Dendritic cell (DC) specification and differentiation are controlled by a circuit of transcription factors, which regulate the expression of DC effector genes as well as the transcription factors themselves. E proteins are a widely expressed basic helix-loop-helix family of transcription factors whose activity is suppressed by their inhibitors, ID proteins. Loss-of-function studies have demonstrated the essential role of both E and ID proteins in different aspects of DC development. In this study, we employed a gain-of-function approach to illustrate the importance of the temporal control of E protein function in maintaining balanced differentiation of conventional DC (cDC) subsets, cDC1 and cDC2. We expressed an E protein mutant, ET2, which dimerizes with endogenous E proteins to overcome inhibition by ID proteins and activate the transcription of E protein targets. Induction of ET2 expression at the hematopoietic progenitor stage led to a dramatic reduction in cDC2 precursors (pre-cDC2s) with little impact on pre-cDC1s. Consequently, we observed decreased numbers of cDC2s in the spleen and lung, as well as in FLT3L-driven bone marrow-derived DC cultures. Furthermore, in mice bearing ET2, we detected increased expression of the IRF8 transcription factor in cDC2s, in which IRF8 is normally down-regulated and IRF4 up-regulated. This aberrant expression of IRF8 induced by ET2 may contribute to the impairment of cDC2 differentiation. In addition, analyses of the transcriptomes of splenic cDC1s and cDC2s revealed that ET2 expression led to a shift, at least in part, of the transcriptional profile characteristic of cDC2s to that of cDC1. Together, these results suggest that a precise control of E protein activity is crucial for balanced DC differentiation. Dendritic cells (DCs) orchestrate a variety of immune responses and are thus important players in responses to microbial infection, tumor immunity and autoimmunity . SubsetsIrf8 transcription. Besides being a terminal selector of cDC1, IRF8 plays critical roles in the maintenance of DC progenitors  , 13. Altcription . E protecription . This coin cDC2s . TherefoTo test this hypothesis, we specifically expressed our gain of E protein function mutant in hematopoietic progenitors and committed DCs and examined the impact on the distribution of cDC1s and cDC2s in lymphoid and peripheral tissue. Sustained E protein activity led to a reduction in cDC2 numbers in both the spleen and lung. Moreover, analyses of pre-cDC progenitors in the bone marrow revealed that a subset of pre-cDC2s that express CD11b was selectively diminished in mice with elevated E protein function. Gain of E protein function also led to impaired cDC2 differentiation in bone marrow cultures supported by FLT3 ligand. These phenotypes were accompanied by high levels of IRF8 expression in pre-cDC2s and cDC2s that normally express low IRF8 levels. These studies illustrate the crucial role of E proteins at multiple checkpoints of DC differentiation and the importance of the dynamic regulation of E proteins for maintaining the balance of DC diversity.CD11c mice are homozygous for the ET2 allele whereas ET2Vav mice have one ET2 allele. Littermate Cre– ET2 mice served as controls.Rosa26-stop-ET2 was generated by knocking ET2 along with IRES-EGFP into the Rosa26 locus downstream of its promoter as previously described . CD11c-C2, 1.8 mM CaCl2. Cell suspension was passed through a 70 μm cell strainer, followed by RBC lysis in buffered ammonium chloride.A single-cell suspension from the spleen was obtained after incubation for 30 min at 37°C in HBSS buffer with calcium and magnesium with Collagenase D (1mg/ml) and DNase (0.1 mg/ml). Lungs were perfused with PBS through the right ventricle, cut into small pieces and enzymatically digested by 45 min incubation at 37°C with Collagenase D (1mg/ml) and DNAse (0.1mg/ml) in HEPES buffer containing 10 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgClAll antibodies were purchased from BioLegend unless specified otherwise: anti- I-A/I-E (M5/114.15.2), anti-CD11c (N418), anti-Siglec H (551), anti-CD8a (53-6.7), anti CD4 (GK1.5), anti-CD45R/B220 (RA3-6B2), anti-CD11b (M1/70), anti-CD103 (2E7), anti-CD24 (M1/69), anti-CD135 (A2F10), anti-CD172a/SIRPα (P84), anti-Ly-6C (HK1.4), anti-IRF4  anti-IRF8 , CD88/C5aR (20/70), and anti-CD26/DPP-4 (H194-112). Antibodies in the lineage (Lin) cocktail are anti-B220 (RA3-6B2), anti-CD3 (17A2), anti-CD19 (6D5), anti-NK-1.1 (PK136), anti-Ly6G (1A8) and anti-TER119 (TER119).Cell sorting was performed on a FACSAria II (BD Biosciences), and flow cytometric analysis was performed on a LSR-II (BD Biosciences). Intracellular staining of transcription factors was done using Foxp3 Staining Buffer kit (eBioscience).Bone marrow-derived DC culture driven by FLT3 ligand was carried out essentially as described . BrieflyMus musculus reference genome (GRCm38/mm10) using STAR v.2.4.0h . Specific tests applied are indicated in each figure legend. Data are presented as mean +/- SEM.via an IRES upon Cre-mediated deletion , which expresses a chimeric protein, ET2, and EGFP deletion . ET2 condeletion , 24. Thedeletion . We crosCD11c and ET2Vav mice were analyzed along with controls that were ET2 mice without the Cre transgene. As shown in hiMHCII+, a fraction which did not differ in numbers among the three strains  to separate monocyte derived DC and macrophage populations from cDCs, as previously described by Nakano et al. (+CD88– subset for the expression of MHCII and CD26 (dipeptidyl peptidase-4 expressed by cDCs). MHCII+CD26+ cells were then dissected into cDC1 and cDC2 subsets using CD11b and CD24 or CD103 surface markers  are thought to give rise to two subsets of pre-cDC intermediates: pre-cDC1 and pre-cDC2  and pre-cDC2s (Ly6C+)  , Cd80, Cd86 and Ccr7 but also have non-redundant roles in cDC specification. We first examined the expression of IRF4 and IRF8 proteins in the pre-cDCs in wild type bone marrow as defined in –pre-cDC2s contained low levels of IRF4 relative to the isotype control, CD11b+pre-cDC2 cells harbored notably higher levels of IRF4, consistent with the role of IRF4 in promoting cDC2 differentiation . Due to the extremely low frequency of CD11b+pre-cDC2s in ET2Vav mice, we could not determine the levels of IRF4 and IRF8 with great confidence. However, we were able to measure the levels of IRF4 and IRF8 in splenic DCs in ET2Vav mice. Although splenic cDC1s in ET2Vav and control mice did not show differences in IRF4 or IRF8 levels (data not shown), CD11b+CD4+cDC2s exhibited changes in expression of these two proteins. Namely, the ET2-expressing cDC2s have lower levels of IRF4 but higher levels of IRF8  expression . We used ET2CD11c as opposed to ET2vav mice to obtain sufficient cDC2s for RNA sequencing. Cells sorted from two individual mice of each strain were processed for RNA sequencing, and data were analyzed using standard bioinformatics tools. Comparing YFP to ET2-expressing cells, we detected 41 and 212 differentially expressed genes for cDC1 and cDC2, respectively  or innate lymphoid cells . We founIrf4 and Irf8 were not among the lists of differentially expressed genes comparing YFP to ET2-expressing cells as in Irf4 and Irf8 expression was specifically examined, we still did not detect any difference between YFP to ET2-expressing cells but observed the expected distinct patterns comparing cDC1 and cDC2, namely high levels of IRF8 in cDC1 and IRF4 in cDC2 . Interestingly, the expression patterns of these genes in ET2-expressing cDC2s were similar to those of YFP-cDC1s but distinct from those of YFP-cDC2s, suggesting that ET2 diminished the transcriptome that is characteristic of cDC2s  stage, exhibited profound defects in cDC2 differentiation and an increase in cDC1s in the lung and spleen. Within the lung resident cDC2 pool, ET2 expression had the greatest effect on the CD11b+CD24hi subset that is dependent on IRF4 for differentiation. ET2CD11c mice showed a similar but less significant reduction in cDC2 numbers. This may stem from our observation that not all CD11c+ pre-cDCs showed evidence of Cre-mediated deletion in mice carrying only the Cre reporter , differentiate to cDC1s and cDC2s in peripheral tissue . We fracre-cDC2s .Vav mice, we detected little impact of ET2 on IRF4 and IRF8 expression in pre-cDC1s or CD11b–pre-cDC2s, and it was difficult to compare the levels of these transcription factors in CD11b+pre-cDC2s due to their scarcity in ET2-expressing mice. However, we did observe reduced levels of IRF4 and increased levels of IRF8 in splenic CD4+ cDC2s of ET2Vav mice. This skewed ratio of IRF4 and IRF8 may be an underestimate of the effect of ET2 because if down-regulation of IRF8 and up-regulation of IRF4 is essential for cDC2 differentiation, the small numbers of cDC2s found in ET2Vav mice may be selected as those that have relatively high levels of IRF4 and low levels of IRF8. The pre-cDC2s with high levels of IRF8 may not progress to the CD11b+ stage and differentiate to cDC2s in the spleen. Our data support the hypothesis that defects in cDC2 differentiation occur when pre-cDCs destined to become cDC2s either aberrantly upregulate or fail to downregulate IRF8.In ET2Irf8 expression .SB, KT, CG, and YZ generated the data. SB, SK, CG, JDW, and X-HS analyzed the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by grants from the NIH (1P20 GM103636-07) to X-HS and JW and (HL119501) to SK, and from the Presbyterian Health Foundation to X-HS. X-HS holds the Lew and Mira Ward Chair in Biomedical Research at the Oklahoma Medical Research Foundation.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.


