Arc and Egr1, and a transcription factor MEF2C. We also observed an increase in MEF2C binding to the promoters of Arc and Egr1. In addition, pups raised in EE show a reduction in HDAC5 and its binding to promoters of Mef2c, Arc and Egr1 genes. With an overexpression of Mef2c, neurite outgrowth increased in complexity. Our results suggest a possible underlying molecular mechanism of EE, acting through MEF2C and HDAC5, which drive Arc and Egr1. This could lead to the observed increased dendritic spine density and complexity induced by early EE.We investigated the effects of environmental enrichment during critical period of early postnatal life and how it interplays with the epigenome to affect experience-dependent visual cortical plasticity. Mice raised in an EE from birth to during CP have increased spine density and dendritic complexity in the visual cortex. EE upregulates synaptic plasticity genes,  Neuronal circuits that process sensory information are shaped by patterns of cellular activity during early brain development called critical periods or CPs . CPs areVisual cortical circuits exhibit maximal plasticity during CP, but this plasticity is lost by adulthood . The losThe visual system is highly amenable and is easily manipulated either by decreasing external sensory experience or by dark rearing and monocular deprivation or increasing external stimuli by environmental enrichment. In the latter paradigm, animals are reared in big cages filled with toys, running wheels, tunnels and nesting materials. Studies have shown that enriched environment (EE) leads to hyperacuity, which enhances their inquisition, social interactions and sensory-motor stimulations ,14. StudArc) and early growth response protein 1 (Egr1) and a transcription factor myocyte enhancer-binding factor 2C (Mef2c) were up-regulated in animals exposed to EE during CP, and we hypothesized that both Arc and Egr1 genes are regulated by MEF2C. We performed chromatin immunoprecipitation (ChIP) assays and found that MEF2C binds to the promoters of Arc and Egr1 in mice raised in EE during CP. In addition, enriched pups showed a reduction in HDAC5 binding to promoters of Mef2c, Arc and Egr1 genes to allow their expressional increase. Taken together, our results elucidate the underlying molecular mechanism of EE through MEF2C and HDAC5 that drive experience-dependent plasticity in mouse visual cortex.In this present study, we ask how environmental stimuli interact with the epigenome to shape cellular function affecting outcomes later in life. We observed that by raising animals exposed to EE from birth to the peak of CP underwent dramatic developmental changes. These animals not only have increased dendritic branching and spine density in the primary visual cortex, but also attained higher visual acuity when tested behaviorally. We sought to unravel the molecular mechanism behind EE that drives these developmental changes. Two candidate synaptic plasticity genes, activity-regulated cytoskeleton-associated protein  at Biopolis Resource Centre, A*STAR, in the Neuroepigenetics laboratory. C56BL/6 mice of mixed sex were maintained on a 12-h light/dark cycle and had access to food and water e strain .. The positions of the toys were changed every week and two filler females are part of EE to promote social interaction. The EE condition selected aims to stimulate cognitive, social and sensory-motor developments that provide an overall enriched experience for visual plasticity to take place during critical period  to obtain the fold enrichment.The perfused tissues were pooled from 11 pairs of visual cortex . Each biological replicate constituted to pooled tissue of pups from different litters. They were quenched, lysed and sheared with the Diagenode Bioruptor for 15 cycles of 30-s intervals. Samples were pre-cleared and incubated with MEF2C, HDAC5, and No Antibody, overnight with rotation at 4°C. The immune-complexes were pulled down with magnetic beads, reversed cross-link and purified with phenol–chloroform. Immunoprecipitated chromatin was quantified by real-time quantitative PCR on FAST7900HT (Applied Biosystems) using SYBR-Green master mix (Applied Biosystems), using 10% input. Primers used were: 5 cells/well on poly-L-lysine coated glass coverslips in 4- or 12-well plates. One day after plating (Day1), cells were transduced with pre-made lentiviral particles expressing Mef2c gene under the suCMV promoter (GenTarget). As a negative control, lentiviral particles with the same lentivector backbone containing a null spacer sequence were used. Seventy-two hours after transduction, protein lysates were collected for Western Blot analysis and dendritic morphology was visualized by immunocytochemistry.P19 cells were differentiated into the neural lineage as described previously. Briefly, cells were differentiated with 1 µM All-Trans Retinoic acid for 4 days and subsequently plated at a density of 2 × 105 cells/cm2 on a 0.56 cm2 12-well dish coated with 75 μg/ml poly lysine (ibidi) and/or on a 9.6 cm2 6-well dish (Nunc). To suppress the growth of proliferative cells, 10 μM cytosine arabinoside was added into culture medium from day 2 to 3. Fresh culture media were added every 3 days and maintained at 37°C in a 5% CO2/95% O2 air-humidified incubator. Subsequently, 25 nM HDAC5 siRNA (Qiagen) was added to the cultures for 24 h. All-Star Negative Control (Qiagen) with a scrambled sequence was used as a negative control. RNA was collected for mRNA expression analysis and dendritic morphology was visualized by immunocytochemistry.Primary neuronal cultures were prepared from 17- to 18-day-old embryonic mice cerebral cortex. The mice cerebral cortices were dissected immediately after killing in accordance with the guidelines of Institutional Animal Care and Use Committee (IACUC). In brief, the cerebral cortices were incubated with Versene (GIBCO), resuspended in culture media , B27 supplement (GIBCO), 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin) and plated at a density 2.5 × 10Cells grown on coverslips were washed, fixed and blocked for 1 h followed by incubation with anti-MEF2C and anti-TUJ1 antibodies for MEF2C transduction experiments or with anti-HDAC5 and anti-MAP2 for HDAC5 siRNA transfection experiments. They were probed with Alexa-Fluor 488 and 546 secondary antibodies and sealed with ProGold stain. Images were visualized on the confocal laser-scanning microscope .t test unless otherwise stated. Analysis for P<0.05. SC and EE were represented by black and white bar respectively, unless otherwise stated.All data are represented using mean±SEM. Results between independent groups were interpreted using two-tailed unpaired n=3 neurons; EE, white bar, 0.95±0.06, n=3 neurons; P=0.0433, two-way ANOVA Sidak’s multiple comparisons test). Next, we assessed the morphological changes between the two environment paradigms by Golgi-impregnated neurons in vivo. Sholl analysis of the Golgi-stained pyramidal neurons was carried out to determine the dendritic length and branching .Enriched environment has been shown to increase dendritic branching, spine density and arborization in the hippocampus, striatum, occipital and motor cortex in rodents . EE alsoranching D. There ranching E . Though it was reported that β-actin expression in cortical circuits changes during postnatal development in an activity-dependent manner [We also checked the amount of cytoskeletal processes, marked by TUJ1 and found that early EE increases TUJ1 expression by 2.4-fold =5.20, P=0.0020, unpaired t test). Although we did monitor the duration mothers spent with their pups in SC and EE, we did not measure other maternal care indicators such as licking behavior that have been found to accelerate visual acuity [n=5; EE, 8.00±2.00, n=2; t(5)=3.21, P=0.0238, unpaired t test). Indeed, we found EE pups to achieve the same trial accuracy of 70% with significantly higher spatial frequency of 0.06 cycles/degree than SC mice  =2.33, P=0.0284, unpaired t test). Together with the results above, we concluded that EE accelerates the organization of the visual cortex as presented in To further substantiate our results above, eye opening of pups was monitored from PND 0 to PND 16. We observed EE pups to experience significantly earlier eye opening than SC pups, leading to earlier experiential exposure that could accelerate visual acuity   while the expression levels of the other members remain relatively unchanged (Mef2a and Mef2d) or below detectable levels (Mef2b). Sensory experience regulates the expression of immediate-early genes such as Egr1 and Arc [Egr1 indirectly regulates synaptic plasticity via its regulation of Arc [Arc and Egr1 upon exposure to environmental enrichment. Mice exposed to EE show significantly increased levels of Arc and Egr1 mRNA in the visual cortex =2.92, P=0.0265; Egr1; 1.87±0.13, n=5, t(6)=5.12, P=0.0022, unpaired t test).We sought to unravel the molecular mechanism behind EE that drives these developmental changes. MEF2 family transcription factors are vastly expressed in the brain and are activated by extracellular stimuli and calcium influx in neurons  to triggve to SC A =2.75, P=0.040; Egr1: SC, 1.03±0.15, n=3; EE, 5.25±1.24, n=3, t(4)=3.40, P=0.0274, unpaired t test).Since ed to SC C =2.58, P=0.016; TUJ1/DAPI: Null vector, 3.07±0.24, n=14, MEF2C, 4.45±0.59, n=15, t(27)=2.10, P=0.045, unpaired t test) after MEF2C over expression. Thus, MEF2C-transduced neurons showed more elaborated dendritic arborization =8.38, P=0.0011, unpaired t test), indicating that EE stimulates the transcription of Mef2c by histone acetylation.EE induces chromatin remodeling via increased acetylation of histones H3 (AcH3) and H4 . We quesl cortex A .Next, we wanted to identify which HDAC is responsible for the increase in histone acetylation on activity  through l cortex C =2.11, P=0.0514, unpaired t test).To ascertain if MEF2C and HDAC5 proteins interact with each other, we carried out a co-immunoprecipitation of HDAC5 and probed with an anti-MEF2C antibody. MEF2C indeed forms a protein-protein interaction with HDAC5 n=3; D. We alsl cortex E =5.29, P=0.0061; Egr1: SC, black bar, 1.56±0.15; EE, white bar, 0.50±0.29, n=3 per group, t(4)=3.23, P=0.0321, unpaired t test). The qPCR analysis for the HDAC5 ChIP was inversely correlated to that of the MEF2C ChIP, suggesting a relationship between these two proteins.To further prove that an inverse relationship exists between MEF2C and HDAC5, the HDAC5 immune complexes were analyzed for binding at the l cortex F  initiates the transcription of Arc and Egr1 genes corresponding to their increase in mRNA levels in EE visual cortex as seen earlier. Arc and Egr1 may contribute to (iv) the increase in total and apical dendritic spine density observed in early EE mice  in EE, the reduction in HDAC5 decreases its occupancy on promoters of Arc and Egr1. This is the first demonstration of the epigenetic dynamics of MEF2C and HDAC5 targeting on Arc and Egr1 genes to regulate experience-dependent plasticity.Despite numerous studies involving EE, the molecular mechanisms underlying its efficacy are still poorly understood. Here, we demonstrated that (1) EE increases dendritic spines tremendously during the CP; EE induces (2) an increase in External stimuli sculpts overall neural circuitry and hence its plasticity. In the clinical setting, EE is being used as a non-invasive treatment for amblyopia ,45. Visuin silico analysis agreed with the genome wide analysis study [mef2c expression in EE mice. Notably, MEF2C is one of the most highly expressed isoforms [Mef2c is also a candidate risk gene for various neurodevelopmental disorders such as schizophrenia [To unravel the molecular basis of EE that led to the increased visual acuity observed, we first carried out a motif-scanning approach on online available EE- related GEO database to predict gene regulatory sequences across the genome. MEF2 proteins have been shown to be key regulators of synapse development and function and their activity are dependent on neuronal stimuli, such as neurotrophins or calcium influx . We hypois study ,49. In tisoforms , crucialisoforms . MEF2C oisoforms . Mef2c iophrenia , major dophrenia  and Alzhophrenia . MEF2C hophrenia ,56.Arc and Egr1. Previous studies demonstrated EE rats have elevated Arc and Egr1 mRNA [Arc with visual acuity has emerged in recent years. Arc gene produces a protein important for memory consolidation, memory formation, and visual cortical development [Arc-/- mice exhibited deficits in visual cortical plasticity [Arc transcription was shown in both adult congenital blind [. Monocular visual stimulation has been found to induce and restore Egr1 expression and protein levels during CPs [Egr1 does not necessarily dictate experience-dependent plasticity [Erg1 and Arc is evident, whereby MEF2 shRNA knockdowns attenuate Arc expression levels [Arc [Arc expression in activity dependent neuronal plasticity in the visual cortex [Arc and Egr1 genes in EE conditions. This could be explained by stimulation-transcription coupling, where EE potentially stimulates neurons to induce Arc and Egr1 gene expressions via MEF2C and regulate synaptic plasticity during CPs [Mef2c KO mice [We next used a candidate gene approach to identify gene targets of MEF2 family transcription factors, narrowing the selection to gr1 mRNA ,39,57,58elopment ,60. Funcasticity  and visual blind  and wildal blind . These ral blind . EGR1 isal blind ,66. Monoring CPs . Howevern levels , activatels [Arc  and MEF2l cortex . In ordering CPs . We then KO mice . MEF2C oMef2c promoter and also the Arc and Egr1 promoter regions. Given the HDAC5–MEF2C interaction and its association to regulate neurite development, we would like to address the signaling cascades of EE via HDAC5 and MEF2C and understand the role of MEF2C and HDAC5 in visual plasticity.The activity of MEF2 gene is regulated by the dissociation of class IIa HDACs from its DNA-binding domain ,44,74,75Arc and Egr1 genes during critical period, there is a possible association between MEF2C activation and visual acuity [Mef2c in deprived knockdown adult mice to see an improvement in their visual acuity.MEF2C protein expression are found to be crucial for visual neuroplasticity during critical period  and thisl acuity ,78. To fClick here for additional data file.


