Xist RNA. In mice, X chromosome inactivation (XCI) is regulated by the antisense RNA Tsix, which represses Xist on the active X chromosome. In the absence of Tsix, PRC2-mediated histone H3 lysine 27 trimethylation (H3K27me3) is established over the Xist promoter. Simultaneous disruption of Tsix and PRC2 leads to derepression of Xist and in turn silencing of the single X chromosome in male embryonic stem cells. Here, we identified histone H3 lysine 36 trimethylation (H3K36me3) as a modification that is recruited by Tsix cotranscriptionally and extends over the Xist promoter. Reduction of H3K36me3 by expression of a mutated histone H3.3 with a substitution of methionine for lysine at position 36 causes a significant derepression of Xist. Moreover, depletion of the H3K36 methylase Setd2 leads to upregulation of Xist, suggesting H3K36me3 as a modification that contributes to the mechanism of Tsix function in regulating XCI. Furthermore, we found that reduction of H3K36me3 does not facilitate an increase in H3K27me3 over the Xist promoter, indicating that additional mechanisms exist by which Tsix blocks PRC2 recruitment to the Xist promoter.One of the two X chromosomes in female mammals is inactivated by the noncoding  Xist initiates chromosome-wide inactivation of one of the two X chromosomes of female cells. On the active X chromosome in males and females, Xist is repressed by several mechanisms. In mice, the Tsix ncRNA is transcribed over the Xist locus in the antisense orientation and functions as a repressor of Xist on the chromosome from which it is transcribed (Tsix has been extensively studied in mouse embryonic stem (ES) cells, which constitute a model for studying the initiation of random XCI   provides dosage compensation between the sexes for X-linked genes . The nonnscribed . The funndom XCI , 3–5. Di studies –9. In moethality . Converslineages , 12. Howome (Xi) , 12.Tsix causes death of male embryos due to initiation of X inactivation in extraembryonic tissues. This lethality can be prevented by complementing the extraembryonic lineages, suggesting that Tsix-independent mechanisms can act to repress Xist in the embryonic lineages  (Eed and Suz12 and the SET domain histone H3 methyltransferase gene Ezh2. Eed is required for PRC2-mediated trimethylation of histone H3 lysine 27 (H3K27me3)  . PRC2 co3K27me3) . Combinehe cells . Althougtivation . HoweverXist have been identified, including the X-linked Rnf12, Ftx, and Jpx genes. Rnf12 inhibits Xist repression in part through targeting Rex1 protein for degradation  regions contributes to the regulation of XCI  at the Xist promoter, which contributes to the repression of Xist expression, among other mechanisms.Here, we investigated repressive mechanisms of Setd2 knockdown are provided in the supplemental material. ES cells were cultured as previously described (Dnmt triple-knockout (Dnmt TKO) ES cells were a gift from Masaki Okano   . For geneviously . The expeviously  was a giThree different chromatin immunoprecipitation ChIP) methods were used: the H2AK119ub1-formulated method see  and the hIP methoXist/Tsix transcription unit  and cDNA was generated by reverse transcriptase SuperScript II . Quantitative real-time PCR for gene expression and ChIP was performed using iQ SYBR green Supermix  with a single-color detection MyIQ i cycler (Bio-Rad), SYBR green PCR master mix , or Thunderbird SYBR qPCR mix  with a StepOnePlus real-time PCR system (Life Technologies). Primer sequences covering the entire unit see  were desunit see , and thoin situ hybridization (RNA-FISH) was performed according to the protocols described previously  as described previously .Xist repression could be discerned from indirect effects on the pluripotent state more readily. We analyzed Xist expression in naive male ES cells that harbor disruptions of Eed (Eed−/−) and Tsix (ΔTsix)  . However, the number of Xist clusters observed with Xist RNA-FISH was not significantly increased in ΔTsix and Eed−/− cells over the control cell line J1:rtTA . In contrast, combined disruption of both Eed and Tsix resulted in the appearance of Xist clusters in 46.2% of E−/− ΔTsix ES cells . Furthermore, the active histone mark H3K4me2 was strongly increased over the Xist promoter in Eed−/− ΔTsix cells , in contrast to cells with mutation of either Tsix or Eed . We conclude that PRC2 function is required for Tsix-independent Xist repression in naive pluripotent cells, a finding that is consistent with earlier observations in serum-LIF-cultured ES cells   by RNA-Faterial)  and B anaterial) . J1:rtTAaterial) . A signiES cells  and highES cells  or ES ceXist activation in Eed−/− ΔTsix cells further, we analyzed the expression of Rnf12, Ftx, and Jpx and two additional genes within the X chromosome inactivation center (Xic), Xpct and Cnbp2. Increased Rnf12 expression is correlated with activation of Xist . This observation excludes upregulation of Rnf12 as a cause of Xist activation in E−/− ΔTsix cells. The observed repression of Rnf12 could likely be a consequence of the activation of Xist in Eed−/− ΔTsix cells. Ftx and Jpx are ncRNAs that are located upstream of Xist and have also been proposed to positively regulate Xist RNA expression . Similarly, Xpct and Cnbp2 expression was also increased, showing that lack of Eed caused derepression of multiple genes within the Xic region .To investigate the mechanisms of  of Xist , 21 but pression –25. We oXic region  . This finding suggests that the expression of Xic-linked genes Xpct, Cnbp2, Ftx, and Jpx is repressed  . An H3K2epressed  by PRC2 epressed . Interes/− cells , suggestTsix prevented H3K27me3 spreading over the Xist promoter from the hot spot in J1:rtTA-naive ES cells. H3K27me3 was enriched at the Xpct, Ftx, and Jpx promoters  but not at the Xist promoter . This observation showed that Tsix prevented the spreading of H3K27me3 from the hot spot into the Xist locus, which is consistent with earlier results in serum and LIF cultures . This observation that Oct4 and Nanog binding is not sufficient for repressing Xist in 2i, where Rex1 and Nanog are homogenously expressed, is further consistent with previous reports that the binding site for Oct4 and Nanog in Xist intron 1 is dispensable for Xist regulation in embryos . To. ToXist  embryos . Althoug embryos , we concXist repression, we disrupted Tsix in ES cells that lack Ring1b (R−/− ΔTsix) . In contrast to Eed−/− ΔTsix ES cells, which showed activation of Xist . H2AK119ub1 was weakly enriched on the Xist promoter in ΔTsix cells and furthermore was reduced in Ring1b−/− ES cells (P < 0.05]) and R−/− ΔTsix cells (P < 0.05]). In Ring1b−/− ES cells, a very small amount of H2AK119ub1 could still be observed  that was not significantly increased upon deletion of Tsix in R−/− ΔTsix cells (Xist promoter [2.48- ± 1.65-fold change {P = 0.20} relative to the mock control]). We cannot fully rule out the possibility that Ring1a catalyzes low levels of H2AK119ub1 in the absence of Ring1b in R−/− ΔTsix cells. However, in R−/− ΔTsix cells, H2AK119ub1 enrichment on the Xist promoter was similar to the Oct4 promoter, which served as a negative control . Therefore, we conclude that establishment of H3K27me3 on the Xist promoter does not lead to efficient H2AK119ub1 recruitment, consistent with earlier results of low enrichment of H2AK119ub1 at the Xist promoter in serum-cultured ES cells . This observation confirmed that Tsix contributed to DNA methylation on the Xist promoter in naive male ES cells, similar to earlier findings in serum-cultured ES cells. To analyze the function of DNA methylation, we used ES cells harboring combined mutations of all three DNA methyltransferases Dnmt1, Dnmt3a, and Dnmt3b (Dnmt triple-knockout (TKO) cells, DNA methylation at the Xist promoter was essentially absent (0.3%) . We further observed that Tsix transcription was slightly increased in Dnmt TKO cells (Tsix) (1.87- ± 0.66-fold change). In Dnmt TKO cells, DNA methylation was lost . We observed only a moderate upregulation of Xist in Dnmt TKO cells (Xist) (3.01 ± 0.33), comparable to the increased Xist expression in single Eed−/− and ΔTsix mutant ES cells  . DespiteKO cells , Tsix . We observed H3K36me3 in both J1 and Dnmt TKO cells at the Xist promoter  and exon 1  or far downstream of Xist , where we observed levels of H3K36me3 that are comparable to the negative-control Sox2 promoter , demonstrating that H3K36me3 is enriched at the Xist promoter and does not require PRC function. Importantly, H3K36me3 was strongly reduced to near background levels in ES cells that lack Tsix . Taken together, these findings show that Tsix transcription is required for establishing H3K36me3 at the Xist promoter.To identify such additional mechanisms, we considered histone marks associated with coding and noncoding transcription. H3K36me3 is correlated with transcriptional elongation and repression of inappropriate initiation of transcription within the gene body –54. We p–promoter . We detepromoter . The enrXist promoter via Tsix transcription prompted us to investigate whether it could exert a repressive function on the Xist promoter in cis. In mammals, to date, at least eight histone H3 lysine K36 methyltransferases (H3K36 HKMTs) have been reported, including those encoded by the NSD1, NSD2, NSD3, SETD2, SETD3, ASH1L, SETMAR, and SMYD2 genes (Xist promoter (P < 0.05]) and Xist exon 1 (P < 0.01]). To test for specificity, we also measured H3K27me3, which remained unchanged globally (Xist promoter (P = 0.82]) and Xist exon 1 (P = 0.34]). We did not measure an increase in the number of Xist clusters by RNA-FISH , but we did observe a significant derepression of Xist in K36M-overexpressing cells by qRT-PCR (Xist) (3.95- ± 1.49-fold change [P < 0.05]), whereas the expression of Tsix remained unchanged (Tsix) (1.15- ± 0.04-fold change [P = 0.39]). To obtain independent evidence by a second method, we aimed to reduce H3K36me3 using an RNA interference-mediated depletion of Setd2. The product of Setd2 is considered a major H3K36 methylase responsible for H3K36me3 . Importantly, under our experimental conditions, the expression of Tsix remained unaffected. Taken together, our data suggest that H3K36me3 is established in a Tsix-dependent manner over the Xist promoter and contributes to a repressive effect on the Xist promoter.H3K36me3 recruitment on the D2 genes . The numD2 genes . We estaD2 genes . Anti-FlD2 genes , Flag anpromoter  (0.48- ±t exon 1  (0.31- ±promoter  (1.04- ±t exon 1  (1.35- ± qRT-PCR , Xist (3nchanged , Tsix (1H3K36me3 . We obseXist promoter. Our results suggest two independent repressive mechanisms: one is mediated by Tsix transcription, and a second and independent mechanism is mediated by PRC2. Although this is a relatively simple scenario, which accurately predicts the derepression of Xist when both repressive mechanisms are impaired in Tsix- and Eed-deficient cells, further insights into the molecular interactions are needed. Here, we dissected the functions of several known and one novel chromatin modification in this system. Combined disruption of Tsix and Eed caused activation of Xist in naive