We demonstrate that the act of transcribing nc-tgp1 over the tgp1+ promoter increases nucleosome density, prevents transcription factor access and thus represses tgp1+ without the need for RNA interference or heterochromatin components. We therefore conclude that tgp1+ is regulated by transcriptional interference. Accordingly, decreased nc-tgp1 transcription permits tgp1+ expression upon phosphate starvation. Furthermore, nc-tgp1 loss induces tgp1+ even in repressive conditions. Notably, drug sensitivity results directly from tgp1+ expression in the absence of the nc-tgp1 RNA. Thus, transcription of an lncRNA governs drug tolerance in fission yeast.Most long non-coding RNAs (lncRNAs) encoded by eukaryotic genomes remain uncharacterized. Here we focus on a set of intergenic lncRNAs in fission yeast. Deleting one of these lncRNAs exhibited a clear phenotype: drug sensitivity. Detailed analyses of the affected locus revealed that transcription of the  The presence of long non-coding RNAs (lncRNAs) is pervasive across genomes, yet few lncRNAs have clearly established mechanisms of action. Here the authors demonstrate that the fission yeast lncRNA nc-tgp1 regulates expression of the drug tolerance gene tgp1+ via+ transcriptional interference. Eukaryotic genomes are pervasively transcribed. Frequently this transcription generates long non-coding RNAs (lncRNAs), which may be transcribed antisense to protein-coding genes, from within introns, or from intergenic regions of the genome. RNA polymerase II (RNAPII) is responsible for generating both messenger RNAs (mRNAs) and lncRNAs2Schizosaccharomyces pombe fbp1+ gene is required to induce fpb1+ expression following glucose starvationSaccharomyces cerevisiae SER3 gene is brought about by lncRNA transcription into the gene promoter, which increases nucleosome density and prevents transcription factor access89The mere act of lncRNA transcription, including accompanying chromatin modifications and resulting changes in nucleosome densityS. pombe, lncRNAs transcribed from centromeric outer repeats are processed by Dicer (Dcr1) into short interfering RNAs, which target the Clr4 H3K9 methyltransferase via Ago1  to establish repressive heterochromatin through the methylation of lysine 9 on histone H3  to dynamically regulate several genes181921S. pombe2324232527lncRNAs can also be processed into smaller regulatory RNAs  H3 refs , 14, 15.Despite rapid advances in lncRNA identification, only a small number have been characterized in detail. A clear challenge in assigning function has been a lack of lncRNA sequence conservation between even the most closely related speciesS. pombe are conserved at the sequence level in three divergent Schizosaccharomyces species, although many retain synteny with flanking genes in at least one other speciesSchizosaccharomyces species. Deletion of one of these loci (SPNCRNA.1343 or ncRNA.1343 for short) exhibited a drug-sensitivity phenotype. We demonstrate that ncRNA.1343 encodes a bidirectional lncRNA promoter and that its deletion causes loss of expression of the divergent unstable transcript nc-tgp1. Our analyses reveal that nc-tgp1 is targeted for Mmi1-directed exosome degradation and is required to repress a downstream phosphate-responsive gene (SPBC1271.09 designated transporter for glycerophosphodiester 1 (tgp1+)). However, rather than involving transient heterochromatin formation as a result of targeted RNA degradation, the regulation of tgp1+ by the nc-tgp1 RNA appears to be mediated by transcriptional interference. Most importantly, tolerance of S. pombe to a broad spectrum of compounds relies on the regulation of tgp1+ by nc-tgp1.Only a few of the ~500 annotated intergenic lncRNAs in S. pombe genome is predicted to encode ~500 intergenic lncRNAsSchizosaccharomyces speciester1+/SPNCRNA.214) is syntenic despite its lack of sequence conservation  displayed a phenotype: hypersensitivity to TBZ, HU and caffeine but not to temperature extremities, ultraviolet-irradiation or oxidative stress  in wild-type cells, cells with ncRNA.1343 replaced by loxP-flanked ura4+ marker (1343Δ::ura4+) and cells with the ura4+ marker subsequently removed  orthologous to the S. cerevisiae permease GIT1. As with S. cerevisiae GIT1, the tgp1+ gene is repressed when cells are grown in the presence of phosphate and induced upon phosphate starvation34tgp1+ was indeed highly expressed in 1343Δ cells but not wild-type cells, both grown in the presence of phosphate . This manipulation restored TBZ, HU and caffeine tolerance to levels comparable with wild-type cells (tgp1+ expression is directly responsible for the drug-sensitivity phenotype of cells lacking ncRNA.1343.To determine whether the drug sensitivity of pe cells . We conctgp1+ (refs ncRNA.1343: one lncRNA transcribed towards the tgp1+ gene (nc-tgp1) and the other in the opposite orientation  cells, but not cells lacking Mmi1 or Red1 (nc-tgp1 was previously detected in rrp6Δ and red1Δ cellsnc-tgp1 transcript and RNA IP (RIP) experiments confirmed a direct interaction between Mmi1 and the nc-tgp1 RNA  mutant cells by RT–qPCR, however, this increase is significantly less than the >50-fold upregulation of tgp1+ observed in 1343Δ cells  that retain its 5′ end did not result in the drug-sensitivity phenotype presented by 1343Δ cells  removes the nc-tgp1 promoter, and the 5′ end of its transcript, resulting in the observed loss of nc-tgp1 expression  prevented nc-tgp1 transcription, induced tgp1+ expression to levels observed in 1343Δ levels and increased sensitivity of these cells to TBZ, HU and caffeine   and phosphate-deprived (−PO4) conditions. As expected, the levels of tgp1+ and the pho1+ control increased upon phosphate starvation  ref. . To detearvation . In conthosphate . The obs nc-tgp1 . Thereforrp6Δ) accumulate non-coding RNAs, some of which have been reported to attract Mmi1-dependent RNA elimination factors, along with RNA interference (RNAi) components and the Clr4 H3K9 methyltransferase, leading to the formation of transiently regulated HOODs (heterochromatin domains)tgp1+ and pho1+ genes are included in HOOD-17 and HOOD-24, respectively, and both form a region of Mmi1-directed transient heterochromatin in rrp6Δ cells24nc-tgp1 transcript is clearly regulated by Mmi1-directed exosome degradation , could be detected on the pho1+ gene and the upstream Mmi1-targeted lncRNA (nc-pho1) within HOOD-24. Moreover, this low level of H3K9me2 did not drop appreciably upon induction of tgp1+ and pho1+  and the highly expressed actin gene (act1+). In contrast, H3K9me2 was ~100-fold enriched over the centromeric outer repeats (dg) in wild-type cells, but reduced to background in clr4Δ cells, indicating that H3K9-methylated chromatin had been efficiently immunoprecipitated. In addition, the transcript levels of tgp1+, nc-tgp1, nc-1343, pho1+ and nc-pho1 were unaffected by loss of RNAi  or heterochromatin components  . Thus, although H3K9me2 accumulates at particular regions in rrp6Δ cells , we conclude that RNAi and heterochromatin play no appreciable role in regulating these genes under normal physiologically repressive conditions or during their induction.Cells with defective exosome function  . Nor werhromatin . In contnc-tgp1 is transcribed into the tgp1+ promoter and suggest that production of this upstream lncRNA represses tgp1+ expression. We therefore investigated if transcription of nc-tgp1 interferes with the induction mechanism of tgp1+ in response to phosphate starvation. The Pho7 transcription factor has previously been shown to engage phosphate-response gene promoters in phosphate-starved cells35pho1+ promoter in phosphate-depleted cells (tgp1+ when activated (nc-tgp1 (1343Δ), higher levels of Pho7–GFP associate with the region upstream of tgp1+ even in repressive conditions . We conclude that loss of nc-tgp1 expression due to phosphate starvation or by preventing production of this lncRNA  allows Pho7 binding and subsequent tgp1+ induction.The above analyses indicate that ed cells . In addictivated . Howevertgp1+ gene and upstream region when it is repressed (+PO4) compared with when it is expressed  were unaffected by phosphate availability. Thus, upstream transcription appears to alter nucleosome density over the tgp1+ promoter and thereby occlude Pho7 binding. Likewise, a considerable drop in H3 levels was observed on the pho1+ gene and nc-pho1 lncRNA region upstream in phosphate-poor conditions, implying a similar mechanism may also operate to regulate the expression of pho1+. We conclude that transcription of the upstream lncRNA inhibits expression of tgp1+ by a transcriptional interference mechanism that alters the chromatin landscape, preventing access to the key phosphate-responsive transcription factor Pho7.Active RNAPII promoters display reduced nucleosome density810ed −PO4; . In conttgp1+ by nc-tgp1 is responsible for tgp1+ repression, we replaced the nc-tgp1 promoter with the strong, thiamine-regulated nmt1 promoter (nmt1-nc-tgp1)  . Transcrlability . In contsitivity . In addithiamine . Lastly,in trans . Collecttion see  and, as S. pombe, for example, the balance of antisense lncRNAs and sense transcription controls various stress-response pathways33S. pombe intergenic lncRNAs. Here we selected and deleted eight stable, discrete lncRNAs in S. pombe that show conserved synteny in at least two of the three other known Schizosaccharomyces species. Excluding the ter1+ control, only deletion of ncRNA.1343 exhibited a definitive phenotype: sensitivity to various compounds due to induction of a nearby phosphate-responsive permease gene (tgp1+). Closer inspection revealed that the ncRNA.1343 promoter is bidirectional. Furthermore, transcription from this bidirectional promoter favours the production of a previously unannotated and unstable lncRNA (nc-tgp1) towards the tgp1+ gene under repressive conditions.An increasing number of lncRNAs have been shown to tightly regulate eukaryotic gene expression following intra-/extra-cellular environment changes that require rapid, integrated responses at the level of transcriptionpho1+ has recently been reported to recruit RNAi-directed heterochromatin to repress pho1+ in response to phosphate availabilitytgp1+ and pho1+, both of which are regulated by upstream lncRNAs that are targeted for exosome-mediated degradation by Mmi1 (rrp6Δ) and not in wild-type cells grown under repressive phosphate-rich conditionsrrp6Δ-dependent heterochromatin at the tgp1+ and pho1+ genes is therefore unclear. Cells lacking Rrp6 accumulate aberrant RNAs and exhibit disrupted heterochromatin globally, including significantly decreased H3K9me2 over centromeric repeatstgp1+ and pho1+ promoters/genes in wild-type cells under repressive (phosphate-rich) conditions. We cannot exclude the possibility that distinct assay conditions in a previous report allowed detection of low H3K9me2 levels on the pho1+ promoter when repressedpho1+ or tgp1+ is unaffected by loss of RNAi/heterochromatin. We note that our findings are in agreement with previous expression profiling analyses, showing unaltered tgp1+ and pho1+ levels in S. pombe cells lacking RNAi/heterochromatinbone fide heterochromatin in centromeric outer repeats are clearly elevated when RNAi/heterochromatin is defective. Thus, our analyses indicate that the repression of both tgp1+ and pho1+ is unlikely to involve regulated heterochromatin in wild-type cells. Instead, we favour a model whereby tgp1+ and pho1+ are repressed by a transcriptional interference mechanism.Recent studies in fission yeast have implicated lncRNAs in directing repression of specific genes by a mechanism involving transient RNAi-dependent heterochromatin formation by Mmi1 ; ref 28,Escherichia coli, the gene encoding the clr transcriptional activator is repressed in response to