ZBTB7A gene has been implicated in fetal hemoglobin silencing, though has a wide range of functions that have not been fully clarified. We thus established the LRF/ZBTB7A-overexpressing and ZBTB7A-knockdown K562 (human erythroleukemia cell line) clones to assess fetal vs. adult hemoglobin production pre- and post-induction. Transgenic K562 clones were further developed and studied under the influence of epigenetic chromatin regulators, such as DNA methyl transferase 3 (DNMT3) and Histone Deacetylase 1 (HDAC1), to evaluate LRF’s potential disturbance upon the aberrant epigenetic background and provide valuable information of the preferable epigenetic frame, in which LRF unfolds its action on the β-type globin’s expression. The ChIP-seq analysis demonstrated that LRF binds to γ-globin genes (HBG2/1) and apparently associates BCL11A for their silencing, but also during erythropoiesis induction, LRF binds the BGLT3 gene, promoting BGLT3-lncRNA production through the γ-δ intergenic region of β-type globin’s locus, triggering the transcriptional events from γ- to β-globin switch. Our findings are supported by an up-to-date looping model, which highlights chromatin alterations during erythropoiesis at late stages of gestation, to establish an “open” chromatin conformation across the γ-δ intergenic region and accomplish β-globin expression and hemoglobin switch.The hemoglobin switch from fetal (HbF) to adult (HbA) has been studied intensively as an essential model for gene expression regulation, but also as a beneficial therapeutic approach for β-hemoglobinopathies, towards the objective of reactivating HbF. The transcription factor LRF (Leukemia/lymphoma-related), encoded from the  HBB locus”) are designated as β-hemoglobinopathies and are mainly inherited by the autosomal recessive pattern. Of the most common genetic disorders among the human population, related to the “HBB locus”, are β-thalassemias and Sickle Cell Anemia (SCA). Clinical practice has shown that despite identical disease-causing mutations, the pathological phenotype of disease may vary significantly between individuals. This is partly due to genetic variants of the “HBB locus” including the remote regulatory region (LCR-Locus Control Region) [HBB locus” [Perturbations in production and operation of the human β-type globin genes (“ Region)  and epig Region) ,3,4. DNAB locus” ,6,7,8,9.Therapeutic schemes aiming to reactivate fetal hemoglobin (HbF) based in pharmacological factors’ administration have been applied to the clinic for the treatment of β-hemoglobinopathies with dubious results, because of the moderate and heterogeneous HbF expression levels produced, the lack of specificity, and the observed cytotoxicity and mutagenicity in the long run ,11. BeneZBTB7A) transcription factor has been characterized as a critical key regulator of HbF in erythroid cells, acting independently from the main repressor, BCL11A (B Cell CLL/Lymphoma 11A) [ZBTB7A-knockout HUDEP-2 cells  exhibit a significantly high γ/β globin ratio [The leukemia/lymphoma-related LRF/ZBTBA transcroma 11A) ,14, and HBB locus”.The present study aimed to determine the influence of LRF to the balance between γ/β-globin production at both LRF-overexpression or LRF-knockout conditions. We thus established stably overexpressing (OE) LRF and LRF-knockdown (KD) K562 human erythroleukemic clones, which were studied before and during response to hemoglobin induction with chemical treatment . K562 ceZBTB7A expression, towards the objective of unraveling the function of the transcription factor LRF, encoded from the ZBTB7A gene, known to act during hemoglobin transversion by inhibiting γ-globin (HBG2/1) genes’ expression [ZBTB7A-GFP for longer than 60 generations during continuous culture of K562 cells in the presence of (400 μg/mL) G418 antibiotic (ZBTB7A knockdown (KD) K562 clones were obtained by a CRISPR/Cas9 system guided to target the first protein coding exon of ZBTB7A, with sequence specific gRNAs . Recombination between donor DNA carrying the Puromycin-GFP cassette and the first exon of the ZBTB7A sequence led to genetically engineered K562 cell clones surviving in the presence of 6 μg/mL Puromycin. Sequencing analysis downstream and upstream from predicted insertion sites revealed heterozygous knockout K562 clones and the insertion of the Puromycin-GFP cassette in exogenous regions as well, which were anticipated due to the high genetic instability nature of the K562 karyotype [We have developed an in vitro study system composed of human transgenic K562 cell clones, either overexpressing or diminishing LRF/pression . LRF-ovetibiotic A. ZBTB7Aaryotype . LRF expZBTB7A-KD K562 clones, were subsequently treated with hemin/erythropoietin (EPO) to urge cell differentiation and hemoglobin production. K562 cell line behaves like undifferentiated early pluripotent hematopoietic progenitors and upon chemical induction with hemin/EPO differentiate towards an erythroid character [HBG2/1) expression still dominates, whereas adult (HbA) hemoglobin production (HBB expression) is totally suppressed [Untransfected and transfected either long-term LRF-OE or haracter . Furtherppressed .HBG2/1 expression was increased by at most 2.7-fold in untransfected and 2.2-fold in LRF-overexpressing K562 clones. HBB expression was elevated at much higher levels. by 10.9-fold in untransfected and 4.5-fold in LRF-overexpressing K562 clones (p < 0.01). Contrariwise, ZBTB7A-KD K562 clones upon hemin/EPO treatment showed 3-fold elevation in HBG2/1 and an insignificant change in HBB expression levels  domain, an ATRX-DNMT3-DNMT3L-type zinc finger (ADD) domain, whereas the smaller C-terminal part, is common for all DNMTs and harbors the active center of the enzyme, which contains six highly conserved cytosine C5-DNA methyltransferase motifs [In an effort to evaluate LRF/e motifs ,22. Due HBG2/1 and HBB expression were estimated in an attempt to study the hemoglobin expression profile during aberrant methylation across the genome.Transfected K562 clones were constantly cultivated for 2 months and studied under the prevailing frame of epigenetic instability, stimulated by the elevated expression levels of DNMT3 and HDAC1. DNMT3A and HDAC1 were upregulated by 6.9- and 4.6-fold respectively E. DNMT3AZBTB7A CpG island 326 has been indicated as a delicate sensor of DNA methylation and associated with the impaired pharmacological induction of HbF upon hydroxyurea treatment of β-hemoglobinopathies patients [In previous studies, patients . Additiopatients . Both geZBTB7A was significantly hypermethylated at positions CpG1, CpG2, CpG5, CpG11, CpG15, CpG19 and CpG22 . To confirm the reliability of our results we have downloaded and compared a relevant ChIP-seq dataset from the Sequence Read Archive (SRA) (Accession number SRR6006200). To identify differentially LRF-enriched regions between experimental conditions and correlate our results to corresponding ChIP-seq data created by other research groups, read normalization was conducted to mitigate technical variance.HBB locus” LRF occupied fetal (HBG2/1) and BGLT3 genes with significant enrichment at post-erythropoiesis condition in untransfected and more evident in LRF-overexpressing K562 clones. Across βLCR, LRF signals were enriched at HS2 and HS4 (DNAse hypersensitive sites) in untransfected and at HS2, HS3 and HS4 in LRF-OE K562 cells, both during hemin/EPO induction. LRF-OE pre-hemin/EPO induction failed to highlight any LRF’s connection to βLCR. LRF-ChIP binding signals were extended to the adult HBB gene at post-erythropoiesis in untransfected and in both LRF-overexpressing clones (pre- and post-hemin/EPO induction). Observed LRF occupancy sites at HBG2/1 were adjacent to BCL11A [HBG2/1 genes. Moreover, LRF can potentially master BGLT3 gene’s up-regulation and as a consequence the enhancement of HBB expression in adults.LRF was shown to be broadly distributed across the genome with a high rate of enriched regions, further increased upon erythropoiesis (hemin/EPO induction) A. Lengtho BCL11A , which cBGLT3 expression during experimental conditions studied, we carried out qPCRs with sequence specific primer sets. BGLT3-lncRNA expression was evident only in hemin/EPO induced K562 clones  within LCR further stimulate the epigenetic events responsible for the activation of the downstream HBB promoter  to adult (HbA), and mainly the role of LRF/pression A,B. Evenpromoter D. The coHBG2/1 genes’ promoters. Apart from BCL11A, the γ-δ putative intergenic region upstream from HBD, encompassing the BGLT3 gene, has been strongly implicated to HBG2/1 silencing. This intergenic region is transcriptionally silent during the fetal stage and is also associated with high HbF levels (HPFH syndrome) in naturally occurring deletions detected in β-hemoglobinopathies patients, implying a strong potential of BGLT3-lncRNA expression to HBB induction [BGLT3-lncRNA production is induced by imatinib treatment in Bcr-Abl  positive K562 erythroleukemic cells and leads to apoptosis. Study by Guo et al., 2018, concluded that Bcr-Abl represents a BGLT3-lncRNA repressor probably acting through DNA hypermethylation of the region, leading to transcriptional silencing [BGLT3-lncRNA transcript elevation during hemoglobin switch reflects several other epigenetic modifications, additional to the already known pleiotropic activities of lncRNAs, such as chromatin remodeling, protein guidance to genomic loci via RNA-DNA interactions, transcription and RNA splicing [HBG2/1 is a perquisite for HbF repression, but to accomplish full repress of HBG2/1 genes, LRF/ZBTB7A occupancy is required, too. Simultaneous binding of LRF at HBG2/1 and BGLT3 advances BCL11A’s suppression efficiency to HBG2/1 genes and the accessible conformation of downstream chromatin up to HBB by BGLT3-lncRNA expression enhancement  is replaced by adult (HbA), a process known to be orchestrated by the BCL11A repressor function to nduction ,28. Addiilencing . Howeversplicing ,31,32. Wancement . Resultspression D, despitd status A, suggesZBTB7A and a reversal of the fetal globin genes’ expression [ZBTB7A, are the KLF1 and GATA-1 binding sites, driving a favorable generation of a longer LRF/ZBTB7A transcript, comprising a 5′ prime-end extension and exclusively expressed during late erythroid differentiation [ZBTB7A gene lacks KLF-1 and GATA-1 binding capacity, the enhancement upon hemin/EPO treatment derives only from the endogenous ZBTB7A gene, which explains the analogous imprinted results from LRF-overexpressing and untransfected K562.Obstructed KLF1 function has been also associated with reduced levels of the γ-globin repressors, BCL11A and LRF/pression ,34,35. WHBB locus, which is composed of almost 300 kb [HBB locus, four MAR elements are predicted [HBE, HBG2 and HBG1 and finally HBB promoters to LCR, leading to a dominant expression of a different hemoglobin at each stage. Looping models between LCR and globin genes’ promoters have long been proposed and further expanded under new enlightened data derived from current techniques [So far, active β-type globin promoters are suggested to form 4D chromatin configurations, such as loops with βLCR tissue-specific enhancer and engagement of transcription factors, accomplishing operative networks to promote spatiotemporal transcriptional programs during distinct developmental stages. Genes in general tend to restrict their contacts with enhancers within the same TAD  , whereast 300 kb . Lengthwredicted  and suggchniques ,46.