Hydra spp. and other pre-bilaterians alike have advanced our understanding of the evolutionary underpinnings governing eumetazoan tissue development, homeostasis, and regeneration. In addition to its regenerative potential, Hydra exhibits continuously active axial patterning due to its peculiar tissue dynamics. These distinctive physiological processes necessitate large scale gene expression changes that are governed by the multitude of epigenetic mechanisms operating in cells. This review highlights the contemporary knowledge of epigenetic regulation in Hydra with contemporary studies from other members of Cnidaria, as well as the interplay between regulatory mechanisms wherever demonstrated. The studies covered in the scope of this review reveal both ancestral and divergent roles played by conserved epigenetic mechanisms with emphasis on transcriptional regulation. Additionally, single-cell transcriptomics data was mined to predict the physiological relevance of putative gene regulatory components, which is in agreement with published findings and yielded insights into the possible functions of the gene regulatory mechanisms that are yet to be deciphered in Hydra, such as DNA methylation. Finally, we delineate potentially rewarding epigenetics research avenues that can further leverage the unique biology of Hydra.Transitions in gene regulatory processes responsible for the emergence of specialized cell types and spatiotemporal regulation of developmental signaling prior to the divergence of Cnidaria and Bilateria are poorly understood. As a sister group of Bilateria, the phylum Cnidaria can provide significant insights into these processes. Among the cnidarians, hydrae have been studied for >250 years to comprehend the mechanisms underlying their unique immortality and robust regenerative capacity. Studies on  Cells are the fundamental unit of life. Cell fate and function are determined by the biomolecular complement at homeostasis, which is responsive to the internal and external stimuli. The microenvironment experienced by a cell provides the cues necessary to determine its role relevant to the niche that it occupies. While the underlying genetic blueprint of life can be altered by mutations, the plasticity of its interpretation promotes the cellular heterogeneity characteristic of multicellular organisms. Once established, cellular physiology is stably inherited for cell type maintenance. Differentiated cells do possess lineage flexibility, evidenced by their direct (transdifferentiation) and indirect re-programmability (via a pluripotent state) to other cell types . EpigeneEpigenetics refers to the alterations in the flow of genetic information other than genetic ones that can be inherited over generations as defined by Histones form the core for DNA compaction in the eukaryotic and Archaeal phyla and contribute in multiple ways toward regulating the activity of the transcriptional machinery. Across the animal kingdom, the level of conservation in the primary sequence and structure of histones is very high indicating their importance and a conserved mechanism of transcription, thereby offering multiple avenues to understand this process using various model organisms . The his5dC and m6dA, respectively. m5dC is a context dependent regulator of transcription and its more commonly studied function is as a transcriptional repressor that commonly occurs at the CpG clusters of promoters of inactive genes and transposons, within the bodies of active genes to suppress spurious internal transcription, and also at insulator elements to block CTCF binding (5dC intermediate (5dC is recognized by Methyl-CpG-Binding Domain protein 2 (MBD2), which then recruits H3K9-specific KMTs and HDACs to repress transcription. Conversely, H3K9me3-bound Hetrechromatin Protein 1 (HP1α) can promote de novo DNA methylation by recruiting DNMT3A/B  knockout mice  in length, called small RNAs, which are generated by cleavage of a transcribed precursor RNA and are partially or fully complementary to their target RNA sequences. miRNAs and siRNAs are 21–23 nt long sRNA species that are recognized by and incorporated into RNA-induced silencing complexes (RISCs) that can bind to complementary mRNAs and either block their translation or inactivate them by cleavage . piRNAs  H3K9me3 . piRNA p H3K9me3 . A graphHydra comprises freshwater polyp species with a broad geographical distribution. Polyps comprise a columnar gastric region ending in the oral and aboral poles that are respectively organized into head and foot structures. The head comprises an oral pore, called the hypostome, through which prey is ingested, surrounded by 5–6 tentacles that harbor nematocytes to immobilize and capture live prey. A basal disk with mucus-producing gland cells forms the foot with which polyps adhere to substrata. The gastric column is composed of epithelial and interstitial stem cells and also harbors a diffused nerve net  has been utilized to understand the transcriptomes in heterogeneous populations of cells in tissues and animals. The single cell RNA sequencing along with transcript localization studies have provided significant insights into the cell type specific transcriptional programs, body plan patterning and also led to the identification of novel cell-types in cnidarians such as Xenia, Nematostella, and Hydra  which eventually give rise to male and female germ cells , KDMs , DNMTs , and HDACs (Hdac4 and Hdac7)  . These ed Hdac7) . ObserveHydra make it a useful model system for tracing back the evolutionary trajectories of the underlying mechanisms of gene expression regulation. Experiments on polyps have yielded insights into how the conserved cellular coordination is affected in a minimalistic body organization, remains conserved in vertebrates, and impacts the unique physiology of Hydra. Studies elucidating the epigenetic mechanisms operating at the transcriptional and post-transcriptional level in Hydra polyps are discussed in this review.The phylogenetic position of cnidarians together with the dynamic tissue turnover and regenerative capacity of Hydra is highly AT-rich (71%) and comprises a large proportion of transposable elements (57%)  . The sizariation . No obvi8 miRNAs . Additio8 miRNAs . The HydHydra so far. The chromatin of interstitial cells in Hydra cauliculata was found to be enriched with canonical histones during somatic and male germline differentiation, concomitant with condensation. Spermatocyte formation is accompanied by a transition from lysine-rich to arginine-rich chromatin, similar to the replacement of nucleosomes by protamines in higher metazoans during spermatogenesis. In Hydra hymnae, which has arginine-rich histones, this transition does not occur. Moreover, protamine transitions were not detected in either species  in Hydra. HyEED, encoding a homolog of the mammalian Embryonic Ectoderm Development (EED), which is a core component of the Polycomb Repressive Complex 2 (PRC2) complex and critical for the H3K27me2/3 mark was upregulated during embryogenesis in H. vulgaris. The modification in addition to the histone variants may serve to silence sperm chromatin in the absence of protamines, thereby playing a conserved role in a divergent trait. The H3K27me2/3-positive population may correspond to primed stem cells owing to an enriched presence in the interstitial cells over their derivative somatic cells  respectively. The presence of a regulatory switch based on the acetylation/methylation on the H3K27 residue which is critical for Wnt/β-catenin mediated axis patterning in Hydra has been elucidated  has been shown to play a key role in the regulation of neurogenesis and also in regeneration. Here, it has been demonstrated that Hdac2 interacts with SoxB2 and regulates the differentiation of neurons from stem cells (Histone H3 lysine 27 acetylation (H3K27ac) was found to be a more reliable signature of enhancers in pitation  and exhipitation . A similase p300 . Histonentenance . In a clem cells . Later iem cells .Hydra. There have been no studies that investigate the expression patterns of the reader and eraser proteins of histone acetylation and few predictions can be drawn from the scRNA-seq data for future studies. The BRD2 protein shows an expression in multiple cell types with higher levels in progenitor cell types which could be a predictor of its function. Similarly, among the various HDAC proteins present in Hydra, the HDAC1 homolog shows expression in various differentiated and stem cell types of all three lineages  could function