FLC is the direct target of both of the transcription factors ABI4 and ABI5, and ABA inhibits floral transition by activating FLC transcription through ABI4. Arabidopsis, flowering time is precisely controlled by extensive environmental and internal cues. Gibberellins (GAs) promote flowering, while abscisic acid (ABA) is considered as a flowering suppressor. However, the detailed mechanism through which ABA inhibits the floral transition is poorly understood. Here, we report that ABSCISIC ACID-INSENSITIVE 4 (ABI4), a key component in the ABA signalling pathway, negatively regulates floral transition by directly promoting FLOWERING LOCUS C (FLC) transcription. The abi4 mutant showed the early flowering phenotype whereas ABI4-overexpressing (OE-ABI4) plants had delayed floral transition. Consistently, quantitative reverse transcription–PCR (qRT–PCR) assay revealed that the FLC transcription level was down-regulated in abi4, but up-regulated in OE-ABI4. The change in FT level was consistent with the pattern of FLC expression. Chromatin immunoprecipitation-qPCR (ChIP-qPCR), electrophoretic mobility shift assay (EMSA), and tobacco transient expression analysis showed that ABI4 promotes FLC expression by directly binding to its promoter. Genetic analysis demonstrated that OE-ABI4::flc-3 could not alter the flc-3 phenotype. OE-FLC::abi4 showed a markedly delayed flowering phenotype, which mimicked OE-FLC::WT, and suggested that ABI4 acts upstream of FLC in the same genetic pathway. Taken together, these findings suggest that ABA inhibits the floral transition by activating FLC transcription through ABI4.During the life cycle of a plant, one of the major biological processes is the transition from the vegetative to the reproductive stage. In  As sessile organisms, plants monitor the changes in both external and internal signals, including photoperiod, temperature, and phytohormonal levels, to decide their flowering initiation . OptimizArabidopsis (FLOWERING LOCUS C (FLC) is a potent integrator, which negatively regulates flowering initiation , FLOWERING LOCUS D (FD), and FLOWERING LOCUS T (FT) through directly binding to the promoter regions of SOC1 and FD or the first intron of FT. Thus these three genes represent further key factors in regulating flowering time -dependent pathways have already been fully elucidated in bidopsis . These fitiation . Consequtructure . As a trHyoscyamus niger) (LEAFY (LFY)  . NumerouFLC transcription by directly binding to its promoter. Thus, ABI5 is an important factor through which ABA inhibits the plant floral transition  is an AP2/ERF domain-containing transcription factor and is an enhancer in the ABA signalling pathway that functions particularly during seed development, seed dormancy, and regulation of germination  Arabidopsis delays the floral transition. Further, we found that the FLC expression level was down-regulated in abi4, but up-regulated in OE-ABI4. Chromatin immunoprecipitation qPCR (ChIP-qPCR), electrophoretic mobility shift assay (EMSA), and tobacco transient expression analysis showed that ABI4 activates FLC expression by directly binding to its promoter. Consistent with these results, genetic analysis demonstrated that OE-ABI4::flc-3 did not alter the flc-3 phenotype, while OE-FLC::abi4 changed the abi4 early-flowering phenotype. This indicates that ABI4 acts upstream of FLC in the same genetic pathway. Taken together, the results of this study suggest that ABI4 is a key factor which negatively regulates flowering through activating FLC transcription directly.Here, we perform further investigation of the roles of ABI4 in the initiation of flowering. Consistent with previous reports  in this study. The point mutant abi4-1 (CS8104) was obtained from the Arabidopsis Biological Resource Center . This mutant originated from the Finkelstein laboratory  had been generated in our previous study  medium. The plates were transferred to tissue culture rooms at 22 ºC under long-day (16h light/8h dark) (LD) or short-day (8h light/16h dark) (SD) photoperiod conditions, depending on the needs of the particular experiments. Ten-day-old seedlings were transplanted into soil and placed in a growth chamber, again under LD or SD conditions, at 22 ºC and 70% relative humidity.ABI4 which had been generated in a previously reported study  35S promoter. This construct was transformed into the abi4 mutant background by the vacuum inﬁltration method using the Agrobacterium tumefaciens strain EHA105  were examined. In this study, flowering time was scored as the days from germination to flowering and the number of total rosette leaves at bolting, according to the protocol of 18S RNA as an internal control. Three biological replicates were performed for each experiment. The primer sequences used for qRT–PCR are shown in Supplementary Table S1 available at JXB online.Preparation of total RNA from the 2-week-old seedlings, ﬁrst-strand cDNA synthesis, and quantitative reverse transcription–PCR (qRT–PCR) were performed as previously described (35S-ABI4-GFP were harvested (1.5g) on 1/2 MS medium and then cross-linked with 1% formaldehyde. The seedlings were ground in liquid nitrogen, and then the nuclei were isolated. Immunoprecipitation assays were performed with the anti-green fluorescent protein (GFP) antibody and protein G beads. Immunoprecipitation in the absence of anti-GFP served as the control (CK). The DNA was precipitated by isopropanol, and dissolved in water containing 20 μg ml–1 RNase. The qPCR analysis was performed using specific primers corresponding to the FLC promoter. TUB4 was used as an internal control. The ABI5 promoter fragment was used as a positive control since a previous study demonstrated that ABI4 could directly bind to the promoter of ABI5 (Supplementary Table S1 at JXB online.ChIP-qPCR assays were performed as previously described (Nicotiana benthamiana leaves as previously described (FLC promoter (Pro-FLC) was amplified from genomic DNA. This promoter fragment was cloned into the pCambia1300-221 vector by replacing the original CaMV 35S promoter, and then pCambia1300-221-ProFLC-GUS was generated. pCanG-ABI4-GFP was the effector construct. The A. tumefaciens-mediated tobacco transient transformation was performed according to our previous protocol  were also generated, in which some key CCAC motif sequences were changed.The transient expression assay was performed in d-glucuronide  as a substrate (β-Glucuronidase (GUS) activity was detected 3 d after infiltration. Leaves were sampled using a hole punch. The total protein was quantified using the Bradford protein assay kit method . GUS activity for each combination was determined using the protocol described previously with 4-methylumbelliferyl-β-ABI4 was inserted into the BamHI/XbaI sites in the pMalC2 backbone vector which contains a maltose-binding protein (MBP) tag. Then the ABI4 fusion proteins were expressed in Escherichia coli (37 °C) and purified. According to the ChIP-qPCR results, we chose the P4 region , according to a previously published protocol  and SD  and SD , E growthenotype , F. Altoabi4 mutant and OE-ABI4 plants. It has been demonstrated that transcription factor FLC is the key repressor in floral transition and links the diverse flowering time regulation pathways  may result from changes in the transcription levels of FLC, FT, and LFY.A previous study revealed that FLC directly represses the flowering identity gene  changes . In the h the WT , and, ac OE-ABI4 . Furtherh the WT . TogetheABI4 transcription. The results showed that the ABI4 expression level was strongly induced by treatment with exogenous ABA . This is in fact consistent with previous studies  and in vitro (EMSA).Previous studies have demonstrated that ABI4 binds to the CCAC motifs in some promoters to regulate the transcription of its targets genes directly  were employed, and similar results were obtained. However, we did not detect the enrichment of the FT and LFY promoter fragments, although there were four and five CCAC cis-elements in their promoters, respectively.We initially explored the promoters of these three genes, as shown in rt codon . In addiFLC promoter in vitro. The recombinant ABI4 fusion protein was expressed and purified from E. coli, and the P4 fragment  in the FLC promoter. The P4 and P5 fragments were chosen as they showed the highest binding activity for ABI4 protein, revealed by ChIP-qPCR and EMSA (Pro-FLC (m1)-GUS and Pro-FLC (m2)-GUS were generated . Using the transient expression system, the results revealed that the activation effect of ABI4 on FLC transcription was impaired in the presence of the mutated promoter constructs . Altogether, combined with the ChIP-qPCR and EMSA results, the transient expression system analysis demonstrated that ABI4 directly promotes FLC expression, and this effect is dependent on some key CCAC elements in the FLC promoter.The reporter plasmid G-HA-GFP , E. Subsficantly , E. Thesand EMSA . SubsequFLC expression. To confirm this conclusion further, the genetic relationship between ABI4 and FLC was explored.The phenotypic analysis, and biochemical and molecular evidence demonstrated that the transcription factor ABI4 negatively regulates flowering time through activation of OE-ABI4 construct was introduced into the flc-3 mutant background by genetic crossing, and the flowering phenotypes of flc-3 and OE-ABI4::flc-3 were examined. In our experimental conditions, we detect the early-flowering phenotype of flc-3 mutant plants, and overexpression of ABI4 did not change the early-flowering phenotype of flc-3 in terms of the number of rosette leaves and the days to flowering  by transgenic assay, and examined the floral phenotype of abi4 and OE-FLC::abi4. The results showed that overexpression of FLC significantly changed the early-flowering phenotype of abi4  mutant screening, and is a point mutation generated by ethylmethane sulphonate (EMS)-induced mutagenesis. It has also been called sugar-insensitive 5 (sis5)   and in previous studies , encoding the rate-limiting enzyme in the triacylglycerol biosynthesis pathway (The SUPPRESSOR OF FRIGIDA 4 (SUF4)-mediated transcription factor complex negatively regulates flowering through promoting tor ABI4 . AnotherFLC is the target of both the the transcription factors ABI4 and ABI5; thus an interesting question arises. How do the plant discriminate between the roles of ABI4 and ABI5 in flowering time regulation? Combined with a previous study, we know that both transcription factors bind to the FLC promoter through different motifs. ABI4 binds the CCAC motif (FLC promoter through ABRE (abscisic acid-responsive element) or ABRE-like elements (CATGCG) (FLC promoter and ABI5/ABI4. Consequently, further bioinformatics analysis is needed to deepen our understanding of the overlap and/or distinct roles of ABI5 and ABI4 in the control of flowering.AC motif , while AFLC expression directly to control plant floral transition precisely. Because ABI4 also binds directly to the ABI5 promoter and activates its transcription (FLC transcription through increasing ABI5 expression. Interesting, it is noted that the model allows for the possibility that other mutants involved in the ABA signalling pathway, such as abi3, may regulate the flowering time, which should be the focus of future investigations.Taking our findings together, we propose a working model illustrated in JXB online.Supplementary data are available at Figure S1. Late-flowering phenotype of OE-ABI4 under long-day conditions.Figure S2. ABA induces ABI4 transcription.Figure S3. The ABI5 promoter was dissected.Figure S4. ABI4 promotes FLC transcription in a CCAC-dependent manner.Table S1. The primers used in this study.