ΖΒΤΒ7A as an indirect epigenetic modifier factor with an evolving role in lncRNAs’ regulation of expression. Further evidence for LRF’s regulatory properties on lncRNAs is established from its designated occupancy sites at 5′ prime-ends of DANCR and NEAT1 lncRNA genes, suggested to have an involvement in erythropoiesis [The proposed looping model derived from the present study  is in coopoiesis ,48, whicZBTB7A has an emerging role as an indirect chromatin modulator during the HbF to HbA switch. The “HBB locus”, including the tissue-specific LCR enhancer, is highly enriched with LRF/ZBTB7A recognition sites, which vary in potency during erythropoiesis and are not affected by variations in genome methylation levels. LRF certainly binds and potentially co-silences HBG2/1 with the BCL11A transcription factor, but also enables BGLT3-lncRNA and apparently other erythroid specific lncRNAs’ expression, triggering HbF to HbA transversion in the adult stage of erythropoiesis.Conclusively, it is apparent that LRF/2. They were passaged every 2 days. Erythroid differentiation was induced by treatment with 30 μΜ hemin  and 5 ng/mL erythropoietin (EPO)  for 3 h at 37 °C and 5% CO2 [® 3000  according to manufacturer’s protocol, and transgenic clones were selected by 800 μg/mL of Geneticin (G418)  or 6 μg/mL puromycin for CRISPR/Cas9. Long-term expression of the reporter gene eGFP in transfected cells was detected by fluorescence microscopy , confirming exogenous genes’ sustainable existence.Human K562 erythroleukemia cells were cultured in Dulbecco’s Modified Eagle Medium , supplemented with 10% fetal bovine serum , 100 units/mL penicillin and 100 μg/mL streptomycin  in a humidified atmosphere at 37 °C and 5% COd 5% CO2 ,50. Celld 5% CO2 . K562 trZBTB7A (1759 bp), DNMT3A (871 bp) and HDAC1 (1489 bp) were synthesized by IDT . LRF/ZBTB7A and HDAC1 encompassed the protein coding transcripts NM_015898.4, and NM_004964.3, respectively. To minimize the length of the DNMT3A gene and favor its insertion in the vector, only the enzymatic center of the coding sequence was synthesized. The LRF/ZBTB7A sequence was ligated into the BspEI restriction site of pEPI-1 episomal expression vector, immediately downstream of the eGFP reporter gene. pEPI-1 includes a functional S/MAR element resulting in successful episomal replication and prolonged retention of the exogenous genes in the nucleus, despite continuous cell mitosis [ZBTB7A-eGFP construct, the eGFP (700 bp) was deleted with double digestion (AgeI and BspEI), followed by Klenow treatment to form blunt ends, which were re-ligated with T4 DNA ligase . HDAC1 and the catalytic domain of DNMT3 were inserted into the episomal vector (with abolished eGFP) as BglII-AseI fragments at relevant sites of the vector. All exogenous genes were flanked by the Kozak consensus sequence (GCCACC) upstream first translation codon and two stop codons at the end of coding sequence  were designed targeting the second exon of LRF/ZBTB7A transcript (NM_015898.4). The donor DNA consisted of a left and a right homologous arm (LHA and RHA) flanking a functional cassette, encoding for the green- fluorescent reporter protein (tGFP) and the puromycin resistance gene  /Cas9 gene editing technology was used through the homology-directed repair mechanism to knockout the LRF/MD, USA) C.−ΔΔCT method or Pfaffl equation with PCR efficiency correction was used for data analysis [GAPDH reference gene and relative to the calibrator sample (untransfected K562).Total RNA was extracted from cultivated (untransfected and transfected) K562 clones using the RNeasy Mini kit  according to the manufacturer’s instructions. 1 μg of total RNA was subjected to cDNA synthesis with the Quantitect Reverse transcription kit . Gene’s expression analysis was performed with specific primers sets  and qPCRanalysis . Fold ch7 cells from studied experimental conditions were cross-linked with 1% formaldehyde. After quenching and cell lysis, chromatin was sonicated with a LABSONIC M sonicator  to generate 150–300 bp DNA fragments. The polyclonal rabbit anti-ZBTB7A antibody (abcam) was utilized in immunoprecipitation reactions and the rabbit serum IgG fraction  as negative control. After reverse crosslinking at 65 °C, immunoprecipitated DNA was recovered/purified via phenol/chloroform extraction and ethanol precipitation. Linear acrylamide  was used for pellet visualization and for increasing the precipitation efficiency.ChIP assay was performed using Protein A/G PLUS Agarose  according to the Santa Cruz Biotechnology protocol. 2 × 10Library preparation from ChIP-isolated DNA was performed using the Ion Xpress™ Plus Fragment Kit and the Ion Xpress™ Barcode Adapters 1–16 Kit . The protocol for 200 bp read-length libraries and for 50–100 ng input DNA was employed with some modifications. Due to the small amount of input DNA (10 ng), and to avoid adapter concatamerization, 1 μL of P1 adapter and IonXpress™ Barcodes was used instead of 2 μL. Adapter ligation and dual bead size selection for 200 bp read length fragments was performed using AMPure XP beads, followed by separation on magnetic rack, two ethanol washes and a final elution of DNA fragments in 25 μL Low TE. The size selected libraries were then amplified for 16 cycles. The quality was assessed on the Bioanalyzer 2100  using a High Sensitivity DNA ChIP , and quantified via qPCR using the KAPA Library Quantification Kit for Ion Torrent™ . TemplatHBB locus that can mask LRF/ZBTB7A occupancy. All datasets were visualised in IGV v2.12.2. For peak calling, only the non-redundant alignments from each BAM dataset were used  and were compared to the negative (rabbit) IgG control via MACS2 callpeak with the following parameters—gsize ‘2,700,000,000’—p value ‘0.05’—bw ‘190’ [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE200135 accessed on 24 May 2022).For downstream analysis, the sequencing data were uploaded to the Galaxy web platform through the usegalaxy.eu public server . The datbw ‘190’ . The Int® Bisulfite Kit  and bisulfite converted DNA was then used as a template for amplification of regions of interest with PyroMark® PCR Kit . PCR products (biotinylated strand) were immobilized with streptavidin-coated sepharose beads  and annealed with a Pyrosequencing primer. Reactions were performed in the PyroMark Q24 instrument using PyroMark® Gold Q24 Reagents , according to the manufacturer’s instructions. Primer sets are listed at DNA methylation was assessed at genomic DNA extracted from K562 cultivated cells using phenol: chloroform: isoamyl acid in 25:24:1 ratio , according to standard protocols . 1.5 μg p values < 0.05 were considered statistically significant.SPSS software version 20.0 was used for data analysis via one-way ANOVA with Bonferroni or Tukey’s multiple comparisons post hoc tests. Results are presented as mean ± SE of at least two biological replicates. Normal distribution of data was assessed by the Shapiro-Wilk test. The


In the present study, cortisol treatment in yellowtail clownfish (Amphiprion clarkii) showed that the expression of kiss (kiss1 and kiss2) and gr (gr1 and gr2) genes were increased significantly. We demonstrated that the yellowtail clownfish Kiss neurons co-express the glucocorticoid receptors in the telencephalon, mesencephalon, cerebellum, and hypothalamus. We further cloned the promoter of kiss2 gene in yellowtail clownfish and identified the presence of putative binding sites for glucocorticoid receptors, estrogen receptors, androgen receptors, progesterone receptors, AP1, and C/EBP. Applying transient transfection in HEK293T cells of the yellowtail clownfish kiss2 promoter, cortisol (dexamethasone) treatment was shown to enhance the promoter activities of the yellowtail clownfish kiss2 gene in the presence of GRs. Deletion analysis of kiss2 promoter indicated that cortisol-induced promoter activities were located between position −660 and −433 with GR1, and −912 and −775 with GR2, respectively. Finally, point mutation studies on the kiss2 promoter showed that cortisol-stimulated promoter activity was mediated by one GRE site located at position −573 in the presence of GR1 and by each GRE site located at position −883, −860, −851, and −843 in the presence of GR2. Results of the present study provide novel evidence that cortisol could regulate the transcription of kiss2 gene in the yellowtail clownfish via GRE-dependent GR pathway.Kisspeptin plays a vital role in mediating the stress-induced reproductive regulation. Cortisol, known as a stress-related hormone, is involved in gonadal development and sexual differentiation by binding with glucocorticoid receptor (GR) to regulate the expression of  Kiss1/Gpr54 in mice has been shown to prevent sexual maturity, cause gonad hypoplasia, hypogonadotropic hypogonadism, and infertility  in vertebrates , 2. Kissertility , 4. Moreertility . Especiaertility , 8.kiss1 and the activity of kisspeptin neuron   . Howeverkiss genes, known as kiss1 and kiss2, are found in some teleosts, such as zebrafish and medaka (Amphiprion clarkii) is a protandrous teleost whose sex is associated with social status within a group, including a male–female breeding pair and some non-breeders (Epinephelus coioides), the female treated with cortisol will change to male  and dorsal part of the nucleus of the lateral recess (NRLd), which are involved in the regulation of reproductive function and environmental cues  in June of 2021. The fish, nine per group, were reared in glass tank  with continuously flowing aerated seawater at 27 ± 1°C. The photoperiod was a 12:12-h light–dark cycle, with lights turn on at 07:00 and off at 19:00. The fish were fed with commercial feed twice a day (09:00 and 18:00) and reared for a period of 1 week before experiment.All animals used in this study were conducted in accordance with the guidelines of the animal welfare of the National Committee and approval of the Institutional Animal Care and Use Committee of Hainan University (HNUAUCC-2021-00014).The cortisol  was dissolved in dimethyl sulfoxide (DMSO) and then diluted in the ratio of 10% cortisol, 40% polyethylene glycol 300 , and 50% saline (0.9%). The immature fish  were anesthetized with MS-222  and then given an intraperitoneal injection with cortisol (10 or 50 mg/g body mass) at a volume of 10 μl/g body mass. The control group was injected the same liquid but without cortisol. We collected the whole brain at 6, 12, 24, and 48 h after injection, respectively.in situ hybridization.The gonads of sexually mature yellowtail clownfish  were isolated and fixed in Bouin’s solution  after anesthesia. The gonadal tissues were embedded in paraffin and cut into 5-μm paraffin sections for indentation of gonadal development. The brain of fish was fixed in 4% paraformaldehyde fix solution  for kiss1, kiss2, gr1, and gr2 were evaluated using comparative threshold cycle (Ct) method with β-actin as internal reference gene and then calculated with