This review synthesizes knowledge on epigenetic regulation of leaf senescence and discusses the possibility of using this knowledge to improve crop quality. This control level is implemented by different but interacting epigenetic mechanisms, including DNA methylation, covalent histone modifications, and non-covalent chromatin remodeling. The genetic and epigenetic changes may act alone or together and regulate the gene expression, which may result in heritable (stress memory) changes and may lead to crop survival. In the review, the question also arises whether the mitotically stable epigenetic information can be used for crop improvement. The barley crop model for early and late events of dark-induced leaf senescence (DILS), where the point of no return was defined, revealed differences in DNA and RNA modifications active in DILS compared to developmental leaf senescence. This suggests the possibility of a yet-to-be-discovered epigenetic-based switch between cell survival and cell death. Conclusions from the analyzed research contributed to the hypothesis that chromatin-remodeling mechanisms play a role in the control of induced leaf senescence. Understanding this mechanism in crops might provide a tool for further exploitation toward sustainable agriculture: so-called epibreeding. In eukaryotic cell nuclei, genomic DNA is packaged into a highly organized nucleoprotein complex known as chromatin. The fundamental unit of chromatin is the nucleosome, which is composed of DNA wrapped around a core of eight histone molecules. Nucleosomes are not simply static structural units. Nucleosomes can be moved, stabilized/destabilized, and disassembled/reassembled at particular genome locations in response to specific environmental signals or developmental cues. The resulting dynamics of the chromatin structure directly modulate the DNA accessibility, thus regulating all DNA template processes  and affecting various processes in plants such as root growth, flowering timing, floral organogenesis, gametophyte or embryo formation, senescence, as well as the response to pathogens or environmental changes ,2. HowevLinaria vulgaris [Solanum lycopersicum [Sinapis alba [Oryza sativa [In the last two decades, researchers have accumulated a wealth of knowledge providing evidence of the prevalence of epigenetic variability  and its potential to influence the phenotype (agronomic traits) and bring large crop improvements. Can the mitotically stable epigenetic information be used for crop improvement? The answer is very similar to that for transcriptional factors. Chromatin changes also control plant morphology and response to the environment, and greater control over traits may be achieved by understanding these mechanisms, which is highly important from the breeding point of view . There avulgaris , Solanumpersicum , Sinapispis alba  and Oryza sativa .Arabidopsis and cereal lines with defects within the genes involved in DNA methylation, modification of histones, and ATP-dependent reorganization of nucleosomes.This review summarizes and synthesizes knowledge on epigenetic regulation of leaf senescence and discusses the possibility of using this knowledge to improve crop quality. It is all based on empirical data, among others from transgenic analysis of mutants of Senescence is a ubiquitous phenomenon in the biological world. From an ontogenetic perspective, senescence is now established as a developmental and genetic program acquired during evolution . In planArabidopsis as a model organism for leaf senescence have revealed several facets of senescence. A theoretical model has been put forward about how the capacity for senescence is formulated during leaf development and possibly how internal and external factors are integrated with age to define the timing of senescence [Arabidopsis, leaves have a very short lifetime and senescence seems to start as soon as full expansion is reached. Moreover, the senescence of the individual rosette leaves may not be closely linked to the developmental stage of the plant. In many plants, such as the pea, removal of the developing flowers and pods will significantly extend the life of the leaves, but in Arabidopsis, male-sterile mutants, or plants from which the developing bolts were removed, did not show any extension to the lifetime of the individual leaves [Studies on nescence . In Arabl leaves ,14,15.In cereals, senescence appears to be regulated at the level of the individual leaf. Nutrients are thus mobilized from the older leaves to the younger leaves and eventually to the flag leaf, which contributes the majority of the nutrients and photoassimilates used for charging of the grain .Arabidopsis: an individually darkened leaf, which supports rapid senescence, and a leaf from a whole darkened plant, characterized by a strong capacity for survival. Several external stimuli, other than darkness, can also induce the onset and progression of senescence, or processes resembling senescence and sharing some common pathways. These include environmental stresses such as extremes of temperature, water stress, nutrient deficiencies, wounding and light conditions, as well as pathogen attack and the hypersensitive response.The study of leaf senescence has been complicated by the lack of coordinated development of the cells within an individual leaf, and various methods have been used to artificially induce senescence to obtain a synchronous process. For example, dark-induced senescence has been used frequently as a useful method to induce synchronous senescence as many typical senescence symptoms such as chlorophyll degradation and loss of protein occur ,18,19,20Plants cannot move away from adverse environmental conditions, and senescence is a mechanism that they have evolved to cope with a problem. In these cases, recycling of nutrients is likely an important concern wherever possible. Senescence refers to the process or condition of growing old . The current physiological understanding of a state of senescence supports a definition that considers senescence (i) to be the developmental phase that is an episode of transient differentiation at the end of growth, (ii) may or may not be replaced by death, and (iii) is entirely dependent on cell viability and expression of specific genes. Green tissues may also pass directly from maturity to death without a clear intervening senescence phase. Such genetic interventions show that death neither requires senescence nor is the inevitable consequence of it .What is more, it is extremely difficult to uncouple senescence regulatory pathways from stress responses, since the genetic program(s) underlying senescence largely overlaps with that of plant defense. Likewise, many senescence-associated genes (SAGs) involved in developmental senescence have also been found to have a role in other biological processes, and gene mutations involved in diverse facets of plant growth and development can alter leaf senescence. This indicates that there is an overlap between the senescence network and other biological networks, or that genes that have evolved for one purpose have been recruited for other purposes . Becker In plants, the cell-specific gene expression scheme determined by the state of chromatin is crucial to establish and maintain the functionality of a particular cell or group of cells, which clearly indicates the presence of various epigenomes differing in the pattern of post-translational modifications (PTMs) conditioned by epigenetic marks . All theThe specific characteristics of the modifications, referred to as the histone code, together with the methylation patterns, condition the plasticity of the complex chromatin structure. Type and location of various PTMs of the histones N-terminal lysine, serine, threonine, and arginine residues determine the availability of the DNA sequence for the transcription factors, thereby controlling the expression of individual genes ,27. The Cytosines in DNA of eukaryotes may undergo methylation mediated by DNA methyltransferases (DNMT). The high level of 5-methylcytosine is specific for the inactive state of chromatin. This mechanism is essential for silencing repetitive sequences and transposable elements (TEs), DNA sequences which can change their position within a genome, to avoid uncontrolled transposition, which allows for genome integrity maintenance . In planATP-dependent chromatin remodeling plays a key role in regulating the accessibility of individual DNA regions, which must be released from the nucleosome tangles to undergo transcription. Chromatin can be changed not only by covalent DNA and histone modifications . The basLeaf senescence, both developmental and stress induced, as mentioned above, is controlled by a multilevel regulatory network, and the dynamics of cooperation among all signal pathways is conditioned by plant phenotypic plasticity under different conditions ,34. EpigArabidopsis mutants and transgenic lines showing senescence-affected phenotype are summarized in There follows a detailed description of changes conditioning the progression of leaf senescence processes that occur under the influence of post-translational histone modifications, DNA methylation, ATP-dependent chromatin remodeling and additional epigenetic regulation mechanisms. Examples of loss-of-function and gain-of-function Leaf senescence initiation conditioned by both cellular signaling and global regulation of transcription is aimed at reducing the activity of genes involved in photosynthesis, while SAGs are sequentially turned on. The complexity of the process requires dynamic epigenetic regulation of chromatin plasticity including histone modifications and the use of these variants in ATP-dependent chromatin remodeling . AlthougArabidopsis plants with expression of the antisense histone deacetylase 1 (AtHD1) gene, or in HDA6 axe1-5 mutant and histone deacetylase 6-RNA-interfering mutant (HDA6-RNAi), both resulting in accumulation of acetylated histones which occurred together with changes in senescence timing in different manners [The global impact of changes in the histone deacetylation profile on the regulation of senescence has been shown in studies on transgenic  manners ,45. Loss manners ,59.Enhancing gene expression by direct histone acetylation was initially shown in the example of acetyltransferase Elongator. Silencing of the gene for acetyltransferase Elongator was correlated with the reduction of small Rubisco subunit  gene transcription and it led to acceleration of tomato leaf senescence .Another well-studied histone modification involved in leaf senescence is acetylation of Lys-9 of H3 histone (H3K9ac). A study based on chromatin immunoprecipitation (ChIP) and RNA analysis showed that H3K9ac marks many upregulated genes during senescence, mainly activating TFs, such as WRKY53, at different phases of leaf development. H3K9ac is similar in nature to H3K4me3. Although the average number of H3K9ac marks increases around genes associated with leaf senescence (such as WRKY53 and probable WRKY transcription factor 41—WRKY41), it is much smaller than that of H3K4me3. The H3K9ac levels are high during the early stages of senescence and decrease toward the end of it. This proportion is contrary to that of H3K4me3 levels, as at first they are below average and increase by a great margin as senescence progresses . TransgeHistone methylation may certainly have an equally strong influence on regulation of leaf senescence as acetylation. The presence of a methyl group/groups on lysine or arginine residues promotes the attachment of regulatory proteins and increases the transcriptional availability of DNA, loosening the structure of the nucleosome .Arabidopsis are related to H3 histone. Histone 3 lysine 4 trimethylation (H3K4me3) has been described as a distinct landmark of leaf senescence. During this process, H3K4me3 significantly increases, mostly within WRKY53, a key regulator of early stages of senescence, regulating the activity of many SAGs and TFs during senescence [Arabidopsis thaliana plants that were growing under dark-induced stress, rather than in plants under normal light conditions, mainly for exon regions. