Schizosaccharomyces pombe.Intriguingly, while Set1 is enriched at highly expressed and repressed loci, Set1binding levels do not generally correlate with the levels of transcription. We showthat Set1 is recruited by the ATF/CREB homolog Atf1 to heterochromatic loci andpromoters of stress-response genes. Moreover, we demonstrate that Set1 coordinateswith the class II histone deacetylase Clr3 in heterochromatin assembly at prominentchromosomal landmarks and repression of the transcriptome that includesTf2 retrotransposons, noncoding RNAs, and regulators ofdevelopment and stress-responses. Our study delineates a molecular framework forelucidating the functional links between transcriptome control and chromatinorganization.Histone modifiers play essential roles in controlling transcription and organizingeukaryotic genomes into functional domains. Here, we show that Set1, the catalyticsubunit of the highly conserved Set1C/COMPASS complex responsible for histone H3K4methylation (H3K4me), behaves as a repressor of the transcriptome largely independentof Set1C and H3K4me in the fission yeast DOI:http://dx.doi.org/10.7554/eLife.04506.001 Genes can be turned on or off at different times in an organism's life. In humans,yeast and other eukaryotes, this is mainly controlled by the way DNA is packaged withproteins—known as histones—in a structure called chromatin. Genes thatare switched on, or only temporarily switched off, are associated with areas of thegenome where the chromatin is loosely packed. In contrast, genes that remain switchedoff for long periods of time are found in regions—known asheterochromatin—where the chromatin is tightly packed.Schizosaccharomycespombe.There are many enzymes that can modify histones to change the structure of chromatin.One enzyme—called Set1—adds a methyl tag to chromatin, which is knownto be associated with genes being switched on. However, Lorenz et al. found that Set1also has other roles in modifying chromatin in the yeast The experiments found that Set1 helps to keep genes switched off and that this roleis largely independent of its ability to add the methyl tag to chromatin. Set1 isrecruited to many sites across the genome by another protein called Atf1, which isinvolved in the cell's response to environmental stresses. Lorenz et al. believe thatthis helps to put these genes in a ‘poised’ off state so that they areready to be switched on rapidly if needed.Set1 also works with another protein that removes acetyl tags—which encouragechromatin to be less tightly packed—from histones. Together, both proteinscontribute to the assembly of heterochromatin and keep genes involved in developmentand stress responses switched off when they are not required.Collectively, these experiments reveal unexpected and important insights into howSet1—which plays critical roles in many aspects of human health includingaging and cancer—works in cells.DOI:http://dx.doi.org/10.7554/eLife.04506.002 Schizosaccharomyces pombe and othersystems show that the euchromatic and heterochromatic landscapes are somewhat fluid,with islands of H3K9me transiently assembled within euchromatin at certain meiotic genesand the 3′ ends of convergent genes (The packaging of eukaryotic DNA with histones into chromatin provides ampleopportunities for chromatin-modifying factors to exert extensive control over manyaspects of genome-based processes . In partnt genes . Conversnt genes .Saccharomyces cerevisiae homolog of Set1 (KMT2) responsible for H3K4methylation (H3K4me) has been implicated in transcriptional silencing at a number ofgenetic elements have orthologs in S. cerevisiae and humans , H3K4me null mutantsH3K4R (histone H3 lysine 4 substituted with arginine) orset1FH3K4me−    affectedsertion) . Profilipression .Importaobserved, with eqash2 and sdc1, which affected a higherproportion of sense strand probes than in other mutants , ‘oxidoreductaseactivity’ , and‘generation of precursor metabolites and energy’   that were little affectedin the other Set1C and H3K4me mutants  . The prod probes . The pro mutants . These rset1Δ mutant atnon-active, stress-response genes or heterochromatic repeats. We therefore performedgenome-wide mapping of Set1 to gain insights into its repressor function. Consistentwith its documented recruitment to active Pol II genes and repressed developmental genes , noticeable Set1binding was detected at the promoters of these genes  fold enrichment ≥2 at three or more adjacent probes) according to theirexpression levels  have previously been shown to betargets of the highly conserved ATF/CREB transcription factor Atf1. In addition tolocalizing to its targets before their activation . We observed that incontrast to wild-type or single set1Δ orclr3Δ mutant strains, a double mutantset1Δ clr3Δ strain exhibited asignificant synthetic slow-growth phenotype and sensitivity to the tubulin inhibitorthiabendazole  and HP1/Swi6 proteins bound to H3K9me . H3K9me/rvations , the losrichment . Loss oflization  and did cregion . H3K9me2cregion . The RNAe siRNAs .We founf siRNAs , the levf siRNAs . Thus, oset1 and clr3 mutant cells. While the majorityof the differentially expressed probes in the set1Δ mutantcorresponded to increased expression, loss of clr3 resulted in 792probes changing significantly in comparison with wild-type, with approximately equalnumbers corresponding to upregulated and downregulated transcripts  did not produce such a drastic change to thetranscriptome compared with set1Δclr3Δ, but only reduced the proportion of downregulatedtranscripts seen in the single clr3Δ mutant. Similarproportions of probes corresponding to the sense or antisense strands of knowntranscripts were differentially expressed across set1Δ andset1Δ clr3Δ mutants, with the exception ofclr3Δ cells, which displayed an increased proportion ofsense strand probes  were further upregulated in the double mutants  produces comparable expression profiles  inS. pombe has only a slight impact on the transcriptome, with mostdifferentially expressed transcripts upregulated. However, there are importantdifferences. Except for the expression profiles of H3K4R andset1FH3K4me− mutants, theprofiles among S. pombe Set1C subunit mutants are notably disparate,which could not be fully explained by their roles as subunits of Set1C orcontributions to H3K4me containing a shorter version of Set1 .Pol II activity is known to be required for transcriptional silencing andheterochromatin assembly at pericentromeric repeats . Other frevisiae . Howeverste11, Set1 may actin concert with transcription factors, including Atf1 together with Clr3 and otherHDACs, to keep the target genes repressed in a steady-state condition. However,unlike heterochromatin, the chromatin states of these loci probably support atranscriptionally poised Pol II and in response to appropriate environmental signalsenable Pol II to rapidly upregulate transcription.In addition to heterochromatic repeats, a significant fraction of the transcriptomeis under repressive control by Set1 and Clr3. Such genome-wide repressive effectstrongly suggests that Set1 behaves largely as a bona fide repressor. Atdevelopmental and stress-response loci such as Null mutants of Set1C subunits were constructed using a kanamycin cassette . Double ringo package (atf1Δ experiments was performed using thelimma (linear models for microarray data) package afterinterarray quantile normalization. Antibodies used for ChIP and ChIP–chipassays were anti-FLAG Set1 , anti-Atf1 , anti Pol II , anti-H3K4me3 , anti-H3K9me2, and anti-Swi6 .Small RNAs were purified from 50 ml culture of logarithmically growing cells usingthe Ambion mirVana miRNA/siRNA isolation kit .Small RNAs (60 µg) were loaded onto a 15% denaturing polyacrylamide gel and runat 300 V until the bromophenol blue dye reached the bottom of the gel (∼1.5hr). Northern transfer was done overnight by capillary blotting in Tris-borate-EDTAbuffer at room temperature onto Hybond-N+ membrane . The membrane was subsequently UV crosslinked twice at 1200 J. Hybridization wascarried out in 10 ml ULTRAhyb-Oligo buffer (Life Technologies) at 40°C overnightwith a 595 ∼0.3–0.6) from mutant and isogenic wild-type strains, reverse-transcribed intocDNA, and labeled with either Alexa Fluor 555 (wild-type sample) or Alexa Fluor 647(mutant sample) using Superscript Indirect cDNA labeling system (Life Technologies).Equal amounts of labeled cDNA (200–300 ng) from wild-type and mutant sampleswere mixed and hybridized on a custom 4 × 44k probe Agilent tiling microarray aspreviously described  enrichment was performed as previously described . Similarly, the author response typically shows only responsesto the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of thepublished articles . An edited version of theletter sent to the authors after peer review is shown, indicating the substantiveconcerns or comments; minor concerns are not usually shown. Reviewers have theopportunity to discuss the decision before the letter is sent . After afull discussion of the study and the reviews, I am happy to report that the reviewersand the BRE member found the study of interest to the journal and therefore we are happyto consider a revised manuscript addressing the following issues:set1 null background in Set1 ChIP-ChIP studies to verify thesignificance of the low signals observed. Additionally, you and co-authors need to showwhat percentages of Set1 localize at active and repressed regions, respectively?1) An essential control in the ChIP-ChIP studies of Set1 is the use2) Further, the result of ChIP-ChIP studies in the paper need to be verified with manualChIP, and in all ChIP studies proper controls such as untagged strains need to be usedand demonstrated.atf1 null backgrounds and also evaluate H3K4me3 levelsgenome-wide in the presence and absence of Atf1.3) Co-localization studies do not represent co-recruitment. Please assess Set1 occupancyin WT and S. pombe, prominent heterochromatin regions includepericentromeres, subtelomeres, rNDA, and the silent mating type locus. Although 4) In clr3set1double mutant is not discussed. One possibility is that double mutant is defective inproduction of small RNAs that are critical for RNAi-mediated targeting ofheterochromatin. Additional experiments including changes in small RNAs might provideinformation about exact cause of the described changes in H3K9me. Moreover, the authorsshow widespread upregulation of genes in the double mutant but the biologicalsignificance of these changes has not been addressed. Is the double mutant defective instress responses or does it show developmental defects (such as untimely meiosis etc.)