Analysis of enhancer-associated lincRNA transcripts shows their efficient and conserved splicing contributes to cognate enhancer activity and cis-regulation of target gene expression. cis. Despite the lack of evidence supporting sequence-dependent functions for most elincRNAs, splicing of these transcripts is unexpectedly common. Whether elincRNA splicing is a mere consequence of cognate enhancer activity or if it directly impacts enhancer function remains unresolved. Here, we investigate the association between elincRNA splicing and enhancer activity in mouse embryonic stem cells. We show that multi-exonic elincRNAs are enriched at conserved enhancers, and the efficient processing of elincRNAs is strongly associated with their cognate enhancer activity. This association is supported by their enrichment in enhancer-specific chromatin signatures; elevated binding of co-transcriptional regulators; increased local intra-chromosomal DNA contacts; and strengthened cis-regulation on target gene expression. Our results support the role of efficient RNA processing of enhancer-associated transcripts to cognate enhancer activity.Transcription is common at active mammalian enhancers sometimes giving rise to stable enhancer-associated long intergenic noncoding RNAs (elincRNAs). Expression of elincRNA is associated with changes in neighboring gene product abundance and local chromosomal topology, suggesting that transcription at these loci contributes to gene expression regulation in  Whereas most enhancers transcribe short noncoding RNAs that are non-polyadenylated, unspliced, and short-lived from both the sense and antisense strands (eRNAs)  , a subse (eRNAs)  and prod (eRNAs) . The asy (eRNAs) .Enhancer transcription can increase local chromatin accessibility , modulatcis , we took advantage of the extensive publicly available data for transcription and chromatin signatures in pluripotent mESC. We considered all intergenic mESC enhancers overlapping a DNase I–hypersensitive region  and annoLoci of single- and multi-exonic mouse embryonic stem cell elincRNAs (mm9) and their respective number of exons.Table S1 Supplemental Data 1 and fully supports the analysis of the stringently annotated set of mESC elincRNAs.Given the relatively small number of the stringently annotated elincRNAs, we also annotated elincRNAs using a less stringent approach. Analysis of this less stringent and more comprehensive set of mESC elincRNA  is described in the Supplemental Data 1.Analysis of the comprehensive set of mESC elincRNAs.of mESC elincRNAs. P < 0.05, two-tailed Mann–Whitney U test, Next, we investigated whether elincRNA splicing is linked to its cognate enhancer activity. Given that most enhancer activity is tissue specific, we first investigated the association between enhancer transcription and putative target expression during embryonic neurogenesis . SimilarConsistent with their stronger association with neighboring protein-coding gene expression, chromatin signatures associated with high enhancer activity were found at enhancers that transcribe multi-exonic elincRNAs compared with those that give rise to either single-exonic elincRNAs or eRNAs. Specifically, multi-exonic elincRNA-producing enhancers were enriched for monomethylation of histone 3 lysine 4 H3K4me1, , acetylaMotifs of transcription factor–binding sites enriched at transcription initiation regions of multi-exonic elincRNAs.Table S2 cis-regulatory interactions are dependent on local chromosomal architecture, we examined whether the observed association between elincRNA splicing and enhanced neighboring gene expression was mediated through the modulation of their local chromosomal organization.Because P < 0.05, Analysis of their relative position within mESC TADs revealed that only multi-exonic elincRNA TIRs were significantly enriched at TAD boundaries and depleted at TAD centers    and DNA ic test) .P < 0.05, two-tailed Mann–Whitney U test, To assess the impact of multi-exonic elincRNA on local chromosomal architecture, we next investigated the relationship between enhancer transcription and splicing and intra-TAD DNA contact density. We found that the frequency of DNA contacts within TADs that encompass multi-exonic elincRNA loci to be significantly higher than those containing other transcribed enhancers . Furthermore, relative to enhancers that transcribe single-exonic elincRNAs, those that express multi-exonic elincRNAs are twofold enriched among conserved enhancers . Importantly, of the conserved enhancers with evidence of transcription in humans , most give rise to multi-exonic elincRNAs in mESCs , consistent with the conservation of the function and transcription of these enhancers during mammalian evolution.We reasoned that if splicing of enhancer-associated transcripts is biologically relevant, multi-exonic elincRNA-producing enhancers should be conserved during evolution. To test this hypothesis, we assessed the extent of enhancer conservation by overlapping the syntenic regions of transcribed mESC enhancers in humans with H1 ESC (hESC) enhancers . We founWe next turned our attention to the mechanisms and sequences underlying the splicing of elincRNAs. Differences in GC content between intronic and exonic sequences are known to facilitate splice site recognition and increase splicing efficiency . The exoP < 0.05, two-tailed Mann–Whitney U test, Table S3). The splicing efficiency of elincRNAs was comparable with that of protein-coding genes  metabolic labeling of RNA for 15, 30, and 60 min. Ribo-depleted total RNA from the total and newly transcribed fractions was sequenced and used to estimate transcriptome-wide rates of synthesis, splicing, and degradation in mESCs using INSPecT  Fig S4CFig S4C. ng genes . No signMetabolic rates of elincRNAs, including their rate of synthesis, processing, and degradation.Table S3 P < 0.05, two-tailed Mann–Whitney U test), which was not detected for mRNAs or oth-lincRNAs . Furthermore, relative to other non-spliced ncRNAs, multi-exonic elincRNA TIRs and gene bodies were enriched in phosphorylated serine 5 (S5P) and serine 2 (S2P)   at RNAPI 2 (S2P) , efficie 2 (S2P) .cis-target expression; and the modulation of local chromosomal architecture. Our results in mouse are also consistent with recent work in human cells, which also supports that multi-exonic lincRNAs are often transcribed from highly active enhancers   to be trMetagene profiles of CAGE reads centered at mESC enhancers and gene TIRs were plotted using NGSplot . Sense aeRNAs were included only in analyses that do not require transcript models because eRNAs, by definition, are non-polyadenylated, unspliced, and shorted-lived .Supplemental Data 1.We annotated a larger set of elincRNAs using a more permissive criterion by considering all mESC lincRNAs whose 5′ end is within 500 bp of an enhancer to be enhancer-derived. Using this approach, we identified 211 multi-exonic and 1,772 single-exonic elincRNAs. Corresponding figures for the analysis of this more comprehensive yet less stringent set of elincRNAs can be found in the Enrichment of histone modifications, transcription factor binding, and gene expression levels were assessed using publicly available mESC ChIP-seq and RNA-seq data sets. Downloaded data sets are listed in Table S4.Publicly available data sets used in the analysis.Table S4 For all downloaded data sets, adaptor sequences were first removed from sequencing reads with Trimmomatic (version 0.33)  and thenMetagene profiles of sequencing reads centered at gene TIRs were visualized using HOMER v4.7 .mESC TADs  were divcis-target. Only pairs where both loci were expressed in at least one embryonic neurogenesis stage were considered. For each gene, the two stages where the locus of interest was most highly or lowly expressed were determined and used to calculate the fold difference between the expression difference of its putative cis-target, as described previously (Level of gene transcription initiation (CAGE-based TPM (transcripts per kilobase million) at TIRs) at each of the three stages of neuronal differentiation (mESC to NPC to neuron) was downloaded from eviously .We predicted DNA motifs for transcription factors enriched at multi-exonic elincRNA TIRs (±500 bp from the center of TIRs) relative to those that transcribe single-exonic elincRNAs and eRNAs. Enrichment of motifs of at least 8mer was predicted using FIMO . EnricheSyntenic regions of mESC (mm9) genetic elements in human (hg19) were determined using liftOver with the following parameters: -minMatch = 0.2, -minBlocks = 0.01 . RegionsWe considered all transcribed mESC ENCODE intergenic enhancers  to be co2 in Knockout DMEM  supplemented with 15% FBS , 1% antibiotic penicillin/streptomycin , 0.01% recombinant mouse leukemia inhibitory factor protein , and 0.06 mM 2-mercaptoethanol , on 0.1% gelatin-coated cell culture dishes. When confluent, the culture was divided into two and passaged eight times. Five million mESCs of two biological replicates were seeded and allowed to grow to 70–80% confluency (∼1 d). RNA was labeled with 4sU  and nascent RNA was isolated after the general procedure as previously described .Hundred-nucleotide-long single-end reads were first mapped to mouse ribosomal RNA (rRNA) sequences with STAR v2.5.0 . On averOnly mESC genes with multi-exonic transcripts (two or more exons) were considered for this analysis. We computed GC content separately for the first and all remaining exons, as well as the introns, for each gene and their flanking intergenic sequences of the same length, after excluding the 500 nucleotides immediately adjacent to annotations, as previously described .We predicted the density of mouse exonic splicing enhancer motifs (identified in The efficiency of splicing was assessed by estimating the fraction of transcripts for each gene where its introns were fully excised using bam2ssj . The splDistribution of RNAPII across the gene TIR and body, commonly used as an indicator of promoter-proximal RNAPII stalling and efficient transcription elongation, was estimated by calculating the travelling ratio and by using mESC RNAPII ChIP-seq data . The traAll statistical analyses were performed using the R software environment for statistical computing and graphics .GSE111951. Most analyses were performed using standard publicly available command-line tools, as detailed in the Materials and Methods section.The raw and processed 4sU sequencing data generated in this study have been submitted to the NCBI Gene Expression Omnibus under accession number


