C11orf95-RELA is an oncogenic driver in ST-EPN  but the molecular mechanisms of oncogenesis are unclear. Here we show that the C11orf95 component of the fusion protein dictates DNA binding activity while the RELA component is required for driving the expression of ependymoma-associated genes. Epigenomic characterizations using ChIP-seq and HiChIP approaches reveal that C11orf95-RELA modulates chromatin states and mediates chromatin interactions, leading to transcriptional reprogramming in ependymoma cells. Our findings provide important characterization of the molecular underpinning of C11orf95-RELA fusion and shed light on potential therapeutic targets for C11orf95-RELA subtype ependymoma.Supratentorial ependymoma (ST-EPN) is a type of malignant brain tumor mainly seen in children. Since 2014, it has been known that an intrachromosomal fusion The online version of this article (10.1007/s00401-020-02225-8) contains supplementary material, which is available to authorized users. C11orf95-RELA fusion was found to be the most recurrent structural variation in approximately 70% of supratentorial ependymomas (ST-EPN) [Ependymoma is the third most common malignant brain tumor in children. It can be classified into three groups based on location of the tumor: supratentorial, infratentorial and spinal. Currently there is no effective chemotherapy identified and treatment is limited to surgery with or without adjuvant radiation therapy. Because a significant portion of ependymoma is found in young children, radiation therapy is not desirable due to the detrimental effects on the developing brain. Thus, there is an urgent need to develop targeted therapy based on the underlying biology. (ST-EPN) , 15. Neu(ST-EPN) . However2. Protein overexpression in G16-2, G16-3 and G16-4 cells were induced by 1 μg/ml doxycycline treatment for 2 days.HEK293T and its derived G16-2, G16-3, G16-4 cells as well as BXD-1425-EPN cells were cultured in Dulbecco’s modified Eagle’s medium (Sigma) with 10% fetal bovine serum (Lonza), 4% Glutamax (Gibco), 1% sodium pyruvate (Gibco) and penicillin–streptomycin (Gibco). Incubator conditions were 37 °C and 5% COfus1-HA, or pCW-C11orf95-RELAfus1-HA) into Lenti-X 293T cells (Clontech), seeded the day before in 6-well plates at a concentration of 1.2 × 106 cells per well, using Lipofectamine 3000 (ThermoFisher). Media was changed 6 h after transfection then incubated overnight. Twenty-eight hours post transfection, the media supernatant containing virus was filtered using 45 μm PES filters then stored at − 80 °C until use.A viral packaging mix of pLP1, pLP2, and VSV-G were co-transfected with each lentiviral vector  was added to each respective well of one replicate plate. Antibiotic selection continued for at least 2 weeks before use in downstream experiments.To make G16-2, G16-3, and G16-4 cell lines, the day prior to transduction, HEK293T cells were seeded into 12-well plates at a density of 1.5 × 10fus1 was obtained as IDT gBlock while HEK293T cDNA served as template for amplifying RELA sequence. PCR and SLIC cloning were used to insert ORF encoding C11orf95fus1, C11orf95-RELAfus1, RELA with C-terminal HA tag into Gateway donor vector . Then LR clonase II reactions were used to shuttle ORFs into pCW-DEST  derived from pCW-Cas9 (Addgene # 50661), and pmax-DEST  vectors, generating pCW-C11orf95fus1-HA, or pCW-C11orf95-RELAfus1-HA, pCW-RELA-HA lentiviral Dox-inducible plasmids and pmax-C11orf95fus1-HA, pmax-C11orf95-RELAfus1-HA, pmax-RELA-HA transient expression plasmids. pmax-Clover and pmax-C11orf95-Clover, and pmax-C11orf95fus1-VP64, pmax-mScarlet-H2A, plasmids were generated with a combination of PCR, restriction-ligation, SLIC and LR Clonase II reactions. GFP-reporter plasmids, 15xUSF1-minCMV-EGFP and 15xC11orf95-minCMV-EGFP, were generated by ligation of annealed oligos containing USF1 or C11orf95 binding sites into minCMV-EGFP plasmid with non-palindromic overhang sites via digestion with BsaI, subsequently screened for the number of binding sites by Sanger sequencing.ORF encoding C11orf955 cells per well the day before transfection. For imaging experiments, 300 ng of pmax-C11orf95fus1-Clover or pmax-Clover (unfused control) was co-transfected with 100 ng of pmax-mScarlet-H2A (nuclear marker) using 1.5 µL Attractene transfection reagent (Qiagen). Microscopy images were taken with the iRiS Digital Cell Imaging System (Logos Biosystems).Cells were seeded into 12-well plates at a density of 1.5 × 10fus1, pmax-C11orf95-RELAfus1, pmax-RELA, pmax-C11orf95fus1-VP64) using 1.5 µl Lipofectamine 3000 (ThermoFisher). 48 h after transfection, cells were trypsinized, suspended in media then analyzed on a LSRFortessa X-20 flow cytometer (BD Bioscience). Fifty thousand events were collected each run.GFP-reporter plasmid DNA (300 ng) containing 15xUSF1 binding sites (15xUSF1-minCMV-EGFP) or 15xC11orf95 binding sites (15xC11orf95-minCMV-EGFP) was co-transfected with 300 ng of plasmid expressing test transcription factor constructs , RELA antibody (Abcam ab32536), and β-actin antibody .BXD-1425-EPN DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen), followed by genotyping PCR using Phusion PCR protocol (NEB) with primer pair of forward: CCTGCACCTGGACGACAT and reverse: TTGGTGGTATCTGTGCTCCTC. PCR amplicons were sent for Sanger sequencing, and analyzed by ApE.https://metascape.org/).Gene ontology analysis was done with Metascape (https://meme-suite.org/tools/meme-chip). Motifs were found de novo by DREAM. Sequences that had specific motif were reported by FIMO.ChIP-seq peak sequences were uploaded and analyzed by MEME-ChIP (RNA was extracted using RNeasy Mini Kit (Qiagen), then sent to Genewiz for library construction and sequencing by Illumina Nextseq sequencer. Each sample had two biological replicates. RNA-seq data was quantified using Salmon (version 0.8.0), followed by differential expression analysis using DEseq2. The cutoff for identifying differentially expressed genes were set as FDR < 0.05 and fold change ≥ 1.5.ChIP was performed using iDeal ChIP-seq kit for Transcription Factors (Diagenode) and protocol provided. HA , RELA (Abcam ab32536) and H3K27ac  antibodies were used for chromatin pull-down. Libraries were constructed using KAPA Hyper Prep Kits (Roche) and NEBNext® Multiplex Oligos for Illumina® (NEB) for Illumina Nextseq sequencing. Each sample had two biological replicates. The reproducibility between replicates was evaluated by Pearson correlation coefficient of read counts using deepTools (version 3.3.0) which was all greater than 0.95. Replicates were then combined for peak calling.Sequencing data was mapped to hg38 reference genome by Bowtie2 (version 2.3.1). Sequences with mapping quality score greater than ten were accepted for the following analysis. Peaks were called by MACS2 (version 2.1.0.20151222) using “–q 0.01 –nomodel –extsize 200” parameter for transcription factor ChIP-seq, and using “–broad −q 0.05, –nomodel –extsize 220” for histone mark ChIP-seq. For peaks that had at least 1 bp overlap, we considered them as overlapped peaks. The peak was considered to locate at gene promoter when it had at least 1 bp overlap with the promoter region. Promoter region was defined as a 5 kb span extending 2.5 kb at both sides from the transcription start site (TSS). Differential H3K27ac peaks were called by MACS2 bdgdiff subcommand. Read density heatmap of ChIP-seq peaks were computed and plotted using deepTools (version 3.3.0). Data was viewed by Integrative Genomics Viewer (IGV).HiChIP was performed following protocol published by Mumbach et al. 2017, with minor modifications. HA  and H3K27ac  antibodies were used for chromatin pull-down. Libraries were constructed using KAPA Hyper Prep Kits (Roche) and NEBNext® Multiplex Oligos for Illumina® (NEB) for Illumina Novaseq sequencing. Each sample had two biological replicates. Replicate data was combined for analysis. Raw data was processed by HiC-Pro  by proviCanonical pathway analysis was done by Ingenuity Pathway Analysis (IPA) using “Core Analysis” function.C11orf95-RELAfus1 was identified as the most frequently occurring fusion subtype in supratentorial ependymoma with C11orf95-RELA fusion (ST-EPN-RELA), containing almost the entire RELA gene except the first two codons fused to a truncated C11orf95 gene fragment (C11orf95fus1) harboring two and a half exons out of five exons of the full-size gene [C11orf95fus1 contains a C2H2 zinc finger domain typically found in transcription factors , implying its DNA binding potential. We hypothesized that the fusion of C11orf95 and RELA create a novel transcription factor whose DNA binding affinity as well as nuclear localization are largely governed by the C11orf95 fragment. Interestingly, fusion of C11orf95fus1 fragment to GFP is sufficient to drive exclusive localization of the fluorescence signal in the nucleus of HEK293T cells, demonstrating the intrinsic propensity of the C11orf95 fus1 fragment to translocate into the nucleus . However, there was previously no direct evidence for the binding of C11orf95fus1 and C11orf95-RELAfus1 to DNA. To address this question and further decipher the regulatory mechanisms of the fusion protein, we engineered HEK293T cell lines to express C11orf95-RELAfus1 (G16-4), C11orf95fus1 fragment (G16-3), or the RELA fragment (G16-2) in order to study the contribution of the fusion protein and each of the partners to epigenetic program and transcriptional regulation. These transgenes are tagged with the HA-epitope and are under the control of a doxycycline-inducible TetO promoter . The HA tag facilitates our molecular experimentations such as Western blot and ChIP, despite the lack of good quality antibodies against the fusion and C11orf95 proteins. We then conducted ChIP-seq using HA antibody to profile genome-wide chromatin bindings of RELA, C11orf95fus1 and C11orf95-RELAfus1 in G16-2, G16-3 and G16-4 cells, respectively. Surprisingly, 32,152 and 38,428 binding peaks were identified for C11orf95-RELAfus1 and C11orf95fus1, respectively, with 16,829 overlapping  analysis showed C11orf95-RELAfus1 unique binding genes were specifically enriched in neuron projection morphogenesis, actin filament process and skeletal system morphogenesis, which are known to be associated with brain or brain disease development [fus1 and C11orf95-RELAfus1 common binding genes were enriched in cell cycle arrest, regulation of autophagy and regulation of mitochondrion organization. However, C11orf95fus1 unique binding genes were enriched in more fundamental biological processes such as translation, carbohydrate derivative biosynthetic process and asparagine N-linked glycosylation  in G16-3 and G16-4 cells, respectively, compared to the controls. In parallel, we compiled a list of 886 ST-EPN-RELA-associated genes  based on published data [fus1) up-regulated genes overlapped with ST-EPN-RELA associated genes, while 67 