Chromatin adopts different configurations that are regulated by reversible covalent modifications, referred to as epigenetic marks. Epigenetic inhibitors have been approved for clinical use to restore epigenetic aberrations that result in silencing of tumor-suppressor genes, oncogene addictions, and enhancement of immune responses. However, these drugs suffer from major limitations, such as a lack of locus selectivity and potential toxicities. Technological advances have opened a new era of precision molecular medicine to reprogram cellular physiology. The locus-specificity of CRISPR/dCas9/12a to manipulate the epigenome is rapidly becoming a highly promising strategy for personalized medicine. This review focuses on new state-of-the-art epigenome editing approaches to modify the epigenome of neoplasms and other disease models towards a more ‘normal-like state’, having characteristics of normal tissue counterparts. We highlight biomolecular engineering methodologies to assemble, regulate, and deliver multiple epigenetic effectors that maximize the longevity of the therapeutic effect, and we discuss limitations of the platforms such as targeting efficiency and intracellular delivery for future clinical applications. Maintenance of cellular identity requires faithful replication of the genome during cell division. However, the balance between ‘stability’ of the genetic information and ‘flexibility’ of the spatiotemporal control of gene expression is highly regulated in eukaryotic cells. Thus, while the genomic ‘infrastructure’ based on the nucleotide sequence is faithfully replicated during cell division, the genomic ‘superstructure’ at the chromatin level exhibits a high degree of conformational freedom . Such adDue to the reversible nature of epigenetic modifications, ‘epidrugs’ have been clinically approved for manipulation of the epigenome. Epidrugs comprise specific inhibitors of DNA methyltransferases (DNMTs), histone-lysine methyltransferases (KMTs), histone-lysine acetyltransferase (KATs), histone-lysine demethylases (KDMs) and histone deacetylases (HDACs) . HoweverXanthomonas genus  guide RNA (gRNA) directs the Cas9 nuclease to cleave or nick DNA. The deactivated or catalytically dead version, referred to as CRISPR/dCas9 technology, has rapidly advanced the field of targeted gene regulation in recent years, due to its ease of targeting and its high versatility. Despite these advantages, CRISPR/dCas9 technology still suffers from significant impediments to its successful applicability.The first ATFs were generated by linking zinc finger (ZF) modules, potentially targeting single genes by the incorporation of six ZFs that can bind to 18-base pair (bp) genomic sites . Howeveras genus , which hIn this review, we discuss the main molecular inducible and repressible systems that have been devised for epigenome engineering, with an emphasis on the new emerging technologies for combining effector domains (EDs) to maximize targeted epigenome engineering for applications in molecular precision medicine.non-homologous end joining (NHEJ) and by microhomology-mediated end joining (MMEJ) mechanisms that lead to the accumulation of small nucleotide insertions or deletions (indels). Alternatively, DSBs can be repaired by error-free homology-directed repair (HDR) when a template that is homologous to the target site is delivered. However, HDR has a lower efficiency than the error-prone mechanisms, and both approaches have been harnessed for genome editing using catalytically active Cas9 proteins.The CRISPR–Cas system is part of an adaptive immunity mechanism present in a multitude of bacteria and archaea that encode a library of DNA fragments from foreign invaders, such as viruses and plasmids, for recognition and defense and ultimately destruction of foreign nucleic acids . All CasStreptococcus pyogenes (SpCas9) comprises 1368 amino acids (aa), and it has been catalytically inactivated by mutation of the Asp10 and His840 positions of the HNH and RuvC domains, respectively, to alanine residues (D10A/H840A). The resulting nuclease dead Cas9 (SpdCas9) is the most commonly employed for epigenetic editing. It recognizes the most simple 5′-NGG-3′ PAM sequence, which occurs every 8–12 base pairs in the human genome  has similarly been catalytically inactivated (D10A/N580A) (SadCas9) for epigenome editing  has been engineered by (D832A and/or E925A) mutations and it has been successfully adapted for gene transactivation  has been characterized more recently and repurposed for genome and epigenome engineering  of transcription activators are assembled . For exaTransactivator domains have been engineered for functional screenings in mammalian cells. Despite binding to multiple targets, large libraries of ZFs-VP64 have enabled the identification of malignant gene signatures in head and neck cancer ,40. In cet al. developed an epitope-based assembly system (SUperNova Tagging (SunTag)) for effective recruitment of multiple EDs -genomic contexts. To this aim, numerous epigenetic enzymes fused to programmable DBDs have recently been developed for precise control of gene regulation.NOS2 promoter induced gene reactivation in fibroblasts (Aberrant DNA methylation (DNAme), i.e., 5-methylcytosine, is associated with many diseases, such as neoplasms and neural degeneration. A ZFP engineered with the catalytic domain (CD) of TET2, Ten-Eleven Translocation methylcytosine dioxygenase 2 (ZFP-TET2), was first reported to precisely direct demethylation and reactivation of epigenetically silenced TSGs in cervical  and ovarroblasts   Binding Protein (GBP) and GFP-TET1-CD represents an additional platform to simultaneously titrate the two molecular constituents, thus minimizing off-target effects  modules, referred to as the Casilio recruiting technology (PUF Binding Site (PBS), while the EDs are fused to PUF modules  or Nei-Like DNA Glycosylase 2 (NEIL2) to complete the DNA demethylation cycle . Multimechnology . This cos Figure -VI-b.et al. have shown that dCas9 fused to the CD of the human acetyltransferase p300, which catalyzes H3K27 acetylation in promoters and in proximal and distal enhancers, induced gene expression with a high degree of specificity across the genome  domain from CREB-binding protein (CBP) was C-terminally engineered with dCas9 as well as with the Casilio PUF modules . The recP. falciparum to hyper-acetylate the transcriptional start site of the silent Rh4 gene, as a way to block the invasion of human erythrocytes by malaria parasites  to either dCas9 or ZFPs is sufficient to re-express silent genes in a context-dependent manner in lung, ovarian and cervical cancer cells. However, H3K79me induced by dCas9 or ZFPs fused to the DOT1-like histone-lysine N-methyltransferase, H3 lysine-79 specific (DOT1L) was found to be essential to maintain durable gene re-expression  is another histone lysine methyltransferase (KMT) that was recently linked to dCas9 to orchestrate chromatin interactions between super-enhancers and the use ESCs . Furtheruse ESCs   by single EDs, either directly fused to or recruited by DBDs, has been shown to be effective for many target genes. However, multiple and mechanistically distinct EDs are required to avoid exhaustion of the recruited endogenous co-factors, as well as to obtain a more durable epigenome manipulation. This can be achieved by different recruiting systems such as SunTag, aptameric, and Casilio technologies. There are several limitations, however, for each system, which include the large size of the SunTag constructs, which can affect delivery efficiencies ms Table .SOX2 in breast cancer  after 3 days of activity . Conversely, activity . SimilarOX2 gene  and ZF-D s s . To redus (DMR)s . This mos (DMR)s . A simils (DMR)s .B2M ; lastly, plant hormones, represented by the classical antagonistic duo comprising the abscisic acid (ABA)-inducible ABI–PYL1 system and the gibberellin (GA)-inducible GID1–GAI system, for which one of the key features is the tightness of regulation  system  and nuclear localization sequences (NLS) fused to N-dCas9-Frb and C-Fkbp-dCas9-VP64, respectively, and rapamycin was used to activate the dimerization  Figure .Different dimerizer-inducible systems can be exploited to generate synthetic epigenetic machinery by fusion of distinct EDs to the dCas9 split. Alternatively, different orthologues, such as Sp and SadCas9 fused to VPR and KRAB, and vice versa, can be employed to achieve simultaneous regulation of orthogonal genes when dynamically controlled by ABA- and GA-inducible systems in the same cell  Figure -II, IV.Hybrid drug Inducible CRISPR/Cas9 Technologies (HIT) have been developed for rapid and reversible modulation of transcriptional activation of the OCT4 and KLF4 genes. The HIT system deploys 4-OHT to chemically induce proximity of either Sp or SadCas9 and the scFv portion of the SunTag molecular entity directly grafted to two copies of a triple-mutated human estrogen receptor (ERT2). ERT2 guarantees low background activity due to its selective affinity for synthetic 4-OHT relative to the endogenous ligand β-estradiol. Without its ligand, scFv-ERT2 is sequestered by heat shock protein 90 in the cytoplasm. VP64 and p65-HSF1 (VPH) are separately attached to scFv-ERT2 constructs, as they have been shown to exhibit more pronounced synergistic activation than V and PH alone or fused together  systems integrating TALEs fused to the light-sensitive protein CRY2 with its interacting partner CIB1, from Arabidopsis thaliana, linked to either VP64 or SIN3 have been used for rapid and reversible optogenetic control of gene activation and repression, respectively, in primary mouse neurons  and VP16 with LOV, two light-inducible dimerizing proteins from ntensity   system. The same optogenetic actuators, CRY2 and CIB1, were attached to VP64 and to SpdCas9, respectively, to tightly regulate endogenous gene transcription in the presence of blue light -derived destabilization domain. The addition of the small molecule trimethoprim (TMP) temporally stabilizes the complex, thereby providing control over degradation of the DHFR fusion construct. However, this system has been shown to exhibit a degree of leakiness due to partial protein degradation  Figure -I.auxin-inducible degron (AID) technology. The AID system involves tagging Sp or SadCas9 with the auxin plant hormone-sensitive domain IAA17 and co-expression of the auxiliary protein TIR1. The administration of TMP allows for drug-tunable gene upregulation, which can be abrogated by the addition of auxin to the system as this results in rapid dCas9 protein proteasomal degradation , by depositing H3K27ac marks, and induction of gene expression from the proximal promoter  Figure -II.As an alternative to chemical approaches, interference and competition with CRISPR/dCas9 DNA recognition represents a valid tool to regulate its epigenetic editing activity. Since the CRISPR–Cas system is a natural bacterial defense against foreign invading elements, such as plasmids or bacteriophage infections , as a coFMR1 gene promoter and its expression were maintained for at least 14 days after inhibition of SpdCas9-TET1 by the potent AcrIIA4 protein. However, the inhibition of DNA demethylation was irreversible  methods (siRNA and shRNAs) , engineeThe binding specificity of genome editing tools depends primarily on the editing platforms employed and on the nature of the effector domains (EDs). Several studies, outlined in this section, have shown different or even partially contradictory results in regard to the exact mapping of DNA-binding events reported for each of these technologies. These discrepancies could, in part, be associated with the methods used to monitor off-target activities, which are determined