Epigenomic modifications are instrumental for transcriptional regulation, but comprehensive reference epigenomes remain unexplored in rice. Here, we develop an enhanced chromatin immunoprecipitation (eChIP) approach for plants, and generate genome-wide profiling of five histone modifications and RNA polymerase II occupancy with it. By integrating chromatin accessibility, DNA methylation, and transcriptome datasets, we construct comprehensive epigenome landscapes across various tissues in 20 representative rice varieties. Approximately 81.8% of rice genomes are annotated with different epigenomic properties. Refinement of promoter regions using open chromatin and H3K4me3-marked regions provides insight into transcriptional regulation. We identify extensive enhancer-like promoters with potential enhancer function on transcriptional regulation through chromatin interactions. Active and repressive histone modifications and the predicted enhancers vary largely across tissues, whereas inactive chromatin states are relatively stable. Together, these datasets constitute a valuable resource for functional element annotation in rice and indicate the central role of epigenomic information in understanding transcriptional regulation. Comprehensive epigenomic maps of various rice varieties are still unavailable. Here, the authors report the development of eChIP as a fast and low-input upgrade of regular plant ChIP-seq protocol for epigenome analysis of 20 rice varieties and annotate over 80% of the genome with different epigenome properties for transcriptional regulation. Oryza sativa) is one of the most important staple crops in the world and a model for plant genomic research. A major challenge in rice biology is understanding how conserved genome information translates and contributes to the formation of important agronomic traits and adaptation to various environmental challenges2. Chromatin states (CSs), which are determined mainly by a variety of epigenomic features, dictate genome activities and transcriptional regulation4. In addition, CSs are instrumental for understanding how genetic variants affect epigenetic information, which leads to variation in gene expression and traits. Epigenomic information, including DNA methylation, posttranslational histone modification, and chromatin accessibility, has been characterized in rice and other plants5. These studies provide insight into the epigenomic features of cis-regulatory elements and coordinative effects of active and inactive histone marks on transcriptional regulation8. Moreover, many studies have revealed the functions of genes encoding various chromatin modification factors11 and characterized genome-wide profiles of some histone marks and DNA methylation15. Despite these significant advancements, comprehensive epigenome maps across different tissues and distinct genetic backgrounds are still lacking for elucidating the dynamics of CSs and their effects on dynamic expression profiles and for assessing genetic variations affecting gene expression in plants. One of the major obstacles is the low efficiency of ChIP-Seq experiments in plants.Rice , Zhenshan 97 (ZS97), Nipponbare (Nip) and 17 other varieties. Our integrative analyses provide a broad overview of epigenomic landscapes, promoter chromatin features, enhancer predictions, epigenomic dynamics in different tissues, and effect of genetic variation on these rice varieties, as comprehensive rice functional DNA elements maps that lay the foundation for rice ENCODE project (The Encyclopedia of DNA Elements).17. The eChIP method reduced the required amount of starting material to 0.01 g for one IP with equal or better data quality, compared to the regular method using approximately 1–5 g of tissue18  method for plants that highly improved the efficiency of chromatin extraction via direct sonication of formaldehyde-fixed tissues Fig. . Most 7, compareBy using the eChIP-Seq method, we characterized the genome-wide enriched regions of five histone modification marks  in four tissues  from MH63, ZS97 and Nip using validated antibodies  data from 20 representative rice tissues Fig. . Genes a3 at 200-bp resolution along the genome for three tissues  from MH63 H63 Fig. . The ricH63 Fig. . The samH63 Fig. .Fig. 2CoThe active states comprised active promoter-associated states , transcription-associated states , an active TTS and flanking state (TtsAFlnk/CS9), and active intergenic states  Fig. . The str21, no corresponding chromatin signature for the 3′ ends of genes in plants had been described. This state covered 3.6% of the genome, on average, with higher enrichment of DNA transposons than other active states and intermediate DNA methylation. Notably, Helitron, one kind of DNA transposons, was also significantly enriched in CS9, indicating its emergence from the transcription of gene 3′ ends.Notably, CS TtsAFlnk/CS9 was characterized by a high frequency of RNAPII occupancy and showed 45% enrichment for TTS and TTS flanking Fig. . AlthougTwo intergenic active states were associated with higher frequencies of H3K27ac and FAIRE signal, respectively Fig. . These sThe repressive states comprised TssBiv/CS4 and ReprPC/CS12 Fig. . In contCopia and Het)  and Copia (36%). This state showed the highest DNA methylation, and the majority (97%) of genes covered with this state were not expressed. CS Copia, which was defined by the active mark H3K4me1 and inactive mark H3K9me2 , bivalent Copia was frequently located within gene body regions, especially in introns, and showed the highest DNA methylation level  Fig. . CS Het me2 Fig. , was a bSeq Fig. , which cSeq Fig.  and exhipia Fig. . Comparevel Fig. . In addiate Fig. . Compareate Fig. .The Quies state (CS15) was devoid of any modification but showed high enrichment of LINE/SINE retrotransposons and DNA transposons Fig. .Taken together, these data revealed distinct genomic region profiles with different biochemical properties and highlighted the complex relationship between DNA methylation, TE enrichment, and gene transcription in various CSs. Except for the Quies state, approximately 81.8% of the rice genomes were annotated with at least one CS.We next explored chromatin features in the vicinity of gene promoters by examining the profiles of different epigenomic marks along ±2 kb regions around TSS. We classified them into 15 clusters based on histone modifications and chromatin-accessible patterns  reporter under the control of truncated promoters in rice protoplasts Fig. . The majTo confirm these results, we used the naturally occurring genomic presence/absence variations (PAVs) and/or insertion/deletions (InDels) in MH63, ZS97, and Nip genomes overlapping with the identified FAIRE element and H3K4me3 modification to evaluate their effects on gene expression Fig. . The majSince H3K4me3 and H3K27ac were frequently co-located in promoters, we generated H3K4me3-ChIP-H3K27ac-reChIP-Seq and H3K27ac-ChIP-H3K4me3-reChIP-Seq data to investigate the co-occupancy of H3K4me3 and H3K27ac in the same nucleosome Fig. . Of the We then systematically characterized the variability of chromatin marks in tissues. We observed greater dynamic changes in the active histone marks H3K4me3 and H3K27ac and the repressive mark H3K27me3 than those in the transcribed mark H3K4me1 and heterochromatin mark H3K9me2 Fig. . NotablyR = 0.67) with gene expression changes, whereas the H3K27me3 changes showed a weak negative correlation (R = −0.38) with gene expression changes  of active genes were classified in the core gene category, while the proportion of repressive and heterochromatin-associated genes were higher in the distributed gene category  and distributed genes (present <99% of 453 rice accessions)ory Fig. . This suory Fig. . Compareory Fig. . Howeverory Fig. . These rCopia), CS14 (Het) and CS15 (Quies) were the least conservative ones across 20 varieties  to CS11 (OpenChr), CS13 (Copia) and CS14 (Het)  group tend to be close to Xian/Indica or Geng/Japonica, respectively  Fig. . In addiely Fig. . We alsoely Fig. . We furtoup Fig. . Taken tArabidopsis thaliana, maize and Brassica napus). With eChIP method, we here present the most comprehensive epigenome maps across four tissues in three rice varieties  for which a high-quality reference genome sequence is available and another 17 representative core rice varieties. The rice ENCODE project will be valuable for elucidating the regulatory effects of DNA elements on transcription, growth and development. Similar projects for human19 and other model organisms32, the Roadmap Epigenomics Program33 and other projects have been implemented to comprehensively annotate functional genomic elements and generate reference epigenomic maps of cells and tissues of interest. One key to achieving these maps is that we developed a highly efficient and low-input eChIP method. With this method, we generated the largest epigenetic resource for rice to gain insight into the epigenomic landscapes, dynamics across tissues and regulatory functions as well as impacts of genetic variants on epigenome. With 510 libraries, we annotated nearly 81.8% of the rice genomes, including active DNA regulatory elements. Promoters-associated active and repressive marks are more dynamic than heterochromatin-associated inactive mark H3K9me2 across tissues. Genomic regions with specific combinations of epigenetic marks possess similar genome properties (such as genomic annotation and TE enrichments), DNA methylation levels, and transcriptional potential in distinct rice tissues and varieties, suggesting that epigenomic annotation has a powerful predictive function for genome activity.The plant epigenome research falls behind of mammalians’ and one of reason is that there is no efficient ChIP-Seq method for plants. Here, we developed an efficient ChIP-Seq method eChIP, which provides a platform for the rapid generation of a large amount of data for epigenome research and works well for all the examined plant tissues and species  in rice with FAIRE and H3K4me3 and validated the results with the LUC reporter assay. Remarkably, regions modified with H3K4me3, with the summits located predominantly (93%) downstream of TSS, are also essential for gene expression, suggesting that the transcription region modified by H3K4me3 serves as a critical component of rice promoters. Thus, the significance of the overlapping region between active promoter and transcription marked by H3K4me3 and the underpinning regulatory mechanism of gene transcription require further investigation, even though H3K4me3 is a well-established promoter feature.We further identified extensive enhancer-like promoters that have function of transcriptional regulation through chromatin interactions in rice Fig. . This fiOverall, our epigenome datasets, regulatory annotations, and integrative analyses have provided the most comprehensive maps of the rice epigenomic landscape. We expect that the eChIP method and the epigenomic map will be widely used by scientific communities for studies on genome interpretation, gene regulation, tissue development, crop genetic improvement, and epigenomic annotations of other plants.OsbZIP23-overexpressing plants34 were also collected. Germinating seeds were obtained by soaking dry seeds in water for 72 h at 37 °C. For roots and young leaves, the germinated seeds were grown in a phytotron with the day/night cycle set at 14 h/10 h and a temperature of 32 °C/28 °C. Roots were obtained 7 d after planting on moist filter paper, and young leaves were obtained from 2-week-old plants