nitrogen starvation by the act of lncRNA transcription from an alternate upstream promoterS. cerevisiae, which lacks RNAi and heterochromatin, transcription of the SRG1 lncRNA into the SER3 promoter, or heterologous promoters, was found to alter nucleosome density and interfere with transcription factor binding89S. cerevisiae, non-coding transcription over the IME1  by transcriptional interference, not by the formation of transient heterochromatin. We base this conclusion on our findings that: (i) tgp1+ and pho1+ expression is unaffected by loss of RNAi/heterochromatin; (ii) H3K9me2 is not associated with tgp1+ or pho1+ loci in wild-type cells; (iii) nc-tgp1 transcription declines when tgp1+ is induced (−PO4); (iv) loss of the nc-tgp1 transcript allows induction of tgp1+ under normally repressive (+PO4) conditions  RNAPII and nucleosome density is increased over the tgp1+ promoter region when the repressive nc-tgp1 RNA is transcribed and (vii) the Pho7 activator binds the tgp1+ promoter region when nc-tgp1 transcription is lost.Transcriptional interference is well-established in many systems. In the bacterium ME1 ref. , GAL7  had an obvious phenotype in the growth conditions tested. Detailed analysis was required to reveal that deletion of ncRNA.1343 actually affected expression of a divergent unstable lncRNA (nc-tgp1) transcribed in the opposite orientation as the annotated locus. Only after further manipulation and analyses could we conclude that the expression of nc-tgp1 interferes with the expression of tgp1+ downstream. The fact that the unstable nc-tgp1 transcript is the functional partner of the apparently non-functional stable nc-1343 RNA transcribed from the same bidirectional promoter demonstrates the importance of comprehensive analyses of ncRNAs and the consequences of their deletion. Based on our analyses. we surmise that the low level expression of nc-1343 represents transcriptional noise, resulting as a byproduct of ample nc-tgp1 transcription. The syntenic conservation of ncRNA.1343 within the Schizosaccharomyces genustgp1+ in related species. Thus, although genome-wide approaches can rapidly catalogue the presence and response of various lncRNAs to different conditions, much more detailed locus-specific analyses is required to pinpoint the function of each individual lncRNA with respect to cis regulation of nearby genes or trans regulation of genes at distal loci.Genome-wide RNA sequencing has allowed the detection of a large number of lncRNAs in a variety of species. However, it remains unclear how many of these lncRNA are functional transcripts that act to influence gene expression and/or chromatin landscapes. Examples such as Xist RNA in mammals and roX RNAs in S. pombe strains used in this study are listed in 2O, and then grown for indicated times in Pombe minimal glutamate (PMG) synthetic medium without Na2HPO4 (−PO4). Genetic deletions and protein tagging were carried out by lithium acetate transformation. All genetic modifications were confirmed by colony PCR. Plasmids were transformed by electroporation. Selections were performed on PMG/agar plates with according auxotrophy or on YES/agar plates with appropriate antibiotic(s) and grown at 32 °C. Serial (1:4) dilutions of equal number of cells were spotted onto YES/agar and grown at 32 °C, unless indicated otherwise. For drug-sensitivity experiments, cells were spotted onto YES/agar or PMG/agar with DMSO or TBZ (20 μg ml−1), HU (10 mM), caffeine (15 mM) and H2O2 (1 mM). For ultraviolet-sensitivity experiments, spotted cells were ultraviolet-irradiated at 80 J m−2 with a Stratalinker UV Crosslinker and grown in the dark at 25 °C. The plasmids containing lacZ under the control of the nc-tgp1 and nc-1343 bidirectional promoter were cloned as follows. The non-coding promoter was amplified from S. pombe genomic DNA in both orientations  in selective media. Cells were permeabolized by SDS/chloroform. Cell extracts were equilibrated at 30 °C for 5 min before the addition of ortho-Nitrophenyl-β-galactoside (ONPG). The reaction was stopped with Na2CO3 once the solution turned yellow and elapsed time was recorded. Cell debris was spun and the OD420 was measured. Units were calculated as follows: Units/OD=1000 × (OD420/Volume × Time × OD595).Assays for 2O before being grown in PMG (−PO4) for 4 h. ChIP was performed essentially as describedCells were grown to mid-log phase at 32 °C in YES. For phosphate starvation experiments, cells in mid-log phase were washed twice in dHS. pombe using RNeasy Mini- or Midi-Kits as per manufacturer’s instructions (Qiagen). For RT–qPCR experiments, first strand complementary DNA synthesis was performed on Turbo DNase (Life Technologies) treated RNA using random hexamers and Superscript III (Invitrogen) as per manufacturer’s instructions. Negative controls lacking RT were performed alongside all RT–qPCR experiments. Northern analysis of long non-coding transcripts was performed using UTP-[a32P]-labelled RNA probes as describedRNA was isolated from act1+). Expression levels were expressed relative to levels detected in wild-type cells. ChIP enrichments were calculated as the ratio of product of interest from IP sample normalized to the corresponding input sample and expressed as ‘%IP’. Error bars represent s.e.m., resulting from at least three independent replicates.Primers used in this study are listed in R.A. and R.C.A. conceived and designed the experiments. R.A. performed the experiments. P.T. performed bioinformatics analysis. R.A., P.T. and R.C.A. analyzed the data. R.A. and R.C.A. wrote the paper.How to cite this article: Ard, R. et al. Long non-coding RNA-mediated transcriptional interference of a permease gene confers drug tolerance in fission yeast. Nat. Commun. 5:5576 doi: 10.1038/ncomms6576 (2014).Supplementary Figures 1-9 and Supplementary Tables 1-2


Dali, aconserved central nervous system expressed intergenic lncRNA. Daliis transcribed downstream of the Pou3f3 transcription factor geneand its depletion disrupts the differentiation of neuroblastoma cells. Locally,Dali transcript regulates transcription of thePou3f3 locus. Distally, it preferentially targets activepromoters and regulates expression of neural differentiation genes, in part throughphysical association with the POU3F3 protein. Dali interacts withthe DNMT1 DNA methyltransferase in mouse and human and regulates DNA methylationstatus of CpG island-associated promoters in trans. These resultsdemonstrate, for the first time, that a single intergenic lncRNA controls theactivity and methylation of genomically distal regulatory elements to modulatelarge-scale transcriptional programmes.Many intergenic long noncoding RNA (lncRNA) loci regulate the expression of adjacentprotein coding genes. Less clear is whether intergenic lncRNAs commonly regulatetranscription by modulating chromatin at genomically distant loci. Here, we reportboth genomically local and distal RNA-dependent roles of DOI:http://dx.doi.org/10.7554/eLife.04530.001 Traditionally genes are considered to contain all the instructions necessary to buildproteins. For these instructions to be followed they need to be‘transcribed’ into molecules called messenger RNA, which are then‘translated’ to form the protein. Messenger RNAs are not the only typeof RNA molecule made in a cell; long non-coding RNAs (or lncRNAs), for example, aretranscribed but never translated into proteins. Instead, some lncRNAs control theexpression of nearby genes and some alter how the DNA is packaged within thecell.Dali is made incells of the nervous system of mammals. In the genome, the gene forDali is situated next to a gene called Pou3f3,which encodes a protein that contributes to the growth and development of nerves andthe kidneys.Several lncRNAs have been found to control their neighbouring genes, but it isunclear how many of these molecules can also regulate genes that are much furtheraway, even on other chromosomes. One lncRNA called Dali lncRNA restricts the development of mouse cells called N2Acells, which are commonly used to study the development of nerve cells. ReducingDali lncRNA levels in these cells caused Pou3f3messenger RNA levels to also decrease, which demonstrates that Daliis a lncRNA that controls its neighbouring gene. The levels of many other genes werealso changed when Dali levels were reduced, including many genesthat are needed to grow working nerve cells.Chalei et al. have now shown that artificially reducing the amount of theDali lncRNA binds to 1427different regions of the genome of N2A cells, most often near to the start of activegenes; Dali could be carried to these sites by the POU3F3 protein.The DNA sequences with which the Dali lncRNA binds were alldifferent. Chalei et al. found that Dali also binds to an enzyme,called DNMT1, that chemically modifies DNA to change how it is packaged into a cell,and they predict that this enzyme helps Dali to find its bindingsites. Furthermore, when Dali lncRNA levels were artificiallyreduced, the chemical modifications that affect the packaging of DNA in thecell—and hence the expression of genes encoded by this DNA—were changedfor several genes. Some of these genes were located far away from the gene thatencodes Dali, indicating that this lncRNA can regulate the packagingand expression of distant genes.Chalei et al. also showed that the Dali are also regulated by thePOU3F3 protein; this suggests that the lncRNA might work together with this proteinto affect the expression of some genes. Further work is now needed to uncover howmany other lncRNAs act away from their sites of synthesis, and how many also formcomplexes with DNA-binding and DNA-modifying proteins.Many genes that are regulated by DOI:http://dx.doi.org/10.7554/eLife.04530.002 Many otivation . Some cicription .Trans-acting lncRNAs that regulate gene expression across multiplechromosomes and on either allele have been documented less frequently. The ability ofsuch lncRNAs to exert widespread effects on gene expression in trans ispoorly understood, in large part because direct transcriptional targets for only veryfew of these transcripts have thus far been identified , owing to its conservation of sequence and transcription across therianmammals and its genomic proximity to a transcription factor gene,Pou3f3 , which encodes a class III POU family transcription factor.Dali is transcribed in the sense orientation, relative toPou3f3, from a locus 50 kb downstream of Pou3f3within the flank of an extended genomic region  down-regulation of the upstreamPou3f3 gene, andlinc-Brn1b-/- mice exhibitedabnormalities of cortical lamination and barrel cortex organization  . To invec region  that is c region . Sauvagenc-Brn1b . Genomicnization . These aelements . The Dalelements .10.7554/Pou3f3 is a single exon gene whose protein binds to DNA in asequence-specific manner. Pou3f3 contributes to both neuronal andkidney development by regulating the proliferation and differentiation of progenitorcells  longer thana previously identified AK034039 cDNA cloned from the telencephalon transcript was identified by RT-PCR and RACE in human foetal brain enhancer in both brain and kidney tissues , olfactory bulb (OB),and dentate gyrus (DG)  cells, but after 3 days of retinoic acid(RA)-induced differentiation, a stage corresponding to the cell cycle exit ofneuronal progenitors and their differentiation into neurons, these transcripts wererapidly up-regulated, their levels subsequently peaking at days 7(Pou3f3) and 8  (ENCODE data indicate that both mouse and human  tissues .Consiste organs . In adulrus (DG)  (Reviewerus (DG) . Dali waicbrain . Both tricbrain . Further8  .Dali , Dali is expressed at a levelat least two orders of magnitude higher than in N2A cells  between days E10.5 to E18.5 , was highly enriched in the nucleus of N2Acells, most