HMS2 in Saccharomyces cerevisiae, sgRNA/dCas9 targeting to the non-template strand for antisense transcription results in antisense transcription termination, premature termination of a proportion of sense transcripts and initiation of a novel antisense transcript downstream of the sgRNA/dCas9-binding site. This redefinition of the transcriptional landscape by CRISPRi demonstrates that it is not strand-specific and highlights the controls and locus understanding required to properly interpret results from CRISPRi interventions.CRISPRi, an adapted CRISPR-Cas9 system, is proposed to act as a strand-specific roadblock to repress transcription in eukaryotic cells using guide RNAs (sgRNAs) to target catalytically inactive Cas9 (dCas9) and offers an alternative to genetic interventions for studying pervasive antisense transcription. Here, we successfully use click chemistry to construct DNA templates for sgRNA expression and show, rather than acting simply as a roadblock, sgRNA/dCas9 binding creates an environment that is permissive for transcription initiation/termination, thus generating novel sense and antisense transcripts. At  Eukaryotic genomes are pervasively transcribed but the effect of this inter- and intragenic transcription is not fully understood . Of partSUT650 at the HMS2 locus to show that SUT650 represses HMS2 sense transcription (SUT650 antisense transcription to ask (i) whether SUT650 represses HMS2 sense transcription without a genetic intervention and (ii) whether CRISPRi is strand-specific using, in addition to HMS2, an engineered GAL1 gene with a well-characterised antisense transcript  premature termination of the sense transcript and (ii) initiation of a new unstable antisense transcript in the vicinity of the sgRNA binding site. As transcription from this new antisense initiation site extends in to the HMS2 promoter, there is no net change in HMS2 sense transcript levels. Thus, CRISPRi redefines the transcriptional landscape at HMS2. This suggests that routine use of CRISPRi for gene expression analysis will require rigorous analysis of transcript integrity and function before conclusions can be drawn.The main conclusion from this study is that CRISPRi at the URA3 selection cassette in the endogenous snR52 locus for expression of a transcript that is then processed to form the mature nuclear-retained sgRNA  initiating within an ADH1 terminator inserted into the GAL1 coding region  strand next to a protospacer adjacent motif (PAM)  Figure . Firstlyg region   (SUT650 TSS (SUT650 TSS. AS+243NT/dCas9 significantly (p=0.011) reduced SUT650  . Note thT650 TSS  and thusd SUT650 . Howevers tested . CRISPR s tested , but we s tested  at each  regions . However regions . These rHMS2, sgRNA AS+243NT/dCas9 was able to reduce SUT650 transcript levels significantly . To map transcript Bt, we used a series of Northern blotting probes across the locus  was created. This is potentially due to changes in the chromatin structure caused by AS+243NT/dCas9 binding to this region .At ficantly . Since Sal block   when the GAL1 AS transcript was blocked , we detected a considerably shortened HMS2 sense transcript (At). Since the combined levels of the truncated and full-length transcripts are similar to those in the strain not expressing an sgRNA, independent of the presence of XRN1 , but the truncated transcript was undetectable using probe H4 downstream of this site. Thus, the transcriptional block caused by AS+243NT/dCas9 is not strand-specific and re-defines the transcriptional landscape at the HMS2 locus by creating a chromatin environment that is suitable for both transcription initiation and termination. To see if we could more directly target and block HMS2 sense transcripts, we designed three sgRNAs to bind the non-template strand adjacent to PAM sites near the HMS2 sense TSS  and monitored their effects on both HMS2 sense and antisense transcripts . To differentiate between these possibilities, we deleted the major cytoplasmic 5’−3’ exonuclease XRN1. Since XRN1 deletion in the TATA mutant strain only slightly increased GAL1 AS levels, this suggests that most repression was at the level of transcription rather than altering transcript stability   . This surference . Whilst  altered . The dis results . Thus, CGAL10 (GAL1 (this work), here we demonstrate that this is not true at HMS2. This may reflect the vastly different transcription levels for the galactose-inducible genes (high) compared to HMS2 (low). Furthermore, a study in human K562 cells found that CRISPRi-induced repression did not correlate with which strand was targeted at 49 genes, although the mechanism behind this observation was not investigated  to minimise any off-target effects .PAM sequences within 300 bp of the TSS in question were identified and the 20 bp immediately adjacent to these were used to design the variable regions of the sgRNAs. These sequences were run through an off-spotter algorithm .Single-stranded DNA oligonucleotides complementary to the constant and variable sgRNA regions were synthesised by standard phosphoramidite oligonucleotide synthesis with 3’-alkyne or 5’-azido modifications respectively  Supplem. The cliint DNAs . PCR wasTDH3 promoter. The DNA templates for sgRNA production were integrated in place of an exogenous URA3 cassette immediately downstream of the splice site in the endogenous snR52 locus using homologous recombination followed by 5-FOA selection. Correct insertion was confirmed by genomic DNA Sanger sequencing. SNR52 is a Pol III-transcribed C/D box small nucleolar RNA (snoRNA) gene and so does not undergo extensive post-transcriptional processing such as capping and polyadenylation, and transcripts from this locus are retained in the nucleus. Additionally, the locus contains a self-splicing site that produces a mature transcript without additional machinery, allowing precise production of a mature sgRNA without any unwanted extensions. Primer sequences for strain construction are listed in dCas9, without fusion to a transcriptional repressor domain, was expressed from a plasmid (# 46920 Addgene ) under tGAL1 locus, yeast were grown to mid-log in rich media  so that the GAL1 induction kinetics were similar to what we had observed previously  was achieved using a Bioscreen spectrophotometer that automatically measures the optical density of cultures at 30°C every 20 min for 24 hr. Doubling times were extracted from the gradient of the curves during logarithmic growth.GAL1 and HMS2, respectively. Primers for these reactions are listed in Northern blotting was performed as before  using asdCas9 expression was confirmed after each experiment using an anti-Cas9 antibody (Diagenode C15310258) at 1:5000 dilution and anti-Tubulin (Abcam ab6160) at 1:3333.Yeast cells were fixed with 1% formaldehyde  followed by the addition of glycine to 125 mM for 5 min. Cell pellets were collected by centrifugation  before washing twice with 10 ml cold PBS. Cells were re-suspended in cold FA-150 buffer  and broken using 1-ml glass beads on a MagnaLyser (Roche) at . Fixed chromatin was sheared by sonication using a biorupter , cleared by centrifugation  and incubated with 5 µl anti-Cas9 antibody (Diagenode C15310258) and 30 µl protein A-dynabeads pre-blocked with 200 µg/ml yeast tRNA, 200 µg/ml BSA and 200 µg/ml glycogen . Beads and attached chromatin were collected using a magnetic rack and washed with TSE-150 buffer  for 3 min, TSE-500 buffer  for 3 min, LiCl buffer  for 15 min and twice with TE, all at 22°C. After washing, chromatin was eluted from the beads for 30 min at 65°C with elution buffer . Addition of 350 mM NaCl and incubation for 3 hr at 65°C reversed the cross-links before treatment of samples with RNase A for 1 hr at 37°C and proteinase K overnight at 65°C. DNA was purified using a PCR-purification kit (Qiagen) and eluted in 400 µl 1 mM Tris-Cl pH 8.0. Input DNA was diluted accordingly. Real-time quantitative PCR (qPCR) was carried out using a Corbett 6000 Rotorgene and Sybr green qPCR master mix (Bioline). Data ([IP - no antibody control]/input) were expressed as a percentage of the input (relative to sgRNA +276 NT). Real-time qPCR primers are listed in GAL1 sense transcript was performed as  appeared as ideally suited for this since it has been shown to be able to trigger transcription termination in a site specific and, most importantly, strand specific manner, without altering the genomic sequence.This short report shows that this strand specificity is actually not true at all loci. In addition, it describes an example in which the binding of the guide RNA actually induces spurious transcription initiation, suggesting that it markedly perturbs the local chromatin environment.I think that the data are suitably convincing. Note, by the way, that this referee has made the similar (unpublished) observations that the termination by this system was not strand specific at yet another locus.eLife might seem appropriate for that.Although one could argue that the main results presented in this manuscript are negative results, I personally think that they are well worth being published to warn further investigators who would wish to use this system and a short report in eLife, 2015) would be located about 60 nucleotides DOWNSTREAM of the AS-44NT sgRNA binding site (position 705184). Thus, as the other SUT650 sgRNA targeting sequences, AS-44NT would actually be located within the SUT650 transcribed region, downstream from its TSS and not upstream. It could thus not be considered as a "control" sgRNA. Could the authors verify this point and, if this is correct, modify the text, The position of the 5'-end of SUT650 seems incorrectly assigned. Indeed, if one refers to the TIF-seq data from the original Pelechano paper, the main transcription start site for SUT650  in wt and Xrn1 deletion strains. Their primary conclusion is that CRISPRi works well in some cases and not in others and requires careful controls. For example, the authors show that the system does repress antisense transcription, but at one of the two studied loci (HMS2), an additional antisense transcript appears from a new initiation site, and the sense transcript is prematurely terminated. In addition, they present a clever tool based on click chemistry for quickly making gRNA. Although I don't find the main conclusion revelatory, I commend the authors for their thoroughness. Certainly, this message is one that bears reporting, although I am not quite convinced this result clears the bar for an 1) The authors show that CRISPRi represses the antisense transcript at a GAL1 model gene. They then check whether this repression has transcriptional effects on GAL1 sense transcription and they conclude there is no difference in galactose. However, previous work has shown that antisense at