Heterocephalus glaber) is one of the mostpromising models used to study genome maintenance systems, including theeffective repair of damage to DNA. The naked mole-rat is the longest livedrodent species, which is extraordinarily resistant to cancer and has a numberof other unique phenotypic traits. For at least 80% of its lifespan, thisanimal shows no signs of aging or any increased likelihood of death and retainsthe ability to reproduce. The naked mole-rat draws the heightened attention ofresearchers who study the molecular basis of lengthy lifespan and cancerresistance. Despite the fact that the naked mole-rat lives under genotoxicstress conditions , the main characteristics of its genome andproteome are a high stability and effective functioning. Replicative senescencein the somatic cells of naked mole-rats is missing, while an additionalp53/pRb-dependent mechanism of early contact inhibition has been revealed inits fibroblasts, which controls cell proliferation and its mechanism ofarf-dependent aging. The unique traits of phenotypic andmolecular adaptations found in the naked mole-rat speak to a high stability andeffective functioning of the molecular machinery that counteract damageaccumulation in its genome. This review analyzes existing results in the studyof the molecular basis of longevity and high cancer resistance in nakedmole-rats.The naked mole-rat ( DNA damage caused by environmental stress and normal metabolic processes occurdaily at a frequency raging from 1,000 to 1·106 per living cell-4.The dHeterocephalus glaber) is one of themost promising models used to study genome maintenance systems, includingeffective repair of DNA damage. The NMR is the longest living of smallburrowing mammals. It’s native to Southeastern Africa  and slightly larger than a mouse. NMR colonies are housed in about 60zoos worldwide and a number of laboratories. It is one of the ~50 knownburrowing herbivorous rodents, a representative of the exceptionally rare trueeusocial mammals [The naked mole-rat  of the genome of a male nakedmole-rat were published in 2011 .Back th−4, which is muchlower than that in mouse and rat populations and is comparable to thenucleotide diversity in humans. The low level of nucleotide diversity mayreflect a low effective size of the NMR population, but it may also be due to ahigh level of inbreeding, a reduced mutation rate, or high efficiency of therepair systems [A total of 1.87 million heterozygous single-nucleotide polymorphisms (SNPs)were also identified using the Genome Analysis Toolkit . The estimated nucleotide diversity (mean pernucleotide heterozygosity) was 7×10 systems . Genome  systems  demonstrTep1 and Terf1 genes involved in telomerelength regulation belong to the set of positively selected genes in NMR, unlikethose in the rat and mouse [Tert gene coding for the telomerase catalytic subunit isstably expressed in the somatic cells of NMR at any age. Meanwhile, thetelomerase activity is low. A comparative study showed that there is a negativecorrelation between the levels of telomerase expression and rodent size, sinceno correlation between telomere length and lifespan has been foundhgTerc) in NMR andother species has revealed two main differences: the A→G replacement inthe first loop of pseudoknot P2b-p3  and the G→A replacement in the CR7-p8b domain . Two transcriptionfactor binding sites were identified in the promoter regions of thehgTerc gene: the ETS family site, which was found to be aconserved element for all the analyzed TER promoters, and the binding site forthe SOX17 transcription factor, which was unique to the NMR gene. The absenceof one Sp1 binding site was an additional specific feature of the Tercgene in NMR [Terc gene has a unique polymorphism and promoterstructure.The nd mouse .Telomere in NMR . Hence, Cyp46a1 andSmad3) [The results of a sequencing of RNA isolated from the brain, liver, and kidneysof a newborn, young (4 year-old), and old (20 year-old) NMRs showed that theexpression level changes with age in a very small number of genes. In the humanbrain, the expression level decreased in 33 genes, while increasing in 21 genes. In NMR,dSmad3) . ThetradSmad3) .Ccne1), uncoupling protein 1(Ucp1), and γ-crystallin, as well as the proteins thatcode for the proteins directly involved in DNA metabolism: multifunctional DNArepair enzyme AP endonuclease (APE1), the large subunit of thereplication/repair factor RFC1, and topoisomerase TOP2A. TOP2A controls thetopologic states of DNA during transcription and, along with TEP1 and TERF1, ispart of a 5-protein complex of alternate lengthening of the telomere pathway. Acomparison to the orthologs present in the genomes of 36 mammals revealed adivergence in the NMR genome, attesting to the existence of 45 unique aminoacid substitutions in the respective proteins[Furthermore, Kim et al.  performeproteins.HR gene but have pelage [HR genes inNMR and mouse/ humans more likely show the phylogenetic divergence from mouse to humans[Spalax galili, but its genomecarries none of the mutations considered to be key ones in NMR[Hence, the first attempted sequencing revealede pelage .The difo humans, 30.Theo humans. Howevero humans. Interess in NMR, 34.Furs in NMRregardins in NMR.Cebpg genecoding for the transcription factor involved in DNA repair regulation is represented bythree copies; and the Tinf2 gene of the shelterin complex component,by two copies. Furthermore, the NMR and human genomes, as opposed to the mousegenome, were found to carry the Rpa4 gene coding for ananalogue of the second subunit of the RPA protein that consists of threesubunits  and is involved in many processes related toDNA conversions. Full-length coding sequences of this gene were previouslyrevealed only in the genomes of apes and horse[in vitro) but exhibits an increased affinityfor damaged DNA and participates in the repair and activation of cell-cyclecontrol (the G2/M stages)[A comparative analysis of a group of the genes involved in genome stabilitymaintenance in humans, mouse, and mole-rat has demonstrated that an elevatedgene copy number is not typical of the NMR genome. Meanwhind horse.The RPA stages)-38.p53 gene that encodes the region involved inapoptosis regulation, as well as in the hyaluronan receptor genes CD44 andHMMR, were revealed. Furthermore, NMR p53 carries the PXXP motifs (P –proline and X – any other amino acid), similar to the PXXP motifs inhuman p53.The higher quality of genome annotation has made it possible to identify ~1,800non-coding and ~42,000 coding DNA regions and approximately the same amount ofproteins using sequencing data. As a result, NMR was found to exhibit a numberof features of the gene sequences associated with cancer resistance and aging. Unique Investigation of the genomes and transcriptomes of nine African naked mole ratspecies has demonstrated that the genes related to tumor suppression, telomereregulation, cell division, RNA repair, and response to stress have been underpositive selection in these species .Modern bioinformatics approaches allow one to perform a full-scale targetedcomparison of the transcriptomes of gene groups in different animal species.The liver is an organ characterized by a high level of oxidative metabolism anda significant number of spontaneous lesions. MacRae et al. performeMost genes encoding DNA repair proteins are constitutively expressed andregulated by post-transcriptional modifications. Nevertheless, transcription ofsome genes in this group is induced upon genotoxic stress, including the genescoding for the key components of the nucleotide excision repair (NER) pathway:DDB1, DDB2, ERCC1, XPC, ERCC4 (XPF), and ERCC5 (XPG).A speciKip1. P27Kip1 binds to cyclin–CDKcomplexes and arrests cell division at the G1 phase of the cell cycle. The keytumor-suppressor pathways, the Rb and p53 pathways, are activated by productsof the Ink4a and Arf genes[NK4a, the Ink4a gene product, binds toand inhibits CDK 4/6, thus activating Rb [Arf gene product activates p53 bybinding to and activating the MDM2 protein. Hence, the Ink4a andArf genes play a crucial role in senescence and resistance to cancer[A vast body of results of studies focused on the biochemical features of NMRand aimed at searching for the mechanisms underlying the unique phenotypictraits of NMR, including its cancer resistance, has been published. The uniquesystem of early contact inhibition of cell growth discovered in 2009 is one ofthese mechanisms . Contactrf genes-46.Protating Rb .The Arfo cancer-48.Kip1 only backs up ECI mediated by thekinase inhibitor p16INK4a [p15a, p15b genes and theInk/Arf locus. The pALTINK4a/b protein was revealed in neithermice nor humans. Expression of pALTINK4a/b is induced upon ECI and understress, such as UV or ionizing radiation, loss of adherence to the substrate,and oncogene expression. Furthermore, pALTINK4a/b is more efficient at inducingcell cycle arrest, thus leaving more time for the cells to overcome theconsequences of genotoxic stress, including DNA-damage repair beforereplication starts. The two-tiered contact inhibition typical of NMR cells (asopposed to mouse and human cells) may contribute to the maintenance of thestability of its genome [Fig. 1).Replicative senescence is not typical of NMR fibroblasts, but in cell culture,the latter grow slowly and arrest at a much lower density, thus showinghypersensitivity to the emergence of intercellular contacts. It was shown thatthere is an additional mechanism that controls cell proliferation termed“early contact inhibition” (ECI). ECI in NMR was initially believedto be associated with increased p16INK4a protein levels. This hyp16INK4a . Later,s genome In accordance with the data reported in ,early cInk4 partaking in ECI[Arf suppression-induced senescence.The coding sequences of the NMR Ink4a and Arf Hgenes were identified using conventional cloning procedures and subsequent Sangersequencing; lentiviral vectors carrying these genes and high-specificitypolyclonal antibodies against the respective proteins were constructed.Endogenous Ink4a and Arf expression in NMRfibroblasts was shown to be upregulated following exposure to DNA-damagingfactors or serial passaging [Ink4a or Arf expression causedcell cycle arrest in NMR fibroblasts. Hence, it was experimentally proved thatthe genes involved in producing the effect of early contact inhibition play aconserved function of cell cycle inhibitors in NMR[Arf oncosuppressor gene and a unique frameshift mutationin the RAS (ERAS) oncogene expressed by stemcells. The upregulated expression of the Arf gene in mouseiPSCs noticeably reduced their tumorigenic potential. The mechanism related toNMR cells that can protect iPSCs and somatic cells against arfsuppression and tumor formation was found. A special type ofsenescence, Arf suppression-induced senescence, was alsorevealed in NMR iPSCs. The Arf-dependent senescence specificto NMR can act as a backup protection method inducing cell senescence andfollowing death by suppressing Arf expression in the cellswhere this gene used to be suppressed under stress[Discovered in 2009, the phenomenon of early contact inhibition of fibroblastgrowth in NMR remains interesting to researchers. In addition to the recentlyrevealed pALTINK4a/b protein, the product of expression of the alternativelyspliced form of g in ECI, anotherassaging . Theuprs in NMR.These rs in NMR. Tumorigr stress.Apoptosis is one of the mechanisms used for resisting the oncotransformation ofcells. The ability of NMR cells to undergo apoptosis in response to genotoxicstress has been insufficiently studied. When investigating the mechanism ofECI, Seluanov et al.  demonstrSpalax genus of blind mole rats  [Spalax, p53 differs from that in mostrelated mammals by having an