the formula 2−ΔΔCt . Primer sequences and primer efficiency for quantitative PCR (qPCR) are listed in a 2−ΔΔCt .in situ hybridization was performed following the manufacturer’s protocol from Advanced Cell Diagnostics (ACD). All steps demanding incubation at 40°C were achieved in the HybEZ Oven . Binding of the specific probes against kiss1 (1044931), kiss2 (1044941), gr1 (1088191), and gr2 (1088201) were detected with RNAscope@ Multiplex Fluorescent Reagent Kit v2 . Probes actb2 (1045881) and dapB (310043) were used as positive and negative controls, respectively. Images were taken by Nikon ECLIPSE Ti2 .Brain samples of female yellowtail clownfish were fixed in 4% paraformaldehyde at 4°C overnight. Samples successively were immersed in 10%, 20%, and 30% sucrose containing phosphate-buffered saline (PBS) for dehydration. Cross-sections (10 μm) were generated after being frozen on dry ice in optimal cutting temperature (OCT) compound. RNAscope kiss2 was isolated in the reference to Universal Genome Walker Kit . The gene-specific primers were designed in the exon 1 based on the sequences of yellowtail clownfish kiss2 (GenBank: MK368702.1) and are shown in kiss2 was determined by our previous result of 5′-rapid amplification of cDNA ends. Transcription factor binding sites were predicted using the online PROMO  and gene-regulation tool .Genomic DNA was extracted from yellowtail clownfish muscle using phenol-chloroform methods. The 5′-flanking region of the kiss2 was obtained from a pair of primers containing two different restriction enzyme sites, respectively, namely, KpnI and XhoI. PCR was performed with PrimeSTAR HS DNA Polymerase . The PCR products and pGL4.10 vector  were digested by KpnI and XhoI restriction endonucleases . After purification, the digested products were ligated using T4 DNA Ligase Kit . The construction of recombinant vector above, namely, pkiss2-1442, was used as template to construct a series of deletion vectors, namely, pkiss2-912, pkiss2-775, pkiss2-660, pkiss2-433, and pkiss2-335. All constructs were sequenced ensuring accuracy. The recombinant vectors were extracted with Omega Endo-Free plasmid DNA mini kit II . Primers used in here are presented in A 1,442-bp 5′-flanking region and 48-bp exon 1 of 2 and passed at least two generations prior to transfection. The healthy cells were seeded into 48-well plates, and approximately 1.5 × 105 cells/well were cultured for 12 h. Cells were then co-transfected with 0.5 μg pkiss2-1442/pkiss2-912/pkiss2-775/pkiss2-660/pkiss2-433/pkiss2-335, 0.05 μg pcDNA3.1-GR1/pcDNA3.1-GR2 (yellowtail clownfish glucocorticoid receptor expression plasmid) and 0.025 μg pRL-CMV in 250 μl Opti-MEM using EZ Trans . After transfection of 6 h, cells were treated with 10−7 M dexamethasone sodium . Luciferase activities were detected 24 h later with Dual Luciferase Kit  in GloMax Discover .HEK-293T cells  were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum at 37°C with 5% COkiss2 promoter were carried out using a series of specific primers .All data are shown as mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test in GraphPad Prism 7.0 . Results were considered significantly different when kiss1, kiss2, gr1, and gr2 in the brain of immature yellowtail clownfish. The highest kiss1 levels were detected at cortisol treatment with concentrations of 10 and 50 μg/g after 6 h , RNAscope in situ hybridization for gr1 and gr2 and kiss1/kiss2 genes were performed in areas of the telencephalon (Te), mesencephalon (Me), cerebellum (Ce), and hypothalamus (Hy). The co-expression of gr1, gr2, and kiss1 was found in the in the dorsal habenular nucleus (NHd), subdivision 2 of the medial dorsal telencephalic area (Dm2), subdivision 3 of the medial dorsal telencephalic area (Dm3), lateral posterior part of the dorsal telencephalic area (DIP), posterior portion of the dorsal telencephalon (DP), corpus of the cerebellum (CCe), and lateral part of the diffuse nucleus of the inferior lobe (NDLIl) , Dm2, Dm3, DIP, DP, CCe, optic tectum (OT), NDLIl, posterior part of glomerular nucleus (NGP), tegmentum (TEG), and periventricular nucleus of the posterior tuberculum (TPp) . In addition, several motifs for other steroid receptors were identified on the kiss2 promoter, such as five androgen response elements, three estrogen response elements, and two progesterone response elements , in combination with expression plasmids for yellowtail clownfish glucocorticoid receptor (GR1 or GR2), to analyze the transcriptional regulation of kiss2 by cortisol. Basal promoter activity was examined for the kiss2 promoter, indicating that functional promoter activity existed in the 5′-flanking region  abolished cortisol-induced promoter activity, indicating that the region from −660 to −433 is relevant with cortisol-induced promoter activity by GR1 (−573 TGTAC−569) was the key regulatory site on kiss2 promoter. Mutation of a GRE at −573 eradicated cortisol-induced promoter activity  or −1,188  did not change cortisol-induced promoter activity (kiss2 promoter activity.Using the full length of the −7 M DXMS treatment significantly upregulated the wild-type kiss2 promoter activity (kiss2 promoter to −775 bp abolished by cortisol-induced promoter activity (kiss2 promoter still responded to the cortisol treatment (kiss2 were removed in mutations of the following four GRE at −883 (−883AGGAT−879), −860 (−860TGTAC−856), −851 (−851AGTTCT−846), and −843 (−843ATCCT−839), indicating that these GRE binding sites contribute to cortisol-/GR2-induced kiss2 promoter activity  in several teleost fish participating in the reproductive regulation (kiss (kiss1 and kiss2) and gr (gr1 and gr2) genes were increased significantly in the brain of yellowtail clownfish. In addition, elevation of two kiss genes expression by cortisol has been reported in zebrafish  levels are higher in the subordinate than in the dominant yellowtail clownfish (Amphiprion ocellaris) showed that the social rank reflects the blood cortisol value , and Kiss1 antagonist injection can reduce AS-evoked fish fear response, indicating that habenular kisspeptin modulates fear in zebrafish  treatment in sea bass  and arcuate nucleus (ARC) by double-labeling immunohistochemistry in female rats . In the ebrafish . The yelal group , 26. Frosea bass . In yellkiss2 promoter in the yellowtail clownfish by silicon analysis, implying that kiss2 could be regulated by cortisol via GR through binding with GRE. In the present study, cortisol injection also enhanced the gr1 and gr2 mRNA levels in the brain. Other binding sites such as AP1, Sp1, and C/EBP were also predicted in the kiss2 promoter region. AP1 could interact with GR for GR-regulated transcription and recruitment to co-occupied AP1 binding site by DNaseI accessibility and chromatin immunoprecipitation with high-throughput sequencing   . Thus, wkiss2 gene were located between position −660 and −433 with GR1, and −912 and −775 with GR2, respectively. Point mutations in the kiss2 promoter were generated by site-directed mutagenesis. Our results showed that in the presence of GR1, cortisol-stimulated promoter activity was only mediated by one GRE site located at the position of −573, whereas in the presence of GR2, promoter activity could be modulated by all four GRE sites located at positions −883, −860, −851, and −843. Therefore, the kiss2 gene is regulated by cortisol through the GRE-dependent mechanism in yellowtail clownfish. It has also been reported that there is a synergistic action between the enhancer binding protein (C/EBP) and GR in the regulation of thymidine kinase promoter activity .S-YB planned and wrote the manuscript, participated in experiments and composed the figures. Y-YZ planned, edited, and drafted the manuscript. XZ, T-XL, D-CZ, and Z-XH participated in experiments. QW planned, edited, supervised, and reviewed the manuscript. All authors read the final article and approved its submission.This work was supported by National Natural Science Foundation of China (NSFC Grant No. 31760759).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Regulation of histone acetylation dictates patterns of gene expression and hence cell identity. Due to their clinical relevance in cancer biology, understanding how human embryonic stem cells (hESCs) regulate their genomic patterns of histone acetylation is critical, but it remains largely to be investigated. Here, we provide evidence that acetylation of histone H3 lysine-18 (H3K18ac) and lysine-27 (H3K27ac) is only partially established by p300 in stem cells, while it represents the main histone acetyltransferase (HAT) for these marks in somatic cells. Our analysis reveals that whereas p300 marginally associated with H3K18ac and H3K27ac in hESCs, it largely overlapped with these histone marks upon differentiation. Interestingly, we show that H3K18ac is found at “stemness” genes enriched in RNA polymerase III transcription factor C (TFIIIC) in hESCs, whilst lacking p300. Moreover, TFIIIC was also found in the vicinity of genes involved in neuronal biology, although devoid of H3K18ac. Our data suggest a more complex pattern of HATs responsible for histone acetylations in hESCs than previously considered, suggesting a putative role for H3K18ac and TFIIIC in regulating “stemness” genes as well as genes associated with neuronal differentiation of hESCs. The results break ground for possible new paradigms for genome acetylation in hESCs that could lead to new avenues for therapeutic intervention in cancer and developmental diseases. Epigenetic regulation of cell identity is a key issue for the use of human stem cells in regenerative medicine and cancer treatment. To date, two distinct multipotent states are generally considered: naïve human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (iPSCs). Whereas the first are derived from the inner cell mass of the blastocyst, the latter offer advantages since they are obtained by somatic cell reprogramming, though they present clinical problems due to their partially differentiated phenotype . RegulatIn order to better understand the relationship of genome-wide occupancy of p300 and its HAT activity with H3K18ac and H3K27ac during hESCs differentiation, we analyzed published ChIP-seq datasets for these factors ,12,13,14TFIIIC, and in particular its largest subunit GTF3C1, has been shown to acetylate H3K18 in vitro . Given tCHD1, KLF4, HELLS and many others  was from GSE120162.Analysis of ChIP-seq data was carried out as previously described  with minhttps://www.rstudio.com, accessed on 29 December 2022).Bed intersection was carried out using bedtools  “intersehttp://cistrome.org/db/#/, accessed on 29 December 2022).Peaks were used for calculating GIGGLE score by using “all peaks” and “chromatin and transcription factor regulation” options in toolkit analysis of Cistrome Data Browser  tool was used.https://biit.cs.ut.ee/gprofiler, accessed on 29 December 2022) [g:Profiler (er 2022)  was usedThis work brings new knowledge about the role of TFIIIC as a putative hESCs HAT for H3K18ac and thereby as a regulator of stem cells fate These results have a direct impact into creating new possibilities for clinical intervention in cancer and other developmental diseases.


