Drosophila and mammals.Disruption of the PcG/TrxG expression is associated with many pathologicalconditions, including cancer, which makes them suitable targets for diagnosisand therapy in medicine. In this review, we focus on the major PcG and TrxGcomplexes, the mechanisms of PcG/TrxG action, and their recruitment tochromatin. We discuss the alterations associated with the dysfunction of anumber of factors of these groups in oncology and the current strategies usedto develop drugs based on small-molecule inhibitors.Polycomb group (PcG) and Trithorax group (TrxG) proteins are evolutionarilyconserved factors responsible for the repression and activation of thetranscription of multiple genes in  Drosophila as regulators of Hox genesexpression. Hox genes are responsible for proper bodysegmentation. Their baseline expression profile is determined by the proteinproducts of the maternal, gap,pair-rule, and segment polarity genes at theearly embryonic stage of development. These proteins activate each other in acascade-like manner . The cPRC1complex can compact chromatin and repress transcription .Kdm2 (KDM2B) is a histone demethylase, which removes the H3K36me2 modificationcharacteristic of active chromatin regions . In addition, the Pc (CBX)protein binds nucleosomes carrying the H3K27me3 modification, which iscatalyzed by PRC2 [62–64].The Sce/RING protein is a catalytic subunit of cPRC1 and ncPRC2 inDrosophilaand mammalian cPRC1 .Drosophila genome, there isno universal DNA-binding recruiter responsible for the binding of all of thePolycomb or Trithorax proteins to the chromatin. The existence of numerous PcGparalogs indicates the possibility of a wide variety of DNA-binding factors aswell. This, taking into account the tendency of DNA-binding factors topartially functionally substitute for each other, creates obstacles for theiridentification. We suppose that combinations of binding sites for differentDNA-binding proteins can play the primary role in the recruitment of thePcG/TrxG factors to the chromatin that form fairly extended regions for stablePcG/TrxG recruitment.Apparently, as in the case of the Epigenetic modifications Drosophila and mice does not lead toa significant loss of Polycomb-dependent repression . This mutation also results in an increased level of H3K27me3.Tumor-suppressive role of PRC2 LOF mutations in the PRC2 complex subunits. LOF(loss-of-function) mutations that disrupt PRC2 activity have been described inall three core components: EZH2, SUZ12, and EED . The EZHIPregion is considered to mimic the H3K27M structure and inhibit PRC2 activity ina similar way.Mechanisms of the oncogenic and tumor-suppressive PRC2 roles The mechanisms that underlie the opposite PRC2 roles in different types oftumors are currently being studied vigorously. In general, these differencesare driven by the PRC2-mediated suppression of either oncogenes or tumorsuppressors in different type of cells.EZH2 has been shown to enhance cellproliferation both in vitro [in vivo [EZH2 accelerate MYC-and BCL-2-mediated lymphomagenesis in mice [CDKN2Atranscription in prostate and endometrial cancer cells, as well as inlymphoid tumors .Overexpression of in vitro , 208 and[in vivo , 284. An[in vivo , cell in[in vivo . GOF mut in mice , 286. Thtails in ).It shoHOXA9and MYC are overexpressed in many types of tumors[EZH2 and EED interact with the Q61Kmutation of the NRAS oncogene and hyperactivate the STAT3signaling pathway, leading to acute myeloid leukemias [EZH2/RUNX1 or EZH2/p53 gene leads to the formation of therapy-resistant myeloidlymphocytic leukemias in mouse models [SUZ12 deletion interacts with the JAK3 factor mutation,leading to acute lymphoblastic T-cell leukemia [EZH2contribute to the development of a myelodysplastic syndrome that isinduced by mutations in RUNX1 [NF1 mutation in tumors of the peripheral nervous system:glioma and melanoma [NF1encodes a GTPase, which activates the ras gene; andmutations in this factor result in Ras-dependent activation of carcinogenesis[ASXL1 [The mechanisms of tumor-suppressive PRC2 action have been less studied.However, such PRC2 targets as, for instance, the oncogenes f tumors, 287. Ineukemias . The come models , 290. Aleukemia . Inactivin RUNX1 . The losmelanoma . NF1encogenesis. A loss s[ASXL1 , or upons[ASXL1  in leukeThus, depending on the mutations or changes in the expression of other genes,PRC2 inactivation can lead to malignant cell transformation , 297. CaSmall-molecule PRC2 inhibitors Fig. 5).The discovery of numerous abnormalities associated with PRC2 hyperactivitystimulated scientists to develop small-molecule inhibitors that suppress theactivity of this complex alsotargets the methyltransferase domain of EZH2. The activity of EPZ-6438 has beendemonstrated on malignant rhabdoid tumors (MRTs). These cells carry the mutant complex .Mutatio complex  and conf complex . Treatme complex .All of the described inhibitors were highly specific to EZH2 and much lessactive against EZH1. However, it had previously been shown that EZH1 canreplace EZH2 if the latter is damaged. Thus, in some cases, when using highlyspecific inhibitors that block the activity of EZH2, the PRC2 complex canretain partial activity thanks to EZH1. Therefore, a series of inhibitors werefurther developed to solve this problem.The inhibitors UNC1999 , OR-S1,High-throughput screening methods can be used to search for the novelsmall-molecule inhibitors of PRC2. For example, screening of approximately250,000 substances allowed scientists to identify 162 that are able to inhibitEZH2 .Determination of the spatial structure of the PRC2 complex allowed scientiststo develop a new approach to the creation of small-molecule inhibitors , 312.First, the SAH-EZH2 peptide, which mimics the EED region required forinteraction with EZH2, was synthesized . TreatmeSecond, a region in the EED protein that specifically interacts with theH3K27me3 modification and is important for the recruitment of PRC2 to chromatinwas used as a target. Two small-molecule substances have been developed: A-395and EED226. Their activity against lymphoid tumor cells is comparable to thatof EZH2 methyltransferase domain inhibitors , 316. ItThird, the hydrophobic tagging (HyT) method is used to suppress PRC2 activity.In this case, the chimeric molecule is created, one part of which binds to thetarget protein, and the other one directs the bound complex to proteasomedegradation . This mePolycombmutation. Tremendous progress has been made in the study of how thePcG/TrxG system functions. The global role of these factors in thetranscription regulation and maintenance of cellular homeostasis is becomingclearer. There is also a growing body of data concerning PcG/TrxG dysfunctionsin various pathologies. However, a number of questions needing to be addressedfor a more complete understanding of the system’s functioning remainopen. The details of PcG/TrxG complexe’s recruitment to specific genomicregions remain unclear. The exact contribution of various factors to theseprocesses, such as the activity of specific DNA-binding factors, epigeneticmarks, non-coding RNAs, as well as other unknown biological processes, has notyet been established. The increasing complexity of the PcG/TrxG system in theprocess of evolution from invertebrates to mammals and the emergence ofnumerous paralogs of these proteins represent a challenge for researchers: towhat extent the currently understood composition of protein complexes ischaracteristic of all types of cells? Are they unique only to a number oftissues and/or developmental stages and do they differ in others? Numerousrecent studies assign a crucial role to the spatial organization of genes inthe nucleus. These processes were also shown to be closely related to thefunctioning of PcG/ TrxG complexes. It is important to determine the extent towhich the spatial organization determines the functions of DNA regulatoryelements or whether it is a consequence of transcriptional complexes’recruitment to chromatin. Much needs to be done to further elucidate thesignificance of PcG/TrxG factors in medicine, in the development of improvedsmall-molecule inhibitors, and in the creation of optimal therapeuticprotocols. At the same time, despite the tremendous progress achieved in thestudy of PcG/TrxG proteins in mammals, the Drosophila remainsan indispensable model organism for studying the details of transcriptioncontrol by PcG/TrxG proteins.It has been more than 70 years since the discovery of the The emergence of biological methods  provides hope that many of thesequestions will be answered in the future. However, what remains quite clear isthat the long road in the study of PcG/TrxG factors is far from beingcompleted.


