Saccharomyces cerevisiae elicit rescue mechanisms mainly associated with reprogramming specific transcriptional responses via the cell wall integrity (CWI) pathway. Regulation of gene expression by this pathway is coordinated by the MAPK Slt2/Mpk1, mainly via Rlm1 and, to a lesser extent, through SBF (Swi4/Swi6) transcription factors. In this review, we summarize the molecular mechanisms controlling gene expression upon cell wall stress and the role of chromatin structure in these processes. Some of these mechanisms are also discussed in the context of other stresses governed by different yeast MAPK pathways. Slt2 regulates both transcriptional initiation and elongation by interacting with chromatin at the promoter and coding regions of CWI-responsive genes but using different mechanisms for Rlm1- and SBF-dependent genes. Since MAPK pathways are very well conserved in eukaryotic cells and are essential for controlling cellular physiology, improving our knowledge regarding how they regulate gene expression could impact the future identification of novel targets for therapeutic intervention.Living cells exposed to stressful environmental situations can elicit cellular responses that guarantee maximal cell survival. Most of these responses are mediated by mitogen-activated protein kinase (MAPK) cascades, which are highly conserved from yeast to humans. Cell wall damage conditions in the yeast  Yeast cells exposed to environmental stress and diverse molecules can elicit cellular responses that guarantee maximal cell survival. Most of these responses are mediated by signal transduction pathways formed by a mitogen-activated protein kinase (MAPK) module, which are highly conserved from yeast to humans. The MAPK module consists of a cascade of three protein kinases, namely, MAPKKK, MAPKK, and MAPK ,2. The fFive MAPKs mediating stimulus-dependent responses are encoded in the yeast genome . Kss1The activation of rapid and efficient transcriptional adaptive responses depends not only on MAPKs and transcription factors, but also on protein complexes that modulate chromatin structure. Transcriptional responses triggered by different stresses share certain regulatory features, but others are unique to each type of stress. In this paper, we review the main molecular mechanisms that regulate gene expression in response to cell wall stress as an example of a regulated transcriptional response mediated by a conserved MAPK pathway, considering the naturally repressive state of chromatin.The regulation of gene transcription in eukaryotic cells involves a dynamic balance between the packaging of regulatory sequences into chromatin and the access of transcriptional regulators to these sequences ,15. ChroTo overcome the naturally repressive state of the chromatin structure and promote transcriptional activation, nucleosome removal at the promoters of stress-responsive genes is a critical step. Nucleosome positioning can influence the accessibility of DNA binding sites in the promoters to specific transcription factors ,24,25 anChromatin modulation via covalent histone modification, such as methylation, acetylation, phosphorylation, SUMOylation, ADP-ribosylation, among others, represents one fundamental way to regulate DNA accessibility during gene transcription and many other cellular processes ,45,46,47Histone acetylation is regulated by the opposing action of histone acetyltransferases (HATs) and histone deacetylases (HDACs). SAGA (Spt-Ada-Gcn5-acetyltransferase) is a highly conserved co-activator complex that controls transcription by modifying histones . It is aBesides histone modifiers, ATP-dependent chromatin remodeling complexes are needed to disrupt DNA-histone interactions using the energy from ATP hydrolysis . The remS. cerevisiae SWI/SNF consists of 11 subunits, with Snf2 serving as the ATPase subunit [There are four different families of chromatin remodeling complexes: SWI/SNF, INO80/SWR1, ISWI, and CHD. They share a similar ATPase subunit that belongs to the SF2 superfamily of ATPases characterized by an ATPase domain split into two parts, the DExx and HELICc regions , althoug subunit ,70. Targ subunit ,72,73,74Transcription initiation is a complicated process that requires the coordinated activities of co-activators and many factors to ensure appropriate gene regulation under stressful situations ,75. ChroMAPKs mainly control gene expression by phosphorylating transcription factors. This modification regulates its activity in different ways, including their protein levels, binding to DNA, translocation between nucleus and cytoplasm, and interaction with regulatory proteins . HoweverSchizosaccharomyces pombe [Genome-wide analyses demonstrated that, in addition to Hog1, Fus3, and Kss1, other kinases of the mating pheromone signaling pathway are physically associated with coding regions of pheromone responsive genes. Strikingly, upstream MAPK proteins can also associate with chromatin regions ,93,94. Ies pombe ,97. Initially, genomic approaches did not find evidence for Slt2 gene occupancy under some specific conditions of Slt2 activation (cell cycle and pheromone exposure) . HoweverSaccharomyces cerevisiae has a main role in the regulation of cellular responses to cell wall damage and has been well characterized with regard to its regulation by cell wall stress [The yeast cell wall is an essential structure surrounding the cell, necessary for maintaining cell morphology and viability. Stressful conditions damaging this structure trigger the CWI pathway coordinated by the MAPK Slt2/Mpk1 (reviewed in ) Figure. As its l stress ,102,103.l stress . Slt2 all stress .Under cell wall stress conditions induced by treatments with cell wall perturbing agents that interfere with the biogenesis of this structure, Mid2 and Wsc1, the primary sensors of this pathway, interact with the guanine nucleotide exchange factor Rom2, activating the small GTPase Rho1, which then interacts with and activates Pkc1. The central role of activated Pkc1 is to trigger a MAPK module comprising MAPKKK Bck1, the redundant MAPKKs Mkk1/Mkk2, and the MAPK Slt2. Phosphorylation of the MAPK Slt2 leads to activation of the transcription factors SBF (Swi4/Swi6)  and Rlm1TPK1 expression in response to heat stress. The CWI pathway may be activated starting from Mkk1 of the MAPK cascade, resulting in an appropriate PKA output. Wsc3 would be the sensor of this lateral input in a way that seems independent of the activation of upstream elements of the CWI route [Although the CWI pathway plays a crucial role in regulating these responses, other pathways are also necessary to overcome cell wall stress situations. The transcriptional response elicited by Congo Red depends almost entirely on the MAPK Slt2 and the transcription factor Rlm1 ,108. MosWI route .The plant natural product poacic acid specifically binds to β-1,3-glucan and triggers a transcriptional response co-regulated by parallel activation of the CWI and HOG signaling pathways . AlternaSLT2 and RLM1-mediated positive feedback mechanisms are also needed for a complete transcriptional activation  is one of the transcription factors involved in CWI signaling. Swi4 is the sequence-specific DNA binding subunit, whereas Swi6 is the transcriptional activation subunit. However, Swi6 is necessary for the binding of Swi4 to DNA by relieving an auto-inhibitory intramolecular association of the Swi4 C-terminal domain with its own DNA binding domain ,124. SBFlleagues ,100,127 A Pol II . Direct FKS2 promoter. This interaction does not require the catalytic activity of the MAPK, however, it does require the presence of Swi4/Swi6 to direct this association with a restricted group of genes regulated by Slt2 and SBF. Slt2 interacts with the Paf1 complex via a docking site motif in the Paf1 subunit, but its catalytic activity is dispensable for this binding. In consequence, mutations in this domain block the transcriptional elongation of FKS2. The recruitment of Pol II (Rpb3) to the FKS2 promoter upon heat stress is not impaired in a paf1-4A mutant; however, the polymerase does not progress to the coding region, indicating a transcription elongation defect. The association between Slt2 and Paf1 blocks the recruitment of the Sen1-Nrd1-Nab3 termination complex and, therefore, avoids premature termination of the FKS2 gene [In addition to their role in transcription initiation, Slt2 and Mlp1 also participate in elongation, traveling along the coding region associated with Pol II and Paf1C. In the model proposed, Slt2 and Mlp1 move from the transcription initiation complex to the transcription elongation complex, leaving Swi4-Swi6 behind . Slt2 asKS2 gene   of RNA Pol II, increasing Tyr1P levels in response to stress [FKS2 promoter is reduced following heat shock in wild-type cells, but increases when the Tyr1 residue changes to Phe, indicating that phosphorylation of Tyr1 by Slt2 is required for Sen1-Nrd1-Nab3 (NNS) complex loss upon heat shock  gene was first identified in a genetic screening for mutants resistant to the growth inhibition caused by a constitutive form of Mkk1 [Rlm1 is the transcription factor responsible for the expression of most of the genes (~90%) induced under cell wall stress ,108,130. of Mkk1 . Rlm1 is of Mkk1 ,132). Rl of Mkk1 . Mutatio of Mkk1 . Rlm1 is of Mkk1 , so its  of Mkk1 ,83,101  and BOX2 (-510/-501). The nucleosome pattern characterized for the MLP1 gene revealed the presence of four nucleosomes positioned within the −699 and +161 region under non-stress conditions, the BOX1 site being exposed in the linker DNA between nucleosomes -3 and -2, or partially exposed at the edge of nucleosome-2, whereas the BOX2 site is completely occluded by nucleosome -3, preventing, or at least hindering, the binding of Rlm1 to this sequence [MLP1 promoter corresponds to that observed in covered promoters or nucleosome-occupied promoters, identified in most stress-induced genes .PHO5 gene has a covered promoter and is one of the first models established for gene regulation via chromatin remodeling complexes [MLP1 gene lacks a TATA box [MLP1 promoter would be jointly regulated by TFIID and SAGA complex [The pattern of promoter nucleosome occupancy is correlated with the capacity of genes to alter their expression. In contrast to housekeeping genes, the promoters of genes that modify their expression levels in response to external and internal signals present nucleosomes covering the transcription start site (TSS), the regions