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis proved that H3K4me3 changes could be observed in dark-induced leaf senescence, as well as during aging. H3K4me3 is proven to be associated predominantly with active genes. Although H3K4me3 has been demonstrated to occur in leaf senescence, some genes with high levels of expression in senescent leaf tissue lacked H3K4me3 marks [Arabidopsis JmjC-domain-containing protein which may act as histone H3 lysine demethylase, delays leaf senescence through repression of WRKY53. Decrease of JMJ16, the specific H3K4 demethylase, is correlated with the increase of H3K4me3 as senescence progresses [Most cases of methylation in nescence ,65. Stude3 marks ,67. Accoogresses .Histone 3 lysine 27 trimethylation (H3K27me3) is considered to be another epigenetic marker of leaf senescence. The number of these marks decreases during senescence, as this methylation prohibits overexpression of key regulatory and functional genes mostly involved in the regulation of developmental leaf senescence. This is caused by genes such as REF6, a plant-unique H3K27 demethylase, which functions as a putative binding protein for the promoter of NYE1 (NONYELLOWING1), the gene encoding senescence-inducible chloroplast stay-green protein, a regulator of chlorophyll (Chl) degradation. REF6 promotes Chl degradation during leaf senescence by up-regulation of NYE1 ,68.Arabidopsis. ATM activity leads to repression of DSB-dependent transcription of SAGs, including WRKY and NAC TFs (one of the largest families of transcription factors in plants). This path of regulation is based on histone lysine methylation. The presented data suggest that accumulation of DSBs may lead to SAG activation connected with modulation of H3K4me3/H3K27me3 marks manifested by accelerated senescence [Leaf senescence involves global alterations in chromatin organization including H3K4me2, H3K4me3, H3K27me2, and H3K27me3 modifications designating marks associated with regions’ activation or inactivation, respectively. Ay et al.  initiallnescence ,70.Arabidopsis methylation is mediated by three enzyme families: methyltransferases (METs), the DOMAINS REARRANGED METHYLTRANSFERASES (DRMs), and the plant specific CHROMOMETHYLASES (CMTs). Research on a transgenic line with an antisense MET1 gene confirmed the relevance of enzyme activity, where delayed senescence and other developmental defects were observed [Arabidopsis plant transcript analysis, which showed a clear decrease in expression of the methyltransferase genes AtCMT3 and AtMETI, while the levels of the demethylase genes AtROS1, AtDME, AtDML2 and AtDML3 rose at least at some phases of plant senescence [Arabidopsis and barley during senescence [Arabidopsis thaliana. Nevertheless, it is not the sole factor regulating PPH expression, since complex regulatory mechanisms are involved in leaf senescence [DNA methylation plays an important role as an epigenetic mark and greatly influences plant development, by changing chromatin structure in a number of ways. The results of many studies confirm that dynamic methylation changes during plant senescence, in contrast to the global range of chromatin adjustments, are local. In observed . Moreoveobserved  suggestenescence . There inescence ,73. TEs nescence . This TEnescence ,75,76.The latest study conducted by Trejo-Arellano et al.  showed scis) as well as between different threads (trans) [Arabidopsis SWI/SNF complex is composed of: one of four SWI2/SNF2-type ATPases ; two of four SWI3-type proteins  and one SNF5-type protein (BSH) [SAG21 ; SAG101 (senescence-associated carboxylesterase); NAC6 (NAC-domain transcription factor 6); SAUL1 (senescence-associated e3 ubiquitin ligase 1); SFP1 (sugar-porter family protein induced during leaf senescence); AOC1; AOC2; AOC3 . Moreover, BRM can colocalize senescence-dependent genes, for example SAG21 or SFP1, with REF6. These supplementary data suggests that BRM containing SWI/SNF complexes can be involved in the nucleosome control of senescence-dependent genes in plants. Moreover, they suggest that BRM-containing SWI/SNF complexes may cooperate with REF6 during epigenetic control of leaf senescence.Chromatin structure can be changed not only by covalent DNA and histone modifications . There i (trans) . Moreove (trans) , change  (trans)  or loose (trans) . Among sin (BSH) ,78. Invein (BSH)  (supplemChromatin remodeling protein 1 (DRD1), whose epigenetic function has been described as a gene silencer from the SWI2/SNF2 subfamily, was also identified  as a leaArabidopsis arf2 mutants. Auxin response factor 2 (ARF2), which binds to the DNA 5’-TGTCTC-3’ sequence, found in the auxin-responsive promoter elements, is responsible for auxin-dependent senescence progression, and it can be knocked out by miR390-mediated production of TAS3—transcriptional silencing complex protein [The influence of microRNAs (miRs) on leaf senescence regulation has been demonstrated in numerous studies to date. It is worth noting that miRNA expression can be spatially dynamic and specific, which determines the diversity of their functions. The nature of the plant’s response also plays a significant role here . Multile protein ,85.Arabidopsis leaf senescence regulation. The main function of the evening complex is to maintain the correct operation of the circadian clock, especially by affecting its gene expression. It also acts as an environmental sensor, conveying information about temperature and light entrainment to growth and developmental pathways [Arabidopsis jasmonate-induced leaf senescence, by binding the promoter of transcription factor MYC2, which encodes a key activator of this signaling pathway, and represses its activation. The study of physiological phenotypes, as well as the measurement of expression level of jasmonate-induced SAGs, showed that the leaf senescence was significantly upregulated in evening complex mutants [prr9 mutant, a research team proved that ORE1 overexpression eliminates mutants’ delayed age-induced leaf senescence [The latest research results reveal the circadian evening complex as a mediator of pathways . It was  mutants . Other rnescence .Arabidopsis through epigenetic control of SAGs. By examining the senescence phenotypes in the loss-of-function mutants for 13 key components of the DNA repair pathway, it was confirmed that the deficiency in ATM results in premature senescence in Arabidopsis. The epigenetic mechanism of this modification relies on ATM repressing the expression of senescence-associated TFs such as ANAC016 , WRKY6 (WRKY transcription factor 6), WRKY53 and WRKY75 (probable WRKY transcription factor), through modulating histone lysine methylation [A different type of epigenetic regulation was observed during a study on the impact of ATM kinase on leaf senescence triggered by DNA DSBs . It was In conclusion, crop senescence involves major reprogramming of gene expression, and recent research has revealed complex regulatory mechanisms, including the hierarchical action of many TFs, but also a higher-order regulation via alterations in chromatin structure. Epigenetic control of the level of senescence-inducing signals, which is connected to the overall environmental cues and the developmental program, is then possible. There is rapidly accumulating evidence that gene regulation of these pathways includes differential changes in chromatin status, switching from transcriptionally inactive heterochromatin to actively transcribed euchromatin, and vice versa. This control level is implemented by different but interacting and often interdependent epigenetic mechanisms, including, described above, DNA methylation, covalent histone modifications, and non-covalent chromatin remodeling. It may allow a rapid response to signaling and stimuli when the expression of a group of proteins (which can range from tens to thousands) needs to be adjusted immediately. The genetic and epigenetic changes may act alone or together and regulate the gene expression, which may result in nonheritable and heritable (stress memory) changes and may lead to survival and/or crop improvement .Arabidopsis expression data reported by Breeze et al. [The barley crop model for early and late events during dark-induced leaf senescence , which ae et al.  and avaie et al. ,90,91, wThe conclusions from the research contributed to the formulation of the hypothesis that chromatin-remodeling mechanisms in response to induced senescence as environmental stimuli control the rate of leaf senescence by: (i) initiation of leaf senescence, (ii) control of senescence-dependent remobilization, (iii) leaf passage from senescence to the death phase.It is desirable to design new breeding strategies in which the epigenetic variability should be taken into consideration . Epigenetic variants in crops might represent an additional and timely resource for crop breeding. Epigenetic variants with potential agronomic interest that have been identified in several crop species are dwarf phenotypes in rice , anthocyIt is thus necessary to deepen our investigation of the epigenetic regulators in crops during stress-induced senescence and their underlying molecular mechanism. Understanding the mechanism of epigenetic regulators and their regulatory networks in this process in crops will be a potential tool for further exploitation toward sustainable agriculture. It seems even more realistic with the advances in genomic technologies and the cost lowering of next-generation sequencing. Like MAS (marker-assisted selection), epigenetic marker-assisted selection could also be initiated.