by a number of ‘OMIC’ approaches, such as ChIP-sequencing (ChIP-seq), to detect the direct genome-wide binding events of the engineered proteins.In the case of CRISPR/Cas nucleases  and baseet al. conducted more comprehensive on- and off-target analyses on a 6ZF protein linked to KRAB, confirming the widespread binding of ZF platforms. Out of ∼6000 promoters bound, mapped by ChIP-seq, only ∼10% of these were differentially regulated (comprising 264 upregulated and 416 downregulated targets), as assessed by RNA-seq. Interestingly, the linkage of the KRAB repressor to the 6ZF arrays led to a ∼5-fold increase in binding sites, and the new sites were predominantly located outside of promoter regions  and downstream (ERMP1) genes  and RNA-seq, respectively. However, 10 off-target deregulated transcripts were reported experimentally, which were not computationally predicted  have proven high specificity, with undetectable genome-wide perturbations, as confirmed by the superimposition of RNA-seq, ATAC-seq, and in silico prediction of off-target binding sites using PROGNOS software  genes . Moreoveredicted . Furthersoftware .dCas9 protein in the absence of ED expression has been referred to as CRISPRi and exploited as gene repression tool, acting by interfering with the endogenous transcriptional activity. The DNA targeting specificity of dCas9 has been comprehensively assessed by ChIP-seq analyses, with off-target binding sites ranging from 10 to >1000  platforms have more consistently confirmed high precision in regulating target genes. For example, the SpdCas9-VP64 system demonstrated zero significantly upregulated off-target genes and one significantly down-regulated off-target gene, as monitored by RNA-seq . HoweverGFP gene driven by the SV40 promoter)  revealed similar results to that of activation platforms. The SpdCas9-KRAB repressor has been particularly studied, and demonstrated high on-target specificity, as evaluated by RNA-seq, for the targeting of a reporter gene (the romoter)  or the e regions . Interes regions ,157.Neisseria meningitidis engineered with LSD1 targeting the Tbx3 distal enhancer. However, gene expression microarray data revealed 174 differentially expressed genes, albeit not exceeding a two-fold change, thus suggesting that limited off-target effects occurred  to investigate the specificity of SpdCas9 proteins fused to either DNMT3A or DNMT3B, identifying >1000 off-target DMRs, with hypermethylated regions mapping in promoter regions, 5′ untranslated regions, CpG islands, and in DNase I hypersensitivity sites. Interestingly, the hypomethylated CpG sites mapped in repetitive sequences, such as Alu and LINE1 interspersed elements   .In summary, both Cas9 and TALE systems demonstrate a degree of off-target activities, which can be exacerbated by certain EDs, in particular DNMTs. Researchers should carefully map and determine potential non-cognate target sites, ideally by integrating several ‘OMIC’ and computational platforms. However, binding to off-target sites does not necessarily produce significant transcriptional perturbations in associated genes since the probability of binding to sequences mapping in proximity to regulatory regions, such as TSSs and enhancers, is relatively low. Validation methods, such as the engineering of multiple DBDs targeting the same regulatory region, as well as functional rescue experiments, are, therefore, recommended to fully validate the application of these tools in cells, tissues, and organisms.Potential off-target activities of genome engineering tools have been attributed primarily to the DNA base-pairing specificity of the DBD, for example the ZF and TALE domains, or the gRNAs interacting with genomic sequences for Cas9 and Cas12a proteins.For protein-based modules, early studies demonstrated that the number of domains or repeats assembled in ZFPs or TALEs increased the specificity by expanding the number of DNA contacts and, therefore, reducing the frequency of DNA binding events genome-wide. For example, 3ZF proteins, which only recognize 9 bp, are expected to recognize thousands of sites in a complex genome, such as the human genome . By contOn the other hand, some residues mapping in key alpha-helical positions of the ZF, such as –1, +3 and +6, can tolerate different amino acids side chains and still bind DNA ,164. ThiIn the case of RNA-guided platforms, such as Cas9 systems, the recognition of the PAM sequence dictates the initial Cas9 binding to the DNA, while the 20 nt target site complementary to the DNA is essential for binding specificity. Within this complementary sequence, the specificity is mainly conferred by the 3′ end of the gRNA, referred to as ‘seed region’, which comprises 10–12 nt upstream of the NGG PAM sequence . TherefoAside from genomic sequences that contain mismatches with the gRNA, off-target binding loci can also originate from targeted DNA sequences that either contain insertions (a DNA bulge) or that have nucleotide deletions (generating a gRNA bulge) relative to the designed gRNA sequence . ImportaFrancisella novicida, including an extended PAM sequence that increases the surface of DNA binding. Moreover, the platform is more permissive therefore more biased towards dT:rU and dG:rG than to dA:rA and dC:rC mismatches . Lastly, the system tolerated mismatches occurring after the 17th nt on the crRNA distal region, and up to three mismatches within the 6 nt of the seed region  for the regulation of targeted genes in heterochromatic contexts  at targeted loci, which can highly influence both the binding and the efficiency of the regulation of different epigenetic editing platforms.To improve specificity, efforts have been directed at re-engineering the DBD or DNA-binding activity of artificial transcription factors. Several strategies, outlined below, focus on: 1) re-engineering and molecular evolution of DBDs/gRNAs; 2) variations on the gRNA design; 3) synthetic Cas9 variants; 4) Cas9 and Cas12a orthologues; 5) cascade systems (CRISPR Class 1 systems); 6) ED recruiting technologies for CRISPR systems; 7) spatiotemporal control of DBDs and EDs.Re-engineering and molecular evolution of DBDs and gRNAsIn the case of ZFs  and TALErepeat-variable diresidue (RVD)  the identification of the diresidue asparagine-histidine (NH), as a more stringent guanine-specific ue (RVD) ; (ii) thue (RVD) ,195; (iiin vitro . (iv) Lain vitro .verification of in vivooff-targets (VIVO) method. In this strategy, the potential off-target cleavage sites from the gRNA/SpCas9 endonuclease ribonucleoprotein (RNP) system are first identified in vitro on the genomic DNA extracted from an animal model, employing the highly sensitive circularization for in vitroreporting of cleavage effects sequencing method (CIRCLE-seq)  . These iCLE-seq) .et al. overlapped the detected off-target sites for six gRNAs targeting cell lines with the genotypes of individuals from the 1000 Genomes Project Consortium  can still bind efficiently to the cognate DNA target site while reducing off-target activities by 5000-fold for particular target sites  or crRNAs (Cas12a) can increase the specificity of the gRNAs by thermodynamically increasing the instability with their off-target sites .Synthetic Cas9 variantsenhanced eSpCas9     (high fidelity HiFi-SpCas9 (R691A)  , the hig, Q926A) , followe, H698A) , and the, R661Q) , all del, R661Q)  and the  (R691A)  variantsV, R1335Q, T1337R), -EQR  and -VRER . These are SpCas9 derivatives that harbor the PAM-interacting (PI) domain mutagenized via bacterial-selection based screening  (Campylobacter jejuni (984 aa)  ,228 and (984 aa)  are natu(984 aa) .Breaks Labeling In Situ and Sequencing (BLISS-seq), which is a sensitive method for detecting genome-wide off-target activity  exhibit higher DNA binding specificity than SpCas9, evaluated by the activity . Additioineering . Lastly,n domain .Cascade systems (CRISPR Class 1)et al. have recently engineered the multi-component Class 1 CRISPR-associated complex for antiviral defense ‘Cascade’ systems from Escherichia coli (EcoCascade) and from Listeria monocytogenes (LmoCascade). Similarly to Cas systems, Cascade proteins can be fused with EDs such as p300 and the KRAB for specific gene activation and repression, respectively.Beyond the canonical Class 2 CRISPR–Cas systems (Cas9 and Cas12a), Pickar-Oliver Pseudomonas aeruginosa (PaeCascade-VPR) guided by 32 nt crRNAs are capable of activating gene transcription of therapeutic genes such as HBB and HBG to treat β-thalassemia, without predicted off-target activities. This recent technology is very sensitive to crRNA-DNA mismatches, including those very distal to the PAM sequence  and 36 nt (LmoCascade-KRAB) in length, respectively, demonstrated high genomic selectivity . For inssequence . DespiteED recruiting technologies for CRISPR systemsIn addition to the nature of the gRNAs, the specific strategy employed to recruit the EDs to dCas9 can highly influence the binding selectivity. For example, direct fusions of SpdCas9-DNMT3A may be more prone to generate off-target activities  comparedSimilarly, several recruiting platforms have been developed for the delivery of DNMTs or other enzymes by the aptameric, Casilio, and SunTag technologies, in order to boost on-target versus off-target DNAme activities. Moreover, recruitment systems can also bypass the tiling of promoter regions with many gRNAs, thus potentially lowering unintended off-target effects.Spatiotemporal control of DBDs and EDset al. observed significantly reduced off-target effects when SpdCas9-DNMT3A-CD direct fusions were induced by doxycycline (Dox) relative to that of the same fusions expressed in a constitutive system  and temporal control of epigenetic editing will be crucial for the development of more selective epigenome engineering tools. While Cas and TALE backbone systems are present naturally in bacteria and plant pathogens, respectively, mammalian cells already utilize several endogenous DBDs/TFs to precisely control cell-type-specific cell fates. Endogenous DBDs/TFs are often tightly spatiotemporally controlled by networks of TFs, which ensure that the right regulator is expressed at the proper threshold (expression levels) and within a given tissue, and in specific cell types. Similarly, future engineered epigenome editing technologies may not only consider on-target ‘genomic’ selectivity, but also ‘cell type’ selectivity. This could in the future be achieved by engineering artificial/synthetic networks of TFs , by usinin vivo.In summary, both TALE and CRISPR platforms have been developed to modify single genes with minimal, in some cases negligible, off-target activities. This can be achieved by modification or direct evolution of the proteins, by using different variants of Cas proteins , by modifications of gRNAs , and by the recognition of more specific and stringent PAM sequences. Moreover, the choice of the method/s to assemble different effectors as well as the spatiotemporal control of the epigenetic editing tools are important, particularly when manipulating DNAme. Lastly, ‘cell type’ in addition to the canonical ‘genomic’ specificity should also be taken into account when designing an effective expression strategy for epigenomic editing de novo induction of these marks by engineered domains can be expected to be ‘read’ and ‘maintained’ by the endogenous epigenetic regulators during DNA replication.An ultimate goal of epigenetic editing is the maintenance of gene expression patterns over cell divisions, which can be achieved by engineering of several epigenetic domains. Some epi-marks, such as DNAme  and H3K9MASPIN  may be due to the transient duration of the epigenetic editing stimulus , liposomes, or other nanotechnology