1-mediated repression, site-specific mutations were made to convert two conserved amino acids (DL) to NV . As expected, the transcriptional repression ability of MaDEAR1 was abolished when the EAR-motif was mutated . These results confirm that MaDEAR1 may act as a transcriptional repressor, and the EAR motif is important for its repression activity.To investigate whether MaDEAR1 possesses transcriptional repression activity MaDEAR1 during banana fruit ripening, we performed quantitative RT-PCR analysis using banana fruit in three different ripening behaviors caused by natural, ethylene-induced, and 1-MCP-delayed ripening treatments. As shown in Figure 3A, MaDEAR1 expression was repressed by ethylene, and its transcript level in natural, ethylene-induced or 1-MCP-delayed ripening was decreased following ethylene production appearance, revealing that MaDEAR1 expression was suppressed by ethylene and ripening.Our previous study showed that fruits in natural ripening group start ethylene production after storage at 22°C and 90% relative humidity for 15 days, which peaks around day-21, and declines thereafter. Ethylene-treated fruit ripened rapidly, with an ethylene peak appearing at day 3 following treatment. In contrast, 1-MCP treatment delayed ripening, with ethylene peaking at day 30 . To inveMaDEAR1 expression modulation, a 976 bp upstream sequence from the start codon of MaDEAR1 was isolated from the genome of M. acuminata using a genome-walking PCR method. Analysis of the promoter using the PLACE and Plant-CARE databases revealed a site for the ethylene-responsive element (ERE), ATTTCAAA, with one nucleotide change found in the promoter at –507 to –515 bp from the initiation codon (Supplementary Data Sheet S1), indicating that MaDEAR1 promoter might respond to ethylene. We then fused the MaDEAR1 promoter in front of LUC in the dual luciferase reporter vector, while the REN driven by the CaMV 35S promoter at the same vector was used as an internal control . The resultant vector was transiently expressed in tobacco BY2 protoplasts with or without ethrel treatment, and the luciferases were assayed thereafter. As shown in Figure 3B, after the treatment with ethrel, the promoter activity of MaDEAR1 was dramatically decreased, as evidenced by the much lower relative LUC/REN ratio compared that of the control, indicating that MaDEAR1 promoter activity was suppressed by ethylene.To better understand the mechanism(s) of MaDEAR1 during fruit ripening is associated with histone acetylation, we examine the histone acetylation levels of MaDEAR1 in fruit of unripe and ripe stages by ChIP-qPCR assays using antibodies such as anti-acetyl-histone H3 (H3ac) and anti-acetyl-histone H4 (H4ac). As shown in Figure 4, as a negative control, the enrichments of IgG in the promoter and coding region of MaDEAR1 were low. In addition, no significant difference of histone H3 or H4 acetylation levels of MaACT2 between unripe and ripe banana fruit was observed. On the contrary, the histone H3 acetylation (H3ac) levels of MaDEAR1 in region B and C as well as histone H4 acetylation (H4ac) levels of MaDEAR1 in region C were decreased in ripening bananas , which is consistent with its decreased level of expression during ripening . These findings, together with the observations of its gene expression and promoter activity, suggest that MaDEAR1 is suppressed by ethylene and ripening, and its decreased expression might be associated with reduced levels of histone H3 and H4 acetylation during ripening.Histone acetylation is a type of chromatin modification facilitating the gene expression, which is closely associated with gene activation. To assess whether the expression of cis-acting dehydration-responsive element/C-repeat (DRE/CRT) in the promoter of their target genes, including RD29A, RD17, COR15A, ERD10, KIN1, and COR6.6 , suggesting that these genes might be the targets of MaDEAR1.To understand the possible roles of MaDEAR1 in banana fruit ripening, we identified the potential targets of MaDEAR1 in relation to fruit ripening. Previous studies indicated that DREB proteins bind to the d COR6.6 . Softenid COR6.6 . Previouoftening , and we E. coli and purified . As shown in Figure 4B, MaDEAR1 can directly bind to the DNA probes containing the DRE-motif in MaEXP1/3, MaPG1, MaXTH10, MaPL3, and MaPME3 promoters, and the bindings were abolished by the addition of increasing amounts of unlabeled competitors with the same sequence. In addition, no parallel band shift was detected with only the GST tag . These results show that MaDEAR1 specifically binds to the DRE motif in the promoters of MaEXP1/3, MaPG1, MaXTH10, MaPL3, and MaPME3, demonstrating that these genes are likely direct targets of MaDEAR1.To test whether MaDEAR1 could directly bind to these promoters, electrophoretic mobility shift assay (EMSA) was performed. DNA fragments containing the DRE/CRT element in the region of these promoters were used as probe. Recombinant glutathione S-transferase (GST)-MaDEAR1 fusion proteins were expressed in MaEXP1/3, MaPG1, MaXTH10, MaPL3, and MaPME3 by MaDEAR1, we performed transient expression assays using the dual luciferase reporter system. The dual luciferase reporter plasmid harbor MaEXP1/3-, MaPG1-, MaXTH10-, MaPL3-, or MaPME3-promoter fused to LUC, and the REN driven by the CaMV 35S promoter, while an effector plasmid carried MaDEAR1 expressed under the control of the CaMV 35S promoter . As shown in Figure 6B, compared with the negative controls, tobacco leaves expressing MaDEAR1 showed significantly lower luciferase activity with the constructs harboring the LUC reporter gene driven by the MaEXP1/3-, MaPG1-, MaXTH10-, MaPL3-, or MaPME3-promoter, which is consistent with the finding that MaDEAR1 is a transcriptional repressor .To further understand the regulation of DREB gene, MaDEAR1, whose predicted protein possesses an APETALA2 (AP2) domain that binds to DREs and an EAR motif that is responsible for transcriptional repression , and thus belongs to A-5 group . Sub-cellular localization and transcriptional activation assays indicated that MaDEAR1 is nuclear-localized and possesses transcriptional repression activity , similar to GhDBP1 in cotton  under constitutive and stress-inducible promoters in transgenic wheat and barley caused dwarfism and decreased frost tolerance, supporting the notion that most DREB members in A-5 sub-group are negative regulators of stress tolerance . It is also important to note that the decline in MaDEAR1 expression showed a concomitant increase in ethylene production during fruit ripening. These findings suggest that MaDEAR1 is a transcriptional repressor associated with fruit ripening, which is consistent with its transcriptional repression activity reported here . Interestingly, we also found reduced levels of histone H3ac and H4ac in MaDEAR1 promoter in the ripening stage of banana fruit , which closely corresponds with its decreased expression during fruit ripening . Several reports have implicated histone acetylation as an important mechanism for controlling DREB gene expression. For example, treatment of maize with the HDAC inhibitor trichostatin A (TSA) under cold stress conditions selectively inhibited the induction of the cold-responsive gene ZmDREB1 through histone modification in the promoter region , suggesting its regulatory role in cell wall degradation during banana fruit ripening. Nuclear localization and promoter activity of MaDEAR1 in banana protoplasts will further substantiate these results. In addition to these fruit softening-associated genes, those involved in ethylene production and aroma formation such as MaACS1, MaACO1 and MaPDC are also associated with banana ripening (Fruit softening is one of the most important features that characterize the ripening process of fleshy climacteric fruits like bananas, and cell wall modification that occurs during the ripening process plays a critical role in the softening process . The prona fruit . It was na fruit , supportna fruit  and AdEIna fruit . Similar (MaEXPs . Our preMaEXP1/2 ,b. Also ripening . In thisripening , but wheripening , MaMADSsripening , MaNACs ripening , MaLBDs ripening ,b, and tMaDEAR1 were repressed by ethylene and ripening, and its expression is likely to be regulated by, at least in part, by histone modification. MaDEAR1 binds to and represses several cell wall-modifying genes, including MaEXP1/3, MaPG1, MaXTH10, MaPL3, and MaPME3. Taken together, our results report new insights into the mechanisms underpinning ethylene-mediated banana fruit ripening, of which MaDEAR1 may be playing a role via transcriptional repression of genes involved in cell wall modification and softening of fruit.The data reported here represent an EAR-motif-containing DREB TF, MaDEAR1, which was found to be a nuclear-localized transcriptional repressor. Expression and promoter activity of JC, JK, WL, PL, and XD designed the research. ZF, WS, YH, YX, and YY performed the experiments. ZF, JK, WL, PL, and JC wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The question of how cells re-establish gene expression states after cell division is still poorly understood. Genetic and molecular analyses have indicated that Trithorax group (TrxG) proteins are critical for the long-term maintenance of active gene expression states in many organisms. A generally accepted model suggests that TrxG proteins contribute to maintenance of transcription by protecting genes from inappropriate Polycomb group (PcG)-mediated silencing, instead of directly promoting transcription.Drosophila between two members of the TrxG, the histone methyltransferase ASH1 and the bromodomain and extraterminal family protein FSH. We investigated this interface at the genome level, uncovering a