?Inclusion of such results may help connect changes in gene expression to biologicalprocesses.5) Set1 is proposed to act in parallel pathway to Clr3 to assemble H3K9meheterochromatin. However, the exact nature of defects in 6) The authors should clarify whether or not this new function of Set1 is trulyindependent of its catalytic activity or methylation of its normal substrate (H3K4).They can either use catalytically dead Set1  orH3K4R mutant (which is preferable).7) Introduction: in the sentence discussing heterochromatin islands, the authors shouldcite a paper by Zofall et al., Science, 335:96, 2012, that discusses dynamicheterochromatin domains in different parts of the genome. Similarly, the next sentencediscussing RNAi and exosome should include reference to Yamanaka et al., Nature,493:557, 2013. 1) An essential control in the ChIP-ChIP studies of Set1 is the use set1 nullbackground in Set1 ChIP-ChIP studies to verify the significance of the low signalsobserved.As pointed out by one of the reviewers, it is often a challenge to detect localizationof enzymatic proteins. However, due to the expected low signals of Set1 at certain loci,we have now performed additional manual ChIP experiments to verify Set1 localization.Because we performed ChIP-chip experiment of Set1 using a FLAG antibody against an epitope tagged Set1, we now include in our manual ChIP verification of Set1targets a control ChIP experiment against an untagged strain. These results are shown inAdditionally, you and co-authors need to show what percentagesof Set1 localize at active and repressed regions, respectively?We have now included analysis of the percentage of Set1 ChIP targets at active andrepressed loci. These results are shown in 2) Further, the result of ChIP-ChIP studies in the paper need to be verifiedwith manual ChIP, and in all ChIP studies proper controls such as untagged strainsneed to be used and demonstrated.atf1null strain. This result is present in atf1+ compared withatf1Δ, suggesting that the antibody binding is specific toAtf1. Moreover, our Atf1 ChIP-ChIP results are in high agreement with recently publishedChIP-ChIP data of Atf1 using antibody against an HA epitope of an Atf1-HA tagged strain.Please see our reply to question 1. In addition, because the Atf1 ChIP-ChIP experimentwas performed using a commercial antibody specific for endogenous Atf1, we now include anegative control ChIP experiment using the same antibody in an 3) Co-localization studies do not represent co-recruitment. Please assess Set1occupancy in WT and atf1 null backgrounds and also evaluate H3K4me3levels genome-wide in the presence and absence of Atf1.atf1Δ. We found that many loci exhibit reduced levels of H3K4me3in an atf1Δ strain. These data are provided in the new atf1 nullstrain compared with wild-type. However, we expect Atf1-mediated repressed loci toexhibit derepression and hence increased H3K4me3 levels in atf1Δstrain. We now include it in fbp1, srk1) that are known to be upregulated inatf1Δ and display increased H3K4me3 levels. A complete listof all such genomic regions are included in In addition to showing reduced Set1 enrichment at pericentromeric repeats, the rDNAarray, and ste11 , we have4) In S. pombe, prominent heterochromatin regions includepericentromeres, subtelomeres, rNDA, and the silent mating type locus.Althoughhas tried to classify the roles of Set1 and COMPASS in different classes ofgenes, it is still difficult to evaluate if any classes of genes are preferentiallyaffected by Set1 only or by COMPASS. The authors need to re-analyze the expressiondata based on those Set1-occupied genes/regions.spp1Δ andshg1Δ or sdc1Δ andash2Δ differ almost completely with upregulated probes in theset1Δ and other experiments), it is not possible to derive acommon subset of genes likely affected by the COMPASS only. This is probably due tocertain COMPASS subunits  having roles outside of the COMPASScomplex that could antagonize the function of COMPASS. However, in order to facilitate aclearer comparison between the classes of genes affected in the individualset1/COMPASS deletion strains summarized in set1/COMPASS deletion mutants.Because most of the upregulated probes are exclusive to individual experiments or groupsof experiments , vs. transcripts within heterochromatin regions. We have revised themanuscript to clarify this point.The most current Gene Ontology mappings provided by The enrichments of Atf1 in heterochromatin regions are quite clear; however, thecolor-coding makes the Set1 signals almost invisible inandWe have reformatted 5) Set1 is proposed to act in parallel pathway to Clr3 to assemble H3K9meheterochromatin. However, the exact nature of defects in clr3set1double mutant is not discussed. One possibility is that double mutant isdefective in production of small RNAs that are critical for RNAi-mediated targetingof heterochromatin. Additional experiments including changes in small RNAs mightprovide information about exact cause of the described changes inH3K9me.set1 and clr3. First, consistentwith the drastically reduced H3K9me levels in cells null for both set1and clr3, the double mutant also exhibits severe depletion of theHP1/Swi6 proteins at pericentromeric heterochromatin accompanied by increased levels ofPol II occupancy. These results are now shown in set1 and clr3 mutants. We found that whereas lossof either set1 or clr3 resulted in increased levels ofsiRNAs, the siRNA level in the set1Δclr3Δ double mutant was dramatically diminished relative towild-type. This is a likely consequence of the failure of the RNAi machinery requiringH3K9me to act in cis to contribute to heterochromatin assembly .This result is shown in We have performed several experiments to further elucidate the nature of functionalinteractions between Moreover, the authors show widespread upregulation of genes in the double mutantbut the biological significance of these changes has not been addressed. Is thedouble mutant defective in stress responses or does it show developmental defects(such as untimely meiosis etc.)? Inclusion of such results may help connect changesin gene expression to biological processes.set1Δ clr3Δ double mutant. These newresults are present in We have performed experiments that reveal defects in mating and sporulation in the6) The authors should clarify whether or not this new function of Set1 is trulyindependent of its catalytic activity or methylation of its normal substrate (H3K4).They can either use catalytically dead Set1 or H3K4R mutant (which is preferable).set1FH3K4me-) or its catalytic activity(set1-SETΔ). These new results are now shown in We have performed additional experiments that show Set1 localization at active andrepressed loci is not impaired due to the absence of H3K4 methylation(7) Introduction: in the sentence discussing heterochromatin islands, the authorsshould cite a paper by Zofall et al., Science, 335:96, 2012, that discusses dynamicheterochromatin domains in different parts of the genome. Similarly, the nextsentence discussing RNAi and exosome should include reference to Yamanaka et al.,Nature, 493:557, 2013.We have now included these references to the indicated sentences in the revisedmanuscript.


Xenopus embryos. Using α-amanitin to inhibit zygotic transcription, we find that the majority of H3K4me3- and H3K27me3-enriched regions form a maternally defined epigenetic regulatory space with an underlying logic of hypomethylated islands. This maternal regulatory space extends to a substantial proportion of neurula stage-activated promoters. In contrast, p300 recruitment to distal regulatory regions requires embryonic transcription at most loci. The results show that H3K4me3 and H3K27me3 are part of a regulatory space that exerts an extended maternal control well into post-gastrulation development, and highlight the combinatorial action of maternal and zygotic factors through proximal and distal regulatory sequences.Histone-modifying enzymes are required for cell identity and lineage commitment, however little is known about the regulatory origins of the epigenome during embryonic development. Here we generate a comprehensive set of epigenome reference maps, which we use to determine the extent to which maternal factors shape chromatin state in  Histone modifying enzymes are required for cell differentiation and lineage commitment during embryonic development. By a comprehensive set of epigenome reference maps of Xenopus embryos, the authors show that H3K4me3 and H3K27me3 exert an extended maternal control well into post-gastrulation development. Drosophila and Caenorhabditis through genome-wide profiling of chromatin marks5Drosophila and