Saccharomyces cerevisiae can be built on Cas12c. We found that, upon fusion to a strong activation domain, Cas12c is an efficient activator. Its functionality was proved as a component of hybrid Boolean gates, i.e., logic circuits that mix transcriptional and translational control (the latter reached via tetracycline-responsive riboswitches). Moreover, Cas12c activity can be strongly inhibited by the anti-CRISPR AcrVA1 protein. Thus, Cas12c has the potential to be a new tool to control the activation of gene expression within yeast synthetic gene circuits.Among CRISPR-Cas systems, type V CRISPR-Cas12c is of significant interest because Cas12c recognizes a very simple PAM (TN) and has the ability to silence gene expression without cleaving the DNA. We studied how new transcription factors for the yeast  CRISPR-Cas is an RNA-based immune system in bacteria and archaea . CRISPR-RNA structures such as riboswitches carry out a post-transcriptional control on gene expression and, to this aim, they have been adopted in both prokaryotic and eukaryotic genetic circuits . RiboswiLogic gates are the fundamental building blocks of digital circuits that permit to realize biosensors . In biolSaccharomyces cerevisiae. Similar to other type V Cas proteins, Cas12c appears more effective as an activator rather than a repressor. Moreover, its activity can be controlled by AcrVA1, whereas AcrVA4 and AcrVA5 (GAL1 promoter). Overall, we identified the best designs for riboswitch-containing hybrid gates and proved that Cas12c worked efficiently as an activator inside Saccharomyces cerevisiae synthetic gene circuits.In this work, we present a first characterization of Cas12c as a transcription factor in the yeast d AcrVA5  have no d AcrVA5 . For a cd AcrVA5  or LexA-d AcrVA5 . AlternaGPD promoter (pGPD). The yeast enhanced green fluorescent protein . Cas12c recognizes a short protospacer adjacent motif   only and put it under the control of the strong constitutive n (yEGFP ) was cho, 5′-TN)  slightly, 5′-TN) . The crRor—SUP4t ). As a por—SUP4t . In a pror—SUP4t , we pointion see . Therefotion see , see Suption see . In termtion see .To improve the action of Cas12c as a repressor, we fused it to the mammalian repression domain Mxi1 that was reported to interact with the histone deacetylase Sin3 homolog in yeast . The chiCYC1 promoter  activation domain . We test removed ) precede removed  , CUP1 , and MET25  promoter , the hormone-binding domain of the human estrogen receptor (HDB(hER)—the docking site for β-estradiol), and an activator domain (AD) such as the strong VP64 (from the Herpes simplex virus)   or the whia coli . We exprhia coli  (20% as  the TSS ) precede the TSS  and bindyEGFP, nor the usage of pGPD and LexA-HDB(hER)-B42 had a beneficial effect on the The best circuit design, which returned 6.57 see , demande2.41 see . We did alue see .CYC1 promoter previously used. However, the full lex2Ops upstream of the two TATA boxes were removed and a short lexOpR was placed between the TATA box starting at position −52 and the TSS. The new promoter, referred to as pCYC1min-lexOpR, was quite weak . Hence, we had to increase its strength in order to see the repression effects on the yEGFP level clearly. To this aim, we added a 40-nt-long upstream activating sequence from the GPD promoter (UAS(GPD-40)) 150 nt upstream of pCYC1min-lexOpR  was expressed under pTEF2. The target, now repressed, promoter was still based on the minimal n-lexOpR . Our finpromoter ). NOR gand 2 see , whereas2.25 see . The latFrom our previous experiments, we concluded that tc2 guaranteed, generally, higher performance than tc1 and N-IMPLY gates could reach  factors . We fuse factors . The samyEGFP gene from position 452 to 472 with respect to the START codon) or Cas12c-Mxi1 binding pTEF2 that was placed upstream of yEGFP. Furthermore, denAsCas12a was expressed under the inducible GAL1 promoter, whereas Cas12c-Mxi1 was constitutively produced by pGPD. Both gates pointed out that the effect of the repressor protein on transcription were less evident than those of tetracycline on translation. Nevertheless, the NOR gate hosting denAsCas12a returned a S. cerevisiae, whereas Cas12c can be easily turned into an effective activator only.We moved, then, to the construction of NOR gates based on the same type V CRISPR-Cas systems. We expressed either the bare denAsCas12a (which targeted the S. cerevisiae (viability coefficient: 0.804). This result is analogous to that we reported for the AcrIIA5-SpCas9 interaction (The anti-CRISPR (Acr) proteins are a response, evolved by phages, to contrast the CRISPR-Cas immune systems . Three teraction .S. cerevisiae synthetic gene circuits. We started from the construction of simple circuits where Cas12c, either bare or fused to an effector domain, was constitutively expressed to enhance or repress the synthesis of the yEGFP. We found that Cas12c fused to the strong VPR activation domain increased transcription considerably, no matter the DNA strand targeted by its crRNA. The bare Cas12c failed to inhibit yEGFP production. However, when fused to the Mxi1 repression domain, Cas12c-Mxi1 could moderately repress transcription by targeting the sense strand of the promoter (pTEF2) upstream of the yEGFP gene. This result showed an opposite behavior to denAsCas12a, the nuclease-deficient type V Cas protein that best serves as a template for engineering transcription factors in yeast. The bare denAsCas12a, indeed, represses fluorescence expression considerably by targeting the middle region of yEGFP.In this work, we aimed to ascertain if Cas12c, a type V CRISPR-associated protein that lacks DNase function, could be used as a transcription factor inside We then tested the working of Cas12c inside hybrid Boolean gates, i.e., logic circuit where both transcription and translation are regulated. In particular, each gate was based on translation inhibition via tetracycline-responsive riboswitches. We built two kinds of hybrid Boolean gates: N-IMPLY, where transcription was activated, and NOR, where transcription was repressed. In general, N-IMPLY gates gave better performance than NOR gates, no matter if the activator was built on Cas12c, denAsCas12a, or LexA-HBD (hER). Moreover, a tandem riboswitch (tc2) usually enhanced the gate performance.Finally, we checked if Cas12c activity could be controlled by type V anti-CRISPR proteins. We found that AcrVA1 drastically reduced Cas12c (fused to VPR) activity, whereas AcrVA4 and AcrVA5 had no effect on the same chimeric protein. AcrVA1 is a potent inhibitor of denAsCas12a and works as a bait by mimicking the PAM. This can explain its strong impact on Cas12c, whose PAM (TN) is like a piece of that of Cas12a (TTTV). As a drawback, we realized that AcrVA1 induces a mild toxicity in yeast cells (viability coefficient = 0.804). A similar trend was detected also on AcrIIA5 that contrast the activity of dSpCas9.On the whole, we can claim that Cas12c, fused to an AD, can be used reliably as an activator, even though denAsCas12a appears more performant. Basic engineering of Cas12c as a repressor did not lead to impressive results. Hence, further attempts with different repression domains or longer pre-crRNA  should be tried.By implementing different hybrid gates, we also showed that Cas proteins, when used as templates to construct new transcription factors, can outperform other proteins such as the bacterial repressor LexA. Interestingly, denAsCas12a, i.e., a dead nuclease, regulates transcription better than Cas12c, which naturally silences transcription. A deeper understanding of the Cas12c-based immune system working is probably necessary to explain this apparent contradiction.All plasmids used in this study are listed in Two methods were used to construct new plasmids. The first method was enzymatic digestion and ligation. First, the backbone and the insert-containing plasmid were digested overnight. The purified DNA fragments were then ligated using T4 DNA ligase (NEB-M0202S) at 16°C for 8 h. Finally, DNA was eluted from the agarose gel using the AxyPrep DNA Gel Extraction Kit #AP-GX-250. The second method was isothermal assembly. First, standard biological parts, such as promoters, coding regions, and terminators, were extracted and amplified by touchdown PCR using Q5 High-Fidelity DNA Polymerase (NEB-M0491S). The purified PCR products were mixed with a cut-open backbone  in equimolar amount and let in a thermal cycler at 50 C for 1 h .E. coli cells  and stored in glycerol storage solution. All DNA sequences are reported in All plasmids constructed in this work were inserted into S. cerevisiae strain CEN.PK2-1C , Euroscarf . For yeast transformation . To measure fluorescence intensity, we used a BD FACSVerse . The FACS machine setting was checked via the QC  procedure by using fluorescent beads (BD FACS quite CS&T Research beads-17495). Each yeast strain was measured three times on different days (independent experiments). In every experiment, for each sample 30000 events were collected at low flow rates (the threshold rate was kept below 2000 events/s). Raw data from the BD FACSVerse were analyzed using the flowcore R-Bioconductor software package .Trypan blue staining assay is a commonly used method to determine cell viability. The assay is based on the principle that trypan blue is excluded from viable cells but penetrates and stains dead cells. In this assay, cells are treated with trypan blue solution, which gives the dead cells a blue color, while viable cells remain unstained. Cells are mixed in a 1:1 ration with 0.4% trypan blue solution. The number of alive and dead cells is then counted under a microscope using a hemocytometer. The percentage of alive cells is calculated by dividing the number of alive cells by the total number of cells.