ES cells. Notably, Xist was activated, although the binding of Nanog and Oct4 in Xist intron 1 was preserved, demonstrating that Nanog and Oct4 binding is not sufficient for Xist repression. This observation is consistent with a recent report showing that the intron 1 binding site is not essential for Xist repression (Xist through recruitment of PRC2. Indeed, a previous study reported a synergistic effect between the loss of the Oct4 binding site in Xist intron 1 and Tsix on Xist activation (Xist that are not critical in male cells.In our study, we addressed the function of chromatin modifications regulating the pression , 22. Howtivation . FurtherXist by Tsix required neither DNA methylation nor Polycomb recruitment, suggesting that additional mechanisms exist by which Tsix exerts its repressive function. We find that H3K36me3 is recruited to the Xist promoter in naive as well as serum-cultured ES cells. We show that H3K36me3 recruitment at the Xist promoter and gene body depends on Tsix transcription but requires neither the PRC complex proteins Ring1b and Eed nor DNA methylation. H3K36me3 is associated with transcriptional elongation and a function in repression of inappropriate transcription (–Xist, we performed two experiments aiming at reducing H3K36me3 globally. The first approach is based on a mutated form of histone H3.3 that is able to sequester histone methylases specifically (Xist but had little effect on Tsix and a control gene. Our second and independent attempt at reducing H3K36me3 was based on RNA interference-mediated depletion of Setd2. Setd2 is considered the major histone methyltransferase catalyzing H3K36me3. This approach was less efficient in reducing H3K36me3 but also resulted in Xist upregulation. Taken together, our experiments support the view that H3K36me3 recruitment contributes to Xist repression. However, we were not able to eliminate H3K36me3 entirely and thus may have observed a partial effect on Xist expression. In addition, depletion of H3K36me3 might also have indirect effects through perturbation of transcription units of other genes. We did not observe Xist clusters after depletion of H3K36me3 by RNA-FISH, despite the fact that H3K27me3 and H3K36me3 were reduced at the Xist promoter, suggesting that additional mechanisms exist through which Tsix represses Xist.In our system, repression of cription –54. To aifically . ExpressTsix, H3K27me3 is recruited to the Xist promoter through PRC2. H3K27me3 apparently spreads from a hot spot that is located 5′ to Xist. When Eed is mutated, H3K27me3 is lost and Xist is activated. We find that loss of H3K27me3 enrichment also leads to derepression of the Ftx, Jpx, Cnbp2, and Xpct genes, suggesting that multiple genes within the Xic are repressed by PRC2. The expression of Ftx (Jpx (Xist activation. Currently, the function of Cnbp2 in XCI is unknown, and overexpression of Xpct does not lead to Xist expression (In the absence of n of Ftx  and Jpx Ftx (Jpx  is elevapression .Xist promoter does not efficiently recruit H2AK119ub1. H2AK119ub1 is reduced in Ring1b-deficient cells to near background levels, but simultaneous loss of Ring1b and Tsix did not result in activation of Xist. This observation could suggest that at the Xist promoter H3K27me3 exerts a repressive function that is largely independent of H2AK119ub1. A PRC1-independent function of PRC2 is presently not regarded as a prominent silencing mechanism. However, genes have been identified in ES cells that are targets of PRC2 but not PRC1, suggesting that this mode of regulation is not entirely specific to the Xist gene promoter (Notably, H3K27me3 at the promoter .Tsix gene, H3K27me3 appears to spread from a hot spot upstream of Xist over several genes within the Xic region but is prevented from entering the Xist locus. We aimed to clarify whether known chromatin modifications can explain how Tsix prevents the spreading of H3K27me3. Previous reports have shown that DNA methylation is recruited by Tsix to the Xist promoter (Xist promoter after depletion of H3K36me3. This observation suggests that H3K36me3 is not critical for restricting spreading of H3K27me3 over the Xist promoter. However, we cannot rule out the possibility that we have not been able to reduce H3K36me3 to low enough levels. Blocking of H3K27me3 spreading by Tsix might involve functional redundancies between H3K36me3 and DNA methylation. Alternatively, additional, as-yet-unidentified mechanisms might exist.In cells carrying an intact promoter , 47, 48.promoter . Using ETsix over the Xist promoter and contributes to Xist repression. This modification can now be considered in future analyses of Xist regulation and will facilitate progress in understanding the chromatin-based mechanisms that contribute to the initiation of XCI. It will also be interesting to see if H3K36me3 is also relevant for gene regulation by noncoding transcription at other gene loci.Our data implicate H3K36me3 as a chromatin modification that is established cotranscriptionally by


HMS2, encoding a growth-regulated transcription factor, switches between sense- or antisense-dominant states that also coordinate up- and down-regulation of transcription at neighbouring genes. Engineering HMS2 reveals alternative mono-, di- or tri-cistronic and antisense transcription units (TUs), using different promoter and terminator combinations, that underlie state-switching. Promoters or terminators are excluded from functional TUs by read-through transcriptional interference, while antisense TUs insulate downstream genes from interference. We propose that the balance of transcriptional insulation and interference at gene clusters facilitates gene expression switches during intracellular and extracellular environmental change.In yeast, many tandemly arranged genes show peak expression in different phases of the metabolic cycle (YMC) or in different carbon sources, indicative of regulation by a bi-modal switch, but it is not clear how these switches are controlled. Using native elongating transcript analysis (NET-seq), we show that transcription itself is a component of bi-modal switches, facilitating reciprocal expression in gene clusters. DOI:http://dx.doi.org/10.7554/eLife.03635.001 A DNA double helix is made up of two DNA strands, which in turn are made of molecules that are each known by a single letter—A, T, C, or G. The sequence of these ‘letters’ in each DNA strand contains biological information.Genes are sections of DNA that can be ‘expressed’ to produce proteins and RNA molecules. To express a gene, the DNA strands in the double helix must first be partially separated so that one of them can be used as a template to build an RNA molecule in a process called transcription. Either of the DNA strands in a helix can be used as an RNA template, but contain different genes and are read in opposite directions. One of the two strands is called the ‘sense’ strand, the other the ‘antisense’ strand.The RNA molecule does not transcribe a whole DNA strand; instead, it transcribes a section of DNA, known as a transcription unit, which contains at least one gene. The end of a transcription unit is marked by certain signals that stop transcription. However, some transcription units in a DNA strand overlap, so there must be some way that the transcription machinery can sometimes ignore these stop signals.The activity of some genes is linked to the activity of their immediate neighbours. Furthermore, some genes are expressed in different amounts in response to changes in environmental conditions. Researchers have previously suggested that there must be some form of switch that controls when these genes are expressed.HMS2 and BAT2, in yeast. When one gene is switched on, the other is switched off and which gene is active depends on the diet of the yeast cells.Nguyen et al. now engineer start and stop signals at a neighbouring pair of genes, called HMS2 is another gene, SUT650. Nguyen et al. show that when this gene is transcribed, the transcription of HMS2 on the other DNA strand is blocked. This has the knock-on effect of turning on BAT2. Conversely, transcribing HMS2 switches off SUT650 and BAT2 because the end of HMS2 overlaps with the beginning of both SUT650 and BAT2. Switching between different genes relies on loops that physically link the start and stop signals of the gene to be transcribed while ignoring the start and stop signals for neighbouring genes.On the antisense DNA strand opposite to HMS2 gene helps to control which DNA strand is transcribed. When transcription factors do not bind to the start of HMS2, antisense transcription—and the expression of SUT650—occurs instead.Proteins called transcription factors can bind to DNA and affect whether a gene is transcribed. Nguyen et al. found that a transcription factor that binds near the start of the Overall, Nguyen et al. show that the transcription process itself makes up part of a switch that can control the expression of several genes on both the sense and antisense strands of a DNA double helix. This may also explain how many other, more complex, gene networks are activated in response to changes in the environment.DOI:http://dx.doi.org/10.7554/eLife.03635.002 Saccharomyces cerevisiae has revealed an extensive array of coding and non-coding transcripts, giving rise to a genome that is heavily interleaved. Individual genes can possess multiple, overlapping transcripts, in the sense and the antisense orientations with respect to the pre-mRNA, as well as poly-cistronic transcripts, which span neighbouring genes , co-expression of clustered genes in S. cerevisiae is largely independent of similarly controlled transcription, gene orientation, and/or shared regulatory sequences. However, members of adjacent gene pairs or clusters are more likely to belong to the same functional pathway than expected by chance - to galactose (GAL)-containing media and show a remarkable enrichment for genes whose transcripts cycle during the YMC. The majority of these genes have no functional associations with transcription factors that might mediate repression or induction in GLU or GAL, but are organised in clusters and subject to overlapping sense and antisense transcription. We exemplify this mode of gene regulation, state-switching by transcriptional interference and insulation, at the + RNA, we show that for the majority (≈88%) of genes, the change in transcript levels on the GLU to GAL shift reflects altered transcription rather than altered transcript stability (−5) for OX phase genes that are mainly involved in ribosome biosynthesis and growth (−5) for genes whose expression peaks in the reductive charging phase (RC) of the YMC  to galactose-containing media (GAL) for 3 h and analysed the native transcripts associated with elongating RNA polymerase II (NET-seq)  to identtability . Gene ontability . The gend growth . By cont the YMC . These g the YMC . In addi the YMC  and 25.7 the YMC .10.7554/We used a computer simulation to obtain the expected number of occurrences of genome-wide features, by randomly shuffling genes, their orientation, YMC status, and expression levels . BAT2 transcripts peak in the RC phase of the YMC and in GAL, reciprocal to HMS2 sense transcripts, which peak in the OX phase of the YMC and in GLU (HMS2 and its antisense SUT650 (transcripts A and B respectively). Dual-labelled RNA FISH revealed a degree of temporal separation in the production of the HMS2 and SUT650 transcripts. In multiple analyses, no examples of FISH signals for both HMS2 and SUT650 at the site of transcription in the nucleus are observed in either GLU or GAL. Cells with nascent transcripts  on the HMS2 sense strand extending through the coding region of BAT2 and beyond, consistent with low-level read-through transcripts initiating at the HMS2 promoter (rrp6Δ) (HMS2 is at the head of a contiguous gene cluster including the downstream genes BAT2 and YJR149W (HMS2 transcript is of variable length. Polyadenylation (pA) occurs either 85 nt  or 426 nt  downstream from the HMS2 stop codon. The distal pA site is 45 nt upstream of the BAT2 initiation codon, such that this longer transcription event is likely to interfere with initiation of BAT2 transcription. Additional transcriptional interference might be mediated through the production of the longer HMS2-BAT2 di-cistronic RNA species.A number of different methods revealed heterogeneity at both the 5′ and 3′ ends of the promoter . These r (rrp6Δ) . Thus HM YJR149W . The 3′ HMS2 and BAT2 supports (i) an overlapping, reciprocally related and temporally segregated, sense–antisense pair (SAP) at HMS2, (ii) reciprocally expressed sense transcripts at HMS2 and BAT2, and (iii) low-level read-through di-cistronic transcripts (HMS2:BAT2 and SUT650:RPS4A). We ask if overlapping transcription in the sense and antisense orientations at HMS2 contributes to the reciprocal regulation of HMS2 and BAT2, by dissociating one transcription unit from the other.In summary, analysis of transcripts around ADH1 transcription terminator (ADH1t) into the middle to HMS2, to separate HMS2 transcription from BAT2, allows us to test directly whether the HMS2 sense transcription over the HMS2:BAT2 intergenic region represses BAT2 transcription (ADH1t efficiently terminates the HMS2 transcript (HMS2 3′ region (ADH1t inserted showed a ≈twofold increase in BAT2 transcript levels compared to WT in both GLU and GAL (HMS2 sense transcription in repressing the BAT2 promoter. We conclude that HMS2 sense transcription interferes with BAT2 transcription.Insertion of the cription . We confanscript  and that′ region . The str and GAL  supportiADH1t into the HMS2 coding region resulted in a ≈fourfold increase in levels of a truncated HMS2 sense transcript (AA)    and blocipt (BA) . We confA)  for SUT650 located between +953 and +1038 relative to the HMS2 ATG, near the end of the HMS2 coding region. We chose to do this by substituting the complete coding region of HMS2 with that of URA3, to ensure a natural ORF between the HMS2 promoter and terminator, rather than mutating the regions containing multiple TSSs for SUT650 within the ORF  relative to the HMS2:URA3 sense transcript (AU) (HMS2:URA3 hybrid gene (UT) (HMS2 promoter remain sensitive to a change in carbon source in the absence of antisense transcription (HMS2:ADH1t (see 1U) relative to the HMS2:URA3 sense transcript (AU) in the absence of antisense transcription suggests a role for antisense transcription in attenuating sense transcription and preventing interference at the BAT2 promoter. Consistent with this, a twofold decrease in BAT2 sense transcript is observed in the antisense-less HMS2:URA3 strain relative to WT in GLU, concomitant with a threefold increase in AU and C1U  analysis at the HMS2:BAT2 intergenic region reveals reduced initiation-related modifications (H3K4me3 and H3K56ac) and increased elongation-related modifications (H3K79me3 and H3K36me3) in the HMS2:URA3 hybrid gene, consistent with transcriptional interference leading to repression of BAT2 transcription initiation (SUT650 antisense transcription (i) changes the proportion of cells expressing HMS2 and (ii) controls the amount of overlapping and read-through transcription from HMS2 and thus levels of BAT2 transcript. We envisage that at any one time in a single cell, formation of the HMS2 and SUT650 transcription units (TUs) is mutually exclusive, but the formation of the SUT650 TU and the BAT2 TU is not. Thus formation of the SUT650 transcription unit insulates BAT2 from interference by HMS2 transcription, as the HMS2 TU cannot form when SUT650 is expressed.Next, we asked whether transcription of  the ORF . We notetability , so we fr SUT650 . Second,ipt (AU) . We confrid gene  and obseene (UT) . We notecription , as we oDH1t see  and cons and C1U . Taken t and C1U  or incre and C1U  HMS2 traitiation . We concHMS2 transcription influences SUT650 antisense transcription, we replaced the HMS2 promoter with the inducible GAL1 promoter (pGAL1)  is evident in GLU and remains until the sense transcript is induced by GAL  and leads to restoration of the antisense transcript (BG) , a region transcribed in both constructs. No changes in induction rate were apparent, with the sense transcript only detectable after 2 h in both strains, signifying that antisense transcription does not impair sense activation kinetics  . When cualactose . SUT650 hift see . Howeverd by GAL . This suechnique  to depleipt (BG) . This copromoter . The senkinetics . Rather,kinetics . Activat of BAT2 , consisttose see . We concGAL1:HMS2; a long species (BG1), dominant until 30 min after the shift to GAL, and a shorter species (BG2), also detectable after 30 min of growth in GAL and which becomes the dominant antisense transcript form after 1 h  in the cbf1Δ and fkh1Δ strains   in a gln3Δ strain in minimal medium (HMS2 promoter (Input) to mediate state-changing (antisense-dominant to sense-dominant) in response to environmental conditions  at promoters of RC and OX genes, using a large data set of 202 factors   and durifactors) . We ablas)  (HMS2 (A), BAT2 (D), and SUT650 (B) transcripts, as expected upon depletion of an essential transcription factor. To explore this further, we used a well-characterised allele of SUA7 (sua7-1) (E62K) (HMS2 transcript (A) and sense read-through transcripts (C), coupled with a decrease in the SUT650 antisense transcript (B)  . However) (E62K) , associa) (E62K) . Remarkaript (B) . Thus, Tript (B) . For insHMS2 locus was engineered by insertion of the pTEF.KanMX.TEFt expression cassette (HMS2:TEF:Kan), there are two promoters in tandem (HMS2 and TEF) and two terminators in tandem (TEF and HMS2). This is sufficient to disrupt the HMS2 and SUT650 transcription units. The SUT650 antisense promoter is functional and produces a truncated antisense transcript (BK) that ends in the vicinity of TEFt , the sense transcript from pTEF, expressing the kanamycin resistance (KanMX) transcript (K), and the sense transcript (AK) initiated at the HMS2 promoter and terminating at TEFt  and considerably reduced levels of antisense transcript (E) (The cassette  at positcassette . In this of TEFt . Thus TE at TEFt . pTEF iscterised  strong b of pTEF . This reript (E) .10.7554/TEF promoter, we would be able to ask how disabling pTEF affects the HMS2 and SUT650 transcription units. Using the HMS2:TEF:Kan construct as a starting point, mutations were introduced into the TEF promoter, including deletions of putative transcription factor binding sites (HMS2.TEFdel1:Kan), removing all the TEF promoter sequences upstream of the TATA box, results in a significant reduction in expression of the KanMX cassette itself (transcript K) and in levels of the antisense transcript (E) (HMS2 that reads through TEFt (BpTEFdel)  the remarkable plasticity of interleaved transcription units. We suggest that the pTEF.KanMX.TEFt expression cassette is strongly expressed  . RemarkapTEFdel) . We confAS probe . Thus, tpromoter . This ext genome .HMS2 and BAT2 that combines the effects of temporal separation, overlapping interfering transcription able to bypass typical termination and promoter signals, and insulation from interference by the formation of an antisense transcription unit . Moreover, antisense transcription arising from divergent promoters may have to bypass the terminator of the upstream gene. This work indicates the capacity of a terminator to function is conditional on the strength of the proximal promoter. We suggest that this reflects the role for promoters in defining the beginnings and ends of TUs ; strongesua7-1 allele. A simple scenario in which 3′end TFIIB directs transcription by promoting both antisense transcription and physically terminating sense transcription is compatible with the interference/insulation model proposed here. It is also plausible that alternative higher order structures linking the sense or antisense promoters to one of a possible number of terminators could also explain dynamic state-switching between sense and antisense or multi-cistronic transcripts  and FKBP12 Rapamycin binding (FRB) domain of human mTOR protein in order to deplete target proteins from the nucleus in a time-dependent manner . Native elongating transcript sequencing, (NET-seq) was done and analysed as described in In Situ hybridization (FISH) protocol was adapted from hms2Δ cells to determine threshold signal for expression. Images displayed as 2D compressions of z-stacks 12–22 (11 stacks), which included most of the nucleus and cytoplasm. Error bars represent standard error of the mean. Chromatin immunoprecipitation (ChIP) was performed as described by All experiments were performed at least in duplicate to ensure that the trends observed were reproducible. Briefly, unless otherwise stated, cells were grown to exponential phase in rich medium (YP) supplemented with 2% glucose (YPD) or 2% galactose . The strains of t manner . Control review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , a Reviewing editor, and 3 reviewers.Thank you for sending your work entitled “Transcription mediated insulation and interference direct gene cluster expression switches during biological rhythms” for consideration at eLife after addressing the concerns listed below.After a thorough discussion, the consensus among the reviewers and the editors was that this is an excellent paper reporting some remarkable findings, which would be worthy of publication in The metabolic cycle has emerged as a fundamental biological phenomenon that is critical for proper cell function. It has been appreciated that oxidative conditions are in conflict with reducing reactions, and vice versa. Consequently, cells have evolved mechanisms to temporarily separate these conflicting conditions, by oscillating both the oxygen consumption as well as the expression of ∼3000 genes. The remarkable precision of the synchrony in maintaining oscillating levels of mRNAs must involve intracellular events and communication, the basis of which remains obscure. This paper provides some solutions to this enigma by demonstrating how transcription of tandemly arrayed genes can regulate a bi-modal switch so that activation of one gene would repress the other and vice versa. This paper will serve the