Drosophila cells. Our results suggest that different rate-limiting steps in the transcription cycle are affected by dCas9-CBP and the SAM system, and that comparing these activators may be useful for mechanistic studies of transcription as well as for increasing the number of hits in genome-wide overexpression screens.Gene overexpression through the targeting of transcription activation domains to regulatory DNA via catalytically defective Cas9 (dCas9) represents a powerful approach to investigate gene function as well as the mechanisms of gene control. To date, the most efficient dCas9-based activator is the Synergistic Activation Mediator (SAM) system whereby transcription activation domains are directly fused to dCas9 as well as tethered through MS2 loops engineered into the gRNA. Here, we show that dCas9 fused to the catalytic domain of the histone acetyltransferase CBP is a more potent activator than the SAM system at some loci, but less efficient at other locations in  Fusion of transcription activation or repression domains to dCas9 has been used to precisely modulate gene expression from gene promoters, both in cell culture and in Drosophila3. dCas9 can also be fused to histone or DNA-modifying protein domains. Such EpiEffectors have been used to identify and characterize functional regulatory elements in mammalian cells1. One such EpiEffector, dCas9-p300core has been used to target proximal and distal regulatory regions in mammalian cell lines4, and has been utilized in gain of function screens for cis-regulatory element activity5. The p300 core domain has also been fused directly to CRISPR/Cpf16, and adopted as a component of the CRISPR-Cas9 based Casilio and SAM systems where it is recruited through modified gRNAs8. Here, we describe a Drosophila dCas9-CBP EpiEffector that proved to be more efficient in activating some genes than the Synergistic Activation Mediator (SAM) system that targets three different transcription activation domains to the genome9. Drosophila CBP, also known as Nejire, is the sole homolog of the related mammalian CBP and p300 proteins10. CBP and p300 are acetyltransferases that target histone 3 lysine 27 (H3K27), H3K18, H4K8, several lysines in H2B, as well as many non-histone proteins12. CBP and p300 function as transcriptional co-regulators and interact with many different transcription factors10. Consequently, p300/CBP are present at many transcriptional enhancer sequences and p300/CBP ChIP-seq is a widely used approach to identify putative enhancers15. Interestingly, we find that both dCas9-CBP and SAM can function from a distance of tens of kb to activate gene expression, but genomic loci respond differently to these two activators. This indicates that dCas9-CBP and SAM target different rate-limiting steps in the transcription cycle, and suggests that dCas9-CBP could be useful for overexpressing genes that are refractory to activation by the SAM system.The type II clustered, regularly interspaced, short palindromic repeat/CRISPR-associated protein 9 (CRISPR-Cas9) system is a versatile genome engineering tool, and the development of nuclease-deactivated Cas9 (dCas9) fusion proteins allow for targeting of effector domains to virtually any genomic regionDrosophila cells, we compared a direct fusion of CBP’s histone acetyltransferase (HAT)-domain to catalytically dead Cas9 (dCas9), with fusions to the MS2 coat protein (MCP). In both cases, the fusions included the bromo-, RING-, PHD-, and catalytic domains from CBP . In the synergistic activation mediator (SAM) system9, MCP fused to the p65 and HSF-1 activation domains is combined with dCas9-VP64 and modified gRNAs containing two MS2 loops  in the CBP catalytic core that we previously showed disrupts the catalytic activity of CBP17. These different constructs were placed under UAS promoters and transiently transfected into S2 cells together with actin5C-Gal4 activator and a gRNA with MS2 loops that targets the twist (twi) gene promoter , dCas9-CBP is also significantly more potent than MCP-CBP combined with dCas9, and the MCP-CBP fusion caused increased expression in the absence of specific gRNA at several loci  promoter, whereas the SAM system did not have a statistically significant effect on this gene  expression were also activated by dCas9-CBP when targeted to the respective promoter, this was not as efficient as the SAM system , hindsight (hnt), and short-gastrulation (sog) genes, dCas9-CBP failed to activate transcription whereas the SAM system could activate them weakly  could be weakly activated by dCas9-CBP bound to the twi promoter  gene whose promoter is located more than 50 kb from the en promoter could be activated by dCas9-CBP with en gRNA  as a mark of active enhancers and promoters, and H3K27 tri-methylation (H3K27me3) as a mark of Polycomb-silenced regions. Both poorly and moderately expressed genes  are decorated with H3K27me3 to different extents in S2 cells, but only the promoters of the moderately expressed genes hnt, sog and Snoo are enriched for H3K27ac  embryos Table . No cons22, we listed the motifs that are present in gene promoters. Interestingly, the sna, hnt, and sog genes all contain a TATA-box and do not respond to dCas9-CBP, whereas twi, wg and en that are activated by dCas9-CBP lack a TATA-box but contain the Initiator (Inr) motif 9, and cloned it into two types of UAS containing plasmids, the pUAST attB vector for S2 cell and somatic tissue specific expression with EcoRI and XhoI restricition sites31, and the pUAS.K10 vector for female germline expression with KpnI and XbaI restricition sites32. We also amplified the DNA region which includes MS2 coat protein (MCP), NLS, p65 activation domain and HSF1 activation domain from the lenti MS2-P65-HSF1_Hygro plasmid (Addgene 61426)9, and cloned it into the pUAST and pUAS.K10 vectors using the same restriction enzymes as above. The 5′primers included the Kozak sequence TCAAC in both cases. To modify this system, we cloned dCas9 and NLS without VP64, as well as MCP and NLS without activation domains into the pUAST and pUAS.K10 vectors. For the pUAS.K10 constructs, the reverse primer had a few extra restriction sites which were added after the NLS sequence. The MCP plasmids were further modified by introducing a 3XFLAG tag by cloning annealed oligonucleotides at the XhoI site for pUAST-MCP (oligos UAST3xFLAGFw and UAST3xFLAGRw) and at AscI for pUAS.K10-MCP (oligos 3xFLAGK10Fw and 3xFLAGK10Rw).We further amplified the dCas9 coding sequence (with D10A/H840A substitutions), nuclear localization signal (NLS) sequence and VP64 activation domain from the plasmid lenti dCAS-VP64_Blast (Addgene 61425)Drosophila CBP (nejire), including the bromodomain, PHD domain, and HAT domain, and cloned it into pUAST-MS2-NLS-3XFLAG and pUAS.K10-MS2-NLS-3XFLAG vectors using primers CBPhatUASTFw and CBPhatRw with Acc65I and XbaI restriction sites for pUAST-MS2-NLS-3XFLAG, and CBPhatK10Fw and CBPhatRw with AscI and XbaI for pUAS.K10-MS2-NLS-3XFLAG. A F2161A substitution that abolishes CBP HAT activity in vitro17, was created by amplification from a mutant plasmid17 with the same primers and restriction sites. We also fused the CBP wild-type and F2161A HAT domains directly to dCas9 using Cas9.CBPhatUASTFw and CBPhatRw primers and KpnI and XbaI sites for pUAST-MS2-NLS-3XFLAG, and CBPhatK10Fw and CBPhatRw with AscI and XbaI for pUAS.K10-MS2-NLS-3XFLAG. All of the resulting plasmids were sequence verified.To make CBP fusion constructs we amplified aa 1696–2329 from twi and en genes in order to compare the effect of dCas9-CBP using gRNAs with or without MS2 loops. The dCas9-VPR activation system (Addgene 78898) was used for comparison, wherein catalytically dead dCas9  is directly fused to the VP64-p65-Rta activation domains under control of the Actin5C promoter33.We also used the unmodified pCFD3 plasmid for targeting the twi, wg, sna, AttC, en and hnt promoters from a published article33. For other genomic regions we used the gRNA design software tools http://tools.flycrispr.molbio.wisc.edu/targetFinder/ and http://crispr.mit.edu/. Primers containing homology to the target genes were annealed and cloned into the pCFD3Aptamer plasmid at the BsmBI site. gRNAs for twi and en were also cloned into pCFD3. All the resulting gRNA targeting plasmids were sequence verified.We adopted the gRNA targeting sites for Drosophila S2 cells were maintained at 25 oC in sterile filtered Schneider’s medium (Gibco) with 10% FBS and 1:100 Penicillin-Streptomycin . For seeding, one million cells in 1600 ul of media per well were plated in six well plates for 24 hours before transfection, and plasmid DNA was added to the cells with Effectene Transfection reagent (Qiagen) at a 1:25 ratio. We co-tranfected 250 ng of pAC-GAL4 (Addgene 24344) with 125 ng each of pUAST-dCas9-VP64 or pUAST-dCas9, and pUAST-MCP-p65-HSF1 or pUAST-MCP-CBP or pUAST-MCP-CBPF2161A, and pCFD3Aptamer gRNA plasmids. We used 125 ng each of pAC-GAL4 and pUAST-dCas9-CBP or pUAST-dCas9-CBP F2161A and pCFD3Aptamer or pCFD3 gRNA plasmids. For dCas9-VPR transfections, we used 125 ng each of pAct-dCas9-VPR and pCFD3Aptamer or pCFD3 gRNA plasmids.Stable cell lines were established by co-transfecting all the components together with a pCoBLast plasmid, and Blasticidin S HCl (10 mg/ml) was added 24 h post transfection. Cells were maintained for one month in Blasticidin media before using them in ChIP-qPCR experiments.RP49 (RpL32). One-way ANOVA with post hoc Tukey test was used to calculate statistically significant differences . The total RNA was purified and concentrated by RNeasy MinElute Cleanup kit (Qiagen). Cleaned RNA was treated with DNaseI (Sigma-Aldrich) and reverse transcribed by the High Capacity RNA-to-cDNA kit (ThermoFisher Scientific) according to the manufacturer’s instructions. The cDNA was used in qPCR with 5X HOT FIREPol Evagreen qPCR Mix Plus (Solis Biodyne) and primers listed in Supplementary Table S2 cells were lysed in ice-cold IP buffer  48 hours after transfection for total protein lysate extraction. Cell lysates were centrifuged at 18000 rpm at 4 °C to collect the supernatant and total protein concentration was estimated by Bradford assay. Blots were probed with the primary antibodies anti-Cas9 , anti-tubulin , anti-FLAG , anti-acetyl lysine , and HRP coupled secondary anti-mouse (P0260) and anti-rabbit (P0448) antibodies . Amersham ECL Select Western Blotting Detection Reagent  was used and signals imaged with a Chemi Doc XRS+ with Image Lab Software (BioRad).twi and sog promoters as well as a sog enhancer were generated by injecting pCFD3Aptamer plasmids into the nos-phiC31; attP40 strain (BL#25709). Transgenic flies were identified by their vermilion+ eye phenotype and verified by genomic DNA PCR. They were made homozygous by crossing with second balancer chromosome flies.Flies with gRNAs targeting vas-phiC31; attP-86Fb flies (BL#24749) were injected with pUAST-dCas9-CBP, pUAST-dCas9-CBPF2161A, pUAS.K10-dCas9-CBP, pUAS.K10-dCas9-CBPF2161A, pUAS.K10-dCas9 or pUAS.K10-dCas9-VP64. The pUAS.K10-MCP-p65-HSF1 plasmid was injected into vas-phiC31; attP-51D flies (BL#24483), whereas pUAS.K10-MCP-CBP and pUAS.K10-MCP-CBPF2161A were injected into nos-phiC31; attP-40 flies (BL#79604). Transformants were identified by their white+ eyes and correct integration confirmed by genomic DNA PCR.To integrate dCas9 fusions, alpha-tubulin-Gal4  maternal driver was combined with UAS-dCas9 and UAS-MCP fusions. Female progenies from this cross were mated with homozygous pCFD3Aptamer gRNA males and embryos collected.The 17.Egg-laying cages with apple juice agar plates were set up to collect and age embryos for 1–5 hours. Embryos were dechorionated in 50% bleach for 3 minutes, fixed and hybridized with digoxygenin-labeled RNA probes as previously described34, with a few modifications. Cells were crosslinked with 1% formaldehyde for 15 minutes at room temperature and the crosslinking reaction was stopped by 125 mM Glycine. Chromatin sonication was done in sonication buffer  using Bioruptor plus (Diagenode) at 30 sec ON/OFF at high power for 30 minutes to shear the chromatin at size ranges of 200–500 bp. Immediately after sonication, chromatin material was centrifuged at 18000 rpm for 10 minutes at +4 °C. Supernatant was transferred to new Eppendorf tube and sonication buffer without SDS and sarkosyl was added to dilute their concentration down to 0.1%. The following antibodies H3 (Abcam ab1791) and H3K27ac (Abcam ab4729) were used for immunoprecipitation. The eluted DNA was purified by using ChIP DNA Clean and Concentrator (Zymogen), and used in qPCR to measure the H3K27ac enrichment at the targeted genomic region. All ChIP-qPCR primer sequences are listed in Supplementary Table Chip-qPCR was performed as described previouslySupplementary Figures and TablesSupplementary Table 2


