Wiz in a mouse screen for epigenetic modifiers. Due to its known association with G9a/GLP, Wiz is generally considered a transcriptional repressor. Here, we provide evidence that it may also function as a transcriptional activator. Wiz levels are high in the brain, but its function and direct targets are unknown. ChIP-seq was performed in adult cerebellum and Wiz peaks were found at promoters and transcription factor CTCF binding sites. RNA-seq in Wiz mutant mice identified genes differentially regulated in adult cerebellum and embryonic brain. In embryonic brain most decreased in expression and included clustered protocadherin genes. These also decreased in adult cerebellum and showed strong Wiz ChIP-seq enrichment. Because a precise pattern of protocadherin gene expression is required for neuronal development, behavioural tests were carried out on mutant mice, revealing an anxiety-like phenotype. This is the first evidence of a role for Wiz in neural function.We previously identified DOI:http://dx.doi.org/10.7554/eLife.15082.001 MommeDs, Modifiers of murine metastable epialleles, Dominant  histone methyltransferase complex . The G9aMommeD30Wiz was identified in the screen because mice heterozygous for the mutant allele showed decreased expression of the GFP transgene. Here, we show at an independent locus known to be sensitive to the dosage of epigenetic modifiers, the Avygouti viable yellow (A) allele, that haploinsufficiency for Wiz also results in decreased expression.It has also been shown that G9a can act as a transcriptional activator, independent of its histone methytransferase activity  and it hMommeD30Wiz mice. We carried out ChIP-seq and RNA-seq in adult cerebellum, a tissue in which Wiz is expressed at high levels . The coat colour of the mouse can be used as a reporter of transcriptional activity at the vyA locus (MommeD30/+Wiz mice (maintained on an FVB/NJ background) were crossed to female vy/aA (pseudoagouti) mice and the coat colours of the offspring were scored at weaning (three weeks). FVB/NJ mice carry a wildtype Agouti locus, A/A, so ~50% of offspring from these crosses will be vy/AA and differ only at the MommeD30Wiz allele, i.e. MommeD30/+; Avy/AWiz or +/+; Avy/AWiz. Offspring were genotyped for the vyA and MommeD30Wiz alleles. Mice that did not inherit the vyA allele we excluded from the analysis. The reciprocal cross was also carried out, i.e. female MommeD30/+Wiz mice were crossed to male vy/aA (pseudoagouti) mice. The pedigrees and outcomes for both crosses are shown in Haploinsufficiency for Wiz resulted in an increase in the probability of silencing at the GFP transgene reporter, suggesting a role in transcriptional activation at this locus . To testvy locus . An actiMommeD30/+Wiz offspring from both crosses displayed a shift towards silencing (i.e. more pseudoagouti mice), compared to +/+Wiz littermates. The shift was statistically significant in both cases; the shift from MommeD30/+Wiz sire cross had a p-value of 0.0002, and from the MommeD30/+Wiz dam cross had a p-value of 0.0458  in mouse tissue, Western blotting was carried out using total protein lysates from  fetuses . The ban fetuses , and con fetuses . In addi fetuses  were in  fetuses .+/+Wiz mice were pooled for ChIP-seq; two cerebellums per sample and two biological replicates. The cerebellum was chosen for two reasons; it is a relatively simple tissue in the brain with respect to cell types and it is an ENCODE tissue, enabling comparisons to be made with ChIP-seq data obtained for other chromatin proteins. Library preparation and sequencing were carried out by Active Motif . Over 30 million reads were generated for each of the two Wiz ChIP-seq datasets and the chromatin Input sample dataset. At least 24 million reads could be aligned to the mouse mm10 genome in all cases, after filtering out PCR duplicates.The cerebellums of four adult male −20 . Reads were aligned to the mouse genome (mm10 build) and differential expression of Ensembl genes between genotypes was calculated using the R package DESeq  and an adjusted p-value of ≤0.05, were considered significantly differentially expressed. A total of 36 were found to change in males and 44 changed in females. The majority of significantly differentially expressed genes, in both cases, decreased in expression in the mutants . As expected, MommeD30/+Wiz samples compared to wildtype samples  gene cluster on Chromosome 18 and a second cluster consisting of mosome 8 . The latmosome 8 . Under tmosome 8 , most arSamd11, psid-ps1, psid-ps2 and Csf2ra, are the last gene on the chromosome. While it is possible that the locations of these genes and the cadherin-11 pseudogene cluster are incorrect in the current genome build (mm10 build), they are likely to remain adjacent to heterochromatic repeats.To see if the other significantly differentially expressed genes in E13.5 brain were related to location, we examined their locations on each chromosome . No otheMommeD30/+Wiz samples compared to +/+Wiz samples  and RNA-seq was carried out on each. A total of 82 genes were significantly differentially expressed; 29 genes decreased and 53 increased in expression in  samples . Of thesnd Rps4l . These aIn general, the down regulation of protocadherin β genes was more pronounced in the embryonic brain than in the adult cerebellum, where significance was seen for only a minority of the genes in the clusters .Pcdhb1, which is the only protocadherin β gene that lacks a canonical protocadherin promoter sequence  was tested and a second cohort  was tested two months later to validate the results from the first. The data from both cohorts showed similar effects and were combined for presentation. Mice normally display a circadian pattern of activity dependent on the light/dark cycle and so data is shown across the 24-hr period after averaging results at each time point across the 12 days. No shift was seen in the circadian pattern, but decreased activity was seen in the first two hours and the fifth hour after lights off in MommeD30/+Wiz mice compared with the +/+Wiz mice  is a test of anxiety-like behaviour in mice . The sam+/+ mice .MommeD30/+Wiz, n = 22; +/+Wiz, n = 21) were subjected to a light-dark box test of anxiety-like behaviour. WMommeD30/+iz mice showed reduced time spent in the light and reduced rearing activity 3–7 and all of these have been shown to bind DNA in vitro using electromobility shift assays  in the MommeD screen . Genotyping was carried out as previously described for the MommeD30Wiz  for ChIP, library preparation, sequencing and initial data QC. Two cerebellums were pooled per biological replicate. The rabbit polyclonal anti-Wiz  was used for ChIP on two biological replicates and an input chromatin sample was made by pooling equal amounts from the two biological replicates. Sequencing was carried out for 75mer read lengths on the NextSeq 500 platform . At least 30 million reads were generated per library. Read alignment to the mouse mm10 genome build was carried out using the Bowtie2 program with default settings (version 2.2.2)  and peakHeat plots and read sequencing density figures were generated using the seqMiner program (version 1.3.3)  using deTo analyse the overlap of Wiz ChIP-seq peaks and ENCODE dataset  peaks, tChIP-qPCR was performed with the Anti-Wiz antibody (NBP180586) on three biological replicates by Active Motif and loci were amplified using primers designed by Active Motif at regions of ChIP-seq enrichment indicated in the Figures. Enrichment was calculated by running qPCR reactions alongside known amounts of DNA to generate a standard curve. These are represented as binding events detected per 1000 cells.The data sets supporting the results of this article are available in the NCBI Gene Expression Omnibus under the accession code GSE76909.MommeD30/+Wiz × MommeD30/+Wiz) and detection of a post coital plug was defined as E0.5 dpc. Dams were euthanized by cervical dislocation and embryos dissected into 1X PBS, tissues were either snap frozen with dry ice for later RNA extraction or lysed overnight for DNA extraction as previously described (Embryos were produced by heterozygous intercrosses (escribed . The sexMommeD30/+/Wiz+/+Wiz) of less than 0.7 or greater than 1.3  and an adjusted p-value of ≤0.05.RNA was purified from 8-weekold male cerebellum or embryonic brain (E13.5) using Triazol reagent , as per manufacture instruction. RNA sequencing was carried out by the Australia Genome Research Facility  and at least 20 million 100 bp single end reads were generated on an Illumina HiSeq platform for each sample, from libraries generated using the Illumina TruSeq RNA Sample Preparation kit . An initial QC analysis was performed by AGRF. Sequencing reads were mapped to the mouse mm10 genome build using the program Tophat (version 2.0.11) , read coProtein lysates from embryos were prepared by homogenising tissue in ten volumes of urea lysis buffer, as described previously . These wNuclear extracts were prepared from either pooled embryonic brains (E13.5) or adult cerebellum using the Nuclear Complex Co-IP Kit  as described previously . For eacad libitum. After one week of acclimatisation mice were put under general anaesthesia and surgically implanted with a biotelemetry device  into their peritoneal cavities. After one week of recovery, the biotelemetry devices were used to monitor body temperature and locomotor activity. A platform receiver sampled the device’s temperature (± 0.1°C) and location at one minute intervals continuously and this was decoded by a software package VitalView . Following a 12 day period of uninterrupted monitoring, mice were exposed to an elevated plus-maze (EPM) test of anxiety-like behaviour (see below). Immediately following the EPM test, the mice were returned to their home cage for approximately 120 min, then anesthetized using a lethal dose of pentobarbital sodium . The telemetry data (activity and body temperature) were collapsed across the 12 days of testing to derive the activity for each hour in the 24 hr cycle. These data were then analysed using a repeated measures analysis of variance (ANOVA) with time as the within subjects factor and genotype and cohort as between subjects factors. A Greenhouse–Geisser correction epsilon (ε) was used to correct for potential violation of the sphericity assumption for the within-subject measure. Where significant main or interaction effects were found, post hoc Fisher’s LSD tests were used to reveal pairwise differences between groups. As there was no main effect for cohort  = 1.313, p=0.268) the data from the two cohorts were combined.Age matched male mice, 8–12 weeks old, were individually housed in standard polypropylene cages (14 × 30 × 13 cm) with shredded paper as bedding enrichment, ambient temperature was 30 ± 1°C, with a 12 hr light/dark cycle and lights-on at 07:00 hr. Mice received standard rodent chow and water Following the 12day monitoring period, mice were exposed to the EPM test. Each mouse was placed on the EPM for a duration of 5 min and behaviour was recorded using a digital video camera fixed above and overlooking the apparatus. Position and movement data was analysed using the Ethovision XT behaviour tracking software. The number of entries in the open arms, a common measure of rodent anxiety-like behaviour was analysed using an independent samples t-test.Light-dark behaviour testing was carried out on mice aged 2–6 months (n = 43) using methodology that was previously described . Each an In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Jessica Tyler as the Senior Editor. One of the three reviewers has agreed to reveal his identity: Michael Stallcup (reviewer #3).Thank you for submitting your article "Wiz binds promoters containing CTCF-binding sites and is required for normal behaviour in the mouse" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has abridged their comments and drafted this decision to help you prepare a revised submission.1) The antibody specificity has been questioned.i) The Western blot  and the ii) A point not being addressed is the ChIP quality/specificity of the antibody. Robust controls are required to demonstrate that the ChIP signals in this manuscript are Wiz dependent: Although Wiz null mice are lethal, 14d embryos have been published  and these embryos have been used for western in this manuscript. Therefore, the WizMD30/MD30 genotype can be and has to be tested by ChIP and qPCR at validating sites (i.e. about 10 positive and negative sites) and compared to wildtype. As a positive control, both genotypes have to be tested for H3 ChIP or similar at these sites. Data may be presented as 2) Isbel et al. convincingly argue that the relevant chromosomal region shows a single mutation in the Wiz gene as identified by exome sequencing. Nevertheless, this neglects mutations outside of exons, which potentially may influence the expression of the 48 genes in this chromosomal region  and/or of non-coding RNA. The authors should discuss this potential combined effect of Wiz and of unknown genes.3) The manuscript may be strengthened if less emphasis is put on the possible transcriptional activator function of Wiz. The transcriptional effects measured in E13.5 are a convolution of direct and indirect effects. Furthermore, only a handful of genes harboring Wiz binding in their promoter are affected, raising unanswered questions about the underlying mechanisms. With the present data it is difficult to conclude about a direct role of Wiz in transcriptional activation, especially in the absence of any biochemical/mechanistic exploration of the phenomenon. This should be commented on.4) It is relevant to consider whether the occupancy of either protein at their shared binding regions depends on the occupancy of the other protein. How many of the differentially expressed genes are bound by Wiz and CTCF in their promoters, respectively? For the down-regulated genes, how many actually have Wiz/CTCF binding at their TSS? How many do not? 5) The title and Abstract are a bit misleading. In their current state they seem to suggest that Wiz specifically binds promoters containing CTCF sites, which is not the case as Wiz binds most CTCF sites as well as most promoters. I suggest rephrasing to reflect the data more appropriately (e.g. "Wiz binds active promoters and CTCF binding sites…". Same throughout the text). 