scientific community in various aspects. First, it will drive the attention of the readers to the complexity of the YMC, an issue that is still underrepresented in the literature. Second, it illustrates the critical roles played by transcription terminators in both terminating transcription and initiating transcription in the anti-sense direction. Third, transcription of anti-sense (AS) as a key modulator of gene expression, in general, and its involvement in the bi-modal switch, in particular. Fourth, and most importantly, the paper demonstrates, in many ways, the intricate relationship between transcription of sense, anti-sense and neighbouring genes in a manner that supports the bimodal switch.The major concerns raised by the reviewers are:1) Generality of findings. The impact of this work would certainly be higher upon defining the prevalence of the key regulatory mechanisms described. The paper focuses on two tandemly arrayed genes (HMS2/BAT2) whose expression switches during the YMC. They provide a detailed mechanism of the switching whereby transcription read-through into the downstream promoter downregulates transcription of the downstream gene and how AS transcription is involved. Their conclusion that the sense and AS transcription are in conflict, and that cells tend to separate the “sense state” from the “AS state” temporarily is by itself remarkable. However, how general is this mechanism? What fraction of YMC-regulated genes is adjacent or anti-sense? More than expected by chance? And then what fraction of adjacent or anti-sense transcripts is co-regulated in the YMC? Moreover, if the mechanism is more prevalent than just HMS2/BAT2, do the anti-sense transcripts always fall into opposite YMC categories, i.e., oxidative vs reductive? Is this locus even representative of other glu-gal clusters? If 10 loci are chosen at random , do the neighbouring genes show potent regulation like the one well-explored region? Reviewers agreed that a more global assessment of the prevalence of the mechanism described would raise the impact significantly. However, it was noted that the bioinformatics involved for a global analysis would not be trivial. Thus, reviewers request that the authors manually mine the NET-seq data regarding transcript isoforms (like the data shown in 2) Weak links to YMC and circadian rhythms. Although the reviewers appreciated the novelty and elegance of the molecular mechanisms discovered, there was agreement that in its current form the manuscript does not make a convincing argument about general relevance to YMC or circadian rhythms and it was agreed that the writing, including the title, should be tempered to focus more on the mechanistic aspects and less on the YMC and circadian biology. It is proposed that the title be changed to “Transcription mediated insulation and interference direct gene cluster expression switches”.3) The choice of sua7-1 is not well communicated. The reader of the “Results” section is puzzled why TFIIB was chosen to begin, and why this specific allele was used, in particular. Only in the Discussion the reader learns that sua7-1 is defective in loop formation.4) 5) The replacement of HMS2 ORF with URA3 requires some clarifications. Why didn't the authors simply remove the region around the TSS, leaving all other sequences unchanged? Why did they prefer to introduce the entire URA3? There are two issues that need to be addressed. (1) The possibility that URA3 sequences, in both orientations, introduce stability biases onto the RNAs. (2) The possibility that the URA3 sequence contains a terminator in the AS direction, explaining why the AS RNA was not detected by H1 probe, located downstream of this hypothetical terminator. These issues can be discussed or addressed experimentally.6) In  1) Generality of findings. The impact of this work would certainly be higher upon defining the prevalence of the key regulatory mechanisms described.We agree with the reviewers about this point and had, in fact, already embarked on a computational genome-wide analysis. Part of this very large dataset has been analysed for this work and is presented in the revised version of the paper.The paper focuses on two tandemly arrayed genes (HMS2/BAT2) whose expression switches during the YMC. They provide a detailed mechanism of the switching whereby transcription read-through into the downstream promoter downregulates transcription of the downstream gene and how AS transcription is involved. Their conclusion that the sense and AS transcription are in conflict, and that cells tend to separate the “sense state” from the "AS state" temporarily is by itself remarkable. However, how general is this mechanism?PHO84 . We also have as yet unpublished evidence that at GAL10, the sense and antisense transcripts are present in different cells during early induction. Given the FISH evidence for YMC regulated genes from the Singer and Botstein labs, we think that switching is likely to be common.From the point of switching sense and antisense states there is now one experiment in a recent paper from the Zenklusen lab supporting the idea of separation of the sense and antisense states in individual cells, in this case at What fraction of YMC-regulated genes is adjacent or anti-sense? More than expected by chance? And then what fraction of adjacent or anti-sense transcripts is co-regulated in the YMC?We have data in the revised manuscript addressing these issues (Tables S5 to S9). The antisense question is hard to address adequately due to the relatively poor annotation of the antisense transcriptome – being limited to CUTs and SUTs. There are 1772 annotated CUTs and SUTs but much more transcription antisense to genes is evident from the NET-seq analysis. A separate analysis suggests that 75% of all yeast genes are subject to some antisense transcription in the vicinity of the sense promoter that influences the chromatin organization around that promoter. Nevertheless, our analysis for this work suggests that YMC genes are much more likely to have antisense transcription, although the number of genes is relatively small because of the high thresholds (>3-fold change in GLU/GAL both the YMC gene and the SUT or CUT). 71 of the 206 genes (34.4%) with regulated sense and antisense have SUTs/CUTs whose transcription also changes 3-fold in the GLU/GAL shift. 26% of all SUTs and CUTs show a >3-fold change on the GLU/GAL shift. Thus regulated SUTs and CUTs are enriched opposite to ORFs that also show a > 3-fold shift.Moreover, if the mechanism is more prevalent than just HMS2/BAT2, do the anti-sense transcripts always fall into opposite YMC categories, i.e., oxidative vs reductive?HMS2.BAT2), 9 (90%) of the OX genes have an antisense (annotated or not) that shows reciprocal transcription to the OX gene.Our selected gene study shows that for the 10 OX.RC tandem genes , do the neighbouring genes show potent regulation like the one well-explored region?Yes, see Table S9 and Reviewers agreed that a more global assessment of the prevalence of the mechanism described would raise the impact significantly.I hope that we have persuaded the reviewers that this is likely to be a more general phenomenon.However, it was noted that the bioinformatics involved for a global analysis would not be trivial. Thus, reviewers request that the authors manually mine the NET-seq data regarding transcript isoforms (like the data shown in) and examine whether transcription of one gene invades the promoter of its neighbouring gene, and whether the AS transcription is anti-correlated. The control should be the same number of tandemly positioned gene pairs that are chosen at random. If the authors have the bioinformatic means to take a whole genome view, all the better.We have used both approaches to provide the data in order to conclude this is a wider phenomenon.2) Weak links to YMC and circadian rhythms. Although the reviewers appreciated the novelty and elegance of the molecular mechanisms discovered, there was agreement that in its current form the manuscript does not make a convincing argument about general relevance to YMC or circadian rhythms and it was agreed that the writing, including the title, should be tempered to focus more on the mechanistic aspects and less on the YMC and circadian biology. It is proposed that the title be changed to “Transcription mediated insulation and interference direct gene cluster expression switches”.We are happy to do this and have removed this from the title and tempered our references to these mechanisms in the text.3) The choice of sua7-1 is not well communicated. The reader of the “Results”" section is puzzled why TFIIB was chosen to begin, and why this specific allele was used, in particular. Only in the Discussion the reader learns that sua7-1 is defective in loop formation.We agree with this point and have included more details in the Results section.4)shows that the insertion of ADH1t abolished the signal detected by probe H2.This is correct for the H2AS probe.From the point of view of the AS transcription, this probe is located downstream of ADH1t.This is correct.The authors concluded that ADH1t blocks SUT650 AS transcription. However, it is possible that ADHt acts in both orientation and simply terminated transcription and therefore H2 could not detect any transcript.This is correct.If the authors know that ADH1t does not act as terminator in the reverse orientation, they should indicate it.GAL10 (see We do know that the ADH1t acts as a terminator in both directions. It does so in its native context (terminating the YOL086AW TU) and when used out of context in AL10 see  NAR.Moreover, even if the ADH1t is not acting as terminator of the AS, it can introduce a destabilization element that leads to its rapid degradation. In order to support this important conclusion, the authors could use a decay mutant strain  and probe the membrane to detect the region between the ADHt and SUT650 promoter.rrp6) using probe H2AS. There are no unstable transcripts detectable in the rrp6 strain.We have included two additional pieces of data in HMS2 sense transcription and SUT650 antisense transcription are prematurely terminated in this strain.Second we use probe H3AS to show that SUT650 is prematurely terminated. Thus both 5) The replacement of HMS2 ORF with URA3 requires some clarifications. Why didn't the authors simply remove the region around the TSS, leaving all other sequences unchanged? Why did they prefer to introduce the entire URA3?HMS2 ORF with the URA3 ORF so as to avoid problems with changing codons, deleting bases, or altering transcript stability while leaving the HMS2 flanking sequences unaltered.We chose to completely replace the There are two issues that need to be addressed. (1) The possibility that URA3 sequences, in both orientations, introduce stability biases onto the RNAs. (2) The possibility that the URA3 sequence contains a terminator in the AS direction, explaining why the AS RNA was not detected by H1 probe, located downstream of this hypothetical terminator. These issues can be discussed or addressed experimentally.ura3Δ and thus the