Alphaherpesviruses comprise three members, herpes simplex virus (HSV) 1 and 2 and varicella zoster virus (VZV). These viruses are characterized by a lytic cycle in epithelial cells and latency in the nervous system, with lifelong infections that may periodically reactivate and lead to serious complications, especially in immunocompromised patients. The mechanisms that regulate viral transcription have not been fully elucidated, but the master role of the immediate early (IE) genes has been established. G-quadruplexes are non-canonical nucleic-acid structures that control transcription, replication, and recombination in many organisms including viruses and that represent attractive antiviral targets. In this work, we investigate the presence, conservation, folding and activity of G-quadruplexes in the IE promoters of the Alphaherpesviruses. Our analysis shows that all IE promoters in the genome of HSV-1, HSV-2 and VZV contain fully conserved G-quadruplex forming sequences. These comprise sequences with long loops and bulges, and thus deviating from the classic G-quadruplex motifs. Moreover, their location is both on the leading and lagging strand and in some instances they contain exuberant G-tracts. Biophysical and biological analysis proved that all sequences actually fold into G-quadruplex under physiological conditions and can be further stabilized by the G-quadruplex ligand BRACO-19, with subsequent impairment of viral IE gene transcription in cells. These results help shed light on the control of viral transcription and indicate new viral targets to design drugs that impair the early steps of Alphaherpesviruses. In addition, they validate the significance of G-quadruplexes in the general regulation of viral cycles.Human  The antntations . A numbentations . UV absontations . The Theop assay  and massop assay ,11. Finaop assay ,12,13.Herpesviridae virus family both at the genomic and mRNA level [Herpesviruses have been classified into three subfamilies, i.e., Alphaherpesvirinae, Betaherpesvirinae and Gammaherpesvirinae. The members of the Alphaherpesvirinae subfamily are characterized by a short reproductive cycle, rapid spread in culture, efficient killing of infected cells, and capacity to establish latent infections primarily but not exclusively in sensory ganglia. This subfamily contains the genera Simplexvirus  and Varicellovirus (VZV). The life cycle of these genera is characterized by a coordinated and sequential cascade of expression of three temporal classes of viral genes [G4s have been shown to regulate key cellular processes, including gene expression and mRNA translation, many of which are in turn linked to significant human disorders ,15,16. TNA level ,25,26,27NA level . These val genes ,30: the al genes , the proHerpesviridae family, i.e., HSV-1, HSV-2, VZV, HHV-4 and HHV-8, are statistically significantly enriched in G4 patterns [Herpesviruses.We have recently demonstrated and visualized the presence of G4s in the HSV-1 genome  and theipatterns . These dAlphaherpesvirinae  genome, in particular in promoters of IE genes, the major regulators of the life cycle of these viruses. The IE promoters contain multiple G4s on both leading and lagging strands; these viral G4s are in general stable and can be further stabilized by G4-ligands. Their folding results in inhibition of transcription. A significant parallelism in conserved G4s in key genomic regulatory regions within members of the same viral subfamily becomes apparent. Our results point for the first time to a G4-mediated regulation of the initial viral steps of this important class of human pathogens and may pave the way to a deeper understanding of Alphaherpesvirinae infection regulation.Here we show that conserved and stable G4s are present in crucial points of the ICP0, ICP4, ICP27 (UL54), ICP22 and ICP47 (US12) [G4s have been reported to regulate transcription, both at the cellular and viral level, when embedded in promoter regions ,24. The 7 (US12) . Our pre7 (US12) . However7 (US12) . The selICP22 and ICP47 genes [http://www.medcomp.medicina.unipd.it/main_site/doku.php?id=g4virus#data_download) in order to discard non-conserved regions. Interestingly, all identified PQS were fully conserved, both in terms of G-tracts and loop-length and -composition, in the entire set of fully sequenced genomes present in the National Center for Biotechnology Information (NCBI) databank. The HSV-1 IE promoters contained multiple PQS ; however, all of them retained the highest degree of conservation. PQS were retrieved on both strands: similarly to HSV-1 ICP22 and ICP47, ORF62 and ORF63 of VZV are transcribed in opposite directions from a 1.5-kb intergenic region that acts as a bidirectional promoter [ORF62 and ORF63 TSSs lie on opposite strands, thus PQS on the leading strand with respect to ORF63, on the lagging strand may act as regulators of ORF62, and vice versa. VZV is also the only virus with a predicted GG-island type PQS (ORF4 4278). Even if the TAATGARAT sequence is not present in VZV promoters, a TAATGARAT-VP16-like transcriptional enhancement can be mimicked by the ORF10 protein [ORF62, but not of ORF61, ORF4 or ORF63 [Alphaherpesviruses IE promoter is reported in HSV-2, has always been considered a close homologue of HSV-1, but recent research highlighted that the two viruses are less related than initially believed . Our anapromoter  and also protein . The ORFor ORF63 ,50. The Alphaherpesviruses is regulated by G4s.These data indicate the possibility that IE gene expression of Alphaherpesvirus IE promoter PQSs to form G4 structures was initially assessed by Circular