Schistosomes are parasitic platyhelminthes that are characterized by passage through two hosts: a mollusk as intermediate host and humans or rodents as definitive host. During its development, the parasite undergoes drastic changes, most notable immediately after infection of the definitive host, i.e. during the transition from the free-swimming cercariae into adult worms.Chromatin structure can control gene expression and can define specific transcription states. For example, bivalent methylation of histone H3K4 and H3K27 is linked to poised transcription in vertebrate embryonic stem cells (ESC). It allows them to rapidly engage specific developmental pathways. We reasoned that non-vertebrate metazoans that encounter a similar developmental constraint  in cercariae, schistosomula and adults (available at http://genome.univ-perp.fr). We saw striking differences in chromatin structure between the developmental stages, but most importantly we found that cercariae possess a specific combination of marks at the transcription start sites (TSS) that has similarities to a structure found in ESC. We demonstrate that in cercariae no transcription occurs, and we provide evidences that cercariae do not possess large numbers of canonical stem cells.We used Chromatin Immunoprecipitation followed by massive parallel sequencing (ChIP-Seq) to analyze genome-wide chromatin structure of i.e. not restricted to stem cells, to poise transcription. Since adult couples must form to cause the disease symptoms, changes in histone modifications appear to be crucial for pathogenesis and represent therefore a therapeutic target.We describe here a broad view on the epigenome of a metazoan parasite. Most notably, we find bivalent histone H3 methylation in cercariae. Methylation of H3K27 is removed during transformation into schistosomula (and stays absent in adults) and transcription is activated. In addition, shifts of H3K9 methylation and acetylation occur towards upstream and downstream of the transcriptional start site (TSS). We conclude that specific H3 modifications are a phylogenetically older and probably more general mechanism,  Schistosoma mansoni causes intestinal bilharzia. The parasite has a complex life cycle in which a freshwater snail serves as intermediate host from which the human infecting larvae hatch. These larvae will actively seek skin contact, penetrate through the epithelium and start developing straight away into adult worms. Development from larvae into adults needs thorough adjustment of gene expression through repositioning or modification of proteins that are associated with DNA (the chromatin). We decided to compare the chromatin of human infective larvae (cercariae), the first developmental stage after infection of the vertebrate host (schistosomula) and adults of S. mansoni. We found that cercariae possess chromatin structures (modifications of histone H3) around the beginning of genes that are very different from schistosomula and adults. We conclude that this structure serves to keep gene transcription in a poised state, i.e. transcription is initiated and can start immediately when the blocking histone modification is removed. A similar type of histone modification was found in embryonic stem cells of vertebrates and our data indicate that it is either a more ancient and/or more general means to poise transcription than previously assumed. Since many parasites possess infective stages that develop rapidly within the host, this particular chromatin structure could be a therapeutic target for a new class of antiparasitic drugs.The blood fluke  Plasmodium falciparum for instance, it is found in subtelomeric and some small intrachromosomal regions containing almost exclusively clusters of genes involved in antigenic variations [i.e. transcription machinery is in position at the TSS, ready for activation upon reception of a signal that triggers differentiation . When En people . In contn people  that devS. mansoni are canonical with 100% identity to human H4.A, 79–99% identity to H3 forms and 62–92% to H2A and H2B [S. mansoni was completely unexplored, and we decided to study the parasite epigenome by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) of DNA associated with the above-mentioned histone isoforms H3K4me3, H3K9ac, H3K9me3 and H3K27me3. The genome of S. mansoni has been recently assembled and most of the gene structure data comes from automatic annotation [The core histone isoforms of  and H2B . The disnotation . Since gHousing, feeding and animal care followed the national ethical standards established in the writ of February 1st, 2013 (NOR: AGRG1238753A) setting the conditions for approval, planning and operation of establishments, breeders and suppliers of animals used for scientific purposes and controls. The French Ministère de l’Agriculture et de la Pêche and French Ministère de l’Éducation Nationale de la Recherche et de la Technologie provided permit A 66040 to our laboratory for experiments on animals and certificate for animal experimentation  for the experimenters.S. mansoni strain SmBre, originally sampled in Brazil was used in this study. The strain was maintained in its sympatric intermediate host strain BgBRE of the mollusk Biomphalaria glabrata, and Mus musculus as definitive vertebrate host. Cercariae were collected from infected snails 30 days after infection by pipetting from spring water and sedimentation on ice. For isolation of adults, infected mice were killed after 60 days of infection by a lethal intraperitoneal injection of sodium pentobarbital, and adult worms were recovered by retrograde perfusions of the hepatic portal system with citrate (7.5%) saline (8.5%) solution administrated through the left ventricle [The entricle . Worms tIn-vitro transformation of cercariae to schistosomula was done as described before [2 for 21 hours.d before . The shed before  preparedd before ,22 and mS. mansoni has mostly been done through prediction software, we performed RNA-Seq on cercariae to unambiguously identify TSS and exon-intron structures. Since published RNA extraction protocols for cercariae did not yield RNA suitable for Illumina sequencing, we developed the following method: 5,000 cercariae were ground with glass beads in liquid nitrogen for 5 minutes, resuspended in 0.5 mL TRIzol (Invitrogen), and 0.1 mL of chloroform was added. The tube was shaken vigorously for 1 minute and left at room temperature for 3 minutes, and was then centrifuged at ≥12,000×g for 15 minutes at 4°C. The upper phase was transferred to a new RNase-free tube, an equal volume of 70% ethanol was added and the tube was vortexed. PureLink RNA Mini kit  was used for further purification following the manufacturer’s protocol. RNA was recovered in 30 μL RNAsecure , left for 1 minute at room temperature, collected by centrifugation for 1 minutes at 8,000xg at room temperature and incubated at 65°C for 10 minutes. To remove remaining DNA, 3 μL of 10X TURBO DNase Buffer  were added to 30 μL of total RNA in RNAsecure, followed by 3 μL of TURBO DNase (2 Units/μL). The preparation was gently mixed and incubated at 37°C for 30 minutes. DNase was removed with an RNA Clean-up system  and eluted in 30 μL RNase-free water. For schistosomula, total RNA extraction, DNase treatment and purification methods were adapted from the protocol described above. 500 ng of this purified total RNA were reverse transcribed using identical concentrations (250 nmol/L) of random hexamers and oligo(dT)18 primers of Maxima H Minus Reverse Transcriptase kit (ThermoSCIENTIFIC). Quantitative PCR was performed using a LightCycler 480 System (Roche Diagnostics). We used 2.5 μL of cDNA (diluted 1/10 in water) in a total volume of 10 μL containing 1X LightCycler 480 SYBR Green I Master Mix (Roche Diagnostics) and 500 nmol/L of each primer. To ensure that a single product was generated, we separated amplification products on a Labchip GX DNA assay system (PerkinElmer). For each studied stage, the level of mRNA was normalized using the mean geometric transcription rate of three reference sequences  previously described by Wang and colleagues [target is the amplification efficiency of the gene of interest; Eref is the amplification efficiency of the reference gene; ΔCp,ref = Cp,ref–Cp,ref(sample); and ΔCp,target = Cp,target–Cp,target(sample). For each reaction, the crossing point (Cp) was determined using the second derivative maximum method applied using Light Cycler Software version 3.3 (Roche Diagnostics). Male cercariae were arbitrarily chosen as calibrator. We obtained male cercariae by infecting host mollusk B. glabrata strain BgBre with single miracidium from SmBre strain. Sex determination was performed as described in [As the genome annotation of lleagues , using etrations 0 nmol/L http://methdb.univ-perp.fr/epievo/Native chromatin immunoprecipitation was done according to an earlier published protocol  and at lFor ChIPseq library construction, the ChIP sample preparation kit  was used according to the manufacturer's protocol except for the indexed adapters. Those adapters came from the Truseq DNA sample preparation kit . Briefly, 30 ng of DNA per condition was blunt ended and adenylated on its 3' ends. Illumina's indexed adapters were ligated to both ends of the adenylated DNA. Ligated DNA was size separated by electrophoresis through a 2% agarose gel and a size selection was performed at 400 bp. Once extracted from the agarose, size selected DNA was enriched by 18 cycles of PCR for 10 seconds at 98°C, 30 seconds at 60°C and 30 seconds at 72°C using Illumina's proprietary primers and Phusion DNA polymerase . Each library was verified using a DNA 1000 Chip on a Bioanalyzer 2100, quantified by real time PCR with the KAPA Library Quantification Kit for Illumina Sequencing Platforms  and then diluted to 10 nM.RNAseq library construction was done with the TruSeq RNA Sample Seq kit  according to the manufacturer's protocol. Briefly, poly-A containing mRNA molecules were purified from 70 to 450 ng of total RNA using oligo-dT magnetic beads. The purified mRNA was fragmented by addition of the fragmentation buffer and was heated at 94°C in a thermocycler for 8 minutes. First strand cDNA was synthesized using random primers. Second strand cDNA synthesis, end repair, A-tailing, and adapter ligation was done in accordance with the manufacturer's instructions. Purified cDNA templates were enriched by 15 cycles of PCR for 10 seconds at 98°C, 30 seconds at 65°C, and 30 seconds at 72°C using Illumina's proprietary primers and Phusion DNA polymerase. Each indexed cDNA library was verified using a DNA 1000 Chip on a Bioanalyzer 2100 and quantified by real time PCR with the KAPA Library Quantification Kit for Illumina Sequencing Platforms  then diluted to 10 nM using Illumina's resuspension buffer.For each lane of sequencing, four libraries (ChIPseq) or 6 libraries (RNAseq) were pooled in equimolar concentrations, denatured using 0.1N NaOH and diluted to a final concentration of 7 pM (ChIPseq) or 8 pM (RNAseq) using Illumina's HT1 buffer. 