Long noncoding RNAs (lncRNAs) perform diverse functions in the regulation of cellular processes. Here we consider a variety of lncRNAs found in the ribosome production center, the nucleolus, and focus on their role in the response to environmental stressors. Nucleolar lncRNAs ensure stress adaptation by cessation of resource-intensive ribosomal RNA (rRNA) synthesis and by inducing the massive sequestration of proteins within the nucleolus. Different cell states like quiescence and cancer are also controlled by specific lncRNAs in the nucleolus. Taken together, recent findings allow us to consider lncRNAs as multifunctional regulators of nucleolar activities, which are responsive to various physiological conditions. Noncoding transcripts longer than 200 nucleotides, known as long noncoding RNAs (lncRNAs) act as specific regulators of gene expression, chromosomal dynamics, RNA modifications and as architectural components assembling protein complexes ,2. In thA fundamentally new stage in cell biology is represented by the emergence of a paradigm, which considers some cellular compartments, including the nucleolus, as biomolecular condensates that are organized by liquid-liquid phase separation (LLPS) and exhibit high sensitivity to environmental cues ,16,17. FXenopus laevi, Mus musculus and Homo sapiens) and invertebrates (Drosophila melanogaster) [0) and downstream (T1–10) of the pre-rRNA coding region. The nucleolus is built around rDNA clusters that include hundreds of tandemly repeated rRNA genes separated by intergenic spacer regions (IGS). Chromosome parts that contain such clusters are termed NORs (Nucleolar Organizing Regions), which in human cells are located on the short arms of the five acrocentric chromosomes . The IGSogaster) ,30,31. IDrosophila rRNA genes carry insertions of the specific retrotransposons R1 and R2 in the 28S rRNA sequence. Although inserted rDNA repeats may contain normal promoters, they are transcribed several orders of magnitude lower compared to non-inserted rRNA genes [The level of rRNA synthesis is extremely flexible. It depends on different environmental conditions, as well as the stage of the cell cycle, and is tightly regulated by various metabolic and signaling pathways ,32,33,34NA genes . General0 (Transcription of rDNA is performed by RNA polymerase I (Pol I) and requires a unique set of basal factors. In mammals, rDNA transcription initiation depends on the binding of the dimeric UBF protein (upstream binding factor) that recruits the SL1 complex (TBP and TAFs) and the initiation factor TIF-IA (RRN3) to the rDNA promoter  43,44,4,46. The 0  and servIn terms of epigenetic marks, promoters of transcriptionally active rRNA genes in mammalian cells are usually characterized by hypomethylated DNA at CpG dinucleotides, acetylation of histone H4, and dimethylation of histone H3 at lysine 4 (H3K4me2). Numerous studies also suggest that the bodies of active rRNA genes contain few nucleosomes or may even be completely free of histones ,48, an eIn addition, promoters of active and silent rDNA repeats contain differently positioned nucleosomes. At active genes, the promoter-bound nucleosome covers nucleotides from −157 to −2 upstream of the transcription start site, facilitating cooperative binding of transcription initiation factors. At silent rRNA genes, the nucleosome is positioned 24 nt downstream, preventing transcription initiation .Although a substantial part of rDNA repeats normally undergo selective heterochromatinization and inactivation, stressful conditions can lead to repression of a much larger number of rRNA genes, or even complete cessation of rDNA transcription. First of all, down-regulation of rRNA synthesis is aimed to limit the resource-intensive processes of ribosome biogenesis and protect cells from energy deprivation. Stress-induced rDNA repression can be initiated by various mechanisms at different levels. For instance, elevated temperature induces phosphorylation of transcription factor TIF-IA, making it inactive and blocking rDNA transcription initiation . AnotherIn studies on murine and human cell lines, the Ingrid Grummt group has found two types of non-coding RNAs which mediate repression of rDNA synthesis: pRNA (promoter associated RNAs) and PAPAS (promoter and pre-rRNA antisense). pRNAs are 150∼300 nt long molecules produced in the same direction as rRNA by RNA Pol I from spacer promoters, which in mice are located ~2 kb upstream of the pre-rRNA transcription start site ,64 Figua. In conpRNAs can associate with 47S pre-rRNA promoters forming RNA-DNA triplex structures by Hoogsteen base pairing between DNA and single-stranded RNA . At the Under normal conditions, the pRNA-NoRC pathway is required for the silencing of a large fraction of rDNA repeats, which usually exist in a repressive state ,64,80. p0 site within the promoter region, initiating transcriptional repression [Importantly, pRNA is able also to induce rDNA silencing by another mechanism that implicates the aforementioned triplex structure a. Formatpression . Moreovepression ,83. Notapression . Althougpression , these tpression , possiblInterestingly, recent data shows that PAPAS lncRNA also forms an RNA-DNA triplex. The 3′ poly(A)-tails of these transcripts interact with poly(T)-sequence (T stretch) at rRNA gene enhancers, which are located at the position −275/−336 upstream of the rDNA transcription start site . ApparenUpon heat stress, PAPAS interaction with CHD4 requires not only an increased number of PAPAS transcripts, but also heat-induced dephosphorylation of CHD4 . Zhao etO-ribose-methylation [O-methylation as a competitor with U3 snoRNA in the process of rRNA maturation. Thus, LoNA is suggested to be a regulator of both rDNA transcription and rRNA methylation. It is conceivable also, that LoNA interaction with fibrillarin and nucleolin can affect LLPS-based nucleolar structure. Small nucleolar RNA (snoRNAs) play an important role in rRNA biogenesis guiding pseudouridylation and fibrillarin dependent 2′-hylation . Recentlhylation . This sphylation ,87 for rA decrease of LoNA level that leads to elevated ribosome production has been shown to correlate with enhanced neuronal activity in the mouse hippocampus . The neuSLERT emerges as a factor promoting rDNA transcription. While in response to environmental stresses the cell suppresses rRNA synthesis, the opposite situation is observed in cancer cells, which seek to maximize the production of ribosomes [SLERT acts as an allosteric repressor inducing “closed” conformation of the DDX21 ATP-binding domain. This conformational change impairs DDX21 interaction with Pol I and thereby allows Pol I-mediated transcription. Contrarily, “open” DDX21 conformation due to SLERT depletion causes an increase of DDX21-Pol I association coupled with diminishing of Pol I transcription that leads to reduced proliferation of cancer cells [Another nucleolar snoRNA-containing transcript called SLERT  was detected in human embryonic stem cells and cancer cells . Contraribosomes . The 694ibosomes . Both ener cells . A lot of proteins that have no known roles in ribosome biosynthesis are sequestered or released by the nucleolus in response to changes in cellular growth conditions or different stresses ,92,93. F28RNAs, due to their origin from a discrete IGS region located 28 kb downstream of the pre-rRNA transcription start site [16RNA and IGS22RNA. Thus, different IGS promoters corresponded to different stress types. Heat shock-inducible transcripts were also able to implement protein detention to the nucleolus, but the set of detained proteins differed from the acidosis-induced set. It was concluded that the IGS comprises different transcriptional units operating independently from each other to recruit nucleoplasmic proteins to the nucleolus in response to environmental cues [Extended  IGSs comprise promoters of the internal spacer transcripts, which can be separated by many kb from 47S promoters . The groart site . These tart site  n/(AG)n repeats lacking predicted folded regions . The RNAThus, IGS transcripts drive protein component concentration, ensuring the start of an amyloidogenic-like program. The meaning of such responses to stress under acidic or high-temperature conditions appears to be the preservation of a significant amount of non-degraded proteins in a dormant state ,75. More36RNA and IGS39RNA), which are specifically activated under certain conditions accompanied by the inhibition of rRNA synthesis [The understanding of the regulation of IGSRNA transcription remains poor. Since different IGSRNAs emerge in human rDNA spacers upon different stresses ,74, actiynthesis .Drosophila melanogaster, heat shock caused a drastic redistribution of the nucleoplasmic protein Piwi to the nucleolus [D. melanogaster. Given that a clear function of IGSRNAs in the process of protein sequestration in the nucleolus has so far only been shown in certain human cell cultures ,73,74,75ucleolus . Importa28RNA described earlier as acidosis-inducible transcript [In addition to the role of IGS transcripts in titrating important nucleoplasmic proteins, their involvement in the formation of the perinuclear compartment (РNC) was recently revealed . The PNCanscript . PNCTRs anscript . PNCTR danscript . Thus, IGS transcripts can have a dual function, providing both the nucleolar response to stressful situations as well as determining PNC body formation in cancer cells.The data discussed here demonstrate the diversity of nucleolar lncRNAs, which originate both from the nucleolar organizer itself  and from remote regions of the genome outside the rDNA cluster . Importantly, most of these RNAs are present at low level or nearly absent in cells under normal conditions. Accumulation of nucleolar lncRNAs is observed in response to different stresses (upregulation of IGSRNA and PAPAS) and unusual cellular states like quiescence (induction of PAPAS) or tumorigenesis (accumulation of SLERT and PNCTR) . HoweverDrosophila melanogaster [Several basic types of nucleolar lncRNA molecular functions can be distinguished. The first role is exemplified by pRNA and PAPAS, which can interact with rDNA by RNA:DNA triplex formation and recruit protein complexes regulating chromatin state and transcription ,69,70,71nogaster . Our knowledge of the various mechanisms of nucleolus-associated lncRNA action is constantly increasing and is not limited to the cases described in this review. For example, a special biological role is performed by the X-linked lncRNA Firre, which is required to anchor the inactive X chromosome in female cells to the nucleolus surface . RecentlAltogether, the discussed studies performed mainly on human and murine cell cultures demonstrate an extreme variety of functions of lncRNAs associated with the nucleolus. In the future, it would be interesting to investigate similar mechanisms at the organism level, as well as exploring the evolutionary aspect, extending the nucleolar lncRNA research to other model organisms.