abundantly in the chromatin fraction  andPou2A cells . Howeverto E18.5 . Therefofraction . Taken tDali regulates neural differentiationby generating three independent stable Dali knockdown N2A cell lineseach showing approximately 50–70% reduction of Dalitranscript levels and inducing neural differentiation using RA  compared to a non-targeting control a region overlapping the 3′ UTR of Pou3f3 and full-lengthAK53590 , aswell as parts of TCONS_00000039 and linc-Brn1b,including a differentially methylated region reported to be important in regulatingPou3f3 expression (TCONS_00000040)  showed that thef3 locus in ES def3 locus  : 1) an pression , and 3) 0000040) . Neitherxpressed . NeverthDaliknockdown can be explained by its effect on Pou3f3, we reduced thelevel of Pou3f3 transcript in N2A cells by 35% using transienttransfection of a Pou3f3 targeting shRNA vector ,and represented 31% of all genes differentially expressed in Daliknockdown cells -Seq -Seq  -Seq . We discome-wide , of whicome-wide .10.7554/Dali binding sites were typically limited to less than 1 kb inlength , neuronal projectiondevelopment , neuronal function (synaptic transmission), and more general cellularprocesses, such as gene expression, intracellular signalling, and cellularhomeostasis  regulated by Dali containedDali binding sites within their regulatory regions  in N2A cells    in paralepletion . The inaDlgap5,Hmgb2, and Nos1 promoters each display increasedCpG methylation in two independent stable Dali knockdown linescompared to control further confirmed these results  acting intrans, as part of a multi-subunit ribonucleoprotein complex, toreduce DNMT1-mediated CpG methylation at a subset of bound and regulated genepromoters away from its site of transcription.Bisulfite sequencing demonstrating that the  results . ImportaNos1, has multiple alternative promoters fallinginto two distinct regions (for simplicity referred to here as Exon 1 and Exon 2)whose differentiated use is proposed to fine-tune its expression in response tovarious physiological and developmental stimuli  alternative promoter potentially through its effect on promoter-associated CpGisland methylation.One of these genes,  stimuli . Only thli  of Pou3f3 targets transcription factor. After72 hr, up-regulation of Dali transcript was shown to significantlyincrease Pou3f3 expression , HOTAIR,NEAT1, TERC, RMST , androx2 (Drosophila)) have been identified thus far of all genes whose expression altered following Dalidepletion were associated with Dali binding sites within 1 Mb  mouse cDNA clone in pCMV Entry vector and inserting it into the multiple cloning site (MCS) ofthe N-terminal pFLAG-CMV-6a vector  betweenEcoRI and EcoRV sites. The sequences of alloligonucleotides used for cloning are shown in We used the Whitehead Institute siRNA selection program to design shRNAs that targetmultiple regions of -CMVeGFP  bylinke5 cells perwell in a six well plate. 16–24 hr later cells were transfected with 1.5μg shRNA expression construct using FuGENE 6  following themanufacturer's instructions. Total RNA was extracted from the cells 48–72 hrlater using TRIzol-chloroform extraction method. For stable transfections, N2A cellswere co-transfected with the pBSU6-shRNA expression vector and pTK-Hyg  at a 5:1 ratio. 72 hr post-transfection 200 μg/ml HygromycinB was added to the cells to select individual drug resistant clones that were laterisolated and expanded under selective conditions. Dali expression inindividual clones was measured by qRT-PCR.Cells were plated at a density of approximately 2 × 10Reverse transcription was performed using the QuantiTect Reverse Transcription Kit. SYBR Green quantitative PCR was performed using a Step OnePlus Real-Time PCR System . For RACE, GeneRacer Kit was used according to the manufacturer's instructions. Human foetalbrain RNA was purchased from Promega. Primers are listed in 2. Biochemicalfractionation, ChIP and UV-RIP experiments was performed exactly as described inMouse N2A neuroblastoma and E14 ES cells were cultured as described in . The N2AAll animal experiments were conducted in accordance to schedule one UK Home Officeguidelines . C57BL/6J, postnatal day P56 male andpregnant females were killed by cervical dislocation; whole brains were dissected inice-cold phosphate-buffered saline (PBS) from adult (n = 2), and intrauterinestages E9 (n = 6), E10.5 (n = 6), E13.5 (n = 6), E15.5 (n = 6)and E18.5 (n = 6) mice. Brains were embedded in 5% agarose  and sectioned using a vibrating microtome  into 200 μmcoronal sections using a chilled solution of 1:1 mixture of RNAlater (Ambion) andPBS. Regions of interest  weredissected from individual sections using 27 gauge needles under visual guidance,using transillumination on a dissecting microscope .Dissected samples were rinsed in RNAse free PBS/RNAlater 1:1, submerged in ice-coldRNAlater kept for 24 hr at 4°C and stored at −80°C in RNAlater untilprocessing.Total RNA was isolated using the Qiagen Mini RNeasy kit according to themanufacturers' instructions. RNA integrity was assessed on a BioAnalyzer . 200 ng RNA was used to produce labelled sense single stranded DNA(ssDNA) for hybridization with the Ambion WT Expression Kit, the Affymetrix WTTerminal Labelling and Controls Kit and the Affymetrix Hybridization, Wash, and StainKit following the manufacturer’s instructions. Sense ssDNA was fragmented andthe distribution of fragment lengths was assessed on a BioAnalyzer. Next, fragmentedssDNA was labelled and hybridized to the Affymetrix GeneChip Mouse Gene 1.0 ST Array. Arrays were processed on an Affymetrix GeneChip Fluidics Station450 and Scanner 3000.Dali andPou3f3 knockdown, as well as upon retinoic acid induceddifferentiation of control and stable Dali knockdown cells, werefiltered to remove genes showing little variation in expression (variance cut off of0.5) before the identification of significant changes. In every case, the LimmaEbayes algorithm was used to identify differential expression between three knockdownand three control samples . 1.3-fold change cutoff was appliedin every case. GOToolbox was used to perform Gene Ontology analyses ((http://genome.crg.es/GOToolBox/). Representative significantlyenriched categories were selected from a hypergeometric test with aBenjamini-Hochberg corrected p-value threshold of 0.05.CEL files were analysed using the Limma, oligo, and genefilter R Bioconductorpackages . Arrays nalyses . Additionally we require peaks to not overlap with those identified inan analogous CHART-sequence experiment using the E. coli lacZsequence (GSE52571) , but no enrichment of the abundant mRNAGapdh  . CompareNAGapdh . Withoutexpected .8 N2A cells perpull down and hybridized overnight with 810 pmol biotinylated oligonucleotidecocktail  and CHART-seq peaks previously analogously identified in N2Acells using two lacZ controls (1928 peaks) (https://github.com/taoliu/MACS/blob/master/README) with the options‘–mfold 10 30 –gsize = 2.39e9 –qvalue =0.01’ using the CGAT pipeline ‘pipeline_mapping.py’ (https://github.com/CGATOxford/cgat). Peak calls were then filteredsuch that only peak calls with a −log10 q value >5 were retained (FDR0.001%).CHART-seq was performed with three independent pull down samples  andsequenced simultaneously with a matched input sample. 50 bp, paired-end reads weremapped to the mouse genome (mm9) using bowtie with the options ‘–m1–v2 –best–strata–a’. For each8 peaks) . Peak ca8 peaks) ; https:/Dali-associated regions genome-wide calledagainst both input and lacZ control samples . 10,000finition  as a basDali peaks were characterised using DNase I hypersensitivity (HS)data generated by the Stamatoyannopoulos lab at the University of Washington andchromatin features identified by the Ren lab at the Ludwig Institute for CancerResearch  for 1 hr at 37°C. Then, SDS was sequestered byadding 1.8% Triton X-100. 1 × 106 nuclei (∼15 μg ofchromatin) were digested with 400 units of BglII restriction enzymeovernight, and the enzyme was inactivated. Nuclei were diluted in 1.15× T4 DNAligation buffer (NEB), and SDS sequestered by adding 1% Triton X-100. The digestedchromatin was ligated using 100 Weiss units of T4 DNA ligase for 4 hr at 16°Cand treated with Proteinase K to reverse cross-links. Samples were further treatedwith RNase A, and DNA was phenol-chloroform extracted and ethanol precipitated.E14 ES cells or day 4 ES-derived neuronal were cross-linked with 2% formaldehyde.Nuclei were prepared and permeabilized with 0.3% SDS in 1.2× restriction buffer. All reactions were performed in triplicate. Themean threshold cycle (Ct) value was calculated and used to calculate relative amountsof PCR products. To normalise for different primer efficiencies, interactionfrequencies were calculated by dividing the amount of PCR product obtained from the3C sample by the amount of DNA obtained from control BAC DNA. Interaction frequencieswere also normalised to Gapdh internal controls prepared fromgenomic DNA in the same manner as the BAC clone sample. All primers used are listedin A RP23-92N4  Bacterial Artificial Chromosome (BAC) clone covering theDali and associated with genes differentially expressed betweenstable Dali knockdown and control cell lines prior to or subsequentto the RA-induced differentiation. 80–350 ng of genomic DNA wasbisulfite-treated using EZ DNA Methylation Gold kit according to the manufacturer'sinstruction and used for PCR amplification. Primers for amplifying bisulfiteconverted template DNA were designed using MethPrimer software accessible athttp://www.urogene.org/methprimer/ (BstUI (NEB), MspI(NEB), TaqI (Thermo Scientific), HpyCH4IV (NEB)) orcontrol (Hsp92II (Promega), BfaI (NEB)) restrictionenzymes overnight. Restriction products were analysed on 3% low melting point agarosegels.We used COBRA to study 9 out of 44 CpG island-containing promoters bound byhprimer/ . PCR proTAL Effector Nucleotide Targeter2.0 website accessible from https://tale-nt.cac.cornell.edu/. Construction of custom TALE-TFsdesigned to target promoter-proximal region of Dali to up-regulatetranscription from the locus was performed as described by Dali: chr1 (mm9):42807019-42807038 ("TGTCCCTTGTCCACATATCT"). The TAL domain sequence used was asfollows: NH NG HD HD HD NG NG NH NG HD HD NI HD NI NG NI NG.Target regions were selected and TAL effector constructs were designed usingsoftware, tools, and information found on the http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE62035).Microarray and CHART-Seq data have been deposited in the GEO database under accessionnumber GSE62035 ( reviewprocess). 