cultured hydroponically as described35. Approximately 20-d-old seedlings were transplanted to the field and managed under normal agricultural conditions on the experimental farm of Huazhong Agricultural University, Wuhan, China. Young panicles and mature leaves at the floral induction stage were harvested. Young leaves of 2-week-old Arabidopsis (Col-0), Brassica napus (B409), and maize (B73) after germinating were harvested for eChIP experiments.Three rice varieties, MH63 and ZS97 , and Nip, were used. The four tissues analyzed in the current study were the root, young leaf, mature leaf, and young panicle. Young leaves of other 17 rice varieties (Supplementary Table 36. Proteins were separated by Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDS-PAGE), blotted, and probed with antibodies (1:1000 dilution) against modified histone according to standard procedures. For dot blot, the indicated polypeptides on nitrocellulose membranes were blocked with 3% bovine serum albumin (BSA) and incubated with the indicated primary antibody (1:1000 dilution) and secondary antibody (1:5000 dilution). After incubating with enhanced chemiluminescence (ECL) reagent , membranes were exposed according to standard procedures.Western blot and dot blot experiments were used to confirm the specificity of modified histones. Nuclear lysate was extracted from MH63 plants. Western blot was performed as reportedg for 10 min at 4 °C, and the supernatant (containing most of the fragmented chromatin) was transferred to a new tube for ChIP. For subsequent analysis, 20 μl of supernatant was used as the input sample. ChIP was performed using antibodies against the following: H3K4me1 , H3K4me3 , H3K9me2 , H3K27me3 , H3K27ac , and RNAPII . In doing so, 50 μl of Dynabeads® protein G beads  were washed with 300 μl of PBST buffer  with 0.1% Tween 20) twice and resuspended in 200 μl of PBST buffer. Antibody solution (5–10 μl) was added to the beads and incubated for 1–6 h on a rotator at 4 °C to form antibody-bead complexes. The antibody-bead complexes were washed with PBST twice and incubated with precleared chromatin for 8 h or overnight at 4 °C on a rotator to immunoprecipitate the target chromatin. The immunoprecipitated chromatin was washed subsequently with low-salt ChIP buffer  (three times), high-salt ChIP buffer  (twice), ChIP wash buffer  (once), and TE buffer  (twice). The protein-DNA complexes were eluted from beads by adding 100 μl of freshly prepared ChIP Elution buffer  for 15 min at 65 °C with agitation at 900 rpm. The supernatant (eluate) was then transferred to a new tube. The beads were washed with 200 μl of 10 mM Tris-HCl (pH 8.5), and the supernatant was also transferred to a new tube. Subsequently, 5 μl of proteinase K was added to the eluate and incubated for 6 h or overnight at 55 °C to reverse-cross-link the protein-DNA complexes. ChIP DNA was extracted with phenol:chloroform:isoamyl alcohol , precipitated with ethanol, and resuspended in TE buffer. ChIP DNA libraries were prepared using an NEBNext® Ultra™ II DNA library prep kit for Illumina® sequencing . Briefly, ChIP DNA was end-repaired, ligated with an adaptor, and followed by 6–10 cycles of PCR amplification, per the manufacturer’s guidelines. Next, library fragments of 250–650 bp were selected using AMPure XP beads . Finally, the DNA fragments were sequenced using an Illumina HiSeq X Ten system .Tissues were crosslinked with 1% formaldehyde for 10–15 min and quenched with 0.2 M glycine at room temperature. Approximately 0.1 g or the indicated amounts of samples were used for each eChIP-Seq assay. Samples were ground in liquid nitrogen into fine powder and then lysed in 300 μl of Buffer S (50 mM HEPES-KOH (pH7.5), 150 mM NaCl, 1 mM Ethylene Diamine Tetraacetic Acid (EDTA), 1% Triton X-100, 0.1% sodium deoxycholate, 1% SDS) for 10 min at 4 °C. The homogenate, containing the entire chromatin from the ground sample, was mixed with 1.2 ml of Buffer F (50 mM HEPES-KOH (pH7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate), and the chromatin was fragmented into 200–600 bp by sonication using a Bioruptor (Diagenode). The lysates were centrifuged at 20,000 × 16 with minor modifications. Young leaves were crosslinked using 1% formaldehyde and quenched with glycine. To isolate the nuclei or chromatin, approximately 1 g of young leaves were ground in liquid nitrogen into fine powder and resuspended in 30 ml of EB1 buffer. The mixture was filtered through Miracloth, and the filtrate was centrifuged at 1800 × g for 10 min at 4 °C. The pellet was washed three times in 1.5 ml of EB2 buffer and centrifuged at 2000 × g for 10 min at 4 °C. Next, the pellet was washed in 0.5 ml of EB3 buffer and centrifuged at 2000 × g for 1 h at 4 °C. The final pellet was resuspended in 0.25 ml of NLB buffer, and the chromatin was fragmented into 200–600 bp by sonication in a Bioruptor (Diagenode). The chromatin sample was then centrifuged at 2000 × g for 10 min at 4 °C, and the supernatant was used for IP. ChIP was performed using H3K4me3 antibody . The remaining steps, including washing the bead-IP complexes, ChIP DNA purification, library preparation, and DNA sequencing, were the same as described for eChIP-Seq.The regular ChIP-Seq experiment was performed as reportedThe protein-DNA complexes were reverse-crosslinked to purify DNA as described above, and the purified DNA was used to evaluate the chromatin content at distinct steps of the assays. The relative chromatin extraction efficiency at distinct steps was normalized to the amount of the starting material.Tissue fixing, chromatin sonication, and IP with the first antibody in the primary ChIP procedure were performed as described in eChIP. The first antibody was crosslinked to the beads using the fixative disuccinimidyl suberate . After washing with low-salt ChIP buffer, high-salt ChIP buffer, ChIP wash buffer, and TE buffer, the protein-DNA complexes were eluted from beads by ChIP Elution buffer for 15 min at 65 °C with agitation at 900 rpm. The supernatant (eluted chromatin) was then transferred to a new tube. A total 5% of the eluted chromatin was retained for the primary ChIP DNA purification. The remaining eluted chromatin was then used for the reChIP. reChIP with the secondary antibody, elution, DNA purification, library construction, and sequencing were performed as described above.38 with minor modifications for rice. Approximately 0.5 g of sample crosslinked with 1% formaldehyde was used for each experiment. Nuclei isolation was performed as described for regular ChIP-Seq. The isolated nuclei pellet was resuspended in 400 μl of Sonic-FAIRE buffer  and fragmented into 200–400-bp pieces by sonication in a Bioruptor (Diagenode). Lysates were centrifuged at 20,000 × g for 10 min at 4 °C, and the supernatant was transferred to a new tube. To prepare FAIRE DNA, 1 volume of phenol:chloroform:isoamyl alcohol was added to the supernatant; the mixture was vortex-mixed and centrifuged at 20,000 × g for 10 min; and the supernatant was transferred to a new tube. These steps were repeated at least twice to remove nucleosome-coated DNA. After ethanol precipitation, FAIRE DNA was resuspended in TE buffer. Subsequently, DNA fragments of 200–400 bp were selected using AMPure XP beads. FAIRE DNA library preparation and sequencing were performed as described above for eChIP-Seq.Open chromatin mapping was performed using the FAIRE-Seq  methodTotal RNA was isolated from the indicated tissues using a RNeasy plant mini kit according to the manufacturer’s instructions . The RNA (2 μg) was used for library construction using an Illumina TruSeq RNA kit, according to the manufacturer’s recommendations. Sequencing was performed using an Illumina HiSeq X Ten system.Genomic DNA was extracted using a DNeasy plant mini kit according to the manufacturer’s instructions . Bisulfite conversion of DNA was conducted using the EZ DNA Methylation-GoldTM kit , and the bisulfite-treated DNA libraries were constructed using the Illumina TruSeq DNA sample prep kit, following the manufacturers’ instructions. Briefly, 2 μg of DNA for each sample was fragmented into 200–400-bp pieces by sonication in a Bioruptor (Diagenode). The fragmented DNA was processed by end-repair, 3′-end adenylation, and adapter ligation. The adapter-ligated DNA fragments were treated with bisulfite, followed by eight cycles of PCR amplification. Finally, the bisulfite-treated PCR products were purified using AMPure XP beads. The paired-end sequencing of bisulfite-treated DNA libraries was performed using an Illumina HiSeq X Ten system.39 were replaced with a multiple clone site (MCS) and 35S terminator (35STor), respectively, to generate the pMCFLMC vector. The MCS fragment containing SacI, NdeI, EcoRI, SphI, HindIII, and SpeI restriction sites was prepared by annealing oligonucleotides. The 35STor fragment was amplified from p2FLGW7 with the 35STor forward and reverse primers, and KpnI and BamHI-XbaI-SalI-PstI-SacII sites were added to the 5′ and 3′ ends, respectively. The attR1-ccdB-attR2 region of p2RLGW739 was replaced with 35STor to generate p2RLMC. Then, DNA containing the assayed promoter region was amplified from MH63 genomic DNA using the corresponding primers; the amplified fragments were cloned into pMCFLMC for the promoter activity assay. The primers used in the current study are listed in Supplementary Table To modify the reporter vector, the coding regions of the 35S promoter and attR1-ccdB-attR2 in the p2FLGW7 vector40 with minor modifications. Briefly, leaf sheaths of approximately 2-week-old MH63 seedlings were digested with 1.5% cellulase R-10  and 0.75% macerozyme R-10  for 4 h at room temperature. After being filtered through nylon mesh, the collected protoplasts were incubated in W5 solution  for 1 h. The protoplasts were then collected and resuspended in MMG solution . Plasmid pMCFLMC containing distinct promoter fragments and plasmid p2RLMC were used to cotransfect rice protoplasts using the PEG-CaCl2 method for 12–15 min. Next, the protoplasts were collected and incubated in WI solution  in darkness for approximately 14 h. Finally, the protoplasts were lysed to perform a luciferase activity assay with the Dual Luciferase reporter assay system . The fLUC activity was normalized to the Renilla luciferase (rLUC) activity in each sample, and the relative ratio was determined by comparing with that of the empty vector. The mean relative ratios were calculated based on three independent experiments.Protoplast isolation was performed as reportedr) for 13 h. The plasmid libraries were extracted using QIAGEN Plasmid Plus Kit. Protoplast isolation and protoplast transformation assay were performed as described above. Total RNA was isolated from the protoplasts using a RNeasy plant mini kit according to the manufacturer’s instructions . First-strand cDNA synthesis was performed with TransScript One-Step gDNA Removal and cDNA synthesis SuperMix  using a specific primer (GACTGGAGTTCAGACGTGTGC). All reactions were pooled. The first PCR , using two reporter-specific primers (R: 5′-GACTGGAGTTCAGACGTGTGC-3′ & F: 5′-GAGCTCTGGTAGAAATCTGAGGGTGTC-3′), one of which spans the splice junction of GFP intron. After cDNA synthesis using EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix , the enhancer activities of the target DNA fragments were quantified using qRT-PCR. The primers used in the current study are listed in Supplementary Table The target DNA fragments were amplified from the genome DNA and then were recombined to linearized screening vector pBI221 with ClonExpress