MYC levels are oncogenic. Originally considered a typical transcription factor recruited to E-boxes (CACGTG), another theory posits MYC a global amplifier increasing output at all active promoters. Both models rest on large-scale genome-wide ”-omics’. Because the assumptions, statistical parameter and model choice dictates the ‘-omic’ results, whether MYC is a general or specific transcription factor remains controversial. Therefore, an orthogonal series of experiments interrogated MYC’s effect on the expression of synthetic reporters. Dose-dependently, MYC increased output at minimal promoters with or without an E-box. Driving minimal promoters with exogenous (glucocorticoid receptor) or synthetic transcription factors made expression more MYC-responsive, effectively increasing MYC-amplifier gain. Mutations of conserved MYC-Box regions I and II impaired amplification, whereas MYC-box III mutations delivered higher reporter output indicating that MBIII limits over-amplification. Kinetic theory and experiments indicate that MYC activates at least two steps in the transcription-cycle to explain the non-linear amplification of transcription that is essential for global, supraphysiological transcription in cancer.Supraphysiological  MYC proto-oncogene drives the pathogenesis of most human malignancy -driven minimal herpesvirus (HSV) thymidine kinase (tk) or thromboxane synthase (ts) promoter, respectively .To interrogate MYC function while avoiding confounding genomic influences, MYC-expression and reporter plasmids were co-transfected into U2OS osteosarcoma cells .   . Upon trr theory . Consistreporter . As the reporter .GREtkLuc when activated by co-transfected progesterone receptor (PR-B) along with the progestin R5020   . This we-F4 (F4)  not onlyGREtkLuc yielding GRE-E-box-tkLuc .Mutant MYC protein levels were checked by immunoblot to rule out simple protein dose explanations. Mutant MBIII-hyperactivation does not result from increased levels of expressed protein. In fact, MBIII-mutant protein levels were markedly reduced compared to wild-type MYC  were plotted . A strikGRE-Ebox-tkLuc and GREtkLuc). With high levels (80 ng) of transfected MYC-EGFP plasmids, the reporter activity profiles were similarly shaped with and without E-boxes, though inclusion of an E-box boosted expression by ~1.5 fold . Importantly the human genome harbors no orthologous sequences for either the Gal4 DBD or the VP16 activating segment. Therefore, U2OS cells are unable to drive Gal4 UAS-reporters unless supplied with exogenous GAL4/VP16. As is typical, five tandem Gal4UASs were inserted upstream of a TATA-box (5xGAL4UAS-TATALuc) to maximize activation by GAL4/VP16. Alone, Gal4UAS-GAL4/VP16-driven luciferase expression saturated at a low dose (2 ng/well) and then declined slowly , perhaps as part of a selective physiological response. To discriminate whether MYC-augmented reporter expression represented specific synergy with GR versus general amplification of transcription, assays were run to test if reporters controlled by synthetic activators could be driven by MYC to levels beyond those achievable by such chimeric activators alone. These experiments used the well-characterized chimeric GAL4/VP16 in which the DNA binding domain (DBD) of the yeast activator GAL4 is fused with an activating segment of the Herpes simplex ICP4 gene . The GALd slowly . Unactivd slowly , which id slowly , just asMYC amplification of GAL4/VP16 activation was tested for the involvement of MYC boxes. Similar to GR and PR, mutation of MBIII dramatically raised the gain on amplification of GAL4/VP16 at high levels of MYC . This reCFP). These chromosomally integrated reporters carried a single GAL4UAS upstream of the CFP transcription start site, and alternative reporter variants either including a single E-box (GAL4UAS/E) or not (GAL4UAS)  . Host ceGAL4UAS) . Thus, iGAL4UAS promoter  increase supra-linearly with varying concentrations of competing F4  of MYC action and its physiological and pathological mechanisms remain controversial. On the one hand, MYC has been considered a sequence-specific transcription factor turning on particular genes via E-boxes and controlling a large set of genes comprising a dense overlapping regulon . On the  studies  that purHere we have interrogated the MYC-amplifier hypothesis using synthetic biology experiments to study the influence of supraphysiological concentrations of MYC at minimal promoters, with or without E-boxes and with or without a separately recruited transactivator to provide an amplifiable input signal.A number of observations support the notion that MYC is a general amplifier of transcription:Basal reporter expression is relatively insensitive to MYC. To increase output, a sufficient signal must first prime the promoter for MYC.MYC can amplify a broad range of transactivators, for example steroid receptors and GAL4/VP16 that transduce a wide range of intra- and extracellular signals.MYC drives reporters to much higher levels than are possible even with saturating levels of transactivator alone. So, supplying MYC is not equivalent to boosting the dose of transactivator. Beyond saturation with transactivator, reporter expression declines due to squelching. MYC suppresses squelching, dramatically elevating reporter output.MYC amplification is auto-limited by MBIII, as evidenced by the much higher gain of MBIII mutants, especially at high levels of MYC. This peculiar property is not only rationalized but required by global amplification. If MYC increased gene transcription according to the level of ongoing promoter activity, then MYC-amplified transcription itself would become the substrate for further MYC-amplification on successive rounds of transcription. Unless checked, such a positive feedback loop delivers an all-or-none output that poorly matches the intensity of the transduced input signal. Suppression of positive feedback allows the output to follow the input across a broader dynamic range.MYC acts kinetically as an accelerator acting at more than one location after the CLS or as a facilitator of an accelerator.cis-elements arrayed along a gene, and the availability and activities of their corresponding transfactors, make the MYC transfer function at each promoter context dependent. MYC acts on top of these combinations of other regulatory molecules. That some promoter-recruited factors, such as MIZ1 , have not been reported to share coactivators nor to funnel directly through a shared regulatory complex onto a single discrete point in the transcription cycle. So MYC amplification of transactivation by GR or GAL4/VP16 is unlikely to reflect synergy driven through a common partner but is more likely to be accounted for by kinetic synergy . More spEZH2 promoter. That co-mutation of MBI blunts this hyperactivation while stabilizing the protein argues for MYC-degradation as a step in MYC action, especially in view of MBI’s proven role in MYC proteolysis  during a single round of transcription. The MBIII mutation that leads to hyperactivity seems also to strip MYC from the teolysis . Degradateolysis . MBII muradation .cis-elements and are insulated from enhancers, hence they are poorly equipped to recruit and deploy the components that expedite transit of the transcription machinery through the transcription cycle. Consequently, these promoters depend upon MYC to supply the complexes that sustain higher expression. In contrast, at high output promoters studded with strong positive cis-elements, multiple transfactors may be recruited that modify and complicate the kinetic landscape upon which MYC acts. A major advantage of the minimal promoters, primed by specific transactivators used here, is that it should greatly reduce the possible sites at which MYC exerts its kinetic activity(s). In fact, if a factor acts at multiple steps in a specific reaction pathway, our model will not give a single mechanistic interpretation from the competition assay  acts as a competitive decelerator at two steps before the CLS, and before MYC, is not unreasonable in the absence of conflicting data. The actions of 10058-F4 can still be specific for MYC if, for example, 10058-F4 prevents the pre-CLS entry of MYC into the reaction pathway or if it negatively affects some factor that is required by MYC at its further downstream site of action. The exposition of the exact biochemical reactions that are associated with the formal kinetic steps regulated by MYC promises to expose therapeutic vulnerabilities to intercept the pathology of this oncogene.Dexamethasone (Dex) was obtained from Sigma  and promegestone (R5020) was from PerkinElmer Life Sciences . Restriction enzymes and DNA polymerase were purchased from New England Biolabs . MYC inhibitor, F4 (5-[(4-ethylphenyl)methylene]−2-thioxo-4-thiazolidinone; 10058-F4)), was from Sigma, St. Louis, MO (Cat. No. F3680). 4-OH tamoxifen was from Sigma-Aldrich, catalog No.94873.xGAL4UAS-TATALuc, is from Stratagene . E-box GRE tkLUC was constructed by amplifying the fragment in GREtkLUC with forward primer 5’-catatgCGGTGTGAAATACCGCGCAC and reverse primer 5’-gtcgacCACGTGCTGCAGGCATGCAAGCTTGACC using Nde I and Sal I respectively to insert the E-box CACGTG. The resulting E-box GREtkLUC was sequenced and confirmed by using sequencing primer 5'-TTCGAGGCCACACGCGTCAC-3. MYC mutants were generated from p-c-Myc-pd4-EGFP-N1 (MYC sequences with G-blocks (IDT) containing MB mutants using Gibson Assembly Cloning Kit . The sequences of G-blocks are shown in Renilla-TS reporter, GREtkLUC, and rat GR (pSG5/GR) have been previously described . The FR--EGFP-N1  by replaPLVX-Gal4UAS-E1bTATA mCherry and PLVX-Gal4UAS-E-box-E1bTATAmCherry.An HIV-1 based, Lentiviral expression vector PLVX-mcherry-N1 was used to generate integrating reporter constructs. To temper reporter expression, the ClaI-XhoI restriction fragment of human cytomegalovirus immediate early promoter Pcmv IE was replaced by a minimal promoter containing a Lex binding site, Gal4 binding site(G), and an E1b TATA box with or without an E-box. The sequences of the fragments are listed below. Each pair of fragments were annealed at 96°C for 5 min with 1x NEB buffer #2 and slowly cooled to the room temperature before further subcloning into the vector yielding two reporter constructs yielding CGATAGGTACTGTATGTACATACAGTACTCGACTGCAGTCGGAGGACAGTACTCCGCTAGCCCGACTAGAGTCGAGTACTGTATATAATC;ClaI-LexGalE1bTATA-XhoI-T:TCGAGATTATATACAGTACTCGACTCTAGTCGGGCTAGTCGGGCTAGCGGAGTACTGTCCTCCGACTGCAGTCGAGTACTGTAGTACATACAGTACCTAT;ClaI-LexGalE1bTATA-XhoI-B:CGATAGGTACTGTATGTACATACAGTACTCGACTGCAGTCGGAGGACAGTACTCCGCTAGCCCGCACGTGAGAGTCGAGTACTGTATATAATC;ClaI-LexGalE-boxE1bTATA-XhoI-T:TCGAGATTATATACAGTACTCGACTCTCACGTGCGGGCTAGCGGAGTACTGTCCTCCGACTGCAGTCGAGTACTGTATGTACATACAGTACCTAT;ClaI-LexGalE-boxE1bTATA-XhoI -B:PLVX-Gal4UAS-E1bTATA mCherry and PLVX-Gal4UAS-E-box-E1bTATAmCherry were replaced by the Hygromycin resistance gene using thermostable assembly. The reporter genes were digested with XbaI and TthIIIi restriction enzymes, gel purified fragment then assembled with PCR products of PGK promoter (554 bp), Hygro I (622 bp), and Hygro2 (459 bp). The resulting constructs were used to make double stable cell lines. The primers used for PCR are in below.In order to make double stable cell lines, the puromycin-resistance genes GTGAGCGGCCGCGACTCTGAGTAATTCTACCGGGTAGG-3’,PKG-F5’-TCATGGTAAGCTTGGGCTGCAGGTCGAAAGG-3’,PKG-R5’-GCCCAAGCTTACCATGAAAAA21GCCTGAACTCACC-3’,Hygro1-F5’-TGTTGGAGCCGAAATCCGCGTGCA-3’,Hygro1-R5’-GGATTTCGGCTCCAACAATGTCCTGACGGACAATG-3’,Hygro2-F5’-GGATTTCGGCTCCAACAATGTCCTGACGGACAATG-3’Hygro2-R5’-FRLUC, GRE-E-box-tkLUC or GREtkLUC) and 10 ng Renilla plus various combinations of other expression vectors  were used. Equal molar amounts of the respective empty expression vectors were included to keep the molar amount of each vector constant, with the total transfected DNA brought to 300 ng/well with pBluescript II SK (Stratagene).U2OS human osteosarcoma cells were from ATTC, #HTB-96. These cells were maintained without further verification or mycoplasma testing, but retained proper growth characteristics, morphology, and properties. Key for these studies, they lacked an intrinsic glucorticoid response. Monolayer cultures of U2OS cells were grown in high glucose (4.5 g/L) DMEM containing 10% FBS in a 37°C incubator supplied