SlGME1, which encodes GDP-Man-3’,5’-epimerase, a pivotal enzyme in the d-mannose/l-galactose pathway. Importantly, SlNFYA10 simultaneously binds to the promoter of SlGGP1, a downstream gene of SlGME1 in the d-mannose/l-galactose pathway. Binding assays in yeast and functional analyses in plants have confirmed that SlNFYA10 exerts a negative effect on the expression of both SlGME1 and SlGGP1. Transgenic tomato lines overexpressing SlNFYA10 show decreased levels of SlGME1 and SlGGP1 abundance and AsA concentration in their leaves and fruits, accompanied by enhanced sensitivity to oxidative stress. Overall, SlNFYA10 is the first CCAAT-binding factor identified to date to negatively regulate the AsA biosynthetic pathway at multiple sites and modulate plant responses to oxidative stress.Ascorbic acid (AsA), an important antioxidant and growth regulator, and it is essential for plant development and human health. Specifically, humans have to acquire AsA from dietary sources due to their inability to synthesize it. The AsA biosynthesis pathway in plants has been elucidated, but its regulatory mechanism remains largely unknown. In this report, we biochemically identified a CCAAT-box transcription factor (SlNFYA10) that can bind to the promoter of  AsA plays an important role in protecting human health and in disease prevention. For example, AsA can effectively control tumor progression2, and intracellular AsA helps maintain the integrity and function of several processes in the central nervous system3. In plants, AsA can remove reactive oxygen species produced by photosynthesis, respiration, biological stress, and abiotic stress so that plants can carry out normal physiological processes. For AsA biosynthesis, the major biosynthesis pathway is the d-mannose/l-galactose pathway (Wheeler & Smirnoff pathway), although an alternative pathway in plants involving galacturonate has been proposed5. However, controversy exists between the d-glucose6 and myo-inositol pathways7. Between them, the pivotal biosynthetic pathway of d-mannose/l-galactose involves ten catalytic steps through which d-glucose is converted to AsA. The first six steps involved the synthesis of active riboside sugars, which are utilized as substrates for AsA synthesis, as well as precursors of cell wall polysaccharides and glycoproteins. The last four steps, starting from GDP-l-galactose, constitute the exclusive steps for AsA synthesis. The genes in the d-Man/l-Gal pathway have been identified; they encode GDP-d-Man pyrophosphorylase (GMP)8, GDP-Man-3’,5’-epimerase (GME)9, GDP-l-Gal phosphorylase (GGP)10, l-Gal-1-P phosphatase (GPP)11, l-Gal dehydrogenase 12, and l-galactono-1,4-lactone dehydrogenase (GLDH)13. GME converts GDP-d-mannose into GDP-l-galactose, yielding an alternative product of GDP-l-glucose. In most plants, there is a single copy of the GME gene14, whereas in tomato (Solanum lycopersicum), two GME genes named GME1 and GME2 have been found15. Overexpression of GME1 and GME2 could increase the AsA content, while knockdown of either GME gene decreased the AsA content16. Simultaneous suppression of the GME1 and GME2 genes by RNA interference (RNAi) could reduce the AsA content by 40–60%, with increasing cell wall mannose content and fragility17. Suppressed expression of GME1 or GME2 separately revealed different functions in cell wall synthesis18, suggesting the divergence of functions of GME family members.Ascorbic acid  contributes to nutritional quality and stress responses in plants and thus has attracted increased amounts of attention. Since humans and higher primates have lost the capability to synthesize AsA, this compound must be obtained from dietary sources rich in AsA, e.g., fresh vegetables and fruits20, temperature21, minerals22, and environmental cues. AsA accumulation is also regulated by hormones such as auxin23, methyl jasmonate24, jasmonic acid25, and Abscisic Acid (ABA)26. Transcription factors or regulators purportedly modulate AsA biosynthesis. amr1 (Ascorbic acid Mannose Pathway Regulator 1), an activation-tagged Arabidopsis  ozone-sensitive mutant, showed decreased AsA content when the expression of genes in the d-Man/l-Gal pathway were negatively regulated27. The photomorphogenic factor COP9 signalosome subunit 5B (CSN5B) could decrease the AsA content by modulating the protein abundance of the AsA biosynthetic enzyme GDP-Man-pyrophosphorylase (VTC1)28. Arabidopsis AtERF98 and the tomato HD-Zip I family transcription factor (SlHZ24) could bind to the VTC1 promoter and increased the AsA content by positively regulating VTC1 expression30. Two nucleotide sugar pyrophosphorylase-like proteins from Arabidopsis, KONJAC1 (KJC1) and KJC2, could stimulate GMP enzymatic activity and increase AsA accumulation31. Moreover, a Cys2/His2-type zinc-finger protein named SlZF3 could directly interact with CSN5B, preventing CSN5B binding to GMP and thus increasing the AsA content in tomato32. More recently, an upstream open reading frame (uORF) in the long untranslated region of GGP was demonstrated to encode a peptide that functions in the feedback regulation of AsA at the posttranscriptional level33. To date, the regulation of AsA biosynthesis largely occurs at the VTC1-catalyzed step32, while the regulation of other genes in the AsA synthesis pathway deserves further investigation.In plants, the AsA content is affected by various factors, such as light34. Sequences associated with the NFY heterotrimer complex have been found in all the sequenced genomes of eukaryotes35, and there are one or two genes encoding NFYs in mammals and yeast36. However, in plants, the NFY family has undergone expansion34. The NFY complex was reported to be involved in the regulation of plant growth and development and the stress response. NFYA in wheat positively responds to low nitrogen and phosphorus: overexpression of TaNFYA-B1 stimulated root development by upregulating nitrate and phosphate transporters and, subsequently, nitrogen and phosphorus absorption in roots37. Overexpression of the transcription factor NFYC9 mediates ABA signaling by targeting ABA-responsive transcription factors such as ABI538. The expression of NFYA5 was strongly induced by drought in an ABA-dependent manner, and NFYA5 overexpression could reduce moisture loss and drought sensitivity39. However, overexpression of the wheat NFYA10 gene increased plant sensitivity to salinity, as judged on the basis of seed germination and root growth40.NFYAs belong to the NFY  complex, which contains NFYAs, NFYBs and NFYCs, and function as heterodimers. After dimerization between NFYB and NFYC in the cytoplasm, NFYA is further recruited to form a heterotrimer. NFYA binds to the CCAAT-box to regulate the expression of downstream genes43, although only a single NFY transcription factor has been reported to be involved in the stress response in plants. However, the regulatory mechanism through which metabolites, e.g., AsA, accumulate has not been elucidated. In this study, an NFYA transcription factor was determined to negatively regulate AsA biosynthesis at multiple sites in the d-mannose/l-galactose pathway.NFY factors have been known to function as heterotrimer complexes during various processes of plant growth and developmenthttp://www.clontech.com/) according to the manufacturer’s instructions. A 2-kb promoter fragment of SlGME1 from the initiation site, as well as a mutated fragment of the SlGME1 promoter were amplified from tomato genomic DNA and then cloned into a pAbAi vector (Clontech). The full-length and truncated NFYA CDSs were amplified from tomato complementary DNA (cDNA) via PCR and cloned into a pGADT7 vector (Clontech); the primer sequences used are listed in Supplementary Table 600 of 0.1, and 2.5 μl of suspension was spotted onto SD/-Leu-Ura media, with or without AbA (Clontech). The plates were subsequently incubated for 3–7 days at 30 °C.A yeast one-hybrid assay was carried out via a Matchmaker One-Hybrid Library Construction and Screening Kit . The Agrobacterium cells with different vectors were mixed together and injected into young tobacco (Nicotiana benthamiana) leaves for transient expression, after which they were were evaluated after a 2-day incubation period44. Luciferase reporter genes were assayed using a Dual-Luciferase Reporter Assay System  using an Infinite M200 Pro instrument (Tecan).There are four CCAAT-boxes in the 2-kb promoter of 45. The CDS of SlNFYA10 was amplified and linked to a pYF503 vector, generating a GDBD-SlNFYA10 effector construct. The effector construct, empty control , and 35S::UAS-GUS reporter plasmid were then transformed into A. tumefaciens strain GV2260. The reporter and two effectors were mixed together, after which the mixture injected into tobacco leaves, followed by GUS staining after a 48 h incubation period. GUS staining was performed as described previously46.A 35S::UAS-GUS reporter system was used to characterize the transcriptional activity of SlNFYA10, as described by Wang et al.A. tumefaciens strain C58 for tomato transformation. The constructs were subsequently transformed into Ailsa Craig (AC) tomato, and transgenic plants were confirmed via PCR using genomic DNA from the leaves; the primers used are shown in Supplementary Table Full-length NFYA ORFs and RNAi fragments were amplified via PCR using Phanta polymerase  from tomato cDNA using gene-specific primers (Supplementary Table http://www.roche.com/) according to the manufacturer’s protocols. The RNA of the transgenic plants and wild type was extracted and reverse transcribed to cDNA to determine the expression of SlNFYA10, SlGME1, SlGME2, and other genes in the AsA biosynthesis pathway. The sequences of the primers used are listed in Supplementary Table −ΔΔCt method, and the actin gene was used as a constitutive internal control.Total RNA was extracted from roots, stems, leaves, and flowers as well as immature fruit, green fruit, breaker-stage fruit, and red ripe fruit using TRIzol reagent. The RNA was then reverse transcribed into cDNA using a HiScript II 1st Strand cDNA Synthesis Kit (Vazyme), and the gene expression in these tissues was measured. Quantitative PCR (qPCR) was performed using a SYBR Light Cycler 480 instrument in conjunction with SYBR Green I Master Kit . The plate was incubated for 20 min at 37 °C to convert the oxidized ascorbic acid into the reduced form. Ten microliters of N-ethylmaleimide  was added to remove the excess DTT, followed by incubation for 1 min at room temperature (approximately 25 °C). Eighty microliters of the color reagent (see below) was then added to the mixture, followed by incubation for 1 h at 37 °C. The absorbance was subsequently recorded at 550 nm using an Infinite M200 Pro instrument . The color reagents were prepared as follows: solution A consisted of 31% orthophosphoric acid, 4.6% (w/v) TCA and 0.6% (w/v) iron chloride (FeCl3); solution B consisted of 4% 2,2-dipyridyl (w/v in 70% ethanol); and solutions A and B were mixed at a ratio of 2.75:1 before use. To assay reduced AsA, the DTT and NEM were replaced with the same volume of 0.4 M potassium phosphate buffer (pH 7.4), while the rest of the procedure was the same as that for the total AsA assay.The AsA levels were measured in TSlNFYA10 was amplified and inserted into the 5’ terminus of GFP in a pCAMBIA-1302 vector under the control of the CaMV35S promoter to generate 35S::SlNFYA10-GFP constructs. Plasmids were extracted and transferred into tobacco protoplasts, and subcellular localization was observed as described previously48.The coding sequence without the terminal codon of SlGME1 and SlNFYA10 were amplified and cloned into a pMV2 vector harboring the GUS reporter gene, and the resultant vectors were transferred into A. tumefaciens C58 for tomato transformation. GUS staining was performed on seedlings, flowers and mature green fruits.The promoters of SlNFYA10-overexpressing and knockdown lines and the wild type, 80 μl of methylviologen  or water consisting of 0.1% Tween-20 (control) was sprayed onto one-month-old seedlings once a