120 μL of the dilution was then transferred into a 200 μL strip tube and placed on ice before loading onto the cBot. The flow cell was clustered using Single Read Cluster Generation Kit , according to the Illumina SR_amplification_Linearization_ Blocking_PrimerHyb recipe. The flow cell was loaded onto the Illumina HiSeq 2000 instrument following the manufacturer's instructions and sequencing was performed with the 50 cycles, single read, indexed protocol. Image analyses and base calling were performed using the HiSeq Control Software (HCS) and Real-Time Analysis component (RTA). Demultiplexing was performed using CASAVA (Illumina). The quality of the data was assessed using fastqc from the Babraham Institute and the Illumina software SAV .http://galaxy.univ-perp.fr). Fastq Groomer was used for verification of the fastqsanger format, and the FASTX-Toolkit  was used for initial quality control. Read quality was judged sufficiently good  and no further quality filter was applied.All data treatment was carried out under a local galaxy instance  using Bowtie2 [quality value 40–42-F 0x0004 - | grep-v XS:i. “. SAM alignment files were converted into the bed format with pyicos [Table 1). Although comparative analysis software usually include a normalization step to handle the different numbers of mapped reads in conditions or replicates when comparing samples, we found that the random sampling step drastically reduces the amount of false positives without lowering the sensitivity. Mostly, this removes background peaks. Identification of peaks was done with ranger of Peakranger v1.16 [http://genome.univ-perp.fr).For ChIP-Seq, reads were aligned to the  Bowtie2  evoking  Bowtie2  (samtoolh pyicos  and sorth pyicos . For peaer v1.16  with P ver v1.16  (http://S1 Table) in 20 randomly chosen regions using the following conditions: denaturation 95°C for 10 seconds, annealing 60°C for 10 seconds, extension 72°C for 20 seconds, over 40 cycles. The kit used for the amplification is the LightCycler 480 SYBR Green 1 Master  and the instrument is a LightCycler 480 (Roche). To compare ChIP-Seq and qPCR results, coverage was calculated with coverageBed of the bedtools suite for the regions whose boundaries were defined by the corresponding qPCR primer positions and divided by the coverage in the alpha-tubulin reference locus . Spearman rank correlation gave values > 0.8, R2 was 0.64 for H3K4me3 and 0.59 for H3K27me3 indicating that both methods gave comparable results .ChIP-Seq results in adults and cercariae were verified by qPCR (primers in ftp://ftp.sanger.ac.uk/pub/pathogens/Schistosoma/mansoni/genome/Assembly-v5/) with Cuffmerge v1.0.0 [For RNA-Seq, reads were aligned to the genome with TopHat v1.4.1 with default parameters and exon-intron structure was reconstructed with cufflinks v1.3.0 evoking-q-I 40000-F 0.100000-j 0.150000-p 8 on each library and data were merged and linked to the gene prediction of the Sanger Centre (e v1.0.0 .number”) with a custom script. EpiChIP was also used to correlate transcription and histone modification, and to quantify the proportion of peaks that contribute significantly to the average profiles. Common peaks were detected from Peakranger region bed-files with multiIntersectBed of the BedTools package.To identify peaks that were different between cercariae and adults, we used the DiffBind library 1.6.1  availablS. mansoni as consensus sequences (available http://methdb.univ-perp.fr/downloads/) [Table 1). After the counts had been normalized, they were compared between samples using the DESeq package of the R software [For the analysis of chromatin structure around repetitive sequences, reads were aligned to a fasta file containing all 3,145 known repeats of nloads/)  using Bonloads/)  evoking nloads/)  . Negative controls without the addition of 5-ethynyl-uridine (EU) and 5-ethynyl-2’-deoxyuridine (EDU) were also performed for every condition. Fixation, development and Hoechst 33342 coloration after incubation were performed following the protocols of the supplier. Imaging was done on a confocal microscope Zeiss LSM 700 at 488 nm (EU and EDU) and 405 nm (Hoechst 33342). For every condition, 100 animals were observed for transcription and replication.For the detection of mRNA synthesis, cercariae were incubated in spring water at 28°C with a concentration of 1mM of 5-ethynyl-uridine  for 2, 18 h and 21 h. Freshly transformed schistosomula were grown in Basch medium at the same EU concentration in an incubator at 37°C and 5% CORNA-Seq and ChIP-Seq reads are available at the NCBI-SRA under study accession numbers SRP034587 (ChIP-Seq) and SRP035609 (RNA-Seq).Fig 1A). We detected on average 7,746 peaks in cercariae and 8,868 peaks in adults (Table 1). For about ~24% of genes, EpiChIP identified transcripts but no significant H3K4me3 peaks (FDR = 0.01). Examples for genes that are free of H3K4me3 peaks are all known micro exon genes (MEGs) supplementary table S5.4 in [Fig 2A), with the exception of Smp_163710 that shows small peaks in adults. MEGs contain unusually small exons of 3–36 bp that are repeated in tandem [et al. estimated trans-splicing events to occur in 11% of S. mansoni genes [Fig 2B), in 2 (1.2%) cases peaks are present in both genes (upstream and downstream), and in 13 (8.2%) genes we did not find any peak, although in 3 cases we suspect that an upstream gene with a peak may be the first of the polycistronic unit (S2 Table). Our data are in line with the view that these genes are indeed transcribed as one unit from a single TSS  and fragmented by trans-splicing. For H3K27me3 we found with Diagenode antibody cat# pAb-069-050 lot# A29900242 enrichment at the TSS , while it was reduced to background level in adults. Since there was anecdotic evidence for cross-reactivity of this antibody with H3K4me3, we repeated the experiment with cat# C15410069 (that replaces pAb-069-050) lot# A1821D. With this antibody we also observe enrichment of H3K27me3 in cercariae compared to adults, but shifted towards the body of the gene . Methylation and acetylation in H3K9 are mutually exclusive at the same histone and H3K9me3 is considered a repressive mark. H3K9me3 shows a peak about 200 bp upstream of the TSS in cercariae, which is reduced to an upstream background in adults . H3K9ac is a hallmark of active transcription. In cercariae we detected this modification primarily around the TSS  while in adults acetylation spans the body of the genes . Taken together, our results indicate that all four studied histone modifications show an unusual enrichment around the TSS in cercariae, while in adults their distribution is similar to that found in other metazoans. We saw no differences between the input fractions  of cercariae and adults .To link transcription and chromatin structure, we considered the correct identification of the TSS as crucial. Anecdotal evidences had suggested that the available automatic annotation had missed a certain proportion of first exons and we decided to perform RNA-Seq with RNA extracted from cercariae. Indeed, our data showed that in about 20% of the genes, TSS had to be corrected. We then performed ChIP-Seq. For H3K4me3, we found relatively sharp peaks with a mean peak maximum located 250 bp (or 1–2 nucleosomes) downstream of the transcription start site (TSS) of genes n tandem . Their fni genes  and had Table 1). For H3K27me3 and H3K9me3 the percentage was higher with 7.60±1.00% and 7.75±0.60%. DESeq [−5 as the selection criterion, we identified 17 (0.54%) repeats that change their chromatin status for H3K4me3, 182 (5.79%) for H3K9ac, 263 (8.36%) for H3K9me3, and 40 (1.27%) for H3K27me3. Results are summarized in S3 Table. Interestingly, not all repeats were concerned equally: in roughly two thirds of these repeats  more than one histone isoform had changed meaning that about 10% of all repeat families undergo important chromatin structure changes during the transformation of cercariae into adults. Two results were particularly interesting . The first is a chromatin change in five regions that are composed of tandemly repeated stretches of class I LTR retrotransposons and class II DNA transposons. These regions are located on the large S. mansoni chromosomes (1 to 4), but there might be similar regions on the three smaller chromosomes that were overlooked. Each of the regions is composed of a different repeat, but they all belong to the same repeat class (i.e. transposon). In these five regions, enrichment of histone forms H3K9me3 and H3K27me3 decreases during cercariae to adult metamorphosis, while enrichment of H3K9ac and H3K4me3 increases. Considering five states , the individual term binomial distribution probability to obtain such a combination by chance is 3.7−36. Our data suggest therefore that the process is not randomly distributed along the genome. Stretches of (retro)transposons could form heterochromatic knobs in the cercariae and their change in chromatin structure