The strengths of StUbi, StGBSS, StPat, and StLhca3 promoters were compared with the conventional constitutive CaMV 35S promoter in various organs  of greenhouse-grown plants. The final amount of gene product was determined at the post-transcriptional level using histochemical analysis, fluorometric measurements, and Western blot analysis. The promoter strength comparison demonstrated that the StUbi promoter generally provided a higher level of constitutive β-glucuronidase accumulation than the viral CaMV 35S promoter. Although the StLhca3 promoter was predominantly expressed in a green tissue-specific manner (leaves and stems) while StGBSS and StPat mainly provided tuber-specific activity, a “promoter leakage” was also found. However, the degree of unspecific activity depended on the particular transgenic line and tissue. According to fluorometric data, the functional activity of promoters in leaves could be arranged as follows: StLhca3 > StUbi > CaMV 35S > StPat > StGBSS (from highest to lowest). In tubers, the higher expression was detected in transgenic plants expressing StPat-gusA fusion construct, and the strength order was as follows: StPat > StGBSS > StUbi > CaMV 35S > StLhca3. The observed differences between expression patterns are discussed considering the benefits and limitations for the usage of each promoter to regulate the expression of genes in a particular potato tissue.Various plant-derived promoters can be used to regulate ectopic gene expression in potato. In the present study, four promoters derived from the potato genome have been characterized by the expression of identical cassettes carrying the fusion with the reporter β-glucuronidase ( The potato is an essential vegetable crop in various climate areas. The global production of potatoes takes first place among dicotyledonous species, second only to monocotyledonous cereal crops such as wheat, rice, and corn . It is gIn recent decades, the advanced methods of genetic modifications have become increasingly important in modern breeding programs. Such technologies as transgenesis and genome editing provide the specific alteration of potato genome that is not easily reached using conventional breeding methods . Many fa35S promoter of Cauliflower Mosaic Virus (CaMV) as a main genetic element for regulating transgene expression. Although the CaMV 35S promoter remains the most popular in potato and other dicotyledonous species, the native promoters have become more in demand to control the temporal and spatial efficiency of expression [To date, the majority of transgenic potato plants have been produced using the constitutive pression ,5,6. Thepression . This, ipression ,9. Various plant-derived promoters have already been validated to provide different transgene expression patterns in potato. The number of promoters derived from the potato genome is still limited, but a few of them were successfully applied for genetic engineering. The best-known potato promoters are promoters that drive tuber-specific expression. To date, several promoters have been characterized, including promoters of such genes as patatin , granule35S promoter is used to achieve the overexpression or knockout of genes involved in the resistance of potato to biotic stresses. In a few cases, the promoters derived from the housekeeping genes, such as ubiquitins, have been characterized and used as alternatives to the CaMV 35S promoter [35S promoter caused some imbalance in transgenic plants, negatively affecting physiological processes and tuber production [Lhca3 gene  was identified and included in expression cassettes to control tuber moth attacks [The promoters expressed in green tissues also play an important role in potato transgenesis. Green tissues, especially foliage, are the main target for viral, pathogenic, and insecticidal attacks in potato. Generally, the viral CaMV promoter ,21,22. Toduction ,23. Unwa attacks  and blac attacks  and to l attacks . Alterna attacks ,28. 35S promoter, while the direct comparison between the promoters is rarely performed. Additionally, it is rather problematic to compare particular reports as varied expression cassettes, potato varieties, and environmental conditions were used to characterize the activity and strength of promoters. The aim of this work was to test and compare the efficacy of several potato-derived promoters with an aim to apply them in various functional and applied studies. It should be noted that in most published reports, the potato promoters were readily compared with the CaMV StPat and StGBSS) and two promoters targeted for expression in foliage, such as the constitutive StUbi promoter and the light-inducible StLhca3 promoter. The activity of promoters was determined at a post-transcriptional level using promoter–gusA fusion and compared with the identical CaMV 35S–gusA construct. In our study, we simultaneously analyzed the activities of four promoters isolated from the potato genome. These include two tuber-specific constitutive promoters  and the StUbi gene  were amplified from genomic DNA of potato cv. “Manhattan”. Promoters of tuber-specific class-I patatin gene (StPat) (GenBank no. A08215) and granule-bound starch synthase (StGBSS) genes (GenBank no. A23740) were cloned from genomic DNA of potato cv. “Chicago”. The proof-reading Phusion high-fidelity DNA polymerase  was used to amplify the promoter sequences by using PCR primers as detailed in The promoter regions of the 35S promoter driving the gusA gene was removed was used to create constructs for transformation. Promoter fragments of the StGBSS, StPat, StLhca3, and StUbi were released from the relevant intermediate vectors and then placed upstream of the gusA reporter gene with the HindIII—XbaI (StGBSS) or HindIII—BamHI  sites. The resulting constructs were designated as pBI-Pat, pBI-GBSS, pBI-Ubi, and pBI-Lhca (Agrobacterium tumefaciens strain AGL0.A modified binary construct pBI121  in whichpBI-Lhca  and wereSolanum tuberosum) cv. “Chicago” provided by the Doka-Gene Technology Ltd.  was used for genetic transformation. Plants were maintained in vitro in plastic containers under controlled conditions (16 h light 40 µmol m−2 s−1 and 8 h dark at 22–25 °C) on phytohormone-free Murashige and Skoog (MS) basal medium supplemented with 3% sucrose and 7% agar, pH 5.8. Stem internodal segments (0.5–1 cm long) of 3–4-week-old micropropagated plants were used as explants for transformation. The inoculation and cocultivation of explants with Agrobacterium were performed as described previously [−2 s−1 and 8 h dark at 22–25 °C) on the MS-basal medium supplemented with zeatin-riboside (3 mg L−1), GA3 (1.0 mg L−1), indoleacetic acid (2 mg L−1), kanamycin (50 mg L−1), and cefotaxime (500 mg L−1). Every 12–15 nodal explants were subcultured on fresh regeneration medium in Petri dishes every 10 days. After the third subculture, the selective concentration of kanamycin was increased to 75 mg L−1. Only one kanamycin-resistant shoot was collected from each explant to avoid duplication of transgenic lines. Independent shoots were multiplicated in culture vessels on phytohormone-free MS medium supplemented with 150 mg L−1 cefotaxime and 75 mg L−1 kanamycin. Potato , 10 mM β-Mercaptoethanol, 10 mM Na2EDTA, 0.1% Sodium lauryl sarcosine (v/v), 0.1% Triton X-100 (v/v). Total protein was extracted for 45 min at 4 °C. After centrifugation for 20 min at 16,000 g at 4 °C, the supernatant was immediately used for analysis. Protein concentration was measured by DC protein assay kit . Four-methyl umbelliferyl β-D-glucuronide  was used in the fluorometric assay; to generate the fluorescence calibrate curve, 4-methyl-umbelliferone  was used as standard. GUS activity was quantitatively determined using fluorometer FLUOstar OPTIMA  and calculated as nmol of 4-methyl-umbelliferone (4-MU) produced per sec per µg of total soluble protein (TSP), noted as units thereafter. For each promoter construct, ten independent transgenic lines were analyzed. Three individual plants of each line were used as biological replicates.The staining of various tissues of transgenic potato was carried out according to . HistochTotal soluble protein (TSP) was extracted as described above for fluorometric assays. A protein sample (25 µg) from each studied line was separated by 12% SDS-PAGE and transferred onto an NC membrane . Rabbit anti-β-glucuronidase  polyclonal antibodies served as the primary antibodies. Anti-rabbit IgG conjugated to alkaline phosphatase was used as the secondary antibody . Blots were treated with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) for visualization.nptII gene (data not shown). The efficiency of Agrobacterium-mediated transformation was in the range of 69–77% for pBI121, pBI-Pat, pBI-Ubi, and pBI-Lhca constructs, while the pBI-GBSS vector displayed weak transformation efficiency of 25% . Surprisingly, the StGBSS promoter strictly directed the gusA gene expression to the “stem cell niche”  of root apical meristem, while the other developing and mature root tissues were lacking GUS activity  than the plants expressing StLhca3 promoter–gusA cassette, although the difference between the highest active transgenic lines was marginal (9752.3 units for StLhca3 promoter and 9592.4 for StUbi promoter) , while the values between the individual independent plants could differ more than 60 times A. In theromoter) A.StGBSS and with StPat promoters, respectively. Due to low fluorometric values, the difference from the untransformed potato (WT) was not statistically significant for the majority of the lines (15 lines) transformed with the pBI-Pat and pBI-GBSS cassettes. Only a few independent lines with residual blue GUS staining in leaf tissues showed significantly higher GUS activity than WT; however, the fluorimetric values were lower than those observed for protein extracts of lines with the weakest gusA expression driven by the CaMV 35S, StUbi, and StLhca3 promoters  was 364.2 units  was generally higher and more stable than the activity of StGBSS promoter (936 bp). Patatin is one of the main storage proteins in potato tubers, and 98–99% of patatin transcripts are accumulated in tubers. In contrast to patatin genes, the expression of granule-bound starch synthase (GBSS) genes is not strictly associated with tuberization. Since the activity of GBSS genes is related to amylose biosynthesis during starch granulation, genes in the GBSS family are known to be expressed in various storage tissues, especially in leguminous seeds and cereal grains [cis-regulatory sequence motives, such as K, M, and D boxes and tandemly arranged sequences [cis-elements responsible for the tuber-specific and sucrose-responsive activity of class-I patatin genes are located between the nucleotides –252 and +14 (relative to the transcription initiation site), thus the deletion of this fragment significantly decreased the expression level [cis-regulatory elements located in the further upstream region may positively influence the strength of activity. To drive the tuber-specific expression of potato GBSSI genes, the 