the GAL locus works mostly under repressive conditions. Have the authors repeated this experiment in glucose or raffinose?2) The authors show that CRISPRi repression at the HMS2 locus results in a changed transcriptional landscape. Replacement of the HMS locus by URA does not have the same effect, and they suggest that CRISPRi binding causes these additional transcripts. However, it is unclear whether the new transcripts at HMS2 appear because of removal of the antisense, which in itself could change the chromatin landscape and thus the transcriptional landscape, or whether it arises because of binding of dCas9. The same authors have previously published that removal of the GAL10 ncRNA by genetic methods (point mutations of the transcription factor binding sites) also results in additional transcripts not visible in the wildtype . Given these findings, it remains unclear whether the new antisense transcript indeed results from the CRISPRi binding. Moreover, it is noteworthy that additional transcripts are also found in some genetic mutants. Control experiments are therefore required for both methods.3) The Xrn1 sensitivity is a confounding factor, and I am not sure that strain should be the gold standard for CRISPRi not working. The whole point is that one can get blocking or perturbation in the absence of genetic manipulation. So, for example one concluding sentence "CRISPRi is not as effective as a genetic mutation in reducing levels of either the GAL1 or HMS2 AS transcripts" should end with "[…] in the Xrn1 deletion."4) In a related point, 5) Lastly, given that CRISPRi does represses the antisense transcript in both loci tested in the study in a strand-specific manner, the title is a bit misleading.In summary, despite technical issues, I nevertheless find their argument that CRISPRi can have locus-specific effects to be convincing. However, the manuscript is mostly of a technical nature. Most experiments described are control experiments that should be done anyways when using a new method. The observations that CRISPRi can result in different transcripts is good to know when designing such experiments, but there is no proposed mechanistic insight, nor new biology that is learned from this study.Reviewer #3:In this manuscript Howe and collaborators in the Mellor and Brown labs explore the molecular basis of CRISPRi transcriptional interference in the context of yeast anti-sense transcription. This reviewer agrees with the authors that understanding the basis of CRISPRi in anti-sense transcription  is an important area of research because it is extremely difficult to establish direct causality with regard to these particular RNAs using genetic perturbations.In the first section of their manuscript, Howe et al., describe synthesis of sgRNA constructs using "click-chemistry" to couple sgRNA variable regions to the constant region. The author's then use knock-in yeast strains generated using these constructs to evaluate the effects of CRISPRi on anti-sense transcription at the GAL1 and HMS2 genes. The authors observe that contrary to behaving as a transcriptional road-block when targeted to the non-template strand, dCas9 CRISPRi complexes result in unpredictable changes in chromatin and transcription, resulting in pre-mature termination of sense transcripts and shifting anti-sense transcription initiation sites. Collectively, these results clearly demonstrate that CRISPRi effects are more complex than previously reported and likely depend on locus-specific chromatin context. These observations have broad implications with respect to design of CRISPRi strategies and argue a strong case for careful characterization of transcript isoforms and promoter/termination sites before and after deploying CRISPRi. However, there are some fundamental issues with controls and some of the conclusions drawn that should be addressed before publication.1) The authors observe that only a subset of sgRNAs targeting the non-template strand are capable of mediating repression, however, the reasons for this phenomenon are unclear. A simple explanation is that some sgRNAs are not capable of mediating stable dCAS9 interaction with chromatin. The author's should test this by performing Chromatin-Immunoprecipitation (ChIP) for dCas9 in their various sgRNA expressing lines to test if dCas9 chromatin occupancy correlates with repression. In my opinion this is an absolutely essential control for interpreting the authors' experiments.2) This reviewer feels that increasing the sgRNA coverage across the sense/antisense transcripts in question would greatly strengthen the manuscript. In its current state, the authors test only 2 sgRNAs towards the GAL1-AS and SUT650 non-template strands, from which they obtain a single functional repressive sgRNA for each anti-sense transcript. As a strand specificity control they test 3 sgRNAs targeting the sense transcript HMS2 and show that none of these has an effect on sense or anti-sense. However, with a hit rate of ~1 in 2, this reviewer is not convinced by authors' claim that sgRNAs targeting the sense transcript have no effect on antisense transcription. Perhaps the coverage was simply too low to obtain a functional repressive sgRNA. The authors should perform high-density sgRNA tiling experiments (targeting both template and non-template) for either GAL1-AS or SUT650 (not necessary to do both loci). I feel this experiment will dramatically improve the manuscript and instill clarity into the context requirements for CRISPRi mediated repression. Reviewer #1:In recent years, transcriptional interference by antisense RNA transcription has been recognized as a relatively frequent means to regulate mRNA expression. Tools enabling antisense transcription arrest without directly affecting sense transcription are required to study the mechanism mediating this effect. The recently reported CRISPRi system, using a modified, partially defective, Cas9 (dCas9) appeared as ideally suited for this since it has been shown to be able to trigger transcription termination in a site specific and, most importantly, strand specific manner, without altering the genomic sequence.This short report shows that this strand specificity is actually not true at all loci. In addition, it describes an example in which the binding of the guide RNA actually induces spurious transcription initiation, suggesting that it markedly perturbs the local chromatin environment.I think that the data are suitably convincing. Note, by the way, that this referee has made the similar (unpublished) observations that the termination by this system was not strand specific at yet another locus.Although one could argue that the main results presented in this manuscript are negative results, I personally think that they are well worth being published to warn further investigators who would wish to use this system and a short report in eLife might seem appropriate for that.We thank the reviewer for recognising the importance of this work as a warning to other researchers.The position of the 5'-end of SUT650 seems incorrectly assigned. Indeed, if one refers to the TIF-seq data from the original Pelechano paper, the main transcription start site for SUT650  would be located about 60 nucleotides DOWNSTREAM of the AS-44NT sgRNA binding site (position 705184). Thus, as the other SUT650 sgRNA targeting sequences, AS-44NT would actually be located within the SUT650 transcribed region, downstream from its TSS and not upstream. It could thus not be considered as a "control" sgRNA. Could the authors verify this point and, if this is correct, modify the text, We thank the reviewer for pointing this out and apologise for our oversight. We note that there is a large heterogeneity in the position of the transcription start site for SUT650 and the one we had chosen previously  is one of the many sites. We have now discussed the transcript heterogeneity in the main body of the text and included a supplementary figure with the Pelechano TIF-seq data to illustrate this . We haveReviewer #2:In this paper by Howe et al., the authors assess the potential of CRISPRi for strand-specific transcriptional perturbation in budding yeast. They study two genes in depth – GAL1 and HMS2 – and compare the effectiveness of CRISPRi to previous approaches (for example deletion of cis-acting motifs such as the TATA box) in wt and Xrn1 deletion strains. Their primary conclusion is that CRISPRi works well in some cases and not in others and requires careful controls. For example, the authors show that the system does repress antisense transcription, but at one of the two studied loci (HMS2), an additional antisense transcript appears from a new initiation site, and the sense transcript is prematurely terminated. In addition, they present a clever tool based on click chemistry for quickly making gRNA. Although I don't find the main conclusion revelatory, I commend the authors for their thoroughness. Certainly, this message is one that bears reporting, although I am not quite convinced this result clears the bar for an eLife paper.eLife, with its reputation for thoroughness and research integrity, would be an ideal journal for publishing this work.We thank the reviewer for their support of the message we are trying to convey to the community. We feel that 1) The authors show that CRISPRi represses the antisense transcript at a GAL1 model gene. They then check whether this repression has transcriptional effects on GAL1 sense transcription and they conclude there is no difference in galactose. However, previous work has shown that antisense at the GAL locus works mostly under repressive conditions. Have the authors repeated this experiment in glucose or raffinose?As presented in 2) The authors show that CRISPRi repression at the HMS2 locus results in a changed transcriptional landscape. Replacement of the HMS locus by URA does not have the same effect, and they suggest that CRISPRi binding causes these additional transcripts. However, it is unclear whether the new transcripts at HMS2 appear because of removal of the antisense, which in itself could change the chromatin landscape and thus the transcriptional landscape, or whether it arises because of binding of dCas9. The same authors have previously published that removal of the GAL10 ncRNA by genetic methods (point mutations of the transcription factor binding sites) also results in additional transcripts not visible in the wildtype . Given these findings, it remains unclear whether the new antisense transcript indeed results from the CRISPRi binding. Moreover, it is noteworthy that additional transcripts are also found in some genetic mutants. Control experiments are therefore required for both methods.The point made here by this reviewer is a valid one – we are unable to infer whether it is the binding of sgRNA/dCas9 or the loss of the antisense that is causing the initiation of the new transcripts. It would be experimentally very difficult to show this and we have now clarified the two possible reasons for new transcripts in the text.However, the main point is that, independent of what is causing the initiation of the new antisense transcript, in the CRISPRi strain, transcription is occurring in the antisense direction into the HMS2 sense promoter and so no conclusions could be made about the function of SUT650 in sense regulation in this strain. The fear is that some investigators may use the CRISPRi system without the proper controls and the current set of experiments is intended to highlight how important these controls are.3) The Xrn1 sensitivity is a confounding factor, and I am not sure that strain should be the gold standard for CRISPRi not working. The whole point is that one can get blocking or perturbation in the absence of genetic manipulation. So, for example one concluding sentence "CRISPRi is not as effective as a genetic mutation in reducing levels of either the GAL1 or HMS2 AS transcripts" should end with "… in the Xrn1 deletion."