zebrafish (Danio rerio) ZGA starts after 8 and 9 mitotic cycles, respectively, in mammals transcription starts at the two-cell stage8Xenopus this happens after the first 12 cleavages at the mid-blastula transition (MBT)1011131415During early embryonic development cells differentiate, acquiring specific transcription and protein expression profiles. Histone modifications can control the activity of genes through regulatory elements in a cell-type-specific manner123Xenopus tropicalis embryos and assessed the need for embryonic transcription in their acquisition. We find a hierarchical appearance of histone modifications, with a priority for promoter marks which are deposited hours before transcription activation on regions with hypomethylated DNA. Surprisingly, the promoter H3K4me3 and the Polycomb H3K27me3 modifications are largely maternally defined (MaD), providing maternal epigenetic control of gene activation that extends well into neurula and tailbud stages. By contrast, p300 recruitment to distal regulatory elements is largely under the control of zygotic factors. Moreover, this maternal-proximal and zygotic-distal dichotomy of gene regulatory sequences also differentiates between early and late Wnt signalling target genes, suggesting that different levels of permissiveness are involved in temporal target gene selection.To explore the developmental origins of epigenetic regulation we have generated epigenome reference maps during early development of We have performed chromatin immunoprecipitation (ChIP) sequencing of eight histone modifications, RNA polymerase II (RNAPII) and the enhancer protein p300 at five stages of development: blastula (st. 9), gastrula st. 10.5, 12.5), neurula (st. 16) and tailbud (st. 30). These experiments allow identification of enhancers 1718191421220.5, 12.51424To determine the maternal and zygotic contributions to chromatin state, we used α-amanitin to block embryonic transcription . α-Amani1128Trimethylation of H3K4 and H3K27 has been associated with CpG density and a lack of DNA methylation. The Set1 and related MLL complexes are responsible for H3K4me3 ref. . Set1 is303113hoxd cluster which harbours a large hypomethylated domain with MaD H3K4me3 and H3K27me3 . The results predict specific transcription factors to recruit p300 in a stage-specific manner . Cluster3940Large enhancer clusters (ECs) are thought to improve the stability of enhancer–promoter interactions, are associated with genes coding for developmental regulators, and have been implicated in cell differentiation4243We next examined how MaD and ZyD p300-bound regions contribute to p300-bound ECs. Approximately 50% of all ZyD p300-bound enhancers are located in ECs at stage 11. Among MaD p300-bound enhancers this fraction is much reduced . Similarcis-regulatory landscapes of genes  genes which are expressed between blastula and tailbud stages but not expressed in oocytes or before the MBT, feature a MaD H3K4me3 promoter . Most ofTo explore the distinctions between expression inside and outside the maternal regulatory space, we analysed Wnt signalling targets. Early Wnt/beta-catenin signalling serves to specify dorsal fates following fertilization, leading to organizer gene expression. This has been shown to depend on Prmt2-mediated promoter poising before the MBTThe H3K4me3 modification poises promoters for transcription initiation by stabilizing Taf3/TFIID binding361347Gene expression outside maternal regulatory space could be mediated by p300-associated enhancers, most of which require new transcription for recruitment of p300. Promoter and enhancer activation in the ZyD regulatory space likely involves binding of specific factors. Indeed, we find that both MaD- and ZyD p300-bound regulatory regions recruit embryonically regulated transcription factors. Enhancers often contain binding sites for many different proteins, which can play different roles in opening up chromatin, recruitment of co-activators and establishing looping interactions with promoters. Future experiments will shed light on the maternal–zygotic hierarchy and the regulatory transitions underlying these events and the roles of maternal and zygotic pioneer factors. We find that ZyD p300-bound enhancers shape enhancer clusters. These form dense hubs of regulatory activity, and EC p300 binding is generally correlated with the expression of the associated genes. The work reported here suggests that recruitment of p300 to ‘seeding' enhancers precedes establishing cluster-wide activity of the local enhancer landscape. Future work will also need to address to which extent seeding causes relaxation and opening of the local chromatin and activity of neighbouring enhancers.Key proteins of the molecular machinery involved in DNA methylation , H3K4me3 , H3K27me3  and enhancer histone acetylation (p300) are not only highly conserved between species but also frequently mutated in cancer495052X. tropicalis embryos were obtained by in vitro fertilization, dejellied in 3% cysteine and collected at the indicated stage. Fertilized eggs were injected with 2.3 nl of 2.67 ng μl−1 α-amanitin and developed until the control embryos reached mid-gastrulation (stage 11). Animal use was conducted under the DEC permission  RU-DEC 2012–116 and 2014–122 to G.J.C.V.X. tropicalis genome JGI7.1, using STAR (RNA-seq) or BWA (ChIP-seq) allowing one mismatch.Chromatin for ChIP was prepared as previously described54Xenopus embryos stages 9 and 10.5 was obtained as described beforeGenomic DNA from odc1, eef1a1o, rnf146, tor1a, zic1, cdc14b, eomes, xrcc1, drosha, gdf3, t, tbx2, fastkd3, gs17  using iQ Custom SYBR Green Supermix (BioRad). We preformed RNA expression PCR (RT–qPCR (quantitative PCR)) and ChIP-qPCR for H3K4me3 and H3K9ac on promoters of q-value of 0.05. Fragment size was determined using phantompeakqualtoolsWe used MACS2 ref.  with staM-values >1 and a −log base 10 >5 (for H3K27me3) or 1.3 (for H3K4me3 and p300) and have a relative RPKM (background corrected) >1 in stage 11 control (no cut-off was used for st.11 control of H3K27me3 rep.1), stage 10.5 (H3K4me3 and p300) or stage 12 (H3K27me3); increased peaks have M-values smaller than -1 and a -log base 10 >5 (H3K27me3) or 1.3 (H3K4me3 and p300) and have a rel. RPKM greater than 1 in stage 11 α-amanitin, stage 10.5 (H3K4me3 and p300) or stage 12 (H3K27me3); unchanged peaks are neither gained nor lost and have a rel. RPKM >1 in stage 11 control (no cut-off was used for st.11 control of H3K27me3 rep.1), stage 11 α-amanitin, stage 10.5 (H3K4me3 and p300) or stage 12 (H3K27me3). Maintained peaks are peaks that are not lost and have a rel. RPKM >1 in stage 11 control (no cut-off was used for st.11 control of H3K27me3 rep.1), stage 11 α-amanitin, stage 10.5 (H3K4me3 and p300) or stage 12 (H3K27me3). Common lost, gained, unbiased and maintained peaks are present in both replicates. All other peaks are considered not defined (ND). Replicate-specific peaks were only used for We used MAnormDNA methylation levels in et al.RNA expression analysis was performed as previously publishedEnhancer clusters were called as previously describedMaD peaks emerge at or before stage 11 and are also acquired in α-amanitin treated embryos in both replicates. Zygotically defined (ZyD) peaks appear at or before stage 11 and are lost in α-amanitin treated embryos in both replicates, or emerge after stage 11. To classify MaD and ZyD H3K4me3 genes we ran MAnorm on promoters (±250 bp from TSS) only, using similar restrictions as described in ‘Detection of enriched regions'. MaD H3K4me3 genes have a maintained promoter in both replicates, ZyD H3K4me3 genes have a lost promoter H3K4me3 peak in both α-amanitin replicates, or a peak that emerges after stage 11. MaD H3K27me3 genes have at least one MaD peak in the vicinity of their promoter (±2.5 kb from TSS). ZyD H3K27me3 genes have at least one ZyD peak in their promoter and lack a MaD peak. ND peaks or genes do meet the criteria for neither MaD nor ZyD. For p300 the total number of ZyD and MaD peaks was counted in GREATChromatin states were discovered and characterized using ChromHMM v1.10 ref. , an implm × n matrix was created, where m corresponds to the 200 base pair intervals and n to the developmental stages (9–30). Each element a represents the chromatin state of interval i at stage j. For each chromatin group occurrences were counted per stage n. The changes between stage n and n+1 were plotted using Sankey diagrams ( https://github.com/tamc/Sankey), a flow diagram closely related to alluvial diagrams.The segmentation files of the seven main groups per stage were binned in 200 base pairs intervals. An et al.z-score activity that was >13 are shown in http://cisbp.ccbr.utoronto.ca/)http://dx.doi.org/10.6084/m9.figshare.1555851 (ref. For the prediction of motif contribution to p300 recruitment  we have 851 ref. .http://simonvh.github.com/fluff)http://cran.r-project.org/web/packages/gplots/index.html). For all heatmap clustering, the Euclidean distance metric was used. Other plots were generated using ggplot2 ( http://ggplot2.org/).All heatmaps were generated using fluff .How to cite this article: Hontelez, S. et al. Embryonic transcription is controlled by maternally defined chromatin state. Nat. Commun. 6:10148 doi: 10.1038/ncomms10148 (2015).Supplementary Figures 1-10 and Supplementary MethodsTranscription start sites of genes that have a MaD or a ZyD trimethylation of H3K4 or H3K27 at stage 11.Genes that are closest to ECs for each cluster of Figure 5d.Transcription start sites of genes that have a MaD, ZyD or ND trimethylation of H3K4 or H3K27 at one or more of the five developmental stages (9-30).


