Chromatin structure is an essential regulator of gene expression. Its state of compaction contributes to the regulation of genetic programs, in particular during differentiation. Epigenetic processes, which include post-translational modifications of histones, DNA methylation and implication of non-coding RNA, are powerful regulators of gene expression. Neurogenesis and neuronal differentiation are spatio-temporally regulated events that allow the formation of the central nervous system components. Here, we review the chromatin structure and post-translational histone modifications associated with neuronal differentiation. Studying the impact of histone modifications on neuronal differentiation improves our understanding of the pathophysiological mechanisms of chromatinopathies and opens up new therapeutic avenues. In addition, we will discuss techniques for the analysis of histone modifications on a genome-wide scale and the pathologies associated with the dysregulation of the epigenetic machinery. Unlike prokaryotic organisms, eukaryotic DNA needs to be compacted to facilitate its localization within the cell nucleus. The level of DNA and chromatin compaction is defined as two different states depending on its permissiveness to the transcriptional machinery. Euchromatin is defined as an “open” permissive state of chromatin and heterochromatin as the “closed”, non-permissive state . The opeIn the 60s, PTMs of the amino-terminal tails of histones have been identified as the targets of different epigenetic factors dubbed writers, readers and erasers. The epigenetic writers affix epigenetic marks on histones amino acids. Epigenetic erasers remove deposited PTMs, and readers are able recognize specific epigenetic marks and recruit chromatin remodeling complexes able to translate the associated information. Genetic defects in genes encoding a number of epigenetic players lead to pathologies defined as chromatinopathies such as Rubinstein Taybi syndrome , Kabuki syndrome 1  or SOTOS syndrome (OMIM #277590) among others [Post-translational modifications of histones play a key role in gene regulation, in particular during neurogenesis and differentiation of the three neural lineages. Neurogenesis occurs during embryogenesis but also in the adult brain . MethylaIn this review, we will discuss the chromatin structure and dynamics, focusing on post-translational modification of histones during neuronal differentiation and the consequences of defects in the epigenetic machinery in pathologies. Finally, we will discuss the methods of analysis of these large-scale histone modifications.9 base pairs (bp), i.e., 6 × 109 bp in the nucleus of a diploid cell [Human genetic information, which is supported by DNA, consists of 3 × 10oid cell . This coThe different states of chromatin play essential roles in all DNA transactions, i.e., transcription, repair and replication of DNA . Two staTwo forms of heterochromatin exist: constitutive and facultative heterochromatin. Facultative heterochromatin can switch to euchromatin, permissive to the transcriptional machinery. The repressive H3K27me3 mark regulated by the Polycomb repressive complexes is enriched at facultative heterochromatin . ConstitMoreover, heterochromatin is preferentially located at the nuclear periphery in interactions with the nuclear envelope. In the 3D hierarchy of the genome, two compartments have been defined: the A compartment (euchromatin) enriched in H3K27ac and H3K39me3 and transcriptionally active, and the B compartment (heterochromatin), associated with a silent state of transcription, enriched in H3K9me3 . In contIn addition to these two conventional compartments, a compartment I has been described in colon cells. In the normal colon, this I compartment is located at an intermediate position between the two canonical A and B compartments, interacts with these two compartments and is associated with a relatively low level of transcription. It is distinguished from the two other compartments by an enrichment in H3K27me3 . The stuThe living chromatin model, which allows the study of coupling between chromatin folding and epigenetic regulation, shows that the maintenance of the epigenome is regulated by 3D compartmentalization. This compartmentalization allows the local concentration of epigenome effectors, increasing their capacity to diffuse an epigenomic signal at a long distance . In addiThe first fundamental level of higher-order chromatin structure is called the nucleosome. This structure is composed of an octamer of histones and DNA wrapped around it ,20,21. TThe nucleosome core particle (NCP) has an axis of symmetry. The axis is in a plane perpendicular to the H2A/H2B dimer and passes through the H3/H4 dimer. The H2A/H2B dimer contains six amino acids of H2A  and two amino acids of H2B  that create a negatively charged environment. This acid patch is involved in chromatin compaction by allowing interactions with the N-terminal tail of the adjacent histone H4 nucleosome ,26.In addition to the nucleosome core particle, the linker histone or internucleosomal histone H1 forms a new chromatin structure called chromatosome . H1 has The second level of DNA compaction involves the formation of the 30 nm chromatin fiber . H1 playAt the 3D level, chromatin fiber can form loops through local interactions that delimitates topologically associated domains (TADs). DNA sequences inside a TAD interact with each other more frequently than sequences outside. These TADs are typically delimited by the interaction of the anchor CTCF protein and cohesin. These regions delineate enhancer/promoter interactions and thus play a role in gene expression . The CTCHistones, like other proteins, are synthesized in the cytoplasm. To prevent histone mismatch, so-called histone chaperone proteins bind to neosynthesized histones involved in the transport of histones to the nucleus . These cHistones from the nucleosome core particle comprise a secondary structure known as the histone fold, amino- and carboxyterminal extensions, an amino-terminal tail and a carboxyterminal tail for H2A. The histone fold is constituted by three helixes connected by loops according to a α1-L1-α2-L2-α3 model. The α1 and α3 helix are relatively short (9 to 14 amino acids), unlike the α2 helix with an average of 29 amino acids ,23. The In addition to the conventional canonical histones, several histone variants, defined as non-allelic isoforms of canonical histones exist . VariantIn the 1960s, it was discovered that histones from the nucleosome core particle may be subject to PTM of amino acids located mainly in their amino-terminal tail  but alsoHistone modifications affect chromatin compaction, thus regulating gene transcription and are therefore associated with epigenetic processes. Enzymes that catalyze PTMs are considered as epigenetic writers; so-called readers are proteins able to read these PTMs, while erasers remove them. Histones undergo different types of PTMs, such as acetylation, methylation, phosphorylation, ubiquination, polyADP-ribosylation, SUMOylation, deimination, citrullination, proprionylation, crotonylation, or isomerization of proline residues ,52,53,54cis-regulatory sequences  has been given . Six famAnother important type of histone PTMs is methylation (CH3) of lysine (K) and arginine (R) or histidine (H) residues . This reKMT can be subdivided into two groups: KMT containing the Su(var)3–9 domain, Enhancer of Zeste, trithorax  and lysine methyltransferases, which do not contain a SET domain . The SET domain is involved in the catalytic activity at the histone tail, while the second group targets the central part of the histone. Methylation readers are composed of different types of domains allowing them to associate with methylated lysine. Examples of characterized domains are: tudor domain, MBT, WD40, ADD, zing finger CW domain, BAH, Ankyrin, PWWP, DCD, TTD and plant homeodomain (PHD) .Lysine demethylases (KDM) are classified into two families: amines oxidase demethylases dependent on the flavin adenine dinucleotide (FAD) (example: KMD1A) and the family of the Jumonji domain proteins . LSD1 acThe combination of different PTMs deposited on histones defines regulatory functions such as enhancers, active promoters, repressed or chromatin in a bivalent state . BivalenRegarding methylation, the characteristic repressive marks correspond to the trimethylation of H3K9 and H3K27 . H3K9me2An implication of these repressive histone modifications was studied in tumoral context. In this case, a disorganization of the three genomic compartments is found with a displacement of compartment B from the nuclear periphery to the interior of the nucleus. The preferential organization of the compartments is lost and heterogeneously distributed throughout the nucleus. Colon tumor cells show downregulation of genes in compartments B and I associated with block hypomethylation. In compartment I, downregulated genes are found enriched in H3K27me3 and those in compartment B by H3K9me3 and/or by methylation at the promoter. These mechanisms of block hypomethylation of B and I compartments are also found during successive passages and are characteristic of cells that have acquired excessive division .The H3K4 methylation mark is associated with transcriptionally active genes. Enhancers are marked with H3K4me1, active genes by H3K4me2 and active promoters and transcription initiation sites with H3K4me3. The presence of H3K4me1 increases the binding of the chromatin remodeling complex BAF at enhancers ,78. MLL,Methylation of H3K79, localized in the globular domain of H3, is an activating mark and DOT1l catalyzes its mono-, di- or trimethylation and appears to be the only enzyme catalyzing this reaction for this residue ,83. TrimH4R3me2a is associated with a decondensed chromatin state promoting histone acetylation. In the case of oxidative stress, OGG1 recruits the arginine methyltransferase PRMT1 and promotes the increase in the H4R3me2a mark at the promoter of c-Myc. The increase in H4R3me2a allows the recruitment of YY1 and promotes c-Myc gene transcription in response to oxidative stress .Different techniques can be used to characterize histone marks. Histone post-translational modifications can be studied using global quantification methods such as western blot, colorimetric or fluorometric techniques and mass spectrometry, based on the observation that a PTM of histones changes its molecular weight ,89. AnalIn addition, many techniques are available to study the distribution of post-translational modifications at a genome-wide scale and to define their target genomic loci. These newer high-throughput techniques are used to analyze the distribution of histone PTMs and associated chromatin structure in two main ways: chromatin accessibility by assay for transposase-accessible chromatin using sequencing (ATAC-seq) or targeted chromatin mapping, such as chromatin immunoprecipitation-sequencing (Chip-seq), CUT and RUN or CUT and TAG ,91,92,93The use of ATAC-seq does not require any prior knowledge of the epigenetic mechanism that regulates a given genomic region as it does not rely on the use of antibodies directed against a specific histone mark . ATAC-secis-regulatory sequences, but also against histone PTMs.Chromatin immunoprecipitation (ChIP), followed by high-throughput sequencing (seq), is now a commonly used method for genome-wide identification of specific post-translational modification-associated loci. ChIP-seq relies on the use of specific antibodies to determine the binding site of a target protein at a genome-wide scale. The antibodies can be directed against transcription factors to identify ChIP can be performed on native (N-ChIP) or cross-linked (X-ChIP) chromatin. X-ChIP consists in stabilizing DNA-protein interactions by bridging with chemical agents such as formaldehyde. The chromatin is then fragmented by ultrasound or enzymatic digestion. The accessibility of chromatin to the