day for three consecutive days. The plant phenotypes were observed one week after spraying, and images were collected.To evaluate the oxidative resistance of 2 transgenic lines and wild-type plants were treated with MV (or water as a control) to investigate the tomato response to oxidative stress. The MV- and water-treated tomato leaves were soaked in 1 mg/ml DAB solution (pH = 3.8) and then incubated in darkness at room temperature for 24 h. The leaves were then transferred to 96% ethanol and placed in a boiling water bath for 10 min to remove the chlorophyll. The ethanol solution was discarded, and the leaves were soaked with 96% ethanol to remove the floating color (the decolorized leaves were stored in 96% ethanol). The reactive oxygen species of the decolorized leaves were evaluated visually according to the brownness of the leaves.MV treatments induce cells to produce excessive ROS, resulting in oxidative damage to plants. Leaves from tomato T2 transgenic lines and wild-type plants were utilized to measure the MDA concentration. Samples were collected and ground to a fine powder in liquid nitrogen, followed by the addition of 3 ml of 5% TCA to 0.2 g of ground tissue; the extraction was performed at room temperature for 30 min. After centrifugation, 2 ml of the supernatant was transferred into a 10 ml tube, after which 2 ml of 0.67% TBA solution was added. After blending, the absorption value of the 200 μl mixture was measured at wavelengths of 450 nm, 532 nm and 600 nm. The supernatant in the standard solution was replaced with ddH2O. The concentration of MDA was calculated using the formula C = 6.45 × (OD532 – OD600) – 0.56 × OD450.Malondialdehyde (MDA), the final product of lipid peroxidation of the cell membrane, reflects the plant response to oxidative stress. Leaves and ripe fruits from T2O2 as an oxidant, POD (peroxidase) catalyzes redox reactions, scavenging H2O2 from cells. POD activity can reflect the antioxidant capacity and stress tolerance of plants. Leaves and ripe fruits from T2 transgenic lines and wild-type plants were harvested to measure POD activity. Samples were collected and ground to a fine powder in liquid nitrogen, followed by the addition of 800 μl of precooled phosphate buffer , mixing and centrifugation. The supernatant (20 μl) was subsequently added to a 1.5 ml reaction mixture, which consisted of 50 ml of phosphate buffer , 28 μl of 2-methoxyphenol and 19 μl of hydrogen peroxide (30%). The absorption value of the mixture was measured at a wavelength of 470 nm.Using HGME1 and GME2 are two homologous genes in tomato. Both contain six exons and five introns, and the first and fourth introns of GME2 are longer than those of GME1. GME1-overexpressing lines showed higher AsA levels than did GME2-overexpressing lines16. Therefore, we used the promoter of GME1 to perform a yeast one-hybrid assay in an equalized tomato cDNA library comprising various tissues of roots, leaves, flowers, and fruits at different stages to identify the candidate proteins binding to the promoter of GME1. The potential transcription factors or binding proteins involved in GME1 promoter binding and AsA regulation were isolated (Supplementary Table http://solgenomics.net/) and mapped onto chromosome 1. It contains an open reading frame of 936 bp, encoding 312 aa, with an NFYB/NFYC interaction domain (A1: 145–163 aa) and a CCAAT-binding domain (A2: 175–198 aa)  that belong to the NFYA family and is a subunit of the NF-Y complex, was further investigated. The sequence of Solyc01g006930.2 was queried via BLAST against the SOL Genomics Network database (aa) Fig. , and itsaa) Fig. . AccordiGME1, we analyzed the 2-kb promoter fragment of GME1 through the PLACE  and PlantCARE (http://bioinfor-matics.psb.ugent.be/webtools/plantcare/html/) databases49. We found four potential target elements, two elements of CCAAT and two of ATTGG, that were named C1 and C2 as well as C3 and C4, respectively  leaves by A. tumefaciens (GV2260)-mediated transformation. Plant agro infiltration showed that the truncation of the promoter successively reduced the promoter capability and luciferase (LUC) activity. When coinfiltrated together with SlNFYA10, the SlGME1 promoter with four boxes (proGME1–1: C1-C4) and three boxes (proGME1–2: C2-C4) showed significantly reduced LUC activity compared to that of the control. However, the promoter fragments with two boxes (proGME1–3: C3-C4), one box (proGME1–4: C4) or no box (proGME1–5) did not show significant differences in LUC activity compared to that of the control  and found that there are two domains: a DNA-binding domain and an NFYB/NFYC interaction domain. According to the protein structure of SlNFYA10, we divided the coding sequence into four segments (N1–N4) and inserted them into pGADT7 vectors. We used a yeast one-hybrid assay to test the interaction of these different SlNFYA10 segments with the C2 box of the SlGME1 promoter. The results demonstrated that N2, N3, and N4 could recognize and bind to the C2 box of the SlGME1 promoter, while the N1 fragment could not  and mixed them together for agro infiltration of tobacco leaves, after which GUS staining and GUS gene expression were evaluated. The tobacco leaves infiltrated with proSlGME1::GUS and pHELLSGATE8 exhibited blue GUS activity, while replacement of pHELLSGATE8 with pro35S::SlNFYA10 led to significantly reduced GUS expression. This was confirmed by qPCR-based detection of GUS gene expression in the infiltrated leaves. Taken together, these results indicated that SlNFYA10 can interact with and negatively regulated the SlGME1 promoter  and RNAi knockdown (CR) tomato lines. The overexpressing lines exhibited significantly higher SlNFYA10 expression than did the wild-type control, while the CR lines showed a decrease in expression. The expression of SlNFYA10 in the leaves of the CO-37–11 and CO-53–9 lines was 5.6- and 5.0-fold that in wild-type leaves, while the CR-6–3 and CR-16–4 lines showed decreased expression   Fig. . The AsAly) Fig. , while tly) Fig. . This miSlGME1 in tomato caused an increase in AsA levels16. We found that enhanced expression of SlNFYA10 modulated its target gene, SlGME1, in SlNFYA10-overexpressing plants. The expression of SlGME1 was significantly decreased in the leaves and red ripe fruits of the overexpression lines compared with the wild type  were predicted to contain CCAAT-boxes that might be bound with NFYA proteins  promoter41.The CCAAT-binding transcription factor also named Nuclear Factor Y (NFY) and Heme activator protein contains three subunits: NFYA, NFYB and NFYCA10 Fig. . NFYs haSlGME1 promoter was used as bait for yeast one-hybrid assays, a CCAAT-box transcription factor (SlNFYA10) was isolated. Among the four CCAAT-boxes predicted in the SlGME1 promoter, only the second CCAAT-box could interact with SlNFYA10, as revealed by luciferase and yeast one-hybrid assays (Figs. SlNFYA10 and SlGME1 (Fig. SlNFYA10 and SlGME1. Compared with the control plants, the SlNFYA10-overexpressing lines presented a lower AsA content in the leaves and fruits (Fig. SlNFYA10 exerts a repressive effect on SlGME1 transcription. The expression of alternative enzymatic genes of the AsA biosynthesis pathway was also evaluated in SlNFYA10 transgenic lines, among which SlGGP1 expression pattern was altered in a manner similar to how the pattern of SlGME1 was altered (Supplementary Fig. SlGGP1 promoter (Fig. SlNFYA10 is located in the nucleus, which is characteristic of nuclear transcription factors (Fig. SlNFYA10 and SlGME1 showed that they are mainly expressed in the same stem, flower and seed tissues (Fig. GME1 and GGP1 in tomato fruit. AsA biosynthesis has been widely investigated, and several transcription factors for biosynthetic genes such as GMP have been isolated30. In the present work, we confirmed that NFYA could simultaneously regulate GME and GGP expression. NFY transcription factors have been reported to be involved in a wide range of biological processes41; however, this is the first evidence that NFYA functions in the regulation of AsA metabolism.When the ys Figs.  and 3. Gys Figs.  and the ME1 Fig.  indicateits Fig. . These rter Fig. . In addiors Fig. . The tisues Fig. . Oxidatiues Fig. , presumaSlNFYA10 by RNAi did not cause significant alterations in terms of AsA content, SlGME1 and SlGGP1 expression, ROS accumulation, MDA concentration or POD activity in the transgenic lines (Supplementary Figs. SlNFYA10 RNAi transgenic lines compared to the wild type (Supplementary Fig. SlNFYA10 RNAi transgenic lines might be due to the functional redundancy of the members of the NFYA gene family. Unlike the NFYAs encoded by one or two genes in animals and yeast, plants tend to have evolved a relatively larger gene family; e.g., the NFY gene family has 10 members in Arabidopsis52, 8 in rice53, 10 in wheat54, and 10 in tomato55. Gene replication may contribute to large numbers of gene families that allow functional compensation or buffering against individual mutations, subsequently leading to functional redundancy56. The F-box protein family also has a large amount of members, with almost 700 predicted genes in Arabidopsis. However, single mutations or combinations of mutations in pollen-specific F-box genes did not lead to gametophytic lethality, suggesting functional redundancy among the F-box protein-coding genes57. In plant viruses such as tobacco etch potyvirus, genotypes with genetic redundancy suffered less from the effects of deleterious mutations and showed relatively minor changes in pathogenicity58. Accordingly, the expression of alternative members of the NFYA gene family, e.g., SlNFYA1.1, SlNFYA3.1, and SlNFYA8, was significantly higher in the SlNFYA10 RNAi lines than in the wild type (Supplementary Fig. SlNFYA10 RNAi transgenic lines might be in part due to functional redundancy of NFYA gene family members in tomato.It is worth noting that the silencing of SlGME1 promoter to isolate a CCAAT-box transcription factor, SlNFYA10, which could modulate AsA biosynthesis at multiple sites. SlNFYA10 exerted a negative effect on SlGME1 and SlGGP1 expression and AsA accumulation. SlNFYA10-overexpressing lines showed a lower antioxidant capacity, with lower AsA content. The identification of potential interacting proteins involved in the heterotrimer complex will provide deeper insight into SlNFYA10 regulation of AsA biosynthesis.We used the Table S1Table S2Table S3Table S4Figure S1Figure S2Figure S3Figure S4Figure S5Figure S6Figure S7Figure S8