VirtUaL ChIP-seq Analysis through Networks (VULCAN) infers regulatory interactions of transcription factors by overlaying networks generated from publicly available tumor expression data onto ChIP-seq data. We apply our method to dissect the regulation of estrogen receptor-alpha activation in breast cancer to identify potential co-regulators of the estrogen receptor’s transcriptional response.VULCAN analysis of estrogen receptor activation in breast cancer highlights the key components of the estrogen receptor complex alongside a novel interaction with GRHL2. We demonstrate that GRHL2 is recruited to a subset of estrogen receptor binding sites and regulates transcriptional output, as evidenced by changes in estrogen receptor-associated eRNA expression and stronger estrogen receptor binding at active enhancers after GRHL2 knockdown.Our findings provide new insight into the role of GRHL2 in regulating eRNA transcription as part of estrogen receptor signaling. These results demonstrate VULCAN, available from Bioconductor, as a powerful predictive tool.The online version of this article (10.1186/s13059-019-1698-z) contains supplementary material, which is available to authorized users. Breast cancer is the most common form of cancer in women in North America and Europe accounting for 31% of all new cancer cases. In the USA, it is estimated that 41,400 deaths will have occurred from the disease in 2018  that ER ChIP-seq enables the identification of potential site-specific interactions at common binding sites between transcription factors and their cofactors; however, to fully characterize all potential cofactors of a single project on this scale is laborious and expensive. To follow up all potential cofactors identified by a chromatin-wide proteomics method, e.g., RIME  or ChIP-To enable discoveries beyond collections like ENCODE, we are proposing a computational framework to integrate patient data in the prediction of functional protein-protein interactions. By applying machine learning methods, we are able to surpass the limitation of current predictive tools that exist to support the interpretation of data. Previous methods provide information in the context of predefined biological pathways and established gene sets , 13 or tOur method, “VirtUaL ChIP-seq Analysis through Networks” (VULCAN), is able to specifically analyze the potential disease-specific interactions of TFs in ChIP-seq experiments by combining machine learning approaches and patient data. Previously, the strategies employed by VULCAN were limited to the analysis of transcription data. By developing VULCAN to overlay co-expression networks established from patient tumor data onto ChIP-seq data, we are able to provide candidate co-regulators of the response to a given stimulus Fig. . FurtherThrough the application of VULCAN to the activation of the ER in breast cancer, we were able to identify multiple previously characterized cofactors of the ER along with GRHL2 as a potential co-repressor of the ER. We then demonstrated experimentally that GRHL2 is able to modulate the expression of eRNA at ER bound enhancers, and the removal of the P300 inhibitory alpha helix results in suppression of the inhibitory effect on eRNA production.VULCAN integrates ChIP-seq data Fig. , step 1 In the following, we first benchmark VULCAN’s performance in a comprehensive comparison to alternative approaches. We then apply it to our data on temporal ER binding, which identifies GRHL2 as a novel ER cofactor, and we explore its function.We generated a mutual information network with ARCANe alongside several partial correlation networks at different thresholds all from the TCGA breast cancer data. To ensure the robustness of our method, we tested the overlap of every partial correlation network with the mutual information network using the Jaccard index (JI) criterion  in our dataset and was therefore applied to all downstream analysis of ChIP-seq data.VULCAN applies gene set enrichment analysis  to identTo further validate our method, we compared the output of our GSEA analysis with different versions of promoter-enrichment approaches implemented by GREAT , ISMARA We performed four replicated ChIP-seq experiments for ER at three time points  after estradiol treatment Fig.  in the Ep value of 0.0029  on ER binding sites detected by differential binding analysis. Our analysis confirmed a strong enrichment for a single element, ERE, bound at both 45 and 90 min, with a corrected 029 Fig. f. When cWe performed a gene set enrichment analysis (GSEA)  and an a.We leveraged the information contained in mutual information networks to establish TF networks enriched in the differential binding patterns induced by estradiol. From our analysis of ER binding, we established four classes of modulation: early coactivators, early co-repressors, delayed coactivators, and transient coactivators Fig. .Using VULCAN, we defined TF network activity of occupied regulatory regions Fig. a accordiWe repeated our TF network activity analysis of ER activation Fig. a–e on anTo ensure the robustness of the results, we performed a joint analysis of data obtained from both networks. At 45  , 23, whiFinally, we compared VULCAN to a classical motif analysis by exploiting the MsigDB C3 collection v6.1  of gene Homo sapiens, but it can be lowered to 100 nt for bacterial chromosomes, to assign peaks that act as representative for the gene.The network generation algorithm uses established methods to optimize the parameters for the RNA-seq input . By default, VULCAN can calculate key settings from the provided ChIP-seq data . Additionally, parameter choice is tunable at the wish of the user. The distance from promoter transcription starting site (TSS) can be tuned to the specific organism investigated by the ChIP-seq experiment. In this manuscript, we used 1000 nt for In our analysis of ER dynamics, the GRHL2 transcription factor was consistently identified as a key player, using both the METABRIC and TGCA networks. GRHL2 is a transcription factor that is important for maintaining epithelial lineage specificity in multiple tissues , 29. It There is only a weak, positive correlation between ESR1 and GRHL2 expression in the TCGA and METABRIC breast cancer datasets  or presence (45 min) of E2 Fig. a. VULCANp value = 1 × 10−179) and the GRHL2 binding motif   Fig. g.Fig. 7ETo establish if the recruitment of GRHL2 was primarily related to a transcriptional function or the previously described interaction with MLL3, we overlapped our GRHL2 data with that of published H3K4me1/3  and P300A more detailed analysis of the GRHL2 overlap with P300 sites showed the greatest co-occupancy of GRHL2/P300 sites was when both TFs were stimulated by E2 Fig. d. Moreovp value = 6.4 × 10−9), TIF1A , PRMT , and GTF3C2 . p values given for estrogen-free and estrogenic conditions were comparable. The only protein differentially bound to GRHL2 in estrogen-free vs estrogenic conditions was the ER.qPLEX-RIME  analysisWe further validated this interaction by co-IP. Our analysis robustly found that GRHL2 and ER interact in both MCF7 and T47D cells . Conversely, eRNA production at the TFF1, XBP1, and GREB1 enhancers was moderately increased 24 h after GRHL2 knockdown . Collectively, these data demonstrate that GRHL2 constrains specific ER enhancer transcription.To further explore how GRHL2 regulates ER enhancers, we measured eRNA expression at the GREB1 , 37, TFFp < 0.05, t test, single-tail, paired).A conserved alpha helix between residues 425 and 437 of GRHL2 has previously been shown to inhibit P300 , in threWe undertook H3K27ac ChIP-seq after knockdown of GRHL2 by siRNA for 48 h. In both MCF7 and T47D cells, we saw a significant change in the acetylation marks surrounding GREB1, and in MCF7, we saw an increase at both XBP1 and GREB1 promoters and a decrease at TFF1 Fig. a. GenomeVULCAN is valuable for the discovery of transcription factors acting as co-regulators within chromatin-bound complexes that would otherwise remain hidden. The challenge of highlighting the cofactors from a ChIP-seq experiment lays in the infeasibility of reliable proteomic characterization of DNA-bound complexes at specific regions. On the other hand, while RNA-seq is arguably the most efficient technique to obtain genome-wide quantitative measurements, any transcriptomic approach cannot provide a full picture of cellular responses for stimuli that are provided on a shorter timescale than mRNA synthesis speed, such as the estradiol administration described in our study. VULCAN, by combining RNA-seq-derived networks and ChIP-seq cistrome data, aims at overcoming the limitations of both. Most notably, our method can work in scenarios where candidate cofactors do not have a well-characterized binding site or do not even bind DNA directly.Through comparative analysis, we have robustly shown that VULCAN is able to outperform other readily available methods for the prediction of on-chromatin interactions of transcription factors. VULCAN achieves this through the integration of ChIP-seq and tumor transcriptional data. The inherent limitation of our method therefore is that tumor transcriptional data must be available in sufficient quantity to build the underlying network for the analysis, whereas tools based on predefined networks have no such limitation. In the majority of cases, this is not a challenge as projects like the TCGA provide transcriptome-wide data for a range of cancers. It is therefore only in the cases of rarer disease types (such as neuroendocrine tumor) and orphan tissues that this limitation will be problematic as these are poorly represented in public data. Even so, in these cases where specific networks cannot be generated, pan-tissue regulatory networks are currently being developed to overcome this limitation and these could be adapted for VULCAN .By developing VULCAN, we have been able to rediscover the known cofactors of the estradiol-responsive ER complex and predict and experimentally validate a novel protein-protein interaction.In the 4T1 tumor model, GRHL2 was found to be significantly downregulated in cells that had undergone EMT . The samThese earlier data combined with the link between GRHL2 expression and patient survival indicate a significant role for GRHL2 in the progression of breast cancer. However, its role in the ER signaling axis has, until now, been unknown. Here, we show that GRHL2 performs its activity at a subset of ER enhancers. Overexpression of GRHL2 resulted in a significant decrease in eRNA production at the TFF1 and XBP1 enhancers, and in agreement with previous studies that correlate eRNA transcription with gene expression –49, we fThese results are consistent with previous findings that GRHL2 inhibits P300  and, whiIn breast cancer, GRHL2 has previously been shown to directly interact with FOXA1, which may contribute to the tethering of the histone methyltransferase MLL3 and, consequently, epigenetic marks at GRHL2/FOXA1 binding sites . Our anaKnockdown of GRHL2 led to a genome-wide remodeling of H3K27ac marks, found at active enhancers, confirming a role of GRHL2 in partially regulating these sites. Detailed inspection of the data showed a significant increase of these marks around the XBP1 and GREB1 