arginine-to-lysine substitution at position 174.This specific mutation is frequently detected in human tumors[Spalax[Spalax, the arginineresidue occupies position 174 in H. glaber p53, as well as innormal human and mouse cells [The necrotic cell death pathway is also typical of the cancer-resistant galili) .In Spaln tumors. The argn tumors, 56.Nev[Spalax-60.As ose cells .et al. focused on the effect of toxicstressors on NMR fibroblasts demonstrated that these cells are more resistantto methyl methanesulfonate, paraquat, and low-glucose media but more sensitiveto H2O2, UV light, and rotenone compared to mousefibroblasts [2O2 at aconcentration ranging from 10-6 to 10-3 M and heat (42°C). The apoptoticresponse of NMR cells to exposure to H2O2 was 3- to10-fold weaker, while their resistance to heat was higher than that in mouseendothelial cells [The study by Salmon roblasts . Labinskal cells .Ctenomys talarum), these sequences arecharacterized by a high degree of sequence conservation. Its 28S rRNA alsolooks like it is split, but highly accurate protein biosynthesis is not typicalof the tuco-tuco [Translational fidelity is one of the key features of the functioning of key NMRsystems. With the translation rates close, the number of misincorporated aminoacids in NMR fibroblasts is fourfold lower than that in mouse fibroblasts. The trauco-tuco . Quite auco-tuco -69.Theruco-tuco .The oxidative stress theory considers the accumulation of oxidative damage incells to be one of the factors behind aging. For this reason, the questionsregarding the level of oxidative damage and the features of the mechanisms ofantioxidant protection in the “long-live” NMR drawresearchers’ attention.Proteins are the main target where oxidative damage emerges. Oxidative eventsmay disrupt the protein structure and functions, in particular by inactivatingenzymes and facilitating the formation of protein aggregates containingcovalent cross-links. The cysteine thiol groups are characterized by highsensitivity to oxidation, since they can form both reversible  and irreversible lesions (sulfinic and sulfonicacids) . Other cGpx1 andthe respective protein also abruptly decrease in NMR [Comparison of the activities of glutathione synthetase, catalase, superoxidedismutases, and glutathione peroxidase (GPX1) demonstrated that the activity ofall enzymes but GPX1 in liver extract from a young naked mole-rat was 1.3- to2-fold higher than that in the extract from the liver of a C57BL/6 mouse ofrespective physiological age. GPX1 activity in the NMR extract was lower byalmost an order of magnitude . Inaccoe in NMR , 73.According to , the levAn analysis of the levels of protein carbonylation in NMR and mouse tissuesdemonstrated that triose phosphate isomerase (TPI) and peroxy redoxin 1 (Prdx1)are the main targets for carbonylation in all samples. NMR proteins arecharacterized by a 1.5-old higher level of carbonyl damage but better retainenzymatic activity.2+) [The level of carbonyl damage in NMR proteins is 1.5-fold higher, but theproteins better retain their enzyme activity. The specific activity of TPI inthe cytosol fraction of a NMR kidney tissue lysate was three times higher thanthat of mouse. Furthermore, NMR TPI and Prdx1 form fewer covalentlycross-linked protein oligomers under oxidative stress(ascorbate/Fe2+) , 74.4,42+) .Comparison of the distribution of carbonylated proteins over subcellularfractions in long-lived (including NMR) and short-lived mammals demonstratedthat the relative level of proteins with oxidative damage in the nucleus inlong-lived animals is lower than that in the cytoplasm , 76. ThiThe actual situation may be disguised by the results of evaluations that do notdistinguish between the types of damage or damaged molecules (proteins or DNA)originating from different cellular compartments. Lack of consistency in thenature of the methods and agents used in different publications also impedesany analysis. One of the reasons for this can be the fact that a high level ofoxidative damage is typical of only certain molecules (molecular classes)and/or cellular compartments .Furthermore, the data regarding the antioxidant status are controversial. Thus,a lack of consistency was observed for the GSH levels in NMR tissue evaluatedin different studies: it was reported to be 1.4-fold lower than that in mouse, while tThe ubiquitin-proteasome system plays a crucial role in maintaining therequired level of active proteins with a proper structure (proteostasis) in thecell .. Specific ChT-L activity of NMR proteasomes was shown tobe 3–5 times higher than that of mouse proteasomes [Evaluation of the proteolytic activity in combination with the results ofWestern blotting revealed a higher chimotrypsin-like (ChT-L) and trypsin-like(TL) protease activity in 26S and 20S proteasomes in liver tissue extracts ofNMRteasomes . Most ofteasomes . This mateasomes . Furtherteasomes . The incThe multifunctional blood plasma protein alpha- 2-macroglobulin (α2m) isalso associated with proteostasis maintenance. Human α2m can bind tovarious cytokines, growth factors ,and it is a universal inhibitor of proteinases . Binding of α2m-proteinase complexes tothe LRP1 (CD91) receptor triggers their quick elimination from the blood andtissues via receptor-mediated endocytosis. This protein is believed to act as achaperone preventing protein aggregation and to facilitate the retention ofzinc in cells  -85. The Another important feature of NMR is the constant activity of the signalingpathway regulated by the Nrf2 factor, which activates the transcription of over200 genes involved in the antioxidant and anti-inflammatory response of theorganism to endogenous and exogenous stressors .Peromyscusleucopus is 2.5-fold higher than that in mouse (Musmusculus) fibroblasts [Efficient “functioning” of DNA repair systems is believed to be oneof the basics of genome stability maintenance. The typical features of the NMRgenome include an increased stability of its structure and function, which aremaintained during the entire lifespan. Its protein system (the proteome) isalso stable. Translational fidelity, upregulated expression of key chaperons,and permanently active proteasomes in combination with a high α2mexpression level facilitate the maintenance of a pool of efficientlyfunctioning proteins in NMR cells. Resistance to denaturing conditions and theability to retain their functional activity under permanent oxidative stresswere experimentally proved for a number of NMR proteins. All the aforementionedfactors and the upregulated expression of a number of genes coding for repairproteins, as well as the intensity of the response of the signaling pathways todamage, provide grounds to expect a high efficiency of the DNA repair system inNMR. In particular, this speculation is consistent with the results of studiesusing cells of mammals with different lifespans. The rate of UV-induced DNAsynthesis in the fibroblasts of the white-footed mouse roblasts .UV-induroblasts . Compariroblasts .It is quite possible that the activity of the poly(ADP-rybosyl)ation processesthat regulate different repair mechanisms  is also The unique phenotypic traits of NMR are obviModel systems of different complexities are employed to study these featuresusing an increasingly broad range of methods . Ma et aFig. 2.However, the lack of studies focused on apoptosis induction under variousgenotoxic stressors and experimental data regarding the function of DNA repairsystems leave a gap in knowledge regarding the real contribution of theseprocesses to the longevity and cancer resistance of NMR. In this connection, acomparative evaluation of the functional activities of DNA repair systems is arather important and topical task.Hence, there is good reason to believe that the molecular machinerycounteracting the accumulation of damage in the NMR genome, including themechanisms of DNA repair, is very efficient. We have made an attempt toillustrate this conclusion with a scheme shownin


The Bromodomain and Extra-Terminal Domain (BET) family of proteins is characterized by the presence of two tandem bromodomains and an extra-terminal domain. The mammalian BET family of proteins comprises BRD2, BRD3, BRD4, and BRDT, which are encoded by paralogous genes that may have been generated by repeated duplication of an ancestral gene during evolution. Bromodomains that can specifically bind acetylated lysine residues in histones serve as chromatin-targeting modules that decipher the histone acetylation code. BET proteins play a crucial role in regulating gene transcription through epigenetic interactions between bromodomains and acetylated histones during cellular proliferation and differentiation processes. On the other hand, BET proteins have been reported to mediate latent viral infection in host cells and be involved in oncogenesis. Human BRD4 is involved in multiple processes of the DNA virus life cycle, including viral replication, genome maintenance, and gene transcription through interaction with viral proteins. Aberrant BRD4 expression contributes to carcinogenesis by mediating hyperacetylation of the chromatin containing the cell proliferation-promoting genes. BET bromodomain blockade using small-molecule inhibitors gives rise to selective repression of the transcriptional network driven by c-MYC These inhibitors are expected to be potential therapeutic drugs for a wide range of cancers. This review presents an overview of the basic roles of BET proteins and highlights the pathological functions of BET and the recent developments in cancer therapy targeting BET proteins in animal models. Drosophila, the maternal effects gene, fsh, plays critical roles in establishing segments and specifying their identities during embryonic development [RING3 gene, found in the class II region of the human major histocompatibility complex (MHC) has substantial homology with the fsh gene [Bdf1 gene encodes a transcription factor that is required for sporulation [ORFX [MCAP [BRDT [RING3 located in the MHC region [Ring3, Orfx, Mcap, and Brdt are simply named as Brd2, Brd3, Brd4, and Brdt, respectively.In elopment . The RINfsh gene . The yearulation . A comparulation . The synrulation . In humaon [ORFX , MCAP [8AP [BRDT  genes haC region ,11. The Drosophila, the brahma (brm) gene encodes a bromodomain-containing protein that is required for transcriptional activation of several Hox genes [In ox genes . Tetrahyox genes . Furtherox genes . Most ofox genes ,15, whicox genes . The broox genes ,18. In mox genes . BRD2 seox genes . BRD4 isox genes ,22. BRD2ox genes . To faciox genes ,25. ThesA genome wide approach for the characterization of nucleosomes decoded by the double bromodomain BET factors shows that direct binding of BRD4 to acetylated nucleosomes associated with transcribed genes is required for their proper expression . Global Drosophila, the roles of mammalian BET proteins, FSH counterparts, have been conclusively determined by a well-defined analysis. On an earlier occasion, interest in the functional analysis of BET was directed toward the elucidation of its basic roles as a transcriptional regulator. However, recent identification of the carcinogenic activity induced by aberrant BET expression and small molecular BET inhibitors has attracted the interest of many researchers in cancer therapy. This review article presents an overview of the basic roles of BET proteins in transcriptional regulation, and highlights the pathological roles of BET in oncogenesis and latent viral infection.Since FSH was first found to be a morphogenetic regulator in BET family genes, are presumed to have arisen by several rounds of genome-wide duplication [Drosophila FSH and Saccharomyces cerevisiae Bdf1 and Bdf2. The exon-intron