The mammalian Ccr4–Not complex, carbon catabolite repression 4 (Ccr4)-negative on TATA-less (Not), is a large, highly conserved, multifunctional assembly of proteins that acts at different cellular levels to regulate gene expression. In the nucleus, it is involved in the regulation of the cell cycle, chromatin modification, activation and inhibition of transcription initiation, control of transcription elongation, RNA export, nuclear RNA surveillance, and DNA damage repair. In the cytoplasm, the Ccr4–Not complex plays a central role in mRNA decay and affects protein quality control. Most of our original knowledge of the Ccr4–Not complex is derived, primarily, from studies in yeast. More recent studies have shown that the mammalian complex has a comparable structure and similar properties. In this review, we summarize the evidence for the multiple roles of both the yeast and mammalian Ccr4–Not complexes, highlighting their similarities. Saccharomyces cerevisiae. In yeast the complex has nine core subunits, comprising Ccr4 (carbon catabolite repression), Caf proteins (Ccr4 associated factor)  and Not proteins  as well as several less strongly associated components which are probably interacting partners [The yeast Ccr4–Not complex is a large 1.9-MDa) and highly conserved multifunctional assembly of proteins, involved in different aspects of mRNA metabolism. These include the repression and activation of transcription initiation, control of mRNA elongation, deadenylation-dependent mRNA turnover, and in ubiquitin-protein transferase activity reviewed .9-MDa an,3,4,5. Tpartners ,7  and C2ORF29 (CNOT11), have been identified in mammals . Four deThroughout this review, we have focused, primarily, on the biological roles of the complex in a variety of contexts. However, it should be noted that some effects can be ‘direct’, caused by the Ccr4–Not proteins themselves, either singly or as a component of the complex, or ‘indirect’ as a result of changes induced in target pathways. For the sake of completeness, we have considered the structure of the complex but a detailed structural description has not been presented here.Schizosaccharomyces pombe, Saccharomyces cerevisiae, Drosophila melanogaster, and Homo sapiens. Limited data are also available for the thermophilic fungus Chaetomium thermophilum complex [Drosophila or humans [Drosophila and human complexes. These are CNOT10 and CNOT11 (C2orf29). TNKS1BP1 (Tab182) also seems to be a member of the human complex [As with any large multi-subunit assembly of proteins considerable effort has been expended to determine the structure of Ccr4–Not. Up to the present, detailed structural information is available for the complex from four species:  complex ,25,26,27 complex . However complex ,14,15,16r humans ,18,29. T complex ,30  and a hinged region present in the centre ,24.The complex is assembled on the NOT1 backbone. How the other components associate has been determined using in vitro protein binding studies, crystallography, and electron microscopy. Binding sites for the other components of the complex on CNOT1 appear to consist of α-helical domains. A number of structural sub-complexes have been delineated-these comprise the deadenylase module , the NOT4 (Not4) the E3 ligase module and the ‘Not module’ . The binding sites of additional subunits have also been mapped-thus, metazoan CNOT10 and CNOT11 bind to the N-terminal domain of CNOT1 and CNOT9 (Caf40 in yeast) binds to the central region of CNOT1 ,20,25,31Drosophila proteins do not. However, it has been shown that the C-terminal region of human CNOT4 (Not4-C) is able to bind to the complex [CNOT4 (Not4) is an evolutionarily conserved E3 ubiquitin ligase ,29. It c complex . It has  complex . As well complex   subunit binds to a central CNOT1 coiled coil domain (termed CN9BD or DUF3819). This is adjacent to the CNOT1 MIF-4G (middle portion of eIF4G) region . The humThe deadenylase module associates with the central HEAT repeat-containing MIF-4G region of Not1. Thus, Caf1 (CNOT7) binds to Not1 through pre-existing structural motifs, made up of conserved hydrophobic residues. This allows full access for RNA molecules to the active site on Caf1 ,16. The Drosophila CNOT11 binds to the N-terminal region of CNOT1 and CNOT10 binds to it [One of the obvious differences between the yeast and metazoan CNOT complexes is the presence of CNOT10 and CNOT11 (C2orf29) in the latter complex. Human and ds to it . The areds to it . It shouds to it .Depletion of CNOT1 results in the destabilization of the whole complex and degradation of some other subunits such as CNOT2, CNOT6L, CNOT7, and CNOT9, but not CNOT3, in HeLa cells . AlthougWhat are considered to be the major constituents of the CNOT complex are described above. However, a number of other proteins have been routinely found to associate with the complex ,7,36. ItSchizosaccharomyces pombe [Interestingly, it has been shown that the Ccr4–Not complex strongly associates with the YTH domain of nucleus-specific RNA binding subunit, Mmi1 (meiotic mRNA interceptor 1), close to the nuclease module, in es pombe . In fisses pombe .Deadenylation-dependent mRNA decay plays an important role in eukaryotic fine-tuning of gene expression and this is achieved by monitoring mRNA translation and initiating degradation under appropriate circumstances ,53,54. MCaf1 and its human orthologues CNOT7 (hCaf1a) and CNOT8 (hCaf1b) belong to the ribonuclease D (RNase D) group of the DEDD superfamily ,57. AminThe EEP-type nuclease family member Ccr4 and its mammalian orthologues CNOT6 and CNOT6L are also largely associated with deadenylase activity. Ccr4 is characterized by the presence of two highly conserved domains: a carboxy-terminal endonuclease-exonuclease-phosphatase (EEP) region and an amino-terminal leucine rich region (LRR) ,62,63. YDeadenylase enzymes are characterized by their ability to promote rapid deadenylation in a 3′ → 5′ direction, resulting in the release of 5′-AMP. The yeast Ccr4–Not complex and the Pan2/Pan3 dimer function as two dominant deadenylase complexes, which are conserved in human cells ,66,67). It has recently been shown that Caf1 and Ccr4 have distinct activities in yeast. Caf1 degrades naked poly (A) sequences but is blocked by PABPC/Pab1. However, Ccr4 is activated by PABPC to trim the PABPC/Pab1-protected regions ,68. The mRNA decay occurs within cytoplasmic ribonucleoprotein (RNP) granules known as processing bodies (P-bodies). Cytoplasmic mRNAs cycle between polysomes and P-bodies. P-bodies are distinct foci formed by phase separation within the cytoplasm and composed of translationally repressed mRNAs and enzymes related to mRNA turnover, including decapping enzymes (DCP1–DCP2 complex and NUDT16), ATP-dependent RNA helicase Dhh1 (yeast)Rck/p54 (DDX6) , 5’-3’ exoribonuclease 1 (XRN1), the Lsm1–7 complex, Pan2/Pan3, the CNOT complex, along with the P-body component GW182 ,74,75,76Formation of P-bodies is induced in response to stress and it has been shown that the localization of Ccr4–Not components to P-bodies is limited to stressed yeast and metazoan cells but not unstressed cells when decay is ongoing ,78,79. IEvidence has been presented to show that components of the yeast Ccr4–Not complex play a role in decapping in P-bodies . Human CATPase activity of Dhh1 is required for the disassembly of P-bodies . Not1 frDifferent types of mRNA decay can be distinguished: generic mRNA deadenylation, nonsense mediated mRNA decay (NMD), targeted mRNA decay and deadenylation-independent decapping. The Ccr4–Not complex is involved in all of these.Generic mRNA decay occurs upon translation termination. It is not clear whether the CNOT complex is present during translation in an inactive form, and is then activated upon translation termination to promote shortening of the poly (A) tail, or is recruited upon translation termination to initiate deadenylation. However, one study has shown a series of events which couple mRNA decay to translation termination starting with the recruitment of translation termination eRF1-eRF3 factors and recruitment of deadenylase complexes Pan2/Pan3, and Ccr4–Not by the BTG/Tob family of proteins via their interaction with PABPC1 [h PABPC1 ,52,68.Nonsense mediated decay (NMD) is a translation-coupled mechanism that recognises and eliminates aberrant mRNAs, generally those containing a premature termination codon (PTC). Interaction between cap binding protein (CBP) and the Upf1 helicase activates the SMG1-mediated phosphorylation of Upf1 [ of Upf1 . Phospho of Upf1 ,89.Targeted mRNA decay is the best-established function of the Ccr4–Not complex and this plays a key role in post-transcriptional gene silencing by its targeted recruitment to the 3′ untranslated regions (UTRs) of mRNAs. Genetic evidence shows that RNA-binding proteins (RBPs) and microRNAs have the ability to tether the Ccr4–Not complex to specific sequences at the 3′ end of the mRNA, thereby promoting poly (A) shortening and repressing translation and/or mRNA turnover [turnover . CNOT1 mediates recruitment of the CNOT7/8 (Caf1/Pop2) and CNOT6/6L (Ccr4) deadenylases to the selected mRNA 3’UTRs through its interaction with specific RBPs thereby triggering degradation of target mRNAs ,91. For Several other examples of specific regulation of biological processes by Ccr4–Not-mediated deadenylation have been described. BTG/Tob adaptor proteins regulate mRNA decay through their interaction with CNOT7 and CNOT8 ,103,104.Drosophila [Drosophila Ccr4 deadenylase acts with two translational repressors, Nanos and Pumilio, to promote germline stem cell (GSC) self-renewal by repressing the expression of differentiation factors such as meiotic P26 (mei-P26) [Drosophila melanogaster oogenesis [The CNOT deadenylase complex is also involved in post-transcriptional control of gene expression during oogenesis and embryonic development in osophila ,112. Dromei-P26) . mei-P26ogenesis . The staogenesis .The miRNA repression complex, including GW182 (TNRC6) and Argonaute (AGO)-binding partners and effectors of miRISCs, has been shown to interact directly with CNOT1 as well as PAN3, leading to degradation of miRNA–target mRNAs ,116,117.Relevant to the role of Ccr4–Not in mRNA decay is the recent observation that the complex is physically associated with the ribosome in yeast . Recruit6-methyladenosine (m6A)-binding protein, has been shown to associate with the SH domain of the CNOT1 subunit and triggers m6A RNAs for degradation [YTHDF2, the Nradation ,119,120.It is important to remember when considering the effects of depletion or mutation of CNOT1, for example, that the deadenylase subunits are reliant on it for their activity. Thus, loss of the ability to reduce poly (A) tails may occur whilst the CNOT6, 6L, 7, and 8 components remain intact. Deadenlyation-independent decapping of certain mRNAs such as EDC1 mRNA, which encodes a protein that enhances decapping, is dependent on yeast Not2, Not4, and Not5 . ATP-depThe steady state level of protein expression is a function of the rate of transcription/translation on the one hand and the rate of protein degradation on the other. The rate of protein synthesis is determined by different factors: firstly, ribosomal protein biogenesis, which is controlled by the rate of ribosomal RNA (rRNA) synthesis; secondly, mRNA synthesis and, thirdly, mRNA deadenylation. Protein degradation is dependent on the ubiquitin-dependent proteasomal degradation pathway. The Ccr4–Not complex influences all these steps, to a greater or lesser extent.The production of ribosomes and ribosomal proteins is restricted by environmental nutrient levels. The Ccr4–Not complex, downstream of mTORC1, regulates the RNA Polymerase I (RNA Pol I)-dependent rRNA expression and couples nutrient signaling to the control of rRNA synthesis. Ccr4–Not localizes to rDNA and physically interacts with the RNA Pol I holoenzyme . Co-tranThe RNA Pol II subunits Rpb4p and Rpb7p form a heterodimer, interact with Nip1 and Hcr1, two components of translation initiation factor 3 (eIF3), and stimulate translation initiation . The intCNOT4/Not4-E3 ligase and its role in protein degradation: The ubiquitin (Ub)-proteasome pathway (UPP) of protein degradation regulates cellular