Cell fate transitions observed in embryonic development involve changes in three-dimensional genomic organization that provide proper lineage specification. Whether similar events occur within tumor cells and contribute to cancer evolution remains largely unexplored. We modeled this process in the pediatric cancer Ewing sarcoma and investigated high-resolution looping and large-scale nuclear conformation changes associated with the oncogenic fusion protein EWS-FLI1. We show that chromatin interactions in tumor cells are dominated by highly connected looping hubs centered on EWS-FLI1 binding sites, which directly control the activity of linked enhancers and promoters to establish oncogenic expression programs. Conversely, EWS-FLI1 depletion led to the disassembly of these looping networks and a widespread nuclear reorganization through the establishment of new looping patterns and large-scale compartment configuration matching those observed in mesenchymal stem cells, a candidate Ewing sarcoma progenitor. Our data demonstrate that major architectural features of nuclear organization in cancer cells can depend on single oncogenes and are readily reversed to reestablish latent differentiation programs. Remodeling of nuclear organization by an oncogenic transcription factor determines tumor cell identity. Perturbations in chromatin regulation are widely recognized to play a critical role in cancer development   with the ability to modify chromatin activation states can drive conformational changes, either at the level of compartmentalization or by reshaping TADs themselves along with intra-TAD interactions , the second most common pediatric bone malignancy, features a low mutational burden and the presence of a fusion between We now show that, in keeping with its powerful role in regulating chromatin states, EWS-FLI1 is also a major determinant of 3D chromatin conformation in EwS. EWS-FLI1 converts repetitive GGAA genomic regions into highly connected chromatin interaction hubs (hereafter EWS-FLI1 enhancer hubs) covering/bridging megabase-sized genomic regions and enabling oncogenic programs. EWS-FLI1 depletion leads to the loss of EWS-FLI1–bound enhancer hubs and the establishment of a different gene-regulatory architecture that mirrors the topological and transcriptional states of primary mesenchymal stem cells (MSCs). In aggregate, our observations support the notion that tumor cells may display a dynamic and reversible nuclear organization and emphasize the critical role of TFs in modeling chromatin topology and activity.The ability of EWS-FLI1 to bind GGAA microsatellite repeats and convert them into de novo active enhancers has been demonstrated in EwS cell lines and primary tumor models . One HiChIP map was generated for SKNMC cells. For A673 cells, HiChIP interactions were called from combined loops from both replicates, and loops identified in both replicates and showing a signal greater than five counts in one of the two replicates were selected for further analysis. Our analysis yielded a total of 38,162 and 31,537 high-confidence, H3K27ac-anchored chromatin interactions in A673 and SKNMC cells, respectively, of which 14,451 and 14,619 were associated with at least one EWS-FLI1 binding site. Both EWS-FLI1 and non–EWS-FLI1 loops were widely distributed across the genome  and profiled differential looping patterns by H3K27ac HiChIP at 96 hours after infection (fig. S2A). Two HiChIP replicates were performed for shGFP and shFLI1 A673 cells, which showed high correlation scores (fig. S2B), whereas one replicate was generated for SKNMC cells. In A673 tumor cells, EWS-FLI1 knockdown resulted in a global reorganization of the genomic interactions, including increases  and decreases  in H3K27ac-mediated contact frequency . SimilarLoss of EWS-FLI1–mediated loops had strong effects on the chromatin states of target promoters and enhancers  at most sites (fig. S4A), indicating that there are no widespread changes in TAD configuration. This is consistent with previous studies showing that TADs tend to remain stable despite major variations in transcriptional programs  signals . Consistent with the observed increases in B-B connections following EWS-FLI1 removal, we identified a moderate decrease in expression for genes located within B compartments (fig. S5L). Together, these observations show that EWS-FLI1 alters compartmental segregation, strength, and connectivity beyond its direct binding sites, which shape gene expression programs in EwS cells.Given these large-scale changes in B-B compartments, we surveyed for transcriptional changes associated to the high-order chromatin alterations induced by EWS-FLI1, independently to its direct binding sites. To this end, we set out to calculate the average expression of genes located within B compartments, showing increased interactions in the knockdown condition and that are not direct EWS-FLI1 targets  and used to generate a heatmap comparing their relative strength between shGFP and shFLI1 A673 cells, as well as wild-type MSCs. A robust subset of the interactions enriched in shFLI1 A673 cells were also present in wild-type MSCs . Primary pediatric MSCs were cultured in Iscove’s modified Dulbecco’s medium containing 10% FBS and platelet-derived growth factor BB , as described previously (The EwS cell lines A673 and SKNMC (American Type Culture Collection) were grown in RPMI containing 10% fetal bovine serum (FBS) at 37°C with 5% COLentivirus-mediated knockdown experiments, RNA isolation, and reverse transcription polymerase chain reaction (PCR) analysis were carried out as previously described , anti–glyceraldehyde-3-phosphate dehydrogenase (MAB374 Millipore), and horseradish peroxidase–conjugated sheep anti-mouse (Cytiva) or goat anti-rabbit secondary antibodies (Dako). Whole-cell protein lysates were prepared by standard methods and quantified using the Bradford reagent (Bio-Rad). Protein electrophoresis was performed in precast Bolt 4 to 12% bis-tris gels in MES running buffer and Mini Gel Tank according to the manufacturer’s protocol (Invitrogen). Blotting was performed by the standard procedure, membranes were developed using the enhanced chemiluminescence substrates SuperSignal West Pico PLUS (Thermo Fisher Scientific) or WesternBright Sirius (Advansta), and proteins were visualized with a FUSION FX camera (Vilbert-Lourmat).et al.  read pairs.et al. . EWS-FLI1 and H3K27ac ChIP-seq results for A673 and SKNMC cells were obtained from the Gene Expression Omnibus (GEO) (GSE61953) and were aligned against hg19 genome using bwa v.0.7.12 with default settings  regions by applying the following parameters: --max-distance 100000000 --read-length 100. Loops with counts greater than 5 and identified in both replicates were kept for further analysis. To eliminate the H3K27ac difference–induced loop bias when comparing shGFP and shFLI1, we performed the hichipper analysis using the union H3K27ac peaks from shGFP and shFLI1. Loops were normalized by using the method described in diffloop   promoters (±1 kb of TSS) bound by EWS-FLI1, (ii) promoters connected to EWS-FLI1 binding sites by loops (with or without bridge anchors), and (iii) promoters connected to bridge anchors that have decreased looping upon EWS-FLI1 loss (twofold decrease in shFLI1). Bridge anchors are defined as EWS-FLI1–connected enhancers.When comparing the loops between shGFP- and shFLI1-infected samples, we annotated the union set of loops into six categories based on the overlapping between loop anchors and the binding of EWS-FLI1 in shGFP sample. EWS-FLI1 connected genes that were down-regulated after EWS-FLI1 depletion with at least 1.5-fold decreases in A673 or SKNMC cells were selected as EWS-FLI1 target genes.Novel H3K27ac loops were defined as H3K27ac loops with more than fourfold increase in shFLI1 and less than two normalized loop counts in shGFP. Genes targeted by novel HiChIP loops that increased more than 1.5-fold expression in shFLI1 were defined as novel loop-targeted genes. The HiChIP networks for A673 and SKNMC were constructed using the R package “Intergraph” v.2.0-2. The vertexes were loop anchors, and the edges were interactions between anchors. A673 and SKNMC HiChIP networks were visualized through R package ggnet v.1.0.Paired-end HiC and MicroC reads were aligned against the hg19 genome using HiC-Pro . Briefly, we calculated the boundary strength for each chromosome from the O/E matrix by using matrix2insulation.pl with default settings. We next used insulation2tads.pl to define TAD boundaries from the information of boundary strength. To compare the HiC and MicroC interactions corresponding to HiChIP loops (EWS-FLI1 loops or loops associated with novel H3K27ac peaks), we used BEDTools pairtopair (v2.17.0) with the default settings. To evaluate the changes in compartment strength between shGFP and shFLI1 samples, we generated saddle plots using the compute-saddle utility of cooltools package .TADs were defined for each chromosome on the basis of 40-kb bin matrix using the analysis method described by ENCODE (www.gsea-msigdb.org/gsea/index.jsp).A673 and SKNMC RNA sequencing (RNA-seq) samples for EWS-FLI1 knockdown experiments were downloaded from the GEO series: GSE94278. The MSC RNA-seq datasets were obtained from GSE94278. Reads were aligned against hg19 using STAR v.2.4.0h  for further analysis. Seurat (v.3.2.2) (z scores for each gene) was used for dimensional reduction. The top 25 principal components were used for visualization with Uniform Manifold Approximation and Projection (UMAP).Treehouse public data were downloaded from HOMER v.4.7 (


