X inactivation-specific transcript (Xist) RNA to initiate X chromosome inactivation (XCI) that eventually results in the formation of the Barr body.Xist encompasses half a dozen repeated sequence stretches containing motifs for RNA-binding proteins that recruit effector complexes with functions for silencing genes and establishing a repressive chromatin configuration. Functional characterization of these effector proteins unveils the cooperation of a number of pathways to repress genes on the inactive X chromosome. Mechanistic insights can be extended to other noncoding RNAs with similar structure and open avenues for the design of new therapies to switch off gene expression. Here we review recent advances in the understanding ofXist and on this basis try to synthesize a model for the initiation of XCI.Female mammals express the long noncoding The choice of the X chromosome to switch off, either maternal or paternal, is widely accepted to be random in placental mammals, and recent evidence in mice suggests a stochastic process for which a few components have been identified3. A mechanism for generating randomness is difficult to establish on a molecular basis but ultimately may inspire technology as well as cell biology. Recent progress in chromatin biology has, however, led to considerable insight into the pathways that accompany the silent fate of the inactive X chromosome (Xi). It is important to state from the outset that once the Xi has been chosen and repressed, its silent state is faithfully maintained and inherited in all progeny of every embryonic cell. This observation requires an epigenetic memory that is established at the Xi but has not been fully deciphered yet5. The process of X chromosome inactivation (XCI) has been dissected into two characteristic phases: 1) a reversible X inactivation-specific transcript (Xist)-dependent initiation step, when X linked genes become repressed, and 2) a maintenance step when gene silencing becomes irreversible and independent ofXist . These studies revealed the requirement of repeat A for the repression of X-linked genes but not for localization or spreading ofXist over the X chromosome13.Close to the 5' end ofXist partners responsible for repressing transcriptional activity were unveiled. Forward genetic screening and biochemical purification ofXist partners, in differentiated and undifferentiated cells, identified SPEN as a top candidate responsible for gene repression20. SPEN is a 400 kDa protein that belongs to theSplit Ends family of transcriptional repressors. Its mutation in mice causes an embryonic lethal phenotype at E12.521. SPEN contains four N-terminal RNA recognition motifs (RRMs) and a C-terminal conserved Spen paralog and ortholog domain (SPOC) that mediates the recruitment of co-repressors and the activation of histone deacetylases (HDACs). Different approaches have confirmed the direct interaction between SPEN and repeat A ofXist23. Biochemical purification has shown that SPEN interacts with the full-length but not the repeat A mutant form ofXist19. Deletion of exons encoding the RRM domains of SPEN abrogates the silencing capacity ofXist20. In addition to a loss of gene silencing,Xist appears to lose the ability to recruit chromatin-modifying complexes of the Polycomb group20. A pathway for gene repression has been proposed based on the known interaction of SPEN with the SMRT co-repressor that activates HDAC317. HDAC3-mutant ESCs show partial gene silencing uponXist expression, suggesting that not all gene repression is mediated by HDAC324. Taken together, these findings make SPEN a well-established silencing factor ofXist, though details of the mechanism in XCI remain to be uncovered.Recently, the identities of some of theXist in a repeat A-dependent manner19. WTAP is a component of the RNA N-6 adenosine methylation (m6A) complex that also contains RBM15, its paralogue RBM15B, and the methyltransferase METTL325. RBM15 has been identified independently as anXist-binding partner by genetic screening and biochemical purification19. Methylation individual nucleotide resolution crosslinking and immunoprecipitation (methylation-iCLIP) experiments in HEK293T cells revealed 78 m6A residues in the humanXIST RNA, some of which are localized in the vicinity of, and two of which are located within, repeat A26. These sites also show prominent binding of the RNA m6A reader YTHDC1. In ESCs, synthetic tethering of YTHDC1 toXist was sufficient to complement the depletion of other WTAP complex members, which impairs gene repression byXist. These findings indicate that m6A acts as a recruitment signal for silencing factors26. However, not all m6A sites are localized at repeat A. In addition, a recent study has investigated the deletion of most m6A sites at the 5' end ofXist and observed only subtle or no effects on X chromosome-wide gene silencing in mouse ESCs27. This suggests that m6A sites in the remainingXist sequence may act redundantly for the recruitment of additional silencing factors. In mice, mutations in WTAP, RBM15, and YTHDC1 result in embryonic lethality30. Although these observations are consistent with a potential role in XCI, female-specific phenotypic dimorphisms have not yet been described. Further evidence would be desirable to establish the precise role of the WTAP complex at the initiation of X inactivation.Additional silencing factors for the initiation of XCI might be inferred from biochemical experiments that also indicate the interaction of RNF20 and WTAP withXist RNA in a repeat A-dependent manner19. The RNF20/RNF40 family of E3 ubiquitin ligases can mediate mono-ubiquitylation of histone H2B lysine 120 (H2BK120ub1), which is required for mouse preimplantation development and ESC differentiation33. H2BK120ub1 has been correlated with both transcriptional activation34 and repression35 and is proposed to play a role in stabilizing nucleosomes and chromatin compaction37. Depletion of either RNF20 or RNF40 did not impairXist-mediated silencing in mouse ESCs19. Although a combined disruption of both RNF proteins has not yet been reported, it is tempting to speculate that RNF20 could contribute to chromatin modification and compaction in XCI.RNF20 is a third factor that has been identified to associate withXist from a heterologous promoter in trophoblast cells caused repression of genes that are not inactivated by theXist repeat A mutant in embryonic cells38. These repressed genes appear to cluster in regions of low gene density and are normally expressed at a low level on the active X chromosome (Xa). One potential explanation for the different requirement of repeat A in ESCs and trophoblast cells might come from considering the timing ofXist expression relative to gene activity: in the mouse embryo,Xist is first upregulated at the four-cell stage, concurring with the zygotic genome activation39. At this stage, not all X-linked genes might be fully activated. It is conceivable thatXist without repeat A is able to repress some X-linked genes by chromatin modifications that normally contribute to the maintenance of gene repression in XCI. Therefore, the repeat A mutantXist would not necessarily use an active silencing pathway but rather could rely on the maintenance pathways. Consistent with this idea is the finding that the expression ofXist containing repeat A has far greater potential for X-linked gene repression in trophoblast cells.Most studies in mouse ESCs have confirmed a key role of repeat A in gene repression. However, studies of imprinted XCI in extraembryonic cells have uncovered the finding that repression of several genes on the Xi is apparently independent of repeat A. Forced expression of a repeat A mutantXist repeat B consists of 32 copies of the cytidine-rich (A/U)GCCCC motif and functions together with repeat C, which contains 14 copies of a 120-nucleotide unit of high similarity40. Recent studies have discovered key roles forXist repeats B and C for the recruitment of Polycomb complexes.MouseXist repeats B and C are required for the recruitment of canonical and non-canonical Polycomb repressive complex 1 (PRC1) and PRC2 to the Xi at the initiation of XCI. Work by the Brockdorff lab has unraveled