The many functions of phosphoinositides in cytosolic signaling were extensively studied; however, their activities in the cell nucleus are much less clear. In this review, we summarize data about their nuclear localization and metabolism, and review the available literature on their involvements in chromatin remodeling, gene transcription, and RNA processing. We discuss the molecular mechanisms via which nuclear phosphoinositides, in particular phosphatidylinositol -bisphosphate P2), modulate nuclear processes. We focus on PIP2’s role in the modulation of RNA polymerase I activity, and functions of the nuclear lipid islets—recently described nucleoplasmic PIP2-rich compartment involved in RNA polymerase II transcription. In conclusion, the high impact of the phosphoinositide–protein complexes on nuclear organization and genome functions is only now emerging and deserves further thorough studies. Phosphatidylinositol (PI) is a negatively charged glycerol-based phospholipid with a hydrophobic acyl tail and a hydrophilic inositol head. The hydrophobic acyl tail composition depends on the organism, as well as the tissue. In animals, the characteristic feature is a high content of stearic and arachidonic acids. All the stearic acid is linked to position sn-1, and all the arachidonic acid is linked to position sn-2. In plants, the main tail composition is of palmitic acid, which is the main saturated fatty acid in position sn-1, while linoleic and linolenic acids are the main unsaturated components in position sn-2. Similarly in yeast, palmitic acid is in position sn-1, with oleic and palmitoleic acids in position sn-2 [The inositol ring, or cyclohexane-1,2,3,4,5,6-hexol, can be reversibly phosphorylated at the 3′, 4′, and 5′ positions, generating seven different phosphorylated phosphoinositides—phosphatidylinositol 3-phosphate (PI(3)P), phosphatidylinositol 4-phosphate (PI(4)P), phosphatidylinositol 5-phosphate (PI(5)P), phosphatidylinositol 3,4-bisphosphate P2), phosphatidylinositol 3,5-bisphosphate P2), phosphatidylinositol 4,5-bisphosphate P2), and phosphatidylinositol 3,4,5-trisphosphate P3). The importance of phosphoinositides in the regulation of important cytoplasmic processes, such as cell proliferation and cell death, regulation of microtubules and microfilaments, membrane dynamics, cell architecture, and motility or modulation of ion channels and transporters, is well established and was reviewed elsewhere ,10,11,12To answer the question about the origin of intranuclear phosphoinositides, one could consider that cytoplasmic phosphoinositides translocate to the nucleus just before the nuclear envelope reassembles during telophase, and then become enclosed by the newly formed nuclear envelope. For example, it was shown that nucleolar PIP2 remains bound to nucleolar organizing regions (NORs) during mitosis . On the We may speculate that PI is imported to the nucleus, where it is subsequently phosphorylated. Indeed, many enzymes involved in phosphoinositide metabolism—kinases, phosphatases, and phospholipases—are present in the nucleus .Due to the lack of tools for in vivo visualization of phosphoinositide synthesis, researchers demonstrated the intranuclear metabolism of phosphoinositides using in vitro studies. Smith and Wells  observedEnzymes able to subsequently generate almost all phosphoinositides species were detected in the nucleus. Nuclear PI3KC2α and PI3KC2β kinases can phosphorylate PI and PI(4)P at the 3′ position of the inositol ring to form PI(3)P and PIP2 ,30. PI3KPI(4)P can be synthesized by two kinases, PI4Kα and PI4Kβ, which both phosphorylate PI at the 4’ position. Their activities were confirmed in the nuclei of human, mouse, rat, and yeast cells ,32,33. MA nuclear kinase that phosphorylates the inositol ring at the 5’ position is yet to be discovered, but nuclear PI(5)P can be formed by the type I phosphatidylinositol -bisphosphate 4-phosphatase P2 4-Ptase I). Inversely, nuclear PI(5)P can be phosphorylated by the type II phosphatidylinositol 5-phosphate 4-kinase β (PIP4KIIβ) to PIP2 . Jones eBoth PI(4)P and PI(5)P may serve as precursors for PIP2 synthesis. Type I phosphatidylinositol 4-phosphate 5-kinases (PIP5KI) utilize PI(4)P, whereas type II phosphatidylinositol 5-phosphate 4-kinases (PIP4KII) utilize PI(5)P as a substrate. Isoforms of both types, type I PIP5KIα and PIP5KIγ_i4, as well as type II PIP4KIIα, PIP4KIIβ, and PIP4KIIγ, localize to the nucleus ,43,44,45Nuclear PIP3 is generated by phosphorylation of PIP2 by class I PI3K kinases PI3Kβ and PI3Kγ or by inositol polyphosphate multikinase (IPMK) ,50,51,52In addition to kinases and phosphatases, PIP2 is a substrate for phosphatidylinositol-specific phospholipase C (PI-PLC). PI-PLC cleaves PIP2 and generates second messengers—inositol 1,4,5-trisphosphate P3) and 1,2-diacylglycerol (DAG) . The eukOne of the biggest challenges in the field is the reduced number of tools for in vivo visualization of phosphoinositides. Therefore, antibodies and phosphoinositide-binding domains are widely used to detect phosphoinositides in cell nuclei via light and electron microscopy ,73. GillHowever, how are amphipathic phosphoinositides retained in the hydrophilic intranuclear environment? We recently showed that PIP2 localizes to a specific, only recently described nuclear compartment—nuclear lipid islets (NLIs) . These gAnother possibility is that the hydrophobic acyl tails interact with hydrophobic protein modifications of intranuclear proteins and hydrophobic protein pockets, or form bigger hydrophobic complexes. Such interactions can shield fatty-acid chains of phosphoinositides from the hydrophilic environment. Indeed, PIP2 interacts with the myristoyl moiety of BASP1 transcription factor and anchors it at promoters of target genes . While aTo understand the different processes that involve nuclear phosphoinositides, a high-throughput study identified was carried out, and over 120 nuclear proteins showed binding to various phosphoinositides with different affinities . These fNuclear PIP3 is an important molecule implied in cell differentiation and anti-apoptotic signaling ,91,92. PThe direct binding of PIP3 to the long non-coding RNA LINK-A, which facilitates AKT recruitment to PIP3 resulting in the changes of AKT conformation and its activation, was described . AKT alsIn addition, PI(5)P affects ubiquitin signaling in the nucleus as it stimulates the activity of the speckle-type POZ domain protein (SPOP)–Cul3 ubiquitin ligase complex. PIP4KIIβ kinase is a key regulator of (SPOP)–Cul3 ubiquitin ligase, and the mechanism behind stimulation of (SPOP)–Cul3 activity is most probably mediated by PI(5)P through a p38-dependent signaling pathway .Due to the negatively charged inositol head, PIP2 interacts directly with positively charged histones H1 and H3 . In vitrAnother mechanism of PIP2 action in chromatin remodeling is through its direct interaction with Brg1, the ATPase subunit of the SWI/SNF-like BAF chromatin remodeling complex . PIPI(5)P can influence chromatin remodeling through proteins which do not contain PHD fingers . Two comHistone acetylation reduces the positive charge of histones and loosens their association with negatively charged DNA. As a result, DNA is more accessible for transcription factors which can facilitate DNA transcription ,107. It It was shown that PI(5)P binds to the PHD finger domain of certain nuclear proteins and modulates their binding to chromatin, as well as having a role in DNA damage response, ubiquitin signaling, and epigenetic regulation of gene expression. In particular, PI(5)P is an important player in DNA damage response through its interaction with the inhibitor of growth protein 2 (ING2) . FollowiArabidopsis plants, PI(5)P affects the level of H3K4me3 [Arabidopsis homolog of trithorax (ATX1) is a trimethyltransferase that acts on H3K4 at the promotor regions, binds to PI(5)P, and causes ATX1 detachment from promoters and translocation from the nucleus to the cytoplasm. These data suggest that the effect of PI(5)P binding to PHD domains can have different impacts on its binding partners [Furthermore, in  H3K4me3 . The Arapartners .Gelato et al.  describePIP2 and PI(5)P regulate transcription during myogenic differentiation. A knock-down of PIP4KIIβ, resulting in accumulation of PI(5)P, increases myotube formation through modulating TATA box binding protein 3 (TAF3). Both PI(5)P and PIP2 modulate TAF3 interaction with H3K4me3 and regulate expression of specific genes . It was Caenorhabditis elegans, depletion of PIP2 results in chromosome structural changes which lead to various defects during meiotic progression. The altered chromosome structure is accompanied by the increased transcription activity, which might indicate the DNA-damage-driven apoptosis in the C. elegans gonad [Both PIP2 and PIP3 can interact with steroidogenic factor 1 (SF-1) ,113, whins gonad . PIP2 forms a complex with the active form of RNA pol II, as shown by immunoprecipitation with anti-PIP2 antibody or a pull-down experiment with PIP2-coupled beads ,80,83. HPIP2 depletion from HeLa nuclear extract by anti-PIP2 antibody inhibits pre-mRNA splicing in vitro. However, addition of PIP2 itself into depleted extract does not restore splicing reaction. This indicates that not only PIP2 but also its binding partners—small nuclear RNAs (snRNAs) and the active form of RNA pol II—are necessary to restore splicing activity . HoweverOnce mRNA is matured, it is exported to the cytoplasm upon association with RNA-interacting protein complexes ,117. PIP2 degradation by PLC results in increased histone binding to the rDNA promoter [Transcription of rDNA by RNA pol I takes place in the nucleolus in specific compartments where tandem repeats of pre-ribosomal RNA genes are assembled ,121. Thepromoter . This suOur group showed that PIP2 also binds to another nucleolar protein—fibrillarin . FibrillMoreover, we showed that PIP2 contributes to the regulation of rRNA gene expression not only at the transcriptional, but also at the epigenetic level ,132. PI.Taking together, all these data indicate that phosphoinositides play multiple specific roles in the nuclear processes depending on their binding partners and physiological state of a cell.In order to regulate the nuclear functions, the processes in the cell nucleus are spatially and temporarily organized. It is, therefore, crucial to understand molecular interactions leading to the formation of the intranuclear order, such as spatial maintenance of chromosomal domains, transcription-dependent movements of gene loci in nuclear volume, and various epigenetic effects regulating gene expression. This review summarizes the origin and essential functions of nuclear phosphoinositides, and describes multiple roles of PIP2 in the regulation of gene expression, which are currently emerging.The available data suggest that PI is transported to the nucleus via PITPs ,17,18, wPhosphoinositides interact with nuclear proteins, resulting in changes of protein conformation, localization, and activity. In such a way, phosphoinositides modulate a variety of important nuclear processes such as chromatin remodeling and modification, DNA transcription, pre-mRNA processing, RNA export, cell-cycle regulation, and DNA damage response ,136,137.The compartmentalization of the nucleus and the existence of the different pools of the same phosphoinositides might allow local fine-tuning of the metabolism of the distinct phosphoinositides in the specific nuclear compartment at exact time points. This permits affecting only the particular process with no or minimal impact on the others. There is growing evidence that the organization of the membraneless intranuclear compartments like nucleoli, nuclear speckles, paraspeckles, and chromatin domains is due to phase separation through specific interactions between different parts of the complex molecules like phosphoinositides, proteins, and nucleic acids ,144,145.