Aureobasidium spp. In this study, a set of 16 superfamilies, 45 PFAM families of TFs with the DNA-binding domains, seven zinc finger families and eight categories of the C2H2 TFs have been identified in Aureobasidium spp. Among all the identified TFs, four superfamilies and six PFAM families are the fungal-specific types in this lineage. The Zn2Cys6 and fungal-specific domain regulators are found to be overwhelmingly predominated, while the C2H2 zinc finger class comprises a smaller regulator class. Since there are currently no databases that allow for easy exploration of the TFs in Aureobasidium spp., based on over 50 references and 2405 homologous TFs, the first TFs pipeline—the Aureobasidium Transcription Factor Database (ATFDB)—has been developed to accelerate the identification of metabolic regulation in various Aureobasidium species. It would be useful to investigate the mechanisms behind the wide adaptability and metabolite diversity of Aureobasidium spp.Transcription factors (TFs) can regulate the synthesis of secondary metabolites through different metabolic pathways in  Aureobasidium is a genus of ascomycete in the family Saccotheciaceae that is found worldwide in water, soil, wood, rock, saline habitats, limestone, coastal waters, deep sea, marine sediments from the Antarctic, desert, honeycomb and mangrove ecosystems ..2Cys6 doAureobasidium spp., it consists of three members: the Fungal trans (PF04082), the Cep3 (PF16846), and the Fungal trans 2 (PF04082 or PF11951). Transcription factors with this domain are involved in the metabolic flow of carbon pool and fatty acids , which has expanded more rapidly in the Ascomycota than in the Basidiomycota . In Aurety acids .Aureobasidium spp. Meanwhile, ankyrins are multifunctional adaptors that link specific proteins to the membrane-associated, spectrin-actin cytoskeleton. The results show that although there are no differences in the KilA-N domain of Swi4/6 among different fungi, in Aureobasidium spp., transcription factor Swi4/6 has a long sequence coverage of the ankyrin repeats (ANK) with at least three consecutive copies, which is obviously different from those in A. fumigatus and S. cerevisiae (The KilA-N domain (PF04383) has been verified to regulate cellular differentiation, melanin synthesis, cell growth, and development in revisiae  25]..AureobasAureobasidium strains, such as the MAT1-1-1 gene (QBQ58041.3) and the MAT1-2-1 gene (QBQ58040.3) (The HMG-box domain (PF09011), the homologues of MATalpha_HMGbox  and the MAT_HMGbox (cd01389) can be clearly identified in each of the investigated 58040.3)  (Table S58040.3)  16]..AureobasAureobasidium spp., which is equivalent to the regulator of phospholipid synthesis in S. cerevisiae. Sre1 (KEQ62711.1) and SreA (KEQ65560.1) are identified in the genome of Aureobasidium spp., of which they are transcription repressors that inhibit the biosynthesis of iron carriers in Aureobasidium spp. The bZIP proteins mainly are primarily involved with the iron acquisition regulator HapX in the synthesis of siderophore  are also classified as yeast-specific TFs . The βHLerophore .Aureobasidium spp. are identified by searching annotated genes and comprehensive results of biochemical and genetic analysis of S. cerevisiae and Aspergillus fumigatus. Generally, the homologous TFs found in Aureobasidium spp. are manually curated according to the literature of previous research , five involved in synthesis of siderophores , four involved in synthesis of polysaccharides , two involved in the glycolysis (Gcr1), and gluconeogenesis regulation (Cat8), while six general TFs are associated with RNA polymerase II transcription , and another six are involved in the signal pathway regulation, such as the HOG, cAMP-PKA, MAPK, and CWI pathways, as well as stress response pathway  and Aspergillus niger (2951). Initially, zinc fingers were categorized according to the different numbers and orders of the residues . Krishna et al. [Aureobasidium spp. as follows (Aureobasidium spp. (The Zinc finger (PFAM entries: 257) exists in various proteins and has a wide range of functions in various cellular processes . There aa et al.  previous follows . Seven tium spp. , which aium spp. .Aspergillus aculeatus ATCC16872, A. melanogenum P16, A. melanogenum CBS110374, Aureobasidium subglaciale EXF-2481, Aureobasidium pullulans EXF-150 and Aureobasidium namibiae CBS147.97, and the neighbor-joining (NJ) phylogenies for the datasets of sequences were then inferred by MEGA v7.0.26. According to the different domains and functions of the C2H2 TFs, the homologues can be divided into eight categories in different strains of Aureobasidium spp.  and biosynthesis of asper furanone expression [PKS1 gene and CMR1 gene links together and constitutes a gene cluster to catalyze melanin synthesis. The metabolic pathway-specific transcription factor Cmr1 can regulate the expression of the PKS gene in Aureobasidium spp. [Aureobasidium spp. and other melanin-producing fungi , such as the transcription repressor CreA/Mig1 (KEQ62800.1), the transcriptional activator Seb1/Msn2 KEQ58292.1), Crz1 (KEQ62890.1) and Ste12/BrlA KEQ57897.1) A. In Aur2.1, Crz1897.1 A. enum P16 . Crz1 cosphatase . PMA bio pathway ,33, and  genomes . The gloalbicans ,33. Fungalbicans . Anotheralbicans ,36. Metaalbicans ,37. Therium spp. B. ScpR/Apression . In fungium spp. . Cmr1 hang fungi . In contn factor  . This is more accurate than directly using the type strain for sequence alignment, because the genomes of type strains such as A. melanogenum CBS110374 or A. pullulans EXF-150 are the draft genomes with many genes to be incompletely assembled. In addition, the TFs in each of the fifty A. pullulans strains are chosen as the representative gene set for analysis since A. pullulans is the common species of Aureobasidium [To establish a complete gene set of TFs in the basidium ,17.Aureobasidium Transcription Factor Database has been established for TF retrieval. The database involves four sections and works on an intuitive approach: (i) users input the retrieval data; (ii) automatic word matching; (iii) query by exactly matching the database; (ⅳ) process and encapsulate the retrieved data format; (ⅴ) return the encapsulated data and render the returned data to show the results (https://huang.zgsj1.com/picture/literature/distweb/index.html (accessed on 15 April 2022), which also supports multiple inputs for users to retrieve. Meanwhile, all of the TFs can be clicked for information linking to external databases, and the KEGG entries for TFs can also be checked in the KEGG section. The user-friendly web server interface accepts user input and produces results in seconds.After submitting all the data, the  results . All theAureobasidium spp., such as the Zn2Cys6, bZIP and GATA zinc fingers, demonstrating the database’s comprehensiveness and usefulness  and counted the number of TFs contained within different categories. The results showed that in the type-level of TF classification, the NCBI (GenBank) database mostly contained general TFs (2511), but the ATFDB largely compensated for the NCBI (GenBank) database’s faults and weaknesses in the global TFs (1386) and particular TFs (697)  A (Table Fs (697) . In partFs (697) B (Table Fs (697) , suggestAureobasidium spp. can synthesize a variety of natural compounds with high-economy value and potential industrial applications, the majority of which are associated with transcription factor metabolic regulatory networks. The built ATFDB can be applied for multiple purposes. Genes involved in each pathway are comprehensively and accurately presented in ATFDB. First, it holds the potential to be further used for genomic annotation. For example, if all transcriptional regulations involved in one pathway are annotated, we can consider the mechanisms of metabolism potentials of this pathway. In addition, ATFDB may be used for analyzing amplicon sequencing data of functional genes to separate homologous groups. In summary, this study presents a manually constructed database (ATFDB) that aims to profile transcription factor metabolic regulatory networks. It is publicly available without login requirements at https://huang.zgsj1.com/picture/literature/distweb/index.html (accessed on 14 April 2022). The ATFDB contains 16 superfamilies, 45 PFAM families of TFs with DNA-binding domains, 7 zinc finger families, and 8 categories of the C2H2-type TFs. The results demonstrate that ATFDB is a useful tool for studying the transcriptional regulations of Aureobasidium spp. in the environment, and it will be continuously updated.The lineage of


Drosophila, and analyzed them for for the presence and location of 17 known consensus promoter sequences. As a control, we also searched all Drosophila RNA polymerase II-dependent promoters for the same sequences. We found that promoter motifs were readily detected in the MEF2 target dataset, and that many of them were slightly enriched in frequency compared to the control dataset. A prominent sequence over-represented in the MEF2 target genes was NDM2, that appeared in over 50% of MEF2 target genes and was 2.5-fold over-represented in MEF2 targets compared to background. To test the functional significance of NDM2, we identified two promoters containing a single copy of NDM2 plus an upstream MEF2 site, and tested the activity of these promoters in vivo. Both the sticks and stones and Kahuli fragments showed strong skeletal myoblast-specific expression of a lacZ reporter in embryos. However, the timing and level of reporter expression was unaffected when the NDM2 site in either element was mutated. These studies identify variations in promoter architecture for a set of regulated genes compared to all RNA polymerase II-dependent genes, and underline the potential redundancy in the activities of some core promoter elements.To gain understanding into the mechanisms of transcriptional activation of muscle genes, we sought to determine if genes targeted by the myogenic transcription factor Myocyte enhancer factor-2 (MEF2) were enriched for specific core promoter elements. We identified 330 known MEF2 target promoters in  Transcription factors function to influence the accessibility of the core promoter to the transcriptional machinery. Core promoters are characterized by the presence of one or a few of several conserved sequence elements, which in eukaryotes vary in identity depending upon the class of RNA polymerase that transcribes the genes, the location of the sequence relative to the transcription start site, and whether the sequence is restricted to one or both strands of the promoter. For RNA polymerase II-dependent genes, classical studies have underlined the existence and functional significance of promoter elements including the TATA box and downstream promoter element (DPE), each of which, when present, are usually essential for transcription to initiate at another essential consensus sequence, the Initiator (Inr)(reviewed in ).Drosophila genes, including those described above as well as novel motifs, several of which were subsequently shown to interact with core transcriptional machinery . Additi example  and Down example . While tGiven the wide variability of promoter architectures even within RNA polymerase II-dependent genes, an open question had been whether the promoters of genes that are regulated by a certain transcription factor or class of factors are distinct relative to other genes. There is notable evidence for this being the case: of the 15 motifs identified by Fitzgerald et al , most shDrosophila has a single gene, Mef2, that is essential for muscle differentiation [Drosophila MEF2 was shown to bind to the promoters or enhancers of several myofibrillar protein genes [Drosophila MEF2 were identified using chromatin immunoprecipitation-microarray (ChIP-chip) by Sandmann et al [To gain further insight into how muscle gene expression is regulated, we sought to determine if targets of the myogenic regulatory factor Myocyte enhancer factor-2 (MEF2) showed characteristic promoter structures. MEF2 was initially characterized as a nuclear factor interacting with an AT-rich sequence in muscle gene promoters and enhancers , and subntiation , 11. Morin genes , 13. Gennn et al , which csticks and stones (sns) and Kahuli (Kah) that contain a single iteration of the NDM2 sequence, and