Maintenance of the individual patterns of gene expression in different celltypes is required for the differentiation and development of multicellularorganisms. Expression of many genes is controlled by Polycomb (PcG) andTrithorax (TrxG) group proteins that act through association with chromatin.PcG/TrxG are assembled on the DNA sequences termed PREs (Polycomb ResponseElements), the activity of which can be modulated and switched from repressionto activation. In this study, we analyzed the influence of transcriptionalread-through on PRE activity switch mediated by the yeast activator GAL4. Weshow that a transcription terminator inserted between the promoter and PREdoesn’t prevent switching of PRE activity from repression to activation.We demonstrate that, independently of PRE orientation, high levels oftranscription fail to dislodge PcG/TrxG proteins from PRE in the absence of aterminator. Thus, transcription is not the main factor required for PREactivity switch. Drosophila, these factors bind to DNAelements called PREs (Polycomb Response Elements). PRE elements contain sitesfor various DNA-binding factors, the recruitment of which results inassociation of PcG/TrxG complexes with PRE .All constructs were made on the basis of the CaSpeR vector containingte gene) . The enhite gene  was exciite gene  and inselacZ gene with the adhleader sequence and SV40 transcription terminator at 3'-terminus(GenBank: X81643.1) was inserted into a pBluescript SK+ vector cleaved by SmaIand SalI [LacZ-SV40-pSK].A fragment SmaI-SalI of 4324 bp in length from the plasmid vectorCaSpeR-hs43-lacZ carrying the hsp26 gene, 472 bp, was amplified by PCR(primers 5'-ctagaaacttcggctctctca-3' and 5'-gttgaatgaacttgtttgacttgt-3') andinserted into a pBluescript SK+ vector cleaved by EcoRV [hsp26- pSK]. AHindIII–PstI fragment of the hsp26-pSK vector was inserted into theLacZ-SV40-pSK vector at the SmaI site [hsp26-LacZ-SV40-pSK]. A fragmentNotI-SalI of the hsp26-LacZ-SV40-pSK vector was incorporated into theEn-white vector at the BamHI site[hsp26-LacZ-SV40-En-white].The promoter of the hsp70 gene and five GAL4 sites at 5' terminus was excised fromthe pUAST vector [28] and inserted into a pBluescript SK+-sce2 vector at theEcoRV site [sce(UAS)]. The coding region of a eGFP gene of 717bp was amplified by PCR (primers 5'-atggtgagcaagggcgaggagct- 3' and5'-cttgtacagctcgtccatgccga- 3') and cloned into the vector pBluescript SK+ atthe EcoRV site [eGFP-pSK].A fragment HindIII–EcoRI containing a minimal promoter of theA HindIII–EcoRI fragment of the eGFP-pSK vector was inserted in forwardorientation into the sce(UAS) vector at the HincII site [(UAS)sce-eGFP].A XbaI–BamHI fragment of 702 bp in length of the pUAST vector containinga transcription terminator was inserted into a pBluescript SK+-lox2 vectorcleaved by EcoRV [lox(SV40)]. A XbaI–XbaI fragment of the lox(SV40)vector was incorporated into the (UAS)sce-eGFP vector at the XhoI site [(UAS)sce-eGFP-lox(SV40)].linker1828bp-pSK]. A fragment XbaI–BamHI of 222 bp ofthe pGL3basic vector containing the SV40 transcription terminator was insertedinto the linker1828bp-pSK vector at the SmaI site[linker1828-SV40s-pSK].A HincII–HincII fragment, 1828 bp, of the LacZSV40- pSK vector wasincorporated into a pBluescript SK+ vector cleaved by EcoRV[ linker1828-SV40s-pSK at the EcoRV site [(UAS)sce-eGFP-lox(SV40)-linker1828-SV40s-pSK].A fragment NotI–BamHI of the (UAS) sce-eGFP-lox(SV40) vector was insertedinto the vectorbxdPRE of 656 bp(3R:16764122..16764777) was excised from the frt(PRE) vector [29] andincorporated into the vector(UAS)sce-eGFP-lox(SV40)-linker1828-SV40s-pSK at the AorI sitein forward [(UAS)sce-eGFP-lox-(SV40)-linker785frt(PREdir)linker1043-SV40s-pSK]or reverse [(UAS)sce-eGFP-lox(SV40)-linker785frt(PRErev)linker1043-SV40s-pSK]orientation.A fragment HincII–XbaI containing UDTPD construct. A XbaI–XbaI fragment of the(UAS)sce-eGFP-lox(SV40)-linker785frt(PREdir)linker1043-SV40s-pSK vector was incorporated into thevector hsp26-LacZ-SV40-En-white at the BamHI site.UDTPR construct. A XbaI–XbaI fragment of the (UAS)sce-eGFP-lox(SV40)-linker785frt(PRErev)linker1043-SV40s-pSK vector wasinserted into the hsp26-LacZSV40-En-white vector at the BamHI site.All details of the constructs design are available upon request.Drosophila melanogaster embryos andphenotypic analysis of yellow and whiteexpression in transgenic linesTransformation of P25.7wc, which served as a source of transposase 117/TM3,Sb, P; dSfmbt  ;PC to  were obtReal-time PCR with Hot-Start Taq DNA polymerase2, 1% Tween 20), 2 •µl of 25 mM MgCl2, 0.5•µl of 10 mM dNTPs, 1.5 •µl of each primer (at aconcentration of 5 pmol/ •µl), 0.25 •µl of SYBRGreen100× (Sigma), 0.3 •µl of Hot-Start Taq DNA polymerase(SibEnzyme), 11.45 •µl of mQ, 5 •µl of sample. Data wereassessed using the Bio-Rad CFX Manager software and Microsoft Excel. Decimaldilutions of Drosophila genomic DNA at a concentration of 0.1to 100 ng were used as reference standards. The primers used for real-time PCRanalysis of the material obtained using chromatin immunoprecipitation arepresented in Table.Real-time PCR was conducted using C1000tm ThermalCycler (Bio-Rad) in a 25•µl volume according to the following protocol (per one reaction):2.5 •µl of 10× buffer . In both constructs, the UAS promoter is directed towardsbxdPRE. However, the first construct (UDTPD) carriesbxdPRE in forward orientation, while in the second construct(UDTPR) bxdPRE is located in reverse orientation relative tothe UAS promoter .In order to suppress internal transcripts of the transgene, two SV40 terminatorswere used: upstream of the hsp26-lacZ gene and upstream ofthe white gene enhancer. An additional transcriptionterminator, SV40, was inserted at the 5’ side of bxdPREin order to block transcription from the UAS promoter.Two constructs were created containing bxdPRE and the SV40 terminator at the 5’side of bxdPRE in both constructs, were flanked by the LOX orFRT site for site-specific recombinases Cre or Flp, respectively. This approachallows one to excise in vivo the selected DNA fragments and tocompare the expression of the marker gene and functional changes in the systemin the presence or absence of key elements at the same genome position (sitesof transgene integration).Key elements, Fig. 1A) andthree lines for UDTPR were obtained with bxdPRE in repressed state.Repression of the white gene was enhanced in homozygous flies.This effect is characteristic of PRE elements and called PSS (Pairing SensitiveSilencing) [bxdPRE orientation. Deletion of the transcription terminatorlocated between the UAS promoter and PRE did not result in any phenotypicchanges. However, the induction of the UAS promoter by GAL4 led to derepressionof the white gene both in the case of terminator deletion andin intact lines. Thus, GAL4 inactivates bxdPRE in the studiedsystem regardless of orientation and presence of a terminator between the UASpromoter and bxdPRE.As a result of construct transformation, four independent transgenic lines forUDTPD (lencing) . The phebxdPRE does not lead toelimination of Polycomb and Trithorax group factors frombxdPRETranscription through bxdPRE if itis oriented forward in the transgene. We tested the influence oftranscriptional read-through in the case of reverse orientation ofbxdPRE. For this purpose, we conducted immunoprecipitation ofchromatin isolated from adult homozygous flies in the presence or absence ofGAL4 .Immunoprecipitation was carried out usingsamples obtained from the transgenic line UDTPR (№ 2) with a deleted SV40transcription terminator. Six areas of the construct were used for PCRanalysis: 1 –UAS promoter, 2 –eGFP gene codingregion, 3 –bxdPRE, 4 –LacZ genecoding region, 5 –white gene enhancer, and 6–white gene promoter. As a positive control, we used thegenomic region of bxdPRE adjacent to the element utilized intransgenic constructs, while the coding region of the Ras64Bgene was used as a negative control.We have previously shown that even robust transcription does not lead tocomplete elimination of PcG/TrxG complexes from Fig. 2A)and dSfmbt factors recruitment corresponds to bxdPRE in the transgene.Localization of these factors is consistent with the data according to which PHand dSfmbt are found predominantly in PRE elements but not in other regions ofthe repressed domain [It has been shown that the peak of the PH  and H3K27me3 modificationhas been found in the derivative of theUDTPR transgene. Introduction of a GAL4 activator did not lead to completeelimination of PC and H3K27me3, but there was a significant decrease in thelevel of their recruitment to bxdPRE and the surrounding areasof the transgene.Factor PC of the PRC1 complex specifically interacts with histone 3trimethylated at lysine 27 (H3K27me3) , 42, a mFig. 2E) and GAF.It was established that theinduction of transcription through bxdPRE leads to a 2-foldincrease in the recruitment of both factors to bxdPRE.We also analyzed the recruitment of the TrxG factors Trx are associatedwith the domain subjected to repression [invected, engrailed),which indicates the absence of a key role for transcription, at least in thefunctioning of several PRE elements [On the other hand, according to other data, non-coding RNAs from pression , 46. Morelements . bxdPRE does notlead to complete elimination of PcG/ TrxG factors but changes the ratio in thebinding of these proteins: recruitment of PcG decreases, while the recruitmentof TrxG increases. The transcriptional effect was analyzed in detail forbxdPRE incorporated into transgene in direct orientation[vg locus correlate withtranscriptional read-through from different DNA strands [bxdPRE orientation does not leadto a change in the transcriptional read-through effect. Recruitment of PcG/TrxGfactors is not abolished upon transcription. However, the recruitment of theTrxG proteins Trx and GAF increases, while the recruitment of PcG proteins  decreases.Previously, we tested the effect of transcription on GAL4-mediated activityswitch of PRE . As a reentation. At the  strands . TherefobxdPRE also does not prevent abolition of repression.Apparently, the GAL4-binding sites themselves are capable of neutralizingPRE-mediated repression and transcription though PRE does not play a crucialrole in this process.The presence of the strong terminator SV40 between the UAS promoter andPRE elements regulate genes the expression of which is changed duringdifferentiation and development. Thus, a particular gene must be expressed incertain cells at a certain stage of development, and then its expression shouldbe suppressed. Apparently, the recruitment of repressor factors to PRE inactivating state could be required for the quick PRE activity switch to therepressing state and to abort the expression of the target gene at a certainmoment in time. A logically similar mechanism has been described for manyeukaryotic promoters: pausing of RNA polymerase II. In this case, RNApolymerase II binds to a transcriptionally inactive promoter and, if necessary,quickly triggers transcription.The mechanism that allows the recruitment of proteins to PRE duringtranscriptional read-through is unclear. A series of DNA-binding factors withzinc finger motifs are known to be associated with PREs. It is possible thattranscription does not interfere with direct DNA-protein contacts. On the otherhand, there is a possibility that retention of complexes at PRE duringtranscriptional read-through is mediated by the contacts between the PcG/TrxGfactors and histone proteins. In accordance, PcG proteins contain domainscapable of interacting directly with nucleosomes  , 49, 50