of G16-4 (C11orf95-RELAfus1) up-regulated genes overlapped the ST-EPN-RELA associated gene list, including some well-known marker genes such as L1CAM, CHD5 and NOTCH1 . All these observations collectively indicate that C11orf95fus1 alone is not sufficient to drive transcriptomic dysregulation like C11orf95-RELAfus1.Although both C11orf95elopment , 16, 18.hed data , 14, 19.C11orf95 gene, and RELA without the first two and half exons , here referred to as C11orf95-RELA1425. Despite the lack of fusion-specific antibody, the fact that unstimulated RELA is excluded from the nucleus [1425 bound chromatin. RELA ChIP-seq in BXD-1425-EPN cells identified 13,954 chromatin binding sites of C11orf95-RELA1425, with 5,338 commonly shared by C11orf95-RELAfus1 bindings in G16-4 cells. With the binding peak sequences of C11orf95fus1, C11orf95-RELAfus1 and C11orf-RELA1425, we implemented de novo motif discovery using MEME-ChIP tool [fus1 fragment dictates DNA binding specificity of the fusion protein. As the motif consensus was repeatedly enriched by using different cells with different types of C11orf95-RELA fusion and different antibodies, the identified C11orf95 binding specificity is highly faithful. To establish GTGGCCCC as the bona fide DNA binding motif of C11orf95, we performed a reporter assay with 15 copies of GTGGCCCC and a minimum promoter cloned upstream of an EGFP reporter gene  DNA binding motif but not on a control reporter harboring 15 copies of an unrelated USF1 motif. These results suggest that C11orf95fus1 binds to the GC-rich motif GTGGCCCC and may activate gene expression by hijacking the transcriptional activation domain of RELA, as in the case of the C11orf95-RELA fusion. Furthermore, to examine the functional region of C11orf95fus1, we constructed three truncation mutants of C11orf95fus1-VP64 fusions and performed reporter assay to measure their transactivation activities . Deletion of the zinc finger (ZF) domain led to the loss of transactivation activity. In addition, deletion of the N-terminal and C-terminal regions upstream and downstream of the ZF domain also resulted in the loss of transactivation activity , suggesting C11orf95fus1 might be the minimal fragment of C11orf95 required for DNA binding. These results and the observation by Parker et al. [fus1 is the most frequent breakpoint of C11orf95 and is contained in all identified C11orf95-RELA fusion subtypes prompted us to speculate that C11orf95fus1 is the minimal fragment required for a functional C11orf95-RELA fusion.While the HEK293T transgenic cell models allow the dissection of the molecular function of C11orf95-RELA and its constituents, to obtain insights of the epigenomic and transcriptomic effects of C11orf95-RELA in the context of ependymoma, we performed further experiments in an ependymoma cell line, BXD-1425-EPN  that wasfus1 and C11orf95-RELA1425, approximately half of them contain one or more of the C11orf95 DNA binding motif . A two-fold up-regulation of endogenous C11orf95 transcripts was observed in G16-4 but not in G16-3 cells after doxycycline induction, validating the potential of C11orf95-RELA to positively auto-regulate its expression via binding to the C11orf95 promoter .Among the top-scoring ChIP-seq peaks of C11orf95-RELACCND1 and L1CAM, two well-known marker genes of ST-EPN-RELA, exemplify two different chromatin state outcomes upon fusion protein binding. At L1CAM promoter region, fusion protein binding likely resulted in the deposition of H3K27ac marks  or absence (in G16-2 and G16-3 cells) of the fusion protein binding  to identify ST-EPN-RELA gene associated interactions. As a result, a total of 445 common interactions were identified in both G16-4 and BXD-1425-EPN cells. Interestingly, not all the 886 genes harbored chromatin interaction(s) to other locus/loci, but only 156 of them  reached out to one or more promoter or non-promoter (enhancer) regions. Since these ST-EPN-RELA gene associated chromatin interactions were directly mediated by the fusion protein, it is reasonable to speculate that the more interacting partners one gene has, the more regulatory events it is involved in, and the more important roles it plays in ST-EPN-RELA. In other words, the genes that have more interactions with other genomic loci are more likely to be the hub of the fusion protein mediated network and more delicately regulated in fusion-associated transcription, so that they might contribute more to ependymoma development. Hence, we counted the number of interactions each gene promoter is involved in and plotted a histogram ranking the 156 genes by their interaction counts    to charaASPSCR1 gene on chromosome 17, connecting with ASPSCR1, DCXR, MAFG, MAFG-AS1, NPB, PYCR1 and RAC3 genes . More impressively, another distal enhancer had robust interactions with promoters of NOTCH1 and GPSM1 genes in both G16-4 and BXD-1425-EPN cells. Fusion protein binding was intensive and resulted in H3K27ac deposition at the enhancer region in G16-4 and BXD-1425-EPN cells  regions . We then counted the number of gene promoters connecting to these 91 enhancers. Although the majority of the enhancers had only one interacting promoter, some of them interacted with two to seven promoters . The enhancer with the highest number of promoter interactions is located in the intron of fus1 alone in HEK293T cells was not sufficient to drive ependymoma-like transcriptomic transformation, the ChIP-seq with both HEK293T and BXD-1425-EPN cells shows that the C11orf95 portion of the fusion plays a critical role binding to DNA and thus serving as a shuttle for the entry of RELA to the nucleus. By recognizing a specific DNA motif, C11orf95 portion targets particular genes and regulates their expression by hijacking the activation domain of RELA. Other than RELA, there was indication that C11orf95 also fused to other transcription factors such as YAP1 [Informed by our study of epigenetic reprogramming driven by C11orf95-RELA fusion in HEK293T cell models and ependymoma cell line, we gained important knowledge in the molecular mechanisms underlying tumorigenesis driven by a single oncogenic C11orf95-RELA fusion , 15. Alt as YAP1 , 14 in SSupplementary file1 (DOCX 24244 kb)Below is the link to the electronic supplementary material.


Transcription of a chromatin template involves the concerted interaction of many different proteins and protein complexes. Analyses of specific factors showed that these interactions change during stress and upon developmental switches. However, how the binding of multiple factors at any given locus is coordinated has been technically challenging to investigate. Here we used Epi-Decoder in yeast to systematically decode, at one transcribed locus, the chromatin binding changes of hundreds of proteins in parallel upon perturbation of transcription. By taking advantage of improved Epi-Decoder libraries, we observed broad rewiring of local chromatin proteomes following chemical inhibition of RNA polymerase. Rapid reduction of RNA polymerase II binding was accompanied by reduced binding of many other core transcription proteins and gain of chromatin remodelers. In quiescent cells, where strong transcriptional repression is induced by physiological signals, eviction of the core transcriptional machinery was accompanied by the appearance of quiescent cell–specific repressors and rewiring of the interactions of protein-folding factors and metabolic enzymes. These results show that Epi-Decoder provides a powerful strategy for capturing the temporal binding dynamics of multiple chromatin proteins under varying conditions and cell states. The systematic and comprehensive delineation of dynamic local chromatin proteomes will greatly aid in uncovering protein–protein relationships and protein functions at the chromatin template. Many proteins assemble on to DNA to implement gene regulatory programs and ensure the expression of a subset of genes in agreement with the state and environmental cues of the cell. RNA polymerase II (Pol II) is an integral part of this assembly as it catalyzes the DNA-dependent synthesis of messenger RNA (mRNA). Regulation of Pol II occurs at different stages of transcription and involves concerted actions of many proteins and protein complexes and dynamic post-translational modifications of histones and the carboxyl-terminal domain (CTD) of Pol II .The process of transcription involves distinct stages: initiation, elongation, and termination . The wraUnraveling the relationships between proteins and the hierarchies among them at the chromatin template will require measuring the chromatin interactome and monitoring the changes upon perturbation. A common and convenient strategy to perturb transcription is the use of inhibitors of Pol II . Some ofTranscription is also regulated under physiological conditions during development and differentiation. In budding yeast, a global change in transcription is observed when cells enter quiescence, a survival mode that involves a G1-like cell-cycle arrest, with increased resistance to stress, metabolic rewiring, and large-scale reorganization of the genome and cellular machineries . QuiesceHO locus (Epi-Decoder-HO). Of this library, we used a dedicated subset of approximately 700 (putative) chromatin proteins  of which the binding at the reporter locus can be assessed in parallel and in triplicate with three independent BCs in a single sample. This Chrom-3×BC library was used to determine the local chromatin-proteome rewiring at the barcoded transcribed locus in response to transcription inhibition by chemical perturbation and during quiescence.To systematically interrogate the chromatin changes at a transcriptionally active locus upon inhibition of transcription, we used Epi-Decoder, a tag–chromatin immunoprecipitation (ChIP)–barcode (BC)-sequencing technology in budding yeast . Epi-DecKanMX marker gene integrated at the HO locus and flanked by two BCs—BC_UP (promoter region) and BC_DN (terminator region)—that are 1.5 kb apart (HO-Barcoders). The barcoded KanMX cassette is a kanamycin gene controlled by the heterologous AgTEF1 promoter and terminator from Ashbya gossypii, a yeast related to Saccharomyces cerevisiae. This 1.5-kb reporter-gene cassette replaces the coding sequence of the HO gene and is therefore flanked by the endogenous HO promoter and terminator sequences as well as an origin of replication (ARS404) 53 bp downstream from BC_DN. This reporter locus has previously been used in screens for various chromatin- and transcription-related features . Upon pooling, cross-linking, ChIP, amplification of the barcoded regions, and counting the BCs by massive parallel sequencing . The expanded Barcoder-HO library was combined with the TAP-tag library in three different ways (see Methods) such that every TAP-tag is linked to three independent HO-Barcoders  and that a triplicate TAP-tag subset of approximately 700 