vehicles. However, the manifestation of potential higher off-target activities, when a genomic integration choice is pursued, has to be considered carefully and addressed by using newer and more reliable and precise inducible systems.Sustained gene repression of highly overexpressed targets, such as oncogenic drivers, represents a great challenge for epigenetic editing technologies, particularly when using transient vectors. Several works have demonstrated that the assembly of multiple epigenetic domains could be highly effective for increasing the longevity of the repression state, even when using ‘hit-and-run’ transient delivery methods. For instance, the DNMT3A-CD (DA) and DNMT3L (DL) have been combined with either KRAB (K) ,109 or Ede novo DNAme induced by the ZFs were marked by H3K4me3  may still be present in the targeted genes. In fact, a genome-wide study with ZF-DNMT3A-CD found that genes that quickly ‘relapsed’ from  H3K4me3 . Thus, fet al. have found that the combination of two KMTs targeting histone H3, PRDM9 (inducing H3K4me3) and DOT1L (inducing H3K79me), fused to Dox-inducible SpdCas9 proteins, resulted in sustained (20+ days) gene re-expression of hyper-methylated gene promoters  enzymes  using the SpdCas9 Casilio recruiting system  with g system , or TET1g system . It mighIn general, the simultaneous writing and erasing of mutually exclusive epi-marks that co-exist at particular promoters  may be a key requirement for more sustained synthetic epigenetic manipulation. As more becomes known regarding the molecular epigenetics of most cell types and tissues, tailored therapeutic strategies may be devised whereby epigenetic regulators are combined to achieve more effective and sustained ‘reprogramming’ of targeted cells.in vivo delivery of the molecular tools to the proper cell types and tissues to restore the altered epigenome in these cells. Several strategies have been pursued for gene therapy. These comprise viral-based approaches, mainly adeno-associated viruses (AAVs), and non-viral-based approaches, which predominantly involve targeted cationic lipid nanoparticles (NPs) and polymers.Personalized medicine by epigenome editing requires precise For therapeutic epigenome editing, AAVs pose safety concerns due to pre-existing immunity towards them in the majority of the human population . Moreovein vivo, although it did elicit immunological reactions. Additionally, since this system relies on the trans-splicing machinery, it might not be the ideal approach to attain sufficiently high levels of gene activation in vivo to induce physiologically relevant phenotypic changes  with an intein-mediated split at its disordered linker (V713–D718) can be used to facilitate incorporation of the VPR domain (1.6 kb) into various AAV9 vectors. No widespread cellular damage has been noted  changes . To over in vivo . A thera type 1A .Pcsk9 gene, respectively, were systemically administered in a wild-type adult mouse model. Transcriptional repression of Pcsk9, a gene responsible for cholesterol homeostasis, was maintained for 24 weeks after a single treatment in post-mitotic tissue, with a moderate host response and coincidental liver toxicity. This was primarily due to SadCas9-KRAB expression rather than the AAV8 used as gene vector  system exploits the synergy of dCas9-VP64-mediated gene activation and Cas9 endonuclease activity to specifically target cancerous cells in vivo  were developed to efficiently tumor-target SpdCas9-VP64 and gRNAs as plasmid DNA polyplex via systemic administration. MDNPs have been intentionally designed to bypass physiological barriers, such as instability in the circulatory system and dissociation in the tumor microenvironment, followed by internalization into tumor cells  led to a therapeutic effect in a mouse model of liver damage. The authors generated this novel genome editing with designed extracellular vesicles (GEDEX) system to mediate targeted transfer of RNPs based on extracellular vesicles produced and released by HEK293 cells (93 cells .versus genome editing, the former being potentially reversible and the latter potentially prone to unattended and more irreversible changes in the DNA sequence, epigenetic editing approaches hold considerable promise for applications in a clinical setting. To date, there have only been clinical trials based on the use of the endonuclease Cas9. The ‘promise’ of epigenetic memory based on the faithful transmission of some epigenetic marks also provides avenues for long-lasting and durable therapeutic effects. Moreover, the success of epigenetic engineering strategies will be highly dependent on the collateral development of safe, immune-inert, and targeted delivery systems to enable dCas9/gRNA expression in specific cell types, such as cancer cells and other diseased tissues.Epigenome engineering has enormous potential as an innovative treatment for many human diseases, including cancer and neurodegenerative diseases. By strategically combining multiple different epigenetic effector domains and enzymes to rewrite the epigenome in a sustained manner, epigenetic therapy has a promising niche in a wide variety of human diseases. With the help of state-of-the-art genomic technologies, personalized medicine will also be a reality for many patients, minimizing side effects and thus improving overall survival. Nevertheless, many aspects, including off-target effects and ethical concerns, need to be addressed to extensively employ epigenome editing clinically. In light of the greater long-term safety of epigenome engineering gkaa1000_Supplemental_FilesClick here for additional data file.


Plasmodium spp. is still a major threat to public health globally. The various approaches to developing new antimalarial agents rely on the understanding of the complex regulatory mechanisms of dynamic gene expression in the life-cycle of these malaria parasites. The nuclear members of the evolutionarily conserved actin-related protein nuclear (ARP) superfamily are the major components of nucleosome remodelling complexes. In the human malaria parasite Plasmodium falciparum, bioinformatics analysis has predicted three ARP orthologues: PfArp1, PfArp4 and PfArp6. However, little is known about the biological functions of putative PfArp4. In this study, we aimed to investigate the function and the underlying mechanisms of PfArp4 gene regulation.Malaria caused by PfArp4 and PfArp6 genes. The transgenic parasites PfArp4-Ty1-Ribo, PfArp6-Ty1-Ribo and pL6-PfArp4-Ty1::PfArp6-HA were generated by the CRISPR-Cas9 technique. The knockdown effect in the transgenic parasite was measured by growth curve assay and western blot (WB) analysis. The direct interaction between PfArp4 and PfArp6 was validated by co-IFA and co-IP assays. The euchromatic gene expression mediated through H2A.Z (histone H2A variant) deposition and H3K9ac modification at promoters and regulated by PfArp4, was determined by RNA-seq and ChIP-seq.A conditional gene knockdown approach was adopted by incorporating the glucosamine-inducible glmS ribozyme sequence into the 3’ UTR of the PfArp4 inhibited blood-stage development of P. falciparum. PfArp4 and PfArp6 were colocalized in the nucleus of P. falciparum parasites. PfArp4 gene knockdown altered the global transcriptome. PfArp4 protein colocalized with the histone variant H2A.Z and euchromatic marker H3K9ac in intergenic regions. The inducible downregulation of PfArp4 resulted in the depletion of H2A.Z and lower H3K9ac levels at the upstream regions of eukaryotic genes, thereby repressing the transcriptional abundance of H2A.Z-dependent genes.The inducible knockdown of PfArp4 regulates the cell cycle by controlling H2A.Z deposition and affecting centromere function, contributing to the understanding the complex epigenetic regulation of gene expression and the development of P. falciparum.Our findings suggest that  Plasmodium parasites is still a major threat to public health globally. In 2018, there were approximately 229 million malaria cases worldwide, which resulted in 435,000 deaths [6A [Malaria caused by Plasmodium falciparum, the local chromatin structure marked by different histone modifications is the major factor determining the activity of gene transcription during infection, development and pathogenesis in the host. For virulence-associated antigenic variant genes such as var, rifin and stevor [P. falciparum [var genes is associated with the singular expression and switching in the next generation as a genetic memory.In the human malaria parasite d stevor , the hetlciparum , 8. GenoP. falciparum, some chromatin-associated factors have been found to interact with different chromatin markers [ap2-g. In addition, for the euchromatic genes, PfBDP1  and AP2-I have been shown to coregulate the transcription of invasion genes via specific interactions between the bromodomain of PfBDP1 and the acetylated lysine at promoter regions. However, to date, little is known about the regulatory factors that determine the selective deposition and dynamic exchange of H2A.Z at promoter regions of euchromatic genes. In other eukaryotes, Arp6 is an acritical component of the SRCAP (Snf2-related CREBBP activator protein) chromatin remodelling complex, which deposits H2A.Z onto chromatin. In budding yeast, H2A.Z/Htz1 is located at the nucleosome in the transcription start site [P. falciparum.In  markers . For insart site , 11 and art site , 13. Expart site . Moreoveart site  but alsoP. falciparum, a comparative genome analysis predicted only three ARP: orthologues PfArp1, PfArp4 and PfArp6 [Actin-related proteins (ARPs) in eukaryotes are a relatively conserved family of proteins , 17. Thid PfArp6 . Here, wgfp with the 1.5-kb C-terminus of PfArp4 fused with the Ty1-Ribo sequence by in-fusion cloning. The resulting vector, pL6-PfArp4-Ty1-Ribo, was used to transfect P. falciparum 3D7 parasites. For the pL6-PfArp6-Ty1-Ribo construct, the 1.5-kb C-terminus of PfArp4 was replaced with the 1.5-kb C-terminus of PfArp6. For the pL6-PfArp6-Ty1-HA construct, the Ty1-Ribo fragment of the pL6-PfArp6-Ty1-Ribo construct was replaced with a sequence encoding the HAx3-Ty1x3 tag via Asc I/Afl II. The pL6-PfArp4-Ty1::PfArp6-HA plasmid was modified by replacing PfArp4 with the 600 bp C-terminus of PfArp4 fused with the Ty1x3 tag and replacing PfArp6 with the 600 bp C-terminus of PfArp6 fused with the HAx3 tag by in-fusion cloning.A pLN-ENR-GFP plasmid was modified by the replacement of P. falciparum 3D7 strain was cultivated and synchronized as described previously [The eviously . Two huneviously . When thhttps://www.bioedit.com). Distance trees were made using both neighbour-joining and Poisson methods with a MEGA 5.05 software package (https://www.megasoftware.net). A maximum-likelihood (ML) tree of the Arp4 domains and Arp6 domains was constructed using the MEGA package (https://www.megasoftware.net/). Bootstrapping was performed for 1000 replicates. The accession numbers of Arp4 and Arp6 in are listed in Additional file Sequences were aligned using ClustalX v1.83, and alignments were manually adjusted using the BioEdit alignment editor . Since the molecular weight of PfArp4 and PfArp6 is approximately 62 kDa and 121 kDa, respectively, an 8% SDS-PAGE gel was used to isolate the proteins. The antibodies used in the experiment were rabbit anti-HA , mouse anti-Ty1 , and rabbit anti-PfAldolase . An ECL western blot kit  was used to develop the blots.P. falciparum parasites at a parasitaemia level of 2% were collected. The erythrocytes were washed once with incomplete 1640 medium at room temperature, and then, 0.15% saponin was added and mixed well for 10–15 min on ice to lyse the erythrocytes. The purified parasites were fixed with 4% paraformaldehyde (Sigma-Aldrich). After