adjacent to the TSS, and most of the binding sites for transcriptional activators, being highly regulated by different remodeling complexes and/or chromatin modifiers ,135,136.omplexes . The covTATA box , althoug complex . Under cell wall stress conditions, the activated MAPK Slt2 phosphorylates Rlm1, resulting in its binding to specific sequences within target promoters . In factMLP1-NAT1) [MLP1 gene, the exposed Rlm1 binding site would allow Rlm1 access to the promoter in a first step, but nucleosome displacement would be required upon stress to expose the additional occluded site in a two-step model for activation [MLP1 promoter [The assembly of a pre-initiation complex at CWI-responsive genes must require chromatin-modifying activities to remodel or evict nucleosomes positioned along the target promoters . A conneP1-NAT1) . Indeed,P1-NAT1) . The ATPtivation  , it increases the remodeling mediated by the SWI/SNF complex. Indeed, the simultaneous deletion of eletions . As alreeletions . Howevereletions ,144,145.SUC1 gene [Parallel recruitments of different co-activators have been observed in the transcriptional initiation of stress-inducible genes, probably to support the rapid and fine-tuned upregulation required during adverse conditions. For example, simultaneous recruitment of SAGA and SWI/SNF has also been observed at osmotic stress-inducible genes  and the UC1 gene , and eveUC1 gene . The RSCUC1 gene ,149,150.UC1 gene . MoreoveUC1 gene . GAL1-LacZ transcription in response to cell wall stress [MLP1 gene expression in the absence of Rlm1. Remarkably, the transcriptional induction of MLP1 upon stress depends on MAPK phosphorylation, but not on its kinase activity. Thus, Slt2, by itself, can activate the transcription of CWI responsive genes independently of Rlm1, supporting a role for Slt2 in transcriptional activation as a structural component. Nevertheless, the presence of Rlm1 is still necessary for adequate gene induction [In response to cell wall stress, Slt2 binds to promoters and to the coding regions of CWI genes . The assl stress . When Slnduction . FLO genes binding to their promoters via Rlm1, independently of its catalytic activity [FLO genes in flocculating cells. Antagonistic binding of Rlm1 and Tup1, and the presence of overlapping binding sites at the promoters of FLO genes suggest that the Rlm1 and Tup1 interplay is involved in the regulation of FLO gene expression and yeast flocculation [Similarly, it has been shown that the phosphorylation of Slt2 but not its catalytic activity is required for flocculation, suggesting that Slt2 probably acts as a transactivator to induce transcription of activity . The binculation  The ability of Slt2 to drive gene expression by itself is explained by the fact that Slt2 physically interacts with RNA Pol II upon cell wall stress. Slt2 is first recruited to promoters by Rlm1 to interact with RNA Pol II, to move from the transcription initiation complex to the transcription elongation complex at the coding region in a second step, independently of Rlm1 . This kiUbiquitination of H2B influences both transcriptional initiation and elongation ,162 and In addition to cell wall damage conditions, other stimuli have also been found to trigger the activation of the CWI pathway . The mecER, HOG, and CWI pathways are closely related and interact with each other in helping the cell to reduce cadmium toxicity [UPRtoxicity . Molecultoxicity . The CWItoxicity . Thus, RUnderstanding gene regulation in response to stress requires knowledge of the transcription mechanisms mediated by MAPKs. Regulatory mechanisms provide the cells with the necessary tools to respond efficiently to environmental changes. Tight regulation is important to ensure the correct temporal modulation of gene expression in response to stress. Gene expression under stress conditions is quite complex and requires not only the participation of MAPK and specific transcription factors, but also the coordinated action of different co-activators. Recruitment of co-activators and the transcription machinery to promoter regions is a key initial step in activating transcription. Although these regulatory mechanisms are not exclusive to each MAPK, different stress situations may mediate distinct molecular mechanisms at the transcriptional level with a diverse group of co-activators as protagonists to achieve coordinated and highly regulated responses. Even under the same type of stress, transcriptional mechanisms may vary depending on the architecture of the gene target.FKS2 regulated by SBF, via a mechanism that requires activation of Slt2 but not its catalytic activity [In this review, we focus on the transcriptional mechanisms elicited by the MAPK Slt2 to activate gene expression under cell wall stress, highlighting the role of chromatin as an extra level of gene regulation. Nucleosome remodeling is crucial for inducible gene expression as it facilitates transcription activation. Upon cell wall stress, Slt2 phosphorylates and activates the main transcription factor Rlm1 to recruit both to the promoters of CWI-responsive genes in complex with SWI/SNF ,101. SWIactivity .ER signaling pathways. They suggest a particular interplay between MAPK signaling, chromatin, and transcription factors [Despite recent advances in molecular mechanisms controlling gene expression via the yeast CWI pathway, some questions remain unanswered. Studies have focused on the recruitment of transcription and chromatin regulatory complexes to chromatin. Still, little is known about how cells coordinate and discriminate activities and crosstalk between many gene regulatory factors in response to external cues. Combining genome-wide and context-specific approaches with chromosome conformation capture techniques will allow us to further understand the molecular mechanism involved in the genome organization of stress-responsive genes. Particularly, it will be interesting to see how each specific cell in a population responds to cell wall stress signals. Single-cell sequencing methods will allow monitoring the dynamics of mRNA production in single live cells and help elucidate global transcriptional responses and regulation mechanisms at the single-cell level. These mechanisms are beginning to be clarified in response to other stresses and their corresponding signaling pathways, like the HOG and UPR factors , and the factors ,176. A b factors .MAPK pathways are essential for controlling cell physiology in all eukaryotes, and many of the signaling mechanisms mediated by MAPKs in yeasts are conserved in humans. A detailed characterization of the mechanisms required for regulating MAPK-mediated signaling and gene expression would allow the identification of novel targets for possible therapeutic intervention. ERK5, the human ortholog of Slt2, phosphorylates and activates several transcription factors, such as c-Myc, Sap1a, CREB, and the MEF2 family members MEF2A, C, and D, playing a critical role in cardiovascular development and vascular integrity ,179. MorCandida albicans [Aspergillus fumigatus [Candida auris [Cryptococcus neoformans [Treatments with cell wall perturbing agents, including antifungal inhibitors of β-1,3-glucan synthesis, like echinocandins, elicit rescue mechanisms in the budding yeast, particularly compensatory chitin synthesis, to maintain cellular integrity ,102. Thealbicans , Aspergiumigatus , Candidada auris , and Cryoformans , may decoformans ,186,187.oformans . Cercospoformans  is additoformans . An alteoformans ,192. The


Regulation of RNA polymerase II (Pol2) elongation in the promoter-proximal region is an important and ubiquitous control point for gene expression in metazoans. We report that transcription of the adenovirus 5 E4 region is regulated during the release of paused Pol2 into productive elongation by recruitment of the super-elongation complex, dependent on promoter H3K18/27 acetylation by CBP/p300. We also establish that this is a general transcriptional regulatory mechanism that applies to ~7% of expressed protein-coding genes in primary human airway epithelial cells. We observed that a homeostatic mechanism maintains promoter, but not enhancer, H3K18/27ac in response to extensive inhibition of CBP/p300 acetyl transferase activity by the highly specific small molecule inhibitor A-485. Further, our results suggest a function for BRD4 association at enhancers in regulating paused Pol2 release at nearby promoters. Taken together, our results uncover the processes regulating transcriptional elongation by promoter region histone H3 acetylation and homeostatic maintenance of promoter, but not enhancer, H3K18/27ac in response to inhibition of CBP/p300 acetyl transferase activity. Saccharomyces cerevisiae) (In addition to RNA polymerase II (Pol2) pre-initiation complex (PIC) assembly and initiation, the transition from promoter-proximal paused Pol2 to productively elongating Pol2 is an essential step in gene transcription and an important process in the overall multicomponent orchestration of gene expression in metazoans . After tevisiae) . Associaevisiae) .Drosophila CBP ortholog stimulates productive elongation past the +1 nucleosome  to promoters and serves as a Pol2 elongation factor dependent on its interactions with acetylated histone lysines through its bromodomains . In addicleosome . Recruitcleosome .The super-elongation complex (SEC) is a multi-subunit complex comprising P-TEFb (CDK9-cyclin T) along with AF4/FMR2 proteins AFF1/4, ELL family members ELL1/2/3, ELL-associated factors EAF1/2, and one or the other highly homologous proteins AF9 or ENL containing YEATS acetyl-lysine-binding domains . There aWe previously reported that CBP/p300 acetylation of H3K18 and K27 in the two to three nucleosomes spanning the TSS had very different effects on distinct steps in transcription from different human adenovirus 5 (HAdV-5) early promoters . At the To investigate the function of H3K18/27ac in transcriptional elongation at E4, we mapped the association of transcriptionally active Pol2 on the Ad5 genome using Global Run-On sequencing (GRO-seq) . We founWe then extended our studies to the human genome, where we found that 2 hr of A-485 treatment resulted in hypoacetylation of total cell H3K18/27 to a new, extensively hypoacetylated steady state. This was associated with defective pause release at a subset of active genes (~7%) where promoter H3K18/27ac was decreased by the drug. Differences in the sensitivity of transcription