chromatin proteins (Chrom-3×BC) was created that can be processed as one pool owing to nonoverlapping BC pairs in the replicates.To measure local proteome dynamics at a promoter and terminator region upon transcriptional inhibition, we first improved the previously reported Epi-Decoder-HO library to optimize the comparison of multiple time points and conditions. Epi-Decoder is a strategy for decoding the local proteome of a single genomic locus . It relifeatures . The libquencing B, the abquencing . Integraquencing , enablinSupplemental Fig. S1B,C; Supplemental Table S1), and this was further confirmed by the analysis of individual clones (see below). Although it is possible that specific BC sequences could cause a bias for detection of certain proteins, in general our results show that the inferred protein-binding scores are largely independent of the short BC sequences. The use of multiple Tag-Barcode combinations further increases the confidence of single-BC measurements. For example, we have previously shown that Ssl2 and Tfa2, two factors known for their roles in transcription initiation, also show high binding at the HO terminator region . The consistent and robust binding of Ssl2 and Tfa2 at the terminator region in the absence of other basal TFs suggests a noncanonical function of these factors.Epi-Decoder uses DNA BC counting as a reporter for protein binding . With thr region . By repl2+ ions, which are essential for the activity of RNA polymerases  A to deteymerases . The addymerases . AU is aymerases . The treymerases . To inveymerases A. All exSupplemental Fig. S2B), and addition of PH led to induction of ZRT1 mRNA whereas addition of AU led to induction of IMD2 mRNA , which is in agreement with previous observations , in agreement with the observed temperature effects on RNA decay kinetics . To determine the consequences of transcription inhibition for the transcription machinery in more detail, we focused on Pol II and transcription elongation factors. All the Pol II subunits present in our Epi-Decoder library showed a pronounced and progressive loss of binding upon addition of PH at BC_UP and BC-DN. In AU, Pol II binding was also reduced but to a lesser extent , whereas treatment with AU showed a modest decrease . Therefore, the reduced binding of Pol II subunits in PH was not accompanied by increased Pol II protein degradation.The global overview of the dynamic chromatin proteomes (average of three replicates) C revealer extent B. TherefKanMX gene under the control of the AgTEF1 promoter and terminator could be validated by ChIP-qPCR at the 5′ and 3′ ends of the endogenous TEF1 gene . Thus, in addition to the current knowledge that Pol II activity is inhibited directly by PH and indirectly by AU . The conserved elongation factors Elf1, Spn1, Spt6, and DSIF (Spt4/5) were rapidly and progressively evicted from chromatin, closely resembling the dynamics of Pol II upon PH treatment .Several proteins and protein complexes have been biochemically or genetically linked to the process of transcription elongation . HoweverII by PH A and AU reatment B, 4A. ThThe FACT complex, composed of Spt16 and Pob3, is known to be recruited to chromatin by the act of transcription and its associating factors Nhp6a and Nhp6a, two high mobility group (HMG) proteins . At BC_USupplemental Fig S4A,B). However, binding of Ctr9, the largest subunit and a key scaffold protein of Paf1C . The differences in kinetics were not caused by differences in protein abundance, because Ctr9 showed very similar global expression levels as the Paf1 and Pol II subunits . We also tested whether the delayed loss of Ctr9 was caused by a difference in crosslinking sensitivity . Finally, addition of PH did not lead to major changes in the expression level of Rpo21 and Paf1, compared with Ctr9, the latter remaining bound when Rpo21 and Paf1 had already been evicted upon PH treatment , and Spt6 can form an intricate protein network around elongation-permissive Pol II . Indeed,of Paf1C , was larof Paf1C A,B. We cof Paf1C C, as welsitivity . Howeverreatment D. Togethreatment . These bSupplemental Fig. S4A). Analysis of Srm1 by ChIP-qPCR confirmed the efficient binding of this protein and an increase after PH treatment at the reporter locus as well as endogenous loci, including a nontranscribed region , and HMG protein Hmo1 . In contrast, the HMG-like protein Spt2, a chaperone involved in histone recycling over transcribed regions of active genes . The dynamic behavior of Srm1, Ssa2, Hmo1, and Spt2 after PH treatment was not caused by changes in global protein levels . Together, these results suggest that Srm1 is an abundant chromatin protein with the potential to affect chromatin structure and function but that it might not act as a canonical transcription elongation factor.In addition to uncovering protein-binding dynamics within well-known protein complexes, the parallel analysis of hundreds of proteins also offers the possibility to obtain more insight into proteins of which the chromatin functions have been less well annotated. Here we more closely examined Srm1 , a homolog of human Regulator of Chromosome Condensation 1 (RCC1). Srm1/RCC1 is a guanine nucleotide exchange factor that localizes to the nucleus, binds nucleosomes in a dynamic way, and occupies a large part of the yeast genome . In the d region E. This, d region , indicatd region A,E, wherd region C, the heve genes , was decHO locus was altered by the strong physiological transcriptional shut down during quiescence. Chrom-3×BC Epi-Decoder library pools were grown in YEPD for 7 d at 30°C , which causes cells to arrest in saturation with G1 DNA content. Stationary phase cultures consist of two populations: Q cells, which are more uniform, long-lived, and stress resistant, and nonquiescent (NQ) cells, which are more heterogeneous, short-lived, and stress sensitive . Overall, the magnitude of the changes was higher than that observed in the G1 cells treated with PH with several exceptions. The FACT-associating factors Nhp6a and Nhp6a showed increased chromatin binding in Q cells , in contrast to PH-treated cells . Tho1, a protein associated with transcribed chromatin, showed increased abundance in Q and NQ cells but not in PH , suggesting that Tho1 might have Q/NQ cell–specific functions and that its recruitment is independent of ongoing transcription, in contrast to canonical transcription elongation proteins.Analysis of the RNA Pol II machinery, transcription elongation factors, basal TFs, and transcription termination and RNA processing factors showed a pronounced loss of the core transcription machinery in Q and NQ cells . Q cells showed binding of the quiescence-specific transcription repressor Xbp1, which was absent from mid-log cells and during PH treatment. This protein is known to recruit the HDAC Rpd3, mediating the global shutdown of gene expression in Q cells . This well-established repression pathway was accompanied by increased abundance of several TFs such as Mcm1, Mbf1, Tbf1, Abf1, and Swi6 .To gain more insight into the transcriptional repression of the barcoded  Q cells . Indeed,Supplemental Fig. S5B–G). Yeast Histone H1 (Hho1) showed increased abundance in Q and NQ cells . HMG protein 1 (Hmo1), which has been shown to function as a linker histone and promote chromatin compaction . In contrast, the negative regulator of transcription Spt2 left the chromatin in Q and NQ cells, as it did in PH-treated cells. These changes were paralleled by the recruitment of the ATP-dependent chromatin remodeling complexes INO80 and RSC, as well as several heat shock proteins and other factors involved in protein folding . In addition, changes were seen in the binding of metabolic enzymes . Future functional studies will be required to determine the role of these dynamic interactions in the global transcriptional repression occurring in Q cells.Besides the core transcriptional machinery, TFs, and Rpd3L, we observed several other changes in the chromatin proteomes  proximal to BC_DN D. In cones pombe . Our resDetermining the changes to chromatin composition in response to cell signaling or stress can reveal insights into common mechanisms of gene regulation. Obtaining a comprehensive understanding of chromatin changes, although highly informative , is ofteHO locus , and differential binding of several heat shock proteins  . We note that some of the observed dynamics in Q and NQ cells might, at least in part, be driven by altered expression of the proteins in the different cell states. Determining the functional roles of these Q-cell-specific and NQ-cell-specific changes will require further mechanistic studies.During quiescence, global changes in histone modifications have been observed , as wellproteins C, Hho1, Although Q cells and NQ cells showed overlap in the changes compared with mid-log cells, we also observed important differences, in agreement with the different fates of the long-lived Q and short-lived NQ cells  and the Our results show that Epi-Decoder provides a powerful strategy for capturing the temporal binding dynamics of chromatin proteins under varying conditions. We expect that future studies on local chromatin-proteome maps under different conditions and at other genomic loci will offer powerful resources for detailed molecular and functional annotation of chromatin proteins and their interactions and relationships at the genome.Supplemental Table S4. Library manipulations on solid media were performed using synthetic genetic array (SGA) technology  using the protocol for yeast cells, with a few modifications, essentially as previously described  and withSupplemental Table S5). Libraries of the PCR products were mixed in an equimolar fashion and sequenced  on a HiSeq 2500/MiSeq platform (Illumina), using one or a mix of custom sequencing primers . Details of the Epi-Decoder protocol and analysis and the BC counting can be found in the Supplemental Materials and Methods.Epi-Decoder was performed and analyzed essentially as described previously . ProteinSupplemental Materials and Methods. Each ChIP was performed in triplicate.ChIP-qPCR experiments were performed with IgG Sepharose 6 fast flow beads or epoxy-activated Dynabeads as described previously  and in tSupplemental Materials and Methods.For immunoblotting, strains were grown to mid-log phase or arrested in G1 and processed as described previously . AntibodSupplemental Tables S6–S11) have been submitted to the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/) under accession number PRJNA610036, and to the NCBI BioSample database (https://www.ncbi.nlm.nih.gov/biosample/) under accession numbers SAMN14271070, SAMN14271071, and SAMN14271072. All processed data are within the paper and the Supplemental Material.All raw sequencing data generated in this study and corresponding reference tables (The authors declare no competing interests.