determined the functional significance of the element by analyzing the activities of wild-type and NDM2-mutant promoter-lacZ reporter constructs in transgenic animals. Both of the control promoter elements showed strong skeletal muscle-specific activity, and activity was not affected when the NDM2 site in either promoter was mutated. Our studies test for the first time the functional importance of this conserved promoter sequence, and its apparent dispensability emphasizes the occurrence of redundant elements within eukaryotic promoters.We used this collection of 169 MEF2 target genes to determine if their promoter architecture showed unique enrichment for core promoter elements. We note that transcription factors are known to bind to both distant enhancers and proximal promoter sequences, and promoters themselves are often minimally active in the absence of enhancer activity. Here we consider the totality of high-confidence MEF2 targets, without consideration of the proximity of the MEF2 sites to the promoter. We found that several canonical promoter sequences were mildly enriched in MEF2 targets compared to a background collection of all RNA polymerase II-dependent genes. More striking was the prominent occurrence in over 40% of MEF2-regulated genes of an element named NDM2. We selected two MEF2 target genes, Drosophila DPE [We searched for consensus promoter sequences in MEF2 target genes and in a control set of genes. Consensus sequences were the 15 sequences identified by Fitzgerald et al , plus thDrosophila melanogaster genome sequences (version BDGP6.22), downloaded from the FTP site of Ensemble release 98 (Sept. 2019) [Direct MEF2 target genes were derived from Sandmann et al : rather t. 2019) . AnnotatFor both MEF2-target and background control promoters, we obtained for analysis sequences corresponding to -200 to +200 relative to the transcription start site, using in-house Perl scripts. Sequences of the MEF2 target promoters are provided in The consensus sequences of the 17 promoter motifs indicated above were scanned across promoter regions of both MEF2 target and background target genes, using Regulatory Sequence Analysis Tools .Graphs summarizing the distributions of identified promoter motifs relative to the transcription start sites (bin size = 5 bp) were generated using R package ggplot2 version 3.3.5 .sns and Kah genes were generated using GeneBlocks (IDT). Sequences corresponding to -1000 to +100 relative to the transcription start site were used for sns (2R 8797489–8798588), and -640 to +100 were used for Kah (3L 589914–589175). Coordinates correspond to Release 6 of the Drosophila genome. Promoter elements for both wild-type and mutant sequence were generated as dsDNA, and had engineered restriction sites at either end to facilitate cloning: NotI at the 5’ end and PstI at the 3’ end. DNA supplied by the manufacturer was digested with these enzymes (New England Biolabs), and ligated with placZ-attB [NotI and PstI removed the minimal heat shock promoter and allowed replacement with the promoter from the gene under investigation. Positive clones were confirmed by sequencing before preparing for injection to generate transgenic animals. The NDM2 site  was mutated from 5’-cgatgccg to 5’-cCCGgGcg for sns, and from 5’-ggacgccg to 5’-ggCcCGGg for Kah. Uppercase letters indicates nucleotides changed relative to wild-type. The 5’ nucleotide of the NDM2 sites listed above correspond to nucleotides -77 for sns and -78 for Kah relative to their respective transcriptional start sites.Wild-type and mutant promoter fragments from the acZ-attB  also cutDrosophila were maintained at 25°C and raised on Jazz Food (Genesee Scientific). Transgenic lines carrying the promoter-lacZ fusion constructs were generated by injecting the constructs described above into the third chromosome Phi C31 Integrase site M{3xP3-RFP.attP}ZH-86Fb [}ZH-86Fb  present Harvesting and staining of embryos was carried out essentially as described by Patel . The priDrosophila promoters. We searched for the presence of each of the 15 motifs identified by Fitzgerald et al (2006), plus the Ohler motif 10  on background genes, we observed enrichment of each motif in the background set of genes at their appropriate locations relative to the transcriptional start site and on the coding strand but not the non-coding strand . In general, these motifs were observed at lower frequencies in the background dataset  and Kahuli (Kah). The sns gene encodes a transmembrane protein expressed in fusion-competent myoblasts that is essential for myoblast fusion [sns expression is still detected in Mef2 mutant embryos [sns at multiple stages of embryonic development [Kah encodes a relatively uncharacterized DNA binding protein expressed in skeletal myoblasts [lacZ reporter and generated transgenic lines carrying the constructs.While NDM1 is thought to interact with GAGA factor, the role of NDM2 in gene expression has not been investigated. NDM2 was observed in the -100 to -50 region of 74 of 169 tested genes (44%), with 38 of these genes containing a single predicted NDM2 site. Given this enrichment for NDM2 sites in MEF2-regulated genes, we hypothesized that activation of these promoters by MEF2 might be dependent upon the NMD2 motif, and tested if NDM2 was required for activity of t fusion . While s embryos , we stilelopment . Kah encyoblasts . For eacsns-lacZ and Kah-lacZ wild-type constructs, we observed robust myoblast-specific reporter expression  and NDM1/GAGA (-75 to -125), each of which are bound by factors other than the holoenzyme , 32. FutS1 File(FASTA)Click here for additional data file.


The mechanisms of CFS formation have been linked to DNA replication timing control, transcription activities, as well as chromatin organization. However, it is unclear what specific cis- or trans-factors regulate the interplay between replication and transcription that determine CFS formation. We recently reported genome-wide mapping of DNA DSBs under replication stress induced by aphidicolin in human lymphoblastoids for the first time. Here, we systematically compared these DSBs with regards to nearby epigenomic features mapped in the same cell line from published studies. We demonstrate that aphidicolin-induced DSBs are strongly correlated with histone 3 lysine 36 trimethylation, a marker for active transcription. We further demonstrate that this DSB signature is a composite effect by the dual treatment of aphidicolin and its solvent, dimethylsulfoxide, the latter of which potently induces transcription on its own. We also present complementing evidence for the association between DSBs and 3D chromosome architectural domains with high density gene cluster and active transcription. Additionally, we show that while DSBs were detected at all but one of the fourteen finely mapped CFSs, they were not enriched in the CFS core sequences and rather demarcated the CFS core region. Related to this point, DSB density was not higher in large genes of greater than 300 kb, contrary to reported enrichment of CFS sites at these large genes. Finally, replication timing analyses demonstrate that the CFS core region contain initiation events, suggesting that altered replication dynamics are responsible for CFS formation in relatively higher level of replication stress.Common fragile sites (CFSs) are specific regions of all individuals’ genome that are predisposed to DNA double strand breaks (DSBs) and undergo subsequent rearrangements. CFS formation can be induced  CFSs are genomic regions that are prone to DNA strand breakage, observable as gaps or other abnormalities on the metaphase chromosomes. The manifestation, or expression, of CFSs is induced by mild level of DNA replication stress such as DNA polymerase inhibition or nucleotide pool limitation, as reviewed in . There aCFSs are an intrinsic feature of the human genome and are hot spots for large scale amplification, deletion, and rearrangements, which are thought to underlie genome instability that are prevalent in cancer as well as neurological disorders . TherefoWe systematically examined the relationship between DSBs and select key genomic features mapped by published studies, the majority of which were curated by the Encyclopedia of DNA Elements (ENCODE) project. We specifically focused on data sets generated from the same cell line (GM06990) as our data were, to minimize confounding genetic factors. These data sets included histone modification sites—specifically histone 3 lysine 4 trimethylation (H3K4me3), H3K36me3, and H3K27me3—DNaseI hypersensitive sites (DNaseI HSS), and topologically associated domain (TAD) architectural protein CTCF binding sites. Each of these elements has been implicated in replication and/or transcription regulation . Of noteDNaseI HSS_1: bigWighttps://www.encodeproject.org/files/ENCFF529JFV/@@download/ENCFF529JFV.bigWig.DNaseIHSS2:bigWigDNaseI HSS_1: bigWighttps://www.encodeproject.org/files/ENCFF709PEX/@@download/ENCFF709PEX.bigWigDNaseIHSS_1: bigBed narrowPeakhttps://www.encodeproject.org/files/ENCFF735IVN/@@download/ENCFF735IVN.bigBedDNaseIHSS_2: bigBed narrowPeakhttps://www.encodeproject.org/files/ENCFF043DMN/@@download/ENCFF043DMN.bigBedCTCF ChIP-seq: bigWighttps://www.encodeproject.org/files/ENCFF469OOI/@@download/ENCFF469OOI.bigWigCTCF ChIP-seq: bed narrowPeakhttps://www.encodeproject.org/files/ENCFF276JDQ/@@download/ENCFF276JDQ.bed.gzH3K4me3 ChIP-seq: bigWighttps://www.encodeproject.org/files/ENCFF965GIX/@@download/ENCFF965GIX.bigWigH3K4me3 ChIP-seq: bed narrowPeakhttps://www.encodeproject.org/files/ENCFF357ALO/@@download/ENCFF357ALO.bed.gzH3K27me3 ChIP-seq: bigWighttps://www.encodeproject.org/files/ENCFF533NLA/@@download/ENCFF533NLA.bigWigH3K27me3 ChIP-seq: bed narrowPeakhttps://www.encodeproject.org/files/ENCFF554UCC/@@download/ENCFF554UCC.bed.gzH3K36me3 ChIP-seq: bigWighttps://www.encodeproject.org/files/ENCFF324YFT/@@download/ENCFF324YFT.bigWigH3K36me3 ChIP-seq: bed narrowPeakhttps://www.encodeproject.org/files/ENCFF372NOF/@@download/ENCFF372NOF.bed.gzBubble-seq origins of replication data (GSE38809_GM_combined_RD_bubbles.bedgraph) were downloaded from GEO accession number GSE38809.The commands used for the following search engines are listed below.AME: ame--verbose 1 --oc. --scoring avg--method fisher--hit-lo-fraction 0.25 --evalue-report-threshold 10.0 --control--shuffle-- --kmer 2 DSBFILE.fasta db/HUMAN/HOCOMOCOv11_core_HUMAN_mono_meme_format.memeSTREME: streme--verbosity 1 --oc. --dna--totallength 4000000 --time 14400 --minw 8 --maxw 15 --thresh 0.05 --align center--p DSBFILE.fastaTomtom: tomtom -no-ssc -oc. -verbosity 1 -min-overlap 5 -mi 1 -dist pearson -evalue -thresh 10.0 -time 300 query_motifs db/HUMAN/HOCOMOCOv11_core_HUMAN_mono_meme_format.memep value.The association between a DSB and a chromatin feature was determined by whether the two regions overlap by at least 1 bp using intersectBed function in BEDTools. Random DSB sequences were generated by shuffleBed with the same number of DSBs per chromosome. The shuffled DSBs were then compared to the chromatin feature for overlaps. The random simulation was performed 1000 times in each test. The number of simulations in which the shuffled DSBs overlapped with the chromatin feature at an equal or higher frequency than the real data was divided by the number of simulations to calculate the GP-seq scores were downloaded from the GEO database under the accession number GSE135882 for experiment 5 (GSE135882_Exp5.1 Mb). GP-seq scores in each unique DSB region were tallied, averaged, and plotted.We used the R package “randomForest” to build and train the random forest models employed in this study. We left mtry on default and used a value of 700 for ntree which represents the number of decision trees used by the model. We set importance to TRUE in order to access the MeanDecreaseAccuracy and MeanDecreaseGini values as well as the variable importance plots. The ‘pROC’ package was installed to plot the ROC (receiver operating characteristic curve) and PR  curves. We used the BigWigAverageOverBed function to calculate the feature signals over DSB regions and used the mean values as input variables. Random sampling of DSBs from the genome was accomplished using the shuffleBed function from the BEDTools suite.6 cells were used for each Repli-seq experiment. Cells were