genes, supporting our hypothesis that GRHL2 has a partial inhibitory role within the ER regulon. The result was further supported by finding enrichment of ER binding events at H3K27ac marks altered by GRHL2 knockdown  across four 15-cm-diameter plates in DMEM with 10% FBS, glutamine and penicillin/streptomycin (Glibco). Five days before the experiment, the cells were washed with phosphate-buffered saline (PBS) and the media were replaced with clear DMEM supplemented with charcoal-treated serum. The media was refreshed every 24 h, which halted the growth of the cells and ensured that majority ER within the cell was not active. On day 5, the cells were treated with estradiol (100 nM). At the appropriate time point, the cells were washed with ice-cold PBS twice and then fixed by incubating with 10 mL per plate of 1% formaldehyde in unsupplemented clear media for 10 min. The reaction was stopped by the addition of 1.5 mL of 2.5 M glycine, and the plates were washed twice with ice-cold PBS. Each plate was then scraped in 1 mL of PBS with protease inhibitors (PI) into a 1.5-mL microcentrifuge tube. The cells were centrifuged at 8000 rpm for 3 min at 4 °C and the supernatant removed. The process was repeated for a second wash in 1 mL PBS+PI and the PBS removed before storing at − 80 °C.MCF7 cells were obtained from the CRUK Cambridge Institute collection, authenticated by STR genotyping and confirmed free of mycoplasma. All cells were maintained at 37 °C, 5% COFrozen samples were processed using established ChIP protocols  to obtaiSequencing data was aligned using BWA  to the hp value correction.Gene set enrichment analysis (GSEA) was performed as described by Subramanian et al.  using thMotif analysis of the binding regions was undertaken with Homer v4.4  using deVIPER BioConductor package, and it was interrogated using the differential binding profiles from our ChIP-seq experiment as signatures, 45 min vs control and 90 min vs control. The peak-to-promoter assignment was performed using a 10-kb window with respect to the transcription starting site (TSS) of every gene on the hg19 human genome. The algorithm msVIPER  was then applied, leveraging the full set of 8 replicates per group, with 1000 signature permutations and default parameters.We reconstructed a regulatory gene network using ARACNe-AP as described by Alvarez . RNA-seqSamples were prepared as previously described for RIME ; the proPublicly available data was downloaded as described in the source publication , 34, 35,MCF7 cells were transfected with Smart Pool siRNA , siControl, GRHL2 overexpression vector , GRHL2 Δ425–437 (Origene), or empty control vector using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol in 6-well format. Expression was monitored by rtPCR using TaqMan assay with GAPDH as a control transcript. Knockdown efficiency was ~ 75%, and the GRHL2 overexpression vector led a 730-fold increase in expression over control plasmid. One microgram of purified RNA was reverse transcribed with Superscript III reverse transcriptase  using random primers  according to the manufacturer’s instructions. eRNAs were quantified with qPCR using Power SYBR™ Green PCR Master Mix  and denoted as relative eRNA levels after normalizing with UBC mRNA levels.ERα (F10) antibody, Santa Cruz (sc-8002), was cleaned using Amicon 10K Buffer Exchange Column  to remove the sodium azide. 2.5 μg ERα (F10) antibody rotated overnight at 4 °C with 100 μL Dynabeads Protein A, Invitrogen (10001D).Nuclear lysate was harvested via cell lysis  followed by nuclear lysis . The uclear lysate was then incubated overnight at 4 °C with the Dynabeads Protein A. Elution via 10 min incubation at 70 °C with 1× NuPAGE LDS Sample Buffer, Invitrogen (NP0007), and 1× NuPAGE Sample Reducing Agent, Invitrogen (NP0004), and subjected to western blotting.Knockdown of GRHL2 was undertaken using ON-TARGETplus SMARTpool Human GRHL2, Dharmacon (#L-014515-02-0050) and Lipofectamine® RNAiMAX Reagent Protocol (Thermo) according to the manufacturer’s protocol. Control samples were prepared following the same method using ON-TARGETplus Control pool Non-targeting pool, Dharmacon (#D-001810-10-50) in place of siGRHL2.Additional file 1:Supplemental figures and tables. (PDF 3249 kb)


Recently, it has become clear that some promoters function as long-range regulators of gene expression. However, direct and quantitative assessment of enhancer activity at long intergenic noncoding RNA (lincRNA) or mRNA gene bodies has not been performed. To unbiasedly assess the enhancer capacity across lincRNA and mRNA loci, we performed a massively parallel reporter assay (MPRA) on six lincRNA loci and their closest protein-coding neighbors.Lincp21, LincEnc1, and Peril, and one mRNA locus, Morc2a, display significant enhancer activity within their gene bodies. We hypothesize that such peaks may mark long-range enhancers, and test this in vivo using RNA sequencing from a knockout mouse model and high-throughput chromosome conformation capture (Hi-C). We find that ablation of a high-activity MPRA peak in the Peril gene body leads to consistent dysregulation of Mccc1 and Exosc9 in the neighboring topologically associated domain (TAD). This occurs irrespective of Peril lincRNA expression, demonstrating this regulation is DNA-dependent. Hi-C confirms long-range contacts with the neighboring TAD, and these interactions are altered upon Peril knockout. Surprisingly, we do not observe consistent regulation of genes within the local TAD. Together, these data suggest a long-range enhancer-like function for the Peril gene body.For both gene classes, we find significantly more MPRA activity in promoter regions than in gene bodies. However, three lincRNA loci, A multi-faceted approach combining high-throughput enhancer discovery with genetic models can connect enhancers to their gene targets and provides evidence of inter-TAD gene regulation.The online version of this article (10.1186/s13059-018-1589-8) contains supplementary material, which is available to authorized users. While a number of lincRNAs function by RNA-based mechanisms , recentlLacZ reporter that is downstream of the endogenous promoter [cis-like regulation) in tissues where the reporter is not expressed implicates DNA and not the act of transcription or RNA product as the regulatory driver. This is because when a region is transcriptionally silent, neither transcription nor an RNA product is present to influence gene expression. Thus, such models allow for the direct detection of DNA-based regulatory elements.To disentangle the contributions of RNA and DNA at a set of developmentally regulated lincRNA loci, we previously generated 18 lincRNA knockout mouse models , 57. In promoter . Thus, rCrnde, LincEnc1, Lincp21, and Tug1, which appear to function as cis-regulators controlling expression in their genomic neighborhoods [cis-acting lincRNA loci  and two lincRNA loci with undetermined regulatory function (Peril and Fendrr), as well as their closest protein-coding neighbors [Morc2a (mRNA), Lincp21, LincEnc1, and Peril (lincRNAs), which harbor high MPRA activity in their gene bodies, consistent with promoter-independent enhancer activity.Using these mouse models, we identified several lincRNA loci, including borhoods , 22. Howeighbors , 21. UsiPeril knockout mouse model to dissect the DNA regulatory roles of the gene body, excluding the promoter, at this locus. We focused on Peril because it has one of the highest gene body MPRA peaks and because it overlaps with a super-enhancer for Sox2, which is a critical stem cell regulator [Peril, we do not observe significant dysregulation of nearby genes . Instead, we find that two genes, Exosc9 and Mccc1, which are distally located (~ 1.5 Mb away) from the Peril locus in the neighboring TAD, are significantly downregulated in all four tissues examined. The downregulation of these genes occurred whether the Peril region was transcribed or silent. Moreover, comparison of high-throughput chromosome capture (Hi-C) data from wild-type and Peril knockout murine embryonic stem cells (mESCs) revealed alterations in physical long-range interactions between the Peril region and the TAD containing Mccc1 and Exosc9 genes. Taken together, our results quantify the presence of gene body DNA regulatory elements within lincRNA loci and identify their corresponding candidate target genes.To determine whether gene body MPRA peaks indicate enhancer activity in vivo, we used a previously generated egulator –25. SurpCrnde, Fendrr, LincEnc1, Lincp21, Peril, and Tug1. To directly compare lincRNA with mRNA loci, we also included the nearest protein-coding gene to each respective lincRNA: Irx5, Foxf1a, Enc1, Cdkn1a, Sox2, and Morc2a, respectively . Using this threshold, we found 45 unique peaks in our lincRNA loci and 28 peaks in our mRNA loci, highlighting that both types of loci can contain enhancers  or gene body-specific  > 2). Morc2a has one 2019-bp high-activity region located in the middle of its gene body  murine tissues and embryonic stem cells -qPCR experiment in Peril WT and KO mESC. We designed multiple sets of primers targeting the promoter regions of both candidate target genes and performed qPCR on Pol II-bound chromatin fragments. Interestingly, we found Pol II to be significantly depleted at both the Mccc1 and Exosc9 promoters in knockout mESC  and CTCF at the very beginning or immediately before the high-activity peak, as well as binding of Rxra , Nr4a1, and Fgfr1 within the peak  on WT and Peril KO mESC  are smaller in size: ~ 900 kb and ~ 1 Mb, respectively  and contains two separate chromosomes: one containing the deleted region sequence and one containing the nce Fig. b. We sounce Fig. ” sectionMccc1 and Exosc9 are located within the + 1 TAD relative to Peril. To gain insight into potential long-range regulatory effects of Peril deletion, we generated digital 4C-like plots anchored on either Mccc1 or Exosc9. Indeed, upon loss of Peril, we observe an alteration of contacts between these loci and the region just upstream of the Peril deletion , we are able to identify novel enhancers as well as their candidate target genes. Together, these results underscore the importance of gene body DNA regulatory elements contained in both lincRNA and mRNA loci.Recent studies have suggested that promoters may play a larger role in reciated –12. SpecPeril locus as a model. The Peril locus overlaps with a Sox2 super-enhancer and forms a tightly associated sub-TAD with the Sox2 locus , we cannot exclude an additional role for the RNA in trans. Consistent with this possibility, we observed numerous gene expression changes in the Peril knockout mESC line which could be due to loss of the RNA product, though this effect could also be due to a cell type-specific effect of reduced Mccc1 or Exosc9 transcript levels.Consistent with our previous report of perinatal lethality among out mice , loss ofethality –50. We fPeril, our study further highlights the DNA regulatory potential contained in promoters and gene bodies regardless of coding classification . We found that both lincRNAs and mRNAs contain cis-regulatory potential in their promoters, as described previously [Lincp21, LincEnc1, and Peril) than mRNA (Morc2a) gene bodies we queried.Beyond