0.4 kb region (–346 bp until +54 bp) of the promoter sequence was found to be required [cis-acting elements, responsible for the grain-specific gbss1 transcription, are located −1.9 kb upstream of the promoter [StGBSS promoter successfully controlled tuber-specific accumulation of the gusA protein with rather low non-tuber-specific activities; its strength, however, was lower than the activity of the 1738 bp StPat promoter. The StPat promoter also showed a higher level of accumulation in other tissues. Owing to the increased demand for exploiting potato tubers as a production platform for nutrients with pharmaceutical or industrial interest, the choice of controllable promoter displaying tuber-specific expression is important for the creation of “factory” plants. Though both purposes ,17,18,19l grains . The proequences ,33,34. Ion level ,35. On tromoter 9 bp. Patapromoter . In our StPat and StGBSS promoters contributed to the leakiness of GUS activity. The unspecific activity was primarily found in old stem tissues, and as we indicated earlier, StGBSS promoter was less active, especially in the young stem tissues. These data are in agreement with those of Visser et al. [StGBSS promoter was used to drive the gusA gene. In contrast, Bansal et al. did not observe StGBSS-driven gusA expression in stems, or in roots and leaves, at least upon histochemical analysis [StGBSS promoter–gusA cassette still displayed a low GUS accumulation. At the same time, the root-specific activity of StGBSS promoter was much lower than reported earlier [StPat promoter, we observed the accumulation of GUS protein in stems and a certain activity was also detected in leaves, but we could not find any histochemical activity of β-glucuronidase in roots. The different genotypes, structural variation in promoters and expression cassettes used in earlier reports, and a sometimes insufficient number of analyzed transgenic lines could explain the contradictory results.In theory, the ideal tuber-specific promoter should not display functional activity in other tissues. In the present study, both r et al. , who preanalysis . In thei earlier , and it  earlier ,39, stol earlier ,39, and  earlier . AminediStGBSS promoter has a clear tendency for silencing as half of the analyzed lines displayed no functional activity of a driven gene. Previously it was shown that different regions of the StGBSS promoter used in RNAi cassettes may influence the level of internal silencing of the StGBSS gene, and the various sequence motives may induce silencing effects in up to 57–60% of produced plants [StGBSS-promoter-based construct in the present study significantly affected the overall output of independent transgenic events as well, since the transformation efficiency with pBI-GBSS vector was considerably lower compared to the other constructs. For the StPat-based construct, the difficulties with the production of functionally active transgenic plants were also found. After the transformation with pBI-Pat vector, a significant part of generated potato plants had truncated variants of StPat promoter–gusA sequence, so a higher number of independent lines was required to identify the events with the correct insertion of StPat promoter–gusA fusion. Interestingly, such insertional and transformation problems were not observed for other constructs, indicating that some additional aspects are present, which are associated with the natural homology between the endogenic allelic sequences of tuber-specific promoters.In the present study, the construct with the d plants . The appStUbi or StLhca3 potato promoters into the transformation vector affected neither the transformation frequency nor the functional activity of transgenic lines. Previously, the promoter of the Lhca3.St.1 gene was shown to predominantly express in foliage and stems and was not expressed in tubers or roots of potato [StLhca3 promoter–gusA construct, though the evident “leakage” phenomenon was discovered in the tubers  and truncated insertions (for constructs with the StPat and StGBSS promoters), should be also taken into account. The results of this study provide important comparative information for designing constructs to produce transgenic and intragenic potato plants for basic research and commercial plant genetic engineering using promoters with a predicted tissue-specific expression.In summary, we compared the level of activity and the expression patterns of a popular viral CaMV


The biosynthesis of diphthamide represents one of the most complex modifications, executed by protein factors conserved from yeast to humans. Diphthamide is not only essential for normal physiology (such as ensuring fidelity of mRNA translation), but is also exploited by bacterial ADP-ribosylating toxins  as their molecular target in pathogenesis. Taking advantage of the observation that cells defective in diphthamide biosynthesis are resistant to ADP-ribosylating toxins, in the past four decades, seven essential genes (Dph1 to Dph7) have been identified for diphthamide biosynthesis. These technically unsaturated screens raise the question as to whether additional genes are required for diphthamide biosynthesis. In this study, we performed two independent, saturating, genome-wide CRISPR knockout screens in human cells. These screens identified all previously known Dph genes, as well as further identifying the BTB/POZ domain-containing transcription factor Miz1. We found that Miz1 is absolutely required for diphthamide biosynthesis via its role in the transcriptional regulation of Dph1 expression. Mechanistically, Miz1 binds to the Dph1 proximal promoter via an evolutionarily conserved consensus binding site to activate Dph1 transcription. Therefore, this work demonstrates that Dph1-7, along with the newly identified Miz1 transcription factor, are likely to represent the essential protein factors required for diphthamide modification on eEF2.Diphthamide is a unique post-translationally modified histidine residue  found only in eukaryotic elongation factor-2 (eEF-2). Mice that are deficient in diphthamide biosynthesis are embryonic lethal, attesting to the importance of diphthamide in normal physiology. It has taken four decades to identify the seven non-redundant genes in diphthamide biosynthesis, but whether additional factors are required and how the pathway is regulated remained elusive. To address these issues, we performed two saturating, independent, and unbiased genome-wide CRISPR knockout screens. The screens concluded independently that Dph1-Dph7 and additionally transcription factor Miz1 are the key factors required for diphthamide biosynthesis. Mechanistically, Miz1 binds to the Dph1 proximal promoter via an evolutionarily conserved consensus binding site to activate Dph1 transcription. While diphthamide biosynthesis machinery (Dph1-Dph7) exists across eukaryotes, Miz1 orthologues do not exist in lower species such as yeast, C. elegans, and Drosophila, indicating that the regulation of diphthamide modification by Miz1 emerged much later in evolution. The work opens a new avenue for understanding the role that diphthamide modification plays in normal physiology and bacterial toxin pathogenesis.Diphthamide is a unique post-translationally modified histidine residue (His Corynebacterium dipheriae diphtheria toxin (DT) ) ≥ 3; Miz1-, Dph1- (as a positive control), as well as the top four hits by single sgRNAs–ST3GAL1-, SDF2-, hsa-mir-5009-, and VPS41-gene edited HT1080 cells  exists in all these proximal promoter sequences (Dph1 promoter by performing a Chromatin Immunoprecipitation (ChIP) assay using B16F10 WT, Miz1-KO, and Miz1-KO (+Miz1) (transfected with a Miz1-expression plasmid) cells. We found that the Dph1 promoter fragments surrounding the Miz1-binding site could be co-immunoprecipitated with Miz1 in B16F10 WT and Miz1-KO (+Miz1) cells but not in Miz1-KO cells  to the cells, and that inactivation of potential weaker regulators of the diphthamide pathway was unlikely to confer the extents of toxin resistance allowing cell survival through the screens, we expected to identify only the non-redundant essential factors and strong regulators of the pathway. As such, our screens appeared to have missed other potential modulators that regulate milder phenotypes or are essential for cell viability. We did not identify the othologue (SERGEF) of the yeast protein Kti13, which is involved in diphthamide modification in yeast via association with Kti11/Dph3 [Atp6v1c1, Atp6v1e1, Atp6v1h, Ccdc115, Tmem199, Wdr7, quickly lost viability during culture.There were a number of genes that play a role in anthrax toxin’s pathogenicity that were not uncovered in our screen. For instance, we used a modified anthrax lethal toxin for these screens, which needs the toxin receptor TEM8 or CMG2 to gain entry into target cells. The reason that we likely did not identify TEM8 or CMG2 from either screens is that HT1080 cells express both TEM8 and CMG2, with each receptor able to independently mediate toxin entry. Therefore, the genome-wide CRISPR KO screen is expected to only identify functionally non-redundant genes under the stringent selection conditions we used in this work . Since ti11/Dph3 . One posi11/Dph3 , 38, 39.Myc-interacting zinc finger protein 1), following the identification of its association with Myc, a basic helix-loop-helix transcription factor, in a two-hybrid analysis [p15Ink4b, p21Cip1, and p57Kip2 and a group of genes encoding proteins involved in cell-cell and cell-matrix adhesion [Miz1 is a BTB/POZ domain-containing transcription factor that is ubiquitously expressed. It was named Miz1 . For cytotoxicity assay, cells grown in 96-well plates (30~50% confluence) were incubated with various concentrations of PA (0–1000 ng/ml) in the presence of 100 ng/ml FP59 for 48 h. Cell viabilities were then assayed by an MTT  assay as described previously , expressPA, LF, and FP59 were purified as described previously  .mle analysis module, to obtain p values, FDRs  [The genome-wide CRISPR knockout lentiviral pooled human library-A and library-B developed by Feng Zhang’s MIT laboratory  were use values) . Genes oTo generate HT1080, RAW264.7, and B16F10 CRISPR edited cells, we cloned the indicated sgRNA sequences  into theTotal RNA was prepared from the indicated cells using TRIzol reagent , and was used to synthesize single-strand cDNA using the SuperScript IV First-Strand Synthesis System following the manufacturer’s manual . Full-length Miz1 cDNA was amplified by reverse transcriptase-PCR from HT1080 cells, cloned into pIREShyg-2 mammalian expression vector, and confirmed by DNA sequencing. The primers used for cloning are listed in Dph1 promoter sequences around the Miz1 consensus binding site or a fragment in the exon 2 using the primers listed in B16F10 WT, Miz1-KO, and Miz1-KO (+Miz1) cells were cross-linked with 1% formaldehyde at 37°C for 10 min. Sonication was carried out after swelling in a Bransson sonifier until the majority of fragments show nucleosomal size. An anti-Miz1 antibody (R&D Systems Cat. No. AF3760) coupled