treated with DMSO, or 0.3 µM, or 0.6 µM APH, or nothing at all for 24 h. BrdU was then added at 100 µM and cells were incubated for 2 h before washing with PBS and harvesting, followed by ethanol fixation. Fixed cells were then sorted by flow cytometry into early and late-replicating fractions. BrdU-labeled DNA from each fraction was immunoprecipitated, followed by preparation for sequencing libraries as previously described (At least 2 × 10escribed . Two bioWe recently generated high resolution mapping of genome-wide DSB sites in human lymphoblastoid (GM06990) cells . We usedvs. late fraction. The result showed that 0.3 µM APH, the dosage at which the majority of our Break-seq experiments were conducted, caused S-phase arrest compared to the NT and DMSO controls  probes in fourteen molecularly characterized CFSs (p < 0.001) (p < 0.001)  associated with FRA3B and FRA16D, respectively  . Moreove< 0.001)  did not < 0.001) . We thenectively , with th, in APH . LRP2 ated genes . Thus, dInterestingly, both the Break-seq and BLESS-derived APH-induced DSBs tend to demarcate the boundaries of the mapped CFSs or CFS cores . The relA recent study has shown that APH-induced CFSs correspond to TAD boundaries that are significantly delayed in replication timing . TAD bouy-axis scale on each plot). It is important to note that the association between CTCF binding sites and DSBs was not statistically significant when compared to randomized DSBs, suggesting that the comparison of DSBs to CTCF binding sites alone might not be sufficient to discern the relationship between DSBs and epigenomic features.We took advantage of the ENCODE project which cataloged a large set of genome-scale experiments of mapping chromatin structures. We specifically focused on those data generated from GM06990, the same cell line used in our Break-seq mapping experiments. We systematically analyzed the distribution of DSBs over each of the chromatin features including TSS, CTCF binding sites, DNaseI HSSs, H3K4me3, H3K27me3, H3K36me3, and origins of replication , whose gp < 0.001). Because H3K36me3 is associated with active transcription and is enriched in the gene bodies downstream from TSS . We found 24, 187, and 138 such DSBs in the untreated, DMSO-, and APH-treated samples, respectively. We first asked if there was any chromosomal bias of the DSBs. Remarkably, the chromosome with the highest enrichment of DSBs in all conditions was Chr19. While spontaneous DSBs were only enriched on Chr19, drug-induced DSBs also showed enrichment on Chr5, 17, and 22 for DMSO and Chr15, 17, and 22 for APH treatment. These chromosomes all tend to be located near the radial center of the nucleus based on an elegant study using Genome Positioning (GP)-seq to analyze the 3D chromosome positioning . Among t nucleus . Indeed, nucleus . Girelli nucleus . It has  nucleus . TherefoZNF582, SMARCA5, ZNF770, STAT5B, ZNF121, PAX5, STAT5A, PRDM6, TAF1, and ZNF418 (p < 5.59e-42). The top ten motifs enriched in APH only DSBs are binding sites for ETS2, EGR2, EGR1, NFATC1, ETV5, LEF1, TBX21, ZNF341, BCL11A, and ZNF121 (p < 5.16e-124). Importantly, CTCF binding motif was also found as enriched in APH only DSBs (p = 5.74e-5). Many of the proteins above  have been implicated in chromatin modifications and remodeling. Others have been implicated in the maintenance of 3D chromatin architecture. For instance, ZNF770 and ZNF121 are significantly enriched at “insulator loops” mediated by CTCF to protect genes from emanating potentially harmful signals  identif signals . PAX5, a signals . Yet oth signals . We also signals  to disco signals   using feature signals over DSBs . Motivated by Mourad et al.'s use of a machine learning method, Random Forest, to distinguish spontaneous DSBs from randomly sampled genomic regions, we developed a similar pipeline to validate features for replication stress-induced DSBs . To best project . We thenver DSBs , and witver DSBs . By far ver DSBs . This rever DSBs , indicatver DSBs . The besver DSBs . Among tIn this report, we detail the computational analysis of spontaneous and replication stress-induced DSBs in GM06990 lymphoblastoids, which were recently mapped by Break-seq . We focuThe first key finding was that DSBs were closely associated with active transcription histone marker H3K36me3 and TAD boundaries. Specifically, spontaneous DSBs are associated with CTCF binding sites. In contrast, we observed a correlation between DMSO-induced DSBs and markers associated with active transcription, including TSSs, H3K36me3 and CTCF binding sites. Upon APH treatment, there was a dampening of the transcriptional response as evidenced by a decrease in the levels of all markers above, as well as a decrease of DSBs in genic regions compared to DMSO treatment. Yet, the APH-induced DSBs showed strong association with H3K36me3, suggesting that despite an overall dampening of transcription by APH DSBs nevertheless took place at actively transcribing regions of the genome.The second key finding related to the first one was that APH-induced DSBs in our study as well as the BLESS study  were notOverall, these findings led us to hypothesize that DSBs were the result of CTCF-mediated chromosome remodeling due to transcription. We propose the following model to explain spontaneous and drug-induced DSB formation, specifically at the large genes where CFSs tend to reside . In untrIn summary, our study provided a comprehensive overview of genomic and epigenomic features associated with replication stress-induced DSBs. It also laid out a framework for future studies to expand DSB mapping to more well-chosen cell lines and with simultaneous queries for active origins of replication and epigenomic features. Such an experimental design will promise to deliver important insights into the mechanisms of replication stress-induced DSB formation.


In eukaryotes, the heat shock response is orchestrated by a transcription factor named Heat Shock Factor 1 (HSF1). HSF1 is mostly characterized for its role in activating the expression of a repertoire of protein-coding genes, including the heat shock protein (HSP) genes. Remarkably, a growing set of reports indicate that, upon heat shock, HSF1 also targets various non-coding regions of the genome. Focusing primarily on mammals, this review aims at reporting the identity of the non-coding genomic sites directly bound by HSF1, and at describing the molecular function of the long non-coding RNAs (lncRNAs) produced in response to HSF1 binding. The described non-coding genomic targets of HSF1 are pericentric Satellite DNA repeats, (sub)telomeric DNA repeats, Short Interspersed Nuclear Element (SINE) repeats, transcriptionally active enhancers and the NEAT1 gene. This diverse set of non-coding genomic sites, which already appears to be an integral part of the cellular response to stress, may only represent the first of many. Thus, the study of the evolutionary conserved heat stress response has the potential to emerge as a powerful cellular context to study lncRNAs, produced from repeated or unique DNA regions, with a regulatory function that is often well-documented but a mode of action that remains largely unknown. The cellular response mediated by Heat Shock transcription Factor 1 (HSF1) is involved in a large number of biological processes and is widespread within the eukaryotic kingdom. Initially described in the context of the cellular response to thermal stress, the involvement of HSF1 has been rapidly extended to a large number of other environmental and cellular stresses and physio-pathological contexts ,3,4. GenSATIII) emanating from pericentric heterochromatin . This non-coding response may be involved in the rapid and coordinated reshaping of protein-coding gene expression through a potentially large range of mechanisms . Indeed,hromatin A 11,12],12SATIIISATIII and TERRA transcripts expressed from DNA repeats in pericentric [SINEs) transcripts that are expressed from repetitive sequences scattered throughout the genome [eRNAs) [NEAT1.1 and NEAT1.2, expressed from the single-copy NEAT1 gene [This review focuses primarily on the known mammalian lncRNA effectors of the HSF1 response, with the aim of providing an updated view of their role and mode of action in the HSF1-dependent stress response. The first part reports and briefly describes the lncRNAs whose production is directly activated by HSF1 A,B. Thesicentric ,12, and icentric ,14, respe genome ,16, enha [eRNAs) ,9 and thAT1 gene . The secSeveral lines of evidence indicate that HSF1 directly controls expression of a set of genes not coding for proteins. These assumptions rely on computational genomic analyses allowing an identification of putative HSF1-binding sites known as Heat Shock Elements (HSE), Chromatin Immuno-Precipitation (ChIP) to evaluate the binding of HSF1 to their promoter and, finally, comparative analysis of their levels of expression in cells expressing HSF1 or not. We will provide here a short description of these lncRNAs and the findings indicating that the genes encoding these lncRNAs are directly upregulated by HSF1 .Satellite DNA sequences, present at pericentric regions, are enriched in tandem repeats organized in a “head-to-tail” orientation.satellites I, II and III. Of these, SATIII are characterized by the high frequency of the sat3 motif [In humans, the size and sequence of pericentric regions varies between chromosomes. The majority of them contain, as a major component, a single family of simple repeated satellite (SAT) sequences formed by the classical t3 motif ,20,21 . SATIII are transcribed by RNAPII into a pool of SATIII lncRNAs ranging in size from 2 Kb to 5 Kb and beyond [SATIII DNA repeats are predominantly transcribed in one orientation (enriched in the pentanucleotide repeat motif GGAAU). Interestingly, a slight increase in the level of SATIII lncRNAs in an antisense ((AUUCC)n-rich) orientation was also observed in stressed cells [SATIII lncRNAs.Human  by HSF1 ,12. In h by HSF1 ,12,36 an by HSF1  were bot by HSF1 . The totd beyond ,12. SATIed cells ,12,39, bSATIII, was also observed in a variety of human tumor tissues [In addition, different cellular contexts involving pericentric satellite lncRNAs’ accumulation have also been described, but for which a direct role of HSF1 has not been demonstrated so far. For instance, in mouse, an accumulation of major satellite RNA from pericentric heterochromatin has been reported during late G1/S and mitotic stages of the cell cycle  and duri tissues ,45,46.TERRA), which range in size from 100 bases to >100 Kb [TERRA production is initiated in specific regions of the chromosome located approximately 1 Kb upstream of the telomeric tract. TERRAs therefore contain chromosome-specific sequences in addition to a common telomeric tract [TERRAs have been found to be expressed from several human chromosome ends [TERRA are associated with telomeric DNA repeats [Telomeric heterochromatin consists of tandem repeats of the TTAGGG hexanucleotide motif ,48. Thes >100 Kb ,50. TERRome ends ,52,53,54 repeats ,56.TERRA was then reported in human [The first observation that heat shock increases transcription at telomeres was reported in Chironomus thummi  then conin human . In humain human .SINEs are present as thousands of copies interspersed in the genome of mouse and higher primates [B1- and B2-like elements. B1-like elements are ancestrally derived from the 7SL gene, which is a component of the signal-recognition particle (SRP) ribonucleoprotein complex with a role in protein synthesis [B2-like elements are derived from tRNA precursors and are present in mouse and rabbit. In human, B1-like elements are known as Alu elements [Alu elements are approximately 280 bases in length, and are formed by two tandem repeats separated by a central A-tract [Alu elements have an internal RNA polymerase III (RNAPIII) promoter that has the potential to initiate transcription of the Alu sequence. Since Alu sequences lack a termination site for transcription, they are transcribed into RNAs of heterogenous length depending on the location of nearby transcription terminator [Alu sequences are not transcribed, unless they are fortuitously positioned near appropriate strong enhancers [SINE sequences, such that only a very low level of SINE RNAs is produced from the internal RNAPIII promoter, at