Transcription and translation are coupled in bacteria, meaning that translation of the nascent mRNA by ribosome happens simultaneously with its synthesis by RNA polymerase (RNAP). Uncoupling of transcription from translation may lead to arrest of transcription via backtracking of RNAP along the template, leading to conflicts with DNA replication, although the molecular mechanisms involved are unclear. We assembled an in vitro coupled transcription–translation system in which both machineries can be controlled with single-nucleotide precision. We showed that through a direct interaction, the coupled ribosome, physically pushes RNAP from the backtracked state, thereby reactivating transcription. This rescue ability of the ribosome prevents inhibition of translation by transcriptional backtracking and mitigates conflicts of backtracked RNAPs with replication at protein-coding genes. In bacteria, the first two steps of gene expression—transcription and translation—are spatially and temporally coupled. Uncoupling may lead to the arrest of transcription through RNA polymerase backtracking, which interferes with replication forks, leading to DNA double-stranded breaks and genomic instability. How transcription–translation coupling mitigates these conflicts is unknown. Here we show that, unlike replication, translation is not inhibited by arrested transcription elongation complexes. Instead, the translating ribosome actively pushes RNA polymerase out of the backtracked state, thereby reactivating transcription. We show that the distance between the two machineries upon their contact on mRNA is smaller than previously thought, suggesting intimate interactions between them. However, this does not lead to the formation of a stable functional complex between the enzymes, as was once proposed. Our results reveal an active, energy-driven mechanism that reactivates backtracked elongation complexes and thus helps suppress their interference with replication. Unlike in eukaryotes, where transcription and translation are separated by a nuclear envelope, in bacteria these processes are coupled. This coupling not only is important for synchronization of the processes and regulation of gene expression –3, but aEscherichia coli; many bacteria have only one Gre factor) can reactivate backtracked ECs by promoting a transcript cleavage reaction at the active site that generates a new 3′ end (The elongation factors GreA and GreB (in w 3′ end . Howeverw 3′ end  and thusfMet in the ribosome P-site) was formed on a 5′-radiolabeled mRNA (mRNA25). The purified M complex was then coupled to transcription by using its mRNA as a transcript in the assembly of a transcription EC with RNAP and complementary template and nontemplate DNA strands. The coupled system was immobilized on streptavidin beads via a biotin tag on the 3′ end of the nontemplate DNA strand, thereby ensuring the presence of only fully assembled ECs and allowing exchange of soluble components by washing of the beads.To investigate the consequences of the encounters of backtracked ECs with a coupled translating ribosome, we developed an in vitro coupled transcription–translation system in which both machineries can be translocated in a controlled stepwise manner and monitored with single nucleotide precision A and B. SI Appendix, Fig. S1A), because of the latter’s attachment to the solid phase (streptavidin bead), as described previously . These results indicate that the backtracked EC does not inhibit coupled translation of the nascent mRNA, but instead, the translating ribosome can actively reverse backtracking.Next, the ribosome was allowed to translate toward the backtracked EC by the addition of individual aminoacylated tRNAs , translocation factor EF-G, and GTP. Because RelE cleavage is most efficient when the A-site contains a UAG stop codon , we placSI Appendix, Fig. S1A). A similar approach was previously used in vivo to measure the proximity of the ribosome to a termination hairpin required to disrupt the hairpin, with the expression level of the gene downstream of the hairpin reporting on the interaction , which stabilized the EC in the posttranslocated state . However, the EC stalled at the abasic site was still able to incorporate an extra nucleotide during walking , and extend mRNA in the presence of the high levels of GTP required for translation , which would affect measurement of the precise distance between the ribosome and RNAP. Therefore, we used 3′-dAMP as the last incorporated nucleotide before the abasic site, which fully abolished unwanted mRNA extension . The presence of 3′-dAMP at the 3′ end of mRNA did not change the translocation state of the EC .The coupled systems on these mRNAs were prepared as shown in SI Appendix, Fig. S1A) or translation was uncoupled , cleavage at this stop codon was restored. The effects of one, two, and three nucleotide deletions and insertions in mRNA22 and mRNA25  revealed that 25 nucleotides between the ribosome’s PTC and RNAP active center was the minimal distance before translocation of the ribosome was physically blocked by the EC.Note that in this experiment, the mRNAs were radiolabeled during transcription via incorporation of radioactive nucleotides into the mRNAs  and “nonstrained” (when ribosome translocation pushes the backtracked RNAP) contacts between them  were performed as described previously  were synthesized and purified as described previously  were obtained from IDT, and NTPs were obtained from GE Healthcare.Primers and the single-stranded DNA template (IDT) used to make a double-stranded template for mRNA synthesis by T7 RNAP are shown in eviously –18. mRNAeviously –18 or rafMet, 200 pmol IF-1, 200 pmol IF-2, 200 pmol IF-3, and 4 mM GTP in 50 μL of coupling buffer  for 10 min at 37 °C. For the experiment shown in Phe TC, 200 pmol EF-G, and 4 mM GTP for 4 min at 37 °C. M or MF complexes were purified by centrifugation through a sucrose cushion as described previously . For the experiments shown in 32P]-UTP , were added for 10 min to radiolabel the mRNA and walk the EC to the abasic site of the template shown in SI Appendix, Fig. S1A. After transcription elongation, the coupled complexes were washed four times with 1 mL of CB. The volume of this master mix was adjusted to allow separation into a number  of 10-μL reactions. For the experiment shown in SI Appendix, Fig. S3A, reactions were supplied with 50 μM inorganic pyrophosphate (Sigma-Aldrich) or with 1 μM GreA or 1 μM GreB for the indicated times at 37 °C.For the experiment shown in SI Appendix, Fig. S4, the reactions corresponding to lanes 3 and 7 of Where indicated, after transcription, translation toward the EC was performed by the addition of 50 pmol of each corresponding TC, 200 pmol EF-G, and 4 mM GTP for 4 min. When indicated, 150 pmol NusG and 40 pmol NusA were added before translation. Then all coupled reactions (including those in which translation was not performed) were washed three times with 1 mL of CB. When indicated, 5 pmol GreB was added for 5 s, and reactions were washed again. Also where indicated, 20 pmol RelE was added for 10 min at 37 °C. For the experiment shown in The efficiency of RelE cleavage at stop codons on different templates is the same and depends only on the availability of a UAG stop codon in the A-site of the ribosome. However, the observed cleavage efficiency also depends on the efficiency of coupling of transcription and translation complexes on each mRNA. Therefore, RelE cleavage before (at the V codon in the MF complex) and after (at the UAG stop codon) translation were quantified as a percentage of the total signal in the corresponding lane, and RelE cleavage at the stop codons was normalized between different mRNAs by dividing them by the efficiencies of RelE cleavage at the V codon in MF complexes on corresponding mRNAs. The data plotted are means, and error bars represent SDs from 3 to 13 experiments.SI Appendix. The plasmids and strains constructed in this study are available on request.All data are presented in the main text and Supplementary File


On the other hand, interactions across different chromosomes  have received less attention, being often regarded as background noise or technical artifacts. This viewpoint has been challenged by emerging evidence of functional relationships between specific trans chromatin interactions and epigenetic control, transcription, and splicing. Therefore, it is an intriguing possibility that the key processes involved in the biogenesis of RNAs may both shape and be in turn influenced by inter-chromosomal genome architecture. Here I present the rationale behind this hypothesis, and discuss a potential experimental framework aimed at its formal testing. I present a specific example in the cardiac myocyte, a well-studied post-mitotic cell whose development and response to stress are associated with marked rearrangements of chromatin topology both in cis and in trans. I argue that RNA polymerase II clusters  and foci of the cardiac-specific splicing regulator RBM20  exemplify the existence of trans-interacting chromatin domains (TIDs) with important roles in cellular homeostasis. Overall, I propose that inter-molecular 3D proximity between co-regulated nucleic acids may be a pervasive functional mechanism in biology.Three-dimensional (3D) genome organization has emerged as an important layer of gene regulation in development and disease. The functional properties of chromatin folding within individual chromosomes (i.e., intra-chromosomal or in  N6-methyladenosine (m6A), but also N1-methyladenosine (m1A), 5-methylcytosine (m5C), and pseudouridine  are 5'-capped, spliced, and finally 3'-polyadenylated and cleaved, altogether generating mature mRNAs. It is well established that these central steps of mRNA biogenesis occur in close proximity and at the same time and place as transcription  involving loci on different chromosomes whose nucleic acids are co-regulated by the same set of protein factors. I further propose that the resulting architecture is not just practical  but also functional, by which I mean that it promotes the efficacy and fidelity of RNA biogenesis.With regards to mRNA biogenesis, over 20years ago Peter Cook and Ana Pombo proposed that transcription may be largely spatially restricted to “transcription factories” . These ctrans, and with some specific examples borrowed from cardiomyocyte biology. Having outlined the supporting evidence, I then flesh out my hypothesis and propose an experimental framework for its formal testing. I conclude by discussing open questions in the area. Throughout I focus on mammalian genome regulation and architecture since it is well established that it involves unique mechanisms not seen in other model organisms such as invertebrates, plants, and yeasts (which in turn have their own peculiarities). Nevertheless, I present selected examples from the broader spectrum of eukaryotic life, as I speculate that the general principle behind the notion of TIDs may be broadly applicable.I begin by extensively reviewing the main features of inter-chromosomal genome architecture. I follow this with a shorter discussion of the possible role of genetic variants in cis) interactions resulting from a variety of hierarchical features at different genomic scales. These include various kinds of DNA loops (such as promoter-enhancer pairing), topologically-associating domains , and A/B compartmentalization (chromosome-wide separation of active/inactive chromatin due to inter-TAD contacts). A large body of work has recently emerged to show that while a large fraction of intra-chromosomal genome structure is largely invariant across cell types and states, dynamic changes do occur at all levels and can contribute to gene regulation both during development and in disease  interactions are much less understood and studied. The motivations are at least three-fold. First, there is a widespread belief in the community that most inter-chromosomal interactions are just noise resulting from Brownian movements of chromatin, and/or technical artifacts arising from proximity ligation-based methods . Indeed, one commonly used quality control measure for 3C-type studies is the ratio of cis/trans interactions, which is expected to be high. While trans interactions are certainly more noisy than cis ones, as discussed below, abundant evidence indicates that there is nevertheless signal worth measuring. Which brings us to the second problem, namely the relative immaturity of statistical and computational approaches to confidently identify trans interactions. Indeed, due to their different biophysical nature trans interactions cannot be simply treated analytically with the now well-established methods developed for cis ones . Finally, compounding on this limitation is the fact that trans interactions are less frequent than cis ones by a factor of ~2–5 (depending on the cell type and assay), while being also spread across a much wider contact matrix 2D space (and even a broader one if we consider three-way interactions). This results in data that is very sparse unless a prohibitively expensive sequencing depth is utilized, particularly in the case of genome-wide studies. Statistical approaches to detect trans interactions have therefore either low power (few data points) or low resolution (genomic bins of large size are used to aggregate the sparse interaction counts). As a result of these three main factors, trans interactions are often dismissed early during the analysis or only used for very basic broad assessments of chromosome territories.In contrast to