from different promoters to H3K18/27ac correlated with differences in SEC component association with the genes after A-485 treatment. This was similar to what we had observed for the HAdV-5 E4 promoter during activation by the multi-site E1A mutant (DM-E1A) with mutations in the E1A-AD acidic peptides required for CBP/p300 binding to the E1A-AD. We also found that at a subset of enhancers with greatly decreased H3K18/27ac in response to A-485 treatment, H3K9ac is sufficient for BRD4 binding and stimulation of Pol2 pause release. Based on these results, we propose mechanisms of BRD4 and SEC recruitment by histone H3 acetylation during the transition from promoter-proximal paused to productively elongating Pol2 and report a homeostatic process that maintains promoter H3K18/27ac.CBP/p300 acetylation of promoter histone H3K18 and K27 stimulates paused Pol2 release at the HAdV-5 E4 promoter, but is not required at the E3 promoter.Transcription from HAdV-5 early promoters is activated by the first viral proteins expressed following infection, the E1A isoforms, primarily large E1A . While tTo analyze the effects of promoter H3K18/27ac on Pol2 elongation through the early Ad5 genes, we applied the GRO-seq method, which reveals the position and direction of transcribing Pol2 by BrU-labeling of 3′-ends of nascent RNA transcripts in isolated nuclei . The nucdl312 to maintain the same number of viral DNA molecule templates for the viral early regions  in cells expressing the same level of wt E1A and the DM-E1A protein  or A-485 treatment. As with cells expressing DM-E1A, A-485 treatment greatly decreased AF9 association at the E2early, E3, and E4 promoter regions, and decreased total ENL ChIP-seq counts from the E2early TTS to the right end of the genome from 13,712,875 to 10,804,279 reads, an ~20% decrease . We alsoTo determine if the effects of H3K18/27ac on Pol2 pause release at E4 is a general mechanism that also applies to transcription of cellular genes, we shifted our study to the human genome. First, we characterized the changes in H3K18/27ac in HBTECs after 2 hr treatment with 10 µM A-485. Western blotting demonstrated an extensive decrease in total cellular H3K18ac, which approached steady state by 2 hr after addition of A-485, as expected . H3K18acincrease in the average level of H3K18ac and H3K27ac at all TSSs in cells treated with A-485  and A-485-treated cells were aligned to the human genome to determine the fraction of genes affected by A-485 at different stages in transcription. We limited our analysis to protein-coding genes with active promoters containing at least 20 GRO-seq counts in the promoter region (TSS to +200) and a significant promoter H3K9ac peak . Out of 10,062 such active protein-coding genes, we found 686 (6.8%) with defective pause release after A-485 treatment . In total, 885 genes (8.8% of active genes) were inhibited for initiation (<50% the GRO-seq counts in the promoter region [TSS – 200 bp] compared to control DMSO-treated cells). We refer to these genes as ‘2XIn’ genes for twofold decreased initiation from A-485. Also, 217 genes passed the criteria for both groups, indicating that both transcription initiation and promoter-proximal pause release were reduced by A-485 treatment for this small set of genes  were >2.5 kb from the nearest TSS in control cells treated with DMSO (dimethyl sulfoxide). Of these distal peaks, 84% overlapped with peaks of H3K27ac, indicating that these peaks were primarily at enhancers. We subsequently clustered all BRD4 peaks >2.5 kb from a TSS  based on whether BRD4 association decreased to <50% of DMSO (dimethyl sulfoxide) control after A-485 treatment for 2 hr. There were 7554 peaks where BRD4 decreased to <50% of control after A-485 treatment and 6324 peaks where BRD4 association remained unaltered or was not reduced to <50% of control  of active cellular promoters in primary human airway epithelial cells.Initially we observed that SEC recruitment through association with promoter region histone H3 acetylated at K18 and K27 stimulates paused Pol2 release at the HAdV-5 E4 promoter . To do tWe observed a correlation between defective paused Pol2 release at the viral E4 promoter as assayed by GRO-seq and decreased association of SEC subunits CDK9, AF9, and ENL assayed by ChIP-seq, indicating that H3K18/27ac is necessary for maximal SEC recruitment and Pol2 pause release in the E4 promoter-proximal region. This is similar to a proposed function of H3K9ac as a binding site for AF9 and ENL, thereby promoting paused Pol2 release by directly recruiting the SEC . SEC recBRD1-4) have two BRD domains, at least one of which binds acetylated lysines with moderate affinity, potentially participating in cooperative protein binding to a region of chromatin with multiple acetylated lysines  in the promoter regions of cellular genes correlated with increased pausing for 2XPI genes and decreased initiation for 2XIn genes in response to A-485 . TATA boAd5 mutant vectors expressing wt E1A and DM E1A were constructed as previously described .2 incubator until they reached confluence. Cells were then incubated 3 days more without addition of fresh medium and were infected for 12 hr with the indicated HAdV-5 mutants in the conditioned medium. A-485 (MedChemExpress) was added to a final concentration of 10 µM, or the same volume of DMSO (dimethyl sulfoxide) vehicle was added, and cells were incubated for an additional 2 hr.HBTECs  were grown at 37°C in a BronchiaLife medium complete kit  in a 5% CO2, 3 mM CaCl2). Nuclei were isolated with lysis buffer . Nuclear run-on was performed at 30°C for 7 min in 10 mM Tris-HCl pH 8, 5 mM MgCl2, 300 mM KCl, 1 mM DTT, 500 µM ATP, 500 µM GTP, 500 µM Br-UTP, 2 µM CTP, 200 U/mL Superase In RNase Inhibitor (Invitrogen), and 1% sarkosyl. Nuclear RNA was isolated with Trizol (Invitrogen). DNAse treatment was performed with Turbo DNA-free kit (Invitrogen). RNA was purified with Micro Bio-Spin P-30 Gel Columns (Bio-Rad), fragmented with RNA Fragmentation Kit (Invitrogen), and treated with 10 units RppH (NEB) and 30 units T4 PNK (NEB). RNA immunoprecipitation was performed with anti-BrdU-conjugated agarose beads (Santa Cruz Biotechnologies). Library preparation was performed with TruSeq Small RNA Library Preparation Kit (Illumina). GRO-seq reads were aligned with HISTAT2 software to Ad5 and human (hg19) genomes and normalized to the number of reads aligned to hg19. Pause indexes (TSS to +200 counts)/(+201 to TTS counts) were calculated using HTSeq software. Genes determined to be affected by A-485 underwent additional profiling using Homer software  was reverse transcribed with random hexamer priming using Superscript III (Invitrogen). RNA was treated with DNase I with Turbo DNA-free kit (Ambion). Quantitative reverse transcription-PCRs (qRT-PCRs) were carried out with the Applied Biosystems 7500 real-time PCR system with FastStart universal SYBR green master mix (Roche). All values were normalized to 18S RNA levels.Preparation of cross-linked HBTEC chromatin, sonication, and immunoprecipitation was as described in ChIP-seq libraries were sequenced using HiSeq 4000 or NovaSeq 6000. For analysis on the Ad5 genome, sequence tags were aligned using Bowtie2 software and normalized to the following formula:  × . For analysis on the human genome, reads were mapped to the hg19 human genome reference using Bowtie2 software. Only reads that aligned to a unique position in the genome with no more than two sequence mismatches were retained for further analysis. Duplicate reads that mapped to the same exact location in the genome were counted only once to reduce clonal amplification effects. MACS2 software was used for peak calling . The total counts of the input and ChIP samples were normalized to each other. Samples were normalized for equal number of uniquely mapped reads. The input sample was used to estimate the expected counts in a window. Wiggle files were generated using a custom algorithm and present the data as normalized tag density as seen in all figures with genome browser shots. Metagene plots displaying normalized average relative ChIP-seq signals were generated using CEAS software.Antibodies included H3K18ac (814), prepared and validated as described previously , H3K9ac  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This study addresses the role of histone acetylation on the release of paused RNA polymerase II from Adenovirus early promoters and from cellular promoters. By combining measurement of nascent polymerase II generated RNA and chromatin immunoprecipitation for RNA polymerase II, BRD4 and components of the super elongation complex in the presence or absence of A-485 a P300/CBP inhibitor, the authors identify both Adenoviral and cellular promoters where P300/CBP inhibition affects RNA polymerase II initiation or more specifically release of RNA polymerase II pausing. The results also revealed that P300/CBP inhibition has differential effects on histone acetylation at super enhancers, standard enhancers and proximal promoters suggesting a novel homeostatic mechanism that regulates acetylation and/or its turnover at these different regions. The study specifically uncovers a group cellular promoters that are particularly sensitive to P300/CBP inhibition leading to decreased released of paused RA polymerase II. This is a well performed study that will be of general interest to researchers investigating the mechanisms regulating gene expression, in particular how chromatin acetylation regulates release of RNA polymerase II pausing, a major regulatory step in transcription.Decision letter after peer review:eLife. Your article has been reviewed by two peer reviewers, including Irwin Davidson as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Kevin Struhl as the Senior Editor.Thank you for submitting your article "Promoter-specific changes in initiation, elongation and homeostasis of histone H3 acetylation during CBP/p300 inhibition" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at Summary:This study addresses the role of histone acetylation on the release of paused Pol II first from Adenovirus early promoters and then extended to all cellular promoters. By combining Gro-Seq to assess Pol II nascent transcription and ChIP-seq for modified Pol II, BRD4 and SEC components in the presence or absence of A-485 a P300/CBP inhibitor, the authors identify both Adenoviral and cellular promoters where P300/CBP inhibition affects Pol II initiation