protein turnover. Protein destruction requires an ATP-dependent process that involves three enzymes: an E1 (ubiquitin activating), an E2 (ubiquitin conjugating) and an E3 (ubiquitin ligating) enzyme. E1 first binds to an ubiquitin molecule and transfers it to E2. E2-ubiquitin interacts with the E3 and ubiquitin is transferred to the substrate. The ubiquitylated proteins are recognized by the 26S proteasome intracellular protease [protease ,129.CNOT4/Not4 is a RING finger E3 ligase and capable of auto-ubiquitylation ,131. TheA synthetic lethal screen first linked Not4 to the ubiquitin proteasomal pathway and, subsequently, genetic and biochemical studies confirmed that Not4 associates with the proteasome in vivo ,140. CelSaccharomyces cerevisiae . NAC is an important component of the proteostasis network that assists with the folding of nascent proteins and functions as a sensor for downstream protein folding conditions. Both Egd1p and Egd2p are ubiquitylated proteins, whose ubiquitylation status is regulated by Not4 [Association of Not4 with Ecm29, a proteasome chaperone, is important for the correct assembly of the proteasome, and co-translational quality control ,133,141. by Not4 . EGD ubi by Not4 ,136.Ribosomal protein Rps7A, but not its paralogue Rps7B, is ubiquitylated by the Not4 E3 ligase. Ub-Rps7A is only present in the large 80S ribosomal subunit and polysomes and not in the free 40S ribosomal fraction . AlthougThe Ccr4–Not complex regulates transcript buffering in yeast . It contThe Ccr4–Not subunits associate with transcription regulatory factors on the 5’ regions of mRNAs, such as TFIID, the SAGA (Spt–Ada–Gcn5 acetyltransferase) histone acetyltransferase complex, TATA binding protein associated factor (TAF), TATA-binding protein (TBP), SRB/Mediator complex and PAF (RNA polymerase II-associated factor) complexes both genetically and physically, reflecting a role for the Ccr4–Not complex in transcription initiation as well as in elongation ,153,154.Initially it was shown that four yeast Not proteins were involved in the global repression of transcription initiation ,155. HowSAGA is a chromatin-modifying complex that possesses two distinct enzymatic activities, acetylation of histones H3 and H2B via the Gcn5 histone acetyltransferase (HAT) and deubiquitylation of histone H2B via enzymatic activity of Ubp8 [ of Ubp8 ,162. Ind of Ubp8 . The yea of Ubp8 . In addi of Ubp8 . Ccr4–No of Ubp8 ,165,166. of Ubp8 .The role of Ccr4–Not and SAGA is illustrated by consideration of the yeast HIS3 promoter region that contains two functionally distinct TATA elements, constitutive (TC) and regulatory (TR). They can be distinguished by their interactions with upstream promoter elements, by their effects on the selection of initiation sites, and by their physical structure in nuclear chromatin . TC is lSAGA-TBP interaction, is principally provided by SAGA coactivator subunit Spt8, in conjunction with Spt3 . Spt3 muSRB/mediator that is associated with RNA polymerase II holoenzyme can act as both a positive and negative regulator of transcription [Saccharomyces cerevisiae [2O2-induced oxidative stress [The cription ,178. A drevisiae . An interevisiae . In resprevisiae . Not4 ube stress . Mutatioe stress .In mammals, the Not-Box motifs of the CNOT2 and CNOT9 subunits can directly repress the association of RNA Pol II with TATA-box promoter sequences ,183,184.c-Myc in MCF-7 human breast cancer cells [Different subunits of the Ccr4–Not complex exhibit variations in their effects on gene expression . Additioer cells ,183.c-Myc stimulates the universal amplification of gene expression by all three RNA polymerases [c-Myc mRNA has been reported following depletion of CNOT9 [c-Myc and Zfx [Overexpression of ymerases ,194. Howof CNOT9 . CNOT3 a and Zfx . The obsIn other studies, it has also been shown that the same subunit can have varying effects in different experimental systems. For example; in asymptomatic carriers of retinitis pigmentosa, a group of rare, genetic disorders that involve a breakdown and loss of cells in the retina, low level expression of CNOT3 leads to higher level of the wild-type splicing factor PRPF31 mRNA, confirming a repressive regulation by CNOT3 on PRPF31 gene expression in human cells ,196. A dThe positive role of the Ccr4–Not complex in transcription activity is supported by evidence showing that the Ccr4 protein is present in a large cluster of proteins involved in transcription elongation by RNA Pol II at promoter regions ,197. ThiThe interaction between Spt6, a histone chaperone, and RNA Pol II maintains chromatin structure during transcription elongation, thereby repressing transcription initiation by mediating the reassembly of nucleosomes onto the promoters ,200,201.Saccharomyces cerevisiae [Not4 appears to contribute to different aspects of transcription from initiation to elongation. It indirectly controls histone H3K4 tri-methylation, an active marker of transcription, by two mechanisms: firstly, by regulating the activity of the PAF1 complex (PAF1C), on the one hand, by facilitating the loading of PAF1C on to promoters and, on the other hand, by ubiquitin-dependent proteolysis of PAF1 not bound to chromatin through the 26S proteasome ,206. Setrevisiae . This rerevisiae . It seemrevisiae . Transcrrevisiae ,212,213)revisiae . The ribrevisiae ,215).Drosophila germline [The similarity between the phenotypes of Ccr4–NotΔ and Rpb4Δ mutants suggests that they overlap in their roles in controlling gene expression and decay rates. For example, they both show increased sensitivity to the transcription elongation inhibitor 6-azauracil, impaired transcription through long GC-rich regions, impaired transcription-coupled repair, and decreased mRNA turnover ,216,217.germline . Transcrgermline ,220,221.Transcription increases recombination rate by a phenomenon known as “transcription-associated recombination” (TAR) ,223. TARTranscription may affect DNA replication if co-transcriptional products such as R-loops (three-stranded DNA–RNA hybrid structures) are not efficiently removed behind the transcription machinery. The collision of replication and transcription complexes with R-loops threatens genome integrity by increasing the rate of mutagenesis, particularly over highly active promoters, such as those that occur in ribosomal genes (rDNA) ,230. GenTFIIS directly induces the resumption of elongation and helps RNA Pol II to pass through trapping sites by increasing the intrinsic nuclease activity of RNA Pol II and displacing transcripts . As discThe THO/TREX complex is able to suppress R-loop formation by binding to the transcript or assembling hnRNPs onto the RNA, thereby preventing RNA-DNA hybrid formation ,241,242.Transcription-coupled repair (TC-NER) nucleases XPG and XPF can excise R-loops that block transcription, leaving a ssDNA gap which can collapse into DNA DSBs . TC-NER Mutations and agents that affect transcription elongation or mRNA processing can be detected by the GLAM reporter system . Ccr4–NoWhile R-loops are a known source of genome instability they can also contribute to developmentally regulated processes, such as immunoglobulin (Ig) class switch recombination (CSR). CSR is needed for antibody maturation by an activated B cell. Interestingly, CNOT3 contributes to early B cell differentiation. In CNOT3-null mice, there is a block in the transition from pro-B (the phase where IgH class switch recombination occurs) to the pre-B stage (that express the pre-B cell receptor (pre BCR)) during the early B-cell development . In addiThe Ccr4–Not complex is strongly associated with the mRNA export system, which bridges the production of mRNA in the nucleus to its translation and degradation in the cytoplasm; in addition, the complex is important in nuclear RNA surveillance. This raises the possibility that Ccr4–Not may remain bound to the mRNA during nuclear export. The CNOT complex and BTG heterodimer have been reported to associate with, and regulate, the arginine methyltransferase PRMT1 in a substrate-dependent manner ,255,256.Schizosaccharomyces pombe, a nuclear isoform of the CNOT complex interacts with the RNA-binding protein Mmi1 and promotes the exosome-mediated elimination of meiotic transcripts during vegetative proliferation [Several nuclear exosome subunits and their co-factor TRAMP complex have been found to co-purify with the Ccr4–Not complex . The TRAferation ,270,271.As mentioned previously, the Ccr4–Not complex also interacts with Thoc5, a component of the human THO/TREX mRNP packaging complex ,147,218.The CNOT/Ccr4–Not complex affects cell cycle progression through several different mechanisms in both mammalian and yeast cells. Many proteins regulate the timing and frequency of DNA duplication and cell division during the cell cycle. A novel role for the Ccr4–Not protein complex in the regulation of mRNAs involved in cell cycle progression in yeast has been demonstrated . These aDrosophilamelanogaster oogenesis [Drosophila embryo, maternal cyclin B mRNA in the germline is directly repressed by binding of RNA-binding proteins Nanos2 and Pumilio to an element in its 3′ UTR. Nanos2 is responsible, at least in part, for recruiting the Ccr4-Pop2-Not deadenylase complex, interacting directly with the Not4 subunit [Xenopus oocytes, the shortening of cyclin B mRNA is directed and controlled by cytoplasmic polyadenylation element binding protein (CPEB), poly (A)-specific ribonuclease (PARN), and embryonic poly (A)-binding protein I (ePAB I) [Twin, the homologue of the yeast Ccr4 deadenylase, regulates the length of cyclin A poly(A) tails to permit ogenesis . In the  subunit . However(ePAB I) ,276. Mam(ePAB I) . During (ePAB I) ,280,281.(ePAB I) . As a re(ePAB I) .The transcriptional profile analysis of yeast cells containing mutant Not4 RRM-C (conserved RNA recognition motif and C3H1 domain) has shown a reduction in cyclins PCL9, PCL5 and M-phase cyclin CLB1  . Cyclin In an early study it was demonstrated that, during telophase/G1 phase transition, Dbf2, which is a cell cycle-regulated Ser/Thr protein kinase and a possible member of a larger Ccr4–Not complex, is involved in transcription of genes required for cell cycle progression . Dbf2p aCyclin G2 is an unconventional cyclin, highly expressed in post-mitotic cells. Unlike classical cyclins that promote cell cycle progression, increased cyclin G2 expression inhibits cell cycle entry and contributes to maintaining the quiescent state of terminally differentiated cells . The knoYeast G1-specific cyclins CLN1, CLN2, and CLN3  in complex with Cdc28 (Cdk1) are required for G1/S transition at the START point . In postSaccharomyces cerevisiae [Down-regulation of canonical histone proteins , which are responsible for packaging of newly synthesized DNA, disrupts cell cycle progression through S phase and beyond . It has revisiae . One rearevisiae . Another relevant point is that the 3′ UTR RNA binding proteins Puf5/Mpt5, involved in Ccr4–Not-mediated deadenylation in yeast, are recruited to the mRNAs of many chromatin regulators, including those of the two major histone deacetylases Hda1 and Rpd3 ,315,316.Methylation of histone H4 arginine 3 by PRMT1 is essential for the establishment and retention of both histones H3 and H4 acetylation patterns across the β-globin locus during erythroid differentiation . Human CFollowing irreversible DNA damage or other cellular triggers, cells either adapt to stress through a permanent cell cycle arrest (senescence) or are eliminated through programmed cell death (apoptosis) or the lysosomal degradation pathway (autophagy) ,319. TheOne of the most significant findings in modern cell biology was the observation that primary mammalian cells have a limited, very reproducible lifespan in culture under defined conditions ,321. AftCellular senescence is marked by increased DNA content and reduced histone synthesis, particularly in terms of the nucleosome histones H3 and H4 and the linker histone H1, as their expression predominantly occurs in S phase, ,324,325.In addition, portions of chromatin fragments are exported from the nucleus for autophagy/lysosomal degradation during senescence, which results in greater chromatin disorganization and disruption