PHO5 gene, a paradigmatic case for gene regulation through promoter chromatin remodeling. A negative role for antisense transcription at the PHO5 gene locus was demonstrated by leveraging the level of overlapping antisense transcription through specific mutant backgrounds, expression from a strong promoter in cis, and use of the CRISPRi system. Furthermore, we showed that enhanced elongation of PHO5 antisense leads to a more repressive chromatin conformation at the PHO5 gene promoter, which is more slowly remodeled upon gene induction. The negative effect of antisense transcription on PHO5 gene transcription is mitigated upon inactivation of the histone deacetylase Rpd3, showing that PHO5 antisense RNA acts via histone deacetylation. This regulatory pathway leads to Rpd3-dependent decreased recruitment of the RSC chromatin remodeling complex to the PHO5 gene promoter upon induction of antisense transcription. Overall, the data in this work reveal an additional level in the complex regulatory mechanism of PHO5 gene expression by showing antisense transcription-mediated repression at the level of promoter chromatin structure remodeling.Pervasive transcription of eukaryotic genomes generates non-coding transcripts with regulatory potential. We examined the effects of non-coding antisense transcription on the regulation of expression of the yeast  S. cerevisiae PHO5 gene that encodes a periplasmic acid phosphatase. Furthermore, we show that the repressive effect of antisense transcription at the PHO5 gene locus is mediated by chromatin-modifying complexes, such as the histone deacetylase Rrp3 and the chromatin-remodeling complex RSC. Consequently, elongation of the antisense transcript through the PHO5 promoter region results in a chromatin conformation that is more difficult to remodel upon gene induction and therefore in slower kinetics of gene expression. Overall, this work paints a more complete picture of the regulatory process involved in repression of the yeast PHO5 promoter, a well-studied model for gene regulation through chromatin structure remodeling.Non-coding transcripts synthesized by RNA Poll are short-lived because they are rapidly terminated by the NNS complex and degraded by the RNA exosome. However, some non-coding transcripts can regulate the expression of coding genes at whose loci they are initiated. In this work, we show that a non-coding antisense transcript regulates the expression of the yeast  The canonical view of eukaryotic transcription has evolved from being considered a highly regulated process initiated from specialized genomic regions, such as gene promoters, to a process that permeates the entire genome . In addii.e. the packing of DNA with histone proteins into nucleosomal arrays [bona fide effects of specific AS RNAs on transcriptional regulation of their respective genes, such as the yeast PHO84 gene.In eukaryotic cells, promoter activation occurs in the context of a repressive chromatin structure, l arrays . Since cl arrays ,5. Dynaml arrays . In genel arrays . AS readl arrays –10. Genol arrays ,12. Our l arrays . HoweverPHO84 gene have been highly instructive in elucidating the mechanisms of transcriptional regulation through AS non-coding RNAs [PHO84 AS transcription is rapidly terminated in wild-type cells by the NNS (Nrd1-Nab3-Sen1) complex and degraded by the activity of the Rrp6-containing nuclear RNA exosome. Inactivation of any of these crucial factors, such as in rrp6Δ mutant cells, leads to transcriptional read-through of PHO84 AS transcripts, allowing recruitment of histone deacetylases (HDACs) Hda1 or Rpd3 to the PHO84 promoter. Histone deacetylation is thought to lock the chromatin structure of the promoter in a repressed conformation, thereby negatively regulating transcription of the sense transcript, i.e. PHO84 mRNA. This mechanism was subsequently explored genome-wide in yeast, which revealed a group of genes that accumulate AS RNAs in the absence of Rrp6 and are silenced in an HDAC-dependent manner [PHO5 gene, which is a member of the same (PHO) regulon as PHO84 [Studies with the ing RNAs –16. Thest manner . Genes ot manner ,18. A paas PHO84 .PHO5 gene encodes the secreted non-specific acid phosphatase which is located in the periplasmic space and has a role in phosphate metabolism. Accordingly, expression of the PHO5 gene is regulated in response to intracellular phosphate concentration through the PHO signalling pathway, so that it is repressed when the intracellular concentration is abundant and induced under phosphate starvation conditions [PHO5 gene. In low phosphate, Pho4 is imported into the nucleus and activates transcription. From the early days of chromatin research in the 1980s until now, the PHO5 gene promoter has been and continues to be instrumental in the discovery of numerous fundamental principles and mechanisms of chromatin structure remodeling  site, which is located in the short nucleosome-depleted region between nucleosomes -2 and -3 of the PHO5 gene promoter. Pho4 recruits histone acetyltransferases, such as the Gcn5-containing SAGA complex, which establish a hyperacetylated promoter configuration [PHO5 promoter by direct interaction with Pho4 [PHO5 promoter [The nditions . This re ∼600 bp . This mapromoter ,22. Anotpromoter . When th nucleus . The firguration ,26. Acetguration ,28. Alteith Pho4 . Five repromoter ,31, withpromoter –34.PHO5 promoter regulatory complexity was revealed upon mapping of the PHO5 AS transcript, CUT025 [PHO5 ORF and extends through its promoter region, spanning ∼2.4 kb in size. It is produced only in cells growing under repressive (phosphate-rich) conditions and is more abundant in rrp6Δ mutants compared to wild-type cells, indicating its degradation by the nuclear RNA exosome [PHO5 gene is among the rare examples for which AS transcription is proposed to have a positive effect [PHO5 gene expression by enhancing or impairing elongation of the PHO5 AS transcript. In both cases, our results argue in favour of antisense transcription having a negative effect on PHO5 gene expression. Moreover, we provide evidence that this negative effect occurs through a chromatin-remodeling based mechanism mediated by AS transcription which decreases the accessibility of the chromatin structure at the PHO5 gene promoter.Another level of , CUT025 ,36. This exosome . AS trane effect . In thisYeast strains and primer sequences used in this study are listed in Saccharomyces cerevisiae strains used in this study are listed in i), yeast strains were grown in YNB medium supplemented with 1 g/l KH2PO4 (YNBP) with or without lack of amino acids for plasmid selection. For gene induction by phosphate starvation (-Pi), cells were washed in water and resuspended in the phosphate-free synthetic medium with or without lack of amino acids for plasmid selection [RRP6 gene was deleted using a disruption cassette generated by PCR with the primer pairs RRP6-Kan1 and RRP6-Kan2 or RRP6hph_fwd and RRP6hph_rev and the BMA41 rrp6::KanMX4 genomic DNA or the hph-carrying pYM16 plasmid from [GCN5 gene was deleted using a disruption cassette generated by PCR with the primer pair gcn5HIS_fwd and gcn5HIS_rev and the SpHIS5-carrying pKT101 plasmid from [TEF1-PHO5 AS strain was constructed by transformation with a cassette generated by PCR with primers TEF1PHO5AS_fwd and TEF1PHO5AS_rev and the pYM-N18 plasmid from [PHO5 promoter-lacZ gene fusion and is described in [PHO5 AS gDNA-URA3 was then obtained by Gibson assembly reaction (NEB) using OFS_2886 and OFS_2887 to amplify pFS3892 backbone and OFS_2888 and OFS_3095 for gDNA cloning. To test the putative in trans activity of the PHO5 AS RNA, strains were designed as following. First, the PHO5 ORF was replaced by the URA3 marker in either a MATA FSY6857 or a MATα FSY5439 strain (see URA3 marker from the pUG72 plasmid with OFS5084 and OFS5085 primers and the resulting amplicon was transformed in FSY6857 and FSY5439 strains following selection in a -Ura medium. The MATA and MATα strains deleted for PHO5 were named FSY9286 and FSY9287. We then amplified the wild-type PHO5 gene with either the OFS5086 and OFS5087 primer pair or OFS5088 and OFS5089 primer pair in order to insert a terminator for the PHO5 mRNA (sense) or the AS transcript, respectively. The PCR products targeting either the sense or the antisense transcription were transformed in the FSY9286 and FSY9287 and counter-selected on a 5-FOA medium. The strains targeting the PHO5 mRNA or AS RNA were named FSY9288 and FSY9291. Finally, the 3 different diploids  were generated after crossing FSY6857 with FSY9287, FSY9286 with FSY9291 and FSY9288 with FSY9291, respectively, and selection on -His-Trp medium.Yeast election ,31,37. Amid from  as templmid from  as templmid from  as templribed in . The pCEribed in  was obtarain see . This waPMA1, ACT1 or SCR1 RNAs amplification, which were used as internal controls. To test the putative in trans activity of the PHO5 AS RNA, OFS2522 and OFS2523 were used to measure PHO5 mRNA and AS RNA levels. Amplifications were done in duplicate for each sample, and three independent RNA extractions were analysed. For the Northern blot, total RNA (10 μg for each sample) was run on a 1% denaturing formaldehyde agarose gel and transferred to nylon membranes (Amersham Hybondtm-N+). Membranes were crosslinked and incubated overnight at 60°C with 100μg/ml boiled salmon sperm DNA in 50% formamide, 5x standard saline citrate (SSC), 20% dextran sulfate sodium, 1% sodium dodecyl sulfate (SDS). Subsequently, membrane wered hybridized with 32P-labeled SP6/T7 riboprobes in 50% formamide, 7% SDS, 0.2 M NaCl, 80 mM sodium phosphate (pH 7.4), and 100 μg/ml boiled salmon sperm DNA for 6h. All blots were washed with 2X SSC and 0.1% SDS for 5 minutes at60°C and then with 0.5X SSC and 0.1% SDS for 45 minutes at 60°C. Riboprobes were obtained by SP6/T7 in vitro transcription of gene-specific PCR fragments containing an SP6/T7 promoter. Quantifications were performed with a Phosphor Imager machine.Acid phosphatase and beta-galactosidase activity assays were done with intact yeast cells, exactly as described in . Total RPHO5-adjacent control region which does not show chromatin signatures similar to the PHO5 gene promoter, as described in [S. pombe spike-in. An anti-Cas9 antibody (Diagenode #C15310258) was used for the immunoprecipitation step.For anti-histone H3 chromatin immunoprecipitation (ChIP), forty millilitres of cells were fixed with 1% formaldehyde for 20 min. After quenching with 400 mM glycine to stop the reaction, the cells were washed and lysed with glass beads to isolate chromatin. Sonication of cell lysates was performed with a Vibra-Cell sonicator in 1.2 mL of FA150 buffer  to reduce average fragment size to approximately 500 base pairs. The samples were centrifuged at 2500 g and the supernatant recovered. Chromatin fractions of 400 μl were taken for each immunoprecipitation reaction and incubated with 4 μl of anti-histone H3 antibodies  at 4°C overnight. After incubation, 40 μl of protein G PLUS-agarose beads  were added and incubated at 4°C for 2 h. The beads were washed extensively by successive washing steps: 3 times with FA150 lysis buffer, 3 times with FA500 lysis buffer (similar to FA150 but with 500 mM NaCl), 1 time with washing buffer 1  and 1 time with washing buffer 2 . Chromatin was eluted at 80°C in elution buffer  during 20 minutes. Samples (regardless of Input or IP) were reverse cross-linked at 65°C overnight. Eluted supernatants (output) and the input controls were hydrolysed with Pronase  at 42°C for 2 h, followed by incubation at 65°C for 7 h to reverse cross-linked DNA complexes. DNA was extracted using the Macherey Nagel Nucleospin Gel & PCR Cleanup Kit. The immunoprecipitated DNAs (output) were quantified by qPCR in Roche LightCycler 480 with the Maxima SYBR Green qPCR Master Mix detection kit (Thermo Scientific). Amplifications were done in triplicate for each sample. Immunoprecipitated samples (output) were normalized to input and to a ribed in . Chromatribed in ,37,44. 