micrococcal nuclease varies according to the cell type, and different batches of enzymes may present variable activity, which may lead to biases. The size of the fragments obtained, following fragmentation, is important since it correlates with the resolution of ChIP .cis-regulatory regions but also to analyze PTMs of histones [2+ and will allow the cleavage on both sides of the protein binding site. The DNA fragments will be submitted to sequencing by sequencing adapter ligation [Cleavage Under Targets and Release Using Nuclease is an another high-throughput technique more recent than ChIP or ATAC-seq. This technique maps the interactions between DNA and proteins, but, unlike ChIP-seq, does not require pre-labelled fragmentation of the DNA and tends to better preserve DNA/protein interactions. Therefore, this method can be used to analyze the binding of transcription factors to their histones . No crosligation .This technique also uses antibodies specifically directed against transcription factors or PMTs of histones. The Tn5 transposase, also used in ATAC-seq, is coupled to protein A. This fusion protein will guide the Tn5 transposase to the binding site of the antibody directed against the protein of interest. The transposase is coupled with adapters for subsequent sequencing .The coupling of different techniques allows the visualization of different activating or repressive histone marks correlated with the chromatin opening state ,98.Post-translational modifications of histones in the modulation of chromatin compaction and access to the transcriptional machinery are involved at the early stages of development, and particularly during neuronal differentiation.During embryogenesis, the neuroepithelium gradually expands to form the neural tube. The cells lining the neural tube are neural stem cells (NSC). These NSCs are multipotent, i.e., able of self-renew and differentiate. They allow the development of the central nervous system, including the spinal cord and brain by differentiating to neuronal, astrocytic and oligodendrocytic lineages . In embrNf1) in radial glial cells from the dentate gyrus of adult mice restores this tripotent potential by generating the three neural lineages [Neurogenesis is not limited to the embryonic stage. Neurogenesis also occurs in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus of the hippocampus in the adult mammalian brain ,103,104.lineages . Adult nlineages .Animal models  as well as human induced pluripotent stem cells (hIPSC) have made it possible to overcome the limited inaccessibility of human neurons to study epigenetic mechanisms during neuronal differentiation. Several protocols have been developed to obtain NPCs from hIPSC with neuronal rosettes obtained in cultures resembling the neural tube. These NSCs can be differentiated into different types of neurons  using appropriate protocols ,109,110.OCT4, NANOG) and promote access to the specific differentiation program associated with an increase in NPC markers  [The mechanisms of NSCs differentiation are spatio-temporally regulated by changes in the accessibility of transcription factors to the promoter of genes that regulate the differentiation to a specific lineage. Changes in chromatin will gradually limit access to multipotency genes, with downregulation of pluripotency genes (et OTX2) .HDAC are essential for the maintenance of self-renewal and proliferation of the neural stem cell by maintaining, through their catalytic activity, transcriptional suppression on target genes. Several studies have been conducted to elucidate the role of HDACs in neuronal differentiation. Transient and incomplete HDAC inhibitors such as 2-propylpentanoic acid , trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA) and sodium butyrate (NaB) have been used for this purpose. HDAC inhibition is associated with histone hyperacetylation and excessive inhibitor concentrations, leading to cell death .Ngn1, Math1 and p15 through an increase in H4 acetylation at their promoter. Similar results were observed in vivo in mice where VPA-treated animals showed an increase in H4 acetylation at the Ngn1 promoter inducing its expression [Ngn1 promoter together with a decrease in the repressive H3K27me3 and H3K9me3. These modifications are independent of the AKT/mTOR pathway activated after VPA treatment [The use of VPA induces neuronal differentiation by affecting histone acetylation. In rat embryonic hippocampus neural progenitor cells, VPA reduces cell proliferation and initiates neuronal differentiation. This is mediated by an increase in the expression of pression . In NSCsreatment .PAX6 promoter, which promotes the induction of neural progenitor cells [VPA also promotes neuronal differentiation in H9 human embryonic stem cells (HUES). Addition of VPA to the differentiation medium increases the expression of neuronal markers  and those specific to the GABAergic and dopaminergic subtypes. The markers of mature neuron, NEUROD1, and NeuN are expressed 30-times higher after VPA addition. This increase is also observed for oligodendrocyte markers (MBP) but not for astrocyte markers (S100β). Differentiation toward the astrocytic lineage is decreased under these conditions, which is usually observed during differentiation to the neuronal lineage. Length and connections established by neurites are also significantly increased by VPA addition . Besidesor cells .PAX6, SOX1, ZIC2 and ZNF521). In addition, the use of TSA inhibits the expression of early neural genes, PAX6 and SOX1, between days zero to four but increases their expression between days four to eight. This is evidenced by an increase in the proportion of NPCs. TSA inhibits HDAC1/5 and 8, which allows the increase in H3K9ac and the differentiation to the neural lineage. The expression of early neural genes such as PAX3/6, SOX1, POU3F2 is improved by HDAC1/5 and 8 knockdown, while pluripotency genes are downregulated. These three enzymes allow an appropriate neural differentiation by preventing an early differentiation [H3K9ac is also involved in the kinetics of neural differentiation. Qiao et al., showed that during the first four days of neural differentiation of HUES-9, H3K9 acetylation decreases and then gradually increases between days four to eight. The kinetics of H3K9 acetylation at specific promoters allows the silencing of pluripotency genes and the expression of early neural genes. This is supported by experiments on p300 and the use of TSA. Indeed, shRNA depletion of p300, involved in H3K9 acetylation, inhibits the expression of early neural genes , in a bivalent conformation in mESCs, a resolution of the bivalent state by methylation of the transcription start site H3K4 is observed [On the other hand, genes associated with differentiation and development are found in a bivalent state with trimethylated marks of H3K27 and H3K4 at the mESC stage. These bivalent domains are reduced in differentiated cells and developmental genes acquire either repressive or activating marks. For genes induced during neural differentiation  and upregulation of postmitotic neuron-specific markers.Another KMT, MLL1 plays an important role in neuronal differentiation by methylating H3K4  and by bpression . Studiespression . This leThe EHMT2 methyltransferase is involved in the demethylation of mono- and dimethylated H3K9  and alsoSox19b transcription factor during embryonic development in zebrafish, leads to a decrease in H3K27me3 at the Ngn1 and ascl1a promoters, without affecting the histone acetylation state. This decrease is due to a decreased expression of EZH2, that catalyzes H3K27 trimethylation. Premature entry of neural tube NSCs into neuronal differentiation is then induced by an increase in the level of Ngn1 and ascl1a transcription, showing a role for the repressive H3K27me3 mark during neuronal differentiation. Thus, the elimination of this repressive marker controls the balance of self-renewal/differentiation of NSCs in zebrafish. The importance of a proper functioning of this balance is further highlighted by the neural tube abnormalities associated with a decrease in the surface area of the diencephalon and telencephalon in SOX19B-deficient embryos [The use of a morpholino antisense oligonucleotide against the  embryos .Nestin-positive precursors. The generated neurons are predominantly glutamatergic and form excitatory synapses. NeuroD1 initiates this process of neuronal differentiation by binding to bivalent chromatin regions (enriched in H3K4me3 and H3K27me3) present in microglia at genes involved in neuronal development and differentiation such as Bhlhe22, Brn2 or Pou3f3. Genes upregulated after NeuroD1 binding show a decrease in H3K27me3 and an increase in H3K4me3. Furthermore, microglial identity is suppressed by modification of histone marks at the promoters of microglia-specific genes. A decrease in H3K4me3 and an increase in H4K27me3 on its promoter regions is observed [Lentiviral-induced overexpression of the neurogenic factor NeuroD1 is able to reprogram mouse microglia and oligodendrocytes into mature neurons both in vitro and in vivo. These neurons are β3 tubulin-positive and respond to N-methyl-D aspartate (NMDA) stimulation. NeuroD1-mediated conversion proceeds directly from microglia to mature neurons without intermediates such as neural stem cells or observed .As previously discussed, 3D compartmentalization that plays a role in the maintenance and regulation of the epigenome and by corollary, also influences neuronal differentiation processes. Comparison of Drosophila embryonic and neuronal cells indicates that three-dimensional organization is modulated during neuronal differentiation with dynamic changes in enhancer/promoter interactions . A numbeFor a more global approach for understanding neuronal differentiation, it is now possible to reconstruct the 3D organization of the genome using predictive methods. An improved population-based modeling approach and a probabilistic framework to model a population of 3D structures of entire diploid genomes was tested on human lymphoblastoid cells. The predicted 3D structures correctly identified many of the features of the lymphoblastoid genome obtained from imaging experiments, including interchromosomal distances between gene loci, their interactions and preferred nuclear locations of chromosomes . HistoneA number of neurodevelopmental disorders and cancers result from mutations in components of the epigenetic machinery ,141,142.Among rare diseases, chromatinopathies account for 5% to 10% of Mendelian disorders . More spKmt2d, present an alteration of neurogenesis with a decrease in DCX+ cells, a marker of immature neurons. These mice exhibit memory defects and visuospatial learning deficits associated with H3K4 hypomethylation in the hippocampus. These defects could be reversed by the use of TAK-418, a KDM1A inhibitor [Mecp2-mutated mouse model revealed that an improvement of the phenotype is observed after restoration of systemic administration of MECP2 [Cpb+/− mice, the defect in CREBBP HAT activity results in long-term memory defects associated with H2B hypoacetylation in the hippocampus [Numerous models have been developed for the study of chromatinopathies and in particular those leading to neurodevelopmental disorders . Mouse mnhibitor . Regardiof MECP2 . Furtherof MECP2 . In Rubipocampus . Similarpocampus .The development of hiPSC-based models represents an interesting alternative to animal models by mimicking steps of human neuronal development ,154,155.The complexity of the human brain and neuronal development cannot be limited to single layer models. To mediate these issues, 3D models have been developed. Brain organoids reproduce part of the development of the nervous system and model the sequential generation of neurons and glial cells . InteresIndeed, it was shown that methylated cytosines outside of CG contexts, called mCH, were similar between brain organoids at Day 40 and Day 60 of differentiation