1) The antibody specificity has been questioned.i) The Western blot  and the A large, ~160kDa isoform of Wiz has previously been reported in adult cerebellum  and we have detected this with our antibody on a Western using protein extracted from this tissue but this was not included in the original version of the manuscript. This data is now included in “A larger ~160 KDa isoform has previously been reported in adult cerebellum  and consistent with that report a band of this size is detected with our antibody in protein extracted from this tissue .”ii) A point not being addressed is the ChIP quality/specificity of the antibody. Robust controls are required to demonstrate that the ChIP signals in this manuscript are Wiz dependent: Although Wiz null mice are lethal, 14d embryos have been published  and these embryos have been used for western in this manuscript. Therefore, the WizMD30/MD30 genotype can be and has to be tested by ChIP and qPCR at validating sites (i.e. about 10 positive and negative sites) and compared to wildtype. As a positive control, both genotypes have to be tested for H3 ChIP or similar at these sites. Data may be presented as We are not able to ChIP Wiz in the developing embryo due to embryonic lethality. The 14 day old embryos mentioned above are grossly abnormal, small  and not suitable for ChIP. Unfortunately, primary cells cultured from these homozygous embryos have limited viability (not shown). To address the issue, we have used an unbiased tandem affinity purification + mass spectroscopy approach to purify the Wiz complexes in wildtype embryonic and adult neural tissues. We identified 45 proteins that were present in anti-Wiz antibody pull down samples and absent in anti-IgG antibody controls. Ranking these by the semi-quantitative number of peptides recovered from embryonic brain, where expression of Wiz is high, shows that four members of Wiz-Zfp644-EHMT1-EHMT2 complex are in the top five proteins identified in the dataset.This mass spectroscopy data has now been included as “In addition, we used an unbiased tandem affinity purification approach and compared mass spectroscopy data with previously published datasets analysing Wiz binding partners. […] Four members of the Wiz-Zfp644-EHMT1-EHMT2 complex  were in the top five proteins in the dataset, ranked by peptide count in embryonic brain .”2) Isbel et al. convincingly argue that the relevant chromosomal region shows a single mutation in the Wiz gene as identified by exome sequencing. Nevertheless, this neglects mutations outside of exons, which potentially may influence the expression of the 48 genes in this chromosomal region  and/or of non-coding RNA. The authors should discuss this potential combined effect of Wiz and of unknown genes.It is unlikely that mutations inside the linked interval but outside exons are responsible for the effects for two reasons:A) The frequency of ENU mutations  make this unlikely;B) The absence of changes in the transcription of genes in the linked interval (excluding the Wiz gene itself). What was not mentioned in the previously version of the manuscript was the fact that no genes within the linked interval were miss-expressed in any of our mutant RNA-seq datasets. The following paragraph has been added to the Discussion:Wiz mutation in the MommeD30 strain . This was the only exonic mutation within the linked interval and in agreement with this being the causative MommeD30 mutation, RNA-seq data presented here show no other genes within the linked interval were mis-expressed in mutants.”“We previously described a 3) The manuscript may be strengthened if less emphasis is put on the possible transcriptional activator function of Wiz. The transcriptional effects measured in E13.5 are a convolution of direct and indirect effects. Furthermore, only a handful of genes harboring Wiz binding in their promoter are affected, raising unanswered questions about the underlying mechanisms. With the present data it is difficult to conclude about a direct role of Wiz in transcriptional activation, especially in the absence of any biochemical/mechanistic exploration of the phenomenon. This should be commented on.The following sentence has been modified in the Abstract:“Wiz is generally considered a transcriptional repressor. Here we provide evidence that it functions as a transcriptional activator.” has been changed to “Wiz is generally considered a transcriptional repressor. Here we provide evidence that it may also function as a transcriptional activator.”The following sentence has been modified in the Introduction section:MommeD30 was identified in the screen because mice heterozygous for the mutant allele showed decreased expression of the GFP transgene, consistent with a role for Wiz as a transcriptional activator.” has been changed to “MommeD30 was identified in the screen because mice heterozygous for the mutant allele showed decreased expression of the GFP transgene.”“The following sentence has been modified in the Results section:vyA locus is similar to the effect at the GFP transgene reporter, i.e. reduced levels of Wiz were associated with increased silencing, suggesting that the wildtype protein acts as an activator of transcription.” has been changed to “The effect of haploinsufficiency at the vyAlocus is similar to the effect at the GFP transgene reporter, i.e. reduced levels of Wiz were associated with increased silencing.”“The effect of haploinsufficiency at the We have already commented that effects could be indirect and this remains in the Discussion:“While any instance of decreased expression could, theoretically, be an indirect effect…”.4) It is relevant to consider whether the occupancy of either protein at their shared binding regions depends on the occupancy of the other protein. How many of the differentially expressed genes are bound by Wiz and CTCF in their promoters, respectively? For the down-regulated genes, how many actually have Wiz/CTCF binding at their TSS? How many do not? As suggested, we have changed the read tag density plots  to heat 5) The title and Abstract are a bit misleading. In their current state they seem to suggest that Wiz specifically binds promoters containing CTCF sites, which is not the case as Wiz binds most CTCF sites as well as most promoters. I suggest rephrasing to reflect the data more appropriately (e.g. "Wiz binds active promoters and CTCF binding sites…". Same throughout the text).This point has now been addressed throughout the manuscript. The title has been changed from "Wiz binds active promoters containing CTCF binding sites…" to "Wiz binds active promoters and CTCF binding sites…". The sentence in the Abstract – “…Wiz peaks were found at promoters showing enrichment for the transcription factor CTCF” – has been changed to: “ChIP-seq was performed in adult cerebellum and Wiz peaks were found at promoters and transcription factor CTCF binding sites”. The sentence in the Discussion – “Our ChIP-seq studies suggest that in adult cerebellum, Wiz binds at promoters at which CTCF, a recognized transcriptional activator, is also known to bind” – has been changed to: “Our ChIP-seq studies suggest that in adult cerebellum, Wiz binds at promoters and at sites which CTCF, a recognized transcriptional activator, is also known to bind”.


Recent genome-wide studies have revealed the presence of thousands of long non-protein-coding RNAs (lncRNAs), some of which may play critical roles in the cell. We have previously shown that a large number of lncRNAs show differential expression in response to interferon (IFN)α stimulation in primary human cells. Here, we show that a subset of IFN-induced lncRNAs are positioned in proximity of protein-coding IFN-stimulated genes (ISGs). The majority of gene pairs originated from bidirectional promoters and showed positively correlated expression. We focused our analysis on a pair consisting of the known protein-coding ISG, BST2, and an un-studied putative lncRNA originating from the promoter region of BST2 in a divergent orientation. We showed that this transcript was a multi-exonic, polyadenylated long RNA that lacked protein-coding capacity. BST2 and the lncRNA were both induced in response to IFNα in diverse cell types. The induction of both genes was mediated through the JAK–STAT pathway, suggesting that IFN-stimulated response elements within the shared promoter activated the transcription of both genes. RNAi-mediated knock-down of the lncRNA resulted in down-regulation of BST2, and we could show that this down-regulation occurred at the level of transcription. Forced overexpression of this lncRNA, which we named BST2 IFN-Stimulated Positive Regulator (BISPR), resulted in up-regulation of BST2, indicating that the regulation of expression of BST2 by BISPR is mediated through interactions involving BISPR RNA itself, rather than the impact of its transcription from an adjacent locus. Importantly, upon IFN stimulation, transcriptional activation of BISPR preceded the induction of BST2, suggesting that expression of BISPR facilitated the initiation of transcription in its paired protein-coding gene. The lncRNA-mediated transcriptional regulation described in this study may help govern the expression of additional protein-coding RNAs involved in IFN response and other cellular processes. The interferon (IFN) response is a critical arm of innate immunity and constitutes a major host defense mechanism against invasion of pathogens such as viruses and bacteria –3. ReleaRecent large scale transcriptome analyses have revealed the existence of tens of thousands of long non-protein-coding RNAs (lncRNAs), which play crucial roles in diverse aspects of cellular function including several steps in both innate and adaptive immune responses –14. HoweCurrent studies of the function of lncRNAs indicate that many of them regulate the expression of other genes through diverse mechanisms including induction of changes in the structure of chromatin and recruitment of various effector molecules such as transcriptional complexes, or direct interactions with other RNAs such as miRNAs or protein-coding RNAs , 16–19. As a first step toward understanding the role of protein-coding/lncRNA pairs in regulation of the IFN response, we identified lncRNA and protein-coding RNAs which were located in proximity of each other and showed differential expression in response to IFN stimulation. All identified pairs showed a correlated transcriptional response to IFN, and included a number of known protein-coding ISGs. Functional analysis of one such loci indicated the presence of a stable, spliced, and polyadenylated long non-coding RNA resulting from apparent bidirectional transcription from the BST2 (Tetherin) promoter. The expression of the lncRNA in response to IFN was mediated through the JAK–STAT pathway and correlated with the expression of BST2 in diverse cell types. Interestingly, forced overexpression and knock down of the lncRNA resulted in an increase and decrease in expression of BST2, respectively, suggesting that the expression of lncRNA, which we name BST2 IFN-stimulated positive regulator (BISPR), was needed for efficient transcription of its paired protein-coding gene. We could show that this regulation was at the level of transcription, and importantly, that upon IFN stimulation, the rise in the level of BISPR preceded the induction of BST2, suggesting that the transcription of BISPR facilitated the initiation of transcription in its paired protein-coding gene. Intriguingly, the putative bidirectional promoter giving rise to BISPR and BST2 overlaps an enhancer, raising the possibility that both RNAs may have evolved from enhancer-associated (eRNA) transcripts.The paired-end 100 bp-long RNA-seq reads that formed the basis for this study originated from high-throughput transcriptome analysis of human primary hepatocytes obtained from five human donors (SRA accession number SRP045406) . Data we2 as described previously  were incubated with 100 μg/ml cycloheximide for 5 min before harvesting. Cells were lysed in polysome lysis buffer ] and homogenized by passing the lysate through a 23G needle 15 times. Lysates were centrifuged at 17,000 × g for 10 min; the supernatants were collected, and absorbance was measured. About seven A254 units of the cytosolic extracts were layered over 10–50% sucrose gradients and centrifuged at 17,000 rpm in a Beckman SW28 rotor for 15 h at 4°C. After centrifugation, fractions (~1.2 ml each) were collected. RNA was extracted with TRIzol reagent (Invitrogen) and used to determine the distribution of ribosomal RNAs (by agarose gel electrophoresis) and BISPR, BST2, and GAPDH transcripts (by RT-qPCR).To investigate the association of BISPR with the translational machinery, we used human corneal epithelial cells, which showed a robust expression of BISPR and BST2 and allowed for unambiguous identification and quantitation of the RNAs in different polysome fractions. The 10.014 pRSVT cells  and random hexamers . For str® RNAiMax Reagent (Invitrogen) according to the manufacturer’s protocol. The transfected cells were incubated for 24 h in RPMI 1640 supplemented with 10% FBS and used for the downstream experiments. EZH2 knock down studies were performed on THP1 cells using shRNA constructs TRCN0000040074 and TRCN0000040077 targeting EZH2 and the non-targeting SHC002 shRNA construct from Sigma. Lentiviral preparation, cell transfection, and RNA harvest was performed as described  prevents us from deriving statistically significant conclusions from these data, the level of BST2 and BISPR in both primary cells and the cell lines studied above showed a modest correlation, with a Spearman’s correlation coefficient of 0.59 for both groups  –54. It hSaba Valadkhan and Hiroto Kambara designed the study. Saba Valadkhan performed the computational analysis of the data. Hiroto Kambara and Lalith Gunawardane performed the experiments, except as noted below. Elizabeth Zebrowski, Lenche Kostadinova and Donald D. Anthony collected blood samples from human donors and prepared NK and PBMC fractions for analysis. Raul Jobava, Dawid Krokowski and Maria Hatzoglou optimized the sucrose gradient fractionation of polysomal complexes, performed gene expression assays and analyzed the data and prepared the relevant figure. Saba Valadkhan re-analyzed all data for accuracy and wrote the manuscript. All authors read the manuscript and commented on it prior to final submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Journal/10.3389/fimmu.2014.00676/abstractThe Supplementary Material for this article can be found online at Click here for additional data file.Click here for additional data file.