Long non-coding RNAs (lncRNAs) are a large class of RNA molecules known to regulate the expression of genes. Although expressed in a highly tissue-specific manner, they appear to modulate virtually all pathways in the cell, either in normal or disease states. LncRNAs are often aberrantly expressed in cancers and can act as oncogenes or tumor suppressors. Here, we describe various mechanisms by which lncRNAs influence gene expression as they bind other RNAs, genomic DNA, or proteins. Particularly, we discuss their relationship with transcription factors as they act on pathways relevant to cancer. While much remains to be discovered about how they regulate genes, lncRNAs hold great therapeutic potential, especially in their transcription factor interactions.As a group of diseases characterized by uncontrollable cell growth, cancer is highly multifaceted in how it overrides checkpoints controlling proliferation. Amongst the regulators of these checkpoints, long non-coding RNAs (lncRNAs) can have key roles in why natural biological processes go haywire. LncRNAs represent a large class of regulatory transcripts that can localize anywhere in cells. They were found to affect gene expression on many levels from transcription to mRNA translation and even protein stability. LncRNA participation in such control mechanisms can depend on cell context, with given transcripts sometimes acting as oncogenes or tumor suppressors. Importantly, the tissue-specificity and low expression levels of lncRNAs make them attractive therapeutic targets or biomarkers. Here, we review the various cellular processes affected by lncRNAs and outline molecular strategies they use to control gene expression, particularly in cancer and in relation to transcription factors. Genome-wide transcriptomic analyses revealed that over three quarters of the human genome can be transcribed and that approximately fifty percent of it may be read at a given time in a single cell . RNAs arWhat makes lncRNA genes difficult to catalogue comes partly from their high cell type specificity and rather low expression levels compared to mRNAs, both of which may be linked to the sequence and structure of their genes . One conCancer refers to a group of diseases where cells evade regulatory checkpoints controlling growth, proliferation, and tissue invasion as natural processes go haywire, including controlled lncRNA expression. MALAT1, H19, and PCA3 are amongst the first lncRNAs found overexpressed in cancer, and along with many other transcripts, they were later confirmed to have important oncogenic roles  and transcription by RNA polymerase III (RNA POL III) does not make it a lncRNA by conventional definition, it employs a mechanism of action analogous to that of lncRNAs. The B2 RNA was found to play an important role in the transient, general repression of POL II activity that occurs immediately upon heat shock and before heat shock-specific genes are induced ,78. B2 RLncRNAs can also influence the likelihood of a gene’s transcription through epigenetic mechanisms that are not via direct interactions with POL II or TFs. The examples presented below are grouped based on their general influence on the chromatin landscape or three-dimensional (3D) architecture.Since the discovery that HOTAIR regulates gene transcription by recruiting histone-modifying complexes , a plethGATA3 locus by GATA3-AS1 involves such a mechanism as the GATA3-AS1 lncRNA transcribed antisense to the GATA3 gene forms an R-loop that tethers an MLL methyltransferase to activate transcription [TARID gene generates an R-loop at the TCF21 promoter. The tethered TARID then binds GADD45A to recruit the DNA demethylating factor TET1, ultimately leading to TCF21 activation and tumor-suppressor activity [LncRNA–PcG complexes can reach their target genes via different means , includicription . The lncactivity ,33.SPHK1 protein-coding gene is known to form triplex-DNA with that gene’s promoter and activates its expression by recruiting histone acetyltransferases CBP/p300 [TGFBR1 in trans by facilitating H3K27me3 deposition and silencing through the formation of triplex-DNA and recruitment of the polycomb repressive complex 2 (PRC2) at one of its distal regulatory regions found to loop with its promoter [Another way in which lncRNAs can directly interact with genomic DNA to recruit chromatin-modifying complexes is through the formation of DNA–RNA triplexes (triplex-DNA): Rather than base-pairing with one genomic DNA strand, the RNA nestles into the groove of the DNA helix through Hoogsteen base-pairing . The proCBP/p300 . Epigenepromoter .HOXA cluster 5′ end, was found to upregulate the transcription of several surrounding genes by associating with the activating WDR5/MLL complex [LncRNAs can indeed act over long distances, with their influence found to spread beyond one gene, as HOTTIP, expressed from the  complex . Further complex .SOX2, which was partly through H3K27me3 deposition facilitated by binding the EZH2 component of PRC2 and hnRNPK to the SOX2 promoter via triplex-DNA formation. While hnRNPK—an essential RNA/DNA-binding protein with critical roles in several cancers [In addition to influencing the recruitment of chromatin-modifying proteins to genomic DNA, lncRNAs also determine the functional composition of RNPs as they can have more than one protein-binding domain and thus recruit varying combinations of distinct proteins. HOTAIR was the first reported lncRNA to bind more than one chromatin-binding complex; it was found to associate with PRC2 at its 5′ end and the LSD1/coREST/REST complex at its 3′ end, both of which contribute to transcription inhibition by coordinating H3K27 methylation with H3K4 demethylation, respectively . Since t cancers —was prev cancers , binding cancers , exempliKey research developments have established an important role for lncRNAs in 3D chromatin organization as both influence transcription as well as various other genome activities including X-chromosome inactivation (XCI), imprinting, DNA damage repair, and DNA replication. First, genome organization is now studied both by microscopy and with genome-wide high-resolution mapping techniques, including Hi-C and ChIA-PET. These techniques were designed to capture chromatin organization from pair-wise DNA contacts—or with more recently developed methods that additionally measure multivalent contacts, such as GAM (genome architecture mapping) and SPRITE (split-pool recognition of interactions by tag extension) ,90,91,92At lower resolution, lncRNAs can act as nuclear organization factors on their own or as part of nuclear subcompartments. For example, NEAT1 (nuclear paraspeckle assembly transcript 1) is a lncRNA that localizes to paraspeckles, which are transcription-dependent, self-organizing, membrane-less nuclear organelles . ParaspeXist (X-inactive specific transcript) is yet another important nuclear lncRNA that controls gene expression partly through changes in 3D chromatin organization . Xist isFirre genomic locus to several regions on other chromosomes [As illustrated by the examples above, lncRNAs can influence 3D chromatin organization in different ways. For instance, the Firre lncRNA encoded on the X chromosome escapes XCI to act as an important genome organizer, reportedly anchoring the Xi to the nucleolus , helpingomosomes . Conversomosomes . At highomosomes . They caomosomes , alter tomosomes , drive Lomosomes ,105,129,omosomes .The mechanisms by which lncRNAs alter chromatin conformation is either through direct DNA recruitment or through protein association. Changes in chromatin organization have been linked to R-loop formation  and to tAfter transcription, lncRNAs continue to control gene expression by influencing alternative splicing (AS) . In humaFGFR2 is a good example where in mesenchymal cells, exon IIIb is excluded because the negative splicing regulator PTB is recruited by the H3K36me2/3-binding protein MRG15. However in epithelial cells, this exon is retained due to the production of a nuclear antisense lncRNA from within FGFR2 that recruits PcG protein and the H3K36me2/3 demethylase KDM2a, which prevents binding of the MRG15-PTB complex [Epigenetic chromatin modification is known to influence AS, and cases have arisen where lncRNA-directed epigenetic changes play a part . The cel complex .Protocadherin a gene cluster, where first exon selection is dependent on the expression of its antisense lncRNA, which by promoting local DNA demethylation allows CTCF binding and long-range looping with a distal enhancer that will drive transcription from the selected site [LncRNAs can also affect AS by altering 3D chromatin organization. One way by which such a mechanism has been used is by altering CTCF chromatin association. Although CTCF binding on its own can affect AS by inducing POL II pausing and exon inclusion , whetherted site . Given tted site ,150, it SEPALLATA3 gene in Arabidopsis thaliana, where a circRNA derived from its exon 6 forms an R-loop with the corresponding DNA sequence, causing transcriptional pausing, specific SF recruitment, and exon 6 skipping in a shorter isoform responsible for homeotic phenotypes [To date, a subclass of lncRNAs—the circular RNAs (circRNAs)—was reported to directly regulate AS by interacting with genomic DNA. CircRNAs are produced by a non-canonical AS back-splicing process that occurs mostly at the middle exons of pre-mRNAs . Like otenotypes . While cenotypes .LncRNAs also can have a broader impact on AS by modulating the activity and/or localization of SFs. One such example is the regulation of the SR family SFs phosphorylation and localization by MALAT1 in nuclear speckles . MALAT1 LncRNAs can modulate splicing using different mechanisms involving direct contacts with RNAs. A lncRNA can directly bind a pre-mRNA, as does the SAF lncRNA shown to bind its antisense pre-mRNA encoding FAS as well as splicing factor 45 (SPF45) to induce exon skipping. The resulting FAS mRNA variant lacks exon 6 and translates into soluble Fas that retains its ability to bind the Fas ligand and blocks interaction with the Fas-receptor and pro-apoptotic signaling in human cancer cells . RNA–RNARNA contacts are not restricted to the specific splicing regulation of certain mRNAs, as exemplified by MALAT1′s partial hybridization to many nascent pre-mRNAs to participate in splicing through SF recruitment . InteresA most frequently reported activity for cytoplasmic lncRNAs is their action as competing endogenous RNAs (ceRNAs) to effectively “sponge” microRNAs away from target mRNAs. As miRNAs can induce translation repression and/or promote mRNA decay, miRNA sponging by lncRNAs generally makes them positive mRNA regulators that lead to higher protein levels . InitialMost studies report derepressed translation as a result of lncRNA sponging of miRNAs, yet it can also potentially lead to