widespread co-localization of both proteins at promoters and PcG-bound intergenic elements. Our integrative analysis of chromatin maps and gene expression profiles revealed that the observed ASH1-FSH binding pattern at promoters is a hallmark of active genes. Inhibition of FSH-binding to chromatin resulted in global down-regulation of transcription. In addition, we found that genes displaying marks of robust PcG-mediated repression also have ASH1 and FSH bound to their promoters.Here we report a physical and functional interaction in Our data strongly favor a global coactivator function of ASH1 and FSH during transcription, as opposed to the notion that TrxG proteins impede inappropriate PcG-mediated silencing, but are dispensable elsewhere. Instead, our results suggest that PcG repression needs to overcome the transcription-promoting function of ASH1 and FSH in order to silence genes. Drosophila melanogaster but present in all higher eukaryotes, cooperate to sustain gene expression states by establishing and organizing information contained in the chromatin template. This epigenetic information layer assures the appropriate usage of the genetic blueprint, according to the developmental history of any given cell. TrxG proteins are required in order to maintain active states. This was discovered because of their essential trans-activating function for homeotic gene expression in flies [Gene expression programs specify diverse cellular identities during metazoan development, ultimately allowing cells to form tissues or organs. The ability to maintain expression states is critical, since inappropriate loss or gain of gene activity may lead to developmental anomalies, tissue dysfunction, or uncontrolled cell growth such as cancer. The Trithorax group (TrxG) proteins and the Polycomb group (PcG) proteins, originally identified in in flies . Despitein flies . Geneticash1) and trithorax (trx) represent histone modifying activities within the TrxG. Both HMTs have attracted particular attention, because they were considered to specifically counteract PcG silencing, rather than being coactivators of transcription [in-vitro, whereas association of SWI/SNF chromatin remodeling complexes incorporating TrxG subunits  is preferred [in-vivo relevance of this observation remains unclear, since the targeting of PRC1 seems to primarily depend on transcription factors and non-coding RNAs (ncRNAs) [The histone methyltransferases (HMTs) absent, small, or homeotic discs 1 (cription . Studiesreferred . However(ncRNAs) . In addi(ncRNAs) -8.Ultrabithorax (Ubx) in active and repressed conditions supports this notion: PRC1 and PRC2 binding patterns at the Ubx locus do not change between Ubx ON or OFF states, whereas ASH1 is only located downstream of the active Ubx promoter in embryonic discs [Ubx repression in the absence of ASH1 is accompanied by the formation of an ectopic H3K27me3 domain, a mark associated with mature PcG-mediated repression. Taken together, these data suggest that ASH1 actively prevents PcG silencing, however not by simply impairing PRC1/2 recruitment to chromatin.Alternative explanations on how ASH1 prevents PcG-mediated silencing propose that ASH1 blocks PRC-activity downstream to chromatin recruitment. A comparison of the HOX gene ic discs . In addiDrosophila tissue culture cells. Both investigations support the proposed anti-repressor function of ASH1, either by showing that fully activated PcG target genes miss typical marks of PcG-mediated repression and instead become embedded in broad ASH1 domains [ash1 mutant flies only identified few deregulated genes, again supporting the idea that ASH1 is only needed at a subset of developmental regulators, which need to be protected from ectopic PcG repression [The functional relationship between PcG proteins and ASH1 has been addressed on a genome-wide scale by two recent ChIP-chip studies in  domains , or by r domains ,11. In bpression .Ubx pattern formation in embryonic disc stages suggest ASH1 to act as a general transcription-promoting factor in analogy to what has been shown for TRX. Briefly, ncRNAs, originating from the bxd region upstream of the Ubx promoter, inhibit Ubx expression in cis presumably by transcriptional interference [bxd ncRNA promoters correlates with the onset of ncRNA expression [bxd ncRNA expression, thereby silencing Ubx indirectly [Ubx ON situation, ASH1 was found to be enriched in the coding region of Ubx and a related GFP reporter gene, but absent from bxd ncRNAs. Hence, the correlation between ASH1 binding and transcriptional activity at functionally diverse genes  is clearly indicative of a more general involvement in transcription.Curiously, studies analyzing rference . ASH1 lopression . This bedirectly . In the Ubx regulation, is female sterile (1) homeotic (fs(1)h) [ash1 and fs(1)h show strong genetic interactions with respect to Ubx activity. These interactions have contributed to the proposal of a 'trithorax gene set' by Shearn, following the earlier idea of a repressive gene cohort around Polycomb [fs(1)h gene products belong to the BET family of proteins, named after their characteristic arrangement of a tandem bromodomain and the extra-terminal domain. Mammalian BET proteins have been shown to serve as chromatin adapter proteins by binding to acetylated histone tails and to facilitate gene expression [C. elegans BET-1 mutants and Drosophila fs(1)h mutants, demonstrate that BET function is crucial for the establishment and maintenance of cell fates [Another TrxG member, explicitly implicated in (fs(1)h) ,17. AllePolycomb . Howeverpression . Lately,pression -23. How ll fates ,24,25.Drosophila cell line, we discovered a biochemical interaction between ASH1 and FSH, as previously suggested from genetic studies in fly mutants. In addition, we were able to show that both proteins extensively co-localize on chromatin, especially at sites bound by PcG complexes and gene promoters. The observed co-localization at promoters is a hallmark of active genes, but independent of gene type and function. These findings are at odds with the prevalent model that TrxG proteins function primarily as PcG-specific anti-repressors, but are dispensable in the absence of PcG-mediated repression. Our additional finding, that a set of PcG-repressed genes displays ASH1, FSH, and TRX-C signals comparable to their active counterparts, further questions the anti-repressor hypothesis. In agreement with a general transcription promoting function, we could show that BET inhibition in Drosophila tissue culture cells leads to an immediate and widespread gene repression, as well as delayed gene activation in reaction to environmental stimuli.By purifying ASH1 from a stable Drosophila cell line and identified copurified proteins by tandem mass spectrometry (MS/MS). Since no full-length cDNA was available to generate bait expression constructs, we cloned the ash1 coding sequence from S2 cells by assembling four subcloned RT-PCR products (A-D) tilling the ASH1 open reading frame (ORF) from start to stop codon . An ASH1 ChIP-chip profile has already been published by modENCODE [In order to screen for ASH1-interacting proteins, we purified tandem-tagged ASH1 from a stable n Figure . We verin Figure . Sanger n Figure . In spitodENCODE . As a meBased on our novel ASH1 cDNA we created a double-affinity tagged expression construct under the control of the inducible metallothionein promoter Figure . Employibona fide TrxG gene fs(1)h. Alternative splicing of the primary fs(1)h transcript results in two polypeptides: a short isoform denoted FSH-S, and a long isoform denoted FSH-L. The second is a C-terminal extension of its shorter relative. In order to approximately map the ASH1-FSH interaction, we co-expressed the ASH1 fragments A-D in combination with FSH-S in S2-DRSC cells. Immunoprecipitation of ASH1 A proved to be sufficient to pull-down FSH-S  at the very end of the protein. FSH-S matches BRD2, 3, and BRD4-S with respect to the missing tail region.The unique domain arrangement identifies FSH as members of the bromodomain and extraterminal (BET) family. BET proteins are conserved from humans to lower eukaryotes and have been shown to regulate gene expression by means of chromatin Figure . Their NDrosophila model system. Analogous to the situation in humans, the fruit fly proteome contains several bromodomain proteins, which have been implicated in transcriptional control. We approached this question by a phylogenetic analysis including human bromodomains of known drug ability and their Drosophila homologs. We found that FSH bromodomains are highly conserved with respect to BRD proteins , but well separated from bromodomains of other families  creating a hydrophobic acetyl lysine binding pocket , polyhomeotic (PH), posterior sex combs (PSC), and the C-terminal TaspaseI cleavage product of Trithorax (TRX-C), generated previously by us [fs(1)h knockdown experiments in S2-DRSC cells . In order to further fine-map ASH1, FSH binding relative to TSSs, we calculated binding profiles across non-overlapping 1 kb windows centered at TSSs. For both proteins we obtained unimodal coverage distributions with a maximum very close to the aligned TSS positions  to the ChIP-seq enrichments at promoters . This approach is related to k-means clustering. Our choice to solely focus on the promoter region was based on our previous finding that all of the TrxG/PcG factors mapped by our laboratory preferred binding close to the TSS. In addition, this special constrain allows for an intuitive correlation between ChIP signals and gene expression.Spf45 displays strong ASH1 and FSH ChIP-seq signals at the transcription start site. The RNA-seq and H3K4me3 ChIP-seq profiles reflect high expression as expected