washing with 1× PBS, the cells were resuspended for immunofluorescence analysis. The antibody was diluted with 0.1% BSA. The mouse anti-Ty1 antibody was diluted to 1:500, the rabbit anti-HA antibody was diluted to 1:500, and the Alexa Fluor 488 combined anti-rabbit antibody was diluted to 1:500.Immunofluorescence assays were performed as previously described . SynchroCo-IP was performed as described previously . BrieflyRNA isolation, mRNA enrichment and library construction were performed as described using 15 cycles of library amplification . BrieflyP. falciparum 3D7 genome  using HISAT2 strand-specific mode (v2.1.0) [https://www.r-project.org). Genes with a minimum of twofold changes (PfArp4 KD versus G7) were considered differentially expressed genes in the two samples.Low-quality and adaptor sequences were trimmed from the reads using Cutadapt (v1.16)  with the(v2.1.0)  with thehttps://plasmodb.org/plasmo/). GO terms with a P-value ≥ 0.05 (Fisher’s exact test) and an enriched gene number ≥ 5 were considered to be enriched. We next classified our GO terms into different functional categories for better understanding.Gene ontology (GO) enrichment was performed using R (v3.5.1). The GO term database was downloaded from PlasmoDB  using Bowtie2 (v2.3.4.3) [Low-quality and adaptor sequences were trimmed from the reads using Cutadapt (v1.16)  with the2.3.4.3)  with fol2.3.4.3)  was used2.3.4.3) . Then, t2.3.4.3)  with the2.3.4.3)  was used2.3.4.3)  using thThe distribution of HMs and Arp4 signals around the gene body and centromere were generated by compute Matrix and plot Profile in the deep Tools suite (v3.1.3) . To geneP. falciparum 3D7 strain, two genes were predicted to encode the highly evolutionarily conserved PfArp4 (PF3D7_1422800) and PfArp6 (PF3D7_0719300) proteins. We constructed phylogenetic trees to trace the evolutionary relationship of PfArp4 and PfArp6 with their orthologues in other eukaryotes. The two Arp proteins in the genus Plasmodium formed a unique clade among eukaryotic organisms  and untreated (GlcN-). The parasites at the ring, trophozoite and schizont stages that retained normal morphology in the cell cycle after drug treatment were harvested for RNA-seq  with and without GlcN treatment. The parasites were pre-synchronized twice by sorbitol treatment followed by Percoll enrichment of the late-stage parasites. In the third cycle, the culture was divided into two groups for drug treatment: treated  parasites from the PfArp4-Ty1-Ribo line through the use of an anti-Ty1 antibody. The genome-wide distribution of PfArp4 ChIP signals showed that the majority of PfArp4 proteins were enriched at the intergenic regions, which generally corresponded to the promoter regions of 762 target genes  genes on the basis of the ChIP signal distribution  and actin-related proteins (ARPs) that are highly conserved in the composition and sequences in various eukaryotes. To date, multiple members that are localized predominantly in the nucleus were identified after they were initially found in various ARPs in the cytoplasmic compartment of cells. While these cytoplasmic ARPs are involved in structural functions, nuclear ARPs execute additional functions, such as gene regulation, by participating in ATP-dependent chromatin remodelling and histone acetyltransferase complexes . In addiP. falciparum [var) promoters in P. falciparum [Importantly, our data strongly support PfArp4 as a critical regulator positively correlated with euchromatic gene expression because it mediates H2A.Z deposition and H3K9ac modification in lciparum . In othelciparum . Study olciparum . Thus, tlciparum , they lilciparum , 49. HerPfArp4 gene knockdown further supports the function of the PfArp4 protein during the nuclear division of malaria parasites.In addition, we found that PfArp4 also occupied the two flanking sites of centromeres, likely as a boundary mark for this special chromatin structure, which is enriched by H2A.Z instead of heterochromatic markers, in contrast to the marks in other eukaryotes . The knoHere, we discovered that PfArp4 correlated positively with the dynamic expression of eukaryotic genes. The PfArp4 protein colocalized with the histone variant H2A.Z and euchromatic marker H3K9ac in the intergenic regions. Inducible downregulation of PfArp4 resulted in the depletion of H2A.Z and lower H3K9ac levels at the upstream regions of eukaryotic genes, thereby repressing the transcriptional abundance of H2A.Z-dependent genes. Intriguingly, PfArp4 was localized at the flanking sites of all centromeres, likely shaping the H2A.Z-enriched centromeric chromatin structure as a boundary. PfArp4 depletion triggered the loss of H2A.Z at all centromere regions and arrested blood-stage development, probably by interfering with nuclear division in the schizogony process. Taken together, our findings suggest that PfArp4 regulates the cell cycle by controlling H2A.Z deposition and affecting centromere function, which contributes to the understanding the complex epigenetic regulation of gene expression and development of malaria parasites.Additional file 1: Table S1. Accession numbers of Arp4 and Arp6.Additional file 2: Figure S1. Phylogenetic trees of PfArp4 and PfArp6 orthologs in eukaryotes. The unique clade of the genus Plasmodium is indicated by a red dash box.Additional file 3: Figure S2. A multiple sequence alignment of Arp4.Additional file 4: Figure S3. A multiple sequence alignment of Arp6.Additional file 5: Figure S4. Knockdown approach of PfArp6 a Schematic representation of generation of PfArp6-Ty1-Ribo transgenic parasites with the same strategy shown in Fig. b PCR analysis of PfArp6-Ty1-Ribo lines. The sequences of primer pairs were listed in Additional file 10: Table S4. c Growth curve assay of PfArp6-Ty1-Ribo lines with versus without GlcN drug in the culture. The parasitaemia were measured by microscopy . d Western blot of ring , trophozoite  and schizont  stage parasites of PfArp6-Ty1-Ribo line with or without GlcN in culture for one cycle. e Western blot analysis of PfArp4-Ty1::PfArp6-HA lines with antibody against Ty1 and HA epitope, respectively. The protein extracts of PfArp6-HA-Ty1 and PfArp4-Ty1-Ribo were used as controls. The aldolase signal was used as the internal control.Additional file 6: Figure S5. Reproducibility of RNA-seq data of PfArp4-Ty1-Ribo line throughout the blood-stage developmental cycle a Correlation of FPKM at R, T, S stages of parasite with wild-type PfArp4 between two biological replicates. Pearson correlation between biological replicates are displayed at top left corner. Correlation: P-value < 2.2e-16, r(5712) = 0.953; P-value < 2.2e-16, r(5712) = 0.979; P-value < 2.2e-16, r(5712) = 0.980. b Correlation of FPKM at R, T, S stages upon PfArp4 knockdown between two biological replicates. Pearson correlation between biological replicates is displayed at top left corner. Correlation: P-value < 2.2e-16, r(5712) = 0.912; P-value < 2.2e-16, r(5712) = 0.959; P-value < 2.2e-16, r(5712) = 0.895.Additional file 7: Table S2. FPKM values of RNA-seq analysis for PfArp4-Ty1-Ribo line with or without GlcN in culture for knockdown.Additional file 8: Figure S6. Reproducibility of PfArp4 ChIP-seq at ring stage. Heatmap showing the correlation of genic ChIP signals between three biological replicates. Pearson correlation is displayed in the heatmap.Additional file 9: Table S3. A list of genes enriched for PfArp4 in the promoter region.Additional file 10: Table S4. Sequences of primer pairs used to construct and verify PfArp4 or PfArp6 transgenic parasite lines.


Grass carp SLC20s proteins possessed conservative domains and amino acid sites relevant with phosphorus transport. The mRNAs of three slc20s appeared in the nine tissues, but their expression levels were tissue-dependent. The binding sites of three transcription factors  were predicted on the slc20s promoters. The mutation and EMSA analysis indicated that: (1) SREBP1 binding site (−783/−771 bp) negatively but VDR (−260/−253 bp) binding site positively regulated the activities of slc20a1a promoter; (2) SREBP1 (−1187/−1178 bp), NRF2 (−572/−561 bp) and VDR(615/−609 bp) binding sites positively regulated the activities of slc20a1b promoter; (3) SREBP1 (−987/−977 bp), NRF2 (−1469/−1459 bp) and VDR (−1124/−1117 bp) binding sites positively regulated the activities of the slc20a2 promoter. Moreover, Pi incubation significantly reduced the activities of three slc20s promoters, and Pi-induced transcriptional inactivation of slc20s promoters abolished after the mutation of the VDR element but not SREBP1 and NRF2 elements. Pi incubation down-regulated the mRNA levels of three slc20s. For the first time, our study elucidated the transcriptional regulatory mechanisms of SLC20s and their responses to Pi, which offered new insights into the Pi homeostatic regulation and provided the basis for reducing phosphorus discharge into the waters.It is important to explore the regulatory mechanism of phosphorus homeostasis in fish, which help avoid the risk of P toxicity and prevent P pollution in aquatic environment. The present study obtained the full-length cDNA sequences and the promoters of three SLC20 members ( Aquaculture was considered to be one of the most important food-producing industries and provided enormous excellent protein sources for people over the world. However, the ever-increasing aquaculture is producing some adverse influences on the aquatic environment. One of the main concerns is the discharge of wastes, especially phosphorus. Inorganic phosphate (Pi) is essential for skeletal mineralization, oxidative response and cellular metabolism. Aquafeeds need dietary Pi addition in the amount that meets the requirement of fish. However, phosphorus is also the rate-limiting nutrient for the eutrophication of waters, and excess phosphorus will be excreted by the fish to the aquatic environment and result in eutrophication . Thus, islc20a1 and slc20a2 expression in many cells [Pi absorption and transport are mediated by sodium-phosphate cotransporters, containing three families of SLC17/type I NaPi, SLC34/type II NaPi and SLC20/type III NaPi. At present, the structure and functional role of the SLC34 members were established for the vertebrates; in contrast, relatively few studies have been performed for SLC20 proteins . Moreoveny cells ,7, suggeny cells . Howeverslc20s have been cloned in humans and mice [slc20s promoters.The regions of gene promoters contain many important transcriptional regulatory elements which modulate their expression . At presand mice ,10 and sand mice ,11. For and mice . Nuclearand mice . Sterol and mice  and vascand mice ,16. Howeslc20s genes from grass carp (Ctenopharyngodon idella). Their mRNA tissue expression, transcriptional regulation and their function in Pi homeostasis were determined. Grass carp, a herbivorous freshwater fish, is one of the most important aquaculture species in the world and is selected as the experimental animals. The present study will increase our knowledge into the Pi homeostatic regulation and provide new knowledge for improving phosphorus utilization and reducing phosphorus pollution in the waters.Teleost fish have approximately 30,000 species and they possess the most diverse group among the vertebrates ,18, whicslc20a1a (Genbank No. MT561019), slc20a1b (Genbank No. MT561020) and slc20a2 (Genbank No. MT561021) from grass carp  sequences of SLC20A1A and SLC20A1B showed 61.9% and 60.0% identities with SLC20A2, respectively . Moreoveslc20a1a mRNA amounts were the highest in the kidney and liver, followed by the spleen and brain, and the lowest in other tissues. The slc20a1b mRNA expression was predominant in the kidney, and no significant differences were found among