this, inter-chromosomal , this would still account for 1/20th of the data. Is it not worth to analyze it beyond just confirming the well-established concept of chromosome territories?The well-established concept of chromosome territories is perhaps the motivation leading many to dismiss inter-chromosomal architecture, relegating its possible relevance to “exotic” examples such as sperm cells , this same feat can now be achieved from “conventionally-sized” datasets with moderate coverage using the SNIPER algorithm  each human chromosome possesses a few regions collectively explaining the majority of trans interactions; (2) such regions are characterized by high transcriptional activity; and (3) they interact with each other in a largely indiscriminate fashion. This led the authors to speculate that such domains intermingle chiefly because they happen to all be accessible within the nuclear interior, “escaping” the constraints of their respective chromosome compartments. However, refinements in the quantitative analyses of 3D structure population models showed that some inter-chromosomal interactions are actually relatively specific and liked to gene co-regulation , can extrude to the surface and engage with each other in the proximity of transcription factories, tRNA factories, polycomb domains, the nucleolus, nuclear speckles, and/or other yet-to-be clarified nuclear subcompartments .In the following subsections, I provide a more in-depth review of the nuclear subcompartments that are most relevant to the hypothesis of this article. For this, I focus on structures involved in the biogenesis of mRNAs. The relationship between chromatin organization and other nuclear subcompartments involved in non-coding RNA biogenesis, such as the nucleolus and tRNA factories, is reviewed extensively elsewhere .Trans interactions between active genes may be indiscriminate and dictated by the random motion of open chromatin in the euchromatic nuclear interior, or could be highly specific. A few examples of the latter category have accumulated over the years, indicating that both mechanisms likely contribute to inter-chromosomal architecture.As just discussed, highly transcribed genomic domains are those most often involved in inter-chromosomal interactions. Accordingly, inhibition of transcription reduces chromosome intermingling . Moreovecis- and in trans- often colocalize at the site of transcription factories (Hbb (beta-globin) gene, and 7% for the inter-chromosomal interaction between Hbb and Hba , which is substantially higher than the expected frequency for a random interaction (~1%). In this context cis interactions involving Hbb require initiation of transcription, arguing for an active recruitment model . Remarkably, both the recruitment of Hbb to transcription factories and its colocalization with its trans partners Hba and Epb4.9 requires the erythroid transcription factor Klf1  and a third gene on a different chromosome (SLC6A5) can associate as part of a NF-κB-dependent multigene cluster  had no effect on the formation of RBM20 foci and on the splicing of CAMK2D and CACNA1C. This excludes the possibility that titin is somehow involved in the control of RBM20 localization and/or activity, and indicates that the TTN pre-mRNA has specific trans-acting effects.While much less established, the role of mRNA biogenesis steps besides transcription in inter-chromosomal regulation is beginning to emerge. Indeed, my colleagues and I recently showed that in cardiomyocytes derived from hESCs (hESC-CMs) foci of the muscle-specific splicing factor RBM20 mediate inter-chromosomal interactions between some of its key target loci . We alsong sites . In celltrans-acting “zip coded splicing factory.” Whether this is an exceptional case or the first of many examples of this type of regulation is yet to be determined.Overall, we concluded that RBM20 foci represent the first example, to the best of our knowledge, of a nuclear subcompartment specialized in the regulation of splicing for a defined subset of genes located across multiple chromosomes, in other words, a trans interactions involving a single gene and one or more regulatory sequences that either repress or promote its expression. Repressive regulations include the inter-chromosomal interaction between the T helper cell 2 locus control region (TH2 LCR) and the IFN-γ gene  to an active intra-chromosomal engagement with Sox17 , an important regulator of gene silencing through the deposition of the repressive histone mark histone 3 lysine 27 trimethylation (H3K27me3). PcG factors such as the catalytic subunit EZH2 are involved in both intra- and inter-chromosomal interactions  identified by genome-wide association studies (GWAS) representing non-synonymous substitutions in protein-coding genes . It is gis eQTLs . Neverth30 genes . An even58 genes .trans eQTLs remains unclear, but three major possibilities have been proposed so far (trans eGene), perhaps as a result of an inter-chromosomal regulatory interaction such as those described in the above section “Trans-Acting Regulatory Regions”. Secondly, the gene variant may act indirectly through a third actor: a proximal gene (cis eGene) whose expression is directly regulated by the SNP and that in turn can directly modulate expression of the trans eGene. Proposed mechanisms behind the mediating action of such a cis eGene include its function as a transcription factor, micro RNA, or another regulatory non-coding RNA  or through modulation of a cis eGene. Indirect regulation or reverse causality can be tested through mediation analysis, which assesses whether a third factor (either a cis eGene or a phenotype) significantly accounts for the observed trans eQTL-trans eGene relationship. Using this approach it was found that 20–35% of trans eQTLs can be explained by the alteration of a mediating cis eGene  consortium reported similar observations, as 31.6% of lead trans eQTL identified across 27 tissues proved to be also cis eQTL, and 77% of these were predicted to contribute to the trans effect by mediation analysis . On the other hand, the authors found that approximately one third (13/37) of these cis eGenes shared the same regulatory motif with the trans eGenes. A similar enrichment of transcription factor motifs in the promoter of trans eGenes linked to trans eQTL has also been reported elsewhere  some TRHs mediate trans eQTL effects. Along these lines, trans eQTLs identified by the GTEx consortium are specifically enriched for CTCF binding sites , suggesting that trans contacts were not inflated by suboptimal capture of the native chromatin environment. A high proportion of trans interactions in cardiomyocytes was observed also in other in situ Hi-C experiments in cardiomyocytes from other hESC lines . Such correlation was even more remarkable for left ventricle trans eQTL gene clusters, reaching 77.2%. Therefore, cardiomyocyte gene expression seems to be particularly affected by genetic variation in trans, providing further evidence for the possible role of inter-chromosomal genome organization in this cell type.As discussed in the above section “ samples . On the 50 genes . A simil samples . Notablyper se. In fact, the contribution of transcription factories to 3D genome topology has been hypothesized at least a decade ago  since some of the underlying mechanisms (described in the next subsection) likely translate to the specific regulations involved in the biogenesis of various classes of RNAs. Moreover, while this hypothesis is articulated specifically for inter-chromosomal regulations, it likely also extends for intra-chromosomal ones. Indeed, there is abundant evidence that ed genes .Trans-Acting Regulatory Regions” and “Polycomb Domains”) and of “zip coded” transcription factories (section “Transcription Factories”), but are not limited to this. Further regulation can be achieved by pre-mRNA processing steps in both specialized nuclear speckles (section “Nuclear Speckles”) and tissue-specific splicing factories (section “Splicing Factories”). Moreover, I anticipate that future studies will reveal how other aspects of RNA biogenesis, such as RNA post-transcriptional modifications, are similarly involved in regulating inter-chromosomal architecture.With regards to the first statement, relevant RNA biogenesis steps include the regulation of transcription, as exemplified by the existence of specific regulatory inter-chromosomal interactions , chromatin modifiers , genome organizers , the transcriptional machinery , splicing regulators , and other protein factors involved in various steps of RNA biogenesis. RNA species with a structural and/or enzymatic role are also likely involved, though this will need to be determined by further studies.trans-interacting (chromatin) domain” (TID). This acronym purposely reminisces TAD in order to stress the conceptual similarity between these structural features of the genome. Similarly to TADs, TIDs are statistical constructs that can be observed only at a population level , the probability of intra- and inter-chromosomal interactions becomes similar. Therefore, very long-range intra-chromosomal interactions may be effectively considered analogous to inter-chromosomal ones when computing and studying TIDs.While the term “RNA factory” describes the overall subnuclear structure, I propose that the genomic loci involved could be referred to as a “on level . In indiI propose a three-step process :Trans-acting regulatory factors bind onto core co-regulated nucleic acids.Regulatory factors from multiple nucleic acids aggregate to form new clusters and/or enrich pre-existing ones.Accessory nucleic acids are brought into proximity the cluster until a dynamic equilibrium is reached.trans-acting regulators include transcription factors, epigenetic remodelers, genome organizing proteins, and Pol II and its cofactors, which engage with DNA either directly or through its associated histones. For splicing factories, the key trans-acting factors, namely splicing regulators, require prior initiation of transcription to generate the target RNA molecules. In both cases, multiple binding sites are found on either the same or multiple molecules (in the case of RNAs). The model stipulates that one or few nucleic acids with the highest density of binding sites function as “core” element(s), dictating the local accumulation of the regulators.Step one involves different factors for distinct classes of RNA factories. For transcription factories, Step two is the result of either active mechanisms  and/or Brownian movements of nucleic acids extruding from the core of chromosomal territories. Changes in the local chromatin environment may facilitate this process and increase intermixing in the inter-chromosomal space. Stabilization of core inter-chromosomal interactions involve either high-affinity interactions between factors that can form dimers and/or higher-order multimers , as well as weaker interactions such as those involving low-complexity regions characteristic of proteins that can undergo liquid-liquid phase separation . In someIn step three, large and growing clusters of regulators stabilize additional inter-chromosomal interactions with co-regulated nucleic acids characterized by lower binding density for the regulators. These “accessory” DNA or RNA molecules are those that most strongly benefit from interfacing with the RNA factory, which augments the efficiency of their regulation. As the RNA factory grows in size, it eventually hits steric and thermodynamic limitations that stabilize its further growth to a state of dynamic equilibrium.Throughout the process, regulators can be either general and housekeeping, binding to nucleic acids with low degree of specificity or no specificity at all , or highly specific and responsive to cell states . The resulting RNA factory is more or less cell state-specific depending on the relative balance of these two classes. “Zip coded” transcription and/or splicing factories result from highly specific regulators enriching for their target