for a splicing factor.The fitted sGMM partitioned genes as follows: class 1 genes do not show enrichments for any of the considered TrxG/PcG proteins at their promoters Figure . Judged H15 TSSs is bound by ASH1, FSH, PRC1, and TRX-C. According to RNA-seq and H3K4me3 signals it is completely repressed. This observation makes sense, since H15 encodes for a transcription factor involved in cardioblast cell fate commitment.Class 3 genes are characterized by extraordinary high signals in the PC, PH, PSC, and TRX-C dimensions, but their promoters are also occupied by ASH1 and FSH, notably to the same extend as class 2 genes Figure . Approxi2 FPKM cut-off = 2.5) and inspected label frequencies within classes 2 and 3. The cut-off was chosen according to the bimodal distribution of expression values as shown in Figure To answer the question how accurately our classification predicts gene expression in a simplified binary manner (ON or OFF), we labeled genes according to their mRNA score as silent or transcribed . The genome-wide correlation coefficient of 0.7 indicates that the appearance of class 2 entities and transcription are strongly coupled. To visualize the spatial correlation we co-plotted smoothed density estimates for mRNA production and class 2 predictions along chromosomes Figure . As suggversus FSH enrichments at promoters visualized that down-regulated genes are bound by FSH in the absence of drug treatment , which is known to trigger the expression of genes coding for antimicrobial peptides (AMPs). Cells pretreated with JQ1 showed a slower accumulation of the AMP transcripts n Figure .Drosophila cells showed a fast and widespread down-regulation of transcription. In contrast to mammalian cell culture systems, Drosophila offers the opportunity to knockdown BET function by targeting a single gene. We therefore decided to compare the transcriptional response between FSH knockdown and small molecule inhibition. RNAi effectively depleted FSH isoforms from dsRNA-treated S2-DRSC cells within 3 days .ash1 and fs(1)h demonstrated a strong genetic interaction, whereas attempts to demonstrate a biochemical interaction remained unsuccessful so far. By purifying tagged ASH1 from a stable cell line, we discovered the missing physical connection between the two transcriptional activators. Indeed, the study by Chang et al. [Experiments using double mutants of the TrxG genes g et al.  attemptiUbx activation in the absence of PcG-mediated repression [Cyp12c1  that was developed to map human transcription factor (TF) binding sites in ChIP-seq data . Sequence specific TFs typically generate sharply restricted, Gaussian-like ChIP-seq signals. Therefore, the algorithm was ideally suited to detect ASH1, FSH signals at TSS. At the same time, it tends to break up more widely distributed signals, like the ones we described for intro-rich active genes, into closely spaced peak clusters. Hence, the absolute number of peaks reported in this study does not reflect the number of functional elements bound by ASH1 and FSH in a one-to-one correspondence.Ubx activity in ash1/E(z) double-mutant mitotic clones, located in- and outside of the endogenous Ubx expression domain [Ubx activity in embryonic tissues devoid of ASH1 protein already argues against such a strict requirement [trx/E(z) double-mutant clones show Ubx reactivation patterns similar to their ash1/E(z) counterparts [The second major argument against a general involvement of ASH1 in transcription was provided by the n domain . In our uirement ,29, and uirement . The latterparts .et al. [in-vivo biotinylation tagging [Drosophila embryo. Our current analysis of FSH chromatin occupancy now shows that PRC1 and FSH co-localize at repressed promoters and intergenic gene regulatory elements, although they are known to fulfill antagonistic functions. Mechanistically this might suggest that PRC1 silences genes by blocking the elongation-promoting functions of FSH, for instance via the pTEF-b pathway. In the light of these deliberations it is sensible that PcG-bound promoters do show elevated levels of promoter proximal pausing, as we have reported earlier [in-vitro transcription systems in conjunction with reconstituted PRC1 and FSH might shed light on the mechanistic details of this interaction.Our unsupervised clustering of genes according to their combined TrxG/PcG binding profiles identified a class of transcription units with concurrent high PRC1 and TrxG enrichments at the promoter. In contrast to the 'balanced' state introduced by Schwartz et al. , these g tagging . Unexpec earlier . Future Drosophila have shown that pausing represents a widespread gene regulatory strategy. Our data are in line with this possible mechanism, since displacement of FSH leads to a widespread down-regulation of transcription. A recent study has shown that BRD4 is also able to directly phosphorylate Serine 2 of the Pol II CTD [in-vitro transcription system, it has been shown that the human BRD2, BRD3 render nucleosomes marked by acetylation permissive to the passage of elongating RNA polymerase II, thereby bypassing FACT requirement [Our data provides evidence that FSH promotes the transcriptional activity of genes. How might this work at a mechanistic level? The human FSH-L homolog BRD4 proteins contain a conserved pTEF-b interacting domain in the very C-terminus, which is sufficient to directly recruit pTEF-b to target genes . Followil II CTD . A kinasl II CTD . Using auirement . Taken tuirement .Drosophila tissue culture cells provides evidence, that transcriptome homeostasis is heavily affected by small molecule inhibition of BET proteins. This observation corresponds to our FSH chromatin map, since we observed a high correlation between FSH localization and gene activity. Exactly the same results have recently been described with respect to BRD4 in human CD4+ T cells and ES cells. ChIP-seq revealed that BRD4 localizes to active promoters and enhancers, and that expression levels are strongly correlated with BRD signal intensity [Drosophila cells. The degree of correlation between BRD4 localization and transcriptional activity even encouraged the authors to predict that 'BRD4 could potentially be used as a genome-wide hallmark of active or poised genes'. Our gene clustering according to TrxG/PcG promoter binding supports this idea. Interestingly, our clustering into active and PcG-repressed genes parallels the human classification into active and poised genes in a sense that poised genes are occupied by BET proteins, but do not produce mRNA. Disruption of BRD4 chromatin binding by JQ1 in CD4+ T cells resulted in reduced expression of more than a thousand genes in CD4+ T cells [Our JQ1 treatment of ntensity . BRD4 pr T cells . Again, A recent study in erythroid cells shows, that BRD3 is recruited to target promoters by reading an acetyl mark on the transcription factor GATA1, instead of being primarily dependent on histone acetylation . The conDrosophila HOX genes cause homeotic transformations' [There is substantial evidence that TrxG proteins play global roles in transcription. In the future it will be interesting to find out, why this group of proteins genetically scores as being important for the maintenance of the active state of HOX genes, or in other words, what discriminates them from other factors in functional proximity to RNA polymerase. More mechanistic insight how TrxG complexes are connected to transcription machinery will be necessary to answer this question. It might indeed turn out that TrxG complexes 'are among those that have emerged as being important for maintenance for a relatively uninteresting reason e.g., because even relatively subtle changes in the expression of mations' .ash1 coding sequence was PCR-amplified from S2 cell derived cDNA in four overlapping parts (A-D) using the primers listed in Additional file ash1 coding sequence was reassembled from these primary clones by three directional subcloning steps, utilizing the restriction sites indicated in Figure ash1 mRNA in S2 cells  was inserted into the AccI/SapI-cut pMT/V5-His A vector backbone (Invitrogen). In addition, the sequence between EcoRV/HpaI encompassing MCS to SV40 polyA was replaced by the EcoRV/PmeI-excised fragment from pAFW . In a subcloning step, this fragment was previously modified by inserting the 8xHis-TEV coding sequence .In order to generate the ASH1 expression construct outlined in Figure −1) and passaged for 4 weeks in the presents of the selecting agent.In order to generate the stable cell line, S2-DRSC cells grown in Schneider's medium incl. 10% FCS were lipotransfected using pMT/FH-ASH1 HygRes according to standard Effectene protocols (Quiagen). Two days post-transfection transformants were selected by Hygromycin B addition to the growth medium (500 µg mL-1 Benzonase (Novagen) for 10 min and cleared by ultra-centrifugation .Expression of tandem tagged ASH1 was induced by adding 500 µM copper(II) sulfate to the growth medium for approximately 16 h. Afterwards cells were scraped in ice cold PBS, pelleted by low speed centrifugation (500 × g) and suspended in N buffer . Resuspended cells were lysed by adding Triton X-100 to 0.5% (vol./vol.) for 10 min. Next, nuclei were separated from cytoplasm by passing the lysate through a sucrose cushion (20% sucrose in N buffer) and washing nuclei twice in N buffer. In order to extract nuclear proteins, washed nuclei were transferred to lysis buffer  and sonicated in the Bioruptor (Diagenode) for five to 10 cycles . Afterwards nuclear extract was treated with 150 u mL-1 3xFLAG peptide (Sigma) dissolved in wash buffer. Eluted proteins were precipitated in 20% TCA (w./vol.) by high-speed centrifugation, washed using 10% TCA and ice-cold Acetone, and