Non-coding antisense transcripts arise from the strand opposite the sense strand. Over 70% of the human genome generates non-coding antisense transcripts while less than 2% of the genome codes for proteins. Antisense transcripts and/or the act of antisense transcription regulate gene expression and genome integrity by interfering with sense transcription and modulating histone modifications or DNA methylation. Hence, they have significant pathological and physiological relevance. Indeed, antisense transcripts were found to be associated with various diseases including cancer, diabetes, cardiac and neurodegenerative disorders, and, thus, have promising potentials for prognostic and diagnostic markers and therapeutic development. However, it is not clearly understood how antisense transcription is initiated and epigenetically regulated. Such knowledge would provide new insights into the regulation of antisense transcription, and hence disease pathogenesis with therapeutic development. The recent studies on antisense transcription initiation and its epigenetic regulation, which are limited, are discussed here. Furthermore, we concisely describe how antisense transcription/transcripts regulate gene expression and genome integrity with implications in disease pathogenesis and therapeutic development. Eukaryotic transcription of the protein-coding genes is a highly coordinated and complex process initiated by an assembly of general transcription factors and RNA Polymerase II at the promoter by an activator protein (activator), followed by elongation, and, finally, termination ,4,5,6,7.Antisense transcripts were originally identified in bacteria . Later, GAL gene cluster in yeast to study the mechanisms of antisense transcription initiation in a dextrose-containing growth medium that is permissive to antisense transcription but not to sense transcription [GAL1, GAL7, and GAL10 constitute the GAL gene cluster, a galactose-inducible genetic unit. In this cluster, GAL1 and GAL10 are divergent genes with a bidirectional promoter, while GAL10 and GAL7 are tandem genes. Such organization has significant implications in gene regulation through transcriptional interference [GAL10 coding sequence in a dextrose-containing growth medium that represses GAL10 sense transcription. Such GAL10 antisense transcription attenuates GAL1-GAL10 sense transcription. Using this system, we analyzed the mechanisms of antisense transcription initiation, from the 3′-end of the GAL10 coding sequence, in a dextrose-containing growth medium [GAL10 antisense transcription was found to be dependent on a Myb-related protein Reb1 that binds to the 3′-end of the GAL10 coding sequence [GAL10 coding sequence and 380 bp downstream of the translational stop codon [GAL10 coding sequence for histone H4 acetylation targeting TBPs (TATA-box binding proteins) and TBP-associated factors (TAFs). This forms the pre-initiation complex (PIC) in recruiting RNA polymerase II , while its sense transcription does not require TFIID (GAL10 antisense transcription does not depend on the 19S proteasome complex or 19S regulatory particle (19S RP) that is required for sense transcription  complex, is dispensable for GAL10 antisense transcription [GAL10 antisense transcription. Antisense transcription from other loci is likely to be similarly epigenetically regulated, something which needs to be elucidated further.As DNA is packaged into chromatin within nucleus, chromatin structure is likely to play an important role in regulating antisense transcription. Indeed, our recent studies demonstrated that NuA4 KAT was targeted to the antisense transcription initiation site at the 3′-end of the tylation . Such tatylation , similarn [PHO84 ,102,103.sequence ) promotehylation ,106. Thuhylation  is likelAL genes ,110,111.h medium ,96 that tor Gal4 ,89,96. Tcription ,113. HowAn antisense transcript can function by itself and/or by the act of its transcription in cis  and/or in trans (which regulates genes on other DNA strands). The trans effect is usually mediated by the antisense transcript, while the cis effect is generally due to the act of antisense transcription . Three-dHBA1) in α-thalassemia patients is repressed by antisense transcription, where an aberrant LUC7L (putative RNA-binding protein Luc7-like) RNA runs antisense to the HBA1 locus and methylates the CpG island at the promoter to repress the HBA1 gene expression [IGF2R through transcriptional interference and DNA methylation in mice [FLC (flowering locus C), namely COOLAIR (cold-assisted intronic non-coding RNA), increases histone H3 K27 methylation levels through recruitment of polycomb proteins to repress FLC expression in response to cold [FLC locus [PHO84 is upregulated upon chronological ageing and represses PHO84 sense transcription via histone deacetylation [Antisense transcription regulates sense transcription by affecting DNA methylation at the CpG islands at the promoter ,115. Forpression . Antisenpression ,117. For in mice ,119. Sen in mice . In plan to cold . In addiLC locus . In buddtylation . Furthertylation ,86,87. Itylation ,125,126.tylation ,117,127,tylation ,132,133,tylation ,134,135.Besides the functions discussed above, antisense transcription also controls mRNA splicing ,137,138,In addition to controlling sense transcription, chromatin structure, mRNA splicing and stability, translation, and generation/expression of non-coding RNAs, antisense transcription/transcripts are involved in the regulation of DNA damage response and repair. Cells are continuously attacked by genotoxic factors, and DNA lesions and damage are repaired by various cellular mechanisms, including transcription-coupled DNA repair ,155,156.CCND1  gene in response to genotoxic stress [CCND1 sense expression [CCND1 is associated with cell cycle arrest and check point activation for DNA repair. Another example is the long intergenic non-coding RNA-p21 (or lincRNA-p21) that is transcribed from the opposite strand of p21 (CDKN1A), a cell cycle regulator, in response to DNA damage [CCND1 that acts locally, lincRNA-p21 functions globally to repress transcription of the genes that are associated with apoptosis and DNA repair [INK locus is ANRIL . This is also induced by the transcription factor E2F1 (E2F transcription factor 1) in an ATM (ataxia-telangiectasia mutated)-dependent fashion in response to DNA damage [INK4A , INK4B  and ARF  [INK4A, INK4B and ARF permits cells to go back to their normal state following DNA repair, via the impediment of cell cycle checkpoints and stimulation of cell cycle progression [CDKN1A (p21) transcriptional start site. It is also induced in response to p53-dependent DNA damage [A number of studies have indicated that the expression of antisense transcripts is altered in response to DNA damage in order to control downstream gene expression for DNA repair. For example, transcription of an antisense non-coding RNA is induced from the upstream region of the c stress . Such dapression . The redA damage . Unlike A repair . AnotherA damage . The tra as p14) . Such algression . In addigression . Anothergression ,164,165.A damage . PANDA iA damage . Thus, DA damage ,168,169.HOXC locus, antisense to the HOXC11 and HOXC12 genes [HOXD locus, and silences transcription of the HOXD genes by altering the chromatin structure. Overexpression of HOTAIR induces genome-wide re-targeting of PRC2 to several hundred genes, leading to altered histone H3 K27 methylation, cancer progression and malignancy [H19/IGF2 on chromosome 11. Its overexpression is linked to cellular migration and invasion in various cancers including stomach, breast, liver, lung, and pancreas cancers [Antisense transcription/transcripts represent potential prognostic and diagnostic markers for therapeutic development for cancer. A promising candidate, HOTAIR, is significantly overexpressed in multiple tumors, including breast, colorectal, hepatocellular and pancreatic cancers ,175,176.12 genes . This an12 genes . HOTAIR lignancy ,175,176.lignancy ,175,176. cancers . Yoshimu cancers  reported cancers . MAPT-AS cancers .HOXA cluster antisense RNA 2), HOXA11-AS (HOXA11 Antisense RNA), PANDA, and ANRIL. WRAP53 regulates the tumor suppressor p53 and is overexpressed in a variety of tumor cell lines [There are other antisense RNAs that could be potential targets for cancer therapy. These include WRAP53 (WD repeat containing antisense to TP53), HOXA-AS2 , is associated with Alzheimer’s disease. BACE1-AS is a 2 kb long transcript originating from BACE1 (β secretase 1) in the antisense orientation [PINK1 antisense RNA), is transcribed from the antisense direction of the PINK1 gene that encodes PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced putative kinase 1. Mutation in the PINK locus causes the early onset of Parkinson’s disease. The PINK1-AS stabilizes the expression of a PINK1 splice variant, svPINK1, via a double strand RNA-mediated mechanism [In addition to their association with cancer, antisense transcripts are also involved in neurological disorders. The characterization of these transcripts and their modes of action may allow them to be used for diagnosis, monitoring disease progression and targeted therapies in neurological disorders. One important antisense transcript, BACE1-AS 3-9 homolog 1) to the binding site of the muscle differentiation regulator MyoD, and causes trimethylation of the histone H3 K9, thus repressing the expression of the MyoD target genes involved in muscle differentiation [MALAT1 promotes myogenic differentiation in cultured cells [MALAT1 knockout mice [+-dependent histone/protein deacetylase, SIRT1, via the formation of RNA duplexes and the facilitation of SIRT1 translation, by competing with miR34a (that can bind with SIRT1) to inhibit muscle formation [Antisense transcripts are also involved in regulating muscular diseases. For example, MALAT1 is linked to muscular dystrophy ,241. In ntiation . On the ed cells . In agreout mice . Recentlout mice . Thus, Mout mice , and theout mice ,241. Anoormation ,244. Thuormation ,245,246.ormation ,248,249.ormation . HOTAIR Since gene expression is central to cellular processes, the regulation of protein-coding gene expression by antisense transcription/transcripts has a significant impact on cellular gene expression and health. The misregulation of antisense transcription or antisense transcripts would be associated with various diseases, some of which are discussed above. However, other diseases/conditions such as aging, metabolic disorders, stress, thalassemia and spinocerebellar ataxia are also associated with antisense transcription/transcripts ,211,212.Antisense transcripts are wide-spread throughout eukaryotic genomes and are generated from independent, bidirectional or cryptic promoters. Antisense transcription/transcripts regulate gene expression and genome integrity via transcriptional interference, histone modification, and/or DNA methylation. Antisense transcripts can bring different macromolecules together within the three-dimensional context of the cell to coordinately execute transcriptional, post-transcriptional, and epigenetic processes. Thus, antisense transcription/transcripts are involved in many biological processes and are misregulated in a variety of diseases including cancer, neurological diseases, diabetes and cardiovascular disorders. Therefore, an understanding of the regulatory mechanisms of antisense transcription, characterization of antisense transcripts and their modes of actions would be useful for the diagnosis, monitoring and targeted therapies of various diseases. Indeed, several antisense oligonucleotides are in clinical trials for the treatment of various diseases . Two antAlthough antisense transcription/transcripts play crucial roles in the regulation of gene expression and genomic integrity due to their involvement in various diseases, it remains unclear how antisense transcription is initiated and epigenetically regulated. Here, we began to develop an understanding of how antisense transcription is initiated and regulated by histone covalent modifications. Further studies are needed for a thorough understanding of antisense transcription initiation and its epigenetic regulation. Such knowledge will provide new insights into the regulation of antisense transcription/transcripts and will aid in understanding the etiologies of various diseases, therefore promoting the discovery of diagnostic markers and therapeutic interventions.