Bones serve mechanical and defensive functions, as well as regulating the balance of calcium ions and housing bone marrow.. The qualities of bones do not remain constant. Instead, they fluctuate throughout life, with functions increasing in some situations while deteriorating in others. The synchronization of osteoblast-mediated bone formation and osteoclast-mediated bone resorption is critical for maintaining bone mass and microstructure integrity in a steady state. This equilibrium, however, can be disrupted by a variety of bone pathologies. Excessive osteoclast differentiation can result in osteoporosis, Paget’s disease, osteolytic bone metastases, and rheumatoid arthritis, all of which can adversely affect people’s health. Osteoclast differentiation is regulated by transcription factors NFATc1, MITF, C/EBPα, PU.1, NF-κB, and c-Fos. The transcriptional activity of osteoclasts is largely influenced by developmental and environmental signals with the involvement of co-factors, RNAs, epigenetics, systemic factors, and the microenvironment. In this paper, we review these themes in regard to transcriptional regulation in osteoclastogenesis. Bone is not just a solid physical support structure, but a dynamic, constantly remodeling tissue, which serves as a keeper for marrow cells, a location of blood cell synthesis, and an organ for the regulation of calcium ion homeostasis and endocrine ,2,3. BonOsteoclasts, as important cells involved in regulating bone metabolism, are mainly responsible for coordinating bone resorption with bone formation to regulate bone remodeling . RegulatIn the 1970s, studies provided convincing evidence that osteoclasts arise from hematopoietic cells ,25. In 1EMP-derived monocytes/macrophages mainly produce embryonic osteoclasts, whose role is gradually replaced later. Early EMPs start to generate on embryonic day (E) 7-7.5, which can develop into colony-stimulating factor 1 receptor (CSF1R) + yolk sac macrophages at E8.5, and give birth to a population of tissue-resident macrophages, such as brain microglia ,29. ArouAs the foundation of the adult hematopoietic system, HSCs play a key role in the long-term maintenance and generation of all mature blood cell lineages throughout the lifespan of an organism. The hemogenic endothelium, which goes through endothelial-to-hematopoietic transition, is the same source of HSCs as it is for EMPs . DespiteBefore generating multinucleated osteoclasts with the potential to absorb bone, HSC- or EMP-derived precursor cells must undergo migration, fusion, and differentiation. The presence of monocytes in the bone marrow cavity is close to the collagen fibers and vascular network . SubsequThe differentiation of osteoclasts is primarily dependent on a tumor necrosis factor (TNF) family, RANK, RANKL, osteoprotegerin (OPG), and M-CSF ,54. HemaThe mechanisms of bone development, repair, and regeneration depend on the interaction and communication between the osteoclast and other surrounding cells, while osteoclastogenesis is also regulated to a large extent by other cells. To further explore the regulatory mechanisms of osteoclastogenesis, we investigated the major cells involved in osteoclastogenesis, including osteoblasts, osteocytes, adipocytes, and immune cells.Through direct cell-to-cell contact or secretion of proteins and RNA, osteoblasts and osteoclasts communicate to manage cellular activity, survival, and differentiation. Direct interactions between osteoblasts and osteoclasts regulate the differentiation and survival of osteoblasts or osteoclasts mainly through bidirectional regulation of the EFNB2 (Ephrin B2)-EPHB4, FAS Ligand (FASL)-FAS, or Semaphorin 3A (SEMA3A)-NRP1 signaling pathways  Figure .Activation of the osteoblast surface molecule EPHB4 is initiated by binding to the cell surface molecule Ephrin B2 of osteoblasts and inhibits osteoclast differentiation by blocking osteoclast-generated c-Fos/NFATC1 . In contThe cytokines M-CSF and RANKL play a crucial role in the development of osteoclasts and their expression is common among a variety of cell types, including osteoblast lineage cells. They regulate significant aspects of osteoclast formation and operation. M-CSF, a hematopoietic growth factor, is secreted by osteoblasts and bone marrow stromal cells and promotes osteoclastogenesis by binding to its cognate receptor C-FMS on the surface of osteoclasts and monocytes/macrophages . RANKL iRunx2 . IP3 then induces calcium oscillations, which are essential for NFATc1 activation . In the In conclusion, NFATc1 appears to be a prime target for anti-osteoclast therapy because it is implicated in every stage of osteoclast production and activation. Thus, to better understand the molecular mechanisms behind NFATc1 regulation in osteoclasts, novel therapeutic strategies for bone illnesses defined by an excess of osteoclast production and activity may be developed.MITF is recognized to have a role in melanocyte development, and mounting research supports its significance in aging and senescence, notably in melanocyte senescence. MITF primarily controls senescence-associated secretory phenotype (SASP) synthesis, oxidative stress, DNA repair, cell cycle, and melanocyte senescence . Like alIn osteoclast precursors and osteoclasts, C/EBPα expression was high, and the combined presence of M-CSF and RANKL could significantly induce high C/EBPα expression . Two impThe Sfpi1 (Spi-1) gene encodes PU box-binding protein (PU.1), which produces a protein with 272 amino acids (predicted MW of 31 kDa) connected to the Spi-B and Spi-C Ets-family factors (both share around 40% amino acid similarity) . The traNuclear factor-κB (NF-κB) is a transcription factor involved in signaling pathways essential for normal cell function and development. Osteoclast differentiation requires NF-κB activation. NF-κB consists of five transcription factors: p50/p105, p52/p100, p65 (relA), c-Rel, and RelB . It has Proteins from the Activator Protein-1 (AP-1) family, especially in the Fos proteins, play a significant role in osteoclast regulation . The exp2+) mobilization, and promoting actin reorganization  in response to RANCL stimulation . Bcl6 inThe C/EBP family member DNA damage inducible transcript 3 (DDIT3) is implicated in apoptosis and differentiation and inhibits osteoclast differentiation via modulating the C/EBPα-CTSK axis . DDIT3 e2+) mobilization through activation of PLCγ2. In the nucleus, NFATc1 cooperates with other transcription factors, such as AP-1, PU.1, and MITF to induce various osteoclast-specific genes. Bcl6 can inhibit NFATc1 expression, and this inhibition is negatively regulated by Blimp1. Each of these transcription factors has the potential to be a key in the regulation of bone homeostasis.Overall, osteoclast transcription factors play a crucial role in osteoclastogenesis. At the same time, osteoclast transcription factors serve as key nodes that constitute the pathways regulating osteoclastogenesis . BindingEpigenetics refers to the study of mechanisms that can affect gene expression in a stable and potentially genetic manner without changing the DNA sequence. It describes three main phenomena: non-coding RNA sequences, methylated DNA modifications, and histone side chain modifications . EpigeneIn both healthy and pathological contexts, non-coding RNAs (ncRNAs) fine-tuning gene expression programs are involved in bone homeostasis. Non-coding RNAs in osteoclast differentiation mainly include micro RNA (miRNA), long-non-coding RNA (lncRNA), and circular RNA (circRNA). Although each class of ncRNAs functions in a unique way, there are emerging connections between their methods for controlling gene expression. On the basis of newly acquired knowledge on the epigenetic regulation of gene expression for skeletal ncRNA regulation, new paths for translating miRNAs, lncRNAs, and cricRNAs into therapeutic targets for skeletal illnesses may be possible.Due to its capacity to regulate gene expression and provide post-transcriptional epigenetic modifications, miRNA has recently attracted attention. The class of non-coding RNAs known as miRNAs makes up only 1-5% of our genome and ranges in length from 18 to 25 nucleotides ,186. It fl/fl mice [The role of miRNAs in osteoclastogenesis includes both inhibition and promotion, involving the expression of multiple pathways and genes . MiRNA-2/fl mice . MiR-31 /fl mice . The mos/fl mice . The exo/fl mice . Several/fl mice ,200. MiRLncRNAs are large non-coding RNAs with transcripts longer than 200 nt. LncRNAs play a crucial role in various activities of life by interacting with proteins, RNA, and DNA to regulate gene expression ,213. TheDuring osteoclast differentiation, LncRNA TUG1 is excessively expressed and controls the protein levels of anti-tartrate phosphatase (TRAP), NFATc1, and osteoclast-associated receptor (OSCAR). Meanwhile, it targets increased protein levels of V-maf muscular neurofibrosarcoma homolog B (Mafb) to promote osteoclast differentiation positively . Lnc-AK0Although research on the regulatory mechanisms of lncRNA in osteoclast differentiation is still lacking, it has been shown that these molecules can serve as markers for diseases related to the bones and as therapeutic tools.CircRNAs are a class of RNA molecules with a stable ring-like structure discovered more than 40 years ago, which are produced by reverse splicing of linear RNAs covalently linked ,231. CirExcept for a few CircRNAs that can regulate osteoclast transcription factors, the rest mostly play a role in regulating osteoclasteogenesis by regulating MiRNAs . CircCHEIn general, CircRNAs are frequently combined with miRNAs to regulate the differentiation of osteoclasts. The combination of circRNAs with miRNAs shows significant potential for treating bone-related diseases.DNA methylation, as an epigenetic event, plays a key role in the pathogenesis, progression, and treatment of human diseases, as well as in the osteoclast differentiation process ,239. In DNA of each cell is wrapped around histone octamers that form what are known as “nucleosomal core particles” . The taiRANKL increases NFATc1 transcriptional activity during subsequent osteoclast differentiation by inducing HAT-mediated NFATc1 acetylation and stabilization . HDAC1 iIt could be a novel strategy to treat bone-related disorders by using epigenetic regulators to modify osteoclast gene expression, therefore regulating osteoclast differentiation, function, and apoptosis. Proteolysis of the histone H3 N-terminal tail (H3NT) via matrix metalloproteinase-9 (MMP-9) is a critical mechanism for activating gene expression during osteoclast differentiation. The activity of MMP-9 on H3NT is tightly controlled by histone modifications, including H3K18 acetylation (H3K18ac) and H3K27 monomethylation (H3K27me1). These modifications are essential for the stable interaction of MMP-9 with nucleosomes during H3NT proteolysis . TreatmeA novel DNA methyltransferase 3a (Dnmt3a)-mediated S-adenosylmethionine (SAM)-mediated DNA methylation regulates osteoclastogenesis through epigenetic suppression of antiosteoclastogenic genes, the defect of which leads to inhibition of osteoclast differentiation . In addiEpigenetic studies have provided new strategies for regulating osteoclastogenesis, which is inextricably linked to transcription factors such as NFATc1. Nevertheless, further comprehensive and systematic research is essential to investigate the epigenetic regulation of osteoclastogenesis more profoundly, and reliable approaches to control transcription factors connected with osteogenesis remain to be deeply studied.Osteoclasts are key cells with a bone resorption function, derived from HSCs and EMPs, and are essential for bone remodeling and bone homeostasis. The process of osteoclastogenesis is tightly regulated by several transcription factors, of which NFATc1 is key. The expression of transcription factors in osteoclasts can be regulated through three major aspects of epigenetics: methylated DNA modifications, histone side chain modifications, and non-coding RNA sequences. Intercellular communication leads to changes in the micro-environment, during which substances such as cytokines are produced that can have a major impact on cell differentiation, function, and survival. Communication between osteoclasts and related cells, mainly osteoblasts, osteocytes, immune cells, and adipocytes, also exerts a regulatory effect on transcription factors of osteoclasts. The mechanisms of action of transcription, as well as the targets and pathways that regulate transcription factor expression, addressed in this review may become important directions for the treatment of bone-related diseases in the future.