Cellular antiviral activities are critically controlled by transcriptional activation of interferon-inducible genes, involving interferon regulatory factors (IRFs). Previous data suggested that IRF1 is an activator and IRF2 is a repressor, which functionally antagonize each other in transcriptional regulation. However, it is not clear how these two factors function to regulate cellular antiviral activities.We show that IRF2 is critically required for the induction of the TLR3 and other interferon-inducible genes in a chromatin environment. While both IRF1 and IRF2 directly interact with the BAF chromatin remodeling complex, IRF2 is associated with the TLR3 promoter in the unstimulated state and IRF1 binding to the promoter is strongly induced by stimulation with interferon, suggesting that these two factors may function at different stages of gene induction in the recruitment of the BAF complex. IRF2 acts to maintain the basal level expression, an open chromatin structure, and active histone modification marks  of the TLR3 promoter in the unstimulated state, while IRF1 serves to rapidly activate the promoter upon stimulation.IRF1 and IRF2 of the IRF family of transcription factors play distinct roles in cellular response to viral infection. IRF2 binds to TLR3 and other IFN-inducible gene promoters and maintains an active chromatin structure in the unstimulated state, which is required for their induction, while IRF1 binding to these promoters activates their transcription upon viral infection. Thus, the division of labor between the IRF transcription factor family members plays a pivotal role in coordinating the transcriptional activation in the cellular antiviral response. It haans, has . Gene duans, has . IRF1 anans, has -8. Previans, has .TLR3 is a critical regulator of cellular antiviral activities . It recoTo gain more insight into how the innate and induced antiviral activities are controlled on gene expression levels, we studied the molecular mechanisms that control the expression of the TLR3 gene that is induced by viral infection. Both the basal level expression and induction of TLR3 require the chromatin remodeling activity of the SWI/SNF-like BAF complexes, since the knockdown of an essential subunit of the complex, BAF47, severely inhibited its expression -15. To cTo identify the DNA elements that mediate the BAF complex activity, 5′-deletion analysis of the TLR3 promoter was performed. The 509 bp DNA fragment of promoter was responsive to IFN-α stimulation and to the BRG1 expression  into appropriate restriction sites in the pREP4-luc  and pGL3The oligonucleotides for Sp1 and IRF-E site mutations are listed below:The Oligonucleotides used for shRNA constructs are listed below:Anti-IRF1, anti-IRF2, anti-TLR3, anti-BRG1, anti-hBRM, anti-BAF47(home made see paper), anti-p48, anti-Stat1, anti-Stat2, anti-PloII , anti-CBP, anti-H3K4me3, anti-H3k9ac/14 ac , anti-H3.SW-13 cells and HeLa cells were maintained in DMEM supplemented with 10% fatal calf serum and 1% penicillin-streptomycin. The THP-1 cells were maintained in ATCC-formulated RPMI-1640 Medium  supplemented with 10% fatal calf serum and 1% penicillin-streptomycin. Transfections of SW-13, HeLa cells, and THP-1 cells were performed using Superfect (Qiagen) as instructed by manufacture. The cells were selected in 1 μg/ml puromycin for 48 hours before stimulation with 500 Units/ml IFN-α for 12 hours for chromatin immunoprecipitation and TLR3 gene expression analysis.TaqMan® Gene Expression Assays for TLR3 and IFN-β gene were ordered from ABI . RT-PCR analyses were performed as described previously , by usinCCGCCCACATCAAATGGT-3′, Reverse primer: 5′-GAAAGGGTCACAGATTTAGCAACA-3′, Probe: 5′-CCCACTTTCAACTTTAG-3′ were used. For the LTR3 gene extron II region: Forward primer 5′-GTGCATCCTCCACCACCAA-3′, Reverse primer 5′-TCGGGTACCTGAGTCAACTTCA-3′, and Probe: 5′-TGCACTGTTAGCCATGAAGTTGCTGACTG-3′ were used. For β-globin gene locus:ACAGTGTGGCGATTCCTCAAG-3′, Reverse primer: 5′-GTAATGGGATTGCTGGGTCAA-3′, Probe: 5′-ATCTAGAACCAGAAATACTG-3′ were used.Forward primer: 5′-The chromatin immunoprecipitation assays were formed as described . For the


Candida glabrata has become the second cause of systemic candidemia in humans. However, relatively few genome-wide studies have been conducted in this organism and our knowledge of its transcriptional regulatory network is quite limited. In the present work, we combined genome-wide chromatin immunoprecipitation (ChIP-seq), transcriptome analyses, and DNA binding motif predictions to describe the regulatory interactions of the seven Yap (Yeast AP1) transcription factors of C. glabrata. We described a transcriptional network containing 255 regulatory interactions and 309 potential target genes. We predicted with high confidence the preferred DNA binding sites for 5 of the 7 CgYaps and showed a strong conservation of the Yap DNA binding properties between S. cerevisiae and C. glabrata. We provided reliable functional annotation for 3 of the 7 Yaps and identified for Yap1 and Yap5 a core regulon which is conserved in S. cerevisiae, C. glabrata, and C. albicans. We uncovered new roles for CgYap7 in the regulation of iron-sulfur cluster biogenesis, for CgYap1 in the regulation of heme biosynthesis and for CgYap5 in the repression of GRX4 in response to iron starvation. These transcription factors define an interconnected transcriptional network at the cross-roads between redox homeostasis, oxygen consumption, and iron metabolism.The yeast  Candida glabrata is a unicellular yeast from the Hemiascomycetes phylogenetic group and a commensal host of the human mucosal microbiota. However, in patients with severe immunodeficiency, it can cause invasive systemic infections, with high mortality rates (about 40–60%). While Candida albicans remains the main cause of systemic candidiasis (50–70%), C. glabrata ranks second (20–25%), and its prevalence has increased in the last decades  have been analyzed by genome-wide approaches and comprehensive network-based models are available for ribosome biogenesis, iron homeostasis, or biofilm formation  family in C. glabrata has 7 Yap members, which slightly differ from the S. cerevisiae family (Supplementary File CAGL0H04631g), Yap2 , Yap5 , and Yap7  are present, but Yap8 is absent. Two versions of Yap3  and only one ortholog for the Yap4 and Yap6 pair  are found. Only three of these Yaps have been studied previously in C. glabrata. CgYap1 has been shown to be involved in oxidative stress response, with a set of targets which is significantly conserved compared to ScYap1, but with different DNA binding preferences .All cultures were conducted in a rotative shaker at 30°C in YPD . Stress conditions used were: 1 mM sodium selenite, 1 M NaCl, 2 mM cadmium chloride, 5 mM iron sulfate, or 0.5 mM bathophenanthroline disulfonate (BPS). These doses were chosen, based on preliminary microarray and growth assay experiments, to induce a transcriptional response in the wild type without causing significant differences of growth rates between the wild type and the CgYAP strains were grown in YPD until exponential phase (OD = 0.8) and then stressing agents were added for 30 min. Cross-linking of the cells and ChIP were performed as described previously .For ChIP, myc-tagged C. glabrata Agilent arrays in an 8 × 60 k format (array express accession number: A-MEXP-2402). After overnight hybridization and washing, the slides were scanned using a 2-micron Agilent microarray scanner. The images were analyzed using the feature extraction software (Agilent technologies) and normalized using global LOESS  and then stressing agents were added. After 30 min, 20 mL of each cell cultures were flash-frozen in two volumes of cold ethanol and collected by centrifugation. The OD of the cultures were monitored before the stress treatment and every 30 min for 2 h after stress treatment. Only samples from wild type and knock-out cultures which showed similar growth rates (±10%)  using the “getfasta” function from the BEDTOOLS suite  for Yap1 : ScYap1, ScYap2, ScYap5, and ScYap7 bind to YRE-O (TTACTAA) motifs while ScYap3, ScYap4, and ScYap6 rather recognize YRE-A (TTACGTAA) motifs  Figures . This prBecause we included transcriptome data in our network, we could determine the directionality  of some of the regulatory interactions set up by the different Yap proteins Figure . This alTRR1, TRX2, OYE2, GPX2, TSA1, CTA1, SRX1, …). Its target set was also enriched in genes involved in heme biosynthesis . CgYap5 target list was clearly associated to iron sulfur cluster binding  and iron homeostasis . The only GO category to be enriched in CgYap7 targets was iron sulfur cluster metabolism. This includes genes involved in the cytosolic and mitochondrial iron sulfur assembly pathway  and in iron sulfur cluster binding . CgYap7 also targets 10 genes encoding oxidoreductases  and genes encoding heme containing proteins  or related to heme metabolism . The targets of CgYap1 and CgYap7 included many genes encoding transcription factors. For instance, several transcription factors involved in oxygen homeostasis and oxidative stress responses are targeted by CgYap1 . Remarkably, CgYap1 and CgYap7 both bound their own promoter, suggesting auto-regulation (Supplementary Table We performed Gene Ontology (GO) term enrichment analyses on the whole set of genes in the network and on the individual sets of target of each CgYap Figure . The whoS. cerevisiae and C. albicans .The functional annotation presented above pointed out a CgYap1/CgYap5/CgYap7 network centered on iron and redox homeostasis. To assess the conservation of this sub-network we compared the targets of CgYap1, CgYap5, and CgYap7 with the targets of their orthologues in a Figure . Remarka3 Figure . Hap43 aOYE2, TRX2, TRR1, CTA1, GPX2, GSH1, ZWF1, CCP1), the MFS permease FLR1  and, remarkably, Yap1 itself, suggesting that the auto-regulation of Yap1 may actually play a significant role in its function. Besides this relatively high conservation of the Yap1 regulon in yeasts, a remarkable specificity of CgYap1 is its role in the direct regulation of several genes encoding enzymes of the heme biosynthetic pathway, a feature which was documented neither in S. cerevisiae nor in C. albicans.Among the 82 targets that we identified for CgYap1, 28 are orthologous to targets of ScYap1 and 20 to Cap1 targets Figures . Of thesC. glabrata wild type cells to various environmental stresses. The list and origin of the transcriptome data which were used can be found in the methods. As could have been expected from the results presented in the previous chapters, the CgYap1 targets were significantly enriched among the genes induced by oxidative stress causing agents  or through heme biosynthesis (HEM3).Similarly to CgYap1, CgYap5 has a conserved role in the iron stress response. In GRX4 in these conditions. As mentioned above, Grx4 inhibits the iron starvation response by promoting the nuclear export of Aft1/2 when iron-sulfur clusters are abundant  and CgYAP1 deletion clearly impacted the expression of three of them . Hence, we defined here a CgYap1/CgYap5/CgYap7 network deeply involved in oxygen and iron sensing by tuning redox homeostasis, heme biosynthesis, iron storage, and iron sulfur cluster metabolism  came out as a “hub” in this network, being positively regulated by CgYap1 and CgYap5 in response to oxidative stress or iron excess, respectively, and repressed by CgYap7 in standard growth conditions (Figure CCP1, encoding the cytochrome c peroxydase which acts as a heme-based sensor of the mitochondrial oxidative stress (Martins et al., In S. cerevisiae for being involved in the regulation of hypoxic genes and oxygen consumption (Rox1 and Ixr1 for CgYap1 and Mot3, Hap1 and Sut2 for CgYap7; Figure SKN7, which encodes a regulator of oxidative stress response known to cooperate with Yap1 both in S. cerevisiae and in C. glabrata (Morgan et al., C. glabrata hierarchy of transcription factors (Jothi et al., Another interesting feature of CgYap1 and CgYap7 is the strong enrichment for transcription factors among their targets. For instance, they potentially control the expression of several transcription factors known in JM constructed the strains and performed the ChIP-seq and transcriptome analyses. AT performed transcriptome analyses. CB, JP, MA, and SL performed the high-throughput sequencing. GL and JC performed the bioinformatics analyses and the network building. FD designed the experiments and wrote the manuscript.This work was supported by the Agence Nationale pour la Recherche  and the France Genomique infrastructure as part of the “Investissements d'Avenir” program [ANR-10-INBS-0009].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