nucleic acids.Besides descriptive investigations, at least three types of perturbations can be used to test the model -D:Depletion of core nucleic acids.trans-acting regulatory factors.Addition of clustered binding sites for trans-acting regulatory factors.Depletion of trans-acting regulatory factor(s) to one or more of the core co-regulated nucleic acids. For DNA loci, this can be achieved by mutations or larger deletions. For RNAs, this can be achieved by mutations of the originating genes to prevent their transcription, or through epigenetic silencing. For both DNA and RNA perturbations, an important control experiment is to reduce the protein levels of the gene(s) of interest by a similar magnitude as the test perturbation, but without affecting the abundance of binding sites for the trans-acting regulatory factors. This is key to exclude that any observed effect is not simply due to impairment of intermediate protein-mediated regulations. For instance, one can generate knockout and/or hypomorph mutations that do not alter the DNA sequence in the regions of interest and/or do not alter RNA levels. The predicted outcomes of this perturbation are: (1) impaired clustering of the trans-acting regulators; (2) impaired inter-chromosomal interactions between loci in the TID; and (3) decreased efficiency in the regulation for accessory nucleic acids (The first experiment involves reducing or completely preventing the binding of ic acids .trans-acting regulatory factor(s). For instance, one can express transgenic copies of a core nucleic acid or an artificial sequence containing arrayed binding sites. In the former case, the transgene can be mutated to prevent protein translation and bypass the need to control for this aspect. An RNA can also be upregulated through epigenetic mechanisms. The predicted outcomes of this perturbation are opposite to those outlined above . Unfortunately, most of the analytical tools for 3C-type data developed so far have focused only on intra-chromosomal interactions (reviewed in cis and trans interactions (trans interactions in mammalian systems, such as their enrichment for loci in the A compartment and their different distribution across small and large chromosomes. A similar limitation applies to existing network-based strategies to study inter-chromosomal interactions both from bulk Hi-C (trans interactions resulting from the random intermixing of neighboring chromatin domains.The identification of significant inter-chromosomal interactions must rely on statistical approaches that are specifically designed for this task. Indeed, approaches originally devised for the analysis of ractions . This apulk Hi-C  and singulk Hi-C . StatistAnother promising venue is the modeling and study of 3D genome structure populations, which as discussed in the section “Nuclear Subcompartments” has already provided important insights in inter-chromosomal genome architecture . Neverthtrans-acting factors that contribute to their formation and/or function. For transcription factories, the enrichment of DNA binding motifs within the regulatory regions proximal to the involved loci may provide testable hypotheses as to the transcriptional regulators involved. RNA binding motifs enrichment on pre-mRNAs may similarly help in the context of RNA splicing factories. Further evidence could be gathered by integration of data modalities directly probing for DNA and/or RNA binding of candidate factors. However, these analyses are limited by the availability of well-annotated binding motifs and/or of experimentally determined binding profiles for the cell state of interest.A second key challenge towards clarifying the role of RNA factories is the identification of the in situ proximity labeling, for instance by relying on promiscuous biotinylating enzymes to covalently tag nearby proteins for subsequent streptavidin-based purification and identification through mass spectrometry. These methods have been successfully used to determine protein-protein interactions, and their implementation to study locus-specific regulations are also emerging (reviewed in Robust methods to unbiasedly identify factors that interact with specific nucleic acids are direly needed. Biochemical purification approaches relying on direct capture of nucleic acids have been developed, but so far showed limited sensitivity and specificity (reviewed in Last but not least, the functional characterization of RNA factories is key to determining their physiological relevance. As elaborated in the section “Experimental Testing Strategies and Predicted Outcomes”, mechanistic studies may involve various means of perturbing RNA factories through loss- and/or gain-of-function. While these studies do not pose unsurmountable challenges due to recent advancements in genome editing technologies, they remain time-consuming. Characterization of putative RNA factories may be performed with more throughput by leveraging on functional screening strategies based on the combination of CRISPR/Cas9 perturbations and transcriptional phenotyping through single-cell RNA-sequencing (reviewed in trans-acting regulators onto core nucleic acid targets, leading to their aggregation and to the subsequent recruitment of accessory co-regulated nucleic acids. Future studies will prove, disprove, or modify this model. It will also be key to test the pervasiveness of such regulation to determine whether it involves only a few specific nucleic acids or it is a general property controlling many loci.In summary, emerging evidence suggests a functional bidirectional link between the regulation of RNA biogenesis and inter-chromosomal 3D genome architecture. In other words, the formation of RNA factories organized around TIDs may be not just an emergent property of gene regulation, but also a feature needed to maximize the fidelity and efficiency of such regulations. This is a testable hypothesis based on a clear mechanistic model: the clustered binding of via a combination of specific and non-specific interactions, possibly leading to liquid-liquid phase separation (reviewed in Many key open questions remain in this area. First off, what is the biochemical and/or biophysical mechanism that explains the co-localization of certain nucleic acids from different chromosomes? Besides transcription factors , 2018, sIs dysfunction of RNA factories and/or TIDs involved in the pathogenesis of human disease? Cancer has been linked to alteration of chromosome territories  and of sMoving beyond RNA biogenesis, is proximity between co-regulated nucleic acids a general phenomenon in biology? It is tempting to speculate that other key biochemical reactions such as DNA repair and replication may also benefit from the increased efficiency and fidelity led by local accumulation of relevant regulators. Overall, increasing our spatial awareness of nuclear regulations may prove a worthy and impactful pursuit.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This review summarizes and highlights the latest advances of histone acetylation dynamics in various systemic and environment-responsive signaling pathways in plants. As sessile organisms, plants face versatile environmental challenges and require proper responses at multiple levels for survival. Epigenetic modification of DNA and histones is a conserved gene-regulatory mechanism and plays critical roles in diverse aspects of biological processes, ranging from genome defense and imprinting to development and physiology. In recent years, emerging studies have revealed the interplay between signaling transduction pathways, epigenetic modifications, and chromatin cascades. Specifically, histone acetylation and deacetylation dictate plant responses to environmental cues by modulating chromatin dynamics to regulate downstream gene expression as signaling outputs. In this review, we summarize current understandings of the link between plant signaling pathways and epigenetic modifications with a focus on histone acetylation and deacetylation. Within the nucleus of eukaryotic cells, DNA wraps around histone octamers in a left-handed superhelix to form highly conserved repeating nucleoprotein complexes called nucleosome cores. Each nucleosome core contains a 145–147 bp stretch of DNA and two copies of each core histone, H2A, H2B, H3, and H4 . The priPseudomonas syringae DC3000 (Pst DC3000) triggers both DNA hypomethylation and hypermethylation at thousands of genes, among which many are correlated with expression changes in Arabidopsis (Oryza sativa) (FLOWERING LOCUS C (FLC) locus, thus promoting FLC expression and inhibiting flowering  domain-containing protein POWERDRESS (PWR) and the WD40 repeat protein HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES15 (HOS15) . A notabpwr and hda9 mutants show attenuated warm temperature-induced hypocotyl elongation. Treatment with the HDAC inhibitor trichostatin A (TSA) diminishes temperature-induced hypocotyl elongation, suggesting that thermomorphogenesis requires deacetylase activity. At high temperatures, the PWR–HDA9 complex maintains low H3K9/K14 acetylation levels to facilitate the eviction of the histone variant H2A.Z from the nucleosome, thus providing a permissive chromatin environment for the basic helix–loop–helix (bHLH) factor Phytochrome-Interacting Factor4 (PIF4). PIF4 binds to the promoter of YUCCA8 (a key auxin biosynthesis gene) and activates YUCCA8 expression and auxin biosynthesis  and PWR  genes. Upon cold treatment, HD2C is ubiquitinated by an E3 ubiquitin ligase complex containing HOS15 and degraded via the proteasome system, leading to COR chromatin hyperacetylation , coupled with increased SA content and resistance to the Pst DC3000 infection in hda19, suggest that HDA19 acts as a negative regulator of SA-dependent pathogen resistance genes such as PR1  interacts with and phosphorylates HD2B. Phosphorylated HD2B shuttles from the nucleolus to the nucleoplasm -triggered immunity signaling (leoplasm  (LatrassATG5 and ATG8e) to initiate H3K9/K27 deacetylation and transcription suppression. Darkness induces HY5 degradation and dissociates HDA9 from ATG5 and ATG8e chromatin to release the transcriptional inhibition  showed reduced BR sensitivity and suppressed the gain-of-function bzr1-1D mutant phenotype. Consistently, TSA treatment decreases sensitivity to BR, suggesting an essential role for HDAC in BR response (CUP SHAPED COTYLEDON3 (CUC3), BRASSINOSTEROIDS AT VASCULAR AND ORGANIZING CENTER (BRAVO), and DWARF4 (DWF4) via BES1/BZR1 to regulate organ boundary formation, root quiescent center cell division, and the BR feedback regulation, respectively (TPL/TPR co-repressors function through interactions with Ethylene response factor-associated Amphiphilic Repression (EAR) motifs that are found in many transcription factors  Long et. In the ectively   to maintain the undifferentiated status of root stem cells  functions with TPR/TPL and HDA19 to silence the differentiation factor HDA6, a close homolog to HDA19, is also involved in hormone signaling. HDA6 interacts with both ethylene-stabilized master transcription factors, ETHYLENE INSENSITIVE 3 (EIN3) and EIN3-LIKE 1 (EIL1), and JA-degraded JAZ to regulate JA-induced derepression of ethylene-responsive genes . As HDA6RHO OF PLANTS (ROP) genes through promoting H3 and H4 deacetylation at their promoters  after its peak expression during the evening. HDA9 specifically binds to the TOC1 promoter through the interaction with ELF3 to deacetylate H3 at the TOC1 locus and inhibit TOC1 transcription at night (GIGANTEA (GI) promoter to regulate the photoperiodic flowering pathway (CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) to restrict their expression to near dawn , PtrAREB1 interacts with and recruits the ADA2b–GCN5 complex to drought-responsive genes , resulting in enhanced H3K9ac and RNA Pol II enrichment and activated transcription  . ADA2b iruitment . In ABA cription  , POLYGALACTURONASE INVOLVED IN EXPANSION3 (PGX3), and MYB54 to mediate histone acetylation and transcriptional activation -induced