1ribed in ,37,45. Qribed in  without For RNA-seq and RNAPII PAR-CLIP, data were retrieved from  , is initiated at the 3’ end of the gene ORF in the antisense direction and extends through the PHO5 promoter region (rrp6Δ mutant cells (PHO5 AS transcript at the PHO5 promoter region in rrp6Δ compared to wt cells by strand-specific reverse-transcription quantitative PCR (RT-qPCR) upon shifting the cells from repressive  to inducing  conditions. PHO5 AS accumulation in rrp6Δ was most pronounced under repressive conditions  signal, which measures only nascent transcription, is comparable or even higher for the AS transcript than for the mRNA transcript under same growth conditions (4 mM Pi), showing that the AS transcript is being produced to a potentially significant level  in wild-type and rrp6Δ cells  also showed delayed kinetics compared with the corresponding wild-type cells (dis3Δ + pDis3) and with the mutant for its endoribonuclease activity (dis3Δ + pDis3-endo-)  . These rrrp6Δ and other RNA exosome deletion mutant backgrounds, AS transcription is constitutively induced due to sequestration of the NNS (Nrd1-Nab3-Sen1) termination complex by stabilised non-coding RNAs. The NNS complex cannot be efficiently recycled to sites of transcription, inducing termination defects at non-coding RNA loci and resulting in their increased elongation frequency [PHO5 gene due to gene deletion mutant backgrounds in which AS transcription is constitutively elongated, we turned to a system in which AS elongation is inducible. To this end, we used the Anchor Away (AA) system to rapidly deplete Nrd1 protein from the nucleus by rapamycin treatment [PHO5 AS transcript production, clearly demonstrating that the NNS complex is important for its early termination in wild-type cells. Importantly, even under PHO5 repressive conditions, induction of PHO5 AS transcript production through the Nrd1-AA system was accompanied by downregulation of PHO5 mRNA levels, as shown by Northern blot (PHO5 inducing conditions (i.e. phosphate free medium)   or an ho medium) . Consist medium) . The resPHO5 AS transcript without using RNA degradation/termination mutant backgrounds, we inserted the strong constitutive TEF1 promoter in the antisense configuration downstream of the PHO5 gene ORF , in trans (from the opposite chromosome) and another one in which AS transcription in cis was blocked  in whic blocked . Insertid in cis . CrucialPHO5 AS transcript negatively affects PHO5 gene transcription kinetics, blocking AS transcript production should enhance it. To specifically target PHO5 AS transcription, we undertook a CRISPRi approach in which a catalytically dead Cas9 protein (dCas9) is directed by a guide RNA (gRNA) to interfere with AS transcription at the PHO5 gene locus. The CRISPRi system blocks transcription due to physical collision between the elongating RNA Polymerase and the dCas9:gRNA complex [PHO5 gene locus to block only AS transcription. First, we confirmed the presence of the dCas9 protein at the PHO5 ORF by anti-Cas9 chromatin immunoprecipitation (ChIP). Notably, a strong peak of dCas9 binding at the PHO5 gene ORF compared to a control strain not expressing the gRNA was observed . These results are consistent with a dCas9-mediated transcriptional roadblock of AS transcription at the PHO5 gene locus. After the addition of rapamycin, PHO5 AS levels were increased in both the CRISPRi Nrd1-AA strain and the control Nrd1-AA strain. However, its levels in the CRISPRi strain remained significantly lower, maintaining the difference in levels already observed without the addition of rapamycin PHO5 genent cells . Consistnt cells . These rpromoter ,26. We rn period . Taken tS. cerevisiae [PHO5 promoter [sth1td, resulted in a strong delay in promoter chromatin structure opening and, consequently, delayed kinetics of acid phosphatase expression upon PHO5 gene induction.Histone acetylation plays two important roles in transcriptional activation. It neutralizes the positive charge of lysine groups, thereby weakening histone-DNA interactions, and it also provides docking sites for the bromodomains of proteins involved in transcriptional regulation. RSC (Remodels Structure of Chromatin) complex is the most abundant and the only essential remodeler in yeast and contains seven of the fourteen bromodomains identified in revisiae ,55. RSC promoter . Its parPHO5 gene promoter upon induction, we used the anchor away system to deplete its catalytic subunit Sth1 from the nucleus in Sth1-AA and corresponding gcn5Δ mutant cells. Because Sth1 is essential for cell viability, we first attempted to induce its depletion in parallel with the induction of the PHO5 gene. Addition of rapamycin upon shifting the cells to phosphate-free medium caused a delay in acid phosphatase expression kinetics similar to the partial depletion through sth1td  cells  cells . This rerrp6Δ mutant cells, we monitored acid phosphatase expression kinetics upon addition of rapamycin , Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and Sth1-Chromatin Endogenous Cleavage-sequencing (Sth1-ChEC-seq) datasets, which give us information about the chromatin conformation and Sth1 binding at the PHO5 promoter upon induction of AS transcription (+Rap/-Rap) and depending on the presence of Rpd3  . These data argue in favour of a model in which read-through of AS transcription acts via recruitment of histone deacetylases to the PHO5 gene promoter, the activity of which results in decreased recruitment of the RSC complex (To directly answer the question of whether AS-induced deacetylation of the apamycin . We exam of Rpd3 . The ChEin -Rap) . Converss region . When co complex .i.e. from the same strand as the gene, or from the opposite strand, resulting in production of antisense (AS) non-coding RNAs. Apart from a few isolated examples, production of AS non-coding RNAs is generally thought to have a repressive cis-regulatory effect on the expression of associated mRNAs [PHO5 gene locus, which was originally suggested to support gene activation [PHO5 gene expression. By leveraging mutant backgrounds in which AS transcription is constitutively enhanced or inducible and artificially driving its expression from a strong promoter in cis, we show that increased PHO5 AS elongation frequency correlates with decreased expression of the corresponding mRNA. Furthermore, we demonstrate that the use of a CRISPRi system that specifically blocks AS transcription at the PHO5 gene locus increases the level of PHO5 mRNA and enhances its induction kinetics upon phosphate depletion. Importantly, these observations show that AS RNA transcription has an impact on PHO5 gene expression in wild-type cells, and not only upon enhanced AS RNA stabilisation in strains mutant for RNA degradation factors. We also show that AS RNA transcription regulates expression of the PHO5 gene only when transcribed in cis, and not in trans. The role of PHO5 AS transcription is therefore reminiscent of the role of AS transcription in maintaining the tight repression of quiescence-related transcripts during the exponential growth phase, recently demonstrated by Nevers et al. [PHO5 AS transcription achieved AS inactivation by incorporating a full-length marker gene sequence with its promoter region in the middle of the PHO5 gene ORF [PHO5 gene locus may have resulted in experimental artefacts, highlighting the need for precise interventions, such as those achieved by the CRISPRi system, to perform functional analyses of AS transcripts [The role of non-coding RNAs in regulation of gene expression could not be appreciated until recent advances in high-throughput methods facilitated their detection and characterization. From a gene-centered view, non-coding RNAs can be transcribed in tandem with genes, ed mRNAs ,9,56. Thed mRNAs ,15,57. Itivation . In thiss et al. . A previgene ORF . This manscripts .SRG1 lncRNA/SER3 protein-coding gene locus, non-coding transcription has been shown to cause nucleosome deposition at the gene promoter, thereby repressing SER3 transcription [PHO84 gene locus silences the corresponding gene by recruiting HDACs to its promoter region [PHO5 AS under repressive conditions leads to increased histone occupancy at the PHO5 gene promoter and slower histone removal upon gene induction. Moreover, the negative effect of AS RNA elongation on PHO5 gene activation is mitigated by inactivation of Rpd3, suggesting a histone acetylation-based regulatory mechanism that may affect the recruitment of RSC, a chromatin remodeler that plays an important role in PHO5 gene promoter opening [PHO5 gene promoter upon induction of antisense transcription, that is suppressed by inactivation of Rpd3.There are now several well-described examples of yeast gene loci at which either antisense or upstream non-coding transcription that extends through a coding sense promoter has an inhibitory effect on its transcription initiation ,59–62. Icription . As anotr region . The AS r region . Consistr region . We have opening . This isPHO5 belongs to a group of ~100 genes that are more transcribed in AS direction as a non-coding transcript than in the sense orientation as an mRNA in a standard medium . These modeling  and our S1 Fig(A) A heatmap summarising tiling array expression data at the PHO5 gene locus in wild-type W101 (wt) and corresponding rrp6Δ cells. Data is from [(B) Levels of SUT446 in wild-type BMA41 (wt) and corresponding rrp6Δ mutant cells upon induction through phosphate starvation. RT-qPCR values were normalized to PMA1 RNA and expressed relative to transcript abundance in wild-type cells at repressive conditions (0 h of induction), which was set to 1. is from  and is v is from . (B) Lev(TIFF)Click here for additional data file.S2 Fig(A) Acid phosphatase induction kinetics in wild-type BMA41 (wt) and corresponding mutant cells upon induction through phosphate starvation. The strain rrp6Y361A carries a point mutation at the RRP6 genomic locus which abolishes exonuclease activity of Rrp6. Plasmid pCEN-RRP6 is a centromeric plasmid which carries the RRP6 gene under regulation of its native promoter. Reported values represent the means and standard deviations of three independent experiments (n = 3). (B) Same as (A), but for wild type and corresponding rrp6Δ mutant cells from the BY4741 genetic background. (C) Same as (A), but for wild type and corresponding rrp6Δ mutant cells from the LPY917 genetic background. (D) Beta-galactosidase induction kinetics in wild-type BMA41 (wt) and corresponding rrp6Δ cells transformed with a reporter plasmid pP5Z carrying the lacZ gene under the control of the PHO5 promoter upon induction through phosphate starvation. Reported values represent the means and standard deviations of three independent experiments (n = 3).(TIFF)Click here for additional data file.S3 FigPHO5 AS transcription, upon induction through phosphate starvation. Reported values represent the means and standard deviations of three independent experiments (n = 3).Acid phosphatase induction kinetics in wild-type BMA41 (wt) and corresponding deletion mutant cells for Rrp6 and Rpd3, with and without expression of the CRISPRi system which blocks (TIFF)Click here for additional data file.S4 Figrrp6Δ cells upon induction through phosphate starvation without (-Rap) or with addition of rapamycin (+Rap) at indicated times. Reported values represent the means and standard deviations of three independent experiments (n = 3).Acid phosphatase induction kinetics in Sth1-AA and the corresponding (TIFF)Click here for additional data file.S1 TableTable includes names of strains, their genotypes and sources.(PDF)Click here for additional data file.S2 TableTable includes names and sequences of oligonucleotides used for strain construction, plasmid construction and RT-qPCR.(PDF)Click here for additional data file.