Histone H1 is the most mobile histone in the cell nucleus. Defining the positions of H1 on chromatin in situ, therefore, represents a challenge. Immunoprecipitation of formaldehyde-fixed and sonicated chromatin, followed by DNA sequencing (xChIP-seq), is traditionally the method for mapping histones onto DNA elements. But since sonication fragmentation precedes ChIP, there is a consequent loss of information about chromatin higher-order structure. Here, we present a new method, xxChIP-seq, employing antibody binding to fixed intact in situ chromatin, followed by extensive washing, a second fixation, sonication and immunoprecipitation. The second fixation is intended to prevent the loss of specifically bound antibody during washing and subsequent sonication and to prevent antibody shifting to epitopes revealed by the sonication process. In many respects, xxChIP-seq is comparable to immunostaining microscopy, which also involves interaction of the primary antibody with fixed and permeabilized intact cells. The only epitopes displayed after immunostaining are the “exposed” epitopes, not “hidden” by the fixation of chromatin higher-order structure. Comparison of immunoprecipitated fragments between xChIP-seq versus xxChIP-seq should indicate which epitopes become inaccessible with fixation and identify their associated DNA elements.We determined the genomic distribution of histone variants H1.2 and H1.5 in human myeloid leukemia cells HL-60/S4 and compared their epitope exposure by both xChIP-seq and xxChIP-seq, as well as high-resolution microscopy, illustrating the influences of preserved chromatin higher-order structure in situ. We found that xChIP and xxChIP H1 signals are in general negatively correlated, with differences being more pronounced near active regulatory regions. Among the intriguing observations, we find that transcription-related regions and histone PTMs  exhibit significant deficiencies (depletions) in H1.2 and H1.5 xxChIP-seq reads, compared to xChIP-seq. These observations suggest the existence of in situ transcription-related chromatin higher-order structures stabilized by formaldehyde.Comparison of H1 xxChIP-seq to H1 xChIP-seq allows the development of hypotheses on the chromosomal localization of (stabilized) higher-order structure, indicated by the generation of “hidden” H1 epitopes following formaldehyde crosslinking. Changes in H1 epitope exposure surrounding averaged chromosomal binding sites or epigenetic modifications can also indicate whether these sites have chromatin higher-order structure. For example, comparison between averaged active or inactive promoter regions suggests that both regions can acquire stabilized higher-order structure with hidden H1 epitopes. However, the H1 xChIP-seq comparison cannot define their differences. Application of the xxChIP-seq versus H1 xChIP-seq method is particularly relevant to chromatin-associated proteins, such as linker histones, that play dynamic roles in establishing chromatin higher-order structure. Histone H1 plays a distinctly different structural and functional role in eukaryotic nuclei compared to the inner (core) histones H4, H3, H2A and H2B. Whereas the inner histones form a defined octamer complex surrounded by a DNA wrapping (the nucleosome), H1 is positioned outside the nucleosome. The central H1 globular domain is at/near the dyad axis, flanked by N- and C-terminal peptide tails, believed to be associated with linker DNA connecting adjacent nucleosomes . The innThe stoichiometry of histone H1 per histone octamer has been estimated to be ~ 0.8–1.0 in somatic cells , 14. GenSeveral studies have examined the in situ enrichment (or depletion) of DNA functional elements at the binding sites of various H1 isotypes –22. ThesIn the present study, the chromatin distributions of two isotypes (H1.2 and H1.5) were examined within the nuclei of the human myeloid leukemia cell line HL-60/S4 in situ. Two chromatin immunoprecipitation methods were employed, and their results compared; i.e., xChIP-seq and xxChIP-seq, see Fig. The human myeloid leukemia cell line HL-60/S4  was maintained in RPMI-1640 medium plus 10% FCS and 1% Pen/Strep/Glut. Cells were grown in 6 ml of medium in T-25 flasks, generally split (1:6–1:12) 2–3 times/week. For large-scale preps, cells were grown in T-75 flasks with up to 30 ml media. Cell concentrations were monitored using a hemocytometer. Rabbit polyclonal ChIP-grade antibodies were obtained from Abcam: anti-histone H1.2 (ab4086) and anti-H1.5 (ab18208). Both antibodies are directed against antigenic determinants within the N-terminal 1-100 aa residues.Images of immunostained cells were obtained on a Leica SP8 confocal microscope and on a home-built STED microscope, as described previously . H1.2 an7 cells/cryovial) in liquid Nitrogen. Prior to storage, the cells were harvested from growth medium at ~ 106 cells/ml, centrifuged and washed with PBS, fixed in 1% HCHO/PBS for 10 min at room temperature (RT), stopped with 0.125 M glycine for 5 min, washed with PBS, followed by PBS + 0.1 M PMSF. The fixed cells were permeabilized for 10 min at 4 °C in a lysis buffer containing 25 mM HEPES buffer (pH 7.8), 1 mM MgCl2, 10 mM KCl, 0.1% NP40, 1 mM DTT and 0.5 mM PMSF. Following centrifugation and removal of supernatants, cell pellets were frozen in residual lysis buffer at liquid Nitrogen temperature.All ChIP-Seq experiments were performed on undifferentiated HL-60/S4 cells that were fixed, permeabilized and stored in cryovials  was added to a final BSA concentration of 5%.For both single-fixation and double-fixation ChIP  or 60 µl anti-histone H1.5 (0.5 mg/ml). The cells plus antibody were rotated for 4 h at RT. Following antibody incubation, the cells were washed several times with PBS to remove unbound antibody. For the second fixation, the washed cells were made 1% HCHO/PBS and rotated 2.5 min at RT. Fixation was stopped with 0.125 M glycine for 5 min, cells washed with PBS and dispersed in 1.0 ml of Covaris Sonication Buffer , followed by sonication at optimized conditions . The sonication buffer was replaced with PBST, employing centrifugal concentration, as described above.In xxChIP, the once-fixed frozen cell pellets were dispersed in a buffer reminiscent of the permeabilizing buffer used in immunostaining reactions  for 20 min at RT. After PBS washes, the permeabilized cells were suspended in PBST + 5% IgG-free BSA (PBSTB) and rotated for 90 min at RT. To 300-µl aliquots containing ~ 6 × 103 + 1% SDS, tumbling for 15 min at RT. After centrifugal recovery, a second elution with 50 µl was performed, yielding ~ 100 µl of pooled eluate. The IP eluates were digested with RNAse and proteinase K, overnight at 65 °C. DNA was purified employing Sigma Gene Elute (NA1020-1KT). By contrast, xxChIP preparations, having the anti-H1 antibodies already bound and crosslinked to the chromatin fragments and in PBSTB buffer, were “cleaned” on the control agarose minicolumns, an aliquot removed for “Input” and the remainder incubated with Protein A/G agarose minicolumns overnight, rotating at 4 °C, followed by washing to remove unbound chromatin. Elution of the bound chromatin fragments and DNA purification were similar to the xChIP procedures.Of necessity, the immunoprecipitation (IP) protocols differed slightly, comparing xChIP to xxChIP. The xChIP preparations in PBSTB buffer were incubated with control agarose  and recovered from the minicolumns by centrifugation at 4 °C. Samples of these “cleaned” sonicates were retained as “Input”. Simultaneously, Protein A/G agarose minicolumns, equilibrated in PBSTB, were incubated for 4–5 h with 4 µg of rabbit anti-histone H1.2 or H1.5, rotating at RT, followed by washing with PBSTB. The equilibrated and “cleaned” sonicates were incubated with the antibody-bound Protein A/G agarose minicolumns overnight, rotating at 4 °C. Subsequently, the sonicate-bound columns were washed 5 times with PBSTB and 2 times with PBST, to remove unbound chromatin. Elution of the bound chromatin fragments was accomplished by addition of 50 µl of 100 mM NaHCOintersectBed and shuffle [xChIP-seq and xxChIP-seq with antibodies against histone H1.2 and H1.5 were each performed in triplicate. Paired-end sequencing was conducted by the sequencing facility of the German Cancer Research Center (DKFZ) using Illumina HiSeq 2000 and processed with manufacturer’s software HCS 2.2.58 and RTA 1.18.64. xChIP and xxChIP sequencing data were aligned to the human genome hg19 using Bowtie2  allowing shuffle  as a rat shuffle . The proThe whole-genome bisulfite sequencing reported in our recent study  is availPrevious studies , 24 haveCurrently, analysis of the genome-wide distribution of DNA-binding proteins  is frequently performed by chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq). Usually the intact cells are fixed once with formaldehyde, followed by sonication to nucleosome-size fragments and subsequent IP (xChIP). However, information about the influence of in situ chromatin higher-order structure on protein (epitope) distribution is largely lost during the sonication step, which precedes the IP step. We developed a modified ChIP-seq protocol to preserve information about the influence of in situ chromatin higher-order structure on protein (epitope) distribution. The method involves two formaldehyde fixation steps: the first, of intact cells; the second, following permeabilization and antibody binding, but before sonication, IP and DNA sequencing (xxChIP-seq) . A schemEmploying xChIP-seq and xxChIP-seq with rabbit anti-histone H1.2 and H1.5 antibodies, we determined genome-wide distributions of these signals and using peak calling software MUSIC  determinFigure As it is clear from Fig. Next, we analyzed the genome-wide distribution of regions enriched with xChIP and xxChIP H1 signals in relation to different genomic features defined using our previous ChIP-seq of histone modifications in HL-60/S4 cells (25). Figure The apparent occlusion of H1 epitopes, due to the preservation of chromatin higher-order structure surrounding various chromatin protein-binding sites, is presented in Fig. Figure We then analyzed xChIP profiles around “open” chromatin regions in general. Figure Figure NRL is defined as the average distance (bp) between the dyad axes of adjacent nucleosomes and is traditionally used as an integrative parameter characterizing local nucleosome packing. Previous publications suggest that NRL is different near binding sites of transcription factors  and affeTo investigate the relationship between linker histones and DNA methylation, we performed whole genome bisulfite sequencing profiling in HL-60/S4 cells . From ouFigure 3 or more nucleosomes (corresponding to ~ 2 × 105 or more bp). The peaks of H1 enrichment that we identify by xxChIP-seq are much smaller , together with traditional xChIP-seq Fig.  to study3, togethOur results indicate that H1.2 xChIP-seq and H1.5 xChIP-seq signals are positively correlated; H1.2 xxChIP-seq and H1.5 xxChIP-seq signals are also positively correlated; but H1 xChIP-seq versus H1 xxChIP signals are negatively correlated Fig. a, b. TheIt is important to emphasize that all types of transcriptionally active or “open” regions such as CpG islands were more enriched by H1 xChIP than xxChIP, whereas Alu and L1 repeats were more enriched with H1 xxChIP Fig. . EpichroSeveral chromatin-bound proteins exhibited clear preferences for “association” with regions enriched in different histone H1 variants Fig. . In partThe investigation of the relationship between active/inactive gene promoters and H1 binding revealed an unexpected observation. The depletion of H1.2 and H1.5 determined by xChIP is quite narrowly localized within ~ 200 bp of the active (but not inactive) TSS. This is consistent with the recently reported H1 xChIP-seq profiles in Drosophila embryonic development . On the Our analysis also indicates some changes of nucleosome packing, characterized by an NRL change from 190 to 184 bp for the areas enriched with H1.2 versus H1.5, respectively (Fig. Given the acknowledged high in vivo mobility and structural redundancy of different H1 variants, the presently described localizations and nuclear element enrichments of H1.2 and H1.5 cannot be regarded as universal to other mammalian cells. Since undifferentiated HL-60/S4 cells are the object of interest within this study, the next logical step could be to examine H1 xChIP-seq versus xxChIP-seq in the differentiated granulocyte and macrophage cell states , to idenAdditional file 1: Additional Figures.Additional file 2: Additional Table S1.Additional file 3: Additional Table S2.


