Unstable repeat expansions and insertions cause more than 30 neurodegenerative and neuromuscular diseases. Remarkably, bidirectional transcription of repeat expansions has been identified in at least 14 of these diseases. More remarkably, a growing number of studies has been showing that both sense and antisense repeat RNAs are able to dysregulate important cellular pathways, contributing together to the observed clinical phenotype. Notably, antisense repeat RNAs from spinocerebellar ataxia type 7, myotonic dystrophy type 1, Huntington’s disease and frontotemporal dementia/amyotrophic lateral sclerosis associated genes have been implicated in transcriptional regulation of sense gene expression, acting either at a transcriptional or posttranscriptional level. The recent evidence that antisense repeat RNAs could modulate gene expression broadens our understanding of the pathogenic pathways and adds more complexity to the development of therapeutic strategies for these disorders. In this review, we cover the amazing progress made in the understanding of the pathogenic mechanisms associated with repeat expansion neurodegenerative and neuromuscular diseases with a focus on the impact of antisense repeat transcription in the development of efficient therapies. In the last few decades, an increasing number of neurological and neuromuscular diseases has been associated with expansions of microsatellites in coding and noncoding gene regions. Microsatellites are polymorphic repeat sequences of 1 to 9 nucleotides distributed throughout the genome, composing about 3% of the human genome. These polymorphic repeats are usually pathogenic when they expand in size above a given threshold . In 1991Depending on the repeat motif and its location in a gene, three different pathogenic mechanisms have been identified in repeat expansion diseases, consisting in protein gain-of-function, gene loss-of-function and/or RNA gain-of-function  Figure . In codiC9ORF72 frontotemporal dementia (FTD)/amyotrophic lateral sclerosis (ALS) C9ORF72 fis (ALS)    and oculis (ALS)  58 expansion, in Purkinje cells, showed motor deficits, progressive loss of dendritic arborization, cell loss and cytoplasmic ataxin-2 aggregates [ATXN2 cDNA with a larger expansion, (CAG)127, in Purkinje cells, which presented earlier motor dysfunction accompanied by a significant decrease in expression of Purkinje cell genes such as calbindin 28K, indicating that an expanded ataxin-2 gain-of-function is involved in SCA2 [SCA2 is one of the most common SCAs worldwide ,27,28,29 protein ,31,32 82 under its endogenous promoter corroborated the decreased expression of Purkinje cell-specific genes. These mice have shown decreased cerebellar mRNA and protein levels of the highly expressed regulator of G-protein signaling 8 (RGS8), supporting ataxin-2 function in mRNA stabilization and translation [42 [ATXN2 mRNA and protein was detected in the SCA2 BAC-transgenic mice, contrarily to BAC-transgenic mice expressing (CAG)22 [.Ataxin-2 is expressed in the endoplasmic reticulum and Golgi complex, playing a role in RNA metabolism and endocytosis ,39. Sevenslation . These Snslation . Moreovetion [42 , a decreAtxn2 (CAG)42 knockin mice Purkinje cells, the expanded ataxin-2 colocalizes with STAU1 and PABPC1 proteins, respectively, into ubiquitin-positive cytoplasm granules, further corroborating an mRNA processing dysregulation in SCA2 [n tracts in ATXN2 increase this interaction and, in conformity, as TDP-43 is a protein implicated in ALS, intermediate (CAG)31–32ATXN2 alleles increase the risk of ALS [Considering that ataxin-2 is key for the assembly of stress granules and P-bodies, which are important structures involved in mRNA transport, splicing and degradation in the cytoplasm, the expanded ataxin-2 interacts abnormally with several RBPs in these granules, like Staufen 1 (STAU1), Fox-1/ataxin-2-binding protein 1 (FOX/A2BP1) and DEAD-Box helicase 6 (DDX6), which suggested that these cellular processes are also dysregulated in SCA2 ,44,45 T. In fact in SCA2 ,43. Rema in SCA2 . Longer k of ALS . In ALS,k of ALS . ATXN2 (CAG)n is bidirectionally transcribed. Li and colleagues have identified an antisense (CUG)n (ATXN2-AS) transcript in brain tissue of both unaffected and SCA2 affected subjects [expATXN2-AS transcript leads to cell death of primary mouse cortical neurons and forms RNA foci in SCA2 human cerebella and BAC-transgenic mice, which likely colocalize with Muscleblind-Like Splicing Regulator 1 (MBNL1), as detected in neuroblastoma cells transfected with the expanded (CAG)n [ATXN2-AS with a (CUG)>37 is expressed in SCA2 human cerebella, both normal and expanded ATXN2-AS RNAs do not seem to be translated by RAN [ATXN2-AS transcripts with intermediate (CUG)31–32 have been identified in ALS lymphoblastoid cell lines, suggesting an involvement of ATXN2-AS in ALS [ATXN2-AS in SCA2 and ALS is an important issue that needs further investigation.Interestingly, the subjects  n . Althougd by RAN . FurtherS in ALS . Howevern expansion in exon 3 of the ATXN7 [>37 mirror the progressive ataxia and the cone-rod dystrophy seen in SCA7 individuals [ATNX7 cDNA in rod-photoreceptors, Purkinje cells and other neurons [atxn7 translation inhibition also causes an early developmental impairment, leading to abnormal differentiation of photoreceptors and cerebellar neurons [SCA7 is caused by a (CAG)he ATXN7 ,48. Indihe ATXN7 . Severalividuals ,52,53,54ividuals ,56,57. Rividuals ,58. The  neurons ,53,57,58 neurons ,60  [SCAANT1 is responsible for ATXN7 transcriptional regulation. The authors found that, in normal size alleles, the CTCF-binding activates SCAANT1 transcription, yielding convergent transcription with ATXN7, and consequently limiting ATXN7 expression due to epigenetic modifications [n alleles. The repeat expansion leads to a decrease in CTCF-binding and, consequently, a decrease in SCAANT1 transcription, which derepresses ATXN7 [In contrast with transgenic mice expressing the human full-length N7 locus . In parttability . In thisSCAANT1) . Notablyications . This tres ATXN7   [Drosophila transgenic lines expressing normal and expanded (CUG)n RNAs in photoreceptor neurons showed retinal neurodegeneration, strongly suggesting that the overexpression of RNA containing CUG repeats is sufficient to drive neuronal degeneration [ATXN8OS with (CTG)104 under its endogenous promoter, recapitulating the motor dysfunction and cerebellar deficits seen in SCA8 [n translation, which was also verified in human SCA8 brain tissue [ATXN8OS, the expanded (CTG)n is transcribed in a noncoding transcript, suggesting that both protein and RNA gain-of-function mechanisms contribute to SCA8 [n forms ribonuclear inclusions that colocalize with MBNL1 in cerebella of SCA8 subjects and BAC-transgenic mice, leading to misplicing [n expanded transcripts are RAN-translated generating homopolymeric peptides of polyA and polyserine (polyS) (SCA8 is caused by a combined polymorphic CTA with a (CTG)ATXN8OS) ,64,65,66neration . To furt in SCA8 . Remarkan tissue   [KLHL1AS) that holds the ATXN8OS (CUG)n at the 3′end. Interestingly, it has been suggested that KLHL1AS regulates KLHL1 expression in the cerebellum, raising the hypothesis that the function of KLHL1AS and KLHL1 could be dysregulated in SCA8 [Klhl1 gene and Klhl1as promoter showed gait abnormalities, motor incoordination and dendritic atrophy, supporting the hypothesis that KLHL1 and/or its antisense have a role in SCA8 [KLHL1 loss-of-function in SCA8, the downregulation of human KLHL1 needs to be further investigated in human tissues of SCA8 subjects [KLHL1AS RNA harbors the (CUG)n, but the mouse Klhl1as RNA ends upstream the (CUG)n [KLHL1AS on KLHL1 expression and the role of the (CTG)n in this regulation [In the human genome, the SCA8 (CAG) (KLHL1) . This ge in SCA8 ,71. In f in SCA8 . Althougsubjects . Moreovegulation . Altogetn insertion causing SCA31 [Brain expressed associated with NEDD4-1 (BEAN1) and Thymidine kinase 2 (TK2) genes, which are transcribed from opposite DNA strands [BEAN1-orientation, the transcribed (TGGAA)n insertion forms nuclear RNA aggregates in Purkinje cells from SCA31 individuals [Drosophila lines expressing (TGGAA)80–100 in the nervous system showed an early lethality and progressive motor abnormalities [n insertion could impact neuronal development. In the Drosophila eye, (TGGAA)80–100 expression led to eye degeneration, accumulation of nuclear (UGGAA)n aggregates and poly-tryptophan-asparagine-glycine-methionine-glutamic acid (polyWNGME) pentapeptide repeat proteins (PPR) in imaginal discs [n insertion binds in vitro to serine/arginine-rich splicing factors (SRSFs) 1 and 9, and in vivo to C9ORF72 FTD/ALS-related proteins such as Fused in sarcoma (FUS), heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1) and TDP-43 [n and TDP-43 protein rescued eye degeneration in transgenic flies by decreasing RNA foci formation and PPR translation, suggesting that TDP-43 could function as an RNA chaperone [n is transcribed within an intron of TK2 gene and though no RNA foci or PPR proteins have been reported, rescue of SCA31 phenotype could require targeting both sense and antisense repeats.In 2009, Sato and colleagues found a (TGGAA)ng SCA31   gene [n causes a decrease of FXN mRNA and protein through histone modifications, possibly associated with heterochromatin formation in the repeat flanking region, in human FRDA brain tissue [FXN promoter and GAA-upstream regions, agrees with the FXN transcriptional repression and FXN protein deficiency [FXN transcription. In FRDA lymphoblasts, overexpression of SRF and TFAP2 leads to an increase of FXN transcription [n expansion could cause alterations in the binding of these transcription factors. Friedreich ataxia (FRDA), an autosomal-recessive ataxia with a high prevalence worldwide, is mainly caused by a biallelic (GAA)XN) gene . FRDA alXN) gene ,78,79 , whose expression is 2-fold increased, in FRDA fibroblast cells [FXN transcription concomitant with an increase of FAST-1 mRNA, recapitulating the FXN and FAST-1 mRNA levels detected in FRDA fibroblasts [FAST-1, in HeLa transfected cells, led to FXN mRNA and protein decrease, CTCF-binding depletion and