and mid-fetal cortex. The accumulation of mCH in cerebral organoids and the fetal cortex is associated with loci that overlap with putative super-enhancers. Corresponding regions are actively expressed during early fetal stages and then become repressed during the neonatal/postnatal transition. In addition, comparative transcriptomic analyses showed a strong correlation between brain organoid differentiation and the human mid-fetal neocortex. The genetic pathways are similar to those of the fetal cortex, including NPC proliferation and self-renewal, migration, adhesion, delamination and differentiation suggesting that in vitro organoids tend to recapitulate embryonic development ,161. OrgThe large phenotypic overlap between different chromatinopathies , Cornelia de Lange ) can be explained by epigenetic modulations occurring within interconnected networks ,164. ThiFMR1 locus, which is hypermethylated in this disease. This leads to a silencing of the FMR1 gene in IPSCs maintained in derived neurons [Based on a better understanding of the pathophysiological mechanisms leading to these pathologies, new therapeutic perspectives have emerged. HDAC inhibitors have shown promising results in Rubinstein Taybi syndrome, being able to reverse the memory defects in mouse models, and to improve morphological and electrophysiological defects of iNeurons ,169. Fur neurons . This apEpigenetic mechanisms have been shown to be essential for modulating gene expression and enabling the acquisition of cellular identity. Here, we focused on the importance of epigenetic modulations in neuronal differentiation, but also on the pathological consequences associated with deficits in the epigenetic machinery. Neurodevelopmental disorders represent about 75% of chromatinopathies ,166. A bA multi-omics integration of these episignatures coupled with data from transcriptomic and chromatin dynamics high-throughput techniques would allow to refine the pathophysiology and the biological pathways deregulated in these chromatinopathies. The joint development of brain organoid models  allows n


Pseudomonas stutzeri A1501, a plant-associated diazotrophic bacterium, prefers to conform to a nitrogen-fixing biofilm state under nitrogen-deficient conditions. The extracytoplasmic function (ECF) sigma factor AlgU is reported to play key roles in exopolysaccharide (EPS) production and biofilm formation in the Pseudomonas genus; however, the function of AlgU in P. stutzeri A1501 is still unclear. In this work, we mainly investigated the role of algU in EPS production, biofilm formation and nitrogenase activity in A1501. The algU mutant ΔalgU showed a dramatic decrease both in the EPS production and the biofilm formation capabilities. In addition, the biofilm-based nitrogenase activity was reduced by 81.4% in the ΔalgU mutant. The transcriptional level of pslA, a key Psl-like (a major EPS in A1501) synthesis-related gene, was almost completely inhibited in the algU mutant and was upregulated by 2.8-fold in the algU-overexpressing strain. A predicted AlgU-binding site was identified in the promoter region of pslA. The DNase I footprinting assays indicated that AlgU could directly bind to the pslA promoter, and β-galactosidase activity analysis further revealed mutations of the AlgU-binding boxes drastically reduced the transcriptional activity of the pslA promoter; moreover, we also demonstrated that AlgU was positively regulated by RpoN at the transcriptional level and negatively regulated by the RNA-binding protein RsmA at the posttranscriptional level. Taken together, these data suggest that AlgU promotes EPS production and nitrogen-fixing biofilm formation by directly activating the transcription of pslA, and the expression of AlgU is controlled by RpoN and RsmA at different regulatory levels. When bacteria adhere to solid surfaces, they produce many extracellular polymeric substances in the extracellular matrix in response to various environmental signals, such as stress signals, and then the bacteria are embedded in a complicated three-dimensional structure called a biofilm ,2. It isPseudomonas aeruginosa is a model organism for studies of biofilm formation and exopolysaccharide production [P. aeruginosa. P. aeruginosa strains isolated from chronic infection patients with cystic fibrosis (CF) usually overproduce alginate and are called mucoid strains. In non-mucoid strains, alginate production is repressed, and psl and pel are the primary exopolysaccharides [algU-mucABCD) encodes the sigma factor AlgU and the anti-sigma factor MucA [algD and other alginate biosynthetic genes by directly binding the AlgU-binding sites (GAACTT-N16/17-TCCAA) in the promoters [P. aeruginosa PAO1; however, promoter mapping indicated that AlgU is probably not directly responsible for the transcription of the psl operon [P. aeruginosa also contributes to environmental stress tolerance and virulence [Pseudomonas species, the functions of AlgU are also characterized. It was reported that AlgU regulates the motility and translocator expression in P. fluorescens and controls the abiotic stress tolerance and virulence in P. syringae [oduction ,6. At lecharides . Alginattor MucA ,9. AlgU romoters . It is al operon . In addiirulence ,13. In osyringae ,15,16.P. stutzeri A1501 is a plant-associated bacterium that colonizes the root surface and endophytically invades the root tissues of host plants [nif genes in response to nutrient stress have been reported [t plants ,18. A150t plants ,20. Analreported ,22,23,24pslA plays an essential role in the production of Psl-like exopolysaccharides, and RpoN directly activates the transcription of pslA [algU homologous gene, encoded by PST1223, was identified in A1501, however, its function is still unclear. In the present study, an algU mutant strain and an algU overexpression strain were constructed and an AlgU dependent regulation network on biofilm formation was established. The phenotype analysis indicated that AlgU played important roles in exopolysaccharide production, biofilm formation and nitrogen fixation. Results from qRT-PCR assays and β-galactosidase activity assays, together with DNase I footprinting assays confirmed that AlgU directly activated the transcription of pslA and AlgU was positively regulated by RpoN at the transcriptional level and negatively regulated by the RNA-binding protein RsmA at the posttranscriptional level.Recently, biofilm formation was linked to nitrogen fixation in A1501. Wang et al. reported the effect of physiological conditions on biofilm formation and the architecture of nitrogen-fixing biofilms. In addition, biofilms could enable bacteria to fix nitrogen under aerobic conditions . Compara of pslA . An algUP. stutzeri wild-type A1501 and its derivatives were grown in LB medium or minimal K medium 3·H2O 0.01 g·L−1, Na2MoO4·H2O 0.01 g·L−1 pH 6.8) supplemented by the desired carbon and nitrogen sources indicated in the text at 30 °C in shake flasks at 220 rpm. Escherichia coli strains were grown at 37 °C in LB medium. Antibiotics were used at the following concentrations: kanamycin (Km): 50 μg·mL−1, tetracycline (Tc): 10 μg·mL−1, and chloromycetin (Cm): 34 μg·mL−1.The bacterial strains and plasmids used in this study are shown in algU gene in P. stutzeri A1501 was inactivated with the suicide plasmid vector pK18mob [algU gene by PCR  was cloned into pLAFR3, a derivative of the low-copy-number wide-host-range plasmid RK2 [algU and individually introduced into the algU mutant and A1501 via triparental mating, generating the algU-complemented strain and algU overexpression strain.The  pK18mob . The prie by PCR . The PCR3 vector , generatsmid RK2 . The conP. stutzeri wild-type A1501 and its derivatives were grown  in a minimal K medium containing 50 mM lactate and 6 mM NH4Cl. The cultures were centrifuged to collect the supernatants, and then exopolysaccharides were precipitated and isolated from the supernatants by adding two volumes of cold absolute ethanol. The purified exopolysaccharide was dissolved by ddH2O, and the exopolysaccharide content of samples was measured quantitatively by using the phenol-sulfuric acid method [P. stutzeri wild-type A1501 and selected strains were grown  overnight in LB medium. The cells were collected by centrifugation and resuspended in a nitrogen-free minimal K medium containing 50 mM lactate. The OD600 of the cell suspensions was adjusted to 0.2 with a nitrogen-free K medium containing 50 mM lactate. A total of 160 μL of each suspension was dropped into the wells in a 96-well PVC microtiter plate. The microtiter plate wrapped with parafilm was placed without agitation in an incubator for 48 h. At the end of incubation, the non-adhered planktonic cells were gently removed using a pipette, and the wells were washed twice with ddH2O. Then, 0.1% CV solution in ethanol was used to stain the biofilm in the well. The free CV in the wells was washed away with ddH2O, and the well-adhered CV was solubilized with 30% acetic acid and quantified by measuring the OD560 of the resulting solution using a microplate reader.A biofilm formation assay using the crystal violet (CV) method was performed according to a previous method . P. stutP. stutzeri strains were grown overnight in LB medium, and then, cells were collected and washed twice with nitrogen-free K medium containing 50 mM lactate. The OD600 of the cell suspension was diluted to 0.2 with nitrogen-free K medium containing 50 mM lactate, and 10 mL of the cell suspension was removed into a 110 mL flask under aerobic conditions (21% oxygen) and was incubated for 48 h at 30 °C under static conditions and then 10% acetylene was added. The gas samples (0.25 mL) were taken from the flask at regular intervals  and were analyzed on a poly divinylbenzene porous bead GDX-502 column using a gas chromatograph SP-2100 with a flame ionization detector . The ethylene content in the gas sample was calculated by reference to an ethylene standard. The nitrogenase activity was expressed as nanomoles of ethylene (mg protein × h)−1. The concentration of total protein was measured using the Bradford protein assay kit .Biofilm-based nitrogenase activity was measured with the acetylene reduction test according to a previous method, with modifications . P. stutalgU or algU-overexpressing strain A1501, bacteria were grown under tested conditions as described. Cells were harvested and centrifuged for 10 min at 4 °C, and pellets were quick-frozen in liquid nitrogen and stored at −80 °C. Total RNA was isolated with an innuPREP RNA Mini Kit  following the manufacturer’s instructions. RNA reverse transcription was conducted using a reverse transcription kit (HiScript® II 1st Strand cDNA Synthesis Kit (+gDNA wiper), Vazyme Biotech Co., Ltd., Nanjing, China). Genomic DNA contamination was quickly and completely removed by treatment with gDNA wiper Mix. The genomic DNA contamination was assessed by using primer pair nifL-rnfA F/R  overnight in an LB medium. The cells were collected by centrifugation and washed twice with a nitrogen-free K medium containing 50 mM lactate. Then, the OD600 of the cell suspensions was adjusted to 0.1 with a nitrogen-free K medium containing 50 mM lactate. Ten millilitres of the cell suspension were removed into a 60 mL flask. The cell suspension was incubated for 4 h at 30 °C with shaking under a nitrogen fixation atmosphere containing 0.5% oxygen. After incubation, the cells were collected by centrifugation and resuspended in 1.0 mL of Z-buffer in 2.0 mL EP tubes. Two to three drops of chloroform were added into the tubes and mixed by inverting several times to lyse cells. The tubes were placed with tops open in an incubator at 30 °C for 40 min. Two hundred microliters of ONPG solution (4 mg/mL) were added to the tubes, and the time of colour change was recorded. The reaction was stopped with 500 μL of 1 M Na2CO3 solution. Then, 150 μL of the reaction solution was added to a 96-well microplate. The OD420 and OD550 were