II One Step Cloning Kit at 37 °C for 30 min, followed by 4 °C for 10 min. All the constructs were introduced into Trans1-T1 Phage Resistant Chemically Competent Cells according to the manufacturer’s protocol. The transformants were transferred to 4 l LB medium 42 to ensure high-quality aligned data. For analysis of H3K4me3, H3K27ac, and RNAPII libraries, narrow-peak calling settings were used in MACS2 (2.1.0)43: macs2 callpeak –t <input file>-c <control file>-f BAM –n <output peak file>-B -g 3.6e+8. For analysis of H3K9me2, H3K4me1, and H3K27me3 libraries, broad-peak mode was used in MACS2 with FDR < 0.1. RSC was set to >0.8, and NSC was set to >1.05 to assess the quality of each eChIP-Seq dataset; FRiP was used to assess the peak quality in the dataset44. For FAIRE-Seq data, two independent FAIRE-Seq libraries were created for each tissue or variety (MH63RS1 or ZS97RS1). The alignment process was similar to that for eChIP-Seq. FAIRE-Seq peaks were identified using MACS2 with the following settings: macs2 callpeak -t < input file>--nomodel--shift -100--extsize 200 -f BAM –n <output peak file>-B -q 0.05 -g 3.6e+8. Statistics for the data are provided in Supplementary Table 45. Signal levels were determined by FPKM (eChIP)/FPKM (input) for regions on both sides (2 kb) of genes, and data were sorted according to expression level 46, and gene expression was quantified using Cufflinks (2.2.1). Expressed genes were defined as those with FPKM > 1. Differentially expressed genes were evaluated using the DESeq2 package47 in R with an adjusted p value < 0.05 and log2 fold-change > 1.5.Sequence quality of RNA-Seq libraries was evaluated using FastQC, and the adapter sequences and low-quality reads were filtered using Trimmomatic (0.36)48. The uniquely mapped reads were used for further analysis. Individual cytosines with coverage at least 3 were considered for methylation-level calling. The DNA methylation level of each cytosine was obtained by dividing the depth of methylated reads by the total coverage of individual cytosines.The sequence quality of the whole-genome bisulfite sequencing (WGBS) libraries was evaluated using FastQC, and the adapter sequences and low-quality reads were filtered using Trimmomatic. The cleaned reads were then mapped to the MH63/ZS97 reference genomes using BatMeth2q value between the average region methylation level was 0.05 or less.To identify the DMRs, the whole genome was divided into 200-bp bins. The DMR between two samples was examined by a binomial test and defined by two conditions: (1) the difference in DNA methylation between two samples was 0.6 or above, and (2) the adjusted 3, a multivariate Hidden Markov Model, was used for unsupervised segmentation of the MH63 genome into a certain number of states based on the combination of histone modifications. The genome was divided into 200-bp bins. Since RNAPII is hard to detect in the root, five histone modifications, RNAPII, and FAIRE-Seq signals were selected in the three remaining tissues  to divide the MH63 genome into 15 states. Multiple models were trained on these data, with CS numbers ranging from 8 to 40. The 15-state model was chosen for all further analyses because it captured all the key information of CS. Greater numbers of CSs did not sufficiently capture significant information. In previous studies12, 15-state models were similarly trained on human tissue data.ChromHMM v1.12Enrichment analysis of the overlap of different histone modification combinations with specific genomic features was performed using ChromHMM. Based on 200-bin segments in three tissues presented by ChromHMM, the expression level of each CS was evaluated by overlapping with genes. Similarly, the distribution of DNA methylation (percent CpG methylation from WGBS data) was computed using regions belonging to each of the 15 CSs based on the core set of six marks and FAIRE-Seq regions.49 (http://rice.hzau.edu.cn/rice/). ChIPseeker50 in the R package was used to analyze the genome-wide distribution of the three TE types  and to combine different TE distributions with 15 core CSs, especially CS 13 (Copia-enriched state). CS 13 was mapped to a gene if it exhibited a 1-bp overlap with that gene and compared with the Copia distribution in CS 13. In the distribution comparison, if the intron and exon of the gene were simultaneously covered by TE, we defined TE as covering the exon region. The overall distribution of TEs close to histone modifications in the MH63 genome was visualized if 1-bp overlaps with peaks were observed, and different preferences were noted. To determine the methylation density of a TE region, methylation of all cytosine sites in the TE region was averaged. Genes with FPKM > 0.15 were analyzed to assess the relationship between different TE locations and the overlap of H3K9me2 with gene expression.MH63 and ZS97 TE annotations are available at the Rice Information GateWayConsidering the histone characteristics of the promoter region, unsupervised clustering of four histone modifications and open chromatin signals around TSS (2 K) in young leaves was performed. The analysis revealed 15 typical categories of histone combinations around TSS. Histone signal level was determined based on the FPKM (eChIP)/FPKM (input) ratio every 10 bp in the vicinity of TSS. Each cluster contained at least 2000 genes and showed a unique gene expression and classification. These clusters were also relatively stable in other tissues. Further, DNA methylation profiles on both sides of TSS were evaluated using these clustering genes. The results corresponded to the histone pattern  had very high expression levels and genes in cluster 13 (modified by H3K9me2) were located in the heterochromatic region. It is worth noting that cluster 4 marked the boundary between euchromatin and heterochromatin, with the transition from high-H3K9me2 and low-H3K4me3 profile to low-H3K9me2 and high-H3K4me3 profile.Using the 15-state model, the entire genome was divided into 200-bp segments, and the switching counts for any pair of states in all pairs of epigenomes in three different tissues were computed. For example, for a given pair of states A and B, the numbers of genomic bins that were marked as  or  in all pairs of epigenomes were counted. A 15 × 15 relative state-switching matrix was computed for every two-tissue combination. The switching matrix was transposed and summed with the original matrix to obtain a symmetric matrix. To obtain the overall conversion frequency in the three tissues, the switching matrices of each pair of tissues were added. The switching matrix (which was symmetric) was then row-normalized to convert the switching counts to switching probabilities. This was done to avoid dependence on the numbers of different CSs.As a general resource of epigenomics, the comparison of all epigenomes is available for methylation data. The DMR resolution was set to 200 bp. The same process as in CS variability and switching was used to construct the transformation matrix to combine the changes in the overall genome DMR regions with histone modifications. Each element in the DMR matrix was divided by the number of CS transitions, relative DMR switching counts was obtained, and the matrix was row-normalized. The above calculation method (CG switching matrix) was used to process the CHG and CHH DMR switching matrices. The three kinds of DMRs exhibited the same pattern throughout the histone change at the overall level. There was a very small chance of a DMR relative occurring if a histone was not modified. However, some CSs switched in the respective DMRs of CHH, CG, and CHG had particular features.p value of 0.05 and log2 fold-change of >1.5 were used to find significantly differential histones. A comparison of peaks between any two tissues is shown in Supplementary Table Histone modification regions were defined based on the merged peak for each duplicate between tissues. The differential histone signal was also calculated using the DESeq2 package in R; an adjusted R > 0.52) with the expression level in tissues, in addition to young leaves and mature leaves, and the same was observed for H3K27ac (R > 0.3). However, H3K27me3 showed a negative correlation with gene expression in different tissues.Since changes in some histone modifications in tissues are not obvious, only three histones  were selected for further dynamic analysis. Using the distribution of different histones, if a 1-bp overlap existed between a histone and a promoter region of a gene, the changes in histone and gene pairs between any two tissues were identified. Among these, H3K4me3 showed a positive correlation  and candidate regions was performed by deepTools (2.5.3). DREs were linked to putative target genes based on nearby genes. Then, FAIRE-Seq signals of DRE were clustered in the three tissues. Based on these clusters, expression levels of the putative target genes were examined and found to be consistent with the determined clustering patterns. In order to analyze the PRE, we used the H3K4me3 loop from the H3K4me3 ChIA-PET datap value < 0.05) between each variety pair were compared using DESeq2. The percentages of histone differences among 20 varieties were compared. We randomly extract ten comparing combinations to simulate the saturation curve from each histone mark. Genes were divided into two categories, core (candidate core) genes and distributed genes, based on the annotations of core (candidate core) genes and distributed genes downloaded from the pan genome website (http://cgm.sjtu.edu.cn/3kricedb).All histone mark libraries were aligned to the Nipponbare (MSU v7.0) genome. Histone modification signal differences  with Xian/Indica or Geng/Japonica which is also in the 200-bp resolution. The CS resolution was set to 200 bp when we calculated the CS transfer from Xian/Indica to Geng/Japonica and heatmap of switch matrix was normalized by proportion of CS in genome. We computed each CS genome coverage of 20 varieties and normalized by Z score to get the genome coverage plot. NMDS plot was used, vegan R package and epigenetic signals were counted in 20 rice varieties.The distribution of SNPs within 2 kb on each side of a histone modification region in 10-bp units was calculated. To identify the boundaries between SNPs more precisely and to exclude the peak length interferences, the 1-kb region before the peak region was divided into 20 units, and the density of SNPs was calculated. We used the same CS model as MH63 Fig.  to trainFurther information on research design is available in the Supplementary InformationReporting Summary