BMP7 gene for bioinformatics analysis. Dual-luciferase reporter system and overexpression were used to analyze the key regions and transcription factor binding sites. There was high activity between −758 bp and −545 bp in the core region of the gene and a possible binding site for transcription factors SP1 and EGR1.Bone morphogenetic protein 7 (BMP7) is one of the largest secretory signal conductive molecules and is in the TGF-β superfamily. It plays an important role in the growth and development of hair follicles. We cloned the proximal promoter of the BMP7 gene is involved in the growth and development of hair follicles but its regulation mechanism is unclear. We studied the regulation mechanism of the BMP7 promoter by cloning the proximal promoter of BMP7 for bioinformatics analysis. A series of missing vectors was then constructed for dual-fluorescein activity detection based on the bioinformatics analysis results. We tested transcription-factor binding-site mutations and transcription factor over-expression to analyze the transcriptional regulation principle of the BMP7 promoter region. The upstream transcriptional regulatory region of the BMP7 gene proximal promoter was predicted by bioinformatics. There were −1216 bp to −1166 bp and −632 bp to −582 bp transcription initiation sites in the upstream transcriptional regulatory region of the BMP7 gene proximal promoter. The CpG islands’ distribution showed that there were many CpG islands at −549 bp to 1 bp. A dual-luciferase assay revealed high activity between −758 bp and −545 bp in the core region and a possible binding site for transcription factors SP1 and EGR1. The transcriptional activity of BMP7 was significantly decreased in the transcriptional regulatory region of the BMP7 after EGR1 and SP1 mutation. Transcription was significantly enhanced by over expression of the EGR1 transcription factor, which strongly suggests that EGR1 and SP1 play important roles in BMP7 regulation.The  BMP7 gene is highly expressed when hair follicles are in their growth phase, especially when the cells begin to proliferate at the basement membrane. When hair follicle enters the degenerative phase or the resting phase, BMP7 is low or undetected [BMP7 can inhibit wool growth in fine wool sheep. These results indicate that BMP7 may promote the proliferation of hair follicle cells in the hair follicle growth phase.Bone morphogenetic protein (BMP) belongs to the transforming growth factor-β (TGF-β) superfamily . The BMPdetected . Wang [9BMP7 and predicted the transcription initiation site and CpG island . A series of deletion vectors were constructed to determine the core region using a dual-luciferase assay. Based on the core region, we analyzed the transcription factor sites by bioinformatics analysis methods. Site-directed mutagenesis and transcription factor over-expression tests were performed to verify the authenticity of the candidate BMP7 transcription factor. Finally, we identified the transcription factor of BMP7.High-throughput sequencing allows the screening of trait-related genes. Research on the regulation of gene expression is a current research emphasis area. Research on small RNA is another research focus, but promoters are also key components of the expression regulation. Understanding the promoter structure is the first step in determining transcriptional regulation. The eukaryotic promoter is located upstream of the 5′ end of the gene and it can bind to RNA polymerase II to regulate gene transcription. This regulation affects cell growth, differentiation, and apoptosis and is the main method of regulating gene expression. To clarify the molecular mechanism of gene transcription regulation, understanding the transcriptional regulation of the promoter is necessary. In this study, we cloned the proximal promoter region of The Institutional Animal Care and Use Committee (IACUC) of the government of Jiangsu Province (Permit No. 45) and the Ministry of Agriculture of China (Permit No. 39) approved the animal study proposal. All experimental procedures were conducted in strict compliance with the recommendations of the Guide for the Care and Use of Laboratory Animals of Jiangsu Province and of the Animal Care and Use Committee of the Chinese Ministry of Agriculture. Efforts were made to minimize animal suffering.2 sample of ear tissue was collected from each sheep. Tissue was frozen with liquid nitrogen and ground to a powder. An animal Tissues/Cells Genomic DNA Extraction Kit  was used to extract DNA from the ground ear tissue. Extracted DNA was stored at −20 °C.Sheep were obtained from the Suzhou stud farm in China. A 0.5 cmBMP7 promoter sequence, bioinformatics software and websites were used to predict basic BMP7 information. Promoter 2.0 Prediction Server [BMP7. MethPrimer [According to the n Server  and NNPPn Server  were usethPrimer  was usedthPrimer  and JASPthPrimer  were use2O. The amplification program was as follows: initial denaturation at 94 °C for 1 min, followed by an optimal number of cycles (33 cycles) of denaturation at 94 °C for 30 s, annealing at 68 °C for 30 s, extension at 72 °C for 60 s, with a final extension at 72 °C for 5 min. The final PCR product was stored at 4 °C.According to the DNA sequence in NCBI (Gene ID: 443175), Oligo7 was used to design different primers for deletion fragments to amplify the BMP7 promoter region. The restriction sites (KpnI and HindIII) were added to the 5′ ends of the forward and reverse primers. The volume of the PCR amplification system was 50 μL, including 25 μL of 2 × GC Buffer I (II) , 8 μL of dNTP Mix , 2 μL of 10 mmol/L primer (forward and reverse), 0.5 μL of LA Taq , 2 μL of DNA, and 10.5 μL of ddHBMP7 (−2000 bp to +500 bp), and different fragments of the promoter region were amplified, including B1(−1500 bp to +257 bp), B2 (−985 bp to +29 bp), B3 (−758 bp to +29 bp), and B4 (−545 bp to +29 bp). Then, we constructed B5 (−758 bp to −545 bp) fragment vectors for regions with high transcriptional activity  was used to recover the PCR product. Connection system: Recycling PCR product 4 μL, pGL3-Base vector 1 μL, Solution I 5 μL, connection at 16 °C for 16 h; 2 μL ligation product was added to a 50 μL of DH5α competent cell and suspended on ice for 30 min at 42 °C for 45–60 s; after 2 min, ice bath Liquid LB medium was added (948 μL), and the mix was shaken with 120 rpm at 37 °C for 1 h. Then, 50 μL of LB medium with DH5α was applied on the LB plates with Amp culturing at 37 °C for 12–16 h. A single colony was selected for culturing and plasmid purification. Polymerase Chain Reaction(PCR) and enzyme digestion experiments were used to perform colony selection. The positive plasmids were sent to Sangon Biotech Co. Ltd.  for sequencing. The EndoFree Maxi Plasmid Kit (DP117)  was used to extract the plasmids and they were stored at −20 °C. We selected the sequence from 2000 bp upstream and 500 bp downstream of the initiation codon ATG of activity .5 with DMEM/F12  supplemented with 10% FBS  with 5% CO2 in air at 37 °C. After 24 h, the cells were cultured to 80% confluence and transfected using FuGENE® HD Transfection Reagent , according to its manufacturer instructions. The target plasmid and the internal reference plasmid pGL-TK were co-transfected into the cells at a ratio of 15–20:1, and negative and positive controls were set. After 24 h, 10 μL of cell lysate were added to the non-transparent 96 well plate, and 50 μL of Luciferase Reagent were added to immediately measure the Firefly luciferase activity with the Synergy 2 chemiluminescence detector . The corresponding Renilla luciferase activity values were measured immediately after adding 50 μL of 1× Stop & Glo® Reagent.The HEK293T cells were cultured in a 24 well plate at a density of 5 × 10EGR1 and SP1 with high scores. To verify whether these two transcription factors have significant effects on transcription activity, transcription factor binding sites were mutated. According to the primer design requirements of the Fast Site-Directed Mutagenesis Kit , the transcription factor point mutation primers (EGR1 binding site) and BT2 (mutation of SP1 binding site) were constructed using the B5 fragment as a template, respectively. The EGR1 overexpression plasmid was constructed by Suzhou Jinglun Biotechnology Co., Ltd., Suzhou, China.Bioinformatics analysis of the regions with high transcriptional activity revealed two transcription factors  primers  were desp < 0.05 was considered to be a significant. The statistics software used was SPSS version 16.0.The relative activity of luciferase  was analyzed using one-way analysis of variance (ANOVA). A BMP7 from translation initiation codon (ATG) were retrieved from the NCBI database. We predicted the possible transcription initiation site. The results showed that there were two possible locations including −1216 bp to −1166 bp and −632 bp to −582 bp. The CpG island prediction of the BMP7 promoter was performed using CpG finder and MethPrimer. The prediction was based on Island size > 100, GC Percent > 50.0, Obs/Exp > 0.6. The results showed CpG-rich islands at −549 bp to 1 bp, with a fragment length of 549 bp . Compared to B1, B2, and B3, the activity of B4 significantly decreased (p < 0.01). The promoter activity was shown between B3 and B4, specifically in the −758 bp to −545 bp region. The total activity of B4 and the −758 bp to −545 bp region was not different than that of the B3 (BMP7 promoter activity.In order to identify the core region of the activity . The actf the B3 . TherefoBMP7 and the published sequences in Genbank Homo sapiens (NM_001719.3), Mus musculus (NM_007557.3), Rattus norvegicus (NM_001191856.2), Sus scrofa (NM_001105290.1), Bos taurus (NM_001206015.1), Canis lupus familiaris (NM_001197052.1), Pan troglodytes (XM_001170064), Canis lupus familiaris (NM_001197052.1); we then constructed a phylogenetic tree (BMP7 promoter had the highest homology (99%) with Bos taurus. However, the promoter transcriptional core sequences of sheep and other species  have very low homology. Sequence analysis showed that homology of the transcriptional core sequences (−758 to −545 bp) between sheep and other species was low, except for Bos Taurus.To identify the conservation of the transcriptional core sequences (−758 to −545 bp), MEGA X software was used for homology analysis between the transcriptional core sequences of tic tree . HomologBMP7 promoter. There were no typical RNA polymerase II binding elements, such as CAAT and TATA boxes. In the prediction results, ZNF263 had the highest score, and SP1 had the highest frequency of occurrence. The