measured using a microplate reader. The β-Galactosidase activity was expressed as Miller units [To test the transcriptional expression of the native or mutant promoter of d pGD926 , and thes method . P. stuter units .algU gene region containing the 500 bp native promoter was cloned by PCR with the designed primers  . Protein concentrations were determined using the Bio-Rad protein assay reagent kit. The separated protein bands were transferred to a polyvinylidene difluoride (PVDF) membrane . The membrane was incubated with a monoclonal antibody against the flag peptide  for 12 h at 4 °C and then washed three times in TBS/Tween buffer before being incubated with an anti-mouse secondary antibody  for 2 h. Detection was performed using an HRP-DAB chemistry kit .The lag-F/R) . The algalgU. First, first-strand cDNA was synthesized with the GSP1 primer  method  was used to determine the transcriptional start site for 1 primer , which wimer AAP . The 5′ pslA promoter region was PCR amplified from the plasmid pJET1.2-pslA using primers of pJET1.2F2 and pJET1.2R2 (FAM). For each assay, 450 ng probes were incubated with different amounts of protein AlgU in a total volume of 40 µL. After incubation for 30 min at 30 °C, 10 µL solution containing about 0.015 unit DNase I (Promega (Beijing) Biotech Co., Ltd., Beijing, China) and 100 nmol freshly prepared CaCl2 was added and further incubation was performed at 37 °C for 1 min. The reaction was stopped by adding 140 µL DNase I stop solution . Samples were firstly extracted with phenol/chloroform, then precipitated with ethanol. Pellets were dissolved in 30 µL MilliQ water. The preparation of the DNA ladder, electrophoresis and data analysis were the same as described before [DNase I footprinting assay was performed by Tolo Biotech according to a method previously described . For thed before , except algU promoter region using the primers EMSA-algU-F/ EMSA-algU-R, 300-bp nifA promoter region using the primers EMSA- nifA-F/ EMSA- nifA-R, 300-bp negative control DNA using the primers EMSA-nc-F/ EMSA-nc-R)  and 10% glycerol). After incubation for 30 min at 30 °C, the reaction system was loaded into 4% PAGE gel buffered with 0.5 × TBE. Gels were scanned with ImageQuant LAS 4000 mini .The His-tagged RpoN was produced by the pET-28a expression system based on the previous method . The DNASA-nc-R)  and was SA-nc-R) . For eacalgU 3′-UTR ssRNA oligonucleotides were synthesized in GenePharma Co., Ltd., Shanghai, China. The binding reaction for purified RsmA protein and target RNA was carried out at room temperature in the binding buffer ; 10 μL binding reactant was loaded into the 5% non-denaturing polyacrylamide gel and the electrophoresis was performed at 4 °C. The RNA was removed from the gel to the nylon membrane with the SEM-DRY transfer cell . Then, RNA was cross-linked into the nylon membrane and was hybridized with the specified ssDNA oligonucleotides labelled with digoxin. The RNA detection was carried out using the X-film with the DIG northern starter kit  according to the manufacturer’s instructions.The RsmA protein was expressed and purified according to a previous method . The 28 p-values of 0.05 or less were considered statistically significant. Error bars represent the standard deviation (SD) of the three biological replicates from a single experiment.Statistical analysis was performed by one-way ANOVA. Pseudomonas, the effect of algU mutation and overexpression on exopolysaccharide production was tested in P. stutzeri A1501. First, we constructed the algU insert mutant strain with the suicide vector pk18mob  in the algU mutant, the major regulator rpoN did not have changed expression in the algU mutant. No AlgU-binding motif was found in the upstream region of the nifLA and nifHDK operons. Therefore, it is unlikely that AlgU affects nitrogenase activity by directly activating the expression of nif genes.Reports have shown that zosphere . In addimutation A. In addU mutant B. AlthoualgU abolished the transcriptional expression of pslA. To test whether algU directly regulated the transcription of pslA, we inspected the nucleotide sequence upstream of the initiation codon ATG. Interestingly, a 27 bp DNA sequence (GAACTCATCCGGAGAGGCACGGTCGGA) that highly matched the AlgU-binding motif (GAACTT-N16/17-TCCAA) was identified in the promoter region of pslA (pslA was determined by DNase I footprint assays. As shown in GAACTCATCCGGAGAGGCACGGTCGGAGCAGGAGTCC) of the pslA promoter, which overlapped the putative AlgU-binding site located at positions ranging from −10 to −35 from the transcription start site. In addition, four 500-bp DNA fragments containing the putative wild-type (WT) or mutant , which contained mutations in genes that mainly control nitrogen fixation. The expression of algU significantly decreased by 3-fold in the rpoN and nifA mutants. The determination of the algU transcription initiation site with a 5′RACE assay showed that the transcript started from an adenine residue, which is the 50th nucleotide upstream from the ATG initiation codon. A putative AlgU-binding site (GAGAACTTTTGCGTAAAGCCCGGGTCTATTC) and two putative RpoN-binding sites (GGN10GC) were predicted in the promoter region of the algU gene; however, no binding site for NifA was found. The locations of the two putative RpoN-binding sites were both atypical . The gel mobility shift analysis was used to detect the interaction between RpoN protein and algU promoter DNA, and it was found that the RpoN protein could specifically bind to the algU promoter DNA  was predicted, which overlapped with the ribosome-binding site in the algU 5′UTR sequence. The purified RsmA protein was also found to significantly change the mobility of the RNA band, the binding between RsmA protein and AlgU RNA gradually increased as the concentration of RsmA increased  the quorum-sensing pathway, (2) the Gac-Rsm pathway, and (3) the c-di-GMP signalling pathway. Recently, the Rpo-Gac-Rsm network was reported to regulate nitrogen-fixing biofilm formation in A1501, and RpoN plays a key role [P. aeruginosa. First, AlgU acts as a regulator to directly activate the transcriptional expression of rsmA [algD expression is regulated by AlgU and RpoN [algD transcription by directly binding the algU promoter, and the binding sites for AlgU and RpoN overlap upstream of the algD transcription start site. Notably, RpoN is required as a positive factor for algD expression in the P. aeruginosa muc23 strain, in which AlgU affects the transcription of algD [pslA transcriptional start site, and these two sites overlapped (pslA in A1501 [pslA. Under some conditions or in some specific strains, the possible regulatory mechanism of AlgU and RpoN competing with each other to activate or repress the expression of pslA could not be excluded.Although exopolysaccharide composition, construction, and biofilm formation regulation vary greatly among the different key role . Crossta of rsmA . Second, of rsmA . Third, and RpoN . RpoN re of algD . Intereserlapped A. RpoN win A1501 . In thisP. stutzeri A1501 is similar to that in P. aeruginosa: AlgU upregulated the expression of rsmA (rsmA, it could not regulate Psl production or biofilm formation through mainly RsmA because RsmA was previously reported to repress pslA expression and biofilm formation in A1501 [P. aeruginosa and the quantitative analysis of sigma factor regulons indicated RpoN and AlgU co-regulate the expression of 49 genes [algU reduces by more than 2 times in rpoN mutant, however, no RpoN-binding site is found in the promoter region of algU [algU was partly controlled by RpoN, interestingly, two RpoN-binding sites were predicted. The locations of predicted RpoN-binding sites are not the −12 and −24 positions, which implies that these RpoN-binding sites are untypical or fake. The purified RpoN specifically binds the algU promoter DNA indicated in algU. It is not clear which pathway AlgU is involved in that mainly affects biofilm-based nitrogenase activity. Because the biofilm formation could enable A1501 to fix nitrogen under the aerobiotic condition and the algU mutation reduced the biofilm formation, it is reasonable to speculate the AlgU could protect the nitrogenase from oxygen or active oxygen species. The significant change in the expression of several oxidative stress response genes in the AlgU mutant implies the possibility that the AlgU mutation increases the amount of active oxygen that would repress nitrogenase activity (Pseudomonas species, pslA is the new exopolysaccharide synthesis gene that has been determined to be directly activated by AlgU. The pslA gene is conserved and widespread in P. aeruginosa, P. stutzeri, P. protegens, P. fluorescens and so on. The expression of pslA is reduced in the P. aeruginosa AlgU mutant; however, no obvious match to the AlgU consensus promoter element is found in the upstream region of pslA [Pseudomonas species is worth further study.The complex regulation mode between AlgU and RsmA in  of rsmA B, and Rs of rsmA . Althougin A1501 . Schulz 49 genes . In addi of algU . In thisactivity . To our  of pslA , and the


FLOWERING LOCUS C (FLC) is the major determinant of flowering in Arabidopsis thaliana. In rapid-cycling A. thaliana accessions, which bloom rapidly, FLC is constitutively repressed by autonomous pathway (AP) genes, regardless of photoperiod. Diverse AP genes have been identified over the past two decades, and most of them repress FLC through histone modifications. However, the detailed mechanism underlying such modifications remains unclear. Several recent studies have revealed novel mechanisms to control FLC repression in concert with histone modifications. This review summarizes the latest advances in understanding the novel mechanisms by which AP proteins regulate FLC repression, including changes in chromatin architecture, RNA polymerase pausing, and liquid–liquid phase separation- and ncRNA-mediated gene silencing. Furthermore, we discuss how each mechanism is coupled with histone modifications in FLC chromatin.Proper timing of flowering, a phase transition from vegetative to reproductive development, is crucial for plant fitness. The floral repressor  Arabidopsis thaliana accessions are classified into winter-annual and rapid-cycling types based on the requirement of long-term winter cold for rapid flowering (FLOWERING LOCUS C (FLC) and FRIGIDA (FRI) genes  and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1  genes . FLC, whNSTANS 1 . FRI, a S 1 (FLC .FLC expression (LUMINIDEPENDENS (LD) was first isolated  induces early flowering by repressing pression . Since Lisolated , severalAP genes . For theve state . Additioanscript . HoweverEmerging evidence suggests that multiple layers of transcriptional processes determine transcript level . In addiFLC has been re-examined based on its chromatin architecture.The 3D property of chromatin plays a vital role in transcriptional regulation . AlthougArabidopsis  mutant line exhibits increased FLC expression and delayed flowering  consistently show reduced FLC looping and decreased FLC level -type ATP-dependent chromatin remodeling complex, also participates in FLC repression by affecting FLC gene looping (BAF60 (BAF60 RNAi) display an increased number of FLC gene loops and upregulated expression of FLC, thereby producing the late-flowering phenotype in long days. This finding suggests that BAF60 plays a negative role in loop formation. Histone modifications including H3K27me3, H3K9Ac, and H2A.Z replacement, are also altered in the BAF60 RNAi lines; thus, the effect of BAF60 on FLC gene looping may be mediated through histone modifications. One caveat is that BAF60 is not a typical AP gene because the BAF60 RNAi lines do not show delayed flowering in short days. The increased FLC level caused by BAF60 RNAi is probably masked by the additional targets of BAF60. Therefore, BAF60 may also be an FLC repressor which acts on gene looping. The functional interdependency between GH1-HMGA family