to Protein G-Dynabeads (Invitrogen) was used for immunoprecipitation. DNA was purified with Qiagen PCR purification kit after elution of the bound chromatin with 1% SDS and reversion of the crosslink. The control antibodies included an isotype control IgG  and an anti-Bcl6 . The DNA was used for quantitative PCR analyses of the Dph1 proximal promoter.Bisulfite sequencing analysis was performed using the EpiJET Bisulfite Conversion Kit (Thermo Scientific Cat. No. K1461). Two μg of genomic DNA was subjected to bisulfite conversion following the manufacturer’s manual. The primers avoiding CpG islands-containing regions as listed in + (Trevigen). Samples were then mixed with a SDS sample buffer, boiled for 5 min and separated on 4−25% SDS-PAGE gels (Invitrogen). The proteins were transferred to nitrocellulose membranes and Western blotting was performed using streptavidin-IR conjugate .Cell extracts were prepared in the modified RIPA buffer containing protease inhibitors as described previously . ADP-ribosylation reactions were performed at 37°C for 1 hour in a volume of 50 μl ADP-ribosylation buffer  containing 50 μg of extract, 100 ng FP59, and 10 μM 6-Biotin-17-NADt-tests was used to identify significant (P < 0.05) differences.Cell culture experiments were repeated a minimum of three times in triplicate. In comparisons between two groups with equal variance, unpaired two-tailed Student's S1 Fig715 residue  of eEF2 by Dph1 to Dph4, yielding ACP-eEF2. Step 2: the ACP-eEF2 undergoes tetra-methylation by Dph5, yielding the methylated diphthine. Step 3: Dph7 catalyzes the demethylation, generates diphthine. Step 4: diphthine is converted to diphthamide by amidase Dph6. N-1 (arrow head) of the histidine imidazole ring of diphthamide is the site for ADP-ribosylation by DT, ETA and other ADRTs. Ado-S-Me, methylthioadenosine; Ado-Hcy, S-adenosylhomocysteine; NAD+, nicotinamide adenine dinucleotide. In “ADP-ribosyl diphthamide”: A, adenine moiety; R, ribosyl moiety.Diphthamide biosynthesis is carried out in four steps by the seven highly conserved proteins Dph1-7 from yeast to humans. Step1: transfer of the 3-amino-3-carboxypropyl (ACP) group from S-adenosyl methionine (AdoMet) to the imidazole ring of His(EPS)Click here for additional data file.S2 FigDph1 expression is diminished in all Miz1-KO cells. Three independent HT1080 Miz1-KO clones were analyzed. Note, HT1080 Miz1-KO cells exhibited consistent diminished Dph1 expression. Data are represented as mean (triplicate) ± SD. Unpaired two-tailed Student's t-tests.Eukaryotic translation initiation factor EIF35S was used as an internal normalization control. Of note, (EPS)Click here for additional data file.S3 FigA. DNA sequence alignment of the Dph1 proximal promoters from human, mouse, rat, and Chinese hamster. The alignment was generated using the EMBL-EBL MUSCLE program (https://www.ebi.ac.uk/Tools/msa/muscle/). A consensus Miz1 binding site  exists in these highly homologous Dph1 promoter sequences. Start codon were labeled in green. * indicates the same residue. B. The CpG islands in mouse Dph1 proximal promoter are not methylated in both B16F10 WT cells and Miz1-KO cells. Bisulfite-sequencing examples of a promoter fragment from the B16F10 WT cells and Miz1-KO cells are shown. The original unconverted sequence and theoretically converted sequence except for the CpG islands are also shown at the bottom. Of note, all the cytosine residues in the indicated CpG islands were converted to thymidine residues.(EPS)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Following signaling events that mediate phosphorylation of MYC at Serine 62, PIN1 establishes structurally distinct pools of MYC through its trans-cis and cis-trans isomerization activity at Proline 63. Through these isomerization steps, PIN1 functionally regulates MYC’s stability, the molecular timing of its DNA binding and transcriptional activity, and its subnuclear localization. Recently, our group showed that Serine 62 phosphorylated MYC can associate with the inner basket of the nuclear pore (NP) in a PIN1-dependent manner. The poised euchromatin at the NP basket enables rapid cellular response to environmental signals and cell stress, and PIN1-mediated trafficking of MYC calibrates this response. In this perspective, we describe the molecular aspects of PIN1 target recognition and PIN1’s function in the context of its temporal and spatial regulation of MYC.PIN1 is a phosphorylation-directed member of the peptidyl-prolyl  While proline residues exhibit an intrinsic ability to isomerize, this process occurs on a very slow biomolecular timescale as a result of the high-energy barrier associated with this conformational change. This high-energy barrier isolates the cis and trans protein states, and rapidly switching between these two conformational states requires a catalyst. The evolutionarily conserved peptidyl-prolyl cis/trans isomerases (PPIases) catalyze this conformational change and are required to drive isomerization in a timeframe relevant to dynamic signaling cascades within the cell  is the only known PPIase that specifically recognizes phosphorylated serine or threonine residues that immediately precede a proline (pSer/pThr-Pro). This pSer/pThr-Pro motif accounts for over 25% of all phosphorylation sites identified in a global phosphorylation study . The pro0 arrest . This reLoss of PIN1 also renders cells resistant to transformation and, strikingly, PIN1 knockout mice have delayed tumor formation when crossed with tumor-driving mutants of HER2 or RAS . PhosphoHere, we discuss the role of PIN1 as a critical controller of dynamic phosphorylation signaling cascades in response to extrinsic signals that governs gene transcription to alter phenotypic responses in normal and diseased states. PIN1 affects a variety of target transcription factors in such cascades, but we focus on work describing PIN1’s temporal and spatial control of the bHLH-LZ transcription factor c-MYC (hereafter MYC), which PIN1 functionally regulates in both physiologic and pathologic responses. We will describe how PIN1-dependent isomerization temporally and spatially influences the phosphorylation cascade that affects MYC stability and activity in the nucleus. Together, these roles frame PIN1 as a promising therapeutic target for controlling oncogenic MYC.MYC encodes a critical transcription factor that influences transcription across the genome to control a multitude of cellular processes including proliferation, survival, metabolism, and morphology (trans (trans-specific phosphatase, PP2A-B56α, to remove the activating S62 phosphate (The proto-oncogene rphology . In physrphology . Brieflyy (trans . Phosphoy (trans . Phosphohosphate . pT58-MYhosphate .trans-specific enzymes. Thus, PIN1 can interrupt the progression of pS62-MYC through its degradation cascade by stabilizing Pro63 in the cis-conformation. This sterically protects the Ser62 phosphate from PP2A-mediated dephosphorylation, allowing for prolonged pS62-MYC interaction with DNA and increasing target gene transcription (trans-specific phosphatase, PP2A-B56α (trans isomer helix (cis-trans isomerization of Pro63, allowing for the function of PP2A at pSer62. However, additional research is required to understand precisely how Thr58 phosphorylation promotes the dephosphorylation of pSer62, and how this additional phosphorylation affects PIN1’s activity on MYC.As depicted in cription . HoweverP2A-B56α . Like Seer helix . Thus, wcis. This dynamic interaction may provide a rational role for the additional phosphorylation at Thr58 to re-engage PIN1 with MB1 to mediate a second isomerization event from cis to trans at the more flexible Pro63. The dual function of PIN1 in promoting both MYC’s activity and degradation through two isomerization events is supported by experiments assessing the effects of point mutations in the MB0 domain that disrupt PIN1 pre-anchoring or of PIN1 knockdown. Both conditions result a reduction in MYC DNA binding and a corresponding decrease in target gene activation, cellular proliferation, and cellular transformation, even though there is an increase in pS62-MYC and MYC stability (Structural studies into PIN1’s substrate interactions indicate that a flexible interdomain, which connects PIN1’s WW phospho-substrate binding domain to its PPIase catalytic domain, can exist in different rigidity states that influence PIN1 target binding and isomerase activity . Furthertability .In addition to directly controlling the conformation of MYC to affect its activity vs. ubiquitination, other proteins regulate and are regulated by PIN1 that contribute to the MYC degradation pathway. For example, PIN1 can downregulate the E3 ubiquitin ligase FBW7 , which cUnderstanding the dynamics of MYC regulation is critical in order to elucidate the pleiotropic effects of MYC in the genome and its control of diverse cellular phenotypes. PIN1 plays a key role in this regulation by imparting both temporal and spatial regulation of MYC activity in the nucleus. Temporal studies of MYC DNA binding revealed that MYC oscillates on and off DNA at E-box containing promoters in response to cell growth signaling . This dyIn addition to temporally regulating MYC activity, PIN1 regulates the subnuclear localization of MYC under normal mitogen stimulation conditions, during wound healing, and in cancer cell lines . InitialThe PIN1-driven spatial reorganization of MYC to specific chromatin domains at the NP suggests that post-translational control of transcription factors in response to environmental signals may dictate their involvement in regulating specific topologically associated domains or TADs. Interestingly, the number and composition of NPs is increased and altered in cancer cells . In addiThese findings suggest that in response to extrinsic signals, PIN1 facilitates the generation of a distinct pool of post-translationally modified MYC that associates with chromatin near the inner basket of the NP. This pool may be distinct from the population of MYC within the nuclear interior that binds promoter regions in open chromatin. There is much discussion in the field for whether oncogenic MYC acts as a global transcriptional amplifier or if there is a more specific MYC-driven gene program that drives malignancies . Our datHere we present a perspective of the role of PIN1 in regulating dynamic response phenotypes, focusing on its isomerization of MYC in multiple cellular contexts. PIN1’s interaction with and isomerization of MYC supports the physiologic and oncogenic activity of MYC . MechaniMultiple efforts to therapeutically reduce or control MYC’s oncogenic activity have been unsuccessful for several reasons, including an inability to specifically control MYC expression and the lack of an enzymatic region to target with small molecules . The dirGC contributed to writing all sections of the manuscript. DL and EL wrote sections of the manuscript. EL organized and oversaw the conceptual approaches. RS oversaw conceptual approaches, edited all sections of the manuscript, and approved the