genes and basal resistance, as the repetitive stress-induced priming is lost in the hac1 mutant (GPT2), DIHYDROFLAVONOL 4-REDUCTASE (DFR), and CHALCONE SYNTHASE (CHS), resulting in H3K27 hyperacetylation and transcriptional activation   accumulation after UV-B exposure, suggesting that HAM1 and HAM2 are required for DNA repair (HAG3-RNAi plants displayed low leaf and root growth inhibition by UV-B irradiation, high levels of UV-B-absorbing compounds, and little UV-B-induced DNA damage (haf1 mutants show less growth inhibition by UV-B than the wild type (haf2 mutants show decreased chlorophyll accumulation and light-induced gene expression (HAM1 and HAM2 are also required for DNA repair after damage by UV-B (A damage . Similargcn5 mutant shows hypersensitivity to ethylene treatment and elevated H3K9ac and H3K14ac levels in the promoter regions of ethylene response genes that are accompanied by increased gene expression (hac1 hac5 also shows hypersensitivity to ethylene constitutive triple response with elevated levels of transcription of ethylene-responsive genes (Ethylene has been reported to trigger the elevation of histone acetylation at H3K14, H3K23, and H3K9, especially at ethylene-responsive genes . The incpression . The douCOR genes (ada2b and gcn5 mutants show similar H3 acetylation increases to wild-type plants after cold acclimation (Histone acetylation has also been implicated in cold response. The cold-responsive transcription factor CBF1 interacts with ADA2b, and overexpression of CBF1 results in H3 hyperacetylation at Through intensive and fruitful studies over the past several decades, it has become clear that epigenetic modifications and chromatin dynamics are integral parts of signaling pathways that bridge the gap between transcription factors, downstream gene regulation, and transcription machinery for signaling output. This review summarizes the recent studies articulating the roles of HDACs and HATs in various signaling pathways (for a list of histone deacetylases and acetyltransferases in this review, see


Transposable elements constitute about half of the mammalian genome, and can be divided into two classes: the class I (retrotransposons) and the class II (DNA transposons). A few hundred types of retrotransposons, which are dynamic and stage specific, have been annotated. The copy numbers and genomic locations are significantly varied in species. Retrotransposons are active in germ cells, early embryos and pluripotent stem cells (PSCs) correlated with low levels of DNA methylation in epigenetic regulation. Some key pluripotency transcriptional factors  bind retrotransposons and regulate their activities in PSCs, suggesting a vital role of retrotransposons in pluripotency maintenance and self-renewal. In response to retrotransposons transposition, cells employ a number of silencing mechanisms, such as DNA methylation and histone modification. This review summarizes expression patterns, functions, and regulation of retrotransposons in PSCs and early embryonic development. Pluripotent stem cells (PSCs) are cells that have potential to differentiate into various cells and tissues. The most important properties of PSCs are self-renewal and differentiation into adult stem cells or multiple somatic cells during development  , also known as transposons, were first discovered in 1948 by geneticist Barbara McClintock Mc, . She fouRetrotransposons are commonly classified into two subclasses with variant characters and functions. Subclass I consists of viral retrotransposons characterized by long terminal repeats (LTRs) on both terminals. They encode reverse transcriptase and change position like retroviruses, but lack a functional envelope gene (Env). They constitute about 8% of the human genome and approximately 10% of the mouse genome , suggesting specific roles during fertilization   until adulthood  can be detected in both ESCs and iPSCs, caused by an overall decrease in CpG methylation in the L1 promoter region  display a fully repressed state, similar to expression patterns in ESCs . DNA methylation levels regulate retrotransposon expression in ESCs and iPSCs. DNA methylation enzymes  are responsible for DNA methylation. Double knockout of DNMT3a/3b causes global loss of DNA methylation, and over 98% of the loss overlaps with repeat sequences  and KRAB-associated protein 1 -regulating retrotransposon silencing has been explored extensively. KRAB-ZFPs recognize specific retrotransposon sequences and recruit KAP1 to repress transcription of the targeted retrotransposon and adjacent genes. KAP1 repressed genes by recruiting histone methyltransferase SETDB1, HP1, and the NuRD histone deacetylase complex. KAP1 deletion decreased the level of H3K9me3, and upregulated ERVs, partial IAP elements, and genes such as In post-transcription regulation, the functions of silencing TEs by microRNA (miRNA), short interfering RNA (siRNA), and the P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA) pathways have been confirmed Fig. b. TissueTEs comprised a great part of the genome, but were previously considered useless. With more and more researches, we have recognized that few retrotransposons still work even though a majority of them have been truncated during genome evolution. Although retrotransposons are usually regulated by small RNAs, DNA methylation, and histone modifications, the global genome is in a more open state in PSCs compared to somatic cells, providing opportunities for repeat sequence transcription, and retrotransposons take part in gene regulation by actively reshaping chromatin structure or providing transcription factor binding sites.The vast majority of retrotransposon functions are not very clear, but specific expression patterns of each family and subfamily and their effect on the genome can be observed. It is worth mentioning that different iPS cell lines have different transcription levels in some transposon classes, indicating heterogeneity of cell lines. Moreover, although ESCs and iPSCs share similar repetitive element expression, differences may exist due to variations in epigenetic modifications. As of yet, we still do not have a detailed protocol to evaluate a good iPS cell line. Retrotransposon transcription level may be considered as a criterion if it proves to work well in cell therapy. Furthermore, active or repressive types of transposon expression may be a new way to induce cells into the desired stage or condition, thus having potential in model construction and tissue differentiation.Despite having benefits, the random insertion of transposons may cause genome instability and possibility of affecting promoter function, gene transcription, and pre-mRNA slicing, which happens more frequently in germ cells and early embryos because of the low level of repressive epigenetic regulation. LINE1 is associated with tumorigenesis in several cancers and human genetic diseases caused by Alu element homologous recombination (reach up to 0.3%) (Ahmed et al., Aberrant transposon activation leads to genome instability and carcinogenesis, thus, the mechanism of transposons regulation needs to be analyzed in detail. Apart from the classic regulation mechanism, more and more epigenetic modifications have been discovered, such as histone succinylation, crotonylation, 6 mA DNA and m6A mRNA modifications. As there are no detailed studies about the relationship between these new modifications and TE regulation, more regulation mechanisms need to be elucidated.To date, many TEs are still not recognized, and the sequences may have changed with genome evolution. Functions of enhancers are not easy to detect because of the complex spatial structures of chromatin. TEs are a large family and have multiple functions and with the development of new technologies, we will be one step closer to understanding them and their regulation.


Escherichia coli, the secondary channel factors (SCFs) GreB and DksA both repress ribosomal RNA (rRNA) transcription, but SCF loading and repression mechanisms are unclear. We observed in vitro fluorescently labeled GreB molecules binding to single RNAPs and initiation of individual transcripts from an rRNA promoter. GreB arrived and departed from promoters only in complex with RNAP. GreB did not alter initial RNAP-promoter binding but instead blocked a step after conformational rearrangement of the initial RNAP-promoter complex. Strikingly, GreB-RNAP complexes never initiated at an rRNA promoter; only RNAP molecules arriving at the promoter without bound GreB produced transcript. The data reveal that a model SCF functions by a ‘delayed inhibition’ mechanism and suggest that rRNA promoters are inhibited by GreB/DksA because their short-lived RNAP complexes do not allow sufficient time for SCFs to dissociate.RNA polymerases (RNAPs) contain a conserved ‘secondary channel’ which binds regulatory factors that modulate transcription initiation. In  Escherichia coli uses at least five distinct GreA-like SCFs . GreB stimulates cleavage of RNA that has reverse threaded through the active site (backtracked) by multiple nucleotides, for instance after nucleotide misincorporation at the RNA 3′ end (70-paused ECs (Multi-subunit RNA polymerases (RNAPs), the molecular machines responsible for the synthesis of cellular mRNAs, contain catalytic subunits that are conserved across all domains of life . In addiike SCFs . Althougructures , they haA 3′ end . By causA 3′ end  and by aused ECs .inhibit initiation by RNAP prior to phosphodiester bond formation at certain promoters with short-lived RNAP-promoter complexes such as the ribosomal RNA (rRNA) rrnB P1 promoter  or positively at other promoters   also can promoter . This Grongation , but it r PhisG) . Productr PhisG) . DksA biresponse ; this efresponse . There aresponse .Although much study has focused on stringent regulation of rRNA production, cells must also maintain appropriate levels of rRNA during rapid-growth  conditions, conditions in which rRNAs are the most abundant transcripts produced. A growth rate-dependent control  system mrrnB P1 promoter complexes at different stages in the process of initiation. Here we used both fluorescence correlation spectroscopy (FCS) and colocalization single-molecule spectroscopy (CoSMoS)  cells. Our results suggest that GreB binds to core RNAP and σ70RNAP holoenzyme an order of magnitude more tightly than previously thought and that GreB/DksA competition for binding to RNAP free in the cytoplasm likely determines the relative importance of these two factors in modulating rRNA initiation under rapid growth conditions. Using GreB as a model to investigate the mechanism(s) of SCF•RNAP binding and action at rrnB P1, we reveal the ‘delayed inhibition’ mechanism for SCF loading onto RNAP and suggest how short-lived promoter-RNAP complexes are selectively inhibited by SCFs.To elucidate the mechanism(s) of rRNA initiation regulation by DksA and GreB, it is essential to define how these SCFs load onto RNAP and modulate the kinetics of (CoSMoS) , a multi70RNAP holoenzyme are lacking. To begin investigating the effects of GreB on transcription initiation, we first measured the equilibrium dissociation constant (KD) for the GreB•σ70RNAP complex (Cy3 construct shown to be fully active in transcript cleavage assays (Cy3 (fast-diffusing species) from σ70RNAP•GreBCy3 complex (slow-diffusing species) . Autocorrelation curves obtained for σ70RNAP yielded  the diffusion constant of GreBCy3 bound to σ70RNAP (10–20 μm2 s−1) and fraction GreBCy3 bound. A hyperbolic fit to the fraction bound data at varying σ70RNAP concentrations revealed tight binding with KD = 10 ± 1 nM  , indicating that the high binding affinity is not an artefact of dye labeling.Although the dynamics of GreB interactions with ECs have been directly observed and quantitatively characterized , analogo complex  using fle assays . The larspecies) . The aut0 ± 1 nM . Similar0 ± 1 nM . These m binding  and an o binding ; Table 1oenzyme) . GreBCy3Cy3•σ70RNAP complexes by addition of 100-fold excess unlabeled