Dichroism (CD), as this technique is considered a reliable tool to study nucleic acids conformation [+ to establish G4 folding, topology and thermal stability. The actual ability of ormation . All seqm) were calculated according to the van ’t Hoff equation  maintained their G4 folding up to the maximum tested temperature (90 °C). For hybrid structures, where two transitions were visible, Tm values were calculated at both wavelengths.The stability of all G4s was assessed by melting experiments monitored by CD. The melting temperatures , which has been reported to specifically recognize and stabilize G4 structures over double- and single-stranded nucleic acids [m higher than 10 °C : the first one was a putative bulged G4 that displayed G4-representative CD and TDS profiles, the second with long G-tracts and the possibility to form GC stem loops, had showed an ambiguous TDS profile. The Taq polymerase stop assay indicates the ability of a G4 to block or pause the enzyme activity and allows evaluation of G4 structures within an extended DNA environment. Samples were incubated in the absence and presence of increasing concentrations of KCl  were cloned upstream of the firefly luciferase gene in a promoterless plasmid. The two promoters were studied in U-2 OS cells, a human osteosarcoma cell line that withstands HSV-1 infection [50 U2-OS > 100 µM) to avoid luciferase signal variation due to cytotoxicity. As shown in ICP0 and up to ~55% in ICP27, proving that in this case G4s act as suppressors of transcription.The biological role of G4s at the promoter level is still matter of debate since little is known on how G4 structures and their interaction with transcription factors and other proteins may contribute to regulate viral promoters. Here, we decided to study the activity of two representative promoters, i.e., HSV-1  latency . In cont latency . The twonfection , in the Alphaherpesvirus IE promoters, as indicated by the B19-mediated G4 stabilization, which down-regulates ICP0 and ICP27 transcription.These findings support the hypothesis that G4s tune the activity of In the past few years, interest in the characterization of G4 structures and their role within viral genomes has greatly increased, providing possibly innovative antiviral targets against many human pathogens. In this context, our group proved that HIV-1 transcription is modulated by the tuned folding/unfolding of G4s located in the U3 region of the LTR promoter ,57. We sThe HSV-1 cycle is strictly regulated by five IE proteins , that are also responsible in vivo for the establishment of latency at the neuronal level ,60. The Alphaherpesvirus subfamily. The promoters were unambiguously identified based both on the presence of characterizing motifs, such as the TATA box and the TAATGARAT sequence and on previously reported biological evidence. In each promoter, we found several multiple and non-overlapping G4 folding motifs, interestingly located both on the leading and lagging strands. At the promoter level, G4 structures on the lagging strand have been shown so far only in few examples, in particular in the promoter region of the REarranged during Transfection (RET) proto-oncogene [Herpesviruses controls gene transcription at the promoter level through G4-mediated mechanisms, as reported for the human genome. ICP4 is the major viral transcription factor, and its promoter presents 5 G4s. The G4 most distant from the TSS is embedded in the lagging strand close to the TAATGARAT sequence, which is a recognition signal for the viral protein VP16 [ICP22 and ICP47 promoters, which share the same sequence albeit in two different locations along the genome, display a high number of G4s, also in this case located on both strands: we envisage the possibility of a control on both transcription and replication, since in the same region resides oriS, one of the origins of viral genome replication [ICP27 promoter is the one that contains fewer G4s. Four out of five ICP0 G4s, displaying low Tm (50 < Tm < 68 °C), were significantly stabilized by the G4-ligand, whereas ICP27 G4s were very stable even in the absence of B19 and thus were less affected by the G4-ligand. These features are likely the reason why we observed a lower degree of response to the G4-ligand B19 in cells for ICP27 compared to ICP0.In this work, we investigated the presence of G4s within the promoter regions of IE genes of all members of the oncogene ,62 and aoncogene  in mammaoncogene ,64. Promoncogene ,66,67, hein VP16 : it is plication . ICP0 pllication ,70,71. TAs HSV-1, HSV-2 and VZV are closely related viruses, all displaying a wide host-cell range, similar pathogenesis and analogous genome organization, we evaluated the presence of G4s also in HSV-2 and VZV IE promoters. The vast majority of the retrieved sequences, besides being fully conserved within all sequenced strains, were experimentally proven by at least two techniques to fold into G4 structures.Alphaherpesvirus IE promoter G4-forming sequences described in this work formed “non-canonical” G4s: they were able to fold even in the absence of loops  or in the presence of putative stem-forming sequences in the loop  or with bulges . This fact indicates that the classic prediction methods based on motif recognition may overlook many non-canonical G4s that actually form. In this view, the newest machine learning models trained with sequences of experimentally validated G4 may yield more reliable results [ICP4, HSV-1 ICP22/ICP47, HSV-2 ICP0, HSV-2 ICP27 and VZV ORF62/ORF63) presented 5 G-tracts; multiple G-tracts have been previously described for another strictly regulated