other tissues. The slc20a2 mRNA amounts were predominant in the brain, followed by the skin and heart, and were lowest in other tissues  increased but the mutation of VDR (−260/−253 bp) binding site reduced the luciferase activities of slc20a1a promoter, indicating that the SREBP1 (−783/−771 bp) negatively but VDR (−260/−253 bp) binding site positively regulated slc20a1a promoter activities. However, the mutation of NRF2 (−1688/−1679 bp) and VDR sites (−1083/−1076 bp) did not influence the luciferase activities of slc20a1a promoter , NRF2 (−572/−561 bp) and VDR (−615/−609 bp) binding sites significantly decreased the luciferase activities of slc20a1b promoter, implying that the SREBP1 (−1187/−1178 bp), NRF2 (−572/−561 bp) and VDR (−615/−609 bp) binding sites positively regulated slc20a1b promoter activities. However, the mutation of SREBP1 (−1354/−1345 bp and −212/−203 bp) and NRF2 (−391/380 bp) binding sites did not affect the luciferase activities of slc20a1b promoter , NRF2 (−1469/−1459 bp) and VDR (−1124/−1117 bp) binding sites decreased the activities of slc20a2 promoter, suggesting that the SREBP1 (−987/−977 bp), NRF2 (−1469/−1459 bp) and VDR (−1124/−1117 bp) binding sites positively regulated slc20a2 promoter activities. However, the mutations of SREBP1 (−1772/−1763 bp) and VDR (−1172/−1165 bp) binding sites did not affect the luciferase activities of slc20a2 promoter  as the probe, while the 100-fold unlabeled mutated SREBP1 binding sequences markedly reduced this competition, indicating that the SREBP1 binding sequence (−783/−771 bp of slc20a1a promoter) was functionally bound by SREBP1  as the probe, while the 100-fold unlabeled mutated NRF2 binding sequence markedly reduced this competition, indicating that NRF2 binding sequence (−572/−561 bp of slc20a1b promoter) was bound by NRF2 (slc20a2 promoter) as the probe, suggesting that this sequence was not bound by any factors  as the probe, while the 100-fold unlabeled mutated VDR binding sequence markedly reduced this competition, indicating that VDR binding sequence (−260/−253 bp of slc20a1a promoter) was bound by VDR  from grass carp. Thus, grass carp show more slc20a1 isoforms than mammals, which is attributable to the TSGD events [In mammals, the SLC20 family includes two members (SLC20A1 and SLC20A2), which are encode by a2 genes . In contD events . MoreoveD events ,24. ManyD events ,26,27. AD events  suggesteslc20a1a, slc20a1b and slc20a2 mRNA amounts were ubiquitously existent in tissues but varied  and TATA-box (−12 bp), the core promoters of slc20a1b only had TATA-box (−14 bp), but the core promoters of slc20a2 had CCAAT-box (−93 bp) and Sp1 sites , indicating that three slc20s had different regulatory mechanism for their transcriptional initiation  and transported to our wet laboratory in Huazhong Agricultural University (HZAU) for the culture. CIK and HEK293T cell lines came from the Cell Resource Center in Fishery College of HZAU. All these media and reagents for cell cultures were purchased from Gibco company . All these animal experiments followed the guideline of the Animal Experimentation Ethics Committee of HZAU  and were approved by Ethics Committee of HZAU .slc20a1a, slc20a1b and slc20a2 were obtained by RT-PCR, with the reference of the genome of grass carp [Total RNA and genomic DNA were extracted from nine tissues of grass carp  by using TRIzol RNAiso reagent  and commercial DNA extracted kit . Total RNA was then reverse-transcribed to cDNA by using the oligo-dT primers and commercial cDNA Synthesis Kits . Core cDNA and promoter sequences of ass carp . The 3′-slc20a1a, pGl3-1804/+143 of slc20a1b and pGl3-1775/+221 of slc20a2, respectively. Then, we used the templates of pGl3-1813/+226 of slc20a1a vector to produce the plasmids pGl3-1355/+226, pGl3-795/+226, and pGl3-263/+226 of slc20a1a vectors. Similarly, the plasmids pGl3-1374/+143, pGl3-899/+143, and pGl3-383/+143 of slc20a1b vector, pGl3-1296/+211, pGl3-835/+211 and pGl3-191/+211 of slc20a2 vector were produced by using the pGl3-1804/+143 of slc20a1b vector and pGl3-1775/+221 of slc20a2 vector as the templates, respectively. We performed the PCR via the TaKaRa PrimeSTAR® HS DNA Polymerase kit . Finally, we sequenced all these plasmids in the Tsingke company . The primers for the plasmid construction were presented in In order to generate the luciferase reporter constructs, we used Sac I and Hind III sites as the restriction sites of PCR products and pGl3-Basic vectors. They were purified and digested using corresponding endonucleases. Then, we ligated the products via the One-Step Cloning Kit . According to the distance from their transcription start sites (TSS), we named these plasmids as the vectors of pGl3-1813/+226 of http://blast.ncbi.nlm.nih.gov/) to compare the nucleotide sequences with DNA sequences from the GenBank database. TMpred (http://www.ch.embnet.org/software/TMPRED_form.html) was utilized to predict the putative transmembrane regions, and the Clustal-W multiple alignment algorithm for analyzing the sequence alignments and percentage of amino acid conservation. Several online software programmes, such as MatInspector (http://www.genomatix.de/), the JASPAR database (http://jaspar.genereg.net/) and the TFSEARCH database (http://www.cbrc.jp/research/db/TFSEARCH.html), were utilized to analyze the potential transcription factor binding sites (TFBS).We used BLAST network service at the NCBI (2 and 37 °C). Prior to the transfection, HEK293T cells were seeded at a density of 1.2 × 105 in 24-well plate. They were cultured for 24 h to reach 70–80% confluence. Lipofectamine™2000  was utilized to transfect all these plasmids into HEK293T cells, based on the manufacture’s protocol. The 400-ng reporter plasmids were co-transfected with 20 ng pRL-TK, the internal control with a Renilla luciferase reporter vector. HEK293T cells were incubated for 24 h and collected to analyze the luciferase activity . For an internal control, we added 20 ng of Renilla luciferase vector (pRL-TK) per well to normalize the efficiency of the transfection. After 4h, we replaced the transfection medium by 10% FBS-DMEM or 10% FBS-DMEM + 3 mM Pi . The form and the concentration of Pi was selected according to our pilot trial and to previous in vitro studies [Firefly luciferase activity to Renilla luciferase activity. We conducted all these experiments in triplicates.HEK293T cells were cultured in DMEM medium with 10% fetal bovine serum (FBS)  in an incubator  to undertake the site-directed mutagenesis, and pGl3-1813/+226 of the slc20a1a vector, pGl3-1804/+143 of the slc20a1b vector and pGl3-1775/+221 of the slc20a2 vector as the templates, respectively. Several mutations were performed, as shown below: (1) at the sites of −783/−771 bp, −1688/−1679 bp, −1083/−1076 bp and −260/−253 bp sites for the slc20a1a promoter; (2) at the sites of −1354/−1345 bp, −1187/−1178 bp, −212/−203 bp, −572/−561 bp, −391/380 bp and −615/−609 bp sites for the slc20a1b promoter; (3) at the sites of −1772/−1763 bp, −987/−977 bp, −1469/−1459 bp, −1172/−1165 bp and −1124/−1117 bp sites for the slc20a2 promoter. The primers used for mutagenesis were shown in 2PO4 and Na2HPO4, pH7.4). After 24-h incubation, we harvested the cells to determine the luciferase activities, based on the procedures mentioned above.In order to characterize the SREBP1, NRF2 and VDR binding sites on the 2, 0.5 mM DTT, 4% Ficoll, 110 mM KCl, 0.2 μg Poly(dI-dC)). Then, the biotin-labeled double-stranded oligo nucleotides . The CIK cells were seeded at a density of 1.2 × 105 in the 6-well plate. They were cultured until the 70–80% confluence was reached. Then we replaced the medium by 10% FBS-DMEM or 10% FBS-DMEM + 3 mM Pi . After 24 h, we collected the CIK cells. The total RNAs were isolated from these CIK cells and reverse-transcribed to cDNA as the templates.We cultured grass carp CIK cells in DMEM medium plus 10% FBS  in a SANYO incubator  were selected to analyze their transcription stability. Two most stable control genes (gapdh and 18s-rrna) were selected by geNorm software analysis [The rotocols ,61. All analysis . The mRNanalysis .slc20 tissue mRNA expression patterns analysis, the data were subjected to one-way ANOVA and Tukey’s multiple range test. For assay of promoters’ activities, Student’s t-test was undertaken to detect the differences between two treatments. The significant level was set at p < 0.05. All of these data were analyzed by using the SPSS 19.0 software .We showed the results with the means ± standard error of mean (SEM) at three independent experiments. Prior to statistical analysis, all data were tested for normality of distribution using the Kolmogornov–Smirnov test. The homogeneity of variances was tested using Barlett’s test. For the slc20a1a, slc20a1b and slc20a2) were isolated and characterized from fish. They shared similar TMDs to mammals and their mRNAs were existent among various tissues. Three transcription factors  regulated the transcription activities of three slc20s promoters and the transcriptional inactivations of slc20s were mediated by VDR under Pi treatment. Our study partially elucidated the mechanism of Pi homeostatic regulation of vertebrates and provided a good basis for dietary Pi addition and for the reduction of Pi discharge into the waters.For the first time, the full cDNA lengths and the promoters of three slc20 members (


Il33), which controls anti-tumor immunity. Analysis of the accessibility peaks on the Il33 gene promoter/enhancer regions revealed sequences for several transcription factors, including ETS and AP-1 motifs, and we show that c-Jun, a component of AP-1, regulates Il33 gene expression by binding to its enhancer in a FAK kinase-dependent manner. This work provides the first demonstration that FAK controls transcription via chromatin accessibility, identifying a novel mechanism by which nuclear FAK regulates biologically important gene expression.Focal adhesion kinase (FAK) localizes to focal adhesions and is overexpressed in many cancers. FAK can also translocate to the nucleus, where it binds to, and regulates, several transcription factors, including MBD2, p53 and IL-33, to control gene expression by unknown mechanisms. We have used ATAC-seq to reveal that FAK controls chromatin accessibility at a subset of regulated genes. Integration of ATAC-seq and RNA-seq data showed that FAK-dependent chromatin accessibility is linked to differential gene expression, including of the FAK-regulated cytokine and transcriptional regulator interleukin-33 ( In addition to well-known localization at integrin-mediated cell–matrix adhesion sites , FAK can localize to the nucleus, where it binds a number of transcription factors, including p534, Gata45 and Runx16, to regulate the expression of Cdkn1a (which encodes p21), Vcam1 and Igfbp3, respectively. By binding to these transcription factors, FAK has been linked to cancer-associated processes such as inflammation5, proliferation6 and survival4.Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is overexpressed in many cancers, including squamous cell carcinoma (SCC)Ccl5, to drive recruitment of regulatory T cells into the tumor microenvironment, resulting in suppression of the antitumor CD8+ T cell response and escape from anti-tumor immunity7. FAK regulates biologically important chemokines via its kinase activity and adaptor functions in the nucleus; briefly, nuclear FAK interacts with many transcription factors and accessory proteins in a gene expression-regulatory network7. Crucially, this includes the pro-inflammatory cytokine IL-33 that can either be found in the nucleus or be released from cells that are damaged or dying 8. In SCC cells however, IL-33 is not secreted, but instead is translocated to the nucleus and functions there; in turn, nuclear IL-33 drives expression of immunosuppressive chemokines, such as Ccl5 and Cxcl10, and we showed that IL-33 functions exclusively downstream of FAK in promoting pro-inflammatory gene expression and tumor growth9. However, the mechanisms by which FAK activity controls Il33 gene expression are not understood, and this important extension of previous work is investigated here.Our previous work demonstrated that, in mutant H-Ras-driven murine SCC cells, nuclear FAK controls expression of cytokines and chemokines, for example 7. Therefore, we wanted to investigate whether FAK controls genome-wide chromatin accessibility changes and if these chromatin changes contribute to FAK-dependent gene expression, which to our knowledge has never been explored. To understand potential molecular mechanisms by which FAK regulates expression of genes like Il33, we examined FAK-dependent chromatin accessibility changes and transcription factor motif enrichment across the genome using ATAC-seq and integrated those data with RNA-seq. This revealed that FAK regulates chromatin accessibility of a subset of genes, and a number of these were differentially expressed in a FAK- and FAK kinase-dependent manner, including the previously identified FAK downstream effector Il33. Motif-enrichment analysis indicated that there was enrichment of sequence motifs known to bind ETS and c-Jun/AP-1 transcription factor family members on the Il33 promoter and enhancer regions which are affected by FAK. Validation experiments confirmed that c-Jun is a key regulator of IL-33 expression in SCC cells by binding to the Il33 enhancer, and that FAK’s kinase activity is important for regulating chromatin accessibility at this site. Analysis of genome-wide motif enrichment indicated that FAK likely regulates many more transcription factors beyond those already reported. Taken together, our data suggest that FAK is a common regulator of gene expression via modulating transcription factor binding to biologically relevant target gene promoters/enhancers by controlling chromatin accessibility, such as we demonstrate here using Il33 as an exemplar. In turn, FAK-dependent gene expression changes, including Il33, are critically associated with cancer-associated phenotypes9. This is the first demonstration of how nuclear FAK can contribute to gene expression, and we report a new activity in the nucleus for a classical adhesion protein.Our previous findings have suggested that FAK is present in the chromatin fraction and interacts with chromatin modifiers in the nucleusCre-lox-mediated Ptk2 (which encodes FAK) gene deletion (FAK−/− cells) with the same cells re-expressing wild-type FAK (FAK-WT-expressing cells) or FAK mutants that were impaired in nuclear localization (FAK-nls-expressing cells) or deficient in kinase activity (FAK-kd-expressing cells) (cell lines generated previously10). These permitted the identification of FAK-dependent, FAK nuclear localization-dependent and FAK kinase-dependent alterations in chromatin accessibility and transcription factor motif enrichment in accessible regions of chromatin across the genome.FAK does not contain a DNA binding sequence and therefore likely regulates transcription indirectly, potentially through interactions with transcription factors and co-factors. To identify which transcription factors may be candidate mediators of FAK-dependent gene expression, we used ATAC-seq to analyze active (accessible) chromatin in FAK-deficient SCC cells or cells expressing different FAK mutant proteins. Specifically, we compared SCC cells in which FAK had been depleted by 11).Chromatin accessibility analysis of ATAC-seq data detected 20,000–60,000 peaks (accessible regions) per sample   and FAK mutant (FAK-nls- and FAK-kd-expressing) cell lines. From this analysis, it was apparent that a subset of genes are regulated by FAK-dependent changes in chromatin accessibility (discussed further below).We identified differentially accessible gene regions using the DiffBind package15 to identify motif binding sites (i.e. genomic regions that match known transcription factor motifs) in the differentially accessible peaks identified by DiffBind analysis. Motifs in FAK-WT-enriched peaks were statistically compared to all the motifs identified in peaks called in the FAK-nls-, FAK-kd-expressing or FAK−/− cells. This was expressed as a proportion of target sequences containing that motif (motifs in peaks enriched in FAK-WT-expressing cells) compared to the proportion of background sequences containing that motif . This analysis detected multiple statistically significant changes in motif enrichment in differentially accessible peaks from FAK-WT vs FAK-deficient (FAK−/−) SCC cells (196 transcription factor motifs), FAK-WT- vs FAK-kd-expressing SCC cells (118 transcription factor motifs) and FAK-WT vs FAK-nls-expressing SCC cells (205 transcription factor motifs) (Benjamini–Hochberg-corrected P ≤ 0.05) (Supplementary Data 16). These findings suggest that FAK regulates transcription factor motif enrichment in accessible regions of chromatin across the SCC genome.We next analyzed the transcription factor motif sequences present in FAK-dependent differentially accessible peaks . Motif-enrichment analysis allowed us to predict which transcription factors were regulating genes across the genome by analyzing the motif sequences in ATAC-seq peaks. We used the HOMER toolFAK−/− cells and FAK-WT- vs FAK-nls-expressing cells, the two most highly enriched transcription factor motifs were for Jun-AP-1 and Fosl2 , which exhibited an almost two-fold enrichment in motifs in the target  compared to the motifs identified in the background  , which likewise revealed a two-fold enrichment in these motifs in the target  compared to the background motifs   and PNT domains . We used FAK RNA-seq data to confirm whether the genes that were regulated by FAK-dependent changes in chromatin accessibility were also differentially expressed in a FAK- and FAK kinase-dependent manner  to understand which genes are regulated by FAK-dependent accessibility changes. To assign each ATAC-seq peak to genes, we used ChIPseekerner Fig. . Set ananer Fig. B. We founer Fig. B. Comparner Fig. B.Il33, because we had previously reported it as a FAK-regulated cytokine of biological significance in mediating FAK-dependent anti-tumor immunity9. Using ATAC-seq data to investigate whether chromatin accessibility was one mechanism by which FAK regulates Il33, we found that there were ATAC-seq peaks in FAK-WT-expressing SCC cells on Il33 enhancer (− 3199 from TSS) and promoter (+ 821 from TSS) regions  did not have sufficient read count to be identified as an ATAC-seq peak, and therefore the peak was not called. We conclude that FAK regulates chromatin accessibility at a subset of gene promoters, and some of these are differentially expressed in a FAK-dependent manner, as exemplified by Il33. This suggests that FAK-regulated, biologically important gene expression alterations may be controlled by FAK-dependent chromatin accessibility changes.As an exemplar, we next focused on one of these genes, Il33 expression in mouse SCC cells, we performed motif-enrichment analysis on the ATAC-seq peaks proximal to the Il33 promoter and enhancer regions in FAK-WT-expressing cells using HOMER  and used interactome analysis to contextualize these with regard to transcription factors that may bind to the identified sequence motifs in the Il33 gene where accessibility is FAK-regulated. This enabled the prediction of putative Il33 transcription factors that have functional connections with nuclear FAK binders and thereby may be regulated by FAK in a direct manner  used in our studies. We performed siRNA-mediated depletion of Jun mRNA (which encodes c-Jun)  in the Il33 enhancer, and whether or not FAK-dependent chromatin accessibility changes on the Il33 enhancer are linked to perturbed c-Jun binding. Our HOMER15 analysis identified a cAMP response element (CRE) (5′-TGACGTCA-3′) within the Il33 enhancer peak, which are known to bind c-Jun–Atf dimeric complexes18. We therefore used ChIP to show that c-Jun binds to the CRE motifs at the Il33 enhancer in a FAK-dependent manner via accessibility. Primers were designed around the region containing the CRE sequence motif in the Il33 enhancer and an unrelated region upstream of this site to control for background binding -qPCR analysis to confirm that c-Jun binds to the predicted c-Jun sequence-binding motif at the cer Fig. C. Followred see “”. Using lls Fig. F. Furthells Fig. F. These 27. Treatment of FAK-WT SCC cells with the FAK kinase inhibitor VS4718 resulted in a significant loss S73-c-Jun phosphorylation Il33. FAK/IL-33 downstream effectors significantly influence tumor biology9.Thus, we conclude that FAK, which is classically thought to be primarily an integrin adhesion protein, can function in the nucleus to control chromatin accessibility at specific gene promoters/enhancers. In turn, this leads to FAK-dependent transcription of specific genes, an example of which is the cytokine Il33 enhancer region via chromatin accessibility changes to control Il33 expression. As IL-33 is an important regulator of cytokine expression and tumor growth8, FAK-dependent c-Jun regulation of IL-33 expression would be predicted to influence cancer cell biology, such as that we described previously9. It is perhaps not surprising that FAK can regulate c-Jun, since cytoplasmic-localized FAK is known to transduce signals through pathways such as MAPK28 and Wnt30, which are known to control c-Jun expression and its transcriptional activity31; however, what is surprising is the more direct link we have uncovered here between nuclear FAK function and its regulation of c-Jun transcriptional activity at the Il33 enhancer via chromatin accessibility. Consistent with the links between nuclear FAK and c-Jun activity being more common, nuclear FAK is reported to regulate the expression of Jun (which encodes c-Jun) in response to ‘stretch’ in cardiac myocytes by binding to, and enhancing, the transcriptional activity of MEF232.In this study, we have discovered an undescribed function of nuclear FAK as a key regulator of chromatin accessibility changes and transcription factor binding. Furthermore, we have confirmed that nuclear FAK regulates c-Jun binding at the 33 and Hic-534, which are believed to function as transcription factor co-regulators35. Furthermore, the focal adhesion protein paxillin can also translocate to the nucleus36, where it contributes to proliferation37, and we believe that there are other integrin-linked adhesion proteins capable of translocating to the nucleus and functioning at the nuclear membrane or inside the nucleus38. Relevant to the work we present here, a number of consensus adhesome components containing LIM (Lin11–Isl1–Mec3) domains have been directly linked to the regulation of chromatin accessibility and dynamics. For example, Hic-5 can inhibit the binding of the glucocorticoid receptor to the chromatin remodelers chromodomain-helicase DNA binding protein 9  and brahma , resulting in a closed chromatin conformation and transcriptional repression of a subset of glucocorticoid receptor target genes39. Also, paxillin can regulate proliferation-associated gene expression by controlling promoter–enhancer looping via nuclear interactions with the cohesin and mediator complex37. Taken together, these reports suggest that focal adhesion proteins in the nucleus are capable of scaffolding chromatin remodeling complexes to regulate chromatin structure and gene expression changes.Focal adhesion proteins other than FAK have been detected in the nucleus, such as Lpp7. These include the Smarcc2 and Actl6a components