air-dried. Next, dried proteins were resuspended in reduction buffer , reduced and alkylated by subsequent addition of TCEP (100 mM) and Iodoacetamid (250 mM), respectively. Then proteins were digested in digestion buffer  by addition of 0.1 mg mL-1 Trypsin Gold (Promega). Tryptic peptides were separated using online reverse-phase chromatography and electrosprayed into a LTQ Orbitrap Velos tandem mass spectrometer (Thermo Scientific). MS spectra were recorded at 60,000 R and most intense peptide ions were selected for CID fragmentation and recording of product ion spectra. MS/MS spectra were matched against UniProtKB release 2011.08 using the Mascot search engine (Matrix Science), assuming the digestion enzyme Trypsin and up to one missed cleavage. Mascot was searched with a fragment ion mass tolerance of 0.5 Da and parent ion tolerance of 5 ppm. Iodoacetamide derivative of cysteine was specified in Mascot as a fixed modification. S-carbamoylmethylcysteine cyclization (N-terminus) of the n-terminus, deamidation of asparagine and glutamine and oxidation of methionine were specified in Mascot as variable modifications. Scaffold 3.3.1 (Proteome Software) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at >95.0% probability as specified by the Peptide Prophet algorithm [In order to capture 8xHis tagged ASH1 protein, nuclear extract was diluted in IMAC buffer A , adjusted to 300 mM NaCl, and loaded onto a Ni-Sepharose column  connected to a FPLC system . Afterwards non-specifically bound proteins were eliminated by washing the column with 4% IMAC buffer B  and bound proteins were eluted at 60% buffer B. Eluate fractions were probed for FLAG-tagged ASH1 by western blot, pooled accordingly, and adjusted to 100 mM NaCl by diluting pooled material in IEX buffer A . Next, material was loaded onto an anion exchange column  and washed using 10% IEX buffer B . Bound material was eluted by a linear gradient from 10 to 100% IEX buffer B over 10 column volumes again monitoring FLAG-tagged ASH1 by western blot. ASH1-containing eluate fractions were pooled, adjusted to 0.05% NP-40 and incubated overnight with 100 µL FLAGM2 agarose (Sigma). Next, agarose was washed several times using TBS, 0.05% NP-40 and transferred to Mobicol spin columns for eluting bound material by 200 µg mLlgorithm . Proteinlgorithm . For comThe FSH-S coding sequence was PCR-amplified from the Drosophila Gold Collection clone LD26482 (available through DGRC) using primers listed in Additional file in-vitro transcription were generated from pENTR/D-TOPO FSH-S using T7 recognition sequence flanked primers listed in Additional file PCR products serving as templates for Serum from rabbits immunogenized with FSH-specific antigens was kindly provided by Igor Dawid . The antigen used for rabbit ID166 corresponds to the XbaI/PstI fragment from clone e1.20 and the antigen injected into rabbit ID173 is derived from the EcoRI/HindIII fragment of clone e4.1, both described in , Basic P-1) cells were lysed at indicated time points using Trizol (Invitrogen). In order to quantify the transcriptional response of known LPS-inducible genes  total RNA was extracted, reverse transcribed , and TURBO DNase (Ambion) treated as outlined in the manufacturer's instructions. Quantification of cDNA was done by qPRC using SYBR Green chemistry and primers listed in Additional file ATPsyn-Cf6 the ΔΔct method including efficiency correction was used. Determination of ct values was done by calculating the second derivative maximum of the SYBR green signal.One hour before lipopolysaccharide (LPS) was added to the growth medium S2-DRSC cells were pretreated using either JQ1 or DMSO as described before. After LPS addition (5 µg mL6 cells) was diluted in binding buffer  and incubated with 20 µg Acetyl-Histone H4  peptides  in the presence or absence of 1 µM JQ1. Afterwards biotinylated peptides were precipitated using Streptavidin Dynabeads M-270 (Invitrogen) and washed several times in binding buffer. Peptide-bound protein was eluted by boiling Dynabeads in 1xLDS sample buffer (Invitrogen) and immunoblotted together with nuclear extracts as described for coIP assays. V5-tagged protein was detected using anti-V5 antibody (Invitrogen) at 1:5,000 in PBS, 0.1% Tween-20, 5% dry milk powder, followed by anti-mouse IgG-HRP  at 1:10,000 in PBS, 0.1% Tween-20, 5% dry milk powder, ECL substrate, and Hyperfilm .The V5-tagged FSH-S expression construct was created by transferring the FSH-S ORF from pENTR/D-TOPO to pcDNA6.2/V5-DEST using gateway cloning according to manufacturer's instructions (Invitrogen). Nuclear extract from HEK293 cells lipotransfected with pcDNA6.2/V5-FSH-S was prepared 2 days post-transfection using Active motif's Nuclear Extract Kit as outlined in the corresponding manual. For histone peptide pull-down assays 10 µL of nuclear extract  applying ligation independent cloning (LIC). The resulting plasmid was used to express 6xHis-tagged BD1-BD2 in BL21(DE3) E. coli. Cell lysate was prepared by sonicating cells in 20 mM Tris-HCl pH 8.0, 1 mM PMSF, 1mg/ml Lysozyme, 0.05% Triton X-100 for 15 min (30 s ON/30 s OFF) in a water bath sonicator followed by high speed centrifugation. BD1-BD2 was purified from cleared lysate by IMAC on Ni-NTA Superflow (Qiagen) deploying an imidazole step-gradient . BD1-BD2 containing eluate fractions were monitored by Coomassie-stained SDS-PAGE and concentrated in 25 mM HEPES pH 8.0, 150 mM Nacl using 10 NMWL centrifugal filter devices (Millipore). For BD1-BD2 pull-downs approximately 3 µg of purified protein was incubated with 35-40 µM of histone tail peptide in 25 mM HEPES pH 8.0, 150 mM NaCl in the presence or absence of 10 µM JQ1. After 1 h of incubation at 4°C biotinylated peptides were captured on streptavidin magnetic beads and washed three times in binding buffer. Precipitated proteins were eluted by boiling magnetic beads in 1x LDS buffer. Eluted proteins and input samples were immunoblotted using anti His-probe (H-3) HRP (Santa Cruz Biotechnology) and standard ECL reagents.7 S2-DRSC cells per ChIP. In order to assure data comparability across ChIP-seq experiments, all chromatin samples were taken from the same chromatin batch. This chromatin batch is identical to the one we used previously to generate ChIP-seq data for PC, PH, PSC, and TRX-C.Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) was essentially done as described in  using foASH1-bound chromatin was enriched using two antibody preparations provided by the lab of F. Sauer . Anti-ASH1-N recognizes an antigen located in the amino-terminus of ASH1, while the anti-ASH1-C specific antigen resides in the carboxy-terminal portion of the protein. Both polyclonal antibodies have been raised in rabbits. Since different dsRNA treatments of S2-DRSC cells did not induce detectable ASH1 knockdown (data not shown), we investigated antibody specificity by expressing ASH1-GFP fusion proteins in S2-DRSC cells and immunoblotting of cell lysates Figure . FSH-bou6 quality filtered reads per library (Illumina Chastity Filter), corresponding to a mean genome coverage of approximately 5x. Short reads (36 bp) were aligned to the Drosophila reference genome (BDGP Release 5) using Bowtie 0.12.7 and the following parameters: -n 2, -m 20, -k 1, --best [P value cut-off, 1x10-5 [Sequencing libraries were prepared from 10 ng of immunoprecipitated DNA using the ChIP-seq DNA Sample Prep Kit (Illumina), including size selection of preamplified fragments on agarose gels (200 bp +/- 30 bp). For quality control, the size distribution of the final libraries was assayed on the Agilent BioAnalyzer 2100 using High Sensitivity DNA microfluidic chips. Each library was sequenced for 36 cycles in a single-end run on the Genome Analyzer IIx (Illumina). Sequencing yielded approximately 20 × 10, --best . Since a, --best . Regions, 1x10-5 . Read co, 1x10-5 . Interva, 1x10-5 . The com, 1x10-5 . Metagen, 1x10-5 .For JQ1 treatment of exponentially growing S2-DRSC cells lyophilized JQ1 was dissolved in DMSO (10 mM) and added to the culture medium at a concentration of 10 µM. RNA extraction from S2-DRSC cells was done using Trizol reagent according to manufacturer's instruction for adherent cells (Invitrogen). Barcoded sequencing libraries were prepared from 4 µg of total RNA according to Illumina's TruSeq protocol including polyA-enrichment, mixed in equal proportions and sequenced together on a single HiSeq2000 lane (Illumina) using a 50-cycle single-end run.Drosophila gene models originating from Ensembl release 64 (default parameters). All reads uniquely mapping to gene models were counted by the HTSeq-count script  and the count statistic was forwarded to bioconductor package DESeq [Alignments of RNA-seq reads were generated using the splice-junction-aware aligner TopHat  and Drosge DESeq . Since tDemultiplexed RNA-seq reads and raw gene counts have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Super Series accession number GSE36450 .N = 13,254 unique TSS defined in the ensGenes table of the UCSC browser with the pysam python module. Subsequently, the relative frequencies i p= i/m Xof ChIP counts i Xon TSS i were normalized by a rescaling of the library size m to maximize the number of TSS where the relative ChIP frequency was identical to that of the corresponding value i q= i/n Yin the input experiment. The symbol i Ydenotes the read count in interval i in the input experiment and n the input library size. For each TSS and ChIP experiment, a binomial likelihood ratio test was computed testing the null hypothesis i = qi pversus the alternative i ≠ qip, and the sum over all TSS of the resulting P values was maximized numerically over m. Normalized enrichments were then computed as i/qip. Data normalization and enrichments were computed