viral promoter, the LTR sequence of HIV-1 virus [Finally, we found that many of the  results . Moreove-1 virus . These d-1 virus .Alphaherpesviruses.The described data provide a possible new direction for active antiviral design. Up to date, only polymerase inhibitors have been found active against HSV-1, HSV-2 and VZV. However, because of the emergence of resistant strains that may be extremely dangerous especially in transplant and immunocompromised patients , new antAlphaherpesvirus genome sequences were downloaded from GenBank. The first analysis was performed with free G4 hunting software (QGRS http://bioinformatics.ramapo.edu/QGRS/index.php and Quadbase http://quadbase.igib.res.in/), using the standard parameters of the two programs . Since Herpesviridae are dsDNA viruses, the presence of PQS was analysed on both genome strands.The complete set of human 2.Desalted oligonucleotides were purchased from Invitrogen and from Sigma-Aldrich, Milan, Italy , Table 3m, spectra were recorded over a temperature range of 20–90 °C, with temperature increase of 5 °C. The reported spectra are baseline-corrected for signal contributions due to the buffer. Observed ellipticities were converted to mean residue ellipticity (θ) = deg × cm2 × dmol−1 (mol ellip). Tm values were calculated according to the van’t Hoff equation, applied for a two-state transition from a folded to unfolded state, assuming that the heat capacity of the folded and unfolded states are equal [DNA oligonucleotides were diluted to a final concentration (4 μM) in phosphate buffer  and KCl 70 mM. All samples were annealed at 95 °C for 5 min and gradually cooled to room temperature. The G4-ligand Braco-19  was added from stock at final concentration of 16 µM. CD spectra were recorded on a Chirascan-Plus  equipped with a Peltier temperature controller using a quartz cell of 5 mm optical path length, over a wavelength range of 230–320 nm. For the determination of Tre equal .Taq polymerase stop assay was carried out as previously described [escribed . BrieflyHindIII sites. The resulting pGL4.10-ICP0 and pGL4.10-ICP27 vectors contained the sequenced regions corresponding to nts 1551-2261 (ICP0) and nts 113451-113734 (ICP27) in the HSV-1 reference genome (NC_001806.2), fused to the luciferase-coding region.The ICP0 and ICP27 promoter regions were amplified by PCR on the HSV-1 genome (GU734771.1) extracted from U2-OS infected cells. The promoter amplicons were subcloned into pGL4.10-Luc2 (Promega) within XhoI and 5 U2-OS cells per well onto 12-well plates, using Lipo3000 transfection reagent . B19 was added to the cell medium 1 h after transfection at increasing concentrations (5–20 µM), to avoid interference, if any, with transfection. Expression of firefly luciferase was determined 24 h after transfection using the Britelite plus Reporter Gene Assay System  at a Victor X2 multilabel plate reader , according to the manufacturer’s instructions. Cells were lysed in 0.1% Triton-X100-PBS and protein concentration was determined by BCA assay . Luciferase signals were subsequently normalized to total protein content, according to the manufacturer’s protocol . Each assay was performed in duplicate and each set of experiments was repeated at least three times.Vectors pGL4.10-ICP0 and pGL4.10-ICP27 (150 ng each) were transfected in 1.2 × 10https://www.lgcstandards-atcc.org). Cells were plated into 96-microwell plates to a final volume of 100 μL and allowed an overnight period for attachment. The following day, the tested compound (B19) was added to each well and tested in triplicate. Control cells were treated in the exact same conditions. Cell survival was evaluated by MTT assay, 24 h after treatment: 10 μL of freshly dissolved solution of MTT (5 mg/mL in PBS) were added to each well, and after 4 h of incubation, MTT crystals were solubilized in solubilization solution (10% sodium dodecyl sulphate (SDS) and 0.01 M HCl). After overnight incubation at 37 °C, absorbance was read at 540 nm. Data were expressed as mean values of at least three individual experiments conducted in triplicate. The percentage of cell survival was calculated as follows: cell survival = (Awell − Ablank)/(Acontrol − Ablank) × 100, where blank denotes the medium without cells. Each experiment was repeated at least three times.Cytotoxic effects were determined by MTT assay. U-2 OS cells were grown and maintained according to manufacturer’s instructions (Alphaherpesviruses that infect humans. In these viruses, regulation of gene expression has been largely debated in the last decades but clear data are still lacking. Thus, the fact that in all Alphaherpesviruses the promoters of all genes involved in the first steps of infection and in the control of expression of the later genes contain 100% conserved G4 forming regions is a potent indication of their significance. Our data broaden the boosting recognition of G4s as molecular switches of gene expression at the viral level [Alphaherpesviruses infection and latency in the human nervous system, with the subsequent possibility to design innovative drugs to manage the infection of some of the most widespread latency-associated human pathogens.The work presented here provides for the first time a comprehensive analysis on the presence of G4s in the IE promoter regions of the three al level . The in-