dispersed loci in mouse and human [SINEs are also abundantly expressed as short-lived RNA as part of intronic sequences of nuclear transcripts [primates ,59. In mynthesis . B2-likeelements . Alu ele A-tract . Sequencrminator . Under nnhancers . Furthernd human . Howevernscripts ,64.Alu elements and Alu retrotransposition events has been described in response to genotoxic stresses [Alu sequences by HSF1, inducing their expression upon heat stress, was demonstrated by means of primer extension upon exposure to mild heat shock [Alu sequences [An increase in stresses . Importaat shock . Likewisequences .eRNAs belong mostly to the category of lncRNAs.Enhancer RNAs represent an emerging class of ncRNAs produced at distal Transcription Regulatory Elements (dTREs), which are defined as genomic regions, localized outside of gene promoters, that increase transcription of neighboring genes . dTREs aeRNAs in both mouse and human cells [Interestingly, two recent papers report HSF1 binding to enhancers that produce an cells ,9. In a an cells . Noticeaan cells , suggestNEAT1-1 and NEAT1-2, respectively, which are generated from the same promoter [NEAT1 lncRNAs and various proteins. NEAT1-2 RNA is essential for the formation and maintenance of paraspeckles [NEAT1-1 lncRNA is not essential for paraspeckles formation but enhances paraspeckles formation when NEAT1-2 is present [NEAT1-2, NEAT1-1 can be found outside of paraspeckles in tissue culture cells, further suggesting that it may have different biological functions than NEAT1-2 [NEAT1 gene sequence is not well conserved between human and mouse but in both organisms, a common strong propensity for long-range intramolecular base-pairing was recently found to contribute to paraspeckle scaffolding [The human NEAT1/Men ε/β gene codes for two overlapping 3.7 Kb and 22.3 Kb lncRNA isoforms called promoter . In mammpromoter ,72,73. Pspeckles ,75. NEAT present . Unlike  NEAT1-2 . The NEAffolding . An averffolding .NEAT1-1 and NEAT1-2 lncRNAs was described in human breast cancer MCF7 cells submitted to a mild 30-min heat shock at 43 °C followed by a recovery period of 2 h at 37 °C [NEAT1 promoter were also present within NEAT1 promoters in other eukaryotes [Recently, a direct HSF1-dependent up-regulation of at 37 °C . Intereskaryotes .NEAT1 lncRNAs, they are transcribed from a large number of genomic repeats, either distributed along all chromosomes , or at more specific and relatively large genomic loci . In addition, two of them  consist of a high density of tandem repeats units of a short nucleic acid motif. At least two of them  are essential scaffolding elements for the formation of sub-nuclear and membrane-less compartments [In this second part of the review, we present how this non-coding fraction of the genome directly targeted by HSF1 for transcription activation may act in the heat shock response. What are the common features, if any, of the lncRNAs identified so far? With the exception of artments ,80 . The nuclear structures that form at sites of SATIII transcription are referred to as nuclear Stress Bodies (or nSBs) [In human stressed cells, or nSBs) . This naor nSBs) ,82,83,84SATIII DNA repeats, could represent an efficient way of promoting a genome-wide reduction in acetylation and, thus, controlling genome-wide expression. Several HATs  [SATIII-enriched 9q12 locus [N-terminal or histone fold regions. Histone acetylation loosens histone–DNA interactions by neutralizing the positive charges of lysine and also provides docking sites for histone-binding proteins that play an important role in the recruitment or activity of essential transcription factors [SATIII lncRNAs partially releases the heat-induced repression of a series of genes, overexpression of ectopic SATIII lncRNAs triggers gene repression in both unstressed and stressed cells and results in a significant decrease in the survival of stressed cells [The accumulation of transcriptional and co-transcriptional factors at nSBs may contribute to a global deacetylation of the two core histone proteins H3 and H4 ,86 detecd Tip60) ,87, and d Tip60) ,87, are d Tip60) ,87  proteins, including (SF2/ASF-SRSF1), Srp30c (SRSF9), 9G8 (SRSF7) and heterogeneous nuclear ribonucleoprotein (hnRNPs-hnRNPA1)-interacting proteins (HAP/SAF-B and hnRNPM), with essential roles in constitutive and alternative pre-mRNA splicing, are massively recruited to HSF1-dependent nSBs in human stressed cells. The recruitment of these alternative splicing factors to nSBs is selective and proteins with constitutive roles in splicing are not recruited [SATIII lncRNAs is potentially direct [6-methyladenosine (m6A) RNA modification on SATIII lncRNAs may also recruit proteins such as YTHDC1 [SATIII lncRNAs [In addition, other serine-rich  is recruited to nSBs. exposure .SATIII, NEAT1 lncRNAs also seem to play a role in RNA processing. Indeed, NEAT1 lncRNAs have been found to anchor the DROSHA-DGCR8 microprocessor complex to paraspeckles via a pri-miR-612  stem-loop structure(s) present within the NEAT1 sequence [NEAT1 could also be involved in the control of gene expression in stressed cells (64). Indeed, a reduction in almost all expressed miRNAs was observed in cells lacking NEAT1 [NEAT1 [NEAT1 up-regulation in terms of miRNA-mediated changes in gene expression in heat-shocked cells, however, remains to be determined.As with sequence . Other psequence ,104. Up-1 [NEAT1   gene, encoding hsrω lncRNAs, is upregulated in heat-shocked cells [SATIII and hsrω lncRNAs may represent functional analogs in the regulation of RNA processing and function upon stress [What might be the advantage of regulating these RNA-based processes through the sequestration of key players? Transient sequestration of RNA processing factors and RNA regulators could be less metabolically demanding than the degradation of these factors followed by their synthesis and could allow rapid adaptation to stressful environments. Such regulation might actually be conserved through evolution. In Drosophila, the heat shock RNA omega  [HSF1-dependent transcriptional activation is primarily modulated at the level of promoter-proximal pause and release, with HSF1 increasing RNAPII release from promoter-proximal pause ,9 and di (pTEFb) . DiffereThe production of Enhancer RNA, at HSF1-binding sites located in the enhancer regions of stress-induced genes, is thought to bring together transcription factors present in the distal promoter region’s elements and in the core promoter  or to maSINE RNAs have been found to impact RNAPII-dependent transcription at different levels. SINE RNAs have been shown to affect RNAPII-recruitment at the preinitiation complex. Observations in human and mouse heat-shocked cells revealed that SINE transcripts (produced by RNAPIII and not RNAPII) establish a direct interaction with RNAPII [B2 RNA (mouse) and Alu RNA (human) disrupt contacts between RNAPII and promoter DNA [B2 RNA can also control transcriptional elongation levels by another mechanism. Indeed, B2 RNA-binding sites have been identified at many genic and intergenic targets, including at heat-responsive genes in mouse [B2 RNA, allowing RNAPII elongation  are essential in setting up pericentric heterochromatin and chromocenter formation in early embryonic development and play an important role in the deposition of the repressive H3K9me3 histone mark to pericentric heterochromatin regions at that stage [SATIII lncRNAs may participate in the reformation of pericentric heterochromatin in the recovery period from stress is, therefore, attractive at a stage when repressive marks need to be restored.Non-coding transcripts produced from pericentric heterochromatin are known to be essential actors in the maintenance of heterochromatin in mouse ,122. Majat stage ,123. In Schizosaccharomyces pombe, a model of choice to study heterochromatin, RNA of pericentric origin is required to initiate and maintain heterochromatin. In this case, pericentric lncRNAs form double-stranded RNAs that are diced into small interfering RNAs  and also serve as a platform to recruit a repressive complex. So far, we have no evidence that RNAi mechanisms involving SATIII RNA also occur in early development or in the recovery period of stress. In addition, the precise biological context in which the RNAi pathway participates or not in the maintenance of heterochromatin at centromeric regions is still unclear in higher eukaryotes. In unstressed mouse ES cells, small centromeric and pericentric double-stranded RNA (dsRNA) molecules, with predominant signals at 150 nt and 25–30 nt, are only detected in DICER-expressing cells [SATIII RNA in human HeLa cells [SATIII RNAs molecules ranging in size from 25 to 75 nt (detected as a ladder with a regular 5 nt increment) was also described in the recovery period from stress [SATIII RNA in stressed cells, a rapid accumulation of small 21–30 nt siRNAs originating from major DNA components of centromeric and pericentric regions (TCAST) was reported in the beetle Tribolium castaneum submitted to a transient heat shock stress [In the fission yeast ng cells . Still ing cells ,125, thung cells , while sLa cells . In HeLam stress . While ik stress . This ack stress . Based oXist (X inactivating specific transcripts) lncRNAs that coat the inactive X chromosome within the Barr body in female mammals [Another non-mutually exclusive possibility is that heterochromatin reformation after stress may involve the use of lncRNAs without the involvement of a process such as RNAi. These lncRNAs would then solely act as platforms to recruit proteins necessary for the formation of heterochromatin, similar to what has been described for the  mammals . In the  mammals ,52,130.  mammals ,132,133.cis [Alu elements containing HSE-binding sites are frequently positioned downstream of the transcription start site of genes, suggesting that HSF1 may contribute to the down-regulation of sense transcripts in heat-shocked cells by promoting the production of antisense lncRNAs. In support of this hypothesis, a knockdown of antisense transcripts produced at several of these loci was found to increase the expression of the corresponding sense RNA [The discovery of the accumulation of SINE transcripts in heat-shocked cells also raised the possibility that these transcripts may regulate gene expression in cis . Indeed,ense RNA   caused by the transcription of identity ,140,141.identity ,143 or aidentity  of centrproteins , and a k defects .SATIII lncRNAs accumulation on the structure and function of centromeres in human cells appears to be an important issue. We have no evidence so far that, as observed in Drosophila, human SATIII lncRNAs play a role in the stability of kinetochore. However, a recent publication reports that a transient prometaphase arrest occurs in stressed HeLa cells, the duration of which correlates with the level of SATIII lncRNAs accumulation [SATIII lncRNAs has returned to normal show chromosome-9-specific segregation defects [SATIII lncRNAs accumulation appears to be a double-edged sword, as SATIII lncRNAs’ accumulation may be part of a checkpoint, which is eventually bypassed in cancer cells, and thus provide a source of chromosomal instability. The impact of heat stress on chromosome instability may also result from an alteration of DNA replication. Indeed, several publications point to the impact of SATIII RNAs on DNA replication. For example, loss of BRCA1 in breast tumor correlates with an upregulation of SATIII RNA resulting in chromosome instability [SATIII RNAs in breast cancer deficient for BRCA1 was found to destabilize replication forks through the interaction of SATIII RNA with the members of the complex required for DNA replication composed of BRCA1-interacting protein [The need for a better characterization of the impact of stress-induced mulation . Noticea defects . In thistability . Overexp protein . The tit protein .SATIII lncRNAs appear as interesting therapeutic markers in cancer therapy. Together with heat-shocked cells, cancer cells overexpressing SATIII RNA as a consequence of DNA hypomethylation at pericentric heterochromatic regions are resistant to etoposide, a topoisomerase IIa (TOP2a) inhibitor. The interaction of TOP2a with SATIII RNA at nSBs protects TOP2A against interaction with etoposide, preventing increased DNA damage in