COX-2 expression. Using primary human mammary epithelial cells and monocyte/macrophage cell lines, we show that the chromatin boundary/insulator factor CTCF establishes an open chromatin domain and induces expression of a long non-coding RNA within the upstream promoter region of COX-2. Upon induction of COX-2 expression, the lncRNA associates with p50, a repressive subunit of NF-κB, and occludes it from the COX-2 promoter, potentially facilitating interaction with activation-competent NF-κB p65/p50 dimers. This enables recruitment of the p300 histone acetyltransferase, a domain-wide increase in histone acetylation and assembly of RNA Polymerase II initiation complexes. Our findings reveal an unexpected mechanism of gene control by lncRNA-mediated repressor occlusion and identify the COX-2-lncRNA, PACER, as a new potential target for COX-2-modulation in inflammation and cancer.Deregulated expression of COX-2 has been causally linked to development, progression, and outcome of several types of human cancer. We describe a novel fundamental level of transcriptional control of DOI:http://dx.doi.org/10.7554/eLife.01776.001 To produce a protein a cell must first transcribe the DNA in a gene to make a messenger RNA molecule, which is then translated to make the protein. However, cells also produce other types of RNA molecules which do not become proteins. MicroRNAs, for example, regulate the expression of genes as proteins, while the role of other RNA molecules called long non-coding RNAs (lncRNAs) is not well understood.COX-2 that is often implicated in breast, colon, prostate, and lung cancer. This RNA molecule, which is called PACER, originates near the start of the COX-2 gene, but it cannot be messenger RNA because it does not contain the instructions to make the COX-2 protein, and it is too long to be a microRNA. Further experiments showed that the newly discovered lncRNA activates the expression of the COX-2 gene.Now Krawczyk and Emerson have found an lncRNA that controls a gene called COX-2 by keeping away the molecules that might switch it off.Krawczyk and Emerson found that PACER attracts enzymes that spotlight genes that need to be activated, thus increasing the transcription of these genes to make messenger RNA. Genes can also be switched on and off by various molecules binding to nearby DNA, and PACER encourages the activation of COX-2 gene.In addition to shedding new light on the role of lncRNAs, these results suggest that PACER could be a suitable therapeutic target in cancers that involve the DOI:http://dx.doi.org/10.7554/eLife.01776.002 Long non-coding RNAs (lncRNAs) are a highly heterogeneous class of RNA molecules which are >200nt long and, apart from the lack of protein coding potential, share many characteristics with mRNAs in that they are transcribed by RNA polymerase II (RNAP II) and can be capped, spliced, and polyadenylated . LncRNAsLncRNAs can utilize a variety of mechanisms to regulate gene activity, where perhaps the most widespread involves interacting and recruiting repressive histone methyltransferase or demethylase complexes such as polycomb repressive complexes 1 and 2 , LSD1/CoREST/REST or G9a to target genes . ConversGiven that lncRNAs have gained increasing appreciation as key regulators of gene expression, it is not surprising that they are frequently deregulated during tumorigenesis . ExampleThe CCCTC-binding factor CTCF is a highly conserved 11-zinc finger DNA binding protein. Its primary function is believed to involve formation of chromatin boundaries/insulators through chromosomal looping. However, CTCF has been implicated in a variety of other processes, including transcription, enhancer blocking, insulation, splicing, nucleolar maintenance, DNA methylation, and imprinting . GenomicCyclooxygenase 2  is one of two COX isomers that are key enzymes in prostaglandin (PG) biosynthesis. COX enzymes catalyze conversion of arachidonic acid to PGH2 that acts as a substrate for a number of eicosanoid derivatives, such as PGE2, PGI2 and thromboxane A2, which are important mediators of many biological processes including inflammation, fever, pain, gastric and kidney function . Unlike Induction of COX-2 expression can be mediated by a number of intracellular signaling pathways in different cell types . Indeed,COX-2 transcriptional regulation in cells relevant to human tumorigenesis. Using primary human mammary epithelial cells (HMECs) and a PMA-driven human monocyte-macrophage differentiation system, we demonstrate that a nuclear antisense long non-coding RNA PACER (P50-Associated COX-2 Extragenic RNA) is expressed in the upstream region of COX-2 and functions to directly sequester the repressive NF-κB p50 subunit from the COX-2 promoter. This facilitates the recruitment of p300 histone acetyltransferase and a domain-wide increase in chromatin accessibility, as well as assembly of RNA Polymerase II pre-initiation complexes. We also show that PACER expression is controlled by two CTCF/cohesin complexes assembled in the 5′ UTR region and at a distal upstream site. CTCF/cohesin induces lncRNA expression by establishing a chromatin domain marked by increased H3K4 methylation, H4K8 acetylation and decreased H4K20 trimethylation, thus creating a permissive chromatin environment and protecting the COX-2 regulatory region from surrounding repressive chromatin. These findings offer unexpected insights into the mechanisms of gene regulation by CTCF/cohesin and by lncRNA-mediated repressor eviction.In this study, we describe a novel layer of COX-2 expression might be regulated by a non-coding antisense RNA overlapping the gene or its promoter. Our search failed to identify lncRNAs within the COX-2 coding region, so we examined the region upstream of the COX-2 transcription start site. This region contains numerous putative binding sites for transcription factors but no additional annotated genes. We performed chromatin immunoprecipitation (ChIP) experiments using antibodies directed against RNA Polymerase II (RNAP II), which revealed high levels of RNAP II as far as ∼1 kb upstream of the COX-2 transcribed region. Intriguingly, we detected a peak of RNAP II binding at approximately −0.45 kb . Primer walking indicated that the COX-2 upstream region is transcriptionally active roughly from −0.3−0.4 kb to approximately −1.5−2 kb . To deteA survey of a recent study utilizing strand specific Global Run-On sequencing (GRO-seq) confirmed that the upstream region of COX-2 is indeed transcribed in an antisense orientation . A stronTo corroborate the finding that two transcriptional units possessing separate RNAP II promoters exist within the COX-2 locus, we used reporter gene assays . 400 or P50-Associated COX-2 Extragenic RNA . The entire nucleotide sequence of PACER is shown in Bioinformatic analysis of the nucleotide sequence failed to support the hypothesis that the novel RNA encodes a functional protein. The longest detected Met-initiated putative ORF is 43 aa and this peptide contains no identifiable protein domains and bears no similarity to any protein in protein databases (not shown). In addition, polysome profiling indicated that the ncRNA does not co-sediment with the poly-ribosome fraction . While we cannot exclude that the RNA might encode a small peptide of unknown function, we conclude with high confidence that it represents a novel species of long non-coding RNA (lncRNA). We will continue to refer to it as During the preparation of this manuscript, Carpenter and colleagues reported that a species of lncRNA located ∼50 kb downstream of the mouse COX-2 gene affects expression of many immune-related genes both positively and negatively, likely through interactions with RNA binding proteins hnRNP A/B and hnRNP A2/B1 in murine macrophages . HoweverBinding sites for known mediators of COX-2 activation, such as NF-κB, AP1, NF-IL6, CREB, TCF4/LEF1, cluster between the COX-2 transcription start site and approximately −1.5 kb upstream of the promoter  de Mora. This clCOX-2 gene and 50 kb flanking sequence on each side. Numerous other genes were also included on the array (data not shown). Chromatin immunoprecipitation (ChIP) experiments were performed with extracts from primary human mammary epithelial cells (HMECs) using antibodies directed against CTCF. IP DNA was purified, amplified, and hybridized to DNA arrays. Before hybridization, enrichment of known genomic CTCF binding sites in IP DNA was verified by qPCR.We addressed this possibility using ChIP-on-chip experiments. A custom genomic DNA tiling array was designed to include the entire human COX-2 locus, one located in the promoter region and the other at approximately −2.5 kb, precisely at the interface between the region of repetitive DNA and the putative COX-2 regulatory region  and COX-2 expression can be efficiently induced in U937 macrophages with a variety of stimuli, including LPS  targeted against PACER using lentiviral vector delivery. U937-sh-lncRNA monocytes showed no detectable COX-2 mRNA or lncRNA expression, as did control monocytes carrying shRNA directed against the Luciferase gene . Upon PMnduction . In knocnduction . Finallynduction  and durinduction . InteresWe analyzed the relative abundance of PACER within the cell using biochemical fractionation of U937 macrophages followed by qPCR. The vast majority (more than 75%) of the lncRNA was found in the nucleus, with one third being stably integrated into chromatin and the rest occupying nuclear space; the nuclear fraction increased even more upon LPS stimulation . This diCOX-2 transcription through chromatin modification status, transcription factor interaction, and co-activator recruitment. First, we analyzed binding of CTCF, cohesin, Sp1 and AP1 to the COX-2 upstream region upon PACER knockdown. Sp1 and AP1 have been previously implicated in mediating COX-2 activation in various cell types, including macrophages  and a modest effect on H3K4 dimethylation . By contCOX-2 locus, we performed ChIP-qPCR experiments. p300 was absent from the COX-2 promoter in non-induced macrophages but was recruited to the promoter upon LPS stimulation  and PACER promoter , as well as across the COX-2 gene (0 to 4 kb) and PACER gene (- 1 kb to −0.25 kb) (To explore the possibility that PACER could affect (directly or indirectly), the assembly of RNA Polymerase II preinitiation complexes and/or later steps in transcriptional activation, we used ChIP-qPCR to assess the association of bulk RNAP II and RNAP II modified at serine 5 or serine 2 within its C-terminal domain across the entire 0.25 kb) . Elongat0.25 kb) . Finally0.25 kb) . These rHow does PACER recruit RNAP II initiation complexes and p300 to the COX-2 promoter? To explore the possibility that the lncRNA might directly interact with p300, we carried out RNA immunoprecipitation (RIP) experiments. Antibodies against p300 were incubated with extracts containing intact cellular RNAs, RNA was then purified from immunoprecipitates and analyzed by RT-qPCR. These experiments failed to detect the presence of lncRNA in p300 RIP samples . SimilarCOX-2. NF-κB-mediated induction of COX-2 has been previously shown to involve p300 recruitment to DNA  involving occlusion of a repressor complex (NF-κB p50) from promoter association which then promotes assembly of co-activators (p300 HAT) and RNAP II pre-initiation complexes. PACER directly interacts with p50, which can form both active heterodimers with p65/RelA during the normal course of NF-κB pathway activation, and inactive p50/p50 homodimers which lack transcription activation domains present in p65/RelA. The repressor role of p50 at er cells . Moreoveer