Using four datasets of condition-dependent genes from RNA-seq and RT-qPCR experiments, we performed unsupervised motif discovery and found two short motifs resembling the proposed binding consensus for XYR1 and CRE1. Using these motifs, we analysed the presence and arrangement of putative cis-regulatory elements recognized by both regulators and found that shortly spaced sites were more associated with XYR1- and CRE1-dependent promoters than single, high-score sites. Furthermore, the approach used here allowed the identification of the previously reported XYR1-binding sites from cel7a and xyn1 promoters, and we also mapped the potential target sequence for this regulator at the cel6a promoter that has been suggested but not identified previously. Additionally, seven other promoters  presented a putative XYR1-binding site, and strong sites for CRE1 were found at the xyr1 and cel7b promoters. Using the cis-regulatory architectures nearly defined for XYR1 and CRE1, we performed genome-wide identification of potential targets for direct regulation by both proteins and important differences on their functional regulons were elucidated. Finally, we performed binding site mapping on the promoters of differentially expressed genes found in T. reesei mutant strains lacking xyr1 or cre1 and found that indirect regulation plays a key role on their signalling pathways. Taken together, the data provided here sheds new light on the mechanisms for signal integration mediated by XYR1 and CRE1 at cellulase promoters.In this work, we report the  Trichoderma reesei is a filamentous fungus extremely relevant to biotechnology due to its remarkable capability to produce a wide number of cellulolytic enzymes T. reesei has been extensively studied in the past decades as a model of cellulases and hemicellulases producing organism T. reesei with enhance enzyme production levels, a great interest has been to elucidate the molecular mechanisms operating at the transcriptional network that controls the expression of cellulase genes in response to the cognate environmental conditions cis-regulatory elements located in the region occupied by the nucleosome T. reeseiIn order to allow the ration engineering of new strains of T. reesei has allowed the identification of several TFs related to each of the mechanisms described above. For instance, the XYR1 (xylanase regulator 1) transcriptional factor is the main positive regulator of cellulase expression in T. reeseiAspergillus niger and Neurospora crassaSaccharomyces cerevisiaeT. reeseiThe investigation of the regulatory network for cellulase expression in T. reesei clearly evidence sophisticate interplay between the characterized and unknown TFs at the target promoters to provide fine-tuning of enzyme production levels in this organism cis-regulatory elements at each particular promoter are only beginning to be elucidated cis-regulatory elements associated with the interaction of XYR1 and CRE1 with their target promoters is crucial to improve engineering attempts to construct new cellulase responsive promoters T. reesei at the global scale T. reesei cells grown in the presence of inducers (cellulose and sophorose) and a repressor (glucose) carbon sources. Separated analysis of the different datasets allowed the identification of new DNA motifs that were associated with the regulation by XYR1 and CRE1 in this organism. The nearly revealed motifs were used to generate, by the first time, Position Weight Matrixes (PWMs) representing the putative cis-regulatory elements recognized by both regulators. Furthermore, these PWMs were used to inspect the architecture of target promoters in co-expressed genes at the genomic scale, allowing the identification of the functional regulons of XYR1 and CRE1 in T. reesei. The PWMs generated here were able to successfully identify two previously characterized cis-regulatory elements at the cel7a and xyn1 promoters cel6a promoter that agrees with previous in vivo data T. reesei at the genomic scape, revealing some important differences with other filamentous fungus Current available experimental data related to cellulase regulation in cis-regulatory elements was performed using four groups of co-regulated genes identified using different experimental setups. Three groups represent genes encoding TFs that were identified using RNA-seq S3 from Supporting Information. The fourth group is represented by 22 cellulases-encoding genes whose promoters are regulated by the XYR1 regulator T. reesei available at the JGI homepage (http://genome.jgi-psf.org/Trire2/Trire2.home.html) using ad hoc Perl scripts. These sequences are expected to contain the cis-regulatory elements for the different TFs acting on each gene. The four groups of fasta sequences where then used to identify conserved DNA motifs as described below. The overall approach used in this work is represented in The analysis of cis-regulatory elements in the four regulons assayed, we analysed the promoter sequences using the MEME tool T. reesei were selected. In cases where similar DNA consensus for the regulators appeared on different motif outputs, the aligned sequences were merged to create a single motif dataset. The resulting datasets were used to construct Position Weight Matrixes (PWM) by extracting the information content of the sequence alignments as described previously XYR1 for XYR1 and PWMCRE1 for CRE1 regulators) were used for further analysis.In order to identify new T. reeseiT. reesei were analysed to identify the best motif for both PWMs on each promoter. Next, the same dataset was re-analysed to identify multiple motifs per promoter with a score above a specific threshold, which was set to 6.2 for PWMXYR1 and 8.0 for PWMCRE1. The resulting identified sites were then analysed to identify adjacent cis-regulatory elements located within short distances (lower than 30 bp) with architectures similar to previously related functional sites for XYR1 and CRE1 regulators Δxyr1 and Δcre1 mutants of T. reesei, were inspected as described above to determine genes potentially and directly regulated by XYR1 (in the case of cellulose and sophorose growth conditions) and CRE1 (for glucose growth).The two PWMs generated using the motifs discovered with MEME were used to analyse the promoters of all annotated genes in the genome of cis-regulatory elements of XYR1 and CRE1 in T. reesei, we used four different datasets of co-regulated genes to search for short repetitive DNA motifs potentially recognized by these regulators. For this, we used RNA-seq data from T. reesei cells grown on cellulose, sophorose and glucose as sole carbon sources S3). It is worth mentioning that we focussed on TF-encoding genes since they could mediate the regulation by XYR1 or CRE1 in an indirect way T. reesei lacking a functional xyr1 gene T. reesei  that resembled the reported consensus sequence for CRE1 Fig. 2C.T. reesei, we decided to decipher how these regulators recognize their target promoters. While several works have tried to understand this process at the global scale, a unified model for protein-DNA interaction for these regulators is still not available After identifying the putative binding consensus of XYR1 and CRE1 in T. reesei genome  and repression mediated by a preferred carbon source such as glucose in vivo and in vitro evidence for direct interaction between XYR1 and CRE1 have been reported for promoters such as cel7axyn1cel6axyr1 gene, since CRE1 has been reported to affect its expression cel7b promoter displayed two sites that were shortly spaced and located about 600 bp upstream of the gene start codon  between positions −234 and −245 that is essential for the induction of this promoter in response to the presence of cellulose and sophorose cel6a promoter, Zeilinger and colleagues (2003) found that the CAE is located in a nucleosome-free region and that proteins binding to this element controls the assemble of a nucleosome (named nucleosome −1) covering a region from −192 to −49 that includes the TATA-box cel6a promoter in response to cellulose and sophorose. However, the target sequence of the XYR1 regulator at this promoter has not yet been identified, although the available data suggest that it would bind somewhere downstream the CAE region cel6a promoter at the position −160 to −184, which agrees perfectly with the current available information on the regulation of this promoter. It is worth to notice that everted binding sites are recognized by other zinc finger proteins cel6a promoter architecture, including the nearly identified potential XYR1 binding site (named UASXYR1) and the cis-regulatory elements characterized previously Comparison of the binding sites found cel7a promoter revealed an element that passed the stringent used criteria . Quite surprisingly, the searching for CRE1 binding sites at the xyr1 promoter retrieved just a cis-regulatory element  and 310 candidates for the latter (Table S5). Next, we classified the identified genes according to their functional categories (KOG), and then we compared the regulons to identify the difference in the functional scope of both regulators. As shown in in vivo expression data, as is described below.Once we defined the T. reesei, we carried out a search for binding sites of these proteins in 6 sets of genes differentially regulated under different growth conditions. For this, we searched for XYR1 sites in the promoters of genes up and down regulated under growth in the presence of cellulose and sophorose in a strain of T. reesei lacking the functional xyr1 gene, as determined through RNA-seq experiments . In the same way, we surveyed CRE1 sites in promoters of genes up and down regulated during growth on glucose in a strain lacking the cre1 gene . Using these datasets, we found that between 13.6 and 15.8% of the genes differentially regulated in the wild type and xyr1 minus strain presented a putative XYR1 binding site. In the case of the cre1 mutant experiments, between 8.9 and 13.9% of the promoters were endowed with a putative binding site for CRE1. The list of genes identified using this analysis along with their expression values determined using RNA-seq is provided in the Supporting Information (S11). Taken together, these data strongly indicated that indirect regulation plays an important role on control of target genes by XYR1 and CRE1 proteins in T. reesei.In order to get an insight into the functioning of the regulon of XYR1 and CRE1 in T. reeseiT. reesei, eluding the bias generated by using consensus sequences determined in other organisms cel7a and cel6a promoters as well as a strong CRE1 site in the xyr1 regulatory region Click here for additional data file.Tables S2Dataset of TFs up regulated in sophorose growth condition.(PDF)Click here for additional data file.Tables S3Dataset of TFs up regulated in glucose growth condition.(PDF)Click here for additional data file.Tables S4Genome-wide prediction of XYR1 binding sites in T. reesei.(PDF)Click here for additional data file.Tables S5Genome-wide prediction of CRE1 binding sites in T. reesei.(PDF)Click here for additional data file.Tables S6Prediction of XYR1 binding sites on genes down regulated in a Δxyr1 mutant induced withcellulose.(PDF)Click here for additional data file.Tables S7Prediction of XYR1 binding sites on genes up regulated in a Δxyr1 mutant induced withcellulose.(PDF)Click here for additional data file.Tables S8Prediction of XYR1 binding sites on genes down regulated in a Δxyr1 mutant induced withsophorose.(PDF)Click here for additional data file.Tables S9Prediction of XYR1 binding sites on genes up regulated in a Δxyr1 mutant induced with sophorose.(PDF)Click here for additional data file.Tables S10Prediction of CRE1 binding sites on genes down regulated in a Δcre1 mutant induced withsophorose.(PDF)Click here for additional data file.Tables S11Prediction of CRE1 binding sites on genes up regulated in a Δcre1 mutant induced with sophorose.(PDF)Click here for additional data file.