or more specifically release of Pol II pausing. The study specifically uncovers a group of around 1000 cellular promoters that are particularly sensitive to P300/CBP inhibition leading to decreased released of paused Pol II. The results also indicate that P300/CBP inhibition has different effects on histone acetylation at enhancers and proximal promoters/TSS.Life.The reviewers think this is a well-conducted study that yields important novel insights in the connection between histone acetylation by the CBP/p300 coactivators and Pol II pause release by the SEC. This manuscript is well suited to the general readership of eHowever, before acceptance, several issues should be addressed in a revised version.Essential revisions:1) The effect of A-485 on ENL recruitment to the E4 promoter in Figure 4A are minimal and much weaker than on AF9 binding. The authors should strengthen their case by including a CDK9 ChIPseq on A-485 treated Ad-infected cells.2) In the subsection “Decreased Pol2 pause-release in the E4 promoter-proximal region correlates with decreased association of SEC subunits CDK9, AF9, and ENL”, the authors speculate that decreases in pol II elongation would lead to increased phosphor-CTD signals. To provide data for this, the authors could perform a ChIPseq on a core pol II subunit.3) The authors should provide a statistical test of the π differences mentioned in the subsection “CBP/p300 acetyl-transferase activity is required for efficient Pol2 pause-release and recruitment of AF9, ENL, and BRD4 at E4”.4) In the context of the Adenovirus early promoters, the authors discuss that the presence of a consensus TATA-element can account for resistance to histone hypo-acetylation, but they do not extend this analyses to the cellular genes. Have they investigated the relationship between consensus TATA-elements and sensitivity of Pol II initiation/pause release at cellular genes to inhibition by A-485, in particular as it has been shown that only a limited subset of cellular promoters comprises consensus TATA-elements?5) When analyzing the cellular promoters and their differential responses to A-485, there is no investigation of “super-enhancers” that are characterized by extended regions of histone acetylation and BRD4 occupancy, key actors in this study. In Figure 7—figure supplement 4, there seems to be a “super-enhancer” region where histone acetylation is strongly affected. Can the authors comment on whether “super-enhancers” are more affected than other enhancers by A-485 and what are the effects on transcription of the associated genes.6) While the study identifies cellular genes where release from Pol II pausing is modulated by histone acetylation, there is no investigation of the underlying mechanism that would explain the differential sensitivity of these 2XPI promoters. Why is H3 acetylation lowered at these promoters but increased at the majority of the others? For example, have they investigated the sequence motifs seen at these promoters or associated enhancers to assess whether binding motifs for specific factors are enriched at promoters where Pol II release is sensitive/resistant to acetylation levels and recruitment of SEC components, have they assessed a relationship to GC content and/or position of the +1 nucleosomes? It is important that the authors provide some data or at least some discussion of which characteristics set these promoters apart and would account for their differential sensitivity to A-485 treatment and release of paused Pol II. Essential revisions:1) The effect of A-485 on ENL recruitment to the E4 promoter in Figure 4A are minimal and much weaker than on AF9 binding. The authors should strengthen their case by including a CDK9 ChIPseq on A-485 treated Ad-infected cells.We would prefer to not pursue an explanation for the difference between the magnitude of the effect of A-485 on AF9 versus ENL association with the Ad5 genome. This is because the principal importance of the ChIP-seq results for AF9 and ENL on the Ad5 genome was that they led us to do the analysis of AF9 and ENL association with cellular genes shown in Figure 6B. We observed an unequivocal and much more extensive decrease in both AF9 and ENL ChIP-seq reads on cellular DNA after A-485 treatment than on the Ad5 genome. This may be because the increase in π caused by A-485 was much greater on the 2XPI cellular genes (≥ 2 fold) than on the Ad5 E4 region (1.4 fold).In the revised manuscript, in order to be clear about the relatively small effect of A-485 we now present quantitation based on the total ChIP-seq read counts under the peaks in the ENL ChIP-seq showing that the ENL read counts were reduced only ~20% by A-485 treatment, a relatively small effect as the reviewers note.New text: "To determine if inhibition of CBP/p300 HAT activity resulted in defective SEC recruitment at E4, we performed AF9, ENL, and BRD4 ChIP-seq in cells infected with the wt E1A vector after DMSO or A-485 treatment. As with cells expressing DM-E1A, A-485 treatment greatly decreased AF9 association at the E2early, E3, and E4 promoter regions, and decreased the number of counts in ENL peaks from the E2early TTS to the right end of the Ad5 genome from 13,712,875 to 10,804,279, an ~20% decrease ."2) In the subsection “Decreased Pol2 pause-release in the E4 promoter-proximal region correlates with decreased association of SEC subunits CDK9, AF9, and ENL”, the authors speculate that decreases in pol II elongation would lead to increased phosphor-CTD signals. To provide data for this, the authors could perform a ChIPseq on a core pol II subunit.We did not mean to “speculate that decreases in pol II elongation would lead to increased phosphorCTD signals.” In this portion of the submitted manuscript we were discussing the small peaks of Pol2 Ser2-P and Pol2 Ser5-P observed just upstream of the short E2 second exon in Figure 2A. These peaks were not a major subject of this paper. The relevant passage reads:“We observed two Ser2-P peaks in the E2early gene body, one just downstream of the TSS, likely indicating paused Pol2. Another Ser2-P peak occurred over the E2early second exon. […] Such a decrease in Pol2 elongation rate over the short E2 second exon would cause an increase in the steady-state level of Pol2 over the exon, potentially leading to the increase in the Pol2 ChIP-seq signal observed over the E2 second exon.”Contrary to the statement in the Review point (2), we did not “speculate that decreases in pol II elongation would lead to increased phosphorCTD signals.” We were simply providing a potential explanation for the small Ser2-P and Ser5-P peaks over the E2 second exon, which were not important points of the paper, and were not discussed further.To clarify our point here and avoid confusion, we changed the wording to:“These Pol2 peaks just upstream of the E2 second exon may be explained by a previously reported reduction in elongation rate over some exons, proposed to influence splice site recognition and spliceosome assembly .”3) The authors should provide a statistical test of the π differences mentioned in the subsection “CBP/p300 acetyl-transferase activity is required for efficient Pol2 pause-release and recruitment of AF9, ENL, and BRD4 at E4”.We have now performed three independent biological replicates of the GRO-seq assays on the Ad5 genome in HBTECs infected with the wt E1A and DM E1A vectors. From this data we calculated the fold change in pause index (PI) at the viral early promoters between wt E1A which makes the E1A-AD interaction with p300/CBP and DM E1A which is blocked specifically for the p300/CBP interaction . We calculated the average change in π and the statistical significance  of the differences between wt E1A and DM-E1A. The p-values are now shown in the new Figure 1B, and the figure legend has been modified accordingly:“(B) Average fold change in pause index for E2early, E3, and E4 in cells expressing DM-E1A compared to wt E1A. Pause index is the ratio of reads in the promoter region (TSS to +200) to reads in the gene body (+200 to TTS). […] * indicates a significant difference  between cells expressing wt E1A and DM-E1A. “ns” indicates no significant difference.”4) In the context of the Adenovirus early promoters, the authors discuss that the presence of a consensus TATA-element can account for resistance to histone hypo-acetylation, but they do not extend this analyses to the cellular genes. Have they investigated the relationship between consensus TATA-elements and sensitivity of Pol II initiation/pause release at cellular genes to inhibition by A-485, in particular as it has been shown that only a limited subset of cellular promoters comprises consensus TATA-elements?We are grateful to the reviewers for asking us to clarify this point. The new Figure 5—figure supplement 1 shows that ~4.5% of 2XPI promoters and ~4.8% of 2XInitiation promoters have consensus TATA-boxes (TATAWAAR), compared to ~1.8% of all active promoters.5) When analyzing the cellular promoters and their differential responses to A-485, there is no investigation of “super-enhancers” that are characterized by extended regions of histone acetylation and BRD4 occupancy, key actors in this study. In Figure 7—supplement 4, there seems to be a “super-enhancer” region where histone acetylation is strongly affected. Can the authors comment on whether “super-enhancers” are more affected than other enhancers by A-485 and what are the effects on transcription of the associated genes.Again, we are grateful to the reviewers for asking us to clarify this point. The new Figure 5—figure supplement 2 shows the total number of peaks and the percentage of total peaks of H3K27ac, H3K18ac, H3K9ac, and BRD4 in super-enhancers, typical enhancers, and promoters in primary human tracheal epithelial cells and the changes in these values in A-485 treated and control DMSO vehicle treated cells. As the reviewers suggested, these plots and numbers of peaks show that “super-enhancers” are more affected than other enhancers by A-485. This is discussed in the revised manuscript:“Comparing the average signals for H3K18ac and H3K27ac at all TSSs and enhancer peaks (defined as peaks >2.5kb from the nearest TSS), we observed the expected decreases in H3K18ac and H3K27ac by A-485 at enhancer peaks . […] Furthermore, the percentage of total H3K27ac and H3K18ac peaks increased at promoters and decreased at enhancers after A-485 treatment, while the effects on H3K9ac peaks were minimal .”