eviously –12, 15. Dync1l1 (an mRNA locus) harbors exonic enhancers which regulate nearby genes Dlx5/6, and removal of these regulatory sequences can lead to disease [Lmbr1 acts over 1 Mb to regulate Shh expression in the developing limb [Knockout Mouse Project (KOMP) and the International Mouse Phenotyping Consortium (IMPC)) [The presence of clear enhancer activity in these loci has important implications for our current understanding and interpretation of lincRNA knockout models, which have been the focus of intense debate , 22. Spe disease . Similaring limb . Current (IMPC)) , 52–54. Peril regulates two distally located genes (Mccc1 and Exosc9) and not its neighbor Sox2. Despite close proximity of the Peril locus with Sox2, we find that neither the DNA nor the RNA in the Peril locus affect Sox2 expression. Moreover, we find that ablation of Peril does not affect the local chromatin architecture, but does affect long-range interactions with Mccc1 and Exosc9 in the neighboring TAD. Together, our combined approach is able to identify DNA regulatory elements in lincRNAs and their candidate regulatory targets in vivo.Here, we conclude, by combining MPRA, in vivo deletion/reporter models, and Hi-C, that both mRNAs and lincRNAs can contain DNA regulatory elements in their gene bodies. Further investigation of one of these DNA elements in vivo revealed a surprising finding: that The MRPA pool was designed as previously described , 55, witC2C12 cells were acquired from ATCC (CRL-1772) and cultured in DMEM supplemented with FBS and antibiotics.p values were corrected using the Benjamini-Hochberg method with an FDR of 1%. All analysis was performed using the aid of in-house python and R scripts, with aid from biopython and R’s bioconductor packages. Oligo annotation is available as Additional file https://github.com/agroff11/PerilPaperAnalysis) and on zenodo (10.5281/zenodo.1477113) [Analysis was performed largely as described previously  with thr1477113) .Peril knockout strain was originally generated by Regeneron Pharmaceuticals [Mice were housed under pathogen-free conditions at Harvard University. A description of the mice used in this experiment is available as Additional file euticals , 57.Peril knockout murine ESCs were generated by the Harvard University Genome Modification Facility from D3.5 embryos. After receiving clones from the core, mESC colonies were transitioned to 2i on feeder MEFs and cultured for six passages. Three individual wells  were collected as biological replicates.Wild-type and Three biological replicates of the kidney and two biological replicates of microdissected ganglionic eminence and olfactory bulb (GE/OB) were harvested from E14.5 progeny of heterozygous matings. Tissues were pulverized in TRIzol and snap frozen.+ libraries from 250 ng total RNA for each tissue replicate and 500 ng total RNA for each mESC replicate. Samples were sequenced on a Hiseq 2500 at Harvard University’s Bauer Sequencing Core.The Illumina TruSeq kit was used to create polyAPeril were assessed visually to confirm knockout status in combination with expression status of LacZ. All RNAseq figures were generated in R with the help of packages available on bioconductor. Code is available as Additional file https://github.com/agroff11/PerilPaperAnalysis) and on zenodo (DOI: 10.5281/zenodo.1477113) [Reads were aligned to mm10 and quantified using rsem with nonstandard parameters --paired-end --bowtie2 --append-names and -p 8 . Read co1477113) .cDNA was generated using SuperScriptIII (Thermo Fisher), and qRT-PCR analyses were performed using an ABI7900HT Fast Real-Time PCR system (applied biosystems). Data was analyzed using qPCR-miner , and thePeril gene body for matches to any of the expressed transcription factors on a per-sample-type basis using FIMO, a motif-finding tool publically available through the meme suite [q value < 0.05 and plotted the binding profiles of the common set of expressed hits. Additionally, we assessed publically available TF binding via the ChIP-atlas [We downloaded the entire set of motifs available through JASPAR  and filme suite . We filtIP-atlas .Peril knockout mESC were crosslinked for 10 min in 1% formaldehyde. Chromatin was sheared with the following parameters: 15 min of 30 s “on” and 30 s “off” using the Bioruptor (Diagenode). ChIP experiments were performed using 2 μL of Pol II monoclonal antibody  per ChIP, and as negative control, 1 μg of IgG was used in a separate ChIP. For downstream enrichment calculation, 1% of the input chromatin was retained. The occupancy of Pol II at the promoter of Mccc1 and Exosc9 was measured by real-time qPCR using two primer sets targeting different regions of the promoters. qPCR was performed using FastStart Universal SYBR Green Master (Rox) , and reactions were run on ViiA7 Real-Time PCR System (ABI). Primer sets used in the experiment targeted Pol II-enriched regions near the target gene promoters as well as control regions: F1_Mccc1: CAAGTAGGCGTCTCCGCAA; R1_Mccc1: GGTCCAATCAGCCAATCGGTA; F2_Mccc1: ATTCTTGAAAGCGTCCCTCCC; R2_Mccc1: CTTGCGGAGACGCCTACTTG; F3_Exosc9: ACATTGTCAGTTCGGCCTGT; R3_Exosc9: AATACTGCCCTGAGGCTTGG; F4_Exosc9: GTGTGGACTCTCCCCATTCC; R4_Exosc9: TCCCTGCCACTGAATACTGC; F_Pol2_positive_region: CTGGCACTGCACAAGAAG; R_Pol2_positive_region: GGGTTCCTATAAATACGG; F_Pol2_negative_region: CTGGCCTCCATACACACATA; R_Pol2_negative_region: AGTCAGCAGGATCCACACTT. Enrichment was calculated by the percent input method.ChIP experiments were performed using the iDeal ChIP-qPCR kit , and experiments were carried out according to the manufacture protocol. Briefly, approximately 16 million wild-type or 16 million HindIII restriction enzyme [HindIII, end-labeled with biotin-14-dCTP, followed by an in situ ligation procedure. After DNA extraction, biotin was removed from unligated ends, and the sample was sheared by using a Covaris S220 instrument (100–500 bp range). After A-tailing, biotin pull-down and adapter ligation, paired-end sequencing was performed on a HiSeq instrument. Each Hi-C library was generated in two biological replicates, and each replicate was sequenced to a depth of ~ 80 million reads. Hi-C mapping, filtering, iterative correction, and binning were performed with the HiC-Pro software v2.8 [Peril region with Ns and then included this sequence as well as the LacZ reporter sequence as two separate chromosomes.In situ Hi-C libraries were generated using the n enzyme , 33. Briare v2.8 . We usedcworld package (https://github.com/dekkerlab/cworld-dekker) [cworld package “matrix2anchorplot.pl” script and with the HiC-Pro “make_viewpoints.py” script by using either the Mccc1 or the Exosc9 loci as anchor points. To do this, we calculated the Hi-C interaction frequency between the Mccc1/Exosc9-containing bins and the neighboring 20-kb bins. WT interactions were mapped to the mm10 reference genome while KO interactions were mapped to our custom mm10 genome build. For each distance, the ± 1 standard deviation of all the Hi-C interaction frequencies of that distance (dashed lines in the figure) were also calculated. The interactions that were above the + 1 standard deviation line  were considered as enriched interactions. To investigate the interactions between the Peril region and the neighboring TAD, we first investigated the HindIII restriction sites in the deleted region or LacZ “chromosomes.” We found that the “deleted region chromosome” contains three cut sites while the “LacZ chromosome” contains none. We thus selected the 3′ shoulder region . This region is shared between WT and KO and is not affected by the deletion. While not specific to the deleted sequence, it can be used as a proxy for alterations in interactions between WT and KO in the deleted region. To generate digital 4C-like bedgraphs anchored on this shoulder region, we plotted either raw reads originating in this region and ending on chromosome 3, or a boxplot of all normalized reads originating from this region and ending in the + 1 TAD. The boxplot was generated using the custom genome for both WT and KO samples.There was a high correlation among the Hi-C biological replicates, and so we pooled all biological replicates for each condition and analyzed them as a single Hi-C dataset. This pooled dataset was used for all the analyses. The TAD analysis was performed with the insulation method, as previously described , by usin-dekker)  “matrix2Additional file 1:MPRA oligo annotation - File containing annotation information used to associate unique barcode tags to synthesized genomic elements in the MPRA experiment. (TXT 7035 kb)Additional file 2:TSS annotation – File containing gencode TSS information for loci used in this study. (TXT 891 bytes)Additional file 3:Supplemental Figures - File contains Figures S1-S4 and their legends. (PDF 5159 kb)Additional file 4:MPRA analysis code - R markdown file containing all code used in the MPRA analysis. (RMD 38 kb)Additional file 5:Mouse master sheet - File containing description of mice used in this study, and information about generation of RNA sequencing libraries. (XLSX 52 kb)Additional file 6:RNA seq analysis code - R markdown file containing all code used in the RNA-sequencing analysis. (RMD 15 kb)


Glutamine synthetase (GS) features prominently in bacterial nitrogen assimilation as it catalyzes the entry of bioavailable nitrogen in form of ammonium into cellular metabolism. The classic example, the comprehensively characterized GS of enterobacteria, is subject to exquisite regulation at multiple levels, among them gene expression regulation to control GS abundance, as well as feedback inhibition and covalent modifications to control enzyme activity. Intriguingly, the GS of the ecologically important clade of cyanobacteria features fundamentally different regulatory systems to those of most prokaryotes. These include the interaction with small proteins, the so-called inactivating factors (IFs) that inhibit GS linearly with their abundance. In addition to this protein interaction-based regulation of GS activity, cyanobacteria use alternative elements to control the synthesis of GS and IFs at the transcriptional level. Moreover, cyanobacteria evolved unique RNA-based regulatory mechanisms such as glutamine riboswitches to tightly tune IF abundance. In this review, we aim to outline the current knowledge on the distinctive features of the cyanobacterial GS encompassing the overall control of its activity, sensing the nitrogen status, transcriptional and post-transcriptional regulation, as well as strain-specific differences. This enzyme reduces the inert triple bond of N2 by providing enormous reducing power to yield bioavailable, dissolved inorganic N in the form of ammonium, which can be taken up and assimilated into organic forms by most microorganisms [Nitrogen (N) is an essential element to all life on earth as it is a significant fraction of crucial biomolecules such as amino acids, nucleic acids and a myriad of derivatives. Despite being the most common pure element in the atmosphere, molecular dinitrogen  catalyzes the reductive, NAD(P)H/H+-dependent amination of 2-oxoglutarate (2OG) to yield glutamate and NAD(P)+, and was regarded as the main enzyme of ammonium assimilation in bacteria for a certain period [+-dependent), and