However, its role as a transcription factor in vivo has remained underexplored. Here we show that in mouse embryonic stem cells, EloA localizes to both thousands of Pol II transcribed genes with preference for transcription start site and promoter regions and a large number of active enhancers across the genome. EloA deletion results in accumulation of transcripts from a subset of enhancers and their adjacent genes. Notably, EloA does not substantially enhance the elongation rate of Pol II in vivo. We also show that EloA localizes to the nucleoli and associates with RNA polymerase I transcribed ribosomal RNA gene, Rn45s. EloA is a highly disordered protein, which we demonstrate forms phase-separated condensates in vitro, and truncation mutations in the intrinsically disordered regions (IDR) of EloA interfere with its targeting and localization to the nucleoli. We conclude that EloA broadly associates with transcribed regions, tunes RNA Pol II transcription levels via impacts on enhancer RNA synthesis, and interacts with the rRNA producing/processing machinery in the nucleolus. Our work opens new avenues for further investigation of the role of this functionally multifaceted transcription factor in enhancer and ribosomal RNA biology.Elongin A (EloA) is an essential transcription factor that stimulates the rate of RNA polymerase II (Pol II) transcription elongation  Transcription by RNA polymerase II (Pol II) is a multistep process, facilitated by a large number of auxiliary factors that modulate the activity of Pol II , 2. In ain vitro elongation activity , was discovered as a biochemical activity that enhances Pol II elongation rate by decreasing the frequency of Pol II arrest and transient pausing in run-off transcription assays , 14, 15.activity . In vivo stimuli , 19, 20. stimuli .in vitro  at a subset of these, as well as elevated levels of nascent RNA at adjacent genes. Furthermore, we show that EloA is associated with RNA polymerase I transcribed ribosomal RNA gene, Rn45s, and is present in the nucleoli. EloA is a highly disordered protein, and we demonstrate that purified EloA forms phase-separated condensates in vitro and that truncations within the highly disordered region interfere with targeting and localization of EloA to the nucleoli. Our work here uncovers new aspects of EloA function by showing its widespread association with genic and intergenic transcribed regions, its impact on eRNA synthesis, and its presence on rRNA genes and in the nucleolus.While the role of EloA in transcription elongation by RNA polymerase II has been examined in great detail in vitro , 15, 16,n domain . Here weEloA−/− mESCs. We characterized EloA protein levels in wild-type and EloA null mES cells by fractionation and by immunoblotting. We found that, as expected, EloA is predominantly a nuclear protein (A). While ChIP-seq and Cut&Run-seq peak calling algorithms both identified many of the robustly expressed genes as being bound by EloA, Cut&Run-seq offered a superior signal/noise ratio for the remainder of genes, allowing for a more comprehensive profiling of EloA binding sites (A–B).The association of EloA with Pol II transcribed genes has been tested at a small number of housekeeping and stress response genes in cells expressing tagged EloA . To gain protein A. While ng sites , A–B.FigB). We established that the EloA CUT&RUN-seq signal is specific by comparing it with that of EloA and IgG CUT&RUN-seq signals in null and wild-type cells, respectively (C). EloA was present at the majority of expressed genes , while 4464 expressed genes were not bound by EloA. Association of EloA at many highly expressed genes exhibited a bimodal distribution pattern, with one strong peak around the TSS/Pol II pausing site and another peak at the 3ʹ-end, downstream of cleavage/polyadenylation signal site (C–D). We grouped EloA-bound genes into three classes based on enrichment levels (D) and found a strong positive correlation with transcription activity of target genes, as determined by profiling nascent transcription by Bru-seq (E). These observations are consistent with an independent report in human DLD1 cells . Expressed genes enriched for EloA also had higher levels of histone modification marks associated with active genes in both the promoter regions  and gene bodies  (F). Molecular pathway analysis of EloA positive genes found that mRNA processing, translation, and pluripotency gene classes were enriched; all categories showing high expression in stem cells (G). We also examined nascent transcription by Bru-seq in wild-type and null EloA mESCs and found a small effect on gene expression with 679 upregulated and 198 genes downregulated in null cells (FC > 1.5) (C). We conclude that EloA binds to a large number of actively transcribed genes and that loss of EloA has a rather minor effect on gene expression under steady-state conditions, predominantly leading to upregulation of a subset of genes.CUT&RUN-seq of EloA identified 17,753 peaks  throughout the genome, more than 70% of which localized to different regions of annotated genes, with a notable bias toward the promoter region B. We estectively C. EloA wnal site , C–D. Wet levels D and fou Bru-seq E. These 3K79me2) F. Molecuem cells G. We alsC > 1.5) C. We conD–E). We also noted a moderate decrease in the levels of Pol II-Ser2P in EloA−/− cells (F).In mESCs, roughly half of actively transcribed genes have been shown to be regulated by Pol II pausing early in the transcription process . We also/− cells F.B) and asked whether these intergenic EloA binding sites show any overlap with annotated intergenic enhancers in mESC. Interestingly, out of 4573 intergenic EloA peaks, 40% (1841) overlapped with annotated enhancers, with 361 of these sites localizing to clusters of enhancers, commonly referred to as superenhancers (A) (B). Interrogation of the chromatin landscape at EloA-bound enhancers also revealed positive correlation between EloA levels and intensity of H3K27ac and H3K4me3 at these sites (B–C), both modification marks delineating active enhancers (B–C).Enhancers are mainly distal regulatory elements that harbor binding sites for transcription factors and which, irrespective of orientation, are capable of activating gene expression over large distances , 27.We wFig. 1nhancers A 28). W. WB) andcers (A) B. Interrse sites , B–C, bonhancers . Whereasnhancers , B–C.FigActive enhancers are transcribed and generate unstable, short-lived RNAs termed enhancer RNAs (eRNAs) , 30, 31.EloA null cells. Strikingly, the majority of these differentially expressed enhancer elements (76.6%) had elevated levels of eRNA transcription in EloA−/− mES cells  (D). In contrast only 3.6% of EloA-bound genic regions show differential expression after loss of EloA (225/6198) (G). The observed upregulation of transcription at EloA-bound intergenic loci was statistically significant for only annotated enhancers and not for other intergenic EloA-bound sites (E). We also noted that genes in the immediate vicinity (<20 kb) of these upregulated enhancers had higher levels of nascent transcription in EloA null cells (F). We asked whether these differentially expressed enhancers in EloA null cells are adjacent to any specific class of genes and performed Genomic Regions Enrichment of Annotations Tool (GREAT) analysis to address this. Genes adjacent to downregulated