in R.ChIP-seq and input reads were counted on 1kb windows +/- 500 bp around L1-penalty favoring sparse solutions [The sparse Gaussian mixture model (sGMM) is a probabilistic mixture model of multivariate Gaussian distributions, in which the covariance matrices are estimated under an olutions . The modi ∈ Rn xbe a vector of ChIP-seq enrichments from different antibodies in TSS window i and i; µ, Σ) ϕ(xdenote the density of a multivariate Gaussian with mean μ and covariance Σ. An sGMM with m mixture components (classes) is defined by the likelihood functionLet k-1||1 is the L1-norm of the inverse covariance matrix, and where ρ ≥ 0 defines the penalization strength, ||Σkπ, kμ, and Σk were estimated iteratively by the expectation maximization (EM) algorithm, where in each step the inverse covariance matrices Σk-1 are computed from the weighted empirical covariances i xbelonging to class k, and N is the number of TSS. For ρ = 0, the sparse GMM is equivalent to a regular Gaussian mixture model with full covariance matrices, while for large values of ρ one enforces a GMM with diagonal covariance matrices, similar to the k-means algorithm. Clustering of the TSS is achieved by the Bayes estimate assigning i xto the class k that maximizes kiγ. This procedure also allows for predicting the classes of new TSS by computing their class probabilities according to Eq. (1).The model parameters lgorithm ,61. Hereρ = 0.5, the model yielded three distinct classes with diagonal covariance matrices, except for a cluster of correlated high PRC1 enrichments. These classes overlapped 94% with the results of a k-means clustering, confirming the robustness of the sGMM estimation. TSS clusterings were computed in R.A three-component sGMM was fitted to normalized ChIP enrichment values as stated above. The unpenalized sGMM had a tendency to overfit the third component into the shoulder of the cluster of highly enriched Ash1/Fsh TSS, instead of separating highly enriched PRC1 TSS. Under a penalization of aa: amino acid; AMP: antimicrobial peptide; BET: bromodomain and extraterminal proteins; BX-C: bithorax complex; cDNA: complementary DNA; ChIP-chip: chromatin immunoprecipitation followed by microarray analysis; ChIP-qPCR: chromatin immunoprecipitation followed by quantitative polymerase chain reaction; ChIP-seq: chromatin immunoprecipitation followed by next-generation sequencing analysis; CTM: C-terminal motive; DGRC: Drosophila Genomics Resource Center; DRSC: Drosophila RNAi Screening Center, Havard Medical School; ES: embryonic stem; FH-ASH1: 3xFLAG, 8xHis-tagged ASH1 protein; FLAG-AC: FLAG affinity chromatography; HMT: histone methyltransferase; IEX: ion exchange chromatography; IMAC: immobilized metal affinity chromatography; LPS: lipopolysaccharide; MS/MS: tandem mass spectrometry; MW: molecular weight; ncRNA: non-coding RNA; ORF: open reading frame; PcG: Polycomb group; Pol II CTD: RNA Polymerase II C-terminal domain; PRC: Polycomb repressive complex; qPCR: quantitative polymerase chain reaction; RNAi: RNA interference; RNA-seq: next-generation sequencing of RNA; sGMM: sparse Gaussian mixture model; TSS: transcription start site; TrxG: Trithorax group.TK carried out all experiments if not stated otherwise, performed computational analysis of ChIP-seq and RNA-seq data, participated in statistical data analysis, and drafted the manuscript. MG performed statistical data analysis with assistance by NB and participated in manuscript preparation. TS performed phylogenetic analysis and ZH contributed coIP experiments. DH performed MS/MS measurements and data analysis. CB conceived the study and helped TK to acquire and interpret data. RP helped to interpret data and revised the manuscript. All authors read and approved the final manuscript.ESI-MS/MS analysis overview. Spreadsheet contains analysis results of ESI-MS/MS experiments.Click here for fileDNA oligonucleotides used in this study. Tables listing oligos used for PCR, molecular cloning, and qPCR.Click here for file


Living cells implement complex computations on the continuous environmental signals that they encounter. These computations involve both analogue- and digital-like processing of signals to give rise to complex developmental programs, context-dependent behaviours and homeostatic activities. In contrast to natural biological systems, synthetic biological systems have largely focused on either digital or analogue computation separately. Here we integrate analogue and digital computation to implement complex hybrid synthetic genetic programs in living cells. We present a framework for building comparator gene circuits to digitize analogue inputs based on different thresholds. We then demonstrate that comparators can be predictably composed together to build band-pass filters, ternary logic systems and multi-level analogue-to-digital converters. In addition, we interface these analogue-to-digital circuits with other digital gene circuits to enable concentration-dependent logic. We expect that this hybrid computational paradigm will enable new industrial, diagnostic and therapeutic applications with engineered cells. Digital and analogue gene circuits each have distinct advantages in natural and engineered cells. Here, Rubens et al. engineer synthetic gene circuits that implement mixed-signal digital and analogue computations in living cells. Analogue and digital computation each have distinct advantages for cellular computing23456711121315+ and V−) and output a digital OFF or ON signal (or ‘LO' or ‘HI') if V+<V− or V+>V−, respectively20212223252627Our approach is to process signals from front-end analogue sensors with composable input-discretization devices that are analogous to electronic comparators. The outputs of these devices can then be processed in a digital manner with downstream circuits. This strategy of explicitly digitizing analogue signals followed by digital computing stages is conceptually different than other mixed-signal computing approaches, such as fuzzy logic, neural networks and hybrid automata, in which analogue and digital processing are intricately coupled. However, the components developed here may be useful for future gene circuits implementing these other forms of hybrid computing. Electronic comparators compare analogue voltages between two terminals (V2O2). H2O2 plays intricate biological roles across all kingdoms of life, and its regulation is linked to human health and disease2O2 oxidizes and activates the Escherichia coli transcription factor OxyR303132oxyR is auto-negatively regulated, and we placed gfp under the control of the OxyR-regulated oxyS promoter (oxySp) on the same low-copy plasmid . The second module is the digitization module, which is composed of a recombinase whose expression is controlled by the threshold module. The recombinase digitizes the input value by inverting the orientation of a targeted DNA segment maintained at a very-low-copy number. The third module is the DNA that is inverted by the recombinase, which can contain a gene or gene-regulatory elements, such as transcriptional promoters or terminators, to alter expression of the desired output(s).We then created genetic comparators , which cattB and attP recombinase-recognition sites, and their activity is unidirectional unless co-factors are present2O2 input levels, we placed the Bxb1 recombinase under the control of the oxySp promoter on a low-copy plasmid. To keep the basal level of bxb1 minimal such that there is little recombinase activity in the cell in the uninduced state, we added a ClpXP-mediated degradation tag to the 3′ end of the bxb1-coding sequencegfp gene flanked by oppositely oriented attB and attP sites.The digitization aspect of the comparator relies on recombinases, and thus we explored how the number of sites targeted by recombinases affects signal digitization into two distinct gene expression states within individual cells. The serine integrases (recombinases) we used flip, excise or integrate DNA depending on the orientation of bxb1 expression at different concentrations of H2O2 and measured GFP expression via flow cytometry , and we calculated the ‘relative input range' of the transition band to define its width  by assembling combinations of promoters with different transcription factor affinities, RBSs and recombinases . We defits width . A narro2O2 concentrations. We screened different RBSs in this construct and found that none of these circuits turned ON below 1 μM H2O2 without also exhibiting a high basal level of recombinase activity  and randomly mutated the −10 region of the oxySp promoter to create a low-threshold comparator that had a transition band between 0.91 and 6.44 μM H2O2, giving it a relative input range of 7.10 , an intermediate concentration (‘0') or a confirmed high concentration (‘+1'). To construct this circuit , or to encode distinct outputs at low or high input levels to actuate downstream circuits.Higher-order signal-processing circuits can be designed to convert a single analogue input into multiple distinct outputs. For instance, we built ADCsle genes . For exa circuit , we altetrations . In futu2O2 concentrations into one of four gene expression states in each cell  due to successive Bxb1, PhiC31 and TP901 expression with increasing H2O2, thereby encoding 2 bits of information tp901 to control the direction of the promoter driving transcription of gfp. We assayed GFP levels at different H2O2 concentrations in the presence and absence of aTc, and found a majority of GFP-positive cells only at intermediate concentrations of H2O2 and when aTc was absent : ADCs convert an analogue input signal into discrete output signals, whereas DACs convert discrete input signals into analogue output signals . For exaThese