manuscript. All authors contributed to manuscript revision, read, and approved the submitted versions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Saccharomyces cerevisiae upon hyper-osmotic shock. We find that the transient activity of the MAPK Hog1 opens a temporal window where stress-response genes can be activated. We show that the first minutes of Hog1 activity are essential to control the activation of a promoter. Chromatin repression on a locus slows down this transition and contributes to the variability in gene expression, while binding of transcription factors increases the level of transcription. However, soon after Hog1 activity peaks, negative regulators promote chromatin closure of the locus and transcription progressively stops.Precise regulation of gene expression in response to environmental changes is crucial for cell survival, adaptation and proliferation. In eukaryotic cells, extracellular signal integration is often carried out by Mitogen-Activated Protein Kinases (MAPK). Despite a robust MAPK signaling activity, downstream gene expression can display a great variability between single cells. Using a live mRNA reporter, here we monitor the dynamics of transcription in  Saccharomyces cerevisiae upon hyper-osmotic shock and characterize the influence of MAPK signalling, chromatin and transcription factors on the dynamics of transcription.Mitogen-Activated Protein Kinases integrate extracellular information in all eukaryotic cells. Using a live mRNA reporter, here the authors monitor transcriptional bursting in  Tight regulation of signaling is thus crucial to ensure the correct temporal modulation of gene expression, which can otherwise alter the cell physiology6. Interestingly, single-cell analyses have revealed that genes regulated by an identical signaling activity can display a high variability in their transcriptional responses10. This noise in transcriptional output questions how signal transduction can faithfully induce different loci and which molecular mechanisms contribute to the variability in gene expression.A crucial function of all cellular life is the ability to sense its surroundings and adapt to its variations. These changes in the extracellular environment will induce specific cellular responses orchestrated by signal transduction cascades, which receive cues from plasma membrane sensors. This information is turned into a biological response by inducing complex transcriptional programs implicating hundreds of genes12. They control a wide range of cellular responses such as cell proliferation, differentiation, or apoptosis. In Saccharomyces cerevisiae, a sudden increase in the osmolarity of the medium is sensed by the High Osmolarity Glycerol (HOG) pathway, which leads to the activation of the MAPK Hog1, a homolog of p38 in mammals14. Upon hyper-osmotic stress, the kinase activity of Hog1 promotes the adaptation of the cells to their new environment by driving an increase in the internal glycerol concentration, thereby allowing to balance the internal and the external osmotic pressures. In parallel to its cytoplasmic activity, Hog1 also transiently accumulates into the nucleus to induce the expression of hundreds of osmostress-responsive genes  cascadesnes Fig. . The MAP15. In parallel, single-cell measurements have uncovered the large variability present in their expression. In particular, translational reporters and RNA-FISH measurements have identified that slow chromatin remodeling promoted by the MAPK at each individual locus is generating strong intrinsic noise in the activation of many stress-responsive genes17.Biochemical analyses of this pathway have identified the key players implicated in gene expression and the central role played by the MAPK in all these steps20. These experiments contributed to revealing the bursty nature of transcription, whereby a set of polymerases simultaneously transcribing a gene generates a burst in mRNA production, which is followed by a pause in transcription23.In order to get deeper insights into the regulation of osmostress-genes expression kinetics, we aimed at monitoring the dynamics of mRNA production in live single cells. Phage coat protein-based assays, like the MS2 or PP7 systems, have been used to visualize mRNA in live single cellsIn this study, we dissect the kinetics of transcription of osmostress genes. The production of mRNA is monitored using the PP7 phage coat protein assay. This reporter allows us to measure with high temporal resolution and in a fully automated manner, the fluctuations in transcription arising in hundreds of live single cells. This analysis enables to dissect the contribution of various players to the overall transcriptional output. We show that the first few minutes of MAPK activity will determine if a gene is transcribed. We also demonstrate that the chromatin state of a promoter will control the timing of activation and thus the variability in the transcription, while the TF binding will influence the level and duration of the mRNA production.Saccharomyces cerevisiae via the HOG signaling cascade, which culminates in the activation of the MAPK Hog1  induce osmostress-genes expression, with approximately 250 genes being up-regulated upon osmotic shock29. The activity of the pathway is limited to the cellular adaptation time, which coincides with the nuclear exit of Hog1 and the recovery of the cell size . In our settings, this PP7 reporter construct is placed under the control of a promoter of interest and integrated in the genome at the GLT1 locus  and expressing a fluorescently-tagged PP7 protein . Upon activation of the promoter, local accumulation of newly synthesized transcripts at the Transcription Site (TS) leads to the formation of a bright fluorescent focus due to the enrichment in PP7-GFP fluorescence above the background signal cus Fig. 30 in a s33. The mean intensity of the 20 brightest pixels in the nucleus, from which the median cell fluorescence was subtracted, was used as a measurement of TS intensity and thus as proxy for transcriptional activity ; however, each strain displays a different transcriptional response following a 0.2 M NaCl stimulus , while pALD3 and pSTL1 require more than 4 min for activation  while the full adaptation time takes 14 min at 0.2 M NaCl.The period of Hog1 activity provides a temporal window, where transcription can potentially be initiated. However, the switch to a transcriptionally active state takes place almost exclusively within the first few minutes after the stimulus. When comparing the characteristic timing of Hog1 nuclear enrichment to the CDF of Start Times for cells bearing the pter Fig. , we obseter Fig. . For pALSTL1 earlier display a larger integral over the PP7 signal and a higher maximum intensity compared with cells that initiate transcription later  influence the promoter output44. Unfortunately, osmostress promoters display a wide diversity in numbers and affinities of TF binding sites and no obvious prediction of the transcriptional activity can be drawn , while pGPD1 activity is barely affected  strongly alters its capability to induce transcription. In comparison, the pGPD1 is less perturbed by these same defects. These results suggest that TFs act in a cooperative manner on pSTL1, while they act independently of each other on pGPD1.Because the induction of the GLT1). Based on a transcription speed of 20 bp/s30, the expected lifetime of a transcript at the TS is 6.6 min. This corresponds well to the mean duration observed for the pALD3, pCTT1, pSTL1, and pGRE2 reporters . In 20–30% of the traces, two or more peaks are identified Fig. . The toters Fig. . HoweverHSP12 and pGPD1, the average peak duration is longer than 11 min  terminate transcription earlier. This shorter transcriptional window may reflect an inferior recruitment of transcriptional activators to the promoter, enabling an earlier inhibition of transcription due to chromatin closure. In addition, promoters with basal activity display an extended period of transcription after adaptation . The unexpectedly high signal obtained from the PP7 reporter at the endogenous locus may be indicative of global difference in transcription rates between the GLT1 and STL1 ORFs alternatively, the smaller STL1 ORF might enhance transcription efficiency via gene-looping51.In this study, we have constructed PP7 reporter strains to monitor the transcription dynamics of osmostress promoters. The second exogenous copy of the promoter is integrated at the 40. Once initiated, transcription seems highly processive and only in a small fraction of traces, we are able to detect a pause in transcription. However, it has been shown that PolII recruits additional chromatin remodelers, including the Ino80 complex and Asf1 that will redeposit nucleosomes after acute transcription52. These conflicting activities will determine the overall duration of transcription at a locus. Indeed, promoters with lower transcriptional activity, such as pGRE2 and pCTT1, recruit fewer positive activators and will be repressed faster by the negative regulators.Our data illustrate the complex balance that exists between positive and negative regulators taking place at the stress-induced loci. At each locus, positive and negative regulators will control the level and duration of transcription. We have shown that the first few minutes of Hog1 activity are essential to initiate the transcription. Transcription factors, chromatin remodelers such as the SAGA and RSC complexes, together with Hog1 will contribute to open and maintain an accessible chromatin environment at the stress-response lociGPD1, which is essential for survival to osmotic stress, has an important basal expression level and can thus be rapidly induced upon stress. Interestingly, the chromatin state, encoded in part by the promoter sequence, can be tuned by external growth conditions. Thus, the noise generated by a promoter is not rigidly set by its DNA sequence but fluctuates based on the environment.The repression level of a promoter during log-phase growth will determine the speed and the noise of the transcription activation process. Thus, for each promoter, a trade-off has to be found between these two contradictory requirements. For instance, 54. Early genes such as c-Fos or c-Jun, are induced within minutes after activation of signaling55. Interestingly, these loci display basal expression and require minimal chromatin modification for their induction57. Conversely, delayed primary response and secondary response genes require more chromatin remodeling to induce their activation58. These similarities with the regulation of Hog1-dependent genes induction suggest a high conservation in the mechanisms used by MAPK in eukaryotes to regulate the dynamics of gene expression.In higher eukaryotes, the stress response MAPKs p38 and JNK relocate to the nucleus upon activationW303 background. Transformations were performed with a standard lithium-acetate protocol. Gene deletions and gene taggings were performed with either pFA6a cassettes60 or pGT