VgR gene from Bombyx mori (BmVgR), and predicted many POU cis-response elements (CREs) in its promoter. Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that the POU transcription factor POU-M2 bound directly to the CREs of the promoter. Overexpression of POU-M2 in an ovarian cell line (BmNs) enhanced BmVgR transcription and promoter activity detected by quantitative reverse transcription PCR and luciferase reporter assays. Analyses of expression patterns indicated that POU-M2 was expressed in ovary at day two of wandering stage initially, followed by BmVgR. RNA interference of POU-M2 significantly reduced the transcription of BmVgR in ovary and egg-laying rate. Our results suggest a novel function for the POU factor in silkworm oogenesis by its involvement in BmVgR regulation and expands the understanding of POU factors in insect VgR expression.Vitellogenin receptors (VgRs) play critical roles in egg formation by transporting vitellogenin (Vg) into oocytes in insects. Although the function of VgR in insects is well studied, the transcriptional regulation of this gene is still unclear. Here, we cloned the promoter of the  VgR gene expression has not been well studied.Vitellogenin (Vg) is one of the main nutrients for oviparous animals in the embryonic stage ,2. The vVgR has been reported in only a few species. In 2004, the juvenile hormone (JH) analogue was reported to upregulate the expression of VgR in Solenopsis invicta in vitro [Aedes aegypti VgR (AaVgR), and a 1.5-Kb region upstream of its transcription start site was reported to be sufficient to drive tissue- and stage-specific expression of a reporter gene in transgenic animals [VgR transcript in the ovary, and an estrogen receptor subtype (Esr1) was found to inhibit the expression of VgR in the presence of 17β-estradiol (E2) in Micropterus salmoides [VgR in Nilaparvata lugens [Colaphellus bowringi [VgR, but also suggest that its regulatory mechanism varies between species. Bombyx mori is a well-studied model insect [B. mori (BmVgR) [Bombyx mori (BmVgR) gene. In addition, since VgR has a highly conserved modular structure and is critical for insect reproduction, it is of interest to investigate whether any conserved factors are involved in its regulation.So far, transcriptional regulation of almoides ,7. In 20a lugens  and Colabowringi . These sl insect ,12,13,14l insect ,16. Receunc-86 in Caenorhabditis elegans (C. elegans), were first identified and named as POU domain transcription factors [S), and a conserved homeobox domain (POUH). Generally, POUs binds to an ATGC sequence, while POUH associates with an A/T-rich sequence [B. mori. POU-M1 was initially cloned from the silk gland and binds to the octamer element of the sericin 1 gene, regulating its expression [diapause hormone and pheromone biosynthesis-activating neuropeptide (DH-PBAN) [steroidogenic enzyme genes, controlling insect ecdysteroidogenesis [et al. have found that POU-M2 is also involved in the regulation of the B. mori vitellogenin gene (BmVg) [VgR transcription in B.mori, or even in other insect species.The POU domain transcription factor is found to be highly conserved throughout the Bilateria . In 1988 factors . POU trasequence . In the sequence . Ventralsequence . To datepression ,26. AnotDH-PBAN) ,28,29. TDH-PBAN) . POU-M2 ogenesis . Recentle (BmVg) . HoweverVgR of six insect species. Then, in B. mori, we verified that a POU transcription factor, POU-M2, directly bound to these POU CREs, regulating BmVgR expression.Here, we found that many POU transcription factor cis-response elements (POU CREs) were located in the upstream regulatory region of B. mori strain Dazao was maintained at the Silkworm Gene Bank in Southwest University . Fertilized eggs were incubated at 25 °C and 85% relative humidity for hatching. Larvae were fed with fresh mulberry leaves (Morus sp.) at 25 °C, with a photoperiod of 12 h light/12 h dark and 75% relative humidity. Ovary samples were dissected from day 6 of the 5th larval instar (L5D6) to pupal day 3 (P4D) for RNA isolation and transcription analysis.The BmVgR translation initiation site was obtained from the silkworm genome database [BmVgR were analyzed for the presence of transcription factor-binding sites using the MatInspector program [2020 [A 2.5 kb upstream region of the database  as a pot program  and JASPam [2020 .BmVgR promoter fragment was inserted into the pGL3-basic luciferase vector  (named p-VgRP-2.5k) for analysis of the promoter activity analysis. Three deletion and mutated constructs of the BmVgR promoter reporter vectors were produced, i.e., p-VgRP-2.0k, p-VgRP-0.7k, p-VgRP-0.1k, p-VgRP-0.1kM. Mutation at predicted POU CREs in the BmVgR 0.1 kb promoter were synthesized by TSINGKE Biological Technology, Beijing, China.The entire 2.5-kb POU-M2 and EGFP genes were cloned into the psl 1180-Hr3-A4 SV40 expression vector (stored in our laboratory) for overexpression assays . Primers for the BmVgR promoter and POU-M2 amplification are shown in The open reading frames (ORFs) of the B. mori (stored in our laboratory) was cultured at 27 °C in IPL-41 medium  containing a 10% FBS . Transfection of overexpression and luciferase reporter vectors was performed using the X-treme GENE HP DNA transfection reagent . Luciferase activity was measured using commercially available kits  according to the manufacturer’s instructions. The vector p-IE1-Rlucp (stored in our laboratory) was used to drive the expression of Renilla luciferase via the B. mori nuclear polyhedrosis virus IE1 promoter as an internal control. The vector ratio (w/w) between p-VgRP-2.5k and p-IE1-Rlucp was 1:0.1. The ratio (w/w) between the reporter vector and overexpression vectors was 1:1. The measurement of luciferase activity was performed with the Dual Luciferase Reporter Assay kit , using the GloMax-Multi Detection System Photometer .The BmNs (corresponding to BmN-SWU1) cell line derived from the ovary of eukaryotic translation initiation factor 4A (BmTIF4A) gene was used as an endogenous control [-△△C (T) method [Total RNA was extracted using Trizol , and genomic DNA contamination was removed by RNase-free DNase I . The TransScript® One-Step gDNA Removal and cDNA Synthesis Super Mix  was used for constructing the cDNA library, according to the manufacturer’s instructions. qRT-PCR was performed using SYBR Premix ExTaq II  on a ABI7500 system . The following program was used for the amplification reaction: 95 °C for 3 min, followed by 40 cycles of 95 °C for 3 s, 60 °C for 30 s. The silkworm  control ,36. The ) method . Three i2). Migration of the biotin-labeled probe was detected by chemiluminescence, using the Super Signal West Femto Maximum Sensitivity Substrate .Electrophoretic mobility shift assay (EMSA) was performed using the Chemiluminescent EMSA Kit , following the manufacture’s protocol. In brief, DNA target probes including the POU elements were designed and labeled with biotin . Probes were annealed by incubating the complementary strands at 100 °C for 10 min, followed by slow cooling to 25 °C. To perform the EMSA, 2.5 μM of the annealed labeled probes were incubated with the supplied buffer, the purified POU-M2 protein (as described in ), and thTo confirm the binding of transcription factors to CREs, ChIP assays were performed with the EZ-ChIP kit , according to the manufacturer’s instructions. Briefly, BmNs cells transfected with the overexpression vectors and control BmNs cells were fixed with 37% formaldehyde to cross-link chromatin, and then sonicated to shear the cross-linked chromatin into fragments of 200-1000 bp in length. Immunoprecipitation assays were performed using mouse monoclonal antibody against Myc (anti-Myc) , anti-POU-M2, or nonspecific mouse/rabbit IgG antibodies. The DNA purified from the immunoprecipitated chromatin was used as a template for PCR amplification. The primers used to amplify the specific region near the potential POU-M2 CRE, as well as nonspecific regions, are listed in EGFP gene) were designed and synthesized by GenePharma  (POU-M2 and BmVgR. Injections were performed on days 0/1 of the wandering stage (W0D and W1D). Five micrograms siRNA for each gene were injected into the abdominal cavity of female larvae/prepupae with a glass needle. We used 30 larvae for each gene, and the ovaries were dissected on pupal day 3 (P3D) to verify gene expression. After eclosion, all injected female moths were mated with wild-type males, and the egg-laying rate was determined for statistical analysis.siRNAs targeting POU-M2 (nucleotide positions 441–459) and negative-control siRNA  . The siRt test with the following significance thresholds: * p < 0.05, ** p < 0.01 and *** p < 0.001. Statistical analyses were performed using Graph Pad Prism 6.0 . Figures were assembled using Adobe Photoshop CS5  and Adobe Illustrator CS5 .Statistical values are shown as means ± SEM. Mean values were compared using the Student’s BmVgR gene, we cloned a 2.5 kb sequence from its upstream regulatory region (Luc) by a dual luciferase reporter assay (DLR assay)  elements were located in the six promoter regions of VgR, especially in Lepidoptera  B,C. The R assay) D. A totaR assay) , includiR assay) E. In addidoptera . These fBmVgR expression, we compared the expression patterns of POU-M2 and BmVgR genes in the ovary at different developmental stages. Previous reports using reverse transcription PCR (RT-PCR) showed the expression of BmVgR mRNA was high in ovarian tissue at the wandering and pupal stages, when the translated VgR protein started to transport yolk protein for ovarian development [POU-M2 reached a peak at W2D  were co-transfected, along with the 1180-POU-M2 expression vector, containing the POU-M2 open reading frame into BmNs cells for assessment of luciferase activity. The BmVgR promoter activities were significantly enhanced by excess POU-M2 B. In add program  for prob program A,C. The  program C,E. We o program , and the program B. As sho program D. When t program . Subsequ program A,A’,A’’  program B,B’ coul program C,C’. ThePOU-M2 genes have been reported to regulate a cuticle protein gene (BmWCP4) [BmVg gene [B. mori treated POU-M2 dsRNA, the effect of POU-M2 on the BmVgR gene has not been reported in vivo. So, we conducted RNA interference (RNAi) of POU-M2 using siRNA. POU-M2-specific siRNA (POU-M2 was significantly reduced by POU-M2-specific siRNA injection (BmVgR upregulation was prevented (POU-M2 and BmVgR cause a significant impairment of ovary development.Although (BmWCP4)  and a BmmVg gene  in B. moic siRNA  was injeic siRNA A, and 24ic siRNA B, and thic siRNA C,C’. Mornjection D and BmVrevented D’. FurthVgR is known to be highly conserved among insects and other species. Yet, at present, there are few studies on its transcriptional regulation. In our study, we found that many POU CREs were located in the VgR promoter region of six insect species representing Lepidoptera, Diptera, and Hymenoptera. Moreover, we verified that a POU domain transcription factor, POU-M2, directly bound to the regulatory region upstream of BmVgR and activated its transcription in B. mori. Analyses of expression patterns suggested that POU-M2 was expressed initially in ovary at W2D, followed by BmVgR. Decreased BmVgR expression was observed in the ovary when POU-M2 expression was downregulated at the wandering stage of silkworm. There observations support the hypothesis that POU-M2 acts as a molecular switch to trigger the expression of BmVgR at the wandering stage.D. melanogaster, the POU domain transcription factor VVL has been shown to bind three distinct VVL recognition elements that are only weakly related to the octamer consensus motif and to each other [B. mori, POU-M1 has been reported to bind to the A/T-rich elements of the POU-M1 gene, but cannot recognize any of the octamer-like sequences in the upstream region of the POU-M1 promoter itself [sericin-1 promoter, indicating that such a binding specificity is probably important for its functional interaction [et al. reported that POUM2 could bind to the octamer-like sequences (CTTTACAT) of the silkworm wing disc cuticle protein (BmWCP4) gene [et al. reported its binding sequence as the A/T-rich elements (TTATTAAA) upstream of the BmVg gene [H subdomain. In other words, the DNA sequence recognition of POU domain proteins is flexible. In our case, POU-M2 can bind to the A/T-rich elements (P1/P5/P6) of the BmVgR gene with a lower affinity, and also bind to the octamer-like elements (P2/P3/P4/P7) with a higher affinity, indicating that