Many crucial epigenetic changes occur during early skeletal development and throughout life due to aging, disease and are heavily influenced by an individual’s lifestyle. Epigenetics is the study of heritable changes in gene expression as the result of changes in the environment without any mutation in the underlying DNA sequence. The epigenetic profiles of cells are dynamic and mediated by different mechanisms, including histone modifications, non-coding RNA-associated gene silencing and DNA methylation. Given the underlining role of dysfunctional mesenchymal tissues in common age-related skeletal diseases such as osteoporosis and osteoarthritis, investigations into skeletal stem cells or mesenchymal stem cells (MSC) and their functional deregulation during aging has been of great interest and how this is mediated by an evolving epigenetic landscape. The present review describes the recent findings in epigenetic changes of MSCs that effect growth and cell fate determination in the context of aging, diet, exercise and bone-related diseases. Almost opoiesis ,17,18,19opoiesis ,20. Moreopoiesis  and Gremopoiesis  have beeopoiesis ,24,25,26opoiesis ,28.The skeleton is the major tissue in supporting human structure and movement. It is also considered an endocrine organ in maintaining calcium and glucose homeostasis . Bone heDNA damage, cellular stress responses and alterations in both the genome and epigenome can cause a decline in healthy tissue homeostasis and lead to a host of changes at both the cellular and tissue level. The epigenetic landscape within stem cells is a dynamic process ,34, wherIn postnatal organisms, BMSC retain their ability to regenerate tissues following bone fractures, articular cartilage injuries and other traumatic injuries of connective tissue. Osteoarthritis, osteoporosis and systemic inflammatory conditions such as rheumatoid arthritis are among the most common and disabling musculoskeletal degenerative disorders often associated with aging . Other rEpigenetics refers to heritable alterations in gene expression that cannot be attributed to alterations in the DNA sequence and range from DNA methylation and histone modifications to alterations in the non-coding RNA expression and chromatin structure . These eBrg-1) and Brahma (BRM). These proteins can slide nucleosomes to either promote open or closed chromatin [Nanog expression due to the recruitment of DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1), which methylates DNA and deacetylates histones, respectively, altering the nucleosome architecture [Nanog expression [The relationship between the chromatin structure, nucleosome positioning and BSC aging is a recent phenomenon that is gaining traction, as its importance in transcription, DNA damage and genomic stability is essential. Recent studies have examined genome-wide chromatin accessibility in adipose-derived MSC (AMSC) using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), which found that chromatin is generally more open in AMSC compared to somatic cells , especiahromatin ,57. Any itecture . In agrepression .CBX4 knockout BMSC, and differentiation proceeded normally; however, BMSC underwent senescence, where the RNAseq analysis identified 306 upregulated genes and 332 downregulated genes. Identification of the binding partners by mass spectrometry identified 262 proteins, most of them nucleolar proteins. CBX4 was found to bind to rDNA repeats, and its loss led to reduced heterochromatin formation in the nucleolus, together with downregulation of histone 3 lysine 9 tri-methylation (H3K9me3) and increased ribosome expression.Another chromatin structural protein is CBX4, which is part of the polycomb repressive complex 1 (PRC1) and was found to be downregulated in aged human BMSC and in BMSC from Werner syndrome patients who exhibit premature aging . SurprisGene expression profiling of old BMSC identified another chromatin structural protein, nucleosome assembly protein 1-like 2 (NAP1L2), where increased expression correlated with senescence and impaired osteogenesis . NAP1L2 ZKSCAN3) to be downregulated in aged BMSC, resulting in senescence. ZSCAN3 was found to maintain heterochromatin via its interaction with lamina proteins. Its downregulation results in the detachment of lamin-associated domains with the nuclear lamina, more accessible chromatin and the expression of repetitive sequences [More recent studies identified zinc finger protein with KRAB and SCAN domain 3  family of DNA hydroxymethylases, controls self-renewal . The thry in ESC  whilst ay in ESC . Conversy in ESC , with thone loss , with fup21 expression via the upregulation of DNMT, leading to increased methylation of the p21 promoter and inhibition of cellular senescence in hair follicle mesenchymal stromal cells. Furthermore, the role of DNMTs in cellular senescence and aging has been supported, with further studies implicating decreased methylation in senescent cells [DNMT1 and DNMT3b. Other investigations reported that DNMT inhibition in MSC led to an increase in activity of cyclin-dependant kinase inhibitors, most notably p16INK4A and p21CIP1, and, ultimately, a reduction in EZH2 and BMI1 expression, associated with replicative senescence in MSC [The activity of DNA methyl transferases (DNMT) has been shown to play a role in BMSC aging, highlighting a constantly changing methylome throughout human development. Pluripotency factors such as OCT4, involved in BMSC self-renewal and growth, are impacted by DNMT activity . This stnt cells  via the e in MSC .6-methyladenosine (N6-mA) demethylase, Alkbh1, is reduced in aging stem cells populations [Alkbh1 resulting in enhanced bone loss and increased bone marrow adiposity. A significant reduction in Osteopontin expression, a known regulator of MSC fate determination in aging [Alkbh1 null animals. Conversely, overexpression of Alkbh1 was reported to alleviate age-related bone loss by promoting osteogenesis. Moreover, an increase in N6-mA was observed around the promoter region of this gene, indicating Alkbh1 may regulate BMSC lineage determination through adenosine methylation of the Osteopontin promoter region. As well as acting as a DNA methyl mark, N6-mA also acts as an abundant mRNA modification and has been linked to cancer progression. Conversely to Alkbh1, methyl transferase-like 3 (METTL3) is a major factor in the adenosine methylation of RNA molecules and has been recently implicated in BMSC osteogenesis via post-transcriptional modification of the Runx2 transcript [METTL3 and METTL4. Upon the overexpression of METTL3 in BMSC, it was found that osteogenic differentiation markers were upregulated, and functional differentiation assays showed an increase in ALP activity and mineral formation. The immunoprecipitation analysis showed an enrichment of a variety of miRNAs, with miR-320 being the most strongly N6-mA methylated of the cohort. Further elucidation of miR-320 targets identified Runx2 as being a key target gene, with the overexpression of miR-320 causing a pronounced decrease in Runx2 expression. These findings proposed METTL3 as a pro-osteogenic or anti-osteoporotic factor, with overexpression of this methyltransferase leading to the downregulation of miR-320, a now established silencing element targeting known osteogenic biomarkers such as Runx2.Recent studies have reported that the expression of the DNA Nulations ,63, within aging , was alsanscript . In an aSOST by Osterix and Runx2 [SOST promoter regulated the osteoblast–osteocyte transition [SOST promoter methylation was found to disrupt the transactivation of key transcription factors Osterix, ERa and RUNX2 via the impairment of transcription factor binding [SOST promoter, with a decreased binding ability found in osteoporotic patients due to elevated levels of CpG methylation.Sclerostin (SOST) is a glycoprotein secreted by osteocytes in response to mechanical stress on the bone and has been shown to play a role in bone formation and remodelling, with the loss of SOST resulting in Van Buchem disease, associated with endosteal hyperostosis of the mandible, skull, ribs and clavicles, as well as diaphysis of the long bones ,78. Studnd Runx2 , have beansition , as well binding . The chrRANKL transcript in osteoporotic samples, coupled with a depletion of OPG, leading to a balance favouring bone resorption through osteoclast activity. It was found that 32 CpG sites of RANKL become highly demethylated in the osteoporotic group compared to the control. Accompanying this, it was found that OPG was largely hypomethylated in both groups, but the levels of DNA methylation in the osteoporotic group were significantly greater. These findings suggest that the imbalance of the RANKL-OPG system, caused by DNA methylation, leads to a favouring of osteoclast development and, thus, bone resorption and a bone loss phenotype.When it comes to bone remodelling, the balance between osteoclast and osteoblast activity is critical to maintain healthy bone formation. Receptor Activator of Nuclear Factor-kappa B (RANK) and binding of its ligand (RANKL) are crucial in the development and maturation of osteoclast cells in the bone microenvironment. Conversely to this, osteoprotegrin produced by osteoblasts is able to sequester RANKL by acting as a decoy receptor in order to temper the bone resorption. The role of DNA methylation in this system has been elucidated in a study using osteoporotic patient samples . The stuHistone modification changes are part of the epigenetic drift that occurs in aging stem cells, which is the centrepiece of the information theory of aging . This evWnt12, Wnt6, Wnt10a, Runx2 and Bglap in ovariectomised mice, which mimics aging by altering the differentiation to favour adipogenesis at the expense of osteogenesis [Ezh2 inhibited BMSC osteogenesis in an in vivo ectopic bone formation model [Ezh2 in BMSC leads to increased bone volume and bone length [ZBTB16, MX1 and FHL1 involved in osteogenesis and adipogenesis [P15INK4b, P16INK4a, P21CIP1 and P27 KIP1 [H3K27 trimethylation (H3K27me3) is a repressive mark and essential for embryonic development. As part of the bivalent domain with H3K4me3, it is crucial for the stem cell renewal and proper differentiation . We and ogenesis . The enfon model , wherease length . In conte length . Ezh2 waogenesis , as wellP27 KIP1 .Ezh2 using the Nestin promoter as the driver results in the premature senescence of MSC, where aged MSC express reduced Ezh2 levels compared to their younger counterparts [Ezh2 results in less H3K27me3 on the promoters of p15INK4b, p16Ink4a, p27 and p21, promoting senescence and decreasing self-renewal [p16Ink4a locus, repressing transcription and senescence [Twist-1 decrease and Ezh2 levels on the p16Ink4A locus decrease, leading to increased p16 expression and senescence [Foxo1, inhibiting the antioxidant defence system [Conditional deletion of terparts . It was -renewal . In addinescence . During nescence . In anote system . By blocKDM6A in human BMSC promotes adipogenesis and reduces osteogenesis and has been shown to bind to the transcription start site (TSS) of Runx2 and Bglap [KDM6A in early mesenchyme has shown subtle defects in bone formation in the cranium and long bones (unpublished data), suggesting redundancy with other KDM6 family members. KDM6B has been found to be reduced in BMSC from aged mice, and the knockout of KDM6B leads to severe bone defects [The examination of epigenetic changes in aged human BMSC has shown that regions of the genome that acquire DNA hypermethylation are associated with repressive histone marks H3K27me3, H3K9me3 and Ezh2 enrichment . DNA hypnd Bglap . The con defects . These sWnt promoters and Runx2, with its deletion leading to elevation of the H3K9me3 levels [DLX2 and DLX3 in response to Bmp4/7, and the GO analysis confirmed KDM4B controls the genes involved in skeletal development and osteoblast differentiation. KDM6B controlled the expression of Hox genes, and the GO analysis confirmed its regulation of skeletal development and matrix organisation. A similar study found that the loss of KDM4B accelerated aging and high-fat diet-mediated bone loss due to increased senescence and BMSC exhaustion [H3K9 methylation is also a repressive mark associated with heterochromatin, and increasing levels are associated with MSC aging . An impo3 levels  and supp3 levels . This cohaustion . Profilihaustion , which aHoxa10, Runx2, Osterix and Sox9 promoters, which displayed low levels of H3K4me3 [Hoxa10, Runx2, Osterix and Sox9 [The transcriptional activator H3K4me3 has been shown to be downregulated in osteoporotic murine BMSC . It is r H3K4me3 . The knoand Sox9 .H3K36me3 is important for transcriptional elongation and restoring the proper chromatin structure following gene transcription. The SET domain-containing 2 protein (SETD2) is responsible for H3K36me3, and its conditional deletion promotes adipogenesis and impairs osteogenesis, resulting in bone loss , resemblWnt promoters [Epigenetic rejuvenation of BMSC is becoming a new strategy to alleviate the aging phenotype. Inhibitors to HMTs and HDMs are used in the clinic in the cancer field, displaying safety and efficacy. It has been demonstrated that inhibiting Ezh2 activity using DZnep and SUV39H1 activity with chaetocin can alleviate osteoporosis. DZnep treatment can increase trabecular bone in ovariectomised mice , and charomoters , demonstTert, Oct4 and Sox2 decreases together with the gene expression, resulting in a decrease in stem cell self-renewal [KDM6B promoter, which was verified by the knockdown of HDAC1 and 2. The increase in KDM6B results in a decrease in H3K27me3 along the p16Ink4A locus increasing expression and senescence [Ezh2, BMI1 and SUZ12 and downregulate KDM6B [Ezh2, BMI1 and SUZ12 and increases KDM6B, reducing the H3K27me3 levels on the p16Ink4A promoter, promoting senescence.The aging of BMSC is also associated with changes in histone acetylation —in parti-renewal . Studiesnescence . During te KDM6B . The dowGCN5), p300/CBP-associated factor (PCAF) and removed by HDAC9. H3K9 acetylation is a primary target for GCN5, which has been shown to reduce dramatically in aged BMSC, and in osteoporotic BMSC from ovariectomised mice [BMP4 and Runx2 promoters [ATG7, BECN1, Lc3a and Lc3b in aged BMSC [Oct4, Sox2 and Tert, resulting in decreased expression [H3K9 acetylation has been shown to be drastically downregulated in aged BMSC  and is dsed mice . Furthersed mice . Althougromoters , its rolged BMSC . This shpression  and compHO-1, as SIRT6 is responsible for H3K56 deacetylation on this promoter; an increase in ROS, oxidative stress and impaired osteogenesis and chondrogenesis. Senescence is accelerated due to the increased expression of p16 and p21 [Another important acetylation mark is H3K56 acetylation, which increases in aged BMSC. One of the key enzymes essential for depositing this mark is the NAD+-dependent Sirt6 enzyme . The del and p21 .KAT7 reduced senescence and restored the ability of the passaged cells to undergo osteogenic differentiation. Heterochromatin formation was also restored, resembling young BMSC. One-third of the deregulated genes were restored following KAT7 knockdown, including the genes involved in chromosome organisation and DNA packaging. In contrast, the overexpression of KAT7 induced heterochromatin loss, increased p16 and p21 expression and was found to acetylate H3K14 of the p15Ink4b promoter. Moreover, KAT7 and p15Ink4b were found to be increased in aged human BMSC (79–81 years old) versus young BMSC (16–28 years old). Interestingly, lentiviral-mediated deletion of KAT7 in aged mice targeting the liver increased the overall health and lifespan of these animals. Similar screens have identified the acetyltransferase p300 [SIRT6 leads to an increase in H3K9Ac and H3K56Ac in association with increased rates of aging [Since senescence is a hallmark of aging, and the ablation of senescent cells in a whole animal prolongs the overall lifespan ,112. A rase p300  to proloof aging ,115.Following transcription, the closed chromatin state is restored by various complexes, including the histone demethylase Kdm5b ,117 and Non-coding RNAs, such as miRNAs and long non-coding RNA (lncRNA), are functional RNA molecules that are not translated into proteins but are able to regulate the expression of genes post-transcription . These nSox9 to enhance osteogenic differentiation over chondrogenesis [Various miRNAs are able to exhibit control over MSC differentiation during a number of stages in the process through regulation of the master regulators  ,121,122 SOD2 was found to be dysregulated upon DGCR8 knockdown, playing a role in enhanced ROS production and premature senescence. Elucidation of the mechanism discovered hypermethylation at the SOD2 promoter after DGCR8 loss, and upon further investigation, it was found that DNMT3A was upregulated in these cells, suggesting that DNMT3A is the intermediate regulator of SOD2 expression. Using predicted conserved miRNA families, it was found that miR-29 and miR-30 share high sequence homology, and upon exogenous expression in DGCR8 KO cells, were able to reduce the ROS, as well as decrease the senescence factors and increase the cell proliferation rate. Through the regulatory control of BMSC proliferation and ROS production, miR-29a-3p and miR-30c-5p can elicit control over the early stages in differentiation.Investigations into miRNA since their discovery  have beeRunx2, ALP, OPN and OCN. This presents miR-376c-3p as a regulator of mature osteoblast function and differentiation from the undifferentiated MSC form all the way until the mature osteoblast stage.A number of miRNAs have been directly implicated in regulating osteogenesis, targeting many different genes and signalling pathways. Investigations have shown the relationship between different miRNAs and the activity of TWIST-1, a key transcriptional regulator of BMSC self-renewal and growth, as a suppressor of osteo/chondrogenic differentiation ,129. TWIALP and overall osteogenesis through chromatin remodelling. A further study implicated the role of miR-181a/b-1 in osteogenesis and bone development, albeit through a different mechanism of action [PTEN knockout mice display an enhanced bone formation phenotype, where PI3K/Akt signalling is central in osteoblast differentiation and, thus, bone development [Other miRNAs have also been shown to play direct roles in bone development and during osteoporosis. Studies have described miRNA-19a-3p as playing a role in osteogenesis during normal and pathological conditions . Prior tf action . The datelopment ,139. Anoelopment . Similarelopment  at the MBmncr (bone marrow stem cell-related lncRNA) [Bmncr was found to be highly expressed in bone and adipose tissue, displaying a high expression in the BMSC of young mice, with a decrease in older mice. The deficiency of Bmncr produced a premature bone aging phenotype in a KO model, with the overexpression leading to increased bone density and reduced bone marrow fat. It was revealed that Bmncr upregulates the extracellular matrix molecular fibromodulin to anchor BMSC in trabecular-rich regions via activation of the BMP2 pathway. Furthermore, it was elucidated that Bmncr interacts with TAZ and ABL, facilitating the TAZ and RUNX2/PPARy transcriptional complex, further promoting osteogenesis whilst inhibiting adipogenesis in bone. These findings demonstrate that non-coding RNA species play important roles in bone development and have helped identify potential targets for the rescue of osteoporosis and other bone phenotypes, with a multitude of pathways and factors involved in these complex systems.Comparatively to miRNA, long non-coding RNA (lncRNA) are longer transcripts of more than 200 nucleotides associated with gene regulation in many cellular processes, such as proliferation, differentiation and cancer development ,143. The lncRNA) , promotiType II diabetes mellitus (T2DM) is among the most common global chronic diseases, with a prevalence of 8.5% in adults . The glop53 as a hallmark of senescence associated with a significant reduction in glucose metabolism [Maternal obesity in mice caused by high-glucose/fat diets has been shown to compromise the skeletal health of dams and during foetal development and postnatal growth in offspring, increasing the risk of low bone mass in adulthood ,153,154.tabolism . These fDuring prenatal and postnatal development, epigenetic modifications have a crucial role in regulating the fate of different cells. Diet controls the epigenetic regulations of gene expression. Offspring born to HFD-fed mothers have altered epigenetic regulation in bone development and formation. Various studies have shown the effect of maternal nutrition on the metabolism status of offspring, which mainly occurs through epigenetic alterations and the gene expression of key metabolic pathways, including glucose metabolism ,156,157.Pthlh and Col2α1 was synergistically decreased through significantly enriched H3K27me3 and H3K27ac in dams of HFD-fed maternal mice [H3K27me3 has been shown to be a repressive epigenetic histone modification of RUNX2, the master transcriptional regulator of BMSC osteogenic differentiation . Maternanal mice . The dienal mice .Diet and, more specifically, glucose implement their effects on bone formation through “the bone–pancreas loop”. Osteocalcin (OCN) is a major bone-specific protein secreted by osteoblasts in a carboxylated form. The acidic environment created by the maturation and activity of osteoclasts promotes the decarboxylation and activation of OCN and its release into the circulation. Upon binding to its receptors on pancreatic β cells, OCN triggers insulin release into the circulation. It stimulates β-cell proliferation and increases insulin sensitivity in liver, muscle and adipose tissue, in addition to its role as a metabolic hormone. Insulin binds to its receptors on osteoblasts (InsR) and increases the glucose uptake ,163,164.Ocn-deficient mice showed glucose intolerance, hyperglycaemia and diabetes-like characteristics in these mice [Studies of ese mice . Thus, iese mice ,38,39,83ese mice ,41. Moreese mice . In addiese mice . AlthougA high protein intake is directly associated with a high bone density. The positive impact of a high protein intake on bones occurs through several mechanisms such as calcium absorption and IGF-1 secretion. However, the epigenetic role of a high-protein diet and HFD on the fate of MSC has yet to be fully investigated. Studies on both human and animal BMSC have demonstrated the essential role of protein intake on bone health during aging through regulation of the calcium balance and metabolism in the presence of both high and low-protein intakes . EssentiCaloric intake has a significant impact on stem cell self-renewal and lineage differentiation, as well as aging. While caloric restriction (CR) has been demonstrated to increase the lifespan, stemness and delay aging, HFD has been associated with the acceleration of aging in stem cell populations. In addition, the formation of ROS and DNA damage, as the result of HFD and the metabolic status, is also a major contributor to the acceleration of cellular senescence and the aging process. In contrast to HFD, CR has been reported to enhance stem cell function and decelerate cellular senescence in mouse HSC .Obesity and aging are considered important intrinsic and extrinsic stressors that apply epigenetic effects on the gene expression of stem cells and alter their fate. Adult stem cells are capable of acquiring epigenetic memory from these stimuli over time. Aging is uniquely linked to the hypermethylation of CpG islands and hypomethylation of global DNA in all cell types ,180. One+ levels, a cofactor of Sirtuins (SIRT1–7) deacetylases, decreases with age, causing a reduction in cellular histone acetylation [The metabolic state plays a vital role in MSC function and survival. Age-associated changes in the MSC metabolic clock occur via mitochondrial dysfunction and accelerated anabolic signalling, causing an increase in the production and accumulation of ROS and cellular senescence ,182. In tylation ,186. As tylation ,188. Protylation . Higher tylation ,190.Hox genes as a regulator of DNA methylation. Consequently, DNA was hypomethylated, changing the differentiation and regeneration potential of these cells [NAD is also a cofactor of histone modifiers, including H3K4me3, H3K36me3 and H3K27me3, influencing epigenomic regulation ,183. Furse cells .Exercise or mechanical loading is a key factor in improving the general body health through balancing metabolism and cardiovascular