wild-type GreB , and that dissociation was 0.2 s−1 or slower. The KD values derived from the kinetic parameters for the surface-tethered RNAP are close to those measured in solution by FCS  or very high affinity  binding of the proteins to σ70RNAP inhibits rrnB P1 initiation, we measured the rate of the overall initiation pathway  at low SCF concentrations equal to these KDs. In a CoSMoS experiment  concentrations GreB and DksA equally inhibited initiation in vitro from the rRNA promoter µM ppGpp . HoweverµM ppGpp  and thesperiment , templatand NTPs . The reaand NTPs . As expeand NTPs , yieldinKD in place of GreB.  and 10 nM GreBCy3B to the surface-tethered rrnB P1 DNA. The experiments  of the eriments  were coneriments . As expeomplexes , red. Coomplexes . Separatproteins .70RNAP647 molecules arrived at DNA locations in complex with GreBCy3B molecules (orange highlights in Cy3B fluorescence (purple highlights). Even though essentially all GreBCy3B molecules are labeled , this result is nevertheless expected since the experiment is conducted at 10 nM GreBCy3B, the concentration at which only 50% of the polymerase molecules in solution have GreB bound. The fraction of σ70RNAP647 molecules observed to arrive simultaneously with GreBCy3B agrees with that expected ×107 M−1 s−1). This binding is specific -- it is much larger than the rate constant for background GreBCy3B binding to DNA in the absence of σ70RNAP ×104 M−1 s−1) – and is comparable to the rate constant for GreB binding to holoenzyme , which would also eliminate the recruitment pathway. Thus these data suggest that GreB suppression of rrnB P1 initiation is essentially completely controlled by GreB binding to σ70RNAP off DNA rather than GreB binding to initiation complexes, and that this conclusion holds in both high (>>KD) and low (<<KD) GreB concentration regimes.Of the holoenzyme molecules that arrived at the promoter without bound GreB, very few recruited GreB after arrival. In particular, at 10 nM GreBloenzyme . However70RNAP dissociated  of the initiation pathway. However, we also saw no substantial alteration of σ70RNAP647 dwell times on rrnB P1 when GreB or DksA was present  such as promoter complex isomerization, initial RNA synthesis, or promoter escape . In thisociation . Insteadex decay . The datex decay  in whichrrnB promoter complexes during those stages. In our experiments, GreB was almost never observed to bind to or depart from σ70RNAP-promoter complexes. Instead, GreB arrived only at promoters that were not already bound by σ70RNAP and arrived only in complex with σ70RNAP. Similarly, it left the promoter only as a σ70RNAP•GreB complex. However, rRNA promoter complexes are atypically short lived  selectively inhibits initiation at promoters that have short-lived promoter complexes (Perhaps unexpectedly for a factor affecting only later stage(s) of initiation, we find that GreB does not function by binding to rt lived . On moreomplexes . On promrrnB suggests that competition between SCFs for binding to σ70RNAP in solution  may determine which SCFs are most important for rRNA transcription regulation under various growth conditions. Here we used three different methods to assess the affinity with which GreB binds to both core RNAP and σ70RNAP. First, we used FCS to measure the affinity of GreB for core and σ70RNAP  and the ratios of unbound  SCF concentrations, which are not known. The two proteins may have redundant functions in growth-rate dependent rRNA regulation, but the phenotypes of genetic deletion strains  from the value of a in Interestingly, the interaction of GreB with free RNAP may be more complex than a simple one-step binding mechanism. Single-molecule kinetic measurements show a biexponential dwell time distribution of GreB on surface-tethered core and σ70RNAP , indicat70RNAP, rather than to initiation complexes, is the critical factor that determines the relative importance of each SCF in inhibiting initiation from rRNA promoters. The data reveal that GreB functions by a ‘delayed inhibition’ mechanism, and imply that DksA functions by the same mechanism. Based on this mechanism, we propose that rRNA promoters are selectively inhibited by SCFs because their short-lived RNAP complexes do not allow sufficient time for SCFs to dissociate.All stages of the transcription cycle are thought to be modulated by SCFs, and each SCF is presumed to compete for the same binding site on RNAP. Therefore, knowing the steps in the transcription mechanism at which SCFs load onto and dissociate from RNAP, as well as the dynamics of this interaction and the manner in which SCF presence affects the kinetic properties of RNAP while bound, can illuminate how these factors are able to exert specific regulatory effects at precise stages in the cycle. Using GreB as a model SCF, we propose based on our data that transcription factor binding to free σ5′-/56-TAMN/gcgtgtttgccgttgttccgtgtcagt-3′ and 5′-/56-TAMN/tctcaggagaaccccgctgacccggcg-3′), or with AlexaFluor488 (AF488) (5′-/5Alex488N/gcgtgtttgcc gttgttccgtgtcagt-3′ and 5′-/5Alex488N/tctcaggagaaccccgctgacccggcg) .Transcription initiation was detected by hybridization of two fluorescently-labeled oligonucleotide probes to the nascent transcript. Probes were fluorescently labeled with either 5(6)-Carboxytetramethylrhodamine (TAMRA) (E. coli rrnB gene from positions −65 to +249 (relative to the transcription start site defined as +1) and included the full rrnB P1 including the upstream promoter (UP) element. The template was synthesized by PCR using AF488- and biotin-labeled primers 5’-/AF488/GCGGTCAGAAAATTATTTTAAATTTCC-3’ and 5’-/5Biosg/CGTGTTCACTCTTGAGACTTGGTATTC-3’ (IDT DNA), E. coli genomic DNA from strain MG1655 (ATCC), and Herculase II Fusion DNA polymerase (Agilent) according to the manufacturer’s protocol.The 314 bp AF488-labeled transcription template consisted of the 2)-PEG-biotin and commercially available ATTO 488 NHS ester . The synthetic intermediate BG-Lys(NH2)-PEG-Biotin was obtained from successive couplings of commercially available α-N-Fmoc-ε-N-Dde-lysine  with BG-NH2  and N-(+)-biotin-6-aminocaproic acid N-succinimidyl ester  by the synthetic route described previously (2)-PEG-biotin  was dissolved in anhydrous DMF (1 mL). ATTO 488 NHS ester  and triethylamine  were added and the reaction mixture stirred overnight at room temperature. The solvent was removed under vacuum and the product purified by reversed-phase HPLC on a VYDAC 218TP series C18 column  at a flow rate of 20 mL/min using a 0.1 M triethyl ammonium bicarbonate/acetonitrile gradient. HPLC fractions containing the product were lyophilized to afford 1.13 µmol (43% yield) of BG-488-PEG-biotin. A high-resolution mass spectrum of BG-488-PEG-biotin was recorded by electrospray ionization (ESI) on an Agilent 6210 Time-of-Flight (TOF) mass spectrometer: ESI-TOFMS m/z 756.7879 [M + 2H]2+ .The bifunctional benzylguanine (BG) adduct BG-488-PEG-biotin (which includes both an ATTO 488 dye and a biotin moiety) was prepared by coupling of synthetic intermediate BG-Lys(NHeviously . BG-488-E. coli core RNAP (α2ββ′ω) with a SNAP-tag on the C-terminus of β′ ; labeled protein was subjected to two cycles in which the protein was concentrated ~4 fold and then re-diluted to its original volume. To prepare σ70RNAP647 or σ70RNAP488-PEG-biotin, labeled core RNAP was incubated with σ70 as described  or contained a relocated cysteine  and were purified as previously described . rcGreB eviously  and GreBescribed  with thee assays  and its eriments .6 Hz and 1–2 × 106 photons were collected per run.All FCS data was collected in a custom one-photon confocal instrument . In brieo) and height (2zo) were determined by acquiring data with a standard dye, rhodamine-6G (Rh6G) using a diffusion coefficient, DRh6G, of 280 μm2/s (Rh6G may be closer to 400 μm2/s (Rh6G has no effect on our determinations of the fraction of bound GreB (boundf) or the associated dissociation constant (DK), but could result in underestimation of the diffusion coefficients of free and bound GreB. For all FCS acquisitions with GreBCy3, ωo and S = oz/ωo were held constant when fitting the autocorrelation traces.The beam width  of Cy3 does not change upon binding, the correlation curves, G(τ) may be fit to<C> is the time averaged concentration in the observation volume, fi is fraction of species i and Di is the diffusion coefficient of species i. The diffusion coefficient of GreBCy3 (DGreB) was obtained by fixing f = 1.For equilibrium binding measurements, fluorescence fluctuation data was first collected for GreBreagents . Autocor70RNAP was then added at a concentration (RT) ranging from 1 to 100 nM to a fixed C = 10 nM concentration of GreBCy3. Fluorescence records were collected at each RNAP concentration, and the autocorrelation was obtained as described above. Curves were then fit to DGreB to obtain N, fbound, and the diffusion constant of the GreB·RNAP complex DGreB·RNAP. Standard errors of the fit parameters were obtained by bootstrapping.Core RNAP or σRbound, isThe concentration of RNAP bound, Rfree, was calculated asTherefore, concentration of free RNAP, KD was measured by performing a standard error weighted fit of the pairs of fbound and Rfree values from measurements at different RT to the Langmuir equationSingle-molecule fluorescence microscopy experiments were conducted using a multi-wavelength micromirror total internal reflection fluorescence microscope  equippedCoSMoS experiments were conducted in flow chambers  using glInitiation experiments were performed essentially as described previously  except tCy3B to surface-tethered RNAP was measured by incubating the chamber with ~20 pM RNAP488-PEG-biotin until a sufficient surface density of fluorescent spots   . The acceviously , except rrnB P1 templates tethered to the chamber surface was measured as described previously (kinit) were corrected for the rate of nonspecific TAMRA-probe or AF488-probe binding (kbkgnd) determined by analyzing randomly selected surface locations without AF488-labeled DNA molecules from the same recording. In Af of these curves was fixed to the value obtained in the experiment without SCF.The rate of transcription initiation on eviously . Initiat70RNAP647 to rrnB P1 DNA locations in the absence and presence of SCFs was determined in the absence of NTPs essentially as described previously , the second-order association rate constant for promoter DNA binding (ka), and the fraction of DNA molecules that were active in binding (Af)  . The disescribed . Photobleviously  and were In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.eLife. Your article has been reviewed by Kevin Struhl as the Senior Editor, a Reviewing Editor, and two reviewers. The reviewers have opted to remain anonymous.Thank you for submitting your article "Delayed inhibition mechanism for secondary channel factor regulation of ribosomal RNA transcription" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. As you can see from their comments, both reviewers are enthusiastic about the work. None of the issues they raise were seen as critical to the study, but would strengthen the manuscript if addressed.Summary:rrnB P1 promoter. Using FCS and single-molecule surface assays the authors show that GreB binding to core or holoenzyme RNAP is tighter than previously thought (dissociation constant 10 nM). Using multiwavelength single-molecule assays the authors then demonstrate identical binding of both RNAP holoenzyme and RNAP holoenzyme complexed with GreB to the rrnB P1 promoter in the absence of nucleotides, and complete inhibition of escape from the rrnB P1 promoter of GreB-bound RNAP holoenzyme. The authors also examine the similar role of DksA and the synergistic, inhibitory role of ppGpp.Stumper et al. use single-molecule fluorescence experiments to study RNAP control through the secondary channel, providing a detailed view of the regulatory mechanism that could not be obtained from bulk experiments. The work is an elegant demonstration of the inhibitory role of GreB in transcription initiation