multiple POU-M2-binding sites might be involved in the precise control of BmVgR gene transcription.A characteristic of POU-domain proteins is that they rely on their two specific subdomains to recognize diverse sets of DNA sequences. In general, POU domain transcription factors interact with promoter sequences based on the octamer consensus element ATGCAAAT ,44. Howech other ,46. In Br itself . Howevereraction . In 2012P4) gene , and in mVg gene , which mBmVgR expression was observed in the ovary, together with a low egg-laying rate. Although the reduction of the BmVgR gene by dsRNA injection can lead to abnormal egg development and low egg-laying rate [BmVg expression in the fat body [POU-M2 only in the ovary through genetic approaches may be possible to further determine the effect of POU-M2 on ovarian development. Nevertheless, the fact that POU-M2 suppression caused BmVgR downregulation in the ovarian tissue is an important reason for the reduction of the egg-laying rate.When POU-M2 expression was downregulated by siRNA injection, decreased ing rate , we cannfat body , as wellfat body . In the VgR expression in B. mori. VgR regulation by POU domain transcription factors may be a common phenomenon in insects, as well as in other oviparous species. In the past three decades, POU domain transcription factors were identified in insects and many other oviparous species, such as the fishes M. salmoides and Paralichthys olivaceus, the amphibian Xenopus laevis, and the nematode C. elegans [VgR promoters of six insect species representing three diverse taxonomic groups. It was also reported that POU CREs exist on the upstream regulatory region of the VgR gene in M. salmoides [VgR transcription.In this study, we verified that a conserved POU domain transcription factor, POU-M2, can upregulate  elegans ,50,51,52almoides . These cVgR is regulated by POU domain transcription factors. The lack of reports about the involvement of POU domain factors in oogenesis may due to their role as vital regulators of insect metamorphosis, such as that their mutation or knockdown may result in embryonic lethality or abnormal metamorphosis, which occurs before egg development. When we injected the siRNA of POU-M2, we chose the ninth abdominal segment of the silkworm for siRNA injection, not the second thoracic segment for dsRNA injection, as described by Deng et al. [BmVgR.Although transcriptional regulation during insect oogenesis has been extensively studied , there ig et al. . TherefoBmVgR promoter, thus inducing BmVgR expression and activating oogenesis in B. mori. This method of regulation may be conserved in insects. These results suggest a new function of the conserved POU transcription factor family in BmVgR regulation, and expand the understanding of the POU transcription factor in insect VgR regulation.We have found that conserved POU domain transcription factors directly bind to CREs in the


Regulation of oncogenic gene expression by transcription factors that function as tumor suppressors is one of the major mechanisms that regulate leukemogenesis. Understanding this complex process is essential for explaining the pathogenesis of leukemia as well as developing targeted therapies. Here, we provide an overview of the role of Ikaros tumor suppressor and its role in regulation of gene transcription in acute leukemia. Ikaros (IKZF1) is a DNA-binding protein that functions as a master regulator of hematopoiesis and the immune system, as well as a tumor suppressor in acute lymphoblastic leukemia (ALL). Genetic alteration or functional inactivation of Ikaros results in the development of high-risk leukemia. Ikaros binds to the specific consensus binding motif at upstream regulatory elements of its target genes, recruits chromatin-remodeling complexes and activates or represses transcription via chromatin remodeling. Over the last twenty years, a large number of Ikaros target genes have been identified, and the role of Ikaros in the regulation of their expression provided insight into the mechanisms of Ikaros tumor suppressor function in leukemia. Here we summarize the role of Ikaros in the regulation of the expression of the genes whose function is critical for cellular proliferation, development, and progression of acute lymphoblastic leukemia. IKZF1 have shown to have a poor prognosis in precursor B-cell acute lymphoblastic leukemia (B-ALL) [IKZF1 is a gene that encodes the Ikaros transcription factor that helps regulate genes controlling cell cycle progression and cell survival [IKZF1-inactivating mutations and/or deletions are seen more commonly in patients with other poor prognostic features including elevated white blood cell count, an age over 10 years old, and the Philadelphia chromosome. B-ALL patients with IKZF1 abnormalities have a reduced 5-year event free survival of 61% compared to the 87% for those without this abnormality. IKZF1 mutations and deletions are more commonly seen in B precursor ALL compared to T precursor ALL [IKZF1 genetic alterations occur both in childhood and adult B-ALL. ALL is the most common pediatric malignancy, and about 60% of ALL cases occur in patients that are younger than 20 years old. In adults, ALL represents only 20% of all acute leukemias, but it has much worse prognosis as compared to pediatric ALL. It was reported that approximately 50% of adult patients have IKZF1 genetic alterations, including over 80% of patients with BCR-ABL1 positive (Ph+) ALL [IKZF1 genetic changes are seen in approximately 15% of childhood B-cell ALL, including up to 70% of patients with BCR-ABL1 positive (Ph+) ALL [IKZF1 deletions to various degrees. Patients with IKZF1 deletions have been shown to have higher rates of induction failure , leading to poorer outcomes [IKZF1 deletions have also been found to have an increased risk of relapse and a reduction in overall survival [IKZF1 deletions treated according to standard therapy had a 12-fold increased risk of relapse [IKZF1 deletion was used to risk stratify and intensify therapy, patients with B-ALL had improved outcomes [IKZF1 to further risk stratify patients with B-ALL with the hopes of reducing relapse and improving long-term cures. The role of IKZF1 is less understood in T-cell ALL. IKZF1 mutations have been shown to play a role in up to 5% of T-ALL and as high as 11% of early T cell precursor (ETP) ALL [IKZF1 genetic and functional abnormalities were also studied and considered as novel prognostic biomarkers for high-risk leukemia in several clinical trials .Acute lymphoblastic leukemia (ALL) is the most commonly encountered malignancy in childhood with a long-term cure rate approaching 90% . Althoug (B-ALL) ,3,4,5. Isurvival ,3,4,5. Isurvival . IKZF1-ioutcomes ,12. Patisurvival . Those i relapse . In a stoutcomes . This shIkaros is a zinc finger protein with N-terminal DNA binding domains and C-terminal dimerization domains ,17. The Ikaros binds to the promoter regions of its target genes, repressing gene expression by formation of repressive chromatin through two distinct mechanisms ,21. DireThe phosphatidylinositol-3 kinase (PI3K) signaling network is a highly regulated pathway important for various physiological processes. This pathway has been implicated in various diseases, such as cancer, as well as in resistance to cancer due to its aberrant activation . DysreguThe cell cycle is a highly orchestrated process regulated by cyclins and cyclin-dependent kinases (CDKs). These proteins play crucial roles in differentiation, apoptosis and epigenetic regulation. Genetic defects in CDKs cause cancers including leukemia, and pharmacological inhibition of CDKs has become a therapeutic option . A studyKDM5B, also known as JARID1B, is a histone lysine demethylase involved in demethylation of histone 3 lysine 4 (H3K4). It has been shown to be mutated and overexpressed in several tumors ,27,28. IIKZF1 have significantly lower levels of PHF2 and that Ikaros positively regulates expression of PHF2. Biochemical experiments showed that expression of PHF2 is upregulated by Ikaros via chromatin remodeling, as evidenced by increased H3K4me3 occupancy at the promoter of the PHF2 gene. Overall, results of this study showed that PHF2 is downregulated in ALL, and that deletion and/or functional inactivation of IKZF1 might be one of the reasons for low levels of PHF2 in high-risk ALL. Low levels of PHF2 in combination with Ikaros deletion are the possible markers of high-risk ALL. Plant homeodomain finger 2 (PHF2) belongs to the Jumonji C (JmjC) class of proteins and encompasses a JmjC domain and plant homeodomain finger. PHF2 is involved in the demethylation of H3K9me2 . PHF2 isIKZF1 deletions have been observed in 43% of pediatric ALL with overexpression of CRLF2 [IKZF1 may be one of the reasons for elevated levels of CRLF2 in high-risk ALL with no CRLF2 rearrangement. Elevated levels of CRLF2 might co-operate with IKZF1 deletion, driving oncogenesis in ALL. The study demonstrated for the first time the existence of an IKZF1-CRLF2 pathway in high-risk ALL and suggested that targeting this pathway could be used as a therapeutic approach for high-risk B-ALL.Cytokine receptor-like factor 2 (CRLF2) is an IL-7 like cytokine. It plays a crucial role during normal hematopoiesis ,37,38,39of CRLF2 . Of lateof CRLF2 ,24,45,46of CRLF2 . In highJumonji (jmj) domain which plays a crucial role in histone modification and transcription. An important member of the ARID family of proteins is ARID5B, which recognizes the core DNA motif AAT(C/T) [ARID5B have shown that single nucleotide polymorphisms (SNPs) within ARID5B are critically affiliated with high-risk B-ALL [IKZF1 and ARID5B SNPs are positively correlated with ALL [ARID5B SNPs were missing. Ge et al. (2018) demonstrated that the deletion of a single copy of IKZF1 correlates with low expression of ARID5B and that ARID5B expression is positively regulated by Ikaros [IKZF1 and ARID5B SNPs with increased risk of ALL and suggest that alterations in SNPs may be associated with ALL due to reduced expression of Ikaros and ARID5B. Overall, the data suggest that the oncogenesis of high-risk ALL involves low levels of ARID5B expression, and that low expression of ARID5B and PHF2, along with haploinsufficiency of Ikaros, represents a high-risk subgroup of ALL.One of the most commonly dysregulated families of DNA-binding factors across multiple cancers is the AT-rich interactive domain (ARID) family ,49. The AAT(C/T)  and playAAT(C/T) . StudiesAAT(C/T) ,54,55. Osk B-ALL ,57. Furtsk B-ALL . Multiplwith ALL ,69,70,71y Ikaros . The resc-myc is a well-known oncogene whose overexpression is associated with various types of malignancies, including leukemia and lymphoma. MYC binding protein 2 (MYCBP2), a member of the PHR family of proteins is an E3 ubiquitin ligase . IncreasB-cell Lymphoma 6 (BCL6) is a proto-oncogene primarily identified in diffuse large B-cell lymphomas (DLBCLs) ,80. BCL6The increased expression of BCL6 regulates the protection and maintenance of leukemia stem cells and the survival of pre B-cells . In ALL BTB and CNC Homology 1 Basic Leucine Zipper Transcription Factor 2 (BACH2) is a transcriptional factor associated with the germinal center formation and affinity maturation of B-cells ,88,89,90IKZF1 deletion is associated with high BCL6 and low BACH2 expression in adult B-ALL patients [Recent studies showed that patients . Strong DNM2 were identified in pro-B ALL, with all of the mutant variants centric to the middle domain [Dynamin 2 (DNM2) regulates a variety of cellular processes including intracellular