to silence oocyte chromatin , determined an m5dC frequency of ∼1%. DNMT and TET orthologs were expressed during early developmental stages, with high TET expression also found in adult combs. This may be functionally correlated with the presence of its intermediate product–hm5dC–in the P. bachei genome  similar to vertebrates indicating convergent evolution  complements of 13 metazoan species, including H. magnipapillata. Four miRNAs were found in the freshwater cnidarian, all novel, including the cnidarian-specific miR-2022 in common with N. vectensis , were in common with N. vectensis. However, barely half the sRNA reads mapped to the draft H. magnipapillata genome, which may be attributed to inadequate coverage of the latter and could explain why only 17 miRNAs were previously identified –found by BLAST in H. magnipapillata–shared only ∼33% sequence identity with a query cover of 42%. It is thus possible that miR-2030 regulates a different protein in Hydra, which could explain the divergence in its localization between the two cnidarians if found to be the case. miRNA pathway components (including HYL1) have also been detected in corals, with predicted roles in endosymbiosis, biomineralization, thermal, and pH stress responses  negatively impacts the stability of its miRNAs and piRNAs, and phenocopies the developmental arrest  observed on RNAi of Dicer1 and PIWI2  facilitate coordinated regulation of various sets of genes required for development and physiology -seq and histone marks to identify the enhancers involved in the regeneration process. This study identified ∼3000 enhancer elements that might contribute to gene regulation during regeneration. HyWnt3a gene locus occurring as early as 4 h post-amputation. Notably, enrichment of CnGsc binding motifs was observed on the enhancer elements found during the regeneration time course which is important in head patterning in Hydra. Based on the initial studies, H3K27ac has proven to be a reliable histone modification mark to identify the enhancer elements with more confidence. However, in Hydra, H3K4me3 also exhibits colocalization at the enhancer elements which needs to be further studied. Additional efforts are required toward the identification of the enhancer elements involved in precise functions such as cell type determination and their functional validation. The conservation of the occupancy of histone marks is depicted in A first step toward understanding the evolution of enhancers is the identification of CREs in genomes of early diverging eumetazoan phyla such as Cnidaria. Characteristic features such as open chromatin and histone marks including H3K27ac, H3K4me1, and H3K4me3 facilitate the identification of CREs. Their functional relevance can be elucidated by studying their dynamics under different experimental conditions. Toward this, a study was performed in ectensis . Among tectensis . In Hydr changes . This ph changes . Furtherer genes . Hydra iHydra spp.  homologs were identified in H. vulgaris, suggestive of the mechanistic origin of RNA Pol II pause in cnidarians  are detected only in the interstitial cells, and therefore the detection of Polycomb proteins in ectodermal epithelial cells is counterintuitive , they have not been characterized so far.The presence of chromatin loops and higher-order assemblies such as the topologically associating domains (TADs) is yet to be determined in polyps. RNA Pol II was detected at putative enhancers in  looping . Cnidari looping  and SATB looping . The rol looping . TADs mantuitive . KLF3, Kem cells . Phylogerthologs . HoweverHydra scRNA-seq data toward identifying cell type-specific regulatory elements. Wherever information in addition to the scRNAseq is available for many proteins and modifications, we have speculated their role in the unique physiology of Hydra and its regenerative capacity. Further validations involving protein levels, the activity of the proteins, the dynamics of target modifications, and the post-transcriptional mechanisms are necessary to understand the complex epigenetic regulation of the developmental processes and regeneration in Hydra. The landmark studies in Hydra alluding to the above are listed in Hydra is provided in Most of the studies on epigenetic modifiers focus on monitoring the transcript levels of the proteins with an evident disparity in mRNA-protein localizations for a few of them. Studies that investigate protein localization and interactions directly will provide better insight into the role of the resultant modification and regulatory nucleic acids. Additionally, single-cell ATAC-seq could provide the chromatin accessibility profiles of individual cells , which cHydra:Apart from the studies suggested in each section of this review, the following investigations may further our understanding of the epigenetic regulation and its transgenerational inheritance in 1.Hydra sperm and oocyte to identify the conserved determinants of transgenerational inheritance. Here, external fertilization is advantageous toward the collection of gametes.Mapping the epigenomes of 2.Delineating the epigenetic regulation of cell fate determination and axis patterning in homeostatic and regenerating polyps could provide evolutionary insights into the process.3.Performing high-resolution proteomic analyses at a single cell level  will fac4.Hydra. The continuous proliferation and differentiation of cells in Hydra offers an excellent paradigm to gain insights into the ancestral roles of the DNA modifying machinery during metazoan evolution. DNA methylation changes are associated with aging and age-related diseases. Hydra is an immortal organism and it does not exhibit typical aging-related disease phenotypes. Thus, understanding the maintenance of DNA methylation in Hydra will provide insights into the aging process.The dynamics and effects of DNA methylation have not been investigated in detail in 5.cis-regulatory DNA elements in the regulation of developmental genes across multiple canonical model organisms (Hydra enhancer elements in higher organisms will allow us to understand the fundamental roles of these regulatory regions and how they were adapted into distinct signaling pathways as the processes diverged. The redundancy of enhancer-mediated regulation can also be addressed well in organisms such as Hydra. They exhibit the processes of embryonic development, budding, and regeneration which offer an opportunity to look at the regulation of the same genes in diverse physiological contexts. Especially, close phylogenetic relationship with Bilateria and occurrence of conserved molecular machinery, cell types, and developmental processes provides a unique opportunity to identify the conserved cis-regulatory elements responsible for the evolution of eumetazoan cell types and functions.Recent studies have elucidated the conservation of rganisms . Investi6.Hydra lack the CTCF-based higher-order compartmentalization mechanisms. However, other mechanisms based on the PcG components, transcription-based organization, and arrangement of the genomic regions in an alternating active-inactive format contribute toward achieving this (Hydra genome.The organization of chromatin into active and inactive compartments aids in the efficient regulation of gene expression in a spatiotemporally appropriate manner. Early metazoans including ing this . It is o7.ADAR machinery has been identified in organisms at the root of animal phyla such as Ctenophores, Placozoans, sponges, and Cnidarians (6A on different RNA species in the cell which targets it for degradation or stabilizes it for translation (Hydra have not yet been identified and the role of these molecules and the modified RNA in the physiology of Hydra will be interesting to study in the context of cellular differentiation during regeneration and budding.Along with the chromatin level regulation of gene expression, there is an additional level of post-transcriptional checkpoints in the cells. A part of this process has been discussed in the review and the other part involves chemical modification of the RNA species inside the cells. Various types of RNA modifications are reported, and the occurrence of the idarians . Among tnslation . The varAP and AG contributed to design concept, collected and analyzed literature, wrote bulk of the manuscript, and prepared table and figures. PCR and SG provided critical inputs as the corresponding authors, wrote sections of the manuscript, and obtained funds. All authors 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.