In biological cells, promoters drive gene expression by specific binding of RNA polymerase. They determine the starting position, timing and level of gene expression. Therefore, rational fine-tuning of promoters to regulate the expression levels of target genes for optimizing biosynthetic pathways in metabolic engineering has recently become an active area of research.Yarrowia lipolytica, and constructed an artificial hybrid promoter library that covers a wide range of promoter strength. The results indicate that the hybrid promoter strength can be fine-tuned by promoter elements, namely, upstream activation sequences (UAS), TATA box and core promoter. Notably, the UASs of Saccharomyces cerevisiae promoters were reported for the first time to be functionally transferred to Y. lipolytica. Subsequently, using the production of a versatile platform chemical isoamyl alcohol as a test study, the hybrid promoter library was applied to optimize the biosynthesis pathway expression in Y. lipolytica. By expressing the key pathway gene, ScARO10, with the promoter library, 1.1–30.3 folds increase in the isoamyl alcohol titer over that of the control strain Y. lipolytica Po1g KU70∆ was achieved. Interestingly, the highest titer increase was attained with a weak promoter PUAS1B4-EXPm to express ScARO10. These results suggest that our hybrid promoter library can be a powerful toolkit for identifying optimum promoters for expressing metabolic pathways in Y. lipolytica.In this study, we systematically detected and characterized the common promoter elements in the unconventional yeast We envision that this promoter engineering strategy and the rationally engineered promoters constructed in this study could also be extended to other non-model fungi for strain improvement.The online version contains supplementary material available at 10.1186/s13068-021-02002-z. Promoters are one of the most important components in synthetic biology, and well-controlled promoters are very critical for regulating gene expression in eukaryotes. The activity of a promoter is co-regulated by various elements. In yeast, the common promoter elements usually include upstream activation sequences (UAS), TATA box and core promoter , 2. By rYarrowia lipolytica alkaline extracellular protease 2 (XPR2) gene promoter, PXPR2, Madzak et al. identified UAS1B to be the most significant functional element that activates the promoter [LEU fragment and different copy numbers of the UAS1B indicates that enhancement in promoter strength is correlated to increased copy number of UAS1B [Y. lipolytica, the expression level of a constructed promoter was sevenfold higher than the wild-type promoter [At the beginning of transcription, regulatory signals are transmitted from the UAS to the core promoter, the site where the transcription factors and the RNA polymerase II assembles to form the transcription preinitiation complex (PIC) . The corpromoter . Subsequof UAS1B . When prY. lipolytica, the promoter elements were characterized and rearranged. Consequently, a library of hybrid promoters that enables stable expression and covers a wide range of promoter strength was constructed. Subsequently, we employed the hybrid promoters for promoter engineering of the pathway genes in isoamyl alcohol biosynthesis. Isoamyl alcohol, an important platform chemical, is widely applied in the production of biofuels, fragrances, medicines and fine chemicals [Escherichia coli, Corynebacterium glutamicum and Aspergillus oryzae [Y. lipolytica by metabolic engineering, a native promoter was employed [Y. lipolytica.In this study, to explore the mechanism of synergy between various elements in hemicals  and has s oryzae –16. Whilemployed . TherefoY. lipolytica Po1g KU70∆, which was the host strain for this work. The results show that hrGFPO was the most suitable reporter gene, as its fluorescence was high and consistent. Thus, hrGFPO was utilized for subsequent promoter characterization  gene promoter PLEU, export protein (EXP1) gene promoter PEXP and translation elongation factor-1α (TEF1) gene promoter PTEF. Based on the results of our experiments  [S. cerevisiae and Y. lipolytica were used to replace UAS1B4 in PUAS1B4+LEUm at the same copy number to explore the influence of UAS types and origin on hybrid promoter activity. By expressing the hrGFPO gene under the hybrid promoters with different UASs, the activities of the promoters were proved to be significantly affected by the variation in UAS. The relative fluorescence intensity from the GFP expressed from the promoters containing various UASs, from high to low, is UAS1B > UASTEF Y.l. > UASCIT S.c. > UASCLB S.c. > UASTEF S.c. , such as the recognition of UASs by transcriptional activators and upstream repression sequences (URSs) by repressors. Many studies have shown that UAS has a strong influence on transcriptional regulation. Several UASs have been identified in s UASTEF , UASCLB s UASTEF  and UASCs UASTEF . However as well , 12. Fou1B4+LEUm . We incr1B4+LEUm . In addipolytica , there iEF Y.l.) –25 from Taken together, we have constructed a library of hybrid promoters with different promoter strengths using various combinations of UASs, TATA boxes and core promoters, as summarized in Fig. Y. lipolytica is quite low natively at a mere 0.37 mg/L , decarboxylases  and alcohol dehydrogenases  were selected and individually overexpressed to determine the key genes of isoamyl alcohol biosynthesis in the Ehrlich pathway. For this purpose, twelve strains overexpressing native and heterologous genes in the Ehrlich pathway were constructed. All genes were individually integrated into the genome of Y. lipolytica Po1g KU70Δ and driven by the constitutive promoter PUAS1B4+LEUm. After 3 days of cultivation, individual overexpression of the pathway genes enhanced the isoamyl alcohol titer in the engineered strains compared to that of the control strain Po1g KU70Δ  had higher titers of isoamyl alcohol, suggesting that the elements of the PEXP have greater beneficial effects to the expression of the ARO10 gene. It is unclear why the expression of ARO10 benefitted from the elements of PEXP, which drives the expression of a necessary gene YALI0C12034g that is highly homologous to an S. cerevisiae encoding a non-classical export protein 2 [ARO10) but more studies are needed to better understand the mechanism between the PEXP and gene expression which resulted in the improved production titer. Nevertheless, our results indicate that different promoters can affect the titer of isomayl alcohol and demonstrate the capability of using our hybrid promoter library for pathway optimization. Attempts to further improve isoamyl alcohol titer may be achieved by applying the hybrid promoter library to other key genes in the pathway. Further understanding of the relationship between promoter elements, genes and gene expression will offer valuable insights to facilitate high production of the target compounds.In metabolic engineering, studies have shown that the yield of the target product can be increased by replacing promoters of pathway genes with stronger ones , 29. Theied Fig. . It can rotein 2 . It is pY. lipolytica, and subsequently constructed a series of promoters which are stable and efficient in this study. This is the first time that the T content upstream of the TSS has been shown to positively correlate with the hybrid promoter strength in Y. lipolytica, although it is worth noting that some core promoter elements, such as POX2m and LEUm, did not conform to the trend. Therefore, the relationship between the T content upstream of the TSS and the promoter strength in Y. lipolytica needs to be further studied. Next, the effects of different UAS elements from S. cerevisiae and Y. lipolytica on promoter strength were investigated and we discovered for the first time that UAS elements can be transferred between yeast species. However, the functions of the UASs from S. cerevisiae were weaker than the native ones from Y. lipolytica , and supplemented with ampicillin to final concentration of 100 μg/mL for plasmid propagation. Yarrowialipolytica strain Po1g KU70Δ, a leucine auxotroph devoid of any secreted protease activity, was used as the base strain in this study. The original plasmid pYLEX1, a pBR322-based monocopy integrative vector [Y. lipolytica Po1g KU70Δ was fitted with an integrated pBR322 docking platform. The vector pYLEX1 digested with Spe I or Not I will be inserted to the pBR322 locus of the Po1g KU70Δ strain. After transformation, the positive Y. lipolytica KU70 Δ transformants were selected on YNB-leu plates and subsequently confirmed by genomic DNA PCR analysis. Yarrowia lipolytica Po1g KU70 Δ containing plasmid was routinely cultivated at 28 °C and 225 rpm with YPD media consisting of 20 g/L glucose, 20 g/L peptone, and 10 g/L yeast extract. In this study, PCR primers were synthesized by Genewiz  and are listed in Additional file e vector , 36, is ® max master mix, 2× Rapid Taq master mix, ClonExpress® II one step cloning kit, FastPure® Plasmid Mini Kit and FastPure® Gel DNA Extraction Mini Kit were purchased from Vazyme Biotech Co., Ltd. , peptone and yeast extract were purchased from Thermo Scientific Oxoid Microbiology Products , isoamyl alcohol and n-dodecane were purchased from Aladdin® .All restriction enzymes were purchased from New England Biolabs , 2× PhantaGFPuv gene was preserved in this laboratory, and cloned into pYLEX1 with primers GFPuv-F/GFPuv-R , decarboxylase gene  and alcohol dehydrogenase gene  from S. cerevisiae S288C were codon-optimized and synthesized and cloned into pYLEX1 to yield plasmids pYLSCBAT1, pYLSCARO10 and pYLSCADH2 , then resuspended in PBS. A cell count of 10,000 was analyzed with a BD Accuri C6 flow cytometer (BD Biosciences) using 488-nm excitation wavelength and FL1 channel for fluorescence detection. The CFlow software was used to analyze the data and compute mean fluorescence values. The background fluorescence was normalized when calculating the relative mean fluorescence.The green fluorescent proteins GFPuv, hrGFP and hrGFPO were selected as reporter proteins. At least three biological replicates were measured by flow cytometry. The colonies of transformants were selected from plates and grew in 5 mL of fresh YPD medium in tubes for 24 h. After that, the seed culture was diluted to ODY. lipolytica transformants were selected from plate and prepared in 5 mL of fresh YPD medium in tube for 24 h. The seed culture solution was inoculated to 250 mL flasks containing 40 mL of YPD medium, starting from OD600 0.1. The cultures were shaken at 225 rpm and 28 °C for 3 days. To extract isoamyl alcohol from the cultures, 10% n-dodecane was added to the cultures, and the mixture was vortexed for 3 min, then centrifuged at 7500 rpm for 5 min. The organic phase of 1ul was detected by GC/MS using an Agilent 7890B GC with an 5977B MSD equipped with a HP-5MS column . GC oven temperature was initially held at 60 °C for 2 min, and then ramped to 140 °C at a rate of 5 °C/min. It was then subsequently ramped at 10 °C/min to 280 °C and held for 5 min. The split ratio was 10:1. Helium was used as the carrier gas, with an inlet pressure of 13.8 psi. The injector was maintained at 280 °C and the ion source temperature was set to 230 °C. Final data analysis was achieved using MassHunter Workstation Software .The engineered Additional file 1: Figure S1. Screening of reporter genes. Figure S2. Characterization of different green fluorescent proteins expressed under the promoter PUAS1B4-LEUm. Figure S3. Schematic of theTEF core promoter. Figure S4. Time course of fluorescence by Po1g PUAS1B4-LEUm + hrGFPO. Table S1. Primers used in PCR. Table S2. Plasmids used in this study. Table S3. Strains used in this study.