EC 1.4.7.1 (ferredoxin-dependent)). In the GS/GOGAT cycle GS facilitates the first step of N assimilation, the conversion of inorganic into organic N by incorporating ammonium into glutamate and yielding glutamine , and Streptomycetaceae families [The family of GSs constitutes one of the oldest existing and operational gene families . GSs arefamilies ,17,18, Gfamilies . The thi+, serine, and alanine, as well as glycine. These intermediates orchestrate a gradual inhibition of GS that mounts depending on the number of bound inhibitors, which is designated cumulative feedback inhibition [GSI (hereafter referred to as GS) consists of two superimposed hexameric rings, which are arranged in a centrosymmetric structure. The GS of enterobacteria is one of the best investigated and most comprehensively regulated enzymes known and represents the prevailing paradigm of bacterial GS regulation, which is addressed in most scientific textbooks. Enterobacterial GS is subject to feedback inhibition by several molecules of N and energy metabolism, which include among others tryptophan, histidine, carbamoyl phosphate, CTP, AMP, glucosamine-6-phosphate, NADhibition ,20,21. Thibition  and AMP hibition  while thII switches between uridylylated and unmodified states [II is catalyzed by the bifunctional uridylyltransferase (UTase) and depends on 2OG [II by UTase requires glutamine [In addition, the enterobacterial GS is subject to posttranslational regulation by reversible adenylylation of specific tyrosine residues in each of the 12 enzyme subunits, which in turn raises the sensitivity of GS for feedback inhibition. The corresponding regulatory circuits, which govern the modification state of GS, are complex and involve two connected cycles of reversible protein nucleotidylation . In the d states ,25, whild states ,27. Urids on 2OG ,25,28,29lutamine . In an alutamine ,32,33,34In general, sensing of the cellular C/N status is of utmost importance for regulating N uptake and assimilation to ensure the maintenance of metabolic homeostasis. Since 2OG provides the carbon skeleton for the amidation reaction in the GS/GOGAT cycle but also participates in the tricarboxylic acid (TCA) cycle, it constitutes a branching point between carbon (C) and N metabolism, and is subject to fluctuations according to the cellular N status. Hence, 2OG is an indicator of C as well as N availability and its level accumulates in response to N limitation . MoreoveII and GS nucleotidylation cycles is ensured by the modification state of PII: adenylylation, and thus inactivation of GS by ATase strictly depends on the binding of unmodified PII, which accumulates during N-excess and high glutamine levels. On the other hand, deadenylylation and thus activation of GS by ATase only occurs upon binding of covalently modified PII(UMP), which accumulates during N limitation promoted by 2OG [A tight connection of the Pd by 2OG ,29,36. Hd by 2OG ,37.glnA gene, which encodes the GS monomers . In sumview see ).Bacillus subtilis, which functions as both enzyme and regulator, is subject to feedback inhibition but devoid of posttranscriptional control [II-like protein. TrnA binding to GS triggers partial inhibition of enzymatic activity and ameliorates the impact of feedback inhibitors, especially of glutamine. In addition, GS binding obstructs DNA binding of TrnA. Under N-limiting conditions, GlnK forestalls interaction of TrnA with active GS, rendering the enzyme fully active [Clostridium saccharobutylicum NCP262, an endospore-forming obligate anaerobe, regulation of ammonium assimilation underlies a yet not fully understood mechanism encompassing post-transcriptional regulation of core enzymes via antisense RNAs as well as a specific RNA-binding regulatory protein of the ANTAR family, termed NitR [II-like protein GlnK was shown to increase GS activity in the presence of 2OG, while low 2OG levels thereof triggered GS inhibition by GlnK [GS regulation in enterobacteria appears relatively uniform, though investigation of an increasing number of bacteria beyond the enterobacterial family revealed striking differences in GS regulation among virtually every family investigated during the last two decades. Nevertheless, the assimilatory enzymes and several regulatory proteins involved appear to be mostly conserved in prokaryotes. For instance, the bifunctional GS of low G+C Gram-positive bacteria like  control . During y active ,43. In Cmed NitR , which imed NitR . Another by GlnK ,47. TakeProchlorococcus strains, for instance, account for 25% of the ocean’s net primary production [2 and simultaneous depletion of the majority of atmospheric carbon dioxide, which fundamentally affected living conditions on earth and paved the way for the evolution of aerobic metabolism and complex life [As the evolutionary ancestors of chloroplasts, cyanobacteria represent a very ancient lineage and are the only bacterial group capable of performing water-splitting, oxygenic photosynthesis by combining the reductive power of two photosystems. This phylum shows substantial genomic and morphological divergence with unicellular and multicellular lifestyles, the latter showing a high degree of organization and differentiation. Cyanobacteria can be encountered in nearly any light-exposed habitat ranging from arctic regions, desert soils, rock surfaces, hot wells, and hypersaline lakes to fresh-water ecosystems, as well as ultraoligotrophic marine realms ,50,51,52oduction . Considelex life .2 fixation, tightly regulate their N assimilatory machinery and undergo substantial adaptations with respect to N availability [13N-labeling experiments unequivocally established GS/GOGAT as the main N assimilatory pathway [For cyanobacteria, N represents a vital nutrient and accounts for up to 10% of their dry weight . This islability . For inslability . Cyanobalability . Notwithlability . Shortlylability , the occlability ,60,61 an pathway ,63,64. A pathway .glnA-deficient E. coli mutant [E. coli, GS of various cyanobacteria showed lower Km values and a higher sensitivity towards common GS inhibitors such as a variety of amino acids [The cyanobacterial GS shares substantial similarities with the GSI of other bacteria, illustrated by the fact that it is able to complement a i mutant . Neverthi mutant ,68,69. Mno acids .glnA gene is regulated with respect to the nature and abundance of the N source. N excess, e.g., induced by ammonium supplementation, results in low glnA expression, while cells grown on nitrate as sole N source feature elevated glnA levels only exceeded by N-starved cells [glnA in a wide range of bacteria, is absent in cyanobacteria [amt1 encoding an ammonium transporter) and other N-related genes including glnA as well as ntcA itself [In cyanobacteria, the expression of the ed cells ,73,74,75bacteria ,66. Instbacteria ,77. NtcAbacteria . Under NA itself ,78,79,80II with the co-activator protein PipX, which modulate NtcA activity with respect to the cellular N status [glnA gene is summarized in 8-TAC [II protein [glnA  [gifA and gifB (glutamine synthetase inactivating factor A and B) with IF7 bearing remarkable resemblance to the C-terminal moiety of IF17 [In the early 1990s, GS in cyanobacteria was shown to be subject to a pronounced short-term inactivation upon addition of ammonium to nitrate grown cells . Neverthocystis) . These t of IF17 . The regSynechocystis unequivocally demonstrated that both factors act independently from each other and do not require additional modifications to exert a concentration-dependent inactivation of GS [ΔgifA and ΔgifB single as well as a ΔgifAgifB double knockout strains rendered GS partially or fully active when grown with ammonium, demonstrating that the proteins encoded by these genes are exclusively responsible for GS inactivation and thus constitute a major layer of GS regulation in cyanobacteria [In vitro analysis of heterologically expressed IF7, IF17, and GS of on of GS . Remarkaon of GS . Generatbacteria .Synechocystis IF7 with GS both in vivo and in vitro [In light of the reactivation of ammonium-inactivated GS by increasing pH values  and the in vitro . Substitin vitro . In contin vitro .Synechocystis vary in several aspects. IF17 is subject to proteolytic degradation stimulated by the absence of ammonium [gifB transcription. The mechanisms involved in the different IF17 degradation pattern, however, were not identified but may include the 82-residue N-terminal moiety, which is not present in IF7 [The stability of both GS inactivating factors is promoted by the interaction with GS in vivo . Neverthammonium . In contammonium . Interest in IF7 ,97. Thist in IF7 . Even tht in IF7 . This ist in IF7 . Synechocystis and the filamentous, dinitrogen-fixing strain Anabaena sp. PCC 7120 (hereafter referred to as Anabaena) suggested a possible coevolution of cyanobacterial GS and their corresponding IFs [Recently, analysis of chimeric enzymes of ding IFs . Each ending IFs . Furtherding IFs . Complexding IFs .Synechocystis revealed that expression of gifA and gifB was strongly increased upon ammonium addition while diminished expression of both genes was observed under N deficiency [gifA and gifB transcript accumulation is in accordance with the observed inactivation of GS in the presence of ammonium. Consequently, sequence analysis of the upstream regions of the gifA and gifB genes revealed potential binding sites for the global N transcriptional regulator NtcA, which center at the core promoter elements at −30.5 and −7.5 bp, respectively, thereby potentially mediating repression of the downstream genes. This assumption was confirmed as a non-segregated NtcA mutant featured constitutive expression of gifA and gifB, independent from the N source [gifA and gifB transcription is repressed by NtcA binding and therefore inversely regulated to the GS structural gene glnA, which is activated by NtcA simultaneously [Transcriptional analyses in ficiency . TherefoN source . Upon N-aneously . Thus, NSynechocystis in which GS inactivating factors were discovered first, cyanobacterial GS inactivating factors can be subsumed into IF7-like and IF17-like classes with the exception of genome-streamlined marine picocyanobacteria like Prochlorococcus, which appear to lack full-length homologs [Thermosynechococcus elongatus features two proteins homologous to IF17 while Anabaena harbors only one IF7-like inactivating factor (IF7A). Functional characterization of the IF7/gifA homolog of Anabaena confirmed that GS is also subject to IF-mediated inhibition in this cyanobacterium [Synechocystis. Expression of the IF7A-encoding gifA gene is also regulated by the global N transcriptional regulator NtcA in Anabaena but in contrast to gif promoters in Synechocystis, the upstream region harbors two NtcA binding sites. One site is centered at the −28.5 position, indicating a repressive role of NtcA for this promoter as the binding site features the very same distance to the −10 box in the NtcA repressed gifA promoter of Synechocystis [gifA gene expression as several NtcA-activated promoters were shown to feature motifs upstream of −41.5 [gifA expression upon NtcA binding in Anabaena is conflicting and the biological meaning is not understood. Nevertheless, gifA expression