Drosophila melanogaster the archetypal Runx protein, Runt, functions in numerous processes including segmentation, neurogenesis and sex determination. During primary sex determination Runt acts as one of four X-linked signal element (XSE) proteins that direct female-specific activation of the establishment promoter (Pe) of the master regulatory gene Sex-lethal (Sxl). Successful activation of SxlPe requires that the XSE proteins overcome the repressive effects of maternally deposited Groucho (Gro), a potent co-repressor of the Gro/TLE family. Runx proteins, including Runt, contain a C-terminal peptide, VWRPY, known to bind to Gro/TLE proteins to mediate transcriptional repression. We show that Runt’s VWRPY co-repressor-interaction domain is needed for Runt to activate SxlPe. Deletion of the Gro-interaction domain eliminates Runt-ability to activate SxlPe, whereas replacement with a higher affinity, VWRPW, sequence promotes Runt-mediated transcription. This suggests that Runt may activate SxlPe by antagonizing Gro function, a conclusion consistent with earlier findings that Runt is needed for Sxl expression only in embryonic regions with high Gro activity. Surprisingly we found that Runt is not required for the initial activation of SxlPe. Instead, Runt is needed to keep SxlPe active during the subsequent period of high-level Sxl transcription suggesting that Runt helps amplify the difference between female and male XSE signals by counter-repressing Gro in female, but not in male, embryos.Runx proteins are bifunctional transcription factors that both repress and activate transcription in animal cells. Typically, Runx proteins work in concert with other transcriptional regulators, including co-activators and co-repressors to mediate their biological effects. In  Four Xelopment . The molSxl expression but differ in their sensitivities to gene dose and in their molecular effects on SxlPe  family of proteins , and byrunt functions to regulate SxlPe. We build on the experiments of Kramer et al.  (Hes)-family, including Deadpan  is a potent co-repressor of pression . Reasoni Stat92E , functio1118w flies were received from Bloomington stock center. The w f run3/Binsinscy/Dpymal108(run+) stock was obtained from T.W. Cline. Null run3 mutant embryos were generated from the cross between w f run3/Binsinscy females and run3+/Yymal108+) background.Flies were grown at 25° on a standard cornmeal and molasses medium. runt+-VWRPY 10,050 bp genomic fragment, was amplified from 1118w fly genomic DNA using Expand Long Template PCR System (Roche) and cloned into pCR II-TOPO TA vector (Invitrogen). An AvrII site was introduced abutting the runt stop codon. The fragment ends are defined by primers: 5′-GGAAAAGTGTGTGGAAAACGGTGGA and 5′-GCAACCCAAATGTCTTGTGAAATGAA. The runt+-VWRPY construct was modified to runt-∆WRPY and runt-WRPW using PCR to amplification to change the C-terminal amino acids. The entire runt coding sequences, including modifications, were introduced into the genomic clone using an AscI site located in the runt 5′ UTR and the introduced AvrII site and confirmed by DNA sequencing. All Runt domain mutations: Cys-127-Ser and Lys-199-Ala, Arg-80-Ala, Arg-139-Ala, Arg-142-Ala, Arg-174-Ala, Arg-177-Ala mutants were generated in pCR II-TOPO TA vector using QuikChange site directed mutagenesis kit (Agilent). The wild type and the respective modifications were confirmed by DNA sequencing. All constructs were cloned, using vector derived EcoRI sites, in the pattB transformation vector kindly provided by Johannes Bischof, Basler lab, Zurich. Transgenic injections were carried out by Genetic services Inc. MA. Constructs were inserted into fly genomic attP2 site on the third chromosome by targeted φC31 mediated specific insertion . Probe was detected using anti-Digoxygenin antibody (Roche) that cross react with NBT-BCIP solution staining the embryos. Primers used to in vitro templates were: Sxl forward 5′-CCCTACGTCGACGGCATTGCAGC-3′, Sxl reverse 5′-TAATACGACTCACTATAGG-GAATGACCCAATGGAATCG-3′ and runt forward 5′-AACGACGAAAACTACTGCGGCG-3′, runt reverse 5′-AATTAACCCTCACTAAAACGGTCACCTTGATGGCTTTGC-3′.Embryos were collected 0 to 3hr 30 min after the egg laying. Fixation of embryos and Strains and plasmids are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables.runt function eliminates Sxl protein and SxlPe activity, as measured by SxlPe-lacZ transgenes, in a broad central region in early embryos but has no apparent effect on Sxl at the anterior and posterior poles  females during nuclear cycle 12  males. Stained progeny were run33/ run and run3/+ female embryos in equal proportions. Male embryos with either one or two copies of runt+ do not express SxlPe and were unstained. We found over multiple experiments that female embryos completely lacking Runt (run3/run3) had obvious defects in Sxl expression during cycles 13 and 14. In early cycle 13 run3/run3 mutants, the defects were evident as a loss of nuclear dots, and thus of expressing nuclei, in the central portions of embryos with the non-expressing regions expanding as cycle 13 progressed , whereas their run3/+ siblings expressed SxlPe in a normal pattern. We could not identify any defects in SxlPe expression during nuclear cycle 12 even though one half of the female embryos were run33/run mutants. To confirm that loss of zygotic Runt had no detectable effects on SxlPe during cycle 12, we examined all the cycle 12 embryos present in two additional experiments. Out of 47 cycle 12 embryos observed, we found 25 stained female and 22 unstained male embryos. As expected given the mosaic nature of the onset of SxlPe expression in cycle 12, the staining patterns of the 25 female embryos were varied, but none appeared outside the norm of wild-type Sxl expression, and none exhibited evidence of central region defects as seen in cycle 13 and 14 run33/run females. While these data suggest that Runt is not needed to express SxlPe in cycle 12, we cannot exclude the possibility that a low-level of maternally-supplied Runt might promote the initial activation of SxlPe in the absence of zygotically provided Runt. We note, however, that runt mRNA is not detectable prior to nuclear cycle 10 by RNA-seq in staged single embryos (in situ hybridization (data not shown).To examine the effects of loss of ogressed . By earlrunt is not required for the initial activation of SxlPe, but is instead needed to keep the promoter fully active during cycles 13 and 14, but only in the central regions of the embryos. In this sense, runt is similar to the XSE upd and its associated Stat92E transcription factor, which are likewise dispensable for SxlPe activation but required to maintain full SxlPe activity after cycle 12  males carrying the two copies of the runt+-VWRPY transgene. All embryos at or before nuclear cycle 13 expressed runt mRNA in patterns indistinguishable from that seen in wild type . Taken together, the complete rescue of SxlPe activity in runt null mutants and the normal transgenic runt expression pattern , predicted to disrupt DNA binding without greatly perturbing Runt structure, was unable to activate Sxl when overexpressed after microinjection of runt mRNA into embryos. To confirm that this finding applied to more normal levels of Runt, and to guard against the possibility that the CK amino acid replacements might otherwise alter Runt structure, we introduced the same C127S and K199A changes, as well as five single amino changes  predicted to inhibit DNA binding without altering structure (runt+-VWRPY transgenes creating runtm).A requirement for Runt DNA binding in tructure  into ourSxlPe as the female progeny of crosses between run3/Binsinscy females and run3+/Yymal108; runtm). In no case did we observe evidence for partial complementation confirming that Runt’s DNA binding motif is needed for its XSE function.We found that each of the amino acid changes abolished the ability of the Runt transgenes to activate SxlPe activation, the WRPY portion of the motif was precisely deleted from the transgene to produce a runt-∆WRPY derivative.  as previous work has shown that transcription of ftz is partially dependent upon runt activity in precellular embryos  (HES) family. HES proteins bind Gro through their C-terminal ‘WRPW’ motif and recruit it to target gene promoters , a finding in stark contrast to what was seen with sc or sisA which are strongly male-lethal if either one is present in three copies . A two- effects .SxlPe known to be important for transcription  where Runt interacts with the transcription factor Opa to bind the slp1 DESE enhancer to drive expression in odd numbered slp1 stripes  bound a variety of SxlPe fragments, but we observed that binding was efficiently competed in every case by low concentrations of non-specific (poly dI-dC) competitor (unpublished data). Given the absence of obvious matches to the Runt binding site consensus at SxlPe and the inability to identify specific in vitro binding sites, it suggests that Runt may bind to SxlPe only in combination with other protein complexes.A remaining mystery is where Runt binds at SxlPe has been activated offers a possible explanation for how the sparingly dose-sensitive, runt protein could play an important role in amplifying the twofold difference in male and female XSE doses into a reliable developmental signal. We previously proposed a model in which female-specific dampening of Gro-mediated repression was a central part of X-chromosome signal amplification (Sxl reduced Gro-mediated repression of SxlPe. In the modified version of the model (SxlPe activation during cycle 12, but that their combined concentrations are insufficient to keep the promoter active in the face of increasing repression as the zygotic repressor Dpn accumulates and translation of maternal Gro mRNA continues. The “weak” XSEs function to counteract repression after Sxl transcription begins, either by further enhancing SxlPe, as may be the case if Stat92E functions as an activator, or by directly inhibiting Gro function by counter-repression as we propose for Runt. Signal amplification would occur because the increasing XSE protein concentrations in 2X embryos maintains the promoter in an active state, whereas the 1X dose of XSEs can never overcome the ever-increasing repression in males.The notion that Runt might target Gro function only after fication . Our foche model , Runt, aSxl exist for other genes? Interestingly, McLarren et al. (Might the kind of counter-repression mechanism we propose for Runt at