of chromatin–lamin interactions . Lamins It is now clear that there are a number of mechanisms by which cells can die . The priSaccharomyces cerevisiae lacking Ccr4 exhibit typical markers of apoptotic phenotypes such as enhanced cellular reactive oxygen species (ROS) levels, externalization of phosphatidyl serine, chromatin fragmentation, and increased caspase gene expression, suggesting that longer poly (A) tail length of the accumulated mRNAs in general contributes to enhanced cell death [Strains of ll death . The phenomenon of autophagy has been recognized for well over half a century. Its most obvious phenotype in mammalian cells is the presence of large vacuoles present in the cytoplasm of autophagic cells ,336. TheEvidence in support of the role of the Ccr4–Not complex in the regulation of autophagy in yeast first came from the observation that Caf1 was associated with Dhh1, a component of the P-bodies . A more The Ccr4–Not complex may function as an effector of the Ras/cAMP pathway that negatively controls the transcription from stress-regulated promoters, by preventing activation of transcription factor Msn2 ,341. MsnThe extracellular signal-regulated kinases (ERK1/2) signaling pathway regulates expression of autophagy and lysosomal genes ,345,346.c-Myc, where it inhibits their expression as discussed in The link between oestrogen receptor (ER) expression and autophagy has been reported in a number of different cancers ,353,354.Autophagy functions as a survival response for hypoxic cells through removal of damaged cellular compartments and recycling of components ,357. As S. pombe haploid deletion library for mutants sensitive to DNA damaging agents identified novel genes involved in DNA repair, in checkpoint response pathways as well as in the establishment and maintenance of chromatin architecture , transcription , and microtubule related processes (including the DASH complex) [Each cellular genome is subjected to many thousands of DNA lesions per day, arising from both endogenous and exogenous environmental sources. In order to maintain the integrity of the genome cells have evolved mechanisms known collectively as the DNA damage response (DDR). The DDR employs multiple protein pathways to monitor and repair various types of DNA damage . Screenicomplex) .Yeast repair genes, including Rad9, Rad52, Rad6, Rad27, and MUS81, have been shown to interconnect genetically and/or physically, in an ionizing radiation (IR)-mediated damage response network, with Ccr4 . IR-sensIn further studies Ccr4 and Chk1 have also been shown to act in the same epistatic pathway with respect to the response to HU-induced DNA replication stress . In mammCRT1, a DNA binding protein, is phosphorylated by the yeast Mec1-Rad53-Dun1 DNA damage sensor complex following HU-induced replication stress. After activation of CRT1, it recruits the general transcription repressors Ssn6 and Tup1 to the promoters of the HU-inducible genes: ribonuclease reductase 2 (RNR2), RNR3 and RNR4, subunits of the yeast RNR complex ,366. CRT6 -methylguanine-DNA methyltransferase), key enzymes in the alkyl transfer repair mechanism and in resistance to cisplatin-mediated apoptosis [Mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell proliferation, autophagy and apoptosis by participating in multiple signaling pathways . Reducedpoptosis . CNOT1 apoptosis ,374. Use of a CRISPR-Cas9-mediated knockout HAP1 cell line, with individual mutations in five Fanconi Anemia (FA) genes, has shown that CNOT1 was among those genes whose inactivation reduces the sensitivity to Mitomycin C (MMC)-mediated DNA damage . SimilarThe homeodomain-interacting protein kinases (HIPKs) enhance apoptotic functions in response to various stress signals, including DNA damage, reactive oxygen species, and hypoxia . CNOT2 aC. albicans revealed an interconnection with the early response to oxygen deprivation (hypoxia), suggesting a role for Ccr4 in low oxygen conditions. This response may be initiated by a drop in ATP generation [The Ccr4–Not complex is intimately involved in the cellular response to hypoxia. Transcriptional profiling analysis of a ΔCcr4 mutant in neration . The mitneration . Hypoxic2O2, for example [2O2-induced oxidative stress [Saccharomyces cerevisiae Yap1 is a transcriptional regulatory protein that activates the expression of genes required for oxidative stress tolerance [Saccharomyces cerevisiae protein Caf1β/P0P2  increases the mRNA level of the stress-regulated gene PGK1 5–10-fold compared to wild-type cells [Paradoxically, acute hypoxia induces production of reactive oxygen species (ROS), in the form of H example . Not4 ube stress . The cyce stress . Srb10 pe stress . Hypoxiae stress . CNOT4 ie stress . Saccharolerance ,385. Notolerance . Hypoxiaolerance . Changesolerance . Since 8olerance ,159,341.olerance . Directlolerance ,390. Conolerance . As we dolerance . Reductipe cells . Tab182 (TNKS1BP1), a member of a larger mammalian, but not yeast, CNOT complex and contributes to radiation-induced DNA double-strand break repair through facilitating the interaction between DNA-PKcs and PARP1. The PARP1-mediated PARylation of DNA-PKcs leads to activation of DNA-PKcs auto-phosphorylation during DNA DSB repair . MoreoveAs with any large multi-functional assembly of proteins mutations, deletions and aberrant protein expression have been linked to various human diseases-this is also the case for the CNOT complex . In somehttps://stuart.radboudumc.nl/metadome). Lesions in the CNOT1 gene have also been linked to other conditions. In recent detailed studies of patients with heterozygous missense, splice site and nonsense variants of CNOT1 it was found that they suffered from a range of neurodevelopmental phenotypes including intellectual disability, motor and speech delay, seizures, hypotonia, behavourial problems and holoprosencephaly [Drosophila model it was noted that the genetic interaction with autism-spectrum genes was impaired in the presence of mutated CNOT1 [As CNOT1 is essential for CNOT function it is not surprising that CNOT1 knock out mice die during development . Howeverncephaly ,399,400.ed CNOT1 . Drosophila was either lethal or resulted in dilated cardiomyopathy, reduced contractility, or a propensity for arrhythmia [In a genome-wide association screen, 35 common variant myocardial repolarization interval loci have been identified that collectively account for ~8–10% of QT variations observed . Some ofrhythmia ,402. Simrhythmia ,402. Therhythmia . Signifirhythmia . Similarrhythmia . A more rhythmia . In hearrhythmia .Drosophila melanogaster model [Several other diverse diseases have been associated with mutations in the CNOT3 gene. A link has been established between CNOT3 and susceptibility to ankylosing spondylitis . CNOT3 wer model . In a seer model . Intereser model . PRPF31 is an RNA binding protein and splicing factor, which forms part of the spliceosome. Mutations in the gene cause retinitis pigmentosa, a condition marked by progressive retinal degeneration. The CNOT3 gene is located very close to PRPF31. It has been shown that expression of CNOT3 is inversely proportional to PRPF31 expression . As CNOTThe deadenylase subunits have also been linked to susceptibility to cancers of various kinds. CNOT6L was shown to have concordant expression patterns with tumour suppressor PTEN and display significant copy number loss in prostate cancer and glioblastoma . Indeed,Links have also been established between the Ccr4–Not complex and adipose and liver function. Targeted loss of CNOT3 in adipose tissue of knock out mice results in a decrease in white adipose tissue and inflammation. The mice also exhibit hyperinsulinemia, hyperglycemia, insulin resistance, and glucose intolerance coupled with difficulties in the maintenance of body temperature . In otheDisruption of the complex in the liver leads to loss of metabolic homeostasis, at least partially through an increase in level of FG21, a major regulator of whole-body metabolism ,413,414.The Ccr4–Not complex was originally recognised as an important regulator of mRNA deadenylation and gene expression in yeast. However, over the past decade it has become clear that it functions at numerous levels and is of central importance in mammalian cells as well. This review has set out to survey these multiple modes of action, gathering recently published insights and well-established material. We have considered recent advances in understanding the potential roles of the CNOT complex separately in cell cycle regulation, transcription-dependent replication stress, replication stress response, DNA damage repair, mRNA transport, and the control of steady state level of protein expression. For example, in 2001, Parker and colleagues hypothesized that certain mRNAs are marked for degradation in the nucleus during transcription. This suggestion was based on the premise that the CNOT complex could regulate both transcription and deadenylation. By 2018, Dronamraju and colleagues were able to confirm that the CNOT complex is recruited to the site of transcription in order to facilitate the degradation of a range of mRNAs, including mRNAs required for cell-cycle progression . SimilarOur knowledge of the mode of action of the CNOT complex has been greatly enhanced by a number of structural and interaction studies, which have helped to demonstrate the contribution of each subunit, although the roles of some are still clearer than others. For example, it appears that the regulation of deadenylation and gene expression in yeast requires less components than are present in mammalian cells. Furthermore, two components, CNOT10 and CNOT11, absent from the yeast complex, stimulate deadenylation through stabilization of the RNA substrate. However, it seems likely that they may have other as yet unknown functions or, perhaps, provide additional subtlety of control of deadenylation required in metazoan cells. Future investigations should shed more light on the precise roles of CNOT10 and 11.Over the past decade, interest in the mechanism of the formation and resolution of R-loops has increased markedly. There is considerable evidence to suggest a role for the Ccr4–Not complex in the process. We suggest that the complex functions at a number of points in the cell’s response to R-loops and it is hoped that this possibility will be clarified in the near future.Although the constitution of the complex is fairly well understood in yeast and in humans it is possible that some of the well-defined binding partners might be considered Ccr4–Not complex components. For example, in fission yeast Mmi1 is very strongly associated with the complex. Mmi1 is essential for viability and represses the expression of transcripts required for entry into meiosis during normal vegetative growth; it seems, therefore, that the interaction between the complex and Mmi1 defines a function specific to fission yeast. It is possible that other binding proteins/possible components could also define specific functions. For example, Tab182 (TNKS1BP1) has been closely associated with the mammalian complex (it has been suggested to be denoted as CNOT12) although its functions are largely unresolved. All these points will have to be addressed before we have a full understanding of the CNOT complex.Deletions and mutations in CNOT components have been associated with various clinical conditions. Because of the multiplicity of functions of the complex, it is not surprising that the loss of CNOT genes is lethal in embryonic mice. However, mutations in CNOT1 and CNOT3 have both been shown to be relatively common in certain ALLs and to be associated with cardiac functioning. Based on Kaplan–Meier plots, it has been observed that variations in the level of expression of CNOT components is associated with enhanced or reduced survival in different cancer types. For example, a high level of expression of CNOT7 is associated with appreciably enhanced survival in lung and gastric cancers, although it has little effect in breast cancer. On the other hand, low-level expression of CNOT3 is associated with enhanced survival in ovarian and gastric cancers. Exactly what role the complex plays in these cases is not yet understood.