possible transcription factors in this region also included EGR1, NRF1, KLF5, FOXI1, FOXB1, and FOXC1. Analysis of the prediction results showed that the transcription factors SP1 and EGR1 had higher scores, and they were involved in cell growth and development. We selected these factors for verification and analysis and believe that they play important roles in transcriptional regulation  after EGR1 and SP1 mutations, and there was a particularly more significant difference between the B5 and the EGR1 mutant plasmids  fragment is high. Transcription factors plasmids . TherefoEGR1 transcription factor in the BMP7 promoter, the EGR1 overexpression plasmid was co-transfected with the B5. The dual-luciferase activity in the overexpression group was significantly higher (p < 0.01) than in the control group . Similar to our results, the tongue sole BMP7 promoter contained one potential region located upstream of the initiation codon (−740 bp to −396 bp) [BMP7 promoter [BMP7 5′-flanking region has a high activity. We found that the sequence within 1500 bp BMP7 5′-flanking region has a high activity in human and tongue sole. The Hu sheep BMP7 promoter contained two main transcription initiation sites (−1216 bp and −632 bp). The human BMP7 promoter contains a single transcription initiation site (−764 bp) [Among molecular regulatory processes, transcription regulation has a significant impact on gene expression. The gene and skin , and it and skin ,16,17. H−396 bp) . Howeverpromoter . In addipromoter  found th−764 bp) .SP1, and AP1. Transcription factors are trans-acting factors that are located on promoter sequences and bind to cis-acting elements to regulate transcriptional regulation [BMP7 in mice [Gene transcriptional regulation by a promoter occurs in a system in which the transcription factor and regulatory elements are the most important components . There agulation . In the  in mice . Previou in mice ,24.BMP7, and we screened transcription factors related to transcriptional regulation. The transcription factors SP1, SP2, ZNF263, EGR1, NRF1, KLF5, FOXI1, FOXB1, and FOXC1 were predicted from −758 to −545 bp of the Hu sheep BMP7 promoter. At present, some reports showed that SNPs on promoters could affect the binding of transcription factors. Four SNPs were found on a porcine BMP7 promoter, including T-3722C, A-3684G, C-3522G, A-1027G. T-3722C, C-3522G, and A-1027G were located at transcription factor binding site and affected the transcriptional activity [EGR1 and SP1 with high scores were selected by site-directed mutagenesis and transcription factor overexpression experiments. The SP1 has also been in the BMP7 promoter of other mammal species [EGR1 appeared in the BMP7 promoter core region of Hu sheep. We speculate that EGR1 might participate in the transcriptional regulation of BMP7.We predicted transcription factor binding sites were in the core region of the promoter of activity . Therefo species ,20, whilSP1 and SP2, belong to the SP/KLF family. It is known that SP1 binds to the GC/CT cassette in the promoter region of the gene, activates the transcription process, and is abnormally expressed in various tumor groups [SP1 in human dermal papilloma was significantly higher than that of normal cells [SP2 has both activation and inhibitory effects; it competes with SP1 or SP4 to bind to the GC/CT cassette, thereby inhibiting the onset of transcription [EGR1) belongs to the immediate early gene family. It is an important transcription factor that activates or inhibits the transcription of genes and, thus, participates in the regulation of cell proliferation and cell apoptosis, growth, and differentiation [HCT116 promoters promote the EGR1 binding promoter and inhibits cell growth. Kruppel-like factor 5 (KLF5) is a family of Sp/Kruppel transcription factors that regulates transcription by binding to a promoter. These transcription factors are abundantly expressed in epithelial tissues and are involved in cell proliferation, embryonic development, and tumorigenesis [The transcription factors r groups ,27,28. Tal cells . In addicription ,31. Earlntiation ,33. Kwonntiation  found thigenesis ,36,37,38BMP7 promoter region between −758 bp and −545 bp and its homology between sheep and other species was low, except for Bos Taurus, and CpG-rich islands at −549 bp to 1 bp, with a fragment length of 549 bp. The transcription factors SP1 and EGR1 may have binding sites that regulate the BMP7 gene promoter. Both EGR1 and SP1 could be used as candidate transcription factors for future studies (This study found a high degree of activity in the  studies .


We show that BRD2 is required for the recruitment of the MYST-type acetyltransferase KAT7 to rDNA loci, resulting in enhanced local acetylation of histone H4. In addition, LYAR binds a complex of BRD4 and KAT7, which is then recruited to rDNA independently of the BRD2-KAT7 complex to accelerate the local acetylation of both H4 and H3. BRD2 also helps recruit BRD4 to rDNA. By contrast, LYAR has no effect on rDNA methylation or the binding of RNA polymerase I subunits to rDNA. These data suggest that LYAR promotes the association of the BRD2-KAT7 and BRD4-KAT7 complexes with transcription-competent rDNA loci but not to transcriptionally silent rDNA loci, thereby increasing rRNA synthesis by altering the local acetylation status of histone H3 and H4.Activation of ribosomal RNA (rRNA) synthesis is pivotal during cell growth and proliferation, but its aberrant upregulation may promote tumorigenesis. Here, we demonstrate that the candidate oncoprotein, LYAR, enhances ribosomal DNA (rDNA) transcription. Our data reveal that LYAR binds the histone-associated protein BRD2 without involvement of acetyl-lysine–binding bromodomains and recruits BRD2 to the rDNA promoter and transcribed regions  The increased activity of casein kinase II or of complexes formed between the mitotic cyclins and the cyclin-dependent kinases  or the mitogen-activated protein kinase ERK  is responsible for this upregulation in certain cell types  by RNA polymerase (RNAP) I, a 5S rRNA by RNAP III, and translation of ribosomal proteins from mRNAs transcribed by RNAP II . In addiogenesis ,2. Becaual cells ,7. Upon al cells . In certll types . Failurell types . AnotherDNA loci . In addiDNA loci ,13 and/oDNA loci .LYAR mRNA is detectable in immature spermatocytes and testes in early embryos and to a lesser extent in fetal liver and thymus tissue  and MCF cells (human breast cancer) , is a 45-kDa protein with 379 amino acid residues including a zinc-finger motif and three nuclear localization signals . LYAR iss tissue . In aduls tissue . Mouse fer cells . In supper cells , and it er cells . Moreove cancer) .via its ability to associate with protein arginine methyl transferase 5 (TP53 (encoding p53) is experimentally disrupted  at 120 mJ/cm2, soaked in 10% acetic acid, and stained with methylene blue. Biotinylated 47/45S pre-rRNA was detected with horseradish peroxidase (HRP)–conjugated streptavidin using the Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific). Signals were detected with the LAS-4000 system. Signal intensities of RNA bands were measured using NIH ImageJ software. To ensure equal loading, control RNA in each gel was stained using SYBR GOLD (Invitrogen) and quantified after the detection with the LAS-4000 system.The incorporation of 4-thiouridine and subsequent biotinylation were performed as described  with somAll other materials and methods are provided in supplementary materials including the list of antibodies  and thatLYAR mRNA in tissues from normal/control subjects and cancer patients. In many of the tumor samples from cancer patients, including those with colorectal carcinoma or small-cell lung carcinoma, LYAR mRNA levels were higher than in normal tissues  and lymph-node metastases (SW620) of a single colorectal cancer patient -inducible Flp-In T-REx 293 cell line expressing HBF (His6-biotinylation sequence-FLAG tag)-LYAR (HBF-LYAR-TO cells). The expression of HBF-LYAR increased gradually over time after the addition of Dox, reaching a steady state at a level 10-fold greater than that of endogenous LYAR . The indhttp://www.geneontology.org/). The top three most abundant proteins had Gene Ontology terms for transcription  and those related to protein-binding, poly(A) RNA binding, and DNA binding (under Molecular function)  . Among tunction) ,52, FACTunction) ,53,54, aunction) ,56 (Tablunction) . The othunction) . In addiunction)  14,57).,57.http:via the GGTTAT sequence that is located in five different regions of rDNA : nucleotides 42992–42997 (promoter), 3955–3960 , 32632–32637 and 34176–34181 , and 37256–37261 (intergenic spacer). However, the locations of these LYAR-binding sequences were not consistent with the regions of rDNA bound to LYAR observed by ChIP analysis; i.e., LYAR was not present in intergenic spacer. In fact, an H0-specific oligonucleotide containing the 42992–42997 sequence present in the promoter region did not bind LYAR as determined with electrophoretic mobility shift assay  inhibits the elongation of rDNA transcription  with 1:1 stoichiometry .,74.via tvice versa  . Given tvia binding to UBF, and we serendipitously found that SPT5 regulates UBF binding to the rDNA promoter, and thus SPT5 probably regulates LYAR binding to that promoter. In addition, we demonstrated that SPT5-UBF and BRD2-KAT7-JADE3 (or BRD4-KAT7-JADE3) form complexes with LYAR independently of each other (Figure via histone acetylation by KAT7/BRD4 at the rDNA promoter or phosphorylation of UBF via the kinase activity of BRD2.Another issue with this mechanism is whether LYAR binds rDNA directly or indirectly. Our data show that LYAR binds rDNA indirectly r Figure –C. Givenr Figure ,54,81, tr Figure  nor the r Figure . Howeverr Figure . Thus, wLYAR at a high level can maintain their tumorigenic potential upon suppression of LYAR function in rRNA synthesis. Recent research revealed that the cell-permeable molecule JQ1 inhibits the binding of BRD proteins with acetylated histones and is a potential therapeutic agent for the treatment of acute myeloid leukemia (Based on our current results and those of our previous study , we propleukemia ,83. Our Drosophila (Finally, it has been shown that the modulation of rRNA synthesis is closely linked to cell differentiation of ovarian germline stem cells in osophila  and in hosophila  and promosophila . Therefoosophila ,86 may bThe Ethics Committee of The University of Tokyo approved the use of human tissues, and all patients gave informed consent.gkz747_Supplemental_FilesClick here for additional data file.