TERT changes such as gene amplifications, TERT structural variants, TERT promoter germline and somatic mutations, TERT epigenetic changes, and alternative lengthening of telomere. All of them are cancer specific at tissue histotype and at single cell level. TERT expression is regulated in tumors via multiple genetic and epigenetic alterations which affect telomerase activity. Telomerase activity via TERT expression has an impact on telomere length and can be a useful marker in diagnosis and prognosis of various cancers and a new therapy approach. In this review we want to highlight the main roles of TERT in different mechanisms of cancer development and regulation.Telomerase reverse transcriptase (TERT) is a catalytic subunit of telomerase. Telomerase complex plays a key role in cancer formation by telomere dependent or independent mechanisms. Telomere maintenance mechanisms include complex  In most human cancers, telomerase is reactivated during carcinogenesis by expression of the catalytic subunit telomerase reverse transcriptase (TERT). TERT plays a key role in cancer formation, ensuring chromosomal stability by maintaining telomere length, and allowing cells to avert senescence. It constitutes a limiting factor for formation of the telomerase complex in cancer cells . TERT isTERT gene is situated at chromosome 5p15.33 in humans, and is an integral and essential part of the telomerase holoenzyme. TERT gene is 42 kb long and consists of 15 introns and 16 exons with a 260 bp promoter core (TERT promoter (TERTp) region contains GC boxes that bind the zinc finger transcription factor Sp1, which increases TERT transcription, and E-boxes that bind both transcriptional enhancers and repressors. TERTp lacks a TATA box but it contains binding sites for many different transcription factors (The  factors .Another major component of the telomerase complex is telomerase RNA component (TERC). It is an RNA sequence, which functions as a template for synthesis of telomeres by TERT. These two main components of telomerase are accompanied by a host of auxiliary proteins, including dyskerin (DKC1), telomerase Cajal body protein 1 (TCAB1), non-histone chromosome protein 2 (NHP2), nucleolar protein 10 (NOP10), glycine arginine rich 1 (GAR1), heat shock protein 90 (HSP90) and serine and arginine rich splicing factor 11 (SRSF11) . This coTERT gene rearrangements and TERT and TERC gene amplification, (2) TERTp somatic mutations, (3) epigenetic alterations, (4) transcription factor binding, (5) polymorphic variants within TERT gene body and TERTp, and (6) alternative splicing . They maintain telomere length by reactivating telomerase, and therefore are known as TERT canonical functions . Individsplicing Figure 1Approximately 10–15% of tumor cells acquire immortality through a telomerase-independent mechanism, namely alternative lengthening of telomeres (ALT) . On the TERT gene copy number and are a known TMM. They may involve insertion of active enhancers close to the TERT gene and increasing TERT expression. A common process is TERT amplification, which can arise from telomere dysfunction . They are also referred to as C228T and C250T, respectively  are characterized by low proliferative potential  is the most frequent. This mutation leads to increased GABPA-GABPB complex formation and activation of TERT expression  is another example of genetic alterations interacting with TERTp. Its mutation is well described in urothelial carcinoma    . These sH2R172K) . Accordi gliomas . A previpatients .TERTp mutation status, alone or in combination with mutations in other genes, can be used to characterize distinguish various types of tumors, as well as predict prognosis and outcome. While TERTp mutation status appears to significantly impact cancer development, some cancers, such as prostate, lung, breast, colorectal, and hematological malignancies display telomerase activity, even though they contain few TERTp mutations . It is located distal to the TSS and is composed of 52 CpG islands  in heterozygous cells , has not been expensively studied. miRNAs are a class of small non-coding RNAs (~22–24 nucleotides) (Most recent studies focus on eotides) . miRNA reotides) 2. miRNAeotides) , 80.TERT. In particular let-7g-3p, miR-128, miR-133a, miR-138-5p, miR-498, miR-541-3p, and miR-1182, downregulate expression of TERT and telomerase activation  transcriptional factor called c-MYC  binding sites that are necessary for TERT expression. Sp1 belongs to the family of nuclear proteins called Sp/KLF (specificity protein/Krüppel-like factor) that binds GC-(GGGGCGGGG) and GT-(GGTGTGGGG) rich elements  significantly decrease transcriptional activity of TERTp, suggesting that Sp1 protein is involved in TERT transcription  and tumor necrosis factor alpha (TNF‐α) that are critical for inflammation and cancer progression  family of cytoplasmic proteins are direct mediators of signaling from the extracellular environment to the nucleus . Seven STERT expression in tumor and normal cells  have been observed to regulate promoter activity in human rhabdomyosarcoma cells (The adipocyte protein 2 (AP-2) family of transcription factors contains five isoforms: AP-2α, AP-2β, AP-2γ, AP-2δ, and AP-2ϵ , 110. Thelopment . AP-2β sTERT expression  may be a new potential activator of pression . It is npression . It was pression , and waspression . An anald cancer .TERT transcription, such as MAD1/2, p53, WT1, CTCF, and MZF-2.Transcriptional repressors are proteins that attach to DNA at specific silencer sites and block transcription of nearby genes. In the following section, we are going to briefly discuss repressors that have been shown to downregulate TERT expression because they bind to the same promoter sites (E-box) to activate TERT expression  tumor suppressor (TERTp (−352 upstream of the TSS), and its mutation significantly reduced telomerase activity and TERT mRNA expression in 293 embryonic kidney cells but not in HeLa cells  is a zinc finger transcription factor which is ubiquitously expressed in human . Its binse cells . Hypermese cells . Interesse cells .2H2 zinc finger protein expressed predominantly in myeloid progenitor cells and involved in growth, differentiation, and tumorigenesis -2 is a Krüppel-like Cigenesis . The mec unclear . There a of TERT , 137.TERT, as well as in TERTp. Some common TERT SNPs found may modify survival and prognosis of certain cancers. A number of studies have recently been conducted to identify new SNP loci related to telomere length, which have shown a relationship between the risk of disease, its severity and the survival time in various cancers (TERT polymorphisms that may be associated with gene expression.Single nucleotide polymorphisms (SNPs) have been described as being associated with increased risk for developing various cancers. They may be located both in intronic and exonic sequences of  cancers –140. In TERT gene, rs2736100 A>C is a important non-coding SNP  haplotype are major factors predisposing to MPN  polymorphism named MNS16A  and long (L). The L allele correlates with higher promoter activity in the antisense strand and increased expression of the antisense TERT transcript. This increased expression of antisense TERT leads to silencing of functional TERT  and 2 (ND2) and protects mtDNA from environmental damage  it contributes to cancer formation and progression. There are many regulatory mechanisms involved in telomerase reactivation and adjustment of TERT expression, among which TERTp mutation is perhaps the most important. Other major TERT regulation mechanisms  are: chromosome rearrangements, methylation, miRNA interference, binding of transcription factors, genetic polymorphism, and alternative splicing. Some of these mechanisms may interact with each other, having a synergistic effect on TERT expression. Aside from the better-known telomere lengthening function, TERT also has many secondary, telomere-independent roles . Taking in to account its major importance in cancer, TERT has become a target of various therapeutic strategies in cancer treatment and continues to be an interesting object of research.The following features of TERT described in this manuscript can be highlighted:TERT is a functional catalytic protein subunit of telomerase, which lengthens telomeres by adding short DNA repeats, consequently averting chromosomal instability;Its regulation is a multifarious process where both transcriptional and posttranscriptional mechanisms are involved;TERT is also a major component of various oncogenic signaling pathways, and its overexpression often contributes to tumorigenesis;TERT gene is often overexpressed in cancers, and this overexpression can be induced by a variety of mechanisms, such as: TERT gene amplification, TERT gene polymorphism, TERTp mutation and methylation, and miRNA interference, alternative splicing of the TERT;Aside from its primary nuclear localization, TERT can also be transported to cytoplasm and mitochondria;It has many non-canonical, i.e. telomere-unrelated, functions these include: interaction with signaling pathways, stress protection, regulation of chromatin structure, binding to and protection of mitochondrial DNA;TERT and its gene may also act as an attractive target for therapeutic interventions with a diagnostic and prognostic impact.MD and KB-K contributed to the conception and design of the review, drafted, and finalized the manuscript. BW contributed to the conception and design of the review and its draft version. PŁ contributed to the writing, reviewing, and editing of the final version of the manuscript. TK contributed to the final version of the manuscript and drew all the figures. All authors contributed to the article and approved the submitted version.This work was supported by the TARGETTELO project No. STRATEGMED3/306853 from the National Centre for Research and Development, Warsaw, Poland.