translation inhibition as some miRNAs act as decoys to translation inhibition proteins. Such is the case for miR-328 shown to bind hnRNP E2 in a way that prevents it from binding to and repressing translation of the CEBPA mRNA in leukemic blasts . While nLncRNAs can bind other RNAs beyond miRNAs, such as mRNAs which influence their subcellular localization and translational output . SeveralSeveral reports describe lncRNAs directly binding translation factors to regulate mRNA translation. In most cases, lncRNAs target the rate-limiting step of translation—the initiation phase—where it often interferes with its limiting factor: eIF4E . GAS5 waLncRNAs use various mechanisms to alter the stability of mRNAs and proteins as gene expression regulation. As previously mentioned, mRNAs under the influence of miRNA-mediated degradation may be controlled indirectly by ceRNAs. Direct mRNA cleavage by miRNAs seem to be more the exception than the rule, as it occurs for ~6% of mRNAs targeted by miRNAs, while the rest appear to become destabilized due to translation inhibition ,171.LncRNAs can base-pair with their mRNA targets to also recruit protein complexes that impact their stability. Staufen1 (STAU1) is one such protein, which is recruited through base-paired Alu sequences to bind resulting double-stranded lncRNA/mRNA hybrids, leading to STAU1-mediated mRNA decay . While iLncRNAs were also found to alter mRNA levels without directly binding them but by interacting with proteins that customarily control mRNA stability. An example is how CAAlnc1 acts as decoy for HuR; in binding HuR, CAAlnc1 prevents the degradation of hundreds of adipogenesis-related transcripts, some of which encode adipogenic TFs . AnotherWhile lncRNAs can bind proteins to alter mRNA stability, they can also affect the stability of proteins themselves. This can occur directly, as for lncRNA SNHG15, as it binds the Slug protein’s zinc finger domain to prevent its ubiquitination, thereby protecting it from proteasome-mediated degradation and promoting colon cancer cell proliferation . ProteinIn addition to MALAT1, which as detailed above controls SR splicing factor distribution, some lncRNAs can regulate the cellular localization of specific proteins. For example, while the NRON lncRNA keeps NFAT in the cytoplasm in resting T-cells, it migrates to the nucleoplasm upon T-cell activation . AnotherCDKN1A/p21 promoter region, PANDA is induced by p53 upon DNA damage and is often overexpressed in human cancers, granting its use as a diagnostic biomarker [As POL II-transcribed genes, lncRNAs are subject to much of the same transcriptional regulation as their protein-coding counterparts. Indeed, despite minor, but distinguishing, structural differences between lncRNA and mRNA genes ,176, theiomarker . PANDA wiomarker . PANDA wiomarker . As suchKeeping with the ubiquity of feedback regulation in cellular biology, lncRNA promoters can also be enriched in binding sites for the TFs they in turn regulate. This is the case for the AK028326/MIAT/Gomafu lncRNA, which is both induced by and co-activates the Oct4 TF in embryonic stem cells . This lnPVT1 is a lncRNA gene known to regulate the expression of MYC, which is located around 55 kb upstream on the same chromosome. PVT1’s normal expression is monoallelic, and as its promoter competes with the MYC promoter for intragenic PVT1 enhancers on its single expressed allele, in doing so, it acts as a tumor suppressor by regulating the release of MYC transcriptional pausing [PVT1 gene has been found to act as an oncogene under certain conditions, including its amplification in extrachromosomal DNA (ecDNA), and it drives MYC transcription in trans [LncRNA–TF crosstalk can go beyond transcript-mediated regulation and involve the lncRNA genes themselves; as exemplified by PVT1, lncRNA promoters and/or their gene bodies can regulate TF expression.  pausing . Howeverin trans .C16orf74 promoter to downregulate its expression and repress cervical cancer progression. In contrast, HNF1A-AS1 was found to bind PBX3 to increase OTX1 expression and promote angiogenesis through ERK/MAPK signaling in colon cancer [Although lncRNAs and TFs have been independently implicated in cancer, increasing evidence suggests that they also do so together as RNPs. The identification of functional lncRNA–TF complexes frequently come from studies of aberrantly expressed lncRNAs, where gene targets were identified and RNA immunoprecipitation assays were used to validate physical interactions between the lncRNA and a TF also regulating those genes. Such studies usually proceed with the knockdown and/or overexpression experiments that demonstrate cooperativity between the lncRNA and TF for proper gene expression, roles in cancer pathways, and effects on cell proliferation, differentiation, and migration. LncRNA–TF RNPs can either positively or negatively regulate target gene transcription. For example, the HAND2-AS1 lncRNA was found to bind E2F4 at the n cancer ,182. WhiACY1 promoter region [The binding of a lncRNA to a TF can prevent the protein from binding either another nucleic acid or protein. In addition to the aforementioned examples of linc-YY1/YY1 and GAS5/GR, where lncRNAs regulate transcription factor binding to chromatin, the MAGI2-AS3 lncRNA was found to exert tumor-suppressive and anti-angiogenic activities in clear cell renal cell carcinoma by interacting with and reducing the enrichment of the HEY1 TF at the r region . Furtherr region .TCF21 was reported for the lncRNA SATB2-AS1 at the SATB2 gene, where it recruits WDR5 and GADD45A, eliciting gene activation that is thought to inhibit metastasis in colorectal cancer [LncRNAs can help TFs find genomic DNA target regions as well as prevent it. Whereas MAGI2-AS3 reduces the chromatin association of TF HEY1 , lncRNA-l cancer .PROX1 TF promoter, where it also physically associates with hnRNPA2/B1 to promote PROX1 transcriptional activation in bladder cancer [BRCA1 promoter and simultaneously bind the GABPA TF; however, the binding of GABPA to CISAL actually prevents the TF from binding the BRCA1 promoter. As such, CISAL contributes to the repression of the tumor-suppressor gene and better disease outcome in tongue squamous cell carcinoma. This study proposes a novel model by which lncRNAs prevent TFs from binding target DNA that does not involve competitive binding or acting as decoys [As previously discussed, lncRNAs can also directly bind genomic DNA as triplex-DNA, as does the LNMAT2 lncRNA at the r cancer . Interess decoys .Alternatively to R-loop or triplex-DNA formation, RNA–RNA interaction may be a potentially important mechanism underlying lncRNA–TF recruitment to target genomic loci. Steitz and colleagues first demonstrated this with the ncRNA EBER2, which is a ~170 bp ncRNA expressed by the Epstein–Barr virus that interacts with the PAX5 TF. Using CHART, they found that PAX5 and the ncRNA EBER2 co-localize at PAX5 binding sites, and that RNA–RNA interaction with nascent transcripts guided such localization, which was later supported by demonstrating an in vitro interaction between ncRNA and nascent transcripts from such loci ,201,202.CDKN2A/B, thereby affecting cell senescence, proliferation, aging, and apoptosis [INK4a locus from which the lncRNA is expressed [KLF2 and p21 promoters to silence them in NSCLC [IL6 and IL8 promoters in human endothelial cells [LncRNAs can act as architectural platforms to facilitate the interaction of multiple proteins and/or nucleic acids; they have been shown to do so with TFs, such as DINO, which supports p53 tetramerization and recruitment at p53 target genes . Similarpoptosis ,204. In xpressed . In tranin NSCLC . ANRIL ial cells . This dual cells . As lncRMYC locus. CCAT1-L is transcribed from a super-enhancer region located 515 kb upstream of the MYC gene. It upregulates MYC by mediating long-range chromatin contacts with its promoter by binding to CTCF and either locally concentrating or allosterically modifying its association at the MYC locus [MYC enhancer–promoter interactions: the MYMLR lncRNA transcribed antisense to MYC also facilitates these contacts to upregulate the proto-oncogene in colorectal cancer. This interaction, between the MYC promoter and a super enhancer region located 550 kb upstream of MYC, relies upon MYMLR’s binding of the oncogenic PCBP2 splicing factor. Interestingly, it might be the same upstream enhancer region that is looping with the MYC promoter and implicated with both MYMLR and CCAT1-L activity [As with general transcription, the act of lncRNA transcription itself likely plays an important role in modulating the 3D chromatin landscape to impart transcriptional regulation . As alsoYC locus . CCAT1-Lactivity . As CCATTEs are mobile repetitive DNA sequences capable of moving from one genomic location to another. This migratory ability has enabled them to expand in our genome over the last 80 Myr of primate evolution, such that they now represent at least 45% of our DNA sequence . TEs havDux TF gene and induce rRNA expression by interacting with a Nucleolin-KAP1 protein complex [LINE1 hypomethylation in fact sometimes used as a cancer biomarker [The ways TEs contribute to the emergence of lncRNA features, regulation, and regulatory functions seem to vary greatly, and to date, at least two TEs themselves were shown to have lncRNA-like activities: human long interspersed nuclear element 1 (LINE1)  and the  complex . This ln complex , with LIiomarker ,222,223,Although the concept of personalized patient therapy is not new, precision oncology only recently became feasible through the development of genomics approaches that molecularly profile tumors and identify targetable alterations. Thus far, much emphasis has been placed on proteins in cancer therapeutics. However, proteins are not necessarily ideal therapeutic targets, as they often have ubiquitous and essential functions in tissues. LncRNAs could be attractive diagnostic or prognostic biomarkers given their expression specificity, functional diversity, easy detection in bodily fluids, and their deep involvement in cancer regulatory networks. Their regulatory relationship with TFs occur for ~20% of known oncogenes, making this an especially promising vulnerability for therapeutics. Thus far, however, while many ongoing clinical trials are exploring the value of specific lncRNAs as biomarkers, such as CCAT1 in colorectal cancer , only prLncRNAs are also being considered as potential therapeutic targets, with a few methodologies having been developed for such treatment: antisense oligos (ASOs), nanoparticle-delivered siRNAs, and small-molecule inhibitors. ASOs and siRNAs bind target lncRNA sequences to promote degradation, making them useful in cancer contexts in which their aberrant upregulation promotes pathology. Small molecule inhibitors, however, target lncRNA’s secondary and tertiary structures, and they have the potential to impinge on their interactions with other macromolecules, such as interfering with lncRNA–TF complexes. Many pre-clinical studies on cancer treatment through lncRNAs are ongoing. For example, MALAT1 is aberrantly expressed in many cancers, and the benefits of targeting it through ASOs and small molecule inhibitors are being investigated ,228,229.LncRNA involvement in virtually all aspects and levels of gene expression regulation is pervasive and undeniable—moreover in cancer. However, the impact of their mis/expression in different contexts, whether it significantly disrupts cell order, and if it can be harnessed for clinical use remains largely unknown. Answers to these questions will require first identifying the full repertoire of lncRNA genes, followed by substantially more individual characterization of their function, the regulatory networks they infiltrate, and the molecular mechanisms underlying their control. It will also require a more fully realized understanding of their potential crosstalk: not merely between lncRNAs and interactors but also between the different functions they might exert across the cell to synergize regulatory responses. Such responses may proceed from the transcriptional output of sole lncRNA genes exhibiting different subcellular localizations. For example, HOTAIRM1 can be stably expressed in its unspliced (nuclear) and spliced (cytoplasmic) forms where they appear to regulate transcription and miRNA sponging, respectively ,236. Ano