Pmepa1 and Wnt3, respectively, and demonstrate their functionality. Finally, we determine a mechanism by which NODAL/Activin signalling induces delayed gene expression, by uncovering a self-enabling transcriptional cascade whereby activated SMADs, together with ZIC3, induce the expression of Wnt3. The resultant activated WNT pathway then acts together with the NODAL/Activin pathway to regulate expression of delayed target genes in prolonged NODAL/Activin signalling conditions.SMAD2, an effector of the NODAL/Activin signalling pathway, regulates developmental processes by sensing distinct chromatin states and interacting with different transcriptional partners. However, the network of factors that controls SMAD2 chromatin binding and shapes its transcriptional programme over time is poorly characterised. Here, we combine ATAC-seq with computational footprinting to identify temporal changes in chromatin accessibility and transcription factor activity upon NODAL/Activin signalling. We show that SMAD2 binding induces chromatin opening genome wide. We discover footprints for FOXI3, FOXO3 and ZIC3 at the SMAD2-bound enhancers of the early response genes, This article has an associated First Person interview with the first author of the paper. Summary: ATAC-seq and footprinting analysis reveals the transcription factor networks underlying NODAL/Activin signalling responses and a self-enabling regulatory mechanism that explains the expression of delayed NODAL/Activin target genes. During embryonic development, extracellular signals induce programmes of gene expression via activation of transcriptional effectors functioning as signal-driven transcription factors (SDTFs) . In thisin vitro to mimic the functional activities of NODAL, are members of the transforming growth factor β (TGF-β) ligand family. During embryonic development NODAL is required both for maintaining the pluripotency state in early embryos and for inducing mesendoderm differentiation and left–right patterning at later developmental stages , P19s express pluripotency and mesendodermal genes in response to NODAL/Activin signalling; however, they do not differentiate when treated with the NODAL/Activin type I receptor inhibitor SB-431542 . Using Pll types . FinallyWnt3 via ZIC3 and FOXH1, and that WNT3 then signals via the β-catenin-TCF/LEF axis and acts with pSMAD2 to synergistically activate expression of delayed NODAL/Activin target genes. Our work thus describes an innovative approach to identify novel cofactors mediating SMAD chromatin binding and unmasks a self-enabling regulatory mechanism shaping long-term NODAL/Activin transcriptional responses.Here, we have used the assay for transposase-accessible chromatin using sequencing (ATAC-seq) followed by TF footprint analysis to discover the transcription factor networks regulating the temporal transcriptional responses to NODAL/Activin signalling. We have performed ATAC-seq on P19 cells over a time course of NODAL/Activin treatment and have integrated it with the RNA-sequencing (RNA-seq) data and SMAD2 chromatin immunoprecipitation sequencing (ChIP-seq) data that we previously generated . We idenFig. S1A–C).Our previous work demonstrated that the complex programme of gene regulation downstream of NODAL/Activin signalling relies on chromatin remodelling events and a network of TFs that are still poorly characterised . To inveLefty1 and Lefty2 are two genes that were rapidly induced by Activin at the 1 h time point and whose expression was then sustained. The ATAC-seq signal was enriched at SMAD2-binding sites (SBSs) and additionally at the promoters/transcription start sites (TSSs) of Lefty1. Importantly, the intensity of the peaks at Lefty1 and Lefty2 SBSs substantially increased upon NODAL/Activin treatment compared to in the SB-431542 condition .We first inspected the ATAC-seq data over time at some representative loci to understand how SMAD2-binding dynamics related to chromatin accessibility. ondition A. This cd Lefty2 B. Of note course A,B. FurtSmad7 is an example of a transiently induced gene target of SMAD2 signalling. Here, SMAD2 binding occurred at an already accessible site, which further opened at later time points, correlating with increased SMAD2 occupancy . The ATAC-seq signal also reflected the SMAD2 occupancy in the case of repressed genes, as exemplified by the Tbx3 locus . Importantly, we observed that distinct modes of SMAD2 binding could be identified not just in response to acute stimulation, but also at later time points. In the case of the delayed gene Eomes, SMAD2 binding occurred at both open  and closed (SBS 2) chromatin regions 8 h after Activin treatment  (reatment D. A simias Tbxt) E. We thuas Tbxt) A–E.Fig. S2). Of note, the enhancers for Nodal and Lefty2 occupied by SMAD2 in response to Activin in P19 cells are also the same ones previously described as NODAL-responsive enhancers in mouse embryos , suggesting that even though many SBSs, such as those regulating Lefty1 and Pmepa1, are occupied by nucleosomes in absence of signalling . We then quantified changes in chromatin accessibility over time at the high confidence set of SMAD2 binding sites, using the DiffReps package to achieve statistical relevance . The SBSs that showed temporal changes in chromatin accessibility were grouped according to the kinetic category of the genes they were associated with, and the temporal changes in ATAC-seq were displayed in a heat map adjacent to the time at which the SMAD2 peak was detected at each site (Table S1). Overall, the chromatin accessibility of SBSs tended to increase upon NODAL/Activin signalling, regardless of the dynamics of induction/repression of the associated target gene .In our previous analysis of nucleosome occupancy during the same NODAL/Activin time course we showed that SMAD pathway activation induced nucleosome displacement at a subset of sites associated with so-called ‘baseline off’ genes (defined as genes that were not expressed in the SB-431542 condition) . When coIn conclusion, this quantitative analysis shows that SMAD2 binding directly increased chromatin accessibility at many target sites. These chromatin remodelling events do not exclusively occur at loci in a closed conformation, but also occur at many SBSs that are already partly open in the absence of signalling.a priori set of motifs and thus provided us with the additional advantage of identifying de novo TF–DNA interactions  were detected 1 h after Activin treatment, and notably, two of them colocalised with FOXH1 motifs. In particular, the second footprint (F.4) was both highly conserved and positioned directly under the SMAD2 peak , indicating that this SBS was devoid of any bound TFs in the absence of signalling RNAs against members of the FOX family most highly expressed in P19 cells  assay. We found that both FOXO3 and FOXI3 bound the footprint sequence in the presence and absence of Activin stimulation, and this did not require the presence of intact neighbouring SBEs D. SimilaPmepa1 SBS, inducing chromatin remodelling and activating transcription.Taken together, these experiments identify FOXI3 and FOXO3 as SMAD2 cofactors required for binding to closed chromatin enhancers, such as the Fig. S4A). When we performed the same analysis with FOXH1 genome wide, we could readily detect a footprint at a subset of loci . However, when we repeated the analysis to understand how occupancy of FOXH1 sites that reside in SMAD2 peaks changed over time, the heatmaps showed a much less well-defined pattern . Thus, despite the fact that the FOXH1-binding site in the Lefty1 SBS is part of a larger footprint . To identify candidate TFs responsible for recruiting SMAD2 to specific enhancers in response to NODAL/Activin signalling, we repeated the analysis on the 456 SMAD2 peaks overlapping with an ATAC-seq peak. The known SMAD-interacting cofactors FOXH1 showed increased activity at both time points compared to the SB-431542 condition  and footprint accessibility (FA) of flanking motifs A, ‘bivarondition B,C. Striondition B–D. We tWnt3 and Wnt8a, were upregulated in response to NODAL/Activin signalling, although with different dynamics. Wnt3 was directly induced immediately after 1 h of Activin, whereas Wnt8a mRNA was only upregulated after 8 h of signalling . Wnt8a and the WNT-target gene Axin2 expression was also affected by the treatment with LGK974 and IWR-1 , consistent with our siRNA knockdown data. As expected, these inhibitors had no effect when combined with the WNT pathway activator, CHIR99021 . Finally, to address to what extent the NODAL/Activin pathway synergised with the WNT/β-catenin cascade, P19 cells were treated CHIRON along with Activin, or with SB-431542 . Both T and Wnt8a were induced with similar dynamics when treated with Activin or with CHIRON in the presence of SB-431542, suggesting that both WNT and NODAL/Activin signalling cascades targeted these genes independently . Furthermore, T expression was greatly increased when cells were simultaneously exposed to both Activin and CHIRON, whereas this was not the case for Lefty1, Eomes or Wnt3, indicating the existence of gene-specific responses to different levels of activation of the WNT pathway . Finally, as expected, induction of the WNT target genes Axin2 and Notum was almost entirely independent of NODAL/Activin signalling .These observations suggested that early NODAL/Activin-dependent pression B. To tespression , in the  CHIRON; , which aWnt3 triggers the activation of the WNT/β-catenin pathway, which is necessary for the full induction of delayed NODAL/Activin target genes like Eomes, T and Wnt8a . Clones were screened by genomic sequencing and the low levels of Foxh1 or Zic3 mRNA were used as a bona fide indicator of the lack of functional proteins, alongside the loss of expression for well-established downstream target genes . As expected from our previous work, all clones with a FOXH1 loss-of-function mutation failed to express Lefty1 in response to Activin, whereas Pmepa1 induction was not affected  (Lefty1 or Pmepa1 was observed in the ZIC3-null clones compared to wild type (WT) . Importantly, loss of FOXH1 or ZIC3 function across multiple clones resulted in a strong reduction of Wnt3 transcription after 2 h of Activin signalling, as well as after prolonged signalling  . Furthergnalling A,B. We t deleted A,B. Eomeor FOXH1 A,B.Fig.Wnt3 induction in response to NODAL/Activin signalling, and that this is due to direct transcriptional regulation occurring at the level of the Wnt3 enhancer, where ZIC3 binds together with FOXH1 and activated pSMAD2–SMAD4 complexes.We therefore conclude that ZIC3 is required for Pmepa1 and Wnt3, analyses of TF footprint dynamics revealed DNA motifs bound by hitherto uncharacterised SMAD cofactors. We have demonstrated that DNA sequences recognized by FOXO3 and FOXI3 and by ZIC3 are necessary for NODAL/Activin-induced transcription via Pmepa1 and Wnt3 enhancers, respectively  and at the Wnt3 SBS . The technique of using ATAC-seq data for footprinting is still in development and two major challenges should be taken into consideration – the detection of the footprints themselves and the assignment of footprints to matching TFs. ATAC-seq footprint analysis, such as the Wellington algorithm is mostly based on adapting tools originally developed for DNase I data and a consensus ‘gold standard’ in the data processing workflow is yet to be achieved  in epithelial cells in response to TGF-β, and FOXO3 has previously been shown to cooperate with the SMADs to regulate cyclin induction in ovarian cancer  targeting Foxh1 or Zic3 were cloned into pSpCas9(BB)-2A-GFP (PX458) (Table S2). The PCR fragments of positive clones were sequenced to identify the CRISPR/Cas9-mediated deletions/insertions. For FOXH1, we targeted the C-terminal SMAD-binding motif  , and theng motif , and forTable S2.NODAL/Activin signalling was inhibited by overnight incubation with 10 μM SB-431542 (Tocris Bioscience), which was washed out three times with PBS prior to stimulation with 20 ng/ml Activin A (Peprotech) in full medium for different times. For the SB-431542 condition, after washout, cells were incubated for 1 h in 10 µM SB-431542 in full medium, providing a control for a possible transient effect of serum stimulation in the 1 h Activin samples. The untreated condition represents a chronic signalling state as a result of autocrine production of NODAL and GDF3 . CHIRON Table S2 cloned into the pGL4.27[luc2P/minP/Hygro] Vector (Promega). Note that in the pGL4.27-Pmepa1 SBS luciferase reporters there are three copies of the WT or mutant SBS sequences and in the pGL4.27-Wnt3 SBS luciferase reporters there are two copies of the WT or mutant Wnt3 SBS sequences. The plasmid encoding TK-Renilla was also from Promega.The pGL4.27-Pmepa1 SBS WT-luciferase reporter, the pGL4.27-Wnt3 SBS WT-luciferase reporter and their mutated versions were generated using the synthesised sequences listed in Cells were transfected with the appropriate plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The P19 line stably expressing the pGL4.27-Pmepa1 SBS WT-luciferase reporter together with TK-Renilla was generated by transfecting the cells with the appropriate plasmids together with a plasmid carrying the puromycin resistance gene . Cells were then selected with 2 μg/ml puromycin (Sigma).Table S2. At 48 h after plasmid transfection or gRNA-Cas9 complex electroporation, cells were treated with drugs or growth factors for the appropriate times and luciferase reporter assays were performed as previously described, using the Dual-Glo assay system (Promega) following the manufacturer's instructions  and electroporated using the Neon system (Invitrogen) with 1275 V, 30 ms pulse width and 1 pulse time. The Edit-R crRNA (Dharmacon) and Edit-R tracrRNA  used are listed in ructions .Table S2. All qPCRs were performed with the PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) with 300 nM of each primer and 2 µl of diluted cDNA. Fluorescence acquisition was performed on either a 7500 FAST machine or QuantStudio 12 Flex (Thermo Fisher Scientific). Calculations were performed using the ΔCt or ΔΔCt method, and levels of mRNA are expressed, respectively, as fold change relative to Gapdh mRNA or 8-h Activin-treated control cells. Unless otherwise stated in the figure legends, means±s.e.m. from at least three independent experiments are shown.Total RNA was extracted using Trizol (Thermo Fisher Scientific) according to manufacturer's instructions. cDNA synthesis and quantitative (q)PCRs were performed as described previously . Primer Pmepa1 and Wnt3 SBSs are shown in the relevant figures and all oligonucleotide sequences are given in Table S2. Western blotting was carried out using standard methods. The list of the antibodies used is shown in Table S2.DNA pulldown (DNAP) assays were performed as previously described with some modifications . Nuclearg for 5 min at 4°C. Pellets were washed once with 50 μl of cold PBS, and the cell suspensions were centrifuged at 180 g for 5 min at 4°C. To lyse the cells, samples were resuspended in 50 μl of cold lysis buffer  and immediately centrifuged at 500 g for 10 min at 4°C. Supernatants were then discarded and the nuclei gently mixed with 50 μl of transposition reaction mix. The transposition reaction was incubated at 37°C for 30 min, directly followed by a column purification step carried out using the PCR purification MinElute Kit (Qiagen). Here, 10 μl of Elution Buffer (Qiagen) were used for eluting the transposed DNA from the columns. To generate the libraries for next-generation sequencing, the transposed DNA fragments were amplified by PCR using the Veriti 96-well Thermal cycler (Applied Biosystems). Samples were then cleaned up with the PCR purification MinElute Kit (Qiagen) and eluted in 20 μl of Elution Buffer (Qiagen). Finally, an additional clean up step was performed using AMPure beads  to remove excess primers. Since the size of the library fragments provides a good indication of the success of the transposition reaction, the High Sensitivity DNA assay was performed on all samples using the 2100 Bioanalyzer (Agilent). For each biological replicate, two libraries were generated for each treatment condition to function as technical replicates, for a total of eight samples for each individual experiment. Upon Bioanalyzer analysis, for each biological replicate one sample per condition was picked amongst the technical replicates based on the fragment size distribution. A total of eight samples  was then submitted for next generation sequencing.Samples for ATAC-seq were obtained as described previously, with minor adaptations . P19 cel10 scale after quantile normalization using the ‘normalize.quantiles’ function in R version 3.3.1. When individual samples were combined to increase the read depth, the filtered alignments from each library were merged using the picard MergeSamFiles command. Duplicate marking was re-performed on the merged alignments, and they were subsequently filtered for duplicate reads, leading to 500–600 million reads for merged sample.The ATAC-seq experiment was performed in biological duplicate. Samples were prepared as described, and 51-bp pair end reads were generated on an Illumina HiSeq 2500. Raw reads from each sample were adapter-trimmed using cutadapt 1.9.1 with parameters ‘-a CTGTCTCTTATA -A CTGTCTCTTATA, minimum-length=25 – quality-cutoff=20’. BWA 0.6.2  with defGenome-wide ATAC-seq peaks and normalized BedGraph coverage tracks for the merged samples were obtained using MACS2 callpeak 2.1.1.20160309  with thehttp://bioinfogp.cnb.csic.es/tools/venny/).To intersect or subtract different BED files, BEDTools or the Table browser found within the UCSC Genome browser were generally used . To iden2FC greater than 0.5 as an absolute value. Hierarchical clustering using Euclidean distance was employed to group the different SBSs on the basis of the diffReps interval log2FC values.The individual replicate samples were employed to call differential ATAC-seq sites between conditions. Intervals of differential ATAC signal were obtained using Diffreps 1.55.4  with theFootprint analysis was performed across the union set of ATAC-seq peaks on the unshifted, merged alignments using the ‘wellington_footprints.py’ command in the pyDNAse package 0.2.5  with par−9 (i.e. 1 bp). This analysis allowed calculation of motif footprint frequency, and the generation of different kinds of footprint heatmaps and plots for each of the known motifs in the HOMER database. First, motif centric heatmaps were obtained using the pyDNAse ‘dnase_to_javatreeview.py’ command with parameters ‘--window_size 100 -A -n’. Then, to identify changes in motif accessibility over time, differential heatmaps were generated by calculating the absolute value of the difference between each signalling condition compared to the SB-431542 sample. Additional differential heatmaps were also produced to conserve the strand information. This was achieved by extracting the absolute difference between each signalling condition and the SB-431542 sample, and then weighting it by ±1 depending on whether a change in sign was observed. Finally, the average profile plots around motif sites were generated using the pyDNAse ‘dnase_average_profile.py’ command with parameters ‘--window_size 100 -A’.To retrieve the genome-wide locations of all known motifs in the HOMER database, the ‘scanMotifGenomeWide.pl’ command was used with parameters ‘-bed -int -keepAll’. The resulting files were then intersected with the merged footprint intervals present in both ATAC-seq and SMAD2 peaks, using the BEDTools command intersectBed and setting the percentage of overlap to 10Pmepa1 SBS and the motif matrices generated by the BaGFoot package described above.The find individual motif occurrences (FIMO) analysis in Changes in TF activity between biological conditions in the experiment were assessed using the BaGFoot software package  under the accession number escribed .t-test.Statistical analysis was performed in Prism 8 (GraphPad). At least three independent experiments were performed for statistical analysis unless otherwise specified in the Figure legends. For qPCRs, statistics were performed using an unpaired two-tailed


We show that the chromatin remodelling complex RSC and general regulatory factors (GRFs) limit transcription in the noncoding divergent direction, thereby increasing the directionality of promoters. The directionality of gene promoters—the ratio of protein-coding over divergent noncoding transcription—is highly variable. How promoter directionality is controlled remains poorly understood. Here, we show that the chromatin remodelling complex RSC and general regulatory factors (GRFs) dictate promoter directionality by attenuating divergent transcription relative to protein-coding transcription. At gene promoters that are highly directional, depletion of RSC leads to a relative increase in divergent noncoding transcription and thus to a decrease in promoter directionality. We find that RSC has a modest effect on nucleosome positioning upstream in promoters at the sites of divergent transcription. These promoters are also enriched for the binding of GRFs such as Reb1 and Abf1. Ectopic targeting of divergent transcription initiation sites with GRFs or the dCas9 DNA-binding protein suppresses divergent transcription. Our data suggest that RSC and GRFs play a pervasive role in limiting divergent transcription relative to coding direction transcription. We propose that any DNA-binding factor, when stably associated with cryptic transcription start sites, forms a barrier which represses divergent transcription, thereby promoting promoter directionality. Transcription is highly pervasive and results in intergenic and intragenic noncoding transcription events. Noncoding transcription and the produced noncoding RNAs play diverse roles in gene and genome regulation . TranscrOur understanding of the function of divergent transcription is still incomplete. There is evidence that the noncoding transcripts emanating from divergent transcription events can help to promote gene transcription in the coding direction and to facilitate cell fate control . Other sSaccharomyces cerevisiae. Hence, there are various molecular mechanisms that limit the accumulation of noncoding RNAs or noncoding transcription itself. Indeed, to reduce the accumulation of divergent RNAs, these noncoding transcripts are often rapidly degraded via the RNA degradation machinery (Preprint). Lastly, general regulatory factors (GRFs) can repress initiation of aberrant noncoding transcription events  and treated the cells with indole-3-acetic acid  for 2 h  and on polyadenylated RNAs (mRNA-seq) after depletion of both RSC and Rap1. We determined the levels of nascently transcribed RNA by measuring RNA polymerase II (Pol II)-associated transcripts using an adapted native elongating transcript sequencing protocol . In shor for 2 h . Rap1 anA Pol II . The repRAP1-AID + IAA versus RAP1-AID + DMSO)   and S1C. + DMSO) . We obseRPL43B and RPL40B) at which Rap1 is known to repress divergent transcription (IRT2 and iMLP1) was up-regulated upon Rap1 depletion, whereas coding transcription was decreased. Upon co-depletion of Rap1 and Sth1, we observed that IRT2 and iMLP1 levels were reduced in mRNA-seq. Although Sth1 depletion by itself did not affect IRT2 levels, levels of iMLP1 were already up-regulated in Sth1-depleted cells Fig 2A). 