cassettes61. Transformants were selected with auxotrophy markers  and gene deletions were performed with antibiotic resistance to Nourseothricin (NAT) or Kanamycin (KAN). In order to generate the membrane-anchored Hog1, the pGTT-mCherry vector was modified by inserting annealed oligos (oSP1648/9) encoding the last 30 bp of the Ras2 sequence to obtain the pGTT-mCherry-CaaX plasmid. A strain possessing the Hog1-mCherry:LEU2 modification was transformed with the pGTT-mCherry-CaaX plasmid linearized with XbaI and SacI to induce a marker switch and introduce the membrane anchoring motif.Plasmids, yeast strains and primers used in this study are listed in Supplementary Tables 32. The PP7 protein was derived from Larson et al.30 (Addgene# 35194) with an additional truncation in the capsid assembly domain (PP7∆FG residues 67–75: CSTSVCGE31). The expression of the PP7 construct is controlled by an ADH1 promoter and a CYC1 terminator. The final construct pVW284 was cloned in a Single Integration Vector URA3 (pSIVu61). The PP7-mCherry was cloned by replacing the GFP by the mCherry sequence. The MS2-GFP was generated by using the original MS2 sequence from Betrand et al.18, which also lacks the capsid assembly domain (Addgene# 27117), inserted into the pVW284. The PP7 stem-loops plasmids are based on the previously published pPOL1 24xPP7sl integrative plasmid30 (Addgene #35196). The stress-responsive promoters replace the POL1 promoter in the original construct using 1 kb  upstream of the start codon. The pSTL1-24xMS2sl was generated by replacing the PP7 stem-loops with the MS2 stem-loops obtained from Betrand et al.18 (Addgene# 31865).The PP7-GFP plasmids are based on the bright and photostable GFPenvy fluorescent proteinGLT1 ORF, as previously published30. Correct integration into the GLT1 locus was screened by colony PCR with primers in the GLT1 ORF  and in the TEF terminator (oSP062) of the selection marker on genomic DNA extractions. The integrity of the PP7 stem-loops array was assessed with primers within the TEF terminator (oSP062) and in GLT1 ORF  for all the promoters and deletions, after each transformation performed. For all the strains used in the study, at least two clones with correct genotypes were isolated and tested during a salt challenge time-lapse experiment. From the data analysis, the most frequent phenotype was isolated and the strain selected.A strain bearing the Hta2-mCherry nuclear marker and expressing the PP7-GFP was transformed with plasmids containing the different osmostress promoters driving the PP7sl production, linearized with a NotI digestion and integrated upstream of the STL1 with the 24xPP7sl, the plasmid pSP264 with the STL1 promoter was modified by replacing the GLT1 ORF sequence by a 500 bp sequence starting 100 bp after the start codon of STL1. The plasmid was digested SacI-NotI and purified over a gel to isolate a fragment that contains the pSTL1-24xPP7sl-STL1100-600. A double-strand break was generated in the STL1 ORF using Cas9 and a sgRNA targeting the PAM motif GGG 62 bp upstream of the start codon. The Cas9 and sgRNA are expressed from a 2 µ plasmid (Addgene #3546462) slightly modified from the work from Laughery et al. (Addgene# 6763963). The purified DNA fragment containing the stem-loops was used as repair DNA to promote homologous recombination at the STL1 locus .In order to generate the diploid reporter strain, a MATa strain containing the The plasmids generated for this study are available on Addgene.600 0.025 in the morning and grown for roughly 8 h to reach OD600 0.3–0.5. In the evening, cultures were diluted by adding (0.5/OD600)µl of cultures in 5 ml SD-full for an overnight growth that kept cells in log-phase conditions. Cultures reached an OD600 of 0.1–0.3 in the morning of the second day and were further diluted when necessary to remain below an OD600 of 0.4 during the day. To prepare the samples, these log-phase cultures were further diluted to an OD600 0.05 and sonicated twice 1 min (diploids were not sonicated) before placing 200 µl of culture into the well of a 96-well glass-bottom plate  previously coated with a filtered solution of Concanavalin A diluted to 0.5 mg/ml in water 64. Cells were let to settle for 30–45 min before imaging. Osmotic shock was performed under the microscope, by adding 100 µl of a three times concentrated SD-full+NaCl stock solutions to the 200 µl of medium already in the well, to reach the final desired salt concentration.Yeast cells were grown in YPD medium  for transformation or in Synthetic Defined (SD) medium  for microscopy experiments. Before time-lapse experiments, cells were grown at least 24 h in log-phase. A saturated overnight culture in SD medium was diluted into fresh SD-full medium to OD65.Images were acquired on a fully automated inverted epi-fluorescence microscope  placed in an incubation chamber set at 30 °C. Excitation was provided by a solid-state light source  and dedicated filter sets were used to excite and detect the proper fluorescence wavelengths with a sCMOS camera . A motorized XY-stage was used to acquire multiple fields of views in parallel and a piezo Z-stage  allowed fast Z-dimension scanning. Micro-manager was used to control the multidimensional acquisitionsExperiments with PP7 stem-loops were acquired with a 60X oil objective. For strains with PP7-GFP and Hta2-mCherry, GFP  and RFP (20 ms), along with two bright field images were recorded every 15 s for the GFP and every minute for the other channels, for a total duration of 25 min. Six z-stacks were performed on the GFP channels covering ±1.2 µm from the central plane with 0.4 µm steps. An average bleaching of 32% for the GFP and 26% for the RFP for the whole time-lapse was quantified in a strain without the PP7 stem-loops, to avoid artifacts from the appearance of bright fluorescent foci. For all time-lapse experiments, media addition was performed before time point 4, defined as time zero. All microscopy experiments were performed in duplicate for non-induced control experiments and at least triplicate for the NaCl induced experiments.67. Periods of 4 s were used and within this time, the valve controlled the fraction of time when SD-full versus SD-full + NaCl was flowing. TTL signals generated by an Arduino Uno board and dedicated scripts were used to control precisely the switching of the valve. The fluorescein present in the SD-full medium quantified outside the Cell object provided an estimate of the NaCl concentration in the medium. Some strong fluctuations in this signal were probably generated by dust particles in the imaging oil or FLSN-dextran aggregates in the flow chamber. Following 24 h log-phase growth, cells bearing the pSTL1-PP7sl reporter, Hog1-mCherry, and Hta2-tdiRFP tags were diluted to OD 0.2, briefly sonicated and loaded in the ibidi channel previously coated by Concanavalin A. Cells were left to settle in the channel for 10 min before SD-full flow was started.The flow experiments were performed in Ibidi chambers . Two 50 ml Falcon tube reservoirs containing SD-full +0.5 µg/ml fluorescein-dextran  and SD-full +0.6 M NaCl were put under a pressure of 30 mbar . The media coming from each reservoir were connected using FEP tubing  to a 3-way valve . The concentration of NaCl in the medium was controlled using a Pulse-Width Modulation strategySTL1-PP7sl induction in glucose versus raffinose, cells were grown overnight to saturation in SD-full medium. The cultures were diluted to OD 0.025 (Glucose) or 0.05 (Raffinose) and grown at 30 °C for at least four hours. In the raffinose medium, the expression level of the PP7-GFP was twofold lower than in glucose. Because of this low fluorescence intensity, cells were imaged with a 40X objective, and a single Z-plane was acquired. Manual curation of the images was performed to define the Start Time in more than 250 cells. This experiment was performed in duplicate.For the experiments comparing p33 based on Matlab. Summary of the dataset, strains, and cell numbers are provided in Supplementary Table Time-lapse movies were analyzed in an automated way: cell segmentation, tracking, and feature measurements were performed by the YeastQuant platformThe Hta2 signal combined with the two bright field images allowed to define the nucleus and cell borders. The GFP z-stacks were converted by a maximum intensity projection in a single image that was used for quantification. In order to avoid improper quantification of transcription sites at the nuclear periphery, the Nucleus object defined by the histone fluorescence was expanded by 5 pixels within the Cell object to define the ExpNucl object.findpeak algorithm was used to identify in the trace all the peaks larger than a threshold of seven counts within the Start and End Times. Following this first process, a set of conditions were defined to retain only the more reliable fluctuations: the drop following the peak has to be larger the fourth of the peak intensity; the intensity of the following peak has to rise by more than a third of the value at the trough. In addition, the value of the peak has to be at least one-fifth of the maximum intensity of the trace in order to remove small intensity fluctuations being considered as peaks.Further analysis was performed by dedicated Matlab scripts. The transcription site intensity was quantified by the difference between the mean intensity of the 20 brightest pixels (HiPix) in the ExpNucl and the median intensity from the whole cell. This provides a continuous trace that is close to zero in absence of TS and increases by up to few hundred counts when a TS is present. To identify the presence of a transcription site, a second feature named ConnectedHiPix was used (Supplementary Fig. Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary InformationSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary SoftwareReporting summary