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A newly identified class of miRNAs termed nuclear activating miRNAs (NamiRNAs), transcribed from miRNA loci that exhibit enhancer features, promote gene expression via binding to the promoter and enhancer marker regions of the target genes. Meanwhile, activated enhancers produce endogenous non-coding RNAs  to activate gene expression. During chromatin looping, transcribed eRNAs interact with NamiRNAs through enhancer-promoter interaction to perform similar functions. Here, we review the functional differences and similarities between eRNAs and NamiRNAs in myogenesis and disease. We also propose models demonstrating their mutual mechanism and function. We conclude that eRNAs are active molecules, transcriptional regulators, and partners of NamiRNAs, rather than mere RNAs produced during enhancer activation. Caenorhabditis elegans in 1993 [The identification of lin-4 miRNA in  in 1993 , and the in 1993 , servingAlternatively, the eRNAs are small non-coding RNA (ncRNA) transcribed by RNA polymerase II (Pol II) from enhancer loci in a similar way as NamiRNA . eRNAs aThe discovery of eRNAs and NamiRNAs has paved a new path in modern cellular genomics, but the differences and similarities in their mechanisms remain unsolved. Here, we review the regulatory effects of NamiRNAs and eRNAs in cellular transcription and their repercussions in myogenesis, diseases, and therapeutics.The biogenesis of eRNA and NamiRNAs may co-occur due to their nuclear associated molecular activities in the nucleus. They are transcribed from enhancers and NamiRNAs from miRNA coding genes. However, they end up being close to target enhancers and genes due to chromatin looping and gene activation. NamiRNAs trigger the transcription of eRNAs, and subsequently, the transcribed eRNAs and the NamiRNA activate the target gene and enhancer.The first enhancer (~ 72 bp) was cloned out of simian virus 40 (SV40), which is associated with an increase in expression of the human β-globin gene . EnhanceeRNAs are transcribed from an active enhancer by Pol II before transcription Fig. . NamiRNAThe cleaving terminates transcription and unbinds eRNAs from Pol II, decreasing the transcript’s population at both strands . eRNA trNuclear AGO2 (nAGO2) and RNAi factors like Dicer and TRBP are proposed to process NamiRNAs in the nucleus with a similar mechanism as the canonical miRNA’s biogenesis . During Alternatively, NamiRNA may be produced in the canonical miRNA processing manner. Some pre-miRNA may escape the nuclear Ago2, Dicer, TRBP, and PACT and move to the cytoplasm with the transporter protein Exportin 5 through the nuclear pore. Upon reaching the cytoplasm, the pre-miRNA is cleaved into ds-miRNAs by the cytoplasmic Dicer-TRBP/PACT complex. The resulting ds-miRNA unwinds and releases a single strand to bind to AGO2. Together with AGO2, this strand binds to other proteins to form a RISC . HoweverUncertainties seldom occur during eRNA transcription. However, diminution of the integrator subunits causes a decrease in eRNA’s signal-dependent induction and retracts stimulus-induced enhancer-promoter chromatin looping. These increase the spreading of GRO-seq reads throughout the body of eRNA transcripts at both enhancers and super-enhancers (SE) but make it difficult to detect . Also, sThere are some uncertainties related to the biogenesis of NamiRNAs. For example, Dicer-involved proteins, such as TRBP, can change the cleavage site of Dicer within some pre-miRNAs  . Like prIn the nucleus, eRNAs either activate or repress their target genes, and therefore their dysregulation affects transcription. For example, knockdown of specific eRNAs reduces its target genes’ expression, inferring their transcription roles . AlternaDRReRNA; an eRNA transcribed from a distal enhancer of MyoD1 controls the expression of MyoD via a cis mechanism  but not the promoter region . Such siMyoD enhances the expression of typical enhancers-associated eRNAs (teRNA) and super-enhancer RNA (seRNA) in the myoblast [Master TFs are examples of genes with multiple eRNA-assigned regions . The dismyoblast . These emyoblast . Similarmyoblast .Yin-Yang (YY)1 is a TF that regulates several genes’ transcription. It is recruited by regulatory element RNAs associated with promoter and enhancer sequences . When haNamiRNAs have enhancer signatures  and activate gene transcription as enhancer triggers . Over 40Moreover, NamiRNA enhancer activation alters some of the enhancer properties, such as affecting transcription and chromatin looping when forming a complex with nAGO2 and Pol II and recruiting p300 to catalyze H3K27ac at activating enhancers . For insNamiRNAs mainly orchestrate target enhancers and gene activation . The humDuring TGA, NamiRNAs recruit an nRISC-like structure (containing up to 7 nt)  and bindCaenorhabditis elegans, which binds to its pri-miRNA to form a positive feedback loop during its processing [NamiRNAs are stored and exist as pri-miRNA and mature miRNAs in the nucleus  and can ocessing . The mouocessing . This coocessing  and posiocessing , which aocessing . The miRocessing , but it CERNA) and associated enhancer expression. Alternatively, CERNA activates MyoD expression in cis and DRReRNA  in trans induces MyoG transcription and muscle differentiation  members, including myogenic factor 5 (Myf5), MyoD, MyoG, and myogenic regulatory factors MRF4), are responsible for orchestrating myogenesis. Other myogenic genes such as Paired box gene 3 (Pax3) and Paired box gene 7 (Pax7) mark the presence of these muscle progenitors . Though , are resCERNA and DRReRNA enhance skeletal muscle cell differentiation and Pol II residency at MyoD and MyoG loci, respectively [DRReRNA exhibits enhancer function in early differentiation while CERNA is transcribed in proliferating myoblasts [DRReRNA in trans via interaction with cohesin complex activates MyoG expression and the rest of the myogenic gene regulatory network without influencing MyoD transcript levels [DRReRNA recruits nascent transcripts of MyoG and associates with protein complexes involved in eRNA biogenesis, such as Integrator and WD Repeat Domain 82 (WDR82) [DRReRNA via the formation of RNA:RNA interaction or via an RNA/DNA triple helix at the MyoG locus recognizes target intronic regions of the MyoG nascent transcripts [DRReRNA then represses the expression of MyoG in trans. Simultaneously, DRReRNA safeguards proper cohesin loading in trans to regulate gene expression and increase chromatin accessibility. A transcribed eRNA from an enhancer region on mouse chromosome 7 (eRNA) recruited cohesin to regulate the MyoG gene’s transcription on chromosome 1 [ectively  Fig. 4)CERNA andyoblasts . During t levels . DRReRNA (WDR82) . Followinscripts . After smosome 1 .Moreover, NamiRNAs enhance some myogenic genes in their myogenic activities. For example, the conserved miR-675-3p and miR-675-5p encoded by exon 1 of H19 empowers H19 to enhance myogenic differentiation . The forCERNA and DRReRNA, some lncRNAs regulate myogenesis [Apart from ogenesis –80 via oogenesis . For exaogenesis . The musogenesis . LincRNAogenesis .eRNAs and NamiRNAs work simultaneously to activate target genes in this proposed model. Chromatin looping and high alteration of chromatin structure before replication proximate target genes and enhancers of both NamiRNA and eRNA, inducing an interaction between the enhancers and genes of eRNAs and NamiRNA. NamiRNAs target the enhancer promoters and subsequently activate them, leading to the transcription of eRNAs . NamiRNANamiRNAs and eRNAs located on different chromosomes may interact and perform similar functions. For example, miR-17 from miR-17HG interacts with NET1e, an eRNA transcribed from a close enhancer of the NET1 gene to induce drug resistance Table . OverexpMoreover, NamiRNAs and eRNAs contribute to cancer drug resistance through enhanced drug efflux, altered drug metabolism, and enhanced anti-apoptosis pathways . In tumoRecently, SEs, enhancers, and eRNAs have been used as factors for analyzing, mapping, and studying a broad range of conditions, including autoimmunity , cancer Moreover, eRNAs and NamiRNAs explore autophagy, apoptosis, and signaling pathways to regulate diseases. For instance, overexpression of the NamiRNA miR-378/378 enhances autophagy and represses apoptosis by targeting caspase 9, while the opposite reduces autophagy and accumulates abnormal mitochondria, and enhances apoptosis. These miRNAs target the rapamycin (mTOR)/unc-51-like autophagy activating kinase 1 pathway to inhibit apoptosis, and target phosphoinositide-dependent protein kinase 1 to maintain autophagy via Forkhead box class O (FoxO)-mediated transcriptional reinforcement . AlternaNamiRNA and eRNA are the next options in diagnosing, treating, and studying the pathogenesis of diseases that are not associated with current biomarkers . People CERNA, and DRReRNA effectively regulate myogenic genes and factors during myogenesis (Fig. Briefly, NamiRNAs activate enhancers to initiate eRNA transcription. The transcribed eRNAs then interact with NamiRNA through chromatin looping to orchestrate the expression of their target genes via similar TFs and mechanisms such as eRNA-NamiRNA dual gene activation, enhancer-promoter interactions, chromatin looping, and signaling pathways. eRNAs and NamiRNA use a similar tool in their activities to associate with enhancers and their target genes, which gives them functional similarities concerning their association with myogenesis Table , myopathsis Fig. . Hence, The building of data portals for biomolecular markers such as eRic for eRNAs  can be uSome current challenges associated with eRNA and NamiRNAs need to be explored to understand these molecules better. Firstly, most proposed models have not been tested; hence the field requires more research. Some enhancers lack H2K27ac; this results in Pol II biased transcription; therefore, a solution to this problem would be beneficial . Moreove