We identified 86 differentially expressed transcription factors that are uniquely enriched, or uniquely depleted, in a specific neurotransmitter type. Some transcription factors show a similar profile across development, others only show enrichment or depletion at specific developmental stages. Profiling of Acj6 (cholinergic enriched) and Ets65A (cholinergic depleted) binding sites in vivo reveals that they both directly bind the ChAT locus, in addition to a wide spectrum of other key neuronal differentiation genes. We also show that cholinergic enriched transcription factors are expressed in mostly non-overlapping populations in the adult brain, implying the absence of combinatorial regulation of neurotransmitter fate in this context. Furthermore, our data underlines that, similar to Caenorhabditis elegans, there are no simple transcription factor codes for neurotransmitter type specification.The remarkable diversity of neurons in the nervous system is generated during development, when properties such as cell morphology, receptor profiles and neurotransmitter identities are specified. In order to gain a greater understanding of neurotransmitter specification we profiled the transcription state of cholinergic, GABAergic and glutamatergic neurons This article has an associated First Person interview with the first author of the paper. Summary: Transcriptome profiling of cholinergic, GABAergic and glutamatergic neurons in Drosophila identified multiple transcription factors as potential regulators of neurotransmitter fate. The human brain is perhaps the most complex system known to mankind. It consists of approximately 85 billion neurons , which pCaenorhabditis elegans, mice and Drosophila have provided a wealth of information about factors and mechanisms involved in neurotransmitter specification. Comprehensive neurotransmitter maps  and Etv1 (vertebrates) to a phylogenetically conserved DNA motif to specify dopaminergic fate (acj6 (Drosophila), unc-86 (C. elegans) and Brn3A/POU4F1 (vertebrates) all have roles in cholinergic specification (PITX2 (vertebrates) and unc-30 (C. elegans) both control GABAergic differentiation  and Ets65A (cholinergic depleted) both directly bind the choline acetyltransferase gene (ChAT) required for cholinergic fate.In order to identify novel candidate genes, and investigate the dynamics of neurotransmitter specific transcription factors throughout development, we have performed cell specific profiling of RNA polymerase II occupancy, ChAT (choline acetyltransferase), Gad1 (glutamic acid decarboxylase 1) and VGlut (vesicular glutamate transporter) . To investigate the global differences in Pol II occupancy between neuronal types and developmental stages, we generated a correlation matrix (Table S2). Encouragingly, a strong enrichment of Pol II occupancy is evident at ChAT, Gad1 and VGlut, the genes encoding the key enzymes involved in the biosynthesis of these neurotransmitters . Transcription factors and non-coding RNAs make up a large proportion of all the enriched genes, at each developmental stage (beaten path (beat) and Down syndrome cell adhesion molecule (Dscam) genes, which play roles in axon guidance and dendrite self-avoidance , five for GABAergic  and two for glutamatergic (VGlut and oc) (Table S2). There is consistent enrichment of the GAL4-trapped genes   that provide type specific expression for the TaDa experiments.In order to investigate which genes participate in the specification of neuronal properties, namely, neurotransmitter choice, we applied the cell specific profiling technique Targeted DamID (TaDa). TaDa is based on DamID  and allosporter) . During rons see B. Windown matrix A. We foun matrix B. For eaal stage C. In thevoidance . Glutamajunction . Interesjunction C. Also, er types C. Very fFig. S2A)  . This shows that across the population of neurons for each neurotransmitter type, major changes in accessibility are limited to genes involved in the respective neurotransmitter synthesis, with none of open regions directly corresponding to transcription factor loci. Differential accessibility is also present at sites outside of the gene and promoter for Gad1 and VGlut . Weaker differences in accessibility are also observed at some of the differentially expressed transcription factor loci .CATaDa, an adaption of TaDa, allows profiling of chromatin accessibility without the need for cell isolation using an untethered Dam protein (i.e. the control experiment in TaDa) (ig. S2A) . CATaDa ig. S2A) A,B but sig. S2A) A,B. WhenTranscription factors play the major role in neurotransmitter specification and we have identified many with enriched Pol II occupancy in specific neurotransmitter types A. Uniquekn, peb, rib and ss are cholinergic enriched in embryo and larva, but not in adults. Dll is an unusual case, as it is cholinergic enriched in larvae, however, switches to being GABAergic enriched in adults .To investigate the expression pattern dynamics of both uniquely enriched and uniquely depleted transcription factors, we examined how their expression patterns transitioned across the stages of development . We obsen adults ; Fig. S3acj6 , Dbx (GABAergic – see oc (glutamatergic), which are enriched in their respective neurotransmitter type throughout all stages. In support of our data, Acj6 is known to promote cholinergic fate in the peripheral nervous system . This agrees with the low-level signal observed in RNA Pol II occupancy plots for acj6 gene in third instar larva glutamatergic neurons .Exceptions to this are s system  and Dbx s system . We checmic data B, we onlmic data C. We obsapterous (absent in GABAergic), and the longer transcript isoforms of Ets65A (absent in cholinergic) , as predicted by the transcriptomic data . As for the longer transcripts of Ets65A-RA/C/D/E in larval neurons, we did identify their presence in a small number of cholinergic neurons , which could reflect the very low signal in the RNA Pol II occupancy plot (within the unique region of the long transcripts) .Candidate repressors of neurotransmitter fate (uniquely depleted transcription factors) also demonstrate dynamic changes in expression pattern across development . Promineinergic) D,F. We ut brains E,G. In aDbx, en, collier and CG4328) and candidate repressor transcription factors  during embryonic development, and larval stages did not result in any obvious changes in neurotransmitter expression patterns .We have identified transcription factors with potentially novel roles in regulating neurotransmitter identity. Therefore, we investigated candidate activators and candidate repressors for their potential to elicit pan-neural reprogramming of neurotransmitter identity. Pan-neural expression and RNAi knockdown of candidate activator transcription factors , GABAergic enriched Ptx1 (PITX1 and unc-30) and glutamatergic enriched oc (OTX1/2 and ttx-1) have all shown to have a role in promoting cholinergic, GABAergic and glutamatergic fate, respectively. However, there are many that have not been investigated in this context (38%), or that are only supported by indirect evidence (38%). These include Dll , sox21a , hbn  and otp . Given the strong conservation of neurotransmitter specification mechanisms, many of these newly highlighted factors provide promising research avenues for expanding our knowledge in this field.Focusing on candidate transcription factors demonstrating binary differences (clear on and off), we performed literature searches to examine whether they have been previously shown, or implicated in regulating neurotransmitter identity . This inFig. S8A). These include non-characterised lncRNAs and GABAergic enriched iab8, which is located in the Hox cluster and plays a role in the repression of abd-A (mir-87 (cholinergic), mir-184 (GABAergic) and mir-190 (glutamatergic), which are enriched during the developing states but not in the adult. Although annotated separately, mir-184 is embedded in CR44206 .While non-coding ribosomal RNAs and tRNAs are transcribed by RNA polymerase I and III, micro RNAs (miRNAs) and long non-coding RNAs (lncRNAs) are primarily transcribed by Pol II. Our Dam-Pol II data identifies a set of differentially bound miRNAs and lncRNAs, between the neurotransmitter types . They co-bind 926 genes, which are highly enriched for nervous system genes, including genes involved in axon development [GO:0061564] and chemical synaptic transmission [GO:0007268] (Acetylcholine esterase (Ace) gene (nicotinic Acetylcholine Receptor α4 (nAChRα4)   (FDR<0.0001) (0007268] B. While ce) gene C, Acj6 unAChRα4) D, and Etnd slpr) E. TherefThere are multiple transcription factors that show enriched expression in adult cholinergic neurons . To inveDrosophila. We profiled the transcription state of cholinergic, GABAergic and glutamatergic neurons in the developing and adult brain of Drosophila. We observe enriched Pol II occupancy at the relevant neurotransmitter synthesis genes  and other genes associated with the activity of the specific types (Table S2). The monoamine neurotransmitter related genes Vmat, DAT and Tdc2 are enriched in glutamatergic neurons  (Dbx (GABAergic) , is a terminal selector for cholinergic motor neuron differentiation  predominantly generate GABAergic interneurons (ARX (hbn orthologue) causes remodelling and increased activity of glutamatergic neurons in vertebrates   and Dbx BAergic) . Also, sntiation . In addir type 3 . Howeverrneurons , and a ptebrates . Acj6 is neurons  and Dbx  neurons . To the  elegans , there aFig. S7). No obvious changes were observed, however, this is not particularly surprising considering the importance of cellular context for the reprogramming of neuronal properties  genes (required for the synthesis of GABA), or indirectly, through another transcription factor, is unclear. We have identified several transcription factors that are expressed in two neurotransmitter types, but absent from the other. These include apterous (ap), Ets65A (long transcripts) and orthopedia (otp), which we hypothesise to be candidate repressors, given their absence from cells with a specific neurotransmitter identity. Our profiling of Ets65A-PA binding in vivo, reveals that it directly binds ChAT , however, again, this might be because they can only act as a repressor in specific contexts (e.g. when a co-repressor is present), or that they regulate genes associated with specific types but do not directly regulate neurotransmitter identity.The majority of transcription factors identified as directly regulating neurotransmitter fate act in a positive manner, whereas only a handful of studies describe the role of repressors. Incoherent feedforward loops exist in gic fate . Whethernds ChAT A, and thDrosophila tissues, including the whole adult brain (The development of single cell RNA-seq (scRNA-seq) technology has led to the profiling of several lt brain , the cenlt brain  and the lt brain . Here welt brain  to compalt brain A. Furthelt brain B. This ilt brain . While blt brain . It is clt brain , which mFig. S2B). The enriched accessibility is not just restricted to the gene bodies of the neurotransmitter genes, and peaks are present upstream (Gad1) and downstream (VGlut) , which are likely enhancers. Accessibility at the ChAT gene is clearly higher in cholinergic neurons at the embryonic and adult stages, however, in third instar larvae, the difference is less pronounced . This could reflect increased plasticity at this stage, possibly linked to the dramatic remodelling of larval neurons during metamorphosis . This could be due to transcription factors being expressed at relatively lower levels and/or that they are only expressed in a subset of the cells, therefore the difference is less prominent.Apart from the neurotransmitter synthesis genes, the chromatin accessibility of the different neuronal types, at a given stage, is surprisingly similar, as demonstrated in embryos , which is intriguing, as mir-184 has been shown to downregulate GABRA3 (GABA-A receptor) mRNA (possibly indirectly) in vertebrate cell lines , and when mutated causes larval locomotion defects in Drosophila  of genes governing neuronal identity. MAP kinase signalling genes are enriched in cholinergic neurons . w;; ChAT-GAL4 [MI04508] / TM3 act GFP, (Bloomington #60317). w;; Gad1-GAL4 [MI09277] / TM3 actin GFP . w[*]; Mi{Trojan-lexA:QFAD.0}ChAT[MI04508-TlexA:QFAD.0]/TM6B, Tb[1], (Bloomington #60319). w[*]; Mi{Trojan-lexA:QFAD.2}Gad1[MI09277-TlexA:QFAD.2]/TM6B, Tb[1],  .ctin GFP . w[*]; Mts; UAS-LT3-NDam-RNA Pol IIUAS-LT3-NDam, tub-GAL80 (from Andrea Brand). Ets65A-RA/C/D/E-GAL4 [MI07721] (this study). apterous-GAL4; UAS-GFP (from F Jiménez Díaz-Benjumea). acj6-GAL4-UAS-mCD8-GFP/FM7c; Pin/CyO (from DJ Luginbuhl) . elavG4;; Mi{PT-GFSTF.0}ChAT [MI04508]/TM3 actin-GFP .ginbuhl) . elavG4;UAS-Dbx . UAS-apterous (from F Jiménez Díaz-Benjumea), UAS-collier (from F Jiménez Díaz-Benjumea), UAS-engrailed [E9] (from Andrea Brand). UAS-otp (Fly ORF #F000016). UAS-CG4328 . UAS-Dbx sh RNAi attP40 (VDRC #330536). UAS-ap sh RNAi attP40 (VDRC #330463). UAS-Ets65A-RA RNAi attP2 (Bloomington #41682). UAS-Ets65A-RA attP2 (this study). yw, hs-Flp 1; +; Dr/TM6B. yw, hs-Flp 1; +; UAS-Ets65A-RA. AyGal4, UAS-mCD8-GFP/(CyO); Cha lexAQF, mCherry /TM6B.Supplemental Material. pUAST-LT3-NDam-acj6-RF and pUAST-LT3-NDam-Ets65A-RA were generated by PCR amplifying acj6-RF and Ets65A-RA from an embryonic cDNA library. The resulting PCR products were cloned into pUAST-LT3-Dam plasmid  using the triplet donor in vivo system described in  for 20 h. After this time, first instar larvae were collected and stored in 1× PBS. Samples were flash-frozen in dry ice, and stored at −80°C until the appropriate amount of tissue was enough to start the experiment. No selection for the right genotype was done, and 20 µl worth of volume of tissue was used as a proxy to determine the appropriate amount of material for each replicate. Three replicates were done for each experiment. With this husbandry protocol, the collected first instar larvae were around 12 h after larvae hatching (ALH), just before the first larval neurons are being generated, then providing the transcriptome of embryonic neurogenesis.Crosses of the right genotypes were allowed to lay eggs for 6 h at 25°C in fly food vials. These vials were then transferred to 18°C (restrictive temperature) for 7 days. They were then moved to 29°C (permissive temperature) for 24 h. Wandering stage larvae, around 96 h ALH, were selected with a GFP scope for the right genotype. Larvae were dissected in 1× PBS, leaving the anterior half of the larvae partly dissected, containing the CNS, but removing the gut and all the fat tissue. Samples were flash-frozen in dry ice, and stored at −80°C until the appropriate amount of tissue was enough to start the experiment. 