URA3 gene is not in our datasets to test for the presence of an early terminator. Given that the promoter of a gene is reported to determine sense RNA stability , we felt the URA3 ORF replacement was an appropriate strategy. We have discussed these potential problems in the text and how we have interpreted our data given these provisos.We cannot address this using experimental data as BY4741 is 6) In, the authors used TFs with putative binding sites at intergenic region between HMS2 and BAT2 (no references were included). The membranes were probed only with the H1 probe. This case might be an opportunity to examine if any of these factors play a role in the transcription of BAT2, by probing the membrane with an appropriate probe. URA3 ORF can be replaced back to HMS2 ORF by homologous recombination and selection on 5-FOA, thus resulting in a precise deletion of just the region around the TSS. Maybe one of these TFs is required for BAT2 transcriptional repression by C1 transcription .HMS2 sense and SUT650 antisense suggest that ablation of some factors coupled to mild nutrient limitation leads to cells where transcription in the HMS2:SUT650 region is predominantly in sense or antisense orientations. This provides evidence for reciprocal switching between HMS2 sense and SUT650 antisense and suggests a signalling input regulates this only at the HMS2 promoter. We have a number of experiments in the paper to support this, particularly those in SUT650 and BAT2) is constitutive in the absence of sense transcription, regardless of growth conditions (GLU or GAL). Normally SUT650 transcription increases on the GLU to GAL shift but this reflects the reduction in HMS2 sense transcription in GAL. Without sense, SUT650 levels remain high in GLU. Similar arguments apply to BAT2. Elimination of sense transcript in the pGAL1:HMS2 anchor away experiment in BAT2 transcript levels. Thus the primary BAT2 regulation is transcriptional interference by the sense transcription in GAL (in this experiment only as the inserted GAL1 promoter is GAL-regulated).We apologise for not including references to the putative TF binding sites – this is now done. Putative factor binding sites, and even ChIP data with tagged factors, does not provide evidence that a particular factor is functional at a particular site, for a variety of reasons . Indeed, many of the experiments linking transcription factors to changes in particular transcriptional responses at genes are likely to be artefacts resulting simply from altered growth rate as a result of the genetic intervention , (and the different times spent in the OX and RC phases of the YMC). We think this criticism applies to all the experiments we have done with TF KO strains in HMS2 switches on and interferes with the transcription of SUT650 and BAT2 as long as the growth signal remains. If there is no growth signal, transcription of BAT2 and SUT650 is constitutive, as if cells are in the quiescent state that occurs naturally when nutrients are limited. In fact all the changes that occur when yeast naturally enter stationary phase due to nutrient depletion also occur in RC cells . This does not rule out TFs in the regulation of RC genes, but it does suggest they are not condition-specific. This is supported by our two new genome-wide analyses now included in the manuscript (Table S10). We have examined the nature of the transcription factors’ association with the promoters of RC or OX genes (in GLU) from the Venters and Pugh work (2011) (now included in reference list). They looked at the association of 202 factors at yeast promoters. We used their dataset to ask whether OX and RC genes are enriched for particular factors, as both will be expressed in GLU-grown cells. We show significantly different (p<0.01) ChIP-Chip binding patterns for factors at the two groups of genes. The RC genes are enriched for cohesion, the CCR4/NOT complex, elongator, chromatin and chromatin remodelling factors and four TFs, . By contrast, OX genes are associated with GTFs, TEFs, TFIID, and the TFs Ifh1 and Rap1. Although “stress” related transcription factors may play a role at the RC genes, these data suggest chromatin-mediated regulation as a central feature for RC genes. Our data support TI-mediated chromatin changes at the BAT2 promoter.Our view is that the (semi-) quiescent RC phase in the YMC is likely to be the default and RC genes would be expressed unless a specific growth signal is received. HMS2 and BAT2 regulate BAT2 expression or more simply, contribute to limiting HMS2 read-through  but do see a role for Gln3  in preventing transcript C (HMS2:BAT2 read through) and a reduction in SUT650 levels (supporting increased TI at the HMS2:BAT2 intergenic region). Given the literature supports a role for TFs and promoters in determining transcript stability, another explanation for the effect of loss of Gln3 is that transcript C is stabilised, but there is no change in transcription per se and thus no change in the transcript ratios. How RC genes are regulated is clearly complicated and beyond the scope of what we can do for a 2 month revision. We think the suggestion of the referee is excellent but have a number of reservations about this type of approach, especially as some of the putative TF binding sites are within the HMS2 ORF (similar arguments to the URA3 question above). Instead we have provided an Anchor-Away experiment to deplete nuclear Gln3 in Galactose (conditions expected to increase BAT2 and SUT650 expression). In the same experiment nuclear depletion of Sua7 results in loss of both BAT2 and SUT650 transcripts. We find no evidence that Gln3 is required for BAT2 or SUT650 expression in these conditions and the Sua7 control suggests this is not simple a result of the BAT2 transcript being particularly stable in GAL. These data are now included in So to the question of whether the putative TF binding sites between -through . As it s


Aspergillus nidulans genomic library that bound specifically to AmyR, a TF from A. nidulans. High-throughput sequencing data were obtained for the DNAs enriched through the selection, following which various in silico analyses were performed. Mapping reads to the genome revealed the binding motifs including the canonical AmyR-binding motif, CGGN8CGG, as well as the candidate promoters controlled by AmyR. In parallel, differentially expressed genes related to AmyR were identified by using RNA-Seq analysis with samples from A. nidulans WT and amyR deletant. By obtaining the intersecting set of genes detected using both gSELEX-Seq and RNA-Seq, the genes directly regulated by AmyR in A. nidulans can be identified with high reliability. This analytical pipeline is a robust platform for comprehensive genome-wide identification of the genes that are regulated by a target TF.For identifying the genes that are regulated by a transcription factor (TF), we have established an analytical pipeline that combines genomic systematic evolution of ligands by exponential enrichment (gSELEX)-Seq and RNA-Seq. Here, SELEX was used to select DNA fragments from an  Peaks were called using MACS (v1.4.2)  by using the default settings. Further analysis and normalization were performed using SeqMonk (http://www.bioinformatics.bbsrc.ac.uk/projects/seqmonk/). DEG lists were generated using a statistical significance test (P < 0.05) and DESeq2 and EdgeR software.The DNA sequences obtained were mapped onto the reference genomic sequence of In vitro characterization of the DNA-binding sites of TFs neither requires a culturing step nor depends on particular cell types or growth conditions. Therefore, in vitro technologies provide highly valuable support for the in vivo identification of TF-binding sites and are suitable for high-throughput analysis. SELEX-Seq is a high-throughput method that is suited for systematically characterizing the DNA-binding specificities of TFs. In a standard SELEX-Seq strategy, DNA targets are selected using an electrophoresis mobility shift assay (EMSA) [1-411 in E. coli [y (EMSA) , 14, 34 y (EMSA) , 11 or My (EMSA) . In this E. coli , 29.A. nidulans genomic library  by using semi-quantitative RT-PCR analysis [8CGG motif in all 8 promoter regions of the previously reported amylolytic genes  and an amyR deletant (Δ50), with or without isomaltose induction -selected RNA samples from nduction . DEGs weated one . By combependent . Howeverpendence , was notpendence .p < 0.05 Chi2 test). Notably, the promoter regions of most of the DEGs contained >1 CGGN8CGG motif .The 41 DEGs were also compared with the promoter candidates obtained from gSELEX, and these candidates were found to include a subset 23 DEGs . Thus, tGG motif . AlthougglaB and 16 genes newly identified as AmyR-regulated genes (freA) [atrA) [treA) [xanA) [treA is required for growth on trehalose used as a carbon source [In RNA-Seq analysis, all identified DEGs should be genes that are affected, either primarily or secondarily, by the expression of the target TF. Thus, the TF would be expected to directly regulate only a subset of the DEGs. Our results strongly indicate that the 23 identified DEGs are regulated directly by AmyR; these 23 DEGs included all aforementioned amylolytic genes except ed genes . Among ts (freA) , a putat) [atrA) , alpha-t) [treA) , and an ) [xanA) , respectn source , which samyR, were not included among the promoter candidates identified using gSELEX-Seq . Blue squares indicate the summits of the peaks. Red square frames indicate CGGN8CGG.gSELEX-Seq peaks were detected using (TIFF)Click here for additional data file.S2 FigA. nidulans WT (BPU7) and an amyR deletant (Δ50), with or without isomaltose induction.RNA-Seq analysis was performed using poly(A)-selected RNA samples from (TIFF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Pseudomonas aeruginosa produces rhamnolipid biosurfactants involved in numerous phenomena including virulence. The transcriptional study of the rhlAB operon encoding two key enzymes for rhamnolipid synthesis led to the discovery of the quorum sensing system RhlRI. The latter positively controls the transcription of rhlAB, as well as of rhlC, which is required for di-rhamnolipid synthesis. The rhlG gene encodes an NADPH-dependent β-ketoacyl reductase. Although it was reported to be required for the biosynthesis of the fatty acid part of rhamnolipids, its function in rhamnolipid synthesis was later questioned. The rhlG transcription and its role in rhamnolipid production were investigated here.luxCDABE-based transcriptional fusion, and quantitative reverse transcription-PCR, we confirmed two previously identified σ70- and σ54-dependent promoters and we identified a third promoter recognized by the extra-cytoplasmic function sigma factor AlgU. rhlG was inversely regulated compared to rhlAB and rhlC: the rhlG transcription was down-regulated in response to N-butyryl-l-homoserine lactone, the communication molecule of the RhlRI system, and was induced by hyperosmotic stress in an AlgU-dependent manner. Consistently with this transcriptional pattern, the single or double deletions of rhlG and PA3388, which forms an operon with