RSC and ISW1a largely co-localize, and genomic nucleosome studies using rsc isw1 mutant combinations revealed opposing functions: promoters classified with a nucleosome-deficient region (NDR) gain nucleosome occupancy in rsc mutants, but this gain is attenuated in rsc isw1 double mutants. Furthermore, promoters lacking NDRs have the highest occupancy of both remodelers, consistent with regulation by nucleosome occupancy, and decreased transcription in rsc mutants. Taken together, we provide the first genetic and genomic evidence for RSC-ISW1a antagonism and reveal different mechanisms at two different promoter architectures.ISWI family chromatin remodelers typically organize nucleosome arrays, while SWI/SNF family remodelers (RSC) typically disorganize and eject nucleosomes, implying an antagonism that is largely unexplored in vivo. Here, we describe two independent genetic screens for DOI:http://dx.doi.org/10.7554/eLife.06073.001 The genome of an organism can contain hundreds to thousands of genes. However, these genes are not all active at the same time. Instead, mechanisms exist that control when genes are switched off or on. One such mechanism alters how tightly DNA is packaged into a structure called chromatin. To form chromatin, DNA is wrapped around histone proteins at different points along its length; these structures are known as nucleosomes. Once formed, chromatin can either adopt a tightly packed form that represses gene activity or a less compact form associated with gene activation.The proteins that control how chromatin is packed are called ‘chromatin remodelers’. These proteins work in complexes that either disassemble chromatin—for example, by repositioning nucleosomes or removing histones—or organize chromatin by replacing nucleosomes and making it more compact.Saccharomyces cerevisiae. The results suggest that this is indeed the case.Studies in many organisms have identified two key families of chromatin remodelers. In yeast, the ISWI family of complexes assembles chromatin and a protein complex called RSC disassembles chromatin. Parnell, Schlichter et al. used a range of genetic techniques to investigate whether these two chromatin-remodeling complexes work against each other in a species of yeast called Both the ISWI complex and the RSC complex bind to regions of DNA called promoters, which are found at the start of a gene and help to regulate its activity. Parnell, Schlichter et al. found that the RSC complex helps to activate genes by establishing or maintaining regions of nucleosome-poor chromatin at a promoter. The chromatin is relaxed in these regions, which allows the proteins that activate genes to access the DNA. This effect is partially counteracted by the ISWI complex, which repositions nucleosomes across the promoters to fill the gaps created by the RSC complex.In comparison, Parnell, Schlichter et al. found that promoters that do not have regions of nucleosome-poor chromatin contain a larger number of both of the remodeling complexes and have a high turnover of histone proteins. This suggests that at these sites, the RSC proteins are needed to overcome the assembly of nucleosomes by the ISWI complex in order to activate the gene. Thus, these two chromatin remodelers, ISWI and RSC, compete at promoters to determine whether they contain or lack nucleosomes, which helps determine whether the gene is silent or active, respectively. Future work will look further at how the ‘winner’ is determined: how transcription factors or signaling systems within the cell bias the recruitment or activity of RSC or ISWI at particular promoters, to determine gene activity.DOI:http://dx.doi.org/10.7554/eLife.06073.002 Genomic DNA is packaged into chromatin, a dynamic material that exhibits numerous changes in post-translational modifications, composition, and protein interactions. One aspect of chromatin modulation involves the assembly or disassembly of chromatin through active remodeling, which can confer either occlusion or access to the DNA—a process that is associated with virtually all DNA-mediated transactions, including transcription, replication, and repair. Each remodeling action, either assembly or disassembly, is mediated (in part) by specialized ATP-dependent chromatin-remodeling complexes .Certain chromatin remodelers align with these two general categories: those that restrict DNA access by chromatin assembly and organization and those that promote DNA access by chromatin disassembly and disorganization. This broad separation in function can be partially illustrated by studies of individual chromatin remodelers and their effects on gene expression ; in geneThe SWI/SNF family of chromatin remodelers provides a well-studied example of remodelers associated with nucleosome disorganization and/or disassembly. In yeast, the RSC chromatin-remodeling complex is an essential and abundant paralog of the canonical SWI/SNF remodeler . The cenISW1 and ISW2, related to the Drosophila ‘Imitation SWitch’ (ISWI) protein, which is the catalytic component of multiple chromatin-remodeling complexes with roles in nucleosome assembly and gene repression . Double mutants were isolated and screened for growth at a restrictive temperature. Four viable combinations were obtained, including combinations of rsc7Δ with rpl20bΔ, lsm7Δ, bud31Δ, or isw1Δ  and RSC. Suppression was linked to isw1Δ, as the rsc7Δ temperature sensitivity returned with the introduction of a wild-type copy of ISW1 on a plasmid  that was covered by a URA3-marked plasmid bearing wild-type histones, HHT2-HHF2. We then introduced TRP1-marked plasmids containing hydroxylamine mutagenized HHT2-HHF2 genes and screened for suppression of the temperature sensitivity phenotype upon loss of the wild-type histone plasmid (using 5-FOA negative selection). From 20,000 transformants screened, we isolated seventeen suppressors that were verified by isolating and retransforming the plasmid containing the histone mutation. Of these, most contained single mutations: eight had either H3 A7V or H3 A7T mutations, seven had an H3 T6I mutation, and one bore an H3 G33V mutation. However, one mutant bore an H4 RH17,18CY double mutation , suggesting that these mutations generally suppress RSC defects and not a specific defect of rsc2-V457M. We focused on the H4 RH17,18CY mutant for subsequent studies as it caused the most robust suppression  of ISW1 , suggesting that they act through the same pathway. We also directly tested whether the ISWI paralog, ISW2, might also suppress rsc2 alleles. Combining the isw2Δ mutation with rsc2-V457M or rsc2-D461G did not confer suppression of the temperature growth defect, although some partial suppression of other phenotypes was observed (isw1Δ and isw2Δ were combined (data not shown). We therefore conclude that the rsc2 mutation suppression is due primarily to the loss of Isw1 activity, with minimal contributions from of loss Isw2 activity.The results of these two genetic screens strongly suggested a functional antagonism between RSC and Isw1. To directly test ormamide  as well ormamide . Growth  of ISW1 . Furtherobserved . We alsoisw1Δ suppression was specific to rsc2 and rsc7 mutations or could extend more generally to RSC mutations. To test this, we combined isw1Δ with two additional RSC mutations in separate subunits, rsc3-3 and rsc4-2. The isw1Δ allele suppressed both RSC mutations tested , and conversely, synthetic lethality with rsc2 set1Δ combinations, suggesting that H3K4me3 either promotes or partially bypasses RSC activity . These results suggest that suppression by loss of Isw1a is epistatic to the effects of Set1 loss and can overcome the reliance of RSC on H3K4 methylation.Our prior work revealed moderate activity . Howeveractivity , an alteethality . We alsoGCN5 is a histone acetyltransferase responsible for much of the H3K14ac in vivo , using the RSC subunit Rsc8 and the ISW1 subunit Ioc3, both tagged with C-terminal Myc epitope tags. We chose the Rsc8 subunit of RSC because it exists as a dimer in the RSC complex, minimizing the low ChIP efficiency observed with chromatin remodelers . We analRSC occupancy was scored across gene promoters (−800 to +800 bp), and promoters were then sorted into six clusters using a k-means algorithm to visualize those with and without enrichment . Using tIn comparison to RSC, the Ioc3 enrichment was less robust, perhaps reflecting a difference in chromatin association or difficulty in capturing complexes. We identified 137 or 230 Pol II promoters at an FDR of 1% or 5%, respectively. Strikingly, 224 of these latter promoters also pass the 1% threshold for RSC enrichment. Visual comparison of the enrichment pattern (log2 fold ChIP/Input) across all Pol II promoters reveals a high degree of overlap , while aTo extend these results, we repeated Rsc8, Ioc3, and Sth1 (the ATPase subunit of RSC) ChIP using micrococcal nuclease-digested chromatin analyzed by paired-end sequencing. We compared the log2 fold enrichment values obtained from both microarray and sequencing technologies . Despitersc2) may result simply in an attenuation of transcription of many or all genes, leading to a general phenotype such as temperature sensitivity. This transcription attenuation, as well as any suppression by ISWI, should be evident by expression analysis. To determine whether this suppression is global in nature or restricted to a subset of genes, we performed a HybMap analysis on a sampling of genes in the genome. The HybMap technique measures both sense and anti-sense RNA levels across a genome . We also did not observe general aberrant transcription from promoters as reported previously in an rsc3 mutant  commonly found near the TSS of genes  and paired-end sequencing (rscΔ only). As a reference, we also analyzed mono-nucleosomal DNA from control strains that cannot degrade Sth1 protein. To analyze the chromatin structure around the TSS, we generated nucleosome profiles around the TSS for every promoter by scoring the nucleosomal occupancy for rscΔ and RSC strains. Promoters were organized into clusters based on their rscΔ/RSC ratio profile using a k-means algorithm . There is also a clear ‘leftward’ shift in nucleosome positions over the body of the gene, towards the NDR. This nucleosome shift is particularly prominent in clusters 3 and 4, while the NDR is filled into a lesser extent and the +1 nucleosome peak is not as depleted (compared to cluster 1). To confirm that these effects were not due to limitations in the sensitivity and resolution of microarray analyses, the nucleosome profiles from the microarray data were directly compared to those from the sequencing analysis .Above, we showed that nucleosome architecture at clusters 1–4 shows a strong response to RSC loss, whereas clusters 5 and 6 show apparently limited changes. Clusters 1–4 display a prototypical promoter nucleosomal architecture . In contrast, clusters 5 and 6 lack this stereotypical organization; here, RSC and/or ISW1a may indeed impact nucleosome occupancy and/or positioning, but the effect may be obscured due to architectural heterogeneity. Notably, these two types of architectures have previously been designated as open (or structured) vs closed (or unstructured) and have been largely correlated with either constitutive or highly regulated gene types, respectively . We verirsc2 mutants (consistent with isw1Δ mutants showed little impact on the bulk expression of these genes (rsc2Δ (isw1Δ) are seen with ‘open’ promoters, the largest effects on transcription are more closely associated with ‘closed’ promoters . Here, we plotted the mean occupancy profile for both Rsc8 and Ioc3  across tTogether, these observations coalesce around the idea that these gene clusters identified in cis regulatory sequences. While there are other remodelers that also act at promoters, we consider the interactions described herein as the strongest evidence to date exemplifying chromatin remodeler antagonism.Chromatin remodelers represent a set of complexes with different functional roles; some remodelers are primarily involved in transcriptional activation, while others are more dedicated to chromatin assembly and/or transcriptional repression. Here, we describe an antagonistic relationship between two such chromatin remodelers, RSC and ISW1, through a combination of genetics, gene expression, and genome-wide nucleosome positioning studies. At genes, RSC is primarily utilized for gene activation, providing this function, at least in part, by establishing or maintaining the NDR structure at promoters. We find that this function is partly counteracted by Isw1 activity, which re-positions nucleosomes to ‘fill in’ the NDR and positions nucleosomes over rsc7Δ and revealed isw1Δ as the strongest of four identified gene suppressors and the only gene with a chromatin-related function. Indeed, combinations of rsc mutants with mutations in chromatin factors are almost invariably lethal . The seand Dot1 . Furthergcn5Δ mutations combined with loss of another H3 acetyltransferase, Sas3  demonstrating antagonism between these remodelers regarding the depth of the NDR, the occupancy and positioning of the +1 nucleosome, and the phasing of proximal nucleosomes in the coding region  and crossed in, or made by PCR disruption, and confirmed by PCR and complementation.Rich media (YPD), synthetic