organization of mammalian BET genes and the primary structure of BET proteins are shown in Drosophila, and yeast. The coding region of the Brd2 gene consists of 11 exons, spanning more than 6 kb of genomic DNA [Brd3 consists of 12 exons spread over more than 20 kb of genomic DNA [Brd4 and 17 coding exons of Brdt span more than 39 and 52 kb of genomic DNA, respectively [Drosophila FSH  possess the C-terminal domain (CTD) at their carboxy terminal region that is a conserved region of ~40 amino acids. The CTD interacts with the positive transcription elongation factor b (P-TEFb), which phosphorylates serine residues of the RNA Pol II C-terminal motif (CTM) [Paralogous genome regions in vertebrates, including the lication ,11. In momic DNA ,36. The omic DNA . Nineteeectively ,9. The Bectively ,38. The ectively . The ET if (CTM) ,42. Brd2if (CTM) ,44.BET genes. The protein encoded by this gene possesses two bromodomains and an extra-terminal domain  cell line in which the gene-trap vector is inserted into the Brd2 locus, exhibit embryonic lethality and abnormalities in the neural tube where the gene is highly expressed [BRD2 is a major susceptibility locus for juvenile myoclonic epilepsy (JME), a group of neurological disorders characterized by epileptic seizures [Brd2+/− mice are viable and overtly normal, but have a decreased tonic-clonic seizure threshold compared to Brd2+/+ wild type mice. Further, anatomical analysis of the brain shows that the number of GABAergic neurons in the neocortex and the striatum of Brd2+/− mice are decreased, compared to those in Brd2+/+ mice [Mice lacking xpressed ,50. Embrxpressed . Associaseizures . Heteroz+/+ mice . These fFluorescence resonance energy transfer (FRET) is a measurable physical energy transfer phenomenon occurring between appropriate fluorophores in sufficient proximity. When a high-energy donor fluorescent molecule transfers energy to a low-energy acceptor fluorescent molecule, a photon of a specific wavelength emitted by the acceptor can be detected as the FRET signal . FRET prChromatin immunoprecipitation (ChIP) of nuclear extracts from HEK293 cells expressing FLAG-tagged BRD2 or BRD3 shows that BRD2- and BRD3-bound chromatin is significantly enriched in several acetylation marks associated with transcribed genes, including H4K5, H4K12, and H3K14 acetylation, but contains little dimethylated H3K9, which is a characteristic of transcriptionally inactive heterochromatin . An in vHOX genes [HOX genes that are arranged in four clusters on the mammalian genome encode transcription factors that determine cell and tissue identities in the embryo during development and in the adult [Drosophila FSH, a counterpart of mammalian BET proteins, has been reported as crucial for the activation of the UltrabithoraxHOX gene [HOX expression by BET proteins might be conserved across species. Collectively, these findings support the idea that BET proteins fulfill key roles as “readers” of the histone code to induce transcriptional activation of various genes that are required for determining cell identities.To fully elucidate how BET proteins decipher the histone codes, which are combinations of histone modifications such as acetylation and methylation on nucleosomes, ChIP of the nuclear extract from HEK293 cells expressing FLAG-tagged BET proteins and quantitative mass spectrometry for immunoprecipitated core histones have been conducted. Consequently, it was confirmed that nucleosomes associated with BRD2, BRD3, and BRD4 are mostly enriched in at least three acetylations of H4K5, H4K8, H4K12, and H4K16, and H3K4 dimethylation or H3K4 trimethylation . To furtOX genes . Thirty-he adult ,57,58. BHOX gene ,59, the GATA1-estrogen receptor fusion gene, termed as G1E-ER cells, express GATA1 in the presence of exogenous estradiol [Hbb [BRD3 has been reported to directly interact with acetylated lysine residues of the transcription factor GATA1, which regulates the expression of all erythroid and megakaryocyte-specific genes . Pull-dostradiol . ChIP-seiol [Hbb . An incriol [Hbb . Howeveriol [Hbb .0/G1 transition in lymphocytes, prior to entry into the S phase [2/M transition [BRD4 is a nuclear protein widely expressed in mammalian tissues, and its expression is induced by mitogen stimulation during the G S phase . BRD4 lo S phase . Furtheransition . In a teansition ,65. The ansition . Furtheransition . These fBrd4 gene was established [Brd4 heterozygotes display prenatal and postnatal growth defects associated with a reduced rate of cell growth. Embryos homozygous for a Brd4 loss-of-function mutation die shortly after implantation because of their disability to form the inner cell mass (ICM) in blastocysts [Using mouse embryonic stem (ES) cells transfected with a gene-trap vector for screening insertional mutations in developmentally regulated genes, a mouse mutant line in which an insertion generates a null allele of the ablished . Brd4 hestocysts . BRD4 knstocysts . These rc-Myc and c-Jun in a dose-dependent manner. These results indicate that BRD4 has a positive effect on Ser2 phosphorylation of the RNA Pol II CTM by enhancing the recruitment of P-TEFb [Immunoaffinity purification of nuclear extracts from HeLa cells expressing FLAG-tagged and HA-tagged BRD4 with an anti-FLAG or anti-HA antibody results in the precipitation of complexes composed of BRD4, Cdk9, and cyclinT1 ,22. Thisf P-TEFb . Immunopf P-TEFb . BRD4 haf P-TEFb . In vitrf P-TEFb .JMJD6 or BRD4 by siRNA in HEK293T cells significantly increases the Pol II occupancy rate at promoter-proximal regions and dramatically decreases its rate along the gene body, suggesting that association of JMJD6 and BRD4 promotes the Pol II promoter-proximal pause release [Mass spectrometry analysis of affinity-purified proteins from cell lysates of 293T cells stably expressing BRD4 Flag-HA-tagged fragments identified NSD3 and JMJD6 as interactors of the BRD4 ET domain . NSD3 be release . Further release , H3Ac, H release . In concc-Myc and Klf-4 are BRD4-dependent genes whose transcript levels are reduced upon BRD4 knockdown or JQ1 treatment. In BRD4-knockdown cells, wild type YFP-tagged BRD4 effectively rescues c-Myc and Klf-4 expression, whereas YFP-BRD4 whose bromodomains are mutated does not rescue their expression. Analysis of nascent chromatin-bound RNA-seq reads across the c-Myc and Klf-4 gene loci in NIH3T3 cells reveals that transcript elongation on these genes is clearly inhibited by JQ1. An in vitro transcription assay shows that BRD4 allows Pol II to transcribe through nucleosomes in a histone hyperacetylation dependent manner, and that JQ1 specifically inhibits the activity of BRD4 but not that of the histone chaperone FACT (facilitates chromatin transcription). In BRD4-knockdown cells, the short form of BRD4 lacking the C-terminal P-TEFb interacting domain (BRD4 short) rescues the expression of c-Myc and Klf-4, while the BRD4 short form whose bromodomains are mutated (BRD4 short mBD mutant) does not rescue the expression of these genes. ChIP and quantitative PCR (ChIP-qPCR) assays with an antibody specific for Ser2 CTM phosphorylation in RNA Pol II (Ser2P Pol II) reveal that Ser2P Pol II distribution on the Klf-4 locus is shifted towards and beyond the transcription end site in BRD4-knockdown cells reconstituted with the short BRD4 compared with those reconstituted with the BRD4 short mBD mutant. These observations collectively support the idea that BRD4 facilitates Pol II progression along the gene body independently of P-TEFb as it traverses hyperacetylated nucleosomes by interacting with acetylated histones through its bromodomains [In addition to the role of recruiting P-TEFb to release RNA Pol II from pausing in the promoter-proximal region, BRD4 has been reported to play a role in supporting the progression of Pol II through hyperacetylated nucleosomes by interacting with acetylated histones via its bromodomains ,77. Assaodomains . This asIEGs) in response to external signals is critical for the consolidation of synaptic modifications underlying synaptic plasticity and memory formation [Rapid activation of immediate early genes  is a technique used to study protein mobility in living cells by measuring the rate of fluorescence recovery at the bleached site . FRAP reTransmission of transcriptional memory from mother to daughter cells is reported to be mediated by chromatin-based epigenetic bookmarks ,88,89. IBrdt is exclusively expressed in the testis [∆BDIBrdt, which lacks only the first of the two bromodomains, into the Brdt locus. ∆∆BDIBDI/Brdt homozygous male mice are sterile and show impaired testicular histology with severely reduced sperm concentrations and abnormal sperm morphology, suggesting that Brdt is essential for normal spermatogenesis [Brdt alleles results in meiotic arrest at the end of the prophase when the pairing chromosomes should undergo compaction in preparation for the first meiotic division [Brdt−/− pachytene spermatocytes show that BRDT governs the expression levels of more than 3000 genes, activating approximately two-thirds of these genes and repressing one-third of them [Ccna1 (Cyclin A1), one of the genes markedly activated by BRDT, is exclusively expressed in the male germ cell lineage, and is essential for spermatocytes to enter the first meiotic division [e testis ,43,44. Ie testis . Targeteogenesis . Furtherdivision . Transcr of them . Ccna1  gene on the long arm of chromosome 15, with BRD4 on chromosome 19p13.1 ) or, in rare cases, with BRD3 on chromosome 9q34.2 ), leading to BRD4-NUT or BRD3-NUT fusion protein production by NMC cells [BRD4-NUT translocation lung cancer patient [NUT midline carcinoma (NMC) is a rare but poorly differentiated and highly aggressive cancer of the squamous cell lineage that arises in midline structures ,105. NMCMC cells ,106. KnoMC cells . Further patient , induces patient . These rImmunohistochemical staining of Cos7 cells transfected with a BRD4-NUT expression vector and of HCC2429 cells using an anti-acetylated histone H4 antibody shows that the BRD4-NUT fusion protein forms speckled nuclear foci that contain hyperacetylated chromatin domains ,109. Immp21 gene that is a direct target of the p53 transcription factor, whereas treatment of A549 lung cancer cells with etoposide causes p21 expression. Knockdown of BRD4-NUT in HCC2429 cells induces efficient restoration of p21 expression, accompanied by accumulation of caspase 3, a member of the cascading reaction associated with apoptotic cell death, and E-cadherin, a marker of epithelial cell differentiation [Treatment of HCC2429 cells with etoposide, an inducer of single- or double- strand DNA breaks, does not induce expression of the ntiation . MoreoveExpression of BioTAP  -tagged Kaposi’s sarcoma-associated herpesvirus (KSHV) causes Kaposi’s sarcoma (KS), a cancer commonly occurring in AIDS patients, as well as primary effusion lymphoma, and some types of multicentric Castleman’s disease. Its latent nuclear antigen (LANA), which is expressed in the nuclei of latently infected cells, mediates episomal replication and persistence of viral genomes . Yeast tPapillomaviruses constitute a group of small DNA viruses that cause benign tumors in a variety of higher vertebrates. During the latent infection period, viral genomes persist as episomes in the nuclei of proliferating host cells. The papillomavirus genome includes an early region (E) encoding six  open reading frames (ORF) that are expressed immediately after initial infection of the host cell. The E2 protein, which binds to the viral replication origin cooperatively with the E1 protein, plays a crucial role in long-term episomal maintenance and DNA replication of the viral genome ,117. TheCellular E2-interacting