could be an indication of large scale chromatin rearrangements or chromosome displacements in the nucleus during transformation into adults. Another peculiar observation during transformation from cercariae to adults is the nearly complete loss of H3 modifications in 22 of the 36 W-specific repeats (61%). We had shown earlier that transcription of these repeats can only be observed during larval stages and is undetectable in adults [S. mansoni (Smp_040020) [In cercariae and adults, both H3K4me3 and H3K9ac showed globally low association with repeats  (details in %. DESeq  identifi%. DESeq . We usedn adults . From qPn adults . A homol_040020)  but homoS. mansoni epigenome was revealed when we analyzed the distribution of the heterochromatic H3K27me3. In cercariae, H3K27 trimethylation is high in the gene body . H3K27me3 and H3K4me3 are also statistically significantly enriched around the TSS in 150 genes. This number was reduced to 121 after visual inspection (S4 Table). Thirty-five days of development separate cercariae and full-grown adults worms. To determine more precisely the time window during which chromatin is remodeled we performed ChIP-Seq on schistomula. Our results show that the adult chromatin structure is already established in schistosomula by 18 h after infection . Most importantly, in schistosomula, bivalent methylation is absent in the 121 genes that were significantly enriched in cercariae.While PeakRanger identify 1,122 more peaks in adults than cercariae for H3K4me3, Diffbind considers only 224 differences to be statistically significant. After visual inspection, we confirmed differential enrichment in only 124 regions along the genome with 95 (77%) hypermethylated in cercariae and 29 (23%) in adults. This means that less than 2% of H3K4me3 peaks are different, while gene transcription patterns of cercariae and adults are clearly distinct . A peculin vivo. At 28°C, the maximum life span of a cercaria is 32 h . In contrast, schistosomula, the first developmental stage after infection of the vertebrate host, showed consistent staining in the 100 arbitrarily chosen individuals and staining was more dispersed. We conclude that there is very little transcription in cercariae and that transcription is rapidly activated upon transformation into schistosomula. Our observation that in cercariae the repressive mark H3K9me3 is slightly upstream and the permissive mark H3K9ac is slightly downstream but close to the TSS is in accordance with stalled transcription. In contrast, in adults H3K9 acetylation and methylation are spread out with H3K9ac occurring along the gene body and H3K9me3 in the upstream regions separating genes .While RNA is clearly present in cercariae, the co-localization of all four histone modifications at the TSS made us wonder if transcription is indeed ongoing or if it is poised. Cercariae are short lived and they do not grow outside the host. It is therefore conceivable that their RNA was produced during their development in the snail. We used incorporation of the cytosine analog 5-ethynyl-uridine (EU) and fluorescence labeling to detect active transcription ast 26 h ) but infe.g. mice [S. mansoni but they are restricted to a small germinal cell cluster in cercariae [Bivalent H3K4/H3K27 trimethylation and absence of transcription are reminiscent of what occurs in ESC of vertebrates, .g. mice , human [.g. mice  and zebr.g. mice . There, .g. mice ). ESC do.g. mice ) to the .g. mice . Recentl.g. mice ,54. Anotercariae . Chromatercariae ,56 and aercariae . Thus, wWe used 5-ethynyl-2’-deoxyuridine (EDU) incorporation to detect replication in cercariae and schistosomula. While the latter showed at least one replicating cell per individual in 27% (36 out of 135) after 2 h EDU pulse and 86% (156 out of 181) after 18 h, in none of the cercariae did we detect replication. Consequently, cercariae do not contain large amounts of rapidly replicating ES-like cells or neoblasts that could explain the ChIP-Seq profiles.S. mansoni using four major histone modifications. We analyzed unique sequences, in particular those containing genes, but also the repetitive compartment of the genome.We describe here the epigenome of S. mansoni there is a clear difference between cercariae and adults with H3K9ac spanning the gene body only in the latter. H3K9me3 is a landmark of constitutive heterochromatin. In many organisms it is highly enriched at telomeric, pericentromeric and repeat-rich regions, and is responsible for stable large-scale repression [Fig 6B). Nevertheless, H3K9me3 can be present at a very high level over transcribed regions of active mammalian genes, especially 1.5 kb after the TSS, and at a low levels at the promoter [S. mansoni, a combination of H3K9me3 and H3K9ac allows the prediction of the direction of transcription  and it seems that blocks of H3K9me3 separate promoters of genes that are transcribed back-to-back . This might be a signature of protein-coding genes. The fourth studied histone isoform, H3K27me3 is known to maintain facultative heterochromatin and is involved in the regulation and sequential expression/repression of developmental genes [Plasmodium falciparum this mark can even be absent [S. mansoni, the mark is weak in genes of schistosomula and adults, but present in cercariae. Similar to ESC, bivalent trimethylation of H3K4 and H3K27 is present in at least 121 genes. Bivalent methylation is often considered as an ESC specific mark that leads to “poised” transcription that can rapidly be activated when the cells enter into differentiation. Our data indicate that there are no rapidly proliferating stem cells in cercariae, and we show that there is no transcriptional activity. Consequently, neoblasts of cercariae are either in a dormant state, or somatic cells of cercariae use the same mechanism as ESC to poise transcription that is rapidly activated after infection, upon transformation into schistosomula (at least during the first 18 h after penetration in the host). Bivalent H3K4/H3K27 trimethylation could therefore be a phylogenetically very old and/or more general mechanism to poise transcription than currently thought. However, as only 121 genes were detected to have these bivalent marks, another mechanisms could be involved in the transcription silencing observed in cercariae. At the moment, we can only hypothesize that the major shift of H3K27me3 enrichment  and/or the strong enrichment of H3K9me3 and H3K9ac at the TSS of cercariae play a role in this phenomenon.The enrichment of histone forms around TSS shares similarities with other metazoans, but also shows particularities. Similar to what is found in model organisms, H3K4me3 is enriched at transcription start sites (TSS) and promoter regions of genes with actual or potential transcription ,5,57–59.pression –11. In hpression . This ispromoter . H3K9me3promoter . In our al genes . It is pal genes . In someS. mansoni genome is composed of repeats [S. mansoni. We show that in five mini-satellite regions composed of retrotransposons H3K4me3 and H3K9ac increase while H3K9me3 and H3K27me3 decrease during the transition from cercariae into adults. It is difficult to evaluate the functional importance of this chromatin structure change. It was shown in cultured human cells that another type of satellite DNA (gamma satellites) is enriched for H3K4me3, and that it shields adjacent genes from heterochromatisation [While the importance of repetitive sequences for genome structure and evolution is generally accepted, they are often considered too difficult to analyze by genome-wide approaches. Almost half of the  repeats  and we dtisation . AcetylaS. mansoni for three life cycle stages. We show that in cercariae, bivalent H3K4/H3K27 trimethylation exists. To the best of our knowledge, this bivalent trimethylation has, in the animal kingdom, so far only been found in vertebrate ESCs. We also show that TSS occupancy by H3K4me3 is stable between cercariae and adults in the large majority of genes. It changes drastically for H3K9 trimethylation and acetylation. In addition, we identified at least five repeat locations that gain euchromatic status during development into adults. Our findings support a model in which transcription is poised in cercariae due to the presence of both activating and repressive marks. It is reactivated, presumably through the removal of the repressive methylation at the TSS and in the gene body, and in conjunction with large scale chromatin remodeling (indicated by changes in repeat regions). In this model, histone-modifying enzymes, in particular H3K27 and H3K9 demethylases would act within 18 h after skin penetration to release the poised state and allow for rapid activation of the transcription machinery that is already in place around the TSS. We had shown before that histone-modifying enzymes are indeed essential for transformation and infection success of another life cycle stage, the miracidia [S. mansoni genome (Smp_034000) and by q-RT-PCR we found that mRNA for this gene is present in cercariae, all four stages of schistosomula in vivo, and in male and female adults. Further studies will show if blocking this candidate will result in developmental arrest. Besides being of academic interest, such enzymes would also be promising new drug candidates, especially in the light of the fact that the only drug currently available to cure schistosomiasis, praziquantel, is ineffective against the larval forms [In summary, we present here the epigenomes of iracidia . Others iracidia . In zebriracidia . An orthal forms . In addiS1 Table(DOCX)Click here for additional data file.S2 Tablehttp://www.plosntds.org/article/info%3Adoi%2F10.1371%2Fjournal.pntd.0001455#s5 (S2 Table).Upstream indicates the transcript that is located upstream of the trans-splicing acceptor site. Down-stream indicates the putative trans-spliced transcript. Polycistron list from (XLS)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S1 Fig(TIFF)Click here for additional data file.