histone methylation enrichment in the FXN promoter region [FAST-1 mRNA originated increased FXN transcription and mitochondrial aconitase activity, restoring frataxin function [FAST-1 transcript, which does not span the repeat expansion, seems to influence this mechanism. FAST-1 could play a role in FXN transcription regulation, as occurs in SCA7, and be a successful target for therapeutic intervention.A CTCF-binding site in the ormation . Spanninst cells . Intriguroblasts . Accordir region . In FRDAfunction . The patC9ORF72 FTD/ALS and SCA36 are autosomal-dominant neurodegenerative diseases, characterized by dementia and motor neuron degeneration or ataxia, respectively. Although these diseases present differences in clinical presentation, both are caused by similar noncoding hexanucleotide repeat expansions. A (GGGGCC)n expansion, of >30 repeat units, in intron 1 of chromosome 9 open reading frame 72 (C9ORF72) causes C9ORF72 FTD/ALS, whereas SCA36 is caused by a (TGGGCC)n expansion of 650 or more repeats in intron 1 of the nucleolar protein 56 (NOP56) [ (NOP56) ,89,90,91C9ORF72 and NOP56 hexanucleotide repeats initiates a cascade of parallel pathogenic mechanisms that include the sequestration of essential neuronal RBPs in RNA foci and the production of toxic dipeptide repeat proteins (DPRs) by RAN translation [C9ORF72 transcripts in brain tissue of C9ORF72 FTD/ALS individuals, together with the characteristic neuropathology observed in animal models for gene loss-of-function, indicate that also C9ORF72 haploinsufficiency is implicated in this disease [NOP56 transcript and protein discarding gene haploinsufficiency [The transcription of nslation ,16,92,93 disease ,95. In Sficiency . C9ORF72 FTD/ALS and SCA36 is not triggered only by transcription of expanded hexanucleotide repeats in C9ORF72 and NOP56 gene expression context, but also by antisense transcripts [C9ORF72 has five transcript start sites (TSSs) in sense orientation and three in antisense orientation [n is transcribed from two C9ORF72 TSSs, whereas the antisense (GGCCCC)n is transcribed from one TSS [C9ORF72 gene and containing gene flanking sequences have presented both sense and antisense RNA foci and RAN DPRs with a brain distribution similar to C9ORF72 expansion carriers, but no detectable neurodegeneration or behavioral abnormalities [C9ORF72 BAC-transgenic mouse containing ~500 repeats, but without 3′ gene sequences have also shown RNA foci and RAN DPRs, but again no neurodegeneration or behavioral changes [C9ORF72 flanked by regulatory regions, driving sense and antisense expression using the endogenous human promoters, presented neurodegeneration and motor dysfunction. In this mouse model, antisense expression was upregulated in frontal cortex, as seen in autopsy tissue from C9ORF72 expansion carriers, indicating that the expansion regulates antisense expression [C9ORF72 FTD/ALS cell populations like the upper motor neurons of the motor cortex. This BAC mouse model shows decreased survival, paralysis, muscle denervation, motor neuron loss, anxiety-like behavior, and cortical and hippocampal neurodegeneration. What seems to distinguish these latter BAC-transgenic mice from the previous mice, leading to the manifestation of the expected neurological phenotype, is the large region flanking both sense and antisense promoters that could facilitate interaction of regulatory regions with promoters, originating robust expression of transcripts. The relevance of antisense transcripts in the severity of the disease is supported by the milder phenotype when only overexpression of sense (GGGGCC)66 throughout the central nervous system is achieved by means of somatic brain transgenesis mediated by adeno-associated virus [The mechanism of RNA-mediated toxicity implicated in nscripts ,14,96,97entation . The n expansion in brain tissue of SCA36 affected individuals, RNA foci containing the antisense expansion have been detected in brains of transgenic mice for the (TGGGCC)n expansion, suggesting that antisense RNA may have a role in SCA36 pathology [The athology ,103.C9ORF72 hexanucleotide repeat RNA in the neurodegenerative phenotype [Drosophila model expressing ~800 GGGGCCs, interrupted with stop codons to avoid RAN translation, showed that the expanded repeat RNA sequesters RBPs, though it is not toxic in this organism. Similarly, an antisense RNA with ~100 CCCCGG repeats alone does not trigger a neurodegenerative phenotype [Interestingly, it has been suggested a modest role for henotype . A Drosohenotype .C9ORF72 FTD/ALS individuals and BAC-transgenic mice [C9ORF72 FTD/ALS subjects [C9ORF72 antisense orientation, the translation in polyPR and polyGP may occur from one and three putative ATG-start codons, respectively, however, in vitro studies using cell lines transfected with a plasmid containing a (GGCCCC)n expansion have confirmed that RAN translation from the antisense strand drives polyPR and polyGP production [The transcription of both sense and antisense expanded repeats leads to translation in RAN DPRs in neuronal brain tissue of nic mice ,100,101.nic mice ,96,97. Asubjects ,96. In CC9ORF72 FTD/ALS and SCA36 go beyond neurodegeneration. The hexanucleotide repeats share five nucleotides by each repeat unit, which has set the stage for the investigation of DPRs resulting from RAN translation in SCA36 [C9ORF72 FTD/ALS and SCA36 brain tissue and both are encoded from antisense repeat strand [Drosophila and zebrafish models using codons, other than the C9ORF72 hexanucleotide repeat, to encode the DPRs showed that the arginine-rich dipeptide species, including polyPR, are highly toxic in these organisms [C9ORF72 FTD/ALS subjects [C9ORF72 FTD/ALS subjects, however, contrarily to polyGP insoluble cytoplasmic aggregates found in C9ORF72 FTD/ALS, they remain soluble in the cytoplasm of neuronal cells in SCA36 [The similarities between in SCA36 ,14. Remat strand ,14  gene [>200 expansion spanning the promoter region triggers its hypermethylation, leading to FMR1 gene silencing [FMR1 loss-of-function in FXS, a slight increase of FMR1 mRNA levels has been detected in FXTAS lymphoblastoid cells, supporting an RNA gain-of-function leading to the disease [FMR1 mRNA and, therefore, identical levels of FMR1 protein (FMRP) have been detected in FXTAS lymphoblastoid cells compared with controls [FMR1 mRNA forms ubiquitin-positive intranuclear inclusions in neuronal and glial cells in FXTAS brain tissues [98-knockin mice [n premutation expressed in Purkinje cells leads to neurodegeneration in mice, independent from FMR1 gene context [Drosophila model expressing a human 5′UTR fragment with (CGG)90 evidenced FMRP reduction, presenting ubiquitin-positive intranuclear inclusions in photoreceptor neurons and progressive neurodegeneration [FMR1 RNA is also RAN-translated into polyA and polyglycine (polyG) peptides, which originate ubiquitin-positive intranuclear inclusions colocalizing with p62 that could also contribute to neurodegeneration in Drosophila and FXTAS brain tissue [FXS and FXTAS result from a (CGG)R1) gene ,108. FXSR1) gene ,108 n and one antisense transcript (ASFMR1) spanning the repeat [FMR1 mRNA, the ASFMR1 RNA levels are increased mainly in FXTAS autopsy brain tissue, endorsing a role for ASFMR1 in FXTAS [Drosophila model expressing a (CCG)90 has shown retinal neurodegeneration, suggesting a toxic role for the ASFMR1 premutation [ASFMR1 RNA with the (CCG)n premutation is transported to the cytoplasm and translated into a polyproline (polyP) peptide from an AUG-start codon [FMR1 mRNA upregulation or silencing is poorly understood. Interestingly, the co-expression of expanded (CCG)n and (GGC)n in Drosophila decreased their RNA levels through Dicer and Argonaute-2, reverting the neurodegenerative phenotype [In FXTAS, in addition to (CGG)e repeat  , leading to sporadic and familial NIID [NOTCH2NLC gene, highly expressed in the brain, is thought to play a role in neuronal proliferation and differentiation [n region, in fibroblasts of NIID individuals [n allele could lead to this antisense transcription, however, the authors did not find significant differences in CpG methylation profiles between leukocytes and fibroblasts from NIID subjects and controls [n could account for the differences in DNA methylation seen in these two studies. Although no NOTCH2NLC expression changes have been detected, several genes with neuronal functions have shown differences in fibroblasts [n could dysregulate neuronal gene expression in NIID.Novel noncoding CGG repeat expansions have recently been discovered causing NIID and OPML1, two neurodegenerative diseases that share with FXTAS the symptoms of ataxia and tremor, and the presence of ubiquitin-positive neuronal inclusions ,21. Broaial NIID  ng OPML1   gene [>40 in exon 2A of junctophilin-3 (JPH3) gene [HTT mRNA with the expanded (CAG)n gains the ability to sequester RBPs, such as MBNL1 in HD fibroblasts and nucleolin in HD transgenic mouse brains, possibly causing a splicing misregulation of MBNL1 target pre-mRNAs, nucleocytoplasmic transport impairment and nucleolar stress [HD and HDL2 are two clinically and pathologically similar neurodegenerative diseases, characterized by movement, cognitive and psychiatric disturbances . The indTT) gene , whereasH3) gene ,138  gene ,143,144.H3) gene ,140,141.H3) gene , the expH3) gene . ConcomiH3) gene ,149,150 r stress ,153,154.n across the HD locus (HTTAS1) [HTTAS1 transcripts and identical HTT RNA levels in HD brain tissues [post-mortem brain tissues from subjects with an adult HD onset and controls, but HTT protein is reduced in brain protein extracts of juvenile onset individuals [HTTAS1 transcript. Chung and colleagues have also shown that HTTAS1 is able to regulate HTT RNA degradation on a RISC-dependent manner [HTTAS1 promoter [ATXN7.In 2011, Chung and colleagues discovered the existence of a natural antisense transcript spanning the (CTG)(HTTAS1) . The aut tissues . Unexpecividuals . Therefot manner   have been identified [HTT mRNA forms hairpins that are cleaved by a Dicer-dependent mechanism and, consequently, the resultant neurotoxic sCAGs of approximately 21 nucleotides could silence other RNAs containing CUG repeats, disrupting