Chd7 is absent from cardiopharyngeal mesoderm (CPM). Here, we investigate altered gene expression in the CPM and identify specific CHD7-bound target genes with known roles in the morphogenesis of affected structures.Haploinsufficiency of the chromo-domain protein CHD7 underlies most cases of CHARGE syndrome, a multisystem birth defect including congenital heart malformation. Context specific roles for CHD7 in various stem, progenitor, and differentiated cell lineages have been reported. Previously, we showed severe defects when Chd7 in CPM and analysed cardiac progenitor cells using transcriptomic and epigenomic analyses, in vivo expression analysis, and bioinformatic comparisons with existing datasets. We show CHD7 is required for correct expression of several genes established as major players in cardiac development, especially within the second heart field (SHF). We identified CHD7 binding sites in cardiac progenitor cells and found strong association with histone marks suggestive of dynamically regulated enhancers during the mesodermal to cardiac progenitor transition of mESC differentiation. Moreover, CHD7 shares a subset of its target sites with ISL1, a pioneer transcription factor in the cardiogenic gene regulatory network, including one enhancer modulating Fgf10 expression in SHF progenitor cells vs. differentiating cardiomyocytes.We generated conditional KO of We show that CHD7 interacts with ISL1, binds ISL1-regulated cardiac enhancers, and modulates gene expression across the mesodermal heart fields during cardiac morphogenesis.  Considerable emphasis has been placed on determining roles in various compartments and progenitor pools of the central nervous system. Important CHD7 functions in differentiation of neurogenic progenitors have been described,3 and CHD7 is one of the set of genes encoding chromatin modifiers where loss of function mutations are overrepresented in patients with autism (or executive dysfunction more broadly) and/or congenital heart defect.4–6CHARGE syndrome  is a birth defect with significant morbidity. It is associated with deletion or point mutation of chromodomain helicase DNA binding protein 7 (CHD7), a chromatin remodeller with adenosine triphosphatase (ATPase) activity.7–9 pharyngeal-epithelial-specific conditional mutants11 and neural crest cell (NCC)-specific mutants. These NCC mutants have outflow tract (OFT) and other defects.12 Our own work described cardiogenic mesodermal-specific  null mutants  of Chd7 which similarly had intracardiac [atrioventricular septal defects (AVSDs)] and outflow defects, together with interruption of the aortic arch, which recapitulate abnormalities seen in the human syndrome.13 Thus, CHD7 has multiple, important tissue specific roles that likely act through both cell autonomous and non-autonomous actions. This study aimed to identify the cardiogenic transcriptional circuitry disrupted by loss of CHD7 in cardiogenic mesoderm, and identify which, amongst these dysregulated genes, had genomic sites binding CHD7. The location of these sites provided insight into co-regulating transcriptional regulators and associated regulatory elements involved in heart development.In terms of cardiovascular development, investigators focused on the constitutive heterozygous mouse model of the human syndrome,14–16Mesp1-Cre cKO mutants were embryonic lethal by E15.5 and displayed defects in both heart field derivatives.13 Regarding the SHF, embryos had a range of outflow defects including common arterial trunk, double outlet right ventricle (DORV) and overriding aorta. Hearts also had AVSDs, sometimes with double inlet left ventricle (the Holmes heart), and hypoplastic ventricular walls. The transcriptome of mutant embryonic day (E) 11.5 and E13.5 hearts suggested abnormal cardiomyocyte (CM) maturation and abnormal excitation contraction coupling, a defect confirmed following calcium imaging of paced primary CMs from E13.5 embryos. Together, these observations suggested a role for CHD7 in progenitors of both mesodermal heart fields providing the foundation for investigations described here.MESP1 is a pioneer transcription factor (TF) expressed in the cardio-pharyngeal mesoderm, a lineage forming various head and neck muscles, and much of the heart via the first and second heart fields (F/SHF).17 However, CHD7 also has non-ATPase-dependent regulatory activity12 possibly mediated through interactions with histone modifiers. For example, it has been proposed to be a sometime constituent of PBAF complexes in NCCs,18 interacting with BRG1 .20 Other reported interactions include the WAR complex (WD repeat domain 5 (WDR5), Absent-Small-Homeotic-2- Like protein (ASH2L), and Retinoblastoma Binding Protein 5 (RbBP5)),21 CHD8 (its close orthologue),23 and SMAD family member 1 (SMAD1).24 In this way, CHD7 has the potential to work in concert with TF binding and histone modification to modulate gene expression. It can also interact with splicing factors and abnormalities of gene splicing were reported in Chd7 mutants.26At the biochemical level, CHD7 operates as an ATPase-dependent chromatin remodeller whereby nucleosome sliding is effected at target sites, thus secondarily affecting availability of target DNA sequences for TF binding.in vivo single cell genomics and in vitro differentiation of stem cells towards CMs have been helpful in elucidating gene regulatory networks (GRNs) underlying cardiac morphogenesis and disease.27–32 A general feature of these networks is the combinatorial binding of TFs to enhancer elements, which can result in enhancers moving through inactive/poised, and active states, and the reverse, as defined by certain histone modifications.33Both Isl1 itself. Moreover, CHD7 physically interacts with ISL1 in CP cells. These results provide new insights into CHD7 function in CPM, where it modulates gene expression across the mesodermal heart fields, likely through its activity at key enhancers of CPM genes.In sum, the mutant phenotypes and functional analysis of CHD7 suggest a vital but poorly understood role for control of gene expression during cardiac morphogenesis. In this paper we explore further the role of CHD7 in cardiogenic mesoderm, identifying differentially regulated genes (DEGs) in the developing heart and caudal pharyngeal mesoderm at E9.5 upon loss of CHD7. Hybridization chain reaction (HCR) expression analysis was used to validate the targets and identify domains of expression affected by CHD7 loss. Overall, we found that certain genes expressed in the posterior SHF had increased expression, with decreased expression of genes expressed in the anterior SHF. There was a significant overlap of the DEGs with genes bound by CHD7 in murine embryonic stem cells (ESCs) differentiated to the cardiac progenitor (CP) stage. 62% of the CHD7 binding sites were >10 kb from the transcription start site (TSS) and had chromatin features associated with enhancer activity, particularly enhancer histone modification marks that change during mesoderm to CP and CP to early CM stages of mESC differentiation. Notably, there was significant overlap between ISL LIM homeobox 1 (ISL1) and CHD7 binding sites, with one such site upstream of 2.2.1flChd7 WtsiChd7) and Mesp1-Cre YsaMesp1). For timed matings, the date of the vaginal plug was designated as E0.5. Ear biopsies or embryonic yolk sacs were used for genotyping.The mouse lines used were: 2.2All animal work was carried out according to UK Home Office regulations, under project license number PPL P168131D1 and conform to the guidelines from Directive 2010/63/EU of the European Parliament. The project license application was approved by the UCL Animal Welfare and Ethical Review Body before submission to the Home Office.2 chamber followed by cervical dislocation.No anaesthetic/analgesic agents were used. Euthanasia was performed in CO2.3‘SHF’ or ‘HEART’ tissue was micro-dissected from E9.5 embryos and stored at −80°C, until genotyping was complete. Tissue from each embryo was processed individually and sequenced as a single sample. Each ‘SHF’ sample originated from a single embryo, and similarly each ‘HEART’ sample originated from a single heart and OFT, as described in the text. Total RNA was isolated using RNeasy Plus Micro kit, with gDNA eliminator column.2.4Mesp1-Cre) and six cKO  samples were processed using the KAPA RNA HyperPrep Kit with RiboErase according to manufacturer’s instructions. ERCC RNA Spike-In Mix was added to all RNA samples. Libraries were pooled in equimolar quantities, calculated from Qubit and Bioanalyser fragment analysis, and multiplexed in the same run. Samples were sequenced on the NextSeq 500 instrument (Illumina) in a 43 bp paired end run.Five control (Raw data were demultiplexed and converted to fastq files using Illumina’s bcl2fastq Conversion Software (v2.18) on BaseSpace. Reads were then aligned to the mouse reference genome (UCSC mm10) using STAR (v2.5.0b) on the BaseSpace RNA-seq alignment app . Reads per transcript were counted using HTSeq (RnaReadCounter by Illumina) and differential expression was estimated using the BioConductor package DESeq2 on BaseSpace app (v1.0.0).2.5Mesp1-Cre) and four cKO  samples were processed using the KAPA mRNA HyperPrep kit. Libraries were pooled in equimolar quantities and sequenced on the NextSeq 500 instrument (Illumina) in a 43 bp paired end run.Four control  before aligning reads to the mouse genome (UCSC mm10) with RNA STAR (v2.7.2b). Once aligned, reads were counted for each gene using featureCounts (v1.6.4). All sequence and annotation were obtained from the Illumina iGenomes repository. Counts were then used to perform DESeq2 differential expression analysis using the BioConductor tool (v1.3.2).2.66 cells (CP cells at day 5 during CM differentiation) with the CUT&RUN assay kit , according to the manufacturer’s protocol. Three biological replicates were processed for each condition, using the following antibodies: anti-CHD7  and anti IgG  as negative control. DNA fragments were purified using DNA Purification kit . Library preparation was performed with the NEB DNA Ultra II assay with some deviations, as described in CUT&RUN was performed using 0.5 × 10Library concentration was assessed with the Qubit DNA High Sensitivity assay and size distribution was assessed with the DNA 1000 High Sensitivity TapeStation assay. Samples were equimolar pooled before sequencing on the NextSeq 2000 (Illumina) according to manufacturer’s instructions. A P2 flow-cell and 100 cycle reagent kit were used to read with a 56 bp paired-end recipe along with 2 × 8 bp unique dual indexing plus an 8 bp UMI.2.734 (v2.1.2) for each sample with IgG as controls and overlapping peaks in at least two replicates were used as the final peaks.Data were UDI demultiplexed using BCL Convert (v03.75) before pre-processing with fastp (v0.20.1) to remove Illumina adapter, polyG + sequences and trim low-quality bases. Processed reads were aligned to the mouse genome (UCSC mm10) with bwa (v 0.7.17.4) before being UMI deduplicated with JE-Suite (v1.2.1). Peaks were called by MACS22.8https://www.molecularinstruments.com/). DNA probe sets were designed and manufactured by Molecular Instruments, using the Mus Musculus accession number for the target mRNA sequences from the NCBI database. In situ HCR was performed using the whole-mount mouse protocol.35 Following HCR embryos were counter stained with DAPI and mounted using CitiFluor AF1 mounting media into a ‘homemade chamber slide’ that provided enough space for the embryos to retain their 3D structure for imaging. Each HCR was repeated on at least four stage-matched embryos. Details on image acquisition and analysis are available in Embryos E9.5 were dissected and processed for whole mount ISH. For each target mRNA a DNA probe set, DNA HCR amplifier, and hybridization, wash and amplification buffers were purchased from Molecular Instruments , mesoderm (MES) , cardiac precursor CP , and CM (Day 10). For the CUT&RUN experiments, we selected to collect the cells at Day 5, based on principal component analysis (PCA) , as described in the results section. Our ‘Day 5’ timepoint is 24 h after plating at MES stage (Day 4) and 8 h prior to CP stage .Mouse ESCs were differentiated to CMs as described by Wamstad et al. data and PCA showed a good matching trajectory .Transcriptional changes during CM differentiation of our ESCs were compared with Wamstad 2.10in vitro CM differentiation were used for the co-IP experiments. 5 × 106 cells were used for each antibody. Cells were lysed with IP lysis buffer , supplemented with protease inhibitor cocktail  on ice. After sonication and centrifugation, the protein lysates were incubated overnight with 2 μg of ISL1 antibody (ab109517) or 2 μg of IgG (CST 66362) at 4°C, followed by 3 h incubation with Dynabeads protein A (ThermoFisher) at 4°C the next day.CP cells at day 5 of The immunoprecipitated samples were analysed by western blot, using antibodies against CHD7 (CST 6505) and ISL1 (DSHB 39.05D).3.3.1Chd7 in the CPM we performed RNA-seq on two microdissections from fl/flMesp1-Cre; Chd7  (referred to as cKO) and control (Mesp1-Cre) embryos. The first involved the distal pharyngeal apparatus, i.e. arches two through six including the dorsal pericardial wall (DPW) which encompasses the SHF before these cells migrate to the poles of heart.38In vivo, CP cells (Mesp1+) found in the pharyngeal arch 2 (PA2) are reported to be a major source of cells contributing to the SHF.39 The region also contains less specified mesodermal multilineage primed progenitor cells (MLPs32). For simplicity we refer to this dissection as ‘SHF’ (we continue to use inverted commas in this denotation to recognize the presence of other cell types) as shown in Figure . We sequenced ‘SHF’ samples from five control and six cKO single embryos. We identified 313 differentially expressed genes , with 91 genes upregulated and 222 downregulated in cKO ‘SHF’ . Gene ontology (GO) analysis of down-regulated genes revealed enrichment of terms linked to the contractile apparatus, heart development, and cardiac ventricle morphogenesis . These genes were also involved in cardiomyopathies and congenital heart disease, based on Jensen disease enrichment analysis . By contrast, up-regulated genes were involved in glycolysis , expression which normally decreases as CPs differentiate.40To investigate the role of Chd7 cKO, we compared the expression of all the ‘SHF’ DEGs among cell types (i.e. clusters) identified in a previously published single cell RNA-seq data.32 There, the authors dissected the pharyngeal apparatus and heart from E9.5 Mesp1-GFP embryos f/+), suggesting that CHD7 may control normal differentiation paths during CPM development, resulting in a reduced CM population or delayed CM differentiation in the cKO embryos.To gain insight to what cell types may be affected more functionally by Figure  (labelled ‘HEART’). We sequenced four control and four cKO single embryos and identified 987 genes significantly dysregulated (FDR < 0.1), with 561 genes up and 426 down-regulated . GO analysis of down-regulated genes revealed processes involved in cardiac development and muscle contraction , terms similar to those found in ‘SHF’ DEGs. Analysis of up-regulated genes showed enrichment for extracellular matrix organization and cell migration . We compared DEGs of ‘SHF’ with ‘HEART’ tissue DEGs and found significant overlap . The significance of overlap was tested using hypergeometric test and the P-value is provided in the figure. Most gene expression changes were in the same direction .To study transcriptional changes in cells that have entered the heart  and parts of the atrium, and FHF-derived left ventricle and atria), we also performed RNA-seq of the OFT and heart tissue from the same embryos, as shown in Chd7 Mesp1 cKO hearts13 revealed abnormal CM maturation, supported by functional impairment of Ca2 + transients in paced CMs from E13.5 hearts. Our RNA-seq analysis at E9.5 showed that early CM markers were reduced in ‘SHF’ and ‘HEART’ tissue of cKO embryos, so we compared with E11.5 and E13.5 data. In E11.5 cKO hearts 102 genes were significantly changed (FDR < 0.1), with 42 (41.2%) significantly changed in E9.5 cKO ‘HEART’ . GO (cellular components) analysis showed involvement of these genes in the sarcoplasmic reticulum membrane, endoplasmic reticulum lumen and cation channel complex . In E13.5 hearts 24 genes were significantly changed (FDR < 0.1), and 15 out of the 24 (62.5%) were differentially expressed in E9.5 ‘HEART’ . This overlap was observed from E9.5  until E13.5 , suggesting that effects of gene expression may persist once CHD7 protein itself has largely disappeared from the heart at the later stage.13Previous microarray analysis of E11.5 and E13.5 Taken together, these data indicate that CHD7 plays a significant role in transcriptional regulation of genes important for heart tube and pharyngeal mesoderm development, with particular effects within the SHF.3.2in vitro approach of directed differentiation of mouse ESCs towards CMs in order to overcome technical difficulties encountered with in vivo derived tissues. Furthermore, in vitro CM differentiation allows comparison of our data with several published datasets from equivalent in vitro systems that map the dynamic enhancer landscapes along CM differentiation.To identify primary and direct targets of CHD7 during the early stages of cardiac commitment and differentiation, we decided to map the genome-wide binding sites of CHD7. We used an in vitro differentiation that best reflects the stage of ‘SHF’ cells in vivo we first compared our control RNA-seq data with the RNA-seq datasets published from Wamstad et al.37 In that study, the authors selected four ‘key’ stages during in vitro CM differentiation  and performed RNA-seq and other genomic analysis. PCA revealed that our in vivo ‘SHF’ samples most closely aligned to the transition between MES and CP, but closer to CP as shown by the first principal component . We therefore selected a time point for the in vitro experiments 8 h prior to their CP stage, and refer to this as ‘Day 5’ . We used a CUT&RUN (‘Cleavage Under Targets & Release Using Nuclease’) technique, followed by NGS.42 This allowed us to use the low number of cells available after replating , and avoid limitations arising from crosslinking, sonication and solubilization.42 The assay was performed in three biological replicates using anti-CHD7, and IgG as negative control.In order to identify a suitable timepoint for Figure ), which were associated with 4975 genes by the software GREAT using default setting . Using ChIP-Enrich we examined the distribution of the peaks compared to the nearest TSS . We found 13.1% of peaks within proximal promoters (up to 1 kb from the TSS) and 26.3% within greater promoters (up to 5 kb from TSS). The majority of peaks (62.9%), however, were located over 10 kb from TSS, with 26.2% located over 50 kb from TSS . Therefore, CHD7 was mainly associated with elements distant from the TSS but had relatively low enrichment at promoters.We identified 3761 peaks enriched in CHD7 Ab samples . CHD7 binding targets play significant roles in important developmental pathways such as cellular responses to various growth factors . To define direct transcriptional targets of CHD7, we intersected CHD7-bound genes with DEGs in ‘SHF’. We found 35% of cKO ‘SHF’ DEGs were bound by CHD7 . GO analysis of these genes shows enrichment for heart development, circulatory system development, cardiac ventricle morphogenesis and skeletal system development processes . Both downregulated and upregulated ‘SHF’ genes independently showed significant CHD7 binding . The intersection of CHD7 peaks with DEGs in ‘HEART’ tissue displayed a similarly significant overlap . GO analysis revealed enrichment for axon guidance and axonogenesis, potassium ion transport, cell migration and signalling pathways involved in cardiac muscle development and function . The term heart development was the most significant when considering ‘HEART’ down DEGs with CHD7 peaks .GO analysis of the genes associated with the CUT&RUN peaks shows that CHD7-associated genes are enriched