Transcriptional heterogeneity is extensive in the genome, and most genes express variable transcript isoforms. However, whether variable transcript isoforms of one gene are regulated by common promoter elements remain to be elucidated. Here, we investigated whether isoform promoters of one gene have separated DNA signals for transcription and translation initiation. We found that TATA box and nucleosome-disfavored DNA sequences are prevalent in distinct transcript isoform promoters of one gene. These DNA signals are conserved among species. Transcript isoform has a RNA-determined unstructured region around its start site. We found that these DNA/RNA features facilitate isoform transcription and translation. These results suggest a DNA-encoded mechanism by which transcript isoform is generated. Promoter DNA sequence features are indicative of gene activity13478101214The dynamic usage of transcript isoforms is a pervasive mechanism in gene regulation. Its functions have been extensively studied for several individual genes. The diversity in transcript isoforms, mediated through alternate promoter usage and alternative polyadenylation, plays a role in messenger RNA transcription, stability and translation1617182022Saccharomyces cerevisiae by RNA sequencing27Saccharomyces cerevisiae27Human genes have multiple TSSs, which have epigenetic and genomic relevanceS. cerevisiaeWe used genome-wide identified transcript isoforms covering the intact ORF in Second, we asked whether the observed TATA box is associated with transcription initiation. To this end, we tested whether PIC is enriched in upstream regions of other isoforms. To avoid the shared PIC between other isoforms and main isoforms, we restricted the analysis to other isoforms whose 150 bp upstream region has no overlap with the 150 bp upstream region of their corresponding main isoforms. We found that TBP occupancy in upstream region is comparable between other isoforms and main isoforms . TFBSs are generally enriched 50–150 bp upstream of the gene in in vitro nucleosome map is determined only by the intrinsic sequence preferences of nucleosomes. Other isoforms show comparable in vitro nucleosome occupancy in upstream regions with main isoforms  we identified above, which have no other isoform in YPD medium but have at least one other isoform in galactose condition, only show small overlap (12 genes) with OPN genes (N = 544)  region immediately upstream of the start codon, which might facilitate ribosome binding and translation initiation41In this study, we performed a genome-wide analysis and investigated into the cause and consequence of transcript isoforms. We found that a gene’s main isoform and other isoforms, produced from alternative transcriptional start sites, show similar patterns within their DNA promoter sequences. In particular, we found TATA box features, nucleosome-disfavored sequence signals, and DNA-encoded RNA unstructured regions near the respective transcriptional start sites. These patterns facilitate isoform transcription and translation. Main results in this study could be reproduced using another independent TSS dataOne key finding in this study is that DNA sequence upstream of other isoforms is conserved among species. DNA signal facilitating isoform transcription is under selective constraint. Transcription initiation includes two steps: chromatin remodeling and PIC assembly. Although there is no low nucleosome occupancy upstream of isoform, the intrinsic nucleosome-disfavored DNA signals upstream of isoform could facilitate nucleosome repositioning by chromatin remodelers. TATA box upstream of isoform recruit PIC for transcription initiation.Genes with more other isoforms have more TSSs . Multiplet al.et al.et al.P value cutoff of 0.001 was used to define the set of genes bound by a particular TF. Genome-wide nucleosome occupancy data in vivo and in vitro measured with high resolution were taken from Kaplan et al.et al.et al.et al.et al.Yeast genome-wide transcript isoform coordinate data in YPD medium were taken from Pelechano Saccharomyces Genome Databaseet al.1) We restricted the analysis to isoforms covering the whole ORF. In this way, these isofroms are transcripts of ORFs, and are not noise of transcription. ORF and their coordinate (start and stop codons) data were taken from S. cerevisiae and S. mikatae, between S. cerevisiae and S. kudriavzevii were taken from Wapinski et al.We classified gene promoter regions not covered by isoforms into two catalogues: one covered by 150 bp upstream region of isoforms, and the other not. Orthologous genes between We compiled available gene expression data from the Stanford Microarray DatabaseThe minimum free energy for the RNA sequence was computed as previous studies50Given two samples of values, the Mann-Whitney U-test (function ‘ranksum’ in software ‘Matlab’ version R2012b) is designed to examine whether they have equal medians. The main advantage of this test is that it makes no assumption that the samples are from normal distributions. Error bars in figures were calculated by bootstrapping: Data points in a data set are randomly resampled to create 1000 different data sets , and the mean value is computed for each data set, and standard deviation is computed for the 1000 mean values.How to cite this article: Dai, Z. et al. DNA signals at isoform promoters. Sci. Rep.6, 28977; doi: 10.1038/srep28977 (2016).


Cardiac myocytes (CMs) proliferate robustly during fetal life but withdraw permanently from the cell cycle soon after birth and undergo terminal differentiation. This cell cycle exit is associated with the upregulation of a host of adult cardiac-specific genes. The vast majority of adult CMs (ACMs) do not reenter cell cycle even if subjected to mitogenic stimuli. The basis for this irreversible cell cycle exit is related to the stable silencing of cell cycle genes specifically involved in the progression of G2/M transition and cytokinesis. Studies have begun to clarify the molecular basis for this stable gene repression and have identified epigenetic and chromatin structural changes in this process. In this review, we summarize the current understanding of epigenetic regulation of CM cell cycle and cardiac-specific gene expression with a focus on histone modifications and the role of retinoblastoma family members. The fetal heart increases in size throughout development via proliferation of CMs but switches to mainly hypertrophic growth of CMs with limited proliferation soon after birth, undergoing terminal differentiation which is associated with permanent cell cycle exit . TerminaEpigenetic mechanisms regulate chromatin structure , which mEpigenetics is typically defined as the regulatory mechanisms of gene activity that are not due to changes in DNA sequence. These include modifications of DNA and histone proteins, which affect chromatin structure, and microRNA. In the nuclei of eukaryotic cells, DNA is wrapped around an octamer of histone proteins that are packed into higher-order chromatin structures. Epigenetic regulation involves covalent modification of either of DNA (DNA methylation) or of nucleosomes, which is primarily through post-translational modification of histones . In addition, expression of cardiac-specific genes such as atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) correlates with p300 occupancy and histone acetylation on their promoters .Histone methyltransferases have also been implicated in cardiac health and disease. PRC2 is a HMT complex, which consists of four components: catalytic subunit enhancer of Zeste 1 (Ezh1)/Ezh2, suppressor of Zeste 12 (Suz12), embryonic ectoderm development (Eed), and RbAp46/48 . PRC2 meJumonji, coded by the Jarid2 gene, is a nuclear factor that plays an essential role in the development of multiple tissues, including the heart. Jmj has a DNA binding domain, ARID, and two conserved Jmj domains .The ability of Rb to stably repress transcription is related to its capacity to recruit HP1s to target gene promoters resulting in their incorporation into heterochromatin . HP1 is The role of H3K27me3 in terminal differentiation is not clear although the promoters of proliferation related genes displayed higher levels of H3K27me3 in ACMs and skeletal myotubes . H3K27meRb is the prototypical member of a gene family encoding three structurally and functionally similar proteins, Rb, p107, and p130 . Rb alsoThe major effect of Rb on the histone acetylation pathway is facilitating deacetylation of target gene promoters by recruiting HDACs . Class IIt has been demonstrated that Rb is also required for establishment and maintenance of H3K27me3 . Rb physImportance of Rb on CM terminal differentiation through epigenetic mechanism was demonstrated . AlthougCommitment to a particular lineage requires both the repression of unnecessary genes while simultaneously up-regulating lineage-specific genes. High-throughput DNA sequencing technology has enable the search for the binding sites of cardiac TFs, enhancers, epigenetic marked histones and chromatin modifying factors on a genome-wide level by ChIP-sequencing . Core caThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