mixed-signal circuits constitute a first step towards advanced analogue–digital hybrid computational approaches. For instance, to implement an artificial neural network circuit, multiple analogue inputs could be fed into the promoter controlling recombinase expression, and the weights of the analogue inputs could be tuned via their binding affinity to the promoter. Linking these artificial circuits together could allow the creation of artificial neuronal networks in living cells. The comparators could also be used in a hybrid automaton if they were integrated with state machines, wherein states switch based on analogue thresholds. In addition, the ternary logic circuit  could beWe envision that mixed-signal processing will enable a wide range of industrial47In addition, cells could be designed to detect continuous quantities of multiple biomarkers, integrate these signals to diagnose disease conditions and produce reporter output(s) for non-invasive biosensing applications. For instance, probiotic or commensalMixed-signal circuits could also be useful for engineering cell therapies whose therapeutic outputs are regulated by quantitative levels of disease biomarkers. For example, mammalian gene circuits could be designed such that blood glucose levels below the normal region (‘−1' in a ternary logic system) would switch on glucagon secretion, blood glucose levels in the desired region (‘0' in a ternary logic system) would result in no hormone secretion and blood glucose levels above the normal region (‘1' in a ternary logic system) would trigger insulin secretion. The ability to trigger distinct outputs in response to different conditions could enable new ‘homeostatic' therapies. As a first step towards such applications, we tested the temporal response of the ternary logic circuit  and founIn summary, mixed-signal gene circuits merge analogue and digital signal processing to enable both continuous information sensing and robust multi-signal integration, and computing in living cells. Ultimately, we expect that this hybrid analogue–digital computational paradigm will allow synthetic biological systems to begin to approach the nuanced complexities found in natural biological systems1415184950515253545556575859E. coli EPI300 (F−mcrA Δ(mrr-hsdRMS-mcrBC) Φ80dlacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ7697 galU galK λ−rpsL (StrR) nupG trfA dhfr) was used for all experiments. Parts and plasmids used in this study are detailed in All plasmids were constructed using PCR and Gibson assembly starting from DNA sources as referenced in E. coli EPI300, plated on LB medium with appropriate antibiotics and grown overnight at 37 °C. Antibiotic concentrations were carbenicillin (50 μg ml−1), kanamycin (30 μg ml−1) and chloramphenicol (25 μg ml−1). The next day, single colonies were inoculated into Teknova Hi-Def Azure Media with appropriate antibiotics and 0.2% glucose, and incubated shaking aerobically for 16–18 h at 37 °C. Cultures were then diluted 2,500 × into fresh Hi-Def Azure Media with appropriate antibiotics and 0.2% glucose, and shaken for 20 min aerobically at 37 °C. After 20 min, 200 μl of culture was transferred to a 96-well plate, and H2O2 (Sigma–Aldrich H1009-100ML) was added at appropriate concentration via serial dilution. For the experiment in −1. Plates were incubated aerobically with shaking for 20 h at 30 °C for all experiments except those in E. coli via forward scatter area and side scatter area, and then analysed as in Plasmids were transformed into chemically competent phenicol 5 μg ml−1Plasmids described in this study have been deposited into the Addgene repository under Addgene ID numbers 78211–78229. The authors declare that all other data supporting the findings of this study are available within the article and its How to cite this article: Rubens, J. R. et al. Synthetic mixed-signal computation in living cells. Nat. Commun. 7:11658 doi: 10.1038/ncomms11658 (2016).Supplementary Figures 1-17, Supplementary Tables 1-3, Supplementary Note 1 and Supplementary ReferencesPart sequencesPlasmid sequences


Polycomb group proteins form multicomponent complexes that are important for establishing lineage-specific patterns of gene expression. Mammalian cells encode multiple permutations of the prototypic Polycomb repressive complex 1 (PRC1) with little evidence for functional specialization. An aim of this study is to determine whether the multiple orthologs that are co-expressed in human fibroblasts act on different target genes and whether their genomic location changes during cellular senescence.Deep sequencing of chromatin immunoprecipitated with antibodies against CBX6, CBX7, CBX8, RING1 and RING2 reveals that the orthologs co-localize at multiple sites. PCR-based validation at representative loci suggests that a further six PRC1 proteins have similar binding patterns. Importantly, sequential chromatin immunoprecipitation with antibodies against different orthologs implies that multiple variants of PRC1 associate with the same DNA. At many loci, the binding profiles have a distinctive architecture that is preserved in two different types of fibroblast. Conversely, there are several hundred loci at which PRC1 binding is cell type-specific and, contrary to expectations, the presence of PRC1 does not necessarily equate with transcriptional silencing. Interestingly, the PRC1 binding profiles are preserved in senescent cells despite changes in gene expression.The multiple permutations of PRC1 in human fibroblasts congregate at common rather than specific sites in the genome and with overlapping but distinctive binding profiles in different fibroblasts. The data imply that the effects of PRC1 complexes on gene expression are more subtle than simply repressing the loci at which they bind. Drosophila but their mammalian counterparts are implicated in a wide variety of physiological processes, including pluripotency, imprinting, differentiation and senescence [CDKN2A tumor suppressor [Polycomb-group (PcG) proteins were originally identified by their developmental effects in nescence -3. Senesnescence ,5. Genetppressor ,7.PcG proteins operate within multi-component complexes, the best characterized of which are termed Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) . PRC2 isDrosophila comprises equimolar amounts of Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph) and Sex combs extra (Sce) [In contrast, the PRC1 components have undergone considerable expansion during evolution. The prototypic PRC1 complex in ra (Sce) ,10. Howera (Sce) ,8,11. Bira (Sce) ,12-15 anra (Sce) .The Psc and Sce subunits combine to form an E3 ubiquitin ligase that mono-ubiquitinates histone H2A on lysine 119 (H2AK119), a modification that is also associated with transcriptional repression -20.The reason for the expansion of PRC1 families is unclear as there is currently little evidence for functional diversification ,21-23. IHere we investigate the genome-wide distribution of the PRC1 components expressed in two strains of normal human fibroblasts (HFs), a somatic cell type required for the physical integrity of multiple tissues and the classical model for the study of cellular senescence. Contrary to expectations, we find that three Pc orthologs  and both Sce proteins (RING1 and RING2) have very similar binding patterns throughout the genome with little evidence for target gene specificity. Moreover, by assembling a panel of antibodies that support chromatin immunoprecipitation (ChIP) of the endogenous proteins, we find that 11 of the PRC1 core components expressed in HFs display virtually identical binding patterns at representative loci. Importantly, sequential ChIP with antibodies against different members of the Pc, Ph and Sce families implies that multiple variants of PRC1 associate simultaneously with the same chromatin. At many loci, the binding patterns have an inherent architecture that is faithfully conserved between the two strains of HF but at an equivalent number of loci the binding is strain specific. Surprisingly, there is no strict correlation between the presence and position of the PRC1 peaks and RNA expression from adjacent gene(s). Moreover, the genomic location of the Pc orthologs remains unchanged in senescent HFs. The findings are consistent with the idea that mammalian PRC1 proteins congregate as complexes of complexes and that their control over gene expression is more subtle than simply blocking transcription of the genes to which they bind.in vitro models of differentiation. We chose to investigate the situation in HFs, in part because of the evidence linking PRC1 proteins with senescence, via effects on the CDKN2A locus [Most studies of mammalian PcG proteins and their potential target genes have focused on ES cells and 2A locus , but als2A locus ,25, the CDKN2A locus as a test bed for antibody evaluation, we assembled a panel of custom-made and commercially available reagents that support ChIP of endogenous PRC1 components in primary HFs. A number of PRC1 components have been previously located at CDKN2A using a validated set of PCR primers  with antibodies against three of the Pc orthologs and both Sce orthologs for the BF and Hs68 fibroblasts. We reasoned that by comparing the binding profiles of CBX6, CBX7 and CBX8, we could ascertain whether they regulate distinct or overlapping sets of target genes in HFs. Conversely, as every catalytically active PRC1 complex should contain either RING1 or RING2, the combined dataset should provide an impression of all PRC1 binding sites in the genome, which would complement the CBX profiles. For reference purposes, we also generated ChIP-seq profiles for H3K27me3 and H3K4me3 for the same cells. GATA6, UNCX and NKX2-3 in Figure Unlike the inferences