enhancers in EloA−/− did not show a statistically significant correlation with any GO biological process; however, positive correlation was observed between eRNA misaccumulation in EloA−/− mES cells and genes involved in maintenance of pluripotency as well as neural tube and embryonic development (G). In addition to enrichment of EloA at many of these distal intergenic enhancers, the CUT&RUN-seq identified EloA peaks at 469 out of 1912 intragenic enhancers described by Cinghu et al. (A–B). We conclude that EloA is recruited to both intergenic and intragenic enhancers and that its loss results in increased eRNA nascent transcription at several of these sites as determined by Bru-seq. This increased eRNA synthesis correlated with upregulation of adjacent protein coding genes.Interestingly, our analysis of nascent RNA transcription at EloA-bound annotated enhancers revealed misregulation in eRNA levels at 40.8% (751/1841) of these sites in C > 1.5) D. In con25/6198) G. The obnd sites E. We alsll cells F. We askelopment G. In addu et al.  in mESC u et al. , A–B. Wein vitro , a potent P-TEFb inhibitor blocking Pol II C-terminal domain (CTD) Ser2 phosphorylation (A). We estimated the position where the read density of nascent transcription approaches background levels on the gene body to be 10–12 and 22–24 kb downstream of the TSS for the 5 and 10-min recovery samples, respectively . This corresponds to an elongation rate of 2–2.4 kb/min, which is consistent with previously reported Pol II elongation rates for genes in mESC ; however, we noted a discernable, yet modest (∼10%) decrease in the density of reads toward the “wave front” of transcription in the 5-min recovery sample. At 5 min the pioneering wave of Pol II appeared to be lagging by about 1 kb past the 10 kb mark in EloA−/− cells (B). A decrease in nascent RNA signal in null cells was also observed in the 10-min recovery samples (C); however, this decrease did not appear more prominent near the leading edge of Pol II transcription (∼+20 kb) but was more notable between the 5 and 10 kb mark. This latter phenotype is not consistent with a substantive role for EloA in modulation of Pol II elongation rate; a more striking difference toward the “wave front” of transcription would be expected. Rather, this suggests that in addition to a potential role in transcription elongation, EloA might also be acting to increase the efficiency of a step upstream of productive elongation such as release form pause, Pol II loading, or initiation. Further, a defect in Pol II processivity would be predicted to cause a drop in the density of transcribing Pol II toward the end of the gene. To examine this issue, we compared the TSS distal to TSS proximal steady-state nascent RNA levels for the same set of genes and did not detect a larger decrease in the density of nascent transcription toward the middle and end of the gene body in null cells (D–E). Figure 3F depicts movement of Pol II along the body of two representative genes, Eed and Dhx9, at various time points after transcription reactivation. From these experiments we conclude that the loss of EloA does not cause a significant global change in RNA polymerase processivity. Furthermore, the effect of EloA on Pol II elongation is limited and that the slight decrease in the density of pioneering Pol II wave upon transcripiton reactivation might be mediated at a stage upstream of productive transcription elongation. Our observation regarding the limited effects of EloA on Pol II elongation rate is consistent with an independent study in human DLD1 cells line .The density profile of nascent RNA (transcribing Pol II) immediately downstream of the TSS (+1 kb) at EloA-enriched genes was similar or even slightly higher in  B–C and C; howeve/− cells B. A decr samples C; howevell cells , D–E. FiC–D). To examine whether EloA plays role in Pol II termination, we also looked at the levels of nascent RNA up to 10 kb downstream of transcripiton end site and noted no difference in the density of Bru-seq signal (Pol II levels) between WT and EloA null cells (F).EloA also shows strong enrichment downstream of polyadenylation signal (PAS) , C–D. Toll cells F.A). As expected, EloA showed the strongest interaction with EloC and EloB, the other subunits of the heterotrimeric Elongin complex (A), as well as the putative RNA exonuclease Rexo1 (EloA-BP1), a previously reported major binding partner of EloA (A–B), all components of the multifunctional polymerase-associated factor 1 complex (Paf1C), which is involved in regulation of various stages of transcription by Pol II (C). This observation is concordant with recent reports where pull-down of Paf1C identified EloA as a major interacting partner of the complex in murine myoblast and ESC (A–B). There were also interactions between EloA and nucleolar proteins , a finding relevant to the studies of nucleoli we describe below. We found multiple components of the Xeroderma pigmentosum group A (XPA)-binding protein 2 (XAB2) complex , a multifunctional complex involved in splicing and transcription-coupled DNA repair among EloA interacting proteins .To better understand the biological processes that EloA may be participating in, we sought to identify the protein interaction network of EloA. We carried out coimmunoprecipitation mass spectrometry (Co-IP/MS) analysis on endogenous EloA from MEF-depleted mESC nuclear extract A. As exp complex A, as wel of EloA . Our res of EloA , A–B, aly Pol II . We valiy Pol II C. This o and ESC , 36. The and ESC , A–B. Thproteins . The IP Rn45s, a 45S coding gene on chromosome 17. The EloA CUT&RUN-seq signal was specific for wild-type mES cells and close to IgG background levels in EloA null cells, and we did not detect enrichment of Ser2P Pol II at this rRNA gene site (A). We carried out coimmunofluorescence microscopy on 3T3 fibroblast cells and observed colocalization between EloA and NOLC1 (Nopp140) (B), a nucleolar factor, present at the fibrillar center (FC) and dense fibrillar component (DFC), which are sites for rRNA transcription and processing, respectively (A–B).The 45S pre-rRNA is transcribed by RNA polymerase I (Pol I) and processed into 18S, 5.8S, and 28S rRNAs. Interestingly, we observed association of EloA with segments that map to ene site A. We carNopp140) B, a nuclectively , 39. Thiectively , A–B.Figin vivo dynamics related to the known liquid–liquid phase separating properties of nucleoli (B). Consistent with the immunofluorescence results on endogenous EloA, mCherry-EloA was present in the nucleolus in addition to the nucleoplasm . We examined the exchange kinetics of mCherry-EloA in 3T3 cells to determine whether it showed exchange characteristics similar to that of other nucleolar proteins. Fluorescence recovery after photobleaching (FRAP) analysis of EloA molecules in the nucleoli showed rapid recovery of photobleached EloA (10–15 s) post photobleaching (D–E), demonstrating the dynamic nature of EloA in the nucleolus. Nucleolar EloA recovery was slower compared with the nucleoplasmic mCherry-EloA recovery profile (5 s), which might represent the diffusion dynamics of nucleoplasmic EloA. The slower recovery rate of nucleolar mCherry-EloA might reflect interaction of EloA with binding partners within the nucleolus. We conclude that the nucleolar fraction of EloA possesses dynamics in cell culture that are consistent with those of previously described nucleolar proteins . Given the localization of EloA in the nucleolus, we examined the ability of EloA to exhibit properties of phase separation in vitro and whether this might be related to nucleolar localization.The liquid–liquid phase separation properties (LLPS) of the nucleolus have been proposed to be driven in part by proteins with large intrinsically disordered regions (IDR) that facilitate multivalent interactions and formation of phase separated condensates . Many ofFig. 