cells . ImportaCOX-2 locus upon stimulus-induction. We postulate that a large class of activation-related lncRNAs, such as enhancer-associated RNAs (eRNAs), might employ this mode of gene regulation, utilizing NF-κB or other transcription factors as baits to recruit CBP/p300. In this regard, it is interesting to note that enhancers are often co-inhabited by CBP/p300 and lncRNAs . Human histiocytic lymphoma monocyte cell line U937  for 48 hr, followed by 24 hr incubation without PMA. To induce COX-2 expression, U937-derived macrophages were treated with LPS (2 µg/ml) for 6 hr.Early passages of human mammary epithelial cells were obtained from Martha Stampfer. Cells were grown in MEGM BulletKit  media at 37°C in 10% Oine U937  was kindg for 5 min. Suspension cells (U937) were washed two times and collected by centrifugation at 300×g for 5 min at 4°C. Cells were lysed by rotating for 10 min on ice in 50 mM K-Hepes pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, and protease inhibitors and then centrifuged at 600×g for 10 min at 4°C. Chromatin pellets were washed once in 10 mM Tris–HCl pH 8.0, 0.2M NaCl, 0.1 mM EDTA, 50 µM EGTA and resuspended in 10 mM Tris–HCl pH 8.0, 0.1% NP-40, 0.1 mM EDTA, 50 µM EGTA. Sonication (20 rounds of 10s at power setting 3.5) was performed with a Branson sonicator, followed by centrifugation to clear the chromatin solution from debris. An aliquot of chromatin corresponding to 106 cells was brought up to 1 ml with 20 mM HEPES, pH7.9, 0.2M NaCl, 2 mM EDTA, 0.1% Na-DOC, 0.5% Triton X-100, 1 mg/ml BSA, and protease inhibitors and incubated with 2 µg of antibodies overnight at 4°C. To capture immune complexes, 10 µl of protein G beads was added and incubated for 2 hr at 4°C. Beads were washed , immune complexes were detached from beads by 10 min incubation at 65°C in 0.1M Tris–HCl, pH 8.0, 1% SDS, followed by adding 140 µg of Proteinase K  and NaCl to 0.1M, and incubation for 2 hr at 42°C and then overnight at 65°C. DNA was purified by phenol/chloroform extraction and analyzed by qPCR. IP efficiencies were calculated as a ratio between qPCR signals obtained with IP material and input DNA processed in parallel with ChIP samples. The following antibodies were used in ChIP experiments: CTCF , p50 , Rad21 (Abcam), RNAP II, H2AZ , H3K4me2, H3K4me3, H3K9me3, H4K20me3, H4K8Ac, H3Ac (Abcam), p300-Ac . qPCR primers are listed in Cells in exponential growth phase  or differentiated (macrophages) were cross-linked with 1% formaldehyde in 5 mM Hepes pH 8.0, 10 mM NaCl, 0.1 mM EDTA, 50 µM EGTA for 10 min at RT. Cross-linking was stopped with 0.125M glycine. Adherent cells were washed two times with PBS and collected by scraping into ice-cold PBS followed by centrifugation at 300×g for 5 min at 4°C, resuspended in 5 vol of cytoplasmic extract buffer  and kept on ice for 5 min. After centrifugation at 600×g for 5 min at 4°C, the cytoplasmic fraction was collected and the nuclear pellet was resuspended in nuclear extract buffer , kept on ice for 10 min and centrifuged for 10 min at max speed. The nucleoplasmic fraction was collected and the pellet was resuspended in the same buffer, sonicated five times for 10 s at power 3 and cleared by centrifugation to produce the chromatin-bound fraction.Cells were scraped into ice-cold PBS, collected by centrifugation at 300×RNA purification was performed with Trizol  according to the manufacturer's protocol. cDNA was produced using the Superscript III system (Life Technologies) using random hexamers or strand-specific primers listed in ChIP DNA and reference input DNA were amplified by ligation-mediated PCR according to NimbleGen  recommendations. Briefly, 1 µg of DNA was blunted with T4 DNA polymerase (NEB), double-stranded staggered oligonucleotide adaptors were ligated, and PCR amplification was performed with oligos specific to the adaptors using a mixture of Taq polymerase (NEB) and PfuTurbo . 4 μg of each DNA was purified and sent to NimbleGen for probe preparation and hybridization. LM-PCR amplified DNA was verified to be enriched for target sequences that were detected by ChIP.Custom high density genomic tiling arrays contained ∼80 regions covering selected genes and 50 or 100 kb flanks on either side. Array manufacturing, probe labeling, hybridization, and signal acquisition were conducted by NimbleGen. Data were visualized using SignalMap software .Constructs were cloned into the pGL3basic vector . Plasmids were transfected in a 20:1 ratio (pGL3 to control Renilla Luc pRL-TK vector) using Lipofectamine 2000 (Life Technologies) into HEK293T cells in 96-well dishes and assayed 24 hr post transfection using the Dual-Glo Luciferase assay system (Promega). All transfections were done in five replicates.Western blotting was performed using standard protocols using the following antibodies: CTCF , COX-2 , p50 (Abcam), TFIIB (Santa Cruz), actin (Sigma).http://insulatordb.uthsc.edu/.Computer-based site prediction was performed using the online tool at 5′ and 3′ ends of COX-2 lncRNA were mapped using Rapid Amplification of cDNA ends (RACE) kit (Roche) according to the manufacturer's recommendations. Sequences of primers used for strand-specific cDNA synthesis and RACE amplification reactions are listed in RIP experiments were performed using MagnaRIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer's recommendations. Antibodies were purchased from Santa Cruz Biotechnology  and Millipore (CTCF). qPCR primers are listed in 5/ml HMECs were transfected according to the manufacturer's protocol using DharmaFect I reagent. For PACER, four siRNA were designed and tested, from which two efficiently knocked down lncRNA expression . The siPACER-753 was most efficient and was used in all experiments. Cells were harvested 72 hr after transfection.siRNA against CTCF, custom-designed siRNA against PACER and control-scrambled siRNAs were purchased from Dharmacon . siRNAs against SMC3 were purchased from Ambion (Life Technologies). Sequences of custom siRNA are listed in Short hairpin sequence against COX-2 lncRNA was created by linking sense siRNA 870, loop, antisense siRNA870 and RNAP II terminator sequences; and cloning into BamHI and EcoRI sites in pGreenPuro lentiviral vector  downstream of the H1 promoter. shRNA against the Luciferase gene was used as a control. shRNAs against p50 and p65 were purchased from Sigma. U937 monocytes were transduced with lentiviral particles and selected with puromycin. Once stable lines were established, puromycin was removed at least 48 hr before harvesting or differentiation. review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  activates The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission:eLife, but they also concurred in that additional work is required to strengthen the main conclusions of this interesting paper.The three reviewers agreed that the paper is exciting and interesting, as it describes a novel lncRNA involved in control of a gene locus of significant biomedical relevance by an unprecedented mechanism. The manuscript reports the characterization of the lncRNA dubbed PACER, which originates from the upstream region of the COX-2 gene in the antisense direction. It is demonstrated that PACER positively regulates COX-2 expression. Mechanistically, PACER promotes histone modifications associated with gene activation and RNAPII association with the COX2 promoter region. PACER binds to the DNA binding protein p50, and it is proposed that this binding leads to gene activation by favoring formation of active p50-p65 NFKB heterodimers, which in turn would drive COX2 transcription. The reviewers agreed that these results merit publication in a high-impact journal such as The reviewers agreed to summarize their request for new work as follows:1) A better characterization of PACER is needed. Key questions raised by the reviewers are: What is the size, exact sequence, relative abundance and exact intracellular distribution of PACER? Does PACER originate from its own unique promoter, is it a divergent transcript from the COX-2 promoter, or is it an eRNA? To address these issues, the reviewers propose the following work:1.1) Full sequence data from the RACE experiments should be provided.1.2) A Northern blot analysis and/or qPCR analysis should be performed to determine the abundance of PACER relative to COX-2 mRNA.1.3) A better characterization of the PACER transcription start site is needed. ChIP analysis of RNAPII and chromatin modifications are provided in the manuscript, but these assays are correlative in nature and could not fully elucidate whether PACER originates from its own unique promoter, if it is a divergent transcript from the COX-2 promoter, or if it is an eRNA. This issue can be resolved with promoter reporter assays to define the DNA sequence that drives PACER transcription.1.4) Both the RNA and RNAPII ChIP profile suggest a longer transcript than what was cloned by RACE, up to -2 kb. The authors should explain how this matches the suggested 3' end of the RNA at -1022 and show the RACE data rather than just describing it. Is there a canonical cleavage and polyadenylation signal around -1022?1.5) Strand-specific amplification of the plus strand should include an oligo within the COX-2 gene as a positive control.1.6) 1.7) The reviewers wonder whether PACER has been detected by recent genome wide Global Run-On deep sequencing experiments (GRO-seq) done in epithelial cells. Publicly available GRO-seq datasets that could be mined to answer this question include Galbraith MD et al, Cell. 2013 Jun 6;153(6):1327-39 (performed in HCT116 colon cancer cells) and the various papers from the Kraus team using MCF7 breast cancer cells such as Danko CG Mol Cell. 2013 Apr 25;50(2):212-22. GRO-seq results for the COX-2 (PTGS2) locus could be displayed to further illuminate the site of PACER transcription.2) The model of PACER action needs further experimental validation.2.1) Whereas the repressive role of p50 is well demonstrated, the positive role of p65 NFKB in driving COX2 expression is not demonstrated in the context of PACER action. As currently depicted in 2.2) Additionally, how does PACER knockdown or CTCF knockdown affect p65 binding to the COX2 promoter? Reviewers propose that ChIP assays for p65 be performed to answer this question.3) A better presentation and interpretation of the ChIP data is needed.3.1) CTCF ChIPs. Regarding the ChIP-on-chip experiment, the authors should show at least one negative control and one positive control locus out of the “numerous other genes” on the chip and extend the COX-2 analysis several kb up and downstream to demonstrate the specificity of the IP. There is a discrepancy between the ChIP-on-chip and ChIP-qPCR analysis for CTCF binding sites. In the ChIP-on-chip, one sees a clear enrichment of CTCF binding at least to -500bp, which is not confirmed by the qPCR. Also, the peak for CTCF binding around TSS is much more pronounced than the one around -2.5kb. Again, this is not recapitulated in the ChIP-qPCR. In addition, the claim that Rad21 binds around the TSS along with CTCF is not substantiated by the ChIP presented in 3.2) Do the two described peaks overlap with a CTCF consensus motif?3.3) It is stated in the text that H4K20me3 is reduced upon CTCF knockdown; 3.4) Chromatin modifications are evaluated upon PACER knockdown. Although the manuscript claims “no significant changes in H3K4me2”, there are clear differences in Figure S5 , especially at the important promoter and -2.5 kb sites. Please revise the description of this result.3.5) The conclusion of PACER directly recruiting RNAP II is not supported by the data shown. Instead, the