+ background, the transcript could not be detected. This is important mechanistically because often it is the act of antisense transcription that may cause the effect on neighbouring/overlapping transcription units rather than the antisense transcript itself . In We apologise that we were not clear about the reason for performing the experiments in the XRN1 delete and have now clarified this in the text. We chose to use the deletion of XRN1 to stabilise any transcripts that may be being produced in the CRISPRi strains but then rapidly degraded so that, if the experiment is done in an XRN14) In a related point, We have found it very difficult to quantify blots with very low signal levels so we apologise that the image did not appear to be representative of the accompanying quantification. We have now included a more rigorous background subtraction step for the GAL1 AS Northerns and hope that the reviewer is satisfied with the new 5) Lastly, given that CRISPRi does represses the antisense transcript in both loci tested in the study in a strand-specific manner, the title is a bit misleading.We have shown that CRISPRi only works strand-specifically at GAL1, because at HMS2, both the sense and the antisense transcripts are blocked. However, we agree that the original title perhaps didn’t reflect this fully so have now changed it to include the words ‘at all loci’: ‘CRISPRi is not strand-specific at all loci and redefines the transcriptional landscape’.In summary, despite technical issues, I nevertheless find their argument that CRISPRi can have locus-specific effects to be convincing. However, the manuscript is mostly of a technical nature. Most experiments described are control experiments that should be done anyways when using a new method. The observations that CRISPRi can result in different transcripts is good to know when designing such experiments, but there is no proposed mechanistic insight, nor new biology that is learned from this study.We agree that most of the experiments we have done should be performed as controls for any CRISPRi experiments but are aware that, especially if CRISPRi is being used for a large-scale screen, this probably won’t be done . We wish to publish this work to highlight how important it is that these controls are done or perhaps to suggest that CRIPSRi may not always be a suitable technique if the correct controls cannot be performed.We have also added mechanistic insight in the new Reviewer #3:In this manuscript Howe and collaborators in the Mellor and Brown labs explore the molecular basis of CRISPRi transcriptional interference in the context of yeast anti-sense transcription. This reviewer agrees with the authors that understanding the basis of CRISPRi in anti-sense transcription  is an important area of research because it is extremely difficult to establish direct causality with regard to these particular RNAs using genetic perturbations.In the first section of their manuscript, Howe et al., describe synthesis of sgRNA constructs using "click-chemistry" to couple sgRNA variable regions to the constant region. The author's then use knock-in yeast strains generated using these constructs to evaluate the effects of CRISPRi on anti-sense transcription at the GAL1 and HMS2 genes. The authors observe that contrary to behaving as a transcriptional road-block when targeted to the non-template strand, dCas9 CRISPRi complexes result in unpredictable changes in chromatin and transcription, resulting in pre-mature termination of sense transcripts and shifting anti-sense transcription initiation sites. Collectively, these results clearly demonstrate that CRISPRi effects are more complex than previously reported and likely depend on locus-specific chromatin context. These observations have broad implications with respect to design of CRISPRi strategies and argue a strong case for careful characterization of transcript isoforms and promoter/termination sites before and after deploying CRISPRi. However, there are some fundamental issues with controls and some of the conclusions drawn that should be addressed before publication.1) The authors observe that only a subset of sgRNAs targeting the non-template strand are capable of mediating repression, however, the reasons for this phenomenon are unclear. A simple explanation is that some sgRNAs are not capable of mediating stable dCAS9 interaction with chromatin. The author's should test this by performing Chromatin-Immunoprecipitation (ChIP) for dCas9 in their various sgRNA expressing lines to test if dCas9 chromatin occupancy correlates with repression. In my opinion this is an absolutely essential control for interpreting the authors' experiments.We are not the first to observe that, as for CRISPR, some sgRNAs are not effective for CRISPRi e.g. Lenstra et al., 2015. We do not aim to address the question of why only some sgRNAs are repressive in this paper. However, we have now performed a ChIP experiment for (d)Cas9 see  to show 2) This reviewer feels that increasing the sgRNA coverage across the sense/antisense transcripts in question would greatly strengthen the manuscript. In its current state, the authors test only 2 sgRNAs towards the GAL1-AS and SUT650 non-template strands, from which they obtain a single functional repressive sgRNA for each anti-sense transcript. As a strand specificity control they test 3 sgRNAs targeting the sense transcript HMS2 and show that none of these has an effect on sense or anti-sense. However, with a hit rate of ~1 in 2, this reviewer is not convinced by authors' claim that sgRNAs targeting the sense transcript have no effect on antisense transcription. Perhaps the coverage was simply too low to obtain a functional repressive sgRNA. The authors should perform high-density sgRNA tiling experiments (targeting both template and non-template) for either GAL1-AS or SUT650 (not necessary to do both loci). I feel this experiment will dramatically improve the manuscript and instill clarity into the context requirements for CRISPRi mediated repression.We do not claim that, if we were to find an sgRNA to block HMS2 sense, that this would not also affect the antisense transcript rather that the 3 sgRNAs we tested against HMS2 sense did not repress either the sense or antisense transcripts. We apologise if this was confused in the text and have clarified this section. We included the (negative) results from the 3 sense-targeting sgRNAs again to reiterate the fact that not all sgRNAs are effective and this is an important control to do. We included the HMS2 AS data from the sense sgRNA strains as a control for the sgRNAs also not working on the template strand.As stated above, we are not aiming to investigate what makes some sgRNAs cause effective transcript repression and others not. We do not feel that testing more sgRNAs would significantly add to the conclusions of the paper, which were that a) CRIPSRi does not always repress transcription b) CRISPRi is not always strand-specific and c) CRISPRi can induce novel transcripts that would confound any analysis. It is also relevant that the highest CRISPRi activity with unfused dCas9 was observed using PAM sites near the relevant TSSs  as we have done here. Using PAM sites that are more distant from the TSS is unlikely to add any insight into the conclusions we have made here.


Our view of the nuclear pore complexes (NPCs) as gateways between the nuclear and cytoplasmic compartments has been largely expanded in recent years. NPCs have now demonstrated roles in genome regulation and maintenance from single cells to multicellular organisms. Both NPC proteins as well as components of the NPC basket act as dynamic scaffolds for silencing factors, and chromatin and cell cycle regulators. Components of the NPC basket also couple mRNA production and export, and prevent the exit of unprocessed mRNAs from the nucleus. Our recent work describes a novel function of the fission yeast nuclear basket component - the translocated promoter region (TPR) nucleoporin Alm1 - in proper localization of the proteasome to the nuclear envelope. Here we discuss how regulation of proteasome localization to the nuclear envelope by Alm1 is key to maintain kinetochores homeostasis and proper chromosome segregation. The nuclear envelope (NE) is a bilayer membrane that separates the DNA-containing nucleoplasm from the cytoplasm. Communication through these two cellular compartments is achieved through the nuclear pore complex, a macromolecular channel that transverses the NE. NPCs act as a highly selective gate for the bi-directional exchange of proteins and the export of mRNA from the nucleoplasm. However, a growing body of evidence indicates that both NPCs and NE have a pivotal role in maintaining nuclear organization and thus, they are also essential to maintain genome integrity.The NPC is composed of multiple copies of roughly 30 different nucleoporins (nups) organized in a channel with eight-fold symmetry. Eight filaments emerge from both the cytoplasmic and nucleoplasmic face of the NPC. The nucleoplasmic filaments are organized in a basket-like structure that projects from the NPC towards the nuclear interior called "Nuclear Basket". The major constituents of the nuclear basket, the TPR nups, are evolutionarily conserved and consist of large (~200kD) coiled-coil proteins that function as a dynamic scaffold for different protein complexes at the NPC.Saccharomycescerevisiae expresses two similar but functionally distinct TPR orthologues, Mlp1 and Mlp2. In S. cerevisiae, active transcription of GAL genes occurs at the nuclear periphery. The assembly