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  and 2 reviewers, one of whom is a member of our Board ofReviewing Editors. The Reviewing editor and the other reviewer discussed their commentsbefore we reached this decision, and the Reviewing editor has assembled the followingcomments to help you prepare a revised submission.Thank you for sending your work entitled "The DNMT1 associated lncRNADali and itsregulation not only of its neighboring transcription factor gene Pou3f3, but importantlyalso at distal sites through the methylation of these sites. The topic of the functionalroles of lncRNAs is of keen interest and most importantly the mechanism of how theseroles are achieved is of critical importance. Thus, this manuscript is both timely andimportant. However, there are several issues that the authors should make clearer forthe readers in order for an audience to understand the messages being communicated andto provide a clear and supportable set of conclusions. These issues are:Your work focuses on the functional roles of the lncRNA Dali via a complex of DNA binding proteins that include CTCF is aninteresting hypothesis, this collection of data can be interpreted by alternativemechanisms such as the coincident binding of CTCF (due to CTCF genome-wide and abundantbinding).1) Several places in the manuscript the authors' interpretation of their dataresults in a set of specific conclusions about mechanisms or cause and effectrelationships that are not the only explanation or conclusion. Importantly, there is noacknowledgement of other interpretations or explanations of why other interpretationscannot be concluded. For example, while the suggestion of recruitment ofDali binding sites? What changes,if any occur, at the other 5 sites? Given that half of the results obtained areconsistent with the model of Dali or Dali:Pou3f3complex acting in trans to reduce DNMT1 methylation, there appears to be the likelihoodthat the mechanism may minimally involve a more complex set of interactions. This shouldbe acknowledged.2) What was the basis of choosing 10 Dali acting in trans to reduceDNMT1-mediated CpG methylation at a subset of bound and regulated gene promoters awayfrom its site of transcription”: this is speculation since the effect on the useof the 5' most CpG site could be achieved through a secondary effect of theDali KD.3) “…we performed Combined Bisulfite Restriction Analysis (COBRA)  in paral4)  1) Several places in the manuscript the authors' interpretation of theirdata results in a set of specific conclusions about mechanisms or cause and effectrelationships that are not the only explanation or conclusion. Importantly, there isno acknowledgement of other interpretations or explanations of why otherinterpretations cannot be concluded. For example, while the suggestion of recruitmentof Dali via a complex of DNA binding proteins that include CTCF is an interestinghypothesis, this collection of data can be interpreted by alternative mechanisms suchas the coincident binding of CTCF (due to CTCF genome-wide and abundantbinding).Dali and CTCF “might independently bind adjacent sequence,or compete for binding to the same region”.We have now edited the main text to include additional explanations that are alsoconsistent with the data as suggested by the reviewers. This specific example now statesthat 2) What was the basis of choosing 10 Dali binding sites? Whatchanges, if any occur, at the other 5 sites? Given that half of the results obtainedare consistent with the model of Dali or Dali:Pou3f3complex acting in trans to reduce DNMT1 methylation, there appears to be thelikelihood that the mechanism may minimally involve a more complex set ofinteractions. This should be acknowledged.Dali binding sites to maximise the likelihood ofdetecting DNA methylation changes using COBRA. We ensured that each region containedseveral COBRA-compatible restriction enzyme sites and could be efficiently amplifiedfrom bisulfite-converted template.We selected these 10 Dali depletion or that those changes that occurred were undetecteddue to technical limitations of the COBRA assay.We also now indicate that the failure to detect changes at all regions tested couldreflect either that the DNA methylation status of the remaining regions did not changeupon Dali mediated DNA methylation changesin trans may minimally involve a more complex set of interactions thatare in addition to Dali, POU3F3 and DNMT1. This is acknowledged in therevised manuscript as suggested. We now state: “Although other unidentifiedfactors are also likely to play a role, our results are consistent withDali  acting intrans, as part of a multi-subunit ribonucleoprotein complex, toreduce DNMT1-mediated CpG methylation at a subset of bound and regulated gene promotersaway from its site of transcription.”We agree with the reviewers that 3) “…we performed Combined Bisulfite Restriction Analysis (COBRA) in parallel at 10 different CpG islands anddemonstrated that five of these regions (corresponding to 4 genes) exhibited alteredrestriction profiles indicative of altered DNA methylation status after Dalidepletion […] These results are consistent with Dali  acting in trans to reduceDNMT1-mediated CpG methylation at a subset of bound and regulated gene promoters awayfrom its site of transcription”: this is speculation since the effect on theuse of the 5' most CpG site could be achieved through a secondary effect ofthe Dali KD.Dali knockdown.Nevertheless, the observation that this site is bound by Dalitranscript suggests that Dali may function by promoting thepreferential use of a distantly located (and more rarely used) alternative promoterpotentially through its effect on promoter-associated CpG islandmethylation.”Thank you. We now have added: “The preferential use of the 5́’ mostCpG site could reflect a secondary effect of 4)(heat map); are these the results of all 3 KDs or only1? Given the results seeninwhat data indicates that some or most of these expression changes are notexplained by "off-target" effects by the sequences used in the KDs?Dali knockdowns. To assess whether these expression changesare likely to be explained by ‘off-target’ effects we used two furthershRNA expression constructs targeting different regions of the Dalitranscript to deplete Dali expression and validated expression changesin 14 out of 15 Dali targets identified in the microarray. Theseresults are shown in The data in


The myocyte enhancer factor 2 (MEF2) transcription factor requires interactions with co-factors for precise regulation of its target genes. Our lab previously reported that the mammalian MEF2A isoform regulates the cardiomyocyte costamere, a critical muscle-specific focal adhesion complex involved in contractility, through its transcriptional control of genes encoding proteins localized to this cytoskeletal structure. To further dissect the transcriptional mechanisms of costamere gene regulation and identify potential co-regulators of MEF2A, a bioinformatics analysis of transcription factor binding sites was performed using the proximal promoter regions of selected costamere genes. One of these predicted sites belongs to the early growth response (EGR) transcription factor family. The EGR1 isoform has been shown to be involved in a number of pathways in cardiovascular homeostasis and disease, making it an intriguing candidate MEF2 coregulator to further characterize. Here, we demonstrate that EGR1 interacts with MEF2A and is a potent and specific repressor of MEF2 transcriptional activity. Furthermore, we show that costamere gene expression in cardiomyocytes is dependent on EGR1 transcriptional activity. This study identifies a mechanism by which MEF2 activity can be modulated to ensure that costamere gene expression is maintained at levels commensurate with cardiomyocyte contractile activity. Members of the myocyte enhancer factor 2 (MEF2) family of transcription factors play essential and diverse roles in tissue development and function as exemplified by mutant phenotypes in mice and other animal model systems . The tracis-elements that may function as binding sites for transcriptional co-regulators of MEF2A-dependent costamere genes. One of these predicted sites belonged to the early growth response (EGR) family of zinc finger transcription factors  which are not conserved in the other MEF2 protein isoforms. Alternatively, the isoform selectivity may be related to encoded functional differences in transcriptional activity given that MEF2A and MEF2D are the predominant isoforms expressed in postnatal cardiomyocytes.Consistent with the notion that EGR1 repressed MEF2, overexpression of EGR1 in NRVMs resulted in significantly decreased expression of MEF2-dependent costamere genes. Interestingly, overexpression of EGR1 also caused cardiomyocyte detachment and significantly reduced survival, which is reminiscent of the MEF2A depletion phenotype in NRVMs . Convers2+-activated NFAT signaling that promotes chamber dilation and loss of contractility [The ability of EGR1 to function as a repressor of gene expression in cardiomyocytes has been documented ,24. Howeactility . In thisactility , to playactility , and to actility . Additioactility  and EGR1actility . Given t


Wnt/β-catenin signaling is often portrayed as a simple pathway that is initiated by Wnt ligand at the cell surface leading, via linear series of interactions between ‘core pathway’ members, to the induction of nuclear transcription from genes flanked by β-catenin/TCF transcription factor binding sites. Wnt/β-catenin signaling is also regulated by a much larger set of ‘non-core regulators’. However the relationship between ‘non-core regulators’ is currently not well understood. Aberrant activation of the pathway has been shown to drive tumorgenesis in a number of different tissues.Mammalian cells engineered to have a partially-active level of Wnt/β-catenin signaling were screened by transfection for proteins that up or down-regulated a mid-level of TCF-dependent transcription induced by transient expression of an activated LRP6 Wnt co-receptor (∆NLRP).141 novel regulators of TCF-dependent transcription were identified. Surprisingly, when tested without ∆NLRP activation, most up-regulators failed to alter TCF-dependent transcription. However, when expressed in pairs, 27 % (466/1170) functionally interacted to alter levels of TCF-dependent transcription. When proteins were displayed as nodes connected by their ability to co-operate in the regulation of TCF-dependent transcription, a network of functional interactions was revealed. In this network, ‘core pathway’ components  were found to be the most highly connected nodes. Activation of different nodes in this network impacted on the sensitivity to Wnt pathway small molecule antagonists.The ‘functional connectome’ identified here strongly supports an alternative model of the Wnt pathway as a complex context-dependent network. The network further suggests that mutational activation of highly connected Wnt signaling nodes predisposed cells to further context-dependent alterations in levels of TCF-dependent transcription that may be important during tumor progression and treatment.The online version of this article (doi:10.1186/s12943-015-0475-1) contains supplementary material, which is available to authorized users. Wnt signaling plays a crucial role in normal development and disease. Deregulated Wnt signaling has been implicated in cancers arising in many different tissues . Wnt ligD. melanogaster, HeLa and colon cancer cells that have identified over 250 genes as regulators of TCF dependent transcription )opicalis . An overopicalis . LuciferXenopus Tropicalis cDNA for the gene Prune. When assayed in the cognate Xenopus laevis animal cap explant system, Prune induced expression of Siamois, a classic Wnt/β-catenin target. In addition, Prune induced partial axis duplication in ventrally injected embryos  also strongly induced expression of Wnt target genes  of the inhibitors reduced TCF-dependent transcription (Benjamini-Hochberg corrected p < 0.01). Five cDNAs (Prune homolog (PRUNE2), Dishevelled (DVL2), Casein kinase 1ε (CSNK1E), Mesoderm posterior (MESPA) and Casein kinase 1δ (CSNK1D)) induced TCF-dependent transcription above background levels  had a corrected p-value of 0.052. Dishevelled and Casein kinases 1ε and δ have previously been implicated in the modulation of Wnt signaling [To determine the capacity of the 45 activators to increase TCF dependent transcription independently of ΔNLRP, they were individually transfected into the 7df3 reporter cells .The conditional dependence of 40 out of the initial 45 activating cDNAs on ∆NLRP expression raised the question as to whether their ability to enhance TCF-dependent transcription was specific to ∆NLRP or whether they could enhance TCF-dependent transcription in combination with other activators or enhancers. To explore this question, the 40 enhancers and 5 activators identified in the screen were co-transfected with each other and with a further 4 constitutive ‘core’ activators Fig. . The addp < 0.01) before a weight was assigned to each combination. The weight quantifies the experimental synergy between two co-transfected cDNAs by comparison with the reporter activity induced by each cDNA of the pair when expressed alone, as described in p-value of <0.03, 315/1170 pairwise cDNA combinations showed significantly higher levels of TCF-dependent transcription and 151 showed significantly lower levels than expected if there were no functional interaction  against regulator combinations that would normally be activated in specific cellular contexts such as cancer cells. To explore this potential, known activators as well as two cDNA pairs  were transfected into 7df3 cells and treated with a number of compounds for 24 h prior to luciferase reporter assay Fig. , c. ICG0To investigate whether the network of functional interactions identified in the 7df3 cells was observed in other cell types a subset of cDNA nodes identified in the 7df3 reporter cells was examined for functional interaction in HCT116 cells Fig. , c. ThisTo derive further mechanistic insight into the reasons for the selective patterns of functional interaction Fig., b, the A prospective analysis suggested that there were more links between communities (candidate protein complexes) than within the same communities than would be expected by chance. This analysis compared the PIN community membership of the connectome’s links to shuffled versions of the connectome, . 