100 partly dissected CNS were used for each replicate. Three replicates were done for each experiment.Crosses of the right genotypes were allowed to lay eggs for 2 days at 18°C in fly food vials. Vials containing those eggs were kept at 18°C (restrictive temperature) until adult flies eclosed. They were then kept at 18°C for 5−10 days. After that, they were selected according for the right genotype, and transferred to 29°C (permissive temperature) for 24 h. Then, adult flies were flash-frozen in dry ice, and stored at −80°C. Around 50 fly heads were used for each replicate. Three replicates were done for each experiment.When preparing the tissue to be used, neither larvae nor flies were sex sorted. It has been recently reported that transcriptomes from males and females, obtained with a cell specific driver combination expressed in neurons in the adult optic lobe, do not present major differences in their transcriptomes. Only a small number of genes, known sex-specific genes showed differences between sexes  preprintOur DamID protocol was based on Libraries were sequenced using Illumina HiSeq single-end 50 bp sequencing. Three replicates were performed for each experiment. A minimum of 25 million reads were obtained from the first instar larvae samples, 30 million reads from the third instar larvae, and 9 million reads from the adults' samples.Crosses producing larvae with the following genotypes were allowed to lay eggs over a minimum of 2 days at 25°C:ts/+; UAS-LT3-NDam/+acj6-GAL4-UAS-GFP; tub-GAL80. ts/+; UAS-LT3-NDam-acj6-RF/+acj6-GAL4-UAS-GFP; tub-GAL80. ts/+; UAS-LT3-NDam/ Ets65A-RA-GAL4MI07721tub-GAL80. ts/+; UAS-LT3-NDam-Ets65A-RA/ Ets65A-RA-GAL4MI07721tub-GAL80.Crosses of the right genotypes were allowed to lay eggs for 2 days at 18°C in fly food vials. Vials containing those eggs were kept at 18°C (restrictive temperature) until adult flies eclosed. They were then kept at 18°C for around 10 days. Then, they were transferred to 29°C (permissive temperature) for 24 h, selected according for the right genotype, flash-frozen in dry ice, and stored at −80°C. A minimum of 150 fly heads were used for each replicate. Two replicates were done for each experiment.The DamID protocol used for these samples is the same as described above, with minor changes, 6 µg of DNA were precipitated, Bioline Polymerase was used in the PCR amplification, and only AlwI was used to remove adaptors from sonicated DNA. Libraries were sequenced using Illumina HiSeq single-end 50 bp sequencing. Two replicates were acquired for each experiment. A minimum of 10 million reads were obtained from these samples.Drosophila genome using a previously described pipeline . Drosophila genome annotation release 6.11 was used, with 1% FDR and 0.2 log2 ratio thresholds. To compare data sets, log2 ratios were subtracted, in this case, producing three replicate comparison files . These data were then analysed as described above to identify genes with significantly different Pol II occupancy. Due to the presence of negative log2 ratios in DamID experiments, these genes were filtered to check that any significantly enriched genes were also bound by Pol II in the experiment of interest (numerator data set). A gene list was generated from the transcript data using the values from the associated transcript with the most significant FDR. Correlation values (https://software.broadinstitute.org/morpheus/). The transition plot (http://gforge.se/). Enrichment GO analysis was performed using the R package clusterProfiler . In brief, a FDR was calculated for peaks (formed of two or more consecutive GATC fragments) for the individual replicates. Then each potential peak in the data was assigned a FDR. Any peaks with less than a 0.01% FDR were classified as significant. Significant peaks present in all replicates were used to form a final peak file. Any gene (genome release 6.11) within 5 kb of a peak (with no other genes in between) was identified as a potentially regulated gene.For acj6 and Ets65A-RA TaDa, peaks were called and mapped to genes using a custom Perl program ;open my $fh, ‘<’, $file or die $!; while(<$fh>){@col = split; if($genenum == $col[0])               {print OUTPUT “$col[0]\t$col[1]\t$col[2]\n”;}} exit;Cells with a transcript count (for the given gene) of less than three were excluded for further analysis.Third instar larval CNS or adult brains were dissected in 1x PBS. They were fixed in 4% formaldehyde (methanol free) 0.1% Triton X-100 PBS (PBST), for 30 min at room temperature. Samples were then rinsed twice with 0.1% PBST, and washed four times for 1 h with 0.1% PBST. 5% normal goat serum in 0.1% PBST was used as a blocking agent for 1 h at room temperature. Brains were then incubated overnight at 4°C with primary antibodies in 5% normal goat serum in 0.1% PBST. The primary antibodies used were: anti-chicken-GFP , and anti-rabbit-DsRed . Brains were rinsed twice with 0.1% PBST, and washed four times with 0.1% PBST for 1 h. Secondary antibodies were diluted in 5% normal goat serum in 0.1% PBST and incubated with the brains for 1 h at room temperature. The secondary antibodies used were: anti-chicken-Alexa 488 , and anti-rabbit-Alexa 546 . Samples were then rinsed twice with 0.1% PBST, and washed four times for 1 h. Brains were mounted on glass cover slides in Vectashield (Vector laboratories). All incubations and washes were performed in a rotator. After dissection of first instar larvae CNS, they were placed in a polylisine coated microscope slide, where we performed all the incubations. Both experimental CNS and wild-type CNS were placed on the same slide. For all the immunostaining experiments, a minimum of five brains were dissected and visualised. Images were acquired using a Zeiss LSM 510 confocal microscope and edited using Fiji/ImageJ.


A crucial feature of gene expression involves RNA processing to produce 3′ ends through a process termed 3′ end cleavage and polyadenylation (CPA). This ensures the nascent RNA molecule can exit the nucleus and be translated to ultimately give rise to a protein which can execute a function. Further, alternative polyadenylation (APA) can produce distinct transcript isoforms, profoundly expanding the complexity of the transcriptome. CPA is carried out by multi-component protein complexes interacting with multiple RNA motifs and is tightly coupled to transcription, other steps of RNA processing, and even epigenetic modifications. CPA and APA contribute to the maintenance of a multitude of diverse physiological processes. It is therefore not surprising that disruptions of CPA and APA can lead to devastating disorders. Here, we review potential CPA and APA mechanisms involving both loss and gain of function that can have tremendous impacts on health and disease. Ultimately we highlight the emerging diagnostic and therapeutic potential CPA and APA offer. In multicellular organisms almost every cell contains the same genome, yet complex spatial and temporal diversity is observed in gene transcripts. This is achieved through multiple levels of processing leading from gene to protein, of which RNA processing is an essential stage . FollowiCPA is carried out by a multi-subunit 3′ end processing complex, which involves over 80 trans-acting proteins , comprisCPA is initiated by this complex recognising specific cis-element sequences within the nascent pre-mRNA transcripts termed polyadenylation signals (PAS) . The PASAs soon as the nascent RNA molecule emerges from RNA polymerase II (RNA Pol II), the CPSF complex is recruited  to the PThe proper formation of the poly A tail in conjunction with the 3′ untranslated regions (3′UTR) in the transcript directs stability, nuclear export, sub-cellular localisation and translation efficiency via cis-acting elements interacting with trans-acting factors, such as microRNAs (miRNA) or RNA-binding proteins (RBP) in given cellular contexts ,39,40,41The intricate, bidirectional coupling of 3′ end formation to transcription and other co-transcriptional events, such as capping and splicing , ensuresAlternative polyadenylation (APA) occurs when more than one PAS is present within a pre-mRNA and provides an additional level of complexity in CPA-mediated RNA processing B. Early Approximately 80% of all APA events occur in the 3′UTR , resultiThe intrinsic strength of a PAS, as regulated by its affinity to CPA factors, determines PAS choice and subsequently APA. This can be modulated by additional elements, for example G-rich DSEs can enhance CPA at a particular PAS ,67. In aAs described previously, CPA and APA are tightly coupled to transcriptional processes involved in the production of a mature RNA, such as capping and splicing ,71. AddiAdditionally, the abundance of individual 3′ end processing factors can affect APA at the global level C. CFI haFinally, transcripts exhibiting dynamic regulation at the 3′ end are typically encoded by phylogenetically ancient genes, which corresponds to the phylogenetic age of most executing APA regulators . Such phRecent studies in human pathologies have shown genetic variants affecting RNA processing are as frequent, and largely independent from, variants affecting transcription . In diseVariations from the canonical PAS hexamer (AATAAA) generally reduce CPA efficiency ,60, althMutations within the sole PAS of genes were the first alterations in CPA demonstrated to result in disease. In thalassemia patients, initial studies identified a non-deletion mutation with homozygotes suffering from moderate to severe α-thalassemia  and resuBoth HBA2 and HBB utilise a single canonical PAS and as such mutations producing the much less efficiently processed AATAAG ,184 and +CD25+ T regulatory cells [Other PAS alterations affecting a sole canonical hexamer have since been identified , Table 1ry cells . Where ary cells . This hary cells .Variant hexamers are found in 30% of genes possessing a single PAS . AlteratThe above section described alterations in the hexamer of genes possessing a single PAS resulting in impaired CPA and reduced gene expression. However for the vast majority of genes that harbour multiple PAS alterations, causing reduced efficiency of one PAS can result in increased use of other competing PASs, ultimately resulting in APA. This can have varying outcomes depending on the relative positions of the affected PAS and other elements within the alternative 3′UTRs produced B. This iInterestingly, as opposed to the transcript lengthening observed when polymorphisms or mutations in proximal PAS hexamers reduce CPA efficiency, alterations in distal PAS hexamers predominately result in 3′UTR shortening. An example of this is seen with the serotonin transporter gene, solute carrier family 6 member 4 (SLC6A4), which possesses two 3′UTR PASs ,246, witAlbeit far less commonly reported, mutations resulting in the de novo creation of cryptic PAS have been identified , Table 1Finally, gain of function variations can arise from altered RNA editing of transcripts. In the neurological disorder amyotrophic lateral sclerosis (ALS), increased RNA editing within an intron of the solute carrier family 1 member 2 (SLC1A2) glutamate transporter creates a cryptic PAS resulting in intron retention with termination of transcription transcripts . ReducedAlthough other CPA sequences are considered to be much more tolerant to alterations than the PAS hexamer , alterat+ and K+ cotransporter ATPase Na+/K+ transporting subunit beta 1 (ATP1B1) undergoes APA due to the use of multiple PASs with the 3′UTR [Alterations in DSE have been associated with disease. The gamma chain of human fibrinogen (FGG) exists in 2 isoforms, FGG-γA formed by all 10 exons, and FGG-γ’ formed by the use of a PAS in intron 9 resulting in a truncated protein consisting of exons 1–9 and the first 60 nucleotides of intron 9 ,263. A rhe 3′UTR ,265, withe 3′UTR ,267,268.he 3′UTR . Variatihe 3′UTR .While alterations in established CPA elements have clear functional outcomes, changes in less defined regions of 3′UTRs affecting CPA have been reported. A SNP (ss52051869) in the 3′UTR of the arginine transporter solute carrier family 7 member 1 (SLC7A1) is involved in predisposition to essential hypertension and has been associated with altered protein expression . SLC7A1 A similar occurrence is found in Parkinson’s disease (PD). The brains of PD patients contain cytoplasmic protein aggregates largely composed of α-synuclein (SNCA) . SNCA mRIn addition, larger 3′UTR lesions have also been reported to affect CPA. This is exemplified in Lynch syndrome, which confers increased risks of multiple cancers and results from mutations in DNA mismatch repair genes, including mutS homolog 6 (MSH6) . A 20 bpApproximately 20% of all APA events fall in the coding region, thereby resulting in proteins with distinct regulatory or functional properties  Figure B. Along Finally, seeming unrelated alterations can have effects on CPA. The fragile X mental retardation 1 gene (FMR1) produces multiple APA transcript isoforms in Fragile X syndrome (FXS) and related diseases, such as fragile X-associated immature ovarian insufficiency ,278 and The presence of 3′UTR alterations that functionally affect CPA/APA outside established CPA elements indicates a lack of knowledge of how 3′UTR sequences are involved in regulating CPA and APA. As 3.7% of the genetic variants detected in GWAS studies are localised within UTRs ,285 and As described in the preceding sections, many alterations in cis, both at the DNA and RNA level, can result in aberrant CPA and ultimately to disease by affecting individual genes. However, as can be seen in the following sections, global alterations affecting CPA and APA in trans are a significant feature of many diseases.APA is modulated in