MUTE promoter contains a 175-bp region rich in Dof regulatory elements (AAAG) that is necessary and sufficient for initiation of transcription in meristemoids and the stomatal lineage.The MUTE triggers the differentiation of meristemoids into stomata. In this study, we carried out MUTE promoter deletion analysis to define a regulatory region that promotes the initiation of expression in meristemoids. Expression constructs with truncated promoter fragments fused to β-glucuronidase (GUS) were developed. The full-length promoter and promoter truncations of at least 500 bp from the translational start site exhibited normal spatiotemporal expression patterns. Further truncation revealed a 175-bp promoter fragment that was necessary and sufficient for stomatal-lineage expression. Known cis-elements were identified and tested for functional relevance. Comparison of orthologous MUTE promoters suggested DNA binding with one finger (Dof) regulatory elements and novel motifs may be important for regulation. Our data highlight the complexity and combinatorial control of gene regulation and provides tools to further investigate the genetic control of stomatal development.The molecular mechanism underlying the decision to divide or differentiate is a central question in developmental biology. During stomatal development, expression of the master regulator The online version of this article (doi:10.1007/s00425-015-2445-7) contains supplementary material, which is available to authorized users. Stem cells are essential for the production of cellular and tissue diversity across all organisms. Stem cells have the ability to divide asymmetrically to produce two daughter cells with different fates; one cell retains stem-cell characteristics, while the other may differentiate  has also been implicated in meristemoid differentiation. Weak spch mutants produce meristemoids that undergo fewer divisions and prematurely differentiate into a GMC  proteins in the differentiation of stomatal-lineage cell types. One of these proteins, ata Fig. a, b  binding elements within this region in orthologous genes, suggesting they may be important for regulation. Together, these findings further our understanding of the molecular mechanisms involved in gene regulation and provide tools to identify specific proteins that bind the MUTE promoter and regulate the differentiation of meristemoids.The molecular basis of expression of some stomatal regulatory components has been investigated and broad microarray analyses have revealed novel stomatal-lineage specific genes and potential DNA binding sites for some stomatal regulators  were used for transformation. Plants were grown under a 16 h/8 h light/dark cycle at 22–24 °C. All transformation constructs were transferred to Agrobacterium tumefaciens strain GV3101 by electroporation. Transformation of Arabidopsis was done by Agrobacterium-mediated transformation using floral dip method  according to manufacturer’s instructions. Briefly, total RNA was dephosphorylated with calf intestinal phosphatase to eliminate RNAs that were not full length. Dephosphorylation with tobacco acid pyrophosphatase was used to remove the cap structure of full-length mRNAs and the GeneRacer RNA oligo was ligated to the 5′ end of the transcript. The RNA was reverse transcribed using the GeneRacer oligo dT primer and Superscript II Reverse Transcriptase. Nested PCR was used for both 5′ and 3′ end amplification. Nested PCR conditions used for amplification were as follows: ; 5 cycles at ; 5 cycles at  and 23 cycles at  using the standard primers from the kit and gene-specific primers listed in Suppl. Table S1. Amplified products were cloned into TOPO pCR4 TA cloning vector according to manufacturer’s instructions and sequenced using an M13F primer.MUTE promoter designated in this study includes 1954 bp of promoter sequence immediately upstream of the translation initiation codon . Oligos were resuspended at the same molar concentration in annealing buffer . For annealing, oligos were combined in equimolar amounts, heated to 95 °C for 5 min followed by slow cooling (1 °C/min) to reach room temperature. Annealed oligos were used directly in a ligation reaction with EcoRI-digested LJP138.For promoter complementation using the 175-bp fragment, primers were designed to amplify −500 to −325 bp of the cis-element were gel-purified using an UltraClean PCR Clean-Up Kit  and cloned into TOPO pENTR (Invitrogen) according to manufacturer’s instructions. To produce GFP reporter constructs, vectors were combined with pGWB4 destination vector  as described previously  and PlantCARE . These two programs are available at http://www.dna.affrc.go.jp/PLACE/ and http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, respectively. Alignment of orthologous promoters was performed using Clustal Omega with manual adjustments.Regulatory elements in the MUTE, we were interested in accurately mapping the TSS and polyadenylation sites of the MUTE transcript. To this end, we performed 5′ and 3′ RACE (rapid amplification of cDNA ends). Our results indicate that MUTE has an invariable TSS (n = 9) 29 bp downstream of the TATA-box consensus sequence (TATAAAT). The 5′ UTR is 85 bp in length, which is consistent with the trend for short 5′ UTRs in TATA-box-containing promoters in Arabidopsis (Kawaguchi and Bailey-Serres Untranslated regions (UTRs) of mRNA transcripts are important contributors to transcript stability, spatial and temporal expression patterns, and translation efficiency  has been analyzed previously and shown to be specific to the stomatal lineage and hydathode. No expression outside the stomatal lineage has been observed using the full-length promoter  reporter gene  to the 35S cauliflower mosaic virus (CaMV) minimal promoter (−46 to +8 bp relative to TTS) and analyzed its ability to drive GUS expression. This region of the CaMV promoter has been demonstrated to function as a core binding site  for its ability to initiate meristemoid-specific expression . This region was chosen based on our promoter deletion analysis, where the 411-bp promoter displayed very weak activity , Dc3 promoter binding factor element (DPBF), GT2, L1-Box, and several Dof binding motifs . For this analysis we focused on elements in the region between −500 and −411 bp because they appear to be necessary for robust expression , has also been shown to bind the MUTE promoter  was conserved among all four orthologous promoters. Clusters of AAAG sites have been shown to additively contribute to guard cell-specificity of AtMYB60 . b 1305 bp of the MUTE promoter driving GFP. Promoter length is relative to the translational start site (+1). Cell borders are stained with propidium iodide. Scale bar 10  µm (TIFF 6131 kb)Suppl.Fig.S2 Regulatory region complementation. a Diagram of the complementation construct. The MUTE promoter fragment from −411 to −325 bp was fused to the 35S CaMV minimal promoter (−46 to +8 bp)  and complementation construct driving GUS expression (c). Scale bar 10 µm for epidermal images (TIFF 8236 kb)Suppl.Fig.S3 Site-directed mutagenesis. a Sequence of the MUTE promoter fragment; location of specific elements are indicated. Nucleotide position is relative to translational start site (+1). b–d Changes made to designated elements are indicated. All nucleotide substitutions were made in the context of a functional >522-bp promoter (TIFF 12019 kb)Supplementary material 4 (DOCX 40 kb)Supplementary material 5 (DOCX 53 kb)Supplementary material 6 (DOCX 63 kb)Supplementary material 7 (DOCX 79 kb)Below is the link to the electronic supplementary material.


Plants commit to flowering based on endogenous and exogenous information that they can remember across mitotic cell divisions. Here, we review how signal perception and epigenetic memory converge at key integrator genes, and we show how variation in their regulatory circuits supports the diversity of plant lifestyles. Flowering at the appropriate season and age is crucial for reproductive success as open flowers are particularly sensitive to adverse climatic conditions and seed maturation is costly in terms of energy and nutrient consumption. Furthermore, cross-pollination depends not only on coordinated flowering between individuals of the same species but sometimes also on the presence of appropriate pollinators. Synchronization of the plant life cycle with the sequence of seasons depends on the detection of environmental signals such as photoperiod and temperature, of which in particular the latter requires integration over several weeks to provide robust information. In many plant species, the response to these external signals is gated by endogenous factors such as sugars and hormone levels that are directly or indirectly related to developmental age –3. Thus,Arabidopsis thaliana. Finally, we explain how plants have diversified the cross-talk and regulatory connections between these main flowering pathways to support their great diversity of lifestyles.In the following, we discuss the pathways dependent on photoperiod, aging and vernalization, which are commonly part of the decision-making process for flowering. We set these pathways in the context of land plant evolution as this sets the frame for the expected conservation of molecular mechanisms contributing to their regulation. We then summarize the state-of-the-art on the regulation of these pathways, mostly based on studies performed in the model plant trans-differentiate from a vegetative state, during which they produce leaves and lateral meristems, to a reproductive state, resulting in the production of inflorescences and flowers. To time flowering towards the appropriate season, photoperiod pathways track either day length or night length and, when a critical value is exceeded, induce florigen, a mobile signal produced in leaves that causes the reproductive transition in the meristems  that can be bound by CO in vitro , which encodes a jumonji domain protein with H3K27me2/me3 de-methylase activity, results in increased FT expression and early flowering [A. thaliana, H3K27me3 at the FT locus is not restricted to the gene body but spreads into the promoter and downstream regions [FT expression in leaves of wild-type but not lhp1 and clf mutant plants [Accurate timing of flowering relies on proper temporal and spatial control of n detail , 44, 45.on of FT , 9, 46. on of FT . LHP1, won of FT , 46. In lowering . Differe regions . While B regions , the prot plants , 51.FT involves at least temporal conversion of chromatin from a repressive to a permissive signature. Dynamic changes in H3K27me3 and the presence of chromatin modifications positively correlated with expression are difficult to detect at FT in wild-type plants probably owing to its restricted expression domain [FT expression involves chromatin factors. First, Morf Related Gene (MRG) chromodomain proteins MRG1 and MRG2 directly bind to the intermediate-proximal FT promoter [FT locus, the interaction establishes an active chromatin signature of upregulation of FT expression by CO in LD conditions  containing a jumonji C (JmjC) domain [FT in phloem companion cells during the night [FT downstream of the photoperiod pathway [FT induction emerges in which conversion of the FT locus from an FLC/FLM–EMF1–JMJ14-dependent repressive state to an active state is mediated through replacement of EMF1c by a CO–MRG1/MRG2–HAM1/HAM2 module at the promoter region.As ) domain . A recenhe night . This no pathway . Taking FT [FT locus. EFM interacts with the H3K36 demethylase JUMONJI 30 (JMJ30) and, by binding to a region of the FT promoter downstream of BlockC, recruits JMJ30 to the FT locus, where it accumulates in the 5′ regions of the gene [jmj30 or efm mutants, the H3K36me2 modification accumulates in the region associated with JMJ30, and FT expression is increased at its peak time in LDs.Chromatin modifications are directly and indirectly involved in repression of FT mediated through the MADS domain factor. First, FLC and FLM interact with EMF1c, which might affect targeting of either complex to FT . Second,the gene . In jmj3FT induction is LDs, FT expresses at sufficient levels to accelerate flowering if plants are grown at high ambient temperature in SDs [FT response to high ambient temperature can in part be explained by the decreased repressive effect of SVP and FLM [FT [Although the main signal for e in SDs . Such a  and FLM –38. In a FLM [FT , 60. Gen FLM [FT .FT in SDs is also observed in the phloem