7df3 cells were maintained under constant antibiotic selection using 200 μg/ml hygromycin (Invitrogen) and 3 μg/ml blasticidin (Invitrogen). The identity of the SW480 and HCT116 lines was confirmed by DNA fingerprinting.All cells were cultured at 37 °C and 5 % COXenopus Tropicalis library, the normalization control plasmid CMV-lacZ or the ‘filler’ plasmid pCDNA3.1 that also contains a CMV promoter. Transfection was performed using the Transfectin (BioRad) reagent according to manufacturers instructions. Assays were performed 48 h post transfection. Luciferase and galactosidase assays were performed using the Bright-Glo or Beta-Glo systems (Promega) respectively according to manufacturers instructions and assayed using a FluoStar Optima plate reader (BMG Labtech). β-catenin antibodies were used at 1/1000 dilution and were from BD Transduction Labs (Clone 14) and Merck Millipore (anti-ABC b-catenin clone 8E7). The anti-Flag (clone M2) antibody was from Sigma.Cells were transfected in 96-well format with 100 ng of plasmid DNA comprising CMV-driven cDNAs from the t-test (p < 0.01).Mean reporter activity was normalised as both a fold of the plate mean, and as the number of standard deviations away from the plate mean. The 100 most activating and 100 most inhibiting pools from each analysis were selected for deconvolution. To identify the responsible cDNA(s) within each well, each plasmid was co-transfected with ΔNLRP in triplicate. Hits that were significantly different from 36 ΔNLRP control wells (12 wells/96 well plate) were identified using a student’s two-tailed n = 6–12 per 96-well plate).To identify and investigate synergistic interactions of co-expressed genes on the integrated β-catenin/TCF-reporter present within the 7df3 cells, we adopted a methodology based on that of Mani et al. for enumeration of genetic interaction . Our obsp < 0.01) including those with means higher than background were set to the background level. Datasets significantly different from background but with means lower than background were also set to the background level. This prevented the propagation of anomalies deriving from low-level fluctuations in reporter activity.The sets of values under scrutiny when evaluating the synergistic interaction between cDNAs A and B comprise the data obtained when A is expressed in isolation, when B is expressed in isolation and when both A and B are co-expressed. Additionally, we also recorded a background (control) scenario where no expression plasmid was expressed (other than the co-tranfected CMV-lacZ and ‘filler’ pCDNA3.1 control). We first computed the mean luminescences μA, μB and μA, B for each cDNA expressed in isolation and the pairwise co-expression (3–6 replicates). The datasets for each individual and the pairwise luminescences were compared to the control luciferase reporter activity. Those datasets that were not statistically significantly different (Kolmogorov-Smirnov (K-S) test; A network link weight (strength) for the synergistic interaction between nodes (cDNAs) A and B when co-expressed was then written:This enumeration of interaction has a number of salient properties. It prescribes positive link weights for synergistic interactions and negative weights for antagonistic interactions. A synergistic interaction is one in which pairwise co-expressed activity was greater than both individuals (i.e. the presence of one of the cDNAs enhanced the activity of the other). If only one of the pair activated when individually expressed, any increase above this would constitute a positive interaction. Similarly, if neither cDNA led to an increase in reporter activity above background levels when individually expressed, any reporter activity above background levels upon co-expression would constitute a synergistic interaction.A negative link weight was prescribed for suppressive interactions in which pairwise activity is less than either one of the individuals. Consequently, if two cDNAs were co-expressed and either (or both) was capable of inducing reporter activity individually and the pairwise activity was lower than the maximum individual level, a negative link weight was assigned. The use of the logarithm ensured that the magnitudes of the multiplicative interactions are consistent between positive and negative synergies and that where no enhancement or suppression was observed; a zero edge weight was assigned (see Additional file A protein interaction network was generated from the list of Wnt-regulators (Additional file Each functional connectome link was either between nodes in different PIN communities or lay within a community. To compare the number of connectome links that lay within and between PIN communities, the experimental connectome links were repeatedly randomized amongst the connectome nodes, in a fashion that preserved the degree of individual nodes (Maslov Sneppen method . From thWe further extended this comparison by considering only the strongest connectome links. We chose a threshold, τ, and retained only those connectome links with a weight greater than this threshold before repeating the randomization procedure.Candidate inhibitor cDNAs were co-transfected with the TOPflash luciferase reporter in triplicate and were normalised to FOPflash (A control vector corresponding to TOPflash but instead containing scrambled TCF binding sites , 3. A stX. laevis embryos were obtained, cultured and injected as described [escribed , 3–5. Exescribed , 6.


MUC4 is a type I membrane-bound mucin expressed at the apical pole of normal polarized epithelial cells. MUC4 apomucin is characterized by a long hyperglycosylated extracellular domain, Epidermal Growth Factor (EGF)-like domains, a hydrophobic transmembrane domain, and a short cytoplasmic tail. MUC4 also contains NIDO, AMOP and vWF-D domains that are unique in the apomucin family. In cancers, MUC4 and the oncogenic receptor ErbB2 interact physically via the EGF-like domains . MUC4 plPancreatic Ductal Adenocarcinoma (PDAC) is the 4th leading cause of death by cancer worldwide. Its poor survival rate at 5 years (3–5%) and median survival curve (6 months) are the consequences of a late detection and a lack of efficient therapies [MUC4 transcription is complex, tightly regulated and involves many signaling pathways  and Med-1) and a distal promoter characterized by a TATA box and containing numerous binding sites for both ubiquitous and specific transcription factors  [MUC4 regulation is highly complex and involves a wide range of specific factors such as AP-2, PEA3, IFN-γ and IL6 inflammatory pathways (via STAT1) and CDX-1/-2, HNF-1α/-1β, FOXA1/A2, HNF-4α/-4γ, and GATA-4/-5/-6 endodermal transcription factors [G12D mutation and downstream signaling via both transcriptional and post-transcriptional mechanisms (unpublished). In healthy pancreas, MUC4 expression is repressed by epigenetic mechanisms and heavy methylation of the CpG islands present in its promoters [s Figure . This reG12D transgenic mouse model [Post-transcriptional regulation by microRNAs (miRNA) is a new promising strategy to control gene expression in cancers that may lead to emerging circulating biomarkers and therapeutic targets . We recese model . We alsose model .Based on our recent work, we propose miR-219-1-3p as a good tumor-suppressor candidate to inhibit MUC4 expression, MUC4-mediated downstream signaling pathways, and MUC4-independent cellular tumor suppressor mechanisms highlighting the therapeutic potential of this miRNA in pancreatic cancer (Figure


Musculin (MSC) is a basic helix-loop-helix transcription factor that inhibits myogenesis during normal development and contributes to the differentiation defect in rhabdomyosarcoma. As one of many transcription factors that impede myogenesis, its binding on a genome-wide scale relative to the widespread binding of the myogenic factor MyoD is unknown.Chromatin immunoprecipitation coupled to high-throughput sequencing was performed for endogenous MSC in rhabdomyosarcoma cells and its binding was compared to that of MyoD in the same type of cells.MSC binds throughout the genome, in a pattern very similar to MyoD. Its binding overlaps strongly with regions enriched for acetylated histone H4, as well as regions that score high for DNase hypersensitivity in human myoblasts. In contrast to MyoD, MSC has a more relaxed binding sequence preference in the nucleotides that flank the core E-box motif.The myogenic inhibitor MSC binds throughout the genome of rhabdomyosarcoma cells, in a pattern highly similar to that of MyoD, suggesting a broad role in buffering the activity of MyoD in development and rhabdomyosarcomas. The advent of high-throughput sequencing coupled to chromatin immunoprecipitation (ChIP-seq) has permitted the global assessment of DNA binding of numerous transcription factors. While some factors show a relatively restricted binding pattern near their regulated genes, others bind widely throughout the genome . The basMyoD and Myf5[Drosophila ortholog of musculin is required for the specification of certain gut muscle cells [musculin might have either positive or negative activities in gene transcription depending on a variety of factors and cellular context.The myogenic activity of MyoD can be inhibited by a variety of transcription factors, including other members of the bHLH protein family . Inhibit and Myf5. A simil and Myf5, and thele cells . There ile cells -17. TogeRecently, we have shown that MSC competes with MyoD for the available pool of E-proteins in rhabdomyosarcoma cells , and thaWe have previously performed ChIP-seq for MyoD in a cell culture model of embryonal RMS, RD cells , and we RD cells were obtained from the American Type Culture Collection (ATCC), and all analyses were performed on cells that originated from low passage number frozen aliquots. RD cells were maintained in DMEM with 10% bovine calf serum and 1% Pen-Strep (Gibco). MSC with a tandem affinity purification (TAP) tag was constructed by cloning the coding sequence for MSC in-frame with a TAP-tagged pBabe plasmid so that the TAP tag is N-terminal to MSC.Chromatin immunoprecipitation (ChIP) was performed in RD cells with an approach that has been described previously . AntibodThe isolation of complexes associated with TAP-tagged MSC was performed identically