6B). We also expressed and purified monomeric enhanced GFP (mEGFP) tagged EloA (mEGFP-EloA WT) (A) and found that upon centrifugation, mEGFP-EloA separates from solution and forms condensate pellets (C). To examine whether the IDR region of EloA plays a role in phase separation, we purified a truncated EloA construct where three highly disordered stretches of amino acids within the IDR were deleted (mEGFP-EloA IDR-skip). This truncation interfered with pellet formation and the protein predominantly remained in solution (C). Furthermore, using fluorescence microscopy, we demonstrated that, in the presence of a volume excluder, mEGFP-EloA forms protein-rich spherical droplets in a concentration-dependent manner (D), whereas the IDR truncation mutant failed to form these protein-rich foci and had diffuse distribution even at the higher tested concentration (D). We conclude that EloA is able to form phase-separated condensates in vitro and that truncations within the highly disordered stretches of EloA IDR appear to interfere with phase separation properties of the protein. We noted the presence of a smaller protein band in our mEGFP-EloA IDR-skip protein preparations that might reflect stability issues of the truncated protein and may have impacted our in vitro results .To determine whether EloA has any of the properties of phase separating proteins, we purified full-length EloA and demonstrated that EloA forms a turbid solution in a concentration-dependent manner B. We alsEloA WT) A and fou pellets C. To exasolution C. Furthet manner D, whereantration D. We conEloA null 3T3 fibroblast cells using lentiviral transduction and determined the doxycycline concentration required for expression of the constructs at the endogenous levels (C–D). Similar to endogenous EloA (B), Dox-induced wild-type EloA localized to the nucleoli . Interestingly, EloA IDR-skip appeared to be partly excluded from the nucleoli and displayed a more diffuse nuclear distribution pattern . We conclude that the highly disordered stretches of amino acid within EloA IDR play a role in localization of EloA to the nucleolus. This might reflect direct effects of these disordered domains on phase separation, as noted above, and also could be influenced by possible impact of this mutation on interactions with other proteins such as EloB and EloC, although the interacting BC box remains intact in the truncated EloA construct.Next, we introduced doxycycline (Dox)-inducible wild-type and IDR-skip mutant constructs in s levels , C–D. Sious EloA B, Dox-inin vitro has been examined in great detail , as well as previous immunofluorescence (IF) analysis showing greater overlap between EloA and Ser5P form of Pol II, as opposed to Ser2P . While loss of EloA has little effect on steady-state gene expression, it has been shown to play a critical role in transcription induction in response to thermal and genotoxic stress , and we observed that its loss also leads to accumulation of nascent eRNA. Future experiments that examine the extent of interplay between EloA, PAF1, and the integrator complex would further elucidate the mechanism of enhancer RNA transcription and regulation.Notably, we observed that EloA is enriched at intragenic and intergenic enhancers, which are regulated by Pol II pausing , 32 and cription . Moreovecription , 49. EloC–D), but not toward the TES in null cells (D–E). One explanation for this could be that EloA negatively regulates Pol II pausing and residence time of paused Pol II and that its loss may lead to rapid turnover of paused Pol II and an increase in nascent transcription immediately downstream of the TSS. We also noted strong enrichment of EloA over the promoter and regulatory regions of actively transcribed genes (D and S2E), suggesting that EloA might have preference for the upstream nucleosome depleted region (NDR), which harbors regulatory elements. The presence of EloA at these sites might also reflect its role in transcription from upstream divergent (antisense) sites, as loss of EloA also leads to upregulation of transcription from the negative strand at upstream divergent transcripiton sites (D). This observation indicates a possible role for EloA in regulation of antisense transcription.We observed an increase in the level of nascent transcription immediately downstream of genic TSS (+0.5 kb), as well as upstream of divergent antisense transcription units in null cells , C–D, bull cells , D–E. Oned genes D and S2Eon sites D. This oin vitro. However, we observed a rather modest (∼10%) drop in the elongation rate of Pol II in EloA null cells upon reactivation of transcription (EloA null cells in the 10-min sample did not appear accentuated around the “pioneering wave” of transcription and was more prominent upstream. This is not consistent with the phenotype expected for a factor that modulates elongation rate of Pol II (C). Therefore, it is possible that EloA also plays role in steps upstream of productive elongation. There could be several explanations for this discrepancy between in vitro and in vivo effects of EloA on transcription elongation rate. First, in vitro transcription assays are performed at limiting NTP concentrations and monovalent salt concentrations that are below physiological levels .Under our experimental setting, we estimated the global elongation rate of Pol II to be in the range of 2–2.4 kb/min. This is concordant with previously reported Pol II elongation rates in mESC by studying the “receding wave” of Pol II upon transcription shutdown . Elongincription . Moreovef Pol II C. Therefl levels . The latRn45s (B). Our results also show that EloA is capable of forming phase-separated condensates in vitro, a feature shared among tested nucleolar proteins . This is consistent with a study showing truncations within the IDR of EloA interfere with localization of EloA to sites of DNA damage  and wild-type mES cells were kind gift of Dr Teijiro Aso  (3 Units/ml of mLIF (Millipore), 55 μM 2BME, 1 μM and 3 μM of PD0325901 and CHIR99021, respectively. NIH/3T3 (ATCC CRL-1658) mouse embryonic fibroblast cells were grown in DMEM supplemented with 10% FCS.Elongin A null ( School) . Cells w School) . In brie6 cells were resuspended in 950 μl of wash buffer. Fifty microliters of BioMag-Plus Concanavalin A beads (Polysciences Inc) was also washed gently twice with binding buffer  and resuspended in binding buffer at the initial volume (50 μl). Concanavalin A beads (50 μl) were added to 5 × 106 cells in 950 μl of wash buffer and incubated rotating for 15 min at RT. The cells were divided into five 200 μl aliquots for each antibody used (1 × 106 cells/antibody) and after removal of wash buffer, 50 μl of ice cold antibody buffer (wash buffer containing 0.05% Digitonin and 2 mM EDTA) containing 0.5 μg of antibody was added to 1 × 106 bead-bound cell pellet, and the mixture was gently resuspended by pipetting up and down 25 times. Resuspended beads were placed on a vortex at the lowest setting and incubated at room temperature for 15 min. For the EloA cut&run experiment using goat anti EloA , a bridging rabbit anti-goat antibody was used for efficient binding to protein A. Cells were washed once after primary antibody incubation with 1 ml of Digitonin buffer (Wash buffer containing 0.05% Digitonin) resuspended in 50 μl of digitonin buffer with 0.5 μg of rabbit anti-goat IgG (rabbit anti-goat IgG [ab6697]). Left on the lowest setting of the vortex and incubated at RT for 15 min. Next, permeabilized, antibody-bound cells were washed once in digitonin buffer containing 1 mM EDTA, and 50 μl of digitonin buffer containing Protein A-MNase (pA-MN) at a final concentration of 700 ng/ml was added to the pellet, resuspended, and incubated at room temperature for 15 min. Cells were washed twice with digitonin buffer containing 1 mM EDTA. Next, 150 μl of ice cold digitonin buffer was added to the pA-MN bound cells, resuspended, and digested on ice (0 °C) for 30 min after addition of CaCl2 to a final concentration of 4 mM. Hundred microliter of 2× stop buffer was added as described in , placed in a styrofoam container, and frozen O/N (this step was included to minimize cell membrane rupture and DNA