functions, as well as promoting good skeletal health. Physical activity can prevent bone deterioration by antagonising RANKL signalling, leading to decreased bone resorption by OC and increasing bone formation and strength with reduced adiposity in the marrow cavity ,193,194.PANK3 are downregulated following mechanical stimulation in BMSC, which stimulates the expression of the Runx2 protein [The effect of mechanical force on BMSC gene expression profiles through epigenetic modifications has been explored in recent years. Actin polymerisation has been shown to regulate epigenetic gene regulation by changing the physical shape of nuclei, which affects specific signal transduction . Moreove protein . In addi protein . Further protein . Similar protein . Similar protein .Nrf2 promotor, making this epigenetic feature a potential target of the pathogenesis of osteoporosis [The effect of a running exercise was further investigated in osteoporotic patients and in an ovariectomised mouse model of osteoporosis. The findings identified nuclear factor erythroid-derived 2-related factor-2 (Nrf2) as a critical anti-osteoporotic factor that was heavily suppressed by the activity of DNMT in its promoter. This study claimed that a running exercise could correct the effect of DNMT hypermethylation of the oporosis .ASC gene revealed that, although the expression on the ASC gene is upregulated by age, six months of intermittent walking reduced ASC expression by increasing DNA methylation, which consequently lowered the level of proinflammatory cytokines [It has also been proposed that exercise can control bone formation through epigenetics by regulating inflammation and triggering changes in the gene expression patterns of neutrophils and peripheral blood mononuclear cells, which can lead to the inhibition of bone resorption . Anotherytokines . TherefoThe information theory of aging relates to the erosion of the epigenetic landscape as cells age. This contributes to the deregulated function of cells and their loss of identity. Investigation of the epigenetic changes in MSC as they age over the years has demonstrated dynamic changes in DNA methylation, hydroxymethylation, histone modifications and non-coding RNA. These changes result in senescence, skewed differentiation favouring adipogenesis at the expense of osteogenesis and the loss of heterochromatin. The chromatin structure is now recognised as an important guardian of the genome and a transducer of epigenetic information that is altered during aging, where MSCs have a higher level of accessible chromatin compared to somatic cells to ensure plasticity. Interestingly, MSCs show remarkable nucleosome position stability as they age, with changes occurring along stress response genes to ensure protective mechanisms are in place. Changes in the chromatin structure and suppression of stemness genes through decreased chromatin accessibility, especially along differentiation genes enriched for bivalent chromatin domains, drive MSC aging. The advantage of assessing chromatin accessibility is amplified with the discovery that chromatin changes seem to occur in regions with identified SNPs for multiple diseases, including obesity, immunity and bone-related disorders, emphasising another level of exploration for potential therapeutic targets to counter stem cell aging and disease.KDM4B results in repression of the osteogenic-related genes, increased MSC senescence and accelerated aging. Many studies have confirmed a global decrease in H3 and H4 acetylation mainly due to an increase in HDAC4, 5 and 6, resulting in a decrease in stemness gene expression and self-renewal. In particular, H3K9 acetylation decreases during aging and is associated with the decreased expression of Wnt, Bmp genes and Runx2, compromising MSC osteogenesis and autophagy. HDAC1 and 2, on the other hand, increase in expression, leading to increased p16 expression and MSC senescence. These examples illustrate how the erosion of the epigenetic landscape alters transcription at multiple levels in aging MSC.Histone and DNA modifications are the key mediators of the chromatin structure and genomic regions associated with MSC age-related increases in DNA methylation in association with repressive histone marks H3K27me3 and H3K9me3, which also appear to play important roles during osteoporosis. Of note, Ezh2 has been shown to deposit H3K27me3 on bone-associated genes in MSC derived from osteoporotic samples, as well as deactivate the antioxidant defence system, reducing osteogenesis. Interestingly, supplementing antioxidants or inhibiting Ezh2 can reverse the effects on aging MSC and restore differentiation. Moreover, the H3K9me3 levels also increase during MSC aging, and loss of the H3K9me3 demethylase The additional layer of epigenetic modifiers in the form of miRNA and long non-coding RNA has also advanced considerably in recent times, as they have been shown to be deregulated in aged MSC. As highlighted in this review, miRNAs are able to regulate the factors involved in DNA methylation, as well as histone methylation/acetylation, imposing a broad range of control over the changing epigenetic landscape within aging MSC. Further works into elucidating the roles of different miRNA species and implicating them in not only aging but many different diseases will provide a greater insight into the ever-evolving epigenetic landscape.Given the influence the environment has on our epigenome, future studies will increase dramatically, showing how diet and lifestyle affects the epigenetic drift and aging. Studies on high-fat/sugar diets have recently shown that a high-fat maternal diet can compromise the skeletal integrity of offspring, giving rise to low bone mass. MSC display senescence, reduced osteogenesis, increased adipogenesis and reduced glucose metabolism. Increased H3K27Ac on senescent genes and the repressive H3K27me3 on osteogenic genes, together with DNA methylation changes, drive senescence and inhibit osteogenesis. Further studies are needed to examine the plethora of histone and DNA modification changes in response to different diets and the effects of different metabolites that act as cofactors for epigenetic enzymes. One of the most effective treatments for bone loss is mechanical loading and resistance exercise, which has shown to result in reduced DNA methylation along osteogenic genes and the expression of long non-coding RNA species, enhancing osteogenesis. The information instructing the chromatin architecture and, therefore, cellular function and longevity is complex and interdependent. Just how all these layers change and contribute to aging represents a field that is now rapidly expanding with an effort to reboot the epigenetic code safely as a strategy to rejuvenate stem cells.


Oryza sativa genome from the Database of Potential Promoter Sequences predicted using a mathematical algorithm based on the derivation and calculation of statistically significant promoter classes. In this paper TATA motifs and cis-regulatory elements were identified in the predicted promoter sequences. We also verified the presence of potential transcription start sites near the predicted promoters by analyzing CAGE-seq data. We searched for unannotated transcripts behind the predicted sequences by de novo assembling transcripts from RNA-seq data. We also examined chromatin accessibility in the region of the predicted promoters by analyzing ATAC-seq data. As a result of this work, we identified the predicted sequences that are most likely to be promoters for further experimental validation in an in vivo or in vitro system. The exact identification of promoter sequences remains a serious problem in computational biology, as the promoter prediction algorithms under development continue to produce false-positive results. Therefore, to fully assess the validity of predicted sequences, it is necessary to perform a comprehensive test of their properties, such as the presence of downstream transcribed DNA regions behind them, or chromatin accessibility for transcription factor binding. In this paper, we examined the promoter sequences of chromosome 1 of the rice  Rice is a tropical plant from of the genus of annual and perennial herbaceous plants of the grass family; a cereal species. Lots of nations treat it as a second bread. According to the time of cultivation and its valuable qualities, it is rightfully considered the most popular cereal in the world. Due to its high agricultural importance, the problems and risks associated with rice cultivation are under the observation of scientists in a wide range of fields, from agronomic sciences to molecular biology and biotechnology. In the field of agronomy, different approaches to rice cultivation are being explored, and there is active research into the genetic differences between domesticated rice species and their wild relatives in order to develop loci that can be used to create a new generation of resistant rice varieties. Molecular biologists and biotechnologists are exploring the possibility of the genomic editing of rice to improve its nutritional properties and increase its environmental resilience .O. sativa Nipponbare genome [https://rapdb.dna.affrc.go.jp/, accessed on 20 May 2022) [The rice genome is quite well studied, sequenced, and annotated. In 2005, the International Rice Genome Sequencing Project (IRGSP) group provided, for the first time, a reference rice genome sequence covering 95% of the ay 2022)  and a daay 2022) . In addiA promoter is a sequence of DNA nucleotides in the vicinity of the transcription start site, necessary for the formation of the preinitiator complex. The length of promoters usually varies from 100 to 1000 bp; it can vary significantly from gene to gene. Eukaryotic promoters are usually divided into three regions: core/basic, distal, and proximal regions . The corProper identification of promoter sequences plays an important role in understanding the dynamics, patterns, and regulation of gene expression. In addition, when genome editing or combining DNA fragments into new synthetic sequences, it is critical for predicting the potential formation of new promoter sequences. In recent years, a great abundance of bioinformatics tools have been created to predict promoter sequences in the genome or in a DNA region of interest. But the exact identification of promoter sequences remains a major challenge in computational biology, as even the best algorithms can generate false results that are indistinguishable from true ones.To detect in the genome, and characterize and quantify the activity of promoters, various sequencing-based analysis methods have been developed, such as: CAGE , which allows for measuring the expression of eukaryotic capped RNAs and simultaneously displaying promoter regions ; PRSeq , predicted to be promoter elements using a mathematical sequence prediction method based on multiple alignments [−8 per nucleotide [−4 per nucleotide. Therefore, all other theoretical databases are at least four orders of magnitude worse than this database, because the number of false positives is less than 10−8 for this database. Thus, the potential promoters included in the database we used are practically unaffected by random processes. In this sense, the results of this database are the most promising for experimental verification.In this work, we studied the sequences of the first chromosome of the ignments . Multiplignments , a mathecleotide . GeneralFirstly, all selected promoters under study were analyzed for the absence of an intersection with annotated promoters and transcripts within 1000 bp downstream of the annotated transcripts.Next, TATA-motifs regions were identified in the selected promoters under study . Also, cAs a result of comparing the studied promoters from the Database of Potential Promoter Sequences with the promoters from the PlantRegMap database, it was found that only nine of the studied promoters—14, 15, 29, 51, 80, 90, 92, 105, and 116—intersect with the promoters from PlantRegMap. This may indicate the correctness of the prediction of the function of these sequences, since they were predicted to be promoters by two different independent algorithms.While analyzing the CAGE-seq data, we found CAP sites mapped to regions of the rice genome free of known annotated