complete (SC), minimal synthetic defined (SD), and sporulation media were prepared by standard methods. Standard procedures were used for transformations, sporulation, and tetrad analysis. All strains are derivatives of S288C, and full strain genotypes are listed in rsc2 TS− mutant, p1411 [HHT2-HHF2.TRP1] was mutagenized with hydroxylamine and transformed into YBC2140 (rsc1Δ rsc2-V457M hht1Δ-hhf1Δ hht2Δhhf2Δ [HHT2-HHF2.URA3]). Approximately 20,000 transformants were plated to SC-TRP + 5FOA medium, incubated at 33°C, and screened for colony growth. Resident plasmids conferring suppression were isolated, retransformed, and sequenced.To isolate mutations in Histone H3 or Histone H4 that could suppress an rsc7Δ [RSC7.URA3] (YBC2039) with the yeast haploid deletion set (BY4741) from Invitrogen and isolating double mutants as described in RSC7 plasmid loss on 5FOA.The SGA screen was performed by mating RSC8, SNF2, and IOC3 genes were tagged endogenously with 13xMyc tags as described  using Illumina sequencers.The HybMap microarray was custom designed to represent genes with a range of RSC and ISW1 occupancies and included 448 coding genes, 93 tRNA genes, and 52 non-coding genes. Gene regions were extended by either 300 bp (coding and non-coding) or 150 bp (tRNA). Probes were selected from a pool of tiled 60 mers and adjusted for length to match melting temperatures as necessary. Both strands for each probe were included in the design. Probes have a mean spacing of ∼50 bp. As a control, 502 probes with sequences from zebrafish were included as non-hybridizing control probes; sequences were confirmed not to have significant homology to yeast sequences. Microarray designs were submitted to Agilent Technologies for production as 4 × 44K arrays. Total RNA from three biological replicates was prepared from each yeast strain, hybridized to the array, and detected as described .Yeast strains were grown under degron-inducing conditions, and mono-nucleosomal DNA was isolated as described previously . DNA fraSaccharomyces cerevisiae genome version 64 (Saccharomyces Genome Database). Gene transcript models were based on whole-genome transcriptome data (https://github.com/taoliu/MACS) to generate fold enrichment data.Raw microarray data were quantile normalized, averaged, median scaled, and assigned to genomic coordinates. For the HybMap protocol, probe values were median scaled to the median intensity from the zebrafish control probes. Probe sequences were mapped to the ome data . Transcrhttps://github.com/tjparnell/biotoolbox). Cluster analysis was visualized with Java Treeview . Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and three reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for sending your work entitled “RSC and ISW1 Chromatin Remodelers Display Functional and Chromatin-based Promoter Antagonism” for consideration at The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.rsc mutants, the authors discovered that another remodeling protein, Isw1, specifically ameliorates the rsc mutant phenotypes. Nucleosome mapping studies revealed global effects of RSC and ISW1a complexes acting against each other in shaping the promoter chromatin architecture. This work appears to be a promising candidate for eLife. There are, however, a number of specific questions and concerns that need to be addressed.This paper addresses the functional antagonism between the RSC and ISW1a remodeling complexes, a subject that is of emerging interest in the chromatin field. By using a synthetic genetic array (SGA) approach to screen for suppressors of ioc3Δ or isw1Δ, the authors should also reveal the corresponding nucleosome maps so that readers can assess the relationship between nucleosome occupancy/position and transcription, and whether there is any correlation between the chromatin and transcription in this respect. Whatever the correlation, the issue should be articulated in the text, and possible explanations provided. Likewise it would be useful to know where the functionally affected genes fall in the categories of open and closed promoters. Do any rules emerge, or is it just random?1) It was somewhat surprising that the general changes in nucleosome arrangement were accompanied by very few changes in RNA levels . For the2) There appears to be a contradiction between 3) The summary diagram  1) It was somewhat surprising that the general changes in nucleosome arrangement were accompanied by very few changes in RNA levels . For the few genes  that do show clear gene expression changes upon suppression by ioc3Δ or isw1Δ, the authors should also reveal the corresponding nucleosome maps so that readers can assess the relationship between nucleosome occupancy/position and transcription, and whether there is any correlation between the chromatin and transcription in this respect. Whatever the correlation, the issue should be articulated in the text, and possible explanations provided. Likewise it would be useful to know where the functionally affected genes fall in the categories of open and closed promoters. Do any rules emerge, or is it just random?isw1 or ioc3. Ideally, one could link changes in nucleosome positioning to expression by using high-resolution data to show that a nucleosome has moved with respect to an important cis-controlling element, and then infer that the movement might underlie the expression change. However, our profiles are derived from a microarray platform, and although these arrays proved to be very reproducible and provide great ‘class average’ maps of gene/promoter cohorts, their resolution does not allow us to state with confidence that a single nucleosome underlies the expression change. This is indeed a limitation worth discussing, so we have added text in the Results section to reflect this limitation, and we have also included how these genes partition into the open and closed categories. This is one reason why we did not focus our paper on these single genes, but instead focused the work on our clear genetic and genomic (cohort average) evidence for antagonism between these two Remodeler families and how this interplay affects promoter/gene architecture at cohorts of genes. Future work by us and others will indeed address their interplay at particular genes.As requested, we examined the genic nucleosome profile analysis for the 20 genes identified as suppressed by either 2) There appears to be a contradiction between. In, it appears that clusters 5 and 6 have the lowest RSC occupancy, whereas in, it appears that clusters 5 and 6 have the highest RSC occupancy. This point needs to be clarified.In retrospect, we see how this was a major source of confusion, but it is easily explained and corrected. The six clusters in 3) The summary diagram“Open” can be easily interpreted, but“Closed” is confusing.We agree, and have revised the right side of the diagram figure fairly extensively. We have removed some of the action arrows with the same color as the remodeler, as we believe this was confusing as to what function these arrows portray. Instead, all action arrows are the same color as the nucleosomes to indicate what direction nucleosomes are moving.


Intracellular zinc concentration needs to be maintained within strict limits due to its toxicity at high levels, and this is achieved by a finely regulated balance between uptake and efflux. Many bacteria use the Zinc Uptake Regulator Zur to orchestrate zinc homeostasis, but little is known regarding the transport of this metal across the bacterial outer membrane.Caulobacter crescentus Zur regulon by global transcriptional and in silico analyses. Among the genes directly repressed by Zur in response to zinc availability are those encoding a putative high affinity ABC uptake system (znuGHI), three TonB-dependent receptors  and one new putative transporter of a family not yet characterized (zrpW). Zur is also directly involved in the activation of a RND and a P-type ATPase efflux systems, as revealed by β-galactosidase and site-directed mutagenesis assays. Several genes belonging to the Fur regulon were also downregulated in the zur mutant, suggesting a putative cross-talk between Zur and Fur regulatory networks. Interestingly, a phenotypic analysis of the znuK and znuL mutants has shown that these genes are essential for growth under zinc starvation, suggesting that C. crescentus uses these TonB-dependent outer membrane transporters as key zinc scavenging systems.In this work we determined the C. crescentus Zur regulon showed that this regulator coordinates not only uptake, but also the extrusion of zinc. The uptake of zinc by C. crescentus in conditions of scarcity of this metal is highly dependent on TonB-dependent receptors, and the extrusion is mediated by an RND and P-type ATPase transport systems. The absence of Zur causes a disturbance in the dynamic equilibrium of zinc intracellular concentration, which in turn can interfere with other regulatory networks as seen for the Fur regulon.The characterization of the The online version of this article (doi:10.1186/1471-2164-15-734) contains supplementary material, which is available to authorized users. Zinc, as it occurs with other transition metals, entails a paradox to living cells. It is a scarce micronutrient in the environment, essential for a substantial number of biological processes . On the Escherichia coli is regulated by the MerR-like transcriptional regulator ZntR  and later for C. glutamicum [09]. Hence, as in those bacteria, Zur is a transcription factor responsible for direct regulation of both zinc uptake and extrusion in C. crescentus.Studies performed with many other bacteria have already described that high affinity-ABC transport systems are regulated by Zur and, more recently, studies using transcriptomics analysis , 32, 33  systems , 12. Thein vitro binding of Zur to the promoter of CC2720-26 operon, but the existence of a Zur-binding site on this promoter was confirmed by site-directed mutagenesis. Although all Zur-binding sites in promoters of Zur-repressed genes overlapped the promoter elements as typically observed for transcriptional repressors, the position of the Zur-binding site in CC2720-26 was upstream of the −35 promoter element in agreement with its role as a positive transcriptional regulator. In the regulatory region of CC2720-26 there is an imperfect inverted repeat 11-4-11 (GCCGGATGCGA-4 N-TCGCAACCGGC) located 13 nt upstream of the Zur-binding site. In X. campestris, Zur activates a gene encoding a transporter belonging to the CDF family in response to zinc, and an imperfect inverted repeat of 20-bp is found on the promoter region which is essential for binding of Zur both in vivo and in vitro, indicating that Zur recognizes simultaneously that inverted repeat and the Zur binding sequence [08]. We speculate that the inability of C. crescentus Zur to bind the CC2720-26 promoter in vitro could be due to the requirement for an accessory protein that binds to the inverted repeat sequence and stabilizes Zur binding.It was not possible to obtain in silico analysis revealed that most of the genes downregulated in MM69 strain with no predicted Zur-binding sites belongs to the Fur regulon, being repressed by iron-bound Fur in the presence of iron , Yersinia pestis respectively were cloned in tandem into the suicide vector pNPTS138 (a kind gift of D. Alley), and the construction was used for deletion of zur, generating strain MM69. Gene deletion was confirmed by both PCR and Southern blotting (not shown). Complementation of the zur mutation was obtained by introduction of plasmid pUJ142  were aligned in order to identify conserved sequences, which were in turn used to perform a genome-scale screening for putative Zur-binding sites in the  website . Additio website . Sequenc website .EcoRI/BamHI fragment into pRKlacZ290. This construction was then used for β-galactosidase activity assays.Site-directed mutagenesis of the CC2720 promoter region was performed using primers CC2720-F (5′-GCGATTGGCTAACG-3′) and CC2720-Zur-boxmut-R (5′-AATCTGTTACTTCGCGGTTGCGCGCCGCGCG-3′), CC2720-Zur-boxmut-F (5′-CGCGCGGCGCGCAACCGCGAAGTAACAGATT-3′) and CC2720-R (5′-GACCAACGCAACCAAG-3′) as described previously for Fur-box mutagenesis . The resC. crescentus zur coding region was amplified from genomic DNA by PCR, using primers Zur-expresstag1 encoding a 5′-FLAG sequence (5′- TTCATATGGATTACAAGGATGACGATGACAAGATGAGCATGGCCAACGCTCCC-3′)/Zur-express2 (5′-TTGGATCCTCAGCTTCGGCAATCCGCGC-3′), and the 419-bp NdeI/BamHI fragment was cloned into pET28a plasmid (Novagen). This construction was introduced into E. coli BL21(DE3) strain and His-FLAG-Zur protein expression was induced for 3 h with 0.5-mM IPTG added to midlog cultures grown at 30°C in M9 medium. The recombinant protein was purified from soluble extract by NTA-resin affinity chromatography (Qiagen).The 2, 1 mM dithiothreitol, 0.1 mM MnCl2, 0.1 mg ml−1 bovine serum albumin, 5% glycerol), containing salmon sperm DNA (0.1 mg ml−1), labeled DNA probes and increasing amounts of purified His-FLAG-Zur protein . In competition assays, a 30-fold excess of cold probe was used to challenge each of the labeled probes. The probe containing the coding region of the stationary phase-induced gene cspD was used as the non-specific competitor. After incubation at 30°C for 30 min, the samples were loaded onto a native 4%, 5% or 8% polyacrylamide gels (depending on probe size) in 40 mM Tris-acetate buffer pH 8.0 (with no added EDTA), containing 2 mM MnCl2. Gels were dried and radioactive species were detected by autoradiography.PCR fragments containing the promoters of selected genes CC0214, CC0320, CC0663, CC1517/CC1518 were purified and labeled with [γ-32P]-ATP using T4 Polynucleotide Kinase (Fermentas). Unincorporated nucleotides were removed with Qiaquick PCR purification kit (Qiagen). DNA binding was performed in a 20 μl reaction in binding buffer  containing 500 μM EDDS (Sigma) to remove externally bound or free remaining zinc. After washes, cultures were harvested, dried and then metal content was determined by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) method, normalized to dry mass.Overnight cultures were diluted to OD 0.1 in 500 ml M2 medium and incubated at 30°C under agitation. When cultures reached OD 0.4, 200 μM ZnCl−5 dilution were spread on four M2 agar plates, containing either 500 μM, 850 μM EDTA, 850 μM EDTA plus 1.7 mM ZnCl2 or no additions. Plates were incubated for 72 h or 120 h and then photographed.To determine the effect of zinc starvation on viability of NA1000, MM69, MM72, MM73 and MM78 strains, they were grown in M2 medium at 30°C up to midlog phase (OD600 nm 0.6) and aliquots were taken for serial dilutions. 20 μl-aliquots of a 10C. crescentus Zur with other regulators of the Fur family. (PDF 195 KB)Additional file 1: Figure S1: Amino acid alignment of