The Elongin complex is thought to be required for transcriptional induction in response to cellular stimuli and to ubiquitinate RNAPII in response to DNA damage. Yet, the impact of the Elongin complex on transcription in vivo has not been well studied. Here, we performed comprehensive studies of the role of Elongin A, the largest subunit of the Elongin complex, on RNAPII transcription genome-wide. Our results suggest that Elongin A localizes to actively transcribed regions and potential enhancers, and the level of recruitment correlated with transcription levels. We also identified a large group of factors involved in transcription as Elongin A–associated factors. In addition, we found that loss of Elongin A leads to dramatically reduced levels of serine2-phosphorylated, but not total, RNAPII, and cells depleted of Elongin A show stronger promoter RNAPII pausing, suggesting that Elongin A may be involved in the release of paused RNAPII. Our RNA-seq studies suggest that loss of Elongin A did not alter global transcription, and unlike prior in vitro studies, we did not observe a dramatic impact on RNAPII elongation rates in our cell-based nascent RNA-seq experiments upon Elongin A depletion. Taken together, our studies provide the first comprehensive analysis of the role of Elongin A in regulating transcription in vivo. Our studies also revealed that unlike prior in vitro findings, depletion of Elongin A has little impact on global transcription profiles and transcription elongation in vivo.Elongin is an RNA polymerase II (RNAPII)-associated factor that has been shown to stimulate transcriptional elongation  The synthesis of mRNA by RNA polymerase II (RNAPII) requires the highly orchestrated action of a large cohort of factors. During the transcription cycle, general transcription factors recruit RNAPII to promoters to initiate transcription, and the RNAPII complex pauses ∼50 to 100 nt downstream of transcription start sites (TSSs). Pausing is enforced by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor, which includes SPT4 and SPT5, as well as negative elongation factor. The release of paused RNAPII into productive elongation is mediated by positive transcription elongation factor b and cyclin-dependent kinase 12, which phosphorylates DRB sensitivity-inducing factor, negative elongation factor, and serine 2 (Ser2) on the C-terminal domain of the largest subunit of RNAPII , maintaiin vitro. Previous studies suggested that Elongin can stimulate RNAPII elongation in vitro, potentially by overcoming the transient pausing of RNAPII on a DNA template. However, the role of this complex during transcription elongation has not been tested in vivo.Several factors have been shown to mediate proper transcription elongation, including PAF1 complex, SPT6, and SPT5 , 8, 9. AThe Elongin complex is a trimer consisting of Elongin A, B and C, and Elongin A is the largest subunit. The N-terminus of Elongin A shares homology with elongation factor transcription elongation factor IIS, and the C-terminus of Elongin A contains a conserved motif, BC-box, that allows the Elongin B and C subunits to bind. Elongin A is the transcription-stimulatory subunit, and Elongin B and C can enhance the activity of Elongin A , 11. Apain vitro studies more than 2 decades ago. Since then, several studies have been performed to investigate the role of Elongin in vivo. It was shown that Elongin A is required for transcriptional induction in response to multiple stimuli in mammalian cells . To overcome this obstacle, we used an alternative approach, cleavage under targets and release using nuclease (CUT&RUN) (B) and immunoglobulin G (IgG) controls. CUT&RUN allowed for a robust signal-to-noise ratio, resulting in the reproducible identification of 8720 Elongin A binding peaks (A). After comparison with Refseq annotations, we found that these peaks corresponded to Elongin A occupancy of 2064 genes. Molecular pathway analysis of Elongin A occupied genes showed that ribosomal proteins, mRNA processing, ciliary, and VEGF signaling pathway proteins, as well as translation factors are among the top enriched categories (C).We first attempted to identify Elongin A binding sites genome-wide by ChIP-seq. However, the enrichment was not efficient, and after peak-calling, we identified 186 peaks A. To oveCUT&RUN) , 20. To CUT&RUN) B and immng peaks A. After tegories C.A and Fig. S1D). We found that ChIP-seq peaks were also observed over some regions that exhibited strong Elongin A enrichment in CUT&RUN experiments . Metagene analysis of annotated Refseq genes suggests that Elongin A is enriched at TSS regions, gene bodies, and transcript end sites (TESs) . Interestingly, we found that we were more likely to observe Elongin A enrichment on gene bodies and regions downstream of the TES on short genes, whereas on longer genes, we were more likely to observe Elongin A enrichment only at TSS-proximal regions . These results suggest that Elongin A may only travel with the RNAPII complex up to a certain distance downstream of TSS or that Elongin A has more important roles near TSS. Interestingly, when we plotted Elongin A CUT&RUN signal in the immediate vicinity of the TSS, we found that Elongin A enrichment is stronger immediately upstream of the TSS than downstream . Our analysis also suggested that Elongin A enrichment correlated with gene expression levels. We ranked Refseq genes based on their expression levels (fragments per kilobase per million mapped reads [FPKM]) and divided them into four groups. We then quantified average Elongin A enrichment on each group of genes and found that genes with higher expression levels tended to show stronger Elongin A enrichment . Plotting individual genes on heat maps likewise showed a clear correlation between gene expression and Elongin A enrichment . We also ranked Refseq genes on heat maps based on their Elongin A enrichment and simultaneously plotted enrichment of RNAPII on individual genes. The results suggest that genes with greater Elongin A enrichment also tend to be more highly transcribed . Our results therefore provide the first genome-wide maps of Elongin A occupancy and indicate a clear correlation between the binding of this factor and gene expression levels. These observations are consistent with results from an independent study , and this observation prompted us to further investigate these binding events. First, we investigated whether these regions were actively transcribed by analyzing nascent transcription genome-wide using a pulse of 4-thiouridine (4sU) and high-throughput sequencing (4sU-seq). Interestingly, many of the intergenic peaks bound by Elongin A also exhibited prevalent nascent transcription , and at some regions, the transcription activity appears to be bidirectional (A). Thus, we speculated that these regions could be active enhancers. We also compared these intergenic peaks with RNAPII ChIP-seq data and observed strong enrichment for Pol II over these peaks (B–D). Because ChIP-seq data are not publicly available for DLD1 cells, we took advantage of ChIP-seq data for H3K27ac and H3K4me1 in HCT116, another widely used colon cancer cell line, available through ENCODE, because enhancers tend to be conserved in a lineage-specific and cell type–specific manner. We compared these two enhancer marks with intergenic Elongin A peaks and observed strong enrichment for H3K27ac and H3K4me1 near these intergenic peaks . In contrast, a repressive mark, H3K27me3, did not show any enrichment at these peaks . Although DLD1 cells do not have enhancer annotations available, our results suggest that the intergenic Elongin A is recruited to potential enhancer regions and that Elongin A may participate in the transcription of enhancer RNAs (eRNAs). Importantly, independent studies in mouse embryonic stem (ES) cells, which have well annotated enhancers, confirmed that Elongin A localizes to enhancers . We confirmed a subset of interacting proteins by immunoprecipitation and Western blotting . Consistent with previous studies, we identified RNAPII subunits and found that a large number of peptides originated from proteins related to transcription elongation and RNA processing. Among the top hits, we identified nearly all subunits of the PAF1 complex, except for RTF1, which does not stably associate with mammalian PAF1 . We first performed RNAPII ChIP-seq in control and Elongin A–depleted cells, and we monitored total RNAPII as well as Ser5- and Ser2-phosphorylated forms. Ser5 first appears during the initiation phase at the TSS and is most strongly enriched over early gene bodies, whereas Ser2 phosphorylation correlates with elongating polymerase and is more highly enriched on gene bodies . When we ranked Refseq genes based on their Elongin A enrichment, we found that genes with higher Elongin A occupancy also exhibited stronger reductions in phosphorylated RNAPII . We also observed a similar pattern at intergenic Elongin A peaks (E).Given that Elongin A associates with RNAPII and a large group of elongation/processing factors, we asked how Elongin A modulates RNAPII transcription after successfully depleting Elongin A with an siRNA pool A. We fire bodies . AlthougFig. 3B, , B–C. WhFig. 3C, D. We als A peaks E.B) in Elongin A–depleted cells, and (2) Elongin A is highly enriched near the TSS, we examined the relationship between Elongin A enrichment and RNAPII pausing. To address this, we used RNAPII ChIP-seq data to calculate the “pausing index,” defined as the ratio of promoter signal to gene body signal. We then ranked, and divided into four groups, all Elongin A–bound genes based on the levels of factor recruitment and compared Elongin A enrichment with the pausing index. We found that genes with stronger Elongin A enrichment tended to have a higher pausing index (D), consistent with independent observations in mouse ES cells (E). These results suggest that Elongin A may participate in the release of paused RNAPII into a productive elongation complex.Given that (1) we observed strong reductions in ChIP-seq experiments detecting enrichment of Ser2-phosphorylated, but not total, RNAPII  B in Elonng index D, consisES cells . Next, wES cells E. These A, A), as 264 genes were upregulated, and 257 genes were downregulated. To monitor nascent transcription, we also performed RNA-seq using a short 4sU pulse (10 min). Analysis with 4sU-seq suggested that, overall, there were no significant or consistent changes in nascent transcription profiles in Elongin A–depleted cells as compared with controls . In addition, we did not observe significant changes in nascent transcription levels near the TSS , suggesting that transcription initiation is not affected in the absence of Elongin A.We then asked whether loss of Elongin A alters the transcriptome and performed RNA-seq on control and Elongin A–depleted cells. Our analyses revealed that a relatively small group of genes exhibited a significant change  upon Elongin depletion Fig. 4A, A, as 264Fig. 4B, B. In addFig. 