F80L) were viable although proliferated more slowly. Cobalamin disorder is an inborn vitamin deficiency characterized by neurodevelopmental abnormalities, most often owing to biallelic mutations in the MMACHC gene, whose gene product MMACHC is a key enzyme in the cobalamin (vitamin B12) metabolic pathway. We show that THAP11F80L selectively affected promoter binding by THAP11, having more deleterious effects on a subset of THAP11 targets, and resulting in altered patterns of gene expression. In particular, THAP11F80L exhibited a strong effect on association with the MMACHC promoter and led to a decrease in MMACHC gene transcription, suggesting that the THAP11F80L mutation is directly responsible for the observed cobalamin disorder.Twelve human THAP proteins share the THAP domain, an evolutionary conserved zinc-finger DNA-binding domain. Studies of different THAP proteins have indicated roles in gene transcription, cell proliferation and development. We have analyzed this protein family, focusing on THAP7 and THAP11. We show that human THAP proteins possess differing homo- and heterodimer formation properties and interaction abilities with the transcriptional co-regulator HCF-1. HEK-293 cells lacking THAP7 were viable but proliferated more slowly. In contrast, HEK-293 cells were very sensitive to THAP11 alteration. Nevertheless, HEK-293 cells bearing a THAP11 mutation identified in a patient suffering from cobalamin disorder (THAP11 The THAP family of gene paralogs encodes 12 proteins in human, named THAP0 to THAP11. These are defined by their N-terminal THAP (for Thanatos—referring to the Greek God of Death—Associated Proteins) domain, an atypical zinc-finger DNA-binding domain –7. StudiTHAP7 represses gene transcription, both by promoting histone deacetylation and by masking histone tails from histone acetyltransferase complexes ,9. It isTHAP11 missense mutation is associated with cobalamin disorder, an inborn vitamin deficiency characterized by neurodevelopmental abnormalities [THAP11  metabolism, defining several complementation groups. The cblC group, accounting for 80% of cobalamin-disorder patients, is owing to biallelic mutations in the MMACHC gene, whose gene product MMACHC is a key enzyme in the cobalamin metabolic pathway [MMACHC gene [HCFC1 gene encoding HCF-1 are responsible for the so-called cblX complementation group and result in both decreased MMACHC mRNA and MMACHC protein levels [cblX-like disorder but without either any MMACHC or HCFC-1 mutation. Instead, this patient had a homozygous missense mutation in the THAP11 gene resulting in a phenylalanine to leucine change at position 80 (referred to here as THAP11F80L) at the C-terminus of the THAP domain [Different human mutations can result in disorders of intracellular cobalamin  sequ sequD/EHif , we detIn an extended study of THAP proteins , we analTHAP7 and THAP11 expression constructs, and whole-cell lysates (wcl) subjected to anti-HA-epitope immunoprecipitation and analyzed by immunoblot. In We first probe the formation of THAP7 and THAP11 homodimers and THAP7/THAP11 heterodimers. For this purpose, HEK-293 cells were co-transfected with Flag- and/or HA-epitope-tagged As shown in N) and C- (HCF-1C) terminal subunits [N. As shown in N as indicated by the recovery of HA-tagged HCF-1N after immunoprecipitation of co-synthesized Flag-tagged THAP7  took advantage of the well-characterized HCF-1 Kelch-domain point mutation called P134S, which prevents its association with HBM-containing target proteins , and , at least one HBM in a double HA and Flag epitope-tagged THAP11 "heterodimer" can be disrupted without preventing THAP11 dimer formation as shown in null), in which we created nonsense mutations at both codons 13 and 14 of the THAP7 coding sequence to disrupt THAP7 synthesis    and high-throughput RNA sequencing (RNA-seq) with both parental and THAP11F80L mutant cells. Using this information, we first analyzed the effects of the THAP11F80L mutation on THAP11 itself. As previously described [F80L mutation  caused by the THAP11escribed , THAP11  patient , THAP11FT levels . In cont protein . The difF80L HEK-293 cells revealed 2341 THAP11WT and only 1473 THAP11F80L peaks, with about half of the peaks in each case being located within +/– 250 bp of an annotated transcription start site (TSS)  . S2 TablWT parental and the THAP11F80L mutant samples called “common”, (ii) peaks missing  in the THAP11F80L sample called “F80L-absent”, and (iii) peaks present exclusively in the THAP11F80L sample called “F80L-only”. As shown in the Venn diagram in WT peaks. Thus, the THAP11F80L mutant protein binds to only a subset of the THAP11WT promoter targets and does no exhibit any evident de novo promoter binding compared to the THAP11WT protein.Being particularly interested in THAP11 regulation of transcription and to focus the study, we present the analysis of the TSS-associated THAP11 peaks. To characterize these peaks, we classified them into three categories : (i) peaWT vs. THAP11F80L DNA-site specificity with a 3.3 Mb view of Chromosome 1 that includes the MMACHC gene. The view covers nine THAP11WT TSS-associated peaks (1–9)—of which four  were selected for close-ups to illustrate the points illuminated below—and one non-TSS-associated peak (labeled *): two TSS-associated peaks (peaks 7 and 8) and the one non-TSS-associated THAP11 peak fell into the THAP11F80L-absent category and the remainder in the common category  were smaller albeit still present in the THAP11F80L sample. In the subsequent analyses described below, we do not distinguish between these two common peak subcategories. In conclusion, the THAP11F80L mutation results in a selective disruption of THAP11 DNA binding at specific promoters in HEK-293 cells, and does not create de-novo THAP11 promoter-binding sites.gory see . Among tgory see  remain lF80L mutation, we calculated size scores for each peak . This tendency was also reflected at the level of the THAP11WT scores as the common peaks (dark-red solid line) scored higher than the F80L-absent peaks (light-red solid line). The peak-score difference between the THAP11WT and THAP11F80L common peaks (dark-red lines) was less pronounced, however, than the difference between the THAP11WT and THAP11F80L F80L-absent peaks (light-red lines). Together, these observations suggest that: (i) the F80L-absent peaks represent lower affinity THAP11WT binding sites and (ii) binding to these sites is hypersensitive to the THAP11F80L mutation.To examine more broadly the nature of the effect of the THAP11 samples . ConsistTo understand the reason for these differences, we examined the DNA sequences underlying the common and F80L-absent peaks. The most prominent motif to emerge from this analysis of both sets of peaks was the THAP11-associated motif (TAM) also referred to as RBM  or SBS2 F80L protein is more sensitive to changes in the 3' half of the consensus than the THAP11WT protein.We probed this possibility by separately generating TAM consensus sequences  for the F80L promoter recognition.In addition to a weaker TAM consensus sequence associated with the F80L-absent peaks, fully one-quarter of the F80L-absent peaks had no discernible nearby TAM , whereas essentially all common peaks were associated with one or more TAMs . These aF80L mutation; it may be disruption of such a core function of THAP11 that makes the aforementioned THAP11null, THAP11HBM and THAP11ΔCC mutations lethal.In addition to examining the TAMs of THAP11-peak-containing promoters, we asked if the nature of the genes associated with the common and F80L-absent peaks differ through gene-ontology (GO) analysis as summarized in F80L mutation at the gene-expression level we analyzed the RNA-seq results (WT cells (X axis) against their fold change in THAP11F80L cells (Y axis). More often transcript levels considered to be changing  had decreased in THAP11F80L cells compared to THAP11WT cells: 523 genes were downregulated (red) whereas 279 genes were upregulated (blue). Furthermore, the magnitudes of decrease were greater than those for increases. Thus, the THAP11F80L mutation apparently affects activation of transcription—either directly or indirectly—more often than repression.To examine the consequences of the THAP11 results . Fig 9A F80L cells, fully one-half were canonical S-phase histone-encoding genes . These genes included 11 upregulated and 44 downregulated genes as shown in the MMACHC and MUTYH). By GO analysis . This observation suggests that THAP11 activates MMACHC gene transcription and that the THAP11F80L mutation impairs this activation, consistent with the cobalamin-disorder phenotype of the patient from whom the mutation was identified [entified .We have studied the THAP family of DNA-binding proteins extending from an evolutionary perspective of the 12 human members to a detailed analysis of a human disease associated THAP11 mutation.THAP genes in human. Nevertheless, two non-THAP domain sequence elements, the coiled-coil domain and HBM, are shared among the large majority of the 12 human THAP proteins, illuminating their importance for this class of proteins.The sequence comparison in The coiled-coil domain is implicated in association of THAP proteins ,48,49. AThe HBM permits many THAP proteins to extend their regulatory network to association with the transcription co-regulator HCF-1. We have shown that both THAP7 and THAP11 interact with HCF-1 via their respective HBM. Nevertheless, the presence or absence of an HBM in a THAP protein does not determine whether it will or will not associate with HCF-1: THAP5 contains an HBM but failed to associate with HCF-1, and THAP8 lacks an HBM and yet associated with HCF-1  deletion [To probe regulatory roles of THAP proteins, in particular in cell proliferation, we designed targeted mutations for the null and THAP7ΔCC mutant cells. As we did not observe a decrease in viability, the decrease in cell accumulation probably reflects a defect in cell-cycle progression. Thus, these two THAP proteins appear to play important and in one case—THAP11— an essential role in HEK-293-cell viability.Although THAP7 was not essential for HEK-293-cell viability, we observed consistent retarded cell proliferation for the THAP7THAP11 is particularly sensitive to mutagenesis, as we did not obtain any viable THAP11null, THAP11HBM or THAP11ΔCC mutants. This mutational sensitivity is also highlighted by the ExAC database of human exon sequence variants  no missense mutations were reported in the THAP11 sequences encoding the THAP domain. This restricted number of genetic variants altering the THAP11 protein highlights the importance of THAP11 functions. And yet an individual homozygous for the THAP11F80L mutation survived for 10 years [THAP11 mutation, located in the THAP domain, can still be tolerated for viability.We observed that 0168286, ) where  on day 0, and for each cell line, 2 plates used for counting every 24 hours from day 1 to day 8 (except days 2 and 3). THAP7HBM, THAP7ΔCC, and parental cell lines were tested in parallel in a first experiment, whereas THAP11F80L, THAP7null and parental cell lines were tested in parallel in a second experiment.Cells were seeded at the same density .CRISPR/Cas9-mediated mutagenesis in HEK 293 cells was performed as recommended  with minTotal RNA was used to prepare ribosomal RNA-depleted libraries for 50-nucleotide single-read high-throughput sequencing using an Illumina HiSeq 2100 device with 6 samples per lane (multiplexing).For each ChIP, 5 IPs were performed in parallel and subsequently pooled, each time by incubating 9 μg of crosslinked and sonicated chromatin at 4°C with 2 μg of anti-THAP11 antibody and further with protein G agarose beads. Pre-IP samples  were prepared in parallel. 