However, there has been no report showing the effects of RXR agonists on Cushing’s disease, which is caused by excessive ACTH secretion in a corticotroph tumor of the pituitary gland. Therefore, we examined the effects of synthetic RXR pan-agonists HX630 and PA024 on the proliferation, apoptosis, ACTH secretion, and pro-opiomelanocortin (Pomc) gene expression of murine pituitary corticotroph tumor AtT20 cells. We demonstrated that both RXR agonists induced apoptosis dose-dependently in AtT20 cells, and inhibited their proliferation at their higher doses. Microarray analysis identified a significant gene network associated with caspase 3 induced by high dose HX630. On the other hand, HX630, but not PA024, inhibited Pomc transcription, Pomc mRNA expression, and ACTH secretion dose-dependently. Furthermore, we provide new evidence that HX630 negatively regulates the Pomc promoter activity at the transcriptional level due to the suppression of the transcription factor Nur77 and Nurr1 mRNA expression and the reduction of Nur77/Nurr1 heterodimer recruiting to the Pomc promoter region. We also demonstrated that the HX630-mediated suppression of the Pomc gene expression was exerted via RXRα. Furthermore, HX630 inhibited tumor growth and decreased Pomc mRNA expression in corticotroph tumor cells in female nude mice in vivo. Thus, these results indicate that RXR agonists, especially HX630, could be a new therapeutic candidate for Cushing’s disease.Various retinoid X receptor (RXR) agonists have recently been developed, and some of them have shown anti-tumor effects both  Cushing’s disease is caused by excessive ACTH secretion in a corticotroph tumor of the pituitary gland, or rarely corticotroph hyperplasia, and is the most frequent cause of endogenous hypercortisolism. Surgical removal of the tumor is the current first line therapy, leading to remission in 70–90% of cases. However, the risk of recurrence of Cushing’s disease reaches 20–25% at 10 years after surgery . Thereafter, the mice were euthanized and their tumors were removed to isolate total RNAs for Pomc mRNA expression analyses. Blood samples were also collected from the mice for the measurement of their plasma ACTH levels. All animal experiments were performed in accordance with the institutional regulations for animal care of Tohoku University Graduate School of Medicine. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Tohoku University. Animals were sacrificed by sevoflurane anesthesia, and all efforts were made to minimize suffering.Eight-week-old female BALB/c-nu mice (nu/nu) were subcutaneously inoculated with AtT20 cells . The mice were housed in temperature- and humidity-controlled cages with a 12-hour/12-hour light/dark cycle, and were given standard chow diet and tap water P<0.05 was considered statistically significant.All data except for microarray analysis are presented as mean ± SEM. Statistical analyses were performed with ANOVA followed by post hoc Tukey test. We examined the effects of RXR agonists on AtT20 cell proliferation using a WST-8 assay after incubation with various concentrations of agonists for 96 hr. RXR pan-agonists HX630 and PA024 significantly suppressed AtT20 cell proliferation at 10 μM . We nextRXR isoform mRNA in AtT20 cells was confirmed by quantitative real-time PCR. RXRα and β mRNAs were expressed in AtT20 cells  [Pomc promoter activity.To explore the molecular mechanisms of the  mutants . There e04/-383) , 30 and 04/-383) , 32  to confirm whether HX630 could affect the recruiting of Nur77/Nurr1 heterodimer or NeuroD1 to the Pomc promoter. The conventional ChIP assay demonstrated that Nur77/Nurr1 heterodimer and NeuroD1 under the basal condition (control) were obviously recruited to NurRE and E-box, respectively . siRNA for RXRα significantly abolished the HX630-mediated suppression of Pomc promoter activity  HX630 (Pomc mRNA expression (Pomc transcription by HX630 is most likely mediated via RXRα.We examined the involvement of RXRα on the activity . The decM) HX630 . Furtherpression . These dP-value<0.01 in AtT20 cells treated with 10 μM HX630, were identified. Both the differentially expressed up-regulated and down-regulated genes were subjected to IPA [in vitro studies. Specifically, CASP3 (caspase 3) and CASP8 (caspase 8), which play a crucial role in apoptosis, were up-regulated, and TNFSF10 , which triggers the activation of caspase 3 and caspase 8, was also up-regulated. Anti-apoptotic Bcl2 (B-cell leukemia/lymphoma 2) was down-regulated. Among tumor suppressor genes, CDKN2B , CDKN2C , and BRCA2 (breast cancer 2) were up-regulated, while CDKN1A  was down-regulated. Additionally, HX630 negatively regulated other growth regulatory biomarker genes, such as EMP3 , FOS (FBJ osteosarcoma oncogene), MAP3K8 (mitogen-activated protein kinase kinase kinase 8), MAPK12 (mitogen-activated protein kinase 12), PDGFC (platelet-derived growth factor C), PDGFRB  and TGFB3 . On the other hand, 25 networks of significantly expressed genes regulated by 10 μM HX630 were identified . Among them, a significant gene network associated with apoptosis is presented in To gain further insights regarding the functional mechanisms of HX630 on AtT20 cells, especially concerning cell proliferation and apoptosis, we conducted DNA microarray analyses to identify gene expression patterns, networks, and pathways in AtT20 cells treated with 10 μM HX630. A total of 2,173 up-regulated genes and 3,169 down-regulated genes, which demonstrated fold-changes of at least 1.5 compared with the control and a d to IPA . IPA groin vivo, we injected nu/nu mice with HX630 or vehicle intraperitonealy 3 times a week for 3 weeks after subcutaneous inoculation with AtT20 cells. Although there were no significant differences in body weights and plasma ACTH levels between the control group and the HX630 treated group (data not shown), HX630 significantly decreased the tumor volumes  used did not contain any RXR binding elements including direct repeat (DR) 1, DR 4, and DR5 [Pomc including transcription factors via RXR. The functional analyses of the Pomc promoter activity using Pomc 5’-flanking region deletion mutants indicated that several transcription factors affecting the promoter region might be involved in the HX630-mediated transcriptional suppression. Indeed, there are several binding sites of transcription factors in the Pomc promoter region [Pomc promoter. The proximal binding sequence termed Nur77-binding response element (NBRE) (-69/-63) binds to Nur77 monomer [Pomc promoter activity mediated by RXR agonists, although a natural RAR agonist, ATRA, inhibited ACTH secretion in vitro by inhibiting the transcriptional activity of the transcription factors Nur77/Nurr1 on the Pomc promoter [Pomc promoter activity at the transcriptional level due to the suppression of Nur77 and Nurr1 mRNA expression and the reduction of Nur77/Nurr1 heterodimer recruiting to NurRE in the Pomc promoter region. Nur77/Nurr1 heterodimer was reported to enhance NurRE transcription synergistically in comparison to that seen with Nur77 homodimer [Pomc promoter activity, resulting in the suppression of Pomc mRNA expression and ACTH secretion in AtT20 cells.On the other hand, we also demonstrated that HX630 decreased the  and DR5 , 12. Ther region . Among t monomer , 46. On  monomer , 30, 45. monomer , 32, 45, monomer , and NF- monomer . There hPomc promoter activity, Pomc mRNA expression, and ACTH secretion between HX630 and PA024, although they are both RXR-selective pan-agonists. RXRs interact with various factors, including heterodimer partners of nuclear receptors, co-repressors, and co-activators. Owing to such complex protein-protein interactions, different RXR agonists do not necessarily exhibit the same biological activities [ABCA1) expression in macrophage cell lines, RAW264 cells and undifferentiated THP-1 cells through LXR-RXR heterodimer activation, while HX630 failed to induce ABCA1 expression because of the inability to activate LXR-RXR heterodimer. Instead, HX630 was able to activate PPARγ/RXR, and induced ABCA1 expression in differentiated THP-1 cells [Pomc expression and ACTH secretion.There were differences in the effects on tivities . For exa-1 cells . Another-1 cells . The difPomc promoter activity at the gene transcription level due to the suppression of Nur77 and Nurr1 mRNA expression and the subsequent reduction of Nur77/Nurr1 heterodimer recruiting to the Pomc promoter region, resulting in the suppression of Pomc mRNA expression and ACTH secretion. These effects of HX630 were shown to be exerted via RXRα. Furthermore, we confirmed that HX630 indeed inhibited tumor growth and decreased Pomc mRNA expression in corticotroph tumor cells in female nude mice in vivo. Thus, these results indicate that RXR agonists, especially HX630, might be a new therapeutic candidate for Cushing’s disease. Further structural optimization of them to reduce the effective concentration for decreasing Pomc expression and ACTH secretion, inducing apoptosis, and inhibiting cell proliferation may be necessary for the introduction into the clinical application.In conclusion, we demonstrated that HX630 at high concentrations suppressed cell proliferation and induced apoptosis, most likely through the activation of caspase 3, in AtT20 cells. We also provide new evidence that HX630 negatively regulates the S1 File(DOC)Click here for additional data file.