Cis-elements in lycopene β-cyclase genes (CpCYC-B and CpLCY-B) were identified, and followed by genome-wide analysis to identify transcription factors binding to these cis-elements, resulting in the identification of CpbHLH1 and CpbHLH2, two bHLH genes. The expressions of CpbHLH1/2 were changed during fruit development, coupled with transcript increase of carotenoid biosynthesis-related genes including CpCYC-B, CpLCY-B, CpPDS2, CpZDS, CpLCY-E, and CpCHY-B. Yeast one-hybrid (Y1H) and transient expression assay revealed that CpbHLH1/2 could bind to the promoters of CpCYC-B and CpLCY-B, and regulate their transcriptions. In response to strong light, the results of elevated expression of carotenoid biosynthesis-related genes and the changed expression of CpbHLH1/2 indicated that CpbHLH1/2 were involved in light-mediated mechanisms of regulating critical genes in the carotenoid biosynthesis pathway. Collectively, our findings demonstrated several TF family members participating in the regulation of carotenoid genes and proved that CpbHLH1 and CpbHLH2 individually regulated the transcription of lycopene β-cyclase genes (CpCYC-B and CpLCY-B). This study yielded novel findings on regulatory mechanism of carotenoid biosynthesis during papaya fruit ripening.The ripening of papaya is a physiological and metabolic process associated with accumulation of carotenoids, alternation of flesh color and flavor, which depending on genotype and external factors such as light and hormone. Transcription factors regulating carotenoid biosynthesis have not been analyzed during papaya fruit ripening. RNA-Seq experiments were implemented using different ripening stages of papaya fruit from two papaya varieties.  Many fleshy fruits, including papaya and tomato, have a dramatic flesh color change resulted from synthesized carotenoids accumulating in chromoplast during fruit ripening. The content of lycopene and β-carotene would have a significant increase from color-break stage to mature fruit, respectively, in red and yellow-fleshed papaya and tomato. As an adaptive characteristic, animals can be attracted by the changed pigments and disperse the matured seeds which are therefore able to germinate3. In papaya carotenoid biosynthesis pathway, with phytoene synthase (PSY) functioned, phytoene is synthesized by condensing two molecules of geranylgeranyl diphosphate (GGPP) in the plastids. Combining the phytoene desaturase (PDS) with ζ-carotene desaturase (ZDS), phytoene is further converted into lycopene which is the precursor of carotene. In nature, carotenoids usually exist in trans-structure. Only trans-lycopene can be catalyzed by downstream cyclase4. ZISO and CRTSO are key isomerases catalyzing the transformation of carotenoid cis-structure to trans-structure in plants. And the catalytic process also requires the participation of auxiliary factor flavin adenine dinucleotide (FAD)6. Afterward, one pathway lead to α-carotene via lycopene ε- and β-cyclase and the other result in β-carotene though chromoplast lycopene beta cyclase (CYC-B) or chloroplast lycopene beta cyclase (LCY-B) and further to zeaxanthin and lutein  for regulating tomato fruit ripening resulting from mutations in these TF genes which inhibit fruit ripening process, such as softening and color development10.During fruit ripening, carotenoid biosynthesis pathway is regulated by an genetically programmed mechanism that involves phytohormones and environmental factors such as, light, water, and temperatureSIAP2a and SIERF6 results in carotenoid accumulation and ethylene biosynthesis in tomato, suggesting that SIAP2a and SIERF6 act as negative regulators for carotenogenesis13. However, in Arabidopsis, these ethylene-responsive factors such as ERFs and RAP2.2 promote carotenoid biosynthesis by binding to PSY promoter14. In NAC family, carotenoid accumulation and ethylene synthesis can be positively regulated since SlNAC4 actively regulate expression of the ripening regulator RIN and cannot be induced by ethylene15. In Citrus, through directly binding to promoters, CsMADS6 upregulates the expression of PSY, PDS, and CCD1, implying CsMADS6 regulate the multi-targeted carotenogenesis16. Loss-of-function mutations in RCP1 belonging to MYB family led to downregulation of all carotenoid biosynthetic genes, suggesting RCP1 boosts carotenoid biosynthesis during flower development in Mimulus lewisii17. Many other important TF families, including HD-ZIP HOMEOBOX PROTEIN-1 (HB-1) and TAGL1, have been proven to play important roles in fruit development in many plant species19.Ethylene related TFs (EIN3/EILs and ERFs) regulate fruit development via implementing a transcriptional domino effect associated with ethylene-responsive genes. Reduced expression of 20. In high-pigment tomato mutants, such as hp1 and hp2, exposing to stronger light, anthocyanin and carotenoid will be accumulated. Meanwhile, in Arabidopsis, homologs of DDB1 and DET1 genes of hp1 and hp2 mutants showed negative regulation in light-mediated signaling pathway22. During tomato development, constitutive downregulation of PIF1 levels resulted in increased accumulation of carotenoids in the fruit because PIF1 directly repressed PSY gene expression23. Other results also showed that PIF1 and other PIFs promote PSY gene expression and carotenoid accumulation through responding to light signals during daily cycles of light and dark in mature Arabidopsis24.bHLH is a transcription factor family in fruit ripening because they can interact with environmental cues driving multiple aspects of downstream morphogenesis25. PAR1 and HFR1 interact with PIFs and keep them from matching the target genes’ promoters28. In recent decades, papaya fruit flesh has gained increased attention from breeders and consumers. Due to carotenoids content and composition, flesh color and nutritional quality have become increasingly important for fruit crop