Teladorsagia circumcincta. Infection over 12 weeks identified susceptible  and resistant sheep . Resistance is mediated largely by a Th2 response and IgA and IgE antibodies, and is a heritable characteristic. The polarization of T cells and the development of appropriate immune responses is controlled by the master regulators, T-bet (TBX21), GATA-3 (GATA3), RORγt (RORC2) and RORα (RORA); and several inflammatory diseases of humans and mice are associated with allelic or transcript variants of these transcription factors. This study tested the hypothesis that resistance of sheep to T. circumcincta is associated with variations in the structure, sequence or expression levels of individual master regulator transcripts. We have identified and sequenced one variant of sheep TBX21, two variants of GATA3 and RORC2 and five variants of RORA from lymph node mRNA. Relative RT-qPCR analysis showed that TBX21, GATA3 and RORC2 were not significantly differentially-expressed between the nine most resistant  and the nine most susceptible sheep . Absolute RT-qPCR on all 45 animals identified RORVv5 as being significantly differentially-expressed (p = 0.038) between resistant, intermediate and susceptible groups; RORCv2 was not differentially-expressed (p = 0.77). Spearman’s rank analysis showed that RORAv5 transcript copy number was significantly negatively correlated with parameters of susceptibility, AWC and FEC; and was positively correlated with BW. RORCv2 was not correlated with AWC, FEC or BW but was significantly negatively correlated with IgA antibody levels. This study identifies the full length RORA variant (RORAv5) as important in controlling the protective immune response to T. circumcincta infection in sheep.This study used selected lambs that varied in their resistance to the gastrointestinal parasite  Teladorsagia circumcincta is a major cause of sheep parasitic gastroenteritis [The abomasal strongylid nteritis , 2. The nteritis , but mannteritis  through nteritis –7. The anteritis , 8, 9 annteritis .T. circumcincta or the related parasite Haemonchus contortus [The candidate gene approach for the identification of molecular markers for selection aims to evaluate the relationship between phenotype and a variation in a gene . Severalontortus –14. HoweT. circumcincta [This current study exploited parasite-naïve Blackface lambs with diversity in their predicted genetic resistance to umcincta . Both imumcincta  and micrumcincta  of ALN lumcincta  associatumcincta . Uncontrumcincta . ConsequTBX21) promotes Th1 differentiation by transactivating IFNG and increasing IFNγ production, and by repressing Th2 activation [GATA3) regulates Th2 development [IL4, IL5 and IL13) and inducing their expression; it also inhibits Th1 development [RORC2) [RORA) [T. circumcincta-infected sheep [FOXP3 in resistant and susceptible sheep.The multiple effector functions of CD4 T cells are achieved by the differentiation of multipotential precursors into distinct polarized subsets, which is largely regulated by the master regulators T-bet, GATA-3 and RORγt; transcription factors that transactivate the genes and mediate the subset-specific functions . T-bet  . Maximum) [RORA) . RORα is) [RORA)  subset o) [RORA)  in mice.) [RORA) , howevered sheep  has showGATA3 gene variants and deletion mutants have been linked to a number of other inflammatory pathologies, including asthma and IgE-mediated allergy [In addition to controlling T cell differentiation, all four transcription factors contribute to the pathogenesis of chronic inflammatory diseases. T-bet plays a role in the abnormal expression of Th1 cytokines in human Crohn’s disease  and GATA allergy , 34. The allergy . Consequ allergy , 37.T. circumcincta.In this study we characterise the different transcript variants of the four master regulators expressed in sheep and then compare the expression of these individual variants in animals of defined resistance status. Finally we quantify expression levels of variants to enable their correlation with quantitative phenotypes of resistance to T. circumcincta larvae three times a week for 12 weeks. At post mortem, two days after the last infection, the abomasal AWC ranged from 0 to 11300 and FEC from 0–950 eggs per g , from a flock previously used for quantitative genetic and QTL analyses , were hogs per g . The lam2 each and the uninfected lambs were in a pen of approximately 50 m2. All animals were bedded on clean straw and had ad libitum access to hay and water supplemented twice daily with Maize Lamb Pellets . Animals were examined at least daily. All lambs were vaccinated with Heptavac P Plus at 5 and 6 weeks and Scabivax  at 6 weeks. Most lambs were administered 2 ml Hexasol  to treat respiratory infections. Infected animals showing mild symptoms of visceral pain associated with parasite infection were treated, under veterinary instructions, with 2 ml Finadyne . Lambs were killed by intravenous administration of Euthetal .Animals were housed in a large open barn in three pens; infected animals were in two adjacent pens of approximately 180 m® DNase I treatment (Ambion). The quantity, quality and integrity of the RNA samples were determined using a NanoDrop ND-1000 spectrophotometer and Agilent 2200 TapeStation system; all had an RNA Integrity Number of > 7.5.Abomasal (gastric) lymph nodes (ALN) were removed immediately post mortem and stored at –80˚C in RNAlater . Total RNA was isolated using the Ribopure Kit (Ambion) according to the manufacturers’ instructions. Contaminating DNA was removed by On-column PureLinkhttp://www.livestockgenomics.csiro.au/sheep/oar3.1.php/). Primers were selected using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) and reanalysed using Net Primer (http://www.premierbiosoft.com/netprimer/). The primers used to amplify overlapping sections of genes are shown in TM Kit for full-length, RNA ligase-mediated rapid amplification of 5’ and 3’ cDNA ends (Invitrogen) was used to sequence the 5’ and 3’ untranslated region (UTR) of the transcription factors. Primers and nested primers (TM III protocol for reverse transcription reaction and Platinum(R) Taq DNA Polymerase High Fidelity protocol for the amplification of the final product, which were cloned as described above.cDNA was synthesised from 1 μg RNA using SuperScript™ II RT with RNaseOUT  and oligo-dT(15) primer . The predicted sequences of the full length sheep genes were obtained by NCBI-BLAST of the bovine sequences against the Oar v3.1 sheep genome assembly  were designed and optimized for all gene variants ; primer in liver . RT-qPCRin liver  and nuclin liver  was follGAPDH and SDHA. Fold changes were calculated from ΔCq values using GenEx. To calculate copy number of RORAv2 and v5 in all 45 infected animals, a standard curve of linearized plasmid DNA was used with a dynamic range that spanned at least five orders of magnitude. For each point on the standard curve, copy numbers were calculated from Cq values using the following formula:Relative gene expression levels were calculated in GenEx 5 Standard Programme  using the comparative 2-(ΔΔ Cq) method and normalized to the geometric mean of GAPDH and SDHA. RT replicates were averaged per animal and multiplied by the dilution factor (x100) to calculate the copy number per μg of total RNA.M = size of plasmid x 660g/mol per bp. The expression levels were normalized by dividing the copy number derived from the standard curve by the calculated normalization factor for each individual sample. The normalization factor was calculated using the method described by Vandesompele , using ts). P-values ≤ 0.05 were considered statistically significant.Relative gene expression levels were analyzed in GenEx using an unpaired, 2-tailed t-test to determine the difference between groups. Graph Pad Prism 6 for Windows  was used for statistical analysis of the absolute expression data. The data were grouped into resistant, intermediate and susceptible (n = 15 per group) and a one-way ANOVA was performed to determine overall significance and Tukey’s multiple comparisons test (within ANOVA) was used to determine significance between groups. The correlations between transcript levels and quantitative phenotypes were analyzed with Spearman’s correlation coefficient . The difference between full length GATA3 (LN848231) and GATA3v1 (LN848232) is the deletion of a single codon (g.806_808delGAA) at the 5’ end of exon 3  which represents the deletion of a glutamic acid (E) at position 260 within the protein (RORC2 is encoded on the minus strand of chromosome 1 (NC_019468) and, in comparison to the full length gene (LN848233), RORC2v1 (LN848234) has a 36 bp deletion (g.1237_1272del) at the 3’ end of exon 7 , which represents the deletion of 12 amino acids (GKYGGVELFRAL) at position 359–370  where five transcript variants (LN848235 –LN848239) were identified in the region spanning Chr7: 46,097,028–46,731,648. All five variants are identical from position 46 of RORAv1 , which represents the 5’ end of exon 6  and the nine most susceptible (S) lambs  lambs . The resRORAv5 as it was the only transcription factor variant that showed evidence of differential expression between the R and S groups; RORAv2 was also quantified as the control RORA variant. RORAv2 expression in all 45 animals was 162938 ± 14799 (transcript copy number per μg RNA ± SD), in comparison to 108806 ± 11211 (p = 0.0045) for RORCv5.Absolute quantitative analysis was performed on RORAv2 and RORAv5 expression in 15 most resistant , 15 intermediate  and the 15 most susceptible  sheep , with one-way ANOVA p = 0.77. In contrast RORAv5 expression levels were significantly different between the infected groups (p = 0.038); mean levels in the resistant sheep were 148439 ± 91397 in comparison with the intermediate  and the susceptible group .Comparison of 8) sheep  shows thRORAv2 and RORAv5 in the uninfected controls were 14336 ± 12927 and 11308 ± 6571 respectively and not significantly different (p = 0.52). RORAv2 was significantly up-regulated 9–11 fold in all three infected groups (p < 0.0003) in comparison with the uninfected controls, with no significant discrimination between resistant, intermediate and susceptible animals. In contrast RORAv5 was significantly increased 13 fold (p < 0.0001) in the resistant sheep but only 7 (p ≤ 0.005) and 6 (p ≤ 0.005) fold in the intermediate and susceptible sheep, in comparison to the controls.Expression levels of RORAv2 and RORAv5 in relation to the quantitative phenotypes, AWC, FEC, BW and IgA antibody levels . In contrast RORCv5 was significantly negatively correlated with AWC  and FEC , and significantly positively correlated with BW ; but was not significantly correlated with IgA antibody .Spearman’s rank analysis was used to quantify the correlation of expression levels of y levels . RORAv2 TBX21, identified two transcript variants each of GATA3 and RORC2, and five transcript variants of RORA. TBX21 has been unambiguously identified in relatively few species, including humans, cattle and mice, and in all these species only a single gene has been characterized.This study represents the first systematic attempt to assess the role of the four major immune system transcription factors in an ovine infectious disease context. Initial characterization of mRNA from abomasal lymph node failed to find any splice variants of sheep GATA3 variants is also found in the two variants of human GATA3 with no reported phenotypic consequence. The deleted aa260 is not within either of the two conserved zinc-finger domains (aa264–286 and aa318–342) nor the functionally-critical DNA-binding YxKxHxxxRP motif (YYKLHNINRP) at aa345–354 [2-zinc finger and could possible affect protein structure. Only one Bos taurus GATA3 sequence has been identified (NM_001076804.1) that encodes an identical protein (isoform X2) to full length sheep GATA3. There are also four predicted Bos