CRISPR-based epigenome editing uses dCas9 as a platform to recruit transcription or chromatin regulators at chosen loci. Despite recent and ongoing advances, the full potential of these approaches to studying chromatin functions in vivo remains challenging to exploit. In this review we discuss how recent progress in plants and animals provides new routes to investigate the function of chromatin regulators and address the complexity of associated regulations that are often interconnected. While efficient transcriptional engineering methodologies have been developed and can be used as tools to alter the chromatin state of a locus, examples of direct manipulation of chromatin regulators remain scarce in plants. These reports also reveal pitfalls and limitations of epigenome engineering approaches that are nevertheless informative as they are often associated with locus- and context-dependent features, which include DNA accessibility, initial chromatin and transcriptional state or cellular dynamics. Strategies implemented in different organisms to overcome and even take advantage of these limitations are highlighted, which will further improve our ability to establish the causality and hierarchy of chromatin dynamics on genome regulation. Chromatin mechanisms play a major role in fine-tuning gene regulation in eukaryotes. The main feature of chromatin is the wrapping of genomic DNA around a protein octamer of four pairs of histones H2A, H2B, H3 and H4, altogether constituting the nucleosome. Nucleosomes prevent DNA from supercoiling and have evolved as a non-specific, passive barrier to limit DNA accessibility . ChromatStreptococcus aureus, or even different systems such as the Cas12a derived from Francisella novicida  . A. ANOS prbidopsis . dCpf1 abidopsis . Parametbidopsis .Two recent studies report the use of the 300 amino acids N-terminus (N300TPL) of the plant transcriptional co-repressor TOPLESS as another repressive domain for CRISPRi strategies. In both cases local recruitment of N300TPL via dCas9 inhibited target gene transcription c 32,33],3332,33]Chromatin homeostasis results from a dynamic equilibrium between multiple pathways that involve a plethora of DNA regulatory factors, histone variants and modifiers as well as nucleosome remodellers. Disentangling these different mechanisms to understand the function of each one in vivo, and its relationship with the others, remains quite challenging. By enabling local recruitment of a given chromatin regulator (CR), CRISPR/dCas9 tools have already started to provide precious insights about the causality of the function of specific epigenetic factors in relation to transcription, in a given genomic and cellular context.Arabidopsis FWA locus through multiplex recruitment of the Nicotiana tabacum DRM methyltransferase catalytic domain (NtDRMcd) via the SunTag system fused to dCas9 [FWA locus. The immediate drawback of this approach, also reported for similar designs in animal cells [In plants, cytosine methylation is found in the CG, CHG and CHH sequence contexts  and is controlled by the action of de novo and maintenance DNA methyltransferases as well as demethylases. DNA methylation is highly enriched over repeated sequences and present over some gene regulatory regions, where it is associated with transcriptional silencing . In contto dCas9  , DEMETER (DME), DEMETER-LIKE 2 (DML2) and DEMETER-LIKE 3 (DML3). These enzymes directly remove methylated cytosines regardless of the sequence context [FWA and the DNA-type transposon CACTA1 loci, was successfully reactivated without detecting ectopic demethylation events in the locus vicinity or elsewhere in the genome [FWA promoter could be maintained after the Sun-Tag TET1 cd construct was segregated out, demethylation was incomplete for the CACTA1 elements, and re-methylation and re-silencing occurred after removal of the targeted demethylation construct. One promising direction to develop targeted DNA demethylation could be through the use of third generation sgRNAs. In this system, an RNA aptamer that directly inhibits endogenous DNA methyltransferases is grafted onto the sgRNA stem-and tetraloops. The efficiency of this strategy was recently demonstrated in mammalian cells for DNMT1 inhibition using the CRISPR-DIR system [Demethylation can occur either passively through DNA replication in the absence of maintenance machinery or actively through enzymatic modules derived from different classes of demethylases such as the mammalian TEN-ELEVEN TRANSLOCATION1 (TET1) protein family that promotes demethylation through a base excision repair pathway . In Arab context . The eff context   locus, a major regulator of flowering time. However, the resulting increase in H3K27ac had little effect on FT mRNA levels and slightly affected flowering time [AREB1 gene , E(z), and Trithorax) domain exist but only one experiment using a dCas9-SET fusion has been reported so far. The SET domain of KRYPTONITE (KYP), an H3K9 methyltransferase from fication . Still vfication ,42, withfication ,23,38.Although experimental data remain scarce in plants and are mostly restricted to proof-of-concept studies, epigenome editing technologies implemented in animals have raised interesting insights that could be directly transposed in plants. Furthermore, in addition to studying the function of individual chromatin factors in regulating transcription, CRISPR/dCas9 also enables to assess the functional relationship between chromatin regulators (CRs) and DNA binding proteins and identify synergies or antagonisms between and among them.Arabidopsis, recruiting the mammalian p300 histone acetyl transferase to the FT locus allowed local doubling of the H3K27ac level, with no significant effect on FT transcript accumulation, which suggests that additional activating inputs on the FT regulatory sequences are needed to increase transcription [AtMED21 promoter, although the resulting acetylation levels were not tested.In CRISPRa experiments, co-targeting of histone acetylation and activating transcription factors (TFs) has a higher effect on transcription than targeting only the transcriptional activator . In Arabcription . Converscription , negativOCT4 locus in embryonic HEK293T cells further enhanced gene expression when compared to the effect measured for the single dCas9-VP64 trans-activation [In keeping with this synergic mode of action, co-recruitment of dCas9-P300 with the dCas9-VP64 transcription activator at the tivation . This suSimilarly, combination of the repressive activities of a CR and a DNA-binding repressor protein can significantly improve CRISPRi efficiency. For instance, local recruitment of the KRAB repressor domain alone only led to short-term repression of the targeted locus in animal cells. In contrast, co-recruitment of the KRAB repressor domain and the DNMT methyltransferase fused to the dCas9 ensured stable silencing in somatic cells . Such a Arabidopsis, in which there is no H3K79 methylation pathway [As in mammals, transcriptionally active genes in plants are usually marked by acetylated histones H3 and H4, monoubiquitinated histone H2B (H2Bub), and di/trimethylated histone H3 at lysine residues such as Lys-4 and Lys-36  . Interde pathway , suggest pathway .In mammals, the use of CRISPR-dCas9 engineering has been instrumental in determining that deposition of H3K4me3 actively promotes transcriptional activity. Local recruitment of PRDM9, a histone methyltransferase specifically expressed during mammal meiosis, via a direct and inducible dCas9-PRDM9 fusion increased H3K4me3 deposition and upregulated the expression of several genes with different chromatin contexts . WhereasPolycomb repressive complex (PRC2) is a conserved, multimeric chromatin modifier that catalyses H3K27me3 deposition via the SET-domain containing E(Z) subunit. PRC2 activity is involved in maintaining transcriptional repression .Her2 promoter but this was not sufficient for long-term silencing [In a human cancer cell line, Ezh2-dCas9 was sufficient to generate H3K27 trimethylation at the ilencing . Only [5ilencing  the combilencing . Thus, iMedaka embryos, dCas9-OlEZH2 ubiquitous expression was sufficient for site-specific accumulation of H3K27me3 and long-term target gene repression [FWA locus triggers ectopic CG methylation at numerous loci over gene bodies. Ectopic CG methylation was shown to reduce the accumulation of H3K27me3 as well as H2A.Z in gene bodies, which supports that CG methylation in plants affects H3K27me3 and H2A.Z accumulation [In pression . Interespression . Anticorpression  as well pression ,62 and ppression ,64. A remulation .PRC2 activity can be locally inhibited. A genetically encoded PRC2 inhibitor named EED-binder was used to compete with EZH2 activity in vivo, likely through the titration of EED, another core subunit of PRC2 . When taFWA locus in Arabidopsis could be obtained through targeted DNA methylation [Arx and Pax6 promoters, possibly because the absence of DNA methylation favoured the action of a repressor TF at Arx and of an activator TF at Pax6. Partial rescue of motoneuron identity was obtained by simultaneously targeting dCas9-DNMT3A fusion both at PAX6 and ARX regulatory regions [As described above, efficient silencing of the hylation , which shylation . Knockin regions .Arabidopsis transcription factors (248 TFs out of 327 tested) is sensitive to DNA methylation, which either inhibits (72%) or favours (4.3%) its binding to a DNA motif [This study highlights the huge interest in locus-specific manipulation of DNA methylation to establish direct relations between chromatin regulation and transcription, and also illustrates the intrinsic complexity of predicting how targeted modulation of DNA methylation dynamics might affect the expression of a gene. In line with this, in vitro assays in plants showed that the binding of over three quarters of NA motif .Arabidopsis reveal how DNA methylation can modulate transcription in opposite ways. A recent screen for methyl readers identified readers with positive [Methylation-sensitive differential TF binding is not the sole mechanism which can explain contrasting transcriptional outputs of DNA methylation. Two studies in positive  as well positive . Thus, lOCT4 promoter and enhancer regions using a dCas9–SunTag–p300core fusion in cultured fibroblasts showed that targeted recruitment of P300 to both regulatory regions is sufficient to trigger sustained expression, with maintenance of transcriptional activation after 10 days in culture [Simultaneous manipulation of histone H3K27 acetylation on the  culture . The samIn keeping with this notion of transcription timing, CRISPR-mediated acetylation via dCas9–p300 was used to study the link between periodic histone acetylation and transcriptional bursting during the circadian rhythm. Heterochronic acetylation of histones was sufficient to alter the frequency of gene expression revealing a direct correlation between acetylation levels at regulatory regions and rhythmic transcriptional burst . SimilarThe advent of dCas9 technologies has started to provide efficient tools to disentangle complex and interrelated chromatin functions in different organisms. These approaches also revealed some apparent limitations, the exploitation of which could bring deeper insights into chromatin dynamics in vivo and calls for the design of next generation CRISPR/dCas9-based tools.Medaka, where dCas9-OlEZH2 which was recruited to target loci with methylated cytosines. However, in this study, cautious design of sgRNAs was performed to target only DNAse I hypersensitive sites obtained from DNAse I-seq data from Medaka blastula [Locus accessibility is an important parameter when conceiving transcriptional or epigenome engineering experiments. Indeed, nucleosomes are a direct physical barrier for Cas9 target binding, both in vitro  