2(FC) > 1, Padj < 0.05]), noncoding transcription of CUTs/SUTs/XUTs/NUTs was substantially up-regulated  > 1, Padj < 0.05]) (2(FC) > 1) in divergent transcription (2(FC) > 1) in directionality score upon Sth1 depletion (STH1-AID + IAA) compared with WT or control cells (STH1-AID + DMSO) (Divergent noncoding transcription is an inherent feature of active promoters across species . Accordi< 0.05]) . Employicription . Consequ + DMSO) . Notably + DMSO)  and S3. STH1-AID +DMSO) displayed a marginal increase in divergent transcription in the most highly expressed and most directional promoters  compared with WT cells, which was consistent with the observation that the AID tag partially affected RSC activity and had a small negative impact on promoter directionality  displayed more defined nucleosome peaks, that were narrower in width at the +1, −1, and −2 positions compared with gene promoters with the lowest directionality score (Q1) (To determine whether the effect at the ore (Q1) . Secondlore (Q1)  (Klein-Bore (Q1) .This work suggests that RSC contributes to limiting relative divergent transcription levels at promoters with high directionality. One possibility is that RSC is more present at highly directional promoters compared with other promoters. To test this, we analysed RSC binding profiles from a published CUT&RUN dataset . We founEnrichment analysis for A-tracks and the CG(C/G)G motif in promoters.Table S1 BAP2 and CSG2 promoters  with both the coding and the divergent noncoding core promoters driving the transcription of fluorescent reporter genes encoding mCherry and YFP, respectively (SUT129/YFP TSS in the PPT1 reporter (REB1-AID + IAA), whereas for the reporter construct with Cbf1 binding sites, we measured the YFP/mCherry levels in WT and cbf1Δ cells.Based on our observations, we predict that Abf1 and Reb1 can repress divergent transcription in promoter regions proximal to their DNA-binding sites . Previouectively . We clonreporter . We selecbf1Δ cells and depletion of Abf1 and Reb1 (ABF1-AID or REB1-AID + IAA) decreased the YFP signal (noncoding) compared with control cells and increased promoter directionality , which is a bulky protein (over 150 kD) and has been widely used as tool to modulate transcription and chromatin    and S6. Transcription from promoters is bidirectional, leading to sense coding and divergent noncoding transcription. However, the directionality (sense over divergent transcription) of promoters greatly varies, indicating that there are mechanisms in place to control directionality of promoters . Why somBecause RSC controls global transcription by Pol II, it is important to emphasise the relative nature of the nascent RNA-seq measurement . For thiThe RSC complex, part of the SWI/SNF family of chromatin remodellers, contributes to generating NDRs by “pushing” the nucleosomes positioned at +1 and −1 positions “outwards,” thus widening the NDR . RSC alsRSC-mediated nucleosome positioning also functions in suppressing other aberrant transcription events. For example, RSC represses antisense transcription initiating from the 3′ ends of gene bodies, which is spatially distinct from the divergent noncoding transcription events we report here . In addiThe opening of chromatin controlled by RSC facilitates the recruitment of sequence-specific TFs to promoters . TherefoPreprint). The GRF Rap1 can repress divergent transcription at gene promoters by occupying cryptic divergent promoters . The RSC related mammalian esBAF complex has comparable functions in repressing noncoding transcription to some extent, suggesting that the role of RSC in controlling divergent transcription and promoter directionality is also likely conserved  were used for this investigation. A one-step tagging procedure was used to generate endogenous carboxy (C)-terminally tagged auxin-inducible degron (AID) alleles of Rap1 and Sth1 . The single-copy integration plasmids for expression of Oryza sativa TIR1 (osTIR1) ubiquitin E3 ligase were described previously (Strains isogenic to the and Sth1 . The RAP7 degron . For tageviously .AID) depletion experiments, induction of AID-tagged protein depletion was performed with 3-indole-acetic acid  (Sigma-Aldrich) during the exponential growth phase (approximately OD600 0.8).For auxin-inducible degron  fluorescent reporter plasmid was described previously   . The tarS. pyogenes–dCas9   experiments, yeast expression constructs for nuclease-inactivated Cas9 (D10A/H840A mutations) from Addgene)  were subAddgene)  were genAddgene) . sgRNA eGenotypes of strains used in this study.Table S2 Plasmids used in this study.Table S3 Oligonucleotide sequences used in this study.Table S4 Yeast cells were harvested from cultures for RNA extraction by centrifugation and then washed once with sterile water before snap-freezing in liquid nitrogen. RNA was extracted from frozen yeast cell pellets using acid phenol:chloroform:isoamyl alcohol  and Tris–EDTA-SDS (TES) buffer , by rapid agitation . After centrifugation, the aqueous phase was collected, and RNA was precipitated at −20°C overnight in ethanol with 0.3 M sodium acetate. After centrifugation and washing with 80% (vol/vol) ethanol solution, dried RNA pellets were resuspended in DEPC-treated sterile water and subsequently stored at −80°C.IRT2 performed as described previously . The result of n = 3 biological replicates are shown. Signals were normalised over ACT1. Primer sequences are listed in Table S4.Reverse transcription and quantitative PCR for eviously . In shorWestern blots were performed as described previously . ProteinSDS–PAGE (polyacrylamide gel electrophoresis) was performed using 4–20% gradient gels (Bio-Rad TGX), and samples were then transferred onto PVDF membranes by electrophoresis . Membranes were incubated in blocking buffer  before primary antibodies were added to blocking buffer for overnight incubation at 4°C. For probing with secondary antibodies, membranes were washed in PBST and anti-mouse or anti-rabbit IgG HRP-linked antibodies were used for incubation in blocking buffer . Signals corresponding to protein levels were detected using Amersham ECL Prime detection reagent and an Amersham Imager 600 instrument .i] buffer [pH 6.8]) at 70°C for 10 min, loading buffer  was added, and RNA samples were subjected to electrophoresis (2 h at 80 V) on an agarose gel . Capillary transfer was used to transfer total RNA onto positively charged nylon membranes (GE Amersham Hybond N+). Bands corresponding to mature rRNA were visualised by staining with methylene blue solution .Northern blots were performed as described previously . BrieflyIRT2 and SNR190 Northern blot probes by PCR can be found in Table S4.The nylon membranes were incubated for at least 3 h at 42°C in hybridisation buffer  or ULTRAhyb Ultrasensitive Hybridization Buffer (Thermo Fisher Scientific) to minimise non-specific probe hybridisation. Probes were synthesised using a Prime-it II Random Primer Labelling Kit (Agilent), 25 ng of target-specific DNA template, and radioactively labelled with dATP (α-32P) . The oligonucleotides used to amplify target-specific DNA templates for Blots were hybridised overnight with radioactively labelled probes at 42°C and then washed at 65°C for 30 min each with the following buffers: 2× saline-sodium citrate (SSC) buffer, 2× SSC with 1% wt/vol SDS, 1× SSC with 1% SDS, and 0.5× SSC with 1% SDS. For image acquisition, membranes were exposed to storage phosphor screens before scanning on the Typhoon FLA 9500 system . To strip membranes before re-probing for different transcripts, membranes were washed with stripping buffer  at 85°C until negligible residual signal remained on the blots.i [pH 7], 0.05 M MgCl2, 1.2 M sorbitol) and resuspended in the same buffer before imaging. Images were acquired using a Nikon Eclipse Ti-E inverted microscope imaging system (Nikon) equipped with a 100× oil objective (NA 1.4), SOLA SE light engine (Lumencor), ORCA-FLASH 4.0 camera (Hamamatsu), and NIS-Elements AR software (Nikon). 500-ms exposure time was specified and GFP, and mCherry filters were used to detect YFP and mCherry signals, respectively.Yeast cells were grown in YPD media  to the exponential growth phase and fixed with formaldehyde (3.7% wt/vol), incubating at room temperature for 15 min. Fixed cells were washed with phosphate-sorbitol buffer   for the S. cerevisiae pellets spiked-in with S. pombe cells in a 10:1 ratio of S. cerevisiae:S. pombe as described above, treated with rDNase in solution (Machery-Nagel), and purified by spin column (Machery-Nagel) before preparation of sequencing libraries. 500 ng of RNA input material was used for polyadenylated RNA (mRNA) sequencing. Libraries were prepared using the TruSeq Stranded mRNA kit (Illumina) according to the manufacturer’s instructions (10 or 13 PCR cycles). The libraries were multiplexed and sequenced on either the HiSeq 2500 or 4000 platform (Illumina) and generated ∼45 million 101-bp strand-specific paired-end reads per sample on average.Total RNA was extracted from 2, 1× Roche cOmplete EDTA-free protease inhibitor, and 50 U/ml SUPERase.In RNase Inhibitor. Chromatin-bound proteins were solubilised by incubation with 1,320 U of Dnase I  on ice for 20 min. Lysates containing solubilised chromatin proteins were clarified by centrifugation at 20,000g for 10 min (at 4°C), and the supernatant was taken as input for immunoprecipitation using 500 μl of anti-FLAG M2 affinity gel suspension  per sample (2.5 h at 4°C). After immunoprecipitation, the supernatant was removed, and beads were washed four times with 10 ml of cold wash buffer each time (1× lysis buffer with 50 U/ml Superase.In Rnase inhibitor and 1 mM EDTA). After the last wash, agarose beads were transferred to small chromatography spin columns , and competitive elution of protein complexes containing Rpb3-3xFLAG protein from the resin was performed by incubating beads with 300 μl of elution buffer (1× cold lysis buffer with 2 mg/ml 3xFLAG peptide) for 30 min at 4°C . Elution was performed twice and 600 μl of eluate was subjected to acid phenol:chloroform RNA extraction and ethanol precipitation. A significant amount of 3xFLAG peptide co-precipitates with the RNA as a contaminant and is later removed by spin column purification.For nascent RNA sequencing (nascent RNA-seq), RNA fragments associated with RNA polymerase II subunit Rpb3 endogenously tagged with 3xFLAG epitope at the C-terminus were isolated by affinity purification as described previously . Small bPurified RNA was fragmented to a mode length of ∼200 nucleotides using zinc ion–mediated