Primary cells enter replicative senescence after a limited number of cell divisions. This process needs to be considered in cell culture experiments, and it is particularly important for regenerative medicine. Replicative senescence is associated with reproducible changes in DNA methylation (DNAm) at specific sites in the genome. The mechanism that drives senescence-associated DNAm changes remains unknown - it may involve stochastic DNAm drift due to imperfect maintenance of epigenetic marks or it is directly regulated at specific sites in the genome.In this study, we analyzed the reorganization of nuclear architecture and DNAm changes during long-term culture of human fibroblasts and mesenchymal stromal cells (MSCs). We demonstrate that telomeres shorten and shift towards the nuclear center at later passages. In addition, DNAm profiles, either analyzed by MethylCap-seq or by 450k IlluminaBeadChip technology, revealed consistent senescence-associated hypermethylation in regions associated with H3K27me3, H3K4me3, and H3K4me1 histone marks, whereas hypomethylation was associated with chromatin containing H3K9me3 and lamina-associated domains (LADs). DNA hypermethylation was significantly enriched in the vicinity of genes that are either up- or downregulated at later passages. Furthermore, specific transcription factor binding motifs  were significantly enriched in differentially methylated regions and in the promoters of differentially expressed genes.Senescence-associated DNA hypermethylation occurs at specific sites in the genome and reflects functional changes in the course of replicative senescence. These results indicate that tightly regulated epigenetic modifications during long-term culture contribute to changes in nuclear organization and gene expression.The online version of this article (doi:10.1186/s13148-015-0057-5) contains supplementary material, which is available to authorized users. For exaotential ,3. AdditA reduction in telomere length has a definitive role in the loss of chromosomal integrity during culture expansion ,5. The nin vitro is associated with global DNA hypomethylation, whereas local DNA hypermethylation occurs at specific CpG sites [Recent evidence suggests that replicative senescence is accompanied by epigenetic modifications at specific CpG sites -14. SenepG sites . SA-DNAmpG sites ,15. SA-DpG sites , coincidpG sites ,19 and wpG sites ,20. HoweTwo non-exclusive mechanisms may influence SA-DNAm changes: 1) compatible with the perception of epigenetic drift ,22, theyin situ hybridization (Q-Fish) with telomere repeat probes . Furthermore, nuclei acquired an elongated morphology  and corresponding late passages P21 to P40) with regard to nuclear area and by quantitative fluorescent  to P40 wHOXC locus revealed prominent DMRs, particularly in donor 2  and late passages (P30 or P33). To this end, we used MethylCap-seq, which is based on capturing methylated DNA with the methyl-CpG-binding domain (MBD) of the methyl-CpG-binding protein 2 (MeCP2) and subsequent next-generation sequencing of salt-eluted DNA . This anP value < 0.05 and DNAm change > 20%). MethylCap-seq signals were then analyzed in a 600-bp window around these SA-DNAm changes identified by BeadChip technology. Overall, differential MethylCap-seq signals had the same tendency of SA-DNAm changes as observed with the 450k IlluminaBeadChip data  DNAm changes in MSCs using 450k IlluminaBeadChips . With IlSubsequently, we analyzed whether SA-DNAm changes were restricted to individual CpGs or if adjacent CpGs were also affected. We focused on the most significant CpGs of the 450k IlluminaBeadChip data  and found that SA-hypermethylation and hypomethylation was not only restricted to individual CpGs but also occurred in upstream and downstream CpGs, usually within a region of 500 bp  and lamin B2 . Further, downregulated genes included the lamin B receptor , which anchors the lamina and heterochromatin to the membrane; thymopoietin , which may play a role in the assembly of the nuclear lamina and thus help maintain the structural organization of the nuclear envelope; and spectrin repeat containing nuclear envelope 2 , whereas SYNE1 was upregulated (P = 3.1*10−18). These results indicate that differential expression of genes involved in the nuclear lamina may contribute to reorganization of chromatin during long-term culture.To further correlate DNAm changes with gene expression changes, we sequenced the transcriptome of three MSC preparations at early (P3) and late passages . Six huSubsequently, we analyzed whether genes localized within the LADs are particularly affected by senescence. Overall, these genes were less expressed, especially at the border of LADs Figure B, which Subsequently, we performed gene ontology (GO) analysis of differentially expressed genes to gain better insight in the functional changes during long-term culture: downregulated genes revealed a highly significant enrichment in categories involved in cell division and DNA repair whereas upregulated genes were enriched in cell adhesion, development, and extracellular matrix organization Figure E. This iP value < 0.05; Fisher’s Exact test) in senescence-associated DMRs of at least one fibroblast sample or of MSCs. Most of these TF binding motifs were significantly enriched in both hypermethylated and hypomethylated regions  binding site analysis in regions with SA-DNAm changes (450k BeadChip and MethylCap-seq data): 51 motifs were significantly enriched . This enrichment of specific TF binding sites indicates that corresponding factors are relevant for the functional changes during long-term culture.Therefore, we analyzed enrichment of TF binding sites in the promoter regions (1 kb upstream) of the differentially expressed genes: 64 motifs were enriched, and most of these were enriched in promoter regions of both up- and downregulated genes Figure C. There In this study, we demonstrate various facets of the impact replicative senescence has on nuclear organization Figure . This covice versa.Telomere length is well known to decline before cells enter replicative senescence. However, the intranuclear positioning of telomeres, which again reflects a major change in genomic organization, is less clear. It has recently been demonstrated that telomeres are enriched at the nuclear periphery during postmitotic nuclear assembly and become localized at the nuclear center during cell cycle arrest . We alsoThe DNAm pattern changes during culture expansion in a highly reproducible manner. In fact, an Epigenetic-Senescence-Signature based on DNAm at six specific CpGs even facilitates reliable prediction of passage numbers and cumulative population doublings for quality control of cell preparations ,30,31. SWe have previously suggested that senescence-associated DNAm changes are related to specific histone modifications by characterizing the promoter regions of genes with SA-CpGs . FurtherIn all datasets analyzed, we observed significant enrichment of SA-hypermethylation in regions with the activating H3K4me3 and H3K4me1 marks. This is in contrast to a recent study by Fernández and coworkers, who demonstrated that particularly H3K4me1 corresponds to regions that become hypomethylated in MSCs upon aging of the organism . FurtherLMNA gene, and lamin B1 and lamin B2, encoded by LMNB1 and LMNB2, respectively [LMNB1 and LMNB2 were among the most significantly downregulated genes during culture expansion. In fact, it has been demonstrated that the loss of LMNB1 is a biomarker for senescence [LBR) was also significantly downregulated. LBR interacts with methyl-CpG-binding protein 2 (MeCP2), the same methylation binding domain we used to capture methylated DNA for MethylCap-seq. This interaction has been suggested to have a role in the localization and/or stabilization of transcriptionally silent heterochromatin adjacent to the nuclear envelope [The inner layer of the envelope consists of filamentous proteins, lamin A and C, which are splice variants of the ectively . Mutatioectively ,43, and ectively  and premectively . LMNB1 anescence , whereasnescence . Furthernescence . The lamenvelope . LADs arenvelope . Therefoenvelope . We demoenvelope . It may envelope . This miCaenorhabditis elegans [In contrast, SA-hypermethylation seems to be associated with differential gene expression of both up- and downregulated genes. These functional changes are reflected by highly specific enrichment of upregulated genes in categories of cellular organization and development, whereas downregulated genes are involved in cell division. Association of DMRs with differential gene expression, even though not necessarily negatively correlated, implies that the SA-hypermethylation may be relevant for these gene expression changes. In fact, several TFs predicted to bind to DMRs and differentially expressed genes upon senescence have been implicated in replicative senescence before: EGR1, also known as zinc finger protein 225, has been shown to play a central role in aging ,52 and r elegans . Concret elegans , indicatIn this study, we provide further evidence that epigenetic changes during long-term culture reflect changes in nuclear organization. It remains unclear whether SA-DNAm alterations are due to epigenetic drift or to a tightly regulated process with the possibility that both mechanisms are involved in this process. The finding that SA-hypomethylation is enriched in LADs and H3K9me3 marks without association to specific gene expression changes is compatible with passive and stochastic changes in DNAm level. In contrast, specific SA-hypermethylation is reflected in differential gene expression. Furthermore, the association of SA-DNAm changes with TF binding sites indicates a functionally relevant and controlled process. Notably, both SA-hypermethylation and SA-hypomethylation are reversed when reprogrammed into iPSCs, which may reflect rejuvenation also on the epigenetic level . In this2. Cells were culture expanded until replicative senescence as determined by ultimate growth arrest. Late passages (as indicated in the text) were within the last three to five passages before entering senescence.Human dermal fibroblasts were isolated from patients undergoing surgical interventions after written consent, using guidelines approved by the Ethic Committee on the Use of Human Subjects at the University of Aachen (Permit Number EK163/07) as described in detail before . Cells w2. All cell preparations were characterized with regard to immunophenotype and in vitro differentiation potential towards osteogenic and adipogenic lineages as described before [Mesenchymal stromal cells were isolated from the bone marrow of caput femoris upon hip replacement surgery after written consent using guidelines approved by the Ethic Committee on the Use of Human Subjects at the University of Aachen (Permit Number EK128/09) as described before . MSCs wed before ,19.in situ hybridization (Q-FISH) was performed on cytospins of three fibroblast preparations of early (P3-5) and corresponding late passages (P 21-40). Staining with a telomere probe labeled with Cy3  and counterstaining with DAPI was performed as described previously [z-stack image with the largest nuclei area was analyzed. Nuclei were defined in three different zones as recently described [Quantitative fluorescent eviously ,59. Cellescribed . Three z7 cells was harvested for subsequent analysis. There was a slight difference in the number of early passages (P3 or P5) due to differences in cell growth, which might be partially attributed to different starting material, and requirement of additional cells for other experiments and long-term culture. The number of corresponding late passages (P30 and P33) was chosen by their growth performance as we wanted to analyze the cells close to senescent state but required residual capability for large-scale expansion. DNA was isolated with the Qiagen DNA Blood Midi-Kit , and quality was assessed with a NanoDrop ND-1000 spectrometer  and gel electrophoresis. DNA was sheared with an S220 focused ultrasonicator  to a size range of 200 to 400 bp and then incubated with 2 μg of recombinant MBD2-glutathione-S-transferase fusion protein with a histidine tag(H6) [http://www.ncbi.nlm.nih.gov/geo/; GSE59960). In addition, we used our previously published DNAm profiles of MSCs during long-term culture (GSE37066) [DNAm profiles were analyzed by methyl-capture sequencing (MethylCap-seq), which is based on precipitation of methylated DNA by recombinant methyl-CpG binding domain of MeCP2 protein. Fibroblasts from two female donors (both 43 years old) were expanded in culture, and DNA from 10 tag(H6) . MethylaSE37066) .6 cells of three MSC donors  at passage 4 and passage 13 using the miRNeasy Mini Kit . Quality control and measurement of RNA concentration were done with a NanoDrop Spectrophotometer , and the Agilent 2100 Bioanalyzer . Multiplexed library preparation of total RNA and deep sequencing with IlluminaHiSeq 2000 technology  with a read length of 50 bases were performed at EMBL gene core facility . RNA-Seq profiles have been deposited at GEO (GSE59966).RNA was isolated from 10http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and Flexbar [Methylcap-seq and RNA-Seq data were subjected to quality control check and preprocessing steps using fastQC  [For Methylcap-Seq, alignment to the human genome built 37 (hg19) was done with Burrows-Wheeler Transform (BWA) . More thameters) . For obtameters) .The RNA-Seq reads were mapped to the human genome (hg 19) using Bowtie2  and Tophhttp://www.regulatory-genomics.org). Regarding DMRs, we extended or shortened the regions to have a length of 40 bps. For up-/downregulated genes, we used 1-kb regions upstream of the transcription start sites as promoter regions . Next, we performed motif match analysis with a false discovery rate (FDR) of 0.0001 [P values were corrected by the Benjamini-Hochberg method [Transcription factor enrichment analysis was performed with the Regulatory Genomics Toolbox .Data have been deposited at NCBIs Gene Expression Omnibus (GEO,