changes in chromatin modification might indirectly influence RNAP II association. The text should be revised to acknowledge this. 1) A better characterization of PACER is needed. Key questions raised by the reviewers are: What is the size, exact sequence, relative abundance and exact intracellular distribution of PACER? Does PACER originate from its own unique promoter, is it a divergent transcript from the COX-2 promoter, or is it an eRNA? To address these issues, the reviewerspropose the following work:1.1) Full sequence data from the RACE experiments should be provided.The full sequence of PACER, as well as sequencing data  for several independent 5’ and 3’ RACE reactions are now provided in 1.2) A Northern blot analysis and/or qPCR analysis should be performed to determine the abundance of PACER relative to COX-2 mRNA.We used qPCR to accurately measure relative amounts of PACER and COX-2 mRNA in HMEC and U937 macrophages. The results for HMEC are depicted in 1.3) A better characterization of the PACER transcription start site is needed. ChIP analysis of RNAPII and chromatin modifications are provided in the manuscript, but these assays are correlative in nature and could not fully elucidate whether PACER originates from its own unique promoter, if it is a divergent transcript from the COX-2 promoter, or if it is an eRNA. This issue can be resolved with promoter reporter assays to define the DNA sequence that drives PACER transcription.We now provide additional pieces of evidence and explanation demonstrating that PACER is a nuclear lncRNA synthesized from its own RNAPII dependent promoter. We performed reporter gene assays which indicate that a separate promoter located ∼0.3-0.5kb can drive expression of PACER independently of the COX-2 mRNA promoter . In addi1.4) Both the RNA and RNAPII ChIP profile suggest a longer transcript than what was cloned by RACE, up to -2 kb. The authors should explain how this matches the suggested 3' end of the RNA at -1022 and show the RACE data rather than justdescribing it. Is there a canonical cleavage and polyadenylation signal around -1022?Indeed, the COX-2 upstream region is transcriptionally active extending to around -2kb, as suggested by our RT-qPCR analyses, RNAPII ChIP and available GRO-SEQ data. However, we believe that PACER is the most abundant well-defined message based on the GRO-SEQ results and considering that we failed to clone additional transcripts further upstream using RACE (data not shown). PACER sequence contains a well conserved polyadenylation signal at position -999  and a we1.5) Strand-specific amplification of the plus strand should include an oligo within the COX-2 gene as a positive control.This control is now included as 1.6)shows the subcellular localization of PACER and COX-2 as determined by cellular fractionation. The authors should perform controls of transcripts present only in the nucleus and only in the cytoplasm to account for cross-contamination of the fractions.1.7) The reviewers wonder whether PACER has been detected by recent genome wide Global Run-On deep sequencing experiments (GRO-seq) done in epithelial cells. Publicly available GRO-seq datasets that could be mined to answer this question include Galbraith MD et al, Cell. 2013 Jun 6;153(6):1327-39 (performed in HCT116 colon cancer cells) and the various papers from the Kraus team using MCF7 breast cancer cells such as Danko CG Mol Cell. 2013 Apr 25;50(2):212-22. GRO-seq results for the COX-2 (PTGS2) locus could be displayed to further illuminate the site of PACER transcription.HIF1A employs CDK8-mediator to stimulate RNAPII elongation in response to hypoxia. Cell, 2013. 153(6): p. 1327-39.]. These signals extend up ∼1100bp upstream, therefore perfectly correlating with the presence of PACER in this region. A genome browser picture incorporating these data is now included as Antisense transcription upstream of the COX-2 transcription start site was indeed detected in HCT116 cells in the recent study . Only upon activation of the NF-κB signaling pathway p65 relocates into the nucleus and activates its target genes . Contrary to this, the activation role of PACER on COX-2 expression is evident both in non-induced macrophages  Additionally, how does PACER knockdown or CTCF knockdown affect p65 binding to the COX2 promoter? Reviewers propose that ChIP assays for p65 be performed to answer this question.Characterization of genome-wide binding of NF-kappaB in TNFalpha-stimulated HeLa cells. Gene, 2013. 526(2): p. 142-9; Lim, C.A., et al., Genome-wide mapping of RELA(p65) binding identifies E2F1 as a transcriptional activator recruited by NF-kappaB upon TLR4 activation. Mol Cell, 2007. 27(4): p. 622-35; Schreiber, J., et al., Coordinated binding of NF-kappaB family members in the response of human cells to lipopolysaccharide. Proc Natl Acad Sci U S A, 2006. 103(15): p. 5899-904; Kaikkonen, M.U., et al., Remodeling of the enhancer landscape during macrophage activation is coupled to enhancer transcription. Mol Cell, 2013. 51(3): p. 310-25]. We attempted to perform similar analyses using the antibodies that were used by other investigators, including, but not limited to, sc-372X from Santa Cruz or Ab7970 from Abcam, all without success. We consistently observed background signals at COX-2, as well as at other regions where p65 binding was also expected, including numerous “positive control” regions that exhibited particularly strong signals for p65 in previous studies . Since our laboratory possesses extensive expertise in ChIP and these types of experiments are performed routinely with success, we were forced to conclude that currently available antibody lots do not permit us to perform p65 ChIP experiments. However, given that the role of p65 in the context of COX-2 expression has been studied extensively  and our knock-down experiments clearly indicate a p65 role in the induction of COX-2 expression, we believe that our experiments provide sufficient support for the model of PACER action.Despite repeated efforts we were not able to demonstrate binding of p65 to the COX-2 locus. We performed these ChIP experiments before the original submission of our manuscript with no success, and repeated again in response to reviewers’ comments. We are well aware that other studies have been performed where ChIP was used to demonstrate p65 binding, including genome-wide studies in mice and humans  These results demonstrate that PACER influences early steps in transcription initiation at the stage of formation of RNAP II preinitiation complexes, likely through p300-induced changes in promoter-associated histone acetylation.” Also, in the Discussion section “Ultimately, the function of PACER lncRNA is to facilitate assembly of RNA Polymerase II pre-initiation complexes. Upon lncRNA knockdown, the levels of elongating RNAP II and initiating S5P-modified RNAP II are significantly lower, thus affecting the ability of the transcription machinery to efficiently initiate transcription of the COX-2 gene. While these effects are indirect, they nevertheless demonstrate that PACER belongs to a novel, unanticipated class of bona-fide transcriptional regulators.”


Kcnq1 domain contains a differentially methylated region (KvDMR) in intron 11 of Kcnq1. The Kcnq1ot1 non-coding RNA emerges from the unmethylated paternal KvDMR in antisense direction, resulting in cis-repression of neighboring genes. The KvDMR encompasses the Kcnq1ot1 promoter, CTCF sites and other DNA elements, whose individual contribution to regulation of the endogenous domain is unknown. We find that paternal inheritance of a deletion of the minimal Kcnq1ot1 promoter derepresses the upstream Cdkn1c gene. Surprisingly, Kcnq1ot1 transcripts continue to emerge from alternative sites, evidence that silencing depends, not on the ncRNA, but on the promoter sequence. Detailed analyses of Kcnq1ot during cardiogenesis show substantial chromatin reorganization coinciding with discontinuous RNA production in both wild-type and mutant mice, with loss of imprinting. We show that CTCF binds to both methylated and unmethylated alleles of the KvDMR. Furthermore, we report a multitude of enhancers within the Kcnq1ot1 region, and present conformational dynamics of a novel heart enhancer engaged in Kcnq1 expression. Our results have important implications on tissue-specific imprinting patterns and how transcriptional mechanisms compete to maximize the expression of vital genes, in addition to shifting our perception on the role of the long ncRNA in regulating this imprinted domain.The imprinted  Kcnq1ot1 has been described historically as a lncRNA of ∼90 kb, emerging from intron 11 of Kcnq1 in the antisense direction  RNAs are produced by the mammalian genome, but few are larger than 10–20 kb. iewed in ,4)  studies in whole embryos and neonatal mouse tissues revealed that expression of Kcnq1 transitions to a biallelic mode after mid-gestation  depromoter . Based oCdkn1c, the closest gene silenced on the paternal chromosome, is also important for regulating proliferation during heart development . This ∼3000 bp region includes elements that control the establishment of Kcnq1ot1 imprinting, although these have not yet been delimited. Two CG islands (CGIs) downstream of the promoter are methylated on the maternal chromosome. This constitutes a primary imprinting mark inherited from the oocyte that inhibits Kcnq1ot1 expression from that allele, allowing maternal expression of the neighboring upstream genes and the sense Kcnq1 gene  in vitro . In contrast to previous studies, this deletion leaves intact the two CTCF binding sites as well as the two CGI . Our data show that, in spite of the absence of the MP, alternative transcripts continued to emerge from alternative sites in the Kcnq1ot1 region. Surprisingly, although the deletion did not ablate Kcnq1ot1 expression, Cdkn1c was no longer monoallelic, suggesting that the residual Kcnq1ot1 transcripts had lost silencing capability or that imprinted Cdkn1c expression required the MP sequence itself.To refine our understanding of the endogenous roles of the sequence elements in the in vitro  was ablaKcnq1ot1 is a single entity or rather a series of overlapping transcripts. Complete elucidation of the transcript structure is required to understand the silencing mechanism attributed to it. Estimates of the length of the Kcnq1ot1 transcript came from reverse transcriptase-polymerase chain reaction (RT-PCR) scans and RNA-sequencing -sequencing data from the Kcnq1ot1 region, we observed many discrete sequences that exhibited features of enhancers or promoters. We reasoned that if there are multiple independent RNAs emerging from the Kcnq1ot1 region, instead of (or in addition to) a 92 kb entity, the transcriptional start sites (TSS) could be identified by existing technologies. By performing targeted 5′ RACE and 3C assays, we show that several such regions are active transcriptionally and at least one is in contact with the Kcnq1 promoter in the heart. Our results completely change previous assumptions on Kcnq1ot1 and lead us to propose that the ncRNA is a by-product of the transcriptional process, which maintains an open chromatin structure allowing access to a variety of enhancers encoded in introns 10 and 11 of Kcnq1. Our experiments also show that deletion of a 200-bp region leads to loss of imprinting of Cdkn1c, strongly suggesting that this sequence is a long-distance silencer. These studies are valuable for understanding the mechanisms that govern imprinting in the context of other co-existing regulatory mechanisms.Unexpected results from our studies raised the