of transcriptional complexes at the nuclear periphery is mediated by the nuclear basket: Mlp1 binds the SAGA (Spt-Ada-Gcn5-acetyltransferase) chromatin-modifying complex, whereas Nup1 interacts with the TREX-2 (Transcription Elongating and RNA Export) complex. Deregulation of any of these elements results in delocalization of GAL genes from the nuclear basket and deregulated transcription. This role of Mlp1 does not seem to be restricted to inducible genes, but also affects constitutive ones. Mlp1 interacts with the poly(A) binding protein Nab2, thus recruiting poly(A) mRNAs to the NPC, where Mlp proteins additionally act as a quality control mechanism to retain in the nucleus unspliced or aberrantly processed mRNPs.One of the best-studied roles for TPR nucleoporins is in transcriptional regulation and mRNP export. Mlps are also involved in transcriptional memory. Inducible genes retain memory of their recent transcriptional activity during intervening periods of repression by creating gene-loops that transiently inhibit transcription. This memory allows rapid recruitment ofRNA polII to promoters and faster reactivation of transcription. Mlp1 and Mlp2 by binding to specific DNA sequences at promoters, specifically recruit a different set of regulated genes to the nuclear periphery and are required for transcriptional memory. Recently, Mlp1/2 have been shown to prevent R-loop formation during transcription. The proximity of transcribed genes to the NPC or "NPC-gene gating", dependent on Mlp1/2, avoids R-loop formation likely by facilitating the export of mRNP out of the nucleus. Thus, the nuclear basket works as a platform to coordinate the function of several multiprotein complexes involved in chromatin regulation, transcription regulation, mRNA biogenesis and proofreading and mRNA export. S. cerevisiae, Aspergillus nidulans, Arabidopsis thaliana, Caenorhabditis elegans,Drosophila melanogaster, and in human cells. During mitosis Mad1-Mad2 accumulate at kinetochores, where they are essential for SAC activation. The requirement of TPR/Mlp proteins for the localization of Mad1 and Mad2 at kinetochores appears to be less conserved. A. nidulans TPR orthologue Mlp1 associates with kinetochores and is required for proper localization and function of Mad1 during mitosis. Human TPR and Megator (Mtor), the D. melanogaster TPR counterpart, are part of a fusiform structure called the nuclear matrix, which surrounds the mitotic spindle, and that is a dynamic structural scaffold for key mitotic regulators. Among these mitotic regulators are the component of the SAC Mph1, Mad1 and Mad2. Human TPR- and Mtor- depleted cells show an accelerated progression through mitosis compared to control cells, decreased levels of Mph1, Mad1 and Mad2 at kinetochores, and weakened response to MT depolymerisation, suggesting that TPR and Mtor are required for proper SAC response.TPR nucleoporins also act as spatial regulators of the Spindle Assembly Checkpoint (SAC), a mitotic surveillance mechanism that inhibits the metaphase to anaphase transition when kinetochores are not properly captured by spindle microtubules (MTs). The core SAC components Mad1 and Mad2 localize at the NPC during interphase in a TPR/Mlp-dependent manner, and this seems to be evolutionarily conserved as it has been found inS. cerevisiae Mlps are required to anchor Mad1 at the nuclear basket. Mad1 has been shown to cycle between the NPCs and the kinetochores during spindle perturbation in order to elicit the Kap121p transport inhibitory pathway (KTIP). This prevents Kap121-dependent nuclear import of cargos during SAC activation. The deletion of Mlps results in loss of the KTIP. It is thought that phosphorylation on both Mad1 and the Mlps by mitotic kinases regulates the cycling of Mad1 and the KTIP.TPR nups are highly regulated. For instance, human TPR is phosphorylated at several residues by ERK2, PKA and CDK1 kinases and these phosphorylations are key for the regulation of the differential localization and function of human TPR. Altogether, these studies show that the nuclear basket also works as a dynamic and highly regulated platform that regulates SAC localization and activity and coordinates SAC signalling with transport through the NPC.Schizosaccharomyces pombe has two members of the TPR family of nups, Nup211 and Alm1. Nup211 is essential for cell viability and is required for mRNA export. In a recent study from our lab, we characterize the function of Alm1. We found that Alm1 is required for proper chromosome segregation: the absence of Alm1 resulted in a delay in the metaphase to anaphase transition due to SAC activation and lagging chromosomes during anaphase. Alm1 is required for proper localization of Mad2 to the NPC during interphase, which indicates that this function of TPR nups is also conserved in S. pombe. In alm1Δ cells Mad2 and Bub1, a key signalling kinase of the SAC, localize properly at kinetochores in normal mitosis and during MT-perturbation, and double mutants between alm1Δ and mad2Δ or bub1Δ showed a negative genetic interaction. This indicated that Alm1 is not required for SAC functionality, but instead SAC function is essential for alm1Δ viability. Consistently, we observed altered kinetochore behaviour during metaphase in alm1Δ cells, which suggests that in the absence of Alm1 kinetochore capture by spindle MTs is affected and SAC is consequently activated. These results led us to perform a synthetic genetic array (SGA) analysis in conditions that affected kinetochore-MT capture (in the presence of the MT-depolymerizing drug thiabendazole). This genomic approach revealed a strong genetic interaction between alm1Δ mutant and mutants in SAC components, ubiquitin-dependent proteolysis, chromatin regulators and kinetochore components.alm1Δ cells Cnp3 degradation was affected.Kinetochores assemble on the centromere, a specialized chromatin domain characterized by the presence of Cnp1, a centromere-specific histone variant and by the non-canonical nucleosomal complexes formed by CENP-S-T-W-X . Cnp1 is considered the landmark for kinetochore assembly and directly interacts with CENP-C/SpCnp3 that, in turn, recruits other kinetochore factors such as CENP-L/SpFta1, CENP-K/SpSim4 or the monopolin complex Pcs1-Mde4. Surprisingly, we found that in the absence of Alm1 several kinetochore components were upregulated such as Mhf2, Cnp20, Mde4, Fta1 and especially Cnp3, that showed a two-fold enrichment at kinetochores compared to wild type cells. Cnp3 enrichment was not the result of altered transcription, which led us to the hypothesis that in S. pombe, the NE protein Cut8 is required to anchor the proteasome to the NE and is also required for proteasome function. Importantly, Cnp1 degradation is regulated locally by the proteasome, which restricts Cnp1 localization to centromeres. Cut8 is also required for Cnp1 degradation, suggesting that proteasome-NE localization has biological significance in the regulation of kinetochore composition.One of the main cellular mechanisms responsible for protein degradation is an evolutionarily conserved large protease complex termed the proteasome. The proteasome functions in the cytoplasm and also is enriched in the nucleus. In alm1Δ compared to wild type cells, suggesting that Alm1 is needed for efficient proteasomal degradation of Cnp3. Finally, we found that Alm1 is required for proper localization of Cut8 and the proteasome subunits Mts2 and Mts4 to the nuclear periphery. We hypothesize that Alm1 might directly regulate or stabilize Cut8 localization at the NE, or alternatively, Alm1 might function as scaffold for proteasomal assembly or regulation, which might indirectly impact Cut8-NE localization .We provided evidence that Cnp3 is a substrate of proteasomal degradation; Cnp3 accumulated at centromeres after proteasome and Cut8 inactivation. Cnp3 became unstable in the presence of the protein synthesis inhibitor cycloheximide, and ubiquitinated forms of Cnp3 accumulated after proteasome inactivation. Furthermore, Cnp3 decay in the presence of cycloheximide was delayed in S. pombe, kinetochores are assembled during the whole cell cycle and during interphase they are clustered and attached to the NE underlying the SPB . Our study shows that Alm1-dependent proteasome localization at the NE is required for kinetochore homeostasis and places proteasomal regulation as a new function at the network of the nuclear basket.The biological role of the proteasome at the nuclear basket remains an open question; however, there is a growing body of evidences that points to a functional relevance of the proteasome at the vicinity of the NE. Mlps interact with Esc1, a NE protein involved in telomere-NE anchoring and silencing, which has also been shown to bind to the proteasome. In


The recruitment of RNA-Pol-II to the transcription start site (TSS) is an important step in gene regulation in all organisms. Core promoter elements (CPE) are conserved sequence motifs that guide Pol-II to the TSS by interacting with specific transcription factors (TFs). However, only a minority of animal promoters contains CPEs. It is still unknown how Pol-II selects the TSS in their absence. Here we present a comparative analysis of promoters’ sequence composition and chromatin architecture in five eukaryotic model organisms, which shows the presence of common and unique DNA-encoded features used to organize chromatin. Analysis of Pol-II initiation patterns uncovers that, in the absence of certain CPEs, there is a strong correlation between the spread of initiation and the intensity of the 10 bp periodic signal in the nearest downstream nucleosome. Moreover, promoters’ primary and secondary initiation sites show a characteristic 10 bp periodicity in the absence of CPEs. We also show that DNA natural variants in the region immediately downstream the TSS are able to affect both the nucleosome-DNA affinity and Pol-II initiation pattern. These findings support the notion that, in addition to CPEs mediated selection, sequence–induced nucleosome positioning could be a common and conserved mechanism of TSS selection in animals. Gene transcription is a complex process that starts with the recruitment and positioning of Pol-II enzyme at the transcription start site (TSS). Specific promoter sequences, known as core promoter elements (CPEs) facilitate this process. Surprisingly, only a fraction of promoters contain them. It is still unknown how Pol-II choses the start site in their absence. A recently proposed alternative mechanism implicates positioned nucleosomes in the TSS selection. Here, we provide new evidence of the existence of such mechanism with a comparative analysis of promoter’s features across the animal kingdom. We analysed the promoter’s DNA sequence composition