However, conventional in vitro transcription assays incorporates radioactive isotopes that cause environmental and health concerns and restricts its scope of application. Here we report a novel non-radioactive method for in vitro transcription analysis by combining primer extension with quantitative real time PCR (qPCR). We show that the DNA template within the transcription system can be effectively eliminated to a very low level by our specially designed approach, and that the primers uniquely designed for primer extension and qPCR can specifically recognize the RNA transcripts. Quantitative PCR data demonstrate that the novel method has successfully been applied to in vitro transcription analyses using the adenovirus E4 and major late promoters. Furthermore, we show that the TFIIB recognition element inhibits transcription of TATA-less promoters using both conventional and nonradioactive in vitro transcription assays. Our method will benefit the laboratories that need to perform in vitro transcription but either lack of or choose to avoid radioactive facilities.Primer extension-dependent  Althocription ,the convin vitro transcription assay is surprisingly low [in vitro. In our previous study, we showed that the TFIIB recognition element (BRE) can inhibit the transcription activity of a core promoter containing a TATA box [in vitro transcription analysis in combination with DNA template elimination, primer extension and qPCR using specially designed primers. Our data demonstrate that the method can be applied to several types of in vitro transcription analyses and that the BRE inhibits the transcription activity of TATA-less promoters.Quantitative real time PCR is a very powerful tool that has been widely used in modern molecular biology including gene expression analysis, pathogen detection, gene contamination detection and clinical diagnosis due to its sensitivity and accuracy –12. It hngly low , 14, andTATA box , howeverAdenovirus E4 core promoter , Adenovirus major late promoter  and theirmutant derivativescontaining a defective TFIIB recognition element (BRE) were cloned into the reporter vector pGL3 basic (Promega). For E4 mutant derivatives, BRE consensus bases downstream of the TATA box were mutated into TFIIB-hatred bases as described before (termed E4mBRE) . The proIn vitro transcription was performed in a cell-free system as described previously [in vitro for nonradioactive method was slightly modified for the study. Briefly, the transcription reaction system contains 25 μL nuclear extract, 3 μL 100 mM MgCl2, 200 ng DNA template, 10 μLddH2O and 10 ng Gal4-AH activator (added only for activated transcription). The reaction was mixed by brieflyvortexing, and incubated at 30°C for 30 minutes. The transcription reaction was then initiated by adding 3 μL of 10 mM NTP mix and maintained at 30°C for 1 hour. When the transcription reaction was complete, 160 μLstop solution containing 5 μL proteinase K and its reaction buffer was added to the reaction mixture and incubated at 55°C for 30 min. For in vitro transcription reactions that included a mutant TFIIB derivative we used HeLa nuclear extract that had the endogenous TFIIB depleted by immunoaffinitychromatography. 10 ngof wild type or mutant derivative TFIIB was added to the reaction system before transcription initiation.Conventional eviously , however2O. The washed phenol was then balanced with an equal volume of 0.1 M HCl buffer on a rocker for 4 hours, the supernatant was changed and pH value was measured 2–3 times during the process until the final pH value of the phenol was below 3.0.Tris.Cl-balanced phenol (pH 8.0) was used to prepare acidic phenol. In brief, the Tris.Cl-balanced phenol was washed twice by excessive ddH2O). The DNA template within the transcription reaction mixture was further removed through DNase I digestion at 37°C for 1 hour followed by precipitation with 3 volumes of 100% ethanol.Following digestion with proteinase K, the terminated transcription reaction mixture was subjected to DNA template depletion, namely, extracted by Trizol and acidic phenol on ice for 20 minutes respectively, followed by precipitation with an equal volume of isopropanol. The dried pellet was then dissolved in 200 μLDNase I digestion solution . Before the initiation of primer extension, the hybridization reaction between the primer and transcriptswas performed at 42°C for 16 hours.Primer extension was performed according to the standard protocol  except t5’-atggaagacgccaaaaacataa-3’(Luc/L);reverse, 5’-gtgaaggtgaaggtgatgcg-3’(GFP/G). In addition, we designed a primer pair for directly detecting luciferase RNA transcript by RT-qPCR or detecting DNA template by qPCR as described in 5’-gaaaggcccggcgccattct-3’(LF); reverse, 5’-ttcatagcttctgccaaccg-3’(LR). The real time PCR data were analyzed by Bio-Rad CFX Manager Software 3.1.Quantitative real time PCR (qPCR) was performed with the Bio-Rad SYBR Green reagent and Real Time PCR Detection System. The primers used for qPCR were specially designed to recognize the primer extension-derived product, in which the forward primer is complementary to the 3’ prime sequence of luciferase gene negative strand, which is immediately downstream of the core promoter; the reverse primer is a GFP oligonucleotide that is contained in the primer used for primer extension, the primer pair give rise to a 121bp product. The primer sequences were as follows: forward, in vitro transcription assay, the radioactive isotope-labeled primer is used to detect RNA transcripts that indirectly reflect the efficiency of transcription in vitro. To achieve direct detection for transcription product in vitro using RT-qPCR, apparently, the DNA template used for transcription must be depleted following the transcription reaction. We first performed an in vitro transcription assay using the Adenovirus E4 promoter in the absence or presence of the activator GAL4-AH. When the transcription reaction was completed, the reaction mixture sequentially underwent proteinase K digestion, DNase I digestion, Trizol extraction and precipitation with isopropanol; the pellet was dissolved in 20 μL ddH2O, 4 μL of which was used for reverse transcription and detected by qPCR using the reporter gene (luciferase) primers. Unexpectedly, our qPCR data were not consistent with the results as described previously [In conventional primer extension-dependent eviously , in whicin vitro transcription assay by adding pGL3 basic-E4 template but excluded addition of NTP, and the remaining DNA template within the ‘reaction’ system was detected using reporter gene primers (LF/LR) and qPCR. In the meantime, a standard curve was generated by qPCR using the same DNA template and reporter primers as described above .It has been proposed that it is necessary to remove 99.999% (i.e. left 1pg out of 100ng) of the DNA template in a transcription reaction to minimize the contribution of the template DNA to the final qPCR reaction . We thered above . As showed above . We haveed above . Using tin vitro such that even the very low level of contaminating DNA template could still interfere with qPCR. To solve this problem, instead of directly detecting theRNA transcript by RT-qPCRwe incorporated a prior step of primer extension. We designed the primer used for primer extension to contain an oligonucleotide fragment complementary to the RNA transcript and an oligonucleotide fragment from the GFP gene (GFP-Luc primer). The primers for the subsequent qPCR include a forward luciferase oligonucleotide (Luc or L) and a reverse GFP oligonucleotide (GFP or G). The primers for qPCRwould therefore specifically recognize the cDNAproduced from the primer extension  or placed in a mock reaction that lacked RT. As shown in in vitro.We supposed that the reason for poor reproducibility in the qPCR-detection of transcripts is likely due to the extreme low efficiency of transcription xtension  as the Gin vitro transcription activity of either E4 or ML promoter-directed reporter gene in both the absence and presence of a transcriptional activator. This method is based on the property that the GFP oligonucleotide-tagged cDNA is specifically recognized by the primers used for qPCR, in which the GFP reverse primer cannot bind to the remaining DNA template within the transcription system after the DNA template depletion  at E4 promoter. The TFIIB mutant derivative (G153Q:R154A) is defective in interaction with theBREd and exhibits decreased transcription activity at the E4 promoter [in vitro.Besides analysis of basal and activator-dependent transcription, the Adenovirus E4 and ML core promoters are often used for the studyof corepromoter element and transcription factor function –6, 16. Tpromoter . Our prevative G13Q:R154A in vitro transcription methods. Mutation of the BRE in the AdML promoter that also has a defective TATA element significantlyincreasedtranscription of the TATA-less promoter. Taken together with our previous work showing that the BRE acts as a negative element in the context of a wild type TATA element [in vitro transcription can substitute for the conventional method.We previously demonstrated and show again above that muta element ,16, thisin vitro transcription assay is a popular approach in the research field of gene transcription because of its strong sensitivity and because it directly detects RNA product by using radioactive isotope-labeled primer, however, its drawbacks are also notable. In this study, we have established a novel nonradioactive method for in vitro transcription analysis using DNA template elimination, primer extension and qPCR techniques. Recently, Park and Magan [in vitro transcription that can directly detect RNA transcripts by DNA template depletion and one-step RT-qPCR. In this method they used a promoter-linked G-less cassette as DNA template and immobilized beads to remove the DNA template after the transcription reaction was completed. We initially sought to use a similar method to analyze transcription of the E4 reporter vector; however, we were not able to achieve stable results, although the DNA template in our experiment was depleted to a similar level [et al [in vitro using AffimetricsQuantigene techniques, but it relies on specialist and facilities to complete the experiment. In our study, the uniquely designed primers have been used to solve the problem; our data indicate that the primers successfully detect low levels of RNA transcript by primer extension and qPCR. Our novel method is distinct from those published recently [in vitro transcription analysis but cannot easily employ approaches that use radioactive isotopes.The conventional primer extension-dependent nd Magan  reportedar level ; it is ll [et al  have recrecently , 17 and