from the classical Essential revisions:NoneMinor points:1) The authors extend their conclusions to other SC factors, arguing that they bind to the enzyme similarly and all effects will be controlled by occupancy. It could be more interesting to point out differences and it is possible that some SC factors are not exchanging that rapidly. In their recent study , the authors found that a cleavage-deficient D41N GreB remained bound to the elongation complex much longer. A "native" Gre-like Gfh, which inhibits catalysis, could have similar properties.2) The authors stress the point that GreB arrives and departs with the holoenzyme, instead of binding to the initiation complexes. This could in part be explained by the low stability of these complexes, and maybe this point could be made clearer. Alternatively, this result may suggest that GreB affinity for the DNA-bound RNAP is lower. Experimentally, this could be tested by including ATP which is expected to stabilize an otherwise absent open complex. This may also allow distinction between GreB effects on open complex vs nucleotide addition.E. coli GreB is present in very low numbers (near KD determined here) and would not be able to shift the balance, particularly in the case of rRNA synthesis carried out by numerous RNAPs. Ten RNAP:GreB nonproductive complexes would not shift the balance – unless GreB is overexpressed under some yet TBD conditions. Even a much more abundant GreA affects initiation from just a handful of promoters. The authors should explicitly discuss this issue and cite known in vivo GreB concentrations.3) While GreB is an excellent model to study control through the SC, the manuscript may convey a notion that GreB makes an important contribution to regulation of rRNA and perhaps other promoters that form very short-lived complexes. This scenario appears to be unlikely in the cell: unless the authors could cite evidence to the contrary, 4) It would be helpful for the authors to comment on the results they presented in Figure 1D of Tetone et al. (2016), in which the PR' promoter was used and RNAP was in the presence of 1 μM of GreB and relate them to this work. In Tetone et al. the authors were able to observe RNA polymerases transcribing, yet according to the arguments here the polymerase is likely saturated with GreB and arrives at PR' promoter with GreB. Should readers expect that GreB dissociates from polymerase bound to the PR' promoter and that this allows transcription to take place, or that polymerase can escape from PR' with GreB bound?5) In Figure 2G, I was expecting a 50% inhibition of transcription initiation given that GreB was at 10 nM (where polymerase is 50% occupied) but it looks to be closer to 75% inhibition. Is this interpretation correct?rrnB P1 in the absence of nucleotides.6) It was somewhat unclear on what basis the authors assert which states are accessible to the polymerase bound to E. coli  and rrnB (subsection “GreB arrives at and departs from an rRNA promoter in complex with σ70RNAP”) are not in italics and a space is missing between # and nM .7) There are only a couple of places that should be in italics =  Essential revisions:NoneMinor points:1) The authors extend their conclusions to other SC factors, arguing that they bind to the enzyme similarly and all effects will be controlled by occupancy. It could be more interesting to point out differences and it is possible that some SC factors are not exchanging that rapidly. In their recent study , the authors found that a cleavage-deficient D41N GreB remained bound to the elongation complex much longer. A "native" Gre-like Gfh, which inhibits catalysis, could have similar properties.We intended our conclusions to apply only to GreB and DksA. We agree that other SC factors may function differently (including possibly having little or no effect on rRNA initiation). We have revised the text of the Discussion section to clarify these points and to explicitly say that understanding the role of other SC factors will require further work. We also changed one sentence in the abstract to avoid the unintended implication that the results apply generally to all SC factors.2) The authors stress the point that GreB arrives and departs with the holoenzyme, instead of binding to the initiation complexes. This could in part be explained by the low stability of these complexes, and maybe this point could be made clearer. Alternatively, this result may suggest that GreB affinity for the DNA-bound RNAP is lower. Experimentally, this could be tested by including ATP which is expected to stabilize an otherwise absent open complex. This may also allow distinction between GreB effects on open complex vs nucleotide addition.We agree with the first possible explanation suggested by the reviewers, and we have edited the Discussion section to make this clearer; the relevant part now reads:70RNAP-promoter complexes. Instead, GreB arrived only at promoters that were not already bound by σ70RNAP and arrived only in complex with σ70RNAP. Similarly, it left the promoter only as a σ70RNAP•GreB complex. However, rRNA promoter complexes are atypically short lived . On more typical promoters, promoter complexes may be sufficiently long-lived to allow association of and dissociation of GreB. Dissociation of GreB during a long-lived promoter complex would prevent the suppression of initiation by the mechanism we observe with rrnB P1, providing a straightforward explanation for why GreB (and DksA) selectively inhibits initiation at promoters that have short-lived promoter complexes .”“In our experiments, GreB was almost never observed to bind to or depart from σ70RNAP holoenzyme (and therefore having a lower affinity for the latter) may contribute to the failure of GreB to bind those complexes during initiation. If that were the case, it would further support our conclusion that GreB loads onto holoenzyme prior to initial binding of RNAP to the promoter.We also agree with the second point , at least to the extent that GreB associating more slowly with promoter complexes than with σWe respectfully disagree with the suggestion that the ATP-only experiment would be informative. We are skeptical that an ATP-stabilized state is a good mimic of the kinetic properties of the open complex intermediate in initiation. To the extent that ATP-only conditions prolong the lifetime of an open complex-like structure, these conditions might artifactually make it appear that GreB can associate with the open complex intermediate whereas under transcription conditions (four NTPs) the lifetime of this intermediate is likely too short for appreciable GreB association, even if the association rate constant were as high as for GreB binding to holoenzyme.E. coli GreB is present in very low numbers (near KD determined here) and would not be able to shift the balance, particularly in the case of rRNA synthesis carried out by numerous RNAPs. Ten RNAP:GreB nonproductive complexes would not shift the balance – unless GreB is overexpressed under some yet TBD conditions. Even a much more abundant GreA affects initiation from just a handful of promoters. The authors should explicitly discuss this issue and cite known in vivo GreB concentrations.3) While GreB is an excellent model to study control through the SC, the manuscript may convey a notion that GreB makes an important contribution to regulation of rRNA and perhaps other promoters that form very short-lived complexes. This scenario appears to be unlikely in the cell: unless the authors could cite evidence to the contrary, We agree that this is an interesting question; the problem is that there are widely differing literature reports of the cellular abundances of DksA and GreB. For example, a modern Super-SILAC mass spectrometry study  reports both proteins sub-stoichiometric with RNAP, with DksA and GreB respectively at ~5% and ~2.5% mole ratios to RNAP subunit rpoC, with relatively small (~2-fold) changes during different growth phases. In contrast, an older analysis of these specific proteins by quantitative western blots  gave rather different results: ~1,140% and ~ 108% respectively, again reportedly largely independent of growth phase. Another recent mass spectrometry study  yielded a still different picture (for growth in glucose media) with DksA and GreB respectively ~210% and ~0.03% of rpoC.  Clearly, there are wide discrepancies between studies even when one is comparing concentration ratios of the same three proteins.rrnB P1 initiation. .Nevertheless, even if DksA and GreB are in fact present at <10% mole ratios relative to RNAP, it is still possible, at least in principle, that these SC factors could efficiently regulate rRNA transcription under rapid growth conditions, in which more than half of the transcriptional output is rRNAs and tRNAs. This is because holoenzyme free in solution  is <~10% of total RNAP under these conditions.* Free holoenzyme is the only one of these species demonstrated to bind DksA and GreB with high affinity, and in our model  free holoenzyme is the only subpopulation of RNAP that GreB need bind to regulate Given the uncertainties about the cellular concentrations of GreB and DksA , we feel that the manuscript’s discussion of this issue is appropriately circumspect. We merely cite the equilibrium competition model proposed by Rutherford et al. and point out that in the framework of this model both binding affinities and free concentrations influence the relative importance of GreB and DksA in regulation. This model is applicable even if the regulators are substoichiometric to RNAP.4) It would be helpful for the authors to comment on the results they presented in Figure 1D of Tetone et al., (2016), in which the PR' promoter was used and RNAP was in the presence of 1 μM of GreB and relate them to this work. In Tetone et al. the authors were able to observe RNA polymerases transcribing, yet according to the arguments here the polymerase is likely saturated with GreB and arrives at PR' promoter with GreB. Should readers expect that GreB dissociates from polymerase bound to the PR' promoter and that this allows transcription to take place, or that polymerase can escape from PR' with GreB bound?In Figure 1 of Tetone et al., the authors allowed open complexes to form on PR' without nucleotides and in the absence of GreB. Transcription was then initiated by washing out the free holoenzyme and simultaneously introducing GreB, NTPs, and hybridization probe. In this design, promoter escape is largely complete before any of the complexes have had a chance to bind GreB.5) In Figure 2G, I was expecting a 50% inhibition of transcription initiation given that GreB was at 10 nM (where polymerase is 50% occupied) but it looks to be closer to 75% inhibition. Is this interpretation correct?Yes, that is the logical expectation. There are a number of possible explanations for this difference. For example, there might be a minority subpopulation of polymerase molecules that are inactive ; this would lead to somewhat greater than half inhibition under conditions when half of total polymerase molecules were occupied by DksA or GreB. This discrepancy does not alter the validity of the conclusion of this section of the manuscript, which is that “GreB and DksA have identical effects on the rate of the full initiation process when the effects are measured at the same fractional occupancy of σ70RNAP”.6) It was somewhat unclear on what basis the authors assert which states are accessible to the polymerase bound to rrnB P1 in the absence of nucleotides.To the presentation of Figure 3 we added citations of key studies on this point  and also corrected an incorrect citation to the relevant review article . We also made minor edits to the Discussion section to clarify this issue.E. coli  and rrnB (subsection “GreB arrives at and departs from an rRNA promoter in complex with σ70RNAP”) are not in italics and a space is missing between # and nM .7) There are only a couple of places that should be in italics = Thanks; we have corrected those.