In embryonic stem (ES) cells, developmental regulators have a characteristic bivalent chromatin signature marked by simultaneous presence of both activation (H3K4me3) and repression (H3K27me3) signals and are thought to be in a ‘poised’ state for subsequent activation or silencing during differentiation. We collected eleven pairs (H3K4me3 and H3K27me3) of ChIP sequencing datasets in human ES cells and eight pairs in murine ES cells, and predicted high-confidence (HC) bivalent promoters. Over 85% of H3K27me3 marked promoters were bivalent in human and mouse ES cells. We found that (i) HC bivalent promoters were enriched for developmental factors and were highly likely to be differentially expressed upon transcription factor perturbation; (ii) murine HC bivalent promoters were occupied by both polycomb repressive component classes (PRC1 and PRC2) and grouped into four distinct clusters with different biological functions; (iii) HC bivalent and active promoters were CpG rich while H3K27me3-only promoters lacked CpG islands. Binding enrichment of distinct sets of regulators distinguished bivalent from active promoters. Moreover, a ‘TCCCC’ sequence motif was specifically enriched in bivalent promoters. Finally, this analysis will serve as a resource for future studies to further understand transcriptional regulation during embryonic development. Embryonic stem (ES) cells have the unique ability to self-renew indefinitely as well as to differentiate in response to internal as well as external stimuli69Bivalency of chromatin has therefore become an important property to investigate the functional relevance of a gene through development, and the presence of bivalent genes in human and mouse ES cells has been validated by many studies independently9121314ChIP sequencing raw data for H3K4me3 and H3K27me3 histone marks in murine and human ESCs was obtained from Gene Expression Omnibus (GEO)The one2one orthologous regions between human and mouse  were obtained from Ensembl BioMartFor the clustering of bivalent promoters into four groups, we gathered chip sequencing data for three different forms of RNAPII: RNAPIIS5P, RNAPIIS7P and 8WG16We calculated the CpG density as the ratio of observed to expected CpG countsFor identification of factors enriched at bivalent and H3K4me3 promoters, we used data from 49 and 99 ChIP-seq experiments for several factors in human and mouse embryonic stem cells respectivelyRNA sequencing data in murine ES cellsThe sequence motif enrichment analysis was performed with the command findMotifs.pl from HOMERr) = 0.75, for H3K4me3 r = 0.84), but not across human datasets . There are other factors contributing to the variation between samples, for example ES cells were grown in diverse culture conditions, and using different cell lines as well as various antibodies across datasets  of H3K4me3 and H3K27me3 ChIP sequencing (ChIP-seq) datasets for human ES cells and 8 pairs for mouse ES cells from the Gene Expression Omnibus (GEO) database and the Roadmap Epigenomics Project . After adatasets . We therdatasets . Eight Hdatasets . Adding datasets . There wdatasets .rd of H3K27me3 top promoters in any individual dataset overlapped with HC bivalent promoters   and S7, romoters .We also checked whether the peaks of H3K27me3 and H3K4me3 modifications were present at the same genomic location within a promoter region and found that over 95% of H3K27me3 and H3K4me3 peaks overlapped in each pair of samples at HC bivalent promoters. Both chromatin modifications were indeed present at the same genomic location . We compIn summary, by integrating data from multiple studies we identified HC human and murine bivalent promoters, which could not be identified by simply selecting the top peaks from individual samples. The HC bivalent promoters were highly enriched for developmental regulators compared to non-HC bivalent promoters.Bivalent promoters are known to show variation in their levels of occupancy by RNA polymerase II−51) while cluster 4 promoters were enriched for developmental functions such as organ morphogenesis . Cluster 3 promoters contained transcription factors important for specific lineages like haematopoiesis factors Gfi1 and Meis1, whereas cluster 4 contained multiple members of transcription factor families controlling development such as winged helix/forkhead box (Fox) and Hox families.HC bivalent promoters could be classified in four distinct clusters based on the presence of PRC1 components and forms of RNAPII . The firet al. (12) suggested that PRC1 was absent in our PRC1 low bivalent promoter  and PRC1 high (cluster 3 & 4). Ku promoter . Ring1b promoter .In summary, all HC bivalent promoters are occupied by components of both PRC1 and PRC2. There exists a distinct set of metabolic genes (cluster 2) which though bivalently marked has RNAPII (S7P) and is expressed at a higher level than other bivalent genes.RNA polymerase II (PolII) may be present but stalled at the promoters of bivalent genes and short (abortive) transcripts may be detected at their promotersAs bivalent genes are thought to be poised for activation or repression, we hypothesised that these genes might be more likely to be differentially expressed upon perturbation of ES cells. We therefore used a collection of differentially expressed genes upon deletion or over-expression of 91 transcription and epigenetic factors in mouse ES cells, and found that 98% of differentially expressed gene sets by the overexpression of at least one TF significantly overlapped  with bivalent genes, and 89% differentially expressed gene sets by the down-regulation of at least one TF . To chec−71) and transcription factor activity ; whereas species-specific promoters were not enriched for the two above terms (−16) and glycoprotein  and the human-specific for plasma membrane part  and alternative splicing .To perform a systematic comparison of chromatin status between human and mouse promoters in ES cells, we used 16,639 one-to-one orthologous genes between the two speciesve terms . SpecifiTo check whether the chromatin status across species is reflected in the gene expression status, we focused on five groups of promoters : three gAs shown in the first section, the bivalent status of promoters is primarily determined by the detection of an H3K27me3 modification . CpG isl3738Over 90% of our HC bivalent promoters in ES cells in both species overlap with at least one CGI region, whereas only 8% (37 of 397) of human H3K27me3 only promoters contained a CGI and no mouse H3K27me3 only promoters (none of 152) contained a CGI . Previou41The loss of H3K27me3 in rodents (mouse and rat) compared to human ES cells at many developmental genes has been associated with depletion of CGIs; mouse CGI erosion has been characterised at MYO1G, CLEC4G and MYF6 gene loci with corresponding H3K27me3 lossIn summary, the H3K27me3-only CpG-poor promoters demonstrate that polycomb recruitment does not only depend on CpG density. Although the CpG density largely correlates with H3K4me3 and H3K27me3 profiles across promoters, the loss of CGI on a promoter does not always imply a corresponding loss of the H3K4me3 and/or H3K27me3 modification on that promoter.As both active (H3K4me3-only) and bivalent promoters are CpG-rich, we investigated possible modes of distinction between the two in ES cells. Voigt, Tee, and Reinberg−256). Moreover, the co-repressor c-terminal binding protein 2 (CTBP2), required for PcG recruitment in Drosophila4647−3). Four other epigenetic regulators, Utf1, Tet1, Rest and Setdb1 were highly enriched at mouse bivalent regions. Utf1  was recently identified as a component of bivalent chromatin by acting as a buffer against full activation of bivalent genesTo identify factors preferentially binding to bivalent promoters, we calculated the overlap between transcription and epigenetic factor binding sites (peaks) and bivalent promoters. Four out of 49 and eleven out of 99 factors characterised by ChIP-seq preferred bivalent promoters in human and mouse respectively . As expeAs expected, many TFs (33 out of 49 factors in human and 39 out of 99 factors in mouse) were enriched at active (H3K4me3-only) promoters. This included known regulators of pluripotency in ES cells such as Klf4, Esrrb, Oct4, Sox2, and Nanog . Only twde novo motif identification on bivalent promoters by providing active promoter sequences as background in HOMER softwarede novo motif discovery). The ‘TCCCC’ motif was most similar to the known binding sequence of the Mzf1 transcription factorde novo motif enrichment was performed on active human and mouse promoters using bivalent promoter sequences as background, they were enriched for a ‘CGGAA’ motif found in 40% of the active promoter sequences, which was not enriched in bivalent promoters. This motif is the most similar to the known motif for Elk1 transcription factor  promoters, but more than H3K27me3 only and latent promoters. Active promoters were preferentially occupied by pluripotency factors. On the other hand, bivalent promoters were enriched for Polycomb factors as well as other chromatin modifiers. The factors enriched at bivalent promoters show very little overlap with the ones enriched at active promoters. These findings are consistent with the observed spatial segregation of transcriptional networks in ES cells where Nanog and Polycomb proteins were shown to occupy distinct nuclear spacesBivalent chromatin domains bearing both H3K4me3 and H3K27me3 modifications have been shown to be a key feature of developmentally regulated genes in ES cells8913149149Bivalent promoters are thought to be poised for rapid activation or inactivation during differentiation13We computed binding profiles of PRC components (PRC1 and PRC2) and various forms of RNA polymerase II at bivalent promoters in murine ES cells. All HC bivalent promoters were marked by Suz12, Jarid2, Ring1b and Cbx7. To note, the PRC2-only group defined byMore than half of high-confidence bivalent promoters were conserved between human and mouse, suggesting the existence of a set of genes bivalently marked across most mammalian ES cells . These g54et al. (2014) recently suggested that bivalency is the default chromatin structure for CpG-rich, G+C-rich DNASince a high density of un-methylated CpG is sufficient for vertebrate polycomb recruitment3839On bivalent promoters, the CpG density and H3K27me3 modification are highly correlated. By performing a cross-species comparison, a small fraction (~5%) of human CpG-rich HC bivalent promoters has the corresponding CpG eroded in the mouse genome, while no CpG-rich bivalent promoters in mouse are eroded in human. This erosion of CpG density was correlated with the loss of H3K27me3 and H3K4me3et al.It is intriguing how bivalent domains are established in ES cells. Voigt De novo motif discovery at HC bivalent promoters identified a ‘TCCCC’ motif in both human and mouse ES cells which was not enriched at active promoters. This motif was present in about half of the HC bivalent promoters and is similar to the sequence motif of MZF1As active (H3K4me3-only) and bivalent promoters are both CpG rich, it is key to unravel the distinguishing factors between these two groups. In summary, this meta-analysis revealed several novel aspects of bivalency in mammalian ES cells and will serve as a resource for future studies to further understand transcriptional regulation during embryonic development. Further work will be aimed at understanding how the HC bivalent promoters identified here are resolved in different cellular lineages during differentiation.How to cite this article: Mantsoki, A. et al. CpG island erosion, polycomb occupancy and sequence motif enrichment at bivalent promoters in mammalian embryonic stem cells. Sci. Rep.5, 16791; doi: 10.1038/srep16791 (2015).