vesicle formation and trafficking, receptor endocytosis, interactions of actin and microtubule, cytokinesis, cell invasion and migration, and regulation of apoptosis ,103,104.e domain . Loss ofe domain . Ikaros binding to the promoter of DNM2 was observed in both B-ALL and T-ALL cells . OverexpIL7R is responsible for the differentiation of hematopoietic cells into lymphoid progenitor cells, and its level is altered during the development of T- and B-cells ,114. TheThe SH2B adaptor protein 3 (SH2B3), also known as lymphocyte adaptor protein (LNK), is a key negative regulator of the cytokine and tyrosine kinase signaling pathways, thus playing a crucial role in hematopoiesis ,127,128.IKZF1 due to deletion of one IKZF1 allele. Analysis of genome-wide occupancy in B-ALL showed that Ikaros binds to the promoters of both the IL7R and SH2B3. Gain-of-function and loss-of-function experiments demonstrated that Ikaros negatively regulates expression of IL7R and activates transcription of the SH2B3. Ikaros binding to the promoter of the SH2B3 is associated with enrichment in H3K4me3 occupancy. These data suggest that Ikaros directly regulates expression of IL7R and SH2B3.Recently, a subtype of adult B-ALL that has high expression of IL7R coupled with low expression of SH2B3 has been identified and found to be associated with a more severe clinical picture and poor prognosis . FurtherOverall, the above-summarized data show that Ikaros plays a highly important and potentially a critical role in regulation of IL7/JAK/STAT5 signaling by directly regulating transcription of at least three genes that are a critical part of this signaling pathway: CRLF2, IL7R and SH2B3.IKZF1 haploinsufficiency due to deletion of one IKZF1 allele). Identification of other Ikaros target genes would be important, both to gain insight into Ikaros function as a tumor suppressor, as well as to identify additional therapeutic targets for high-risk acute lymphoblastic leukemia.In summary, Ikaros is a potent transcription factor that regulates expression of a large number of genes in normal hematopoietic cells and in leukemia. Since Ikaros target genes have diverse roles in various cellular activities, Ikaros plays important roles in regulation of multiple signaling pathways and cellular functions via regulation of expression of its target genes . Some of


VDAC genes (VDAC1–3), their expression profiles, promoter activity, and potential transcriptional regulators. VDAC isoforms are broadly but also specifically expressed in various human tissues at different levels, with a predominance of VDAC1 and VDAC2 over VDAC3. However, an RNA-seq cap analysis gene expression (CAGE) approach revealed a higher level of transcription activation of VDAC3 gene. We experimentally confirmed this information by reporter assay of VDACs promoter activity. Transcription factor binding sites (TFBSs) distribution in the promoters were investigated. The main regulators common to the three VDAC genes were identified as E2F-myc activator/cell cycle (E2FF), Nuclear respiratory factor 1 (NRF1), Krueppel-like transcription factors (KLFS), E-box binding factors (EBOX) transcription factor family members. All of them are involved in cell cycle and growth, proliferation, differentiation, apoptosis, and metabolism. More transcription factors specific for each VDAC gene isoform were identified, supporting the results in the literature, indicating a general role of VDAC1, as an actor of apoptosis for VDAC2, and the involvement in sex determination and development of VDAC3. For the first time, we propose a comparative analysis of human VDAC promoters to investigate their specific biological functions. Bioinformatics and experimental results confirm the essential role of the VDAC protein family in mitochondrial functionality. Moreover, insights about a specialized function and different regulation mechanisms arise for the three isoform gene.VDACs (voltage-dependent anion-selective channels) are pore-forming proteins of the outer mitochondrial membrane, whose permeability is primarily due to VDACs’ presence. In higher eukaryotes, three isoforms are raised during the evolution: they have the same exon–intron organization, and the proteins show the same channel-forming activity. We provide a comprehensive analysis of the three human  The VDAC (voltage-dependent anion-selective channel) is the prototype of the family of subcellular pores responsible for the permeability of the mitochondrial outer membrane . This prVDAC genes codify proteins with apparently the same function, differences in their amino acid content, the mitochondrial outer membrane localization [In general, the distribution of VDAC isoforms is more or less ubiquitous in tissues but with a prevalence of VDAC1 isoform. Although the three lization , the chalization ,7, and tlization , lead toVDAC1−/−) are characterized by increased size, irregular and compacted cristae, and altered oxygen consumption either in oxidative either in glycolytic muscle fibers [VDAC2−/− knock-out were obtained and have been used to generate knock-out mice. However, the lack of VDAC2 determined embryo lethality [VDAC1/3 −/− mice; and VDAC3 −/− deficient male mice show the peculiarity of infertile, with a disassembled sperm tail, the flagellum essential for sperm motility [Results from various groups highlighted specific functions for each isoform, and it is considered a common notion that, for example, VDAC1 is a pro-apoptotic actor , while Ve fibers . Other ae fibers ,16. Impae fibers ,18. Embrethality . A signimotility . More remotility . The cysmotility . The thrmotility .Drosophila melanogaster VDAC1 mutants [Saccharomyces cerevisiae, two porin genes were found, but only POR1 coding VDAC1 is essential for life [Similar to VDACs knock-out mice, defects in the brain, muscle, and germinal tissues function were observed in  mutants . In the for life .A peculiar expression of VDAC isoforms was observed in cells and tissues of the germinal lines of different organisms. While VDAC1 is mainly located in cells of reproductive organs necessary to support the development of gametes ,25, VDACVDAC gene expression regulation have never been explored in a comparative and systematic way. Only one paper has been published, describing the organization and activity of mouse VDAC gene promoters [VDAC genes shows that the promoters lack the canonical TATA-box and are G+C-rich. The transcription start site (TSS) was identified, and the mouse putative VDAC promoters tested for their activity. In more recent years, few other reports have been published on VDACs promoter regulation for specific germinal lineage. The activity of VDAC2 promoter in a mammal’s developing ovary triggered by GATA1 and Cellular and viral myb-like transcriptional regulators (MYBL2) transcription factors lead to autophagy inhibition confirming its relevant role in cell survival [VDAC2 promoter correlated with idiopathic asthenospermia while in complete unmethylation or mild hypermethylation, sperm motility improved confirming the role of VDAC2 expression in human spermatozoa [VDAC genes structure and transcription from the main publicly resources. Once VDAC gene promoters were identified, we analyzed the sequences using bioinformatics software to identify transcription factor binding sites (TFBSs) distribution. We performed experimental tests using a molecular biology approach to confirm the insights obtained and to assess the transcriptional activity. We believe that understanding the molecular mechanisms triggering VDAC genes transcription in physiological and altered conditions might highlight the biological role of each isoform inside the cells and in different biological contexts.Mechanisms of romoters . A predisurvival . In humarmatozoa . The incrmatozoa . The preVDAC isoform, and to study the transcriptional control of the promoter structure via the analysis of TF-binding sites specificity and co-association patterns with other TFs.The vast amount of large-scale genomic projects, high-throughput sequencing, and transcriptomic data, as well as the plentiful supply of promoter resources, assist in the comprehensive reconstruction of a transcriptional regulatory region. The intersection of this data enabled us to provide a framework of the main functional DNA elements for the identification of active biochemical regions, which are commonly understood as gene promoter sequences of each VDACs, which helps to investigate the co-expression relationship and the context-dependent regulation of VDAC genes.The present work combines this evidence with information from gene expression profile datasets and with analysis of the promoter-specific activity of VDAC isoform to defined 600bp segment for PVDAC1 , PVDAC2 , and PVDAC3 , whose range is from −400 bp to +200 bp relative to TSS of annotated promoter sequences in EPD new (v.006). The boxed area in VDAC1, PVDAC2, and PVDAC3 matching with functional elements positioned within the region of accessible chromatin that define the nearest active promoter sequence. The structural organization of VDAC genes, transcripts, and the surrounding regions were analyzed.Our approach narrowed down the analysis of the promoter region of each The transcripts coding the three VDACs functional proteins are reported with the codes ENST00000265333.8 for VDAC1, ENST00000543351.5 for VDAC2, ENST00000022615.9 for VDAC3 corresponding respectively to NM_003374.2, NM_001324088.1, and NM_005662.7 in the Refseq database of NCBI.VDAC isoforms. Most of the splice variants have the same exons composition compared to the coding transcript but differ in the length of the 5′ and 3′. Some of them are processed transcripts, other features retained intron, and for VDAC3, two are involved in the nonsense-mediated decay mechanism. It is not known whether the other splice variants identified have any functional biological role. However, gene expression data collected from the NIH Genotype-Tissue Expression project (GTEX), reports their expression, including the non-protein-coding transcripts.Several transcript variants from the Ensemble database are also indicated for each The 600 bp-selected promoter region shows a high degree of overlap with the identification of the CpG island and the RNA polymerase II binding site close to the TSS, also confirmed by ChIP-seq data of chromatin-state model and the enrichment levels of the H3K4me1 and H3K4me3 histone marks, were chosen as the best predictors of transcription and open chromatin elements available among the UCSC regulation tracks.VDAC isoforms depict the level of VDAC transcripts distribution in different human tissues.The gene expression profile was highlighted through the analysis of available high-throughput data that have been included in international collaborative projects aiming to characterize human genome expression and regulation. In this work, data revised and curated by the Expression Atlas of EMBL-EBI , from GTThe RNA-Seq expression data from GTEx obtained using human tissue samples from post-mortem individuals, show that the level of VDAC mRNA expression seems comparable among the three isoforms but with a prevalence of VDAC1 a,b. A reRNA-seq cap analysis gene expression (CAGE) VDAC transcript expression was selected from the RIKEN FANTOM 5 project. It is well known that the enrichment of mRNA obtained using this technique is a map of TSS and gene promoter activity. In Although the data from the banks are based on experimental results, some comparison between them might look unexpected. The CAGE approach, indeed, is