Cellular senescence provokes a dramatic alteration of chromatin organization and gene expression profile of proinflammatory factors, thereby contributing to various age-related pathologies via the senescence-associated secretory phenotype (SASP). Chromatin organization and global gene expression are maintained through the CCCTC-binding factor (CTCF). However, the molecular mechanism underlying CTCF regulation and its association with SASP gene expression remains to be fully elucidated. A recent study by our team showed that noncoding RNA (ncRNA) derived from normally silenced pericentromeric repetitive sequences directly impair the DNA binding of CTCF. This CTCF disturbance increases the accessibility of chromatin at the loci of SASP genes and caused the transcription of inflammatory factors. This mechanism may promote malignant transformation. In aged individuals, inflammatory SASP factors promote numerous age-related diseases, including some cancers [et al. reported that cellular senescence causes a dramatic alteration of chromatin organization [Cellular senescence is a state of irreversible cell cycle arrest induced by many stressors, i.e., aging, obesity, radiation, and chemotherapy . Senesce cancers . Therefo cancers . Additionization ; howevernization .First, the authors have hypothesized that an aberrant chromatin architecture observed in senescent cells might be associated with SASP and have conducted the analysis of genome-wide chromatin accessibility  followed by gene expression (RNA-Seq) by comparing the proliferating and X-ray-induced senescent human diploid fibroblasts (IMR-90). The combinational analysis showed that the loci containing pericentromeric repetitive sequences called human satellite II (hSATII), which is epigenetically silenced in normal somatic cells, were highly accessible, and hSATII ncRNA expression were markedly upregulated in senescent IMR-90 cells compared with proliferating cells. Conversely, it has been reported that hSATII ncRNA expression is upregulated in senescent cells and many types of cancer ; the funet al. reported that enhancer RNA transcripts up on 17β-estradiol (E2) could be functionally essential for the actions of E2-regulated gene enhancers, at least in part by contributing to the dynamic generation or stabilization of enhancer-promoter looping in human breast cancer cells [et al. reported that murine pericentromeric satellite RNA in cis stabilizes heterochromatin retention of Suv39h [et al. reported that murine centromeric satellite RNA in trans regulates telomerase activity in embryonic stem cells [Accumulating evidence uncovers that diverse noncoding RNAs (ncRNAs) are involved in various biological processes. Especially, a subset of ncRNAs epigenetically regulates gene expression through chromatin remodeling. Li er cells ,24. Intef Suv39h . Additioem cells . These rem cells . The ectem cells . These eTo uncover the molecular mechanism of how hSATII ncRNA promotes SASP-like inflammatory gene expression, the authors attempted to identify hSATII ncRNA-binding proteins. RNA pull-down followed by using mass spectrometry analysis identified CCCTC-binding factor (CTCF) as a hSATII ncRNA-binding protein, and RNA immunoprecipitation analysis showed that the zinc finger (ZF) DNA- and RNA-binding domains of CTCF are essential for their binding to hSATII ncRNA. Significantly, the upregulation of SASP-like inflammatory gene expression induced by hSATII ncRNA was canceled in cells expressing excessive CTCF. Conversely, CTCF depletion caused SASP-like inflammatory gene expression in proliferating cells. Interestingly, the authors found that CTCF depletion increases the expression of not only SASP-like inflammatory genes but also hSATII RNA. Moreover, the expression of CTCF decreases during cellular senescence. Together, although it is not clear whether CTCF is the direct or indirect trigger for hSATII RNA expression, these findings imply that the loss of CTCF drives hSATII RNA expression during cellular senescence. Furthermore, the authors investigated the expression of mouse major satellite (MajSAT) RNA, which is located at the pericentromeric locus of chromosomes, as well as human hSATII ncRNA. The expression of MajSAT RNA also increased in senescent mouse embryonic fibroblasts. Additionally, MajSAT RNA, but not mouse centromeric minor satellite (MinSAT) RNA, bound to CTCF, leading to upregulation of SASP-like inflammatory genes. Altogether, these findings indicate that the functional impairment of CTCF by pericentromeric satellite ncRNAs induces the expression of SASP-like inflammatory genes during cellular senescence.Because the DNA-binding domain of CTCF is essential for the maintenance of genomic integrity and was relevant to its binding to hSATII ncRNA, the authors hypothesized that hSATII ncRNA disturbs the DNA-binding capacity of CTCF through direct binding to its ZF domains. Chromatin immunoprecipitation (ChIP)-seq analysis showed that ectopic expression of hSATII ncRNA altered the distribution of CTCF. Remarkably, ChIP-qPCR and electrophoretic mobility shift assay showed that hSATII ncRNA inhibited the DNA-binding capacity of CTCF to an imprinting control region, a representative CTCF binding site . Moreoveet al. showed that nuclear depletion of the high-mobility group B protein (HMGB2) provokes the alteration of CTCF distribution during cellular senescence [et al. reported that different stressors alter CTCF-RNA interaction [Cellular senescence causes a dramatic alteration of chromatin organization ,30; howeeraction . These reraction . Howevereraction . TherefoSmall extracellular vesicles (EVs) secreted from cancer and stromal cells dynamically contribute to tumor incidence and progression in a non–cell-autonomous manner in the tumor microenvironment . Intriguin vivo could be toxicity because SASP factors play important roles in various physiological processes [These findings highlight the new role of the pericentromeric satellite RNA, which supports tumor development in a non–cell-autonomous manner through the secretion of SASP-like inflammatory factors and small EVs. The SASP factors are thought to promote multiple age-related diseases including some cancers, such as breast, liver and colon cancers. However, blockage of all SASP factors rocesses . In addirocesses , suggest


However, these variants associated with cognition and mental activity have rarely been explored in healthy adolescents. In this study, we screened for SNVs in the glutamatergic signaling pathway to identify genetic variants associated with cognitive ability. We found that SNVs in the subunits of ionotropic glutamate receptors, including GRIA1, GRIN1, GRIN2B, GRIN2C, GRIN3A, GRIN3B, and calcium/calmodulin-dependent protein kinase IIα (CaMK2A) are associated with cognitive function. Plasma CaMK2A level was correlated positively with the cognitive ability of Taiwanese senior high school students. We demonstrated that elevating CaMK2A increased its autophosphorylation at T286 and increased the expression of its downstream targets, including GluA1 and phosphor- GluA1 in vivo. Additionally, methyl-CpG binding protein 2 (MeCP2), a downstream target of CaMK2A, was found to activate the expression of CaMK2A, suggesting that MeCP2 and CaMK2A can form a positive feedback loop. In summary, two members of the glutamatergic signaling pathway, CaMK2A and MeCP2, are implicated in the cognitive ability of adolescents; thus, altering the expression of CaMK2A may affect cognitive ability in youth.The glutamatergic signaling pathway is involved in molecular learning and human cognitive ability. Specific single variants (SNVs, formerly  NMDARs form tetrameric complexes that consist of two glutamate ionotropic receptor NMDA type subunit 1 (GluN1) subunits and two GluN2 or GluN3 subunits [N-methyl-subunits –8. Approsubunits , 10. Glusubunits . By contsubunits .2+ transport, thus activating intracellular signaling cascades to alter synaptic efficacy and induce LTP [Synaptic proteins play a crucial role in synaptic activity and dendritic spine morphogenesis, and variations in synaptic proteins lead to cognitive deficits . Becauseduce LTP .2+ influx involves Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII), a serine/threonine kinase enriched at excitatory synapses and postsynaptic densities [2+/CaM, CaMKII phosphorylates numerous substrates responsible for LTP, including voltage- and ligand-gated Ca2+ channels, cAMP-response element-binding