proteins have been explored using the bovine papillomavirus (BPV) E2 protein. A proteomic analysis of nuclear lysates from human cervical carcinoma C33 cells transduced with a recombinant retrovirus expressing FLAG-HA-tagged BPV E2 shows that BRD4 is co-immunopurified with anti-FLAG and anti-HA antibodies, suggesting that BRD4 is a member of the cellular interacting partners of BPV E2 . The oveThe E2 protein encoded by human papillomaviruses (HPVs) has also been reported to be associated with BRD4. Using nuclear extracts of human embryonic kidney-derived 293 cells that express FLAG-tagged HPV E2, anti-FLAG immunoaffinity purification followed by mass spectrometry identified BRD4 as a cellular protein that is naturally associated with HPV E2 . In vitr18F-fluorodeoxyglucose (FDG) uptake shows that JQ1-treated mice exhibit marked reduction of FDG uptake, whereas vehicle-treated mice develop progressive cancer. In fact, remarkable tumor regression and prolonged survival are observed in mice treated with JQ1 compared to that in the vehicle control [To identify and optimize therapeutic lead compounds for translation of small-molecule modulators of epigenetic targets as cancer therapeutics, high-throughput screening has been conducted. JQ1, a thienotriazolodiazepine, is identified as a potent and selective small-molecule inhibitor of BET proteins that competitively binds to the acetyl-lysine recognition pocket of their bromodomains . FRAP as control . These oMYCN-amplified neuroblastoma [c-MYC is a transcription factor that regulates cell proliferation. Aberrant c-MYC expression leads to cancer by the coordinated activation of transcriptional pathways involved in cell division, metabolic adaptation, and survival. c-MYC is thus viewed as a promising target for anti-cancer drugs. To investigate whether BET inhibition specifically abrogates MYC-dependent transcription, global transcriptional profiling and unbiased gene set enrichment analysis (GSEA)  was perfblastoma , castratblastoma , Ewing’sblastoma , and colblastoma . These rblastoma .MYC transcription [PIK3CA  mutation, which is supported by the fact that PIK3CA overexpression confers JQ1 resistance to JQ1-sensitive cells whereas a PIK3CA-specific inhibitor increases JQ1 sensitivity in resistant cells [PIK3CA mutation leading to BET-inhibitor resistance in luminal breast cancer.To optimize the clinical efficacy of BET inhibition, mechanisms of drug resistance have been evaluated. Comparison between I-BET-resistant MLL cells and vehicle MLL cells by GSEA reveals the enrichment of leukemia stem cell signatures and significant upregulation of Wnt/β-catenin signaling in the resistant clones. Negative regulation of the Wnt/β-catenin pathway results in restoration of sensitivity to I-BET both in vitro and in vivo . A chromcription . These rnt cells . These fdK values and this protein-protein interaction is directly inhibited by JQ1 [An ITC study, which has been performed to confirm the binding of a synthetic H4Kac4 peptide to human BRDT bromodomains in the presence and absence of JQ1, shows that the peptide binds to modules with sufficiently low d by JQ1 . Intraped by JQ1 . To defid by JQ1 . These fThis review focuses on the basic functions of the four paralogous BET proteins, which are summarized in Secondly, the review focuses on the pathological functions of BET family proteins. BRD2 and BRD4-S (a BRD4 short variant) interact with the LANA of KSHV, which is required for episomal replication and persistence of viral genomes, through their ET domain. The association of the HPV E2 protein with BRD4 is required to tether the viral genome to host mitotic chromosomes, and BRD4 is essential for E2-mediated transcriptional regulation. The BRD4-NUT fusion protein, which is produced by a gene reconstructed by a chromosomal translocation, activates multiplication related genes by hyperacetylating their chromatin with recruited p300, which causes NUT midline carcinoma (NMC). Treatment of NMC-patient derived cells with JQ1, a potent and selective small-molecule inhibitor of BET proteins, arrests proliferation at the G1 cell-cycle stage and prompts terminal squamous differentiation. Global transcriptional profiling of human multiple myeloma cells treated with JQ1 reveals a strong correlation between the genes downregulated by JQ1 and the genes comprising MYC-dependent transcriptional pathways. Small-molecule BET inhibitors including JQ1 have immediate therapeutic potential for a wide range of malignancies. Although BET inhibitors have a dramatic effect on suppression of tumor growth, there is a possibility that they cause critical damage to normal cell proliferation that is controlled by the MYC-dependent transcriptional pathway and the Wnt signaling pathway. Furthermore, as revealed by the administration of JQ1 to mice, BET inhibitors might have deleterious effects on the transcriptional regulation underlying human learning and memory. Further basic studies should be required to investigate various effects of BET inhibitors. Of many derivatives of BET inhibitors, compounds without harmful effects should be strictly selected. For example, a compound that cannot cross the blood-brain barrier would be available as an anti-cancer drug. To further develop BET inhibitors as anti-cancer drugs towards clinical application, the biological pathway leading BET inhibitor resistance needs to be investigated through a detailed molecular study.


Here, we described the cloning and characterisation of the zebrafish fads2 promoter. Using 5′-deletion and mutation analysis on this promoter, we identified a specific region containing the sterol regulatory element (SRE) which is responsible for the activation of the fads2 promoter. In tandem, two conserved CCAAT boxes were also present adjacent to the SRE and mutation of either of these binding sites attenuates the transcriptional activation of the fads2 promoter. An in vivo analysis employing GFP reporter gene in transiently transfected zebrafish embryos showed that this 1754 bp upstream region of the fads2 gene specifically directs GFP expression in the yolk syncytial layer (YSL) region. This indicates a role for LC-PUFA in the transport of yolk lipids through this tissue layer. In conclusion, besides identifying novel core elements for transcriptional activation in zebrafish fads2 promoter, we also reveal a potential role for fads2 or LC-PUFA in YSL during development.The front-end desaturases (Fads) are rate-limiting enzymes responsible for production of long-chain polyunsaturated fatty acids (LC-PUFA). The full spectrum of the transcriptional regulation of  Long-chain polyunsaturated fatty acids (LC-PUFA) such as arachidonic acid , eicosapentaenoic acid  and docosahexaenoic acid  are generated from shorter polyunsaturated fatty acids (PUFA) through the actions of Fads and elongases (Elovl). LC-PUFA are crucial for maintenance of cellular membrane integrity, as precursors for eicosanoids, regulation of gene expression and signal transduction pathways. In aquaculture, the interest to decipher the activities of the LC-PUFA biosynthesis enzymes and transcription factors involve in LC-PUFA biosynthesis is driven by the pressing need to improve performance of vegetable oils in aquafeeds1. These oils are lacking in LC-PUFA but rich in C18 PUFA. Understandably, a greater understanding of distinctive bioconversion capacities of C18 PUFA to LC-PUFA in different farmed species could theoretically improve the strategy of employing vegetable oil as feeds2.In eukaryotic cells, the front-end desaturases (Fads) catalyse the introduction of a double bond at fixed number of carbons from the carboxyl group. A typical Fads possesses characteristic features such as three histidine-rich boxes, transmembrane regions, and an N-terminal cytochrome b1. Fads from mammals are principally mono-functional, with Δ5 and Δ6 desaturations being carried out by two separate genes, Fads1 and Fads2, respectively3. In fish, fads1 have so far been isolated from only one species, a basal gnathostome, leading to the hypothesis that a complete loss of fads1 in teleosts have occurred following gnathostome radiation4. Isolation and characterisation of teleost fads2 to date have reported a broad range of substrate specificities, with Δ4, Δ5, Δ6 and Δ8 desaturation capacities reported. In addition, both mono-functional and bi-functional fads2 have been isolated from a myriad of teleost species7. As compared to freshwater species, marine teleosts seemed to display a lesser capacity for bioconversion of C18 PUFA to LC-PUFA, due to the loss of crucial fads from its genome or low activities of fads2 or elovl8. This was postulated to be a result of richer LC-PUFA content in the marine environment, causing diminished capacity for LC-PUFA biosynthesis. Subsequent works however advocated the influence of feeding niche on LC-PUFA biosynthesis, when a catalogue of fads2 and elovl were isolated from marine fish species which possess a diet with limited DHA intake9.Among the vertebrates, numerous studies have reported the molecular cloning and characterisation of Fads from different fish speciesfads in teleost, there is a paucity of understanding on its regulation at the transcriptional level. It is conceivable that differences in regulatory activities by dissimilar transcription factors responding to different cues including nutritional status is responsible for the different LC-PUFA biosynthesis capacity in various fish species. These transcription factors themselves are subjected to regulation by the nutritional status of the animal. Therefore, efficient use of vegetable oils in aquafeeds will benefit from the ability to decipher the link between the overall lipid metabolic pathways modification caused by the diet and the subsequent influence on lipid homeostasis genes at the transcriptional level. To date, promoter sequences from teleost fads2 have been isolated from Atlantic salmon10, Atlantic cod10, rainbow trout, European sea bass11, Japanese seabass12, large yellow croaker13 and rabbit fish14. Firstly, these works collectively showed the presence of binding sites for transcription factors known to play a role in mammalian cholesterol and lipogenic synthesis pathways such as sterol regulatory element-binding protein (Srebp), nuclear factor Y (NF-Y) and ubiquitous transcription factor Sp1 in the fads2 promoter14. Secondly, these studies suggested an interplay between different transcription factors to mediate the regulation of fads2 transcription.Despite the multiple interests in molecular characterisation of 16. Largely driven by the existence of multiple homologous genes involved in conserved lipid and lipoprotein metabolism network, zebrafish have been used to gain insight on lipid adsorption, atherosclerosis, fatty liver disease and obesity17. Zebrafish tissues are reported to contain significantly higher concentrations of PUFA than equivalent mammalian tissues19. Molecular cloning, functional characterisation and expression profile of fads2 and several elovl genes during embryogenesis have been reported in zebrafish23. However, our knowledge on the function of fads2/elovl and LC-PUFA during development is still fragmentary. Given how important transcriptional regulation in influencing the activities of fads2 may be and how little is known about the regulators and cellular conditions for expression, it is opportune to use zebrafish as a model to fill these gaps.The zebrafish has gained reputation as a useful model in understanding the role of lipids and fatty acids during developmentfads2 promoter. This includes identification and functional characterisation of pivotal regulatory elements responsible for driving transcriptional activities using luciferase