of developing siliques (fruits) [FT expression in siliques does not require the distal FT enhancer BlockC, indicating that a different enhancer region might be controlling FT expression at this late developmental stage [Expression of (fruits) . Express(fruits) , 62. Simal stage .FT paralogs , lacks the distal enhancer BlockC but shows extensive conservation of the proximal promoter [TSF contributes only marginally to flowering time under controlled greenhouse growth conditions but colocalizes with quantitative trait loci (QTLs) for flowering time under field conditions [TSF is generally co-regulated with FT but expressed at much lower levels, which might be explained by either the absence of the enhancer or the presence of several heterochromatic transposable elements (TEs) in close proximity downstream [FT ortholog in Brassica rapa, which features six paralogs [In the course of evolution, plant families show a tendency to increase the number of genes encoding ews, see , 20). Nopromoter . TSF connditions , 64. TSFwnstream . TE inseparalogs .FT paralog neo-functionalization has been reported in sugar beet, where a limited number of point mutations converted one Beta vulgaris FT paralog from florigen to anti-florigen [FT1 is expressed in SDs and in biennial accessions also in LDs before vernalization. The florigen BvFT2 is expressed only in LDs after BvFT1 expression has been permanently suppressed by vernalization.An interesting case of florigen . The negA. thaliana.Floral induction by photoperiod and/or high ambient temperature can be counteracted by the age-dependent pathway, which prevents flowering in juvenile plants that have not yet acquired enough resources and by the vernalization pathway that prevents flowering in late summer or autumn before vernalization. In the following section, we outline the molecular mechanisms underlying the vernalization and the age-dependent pathways in miR156, which target mRNAs of several SQUAMOSA-BINDING-PROTEIN-LIKE (SPL) transcription factors for degradation and/or suppress their translation [A. thaliana, SPL transcription factors can promote flowering directly by inducing the expression of positive regulators of the reproductive transition, such as the MADS domain transcription factors SOC1, FUL and AP1, which are most effective in meristems [FT in the phloem companion cells of the leaves [SOC1, FUL and AP1, miR156 is the basis for a feed-forward loop inducing the reproductive transition [FT, SOC1, FUL and AP1 are also indirectly regulated by SPLs, which induce the expression of genes encoding miR172 [miR172 RNAs target AP2 and the related factors SMZ, SCHNARCHZAPFEN (SNZ) and TARGET OF EAT 1 to TARGET OF EAT 3 (TOE1–TOE3), which collectively act as direct transcriptional repressors of genes promoting the floral transition [miR156 genes is gradually downregulated during the first weeks of development [A. thaliana, the MADS domain transcription factors AGL15 and AGL18 form a heterodimer that might directly activate transcription of primary (pri)-mirRNA156a/c. Accordingly, agl15;agl18 double mutants show an early-flowering phenotype [Two connected microRNA (miRNA) and target transcription factor modules act as developmental timers in many plant families Figs.  and 4 6. Severalnslation , 71, 72.eristems . SPLs ale leaves . As the ansition . Furtherg miR172 . miR172 ansition , 76, 77.elopment , 78–80. henotype . As AGL1miR156 and miR172 as well as their target loci is likely to be influenced by the PcG pathway, given the high number of H3K27me3-positive loci within the network [miR156a and miR156c are dependent on the function of AtBMI1A and AtBMI1B, which are part of canonical PRC1 complexes. Age-dependent silencing of pre-miR156a and pre-miR156b is impaired in atbmi1a atbmi1b double mutants, leading to a prolongation of the juvenile phase [miR172 repression in the juvenile phase [Expression of  network . A recenle phase . By contle phase .SOC1 in the shoot apex and FT in the phloem companion cells of leaves [FLC expression are dependent on the activity of the FRIGIDA complex (FRI-C) that is recruited by EARLY FLOWERING IN SHORT DAYS (EFS) [FLC [FLC transcription requires recruitment of the pre-initiation complex (PIC) comprising RNA polymerase II (RNAP II) as well as general transcription factors and accessory factors [FLC also requires the catalysis of the co-transcriptional H3K36me2/3 mark at the gene body, implemented by SET2 domain histone methyltransferases [To prevent flowering in unfavorable seasons, such as cold winters or dry summer periods, many plant species possess an effective block for their response to promotive signals that has to be removed by vernalization . In the f leaves  Fig. 4)SOC1 in tYS (EFS)  and consYS (EFS) , 86. FRIFS) [FLC , 87. Ind factors . Assembl factors , 89. Tra factors , 90. H2B factors . An actisferases .FLC mRNA transcriptional activity is accompanied by transcription of noncoding RNAs (ncRNAs) from the FLC locus  is downregulated during vernalization, which allows ‘mature’ shoot meristems to transition to inflorescences that form floral primordia during the cold period [PEP1 and AaSOC1 in the cold. The inability of SOC1 to trigger the transition is in part explained by higher levels of the anti-florigen AaTFL1 in young versus more mature meristems [PEP2, a paralog of miR172-controlled AP2, is involved in the regulation. Loss of PEP2 function causes a phenotype similar to that of pep1 mutants, namely flowering without a requirement for vernalization and without seasonal cessation — thus, perpetually [A. alpina plants, AP2 and miR172 are controlled by both age and vernalization as prolonged cold increases miR172 levels only in mature plants but not in juvenile plants that express still high levels of miR156. A decrease in miR156 and a subsequent increase in a group of SPL genes are observed as meristems age. Thus, in A. alpina, the decrease of miR156 is not directly coupled to an increase in miR172 but further requires vernalization, whereas vernalization is ineffective before the decrease of miR156.Expansion of research to other Brassicaceae has revealed common principles as well as different strategies of flowering time regulation in the d period , 112. And period . Althougeristems . In addipetually . In wildC. flexuosa shows a variation in the connection between the aging and vernalization pathways. Here, the coupling between the miR156–miR172 regulons seems direct and not further gated by vernalization [C. flexuosa, increased SOC1 expression is correlated with inflorescence development, in contrast to A. alpina, where both can be uncoupled.Regulation in the related perennial species lization . Furtherpep mutants flower continuously once they have passed the juvenile stage. Thus, PEP1 and PEP2 play a dual role in preventing the floral transition before vernalization as well as beyond the end of the reproductive season. PEP1, in contrast to its annual ortholog FLC, is not stably silenced after vernalization but upregulated after the return of warmer temperatures [COOLAIR antisense transcripts are induced in both species in the cold, whereas COLDAIR is not detected in A. alpina [PEP1 shows a much higher and broader increase of H3K27me3 during the cold than FLC, for which the increase is mainly restricted to the nucleation region at the TSS [PEP1, whereas they increase further across the gene body at FLC [As indicated by their name, perpetual flowering eratures . Differe. alpina . However the TSS . Differey at FLC .PEP1 and FLC regulation are explained by cis or trans effects. A genomic PEP1 fragment that was stably repressed after vernalization was poorly expressed in A. thaliana even before vernalization, suggesting that both cis and trans differences exist between the species. Candidates for trans-components are, for example, the more specialized PcG components VRN2 and VRN1 [FLC expression. In A. thaliana, resetting of the epigenetic memory of FLC occurs in the embryo and is dependent on the presence of TrxG components [It is not yet known whether differences in and VRN1 , as wellmponents –117 and mponents .Brachypodium distachyon , making it worthwhile to discuss differences and commonalities. The impact of the aging pathway on flowering is less studied in temperate grasses, but there is some indication that it participates in the response to ambient temperature in B. distachyon. In this species, the expression of miR156 is upregulated in transgenic lines possessing reduced levels of BdVIN3-LIKE 4 [miR156 was stronger in plants grown at low ambient temperature and correlated to late flowering. BdVIL4 encodes a PHD domain protein related to A. thaliana VIN3, which is required for the epigenetic memory of FLC repression during vernalization [Flowering pathways in temperate grasses (Poaceae) diverge from the established Brassicaceae models. The requirements for photoperiod induction and vernalization are common and well investigated in the grasses barley, wheat and 3-LIKE 4 . The uprlization .CO regulation is mediated through the circadian clock, involving PPD1, and CO protein levels are highest and sufficient for VRN3 induction at the end of LDs, which is similar to the situation for A. thaliana [AP1 (VRN1), CCT domain proteins (VRN2) and FT (VRN3) is involved in the vernalization response [VRN3 induction in LDs is prevented by VRN2. Wheat VRN2 interacts with NF-Y complexes, similarly to the A. thaliana CCT domain protein CO [VRN2 or VRN3–VRN1 [VRN1, which gradually increases in expression during the cold, resulting in downregulation of VRN2 in leaves. This allows VRN3 to respond to LDs, leading to a ‘secondary’ strong upregulation of VRN1.The three main players of the photoperiod-mediated flowering response in barley are the pseudo-response regulator CCT domain protein PPD1, a barley CO homolog and the FT-related protein VRN3. Barley thaliana . Winter response . Before otein CO . Thus, iRN3–VRN1 . The intVRN3 [VRN1 alleles in spring wheat and barley cultivars are expressed before vernalization [VRN1 slowly decreases, whereas H3K4me3 and H3 acetylation increase, concomitant with increased VRN1 expression. The active chromatin state and increased VRN1 levels are maintained after a return to warm temperatures. This could be attributable to an effect downstream of the induction of VRN1 by VRN3 in LDs, the repression of VRN2 in SDs or explained by an epigenetic memory due to the presence of TrxG complexes. From an evolutionary perspective, it is interesting to note that the vernalization memory system in cereals promotes an active, instead of a repressive, epigenetic state as in Brassicaceae. This exemplifies that evolution of vernalization memory systems cannot only involve different players of the genetic flowering time network but also engages ambient temperature-sensitive chromatin-modifying pathways in a versatile manner.Barley and wheat variants not requiring vernalization have been selected to allow planting in spring. The dominant wheat TaVRN3-Hope allele is associated with a TE insertion in the promoter region that bypasses the repressive effect of VRN2 on VRN3 . In addilization . Small ilization . During Data from an increasing number of species are becoming available, and we find it helpful to consider evolutionary aspects to detect common principles and separate them from family-specific or species-specific complexity that can become quite confusing. A judicious choice of closely related annual and perennial species within the Brassicaceae has given insight into the connection between the different modules engaged in the regulation of flowering time. However, more models, in particular representing basic angiosperms and seed plants, could further advance our knowledge of the control of flowering. Vernalization pathways, as the latest addition to flowering time control, appear to recruit different key regulators and show considerable variation even within plant families. However, molecular memories implemented by PcG and TrxG complexes are a common denominator of the vernalization response in families as distant as Poaceae and Brassicaceae. Vernalization pathways evolved rapidly also in other families, where fewer molecular details are known. This rapid adaptation of plants to temperate climates could have been facilitated by an inherent sensitivity of chromatin and chromatin-modifying pathways to fluctuations in ambient temperature. The possibility to study and compare the response of chromatin to ambient temperature on a genome-wide and comparative level will answer whether, and to what extent, the chromatin is a holistic plant ‘thermometer’.