in Anabaena is strongly increased upon addition of NH4+, similar to the situation in Synechocystis [gifA gene in the ntcA mutant strain CSE2. This mutation leads to a premature stop codon, preventing synthesis of IF7A. Complementation of a WT gifA gene in the ntcA mutant strain resulted in poor growth, which points towards IF7A toxicity if gifA is not repressed by NtcA und thus de-regulated. Hence, the existence of two independent IFs in Synechocystis might explain why there is no fully segregated ntcA knockout mutant available for that strain thus far because single mutations, e.g., in gifA would not compensate the steady inactivation of GS.Derived from the IF7/IF17 system of homologs . Most cyhomologs  but otheacterium . Neverthhocystis . Intereshocystis . Despiteof −41.5 ,100. Thehocystis . In addiSynechocystis for instance, the joint action of IF7 and IF17 during N-rich conditions rapidly inhibits GS activity and thereby, quickly restore the intracellular 2OG levels, which leads to repression of the gif genes once again. On the contrary, in strains harboring solitary IFs such as Anabaena this process is less effective, thus a rather continuous expression of gif genes is required to maintain appropriate levels of GS activity [In general, it can be stated that inactivation of GS is more efficient in strains, which possess two IF homologs. In activity ,101.In addition to transcriptional regulators, bacteria possess numerous and diverse means of RNA-mediated gene regulation. These regulatory RNA elements do not encode proteins and thus are non-coding. A major group of those non-coding RNAs (ncRNAs) can activate or repress gene expression at the post-transcriptional level by complementary base pairing with mRNAs. Those small regulatory RNAs (sRNAs) show short, imperfect base pairing interactions but frequently overlap with sequences required for translation initiation and hence, contribute to the specific and customized synthesis of the respective proteins ,103,104.Anabaena and named N stress-induced RNAs . Nevertheless, their detailed functions are yet to be elucidated [Synechocystis, the sRNA NsiR4 was verified as one of the most strongly induced transcripts during N-limiting conditions [Anabaena [Synechoccoccus sp. PCC 7002 [Synechocystis sp. PCC 6714 [Synechocystis and Anabaena [In recent years, biocomputational predictions and studies mapping TSSs on a genome-wide scale gave rise to a plethora of sRNAs in cyanobacteria ,107,108.ucidated ,109,110.nditions . Albeit nditions . These fAnabaena , SynechoPCC 7002  and SynePCC 6714  revealedAnabaena .gifA gene was computationally predicted as the most promising NsiR4 target and the direct interaction of both RNAs was experimentally verified [gifA mRNA abundance while NsiR4 overexpression decreased gifA transcript abundance compared to the WT, i.e., after adding ammonium. Subsequent analysis clearly demonstrated that the interaction of NsiR4 with the gifA transcript negatively affects IF7 protein synthesis and in turn also impacts GS activity [gifA by NtcA is obviously not sufficient to control GS activity and hence, an additional post-transcriptional mechanism has evolved that helps to determine whether or not IF7 is produced  motifs and annotated as RF01739 and RF01704 in the Rfam database [glnA motif of Synechococcus elongatus as well as the DP motif of Synechococcus sp. CC9902 were shown to alter their secondary structure upon selective binding of l-glutamine in vitro, which classified them as glutamine-binding aptamers [Yet another type of ncRNAs are riboswitches, which fulfill integral regulatory tasks in bacteria ,118,119.database , respectaptamers . For theII, and ammonium transporters as reported previously [Synechocystis it was experimentally proven that the glutamine binding aptamer present in the 5’UTR of gifB undergoes structural modulations, which in turn impact the production of IF17 [glnA aptamer represents the metabolite-sensing domain of a glutamine-binding riboswitch, thus renaming to type 1 glutamine riboswitch was proposed. Investigation of a DP aptamer of a Prochlorococcus strain revealed that these elements are also part of functional glutamine riboswitches and activate gene expression upon glutamine binding similar to the type 1 glutamine riboswitch. Hence, for those containing the DP aptamer the designation type 2 glutamine riboswitch was suggested [Recently, detailed analysis revealed a widespread occurrence of the glnA aptamer in cyanobacterial strains. The DP aptamer, however, is apparently restricted to the monophyletic group of marine picocyanobacteria where it frequently exists in multiple copies . Within eviously  appear t of IF17 . These ruggested .gifA and gifB by NtcA was considered to be exclusively responsible for the control of GS activity in cyanobacteria [The glutamine riboswitches are restricted but common to cyanobacteria and their discovery added an important piece in the puzzle of glutamine synthetase regulation in this bacterial group . Previoubacteria ,99. Nevebacteria . Hence, Prochlorococcus [Prochlorococcus diverge in genetically distinct ecotypes along the water column, mainly caused by the aptitude to cope with different light intensities resulting in a tailor-made outfit of photosynthetic pigmentation for the given light conditions [Prochlorococcus strains to flourish in N-poor environments of the open ocean.Marine picocyanobacteria constitute a monophyletic group within the cyanobacterial realm, which displays several remarkable traits. One representative that attained the reputation of a model organism for marine ecology is the genus rococcus . Prochlonditions . While tnditions ,131 it inditions . The extnditions ,133 acconditions  and a londitions . N limitnditions . Thus, Nnditions  and the nditions  allow PrII, the key proteins for N sensing and regulation of N assimilation in cyanobacteria [Prochlorococcus illustrated that N shortage causes a different response compared to other cyanobacteria [Prochlorococcus MED4 was shown to be substantially enhanced during long-term N starvation, while GS protein levels remained relatively constant under these conditions [Despite the cost-minimizing loss of proteinaceous regulators, these strains retained NtcA and Pbacteria . Albeit bacteria ,140. Fornditions .Prochlorococcus. These display rather stable N supplies rendering excessive GS regulation superfluous and costly [glnA gene was shown to be one of the top responding transcripts during N stress in Prochlorococcus and appears to be regulated by NtcA, consistent with observations made in other cyanobacteria [Prochlorococcus GS for metal-catalyzed oxidation (MCO) prior to degradation upon N limitation [E. coli and B. subtilis [Prochlorococcus strains.The steady GS abundance in response to N availability probably resulting from an inexpensive, less sophisticated regulation of this pivotal enzyme may impart evolutionary advantages in habitats occupied by d costly . Neverthbacteria . These cmitation ,145, whisubtilis ,147. ThiSynechocystis [Anabaena could not be inhibited by IF7 and IF17 of Synechocystis in vitro [PMM1846 of Prochlorococcus strain MED4  encodes a small protein of 7 kDa that exhibits striking resemblance to IFs of other cyanobacteria. It bears DUF4278, homologous to the N-terminus of IF17, and harbors a glutamine riboswitch in its 5’UTR that boosts gene expression during N excess [PMM1846 shares several physicochemical properties with IFs, which are characteristic for proteins that partake in electrostatic protein-protein interactions such as the prevalence of “electrostatic targeting residues” like arginine [Most compelling, the previously described type 2 glutamine riboswitch, which always occurs in multiple copy numbers and appears restricted to marine picocyanobacteria, is functionally associated to genes encoding for DUF4278 containing proteins. Intriguingly, these proteins share substantial similarities with the N-terminus of IF17 , which whocystis . Previouhocystis ,142. Nevin vitro . For insN excess . Moreovearginine . NeverthglnA-deficient mutant of Synechococcus sp. PCC 7002, which was able to grow without glutamine and retained most of the wild-type glutamine biosynthetic activity [Synechocystis glnA mutant strain, which exhibited WT like growth in nitrate-containing media, devoid of glutamine supplementation strongly supported the idea of an additional GS enzyme, which facilitates N assimilation during glnA deficiency [glnA deficient E. coli mutant strain with a Synechocystis library led to the discovery of the glnN gene encoding a GS enzyme, which only shares a minor resemblance with the amply investigated GSI and GSII mostly restricted to 17 strongly conserved residues in the predicted ATP and cation binding sites. However, the protein was found homologous to the GSIII, which was previously described in the family of Bacteroidaceae with a sequence-derived subunit size of 75 kDa and a putative hexameric arrangement [glnN expression is substantially affected by the N source as ammonium supplementation suppresses glnN expression while N-free media strongly induced glnN transcript accumulation. Being dispensable for N assimilation and WT-like growth rates, GSIII accounts for 3% of total GS activity in nitrate-grown Synechocystis cells [glnN-deficient strains [Synechocystis glnA/glnN double mutant was not viable illustrates the fact that there are most likely no other GS enzymes present in this strain [glnN gene whose expression is induced upon N starvation, consistent with observations made in Synechocystis, while diazotrophic cyanobacteria apparently lack the glnN gene [glnN promoter sequence features an imperfect NtcA consensus motif as well as N-dependent expression patterns, first attempts to show E. coli-expressed NtcA binding failed and it was assumed that interaction requires additional factors [glnN is another NtcA regulated gene in Synechocystis [Synechococcus sp. PCC 7942 GSIII was shown to promote the synthesis of phycobiliproteins when chlorotic cells were supplied with limiting amounts of combined N [Synechocystis and other GSI bearing strains is regulated by IFs. Given that GSIII in Synechocystis only occurs upon prolonged N starvation in physiologically relevant levels, a circumstance that causes fully active enzymes [Synechococcus sp. WH7803 revealed an unusual regulation of this enzyme as GSIII activity in this strain is highly responsive to light-dark changes while being unaffected by N availability [Synechococcus sp. WH7803. Given that all sequenced Prochlorococcus strains lack the glnN gene, it appears that GSIII was lost in the course of streamlining in environments with stable nutrient availability [Synechococcus sp. WH7803 could be on the verge of losing its GSIII and the loss of its regulation by N availability might be the first step in this process.As mentioned, GS splits in three distinct classes, which significantly differ in subunit size, subunit arrangement and sequence. First assumptions that another glutamine synthesizing enzyme exists in cyanobacteria were furnished by a activity . Shortlyficiency . Subsequangement ,148,149.is cells . In cont strains . Moreoves strain . Subsequs strain ,150. Furs strain ,150. Con factors . Subsequhocystis ,99. In