their functions [Drosophila [In HD mice expressing exon 1 of entified . In factunctions ,158. Indosophila .Jph3 knockout mice, these animals have shown motor dysfunction, supporting a role for reduced JPH3 protein in HDL2 [Due to clinical and neuropathological similarities between HD and HDL2, the same neurodegenerative mechanisms could underlie both diseases. JPH3 protein is highly expressed in Purkinje cells and is involved in the formation of junctional membranes, therefore, the loss of JPH3 could partially explain motor dysfunction in HDL2 ,161,162. in HDL2 ,162. JPH3 transcripts form RNA aggregates with MBNL1, with consequent misplicing of MBNL1 mRNA targets in frontal cortex [JPH3 locus with a (CTG)116 has also shown cortical (CUG)n RNA foci colocalizing with Mbnl1 accompanied by cortex atrophy and motor impairment, mimicking the HDL2 phenotype [HDL2 antisense CAG transcript translated into an aberrant polyQ expanded protein, forming ubiquitin-positive intranuclear inclusions in cortex, hippocampus and striatum of HDL2 BAC-transgenic mice [n is transcribed, however, the expanded (CAG)n transcript and the polyQ protein were not detected in autopsy brain tissue from HDL2 individuals [JPH3 repeat in unaffected chromosomes. Further investigation of a potential regulatory mechanism involving both JPH3 sense and antisense expression, as occurs in HD, may contribute to explain how JPH3 protein expression is regulated >250, mimicking the location of the repeat expansion in DMPK, in a muscle-specific gene, developed limb difficulties, myotonia and myopathy, indicating that the expanded (CUG)n RNA is sufficient to cause DM1 clinical features in muscle [DM1 and DM2 are two clinically similar neuromuscular diseases . While auses DM1 ,167,168, for DM2  480 in the eye and muscle also mimicked the degeneration of these tissues and presented nuclear RNA foci colocalizing with MBNL proteins [3′DMPK RNA holding a (CUG)91 also formed nuclear RNA foci, developed morphological and behavioral abnormalities and showed transcriptional dysregulation during early development [The hypothesis of elopment ,178. Theelopment , whereaselopment ,180. In ividuals . Nuclearividuals . Interesividuals  n substantially reducing binding of CTCFs [ATXN7, Nakamori and colleagues discovered a DMPK alternative promoter upstream the (CTG)n, that is regulated by an antisense CAG repeat transcript [DMPK transcription, leading to increase in (CUG)n RNA and enhancing RNA toxicity (n and (CAG)n DM1 transcripts form distinct ribonuclear foci, both potentially contributing to the disease [n toxicity, decreasing the (CUG)n RNA foci formation and splicing abnormalities [DM1 locus transcriptional dysregulation with a consequent decrease of antisense CAG siRNAs and increase of expanded (CUG)n-mediated toxicity, whose downstream effects lead to muscle deficiencies in congenital DM1 [Bidirectional transcription of a repeat expansion has first been discovered by Cho and colleagues, for DM1. These authors demonstrated that the antisense transcript spanning the repeat is converted into siRNAs, able to recruit DNA and histone methyltransferases, in particular the heterochromatin protein 1γ (HP1γ), leading to heterochromatin formation . AbnormaIX5 gene ,195,196.of CTCFs . Similaranscript . These atoxicity . Interes disease . Moreovemalities . Altogetital DM1  n are upregulated in DM2 human brain tissue [n and antisense (CAGG)n expanded RNAs are RAN-translated producing toxic poly-leucine-proline-alanine-cysteine (polyLPAC) and poly-glutamine-alanine-glycine-arginine (polyQAGR) tetrapeptides, respectively, able to induce cell death in human transfected cells n also binds to hnRNP A1, an RBP implicated in RNA processing [In DM1, the expanded CAG transcript is RAN-translated into polyQ peptides in cardiac myocytes of DM1 mice  Table 1Table 1. n tissue . Both seocessing . Taken together, this emerging evidence in DM1 and DM2 strongly indicates that antisense repeat RNAs are key players in several molecular pathways that when dysregulated cause disease, being important targets for the development of therapeutic strategies.HTT mRNA delivered using an AAV5 has been tested in the striatum of HD rodents and minipig models, leading to mutant mRNA and protein reduction, with consequent reduction of abnormal protein aggregation, thereby ameliorating the HD phenotype [A potential therapeutic strategy to target mutant repeat RNAs is based on RNA interference (RNAi) molecules that are complementary RNAs to the mutant transcripts, leading to the formation of double-stranded RNAs and, consequently, to their RISC-mediated degradation or translation repression. These molecules can be delivered directly into the cell as miRNAs or siRNAs, or indirectly as short hairpin RNAs (shRNAs) that are delivered generally by adeno-associated viruses (AAV) . Severalhenotype ,205. Thihenotype ,212. TheC9ORF72 FTD/ALS [C9ORF72 FTD/ALS. In SCA2, ASOs decrease the toxic RNA and protein, improving the motor function in mice [HTT RNAs, in which no pathogenic effect caused by wild-type HTT protein deficiency was detected [HTT, using single nucleotide polymorphisms associated with the disease haplotype [DMPK RNA is currently in phase I/II clinical trial, due to the observed successful rescue of muscle dysfunction in mouse models [C9ORF72 FTD/ALS [C9ORF72 FTD/ALS transgenic mice, has been in clinical phase I studies. The presented therapeutic strategies are based on ASOs that specifically target the toxic sense transcripts. However, in several diseases, including HD, SCA2, DM1 and C9ORF72 FTD/ALS, both sense and antisense repeats contribute with toxic species, thus targeting both mutant sense and antisense RNAs could be crucial to improving the success of therapies designed to revert the disease phenotype and/or delay disease progression. Other promising therapies to target mutant repeat RNAs are based on antisense oligonucleotides (ASOs) . These A FTD/ALS ,224,225. FTD/ALS ,218, and FTD/ALS  and C9OR in mice . Moreovedetected , and theaplotype . In DM1,e models . More re FTD/ALS , which rAntisense repeat RNAs have been identified in several genes associated with neurodegenerative and neuromuscular diseases caused by repeat expansions. Although enormous efforts have been made to understand the complex mechanisms underlying these diseases, the contribution of mutant antisense repeat RNAs remains elusive. This is due to challenges regarding the assessment of their expression levels that tend to be low, the recognition of their cell and tissue-specific expression that probably varies from development to aging hindering the discovery of their biological function and role in disease. In addition, the scarcity of animal models with bidirectional transcription of repeat expansions has also limited the understanding of the role of antisense repeat RNAs. It remains challenging to weight the contribution of each transcript, sense or antisense, in the disease process. So far, it is known that some mutant antisense repeat transcripts can modulate gene transcription, dysregulate the activity of sense repeat transcripts or other complementary RNAs, sequester RBPs and be translated into toxic peptides. Intriguingly, a dysregulation of gene expression has arisen associated with antisense repeat transcription in at least four neurological diseases . Therefo


Oct4 pseudogene lncRNA that is able to direct epigenetic silencing of the parental Oct4 gene via a 2-step, lncRNA dependent mechanism. The murine Oct4 pseudogene 4 (mOct4P4) lncRNA recruits the RNA binding protein FUS to allow the binding of the SUV39H1 HMTase to a defined mOct4P4 lncRNA sequence element. The mOct4P4-FUS-SUV39H1 silencing complex holds target site specificity for the parental Oct4 promoter and interference with individual components results in loss of Oct4 silencing. SUV39H1 and FUS do not bind parental Oct4 mRNA, confirming the acquisition of a new biological function by the mOct4P4 lncRNA. Importantly, all features of mOct4P4 function are recapitulated by the human hOCT4P3 pseudogene lncRNA, indicating evolutionary conservation. Our data highlight the biological relevance of rapidly evolving lncRNAs that infiltrate into central epigenetic regulatory circuits in vertebrate cells.The resurrection of pseudogenes during evolution produced lncRNAs with new biological function. Here we show that pseudogene-evolution created an  Scarola et al. identify a conserved OCT4 pseudogene mechanism of action and demonstrate that the OCT4 pseudogene lncRNA is required for FUS-dependent loading of the SUV39H1 histone methyltransferase to the promoter of the parental OCT4 gene. In many cases, sequence similarity of pseudogene derived lncRNAs with parental gene transcripts provides the rational basis for pseudogene dependent control of ancestral gene expression. Pseudogene lncRNAs have been reported to compete with parental gene transcripts for miRNAs or RNA binding proteins or, alternatively, can give rise to endo-siRNAs8. Antisense transcription of pseudogenes can mediate epigenetic silencing of ancestral genes in trans, presumably by pairing with ancestral sense gene transcripts10. Remarkably, pseudogene derived lncRNAs have also been demonstrated to act as scaffold for chromatin modifying complexes that can modulate gene expression at multiple loci across the genome12.Pseudogenes are non-functional gene copies that have lost protein coding potential. Precise annotation and integration of functional genomics data revealed a high number of pseudogenes that have evolved to new functional elements, producing long noncoding RNAs (lncRNAs) in a tightly controlled manner23. During evolution, the murine and human Pou5f1/POU5F1 genes, that encode OCT4, gave rise to five processed murine (Pou5F1P1–Pou5F1P5) and eight processed human pseudogenes (POU5F1P1–POU5F1P8), with validated lncRNA expression26. Hereinafter, Pou5f1 and POU5F1 pseudogenes will be referred to as Oct4/OCT4 pseudogenes. Murine Oct4 pseudogene derived lncRNAs show defined pattern of expression during mouse embryonic stem cells (mESC) differentiation and specific cytoplasmic or nuclear localization, supporting evidence for the acquisition of new biological