rhlG, did not dramatically impair rhamnolipid synthesis.Using 5′-RACE PCR, a rhlG transcription reveals a complex regulation involving three sigma factors and N-butyryl-l-homoserine lactone. We furthermore present evidences that RhlG does not play a key role in rhamnolipid synthesis.This first detailed study of  Pseudomonas aeruginosa is a ubiquitous Gram negative bacterium and an opportunistic human pathogen, in particular responsible for the chronic lung infection of cystic fibrosis patients. P. aeruginosa produces rhamnolipids, which are glycolipidic biosurfactants consisting of one or two hydrophilic l-rhamnose molecules  and of a hydrophobic fatty acid moiety, see [l-rhamnose synthesis from glucose-1-phosphate, while RhlA supplies the acyl moieties by converting two molecules of β-hydroxylacyl-Acyl Carrier Protein (ACP) in one molecule of β-D- alkanoic acid (HAA). Finally, the rhamnosyltransferase RhlB links one l-rhamnose molecule to one HAA to yield one mono-rhamnolipid, which either will be the final product or will be the substrate of the second rhamnosyltransferase RhlC to obtain one di-rhamnolipid. RhlG was described as an NADPH-dependent β-ketoacyl reductase specifically involved in rhamnolipid synthesis [rhlG mutant of P. aeruginosa PAO1; and iii) similarities between the promoters of the rhlG gene and of the rhlAB operon, suggesting a coordinated expression of the genes involved in rhamnolipid synthesis [Escherichia coli and that rhlG mutants of P. aeruginosa PA14 and PAO1 were not affected in rhamnolipid production. These authors concluded that RhlG plays no role in rhamnolipid formation and that its physiological substrate remains to be identified [rhlG gene has not been so far studied in more details than in [rhlAB operon was the first and most extensively studied at the transcription level. These works led to the discovery of the RhlRI quorum sensing (QS) system, which is encoded by genes lying just downstream of rhlAB and is required for rhlAB transcription [N-butyryl-l-homoserine lactone (C4-HSL) which binds to the transcription regulator RhlR. Medina et al. [rhlA, regardless of the presence or not of C4-HSL. Without C4-HSL, RhlR would act as a transcriptional repressor of rhlAB, whereas RhlR/C4-HSL would activate transcription. It should be noted that the RhlRI system is embedded within a complex QS network including the LasRI system with its autoinducer N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL) and the Pseudomonas Quinolone Signal (PQS) system [rhlAB transcription [rhlA could result from two putative promoters, one of which would dependent on the alternative sigma factor σ54 (RpoN) and the other on the primary sigma factor σ70[rpoN mutants [4-HSL complex activates the rhlA promoter independently from σ54[rhlAB transcription. Determining the 5′ end of rhlG mRNAs by primer extension led to the identification of two overlapping promoters likely dependent on the sigma factors σ70 and σ54[rhlG mRNA concentration was only slightly lower in a lasR mutant than in the wildtype strain, it was concluded that LasR is not a direct activator of rhlG transcription, but it remained possible that RhlR plays this role [rhlG was thus proposed to be regulated similarly as the rhlAB operon [rhlC and the rmlBDAC operons is also mainly dependent on RhlR/C4-HSL, and the PA1131-rhlC promoter was proposed to be σ54-dependent [ety, see  for reviety, see , and areety, see ,3: RmlA,ynthesis . It was ynthesis . These cynthesis . Howeverynthesis . Zhu andcription -10. RhlRcription , RhlI sya et al.  showed t) system ,14, but  mutants , but subctone 3OC-HSL and ependent ,16.rhlAB and rhlC and prevented rhamnolipid production. These works prompted us to investigate in more details the transcriptional regulation of rhlG and to compare its transcription pattern to the rhlAB and rhlC ones. Here, we mapped the rhlG promoters, confirming that the σ70-dependent promoter is functional and identifying a third promoter dependent on the alternative sigma factor AlgU. On the contrary to rhlAB and rhlC, rhlG was down-regulated by quorum sensing and induced under hyperosmotic stress. We constructed single PAO1 mutants with deletions in rhlG or PA3388 (which is co-transcribed with rhlG), and the double rhlG/PA3388 mutant. The phenotypes of the mutants confirmed that RhlG is not involved in rhamnolipid biosynthesis.In previous works, we examined the effect of hyperosmotic stress on rhamnolipid production, accumulation of QS communications molecules, and expression levels of related key genes ,18. We orhlG transcription when cells were grown during 24 h in PPGAS medium, which favors rhamnolipid production [P. aeruginosa PAO1 strain containing pAB134, which carries the luxCDABE operon under the control of the rhlG promoter region (prrhlG), extending from − 413 to −23 relative to the first base of the rhlG translation initiation codon. We chose this strain since the multi-copy pAB134 plasmid led to higher amounts of mRNAs than the genomic mono-copy rhlG gene, thereby facilitating the experiment. Three internal luxCDABE primers were used to synthesize cDNAs and amplify them by PCR. A mix of two DNA fragments, both of ~ 400 pb was obtained after the last PCR. They were sequenced, identifying two different transcription start sites at positions −113 and −55 relative to the rhlG translation initiation codon  revealed a novel transcription start site preceded by the sequence CAACCT − N16 − TCTG, which is similar to the consensus sequence for AlgU-dependent promoters, GAACTT − N16–17 − TCTG [We used 5′-RACE PCR to determine which promoter mainly controls oduction . We used7 − TCTG . AlgU is7 − TCTG -23.prrhlG activity in the course of bacterial growth by measuring the luminescence resulting from the LuxCDABE proteins. To verify the role of AlgU in the transcription of rhlG, P. aeruginosa PAO1 and its algU mutant strain PAOU [luxCDABE operon, used to quantify the luminescence baseline) and pAB134. Strains were grown in PPGAS medium and luminescence was followed during 30 h. Figure prrhlG activity was ~3-fold lower in P. aeruginosa PAOU than in PAO1 during stationary phase  in plasmid-free PAOU and PAO1 strains at 20 h of growth. The rhlG mRNAs were 3-fold less abundant in PAOU than in the wildtype strain PAO1 , are thus involved in rhlG transcription.We did not identify the transcription start site at position −65 Figure  resultinoding σ54. The lumh Figure B, indicarhlG promoter region . This led to luminescence levels similar to those of PAO1(pAB134)  and in C4-HSL synthesis (rhlI) [rhlG mRNA was 3.7-fold higher after 20 h of growth in hyperosmotic condition (0.5 M NaCl in PPGAS medium)  abolishes rhamnolipid production and inhibits the transcription of genes involved in rhamnolipid synthesis (s (rhlI) ,18. In PrhlG promoters is responsible for this response to hyperosmotic condition, we used the PAO6358 (RpoN mutant) and PAOU (AlgU mutant) strains. No significant difference was observed when comparing the prrhlG activity in the PAO1 and PAO6358 strains, showing that σ54 is not involved in prrhlG induction in hyperosmotic condition , we constructed our own rhlG mutant (PAOGAB) of PAO1 in order to clarify the RhlG involvement in rhamnolipid production. Rhamnolipids produced by the strains were then quantified both intra- and extra-cellularly. In PAOGAB compared to PAO1, we observed a slight decrease (~20%) of extra-cellular production that complementation by rhlG did not restore. No difference at all was observed in the intracellular fraction  [et al. observations could result from a defective expression of PA3388, or of both rhlG and PA3388. We therefore constructed a PA3388 single deletion mutant and a double rhlG/PA3388 mutant. These two mutants displayed similar levels of rhamnolipid production as the PAOGAB and PAO1 strains  . We suspSince β-ketoacyl-ACP, a potential substrate of RhlG, is a precursor for both rhamnolipid and PQS biosynthesis ,27, we fP. aeruginosa, only few reports investigated the involvement of rhlG in this biosynthesis pathway. We focused our study on transcriptional regulation. A previous study [rhlG transcription, σ70 and σ54. Promoter mapping led us to discover an additional promoter and a third sigma factor involved: AlgU. Since rhlG has been found to be involved in rhamnolipid production [4-HSL, it was suggested that rhlG was regulated similarly as the other genes involved in the rhamnolipid biosynthesis (rhlAB and rhlC). Here we found that it was not the case. Whereas C4-HSL is required for rhlAB transcription [rhlG promoter activity. The “lux box” overlaps the AlgU-dependent promoter   and is closely related neither to an rhl-responsive nor to a las-specific binding sequence as defined in [4-HSL acts indirectly on rhlG transcription, for example by inducing the expressing of a gene encoding an unknown rhlG repressor.Although rhamnolipid production is well described in us study  identificription , we obser Figure  and it i) Figure A and B, ntration ,18. We ac stress . We confbolished ,18. It sfined in . The conrhlG and the genes involved in rhamnolipid synthesis, rhamnolipid production was not dramatically impaired in the rhlG null mutant that we constructed in P. aeruginosa PAO1, in agreement with Zhu and Rock [rhlG transcription under hyperosmotic stress, we examined if rhlG was involved in osmotic stress response, but no difference was observed in terms of growth and survival between the rhlG mutant and its parental PAO1 strain after osmotic stress (data not shown). We furthermore tested a number of phenotypes related to rhamnolipids production , but the rhlG mutant displayed no difference compared to PAO1 .Consistently with the inverse regulation of and Rock  data. Thand Rock . Since wfunction , we furtrhlG transcription, revealing a complex regulation since it relies on three sigma factors and is negatively affected by cell-to-cell communication molecule C4-HSL. rhlG transcription is induced by hyperosmotic stress via the ECF sigma factor AlgU and inversely regulated compared to the genes involved in rhamnolipid synthesis. Finally, we definitely ruled out that neither rhlG nor the downstream PA3388 gene are required for rhamnolipid production, but we failed to identify a function in which these genes are involved.We present here the first detailed study of −1; yeast extract 5 g.l−1; tryptone 10 g.l−1) and in PPGAS  . Hyperosmotic conditions were obtained by including 0.5 M NaCl into the medium before inoculation. Glycine betaine (GB)  was used at a final concentration of 1 mM. When indicated, C4-HSL (Sigma-Aldrich Co.) was added at a final concentration of 10 μM. Antibiotics were used at the following concentrations when necessary. For E. coli: 50 μg.ml−1 kanamycin (Km), 35 μg.ml−1 gentamycin (Gm), 100 μg.ml−1 ampicillin (Amp), and 10 μg.ml−1 tetracyclin (Tc); and for P. aeruginosa: 400 μg.ml−1 Gm, 600 μg.ml−1 carbenicillin (Cb), and 150 μg.ml−1 Tc.Strains and plasmids are listed in Table o pH 7.2 ) media aPQS and the major rhamnolipid species (di-rhamnolipid Rha–Rha–C10–C10) were identified and quantified from culture supernatants and cellular pellet using LC-MS as previously reported ,18.