Thermus thermophilus HB8 TetR-family transcription factor TTHB023. The consensus sequence 5′–(a/c)Y(g/t)A(A/C)YGryCR(g/t)T(c/a)R(g/t)–3′ was found to have a nanomolar binding affinity with TTHB023. Analyzing the T. thermophilus HB8 genome, several TTHB023 consensus binding sites were mapped to the promoters of genes involved in fatty acid biosynthesis. Notably, some of these were not identified previously through genetic approaches, ostensibly given their potential co-regulation by the Thermus thermophilus HB8 TetR-family transcriptional repressor TTHA0167. Our investigation provides additional evidence supporting the usefulness of a biochemistry-based approach for characterizing putative transcription factors, especially in the case of cooperative regulation.Transcription factors are proteins that recognize specific DNA sequences and affect local transcriptional processes. They are the primary means by which all organisms control specific gene expression. Understanding which DNA sequences a particular transcription factor recognizes provides important clues into the set of genes that they regulate and, through this, their potential biological functions. Insights may be gained through homology searches and genetic means. However, these approaches can be misleading, especially when comparing distantly related organisms or in cases of complicated transcriptional regulation. In this work, we used a biochemistry-based approach to determine the spectrum of DNA sequences specifically bound by the  The process of transcription is the primary means by which all organisms control their specific gene expression. This is achieved through the binding of sets of proteins known as transcription factors to specific DNA sequences often located within the promoter regions of regulated genes. Identifying those genes can provide valuable information regarding a transcription factor and its biological role in an organism’s physiology.Advances in DNA sequencing techniques have allowed the determination of genomic sequences for over 370 thousand organisms . Within Escherichia coli, where information regarding its transcription factors, their consensus binding sequences, and the genes that they regulate, has been obtained ) ) .T. thermophilus HB8 were obtained from the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) website (https://www.ncbi.nlm.nih.gov/geo/) [T. thermophilus HB8 grown in rich medium for 360 min and samples GSM904753-5, obtained from TTHB023-deficient T. thermophilus HB8 strains propagated under identical conditions, were analyzed using their NCBI GEO2R program with default settings. Changes in gene expression  and their statistical significance  were determined for each downstream gene within a potentially regulated operon. Likewise, data sets GSM532200-2 (wild-type) and GSM539588-90 in SuperSeries GSE21875 were used to compare gene expression from wild-type and TTHA0167-deficient T. thermophilus HB8 strains, to ascertain its involvement in potentially cross-regulated gene expression. Full data from both GEO2R analyses are provided in To ascertain whether TTHB023 was a regulator of target genes, publicly available microarray data for gene expression profiles in wild-type and TTHB023-deficient ov/geo/) . SuperSe


However, studies using isoform-specific knockout and knockdown strategies revealed that each calpain species carries out specific functions in vivo. To understand the mechanisms that differentiate calpain-1 and calpain-2, we focused on the efficiency and longevity of each calpain species after activation. Using an in vitro proteolysis assay of troponin T in combination with mass spectrometry, we revealed distinctive aspects of each isoform. Proteolysis mediated by calpain-1 was more sustained, lasting as long as several hours, whereas proteolysis mediated by calpain-2 was quickly blunted. Calpain-1 and calpain-2 also differed from each other in their patterns of autolysis. Calpain-2–specific autolysis sites in its PC1 domain are not cleaved by calpain-1, but calpain-2 cuts calpain-1 at the corresponding position. Moreover, at least in vitro, calpain-1 and calpain-2 do not perform substrate proteolysis in a synergistic manner. On the contrary, calpain-1 activity is suppressed in the presence of calpain-2, possibly because it is cleaved by the latter protein. These results suggest that calpain-2 functions as a down-regulation of calpain-1, a mechanism that may be applicable to other calpain species as well.Calpain-1 and calpain-2 are highly structurally similar isoforms of calpain. The calpains, a family of intracellular cysteine proteases, cleave their substrates at specific sites, thus modifying their properties such as function or activity. These isoforms have long been considered to function in a redundant or complementary manner, as they are both ubiquitously expressed and activated in a Ca The primary function of calpains is limited proteolysis of substrate proteins and modulation of their functions, localization or turnover rates. Defects in individual calpain species induce serious pathogenic states at the cell and organ levels, including neurodegenerative diseases and muscular dystrophy  including 2 mM AEBSF as described previously . For theTotal RNA from cultured cells was extracted by TRIzol® reagent (Invitrogen) according to manufacturer’s instruction. First-strand cDNA was synthesized from 0.3 μg of RNA using PrimeScript RT Master Mix . Real-time qPCR (RT-qPCR) was run in the LightCycler 480 II (Roche) using SYBR Premix EX Taq II . Reactions were carried out in duplicate or triplicate and results were normalized according to GAPDH quantitation in the same reaction. The primer sets were listed in Cells were transiently transfected with 20 nM small interfering (si) RNA using Lipofectamine RNAiMAX (Thermo Fischer Scientific) and subsequently cultured for 48 h. The siRNAs for CAPN1 (sc-29885), CAPN2 (sc-41459) and non-targeting siRNA  were purchased from Santa Cruz Biotechnology . At 24 a1TL9 and 3BOW, respectively  1, 2 and 3, respectively.As often observed in slightly , bracketslightly D,G. ThesNext, we analyzed autolytic fragments of C1 and C2 detectable by SDS-PAGE . The N- and C-terminal ends of these fragments generated in the course of autolysis revealed that C1 and C2 differed in terms of the complexity of their autolytic products. Comparison of the deduced structures of autolytic fragments from CAPN1 and CAPN2 suggested that C2 undergoes more heterogenous and aggressive proteolysis at consecutive peptide bonds. Although the exact CuT sites were not detected as the N-termini or C-termini of any of the fragments, proteolysis at nearby sites was indicated for several fragments from CAPN1 , as well as CAPN2 .To determine whether autolysis at CuT sites is related to substrate specificity of C2, we analyzed reciprocal proteolysis between C1 and C2.First, we investigated whether C1 cleaves CuT sites in C2 using proteolytically inactive mutant C2:CS as a substrate. In these experiments, C2:CS, expressed and purified as a complex of CAPN2:CS and CAPNS1, was incubated with C1 or C2 and subjected to SDS-PAGE A and LC-m/z 683.88), which corresponds to the sequence flanked by CuT1 and CuT3; and SEKKADYQAVDDEIE , one of the fragments commonly generated by C1 and C2  was detected in both. Therefore, we concluded that C1 cleaves C2:CS, but not at the CuT sites.LC-MS/MS analysis of the above reactions showed that proteolysis of C2:CS by C1 and C2 was detected with sequence coverage of 15.1% and 37.7%, respectively. Extracted ion chromatograms (XIC) were drawn for two peptides: NEEILARVVPLN . HCT116 have increased levels of CAPN2 in comparison with other colon cancer cell lines , and we To evaluate the role of C2 on down-regulation of C1, CAPN1 or CAPN2 was knocked down. Analysis of mRNA levels by RT-qPCR confirmed the specificity of treatment and denied the possible cross-isoform compensation at the transcript level . At the protein level, specific decrease of CAPN1 or CAPN2 was confirmed A. Althou2+. Increased abundance of the 76 kDa fragment in CAPN2 knocked down cells was also confirmed using an antibody specific to its N-terminal sequence . The prediction is in part contradictory to the experimental result, where C2 recognized CuT1 in the sequence of CAPN1 as well as CAPN2. It is possible that additional parameters that were logically missing in the prediction algorithm reflect autolytic site specificity, e.geviously .2+ requirement of C2 was reduced in the presence of C1 under a low Ca2+ concentration at which C1 was activated and digested the small regulatory subunit of C2, proposing the calpain cascade (2+ requirement for activation [Consistent with their high similarity, C1 and C2 exhibit reciprocal proteolytic activity toward each other. At the level of cleavage site preference, however, the reciprocal proteolysis was not identical to autolysis by each protein. This difference led us to examine the impact of reciprocal action between C1 and C2, in particular, the possibility that the long-lasting activity of C1 was shortened in the presence of C2 , arrow B cascade , arrow Ativation . Therefoin vitro conditions, we showed that the coexistence of C1 and C2 does not necessarily increase net proteolytic activity, but rather results in reduced substrate proteolysis. The substrates used in these assays are technically cleavable by both C1 and C2, but are preferentially cleaved by C1. Therefore, these results suggest that C1 undergoes pseudo-autolysis by C2, which down-regulates the activity of C1 more rapidly than genuine autolysis by C1 itself.Under two different 2+ for activation than C1 does, it might be reasonable that activation of C2 is more tightly regulated in terms of occurrence and duration than that of C1. In addition, sustained activity of C1 and its autolytic product should be appropriately terminated. This hypothesis is consistent with the result that C2-specific knockdown induced an increase in C1 activity in C2C12 cells [As an indication of physiological significance of reciprocal regulation between C1 and C2, we focused on that relative expression levels of CAPN1, CAPN2, CAPNS1 and CAST vary among cell lines. For example, altered expression levels of CAPN1 or CAPN2 have been related to pathogenic phenotypes such as malignancy potency ,45. In t12 cells . Cleavabin vivo may be critical.Our results suggest that in a cellular context in which activation of C2 is enabled, C2 is a candidate quencher of C1. Further, using the cell lysates, it was indicated that the ratio of CAPN1/CAPN2 has some impact on the longevity of activated C1. Our model needs further verification, and identification of a condition where C1 and C2 colocalize to realize reciprocal regulation through inter-isoform proteolysis Click here for additional data file.