function17. In line with this, human OCT4 pseudogene 4 and 5 lncRNAs alter ancestral gene expression by acting as classic ceRNAs, and pairing of the murine Oct4-pseudogene 5 antisense lncRNA with Oct4 transcripts has a role in guiding the histone methyltransferase (HMTase) EZH2 to the OCT4 promoter27.The transcription factor OCT4 is central for vertebrate embryonic stem cell (ESC) pluripotency and cancer cell biology and represents a hallmark model for the multifaceted pathways of pseudogene lncRNA mediated regulation of parental gene expressionOct4 promoter in trans17. Induction of mESC differentiation results in efficient upregulation of the X-linked mOct4P4 gene that encodes the mOct4P4 lncRNA. The resulting nuclear restricted mOct4P4 lncRNA forms a complex with the HMTase SUV39h1 and targets H3K9me3 and HP1 to the promoter of the parental Oct4 gene on chromosome 17, leading to gene silencing in trans. Importantly, this mechanism does not involve pairing of Oct4 sense and pseudogene antisense RNAs. To this end, lncRNA sequence determinants and evolutional importance for mOct4P4 pseudogene lncRNA dependent silencing of Oct4 are not known.We recently reported on a new mechanism of ancestral gene regulation that depends on pseudogene lncRNA dependent recruitment of an epigenetic silencing complex to the POU5F1P3 pseudogene derived lncRNA, hOCT4P3, is a functional homolog of the murine Pou5f1P4 lncRNA in OVCAR-3 ovarian cancer cells, demonstrating evolutionally constraint on pseudogene–lncRNA-mediated epigenetic silencing of OCT4. Performing mOct4P4 lncRNA pulldown experiments and a mOct4P4 lncRNA deletion analysis we demonstrate that the RNA binding protein FUS and a 200 nucleotide mOct4P4/hOCT4P3 region are essential for Oct4/OCT4 silencing in mouse and human cells. Binding of FUS to endogenous, full length mOct4P4/hOCT4P3 lncRNAs allows subsequent binding of SUV39H1 to the 200-nucleotide lncRNA element, forming a silencing complex with target specificity for the parental Oct4/OCT4 promoter. In experimental cell lines, the 200nt mOct4P4/hOCT4P3 lncRNA sequence element is sufficient to guide SUV39H1 dependent Oct4/OCT4 silencing, even in the absence of FUS.Here, we show that the human mOct4P4/hOCT4P3 to SUV39H1 binding, thereby imposing target specificity of the silencing complex towards the parental Oct4/OCT4 gene promoter. Our data highlight the evolutionary relevance of pseudogene lncRNA mediated control of parental gene expression and the role of FUS in instructing the formation of an epigenetic regulatory complex with target site specificity defined by a lncRNA component.We thus propose a model where FUS represents a licensing factor that mediates the accessibility of the 200 nucleotide mOct4P4 lncRNA–SUV39H1 complex targets conserved promoter elements of the ancestral Oct4 gene in trans, mediating gene silencing during mESC differentiation. To support the relevance of pseudogene lncRNA mediated epigenetic regulation of parental gene expression we tested whether this mechanism is conserved in human cells. To date, eight human POU5F1 pseudogenes have been annotated in the human genome25. Similar to mOct4P4, the human hOCT4P1, hOCT4P3, and hOCT4P4 pseudogenes have an exon structure that is similar to the OCT4 mRNA and show 81%, 82%, and 82% overall sequence identity to OCT4, respectively25. We previously showed that OCT4 is frequently expressed in ovarian cancer cell lines and controls cancer relevant pathways in OVCAR-3 cells15. This identifies OVCAR-3 ovarian cancer cells as ideal model system to validate conservation of pseudogene lncRNA mediated silencing of parental OCT4. hOCT4P3 lncRNA displays high sequence similarity to mOct4P4 and reproduces nuclear localization pattern in a series of human ovarian cancer cell lines . Expression of dCAS9-HAKRAB and respective sgRNAs in experimental mESCs and OVCAR-3 cells was validated by western blotting and RT-PCR  differentiation as model system to address the impact of reduced mOct4P4 lncRNA expression on self-renewal and early differentiation markers. dCAS9 empty and dCAS9 sgOct4P4 mESCs were cultivated in hanging drop cultures in the absence of the self-renewal factor leukemia inhibitory factor (see “Methods”). We found that upregulation of mOct4P4 expression was strongly impaired during EB differentiation of dCAS9 sgOct4P4 mESCs . In a subsequent step dCAS9 empty cells were stably transfected with an expression vector encoding short-guide RNAs (sgRNAs) that locate dCas9–HAKRAB to the promoter region of the PCR Fig. . We prevSCs Fig. . This efvel Fig. . Accorditin Fig. . On the vel Fig. . This efene Fig. . Based oOct4/OCT4 by mouse and human mOct4P4 and hOCT4P3 sense lncRNAs. This further implies the existence of defined lncRNA sequence elements essential for site specific targeting of SUV39H1 to the Oct4/OCT4 promoter.Our results demonstrate the evolutionary conservation of H3K9me3 dependent silencing of parental mOct4P4 lncRNA–SUV39H1 complex locates to the promoter of the ancestral Oct4 gene in trans17. In order to identify lncRNA regions essential for mOct4P4 function we used a mESC cell line stably expressing a flag-tagged version of the MS2 phage coat protein (MS2-flag mESCs) as well as mOct4P4 deletion constructs that were tagged with 24 repeats of the MS2 RNA stem loop motif  efficiently reduced Oct4 mRNA levels, as determined by quantitative RT-PCR  tether the flag-tagged MS2 phage coat protein to the Oct4 promoter and (ii) trigger increased H3K9me3 levels at the Oct4 promoter. Anti-flag ChIP experiments revealed that only MS2 RNA tagged full length, Δ200 and Δ400 Oct4P4-24xMS2 lncRNAs were able to locate the flag-tagged MS2 protein to the promoter of the ancestral Oct4 gene and to trigger a local increase of H3K9me3  but also a construct encoding a MS2 RNA motif tagged mOct4P4 lncRNA version that exclusively covers region 984–1183 (200 bp-mOct4P4-24xMS2)  5′ and 3′ located sequences to ensure nuclear lncRNA and (ii) region 984–1183 that directs H3K9me3 to the Oct4 promoter.We conclude that mOct4P4 lncRNA mediated silencing of Oct4 we aimed to identify mOct4P4 lncRNA interacting proteins. MS2-flag cells expressing full-length mOct4P4-24xMS2 and control mESCs expressing only a 24xMS2 stem loop control RNA were used to perform anti-flag RNA immunoprecipitation (RIP) experiments. Obtained control and mOct4P4-24xMS2 RNA-immunoprecipitates where run on denaturing polyacrylamide gels. After Coomassie staining, protein bands specifically appearing in eluates from mOct4P4-24MS2 RIPs were cut out from the gel and subjected to mass spectrometry , indicating that persistent localization of the mOct4P4 lncRNA at the Oct4 promoter is essential to maintain Oct4 silencing in differentiated cells17.We previously showed that the mOct4P4 lncRNA is required for the localization of FUS to the Oct4 promoter we performed ChIP experiments in mOct4P4 lncRNA knock-down pMEFs. Our results show that loss of endogenous mOct4P4 lncRNA displaced FUS from the Oct4 promoter in pMEFs  does not require FUS  or a 24xMS2 tagged hOCT4P3 lncRNA region (200 bp-hOCT4P3-24xMS2) that corresponds to the functional relevant 200 nucleotide mOct4P4 region  in human or mouse cells, respectively. This indicates functional conservation of Oct4 pseudogene lncRNA mediated silencing of parental gene expression in mouse and human cells. High overall sequence identity and conservation of mOct4P4 function in human cells suggested the existence of functionally relevant lncRNA regions.Here, we investigate the molecular mechanism and evolutionary conservation of mOct4P4 and hOCT4P3 lncRNA that is required for targeting of the lncRNA-SUV39H1 silencing complex to the promoter of the ancestral Oct4/OCT4 gene, resulting in local H3K9 tri-methylation. Binding of Oct4/OCT4 pseudogene lncRNA by SUV39H1 is in line with studies demonstrating interaction of SUV39H1 HMTases with pericentric RNAs, telomere repeat containing RNA (TERRA), LINE1 L1MdA 5′UTR elements, SINE B1 repeats and pRNAs of the rRNA cluster42. Direct interaction of mOct4P4 lncRNA with SUV39H1 was recently demonstrated by in vitro EMSA experiments (37). SUV39H1 HMTase–RNA-binding specificity is reported to be promiscuous and characterized by low sequence specificity. This lead to the hypothesis that the formation of lncRNA–SUV39H HMTase complexes with defined epigenetic function may depend on additional proteins or the presence of physiologically functional RNA:chromatin templates44.A deletion analysis identified a 200-nucleotide region in mOct4P4 lncRNA interacting proteins with a potential role in silencing parental Oct4. Here, we demonstrate that the RNA binding protein FUS has a critical role in Oct4/OCT4 lncRNA mediated silencing of OCT4. Loss of FUS prevents the formation of a full length mOct4P4/hOCT4P3 lncRNA–SUV39H1 silencing complex, abrogating the initiation and maintenance of Oct4/OCT4 silencing. Notable, FUS is dispensable for the function of the minimal sufficient mOct4P4/hOCT4P3 lncRNA version . Thus, we conclude that FUS does not have a central role in closing the Oct4/OCT4 promoter.RNA pull-down experiments revealed a series of Oct4 pseudogene lncRNA template to allow the binding of SUV39H1 to the 200-nucleotide region, thereby defining a specialized SUV39H1–lncRNA complex with selective target specificity towards the parental Oct4/OCT4 promoter. Importantly, FUS and SUV39H1 do not bind to the Oct4 mRNA in RIP experiments. This demonstrates that the specific interaction with FUS and the noncoding RNA-guided SUV39H1 HMTase represents a new biological feature of Oct4P4/OCT4P3 lncRNAs, that was acquired during pseudogene evolution. Future experiments will have to validate whether FUS has a more general role in epigenetic gene regulation by controlling the association of lncRNAs with epigenetic writers. In addition, the impact of Oct4/OCT4 promoter associated pseudogene transcripts on transcriptional initiation and Oct4/OCT4 promoter evasion remains an interesting issue to be addressed.We propose that FUS is critical for the structuring the long mOct4P4/hOCT4P3 lncRNA versions to the Oct4/OCT4 promoter. Thus, after FUS dependent silencing complex formation, SUV39H1 and the 200 nucleotide