reporter gene in a zebrafish cell line. In addition, we also carried out an in vivo characterisation of the zebrafish fads2 promoter using a transient approach to track its expression in developing zebrafish embryos.In this present study, we described the cloning and characterisation of the zebrafish fads25 and 1735 bp of the upstream region. For positioning purpose, +1 was given to the putative transcription start site (TSS) derived from comparison of the 5′-end of full-length cDNA (GenBank accession number: NM_131645.2) and DNA sequences of the fads2 gene. Within this fads2 promoter region, a classic transcription initiation element, the TATA box was identified at the location of −26 bp  or Srebp2 (nSrebp2) proteins, respectively, resulted in significantly higher reporter activities than cells transfected with empty pcDNA3.1 expression plasmid , Atlantic salmon10 (GenBank accession number: AY736067.2), European seabass11 (GenBank accession number: FP671139.1), large yellow croaker13 (GenBank accession number: KT781409.1), Japanese seabass12 (GenBank accession number: KT781410.1), Atlantic cod10 (GenBank accession number: FJ859898.1), human24 (GenBank accession number: AP002380.3) and mouse (GenBank accession number: AC135670.3). The alignment disclosed the presence of several conserved putative transcription factor binding sites, namely two inverted CCAAT boxes, an inverted ubiquitous transcription factor SP1 site and a sterol regulatory element (SRE) within the cloned promoter region  by three consecutive nucleotides. The SP1 binding site is less conserved amongst the mammalian and fish species, with two SP1 binding site in the mammalian Fads2 promoters, while teleost fads2 promoters typically contain either a single SP1 site or none.To identify the presence of any potential fads2 promoter fragment capable of activating transcription by fusing a series of 5′–deleted promoter fragments to the luciferase reporter gene. Upon normalisation to values obtained with a promoter-less pGL3.1 plasmid, the intact fads2 promoter (−1735/+19 bp) drove a luciferase activity of approximately 200-fold  and distal CCAAT box (CATd) to the fads2 promoter cis-regulatory elements. Results showed the formation of DNA-protein complex when ZFL cells nuclear extract was incubated with wild type oligonucleotide probe containing CATp binding site. There was no complex formed when the probe was incubated without the nuclear extract or when a probe with mutated CATp sequence was used with the nuclear extract. Similarly, the use of non-labelled wild type oligonucleotides in excess amount completely outnumbered the formation of labelled DNA-protein complex. A comparable pattern was shown with CATd sequence . Taking this and the results from mutagenesis experiment into consideration, we used a longer oligonucleotide consisting of both SRE and the adjacent CATp. Results showed that binding of transcription factors and DNA occurred with the simultaneous presence of both the SRE and CATp binding sites Fig. . Mutatiofads2 promoter fragment containing response elements such as SRE, CATp and CATd proven to be essential for activation of expression is now shown to drive a spatio-temporal expression of fads2 in the zebrafish YSL.We observed GFP signal in the yolk syncytial layer (YSL) of embryos beginning at 26 hpf for both the pZs−244 bp and pZs−1735 bp constructs, respectively. As embryos developed, higher percentage of embryos with YSL-GFP expression were observed, along with increase in GFP signal intensity  and human FADS2 promoters both showed a requirement for putative NF-Y binding sites located adjacent to SRE for transcriptional activation39. Promoters of teleost and amphibian fads genes also contain a conserved region bearing NF-Y and Srebp binding sites at similar positions relative to the transcriptional start site (TSS)10. It is speculated that direct interaction of Srebp and NF-Y, together with the binding of both these transcription factors to neighbouring sites on the DNA lend stability to the DNA-protein complex39. In addition, our results showed that removal of the putative SP1 binding region did not supress the luciferase expression. The interaction between Srebp and SP1 in the regulation of Fads have not been demonstrated this far33, although a synergistic type of activation between these two transcriptional factors have been elucidated in other lipid metabolism genes44. A role for SP1 in Srebp-mediated activation of Fads2 however cannot be ruled out as different isoforms of Srebp or nutritional conditions may utilise different co-regulatory factors to activate expression of Fads, as shown in promoters of fatty acid synthase45.Our present finding suggests that the putative NF-Y binding sites are required for formation of complex between Srebp and SRE, which then activates the transcription of reporter gene. This result resonated with earlier findings wherein Srebp was incapable of driving transcription of target genes if adjacent NF-Y binding sites were mutated46. Together with the head region, yolk sac has been reported to contain majority of the lipids during zebrafish embryonic stages19. While saturated and monounsaturated fatty acids  are consumed for energy, LC-PUFA are essential for cellular membrane integrity, regulation of gene transcription, regulation of signalling pathways and production of eicosanoids. Previously, the yolk was regarded as a non-metabolic active site for nutrient reserve, where molecules such as lipids are stored within yolk cells and transported when needed47. However, emerging findings employing zebrafish as the model organism, are recognising the yolk as site for active lipid metabolism preceding transportation16. Using a transient transgenic approach in zebrafish embryos, we showed that the −1735/+19 bp region of the fads2 promoter directed a specific expression in the YSL beginning at 26 hpf embryos. In support of this, fads2 and elovl transcripts were reported to be expressed in YSL at the 24 hpf stage, alongside brain tissues21. During early embryonic development, some of the blastomeres situated at surface of the yolk collapse and fuse with the yolk cells, leading to the formation of YSL49. It then undergoes a series of changes in terms of thickness and nuclei distribution, before going into the process of degradation upon the exhaustion of yolk supplies50. YSL governs major roles during development, including yolk metabolism, epiboly, specification of germ layers, organogenesis of endoderm organs and innate immunity52. A major role of YSL in teleost development is on the transport of nutrients from yolk to embryonic cells and tissues. Transcripts of genes related to lipid metabolism and transportation58, are expressed in YSL during early development to facilitate the hydrolysis and transport of yolk lipids. When these genes are disrupted, embryos show reduced yolk consumption, delayed embryonic growth and failure in lipid absorption59.Fatty acids from yolk storage are critical for energy and structural components for both fish embryo and larval stagesfads2 promoter activities, in transportation of yolk lipids. Detailed analyses of lipid classes in zebrafish yolk imply active de novo synthesis of lipids, in which fatty acids are resynthesised into major lipid classes between 0–48 hpf stage, followed by packaging into very low-density lipoproteins for supply to whole embryo beyond 48hpf19. In Caenorhabditis elegans, transportation of yolk lipoprotein to oocytes was disrupted in mutants with PUFA deficiency, a malaise restored by feeding worms with n-6 PUFA60. These findings, viewed alongside our results showing localisation of fads2 promoter activity at the YSL, provides the collective interpretation that LC-PUFA is required for transportation of yolk lipids during development.The presence of GFP signal in YSL also suggested a role for LC-PUFA, the product of fads and elovl transcripts being reported in adult zebrafish liver and early larval stages21. Since the injected embryos only showed expression in the YSL, the fads2 promoter fragment lacks the regulatory region responsible for directing expression of fads2 in other organs such as liver, intestine or brain. It is plausible that the regulatory elements for the expression of these tissues are located in more distal regions or/and introns62. Multiple studies have shown that recapitulation of endogenous expression by a promoter in zebrafish embryos depends on the length of the promoter fragment64.In adult teleost, the liver is a major site for LC-PUFA biosynthesis, with both fads2 promoter and identified several response elements critical to drive the activation of transcription. We also showed the capacity of this fragment to direct GFP expression at the YSL region of 26 hpf and older stages embryos. Since zebrafish relies on yolk as sole lipid source prior to onset of exogenous feeding capacity, it potentially provides a platform to further elucidate the role of fads2 or LC-PUFA in transportation of yolk lipids across the YSL.In conclusion, we successfully isolated a 1754 bp upstream region of the zebrafish 2 flask.The zebrafish liver cell line, ZFL (ATCC® CRL-2643™) was purchased from the American Type Culture Collection  and maintained in the complete growth medium at 28 °C following the protocol of ATCC. Routine subculture was performed once the ZFL cells have achieved 80 to 90% confluence in a 25 cmArtemia nauplii. Breeding and embryo collection were carried out according to Westerfield (2000) with adaptation65.Wild type zebrafish (AB line) were purchased from the Institute of Molecular and Cell Biology , and maintained in the ZebTEC Stand Alone System . The fish were kept on a 13:11 h light/dark cycle at 28.5 °C, and fed till visual satiation twice daily with a combination of commercial micro pellet , frozen bloodworms and Husbandry, handling and use of animals in this manuscript comply with the guidelines and requirements of the Animal Ethics Committee, Universiti Sains Malaysia and were approved by the same committee (PA/ACSC/002/2011).fads2 gene was amplified with PCR utilising the i-Taq™ Plus DNA Polymerase . DNA extracted from the ZFL cells was used as the template for the amplification, together with the forward primer F1735 containing the restriction site for KpnI, and a reverse primer R19 containing the restriction site for XhoI  and a common reverse primer (R19).A promoter region of 1754 bp, corresponding to −1735/+19 bp relative to the transcription start site of the zebrafish ® Reagent  according to the manufacturer’s descriptions. First-strand cDNA was generated via reverse transcription PCR using the M-MLV Reverse Transcriptase . PCR fragments corresponding to the N-terminal of zebrafish srebp-1  and srebp-2  were identified through alignment with the human SREBP homologues , and amplified from the first-strand cDNA using forward primers containing the restriction site for KpnI and reverse primers containing a stop codon (TAG) for translation termination and the restriction site for XhoI  and incubated overnight for cell attachment. Transient transfection was carried out using transfection reagent Lipofectamine™ 2000  at a volume of 0.15 µl per 100 ng DNA. For nSrebp overexpression assay, ZFL cells were co-transfected with fads2-1735 promoter-luciferase reporter plasmid (100 ng/well), one of the nSrebp expression plasmids (50 ng/well) and Renilla luciferase reference plasmid, pRL-SV40 (50 ng/well). ZFL cells prepared for the 5′-deletion assay were co-transfected with one of the pGL3 promoter-luciferase reporter plasmids (equimolar to 100 ng fads2-1735 plasmid/well), pcDNA3.1-nSrebp2 (50 ng/well) and pRL-SV40 (50 ng/well). Co-transfection for mutation analysis was performed with