The homeotic protein SIX3 is a transcription factor vital for neurogenesis and has a bivalent promoter. We previously showed that SIX3 can be transcriptionally silenced by DNA hypermethylation, functions as a tumor suppressor gene, and inhibits human glioblastoma transcriptionally. Here, we show that the activation of epidermal growth factor (EGFR) induces DNA methylation of SIX3 promoter through the MAPK pathway. ERK, when activated, binds with ZNF263, consequently abrogating the ubiquitination of ZNF263 and leading to its stabilization. ZNF263 binds to the core promoter region of SIX3 and recruits the KAP1/HATS/DNMT corepressor complex to induce transcriptional silencing of SIX3 through H3K27me3 and methylation of SIX3 promoter. Activation of the EGFR-ZNF263 signaling axis in phenotypically normal astrocytes or glioblastoma cells triggers or enhances tumorigenic activities, while elevated expression of the EGFR-ZNF263 signaling components in glioblastoma tissues is associated with poor prognosis of the patients. Together, our findings demonstrate that epigenetic silencing of SIX3 is controlled by a sophisticated and highly ordered oncogenic signaling pathway and therefore provide new insights into initiation and progression of glioblastoma. During tumorigenesis, cells undergo a genome-wide epigenetic reprogramming process, which contributes to massive overall DNA hypomethylation and specific hypermethylation at certain CpG promoters , 2. DNA Glioblastoma is the most aggressive malignancy in central nervous system (CNS), with 15 months media survival . EpigeneIt is well established that amplification and mutations of epidermal growth factor receptor (EGFR) are the most frequent genetic event in glioblastoma, which promotes tumor growth and survival through uncontrolled activation of signaling networks and metabolic reprogramming , 17–19. SIX3 belongs to the SIX transcription factor family and contains a homeobox domain for DNA binding and a six domain. SIX3 is predominantly expressed in CNS and critical for development –25. SIX3We had shown previously that SIX3 promoter contains higher levels of DNA 5-methylcytosine (5mC) in glioma than in normal brain tissues . In thisWe next examined SIX3 expression in glioma from two independent sets of patients. qRT-PCR and IHC showed that SIX3 was expressed at a much lower level in tumor than in normal brain tissues Fig.  and thatDNMT3A/B are the major de novo writers that directly catalyze the addition of methyl groups onto DNA and bind to DNA . HoweverWe first assessed the expression of ZNF263 in HEB cells and six primary patient-derived glioma cells and discovered an inverse relationship between ZNF263 and SIX3 Fig. . TCGA anAfter revealing ZNF263 binding to and suppression of the transcriptional activity of SIX3 promoter, we next asked whether ZNF263 is involved in the regulation of DNA hypermethylation or histone modifications of SIX3 promoter. ChIP analysis showed that knockdown ZNF263 decreased the enrichment of H3K27me3 in the entire region of SIX3 promoter and specifically reduced the enrichment of H3K9me3 at region 2 and 3 Fig. . OverexpIt is well known that H3K27me3 and H3K9me3 are necessary for chromatin compaction and heterochromatin maintenance. The effects of ZNF263 on H3K27me3 and H3K9me3 led us to ask whether ZNF263 alter chromatin accessibility of SIX3 promoter in glioblastoma. We found that ectopic expression of ZNF263 significantly reduced the fraction of extractable SIX3 promoter, as shown by the formaldehyde-assisted isolation of regulatory elements (FAIRE) assay Fig.  while exHow does ZNF263 regulate SIX3 epigenetically? We employed ZNF263 immunoprecipitation followed by tandem mass spectrometry (LC-MS/MS) to identify protein(s) interacting with ZNF263 Fig. . GO analImmunoprecipitation followed by immunoblotting validated that ZNF263 interact with DNMT1, DNMT3A, HDAC2, KAP1, UHRF1, PHF8, HP1α, HP1β, and MTA2 Fig.  in U251 To elucidate ZNF263 function in recruiting chromatin modifiers to SIX3 promoter, we performed ChIP-qPCR analysis in U251 cells under overexpression or knockdown conditions of ZNF263. SUZ12, EZH2, DNMT3A, and RNA polymerase II antibodies were used for chromatin immunoprecipitation. Overexpression of ZNF263 led to increased enrichment of EZH2, DNMT3A, and SUZ12 to the promoter of SIX3, in accordance with decreased binding of RNA polymerase II Fig. . While kFinally, we asked whether ZNF263 affected the expression of DNMTs, H3K9 methyltransferase, and members of the PRC2 complex. The results from TCGA showed that ZNF263 expression had no significant correlation with the expression of DNMT3A, DNMT3B, SUV29H1, SUV39H2, EED, EZH2 at the mRNA level, while positively correlating with the expression of DNMT1, SETDB1, and SUZ12 Fig. . MoreoveEarlier studies showed that EGFR and RAS induce epigenetic silencing of TSGs , 34, 36.Next, we further detected whether elevated SIX3 expression caused by Trametinib resulted in changes in epigenetic modifications of SIX3 promoter. In keeping with elevated SIX3 expression, Trametinib dramatically reduced the enrichment of H3K27me3 and H3K9me3 in SIX3 promoter and enhanced RNA pol II recruitment Fig. .In contrBecause ZNF263 plays a critical role in epigenetic silencing of SIX3 in glioblastoma, we next examined whether erlotinib or Trametinib induced SIX3 expression through the regulation of ZNF263. We found that erlortinib did not decrease and instead slightly elevated mRNA levels of ZNF263 in glioblastoma cells Fig. . InteresHow does the EGFR/MAPK pathway regulate ZNF263 protein? We first treated glioblastoma cells with cycloheximide to block de novo protein synthesis and then added Trametinib to inhibit the MAPK pathway, which resulted in a dramatic decrease in the half-life of ZNF263 Fig. . How ZNFhttp://scansite.mit.edu), we discovered an ERK Docking domain (ERK D-domain) located between SCAN box and KRAB domain at N terminus of ZNF263 and predicted to bind to ERK1/2  and tagging it with either FLAG or GFP Fig. . We founIt has been well documented that the gain of chromosome 7 where EGFR located is the first event in human glioblastoma development . EGFR-vIWe also investigated the regulatory role of the EGFR-ZNF263 signaling axis in glioblastoma cells. We expressed EGFR-vIII in glioblastoma cells, which expectedly increased ZNF263 levels and decreased SIX3 levels Fig. . KnockdoTo further confirm the relationship of EGFR-ZNF263 signaling components, we investigated the reverse phase protein array data from TCGA which contains the expression data of EGFR, EGFR-pY1068, EGFR_pY1173, and EGFR_pY992 in part of glioblastoma samples. The association analysis of EGFR copy number, EGFR mRNA, EGFR protein, and phosphorylated EGFR showed high consistency by pairs Fig. . In addiWe then extended our studies and determined the expression of EGFR-ZNF263 key signaling components in more glioma samples. We chose 235 glioma tissues and assembled them for tissue microarray analysis. One hundred sixteen patients were subjected to follow-up studies. We found strong positive correlation between the level of p-EGFR (Y1173) and that of ZNF263 Fig. . While SDNA hypermethylation is a frequent mechanism for transcriptional silencing of TSGs in cancer , 10. TheWhile we were analyzing the SIX3 promoter regions, it came to our attention that SIX3 contains a “bivalent” promoter. Bivalent chromatins are characterized by active histone modification H3K4me3 and suppressive H3K27me3 concurrently, leaving SIX3 poised for responding to extrinsic stimuli. The loss of H3K4me3 is more profound in mediating the silencing of bivalent chromatins in cancer than the gain of H3K27me3. Moreover, bivalent chromatins mark a high extent of promoters of genes encoding transcriptional factors known to regulate development , 18. DNAEpigenetically based molecular classification of glioma uncovered that there exists G-CIMP (Glioma-CpG island methylator phenotype) subtype and NON G-CIMP subtype. G-CIMP is mainly found in low-grade gliomas and in subgroup of proneural subtype glioblastoma which was thought to be developed from low-grade glioma . StrikinThe role of transcription factors in mediating the initiation of DNA methylation and histone modifications has been established , 36, 39.Since KRAB-ZNF proteins have been validated as indispensable factors linking signaling pathway with promoter methylation of TSGs, revealing the modulation of these factors is of great importance , 39, 40.2.Primary astrocytoma tissues and matched clinical data were obtained from the Department of Neurosurgery, the Xiangya Hospital, Central South University. Human glioma tissue array from glioma patients, who received surgery in Jining Affiliated Hospital of Jining Medical University from January 2008 to May 2017, was a gift from WW. All samples collected were obtained with the informed consent of the patients, and all experiments using human tissues were approved by the Joint Ethics Committee of the Central South University Health Authority. Astrocytoma cell lines U251, U87, and U118 were purchased from cell banks of Chinese Academy of Sciences . All astrocytoma cell lines were subjected to short tandem repeat test. Human phenotypically normal astrocyte cell line HEB was obtained from Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences . HEB, U251, and U118 were cultured in high glucose DMEM with 10% FBS. U87 cells were cultured in MEM with 10% FBS at 37 °C with 5% CO5-Azacytidine (cat.A2385) and Cycloheximide cat.R750107) were purchased from Sigma-Aldrich. Trametinib (cat.M1759), Chloroquine (cat. M2510), Rapamycin (cat.M1768), RAD001 (cat.M1909), and MG132 (cat.M1902) were purchased from Abmole. Erlotinib HCL (cat. S1023), MK2206 2HCL (cat.S1078), and Ruxolotinib (cat.S1378) were purchased from Selleck. The primary antibodies and their applications are listed in Supplementary Table 07 were pZNF263 cDNA clone and shRNAs were purchased from GemeChem (Accession: NM_005741). We generated full-length and truncated ZNF263-FLAG-tagged fusion proteins using different primers. ERK2-RFP plasmid was constructed as previously described. The promoter regions of SIX3 were cloned from gDNA from normal human tissues and inserted into pGL3-basic and pGL3-enhancer vectors with T4 ligase.Luciferase reporter assay was performed as previously described. To determine the activity of the core promoter region of SIX3, U251 cells were transfected with the firefly luciferase reporter plasmids and pRL-SV40 plasmids (Renilla luciferase) (at the ratio of 5:1). For repression analysis, the firefly luciferase reporter plasmids were transfected together with ZNF263 and pRL-SV40, or treated with Trametinib. Luciferase activity was determined according to the manufacturer’s protocol . The ratio of firefly to renilla was used to analyze the relative luciferase activity.3 cells/ml. After the medium was totally solidified, the plates were placed in cell incubator at 37 °C and 5% CO2 for ~20 days. The colonies per well were counted under the microscope.The soft-agar colony formation assay was performed as previously described . Twenty Genome DNA extraction was performed according to the manufacturer’s protocol . For DNA bisulfite conversion, standard protocol  was used. One microgram gDNA was subjected to bisulfite conversion by using the standard conversion program.MSP was performed using a reaction mixture including 2× Taq MIX (10 μl), gDNA (2 μl), primers (1 μl), and H2O (7 μl) . The PCR program includes degenerate reaction , degenerate reaction , annealing , extension ; and extension .Electrophoretic mobility shift assay was performed as previously described , with slImmunohistochemistry and immunofluorescence analyses were performed as previously described . For imm7 cells) was added to the washed cells, and 500 μl GLB (with prior addition of protease and phosphatase inhibitors) was added to the 10 cm culture dish and then lysed on ice for 10 min. Afterwards, the mixture was transferred to a 1.5 ml centrifugal tube and kept on the ice for 20 min for further lysis. During the period, the mixtures were subjected to several strong oscillations for 15 s or to ultrasonic perturbations for 10 s. After centrifugation at 13,000 × g , the supernatant was transferred to a new centrifugal tube, and the