genes, as shown in -Three potential TSS located no further than 100 bp from the 3′-end of the predicted promoters ;-Nine potential TSS overlapping with the 3′-end of the promoter ;-One potential TSS located no further than 300 bp from the 3′-end of the predicted promoter (promoter number: 77);-One potential TSS that has a distinct peak located no further than 1000 bp from the 3′-end of the predicted promoter (promoter number 81) and lower peaks, the closest of which is no further than 250 bp from the 3′-end of the promoter.Among such unannotated potential transcription start sites there are also those located close to some of the investigated predicted promoters , among tThus, these predicted promoters may indeed be the promoter region of the genome from which transcription starts, as there are potential unannotated transcription start sites behind them. downstream) direction from predicted promoter sequences. However, few transcripts were found in such a range, so we decided to extend the search range to 5000 bp. Theoretically, promoters can be distanced from genes by embedding mobile genome elements. Therefore, we thought it would be interesting to study the assembled transcripts more distant from the potential promoters as well. We then searched for unannotated transcripts in the rice genome via de novo genome assembly. First, we selected transcripts located up to 1000 bp in the 3′ . But at the moment, the occurrence of false-positive results in sequence prediction remains an unsolved problem in all computational biology, including the method we use, in which the number of false positives for a randomly mixed rice genome is <10We saw that prediction tools based on different algorithms—MAHDS and an algorithm based on searching for DNA-binding domains of transcription factors—produced predominantly divergent predictions of promoter regions of the rice genome. As we can see from the results, only 9 of the studied 126 promoters predicted via the MAHDS method were also detected in the Plant Reg Map database based on another prediction algorithm. Can we be sure that these nine sequences are really promoters since each of them was predicted via different independent methods? Probably, yes. However, as we can see from our results, we did not detect unannotated TSSs or transcripts behind any of these nine predicted promoters. We also did not see accessible open chromatin in the region of these sequences. It is well known that the accessible region of chromatin does not physically prevent the binding of transcription factors, which is important to consider when studying the properties of putative promoter sites. In our study, we paid attention to investigating the issue of the degree of chromatin openness in the neighborhood of predicted regulatory sequences. As a result, we obtained a number of predicted promoters located in the open region of chromatin —16 sequeThe presence of a promoter in the genome implies the presence of transcription behind it. One of the options for finding promoters or confirming the role of predicted promoters is to search for transcripts or transcription start sites. In this work we used both strategies. It is worth noting that the TSS search gave us more results, which showed the presence of 5′-end transcripts directly near the predicted promoters . The sinThe theoretically predicted sequences may also be other regulatory elements of the genome, such as enhancers. Enhancers are small regions of DNA that, when transcription factors bind to them, can increase the level of transcription of a gene or group of genes. Enhancers do not need to be in close proximity to the genes they affect. Recently, it has been shown that enhancers can also perform promoter functions . For exaThe lack of visible transcription behind the promoter region may also be due to weak transcription levels or the silencing of genes due to methylation of their promoter region. Methylation of these regions prevents RNA polymerases and/or various other transcription factors from binding to the promoter region of DNA due to steric hindrance, thereby reducing and even inhibiting gene transcription . The stuhttp://victoria.biengi.ac.ru/cgi-bin/dbPPS/index.cgi, accessed on 20 August 2022). This database contains promoter sequences, predicted via the MADHS method, of the following genomes: Capsicum annuum, Homo sapiens, Lactuca sativa, and Oryza sativa. We selected the first chromosome of the rice genome. Further selection was performed among the predicted potential promoters located no closer than 1000 bp upstream from known genes and no closer than 200 bp from transposons and SINE repeats. This selection was necessary to ensure that the predicted potential promoter is not associated with the transposons and promoters of known genes, and that it is more likely that the predicted promoter is associated with previously unknown genes. Of these, potential sequences were selected with the largest argument of the normal distribution, which shows the degree of non-randomness of the created alignment. The length of the selected predicted potential promoters varies from 428 to 599 nucleotides, and the distance from them to the nearest annotated transcripts varies from 1044 to 92,434 bp.In total, 126 potential promoter sequences  were selhttps://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 4 September 2022), a plant cis-acting regulatory element database [TATA-motifs and cis-regulatory elements were searched for within the predicted sequence of potential promoters. The search was performed by analyzing nucleotide sequences of promoters in the FASTA file using PlantCARE  was downloaded from the Ensembl Plants integrative resource . From these data, annotated transcripts of the first chromosome—6437 transcripts—and sequences of the 5′-untranslated regions of the first chromosome—6474 sequences—were extracted.The rice closest [-d -iu to search for transcripts in the downstream direction on the same strand and determine the distance from promoters to transcripts.The search for annotated transcripts closest to the promoters under study was carried out using Bedtools closest  with opthttp://plantregmap.gao-lab.org/download.php, accessed on 4 September 2022). From the file on promoters extracted from PlantRegMap, sequences and promoter IDs of the first chromosome were extracted using Bioawk  and formatted in multi-FASTA format. To obtain the coordinates of these promoters, they were aligned to the genome using bowtie-align-s [-s --wrapper basic-0 -a -m1 -v0. The first chromosome genome  was downloaded from the NCBI Genome. Then, using the Samtools toolkit [view and options -b -o, sorted with command sort and option -o, and reformatted to BED format with promoter coordinates using bedtools bamtobed with option -i. Promoters of interest that intersect with promoters from PlantRegMap were found using bedtools intersect with the -s option and those not intersecting, with options -s and -v.The investigated promoters were compared with promoters from another database, PlantRegMap . This da-align-s  with opt toolkit , the alihttps://github.com/FelixKrueger/TrimGalore, accessed on 2 November 2022) with options --nextera and --length 51, the quality of reads was assessed in FastQC [To search for possible unannotated transcription behind the predicted promoters, the gene expression cap analysis data (CAGE-seq) of control samples of O. sativa rice  were analyzed. Available raw sequencing data were searched for in the NCBI SRA. DRR127211 data were selected for further analysis. Adapters and reads of poor quality were removed using Trim-Galore  with help of the fastq-dump tool. Then, using Trim-galore  with options -illumina -q 28, Illumina sequencing adapters were removed from the reads in addition to low-quality reads .To search potential new transcripts, de novo transcripts were assembled from raw RNA data sequencing available from the NCBI SRA. The following RNA-seq data sets were selected for de novo transcript assembly: SRR11829613, SRR15060372, SRR8469588, SRR11825481, SRR12046251, SRR8146491. They are the results of the RNA sequencing of control samples, deliberately not subjected to stress, infection, or genetic modification. Data loading and conversion from SRA to FASTQ format was performed using the SRA-Toolkit 3.0.2. . To assess the expression level of the assembled transcripts, a method based on the normalization of transcriptome sample data was used, and the number of transcripts per million mapped reads (TPM) was calculated using Salmon .De novo transcript assembly from RNA sequencing data was performed using Trinity  downloaded from the Ensemble Plants Integrative Resource website . From these data, annotated transcripts of the first chromosome—6437 transcripts—and sequences of 5′-untranslated regions of the first chromosome—6474 sequences—were extracted. To select potentially “new” transcripts that had not previously been annotated, a search was made for assembled transcripts that did not overlap with those annotated using Bedtools with the downstream) direction from potential predicted promoters were selected. The selection was made using Bedtools closest [-iu -D a -s.To search for potentially novel transcripts, transcripts that are up to 5000 bp away in the 3′  with options “ATG and alternative initiation codons” and minimal ORF length (nt) = 150; in cases where no ORFs were found, the minimum length was reduced to 75 nucleotides.Selected transcripts were checked for the presence of ORF in ORFFinder  on tomato (taxid:4081), Arabidopsis (taxid:3702), and rice (taxid:4530). Homologs were searched using the blastx algorithms at NCBI —SRR15311718 were taken for analysis. Data quality analysis was performed in FastQC [https://github.com/FelixKrueger/TrimGalore, accessed on 2 November 2022) with options --nextera --paired -q 26 --length 150. Alignment to the rice genome was performed using bowtie2 [very-sensitive option. The alignment in SAM-format data were converted in BAM format with the help of the Samtools command—view. Then, alignment data in BAM format were sorted and indexed using Samtools with the help of the commands sort and index [bamCoverage. At the next stage, peaks intersecting with predicted promoters were selected using the Bedtools command intersection [Chromatin accessibility was analyzed by examining ATAC-seq sequencing data to recognize open chromatin regions available for transcription factor binding. Data from NCBI SRA  which we consider the most promising for further experimental analysis.For in vivo/in vitro testing, constructs with reporter systems such as GUS or GFP behind potential promoter sequences can be used. Y2H/Y1H assays or Bimolecular fluorescence complementation (BiFC) also can be used. But, unfortunately, even such an analysis may not show the real result, as it depends on a number of factors. The tissue specificity of certain classes of spatiotemporal promoters is difficult to properly assess due to the apparent lack of differentially differentiated tissues. Also, the size of the isolated promoter can negatively affect the assay, as the cloning of the promoter may miss deleted essential cis-elements, or may lack the transcription factors necessary for interaction with cis-elements. Additionatly, in in vivo assays, promoter sequences can be randomly integrated into independent genomic events during transformation, which can lead to large expression variability depending on genomic location and may also cause silencing.Despite the difficulties encountered, the study of genomes, and in particular regulatory sequences, is an important task of modern biology. The genomes of many organisms are already fairly well annotated, but there are still gaps and functionally unknown regions remaining. Knowledge of the exact reference sequence of the genome and its functional components can play an important role in genome editing technology. It is important to know all the areas in which editing can take place, as well as sensibly assessing the possible off-target effects of editing. Precise knowledge of the location of functional elements of the genome will allow us to notice and control random mutations in these areas, which can affect the expression of some genes and have a noticeable or imperceptible effect on the whole, living, edited organism.