improvment29. CpCYC-B and CpLCY-B are crucial genes controlling flesh color and carotenoids profile in papaya. The red color of papaya flesh is from accumulating lycopene, while the yellow color is attributed to lycopene conversion to β-carotene and β-cryptoxanthin. Studying the regulatory mechanisms of CpCYC-B and CpLCY-B could lead to potential applications for improving fruit color and quality.Acting as transcriptional cofactors, bHLHs regulate target genes with other transcription regulators. Since bHLHs proteins are absent from an appropriate DNA-binding domain, they usually function as bHLH heterodimerbHLHs regulate CpCYC-B and CpLCY-B during papaya fruit development. To delineate the function of bHLHs on CpCYC-B and CpLCY-B, we conducted genome-wide identification of TFs targeting carotenogenesis by RNA-seq assay coupled with cis-element analysis and isolation for narrowing the numbers of TF families regulating CpCYC-B and CpLCY-B genes. More than 15 TF families were identified and 8 TFs were selected that may have positive or negative role in regulating CpCYC-B and CpLCY-B during fruit ripening. Yeast one-hybrid experiments and dual-luciferase transient expression assays demonstrated CpbHLH1 and CpbHLH2 can directly bind to the promoter upstream regions of CpCYC-B and CpLCY-B and individually inhibit or promote their transcription. Furthermore, we demonstrated that light might also involve in the regulation of CpbHLH1 and CpbHLH2 during fruits ripening.To unravel the molecular mechanism of papaya flesh color, we examined how Carica papaya L., cv. Hongling, SunUp, AU9) fruits at green, color break, and ripening stages were collected from experimental stations in Anxi and Yangzhong in Fujian, China. Two post-harvest treatments have been subjected: dark and light. During dark and light treatment, fruits were kept at 28 °C for 2 days. Fruits with the same morphology were selected, such as shape, maturity, weight, and without virus defects. All experiments will be biologically replicated with three samples after being frozen with liquid nitrogen or −80 °C.Red-fleshed papaya  protocol. The quality and concentration of total RNAs were checked on an Agilent 2100 Bioanalyzer. After matching the qualification, mRNA samples were synthesized as cDNA and further constructed into libraries according to NEBNext Ultra RNA library Pre Kit for Illumine . The cDNA libraries were sequenced using Illumina NovaSeq with paired-end 150nt read length. By analyzing the RNA-seq data, eight differentially expressed genes, named −ΔΔCt 30. CpTBP1 adopted as an internal standard in papaya31. All experiments were implemented with three biological replications. The final values were presented with the mean of three biological replications.The experiments of qRT-PCR were performed with above RNA libraries. The primers applying to qRT-PCR analysis were designed as shown in Supplementary Table CpZDS, CpPDS, CpLCY-E, CpCYC-B, CpLCY-B, and CpCHY-B were amplified from papaya genomic DNA (ftp://ftp.jgipsf.org/pub/compgen/phytozome/v9.0/Cpapaya) . Genomic sequences in promoters of CpCYC-B and CpLCY-B 5′ promotor sequences were amplied based on the annotated papaya genome at positions −0.5, −1.0, −1.5 kb (including −0.2/−0.3/−0.4/−0.5-absent element) and cloned into pDNOR207 vector using Gateway technology (Invitrogen). The targeting promoter fragments were then sub-cloned into pMDC162 and pGWB633 vectors using the Gateway conversion system. The Agrobacterium tumefaciens-mediated transformation system was implemented to transform pMDC162 and pGWB633-deletion promoters’ constructs into Arabidopsis Columbia (Col-0) ecotype.Deletion constructs of CpbHLH1 and CpbHLH2 was analyzed through the double luciferase reporter gene system in tobacco (N. benthamiana). As effectors the CDS sequences of CpbHLH1 and CpbHLH2 were inserted into the pGreenII 62-SK vectors, including a CaMV35S-Rluc and internal reference REN regulated by the 35S promoters (pG62-CpbHLH1 and pG62-CpbHLH2). Meanwhile, the promoters of CpCYC-B and CpLCY-B genes were cloned into the pGreenII 0800-LUC double-reporter vectors32. Through co-transforming the Agrobacterium, respectively, containing effector and reporter constructs into tobacco leaves, the luciferase activities of LUC and REN were obtained using luciferase assay kit (Promega). At least eight biological replications were performed. Luminoskan Ascent Microplate Luminometer (Thermo Fisher Scientific) was used to collect data.The transcription activity of CpbHLH1 and CpbHLH2 were cloned into pGADT7 to fuse with the activation domain in the vector. The promoter fragments and short repeat promoter elements of CpCYC-B and CpLCY-B were cloned into pHIS2 vector. Co-transforming PGADT7 vectors containing transcription factors and pHIS2 vectors embodying promoters into yeast strain Y187. Yeasts were cultivated on SD basic medium without Leu and Trp for 3 days. After successfully growing on double dropout plate, the yeast colonies were picked to plate onto triple dropout minimal medium (SD/-Leu/-Trp/-His). Using positive and negative controls, the possible interactions between CpbHLH1/2 and CpCYC-B/CpLCY-B were defined by their growth status. Y2H experiment protocol was followed. Yeast cells co-transformed PGADT7-CpbHLH1 + PGBKT7-CpbHLH2 and PGADT7-CpbHLH2 + PGBKT7-CpbHLH1 were grown on triple-dropout and four-dropout minimal medium that basic medium lacks Leu, Trp, His, and Ade.Y1H experiments were completed according to the Matchmaker Gold Y1H System protocol (Clontech). The CDS sequence of CpCYC-B, CpCHY-B, and CpLCY-B were significantly upregulated during fruit