lmbined N . Hence,  enzymes , IF-depelability . Thus, tlability  appears lability . TherefoglnN-bearing strains, Pseudanabaena 6903 was shown to lack a glnA gene and thus was the first known cyanobacterium in which only GSIII accounts for the biosynthesis of glutamine [Synechococcus sp. RS9917 revealed that this marine cyanobacterium possesses two GSIII enzymes and lacks GSI [Synechocystis where nitrate-supplementation causes basal GSIII levels, glnN expression is strongly induced in nitrate-grown as well as N-starved cells of Pseudanabaena 6903, while ammonium addition triggers a rigorous inhibition of enzymatic activity accompanied by steady protein levels. This was not observed when N-starved cells were subjected to nitrate [Pseudanabaena 6903 is subject to a yet unknown, rapid, ammonium-dependent inactivating mechanism, as minor GS activity would already be sufficient to ensure proper N supply under these conditions. Interestingly, glnN transcription in Pseudanabaena 6903 was also shown to be controlled by N, which is supported by the fact that the glnN promoter harbors an imperfect NtcA consensus sequence which is bound by NtcA of Synechocystis [glnN expression in Pseudanabaena 6903 is similar to the N-dependent control of glnA expression in Synechocystis as NtcA strongly induces the transcription of N-related genes during growth on nitrate or N-depleted media [glnN transcription, Pseudanabaena 6903 deploys sufficient levels of its sole GS enzyme to meet metabolic demands during N-limiting conditions [Pseudanabaena 6903, and thus the question whether GSIII is subject to protein-mediated regulation or an utterly different mechanism in this strain remains unanswered.Intriguingly, one of the previously discovered lutamine . More reacks GSI . In cont nitrate . These fhocystis . Thus, ted media . Thus, bnditions . Unfortu2. However, their rising importance as microbial cell factories in biotechnology is contradicted by the current state of knowledge about metabolic regulation. As exemplified by the regulation of glutamine synthetase, cyanobacteria differ from other bacterial groups in many aspects. Hence, data that were obtained from genetic models like E. coli or B. subtilis can often not be applied. The intriguing example of cyanobacterial GS regulation suggests that further metabolic reactions might be controlled differently compared to other bacteria and that many regulatory factors still await discovery and/or characterization. Altogether, further basic research is required to make cyanobacteria more definable for metabolic engineering and thus also amenable for biotechnological applications.Due to their photosynthetic lifestyle cyanobacteria are interesting hosts for the production of fuel components and valuable chemicals using light and CO


Indeed, their frequency progressively increases passing from indolent lesions to aggressive and anaplastic forms. TERT promoter mutations create binding sites for transcription factors, increasing TERT expression and telomerase activity. Furthermore, aggressiveness of TCs is associated with TERT locus amplification. These data encourage investigating telomerase regulating pathways as relevant drivers of TC development and progression to foster the identification of new therapeutics targets. Here, we summarize the current knowledge about telomere regulation and TCs, exploring both canonical and less conventional pathways. We discuss the possible role of telomere homeostasis in mediating response to cancer therapies and the possibility of using epigenetic drugs to re-evaluate the use of telomerase inhibitors. Combined treatments could be of support to currently used therapies still presenting weaknesses.Telomere and telomerase regulation contributes to the onset and evolution of several tumors, including highly aggressive thyroid cancers (TCs). TCs are the most common endocrine malignancies and are generally characterized by a high rate of curability. However, a small but significant percentage develops distant metastasis or progresses into undifferentiated forms associated with bad prognosis and for which poor therapeutic options are available. Mutations in  Telomere protection and/or elongation are considered among the primary mechanisms of cancer survival and aggressiveness. Telomeres, shortening at each mitosis, sense somatic cells aging and induce cell death when they become critically short. Thus, progressive reduction of telomere length is a rate-limiting step for uncontrolled and unlimited cell proliferation capacity. Telomerase is the enzyme responsible for telomere maintenance. It is a ribonucleic complex that includes the telomerase reverse transcriptase (TERT) and its RNA template, the Telomerase RNA component (TERC) . TelomerTERT expression and/or activity has been reported in many cancers and associated with increased aggressiveness. Among the mechanisms leading to aberrant telomerase activation, TERT promoter mutations C228T and C250T  are frequent in cancer and associated with increased aggressiveness and metastatic potential in several tumor settings. These alterations cause the formation of ectopic binding sites for specific transcription factors (TFs) within the TERT promoter inducing its overexpression [ATRX chromatin remodeler (ATRX) and death domain associated protein (DAXX) genes in both tumors and cell lines [TERT promoter and ATRX have been reported to be mutually exclusive. However, melanomas with loss-of-function mutations in ATRX have also been found to have TERT promoter mutations [Being the sensor of cell proliferative capacity, aberrant telomere elongation occurs in the majority of cancers due to telomerase reactivation  and/or alternative lengthening of telomeres (ALT) . Alteratpression . Similarll lines ,11,12. Iutations .Thyroid cancers (TCs) are the most common endocrine malignancy. Metastasis and aggressive clinical behavior in these tumors are generally limited to a small but significant group of high-grade lesions  with poor biological characterization and very limited therapeutic options . These tTERT promoter are strongly over-represented in highly aggressive TCs, with the C228T usually being more frequent than C250T [TERT promoter mutations consistently present distinct transcriptomic profiles associated with enhanced cell cycle progression and metabolic activities as compared to wild-type patients [TERT promoter mutations are markers of malignant transformation, since no evidence of their occurrence in benign thyroid lesions has been reported so far. Furthermore, TERT promoter mutation frequency increases dramatically from low-grade microcarcinomas  V600E mutation, considered a major oncogenic determinant of TC. Patients harboring both BRAF V600E and TERT promoter mutations display more severe clinical factors, suggesting that these mutations contribute to different molecular mechanisms resulting in synergic effects [TERT promoter mutations and RAS mutations, which are frequent in PDTCs and ATCs [TERT promoter mutations, but also duplication of TERT locus on chromosome 5 [TERT locus in the progression of TC. Furthermore, THYT1 may represent a prognostic marker to improve risk-based stratification and management of pre-operative PTCs.Mutations in the an C250T ,18,19,20an C250T ,21,22,23patients . As in oiagnosis . In lined tumors . Interes effects ,18,27. Aand ATCs ,19,21. Tmosome 5 . These dTERT promoter mutations occur within non-coding elements, and their mechanism of action has only partially been elucidated. Both of the most frequent mutations (C228T and C250T) introduce within the TERT promoter new binding sites for members of the E-Twenty-Six (ETS) family of transcription factors. The increased activity of these TFs enhances TERT expression, leading to an augmented telomerase activity [TERT transcription activation. Different substitutions in the same position like G228C, G250C or G250T showed no effect on TERT transcription. In contrast, the G228T mutation partially induced promoter activity, being collocated in a position of ETS motif frequently degenerated for A/T. To identify which among the ETS transcription factors was responsible for TERT activation, a small interfering RNA screening of 13 molecules was conducted. The GA-binding protein alpha subunit (GABPA) KD resulted in a substantial decrease in TERT expression. Consistently, only GABPA KD impaired mutant promoter activity, despite having no effect on the wild type sequence in Luciferase assay indicating that GABPA is the only ETS factor able to affect TERT expression in presence of cancer-associated mutations [Differently from the vast majority of cancer driver mutations that fall within the coding region of oncogenes and/or tumor suppressors altering the resulting proteins, utations . In contutations .TERT promoter is also affected by the presence of cancer-associated mutations reflecting the transcriptional activity of this region. The mutant TERT promoter exhibits high levels of H3K4me2/3, markers of opened chromatin and low CpG methylation upstream of the transcription starting site. On the contrary, high levels of H3K27me3 and CpG methylation outline wild-type TERT promoter, indicating a more silenced transcriptional state [TERT promoter mutations and BRAF V600E that perfectly fits in this context. It is known that binding of GABP to mutant TERT promoter, necessary for TERT activation, is influenced by the presence of native ETS motif spanning the two mutations and allowing the binding of tetramers of GABPA and GABPB. These authors demonstrated that BRAF V600E enhances the activation of Mitogen-activated protein kinase (MAPK) pathway, leading to the FOS-mediated expression of GABPB, which, in turn, binding to TERT mutated promoters in tetrameric form, induces TERT overexpression [TERT expression in TC patients is positively correlated with both TERT promoter and BRAF V600E mutations. Mechanistically, this was due to the BRAF-induced upregulation of the PEA3 subfamily of ETS transcription factors , which in turn bind and selectively activate the mutant promoter driving TERT expression [The methylation status of the chromatin surrounding al state . Recentlpression . In agrepression .TERT promoter mutations drive TERT activation and, by circular chromosome conformation capture (4C) assay, they demonstrated that GABP mediates a long-range chromatin interaction that along with an enrichment of active histone marks consequently drives TERT transcription. Indeed CRISPR/CAS9-mediated reversal of TERT promoter mutations to the wild type residue abrogates this long-range chromatin interaction and RNA Polymerase II recruitment, and reduces TERT transcription and telomerase activity. Furthermore, they found that a major player of this complex assembly is Bromodomain-containing protein 4 (BRD4), which is able to interact with the long-range region 300kb upstream of mutant TERT promoter enhancing TERT expression. Accordingly, BRD4 KD led to significant loss of active histone marks and impaired long-range chromatin interaction to an extent similar to GABPA KD. Furthermore, BRD4 regulates GABPA transcription by directly binding its promoter [TERT promoter mutations sparing the ones that are not affected by these mutations, including normal cells. Ankicilar