Loligo pealei (lp) [Note: Loligo pealei has been officially renamed Doryteuthis pealei. For reasons of uniformity and clarity Loligo pealei (lp) is used here]. Of the most abundant ones, we cloned a full length cDNA which encoded the squid ortholog of the paired-type homeodomain proteins Phox2a/b. The homology of lpPhox2 to invertebrate and mammalian Phox2 was limited to the homeodomain. In contrast to mouse Phox2b, lpPhox2 was unable to transactivate the dopamine beta-hydroxylase (DBH) promoter in a heterologous mammalian transfection system. In vivo, lpPhox2 was expressed in the developing stellate ganglion of stage 27 squid embryos and continued to be expressed in the adult stellate neurons where expression was confined to the giant fiber lobe containing the neurons that form the giant axons. The expression of lpPhox was similarly timed and distributed as the Fmrf gene. Furthermore, the Fmrf upstream region contained putative Phox2a/b binding sites. These results suggest a role of lpPhox2 in the developmental specification of neuronal identity and regulation of neurons of the squid giant axon.Homeodomain transcription factors regulate development of embryos and cellular physiology in adult systems. Paired-type homeodomain genes constitute a subclass that has been particularly implicated in establishment of neuronal identity in the mammalian nervous system. We isolated fragments of eight homeodomain genes of this subclass expressed in the stellate ganglion of the North Atlantic long finned squid  The development of the nervous system involves multiple stages of cellular commitment that lead via patterning, proliferation, and fate determination to defined neuronal identities. These processes are driven by complex regulatory networks of transcription factors and signalling molecules . NeuronaSeveral families of transcription factors have been demonstrated to play a key role in the development of the mammalian nervous system. Early stages of commitment to neural cell fates and proliferation involve basic helix-loop-helix factors, while homeodomain proteins provide patterning and neuronal fate specification . For exaLoligo pealei since the 1930s . Screening of a cDNA phage library constructed from adult stellate ganglia by filter hybridization, using labelled PCR fragments as probes, resulted in the isolation of clones for RPF-1 and Phox2, but none of the other homeodomains.To screen for homeobox genes expressed in the stellate ganglion we employed a PCR cloning strategy used previously to identify paired-type homeobox genes expressed in rodent brain . SequencLoligo pealei . There was a variant Kozak sequence present at the translation initiation site 442 (CUCAUCAUGG)  . This prAUCAUGG) .Fig. 1.Sepia officinalis to 92% for Homo sapiens  with 96% overall identity to lpPhox2. Differences were confined to a stretch of repeated Gln residues , GMDSS (residues 20–24) and TxGSCSxxxxxRDH (residues 62–75). Compared to vertebrate Phox2 proteins, lpPhox2 shared a strong homology in the homeobox   was found with Phox2 of other invertebrates, e.g. (ceh-17) . In the etazoans , no evidLoligo pealei expresses a Phox2 transcription factor that is highly conserved in the homeodomain. The homeodomain confers the DNA-binding properties of this class of transcription factors, suggesting that the specificity of DNA-binding is a conserved, and, therefore, essential property for the function of Phox2 proteins.These data indicated that the squid dopamine beta-hydroxylase (DBH) gene. The DBH promoter is an established mammalian Phox2 target gene which contains Phox2 response elements . This indicated that lpPhox2 was expressed in brain structures, but at a low expression level. This observation agreed with a search in available transcriptomes of ganglia and brain lobe of Loligo pealei (http://ivory.idyll.org/blog/2014-loligo-transcriptome-data.html), showing the presence of lpPhox2 transcripts in the optical and buccal lobes, but not in the vertical lobe.In cryosections of stage 30 embryos weak signals of lpPhox2 mRNA were obtained in the adult stellate ganglion . In this way a short putative promoter region could be identified that contained two potential TATA boxes closely together , has been demonstrated in mammals. In mammalian systems two paralogs exist for Phox2: Phox2a and Phox2b. These exert complementary and concerted functions in a spatiotemporal relationship during the development of the neurons of the visceral nervous system  RNA was isolated, size selected and multiple libraries were made in the lambda-ZAP in situ hybridizations were performed according to In situ hybridization with DIG-labelled RNA probes on cryosections was performed as described  contained the 5′ region of the human DBH gene , 15% glycerol, and 2 mM dithiothreitol. The lysate was used to assay luciferase activity.Mouse Neuro2A cells, obtained from the American Type Culture Collection, were cultured in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 4 mM L-glutamine, 20 U/ml penicillin, 20 μg/ml streptomycin, 1:100 (v/v) nonessential amino acids (GIBCO), and 10% fetal calf serum at 37°C in a 5% COpromoter . After 4


NANOG locus in ESCs is necessary.The potential of pluripotent stem cells to be used for cell therapy depends on a comprehensive understanding of the molecular mechanisms underlying their unique ability to specify cells of all germ layers while undergoing unlimited self-renewal. Alternative splicing and alternate promoter selection contribute to this mechanism by increasing the number of transcripts generated from a single gene locus and thus enabling expression of novel protein variants which may differ in their biological role. The homeodomain-containing transcription factor NANOG plays a critical role in maintaining the pluripotency of Embryonic Stem Cells (ESC). Therefore, a thorough understanding of the transcriptional regulation of the NANOG in human embryonic stem cells from data obtained using high-throughput sequencing methodologies. Quantitative real-time PCR following reverse transcription of RNA extracted human ESCs was used to validate the expression of transcripts from a region that extends upstream of the annotated NANOG transcriptional start. Promoter identification and characterization were performed using promoter reporter and electrophoretic mobility shift assays.Regulatory footprints and transcription levels were identified for NANOG. Expression of novel transcripts from this transcriptionally active region confirmed the existence of NANOG alternative splicing in human ESCs. We identified an alternate NANOG promoter of significant strength at this upstream region. We also discovered that NANOG autoregulates its expression by binding to its proximal downstream promoter.Transcriptionally active chromatin marking and transcription factor binding site enrichment were observed at a region upstream of the known transcriptional start site of NANOG in human ESCs, indicating that alternative splicing increases the diversity of transcripts originating from the NANOG locus and that these transcripts are expressed by an alternate promoter. Alternative splicing and alternate promoter usage collaborate to regulate NANOG, enabling its function in the maintenance of ESCs.Our study reveals novel transcript expression from  Embryonic Stem Cells (ESCs), derived from the inner cell mass of the mammalian blastocyst, hold great promise for future therapeutic applications because of their unique ability to both proliferate indefinitely  in an undifferentiated state and retain the potential to give rise to every cell type in the body (pluripotency) –3. DiffeSeveral recent studies using proteomics, RNA interference, loss of function screens, genome scale location analyses and transcriptome profiling have identified transcription factors that serve as the framework for maintaining ESC pluripotency and self-renewal –14. The Nanog gene which enables production of the novel Nanog protein variants Nanog b and Nanog c that exhibit altered capacities for self-renewal and pluripotency in ESCs  dCTP using Roche Klenow for 30 minutes at 37°C. Following this, the radio-labeled ds-oligonucleotides were purified using Roche spin columns. For DNA binding reactions, 6 µg of nuclear extract was added to a 20 µl reaction containing 400 fM of radio-labeled oligonucleotide and 1 µg of poly(dIdC) (Roche) in the binding buffer  and incubated on ice for 45 minutes. Where specified, 60 pM of unlabeled double-stranded competitor was included along with the radio-labeled oligonucleotide. Complexes were separated on pre-run 6% native polyacrylamide gels for 4 hrs at 150 V in 0.5X Tris-borate-EDTA. The gels were subsequently dried and autoradiography was performed.For EMSA, complementary oligonucleotides having an additional G at their respective 5’-ends were annealed in annealing buffer  and the resulting double-stranded (ds) oligonucleotides were end-filled with [γ-All Nanog DNA sequences are from the most recent genomic builds taken from the NCBI public database (July 2012 freeze). DNA alignments were done with CLUSTALW using the AlignX program from the VectorNTI suite. The DNAseI hypersensitivity (HS) peaks for the human H1 and H7 ESCs were obtained from ChIP-Seq data generated by the University of Washington ENCODE group . The hisFor data analysis and peak calling, Fastq files where downloaded from the NCBI SRA and aligned with bowtie 2-2.0.0-beta6 against the hg19 genome using default options. The aligned files where then combined using samtools merge to create one alignment file for each experiment (where multiple fastq files existed). Peak calling was then done with MACS 1.4.2 using default parameters except spacing was changed to 25 and wig file changed to single output. BigWig files were generated with the UCSC tool wigToBigWig.GAPDH standard curve.RNA was prepared from H13 human ESCs using the PARIS kit (Ambion) following the manufacturer’s instructions. An in-column DNAse digestion was performed to remove contaminating genomic DNA. cDNA was synthesized from 1 µg of RNA using the SSIII reverse transcriptase (Invitrogen) in a 20 µl total volume. cDNAs were diluted 40-fold and 5 µl of the dilute cDNA was used in a 25 µl SYBR Green reaction using a Bio-Rad CFX96. Technical replicates were represented from two independent biological replicates. Starting quantities were determined from a Nanog gene in ESCs [NANOG in a human embryonal carcinoma cell line, revealing additional exons at the 5’ end beyond the known gene structure [NANOG gene in human ESCs [cis-regulatory