a hierarchy for Polycomb complex recruitment byXist. Initially,Xist repeats B and C recruit a non-canonical PRC1 that catalyzes the ubiquitination of histone H2A lysine 119. H2AK119ub is bound by RYBP, which is a component of a non-canonical PRC1 complex, and leads to an amplification and presumably spreading of the H2AK119ub mark. PRC2 activity is subsequently recruited and mediates H3K27me3. H3K27me3 in turn is recognized by CBX7, which is a component of canonical PRC1 complexes. Therefore, in XCI, PRC1 is recruited before PRC2 and multiple interactions amplify the spreading of both Polycomb complexes41.Xist repeat B in Polycomb recruitment was provided by the discovery of a function for JARID2 in XCI42. Da Rocha and colleagues tested the recruitment of JARID2 in differentiating ESCs containing inducibleXist mutant transgenes with different deletions.Xist lacking repeats F, B, and C did not recruit JARID2, leading to a decrease in the recruitment of PRC2. Soon after that, JARID2 was identified as an essential component for PRC2 recruitment to PRC1-marked chromatin43. Using autosomalXist-inducible transgenes carrying a deletion of repeat B plus a partial deletion of repeat C, defined as theXist RNA-Polycomb Interacting Domain (XR-PID), researchers reported a strong reduction in the accumulation of Polycomb complexes byXist44. Reassessment of these experiments in an endogenous inducibleXist system in XY ESCs showed that both repeat B and repeat C can efficiently recruit Polycomb proteins to the Xi and that a double deletion of repeats B and C leads to a complete abrogation of the accumulation of H2AK119ub and H3K27me3 at the Xi45.Initial evidence for a role ofXist46. Fluorescence recovery after photobleaching (FRAP) experiments showed PCGF3/PCGF5 to form stable interactions withXist RNA domains, and a doublePcgf3/5 deletion resulted in the loss ofXist-dependent H2AK119ub1 and H3K27me3 deposition46. Notably,Pcgf3/5 double mutant mice present a female-specific lethality at E9.5, consistent with their requirement for dosage compensation46.Xist repeats B and C have been linked to PCGF3/5–PRC1 through hnRNPK, which has been independently identified as anXist interactor47 and shown to be required for Polycomb protein recruitment19. hnRNPK is essential for early embryo development48. It presents several KH RNA-binding domains that are predicted to mediate interaction withXist repeats B and C and a K protein interactive region (KI) required to bind PCGF3/5–PRC149. It has been shown that deletion of repeats B and C abolishes the interaction ofXist RNA with hnRNPK and secondarily with PCGF3/5–PRC145. These experiments establish hnRNPK as the RNA-binding protein that linksXist to non-canonical PRC1, leading to a change of previous views from a direct binding of Polycomb proteins to RNAs51. PCGF3 and PCGF5 associate with hnRNPK and a catalytic RING protein for marking entry sites with H2AK119ub44. The requirement of hnRNPK for early embryo development makes studies of its function difficult. However, synthetic tethering of hnRNPK, but not KI mutant hnRNPK, to transgenicXist that lacks the XR-PID region restores the recruitment of H2AK119ub44. In the future, it might be interesting to more specifically disrupt interactions between hnRNPK andXist or PCGF3/5–PRC1 to understand if repeats B and C could have additional functions that have not yet been discovered. Taken together, these findings provide strong experimental support for a pathway of Polycomb recruitment byXist. A modular architecture ofXist is also suggested, with repeats B and C forming a recruitment site for hnRNPK and Polycomb complexes. More research will be needed to understand to what extent Polycomb complexes contribute to gene repression and in particular if they function in maintaining gene silencing on the Xi.To understand this mechanism in detail, it was crucial to identify the non-canonical Polycomb group ring finger (PCGF) proteins PCGF3 and PCGF5, as they are required for the recruitment of Polycomb complexes byXist might initially localize in spatial proximity to its genomic locus in regions pre-marked with PcG decoration and low transcriptional activity52. From these initial “entry sites”,Xist triggers the formation of a repressive compartment that is devoid of marks of active transcription including RNA polymerase II53. These initial events are followed by the recruitment of Polycomb proteins to the Xi and a conformational change that subsequently permits spreading and silencing of gene clusters with high transcriptional activity53. Notably, theXist mutant lacking repeat A is able to recruit Polycomb complexes to the Xi, suggesting that gene repression is not required forXist localization and Polycomb recruitment4. Recent evidence supports and extends this view, showing that repeat A mutantXist localizes and mediates decoration over most of the Xi except for regions where active genes reside24. Similar observations were made with HDAC3-mutant ESCs showing that spreading of Polycomb complexes is not observed over active gene loci. These results directly compare transcriptional activity with PcG recruitment and suggest that they are mutually exclusive. Zyliczet al. observed that deletion of repeat A causes a reduction in the deposition of H2AK119ub over the Xi. However, this effect was not recapitulated in HDAC3-mutant ESCs where PRC1 recruitment was unperturbed24. These findings might provide a stringent test for the proposed mechanism for Polycomb recruitment and potentially also suggest a contribution fromXist repeat A in Polycomb recruitment.Studies in ESCs indicate thatXist is the recruitment of SMCHD1 to the Xi. SMCHD1 was initially identified in a genetic screen in mice as a key player in XCI. SMCHD1-null embryos present a female-specific lethality between E10.5 and E13.5, with derepression of genes and hypomethylation of CpG islands on the Xi56. The interaction betweenXist and SMCHD1 was also described through proteomics screening in MEFs47, although this is likely not direct, as it depends onhnRNPK57. Recruitment of SMCHD1 is dependent onXist expression58.Xist repeats B and C are required for recruiting SMCHD1 to the Xi in ESCs at the transition between the initiation and the stable maintenance phase of XCI57. Studies in female mouse embryonic fibroblasts (MEFs) suggest that SMCHD1 recruitment byXist is dependent on PRC1 and H2AK119ub57. Notably, the mutation ofSmchd1 results in a stronger recruitment of H3K27me3 to the Xi59. At present, it is not clear how SMCHD1 can be recruited by PRC1 but insulate from the effects of PRC2. Furthermore, additional roles forSmchd1 have been proposed in remodeling the 3D architecture of the Xi61. SMCHD1 has been postulated to regulate the transient reorganization of the Xa chromatin towards a bipartite Xi structure58. Studies inSmchd1-deficient MEFs have shown that the overall structure of the Xi is largely unaffected in the absence of SMCHD1. However, genes that are reactivated become reorganized in a manner that resembles more the Xa than the Xi. Importantly, the Polycomb dependence of SMCHD1 recruitment to the Xi would suggest a requirement of Polycomb complexes for the establishment of a memory for the maintenance of XCI. Taken together, these data demonstrate thatXist repeats B and C can engage different effectors and they do this through hnRNPK and a non-canonical PRC1.One of the downstream effects of the recruitment of Polycomb complexes byXist RNA to the Xi is scaffold attachment factor A (SAF-A/Hnrnpu)62. SAF-A binding has been described at the repeat D ofXist63. Repeat D contains multiple copies of a 290-nucleotide-long motif that can mediate interaction with SAF-A. However, repeat D might not be the only element that contributes toXist localization. Earlier studies indicated that several regions ofXist act in parallel or redundantly toXist RNA localization13. However, it remains to be shown if repeat D is sufficient forXist localization or if other interactions are also required22.One important factor for the precise localization ofXist repeat E consists of 50 highly variable copies of 20–25-nucleotide-long units that have been implicated inXist localization in