transcript variants, X1, X2 and X3 encode isoform X2, and variant X4 encodes a protein (isoform X1) identical to sheep GATA3v1.The sequence difference between the two ovine a345–354  althoughRORC; transcript variant 1 encodes full length RORC (isoform a or RORγ) of 518 amino acids with major functions in regulating embryo development as well as testicular and liver functions [RORC2 variants are homologous to human transcript variant 2, and the 12 amino acid deletion in RORC2v1 is within the predicted ligand binding domain [RORC2 variant also exists in humans; this has a deletion of exons 5–8, also within the ligand binding domain, and functions to suppress Th17 function by the repression of IL17A and IL21 gene transcription [Humans express two transcript variants of unctions . Transcrunctions . The twog domain . One majcription .TBX21, GATA3 or RORC2. A possible explanation for this is that the lymph nodes studied had been responding to T. circumcincta infection for 12 weeks and that the regulatory events controlling T cell polarization occurred much earlier in infection; during initial activation of T helper cells.Previous studies, using RT-qPCR of cytokine transcripts and whole genome microarrays, have shown that a strong Th2 response is associated with resistance and Th1/Th17 activation linked to susceptibility , 17. HowRORA transcript variants in sheep is not an unusual finding, as several species express multiple RORA variants, including four in humans. All four human isoforms have identical zinc-finger DNA and ligand-binding domains but differ in their NH2-terminal sequences  contains both DNA- and ligand-binding domains and a 17 amino acid B domain immediately upstream of the DNA-binding domain, which is identical to that of human Rora isoform d (NP_599024.1). RORαv3 and RORαv5 possess both DNA- and ligand-binding domains and identical B domains, which is the same sequence as that of human isoform a, and distinct from that of RORαv1. RORαv3 possesses an upstream A domain of 13 amino acids and RORαv5 has an A domain of 44 amino acids, different to any human isoform.The identification of five  domains ) and it  domains  and dete domains , 47. Indhenotype . The fivRORA only RORAv5 showed consistent differential expression in the resistant vs susceptible comparison and quantitative levels of RORAv5 were highly significantly correlated with quantitative parameters of resistance. This is likely to be related to the length and identity of the A/B domains. Human Rora isoforms a and b have distinct A/B domains and display different binding specificities. The A/B domains of isoform a mediate almost complete nuclear localization [Of the four measurable variants of lization  making ilization .RORAv5 and parasite resistance. Analysis of the sheep immune response to T. circumcincta shows that resistance is correlated with the development of a Th2 response, including the production of IL-13 and up-regulated transcription of genes associated with wound healing [+ ILC2 cells [This does not fully explain the link between sheep  healing . In huma healing , and it  healing , 17. ROR healing , optimiz healing  and consC2 cells , 50 as tC2 cells , but theC2 cells .RORAv2 expression showed no correlation with AWC, FEC or BW and a low but significant negative correlation with IgA, i.e. a positive correlation with susceptibility. RORαv2 possesses neither the A/B nor DNA-binding domains, but does have the ligand-binding domain. Both RORAv2 and RORAv5 are expressed at high levels only after infection. however RORAv5 showed a 13 fold up-regulation in resistant animals, but only a 7 and 6 fold increase in the intermediate and susceptible groups. In contrast, the infection-associated increase in RORAv2 expression was similar in all three groups. It is possible that RORαv2, which is found at higher levels than RORαv5 in all infected lambs, could compete with RORαv5 for ligand binding and inhibit its function in those animals with lower levels of RORAv5, with consequent pathological effects in susceptible lambs.Analysis of GATA3 and RORC2, five variants of RORA and a single TBX21 transcript. Analysis of expression of each variant in sheep with defined resistance status to the gastrointestinal parasite T. circumcincta showed that only RORAv5 is differentially expressed in resistant compared to susceptible sheep, with expression levels that correlate with the phenotypic parameters of resistance. The largest variant of RORA (RORAv5) seems to be important in controlling the protective immune response to T. circumcincta infection in sheep.In conclusion, we have identified transcript variants of the master regulators of T cell immunity in sheep. There are two variants of S1 FigRORC1 primers, Lanes 1 and 2 mRNA from liver; Lane 3 mRNA from liver (no RT control); Lane 4 mRNA from lymph node; (B) RORC2 primers, Lane 1 mRNA from lymph node (no RT control); Lanes 2 and 3 mRNA from lymph node.RT-PCR products using: (A) (PDF)Click here for additional data file.S2 Fig2-protein sequences (CRI68167.1 and CRI68168.1).(A) 5’ nucleotide sequences (LN848231 and LN848232). (B) derived NH(PDF)Click here for additional data file.S3 Fig2-protein sequences (CRI68169.1 and CRI68170.1).(A) 5’ nucleotide (LN848233 and LN848234). (B) derived NH(PDF)Click here for additional data file.S4 Fig5’ nucleotide sequences of RORA transcript variants .(PDF)Click here for additional data file.S5 FigRORAv1, v2, v3, v4 and v5 intron and exon structures, mapped to Oarv3.1 genome assembly.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(A) Primer sets for amplification of sheep transcription factors. (B) Primer sequences for 5’ and 3’ RACE. (C) Primer sets for RT-qPCR with optimized parameters and volumes.(PDF)Click here for additional data file.


Recent studies have revealed exciting new functions for forkhead transcription factors in cell proliferation and development. Cell proliferation is a fundamental process controlled by multiple overlapping mechanisms, and the control of gene expression plays a major role in the orderly and timely division of cells. This occurs through transcription factors regulating the expression of groups of genes at particular phases of the cell division cycle. In this way, the encoded gene products are present when they are required. This review outlines recent advances in our understanding of this process in yeast model systems and describes how this knowledge has informed analysis in more developmentally complex eukaryotes, particularly where it is relevant to human disease. The accurate duplication of a cell is essential for successful cell proliferation. Many cellular mechanisms control the cell division cycle, and some operate in parallel and show redundancy. This “belt and braces” approach contributes to the incredible fidelity of the process. The control mechanisms range from changes in protein activity  to changes in protein stability or localisation.Another major level of control is through the regulation of gene transcription, where genes or groups of genes are transcribed at particular cell cycle times in order to ensure that protein products are produced when they are required. In some cases, the protein products have structural or enzymatic functions specific to a certain cell cycle phase. In other cases, they have regulatory roles such as activating other proteins important for cell division or controlling the transcription of genes required later in the cell cycle. There are also a few examples of protein products whose presence at the “wrong” cell cycle time is deleterious to the cell.Saccharomyces cerevisiae and the fission yeastSchizosaccharomyces pombe3  the forkhead transcription factors which control expression of two groups of genes in the cell cycle, one during the mitotic (M) phase and the other at the end of the M phase and the beginning of the Gap 1 (G1) period, and (2) the MBF-Nrm1 transcription factor complex which regulates gene expression at the end of the G1 period and the start of the synthesis (S) phase. The remainder of this review will focus on these two systems, first by describing them in more detail in each model yeast and then by explaining their function in human cells and in disease.Strikingly, however, a number of these transcription factors are evolutionarily conserved with related proteins found in the two yeast species. Furthermore, these same transcription factors have been identified in more developmentally complex eukaryotes (including humans) where they too control cell cycle gene expression5 (1.The role of forkhead transcription factors in cell cycle-regulated gene expression was first described in budding yeast. The forkhead transcription factor Fkh2, in combination with the MADS box protein Mcm1, controls gene expression during M phase5 . In addi1 gene expression as well as the subsequent group of gene expression in G1 through the regulation of the Ace2 transcription factor7 (Similar mechanisms have been identified in fission yeast, where the forkhead transcription factor Fkh2, in combination with the MADS box protein Mbx1, regulates M-Gactor7 .9. Furthermore, the medical importance of FOXO1 and FOXO2 has been established, as these transcription factors have roles in a wide range of human medical conditions, including cancer, obesity, diabetes, autoimmune disease, and ageing14. Recently, the formation of blood vessels through the control of the metabolism and proliferation of vascular endothelial cells has been shown to be under the control of FOXO115. Indeed, forkhead transcription factors are now seen as promising therapeutic targets for a wide variety of conditions17.Such conservation of control mechanisms between two distantly related yeasts suggests that forkhead transcription factors are important. Consistent with this suggestion, similar control processes have been identified in more developmentally complex eukaryotes. For example, the human forkhead transcription factors FOXO1 and FOXO2 regulate cell cycle gene transcription that is critical for proliferation3. In budding yeast, it is composed of Swi4 and Mbp1 and controls the expression of a large group of genes in late G1 and early S phase (1/S. MBF in both yeasts is controlled by Nrm121.As one of the first cell cycle transcription factors to be identified in yeasts, the MBF complex has a long historyS phase . MBF in 1/S22. The expression of these genes is deregulated in many types of cancer, as they allow cancer cells to divide in the absence of growth factors, thereby making them insensitive to signals that normally inhibit growth24. E2F family members are now potential therapeutic targets for certain malignancies25.The human functional equivalent to MBF is E2F, which, along with pocket proteins such as the retinoblastoma protein Rb, controls the expression of genes at Get al.26 have shown that in fission yeast deregulation of G1/S gene expression increases DNA replication errors. This demonstrates that faulty transcription of certain genes under the control of MBF can have a profound impact on genome stability. It seems likely that similar mechanisms operate in human cells and that these may be possible targets for therapeutics.Recent experiments have revealed further mechanisms by which deregulated gene transcription can contribute to abnormal cell cycles, the hallmark of cancer and tumorigenesis. CaetanoThe examples described here show how yeast model systems have been used to identify proteins that control the cell division cycle in all eukaryotes, including humans. That these proteins have subsequently been shown to be useful therapeutic targets suggests that this avenue of study will continue to be useful in the future. It seems likely that more transcription factors with cell cycle-specific functions remain to be identified and, moreover, that yeasts will continue to offer an excellent system in which to discover and characterise them.