breakage). On the day of the experiment, cross-linked cells were thawed and washed once with PBS and twice in cut&run wash buffer , Tween20 was included in the wash buffer to minimize concanavalin A magnetic bead aggregation in downstream steps. Next, 5 × 10ribed in  and incuribed in .B). The same annotations were used to calculate the proportions of features in the mouse genome for comparison. Deeptools v3.3.0  according to manufacturer's protocols. We also generated a pool of BrU-incorporated in vitro transcribed Drosophila spike in RNAs using 5-Bromouridine 5′-triphosphate (B7166-5MG) and MEGAscript T7 Kit (AM1333) according to manufacturer's protocol. Names of Drosophila genes and the primer sequences used for synthesis of the templates are provided in in vitro transcribed spike in RNAs were pooled at varying concentrations and added to the total RNA from each sample. RNA immunoprecipitation and washing steps were carried out in the Bru-seq buffer as described above and previously   was added to cells for 10 min. For the elongation rate analysis using flavopiridol (FP), FP  was added at a final concentration of 1 μM for 60 min. Plates were washed with PBS and incubated in media containing 2 mM BrU for 5 or 10 min. Plates were washed once with PBS and total RNA was isolated by addition of 4 ml of Trizol. Bru-seq was performed as described previously , 59 on 1eviously . In brieeviously .All experiments were performed in duplicate. Paired end sequencing reads were aligned to the mm10 and dm3 genomes using bowtie2 and filtered using samtools to keep uniquely aligned reads. Reads from the positive and negative strand were separated using samtools. Browser tracks were generated using Homer v4.10.3 and visualized in IGV. Reads in whole gene of Refseq annotated genes were counted using featureCounts v1.6.1 . edgeR  and R v36 cross-linked mESC cells as described previously  and placed on a glass slide and covered with a coverslip. Images were acquired using the 100× oil objective on Nikon 90i Eclipse epifluorescence microscope equipped with an Orca ER camera and velocity imaging software (Perkin Elmer).Recombinant mEGFP and mEGFP-EloA were diluted to the specified concentration in 20 mM HEPES pH 7.9, 10% glycerol, 1.5 mM MgClCells were grown on coverslips and fixed with 1% formaldehyde for 10 min, washed with PBS, and permeabilized with 0.2% Triton X-100 in PBS for 15 min. Cells were blocked for 30–60 min in Blocking buffer  and incubated with primary antibodies  ) in blocking buffer O/N at 4 °C. Coverslips were washed three times with wash buffer  and incubated with secondary antibody conjugated to Alexa Fluor 568 or 488 for 60 min at room temperature, coverslips were washed three times with wash buffer, once with PBS, rinsed with water, and mounted on slides using DAPI-containing mounting medium. Images were acquired using the 60× oil objective of Nikon 90i Eclipse microscope equipped with Orca ER camera (Hamamatsu) and velocity imaging software (Perkin Elmer). Z-stacks were collected using the 0.2 μm spacing and analyzed using maximum intensity of each section using ImageJ Fiji software.Cells were grown on glass-bottomed fluorodish (WPI) in phenol-red free DMEM medium. Expression of mCherry-EloA was induced using doxycycline at the indicated concentration (50–100 ng/ml for 6 h). All images were acquired using a Ti-2 Eclipse microscope (Nikon). Imaging was performed at 37 °C using a temperature-controlled Tokai-Hit culture dish system. Incubation with Hoechst 33,342  for 2 h was carried out for live-cell DNA staining. A square of 1.5 × 1.5 microns was imaged for 2 s at 1 frame/s before stimulation for photobleaching using a 561-nm laser at 10% power for 2 s. Two distinct time-lapse imaging protocols were used: a fast acquisition at 600 ms/frame for 20 s or a slow acquisition at 2 s/frame phase for 1 min. Images were background subtracted and fluorescence intensity evaluated at bleached areas using Fiji software. Measurements were normalized to values obtained before bleaching (t = 0) and compared with an unbleached area used as a reference. All recovery graphs show relative fluorescence intensity (RFI) as a function of time (s). For nucleoplasm FRAP experiment, squares of 1.5 × 1.5 microns were stimulated and imaged as described above.FLAG-EloA WT or FLAG-EloA skip IDR was cloned into a modified pTRIPZ vector (Dharmacon), described elsewhere . The skiFor characterization of phase separation properties of EloA, FLAG-tagged EloA and FLAG-EloA in complex with EloB and EloC constructs were cloned in pFastBac1 baculovirus expression plasmid and purified and described previously . The conEloA null mESC cells growing on 100 mm gelatinized feeder plates were de-MEFed and 2 × 108 cells were washed once in PBS, resuspended in lysis buffer  and left on ice for 10 min, dounced ten times, and spun down at 2850g for 5 min to separate nuclei from cytoplasmic extract. Nuclear extract was prepared as described elsewhere  and while gently vortexing, equal volume of high salt buffer  was added to the resuspended nuclei. Nuclear extract was obtained by gentle continuous mixing in cold room for 30 min, followed by centrifugation at 20,000g. The concentration of the isolated nuclear extract was brought to 300 mM KCl using the low salt buffer and NP-40 was added to the extract to a final concentration of 0.05% before immunoprecipitation (IP). IP was carried out using 15 μg of goat anti EloA  conjugated to magnetic protein G for 3 h at 4 °C. Beads were washed three times using wash buffer . Following the final washing step, the beads were eluted using 1× SDS gel-loading buffer and separated on a 10% SDS-PAGE gel 1/4 of the way down and stained using coomassie blue. Each immunoprecipitation was excised as 5 gel sections, minced, and digested in-gel using trypsin. Digested gel sections were dehydrated by treatment with acetonitrile and speed-Vac. Rehydration was carried out in 50 mM ammonium bicarbonate supplemented with 12.5 ng/μl of sequencing-grade trypsin (Promega) at 4 °C. Samples were incubated at 37 °C overnight. For peptide extraction, ammonium bicarbonate was removed and washed with a 50% acetonitrile,1% formic solution and dried in a speed-Vac. Dried samples were resuspended in HPLC solvent A  and loaded into a reverse-phase HPLC column . Following formation of gradient, elution of peptides was carried out using increasing concentration of HPLC solvent B . Electroionization was performed on the peptides before entering an LTQ Orbitrap Velos Pro ion trap mass spectrometer (Thermo Fisher Scientific). Detection, isolation, and fragmentation of the peptides were carried out to produce a tandem mass spectrum of fragment ions for each peptide. Sequest (Thermo Finnigan) was used for identification of peptide sequences. The false discovery rate (FDR) filter for peptide identification was set at 1% or less. Processing, preparation, and mass spectrometry analysis of the samples were carried out at Taplin Mass Spectrometry Facility . Verification of EloA PAF1 interaction by immunoprecipitation Western Blot was carried out on 1 mg of nuclear extract using 1–3 μg of the following antibodies: Paf1 , CTR9 (Bethyl A301-395A), Leo1 , Parafibromin/CDC73, , EloA . Immunoblots were performed at a primary antibody concentration of 1:2000. Proteomic data analysis presented in Figure 4A was performed using the CRAPome (crapome.org) analysis pipeline -2A-Puro (PX459)V2.0   was transfected using 5 μl of jetPRIME (Polyplus) transfection reagent. After 24 h, transfected cells were selected using puromycin at a final concentration of 2 μg/ml and 48 h later, cells were washed once with PBS, trypsinized, counted, and diluted down to 5 cells/ml (0.5 cells/100 μl), and 100 μl was plated in each well of a 96-well plate . One week later, wells with cells growing in them were expanded in a 12-well plate and harvested after reaching confluency. Harvested cells were frozen down and also used for verification of EloA KO by western blot using EloA  antibody.Guide RNA (gRNA) oligos targeting the 5ʹ untranslated region and translation initiation site of murine  #62988) . Primer great.stanford.edu) and downloading the GO biological process list. Wikipathways (http://amp.pharm.mssm.edu/Enrichr)  .Published data were downloaded from GEO as follows: H3K4me1 (GSM747542) , H3K4me3FASTQ files were downloaded from GEO using sratools v2.9.1. All reads were processed as described before.GSE151582. Mass spectrometry raw data are accessible under ProteomeXchange: PXD022138 and jPOSTrepo: JPST000951.All sequencing data have been deposited to GEO under accession number: The authors declare that they have no conflicts of interest with the contents of this article.