question of whether quencing , but theKcnq1ot1-MP was constructed by Genebridges  and modified to add the negative selection marker DTA (Diphtheria toxin A fragment) . The targeting vector was linearized with SalI and electroporated into B6-3 ES cells. G418-resistant clones were isolated and restriction enzyme digestions and Southern blots were used to confirm accurate homologous recombination . Two confirmed targeted clones underwent a second round of electroporation with Flp to delete the neomycin-resistance (neoR) marker, and G418-sensitive clones were identified and confirmed with long-range PCR . Two confirmed neoR-deleted clones were injected into BALB/c mouse blastocysts and chimeric mice were generated.Targeting strategy is illustrated in Supplementary Figure S2A. pTV-Kcnq1ot1 MP was excised by crossing heterozygous mutant mice (+/Kcnq1ot1flox-MP) to mice expressing CMV-Cre recombinase . Excision of the MP was confirmed in the progeny by PCR analysis . Positive heterozygous progeny, designated as +/POKcnq1ot1, were maintained by backcrossing to C57BL/6 or bred to homozygosity.All mouse protocols were approved by the Institutional Animal Care and Use Committee at Temple University Medical School. For each chimeric line, germline transmission of the targeted mutation was confirmed by PCR genotyping of progeny from chimeric mice crossed with C57BL/6 mice on DNA isolated from tail biopsies . Nomenclature indicates the maternal allele first. The Mus musculus castaneus chromosome 7 , mutant mice were crossed to C57BL/6(CAST7), a consomic strain homozygous for a mosome 7 . In all +/POKcnq1ot1 and C57BL/6(CAST7) mice. Noon of the day of the plug was designated E0.5. Pregnant dams were killed at the appropriate day of gestation and fetal tissues were dissected and frozen for further analysis. Neonatal tissues were obtained from postnatal day 2 pups.To obtain embryonic tissues, reciprocal crosses were set up between C57BL/6 and C57BL/6(CAST7) mice, or between RNA was extracted from snap frozen 10.5 dpc, 12.5 dpc, neonatal and adult tissues using the Roche High Pure RNA Kit . An additional DNase step was performed using Invitrogen Turbo DNase . GSP cDNA was generated for each specific qPCR analysis using Invitrogen SuperScript III at 65°C incubation for 50 min, with controls including minus reverse transcriptase and minus primers indicating no contaminating DNA nor non-specific cDNA synthesis. Samples were either used immediately for qPCR or frozen at −20°C. Primers are listed in Supplementary Table S1.β-actin, Ppia and Rplp1, three housekeeping genes that show no expression variation across cardiac developmental stages nor between heart and brain. Statistical analyses were performed using Student's t-test with GraphPad Prism software v6. Results are the average of three independent biological replicates (one mouse from each of three different litters), each performed in triplicate.Immediately following GSP cDNA synthesis, RT-qPCR was performed using 1x Power Sybr Green (Invitrogen) according to manufacturer's protocol with primer-optimized reaction conditions (primers and efficiencies listed in Supplementary Table S1) and according to MIQE guidelines. PCR products were analyzed both by melt curve analysis and polyacrylamide gel electrophoresis. Mean Ct values for each triplicate reaction were normalized to geometric mean of expression values of 5′ and 3′ rapid amplification of cDNA ends (RACE) was performed with the 5′ and 3′ RACE systems (Invitrogen). For 5′ RACE, primers were located downstream of the predicted transcription factor binding sites. For 3′ RACE, primers were anchored at 100, 500 and 1000 bp away from the TSS found by 5′RACE. PCR products amplified according to the manufacturer's recommendation were run on 3% agarose gels and bands were extracted, cloned and sequenced. Sequences were aligned with BLAT in the UCSC genome browser. Primers for 5′ and 3′ RACE assays are listed in Supplementary Table S1.For western blot analyses, total proteins were isolated from mouse neonatal hearts using RIPA buffer . Protein samples (30 μg) were separated on 10% SDS–polyacrylamide gels and subjected to a standard western procedure. Antibodies for Kcnq1 and β-actin were purchased from Santa Cruz , and both were used at a 1:400 dilution. Secondary antibody was horseradish peroxidase conjugated and used at a 1/5000 dilution. Blot was visualized on an Alpha Innotech imaging system and quantified with the Alpha FluorChem software.6 nuclei. Micrococcal nuclease digestion in 1 mM CaCl2 was performed [20 U for 1 min] and the reaction was terminated with 2 mM ethylene glycol tetraacetic acid (EGTA), followed immediately by sonication in a 0.1% SDS–PBS solution. Direct ChIP was performed on the soluble nuclear extract at 4°C overnight using either CTCF antibody  or IgG control antibody . Substrates were pulled down with Invitrogen Protein A Dynabeads. After washing and elution, samples were incubated with Proteinase K overnight at 65°C to reverse cross-links and enriched DNA was purified with phenol–chloroform. Ethanol precipitated DNA was resuspended in 10 mM Tris–HCl solution and analyzed by qPCR using the ActiveMotif qPCR Analysis Kit. Each ChIP assay was performed three times and qPCR results represent the mean ± SEM. ChIP primers are listed in Supplementary Table S1.Tissues were extracted from postnatal day 2 (P2) mice and cross-linked in 0.5% formaldehyde in phosphate buffered saline (PBS) and protease inhibitors for 5 min (heart) or 1% formaldehyde for 10 min (brain) and quenched with 0.125 M glycine. Intact nuclei were isolated from tissue digestions and split so that each subsequent reaction had 5 × 101 ug of DNA was mutagenized using the EZ-DNA Methylation Gold Kit (Zymo Research #5005) following the manufacturer's protocol. Amplification was performed with nested primers (listed in Supplementary Table S1) and PCR products cloned into the pCR2.1-TOPO vector using a TOPO-TA Cloning Kit (Life Technologies#K4500-01). The bacterial colonies were cultured and plasmids purified using a Spin Mini Prep Kit (QIAGEN #30020). Sequencing of the plasmid inserts was done by Eurofins MWG Operon. Sequences were analyzed with Geneious Pro 6.5. Data were obtained from three independent biological samples, with at least 10 clones sequenced from each.http://phenome.jax.org/) between M. musculus castaneus and C57BL/6 genomes were used to determine maternal/paternal contributions with Allele Quantification (AQ) assays. For methylation assays, the ratio of C-to-T at cytosine residues within CpG islands was quantified.Biotin-labeled PCR products were amplified from either cDNA or bisulfite-treated genomic DNA using Qiagen PyroMark Custom Assay Primers and Hot Start Taq Polymerase . The list of PCR primers and sequencing primers is provided in Supplementary Table S1. Pyrosequencing was carried out in duplicate on a PSQ96 system with a Pyro-Gold reagent Kit  and the results were analyzed by PyroMark Q96 ID software version 1.0 (Qiagen). Known polymorphisms (IgG and Input controls with the Hot Start Taq Polymerase Kit (Qiagen #203203) and with PyroMark Custom Assay Primers (listed in Supplementary Table S1). The resulting biotinylated PCR products were bound to Strepavidin Sepharose beads  and pyrosequenced in duplicated on the PSQ96 Pyrosequencing Platform with the Pyro-Gold reagent Kit (Qiagen #972804). Resulting Allele Quantification (AQ) data were analyzed by PyroMark Q96 ID software version 1.0 (Qiagen) and percent maternal/paternal contribution was determined by comparing known polymorphisms between M. musculus castaneus and C57BL/6 mouse strains. Successful enrichment of methylated DNA was confirmed by comparing AQ data from immunoprecipitated samples against comparable IgG and Input controls.Genomic DNA was extracted from snap-frozen hybrid neonatal (P2) tissues using the Quick-gDNA MiniPrep Kit (Zymo Research #D3024). Using the EpiSeeker MeDIP Kit (Abcam #ab117133), 1 ug of the extracted gDNA was then subject to sonication and immunoprecipitation according to manufacturer's protocol. PCR was performed on the methylated DNA enriched samples alongside Tbx5 candidate binding site was amplified from C57BL/6J genomic DNA and subcloned into the Hsp68-LacZ vector. For microinjection, constructs were linearized, gel purified by electroelution and run through the Wizard DNA Clean Up columns (Promega). DNA was injected into male pronuclei of C57BL/6J embryos, and pseudopregnant Swiss Webster females (Taconic) were used as recipients. Transgenic embryos were collected at 11.5 dpc and stained with X-gal (Sigma) for 20 min to detect β-galactosidase activity. Images were obtained with an Olympus MVX10 stereoscope, cropped and levels adjusted with Adobe Photoshop.A 500-bp region spanning 5 sequences, including the Kcnq1, Kcnq1ot1 and Cdkn1c genes. The linear range of the control template was determined with a serial dilution of the random ligation mix made in the same amount (500 ng) of digested and ligated BAC DNA. Primer sequences are listed in Supplementary Table S1. All PCR products were cross-linking and ligation-dependent. PCR products were run on 7% polyacrylamide gels. Intensity of the fragments was quantified on a Typhoon 9200 imager (Molecular Dynamics) and normalized between gels. All data points were generated from three different biological replicates, each performed in triplicate. Relative cross-linking frequency for a fragment X was calculated as: γ(x) = [I(A-X)3C/I(A-X)C]/[I(A-NF)3C/I (A-NF)C, where I(A-X)3C is the intensity of PCR products from ligations between the anchor (A) and another fragment in the Kcnq1ot1 region in the 3C substrate; I(A-X)C is the same for the BAC control template; I(A-NF)3C is the intensity of PCR products from ligations between the anchor and the nearest fragment to the anchor in the 3C substrate; I(A-NF) is the same for the BAC control template. Data were expressed as mean ± SD.Chromosome conformation capture (3C) assays were conducted as previously reported  with theKcnq1ot1), spliced reads from RNA-seq data were used to predict putative spliced isoforms and a Sashimi plot was generated with the Sashimi tool in the Integrative Genomics viewer (RNA-seq data were downloaded from the Genome Browser and imported into the Integrative Genomics Viewer (IGV) ,22. AnnoKcnq1ot1, mice carrying a mutant allele at the endogenous locus were generated by homologous recombination in embryonic stem (ES) cells . This allele, designated as POKcnq1ot1 (Promoter-Out), removes 564 bp of sequence and leaves two CTCF sites and two CG-islands (CGIs) intact. A comparison of the POKcnq1ot1 allele with previously generated deletions is shown in Supplementary Figure S1, illustrating that CTCF sites were either eliminated or partially truncated in those studies. Mutant progeny inheriting the deletion maternally or paternally were assayed for defects in Kcnq1ot1, Cdkn1c and Kcnq1 expression and imprinting, CTCF binding and alterations in chromosome conformation. Parental origin of the alleles was distinguished by crossing heterozygous mutant mice with B6(CAST7) mice, a consomic strain in which chromosome 7 is derived from M. musculus castaneus  showed positive occupancy of CTCF  allele. Primers for CTCF-2, which detect both WT and mutant alleles, were also positive for CTCF occupancy and allele-specific restriction digests of the PCR products showed binding to both alleles . Thus, methylation of the maternal allele does not affect CTCF binding to its cognate sites at the KvDMR. Importantly, CTCF binding is not perturbed by the absence of the MP, making it a good model for testing the role of