Transcription factor fusion proteins can transform cells by inducing global changes of the transcriptome, often creating a state of oncogene addiction. Here, we investigate the role of epigenetic mechanisms in this process, focusing on Ewing sarcoma cells that are dependent on the EWS-FLI1 fusion protein. We established reference epigenome maps comprising DNA methylation, seven histone marks, open chromatin states, and RNA levels, and we analyzed the epigenome dynamics upon downregulation of the driving oncogene. Reduced EWS-FLI1 expression led to widespread epigenetic changes in promoters, enhancers, and super-enhancers, and we identified histone H3K27 acetylation as the most strongly affected mark. Clustering of epigenetic promoter signatures defined classes of EWS-FLI1-regulated genes that responded differently to low-dose treatment with histone deacetylase inhibitors. Furthermore, we observed strong and opposing enrichment patterns for E2F and AP-1 among EWS-FLI1-correlated and anticorrelated genes. Our data describe extensive genome-wide rewiring of epigenetic cell states driven by an oncogenic fusion protein.  •Reference epigenome maps identify widespread epigenetic change in Ewing sarcoma cells•EWS-FLI1-regulated genes fall into clusters with characteristic chromatin signatures•Transcriptome response to HDAC inhibitors depends on promoter-specific histone marks•EWS-FLI1 induces global changes in H3K27ac and genome-wide enhancer reprogramming EWS-FLI1 is an oncogenic fusion protein and the main driver of Ewing sarcoma. Tomazou et al. establish comprehensive epigenome maps for an EWS-FLI1-dependent cell line. Based on these data, they identify clusters of epigenetically regulated genes and a unique enhancer signature that is associated with EWS-FLI1 oncogene addiction. Fusion proteins are a common cause of cancer. They often establish a state of oncogene addiction that makes cancer cells vulnerable to losing the fusion protein's biological function . Direct Here, we focus on epigenetic deregulation as a mechanism by which an oncogenic fusion protein may rewire cells for malignancy . We mapphttp://ihec-epigenomes.net). Integrative bioinformatic analysis identified significant associations between EWS-FLI1 binding and the chromatin states of promoters, enhancers, and super-enhancers. We also compared the epigenomes before and after knockdown of the EWS-FLI1 fusion protein, allowing us to define clusters of EWS-FLI1-regulated genes. We validated the relevance of our gene clustering by measuring the transcriptome response to epigenome-modulating drugs, and we identified EWS-FLI1-specific enhancer and super-enhancer signatures that are dependent on EWS-FLI1 expression.To study EWS-FLI1-associated epigenetic changes, we performed comprehensive epigenome mapping in Ewing-sarcoma-derived cells, following the standards of the International Human Epigenome Consortium . This cell line is EWS-FLI1 dependent and was previously engineered to harbor a doxycycline-inducible small hairpin RNA against EWS-FLI1 , open chromatin states, and RNA levels. DNA methylation was mapped both by whole-genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS), histone marks by chromatin immunoprecipitation sequencing (ChIP-seq), open chromatin states by assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), and RNA levels by strand-specific RNA sequencing (RNA-seq). Each experiment was performed in two biological replicates each for both the EWS-FLI1-high state  and the EWS-FLI1-low state (53 hr after adding doxycycline to induce knockdown of EWS-FLI1). We also performed RNA-seq in cells that were treated with each of three histone deacetylase inhibitors to further dissect the relevance of histone acetylation in our model. In total, we generated 61 datasets and sequenced 2.9 billion reads comprising 221 billion base pairs .http://tomazou2015.computational-epigenetics.org). Our dataset can be used to view and investigate the epigenetic changes in EWS-FLI1-dependent cells one gene at a time  than for anticorrelated transcripts A. FurtheThe chromatin-based gene clusters not only showed distinct patters of functional enrichment but also responded differently to EWS-FLI1 knockdown C. Among Based on these promoter-centric analyses, H3K27ac and, to a lesser degree, H3K4me3 and H3K4me1 emerged as the histone marks that were most strongly affected by EWS-FLI1 knockdown. This result was corroborated by western blots for all seven histone marks in additional Ewing sarcoma cell lines , where we observed a global reduction in H3K4me3 and H3K27ac levels upon knockdown of EWS-FLI1 E. These Given that H3K27ac was most variable between the chromatin-based gene clusters and also most dynamic upon EWS-FLI1 knockdown  and 4, wHDAC inhibitors are being evaluated as drug candidates for Ewing sarcoma , but herWe performed RNA-seq to measure the transcriptome response to each HDAC inhibitor at the chosen concentrations D and S4CAmong the EWS-FLI1-correlated genes, there was no global trend in either direction C, but weOur promoter-centric analysis  and 4 esWe observed striking differences between EWS-FLI1-correlated and anticorrelated H3K27ac peaks, which became apparent when we stratified all peaks by the strength of H3K27ac signal difference upon EWS-FLI1 knockdown. Among EWS-FLI1-correlated enhancers, only 10% of the top 20% most differential H3K27ac peaks were located in promoters, whereas 62% of the bottom 20% peaks (which are still significantly different for H3K27ac between the EWS-FLI1-high and low states) were located in promoters B. No suchttp://tomazou2015.computational-epigenetics.org). Overall, more than 10% of H3K27ac peaks in EWS-FLI1-high cells were located in super-enhancers . EWS-FLI1 also appears to contribute to cell-type-specific enhancer signatures by converting broadly active enhancers to a poised state, as illustrated by the enhancer element upstream of the FOSL1 locus , thus providing a genome-wide reference that can guide future work on regulatory mechanisms and epigenetic drug effects in Ewing sarcoma.In summary, the establishment and analysis of reference epigenomes for EWS-FLI1-high and EWS-FLI1-low cells not only constitutes a first comprehensive epigenome dataset for a solid cancer that is driven by an oncogenic fusion protein, but it also provides relevant insights into the mechanisms by which a single oncogenic event has shaped a conductive epigenome. The dataset is available for interactive exploration and data download , using a two-step semi-supervised clustering method. Multinomial logistic regression models were trained based on expert-curated examples and used to classify EWS-FLI1-regulated genes derived from the RNA-seq data. We trained separate classifiers for identifying clusters among the EWS-FLI1-correlated genes and among the EWS-FLI1-anticorrelated genes.http://lola.computational-epigenetics.org), and we identified sets of functional elements that significantly co-localized with the promoters of a given gene cluster. We used Fisher’s exact test to establish significance, and odds ratios as well as p values were used to rank the results. Comparisons were done (1) against the background of all genes identified by our RNA-seq analysis to identify enrichment patterns among EWS-FLI1-regulated genes, (2) against the background of genes in other clusters to identify cluster-specific enrichment patterns separately for EWS-FLI1-correlated and EWS-FLI1-anticorrelated genes, and (3) against the background of genes in the same cluster but with opposite direction of EWS-FLI1 regulation (correlated or anticorrelated) to identify upregulation- versus downregulation-specific enrichment patterns separately for each cluster.To functionally annotate the gene clusters, we identified significant overlap between gene sets derived from our dataset and gene sets obtained from several public databases (see the E.M.T., N.C.S., C.B., and H.K. designed the study. E.M.T. performed the experiments and contributed to the data analysis. N.C.S. performed the data analysis. C.S. and P.D. contributed to the experiments. M.S. and A.S. contributed to the data analysis. S.K. provided relevant materials and drug-related expertise. E.M.T., N.C.S., C.B., and H.K. wrote the manuscript.