proteins and BAF60 needs further analysis.Chromatin loops, defined as the intergenic or intragenic bending of chromatin, are observed genome-wide in bidopsis . The 5′-in loops . Importaecycling . A recenecycling . GH1-HMGecycling . Similarlowering . Given tcription . HoweverFLC gene . ImportaLC level . Therefo looping . It has FLC chromatin has an R-loop around its 3′-end, where the antisense transcript COOLAIR is transcribed  increases the level of R-loops, indicating that the N6-methyladenosine (m6A) modification of RNA is involved in R-loop resolution  family genes  are RNAs that are not translated into proteins. They function in transcriptional or post-transcriptional gene regulation, structural organization of nuclear bodies, and genome integrity control . The FLCCOLDWRAP , all of exposure . Among tproteins .COOLAIR  site in COOLAIR , CPSF100, and FH INTERACTING PROTEIN 1  site  site . This co COOLAIR ,b.COOLAIR-processing complex likely controls the FLC chromatin state through FLD. FLD assembles into a complex with LD and SET DOMAIN GROUP 26 (SDG26), which causes the removal of H3K4me1 deposited at FLC chromatin , could be associated with  COOLAIR . Moreoveechanism . TherefoArabidopsis. Decades of studies have proposed unique mechanisms for FLC regulation, such as control of Pol II pause–release mechanism, modulation of chromatin architecture, and processing of ncRNA triggered by phase-separated machinery. The studies on such mechanisms are still in their infancy and heavily dependent on genome-wide transcriptome analyses. Thus, a large portion of the current models presented in this review has yet to be validated. Further verification of the proposed mechanisms through biochemical, genetic, and molecular work would be valuable to develop a better understanding of AP. In addition, this pathway has been closely linked with the epigenetic modification of FLC chromatin, particularly in relation to the changes in histone methylation patterns in the AP mutants. However, the detailed mechanism connecting the regulatory function of AP proteins described in this review and the epigenetic silencing of FLC remain largely unknown; thus, further studies are required.This review summarizes the latest research progress in the autonomous pathway in FLC expression even in the presence of AP proteins in winter-annual Arabidopsis  grant funded by the Korea Government .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


LCN-2, and alternative splicing (AS) of ubiquitination-related gene WWP-2. Finally, CLIP-seq data analysis indicated that the first UC motif was the critical site for PCBP-1 binding to its targets. In this study, we provided evidence that PCBP-1 could regulate the expression of LCN-2 gene expression associated with neuroinflammation and AS of WWP-2 in relation to protein ubiquitination. These findings thus provided novel insights into the potential application of PCBP-1 as the disease pathophysiological or therapeutic target for neurodegenerative disease.PCBP-1, a multifunctional RNA binding protein, is expressed in various human cell/tissue types and involved in post-transcriptional gene regulation. PCBP-1 has important roles in cellular Iron homeostasis, mitochondrial stability, and other cellular activities involved in the pathophysiological process of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD). However, it remains enigmatic whether PCPB-1 is associated with the pathogenesis of PD. In this study, we cloned and constitutively overexpressed PCBP-1 in rat PC12 cells (PC12 cell is the common cell line studying neurodegenerative disease include PD). RNA-seq was performed to analyze PCBP-1-regulated differentially expressed genes (DEGs) and alternative splicing events (ASEs) between control and PCBP1-overexpressed cells. GO and KEGG pathway analyses were performed to identify functional DEGs and alternatively spliced genes. Consequently, we validated PCBP-1-regulated genes using RT-qPCR. Finally, we downloaded CLIP-seq data from GEO (GSE84700) to analyze the mechanisms of PCBP-1’s regulation of gene expression and ASEs by revealing the binding profile of PCBP-1 on its target pre-mRNAs. Overexpression of PCBP-1 partially regulated the ASE and expression of genes enriched in neuroinflammation and protein ubiquitination, which were also associated with PD pathogenesis. Moreover, RT-qPCR assay verified the PCBP-1-modulated expression of neuroinflammatory genes, like  Parkinson’s disease (PD), a progressive neurodegenerative condition primarily affecting the midbrain, is the second most leading cause of neurodegenerative disorder in the elderly population, following Alzheimer’s disease (AD), while the etiological factors largely remain unknown . To dateRBPs bind to their respective target RNAs through highly conserved RNA-binding domains (RBDs) in order to regulate the functionality of those RNAs. Poly (rC)-binding protein 1 (PCBP-1), a key member of the PCBP family of proteins, is widely expressed in various human tissue types. PCBP-1 plays an important role in the regulation of transcription and RNA metabolism at various levels . The prein vitro studies related to neurodegenerative diseases were performed in PC12 (rat pheochromocytoma) cells because of their inherent capacity to produce neuronal synapses and express key neuronal proteins .2 at 37°C. PCBP-1 plasmid was transfected in PC-12 cells by Lipofectamine-2000 reagent , according to the manufacturer’s protocol, and cells were harvested at 48 h post-transfection for RT-qPCR analysis.PC-12 cells  were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS), 50 μmol/L β-mercaptoethanol, 100 U/mL penicillin, and 100 μg/mL streptomycin with 5% CO–ΔΔCt method  was the house-keeping control for PCBP-1 overexpression analysis. cDNA synthesis and RT-qPCR assay were done using Bestar SYBR Green RT-PCR Master Mix  and Bio-Rad S1000 machine. The primer sequences are listed in the t method , and paiRQ1 DNase  was used to remove genomic DNA contamination from total RNA prior to construction of directional RNA-seq library by KAPA mRNA-Seq Kit . RNA concentration and quality were measured using NanoDrop 2000 . 1.5% Agarose gel electrophoresis was employed to check RNA integrity.Poly-A tailed mRNAs were purified from 1 μg of total RNA and fragmented, followed by conversion into respective cDNA. Then, Roche DNA Adaptors  were ligated to end-repaired and A-tailed double-stranded (ds) cDNA. Ligation products corresponding to 300–500 bps were then processed and stored at −80°C for downstream applications. dUTP incorporated cDNA strand was not amplified for strand-specific sequencing.Illumina Novaseq 150 nt paired-end kit was applied for cDNA library preparation for high-throughput sequencing.2-N bases containing raw reads and reads with less than 16 nt were first eliminated, followed by FASTX-Toolkit v0.0.13 guided trimming of adaptor sequences and low-quality bases. The Ensemb Rat genome (Rnor_6.0) alignment of the clean reads was performed by TopHat2 , allowinDifferentially Expressed Genes (DEGs) were screened by R Bioconductor package edgeR  by applyt-test was performed, and the events with significant P-value and FDR cut-off were finally considered.Alternative splicing (AS) and regulated AS (rAS) events were classified by identification of 10 types of splice-junction events, including alternative 5′ splice site (A5SS), exon skipping (ES), alternative 3′splice site (A3SS), mutually exclusive exons (MXE), intron retention (IR), mutually exclusive 5′UTRs (5pMXE), mutually exclusive 3′UTRs (3pMXE), and cassette exon using the ABLIRC pipeline . To asseThe primers used for individual DEG validation using RT-qPCR assay are listed in In addition, AS and rAS events were validated by qRT-PCR assay. The primer sequences are shown in the Gene Ontology (GO) terms and KEGG pathway analyses were carried out to functionally categorize the identified DEGs using KOBAS 2.0 server . FurtherTo further clarify the target genes of PCBP-1, we downloaded and reanalyzed the Clip-seq data of PCBP-1 from GEO (GSE84700) in Jurkat T cells as described earlier , sequencP < 0.05) were selected. The PCBP-1 peaks overlapping with the input peaks were detected and eliminated by independent simulation analysis. The PCBP-1 target genes were determined by analyzing the locations of PCBP-1 binding peaks on the Rat genome by calling binding motifs of PCBP-1 by Homer software. Finally, we performed overlapping analysis for GSE84700 CLIP-seq data with PCBP-1 overexpression using ABLIRC analysis methods.A computational simulation was used to randomly generate reads similar in number and length as in peak and reads with at least 1 bp were clustered at peaks. The overlapping output reads were then mapped back to the respective gene to generate random max peak height, repeatedly for 500 times. The peak heights which were higher than random max peaks  model in PC12 cells to investigate PCBP-1’s role in gene regulation.  control . In addi control . The six control . SignifiFor investigation of the PCBP-1’s role in the post-transcriptional regulations, we analyzed the RNA-seq data of PCBP1-OE and control samples by adjusting absolute fold change ≥ 2 and FDR ≤ 0.05 with the edgeR package and determined the DEGs . SubsequP ≤ 0.05, To understand the functional roles of PCBP-1 in PC12 cells, KEGG pathway and GO enrichment analyses were carried out. GO analysis showed that responses to hypoxia, protein phosphorylation, metabolic process, response to the drug, regulation of transcription DNA-templated at the molecular function (MF) level were enriched . MoreoveWe found that PCBP-1 overexpression activated the neuroinflammation-related gene expression. Furthermore, we conducted RT-qPCR analysis to individually assess the levels of lipocalin-2(LCN2), Mx1, Mx2, Bcl7a, Ep300, Lfit3, and Oasla mRNAs. Previous studies have shown that  LCN2 are highly expressed in neuroinflammation . Our resWe assessed the RNA-seq data quality for differential splicing in PC12 cells. A total of 49.4 ± 1.5 million uniquely mapped reads were retrieved from both samples, including 33.5% junction reads. The RNA-seq data detected 268438 novel splice junctions and 174170 annotated exons . SubsequThe ABLIRC tool was applied to elucidate the PCBP-1 target genes from the CLIP-seq reads downloaded from GEO (GSE84700). As displayed in In this study, we found that the RNA-binding protein PCBP-1 could mediate the transcriptional and post-transcriptional activities of its several target genes in PC12 cells. However, the underlying regulatory mechanisms were largely unknown. To the best of our knowledge, we established that PCBP-1 partially modulated the differential expression of genes associated with neuroinflammation and neuroimmune mechanisms in PC12 cells, involving 132 up-regulated and 58 downregulated genes, indicating PCBP-1 expression level is critical in the neuronal cells.As an iron-trafficking protein, LCN-2 was the most important gene, down-regulated by PCBP-1 overexpression contributing to both oxidative stress and inflammation. Recently, LCN-2 has been reported to possess various roles in cellular iron transportation and homeostasis. In the central nervous system (CNS), LCN-2 is known to play a role in neuroinflammation under neurodegeneration, glioma, brain injury, and multiple sclerosis conditions . Byung-WRNA-seq analysis showed that PCBP-1 partially modulated AS in PC12 cells. Particularly, PCBP-1 binding was associated with increased RASE numbers. Functionally, these changes in gene expression patterns impacted protein ubiquitination and endocytosis. Specifically, PCBP1 correlated with protein modulation. Notably, ubiquitin signaling plays a pivotal role in protein quality control. Ubiquitinylated and misfolded proteins are aggregated in PD, which includes genes encoding regulatory molecules, such as PTEN-induced kinase 1 (PINK1), Parkin, and FBX07, which has provided an opportunity to dissect the molecular basis of ubiquitin signaling in PD pathomechanism and develop novel therapeutic strategies in PD. These fWWP-2) has been linked to endothelial injury and vascular remodeling as a key regulatory factor  protein is not only the substrate for the PINK1-PRKN-dependent degradation pathway but also the active modulator of mitophagy. Thus, RHOT-1 is assumed to be a potential risk factor in PD  can be found below: The animal study was reviewed and approved by the Ethics Committee of Social Work Department of People’s Hospital of Xinjiang Uygur Autonomous Region.AY drafted the article. AY and HL revised it critically for important intellectual content. CY and SZ contributed to the revision of the manuscript and figure illustrations. SJ and LY made substantial contributions to the conception and design of the review and gave final approval of the version to be published. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Transcriptional deregulation, a cancer cell hallmark, is driven by epigenetic abnormalities in the majority of brain tumors, including adult glioblastoma and pediatric brain tumors. Epigenetic abnormalities can activate epigenetic regulatory elements to regulate the expression of oncogenes. Superenhancers (SEs), identified as novel epigenetic regulatory elements, are clusters of enhancers with cell-type specificity that can drive the aberrant transcription of oncogenes and promote tumor initiation and progression. As gene regulators, SEs are involved in tumorigenesis in a variety of tumors, including brain tumors. SEs are susceptible to inhibition by their key components, such as bromodomain protein 4 and cyclin-dependent kinase 7, providing new opportunities for antitumor therapy. In this review, we summarized the characteristics and identification, unique organizational structures, and activation mechanisms of SEs in tumors, as well as the clinical applications related to SEs in tumor therapy and prognostication. Based on a review of the literature, we discussed the relationship between SEs and different brain tumors and potential therapeutic targets, focusing on glioblastoma. “Superenhancer” regions within the genome help coordinate the malignant growth of brain tumors, and could offer a target for the development of therapies and prognostic biomarkers. These regions contain a dense accumulation of gene regulatory sequences, which operate in collaboration to establish cellular identity. A review by Jian Qu and colleagues at Central South University, Changsha, China, shows how these regulatory sequences get hijacked in various brain cancers and can facilitate tumor cell proliferation and invasion. Many tumor cells acquire mutations that lead to the formation of superenhancers that are absent in healthy cells. However, this process also offers therapeutic opportunities, and researchers have identified numerous drug candidates that can potentially block tumorigenesis by selectively interfering with superenhancer function. Superenhancer activity could also provide a potentially useful clinical signature for assessing tumor aggressiveness. While brain tumors are relatively rare, they still deserve research attention because of their significant mortality and morbidity at all ages2. Currently, treatments for brain tumors include surgery, chemotherapy, and radiotherapy. Despite substantial advances in chemotherapy for different brain tumors, chemotherapy resistance and relapse are still challenges. Delivering therapeutic agents to primary brain tumors is particularly challenging because of the unique brain–blood barrier (BBB) and brain–tumor barrier (BTB), resulting in poor response to treatments4. Moreover, brain tumor cells breach the BBB and adhere to the BTB, and this diffuse mode of invasion is the fundamental reason for brain metastases and relapse4. Therefore, it is necessary to study pathogenesis further and find more effective targets to improve the prognosis of patients with brain tumors.Brain tumors, including glioblastoma, medulloblastoma, diffuse intrinsic pontine glioma, meningioma, ependymoma, and other rare brain tumors, account for approximately 3% of cancer cases worldwide5. Enhancers are a class of regulatory DNA sequences that function as cis-regulatory elements to enhance the transcription of target genes occupied by coactivators and transcription factors (TFs)7. With the expanding concept of enhancers, “superenhancers (SEs)” have been proposed, which span several kilobases and are enriched with a higher density of TFs, coactivators, and epigenetic modifications8. Compared to typical enhancers, SEs can drive higher gene expression and participate in many biological processes9. It is worth mentioning that oncogenes regulated by SEs in tumor cells are not expressed in normal cells, suggesting that SEs play a critical role in the occurrence and development of tumors10. As tumor-associated variants are significantly enriched in SEs, identifying SEs improves the understanding of the mechanisms of tumorigenesis and provides new insights into the diagnosis, treatment, and prognosis of tumors11. Overall, SE-driven transcriptional disorders are associated with the progression of human cancers, including brain tumors.Gene transcription is a complex and highly coordinated process. Transcriptional dysregulation mediated by epigenetic modifications in tumors has attracted significant attention. Over the last decades, epigenetic modifications such as DNA methylation and histone modification have been identified as critical drivers of several types of brain cancerThis review will summarize the characteristics and identification, unique organizational structures, and activation mechanisms of SEs in tumors, as well as the clinical applications related to SEs in tumor therapy and prognostication. Furthermore, we will discuss SE-associated genes, transcriptional regulatory mechanisms, and therapeutic targets in different brain tumors.10. SEs were initially discovered in embryonic stem cells (ESCs), with ESC-specific master transcription factors Oct4, Sox2, and Nanog binding to enhancer elements and recruiting mediators to activate the gene transcription program12. Subsequently, SEs were identified in other cells, including cancer cells14. As novel epigenetic regulatory elements, SEs precisely regulate oncogene transcriptional activation during tumorigenesis and may be potential therapeutic targets16.In 2013, Young et al. first proposed superenhancers: large clusters of transcriptional enhancers that drive the expression of cell-identity genes17. In addition, histone modifications are important features of SEs. Histone H3 lysine 27 acetylation (H3K27ac) and histone H3 lysine 4 methylation (H3K4me1), which make the chromatin structure looser and provide accessible TF binding sites, are labels of active SEs19. H3K27ac and H3K4me1 are required for enhancers to activate target gene transcription, and these active chromatin markers can mediate the recruitment of epigenetic readers, including BET proteins21. In conclusion, these key components are integral parts of SE organization and the functional basis of SEs  as well as RNA polymerase II (RNA pol II), thus forming an SE-promoter loop to initiate downstream transcription22, DNase I sequencing (DNase-seq)23, chromosome conformation capture sequencing (3C-seq)24, and assay for transposase-accessible chromatin sequencing (ATAC-seq), are powerful tools25. SEs can be identified based on the strength of their binding with chromatin regulators and histone modification levels. Histone modifications can be used as indicators to identify SEs based on genome-wide factors such as the ChIP-seq-identified level of H3K27ac. Moreover, as an increasing number of SEs are defined in different types of tumors, various databases have been established to explore SE functions and facilitate SE research, such as SEdb (http://www.licpathway.net/sedb) and dbSUPER (http://bioinfo.au.tsinghua.edu.cn/dbsuper/)40.For the identification of SEs, next-generation sequencing and high-throughput sequencing technologies, including chromatin immunoprecipitation sequencing (ChIP-seq)41. Correspondingly, SEs are also transcribed into seRNAs, including long noncoding RNAs (lncRNAs), microRNAs (miRNAs), and circular RNAs (circRNAs), which play an important role in gene expression and epigenetic regulation44.Enhancers regulate the transcription of target genes and are actively transcribed into enhancer RNAs (eRNAs), and this ability to be transcribed is a general feature of functionally active enhancers45. The seRNAs act synergistically with SEs to regulate gene expression by maintaining the SE-promoter chromatin loop46. For example, the seRNA HPSE interacts with hnRNPU and p300 and then facilitates chromatin looping between the SE and HPSE promoter to enhance HPSE expression, thus promoting cancer progression47.A growing number of studies have revealed the critical roles of seRNAs in regulating target gene transcription. Young’s group proposed an RNA-mediated feedback control model of transcriptional condensates. In the model, low levels of RNAs mediate the formation of transcriptional condensates, while high levels of RNAs cause condensate dissolution, thus affecting the transcriptional outputcis and trans is clear, the molecular mechanism is still controversial. Three models of seRNA mechanisms have been reported: interacting with TFs or coactivators, driving enhancer-promoter looping, and transferring to the cytoplasm to mediate various cell activities48. While most studies have demonstrated that seRNAs promote enhancer-promoter looping, Panigrahi et al. showed that seRNAs do not affect chromatin looping49. They concluded that mutual costimulation of enhancer and promoter transcription is not dependent on seRNAs49. Further studies are required to explain these conflicting observations regarding the molecular mechanism of seRNAs.Although the role of seRNAs in transcriptional regulation in both 46  structure of the genome. In the eukaryotic genome, DNA‒protein complexes fold 3D chromatin loops, called topologically associating domains (TADs), which are structural units for transcriptional regulation64. In 2017, the proposed phase separation model limited SE-specific gene regulation to the membraneless organelle65. The intrinsically disordered regions (IDRs) of TFs, the transcriptional coactivators bromodomain protein 4 (BRD4) and mediator complex subunit 1 (MED1), and RNA Pol II can form phase-separated condensates at SE regions, leading to the transcriptional bursting and simultaneous activation of SE-driven genes67. The phase-separated condensate can separate SEs from other chromatin regions and concentrate SE-associated transcriptional processes at key regions, which explains why the effect of transcription regulation of SEs is greater than the overall effect of individual typical enhancers66. In leukemias, NUP98–HOXA9, a NUP98 fusion oncoprotein containing IDRs, can form LLPS condensates. LLPS of NUP98–HOXA9 is critical for leukemogenesis because it not only induces CTCF-independent chromatin loops but also leads to the formation of a “superenhancer”-like binding pattern at leukemogenic genes to regulate transcriptional activity69. The pharmacological inhibition of LLPS condensates can suppress metastasis and chemoresistance in osteosarcoma, representing a novel therapeutic strategy70. In addition, seRNAs also contribute to the organization of the phase-separated condensate and play a role in transcriptional activation71. Current studies on phase separation mainly focus on the role of biomolecular condensates assembled in tumors. However, such studies have not considered the intrinsic mechanism of the dynamic phase separation process, which still needs further investigation. Therefore, the phase separation model, which can ensure the precise regulation of genes, provides a novel perspective to elucidate the formation and transcriptional regulatory mechanisms of SEs , a dynamic physicochemical