−1 of LB medium) at 37°C as previously described [Biofilms were grown for 24 h in flow cell chambers under dynamic conditions , with a gain set at 1 or 6 and with photomultiplier tubes (PMT) set at 1100. 100 μl of bacterial suspensions were adjusted to the lowest optical density of the different samples, and bioluminescence values of a negative control strain (containing pAB133) were subtracted from values resulting from pAB134-containing strain(s) [Induction of bioluminescence in bacteria carrying rhlG mRNAs were quantified by real-time PCR amplification of their cDNAs with the 7300 Real Time PCR System apparatus and SYBR Green PCR Master Mix (Applied Biosystems), using procedures previously described [RNAs were extracted using RNA protect bacteria reagent, RNeasy Midi Kit, and RNase-Free DNase Set . RNAs were converted to cDNAs using the High Capacity cDNA Archive Kit . escribed  and the E. coli  and P. aeruginosa were transformed by electroporation as described by manufacturer and in [P. aeruginosa was transformed by conjugation [Restriction enzymes, T4 DNA ligase, and alkaline phosphatase were purchased from Invitrogen . PCR reactions were performed using the FailsafeTM PCR reagent with 2x Premix D . Plasmids and RNAs were purified using the QIAprep Spin Miniprep Kit and RNeasy Midi Kit (Qiagen). r and in , respectjugation .rhlG promoter region (prrhlG) and the luxCDABE reporter operon was constructed as follows. The DNA fragment containing prrhlG was amplified from P. aeruginosa PAO1 chromosomal DNA by PCR with the prRhlG1 and prRhlG2 primers . The 5′ end of rhlG mRNAs was amplified using the 5′-RACE System for Rapid Amplification of cDNA Ends, Version 2.0  according to the manufacturer’s instructions. The primers used for cDNA synthesis, and for the first and second PCR reactions are listed in Table Total RNAs were isolated from P. aeruginosa PAO1 were obtained by allelic exchange as previously described [rhlG or PA3388) were PCR-amplified with primer pairs rhlGko1/2 and rhlGko3/4 or PA3388ko1/2 and PA3388ko3/4  (Table Mutants of escribed . The flarhlG mutation, the DNA fragment including rhlG and its promoter region was amplified by PCR using the primers prRhlG1 and rhlGko4 (Table To complement the o4 Table . The ampThe authors declare that they have no competing interests.AB performed all the experiments and co-drafted the manuscript. AD supervised the study and co-drafted the manuscript. Both authors read and approved the final manuscript.Figure S2. Extracellular and intracellular production of di-rhamnolipid. Figure S3. CLSM images of biofilms.Expression levels of rhlG gene. Click here for file


Transcriptional activation is a complex, multistage process implemented by hundreds of proteins. Many transcriptional proteins are organized into coactivator complexes, which participate in transcription regulation at numerous genes and are a driver of this process. The molecular action mechanisms of coactivator complexes remain largely understudied. Relevant publications usually deal with the involvement of these complexes in the entire process of transcription, and only a few studies are aimed to elucidate their functions at its particular stages. This review summarizes available information on the participation of key coactivator complexes in transcriptional activation. The timing of coactivator complex binding/removal has been used for restructuring previously described information about the transcriptional process. Several major stages of transcriptional activation have been distinguished based on the presence of covalent histone modifications and general transcriptional factors, and the recruitment and/or removal phases have been determined for each coactivator included in analysis. Recruitment of Mediator, SWItch/Sucrose Non-Fermentable and NUcleosome Remodeling Factor complexes during transcription activation has been investigated thoroughly; CHD and INOsitol auxotrophy 80 families are less well studied. In most cases, the molecular mechanisms responsible for the removal of certain coactivator complexes after the termination of their functions at the promoters are still not understood. On the basis of the summarized information, we propose a scheme that illustrates the involvement of coactivator complexes in different stages of the transcription activation process. This scheme may help to gain a deeper insight into the molecular mechanism of functioning of coactivator complexes, find novel participants of the process, and reveal novel structural or functional connections between different coactivators. Transcriptional machinery includes hundreds of transcription factors that function coordinately to provide for the progression of the multistage transcriptional process. To date, ample data have been accumulated on the functional properties of multiprotein transcription coactivator complexes that cannot themselves specifically interact with DNA but are nevertheless indispensable for transcriptional activation , 2. UnfoThis review is focused on the data concerning the involvement of different coactivator complexes at particular stages of transcriptional activation, which have been obtained by different authors in experiments on different organisms. Its purpose is to integrate these data into a preliminary model of transcriptional activation, which is not intended to describe this process for any real gene but rather is an attempt to generalize all the relevant information available from different sources. Hopefully, the proposed model will help to find new directions for research on the functions of coactivators and their participation in the transcription cycle.Activation of gene transcription is a multistage process, but for clarity in data presentation, we concentrate here only on its basic steps that are likely to occur during activation of the majority of inducible genes Fig. . In the Transcription activation processes begin with the removal of repressor complexes from the promoter Fig. a. Since At the next step, a DNA-binding transcriptional activator binds the promoter Fig. b. TranscThe transcriptional activator interacts with the promoter to stimulate the subsequent recruitment of chromatin remodeling complexes . Under their influence, the promoter sequence is cleared of nucleosomes Fig. c. This sGeneral transcription factors (GTFs) are recruited at the next step of transcriptional activation, which results in Pol II recruitment and preinitiation complex (PIC) formation. This process has been well studied to date, and its detailed three-dimensional models have been recently proposed by two independent research groups , 11. RecPol II can be stably associated with the promoter as an inactive complex, but its recruitment usually leads to the initiation of transcription Fig. f. This sAt the next step, active transcription elongation takes place Fig. g. The inFor a long time, studies of protein kinetics in transcription have generally been limited to two methods: FRAP and ChIP (fluorescence recovery after photobleaching and chromatin immunoprecipitation) –16. The With time, the “slow” recruitment of transcriptional complexes has found an explanation in a contemporary model of the transcriptional process . The patThe palette of the methods that can be used to investigate transcriptional protein dynamics has greatly expanded in recent times. Contemporary spectroscopic techniques enable research into the actions of the individual proteins in individual living cells or even in their parts , 27. TheFor a long time, scientists investigating the transcription cycle have been searching for additional markers that could help them to distinguish between its different stages. Eventually, covalent chromatin modification has proved to be the most promising marker. More than a decade ago, the correlation between the state of promoter chromatin and gene activity was discovered, and the “histone code” hypothesis was advanced to explain the observed results . AccordiThe correlation between histone modifications and the course of transcription has been evaluated in detail in studies on the transcription activation cycle kinetics, including an analysis of promoter chromatin status at different transcriptional stages. In the pioneering study on the molecular mechanism of pS2 gene activation by estrogen receptor, Metivier and colleagues have shown that several histone modifications (such as histone H4 methylation at Arg3 and histone H3 acetylation at Lys14) are introduced into the promoter chromatin at the beginning of the transcription cycle, immediately after activator recruitment but prior to preinitiation complex formation . HistoneAnother detailed kinetic research paper is focused on the dynamics of coactivators during progesterone receptor-dependent activation and, in particular, on the primary molecular mechanisms of transcription initiation immediately after the input of activation signal . Its resUnfortunately, no other detailed studies are available that describe the kinetic of transcriptional activation and summarize chromatin modifications during the transcription process, but there are several pieces of evidence that support the concept that the introduction of histone modifications is correlated with particular stages of transcription.Drosophila [As noted above, histone H3 trimethylation at Lys4 (H3K4me3) takes place at the earliest stages of progesterone-dependent gene transcription initiation . It has osophila –41. UnfoAcetylation was the first covalent posttranslational modification described for the histones . It was Numerous data confirm the association of histone H3 trimethylation at Lys36 (H3K36me3) with transition from transcription initiation to productive elongation by Pol II . This moAs mentioned above, transcription initiation involves a dynamic circuit of chromatin modifications, with different covalent modifications of histones replacing each other. Another illustrative example is the cross-talk between histone H2B ubiquitination at Lys120 (H2BK120Ub) and histone H3 trimethylation at Lys4 (H3K4me3) in the promoter chromatin. It has been shown that H2BK120Ub is spread along the coding region of the actively transcribed genes, while H3K4me3 is locally concentrated in the distal promoter region and is dependent on histone H2B ubiquitination , 51. BotData regarding the correlation of deposition of covalent histone modifications with individual stages of the transcriptional activation cycle are summarized in Table Mediator is the largest transcriptional coactivator complex described to date . This coAs shown in many model systems, recruitment of the Mediator complex is dependent on transcriptional activators; i.e., Mediator interacts with the promoter immediately after activator binding but prior to preinitiation complex formation . Its recThus, multiple data obtained to date make it possible to identify the exact transcriptional stage at which Mediator is recruited. Its binding to the promoter takes place immediately after activator recruitment but before the removal of promoter-bound nucleosomes. Therefore, Mediator