Saccharomyces cerevisiae, the promoter is one of the most important tools available for controlling gene expression. However, the complex expression patterns of yeast promoters have not been fully characterised and compared on different carbon sources  and across the diauxic shift in glucose batch cultivation. These conditions are of importance to yeast cell factory design because they are commonly used and encountered in industrial processes. Here, the activities of a series of “constitutive” and inducible promoters were characterised in single cells throughout the fermentation using green fluorescent protein (GFP) as a reporter.Predictable control of gene expression is necessary for the rational design and optimization of cell factories. In the yeast PHXT7, PSSA1 and PADH2) varied in induction strength on non-glucose carbon sources ; in contrast to the “constitutive” promoters, GFP expression increased as glucose decreased and cells moved towards the diauxic shift. While lower than several “constitutive” promoters during the exponential phase, expression from the SSA1 promoter was higher in the post-diauxic phase than the commonly-used TEF1 promoter. The galactose-inducible GAL1 promoter provided the highest GFP expression on galactose, and the copper-inducible CUP1 promoter provided the highest induced GFP expression following the diauxic shift.The “constitutive” promoters, including glycolytic promoters, transcription elongation factor promoters and ribosomal promoters, differed in their response patterns to different carbon sources; however, in glucose batch cultivation, expression driven by these promoters decreased sharply as glucose was depleted and cells moved towards the diauxic shift. Promoters induced at low-glucose levels  contains supplementary material, which is available to authorized users. Saccharomyces cerevisiae is widely used as a cell factory for producing biofuels and biochemicals. Economic application of cell factories requires that feed stocks (carbon sources) are efficiently converted to desired products. Metabolic engineering involves over-expressing certain genes to introduce/enhance/optimize the metabolic network to improve strain performance. The strength and pattern of gene over-expressions are primarily controlled by promoters. Most promoters used in yeast metabolic engineering are endogenous and respond to environmental signals by up-regulation or down-regulation through in vivo transcriptional regulatory networks [The budding yeast networks –3. Thesenetworks , 5.S. cerevisiae firstly ferments sugars rapidly into ethanol (the ‘Crabtree effect’) [The carbon source has a significant effect on global regulatory patterns. The effect directly imposed through promoters on the engineered gene expression is of particular importance when developing yeast cell factories –8. When effect’) . When theffect’) , 11, andeffect’) . It is teffect’) , 14. ComPPGK1), glyceraldehyde-3-phosphate dehydrogenase (PTDH3), triose phosphate isomerase (PTPI1), enolase (PENO2) and alcohol dehydrogenase (PADH1) [PTEF1 and PTEF2), which are thought to enable a relatively stable expression level during glucose batch cultivation [Commonly-used promoters can be divided into two main classes. ‘Constitutive’ promoters are considered to give stable expression levels across varying culture conditions, while ‘dynamic’ or ‘inducible’ promoters drive dramatic changes in expression level in response to environmental stimuli. Constitutive promoters that drive high level transcription (strong constitutive promoters) are often used for engineering applications. Well-known examples include: promoters of glycolytic genes, such as 3-phosphoglycerate kinase ( (PADH1) , 15; andGAL1/GAL10 promoter  is induced when cells are grown on galactose [CUP1 promoter can be induced by adding copper(II) to a fermentation [PHXT7) [PADH2) [The constitutive expression of certain proteins and metabolic pathways can be detrimental to cell growth due to product toxicity, and the metabolic burden imposed by the redirection of carbon flux, redox cofactors, and ATP , 17. In alactose , and theentation . Another [PHXT7) , 21 and  [PADH2) , 22.CUP1 promoter, we also characterized the induction profile on a range of different copper concentrations. Our findings reveal the composite activities of various promoters in response to different carbon sources and the diauxic shift.Ideally, gene expression would be tailored towards the specific bioprocess and product requirements for metabolic engineering applications, thereby achieving optimal yields/rates/titres . AlthougSSA1 gene is up-regulated significantly during growth on ethanol [SSA1 promoter a candidate that is automatically induced during the ethanol consumption phase in glucose batch cultivation. The full range of promoters included glycolytic promoters , translational elongation factor promoters , galactose metabolic promoters (PGAL10/GAL1), ribosomal protein promoters , chaperone promoters (PSSA1 and PSSB1), the copper-inducible CUP1 promoter, low-glucose-inducible promoters , and the PDA1 promoter . To detyEGFP) and a destabilized version, yEGFP-CLN2PEST , were used as reporters to compare promoter expression levels. Expression of yEGFP reflects the accumulation level of a stable protein with a half-life of ~7 h, while the destabilized GFP shows the dynamic protein synthesis rate, because of its short half-life (12 min) [TEF1 promoter for pre-evaluation, we observed that for both yEGFP and yEGFP-CLN2PEST reporter genes, the intracellular GFP levels varied throughout the cultivation time  . Using T2 min . U2 min . U−1 glucose using the destabilised GFP in microtitre plate format  was used as reporter to compare the promoter activities on different carbon sources. While GFP levels from the stable yEGFP were ~50-fold higher than that from the destabilized yEGFP, the GFP levels were highly correlated  .In conventional genetic cloning, a restriction site is commonly introduced between the promoter and the ATG start codon. This can potentially interfere with transcription/translation. To examine this, we introduced a −1 sucrose, 20 g L−1 galactose and 2% v/v ethanol , ribosomal , galactose-responsive (PGAL1), chaperone (PSSA1 and PSSB1), copper-responsive (PCUP1) and PDA1 promoters showed no difference in GFP levels between glucose and sucrose. The ‘low-glucose-inducible’ promoters had varying responses: PTPS1 (low levels on glucose) also showed no difference on sucrose, whereas PCYC1 showed a slight increase on sucrose, and PHXT7  was de-repressed on sucrose, resulting in an intermediate level of activity (relative to activity of other promoters on sucrose). The glycolytic promoters (PPGK1 and PTDH3) showed decreased levels of GFP activity on sucrose relative to glucose .Promoter activity was tested on media containing 20 g LGAL1 promoter exhibited the highest activity on galactose ; PSSA1 was about ~5-fold higher relative on ethanol to 20 g L−1 glucose. The ADH2 promoter was repressed on most carbon sources; it was strongly induced on ethanol, and also showed low level activity on galactose. The HXT7 promoter was up-regulated on ethanol, but the GFP level was lower than on sucrose and galactose. The TPS1, CYC1 and CUP1 promoters were up-regulated on ethanol compared to glucose/sucrose, but were still relatively weak. Activity driven by the TDH3 and PGK1 promoters was low on ethanol; it was reduced by ~85% relative to activity on 20 g L−1 glucose and by ~60% relative to 20 g L−1 galactose. Activity driven by the ribosomal promoters, the YEF3 promoter, and the SSB1 promoter was more than 50% reduced compared to activity on 20 g L−1 glucose . However, compared to other promoters investigated, the activity level was relatively low. sources . ConsistPTEF1, PTEF2, PTDH3, PPGK1, PRPL3, PSSA1, PADH2 and PHXT7; Figure Glucose batch cultivation involves a diauxic shift from glucose respire-fermentative metabolism to ethanol respiratory metabolism Figure a, b. TheTEF1, TEF2, TDH3, PGK1 and RPL3 promoters decreased dramatically, and kept decreasing sharply until 16–20 h , although this level of expression was weaker by an order of magnitude than the strong constitutive promoters  provides an instantaneous readout of promoter activity, while the stable GFP integrates activity with a protein half-life of ~7 h [A ‘promoter’ sequence can be loosely defined as the upstream region of a gene that can replicate the observed expression pattern of that gene when fused to a reporter gene with an easily-assayable phenotype. Several reporter genes are available, and each has different characteristics that make it suitable for different applications , 31. The of ~7 h . HoweverTEF1 and TEF2 promoters is to enable stable, high-level expression on different carbon sources . Sun et al. used synthetic complete media with added amino acids, whereas here we used a defined medium without added amino acids. Amino acids might activate the TOR (target of rapamycin) signalling pathway, thereby interfering with the translational blockage phenotype [TEF1 and PGK1 promoters are used to control production pathway gene expression [The advantage of the commonly used henotype , 39 whicpression .SSA1 promoter could be used to automatically increase the expression of production pathways as biomass increases  has been shown to improve the performance of cell factories in glucose-depleted conditions [Another important consideration in metabolic engineering is that high-level expression of certain pathways might result in detrimental effects on cell growth . In thiss Figure . Up-regucription , 26, 36.nditions .HXT7 promoter also gradually increased after the diauxic shift, at high biomass levels .ADH2 promoter showed the strongest activity on ethanol when provided with ethanol as a carbon source  but not in minimal media. This is consistent with our data  feeding  detoxification mechanism in yeast whereby cells can reduce copper(II) to copper(I) [CUP1 promoter is still superior to other promoters because it enables inducible, and high-level GFP expression after the diauxic shift  , and copopper(I) , 44 or at Figure —a patterDifferent carbon sources and the diauxic shift can influence gene expression driven by endogenous promoters, resulting in different response patterns. The analysis of promoter-regulated gene expression presented here can be used to inform the rational design of metabolic pathways and synthetic genetic circuits, and reveals potential limitations in current metabolic engineering strategies. In particular, the relatively low expression levels observed for all of the ‘constitutive’ promoters after the diauxic shift during glucose batch cultivation suggests a novel target for metabolic engineering in yeast and the expression patterns of low-glucose-inducible or copper-inducible promoters reveals potential utility for the dynamic regulation of synthetic genetic/metabolic networks. Our results provide instructive and in-depth information about promoter performance on different carbon sources and during the diauxic shift, and can be used to inform expression pattern design for yeast cell factories. It would be useful to examine promoter responses on other industrially-useful carbon sources—in particular, cellulosic feedstock sugars such as xylose, which yeast