BCA method was used to determine the protein concentrations. A fraction of supernatant was used as input and stored at −80 °C. The rest of the supernatant was divided into several tubes based on protein concentration. Two micrograms of antibodies were added to each tube and the tubes were rotated gently at 4 °C overnight. After centrifugation, the samples were incubated with protein A/G magnetic beads rinsed with GLB buffer. The antigen/antibody mixtures were gently vortexed for 6 h at 4 °C or 1 h at room temperature. After centrifugation, the tubes was placed on a magnetic rack, and after the magnetic beads were separated, the supernatant was removed. The IP wash buffer was used to rinse the beads and was changed every 10 min for four times. The GLB buffer and loading buffer were subsequently used to re-suspend the magnetic bead-antigen/antibody mixtures, which were heated for 5 min at 95 °C and were subjected to immunoblotting as detailed below.Precooled GLB Buffer . Nuclei were pelleted and lysed on ice for 10 min in cell lysis buffer . Lysates were sonicated in a sonic Bioruptor and diluted with 50% v/v dilution buffer . Cell debris was removed by microcentrifugation, and free DNA was extracted from the collected supernatant by phenol/chloroform extraction. Under these conditions, DNA that is not cross-linked with the protein remains in the aqueous phase, while DNA that cross-links with the protein remains in the phenolic phase.The FAIRE and MNase digestion were performed as previously described , 54. Celt test and one-way ANOVA were used for comparison of data between two or more groups, and Kaplan–Meier survival curve was plotted using log rank. All data were calculated using mean ± standard deviation (mean SD). The experiments were repeated at least three times. All tests were two-tailed; p < 0.05 was considered statistically significant.The statistical analyses in this study were performed by the use of SPSS 22.0 and GraphPad Prism 5 software. Student supplement figure and legendsupplement_antibody informationsupplementary information


H3K27ac is well recognized as a marker for active enhancers and a great indicator of enhancer activity. However, its functional impact on transcription has not been characterized. By substituting lysine 27 in histone variant H3.3 with arginine in mouse embryonic stem cells, we diminish the vast majority of H3K27ac at enhancers. However, the transcriptome is largely undisturbed in these mutant cells, likely because the other enhancer features remain largely unchanged, including chromatin accessibility, H3K4me1, and histone acetylation at other lysine residues. Our results clearly reveal that H3K27ac alone is not capable of functionally determining enhancer activity.Supplementary information accompanies this paper at 10.1186/s13059-020-01957-w. Enhancers are critical regulatory elements that control spatial and temporal gene expression , and theDrosophila [Drosophila causes male lethality and infertility and reduced global H3K27ac levels [H3f3a and H3f3b) and producing a homogenous population of H3.3 lysine 27-to-arginine (K27R) mutant histones in mouse ESCs.Enrichment of histone variant H3.3 and high histone turnover are other enhancer features –14. Althosophila , 23. Howosophila . Replacic levels . A similc levels . TherefoH3f3a and H3f3b, and the engineered mouse ESC clones were confirmed by Sanger sequencing  experiments in WT and the two H3.3K27R mutant lines, and all experiments were performed in biological duplicates. H3K27ac occupancy in the mutant lines displayed an extensive genome-wide loss  and Nanog, and observed a substantial reduction in H3K27ac , H3.3 , H3K4me1 , H3K4me3 , H3K9ac , H3K18ac , H3K27ac , H3K27me3 , H3K122ac , histone H4 , H4K5ac , H4K8ac , and RNA Pol II  were obtained commercially.Drosophila genome sequences (dm6) were concatenated to be used as the reference genome. H3.3 ChIP-seq data were downloaded from GEO database under the accession number of GSE117035 [ChIP-seq and ATAC-seq libraries were sequenced PE150) with Illumina NovaSeq 6000 Additional file 2:Figure S1. Strategy for CRISPR-Cas9-mediated gene editing at H3f3a and H3f3b loci. Figure S2. H3.3K27R mutation does not affect the H3.3 protein level and pluripotent state of ESCs. Figure S3. Genome-wide decrease in H3K27ac levels in individual H3.3K27R mutant mouse ESC lines. Figure S4. Averaged profiles of H3.3 at active enhancers and H3K27-occupied promoters in mouse ESCs. Figure S5. H3.3K27R mutation does not affect the genome-wide distribution of H3K27me3 in mouse ESCs. Figure S6. Heatmap clustering of differentially expressed genes in WT and H3.3K27R mutant mouse ESC lines. Figure S7. H3.3K27R mutation does not affect the transcription of genes associated with super enhancers in mouse ESCs. Figure S8. H3K4me1 distribution at enhancers and H3K27ac-occupied promoters in WT and H3.3K27R mutant ESC lines. Figure S9. H3K4me3 distribution at H3K27ac-occupied promoters in WT and H3.3K27R mutant ESC lines. Figure S10. Averaged profiles of H3K9ac, H3K18ac, H3K122ac, H4K5ac, and H4K8ac at enhancers and H3K27ac-occupied promoters in WT and H3.3K27R mutant ESC lines. Figure S11. Barplots show Pearson correlation coefficients between ChIP-seq signal of histone acetylation and chromatin accessibility . (PDF 2893 kb)Additional file 3:Table S1. Sequencing depth and mapping efficiency of ChIP-seq and ATAC-seq experiments. Table S2. Peak numbers called in ATAC-seq and ChIP-seq experiments. Table S3. Stats for mRNA-seq experiments. Table S4 Sequences of DNA oligos used in this study. (PDF 459 kb)Additional file 4.Review history.


It has become impossible to review all the existing literature on Voltage-Dependent Anion selective Channel (VDAC) in a single article. A real Renaissance of studies brings this protein to the center of decisive knowledge both for cell physiology and therapeutic application. This review, after highlighting the similarities between the cellular context and the study methods of the solute carriers present in the inner membrane and VDAC in the outer membrane of the mitochondria, will focus on the isoforms of VDAC and their biochemical characteristics. In particular, the possible reasons for their evolutionary onset will be discussed. The variations in their post-translational modifications and the differences between the regulatory regions of their genes, probably the key to understanding the current presence of these genes, will be described. Finally, the situation in the higher eukaryotes will be compared to that of yeast, a unicellular eukaryote, where there is only one active isoform and the role of VDAC in energy metabolism is better understood. Paramecium extracts was first claimed in 1976 Parameciu in 1976 ,18. The  in 1976 ,12,13. TStudies focused on the biochemical-structural aspects of the protein underwent a strong acceleration following the use of molecular biology technologies that became within the reach of all laboratories. In the case of VDAC, this led to a great expansion of knowledge of the protein’s genetic and cellular activities.The milestones in a twenty years path of achievements, in my opinion, were: (I) the identification of three isoforms of VDAC in the superior metazoa ,22. (II)The discovery of more isoforms of VDAC suggests that evolution developed variants with slight amino acidic differences in its protein armory for precise but still undefined purposes. The production of stem cells and mice knockout for the individual isoforms has allowed us to start providing clues about their function ,39,40,41Most of the literature about VDAC1 was covered in the excellent and extensive review by Shoshan-Barmatz in this special issue . We willIn higher metazoa three VDAC genes with the same exon-intron structure  evolved:We have been studying post-translational modifications of VDAC isoforms for some years now. The initial starting point was the consideration of the different number of cysteines present in VDAC isoforms and the suspicion that this difference was not a mere coincidence but was likely linked to a specific function or structural involvement of these residues in protein folding . In partThe need to investigate this functional result more thoroughly led us to collaborate with the mass spectrometry unit to unequivocally highlight, at a molecular level, the oxidation state of cysteines, in particular, and of other residues in the VDAC3 isoform, first , and in The technique of protein isolation from animal cells and/or tissues has been modified and adapted to eliminate any risk of oxidation or accidental modification due to isolation protocols or electrophoretic techniques. We used enriched extracts rather than purifying proteins after SDS-PAGE or 2D electrophoresis. To this end, reduction and alkylation of sulfur were performed on purified mitochondria, and only later the chromatographic separation was run. The eluted proteins were cleaned by PlusOne 2-D Clean-Up kit , then RapiGest SF , to eliminate non-protein contaminating molecules. The sample was then subjected to proteolytic cleavage and the peptide mixture loaded on UP-nanoLC and then analysed by a highly sensitive Orbitrap Fusion Tribrid (Q-OT-qIT) mass spectrometer. This modified procedure is all the more delicate and important to develop when considering that the main PTM studied was the oxidation of -SH ,54.2H), and sulfonic acid (SO3H) [We initially focused on oxidative post-translational modifications of VDAC3 cysteines ,50 and ld (SO3H) . Except d (SO3H) ,53,54,55d (SO3H) . The sigd (SO3H) .In VDACs other more common post-translational modifications were detected. For example, phosphorylation is very common and dynamic . In our Acetylation was always detected at the N-terminal amino acid of the three isoforms ,51, togeThe studies of the structure, activity, and regulation of VDAC genes aim at obtaining a reliable picture of their differences in tissue expression or sensitivity to specific stimuli . In humaAll three VDAC isoforms are ubiquitously expressed, with the highest levels found in skeletal and heart muscles as determined by RNA-seq GTEx. The level of the VDAC1 and VDAC2 transcripts is comparable, while VDAC3 is lower than the other two isoforms, confirming previous experimental conclusions drawn by RT-PCR . While cThe promoters of the human VDACs genes were also characterized. The organization of the core promoter is similar to that of most TATA-less human promoters of ubiquitously expressed genes, where the presence of abundant GC regions, alternative binding sites Inr, DPE, and BRE assure a basal levels of transcription. Interestingly, a non-canonical initiation site termed the TCT motif (polypyrimidine initiator), which is a target for translation regulation by the mTOR pathway, oxidative, and metabolic stress , was alsThe basal expression level of VDAC genes seems to be subject to quantitative regulation of expression. Using a bioinformatics approach, the main transcription factors regulating the activity of VDAC promoter regions were identified, and a quest for the corresponding binding sites located in the promoters was performed. In all three VDACs promoters, the majority of identified TFs classes belong to the E2FF, NRF1, SP1, KLFS, EBOX families which are prevalently involved in cell proliferation and differentiation, apoptosis, and metabolism regulation ,67,68,69The unique transcription regulators in VDAC1 promoter suggest a prevalent role of this protein in the mitochondrial outer membrane in physiological context and in altered environmental conditions in which cells have to restore the mitochondria energy balance ,71,72. VIn conclusion, the study of the features of the promoters of each VDAC isoform indicates that they evolved different control sequences, requiring transcription factors that link these genes to specific functions. Interestingly, the accumulated evidence points to the same biological area that was involved in the functions of the proteins.The families of transcription regulators identified as unique in VDAC1 promoter suggest that this isoform has the main role of mitochondrial channel protein in a physiological cell context and is the main tool used to maintain mitochondria energy balance . SeveralNeurospora crassa and Saccharomyces cerevisiae do not have the same gene multiplicity for VDAC isoforms. The case of yeast has been studied and definitively clarified by us. After the discovery and characterization of yeast VDAC [Unlike vertebrates, unicellular eukaryotic organisms such as ast VDAC ,81, Fortast VDAC . Our groast VDAC ,84. Whilast VDAC .