protein, extracellular signal-regulated kinase (ERK), and voltage-gated sodium channels [2+-bound CaM, CaMKII autophosphorylates at Thr286/287, thus enhancing its binding affinity for Ca2+/CaM and inducing Ca2+/CaM-independent autonomous phosphorylation [T286A variant proteins impaired LTP, sLTP, and spatial learning and memory [A signaling pathway activated by ionotropic receptor-mediated Caensities . Upon bichannels . After irylation . This aurylation , 21. Blod memory .E831V) variation in the CaMK2A catalytic domain reduces both CaMK2A substrate phosphorylation and regulatory autophosphorylation. Additionally, CaMK2AE831V may inhibit the phosphorylation of CaMK2A in a dominant-negative manner [CaMK2A variation (CaMK2AH477Y) causes growth delay and seizures in humans [CaMK2A and CaMK2B have demonstrated that Thr286/Thr287 plays a pivotal role in neuronal plasticity [Several recent studies have provided novel insights into the synaptic mechanisms of pathological pathways and have demonstrated that de novo variations in CaMK2A disrupt the function of synaptic proteins . For exae manner . Loss ofn humans . Relatedasticity , 26. Actasticity .2+ signaling. One principal substrate of CaMKII is methyl-CpG binding protein 2 (MeCP2). During neuronal activity and the subsequent Ca2+ influx, CaMKII phosphorylates MeCP2 at S421 [BDNF), ras-related GTP-binding protein 3d (Rab3d), vesicle-associated membrane protein 3(Vamp3), and cell adhesion molecule (CADM3). The neuronal activity-induced phosphorylation of MeCP2 might function as a molecular switch regulating the dynamic expression of neuronal genes [CaMKII also functions in the nucleus through nuclear Ca at S421 , 29. Thi at S421 . MeCP2 pal genes .MeCP2 is an X-linked global transcription regulator that binds to methylated sites in DNA, and its dysfunction is implicated in Rett syndrome (RTT) and MeCP2 duplication syndrome (MDS). Well-defined mouse models of both syndromes have resulted in learning and memory impairment –34. AparCaMK2A and the subunits of ionotropic glutamate receptors, including GRIN1, GRIN2B, GRIN2C, GRIN3A, GRIN3B, GRIA1, and GRID1, are associated with student cognitive function. Furthermore, we discovered that CaMK2A levels were elevated in the peripheral blood samples of senior high school students with superior reasoning skills. Elevated CaMK2A increased the pCaMK2A and GluA1 in cells. Moreover, a downstream target of CaMK2A, MeCP2 could increase the expression of CaMK2A, suggesting the CaMK2A and MeCP2 could form an autoregulatory positive feedback signal transduction loop. We believed that alteration in the expression of CaMK2A might affect the cognitive ability of adolescents through altering the expression of the components of the glutamatergic signaling pathway.Signaling modulators, including CaMK2A and MeCP2, impair cognitive function in many neuropsychiatric disorders –39. AlthA total of 832 students  from three senior high schools  were recruited. This study was approved by the Institutional Review Board of National Taiwan University Hospital (Research Ethics identifier: NCT00713570). The volunteers and their parents were explicitly informed, and written consent was obtained.DNA samples from 20 healthy participants were first genotyped in a pilot study. SNVs with a minor allele frequency of > 5% were selected. The selected SNVs were then genotyped for all participants. Genotyping was conducted through DNA sequencing of relevant polymerase chain reaction (PCR) products using Prism_ BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kits and a Prism_ 3730 Genetic Analyzer  following manufacturer instructions.Cognitive ability was assessed with the Multiple Aptitude Test Battery (MAT) , 41. A Cg. Plasma was collected and stored at − 80 °C until use. Platelets were removed through centrifugation for 10 min at 10000g. CaMK2A levels were measured using a quantitative enzyme-linked immunosorbent assay (ELISA) kit following the manufacturer’s instructions .Blood samples were collected from the participants by using ethylenediaminetetraacetic acid (EDTA) as an anticoagulant and placed on ice. The samples were then centrifuged at 4 °C for 10 min at 3000CaMK2A mRNA : 95) was purchased and reverse transcribed to generate CaMK2A cDNA . The CaMK2A cDNA was digested with NotI and ApaI restriction endonucleases and cloned with N-terminal-tagged enhanced green fluorescent protein or C-terminal-tagged DsR into a pcDNA/Flp recombination Target (FRT)/TO cloning vector (Thermo Fisher Scientific Co. MA USA) Fig. .Fig. 1ScThe Flp-In T-REx system was used to stably induce the expression of target genes in the HEK293 and SH-SY5Y cell lines . HEK293-derived Flp-In host cells were purchased . The generation of the SH-SY5Y-derived Flp-In host cells was described previously . In brieg for 10 min at 4 °C, protein concentration was determined  with bovine serum albumin as the standard. Soluble proteins (25 μg) were separated through 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were electroblotted onto nitrocellulose membranes, which were blocked with 10% nonfat milk and probed with primary antibodies. The antibodies and dilutions used were as follows: anti-MeCP2 , anti-pMeCP2 , anti-CaMK2A , anti-pCaMK2A , anti-BDNF , anti-GluA1 , anti-GluA1 , and anti-H3.3B  antibodies. Immune complexes were detected using horseradish peroxidase-conjugated goat anti-mouse, goat anti-rabbit  IgG , and a chemiluminescent substrate .Total soluble protein was extracted from the HEK293-derived and SH-SY5Y-derived cells treated with 3 μM CaM  at designated time points  using a buffer containing 50 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 1 mM ethylene glycol-bis(β-aminoethyl ether)-N, N, N′, N′-tetraacetic acid, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, and a protease inhibitor cocktail . After sonication and centrifugation at 15000GRINI, GRIA1, and GRID1.The SH-SY5Y-derived cells were treated with formaldehyde  for 10 min at 37 °C and quenched with 0.125 M glycine for 5 min at 25 °C. After being washed twice with PBS, the samples were homogenized in lysis buffer  containing a protease inhibitor . Chromatin was sheared using a Bioruptor sonicator for 35 30-s-ON/30-s-OFF cycles in a 4 °C water bath. For immunoprecipitation, 150 μL of chromatin was diluted at a ratio of 1:10 in chromatin immunoprecipitation (ChIP) dilution buffer . Approximately 7% of the diluted sample was used as input control. Protein A/G Magnetic Beads  were incubated with anti-MeCP2 antibodies (GeneTex) or rabbit IgG overnight at 4℃ and constantly rotated in a blocking solution (0.5% BSA in PBS). After washing and resuspension of the antibody–bead conjugates, the chromatin samples were added to the antibody-bead conjugates and incubated for 16 h and rotated constantly at 4℃. After immunoprecipitation, the beads were washed six times (5 min each) with buffer . and washed with TE buffer . DNA-proteins complexes were eluted with a 200-μL elution buffer . The eluted samples were incubated at 65 °C for 4 h, and the input DNA was diluted in elution buffer (to 200 μL) and processed for cross-link reversal. The samples were then digested sequentially in 0.2 μg/mL RNase A for 2 h at 37 °C and 20 μg of proteinase K for 30 min at 55 °C. The DNA was extracted using the phenol/chloroform/isoamyl alcohol method and subjected to quantitative PCR using the ViiA 7 real-time PCR system  with primer pairs for p < 0.05. Bonferroni correction was used for multigroup comparisons. Post hoc Scheffe’s F testing was performed because of its high statistical power [The associations of genotypes with the participants' cognition (MAT scores) were assessed through analysis of variance (One-way ANOVA). Univariate ANOVA was performed to compare MAT subtest scores among the three genotype groups for each SNV. Significance was set at al power . For eachttp://www.ncbi.nlm.nih.gov/gene), HapMap (http://www.hapmap.org), and Ensembl (http://www.ensembl.org/Homo_sapiens) databases. We found that SNVs in genes encoding NMDAR subunits were associated with academic performance and cognitive ability . The GRIN2B rs1805502 variant was significantly associated with abstract reasoning and grammar and language usage (p = 0.022 and p = 0.053), and the GRIN2C rs3744215 variant was significantly associated with mechanical reasoning (p = 0.013). Two SNVs in GRIN3A (rs10989589 and rs3739722) were significantly associated with verbal comprehension and abstract