sequences. The data from two human ESC lines (H1 and H7) showed strong and identical DNAseI HS positioned 1.8 kb upstream from the known transcriptional start site (TSS)  , whereaste (TSS) . Bindingman ESCs . These dnog gene .NANOG gene structure suggested by Eberle et al. [NANOG transcripts originating from the upstream region  gene and form colonies in a chromatinized stable transfection assay that we employed previously to analyze murine Nanog regulatory elements [NANOG proximal promoter has been shown to be highly active in mouse ESCs [R single cell colony assay. For these reasons, we performed the NeoR assay in mouse J1 ESCs. High numbers of neomycin resistant colonies were obtained from cells transfected with either the proximal (P1) or the distal (P2) promoters in comparison to cells transfected with a Hoxb6 promoter containing plasmid (NANOG −2401 to −1702) of the minimal promoter completely abolished the activation potential of the promoter revealing the inhibitory capacity of the SINE. Additionally, the minimal P2 promoter (−1788 to −1737) was orientation-independent as the sequence in reverse orientation also produced similar number of colonies. This orientation independence explains why the mouse counterpart of this regulatory element may have been categorized previously as an enhancer by our group and others [NANOG transcripts.The existence of Seq data  also revanalyzed . Differeelements . The prouse ESCs ; and unl plasmid . These r plasmid . This mid others ,43,44. TNANOG P1 for the presence of the most conserved tetramer ATTA/C reported to be present at the center of Nanog binding sites [catTacaatg, predicted Nanog binding site underlined), only the T spacer nucleotide joining the Oct4 and Sox2 binding sites was mutated to A to ensure that the Oct4 and Sox2 binding sites remained functional. NANOG P1 induced high-levels of luciferase expression. Out of the five predicted Nanog binding sites tested, mutations at sites between −256 to −249, −240 to −233 and the site between Oct4-Sox2 binding had no effect on luciferase expression (data not shown). However, mutation of the sites at −81 to −74 (Nanog site-1) and −60 to −63 (Nanog site-2) resulted in 56% and 22% reductions in luciferase expression  responsible for transcription initiating from this exon. Although there was a more robust DNAseI HS regulatory footprint at P2, we observed that there was a decreased enrichment of Oct4 and p300 at this region that might affect assembly of a robust RNA transcriptional complex. Alternatively, transcripts may be produced with comparable strength at both P1 and P2, but diminished elongation of transcripts from P2 resulting from a less processive RNA polymerase complex or due to post-transcriptional regulatory mechanisms might contribute to the lower levels of fully processed transcripts produced from this alternate promoter. We also determined that Nanog employs a positive autoregulatory mechanism to control its own downstream promoter (P1).The unique self-renewal and pluripotency characteristics of ESCs are regulated by a multi-layered network of post-transcriptional and post-translational modifications . Alternall types ,53. Our ignature . In the The transcription factor Nanog functions collaboratively with the other core pluripotency factors Oct4 and Sox2 to govern the maintenance of pluripotency. Even though Nanog was originally shown to be critical largely for repressing primitive endoderm formation , work byNANOG P1 and P2 are narrowing a genomic gap during evolution as the distance of 5 kb in the mouse has gradually contracted down to 1.8 kb in the human gene locus. The sequence analysis of this region in TRANSFAC predicted binding sites of potent components of the basal transcriptional machinery. The inclusion of TFIID, CBP, CEBPA and NF-1 (An alignment of the most active human P2 region (−1788 to −1737, and NF-1  binding and NF-1 . Two ATTNANOG proximal promoter has been demonstrated [While NANOG binding to the nstrated , the regnstrated . Given tNANOG P1, efficient Nanog recruitment is diminished. As we have shown that Nanog activates P1 activity, less efficient binding could explain its loss of strength.The human P1 resulted in expression of neomycin in our stable transfection assay; however, the promoter strength measured by this assay was significantly weaker in comparison to the P2 minimal promoter . AdditioNANOG locus in human ESCs will be required in the future to reveal the full upstream exon structure and to determine if additional protein variants exist, as suggested by our recent work in mouse ESCs [NANOG promoter serves a dual role in producing novel protein variants and downregulating the predominant NANOG A protein.The discovery of an alternate NANOG promoter that functions in ESCs significantly expands the regulatory repertoire used to control this core pluripotency factor. A comprehensive study of the human use ESCs . The exiuse ESCs . Alternause ESCs  that fre


Previous studies demonstrated PDHB to be positively related to the intramuscular fat (IMF) content. However, the transcriptional regulation of PDHB remains unclear. In our present study, the cDNA of bovine PDHB was cloned and the genomic structure was analyzed. The phylogenetic tree showed bovine PDHB to be closely related to goat and sheep, and least related to chicken. Spatial expression pattern analysis revealed the products of bovine PDHB to be widely expressed with the highest level in the fat of testis. To understand the transcriptional regulation of bovine PDHB, 1899 base pairs (bp) of the 5’-regulatory region was cloned. Sequence analysis neither found consensus TATA-box nor CCAAT-box in the 5’-flanking region of bovine PDHB. However, a CpG island was predicted from nucleotides -284 to +117. Serial deletion constructs of the 5’-flanking region, evaluated in dual-luciferase reporter assay, revealed the core promoter to be located 490bp upstream from the transcription initiation site (+1). Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP) in combination with asite-directed mutation experiment indicated both myogenin (MYOG) and the CCAAT/enhancer-binding protein beta (C/EBPß) to be important transcription factors for bovine PDHB in skeletal muscle cells and adipocytes. Our results provide an important basis for further investigation of the bovine PDHB function and regulation in cattle.The  The pyruvate dehydrogenase complex (PDC) is a “gateway” enzyme for the mitochondrial oxidative metabolism of carbohydrates, catalyzing pyruvate into acetyl-CoA and carbon dioxide, and providing a linkage between glycolysis and the tricarboxylic acid cycle (TCA) pathway . PDC is pyruvate dehydrogenase alpha subunit (PDHA) and the pyruvate dehydrogenase beta subunit (PDHB), respectively. The product of PDHB expression is distributed in mitochondria  for forglycePDHB-P1~P6, PDHB-R, KpnI-SmaI site of the pGL3-basic vector (Promega Corp.). After sequencing verification, we extracted the plasmids with an Endo-free Plasmid Mini Kit . We co-transfected the plasmids (0.8 μg) and the lipofectamine 2000 (2μl) into C2C12 or 3T3-L1 cells grown in 24-well plates with 10 ng pRL-TK (Promega Corp.). At 5 h after transfection, we replaced the media with DMEM with 2% horse serum (HS) (GIBCO-Invitrogen) and incubated for 40 h to induce differentiation of the C2C12 myoblasts into myotubes. We performed all remaining steps as previously described [We cultured the mouse myoblast cell line (C2C12) and the 3T3-L1 cell line in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (GIBCO-Invitrogen) under humidified air containing 5% CO2 at 37°C. 5’-nested PCR primers ))) [We isolated longissimus dorsi muscle and inguinal fat from bulls at 3 days after birth (n = 3). We conducted the ChIP assay using the EZ-ChIP™ Kit  according to the manufacturer’s protocol. We cross-linked the DNA-protein complexes with 37% formaldehyde and neutralized with glycine. After sonication, we added about 200–1000 bp of fragmented chromatin into the ChIP dilution buffer. We immunoprecipitated an equal amount of chromatin overnight at 4°C with 4μg of the myogenin (or C/EBPß) antibodies and normal mouse IgG. We then collected the immunoprecipitated products with Protein A+G coated magnetic beads. We eluted the bound chromatin in ChIP Elution Buffer and digested with proteinase K, then purified the DNA for PCR analysis. We used the primers ))) . As a neAll values are expressed as mean±standard deviation (SD). We analyzed the differences between groups with a Student’s two-tailed T-test. * P<0.05. ** P<0.01. n = 3.PDHBc DNA sequence (GenBank No. NM_001035435.3), we obtained a1576 bp cDNA. It contained an open reading frame (ORF) of 1080 bp, which had 359 amino acids (aa) encoded with a calculated molecular mass of 39.13 kDa and an isoelectric point (pI) of 6.466. Our cDNA consisted of a 5’-UTR of 91 bp and a 3’-UTR of 405 bp with a consensus AATAAA polyadenylation signal 16 bp upstream of the poly (A) stretch. The bovine PDHB gene spanned approximately 5.49 kb on the genome, containing 10 exons and 9 introns , goat PDHB1 (99%), porcine (97%), dog PDHB1 (96%), sheep PDHB2 (94%), goat PDHB2 (94%), mouse (94%), rat (94%), human PDHB1 (94%), human PDHB2 (89%), dog PDHB2 (86%) and chicken (82%). The phylogenetic tree indicated the bovine PDHB to be closest related to goat and sheep, and the lowest relatedness to chicken of all nine species evaluated for this study .In the first and second PCR, we cloned 682 bp and 400 bp fragments, respectively . SequencPDHB gene 5’-regulatory region spanning nucleotides from nucleotides -1724 to +175 and submitted it to GenBank (GenBank No. KJ649747). Several transcription factor-binding sites were recognized via sequence analysis of the 5’-regulatory region, including the Sp1 transcription factor (Sp1), the transcription factor Yin Yang-1 (YY1), the CCAAT/enhancer-binding protein ɑ (C/EBPɑ), myogenin (MYOG), the CCAAT/enhancer-binding protein ß (C/EBPß) and the cAMP responsive element-binding protein (CREB) (We cloned a 1899 bp fragment of the bovine n (CREB) . Interesn (CREB) .PDHB gene 5’-regulatory region (GenBank No. KJ649747), the 5'-flanking sequences of cattle, goat and sheep shared 97%, 94% and 93% sequence similarity. However, it had no significant sequence similarity with the 5'-flanking sequences of porcine, dog, human, rat, mouse or chicken PDHB.Compared to the bovine PDHB gene products in various tissues, it was necessary to provide mRNA expression profiles via real-time PCR analysis. The results showed the highest expression in testis fat, followed