in several biological processes, including heart development and nervous system development  with CHD7 binding sites and (b) that have known importance within the cardiogenic GRNs . Whole mount Fgf10 is expressed in CPM including arterial pole progenitor cells and was the first molecular marker of the murine SHF.38 In control embryos Fgf10 was expressed in the SHF (green bracket), the mesodermal core of the branchial arches (arrowheads) and weakly in the RV . In cKO embryos SHF expression was lost  and some minimal expression was present in the DPW in medial sections . Fgf10 expression in the mesoderm of the branchial arches (arrowheads) and the RV was also reduced in cKOs .We first analysed SHF progenitor markers downregulated in cKOs. Isl1, which is expressed throughout the SHF.43 In control embryos Isl1 is expressed in SHF (green bracket and DPW arrow), branchial arches (arrowheads) and OFT . In cKO embryos Isl1 expression in the branchial arches was reduced, especially in the 2nd arch , but expression at the lateral part of the SHF (green bracket) and medial DPW was not severely affected .Next, we examined Hand2 and Hand1 were reduced in cKO embryos in both dissections. In control embryos, Hand2 is expressed in the distal portion of the branchial arches (arrowheads), in SHF (green bracket), in the OFT, and RV , but this expression was significantly reduced in cKO embryos especially in the SHF and OFT . Hand1 expression was also reduced in cKO embryos, mainly in the OFT .Based on our RNA-seq data, Mef2c this is an important part of the SHF cardiogenic GRN, and a Mef2c enhancer-derived CRE transgene is commonly used as mechanism for targeting this lineage.44 HCR revealed reduced Mef2c expression throughout the SHF of cKO embryos .While we did not detect a CHD7 binding site at Tbx5 is expressed in the pSHF, FHF-derived left ventricle and atria, as shown in control embryos viewed from the left side. In cKO embryos, Tbx5 expression in the heart tube was not affected (consistent with RNA-seq data), but increased expression was observed in the posterior SHF region . Whole mount HCR staining for additional genes in the pSHF—Osr1 , Foxf1  and Wnt4 —showed increased gene expression levels in cKO embryos, confirming the RNA-seq data (quantification in Table S1).We then examined genes highly expressed in venous pole progenitor cells of the posterior SHF (pSHF) and upregulated in the ‘SHF’ of cKO embryos, based on the RNA-seq data. Mesp1 lineage, further supported by expression quantification of structures with no significant change . Tbx1 is a SHF marker with important roles during CPM development.14Tbx1 expression was not changed in cKO embryos based on RNA-seq data, and this was consistent with microarray analysis performed in Chd7 cKO heart samples at E11.5 and E13.5.13 To rule out changes in domain of expression, we performed whole mount HCR to examine the spatial distribution and expression level changes of Tbx1; these were similar in control and cKO embryos. In particular, there was no significant difference in the expression levels of Tbx1 + ve cells in the branchial arches and OFT or within SHF cells in the lateral and medial DPW . Tbx1 is also expressed in a population of cells within the CPM recently described as MLP.32 At E9.5, MLP cells are localized in the lateral part of the caudal pharyngeal mesoderm, adjacent to the forming fourth arch, and these cells express Tbx1, Aplnr (Apelin receptor) and Nrg1 (neuregulin),32 amongst other markers, none of which were altered in ‘SHF’ cKOs. Quantification of relative fluorescence intensity of Tbx1 + ve MLP cells in control and cKO embryos showed no significant difference . We also stained for Aplnr, again finding similar expression levels in control and cKO embryos .Together these data reveal altered patterns of gene expression in the SHF such that genes expressed in arterial pole progenitor cells are downregulated and genes expressed in venous pole progenitor cells upregulated, consistent with our RNA-seq results. Other genes associated with SHF and branchial arch mesoderm were unaffected making a general cell loss unlikely. It is also important to note that these changes were subdomain specific, and not pan the Tbx1 expression appeared normal by HCR, given that a transcriptional boundary between Tbx1-positive progenitors at the arterial pole and Tbx5-positive cells at the venous pole is required for correct AV septation45 we examined TBX1 and TBX5 expression by immunofluorescence of sections containing the DPW. Chd7 cKO embryos showed normal boundary formation (5/6); one embryo displayed some TBX5 positive cells in the anterior domain of the SHF. We concluded that altered TBX1:TBX5 boundary formation was not a major contributor to the AV septation defects in CHD7 cKOs .Although Together these data validate specific changes in gene expression following loss of CHD7 and highlight the role of CHD7 in patterning gene expression in the SHF.3.43.4.1Figure ). To further characterize CHD7 genomic occupancy we compared our CHD7 CUT&RUN peaks with stage-specific enhancers identified in an in vitro model of CM differentiation.37 Wamstad et al. classified the candidate enhancers as ‘active’ , ‘poised’ (H3K4me1 + only) or ‘absent’ (neither mark) at each stage . We found significant overlaps between our CHD7 peaks and enhancers in all categories, supported by large odds ratio (OR) and low P-values by Fisher's test . The OR was particularly high for enhancer elements that are active in MES and CP, indicating the important role of CHD7 in the MES > CP transition. To further investigate, we clustered enhancers according to their states, based on marks described above, during the MES to CP transition for comparison with CHD7 peaks. We found significant overlap with all enhancer clusters, with the highest OR in the clusters that transition from ‘poised to active’ or remain ‘active’ during the MES to CP transition .Most CHD7 binding sites (63%) are located >10 kb from TSSs, with 26% > 50 kb  that have CHD7 binding sites (see Table S2). Isl1 is one example. Isl1 expression is reduced in cKO embryos, mainly in cells migrating through the branchial arches  and has a CHD7 binding site ∼200 kb upstream of the TSS . This site overlaps a putative CP enhancer from Wamstad37 also marked by H3K4me1 in E10.5 mouse embryonic heart 47 , supporting its role as a cardiac enhancer. Detailed analysis of the Wamstad data during CM differentiation indicates that this element was not active in ESC, became poised in MES and active in CP, suggesting that activation of this regulatory element is important for the transition from MES to CP (MES > CP). This peak also had an ISL1 binding site in CP cells (see section 4.3 below). Examination of other cardiac genes with reduced expression in Chd7 cKO (Hand1 and Hand2) revealed a similar pattern, where CHD7 binding overlaps with Wamstad candidate enhancers and ENCODE histone marks in E10.5 heart samples , suggesting these CHD7-bound elements drive cardiac expression of these TFs.We then examined specific CP genes dysregulated in Chd7 cKOs (see Table S2). Tbx5 is increased in Chd7 cKO  and has two CHD7 binding sites: one upstream , and one intronic .We also interrogated genes upregulated in Foxf1 is expressed in the pSHF and its expression is upregulated in Chd7 cKO embryos . Foxf1 has a CHD7 binding site ∼100 kb upstream of the TSS , and this site falls within a Wamstad cardiac enhancer, and is also enriched for H3K4me1, H3K27ac and K27me3 . Osr1 and Wnt4 were also upregulated in Chd7 cKO  and have CHD7 binding sites, with overlap for cardiac enhancers and ENCODE marks .In summary, these data indicate that both up- and down- regulated genes may be targeted by CHD7, which acts primarily at cardiac enhancers dynamically regulated during cardiac morphogenesis.Next, we decided to mine other published datasets for additional evidence of CHD7 interacting transcriptional regulators, especially in CP cells. We took three approaches: we looked for TF motifs enriched at the CHD7 binding sites (for TFs without published ChIP-seq data in CP cells), we unbiasedly searched for TFs exhibiting binding patterns significantly overlap with CHD7 (where ChIP-seq data from CP and other cardiac related cells were available), and we investigated TFs binding at the promoters of CHD7-bound genes (to account for co-regulation and looping without direct co-binding).3.4.248 and RSAT50 to identify TF binding motifs enriched in the Chd7 CUT&RUN peaks in order to study what TFs CHD7 may cooperate with in regulating SHF development.As CHD7 is not a DNA sequence specific binding factor, we used HOMERFigure S9A), further supporting a role in regulating cardiac genes, as well as SP1/KLF zinc finger motifs. HOMER analysis of the same peaks discovered 54 significantly enriched motifs . CHD7 binding sites were enriched for SP11, KLF4 and GATA motifs, consistent with RSAT motifs. Moreover, HOMER showed additional enrichment of CTCF and CTCFL (BORIS) zinc finger motifs. Analysis of the CHD7 peaks associated with DEGs in ‘SHF’  or ‘HEART’  also showed significant over-representation of GATA motifs. ‘SHF’-related motifs also revealed various homeodomain TF motifs, predominantly the ‘Caudal (homeobox) motif’ as the top hit, including CDX4, CDX2, HOXD11, HOXA11 and HOXA13 TFs (based on Homer motif data).RSAT analysis of all CHD7 CUT&RUN peaks showed enrichment for motifs recognized by GATA and MEF2 TF families  among the top five regulators . Interestingly the similarity to published CHD7 data from non-cardiac tissues was not statistically significant (P = 0.17), indicating tissue specificity of CHD7 co-binding. The ISL1 ChIP-seq data were from CP cells (dataset GSE7970152), and we found 150 common peaks between CHD7 and ISL1 datasets. GO analysis of genes associated with these co-binding peaks showed enrichment for heart development, cardiac ventricle formation and RV morphogenesis . We examined the distribution of CHD7-ISL1 peaks (using ChIP-Enrich) and found 33.3% within promoters (<5 kb from TSS) and 61.3% in distal elements (over 10 kb from TSS) . Specific examples of sites occupied by both CHD7 and ISL1 include Isl1 itself , Fgf10 , Hand1 , Hand2  and Tbx5 .As an unbiased approach, we used the BARTweb53 The ‘RNAseq Automatic GEO Signatures Mouse Down’ function of Enrichr showed that there was significant overlap between the ‘SHF’ down DEGs and down DEGs from (FACS sorted Nkx2–5–GFP+) CPCs derived by (in vitro) differentiation of −/−Isl1 mESCs vs. control . Similarly, there was significant overlap of −/−Isl1 CPC down DEGs and ‘HEART’ down DEGs . The second is from additional analysis of genes associated with CHD7 CUT&RUN peaks. Using The ChEA function within Enrichr, containing results of ChIP-seq studies, we searched for TFs bound to the promoter of genes associated with a CHD7 binding site. Third