The study introduces the development of two novel porous organic polymers, namely FEC-Mel and FEC-PBDT POPs, constructed using a simple method based on the ferrocene unit (FEC) combined with melamine (Mel) and 6,6′-bis (PBDT). The synthesis involved the condensation reaction between ferrocenecarboxaldehyde monomer (FEC-CHO) and the respective aryl amines. Several analytical methods were employed to investigate the physical characteristics, chemical structure, morphology, and potential applications of these porous materials. Through thermogravimetric analysis (TGA), it was observed that both FEC-Mel and FEC-PBDT POPs exhibited exceptional thermal stability. FEC-Mel POP displayed a higher surface area and porosity, measuring 556 m2 g−1 and 1.26 cm3 g−1, respectively. These FEC-POPs possess large surface areas, making them promising materials for applications such as supercapacitor (SC) electrodes and gas adsorption. With 82 F g−1 of specific capacitance at 0.5 A g−1, the FEC-PBDT POP electrode has exceptional electrochemical characteristics. In addition, the FEC-Mel POP showed remarkable CO2 absorption capabilities, with 1.34 and 1.75 mmol g−1 . The potential of the FEC-POPs created in this work for CO2 capacity and electrical testing are highlighted by these results.This work focuses on porous organic polymers (POPs), which have gained significant global attention for their potential in energy storage and carbon dioxide (CO The conventional wet cleaning technique, involving the use of aqueous solutions of alkanolamine (known as amine scrubbing), has been widely employed for CO2 capture and separation. However, this method has several drawbacks. It exhibits high energy consumption, is costly to implement, has limited efficiency, poses challenges with solvent renewal, results in solvent loss, and leads to equipment corrosion due to the presence of amines [2 capture in industrial operations while simultaneously reducing emissions. Over the past decade, due to their exceptional characteristics, POPs have attracted substantial attention in both the academic and industrial worlds. These characteristics include high surface areas, suitable pore sizes, excellent thermal stability, low density, diverse composition, and low regeneration energy requirements [2) emissions, there is a growing need to focus on CO2 capture and supercapacitors (SCs). POPs have become desirable candidates for CO2 uptake and as active electrode materials in SCs because of their unique characteristics [2 or I2 [−1 [2 uptake and supercapacitors were the main goal of their development. Numerous analytical techniques, including BET (Brunauer–Emmett–Teller), Fourier-transform infrared spectroscopy (FTIR), solid-state nuclear magnetic resonance (ssNMR), powder X-ray diffraction (XRD), and scanning electron microscopy (SEM), were used to validate the synthesis, morphology, and porosity of the FEC-Mel and FEC-PBDT POPs. A capacitance of 82 F g−1 was discovered on the FEC-PBDT POP electrode. This result shows that it is feasible to use them in supercapacitors. The FEC-Mel POP sample also showed significant CO2 capture capabilities of 1.34 and 1.57 mmol g−1 at 298 and 273 K. Overall, the findings point to the potential of the FEC-POPs produced in this work for use in gas adsorption and energy devices.Numerous energy-collecting techniques have been developed as a result of the rising need for energy that emits no greenhouse gases. Although they are essential, renewable energy sources cannot provide all of our daily energy needs. Investigating reasonable and cost-effective methods of energy collection and storage is therefore important. Due to their excellent qualities including high energy densities, quick charge/discharge rates, and extended lifespan, supercapacitors (SCs) have received a lot of interest in this respect ,2,3. SCspatterns ,16. Consf amines ,18,19. Tirements ,21. Due irements ,30,31,32eristics ,34,35. S[2 or I2 ,37. Vari[2 or I2 ,42,43,44[2 or I2 ,46,47,48[2 or I2 . There a[2 or I2 ,51,52,53[2 or I2 ,56,57,58[2 or I2 . Ma and [2 or I2 . Additio[2 or I2 . Furtherr I2 [−1 . In this3 in DMF as a solvent for 16 h, resulting in crimson crystals  incorporating a ferrocene moiety were synthesized using a polycondensation reaction. The building unit, FEC-CHO, was reacted with Mel and PBDT in DMSO at 180 °C to yield FEC-Mel and FEC-PBDT POPs, respectively see a,b. To ocrystals . The FTIne rings . The 1H ectively . The 13CC moiety . For the in PBDT . The prospectrum  correspo of PBDT  revealed13C NMR spectra and FT-IR measurements. The FT-IR spectra of FEC POPs displayed absorption peaks at 1023 cm−1, which corresponded to the C=C stretching of the ferrocene unit. Notably, absorptions at around 1082, 1554, and 3418 cm−1 were attributed to C-N, C=N and NH units, respectively. Moreover, distinct vibration bands for the aliphatic C-H group originating from the ferrocene unit were observed at 2927 cm−1  were confirmed through solid-state 927 cm−1 a. In themoieties b. Thermomoieties c. The TGmoieties d. The XP2 adsorption and desorption curves of both FEC POPs exhibited a type IV isotherm, indicating the presence of micropores and mesopores. Remarkably, FEC-Mel and FEC-PBDT POPs displayed large SBET of 556 and 428 m2 g−1, respectively , nitrogen (N), and oxygen (O) atoms in FEC POPs a–h. Over2 uptake capabilities of FEC-Mel and FEC-PBDT POPs, CO2 isotherm measurements were conducted at temperatures of 298 K and 273 K, respectively. At 298 K, FEC-Mel and FEC-PBDT POPs displayed a CO2 capacity of 1.34 and 0. 51 mmol g−1, respectively. At 273 K, the CO2 uptake capacities increased to 1.57 mmol g−1 for FEC-Mel POP and 1.53 mmol g−1 for FEC-PBDT POP  measurements at different current densities (ranging from 0.5 to 20 A g−1) and all CGD profiles are approximately in an isosceles triangle shape. The results, depicted in The electrochemical performance of FEC-Mel and FEC-PBDT POPs warrants investigation owing to their considerable BET surface areas and incorporation of triazine moieties, as illustrated in −1 at a current density of 0.5, 1, 2, 3, 5, 7, 10, 15, and 20 A g−1, respectively. Similarly, for the FEC-PBDT POP, the specific capacitance values were recorded as 82, 49, 25, 18, 14, 12, 10, 8, and 7 F g−1 at the corresponding discharge rates of 0.5, 1, 2, 3, 5, 7, 10, 15, and 20 A g−1. In −1, while the FEC-PBDT POPs material has a higher energy density of 11.36 Wh kg−1. These values correspond to a power density of 250 W kg−1. Notably, both FEC-Mel and FEC-PBDT POPs materials outperform other N-doped porous carbon materials, with an energy density of 7.11 Wh kg−1 [−1 [The Ragone plot shown in  Wh kg−1 , and a pkg−1 [−1 . This hi3), sodium acetate, hexane, acetone, NaOH, 2-cyanoguanidine (GD-2CN), tetrahydrofuran (THF), ethanol (EtOH), and methanol (MeOH) were obtained via various trade resources, such as Sigma-Aldrich , and Alfa Aesar .Ferrocene (FEC), 1,4-dicyanobenzene (BZ-2CN), dimethyl sulfoxide (DMSO), potassium hydroxide (KOH), chloroform, dimethyl formamide (DMF), phosphoryl chloride  was then progressively added after DMF  had been chilled in an ice bath. A 15 min break was followed by the addition of 100 mL of CHCl3 to thin the liquid. FEC  was then included in the mixture. The resultant mixture was agitated at 60 °C (for 16 h) and had a dark amber appearance. Upon completion of the reaction, the mixture was allowed to cool down. Ice water (500 mL) was then added gradually, followed by the slow addition of 35 g of NaOH and 107 g of sodium acetate. The product was extracted using chloroform (500 mL) and subsequently washed three times with water. To purify the product, it was passed through a silica column, and any remaining impurities were removed by elution with a hexane/acetone mixture (ratio of 5/1). Finally, crimson crystals of the desired product were obtained  was added into DMF (10 mL). Separately, a mixture of KOH  and GD-2CN  was added into DMF (40 mL). The KOH/GD-2CN solution was then added to the BZ-2CN solution. About 20 h were spent stirring and refluxing the resultant mixture at 130 °C under a Nain PBDT . FTIR  and FEC-CHO  in DMSO (20 mL) were produced and charged into a Schlenk flask. Three cycles of freezing and thawing were performed on the flask. The reaction flask was then heated to 180 °C and held at this temperature while stirring for 3 days. Following the reaction, the flask was allowed to cool to room temperature. After filtering, the finished product was washed with acetone, MeOH, and THF. The resulting black powder is known as FEC-Mel POP a. To predT10 value of up to 353 °C and a char yield of approximately 54.55 wt% at 800 °C, as determined using thermal gravimetric analysis (TGA). For the potential applications for FEC-POPs, we revealed that the FEC-PBDT POP electrode exhibited an exceptional capacitance of 82 F g−1, confirming its suitability for application in supercapacitors. Furthermore, the FEC-Mel POP sample demonstrated impressive CO2 capture capacities, measuring 1.34 and 1.57 mmol g−1 at 298 and 273 K, respectively. These findings highlight the potential of the synthesized FEC-POPs for utilization in energy storage and gas adsorption devices.In summary, we developed two types of porous organic polymers (FEC-POPs) by incorporating the FEC unit with different aryl amines through a polycondensation reaction. The resulting polymers were named FEC-Mel and FEC-PBDT POPs. Both the FEC-Mel and FEC-PBDT POPs exhibited remarkable thermal stability, with a