etoposide-treated cells [Finally, ed cells .TERRA transcripts are thought to play a role in telomere stability, telomere replication and the telomeric DNA damage response [TERRA with the shelterin components TRF1 and TRF2 (Telomere Repeat binding Factors 1 and 2) contributes to telomere length homeostasis and protection [TERRA to chromosome ends, sustaining the enzymatic activities of TERRA-binding factors at telomeres [TERRA production, its distribution and its role [The possibility that the HSF1-induced accumulation of lncRNAs may play a role in genome protection is perhaps best illustrated in the case of TERRA. In unstressed cells, response . In manyresponse . The intotection , and mayelomeres . Yet, maits role ,148.TERRA correlates with the recruitment of LSD1, a lysine-specific demethylase, at telomeres [TERRA and Mre11, a H3K4 methyltransferase subunit of the MRE11/RAD50/NBS1 (MRN) repair complex that may promote telomere fusions [Interestingly, an HSF1-dependent up-regulation of TERRA has been observed at specific telomeres in stressed cells ,14,56. Helomeres . LSD1 di fusions . Whateve fusions . Finally fusions ,130,131,Alu sequences have been found to undergo retrotransposition upon stress [LINE), another class of interspersed repetitive sequences, also present in thousands of copies in AT-rich and gene-poor genomic regions, SINEs are not capable of retrotransposition by themselves. However, with the help of LINE sequences, which provide them with the necessary elements for transposition, transposition of Alu elements has been reported. Retrotransposition of Alu elements was observed in human cells submitted to etoposide [Arabidopsis thaliana, the heat-shock-induced transposition of ONSEN, a Ty1/copia-like retrotransposon [Vigna angularis [Arabidopsis thaliana, this transposition is mediated by the Heat Shock transcription Factor A2 (HSFA2), a functional equivalent of HSF1 [sat2 motifs has been found to expand locally and genome-wide in primary human colon cancer samples [SINE and of SATIII sequences in human cells.Other stress-induced lncRNAs may also have a direct impact on genome stability. For example, n stress . Unlike toposide  and one toposide  and insetoposide . In Arabansposon , and of ngularis  has beenngularis ,156. At  of HSF1 . However of HSF1 . Althoug samples , there iHSP gene expression [NEAT1 transcription [An emerging view of the HSF1-mediated non-coding response to stress is that it plays a global role in controlling gene expression by regulating transcriptional and post-transcriptional processes. The HSF1-mediated non-coding response should also be considered for its impact on genome stability. The non-coding fractions of eukaryotic genomes have long been neglected in genomic and gene expression studies. While only a small number of lncRNAs have been considered so far as direct effectors of HSF1 in stressed cells, it already appears that these RNAs are important actors of the heat shock response. The functional analysis of some of these RNAs is still hampered by the difficulty to efficiently knock-down nuclear RNAs and by the fact that they may be transcribed from multiple genomic loci, e.g., in genomic regions that are highly enriched in Satellite DNA repeats. The development of new tools such as CRISPR-Cas9 to knock out genomic sequences  and CRISpression . Trappinpression . Other tcription , may sercription . Knowingcription ,168.SATIII lncRNAs, brings a new dimension to the orchestration of the cellular response to stress by HSF1. Finally, HSF1 overexpression and activation was also observed in cancer cells while the knock-out of HSF1 was found to protect mice from tumorigenesis induced by mutations of the RAS oncogene or by a hot spot mutation in the tumor suppressor p53 [The extraordinary diversity of the new regulators, issued from the emerging population of ncRNAs accumulating in response to a stress and the HSF1-dependent activation of their transcription, such as ssor p53 . It is,


The cis-regulatory element became increasingly important for resistance breeding. There were many DNA variations identified by resequencing. To investigate the links between the DNA variations and cis-regulatory element was the fundamental work. DNA variations in cis-regulatory elements caused phenotype variations in general.We used WGBS, ChIP-seq and RNA-seq technology to decipher the regulatory element landscape from eight hulless barley varieties under four kinds of abiotic stresses. We discovered 231,440 lowly methylated regions (LMRs) from the methylome data of eight varieties. The LMRs mainly distributed in the intergenic regions. A total of 97,909 enhancer-gene pairs were identified from the correlation analysis between methylation degree and expression level. A lot of enriched motifs were recognized from the tolerant-specific LMRs. The key transcription factors were screened out and the transcription factor regulatory network was inferred from the enhancer-gene pairs data for drought stress. The NAC transcription factor was predicted to target to TCP, bHLH, bZIP transcription factor genes. We concluded that the H3K27me3 modification regions overlapped with the LMRs more than the H3K4me3. The variation of single nucleotide polymorphism was more abundant in LMRs than the remain regions of the genome.Epigenetic regulation is an important mechanism for organisms to adapt to complex environments. Through the study of DNA methylation and histone modification, we found that many changes had taken place in enhancers and transcription factors in the abiotic stress of hulless barley. For example, transcription factors including NAC may play an important role. This enriched the molecular basis of highland barley stress response.The online version contains supplementary material available at 10.1186/s12864-022-09070-x. Hordeum vulgare L.) is one of the major cereals grown worldwide and among the oldest of domesticated crops [Barley  and promotes gene expression via recruitment of the NURF(Nucleosome Remodeling Factor) complex for chromatin remodeling , 18. H3KIn our study, lowly methylated regions (LMRs) were detected using methylome data. Using ChIP-seq technology, the different histone modifications (H3K4me3 and H3K27me3) were detected in the LMRs of tolerant and susceptive varieties subjected to stress. The aim of this approach was to characterize 1) the regulatory landscape of the hulless barley genome; 2) the enhancers that are differentially modified when comparing tolerant and susceptive varieties under stress; 3) the links between the enhancers and target genes; and 4) the gene regulatory network involved in the response to stress.To investigate the relevant regulatory elements in hulless barley under abiotic stress, we examined eight whole-genome bisulfite sequencing (WGBS) libraries (see Methods) for the genome-wide identification of LMRs that are correlated with enhancers. The abiotic stresses included in our study were drought, salinity, cold, and low nitrogen. The libraries were sequenced using Illumina Hiseq X Ten. A total of 5.1 billion 150 bp paired-end reads were generated from sodium bisulfite-treated DNA with a 99% bisulfite conversion rate for each library. On average, about 637 million sequencing reads with an average of 26 × coverage were aligned to the hulless barley reference genome for each sample  genome-wide association study (GWAS) loci occurred in noncoding DNA, suggesting regulatory functions . In our Our study primarily identified cis-regulatory elements in hulless barley via whole-genome bisulfite sequencing. A total of 231,440 lowly methylated regions were identified in our study. Through correlation with expression data, 97,909 enhancer–gene pairs were discovered for all the LMRs. The transcription factor regulatory network was inferred based on the characterization of TFBS in the CREs. The histone marks corresponding to H3K27me3 modification indicated that many LMRs were suppressed under specific abiotic stresses. The cis-regulatory elements identified in our study contained numerous single nucleotide polymorphisms in the natural population.H. vulgare subsp. vulgare) varieties were used for methylome analysis in this study, which includes the drought-tolerant and susceptible varieties XL16 vs. DQ, salt-tolerant and susceptible varieties Z0119 vs. Z0226, cold-tolerant and susceptible varieties NC3 vs. NC6, and low-nitrogen -tolerant and susceptible varieties BQ3 vs. GND7. Eight hulless barley varieties were preserved in the Agricultural Research Institute, Tibet Academy of Agricultural and Animal Husbandry Sciences, Lhasa, China for many years, all the methods involving the plant and its material complied with relevant institutional, national, and international guidelines and legislation.Eight Tibetan hulless barley  6000 solutions for 48 h. For the assessment of salinity tolerance, seedlings were stressed with 200 mM NaHCO2O2 for 40 min, rinsed in sterile water, and then germinated on moistened filter paper at 16–18 °C with 14 h light/10 h dark photoperiods and relative humidity of 80% in a growth chamber. After germination, the seedlings were grown in a growth chamber at 16 °C, relative humidity of 80%, and a photoperiod of 14 h. The roots and leaves were harvested from 10-day-old seedlings belonging to two genotypes under normal and stressful conditions. Two plants from each pot were considered biological replicates. The aforementioned tissue samples were snap-frozen in nitrogen and immediately stored at − 80 °C [For the assessment of cold tolerance, seedlings were grown in a growth chamber at a temperature of 4 °C for 72 h. For the assessment of low-nitrogen tolerance, sterilized seeds were germinated on moistened filter paper deficient in nitrogen in a growth chamber for 128 h. Healthy seeds were sterilized after soaking in 2% H − 80 °C .DNA was isolated using the cetyltrimethylammonium bromide (CTAB) method from hull-less barley leaves. The integrity of DNA was checked by agarose gel electrophoresis and the concentration was measured via a non-ultraviolet method . Wuhan Ihttp://www.igenebook.com). Briefly, 3 g of barley leaves were washed twice in cold PBS buffer and cross-linked with 1% formaldehyde for 10 min at room temperature. They were then quenched by adding glycine . Afterward, samples were lysed, and chromatin was obtained following incubation on ice. The chromatin samples were sonicated to obtain soluble sheared chromatin with an average DNA length of 200–500 bp. A total of 20 μL of chromatin was stored at − 20 °C for input DNA, and 100 μL of chromatin was used for immunoprecipitation using H3K4ME3 and H3K27ME3 antibodies . A total of 10 μg of antibody was used in the immunoprecipitation reactions at 4 °C overnight. The next day, 30 μL of protein beads were added, and the samples were further incubated for 3 h. Afterward, the beads were washed once with 20 mM Tris/HCL (pH 8.1), 50 mM NaCl, 2 mM EDTA, and 1% Triton X-100, 0.1% SDS; twice with 10 mM Tris/HCL (pH 8.1), 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% deoxycholic acid, and twice with 1 × TE buffer (10 mM Tris–Cl at pH 7.5. 1 mM EDTA). Bound material was then eluted from the beads in 300 μL of elution buffer  and treated first with RNase A  for 6 h at 65 °C and then with proteinase K  overnight at 45 °C. Immunoprecipitated DNA was used to construct sequencing libraries following the protocol provided by the I NEXTFLEX® ChIP-Seq Library Prep Kit for Illumina® Sequencing  and sequenced on Illumina Xten using the PE 150 method.ChIP assays were performed according to methods previously described  by WuhanAfter removing low-quality reads, clean data were mapped to the reference genome using the software BitMapperBS (version 2.9), which permits an 8% mismatch per read . Then, tThe raw reads were filtered using Trimmomatic (version 0.38) . Then, tEach of the pupative enhancer-gene relations (the closest 10 upstream genes and the closest 10 downstream genes) was tested for correlation between the methylation level of the enhancer and gene expression. The gene expression data were derived from published data , 51. To P < 0.0001 [Transcription factors were detected using iTAK tools . A total< 0.0001 , which aAdditional file 1:Table S1. Identified enhancer–gene pairs.Additional file 2: Table S2. Genome wide transcription factor identification.