CTCF in the Kcnq1ot1 region.To confirm this result with a different assay, we quantified allelic presence of Kcnq1ot1 MP on imprinted expression of Cdkn1c, we performed quantitative pyrosequencing of expressed strain-specific single nucleotide polymorphisms (SNPs). Although Cdkn1c was monoallelic in all WT tissues, +/POKcnq1ot1 mice showed biallelic expression of Cdkn1c in every tissue tested , indicating that absence of the MP releases the repression of the paternal Cdkn1c allele. However, the maternally biased expression was maintained, suggesting that repressive epigenetic marks were not completely removed. Surprisingly, despite the biallelic expression pattern, total RNA abundance of Cdkn1c does not increase significantly in the +/POKcnq1ot1 heart and brain, as assayed by qPCR. This implies the existence of a feedback mechanism to limit Cdkn1c levels in those tissues. Cdkn1c maintained a monoallelic pattern in all neonatal tissues from progeny of the reciprocal cross (PO/+Kcnq1ot1) .To determine the effect of deleting the d Figure . PaternaCdkn1c gene lies in a CG-rich region that harbors a secondary methylation mark on the paternal allele (Kcnq1ot1 ncRNA is directly responsible for recruiting DNA-methyltransferases to establish this modification (+/POKcnq1ot1 and PO/POKcnq1ot1 mice by bisulfite mutagenesis and pyrosequencing. The Cdkn1c promoter is unmethylated in sperm from WT and mutant (heterozygous or homozygous) mice. In neonatal heart and brain tissues of the +/POKcnq1ot1 and PO/POKcnq1ot1 mice, the Cdkn1c promoter exhibits complete lack of parental methylation compared to WT mice . In the fetal heart, however, absence of the MP results in only partial loss of imprinted expression, with paternal levels contributing less than 20% of the total RNA abundance in 10.5 dpc hearts, as assayed by pyrosequencing , suggesting that transcription from the MP or the MP sequence itself is not required for full activation of Kcnq1.To determine if the d Figure . ProteinCdkn1c expression in the +/POKcnq1ot1 mouse could be due to absence of the Kcnq1ot1 ncRNA or to an intrinsic regulatory activity of the MP. To distinguish between these two possibilities, we compared the levels of Kcnq1ot1 transcript in neonatal hearts and brains of WT and +/POKcnq1ot1 mice, by performing RT-qPCR with sets of primers that spanned the entire 92 kb length of the RNA , but they were similar to WT levels with primer sets further downstream  , and a trend toward lower expression with the remaining primer sets further downstream  that the sequence previously defined as the MP is not essential for transcription throughout the Kcnq1ot1 region and (iii) that in the heart, there are tissue-specific alternative TSS more than 33 kb downstream of the MP that act independently of it.Loss of imprinted A Figure . Contrar) Figure . On the m Figure . These rKcnq1ot1 in +/POKcnq1ot1 neonatal hearts and brains compared to WT, and to test whether parental contributions were similar throughout the locus, we performed pyrosequencing analysis at 2, 33, 44, 60 and 90 kb downstream of the MP. In the heart, loss of imprinting of Kcnq1ot1 was not observed at 2 kb, but was evident with primers from 33 kb onward. This was true in both in both WT and +/POKcnq1ot1 mice in the cardiac tissue . In contrast, paternal-specific expression was maintained in brain independently of the presence or absence of the MP . Thus, the loss of imprinting of Kcnq1ot is a tissue-specific phenomenon. Activation of maternal allele suggests the presence of alternative cardiac promoters not subject to the imprinted KvDMR.To determine the imprinting status of Kcnq1ot1 locus in neonatal hearts. In the WT mouse , a significantly greater contribution of maternal expression is seen with primers located at 44 kb downstream of the MP, relative to that observed at 2 kb (P < 0.0001). In the +/POKcnq1ot1 hearts , all primer sets showed a higher maternal proportion of total expression relative to 2 kb .We quantified the contributions of the maternal and paternal alleles throughout the Kcnq1ot1 transcripts emerge from alternative start sites (TSS) that bypass the methylated CGIs downstream of the MP. We performed 5′ RACE assays to determine if these sites were active on the maternal allele in +/POKcnq1ot1 neonatal hearts. Our results confirm that the maternal-specific TSS are activated normally in the mutant mice , suggesting that the maternal contributions to Kcnq1ot expression are not captured equally with all primer sets, but depend on their location relative to the alternative TSS.In the WT heart, maternal KvDMR, a CG-rich region that is hypermethylated maternally and methylation-free paternally. To determine if the MP is required to prevent establishment of paternal methylation, we assayed DNA methylation by pyrosequencing in PO/POKcnq1ot1 sperm and +/POKcnq1ot1 neonatal heart and brain at the two CG islands (CGI 1 and 2) , the absence of the MP led to an increase in methylation of the region in neonatal tissues. The higher methylation levels were contributed by an increase in methylation of the paternal allele. Thus, the region remains refractive to de novo but not maintenance DNA methylation.Figure ) Figure . AlthougKvDMR in PO/POKcnq1ot1 oocytes . To validate them, we designed primers spanning the predicted introns and sequenced the PCR products. Reverse transcription and PCR of liver, brain and heart RNAs produced expected product sizes, and sequencing confirmed the presence of three exons  hypothesis to explain the variable levels of the g motifs  and/or eg motifs . Some cart cells  and Oct4ES cells , respectES cells . To testKcnq1ot1 region produces multiple transcripts of varying sizes, emerging from a previously defined MP and at least two additional TSS. Transcripts produced from the MP can undergo splicing, with at least two introns removed from the ncRNA.Thus, the Tbx5 and Oct4 sites could be indicative of enhancer activity. We performed transgenic mouse assays with the Tbx5 region. At E11.5, the Tbx5 sequence exhibited positive β-galactosidase staining in heart and limb buds, consistent with the known regulatory activities of Tbx5    in introhe heart . This ins Figure  and sequKcnq1ot1 region are responsible for imprinted expression of Cdkn1c and, to some degree, of early embryonic Kcnq1. This undermines the current paradigm whereby the Kcnq1ot1 ncRNA is a single molecule that spreads and directly silences its neighboring genes. In the absence of the MP, a sequence that contains the main Kcnq1ot1 promoter, Cdkn1c loses imprinted expression, but the ncRNA continues to be transcribed across the Kcnq1ot1 region from multiple initiation sites. We show that Kcnq1ot1 is not a single molecular entity, but rather encodes a complex population of overlapping RNAs, at least one of which is spliced. We propose that the transcription throughout the Kcnq1ot1 locus in the early stage embryo serves to make regulatory elements available to their protein binding partners, setting the stage for their use later in development. In fact, we identify tissue-specific usage of a cardiac enhancer involving chromatin looping to allow physical proximity to other regulatory elements. This conformation is not present in the early embryo, suggesting it requires specific transcriptional factors to be established. We also propose that as the Kcnq1 promoter and/or other genes contact elements within the Kcnq1ot1 locus, alternative TSS are activated, driving expression of several overlapping ncRNAs.In this report, we provide strong evidence that DNA sequence elements and their relative location in the KvDMR was initially defined as a region of differential methylation between parental alleles in intron 11 of Kcnq1, with a ncRNA emerging from the unmethylated paternal copy in antisense direction to Kcnq1. Several experiments in vivo and in vitro identified a MP, an enhancer-like activity, a silencing element, two CTCF binding sites and several repetitive sequences   ,27,40, egure S1) . TogetheCdkn1c accompanied by secondary methylation at its promoter may still be dependent on the Kcnq1ot1 transcript, as posited by several labs  in neonatal heart, CTCF exhibited binding to the maternal allele despite its hypermethylated status; (ii) the Kcnq1ot1 region was transcribed in spite of the lack of the main promoter. These results led us to re-examine the assumptions on the structure and function of the KvDMR and the ncRNA itself in the WT mouse.We were interested in seeing if removal of the KvDMR, and it was hypothesized that maternal methylation impeded CTCF binding, allowing Cdkn1c access to an upstream enhancer element , mutations in specific CpGs allowed binding of CTCF despite the presence of methylation    with a sKcnq1 expression matches the Tbx5 pattern in the limb bud, or if this particular enhancer is used in the heart for Kcnq1 and engages other distant genes in the limb bud. The expression domain suggested by the conformational assays includes the Cdkn1c gene, raising the question of how cell-cycle inhibition is coordinated with activation of cardiac-specific genes within this domain. Furthermore, it will be interesting to refine the timeline for these regulatory events, since there may be temporal differences between the maternal and paternal alleles due to their distinct chromatin status.One pending question is whether Kcnq1ot1 region vary in molecular structure, abundance and complexity as development ensues. The ∼90 kb Kcnq1ot1 molecule differs from other macroRNAs that act directly by silencing other genes, either by a spreading mechanism , we identified a region in Kcnq1 intron 10 overlapping with the Kcnq1ot1 ncRNA that, upon analysis with rVISTA (Tbx5, Nkx2–5 and MyoD, among other cardiac transcription factors (Figure Kcnq1 is associated with long QT syndrome, it will be interesting to determine whether there are mutations in the non-coding portions of the gene that could be responsible for the cardiac phenotypes that have not been associated with mutations in the protein-coding regions.The human h rVISTA , was pres Figure , stronglKcnq1ot1 region exhibiting features of regulatory elements that need to be tested. We propose that the Kcnq1ot1 region downstream of the KvDMR is a repository of enhancers and that the discontinuous levels of the ncRNA are due to activation of these enhancers by transcription factors. This leads to reorganization of the chromatin fiber, favoring induction of genes necessary for lineage determination at later stages. In the heart, for example, enhancers involved in cardiogenesis are engaged by the Kcnq1 promoter, and this coincides with a maximum decrease in the abundance of Kcnq1ot1 at 12.5 dpc, an up-regulation and loss of imprinting of Kcnq1 and the compression of the whole domain (in vivo, and that this region may be an evolutionary nursery for regulatory elements, as suggested by others (Kcnq1 domain, with many genes active at different stages and in different tissues, insulators are likely to be important to ensure that genes are activated by the right enhancer. Ultimately we need to identify all the regulatory sequences, but also their relative locations, their tissue specificity and their three-dimensional architecture to fully understand their developmental role in vivo. The Kcnq1 region is an excellent model to understand mechanistically how enhancers, insulators, ncRNAs and the target genes are deployed to generate the appropriate expression outputs in the endogenous context.There are a multitude of sequences within the e domain ,51 (Figue domain . We alsoy others . In a reSupplementary Data are available at NAR Online.