differentiated cells and maintenance of XCI40. Insight into repeat E function comes from the identification of CIZ1 as a protein that is recruited to the Xi in somatic cells. CIZ1 is a nuclear protein that progressively associates with the Xi in aXist-dependent manner during female ESC differentiation. AlthoughCiz1 mutant mice do not show developmental defects, a highly penetrant lymphoproliferative disorder has been reported in adult females64. This hematopoietic phenotype is consistent with an earlier report of leukemia development in female mice, whenXist is conditionally mutated in the blood system65. The blood cell diseases have further been linked with dosage compensation defects65, suggesting a special context for XCI in blood cells67. Systematic deletion experiments over differentXist regions in ESCs and MEFs unveil that CIZ1 recruitment depends on repeat E68. Deletion ofXist repeat E in ESCs does not impairXist localization or the recruitment of Polycomb complexes69. However, inCiz1-mutant female MEFs,Xist is delocalized and Polycomb complexes are not recruited to the Xi. These defects in somatic cells are accompanied by a partial de-repression of X-linked genes68. Importantly, delocalization ofXist has also been observed in hematopoietic cells fromCiz1-mutant mice.69, but the function of ASH2L for gene repression on the Xi remains largely unclear. Recruitment of ASH2L depends onXist localization but is independent of repeat A5. Taken together, current evidence suggests that repeat E acts as a binding site for factors that contribute to XCI maintenance. The cell-type-specific effects of theCiz1 mutation are intriguing. It would be interesting to explore if a mutation of repeat E would resemble theCiz1 mutation in mice. Alternatively, other repeat E binding factors could be involved in either maintenance or transition to maintenance of XCI.A recent study has uncovered an independent function for repeat E in the recruitment of the ASH2L Trithorax group proteinXist repeat F was not included in the initial description ofXist9 but has been more recently defined as a short fragment that is located just 3' of repeat A. Repeat F is composed of two copies of a 10-nucleotide-long G/C-rich sequence motif (UGGCGGGCUU)40 and appears to function together with a region near repeat E as a binding site for lamin B receptor (LBR)70. LBR is a 60 kDa integral membrane protein embedded in the inner nuclear membrane that associates with lamin B and tethers heterochromatin to the nuclear periphery71. Mutation ofLbr has been described to result in an embryonic lethal phenotype with incomplete penetrance, but a female-specific phenotype has yet to be described73. LBR was identified as a direct binder ofXist through a biochemical screening approach17. RNA interference (RNAi)-mediated depletion ofLbr in ESCs was reported to cause an impairment of gene repression byXist. These observations were further corroborated by showing that deletion of the LBR-binding sites ofXist resulted in a loss of gene repression70. Tethering of LBR toXist lacking the LBR interaction site could restore silencing activity. In addition, tethering a mutant form of LBR that lacked the RNA-binding domain toXist complemented the silencing defect. Notably, LBR does not possess a canonical RNA-binding domain and associates withXist through a novel surface. LBR might contribute to gene repression by mediating an association with the nuclear lamina. It is conceivable that also an association with a repressive heterochromatic environment at the nuclear lamina or reshaping of the 3D architecture of the Xi might play a role in XCI. However, a recent study has investigated a deletion of theLbr gene in ESCs and observed only subtle silencing defects27. In addition, after removal of the entireXist repeat F, only minor defects in gene repression were observed. These observations are conflicting with earlier observations and suggest that additional evidence will be necessary to understand the requirement ofLbr for gene silencing. Nevertheless, repeat F might provide a binding site forXist partners that engage additional mechanisms that are not yet fully understood.Xist function. The current understanding suggests thatXist RNA might act as a link between different functional effector complexes. Engaging a number of molecular activities would then bring about the astonishing properties ofXist, namely its ability to specifically localize over the Xi and its ability to trigger chromosome-wide chromatin modifications and gene repression. This poses the following question: is the current knowledge of knownXist effectors sufficient for reconstitution ofXist function? The identification of the binding sites withinXist could make experimental testing possible as of today (The understanding of chromosome-wide silencing on the molecular level has seen impressive advances over the past few years. Progress has been driven by the development of new methodology in RNA protein biochemistry and mammalian genetics. A large number of factors have been identified that cannot all be discussed in this review for space reasons. We focus here on factors that have been implicated and shown to be relevant for understandingf today . CertainXist can initiate gene repression45. However, this view might change during the maintenance phase of XCI. Without repeats B and C, SMCHD1, and likely other factors, would not be recruited and establishing an epigenetic memory could potentially fail. This idea would suggest a role for Polycomb complexes in establishing a repressive memory in XCI, which is consistent with the traditional view of Polycomb function. Notably, interactions of the PRC2 subunits Ezh250 and Suz1251 withXist repeat A have previously been observedin vitro. However, the relevance of these observations for XCI remains unclear. Although Polycomb proteins might bind RNA directly, the requirement of hnRNPK in XCI suggests that these interactions are not sufficient in cells. Early studies have further proposed a role for repeats B and C inXist localization. Jeonet al. implicated YY1 as a DNA- and RNA-binding protein that bridgesXist repeats B and C RNA to repeat F DNA, facilitating the localization ofXist74. Consistent with this observation, peptide linked nucleic acids and locked nucleic acids complementary to repeat C sequences also induced delocalization ofXist76. These observations are puzzling when one considers that the deletion of repeats B and C does not abrogateXist localization or spreading at the initiation of XCI45. To reconcile these conflicting interpretations, one might consider different cellular contexts or experimental conditions similarly to the cell-type-specific requirement ofCiz1 forXist localization64.For repeats B and C, the situation appears less clear. Would Polycomb engagement be required for initiating gene repression? There is evidence that XCI could initiate without Polycomb recruitment, and a repeat B and C mutantXist77. However, it remains to be shown if repeat D is sufficient forXist localization or other interactions are also required such as with LBR. A prominent role ofXist repeat E in the maintenance but not in the initiation step of XCI has been uncovered through the identification ofCiz1. We speculate that both repeat D and repeat E could be important for the maintenance of XCI, where they might recruit factors independent of gene silencing but dependent on a chromosomal memory that is imposed by Polycomb complexes. Thus, repeats D and E ofXist could act as docking sequences for factors that preserve the integrity of heterochromatin of the Barr body and facilitateXist localization in a cell-type-specific manner. Taken together, the current understanding of molecular interactions ofXist appears to provide a detailed guide for reconstitution ofXist function. Performing these experiments will be important for establishing if the main mechanistic components have indeed been identified or key factors are still missing. In the latter case, a number of unstudied proteins from the list ofXist interactors represent a resource for further exploration.Repeat D could be handy for engineering RNAs that mimic the localization ofXist has