Δpor1 mutant) grown on glucose showed that there is no increase in the expression levels of yVDAC2. Together, these results indicate that there is no coordination between the expression of the two genes; the fact that there is a second VDAC gene could be the result of a gene duplication that has led to the presence of some sort of inactive pseudogene in the yeast genome [Proteomic analysis of the yeast mitochondrion, recently carried out at such high resolution as to allow a realistic estimate of the number of individual proteins present per mitochondrion , showed t genome .Even the analysis of the global permeability of the outer mitochondrial membrane of yeast, while highlighting the possible existence of other minor (quantitatively) proteins capable of forming pores, found no evidence of a yVDAC2 contribution to it . We calcThe main unresolved questions are: how can the yeast mitochondria not be degraded and inactivated in the absence of VDAC1? How can the yeast mitochondria survive when VDAC1 is absent?Δpor1 strain compared to the wild type (WT) strain provided interesting information. There is a marked aliquot of genes whose expression is completely modified in Δpor1: particularly impressive is the full inactivation of the mitochondrial genome. This drastic change can be attributed to the reduced transport capacity of nucleotides in the absence of VDAC1, also due to the impossibility to import carriers, a function recently linked to VDAC1 [The transcriptomic profile of the to VDAC1 . In thesto VDAC1 . This reto VDAC1 . Our resI hope that this quick review of the biological functions related to the onset of VDAC isoforms during evolution supports the hypothesis that they are involved in specific functions, as well as having, all of them, the ability to form large, porous aqueous channels. It is difficult to identify functional specificities based on small structural differences, such as the disposition and reactivity of some residues, the presence of small amino acid traits whose 3D organization and mobility have not yet been precisely defined, as it is the case of N-terminal extensions. These clues will have to be explored and revealed, together with the other major unsolved issue of this protein: the mechanism of voltage dependence. The road is open in this direction, and the end of the knowledge of the functions of VDAC isoforms will make it possible to steer the path of discovery of therapeutic molecules targeting this intriguing pore.


The molecular mechanisms associated with FRDA are still poorly understood and most studies on FXN gene regulation have been focused on the region around the minimal promoter and the region in which triplet expansion occurs. Nevertheless, since there could be more epigenetic changes involved in the reduced levels of FXN transcripts, the aim of this study was to obtain a more detailed view of the possible regulatory elements by analyzing data from ENCODE and Roadmap consortia databases. This bioinformatic analysis indicated new putative regulatory regions within the FXN genomic locus, including exons, introns, and upstream and downstream regions. Moreover, the region next to the end of intron 4 is of special interest, since the enhancer signals in FRDA-affected tissues are weak or absent in this region, whilst they are strong in the rest of the analyzed tissues. Therefore, these results suggest that there could be a direct relationship between the absence of enhancer sequences in this specific region and their predisposition to be affected in this pathology.Friedreich’s ataxia (FRDA) is an autosomal recessive disease caused by an abnormally expanded Guanine-Adenine-Adenine (GAA) repeat sequence within the first intron of the frataxin gene  Friedreich’s ataxia , the most common form of hereditary ataxia among individuals of Indo-European origin, is an autosomal recessive disease clinically characterized by progressive cerebellum and spinal cord neurodegeneration, hypertrophic cardiomyopathy, and predisposition to diabetes ,2. The d29300), tFXN gene [MIM 606829], which encodes for a protein called frataxin. The genetic defect associated with the disease is mostly an abnormally expanded GAA repeat sequence within the first intron of the gene [FRDA, firstly described by physician Nicolas Friedreich in 1863, is caused by mutations in the the gene . Most FRAt the molecular level, the final consequence of GAA expansion is that FRDA patient cells show a severe deficiency in frataxin transcription ; this ulFXN gene  are currently described in the FXN gene : the firFXN gene ; to dateFXN, being considered as the minimal promoter. Although this region lacks TATA-box, it contains an Inr  24 bp downstream from TSS1 and a Downstream Promoter Element (DPE) located 26 bp downstream from TSS1  element, and part of an Alu element, which is where the expansion of GAA repeats derives from  Figure .FXN, there are few data available to date. Previous studies have identified a Serum Response Factor binding site , which is important in neuronal development, an AP2 transcription factor binding site , involved in the development of the neural crest, and an Early Growth Response factor 3 binding site  [FXN expression and it binds to the MyoD and c-myc transcription factors [FXN is activated by p53 [Regarding the transcription factors that regulate the expression of om TSS2) . The delom TSS2) . The exi factors . A CCCTC factors , which i factors . In addid by p53 . Finallyd by p53 , also shA scientific field that has acquired special relevance in recent years is the study of epigenetics, which is an essential mechanism in genome regulation . AlthougFXN transcriptional deficiency initiation is the major cause of frataxin deficiency, and it is related to the extent of repressive chromatin from the GAA expansion [In FRDA patients, xpansion ,30. GAA xpansion ,32 and Rxpansion ,34, and xpansion ,36. The xpansion  (reviewexpansion  (reviewexpansion ). There xpansion ,40,41,42FXN gene regulation have been focused on the region around the minimal promoter [FXN transcripts [FXN locus from an epigenetic point of view. Thus, in this work, we used data generated by ENCODE [FXN gene.Most studies on promoter ,19 and tpromoter . Howeverpromoter . Due to nscripts ,45, the y ENCODE  and Roady ENCODE  to perfohttps://genome-euro.ucsc.edu) is the main tool to access these data [The objective of the ENCyclopedia Of DNA Elements (ENCODE) consortium is to identify all the functional elements present in the human genome ,48. To dese data ,51. FXN locus, although none of them have a neural origin [As was previously mentioned, chromatin plays an important role in controlling DNA access and specific histone marks are related to promoters, enhancers, transcribed regions, and silenced regions ,53. Combl origin : B-lymphThe mapping of the different chromatin states was done using a ChIP-seq method with antibodies against the following histone modifications: histone H3 lysine 4 methylation (H3K4me1), a mark predominantly associated with enhancers; H3K4me2, associated with promoters and enhancers; H3K4me3, related to promoters; H3K36me3 and H4K20me1, associated with transcribed regions; H3K27me3, associated with Polycomb-repressed regions; histone 3 lysine 9 acetylation (H3K9ac) and H3K27ac, associated with active regulatory regions; and CTCF, a sequence-specific insulator protein. The chromatin states were established using a multivariate Hidden Markov Model (HMM) that models the combinatorial patterns of observed modifications; the recurrent combinations of these marks define 15 chromatin states as repressed, poised, and active promoters, strong and weak enhancers, putative insulators, transcribed regions, and large-scale repressed and inactive domains .FXN gene, and the beginning of the TJP2 gene, located downstream from the 3′ end   A. In ordFXN genomic locus  of epigenetic maps in stem cells and ex-vivo tissues, including several human adult brain structures [The US National Institutes of Health (NIH) established The Roadmap Epigenomics Program with the goal of elucidating how epigenetic processes contribute to human biology and disease. They provide a publicly accessible resource  and in the same tissue types  and H3K27ac (strong enhancers), in contrast to the rest of the cultures, where a strong signal can be observed (data not shown).Given that over 95% of the human genome reveals no protein-coding information, studies are forced to shift the focus from genomes to epigenomes, with the aim of explaining how the complex diversity of human cells may be caused only by a limited number of genes. Using already published information, we described how different types of human cells and tissues present a different regulatory pattern, including regions inside the frataxin gene and in the upstream and downstream regions of the gene. The region of intron 4 is of particular relevance, due to the differences observed between affected cells and tissues from FRDA patients compared to other tissues that are not affected by the disease. However, it is important to remark that, despite having access to epigenetic studies in brain tissues , there aThe findings shown in this study highlight the essential role of certain intronic regions in gene regulation ,61 and tTherefore, based on previous evidence and on the results obtained in this bioinformatic study, vectors with complete genomic loci are a promising candidate for gene therapy in FRDA patients, since they contain all the regulatory elements necessary for the physiological expression of frataxin.