by lung, cecum, reticulum, rumen, kidney, spleen, liver, large intestine, heart, small intestine, omasum, subcutaneous fat, abomasums, longissimus dorsi and biceps femoris  in C2C12 cells [PDHB promoter. Mixing 5’-biotin labeled MYOG probes with nuclear extracts of C2C12 myotubes, led to the upshift of two bands. We suspect the reason to be heterodimers-formation between MYOG and other bHLH proteins [PDHB promoter. The EMSA and ChIP results combined with mutation analysis suggest MYOG likely to be an important transcription factor regulating the expression of the bovine PDHB gene in skeletal muscle cells.After analysis with online prediction software, we found the potential transcription factor binding sites MYOG and C/EBPß on the sequence from nucleotides -490 to -190 to be conserved in domestic animals. Myogenin (MYOG) is a member of the family of muscle regulatory factors (MRFs) that also include myogenic differentiation 1 (MYOD), myogenic factor 5 (MYF5) and myogenic regulatory factor 4  –31. MYOG12 cells . Mutatioproteins ,34. The PDHB promoter. Mixing 5’-biotin labeled C/EBPß probes with nuclear extracts of 3T3-L1 cells, led to an upshift of the band. The ChIP assay confirms the transcription factor C/EBPß to also be capable of binding to the C/EBPß binding site in the PDHB promoter. These results reveal the C/EBPß to be a key transcription factor for the bovine PDHB expression in adipocytes.C/EBPβ is a member of the family of CCAAT/enhancer binding proteins (C/EBPs), and binds to promoter regions of target genes to regulate the expression of downstream genes . A recenPDHB gene, and identified its transcription initiation sites. The amino acids and promoter sequences of bovine PDHB gene shared high similarity with the homologs of goat and sheep. We found the gene widely expressed with the highest level intestis fat. MYOG and C/EBPß are likely important transcription factors for the expression of the bovine PDHB gene in skeletal muscle cells and adipocytes, respectively. Our results provide an important basis enabling further understanding of the transcriptional regulation and biological function of the bovine PDHB gene.In summary, we cloned the CDS and promoter sequences of the bovine


Here, we demonstrate a role for a novel lincRNA, linc-MYH, in adult fast-type myofiber specialization. Fast myosin heavy chain (MYH) genes and linc-MYH share a common enhancer, located in the fast MYH gene locus and regulated by Six1 homeoproteins. linc-MYH in nuclei of fast-type myofibers prevents slow-type and enhances fast-type gene expression. Functional fast-sarcomeric unit formation is achieved by the coordinate expression of fast MYHs and linc-MYH, under the control of a common Six-bound enhancer.Thousands of long intergenic non-coding RNAs (lincRNAs) are encoded by the mammalian genome. However, the function of most of these lincRNAs has not been identified  in vivo adult muscle fiber-type specification implicating a long intergenic non-coding RNA, linc-MYH. We demonstrate a three-element genetic partnership, where an enhancer under the control of the myogenic homeoprotein Six1 functions as a regulatory hub to control fibre phenotype. In this partnership, the enhancer controls positively the expression of both the adjacent fast myosin heavy chain (MYH) gene cluster and of linc-MYH. linc-MYH is present only in adult fast type skeletal myofibers and controls their phenotype by suppressing slow-type gene expression. The regulation of linc-MYH could provide a lead for new therapeutic approaches or drug development.Adult skeletal muscles are classified into fast-type and slow-type, which display different resistance to muscle atrophy and metabolic protection against obesity. We identify in this manuscript a new mechanism controlling  The coordination of fast-type and slow-type gene expression in fast myofibers is not currently understood. Distinct intramyofibrillary calcium transients, evoked by slow tonic motor neuron firing, induce a cascade of downstream signaling involving Calcineurin and CamK. This results in the activation of selective transcription activators and repressors in slow myofibres. However, the signaling pathways operating in distinct Myh2, Myh1 and Myh4 myofiber subtypes, which coordinate the activation of the other fast-type genes and the repression of slow-type genes, is less well understood Adult skeletal muscles are composed of slow and fast myofiber subtypes which selectively express the genes required for their specific contraction activity and metabolic properties Six1 KO present severe muscle hypoplasia and die at birth in vivo analysis of the adult phenotype and the ability to investigate the direct or indirect involvement of Six1 in the spatio-temporal control of the expression of genes in the fast Myh cluster.Six homeoproteins are major myogenic transcription factors which directly bind to DNA sequences  to control myogenesis The mammalian genome encodes thousands of long intergenic non-coding RNAs (lincRNAs) which have multiple functions linc-MYH, and the mechanism of its control of adult muscle fast fiber-type specification in vivo. We demonstrate a three-element genetic partnership, where an enhancer element under the control of the myogenic homeoprotein Six1 functions as a regulatory hub to control fiber phenotype. In this partnership, the enhancer positively controls the expression of both the adjacent fast Myh gene cluster and linc-MYH, suppressing slow-type gene expression and facilitating fast fiber-type specialization.In this study we identify a new lincRNA, Myh genes, since higher levels of this transcription factor accumulate in the nuclei of adult fast myofibers than in slow myofibers Six1 could control the expression of fast Myh isoforms, we used computational analysis to locate MEF3 sites at the fast Myh locus (see Myh2 gene (linc-MYH (in vivo by ChIP (Chromatin Immunoprecipitation) experiments with Six1 antibodies on adult fast gastrocnemius plantaris (GP) and tibialis anterior (TA) muscles  assays of adult fast EDL (Extensor digitorum longus) myofibers. These experiments revealed that the enhancer interacts with the promoter of Myh2/1/4 genes in native chromatin of EDL myonuclei  promoter and obtained flox/flox;HSA-CRESix1 conditional knockout mice (hereafter named cSix1 KO) cSix1 KO mice at embryonic day 18.5 (E18.5) and at several post-natal stages (two weeks (P2W), four weeks (P4W) and eight weeks (P8W)) animals  increased during postnatal development in control mice but that of slow-type genes  decreased. The linc-MYH RNA was detected after birth in muscle samples and its expression increased in line with that of Myh4  muscle enriched with slow-myofibers  and a five and to 25 fold increase in the levels of slow-type genes   . Neverthrol mice . These rlinc-MYH is expressed in fast-type skeletal muscles , but not in SOL, brain, kidney, heart or fat tissues, an expression pattern which parallels that of the fast-fiber Myh4  to test the requirement for Six binding on the MYH enhancer to activate linc-MYH expression. These transient transfection assays, performed in adult TA, show that the MYH enhancer activates linc-MYH expression in a Six-dependent manner, as measured two weeks after electroporation , using linc-MYH sense and antisense RNA on isolated myofibers from fast EDL. Intranuclear localization of linc-MYH was observed with the antisense linc-MYH RNA probe, with approximately 10 linc-MYH foci per nucleus . We concluded from these experiments that linc-Myh RNA accumulates at specific sites in the nucleus of fast myofibers.We found that ber Myh4 . This suporation . lincRNA n = 10, , while tlinc-MYH foci observed in fast type nuclei, we hypothesized that linc-MYH could act in trans linc-MYH (shlinc-MYH) in TA muscle and analyzed the transfected samples after fourteen days. This method yielded the efficient knockdown of linc-MYH, with a 90% reduction of its expression  and moderately downregulated the expression of several fast genes,   . We note of MYH4 . Altogetlinc-MYH and of Six1 homeoprotein in adult muscles by the transcriptomic analysis of cSix1 and linc-MYH knockdown  than in cSIX1 mutant myofibers . These non slow-type genes could be exclusively repressed by linc-MYH in adult fast myofibers since there is a stronger downregulation of linc-MYH accumulation after its knockdown than in cSix1 mutant myofibers. Transcriptomic analysis of adult myofibers deprived of either Six1 or of linc-Myh identified a strong qualitative and quantitative correlation in the expression of specific genes between linc-MYH knockdown and cSix1 adult mutant myofibers. The expression of slow muscle genes was 3 to 10 fold higher in linc-MYH knockdown samples, and 5 to 25 fold higher in cSix1KO samples, than in the wild type. We further showed that linc-MYH lies downstream of Six1 in the Six myogenic pathway and helps to repress slow muscle genes in fast myofibers. The downregulation of all fast-type genes (other than Myh4), and the upregulation of slow-type genes, was weaker in the linc-MYH knockdown than in the Six1cKO line. Six1 may control several inhibitory pathways, including the linc-MYH pathway, to prevent slow-type genes expression in adult fast myofibers. During fetal development, at a stage where linc-MYH expression is not yet activated, Six1/4 increases the nuclear accumulation of the slow muscle repressors Sox6 and HDAC4 to repress slow muscle gene expression Myh7, Sln, Tnni1 and Tnnt1 is upregulated in the muscle-specific cSox6 mutant linc-MYH and Sox6, lying both downstream of Six1, directly participate in the downregulation of Sln, Tnni1 and Tnnt1 in fast myofibers. However, the repression of slow Myh7 in fast myofibers acts by a Six1-Sox6 dependent, but linc-MYH independent, repression mechanism. In this study, we observed that the levels of fast muscle gene expression decreased by 2–3 folds in the linc-MYH knockdown, with the highest decrease found for Myh4 expression. The expression of these genes decreased by a factor of 1.3 to 2.5 in cSix1KO, with the highest decrease found for Pvalb expression . Transfection of a 13 kb genomic fragment encompassing the whole linc-MYH gene lead to efficient linc-MYH RNA accumulation in myotubes, but after this forced expression, we were unable to detect any modification in the expression of slow or fast type genes (data not shown). These results suggest that specific cofactors of linc-MYH required for its appropriate functioning are lacking in cultured myotubes in culture, in agreement with the expression of linc-Myh only in adult fast type