various normal physiological circumstances ,13,289. APA shortening  in proliferating cells has been shown to be accompanied by an increased expression of polyadenylation factors ,296,297,This has led to a model where APA shortening of 3′UTR of oncogenes in cancer cells allows evasion of the repressive effects of microRNA and RNA binding proteins ,298,299.The CFI complex is well characterised in the regulation of APA. It has a particularly strong impact on PAS choice through preferential binding to the distal PAS, enhancing its use. For distal non-canonical PASs, the CFI complex can facilitate PAS usage through NUDT21 binding to USE UGUA motifs and stabilise the binding of CPSF complex to the pre-mRNA ,303,304.In glioblastoma patients, NUDT21 expression is reduced, with lower levels being associated with shortened 3′UTRs and worse survival ,117. SupHowever, elevated NUDT21 expression is also associated with disease. In individuals with neuropsychiatric disease, copy-number variations producing elevated NUDT21 levels have been reported to result in the increased usage of the distal PAS and reduced expression of ethyl-CpG binding protein 2 (MECP2), a gene closely associated with neuropsychiatric disease . In chroAltogether this demonstrates an important role of NUDT21 in various disease entities. This reflects its pervasive role in APA regulation  Figure C.Downstream elements (DSE) assist in the selection of non-canonical PASs ,228. CstCSTF2 up-regulation is associated with several cancers. In an examination of seven tumour types, CSTF2 was found to be up-regulated in five  and to exhibit a significant correlation between expression and 3′UTR shortening . AdditioOther CstF components have also been shown to be dysregulated in cancer. In B-cell leukemia/lymphoma samples CSTF3 protein is expressed at significantly higher levels in tumours is associated with 3′UTR shortening . In contFinally, alterations in CstF have been observed in other disorders. In a transverse aortic constriction mouse model of cardiac hypertrophy 3′UTRs were found to be generally shortened and the expression of CstF components CSTF1, CSTF2, and CSTF3 were found to be up regulated . Thus, aAlthough CFII is the least characterised among the CPA complexes, and interacts only weakly and/or transiently with the CPA complex , it contPCF11 is a sub-stoichiometric component of CPA, with levels an order of magnitude lower than other CPA components . It contIn an extensive screen of potential CPA effectors in neuroblastoma cells C PCF11 eWhile the tumour phenotype described above is driven by the pervasive functional impact of PCF11 on APA, there are functionally more complex pathomechanisms illustrating the intricate molecular nature of the CPA machinery. PCF11 also appears to be involved in APA shortening in cancer. Aberrant expression of the ubiquitin ligase adapter MAGE family member A11 (MAGE-A11) in tumours, including prostate ,320 and Finally, outside of cancer, loss of CLP1 results in accumulation of tRNA fragments, which are thought to provoke neurodegenerative disorders ,324.CPSF recognises the PAS hexamer ,19,20 wiInterestingly, although CPSF consists of a large number of factors, each presumably open to mutation, reports of alterations in this complex in disease are lacking. Although reasons for this are currently not clear, potential mechanisms such as functional redundancy within the complex may account for this observation.Polyadenylation of nascent RNA by canonical (PAPα and PAPγ) and non-canonical  or Star-PAP) poly(A) polymerases plays a key role in PAS selection and impacts APA genome-wide . For exaStar-PAP has been shown to affect APA in cardiac hypertrophy through an association with the RNA binding protein RNA binding motif protein 10 (RBM10), which stimulates Star-PAP polyadenylation activity . Down-reAdditionally, poly(A) binding proteins are associated with disease. While initially believed to be important for only the polyadenylation step, PABPN1 has been shown to also play a role in the regulation of PAS choice ,330. PABOculopharyngeal muscular dystrophy (OPMD) is caused by expansion of a triplet repeat in the PABPN1 gene  resultinIn colorectal cancer patients, 3′UTR shortening has been observed to correlate with disease stage ,110. An Regulation at the 3′UTR is an important mechanism in heart function and further conditions, including cardiac hypertrophy and heart failure . In dilaThe previous sections show that, while CPA and APA impact a wide variety of diseases, certain entities, particularly cancers and neurological diseases, appear to be broadly affected. While this may simply reflect biases in investigation, it is tempting to speculate that underlying molecular characteristics may predispose certain tissues to a susceptibility to APA alterations. Given the increasing evidence that CPA and APA impact disease, these findings call attention to the need for research to determine where APA alterations drive disease, and in which cases they simply reflect disease-dependent cellular states.High-throughput sequencing technologies have significantly promoted the elucidation of disease mechanisms, and have equipped us with novel diagnostic opportunities. As mentioned previously, dynamic changes at the transcriptome 3′ end are prevalent ,154,335 APA perturbations, even when resulting in subtle changes of non-coding RNA sequence elements in the 3′UTR, can be functionally most significant . At the As for alternative splicing, identifying global APA patterns can likely have wide diagnostic implications ,341,342.With the advent of high-throughput analyses, the bioinformatical workload has increased dramatically. In contrast to total RNASeq, the sequencing restricted to the transcriptome 3′ end directly uncovers the variability and perturbation occurring at the mRNA 3′ end. This has several advantages. Firstly, it drastically reduces the bioinformatical workload. Furthermore, these data are typically not confounded by other variables that complicate the bioinformatical processing of the data . Finally, restricting the sequencing to the last (approximately) 30 nucleotides of the transcriptome opens up interesting (and first and foremost cost-effective) opportunities for multiplexing, while still keeping a high coverage for a reliable analysis. In depth APA profile studies have recently revealed “aberrant” APA signatures to be associated with more aggressive tumour phenotypes in cancer patients and thereby provided the proof-of-concept that such a determination can reveal prognostic signatures ,78,141. http://shiny.imbei.uni-mainz.de:3838/trend-db/). They provide easy access to APA signatures for non-expert users allowing them to select gene-specific APA signatures to be tested in a targeted approach. This could advance diagnostic strategies for a more thorough understanding of underlying disease mechanisms as well as for a reliable prognostic and possibly therapeutic stratification.It remains to be observed in which disease conditions and to what extent the analysis of APA signatures could further improve diagnostic strategies and possibly allow detecting biological aberrations with higher sensitivity and specificity. Interestingly, selected APA events can confer strong prognostic power beyond common clinical and molecular variables, suggesting their potential as novel prognostic biomarkers ,141. ThuUltimately, ongoing genome sequencing activities will most likely grant us further insights into genomic variations resulting in gene-specific perturbation of APA isoforms with possible detrimental functional consequences. Unlike global aberration in trans , the cause–consequence relationship in this kind of setting is substantively clearer. Further such changes may be directly accessible for specific, targeted therapeutic approaches.The significant role that disturbances in CPA play in the diseases presented above reveals the potential of novel therapeutic strategies targeting these mechanisms . How sucASO therapies involve the targeting RNA via complementary base pairing of oligonucleotides. Initial ASOs were capable of recruiting RNaseH resulting in target RNA degradation. However, the development of phosphorodiamidate morpholino oligomers blocked this activity, improved stability, and lowered toxicity . This alStrategies using ASOs to block access of the CPA machinery to specific gene PASs to modulate CPA and APA have been developed. As described above, FSHD can result from SNPs creating a canonical PAS resulting in stabilisation of the DUX4 transcript and over-expression. ASOs targeting this novel PAS successfully interfered with CPA resulting in mRNA reduction in differentiated immortalized FSHD myotubes ,357, as CPA is tightly bound to splicing and can be regulated by splicing factors ,363. SmaAdditionally it has been demonstrated that through the use of bifunctional U1 adaptors to tether U1snRNP upstream of a PAS, CPA at that PAS can be inhibited ,371,372.In addition to ASO targeting of PASs, it has been reported that siRNA targeting can influence CPA. Here an siRNA binding a proximal PAS of the interleukin 4 receptor was found to be active independent of any argonaute RISC catalytic component (Ago) or other RNA-induced silencing complex (RISC)-associated proteins, and appeared to reduce targeted message in an APA-independent manner through deadenylation or inhibition of polyadenylation and subsequent degradation of the immature mRNA .For alterations occurring in cis, the PAS structure of the affected gene is critical. In pathologies resulting from a gain of function a therapeutic approach could be simply to block the novel PAS directly using ASOs or via bifunctional U1 adaptors. If novel PAS occurs within an intron ASO, blocking of splice junctions may be possible. Similar approaches can be taken for genes with multiple PASs, when blocking a proximal PAS may assist in restoring 3′UTR length to genes with mutated distal PAS and vice versa.However, for conditions where a gene possessing a single PAS is affected, resulting in reduction of expression, options are more limited. Here techniques aimed at correcting the underlaying mutation may be possible. Induced pluripotent stem cells from different cell types from β-thalassemia patients have been used to correct coding region mutations in the HBB using the CRISPR-Cas9 technology ,378,379.While clear approaches are available for the correction of specific gene defects resulting in pathological CPA, the treatment of global APA alteration provides more challenges. However, avenues exist where the CPA machinery itself may be a potential therapeutic target. The availability of CPA factors has a significant effect on APA . SystemaOther cellular processes controlling gene expression influencing APA may serve as therapeutic targets. One such potential target stems from the observation that processivity of RNA Pol II can influence APA, and mutations in RNA Pol II directly slowing down RNA Pol II elongation favour proximal PAS use . TherefoJust as a diverse range of compounds targeting the spliceosome have been developed ,396,397,Additionally, as epigenetic modifications such as genomic imprinting , DNA metTherapeutic intervention can also be achieved on the front of RBPs, which in the future may form the basis of therapies to influence CPA . For exaFinally well-established drugs may have unexpected effects on CPA. As described above, APA of SLC6A4 plays a role in several neurological conditions and interestingly treatment of mice with the SLC6A4-selective antidepressant/anxiolytic drug fluoxetine, which has been shown to reduce anxiety disorders, increases expression of the distal polyadenylation form of SLC6A4 ,413. WitPost-transcriptional gene regulation through mRNA 3′UTR sequences has emerged as a critical process controlling important cellular functions by directing transcript stability, nuclear export, sub-cellular localisation and translation efficiency. In recent years the advent of high-throughput sequencing has revealed the enormous contribution CPA and APA plays in sculpting the 3′UTR. Far from acting as a constant mechanism simply terminating mRNA transcription, CPA has emerged as a complex process, tightly integrated with other transcriptional processes regulating the diverse molecular aspects of mRNA metabolism.APA has been found to represent an important layer of post-transcriptional gene regulation, playing a central role in the establishment of regulated expression networks fundamental to a multitude of biological roles. Modulation of APA in various normal physiological circumstances can influence RNA fate and regulate protein output both quantitatively and qualitatively, directing important cellular programs. Regulation of CPA relies on the precise integration of transcription, with trans-acting CPA factors interacting with specific cis-elements in the pre-mRNA. For individual genes where mutations in these cis elements disturbs CPA, the relationship to pathology is clear. However, although altered APA signatures have been associated with a variety of disorders, the contribution of CPA factors on global APA in human pathologies is still unclear. First evidence is accumulating  that APAGiven this increasing recognition of the prevalence of CPA and APA, further studies defining key components directing these processes are required to decipher their functional contribution and significance in normal cellular processes as well as human disease. Additionally, an understanding of the cellular pathways that regulate CPA and APA and how these molecular mechanisms connect are needed to understand the development of certain pathologies. Finally, in order to determine whether APA can serve as a meaningful therapeutic target, biological model systems as well as comprehensive analysis tools for APA are required.This review highlights the relevance of CPA and APA mechanisms in the correct expression of genes involved in disease development and cell homeostasis, and how this information may impact diagnosis and therapy. It will be interesting to see how these findings translate into the manipulation of APA in clinical settings and result in the identification of disease biomarkers that may be used as diagnostic and therapeutic tools in the future.