in 5 organisms and found conserved and unique consensus sequences used to organize chromatin in the region of the first nucleosome downstream the TSS (N+1). Moreover, we found that all organisms show a strong correlation between the spread of Pol-II initiation and the strength of the DNA-encoded signal in the N+1 region. A detailed analysis of Pol-II initiation sites reveals also the presence of a 10 bp periodicity that is correlated with the intensity of the DNA signal in the N+1 region. Importantly, we report that genetic variants that alter the DNA-nucleosome affinity in that region alter Pol-II initiation spread as well. An essential step in gene regulation is the recruitment of RNA-Pol-II (Pol-II) to the transcription start sites (TSS) at gene promoters –3. This In spite of the CPE’s demonstrated capability to select a TSS with high precision, only a minority of promoters have a CPE  . A centrAn open question in gene regulation is whether the chromatin at promoters is organized by sequence-intrinsic features or indirectly by the transcription machinery occupying the nucleosome-free region and thereby forcing the nucleosome to bind to the nearest free space downstream the TSS. On a genome level, two types of sequence features have been reported to participate in nucleosome positioning: dinucleotide periodicity and base composition . A theorin-vitro and in-vivo nucleosome organization in these regions [As said before, the role of sequence-intrinsic features in chromatin organization around promoters remains a matter of debate . Zhang a regions ,43. Rece regions . A simil regions . The phaHomo sapiens, Mus musculus, Danio rerio, Drosophila melanogaster and Caenorhabditis elegans) confirms that different species have an overall similar chromatin organization with nevertheless some noteworthy species-specific differences. All five organisms have sequence-intrinsic nucleosome-positioning signals that are predictive of in-vivo nucleosome organization, but only in promoters that lack TATA-boxes. Additionally, we show that broad promoters associated with strong sequence-encoded nucleosome +1 have 10 bp periodic initiation patterns. By analyzing the effects of genetic variants on promoter initiation site patterns and dinucleotide periodicity, we provide genetic evidence that rotational nucleosome positioning is mechanistically involved in TSS selection.In this paper, we investigate the molecular mechanisms of TSS selection by jointly analyzing experimentally determined chromatin architectures, DNA-encoded nucleosome signals, Pol-II initiation site patterns and natural genetic variation in promoters stratified by the presence or absence of specific CPEs and/or the breadth of the initiation patterns. The analysis on five model organisms  showed the highest correlation with in-vivo nucleosome signals . The organisms had heterogeneous number of promoters with concordant signals   was characterized for having focused initiation and weak periodicity. All these promoters had a DPE [2 = 0.18, p-value = 0.21)  . Focusedad a DPE  and an I = 0.21) .To further elucidate the relationship between periodic DNA signals and Pol-II, we studied the primary and secondary transcription initiation patterns in promoters. In fact, rotational nucleosome positioning due to a 10 bp periodic signal does not require the occurrence of the nucleosome center at exactly the same base: it tolerates shifting by multiples of 10 bp ,27. To vH. sapiens, each promoter subclass showed a similar level of primary TSS activity with comparable frequencies of micro-peaks at the TSS   produced by natural variants and their effects on Pol-II initiation. To assess this, we scanned the promoter region with a sliding window of 150 bp (10 bp shift) and investigated the linear relationship between the variation in 10 bp frequency for the WW dinucleotide  and the variation in the observed DI for the corresponding promoters. The N+1 region was the only one showing a negative correlation between the variation measured in the nucleosome-DNA affinity and the variation in promoters’ DI, with a minimum centered at base +110  . On a si -0.184) . On the Two pathways for TSS selection by POL-II have been described in the literature. According to the conventional model the TSS position is defined by the presence of CPE . HoweverH. sapiens, M. musculus, D. rerio, D. melanogaster and C. elegans) the position of nucleosomes at the majority of promoters is at least partly determined by DNA encoded signals, with some remarkably species-specific differences. Promoters of all organisms show a 10 bp periodic signal for the four dinucleotides tested . H. sapiens is the only organism showing also a strong signal for YY and RR dinucleotides for a periodicity of 8 bp, that is probably the consequence of the presence of specific CT rich microsatellite sequences in human promoters [in-vivo nucleosome maps is WW H. sapienps is WW . Regardlps is WW , suggesthistones . Althoughistones , our anaD. melanogaster) showing strong affinity in the N+1 region. Moreover, the 10 bp periodicity seen in Pol-II initiation in all promoters, focused and broad, that lack CPEs  from . PromoteCGI coordinates for human and mouse were downloaded from the UCSC genome browser . PromoteH. sapiens: hg19; M. musculus: mm9; D. rerio: danRer7; D. melanogaster: dm3; C. elegans: ce6) and scanned for the presence of four dinucleotide types (identified by IUPAC codes): WW (W = A or T), SS (S = C or G), RR (R = A or G) and YY (Y = C or T). The resulting binary sequences were individually scanned in a sliding window of 150 bp, shifted by 10 bp at a time. A Fourier transform was applied to each window in order to extract the power spectrum. From the resulting spectrum, the value corresponding to a frequency of 0.097 (corresponding to a period of 10.3 bp) was extracted. This value was directly used as a periodicity score.Promoter sequences from position -1074 to position 1075 relative to the TSS were extracted from the corresponding genome assembly  and two Ns. Sequences starting with an N were discarded resulting in a total of 240 sequences. These consensus motifs were then mapped to promoters and MNase-seq enriched regions.For the analysis of nucleosome +1, the region from position -99 to 300 relative to the TSS of the corresponding genome assembly was used for mapping each consensus motif allowing a maximum of 3 mis-matches. Then, the average occurrence frequency for each motif was evaluated from base +50 to +200 relative to the TSS and a Fourier transform was applied in order to identify the intensity of the frequency of 0.097 (corresponding to a period of 10.3 bp). This value was then stored as the motifs’ score for the nucleosome +1 and the procedure was repeated for all consensus motifs. For the genomic nucleosomes a similar analysis was performed. In order to speed-up the analysis, 80.000 inferred positions were randomly selected from each sample. Subsequently, each consensus motif was mapped around the inferred nucleosome position and the average occurrence frequency was calculated from position -75 to +75 relative to it. A Fourier transform was then applied as before and the value for a period of 10.3 bp was used as the motif score in genomic nucleosomes.CAGE data from different samples belonging to the same species were first merged into one file. TSS profiles were then extracted for promoter regions extending from -103 to +104 relative to the dominant TSS using the ChIP-Extract tool from the ChIP-Seq web server . The resTo identify promoters with a strong 10 bp periodicity in micro-peaks signals, promoters were ranked according to the covariance between their micro-peaks distribution and a cosine function of period 10 bp. Promoters with weak micro-peak signal  were selected for having a cumulative covariance equal to 0.Nucleosome distributions for promoter subsets were computed from nucleosome mapping data using the ChIP-Cor program from the ChIP-Seq web server . MNase- N is the total number of tag starts in the window around promoter k, and xi is the mapped position of the 5’ end of tag i. For each species, DI values were calculated for each promoter using CAGE data from individual samples. A DI was calculated only if more then 5 tags mapped in the selected region. The sample–specific DI were then averaged to obtain a final unique and robust DI value for each promoter.The spread of CAGE tags in a window of 100 bp around the TSS was expressed as a Dispersion Index (DI) using the following formula:VCF files of Indels (version 2010_07) and SNPs (version 2010_03) for the GM12878 cell line were downloaded from the 1000Genomes ftp web server. All homozygous variants were extracted from these files and used to generate a GM12878 genome. On the other end the frequencies of these variants were evaluated using the allele frequency calculated by the final version of the 1000Genome project . For each variant, the most frequent allele was stored and used to generate the Most Likely genome that was then used as reference. The final list of SNPs and Indels for GM12878 cell line was restricted to the variants that differ compared to the ML genome.S1 TextFile containing a detailed description of the input data and the R code, properly commented, that has been used to make all the calculations and to generate all the figures. The file contains the R code for the figures and the functions used.(PDF)Click here for additional data file.S1 Fig2 kb region around promoters were scanned with a sliding window of 150 bp and 10 bp shift for the intensity of dinucleotide frequencies of period 2 to 20 bp. For each region, the average frequency intensities across all promoters were plotted against the distance of the region to the TSS. All organisms have a peak of signal intensity in correspondence to a period of 10–11 bp in agreement with the notion that this frequency helps the DNA wrapping around the histone octamer.(PNG)Click here for additional data file.S2 FigH. sapiens and M. musculus: RR; D. rerio, D. melanogaster and C. elegans: WWAverage spectrum intensities for selected dinucleotides evaluated at position +120 bp from the TSS. Each organism shows a peak in correspondence of 10–11 bp frequency. Dinucleotide selected: (PNG)Click here for additional data file.S3 FigThe average intensity of 10 bp frequency for selected dinucleotides in a 2 Kb region around animal promoters. All organisms show signal depletion immediately upstream the TSS followed by a peak downstream.(PNG)Click here for additional data file.S4 FigIntensity of a 10 bp dinucleotide periodicity calculated using a Fourier transform in a sliding window of 150 bp on a 2 kb region around animal promoters compared to the in-vivo nucleosome occupancy profiles derived from MNase-seq reads counts in the same region. Promoters were ordered according to their correlation between the intensity of the 10 bp dinucleotide signal and the average in-vitro nucleosome distribution in the same region.