Enhancer elements control mammalian transcription largely in a cell-type-specificmanner. The genome-wide identification of enhancer elements and their activitystatus in a cellular context is therefore fundamental to understanding cellidentity and function. We determined enhancer activity in mouse embryonic stem(ES) cells using chromatin modifications and characterised their globalproperties. Specifically, we first grouped enhancers into 5 groups usingmultiple H3K4me1, H3K27ac, and H3K27me3 modification data sets. Active enhancers(simultaneous presence of H3K4me1 and H3K27ac) were enriched for binding ofpluripotency factors and were found near pluripotency-related genes. Althoughboth H3K4me1-only and active enhancers were enriched for super-enhancers and aTATA box like motif, active enhancers were preferentially bound by RNA polII(s2) and were enriched for bidirectional transcription, while H3K4me1-onlyenhancers were enriched for RNA polII (8WG16) suggesting they were likelypoised. Bivalent enhancers (simultaneous presence of H3K4me1 and H3K27me3) werepreferentially in the vicinity of bivalent genes. They were enriched for bindingof components of polycomb complex as well as Tcf3 and Oct4. Moreover, a‘CTTTCTC’ de-novo motif was enriched at bivalent enhancers, previouslyidentified at bivalent promoters in ES cells. Taken together, 3 histonemodifications successfully demarcated active, bivalent, and poised enhancerswith distinct sequence and binding features. Collaborative efforts such as the FANTOM2 and Roadmap Epigenomics3 projects have now successfully built enhancer and promoter repertoires acrosshundreds of cell types with an estimated 1.4% of the human genome associated toputative promoters and about 13% to putative enhancers. The vast majority of diseasesusceptibility loci lie in non-coding genomic regions, particularly in enhancerregions5and have been estimated to explain a greater proportion of the heritability for somedisorders than variants in coding regions.7 Despite their known functionalimportance during development and disease, computational approaches towardsgenome-wide identification of enhancer elements remains a major challenge to date.This is due to various factors, including enhancers are typically distal to the TSSwith no fixed distance preference, can range from a few to few hundred bases lengthand tend to be cell-type specific.8 On the other hand, regulatory regions (enhancers and promoters) have sequencefeatures enriched for motifs of known transcription factors and evolutionarilyconserved facilitate computational prediction. Moreover, enhancers can bedistinguished from promoters using the presence of certain regulatory proteinsincluding p300 (an acetyltransferase), BRG1 (a chromatin remodelled), the presenceof histone modifications (H3K4me1 and H3K27ac), and unstable nucleosomes.9 Interestingly, several experiments have recently shown that enhancer regionsget transcribed to form short, often bidirectional transcripts, called enhancersRNAs or eRNAs.10 Enhancer RNA expression is correlated with the expression of target genes,many a times in a stimulus-dependent manner and therefore can be used to predictenhancers and their target genes.11 Several functional mechanisms of gene regulation have been described inliterature including enhancer-promoter looping, recruiting transcriptionalmachinery, and facilitating RNA polymerase pause-release.12 It is important to note that, eRNAs might not always be a predictor ofenhancer activity or localisation, and not all functional enhancers have to betranscribed in eRNAs.Embryonic stem (ES) cells have an indefinite proliferative life span and have apotential for both generating daughter cells with equivalent potential or cellsready for differentiation, that is, to establish unique gene expression profilescharacteristic to different cell and tissue types during development. The delicatebalance between self-renewal and the competency-to-differentiate, is achieved byepigenetic and transcription control. This process is tightly regulated bytranscription factors and chromatin-associated proteins, which respond to signallingpathways which in turn responds to environmental cues.13 Super-enhancers were defined as large genomic regions (>3 kb) near highlyexpressed genes, differing from typical enhancers in size, transcription factordensity and content, ability to activate transcription, and sensitivity to perturbation.14 But, a CRISPR/Cas9-mediated deletion several super-enhancers clusters andisolated enhancers in mouse ES cells demonstrated enhancers and super-enhancers havean equivalent regulatory role in ES cells15 and iPS cells.16 The chromatin state is thought to be more functionally relevant with positivefeedback loop between permissive chromatin and translation during early embryonic development.17 It is important to note that though histone modifications are widely used todefine chromatin functional state, they are only predictions and not a proven fact.Furthermore, the experimental validations have demonstrated that they contain largenumber of false positives.18 Enhancers are marked by putative active (H3K4me1) or putativerepressive (H3K27me3) chromatin modifications according to the cellular context.H3K4me3 (putative active) and H3K27me3 (putative repressive) modifications werefound co-localised at promoters and enriched in developmentally regulated genes inmouse ES cells.19 Bivalent promoters were subsequently identified in human ES cells and weresimilarly enriched for developmental regulators20 and conserved their chromatin signature across species.21 The bivalent state is thought to assure silencing of key developmentalcontrollers in ES cells while keeping them poised for activation during differentiation.22 Similar to promoters, putative enhancers marked with the bivalent enhancersare defined by the presence of the repressive mark H3K27me3 and active mark H3K4me1.Bivalent promoters identified in many mature lineages including T-cells,23 neural progenitor cells (NPCs), and mouse embryonic fibroblast (MEFs).24 Similarly, putative bivalent enhancers were identified in pro-B cells andadult liver,25 fibroblast-derived adipocytes, and bone-marrow macrophages.26 In summary, bivalent domains are present in enhancers and promoters and arethought to prime regulatory regions for their differentiation trajectory.27 An integrative analysis of bivalent promoters in mouse ES cells demonstratedthat most H3K27me3 promoters were bivalent.28 Importantly, most bivalent promoters did not resolve into active or repressedchromatin in differentiated lineages, with many bivalent chromatin domains in factremained bivalent across diverse cell types.29Enhancer regions have been classified into different groups based on their length,motif content, or chromatin status.Similarly, to study the functional relevance of chromatin signature of enhancerelements, we performed systematic analysis of putative enhancers in mouse ES cellsby data integration. Specifically, we first grouped putative enhancers into 5 groupsusing multiple H3K4me1, H3K27ac, and H3K27me3 modification data sets. Putativeactive enhancers (simultaneous presence of H3K4me1 and H3K27ac) were enriched forbinding of pluripotency factors and were found near pluripotency-related genes.Although both putative H3K4me1-only and active enhancers were enriched forsuper-enhancers