one of the mutated pGL3 promoter-luciferase reporter plasmids (100 ng/well), pcDNA3.1-nSrebp2 (50 ng/well) and pRL-SV40 (50 ng/well). Lastly, nuclear extracts for EMSA (refer to section 2.7) were prepared from the ZFL cells seeded in a 25 cm2 flasks and transfected with pcDNA3.1-nSrebp2 (2 µg/ml). Briefly, plasmid DNA-Lipofectamine™ 2000 complexes in 50 µl Opti-MEM were equally distributed to each well and the ZFL cells were incubated for 4.5 h at 28.5 °C. The plasmid DNA-Lipofectamine™ 2000 complexes contained-culture medium was then replaced with fresh culture medium and the transfected ZFL cells were further incubated for 24 h before being proceeded to subsequent assay. The ZFL cells were harvested in 20 µl/well Passive Lysis Buffer and luciferase assay was carried out using the Dual-Glo® Luciferase Assay System  according to the manufacturer’s protocols. All the luminescence measurements were performed in the GloMax® Multi + Detection System . Firefly luminescence reading for each sample was first normalised to Renilla luminescence reading as the internal control of transfection efficiency, before it was further normalised to the control cells transfected with pGL3-Basic plasmid for the comparison of promoter activities.Prior to the day of transient transfection, 2.5 × 10fads2-244 plasmid employing the Muta-DirectTM Site-Directed Mutagenesis Kit  following the manufacturer’s instructions. Complementary mutagenic primers (Supplementary data http://www.bioinformatics.org/primerx/). Multiple site mutations were carried out one after another by using previously constructed mutant plasmids as templates.Multiple base substitution mutations at the SRE site, proximal and distal CCAAT box sites were created in the Prior to electrophoretic mobility shift assay (EMSA), 3′-end of each complementary strand of the oligonucleotides . The injected embryos were incubated in E3 medium at 28.5 °C. The embryos were periodically monitored under the MVX10 Fluorescence Macro Zoom Microscope equipped with ColorView III Soft Imaging System  until 120 hpf. For cryosectioning, zebrafish young larvae were fixed with 4% paraformaldehyde, treated and sectioned at a thickness of 20 µm using the Leica CM1850 UV Cryostat .Supplementary Datasets


DNA methylation (DNAm) has been linked to changes in chromatin structure, gene expression and disease. The DNAm level can be affected by genetic variation; although, how this differs by CpG dinucleotide density and genic location of the DNAm site is not well understood. Moreover, the effect of disease causing variants on the DNAm level in a tissue relevant to disease has yet to be fully elucidated. To this end, we investigated the phenotypic profiles, genetic effects and regional genomic heritability for 196080 DNAm sites in healthy colorectum tissue from 132 unrelated Colombian individuals. DNAm sites in regions of low-CpG density were more variable, on average more methylated and were more likely to be significantly heritable when compared with DNAm sites in regions of high-CpG density. DNAm sites located in intergenic regions had a higher mean DNAm level and were more likely to be heritable when compared with DNAm sites in the transcription start site (TSS) of a gene expressed in colon tissue. Within CpG-dense regions, the propensity of the DNAm level to be heritable was lower in the TSS of genes expressed in colon tissue than in the TSS of genes not expressed in colon tissue. In addition, regional genetic variation was associated with variation in local DNAm level no more frequently for DNAm sites within colorectal cancer risk regions than it was for DNAm sites outside such regions. Overall, DNAm sites located in different genomic contexts exhibited distinguishable profiles and may have a different biological function. Cytosine DNA methylation (DNAm) is a covalent modification of DNA brought about by the addition of a methyl group to the fifth position of the pyrimidine ring of cytosine. In differentiated mammalian cells DNAm occurs almost exclusively at cytosine bases directly upstream of guanine bases (CpG dinucleotide) . Importacis acting genetic variation can explain a substantial proportion of the phenotypic variation for some DNAm sites  in healthy colorectum tissue collected from 132 unrelated Colombian subjects who attended Colonoscopy examination and with diagnosis of hyperplasic polyp, adenoma, f others ,21. We uf others  and estial cells , which aM-value was −2.67 and −3.51, and 1.50 and 1.89 for islands and sea, respectively). Additionally, our results showed that the distribution of the average DNAm level for DNAm sites in the north and south shores were similar to one another and more similar to the distribution of the average DNAm level for DNAm sites in islands rather than DNAm sites in the sea , within 2–4 kb upstream or downstream of an island  or none of the aforementioned categories which we term sea. An island was defined as being composed of one or more adjacent sections of the genome each 500 bp in length with a C and G density >50% and an observed to expected ratio of CpG dinucleotide >0.60 . We grouM-value that was on average 0.15 >DNAm sites located in the TSS of genes expressed in LCM .Unless otherwise specified, the following analyses were based on the two most extreme cases of CpG density: high-CpG density regions (islands) and low-CpG density regions (sea). We tested if the DNAm level of DNAm sites located in the transcription start site (TSS) of genes expressed in WCB was different to those located in the TSS of genes expressed in cells from the colon epithelium collected using LCM. The genes that were expressed in the LCM and the WCB were excluded from the WCB group for this analysis. We found that DNAm sites in the TSS of genes expressed in WCB had an Subsequently, we grouped DNAm sites located within the sea or an island into four mutually exclusive sets based on location (i) in a TSS of a gene expressed in WCB (ii) in a TSS of a gene that is not expressed in WCB (iii) in intragenic DNA, where we do not distinguish between genes expressed or not expressed in colon because the methylation level of intragenic DNAm sites has not been correlated with the expression of the surrounding gene or (iv) intergenic DNA Table . AdditioP < 2.16 × 10−16) and between sea TSS expressed and sea TSS not expressed   and in both cases being located in the TSS of genes not expressed in colon led to an overall greater mean site-specific DNAm level. Additionally, we found that the mean of the distribution of the mean site-specific DNAm level for DNAm sites located in intragenic and intergenic regions was greater than for DNAm sites located in a TSS of a gene  Fig. . We compene Fig. .Figure −3 and 4.91. We observed that on average over all sites the variance of the DNAm levels was significantly higher for DNAm sites located within the sea than for DNAm sites located within an island  for all pair-wise comparisons of these categories. Within the sea the difference in mean residual variance was at least moderately significant (P < 0.001) for all pair-wise comparisons made except for the comparison of intragenic and intergenic. In this case, the mean residual variance for the sea intragenic and sea intergenic was not significantly different (P = 0.240).We explored the variation in the DNAm level by investigating the extent to which residual site-specific DNAm level varied across the 132 samples for the full set of 196080 DNAm sites that passed the quality control procedure. The variance of the residual DNAm level ranged between 3.00 × 10P < 0.05). The percentage of heritable DNAm sites exceeds that expected from a false-positive rate of 0.05 under the null hypothesis that the DNAm level is not associated with local genetic variation. For significantly heritable loci, the proportion of the variance in DNAm under local genetic control ranged between 0.06 and 0.99 with a mean of 0.29 and median of 0.26 . For instance, considering the range of the number of SNPs within a region, at the first decile (304 SNPs) and ninth decile (2733 SNPs), we expect a respective 7.2 × 10−3 and 6.5 × 10−2 increase in the heritability for the DNAm sites found to be significantly associated with local genetic variation. In addition, we found that the variance of the residual DNAm level at each DNAm site explained a proportion of 1.93 × 10−2 of the variance in the heritability estimate (univariate linear regression: P < 2.2 × 10−16). An analysis of variance indicated that genomic context explained a significant proportion (P < 2.2 × 10−16) of the variability in the heritability estimate after accounting for residual variance.We found that at 20239 DNAm sites, 10.32% of the 196080 tested, SNPs within 1 MB explained a significant proportion of the variation in the methylation level . Additionally, the proportion of significantly heritable DNAm sites in the LCM and WCB groups was 0.0723 and 0.0734, respectively, and was not significantly different from one another (P = 0.0834).We investigated the heritability of the site-specific DNAm level for genes expressed in LCM and in WCB excluding those expressed in LCM Fig. . The difP < 2.20 × 10−16, Table P < 2.20 × 10−16); additionally, the proportion of heritable DNAm sites is 1.13 times higher for DNAm sites located in the sea intragenic than the island intragenic contextual group (P = 1.47 × 10−6). There was no significant difference in the proportion of heritable DNAm sites located in intergenic regions when comparing between the sea and island contextual groups (P = 0.125).The proportion of sites with a significant heritability was higher in the sea than in islands (P = 1.55 × 10−14) and for the comparison of sea intragenic and sea TSS not expressed (P = 2.65 × 10−5). The difference was significant at a nominal threshold (P < 0.05) for the sea intragenic and sea TSS expressed comparison (P = 1.52 × 10−2). The proportion of heritable DNAm sites was significantly different for all comparisons made within the island contextual groups. This difference was highly significant (P < 1.00 × 10−8) except for the island TSS not expressed and island intragenic comparison (P = 4.34 × 10−2). Within the island contextual groups the proportion of heritable DNAm sites was as follows: intergenic > intragenic > TSS not expressed > TSS expressed.The proportion of heritable DNAm sites was lower for sea intragenic than the other three sea contextual groups. This difference was highly significant for the comparison of sea intragenic and sea intergenic .The proportion of heritable DNAm sites for each contextual group followed a similar pattern to the mean local genetic variance for each contextual group Fig. . Across The average ar Table . DNAm sicis regions and to determine if there was evidence for genetic effects on the DNAm level in trans. We found enrichment for cis and trans genetic effects on the DNAm level beyond what would be expected by chance assuming the test for the association of each SNP and DNAm site are independent  was paired with the SNP to which it was most significantly associated with in cis. We calculated the proportion of the regional heritability estimate explained by variation of the SNP for each pair  of genetic variants on the DNAm level with nominally unrelated individuals using the regional heritability method. This is because variance in the extent of identify by descent between nominally unrelated individuals is lower across the genome as a whole than it is within a small genomic region. However, in accordance with , we haveWe have identified a subset of DNAm sites genome-wide and measured in healthy colon tissue that are influenced by the local genetic variation. Therefore, we have contributed to understanding