moved the field towards a new frontier: the chromatin. Conceptually,Xist can be seen as a sender of a message. This view would predict a receiver on the chromosome. How X-linked gene promoters are inactivated on the chromatin level remains an open question. Just a few hundred molecules ofXist accumulate over the Xi and are capable of switching active genes and regulatory regions from an open into a closed chromatin configuration along an entire chromosome. This switch implies that the chromatin has the ability to read instructions fromXist and coordinate gene repression. Although some of these instructions come in the form of histone modifications, it remains unclear how silencing is spread. It is enticing to speculate if a domino-like effect might be triggered byXist that perpetuates changes along the chromatin fiber. Would this make histones domino pieces? Histone exchange can facilitate switching an active into a repressive chromatin configuration through the removal of actively marked histones. Besides, gene silencing is accompanied by compaction of the chromatin fiber and prevents regulatory proteins from binding and triggering transcriptional events. We speculate that nucleosome assembly and chromatin dynamics might be important aspects for silencing and spreading of repressive chromatin modifications (Xist remain to be discovered. Identification of these components can be predicted to have wide implications for the understanding of gene regulation in mammals.Progress in understanding the molecular functions ofcations . The facXist for the generation of a silent and stable Barr body are starting to be elucidated, albeit much remains to be understood. Disrupting X-linked gene silencing leads to female embryonic lethality, showing the importance of this phenomenon for the survival of mammalian species. Functional characterization of the different effectors engaged byXist can advance our understanding of the striking mechanisms that were selected by nature to warrant gene silencing for survival. This knowledge can be exploited therapeutically for the treatment of genetic disorders where a gene overdose impairs the correct function of cells, such as Down syndrome cell pathogenesis77, X-linked diseases78, or approaches involving cell reprogramming79. Thus, further unraveling the mechanisms used byXist and its effectors for silencing and heterochromatinization will contribute to advances in science and medicine.The molecular pathways used byXist, X inactivation-specific transcript.ESC, embryonic stem cell; HDAC, histone deacetylation complex; MEF, mouse embryonic fibroblasts; PCG, Polycomb complex group; PRC, Polycomb repressive complex; RRM, RNA recognition motif; Xa, active X chromosome; XCI, X chromosome inactivation; Xi, inactive X chromosome;


Drosophila genome encodes three BEN-solo proteins including Insensitive (Insv), Elba1 and Elba2 that possess activities in transcriptional repression and chromatin insulation. A fourth protein—Elba3—bridges Elba1 and Elba2 to form an ELBA complex. Here, we report comprehensive investigation of these proteins in Drosophila embryos. We assess common and distinct binding sites for Insv and ELBA and their genetic interdependencies. While Elba1 and Elba2 binding generally requires the ELBA complex, Elba3 can associate with chromatin independently of Elba1 and Elba2. We further demonstrate that ELBA collaborates with other insulators to regulate developmental patterning. Finally, we find that adjacent gene pairs separated by an ELBA bound sequence become less differentially expressed in ELBA mutants. Transgenic reporters confirm the insulating activity of ELBA- and Insv-bound sites. These findings define ELBA and Insv as general insulator proteins in Drosophila and demonstrate the functional importance of insulators to partition transcription units.The  Drosophila embryos, finding that ELBA and Insv function as general insulators and partition active chromatin to ensure proper gene activation in Drosophila.The BEN-solo proteins—including Insensitive (Insv), Elba1 and Elba2—function in both transcriptional repression and chromatin insulation. Here, the authors investigate the role of these proteins in  Historically, boundary or enhancer-blocking activities of newly identified insulators were mostly tested on a one-on-one basis in transgenic lines or genetically dissected for individual loci. Recent advances in genomics and chromatin structure capture techniques allowed more systematic identification of insulators and also assigned new properties to them in chromatin architecture organization (reviewed in refs. 8).Proper gene regulation requires coordinated activities of distinct classes of cis- and trans-regulators. Insulators (or boundary elements) are a special type of cis elements that constrain enhancer–promoter interactions7, several of the early studies on mammalian CTCF indicated that it functions in transcriptional repression10. More than a dozen of proteins have been shown to have insulator function in Drosophila11. According to the combinatory co-occupancy patterns of the five insulator proteins CP190, BEAF-32, CTCF, Su(Hw), and Mod(mdg4), Drosophila insulators were divided into two classes12. Class I insulators are mainly bound by CP190, BEAF-32, and CTCF in active chromatin regions proximal to promoters, while class II insulators are mostly bound by Su(Hw) located in distal intergenic loci. However, at the functional level, how these factors cooperate remains unclear.The activity of insulator elements depends upon their associated factors. The zinc-finger protein CTCF seems to be the only insulator protein conserved between vertebrates and invertebrates. In addition to its established roles as an insulator in chromatin organization, long-range regulatory element looping and enhancer segregating13. Several BEN-containing proteins including mammalian BANP/SMAR115, NAC117, BEND318, and the C isoform of Drosophila Mod(mdg4)19 have chromatin-associated functions and have been linked to transcriptional silencing. We and others showed that the BEN domain possesses an intrinsic sequence-specific DNA-binding activity. Mammalian RBB, a BEN and BTB domain protein, binds to and directly represses expression of the HDM2 oncogene through interaction with the nucleosome remodeling and deacetylase (NuRD) complex20. Drosophila Insv binds to a palindromic motif, TCCAATTGGA and its variants (TCYAATHRGAA), and represses genes in the nervous system21. Two other Drosophila BEN proteins, Elba1 and Elba2, along with the adaptor protein Elba3, are assembled in a heterotrimeric complex (ELBA) and associate with the asymmetric site “CCAATAAG” in the Fab-7 insulator22. elba1 and elba3 are closely linked in the genome and specifically expressed during the mid-blastula transition, which restricts ELBA activity to this early developmental window. Interestingly, the genes encoding Insv and Elba2 are also arranged next to each other in the genome, even though their gene products show different tissue specificity in later developmental stages.The BEN  domain is a recently recognized domain present in a variety of metazoan and viral proteins24. Our previous work demonstrated that Insv and ELBA BEN-solo factors share common properties, e.g., binding to the palindromic sites as homodimers and repressing reporter genes in cultured cells, but also display distinct activities, e.g., Insv being the only one that interacts with Notch signaling and its inability to bind to the asymmetric site23. Interestingly, the Fab-7 insulator requires ELBA for its early boundary activity, but also needs Insv in later development25.Most of the BEN-domain proteins contain other characterized motifs. However, Insv, Elba1, Elba2, and several mammalian homologs, such as BEND5 and BEND6, harbor only one BEN domain and lack other known functional domains. Thus, we refer to this sub-class as BEN-solo factorsDrosophila BEN-solo factors and the adapter protein Elba3, by analyzing DNA-binding preferences (symmetric versus asymmetric), chromatin binding inter-dependence (homodimers versus heterotrimeric complex) and mechanisms in gene regulation (repressor versus insulator). Our ChIP-seq (chromatin