(PNG)Click here for additional data file.S5 FigH. sapiens promoters. (B) Correlation between 10 bp long dinucleotide patterns composed of one copy of each SS, WW, YY and RR dinucleotides and 2 Ns evaluated on M. musculus genomic nucleosomes and the N+1 nucleosome; each dot represents the strength of the 10 bp frequency of a consensus sequence in the N+1 region or in genomic nucleosome regions (defined by MNase-seq data); green and red dots mark two classes of patterns  that are known to have high nucleosomes affinities. (C) As B) but for D. rerio promoters and genomic nucleosomes. (D) As for (B) but for D. melanogaster promoters and genomic nucleosomes.(A) Average 10 bp frequency intensity of the consensus sequence YYWWNNRRSS  around (PNG)Click here for additional data file.S6 Fig10 bp frequency intensity for the 240 randomly generated sequences in MNase-seq defined genomic nucleosomes for the 5 organisms under study.(PNG)Click here for additional data file.S7 Fig10 bp frequency intensity for the 240 randomly generated sequences in the region +50 to +200 from the TSS of the organisms tested.(PNG)Click here for additional data file.S8 FigC. elegans promoters. Different shades of blue represent the total number of mismatches allowed in the mapping. (B) Intensity of the 10 bp frequency of the two consensuses on the N+1 region for each C. elegans promoters. Red and green dots highlight promoters characterized for a strong signal of the YYWWNNRRSS consensus  compared to the RRWWNNYYSS consensus respectively. (C) Nucleosome distribution around promoters characterized for a strong signal in one consensus as defined by (B). Each dot represents the average tag count in a widow of 10 bp. Continuous lines are the local polynomial regression fit. (D) Same as (C) but with H3K4me3 and window of 1 bp.(A) Average 10 bp frequency intensities of two consensus classes (represented by the RRWWNNYYSS and YYWWNNRRSS consensuses for the SS-RR-WW-YY and SS-YY-WW-RR classes) around (PNG)Click here for additional data file.S9 FigFor each organism promoters were grouped according to their CPE status (TATA-box and Inr-DPE presence in the expected position). CPE-less promoters were also grouped according to their DI value in groups of 2000 promotes (a similar number as the promoters with the TATA-box). Each dot represents the average value of the intensity of the WW, SS, RR and YY dinucleotide for groups with similar DI and CPE status. Solid line represents the predicted values  whereas dotted line the 99% confidence intervals as evaluated by the linear model.(PNG)Click here for additional data file.S10 FigH. sapiens promoters with TATA-box (TATA-box) and without (TATA-less) (left panel); WW dinucleotide frequencies for the same promoters groups ; and spectrum decomposition in the region +50 to +200 (right panel) for the 4 signals. (B) Same as A but for D. rerio promoters. (C) Similar to A but with D. melanogaster promoters stratified for the presence of the TATA-box (TATA-box), the absence of the TATA-box but the presence of Inr-DPE motif (Inr-DPE) and for the absence of both (CPE-less). (D) Same as (A) but with C. elegans promoters.(A) YY dinucleotide frequencies for (PNG)Click here for additional data file.S11 FigH. sapiens promoters were grouped following the same rules as in (JPG)Click here for additional data file.S12 FigVertical grey lines are separated by 10 bp from position -30 to 30 relative to the dominant TSS. Left panel: raw global CAGE distribution, periodic initiation is not clearly visible. Middle panel: CAGE distribution after a 10 tags count cut-off was applied to each position around each promoter, a 10 bp periodicity is starting to emerge from the data. Right panel: micro peak distribution, 10 bp periodic distribution is evident.(PNG)Click here for additional data file.S13 FigA strong 10 bp periodicity in Pol-II initiation is visible only in CPE-less promoters, reflecting the presence in the N+1 region of a DNA-encoded nucleosome signal.(PNG)Click here for additional data file.S14 Fig(PNG)Click here for additional data file.


Cigarette smoking is the primary cause of chronic obstructive pulmonary disease (COPD) with repeated and sustained infections linked to disease pathogenesis and exacerbations. The airway epithelium constitutes the first line of host defense against infection and is known to be impaired in COPD. We have previously identified Fatty Acid Binding Protein 5 (FABP5) as an important anti-inflammatory player during respiratory infections and showed that overexpression of FABP5 in primary airway epithelial cells protects against bacterial infection and inflammation. While cigarette smoke down regulates FABP5 expression, its mechanism remains unknown. In this report, we have identified three putative c-Jun binding sites on the FABP5 promoter and show that cigarette smoke inhibits the binding of c-Jun to its consensus sequence and prevents LPS-induced FABP5 expression. Using chromatin immunoprecipitation, we have determined that c-Jun binds the FABP5 promoter when stimulated with LPS but the presence of cigarette smoke greatly reduces this binding. Furthermore, cigarette smoke or a mutation in the c-Jun binding site inhibits LPS-induced FABP5 promoter activity. These data demonstrate that cigarette smoke interferes with FABP5 expression in response to bacterial infection. Thus, functional activation of FABP5 may be a new therapeutic strategy when treating COPD patients suffering from exacerbations. Chronic Obstructive Pulmonary Disease (COPD) is the third leading cause of death in the United States and its global prevalence continues to rise . AlthougToll-like receptors (TLRs) are pattern recognition receptors that play a pivotal role in mediating the host innate immune response to infections. Specifically, TLR4 recognizes lipopolysaccharide (LPS) . TLR4 ac-/- mice infected with influenza A have heightened lung inflammation that persists long after wild type mice recovery [FABP5 gene expression is regulated under basal or diseased conditions is poorly understood. In this study, we dissected in more depth the regulation of FABP5 by cigarette smoke and LPS in airway epithelial cells. We focused on the role of cigarette smoke and/or LPS-mediated MAPK/AP-1 activation in FABP5 regulation, as we identified three putative binding sites for c-Jun within the FABP5 promoter. Understanding how FABP5 expression is regulated during infection will open up novel possibilities to enhance COPD patients’ innate immune defense mechanisms against repeated episodes of infections.We have recently identified Fatty Acid Binding Protein 5 (FABP5) as an important anti-inflammatory player during respiratory infections. In human airway epithelial cells, FABP5 expression is down regulated by cigarette smoke, thus leading to a more sustained inflammation . In addirecovery . Althougrecovery , 9. How 2. Approximately 75% confluent monolayers of BEAS-2B cells were exposed to cigarette smoke extract and 24 hours later treated with LPS  for up to 48 hours.The immortalized human epithelial cells isolated from normal human bronchial epithelium obtained from autopsy of non-cancerous individuals, BEAS-2B cells (ATCC CRL-9609) were cultured in BEBM along with all the additives obtained from Lonza/Clonetics Corporation at 37°C, 5% COTwo filtered research grade tobacco cigarettes (3R4F) from the Kentucky Tobacco Research and Development Center  were smoked through an apparatus with a constant airflow (0.2 l/min) driven by an air compressor. The smoke was bubbled through 20 ml of PBS and the pH was adjusted to 7.4. The CSE obtained was filtered and considered 100% CSE. It was further diluted to treat the cells with 2%. The CSE was prepared freshly before each experiment along with an air control extract (AC) consisting of ambient air bubbled into 20 ml of PBS.RNA was extracted (QIAGEN) and Taqman RNA-to-Ct 1-Step kit (Applied Biosystems) was used to perform Real Time RT-PCR as previously described on GAPDH  and FABP5  [Cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors. Ten μg of proteins were electrophoresed on 10% or 15% SDS-PAGE, transferred onto PVDF membrane, blocked with 5% BSA, and then incubated with a rat anti-FABP5 antibody , a rabbit anti-c-Jun antibody , a rabbit anti-phosphoSer63-c-Jun antibody , or a rabbit anti-phosphoSer73-c-Jun antibody  overnight at 4°C. After washes in PBS with 0.1% Tween-20, the membranes were incubated with anti-rat or anti-rabbit IgG conjugated to horseradish peroxidase for FABP5, c-Jun and phospho-c-Jun protein detection. Membranes were stripped and re-probed with a mouse anti-GAPDH antibody . Densitometry was performed to quantify proteins of interest.Cells were homogenized in nuclear protein extraction buffer to extract nuclear proteins following manufacturer’s instructions . Nuclear proteins (20 μg per sample) were used to perform c-Jun ELISA  to quantify c-Jun activation. Briefly, multiple copies of c-Jun specific double-stranded oligonucleotide were immobilized to a 96-stripwell plate. Cell nuclear extracts (protein) were then added to the well where activated c-Jun bound specifically to the oligonucleotide at its consensus-binding site. Thereafter, a primary antibody specific for the activated form of c-Jun was added, followed by incubation with HRP-conjugated secondary antibody and then developing solution. Results were expressed as absorbance read at 450nm with a reference wavelength at 650nm.5’-TTGTGCGCTGGCGAGG-3’, reverse 5’-CCCTCGCCGGACTCGG-3’. The PCR products were subjected to electrophoresis on 2% agarose gels containing ethidium bromide. The expected PCR product is 104 bp.BEAS-2B cells were exposed to cigarette smoke extract or air control for 24 hours and then stimulated with LPS for 1 hour. The ChIP assay was performed using a ChIP assay kit  according to the manufacturer’s specifications. Immunoprecipitation was carried out overnight at 4°C with 2 μg of c-Jun antibody . Polymerase chain reaction (PCR) was performed with input DNA  and immunoprecipitated DNA (5 μl) using FABP5 promoter-specific primers: forward FABP5 gene (accession number NG_028154) and using MacVector, we designed the following primers to clone the FABP5 promoter from BEAS-2B genomic DNA by polymerase chain reaction: forward 5’-CCGCTCGAGCTTTTAATCCAAAGTAAG-3’, reverse 5’-CCTAGGCCAATTCATGTATTCATC-3’ into TOPO vector (Invitrogen). The expected PCR product size is 5,143 bp with the inclusion of an XhoI and EcoRI restriction sites in the forward and reverse primers, respectively. The promoter was then subcloned into pGL4.18 vector . Site-directed mutagenesis was used