and in vblastula . This alFWA promoter, only moderate transcriptional activation could be detected, whereas targeting the non-methylated promoters of APETALA3 or CLV3 led to a strong increase in mRNA accumulation. It is not entirely clear whether these differences resulted from a differential access of the dCas9 fusion protein or to a competition between the transactivating effect of VP64 and the silencing action linked with DNA methylation.Interrogating the epigenome through local perturbation of chromatin dynamics via dCas9–CR fusions can result in variable outputs depending on the target locus chromatin context. In a trivial way, opening up the accessibility of a locus via targeted histone acetylation, for instance, can promote equally the binding of a transcriptional activator or repressor at different loci, hence leading to a different transcriptional response ,12. SimiSox1 gene regulatory regions in mice neural progenitor cells led to a heterogeneous response with many transfected cells escaping CRISPRa [Sox1 TSS and a more homogeneous response was achieved by combining the simultaneous recruitment of the histone acetyltransferase p300 and the DNA demethylase TET1. Plant studies using CRISPR-mediated epigenome engineering so far have been conducted at the whole organism level. Thus, the observed response is likely an average of many different scenarios that can blur interpretations. As single-cell approaches are picking up in plants, especially scRNA-seq [The outcome of targeted epigenome manipulations can also vary depending on cell identity, i.e., on the transcriptional and chromatin status of a given locus at a given developmental time point. Targeting dCas9-VP64 or -VPR to the  CRISPRa . This vacRNA-seq , transcrcRNA-seq ,82. TherOne complementary leverage in exploiting epigenome editing technologies in plants is the use of inducible or cell-specific control of the CRISPR–dCas9 system. Cas9 expression can be rendered inducible and cell-specific ,84 and, AtMED21 gene led to cellular identity reprogramming, consistent with reduced target gene expression. Further work demonstrated that TPL300-mediated gene co-repression involves members of the mediator complex [Conditioning of dCas9 activity to auxin concentration was achieved by fusing the dCas9–C-terminus to an auxin dependent degron (31). In this HACR system e, the dC complex . AccuratThis report summarizes current approaches to locally modify plant chromatin homeostasis through the use of dCas9 and address the function of individual chromatin regulator activity. While in vivo studies based on dCas9-based approaches are slowly cranking up in plants, they should be more generally accompanied by transcriptomic and epigenomic analyses not only to verify target specificity but also to assess efficiency and consequences in distinct cellular and molecular contexts. Considering all proof-of-concept works in plants see , so far


TERT). Improved understanding of the transcriptional control of this gene may provide potential therapeutic targets. Epigenetic modifications are a prominent mechanism to control telomerase activity and regulation of the TERT gene. TERT-targeting miRNAs have been widely studied and their function explained through pre-clinical in vivo model-based validation studies. Further, histone deacetylase inhibitors are now in pre and early clinical trials with significant clinical success. Importantly, TERT downregulation through epigenetic modifications including TERT promoter methylation, histone deacetylase inhibitors, and miRNA activity might contribute to clinical study design. This review provides an overview of the epigenetic mechanisms involved in the regulation of TERT expression and telomerase activity.Maintenance of telomeres is a fundamental step in human carcinogenesis and is primarily regulated by telomerase and the human telomerase reverse transcriptase gene (TERT encodes the catalytic subunit and rate-limiting factor for telomerase enzyme activity. The mechanisms of activation or silencing of TERT remain open to debate across somatic, cancer, and stem cells. Promoter mutations upstream of TERT may promote dysregulated telomerase activation in tumour cells but additional factors including epigenetic, transcriptional and posttranscriptional modifications also have a role to play. Previous systematic analysis indicated methylation and mutation of the TERT promoter in 53% and 31%, respectively, of TERT expressing cancer cell lines supporting the concept of a key role for epigenetic alteration associated with TERT dysregulation and cellular transformation. Epigenetic regulators including DNA methylation, histone modification, and non-coding RNAs are now emerging as drivers in the regulation of telomeres and telomerase activity. Epigenetic regulation may be responsible for reversible silencing of TERT in several biological processes including development and differentiation, and increased TERT expression in cancers. Understanding the epigenetic mechanisms behind telomerase regulation holds important prospects for cancer treatment, diagnosis and prognosis. This review will focus on the role of epigenetics in telomerase regulation.Telomerase was first described by Greider and Blackburn in 1984, a discovery ultimately recognized by the Nobel Prize committee in 2009. The three decades following on from its discovery have been accompanied by an increased understanding of the fundamental mechanisms of telomerase activity, and its role in telomere biology. Telomerase has a clearly defined role in telomere length maintenance and an established influence on DNA replication, differentiation, survival, development, apoptosis, tumorigenesis, and a further role in therapeutic resistance in human stem and cancer cells including those of breast and cervical origin.  TERT which is the rate-limiting component of telomerase activity. A direct correlation between TERT mRNA level expression and telomerase activity is well described [Telomerase, a ribonucleoprotein enzyme, is the main complex responsible for telomere elongation. The telomeric sequence was recognized as an essential structure at the end of chromosomes ,2, then escribed .TERT in cancer through promoter mutations [TERT expression in human tumour cell lines [Genetic mechanisms contribute to the regulation of utations , gene coutations , and genutations . Epigeneutations , histoneutations  all alsoll lines ,20,21.Telomeres are guanine-rich tandem repeats that protect chromosomes from degradation, provide stabilization, and contribute to overall chromosomal organization . Human tIn human cell populations, telomere lengths range from ~5 to 15 kb and each cell division results in a telomere repeat loss of 25–200 bp due to the end replication problem ,30 In DNTelomerase enzyme activity in somatic cells can be detected at very low levels . HoweverTERT locus is located on the short arm of human chromosome 5 (5p15.33), 2 Mb distal from the telomere [TERT locus thereby negatively regulating expression via telomere position effect [TERT via changes in epigenetic status. Telomere shortening alters histone marks and DNA methylation of promoter regions that regulate TERT expression. Human fibroblasts with long telomeres have significantly higher methylation levels in the TERT promoter region than cells with shorter telomeres. Further, aged cells with short telomeres show an increase in both active chromatin marks, H3K4 trimethylation (H3K4me3) and H3K9 acetylation (H3K9ac), across the TERT promoter. These results support the concept that telomere length-associated changes or telomere position effect might affect TERT transcription. Three-dimensional interactions between the TERT locus and the sub-telomeric 5p region are conserved in young somatic cells with elongated telomeres but 5p/TERT looping interactions become separated by gradual telomere shortening [The telomere . Longer n effect ,47. Kim ortening . Telomerortening . HoweverTERT promoter contains a GC-rich sequence and binding sites for many transcription factors but does not contain TATA or CAAT box transcriptional regulatory elements [TERT promoter including activators; SP1 (Specificity Protein 1), MYC, NF-κB (Nuclear Factor κB), AP1, STAT3 , STAT5, PAX (Paired Box Proteins), ER (Estrogen Receptor), HIF1s, and repressors; MAD1, P53, WT1, (Wilms’ tumour 1 suppressor), SP3, CTCF factor and E2F1 [TERT promoter [TERT transcription in various cancer cell lines [TERT promoter in normal human somatic cells [Transcriptional regulation of TERT can be driven through epigenetic mechanisms including DNA and histone modifications around the TSS of the promoter. The elements ,50. A nuand E2F1 . MYC dimpromoter , while tll lines . Howeveric cells . Furtheric cells .TERT in HCC [TERT expression. STAT3 binding to the promoter in breast cancer stem cells [TERT promoter by binding to the proximal region, and indirectly via increased binding of MYC and SP1 [TERT promoter [STAT3 has a key role in the expression of T in HCC , breast T in HCC  and glioT in HCC  where STem cells  along wiem cells ,61. NF-κ and SP1 ,62. Nuclpromoter ,64.TERT promoter has two HIF1 consensus sequences between −165 and +51 [TERT transcription, driven through these two HIF1 binding motifs [TERT promoter activity in renal cell carcinoma cell lines, while HIF2A alone inhibited TERT promoter activity in glioma cell lines [TERT promoter identify the sites of transcription factors binding, frequent mutation, and response elements that outline a complex landscape of activation, inactivation, and dual role regulators  that can both activate and inactivate gene expression [The hypoxia-inducible transcription factor family  are activated in response to sub-ambient oxygen levels and regulate adaptive cellular responses . The TER and +51 . HIF1A ug motifs . HIF1A all lines . DetaileTERT transcription via binding at −1877 and −1240 upstream of the transcription start site (UTSS). Moreover, P21, itself a downstream activation target of P53, has a significant role in regulating P53 dependent TERT suppression [TERT are MYC and MAX which bind the E-boxes consensus sites (5′-CACGTG-3′) at positions −165 and +44 to regulate TERT activity [TERT gene. The cytosine to thymidine transitions at −124 bp and −146 bp are common mutations relative to ATG (start codon) [TERT pre-mRNAs can be spliced into 22 isoforms during development but only the full-length transcript (16 exons) is functionally active [TERT +α+β mRNA levels [TERT –α+β, TERT +α–β and TERT –α–β) decreased telomerase activity due to interference from these non-functional alternate splice variants [TERT –α+β, TERT +α–β and TERT –α–β) expression becomes dominant and telomerase activity is reduced [The P53 transcription factor, with SP1, and potentially others, suppressed pression ,70,71. Oactivity . Furthert codon) ,74. Thest codon) . Mutatiot codon) . Alternat codon) ,79. Humay active . ExpressA levels . Alternavariants . The –α variants ,83. Deve reduced ,84,85.Waddington defined epigenetics as the correlation between phenotypic and genotypic changes during mammalian embryonic development, and tissue-specific gene function, through studies on Drosophila melanogaster ,87. Tradn = 269) and somatic cells demonstrated that a distinct methylation signature distinguished hiPSCs (human-induced pluripotent stem cells) and hESCs from somatic cells [Environmental factors such as pollutants, diet, temperature changes, and other external stresses can modulate the establishment of epigenetic modifications, and thereby influence subsequent gene expression, development, metabolism, and phenotype . DNA metic cells . As highic cells . DNA metic cells . DNA metic cells . DNMT1 cic cells . DNMT3A ic cells . The expic cells ,98. DNMTic cells ,100.Ten-eleven translocation family of dioxygenases (TETs) catalyse the successive oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) . TET famHistone modification is described by covalent post-translational modification of histones including methylation, phosphorylation, acetylation, ubiquitylation, and sumoylation. They are also responsible for gene expression changes via modification of histone structure or disruption of transcription factor access to promoter sequences. Acetylation of histones is regulated by two main enzyme families; histone acetyltransferases (HATs) and histone deacetylases (HDAC) . HDACs aA further contributor to epigenetic modification is non-coding RNAs. These are a cluster of RNAs that regulate gene expression at the post-transcriptional level such as miRNAs (microRNAs), piRNAs (piwi-interacting RNAs), siRNAs , and lncRNAs (long non-coding RNAs). A number of studies have shown that non-coding RNAs also play an important role in the post-transcriptional regulation of epigenetic-modifying enzymes and genes involved in carcinogenesis . lncRNAsTERT is significant in carcinogenesis and, because of that, is a potential target for cancer treatment. However, the underlying epigenetic regulation of this gene remains ambiguous. DNA methylation, histone methylation–acetylation, and non-coding RNAs all play roles in the regulation of TERT expression in various biological processes including ageing and cancer [TERT promoter harbours a large CpG island  within the promoter region, exons 1 and 2 (UCSC Genome Browser). Further, TERT promoter and exon 1 were found to be highly methylated in primary acute myeloid leukaemia cell lines [TERT promoter is an exception to this model due to its hypermethylated promoter pattern found in most tumour cells and accompanying robust expression [d cancer . The TERll lines ,118. DNAll lines . The TERpression .TERT expression levels in tumour cells. For instance, screening of 31 cancer types, including tumours and non-neoplastic tissue samples, revealed that 95% of them displayed telomerase activity and of that 53% displayed altered TERT promoter methylation, 31% TERT promoter mutations, 3% TERT amplification, 3% TERT structural variants, and 5% TERT promoter structural variants. In addition, TERT promoter methylation and mutations were associated with relative telomere shortening when compared to other alterations [TERT expression [TERT promoter rearrangements could potentially change the position of enhancer elements which regulate activation of TERT expression [Epigenetic and genetic factors both have roles in determining erations . Structupression . TERT prpression .TERT during differentiation and stem cell research has the potential to contribute to our knowledge of down-regulation of TERT and other related gene regulations [TERT during differentiation. hESCs provide a vital model for the assessment of endogenous epigenetic gene regulation during differentiation in a non-transformed background. Embryonic differentiation contributes to telomeric attrition and the initiation of aging, however, mechanisms underlying telomerase down-regulation remain elusive. Epigenetic studies in ESC differentiation could indicate whether DNA methylation or histone modification contribute to this process and might also help our understanding of TERT regulation in telomerase-positive cancer cells.Epigenetic mechanisms may be responsible for reversible silencing of the ulations ,121 Retiulations . hESC diulations . These iTERT promoter region includes several GC motifs  where the promoter methylation pattern affects gene expression [TERT core promoter region spans 260 base pairs including several transcription factor binding sites, e.g., MYC, SP1, HIF1, and WT1  located upstream of the TERT promoter contains 52 CpG sites across its 433-bp genomic region [TERT promoter activity regardless of TERT promoter mutations while hypermethylation of this region increased promoter activity in human tumours [TERT promoter reduced the ability of CTCF (CCCTC-binding factor) transcriptional repressor from binding the CCCTC binding region in the first exon of TERT  [TERT [TERT promoter hypermethylation, which in turn activates TERT transcription, which further up-regulates DNMT3B expression with a positive feedback loop. However, there is not enough data to explain how THOR hypermethylation occurs in cancer. De novo DNA methyltransferase DNMT3B aberrant up-regulation during early cancer progression may induce THOR hypermethylation.TERT cooperates with the transcription factor SP1 to promote patients . All-traexposure . Furtherfication ,149. It fication . The inh., 2009) ,151. Fur., 2009) . In summ9) [TERT ,151. A p9) [TERT . AccordiTERT promoter is strongly linked to its regulation in stem and cancer cells [TERT transcription [TERT promoter and activate TERT transcription through histone H3K4 dimethylation or trimethylation in human fibroblasts and cancer cells [TERT mRNA levels were observed after overexpression of SMYD3, while its suppression led to reduced H3K4 trimethylation within the TERT core promoter and also decreased the ability of MYC and SP1 to bind the promoter in cancer cell lines [TERT promoter through SMYD3 provides an optimal binding condition for MYC and SP1. H3K4me3 is considered a positive marker for gene activation.DNA is packaged around histone proteins and modifications of DNA and histone tails regulate chromatin structure and gene accessibility, therefore, modifications within the histone core affect the interactions between the nucleosome and DNA . Epigeneer cells . DNA metcription ,155. Adder cells . Increasll lines . TherefoMethylation of histone tails, occurring on lysine and arginine residues, may induce repression or expression of genes related to methylation level and methylated residue . Mono anTERT promoter are H3K27ac and H3K4me3 active histone marks and epigenetic silencing H3K9me3 and H3K27me3 marks  exhibit increased H3K4me3 and H3K9Ac marks, where CRISPR mediated reversal of mutant to wild-type TERT promoter leads to a reduction of telomerase activity and active histone marks [Common histone modifications in the e3 marks  [149,159er cells . Akincilne marks .TERT promoter region spanning −150 to +150 from UTSS displayed active chromatin remodelling and an overall lack of methylation, enabling transcription of the TERT gene. Nevertheless, the −600 bp UTSS region is intensely methylated in breast, lung, colon cancer cells and immortalized cell lines [TERT promoter [TERT expression in telomerase-negative cell lines, while on the other hand, H3 and H4 hyperacetylation and H3K4 methylation are associated with TERT transcription in telomerase positive cells [Zinn et al. reported that the ll lines . Additiopromoter . H3K4 hyve cells ,163.TERT promoter activity is decreased 5–10 fold after 14 days differentiation of hESCs [TERT locus. However, during differentiation, these active histone marker levels were considerably down-regulated [TERT promoter activities in hESCs and differentiated cells. Moreover, a considerable increase in H3K9me3 and H3K27me3 levels on TERT promoter are observed and these two marks are related to gene silencing in differentiated hESCs [of hESCs . Additioegulated . It was ed hESCs .TERT expression in normal cells [TERT expression were downregulated in brain cancer cells A-172 (glioblastoma) and ONS-76 (medulloblastoma) after exposure to HDAC inhibitors [Histone deacetylation is a repressive feature for al cells ,164. Inhal cells . Consisthibitors . Alternahibitors . As a reNon-coding RNAs are functional RNA molecules transcribed from DNA that modulate gene expression at transcriptional and post-transcriptional levels. miRNAs are 18–25 bp non-coding RNAs. They contribute to numerous biological processes, including carcinogenesis, by acting as tumour suppressors or oncogenes . A largeLncRNAs modulate transcriptional regulation by acting as scaffolds to facilitate the assembly of specific transcriptional complexes or post-transcriptional regulation via serving as sponges to reduce the functional availability of specific miRNAs ,180. lncThe potential clinical application of TERT expression and telomerase activation or telomerase activity inhibition remains at the forefront of a number of cancer therapeutic strategies. Regulation of telomerase via epigenetic modification aids further understanding of the onset of cancer and holds significant potential for therapy. Epigenetics and genetics in concert may provide the necessary framework to resolve the remaining challenges. The continued development of an understanding of TERT regulatory mechanisms will help clarify this complex, multifactorial, process. It is clear that alternate control mechanisms work in a synchronized and orchestrated manner to ensure proper regulation of telomerase activity and maintenance of telomere lengths. Epigenetic manipulation of TERT could be a promising approach for the treatment of cancer.TERT promoter is unique to human cancer and might be useful as a cancer diagnosis [TERT promoter is an attractive target for tumor-specific therapy to selectively kill cancer cells without impacting somatic cell populations due to high levels of telomerase expression in cancer cells [TERT promoter is a potential target for epigenetic-driven telomerase therapy. By combing dCas9  with the Tet1 (Ten-eleven translocation methylcytosine dioxygenase 1) catalytic domain, an enzyme involved in regulating the demethylation of DNA, researchers were able to successfully demethylate a region of DNA and subsequently upregulate the transcription of the target genes [TERT promoter region to reduce telomerase activity. G-quadruplexes (G4s) are important for transcription and replication processes due to their role in both DNA and histone methylation. G4 structures can act as an obstacle to replication fork progression and disrupt histone recycling, which means that original histone modifications can be replaced with new histones resulting in epigenetic reprogramming [TERT promoter has a high propensity of G4-formation [TERT promoter is an emergent concept and the clinical utility remains to be determined. It is suggested that CpG methylation at the TERT promoter induces G4 formation, enriched near TERT TSS, that results in disrupted CTCF protein binding and an increase in transcription [The hypermethylation pattern of iagnosis ,198 and iagnosis  but furter cells . The TERet genes . The appgramming . The TERormation . The lincription .TERT and repressed telomerase activity in non-small cell lung cancer and lymphoma cells [TERT promoter [TERT mRNA and telomerase activity in somatic cells, while, under the same conditions no altered response was noted with cervical cancer cell lines [TERT expression via demethylation and recruitment of CTCF into the demethylated TERT promoter region in colon cancer cells [The current diversity of epigenetic tools available creates an opportunity for novel strategies for reversible telomerase regulation and targeted telomere maintenance pathways. For example, preclinical and clinical information has suggested that histone deacetylase inhibitor (HDACi) can create a new therapeutic concept for biomedical applications . HDACi mma cells ,213. Vorpromoter . HDACi ipromoter , human npromoter , small-cpromoter  and prospromoter . In contll lines . Furtherer cells . It remaer cells . SimultaAlthough telomerase possesses desirable advantages as a target, there are challenges and limitations for the development of successful clinical therapies. For example, targeting telomerase alone requires an extended treatment time to see gradual attrition of telomeres. However, a combination with other targeted therapies might be a solution to overcome challenges and achieve optimal anticancer effects. Moreover, the outcomes of targeting TERT expression are expected to be different from those arising upon the inhibition of the telomerase catalytic activity alone.