immunoprecipitation followed by deep sequencing) analyses show that ELBA and Insv bind many common and distinct genomic regions. Unexpectedly, Elba3 associates with chromatin even in the absence of its DNA-binding partners Elba1 and Elba2. Our ChIP-nexus  assay distinguishes asymmetric heterotrimeric binding pattern of Elba1 and Elba2 from symmetric homodimer pattern of Insv. Although all four factors repress transcription, only the ELBA factors genetically interact with GAF and CP190 and are required for embryonic patterning. Finally, we show that adjacent genes separated by ELBA binding are less differentially expressed in the ELBA mutants. Insv-associated adjacent genes do not show such a global effect, despite individual loci relying on Insv insulation. And ELBA- and Insv-bound elements block enhancer–promoter interaction in transgenic reporters. Collectively, these findings indicate a role of ELBA and Insv as general insulators in partitioning transcription units in Drosophila.It remains to be determined how the ELBA factors regulate gene expression and embryogenesis. In this study, we have comprehensively characterized the three 24. A few individual sites in the Fab-7 and the Fab-8 insulators were known genomic locations of the ELBA complex26. We intended to broaden this perspective by generating ChIP-seq data for each of the three ELBA factors from the blastoderm stage of embryos, which covers the peak expression of the ELBA factors23. For uniformity of comparing peak-calls, we re-made Insv ChIP-seq data in parallel with the ELBA libraries.We previously described genomic binding for Insv whose ChIP-seq peaks cover numerous genomic sites that bear its specific binding motif (CCAATTGG and variants thereof)elba1, elba2, and elba3  Fig. . This raMesh1) by ChIP-qPCR using a different Elba3 antibody than the one used in ChIP-seq  , indicating that Elba3 is an essential component for the endogenous ELBA complex to bind the genome. This was unexpected, given that Elba1 and Elba2 under ectopic expression can form homodimers in cultured cells23. Most of the Elba1 sites are lost in the elba2 mutant with 712 (∼20%) peaks left. Nearly all of the Elba2 sites are lost in the elba1 mutant, with 48 (∼3%) remaining. As the expression of the ELBA factors is not reduced in the non-cognate mutant condition   Fig. . We confely Fig. . The enrSince Elba3 has no DNA-binding domain, we asked whether Insv mediates Elba3 to the genome in the absence of Elba1 and Elba2. The overlapping fraction of Elba3 and Insv peaks is twofold higher in the Elba1/2-independent sites (50%) than that of the dependent ones (25%). However, half of the Elba1/Elba2-independent peaks do not overlap with Insv peaks  according to the signal to background ratio. After applying this cutoff, we defined ChIP-nexus peaks and compared motif occurrence frequencies between the ChIP-seq and ChIP-nexus data sets. ChIP-nexus increased the motif enrichment frequency for Elba3, but not for the other three factors  for the top 500 ChIP-nexus peaks that contain the Insv/ELBA motifs. The OI value is determined as the ratio of the number of reads from the dominant strand to the total number of reads from both strands. Elba3 and Insv peaks are symmetric at a global level as their OIs are mostly close to 0.5 Fig. . In cont23. As Elba3 can target chromatin independent of Elba1 and Elba2, we asked whether Elba3 can repress transcription independent of Elba1 and Elba2. To address this, we tethered Elba3 with the Tet repressor DNA-binding domain (TetR-Elba3) and examined its activity in Drosophila S2 cells on a luciferase reporter driven by an actin enhancer and the tet operator sites. Elba3 represses reporter expression with similar efficiency as the other three proteins  for the targets as a set (FDR < 1E-5 for Elba1/2/3 and FDR < 0.01 for Insv). While the target genes all display a trend of de-repression, the top-bound genes with the Insv/ELBA motifs show the highest fold change, and the genes with the motifs have higher fold change than those without the motifs Fig. . This reELBA mutants. However, the overlapping pattern of the upregulated genes resembles their ChIP-seq peak overlapping pattern  , and fewer with GAF and Su(Hw). The ELBA factors display similar co-occupancy patterns   Fig. , Table 1ELBA and insv mutants are viable and do not exhibit substantial embryonic patterning defects, the dose-sensitive interactions we observe with other insulator proteins support the notion that they impact developmental gene regulation and that insulators in flies are composed of redundant elements.Together, although ELBA and Insv associate with a subset of known insulator proteins, the ELBA factors seem to be selectively needed during early embryonic development in collaboration with other insulator proteins. Importantly, even though 12. As Insv and ELBA bind to class I insulators and are enriched in gene-dense regions, we asked whether these factors insulate adjacent genes to ensure transcription autonomy. As the early embryo contains abundant maternal RNAs, we used PRO-seq assay from 2–4 h wt and mutant embryos to identify real-time transcripts produced by RNA Pol II. We then did de novo PRO-seq peak calling to define actively transcribed genes and determined differential expression between every gene pair that is separated by an ELBA or Insv ChIP peak. In the ELBA mutants, there is a global reduction in the differential expression of paired genes that are separated by ELBA ChIP peaks . 2xPE is an enhancer from the twist gene locus that drives reporter expression in a ventral stripe in the early embryos. iab-5 is an element from the Abd-B region of BX-C and drives reporter expression in the posterior segments of the embryo. In between the two enhancers, there is a restriction site for inserting sequences to be tested for enhancer-blocking activity. As shown in Fig. LacZ and white will be expressed in both the 2xPE and iab-5 domains if the test fragment, for example, the uMar spacer, does not have insulation activity . Insv binding is always symmetric, suggesting that it binds to the sites as homodimers. This evidence shows ChIP-nexus can be a powerful tool to resolve binding symmetry by a heterotrimeric complex. It will be of interest to understand how the BEN domains have evolved in DNA-binding affinity and sequence specificity across species. Our previous and current work well exemplifies the approach to determine the molecular properties of a less studied DNA-binding protein family23.We used a high-resolution ChIP-nexus approach and confirm that Insv and the ELBA factors associate with both types of DNA motifs. The ChIP-nexus analyses also provided evidence that Elba1 and Elba2 in the ELBA complex bind to DNA in an asymmetric configuration. Intriguingly, at some of the loci, such as the asymmetric sites in 22. However, the effect of complete loss of ELBA in embryonic development has not been investigated. We generated the loss-of-function mutants for the ELBA genes and found that they are dispensable for viability. This is not surprising as other chromatin insulator proteins, such as dCTCF35 and BEAF-3236, are not required for viability. One possibility is that Drosophila utilizes multiple backup mechanisms to ensure boundary fidelity. Indeed, when one copy of CP190 or GAF is removed, loss of ELBA led to drastic developmental consequences in this sensitized background. Despite both ELBA and Insv associating with known insulator proteins, such as CP190, BEAF-32, and GAF 43. We show that the neighboring genes that are differentially expressed in wild type become more equally expressed in ELBA mutant embryos. In this case, all three types of promoter configurations, divergent, tandem, and convergent, have similar requirement for ELBA-dependent insulation. Together with the evidence that genomic elements bound by ELBA and Insv are sufficient to block enhancer–promoter interactions in transgene assays, our results suggest ELBA is required to ensure the autonomous regulation of linked transcription units.Genes in the homie-nhomie44 and scs and scs’ loci45. Future studies will be needed to determine the roles of ELBA and Insv in chromatin organization.New