reasoning (p = 0.017 p = 0.033). The other six SNVs in GRIN3B were significantly associated with the eight MAT subtest scores as follows. GRIN3B rs2240154 was significantly associated with abstract reasoning (p = 0.012). GRIN3B rs4807399was significantly associated with abstract reasoning and grammar and language usage (p = 0.035 and p = 0.024). GRIN3B rs2240157 was significantly associated with abstract reasoning (p = 0.025). GRIN3B rs2285906 was significantly associated with mechanical reasoning, abstract reasoning, and perceptual speed and accuracy . GRIN3B rs10417824 was significantly associated with abstract reasoning and perceptual speed and accuracy (p = 0.012 and p = 0.033). GRIN3B rs10401454 was significantly associated with scores on five of the eight MAT subtests, namely the verbal reasoning, spatial relations, abstract reasoning, verbal comprehension, and grammar and language usage  scores, as listed in Table NMDARs are glutamate-gated cation channels that are expressed throughout the brain and play an essential role in physiological and pathological processes in the CNS. The spatiotemporal expression of the diverse subunits imparts distinct channel kinetics, permeation, blockage by divalent cations, and sensitivity to endogenous modulators. The GRIA1 rs548294 and CaMK2A (rs2241694) SNVs were highly associated with cognitive ability, prompting us to study how they affect gene function and how these genes affect cognitive ability. The CaMK2A (rs2241694) variant was significantly associated with perceptual speed and accuracy (p = 0.041). To address this, 120 students were selected for the study based on their MAT scores. Sixty students with MAT scores higher than 80% were included in a high–cognitive ability group, and 60 students with MAT scores lower than 12% were considered a low–cognitive ability group. Plasma CaMK2A levels were measured using ELISA. The plasma CaMK2A levels of the students with high and low cognitive ability were 529 and 271 pg/mL, respectively. Thus, plasma CaMK2A was positively correlated with the cognitive ability of Taiwanese senior high school students  was significantly associated with higher plasma CaMK2A levels and cognitive ability. Elevated plasma CaMK2A levels might not reflect an increase in activity in the CNS. We address this issue in the Section “Discussion”.In correlating SNVs and CaMK2 levels with the cognitive ability of adolescents, we found that ty Table . AdditioTo assess the effect of CaMK2A upregulation in vivo, quantitative immunoblotting was performed using a human-derived neuroblastoma SH-SY5Y cell line because it has been used to study the relations of epigenetic biomarkers with academic performance and neuronal disorders associated with neurocognitive disorders , 46–50. We previously reported the involvement of a homeostatic regulatory control mechanism of MeCP2, similar to that of CaMKII, in both Rett syndrome and synaptic plasticity . CaMKII GRIA1 promoter 548294, GRID1 promoter 3814614, and GRIN1 promoter 4880213). An anti-MeCP2 antibody could pull down the promoters of GRIA1 and GRID1 in SH-SY5Y neurons, indicating that MeCP2 directly regulates GRIA1 and GRID1  and several glutamatergic system genes including GRIA1, GRIN1, GRIN2, GRIN3A, and GRIN3B were associated with cognitive ability. Of all 174 SNVs in our genetic association study, those of GRIN2A and GRIN2D were excluded because of their low minor allele frequency (< 5%). This finding suggests that the major alleles for the GRIN2A and GRIN2D SNVs might be conserved in the Han Chinese population. Furthermore, the GRIN2C rs3744215 SNV was significantly associated with abstract reasoning. In addition to the cerebellum, GluN2C expression was also detected in several first- and higher-order thalamic nuclei, vestibular nuclei, and parvalbumin-positive interneurons [The students in our study were physically healthy and from homogeneous socioeconomic areas; therefore, our results may not apply more broadly to a heterogeneous population. Additionally, although our effective sample size was small, we detected the associations between SNVs in glutamatergic system genes and the cognitive ability of Taiwanese senior high school students. Specifically, variants of rneurons . Lesionsrneurons . Moreoverneurons . These fGRIN3B variants were previously associated significantly with cognitive function [GRIN3B, an unconventional member of the NMDAR family, complexes with the GluN1 and GluN2 subunits and modulates Ca2+ permeability and membrane trafficking [GRIN3B variations affect the function of glutamatergic receptors and the cognitive ability of students is warranted.Consistent with our findings, several function . GRIN3B,fficking , 59. Howfficking . This mehttps://www.proteinatlas.org/ENSG00000070808-CAMK2A/blood). CaMK2A is expressed mostly in the CNS, adrenal gland, stomach, kidneys, liver, and bone marrow (https://www.proteinatlas.org/ENSG00000070808-CAMK2A/tissue). It can also be detected in neutrophils and memory B cells in the circulatory system. Although we do not know the source of plasma CaMK2A or how it is secreted into serum, ELISA is sufficiently sensitive for measuring it in blood samples. Because blood samples are more accessible than neuronal tissue samples, plasma proteins and peptides exported from the brain in blood samples would be ideal for practical exploration of the biomarkers that affect cognitive ability in young people.Using ELISA, we discovered that students who performed well on the MAT had higher plasma CaMK2A, whereas students who scored poorly on the MAT had lower plasma CaMK2A. CaMK2A concentrations quantified through mass spectrometry and ELISA are compatible because both are in picogram per liter ranges , GluA1, and pGluA1 Ser831) were increased was elevated in the stably transfected HEK293 cells expressing CaMK2A, suggesting that CaMK2A could initiate an autophosphorylation cascade, thereby activating the downstream target genes , numerical ability, mechanical reasoning, spatial relations, and abstract reasoning, whereas female students performed better in verbal comprehension and grammar and language usage Table . We cannWe found that plasma CaMK2A levels were correlated with the cognitive ability of students Table . To furt


Caenorhabditis elegans, we determined the genomic positions of DNA-binding motifs for 201 different transcription factors. Surprisingly, we found that almost all examined transcription factors have binding motifs that reside within transposable elements, and all types of transposable elements have at least 1 instance of a transcription factor motif, demonstrating that transposable elements provide previously unappreciated numbers of transcription factor-binding motifs to the C. elegans genome. After determining the occurrence of transcription factor motifs in transposable elements relative to the rest of the genome, we identified DNA-binding motifs for 45 different transcription factors that are greater than 20-fold enriched within transposable elements compared to what would be expected by chance. Consistent with potential functional roles for these transposable element-enriched transcription factor-binding sequences, we determined that all transcription factor motif types found in transposable elements have instances of residing within accessible chromatin sites associated with transcription factor binding. The overwhelming majority of transcription factor-binding motifs located within transposable elements associate with their cognate transcription factors, suggesting extensive binding of transcription factors to sequences within transposable elements. In addition, transposable elements with accessible or transcription factor-bound motifs reside in the putative promoter regions of approximately 12% of all protein-coding genes, providing widespread possibilities for influencing gene expression. This work represents the first comprehensive analysis of transposable element–transcription factor interactions in C. elegans and demonstrates that transposable element-provided transcription factor-binding sites are prevalent in this important model organism.Transposable elements are powerful agents of evolution that can diversify transcriptional programs by distributing transcription factor DNA-binding sites throughout genomes. To investigate the extent that transposable elements provide transcription factor-binding motifs in  While icis . TE-deriectively . Highligcis-acting elements throughout