ripening; CpZDS, CpPDS, and CpLCY-E were also upregulated from stage 3 (50% ripening stage) to stage 5 (100% ripening stage)  and AU9  fruit at different developmental stages. To calculate reads per kilo bases per million reads (RPKM), significant expressed tags in different samples were identified. The differentially expressed transcription factors in SunUp and AU9 samples were identified based on the following criteria: CpCYC-B and CpLCY-B during papaya fruit development, we analyzed the promoter sequences in silico and constructed a series of chimeric genes containing truncated fragments of CpCYC-B and CpLCY-B promoters and GUS reporter gene. Promoters were analyzed for potential TFs binding sites using the PlantCare database. The potential regulatory elements in upstream −1.5 kb promoters of CpCYC-B and CpLCY-B have been summarized in Table cis-elements of CpCYC-B and CpLCY-B promoters. For example, bHLH binding site (-CANNTG-), was found at −113 and −164, respectively, in CpCYC-B and CpLCY-B promoters. The distribution of different regulatory elements in different region of promoters implied that some special elements played special function at different developmental stages.To test whether these transcription factors can bind promoters of cis-elements in transgenic Arabidopsis. Higher expression level of GUS gene was detected at −1.5 and −0.5 kb segments of CpCYC-B . These results indicated that −0.5 kb fragment of CpCYC-B and CpLCY-B promoter, respectively, can activate or inactivate the regulation of GUS gene expression. To further verify GUS gene expression patterns in transgenic lines, Arabidopsis transformed with CpCYC-B/CpLCY-B-GUS fusion gene were analyzed by histochemical staining  in CpLCY-B promoter.By in silico analysis and experimental validation, we identified that −0.5 kb promoter of CpCYC-B and CpLCY-B, we obtained four types of transcription factor families as shown in Fig. CpCYC-B and CpLCY-B, all of their binding sites existed in −0.5 kb CpCYC-B promoter, while, bHLH and ERF binding sites existed in −0.5 kb CpLCY-B promoter. The characterizations of four TF families were shown in Table CpbHLH1 (evm.model.supercontig_131.7), CpbHLH2 (evm.model.supercontig_8.253), CpbZIP1 (evm.model.supercontig_81.160), CpbZIP2 (evm.model.supercontig_9.75), CpNAC3 (evm.model.Supercontig_594.1), CpERF1 (evm.model.supercontig_131.83), CpERF2 (evm.model.supercontig_13.250), and CpbZIP3 (evm.model.supercontig_244.3). The transcription of the eight TFs was validated by RNA-Seq and qRT-PCR. The expression levels of CpbHLH1 and CpERF1/2 were decreased from the initial stage. As shown in Fig. CpNAC3 had been decreased from stage 2; the expression patterns of CpbZIP1/2/3 were random, and for example, the expression level of CpbZIP1 first increased, then decreased and finally increased again. For CpbHLH2, expression level first increased at color break stage, decreased from stage 2, and then increased significantly from stage 4. These results were further demonstrated by RNA-Seq data  experiment was performed. Yeast cells were co-transformed with the pGADT7-CpbHLH1 + CpCYC-B190bp promoter, pGADT7-CpbHLH1 + CpCYC-B190bp promoter mutant, pGADT7-CpbHLH1 + target repeated element, pGADT7-bHLH1 + repeated target mutation element, positive control and negative control would be cultured on basic SD medium lacking Leu and Trp element  failed to act with promoters of CpCYC-B/CpLCY-B  when either the CpCYC-B or CpLCY-B pro-LUC reporter construct was co-transfected with the CaMV35S-CpbHLH1 effectors, implying that CpbHLH1 repressed CpCYC-B and CpLCY-B expression activities  when previous promoters-LUC reporter constructs was co-transfected with the CaMV35S-CpbHLH2 effectors, indicating that CpbHLH2 may involve the activated regulation of CpCYC-B and CpLCY-B  in response to light, we collected SunUp fruit from S1 to S3 stage . Under 28 °C, papaya fruits of each stage were divided into two parts, one part was exposed to strong white light for 2 days, another part was kept in dark. After 2 days of treatment, expression levels of CpbHLH1/2, CpCYC-B, and CpLCY-B were examined by qRT-PCR. In Fig. CpCYC-B and CpLCY-B in light were higher than in dark. In contrast, the expression levels of CpbHLH1/2 were much lower in light than in dark. Especially, at stages S1 and S2 when the carotenoids have not been accumulated, the expression levels of CpCYC-B and CpbHLH2 were twice in light and in dark, respectively. At ripe stage S3, there was no obvious expression difference for CpbHLH1/2, CpCYC-B, and CpLCY-B. In conclusion, CpbHLH1/2 may be among the factors regulating CpCYC-B and CpLCY-B in response to light.To investigate if 35. Lycopene conversion into carotenoids in ripening papaya flesh was regulated by CpbHLH1/2 based on a molecular mechanism that is similar in Arabidopsis leaves or other plants in response to light24. In red grapefruit, lycopene content in shaded fruits is 49-fold than fruit exposing to light, implying shading- promoted carotenoid accumulation36.Carotenoids are very important pigments for plant growth and development, contributing to fruit flesh color and nutritional properties, and protecting the photosynthetic apparatus. In fruit, carotenoid accumulation could be promoted by light, but also could be harmed by excess light. The way their production is regulated by light is commonCpCYC-B and CpLCY-B promoters, these promoters were identified and isolated. By analysis of promoter fragments, −0.5 kb promoter sequences of CpCYC-B and CpLCY-B were considered as the effective region in response to fruit ripening  was 1.5-fold higher than non-transgenic