mOct4P4/hOCT4P3 lncRNA regions hold the information for selective targeting and epigenetic silencing of the parental Oct4/OCT4 gene promoter.In contrast to the selective requirement of FUS for full length pseudogene lncRNA function, we found that SUV39H1 is essential for targeting of both, the full-length and 200 nucleotide mOct4P4/hOCT4P3 lncRNA function may also represent a regulatory mechanism that restricts pseudogene-lncRNA mediated silencing to a defined biological context. Along these lines, PRMT1 dependent arginine methylation of FUS was recently shown to prevent the interaction with the CCND1 gene promoter-associated noncoding RNA-D (pncRNA-D), thereby blocking the repression of the HAT activity of the CBP/p300 HAT complex29. Addressing post-translational modifications of FUS may identify windows of mOct4P4/hOCT4P3 function in development and disease.The requirement of FUS as critical factor to license endogenous mOct4P4/hOCT4P3 also other pseudogene derived lncRNAs, such as DUXAP8 and DUXAP10 have been shown to interact with epigenetic writers46. However, DUXAP lncRNAs rather act as general scaffold for epigenetic regulatory complexes that do not selectively target the parental DUXA gene. In contrast, pseudogene PTENP1 antisense transcripts drive DNMT1 dependent silencing of the parental PTEN gene by paring with the 5′UTR of the nascent, sense PTEN RNA11. We experimentally validated that Oct4 and mOct4P4 are exclusively transcribed in sense orientation, thus excluding extended RNA:RNA interactions17. Thus, mOct4P4 and hOCT4P3 represent pseudogene sense lncRNAs that use a conserved mechanism to target and remodel the chromatin status of the parental gene promoter, located on a different chromosome.In addition to mOct4P4/hOCT4P3 to (ii) allow SUV39H1 binding to the 200 nucleotide region, followed by (iii) sequence specific targeting of the Oct4/OCT4 promoter, resulting in (iv) increasing local H3K9me3 and HP1 levels and Oct4/OCT4 silencing  FUS binds ing Fig. . The speOct4/OCT4 in trans may depend on complex long-range chromatin interaction of involved (pseudo)gene–loci, alternative DNA structures or the recruitment of additional factors. Elucidating mechanisms that functionally connect pseudogenes loci with ancestral genes will provide new insights into the power of pseudogenes encoded lncRNAs in fine-tuning the expression of ancestral genes in development and disease.Silencing of 47. OVCAR-3 cells were obtained from ATCC and cultured in RPMI-1640 medium (Lonza) supplemented with 20% (v/v) fetal bovine serum (FBS) (Lonza), insulin  and 1% (v/v) penicillin/streptomycin (Lonza). Primary mouse embryonic fibroblasts (pMEFs) were generated in house from 13.5 d.p.c. C57BL/6 mouse embryos. pMEFs were maintained in culture in DMEM (Lonza) supplemented with 10% (v/v) FBS (Lonza) and 1% (v/v) penicillin/streptomycin (Lonza). Cell lines were maintained as monolayers at 37 °C in a humidified 5% CO2 atmosphere.Feeder independent mESCs were cultured on 0.2% gelatin-coated plates using mESC self-renewal medium composed by Dulbecco’s modified Eagle’s medium (DMEM) (Lonza) supplemented with 15% knockout serum replacement (Gibco), 1% nonessential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), 0.1 mM β-mercaptoethanol, 1% penicillin/streptomycin (Lonza) and 1000 U/ml mouse leukemia inhibitory factorl-glutamine (Invitrogen), 0.1 mM β-mercaptoethanol, and 1% penicillin/streptomycin (Invitrogen). EBs were generated by cultivating 300 cells in hanging drops culture for 3 days. Subsequently, EBs were transferred to a low-attachment 24-well plates (Euroclone) and grown in suspension for the indicated days. Alternatively, embryoid bodies were transferred to adherent cell culture dishes and cultivated for the indicated time periods to obtain contractile cardiomyocyte structures. All used cells were tested for mycoplasma contamination in regular intervals.mESCs differentiation was obtained with a DMEM supplemented with 15% ES cell certified serum (Invitrogen), 1% non-essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), 1% mOct4P4-deletion constructs pLPC-200 bp-mOct4P4-24xMS2, pLPC-(−200 bp)-mOct4P4-24xMS2, pPLC-hOCT4P3-24xMS2, pLPC-200bp-hOCT4P3-24xMS2, and pMSCV-HA-MS2-Flag were packaged using 293GP cells. Forty-eight hours post transfection 10 ml of supernatants were harvested, filtered through a 0.45 µm filter and used to infect OVCAR-3 or ES cells in presence of polybrene. Twenty-four hours later medium was replaced with selection medium to obtain stable cell pools. Lentiviral vectors pLX-sgOCT4P3, pLX-sgOct4P4, and pHAGE-EF1α-dCas9-HA-KRAB (Addgene plasmid #50919) were packaged using 293T cells. Ten millilitre of supernatants were harvested, filtered through a 0.45 µm filter and used to infect OVCAR-3 or ES cells in presence of polybrene. Twenty-four hours later medium was replaced with selection medium to obtain stable cell pools. ES were transduced with retroviral vectors; OVCAR-3 cells with lentiviral and retroviral vectors. Cell lines infected with pLPC and pHAGE vectors were maintained in culture with 3 µg/mL of Puromycin and pMSCV and pLX vectors with 4 µg/mL of Blasticidine.Retroviral vectors such as pLPC-24xMS2, pLPC-mOct4P4-24xMS2, pLPC-®-LT1 transfection reagent . Transient transfection of siRNA was performed using Lipofectamine RNAiMAX reagent (Invitrogen) according to manufacturer’s suggestions. Following siRNAs were used for transient siRNA experiments: Fus: GCAACAAAGCUACGGACAA (Eurofins Genomics); Suv39h1: CCAAUUACCUGGUGCAGAA (Thermo Scientific Dharmacon); mOct4P4 GAGCAUGAGUGGAGAGGAA (Thermo Scientific Dharmacon). Control siRNA was used as a negative control: Non-Targeting siRNA#1, TAGCGACTAAACACATCAA (Thermo Scientific Dharmacon).Transient transfections of plasmids were performed using TransITExperimental cells were scraped in RIPA buffer , 0.5% sodium deoxycholate, 0.05% sodium dodecyl sulfate, 1 mM EDTA, 150 mM NaCl) and supplemented with protease inhibitors  and RNaseOUT (Invitrogen). After incubation at 4 °C for 20 min, cell lysates were centrifuged. The supernatant was precleared for 1 h at 4 °C with protein A/G PLUS-Agarose . The precleared supernatant was incubated overnight at 4 °C with rabbit polyclonal anti-TLS/Fus , mouse monoclonal anti-KMT1A/Suv39h1  or mouse monoclonal anti-FLAG M2, clone M2  antibodies. RNA–protein complexes were recovered with protein A/G PLUS-Agarose beads and were washed six times in RIPA buffer. An aliquot of beads containing immunoprecipitated samples were saved for western blotting analysis. Remaining beads were used to obtain immunoprecipitated RNA that was analyzed by qRT-PCR. A mouse monoclonal anti-HA antibody, clone HA-7  was used as negative control for immunoprecipitation.t test was performed to calculate p values and statistical significance was set at p < 0.05. Each finding was confirmed by three independent biological replicates, unless differently specified. Error bars represent standard deviation.A one-tailed Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Data 1Reporting Summary


Pseudomonas syringae by antagonizing RNA-directed DNA methylation (RdDM). Furthermore, we demonstrate that ROS1 facilitates the flagellin-triggered induction of the disease resistance gene RMG1 by limiting RdDM at the 3' boundary of a transposable element (TE)-derived repeat embedded in its promoter. We further identify flagellin-responsive ROS1 putative primary targets and show that at a subset of promoters, ROS1 erases methylation at discrete regions exhibiting WRKY transcription factors (TFs) binding. In particular, we demonstrate that ROS1 removes methylation at the orphan immune receptor RLP43 promoter, to ensure DNA binding of WRKY TFs. Finally, we show that ROS1-directed demethylation of RMG1 and RLP43 promoters is causal for both flagellin responsiveness of these genes and for basal resistance. Overall, these findings significantly advance our understanding of how active demethylases shape transcriptional immune reprogramming to enable antibacterial resistance.Active DNA demethylation has emerged as an important regulatory process of plant and mammalian immunity. However, very little is known about the mechanisms by which active demethylation controls transcriptional immune reprogramming and disease resistance. Here, we first show that the Arabidopsis active demethylase ROS1 promotes basal resistance towards  Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000) injects 36 type-III effectors into plant cells to dampen PTI , which are sensed by surface-localized pattern-recognition receptors (PRRs) . Plant Pmpen PTI . Plants mpen PTI . Most R mpen PTI . Plant Nmpen PTI . These impen PTI . Howeverathogens .NLRs often triggers ectopic cell death  pathway, which is directed by short interfering RNAs (siRNAs) (de novo methyltransferase DOMAIN REARRANGED METHYLTRANSFERASE 2 (DRM2) that catalyses methylation in all cytosine sequence contexts  (DNA methylation is an epigenetic mark that negatively regulates the transcription and transposition of transposable elements (TEs). It can also trigger transcriptional silencing of some genes carrying TEs/repeats in their vicinity . Homeost(siRNAs) . These s(siRNAs) . The res(siRNAs) . These s(siRNAs) . AGO4 tht not G) . During t not G) . On the t not G) . Both ROt not G) . ROS1 ist not G) . Mechanit not G) . Importat not G) .Pto DC3000 and the obligate biotrophic oomycete pathogen Hyaloperonospora arabidopsidis  contributes to basal resistance against Pto DC3000, and that ROS1-directed suppression of RdDM at RMG1 promoter ensures a proper induction of this gene during PTI. Furthermore, we retrieve the whole set of flg22-responsive genes that are controlled by ROS1 and show that PAMP-triggered inducibility at two ROS1 targets, namely RMG1 and RLP43, depends on the cis-effect of demethylation at their promoters. Furthermore, ROS1-directed demethylation at a specific sequence of the RLP43 promoter, which carries a functional ‘W-box’ WRKY-binding site, is critical for the DNA binding of PAMP-responsive WRKY TFs. Overall, this study reveals the extent to which ROS1 orchestrates Arabidopsis transcriptional immune reprogramming and unveils a crucial role for this demethylase in