process, forms a membraneless organelle termed a condensate to organize biological processes within cells72. The activities of SEs and TFs affect each other. On the one hand, SEs coordinate with TFs to regulate the gene expression program, and the activity of SEs is affected by TF enrichment. On the other hand, the expression of TFs is often regulated by the activity of SEs, indicating positive feedback regulation between SEs and TFs8. The interconnected autoregulatory loop containing SEs can regulate specific cell-type transcriptional programs. To date, the CRC model and associated master TFs have been identified in multiple cancer types, including esophageal cancer72, acute myeloid leukemia73, multiple myeloma74, and osteosarcoma70. Because of the important role of CRC in malignant tumors, targeting the core TFs may suppress tumor growth.The gene transcription process requires a variety of transcriptional activators at specific DNA regulatory elements. To coordinate transcription programs in normal and malignant cells, a small group of master TFs forms an interconnected core regulatory circuitry (CRC) by directly co-occupying their SEs and each other’s SEs, allowing close contact between SEs and their target promoters11 , chromosomal rearrangement, and viral infection11 Fig. . During TAL1 oncogene, which generates an SE region through the binding of MYC and the recruitment of CBP and other core TFs, driving the aberrant expression of key oncogenes76 , human papilloma virus (HPV), human T-cell leukemia virus (HTLV), and human hepatitis B virus (HBV) are common oncogenic viruses, and their capacity to activate SE-associated genes has been reported in several tumors. HTLV-1 is associated with adult T-cell leukemia/lymphoma (ATLL) and specifically encodes the TF HBZ. HBZ binds to ATLL-specific 88. Thus, targeting SE complexes with small-molecule inhibitors is a promising strategy for tumor therapy , which can decrease the expression of SE-associated genes and suppress tumor growth94. Another BET inhibitor, OTX015, is effective against mouse and human MYCN-driven neuroblastoma in models, as it can selectively disrupt the binding of BRD4 and SEs and lead to the repression of MYCN expression95.BRD4, a member of the bromodomain and extraterminal domain protein (BET) family, plays a role in SE organization and oncogene expression regulation96. During transcriptional activation, BRD4 binds to SEs, followed by the recruitment of the TFIIH/CDK7 initiation complex and P-TEFb elongation complex containing CDK975. Cyclin-dependent kinase 7 (CDK7), an important CDK, can phosphorylate the RNA pol II C-terminal domain (CTD) at serine 5 (Ser5) and Ser7, leading to transcriptional initiation97. CDK7 also phosphorylates and activates CDK9/cyclin T, and CDK9 increases the phosphorylation of RNA pol II at Ser2, thus promoting transcriptional elongation98. Therefore, given that CDK7 and CDK9 are directly or indirectly involved in cellular transcriptional regulation, inhibition of CDK7 and CDK9 may interfere with transcription99. Covalent CDK7 inhibitors (such as THZ1 and THZ2) result in the downregulation of oncogene transcription and can serve as SE blockers to inhibit the expression of SE-driven oncogenes100. THZ2 is a newly developed CDK7 inhibitor with a fivefold longer half-life than THZ1, and its antitumor effects have been demonstrated in triple-negative breast cancer, gastric cancer, and osteosarcoma102. Moreover, the CDK9 inhibitor AZD4573 has shown effective antitumor activity through inhibition of CDK9, and AZD4573 is currently being evaluated in phase I clinical trial for patients with hematological malignancies (NCT03263637)104.In addition, cyclin-dependent protein kinases (CDKs) are protein-serine/threonine kinases that play an essential role in regulating the cell cycle and transcription106. The notable antitumor effects of JQ1 and THZ1 have been demonstrated in multiple types of tumors, including triple-negative breast cancer, diffuse large B-cell lymphoma, and pancreatic cancer108. Simultaneous inhibition of BRD4 and CDK9 with JQ1 and LDC067 suppresses cell growth and migration in medulloblastoma110. The coinhibition of these two molecules also shows a similar antiproliferative effect in malignant rhabdoid tumors110. Combination therapy with JQ1 and the novel CDK7 inhibitor YKL-5-124 shows a synergistic effect in neuroblastoma and can delay resistance to BRD4 inhibition111. In addition, combination of a histone deacetylase (HDAC) inhibitor and JQ1 or OTX015 results in stronger repression of oncogenes and higher expression of tumor suppressor genes, suggesting that these epigenetic drugs have synergistic antitumor effects112. Similarly, when THZ1 is combined with an HDAC inhibitor, sensitivity notably increases113. Given the potential therapeutic effects of small-molecule inhibitors, rational combinational strategies may be more effective for cancer treatment.More importantly, studies have shown that combination of SE inhibitors with traditional therapy enhances efficacy116. Furthermore, drug resistance is a challenge related to small-molecule inhibitors. In MYCN-driven neuroblastoma cells, upregulation of the multidrug transporters ABCB1 and ABCG2 results in resistance to THZ1117. In tumors with resistance to BRD4 inhibitors, hyperphosphorylation of BRD4 and epigenetic plasticity contribute to decreased sensitivity of JQ1119. The safety and efficacy of these small-molecule inhibitors, including potential side effects, off-target effects, and drug resistance, need to be further studied before clinical application.Despite significant inhibitory effects in previous studies, some side effects of BRD4 or CDK7 inhibitors have been reported in animal experiments and phase I clinical trials, such as heart toxicity, gastrointestinal toxicity, fatigue, thrombocytopenia, and impairment of muscle function50. Further analysis revealed that the aberrant SE landscape in HCC is the result of extensive reprogramming. The key components of SEs  are overexpressed in HCC, which is associated with the poor prognosis of patients120. In addition, SEs with broad and high H3K27ac signals have been identified in nasopharyngeal carcinoma and were found to be associated with overexpression of the ETV6 oncogene and poor prognosis121. Xu et al. defined an SE-associated gene risk signature to predict the response to chemotherapy in patients with diffuse large B-cell lymphoma, which may help clinicians make more appropriate treatment decisions122. Moreover, in our previous studies, we constructed prognostic models based on SE-associated genes to predict overall survival for osteosarcoma and multiple myeloma patients, which may be helpful for clinical treatment124. Therefore, oncogenic SEs may promote the malignancy of tumors and have utility in predicting the clinical outcome of tumor patients.SEs can also serve as biomarkers for evaluating the prognosis of tumor patients. In HCC, the SE-associated lncRNA HCCL5 regulates malignant biological behavior and is associated with the prognosis of patients with HCC125. Despite multimodal treatment, including surgical resection, radiation, and standard therapy with temozolomide (TMZ), the prognosis is universally poor due to therapy resistance and recurrence128. Therefore, it is urgent to explore the molecular mechanisms underlying GBM progression and develop novel therapeutic strategies to improve the prognosis of GBM patients.Glioblastoma (GBM), a high-grade glioma , is the most common malignant brain tumor and has a 5-year survival rate of 5.6%129. Researchers identified a translocation of a superenhancer to the 5’ promoter region of TERT, which can activate TERT129. Moreover, another study described 3D genome information through Hi-C sequencing in glioblastoma stem cells (GSCs)130. In GSCs, genomic structural variants lead to SE-promoter interactions, such as physical interactions between the two SEs (SE1 and SE2) and JAK1130. In addition, integration of Hi-C and chromatin data revealed strong H3K27ac signals in stemness genes and identified a region overlapping SOX2 as an SE locus130. Currently, the majority of biopsies are preserved as archived formalin-fixed paraffin-embedded (FFPE) samples. Zhao et al. assessed FFPE tissue with antibody-guided chromatin tagmentation with sequencing (FACT-seq), the first highly sensitive method to describe histone modifications in FFPE tissues131. Using FACT-seq of H3K27ac in FFPE human GBM samples, the researchers identified 492 disease-specific SEs 131. In summary, the development of novel technologies allows the identification of complete SE landscapes and histone modifications in the whole genome, which aids the investigation of the epigenetic regulation and heterogeneity of GBM.In the past few years, several novel epigenetic markers contributing to the pathogenesis of GBM have been reported. In 2014, a population-based single-cell whole-genome sequencing methodology was applied to characterize genomic heterogeneity in EGFR-amplified GBM132. The CDK7 inhibitor THZ1 can disrupt global gene transcription and preferentially target SE-associated genes in GBM cells. For example, five highly expressed SE-associated genes  were found to be associated with sensitivity to THZ1, and their knockdown inhibited the proliferation of GBM cells  located between the Ki67 and MGMT genes can increase MGMT expression and is activated in TMZ-resistant GBM tissues137. Deleting the K-M enhancer reduces MGMT and Ki67 expression, thus increasing sensitivity to TMZ137. Moreover, another study demonstrated that aberrant transcriptional regulation of SEs is a cause of resistance to TMZ138. The H3K27ac status regulates cis-regulatory elements such as SEs. However, the RET finger protein-histone deacetylase 1 (RFP-HDAC1) complex deacetylates H3K27ac marks on core histones, decreasing gene transcription levels138. Disruption of the HDAC complex can increase TMZ sensitivity in GBM cells by affecting the H3K27ac status  is one of the leading causes of TMZ resistance because MGMT is a DNA repair factor that can reverse DNA damage caused by TMZtus Fig. 138,139. 10. SLC30A3 is significantly reduced in GBM tissues and can inhibit the growth and metastasis of GBM cells140. Overexpression of HDAC1 decreases the H3K27ac level in the SE region of SLC30A3, which inhibits the expression of SLC30A3140. HDAC1 is predominantly associated with transcriptional repression, while the transcriptional regulation mechanism of HDAC7 is complex and related to both activation and repression. In stem-like breast cancer cells, HDAC1 and HDAC3 regulate the expression of HDAC7141. In turn, HDAC7 maintains H3K27ac levels by binding to SEs, thereby promoting the expression of SE-associated oncogenes141. HDAC1/3 knockdown increases the overall H3K27ac level, but HDAC7 knockdown decreases the H3K27ac level, and these changes are only observed in cancer stem cells and not in other cells141. These results suggest functional differences between HDAC1/3 and HDAC7. Inhibition of HDAC1/2/3 disrupts SE activity by increasing the accessibility of TF sites and disrupting chromatin loops, an indirect effect144. In GBM models, the HDAC inhibitors panobinostat and romidepsin have been shown to target SEs and elicit metabolic reprogramming through H3K27ac modification, thereby suppressing GBM progression145. Overall, different types of HDACs have different regulatory effects on SE activity.Multiple lines of evidence have reported that the malignant phenotype of GBM is regulated by SEs and HDACs. HDAC1, together with BRD4, RNA pol II, and other key components, is enriched in SEs147; (2) decreasing the binding of RNA pol II at SE regions145; (3) inducing SE looping defects by reducing contact with SE components and promoters142; and (4) reducing the expression of core regulatory circuitry TFs to repress SE activity72. These epigenetic alterations cause the global disruption of SEs and thus inhibit SE-associated transcript and gene