more focused on identifying productive RNA as mRNA and ncRNA. Further work will be necessary to explain in its entirety the regulation of these genes.VDAC3 gene transcription on other isoforms reflecting a higher promoter activity.In With this analysis, we can also confirm that VDAC isoforms are ubiquitously expressed in tissues, even if with different specificity for each isoform.VDAC1 gene was established as a reference gene, and quantification of VDAC2 and VDAC3 mRNA level was reported relative to VDAC1. In VDAC isoforms transcription was analyzed in HeLa cells, and a comparison of their expression level was performed. mRNA of the VDAC genes was identified as a putative promoter named PVDAC1, PVDAC2, and PVDAC3 and utilized for experimental characterization. These sequences were cloned in front of the Luciferase (Luc) reporter gene to study the activity of the human VDAC gene promoters in HeLa cells. Luciferase activity, driven by the indicated VDAC promoters, was compared among the three isoforms, as shown in the histogram of VDAC3 and VDAC2 promoters. These experimental results are consistent with the predominant transcriptional activity of VDAC3 and VDAC2 emerging from the FANTOM 5 project, suggesting a mechanism of fine regulation of VDAC genes expression. A 600 bp genomic region encompassing the TSS of p-value ≥ 0.001), which we schematically represented for PVDAC1 , downstream promoter element (DPE), and B recognition element (BRE). As typically observed in TATA-less promoters, multiple GC-boxes are required, and Inr and DPE are functionally analogous to the TATA box as they cooperate in the binding of TFIID in the transcription [VDAC2 , and the results were overlapped to find the most relevant TF families that regulate VDAC gene expression. We used a search window of −400 to +200 bp around the TSS.Identifying the upstream regulators of VDAC promoters’ sequences that were predicted by MatInspector software (Genomatix v3.10) and experimentally validated by ChIP-Seq data (ENCODE project v3). The ChIP-Seq peaks of V$E2FF, V$EGRF, V$KLFS, V$NRF1, V$MAZF, V$SP1F, V$ZF02, V$ZF5F were numerically the most overrepresented as highlighted by the bioinformatic prediction, confirming the importance of these factors in the regulatory network of VDAC genes. These binding sites are known to participate in several biological processes, such as cell growth, proliferation and differentiation, development, inflammation and tumorigenesis [VDACs promoter sequences and those exclusively present in each VDAC gene promoter, as reported by Genomatix analysis  transcription factors, affecting various processes of cell cycle regulation [The detection of common TFBS clusters indicates that these different classes of TFs participate in many similar activities and are mainly involved in cell proliferation and differentiation, apoptosis, and metabolism regulation. Therefore, it is possible to divide different classes of TFs involved in the control of gulation . The secgulation ,47. The gulation ,49,50.VDAC isoform. We found in the PVDAC1 sequence . The information we gained by bioinformatic analysis suggested the central role of VDAC protein expression in regulating mitochondrial function in fundamental cell processes. We recognized TFBS shared by the three VDAC promoters, as well as single promoters’ unique sites. Among the common ones, the majority of identified TFs classes belonged to the E2F-myc activator/cell cycle (E2FF), Nuclear respiratory factor 1 (NRF1), SP1, Krueppel-like transcription factors (KLFS), E-box binding factors (EBOX) families, which participate in many similar activities but are prevalently involved in cell proliferation and differentiation, apoptosis, and metabolism regulation [VDAC promoters. VDAC promoters were mainly characterized by a large number of recognition sites for E2FF and NRF1 transcription factors, which were found associated by chromatin immunoprecipitation with microarrays (ChIP-on-chip) to a significant subset of genes implicated in mitochondrial biogenesis and metabolism, other than mitosis, cytokinesis, cell cycle control, grow, proliferation [We also characterized the main transcription factors regulating the activity of gulation ,42,43,44feration . Many idferation .VDAC isoforms allowed us to hypothesize their involvement in specialized biological functions. However, the transcription factors specific for each VDAC genes were always correlated to essential functions ensuring cell survival and functionality. In general, these processes require a noteworthy energy cost: metabolism maintenance, development, organogenesis, dysfunction of mitochondria in pathology are some examples.Search for unique transcription factor binding sites in the promoters of VDAC1 promoter suggests that this isoform was probably selected by evolutionary process to have the prevalent role of channel protein in the mitochondrial outer membrane in physiological context and, in particular, when altered conditions force the cells to restore the mitochondria energetic balance [The families of transcription regulators identified as unique in  balance . These o balance .VDAC2 promoter highlighted the presence of different factors specially involved in developing specialized tissues and the organogenesis process as unique among VDAC promoters. Most of these factors are related to nervous system genesis and development.VDAC2 was indicated as the isoform carrying out channel function and governing apoptosis and autophagy in various contexts . AnalysiVDAC3 is controlled by the most active promoter: it is particularly rich in GC repetitions, suggesting an epigenetic control mechanism able to reduce the expression of transcripts. Factors binding sites found in VDAC3 promoter belong to various families, but those involved in the development of germinal tissues, organogenesis, and sex determination are the most abundant. In addition, in this case, the experimental evidence reported in the literature confirms the crucial role of VDAC3 in fertility [ertility .hVDAC1 (NM_003374), hVDAC2 (NM_001324088), and hVDAC3 (NM_005662) genes was carried out by high-quality promoter resource EPDnew version 006 (https://epd.epfl.ch). For study purposes, PVDAC1 , PVDAC2 , and PVDAC3  were the promoter sequences extended from −400 bp to +200 bp relative to annotated Transcription Start Site (TSS) of basal EPD promoter sequences . Tools used to scan for canonical core promoter elements and synergistic combinations were EPD Promoter Elements Page, YAPP Eukaryotic Core Promoter Predictor (http://www.bioinformatics.org/yapp/cgi-bin/yapp_intro.cgi), and ElemeNT (http://lifefaculty.biu.ac.il/gershon-tamar/index.php/resources). The core promoter elements with the higher consensus match and functionally recommended scores  were selected.Human promoter retrieval for Analysis of transcription factor binding site (TFBS) clusters was done by Genomatix software suite (Genomatix v3.10). MatInspector application was used to identify potential binding sites for transcription factors (TFBSs) in input sequence using the Matrix Family Library version 11.0 for core promoter elements in vertebrates with a fixed matrix similarity threshold of 0.85 , and thep-value ≥ 0.001, and UniBind, a comprehensive map of direct TF–DNA in the human genome based on public ChIP-seq datasets [TFBSs enrichment analysis was also performed using JASPAR (JASPAR CORE vertebrates collection 2020), the largest open-access database of position-specific scoring matrices derived from experimentally validated TFBS , by seledatasets .VDAC isoforms were investigated using the UCSC Genome Browser (https://genome.ucsc.edu). DNA regulation tracks of the UCSC Genome Browser and some information on promoter sequence and TFBS predictions are currently available on GRCh37/hg19.In view of implementing the TFBS clusters analysis, all information relevant to the genomic structure and DNA regulatory elements related to three https://www.ebi.ac.uk/gxa/home), a publicly available repository of selected RNA-seq, microarray, and proteomics datasets manually curated and analyzed through standardized analysis pipelines [VDACs genes were selected for the most common tissues and represented by a histogram and reported in a table.Gene expression data were collected from the Expression Atlas of EMBL-EBI open science resource , according to the manufacturer’s instructions. RNA concentration and purity were measured by a spectrophotometer, and 2 µg was used to synthesize cDNA by a QuantiTect Reverse Transcription kit . Real-time amplification was performed in a Mastercycler EP Realplex (Eppendorf) in 96-well plates. The reaction mixture contained 1.5 µL cDNA, 0.2 µM gene-specific primers pairs , and 12.5 µL of master mix . Three independent experiments of quantitative real-time were performed in triplicate for each sample. Analysis of relative expression level was performed by the ΔΔCt method using the housekeeping β-actin gene as an internal calibrator and VDAC1 gene as reference.HeLa cells were plated in 25 cmVDACs genes was selected from GenBank and cloned into pGL3 basic vector (Promega) for transcriptional activity study. The construct PVDAC1 contained the sequence  derived from VDAC1 NM_003374, PVDAC2 contained the genomic trait  from VDAC2 NM_001324088 and for PVDAC3, the sequence was  of VDAC3 NM_005662.A putative promoter region of 600 bp encompassing the TSS of the human 6 cells/well in a 6-well plates. After 24 h, cells were transfected with 800 ng of pGL3 constructs and 20 ng of pRL-TK renilla reporter vector by Transfast transfection reagent according to the manufacturer’s protocol (Promega), and after 48 h, cells were lysed. Luciferase activity of cell lysate transfected with pGL3 promoter constructs was detected with the Dual Luciferase Assay (Promega) according to the manufacturer’s instructions. The activity of firefly luciferase relative to renilla luciferase was expressed in relative luminescence units (RLU). variation of luminescence units of treated samples relative to control, were indicated as fold increase (FI).HeLa cells were plated at a density of 0.3 × 10p < 0.05, ** p < 0.01, and *** p < 0.001.Data are presented as means ± SD of results obtained from three independent experiments. All experiments were performed with n = 3 biological replicates and n = 3 technical replicates. Statistical significance was determined by one-way ANOVA. Significance was determined as reported and indicated as * In the present work, we proposed a general overview of the structural and functional organization of VDAC isoform promoters, cross-referencing public available data sources, bioinformatics prediction, and experimental data. The purpose of this work was, thus, to present experimental data extrapolated from Databases, with our experimental confirmation of the promoter sequences and activity by artificial constructs. From this analysis, emerges the essential function of the family of VDAC proteins in the regulation of energetic mitochondrial metabolism in physiological and pathological cell life. Moreover, we shed some new light on the molecular mechanisms that explain the differences among the three VDAC isoforms. It is becoming increasingly clear that the most known specialized functions of each VDAC isoforms are connected with the organization of the “button room” that decides the transcriptional activity of their genes and were produced by evolution. With this work, we established a starting point to pave the way for a deeper and wider analysis, an aim that we are pursuing.