healthy genetically influenced variation in the DNAm level in colon tissue. A number of the DNAm sites which we report as heritable are located within CRC risk loci and thus have the potential to mediate genetic susceptibility to CRC. We expect further studies will focus on exploring a role for these DNAm sites in disease aetiology.A total of 144 samples from normal colorectal tissue were obtained from Colombian patients diagnosed with hyperplastic polyps, adenomas or adenocarcinoma of the colorectum. The study had ethical approval from the Ethics Board of The National Cancer Institute of Colombia, and participants gave informed written consent.Similarly, 12 people undergoing colonoscopic examination at General University Hospital of Elche (Spain) but without adenocarcinoma or adenomas of the colorectum provided tissue samples from the colon. Written informed consent for inclusion in the study was obtained from every participating individual. The study was approved by the ethics committees of the General University Hospital of Elche.We assayed 144 samples for the DNAm level at 485512 DNAm sites using the HM450K array.P = 0.01) for each sample and found that no samples exceeded our threshold of 5% for exclusion. Two samples where the recorded sex of the individual did not match the sex estimated from the levels of DNAm measured on the X chromosome were removed. A total of 280 469 DNAm probes were removed because they contained a SNP within the target sequence or at the site of single-base extension or they were deemed cross-reactive based on the work of (P < 0.01). This resulted in 133 samples and 196080 autosomal DNAm probes left for downstream analysis. Colour bias was taken into account by comparing for all samples the within sample distribution of total intensities measured by the type I probes in the green channel to those measured in the red channel. A quantile normalization adjustment was applied within the bioconductor package, ‘lumi’  or (iii) if they had a MAF of <0.01 or (iv) if the rate of genotype failure was significantly different in cases and controls (P < 0.00001). This procedure left a total of 682945 autosomal SNPs for analysis. Additionally, the inbreeding coefficient for each sample was calculated from SNPs along the X chromosome. This analysis revealed 3 samples recorded as female that were more inbred than expected (F > 0.98) and 10 samples recorded as male that were less inbred than expected (F < 0.2). These samples were removed from subsequent analysis due to an assumed discrepancy between the recorded and observed identity. This left 132 samples with quality genotype information that overlapped with the samples assayed for the DNAm level and which passed the DNAm level quality control procedures.We genotyped 468 samples at 958178 SNPs genome-wide using the Illumina HumanOmniExpress Exome Chip. We followed a standard quality control procedure , from each slice for each individual and mRNA from the slices was amplified prior to being assayed for gene expression. The gene expression assay on the 24 samples was performed using the HumanHT-12 Expression BeadChip. Quality control indicated failure of four samples  which were removed from subsequent analysis. We then identified for each condition mRNA probes which were detected above background (P < 0.01) in >80% of samples, i.e. 9 or more of 11 samples and 8 or more of 9 samples for the whole-tissue and epithelial conditions, respectively. This yielded 9223 probes in the whole tissue and 4071 probes in the epithelial conditions. Probes were mapped to genes using the Illumina provided manifest file for the HumanHT-12 Expression BeadChip platform, yielding a list of 8114 genes expressed in general colon tissue and 3754 genes expressed in epithelium. As expected a majority of genes identified in the epithelial condition were also detected in the whole tissue, which contains both the epithelial and other cells, with only 10 specific to the epithelial condition. This supports the view that the genes identified form subset of genes enriched for epithelial specific expression.Genes expressed in general colon tissue and specifically in colon epithelial cells were identified based on the analysis of normal tissue from biopsies of 12 people undergoing colonoscopic examination at General University Hospital of Elche (Spain). In order to separate epithelial specific expression, tissue samples were sliced with alternate slices assigned to the A DNAm site was considered to be located within the TSS of a gene expressed in colon tissue if in the manifest file the site was recorded as being located within 200 bp upstream of the TSS (TSS200) or, within 200–1500 bp upstream of the TSS (TSS1500) of a gene in our list of expressed genes. All other DNAm sites located within the TSS200 or TSS1500 region of a gene were considered as being located within a gene not expressed in colon tissue. Intragenic DNAm sites were those documented in the manifest file as located within the 5′UTR, first exon, gene body or 3′UTR. Intergenic DNAm sites were those not documented as residing within a gene. We applied successive filters in the aforementioned order so that each DNAm site fit into one of the four mutually exclusive categories.in situ carcinoma or carcinoma of the rectum or colon. In our analyses, we included both diagnosis and biopsy location as explanatory variables. Biological differences between the right  and left  colon have been identified and they include the tissue of developmental origin and manifestation of CRC  for a single DNAm site, β a vector measuring the effects sex and age and X a design matrix mapping the appropriate explanatory variable to each sample. Then with W a matrix of standardized SNP genotypes from each sample and assuming a vector of SNP effects, I a diagonal matrix and the random error The components of variance were estimated by fitting a mixed linear model using restricted maximum likelihood and the publically available software: REACTA . ConsideN SNPs for n samples. The formula used for calculating the pair-wise relationships from the SNP information can be found in VanRaden  and The Roslin Institute Strategic Grant funding from the Biotechnology and Biological Sciences Research Council (BB/J004235/1). A.T. and C.S.H. also acknowledges funding from the Medical Research Council Human Genetics Unit. Funding to pay Open Access charges was provided by the Biotechnology and Biological Sciences Research Council (BB/J004235/1).This work was supported by


S. cerevisiae, CBC positively controls the expression of respiratory pathway genes. This role involves interactions with the regulatory subunit Hap4. In many pathogenic fungi, CBC interacts with the HapX regulatory subunit to control iron homeostasis. HapX is a bZIP protein which only shares with Hap4 the Hap4Like domain (Hap4L) required for its interaction with CBC. Here, we show that CBC has a dual role in the pathogenic yeast C. glabrata. It is required, along with Hap4, for the constitutive expression of respiratory genes and it is also essential for the iron stress response, which is mediated by the Yap5 bZIP transcription factor. Interestingly, Yap5 contains a vestigial Hap4L domain. The mutagenesis of this domain severely reduced Yap5 binding to its targets and compromised its interaction with Hap5. Hence, Yap5, like HapX in other species, acts as a CBC regulatory subunit in the regulation of iron stress response. This work reveals new aspects of iron homeostasis in C. glabrata and of the evolution of the role of CBC and Hap4L-bZIP proteins in this process.The CCAAT-binding complex (CBC) is a heterotrimeric transcription factor which is widely conserved in eukaryotes. In the model yeast  Many roles have been attributed to fungal CBC but the most extensively studied are its requirement for the regulation of respiration in Saccharomyces cerevisiae on one hand, and its involvement in the regulation of iron homeostasis in many other fungal species  on the other hand , the CBC plays an important role in iron homeostasis19. This role is mediated by the HapX  regulatory subunit. HapX acts by repressing the expression of iron consuming genes in iron starvation conditions and by activating these genes in iron excess20. HapX proteins have few similarity with Hap4 except for the 16 amino acids domain  which is required for their interaction with Hap522. In addition, they also have a bZIP DNA binding domain similar to the one found in the oxidative stress response factor Yap123. In Yap1, this domain is responsible for the specific recognition of the YRE (Yap Response Element) DNA motif (TTACTAA). The role of this domain in HapX proteins remains to be elucidated since HapX and Hap43 clearly bind CCAAT motifs indirectly through their interaction with CBC, rather than YREs24. However, HapX also contributes to the DNA binding specificity of the CBC-HapX complex in A. nidulans and the bZIP domain is absolutely required for the regulation of transcription by Hap43 in C. albicans25.The CCAAT-binding complex (CBC) is a heterotrimeric transcription factor which is conserved from fungi to vertebrates and plants and which specifically recognizes the CCAAT DNA motif. In yeasts, the three subunits of the CBC are called Hap2/3/5. In fungi, the Hap2/3/5 complex is sufficient to bind DNA but it requires a fourth subunit to regulate transcription of some of its target genesS. cerevisiae, in contrast to C. albicans and Aspergillus sp., CBC does not seem to contribute to the global iron starvation response26, but its role in iron homeostasis has actually been poorly investigated in this species27  and transcriptome analyses. We found that Hap5 binds many genes involved in respiration and positively controls their expression. As in S. cerevisiae, this function requires the Hap4 regulatory subunit. More surprisingly, we observed that Hap5 directly controls the iron stress response mediated by the Yap5 bZip transcription factor. We showed that Yap5 cannot bind the promoter sequence of GRX4 in the absence of Hap5 and co-immunoprecipitation experiments suggested that Hap5 interacts with Yap5. Interestingly, Yap5 contains a sequence which resembles the N-terminal part of theHap4L domains of Hap4 and HapX. Full deletion or substitutions of the conserved amino acids in this sequence severely impaired Yap5 binding to its targets and its interaction with Hap5. Our results revealed a key role of CBC in the iron stress response of C. glabrata and identified Yap5 as a new CBC regulatory subunit.In the present study, we analyzed the role of the CBC in the yeast C. glabrata, we analyzed the DNA binding pattern of Hap5 by ChIP-seq. We chose this subunit because it was shown in other species to be essential for the assembly, the DNA binding and the activity of CBC32. Moreover, Hap5 is the CBC subunit which recruits Hap4 through a Hap4 recruitment domain which is present only in fungi32. We performed ChIP-seq on exponentially growing cells in rich glucose media. The peak calling procedure identified 113 bound promoters and 154 potential target genes (Supplementary File −23), ATP metabolism (p = 6.1 × 10−29), oxido-reduction processes (p = 1.7 × 10−20) and TCA cycle (p = 6.2 × 10−10). Half of the targeted promoters were associated to a gene encoding a mitochondrial protein (p = 3.4 × 10−18). These included genes encoding subunits of the respiratory complexes , mitochondrial transporters  and enzymes of the TCA cycle   or iron excess. As observed in our transcriptome analyses, ATP2 levels are decreased in the hap5Δ mutant in all three growth conditions examined. Remarkably, the same pattern was observed for the hap4Δ mutant, while ATP2 levels were independent on the presence of YAP5  was normally