properties have been assigned to insulators recently, especially in chromatin organization and long-range cis-element interactions. In this work, we focus on the functions of ELBA and Insv in active chromatin regions because of their enrichment in close proximity to active promoters. However, we have detected enrichment of ELBA and Insv in several known elements that could mediate long-range interactions, such as the 23B was described previously46. ELBA mutants were created using CRISPR: transgenic flies carrying single-guide RNA targeting the coding sequence of each gene were crossed into the nos-Cas9 transgenic flies. Frame-shift mutations were identified by PCR and Sanger sequencing. The TrlR85 and Trl13C alleles were kindly provided by Dr. Ana Busturia (Centro de Biología Molecular “Severo Ochoa” CSIC-UAM), the CP190P11 allele was from Bloomington Stock Center and used for genetic interaction crosses.All fly stocks were kept at 25 °C. The insv mutant allele insv34). The sequences of cloning oligos are provided in Supplementary table For making the insulator transgenes, selected fragments were amplified and cloned into the insulator transgene backbone  for 20 min. For immunostaining, embryos were rehydrated, washed with PBTween (0.1% Tween in PBS), and blocked with PBSBT . The primary rabbit anti-Elba1 or anti-Elba3 were added to the samples in 1:150 and 1:500 dilution with PBSBT, respectively, and incubated at 4 °C overnight. After extensive washing with PBSBT (5–15 min), the secondary antibody  was added in 1:500 dilution and incubated at room temperature for 2 h. After washing with PBSBT and then PBTween, the embryos were counterstained with DAPI and mounted in Vectashield (Vector Laboratories).LacZ and white probes were generated by transcription from linearized pBluescript template plasmids (kindly provided by Dr. Mattias Mannervik) with T3 or T7 RNA polymerase (Thermo Fisher) and Dig RNA labelling mix (Roche) according to the manufacturer. Embryos were aged and fixed with 9% formaldehyde in fixation buffer  for 25 min. For in situ hybridization, fixated embryos were permeabilized with xylene and rehydrated as well as postfixated with 5% formaldehyde in PBT  for 25 min. Embryos were treated with proteinase K (4 µg/ml) for 8 min, followed by another round of postfixation for 25 min, before hybridization with the probes at 55 °C for overnight in hybridization buffer . Samples were incubated with alkaline-phosphatase-labeled anti-digoxigenin antibody  overnight at 4 °C, and developed with 0.6 mg/ml nitrotetrazolium blue chloride (NBC) and 0.3 mg/ml 5-bromo-4-chloro-3-indolyl phosphate disodium salt (BCIP). Samples were dehydrated by repeated washes in ethanol, rinsed in xylene, and mounted in Permount (Fisher).The elba1SK6, elba2SK2, elba3SK3, and insv23B were collected and aged for 2–4 h. In each IP, 50 µl of embryos were used. Protein extracts were prepared by homogenizing the embryos in lysis buffer . About 5 µg of rabbit anti-Elba1, anti-Elba3, and rabbit IgG were used in the first round of immunoprecipitation. The protein/antibody complex was then precipitated with the Gamma-bind G beads (Thermo Fisher) and separated on 10% of SDS-PAGE for western blotting. The left supernatant was re-precipitated with the antibodies against Elba1, Elba3, and Insv46 . The same antibodies were used in blotting. To minimize unspecific signal from IgG heavy chains, light-chain-specific secondary antibodies of mouse anti-rabbit IgG  were used, and the signals were developed with ECL Plus reagent .Embryos from wild-type (wt), Drosophila S2-R + cells grown in Schneider Drosophila medium containing 10% fetal calf serum. Cells were co-transfected with TetR fusion, 2xTetO-Firefly luciferase and pAc-Renilla plasmids in 96-well plate using the Effectene Transfection kit (Qiagene). Luciferase assays were performed and measured using the Dual Luciferase Assay System (Promega). Expression was calculated as the ratio between the firefly and Renilla luciferase activities. Three rounds of transfections were performed on different days and different cell populations, and considered as three biological replicates. Within each biological replicate, four wells of cells were transfected with the same DNA mixture and measured and thus considered as four technique replicates. In total, for each DNA combination, the values from the 12 wells of transfection were calculated, averaged, and plotted in Fig. To generate the TetR-DBD fusions with the ELBA factors and Insv, the open-reading frames of Elba1, Elba2, Elba3, and Insv were PCR amplified and cloned into the pAC-TetR vector. All transfections were performed using elba1SK6, elba2SK2, elba3SK3, and insv23B were collected and aged for 2–4 h (50 µl of embryos per reaction). After dechorionization in bleach and rinsing with water, embryos were homogenized in 1.8% formaldehyde and 2.5 mM DSG (Di(N-succinimidyl) glutarate, Sigma) in buffer A1 ). Chromatin was extracted by repeatedly spinning at 4000xg and washing in buffer A1 . Cross-linking occurred for 10 min on ice in LysisPlus buffer ). Sonication was done using a Bioruptor (Diagenode) in high-power mode, 15 cycles of 30 s ON/30 s OFF. The samples were pre-cleared with Gamma-bind G sepharose beads  overnight and the beads removed. The ELBA antisera used for IP were tested in ChIP previously26 and kindly provided by Dr. Paul Schedl (Princeton University). Two different sets of antibodies were used for ChIP-qPCR and ChIP-seq, respectively. For each ChIP reaction, 5 µl of antibody were added to the respective sample, and the tubes rotated at 4 °C overnight. New beads were then added to the samples and binding occurred for 4–16 h before transferring the beads in 200 µl LysisPlus buffer (without sarcosyl) onto Ultrafree Filter columns . The beads were washed on the filters four times for 10 min with LysisPlus (no sarcosyl) and twice with TE . Elution of the samples from the beads occurred by first adding 100 µl EB1  for 30 min at 65 °C, then adding 100 µl EB2 (TE + 0.67% SDS) for 30 min at 65 °C, and spinning the whole elute through the filters at 3000 rpm for 2 min in a tabletop centrifuge. All samples as well as input samples (in 200 µl TE + 1%SDS) were reverse cross-linked at 65 °C overnight, digested with 2 µl 20 mg/ml proteinase K (Thermo Fisher) for 2 h at 42 °C, and RNase digested with 2 µl 0.5 mg/ml RNase . The DNA was purified using the QIAquick PCR Purification Kit (Qiagen) with two times elution in 30 µl each.For chromatin immunoprecipitation (ChIP), embryos from wild-type (wt), All qPCR reactions were run in technical triplicates using iTaq Universal SYBR Green Supermix (Bio-Rad). In all, 1 µl of chromatin template was used in each 20 -µl reaction, and amplification was normalized to 0.2% input. qPCR oligos are provided in Supplementary Data Drosophila melanogaster (dm3) genome assembly using Bowtie2 with the default parameters, after the adaptor trimming by Trimmomatic. The uniquely mapped reads with a mapping quality MAPQ > 20 were used for further analysis. For all ChIP-seq samples, we generated coverage tracks at 1-nt resolution and normalized to the library sizes to give read per million (RPM) in “bigwig” format. We further created the coverage differential tracks for four factors by subtracting the mutant from wild-type coverage (log2 wt/mutant).ChIP-seq libraries were made using the NEBNext Ultra™ II DNA Library Prep Kit and sequenced in the Illumina Hi-seq platform. The ChIP-seq reads were mapped to the 47 with default parameters and the confident peaks were determined by an FDR < 1%. The peaks overlapped with Drosophila blacklist were also removed. Peak overlap analysis was performed by “mergePeaks” function in Homer2 package with the default parameters, and a maximum distance was set to “-d given” which requires the peak regions overlap.For each of the four factors, the peak calling was performed by the ChIP-seq reads of wt or a mutant condition to its own mutant ChIP or IgG or Input. The peaks were called using MACS248. We extended the summits of the called peaks by 500 nucleotides in each direction, and searched for 5–15 nt motifs in the central regions (100 nucleotides) using default parameters.The