and a TATA box like motif, putative active enhancers werepreferentially bound by RNA polII s2 form and were enriched for bidirectionaltranscription, while putative H3K4me1-only enhancers were enriched for RNA polII(8WG16) suggesting they were likely poised. Putative bivalent enhancers(simultaneous presence of H3K4me1 and H3K27me3) were preferentially in the vicinityof bivalent genes. They were enriched for binding of components of polycomb complexas well as Tcf3 and Oct4. Moreover, a ‘CTTTCTC’ de-novo motif was enriched atputative bivalent enhancers, previously identified at bivalent promoters in EScells.30 and Roadmap Epigenomics.31 The accession numbers (containing the information about the ES cell linesand culture conditions) for raw data for each sample are provided in Supplemental Table S1. After mapping the reads to mm10 genomeassembly, we called peaks in each sample using SICER.32 Input controls were not used, if they were not available for the samples.Specific parameters for running SICER for H3K4me3 and H3K27acwindow = 200 and gap size = 200 andfor H3K27me3, window = 200 and gapsize = 2 × 300, since H3K27me3 covers wider chromatin domains. Therest of the parameters  wereeffective genome fraction = 0.7, redundancythreshold = 1, fragment size = 150 andE-value = 100. We filtered out GENCODE M2 TSSs, whichinclude both protein-coding and non-coding genes, to select enhancer peaks. Promoter regions were defined as 1 kbregion around GENCODE TSSs.We collected ChIP sequencing raw data for H3K4me1, H3K27ac, and H3K27me3 profileswith 4 samples of each type in mouse ES cells from GEOSupplemental Table S2). The H3K4me1 peaks with an overlap of atleast 25% with one of the peaks where the width of the merged peak was the unionof 2 peaks. The merged list of H3K4me1 peaks was treated as ‘putative enhancers’forming the rows of the matrix described above. The peaks for H3K4me1, H3K27ac,and H3K27me3 in ES cells overlapping at least 25% of the ‘putative enhancer’peaks were considered overlapping and the H3K4me3 peak was defined as putativeenhancer region. We then identified high confidence putative active enhancers bysimultaneous presence of H3K4me1 and H3K27ac in 3 or more sample pairs andabsence of H3K27me3 in all 4 samples and high confidence putative bivalentenhancers by simultaneous presence of H3K4me1 and H3K27me3 in 3 or more samplepairs and absence of H3K27ac in all 4 samples. We also defined H3K4me1-only,H3K27ac-only, and H3K27me3-only peaks by the presence of each chromatinmodification in 3 or more samples and absence of other modifications. We haveprovided the genomic co-ordinates for all predicted enhancers in each enhancergroup as Supplementary Material 2.We created a genomic region by sample matrix by merging all peak files where ‘1’represented a peak present in a given genomic region in a given sample and ‘0’otherwise. Specifically, H3K4me1 peaks across diverse tissue and cell lines fromENCODE mouse data and 4 samples ES cell were merged using the following schemaas well as distance to the nearest TSS was calculated using HOMER.36 The de-novo motif analysis was performed using HOMER with defaultparameters. Mammalian conservation tracks were downloaded from UCSC genomebrowser and conservation status was assigned if more that 75% of regionoverlapped with mammalian conserved regions. We used a stringent threshold toselect the enriched motifs . Gene ontology functionalanalyses for the putative bivalent, active, and H3K4me1-only enhancers were doneusing DAVID.37 The TSSs predicted by the CAGE data as putative enhancers were obtainedfrom the FANTOM consortium.2 The enrichments of overlaps were calculated using a hypergeometric testand P values were corrected using Bonferroni correction. Theanalysis was performed using R and shell scripts. All analysiswas performed in mm10 genome assembly. The data sets provided by authors in mm9were converted to mm10 using the liftover tool.38The promoter regions in mouse ES cells grouped according to chromatin marks asbivalent, active (H3K4me3-only), H3K27me3-only, and latent (absence of bothmarks) were obtained from Mantsoki et al.https://genome.ucsc.edu/encode/downloadsMouse.html . The peaks for the chromatinmarks  during reprogramming by 4 transcriptionfactors Oct4, Sox2, Klf4, and Myc (OSKM) towards pluripotency were obtained fromGEO, GSE67520.39The peaks for the chromatin marks  across mousecell lines and tissues were downloaded from the mouse ENCODE resource Supplemental Table S1). Putative enhancer peaks in each samplewere determined by removing the peaks identified using SICER32 overlapping with GENCODE40 promoters. This resulted into about 120 thousand nonpromoter genomicregions with H3K4me1 modifications in at least 1 of 4 samples  and genes neighbouring ‘putativebivalent enhancers’ were enriched for Wnt Signalling Pathway  and TGF Beta Signalling Pathway . Interestingly, genes neighbouring ‘putative H3K4me1-onlyenhancers’ were enriched for both pluripotency network genes  and Wnt Signalling Pathway  and TGF Beta Signalling Pathway .We mapped regulatory regions to the TSS of the nearest gene resulting into onaverage 2.5 regulatory regions mapped to a gene. We then calculated functionalenrichment and pathway enrichments for each gene list . Gene ne41 used bidirectional expression from CAGE data to determine putativeenhancer regions across multiple human and mouse cell types. We collected 44 507bidirectional TSSs as well as 51 807 (positive-strand) and 46 434(negative-strand) unidirectional TSSs in ES cells from FANTOM5 resource.2 We noted that significantly higher fraction of regulatory regions in allgroups overlapped with bidirectional TSSs compared to unidirectional TSSs  while putative bivalent enhancers were enriched to a lesserextent .We further analysed whether specific genomic regions were preferred by putativeenhancers. Most putative enhancers were present in either intergenic or intronicregions for all putative enhancer types . Given tWe downloaded genomic regions conserved across mammals from UCSC genome browserand noted that more than half of all putative enhancers were conserved acrossmammals. This fraction did not significantly change for any of the enhancercategories .To evaluate whether different putative enhancers showed a bias for specificgenomic distance with respect to the nearest TSS. We calculated distance to thenearest TSS from each putative enhancer and noted that most enhancers werepromoter distal with over half more than 50 kb away from the nearest TSS . About 1Supplemental Table S5). We have previously identifiedhigh-confidence bivalent, active (HK4me3 only), H3K4me3-only and latent (nomark) promoters in mouse ES cells.28 To test whether specific enhancer types are enriched near specificpromoter types, we calculated the gene overlap between enhancer and promotercategories. Putative bivalent enhancers were highly enriched near bivalentpromoters , PRC2components: Suz12, Jarid2, and PRC1 subunits: Cbx7 and Ring1b density atputative bivalent enhancers in mouse ES cells . We noted that similar to promoters, afraction of putative bivalent enhancers indeed showed a strong occupancy ofpolycomb and RNAPII. Never the less, these enhancers formed about 10% ofputative bivalent enhancers , unlike promoters which formed abouthalf of bivalent promoters.Bivalent promoters belong to 2 distinct groups in mouse ES cells. The first classconsists of domains where only PRC2 exists ( ‘PRC2-only’) and the second one,called PRC1-positive, where PRC2 domains are also occupied by PRC1.PRC1-positive, in contrast to PRC2-only, were more expansive bivalent regions,highly conserved, and consisted numerous developmental promoters.Supplemental Table S6). Active enhancers were significantlyenriched for p300 and core pluripotency factors including Oct4, Sox2, Nanog,Smad1, and Stat3. Importantly, these factors were not enriched at putativeH3K4me1-only enhancers  of putative active enhancers. Thiscan likely be due to the number of peaks called in p300 ChIP-seq sample (9429peaks) compared with more