Here, we showed metaRE performance for the identification of low-temperature-responsive cis-regulatory code in Arabidopsis thaliana and Danio rerio. MetaRE identified potential binding sites for known as well as unknown cold response regulators. A notable part of cis-elements was found in both searches discovering great conservation in low-temperature responses between plants and animals. At the molecular level, response to an external factor or an internal condition causes reprogramming of temporal and spatial transcription. When an organism undergoes physiological and/or morphological changes, several signaling pathways are activated simultaneously. Examples of such complex reactions are the response to temperature changes, dehydration, various biologically active substances, and others. A significant part of the regulatory ensemble in such complex reactions remains unidentified. We developed  More than two decades have passed since the establishment of whole-genome expression profiling methods. Nowadays, thousands of transcriptomes are publicly available. Typically, several related experiments studying the same phenomenon can be found, thus, providing a rich set of material for analysis. Meta-analysis is applicable to sets of experiments testing the same hypotheses to extract robust signals and repetitive features that are impossible to derive from the individual experiments.The typical example of meta-analysis is the definition of robust differentially expressed genes (DEGs) over many transcriptomic datasets. This approach is widely used in medical genomics to identify the gene signatures associated with a condition or disease, e.g., in ,2,3. To cis-regulatory code behind the transcriptional response. The simplest way is to analyze the upstream regions of the robust DEGs for overrepresented sequences, e.g., as in  of protein-coding genes from Ensemble BioMart Database  [metaRE annotated the upstream regions by the diversity of nonredundant k-mers. In this study, we searched for hexa-, hepta-, and octamers.On Step (2), o rerio) ,34. metametaRE identified all k-mers associated with the transcriptional cold stress response, separately for Arabidopsis and zebrafish. As the number of datasets for Arabidopsis allowed us to study time-resolved response, these cold-responsive transcriptome datasets were divided into two groups by the time of response: early response (up to six hours of cold exposure), and late response (12–24 h of cold exposure). We tried two multiple testing corrections (Bonferroni-Hochberg or Bonferroni) and set the stringent threshold for adjusted meta-p-value < 1 × 10−10 and adjusted permutation p-value ≤ 1 × 10−3.On Steps (3–5), k-mers , the potential binding sites for CAMTA; (2) CACGTG (p = 1.52 × 10−54), the G-box bound by bHLH and bZIP transcription factors; (3) ACACGT (p = 2.3× 10−53), the motif bound by NAC, BES, bZIP, and bHLH transcription factors; (4) ACGTGG (p = 2.65 × 10−52), potential binding site for bZIP and bHLH; and (5) a group of AT-rich elements (3.47 × 10−11 < p < 2.89 × 10−50). The involvement of transcription factors bound to (1) – (4) with the cold response was known beforehand [The binding sites for C-REPEAT BINDING FACTORs (CBFs) transcription factors from AP2/ERF family  were expected to be found as associated with the transcriptional cold response, as CBFs are the major regulator of cold acclimation ,36,37,38forehand ,42,43,44forehand ,45. PotemetaRE usage in the study of the cis-regulatory code behind transcriptional reprogramming in complex reactions. It allows not only predicting the diversity of involved cis-elements and respective transcription factors but also ranking them and clarifying their role in certain phases of transcriptional response.However, most of the detected AT-rich elements remain unknown; some of these sequences significantly match  the known binding sites for HD-ZIP and MYB families B. Althou−4). In vertebrates, the role of Elk1 transcriptional activator was widely discussed in relation to many developmental processes [Danio rerio, it was only shown that Elk1 and its homologs express around the developing bone [cis-elements and transcription factors from Danio rerio genome are much less annotated comparing to Arabidopsis. We were not able to annotate overlapped hexamers , that show the greatest association with the hypothermia condition, using publicly available data. However, we assume that they compose the binding site for zebrafish’ transcriptional regulator(s) that mediate the low-temperature responses.A similar study for zebrafish yielded 67 hexamers enriched in promoters of hypothermia-induced genes. As for predicted cold-associated elements in Arabidopsis, most of the zebrafish ones are associated with upregulation and there are many A/T-rich hexamers . The onlrocesses ,50, but ing bone . Unfortu−32 < p < 8.09 × 10−22) and the binding sites for Homeobox transcription factors ; (2) potential binding sites for bHLH transcription factors ACATAT (p = 2.19 × 10−22) and CACGTG (p = 4.4 × 10−17); (3) potential bZIPs binding sites ; (4) ACCAAT, the binding site for Nfya (p = 5.28 × 10−18), and many others . Among them: (1) two groups of AT-rich motifs resembling the binding sites for Dmrt2a  identified by metaRE R package is that if the cis-regulatory elements are involved in a transcriptional response, then they should be overrepresented in the promoters of differentially expressed genes. This idea is not new, and there are many approaches facilitating the analysis of cis-elements overrepresentation within upstream regions of pre-compiled gene sets, e.g., in [cis-elements overrepresentation systematically and summarizes the output taken from many independent datasets has been still required, these tasks were solved in the metaRE R package.The main idea behind the method implemented in the e.g., in ,53,54,55metaRE method lies in: (1) taking into account a large number of comparable transcriptome experiments, and (2) the consideration of enrichment significance for an individual cis-element. Usually, authors evaluate the enrichment of cis-elements in one or more gene lists independently; the results of enrichment between the lists are not compared [cis-element in different datasets is leveled, which can lead to over- and underpredictions. The method underlying metaRE solves this problem.The novelty of the compared ,5. In thmetaRE algorithm, is that only robust and significant cis-element association with transcriptional response will be detected across multiple, diverse transcriptomic datasets that test similar experimental variables. This could be considered both as an advantage and as a disadvantage of the systematic analysis. On the one hand, analysis of several datasets excludes a bias that could be caused by separate experiments . Thus, meta-analysis would detect the major cis-elements that operate under a variety of conditions. On the other hand, this approach will miss rare and condition-specific cis-elements. The latter could be solved by separate analysis of the datasets from experiments performed on different tissues, so one can have a tissue-specific cistrome. For example, in this study for cold-stress-responsive cistrome, as well as in [Separate studies showed that systematic analysis of transcriptome datasets is powerful in the identification of the cistrome behind a complex reaction ,8,19. ThCis-elements enrichment analysis is especially powerful when performed using the position weight matrices (PWM) for known transcription factors. E.g., using Homer [metaRE we intentionally used a simpler consensus model for identification of overrepresented elements, making it more versatile and applicable for more organisms. First, for almost all organisms, including the model ones, the binding sites of most transcription factors remain unknown. Moreover, only very few organisms have PWMs for at least a hundred transcription factors  [metaRE could be applied not only to the upstream regions but to any sequences associated with the genes to find the signals unrelated to transcription factor DNA binding and not described by PWMs. For example, analyzing the 3’UTR metaRE could help identify the sites for the miRNA seeds binding. Third, in the present study of cold-responsive cis-elements, consensus search in metaRE with the subsequent analysis of identified sequences using PWMs for known transcription factors in TOMTOM [ng Homer , one cansapiens) . Second,n TOMTOM  was shown TOMTOM .metaRE in the investigation of widely studied processes, cold stress response, in which molecular mechanisms are still full of gaps. We performed an analysis using datasets generated for model objects in plant and animal fields, Arabidopsis, and zebrafish.Here, we employ cis-element in early cold stress response detected by metaRE was the potential binding site for CAMTA  found that, in parallel with CBF genes, 27 other “first-wave” transcription factor genes were highly upregulated at an early stage of cold treatment. Analysis of gene expression in transgenic plants overexpressing 11 of these first-wave transcription factors identified four transcription factors from bZIP family  and heat-shock factor HSFC1 involved in the regulation of cold-stress-responsive genes ,59. metald genes , howevercis-elements overrepresented in the promoters of downregulated by cold genes, which regulatory mechanisms are completely unknown. Here, we found potential binding sites for GATA and TCP transcription factors, as well as many unknown motifs.Another interesting result relates to the cis-elements, they could be rare versions of transcription factors binding sites, or form the biochemical environment for transcription factors binding, or be involved in chromatin structure formation. Anyway, to study these hypotheses experimental investigations are required. The role of candidate genes like GATA, HD-Zip, TCP, and others in the cold stress response still lacks the total understanding and explanation which we need to search for with experimental approaches.A further experimental study is required to clarify the role of predicted unknown cis-regulatory code behind the low-temperature response has been performed [cis-elements overrepresented in the clusters. As a result, they identified 17 octamers, overrepresented in one of the clusters, and experimentally verified two of them, AG(A/C)AACCA and (C/G)AGTCA. Here, we have applied an alternative strategy to search for the systematically enriched cis-elements over the same set of transcriptomes using metaRE. Notably, but not unexpectedly, that the cis-elements identified by [Although we found a great number of transcriptomes generated on zebrafish under suboptimal temperatures, the mechanisms of cold acclimation for this animal appeared to be largely unknown. In one of the few studies, a comprehensive analysis of the erformed . In 16 Rerformed  for cis-ified by  and in tcis-elements detected in two separate metaRE studies for Arabidopsis and zebrafish significantly overlap by 27 hexamers. E-box motif CACGTG was highly overrepresented in promoters of both hypothermia-induced zebrafish’ and cold-stress-induced Arabidopsis’ genes. The E-box elements are known to be bound by bHLH transcription factors in many species including Arabidopsis and zebrafish [An exciting finding was that ebrafish . In zebrebrafish . The expebrafish .metaRE detected the overrepresentation of A/T-rich sequences. Earlier, we got a similar result for auxin-regulated cistrome in Arabidopsis [Danio rerio, A/T-rich motifs were recognized as the potential binding sites of ZF (Zinc Finger) and Homeobox transcription factors. Homeobox transcription factors are known as development regulators [In both searches, bidopsis , but notgulators . Since egulators .cis-elements conservation between plants and animals will help to clarify the mechanisms of low-temperature response. To do that, a more rigorous meta-analysis study on many organisms is in need. metaRE provides a framework of how this can be studied when a sufficient number of transcriptomes is generated.Temperature response is one of the basic stress responses with which primitive organisms had to cope millions of years before the separation of plant and animal kingdoms in evolution. Thus, we believe that comparative studies of the