listed in terms of probability value was again the CP cell ISL1 ChIP-Seq . ISL1 has been shown to bind at promoters as well as enhancers/distal elements in CP cells.53 In addition, ChEA analysis uncovered possible cooperation with WT1 .The ISL1 connection was also supported by two additional results. The first is from RNA-seq data from ISL1 mutants.in vitro CM, same stage as the CHD7 CUT&RUN experiment). Immunoprecipitation with anti-ISL1 antibody followed by western blot with anti-CHD7 confirms CHD7-ISL1 protein interaction .To study whether CHD7 and ISL1 physically interact, we performed co-immunoprecipitation experiments in CP cells , and similarly for the ‘HEART’ dissection , we found that promoters at these genes were significantly enriched for proteins of the polycomb group complex (PcG). Unsurprisingly, ISL1 was also enriched at these promoters .Focusing on the subset of genes with CHD7 binding sites and differentially expressed in ‘SHF’ , is associated with two CHD7 CUT&RUN peaks: one upstream  and one intronic . Both the upstream and intronic peaks coincide with cardiac enhancer elements37 and E10.5 heart H3K4me1 marks . Interestingly, the intronic peak ‘peak2900’ is located within a characterized Fgf10 enhancer, a 1.7 kb regulatory element important for the regulation of Fgf10 expression in SHF vs. differentiated CMs and left ventricle.58 Notably, the intronic peak also contains TBX1 and NKX2.5 binding sites as well as for ISL1. Moreover, the upstream peak ‘peak2899’ is conserved in mammals and maps to the location of the Fgf10 ‘enhancer trap’ cassette Mlc1v-nLacZ-24 that originally led to the identification of the SHF as a source of CPs for the RV and the OFT myocardium.38Finally, one well studied enhancer provides an informative example. Fgf10 expression during heart development.Thus, collectively our analysis implicates CHD7 in the transcriptional control of ISL1-regulated cardiogenic TFs, and the complex switch of 4.Mesp1-Cre; Chd7 cKO embryos we set out to identify genes whose dysregulation are likely to contribute to these phenotypes. We paid particular attention to genes whose loci were bound by CHD7 as these would more likely be direct targets. Examination of existing datasets, TF binding motif analysis and gene ontology were employed to assess the most important TF interactions and pathways involved.Given the varied and severe heart malformations seen in the Mesp1 lineage e.g. pharyngeal epithelia, the dissection to remove the first pharyngeal arch and neural tube overlying the caudal arches should enrich for CPM while not introducing noise from exposure to FACS purification. This dissection includes the DPW from which SHF cells migrate to the arterial poles, and a progenitor cell pool recently described and characterized by expression of Aplnr and Tbx1.32 The ‘HEART’ dissection would contain cells from both heart fields, but at a more advanced stage of differentiation, including early CMs, thus likely capturing longer term and late effects. A significant number of genes were concordantly differentially expressed across both data sets. Included in these were Sema3a and Sema3c , both genes reported to have decreased expression in later stage Mesp1 cKOs,13Chd7 heterozygous whole embryos,59 and, for Sema3a only, a Xenopus model of CHARGE syndrome.60 Notably, the Semaphorin3 receptor PlexinA2 is a BRG1:CHD7 target in NCCs.19 CHD7 has similar phenotypes in the NCC and mesoderm conditional mutants, in particular both have 4th PAA and OFT septation defects; mesodermal mutants have more severe AVSDs. It is known that the SHF and NCC ingressing into the OFT reciprocally interact.61 One possible mechanism is via Semaphorin signalling. For example, NCC derived-Sema3c is required for EMT during OFT septation,13 and the Sema3 receptor PLXNA2 is similarly required in the cardiac NCC.62Although our E9.5 ‘SHF’ bulk RNA-seq contained cells outside the Mesp1 cKOs that we published previously. Of interest, the failure of CM differentiation culminating in abnormal coupling of calcium signalling to pacing13 was foreshadowed by the decreased expression of early CM markers shown most evidently in Figure S1C. Some genes were concordantly differentially expressed in both E9.5 dissections and whole hearts at E11.5 . The reduction of markers associated with CM function was particularly striking.We compared the E9.5 data with E11.5 and E13.5 transcriptomic data from Mef2c, and the control (expression unchanged) genes Tbx1 and Aplnr. The expression levels of SHF arterial pole progenitor cell markers Fgf10, Mef2c, and Hand2 were reduced in cells in the medial and lateral DPW as well as the PAs, while Isl1 expression was only reduced in the cells migrating through the PAs and not cells in the DPW. Of the pSHF genes Wnt4 was increased across its SHF domain, whereas the picture for Tbx5 was more complicated. Medial expression in the DPW, where it establishes a boundary with Tbx1 in concert with retinoic acid signalling, did not appear affected. However, the Tbx5 expression domain was expanded caudally on the lateral side of DPW. Unaltered expression of the SHF MLP pool marker Aplnr32 and Tbx1, which is expressed across the anterior and posterior SHF,63 indicate diminished expression of the aSHF genes listed above rather than loss of cells in which the genes are expressed.To confirm RNA-seq results and to examine overall vs. region-specific changes in gene expression we selected candidate DEGs for validation on the basis of CHD7 binding sites at the genomic locus (discussed below) or because they mark specific cell populations in the case of Mesp1Cre cKO mutants we see malformations of the OFT and interrupted aortic arch, which will likely be due to cell autonomous alterations of gene expression in the SHF  and/or noncell autonomous effects on cardiac NCCs . Disturbance of relative levels of expression of anterior vs. posterior SHF genes could affect both cardiac outflow and inflow patterning. The AVSDs seen in Mesp1 cKOs may be secondary to anomalous SHF or FHF contributions, or both. SHF derivatives are found in the dorsal mesenchymal projection (DMP), a structure vital for atrial and atrioventricular septation. A boundary between TBX1-positive arterial pole and TBX5-positive venous pole progenitors is vital for the DMP contribution45 but is established normally in Chd7 cKOs. Several genes specifically involved in AV canal morphogenesis showed altered expression e.g. Wnt4 . Wnt2, which also displayed increased expression in the ‘SHF’ sample, is expressed specifically in the developing inflow tract mesoderm and required for its remodelling, although we could not obtain good quality HCR data for this gene. GO analysis revealed WNT pathway as an enriched term for genes associated with CHD7 CUT&RUN peaks , and also for the intersect of genes with CHD7 peaks and differentially expressed in ‘SHF’ . Sema6D, which had reduced expression in both ‘SHF’ and ‘HEART’ dissections, is required for atrioventricular septation, compact-layer expansion and trabeculation.65Tbx2, with reduced expression in mutant hearts, is similarly required for AVS formation.66 TBX5 target genes Osr1,67Fendrr and Foxf1 are each upregulated. The TSS for the non-coding RNA Fendrr is approximately 1.3kbp upstream of Foxf1.68 Thus either or both genes might be coregulated by CHD7 binding to the site in Figure S6A. Osr1 and Foxf1 are both involved in remodelling the AV canal via a hedgehog cascade,68 and Fendrr is itself vital for normal heart development.69 Myosin heavy chain gene (Myh) mutations have also been described in human and murine heart defects (reviewed in70). Thus, a substantial number of genes orchestrating atrioventricular septation are dysregulated in Mesp1 cKOs.In 37 or in murine E10.5 whole hearts.71 In the differentiating mESCs, the most significant overlap of CHD7 was at sites with dynamically changing histone modifications during the mesodermal to CP or CP to CM transitions.As there is substantial extant data on regulatory elements modulating cardiac mesoderm differentiation, our results were compared to genome wide datasets to establish whether CHD7 mainly acted at known regulatory sites and TFs likely to be involved in co-regulation. There was significant overlap of DEGs and the presence of one or more CHD7 binding sites at that locus. The majority of binding sites were at sites where histone modifications suggested an enhancer element, either in differentiating mESCs,52 One of these binding sites was at Isl1 itself, was conserved in human sequence and showed dynamic histone mark transitions during CP differentiation from mESCs . Others included Fgf10, Hand1, Hand2, Tbx5, and Sema6d. The effect of loss of Isl1 in developing cardiac lineages is also similar to loss of Chd7 in both our ‘SHF’ and ‘HEART’ dissections from the point of DEGs. Interestingly, 25% of CHD7 binding sites were >50 kb from the TSS; it is notable that a study in β-cells of the pancreas, ISL1-enhancer-promoter loops had a median enhancer-promoter distance of 165kb.72 ISL1 is a pioneer factor capable of binding compacted chromatin. A recent study described how ISL1 cooperates with a BRG1-BAF60c-based SWI/SNF complex to effect epigenetic regulation of the chromatin landscape of genes in the cardiogenic GRN, including CM structural genes expressed once Isl1 itself is extinguished.53 In this paper, four of eleven cardiac GRN genes shown to be downregulated in +/−/Brg1fl/+Isl1-Cre and +/−/Brg1fl/flIsl1-Cre dissected E10.5 OFT and RV (SHF derivatives)  were similarly dysregulated in our E9.5 ‘SHF’ cKO dissections . Thus, these widespread effects mirror what we observe with CHD7 and support a CHD7-ISL1 co-operation, which is further supported by co-immunoprecipitation of the two proteins in CP cells .Another significant finding was the overlap with ISL1 binding as determined by ChIP-seq in CP cells.et al. suggest CHD7 regulates Fgf10 expression in neuroblasts during inner ear neurogenesis through one or both of inhibition of Tbx1 and activation of Isl1/NeuroD/Ngn1.73 In zebrafish, knockdown of CHD7 produces abnormal organization of cranial motor neurons tagged with GFP driven by ISL1 regulatory elements.74 Moreover, we performed HOMER motif enrichment analysis of previously published CHD7 ChIP-seq data from cerebellar granule cells75 and found significant enrichment for the neuronal ISL1 motif . We examined CHD7 binding sites for specific DNA sequence motifs. The GATA motif was the most over-represented. Notably, it has been shown that CHD7 interacts directly with GATA4 protein in CP cells derived from human iPS cells,76 that ISL1 and GATA4 often co-occupy sites with open chromatin,53 and ISL1 and GATA4 co-operate in regulation of Fgf10.77 Also of interest was the over-representation of CTCF and CTCFL (BORIS) binding sites. CTCF/L are 11-Zn finger proteins that bind to