host genomes. Intact functional elements can be inserted into new regions during a single transposition event, circumventing an extended process involving multiple single-nucleotide changes   terms. Finally, orthologous genes shared between C. elegans and Caenorhabditisbriggsae contain similar TF-motif-containing TEs, some of which may represent the conservation of ancient TE insertions. As the first comprehensive analysis of TE-TF interactions in C. elegans, our studies reveal that TEs provide numerous TF-binding sites with widespread potential for influencing gene expression in this important model organism.Previously, we showed that Helitron TEs in the model organism Caenorhabditiselegans TEs were obtained from the RepeatMasker track (Caenorhabditisbriggsae TEs were obtained from WormBase (version WS280) (C. elegans protein-coding genes and one-to-one orthologs shared between C. elegans and C. briggsae were obtained from ParaSite (version WBPS15) (C. elegans operons were obtained from WormBase (version WS280). Coordinates for coding exons, 5’UTRs, 3’UTRs, and introns were acquired from the UCSC Genome Browser using WormBase annotations (version WS245). Promoter regions are defined as the space within 2.5 kb upstream of protein-coding genes or operons. The overlap of TEs with genomic features was determined using the intersect function of BEDTools v2.29.2 (er track  availabl WBPS15) , while C v2.29.2 .Caenorhabditiselegans TF motifs with direct experimental evidence for their preferred binding sequences were obtained from the Cis-BP database v2.00  and C. briggsae (cb4) genomes with a statistical cutoff of P < 1e−04 using the following command: fimo –max-stored-scores 100000000 –max-strand. To account for biases in the genomic distribution of individual bases, a zero-order Markov background model was generated using the fasta-get-markov utility included with the MEME suite and used during motif scanning. To determine whether scanned TF motifs reside within TE sequences, the intersect function of BEDTools (BEDTools  was thenq < 0.05) and bedGraphs of SPMR-normalized signal were generated individually for each available developmental stage  using combined biological replicates of experimental data with MACS2 v2.2.7.1  and bedGraphs of SPMR-normalized signal were generated individually for each available developmental stage using combined biological replicates of experimental and control data with MACS2  is covered by TEs and their remnants  of all protein-coding genes, raising the possibility that gene expression is affected by TE-provided sequences in a widespread manner.Regions derived from TEs make up a substantial portion of protein-coding gene promoters in humans   that influence the expression of adjacent genes during heat shock in database . To idenal test) . This yial test) . For manal test) . For exaequences . Many hiequences , suggestC. elegans, and TEs contain substantial portions of genomic TF motifs. Furthermore, TE-enriched TF motifs reside in the putative promoter regions of numerous genes, where they potentially influence their expression.To determine the extent that TE-enriched TF motifs reside within promoter regions, we compared the positions of these motifs to the promoter regions of protein-coding genes. For this and subsequent analyses, promoters are defined as regions within 2.5 kb upstream of genes and include overlapping genomic features. Through this analysis, we found that a total of 2,443 or approximately 12% of all protein-coding genes have TE-enriched TF motifs in their promoters . These fC. elegans are found within open chromatin sites, we compared the locations of TF motifs to previously published ATAC-seq data  (Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) allows for directly determining genomic regions bound by TFs of interest (P motifs . In addict test) . To focuct test) . To prevct test) . Next, wct test) . Finallyct test) . From thdaf-18 has 2 CELE1-provided LSL-1 motifs  for 5 GO terms  after filtering for biological processes and redundancy using REViGO  for Cis-BP TF-binding motifs . From this, we found motifs for 4 individual TFs  that are shared between orthologs in C. elegans and C. briggsae in significant numbers  reside within Helitron TEs  have at least 20% of their genome-wide binding sites in TE-derived regions, demonstrating that substantial numbers of TE-derived DNA-binding sites for multiple TFs are found within C. elegans. In addition, using regions of accessible chromatin as a proxy for TF binding, we identified an additional 13 TFs with greater than 10% of their accessible binding motifs residing within TE-derived regions. However, it remains possible that these open chromatin sites are due to the binding of unrelated factors or other mechanisms that affect chromatin accessibility. In most cases, TF-binding sites are not restricted to a single TE family but provided by multiple families, revealing that distinct TE families can simultaneously influence the genome-wide binding patterns of individual TFs. The TE-derived sites identified here are probably an underrepresentation of the total number present, as not all developmental stages were included in this analysis, and TE-TF interactions that occur in a limited number of cells or are transient in nature and may not be detectable in the data analyzed here. Whether these TE-provided TF-binding sites influence gene expression or are important for biological functions remains to be determined. This study provides the raw material for future work to determine the functional consequences of individual TE-derived binding sites for specific TFs of interest.Since the groundbreaking work demonstrating that ∼30% of genome-wide human p53 binding sites reside within endogenous retroviruses (ERVs) and influence gene expression (C. elegans and C. briggsae, we found significant numbers of orthologous promoter pairs that contain TE-derived motifs for LSY-2, LSL-1, EFL-1, and LIN-15B. The presence of TE-derived TF-binding motifs in orthologous promoters suggests these genes have a propensity to acquire these motifs or possibly represent conservation. Most of these orthologous regions are derived from different TE families in each species and are therefore likely independent TE insertions rather than conservation, and may reflect “hotspots” where insertions are preferred or well tolerated. Whether these TE-derived motifs were already present within TEs during insertion or subsequently acquired through evolutionary forces is unknown. For orthologous TE-derived TF-binding motifs obtained from similar TE families, we found that the overwhelming majority are part of the Tc1/mariner superfamily, a diverse range of TEs found in a wide variety of species including animals, plants, and bacteria (C. elegans (C. briggsae, which provide 17,643 individual elements that comprise roughly 4% of the genome (C. elegans and C. briggsae, it is unclear whether these orthologous promoter TEs with similar TF-binding motifs represent ancient insertions that have been conserved or independent insertions that occurred at different times.After comparing the genomes of C. elegans and provides a foundation for further investigation into the contributions of TEs to gene expression programs.While largely ignored in many genome-wide studies due to their repetitive nature, regions derived from TEs are major sources of TF-binding sites. Indeed, our prior work determined that roughly half of all protein-coding genes under the control of HSF-1 during heat stress are due to the presence of TE-derived binding sites (All data used in this study have been previously published and are publicly available. Accession numbers for all ATAC-seq data (jkac009_Table_S1Click here for additional data file.jkac009_Table_S2Click here for additional data file.jkac009_Table_S3Click here for additional data file.jkac009_Table_S4Click here for additional data file.jkac009_Table_S5Click here for additional data file.jkac009_Table_S6Click here for additional data file.jkac009_Table_S7Click here for additional data file.jkac009_Table_S8Click here for additional data file.jkac009_Table_S9Click here for additional data file.jkac009_Table_S10Click here for additional data file.jkac009_Table_S11Click here for additional data file.jkac009_Table_S12Click here for additional data file.jkac009_Table_S13Click here for additional data file.jkac009_Supplemental_Tables_LegendsClick here for additional data file.