Active positive transcription elongation factor b (P-TEFb) is essential for cellular and human immunodeficiency virus type 1 (HIV-1) transcription elongation. CTIP2 represses P-TEFb activity in a complex containing 7SK RNA and HEXIM1. Recently, the inactive 7SK/P-TEFb small nuclear RNP (snRNP) has been detected at the HIV-1 core promoter as well as at the promoters of cellular genes, but a recruiting mechanism still remains unknown to date. Here we show global synergy between CTIP2 and the 7SK-binding chromatin master-regulator HMGA1 in terms of P-TEFb–dependent endogenous and HIV-1 gene expression regulation. While CTIP2 and HMGA1 concordingly repress the expression of cellular 7SK-dependent P-TEFb targets, the simultaneous knock-down of CTIP2 and HMGA1 also results in a boost in Tat-dependent and independent HIV-1 promoter activity. Chromatin immunoprecipitation experiments reveal a significant loss of CTIP2/7SK/P-TEFb snRNP recruitment to cellular gene promoters and the HIV-1 promoter on HMGA1 knock-down. Our findings not only provide insights into a recruiting mechanism for the inactive 7SK/P-TEFb snRNP, but may also contribute to a better understanding of viral latency. In eukaryotic cells, RNA Polymerase II (RNA Pol II) is responsible for the transcription of protein-coding genes. On promoter clearance, the enzyme is paused in a promoter proximal position by the negative elongation factor and the 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF), which poises RNA Pol II to promptly react to upstream signals and generate full-length mRNA transcripts . The posIn a recent study, we have identified the chicken ovalbumin upstream promoter transcription factor (COUP-TF) interacting protein 2 (CTIP2) as a novel compound of the 7SK/HEXIM1/P-TEFb snRNP, efficiently contributing to P-TEFb inactivation . CTIP2 iThe non-histone chromatin protein High mobility group AT-hook 1 (HMGA1) is able to interact with the loop2 substructure of 7SK RNA ,23, whicThus, we here set out to investigate whether HMGA1 is involved in the recruitment of CTIP2-inactivated P-TEFb to HIV-1 and cellular target promoters to regulate viral and endogenous gene expression.et al.  as recommended by the manufacturer. Endogenous HMGA1 was immunopurified from nuclear extracts using a specific anti-HMGA1 antibody (Abcam). Immunopurifications with boiled anti-HMGA1 IgG were used as a control. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotting were performed as described previously . AntibodQuantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analyses were performed as described previously ,30. PrimOn shRNA-mediated knock-down or overexpression of HMGA1 and/or CTIP2, CMEH3 microglial cells were transfected with a HIV-1 Long terminal repeat (LTR)-driven luciferase reporter in the presence or in the absence of HIV-1 Tat-FLAG. The efficiencies of the used constructs have been previously validated ,20. AfteFor transcription assays with integrated virus, HIV-latently infected CHME-5 cells  were tra3) for 15 min at room temperature. Eluates and input chromatin were heated to 65°C overnight in the presence of 0.2 M NaCl to reverse formaldehyde cross-linking, treated with 2 μl of proteinase K (10 mg/ml), 10 μl of 0.5 M EDTA and 20 μl of 1 M Tris–HCl (pH 6.5) for 1 h at 45°C. DNA was phenol/chloroform extracted and precipitated with ethanol plus glycogen as carrier. Immunoprecipitated DNA was subjected to real-time PCR quantification on an ABI Prism 7000 Ligthcycler using the Power SYBR Green kit (Applied Biosystems). The specificity of the enrichment has been controlled by amplification of the GAPDH gene. The following primer sequences were used: HIV-1 promoter: 5′-CAG CTG CTT TTG CCT GTA CTG-3′ (forward) and 5′-TCC ACA CTG ACT AAA AGG GTC TGA-3′ (reverse); luciferase gene: 5′-CCG TGA TGG AAT GGA ACA AC-3′ (forward) and 5′-CAT AGA ACT GCC TGC GTC A-3′ (reverse); Epithelial stromal interaction 1 (EPSTI1) promoter: 5′-CGT GGC TGA TCC CAG TTA TT-3′ (forward) and 5′-CCA GAG AAC TTC CCC ACG TA-3′ (reverse); Poly [ADP-ribose] polymerase 12 (PARP12) promoter: 5′-TAC CTC CCA GGT AGG GTT CC-3′ (forward) and 5′-GCC CTT TGA GGA GGT AAT CC-3′ (reverse).Chromatin immunoprecipitation (ChIP) assays were performed as described previously . HEK293 TM RNA Amplification Kit (Ambion) according to the manufacturer’s instructions. For microarray analyses, hybridization and detection were performed following the protocols supplied by Illumina using the HumanHT-12 v4 Expression BeadChip Kit and an iScan system (both Illumina). The raw data were quality controlled (http://mace.ihes.fr) using Accession Nos.: 3037572262 (CTIP2 knock-down), 2166467750 (CTIP2 overexpression), 3164892326 (HMGA1 knock-down), 2365090982 (HMGA1 overexpression), 2401152166 (7SK knock-down), 3197764774 (7SK overexpression) and 2967448742 (dnCdk9 expression).Total RNA from transfected microglial cells was prepared using the RNeasy Midi Kit (Qiagen) as recommended by the manufacturer. The RNA was labelled using the Illumina® TotalPrepntrolled , NeoNORMntrolled  and analntrolled ,35. For ntrolled  with a pHMGA1 interacts with the 7SK/P-TEFb snRNP via the loop2 substructure of the RNA , while CFor that we performed immunopurifications of HMGA1-FLAG from transiently transfected HEK293 cells in the presence and absence of RNase A and subsequently monitored the amounts of co-purified endogenous CTIP2 A. A signOur hitherto results allow for a functional connection of HMGA1 and CTIP2, which might affect the expression of P-TEFb target genes. To test this hypothesis, we used global gene expression profiling to identify and compare CTIP2, HMGA1 and P-TEFb target genes in microglial cells.Supplementary Figure S1A). Thus, CTIP2 predominantly represses target gene expression, which is in line with previously reported roles of CTIP2 . Comparing the HMGA1 and the CTIP2 target genes, we observe a common subset of 1394 genes being significantly regulated in both conditions . The expression of dnCdk9 resulted in the differential expression of 6291 genes, among those, 2667 genes (42%) being up- and 3624 genes (58%) being down-regulated . The target genes of 7SK overexpression and dnCdk9 expression show a statistically highly significant . We defined those genes, whose expression was concordingly affected by 7SK overexpression, 7SK knock-down and dnCdk9 expression in a statistically significant manner .To identify genes, whose expression is affected by CTIP2 and HMGA1, the expression of endogenous CTIP2 and HMGA1 was knocked down using specific shRNAs A and B, of CTIP2 ,9. The Hal cells D and oveal cells E. Becauspression G. Ideallet genes H. Among To assess the role of CTIP2 and HMGA1 during the expression of these 7SK-dependent P-TEFb target genes, we compared their expression changes on 7SK knock-down with their differential expression on CTIP2 I and HMGapproach A. To conapproach B and D. approach C and E.Our findings support a model in which HMGA1 is involved in the recruitment of the P-TEFb repressor CTIP2 and/or the CTIP2-repressed P-TEFb snRNP to target promoters, as HMGA1 interacts with a large variety of transcription factors and also with specific DNA target sites.To answer this question, we randomly chose PARP12 and EPSTI1 as two 7SK-dependent P-TEFb target genes, whose expression is repressed by CTIP2 as well as HMGA1 F. We perHIV-1 gene expression critically depends on P-TEFb function. Thus, we set out to investigate the impact of CTIP2 and HMGA1 on HIV-1 transcription.We performed HIV-1 promoter-driven luciferase reporter assays in microglial cells on shRNA-mediated knock-down of HMGA1 and/or CTIP2 both in the presence and in the absence of Tat . In the As it is the case for host cellular P-TEFb target genes, also the repressive synergistic cooperativity of CTIP2 and HMGA1 in the case of HIV-1 transcription might be a consequence of a HMGA1-mediated recruitment of the CTIP2-inactivated 7SK/P-TEFb snRNP to the HIV promoter.HMGA1 localizes at the HIV-1 core promoter, as verified by ChIP experiments from HEK293 cells on overexpression of HMGA1-FLAG or knock-down of endogenous HMGA1 F. To invWe here establish a global cooperativity of HMGA1 and CTIP2 in terms of gene expression regulation, including the regulation of 7SK-dependent P-TEFb target genes. While CTIP2 contributes to Cdk9 inactivation as a part of the inactive 7SK/P-TEFb snRNP in microglial cells , HMGA1 hTaken together, the HMGA1-mediated recruitment of CTIP2-repressed P-TEFb not only affects endogenous gene expression in microglial cells but, moreover, also modulates the expression of the HIV-1 genome. The identification of host-cellular factors involved in HIV-1 silencing, as accomplished here, contributes to a more detailed understanding of how viral latency is established and maintained and thus may open ways to novel therapeutic approaches for fighting HIV infection ,43,44.Supplementary Data are available at NAR Online.ANRS ; Sidaction, Institut Universitaire de France (to O.R.); the Genopole Evry; the Institut des Hautes Études Scientifiques; and the Centre National de la Recherche Scientifique (CNRS) (to A.B.). C.V.L. is Directeur de Recherches of the Belgian Fund for Scientific Research . Work in C.V.L.’s lab was supported by grants from the FRS-FNRS (Belgium), the Télévie-Programme of the FRS-FNRS, the Programme d’Excellence Cibles of the Walloon region, the NEAT (European AIDS treatment network) integration grant, the International Brachet Stiftung, the Fondation Roi Baudouin (Belgium) and the ANRS. Funding for open access charge: Centre National de la Recherche Scientifique.Conflict of interest statement. None declared.