TAL1 is associated with up to 60% of T-ALL cases and is involved in CTCF-mediated genome organization within the TAL1 locus, suggesting that CTCF boundary plays a pathogenic role in T-ALL. Here, we show that −31-Kb CTCF binding site (−31CBS) serves as chromatin boundary that defines topologically associating domain (TAD) and enhancer/promoter interaction required for TAL1 activation. Deleted or inverted −31CBS impairs TAL1 expression in a context-dependent manner. Deletion of −31CBS reduces chromatin accessibility and blocks long-range interaction between the +51 erythroid enhancer and TAL1 promoter-1 leading to inhibition of TAL1 expression in erythroid cells, but not T-ALL cells. However, in TAL1-expressing T-ALL cells, the leukemia-prone TAL1 promoter-IV specifically interacts with the +19 stem cell enhancer located 19 Kb downstream of TAL1 and this interaction is disrupted by the −31CBS inversion in T-ALL cells. Inversion of −31CBS in Jurkat cells alters chromatin accessibility, histone modifications and CTCF-mediated TAD leading to inhibition of TAL1 expression and TAL1-driven leukemogenesis. Thus, our data reveal that −31CBS acts as critical regulator to define +19-enhancer and the leukemic prone promoter IV interaction for TAL1 activation in T-ALL. Manipulation of CTCF boundary can alter TAL1 TAD and oncogenic transcription networks in leukemogenesis.Aberrant activation of the  T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive fatal disease that affects both children and adults. Approximately 30% of T-ALL cases relapse within the first 2 years following diagnosis . The pooTal1 in T-cells also led to leukemia in mice  transcription factor, is the most frequent gain-of-function mutation observed in T-ALL patients and is found in 40–60% of T-ALL cases resulted from chromosomal translocation (4–5%), interstitial chromosome deletion (25–30%), or an undefined mechanism (60%) . Upregul in mice ,9. Deletpoptosis , implicaTal1 in mice leads to embryonic lethality at embryonic day 9.5 (E9.5) due to a complete loss of hematopoietic cells  are unable to generate both primitive and definitive erythroid cells in vitro and do not contribute to hematopoiesis in vivo in a chimeric mouse  of the TAL1 gene  plays a critical role in organizing enhancer/promoter communications to activate TAL1  female mice as previously described (Primary T-ALL patient samples including TAL1-positive (08H125) and TAL1-negative (08H028) blasts were obtained from the Quebec Leukemia Cell Bank  and expaescribed . All expescribed . All cel−31CBS-KO or −31CBS-INV Jurkat cells were transduced with lentiviral particles expressing the mCHERRY fluorescent protein and luciferase (FUW-CHERRY-puro-LUC)  was then measured 1, 3, 7, 10 and 13 days after injection by invivo bio-imaging using the in Vivo Imaging System . Briefly, mice were anesthetized and given an intraperitoneal injection of 100 mg/kg D-luciferin (Perkin Elmer #122799). After 5–10 min. luciferase activity was measured by IVIS. Animals were used in accordance with a protocol approved by the Institutional Animal Care and use committees of the Ottawa Hospital Research Institute and the Pennsylvania State University.Wild-type (WT) control, uro-LUC) . The viaTotal RNA was extracted using TRIzol Reagent (Invitrogen). A total of 1 μg of total RNA was subjected to reverse transcription with M-MLV Reverse Transcriptase (New England Biolabs) and analyzed by the CFX96™ Real-Time System (Bio-Rad). RNA-seq library was prepared by ‘TruSeq Stranded mRNA Library Prep Kit’ , and paired-end RNA-seq was performed by the Penn State University Genome Sciences and Bioinformatics Facility using the Hiseq2500 (100PE) platform with 40 M reads per sample according to standard protocols. The primers are listed in the CTCF , H3K4me3 , H3K4me2 , H3K27Ac  and H3K9/14ac . ChIP-DNA was recovered using phenol/chloroform/isoamyl alcohol and assayed by quantitative polymerase chain reaction (qPCR). ChIP primers are listed in the 4 cells were harvested and lysed in lysis buffer on ice. Nuclei were extracted and processed by ‘Nextera DNA Library Preparation Kit’ (Illumina). Libraries were purified with the Qiagen MinElute PCR Purification Kit. ChIP-seq and ATAC-seq libraries were sequenced using the Hiseq2500 (100PE) platform with 25M reads per sample.ChIP and ChIP-seq were performed as previously described ,45–46. BRNA-seq, ChIP-seq and ATAC-seq analyses were performed as previously described . In brie6 cells were cross-linked with 2% formaldehyde. Nuclei were extracted and digested with 800U of BamH1 or DpnII (New England Biolabs) at 37°C overnight with shaking. The digested chromatin was treated with 400U of T4 DNA ligase (New England Biolabs) to ligate interacting DNA fragments at 16°C for 3 days. The ligated chromatin was reverse-crosslinked, followed by phenol/chloroform extraction to purify 3C DNA. Purified 3C ligated DNA was amplified using PCR, the products were visualized on a 2% agarose gel with SYBR safe (Life Technologies). The PCR products were cloned into pGEM-T Easy vector (Promega) for Sanger sequencing (Genewiz). Relative crosslinking frequencies were calculated and plotted after normalization to the loading control. The 3C primers are listed in the Chromosome conformation capture (3C) assay was performed as described previously with minor modifications . In brie6 cells were cross-linked with 2% formaldehyde and digested with restriction enzyme overnight. Circular ligated chromatin was reversely cross-linked. Then, 4C DNA library was prepared by ‘TruSeq ChIP Sample Preparation Kit’  for further sequencing.The circular chromosome conformation capture (4C) assay was performed as described previously ,50 with https://arimagenomics.com/) with minor modifications. In brief, 2 × 106 cells were collected and cross-linked in 10 ml of phosphate-buffered saline (PBS) buffer containing 1% formaldehyde at room temperature for 10 min. The reaction was quenched by 0.125 M glycine solution. Cross-linked cell pellet was washed in 1× PBS buffer and collected. Cross-linked cell pellet was treated with lysis Buffer in a tube and incubated at 4°C for 15 min, and then added conditioning solution to incubate at 62°C for 10 min. Add stop solution and incubate at 37°C for 15 min. Cell pellet was digested with reaction buffer and restriction enzyme cocktail to digest chromatin overnight or for at least 2 h at 37°C with rotation. Digested DNA was purified with DNA purification beads (AMPure XP Beads), and then quantified its concentration with Qubit. A total of 750 ng of DNA per sample was sheared through sonication (Bioruptor) with default parameters . Fragmented DNA must then be size-selected to have a size distribution between 200 and 600 bp. 250ng of size-selected DNA was used to generate sequence library with KAPA Hyper Prep Kit . Final libraries were submitted to paired-end sequencing of 100 bp length on an Illumina HiSeq 2500. The detailed Hi-C data analysis protocol is provided in the Hi-C assay was performed to generate a genome-wide interaction as described previously with Arima-HiC Kit (Cat: A410030) (CBS deletion and inversion cell lines by CRISPR/Cas9 technology. Single-guide RNA (sgRNA) oligos (Sigma) were designed using the CRISPR Design Tool (crispr.mit.edu) according to the target genome sequence and cloned into a pLKO5.sgRNA.EFS.tRFP  or pL-CRISPR.EFS.GFP  lentiviral vector. After lentiviral infection, GFP+/RFP+ cells were sorted by flow cytometry. Single cell clones were then selected, and −31CBS deletion and inversion clones were verified by genotyping PCR and Sanger sequencing. The genotyping primers are listed in the We generated −31TAL1 gene, +57CBS, +53CBS, +40CBS and −31CBS relative to TAL1 TSS. Computational analysis revealed that orientations of all four CBSs are point toward TAL1 gene  exhibited very distinct effects on TAL1 expression between K562 and Jurkat cells . Howevn Figure .−31CBS and +53/+57CBS facilitates the +51 enhancer to activate the TAL1 promoter-I in erythroid cells , −/−−31CBS in K562 cells significantly inhibited CTCF binding to all CBSs in the TAL1 locus  leads to diminishment of active chromatin signature in the TAL1 locus in K562 cells  and a TAL1-positive patient sample (08H125), the TAL1 promoter-IV only weakly interacts with the +19 enhancer in the TAL1-negative patient (08H028)   Figure . In cont) Figure . Thus, oCBS orientation reshapes the genome organization and leads to changes in CTCF protected gene expression . Two clones that exhibited inv/inv−31CBS were obtained , and a weak TAD between +53CBS and −31CBS (broken blue cycle dash line) , as well as the larger TAD between +53CBS and −157CBS (broken purple square dash line) encompassing the whole TAL1 locus  Figure , left. Is Figure , right. CBS may change chromatin modifications and accessibility. To address this, we assessed the effects of inv/inv−31CBS on enhancer/promoter chromatin accessibility and histone modifications by ATAC-seq and ChIP-seq assays, comparing vector control and inv/inv−31CBS Jurkat cells. inv/inv−31CBS leads to decreases in chromatin accessibility in −31CBS, -8 Jurkat super-enhancer, TAL1 promoter-I and +53CBS. Consistent with loss of chromatin accessibility, H3K4me3, a promoter mark, is significantly decreased in the TAL1 promoter regions, while enhancer mark H3K27ac is reduced in the -8 super-enhancer, promoter-IV, +19 enhancer and +53CBS  under accession number GSE135320.gkaa098_Supplemental_FilesClick here for additional data file.