CDK4, LIG1, and NEIL3, which are transcriptionally controlled by cell cycle progression and highly acetylated by EP300 in proliferating cells. As previously documented, in dividing cells BRG1 directly activates gene transcription by evicting EP300 modified nucleosomes from the promoters and, thereby, relaxing chromatin. However, the deficiency of BRG1 or EP300 activity for 48 h leads to cell growth arrest and to chromatin compaction, but also to the assembly of RB1/HDAC1/EZH2 complexes at the studied cell cycle-dependent gene promoters. Epigenetic changes include histone deacetylation and accumulation of H3K27me trimethylation, both known to repress transcription. Cell cycle arrest in G1 by inhibition of CDK4/6 phenocopies the effect of the long-term BRG1 inhibition on the chromatin structure. These results suggest that BRG1 may control gene transcription also by promoting expression of genes responsible for cell cycle progression in the studied breast cancer cells. In the current study, we show that BRG1 binding occurs at the promoters of functionally linked genes in proliferating breast cancer cells, revealing a new mechanism by which BRG1 defines gene transcription. Cancer malignancy is usually characterized by unlimited self-renewal. In some types of advanced tumors that are rapidly dividing, gene expression profiles depict elevations in pro-proliferative genes accompanied by coordinately elevated transcription of factors responsible for removal of DNA lesions. In our studies, fast proliferating breast cancer cell lines (MDA-MB-231 and MCF7), BRG1, a component of the SWI/SNF complex, emerges as an activator of functionally-linked genes responsible for activities such as mitotic cell divisions and DNA repair. Products of at least some of them are considerably overrepresented in breast cancer cells and BRG1 facilitates growth of MCF7 and MDA-MB-231 cell lines. BRG1 occurs at the promoters of genes such as  Genomic instability and deactivation of DNA repair genes are often associated with tumor-prone phenotypes and necessary for acquisition of tumor initiating mutations. However, from a certain moment of malignancy, the genetic stabilization and maintenance of genome integrity might be required in order to invade tissues and give rise to distant metastases, but the increased ability to repair DNA damage seems to depend on tumor type . TranscrPARP1, BRCA1, and XRCC1, is controlled by SWI/SNF in a cell cycle-dependent manner [Our recently published results provide evidence that transcription of some of DNA repair genes, such as t manner . In our With respect to other genes and cell types, the role of BRG1 remains ambiguous and a molecular mechanism that turns this enzyme into a gene activator or repressor has not been found. It might be linked with co-operating factors, nucleosome modifications or histone variants, which appear at a given place and time at chromatin ,5,6. DatCDK2, CCNE, and CCND [E2F motifs mark proliferation-sensitive gene promoters, which respond to growth arrest by recruiting retinoblastoma-based repressive complexes comprising  RB1, RBL1, and RBL2 as its basic components. These proteins co-occur with numerous chromatin writers and erasers, including HDACs, PRC2, DNMT1, SUV39H1, or HP1, all capable of suppressing gene transcription ,8,9. Sinand CCND .In the current study, we reveal additional complexity to how BRG1 can modulate E2F-dependent transcription. In our model, BRG1 occurs at E2F/CpG-positive, highly acetylated promoters of genes that are overexpressed in breast primary tumor, and two selected highly invasive breast cancer cell lines: MCF7 and MDA-MB-231. Among BRG1-enriched promoters we found genes encoding factors responsible for cancer cell proliferation and resistance to DNA damage. BRG1 dually activates their transcription: (a) directly by acting at the chromatin level and evicting acetylated nucleosomes from their promoters, and (b) indirectly by potentiating cell proliferation and preventing assembly of RB1-HDAC1-PRC2 repressive complexes at the gene promoters. The E2F binding motif at the promoters of some genes, which are functionally linked to cell proliferation and DNA repair in the studied breast cancer cells, allow BRG1-EP300 complexes to provide a common mechanism of gene transcription control.To test if BRG1 may contribute to transcription regulation of genes in fast proliferating breast cancer cells, we investigated whether this enzyme co-occurs genome-wide with any particular histone mark that is known for its involvement in transcription control. For this analysis, we took publicly available data from ChIP-Seq experiments for BRG1 and selected histone modifications, and calculated Pearson correlation coefficient between their co-distribution in the genome of MDA-MB-231 cells. Genomic occurrence of BRG1 showed it was most strongly correlated with histone acetylation and H3K4me3, which are usually associated with gene promoters and active transcription A. Lack oPromoters characterized by all four considered features—BRG1/H3K27ac/E2F/CpG—were associated with genes involved in key cellular processes, among them DNA repair and mitotic divisions . AnalysiTo check if BRG1 creates a multi-enzyme complex with EP300 and HDAC1 in proliferating cancer cells, we searched for the latter two enzymes in BRG1 co-immunoprecipitates. As shown in CDK2, CDK4, PCNA, CHEK2, CCNB) and removal of DNA damage  [XRCC2 was the only exception and was upregulated in BRG1 deficiency. Although iEP300 causes simultaneous inhibition of EP300 and CBP, the data for siEP300 (Silencer™ Select Pre-Designed siRNA ID:s534247) is missing due to the fact, that substantial EP300 silencing with siRNA induced dramatic cell death. To verify the possible contribution of BRG1-EP300 to transcription regulation of selected genes functionally linked to proliferation and removal of DNA damage, we treated MCF7 and MDA-MB-231 cells with inhibitors of EP300 (iEP300–C646–blocks acetylatransferase activity of EP300 and CBP), BRG1 (iSWI/SNF–PFI3 is a bromodomain inhibitor of BRG1 and BRM) and calculated Log2FC of mRNA level quantified by real-time PCR between inhibitor treated and control cells. A high transcription rate of at least some genes, which were found overexpressed in all cancer cell types according to  and HR) ,13, was Repression of proliferation-relevant genes by the deficiency of BRG1 and EP300 activity led to a substantial reduction in protein proliferation markers  and to cell cycle arrest in G1 B,D,E. SiCDK4 and effective removal of DNA lesions—LIG1 and NEIL3. As shown in To study in detail the molecular mechanism of BRG1 contribution to regulation of transcription and possible functional interdependence with the cell cycle progression, we focused on three promoters of genes, which are crucial for cell proliferation—CDK4, LIG1, and NEIL3   we checked whether the arrest of cell cycle progression triggered by the inhibition of cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) that blocks cells in G1 E suppres island) D. All thus study . As currus study . BRG1 seCDK4 and LIG1. The enzymatically active subunit of PRC2–EZH2 catalyzed methylation of lysine 27 of histone H3, that is known to cause gene suppression. Of note, PRC2 was found only at two of these gene promoters, and did not correlate with enrichment or extrusion of other possible components of the repressive complex. This may indicate that PRC2 targets chromatin in a promoter rather than in a RB1-dependent manner, and that the presence of RB1 is a prerequisite, but not essential, for PRC2 contribution to gene silencing.Increase in BRG1 association with gene promoters induced by G1 arrest was followed by displacement of EP300, which may (in addition to recruitment and activation of HDAC1) further explain reduced nucleosome acetylation. The assembled repressive complex was supported by polycomb repressive complex 2 (PRC2) at the promoters of LIG1 promoter, whereas it faintly methylated lysine residues at the promoters of CDK4 and NEIL3.To search for a molecular mechanism that links BRG1/EP300 activity with cell cycle-dependent gene transcription, we analyzed the impact of BRG1 and EP300 on chromatin structure at the chosen gene promoters A. Long-tCDK4, PCNA, CCNB), we checked their transcription rate and chromatin features which are representative of BRG1/EP300 proximate effects (H3 density and nucleosome acetylation), and also looked for cell cycle-related chromatin remodeling (RB1) after short-term MCF7 cell treatment with inhibitors , checkpoints (CHEK2 responses to genome integrity assessment), DNA replication (PCNA—DNA clamp and scaffold for DNA polymerase) and many others listed in Drosophila brahma, were identified as activators of estrogen and the retinoic acid receptors at the gene promoters which respond to nuclear receptors [PSA) that is transcriptionally controlled by androgen receptor [PSA expression should be also checked in dividing cells. BRG1 may act as gene activator or suppressor at the target gene promoter depending on the cell cycle status, which can possibly switch the BRG1 role from extrusion of acetylated nucleosome to insertion of their deacetylated form. Again, the molecular trigger  in ~20% of human tumors. Mutations associated with gain and loss of function, as well as fluctuation in the expression of SWI/SNF components, are linked to the occurrence of cancer and its progression in several ways ,16. BRG1eceptors . A latereceptors . BRG1-mereceptor . Howeverpromoter ) that caIn the context of possible use of BRG1 inhibitors in anticancer therapies, of particular importance is our finding regarding BRG1’s co-operation with EP300, since only one non-specific inhibitor of BRG1 is commercially available for scientific purposes. Due to a similar degree