Recent advances in genome-wide analysis have revealed that 66% of the genome is actively transcribed into noncoding RNAs (ncRNAs) while less than 2% of the sequences encode proteins. Among ncRNAs, high-resolution microarray and massively parallel sequencing technologies have identified long ncRNAs (>200 nucleotides) that lack coding protein function. LncRNAs abundance, nuclear location, and diversity allow them to create in association with protein interactome, a complex regulatory network orchestrating cellular phenotypic plasticity via modulation of all levels of protein-coding gene expression. Whereas lncRNAs biological functions and mechanisms of action are still not fully understood, accumulating data suggest that lncRNAs deregulation is pivotal in cancer initiation and progression and metastatic spread through various mechanisms, including epigenetic effectors, alternative splicing, and microRNA-like molecules. Mounting data suggest that several lncRNAs expression profiles in malignant tumors are associated with prognosis and they can be detected in biological fluids. In this review, we will briefly discuss characteristics and functions of lncRNAs, their role in carcinogenesis, and their potential usefulness as diagnosis and prognosis biomarkers and novel therapeutic targets. Large-scale genomic technologies  combined with bioinformatics analyses have profoundly changed the genome organization understanding. Unexpectedly, these global transcriptional analyses revealed that 66% of the genome is transcribed and 80% presents biochemical marks of active transcription whereas less than 2% encodes proteins . DiscoveNcRNAs are classified into two categories according to their structural or regulatory properties and size. This length, arbitrarily set to 200 nucleotides (nt), corresponds to the threshold of sensitivity of RNA extraction methods and can differentiate lncRNAs from short and medium ncRNAs such as microRNAs (miRNAs), small nucleolar RNAs (snoRNAs), and piwi RNAs (piRNAs)  Table 1Table 1. BC1, snaR) are included in the lncRNAs subgroup. There is no specific transcription of lncRNAs, the transcriptional machinery being common to lncRNAs and mRNAs. Lack of reading frame cannot also be a discriminating factor between mRNAs and ncRNAs because 50% of lincRNAs contain regions with high translational potential, comparable to those of mRNAs, suggesting that they are actively exported to cytoplasm and translated into short polypeptides, albeit certainly not active. Noncoding characteristic is then not specific to lncRNAs, as bifunctional coding and noncoding lncRNAs and lncRNAs containing other ncRNAs  have recently been discovered. Several mRNAs lose their ability to encode proteins (Xist) while others acquire coding function (SRA) [LncRNAs transcripts are defined by a length greater than 200 nts without any potential of translation . This deon (SRA) . Further Ubxn transcriptional interference-induced repression by trithorax-promoted lncRNAbdx, (ii) sex chromosome dosage compensation and Xist-induced X chromosome, and (iii) inactivation and genomic imprinting promoted by antisense lncRNAs Airn and Kcnq1. These seminal studies established the concept of functional lncRNAs acting as transcriptional regulators involved in important and diverse biological processes. Over the past two decades, systematic global sequencing of cDNA libraries yielded comprehensive knowledge of the transcribed but noncoding component of the genome. These transcriptomic analyses have identified numerous lncRNAs. RNA immunoprecipitation and siRNA-induced inactivation techniques led to functional validation of many lncRNAs.The first lncRNAs were discovered by using functional genetic approaches based on their relation to specific cellular mechanisms: (i)Currently, lncRNAs identification and functional characterization are based on an experimental approach combining (i) discovery of new noncoding transcripts using RNA-seq and ChIP-seq, (ii) annotation of transcribed regions extent and quantity of products transcribed through bioinformatic analysis, (iii) lncRNAs quantification in various cellular and tissue types and conditions, (iv) coexpression or coregulation studies, and (v) gain and loss functional tests on cell lines and xenografts . Advantage of RNA-seq technique resides in its sensibility, allowing most weakly expressed lncRNAs to be identified. However, due to their frequent very low expression and complex exon/intron structures, it can be difficult to identify different transcripts produced from alncRNA gene. It is then necessary to use other techniques through epigenetic analyses (markers of promoter regions or entire gene), targeted transcripts of interest sequencing after capture on a DNA chip (capture-RNAseq) and 5′ and 3′ extremities identification analyses –11.Systematic large-scale projects have contributed a lot to the systematic characterization of mammalian lncRNAs. For instance, the Fantom  is an international project initiated by Japan in 2000 designed to identify and annotate entire transcripts of mouse. Results published identified 15000 lncRNAs in 2002, 23000 in 2005, and 34000 in 2006 with a transcribed genome fraction of 63%. The ENCODE project initiated in 2003 is designed to identify, map, and make public all functional elements of the human genome. This project led to a catalog of lncRNAs with 10000 genes producing 15000 lncRNAs. Very recently, 58648 lncRNAs were identified by using the TCGA database, of which 79% were previously unannotated [LncRNAs genes outnumber those of short ncRNAs and are probably more abundant than mRNA genes. LncRNAs are very heterogeneous in size with some extending over tens of kilobases (kb). They are transcribed in all regions of eukaryotic genome, particularly during development, and are remarkably specific for a given cellular and tissue type.LncRNAs genes share many biochemical characteristics with proteins coding genes: predominant action of RNA polymerase II, epigenetic profiles, cotranscriptional modifications , and exons and introns size. However, when lncRNAs are compared to mRNAs, they appear less stable and shorter, with fewer exons, a less well defined reading frame, and many repeat sequences . A minority group of poorly characterized lncRNAs is represented by nonpolyadenylated lncRNAs synthesized by RNA polymerase III and lncRNAs synthesized via alternative splicing and snoRNAs.Depending on their relative position to the nearest coding genes, several categories of lncRNAs can be identified as follows.(i) Intragenic Regions. Intragenic IncRNAs may be subdivided depending on how they overlap protein-coding genes or their orientation [Long intronic ncRNAs (linRNAs) constitute the major component of ncRNAs transcriptome. Unlike lincRNAs, tens of thousands linRNAs have recently been identified but only few have been analyzed functionally .SRA, ENOD40).Sense lncRNAs are transcribed from the sense strand of protein-coding genes containing exons. They may have some overlap with coding genes or cover entire sequence from an intron. If it has been shown that most have no protein-coding potential, several sense lncRNAs can function as both ncRNAs and protein-coding genes (Antisense lncRNAs (NAT) represent 32% of lncRNAs in humans and are poorly defined. They are transcribed from the antisense strand of protein-coding genes coding. They may also have overlapping with exonic or intronic regions or cover entire sequence from an intron. NATs are observed in many species, including animals, plants, yeasts, prokaryotes, and viruses, but have no sequence or conserved structure that could be indicative of a particular function despite several highly conserved lincRNAs .entation .Long int(ii) Intergenic Regions. Intergenic lncRNAs (lincRNAs) are located in unannotated genomic regions and represent the best studied class of lncRNAs. LincRNAs are functional lncRNAs characterized by stability, K4H36 active domain of transcription, and tissue-specific expression. They act in trans and are primarily involved in epigenetic regulation of protein-coding genes expression and maintenance of stem cells pluripotency [ipotency .(iii) Enhancer ncRNAs (eRNAs). Two types of lncRNA  are derived from enhancer sequences. Enhancer sequences regulate temporal and specific expression of genes via cis or trans mechanisms. The human genome contains approximately one million enhancer sequences most likely controlling the 19000 genes coding for proteins. LncRNAs derived from enhancer sequences play a crucial role in the formation of chromatin loops that allow stabilization and association of enhancer sequences with the promoter regions that initiate transcription of their target genes [et genes .(iv) Promoters. PALR (promoter associated long ncRNA).(v) Telomeric Regions. TERRA (telomeric repeat containing RNAs).NRON), mRNA stability, and translational regulation. Several lncRNAs (MALAT1) are cleaved by RNase P in a structural and regulatory intranuclear component (speckle) and a small cytoplasmic tRNA-like transcript. LncRNAs topography recognition should help in finding new efficient lncRNAs-based targeted therapies [Within cells, lncRNAs are located in the nucleus, according to their main protein-coding genes epigenetic regulatory function. A minority of lncRNAs are involved in nucleocytoplasmic trafficking  from ancestral genes that have lost their coding potential, (ii) by genes or other lncRNAs duplication, transposition, and mutation and (iii) de novo from intergenic DNA , 53. Xist, KCNQ10T1, AIR, HotAir, ANRIL, HOTTIP, MALAT1, TERRA, and HULC.Growing data have demonstrated that lncRNAs exhibit the greatest diversity among functional ncRNAs and play regulatory and structural roles in embryogenesis, stem cells pluripotency, allelic expression, protein-coding genes regulation, apoptosis, cycle control, growth, differentiation, and senescence. In practice, only a very limited number of lncRNAs (1%) have been well characterized functionally in humans, includingLncRNAs play a role in stem cells and differentiation: maintenance of pluripotency and lineage differentiation in embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Adult stem cells are finely regulated by lncRNAs networks that are targets of most of master pluripotency transcription factors  .LncRNAs are considered as crucial regulators coordinating protein-coding genes expression by numerous mechanisms depending on their cellular localization and leading to modifications of chromatin, transcription, and translation.These regulatory mechanisms are located at epigenetic, transcriptional, posttranscriptional, and translational levels as follows. ANRIL, LSD1/PRC2 by HOTAIR) whose subcatalytic units  interact by altering histone code and methylation profile. Cis-acting lncRNAs adjacent to locus where they are transcribed act by transcriptional interference or by modifying chromatin. Transcriptional interference allows inhibition of preinitialization complexes and interaction with transcription factors. Chromatin modifications result from recruitment of complexes inhibiting (Polycomb by ANRIL) or activating (MLL by HOTTIP) gene expression. Trans-lncRNAs operate independently of complementarities sequences and act at distance on many genomic loci via specific DNA motifs. They influence genes expression by recruiting modifying chromatin complexes, binding transcriptional elongation factors, and inhibiting RNA polymerase [(i) LncRNAs constitute a network of epigenetic modulators, recruiting, guiding, and forming platforms that build ribonucleoprotein complexes at specific genomic sites. 20% of human lncRNAs recruit multimolecular repressor, activator, or chromatin remodeling complexes  [(ii) LncRNAs associated with promoters (PaRNAs) and lncRNAs of enhancer type (eRNAs) can directly regulate transcription of target genes by transcriptional activation or suppression. PaRNAs function as protein coactivators facilitating gene transcription. PaRNAs of 50–200 nt are involved in repression of polycomb-targeted genes via mechanisms allowing transcription of these lncRNAs from common promoter regions to those of target genes regulated by these noncoding transcripts. Gene cis-repression is the result of PRC2 complex (SUZ12) recruitment by PaRNAs, attachment to promoter regions, and modification of histone methylation (H3K27me3) on target promoters. ERNAs have transcriptional activation functions of protein-coding genes coding, including genes involved in embryonic development and differentiation (d SNAI1) –58.CDR1-as/ciRS-7, circular RNA sponge for miR-7) [(iii) LncRNAs can also act at posttranscriptional level and are widely involved in biogenesis, stability, and transcriptional activity of mRNAs. They regulate alternative splicing, promote trafficking, direct cellular localization, and promote mRNAs degradation. LncRNAs also synthesize miRNAs and build sponge-like structures to prevent binding of miRNAs to their target mRNAs (r miR-7) .