of the BRG1/BRM-associated factor (BAF) complex7, which have been shown to be recruited to target gene enhancers by AP-1 to regulate chromatin accessibility40. IL-33 is required for the chromatin recruitment of the Wdr82 component of the chromatin-modifying protein serine/threonine phosphatase (PTW/PP1 phosphatase) complex41. IL-33 binds to the Brd4 (bromodomain-containing protein 4) transcriptional coactivator9, which is known to recruit the BAF complex to target genes42. The nuclear FAK binding protein Sp-1 also interacts with the BAF complex to facilitate its recruitment to specific promoters43. Therefore, there is abundant evidence of connections between FAK or FAK binding proteins  and FAK-regulated transcription factors (e.g. AP-1) to chromatin accessibility factors, such as the BAF complex and PTW/PP1 phosphatase complex. It is likely that FAK, and proteins to which it binds, scaffold chromatin remodeling proteins at target genes, such as Il33 as we describe here, in order to determine the state of chromatin accessibility, the binding of transcription factors like AP-1 and transcription  in the nucleus by interacting with a network of chromatin modifiers and transcriptional regulators9.An unanswered question is the mechanism by which FAK controls chromatin accessibility at regulated genes. In this regard, our previous nuclear interactome proteomics revealed that FAK can interact with proteins known to regulate chromatin accessibility15 uses motifs from a number of sources, including human and mouse. Indeed, many of the transcription factor motifs are highly conserved between related organisms, with DNA binding profiles for human and mouse transcription factors being almost identical. This high homology makes the information about transcription factors specifically interchangeable between organisms15. Furthermore, many transcription factors have high protein sequence homology between species. For example, mouse c-Jun is 96% homologous to its human counterpart44. In addition, the information used to predicted the c-Jun/CRE motif upstream of the Il33 enhancer originated from a human ChIP-seq dataset [K562-cJun-ChIP-Seq(GSE31477)]. Therefore, our mouse c-Jun ChIP analysis at this CRE motif has experimentally confirmed that there is homology in AP-1 binding motifs between mouse and human. As such, we believe that the data presented in this manuscript are also applicable to human cell lines.One limitation to this study is that our ATAC-seq analysis was only performed in one species. However, we note that the HOMER toolIl33 we show here. Translocation of FAK to the nucleus, where it can bind to factors that control chromatin accessibility, can therefore communicate extracellular cues to the gene transcription machinery in the nucleus by this route.In summary, we have discovered a completely new paradigm for how FAK may regulate transcription in the nucleus, i.e. as a critical regulator of chromatin accessibility changes at biologically important target genes, such as 10. Briefly, K14CreER FAKflox/flox in FVB mice were subjected to the dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol 13-acetate two-stage cutaneous chemical carcinogenesis protocol. FAK deletion was induced by culturing excised SCC cells in 4-hydroxytamoxifen. FAK-WT, FAK kinase-deficient  or FAK nuclear localization sequence-mutated  constructs were stably expressed in a FAK−/− clone using standard retroviral induction protocols . Cells expressing FAK-WT and FAK mutant constructs were selected using 0.25 mg/ml hygromycin.Generation of the FAK SCC cell model has been described previouslyFAK−/− and FAK-WT-, FAK-nls- and FAK-kd-expressing SCC cell lines were cultured at 37 °C, 5% CO2, in 1 × Glasgow minimum essential medium (GMEM) supplemented with 10% fetal bovine serum, 1% MEM non-essential amino acids, 1% sodium pyruvate, 1% l-glutamine and 1% MEM vitamins .45. The specific ATAC-seq protocol used in this study has been previously reported46. ATAC-seq data were aligned to the Mus musculus reference genome mm10 using the bcbio ATAC-seq pipeline47. Accessible regions (i.e. ATAC-seq peaks) were called from the BAM files using the MACS2 algorithm48 with the following parameters: -B -broad -q 0.05 -nomodel -shift -100 -extsize 200 -g 1.87e9. Differentially accessible regions between the FAK-WT-expressing cells and the FAK−/−, FAK-nls-expressing and FAK-kd-expressing cells were identified by differential peak calling using the R/Bioconductor package DiffBind14, where significantly different peaks were defined as those with a false discovery rate (FDR) of below or equal to 0.05. Motif-enrichment analysis was performed using HOMER15 following default parameters. ATAC-seq peaks were assigned to genes using ChIPseeker17 with the following parameters: tssRegion = c, annoDb = "org.Mm.eg.db", TxDb = TxDb.Mmusculus.UCSC.mm10.knownGene.ATAC-seq samples were prepared similarly to described previously49.Set analysis of ATAC-seq data in Figs. Jun SMARTpool , siGenome Jun siRNA #1, siGenome Jun siRNA #2  or non-targeting control siRNA #2  diluted in 500 μl Opti-MEM Reduced Serum Medium with GlutaMAX (Gibco) to a final concentration of 33 μM. Transfections were carried out using Lipofectamine RNAiMAX transfection reagent (Invitrogen) following manufacturer’s instructions. Cells were incubated in transfection mixes for 48 h before harvesting for RNA extraction or whole cell lysate preparation.FAK-WT SCC cells were transfected with either siGenome 51. FAK-WT- and FAK-kd-expressing cells (4 × 106) were plated on 10-cm dishes (Corning) and then, the following day, were formaldehyde crosslinked and fractionated as described in50. Sonication was carried out using a BioRupter (Diagenode) at high power for 10 min (30-s on/off cycles) to yield DNA fragments of 1000–200 bp in size. The samples were then centrifuged at 13,000 rpm for 15 min at 4 °C. The supernatant was collected and 50 µl reserved for an input sample. For immunoprecipitations, a protein A and protein G Dynabead mixture (1:1) (both from Invitrogen) was added to sonicated lysate along with 0.48 µg of c-Jun antibody  or equal amount of rabbit IgG . Immunoprecipitations were incubated at 4 °C overnight with rotation, prior to washing with 1 × RIPA 150 mM NaCl , 1 × RIPA 500 mM NaCl  and twice with cold Tris–EDTA (TE) buffer , before resuspension in 200 µl of ChIP direct elution buffer . Input samples were made up to 200 μl with 1 × RIPA 150 mM NaCl and 0.4% SDS. Both the input and immunoprecipitation samples were incubated for 5 h at 65 °C. The following day, the input and immunoprecipitation eluates samples were treated with 2 µl of RNaseA/T1 (Ambion) for 30 min at 37 °C and 5 µl of 10 mg/ml proteinase K (Ambion) for 1 h at 55 °C. The DNA was then purified using the QIAquick PCR purification kit (Qiagen) following manufacturer’s instructions.The ChIP-qPCR experiments were performed as described previouslyIl33 enhancer, F: ACCCTGGAGTGTTCTTTGCA and R: TGCCTTCTGAAGCTTACTCGA; negative control region, F: ATGTGTGCTGTGTGTATGCC and R: ACATTAAGGGCAGGAGACGT. ChIP-qPCR analysis was performed using SYBR Green master mix (Thermo Scientific) following manufacturer’s instructions. The following cycling conditions were used: 98 °C for 10 s, 30 ×  and 72 °C for 5 min. The % input method was used for c-Jun ChIP data normalization, whereby the cycle threshold (Ct) values of the ChIP samples are divided by the Ct values of the input sample (starting amount of chromatin used for the ChIP). The input sample Ct value was first adjusted using the following equation: adjusted input = Ct of input sample − log2(dilution factor). Then the % input was calculated for the CRE c-Jun ChIP and the background control region upstream of the Il33 gene using the following calculation: 100 × (PCR amplification factor)). Then the % input of the CRE motif was subtracted from the % input of the background control region to determine the amount of enrichment of c-Jun binding to the CRE motif over the background control region.ChIP and input DNA were amplified using the following primers: c-Jun motif/Il33, F: GGATCCGATTTTCGAGACTTAAACAT and R: GCGGCCGCATGAGACCTAGAATGAAGT; Cxcl10, F: CCCACGTGTTGAGATCATTG and R: CACTGGGTAAAGGGGAGTGA; GAPDH, F: CTGCAGTACTGTGGGGAGGT and R: CAAAGGCGGAGTTACCAGAG. Jun was amplified using pre-designed primers from Qiagen (cat no. QT00296541).RNA extraction was performed using an RNeasy Mini kit (Qiagen) following manufacturer’s instructions. cDNA synthesis was performed using the SuperScript First-strand Synthesis System (Invitrogen) following the manufacturer’s random hexamers protocol. qRT-PCR analysis was performed using SYBR Green master mix (Thermo Scientific) following manufacturer’s instructions. The following cycling conditions were used: 98 °C for 10 s, 30 ×  and 72 °C for 5 min. Primers used were as follows: 9. Antibodies used in this study were as follows: IL-33 , c-Jun , Phospho-FAK (Tyr397) , Phospho-c-Jun (Ser73) , GAPDH .Cell lysis and immunoblotting was performed exactly as described previouslyFAK-WT SCC cells were treated with 250 nM VS4718 (Selleckchem) for 24 h, prior to cell lysis and immunoblotting.FAK−/− and FAK-kd SCC cells using an RNeasy kit (Qiagen) following manufacturer’s instructions. To verify sample purity, the samples were run on a 2100 Bioanalyzer using the Bioanalyzer RNA 6000 pico assay (both Agilent). Samples that achieved an RNA integrity number (RIN) of 8 or above were considered suitable purity for sequencing. Samples were prepared for sequencing using the TruSeq RNA Library Prep Kit v2 (low-sample protocol) (Illumina) and paired-end sequenced using a HiSeq 4000 platform (Illumina) at BGI.RNA was extracted from FAK-WT, 52 on the mouse transcriptome database acquired using the kallisto index, implementing default parameters. Quality control was performed on the kallisto output using MultiQC software (https://github.com/ewels/MultiQC). Transcript abundance was summarized to gene level and imported into the differential expression analysis R package DESeq253 using the R package tximport54. Genes which had zero read counts were removed prior to differential expression analysis.Transcript abundance was determined using the pseudoalignment software kallistoFAK−/− and FAK-WT vs FAK-kd SCC cell line gene read counts were compared. The Wald test was used for hypothesis testing in DESeq2, and all P-values were corrected for multiple testing using the Benjamini–Hochberg method. Transcripts that acquired a Benjamini–Hochberg corrected P-value of 0.05 or below and a log2-transformed fold change in expression of ≥ 1 or ≤ – 1 were considered significantly different between the cell lines. RNA-seq results from FAK-WT and FAK−/− cells were previously analyzed38; RNA-seq data from all cell lines (Supplementary Data 55 (GEO series accession number: GSE147670).Differential expression analysis was performed using DESeq2 using default parameters, where FAK-WT vs 57. All terms that acquired a Benjamini–Hochberg corrected P-value of below 0.05 were considered statistically significant.Protein domain-enrichment analysis was performed for SMART domains using DAVIDhttps://www.qiagenbioinformatics.com/products/ingenuitypathway-analysis). The following parameters were used for network construction: database sources , direct interaction, experimentally observed, protein–protein and functional interactions, mammalian interactions only. All networks were exported into Cytoscape58, and the NetworkAnalyzer plugin59 was used to visualize the most connected nodes in the networks before applying yFiles layout algorithms (yWorks).Network analysis was performed using Ingenuity Pathway Analysis . All Supplementary Data S1.Supplementary Data S2.Supplementary Data S3.Supplementary Information.