5 ng of each ChIP and input purified DNA were used to prepare paired-end sequencing libraries for 100-nucleotide paired-end high-throughput sequencing  with 3 samples per lane (multiplexing).The evolutionary tree  was prepSingle reads were mapped onto the Hg19 human genome annotation using STAR  and rea2 counts, scaled by total tags for the sample and the peak width. Peaks were visualized with the UCSC genome browser [Paired-end reads were mapped onto the Hg19 human genome annotation using STAR (Spliced Transcripts Alignment to a Reference) . Peaks w browser .S1 Raw Images(PDF)Click here for additional data file.S1 FileThis file describes in detail the materials and methods used in the experiments reported in this article.(PDF)Click here for additional data file.S1 FigF80L cobalamin-disorder patient.Sequence alignment of the first 90 amino-acids of the 12 human THAP proteins, with the conserved features highlighted as follows: red, “C2CH signature” of the zinc finger; purple, strictly conserved residues; green box, “AVPTIF box”; blue, phenylalanine residue mutated in the THAP11(EPS)Click here for additional data file.S2 FigC (lanes 3 and 4), HA-HCF-1N (lanes 5 and 6), or HA-HCF-1FL (lanes 7 and 8) constructs and whole-cell lysates  subjected to HA immunoprecipitation  and analyzed by immunoblot with anti-HA (two upper panels) and anti-THAP11 (lower panel) antibodies. Relative to (A) HEK-293 cells were transfected with THAP7-Flag construct, and whole-cell lysate (lane 1) was either directly treated with calf intestinal phosphatase (CIP) (lane 2) or subjected to Flag immunoprecipitation (lane 3) before being treated with CIP (lanes 4 and 5), and analyzed by immunoblot with an anti-Flag antibody. d.CIP, heat-inactivated CIP. Relative to (EPS)Click here for additional data file.S3 Fignull, (B) THAP7HBM, and (C) THAP7ΔCC mutant clones. The mutated nucleotides and resulting amino-acids are depicted in red in the mutant sequences.Details of the mutagenesis (left) and sequencing chromatograms (right) of the (A) THAP7(EPS)Click here for additional data file.S4 FigWT and (A) THAP7null, (B) THAP7HBM and (C) THAP7ΔCC cells over the course of the cell-proliferation experiments, shown as the mean +/- standard deviation of the duplicates. Cell viability is determined as the ratio of the live cell number  over the total cell number. Relative to Cell viability of THAP7(EPS)Click here for additional data file.S5 FigWT and (A) two independent THAP7HBM or (B) four independent THAP7ΔCC cell lines were seeded at the same density (1.25 x 104 cells per ml) on day 0, and for each cell line, 2 plates used for counting every 24 hours from day 1 to day 8 (except days 2 and 3). The ratio of the mean of live cell counts between duplicates (Nt) and the initial cell number (N0), with standard deviation, is plotted. Cartoons of the THAP7WT, THAP7HBM and THAP7ΔCC protein structures are shown. Relative to THAP7(EPS)Click here for additional data file.S6 FigHBM and (B) THAP11F80L mutant clones. The mutated nucleotides and resulting amino-acids are depicted in red in the mutant sequences.Details of the mutagenesis (left) and sequencing chromatograms (right) of the (A) THAP11(EPS)Click here for additional data file.S7 FigF80L cells over the course of the cell-proliferation experiment, shown as the mean +/- standard deviation of the duplicates. Cell viability is determined as the ratio of the live cell number  over the total cell number. Relative to Cell viability of THAP11(EPS)Click here for additional data file.S1 TableWT (columns F and H) and THAP11WT (columns G and I) peaks are indicated. Details about the THAP11-associated motifs are indicated: total number of motifs in a region expanding 1000 bp on each side of the peak maximum (column J), genomic coordinates of the start (column K) and end (column L) of the closest motif to the peak center, motif sequence (column M), motif E-value relative to the consensus motif (column N) and the relative position of the motif to the peak (column O). Details of the genes identified under the peaks are listed, together with their RNA-seq data: number of genes having their TSS in a region expanding 250 bp on each side of the peak boundaries (column P), distance of the TSS gene to the peak , gene strand , gene type , normalized gene mRNA levels (log2(RPKM)) in each of the THAP11WT  and THAP11F80L  biological replicates, the (log2) THAP11F80L versus THAP11WT fold change and associated adjusted p-value of gene expression values . NA, non-applicable, meaning no such feature (motif of gene) relative to the peak. ND, non-determined, meaning gene not expressed in our dataset.Table listing the peaks identified in the ChIP-seq experiment . Each peak has been identified with a unique identifier (column A) and categorized as “common”, “F80L absent” or “F80L only” (see text. Column B). The exact peak position is detailed in columns D and E . The peak scores and counts in the THAP11(XLSX)Click here for additional data file.S2 TableWT peaks.Complete results of the KEGG, GO, BIOCARTA and REACTOME analyses performed on the 1138 expressed genes associated with the 1114 TSS-associated THAP11(XLSX)Click here for additional data file.S3 TableMMACHC gene. Relative to Features of the 10 THAP11 peaks in a 3.3 Mb region of Chromosome 1 including the (PDF)Click here for additional data file.S4 TableComplete results of the KEGG, GO, BIOCARTA and REACTOME analyses performed on the expressed genes associated with the common (first sheet) or F80L-absent (second sheet) peaks. Relative to (XLSX)Click here for additional data file.S5 TableWT (columns E and F) and THAP11F80L (columns G and H) cells; log2(RPKM) values of each biological replicate in THAP11WT (columns I and J) and THAP11F80L (columns K and L) cells; (log2) THAP11F80L versus THAP11WT fold change (column M); and associated p-values (column N) and adjusted p-value (column O).Table listing the expressed genes identified in the RNA-seq experiment, with their respective expression values. Each gene is identified by its Ensembl ID (column A) and its name (column B), the gene type and description being detailed . The results of the RNA-seq data are presented for all expressed genes (see (XLSX)Click here for additional data file.S6 TableF80L cells. Relative to Complete results of the KEGG, GO, BIOCARTA and REACTOME analyses performed on the expressed genes downregulated (first sheet) or upregulated (second sheet) in the THAP11(XLSX)Click here for additional data file.S7 TableF80L cells whose promoter is directly bound by THAP11WT but not THAP11F80L. Relative to Complete results of the KEGG, GO, BIOCARTA and REACTOME analyses performed on the expressed genes changing in the mutant THAP11(XLSX)Click here for additional data file.S8 TableGreen plus, interaction; red minus, no interaction; (—), very weak, if any, interaction; grey dot, not tested. See .(PDF)Click here for additional data file.


Haematopoietic cells arise from endothelial cells within the dorsal aorta of the embryo via a process called the endothelial-haematopoietic transition (EHT). This process crucially depends on the transcription factor RUNX1 which rapidly activates the expression of genes essential for haematopoietic development. Using an inducible version of RUNX1 in a mouse embryonic stem cell differentiation model we showed that prior to the EHT, haematopoietic genes are primed by the binding of the transcription factor FLI1. Once expressed, RUNX1 relocates FLI1 towards its binding sites. However, the nature of the transcription factor assemblies recruited by RUNX1 to reshape the chromatin landscape and initiate mRNA synthesis are unclear. Here, we performed genome-wide analyses of RUNX1-dependent binding of factors associated with transcription elongation to address this question. We demonstrate that RUNX1 induction moves FLI1 from distal ETS/GATA sites to RUNX1/ETS sites and recruits the basal transcription factors CDK9, BRD4, the Mediator complex and the looping factor LDB1. Our study explains how the expression of a single transcription factor can drive rapid and replication independent transitions in cellular shape which are widely observed in development and disease. Significant progress has been made in identifying the transcription factor (TF) networks that are required to specify independent cell lineages during haematopoiesis. Wilson et al. identified a heptad of TFs essential for regulation of human HSPCs including GATA2, SCL/TAL1, RUNX1 and FLI12. A smaller network of RUNX1, FLI1 and NF-E2 is responsible for terminal megakaryocyte differentiation, although other factors are necessary earlier in the differentiation process3.Commitment to a specific cell fate requires a complex regulatory network of transcription factors acting in a stage-specific manner8. RUNX1 plays an essential role in the EHT, since in its absence progenitor cells fail to emerge11. RUNX1 acts as both a repressor and activator of gene expression and both activities are essential for the EHT12. The importance of RUNX1 for the differentiation of multiple haematopoietic lineages suggests that the precise molecular mechanism governing the initiation of different transcriptional programs may derive from its many interaction partners.The first definitive blood cell progenitors arise from specialised endothelial cells within the dorsal aorta which are termed the haemogenic endothelium (HE), and this process is known as the Endothelial to Haematopoietic Transition (EHT)13. In the absence of RUNX1 the HE is formed, but cells are unable to undergo the EHT and to form definitive haematopoietic precursors expressing the surface marker CD41. We showed that many haematopoietic genes required for the EHT are in a ‘primed’ state with TFs FLI, SCL and CEBPβ already bound and with RNA Polymerase II (Pol II) already present at many promoters13. It is widely accepted that TFs binding to enhancers direct the onset of transcription by recruiting co-activators, together with the mediator complex, which interact with the basal transcription machinery containing the positive transcription elongation factor b (pTEFb) complex. Activation of pTEFb by interaction with TFs, with the bromodomain protein BRD4 or the super elongation complex (SEC) causes phosphorylation of the Pol II CTD by the kinase component of this complex, CDK9, resulting in release of the polymerase from a paused state15. Mediated by bridging molecules such as LDB1, promoter and enhancer elements physically and differentially interact in nuclear space providing an additional layer of gene regulation17. We showed that RUNX1 directly orchestrates both processes since its induction rapidly activated the transcription of haematopoietic genes and re-shaped the epigenetic landscape by causing increased histone acetylation and global redistribution of TF complexes13. However, the precise nature and order of factors recruited by RUNX1 are still unclear.Using an established model of Embryonic Stem Cell (ESC) differentiation we previously examined the role of RUNX1 in the onset of a haematopoietic transcriptional program. We employed an inducible RUNX1 (iRUNX1) ES cell line expressing an HA-tagged, doxycycline (Dox) inducible RUNX1 protein in a RUNX1−/− backgroundIn this study, we used the iRUNX1 system to dissect, at the global level, how RUNX1 orchestrates the formation of transcription factor complexes and Pol II recruitment to drive the transcriptional processes that underlie the EHT. We show that after induction, genome-wide redistribution of TF complexes by RUNX1 is coupled with increased enrichment of CDK9 at distal sites, the onset of transcription of genes essential for haematopoietic development and increased deposition of the H3K79me2 mark on histones as a marker for transcriptional elongation. In parallel, the bridging factor LDB1 moves with the other factors bound at distal sites towards RUNX1 bound sequences and leads to recruitment of the Mediator complex. Factor movement is blocked by treatment with the bromodomain inhibitor JQ1, leading to a block in the up-regulation of haematopoietic genes and the EHT. Our data show a direct requirement for RUNX1 to recruit factors associated with transcriptional elongation and to orchestrate the reorganisation of interacting transcription factor complexes, thus facilitating a permissive environment for the rapid activation of genes essential for the EHT.10. Early haemogenic endothelium (HE1) expresses c-Kit and the endothelial marker Tie2 but is negative for CD41, a marker of haematopoietic commitment . A later stage of HE - HE2 - acquires CD41 expression as cells progress through differentiation . As cells progress further towards haematopoietic commitment they lose the Tie2 endothelial marker (Haematopoietic Progenitors (HP): c-Kit+, Tie2−, CD41+). In the absence of RUNX1 the doxycycline (Dox) inducible iRUNX1 ES cell line is unable to progress past the haemogenic endothelium (HE) stage of development during in vitro haematopoietic development  of RUNX1, BRD4 and the histone modification H4K5Ac in HE cells which were either untreated (No Dox) or Dox treated (+Dox). RUNX1 bound at 18,430, mostly distal, peaks and correlated with the presence of RUNX motifs .BRD4 has been reported to contribute to recruitment of the pTEFb complexJQ1 Fig. . While JMyb, Spi1, Gfi1 and Vav1 and includes gene ontology (GO) terms related to haematopoiesis, cell migration and adhesion  which is expressed at early stages of the haematopoietic system and is involved in reorganisation of the cytoskeleton amongst other functions28. Genes normally strongly down-regulated by RUNX1 exhibit a varied response to JQ1 , indicating that cells adopt a fate that is related to that of endothelial cells, again confirming that the EHT did not take place.Global analysis of gene expression by RNA-Seq showed that JQ1 had a dramatic influence on RUNX1 induced transcription. More than 2,000 genes were deregulated across the treatment conditions, with the highest number of down-regulated genes observed between the +Dox and +Dox +JQ1 samples  with and without RUNX1 induction and found that FLI1 binding increased more than two fold at 2648 sites . Similar numbers and distribution of peaks were observed in each of the samples  is shown in Fig. Gfi1 expression as detected by RNA-seq. The gene ontology terms associated with the RUNX1 bound up-regulated genes – within the top half of the heatmap in Fig. To display a comprehensive picture of how complexes form at distal sites in response to RUNX1 induction we integrated all of our ChIP-seq data with motif density plots and gene expression data. In Fig. 13. In our current study, we have used the same system to identify the molecular details of how the reshaping of the transcription factor binding landscape links to the rapid initiation of active transcription at lineage specific genes. From this data we can build a model of how RUNX1 orchestrates the formation of transcription factor complexes at distal sites of genes up-regulated during the EHT  towards RUNX1 does not involve binding of BRD4 to RUNX1, as we were unable to see an interaction between RUNX1 and BRD4. JQ1 affected CDK9 recruitment but only at sites where RUNX1 recruitment was lost as well. Roe et al. found that in addition to acetylated histones, BRD4 could interact with acetylated TFs including FLI1 (RUNX1 was not studied)21. Our ChIP-seq data support this mechanism  which could further stabilize BRD4 binding to RUNX1 binding sites.Prior to RUNX1 induction CDK9 binds to distal sites of genes essential for the haematopoietic lineage such as ism Fig. , with BRism Fig. . RUNX1 ade novo FLI1 binding sites are observed (1615) and these contained RUNX or RUNX::ETS composite motifs. This result would suggest that co-operative binding of RUNX1 and FLI1 is essential for progression of the EHT and that changes in the composition of TF complexes, e.g. loss of GATA motif binding factors and gain of RUNX1, are required to drive such cell shape switches. The binding of different combinations of factors to different cis-regulatory elements is associated with cell lineage specification2. Our study provides a direct mechanism for how the presence or absence of one factor differentially drives such changes.Transcription factor binding at enhancers or promoters is determined by the underlying DNA sequence. We found that the movement of TF complexes mediated by RUNX1 follows a specific binding motif code. In the absence of RUNX1, FLI1 binds to sites with ETS motifs together with AP1 and GATA motifs. Following RUNX1 induction around 2000 of these sites are retained, however, a substantial proportion of et al. demonstrated a function for LDB1 early in blood cell development where they showed defective blast-colony formation from Flk1+ haemangioblast cells from LDB1−/− ES cells. Many of the pathways deregulated in the absence of LDB1 were also deregulated by blocking the EHT with JQ1 treatment, including genes involved in integrin signalling, focal adhesion and the Wnt pathway35. LDB1 has been shown to function by mediating long-range promoter-enhancer interactions17. Based on their observations in the Myb locus36 where multiple enhancers control tissue specific expression of the myb gene, Stadhouders et al. proposed a model for gene activation involving the looping of distal element-bound CDK9 to genic regions16. RUNX1 has been shown to mediate cis-regulatory element interactions at the human CD34 locus37. Together with the strong global association between RUNX1 binding, FLI1/BRD4 relocation, CDK9 and LDB1 enrichment at RUNX1 target genes this suggests that RUNX1 associated transcription factor assemblies mediate the initiation of transcriptional elongation through looping.Mylona RUNX1, in particular in acute myeloid leukemia (AML). For example, in t AML the DNA binding domain of RUNX1 is fused to the repressor ETO (MTG8) turning this protein into a constitutive repressor, causing a block in differentiation at an early myeloid progenitor stage38. Structural and functional studies demonstrated that the fusion protein exists in a complex containing FLI1 and LDB141 and knock-down of the fusion protein leads to cell differentiation and changes in the binding patterns of myeloid transcription factors42. Conversely, induction of the fusion protein in the haemogenic endothelium blocks the EHT and leads to a block in differentiation when induced in progenitors43. It is therefore likely that the fusion protein is unable to orchestrate the regulated movement of transcription factor complexes described here.We suggest that such specific and rapid factor relocation processes mediated by protein-protein interactions, as described here, drive all replication-independent transitions in cellular shape and function that are widely observed in development. It is important to note that our findings are also relevant in a disease context. A number of haematopoietic malignancies are caused by mutations in In summary, the replication independent rapid change in cell shape during the endothelial-haematopoietic transition (EHT) starts out from a primed state and involves the RUNX1-dependent recruitment of BRD4, CDK9, LDB1 and Mediator to RUNX1 binding sites at genes required for haematopoietic differentiation. The formation of transcriptionally competent TF complexes at RUNX1 target genes requires the relocation of BRD4-FLI1 complexes from GATA/ETS to RUNX1/ETS sites. Inhibition by the BRD4 inhibitor JQ1 blocks formation of these complexes, thus preventing the EHT and haematopoietic development.43. This cell line carries an HA-tagged RUNX1 under the control of a doxycycline inducible promoter in a RUNX1 null background10. Briefly, cells were grown on mouse embryonic feeder cells (MEFs) inactivated with mitomycin C (Sigma), in DMEM supplemented with 15% FCS (Stem CellTechnologies), 1 mM sodium pyruvate, 1 mM glutamine, 100 units per ml penicillin and 100 μg per ml streptomycin, 25 mM HEPES buffer, 1 × non-essential amino acids, 0.15 mM MTG and 103 units per ml LIF .The iRUNX1 ES cell line was maintained as described previouslyIn vitro haematopoietic differentiation of the iRUNX1 ES cell line was performed essentially as described in Lichtinger et al.13. A more detailed description can be found in Supplementary Materials.Haemogenic endothelium cells treated with and without Dox in the presence or absence of JQ1 were isolated for flow cytometry by trypsinising adherent cells, and washing with PBS, followed by MACS buffer. Cells were stained with antibodies against c-Kit , CD41-PECy7 (eBioscience 25-0411), Tie2-PE (eBioscience 12-5987). Samples were run on a Cyan Flow Cytometer and analysed using Summit software (Beckman Coulter).31. ChIP-seq libraries were prepared using the Kapa Hyper Prep kit for Illumina platforms according to manufacturer’s instructions. More details including antibodies used and primer sequences for manual ChIP can be found in Supplementary Materials.ChIP was performed as described previously44, and more details including primer sequences for manual qRT-PCR can be found in Supplementary material. RNA-seq libraries were prepared from at least two biological replicates for each sample using the Tru-seq Stranded Total RNA kit (Illumina), according to manufacturer’s instructions. Libraries were sequenced in a pool of 12 indexed libraries using a NextSeq® 500/550 High Output Kit v2 (150 cycles) for paired end sequencing  at the Genomics Birmingham sequencing facility.RNA was extracted as described previouslyCo-IPs were performed using the Pierce Crosslink Immunoprecipitation Kit (Thermo Fisher) according to manufacturer’s instructions. Samples were subsequently run on 4–20% gradient gels (Bio-Rad) and transferred to nitrocellulose membranes using a BioRad Trans-Blot Turbo transfer system. Membranes were blocked in 5% milk powder in 0.05% TBS-Tween and incubated with anti-HA (for RUNX1) , anti-BRD4  and anti-CBFβ  antibodies. Proteins were visualised using Pierce SuperSignal West Pico Chemiluminescent substrate (Thermo Scientific).Western blots showing dose response of RUNX1 protein to Dox were performed as above then protein was detected using primary antibodies directed to AML1 (RUNX1)  or GAPDH  followed by secondary antibodies IRDye® 800CW Donkey anti-Rabbit IgG (Li-Cor Biosciences) and IRDye® 800CW Donkey anti-Mouse IgG (Li-Cor Biosciences) respectively. Proteins were visualised on a Li-Cor Odyssey Imaging system.Methods used to analyse ChIP sequencing and RNA sequencing data are described in the Supplementary Methods.Data were deposited to NCBI under GSE104046.Supplemental MaterialS1S2S3S4S5Original blots