with 5% oxygen-air atmosphere. Cells are transfected for 18 hr using Lipofectamine . For each well of a 24-well plate, 100 ng of reporter . Sixteen hrs. later, the medium was replaced with 14 ml of fresh full medium. Virus was harvested at 48 hr and 68 hr and filtered through 0.45 µm PVDF filters. The viruses were concentrated using the Lenti-X concentrator . Concentrated viruses were then aliquoted and stored in −80°C.The packaging of all escribed . The PLVFor lentivirus reporter experiments U2OS cells were grown in DMSO containing 10% Tet-approved FBS , MEM Nonessential Amino Acids (Corning cellgro REF. 25–025 Cl) and 50 U/ml penicillin G, 50 µg/ml streptomycin sulfate (Gibco REF. 15070–063). All pTripZ containing stable U2OS cell-lines were established by transducing the viruses into U2OS cells followed manufacturer’s protocols as previously described . The Lenhttp://n2t.net/addgene:36209 ; RRID:Addgene_36209) (The lentivirus-expressing MYC (wild type or mutated) and mCherry-reporter double-stable cell lines were plated in glass bottom cultureware plates (MatTek P06G-1.5–14F) the day before transfection with 60–80% confluency. Transfection of 10–20 ng of Gal4-VP16, 35 ng of Palmitoyl-turquoise2   and pBluFor measurement of human EZH2 RNA in pTripZ-EGFP, or pTripZ-lentivirus vector directed wild-type MYC-EGFP and mutant-MBI, II, III or IV-MYC-EGFP U2OS stable cell lines, RNA was prepared using Luna Cell Ready One-Step RT-qPCR Kit (NEB # E3031) following the manufacturer’s instructions. ~ 0.2×104 cells/well of each were seeded on a 96 well plate and grown at 37°C, 5% CO2 overnight. Cells were then treated or not with 0.5 mg/ml of doxycycline. After 8 hr., cell cultures were removed, rinsed with cold 1x PBS and aspirated. 25 μl Luna Cell Ready Lysis Buffer (2X), 5 μl of RNase-free DNase I (10x), 2 μl of Luna Cell ready RNA Protection Reagent (25x), 2 μl of Luna Cell Ready Protease (25x) and 6 μl of Nuclease-free water for total volume of 40 μl / well were added and incubated at 37°C for 10 min. The cell lysis reactions were terminated by adding 5 μl of Luna Cell Ready Stop Solution to each well and mixed well then incubated at 25°C for 5 min. 1.5 μl of the above cell lysis mix were used to program q-PCR reactions using Luna Universal One-Step RT-qPCR kit (NEB # E 3006). For a 20 μl reaction mixture, 10 μl of Luna Universal probe One Step Reaction Mix, 1 μl of Luna WarmStart RT Enzyme Mix, 0.8 μl each of the 10 mM primer, 0.4 μl of 10 mM probe, 1.5 μl of cell lysate and Nuclease-free Water were added. Triplicate reactions for each sample were performed. The PCR programs were set as 1 cycle of reverse transcription at 55°C for 10 min; 1 cycle of initial denaturation at 95°C for I min; 40 cycles of denaturation at 95°C for 10 s followed by extension at 60°C for 30 s for each cycle. The primers used for human EZH2 expression were: 5’-ATG GCA CCT GCA GAA GGA-3’; 5’-TTG GGA AGC CGT CCT CTT-3’. The probe used for the qPCR is Universal probe library # 79. For measurement of Luc and Ren RNA using qPCR, transfections were performed as above in 24-well plates and RNA was prepared with Luna cell ready one-step RT-qPCR kit (NEB # E3030). The cell lysis was as described for EZH2 expression for MYC -EGFP and mutant MB MYC-EGFP except 120 ml of cell lysis mix was used and all other reagent were proportionally increased. Real time qPCR reactions were performed with Luna one-step RT-qPCR kit (NEB # E3005) according to the manufacturer’s instructions. Primers were Firefly luc F 5’- GTG TTG GGC GCG TTA TTT AT-3’, Firefly luc R 5’- CAT CGA CTG AAA TCC CTG GT-3’; renilla F 5’- GTG GTG GGC CAG ATG TAA AC −3’, renilla R 5’- ACC AGA TTT GCC TGA TTT GC-3’. The SYBR scan mode was used on the real-time instrument. The instrument setting for reverse transcription is 55°C for 10 min. The initial denaturation step is at 95°C for 1 min followed by 40 cycles of denaturation at 95°C for 10 s. then 60°C for 30 s extension for each cycle. The melt curve was set for 60°C.For MYC-ER cell total RNA, rat Myc-ER cell line HO15.19 grown as previously  describe2 usually ≥0.95). All errors are ± S.E.M.U2OS cells were treated for 20–24 hr with/without steroid (100 nM in ethanol unless otherwise stated) in the culture media  before being harvested in 1 × Passive Lysis Buffer . Added 10058-F4 to a final concentration of 50 µM (from 100 mM in DMSO) was used as an inhibitor in some MYC amplification experiments. Cell lysates (50 µl per well) in a 96-well plate were used for luciferase activity assays using the Dual-Luciferase Assay System (Promega). Bioluminescence was read by a GloMax 96 Microplate Luminometer (Promega). The fold induction by steroid is defined as (the activity with steroid)/. Each sample was run in triplicate and each experiment was performed at least two times. The luciferase induction curve with steroid was then analyzed using KaleidaGraph  for the best fit by Michaelis–Menten kinetics  equipped with 355 nm, 407 nm, 488 nm, 532 nm, and 640 nm LASER lines using DIVA eight software . Briefly, after excluding cellular debris, using a forward light scatter (FSC)/side scatter plot (SSC), doublet cells were removed on the basis of signal width of FSC and SSC parameters. These debris free single cells were further gated to identify fluorescence signals of GFP, Turquoise, and mCherry proteins. GFP fluorescence was excited by 488 nm LASER and emission was read using 515/20 nm bandpass filter sets on PMT (B515). Turquoise protein’s fluorescence was excited by 407 nm LASER and emission was read using 450/50 nm bandpass filter sets on PMT (V450), whereas mCherry protein’s fluorescence was excited by 532 nM LASER and emission was read using 610/20 nm bandpass filter sets on PMT (G610). GFP-positive, but turquoise-negative (and hence GAL4/VP16-negative) vs. GFP-positive-turquoise-positive (and hence GAL4/VP16-positive) cells were sorted and stored at −80°C for ChIP-PCR analysis.MYC-EGFP plasmids were transfected into U2OS cells in 6-well plates for 48 hr using Lipofectamine . Cell lysates were collected and supernatants were obtained by centrifugation at 13,500 g for 20 min (4°C). Equal amounts of lysates protein were resolved into 3–8% Bis-acetate gels (Invitrogen) at 150 v and transferred onto nitrocellulose membranes (Bio-Rad) at 20 v at room temperature for 1 hr using a semi-dry apparatus (Bio-Rad). MYC or MYC-EGFP protein were detected using anti-MYC antibody from Epitomics (Rabbit mAb Cat.# 1472–1) and a secondary goat-anti rabbit IgG–HRP  antibody was used to amplify the signal. Western blots were visualized by ECL detection reagents as described by the manufacturer (Amersham Biosciences). The relative protein expression levels were compared by using a densitometer (Bio-Rad). For immunoblot of cellular and stably expressed MYC-EGFP, lentivirus expressed MYC-EGFP and MB-mutant MYC-EGFP, the U2OS cell lines with integrated lentivirus vectors expressing pTripZ-EGFP-, MYC-EGFP, or MYC-EGFPs with mutant MBI, MBII, MbIII or MbIV were grown in DMEM standard medium with 10% Tet-system approved FBS . After splitting into 6-well plates (~3×105/well), cells were grown for another 22 hr with or without 0.6 mg/ml doxycycline for 8 hr. Cells were lysed with 200 ml of RIPA buffer with protease inhibitors at 4°C for 20 min. The cell-lysates were centrifuged at 4°C for 20 min and the supernatants were collected. Cell lysate containing 40 mg protein were used for western Blot analysis. Both anti-GFP  and anti-cMyc  were used for detection. Anti-GAPDH antibody  was used as a loading control.For transiently expressed MYC-EGFP, different amounts of Images were acquired on a Nikon TiE inverted fluorescence microscope equipped with an automatic focus correction system, a SOLA LED light source (Lumencor), and a Neo sCMOS camera (ANDOR Technology Ltd). The Nikon TiE fluorescent microscope was adapted for long-term time-lapse microscopy through the addition of an environmental chamber that maintains a constant environment of 37°C, 5% CO2, and 40% humidity. Images were acquired with a 20x plan apo objective (NA 0.75) every 30 min over a 24 hr period. The mCherry filter set contained filters of 550/28 nm for the excitation light, 585 nm for the dichroic beam splitter, and 605/52 nm for the emission light (Chroma). The GFP filter set contained filters of 485/25 nm for the excitation light, 495 nm for the dichroic beam splitter, and 525/36 nm for the emission light (Chroma). The Turquoise filter set contained filters of 434/21 nm for the excitation light, 450 nm for the dichroic beam splitter, and 480/40 nm for the emission light (Chroma). Images were analyzed using NIS-Elements software (Nikon) and MATLAB software (Mathworks), which is available upon request.AAATTCAAAATTTTCGGGTTTATTAC-3’, -R: 5’CGGGGCTAGCGGAGTACTGT-3’. The Universal library probes used for the PCR reaction were PLVX-LGET-mcherry: #56; human EZH2; #82; human BEX: # 52. To compare WT MYC-EGFP and Myc -Box mutant MYC-EGFP binding to promoters, the procedures are the same as previously described  following the manufacturer’s instructions with minor changes. Frozen cells were resuspended and fixed in the growth medium containing 1/10 vol of fixation buffer at room temperature for 15 min. with gently shaking and then 1/20 medium volume of stop solution was added. The chilled cells were lysed with 30 strokes through a 25½ G needle and then centrifuge at 1250 g for 3 min. Chromatin sonication was performed with Qsonica sonicator Q700 systems using microprobes. Sonication was performed at 30% amplitude, pulse 30 s on and 30 s off for a total ‘’on’’ time of 10–20 min, depending on the cell number. Chromatin immunoprecipitation was performed with anti-GFP antibody (Abcam) or pre-Immune normal rabbit IgG  as control. Quantitative PCR was performed with LightCycler480 probe system and the LightCycler 480 Probes master . The primers for BEX and EZH2 are the same as previously described . The priescribed  and abovmax and the Dex dose of half-maximum EC50, are estimated from the dose-response curve. The basic protocol for gene induction  from the same laboratory demonstrating that MYC is required for induction of global mRNA production upon mitogenic stimulation of primary B-cells , revealing that MYC can act as a global transcription enhancer. The current manuscript extends these findings by showing that ectopic expression of MYC enhances transcription from a promoter that is pre-activated by a glucocorticoid receptor, but much less so a basal promoter, suggesting that MYC "amplifies" transcription in this setting. The authors go on to map domains of MYC that either positively nor negatively affect the ability to amplify transcription. A kinetic analysis suggests that MYC acts at more than one stage of the transcription cycle.Essential revisions:1) One issue with this manuscript is that a recent analysis of the same experimental system  challenges some of the central conclusions of the Nie, 2012 paper and argues that the global MYC-dependent enhancement of transcription is a late and indirect consequence of specific effects of MYC on individual genes. Furthermore, work in P493-6 cells, a model B-cell line, in which MYC levels can be regulated over a large concentration range, has concluded that amplification is a weak and serum batch-dependent effect . While these are simply different data and viewpoints in an ongoing and unresolved scientific debate, these studies raise the