fibers. To circumvent the limitations of cultured cells, we turned to in vivo experiments in the Soleus in which linc-Myh is weakly expressed. Two weeks after linc-MYH gene transfection in the Soleus we observed that linc-MYH RNA accumulates up to approximately 80% of its expression level in TA . These genes, which belong to the muscle contractile machinery and are repressed in adult fast myofiber, are positively controlled by Six1 in myogenic C2 cells linc-MYH expression is not detected. This suggests that their expression in adult fast myofiber may be restricted by an additional level of regulation involving the Six1-linc-MYH axis. As a result of our study we suggest that Six1 controls the acquisition of fast-type myofiber mechanical properties by binding to a single enhancer region of the fast Myh locus. It promotes the coordinated expression of fast Myhs and that of a strong repressor of genes controlling slow contractile properties. The modulation of Six activity (depending on fiber-type) facilitates changes in the expression levels of the fast genes Myh and Tnn; these changes are required for the formation of efficient sarcomeric units and the appropriate Ca++ cycling and excitation/contraction/relaxation coupling Myh enhancer element therefore connects distinct regulatory hubs to achieve ultimate muscle fiber specialization. In this context, linc-MYH functions as an end-of-the-chain control element, conveying information on the state of fast Myh enhancer activity to repress slow-type specific genes and coordinates a finer level of regulation. This genomic organization at the fast Myh locus is reminiscent of the slow Myh7 locus where two microRNA miR-208b and miR-499 involved in fast myofiber program repression are co-regulated with Myh7linc-Myh and higher order chromatin modifying complexes remains to be identified, to explain how linc-Myh coordinates the activation of target genes at specific sites in the nucleus, and the repression of others.The commitment and maintenance of muscle fiber fast sub type specialization relies on the specific expression of one of the fast Myosin heavy chain gene present at the fast cSix1KO mice were obtained by breeding the Six1-LoxP mice Animals were bred and handled as recommended by European Community guidelines. Experiments were performed in accordance with the guidelines of the French Veterinary Department. Six1 antibodies  under agitation at +4°C overnight. 20 µl of Dynabeads protein G (Invitrogen) were added to the samples and incubated under rotation at +4°C for 1 hour. The beads were washed with low-salt buffer , high salt buffer , LiCl buffer  and TE buffer . The DNA was eluted with elution buffer  containing 0.1 mg/ml proteinase K (Invitrogen) at 62°C for 2 hours, and, proteinase K was inactivated by incubation at 95°C for 10 minutes. The DNA was finally purified with MinElute PCR purification kit (Qiagen). The amount of specific amplified DNA is normalized by beta-Actin promoter amplification. The sequences of the oligonucleotides used in this study are as follows. Enh 1F, 5′-ATC TCC ACC TCC CTC CAA CT; Enh 1R, 5′-ACC CCC TAG CTT TGA CAG GT; Enh 2F, 5′-AAT CTG ACG ACA GGG TGA GC; Enh 2R, 5′-GGT CGC CTG ACC TGA TAG AG; AldoaF, 5′-CTC TCA AGG CAA ACC AAA GC; AldoaR, 5′-CCA GTG TCC CAG ACC TTC TC; ActbF, 5′-TGT TAC CAA CTG GGA CGA CA; ActbR, 5′-ACC TGG GTC ATC TTT TCA CG, NCF, 5′-ATC CTG CCC CAC TGT GTT AG; NCR, 5′-GCC AGC AAT TTG GTT TGA AT.GP and TA muscles of 2 months old mice were minced with scissors just after sampling and fixed in 1% formaldehyde for 10 minutes. Formaldehyde was quenched by addition of 0.125 M glycine, and muscles were washed twice in PBS. The muscles were incubated on ice in lysis buffer ) for 10 minutes and homogenized with a mortar and, subsequently with a Dounce homogenizer. The nuclei were obtained by centrifugation, incubated in SDS lysis buffer  for 10 minutes, and sonicated in a Bioruptor apparatus (Diagenode). The debris were removed by centrifugation. The sonicated DNA was incubated with 1 µg of Myh locus (RP23-61C14) was digested, ligated and used as control templates. Quantification of the data was performed by quantitative real-time PCR using the Lightcycler 480 probe master (Roche Diagnostic). The sequences of the oligonucleotides used in this study are given in 3C experiments were performed as described cSix1 KO and control mice. Total RNAs were extracted by Trizol Reagent (Invitrogen) according to manufacturer's instruction.TA, back, soleus and GP muscles were collected from Actb. The sequences of the oligonucleotides used in this study are given in RNAs were treated with DNase I  and were reverse transcribed with Superscript III kit (Invitrogen) according to manufacture's instruction. Reverse transcription was performed with 1 µg of total RNA. Quantitative real time PCR  was performed using Light Cycler 480 SYBR Green I Master Kit (Roche) according to the manufacturer's protocols. PCR was performed for 40 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 15 seconds. Genes expression level was normalized by the expression level of the housekeeping gene in situ muscle contraction, as described previously Skeletal muscle function was evaluated by measuring linc-MYH probes were synthesized according to manufacturer's instruction . FISH experiments were performed on isolated EDL myofibres and images acquired on a Leica SP2 confocal microscope.Fluorescent-labeled antisense linc-MYH were designed, called shlincMYH, and inserted into the psiSTRIKE hMGFP system (Promega). The efficiency of each shRNA was established by determination of linc-MYH transcript levels in TA muscles transfected by each shlincMYH. The shRNA against 5′-TTC TGC TCA CCA CCT ACA ATT-3′ sequence was selected for the knockdown experiment. For knock down experiments using shlincMYH, a plasmid coding for shLacZ was electroporated in the contra-lateral TA as a negative control.Five distinct shRNAs targeting mouse In vivo transfections were also carried out on 10-weeks old C57Bl6 mice. For each experimental conditions, three to five Tibialis anterior (TA) or Soleus (Sol) muscles belonging to different mice were used. Under isoflurane anesthesia, legs were shaved and muscles were pre-treated by injection of a sterile 0.9% NaCl solution containing 0.4 U of bovine hyaluronidase/µl two hours before plasmid injection. Ten µg of shRNA-expressing vector were introduced into TA muscles of 8 week-old mice by electroporation as previously described MYH7  (1/1000 dilution), MYH2  (1/20 dilution) and against MYH4  (1/20 dilution) were applied overnight at 4°C to the treated sections. The next day, after three washes with 1× PBS containing 0.05% Tween-20, cryosections were incubated for 1 h with appropriate fluorescent secondary antibodies . After three washes with 1× PBS containing 0.05% Tween 20, samples were mounted in Vectashield mounting medium.TA, soleus and gastrocnemius muscles were embedded in cryomatrix and quickly frozen in isopentane cooled with liquid nitrogen. Cryostat sections (10 µm) were fixed in 4% PFA, washed in PBS, permeabilized with 0.1% Triton X-100 and left for 1 hour in blocking solution . Rabbit poly-clonal antibodies directed against Laminin  (1/100 dilution), and monoclonal antibodies against After validation of RNA quality with the Bioanalyzer 2100 (using Agilent RNA6000 nano chip kit), 50 ng of total RNA were reverse transcribed following the Ovation PicoSL WTA System (Nugen). Briefly, the resulting double-strand cDNA was used for amplification based on SPIA technology. After purification according to Nugen protocol, 5 µg of single strand DNA was used for generation of Sens Target DNA using Ovation Exon Module kit (Nugen). 2.5 µg of Sens Target DNA were fragmented and labelled with biotin using Encore Biotin Module kit (Nugen). After control of fragmentation using Bioanalyzer 2100, the cDNA was then hybridized to GeneChip Mouse Gene 1.0 ST (Affymetrix) at 45°C for 17 hours. After overnight hybridization, the ChIPs were washed using the fluidic station FS450 following specific protocols (Affymetrix) and scanned using the GCS3000 7G. The scanned images were then analyzed with Expression Console software (Affymetrix) to obtain raw data (cel files) and metrics for Quality Controls. The analysis of some of these metrics and the study of the distribution of raw data show no outlier experiment. RMA normalization was performed using R and normalized data was subjected to statistical tests.Six1 full-length mouse cDNA cloned into the pCR3 vector (Clontech) as previously described Six1 protein was obtained with a T7 transcription/translation kit (Promega). The oligonucleotide containing double-stranded myogenin MEF3 site was incubated with recombinant proteins. Competition experiments were performed in the presence of a ten-fold and hundred-fold molar excess of unlabeled identified Myh enhancer MEF3 sites (Enh1 to Enh6) or mutated Myh MEF3 sites (mtEnh1 to mtEnh6), or Myogenin promoter NFI or MEF3 sites. The sequences of the oligonucleotides used are as follows, the MEF3 consensus sequence is underlined; Enh1F 5′-CTC TTG GGT AAC TGG AGC CCC TC-3′. Enh1R TC CAG TTA CCC AAG AG-3′5′-GAG GGG C. Enh2R TT AGA TTT CCT TAT GA-3′5′-GGT TGA C. Enh2F 5′-TCA TAA GGA AAT CTA AGT CAA CC-3′. Enh3F 5′-TGT AAG AGA AAC TGA AAT AAA AT-3′. Enh3R T CAG TTT CTC TTA CA-3′5′-ATT TTA TT. Enh4F 5′-GGG GTA AGA AAT CTG ACG ACA GG-3′. Enh4R TC AGA TTT CTT ACC CC-3′5′-CCT GTC G. Enh5F T CAG GCG ACC TCA GT-3′5′-CTA TCA GG. Enh5R 5′-ACT GAG GTC GCC TGA CCT GAT AG-3′. Enh6F 5′-CGT CAA GGA AAC CTT ATT CCA TC-3′. Enh6R TA AGG TTT CCT TGA CG-3′5′-GAT GGA A. MyogF TCA GGT TTC TGT GGC GT-3′5′-TGG GGG GGC . MyogR 5′-ACG CCA CAG AAA CCT GAG CCC CCC CA-3′. NF1F 5′-TAT CTC TGG GTT CAT GCC AGC AGG G-3′. NF1R 5′-CCC TGC TGG CAT GAA CCC AGA GAT A-3′. mtEnh1F 5′-CTC TTG GGT AGG ATC CGC CCC TC-3′. mtEnh1R 5′-GAG GGG CGG ATC CTA CCC AAG AG-3′. mtEnh2F 5′-GGT TGA CGA ATT CTT GCT TAT GA-3′. mtEnh2R 5′-TCA TAA GCA AGA ATT CGT CAA CC-3′. mtEnh3F 5′-TGT AAG ACC AAC TGA AAT AAA AT-3′. mtEnh3R 5′-ATT TTA TTT CAG TTG GTC TTA CA-3′. mtEnh4F 5′-GGG GTA AGA AGG ATC CCG ACA GG-3′. mtEnh4R 5′-CCT GTC GGG ATC CTT CTT ACC CC-3′. mtEnh5F 5′-CTA TCA GGT CGG ATC CCC TCA GT-3′. mtEnh5R 5′-ACT GAG GGG ATC CGA CCT GAT AG-3′. mtEnh6F 5′-CGT CAA GGA AGG ATC CTT CCA TC-3′. mtEnh6R 5′-GAT GGA AGG ATC CTT CCT TGA CG-3′.EMSA was carried out with Six1KO mice and control mice as previously described Western blots were performed with protein extracts of GP and soleus muscles from cAll graphs represent mean values ± SEM. Significant