to mutate the c-Jun binding site by polymerase chain reaction amplification . The following primers were used to create the mutation in the c-Jun binding site at position -495 on the FABP5 promoter (accession number NG_028154) with two T substitutions for two A: forward 5’-CCGTAGTTAATGTATCTATAAACC-3’ and reverse 5’-GGTTTATAGATACATTAACTACGG-3’. Plasmid DNAs were amplified, confirmed by restriction analysis and by sequencing. BEAS-2B cells were co-transfected with a firefly luciferase reporter gene (F-luc) containing FABP5 promoter sequence (-5072bp/+80bp) (pGL4-FABP5) and renilla luciferase (R-luc) construct (pGL4-CMV) at a 10 to 1 ratio. The cells were then exposed to cigarette smoke or air control and stimulated the following day with LPS (1μg/ml) for 2 hours. Firefly luciferase (F-luc) and renilla luciferase (R-luc) activity were determined using a dual luciferase reporter assay kit . The transfection efficiency was normalized by the ratio of F-luc and R-luc to determine changes in FABP5 promoter activity.Based on the sequence provided for the t test was used when only two groups were compared. Differences were considered statistically significant when p < 0.05.Data are expressed as mean ± SEM. One-way analysis of variance was used for multiple comparisons, and Tukey's post hoc test was applied where appropriate. Student's P. aeruginosa infection increases FABP5 expression in primary normal human airway epithelial cells [P. aeruginosa showed increased levels of FABP5 mRNA expression in a dose-dependent manner  and protein (1.5-fold) levels ; therefoFABP5 gene promoter, causing enhanced FABP5 mRNA and protein accumulation, but also that cigarette smoke can interfere with FABP5 expression in response to bacterial infection.Bioinformatics analysis using TESS (transcription element search software) revealed three putative c-Jun binding motifs in the 5’ flanking region (-5 kb) from the FABP5 transcription start site  . To studBEAS-2B cells were transiently transfected with an FABP5 promoter reporter construct and renilla luciferase as transfection control. Either wild type or mutated FABP5 promoter construct at position -495 were used, as illustrated in COPD is a complex disease characterized by an abnormal lung inflammatory response to cigarette smoke that leads to tissue destruction and airflow obstruction . Acute eFABP5 gene regulation. Our data are showing, for the first time, that LPS increases FABP5 transcript expression in a dose-dependent manner in BEAS-2B lung epithelial cells. We also demonstrate that c-Jun, a subunit of the activator protein 1 (AP-1) transcription factor, binds to a consensus sequence we identified in the FABP5 promoter region. In addition, we established that cigarette smoke impairs c-Jun binding to the FABP5 promoter, providing a molecular mechanism underlying our observation that CSE negatively affects FABP5 expression. Therefore, our experiments with the BEAS-2B lung epithelial cell line suggest that LPS-induced FABP5 up-regulation may be part of an effective innate host response aimed at protecting lung tissue against bacterial infection-induced inflammation. Further in vivo studies will be needed in the future to confirm the physiological impact of our findings.We have previously shown that bacterial infection and cigarette smoke exposure differentially regulate FABP5 expression , 9, howeFABP5 with AP-1 was previously reported [FABP5 gene was cloned, along with other transcription factors including myogenic differentiation factor (MyoD), C/EBPα and β, HNF1, myeloid zinc finger 1 (MZF1) and GATA1. The authors identified 560 binding sites for 40 different transcription factors with a level of 80% identity to the consensus sequence, and suggested that the transcription factors might be involved in regulating the expression pattern of FABP5 [The putative interaction of reported  when theof FABP5 . In addiof FABP5 . FABP5 iof FABP5 . Thus, FBeing at the interface between the host and the environment, the airway epithelium represents the first line of host defense against potential pathogens or irritants such as cigarette smoke. This barrier has to not only serve the physiological function of the lung, but also protect its well-being. The protective role of the epithelium includes recognition  and respMycoplasma pneumoniae or Pseudomonas aeruginosa infections increase FABP5 expression in primary human airway epithelial cells, cigarette smoke reduces FABP5 expression [FABP5 gene regulation. Furthermore, we chose BEAS-2B cells for their sturdier nature and easier manipulation, especially for transfection of FABP5 promoter reporter construct, rather than primary human airway epithelial cells.Airway TLR signaling initiates inflammatory responses and increases the production and secretion of host defense molecules that collectively provide protection against respiratory infections . We havepression , 9. The pression , thus, vWe observed up to 33% increase in FABP5 promoter activity after only 2 hours of LPS treatment indicating that FABP5 transcription is a rapid event following c-Jun activation, which also appears after 2 hours of LPS treatment. On the contrary, cigarette smoke exposure for 24 hours completely suppressed FABP5 promoter activity and gene transcription. In addition, we have shown that site-directed mutagenesis (at position -495) to disrupt c-Jun binding site was sufficient to reduce FABP5 promoter activation following LPS treatment.FABP5 gene regulation and may open up new therapies for the treatment and/or prediction of recurrent exacerbations that are triggered primarily by bacterial and viral infections and are associated with increased airway inflammation.FABP5, a novel anti-inflammatory protein, is significantly reduced in inflammatory lung diseases including COPD , 9, whic


Escherichia coli thiamin pyrophosphate (TPP) thiC riboswitch contains a regulatory pause site in the translation initiation region that acts as a checkpoint for thiC expression. By biochemically probing nascent transcription complexes halted at defined positions, we find a narrow transcriptional window for metabolite binding, in which the downstream boundary is delimited by the checkpoint. We show that transcription complexes at the regulatory pause site favour the formation of a riboswitch intramolecular lock that strongly prevents TPP binding. In contrast, cotranscriptional metabolite binding increases RNA polymerase pausing and induces Rho-dependent transcription termination at the checkpoint. Early transcriptional pausing may provide a general mechanism, whereby transient transcriptional windows directly coordinate the sensing of environmental cues and bacterial mRNA regulation.On the basis of nascent transcript sequencing, it has been postulated but never demonstrated that transcriptional pausing at translation start sites is important for gene regulation. Here we show that the  E. coli thiC riboswitch has a pause site in the translation initiation region that acts as a checkpoint for thiC expression.Riboswitches are non-coding RNA elements that detect metabolites and control expression by regulating mRNA levels or translation. Here, the authors provide evidence that the  Gene expression is a highly regulated process that is vital for cellular homoeostasis. Numerous cellular factors such as RNA molecules regulate gene expression at the transcriptional level and contribute to an ever expanding repertoire of mechanisms governing cellular processes3in vivo, growing evidence indicates that multiple biological processes can be controlled by riboswitch conformational changes. For example, it was recently demonstrated that the lysine-dependent lysC riboswitch controls both translation initiation and RNase E cleavage activity on ligand bindingribB and mgtA riboswitches were shown to regulate transcription termination by using the transcription factor RhomgtA riboswitch was also found to contain a critical RNA polymerase pause site important for Rho transcription termination8Although only a few riboswitch regulation mechanisms have been characterized E. coli and Bacillus subtilisin vivo regulation mechanism of the thiamin pyrophosphate (TPP)-sensing thiC riboswitch in E. coli. We identify a transcriptional pause site occurring within the vicinity of the translation start codon. Using in vitro assays, we find that pause efficiency increases on TPP binding and is required to trigger Rho-dependent transcription termination. Mutational analysis shows transcriptional pausing to be crucial for mRNA control in vivo. Furthermore, we perform biochemical probing on transcription elongation complexes (ECs) to directly monitor TPP binding on nascent riboswitch transcripts. We find that while TPP binding is efficient for transcription complexes located upstream of the pause site, it is severely reduced for complexes stalled at the pause site. Our results suggest that the nascent RNA forms an intramolecular lock that both prevents TPP binding to the aptamer and exposes the RBS/AUG start codon, promoting translation initiation. The pause site acts as an early transcriptional checkpoint, where a decision is made to either repress or allow transcription elongation and translation initiation of the thiC operon. Here we describe a riboswitch-based molecular mechanism, in which transcriptional pause site is involved in both sensing environmental cues and controlling gene expression.Nascent elongating transcripts sequencing recently revealed a 16-nt consensus pause sequence that is enriched at translation start sites in both E. coli thiC riboswitch is located upstream of the thiCEFSGH operon involved in TPP biosynthesisthiC expression. However, the thiC riboswitch was shown to negatively regulate gene expression in TPP-rich mediathiC riboswitch in the absence of TPP, we retrieved intergenic sequences located upstream of thiC in proteobacteria. By aligning 77 sequences, we identified a highly conserved secondary structure describing the TPP-free ON state  was unknown, we first determined the transcriptional +1 residue by performing primer extension analysis on thiC mRNA . While translational fusions report on protein and mRNA levels, transcriptional thiC–lacZ fusions have two independent RBS (thiC and lacZ) allowing us to monitor only thiC mRNA levels  are usually rich in cytosine but poor in guanine, and are relatively unstructuredthiC nucleotide composition indicated that the 100–140 nt region is a good match for a typical rut site  value of 422 nM±27 nM for the structural modulation. A similar Kswitch value of 453 nM±13 nM was obtained using the RBS probe  concentrations  transcriptional pausing has been shown to influence RNA folding in numerous systems72527287thiC riboswitch, we performed single-round in vitro transcription assays and analysed