have been successfully engineered to utilize , 45.URA3 promoter (PURA3) and URA3 terminator (TURA3) were amplified from CEN.PK113-7D [KluyveromyceslactisURA3 (KlURA3) was amplified from plasmid pUG72 [yEGFP) and yEGFP-CLN2PEST sequence were amplified from pFA6a-yEGFP3-CLN2PEST-natMX6 [PURA3- KlURA3- yEGFP3- CLN2PEST- TURA3 was fused together through overlap extension PCR and cloned into SphI/EcoRI sites of pUC19 [yEGFP fragment was cloned into XhoI/SpeI sites of pITGFP3 to replace yEGFP3- CLN2PEST and generate the plasmid pILGFP3  region on promoter strength [BamHI/BglII and self-ligated to generate the control plasmid pILGH4 (without GFP). The tested promoters . YNB with 20 g L−1 glucose (YNBG) was used as the medium without additional buffer. To buffer against the significant changes in pH, 100 mM MES was supplied in YNBG and the pH was initially adjusted to 6.0 with ammonium hydroxide. The tested strains were recovered from glycerol stocks by streaking on the YNBG agar plates. A single colony was used to initiate a seed culture by inoculating into 5 ml YNBG broth (or YNBG with 100 mM MES buffer) to a cell density (OD600) of 0.02. Over-night, seed cells in the mid-exponential phase (OD600 = 1.5–3) were inoculated into 20 mL of YNBG (or YNBG with 100 mM MES buffer) in a 125 mL flask to a starting OD600 of 0.2, and incubated at 30°C at 200 rpm. To examine copper induction of the CUP1 promoter, YNBG with 100 mM MES buffer was used as the medium and copper(II) sulfate stock solution (1 M) was added to the 20 mL flask cultures at 5 h. Cultures were periodically sampled for the measurement of OD600, GFP fluorescence, pH, and/or extracellular metabolites. Each strain was cultivated in duplicate.The minimal media used to analyse the GFP expression level contain 6.7 g LTo test promoter activity in a high-throughput format on different carbon sources, microplate cultivation was performed at 30°C in a 350 rpm shaking incubator using a U-bottom 96-well plate  sealed with a Breathe-Easy sealing membrane . Each strain was cultivated in triplicate for testing. The tested strains were recovered from glycerol stocks by streaking on the YNBG agar plates. To prepare the seed culture, a single colony was resuspended in 100 μL YNBG broth, and 5 μL of resuspended cells were inoculated in 95 μL fresh YNBG (pre-culture 1). The seed cultures were cultivated overnight.EGFP-CLN2PEST), 5 μL overnight pre-culture 1 was inoculated into 95 μL YNBG in a fresh plate (pre-culture 2), and 5.5 h later, 5 μL culture was inoculated into 95 μL fresh YNBG in a fresh plate (test culture). After 6.5 h, the cells were analysed for GFP fluorescence (see below).For the analysis using the destabilized GFP , 2.5 μL of pre-culture 1 was inoculated into 97.5 μL YNB broth with 2% (v/v) ethanol. After 24 h this dilution was repeated to inoculate the test culture. Cells in mid-exponential phase (OD600 = 1–2.5) were analysed for GFP level (see below).For the analysis using the normal y600 was above 10.GFP fluorescence in single cells was analysed, immediately after sampling, using a flow cytometer . GFP fluorescence was excited by a 488 nm laser and monitored through a FL1.A filter (wavelength 530/20 nm). For plate cultures, 5,000 events were counted; for flask cultures, 10,000 events were counted. The particle volume and complexity for each event were monitored by forward scatter detector (FSC.A) and side scatter detector (SSC.A). For flask cultures, the cells were diluted fivefold with water when the ODThe GFP fluorescence signal was corrected for cell size and complexity using a heuristic formula,2SO4 at 0.6 mL/min at 65°C. Glucose, ethanol and glycerol were monitored using a refractive index detector , and acetate was detected using an ultraviolet–visible light absorbance detector  at 210 nm.Extracellular metabolites  were analysed by Metabolomics Australia Queensland Node through ion-exclusion chromatography . Ion-excAll statistical analyses were performed in R. The relative fluorescence (NormFL.A/Ref_NormFL1.A) data were log transformed, in order to stabilise variance across the dataset. A Bartlett test was used to confirm variance homogeneity for each dataset prior to performing linear regression or analysis of variance (ANOVA). Post-hoc analyses for one-way ANOVA were performed using Tukey tests. In the few instances where data failed the Bartlett test for variance homogeneity, the one-way ANOVA was performed using Welch corrections and posthoc analysis was performed with a Games–Howell test instead of a Tukey test.


Candida albicans. Our analyses indicate that, contrary to model systems, each type of repetitive element is assembled into a distinct chromatin state. Classical Sir2-dependent hypoacetylated and hypomethylated chromatin is associated with the rDNA locus while telomeric regions are assembled into a weak heterochromatin that is only mildly hypoacetylated and hypomethylated. Major Repeat Sequences, a class of tandem repeats, are assembled into an intermediate chromatin state bearing features of both euchromatin and heterochromatin. Marker gene silencing assays and genome-wide RNA sequencing reveals that C. albicans heterochromatin represses expression of repeat-associated coding and non-coding RNAs. We find that telomeric heterochromatin is dynamic and remodelled upon an environmental change. Weak heterochromatin is associated with telomeres at 30 °C, while robust heterochromatin is assembled over these regions at 39 °C, a temperature mimicking moderate fever in the host. Thus in C. albicans, differential chromatin states controls gene expression and epigenetic plasticity is linked to adaptation.Transcriptionally silent heterochromatin is associated with repetitive DNA. It is poorly understood whether and how heterochromatin differs between different organisms and whether its structure can be remodelled in response to environmental signals. Here, we address this question by analysing the chromatin state associated with DNA repeats in the human fungal pathogen  Saccharomyces cerevisiae, assembly of subtelomeric heterochromatin is mediated by the Sir silencing complex formed by Sir2, Sir3 and Sir41213Candida albicans. C. albicans normally lives as a commensal in humans but it can become virulent causing systemic life-threatening infections with mortality rates of up to 50%C. albicans undergoes major morphological and genomic changes that can promote adaptation and survival4C. albicans genome is also plastic and capable of remodeling upon environmental changes.Large blocks of DNA repeats are commonly clustered at rDNA loci, telomeres and centromeres and are assembled into heterochromatin. Heterochromatic regions impose a transcriptionally repressive environment that can propagate over long distances up to 50 kb) stochastically silencing native genes as well as reporter genes inserted at these regions independently of the underlying DNA sequence1230 kb stoc1278910C. albicans genome has 8 diploid chromosomes and contains 3 major classes of large blocks of repetitive DNA: the rDNA locus, Major Repeat Sequences (MRS) and telomeres17rDNA locus consists of a tandem array of a ~12 kb unit repeated 50 to 200 times on chromosome R. Each unit contains the two highly conserved 35 S and the 5 S rRNA genes that are separated by two Non-Transcribed Regions (NTS1 and NTS2), whose sequences are not conserved across eukaryotes  of nested DNA repeats found on 7 of the 8 C. albicans chromosomes18C. albicans telomeres are composed of a terminal region composed of tandemly repeating 23 bp units and subtelomeric regions  3–9 orthologue cannot be identified in either C. albicans or S. cerevisiae. In contrast, the C. albicans genome encodes for two putative NAD-dependent histone deacetylases that resemble Sir2 (orf19.1992 and orf19.4762)C. albicans telomeres lack heterochromatin as it has been recently shown for the yeast Clavispora lusitaniaeDNA repeats are central to C. albicans repetitive elements under different environmental conditions. We found that the different types of repetitive elements are assembled in distinct chromatin states. Classical hypoacetylated heterochromatin is associated with the non-transcribed region of the rDNA locus. The histone deacetylase Sir2 (orf 19.1992) is required to maintain this repressive epigenetic state via hypoacetylation of Lysine 9 of Histone H3. Heterochromatin associated with these regions represses non-coding RNA transcription. In contrast, MRS repeats are assembled into a transcriptionally permissive chromatin state bearing both heterochromatic and euchromatic histone marks. Finally, we find that, despite the apparent absence of the Sir silencing complex, telomeric regions are assembled into a Sir2-dependent hypoacetylated and hypomethylated heterochromatin. This chromatin state silences expression of endogenous transcripts as well as inserted marker genes. Telomeric heterochromatin is plastic and affected by environmental conditions with heterochromatin being more robust at higher temperature (39 °C) than at lower temperature (30 °C). Thus, the epigenetic state associated with telomeric repeats switches in response to an environmental change that are linked to C. albicans virulence and pathogenicity.In this study, we investigated the chromatin states associated with C. albicans, the URA3+ marker gene was integrated into the NTS2 region of the rDNA locus (rDNA::URA3+)  medium and in medium lacking uridine (−Uri) in which only cells expressing sufficient Ura3 protein are able to grow. Silencing of URA3+ is expected to result in slower growth in –Uri medium compared to N/S. The rDNA::URA3+ strain displayed a reduced growth rate in selective medium and it is, therefore, silenced (URA3 mRNA levels at the rDNA locus were significantly lower (20 fold) for rDNA::URA3+ than for a URA3+ gene expressed from its own euchromatic locus, as determined by quantitative reverse transcriptase analysis (qRT-PCR)  . We invesilenced . ConsistqRT-PCR) . TherefoC. albicans rDNA repeats to cause transcriptional silencing suggests that the rDNA NTS regions are assembled into heterochromatin. The histone deacetylase Sir2 is a key regulator of heterochromatin in all organisms studied1C. albicans genome contains 5 genes encoding proteins with homology to S. cerevisiae SIR2  and Sir2 (orf19.1992) share the highest homology with the S. cerevisiae Sir2 (46% and 42% identity respectively)  and/or Sir2 (orf19.1992) are required for maintaining the repressive state of the rDNA NTS region, we constructed hst1 Δ/Δ and sir2 Δ/Δ null mutants in strains carrying the rDNA::URA3+ reporter and performed marker gene silencing assay. At the NTS region of the rDNA locus, silencing was not alleviated in hst1 Δ/Δ cells  cells  originating from the NTS region of the rDNA locusC. albicans heterochromatin represses transcription of this non-coding RNA, we isolated RNA from wild-type and sir2 Δ/Δ cells and performed qRT-PCR analyses. Deletion of the SIR2 gene results in a clear upregulation of this non-coding transcript (qChIP)rDNA locus, but not the euchromatic ACT1 locus, showed low enrichment for H3K9 acetylation, H4K16 acetylation and H3K4 methylation  . Given t/Δ cells  but it w/Δ cells . In agreT) cells . Thus, tanscript . Therefohylation , a chrom H4K16Ac  demonstrwt cells . TherefoC. albicans