(iv) LncRNAs can finally bind to inhibiting factors of translation, interact with ribosomes, and allow transport of proteins.MALAT1) paraspeckles (NEAT1), and polycomb body (TUG1).LncRNAs may participate in assembly of specialized intranuclear functional structures, including speckles  and play key roles in maintaining stem cells pluripotency.Moreover, lncRNAs have four known molecular functions : (1) SigBecause lncRNAs are involved in various and important physiological processes, their dysfunction should have important consequences for cell homeostasis. Several recent studies have indeed shown that expression of many lncRNAs varies significantly in different conditions compared to healthy tissue. Significance of these deregulations  is still a matter of debate. However, transcripts of noncoding genome revealed new dimension of the molecular architecture of cancer and lncRNAs are involved in all stages of oncogenesis. Gene expression profiles analysis of various tumors showed that lncRNAs are deregulated and functional studies have demonstrated that lncRNAs are implicated in general mechanisms of carcinogenesis. Moreover, genetic studies have revealed existence of mutations in their primary sequences. Since most genetic variants identified by genome-wide association studies (GWAS) are located outside coding genes, many of these mutations may therefore affect lncRNAs. PINC and PGEM1 were the first oncogenic lncRNAs found overexpressed in breast and prostate carcinomas. Since then, many other lncRNAs have been identified, with oncogenic properties  or oncosuppressive properties . Interestingly, several lncRNAs may show both oncogenic and oncosuppressive activities, depending on cellular context. XIST noncoding transcript is overexpressed in male tumors and underexpressed in female tumors.LncRNAs may regulate signaling pathways involved in initiation, tumor progression, and metastatic spread similar to protein-coding oncogenes and oncosuppressors.(i) Oncogenic lncRNAsSRA (steroid receptor RNA activator) is a coactivator for steroid receptors and acts as an ncRNA found in the nucleus and cytoplasm. SRA regulates gene expression mediated by steroid receptors through complexing with proteins also containing steroid receptor coactivator 1 (SRC-1). The SRA1 gene can also encode a protein that acts as a coactivator and corepressor. SRA levels have been found to be upregulated in breast tumors where it is assumed that increased SRA levels change the steroid receptors' actions, contributing to breast carcinogenesis. While expression of SRA in normal tissues is low, it is highly upregulated in breast, uterus, and ovary carcinomas [rcinomas –63.HOTAIR (HOX antisense intergenic RNA) is a long intergenic noncoding RNA with a length of 2.2 kb in HOXC locus and transcribed in antisense manner. HOTAIR regulates gene expression by modulating chromatin structures. It was the first lncRNA discovered to be involved in carcinogenesis. Polycomb group proteins mediate repression of transcription of thousands of genes that control differentiation pathways during development, pluripotency, and cancer progression. Target of PRC2 is HOXD locus on chromosome 2 where PRC2 in association with HOTAIR promotes transcriptional silencing of metastasis suppressor genes. HOTAIR acts as a molecular scaffold remodeling chromatin by causing histone modifications on target genes. HOTAIR comprises two known chromatin modification complexes with 5′ region binding to PRC2 complex responsible for repressive H3K27 methylation and 3′ region binding to LSD1, which initiates activating H3K4 demethylation [hylation , 65.ANRIL (antisense ncRNA in the INK4 locus) is a natural antisense transcript, which activates the two polycomb repressor complexes PRC1 and PRC2, resulting in chromatin reorganization with silencing of INK4b-ARF-INK4a. This results in p15/INK4b, p14/ARF, and p16INK4a inhibition which are normally implicated in cell cycle negative regulation, senescence, and stress-induced apoptosis. ANRIL overexpression in prostate carcinomas has shown silencing of INK4b-ARF-INK4a and p15/CDKN2B. Repression mechanism is mediated by direct binding to CBX 7 and SUZ12, 2 members of PRC1 and PRC2, respectively [ectively –68.MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) is a lincRNA widely expressed in normal human tissues and overexpressed in a variety of breast, colon, liver, pancreas, prostate, and uterus carcinomas. MALAT1 locus at 11q13.1 has been identified to harbor chromosomal translocation break points and mutations in breast and bladder carcinomas. It is localized in nuclear speckles and recent studies have implicated MALAT1 in splicing, EMT, cell mobility, ECM remodeling, and metastatic spread [c spread .HULC (highly upregulated in liver cancer) has been suggested to act as a “sponge” that inhibits miR-372 by sequestering it away from potential mRNA targets [ targets .PCGEM1 (prostate cancer gene expression marker 1) is overexpressed in prostate cancer cells that promotes tumor cells initiation and progression and protects against chemotherapy-induced apoptosis. Moreover, the reciprocal negative control relationship between PCGEM1 and miR-145 regulates both prostate cancer cells proliferation and tumor growth. These results identify PCGEM1 and associated regulators as possible targets for prostate cancer therapy [ therapy .(ii) Tumor Suppressor lncRNAsMEG3  is a transcript of the maternally imprinted gene normally expressed in pituitary cells. MEG3 loss of expression is observed in pituitary adenomas and meningiomas. MEG3 acts by regulating p53 pathway. P53 levels are usually extremely low due to its rapid degradation via ubiquitin-proteasome pathway. P53 ubiquitination is mainly mediated by E3 ubiquitin ligase MDM2. MEG3 downregulates MDM2 expression, increases p53 protein level, stimulates p53-dependent transcription, and enhances p53 binding to target promoters [romoters .GAS5 (growth arrest-specific 5) is widely expressed in embryonic and adult tissues. GAS5 acts as a starvation or growth arrest-linked riborepressor for glucocorticoid receptors by inhibiting association of these receptors with their DNA recognition sequence. This suppresses several responsive genes activation including gene encoding cellular inhibitor of apoptosis 2 (cIAP2). GAS5 low expression levels have been observed in prostate and breast carcinomas [rcinomas , 74.CCND1/Cyclin D1 is transcribed from the promoter region of the Cyclin D1 gene. Cyclin D1 is a cell cycle regulator frequently mutated, amplified, and overexpressed in carcinomas. CCND1/Cyclin D1 recruits the RNA-binding protein TLS, which undergoes allosteric modification, resulting in Cyclin D1 gene inhibition [hibition , 76.LincRNA-p21 expression is directly induced by p53 signaling pathway. This lncRNA is implicated in global repression of genes interfering with p53 function to regulate cellular apoptosis [poptosis .TERRA (telomeric repeat containing RNA) is expressed at chromosome ends. TERRA upregulation upon experimental manipulation or in ICF  patients correlates with short telomeres. TERRA transcription facilitates the 5′-3′ nuclease activity of Exo1 at chromosome ends, providing a means to regulate the telomere shortening rate. Thereby, telomere transcription can regulate cellular lifespan through modulation of chromosome end processing activities [tivities .(iii) Oncogenic and Tumor Suppressor lncRNAsH19 is expressed from the maternal allele and has a pivotal role in genomic imprinting during cell growth and development. The locus contains H19 and IGF2, which are imprinted. This leads to differential expression of both genes H19 from maternal and IGF2 from paternal allele. This lncRNA presents both oncogenic and suppressive properties although the exact mechanism is still elusive [ elusive .LncRNAs are implicated in cancer epigenetics. Epigenetics refers to events that modulate activity of the genome without changing its sequence. It provides control of genome expression and establishment of tissular and cellular-type specific genes expression profiles. The main epigenetic mechanisms comprise chemical modifications of DNA and histones . These chemical changes are reversible and controlled by enzyme complexes directly connected to metabolic and signaling pathways as well as sensors of extra- and intracellular microenvironments. Deregulation of these epigenetic mechanisms has been demonstrated in cancer cells which have no transcriptional gene or protein expression while being free of DNA damage. Carcinogenesis is very frequently associated with abnormal signaling and epigenetic abnormalities, most of which are considered as significant oncogenic events (aberrant methylation of oncosuppressor genes). LncRNAs interact with numerous proteins and in particular epigenetic regulators, including DNA methyltransferases and enzymatic complexes modifying chromatin and nucleosomes [leosomes .(i) Methylation of DNA was first observed in cancer epigenetic alterations. Cancer epigenome is characterized by global hypomethylation leading to genomic instability and hypermethylation of CpG islands which cause inactivation of genes oncosuppressors implicated in signaling pathways  or transcription factors involved in the control of these genes. LncRNAs bind to DNA methyltransferase, guide it to promoter of oncosuppressor genes, and ensure transcriptional silencing.(ii) Nucleosomes can be destabilized and restructured by remodeling complexes belonging to 4 families . Nucleosomes positioning and remodeling regulate gene expression by perturbating accessibility of DNA regulatory sequences by transcription factors and transcription machinery. Mutations and silencing of subunits of these complexes in various types of cancer have been observed. LncRNAs could reposition nucleosomes by interacting and guiding these remodeling complexes to specific genomic regions where nucleosome restructuring would ensure repression of oncosuppressive genes [(iii) LncRNAs recruit histone modifying enzymes acting near the transcription site of lncRNAs. Histones modifying enzymes read, add, or remove covalent links with change in accessibility of chromatin and fixation of nonhistone protein effectors that decode modified histone code. Alterations in the expression of histone-modifying enzymes  were observed in various carcinomas. These alterations inhibit oncosuppressive genes transcription  or activate oncogenes transcription (demethylases and deacetylases) .SRA1) and inhibition of elongation (RN7SK). LncRNAs allow escape mechanisms from suppressor pathways via competition (PFS), silencing (ANRIL), or ribonucleic repression (GAS5). Several lncRNAs are involved in replicative immortality by inhibiting telomerase (TERRA). LncRNAs promote neoangiogenesis is by inhibiting HIF1A . They induce resistance to cell death by acting on p53 and p21 (PCGEM1) and by reducing expression of proapoptotic genes (PANDA). Finally lncRNAs are involved in mechanisms of invasion and metastasis via deregulation of alternative splicing (MALAT1) or trans-silencing of HOXD locus (HOTAIR).Growing numbers of LncRNAs are implicated in numerous mechanisms characterizing hallmarks of cancer. They can act on proliferation via coactivation  Esophagus. Three lncRNA signatures have been identified recently in oesophageal squamous cell carcinoma, including ENST00000435885.1, XLOC_013014, and ENST00000547963.1. The expression of these lncRNAs classified the patients into two groups with significantly different overall survival [survival .(ii) Stomach. HOTAIR, GCAT1 (gastric cancer-associated transcript 1), H19, and SUMO1P3  are the main lncRNAS reported as overexpressed in gastric carcinomas. They are often associated with lymph node and distant metastasis, suggesting that they might serve as potential diagnostic and prognostic biomarker [ CCAT1 functions as an oncogene and may be used as biomarker and potential therapeutic target in gastric carcinoma [iomarker –17. Moreiomarker . CCAT1 farcinoma , 16.(iii) Lower Digestive Tract. HOTAIR overexpression is observed in colorectal carcinomas with advanced stage and liver metastases [tastases . uc.73 ltastases . These r(iv) Liver. In hepatocellular carcinomas (HCC), HULC (highly upregulated in liver cancer) was the first lncRNA with highly specific upregulation detected in blood [ HULC rates were observed in patients with high grades HCC or with HBV+ status [ HULC promoter to induce HCC via oncosuppressor p18 silencing [Dreh, which can inhibit hepatocellular growth and metastasis in vitro and in vivo.in blood . High pl+ status . Recent ilencing . HBx wasMALAT-1 and HOTAIR have been shown to be overexpressed in large cohorts of HCC patients. Furthermore, HOTAIR is a prognostic biomarker for recurrence after liver transplantation. SiRNA-mediated inhibition of MALAT-1 and HOTAIR suppresses cancer cell viability and invasion, sensitizes TNF-α, induces apoptosis, and increases chemotherapeutic sensitivity of HCC to cisplatin and doxorubicin [ HOTTIP  and HOXA13 were also found to be upregulated in HCC. HOTTIP and HOXA13 levels were associated with HCC tumor progression, metastasis, and survival [ HEIH (high expression in HCC) is another lncRNA overexpressed in HCC and an independent prognostic factor associated with recurrence [orubicin , 83. HOTsurvival . HEIH (hcurrence .