drawn from other cell systems ,24, the To support our interpretation of the ChIP-seq data, we selected representative examples of target loci and performed conventional ChIP followed by quantitative PCR analyses with a series of primers that not only crossed each peak but allowed discrimination between regions of high and low/no enrichment  as the initial criterion. However, a striking feature of the data was that many of the ChIP-seq peaks had a complicated architecture that would be inconsistent with a normal distribution around a single binding site. It was also common to find multiple discrete peaks at the same locus and there was no simple relation between the number of peaks identified by MACS and the number of potential target genes. The bioinformatically compiled lists were therefore edited manually to try to resolve ambiguities  had an H3K4me3 peak associated with the TSS  and quality controlled on the DNA 1000 BioAnalyzer 2100 chip before clustering. Input DNA was used as a control for the ChIP-seq analysis. Detailed protocols for library preparation and genome-wide sequencing are available online -59.The alignments were performed using Novoalign (version 2.07.14)  allowingTotal RNA was extracted using the High Pure RNA extraction Kit (Roche). RNA samples were quality controlled (QC) using the 6000 Nano RNA Chip on a BioAnalyzer 2100 (Agilent) and subjected to poly-A selection using Sera-Mag oligo dT beads (Thermo Fisher Scientific Inc). Libraries were prepared using the Directional mRNA-Seq Library Prep. v1.0. Pre-Release Protocol from Illumina with minor adjustments . The PhuSequenced reads were aligned using RSEM (version 1.2.4) . Each laFor qRT-PCR validation, cDNAs were generated from 0.5 to 1 μg of RNA using MultiScribe reverse transcriptase and random hexamer primers (Applied Biosystems). Of the cDNA, 1/50 was used as a template for quantitative PCR with POWER SybrGreen (Applied Biosystems). RPS17 was used as an internal standard. Relative RNA levels were presented either as normalized to RPS17, or as fold-difference between the normalized values. Primer sequences can be found in Additional file bp: base pair; ChIP: chromatin immunoprecipitation; ChIP-seq: ChIP-sequencing; ES cell: embryonic stem cell; HF: human fibroblast; kb: kilobase; Pc: Polycomb; PcG: Polycomb group; PCR: polymerase chain reaction; Ph: Polyhomeotic; Psc: Posterior sex combs; QC: quality controlled; qRT-PCR: reverse transcription and quantitative PCR; RNA-seq: RNA-sequencing; SA-βgal: senescence-associated β-galactosidase; SAHF: senescence-associated heterochromatin focus; Sce: Sex combs extra; TES: transcription end site; TSS: transcription start site.The authors declare that they have no competing interests.HPe, EA, HC and JS performed the ChIP-seq and re-ChIP analyses. RP contributed to the ChIP validation. SB validated the RNA-seq data. MRN, LdeB and TR evaluated the antibodies for specificity and applicability to ChIP. HPa and AS performed the bioinformatic analyses. NM conducted the DNA sequencing. GP conceived the study and wrote the manuscript. All authors read and approved the final manuscript.(A) Flag-tagged versions of human CBX2, CBX4, CBX6, CBX7 and CBX8 were expressed in 293T cells by transient transfection of pcDNA6-based vectors. Cell lysates were precipitated with an anti-Flag antibody, ractionated by SDS-PAGE and immunoblotted with the indicated antibodies. Although multiple products were detected, presumably reflecting premature termination or post-translational cleavage, the CBX6 antibodies only detected Flag-CBX6, the CBX7 antibodies only detected Flag-CBX7 and the CBX8 antibodies only detected Flag-CBX8. (B) Equivalent analyses with cells expressing Flag-tagged versions of HPH1, HPH2 and HPH3. (C) Similar evaluation of antibodies against RING1 and RING2. (D) Lysate from cells expressing Flag-tagged CBX4 (left) or CBX6 (right) were immunoprecipitated with antibodies against CBX4, 6, 7 and 8 as indicated. Following SDS-PAGE, the Flag-tagged proteins were identified by immunoblotting. Ig refers to the position of the immunoglobulin heavy chain.Evaluation of antibody specificity. Click here for file(A) GATA6 in BF cells, (B) CCND2 in BF cells, (C) MEIS1 in BF cells and (D) NRN1 in Hs68 cells.ChIP-PCR analyses of multiple PRC1 proteins at representative loci. Each dataset includes a screenshot of the CBX7 binding profile across the locus (top), with a diagram showing the position of the PCR primer sets relative to the organization of the suspected target gene. The primer sequences are described in Additional file Click here for fileList of candidate PRC1 target loci in the BF and Hs68 strains of HF. Alphabetic list of loci associated with PRC1 ChIP-seq peaks in HFs, showing the number of PRC1 proteins identified at the locus for the BF and Hs68 strains. TRUE and FALSE indicate whether the locus is transcriptionally active in BF and Hs68, as judged by RNA-sequencing.Click here for file(A) TBX2, (B) TBX4 and (C) RUNX3.ChIP-PCR showing differential binding of PRC1 proteins in BF and Hs68 cells. Each dataset includes a screenshot of the CBX7 binding profile across the locus (top), with a diagram showing the position of the PCR primer sets relative to the organization of the suspected target gene. The primer sequences are described in Additional file Click here for fileChIP-seq and RNA-sequencing profiles of the HOX clusters in BF and Hs68 cells. Upper tracks in each figure show the profiles of DNA sequence tag densities following ChIP-seq with antibodies against CBX6, CBX7, CBX8, H3K27me3 and H3K4me3 in the BF and Hs68 strains of HDF as indicated. The lower tracks show duplicate RNA-sequencing data for the corresponding genomic regions in either BF or Hs68 cells. The tag densities were normalized to the same maximum (numbers on left).Click here for fileTable S3. List of oligonucleotide primers for PCR analysis of immunoprecipitated chromatin. Table S4. List of oligonucleotide primers used to assess RNA levels by reverse transcription and quantitative PCR.List of antibodies used for ChIP. Click here for file


Long non-coding RNAs (lncRNAs) have emerged as important regulators of gene expression in a variety of biological process and in multiple species. In plants, they are transcribed by different RNA polymerases and show diverse structural features. With the aid of next-generation sequencing technologies, a large number of lncRNA have been identified in model plants as well as in crops. This review focuses on the demonstration that lncRNAs control root system architecture, notably in response to phosphate availability, through regulation of transcription, alternative splicing, microRNA activity, messenger RNA stability and translation, illustrating remarkable diversity in their roles in regulating developmental plasticity. COOLAIR and COLDAIR (FLOWERING LOCUS C (FLC). Sequencing-by-synthesis technologies has revealed lncRNAs as a significant component of the transcriptome in a number of plant species , requiring phosphate inputs as fertilizer or else limiting agricultural production. Low Pi availability in the soil results in Pi deficiency that triggers a cascade of developmental responses, which reprogram the root system architecture. This developmental plasticity allows roots to favor exploration of the shallow part of the soil, where phosphate tends to be more abundant and thus maximizing its uptake. This process is under the control of spatiotemporal-regulated gene expression networks . These d COLDAIR , which i species , includiThis review considers the remarkably diverse roles lncRNAs play in environmental regulation of root system architecture.cis-NATs) or from distinct genomic loci  tail. Plant specific RNA polymerases IV and V (pol IV and pol V), which evolved from pol II (Most of lncRNAs identified in eukaryotes are likely to be transcribed by RNA polymerase II (pol II) in a process in plants involving the eukaryotic multi-subunit protein complex Mediator . They alm pol II , also acm pol II , whereasm pol II .7mGTP-capped, but do not have 3′-poly(A) tails  tails . A compaA) tails .Arabidopsis uncovered nearly 6,500 lincRNAs that accumulate across diverse organs of Arabidopsis, of which 2,708 were reconfirmed in a subsequent RNA-seq analysis , which encodes a regulatory kinase that determines the polar localization of the auxin transporter PIN-FORMED 2 (PIN2) in root cells , a member of the Polycomb Repressive Complex 1 (PRC1), whereas pol IV/V transcription of APOLO triggers its methylation, mediating loop closure and thus downregulation of PID transcription by pol II. Hence, this mechanism fine-tunes PID promoter activity and could enable oscillation of gene expression in roots in response to auxin.By use of a system that allows synchronization of the formation of lateral organs in roots, the authors showed that auxin recruits the DNA Demethylation Machinery (DDM) at the Arabidopsis  of a specific subset of genes which are involved in the control of lateral root initiation  was identified in a screen for proteins that bind the short open reading frame mRNA called ENOD40, a highly structured long RNA . This study found ENOD40 overexpression caused the relocalization of MtRBP1/MtNSR from nuclear speckles to the cytoplasm. Interestingly, this phenomenon was recapitulated when ENOD40 was co-expressed with AtNSR in tobacco cells. An Arabidopsis lncRNA that binds AtNSRs called ASCO lncRNA (AS competitor long non-coding RNA) was identified  of bidopsis . The parn Figure . In a prentified . The strPlants have evolved an array of physiological and biochemical responses to cope with dynamics in the moisture, nutrients and microbiome of the soil. This is particularly striking when plants experience limiting Pi