than 21 000 predicted active enhancers.As the number of binding events vary greatly across factors affecting theenrichment, we calculated the fraction of peaks in each group occupied by agiven factor. Ezh2, Kdm2b, Rnf2, and Klf2 were present at about a third ofputative bivalent enhancers while Nanog, Pou5f1, Brd4, and Prdm14 were presentat about a quarter of putative active enhancers . P300, t36 We selected motifs enriched with a P value less that1e−10 and present in at least 10% of enhancers. Both putative active andH3K4me1-only enhancers were enriched for KLF, FLI, and ETS motifs. Importantly,the sequence motif for pluripotency factors enriched at putative activeenhancers was not highly enriched. This suggests that KLF, FLI, and ETS motifsmight facilitate binding of pluripotency factors to the enhancers. Indeed,simultaneous depletion of Klf2, Klf4, and Klf5 lead to ES cell differentiation.46 No known motif was found enriched at bivalent enhancers using stringentcut-offs defined above. The lack of enrichment of known pluripotency factormotifs at putative active enhancers and polycomb comples components at bivalentenhancers might be due to wider peaks called in histone modification data.In the previous section, we noted that putative active and bivalent enhancerswere enriched for different epigenetic and transcription controller. Toinvestigate whether the binding enrichment of these factors is also reflected insequence motif enrichments at putative active and bivalent enhancers, wecalculated known and de-novo motif enrichment using HOMER.28 The second de-novo motif was similar to Myf6 from JASPAR database,47 while the third was present in about half of bivalent enhancers and wassimilar to Egr1 from JASPAR database.47 Of the 3 motifs in putative active enhancers, the first was similar to aPOLII TATA box, the second similar to KLF motif while the third similar toMA0006.1, a bHLH motif from JASPAR database.47 Supporting known motif enrichments, the de-novo motif similar to TATA boxwas present in active and H3K4me1-only enhancers. Putative H3K4me1-only specificde-novo motif was similar to Zbtb3 motif from JASPAR database.47 As POLII TATA box like motif was enriched in putative active andH3K4me1-only enhancers, we calculated the overlap between enhancers and 3 formsof RNA POLII binding  using ChIP sequencing data.48 Putative H3K4me1-only enhancers showed a much higher overlap with 8WG16antibody, recognising nonphosphorylated Pol II while putative active enhancersshowed a higher overlap for s2 antibody, specific for active, elongating Pol II.The de-novo motif search identified 3 motifs enriched in bivalent enhancers, 3 inputative active enhancers and 2 in putative H3K4me1-only enhancers . The firTo study the dynamics of the 5 groups of putative enhancers in ES cells throughdifferentiation, we collected H3K4me1, H3K27ac, H3K27me3 data for 10 tissues andcell types from the mouse ENCODE resource. Similar to the analysis of ES cellsdata, we classified the putative enhancers in each cell type in the same 5groups, namely active, bivalent, H3K4me1-only, H3K27ac-only, and H3K27me3-only.We then calculated the overlap of each groups of putative enhancers in ES cellswith all 5 enhancers groups in each of the 10 tissue and cell types individually, left.P39 Similar to the observation in other tissues and cell types, putativeactive and H3K4me1-only enhancers in ES cells were mostly active or H3K4me1-onlyduring reprogramming. As expected putative active enhancers in ES cells showed ahigh overlap with putative active enhancers in iPSCs and putative H3K4me1-onlyenhancers also behaved similarly. Interestingly, putative bivalent enhancers inES cells lost H3K27me3 and gained H3K4me1 during reprogramming.To explore how the above-noted patterns might establish during differentiation,we collected time-series data during cellular reprogramming by inducedexpression of 4 transcription factors Oct4, Sox2, Klf4, and Myc (OSKM) pushingsomatic cell reprogramming towards pluripotency.18 and have led to otherdefinitions of active enhancers but there is no agreed consensus. Simultaneouspresence of H3K4me1 and H3K27ac remains a common criterion used for identificationputative active enhancers. We note that in this study used only histone modificationdata, as the aim was to identify high confidence (relatively wide) regulatoryregions and not identification of precise regulatory site within them. In lattercase, one would ideally integrate other data including p300 binding or nucleosomedepletion measured via ATAC-Seq or DNAse hypersensitivity assays. Furthermore, wemapped the regulatory regions to the nearest TSS. Although this is the most widelyused approach to map putative targets of regulatory regions, it is important to notethat the nearest neighbour gene is not always the enhancer target. There are manyapproaches to improve the mapping including chromatin capture assays tocomputational models.49Understanding enhancer function is fundamental to understanding cell-type-specificgene control. Here, we have defined putative enhancers based on the presence ofH3K27ac, H3K27me3, and H3K4me1 modifications and characterised them in detail. Wedefined putative active enhancers by simultaneous presence of H3K4me1 and H3K27ac.We note that experimental validations of putative active enhancer predictionsdemonstrate that they contain high rate of false positives25 The higher overlap of putative H3K4me1-only enhancers with RNA POLII 8WG16antibody, with no enrichment for bidirectional expression points out that they mightbe poised. Intriguingly, genes near H3K4me1-only enhancers were also enriched forpluripotency-related genes. Furthermore, genes neighbouring ‘putative activeenhancers’ were enriched for pluripotency network genes  and genes neighbouring ‘putative bivalent enhancers’ wereenriched for Wnt Signalling Pathway , and TGFBeta Signalling Pathway . Interestingly, genesneighbouring ‘putative H3K4me1-only enhancers’ were enriched for both pluripotencynetwork genes  and Wnt Signalling Pathway and TGF Beta Signalling Pathway. We noted that putative H3K4me1-onlyenhancers share the (nearest) genes with putative active and bivalent enhancers,resulting into enrichment for the signalling pathways. H3K4me1 modification hassharp ChIP-seq signal compared to both H3K27ac and H3K27me3 modifications andtherefore more consistent peak detection across data sets. One of the possibilitiestherefore is that at least some of the putative H3K4me1-only enhancers were in-factputative active (missing H3K27ac) or bivalent enhancers (missing H3K27me3). It isalso worth noting that bivalent enhancers had all 3 RNA Pol II peaks present.Putative active enhancers showed a high overlap with FANTOM bidirectional putativeenhancers, ES putative super-enhancers, and binding of many pluripotency factors. Wenote that the FANTOM bidirectional putative enhancers were identified across diversetissue and cell types and therefore limiting the overlap. The properties of activeenhancers were distinct from putative H3K4me1-only enhancers corroborating that thepresence of H3K27ac is sufficient to distinguish putative active enhancers.50 Bivalent regions selected by this definition show a high overlap with our‘putative active enhancers’ as most H3K27ac marked enhancers also carry H3K4me1modification. Finally, we also identified a ‘CTTTCTC’ de-novo motif enriched atbivalent enhancers previously identified at bivalent promoters.We defined putative bivalent enhancers by simultaneous presence of H3K4me1 andH3K27me3 modifications. Putative bivalent enhancers were present near bivalentpromoters and many maintained bivalency through differentiation. Other definitionsof putative bivalent enhancers include simultaneous presence of H3K27ac and DNA methylation.Click here for additional data file.Supplemental material, sj-pdf-1-bbi-10.1177_1177932220974623 for Identificationand Characterisation of Putative Enhancer Elements in Mouse Embryonic Stem Cellsby Anna Mantsoki, Karla Parussel and Anagha Joshi in Bioinformatics and BiologyInsights