expressed  experiments by tagging the chromosomic version of Hap5 with protein A and transforming the resulting strain with a plasmid bearing the wild type or the mutated versions of Yap5-myc under the control of the native ody Fig.  or an anody Fig. . A clearody Fig. . This diody Fig. . This reC. glabrata. As mentioned in the introduction, the main role described for CBC in the model yeast S. cerevisiae is the activation of respiratory pathway genes in the absence of glucose. This also involves the regulatory subunit Hap4. Very recently, it was reported that hap5Δ and hap4Δ strains have a severe growth defect in non-fermentable carbon sources in C. glabrata, indicating that this role may be conserved in this species33. Consistently with this observation, our genome-wide analyses showed that Hap5 positively controls the expression of many mitochondrial protein encoding genes, including subunits of the respiratory complexes and enzymes from the TCA cycle. We also showed that Hap4 is required for this regulation. Hence, this role in cellular respiration seems to be perfectly conserved between S. cerevisiae and C. glabrata.In this work, we identified a part of the transcriptional network associated with CBC, more particularly the Hap5 subunit, in the pathogenic yeast C. glabarata CBC in iron homeostasis. Hap5 was found to bind the promoters of the genes of the Yap5-dependent iron stress regulon  and is necessary for their induction in iron excess conditions  and C. albicans CBC (positive regulation of iron stress response)  in the promoters of these genes35. We showed here that Hap5 is also necessary for this regulation to happen. More precisely, Yap5 is unable to bind and activate its targets in the absence of Hap5 , HapX/Hap43 proteins negatively regulate iron consuming genes in iron starvation and positively regulate them in iron excess20. They directly sense iron concentration through conserved cysteine rich motifs20. Gene expression regulation by HapX involves both binding to CBC and direct interaction with a DNA consensus (TGAC) which is close to a half-YRE site and which may rely on the bZIP domain of HapX25. The Yap5 functioning described here is very close to HapX, except that Yap5 binds a full YRE site35. Interestingly, Yap5 senses iron excess through a cysteine rich domain (CRD) which is very similar to the CRD of HapX37. Moreover, like HapX, C. glabrata Yap5 can act both as a repressor or an activator, depending on iron availability29. Then, CBC’s role in iron homeostasis is globally conserved  but its mechanisms of action have diverged from a Hap4-like functioning in S. pombe to a fully cooperative DNA binding model in C. glabrata. In this model, the CBC-HapX case would be an intermediate situation, in which HapX also contribute to DNA binding specificity and affinity but in which the interaction with CBC is predominant 44. This activity is independent of the regulatory subunits Hap4 and Hap4344. It rather involves cooperative action between the CBC and specific DNA binding transcription factors, such as the GATA factor Gln3 in S. cerevisiae42. Moreover, CBC has been shown to be a negative regulator of ergosterol biosynthesis and azole resistance in Aspergillus fumigatus45, while having a positive role on ERG9 expression in S. cerevisiae46. Finally, CBC plays an important role in the oxidative stress response of Aspergillus nidulans and Hansenula polymorpha47. The overexpression of the Hap4B regulatory subunit of H. polymorpha (which is orthologous to the HapX proteins48) is able to complement the yap1Δ sensitivity to hydrogen peroxyde in S. cerevisiae23. Our ChIP-seq results also suggest that some of these roles may be conserved in C. glabrata. Indeed, the glutamate dehydrogenase encoding gene GDH1 and the ammonium permease encoding gene MEP3 were targeted by Hap5 in our experiments. Also, a handful of oxidative stress response genes were bound by Hap5 in C. glabrata . Further investigations will be required to decipher the role of Hap5 in these processes.Additional roles of CBC have been described in fungi. For instance, in 50 and were verified by PCR before use. The genomic myc-tagging of Hap5 was performed as described previously51. Briefly, myc-tagging cassette was PCR amplified from the M. Longtine’s plasmids52 with oligonucleotides containing homology sequences flanking the desired genomic insertion points in 5′. At least 10 micrograms of purified PCR product was used to transform HTL cells using a standard yeast transformation protocol21. Genotyping of the clones growing on selective media was done by PCR. The correct myc-tagging of Hap5 was verified by sequencing of the gene and western blot21.The lists of the strains and primers used in this study are available in Supplementary Files TAP-TRP1 cassette was amplified from the pBS1479 plasmid53.The genomic TAP-tagging of Hap5 was obtained with the same strategy except that the YAP5, we started from the pGRB2-YAP5myc plasmid21 and used the Quick change site-directed mutagenesis kit from Agilent, following the recommendations of the supplier. The mutant plasmids were controlled by sequencing. After transformation in C. glabrata using the “one-step” yeast transformation protocol21, the correct expression of the Yap5 mutant proteins was tested by western blot . For growth in non-fermentable conditions, glucose was replaced by 2% glycerol. The following stress conditions were used: 2 mM iron sulfate in CSM ) for iron excess conditions and 0.5 mM bathophenanthroline disulfonate (BPS) in YPD for iron starvation conditions. The cells were exposed to the corresponding stress for 30 minutes. The strains harboring 54. The parental HTL (untagged strain) was grown and processed the same way to provide the mock-IP samples. Sequencing of the IPs, Input DNAs and mock IPs samples and primary data analyses  were performed as described previously54. Peak calling was performed with the bpeaks software51, using both the Input DNA and the mock IP as references. For peak calling using the Input DNA as reference, the bpeaks parameters were T1 = 1.9, T2 = 6, T3 = 1, T4 = 0.7. For peak calling using the Mock IP as reference, the bpeaks parameters were T1 = 1.9, T2 = 6, T3 = 1, and T4 = 0. Only the peaks that were found with both analyses were kept for further processing. These peaks were then manually checked on a genome browser55 to discard artefactual peaks  which would have escaped the bpeaks filter. Peaks located outside of a promoter region (i.e. between convergent ORF or inside ORFs) were also discarded from the final list presented in Supplementary File For ChIP, myc-tagged strains were grown in YPD until exponential phase (OD = 0.8). Cross-linking of the cells and ChIP were performed as described previously56. These genomic sequences were used as inputs for the peak-motif tool to search for regulatory motifs57. An additional filtering step was added to the standard peak motif procedure to discard low complexity motifs .DNA sequences of ChIP peaks were retrieved from their genomic locations (BED file) using the “getfasta” function from the BEDTOOLS suite55. The genome-wide search of YRE-CCAAT co-occurrences with a spacing of 10–14 bp in C. glabrata promoters was performed using the DNA pattern search in RSAT58 and a dedicated R-script which automatically identified co-occurrences in promoters and measured the distance between the two motifs.The co-occurrences of YRE and CCAAT motifs in the ChIP peaks shared by Yap5 and Hap5 were confirmed by visual inspection of the peak sequences using IGV51. When a peak was located in a divergent promoter  the two genes were fused in one target in the network named “gene 1/gene 2”. The network was represented using the igraph R library59. The presence of a ChIP peak was used as the parameter to define interactions (arrows) between Hap5 and its targets. The GO criterion was used to differentially color the target promoters . After overnight hybridization and washing, the slides were scanned using a 2- micron Agilent microarray scanner. The images were analyzed using the feature extraction software (Agilent technologies) and normalized using global LOESS61. The mean of the biological replicates was calculated. A gene was considered as being differentially expressed if its mean absolute Log2(fold change) value was more than 0.5 and if its expression variation was considered as being statistically significant using the LIMMA package with a cut-off p-value of 0.0562.Knock-out and wild type strains were grown in 50 mL of YPD until exponential phase (OD = 0.8) and then stressing agents were added. After 30 min, 15 mL of each cell cultures were flash-frozen in two volumes of cold ethanol and collected by centrifugation. Total RNA was extracted, quality controlled and quantified as described previouslyACT1 gene, used as an endogeneous reference, based on the ΔCt method. Finally, the expression values were normalized with the expression of the studied gene in the wild type strain grown in glucose given the arbitrary value 1 , then stressing agents were added for 30 min when required. Cells were then snap-frozen in cold ethanol and collected by centrifugation. Cells lysis was mechanically performed with glass beads using a Fastprep®-24 bead beater . Total RNA extraction was carried out using the RNeasy extraction kit (Qiagen) following the manufacturer’s instructions. The concentration of each sample was determined using NanoPhotometer® spectrometer (IMPLEN). For each sample, 1 μg of the total RNA were DNAse treated using Turbo DNA-free kit (Ambion). After DNAse treatment, 0.2 μg of total RNA were used to perform cDNA synthesis using Superscript II Reverse Transcriptase according to the manufacturer’s instructions (Invitrogen). The resulting cDNA were diluted to three different concentrations . Quantitative PCR reactions were performed on a C1000 TM Thermalcycler (Bio-rad) with a 2X SYBR Green master mix (Promega). The qPCR reaction mixture contained 0.5 μM of each primer and 4 μL of one of the three dilutions of the cDNA. These dilutions served as triplicate for each sample. The primers used for qPCR are listed in Supplementary File YHB1 promoter was used as an endogenous control. Q-PCR was performed as described above. The relative enrichment of a specific locus in the immunoprecipitated DNA relatively to the Input DNA and to the YHB1 promoter enrichment was determined using the ΔΔCt method.For Chromatin-immunoprecipitation followed by qPCR (ChIP-qPCR), three serial dilutions  of immunoprecipitated samples were simultaneously processed together with Input samples used for normalization. The primers used for qPCR are listed in Supplementary File 63 except that we started from 100 mL of cell culture at OD = 0.8 in CSM-His media, that we used DynabeadsTM PanMouse IgG and that all the IP sample was mixed with 2X Laemmli for western blots. Proteins contained in 15 µl of Input or IP samples were separated on 10% SDS-Polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred to Whatman® Protan® BA83 nitrocellulose membrane . Immunoblotting of Yap5-myc wild type and mutant proteins were performed using 1:10000 mouse IgG Anti-cMyc (Roche) and 1:10000 anti-mouse IgG-HRP (Promega) as primary and secondary antibodies. Detection of the signals was performed using G:BOX Chemi XT4 (Syngene) following incubation with UptiLightTM HRP blot chemiluminescent ECL substrate (Interchim).Co-immunoprecipitation using Hap5-Protein A as bait was performed as describedThe ChIP-seq data can be downloaded from the GEO database (accession number: GSE91371). The complete microarray data are available at Array express database under the accession number: E-MTAB-5348.supplementary files S1 and S3 to 7supplementary file S2