Chlamydiae feature a compact regulatory genome that remains uncharted owing to poor genetic tractability. Exploiting the reduced number of transcription factors (TFs) encoded in the chlamydial (pan-)genome as a model for TF control supporting the intracellular lifestyle, we determined the conserved landscape of TF specificities by ChIP-Seq (chromatin immunoprecipitation-sequencing) in the chlamydial pathogen Waddlia chondrophila. Among 10 conserved TFs, Euo emerged as a master TF targeting >100 promoters through conserved residues in a DNA excisionase-like winged helix-turn-helix-like (wHTH) fold. Minimal target (Euo) boxes were found in conserved developmentally-regulated genes governing vertical genome transmission (cytokinesis and DNA replication) and genome plasticity (transposases). Our ChIP-Seq analysis with intracellular bacteria not only reveals that global TF regulation is maintained in the reduced regulatory genomes of Chlamydiae, but also predicts that master TFs interpret genomic information in the obligate intracellular α-proteobacteria, including the rickettsiae, from which modern day mitochondria evolved.Like other obligate intracellular bacteria, the  Chlamydiae encode far fewer TFs in their reduced genomes, making them ideal models for determining the ‘regulatory pan-genome' during intracellular growth. We defined here the regulatory pan-genome as the genomic regulatory sites that are targeted by TFs common to all members of the phylum Chlamydiae. In other bacteria, the regulatory program implements stochastic and/or deterministic cell fate and transcriptional switches that promote productive acute or chronic infections of host cells  transcriptional factors (TFs) that directly coordinate the activity of hundreds of genes by binding to their promoters   have a larger genome and therefore presumably an expanded metabolic capacity  were cultivated in Dulbecco's modified minimal essential medium  supplemented with 10% fetal bovine serum , at 37 °C in the presence of 5% COWaddlia chondrophila strain ATCC VR-1470 was co-cultivated at 32 °C with Acanthamoeba castellani strain ATCC 30010 in 75 cm2 flask containing 30 ml of peptone-yeast-extract-glucose broth. After 7 days of culture, the suspension was filtered on 5 μm filter to eliminate trophozoites and cysts and to isolate bacteria in the filtrate. The filtrate was then diluted at the appropriate dilution in DMEM to proceed to infection of Vero cells.5 Vero cells were seeded per well in 24-well plates. A 1/2000 dilution of W. chondrophila was used to infect Vero cells. This corresponded to a multiplicity of infection 2–3, as estimated by qPCR. This infectious dose led to 50% of infected cells with 2–3 bacteria, as determined by confocal microscopy. The infected Vero cell suspension was centrifuged at 1790 g for 10 min at room temperature and then incubated for 15 min at 37 °C with 5% CO2. To remove non-internalized bacteria to obtain a synchronous infection, cells were washed with the DMEM. Then, infected cells were incubated for different time periods.One day before the infection, 5 × 10m of primers WadF4 and WadR4, 100 nm of the probe WadS2 and 5 μl of DNA. The qPCRs were performed on the Step-One system  using the following cycling conditions: 3 min at 95 °C, 40 cycles of 15 s at 95 °C and 1 min at 60 °C.The number of bacteria at different time post-infection was determined using real-time quantitative PCR (qRT-PCR). Infected cells were recovered after cell scraping. Genomic DNA extraction was performed on 50 μl of cell suspension using the WizardSV Genomic DNA purification kit  and eluted in 100 μl. The quantitative PCR was performed as described previously using iTaq supermix with ROX  hexane  a crosslinking agent. When indicated, 80 ng of PCR amplified DNA fragment was added to the reaction mix. Four μl of 6 × denaturing Laemmli loading dye was added to each sample. The samples were then separated by SDS-PAGE on a 12% polyacrylamide gel (Bio-Rad) and transferred onto nitrocellulose. The His6-Euo was detected by immunodetection using mouse monoclonal anti-His6  (see Immunoblotting section).Five μISME Journal website.All ChIP-Seq data files have been deposited at the GEO database under accession number GSE68059. W. chondrophila TFs in all known chlamydial genera and representatives of two sister clades of the Chlamydiae phylum, the Planctomycetes and Verrucomicrobia. We then conducted pairwise sequence comparisons between the 12 members of the PVC superphylum. This revealed that several of these 10 TFs are conserved within the entire PVC superphylum , while others are not . Euo may have a key role in Chlamydiae as it is the only conserved TF unique to the Chlamydiae and shows a remarkable degree of sequence conservation  sequence identity level. By contrast, the paralogues of the bifunctional replication initiator/TF DnaA and orthologues of the heat-sensitive repressor HrcA are also found outside the PVC lineage. The phosphate response regulator PhoB is also found in other lineages, but it is unusual because it features a high degree of conservation (near 80% identity) across many chlamydial families , except for the Chlamydiaceae family  of Vero cells with W. chondrophila. We observed a peak in the steady-state levels of the dnaA1, phoB, parB, wcw_1223, hrcA, dnaA3 and ytgC transcripts in mid-phase  and stored in EBs, thus allowing immediate translation during the early stage of the infection in the next cycle. A similar peak in euo transcripts has been observed for C. psittaci euo at 15 h p.i.  using polyclonal antibodies to each of these TFs . We obseW. chondrophila with the antibodies to the TFs  with a purified recombinant His6-tagged version of W. chondrophila HrcA (His6-HrcA)  and fluorescently-labelled target promoter fragments  as probes. All five probes were band-shifted by His6-HrcA (15-TGCTAA-3′) matches the CIRCE element-containing inverted repeat that is bound by HrcA in other bacteria and that predicted for C. trachomatis -based transcription interference assay in which a synthetic promoter containing the triple repeat of the HrcA consensus motif directs the expression of a promoterless lacZ on a low-copy plasmid in E. coli. We used this plasmid to test whether W. chondrophila HrcA expressed heterologously in E. coli can downregulate promoter activity (measured as LacZ activity). We observed that LacZ activity dropped by 40% when HrcA was expressed compared with E. coli cells harbouring the empty vector  and includes genes targeted by C. trachomatis HrcA in vitro for example groES1(ct111/wcw_1342), groEL1(ct110/wcw_1343), groES3(wcw_1848), groEL3(wcw_1849) and hrcA(ct394/wcw_1636)  W. chondrophila HrcA target regulon (see Discussion).Next, we used a BBH  approach to compile a list of orthologous target genes of HrcA encoded in 13 mydiales . This licw_1636) . The secAA(G/A)AAANTGN(T/A)AAATTT-3′, C. trachomatis . We observed a reduction in LacZ activity by ±40% comparing the Pwcw_0193 and PphoB-triple-consensus and by ±15% and 30% for Pwcw_1016 versus Pwcw_1714 promoter-probe plasmids, respectively, compared with the empty vector , specifically within the Chlamydia-related bacteria. The other group of PhoB-regulated genes includes W. chondrophila orthologues present also in other members of the Chlamydiae phylum, but with a lower degree of conservation and of unknown function.Next, we validated the predicted target sites of PhoB . The dedy vector . Thus, Wanalysis  revealedW. chondrophila His6-Euo in EMSAs (6-Euo-DNA nucleoprotein complexes (GTAACAACCAAGTACTTGGGTTTT-3′ and 5′-TTTTAAAAAACAATTGATATAATTTTTATT-3′) deduced for C. trachomatis and C. psittaci based on targets identified in vitro  the predicted Euo DNA-binding motif  were band-shifted by in EMSAs . Moreoveomplexes . The Euoomplexes , resemblin vitro . To explng motif . EMSAs rus motif .6-Euo bound this probe, whereas no shift was observed with the NrdR-triple repeat consensus . These results show that Euo binds these promoters in vivo in E. coli.Next, we designed synthetic EMSA probes in which the Euo consensus was repeated three times (Euo-triple consensus) and showed that Hisonsensus . TranscrqueF EMSA probes (A/C)(A/T)TTT-5′, henceforth minimal Euo box). Interestingly, one minimal Euo box was always embedded in the long consensus motif predicted by MEME above , cell division and morphogenesis , general homeostasis/metabolism  and unknown functions . Almost all transcripts showed a mid-phase profile with a peak in abundance between 16 and 24 h p.i.  proteins across the Chlamydiales order, and a second large cluster of less conserved proteins (<40% identity) , hrcA (ct394/ wcw_1636), ftsI (ct682/wcw_1931) and sctR (ct562/wcw_0093)).Interestingly, dentity) . Even thChlamydiae offer a unique opportunity to explore which regulatory systems are dispensable for developmental control. C-di-GMP- and histidine kinase-based regulatory systems that are most commonly used for post-translational regulation in free-living bacteria  bending DNA (nucleoprotein filament) and (ii) positioning recombination integrase proteins for proper formation of the excisive nucleoprotein complex . Thus, tChlamydiae and Planctomycetes, but has not yet been studied, functions as a master TF across the chlamydial and planctomycetes phyla. Wcw_1223 also targets >100 putative sites in W. chondrophila and like Euo controls genes of different functional categories. Moreover, using the Phyre2 prediction server  in isolation or as modules . Such stn server  we notedLentisphaerae, Verrucomicrobia and Planctomycetes (which seems to encode RpoH/σ32)  compared with that of Euo and includes hrcA as well as genes involved in the adaptive response to temperature upshift, such as the (co-) chaperonin encoding genes groEL/ES, dnaK and grpE  encoding a protein featuring a von Willebrand factor type A domain known to act as mechanosensors , we suspect that PhoB may have become functionally specialized as the Chlamydiaceae branched from the ancestral lineage. In support of this, many of the promoters targeted by W. chondrophila PhoB appear to direct expression of hypothetical proteins, and none of the five known members of the C. trachomatis ChxR regulon  with eukaryotic host cells (By contrast to such phylum-specific regulation, PhoB of the