fragmentation . Fragmented RNA was purified using miRNeasy spin columns , which retain RNAs ∼18 nucleotides or more in length. Purified RNA was quantified by Qubit (Thermo Fisher Scientific), and ∼150 ng of RNA was subjected to rRNA depletion using the Ribo-Zero Gold rRNA Removal Kit (Yeast) . Libraries were prepared using the TruSeq Stranded Total RNA kit (Illumina) according to the manufacturer’s instructions (14 PCR cycles). The libraries were multiplexed and sequenced on the HiSeq 4000 platform (Illumina) and generated ∼28 million 101-bp strand-specific paired-end reads per sample on average.S. cerevisiae  and S. pombe  genomes. Genomic alignments were filtered to only include those that were primary, properly paired, uniquely mapped, not soft-clipped, maximum insert size of 2 kb, and fewer than three mismatches using BamTools   with parn 2.4.0; ). Read cS. pombe transcripts were used to assess the size factors, potentially mitigating any impact a global shift in the S. cerevisiae read counts would have on DESeq2’s usual normalisation procedure. PCA was performed with DESeq2 on 500 genes with the highest variance.Differential expression analysis was performed with the DESeq2 package (version 1.12.3) within the R programming environment (version 3.3.1) . The spiS. cerevisiae genes downloaded from the Ensembl genome browser   with parease 90; ). The paS. pombe transcriptome were used for normalisation of the per-sample counts. Genomic annotations of CUTs, SUTs, XUTs, and NUTs (various noncoding RNA species) was obtained from STAR genomic alignments were filtered to include only unspliced, primary, uniquely mapped, and properly paired alignments with a maximum insert size of 500 bp. The featureCounts tool from the Subread package (version 1.5.1)  was usedGSE73337, GSE98260)  (Publicly available datasets were obtained from NCBI Gene Expression Omnibus (GEO) MNase-seq (SE98260) . Adaptorease 90)  with BWAease 90)  using deease 90)  was usedGSE116853    with par 2.26.0)  using pa10) in the WT strain. Analyses were repeated on only promoters with intermediate gene transcription levels (belonging to Q3 according to sense transcription level). Analysis for enrichment and distribution of A-tracks (AAAAAAA) and GC-rich motifs (CG(C/G)G) as defined in test_regexp_enrich and get_regexp_prof functions (Gene promoters were stratified in quintiles (Q1 to Q5) using Matt (version 1.3.0) , accordiunctions .Bar plots, scatter plots, and volcano plots were generated using GraphPad Prism (version 7 or 8). Screenshots of sequencing data were captured using the Integrative Genomics Viewer  . The RStt tests, calculated P-values less than 0.05 were considered significant. Plotted error bars in individual figures are stated in the figure legend, as either SEM or 95% confidence intervals (CI).Information regarding any statistical tests used, number of samples, or number of biological replicate experiments is stated in the corresponding figure legends. For GSE73337 (GSE98260 (GSE49026 (GSE116853 (GSE97913 and GSE97915 (GSE67453 (MNase-seq data were obtained from GSE73337  and GSE9GSE98260 , respectGSE49026 . Rap1-reSE116853 . Pol II GSE97915 . Reb1-ChGSE67453 .GSE179256.The accession number for the RNA sequencing data reported in this paper is


Prokaryotic transcription was extensively studied over the last half-century. A great deal of data has been accumulated regarding the control of gene expression by transcription factors regulating their target genes by binding at specific DNA sites. However, there is a significant gap between the mechanistic description of transcriptional control obtained from in vitro biochemical studies and the complexity of transcriptional regulation in the context of the living cell. Indeed, recent studies provide ample evidence for additional levels of complexity pertaining to the regulation of transcription in vivo, such as, for example, the role of the subcellular localization and spatial organization of different molecular components involved in the transcriptional control and, especially, the role of chromosome configurational dynamics. The question as to how the chromosome is dynamically reorganized under the changing environmental conditions and how this reorganization is related to gene expression is still far from being clear. In this article, we focus on the relationships between the chromosome structural dynamics and modulation of gene expression during bacterial adaptation. We argue that spatial organization of the bacterial chromosome is of central importance in the adaptation of gene expression to changing environmental conditions and vice versa, that gene expression affects chromosome dynamics. Escherichia coli, it is shown that chromosome compaction varies during the growth cycle. For example, during rapid exponential growth characterized by high levels of negative superhelicity, the chromosome is condensed with RNA polymerase (RNAP) mainly accumulated at a few highly active loci such as those encoding ribosomal RNA [nucleoid-associated proteins (NAPs) [The organization of the bacterial nucleoid structure is a compromise between the requirements of the dramatic (about thousand-fold) compaction of DNA and the accessibility of the genetic material to enzymes securing vital functions such as replication, transcription and repair. Recent studies demonstrate that bacterial chromosomes are not only highly compacted but also spatially organized, with distinct genetic loci occupying specific positions in the cell ,2,3,4. Iomal RNA ; by contomal RNA ,10,11,12omal RNA  which esomal RNA . Accordis (NAPs) ,15,16. OSalmonella enterica, and in the plant pathogenic bacteria, Dickeya dadantii, cooperative effects of NAPs and DNA supercoiling coordinate the expression of virulence and adaptive genes required at different stages of pathogenic growth [Chief among the bacterial global regulators are the abundant NAPs, which are thought to influence both the chromatin structure and transcription ,15,17,18c growth ,29,30,31c growth ,15 whilec growth , suggestEscherichiacoli and focused on the five major proteins H-NS, FIS, IHF, Lrp and HU, for which a direct role in transcriptional regulation was clearly established [NAPs thus play a crucial role in the adaptation of bacteria to unfavorable conditions and environmental stress ,32,33,34ablished  and is b(histone-like nucleoid-structuring protein) is considered a global modulator of genes involved in adaptive processes related to changes in environmental conditions. DNA binding by H-NS is sensitive to environmental factors and DNA supercoiling states [2+ concentration-dependent manner: above 5 mM Mg2+, the extension mode is inhibited, and instead, H-NS switches to bridging the DNA duplexes. It remains unclear whether H-NS primarily bridges or extends the DNA in vivo or whether these binding modes are mutually exclusive [H-NS g states ,15,30,35g states ,37. Accog states ,40,41,42g states . Single-g states . Furtherxclusive . A more xclusive . The comxclusive . Interes(factor for inversion stimulation) is a NAP initially characterized as a stimulator of site-specific DNA recombination. FIS activates genes and operons involved in primary metabolism, including those encoding biosynthetic enzymes and stable RNAs [oriC-directed DNA replication and modulates the DNA supercoiling state by repressing DNA gyrase and stimulating topoisomerase I gene expression [E. coli [Shigella flexneri [Salmonella [Vibrio cholera [D. dadantii [FIS ble RNAs . FIS is pression ,48. FIS pression . FIS is pression . The celpression . One imppression . Further[E. coli , Shigellflexneri , Salmonelmonella ,55, Vibr cholera  and D. ddadantii . integration host factor) is a heterodimeric protein composed of IHF α and IHF β subunits, respectively, encoded by ihfA and ihfB genes. Upon binding, IHF bend DNA by as much as 180 °C and thus promote long-range interactions. The influence of IHF on local DNA structure is critical for its contribution to transcriptional regulation [Brucella abortus, Shigella flexneri, Salmonella enterica, enteropathogenic and enterohemorrhagic E. coli, V. cholerae and D. dadantii [IHF  is found as a homo- or heterodimer of the homologous subunits α and β. HU has a preference for binding distorted regions of the DNA, such as kinks or four-way junctions [E. coli, including those involved in primary metabolism and respiration [S. enterica [HU  regulates the expression of more than 10% of the E. coli genome [E. coli and Salmonella [Citrobacter rodentium [Lrp  in adaptation to the changing growth environment. However, NAPs are also involved in maintaining supercoiling homeostasis by modulating the activity of topoisomerases both via direct effects on their substrate topology  and indirectly by regulating topoisomerase gene expression.Transcription is a complex multifactorial process in which the central player is RNA polymerase. The interconnections between the chromosome structural organization and RNA polymerase activity have been extensively reviewed ,69,70,71These transient changes in supercoiling can lead to supercoil diffusion modulating the activity of neighboring gene promoters . Recent E. coli chromosome structure pertinent to transcription were observed, including the rrn functional domain spanning the chromosomal Ori end, large clusters of genes sensitive to “nucleoid-perturbation”, DNA supercoiling-dependent spatial transcript patterns spanning regions from 16 to 800 kb in size, 10–20 kb topological domains and 5–10 kb gene proximity clusters [On the other hand, compelling data was obtained concerning the dynamic organization of the bacterial chromosome. The positioning of the chromosome in growing cells was shown to follow a predetermined choreography. Furthermore, various dynamic organizational features of the clusters ,80,82,83clusters ,85,86, wIn order to reduce the energy spent on gene regulation and orchestrate the production of numerous factors involved in a metabolic pathway or adaptation to a particular challenge, bacteria have set up different synchronization tools and processes. For a long time, studies focused on genetic processes that elucidated the synchronization of genetic expression based on operon structures or the organization of genes in spatial proximity to each other (synteny). In contrast, the coordinated expression of regulons and, in general, coordinated expression of gene clustered at different places in the genome (‘archipelago’ organization) has long remained a mystery, confounded further by the emergence in the 2000s of data indicating a relatively slow diffusion of regulatory proteins from their sites of production in the cell. Consequently, epigenetic-type mechanisms linked in particular to the structural dynamics and organization of the chromosome previously observed in eukaryotes were then explored. Hence, the implementation of tools to assess the long-range patterns on a genome-wide scale prompted the study of co-regulated, isofunctional and evolutionary correlated gene sets ,88,89,90Dickeya and Pectobacterium, responsible for soft rot disease in a wide range of plants. In these bacteria, the major virulence factors are pectate lyases, secreted by a specific type II secretion system [kdgR gene in the chromosome, or by disrupting the periodic spatial distribution of the KdgR regulon by introducing DNA ‘spacers’ between the regulated genes , first documented in E. coli ,116. Thesubtilis  but not lobacter . The maclobacter  and playlobacter . Their plobacter . Overalltranscription-coupled diffusion of supercoils (TCDS) and the formation of the so-called coherent domains of transcription (CODOs) spanning chromosomal regions from several tens to hundreds of kilobases and encoding particular traits [E. coli and the plant pathogenic bacterium D. dadantii. It was found that in E. coli, the genomic sequence organization favors a temporally sequential expression of genes required for growth along the OriC-Ter axis. Thus, the CODOs comprising relatively G/C rich genes required for rapid growth under conditions of high levels of negative DNA supercoiling are located in the vicinity of the origin, whereas those CODOs comprising relatively A/T rich genes less dependent on negative DNA supercoiling and required under conditions less favorable for growth are located closer to the terminus. In Dickeya, a more nuanced sequence organization of the genome is observed, demonstrating CODOs comprising genes coherently responding to different kinds of stress encountered during the infection process. It turned out that in D. dadantii various constellations of CODOs form transiently in response to particular environmental stress, whereby the CODOs demonstrate unique couplings between the dynamical and physicochemical properties of the expressed sequences, their functional content and the specific impacts of H-NS, FIS and IHF. Interestingly, while the mechanistic role of H-NS and FIS in the establishment of CODOs in D. dadantii remains unspecified, the establishment of CODOs by IHF was associated with the DNA supercoiling-dependent modulation of the directionality of transcription selecting the particular orientation of the transcribed genes [Recent studies highlighted the importance of the r traits ,107. Theed genes . Notablyed genes .E. coli fisP-YFP reporter construct in different CODOs in D. dadantii demonstrated that under various growth conditions, the reporter construct behavior reflected that of the targeted CODOs [E. coli chromosome. The quantification of reporter activities revealed a 20-fold variation in transcriptional propensity across the genome leading the authors to propose that this bacterium possesses reporter gene-independent mechanisms for regulating expression from specific chromosomal regions [Importantly, the targeted insertions of the ed CODOs . These oed CODOs ,107. The regions .E. coli and Bacillus subtilis cells grown in rich media, it was reported that the accumulation of RNA polymerase molecules forms dense aggregates, so-called transcription foci, mostly engaged in rRNA synthesis [As mentioned above, the relationship between chromosomal DNA supercoiling and transcription is defined as a “two-way street” , meaningynthesis ,124,125.ynthesis . A potenynthesis ,128 (FigHowever, this hypothesis was questioned by recent studies, which revealed that RNAP is able to form clusters in the presence of a limited number of rrn operons or in the absence of high levels of rRNA synthesis  and thatThere is growing evidence that the different levels of DNA organization play roles in transcriptional regulation and that, in turn, transcription affects chromosome organization ,2,3,4. T


The establishment, maintenance and dynamic regulation of three-dimensional (3D) chromatin structures provide an important means for partitioning of genome into functionally distinctive domains, which helps to define specialized gene expression programs associated with developmental stages and cell types. Increasing evidence supports critical roles for intrinsically disordered regions (IDRs) harbored within transcription factors (TFs) and chromatin-modulatory proteins in inducing phase separation, a phenomenon of forming membrane-less condensates through partitioning of biomolecules. Such a process is also critically involved in the establishment of high-order chromatin structures and looping. IDR- and phase separation-driven 3D genome (re)organization often goes wrong in disease such as cancer. This review discusses about recent advances in understanding how phase separation of intrinsically disordered proteins (IDPs) modulates chromatin looping and gene expression. Development of next-generation sequencing technologies, especially those based on high-throughput chromosome conformation capture (Hi-C)  and deri3D chromatin structure and genome folding are organized at different scales of genomic length ,31–35, w[48]) , and an unusual granular DNA staining pattern [Nipbl depletion leads to genome-wide disappearance of TADs [Loop extrusion is a widely recognized model to explain the establishment of CTCF/cohesin loops and TADs ,38–43. H,48)  . The WAP pattern ,48; on t of TADs , resembl of TADs ,34.FiguBesides loop extrusion, other mechanisms and molecular forces are also critically involved in 3D genome folding ,18–20,49Recently, the increasing amount of evidence points to critical roles of phase separation in determining and/or shaping the landscape of 3D chromatin structures ,23,50–57Phase separation refers to a type of weak, multivalent, and self-associating interaction that leads to the partitioning and condensation of biomolecules such as protein, DNA, and RNA and their complexes, without involvement of a sub-cellular membrane ,57,64–80in vitro setting, Gibson et al. elegantly demonstrated LLPS of chromatin, a process modulated by various factors such as linker DNA, linker histone, and post-translational modifications [First, phase separation has been proposed to be a driving force of compartmentalization ,23,50,51ications . In addiications . The obsications  and otheications  support ications ,90. BRD4ications ; in addiications . More reications .in vitro and form the clustering or puncta patterns in the nucleus [Loop extrusion has been an elegant and widely accepted model to explain key aspects regarding the generation of CTCF/cohesin loops and TADs; however, it does not specifically explain why merely a minor fraction of CTCF/cohesin-bound sites form loops ,20. Addi nucleus . Hansen  nucleus . Compari nucleus . RBRi-de nucleus . Thus, l nucleus . In anot nucleus . Cohesin nucleus . Further nucleus . These T nucleus . These oTranscription factors (TFs), chromatin modifiers, and cofactors often harbor IDRs, which can induce phase separation . A modelFinally, RNA represents another prominent class of contributing factors for 3D genome organization. RNA molecules such as those with N(6)-methyladenosine (m6A) modification ; a wide Recently, in-depth Hi-C/Micro-C mapping studies have demonstrated the existence of diverse classes of chromatin loops, which at least include cohesin/CTCF loops, long-range promoter–enhancer (E–P) loops, promoter–promoter (P–P) loops, and polycomb-associated contacts ,113. AltDistal regulatory elements such as enhancers play crucial roles in controlling gene transcription in a tissue- and cell type-specific manner. How exactly distal enhancers contribute to target gene expression has been a topic of intense investigation. In the classic E–P looping model, a tethering factor transiently brings promoter and enhancer into close proximity, thereby enhancing frequency of contacts between the two . Recent The presence of IDPs is a feature common to all organisms , and the abundance of disordered protein sequences increases proportionally with complexity of the organism, with approximately 52–67% of eukaryotic proteins carrying IDRs longer than 30 amino acids ,119. Unlin vitro [PBX3, HOXA and HOXB proto-oncogenes), and is also required for proto-oncogene activation and leukemogenesis [IDPs/IDRs are frequently involved in cancer-associated mutations such as aberrant fusion, suggesting a common oncogenic mechanism ,63. For in vitro ,120–122.in vitro ,121,122.in vitro . By mutaogenesis . An artiogenesis ), largelogenesis .Such a role of TF LLPS in enhancing loop formation can potentially be generalized to other cancer-related chimeras that display features similar to NUP98-HOXA9  Figure . Indeed,in vivo and in cells [LLPS of IDP can significantly influence the compartmentalization and biological function of interacting partners, as previously proposed in a scaffold–client model ,126. In in cells . A previin cells . Based oin cells . Furtherin cells , in a main cells . It meriIn addition to oncoproteins, IDR and phase separation also regulate the function of tumor suppressors. Recently, Shi et al. reported that an IDR within UTX  forms phase-separated liquid condensates . UTX actUTX represents one of a few tumor suppressors known to escape X inactivation and contribute substantially to a higher rate of cancer susceptibility seen with the male than the female . The homIt is generally thought that TADs are stable across diverse cell types and conserved across species ; howeverin vitro and form nuclear droplets in PSCs [OCT4  harbors  in PSCs ,132. Comin vitro; in animal models, compaction of PRC1 target chromatin also exhibited an IDR dependency [Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) represent a group of gene silencing-associated chromatin modifiers . There ependency . Evidencpendency ,145–150.pendency ,151–154.pendency ,155 or Cin vitro and mediate condensate formation in vivo. Furthermore, classic heterochromatic factors, such as HP1-alpha [As discussed above, emerging evidence shows that IDR-containing gene/chromatin-regulatory factors form condensates and are involved in generation of new chromatin loops, a process dependent on IDR and its LLPS-forming ability (as seen with NUP98-HOXA9  and UTX P1-alpha ,85, PRC1P1-alpha ,139,141,P1-alpha , also coP1-alpha ,55–57, wConceivably, the IDP-/LLPS-associated chromatin loops and the CTCF-/cohesin-formed structural TADs/loops operate independently and can cross-talk as well, in either a cooperative or antagonistic manner. How exactly these two 3D structure classes influence one another seems rather complex. On the one hand, it has been long postulated that CTCF-/cohesin-mediated structural TADs/loops can constrain the communication between a E–P pair separated by CTCF, which has gained much support from genetic manipulation studies of specific CTCF sites/loops ,117; meain vivo need to be developed. Also, how exactly the E–P contact/looping regulates gene expression remains to be more clearly determined in the future [Finally, it is worth mentioning that some previous studies of phase separation relied heavily on non-physiological over-expression and artificial systems, which represents a concern ,168,169.e future . For inse future . Furthere future ; however