possibility that global effects of MYC on gene expression have been overestimated. Therefore, the authors need to show in a physiological setting – such as primary B-cells – that induction of MYC causes a global increase in mRNA synthesis and total mRNA levels ("amplification") and that the MycBox deletion mutants have the expected effects.2) Given expansive prior data on MYC-MAX binding to GC rich regions and non-canonical e-boxes, the authors should provide schematics fully characterizing the reporters used including the presence of all canonical and non-canonical E-boxes, and the GC% and C+G% of regions.3) One of the very interesting observations is that deletion of MycBoxI blocks over-amplification by MYC alleles with a deletion of MycBoxes III and IV. What is the explanation for this?4) The idea that MYC is a general amplifier is closely linked to the notion that it causes global pause-release of RNAPII. The concept put forward here is that MYC must regulate multiple steps in the transcription cycle to amplify transcription. The effects of MYC and the deletion mutants on RNAPII function therefore need to be documented and the authors should clarify whether the effects on reporter genes and the effects of individual deletion mutants can be correlated to distinct steps in the transcription cycle.5) The observation that the interaction of MYC with promoters depends on the presence of other activators is very interesting. There is no clear mechanism that can explain this observation. So how does the deletion of individual MYCBoxes affect binding? Do MYCBoxes III and IV limit MYC binding to promoters? This seems somewhat at odds with the description of WDR5.6) The results presented in Figure 1C are interesting and suggest that Myc can cause transcription activation (by a factor of 3) of a non-Ebox promoter upon co-stimulation. However, it is also possible that this is an indirect effect based on more efficient luciferase translation or changes in the cellular metabolism. The authors therefore should analyse if luciferase mRNA levels are regulated as well and if Myc binding to the reporter (reporter ChIP) follows the luciferase signal at different amounts of transfected MYC.7) There is a huge effect of Dex in Figure 1C but not in Figure3B. No error bars in Figure 3B-H, with the exception of Figure 3F.8) Strikingly, in transient transfection assays, MYC-∆MBIII behaves like a super-amplifier. Why is this behaviour not evident for stably integrated reporters (5D)? Why is MBIII mutant shown twice in 5D?9) The ChIP data in Figure 6 is not nicely presented .10) The two-factor competition assay is not described in a way that I can understand the experiment, neither in the legend, nor in the Materials and methods section.11) Instead of "amplification" the authors should use the neutral term "(reporter) gene activation", whenever possible. E.g. "Driving minimal promoters with exogenous (glucocorticoid receptor) or synthetic transcription factors increased amplification by MYC" could be changed to „Driving minimal promoters with exogenous (glucocorticoid receptor) or synthetic transcription factors increased reporter gene activation by MYC".12) The Discussion section is too long and the topics extend beyond the scope of this article and should be shortened. Overstatements should be avoided. E.g. „These results unambiguously display the transcription amplification driven by MYC". Essential revisions:1) One issue with this manuscript is that a recent analysis of the same experimental system  challenges some of the central conclusions of the Nie, 2012 paper and argues that the global MYC-dependent enhancement of transcription is a late and indirect consequence of specific effects of MYC on individual genes. Furthermore, work in P493-6 cells, a model B-cell line, in which MYC levels can be regulated over a large concentration range, has concluded that amplification is a weak and serum batch-dependent effect . While these are simply different data and viewpoints in an ongoing and unresolved scientific debate, these studies raise the possibility that global effects of MYC on gene expression have been overestimated. Therefore, the authors need to show in a physiological setting – such as primary B-cells – that induction of MYC causes a global increase in mRNA synthesis and total mRNA levels ("amplification") and that the MycBox deletion mutants have the expected effects.The reviewers have asked us to address criticisms of the amplifier hypothesis arising in the papers of Tesi et al., 2019 and Lewis et al., 2018. We address these misguided criticisms below:a) Tesi et al.,  claim that the increase in bulk RNA occurs only late (24 hours) after MYC activation, and so conclude that the accumulation of total RNA is an indirect consequence of early mitogenic programs triggered by MYC. This paper  is too conceptually and technically flawed to justify re-doing many of the experiments we reported in our 2012 paper . They have misananalyzed their data; the conclusions concerning the down- or upregulation of specific MYC targets and the inability to document an increase in total RNA are derived from data analyzed with the algorithm DESEQ-2. This program performs very stringent normalization that wipes-out differences in global expression levels. DESEQ-2 uses median-normalization that will artifactually shift the expression profile of one sample with respect to another when their median expression levels differ by a non-trivial amount. DESEQ-2 also uses a Bayesian approach to shrink the differences between samples along an expression distribution—it implicitly assumes that the expression curves of the different samples are fundamentally similar with only occasional “differentially expressed” (DE) outliers that deviate from this curve. DESEQ-2 will also suppress expression differences when broad zones of the expression spectrum are differentially up- or downregulated. As cited in our manuscript, a discussion archived at the Bioconductor web-site specifically discourages the use of DESEQ-2 for the evaluation of global changes in expression. Even if we re-do the experiments, we will analyze them differently and unless skeptics of the amplifier hypothesis recognize their misuse of DESEQ-2  the controversy will not be quelled. The only way we can assess the global up- or down-regulation of mRNA (versus regulation of subsets of mRNA) is to compare RNA-Seq before and after some sort of acute MYC activation or inactivation. The outcome of such analysis is completely dependent on the choice of statistical algorithms and bioinformatic pipelines used to process the data. The motivation of this manuscript was to use simple, non-genomic methods and conventional “old-school” assays to define the fundamental properties of MYC without arguing about the implicit assumptions of the algorithms and pipelines.b) In the manuscript by Nie et al., (2012) we demonstrated that naïve murine B-cells increase total RNA as early as 8 hours after activation which is 4 hours after MYC peaks. This global increase does not occur without MYC. Because total RNA is 97% rRNA, the global increase in RNA is primarily due to increased rRNA synthesis. Since total RNA and total poly-A RNA move in parallel, total RNA synthesis is a reasonable proxy for mRNA synthesis. Surprisingly, Tesi et al., saw no increase in global RNA transcription until 24 hours post-activation, using an in situ assay. Because of this late rise, they conclude that increased total RNA (and hence rRNA) is an indirect consequence of increased MYC. This is surprising. Ribosomal proteins and rRNA have long been recognized as primary MYC targets from flies  to humans .eLife’s timeframe for revision, that could cleanly switch on MYC activity using MYC-ER/tamoxifen rather than the slower doxycycline induction of MYC expression that might not exclude indirect effects). Therefore we studied HO15.19 MYC-ER cells . These MYC-knockout rat cells stably express MYC-ER. We serum starved cells for 72 hours and then treated with 200 nM tamoxifen. Although these serum-starved cells still contain a large basal amount of RNA, an increase in total RNA was almost statistically significant at 6 hours and became so at 10 and 14 hours after tamoxifen addition . Such an experimentally detectable increase in total RNA must largely represent increased rRNA synthesis reflecting ribosome biogenesis. While planning additional experiments to build on this observation, we reviewed the literature and discovered an experiment from the Dirk Eick lab that pulse-labeled HO15.19 MYC-ER cells that showed an immediate 5-fold increase in rRNA transcription upon addition of tamoxifen . They also documented an immediate increase in the expression of nucleolar proteins and other components required for ribosome biogenesis. In parallel they demonstrated the same immediate increase in rRNA synthesis and nucleolar protein mRNAs in P493-6 B-cells upon MYC activation. Together with other published work from the labs of Ingrid Grummt  with Anthony Wright, it is clear that ribosome biogenesis is an immediate early target of MYC activation and hence the increase in rRNA, and consequently total cellular RNA, commences early. Due to the other exigencies of manuscript revision and because we feel that we cannot improve on the existing literature, we now include our HO15.19 data in Figure 1—figure supplement 1 and cite these earlier studies to show that global RNA levels are rapidly increased by MYC.At the reviewer’s suggestion we sought a system compatible with c) The reviewers also refer to the Replication Study by Lewis et al., of transcription amplification by MYC in P493-6 cells. This study strongly confirms the MYC amplification from the Rick Young lab . I encourage the editors and reviewers to re-read that paper along with the commentary by Eick, 2018. Also, please see the “Annotations” appended to this study, one by Rick Young, and the second by one of the current authors, D. Levens. One issue raised by Lewis is rather trivial and simply relates to where to set the arbitrary threshold separating silent and expressed genes; there is no fundamental disagreement on the results. The second issue raised by Lewis et al., relates to variability between lots of serum in their hands—as noted in the annotation by Levens, the failure of Lewis to use charcoal-stripped serum, leaves the estrogen level of the media uncontrolled. Estrogen will activate an EBNA2-ER fusion resident in P493-6 cells and allow these cells to grow in the absence of MYC-induction. This oversight leaves the replication study potentially compromised in this regard.2) Given expansive prior data on MYC-MAX binding to GC rich regions and non-canonical e-boxes, the authors should provide schematics fully characterizing the reporters used including the presence of all canonical and non-canonical E-boxes, and the GC% and C+G% of regions.We have now supplied the requested information in the new Figure 1—figure supplement 2. It should be noted that canonical E-boxes CACGTG occur rarely. The only ones that occur in our transfected reporters were engineered into the promoter by us. In the lentivirus, there are three canonical E-boxes, one in each LTR, and one engineered into the reporter promoter by us. It should be noted that, in a 50% G-C sequence, a 5/6 E-box will occur by chance once every 228 bp. So, the large number of 5/6 matches is just as expected. We have no evidence