de novo motif search was performed for all the called peaks for each factor by MEME-ChIP12), including ChIP–ChIP data for CP190, BEAF-32, CTCT, GAF, Mod(Mdg4), and Su(Hw). As ChIP-seq peaks are generally narrower than ChIP–ChIP peaks, we extended the ChIP–ChIP summits by 100 nucleotides in each direction for the overlapping analysis. We used a maximum distance of 50 nucleotides between the peak summits for overlapping. The overlap fraction between the two sets was calculated by the number of overlapped peaks divided by the minimum number of peaks of two sets.Pairwise peak overlaps were performed for the ChIP-seq peaks of Elba and Insv factors with the modEncode insulator data sets  was first removed, and the random 4nt barcode was retained for each read. After the 3′ adaptor trimming by Trimmomatic, the sequencing reads were collapsed to only include unique reads. The random 4nt barcode was further removed, and the reads with at least 22 nucleotides were retained for mapping. We mapped the reads using bowtie with the parameter setting “-k 1 -m 1 -v 2 --best --strata”. Similar to ChIP-seq, we generated normalized coverage tracks separately for each strand in “bigwig” format. Similar to the ChIP-seq data, the ChIP-nexus peak calling was performed by MACS2 using the default parameter. To obtain highly confident binding sites for each factor, we set a highly stringent cutoff . We further required the binding sites to be called by both ChIP-seq and ChIP-nexus. Defined high-confident peaks are summarized in Supplementary Data To examine the asymmetry of the binding sites, we calculated an OI for each binding site by ChIP-nexus for each factor. OI was defined by maximum number of reads between two strands divided by the sum of reads of two strands, max /sum , ranging from 0.5 to 1. The OI values for the peaks are included in Supplementary Data The total RNA was extracted from stages 2–4 h embryos using Trizol reagent (Invitrogen). RNA quality was tested by the Agilent Bioanalyzer.23B allele. The qPCR reactions were run in technical triplicates using iTaq Universal SYBR Green Supermix (Bio-Rad). Amplification for each sample was normalized to the housekeeping gene RpL32. Four biological replicates were performed. qPCR oligos are provided in Supplementary Data For RT-qPCR, the total RNA was first cleared from residual DNA using Turbo DNA-free kit (Invitrogen), and were then reverse-transcribed using Superscript III Supermix (Invitrogen). qPCR primers for Elba1, Elba2, and Elba3 were so designed with one primer binding on top of the CRISPR cut site and those for insv are inside of the deletion of the insvRNA-seq libraries were made using the Illumina Truseq Total RNA library Prep Kit LT. Sequencing was performed on the Illumina Hiseq2500 platform.Drosophila melanogaster (dm3) genome assembly using HISAT2. RNA-seq signal was normalized by the TMM method implemented in the Limma Bioconductor package49. The gene annotation was obtained from FlyBase. Differentially expressed mRNAs between BEN factors mutants versus wild type were identified, and FDR (Benjamini–Hochberg) was estimated. Calculated differential expression is presented in Supplementary Data After trimming the adaptor sequences using Trimmomatic, the RNA-seq reads from the replicated wild type (x3), and mutant samples (x3) were mapped to the To test whether a set of genes are significantly changed (up- or downregulated as a gene set) among the differentially expressed (DE) genes from wild-type and mutant RNA-seq data, the gene set enrichment testing function “camera” in the R limma package was used. “camera” is a ranking based gene set test accounting for inter-gene correlation, to test whether the called peaks in five Elba3-binding subsets are significantly changed as a set.50. Drosophila embryos of 3.5–4.5 h age were dechorionated and homogenized in 1 ml of buffer A . The homogenate was filtered through a nylon membrane (pore size 0.8 µm) and spun down at 500×g. The pellet was resuspended in fresh buffer A and spun down several times for washing. Nuclei were then resuspended in buffer D  to achieve a final concentration of roughly 106 cells per 5 µl before freezing in liquid nitrogen. Run-on reaction was performed for 3 min at 30 °C after adding 2x reaction mix to the samples . Nascent RNA was extracted directly after with Trizol, precipitated in ethanol, and fragmented by base hydrolysis in 0.2 N NaOH on ice for 10 min. After neutralization with 1 M Tris-Cl pH 6.8, salts and free NTPs were removed by buffer exchange on a P-30 column (Bio-Rad). To enrich biotin-labeled RNA, the samples were bound to 30 µl M280 streptavidin beads (Invitrogen) according to the manufacturer and washed with twice with high salt buffer , twice with binding buffer  and once with low salt buffer . RNA was extracted from the beads twice with 300 µl Trizol in each round and precipitated with glycoblue in ethanol. RNA pellets were redissolved in 4 µl of 12.5 µM reverse 3′ adaptor dilution  and incubating at 37 °C for 1 h. 5′-hydroxyl was performed by directly adding PNK (NEB) reaction mix to each decapped sample. The total reactions of 100 µl volume  were incubated at 37 °C for 1 h. The RNA was then extracted using Trizol and precipitating in ethanol. Pellets were redissolved in 4 µl of 12.5 µM reverse 5′ adaptor  in 20 µl of final reaction volume. Water, dNTPs  and reverse transcription primer  were added first, samples heated to 70 °C for 2 min, chilled on ice for 2 min and the remaining reaction components except for the enzyme added . After incubation at 37 °C for 5 min, the enzyme was added (15 U/µl) and the reaction run in a PCR machine: 45 °C for 15 min, 50 °C for 40 min, 55 °C for 10 min, 70 °C for 15 min. The ideal amount of amplification cycles was determined by makig serial dilutions of the template and test amplification with primer RP1 TrS, primer RPI TrS short  at 50 °C for 13 h. After spinning down at 10,000xg for 5 min and retrieving the eluted library, a second extraction round was performed with 600 µl buffer for 2 h and the supernatants pooled. Eluates and remaining gel fragments were filtered through Spin-X filters (Corning), and the volume reduced to 500 µl in a speed vacuum centrifuge. The DNA was then extracted using buffered phenol–chloroform, and the concentration measured. Barcoded libraries were pooled and sequenced on the Illumina Hiseq2500 platform with 1 × 50 bp SR configuration.The protocol was adapted from Kwak et al.Drosophila melanogaster (dm3) genome assembly using BWA with the default parameters. We also generated the PRO-seq coverage tracks  with separate strands for each factor. To detect de novo transcripts from PRO-seq, we combined all genotypes and adapted the Homer251 GRO-seq transcript identification method (using a parameter setting “findPeaks -style groseq -tssFold 4 -bodyFold 3”). The pausing regions (promoter region) were defined from the de novo transcript starts to 200 nt downstream, and the gene-body regions were defined from 400 nt downstream to the end of the de novo transcripts. The de novo transcripts having a promoter expression of greater than 1 transcript per million (TPM) were retained for further analysis. Fold difference between adjacent gene pairs is summarized in Supplementary Data The adaptors were first trimmed from the sequencing reads by cutadapt software and the reads with at least 15 nt were retained. We then removed reads that mapped to rRNAs, and the remaining reads were further mapped to the For each Elba/Insv high-confident binding site, which was called by both ChIP-seq and ChIP-nexus (see Methods above), we looked for the adjacent PRO-seq promoter pairs. We then calculated absolute differential expression between the adjacent promoter pairs (abs log2FC adjacent pair). We further classified the adjacent promoter pairs flanking an Elba/Insv peak into three types: convergent, divergent, and tandem.P-values were calculated by dividing the number of instances that show a higher fold change between the random adjacent genes than that between the Elba/Insv-bound genes by 2000 iterations. These were done separately for convergent, divergent, and tandem pairs in each of wt and four mutants.To test whether the expression change between the adjacent pairs is above background, we performed a Monte-Carlo simulation. We randomly located the same number of regions with the same length as the Elba/Insv ChIP peaks in the same chromosome and repeated the random selection and calculation 2000 times. Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6