Plasmodium falciparum tightly orchestrates the rise and fall of transcript levels for hundreds of genes. Considerable debate has focused on the relative importance of transcriptional versus post-transcriptional processes in the regulation of transcript levels. Enzymatically active forms of RNAPII in other organisms have been associated with phosphorylation on the serines at positions 2 and 5 of the heptad repeats within the C-terminal domain (CTD) of RNAPII. We reasoned that insight into the contribution of transcriptional mechanisms to gene expression in P. falciparum could be obtained by comparing the presence of enzymatically active forms of RNAPII at multiple genes with the abundance of their associated transcripts.Over the course of its intraerythrocytic developmental cycle (IDC), the malaria parasite P. falciparum genes across the IDC. We raised highly specific monoclonal antibodies against three forms of the parasite CTD, namely unphosphorylated, Ser5-P and Ser2/5-P, and used these in ChIP-on-chip type experiments to map the genome-wide occupancy of RNAPII. Our data reveal that the IDC is divided into early and late phases of RNAPII occupancy evident from simple bi-phasic RNAPII binding profiles. By comparison to mRNA abundance, we identified sub-sets of genes with high occupancy by enzymatically active forms of RNAPII and relatively low transcript levels and vice versa. We further show that the presence of active and repressive histone modifications correlates with RNAPII occupancy over the IDC.We exploited the phosphorylation state of the CTD to detect enzymatically active forms of RNAPII at most The simple early/late occupancy by RNAPII cannot account for the complex dynamics of mRNA accumulation over the IDC, suggesting a major role for mechanisms acting downstream of RNAPII occupancy in the control of gene expression in this parasite.The online version of this article (doi:10.1186/1471-2164-15-959) contains supplementary material, which is available to authorized users. Plasmodium falciparum undergoes a 48 h life cycle from the moment of red blood cell invasion through to the production and release of mature progeny. In the course of this intraerythrocytic developmental cycle (IDC), the mRNA level for many genes rises and falls once at a point that correlates with the time its protein product is needed. Such results have led to the proposal of a “just in time” model of plasmodial gene expression in which mRNAs accumulate just as their products are required during the IDC [Plasmodium gene expression at the level of transcription has been demonstrated [P. falciparum shows a widespread chromatin opening and histone H2A.Z recruitment in the intergenic regions throughout the IDC. Although this modification has been associated with actively transcribed chromatin in other species, in Plasmodium falciparum this histone variant is also recruited early to genes whose transcripts do not appear until much later [Plasmodium genes regardless of corresponding transcript abundance [The malaria parasite  the IDC  Many facnstrated –5, severch later –8. A simbundance . Moreovebundance  and consbundance . Additiobundance . Last, pbundance . Such fiSaccharomyces cerevisiae. Moreover, the number and sequence of repeats is highly invariant within and even between species. By contrast, the CTD of Plasmodium spp. differs by the inclusion of a lysine at position 7 of the heptad repeat (YSPTSPK), contains fewer repeats and shows much greater variability in repeat number between and within Plasmodium species [During transcription, many steps of mRNA synthesis and processing are integrated through the C-terminal domain (CTD) of the largest subunit of RNAPII (RPB1). A hallmark of RPB1 in most eukaryotes is the presence of a repeating heptapeptide motif in the CTD . In most species , 16.Among other modifications, the serine residues at positions 2 and 5 (and 7 in many organisms), can be phosphorylated and intensive effort has gone into trying to understand the role of these modifications in gene expression. Much attention has focused on the functional consequences of an unphosphorylated CTD, mono-phosphorylation at position 5 (Ser5-P), and di-phosphorylation at positions 2 and 5 (Ser2/5-P). A long-held model proposes that the enzymatic activity of RNAPII is determined by the phosphorylation status of the CTD. In this model, RNAPII bearing a hypophosphorylated CTD is enzymatically inert, while Ser5-P is required for RNAPII to initiate transcription, and Ser2/5-P then confers the elongating and highly processive activity of RNAPII . HoweverP. falciparum genes with the transcriptional machinery across the IDC. This allowed us to assess the extent to which RNAPII occupancy correlates with the mRNA accumulation. Our data indicate that P. falciparum genes are divided into two classes depending on whether peak RNAPII binding occurs early or late during the IDC. When comparing RNAPII occupancy to mRNA abundance, we identified subsets of genes with high occupancy and relatively low RNA levels and vice versa. We find that genes at which RNAPII is detected early (8 to 32 hours post-infection (hpi)) are shorter in length, have more introns and shorter first exons when compared to genes at which RNAPII is detected late (40-48 hpi). Furthermore, we find distinct RNAPII occupancy patterns for the early and late genes along the gene length. Comparison of RNAPII occupancy with various histone modifications identifies chromatin changes that precede, accompany and follow RNAPII recruitment. Together our results show that occupancy by presumed active forms of RNAPII cannot account for the highly orchestrated pattern of mRNA accumulation and loss over the IDC. We suggest that post-initiation mechanisms such as transcriptional elongation and mRNA stability may play predominant roles in determining the dynamics of mRNA abundance during infection of red blood cells.We have exploited the phosphorylation state of the RNAPII CTD to assess the engagement of most P. falciparum RPB1 CTD: unmodified CTD, Ser5-P-bearing CTD, and CTD having the Ser2/5-P double phosphorylation. The phosphorylated forms of RPB1 have been associated with RNAPII molecules that are functionally engaged with bound genes. Unphosphorylated heptad repeats may be associated with inert forms of the polymerase, but could also be present as one or more copies within a CTD that also contains phosphorylated motifs. The specificity of the antibodies was confirmed in several ways. First, the antibodies showed high specificity in ELISA by serial dilution (data not shown) and, second, by competition with the immunizing peptide in a peptide competition assay .Steps in the generation and use of monoclonal antibodies in this study are summarized in Figure P. falciparum anti-RNAPII antibodies in ChIP-on-chip analysis to map the position and enzymatic status of RNAPII across the entire P. falciparum genome over the 48 h of the IDC at 8 h intervals. The commercial anti-CTD antibody, 8WG16, was also used in parallel. We used custom-made microarrays spotted with 70-mer oligonucleotides covering both coding and intergenic regions [We next used these  regions , 20.p <0.05 and ≥1.5 fold change across the life cycle. This cut-off resulted in the retention of a subset of probes for each of the three antibodies: 2,936 probes with the αCTD (unphosphorylated) antibody; 4,462 probes with the αSer5-P antibody; and 4,255 probes with the αSer2/5-P antibody. We addressed the temporal order within the dataset by creating a phaseogram of RNAPII occupancy. The genome-wide profiles reveal a striking periodicity involving the majority of genes  form of RNAPII early in the IDC and four that bind late. Although qPCR reveals a greater dynamic range than the microarray analysis, the results show that all early genes acquire peak RNAPII recruitment early in the IDC at either time point 3 or 4 (24 and 32 hpi), while late genes do not display maximal RNAPII binding until time point 6 (48 hpi)  between the dynamic RNAPII occupancy (p <0.05 and 1.5 fold change) and the expression levels of corresponding mRNAs. We established two subsets Figure . In one P. falciparum chromosomes. All chromosomes were seen to bear potential clusters  to the difference in RNAPII distribution  between early and late binders , Kyoto Encyclopedia of Genes and Genomes (KEGG) and Malaria Parasite Metabolic Pathway (MPMP) Figure D. We fouP. falciparum[Our group and others have shown that chromatin influences gene expression in lciparum, 28–34. lciparum. We idenlciparum, 31, 33,To determine whether some histone modifications may precede or follow RNAPII recruitment, we calculated the PCC (r) between RNAPII occupancy  and histone modification occurring 8 h before or after recruitment Figure . For exaP. falciparum RBP1 CTD in addition to 8WG16, a well-characterized commercial monoclonal antibody raised against wheat germ RNAPII and likewise recognizing the CTD [Plasmodium life cycle is divided into rather stark early vs. late stages defined by a component of the transcriptional machinery.In this study, we performed genome-wide mapping of RNAPII occupancy over the 48 h of the IDC using a ChIP-on-chip approach. We used three highly specific monoclonal antibodies against different forms of the  the CTD , 39. Allin vitro[. In addition, the loss of Ser2 phosphorylation does not affect the transcription of the majority of genes in fission yeast, while the budding yeast kinase Kin28p is not required for transcription but rather to couple transcription initiation to the capping process [per se, but rather to bind and coordinate the activity of proteins exerting co-transcriptional functions such as capping and splicing [Plasmodium species suggests that heptad repeats were first lost in Plasmodium spp and then re-acquired in primate-infecting lineages [P. falciparum play roles entirely unrelated to those of their counterparts in other species, or at least unrelated to transcriptional processes. This seems unlikely for several reasons. First, P. falciparum encodes an almost complete set (13 of 14) of so-called core CTD-interacting proteins with major transcriptional functions [P. falciparum encodes a clear homolog of the yeast Ctk1 kinase in part responsible for Ser2 phosphorylation (PF3D7_0417800) [P. falciparum CTD are phosphorylated in vivo. Fourth, an alternative register and alignment of the functional heptad unit suggests that basal Plasmodium species may harbor more units of full and partial heptads than previously appreciated [P. falciparum CTD may have also acquired novel functions and interaction partners.Considerable controversy has surrounded the role of the CTD heptad repeats and their phospho-serines. While the heptad repeats are necessary for the survival of yeast and human cells, they are dispensable for transcription in vitro. In add process . These asplicing . Apicomplineages , 16. Thiunctions . Second,n PF3D7_07800 [41,0417800)  and anotreciated , suggestAt a minimum for the interpretation of our results, it seems reasonable to expect that the presence of phosphorylated serines in the CTD is indicative of RNAPII that is actively engaged in transcription. We find a similar distribution of RNAPII bearing unmodified and phosphorylated heptads. Similar observations have been made by several groups and can be explained simply by the presence of one or more unmodified heptads in the company of other phosphorylated repeats within a CTD . Becausefollow RNAPII recruitment [P. falciparum are primed for re-initiation by a TBP- and TFIIE-containing scaffold, ready