that any of these are functional. Our earlier work  indicates that E-boxes must be situated close to the transcription start-site to amplify transcription.3) One of the very interesting observations is that deletion of MycBoxI blocks over-amplification by MYC alleles with a deletion of MycBoxes III and IV. What is the explanation for this?We agree; this is indeed a very interesting observation that may provide a strong clue as to the mechanism of MYC transactivation. In out first version we stated, “Co-mutation of MBI and MBIII reduced the excessive gain of the MBIII mutation back to wild-type levels  inviting consideration of MBI as a regulator of mutant MBIII activity.” and did not conjecture further. In the current manuscript we elaborate a bit further: “If MYC participates at multiple points in the transcription-cycle, perhaps by ferrying in different components, then cyclic MBI/phosphorylated T58-directed MYC degradation may help to ratchet the transcription machinery through various stages culminating in pause-release and efficient elongation”. Basically, the degradation of MYC would provide an energetic impulse driving the reaction forward. These concepts build on our observations as well as observations from the Tansey and Sears groups. We now comment on this in the Results section and in the Discussion section.4) The idea that MYC is a general amplifier is closely linked to the notion that it causes global pause-release of RNAPII. The concept put forward here is that MYC must regulate multiple steps in the transcription cycle to amplify transcription. The effects of MYC and the deletion mutants on RNAPII function therefore need to be documented and the authors should clarify whether the effects on reporter genes and the effects of individual deletion mutants can be correlated to distinct steps in the transcription cycle.The reviewers raise an excellent point; however a complete description relating MYC’s biochemical roles in the transcription cycle with the formal kinetic steps that we mathematically defined, we believe to be not only beyond the scope of this manuscript, but technically very difficult if not presently impossible. We explicitly investigate a simple transfected reporter gene vs. endogenous genes, but also we make no claim to be able to identify the precise biochemical steps at which MYC acts. The power of the mathematical analysis lies in its ability to constrain biochemical models and guide future experiments. The fact that we now have evidence that MYC acts at more than the one step cited by the reviewer is by itself a significant advance in understanding MYC action.5) The observation that the interaction of MYC with promoters depends on the presence of other activators is very interesting. There is no clear mechanism that can explain this observation. So how does the deletion of individual MYCBoxes affect binding? Do MYCBoxes III and IV limit MYC binding to promoters? This seems somewhat at odds with the description of WDR5.There is a straightforward mechanism to explain the recruitment of MYC to activated promoters. As shown in Lorenzin et al., 2015, and as summarized in Wolf et al., 2016, MYC at promoters binds both to DNA and with the transcription and chromatin machineries. If an activator shares a protein partner with MYC and has pre-recruited or co-recruited that partner, then energetically that activator must enhance/stabilize MYC binding.To address this question further, we explored how MB mutations  influence MYC-binding with promoters. Using lentivirus encoded, doxycycline-inducible wild-type or mutant MYCs, as described in the manuscript, we monitored both EZH2 expression and MYC-binding at the EZH2 promoter . We (and others) find EZH2 to be a reliable reporter of MYC-driven transcription. The results are stunning, and consistent with the observations and hypotheses of the prior version of our manuscript. We find that whereas wild-type MYC (wtMYC) and MBIII-mutant drove high levels of EZH2 expression, MBII- and MBIV-mutants did not. In contrast, binding was most apparent with wtMYC and with MBII-mutant. How can we reconcile these confusing data? If MYC binds to promoters simply by mass-action, and if promoter output simply reflects the equilibrium or steady state levels of promoter-bound MYC, then these results make no sense. But if several cycles of MYC binding and release/degradation/modification recur for each mRNA as MYC kinetically ratchets the transcription machinery through the various stages of the transcription-cycle, leading to productive elongation, then these results are easily rationalized. Mutation of the different MBs impairs orderly progression through the steps of the transcription-cycle. If MBII mutation were effectively a “wrench in the gears” then the mutant MYC might jam the promoter and remain frozen there unable to increase transcription.EZH2 gene almost as well as wtMYC. Yet, by ChIP-qPCR, mutant-MBIII binding to the EZH2 gene was greatly attenuated compared with wtMYC. The simplest way to reconcile these observations is that mutant-MBIII is consumed at a greater rate during the transcription-cycle than is the wild-type protein, and that such consumption is obligatory for MYC action. As MYC turnover is directed by MBI and coupled with T58 phosphorylation and recruitment of E3s such as Fbxw7, co-mutation of MBI would be expected to blunt the enhanced-turnover of mutant MBIII and canceling its hyperactivity. So the hyperactivity of mutant-MBIII is funneled through MBI.In contrast, though mutant-MBIII is found at levels much lower the wild-type protein, it activates transfected reporter genes better than the latter, it activates the native 6) The results presented in Figure 1C are interesting and suggest that Myc can cause transcription activation (by a factor of 3) of a non-Ebox promoter upon co-stimulation. However, it is also possible that this is an indirect effect based on more efficient luciferase translation or changes in the cellular metabolism. The authors therefore should analyse if luciferase mRNA levels are regulated as well and if Myc binding to the reporter (reporter ChIP) follows the luciferase signal at different amounts of transfected MYC.Reporter assays in general and luciferase reporters in particular have been a mainstay of molecular biological studies of promoter activity—including studies of MYC—for more than four decades and for ~37,500 publications. The vast majority of these report effects at the transcriptional level. Because of its protean influence, global transcription activation by MYC may (and perhaps inevitably will) provoke feedback via its influence on translation and metabolism. But there is every reason to expect, based on many hundreds of papers in the MYC literature, that the primary effect is on transcription. In our studies, MYC activates non-E-box promoters exactly in parallel with E-box promoters, the difference being only of degree not of quality. Neverthless, we performed transient transfection experiments to compare activation of E-box and non-E-box reporters by wtMYC and by MBIII-mutant MYC . Again, we find that MBIII-mutant is a more effective activator than wtMYC on both E-box and non-E-box promoters.GREtkLuc along with empty vector, wtMYC or MBIII-mutant was performed. The results show that much of the increased output of this E-box-less promoter indeed occurs at the transcriptional level . RT-qPCR assays of transfected reporters are inherently noisier than are reporter-enzyme assays; whereas the latter measures primarily correctly initiated mRNAs that sponsor proper translation initiation, the former detects any RNA that includes the reporter-specific primer binding sites (luciferase and Renilla in this case). Background transcription extending throughout the plasmid backbone is an old and well known problem for quantifying mRNAs recovered from transfected cells. It is highly likely that some of background expression that we detect from the empty vector reflects such non-specific transcription.To ask whether the MYC-activates non-E-box reporters at the RNA level, RT-qPCR of cells transfected with 7) There is a huge effect of Dex in Figure 1C but not in Figure3B. No error bars in Figure 3B-H, with the exception of Figure 3F.The Dex effects in Figure 1C and Figure 3B are identical. The y-axis scale in the old-figure 1C (and 1D) was carried over from an earlier version and should have been replaced. Note that the y-axes scales of Figure 1C and Figure 3B are different to accommodate the full range of MBIII-mutant activity. If the reviewer will zoom on the panels of Figure 3, he/she will see that there are error bars, but they are smaller than the symbols used to plot the data.8) Strikingly, in transient transfection assays, MYC-∆MBIII behaves like a super-amplifier. Why is this behaviour not evident for stably integrated reporters (5D)? Why is MBIII mutant shown twice in 5D?The stably-integrated reporters are driven by stably-integrated lentivrus encoded, doxycycline-inducible MYC and MB-mutant MYCs. Comparison of the immunoblots in Figure 1B versus 5D shows that the lentivirus-encoded MYC-EGFP does not reach the highest levels of expression achieved by the transiently transfected MYC-EGFP (note change relative to endogenous MYC). Because of the non-linear amplification by MYC , the lentivirus MYC-EGFP behaves as an amplifier, but not a “super-amplifier”.I don’t believe that MBIII is shown twice in Figure 5D. The panels include MBIII and the MBI-III double mutant, each with and without doxycycline induction.9) The ChIP data in Figure 6 is not nicely presented .The figure has been extensively revised and the axes labelled.10) The two-factor competition assay is not described in a way that I can understand the experiment, neither in the legend, nor in the Materials and methods section.A better description of the two-factor assay has now been provided in the Materials and methods section.11) Instead of "amplification" the authors should use the neutral term "(reporter) gene activation", whenever possible. E.g. "Driving minimal promoters with exogenous (glucocorticoid receptor) or synthetic transcription factors increased amplification by MYC" could be changed to „Driving minimal promoters with exogenous (glucocorticoid receptor) or synthetic transcription factors increased reporter gene activation by MYC".We respectfully disagree. We believe “amplifier” is a better objective descriptor of MYC’s action than “activator”. In these experiments, without glucocorticoid receptor (GR) or Gal4-VP16, MYC does almost nothing. So by itself, in our experiments, MYC is NOT an activator. MYC amplifies activation by these other transcription factors. Perhaps “co-activator” might be appropriate, but coactivation might imply that both MYC and its partner are required for activation—but this is not so—GR and Gal4-VP16 each act solo, but the degree of transcription activation is increased by the presence of MYC. Isn’t “amplify” the appropriate verb to describe this behavior? Nevertheless we have edited the text to try to describe objectively the results of the experiments.12) The Discussion section is too long and the topics extend beyond the scope of this article and should be shortened. Overstatements should be avoided. E.g. „These results unambiguously display the transcription amplification driven by MYC".We have edited the Discussion section for brevity, clarity and objectivity.