differences between mean values were evaluated using two-tailed, unpaired Student's t test  or one-way ANOVA followed by Student Newman-Keuls test (for three or more groups).Myh2/1/4, c57bl6N mouse DNA was first used as a template to clone 1.1 kbp promoters of Myh2/1/4 with forward KpnI/SacI 5′-TTC AGA AAC TGC ATC ACT TAA A-3′ and reverse MluI, 5′-GCA GCT CGG GCA GTG GCC AGT GT-3′, forward KpnI/SacI 5′- CAT ATC TGC ATC TCT AGA TAC C-3′ and reverse MluI, 5′- GGC AGC AGC AGC CAG GAT GTG T-3′, forward KpnI/SacI 5′- ACC GCT AGC CTT GAG CCT TTG-3′ and reverse MluI, 5′- ATA GCG AGA GCC CTT TGT TCT C-3′, respectively. Myh2/1/4 promoter fragments were subsequently inserted into an KpnI-MluI digested pGL3 basic plasmid. For the construction of the pGL3-Enhancer-Myh2/1/4, mouse DNA was first used as a template to clone the enhancer with forward KpnI 5′- GCG TTT CTA ATT CGG CTT GAA C-3′ and reverse SacI, 5′- CAT TTC CTT CCT CTA AAG GCT CTT TATT C-3′. This enhancer fragment was subsequently inserted into KpnI-SacI digested pGL3-Myh2/1/4 plasmids. For the construction of the pGL3-mutEnhancer-Myh2/1/4, the six MEF3 sites of the enhancer were mutated as follows; MEF3 1: 5′-GTAACTGGA to 5′-GTAGGATCC; MEF3 2: 5′-TTAGATTTC to 5′-GAATTCTTG; MEF3 3: 5′-GAAACTGAA to 5′-CCAACTGAA; MEF3 4: 5′-GAAATCTGA to 5′-GAAGGATCC; MEF3 5: 5′-GTCAGGCGA to 5′-GTCGGATCC; MEF3 6: 5′-GAAACCTTA to 5′-GAAGGATCC. All plasmids sequence was confirmed by sequencing.For the construction of the pGL3-linc-MYH, genomic DNA fragment containing linc-MYH was obtained from digestion of a BAC clone containing (RP23-61C14) by BsaWI and AvrII. The 13.3 kbp DNA fragment was subsequently inserted into a SpeI-XmaI digested pSF-pA-CMVe plasmid. For linc-Myh gain of function experiments, the empty pSF-pA-CMVe plasmid was electroporated in the contra-lateral Soleus as a negative control. For the construction of the pGL3-Actb, pGL3-Sln and pGL3-Tnni1, mouse DNA was first used as a template to clone the promoters of Actb, Sln, and Tnni1 with forward 5′- TTT CTC TAT CGA TAG GTA CCT TTG AGC TCC TGA CCC CGT GTG TAG CTC T-3′ and reverse 5′- GAG CCC GGG CTA GCA CGC GTA AGG AGC TGC AAA GAA GCT G-3′, forward 5′- TTT CTC TAT CGA TAG GTA CCT TTG AGC TCT ACC GAC TAT CAT GCC CAC A-3′ and reverse MluI, 5′- GAG CCC GGG CTA GCA CGC GTC AGG CTA CCA AGG ACC TCA G-3′, forward 5′- TTT CTC TAT CGA TAG GTA CCT TTG AGC TCC TGG GAT TTG AAC CCA TGA C-3′ and reverse 5′- GAG CCC GGG CTA GCA CGC GTC CTC ACC ACA GAC TGC AGA G-3′, respectively. Actb, Sln, and Tnni1 promoter fragments were subsequently inserted into a KpnI-MluI site of pGL3 basic plasmid by GeneArt kit (Life Technologies). For the construction of the pGL3-Enhancer-Actb, pGL3-Enhancer-Sln and pGL3-Enhancer-Tnni1, the enhancer fragment was subsequently inserted into an KpnI-SacI site of pGL3-Actb, pGL3-Sln and pGL3-Tnni1plasmids. All plasmids sequence were confirmed by sequencing.For the construction of the pSF-pA-CMVe-Two µg of Luciferase-expressing vector and one hundred ng of pRL-TK vector (Promega) were introduced into TA muscles of 8 week-old mice by electroporation as previously described Myogenin consensus GAAACCTGA de novo motif finder Imogene In order to computationally identify MEF3 binding sites, we built a PWM (position-specific weight matrix) for MEF3, starting from a list of 15 binding sites see  that werFigure S1Myh enhancer. Sequences of P1 and P2 boxes of the Myh enhancer in mouse, rat, human, bovine and equinides species, and showing the sequence conservation of the six MEF3 sites and E boxes. Coordinates are in mm9 assembly.Sequences of P1 and P2 boxes of the (TIF)Click here for additional data file.Figure S2Predicted linc-MYH structure. Predicted minimum free energy (MFE) structure of the 1050 nt long linc-MYH, as determined by RNAfold (TIF)Click here for additional data file.Figure S3Sln mRNA in adult wt TA and Sol. (C). Luciferase assays of adult TA transfected with Sln, Tnni1 and Actb promoters with or without the Myh enhancer. TA sampling was performed two weeks after the electroporation, (n = 4). (D). Primary embryonic fibroblasts were transfected with Luciferase plasmids under the control of Myh4 promoter or Myh4 promoter linked with the Myh enhancer. After transfection, fibroblasts were cultured two days before sampling, (n = 3). *P<0.05, ***P<0.001.(A). Competitive Electromobility shift assays. Competitive Electromobility shift assays performed with recombinant Six1 proteins and labeled Myogenin MEF3 oligonucleotide and 10 or 100 fold molar excess of unlabelled oligonucleotides containing Myogenin MEF3 or NF1 site, with MYH MEF3 sites  or with mutated MYH MEF3 sites whose sequence is presented on (TIF)Click here for additional data file.Figure S4cSix1KO male mice.Immunostaining of MYH proteins. Immunostaining of MYH7 (red), MYH2 (red), MYH4 (red) and laminin (green) in TA of 12 weeks old control and (TIF)Click here for additional data file.Figure S5cSix1 mice and shlinc-MYH treated mice. (A) Venn diagram showing the overlap between genes that are up-regulated more than five fold in shlinc-MYH and two fold in cSix1 muscles. (p = 10−17 as given by a hypergeometric test). (B) Scatter plot of mRNA expression fold change (log2) as determined by microarray analysis of shlinc-MYH and cSix1. Genes that are up-regulated more than five fold in shlinc-MYH and two fold in cSix1 muscles are indicated in red. (C) mRNA expression levels in shlinc-MYH knock-down and cSix1 TA muscles, as measured by qPCR experiments. Ctrl , cSix1 , shLacZ , shlinc-MYH . (D) mRNA expression level of Ankrd1, Zdbf2 and Peg10 in back muscles of cSix1 KO mice at E18.5, P2W, P4W and P8W, as determined by qPCR experiments, . *P<0.05, **P<0.01, ***P<0.001.Comparison between (TIF)Click here for additional data file.Figure S6MEF3 PWM used in this study.(TIF)Click here for additional data file.Table S1Microarrays analysis of TA muscles electroporated by shRNA.(XLS)Click here for additional data file.Table S2Microarrays analysis of GP muscles of cSix1.(XLS)Click here for additional data file.Table S3Sequence of the oligonucleotides used for 3C.(DOCX)Click here for additional data file.Table S4Sequence of the oligonucleotides used for qPCR.(DOCX)Click here for additional data file.Table S5Coordinates (mm9) of the fragments used to learn the MEF3 PWM.(DOCX)Click here for additional data file.Table S6MEF3 frequency matrix.(DOCX)Click here for additional data file.


Drosophila nuclei.A recent super-resolution imaging study by Boettiger et al. elegantly demonstrates that three epigenetically defined, and functionally disparate, chromatin states have distinct folding characteristics in  Chromatin provides a framework upon which the activity state of the underlying DNA sequence may be registered, modified or perpetuated. This is due, in part, to the addition of a range of chemical modifications to DNA and histones, which increase the information content of the genome to form the epigenome. However, the local packing state of chromatin is also thought to be related to the epigenetic state and an understanding of chromatin topology and three-dimensional (3D) chromatin architecture is therefore central to understanding gene regulation.Chromosome conformation capture (3C) techniques have shown that the genome is subdivided into topologically associating domains (TADs), which can range in size from tens to thousands of kilobases and which are defined by an extensive network of internal contacts. TADs appear to segregate genes and their distal regulatory elements into compartments that are crucial to gene regulation . Within Drosophila genome.Investigation of chromatin compaction status in vivo, using DNA fluorescence in situ hybridization (FISH) has confirmed this . AnalysiBased on their histone modification state and bound complement of regulatory proteins, the regions studied were stratified as either transcriptionally active (histone H3 dimethylated lysine 4 H3K4me2) or histone H3 trimethylated lysine 79 (H3K79me3)), polycomb-repressed (histone H3 trimethylated lysine 27 (H3K27me3) or polycomb group proteins) or inactive . Repressed chromatin marked by histone H3 lysine 9 methylation and the heterochromatin protein 1 family of proteins was not investigated. With probes ranging from 10 to 500 kb, the median volumes of transcriptionally active domains were consistently the largest, while polycomb-repressed domains were the most compact , a PRC1 component whose sterile alpha motif (SAM) domain has known self-interacting properties. This resulted in the disruption of all architectural features of the repressed chromatin state, leading to chromatin decompaction, reduced intermixing within the domain, and de novo association with active chromatin domains. These findings are also in agreement with previous observations in Drosophila, showing that polycomb target loci are clustered within the nucleus into visible polycomb bodies. Distally located, polycomb-repressed loci separated by megabases of DNA coalesce within these bodies, albeit with varying frequencies depending on their chromosomal context [The compact chromatin state of polycomb-repressed domains in mammalian cells is caused by polycomb repressive complex 1 (PRC1), and not dependent on the catalytic activity of that complex . In Dros context . However context .Hox loci. Knockout experiments in combination with ChIP-seq (chromatin immunoprecipitation sequencing) identified that these interaction networks were both associated with and primarily dependent on PRC1 [A role for polycomb in scaffolding loci into defined bodies in mammalian cells is less evident. Recently, promoter capture Hi-C in mouse embryonic stem cells identified a marked enrichment for long-range intra- and inter-chromosomal interactions between polycomb target loci, centring primarily on the four paralogous polycomb-repressed  on PRC1 . These frepressed to active). Alternatively, mechanistically distinct phases of decompaction may exist, whereby the loss of polycomb interactions leads to a return to a basal or inactive chromatin conformation, followed by further decompaction to the active state as a result of coincident gene derepression (Fig. Taken together these results paint a picture of the distinctive 3D chromatin architecture characteristic of polycomb-repressed loci. However, the exact mechanism leading to the dissipation of these structures upon the loss of polycomb components, or forced differentiation, remains unclear. Loss of the aggregated PRC1 components could lead directly to the observed chromatin decompaction and altered structure (Fig.