to prior experiments , but MSCShift assays were performed as described previously . ProteinP value cutoff of 10-5 were removed from the analysis.Sequences were extracted by GApipeline-0.3.0. Reads mapping to the X and Y-chromosomes were excluded from our analysis. Reads were aligned using BWA to the human genome (hg19). Duplicate sequences were discarded to minimize the effects of PCR amplification. Each read was extended in the sequencing orientation to a total of 200 bases to infer the coverage at each genomic position. Peak calling was performed by an in-house developed R package, which models background reads by a negative binomial distribution as previously described . Peaks ide novo motif discovery as previously described [P value cutoff of 10-10 and absent for the other with a P value cutoff of 10-4. Shared peaks were present for both factors with a P value cutoff of 10-10.We applied an in-house developed Bioconductor package motifRG for discriminative escribed ,25. To fP values and grouped ranks into bins of 3,000 . Then we computed the fraction of top x peaks in a sample that overlap with the top y peaks in another sample, where x and y vary from 3,000 to 30,000, and y is equal to or greater than x.We adopted a nonparametric rank-based paradigm to compare two ChIP-seq samples as previously described . We rankTo compare the binding pattern of the bHLH myogenic inhibitor MSC to that of the myogenic activator MyoD, ChIP-seq for endogenous MSC was performed in RD cells under growth conditions. MSC binds at a comparable number of sites as MyoD and with a similar genomic distribution, although there was a slightly greater enrichment of MSC binding in the region surrounding the transcription start site (TSS) compared to MyoD Table , possiblMSC heterodimerizes with E-proteins to bind to E-boxes , and we A motif analysis of the binding site preferred by MSC found strong enrichment for binding at a GC core E-box Figure A, top, oP value and the percentage overlap ranged from approximately 40% to 80%   Figure B. MSC biGiven the lack of a global effect on gene expression, we hypothesized that the association with AcH4 might simply reflect binding of MSC at regions of open chromatin. The MyoD-specific, MSC-specific and shared peaks in the RD cells were compared to publicly available DNase hypersensitivity data from human myoblasts. Shared peaks had the highest proportion of peaks that overlapped with DNase hypersensitive sites  Figure C, and thMyoD-specific peaks seemed to have a surprisingly low level of association with hypersensitive sites, but subcategorizing the MyoD-specific peaks based on whether they were unique to RD cells, or common to RDs and human myotubes  revealedElectrophoretic mobility shift assays with in vitro translated proteins were performed to further investigate the sequence preference of MyoD and MSC dimers using the sequence from a MSC-specific peak at the SKI gene that contained the MSC-specific consensus 8-bp motif (CCAGCTGG). Shifts comparing binding of MyoD:E and MSC:E heterodimers demonstrated that MSC heterodimers could bind to the 8-bp motif or a probe in which the −1 position was changed from a ‘C’ to a ‘G’ (−1 mut), making it more reflective of the core MSC motif from Figure To further test this hypothesis, competition assays were performed on MSC and MyoD heterodimers. As expected, MSC was competed off the MSC-specific 8-bp motif equally well by cold competitors with the consensus 8-bp motif, –1 mut, or −1/+1 mut , though it is possible that part of this enrichment is due to the GC-rich nature of promoters and the binding site preference of MSC for an additional flanking ‘G’ compared to MyoD Figure . While aThe myogenic bHLH inhibitor musculin binds widely throughout the genome in RD rhabdomyosarcoma cells and has a broadly overlapping, but non-identical, set of binding sites and peaks as MyoD. Compared to the preferred MyoD E-box sequence, MSC has slightly less stringency for flanking sequence preference, permitting binding to a slightly broader set of E-boxes and potentially overlapping with other bHLH factors. Together with prior studies showing the ability of MSC to modulate MyoD activity at overlapping sites at specific promoters, these results suggest a broad potential for MSC to modulate the activity of MyoD, and perhaps other bHLH proteins, during normal development and in cancers.AcH4: acetylated histone H4; bHLH: basic helix-loop-helix; bp: base pair; ChIP: chromatin immunoprecipitation; ChIP-seq: chromatin immunoprecipitation coupled to high-throughput sequencing; EMSA: electrophoretic mobility shift assay; HSS: hypersensitivity; kb: kilobase; LC-MS/MS: liquid chromatography coupled to tandem mass spectrometry; MRF: myogenic regulatory factor; MSC: musculin; mut: mutation; qPCR: quantitative PCR; RMS: rhabdomyosarcoma; TAP: tandem affinity purification; TEV: tobacco etch virus; TSS: transcription start site.The authors declare they have no potential competing interests.KLM contributed to all experimental designs, performed all non-computational experiments and drafted the manuscript. ZY performed the ChIP-seq and all other computational analyses and contributed to both experimental design and data interpretation. APF performed the RD cell-type control ChIP-seq. SJT conceived the project, contributed to all experimental designs and edited the manuscript. All authors read and approved the final manuscript.y-axis is non-linear. Screenshots are from the UCSC genome browser, and the identity of the factor used in the ChIP, and the number of reads at the peak of occupancy are indicated along the side. ChIP, chromatin immunoprecipitation; ChIP-seq, chromatin immunoprecipitation coupled to high-throughput sequencing; MSC, musculin.MyoD and MSC site-specific ChIP confirms the ChIP-seq results. Biologically independent site-specific ChIP was performed at three sites indicated by the ChIP-seq to be MyoD-specific binding sites, three sites indicated as MSC-specific, and one control location with no significant binding of either factor, as indicated by both the chart and the screenshots. The enrichment was calculated for each location as the percentage of input amplified in qPCR with antibody divided by the percentage of input amplified with no antibody, and the value is indicated at the top of each bar. Note that the Click here for fileLC-MS/MS identification of MSC-associated transcription factors in RD cells.Click here for filey-axis represents the fraction of data that has a value equal to or less than the corresponding x-axis DNase HSS value. (B) MyoD-specific sites bound by MyoD only in RD cells, and not in human myotubes, overlap poorly with HSS sites in human myotubes. The MyoD-specific peaks from Figure MSC binds sites associated with DNase hypersensitivity, and MyoD peaks found only in RD cells, not in normal myotubes, are associated with areas identified in myotubes as DNase-resistant. (A) Shared MyoD and MSC binding peaks are associated strongly with DNase hypersensitive (HSS) sites in human myoblasts. The overlap between ChIP-seq peaks and HSS data is graphed for the entirety of the range of HSS values. Values for a DNase signal of ‘0’ are equal to 1 – the fraction graphed in Figure Click here for file


Adrb1) and downstream targets including Ucp1 in brown adipocytes (BATs). Phosphorylation of JMJD1A by PKA increases its interaction with the SWI/SNF nucleosome remodelling complex and DNA-bound PPARγ. This complex confers β-adrenergic-induced rapid JMJD1A recruitment to target sites and facilitates long-range chromatin interactions and target gene activation. This rapid gene induction is dependent on S265 phosphorylation but not on demethylation activity. Our results show that JMJD1A has two important roles in regulating hormone-stimulated chromatin dynamics that modulate thermogenesis in BATs. In one role, JMJD1A is recruited to target sites and functions as a cAMP-responsive scaffold that facilitates long-range chromatin interactions, and in the second role, JMJD1A demethylates H3K9 di-methylation.Histone 3 lysine 9 (H3K9) demethylase JMJD1A regulates β-adrenergic-induced systemic metabolism and body weight control. Here we show that JMJD1A is phosphorylated at S265 by protein kinase A (PKA), and this is pivotal to activate the β1-adrenergic receptor gene ( Demethylase JMJD1A activates thermogenesis-related target genes in response to β-adrenergic hormones. Here, the authors show that phosphorylation of JMJD1A at S265 increases its interaction with the SWI/SNF chromatin remodeler, leading to long-range chromatin interactions and target gene activation. The coupling of environmental cues with the regulation of gene transcription is a very important step in all cellular adaptive responses. For example, catecholamines released from the sympathetic nervous system in response to cold exposure bind to β-adrenergic receptors (β-ARs), and trigger the downstream cyclic AMP (cAMP) signalling cascade. The cAMP-dependent protein kinase A (PKA) phosphorylates a variety of downstream target substrates , a member of the Jumonji C-domain containing histone demethylase family, catalyses removal of H3K9 mono- and di-methylation  whose product is pivotal for heat generation. This β-adrenergic-induced binding of JMJD1A to a proximal Ucp1 enhancer region is a critical step for subsequent gene activation; however, how β-adrenergic stimulation triggers JMJD1A recruitment to Ucp1 and other genes involved in energy expenditure in BATs has remained elusive.We and another group reported that JMJD1A deficiency results in obesity with defects in brown adipose tissue functions that lead to cold intolerance and decreased oxygen consumption13The chromatin remodelling SWI/SNF (SWItch/Sucrose NonFermentable) complex couples the perturbation of histone–DNA contacts with promoter access by TFs to their cognate DNA elementsPost-translational modifications allow proteins to play multiple roles in different physiological contexts. Thus, histone modification enzymes are feasible targets of post-translational modifications that enable cells to adopt various environmental changes. In the current study, we show that JMJD1A is phosphorylated at serine 265 by PKA downstream from β-adrenergic stimulation. This modification facilitates JMJD1A interaction with SWI/SNF and DNA-bound peroxisome proliferator-activated receptor-γ (PPARγ). This phosphorylation switch in JMJD1A is independent of its demethylase activity, suggesting that it plays a scaffolding role to mediate long-range chromatin interactions that position distal enhancers in close proximity to target gene promoters for key thermogenic genes.Z-score of 62.7 in the JMJD1A-associated regions with the 10,000 highest SICER scores -seq and global gene expression analyses. Immortalized pre-BATs  were differentiated and ChIP-seq was conducted using a newly generated monoclonal anti-mouse JMJD1A antibody at 0 time and 2 h following treatment with the β-AR pan-agonist isoproterenol (ISO). ChIP-seq peak calling by SICER identified 27,397 genomic regions as significant binding sites of JMJD1A in ISO-treated iBATs. JMJD1A localized on proximal promoters (∼13%), intragenic (∼52%) and intergenic regions ∼24%; . The seqR scores .0.8-fold after 1 h ISO treatment . Compared with the top 2,000 ISO-induced JMJD1A binding sites, which were annotated to 1,551 proximal genes, 39 out of 67 ISO-induced genes were found to be occupied by JMJD1A  1–300) at S265  analysis. The immunoprecipitated JMJD1A from HeLa cells treated with ISO for 1 h was excised from an SDS–PAGE gel and subjected to LC/MS/MS . This an at S265 . Approxi at S265 . These dImmunoblot analysis with a newly generated phospho-specific antibody against phospho-S265-JMJD1A detected WT-JMJD1A transiently expressed in iBATs cultured under ISO-plus conditions; however, this antibody failed to detect the S265A-JMJD1A mutant . Immunopin vitro demethylation assay. H3K9me2 demethylation activity of JMJD1A did not differ between phosphorylated and non-phosphorylated full-length recombinant JMJD1A . Human Jmjd1a is resistant to this shRNA designed against mouse Jmjd1a and the retroviral vector is driven by a very weak long terminal repeat promoter. Immunoblot analysis showed that protein level of V5-hJMJD1A was equivalent to that of native JMJD1A  reduced expression  or the control e JMJD1A . Reverse protein . In cont-iBATshs . The pospression . It is aand Ucp1 . ChIP–qPne locus . These rshs lysates confirming the LC/MS/MS analysis. Interestingly, the interaction of these SWI/SNF components with JMJD1A was increased by ISO treatment  of JMJD1A with PPARγ was no longer observed  as previously shownNext, we used ChIP-seq for two major components of the SWI/SNF complex  and the SWI/SNF chromatin remodelling complex that are located at distal E1 and/or E2 enhancers are brought in proximity to the TSS of reatment . 