BTG4 is the last cloned and poorly studied member of BTG/Tob family. Studies have suggested that BTG4 is critical for the degradation of maternal mRNAs in mice during the process of maternal-to-zygotic transition, and downregulated in cancers, such as gastric cancer. However, the regulatory mechanism of BTG4 and its function in cancers remain elusive. In this study, we have for the first time identified the promoter region of the human BTG4 gene. Serial luciferase reporter assay demonstrated that the core promoter of BTG4 is mainly located within the 388 bp region near its transcription initiation site. Transcription factor binding site analysis revealed that the BTG4 promoter contains binding sites for canonical transcription factors, such as Sp1, whereas its first intron contains two overlapped consensus p53 binding sites. However, overexpression of Sp1 has negligible effects on BTG4 promoter activity, and site-directed mutagenesis assay further suggested that Sp1 is not a critical transcription factor for the transcriptional regulation of BTG4. Of note, luciferase assay revealed that one of the intronic p53 binding sites is highly responsive to p53. Both exogenous p53 overexpression and adriamycin-mediated endogenous p53 activation result in the transcriptional upregulation of BTG4. In addition, BTG4 is downregulated in lung and colorectal cancers, and overexpression of BTG4 inhibits cell growth and induces apoptosis in cancer cells. Taken together, our results strongly suggest that BTG4 is a novel p53-regulated gene and probably functions as a tumor suppressor in lung and colorectal cancers.  The mammalian BTG (B-cell translocation gene)/Tob (Transducer of ErbB2) family comprises six members, including BTG1, BTG2/PC3/Tis21, BTG3/ANA, BTG4/PC3B, Tob1/Tob, and Tob2 [The BTG/Tob proteins are structurally characterized by the highly conserved N-terminal BTG domain, which contains two conserved motifs, BOX-A and BOX-B, whereas their C-terminal regions are less conserved. Biochemical data revealed that the conserved BTG domain prominently function as a protein–protein interaction module. It has been reported that the BTG domain can interact with several DNA-binding transcription factors to mediate its role in transcriptional regulation. For example, BTG1 and BTG2 can interact with Hoxb9 , BTG3 inStudies suggest that BTG/Tob proteins play important roles in tumorigenesis and cancer. Suzuki et al. reported that Tob1 inhibits cell growth by decreasing cyclin D1 expression, and phosphorylation-mediated inactivation of Tob1 by Erk1 and Erk2 is required for Ras-mediated cell proliferation and transformation . Mice laBTG4 is the last cloned BTG/Tob family member, and thus remains poorly studied . Two rec2. Human lung cancer cells H1299 and A549 and colon cancer cells HCT116 were obtained from Cell Resource Center, Shanghai Institutes for Biological Science, Chinese Academy of Sciences. H1299 were cultured in RPMI 1640 (Hyclone), and A549 were cultured in DME-F12 (Hyclone), and HCT116 were cultured in DMEM (Hyclone) supplemented with 50 units/mL penicillin, 50 mg/mL streptomycin, and 10% (v/v) FBS (Invitrogen). These cells were cultured in a 37 °C incubator in a humidified atmosphere containing 5% COThe luciferase reporters BTG4-P1837 (−1611/+226), BTG4-P647 (−421/+226), BTG4-P412 (−421/−9), and BTG4-p388 (−397/−9) were established by cloning the BTG4 promoter region into pGL3-basic vector using a seamless cloning kit . The primer sequences as well as restriction enzymes are provided in The luciferase reporters P412-M1, P412-M2, P412-M3, and P412-M4 were constructed by a site-directed mutagenesis kit  based on the parental construct BTG4-P412 (-421/-9) according to the manufacturer’s instruction. For M1 mutant, the putative Sp1 binding site of GGGCGA at − 115 bp was mutated to TCTACT. For M2 mutant, the putative Sp1 binding site of GGGGCG at −80 bp was mutated to GTTGAG. For M3 mutant, the putative Sp1 binding site of GGGGCG at −75 bp was mutated to GCTATG. For M4 mutant, the putative Sp1 binding site of GGGAGG at −48 bp was mutated to GTTCGG. All the mutations were confirmed by DNA sequencing. The primer sequences are provided in ® transfection reagent . The pRL-TK vector  encoding Renilla luciferase was used as an internal control to monitor the transfection efficiency. Forty-eight hours after transfection, cells were lysed, and luciferase activity was determined using the Dual-Luciferase assay system  as described previously [For the luciferase reporter assays, cells were seeded in 12-well plates in triplicate and then co-transfected with the indicated plasmids using Neofect DNAeviously ,25.https://genome.ucsc.edu/). Transcription factor binding sites were analyzed by online software MatInspector. Alignment for the BTG4 gene promoters from the human, mouse, and rat were carried out by online software Clustal Omega . The BTG4 mRNA and promoter sequences were acquired from the NCBI and UCSC database  according to the manufacturer’s instruction. cDNA was synthesized from 500 ng of total RNA using a PrimeScript™ RT Master Mix kit . Real-time PCR was conducted by using the SYBR Premix Ex Taq  as described previously ,26. The HCT116 cells were transfected with empty vector (pcDNA3.0-Flag) and BTG4 expression plasmid (pcDNA3.0-Flag-BTG4). Forty-eight hours later, cells were seeded into 96-well plates at a density of 3000 cells per well. The CCK8 reagent was added at 0, 24, 48, 72, and 96 h after seeding and incubated for 1 h at 37 °C. The resultant optical densities (ODs) were measured at 450 nm. H1299 cells were transfected with empty vector (pcDNA3.0-Flag) and BTG4 expression plasmid (pcDNA3.0-Flag-BTG4). Forty-eight hours later, cells were scratched with a 10-µL pipette tip, and washed twice with PBS, and finally incubated in serum-free RPMI-1640. Images were taken at 0 and 24 h with a Leica light microscope . The migration rate was quantified by calculating the wound area changes at the indicated time points. 6 cells/mL. Annexin V-FITC and propidium iodide (PI) were then added and incubated for 15 min at room temperature in the dark. Cells were finally analyzed for apoptosis by flow cytometry. An annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit  was used to determine apoptosis according to the manufacturer’s instructions. In brief, cells were collected, washed twice in ice-cold 1× PBS, and resuspended in binding buffer at a concentration of 1 × 10http://genome.ucsc.edu/). As shown in To better understand the genomic organization and chromatin state of the human BTG4 gene, we retrieved the UCSC genome browser , BTG4-P647(−421/+226), BTG4-P412(−421/−9), and BTG4-P388(−397/−9), were established by cloning the different sizes of 5′-flanking genomic fragments of the BTG4 gene into a pGL-3-basic promoter-less vector a. As disAs the gene organization analysis revealed that BTG4 and its adjacent miR-34b/c are oriented head to head, we established an overlapped promoter reporter for miR-34b/c to determine whether the intergenic region has bidirectional promoter activity. As shown in To understand the transcriptional regulatory mechanism of the human BTG4 gene, we used the online software MatInspector to predict potential transcriptional factor binding sites in the promoter region of the BTG4 gene. As displayed in As Sp1 binding sites are prominently enriched in the proximal promoter of the BTG4 gene, we decided to further verify their roles in BTG4 transcription regulation. To this end, wildtype BTG4-P412 was used as a template to establish four mutant reporter constructs, P412-Sp1-M1, P412-Sp1-M2, P412-Sp1-M3, and P412-Sp1-M4, in which the four evolutionally conserved putative Sp1 binding sites in the BTG4 proximal promoter region were disrupted by site-directed point mutation, respectively a. As shoTo further explore the transcription factors that regulate BTG4 gene expression, we retrieved the transcription factor binding sites in the whole genomic sequences of the BTG4 gene. The analysis revealed that the first intron of the BTG4 gene contains two overlapped p53 binding sites, namely p53BS1 and p53BS2 a. Of notNext, we decided to determine whether BTG4 transcription can be regulated by p53. As shown in +/PI−) and late apoptotic/secondary necrotic phenotype 48 hours after transfection, whereas only 5.86% and 3.05% of control cells showed this phenotype. As shown in We then determined whether BTG4 could affect cancer cell proliferation, migration, and apoptosis. To this end, HCT116 as well as H1299 cells were transiently transfected with BTG4 expression plasmid. As shown in Finally, we examined whether the expression of BTG4 is aberrantly changed in cancer tissues. As shown in In the present study, we firstly identified the promoter region of the human BTG4 gene, which might not be mainly regulated by Sp1 although it contains several GC boxes. Our results strongly suggest that BTG4 is a novel target gene of p53 as evidenced by the fact that its first intron contains a canonical p53 response element and its transcription could be induced by p53. As a well-known tumor suppressor, p53 mainly functions as transcription factor to regulate the expression of various downstream target genes, consequently participating in diverse biological processes, including DNA damage response, cell cycle progression, apoptosis, autophagy, metabolism, etc. ,28,29. TOur data also found that the expression of BTG4 is downregulated in lung and colon cancers, and overexpression of BTG4 can inhibit proliferation and migration, and induce apoptosis, suggesting that similar to BTG2 and BTG3, BTG4 might also act as a potential tumor suppressor. Our results are consistent with the previous report by Toyota et al., who found the 5′-flanking region of BTG4 is hypermethylated and overexpression of BTG4 inhibits the colony formation ability in colorectal cancer cells . AlthougOf note, two microRNAs miR34b and miR34c exist upstream of the BTG4 gene . Our resHuman BTG4 gene contains several potential Sp1 and E2F binding sites in its promoter region, and one functional p53 consensus binding site in its first intron. Sp1 might not directly regulate BTG4 transcription. Overexpression of BTG4 inhibits cell growth and induces apoptosis in cancer cells. BTG4 is downregulated in lung and colorectal cancers. BTG4 is a novel p53-regulated gene and probably functions as a tumor suppressor in lung and colorectal cancers.