Anthocyanin biosynthesis is regulated by a transcription factor complex. Here, it is determined that the potential bHLH partners in this complex function in a hierarchy to control each other and the anthocyanin biosynthesis pathway. Actinidia sp.) showing a strong activation of the anthocyanin pathway in tobacco (Nicotiana tabacum) was used to examine the function of interacting endogenous bHLH partners. Constitutive expression of AcMYB110 in tobacco leaves revealed different roles for two bHLHs, NtAN1 and NtJAF13. A hierarchical mechanism is shown to control the regulation of transcription factors and consequently of the anthocyanin biosynthetic pathway. Here, a model is proposed for the regulation of the anthocyanin pathway in Solanaceous plants in which AN1 is directly involved in the activation of the biosynthetic genes, whereas JAF13 is involved in the regulation of AN1 transcription.The anthocyanin biosynthetic pathway is regulated by a transcription factor complex consisting of an R2R3 MYB, a bHLH, and a WD40. Although R2R3 MYBs belonging to the anthocyanin-activating class have been identified in many plants, and their role well elucidated, the subgroups of bHLH implicated in anthocyanin regulation seem to be more complex. It is not clear whether these potential bHLH partners are biologically interchangeable with redundant functions, or even if heterodimers are involved. In this study, AcMYB110, an R2R3 MYB isolated from kiwifruit ( Anthocyanins are the major pigmented group of flavonoid compounds responsible for a range of colours varying from pink to blue, through to red and violet in flowers, fruits, or leaves. The anthocyanin biosynthetic pathway is one branch of flavonoid metabolism  and its C1 MYB (Zea mays) was the first transcription factor identified to regulate anthocyanin biosynthesis  and Red1 (R) (AN1 and JAF13 in Petunia (Petunia hybrida) (Delila and Mutabilis in snapdragon (Antirrhinum majus) (TT8 and GLABRA3 in Arabidopsis (VvMYC1 and VvMYCA1 in grape (Vitis vinifera) (A in pea (Pisum sativum) x3Lx6Lx3R, determines the binding specificity of the MYB to the bHLH partner and is responsible for the different ability of ZmC1 and a related R2R3 MYB, ZmP1, to interact with the bHLH R. The ability of the MYB to interact with the bHLH triggers the activation of the biosynthetic gene ZmBz1, with R playing a key role in the target specificity of the MBW complex LxPetunia, the MBW complex AN2–AN1–AN11  regulates anthocyanin biosynthesis in the flower petals . This response enables us to further define the specific roles of the bHLH proteins NtAN1 and NtJAF13 and to present a model that explains the hierarchical interactions of the MYB and bHLH partners within the MBW complex.Here it is shown that Protein alignment used ClustalW within the software package Geneious 6.1.7 , with manual refinement. A phylogenetic tree was formed from the alignment within the Geneious package using PhyML v2.2 and the maximum likelihood method , with thNicotiana tabacum) ‘Samsun’ and Nicotiana benthamiana plants grown in a glasshouse, using natural light with daylight extended to 16h. Transient dual-luciferase and colour assay were executed infiltrating approximately 300 µl of Agrobacterium tumefaciens GV3101 (MP90) mixture into young leaves. Agrobacterium were cultured on Lennox agar plates supplemented with selection antibiotics and incubated at 28 °C. The freshly grown bacteria were re-suspended in infiltration media and incubated at room temperature for at least 2h (Transient assays were performed as described previously (AcMYB110 and NtAN1 were amplified using specific primers (Supplementary Table S1) and cloned into pENTR-TOPO and subsequently a Gateway reaction was performed with destination expression vector pHEX2 (http://www.lifetechnologies.com). PhAN1 expression vector, as described in To generate overexpression vectors the open reading frames of NtAN1 and NtJAF13 (respectively 383bp and 197bp long) with specific primers (Supplementary Table S1) and the cloned products were put into pENTR-TOPO, followed by a Gateway reaction with RNAi destination vector pTKO2 (http://www.lifetechnologies.com).To generate the RNA interference constructs, small fragments were amplified of Agrobacterium transformed with constructs constitutively expressing transcription factors fused to the 35S promoter in pHEX2 and silencing the endogenous bHLHs NtAN1 and NtJAF13 by RNAi using a pTKO2 vector was used. Final colour development was assayed at four days and seven days after infiltration.For the colour assay, a mixture of AN1, CHS, and DFR promoter sequences were obtained by a genome walking strategy; approximately 1kb of target promoter was amplified using specific primers (Supplementary Table S1) and inserted into pGreen 0800-LUC. Promoter activation assays were performed in tobacco leaves. Agrobacterium containing the promoter–LUC fusions were mixed with Agrobacterium containing the transcription factor to be tested (ratio 1:3) and used to infiltrate tobacco leaves (Tobacco –1 of fresh weight.Five independent replicates were collected for each infiltration. Leaf samples were freeze-dried and ground. Anthocyanins were extracted in acidified MeOH (0.1% HCl) and centrifuged at 3000g. Aliquots were dried down and re-suspended in 20% v/v aqueous MeOH (0.2ml). The re-dissolved extracts were analysed by reversed phase HPLC using a method previously described (http://www.lifetechnologies.com) and DNAse-treated with DNA-free:Ambion (http://www.ambion.com/). Reverse transcription was performed using oligo(dT) according to the manufacturer’s protocol . Quantitative real-time PCR was carried out using the LightCycler 480 System, using 480 SYBR® Green I Master (Roche Diagnostics). Reactions were performed in quadruplicate and a non-template control was included in each run. The data were analysed using the LightCycler® 480 software 1.5 normalised to tobacco actin (GQ281246) and calibrated to the control (empty vector) infiltration (primer sequences in Supplementary Table S1).Gene expression analysis was performed on infiltrated leaves, 4 d after infiltration. RNA was isolated using TRIzol®  and transfected into yeast as a fusion to the GAL4 activating domain (AD). MYB110 was cloned using Gateway recombination technology (Life Technologies) in pGW6, a Gateway-adapted version of pTFT1 vector (–1 aureobasidin A was scored after 5 d at 30 °C.Yeast one-hybrid experiments were conducted using the Matchmaker Gold Y1H library Screening System (Clontech). 490bp and 562bp of the Supplementary Fig. S1).For the yeast two-hybrid assays, full length sequences of the candidate genes were inserted in pDEST22 and pDEST32 vectors from the ProQuest™ Two-Hybrid System (Life Technologies) and transfected in the PJ69-4A and PJ69-4alpha yeast strains (P<0.05) was used to separate the treatment means.Analysis of variance (ANOVA) using the Genstat 14th edition, Tukey test (Petunia), R (maize), Delila (snapdragon), and GL3/EGL3 (Arabidopsis), whereas clade B contained AN1 (Petunia), Intensifier (maize), Mutabilis (snapdragon), and TT8 (Arabidopsis). Members of the same subgroups are often involved in the same biological process, although it is not always clear if their functions overlap. For example; GL3, EGL3, and TT8 are partially redundant in the control of anthocyanin biosynthesis in Arabidopsis. However, each of these have also more specific functions such as PA biosynthesis (TT8), trichome formation (GL3/EGL3), and production of seed coat mucilage (TT8/EGL3) (Petunia the lack of AN1 (clade B) cannot be complemented by JAF13 (clade A), despite both being able to regulate anthocyanin biosynthesis  an anthocyanic patch can be generated  was used to inhibit the activity of each of the endogenous tobacco bHLHs known to be involved in anthocyanin biosynthesis: NtAN1a (HQ589208), NtAN1b (HQ589209), and NtJAF13b (KF298397). When NtAN1 was silenced (the same RNAi construct was able to target both NtAN1a and NtAN1b) no red colour occurred, despite constitutive expression of AcMYB110  and NtJAF13b. NtAN1 RNAi lines developed white flowers, whereas the NtJAF13 RNAi lines developed less intense pink flowers compared with the wild type . To further characterize the functional interaction of the two bHLHs, the down-regulated NtAN1 (NtAN1 RNAi) was complemented by constitutively expressing the Petunia hybrida JAF13 . Using the Petunia PhJAF13 to complement the reduction in expression of the tobacco bHLH allows the monitoring by qPCR of expression of the endogenous NtbHLHs, as there is sufficient sequence difference between the genes from the two species. Constitutive expression of PhJAF13 was not able to complement the lack of tobacco NtAN1 (RNAi) and did not restore the ability of AcMYB110 to drive red pigmentation  and dihydroflavonol 4-reductase  were therefore examined, as representative of early and late stages of anthocyanin biosynthesis; as well as the expression of the endogenous NtJAF13b and NtAN1 genes. Ectopic expression of AcMYB110 in the tobacco leaf up-regulated the transcript level of the endogenous NtAN1 and the biosynthetic genes NtCHS and NtDFR  driving the expression of the luciferase reporter gene (LUC). The results are presented relative to the level of the Renilla (REN) reporter gene, which is under control of a 35S promoter. As expected, AcMYB110 was able to activate the promoters of the biosynthetic genes, NtCHS and NtDFR . Nine different variants of the canonical CANNTG bHLH binding motif were identified. When focusing on the 650bp region upstream of the ATG, it was found that the CACGTG variant was only present in the promoters of the two biosynthetic genes whereas two other motifs, CATCTG and its reverse complement CAGATG, were only found in the NtAN1 promoter. The promoter analysis of AN1, CHS, and DFR was extended to three other species of the Solanaceae family  as well as to Arabidopsis. The results confirmed that the CACGTG motif was specific to the CHS and DFR biosynthetic genes and that either, or both, CATCTG and CAGATG motifs were present in the AN1-like promoters in the Solanaceae. However, this did not occur in the Arabidopsis promoters as the CATCTG motif was present in the AtCHS promoter and none of the two AN1-specific motifs were present in the TT8 promoter. Although the AN1-like promoters of the four Solanaceae share a low pairwise identity (48–55% with one another and 81% between tomato and potato AN1), the common CACGTG motif sits in a highly conserved stretch of 27bp which also contains conserved putative MYB binding sites. This suggests that this region is under high selective pressure and so may have an important regulatory function.The promoter sequences of AN1, JAF13, AN2, DPL, and PHZ in Petunia  and those of dicots, but different gene family members within a species can vary in function, as seen from markedly different transgenic phenotypes resulting from over-expression of PhAN2, PhDPL, or PhPHZ in Petunia (Petunia), TT8 (Arabidopsis), and Mutabilis (snapdragon), and a second that contains sequences such as JAF13 (Petunia), Delila (snapdragon), and GL3 from Arabidopsis  AcMYB110, previously shown to activate anthocyanin biosynthesis  and MYB75-PAP1 were shown to enhance the expression of PhAN1 and TT8 in Petunia and Arabidopsis, respectively  was further confirmed by yeast one-hybrid assay, in which AcMYB110 was able to bind the NtAN1 promoter only in the presence of the NtJAF13  Promoter sequence alignment of AN1 (A), CHS (B) and DFR (C) genes from tobacco, Petunia, tomato and potato.Table S1. Oligonucleotide primer sequences.