Endocan, also known as endothelial cell specific molecule-1 (ESM1), is a 50 kDa soluble proteoglycan which is frequently overexpressed in many cancer types. Whether it is dysregulated in head and neck squamous cell carcinoma (HNSCC) has not been investigated.We analyzed the expression of ESM1 using bioinformatics analysis based on data from The Cancer Genome Atlas (TCGA), and then validated that ESM1 was significantly overexpressed in human HNSCC at the protein level using immunohistochemistry. We also analyzed the genes co-expressed with ESM1 in HNSCC.The most correlated gene was angiopoietin-2 (ANGPT2), a molecule which regulates physiological and pathological angiogenesis. Several transcription factor binding motifs including SMAD3, SMAD4, SOX3, SOX4, HIF2A and AP-1 components were significantly enriched in the promoter regions of the genes co-expressed with ESM1. Further analysis based on ChIP-seq data from the ENCODE (Encyclopedia of DNA Elements) project revealed that AP-1 is an important regulator of ESM1 expression.Our results revealed a dysregulation of ESM1 and a potential regulatory mechanism for the co-expression network in HNSCC.The online version of this article (10.1186/s12935-019-0833-y) contains supplementary material, which is available to authorized users. TP53, CDKN2A, PTEN, PIK3CA, HRAS, NOTCH1, IRF6, and TP63 are frequently mutated in HNSCC [Head and neck squamous cell carcinoma (HNSCC) includes many cancers in the head and neck originating from a variety of sub-sites including the lip, oral cavity, nasopharynx, oropharynx, and larynx. HNSCC is the sixth most common cancer worldwide. There are about 650,000 new cases and nearly 350,000 patient deaths from HNSCC annually . The mosin HNSCC . As for in HNSCC  and metain HNSCC , have beEndocan, also called endothelial cell specific molecule-1 (ESM-1), is an endothelial cell-associated proteoglycan . It is uThe differential expression of ESM1 has not been investigated in HNSCC. In this study, we analyzed the expression of ESM1 in cancerous and adjacent normal HNSCC tissue using RNA-seq data from The Cancer Genome Atlas (TCGA) , and we http://xena.ucsc.edu/). In the UCSC-hosted database, TCGA data sets are normalized and can be explored and downloaded.The Cancer Genome Atlas (TCGA) data related to HNSCC were downloaded from Xena public data hubs  was estimated using the GISTIC2 method . GISTIC2The BioXpress database, which also uses TCGA data, was used to query differential expression .2O2 for 10 min, and then EDTA pH 9.0 for 1 min 50 s. For immunohistochemical analysis, the slides were incubated with anti-ESM1  (1:300) for 1 h at 37 °C. After thorough washing with PBS, the slides were incubated with horseradish peroxidase (HRP) conjugated anti-rabbit IgG at 37 °C for 15 min, and then thoroughly washed again. After washing, bound antibody was detected using the 3,3′-diaminobenzidine (DAB) reaction. Nuclear counterstaining was performed with hematoxylin. Control sections were subjected to the same procedure except that the first antibody was eliminated from the incubation. Positive staining was seen as a brown color of varying intensity, and a positivity score was assigned for statistical analysis (Chi squared test).After informed consent had been obtained, all specimens were collected from patients. Twenty-one cases of laryngeal or hypopharyngeal squamous cell carcinoma were studied. Paraffin embedded cancer tissue and peri-cancerous tissue were selected for the immunohistochemical tests. After dehydration, transparent, paraffin embedded, frozen tissues were made into 2 μm serial sections. Slides of tissue were incubated for 40 min at 70 °C, rehydrated in alcohol solution, and then washed with water. Then the slides were treated with 3% HFor immunofluorescence staining of ESM1 and ANGPT2, paraffin-embedded 3 μm serial sections of five cases of laryngeal or hypopharyngeal squamous cell carcinoma samples were deparaffinized and rehydrated. Preheat EDTA 8.0 was used for repairing in the high pressure cooker. Polyclonal rabbit anti-human primary antibodies anti-ESM1/FITC  and anti-ANGPT2/TRITC  (1:100) were applied overnight at 4 °C. After washing, fluorescently conjugated secondary antibodies were used. Nuclear counterstain was achieved using DAPI staining. All fluorescently stained images were taken using an Olympus BX-51 upright light microscope . Each site was imaged in all channels and overlaid in DPViewer version before examination in Photoshop.http://homer.ucsd.edu/) [http://genome.ucsc.edu).The HOMER (Hypergeometric Optimization of Motif EnRichment) program package   was usedESM1 copy number and gene expression in 514 common HNSCC samples. As shown in Fig. ESM1 has frequent heterozygous loss of copy number in HNSCC with a ratio of about 36.97% (193/522) compared to gain of copy number . However, there is no apparent correlation between copy number variation (CNV) and gene expression  data have become an important and widely used resource in cancer research . As for Because these results from TCGA data were at the RNA level, we then detected ESM1 expression at the protein level in the 21 laryngeal or hypopharyngeal cancer samples. As shown in Table ESM1 was not associated with copy number alteration in HNSCC, we next investigated the potential regulatory mechanisms, mainly focusing on transcription factors (TFs). Generally, transcription factor search tools identify potential TF binding sites (TFBSs) by sequence matching, which often results in dozens or even hundreds of candidate TFBSs and thus it is difficult to identify the true transcription factors that have important regulatory roles. Therefore, we first identified the co-expressed genes based on Pearson correlation. In total, there were 85 genes with r ≥ 0.45 and all of these genes were significantly overexpressed in HNSCC based on our analysis  with a correlation coefficient (r) of 0.7133   of the 85 correlated genes. As shown in Fig. We then used ChIP-seq data from the ENCODE project to filter the results. We found that only AP-1, which is a heterodimer composed of proteins belonging to the c-Fos, c-Jun, ATF and JDP families, overlapped in the promoter region of ESM1 Fig. b. We alsEndocan is a 50 kDa soluble proteoglycan secreted by vascular endothelial cells, especially from the inflamed endothelium, thereby it is also thought to play a role in the pathogenesis of vascular disorders, inflammation and endothelium dysfunction . It can An early study showed that Ets-binding motifs were mainly responsible for endothelial-cell-specific expression of ESM1 in vitro, though putative binding sites for GATA, AP1, AP4, NF1, and CREB/ATF transcription factors were also speculated . We alsoBesides AP-1, ChIP-seq from the ENCODE project also suggests that other transcription factors such as STAT3 , TBP (TATA-box binding protein), GATA2 (GATA binding protein 2), RAD21 (RAD21 cohesin complex component) and MYC  are also potential regulators of ESM1. Considering the genes co-expressed with ESM1, AP-1 probably plays a key role but other factors may synergize the regulation. Further details still need investigation.ANGPT2. ANGPT2 can also be regulated by Ets-1 and AP-1 [We identified the genes co-expressed with ESM1 in HNSCC and the most correlated gene is and AP-1 , 40, furand AP-1 , whetherand AP-1 .ANGPT1 is critical for vessel maturation, adhesion, migration, and survival, but ANGPT2 is an antagonist of ANGPT1 promoting cell death and disrupting vascularization;  however,In conclusion, we have identified that ESM1 is overexpressed in HNSCC and investigated the regulatory mechanism of ESM1-centered co-expression. These results provide important functional clues for ESM1 dysregulation and regulation in cancers.Additional file 1. ESM1 expression of 21 paired samples of HNSCC with clinical and pathological features.