The three-dimensional architecture of the genome plays an essential role in establishing and maintaining cell identity. However, the magnitude and temporal kinetics of changes in chromatin structure that arise during cell differentiation remain poorly understood. Here, we leverage a murine model of erythropoiesis to study the relationship between chromatin conformation, the epigenome, and transcription in erythroid cells. We discover that acute transcriptional responses induced by erythropoietin (EPO), the hormone necessary for erythroid differentiation, occur within an invariant chromatin topology. Within this pre-established landscape, Yin Yang 1 (YY1) occupancy dynamically redistributes to sites in proximity of EPO-regulated genes. Using HiChIP, we identify chromatin contacts mediated by H3K27ac and YY1 that are enriched for enhancer-promoter interactions of EPO-responsive genes. Taken together, these data are consistent with an emerging model that rapid, signal-dependent transcription occurs in the context of a pre-established chromatin architecture.  •EPO induces rapid RNA Pol II response at a key subset of genes•YY1 is redistributed in the genome following 1 h EPO stimulation•CTCF and YY1 bind different locations pre and post 1 h EPO stimulation•E-P loops mediated by H3K27ac are largely invariant in response to EPO Molecular Biology; Developmental Biology; Genomics Transcription control is a primary mechanism for regulating gene expression in eukaryotes. Three major steps exist in the transcription cycle: (1) preinitiation complex (PIC) formation, (2) pause release of RNA polymerase II (Pol II) to productive elongation, and (3) transcription termination . MultiplMore recently, high-resolution maps of the three-dimensional (3D) genome have revealed that enhancers exhibit long-range control of transcription. Structural proteins, such as CCCTC-binding factor (CTCF) and Yin Yang 1 (YY1), tether distal TF-bound enhancers to their target gene promoters. CTCF is an evolutionarily conserved zinc finger that co-localizes with cohesin . TogetheOther TFs, such as YY1, are specifically enriched at chromatin loops that connect enhancers to promoters of actively transcribed genes . YY1 is Erythropoiesis has been a useful model system for understanding the interplay between Pol II dynamics , enhanceex vivo in response to EPO stimulation  system enables us to investigate the temporal dynamics of gene regulation and genome architecture in response to hormone stimulation. In this model, systemic treatment of mice with FVA induces ProEB proliferation in the spleen. After 14 days, large quantities of lineage-committed ProEBs can be isolated and purified. Stimulation of ProEBs with the hormone EPO in an h period A  at six time points demonstrated both up- and down-regulation of genes when compared with EPO-naive cells . OverallThe progressive changes in gene expression observed by RNA-seq reflect global transcriptional responses. We set out to investigate the immediate transcriptional response to EPO in purified ProEBs. To assess the acute effect of hormone stimulation on transcription, we performed chromatin immunoprecipitation (ChIP)-exo for RNA polymerase II (Pol II) before and after 1 h of EPO stimulation. Overall, Pol II occupancy is highly correlated when comparing ChIP-exo signal pre- and post-EPO stimulation in this short time frame . This reOverall, gene expression as measured by RNA-seq and transcription as measured by Pol II ChIP-exo are not in concordance with one another, specifically when investigating after 1 h EPO stimulation. There are 450 unique differentially expressed genes with consistent Pol II occupancy at promoters. Gene ontology (GO) analysis of these genes reveals an enrichment for genes involved in regulation of erythrocyte development, tyrosine phosphorylation of STAT protein, and response to hormonal stimulus, among others. This supports the role of the identified differentially expressed genes in response to EPO during erythroid maturation. However, when investigating stably expressed genes that have differential Pol II occupancy at promoters, there is no significant enrichment for specific biological processes. The explanation of the discordance of these features represents an area of gene regulation that requires further investigation.Rapid transcriptional induction may reflect PIC assembly, pause release of Pol II, or a combination of these steps. To investigate the mechanism of EPO-regulated transcription, we next mapped Pol II signal at transcriptional start sites (TSS) or gene bodies of all induced genes. This approach identified that Pol II is more abundant at the TSS of genes before EPO C. After Cish)  D. Cish id by EPO . We alsound gene E. Jund icontexts . Jund, aic cells , highligSignal-dependent activation of Pol II is accompanied by alterations in chromatin organization. However, the impact of EPO on chromatin structure and function during erythropoiesis is not well understood. To begin addressing this question, we first examined the genome-wide occupancy patterns of CTCF and YY1, two TFs known to play key roles in genome organization and gene regulation. CTCF occupancy did not change between pre and post 1 h EPO treatment, as demonstrated by the comparison of global enrichment analysis at each time point  and S3. In contrast to CTCF, YY1 is rapidly redistributed in the genome following 1 h EPO stimulation  and S3. In addition, ranking of H3K27ac signal by YY1 enrichment demonstrated that H3K27ac signal did not change in an appreciable manner compared with YY1 after EPO. This suggests that a subset of YY1 sites were more dynamic than H3K27ac, which is commonly used to identify active enhancers in the genome C and 3D.Stat3 gene, an exemplary locus of EPO-mediated transcription, a strong CTCF peak is evident pre-EPO and does not change after EPO  of invariant E-P contacts that map to UCSC-annotated TSSs in the mm10 reference genome C. We wouFadh1 gene, which is intricately involved in mitochondrial activity and metabolism .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Andrea Perreault , GEO: GSE142004 (HiChIP), and GEO: GSE142005 (RNA-seq).Unpublished custom code is available upon request from the Lead Contact. Summary of sequencing statistics can be found in All methods can be found in the accompanying


Cancer is a genetic disease caused by changes in gene expression resulting from somatic mutations and epigenetic changes. Although the probability of mutations is proportional with cell number and replication cycles, large bodied species do not develop cancer more frequently than smaller ones. This notion is known as Peto’s paradox, and assumes stronger tumor suppression in larger animals. One of the possible tumor suppressor mechanisms involved could be replicative senescence caused by telomere shortening in the absence of telomerase activity. We analysed telomerase promoter activity and transcription factor binding in mammals to identify the key element of telomerase gene inactivation. We found that the GABPA transcription factor plays a key role in TERT regulation in somatic cells of small rodents, but its binding site is absent in larger beavers. Protein binding and reporter gene assays verify different use of this site in different species. The presence or absence of the GABPA TF site in TERT promoters of rodents correlates with TERT promoter activity; thus it could determine whether replicative senescence plays a tumor suppressor role in these species, which could be in direct relation with body mass. The GABPA TF binding sites that contribute to TERT activity in somatic cells of rodents are analogous to those mutated in human tumors, which activate telomerase by a non-ALT mechanism. Within species, however, the incidence of cancer is higher in larger individuals2. To explain this contradiction it was suggested that animal species with larger bodies have developed more strict tumor suppressor mechanisms than those with smaller ones4. One of these mechanisms is the regulation of telomere length through telomerase reverse transcriptase (TERT) enzyme activity9. In human stem cells the telomerase is active, however in somatic cells the catalytic subunit of the enzyme is transcriptionally silenced10. In the absence of functional telomerase, chromosome ends shorten with each cell cycle as a result of the end replication problem12. Depleted telomeres induce apoptosis or cause cell cycle arrest, resulting in a cell state referred to as replicative senescence. The tumor suppressor role of replicative senescence is still debated, however it is supported by numerous wet lab experiments16 (see9 for a review). A recent report on whole genome sequence analysis of a very large number of tumor samples also strengthens the tumor suppressor role of senescence induced by telomere attrition17. The reason for the debate concerning the tumor suppressor role of replicative senescence is its similarity to induced senescence, which occurs from causes other than telomere shortening in cellular homeostasis during ageing of cells. With certain limitations induced senescence can be considered a tumor suppressor mechanism18; however, it is a “double-edged sword” since senescent cells could both improve immune response and promote tumor progression—for reviews on this see20.It has been observed that between different species, animal body size and cancer incidence do not correlate with each other, despite the fact that larger biomasses are assembled from more cells and would, therefore, be expected to incur an increased probability of mutations21. In these rodents, the tumor suppressor effect of telomere shortening may never be essential or take place because their lifespan is short and fewer number of cell divisions are necessary to reach the final body mass compared to large mammals7. In general, in mammalian species the length of telomeres in somatic cells is in correlation with telomerase activity and inversely correlates with body mass, but not with lifespan22. Nevertheless diverse tumor suppressor mechanisms act in small but long lived animals, like naked mole rats or squirrels23.In somatic cells of mice and other small rodents the TERT promoter is active24. However, telomere depleted mice were resistant to skin cancer25. The increased cancer incidence in TERT KO mice could be explained by telomere loss in stem cells compromising the immune system, thereby allowing more cancer cells to avoid immune surveillance. Several other severe phenotypes characterize TERT KO mice, indicating the importance of telomere maintenance in stem cells26. A recent report offers further insight to this puzzle by pointing out the importance of transient upregulation of TERT for appropriate senescence response27. It was found that cells from animals in which the telomere length was shortened due to down-regulation of TERT reached senescence earlier than cells from their wild type relatives; however, a transient TERT up-regulation was necessary for the proper senescence response. Without TERT, cells were more frequently immortalized using the alternative telomere elongation (ALT) mechanism, which caused increased tumor incidence. In contrast, when TERT gene was present, fewer cells were immortalized due to the proper senescence response. Cells only occasionally escaped from senescence, which was associated with telomerase activation. Only 10% of human tumors maintain telomeres by ALT and 90% use reactivation of TERT, which reveals the importance of replicative senescence in tumor suppression31.Replicative senescence resulting from telomere shortening is the consequence of telomerase inactivation in somatic cells. Early experiments with TERT null mutant mice resulted in contradicting data regarding the tumor suppressor effect of telomere shortening. In the absence of TERT expression telomeres indeed shortened in mice, but the overall tumor incidence increased32. It is believed that it became inactivated in early placental mammals but reactivated in a common ancestor of rodents, and it is active in most recent rodent species22. Exceptions are beavers and capybaras, in which the telomerase activity is lower than in other rodents. These species have larger body masses, at which replicative senescence could be beneficial. We hypothesized that telomerase inactivation in beavers and capybaras originates from changes in the promoter of the TERT gene. Revealing the importance of a cis-acting element in TERT gene inactivation highlights not only an evolutionary mechanism that allowed longevity and therefore increased fitness of large bodied animals, but also our vulnerability to acquired mutations providing an ideal cellular environment for the development of cancer.During the evolution of rodents the inactivation and activation of telomerase in somatic cells may have happened multiple times. Telomerase in platypus is found to be activeDue to the rapid development of sequencing methods, genome sequence data are available from a wide variety of mammals. By using these and data on telomerase activity, it is possible to identify potential transcription factor binding sites that could explain the activation or inactivation of TERT expression in different mammals. By identifying these