Two promoters lacked TATA elements, but all supported strong initiation from a single transcription start site. In contrast to results with minimal in vitro systems based on the TATA-binding protein (TBP), TATA promoter templates with a +51 NPE were transcriptionally inhibited in extracts; activity continuously increased as the nucleosome was moved downstream to +100. Inhibition was much more pronounced for the TATA-less promoters: +51 NPE templates were inactive, and substantial activity was only seen with the +100 NPE templates. Substituting the histone variants H2A.Z, H3.3, or both did not eliminate the inhibition. However, addition of excess TBP restored activity on nucleosomal templates with TATA promoters, even with an NPE at +20. Remarkably, nucleosomal templates with histone H3 trimethylated at lysine 4 are active with an NPE at +51 for both TATA and TATA-less promoters. Our results strongly suggest that the +1 nucleosome interferes with promoter recognition by TFIID. This inhibition can be overcome with TBP alone at TATA promoters or through positive interactions with histone modifications and TFIID.A nucleosome is typically positioned with its proximal edge (NPE) ∼50 bp downstream from the transcription start site of metazoan RNA polymerase II promoters. This +1 nucleosome has distinctive characteristics, including the presence of variant histone types and trimethylation of histone H3 at lysine 4. To address the role of these features in transcription complex assembly, we generated templates with four different promoters and nucleosomes located at a variety of downstream positions, which were transcribed  The in Refs. , 12, 13)in Refs. . Consistromoters , which ein vitro , 15. Nucromoters , 15, 16.in vitro studies showed that incorporation of these histones results in reduced nucleosome stability  nucleosome whose proximal edge usually lies just beyond the expected boundary of a PIC , 17. Maptability , 23.in vitro transcription system to determine the functional importance of promoter-nucleosome distances and nucleosome composition for PIC assembly. We tested mononucleosomal templates with TATA or TATA-less promoters located at a series of distances upstream of a precisely positioned +1 nucleosome. The nucleosomes had variable composition, with some containing H2A.Z, H3.3, or H3K4me3. We show that the +1 nucleosome negatively regulates transcription complex assembly by default, an effect that is much more pronounced on TATA-less promoters. Bypassing TFIID through TATA-binding protein (TBP) supplementation can overcome this inhibition but only on TATA-containing promoters. Crucially, substitution of H3K4me3 for nonmodified H3 overcomes the inhibition for both promoter classes. Our results implicate TFIID as a key regulator in modulating assembly of pol II PIC in a chromatin environment similar to that seen in the nucleus.In the experiments reported here, we utilized a nuclear extract–based in vitro approaches. Our experimental design was intended to encompass not only the range of locations downstream of TSS typical for +1 nucleosomes but also different histone types and modifications frequently associated with the initial downstream nucleosome. Particularly in light of potential mechanistic differences in the pathways of PIC assembly for TATA-containing and TATA-less promoters . We selected HNRNPAB as a representative TATA-containing promoter. We also included in the TATA class the core section of well-studied adenovirus major late (AML) promoter, which supports a single TSS and has an engineered G-less cassette downstream (A). Among the TATA-less promoters, we selected FLNB and KLHL15. It is worth noting that both TATA-less sequences have consensus initiator elements . Our template constructs retain the native sequences for at least 96 bp upstream of the TSSs for HNRNPAB, FLNB, and KLHL15.We wished to investigate the effects of promoter-proximal nucleosomes on pol II transcription through romoters , 15, we romoters . Among tr G or T A. We selwnstream A. Among elements   . N. NB) (24B)  on a modB)  biotinylA, all the non-nucleosomal templates were strongly transcribed, including the two TATA-less promoters. We were surprised to find that the nucleosomal 51 templates were either substantially less active (AML and HNRNPAB) or inactive (FLNB and KLHL15) for transcription. This is in sharp contrast to our earlier studies with nucleosomal TATA-promoter templates in which the TSS to nucleosome distance was 50 bp (B). On all templates, the large majority of polymerases that did initiate advanced to two closely spaced major pause points ∼45 to 55 bp within the nucleosome, marked by brackets on each panel. This is consistent with many earlier studies on the barrier imposed to advancing pol II by nucleosomes located on the 601 element and on other positioning sequences : TBP supplementation for either of the TATA promoters does restore transcriptional activity, nearly to that level seen with the naked DNA template. Addition of the same amounts of TBP did not rescue activity on the TATA-less KLHL15 51 promoter (A).In considering the basis for inhibition of transcription on the nucleosomal templates, we focused on the differences in response of the TATA and TATA-less templates to the proximal nucleosome. Assembly of the PIC at TATA-less promoters depends on elements downstream of the TSS, most recently mapped at +17 to +35 , whereasemplates  using TFemplates , supplememplates , A and Cpromoter A.Figure A showed that all these templates were transcriptionally active. To confirm that the surprising 20 template result was not specific to HNRNPAB, we also constructed an AML 20 template. Both these 20 templates reproducibly supported about half the activity of the 51 templates with the same promoter (B and C). The significance of this observation for mechanistic understanding of XPB-driven template melting will be explored in the The TBP-dependent activity of the 51 TATA templates raises the question of how close the +1 nucleosome can approach the TSS while retaining transcriptional activity. Even when PIC assembly is driven primarily by interactions with the TATA element upstream, recent evidence indicates that downstream contacts are still involved at TATA promoters, primarily from the XPB subunit of TFIIH , 4. AccoFigure 3promoter , B and CA) should result from bypassing TFIID. It seems unlikely that the transcriptional stimulation from added TBP is caused by the assembly of additional TFIID by combination of the added TBP with free TAFs in the extract, since TBP addition allows transcription of the otherwise inactive TATA 20 templates . On those DNAs, the NPE would completely occlude the downstream promoter elements required for TFIID binding, which extend out to about +35. A +1 nucleosome with a proximal edge at 51 is strongly (TATA promoters) or completely (TATA-less promoters) inhibitory to transcription when those promoters depend on TFIID, but a +51 TATA template is transcribed at near-naked DNA levels when driven only by TBP . However, there was a modest effect on the level of nucleosome traversal (A). This was only observed for nucleosomes containing H2A.Z and was more apparent when the reactions were chased in 150 mM salt instead of 75 mM used in all other experiments . The traversal increase was approximated by comparing the signal at the major nucleosome barrier to the signal of the run-off products. In reactions chased at 150 mM with nucleosomes containing the usual histone complement, the level of run-off RNA to major pause RNA was about 1:3, but for nucleosomes containing H2A.Z, that ratio increased to nearly 1:1 . This increase in traversal is consistent with the recent observation that incorporation of H2A.Z promotes nucleosome unwrapping  following the manufacturer’s recommended procedure. After amplification, the reaction was then passed through a single column from a Monarch PCR & DNA cleanup kit (NEB) following the manufacturer’s recommendations for double-stranded DNA <2 kb. The column was eluted with 15 μl elution buffer and digested with 10 U AvaI at 37 °C for 2 h. For KLHL15 promoter fragments only, before digesting with AvaI, the eluate was digested with 10 U SmaI at 37 °C for 1 h followed by treatment with 5 U QuickCIP (NEB) for 30 min at 37 °C and heat inactivation for 10 min at 80 °C. The reactions were stopped by adding 4 μl 6× Purple Gel Loading Dye (NEB) and then run on a 0.8% Tris:acetate:EDTA agarose gel. Fragments were gel purified using a QIAquick gel extraction kit (Qiagen) according to the manufacturer’s recommendations and were further concentrated by ethanol precipitation.Histone tetramers containing H4 and H3K4me3 were custom ordered from EpiCypher. All other histone dimers, tetramers, and octamers were purchased from ActiveMotif. Nucleosomes were reconstituted using a microscale reconstitution method performed at 24 °C adapted from previous work  with theTemplates were ligated by mixing promoter DNA fragments with 100 ng reconstituted nucleosome fragments at 2:1 M ratios followed by addition of 10 μl 10× T4 DNA ligase buffer (NEB) and 400 U T4 DNA ligase (NEB) in a final volume of 100 μl. The reactions were incubated for 16 h at 15 °C and diluted to 500 μl with TE100 buffer . About 15 μl of Dynabeads M-280 streptavidin (Invitrogen) were washed according to the manufacturer’s recommendations and resuspended in 15 μl of TE100. These resuspended beads were added to the diluted ligation reaction, gently mixed, and incubated at 24 °C for 2 min. The beads were then washed twice with 100 μl TE250  and resuspended in 35 μl TE100. Successful ligation of templates was verified by phenol:chloroform extracting 5 μl of beads and resolving the ethanol-precipitated DNA on a 4.5% native PAGE. The gel was stained with ethidium bromide and imaged using a GelDoc EZ (Bio-Rad). Bands were quantified using Image Lab, version 6.1.0. Where BstXI digestion was necessary, the beads were resuspended in 50 μl 1× r3.1 buffer (NEB) and incubated at 37 °C for 1 h with 10 U BstXI. After digestion, the beads were washed twice with 100 μl TE250 and then resuspended in 35 μl TE100.Escherichia coli DNA. Final buffer for PIC assembly was 20 mM Tris (pH 7.9), 1 mM DTT, 8 mM MgCl2, 35 mM KCl, 40 mM NaCl, 7% glycerol, and 0.5 mM EDTA. Reactions were initiated by adding 3.5 μl of limiting NTPs to a final concentration of 100 μM ATP, 0.767 μM α32P-CTP (PerkinElmer), and 60 μM UTP (for AML templates only) or 60 μM GTP , followed by incubation at 30 °C for 2 min. The reactions were then washed twice with 70 μl XM75 buffer  supplemented with 0.002% Igepal CA-630, changing to a fresh test tube on the second wash. Reactions were resuspended in 28.5 μl XM75 and then chased by adding 3 μl of 2 mM each of ATP, CTP, GTP, and UTP to bring each NTP concentration to 190 μM, followed by incubating at 30 °C for 5 min. Reactions that were chased at 150 mM salt were resuspended in XM150 instead of XM75 . Reactions were stopped by adding 70 μl of stop solution  and incubated at 30 °C for 5 min. For the experiments that were supplemented with TBP , 9 μl of HeLa cell nuclear extract (prepared as described ), and 25escribed ), reactiescribed  before uAll data that support this study are provided in the article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.