Recent evidence indicates that genomic individuality of neurons, characterized by DNA-content variation, is a common if not universal phenomenon in the human brain that occurs naturally but can also show aberrancies that have been linked to the pathomechanism of Alzheimer’s disease and related neurodegenerative disorders. Etiologically, this genomic mosaic has been suggested to arise from defects of cell cycle regulation that may occur either during brain development or in the mature brain after terminal differentiation of neurons. Here, we aim to draw attention towards another mechanism that can give rise to genomic individuality of neurons, with far-reaching consequences. This mechanism has its origin in the transcriptome rather than in replication defects of the genome, i.e., somatic gene recombination of RNA. We continue to develop the concept that somatic gene recombination of RNA provides a physiological process that, through integration of intronless mRNA/ncRNA into the genome, allows a particular functional state at the level of the individual neuron to be indexed. By insertion of defined RNAs in a somatic recombination process, the presence of specific mRNA transcripts within a definite temporal context can be “frozen” and can serve as an index that can be recalled at any later point in time. This allows information related to a specific neuronal state of differentiation and/or activity relevant to a memory trace to be fixed. We suggest that this process is used throughout the lifetime of each neuron and might have both advantageous and deleterious consequences. The individuality of neurons provides an accepted paradigmatic framework for the nervous systems of invertebrates such as Caenorhabditis elegans or Drosophila melanogaster, where the uniqueness of each single neuron is reflected by its particular spatial and functional position . With cuOne particular aspect that is crucial to our understanding of the individuality of somatic cells is the individual genetic equipment giving rise to genetic mosaicism. Commonly, mosaicism is defined as the presence of genetically different lineages of cells derived from a single zygote, with additional variations arising in the soma of each cell that are usually not inherited by the next generation . GenomicResearch on single-neuronal DNA content in human brain over the last 20 years or so has collected a huge but, to some extent, inconsistent pool of data. Searching for cellular signatures of neurodegenerative diseases such as Alzheimer’s disease (AD) was a particular driving force for early studies .First reports, based on analyses of bulk DNA, derived from a mixture containing neuronal and non-neuronal cells of the human brain, showed alterations of DNA content . SubsequIn a comparison of the predicted number of protein-coding genes in a wide range of phylogenetically related vertebrates, only 16,000–26,000 hits are proposed . Most ofMost approaches to assess a potential function of somatic genomic mosaicisms in both health and disease largely ignore the role of RNA or, at the most, attribute to RNA only a canonical function within the context of transcription and translation of genetic information. There are, however, a few most intriguing studies suggesting a role for RNA in DNA sequence modulation, e.g., transcription-associated mutagenesis or transcription-associated recombination resulting from events like RNA collision with replication machinery or co-transcriptional R-loop formation . While iRecently, a study by A striking observation by Firstly, introns are a characteristic feature of eukaryotic genomes. They are genetic elements that can monitor their own gene transcription or the transcription of functionally clustered genes . FollowiUp to 10% of sequences that appear as pseudogenes in the human genome seem to be transcribed  and coulIntronless genes, which represent less than 5% of the human genome, lack intron-dependent transcription control, leading to a more constant expression level. Such features have been reported for genes that preferably encode metabolically passive proteins .Most intronless genes are evolutionarily relatively young, are expressed at lower levels compared to intron-containing genes, show a higher tissue specificity, and evolve faster than spliced genes . It seem1; An unusually high number of intronless genes have neuron-specific expression  or at leIntronless transcripts circumvent the complex splicing process, thereby saving energy and time and allowing for replication of much shorter genes. Splicing mechanisms always pose a definite risk of inaccurate execution. Thus, a globally impaired exon exclusion and selective loss of splicing factors have been shown for AD brains . MoreoveThe number of somatic mutations in the human brain increases over the lifetime due to various types of stress and an age-related loss of DNA repair efficiency, which itself is comprised of mutations contributing to this genomic instability . Usage oTaken together, several lines of evidence suggest that intronless genes, which, to some extent, may appear as pseudogenes, could substantially contribute to the genetic diversity of cells .A likely source of intronless genes in eukaryotic genomes is the retroposition of cellular mRNAs by retrotransposable elements , though Retrotransposons are mobile elements that account for more than 40% of the human genome . They haThe evolutionary bursts of retrotransposable elements in the human genome gave rise to about 700 human intronless genes, which stabilized biological processes critically required for the survival of the species . GeneratThe long interspersed element  is the only known active autonomous retrotransposon in human cells  and covede novo insertions into genomic locations of germline and somatic cells. TPRT is catalyzed in cis by ORF1p and ORF2p, two proteins translated from a bicistronic 6 kb L1 mRNA  to generate  L1 mRNA . The L1  L1 mRNA . Retrotr L1 mRNA . Synthes L1 mRNA , where O L1 mRNA . After e L1 mRNA . Intrage L1 mRNA . Recentl L1 mRNA . The abi L1 mRNA  and play L1 mRNA  and pers L1 mRNA .in vitro mature neurons express detectable L1 mRNA and ORF1p levels and exert efficiently engineered L1 retrotransposition  activity has recently been detected in normal human brain extracts  and blooReplacement studies demonstrated that a poly(A) sequence is required for LINE-1 directed retrotransposition , where nThe poly(A) tail of mRNA facilitates its export from the nucleus, enhances protein synthesis, and stabilizes mRNA by interacting with poly(A)-binding proteins to prevent exonucleolytic degradation.Interestingly, it has been reported that non-conserved poly(A) sites are associated with transposable elements to a much greater extent than conserved ones . This opAPAs could thus provide a broad basis for the incorporation of selected transcripts into the genome of each single neuron by somatic recombination according to their individual profile. Additionally, a potential LINE-1 insertion candidate RNA can possess poly(A) tails of different lengths, obtained by somatic mutation, which are finally reverse-transcribed into the genome . Data frWhile polyadenylation of RNA is required for labeling RNAs to prevent degradation processes, the primordial role of oligoadenylation is RNA tagging for subsequent destabilization, which blurs the boundary between stabilization and destabilization by adenylations . It mighA further requirement for the generation of somatically recombined transcripts and their DNA integration are DNA double-strand breaks. Among others, DNA double-strand breaks have been linked to tumorigenesis and genetic instabilities . In addiRecently, it has been reported that DNA double-strand breaks linked to neuronal activity are a common, basic, and physiological phenomenon. Exploratory activity in mouse, for example, which is associated with increased neuronal activity, has been shown to cause a significant increase in neuronal DNA double-strand breaks . MoreoveLINE-1 mobilization in brain uses functionally active DNA double-strand breaks to jump into the genomic DNA. The linkage of DNA double-strand breaks to neuronal activity  might thL1 insertions in neurons were proposed to be a mechanism of “genomic plasticity” some years ago . AccordiTaken together, there is thus strong evidence that L1 retrotransposon insertions might be involved in the process of long-term memory formation.de novo genomic LINE-1 insertion has mainly focused on the alteration of mRNA/ncRNA expression levels or relocation of splicing-variant-ratios. However, the detection of hundreds of somatically recombined APP sequences in neurons of both the healthy and diseased human brain , appears to be a characteristic feature of AD . At leasA recent study showed upregulation of the histone demethylase KDM4B in AD brains . This hiMany more mutated APP-RNA variants were detected in single neuronal nuclei derived from prefrontal cortices of sporadic AD brains than in control brains . Some ofIf the proposed mechanism is indeed instrumental to the AD pathomechanism, a moderate influence on LINE-1 activity might ameliorate the deleterious insertion of AD-related and mutated APP transcripts. The inhibition of reverse transcriptase activity, as already proposed elsewhere , might tThus, it is necessary to accumulate more detailed knowledge of these mechanisms before any interferences regarding this mechanism can be envisaged.UU and TA wrote the manuscript.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.


Glioma stem cells (GSCs) are crucial in the formation, perpetuation and recurrence of glioblastomas (GBs) due to their self-renewal and proliferation properties. Although GSCs share cellular and molecular characteristics with neural stem cells (NSCs), GSCs show unique transcriptional and epigenetic features that may explain their relevant role in GB and may constitute druggable targets for novel therapeutic approaches. In this review, we will summarize the most important findings in GSCs concerning epigenetic-dependent mechanisms. GSCs ed NSCs) . In factMGMT) gene can predict good outcomes in TMZ treatment . The chromatin organization and its degree of compaction are modulated by DNA and histone covalent modifications, ATP-dependent chromatin remodeling and certain non-coding RNAs (ncRNAs). Epigenetic mechanisms contribute to the cellular hierarchy of tumoral tissue in GB  should be more relevant. For instance, changes in the patterns of DNA methylation, H3K27me3 and H3K4me3 are important in neural lineage differentiation  due to their high prevalence in these cells , 19 and  stimuli . Genome- stimuli , 21. Los stimuli . Moreove- Enhancers often regulate cell-specific gene expression and are defined by the simultaneous occupancy of H3K27ac and H3K4me1. Although enhancer patterns are relatively conserved between GSCs and NSCs, unique GSC patterns are mainly linked to genes with functions in DNA damage response, p53 signaling and angiogenesis; prominent examples are HOX cluster genes, which acquire enhancer histone modifications in GSCs and become highly expressed despite promoter methylation . In contOverall, GSCs are characterized by an impairment of differentiation due to a permanent epigenetic block that maintains the self-renewal capacity of these cells , 23. NonThe Polycomb repressive complexes, essential for normal developmental processes, have been the most studied epigenetic modulators in GSCs. The most relevant findings are summarized in in vitro and in xenotransplanted mice and enhances radiosensitivity  . Within sitivity –37. Secositivity , which esitivity , 40.GSC characteristics display regional variations depending on the tumor niche. Whereas the regions defined by the disruption of the blood-brain barrier in angiogenesis foci were characterized by a high expression of proneural genes, an enrichment of EZH2/SUZ12/H3K27me3 targets and GSCs primarily positive to the proneural markers SOX2 and OLIG2, the hypoxic necrotic regions contained high expression of mesenchymal genes, a strong association with H2A119ub, an enrichment of BMI1 targets and GSCs primarily positive to the mesenchymal markers CD44 and YKL40 . SelectiBMPR1B by hypermethylation of its promoter, mediated by the EZH2-dependent recruitment of DNMT1  and ciliary neurotrophic factor (CNTF) signaling pathways is impaired due to the silencing of the BMP receptor subtype gene of DNMT1 . Whereasferation . These pferation . To add feration .+ cells, HOTAIR recruits both EZH2 and the lysine demethylase KDM1A/LSD1 to repress the tumor suppressor gene PDCD4  and activation of EZH2 through phosphorylation by NIMA-related kinase 2 (NEK2) , 45. Morne PDCD4 . In addiand NTF3 .H3F3A (H3.3A) and H3F3B (H3.3B). Mutations in H3F3A are present in approximately one-third of pediatric gliomas, affecting either lysine 27 (H3K27M) or glycine 34 (H3G34R/V), although the former mutation can also be found to a much lesser extent in the HIST1H3B (H3C2) gene  gene –53. H3K2C2) gene . In DIPGein GLI2 , a relevein GLI2  , maintaining the self-renewal capacity of GSCs and interfering with their differentiation , HDACs catalyze this removal, which is associated with gene repression. Inhibition of HDACs can induce cell cycle arrest, apoptosis and cellular differentiation and can interfere with cancer angiogenesis  in GB , 67, mosogenesis . One intogenesis .in vivo. First, acetylation increase by HDACis is not exclusive of histones   . Effortsicelles) , 76, 77.icelles) , retrospicelles)  althoughicelles) , 81. VPAicelles)  and can icelles) . Howevericelles) , althougicelles) . VPA is icelles) , leadingpairment . This is in vivo . Side ef in vivo .Research on the epigenetics of GSCs has the potential to elucidate the self-renewal and perpetuation mechanisms of these cells through the identification of the epigenetic program that governs aberrant gene activation and repression in cancer. Less known epigenetic modifications should be further explored, as they can provide further insights into tumorigenesis, as in the case of 5’-formylcytosine (5fC) and 5′-carboxylcytosine (5caC) . In addiAll authors contributed to the article and approved the submitted version.LV is supported by the Plan Propio INiBICA (Grant L19-07IN-CO07) and by the Programa Estatal de Generación de Conocimiento y Fortalecimiento del Sistema Español de I+D+i, financed by the Instituto de Salud Carlos III and Fondo Europeo de Desarrollo Regional 2014-2020 (Grants CP15/00180 and PI16/00722). LV is the recipient of a Miguel Servet I contract (CP15/00180) financed by the Instituto de Salud Carlos III and Fondo Social Europeo 2014-2020, Programa Estatal de Promoción del Talento y su empleabilidad en I+D+i.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.