transcription factors, a mechanism can be proposed to explain the evolution of replicative senescence as a tumor suppressor mechanism in larger animals.In this study we performed comparative TERT promoter analysis of different mammals with known TERT activity in somatic cells. We found that GABPA is a key factor in determining TERT transcription status in mammals of different evolutionary groups, similarly to certain human cancer cells. Our results support the hypothesis that a mutation in the TERT promoter is sufficient for the inactivation of the gene in somatic cells, triggering replicative senescence as a tumor suppressor mechanism. Therefore, the presence or absence of the GABPA binding site in the TERT promoter depicts a mechanism that might solve Peto’s paradox.34. An alignment of the sequences reveals that the TERT promoter sequence is rapidly evolving, therefore a comparison among them on nucleic acid level is challenging  and ELK1 proteins are Ets transcription factor family members. E47 (or TCF3) is a member of the E protein family of helix-loop-helix transcription factors, and GATA3 is a member of the GATA transcription factor family. The E47 (V$E47_01) binding site is more likely to be present in inactive TERT promoters, while the other three sites  are mostly found on active promoters. We performed k-means clustering multiple times on the data using the occurrence of transcription factors and the telomere state as a base. The clustering which best represents phylogenetic relations is presented in Fig. Next, we examined the positional distribution of these transcription factor binding sites on the promoter sequences of different species Fig. . GABPA s36. C to T mutation(s) within either or both of these sites creates a binding site for the GABPA transcription factor. Binding of the GABP complex of GABPA and GABPB results in the spreading of euchromatic chromatin structure to the promoter region and consequent transcriptional activation37.Based on our statistics on binding site presence and binding site distribution, we concluded that the GABPA TF could be the distinguishing factor between TERT active and inactive states. Our conclusion is supported by the notion that in human tumor cells a single mutation either at 146 or at 124 nucleotides upstream of the START codon of TERT is enough to reactivate telomeraseMus musculus) and shrew (Elephantulus edwardii) GABPA have amino acid differences in the DNA binding domain; however, based on the available 3D structures of GABPA-DNA interaction, these differences probably do not affect the DNA binding surface of the molecule , mutation of the GABPA binding site 150–200 bp upstream of the start codon inactivated TERT expression and allowed replicative senescence to take place. The lack of beaver cell cultures and the difficulties of obtaining biological samples prevented us from conducting ChIP experiments on beaver samples. However, we were able to test whether GABPA is able to bind to the beaver TERT promoter. Castor canadensis genomic DNA (kindly provided by Dr. Si Lok) was used to amplify a 500 bp fragment within the TERT promoter region38. This promoter fragment harbours a weak GABPA binding site at 164–158 bp upstream of the translation start codon. The score of this site, according to TFbind prediction, is below the cut-off value. We generated a strong GABPA binding site containing a variant of this promoter fragment (with TFbind score prediction above cut-off) by introducing TT > CG changes at position − 159–158 , and GABPA binding sites were removed from the rat and mouse TERT promoters  64. Surprisingly, under oxidative stress TERT is excluded from the nucleus and transferred to the mitochondria where it plays a protective role against oxidative stress55. This links TERT to the regulation of mitochondrial gene expression, and the common regulation strengthens our earlier proposal that active TERT in small animals is beneficial due to its role in oxidative stress reduction. GABPA sites were predicted in similar positions in different species, suggesting its conservation, while recognition sites of the other Ets family member, ELK1, were more dispersed , it was shown that GABPA selectively binds the de novo binding site and activates TERT expression67. TERT promoter mutations in tumors are very common69, and it is plausible that similar mutations could influence TERT expression during the evolution of rodents. Although GABPA activates TERT in tumors harbouring the promoter mutation, it should not be regarded exclusively as an oncogene. GABPA has tumor suppressor roles as well through the regulation of DICER1, which is responsible for multiple tumor suppressor mechanisms involving the miRNA pathway70.With this prerequisite, we assumed that molecular traces of TERT inactivation and reactivation have remained in the promoters of different species and consequently, comparison of the promoter sequences may unravel such traces. By comparing TERT promoters of 28 species, we found that multiple transcription factor binding motifs showed significant differences in active and inactive promoters, both in respect of their presence or absence and their pattern of location. GABPA (or NRF2), ELK1 and GATA3 transcription factors are more frequent in active promoters, while E47 (or TCF3) factor binding sites are more frequent in inactive promoters. As its binding sites showed the most significant differences between active and inactive promoters, we focused on the GABPA transcription factor. GABPA (NRF2) contributes to mitochondrial biogenesis as a regulator of mitochondrial genes27 and telomere length to the widely accepted tumor suppressor mechanisms of replicative senescence24, we conclude that our data support the initial hypothesis. Therefore, one could assume that in the beaver TERT promoter the loss of the GABPA binding motif reduced the promoter activity and inactivated TERT expression in somatic cells, by which replicative senescence was enabled. Since replicative senescence is a powerful tool to keep tumorigenesis at a low level, it is expected to allow beavers to develop larger body mass than other rodents.The ETS factor binding site that we identified as a GABPA binding site is missing in beavers, where telomerase is inactivated. Thus, TERT inactivation in beavers could be explained with mutation(s) in a strong GABPA binding site. In the beaver promoter only weak motifs were predicted, while strong binding sites can be found in relatives such as in mice. The experiments performed to validate that GABPA indeed binds to the mice TERT promoter and not to that of beavers supported our assumption: GABPA was detected on the TERT promoter in mouse fibroblast cells, but we could not detect interaction between the transcription factor and beaver TERT promoter. The loss of GABPA binding site, however, does not necessarily mean decreased promoter activity, therefore we performed reporter assays where promoter regions with and without GABPA binding sites were tested. For these functional assays, human and beaver promoters were mutated to introduce GABPA binding motifs. The presence of the binding site increased promoter activity. In contrast, when mutations were introduced to mouse and rat promoters to eliminate GABPA binding sites, the promoter activity was greatly reduced. Considering the experimental proof that links telomerase expression levels to telomere lengthSus scrofa) and camels (Camelus dromedarius), which also have GABPA sites in the TERT promoter and active telomerase71.It is important to emphasize that the hypothesis that the elimination of GABPA site(s) led to the loss of TERT promoter activity in somatic cells and by this generated replicative senescence as a tumor suppressor mechanism does not exclude the possibility that other effective tumor suppressor mechanisms are active in large-bodied animals. This could be the case in pigs  and rhinoceros (Ceratotherium simum sinum), which are their closest studied relatives 76. ChIP-Seq data were obtained from available databases  and TFbind software (TRANSFAC R.3.4 database)https://www.graphpad.com/quickcalcs/contingency1.cfm]. We used FIMO and Jaspar2020 database to calculate p-values for GABPA, Elk1, E47,GATA3 transcription factor binding sites identified by TFbind or Match  and 104.32 kg (Mastiffs) according to the American Kennel Club [https://www.akc.org/expert-advice/nutrition/breed-weight-chart/]; we presented the mass of an average German shepherd. Mouse Tert promoter sequences were extracted from Mouse Genomes Project data [https://www.sanger.ac.uk/science/data/mouse-genomes-project]82. Scripts used are available in Supplementary Note The number of samples we could use naturally was limited by the availability of data on TERT promoter sequences and telomerase activity among mammals. We therefore selected statistical methods suitable for small sample sizes to compare active and inactive promoters . For Random sampling bootstrapping we used R, while two tailed Fisher’s exact tests were done by GraphPad [Chromatin was cross-linked and nuclei were isolated using cell lysis buffer . Nuclei were lysed in nuclear lysis buffer , and sonicated in Bioruptor sonicator. 20–30 μg of total chromatin diluted in dilution buffer  to 500 μl was used for each sample. Antibody (Ab) and no antibody (NAC) samples were pre-cleared with Dynabeads M-280 Sheep Anti-Rabbit IgG . 5 μg of primary GABPA Antibody  was used in the Ab sample. Following incubation (1 h 4 °C), 40 μl Dynabeads were added. In the total input control (TIC) no antibodies or magnetic beads were used. After incubation , washing steps were done using freshly prepared Low Salt Wash Buffer , High Salt Wash Buffer , LiCl Wash Buffer  and TE Buffer  at 4 °C. DNA was eluted with elution buffer . The eluted samples were reverse cross-linked, RNase and proteinase treated, and DNA was purified with phenol-chlorophorm-isoamylalcohol (25:24:1) extraction. The regions of interest were amplified with PCR. Primers are listed in Supplementary Table Cloning was done by HiFi assembly . DNA fragments were amplified with Q5 polymerase . The list of primer sequences used for cloning different desired fragments can be found in Supplementary Table E. coli cell line83. Cells were suspended in buffer A  supplemented with proteinase inhibitor cocktail (PIC), and sonicated. The cell extracts were loaded on Nickel-NTA agarose column  and after washing, heterologous proteins were eluted in buffer B . Samples were concentrated and the buffer was changed using Amicon ultra filter units .GABPA proteins were produced in the Six Pack This technique is suitable to detect macromolecule interactions based on the changes in thermophoretic properties of macromolecules. The assay requires the fluorescent labelling of one of the interacting partners. For this we used either GFP-GABPA or GrGreen DNA dye . 16 step dilution series were prepared using wild type and mutant beaver promoter fragments from 3.36 mM and 4.3 mM starting concentrations, respectively. GFP-GABPA (20 nM concentration) and DNA samples were mixed and incubated at room temperature for 20 min. For the reciprocal experiment, wild type and mutant beaver TERT promoter DNA fragments dyed by GrGreen DNA dye and purified his-GABPA protein  were mixed. The measurements were done in buffer containing 20 mM TRIS pH 8.0, 50 mM NaCl, 10% Glycerol, 0.05% NP40, in standard capillaries. The equipment used was Monolith NT 115 .85. Cell lines NIH 3T3 and MCF-7 were obtained originally from ATCC . UACC-257 cell line is from cancer panel NCI-60. Each cell line was maintained and used in tissue culture laboratories regularly at our Department. Mycoplasma infection of the cell lines was tested regularly using MycoAlert Mycoplasma Detection Kit  as described by the supplier. Each cell line used here was free of detectable mycoplasma infection at the time used in the experiments described here. Cell lines were grown in Ham’s F12 (D12), RPMI , and DMEM (NIH3T3) media, supplemented with 10% fetal bovine serum (5% for Ham’s F12), 2 mM of glutamine and antibiotics.The dexamethasone-resistant hepatoma rat cell line (D12) was isolated and described by Venetianer et al.5 cells were seeded into 6 well cell culturing plates. 4 μg of plasmid DNA was measured to 400 μl of serum free media, then 6 μl of transfection reagent was added to the solution and mixed by pipetting. After 20 min of incubation at room temperature the mixture was pipetted on the cells. The plate was gently rocked to mix the solution. Cells were collected 24 h after transfection using cell scratchers. Luciferase expression levels were determined using Luciferase Assay system  as has been described85. The measurements were performed in Orion L type Microplate Luminometer . The protein concentrations were determined by Bradford reaction and the measured expression values were normalized using this data.D12, MCF-7, UACC-257 and NIH 3T3 cell lines used for transient transfection followed by luciferase reporter assay. Transfections were done using Turbofect reagent (Thermo Scientific). For this 2.5 × 10All experiments were conducted in at least 3 biological replicates. Outliers within these experiments were not excluded.Supplementary information.