Background: The therapeutically important DNA repair gene O6-methylguanine DNA methyltransferase (MGMT) is silenced by promoter methylation in human brain cancers. The co-players/regulators associated with this process and the subsequent progression of MGMT gene transcription beyond the non-coding exon 1 are unknown. As a follow-up to our recent finding of a predicted second promoter mapped proximal to the exon 2 , we addressed its significance in MGMT transcription. Methods: RT-PCR, RT q-PCR, and nuclear run-on transcription assays were performed to compare and contrast the transcription rates of exon 1 and exon 2 of the MGMT gene in glioblastoma cells. Results: Bioinformatic characterization of the predicted MGMT exon 2 promoter showed several consensus TATA box and INR motifs and the absence of CpG islands in contrast to the established TATA-less, CpG-rich, and GAF-bindable exon 1 promoter. RT-PCR showed very weak MGMT-E1 expression in MGMT-proficient SF188 and T98G GBM cells, compared to active transcription of MGMT-E2. In the MGMT-deficient SNB-19 cells, the expression of both exons remained weak. The RT q-PCR revealed that MGMT-E2 and MGMT-E5 expression was about 80- to 175-fold higher than that of E1 in SF188 and T98G cells. Nuclear run-on transcription assays using bromo-uridine immunocapture followed by RT q-PCR confirmed the exceptionally lower and higher transcription rates for MGMT-E1 and MGMT-E2, respectively. Conclusions: The results provide the first evidence for transcriptional pausing at the promoter 1- and non-coding exon 1 junction of the human MGMT gene and its activation/elongation through the protein-coding exons 2 through 5, possibly mediated by a second promoter. The findings offer novel insight into the regulation of MGMT transcription in glioma and other cancer types. MGMT weight ~2 kDa, antg agents ,5; this echanism .MGMT gene, located on chromosome 10q26, occupies a region of 303,780 nucleotides and contains 5 exons and 4 introns [MGMT expression is epigenetically silenced through promoter methylation in a large subset of brain tumors [MGMT has emerged as one of the best-studied candidates for its susceptibility to cytosine-phosphate-guanine (CpG) island (CGI) for DNA methylation in glioma and other cancers [MGMT protein expression in gliomas and the methylation level of the promoter region, which is usually about 30 to 60% [MGMT promoter methylation levels are 20–30% for grade I gliomas, 60–80% for grade II, 40–50% for grade III, and 20–45% for grade IV tumors [MGMT expression and the extent of its promoter methylation have also been performed [MGMT promoter methylation and the resulting decrease in MGMT expression in brain tumors are associated with a better response to chemotherapy, greater overall survival, and longer time to disease progression [MGMT expression; therefore, these sequences are probed in clinical assays [The  introns . This pr introns ,10. It in tumors , and MGM cancers ,13. Many0 to 60% ,15. To bV tumors . Studieserformed ,18; theserformed ,20. Becagression ,22, currgression ,24. Methl assays .MGMT transcription is controlled by the MGMT-P1 promoter, NCBI-Nucleotide ID: X61657.1, mapped at the untranslated MGMT exon 1 [MGMT alternative promoters that overlap the MGMT-E1 promoter and motifs for p53, NRF1/2, CTCF, and the Myc-MAD-MAX genetic switch in the regulatory region [MGMT-E2 promoter henceforth. This sequence is located at the 5′-side of MGMT-E2 approximately 70 kilobases downstream of the MGMT-E1 promoter [MGMT transcripts in human cells and whether these events are altered in glioma to promote an active transcription or its attenuation. How exactly the promoter methylation intersects with these processes is also unclear. Therefore, extending our work, the present study investigated the role and significance of the second promoter in MGMT transcription in human glioblastoma cells.The initiation of T exon 1 . Recently region . Furthery region ) showed promoter . No infoMGMT promoters [Public databases NCBI-Nucleotide, Ensembl, UCSC, PrESSTo/FANTOM5, TRED, and EPD were searched for genomic criteria and map locations of romoters . The UCSMGMT promoter: Observed/Expected ratio > 0.60, Percent C + Percent G > 55.00, Length > 200 bp.An in silico search was conducted to identify transcription factor DNA binding sites (TFBSs) involved in the initiation of transcription. The following motifs were included in the analysis: TATA-8  and TATA-532, INR (YYANWYY), CCAAT and its inverted sequence TAACC, BRE (SSRCGCC), DPE (RGWCGTG), MYC/MAX/MAD genetic switch, and the GAGA factor (GAF) motifs The bioinformatic search was conducted using JASPAR, TRED, tfbss.org tools, and Bio-Base. The following parameter sets were used to identify CpG islands in the nucleotide sequence of the 2 atmosphere at 37 °C.Three human cell lines were used in this study. A pediatric glioma SF188 (Grade IV) cell line was purchased from the Department of Neurosurgery, Univ. of California, San Francisco. T98G (Grade IV) and SNB19 (highly invasive glioblastoma) cell lines were obtained from The American Type Culture Collection . All cancer cell lines were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum in a humidified 5% COStandard Western blot procedures were followed . BrieflyMGMT-exons 1, 2, and 5. The primer sets for the MGMT-E1 promoter were F: GCGCTCTCTTGCTTTTCTCA, R: GACACTCACCAAGTCGCAAA. The primers for the MGMT-E2 promoter were F: TGGAGCTGTCTGGTTGTGAG, R: TGGAAAACATGCCGTTATCA. The primers for extension of MGMT-exon 5 were F: CCGTGAAGGAATGGCTTCTG, R: TAGCTCCCGCTCCCTTG. Normalization was performed using the hypoxanthine-guanine phosphoribosyl transferase 1 (HPRT1) as the reference gene [MGMT target gene was carried out using HPRT1 as a reference gene [−∆∆CT method, which was used to analyze relative gene expression data of real-time quantitative PCR [RNA was extracted from 70–80% confluent GBM cells using RNAzol reagent . cDNAs were prepared from 0.5 µg of total RNA using specific primer sets for nce gene  using thnce gene  accordintive PCR .MGMT gene and quantitate the relative rates of transcription initiated from the promoters proximal to MGMT exon 1 and MGMT exon 2, respectively. For these experiments, the non-isotopic bromouridine immunocapture nuclear run-on followed by the RT-qPCR method [6) growing in their logarithmic growth phase were isolated by gentle lysis in 10 mM Tris-HCl (pH 8.0) containing 0.5% NP-40, 0.5 mM MgCl2, 1 mM dithiothreitol, 5% glycerol and low-speed centrifugation. Freshly isolated nuclei (5 × 106/assay) were suspended in ice-cold assay buffer (10 mM Tris-HCl (pH 8.0), 2 mM dithiothreitol, 2.5 mM MgCl2, 150 mM KCl, 1 mM each of the nucleotides, ATP, GTP, CTP, 0.5 mM Bromo-UTP and 0.5 mM UTP. The reactions were incubated at 30 °C for 30 min. Following DNase and protease treatments, the total RNA was extracted using RNAzol and the nascent transcripts were purified by binding to a BrdU monoclonal antibody  and protein-G Dynabeads. The RNA eluted therefrom was converted to cDNA using random primers and a high-capacity cDNA Reverse Transcription Kit  [MGMT promoters (1 and 2) and the corresponding exons as well. The sequences of the primer set for MGMT-E1 promoter, F: CGGGCCATTTGGCAAACTAA, R: CCCTTCGGCCGGTACAA. The primer sequences for MGMT-E1, F: CCCTAGAACGCTTTGCG, R: ACCCAGACACTCACCAA. MGMT-E2 promoter set, F: AAACGAGAAGAATCGGGATACAG, R: CCATTACCTAGAGCAGCCAAC. MGMT-E2 set, F: CAGCCTCTTACCTATACACTTTGTC, R: GGTGCGTTTCATTTCACAATCC. Normalization was performed using HPRT1 as a reference gene [MGMT target sequence were carried out using HPRT1 as a reference gene according to Schmittgen and Livak’s 2−∆∆CT method [Nuclear run-on transcription assays  were perR method  was follCA, USA) . The cDNnce gene . The HPRT method .p < 0.05.The statistical analyses were performed using Excel software. T-tests were used for the analysis of two independent samples, whereas one-way ANOVA was used to analyze the differences among group means of more than two samples. All statistical computations were calculated using GraphPad Prism 7. A significant difference was assessed at MGMT gene locus occupies a region of 303,780 bps in the forward (plus) strand of the long