and is associated with the NTS region of the rDNA locus, we analysed the chromatin state associated with the MRS repeats by assessing silencing of a marker gene inserted into the tandem RPS repeats (MRS::URA3+)  were determined for all the genes proximal to MRS repeats and compared in sir2 Δ/Δ and WT strains. Upon deletion of the SIR2 gene, we did not observe any clear effect on expression of MRS associated (orf C) and proximal genes (orf L and R) as only 3 out of 24 genes were expressed at more than 2 fold of WT in sir2 Δ/Δ isolates (2 fold change = 0)  . As showsilenced . If the isolates . Averaginge = 0) . These dqChIP analyses demonstrated that MRSs are assembled into highly acetylated chromatin where H3K9 and H4K16 are acetylated to a level similar to the active and euchromatic locus actin 1 (ACT1) (sir2 Δ/Δ isolates (ACT1 locus (Acetylated H3K9 and H4K16 1 (ACT1) . Histoneisolates . In contT1 locus . TherefoC. albicans. It is therefore possible that telomeres are not assembled into heterochromatin. To test this hypothesis, we analysed silencing of a URA3+ marker gene integrated into a telomeric region (Tel5::URA3+). The Tel5::URA3+ marker gene displayed a small but reproducible reduction in growth rate on -Uri media compared to N/S media, indicative of weak silencing , mimicking the production of reactive oxygen species by the host’s immune cells, or Fluconazole, the most widely used antifungal drug, does not change the transcriptional state associated with any of the loci tested  . As with the telomere repeat tract on Chr5, silencing of telomeric repeats on Chr7 was much stronger at 39 °C compared to 30 °C   and S6. he host)  indicatihe host) . Therefoto 30 °C . Importa/Δ cells . These rto 30 °C . Consistto 30 °C . Thus teC. albicans DNA repeats. Our data demonstrate that, in C. albicans, differential chromatin states control gene expression and epigenetic plasticity is linked to adaptation to a specific environmental niche.This study provides the first comprehensive analysis of the chromatin state of rDNA locus maintains a bona fide silent heterochromatin state that represses coding and non-coding transcription and is marked by the histone modification pattern typical of heterochromatic regions: hypoacetylation of histone H3K9 and histone H4K16 together with hypomethylation of H3K4 (orf19.1992) as one of the key enzymes necessary to maintain this chromatin state  and Cdc14 (orf 19.4192). Therefore, it is very likely that, as observed in S. cerevisiae, a conserved RENT complex targets heterochromatin at the C. albicans rDNA locus. However, the mode of action of Sir2 in C. albicans differs from S. cerevisiae: while at S. cerevisiae rDNA locus, Sir2 specifically deacetylates K16 on histone H4, C. albicans Sir2, reduces H3K9 acetylation. Therefore, heterochromatin at the non-transcribed regions of the rDNA array seems ubiquitous but it can differ in its structure.We find that the NTS region of the  of H3K4 . We idenin state . In S. cWe demonstrated that the MRS repeats are assembled into chromatin hypomethylated on H3K4, as observed in heterochromatic regions, but highly acetylated, as in euchromatic regions. Low levels of H3K4 methylation are not sufficient to create a transcriptionally repressive environment as a marker gene inserted at MRS repeats is not silenced. In addition, expression of MRS-proximal genes is not regulated by Sir2 .In many organisms, insertion of artificial DNA array is sufficient to seed heterochromatinC. albicans, an organism lacking a canonical sexual cycle and meiosis214345MRS loci remains limited, making it very difficult to assess whether this epigenetic signature controls MRS function. Further studies will reveal whether and how the epigenetic state associated with these repetitive elements contributes to C. albicans biology.Alternatively, genome instability at the MRSs could be beneficial for S. cerevisiae the Sir silencing complex is responsible for the assembly of hypoacetylated telomeric heterochromatinC. lusitaniae appears to lack telomeric heterochromatinCryptococcus neoformans telomeric chromatin is methylated on K27 of histone H3 and silences expression of genes located in a 40 kb subtelomeric regionC. albicans telomeres have also a specialised chromatin structure. Despite the apparent absence of Sir silencing proteins, C. albicans telomeres are associated with transcriptionally repressive heterochromatin (orf19.1992) controls heterochromatin assembly at telomeres by deacetylation of histones. We hypothesise that an as yet unidentified protein complex targets Sir2 to telomeres. We demonstrate that telomeric heterochromatin transcriptionally silences subtelomeric genes as deletion of the SIR2 gene causes their upregulation. This modulation is likely to have major impacts on the biology and the pathogenicity of C. albicans as many subtelomeric genes have key regulatory functions. For example, the subtelomeric TLO genes encode proteins with similarity to Med2, a component of the Mediator complex that regulates transcription by RNA polymerase IITraditionally, telomeric heterochromatin has been described as a repressive chromatin structure that silences expression of subtelomeric geneshromatin . Sir2  mimicking fever in the host than at lower temperatures (30 °C). This enhanced silencing is linked to changes in chromatin structure, as telomeres at 39 °C had lower levels of H3K9 acetylation and Sir2 was required for the increased silencing. This effect is specific for telomeric regions and for temperature shift as other loci are not affected by temperature changes and other physiologically relevant stresses do not lead to chromatin remodelling. Therefore, in C. albicans, adaptation to higher temperature is linked to chromatin remodelling at telomeric regions.We find that telomeric heterochromatin is dynamic: the ability of telomere terminal repeats to repress the expression of an embedded S. cerevisiae, transcriptional silencing at higher temperature is enhanced at telomeres and weaker at the rDNA locusC. albicans remain to be determined. It is possible that changes in telomeric heterochromatin correlate with changes in expression of subtelomeric genes. Alternatively, chromatin remodelling at telomeres could regulate genomic stability of these loci.Dynamic heterochromatin is seen at telomeres in many species: For example, in Candida albicans, the most common human fungal pathogen. We show that in C. albicans differential heterochromatin states control gene expression independently of the underlying DNA sequence and remodelling of heterochromatin is linked to adaptation in a stress condition.In conclusion, this study highlights the diversity and plasticity of chromatin states associated with DNA repeats in TM) or SC Drop-Out media (FormediumTM). Cells were grown at 30 °C or 39 °C as indicated.Yeast cells were cultured in rich medium (YPAD) containing extra adenine (0.1 mg/ml) and extra uridine (0.08 mg/ml), complete SC medium  using the protocol described inStrains are listed in rDNA::URA3+, Tel5::URA3+ and MRS::URA3+ strains were performed using a plate reader  in 24 well or 96 well plate format at 30 °C. When indicated Silencing assays were performed in the presence of 200 ng/μl of fluconazole (Sigma), 1 mM H202 (Sigma) and 39 °C. For each silencing assay in a 24 well plate format, 1 ml of a starting culture was inoculated in SC or SC-Uri media to reach a concentration of 60 cells/μl. Growth was assessed by measuring A600, using the following conditions: OD600 nm, 3600 s cycle time, 30 flashes per well, 400 rpm shaking frequency, double orbital shaking mode, 850 s additional shaking time after each cycle, 0.5 s post delay, for 44 to 60 hours at 30 °C. For each silencing assay in 96 well plate format, 1:100 dilution of an starting culture was inoculated in a final volume of 95 μl of SC or SC-Uri media to reach a concentration of 60 cells/μl. Growth was assessed by measuring A600, using the following conditions: OD600 nm, 616 cycle time, 3 flashes per well, 700 rpm shaking frequency, orbital shaking mode, 545 s additional shaking time after each cycle 0.5 s post delay, for 44 hours.Growth analyses with Graphs represent data from three biological replicates. Error bars: standard deviations of three biological replicates. Data was processed using SpectrostarNano MARS software and Microsoft Excel.TM Real-Time System.All strains were grown in YPAD rich media. RNA extraction was performed using a yeast RNA extraction kit  following the manufacturer’s instructions. RNA quality was checked by electrophoresis under denaturing conditions in 1% agarose, 1× HEPES, 6% Formaldehyde (Sigma). RNA concentration was measured using a NanoDrop ND-1000 Spectrophotometer. cDNA synthesis was performed using iScript™ Reverse Transcription Supermix for RT-qPCR (Bio-Rad) following manufacturer’s instructions and a Bio-Rad CFXConnectsir2 Δ/Δ and sequenced with an Illumina iSeq2000 platform. Illumina Library and Deep-sequencing was performed by the Genomics Core Facility at EMBL . Raw reads were analysed following the RNA deep sequencing analysis pipeline describedhttps://usegalaxy.org/) and Linux platform. Heatmaps and boxplot graphs were generated with R (http://www.r-project.org/). RNA sequencing data are deposited into ArrayExpress (accession number E-MTAB-4488).Strand-specific cDNA Illumina Barcoded Libraries were generated from 1 μg of total RNA extracted from WT and TM (Scientific Industries) for 30 min at 4 °C. Chromatin was sheared to 500–1000 bp using a Bioruptor (Diagenode) for a total of 20 min (30 s ON and OFF cycle) at 4 °C. Immunoprecipitation was performed overnight at 4 °C using 2 μL of antibody anti-H3K4me2 (Active Motif- Cat Number: 39141), anti-H3K9ac (Active Motif- Cat Number: 39137), and anti-H4K16ac (Active Motif- Cat Number: 39167) and 25 μl of Protein G magnetic beads . DNA was eluted with a 10% slurry of Chelex 100-resin (Bio-Rad) using the manufacturer’s instructions.qChIP was performed as describedTM Real-Time System. Data were analysed with Bio-Rad CFX Manager 3.1 software and Microsoft Excel. Enrichments were calculated as the percentage ratio of specific IP over input for qChIP analysis and as enrichment over actin for RT-qPCR. Histograms represent data from three biological replicates. Error bars: standard deviation of three biological replicates.Primers used are listed in Genomic DNA was extracted using glass acid beads (Sigma), phenol: cholorform: isoamyl alcohol (25:24:1) (Sigma) and RNAse A treated (Fisher). Following centrifugation, pellets were precipitated with 0.05 mM Sodium Acetate (Sigma) and Ethanol (Fisher) at −20 °C during 30 minutes and resuspended in water.Genomic DNA was then digested with corresponding enzymes and run in 1% agarose gel. DNA was transferred to a nylon membrane , probed with DIG probes (Roche) and hybridized as describedMicroscopy was carried out using an Olympus IX81 inverted microscope. Images were captured with a Hamamatsu photonics C4742 digital camera, with light excitation from an Olympus MT20 illumination system. Olympus CellR imaging software was used to control the apparatus.How to cite this article: Freire-Benéitez, V. et al.Candida albicans repetitive elements display epigenetic diversity and plasticity. Sci. Rep.6, 22989; doi: 10.1038/srep22989 (2016).