fruits because SlbZIPtrans-activates the asparagine synthase and proline dehydrogenase genes38. In Citrullus colocynthis, higher transcripts of CcNAC1 and CcNAC2 following red light imply that NAC genes might function in light signaling pathway39. Besides, CpNAC1 was found to play a positive regulatory role in carotenoid accumulation through boosting CpPDS2/4 transcription by direct binding to their promoters34. When transferring Arabidopsis thaliana triose phosphate/phosphate translocator (tpt) mutants from low light to high light, transcripts expression level of four AP2/ERF genes, ERF6, RRTF1, ERF104, and ERF105 will absolutely and quickly decreased within 10 min, implying that AP2/ERF-TFs were responsive to strong light40. Through Y1H experiments, CpbHLH1/2 could bind to CpCYC-B and CpLCY-B promoters  CpbHLH2 might co-activate CpCYC-B and CpLCY-B expression with other activators. Protein–protein interaction assays suggested that CpbHLH1 could not physically interact with CpbHLH2, that they could not co-operatively regulate CpCYC-B and CpLCY-B (Supplement Fig. In papaya, the transcriptional regulation mechanism of carotenogenesis mediated by bHLHs is unclear. In current research, three models of transcriptional regulation by bHLHs were proposed. First, in dark or etiolated seedlings, responding to shade or night, PIFs mainly act as constitutive transcriptional activators. Under diurnal conditions, reversely, light will weaken or remove the activation of PIFs. A considerable portion of PIF-associated genes are upregulated in PSY was reported to be the only one gene that was controlled by PIF transcription factors involving light-mediated regulation in carotenoid pathway, because it is considered as the main-rate limiting enzyme of carotenoid pathway23. In current study, bHLH family members, CpbHLH1 and CpbHLH2 bind promoter regions of CpCYC-B and CpLCY-B and regulate their expression during papaya fruit development. Correlating with the decreased expression of CpbHLH1/2, expression level of the CpCYC-B and CpLCY-B was elevated during fruit ripening. We analyzed transcriptional regulation network and identified some potential family members regulating carotenoid biosynthesis genes in response to light or other factors during papaya fruit development in a genome scale. Our results first reported bHLH family members involved in the regulated network of carotenoid biosynthesis-related genes in papaya, which revealed two novel TFs of bHLHs and their regulation mechanism related to carotenoid biosynthesis genes in response to light. These findings contribute to uncover regulatory cascades, which could be directly or indirectly regulate fruit ripening in papaya responding to light.In previous studies, Supplementary figuresSupplementary Tables


The lack of telomerase/TERT expression leads to progressive telomere erosion in dividing human cells, whereas critically shortened telomere length induces a permanent growth arrest stage named replicative senescence. TERT/telomerase activation has been experimentally shown to be essential to cellular immortalization and malignant transformation by stabilizing telomere length and erasing the senescence barrier. Consistently, TERT expression/telomerase activity is detectable in up to 90% of human primary cancers. Compelling evidence has also accumulated that TERT contributes to cancer development and progression via multiple activities beyond its canonical telomere-lengthening function. Given these key roles of telomerase and TERT in oncogenesis, great efforts have been made to decipher mechanisms underlying telomerase activation and TERT induction. In the last two decades since the TERT gene and promoter were cloned, the derepression of the TERT gene has been shown to be achieved typically at a transcriptional level through dysregulation of oncogenic factors or signaling, post-transcriptional/translational regulation and genomic amplification. However, advances in high-throughput next-generation sequencing technologies have prompted a revolution in cancer genomics, which leads to the recent discovery that genomic alterations take center stage in activating the TERT gene. In this review article, we summarize critical mechanisms activating TERT transcription, with special emphases on the contribution of TERT promoter mutations and structural alterations at the TERT locus, and briefly discuss the underlying implications of these genomic events-driven TERT hyperactivity in cancer initiation/progression and potential clinical applications as well.Long-lived species Homo sapiens have evolved robust protection mechanisms against cancer by repressing telomerase and maintaining short telomeres, thereby delaying the onset of the majority of cancer types until post-reproductive age. Indeed, telomerase is silent in most differentiated human cells, predominantly due to the transcriptional repression of its catalytic component  Cancer formation/progression results from the accumulation of genetic mutations in cells . BecauseMechanistically, this telomerase repression and/or shorter telomeres in human cells function to prevent uncontrolled cellular proliferation . The lacTERT gene is stringently repressed in most human somatic cells, which consequently results in telomerase silencing. Thus, TERT is a rate-limiting determinant for controlling telomerase activity. Indeed, numerous studies have unequivocally demonstrated that TERT induction is required for human cells to acquire telomerase activity. Bodnar et al. , 22TERT  to +37) . Remarka to +37) . On the  to +37) . SeveralTERT is a single copy gene with a single