(v) Lung. MALAT1 is a key prognostic biomarker for metastatic spread in lung adenocarcinomas [ TUG1 (taurine upregulated gene 1) is generally downregulated in non-small cell lung carcinomas (NSCLC). In NSCLC patients, TUG1 low expression was associated with high TNM stage, tumor size, and poorer overall survival. Univariate and multivariate analyses revealed that TUG1 expression serves as an independent predictor for overall survival [ HOTAIR was initially reported to be highly expressed in NSCLCs with advanced stage and lymph node metastasis [ BANCR (BRAF activated noncoding RNA) expression is significantly decreased compared to normal tissues. BANCR underexpression is considered as an independent prognostic factor and is associated with larger tumor size, advanced pathological stage, metastasis distance, and shorter overall survival. Recently, BANCR overexpression was found to play a key role in epithelial-mesenchymal transition [ GAS5 (growth arrest specific transcript 5) underexpression was observed compared to normal mesothelial tissue. Conversely, GAS5 was upregulated upon growth arrest induced by inhibition of Hedgehog and PI3K/mTOR signaling in MPM models [rcinomas . TUG1  Breast. Breast cancer progression is correlated with HOTAIR activity in numerous recent studies. The study carried out by Chisholm and colleagues demonstrated HOTAIR overexpression in primary and metastatic breast carcinoma tissues by using RNA in situ hybridization technique [ HOTAIR revealed the dependence of HOTAIR expression on oestradiol production, due to its promoter region via several estrogen response elements. This study also demonstrated that HOTAIR knockdown induced apoptotic pathways in breast cancer cell lines and suggested estrogen receptors as coregulators for HOTAIR expression [ HOTAIR downregulation and an unfavorable disease state, questioning suitability of HOTAIR as negative prognostic biomarker in breast carcinomas [ LincRNA-RoR suppresses p53 in response to DNA damage through interaction with heterogeneous nuclear ribonucleoprotein I (hnRNP I). Recent data demonstrated that hnRNP I can also form a functional ribonucleoprotein complex with UCA1  and increase UCA1 stability. Of interest, the phosphorylated form of hnRNP I, predominantly located in the cytoplasm, is responsible for the interaction with UCA1. Although hnRNP I enhances p27 translation through interaction with the 5′-untranslated region of p27 mRNAs, interaction of UCA1 with hnRNP I suppresses p27 protein expression via competitive inhibition. In support of this finding, UCA1 seems to have an oncogenic role in breast carcinogenesis both in vitro and in vivo. Finally, a negative correlation between p27 and UCA1 was found. Together, these results suggest an important role of UCA1 in breast carcinogenesis [echnique . Anotherpression . Conversrcinomas . LincRNAogenesis .(vii) Glioma. H19/miR-675 signaling was recently identified as a critical pathway in glioma progression. By analyzing gene expression data, H19 increased levels of expression were found in high grade glioma. SiRNA-induced H19 depletion inhibited invasion in glioma cells. Furthermore, H19 expression was positively correlated with miR-675 and H19 inhibition reduced miR-675 expression. Collectively, these data suggest that H19 regulates glioma development by deriving miR-675 [ miR-675 .(viii) ENT Tumors. Recently, HOTAIR was suggested to play a part in nasopharyngeal carcinoma (NPC). HOTAIR is implicated in NPC progression and patients with high HOTAIR levels have poor clinical outcome with tumor recurrence and distant metastasis [ lnc-C22orf32-1, lnc-AL355149.1-1, and lnc-ZNF674-1 has been also observed. High levels of lnc-C22orf32-1 and lnc-ZNF674-1 are associated with advanced tumor stages. Recurrent NPC displayed a distinctive lncRNA expression pattern with increased expression of lnc-BCL2L11-3 and decreased expression of lnc-AL355149.1-1 and lnc-ZNF674-1 [ HOTAIR is differentially expressed in saliva of metastatic oral squamous cell carcinoma patients compared to primary tumors. These findings suggest that detection of lncRNAs in saliva may be used as a noninvasive and rapid diagnostic tool for diagnosis of oral carcinomas [tastasis , 43. In ZNF674-1 . Interes(ix) Bladder. Oncogenic lncRNAs, including UCA1, H19, MALAT1, and linc-UBC1 (upregulated in bladder cancer 1), are overexpressed in bladder carcinomas and activate PI3K-AKT and Wnt/β-catenin pathways. A pilot study took advantage of this to evaluate potential application of UCA1 in urinary sediments from patients with bladder carcinomas. It turned out to be especially valuable for superficial G2/G3 patients at a high risk for muscular invasion, indicating that UCA1 may be a new promising urinary biomarker for the diagnosis of bladder cancer. H19 expression levels are remarkably increased in bladder carcinomas compared to normal tissue and could also serve as another biomarker. More recently, a new lncRNA linc-UBC1 was found to be overexpressed in 60% of invasive bladder carcinomas and correlated with lymph node metastasis and poor survival. MALAT1 is upregulated in bladder cancer and its expression level is correlated with tumor grade and metastatic stage [ic stage , 35.(x) Prostate. Prostate cancer specific lncRNADD3/PCA3 was identified fifteen years ago [ PCA3 is a prostate-specific lncRNA markedly overexpressed in prostate carcinomas. It can be detected in prostate cancer tissue, urine, and/or urine sediments. In recent studies, PCA3 specificity was found even higher than prostate biomarker PSA and PCA3 score can accurately predict tumor volume and pathological features, which may guide treatment [ PCA3 can be used as a noninvasive urine-based test for large-scale screening protocols and for predicting prostate carcinomas aggressiveness. This prominent example of rapid translation of lncRNA research into clinical practice offers a prototype for developing different lncRNAs as biomarkers. Other lncRNAs, such as PCGEM1 and PCAT1 (prostate cancer associated ncRNA transcript 1), are also prostate-specific, posing as attractive biomarkers [ears ago . PCA3 isreatment , 37. Theomarkers , 39.(xi) Melanoma. BANCR (lncRNA BRAF-activated noncoding RNA) plays a potentially functional role in melanoma cells proliferation and migration by activating ERK1/2 and JNK MAPK pathways. BANCR is upregulated in human malignant melanoma and patients with high levels of expression have a lower survival rate [val rate .(xii) Hemopathies. Several recent reports have revealed deregulation of lncRNAs in leukemia, including ANRIL, lncRNA-P21, MEG3, Dleu2, HOTAIRM1, EGO, and lncRNA-a7. Moreover, MEG3, UCA1, and H19 are upregulated in acute myeloid leukemia [leukemia , 88.(xiii) Ovary. HOTAIR plays a pivotal role in epithelial ovarian cancer (EOC) metastasis and could represent a novel prognostic marker and potential therapeutic target in patients with EOC. In recent study, HOTAIR expression was elevated in EOC tissues, and its level of expression is highly positively correlated with FIGO stage, histological grade, lymph node metastasis, reduced overall survival, and disease-free survival [survival .Because of their central role in genes expression regulation, lncRNAs could also represent potential therapeutic targets. Better characterization of lncRNAs  could help in faster development of new anticancer strategies modulating expression levels and functions of deregulated lncRNAs. Targeting lncRNAs offers novel exciting opportunity to treat cancer. Currently, nucleic acid-based methods prevail in targeting RNA, by regulating levels of expression and modifying their structures or mature sequences. Among them, RNA interference (RNAi) based techniques are arguably the most popular methods to inhibit lncRNAs in cancer cells. Both siRNAs and shRNAs exhibit great RNA selectivity and knockdown efficiency. Meanwhile, other established methods in inhibiting cancer-associated RNA, including antisense oligonucleotide (ASO), ribozyme, and aptamer, are also effective to modulate lncRNAs, and they show unique features that can have advantages over siRNAs .In principle, targeting of lncRNAs can be achieved using the several approaches, including siRNA-mediated silencing, functional block using small molecules, or oligonucleotide inhibitors to prevent interactions of lncRNAs with proteins and structure disruption via small molecules or oligonucleotide inhibitors to change or mimic their secondary structure to compete for their binding partners . Small interfering RNAs (siRNA) are short stretched (19–30 nt) double-stranded RNAs that target RNA molecules via complementary to unpaired lncRNA sequences. The RNA duplex of siRNAs must be unwound into single strands before assembling into the active RNA-induced silencing complex (RISC). SiRNAs are fully complementary to their RNA target that is then cleaved at a single phosphodiester bond located near the centre of the sequence complement to the siRNA sequence [ in vitro and in vivo. For instance, depletion of HOTAIR by siRNAs decreases matrix invasiveness of breast cancer cells and inhibits tumor growth of pancreatic cancer xenograft. HULC and MALAT1 siRNA-induced knockdown inhibits HCC cell proliferation and cell cycle progression [ H19 induces apoptosis and inactivates p53 [ EphA2), TKM-080301 (targeting PLK1), and CALAA-01 (targeting RRM2). Therapy combining siRNA drug siG12D LODER and Atu027 with conventional chemotherapy is studied in phase II for its therapeutic effect in advanced pancreatic cancer patients. However, the main obstacle of siRNAs therapeutics remaining is their delivery. SiRNAs using ribonucleic acid as building block are susceptible to be degraded by nuclease and have poor pharmacokinetics.(1)sequence . SiRNAs gression . Furtherates p53 . Phase I Antisense oligonucleotides (ASO) are short, single-stranded DNAs or RNAs (between 8 and 50 nt) designed with sequence specific to target lncRNA [ MALAT1 by ASO attenuates various malignant phenotypes in cancer cells via cycle arrest in cervical cancer cells [ MALAT1 in vivo and blocks metastasis of lung cancer cells [(2)t lncRNA . ASOs dier cells . Injectier cells . Ribozymes are naturally produced RNA molecules that present intracellular catalytic functions. One of their functions is degradation of RNA molecules. Among all types of ribozyme, hammerhead ribozyme (HamRz) has caught major interest as it shows good target inhibitory effect while having the smallest RNA endoribonucleolytic motif as well as function independent to presence of metal ions [(3)tal ions . HamRz ital ions . Aptamers are short DNA or RNA oligonucleotides or peptides that have a stable 3-dimensional structure in vivo. They have broad molecular targets including protein, RNA, and small molecules that rely on fitting 3-dimensional shape of their ligands [(4) ligands . They sp ligands . Some re ligands . Another ligands . Another ligands . MicroRNAs (miRNAs) can interact with lncRNAs. H19/miR-675 signaling is critical in glioma progression. By analyzing glioma gene expression data sets, H19 is found to be increased in high grade gliomas. H19 depletion via siRNA inhibits invasion in glioma cells. Further, H19 is positively correlated with its derivate miR-675 expression and reduction of H19 inhibits miR-675 expression. MiR-675 modulates cadherin 13 expression by directly targeting its binding site within 3′ UTR. These results demonstrate that H19 regulates glioma development by deriving miR-675 and provide important clues for understanding key roles of lncRNA-microRNA functional network in glioma. MicroRNA-based anticancer therapy has great potential as reports show there is apparent lack of adverse event in normal tissues when administrated with microRNA-based agent. Various microRNA delivery strategies, such as cationic lipid and nanoparticle encapsulation, are developed to improve microRNA shuttling into target cancer cells [(5)er cells , 99, 100 Small molecules are synthesized to specifically bind to RNA binding pockets of lncRNAs. They compete with protein factors or intracellular small ligands for binding lncRNAs. Binding of small molecules may also induce conformational change within lncRNA molecules and disrupt formation of important lncRNA structures [(6)ructures .Deregulated oncogenic and oncosuppressive lncRNAs are observed at all stages of malignant tumors development of various origins. Mechanisms implicating lncRNAs in carcinogenesis are dominated by deregulation of signaling pathways and altered epigenetic, transcriptional, and posttranscriptional expression of numerous genes. Their use as biomarkers and potential therapeutic targets could appear promising.Major challenges of the next 10 years remaining are identification, mapping of all lncRNAs belonging to the human genome, and their functional characterization. Although their mechanisms of action are better known, it is still possible that lncRNAs are rather downstream products of particular chromatin structures or deregulated transcriptional processes. Their functionality is currently still debated because of their low level of expression and conservation between species. Unlike proteins, which often have well-defined functional areas, it is currently impossible to predict lncRNAs function from their single sequence. However, many traits exhibited by lncRNAs favor functionality, including 3′polyadenylation, 5′cap, multiple exons, transcriptional activation similar to that of mRNA, K4K36 domain, and alternative splicing. These challenges will only be achieved through combined efforts of functional genomics, epigenomics, and bioinformatics.