expression144. Based on their antitumor effects, HDAC inhibitors should be able to exert synergistic effects in combination with SE inhibitors (such as THZ1 and JQ1) and chemotherapies in neuroblastoma, glioblastoma, and other tumors146.The molecular mechanisms underlying HDAC inhibitor-mediated SE activity are not yet fully understood. Some studies have investigated the regulatory mechanisms of HDAC inhibitors: (1) reshaping epigenetic markers by promoting H3K27 acetylation150. An active chromatin landscape was mapped in GSCs, identifying SE-associated genes and core TFs that establish SEs and maintain GSC identity151. The study found that many genes defining GSC identity are highly expressed and regulated by SEs, such as SOX2, SEPT9, CXXC5, CDK6, SALL3, and EGFR, which are required for tumor cell proliferation151. In addition, another study depicted the core TFs and their interconnected regulatory circuitry in GBMs, showing that many motifs of SE-driven TFs  are enriched in SE regions  relevant in the tumor microenvironment were identified in glioma158. The antiproliferative activity is equivalent to that of TMZ, suggesting that I-BET151 may be beneficial for GBM patients with TMZ resistance156. In addition, another BET inhibitor, OTX015, was found to cross the BBB and selectively penetrate tumor tissues in a preclinical study, indicating its potential efficacy in GBM159. In addition, dBET6, a chemical degrader of BET proteins, can inhibit the proliferation and self-renewal ability of GBM cells by significantly reducing BET protein occupancy, RNA pol II activity, and active histone markers, and dBET6 is superior to first-generation BRD4 inhibitors such as JQ1161. Interestingly, GBM cells exhibit sex-specific responses to the BRD4 inhibitor JQ1, which is correlated with sex-specific gene expression patterns. The sensitivity of male patient-derived GBM cells to BET inhibitors is higher than that of female patient-derived GBM cells162.The BET inhibitor I-BET151 inhibits the proliferation and eliminates the tumorigenicity of GBM cellsAs newly recognized epigenetic elements, numerous SEs have been identified in GBM and have been shown to be related to tumor progression Table . As desc163. Previous genomic studies have revealed four molecular subgroups of MB , Group 3, and Group 4) with different biological and clinical behaviors165. Among these subgroups, Group 3 and Group 4 MBs account for the majority of cases and have the poorest outcomes166.Medulloblastoma (MB) is the most common malignant brain tumor in children and adolescents younger than 15 years old167. The growth factor independent 1 family oncogenes GFI1 and GFI1B are oncogenes of Group 3 and Group 4 MBs that can cooperate with MYC to promote MB formation in vivo167. Genomic structural variants occur in the GFI1 or GFI1B coding sequences proximal to enhancers and superenhancers, leading to oncogenic activation of GFI1 and GFI1B167. In addition, scientists have identified some subgroup-specific SEs in MBs that can activate oncogenes, including ALK, MYC, SMO, and ETV4148. In Group 3 MB, an SE-driven transcriptional regulatory network consisting of 14 SE-associated genes exists168. A BET inhibitor (JQ1) and a CDK7 inhibitor (THZ1) showed synergistic inhibitory effects, with therapeutic potential for Group 3 MB168. Moreover, some adult SHH MB patients present with truncating mutations in the chromatin reader BRPF1, which are absent or rare in pediatric patients169. Mutated BRPF1 increases the accessibility of a subset of SEs associated with key genes involved in cerebellum development and chromatin remodeling, promoting tumorigenesis in adult SHH MB170.A study of MB illustrated that oncogenic drivers were restricted to Group 3 and Group 4171. Mechanistically, MLL4 loss leads to MB by reducing broad H3K4me3 and SE signals, downregulating tumor suppressors (Dnmt3a and Bcl6), and extensively damaging epigenomic signatures171. Therefore, MLL4-established superenhancers play an important role in tumor suppression in normal cells. The antitumor role of SEs has also been reported in other types of tumors, such as chronic myelogenous leukemia and breast cancer172. Superenhancers can drive either oncogene expression or tumor suppressor expression, playing dual roles in tumor progression. SE-associated genes and possible mechanisms in MB are shown in Table Superenhancers not only activate oncogene expression but are also associated with tumor suppressor gene activation. Brain-specific knockout of the H3K4 methyltransferase MLL4 in mice can induce MB173. Because of its diffuse growth and location, DIPG is inoperable, and the standard therapy is radiation therapy, but its effectiveness is extremely limited174. The characterization and development of drugs are challenging due to the scarcity of DIPG samples.Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric brain tumor with a median survival time of 11 months175. H3K27M glioma cells exhibit greater proliferation potential due to the aberrant oncogenic program176. Together with RNA pol II and H3K27ac, H3K27M localizes to transcriptionally active regions and can drive SE formation178. H3K27M inhibits the enzymatic activity of PCR2 through its interaction with the E2H2 subunit, resulting in the loss of H3K27me3 at SE regions180. H3K27M leads to a more accessible chromatin configuration in key regulatory regions, which can expose binding site motifs for key TFs, including ASCL1 and NEUROD1181. These TFs then bind to SEs to activate neurogenesis and NOTCH pathway-related genes, ultimately contributing to glioma formation181. Recent studies of H3K27M DIPG in vitro and in vivo have identified some important druggable targets and found some effective small molecules to reverse epigenetic alterations, including the HDAC inhibitor panobinostat182, the H3K27me2/3 inhibitor GSKJ4182, the BET inhibitor JQ1178, and the selective E2H2 inhibitor EPZ6438183.Approximately 85% of DIPGs are characterized by mutation of lysine 27 to methionine in histone 3 (H3K27M), which leads to oncogenic transcription dysregulation and increased stem-like potential and proliferationSOX10) and genes expressed by oligodendroglial lineage cells during differentiation 105. DIPG is vulnerable to BRD4 and CDK7 blockade, which can impair DIPG cell growth105. For example, the BRD4 inhibitor JQ1 can induce neuron-like differentiation and delay tumor growth in a mouse model of DIPG, and the CDK7 inhibitor THZ1 can disrupt transcription and inhibit DIPG growth184. Hyperacetylation in DIPG favors the action of BDR4 and leads to enhancer landscape reprogramming, activating SE-driven oncogenes in DIPG174. In addition, CBP is related to transcription activation, and its activity can be enhanced by BRD4185. The combination of JQ1 and a CBP inhibitor (ICG-001) can reverse the detrimental SE programs activated by BET or CBP174. Collectively, these findings suggest that SE-driven oncogenes in DIPG can be targeted with SE blockers. SE-associated genes and possible mechanisms in DIPG are shown in Table The analysis of SEs in DIPG has revealed potential cell-identity genes, supporting the idea that DIPG originates from a precursor cell of the oligodendroglial lineage. These SE-associated genes include genes classically associated with oligodendrocyte precursor cells , one of the molecular alterations in GBM, can recruit DNA-binding proteins and is essential for GBM tumorigenesis189. The SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin-remodeling complex is an essential regulator of pluripotency in human embryonic stem cells. Nevertheless, mutation of its core subunit SMARCB1 can lead to AT/RTs due to the disruption of enhancer accessibility190. With noticeable H3K27ac features in enhancer regions, SMARCB1 is required for the integrity of the SWI/SNF complex191, which is inactivated abnormally in most rhabdoid tumors192. Loss of SMARCB1 reduces genome-wide targeting at enhancers, thus impairing the functions of the SWI/SNF complex. However, the small amount of residual SWI/SNF complexes preferentially bind to SEs, including some shared by all subtypes, such as SPRY1, and other lineage-specific SEs, such as SOX2, in brain-derived rhabdoid tumors, which is crucial for maintaining aberrant cell identity. Then, these SEs drive oncogenic transformation by locking cells into a poorly differentiated and highly proliferative state191. Further research found that knockdown of SMARCB1 prevents silencing in SE regions, thus leading to transcriptional upregulation in human embryonic stem cells193. Therefore, SMARCB1 and its regulatory effects on SEs in AT/RT are part of a novel AT/RT tumorigenesis mechanism.Atypical rhabdoid/teratoid tumors (AT/RTs) are aggressive and lethal cancers that can be diagnosed at a young age195. Because there are few research models, mechanistic and therapeutic studies of this rare disease are extremely limited. Recently, an oncogenic circuit driven by hijacked superenhancers in ETMRs was revealed. The C19MC-TTYH1 gene fusion and MYCN DNA interactions create superenhancers. Then, the interaction between C19MC-TTYH1 superenhancers and MYCN enhancers fortifies the C19MC-LIN28A-MYCN circuitry, driving the expression of embryonically restricted DNMT3B6 to promote a primitive malignant epigenetic state in ETMRs196. Interestingly, the BET inhibitor JQ1 can downregulate key components of the C19MC-LIN28A-MYCN circuit, including MYCN, LIN28A, and DNMT3B6, disrupting the circuit and inducing ETMR cell death196. The unique SE-dependent oncogenic circuit protects the ETMR and is vulnerable to BET inhibition. Therefore, inhibition of BET may be a promising therapeutic strategy for this orphan disease. SE-associated genes and possible mechanisms in these pediatric brain tumors are shown in Table Embryonal tumor with multilayered rosettes (ETMR) is a sporadic and difficult-to-treat brain tumor in infants and young children, with rapid progression and only 10–20% overall survival197. A comprehensive investigation of the genomic landscape has revealed the overall genomic instability in aggressive meningioma198. Upregulation of the SE-associated HOXD gene is associated with meningioma aggressiveness198  gene fusion200. ZFTAfus contributes to an oncogenic transcriptional program because it binds TF motifs and recruits transcriptional coactivators , thus driving SE gene expression in ependymoma201. To identify SE-associated genes that ependymoma cells depend on, Mack et al. analyzed the chromatin landscapes of ependymomas202. In two cohorts of ependymoma specimens, they identified that the vast majority of SEs were tumor-specific and enriched in oncogenes. Among the genes, EPHB2 and CCND1 have been previously proven to be ependymoma-related oncogenes204. In addition, ependymoma is sensitive to the BET inhibitor JQ1, which can inhibit the proliferation of ependymoma cells202  are highly enriched in oncogenic SE regions, and their inhibition preferentially affects SE-associated genes in tumor cells. Some inhibitors targeting transcriptional activators have been evaluated in clinical trials. Moreover, combining SE inhibitors with other chemotherapeutic drugs can suppress tumor growth, providing a new strategy for cancer treatment. Since transcription is a basic biological process common to all nucleated cells, targeting SE-associated transcription may lead to general toxicity. Although inhibitors targeting SEs, including JQ1, THZ1, and THZ2, have been studied in many tumors, their potential side effects and off-target effects need further study.As described above, the development of various brain tumors is closely associated with SEs and downstream oncogenes, and SEs may serve as master gene regulators and novel therapeutic targets. Further studies on the underlying mechanisms of SE activation will shed light on the complex pathogenesis of brain tumors. With persistent SE research efforts, breakthroughs in the treatment of malignant brain tumors are expected.