transcripts at various time points  are protected within RNAP31We measured the TPP-binding affinity of transcription complexes by performing RNase H assays using a range of TPP concentrations. We found that TPP sensing is more potent when RNAP is stalled at pause sites 138 (28±1 nM) or 158 27 nM±2 nM)  compared nM±2 nM In contrast to EC-187, we speculated that the presence of RNAP in EC-158 would prevent the formation of the anti-P1 stem, thereby positively influencing TPP binding . As expethiC transcripts using E. coli RNAP and either halted them at position 187 (EC-187) or allowed RNAP to transcribe the full riboswitch sequence. Immediately after transcription, we probed the RNA structure using 2-methylnicotinic acid imidazolide (NAI). Interestingly, SHAPE reactivity was similar for the EC-187 nascent RNA and full-length transcript, with the noteworthy exception of the C7-G16 region 28333435363738396 region . The dec6 region . HoweverthiM riboswitch39thiC riboswitch adopts a TPP-free structure in which the anti-P1 stem is stabilized.We next repeated similar experiments but added TPP cotranscriptionally. For both the EC-187 complex and the full-length transcript, adding NAI resulted in reactivity changes in both the aptamer and expression platform . For eacE. coli riboswitches, we performed transcription kinetics for the thiB, thiM, ribB, btuB and lysC riboswitches  or by delaying the transcription of the anti-P1 competing sequence (ribB)  . The prely large , suggestE. coli thiC riboswitch and show that TPP binding mediates both Rho-dependent transcription termination and repression of translation initiation. We developed an approach in which cotranscriptional TPP-mediated conformational changes were monitored in the context of transcription ECs stalled at specific positions. Our results demonstrate that the thiC riboswitch regulates in a narrow transcriptional window in which the downstream boundary is delimited by a critical RNAP pausing site found in the vicinity of the translation initiation codon. Strikingly, while TPP-binding affinity is high for transcription complexes located upstream of the pause site, ligand binding is impaired by ∼100-fold when transcription complexes are stalled at this regulatory checkpoint. In contrast, TPP-bound transcription complexes reaching the checkpoint undergo transcription termination or translation repression. Such a regulatory checkpoint provides a strategic gate where the outcome of the genetic decision is sealed. Given that transcriptional pausing occurs widely in E. coli and B. subtilisSince their discovery, riboswitches have been extensively studied for their importance in bacterial gene regulation and more recently as potential targets for novel antimicrobial agents43thiC transcriptional pausing is intimately linked to riboswitch TPP sensing and gene regulation, consistent with a recently predicted kinetic modelthiC operon length, explaining the strong preference of the thiC riboswitch to bind ligand cotranscriptionally  and accurate signal transduction (EC-187) to produce the required genetic outcome. Additional studies implied that transcriptional pausing could coordinate aptamer and expression platform folding25E. coli riboswitches. Interestingly, such pauses were also detected in vivo for the lysC and thiM riboswitchesE. coli riboswitch sequences. The presence of multiple transcriptional pause regions might indicate strong evolutionary pressure on riboswitches to maintain specific metabolite-binding capabilities and transcriptional pausing activities. From an evolutionary standpoint, the presence of already existing pause sites allowing efficient translation initiation probably facilitated the establishment of Rho-dependent transcription termination mechanisms. The identification of pause sites in a wide range of bacterial transcripts strongly suggests that transcriptional dynamics is important for the regulation of bacterial mRNAs.The importance of pause sites for riboswitch regulation was first established for the kinetically controlled E. coli MG1665 and are described in thiC transcriptional and translational fusions were constructed using the PM1205 strainthiC riboswitch translational constructs is described in thiC were made using a three-step PCR strategy  was added to induce the expression of lacZ constructs. β-galactosidase experiments assessing the involvement of Rho were performed in 3 ml of culture media as described above. TPP (0.5 mg ml−1) and bicyclomycin (25 μg ml−1) were added as indicated. The experiments were performed in triplicate and the average values with standard deviations (s.d.) are reported. Descriptions of the mutants are in Kinetic assays for β-galactosidase experiments were performed as described previously2, 100 mM KCl, 50 μM EDTA and 10 mM 2-mercaptoethanol at 37 °C for 5 min. Reactions were stopped by adding an equal volume of a stop solution . The experiments were performed in triplicate and the average values with s.d.'s are reported. To monitor co- or post-transcriptional TPP binding, the transcription reaction was mixed with 200 μM DNA and RNase H for 15 s at 15 s, 45 s, 90 s, 2 min and 3 min after TPP addition. The reported errors for the TPP-binding rates are the s.e. in the fitting, which is assumed to be approximated by the standard deviationThe PCR strategy to obtain DNA templates used for transcription reactions is described in 2, DNA template, E. coli RNA polymerase and sigma70 factor at 37 °C for 3 min. Reactions were performed in the absence or presence of 10 μM TPP. Transcription reactions were completed by adding NTP at 37 °C for 10 min. The EC-187 complex was obtained by transcribing a DNA template containing a biotin on the antisense strand 5′-end. Nascent RNA molecules were subjected either to 10% DMSO or 150 mM NAI for 15 min at 37 °C. Transcription reactions were quenched by passing complexes through G50 gel filtration columns and purified RNAs were collected in 0.5 × TE buffer. Selectively acylated RNA was incubated with different radioactively labelled DNA oligonucleotides to cover the complete riboswitch  at 65 °C for 3 min, then cooled to 22 °C for 5 min. Reverse transcription reactions were performed at 55 °C for 15 min. Reactions were next quenched by incubating at 95 °C for 5 min. RNA molecules were hydrolysed using 200 mM NaOH, which was neutralized using a Tris-HCl-buffered solutionThe SHAPE reaction was essentially prepared as previously describedthiC riboswitch in the absence of TPP, intergenic sequences located upstream of the thiC were retrieved in proteobacteria using the RiboGap database (http://ribogap.iaf.inrs.ca). Using these sequences, RNA secondary structure prediction was performed using both LocaRNAthiC riboswitch . The sample was passed through G50 columns to remove any free nucleotides. Transcription reactions were completed by adding all four nucleotides with heparin to allow only one round of transcription. TPP was added at 10 μM when indicated. Time pausing experiments were either performed using 25 or 100 μM NTPs. For reactions done at 25 μM NTP, time aliquots were taken from 10 s to 25 min. Time aliquots of 15 s, 30 s, 45 s, 60 s, 90 s, 2 min, 3 min, 4 min, 5 min, 8 min and 10 min were used for reactions done at 100 μM NTP. A chase reaction was done by adding 1 mM NTP. The half-life of transcriptional pausing was done by determining the fraction of each RNA species compared with the transcription readthrough for each time point, which was analysed with pseudo-first-order kinetics to extract the half-lifeThe PCR strategy to obtain DNA templates used for transcription reactions is described in The description of PCR constructs used for the preparation of ECs can be found in In vitro transcriptions were performed as described above using a transcription buffer containing 40 mM Tris-HCl, pH 8.0, 50 mM KCl, 5 mM MgCl2 and 1.5 mM DTT. Rho (50 nM) was added in the first step of the transcription reaction. Transcriptions were completed by adding 50 μM NTPs and rifampicin (20 μg ml−1). The NusG factor (50 nM) was added where indicated. Resulting reactions were treated with a phenol/chloroform extraction and mixed with an equal volume of a stop solution containing 95% formamide, 10 mM EDTA and 0.1% SDS. Images have been cropped for presentation. Full size images are presented in In vitro translations were performed with the PURExpress Kit from New England Biolabs. Reactions were performed in the presence of transcription and ribosome solutions, 0.1 μM thiC and lacZ DNA template, 0.16 μM E. coli RNA polymerase, 0.32 μM sigma70 factor and [35S]-methionine at 37 °C for 2 h. Reactions were stopped by placing samples on ice for 10 min. Acetone 100% was added to a 4:1 ratio and the resulting solution incubated for 15 min at 4 °C. Precipitation was performed by centrifugation at 15,900 g for 10 min. Pellets were resuspended in denaturing buffer. Samples were resolved on SDS–polyacrylamide gel electrophoresis and gels were visualized using a Typhoon FLA 9500 . For each experiment, the expression of ThiC was normalized to that of LacZ, which acts as an internal control not modulated by the presence of 25 μM TPP. Titrations were performed using TPP concentrations corresponding to 50 nM, 100 nM, 250 nM, 1 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, 50 μM and 100 μM. For uncoupled transcription–translation assays, the reaction was initiated by performing a transcription step for 15 min at 37 °C using a transcription solution without amino acids/transfer RNA. Rifampicin (250 μg ml−1) was next added to the reaction and incubated for 1 min to prevent transcription re-initiation. Translation was initiated by adding a solution containing amino acids and transfer RNA and was incubated for 15 min at 37 °C. Reactions were stopped as described for transcription–translation-coupled assays. Images have been cropped for presentation. Full size images are presented in The description of the PCR strategy to obtain DNA templates used for transcription–translation assays can be found in −1) was added to block transcription. The DNA probe used for the detection is AD113. Images have been cropped for presentation. Full size images are presented in The Northern blot analysis was performed as previously describedThe determination of the TSS was done as previously reportedThe authors declare that all data supporting the findings of this study are available within the article and its How to cite this article: Chauvier, A. et al. Transcriptional pausing at the translation start site operates as a critical checkpoint for riboswitch regulation. Nat. Commun.8, 13892 doi: 10.1038/ncomms13892 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables and Supplementary References.Sequence alignment for the structure in Supplementary Figure 1.