the regulation of WRKY-DNA binding at immune-responsive promoters.Here, we have characterized the Arabidopsis demethylase ROS1 in the context of antibacterial immunity. We first demonstrate that ROS1 positively regulates basal resistance against ros1 mutants exhibit enhanced spreading of Pto DC3000 in Arabidopsis leaf secondary veins (ros1-3 and ros1-4 mutant leaves were wound-inoculated with a GFP-tagged Pto DC3000 (Pto DC3000-GFP) , in which the biogenesis of 22 to 24 nt siRNAs is abolished. Interestingly, we found that both the enhanced vascular propagation and apoplastic growth of Pto DC3000-GFP detected in the ros1-3 mutant were reduced in ros1dcl23 mutants, and almost comparable to the phenotypes of Col-0-infected plants  . In addi3000-GFP . These dt manner . To deted plants . These dRMG1, an orphan TNL that is demethylated by ROS1 in its promoter  and the proximal repeat AtREP11 (At4TE29280) , which is located 216 bp upstream of RMG1 start codon, is unmethylated in Col-0 but hypermethylated in a ros1 mutant, as previously demonstrated by a targeted bisulfite Sanger sequencing approach  datasets generated in rosette leaves of untreated Col-0, ros1-3 and ros1dcl23 mutants. While no siRNA was retrieved at the 3’ boundary of AtREP11 in Col-0 plants, we detected the accumulation of siRNA species of different sizes in the ros1-3 mutant, ranging from low levels in the 21–22 nt species to high levels in the 23–24 nt species  . AtREP4 nditions . By contapproach . These r mutants . We foun0 plants . This re0 plants . To dete species . All thed region . CollectAtREP11 and its 3’ boundary could be required for the proper induction of RMG1 during flg22 elicitation. For this end, we challenged Col-0, ros1-3, dcl2-1 dcl3-1 (dcl23) and ros1dcl23 plants with either mock or flg22 for 6 hr and further monitored the mRNA accumulation of RMG1. We found a compromised induction of RMG1 in the ros1-3-elicited mutant that was not observed for Flg22-induced Receptor-like Kinase 1 (FRK1), which is not hypermethylated in ros1-3 mutants, and thus served as a control  and 115 less-repressed  genes were recovered  method and applied a statistical analysis that retrieved transcripts that are differentially accumulated at 6 hr after flg22 treatment in Col-0 to Col-0 . To idenecovered . To gainecovered . We foun regions . An incr0 plants . By cont mutants . The latros1-3 mutants (RBA1 (AT1G47370), which was previously shown to be repressed by DNA methylation in Col-0 and to recognize the bacterial effector HopBA1 in the Arabidopsis thaliana Ag-0 accession (receptor-like protein 43 (RLP43) (AT3G28890), which presents typical features of RLP PRRs (AT1G08940)  might additionally contribute to this process. Collectively, these data indicate that the flg22-triggered induction of these three genes is facilitated by ROS1, which prevents hypermethylation at discrete regions in their promoters. They also indicate that their promoter hypermethylation detected in ros1-3 mutants can be fully or partially altered upon concomitant removal of DCL2 and DCL3, as found at the phosphoglycerate mutase and RBA1 genes, respectively, or unchanged, as observed at the RLP43 locus.We next selected, from our RNA-seq datasets, candidate genes that were strongly induced by flg22 in Col-0 plants and showing pronounced compromised flg22 inducibility in elicited  mutants . These gccession , the orpRLP PRRs , and a p1G08940) . We furt mutants . The flg0 plants . Further mutants . By cont mutants . It is n mutants . It was  mutants . Because mutants , the aboros1-3 mutants, along with a strong impaired induction in ros1-3-elicited mutants, and subjected their hyperDMR sequences to Genome Association Tester (GAT)  data sets generated in flg22-challenged transgenic seedlings  . This aper (GAT) . By usins tested . By conts tested . Among thylation . Five oueedlings . Importaeedlings . For exa element . This obter gene . We founeporters . These r tissues . Collectros1-3 mutation alters the flg22 induction of RLP43  analysis. More specifically, we have expressed in bacteria Glutathione S-transferase (GST)-tagged AtWRKY18 and AtWRKY40, two well-characterized Arabidopsis WRKYs that are PAMP-responsive  previous DAP-seq and ChiP-seq data showing robust binding of different WRKY TFs at the ROS1-directed demethylated RLP43 promoter  thec region , and (3)promoter . As a poWRKY TFs . Using tomic DNA , while nomic DNA . By contnditions . CollectRLP43 promoter region, carrying a functional W-box cis-element, is required for WRKY TF DNA binding, suggests that the ROS1-directed suppression of promoter hypermethylation must be causal for flg22-triggered gene inducibility. To test this hypothesis, we generated Arabidopsis transgenic lines expressing a chimeric inverted repeat transgene bearing sequence identity to the RMG1 and RLP43 promoter regions that are subjected to ROS1-dependent demethylation  mutant, and this phenotype was exacerbated by knocking-down DME in this background  and down-regulated (115 genes) in response to flg22. These results suggest that ROS1 is equally capable of promoting the induction and repression of genes during flg22 elicitation. Some of these repressed targets might act as plant defence repressors or, alternatively, as modulators of plant developmental or physiological processes, possibly to redirect resources towards immunity and away from growth. It is noteworthy that the role of active demethylation in promoting repression of genes has previously been reported in the context of fruit ripening, whereby the tomato ed genes . Furtherros1dcl23 mutants   . The AtRWRKY TFs . Significkground . Furtherckground . Importaacterium . These d factors . Althougin vitro  and all experiments were performed on 5-week-old rosette leaves.ros1-3 (ros1-4 (SALK_045303), rmg1-1 (SALK_007034) and rmg1-2 (SALK_023944), the double dcl2-1 dcl3-1 (dcl23) (ros1-3 dcl2-1 dcl3-1 (ros1dcl23) mutants were used in this study.Besides WT Col-0, the single mutants ros1-3 , ros1-4  (dcl23) , and theThe pGEX-2TM-WRKY18 and pGEX-2TM-WRKY40 plasmids allow bacterial expression of the N-ter GST-tagged version of full-length WRKY18 and WRKY40 . Full-length cDNAs of each WRKYs were amplified and cloned into Gateway compatible entry vectors and subsequently recombined into the pGEX-2TM-GW destination vector.-RMG1p/RLP43p construct is a chimeric inverted repeat construct composed in each arm of the 180 bp and 137 bp sequences that are demethylated by ROS1 at the RMG1 and RLP43 promoters, respectively . The IR-RMG1p/RLP43p was synthesized and inserted by restriction enzyme digestion into the modified pDON221-P5-P2 vector by GenScript. A double recombination of this vector together with the pDON221-P1-P5r plasmid carrying the Cauliflower Mosaic Virus (CaMV) 35S promoter sequence in the pB7WG Gateway destination vector was performed to obtain the destination plasmid containing the 35Sp::IR-RMG1p/RLP43p construct. The plasmid was further introduced into the Agrobacterium tumefaciens C58C1 strain, which was then used for Agrobacterium-mediated plant transformation  or 1 μM of flg22 during 6 hr for the qRT-PCR and 24 hr for the GUS staining.The 1.5 Kb sequence located upstream of the start codon of d method . PrimaryPto DC3000 at a concentration of 5 × 106 cfu/ml (OD600 of 0.2 corresponds to 10 8cfu/ml). Inoculation was done on six secondary veins per leaf and two sites of inoculation per vein. The number of bacterial spreading events from the wound inoculation sites was quantified after 3 days under UV light using a macrozoom (Olympus MVX10). When the bacteria propagated away from any of the 12 inoculation sites, it was indexed as propagation with a possibility of maximum 18 propagations per leaf. The values from three independent experiments were considered for the comparative analysis. Statistical significance was assessed using a one-way ANOVA test and Tukey’s multiple comparisons test.Bacterial propagation in the secondary veins was assessed as described previously . About 1Pto DC3000 at 5 × 107 cfu/ml supplemented with 0.02% Silwet L-77, and immediately placed in chambers with high humidity. Bacterial growth was determined 3 days post-infection. For the quantification, infected leaves were harvested, washed for 1 min in 70% (v/v) EtOH, and 1 min in water. Leaf discs with a diameter of 4 mm were excised, grinded, and homogenized in 100 µl of water. Each data point consists of four-leaf discs. Fifteen microliters of each homogenate were then plated undiluted and at different dilutions. Bacterial growth was determined after 36 hr of incubation at 28°C by colony counting. The values from three independent experiments were considered for the comparative analysis. Statistical significance was assessed using a one-way ANOVA test and Tukey’s multiple comparisons test.Four plants per condition were dip-inoculated using Total Arabidopsis RNAs from two leaves per individual plant per condition were extracted using Nucleospin RNA plant kit (Macherey Nagel). Five hundred nanograms of DNase-treated RNA were reverse transcribed using qScript cDNA Supermix (Quanta Biosciences). cDNA was then amplified by real time PCR reactions using Takyon SYBR Green Supermix (Eurogentec) and gene specific primers. Expression was normalized to that of the Arabidopsis housekeeping gene Ubiquitin. Sequences of the primers are listed in McrBC digestion was performed as described previously  on 200 nDAP followed by qPCR was performed following a protocol modified from For each condition, genomic DNA was extracted from leaves of three independent 5-week-old plants using the CTAB (hexadecyl trimethyl-ammonium bromide) protocol . Then, 5E. coli Rosetta strains, carrying pGEX-TW-WRKY or empty vector for the GST alone, at a OD600nm = 0.3–0.6 were induced with 1 mM IPTG during 1 hr at 37°C. Expression of the WRKYs was then confirmed on a Coomassie gel.For each TF or control expressing GST alone, 200 ml cultures of Bacterial pellets were lysed and sonicated for 10 cycles of 10 s. The supernatants containing expressed proteins were incubated with washed MagneGST beads (Promega) (25 µl per TF and per DNA sample tested) during 1 hr at room temperature with gentle rotation. After five steps of