Genetic information is loaded on chromatin, which involves DNA sequence arrangement and the epigenetic landscape. The epigenetic information including DNA methylation, nucleosome positioning, histone modification, 3D chromatin conformation, and so on, has a crucial impact on gene transcriptional regulation. Out of them, nucleosomes, as basal chromatin structural units, play an important central role in epigenetic code. With the discovery of nucleosomes, various nucleosome-level technologies have been developed and applied, pushing epigenetics to a new climax. As the underlying methodology, next-generation sequencing technology has emerged and allowed scientists to understand the epigenetic landscape at a genome-wide level. Combining with NGS, nucleosome-omics (or nucleosomics) provides a fresh perspective on the epigenetic code and 3D genome landscape. Here, we summarized and discussed research progress in technology development and application of nucleosome-omics. We foresee the future directions of epigenetic development at the nucleosome level. The nuclear genetic code of eukaryotes  depends on chromatin, which is a highly organized complex of DNA and proteins and is a principal component of the cell nucleus . What isChromatin is the carrier of genetic and epigenetic information about organisms. Under an optical microscope, at the 1400 nM length interval, we could observe some regular-shaped and compact chromosome packages called metaphase chromosomes, and sometimes, loose and seemingly irregular chromatin in other interphase cells . The chrDuring the 1970s, the nucleosome and nucleosome “beads on a string” model were first discovered and proposed a 21]. N. N21]. NAs is known, DNA methylation and histone modification are two important directions of classical epigenetic research a 25,26],2625,26]The packaging of eukaryotic genome into chromatin affects every process that occurs on DNA. Different cells have different nucleosome organization characteristics, and nucleosomes can occupy specific locations in some cells but not in others . The posWith the development of next-generation sequencing technology, it is possible to accurately locate nucleosomes at the genome-wide level and calculate the nucleosome occupancy rate of the whole genome .Micrococcal nuclease sequencing (MNase-seq) is a sequencing technology first developed to study nucleosome localization. This technology represents the starting point for nucleosome-omics technology . MNase iMNase is the endonuclease of choice when isolating nucleosomes from chromatin after the digestion of linker DNA. In history, MNase is widely used for the localization of nucleosomes . Once doMNase-seq and derivatives help scientists discover many phenomena that cannot be obtained by traditional optical microscopy. For instance, Ramachandran et al.  revealedThe genome-wide nucleosome mapping technologies of nucleosome positions, nucleosome occupancy, and chromatin accessibility mainly focus on the one-dimensional (1D) and two-dimensional (2D) levels of genome . How aboInterestingly, in 2015, Hsieh et al. created Micro-C technology , and by using it, mapped nucleosome resolution chromosome conformation in yeast  17]. Mi. Mi17]. Micro-C and its derivatives fill this gap by accessing the nucleosomal fiber at 100–1000 bp resolution . They reBenefitting from Micro-C technology, scientists have discovered many new structures. Technically, the main advantages of Micro-C and its derivatives are as follows. Firstly, Micro-C mainly measures the interaction between nucleosomes, the basic unit of chromatin, rather than the uneven size interactions of genome fragments that are interrupted and generated by interacting protein bodies after restriction enzymes digestion . This enIn addition to using Mnase digestion step as the key to study the chromatin landscape at the nucleosome level, classical epigenetic techniques DNase-Seq , ATAC-SeDeoxyribonuclease I (DNase I) is an enzyme that randomly cuts DNA at every site. Along with DNase I digestion and high-throughput sequencing, DNase I hypersensitive site sequencing (DNase-seq) was developed . In the Interestingly, other omics or biochemical reaction steps can be integrated to develop the brand new Micro-C-based technologies, which would be the trends in the future nucleosome level 3D genomic research. Hua et al.  developeThere is another interesting piece of research work. Masae et al.  establisWith the development and application of omics technology, the supporting bioinformatics analysis algorithms, software, and pipelines have also been gradually coded.Nucleosome positioning is an important piece for studying chromatin. The developing high-throughput sequencing further provides a way to analyze the genome-wide scale nucleosome positioning across different species and data types. A high resolution of nucleosome positions requires up to billions of reads order, which means the storage space will be terabytes . To collNucleosome positioning depends on cell type and state mainly. As another dataset of NGS analysis, the first steps for read mapping and quality control of nucleosome positioning sequencing data require standard NGS software . DivergiIn this paper, we reviewed the scientific discovery, development, and related research of nucleosomes, described the significance of studying chromatin nucleosomes and nucleosome-level DNA, then summarized genomics techniques and progress for studying epigenetic phenomena at the nucleosome level, including genomics and 3D-genomics technologies. The bioinformatics analysis tools and databases were also discussed in the review. We expect this review could enable readers to understand the field of nucleosome-omics, which would open a new perspective on the epigenetic code and 3D genome landscape.Finally, the future perspectives of nucleosome-level genomics technologies and their improvement directions are considered here. Towards scientific experiments, on the one hand, the combination of chromatin capture technology and nucleosome positioning technology helps to analyze the 3D interactions of nucleosomes at a more refined chromatin level and can better understand the relationship between chromatin structure and its function. Learning from the idea of the SPRITE technology , the mul


Just as biophysicists were identifying the component proteins of functional RNA polymerase II (RNAPII) complexes, virologists were identifying viral proteins that control it with peculiar efficiency . PurifieCLN genes (G1 cyclins) [In the early 90s, prior to discovery of Mediator, a cyclin of unknown function, cyclin C, identified in screens for the rescue of yeast deficient in cyclins) –6, was fcyclins) –9. At thcyclins) .CLN-deficient yeast and induced hyperplastic lesions in mice carrying its transgene [This same period saw the sequence of an acutely transforming oncogenic retrovirus, walleye dermal sarcoma virus (WDSV) . The WDSransgene ,13.At this point, many components of Mediator had been identified as parts of a large, thyroid-hormone receptor complex , and cofactor required for Sp1 activation (CRSP)) ,15, and The evolution of a viral cyclin that binds and activates the CDK component of Mediator illustrates the value of Mediator control for virus replication. While RV-cyclin is the only virus protein known to directly activate Mediator CDKs, the TADs of many viral transactivators function via direct contact with Mediator proteins, and additional virus protein contacts are being identified. Mediator offers refined control of host and virus gene expression, and viruses avail themselves of this control to meet their specific needs.RNAPII transcription is initiated by transcription factors bound to enhancer and promoter regions, recruitment of coactivator complexes that modify and remodel chromatin, and assembly of preinitiation complexes (PICs) with general transcription factors and RNAPII at core promoters. The Mediator complex, does just what it was named for: It mediates signals from transcription factors to assembled RNAPII complexes. Mediator is a bridge between a diverse array of DNA recognition factors and RNAPII. It also positions CDK7 at the RNAPII CTD for phosphorylation and transcription initiation –23. MediMediator is composed of approximately 30 proteins in 4 structural modules: the head, middle, and tail modules comprise the core, which can associate with the CDK8 kinase module (CKM) . The MedThe CKM includes the CDK8-activating cyclin, cyclin C, and Mediator proteins, Med12 and Med13. MED12 increases CDK8 activation, and MED13 links the CKM with the core ,34,35. ACDK8 activity regulates nutrient stress response genes in yeast ,46. In mEctopic RV-cyclin increases CDK8 kinase activity for histone H3 and RNAPII CTD in vitro. It also increases and extends the duration of CDK8 and RNAPII occupancy across the loci of serum response genes, prior to and after stimulation. This increases total mRNA levels from these CDK8-dependent genes and significantly increases cell proliferation ,54–56.DNA viruses and retroviruses depend on host transcription machinery and encode proteins that control the extent and timing of their gene expression. This allows coordination of virus replication with capsid construction and packaging. The TADs of viral factors make high affinity contacts with Mediator proteins to activate and maintain high transcription rates of virus genes . These vMany viruses require host machinery to replicate their genome. For small DNA tumor viruses, polyoma, papilloma, and adenoviruses, host–cell proliferation provides DNA polymerase components that replicate viral DNA. Proteins encoded by these viruses target pRb and p53 to trigger cell cycle and block apoptosis and may also contact Mediator to control host gene expression, e.g., E1A .The lymphotropic retroviruses and herpesviruses persist as integrated genomes or tethered episomes, so host cell proliferation replicates the viral genome. These viruses encode proteins that manipulate host transcription factors that contact Mediator tail proteins to promote cell division. Transition to virus production is also maintained via tight control of host transcription and requires Mediator.FOS, JUN, and EGR1. RV-cyclin increases CDK8 activity and chromatin occupancy of these genes to increase their rates of transcription elongation and reinititation [A refined interplay between host proliferative response and virus latency and lytic replication is modeled in the seasonal development and regression of WDSV. Latent provirus produces low levels of RV-cyclin and OrfB transcripts. OrfB protein constitutively activates protein kinase C and AKT  to activtitation . After atitation . This motitation .Viruses evolved many mechanisms to antagonize innate and adaptive immunity, especially interferon response pathways. Virus proteins that target transcription of key antiviral genes include those that contact Mediator. CDK8 and CDK19 regulate transcriptional responses to IFNγ ,65, and Orthobunyaviruses encode a nonstructural protein, NSs, which antagonizes the type I interferon response. Bunyamwera NSs binds Med8 in the head domain of Mediator to prevent phosphorylation of serine 2 in the RNAPII CTD and inhibit elongation of mRNA ,67. HumaVirus infection frequently results in reprogramming of host cell metabolism in ways that support virus replication. As indicated above, Mediator, and CDK8 in particular, are key regulators of metabolic reprogramming. Dengue virus enhancement of glucose metabolism and oxidative phosphorylation is well characterized –72. InfeThe question remains as to a specific role for NS5 and similar viral proteins in the direct control of host gene expression. There are many cell signaling pathways, triggered by virus infection and replication, which would result in metabolic reprogramming without benefit of direct virus control of Mediator. However, as we move into a new era of Mediator control of nuclear condensates to stabilize transcription and splicing components in response to cell signaling , we may