Linker histone H1 is a core chromatin component that binds to nucleosome core particles and the linker DNA between nucleosomes. It has been implicated in chromatin compaction and gene regulation and is anticipated to play a role in higher-order genome structure. Here we have used a combination of genome-wide approaches including DNA methylation, histone modification and DNase I hypersensitivity profiling as well as Hi-C to investigate the impact of reduced cellular levels of histone H1 in embryonic stem cells on chromatin folding and function.We find that depletion of histone H1 changes the epigenetic signature of thousands of potential regulatory sites across the genome. Many of them show cooperative loss or gain of multiple chromatin marks. Epigenetic alterations cluster to gene-dense topologically associating domains (TADs) that already showed a high density of corresponding chromatin features. Genome organization at the three-dimensional level is largely intact, but we find changes in the structural segmentation of chromosomes specifically for the epigenetically most modified TADs.trans-acting factors.Our data show that cells require normal histone H1 levels to expose their proper regulatory landscape. Reducing the levels of histone H1 results in massive epigenetic changes and altered topological organization particularly at the most active chromosomal domains. Changes in TAD configuration coincide with epigenetic landscape changes but not with transcriptional output changes, supporting the emerging concept that transcriptional control and nuclear positioning of TADs are not causally related but independently controlled by the locally associated The online version of this article (doi:10.1186/s13059-015-0857-0) contains supplementary material, which is available to authorized users. DNA in the eukaryotic nucleus is packaged into arrays of nucleosome core particles that are the basic unit of chromatin , 2. EachTo investigate the role of H1 in genome-wide, higher order chromatin structure, we studied the regulatory landscape and overall genome conformation in the H1-depleted TKO ES cells . ES cellLocal DNA demethylation was previously observed in the H1-depleted TKO ES cells particularly at the imprinting control regions of H19-Igf2 and Gtl2-Dlk1 loci  and at tTo investigate whether the DNA methylation changes associated with H1 depletion localized to any specific type of chromatin, we used published ES cell ChIP-seq profiles and a partitioning of the genome into different chromatin states by means of ChromHMM, a segmentation algorithm for the identification of chromatin states based on the presence of combinations of chromatin modifications . We founIn summary, a reduction in the amount of histone H1 protein results in abundant changes in DNA methylation, with some sites gaining but most sites losing methylation. Methylation changes accumulate at the most gene-dense TADs. They occur particularly at enhancer sequences, further indicating that histone H1 plays a role in controlling the DNA methylation status at potential regulatory sequences. In contrast, CpG-rich promoter sequences appear to stably maintain their methylation status in TKO cells, indicating that they control methylation levels in a histone H1-independent manner. A previous observation that H1c and H1d variants are depleted around the transcription start site of active promoters may be in agreement with this idea .Drosophila [To further characterize the consequences of histone H1 depletion on the regulatory chromatin landscape of ES cells, we determined the distribution of DNase hypersensitivity sites (DHSs), H3K4me1, H3K4me3, H3K27me3, and H3K9me3 sites across the genome of H1 TKO ES cell lines and their wild-type counterparts. Overall, the histone modification distributions looked similar between the wild-type, TKO cells and those published by ENCODE for another ES cell line Fig. . Chromatosophila .Fig. 2AlKlf4  and Sox2 (two-fold). This suggests that histone H1 normally serves to occlude these sites, which may be in agreement with the earlier observation that wild-type H1 levels are necessary for normal ES cell differentiation and the concomitant repression of Oct4 expression [p < 1e-6, significance of overlap in hypergeometric test) or H3K4me3 (p < 1e-6) or both, whereas loss of these marks was very infrequently observed at new DHSs , but alpression . Nearly s Figure . Finallys Figure .In summary, the depletion of histone H1 has little impact on the genome-wide distribution of the repressive histone modifications H3K27me3 and H3K9me3, but changes the active chromatin H3K4me1 and H3K4me3 signatures of thousands of sites across the genome. Many of them show concomitant loss or gain of multiple chromatin marks associated with regulatory activity. Nearly invariably these combinatorial changes are either all positively or all negatively contributing to an active chromatin signature, implying that they can cooperate to strengthen or dampen the regulatory potential of a site.p < 10−6) from the most gene-dense TADs and appeared to accumulate in the gene-poorest TADs  cells closely followed that of genes, with all of these marks specifically accumulating in the most gene-dense TADs  , in bothWe previously reported that ES cells harbor a unique 3D genome, hallmarked in general by a more random higher order topology with particularly the inactive chromatin compartment being spatially unorganized. Another feature of the pluripotent 3D genome is the specific clustering of genomic regions dense in binding of pluripotency factors . This prRealizing that the overall topology of chromosomes is unaltered in H1-depleted cells but that domains gain some capacity to reach out and contact other domains elsewhere on their chromosome, we used the recently published TAD cross-boundary ratio to search for more subtle topological changes. The TAD cross-boundary ratio divides the intra-domain contacts over the inter-domain contacts  and as sp < < 10−6) with the amount of epigenetic changes observed per TAD  and were removed from the data for subsequent analyses. Regions significantly enriched for H3K4me1, H3K4me3, H3K27me3, and H3K9me3 compared with matched input samples were identified using the MACS2 peak caller [Chromatin immunoprecipitation (ChIP) was performed as described previously , 13 withk caller  with defk caller  with parp value smaller than 0.05 and an absolute log2 fold-change bigger than 1 to be differentially expressed (598 genes).Total RNAs were prepared from ES cells adapted to gelatinized dishes using RiboPure RNA purification kits (Ambion). Paired-end library construction was performed using Tru-seq kits (Illumina). Resulting libraries were run on the Hi-seq 2000 , generating 2× 100-bp paired-end reads. We aligned reads of two replicate wild-type ES cell RNA-seq libraries and three replicate H1 TKO ES cell RNA-seq libraries to the reference genome (NCBI37/mm9) with TopHat  and usedp value smaller than 1e-6.Genomic DNA was isolated and digested with HpaII and MspI, and Illumina library preparation was performed exactly as described previously . Library2 and with limiting concentrations of the DNA endonuclease deoxyribonuclease I (DNase I). Reactions were stopped by adding stop buffer  and purified fragments were recovered by sucrose ultracentrifugation, end‐repaired and ligated with adapters, followed by sequencing on the Illumina sequencing platform. From an initial collection of 33 different DNase-seq libraries , we filtered out three high quality replicates in each condition according to their SPOT score. Reads were aligned to the reference genome (NCBI37/mm9) and we considered 36-bp reads that aligned uniquely and contained no more than two mismatches as properly mapped reads. We used the Hotspot [DNase I hypersensitivity assay was essentially carried out as described in . In brie Hotspot  algorith Hotspot . We comphttp://genomics.iit.it/groups/computational-epigenomics.html).All statistical analyses were performed under R/Bioconductor  using cuThe authors state that no ethical approval was required for this study.All the raw and processed data for the experiments described in this paper have been submitted to NCBI Gene Expression Omnibus (GEO) under accession number GSE75426.