for RNAPII recruitment in response to appropriate cues. Two of the genes analyzed for TBP and TFIIE binding are included in our data set. msp1 (PFI1475w/ PF3D7_0930300) and rhoph3 (PFI0265c/ PF3D7_0905400) bind TBP and TFIIE at both ring and trophozoite stages [msp1 and the presence of the Ser5-P form at rhop3 is not observed until T4 (trophozoite stage). Both genes then associate with the Ser2/5-P form by T5. Thus, the appearance of enzymatically active forms of RNAPII follows that of TBP and TFIIE, at least in these instances. This is consistent with the presence of TBP and TFIIE in a scaffold-like complex ready for efficient RNAPII recruitment when the time is right.It may be particularly insightful to compare our results with those of two previous studies. One report used the nuclear run-on assay to assess the engagement of RNAPII at several genes across the IDC . Strikinruitment . Howeverruitment . It is tP. falciparum at the point of transcriptional elongation as has been well documented in other eukaryotes [Our data show that RNAPII occupancy and the corresponding mRNA levels of many genes are well correlated, suggesting that the control of transcription is often a determinant of gene expression. Surprisingly, however, a large number of genes achieve significant mRNA levels only several hours following their peak RNAPII Ser2/5-P occupancy (early/negative and late/negative), suggesting that transcription is not a rate-limiting step at these loci. One possibility is that RNAPII is “pre-loaded” onto such genes but kept in a paused (or poised) state. Although RNAPII levels subsequently fall as mRNA levels rise, this may be due to the greater transcriptional activity of fewer polymerase molecules. This would then place a major regulatory control in karyotes . If so, karyotes . Non-exckaryotes .Schizosaccharomyces pombe[The highly similar distribution along the gene length of all forms of the polymerase studied here may be consistent with this notion. On the other hand, the apparent distribution of CTD isoforms along the gene in other organisms varies greatly depending on the antibody in use and the presence/absence of modifications adjacent to the epitope (summarized and extended in ). From tes pombe.P. falciparum.The division of the IDC into early and late phases as evidenced by RNAPII occupancy profiles is paralleled by the binary changes (open/closed) in chromatin structure that occur during the parasite’s life cycle  whereby P. falciparum.We previously carried out genome-wide mapping of 13 histone modifications and correlated their presence at particular genes with transcript accumulation over the IDC. In particular, H4K8ac was tightly linked to mRNA levels and accompanied a promoter inserted at an ectopic site in the genome . Here, wversus the highly orchestrated rise and fall of their transcripts across the IDC suggests that mechanisms downstream of transcriptional initiation may play a predominant role in the control of gene expression in P. falciparum. The recent demonstration that the negative regulation of a var gene subgroup in P. falciparum is mediated by a transcriptionally coupled RNase is consistent with this notion and may have broader implications for gene expression in this species more generally [The striking contrast between the relatively simple pattern of RNAPII recruitment (early vs late) to approximately two thirds of genes enerally . Limitedenerally . A bias This research involved the use of human blood drawn from healthy volunteers by trained healthcare professionals and was approved by the NTU Institutional Review Board and assigned the IRB approval number IRB-2013-07-020. Informed consent was obtained and documented by the signing of an approved informed consent form.P falciparum RNAPII CTD and bearing (1) no phosphate (CTD)- Biot-Ahx-YSPTSPKYSPTSPK and CYSPTSPKYSPTSPK (2) a phosphate at Ser5 (Ser5-P)-Biot-Ahx-YSPTSPKYSPTSPK and CYSPTSPKYSPTSPK or (3) phosphates on Ser2 and Ser5 (Ser2/5-P)- Biot-Ahx-YSPTSPK and CYSPTSPK were used to immunize BALB/c mice. Serine residues in bold typeface were phosphorylated. All three peptides were coupled with Keyhole Limpet Haemocyanin (KLH) using a Imject® Maleimide Activated mcKLH kit. The immunization schedule included a first subcutaneous injection of KLH-coupled peptide resuspended in complete Freund’s adjuvant  followed by a second subcutaneous injection in incomplete Freund’s adjuvant  6 weeks later. Eight weeks after the first immunization, mice were sacrificed and draining lymph nodes collected. Isolated lymph node cells were fused with SP2/0 myeloma cells using polyethylene glycol  according to standard fusion protocols [via a GammaBind Plus Sepharose .Peptides based on the heptad repeat of the rotocols . Cells wPhosphorylated and non-phoshorylated peptides were incubated in a range of concentrations with monoclonal antibodies α-CTD, α-Ser5-P and α-Ser2/5-P at room temperature for 30 min. The peptide antibody mix was then tested for reactivity in an ELISA .Parasite nuclear protein extract was prepared by high salt. The nuclear extract was further diluted, pre-cleared and incubated with RNAPII antibodies overnight at 4°C. Protein complexes were precipitated by incubating with Protein G agarose slurry for 1 h at 4°C followed by extensive washings to reduce the background. Protein complexes were eluted by addition of SDS-PAGE loading buffer and boiling for 5 min at 95°C. Supernatants were resolved in 6% SDS-PAGE and subsequently used for Western blot analysis. Western blots were carried out using antibodies raised against the CTD of RNAPII described in this study and the anti-CTD antibody 8WG16 (Covance) . SecondaBriefly, after immunoprecipitation, the eluent was separated on a SDS-PAGE gel, and silver stained. The gel lane was cut into 5 slices and then processed by a standard in-gel digestion protocol using trypsin. Extracted tryptic peptides of each slice was injected into a LC-MS/MS system  for peptide sequencing using tandem MS. The MS/MS spectra of the raw data was extracted and converted into dta files using extract_msn and then combined into a mascot generic file using an in-house program as previously described .P. falciparum protein database  was used for database searches [The Plasmodium falciparum T996 strain parasites were cultured as described previously [eviously . Highly The chromatin immunoprecipitation assay was performed as described . BrieflyFor transcriptional profiling by microarray total RNA from all the six time points was isolated using the Trizol method and cDNA was synthesized by reverse transcription, purified and labeled as previously described . SamplesP. falciparum genome. These microarrays consisted of14,981 probes, containing 3895 50-mer intergenic oligonucleotide probes and 10,566 70-mer ORF probes covering most of the 5,283 protein-coding genes [P. falciparum genomic database (PlasmoDB) [p were assigned to each spot based on comparing the triplicates of summit and valley time points. Significantly oscillated profiles between the summit and valley time points were defined as p <0.05 and fold change ≥1.5 across the IDC. For ChIP-chip experiments using the monoclonal antibody 8WG16 exceptionally, microarrays were performed twice and only signals showing ≥1.5 change across the IDC in both arrays were included for analysis.Equal amounts of Cy5 and Cy3 labeled probes were used to probe the microarrays representing the ng genes . On averlasmoDB) . The miclasmoDB) . BrieflylasmoDB) ) and follasmoDB)  between p <0.05 using a chi-square test.Pearson’s Correlation Coefficient (PCC or r) was calculated between the RNAPII binding profile and their corresponding mRNA profiles. To compare the distribution of RNAPII along the gene body on the chromosome, histograms were plotted to display the binding positions with 500 bp intervals from -2 kb to 5 kb around coding start site (ATG). A goodness-of-fit test was performed within each bin to calculate the probability of that region being equally bound by RNAPII for different gene groups. Regions with binding bias by group were assigned at PCC (or “r”) was calculated between the RNAPII binding profiles and corresponding histone mark profiles. Modified skewness value (S) was also calculated to measure the asymmetry of correlation distribution. Strictly, a negative S value indicates that the distribution is biased toward a positive correlation. However, for convenience, we have taken the negative of the S value such that a positive S value now represents a distribution biased to a positive correlation. Likewise, a negative S value now represents a bias toward negative correlation.p <0.0005 and S >0.05 and which showed clear positive correlation with Ser2/5-P RNAPII binding.To test how statistically significant the RNAPII levels correlated with a particular histone modification level across IDC, we generated a random dataset which contained ~20 k randomly paired profiles of RNAPII and histone modifications and a two-sample Kolmogorov-Smirnov test was applied to compare the observed correlations and the randomized correlations. In addition, a two-sample Kolmogorov-Smirnov test was also applied to each pair of data to test whether the function of correlations between RNAPII occupancies and histone modifications was less or greater than the function of similar correlations after one time point shift forward or backward in RNAPII profile. In addition, we used the degree of skewness (S) of the PCC distributions as a means of identifying histone marks with maximum probe density showing positive correlation with RNAPII occupancy. We identified 5 histone marks including H3K9ac, H3K56ac, H4ac4, H4K12ac and H4K20me1 with qRT-PCR experiments were performed on immunoprecipiated and input DNA using the SYBR Green PCR master mix (Kappa SYBR Fast Master Mix) according to manufacturer’s instructions. All reactions were run in triplicate. Product-specific amplification was confirmed by performing melting curves for each reaction. For each gene the fold change across the time points was calculated by calculating the difference in the Ct value for each time point.All the primers used in the current study are listed (A) Heat maps showing phaseogram and hierarchical clustering of loci bound by RNAPII. (B) A comparable analysis to those presented in Figure r ≥0.4) while a distinct third is strongly negatively correlated (r ≤ -0.4). (TIFF 818 KB)Additional file 2: Figure S1: Results of ChIP-chip analysis using the commercial anti-CTD monoclonal antibody 8WG16. ChIP-chip was performed in duplicate and only probes with signals showing a fold change ≥1.5 across the life cycle in both experiments were included for analysis. Additional file 3: Figure S2: A hierarchical clustering of the microarray data presented in Figure Additional file 4: Figure S3: A representative example of the chromosomal distribution of genes identified in this study that bind RNAPII either early or late in the infectious cycle. Chromosomal projection of RNAPII Ser2/5-P occupancy for early and late gene groups onto chromosomes 3 (MAL3) and 4 (MAL4). The vertical bars (green) indicate probe position for each gene group. Gene clusters can be seen in red, the results for a “late” cluster on chromosome 4 is shown. Vertical bars denote the positions of late genes, and the bars in red indicate a cluster of 6 such genes spanning a mere 30 kb . (TIFF 242 KB)(A) gene length, (B) percentage GC content, (C) intron length (intron/exon ratio) and (D) length of the first exon for the early and late genes. 