3C-qPCRependent . Togethemulation . Similarmulation .Brg1 abrogated the FSK-dependent chromatin looping between E1 and Adrb1 promoter as well  enhancer–promoter drove appreciable level of luciferase gene expression and the additional two distal enhancers (−13 and −5 kb regions) plus proximal (−2 kb) enhancer–promoter to this construct drove much higher luciferase gene expression  by DOB stimulation, while this induction was markedly blunted in S265A-hJMJD1A-iBATshs   . We next−/− mice . We alsod by ISO . This rein vivo by β-adrenergic stimulation and participates in enhancer–promoter looping in a physiologically relevant context.Taken together, these results provide strong evidence that S265 of JMJD1A is indeed phosphorylated in vivo. Mechanistically, we showed that assembly of the JMJD1A–SWI/SNF complex, recruitment of signature enhancers to a target promoter and transcriptional activation were all dependent on PKA phosphorylation of S265 in JMJD1A. As a result of this PKA-dependent switch in JMJD1A–SWI/SNF interaction, a PPARγ-bound enhancer is brought in proximity of the responsive TSS, which accelerates RNA polymerase recruitment and transcription  is the most upstream component of β-adrenergic signalling pathway that directly couples the cAMP-PKA-mediated phosphorylation cascade with downstream cellular events. We identified Adrb1 as a bona fide target of cAMP-sensing through JMJD1A. Adrb1 is under positive β-adrenergic control and is thus self-inducing under the condition of increased sympathetic tone21Adrb1 is inherently transient because its transcript quickly decays with an mRNA half-life of 17 min  waiting at remote enhancers are immediately recruited to the promoter/TSS via long-range looping to turn transcription ‘On'. On the flip side, enhanced transcription can also be quickly turned ‘Off' via the rapid dephosphorylation of JMJD1A and the disassembly of the JMJD1A–SWI/SNF–PPARγ complex. This spatial regulation of remote enhancer and associated competent proteins (TFs and chromatin remodelers) may have profound merit for a rapid on-off gene transcriptional switch.et al.21Adrb3 but a reciprocal significant increase in Adrb1. Our results show a similar reciprocal expression pattern for the two different β-AR proteins in response to cold in WT mice  or cold exposure after acclimating at thermoneutral conditions may lead to more drastic H3K9me2 changes at enhancers in brown fat as a consequence of the chronic adaption to cold environment.The PKA-dependent scaffold function of JMJD1A is independent of its lysine demethylase function. Previous study showed that JMJD1A is recruited to the proximal enhancer (∼2 kb) of Overall, the current studies combined with previous observations suggest that JMJD1A has dual roles for the adaptation to environmental cues : one for hormone-induced dynamic chromatin remodelling and the other for demethylation that ensures a long-term stable pattern of gene expression. Our data suggest that PKA-dependent DNA looping plays a more dominant role for the acute and dynamic response, while H3K9 demethylation subsequently plays a key role for a more stable long-term change in gene expression.Mouse monoclonal antibodies immunoglobulin G F0618 (IgG-F0618) and IgG-F0231 against mouse JMJD1A (a.a. 843–893) were produced by immunizing separate mice with gp64 fusion protein expressing baculovirus. Mouse monoclonal antibodies IgG-F0026 and IgG-F1628 against hJMJD1A were produced by immunizing mice with affinity-purified recombinant hJMJD1A protein. A rabbit polyclonal phospho-specific antibody against JMJD1A-Ser265 was produced by Operon Biotechnologies with a synthetic phosphopeptide corresponding to residues surrounding Ser265 JMJD1A (Ac- C-KRKS(pS)ENNGS-amide). A list of the other antibodies used in this article is shown in the −1 pepstatin A, 10 μg ml−1 leupeptin, 2.8 μg ml−1 aprotinin, 1 mM dithiothreitol (DTT) and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)) and phosphatase inhibitors (40 mM NaF and 1 mM Na3VO4). For immunoprecipitation, whole-cell lysates or nuclear extracts were immunoprecipitated in cell lysis buffer  containing both protease inhibitors  and phosphatase inhibitors (40 mM NaF and 1 mM Na3VO4) for 6 h wheel rotating at 4 °C as described, using the antibodies described in the legend to figures. For immunoblot analysis, aliquots of proteins were separated by SDS–PAGE and transferred to nitrocellulose membrane (Bio-Rad Laboratories). Immunodetection was carried out with the indicated antibodies , the eluent was incubated with pronase (1 mg ml−1) at 42 °C for 2 h and then incubated at 65 °C overnight to reverse the crosslinks. DNA was purified using QIA quick PCR purification kit (Qiagen) and the concentration was measured by Qubit double-stranded DNA High Sensitivity assay kit (Invitrogen). For ChIP using the other antibodies , iBATs at day 8 of differentiation were crosslinked with 0.5% formaldehyde for 10 min and chromatin DNA was sonicated to be 0.5 kb average in size. ChIP-sequencing was done with an Illumina/Solexa sequencer as previously described829For ChIP using JMJD1A, ARID1A, BRG1, C/EBPα, C/EBPβ and C/EBPδ antibodies, iBATs were crosslinked with 1.5 mM ethylene glycol bis(succinimidylsuccinate) (Thermo Scientific) for 30 min at room temperature (RT) and then directly a second crosslinking was performed by addition of 1% formaldehyde for 10 min. Fixation was stopped by adding 0.2 M glycine. After crosslinking, nuclear pellets were prepared for ChIP as described previously with the following modifications as described before829ChIP samples were also analysed by gene-specific qPCR analyses. The results were presented as input per cent or normalized to precipitated DNA (fold enrichment). All primer sequences used in this article are listed in The method for qPCR has been described31Formaldehyde-assisted isolation of regulatory elements was performed as described previously33http://support.illumina.com/sequencing/sequencing_software/casava.html) based on the 5′-side 36 bp sequences.All bound DNA fragments were mapped to UCSC build mm9 (NCBI Build 37) assembly of the mouse genome by a mapping programme ELAND  based on the Z-scores.To adjust the distribution of the number of DNA sequence tags mapped in the top 10,000 ‘significant binding sites' of JMJD1A, http://www.genomatix.de/). The top 10 known TF binding motifs were selected as the putative binding motifs based on the Z-scores representing scores of motif enrichment on the basis of random promoter sequences. To reconstruct exact binding motifs, all sequences corresponding to the identified putative motifs were assembled using a scoring method named matrix similarity score (MSS)To gain insights into the mechanism by which JMJD1A recognizes is recruited to its cognate DNA sequences, we studied the frequency of known TF binding motifs in the JMJD1A-associated regions with the top 10,000 highest JMJD1A-associated regions with higher SICER scores. DNA sequences that JMJD1A bound to were examined for motifs that are known to be binding sites of TFs using Genomatix .Putative PKA phosphorylation sequences located in JMJD1A were surveyed by the Scansite Motif Scanner (JMJD1A and/or JMJD1A-associated proteins that were immunoprecipitated from HeLa cells or 3T3-L1 cells treated with ISO (10 μM for 1 h) with anti-hJMJD1A (IgG-F0026) or anti-mJMJD1A (IgG-F0231), respectively, were separated on SDS–PAGE, stained by SYPRO Ruby (Invitrogen), and the excised band was digested in-gel as previously described2 according to the method described previously−1. The immortalized cell line (pre-iBATs) was tested for differentiation capacity and expression levels of Ucp1 as described−1) and indomethacin (125 μM) in basal medium. The cells were then returned to the basal medium containing insulin , which was replenished every other day. The cells were stained with Oil Red O as described29Stromal vascular fractions (SVFs) were isolated from interscapular BATs of C57BL/6 mouse at P3 by digesting the brown adipose tissue depots with collagenase D (Roche) and dispase II (Roche) in PBS supplemented with 10 mM CaClshs), iBATs were transduced with retroviral vector expressing shRNA targeting mouse Jmjd1a and were selected by G418 (1 mg ml−1) in basal medium for 6 days. To establish iBATshs ectopically expressing V5-tagged WT or mutants JMJD1A, iBATshs were infected with retrovirus expressing WT, S264A, S265A, S341A, 3SA, H1120Y, H1120F -hJMJD1A or empty vector followed by Zeocin selection (0.1 mg ml−1) for 12 days. The resultant iBATs transformants were designated WT, S264A, S265A, S341A, 3SA, H1120Y, H1120F -hJMJD1A-iBATshs and empty -hJMJD1A-iBATshs, respectively.To establish shRNA-mediated JMJD1A knockdown iBATs  containing protease inhibitors  and phosphatase inhibitors (40 mM NaF and 1 mM Na3VO4), and incubated for 90 min at 4 °C. The swollen cells were centrifuged at 3,300g for 1 min at 4 °C in microcentrifuge. The pellet was dissolved in digestion buffer containing 0.3% SDS, incubated for 1 h at 37 °C, digested with 600U Hind III (Takara) containing 1.8% Triton X-100 overnight at 37 °C, followed by random re-ligation with 350U T4 DNA ligase (Takara). After reversing crosslinks by incubation with proteinase K (0.1 mg ml−1) at 65 °C for 4 h, genomic DNA was purified by phenol/chloroform extraction and ethanol precipitation. The ligated DNA (50 ng) was assessed by qPCR using ABI 7900HT system (Applied Biosystems) and TaqMan Universal PCR Master Mix (Applied Biosystems). All primer sequences used in this article are listed in g for 1 min at 4 °C. The pellet was processed for Hind III digestion as described above.3C assay was performed following a protocol as described previouslyt-test was performed for the comparison of two groups. For multiple comparisons, analysis of variance were performed followed by Tukey's post hoc comparison. *P<0.05, **P<0.01 and ***P<0.001.All data are presented as mean±s.e.m. Student's T.I. and J.S. designed the study and wrote the paper. Y.A., R.R., Y.M., T.K., Y.T, K.T.-I., A.S., K.M., K.N. and S.O. carried out experiments. R.N carried out analyses. S.K. and H.K. provided materials.Accession codes: The GEO accession numbers for gene expression microarray data and ChIP-seq data reported in this paper is GSE58936 and GSE67586, respectively.How to cite this article: Abe, Y. et al. JMJD1A is a signal-sensing scaffold that regulates acute chromatin dynamics via SWI/SNF association for thermogenesis. Nat. Commun. 6:7052 doi: 10.1038/ncomms8052 (2015).Supplementary Figures 1-15, Supplementary Tables 1-4, Supplementary Methods and Supplementary References


Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis (Bt) are spore-forming members of the Bacillus cereus group. Spores of B. cereus group species are encircled by exosporium, which is composed of an external hair-like nap and a paracrystalline basal layer. Despite the extensive studies on the structure of the exosporium-related proteins, little is known about the transcription and regulation of exosporium gene expression in the B. cereus group. Herein, we studied the regulation of several exosporium-related genes in Bt. A SigK consensus sequence is present upstream of genes encoding hair-like nap proteins (bclA and bclB), basal layer proteins , and inosine hydrolase (iunH). Mutation of sigK decreased the transcriptional activities of all these genes, indicating that the transcription of these genes is controlled by SigK. Furthermore, mutation of gerE decreased the transcriptional activities of bclB, bxpB, cotB, and iunH but increased the expression of bxpA, and GerE binds to the promoters of bclB, bxpB, cotB, bxpA, and iunH. These results suggest that GerE directly regulates the transcription of these genes, increasing the expression of bclB, bxpB, cotB, and iunH and decreasing that of bxpA. These findings provide insight into the exosporium assembly process at the transcriptional level. Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis (Bt) are spore-forming members of the Bacillus cereus groupBacillus encompasses species capable of forming highly resistant dormant endospores as a response to harsh environmental conditions. Spores of the B. cereus group are complex, multilayered structures. The nucleoid-containing core is enclosed within a peptidoglycan cortex, which is surrounded by the spore coatB. cereus group species are encircled by an additional loose-fitting layer called the exosporiumBacillus subtilis, for which the coat constitutes the outermost layer of the mature sporeThe genus Many characteristics of the exosporium have been previously elucidated. The exosporium is separated from the spore coat by a region known as the interspace and is the final layer of the spore to be assembled9101113149109161011212628B. cereus group. Herein, we demonstrate that the transcription of bclA, bclB, bxpA, bxpB, cotB, exsY, and iunH are controlled by RNA polymerase sigma factor SigK in Bt HD73. Furthermore, the expression of bclB, bxpA, bxpB, cotB, and iunH is directly regulated by GerE. gerE encodes the terminal transcription factor in the sporulation regulatory cascade in Bacillus subtilis. GerE is a small DNA-binding protein that is both an activator and a repressor in the mother cell that regulates the transcription of many genes involved in spore coat synthesis and assembly in the late stages of sporulation and germination3031Despite the extensive studies on the structure of the exosporium-related proteins, little is known about the transcription and regulation of exosporium gene expression in the B. cereus and B. anthracis in Bt HD73 (HD73_1438 (bclA) and HD73_2664 (bclB) encode BclA and BclB and have 67.8% and 90.0% identity, respecively, to homologous genes in B. anthracis Sterne strain 7702B. cereus ATCC 10876bclA and bclB, 5′-RACE analysis was performed as described in the Methods. The TSSs of bclA and bclB were confirmed to be a single 5′-end nucleotide residue C and G located 120 bp and 150 bp upstream of the start codon according to the sequences of 20 random clones, respectively (bclA and bclB promoter sequences identified sequences CAC(-N16-)CATATGTTA and AGC(-N16-)CATATAATT upstream of the bclA and bclB TSS, respectively, which are similar to the consensus sequences recognized by SigK-containing RNA polymerasebclA and PbclB, PbclA-lacZ and PbclB-lacZ fusions were constructed and transformed into Bt wild-type strain HD73 and mutant strains, HD(ΔsigK) and HD(ΔgerE). The β-galactosidase assay showed that the transcriptional activity of PbclA was sharply decreased from T10 to T23 in HD(ΔsigK) (10 to T18 in HD(ΔgerE), and with no significant difference from T18 to T23 compared with that of wild-type strain HD73 (bclB was sharply decreased from T9 to T23 both in HD(ΔsigK) and HD(ΔgerE) compared with that of HD73  and PbclB (276 bp) promoters was examined by EMSA. FAM-labeled fragments containing the promoter regions of bclB were incubated with different amounts of GerE and assayed for the formation of protein-DNA complexes. Slower-migrating probe-protein complexes were observed upon incubation with increasing amounts of GerE . This may result from the lack of direct binding, from a purified GerE protein partially defective in binding or from unfavorable in vitro binding conditions. These results indicated that transcription of PbclA and PbclB are controlled by SigK in the late stage of sporulation and that PbclB is directly activated by GerE, while PbclA is negatively regulated by GerE.We identified 17 exosporium homologous genes with known functions in  Bt HD73  comprisiectively A and 2A.D(ΔsigK) . It was ain HD73 . However of HD73 . To dete of GerE . It indithe EMSA . A 23-bpr region  was protbxpA (HD73_2410), bxpB (HD73_1452), cotB (HD73_0469), and exsY (HD73_1449). These genes have 75.4%, 97.0%, 76.9%, and 87.0% identity, respectively, to homologous genes in B. anthracis or B. cereus  and decreased in HD(ΔgerE) compared with those of wild-type strain HD73 , whereas it was increased in HD(ΔgerE) compared with HD73  but showed no significant difference in HD(ΔgerE)  . These riunH (HD73_3089) in Bt HD73, which has 93.1% identity to the homologous gene bas2693 in the B. anthracis Ames strainiunH was confirmed to be a single 5′-end nucleotide residue G residue located 10 bp upstream of the start codon (iunH (iunH was abolished from T8 to T22 in HD(ΔsigK) and lower in HD(ΔgerE) than in HD73  controls gene expression in the mother cell during sporulation314B. subtilis sporeB. cereus group species are encircled by the exosporiumB. cereus group. Indeed, only exsB is known to undergo SigK-mediated transcription and is positively regulated by GerE, as shown in our pervious studybclA, bclB, bxpA, bxpB, cotB, exsY, and iunH are controlled by SigK using a β-galactosidase assay. The SigK consensus sequence is located upstream of these and ten other exosporium-related genes in Bt and is predicted to be present in most B. cereus group strains . This finding suggested that the transcription mechanisms of exosporium genes are similar throughout the B. cereus group.In a B. subtilis cascade, the synthesis of each factor depends upon the activity of the prior factor, and there is a feedback loop in which SigK RNAP transcribes gerE, which then negatively regulates transcription of the sigK gene31oxdDgerT cotBcotCyxeEyeeKB. subtilis. We observed similar effects under the current conditions. The transcription of bclA and bxpA is negatively regulated by GerE, which could bind to the promoter of bxpA. Furthermore, the transcription of bclB, bxpB, cotB, and iunH is positively regulated by GerE, and their promoters could bind to GerE.In the 10). BxpA is located below the spore coat associated with the cortex and is synthesized during sporulation and assembled into the spore before mother cell lysis, but it is not found in vegetative cells in B. anthracis AmesbxpAbxpA initiates at T8 and is abolished in the sigK mutant. ExsY is a homologue of B. subtilis cysteine-rich spore coat proteins CotY and CotZexsY begins at T7 under the control of SigK and is similar to the transcriptional mechanism of cotYZ in B. subtilisB. anthracisB. subtilis spore coat protein CotBcotB begins at T10 under the control of SigK, and is regulated by GerE in Bt. The manner of transcription and regulation is similar between Bt and B. subtilis31bclA, bxpB, cotB, bxpA, exsY, and iunH in wild-type HD73 is very similar, increasing from T8 to T17 and decreasing thereafter, suggesting that these proteins are assembled into the basal layer and hair-like nap simultaneously and are nearly complete at T17. However, the transcription of bclB is significantly higher than that of blcA after T17 with continuous transcriptional activity from T8 to T23. These transcriptional data are differ to previous reports, which have suggested that bclB and bclA are transcribed at an identical stage in sporulation, but with bclB transcribed at an approximately two-fold lower level9The collagen-like glycoproteins BclA and BclB require BxpB to assemble the hair-like nap of exosporium, and the assembly timing of the three proteins is similarEscherichia coli strain TG1 was used as the host for cloning experiments. The Dam-/Dcm-E. coli ET12567 strain (laboratory stock) was used to generate unmethylated DNA for the electrotransformation assay. Bt strains were transformed by electroporation, as described previously51E. coli and Bt strains were cultured in Luria-Bertani (LB) medium, with 220 rpm shaking at 37 °C and 30 °C, respectively. The antibiotic concentrations used for bacterial selection were as follows: 100 μg/ml kanamycin and 10 μg/ml erythromycin for Bt, and 100 μg/ml ampicillin for E. coli.The bacterial strains and plasmids used in this study are listed in Taq and KOD DNA polymerase . Amplified fragments were purified using purification kits . Bt chromosomal DNA was extracted with the Puregene kit . Restriction enzymes and T4 DNA ligase  were used according to the manufacturer’s instructions. Oligonucleotide primers .PCR was performed using  primers  were synTM RACE cDNA Amplification Kit  according to the manufacturer’s instructions. Gene-specific primers and the universal primer mix (UPM) ix (UPM)  were useS-transferase (GST) tag was purified from E. coliE. coli BL21(DE3) strain carrying pGEXgerE plasmid was incubated in LB medium. When the optical density at 600 nm (OD600) reached 0.6, IPTG was added to a final concentration of 1 mM. After 4 h of induction at 37 °C, the bacterial cells were harvested by centrifuging the culture at 13,000 × g for 10 min. The pellet was resuspended in phosphate-buffered saline (PBS) and sonicated on ice. All subsequent procedures were carried out at 4 °C. The supernatant was collected by centrifuging the lysate at 13,000 × g for 20 min and loading it onto a glutathione-Sepharose 4B column previously equilibrated with PBS buffer. The column was washed with 50 mM Tris-HCl containing 10 mM reduced glutathione (pH 8.0). The fractions were analyzed by SDS-PAGE. Fractions with the target protein were pooled and dialyzed against PBS buffer. The purified GST-GerE protein was analyzed by SDS-PAGE on a 12% polyacrylamide gel with a protein molecular standard. All the steps described above were performed according to the manufacturer’s instructions .GerE protein with a glutathione cry1Ac promoter  was used as the negative control.The DNA fragment was obtained by PCR of strain HD73 genomic DNA using specific primers  labeled DNase I footprinting assays were performed based on a fluorescence labeling procedurelacZ geneΔsigK and ΔgerE mutant strains. The resultant strains, HD73(Pn), ΔsigK(Pn), and ΔgerE(Pn), were selected by resistance to erythromycin and tested by PCR to confirm the presence of the promoter fragments in the plasmids.The promoters of exosporium genes were amplified from Bt HD73 genomic DNA using specific primers. Promoter restriction fragments were then ligated into the pHT304-18Z vector containing a promoterless lacZ transcriptional fusions were cultured in Schaeffer’s sporulation medium (SSM)8 to T22 , from which cells were harvested by centrifugation for 1 min at 10,000 × g. The supernatant was removed, and the pellet was stored at −20 °C or resuspended in 500 μl Buffer Z  with 1 mM dithiothreitol. The β-galactosidase activity was determined as previously describedBt strains containing How to cite this article: Peng, Q. et al. The Regulation of Exosporium-Related Genes in Bacillus thuringiensis. Sci. Rep.6, 19005; doi: 10.1038/srep19005 (2016).