4C, C, suggesin vitro . Because DRB inhibits the release of paused RNAPII into a productive elongation complex, this method allowed us to track the clearance of existing elongating RNAPII complexes over gene bodies with time. We plotted the signal at each time point as a function of the 0-min time point (no DRB). Thus, a negative log2 value suggests decreased signal (clearance). Our analysis suggested that this method is robust, as we can clearly observe clearance of 4sU signal from the TSS with time (D–E). Although we observed modest delays in 4sU clearance at some early time points (5 min) upon Elongin A depletion , the effect did not appear to be dramatic. More importantly, we did not observe similar delays upon Elongin A depletion at other time points. . These results suggest that Elongin A does not significantly enhance RNAPII elongation rates, as it does under certain in vitro conditions . This enrichment is different from the canonical RNAPII profile, wherein RNAPII is skewed toward regions downstream of the TSS. This could suggest that Elongin A plays a more important role in antisense transcription or that Elongin A is loaded onto genes before RNAPII. However, we were not able to detect changes in antisense transcription or transcription directionality with our 4sU-seq data (data not shown). Our immunoprecipitation results suggest that Elongin A associates more robustly with RNAPII–Ser5p than with RNAPII–Ser2p (A). However, RNAPII ChIP-seq results suggest that Elongin A–depleted cells show enhanced promoter RNAPII pausing and dramatically reduced RNAPII–Ser2p levels (B and E). In addition, we found that genes with stronger Elongin A enrichment also tended to have a higher pausing index (D). These results suggest that Elongin A is potentially involved in the release of paused RNAPII into productive elongation complexes. This is consistent with the fact that Elongin A is more highly enriched at the TSS than other genic regions. Accordingly, we observe strong reductions in the level of RNAPII–Ser2p genome-wide upon Elongin A depletion, whereas the levels of total RNAPII did not change significantly.Prior studies by Kawauchi et al.  showed uet al. F. This eII–Ser2p A. Howevep levels , B and Eng index D. These A), which is a feature of eRNA , and we also did not observe significant changes in nascent transcription levels or transcription initiation by 4sU-seq. Whether Elongin A is required for transcription induction in response to stimuli in vivo, however, needs to be further investigated.Previous studies suggested that Elongin A is required for transcriptional induction of certain genes in response to different stimuli. Here, we did not observe many differentially expressed genes upon Elongin A depletion in DLD1 cells under normal culture conditions. Even when we set the fold-change threshold to 1.5-fold, we only observed a small group of differentially expressed genes A, and weDLD1 cells were purchased from ATCC (CCL-221) and grown in RPMI (Corning) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. Elongin A siRNA was purchased from Horizon Discovery . All siRNAs  were delivered using Lipofectamine RNAiMax (Invitrogen) as per the manufacturer’s protocol. Cells were harvested 48 h after transfection for downstream analysis.ChIP was performed as previously described . BrieflyThe following antibodies were used for ChIP in this study: PolII-ser2p , PolII-ser5p , and PolII .et al.  was used for permeabilization and 1.5-μg Elongin A antibody or normal goat IgG was used in each reaction. The final DNA product was extracted with phenol–chloroform and used to make sequencing libraries using methods similar to ChIP-seq library preparation with 14 PCR cycles. The CUT&RUN libraries were sequenced by paired-end Illumina sequencing.CUT&RUN was performed as described by Skene et al.  with theThe following antibodies were used in CUT&RUN experiments in this study: Elongin A , rabbit anti-goat IgG , and goat IgG .2, 0.34 M sucrose, 10% glycerol, and 1-mM DTT) supplemented with 0.1% Triton X-100 and incubated on ice for 5 min. The lysate was then centrifuged at 4 °C, 1400g, for 5 min. The supernatant was discarded. The pellet (nuclei) was resuspended with buffer A supplemented with 1-mM CaCl2 and 5 U/μl MNase and incubated at RT for 30 min. MNase digestion was stopped by adding EGTA to a final concentration 1 mM, and the lysate was centrifuged at 4 °C, 1400g, for 5 min. The supernatant was discarded. The pellet was then resuspended in solution B  and incubated on ice for 30 min. The lysate was then centrifuged at 4 °C, 1700g, for 5 min, and the supernatant was collected and used for purification (immunoprecipitation). 1 mg of the chromatin lysate was used for each IP. Hepes, pH 7.6, NaCl, and NP-40 were added to the lysate at a final concentration of 25-mM Hepes, pH 7.6, 150-mM NaCl, and 0.1% NP-40. The lysate was precleared with protein A/G beads for 1 h at 4 °C and then incubated with Flag-M2 beads  for 4 h at 4 °C with rotation. Beads were then washed with a wash buffer  5 times. For mass spectrometry, beads were eluted with an elution buffer  supplemented with 2 mg/ml 3X Flag peptide for 45 min at 4 °C. For immunoblots, beads were eluted with Laemmli buffer and boiled for 5 min. All buffers were supplemented with protease and phosphatase inhibitors.WT DLD1 cells or DLD1 cells stably expressing Flag-tagged Elongin A were used for immunopurification of soluble chromatin. To extract the chromatin fraction, cell pellets were first resuspended in solution A , Flag , PAF1 , CTR9 , CDC73 (homemade), SPT6 , SPT16 , SPT5 , PolII , PolII-ser2p , and PolII-ser5p .Homo sapiens reference proteome database (downloaded 02/2019) containing common contaminant proteins using Proteome Discoverer 1.4.0.288, and 30,993 entries in the database were searched. Sequest HT search engine was used. The search parameters were as follows: precursor mass tolerance ± 10 ppm, fragment mass tolerance ± 0.02 Da, digestion parameters trypsin allowing two missed cleavages, fixed modification of carbamidomethyl on cysteine, variable modification of oxidation on methionine, deamidation on glutamine and asparagine. The threshold for accepting individual spectra was minimum peptide length of 6 and maximum peptide length of 144 with an signal to noise threshold (FT only) 1.5. The data were filtered using a 1% peptide and protein false discovery rate cut-off searched against a decoy database.Mass spectrometry analysis was performed at NYU School of Medicine Proteomics Laboratory. Eluted samples were reduced with DTT and alkylated with iodoacetamide before loaded on NuPAGE 4 to 12% Bis-Tris Gel (Life Technologies). Gel was run for 30 min at 200 V and stained with GelCode Blue Stain Reagent (Thermo Fisher Scientific). The whole lane was excised out and destained with 1:1 mixed methanol and 100-mM ammonium bicarbonate. Destained gel was then dehydrated and digested with trypsin before subjected to peptide extraction. Peptides were gradient eluted from the column directly to the Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific) using a 1-h gradient. High resolution of full mass spectrometry spectra was acquired with a resolution of 45,000, an automatic gain control target of 3e6, a maximum ion time of 45 ms, and scan range of 400 to 1500 m/z. After each full mass spectrometry, twenty data-dependent high-resolution higher-energy collisional dissociation mass spectrometry (HCD MS/MS) spectra were acquired. All MS/MS spectra were collected using the following instrument parameters: resolution of 15,000, automatic gain control target of 1e5, maximum ion time of 120 ms, one microscan, 2 m/z isolation window, fixed first mass of 150 m/z, and normalized collision energy of 27. MS/MS spectra were searched against the UniProt Saccharomyces cerevisiae RNA labeled with 4sU was used as a spike-in control. 100 μg mammalian RNA was mixed with 1-μg S. cerevisiae RNA and fragmented by adding ice-cold NaOH to the final concentration of 0.2 N and incubating on ice for 20 min. Fragmentation was stopped by adding an equal volume of 1 M Tris, pH 6.8, and RNA was cleaned up performing buffer exchange with P-30 Micro Bio-Spin column . Biotinylation of 4sU-labeled RNA was carried out in biotinylation buffer  supplemented with 0.2 mg/ml EZ-Link HPDP-biotin  at RT for 2 h with rotation. RNA was then purified by phenol:chloroform extraction, and biotin RNA was enriched by incubating RNA with 50-μl Streptavidin C1 beads  in a binding buffer  for 20 min at RT with rotation. Beads were then washed with a high-salt buffer  twice, binding buffer  twice, and a low-salt buffer  once. Biotin RNA was eluted twice by directly adding 300-μl TRIzol to beads. RNA was then extracted as per the manufacture’s protocol. Purified RNA was used to make sequencing libraries with a NEBNext small RNA library prep kit for Illumina  or NEBNext Ultra II directional RNA library prep kit for Illumina . Libraries were sequenced by paired-end Illumina sequencing.For nascent RNA sequencing experiments in this article, DLD cells were treated with or without 100-μM DRB  for indicated time. Nascent RNA was labeled by adding 1-mM 4sU  for 10 min. Labeling was stopped by adding TRIzol  directly to the cell culture plate, and total RNA was extracted as per the manufacture’s protocol. 100-μg total RNA was used for purification of nascent RNA. Paired-end reads were aligned to hg19 reference genome with Bowtie2 (v2.3.4.1)  with opthttp://amp.pharm.mssm.edu/Enrichr/)  , 33, andS. cerevisiae was used as a scaling factor. Differential expression analysis was performed using DESeq2.Paired-end reads were aligned to hg19 reference genome with STAR (V2.5.0c) . For 4sUTo plot metagene plots and heat maps, we selected Refseq genes that (1) have FPKM >5 in DLD1 WT RNA-seq and (2) have no overlapping transcripts within 5 kb upstream or downstream of the gene. 4544 genes were used for plotting. Metagene plot and heat map were plotted with deepTools (v3.1.0) . For ChITotal pol II ChIP-seq data were used to calculate the pausing index. We selected genes that (1) have an FPKM >5 in DLD1 WT RNA-seq, (2) are longer than 300 bp, and (3) have no overlapping transcripts within 1 kb upstream or downstream of the gene. 5939 genes were used for calculation. The TSS region is defined as 100 bp upstream to 300 bp downstream of TSS, and the gene body region is defined as 300 bp downstream of the TSS to TES. Pol II ChIP-seq reads within TSS and gene body were counted with bedtools (v2.27.1) “bedtools coverage” function, and FPKM was calculated. The pausing index was defined as log2[(TSS_FPKM +1)/(genebody_FPKM +1)].All next-generation sequencing data have been deposited to Gene Expression Omnibus under the accession number GSE145368. Mass spectrometry raw files are accessible under MassIVE ID: MSV000085926 and ProteomeXchange: PXD020796.The authors declare that they have no conflict of interest with the contents of this article.