washing with 1× PBS + NP40 (0.005%) and three steps with 1× PBS, beads bound with GST-WRKYs were mixed with 200 ng of sonicated DNAs and incubated 1 hr at room temperature with gentle rotation. Samples were then washed five times with 1× PBS + NP40 (0.005%) and two times with 1× PBS. Beads were then resuspended in 25 μl EB  and the samples heated during 10 min at 98°C. Supernatant was kept for further qPCR analysis.The equivalent of 1 μl of immunoprecipitated DNA was then amplified by real time PCR reactions using Takyon SYBR Green Supermix (Eurogentec) and gene specific primers, and normalized to the input DNA.Accumulation of low molecular weight RNAs was assessed by low molecular weight northern blot analysis as previously described . Total RGUS staining was performed as described previously . The staros1-3 and ros1dcl23 mutants were used for small RNA deep-sequencing . Custom libraries for ~16–40 nt RNAs were constructed and sequenced by Fasteris. We filtered all six libraries based on base-call quality of Q20 . We then selected a subset of read sizes comprised between 16 and 29 nt for further analyses. Reads were mapped to TAIR10 genome using bowtie  or 1 μM of flg22 for 6 hr, and mRNA-seq experiments further carried out . Reads were mapped to the TAIR10 genome using TopHat (v2.0.8b)   and allov2.0.8b) . An annoros1-3 samples in elicited vs mock conditions. For each gene, we kept the information of the type of regulation: ‘induced’ when the p-value after correction is ≤0.05 and the log2 fold-change >0, ‘repressed’, when the p-value after correction is ≤0.05 and the log2 fold-change <0, or ‘none’ for the rest of the genes.We identified differentially expressed genes separately for Col-0 and ros1-3 or the regulation in ros1-3 is at least two times less important than that of Col-0 (based on fold-change), the gene will be considered as ‘less-induced’. Likewise, a ‘repressed’ gene in Col-0 will be considered as ‘less-repressed’ if its repression is at least two times less important or if there is no regulation in ros1-3.In the following analysis, we considered four cases. If there is an induction in Col-0 and there is no regulation in ros1-3 and ros1dcl23 were extracted using DNeasy plant mini kit (Qiagen) and used for bisulfite sequencing (Bs-Seq) analyses . We filtered-out low quality reads using trim galore and aligned to TAIR10 genome using Bismark . Using this method, we selected 2943 protein coding genes. This list was then crossed with the list of ‘less-induced genes’, which allowed us to retrieve 102 candidates for further analyses.For this analysis, we focused on protein-coding genes having a hyper-DMR ‘within 2 Kb’ . We used the Genomic Association Tester (GAT)   to find er (GAT) . A TF wa In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This manuscript describes a carefully conducted study about the regulatory function of DNA (de)methylation in Arabidopsis defence gene expression. The authors show that DNA demethylation positively controls defence-related expression of a disease resistance gene, thereby contributing to basal resistance to bacterial infection. This paper clearly establishes a causal relationship between DNA methylation, transcription factor binding, and pathogen-responsive gene expression in plants.Decision letter after peer review:cis-regulates defense genes by erasing DNA methylation at promoter-regulatory regions" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Daniel Zilberman as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Detlef Weigel as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Jian-Kang Zhu (Reviewer #2); Jurriaan Ton (Reviewer #3).Thank you for submitting your article "The Arabidopsis demethylase ROS1 The Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:P. syringae pv. tomato (Pst). The current study focuses largely on two defence-related genes, RMG1 and RLP43, which are negatively controlled by ROS1. The authors show that ROS1-dependent DNA demethylation of a RdDM-targeted AtREP4 element in the RMG1 promoter positively controls defence-related expression of this resistance gene, thereby contributing to basal resistance against Pst. Subsequently, the authors selected genes displaying repressed induction by the bacterial PAMP flg22 and increased DNA methylation in the ros1-3 mutant. Using the epicistrome dataset of the O'Malley et al., 2016 study, a subset of these Flg22-inducible genes was analysed for methylation-sensitive TF binding sites, revealing enrichment of WRKY-binding W-box elements. In the final part of the manuscript, the authors employ state-of-the-art DAP-qPCR and artificial siRNA-directed re-methylation techniques to establish a causal relationship between ROS1-dependent DNA methylation of the W-box in the RLP43 gene promoter, its binding to WRKY18 and WRKY40 TFs, and Flg22-responsive expression.This interesting manuscript describes a carefully conducted study about the regulatory function of DNA (de)methylation in WRKY-dependent defence gene expression in Arabidopsis. The work follows a previous pioneering study by this group from 2013 , which established a role for RdDM and ROS1-dependent DNA de-methyaltion in Arabidopsis resistance against The authors generate compelling evidence that ROS1-dependent DNA demethylation at some defence gene promoters is required for WRKY-dependent induction following PAMP perception. The manuscript is clearly written, follows a logical narrative, and includes all details and data necessary to repeat experiments and re-analyze data.Essential revisions:ros1-3 mutant, of which 102 were "less- induced" and 115 were "less-repressed". These numbers don't add up though: 102 + 115 = 217. Furthermore, the hyperDMRs at these 219 (or 217?) genes are reported to be within the gene bodies plus 2Kb upstream/downstream regions. It would be helpful if the authors could show these data for the 115 less-repressed and 102 less-induced genes separately, and indicate the distribution of ROS-dependent methylation between gene bodies, 2 Kb upstream sequences and 2 Kb downstream sequences.1) Subsection “ROS1 directs promoter demethylation of flg22-induced ROS1 targets, mostly by antagonizing DCL2 and/or DCL3 functions”: The authors state that there were 219 Flg22-responsive candidate genes that are associated with regions that are hyper-methylated in the ros1-3 mutant? For these genes, one would expect that ROS1-dependent DNA demethylation facilitates binding of repressive TFs. Are the promoters of these Flg22-repressed genes also associated with methylation-sensitive W box elements?2) Related to the above point, could the authors comment on how ROS1-dependent DNA demethylation may control the 115 genes showing less flg22-induced repression in the RLP43 promoter of the ros1-3 mutant. Is this also true for the W-box element in the promoter of the RMG1 gene?3) Using their DAP-qPCR system, the authors convincingly demonstrate in Figure 5 that WRKY18-GST and WRKY40-GST have lower affinity to the methylated W-box within the  Essential revisions:1) Subsection “ROS1 directs promoter demethylation of flg22-induced ROS1 targets, mostly by antagonizing DCL2 and/or DCL3 functions”: The authors state that there were 219 Flg22-responsive candidate genes that are associated with regions that are hyper-methylated in the ros1-3 mutant, of which 102 were "less- induced" and 115 were "less-repressed". These numbers don't add up though: 102 + 115 = 217. Furthermore, the hyperDMRs at these 219 (or 217?) genes are reported to be within the gene bodies plus 2Kb upstream/downstream regions. It would be helpful if the authors could show these data for the 115 less-repressed and 102 less-induced genes separately, and indicate the distribution of ROS-dependent methylation between gene bodies, 2 Kb upstream sequences and 2 Kb downstream sequences.The number of flg22-responsive candidate genes is indeed 217. This mistake has been corrected. To address reviewers’ suggestions, we have added the distribution of ROS1-dependent demethylation regions between gene bodies, 2 Kb upstream sequences and 2 Kb downstream sequences. Furthermore, we now provide the hyperDMRs location for each ROS1 target from the 115 less-repressed and 102 less-induced gene sets.This information has been added in the Results section as well as in Supplementary file 1:“Among them, 102 less-induced  and 115 less-repressed  genes were recovered .”2) Related to the above point, could the authors comment on how ROS1-dependent DNA demethylation may control the 115 genes showing less flg22-induced repression in the ros1-3 mutant? For these genes, one would expect that ROS1-dependent DNA demethylation facilitates binding of repressive TFs. Are the promoters of these Flg22-repressed genes also associated with methylation-sensitive W box elements?ros1-3 mutants. By doing this analysis, we found that WRKYs enrichment was detected at only 2 candidate demethylated promoter regions. These data indicate that the flg22-triggered repression of these genes occurs mostly independently of WRKYs DNA binding. In addition, we noticed that other classes of TFs can bind to the demethylated promoter regions of flg22-repressed ROS1 targets. As proposed by the reviewers, ROS1 might direct demethylation of these promoters to facilitate binding of various repressive TFs, thereby ensuring a proper repression of these genes during PTI. We have now included a new figure presenting these results . Furthermore, we have added the following sentences in the Results and Discussion sections of the revised manuscript to describe and discuss these new results:To address this point, we performed a GAT analysis on 28 less-repressed ROS1 targets, which exhibit dense hyperDMRs at their promoter regions in “By contrast, only 2 out of 28 less-repressed stringent targets displayed WRKY TFs enrichment at their demethylated promoter regions ”.