of repression of BRG1-dependent genes by EP300 inhibition A–D, suchEven at the genomic level, chromatin features or DNA sequence that guide BRG1 distribution and association with particular gene promoters or regulatory regions remain poorly defined. It is well acknowledged that this enzyme marks actively transcribed genes and correlates with nucleosome acetylation and trimethylation of H3K4me3, but spatiotemporal mutual interdependence between BRG1’s occurrence and covalent modifications of nucleosomes are also unknown. Since in our study’s SWI/SNF complexes turned out to be readers of nucleosome acetylation governed by EP300-HDAC1 balance intwo distinct cell types (human macrophages and two breast cancer cell lines), one might think that BRG1 confers active gene transcription in other cancer types characterized by BRG1 overexpression. Apart from the documented BRG1 role at gene promoters, cancer cell divisions that are transcriptionally controlled by this enzyme regulate expression of numerous functionally-related genes, which may set the phenotype of breast cancer cells. Their link with proliferation is primarily through the presence of the E2F motif, which responds to cell cycle status by recruiting or releasing retinoblastoma-based complexes. However, only ~60% of BRG1/H3K27ac/CpG/E2F-positive genes are overexpressed in fast dividing cells E, suggesAs long as the abundance of DNA repair enzymes might not be of particular importance or even antagonize cancer development, the described functional dependence of cancer cell growth on transcription of enzymes capable of removing DNA lesion might be of key importance, not only for cell protection from anticancer therapeutics, but for the maintenance of cancer cell viability and metastases since production of proliferation-associated reactive oxygen species challenges genome integrity and might limit tumor progression. Therefore, BRG1 turns out to be considerable regulator of breast cancer cell physiology. Malignancy-relevant cell cycle re-entrance from a differentiated cell state may globally activate the desired gene expression in a unified mode due to the placement of BRG1-EP300-HDAC1 complexes at promoters that have CpG islands  were from Biowest , L15 Medium, iEP300 (C646), iSWI/SNF (PFI-3), iCDK4/6 ; anti-rabbit IgG (A0545) and anti-mouse IgG (A4416) (whole molecule)–peroxidase antibody produced in goat, BLUeye prestained protein ladder (#94964), oligonucleotides for real-time PCR were from Sigma Aldrich . Anti-HDAC1 (PA1-860), anti-EP300 (PA1848), Lipofectamine RNAiMAX, OptiMem, Dynabeads™ Protein G, High-Capacity cDNA Reverse Transcription Kit, Click-iT™ Nascent RNA Capture Kit for gene expression analysis, SuperSignal™ West Pico Chemiluminescent Substrate, TRI Reagent™, Silencer™ Select Pre-Designed siRNA ID:s13141 (BRG1), RNase A were from Thermofisher Scientific , while iCDK4/6 (PD0332991) was from Cayman Chemical .® Dye, 20X in water was purchased from Biotium . WB antibodies: anti-BRG1 (sc-17796), anti-DNA Ligase I (sc-271678), anti-CDK4 (sc-23896), anti-NEIL3 (sc-393703), anti-PCNA (sc-56), anti-Ki-67 (sc-23900), and anti-cyclin B  were purchased from were Santa Cruz Biotechnology . ChIP grade antibodies: normal rabbit IgG (#2729), BRG1 (#49360), H3K27ac (#4353), anti-histone H3 (#4620), anti-RB1 (#9313), anti-H3K27me3 (#9733), anti-EZH2 (#5246) were purchased from Cell Signaling Technology .KAPA HiFi™ HotStart ReadyMix (2X) from KapaBiosystems and Takyon™No ROX SYBR Core Kit blue dTTP from Eurogentec were purchased from Polgen . EvaGreen2, whereas MDA-MB-231 in F15 medium supplemented with 15% FBS, penicillin/streptomycin  without CO2 equilibration. Both cell lines were maintained in a logarithmic growth phase in a culture and prior to the treatment with any compound. iEP300 , iSWI/SNF  and iCDK4/6  were added to cells 48 h prior to analysis. Concentration of the studied inhibitors and the time required to induce enzyme inhibition were chosen based on our and other reports. iEP300 and iCDK4/6 at the doses higher than tested induced dramatic cell death in response to longer incubation periods. MCF7 were cultured in DMEM supplemented with 10% FBS, penicillin/streptomycin  in 5% COCDK2, 5′-CAGGATGTGACCAAGCCAGT-3′ (forward) and 5′-TGAGTCCAAATAGCCCAAGG-3′ (reverse); CDK4, 5′-CTGGTGTTTGAGCATGTAGACC-3′ (forward) and 5′-AAACTGGCGCATCAGATCCTT-3′ (reverse), XRCC2, 5′-TCGCCTGGTTCTTTTTGCA-3′ (forward) and 5′-TCTGATGAGCTCGAGGCTTTC-3′ (reverse), BRCA2, 5′-CTTGCCCCTTTCGTCTATTTG-3′ (forward) and 5′-TACGGCCCTGAAGTACAGTCT-3′ (reverse), LIG1, 5′-CAGAGGGCGAGTTTGTCTTC-3′ (forward) and 5′-AGCCAGTTGTGCGATCTCTT-3′ (reverse), EXO1, 5′-AAACCTGAATGTGGCCGTGT-3′ (forward) and 5′-CCTCATTCCCAAACAGGGACT-3′ (reverse), NEIL3, 5′-GGTCTCCACCCAGCTGTTAAAG-3′ (forward) and 5′-CACGTATCATTTTCATGAGGTGATG-3′ (reverse), PCNA, 5′-TCTGAGGGCTTCGACACCTA-3′ (forward) and 5′-TTCTCCTGGTTTGGTGCTTCA-3′ (reverse); CHEK2, 5′-CAGGTTCTAGCCCAGCCTTC-3′ (forward) and 5′-ACGGAGTTCACAACACAGCA-3′ (reverse); CCNB1, 5′-TGGAGAGGTTGATGTCGAGC-3′ (forward) and 5′-AAGCAAAAAGCTCCTGCTGC-3′ (reverse); BRG1, 5′-AAGAAGACTGAGCCCCGACATTC-3′ (forward) and 5′-CCGTTACTGCTAAGGCCTATGC-3′ (reverse), BRCA1, 5′-TGCCCACAGATCAACTGGAA-3′ (forward) and 5′-CACAGGTGCCTCACACATCT-3′ (reverse); XRCC1, 5′-CGGCGGAAACTCATCCGATA-3′ (forward) and 5′-CCATCAGGGCCTCCTCAAAG-3′ (reverse); ACTB, 5′-TGGCACCCAGCACAATGAA-3′ (forward) and 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′ (reverse). GAPDH and B2M were from Human Toll-like Receptor Signaling Primer Library (HTLR-I). ACTB, GAPDH, and B2M (HSKG) were used for normalization. Data in figures are shown as Log2FC with respect to untreated control. For mRNA quantification the total RNA was extracted with TRI Reagent™, reverse transcribed with High-Capacity cDNA Reverse Transcription Kit and selected cDNA fragments were amplified in real-time PCR  using the following primer pairs: CDK4, NEIL3, and LIG1 mRNA were measured 8 h after cell treatment with iEP300 and iSWI/SNF using Click-iT® Nascent RNA Capture Kit as described previously and was normalized to ACTB [Nascent  to ACTB . For protein detection cell lysates were separated with SDS-PAGE, transferred to nitrocellulose membranes and stained overnight with primary antibodies  at 4 °C. After staining with HRP-conjugated secondary antibodies , the signal was developed using SuperSignal™ West Pico Chemiluminescent Substrate and acquired with ChemiDoc-IT2 .Cell cycle progression in MCF7 and MDA-MB-231 cells treated with iCDK4/6, iEP300 and iSWI/SNF for 48 h was analyzed by flow cytometry as described previously [® Dye and 4% DMSO and the following primers: CDK4 prom, 5′-ATAACCAGCTCGCGAAACGA-3′ and 5′-AGAGCAATGTCAAGCGGTCA-3′, LIG1 prom, 5′-AACACACTCAGATCCGCCAG-3′ and 5′-GCTTCCACCGATTCCTCCTC-3′, NEIL3 prom, 5′-GTAGGGAGCGACCTCAACAG-3′ and 5′-AGTACAGCCTGGTCCTTCCA-3′.Chromatin immunoprecipitation was carried out according to the protocol previously described ,19. FragFor BRG1 silencing MCF7 and MCD-MB-23 were seeded at the density of 100,000 cells per well and transfected on the following day with RNAiMAX-siRNA complexes prepared in OptiMem according to the following ratio: 20 nmol siRNA and 3 µL of transfection reagent. BRG1 silencing versus non-template control siRNA was confirmed by real-time PCR and western blot 48 h after cell transfection. −3 [Publically available, generated by other groups and deposited in the PubMed Central Database data from MDA-MB-231 cells were taken for ChIP-Seq analysis in Galaxy Version 19.05.dev : BRG1—GS−3 . Co-distribution of BRG1 and selected histone modifications in the whole genome was studied by MulitBamSummary/plotCorrelation . In briehttp://www.interactivenn.net/ from gene lists. Annotation of differentially expressed genes to gene ontology terms was carried out in GOrilla (using two unranked lists of genes and complete list of genes expressed in MDA-MB-231 as a background). Gene promoters enriched in BRG1, H3K27ac, CpG islands, and E2F were identified by returning intersects of recalled peaks and genomic regions ±2000 bp centered on TSS  . GenomicThe following data from normal breast, primary tumor, MCF7 and MDA-MB-231 cells were taken for RNA-Seq analysis: normal breast—GSM1695870 (SRR2040339), GSM1695872 (SRR2040341), GSM1695873 (SRR2040342), GSM1695874 (SRR2040343), GSM1695877 (SRR2040346), GSM1695878 (SRR2040347); primary tumor—GSM1695891 (SRR2040360), GSM1695898 (SRR2040367), GSM1695899 (SRR2040368), GSM1695882 (SRR2040351), GSM1695890 (SRR2040359), GSM1695894 (SRR2040363); MCF7—GSM2422725 (SRR5094305), GSM2422726 (SRR5094306), GSM2422727 (SRR5094307), GSM2422728 (SRR5094308), GSM2422729 (SRR5094309), GSM2422730 (SRR5094310); MDA-MB-231 - GSM2422731 (SRR5094311), GSM2422732 (SRR5094312), GSM2422733 (SRR5094313), GSM2422734 (SRR5094314), GSM2422735 (SRR5094315), GSM2422736 (SRR5094316).Having FASTQ quality formats unified to sanger formatted with FASTQ Groomer reads were mapped to Human Genome version 19 using TopHat . Transcrt-test was used to determine statistically significant differences between two means , while one-way analysis of variance (ANOVA) was carried out in GraphPad Prism 5 to compare means in several groups . Data in Summarizing, activity of one chromatin-associated enzyme defines the expression profile of numerous genes in breast cancer cells by acting directly on chromatin, and by promoting cell cycle progression. BRG1 removes acetylated nucleosomes, thereby facilitating transcription and preventing recruitment of retinoblastoma-based repressive complexes to E2F-driven promoters. Thus, BRG1 defines key breast cancer features in the cell lines we have investigated. Inhibitors of BRG1 and EP300 can be considered as future anticancer drugs that can arrest cell growth and/or render them sensitive to DNA damaging agents.