arm of chromosome 10 at 10q26.3 and genomic coordinates (GRCh38/hg38): chr10: 129,467,184–129,770,983. The MGMT-mRNA (NM_002412) space of 1372 bp is comprised of five exons and one MGMT promoter X61657.1, whereas other databases showed five more overlapping promoter sequences that were combined by us to form a 2019 bp sequence called the MGMT-E1 promoter, mapped at 5′ of the untranslated MGMT-E1 [MGMT-E1 promoter revealed the presence of a genetic switch MYC/MAX/MAD, and transcriptional pausing factor GAF motifs. This study analyzed the molecular features of the unexplored but predicted MGMT-E2 promoter (TRED-5071) located at 5′ of MGMT-E2 , a website listing experimentally validated enhancers. Enhancers are cis-acting regulatory sequences that function independently of orientation and present at various distances from their target promoters and act to increase gene transcription [MGMT introns 2 and 3 and an additional four enhancers mapped in the genomic space between MGMT and MKI67 loci  motifs spread all over the 1 kb sequence and at the upstream and downstream of the transcription start site (TSS)  B. An ATFignature . The seqMGMT gene is rather unusual, with DNA methylation elements clustered in the promoter 1 and a non-coding exon controlling the expression of a transcript, with its first codon 67 kb away in exon 2. The molecular events regulating the initiation and elongation of the MGMT transcript are not known. In the context of our findings of a promoter-like sequence 5′ of exon 2, we performed a series of experiments to investigate the relative expression of exon 1 and exon 2 in three glioblastoma cell lines. Two MGMT-proficient and TMZ resistant (SF188 and T98G) and an MGMT-deficient/TMZ sensitive SNB19 cell line were selected for these studies. SNB19 cells have no detectable MGMT expression due to promoter methylation [The human hylation ,34. WestMGMT for the first time using both conventional and real-time PCR procedures. While a whole-gene expression analysis provides overall information on transcription, measuring the activity of each exon can shed light on the nature of transcriptional initiation, elongation, and pause events [MGMT’s exon 1 and exon 2 in the three GBM cell lines; the DNA products obtained after routine RT-PCR and their relative expression are shown in MGMT-E2 compared to the feeble bands for MGMT-E1  value measurements for the transcription of three MGMT exons, MGMT-E1, MGMT-E2, and MGMT-E5. The CT values are inversely proportional to the amount of target nucleic acid in the PCR reaction sample, i.e., the lower the CT level indicates, the greater the target DNA amount in the PCR reaction sample. The calculated 2−∆∆CT values [MGMT exons 1 and 2 relative to the expression of the calibrator reference gene, HPRT1. The data in MGMT-E1 and MGMT-E2 promoters that reflect their original content in the whole transcript population in the three GBM cell lines. The results unambiguously reveal the poor and lower expression of MGMT-E1 compared to the strong and robust expression of MGMT-E2 in SF188 and T98G cells. The data also confirmed an exceptionally weak expression of the MGMT-E1 and MGMT-E2 in SNB19 cells, explaining the observed lack of MGMT mRNA and protein in these cells [MGMT E-1 compared with that of E2 and E5 in all three GBM cells remained significant at p < 0.05. Initiation of transcripts is always considered a rate-limiting step, and the evidence provided was strongly suggestive of transcriptional pausing at MGMT’s exon 1 and a rapid elongation along with exons 2 through 5 to generate a complete unprocessed transcript.Further confirmation of the critical role played by the unexplored T values  were usese cells ,34. The MGMT’s exon 1 and exon 2 was performed by incubating freshly isolated nuclei from SF-188 cells in the presence of ribonucleotides including Bromo-UTP. The nascent transcripts generated in these reactions were selectively purified on anti-BrdU affinity matrices, reverse transcribed to their cDNA, and the sequences corresponding to MGMT’s E-1 and E-2 were amplified by quantitative RTq-PCR. The primers for real-time PCR were carefully designed and included parts of the promoters and exons [MGMT exon 1 and exon 2 amplicons are shown in MGMT-UE1 (P1), MGMT-DE1 (E1), MGMT-UE2 (P2) and MGMT-DE2 (E2), respectively.The in vitro run-on transcription in nuclei is an established procedure to assess the direct activation of transcription. In this method, cells are lysed, and nuclei are prepared, halting the transcription by RNA polymerase. The transcripts already initiated in the nuclei are elongated in vitro up to 500 nucleotides, and the amount of labeled, nascent RNA at a given region corresponds to the abundance of RNA polymerase at the target site and provides accurate data on the rates of transcription ,36. Run-nd exons  to ampli−∆∆CT [The P1, E1, P2, and E2 transcripts in NRO bromouridine-labeled nascent RNA derived from nuclei of SF188 cells were measured by RT qPCR. The NRO itself and subsequent real-time PCR reactions were repeated twice, and similar results were obtained. We used the HPRT1 gene as the control and relative quantitation method, 2−∆∆CT  as descrMGMT remains a critical DNA repair gene at the crossroads of carcinogenesis, malignant progression, and anticancer therapies, particularly for brain cancers. We investigated the molecular aspects of MGMT transcription in human GBM cells and provided the first evidence for a tight transcriptional initiation, pausing, and a rapid elongation. An earlier bioinformatic and experimental work from our laboratory [MGMT expression by CTCF, laid the foundation for the current study. Much information has established the core promoter extending into a non-coding exon of MGMT first described by Harris et al. [MGMT promoter, namely, the estrogen receptor element (ERE), antioxidant response element (ARE) which binds the transcription factors, NRF1 and NRF2, sites for the binding of GAGA pausing factor (GAF), a MYC/MAX/MAD genetic switch, CTCF and p53-binding consensus motifs in intron 1 [MGMT gene has been considered a non-inducible constitutionally active gene [MGMT expression. However, the mechanics of how this tight initiation is integrated with a productive elongation of MGMT mRNA and which genetic elements determine the elongation efficiency are yet unclear.Human boratory , demonsts et al.  to be ths et al. . Researcintron 1 ,38. Thouive gene , the aboMGMT’s exon 2 [MGMT expression. The in silico analysis showed that the MGMT-E2 promoter is characterized by the absence of CGI, motifs for GAF factor, and MYC/MAX/MAD element, which are all associated with silencing of gene expression and transcriptional pausing [MGMT-E2 promoter showed abundant binding sites for INR (initiator element), ATF1 (activating transcription factor 1), BRE (the TFIIB recognition element), and a large number of TATA box motifs  do provide a good explanation of why it is a preferred location for transcriptional pausing. There are three copies of GAGA-associated factor (GAF) motifs (GAGAG and GAGAGA) in the MGMT promoter, of which two are located upstream and one just downstream of the TSS [MGMT promoter also hosts a motif for a MYC/MAX/MAD repression network [MGMT gene is well established [MGMT promoter 2 proximal to exon 2 does not have negative regulatory elements and, therefore, is well-positioned to support continued mRNA elongation.The regulatory elements and motifs present in  the TSS . The pri the TSS ,46, and  the TSS . The MGM network  where MA network . Furtherablished ,13. In cMGMT promoter 1-exon 1 are required to pinpoint the exact location of RNA polymerase pausing and its subsequent release. We did not perform experiments to evaluate the direct role of the E2 promoter; however, the data reported indeed suggest an important function for this sequence. Further studies are also required to define the nature, role, and fine interplay of transcription regulatory proteins at the second promoter using ChIP and Cap Analysis of Gene Expression (CAGE) [MGMT-deficient did express small amounts of exon 1 and exon 2, perhaps indicating the instability of MGMT mRNA in these cells   analysesse cells . FinallyMKI loci  adds to