insulator elements, interfering with enhancer-promoter interactions thereby repressing or activating transcription at long range, and establishing domains permissive (or not) for transcription. Notably, CHD8 directly interacts with CTCF, and ChIP of CHD8 (the most closely related chromo-domain remodeller) showed it localized to many of the same sites as CTCF in Hep3B cells.78 CDX motifs were prominently over-represented; CDX TFs have important roles during early mesoderm development; more specifically CDX2 interacts and colocalizes with BRG1  at several cardiogenic loci.79There are indications an ISL1-CHD7 relationship exists outwith the cardiovascular system. Hurd 81 In G4 human medulloblastoma cells, CHD7 creates a less accessible chromatin conformation thereby preventing BMI1 repression of DUSP4,82 and more recent studies demonstrated the presence of a CHD7-BMI1-MAPK regulatory axis,83 so one specific example of PcG-CHD7 interaction already exists.As enhancers are known to loop to promoter sequences in a higher order chromatin structure, we wondered what transcriptional regulators might be over-represented at DEGs bound by CHD7: as CHD7 is likely to be part of multimeric complexes first bound at enhancers its fixation to promoters during our experiments might be weakened leading to underrepresentation. Here too, we found the presence of ISL1, but the most intriguing observation was the presence of many members of the polycomb group of proteins (PcG). Polycomb proteins are generally, but not always, associated with repression of transcription and H3K27me3 modification especially at genes regulating development. While we have not taken the observation further it is worthy of additional study since the fly ortholog of CHD7 (and CHD8), kismet, is known to antagonize PcG-mediated repression during development.Fgf10 is of especial interest, Fgf10 expression being downregulated in the aSHF of CHD7 cKOs. This element is instructive since it contains binding sites for ISL1, NKX2.5, GATA and TBX1. Enhancer transgenesis showed it activates Fgf10 expression in the SHF via ISL1 activity, but represses, via NKX2.5, in myocardium.58 CHD7 also binds an upstream FGF10 element  and this site is coincident with a nLacZ enhancer trap that was used in the initial description of a ‘second heart field’.38 This binding site shows mammalian sequence conservation and has GATA and MEF2a/c motifs. As an ATP-dependent nucleosome sliding protein CHD7 binding could modulate transcription in various ways as depicted in the context of ISL1 co-regulation in the model of Figure 7.As an insight into how CHD7 may function at a specific regulatory element the intragenic CHD7 binding site within 5.Isl1, Hand1, Hand2, Tbx5, Foxf1, Osr1 as well as signalling proteins such as Fgf10, Wnt4, and Semaphorins. We present evidence that CHD7 acts at sites bound by the pioneer TF ISL1, and at enhancers >50 kb from the TSS, a feature also noted for ISL1. CHD7 physically interacts with ISL1 in CP cells, we therefore suggest important co-operation during mesodermal through CM differentiation. TF-CHD7 interactions may be important where dynamic changes in expression level are required during mesodermal differentiation as exemplified by Fgf10 expression within a SHF domain and its repression in FHF lineages contributing to the left ventricle. In conclusion, it is clear that CHD7 has vital functions in the SHF as well as mesoderm more generally during heart morphogenesis. Issues such as CTCF, CHD8, and polycomb interactions may prove fruitful avenues for future research.Our data reveal that CHD7 fine-tunes the expression of genes in the cardiogenic GRN during cardiac morphogenesis. This includes critical TFs such as Cardiovascular Research online.cvad059_Supplementary_DataClick here for additional data file.


Collectively, the MYC family of oncoprotein transcription factors is overexpressed in more than half of all malignancies. The ability of MYC proteins to access chromatin is fundamental to their role in promoting oncogenic gene expression programs in cancer and this function depends on MYC–cofactor interactions. One such cofactor is the chromatin regulator WDR5, which in models of Burkitt lymphoma facilitates recruitment of the c-MYC protein to chromatin at genes associated with protein synthesis, allowing for tumor progression and maintenance. However, beyond Burkitt lymphoma, it is unknown whether these observations extend to other cancers or MYC family members, and whether WDR5 can be deemed as a “universal” MYC recruiter. Here, we focus on N-MYC amplified neuroblastoma to determine the extent of colocalization between N-MYC and WDR5 on chromatin while also demonstrating that like c-MYC, WDR5 can facilitate the recruitment of N-MYC to conserved WDR5-bound genes. We conclude based on this analysis that N-MYC and WDR5 colocalize invariantly across cell lines at predicted sites of facilitated recruitment associated with protein synthesis genes. Surprisingly, we also identify N-MYC-WDR5 cobound genes that are associated with DNA repair and cell cycle processes. Dissection of chromatin binding characteristics for N-MYC and WDR5 at all cobound genes reveals that sites of facilitated recruitment are inherently different than most N-MYC-WDR5 cobound sites. Our data reveals that WDR5 acts as a universal MYC recruiter at a small cohort of previously identified genes and highlights novel biological functions that may be coregulated by N-MYC and WDR5 to sustain the neuroblastoma state. MYC family of oncogenes encodes three related transcription factors  that are commonly overexpressed or deregulated in diverse types of cancers [MYC alteration place MYC proteins at the top of the list for targeted anti-cancer therapy pursuits.The  cancers . The per cancers , a funct cancers , 4. MYC-Recently, the paradigm for how MYC-MAX recognizes its target genes has undergone reevaluation, spurred on by the identification of additional factors beyond MAX that are important for MYC to engage its target genes –9 and suMYCN [MYCN for MYC in mice is incomplete for some functions [Beyond Burkitt lymphoma, WDR5 is thought to play a pro-tumorigenic role in various other cancers, including high-risk neuroblastoma , 17, whiMYCN . OverexpMYCN , 20. MorMYCN , 19, 21.MYCN  most of unctions . Therefounctions –25, unraunctions  and may unctions .To that end, in this study we find that the ability of N-MYC to bind chromatin at known conserved WDR5-bound genes is impaired if N-MYC cannot interact with WDR5, indicating that WDR5 can facilitate recruitment of N-MYC to specific loci. Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) of N-MYC and WDR5 in two N-MYC amplified neuroblastoma cell lines reveals that colocalization of N-MYC and WDR5 occurs invariably at genomic regions associated with genes involved in protein synthesis and includes the sites in which WDR5 can recruit N-MYC to chromatin. Interestingly, we find that N-MYC and WDR5 also colocalize across the genome at genes seemingly unrelated to protein synthesis processes, suggesting that there are genes cobound by N-MYC and WDR5 that are regulated through a non-recruitment mechanism. Overall, our data suggest that WDR5 acts as a universal MYC recruiter at a cohort of previously identified conserved WDR5 target genes and broadens the significance of the MYC–WDR5 interaction. for endogenous N-MYC in the N-MYC amplified neuroblastoma cell line, CHP-134. Western blots of co-immunoprecipitated material were positive for the presence of both MAX and WDR5 Fig. . To furtFig. . To probTo begin to dissect the interplay between N-MYC and WDR5 we performed ChIP-QPCR in the N-MYC amplified cell line, IMR-32, using antibodies targeted against N-MYC or WDR5. At conserved WDR5-bound sites, N-MYC and WDR5 co-localize as expected Fig. . To assehttps://david.ncifcrf.gov/) shows cobound sites are associated with genes involved in various important biological functions such as RNA processing, ribosomes, and translation all of which are known conserved WDR5-related functions [To gain insight into the degree of N-MYC and WDR5 colocalization beyond conserved WDR5-bound sites that we can track by QPCR, we performed ChIP-seq for N-MYC and WDR5 in CHP-134 cells. We detected ~1600 peaks for WDR5 and 10,000 peaks for N-MYC Fig.  each of unctions , 30, 31.Facilitated recruitment of c-MYC to chromatin by WDR5 occurs at <100 WBM-sensitive genes  predominAs a second point of comparison for understanding how N-MYC and WDR5 feature in N-MYC-amplified cell lines, we performed ChIP-seq for N-MYC and WDR5 in IMR-32 cells. Overall, we detected ~990 peaks for WDR5 and 4000 peaks for N-MYC in this cell line. ~63% of detected WDR5 peaks and almost all of the peaks detected for N-MYC were also peaks called in CHP-134 cells  sample. To crosslink DNA–protein complexes, 1% formaldehyde was added to each plate for 10 min, followed by the addition of 0.125 M glycine to quench the reaction. Cells were washed twice with ice-cold PBS and collected from the plate. Fixed cells were pelleted by centrifugation and nuclei were extracted by incubating the cells for 5 min on ice in a nuclear lysis buffer  supplemented with PMSF and protease inhibitor cocktail (Roche). Nuclei were pelleted at 1500 RPM and lysed for 15 min in FALB buffer  containing 1% SDS, PMSF, and protease inhibitor cocktail (Roche). Chromatin was sheared using a Diagenode Bioruptor Plus instrument and then debris was clarified through centrifugation. Each chromatin sample was stored at −80 °C until ChIP was performed. For DTWDR5 CHP-134 cells, chromatin was collected in the same manner following a 4 h treatment with 500 nM dTAG47 or DMSO-matched control. For ChIP-QPCR, chromatin from each sample was diluted in ice-cold FALB supplemented with PMSF and protease inhibitor cocktail (Roche) but containing no SDS. Immunoprecipitation was performed with 5 µl of an antibody against N-MYC , WDR5 , or 800 ng of normal rabbit IgG control  overnight at 4 °C. The following day, protein A agarose (Fisher Scientific) was blocked for 30 min with 1% BSA in FALB containing no SDS and allowed to bind immunocomplexes for 2–4 h at 4 °C. All ChIP samples were washed extensively as previously described [6 IMR-32 cells or CHP-134 cells treated with 0.005% dimethyl sulfoxide was extracted as described above. ChIP was performed using 5 µl of anti-WDR5 , 800 ng of normal rabbit IgG control , or 5 µl of custom serum to target N-MYC [Approximately 7.5–10 × 10escribed  and thenet N-MYC , which wq-value of 0.05 [http://homer.ucsd.edu/homer/) and Homer: findMotfisGenome. GO term enrichment analysis was performed using functional annotation clustering through DAVID (https://david.ncifcrf.gov/).After adapter removal via Cutadapt , alignme of 0.05 . DiffBin of 0.05  was thenSupplementary MaterialSupplementary Table 1Supplementary Table 2Supplementary Table 3