“It is noteworthy that the proposed mechanism might also hold true for other TF families, which exhibit in vitro binding at the demethylated promoter regions of flg22-induced ROS1 targets . […] Consistent with this hypothesis, different classes of TFs were found to preferentially bind to the demethylated promoter regions of the flg22-repressed ROS1 targets .”3) Using their DAP-qPCR system, the authors convincingly demonstrate in Figure 5 that WRKY18-GST and WRKY40-GST have lower affinity to the methylated W-box within the RLP43 promoter of the ros1-3 mutant. Is this also true for the W-box element in the promoter of the RMG1 gene?RMG1, which was difficult to reproducibly detect using DAP-qPCR analysis. Furthermore, the presence of W-box cis-elements in the close vicinity of the RMG1 hyperDMR is expected to complexify the interpretation of DAP-qPCR results as the primers flanking this region also amplify W-box sequences that are not subjected to ROS1-dependent demethylation. By contrast, the DAP-seq datasets at the RLP43 demethylated promoter region revealed robust in vitro binding of several WRKY TFs, which was convincingly detectable by DAP-qPCR analysis. Furthermore, only a single –and functional– W-box cis-regulatory element is present right in the middle of the RLP43 hyperDMR, which is devoid of W-box in its surrounding. The RLP43 promoter is therefore the most appropriate candidate to investigate the relevance of demethylation in WRKY TFs binding using our DAP-qPCR system. In the revised manuscript, we are now providing an explanation for why we made this choice. In the future, we intend to conduct DAP-seq analyses in Col-0 versus ros1 mutants, which should address reviewers’ comments at RMG1 but also at other ROS1 targets showing similar features. This approach will also determine the extent to which ROS1-directed demethylation facilitate WRKY TFs binding at the whole genome level.The available DAP-seq datasets revealed a weak in vitro binding of WRKY TFs at the demethylated region of The following sentence has been added in the Results section of our revised manuscript:RLP43 promoter for this specific analysis was based on (1) previous DAP-seq and ChIP-seq data showing robust binding of different WRKY TFs at the ROS1-directed demethylated RLP43 promoter , (2), the presence of a single W-box cis-element located right in the middle of this genomic region  and, (3) the central role of this W-box in the flg22-responsiveness of the RLP43 promoter .”“The rationale for using


We have previously reported that Brahma related gene 1 (BRG1) directly interacts with SREBP1c and SREBP2 to activate pro-lipogenic transcription in hepatocytes. We report here that BRG1 deficiency resulted in reduced processing and nuclear accumulation of SREBP in the murine livers in two different models of non-alcoholic steatohepatitis (NASH). Exposure of hepatocytes to lipopolysaccharide (LPS) and palmitate (PA) promoted SREBP accumulation in the nucleus whereas BRG1 knockdown or inhibition blocked SREBP maturation. Further analysis revealed that BRG1 played an essential role in the regulation of SCAP expression. Mechanistically, BRG1 interacted with Sp1 and directly bound to the SCAP promoter to activate SCAP transcription. Forced expression of exogenous SCAP partially rescued the deficiency in the expression of SREBP target genes in BRG1-null hepatocytes. In conclusion, our data uncover a novel mechanism by which BRG1 contributes to SREBP-dependent lipid metabolism.Sterol response element binding protein (SREBP) is a master regulator of cellular lipogenesis. One key step in the regulation of SREBP activity is its sequential cleavage and  Lipid is a major biological macromolecule that plays diverse roles in orchestrating differentiation, proliferation, migration, communication, survival, and death of mammalian cells . On the in vivo (Sterol response element binding protein (SREBP), initially identified and characterized by the Brown and Goldstein laboratory, represents a family of transcription factors considered master regulators of lipid metabolism . Three min vivo . The relin vivo . On the in vivo .trans-membrane factors  utilizes the energy derived from ATP hydrolysis to mobilize nucleosomes along the chromatin and modulate chromatin accessibility. We have previously shown that BRG1 is an important moderator of liver injury linking specific transcriptional events to the alteration of liver function . ImportaSmarca4f/f mice  with freshly added protease inhibitor (Roche) as previously described , b. NuclSCAP, 5′-TCACGTTGCAGCCGTCTTCCTT-3′ and 5′-CAGGATGCCA ATCCAGACAACG-3′; human BRG1, 5′-TCATGTTGGCG AGCTATTTCC-3′ and 5′-GGTTCCGAAGTCTCAACGATG-3′; human FASN, 5′-CTTCCGAGATTCCATCCTACGC-3′ and 5′-TGGCAGTCAGGCTCACAAACG-3′; human LDLR, 5′-GAC GTGGCGTGAACATCTG-3′ and 5′-CTGGCAGGCAATGCTT TGG-3′; mouse Scap, 5′-CCGAGGATGACCCTGACTGA-3′ and 5′-AGAGCAGCCCATGGTTGTAGA-3′; mouse S1p, 5′-CT ACTATGGAGGAATGCCGACAG-3′ and 5′-CTCCGTTCTGT GGCAAATAGGG-3′; mouse S2p, 5′-ACGGCGGAAAGCAA GGATGCTT-3′ and 5′-GTGCCAAAGTCTGCATCAGCGT-3′; mouse Fasn, 5′-TTTAAAGGGAGGGAGGGAGA-3′ and 5′-GG CAGGATAGGGAAACACTGA-3′; mouse Ldlr, 5′-TGTGAAT TTGGTGGCTGAAAAC-3′ and 5′-AATAGGGAAGAAGATG GACAGGAAC-3′. Ct values of target genes were normalized to the Ct values of housekeekping control gene  using the ΔΔCt method and expressed as relative mRNA expression levels compared to the control group which is arbitrarily set as one.RNA was extracted with the RNeasy RNA isolation kit (Qiagen). Reverse transcriptase reactions were performed using a SuperScript First-strand Synthesis System (Invitrogen) as previously described . Real-tiSCAP proximal promoter, 5′-ATACTTCCCTCCGGTGTCCAC-3′ and 5′-ACCTCTCACCTCCACCTTTAC-3′; SCAP distal promoter, 5′-AAATGCGAGGACATGTACAATAC-3′ and 5′-ATTTAAAAGCTAAGTTGAC-3′. A total of 10% of the starting material is also included as the input. Data are then normalized to the input and expressed as % recovery relative the input as previously described (Chromatin immunoprecipitation (ChIP) assays were performed essentially as described before . In brieescribed , c. All t-test was performed using an SPSS package. Unless otherwise specified, p values smaller than 0.05 were considered statistically significant.Sample sizes reflected the minimal number needed for statistical significance based on power analysis and prior experience. Two-tailed Student’s Smarca4-flox mice with the Alb-Cre mice. In the first model in which the mice were fed a high-fat diet (HFD) for 16 weeks, it was observed that nuclear SREBP1/2 levels were appreciably increased in the livers of the HFD-fed mice compared to the control diet-fed (chow) mice; up-regulation of nuclear SREBP1/2 by HFD feeding was more modest in the LKO livers than in the WT livers  mice by crossing the T livers . In the FASN, a prototypical SREBP1 target gene, and the expression of LDLR, prototypical SREBP2 target gene, in HepG2 cells , which has been reported to induce SREBP target gene expression in macrophages , inducedG2 cells . Knockdoactivity . Consistactivity . Similaractivity . Finallyactivity .Because BRG1 deficiency was associated with impaired SREBP maturation in hepatocytes, we hypothesized that BRG1 might play a role regulating the expression levels of SREBP processing enzyme(s). We therefore compared the expression of the three SREBP processing enzymes, SCAP, S1P, and S2P, in WT and LKO livers. As shown in Next, we evaluated the effect of BRG1 depletion or inhibition on SCAP expression in cultured hepatocytes. Exposure of HepG2 cells to LPS + PA treatment robustly augmented SCAP expression at both mRNA  and protNext, it was determined whether regulation of SCAP expression by BRG1 in hepatocytes occurred at the transcriptional level. An SCAP promoter-luciferase fusion construct  was transfected into HepG2 cells. Treatment with LPS + PA stimulated the SCAP promoter activity by more than twofold and BRG1 over-expression further augmented the SCAP promoter activity in a dose-dependent manner . When thFasn expression and a 36% increase in Ldlr expression in the SCAP over-expressed LKO cells compared to the mock over-expressed LKO cells (Finally, exogenous SCAP was introduced into BRG1-deficient hepatocytes to address the question as to whether it could rescue the expression of SREBP target genes. As shown in KO cells . AccordiKO cells .trans-location and subsequent cleavage of nascent SREBPs catapults these master regulators of lipid metabolism into the nucleus to orchestrate pro-lipogenic transcription. This process is mediated by the well-conserved SCAP-S1P-S2P axis. We have previously shown that the chromatin BRG1 contributes to hepatic lipid metabolism by functioning as a co-activator for SREBP1/2 (via activating SCAP transcription (The SREBP1/2 . Here wecription . Our datcription . Metabolcription . Genomewcription . It is icription  and in ccription , suggestSCAP promoter appears to be influenced by epigenetic modifications. Another noteworthy finding is that although SCAP re-introduction in the BRG1 LKO cells largely restored SREBP processing , SREBP tin cis. Because BRG1 can interact with both SREBP1 (There are several lingering issues that deserve further attention in future studies. First, the broader biological/pathobiological significance of our finding, i.e., whether BRG1 can contribute to SREBP maturation in other pathophysiological conditions such as insulin stimulation or fast-feed cycle remains unclear. Of note, it has been previously shown that insulin-induced transcription of SREBP target genes in pre-adipocyte correlates with increased recruitment of BRG1 to the SREBP target promoters although it was not determined whether BRG1 could directly promote insulin-induced SREBP nuclear translocation . Second,h SREBP1  and SREBh SREBP1 , it is pIn summary, we present evidence to demonstrate that BRG1 can contribute to cellular lipid metabolism by, in addition to acting as an SREBP co-factor, promoting SCAP-dependent SREBP maturation. These data not only place BRG1 in the center of metabolic programming but provide renewed rationale for targeting BRG1 in the intervention of human diseases related to lipid metabolic disorders.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Nanjing Medical University Ethics Committee on Humane Treatment of Experimental Animals.ZF and YX conceived the project. MK, YZ, and JS designed the experiments, performed experiments, collected data, and analyzed data. YX wrote the manuscript. ZF handled the funding. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.