Despite their clear T7-bacteriophage origin, mitochondrial RNA polymerases have evolved to require transcription factors. All mitochondrial polymerases contain an extra N-terminal domain that has no counterpart in the self-proficient phage enzyme, which is therefore hypothesized to interact with transcription factors. We studied a series of N-terminal deletion mutants of yeast mitochondrial RNA polymerase, Rpo41, and have found that the N-terminal region does not abolish the effects of Mtf1; rather it contributes directly to enzyme catalysis. Mtf1 can rescue the defective Rpo41 enzymes resulted from N-terminal domain deletions. Although Rpo41 appears to have retained all promoter recognition elements found in T7 RNAP, the elements are not independently functional, and Mtf1 is necessary and sufficient for holoenzyme promoter-directed transcription activity. An important function of mitochondria is supplying energy to cells via oxidative phosphorylation where the proton potential is converted to chemical energy. Genes for oxidative phosphorylation pathway components are both nuclear- and mitochondria-encoded and coordinated gene expression from both organelles is therefore essential. In addition, mitochondrial RNA polymerase can provide primers for DNA replication ,2, therein vivo invariably initiates with ATP.The yeast mitochondrial transcription system is a two-subunit RNA polymerase holoenzyme contains a core polymerase, Rpo41, and a transcription factor, Mtf1. Rpo41 exhibits non-specific activity because it is able to transcribe poly dA/dT templates , but theE. coli rRNA methyltransferase ErmC' ATP on the pre-melted 14S rRNA promoter.While studying the transcription activities of Rpo41 variants on the pre-melted promoter DNA, we observed unexpectedly high activity on a control, non-promoter-containing DNA. The design of this control template potentially allowed transcription from both strands and also initiation with GTP. We found that Rpo41 initiates efficiently with either GTP or ATP and we found transcripts originating from positions other than +1 (data not shown). Even more unexpectedly, the pre-melted 14S rRNA promoter also showed aberrant transcription from sites other than the +1 position. Consequently, we tested for incorporation of [γ-32P]ATP, Rpo41 makes two RNAs of about the expected size for a run-off transcript, and a less abundant transcript of ~45 nucleotides  is an ideal case for understanding the interplay between the RNA polymerases and transcription factors, as the system contains a catalytic subunit Rpo41 and only single transcription factor Mtf1.Previously, Rpo41 was previously reported to possess intrinsic promoter-specific transcription activity. However, the experiments used only three NTPs, which precluded detection of non-specific transcripts. Upon a more detailed examination, we found that this activity of Rpo41 is not sequence-specific but is actually template-structure dependent, as Rpo41 has high affinity to pre-melted bubble DNA, and initiates efficiently with either GTP or ATP on a single stranded DNA within the bubble, predominantly from a site that is two nucleotides from the downstream duplex. The activity can be affected by the size of the bubble . CautionBecause Rpo41 was thought to possess intrinsic promoter specific activity, the function of Mtf1 was assumed to be restricted to promoter binding and unwinding. In this study, while we confirmed that Mtf1 is absolutely required for promoter-specific activity, we also show that Mtf1 increases promoter specific transcription by suppressing Rpo41 non-specific RNA synthesis. On a pre-melted promoter bubble template, Mtf1 biases Rpo41 transcription to that on the template strand by preferentially inhibiting reactions initiated on the NT strand. This could be accomplished by specific interactions between Mtf1 and the NT strand , 18, whiThe selective inhibition of non-specific transcription by Mtf1 appears to be intimately related to the N1 subdomain of Rpo41, because holoenzyme with deletion mutant Rpo41-ΔN160 is less sensitive to Mtf1 than wild-type. The N1 subdomain and Mtf1 may jointly interact with the NT strand to inhibit non-specific transcription. Therefore, holoenzyme containing Rpo41-ΔN1 deletion would result in easier promoter release and higher efficiency to transition into elongation phase. The trade-off for N1 deletion is relaxed specificity, as the mutant holoenzymes have elevated both non-specific and specific activities. This, in turn, exemplifies the necessity of Rpo41 N1 and Mtf1 for promoter specific transcription. A summary of our findings is presented in The defects of the Rpo41-ΔN2 mutant Rpo41 has reduced affinity to both Mtf1 and promoter DNA than wild-type, indicating although this region is spatially homologous to the AT-rich minor groove recognition loop of T7 RNAP, the N2 region forms part of the Mtf1 binding site and performs a distinct function.Our findings that Mtf1 restores Rpo41-ΔN1 are in good agreement with previous studies . Yeast cTo synthesize mature RNAs, all transcription reactions have to transition from initiation and elongation phases, where RNAP releases the promoter and synthesizes RNA processively. Nevertheless, the mechanisms differ: Single subunit RNAP such as T7 RNAP undergoes conformational changes to refold in the NTD promoter-binding site to RNA exit tunnel for processivity enhancement , 28, 29,Rpo41 deletion mutants were constructed where 52, 96, 160, 254, 321 or 393 amino acids from the N-terminus were systematically removed, yielding N-terminal his-tagged variants Rpo41-ΔN52, ΔN96, ΔN160, ΔN254, ΔN321 and ΔN393. pProExHTb-RPO41 DNA was used as template for mutant generation using PCR method, and the unique restriction sites StuI and BsmI were used for in-frame ligation of a mutant construct. Four forward primers, one for each Rpo41 variant were synthesized:StuI-52aa-rpo41 Fwd 5’-GAAGGCCTACCTCATCTACAACATCAGG,StuI-96aa-rpo41 Fwd 5’-GAAGGCCTAAATCGCATGCCTCAGCAG,StuI-160aa-rpo41 Fwd 5’-GAAGGCCTAAAAATGACCCTAATTTCCC,StuI-254aa-rpo41 Fwd 5’-GAAGGCCTTCGCAACTGCCAAATTCTG,To construct an Rpo41 variant, the corresponding forward primer is paired with an invariant reverse primer 5’-GCTTTCGAATGCCTTCTTTTCC 3’ in a PCR reaction. The yield PCR product was purified using QIAGEN PCR cleaning kit), and was subject to restriction enzymatic digestion with StuI and BsmI. The gel purified digested PCR fragment was ligated to pProExHTb vector.The clone pTrcHisC-MTF1 coding for gene of a N-terminal His-tagged Mtf1 was subcloned from the parental clone pGEX2TK-MTF1 that contains gene of a GST-Mtf1 fusion protein . The DNAClones of all Rpo41 variants and Mtf1 constructs were confirmed by DNA sequencing at the ICMB Core Facilities of The University of Texas at Austin, and expressed in Rosetta cells.Escherichia coli was as described previously ATP, [γ-32P]GTP, or [α-32P]ATP  was used in reactions as indicated. The reactions mixtures were incubated for 10 min at 20°C, and stopped by the addition of 10 μl of stop-buffer . Each sample was then boiled at 95°C for 5 min prior to being loaded on an 20% polyacrylamide gel with 7 M urea to resolve RNA products. After electrophoresis, the gel was exposed to a phosphor screen, scanned on Typhoon Trio instrument  to quantify RNA products, and quantified using Quantity One (BioRad).Transcriptions were conducted using 10 μl of reaction buffer Click here for additional data file.