Oligodendrocytes generate myelin in the vertebrate central nervous system and thus ensure rapid propagation of neuronal activity. Their development is controlled by a network of transcription factors that function as determinants of cell identity or as temporally restricted stage-specific regulators. The continuously expressed Sox10 and Myrf, a factor induced during late development, are particularly important for terminal differentiation. How these factors function together mechanistically and influence each other, is not well understood. Here we show that Myrf not only cooperates with Sox10 during the induction of genes required for differentiation and myelin formation. Myrf also inhibits the activity of Sox10 on genes that are essential during earlier phases of oligodendroglial development. By characterization of the exact DNA-binding requirements of Myrf, we furthermore show that cooperative activation is a consequence of joint binding of Sox10 and Myrf to the same regulatory regions. In contrast, inhibition of Sox10-dependent gene activation occurs on genes that lack Myrf binding sites and likely involves physical interaction between Myrf and Sox10 followed by sequestration. These two opposite activities allow Myrf to redirect Sox10 from genes that it activates in oligodendrocyte precursor cells to genes that need to be induced during terminal differentiation. In the vertebrate central nervous system (CNS), oligodendrocyte-dependent myelination ensures rapid saltatory conduction along axons. If myelin is defective or damaged, severe cognitive and motor disabilities result. Oligodendrocytes acquire their ability to form myelin sheaths around axonal segments during terminal differentiation from committed oligodendrocyte precursor cells (OPCs).This process is regulated by a complex regulatory network that is constructed around central transcriptional regulators and additionally includes chromatin modifying proteins and regulatory RNAs . Among tMany genes that are activated during terminal differentiation in oligodendrocytes and required for myelination have been identified as direct target genes of Sox10 . It has Pdgfra and NG2/Cspg4 as potential targets  , mouse NTotal RNA was prepared from independent OLN93 cell clones A and B that had undergone CRISPR/Cas9-dependent genome editing to inactivate Sox10 with different guide RNAs and two control clones using the RNeasy Micro Kit (Qiagen). RNA samples were treated with DNase I to remove contaminating DNA. Quality and purity of samples were evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies Germany). 100 ng total RNA were used for library preparation (Ilumina Stranded mRNA Kit). Approximately 52 million reads were generated per library using an Illumina Hiseq 2500 platform sequencer  and mapped onto rat genome rn5 using STAR (version 2.5.1.b). Unique mappings were detected using HTSeq count based on ENSEMBL Gene identifier Version 75. Statistical analysis was carried out using DESeq2 R Version 1.8.1. Gene expression values are deposited in GEO under accession number GSE136659.P ≤ 0.05). A second list was generated that contained all genes associated with CNS-specific Sox10 ChIP-Seq peaks (GSE64703) (http://bioinformatics.psb.ugent.be/webtools/Venn/). The resulting 203 genes with Sox10-dependent expression and nearby genomic Sox10 binding site were defined as potential Sox10 target genes. Using lists of genes with preferential expression in OPCs or differentiating oligodendrocytes (http://cbl-gorilla.cs.technion.ac.il/) in combination with semantic clustering by REViGO (http://revigo.irb.hr/)   as deterdrocytes  and compirb.hr/) . The IntId4, Tgfb2, Wnt7a genes 3(c) .Regulatory regions from the ) Figure . The pTA) Figure  in front) Figure  and 6A. d before ,12,19,24d before  was perfMyrf variants . Overall amounts of plasmid in a particular experiment were kept constant by adding empty pCMV5 where necessary. Whole cell extracts were prepared 48 h after transfection by lysing cells in buffer containing 0.1% Triton X-100 and 2.5 mM ATP. Luciferase activities were determined after addition of luciferin substrate by chemiluminescence.For preparation of protein extracts, HEK293 cells were transfected on 10 cm plates with 10 μg of pCMV5-based expression plasmids using polyethylenimine. 48 h after transfection, whole cell extracts were prepared by using 1% NP-40 to initiate cell lysis and 400 mM NaCl to extract nuclear proteins .32P-labeled double-stranded oligonucleotides containing putative Myrf binding sites  or protein interaction studies. EMSA were performed with extracts and For co-immunoprecipitation experiments, HEK293 cell extracts were incubated with rabbit antibodies against Sox10 (home made)  or mouseTgfb2, Id4, Wnt7a, Mag, Aatk and Mbp genes in precipitated DNA was by qPCR on a Bio-Rad CFX96 thermocycler with each reaction performed in triplicates. The ΔΔCt method was used to calculate the percent recovery of a given DNA segment relative to the total input. After normalization, the enrichment of chromatin in the various precipitates over the input was determined. The sample with highest enrichment in a specific experiment was set to 1 and all other values were expressed relative to it.Chromatin was prepared from rat oligodendroglial cells cultured three days under differentiating conditions. Chromatin was cross-linked with 1% paraformaldehyde and sheared to fragments of ∼200–400 bp in a Bioruptor (Diagenode) . After pTgfb2 regulatory region, 5′-GATAAAGGAAGGCTTTCAGCAA-3′ and 5′-TTGGATGGTGTGGTGCAG -3′ to amplify the Id4 regulatory region, 5′-GAGCGAAAATCCAGGATGAA-3′ and 5′-GGATGGATGGGAAGTCCTTT-3′ to amplify the Wnt7a regulatory region, 5′- TTGGATGGTCTGGCTTCTG-3′and 5′-CCCATCTTCTCCAGGAAGG-3′ to amplify the Mag promoter region, 5′-AGCAAGAAGCTGGTGCTGAG-3′and 5′- GAGTGGGTGCAGTGAGTCCT-3′ to amplify the Aatk regulatory region, 5′-TTTGCTCACTCGAAGGGACT-3′and 5′-TTAGGTCCTTCTGGGGACAGT-3′ to amplify the Mbp regulatory region, and 5′-AGAGACTGGTTGCCAGGAAG-3′ and 5′-GGTGGAGACAGACTCGGAAC-3′ to amplify a negative control region (NCtrl).The following primers were used for the detection of specific DNA regions: 5′-AAGTTCACATTCAGCGAAACG-3′ and 5′-GAGGACCCCTGAGTGAACAA- 3′ were used to amplify the Primary oligodendroglial cells were cultivated for two days under proliferative conditions or for 6 days in differentiation medium on cover slips before fixation in 4% paraformaldehyde for 10 min. In some experiments, BrdU was added at a final concentration of 4 mM to proliferation medium 2 h before fixation. Cells on cover slips underwent immunocytochemistry using the following primary antibodies: rat anti-Mbp monoclonal , guinea pig anti-Sox10 antiserum  , rabbit n ≥ 3 for all molecular biology experiments and experiments using cell cultures as common for this kind of study. No data were excluded from the analysis. GraphPad Prism6  was used to determine whether differences in cell numbers, luciferase activities or immunoprecipitated DNA were statistically significant by one way Anova with Bonferroni correction or two-tailed Student's t tests . Variance between statistically compared groups was similar.Results from independent experiments were treated as biological replicates. Sample size was We have previously used CRISPR/Cas9-dependent genome editing to inactivate Sox10 in the immortalized rat oligodendroglial cell line OLN93 . Here weTgfb2, Id4 and Wnt7a caught our attention  w w20) were Figure .Pdgfra promoter or an intronic enhancer of the Cspg4 gene were also tested as these two regions represent the only other regulatory regions known to be under control of Sox10 in OPCs . ,19. Pdgfdrocytes .Mag, Aatk and Mbp genes furthermore responded to the joint presence of Myrf and Sox10 in transfected cells with a substantially higher activity. For the Mag promoter, induction rates increased from 13 ± 3-fold in the presence of Sox10 to 39 ± 8-fold in the presence of Sox10 and Myrf  in Figure To further investigate the exact spacing requirements, we generated a set of oligonucleotides in which we changed the distance of the two consensus motifs by inserting up to five additional basepairs between them Figure . AlreadyTo look into the effects of motif orientation, we also generated oligonucleotides with two consensus motifs spaced one or two helical turns apart and opposite (head-to-head) orientations Figure . In EMSAWith a better knowledge of the effects of motif orientation and spacing, we generated additional oligonucleotides with three consensus motifs in various orientations and spacings compatible with Myrf binding Figure  and compTo be able to predict genomic Myrf binding sites we next analyzed the impact of mismatches in one consensus motif on the ability of Myrf to bind. For that purpose, we used the oligonucleotide as template that contained two motifs in head-to-tail orientation spaced one helical turn apart, and generated a series in which single positions were exchanged in the lateral motif Figure . ChangesWe next asked whether it would even be possible that both binding motifs contain a mismatch. For this set of experiments, we started out with the mutant oligonucleotide from the previous series that contained a mismatch at position 4 of the lateral motif, and now introduced additional mutations at single positions in the central motif employing the same principles as previously used in the study of the lateral motif Figure . As evidTo analyze how the determined DNA-binding characteristics influence the transcriptional activity of Myrf, we generated a series of artificial reporter gene constructs. All of them contained a luciferase gene under control of a minimal promoter and a well-characterized dimeric Sox10 binding site .