et al. investigated how promoter . BRD4 bepromoter . Furtherpromoter . In linepromoter . Thus, tTERT promoter contains binding sites for several known regulatory factors, which include GC-motif and E-boxes. It has been widely demonstrated that MYC proto-oncogene, bHLH transcription factor (c-Myc) directly regulates TERT expression by binding to E-boxes within the TERT promoter [BRAF V600E - MAPK pathway axis may activate TERT expression through c-Myc in a TERT promoter mutation-independent manner, even if this mechanism seems to be less robust than that previous cited in TERT promoter mutated lesions [TERT expression and the cooperation with Specificity Protein 1 (Sp1), a transcription factor that binds GC-motifs next to E-boxes, seems to be required for an efficient TERT promoter activation. Intriguingly, BETi, which we previously described as a potential therapeutic options for aggressive TCs harboring TERT promoter mutations, have been shown to drastically suppress c-Myc expression, and this effect has been widely documented in human patients in several clinical studies for solid and hematological tumors [TERT promoters and indirectly by restraining the expression and transcriptional activity of c-Myc on TERT regulation. promoter . Severalpromoter ,40. Stil lesions . c-Myc al tumors ,42. Thusc-Myc and Sp-1 gene expression driving TERT activation in several hematological cell lines [TERT promoter 600 bp upstream of the transcription starting site [It has been reported that the nuclear factor kappa B (Nf-KB) pathway is able to enhance ll lines ,44. Furting site . Howevering site . Indeed,ing site . TERT promoter also presents binding sites for signal transducer and activator of transcription 3 (STAT3), located about 2000 bp upstream of the transcription starting site. Published studies have suggested that STAT3, a TF activated in response to various cytokines and growth factors, in TC may function as onco-suppressor rather than as cancer promoting factor. Indeed, although STAT3 expression is detected in PTCs [STAT3 expression and tumor size [ in PTCs , the majmor size ,50,51. TERT promoters, and it has been demonstrated that this TF positively regulates the expression of TERC raising the hypothesis that it plays a role in telomere regulation [Paired box 8 (PAX8) is an important TF in thyroid tumorigenesis. Additionally, it is known to be a marker of thyroid cell differentiation; PAX8 is among the more reliable markers of thyroid specification in advanced TCs, and indeed its expression has been detected in biopsies of ATC patients . PAX8 regulation . Runt-related transcription factor 2 (RUNX2). AP-1 is a leucine zipper heterodimeric complex and the Jun-Fos dimer is the most common form of AP-1 protein in human cells. We reported the collaboration of c-JUN and BRD4 in the regulation of RUNX2 where they orchestrate the cooperation of three distal active enhancers (ENHs) with the RUNX2 promoter. Two hypotheses for their interplay have been formulated: either BRD4 binding to acetylated regions may function as a docking site for the recruitment of c-JUN on RUNX2 regulatory regions or c-JUN binding to ENHs favors BRD4 recruitment [TERT promoter. Indeed, AP-1 can directly bind to the TERT promoter through binding sites at positions −1655 and −718 [TERT promoter by long-range chromatin interaction, the same ability BRD4 has to modify chromatin structure observed in the RUNX2 locus [TERT promoter seems to have an inhibitory effect on TERT expression. Consistently, the mutations at both AP-1 binding sites eliminated about 70% of the suppressive effect caused by AP-1 [AP-1 expression to TC features in vivo. Some reports indicate that the expression of AP-1 was negative correlated with tumor size in PTCs [Recently, we described a relevant role for activator protein-1 (AP1) in driving thyroid tumorigenesis by controlling the expression of the pro-oncogenic factor ruitment . A similX2 locus . However in PTCs . Convers in PTCs . Thus, iTERT promoter consensus sequences has been reviewed by Ramlee and colleagues, and may be used as a further hint to study possible players in telomerase regulation in aggressive TCs [A comprehensive description of sive TCs .TERT expression were observed in PTCs as in other human cancer tissues [TERT promoter mutations [In the spectrum of thyroid tumors, familial forms of non-medullary TCs (NMTCs) have also been reported. Familial NMTC patients show progressively increased disease aggressiveness in subsequent generations. This phenomenon is called “genetic anticipation”, and it was defined and associated with several inherited benign and malignant disorders. In the case of NMTCs, the later generations when compared to the first one showed an earlier age of insurgence, higher rate of lymph node metastases at surgery, and a worse outcome ,59. Fami tissues ,68. Thus tissues . Overallutations ,70; subsCDH6 expression in human PTCs is restricted to highly metastatic lesions, in which we also found a significantly higher incidence of TERT promoter mutations and TERT locus amplification, raising the hypothesis of a functional interconnection between these molecular events [Telomere-independent activities of TERT have been reported, and several studies have shown that this protein is engaged in processes such as regulation of DNA damage response, repression of apoptosis, regulation of chromatin state, and enhanced cell proliferation ,72,73. Fr events ,23. Inder events . TERT promotes the acquisition of mesenchymal features in gastric cancer. Conversely, its knockdown causes loss of mesenchymal markers and reduced invasiveness. In this model, TERT mediates the TGF-β signaling and in TERT KD cells TGF-β induction of target genes expression is impaired. Indeed, TERT interacts with beta-catenin  and together associate with mesenchymal marker promoters to drive their expression. This effect was largely independent of the telomere-lengthening activity of TERT, since overexpression of TERT mutants lacking these properties are still able to induce expression of EMT associated genes [E-Cadherin promoter and represses its transcription leading to EMT [Snail1 overexpression leads to a strong inhibition of TElomeric Repeat-containing RNA (TERRA) and TERT expression, coherently with the increased telomerase activity observed in Snail1-depleted cells [Snail1 upregulation. Furthermore, RNA-sequencing profiling in a TERRA overexpressing model of mammary epithelial cells, showed that TERRA impaired TGF-β signaling by altering the response of a large number of EMT-related genes to the TGF-β stimulation. Together, these data indicate that expression of TERRA prevent activation of EMT and that the Snail1-mediated inhibition of this lncRNA is required to drive EMT [Liu et al. reported that overexpression of ed genes . The trag to EMT . The actg to EMT . TERRA ig to EMT . In the ed cells . In partrive EMT .For many years, therapeutic options for TC patients were limited to surgery and radioiodine ablation (RAI). However, 15–20% of well-differentiated cases that generate distant metastasis, as well as most of ATCs, remain resistant to standard treatments or present contraindication for surgery ,85. BRAF and other members of the MAPK pathway are frequently mutated in TCs. BRAF V600E specific (Vemurafenib) and BRAF-MEK dual inhibitors (Dabrafenib) were recently proposed as potential therapies for advanced forms of TC, including ATCs [TERT promoter, and therefore the use of these drugs may impact TERT expression and consequently the biology of telomeres [However, in recent years, several new drugs have been approved for the use in TCs. In this section, we intend to discuss some of these new options and their possible interplay with telomere biology. Mutations in ing ATCs ,87. Furting ATCs ,90,91,92ing ATCs ,94. Bothelomeres ,55.TERT regulation and immunotherapy response. However, in this context, telomere biology should be exploited both as potential biomarker for response as well as potential co-target to improve immunotherapy response in combination therapy schedules. Immunotherapy, aiming to exploit immune response and restore its ability to eliminate tumor cells, represents one of the more recent frontiers for tumor treatment. Among others, Pembrolizumab, targeting PD-1 by avoiding the interaction with its ligand, was tested in a clinical trial involving different solid malignancies including thyroid tumors ,96. ThisTERT expression, but may also indirectly condition its transcription by repressing other BRD4-dependent TFs like c-Myc. Furthermore, the involvement of BRD4, as well as HDAC , in telomere regulation suggests the use of epigenetic drugs (such as BETi and HDAC inhibitors) to improve the effectiveness of telomerase-inhibiting compounds. Both of these drugs, when employed alone, failed to show substantial effects in clinical trials [TERT expression and telomeres regulation may synergize with the one of telomerase inhibitors and improving their effectiveness. This could potentially allow the use of telomerase inhibitors at lower doses, thus limiting the toxicity of these drugs.Targeting telomerase and mechanisms regulating telomere homeostasis may be considered a promising anti-cancer strategy. However, drugs directly targeting the telomerase complex have shown effectiveness in preclinical studies, but in clinical trials show lower performance and high side effects of toxicity ,112,113.l trials ,116. Howl trials . We may TERT promoter are among the more common genetic abnormalities observed in cancers and the most frequent alterations observed in aggressive thyroid tumors. Mutations in TERT promoter are generally associated with increased TERT expression and Telomerase activity and although they are not always connected to increased telomere length [TERT promoter and the possible still poorly understood connections with the biological mechanisms already described in thyroid cancer biological settings. Overall, this evidence highlights the feasible employment of BETi for the treatment of thyroid tumors. Indeed, these drugs seem to hamper tumor spreading by different sides and having beneficial effects both in tumor harboring TERT promoter mutations and in the wild type ones. Here, we also discussed the available evidence showing the ability of BETi to disrupt long-range chromatin interaction on mutated TERT promoter such as to inhibit c-Myc and AP-1 transcriptional activities independently from tumor genetic background (TC represents a paradigmatic example of the importance of telomere regulation in malignancies. Currently, mutations in e length , they are length ,76,106. ckground  [34,41,4ATRX and DAXX, are not reported in non-medullary cells derived thyroid cancers in many different studies [The role of Shelterin proteins in the regulation of telomeres has not been studied in deep in TC setting. A single paper reported no mutations or polymorphisms in Shelterin complex genes in familial PTCs . However studies ,11,118, In conclusion, these data enforce the role of telomerase dependent mechanisms in sustaining TC progression. Thus, telomerase regulation represents a major challenge and an interesting hint for studies investigating the pathogenesis of TC and/or the employment of new combined treatments.