functions as a transcriptional coactivator that is important for the recruitment of chromatin remodeling complexes and preinitiation complex formation, i.e., for the progression of the most important steps of transcription initiation.The Mediator complex undergoes serious structural rearrangements in the course of transcriptional activation. It has been shown that the complete form of the Mediator complex, which includes the CDK8 module, interacts with the SAGA histone modifying complex but has no effect on the transcriptional process in vitro . Whereas its truncated form which lacks the CDK8 module but contains an additional Med26 subunit is capable of stimulating in vitro transcription . The CDKThe presence of the Mediator complex on the promoters of yeast genes is negatively correlated with chromatin acetylation at Lys16 of histone H4 (H4K16Ac) . As noteRecent studies provide evidence that an important role in the removal of Mediator complex from the promoter is played by CDK7/Kin28 kinase, which is also responsible for phosphorylation at Ser5 of the Pol II C-terminal domain . CDK7/KiThe above data on the Mediator complex point out the importance of timely removal of coactivators in the course of the transcription cycle. We have previously noted that failure to remove chromatin modifications (the example of dKDM5/LID demethylase acting on H3K4me3) can also interfere with the transition into the elongation stage . To all The removal of nucleosomes from the promoter is one of the key stages of the transcription activation cycle. This stage is indispensable for the correct formation of the preinitiation complex and subsequent recruitment of Pol II. A number of coactivator complexes capable of chromatin structure remodeling have been described to date and, based on type of enzymatic activity of their ATPase subunits, classified into four families: SWI/SNF, ISWI, CHD, and INO80. Chromatin remodeling complexes have previously been regarded as frontline coactivators that are recruited at the earliest stages of transcription cycle and are important for clearing the promoter from heterochromatin and recruiting other coactivators. The results of recent studies show, however, that these complexes also participate in later stages of the transcription cycle, including transcription elongation. This is evidence for the insufficiency of our knowledge about chromatin remodeling coactivators. Apparently, they have additional, as of yet unknown functional characteristics that are realized after the basic function of a given coactivator is accomplished.Drosophila and humans). Mammals also have an additional ATPase subunit, named Brg1, which is structurally very close to Brahma but significantly differs from it functionally [SWI/SNF is a special type of chromatin remodeler that can affect nucleosomes in all known ways, causing their removal, sliding, loss of H2A/H2B dimers, and disruption of nucleosome interaction with DNA . The SWItionally .DNA-binding transcriptional activators are considered to play the leading role in SWI/SNF recruitment during promoter activation . SeveralSeveral studies have shown that the SWI/SNF remodeler and the transcriptional activator are recruited to the promoter simultaneously, as a preformed stable complex, and that this complex can also interact with other transcriptional coactivators , 18. In The exact mechanism of SWI/SNF removal from the promoter sequence is still unclear. Recent studies have provided data on the involvement of the SWI/SNF complex in the Pol II elongation process and on its physical association with the nascent pre-mRNP –85. HoweA distinctive feature of the ISWI coactivator family is a high diversity of complexes formed by the ATPase enzymatic subunit (named ISWI) shared by all members of this family. ISWI contains the catalytic DEXD ATPase domain together with two DNA-binding domains (HAND and SLIDE) and the histone-binding SANT domain and can interact with different proteins, forming various multiprotein complexes. The sets of such complexes differ between species. The family of the ISWI complexes described for humans is the most diverse and includes the ACF1, CHRAC, RSF, CERF, NoRC, WICH, WCRF, and NURF complexes . It has Drosophila ecdysone cascade [Although ample data point to the high biological significance of the NURF complex, the molecular mechanism of its action during transcriptional activation has not been studied in detail . There i cascade , 89. The cascade .The role of histone modifications in the recruitment of chromatin remodelers has been best demonstrated for the NURF complex. It has been found that the BPTF/NURF301 subunit of NURF contains a PHD finger domain that binds to histone H3 trimethylated at Lys4 (H3K4me3) and that a decrease in the level of this modification results in the impairment of BPTF/NURF301 recruitment and consequent reduction in the level of chromatin-bound ISWI ATPase . The resThus, both the interaction of NURF with the activator and its binding to modified histone H3K4me3 may account for the recruitment of this complex, and further studies are needed to find out which of these factors plays the leading role.In coactivators of the CHD family, the enzymatic ATPase subunit contains the DEXD domain, as in other chromatin remodelers, but its distinctive feature is the presence of two additional chomodomains. In contrast to the families described above, the CHD family comprises complexes in which ATPase subunits slightly differ structurally . On thisThe CHD1 complexes are the only group in the CHD family that has been shown to participate in transcriptional activation processes that take place at the promoters. CHD1 homologs in yeast have been found at the promoters of actively transcribed genes and supposed to be important for the stabilization of the open chromatin structure . MutatioDrosophila Mi2 ATPase is recruited to the coding regions of actively transcribed heat-shock-dependent genes and is important for enhancing their transcription to the full extent [Complexes of the CHD3-5 (Mi2) subfamily contain specific subunits that have deacetylase activity, and their main function is considered to be related to transcriptional repression . Indeed,l extent . The recKismet gene mutation dramatically reducing the level of Pol II at the gene bodies while not affecting the level of its promoter-bound fraction [According to available data, complexes of the CHD6-9 subfamily are not directly involved in the processes of gene activation at the promoters but specifically associate with transcriptional enhancers , 104. A fraction .Molecular mechanisms that control the recruitment of CHD complexes to the actively transcribed genes have not been studied adequately. In cases of Mi2 and CHD1, which participate in Pol II elongation, this process is mediated by PARP and PAF complexes, respectively, but the nature of their interaction with the promoter region during transcriptional activation is still obscure.The INO80 family is composed of multiprotein complexes formed by SNF-2-like ATPases INO80 and SWR1 . A distiPHO5 and INO1. In the former, INO80 is recruited to the promoter region immediately after transcription induction, in a complex with SWI/SNF chromatin remodeler and SAGA histone modifier. These coactivator complexes are probably recruited to the chromatin via interaction with the corresponding DNA-binding transcriptional activator. The INO80 complex assists the SWI/SNF remodeler in clearing the promoter of nucleosomes and is important for complete transcriptional activation of PHO5 [INO1 gene, where its recruitment to the promoter chromatin is also mediated by a DNA-binding activator [The results of in vitro experiments show that the INO80 complex causes nucleosome sliding and arrangement into a specific pattern, different from those obtained with other chromatin remodelers, and facilitates transcription, increasing its level up to tenfold , 109. Ho of PHO5 . The invctivator . EvidencA major function of the SWR1 complex is to substitute the H2A/H2B dimer by a dimer containing histone H2A variants H2AZ . EnrichmAn analysis of available information on the involvement of coactivator complexes in individual stages of transcriptional activation has allowed us to propose a scheme illustrating the functions of coactivators at different stages of this process. Although consideration has been given to multiple data obtained with different gene models and organisms, we have revealed a substantial lack of information regarding recruitment/removal mechanisms for most of the analyzed complexes. The recruitment methods during the transcriptional activation cycle have been well described for some coactivator complexes. Transcriptional activators  are responsible for the binding of the SWI/SNF and NURF complexes, which mediate promoter clearance of occupied nucleosomes. Recruitment of the Mediator complex, a key structure organizing cross-talk between coactivators and general transcriptional factors, also occurs with participation of transcriptional activators. The involvement of the Mi-2 chromatin remodeler in transcription at active genes is mediated by the activity of PARP. Nevertheless, evidence for the participation of some coactivator complexes  at the particular stages of this cycle is based only on scarce experimental data or even indirect evidence. The molecular mechanisms responsible for the removal of certain coactivator complexes after the termination of their functions at the promoters also remain unclear. Altogether, the proposed scheme of the transcription activation cycle can give an idea about the current state of knowledge in this field of molecular biology and point out the least studied issues for future research on coactivator complexes and the molecular mechanisms of their functioning. We believe that future investigation of the stages in which coactivators are recruited and removed will allow for significant improvement of integrated scheme of transcription activation process.Kinetic studies of transcriptional activation will help to determine additional participants in the process. Even analogous molecular functions could be realized during gene activation by different coactivator complexes (just as the chromatin remodeling process at the promoter has been shown to be executed by different remodeler families). That is why the action of the coactivator complex recruited at the latter stages of the cycle is frequently hidden by the previous player with properties similar to those of the initial coactivator. Research on coactivator dynamics could uncover an answer to one of the current mysteries in the field: the accumulation of different transcriptional complexes in the same genomic regions. The temporal separation of the molecular functions of these complexes would help to determine the functional scheme of the particular genome region.Recent advances in the visualization of the individual transcriptional processes in living cells have brought much clarity to the field. The real timing of transcription and the duration of its individual stages have become much clearer , 114. Un