availability, which promotes the root system to undergo profound changes in morphology and architecture. This is accompanied by metabolic reprogramming to maintain intracellular Pi homeostasis.At4 and IPS1 (INDUCED BY PHOSPHATE STARVATION 1) and their orthologous in Medicago (Mt4) and tomato (TPS1) respectively, are strongly induced by Pi starvation. Loss-of-function mutation of At4 results in defects in Pi redistribution between root and shoot under Pi deficient conditions  was shown to act as a translational enhancer of OsPHO1;2 and to contribute to Pi homeostasis  regulates UCHL1 synthesis without changes at the transcript level (Uchl1 mRNA. However, PHO1;2cis-NAT does not have features of a SINE or other repetitive element. Nonetheless, subcellular transcriptomic analysis in human cell lines revealed a large fraction of lncRNAs is enriched in the cytosol as well as polysomal fractions, whereas only a small subset was found in the nucleus (Arabidopsis included about 10% of the annotated lncRNAs (It was hypothesized that s Figure . Stimulapt level . These a nucleus . Nucleas lncRNAs , with moThe recent burst of papers describing roles of lncRNAs in plant development and environmentally-regulated developmental plasticity highlights the need for deeper study of the contributions of lncRNA to dynamics of gene expression networks. This is particularly relevant in the control of the root system response to changes in nutrient and water availability where fine-tuning of developmental regulator genes is required to trigger the rapid and reversible developmental and physiological alterations needed to obtain moisture and nutrients.Functional analysis of lncRNAs is challenging. In many cases, their activity may rely only on secondary structure rather than primary nucleotide sequence. However, the recent advances in genome engineering using designer site-specific nucleases such as CRISPR/Cas9 and TALENs, as well the ability to access to cell-specific and/or subcellular RNA populations using technologies such as isolation of nuclei tagged in specific cell types INTACT;  and tranThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Escherichia coli and govern intraspecies competition. In a previous report, we demonstrated that the global transcriptional factor IscR, co dependently with the master regulator of the DNA damage response, LexA, delays induction of the pore forming colicin genes after SOS induction. Here we show that IscR is not involved in the regulation of nuclease colicins, but that the AsnC protein is. We report that AsnC, in concert with LexA, is the key controller of the temporal induction of the DNA degrading colicin E8 gene (cea8), after DNA damage. We demonstrate that a large AsnC nucleosome-like structure, in conjunction with two LexA molecules, prevent cea8 transcription initiation and that AsnC binding activity is directly modulated by L asparagine. We show that L-asparagine is an environmental factor that has a marked impact on cea8 promoter regulation. Our results show that AsnC also modulates the expression of several other DNase and RNase colicin genes but does not substantially affect pore-forming colicin K gene expression. We propose that selection pressure has “chosen” highly conserved regulators to control colicin expression in E. coli strains, enabling similar colicin gene silencing among bacteria upon exchange of colicinogenic plasmids.Colicins are plasmid-encoded narrow spectrum antibiotics that are synthesized by strains of  Colicins are considered model proteins for studying bacterial toxins. These narrow spectrum antibiotics can kill by a variety of mechanisms, e.g. by forming pores in the membranes of susceptible cells or by degrading their nucleic acids. Colicin genes are plasmid-encoded and repressed by the master regulator of the DNA damage response, LexA. Induction of several pore-forming colicin genes is also repressed by IscR, which ensures that colicin genes are switched on as a last resort in DNA damaged cells, when nutrients are depleted. Here we show that nuclease colicin genes are not controlled by IscR but that the AsnC protein, in concert with LexA, is directly responsible for uncoupling the immediate expression of the DNase colicin E8 from the main induction of the SOS response. AsnC wraps the DNA of the colicin E8 promoter into a complex nucleoprotein assembly and the architecture of this complex is altered by the presence of the amino acid L-asparagine. Thus, repression by metabolite-responsive and DNA-damage responsive regulators operates at the regulatory regions of different colicins. Hence, the response to several environmental signals have been integrated to ensure that, following DNA damage, colicin synthesis is tightly repressed and induced only in terminally damaged cells. Escherichia coli and its close relatives -ATP using polynucleotide kinase (NEB). Approximately 0.5 ng of DNA fragment was incubated with varying amounts of purified proteins, as indicated. The reaction buffer contained 20 mM Hepes (pH 8), 5 mM MgCl2, 50 mM potassium glutamate, 1 mM DTT, 5% (v/v) glycerol and 0.5 mg/ml BSA and the final reaction volume was 10 μl. Where AsnC was used, the EMSA buffer contained 5 mM L-asparagine (L-asn), where indicated. Samples were incubated at 37°C for 15 min before electrophoresis. For competitive EMSA experiments, DNA fragments were first incubated with various concentrations of LexA (for 15 min at 37°C) followed by the addition of RNAP and incubated for another 15 min at 37°C. Herring sperm DNA was included at a concentration of 6.5 μg ml-1 for these experiments. After incubation, all samples were immediately run on a 5% polyacrylamide gel at 12 V cm-1 in 0.25 x TBE, running under tension, and were visualised using a Bio-Rad Molecular Imager FX and Quantity One Software (Bio-Rad).EMSA analysis, using purified LexA, IscR, AsnC and RNAP, with the ribed in . DNA fracea8 promoter region was performed as described [cea8 fragment that had been 32P-end labelled at the HindIII site using polynucleotide kinase and [γ-32P]ATP.DNase I footprinting of AsnC and LexA at the escribed , using pS1 Figlac transcriptional fusions. The dashed lines represents OD600.Measured β-galactosidase activities of strain BW25113, harbouring pRW50, with colicin promoter-(DOCX)Click here for additional data file.S2 FigiscR cells, carrying naturally occurring plasmids, which encode either pore-forming or nuclease colicins. The arrow indicates the time of addition of nalidixic acid.Growth curves of wild-type BW25113 (wt) or Δ(DOCX)Click here for additional data file.S3 FigiscR strain, harbouring plasmids encoding pore-forming or nuclease colicins. The indicator strain used was DH5α, harbouring pBR322. Samples were taken before and 1, 2, and 3 h after the induction of the SOS response by nalidixic acid.A ten-fold dilution series of colicin extracts of the wild-type strain BW25113 or the isogenic Δ(DOCX)Click here for additional data file.S4 Figcka promoter region. Above the sequences »x« denotes position of the AsnC-induced hypersensitive sites observed by DNAse I footprinting at the colicin E8 promoter region (http://dna.macrogen.com/) and were identical to the deposited sequences in GeneBank: ID numbers for colicin K, E2, E5, E6 and E8 are AY929248.1, M29885.1, KF925332.1, M31808.1 and FJ985252.1, respectively.The promoter -35 and -10 elements, the SOS box targets, the Shine-Dalgarno sequence and the translation start sites are indicated in red, yellow, blue and orange, respectively. The green box indicates the IscR binding site at the r region  and the r region . Nucleot(DOCX)Click here for additional data file.S5 Fig32 end-labelled colicin K promoter fragment in the presence and absence of L-asparagine (± L-Asn). The concentration of AsnC in lanes 2–7 and 9–14 was 0.5, 1.05, 2.1, 4.2, 8.4 and 12.6 μM, respectively. The location of free DNA, the position of the wells and the various AsnC/DNA complexes is indicated. B) Growth curves of BW25113 (wt) and ΔasnC cells harbouring the naturally occurring plasmid, which encodes the pore-forming colicin K (pColK). The arrow indicates the time of addition of nalidixic acid. Experiments were performed in duplicate and representative growth curves are shown. C) Colicin synthesis was measured in BW25113, ΔasnC and ΔiscR cells carrying a colicin K-encoding plasmid. Cells were collected at hourly time points after the of addition of nalidixic acid (0 h) and a five-fold dilution series of cell extracts were applied on an agar plate supplemented with tetracycline and overlaid with the colicin sensitive strain DH5α pBR322. Results illustrate that in comparison to the colicin K production in the wild-type cells, an hour after SOS induction 5- and 125-times more colicin K is synthesized in the ΔasnC and the ΔiscR mutant, respectively. The experiments were performed in duplicate and representative results are shown.A) EMSA analysis of the binding of purified AsnC protein to a P(DOCX)Click here for additional data file.S1 TableE. coli database using Mascot (Matrix Science) software; MW (kDa) – protein molecular weight.The numbering of bands are the same as those indicated in (DOCX)Click here for additional data file.S2 Table-1) and 37 μM of nalidixic acid. After 12 h of growth the β-galactosidase activity of the cultures was determined (presented in Miller units (U)). The β-galactosidase ratio is the β-galactosidase value of the cea8::lac activity observed in each mutant in comparison to the wild-type strain BW25113 (wt). Potential candidates, which were analysed further, are shown in bold.Overnight cultures of each strain were inoculated 1:100 in 10 ml of LB broth supplemented with tetracyclin (12.5 μg ml(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.