1, early/negative, 2, late/negative, 3, early/positive and 4, late/positive. p value for the two group comparisons is calculated using Wilcoxon two-tailed test and the p-value for the four group comparison is calculated using “Kruskal-Wallis test”. (TIFF 1 MB)Additional file 5: Figure S4: An analysis of the genomic features  in the four classes of genes identified in this study. Distinct genomic properties of early vs late genes. Box plots show the distribution of (A) Correlations with histone H3 modifications. (B) Correlations with histone H4 modifications. Correlation distributions were plotted as histograms for each histone modification, with bins of PCC ranging from -1 to +1 on x-axis plotted against the total number of probes falling into each bin on the y-axis. A two-sample Kolmogorov-Smirnov test was applied to each pair of data to test whether the function of correlations between RNAPII occupancies and histone modifications was less or greater than the function of similar correlations after one time point shift forward or backward in RNAPII profile. The statistical significance (p) of the correlation after the shift can be seen at the bottom of each graph. Control: correlations between RNAPII binding and histone modification within a single time point (e.g. T2). Shift HM -8 h: Comparison of RNAPII binding to histone modification (HM) in place 8 hours earlier. Shift HM +8 h: Comparison of RNAPII binding to histone modification (HM) in place 8 hours later. (TIFF 2 MB)Additional file 6: Figure S5: Distribution of correlation between the binding of the unmodified-CTD form of RNAPII and the occupancy profiles of 13 histone modifications assessed before during and after RNAPII binding. Distribution of correlation between the binding of the unmodified-CTD form of RNAPII and the occupancy profiles of 13 histone modifications assessed at 8 h earlier (-8 h) or later (+8 h) than RNAPII binding. PCC was calculated between RNAPII profiles for all the probes with p <0.05 and ≥1.5 fold change and the corresponding histone mark after one-time point shift. For example, RNAPII binding at T2 was correlated to histone marks in place at T1 (-8 h) and T3 (+8 h). (A) Correlations with histone H3 modifications. (B) Correlations with histone H4 modifications. Correlation distributions were plotted as histograms for each histone modification, with bins of PCC ranging from -1 to +1 on x-axis plotted against the total number of probes falling into each bin on the y-axis. A two-sample Kolmogorov-Smirnov test was applied to each pair of data to test whether the function of correlations between RNAPII occupancies and histone modifications was less or greater than the function of similar correlations after one time point shift forward or backward in RNAPII profile. The statistical significance (p) of the correlation after the shift can be seen at the bottom of each graph. Control: correlations between RNAPII binding and histone modification within a single time point (e.g. T2). Shift HM -8 h: Comparison of RNAPII binding to histone modification (HM) in place 8 hours earlier. Shift HM +8 h: Comparison of RNAPII binding to histone modification (HM) in place 8 hours later. (TIFF 1 MB)Additional file 7: Figure S6: Distribution of correlation between the binding of the Ser5-P form of RNAPII and the occupancy profiles of 13 histone modifications assessed before during and after RNAPII binding. Distribution of correlation between the binding of the Ser5-P form of RNAPII and the occupancy profiles of 13 histone modifications assessed at 8 h earlier (-8 h) or later (+8 h) than RNAPII binding. PCC was calculated between RNAPII profiles for all the probes with p <0.05 and ≥1.5 fold change and the corresponding histone mark after one-time point shift. For example, RNAPII binding at T2 was correlated to histone marks in place at T1 (-8 h) and T3 (+8 h). (A) Correlations with histone H3 modifications. (B) Correlations with histone H4 modifications. Correlation distributions were plotted as histograms for each histone modification, with bins of PCC ranging from -1 to +1 on x-axis plotted against the total number of probes falling into each bin on the y-axis. A two-sample Kolmogorov-Smirnov test was applied to each pair of data to test whether the function of correlations between RNAPII occupancies and histone modifications was less or greater than the function of similar correlations after one time point shift forward or backward in RNAPII profile. The statistical significance (p) of the correlation after the shift can be seen at the bottom of each graph. Control: correlations between RNAPII binding and histone modification within a single time point (e.g. T2). Shift HM -8 h: Comparison of RNAPII binding to histone modification (HM) in place 8 hours earlier. Shift HM +8 h: Comparison of RNAPII binding to histone modification (HM) in place 8 hours later. (TIFF 1 MB)Additional file 8: Figure S7: Distribution of correlation between the binding of the Ser2/5-P form of RNAPII and the occupancy profiles of 13 histone modifications assessed before during and after RNAPII binding. Distribution of correlation between the binding of the Ser2/5-P form of RNAPII and the occupancy profiles of 13 histone modifications assessed at 8 h earlier (-8 h) or later (+8 h) than RNAPII binding. PCC was calculated between RNAPII profiles for all the probes with p <0.05 and ≥1.5 fold change and the corresponding histone mark after one-time point shift. For example, RNAPII binding at T2 was correlated to histone marks in place at T1 (-8 h) and T3 (+8 h). Additional file 9: Table S2: Oligonucleotides used in this study. (DOCX 25 KB)


Sigma (σ) factors are the predominant constituents of transcription regulation in bacteria. σ Factors recruit the core RNA polymerase to recognize promoters with specific DNA sequences. Recently, engineering of transcriptional regulators has become a significant tool for strain engineering. The present review summarizes the recent advances in σ factor based engineering or synthetic design. The manipulation of σ factors presents insights into the bacterial stress tolerance and metabolite productivity. We envision more synthetic design based on σ factors that can be used to tune the regulatory network of bacteria. While, one or more alternative σ factors control the transcriptional initiation of a subset of genes with shared functions that varies between species. For example, an intracellular pathogen Mycoplasma genitalium encodes 1 σ factor, E. coli encodes 7 σ factors, B. subtilis has 18 σ factors, Pseudomonas putida encodes 24 different σ factors, and the soil bacterium Streptomyces coelicolor contains 65 σ factors including 53 alternative σ factors  belong to the σ70 family   encoded by the rpoH gene, which regulates the transcription of heat shock genes  that regulates cell envelope response and σFecI (FecI) controlling the iron citrate transport system, respectively  regulates the expression of the genes essential for synthesis of the exopolysaccharide alginate, which is responsible for biofilm formation and provides the bacterium better fitness against environmental stresses. AlgU is structurally similar to σE and recognizes similar promoter sequences  , high pressure, high salt concentration, toxic metals, and increased radiation  is found in Vibrio parahaemolyticus, a Gram-negative bacterium inhabiting coastal waters. The rpoS deletion mutant strain had significantly reduced survival under acid stress conditions, suggesting its role in cell survival under stress conditions, such as oxidative stress and exposure to acid , an ECF σ ortholog Sig1, and a third putative ECF σ ortholog Sig2. Sig 1 was found to play a major role in the regulation of heat shock genes for survival against heat and ethanol stresses, while Sig2 likely controls the expression of a smaller set of heat shock genes. Other alternative σ factors such as σS, σH, and σN were not found in D. radiodurans  was applied to Actinoplanes teichomyceticus L-27 was recently demonstrated. For this strain, the exact principal sigma factors σHrdB are not well understood, but the actinomyces genera shares high similarity in σHrdB amino acid sequences. Thus, three σHrdB genes from A. missouriensis 431, Micromonospora aurantiaca ATCC27029, and Salinispora arenicola CNS-205 were rationally selected and engineered by random mutagenesis, DNA shuffling, and point mutation, and the resulting library transferred in A. teichomyceticus L-27. Screening yielded a recombinant strain with a twofold increase in teicoplanin production in a pilot scale fermentation (5.3 mg L−1). The mutants showed significant diversity in the region 1.1 of σHrdB, apparently generated by DNA shuffling  enhanced the tricarboxylic acid cycle and the glyoxylate shunt , which theoretically stimulated the production of the common precursor 2-ketoisovalerate. The mutant strain NV3 produced an improved isobutanol level of about 8 g L−1 in comparison to the wild-type strain with 5 g L−1 in 24 h. A truncation mutation was found in the rpoS gene of the mutant strain. Repair of this gene further elevated the yield of isobutanol to 21.2 g L−1 in 99 h  can disrupt this loop and overexpression of these sRNAs increased acid tolerance supra-additively up to 8500-fold during active cell growth . The engineered strain produced PHB up to 85.8% of cell dry weight in a glucose medium without the addition of any additional inducer  production by placing the synthesis genes under the control of the 5′ untranslated region and the promoter of Synechocystis sp. PCC 6803, overexpression of sigE enhanced the transcription of genes for the oxidative pentose phosphate pathway and also for glycogen catabolism, and altered the metabolite levels  of the TCA cycle  in Thermoanaerobacter sp. X514. Long-term exposure to ethanol caused the ethanol sensitive wild-type to develop a low ethanol tolerance of 2% (XI) and subsequently a higher tolerance of 6% (XII). Genomic and transcriptomic analyses identified an iron containing alcohol dehydrogenase (ADH) and the ECF σ24 as the key factors for improved tolerance for XI and XII, respectively. The adh over-expressing strain showed a 33% improvement in the ethanol productivity over the control, and a 31.8-fold enhanced growth under 1% ethanol. Under the same condition, ethanol tolerance was improved by 102-fold by over-expressing σ24, with a 21% higher ethanol productivity than the control Re-design of the different domains of σ factors, including the helix-turn-helix (HTH)-mediated DNA binding motifs. This is in part demonstrated by the pioneering work of Rhodius et al. . Design (2)Editing the promoter sequences for σ factors. Engineered promoters of different strength for both exponential and stationary phases have been available Rewiring of the regulatory network for σ factors, which are part of the cellular transcriptional regulatory network that is interconnected, multi-layered, and hierarchical Construction of sRNA molecules or sRNA circuits that regulate the translation of σ factors, as shown by Jin and Huang . Na et a(5)P. putida enhanced thermo-tolerance and ethanol tolerance in E. coli, and the groESL from the thermophilic Thermoanaerobacter tengcongensis provided better tolerance toward corn cob hydrolyzates and better productivity for Clostridium acetobutylicum (Luan et al., Finally, utilization of extremophilic σ factors in mesophilic organisms, which has not been explored so far. Along this line, it is interesting to note that the GroESL chaperonins from the solvent-tolerant 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.