transcription start site, but subject to alternatively splicing regulation [The gulation , 28. Morgulation , but theThe cloning of the TERT promoter and identification of its binding motifs for various transcription factors has contributed to profound insights into TERT/telomerase regulation in human cells , 27. TheTERT gene occurs at multiple levels by various positive and negative factors or signaling pathways. The TERT promoter is bound by different transcription factors, responds to numerous signals and integrates these diverse and dynamic inputs to set the TERT expression output [TERT transcription [TERT transcription by cooperating with transcription factors or other regulatory elements in a context-dependent or independent manner. All these regulators coordinately and tightly control the TERT gene to ensure its silence in most normal cells, while its expression at the right time, right place and right quantity only in a small subset of cells, such as activated lymphocytes and stem/progenitor cells [TERT transcription [TERT gene trans-activation by endogenous c-Myc during the transformation process of human fibroblasts, and they observed that the endogenous c-Myc expression resulted in a switch from Mad1/Max to c-Myc/Max binding to E-boxes on the TERT promoter, TERT expression and telomerase activation [The findings accumulated in the last 20 years have shown that the transcriptional regulation of the n output . Furthercription . In addior cells . Howeveror cells . The mosor cells –33. MYC or cells . Ectopicor cells . In leukcription . Casillativation . In additivation , 35–44.TERT transcription regulation. However, gene transcription involves not only the assembly of transcription factors at promoter/enhancer regions, but also the regulation of accessibility to DNA, a process controlled by an epigenetic mechanism [TERT transcription. DNA methylation, histone acetylation, methylation, and phosphorylation have all been shown to be involved in the TERT transcription regulation [TERT transactivation/repression in human cells [TERT transcription is activated. SMYD3-mediated H3-K4 tri-methylation is required for optimal occupancy of c-MYC and Sp1 on the TERT promoter [TERT transcription.The presence of transcription factors is essential to the echanism , 42, 45.gulation . The TERgulation –26. Histan cells , 42, 45.an cells , 32, 42.TERT transcription. These exogenous regulators include Epstein-Barr virus (EBV), cytomegalovirus (CMV), Kaposi sarcoma-associated herpesvirus (KSHV), human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell leukemia virus-1 (HTLV-1), and others [TERT transcription. E6 forms a tertiary complex with E6AP and c-Myc, and such complex then binds to the E-box in the TERT core promoter and subsequently induces promoter activation [TERT transcription by interacting with Sp1 [TERT transcription is one of the key mechanisms for virus-mediated carcinogenesis.In addition to the endogenous TERT regulators discussed above, certain tumor viruses encode proteins that stimulate d others , 48. Thetivation . The CMVTERT transcription.Advances in high-throughput sequencing technologies have enabled comprehensive genomic characterization of various human malignancies and numerous genomic aberrations have been unraveled. Barthel et al.  recentlyTERT promoter mutations occur predominantly at two hotspots of chromosome 5  with a cytidine-to-thymidine (C > T) dipyrimidine transition, thus named C228T and C250T, respectively Fig. . These mTERT transcription was shut down following cellular differentiation [The C228T mutation is more prevalent than C250T among various malignancies and their presence is mutually exclusive, which suggests their functional redundancy. Indeed, primary tumors bearing either mutation tend to express higher levels of TERT mRNA and telomerase activity, implying a stimulatory effect on TERT expression , 49. Crentiation . Moreoventiation . Similarntiation . Thus, tTERT transcription. It has now been clear that these mutations create a de novo binding site for ETS transcription factors [TERT transcription and telomerase . These observations raise a number of critical questions: How does GABPA regulate TERT expression in wt TERT promoter-carrying TC cells? Whether the effects of GABPA on the wt TERT promoter and cancer development/progression are context-dependent? Whether GABPA could serve as a therapeutic target for cancer? Elucidating these issues should be both biologically and clinically important.Surprisingly, however, our results obtained from thyroid cancer (TC) studies question it as a therapeutic target . First, TERT transcription [In addition to C228T and C250T mutations, CC > TT tandem mutations at position −124/−125 bp and −138/−139 (from ATG) were found in a subset of cancer , 60. Thecription . Tumors cription . HoweverTERT promoter mutations was selected for in blood cells from non-tumor individuals with very short telomere due to germline defects in the TERT protein or other telomerase components [TERT locus significantly affected the TERT promoter mutation rate in HCC patients, and the TERT rs2736100-CC cancer risk genotype was more frequently seen in patients with a wt TERT promoter [The TERT promoter mutation rate varies significantly from undetectable to more than 90% among studied human malignancies, and it remains poorly understood what cause such differential mutation distributions among different types of cancer . We as wmponents . We founpromoter . This vapromoter .TERT locus as novel mechanisms to upregulate TERT expression by hijacking enhancers  using multi-color FISH unraveled the TERT rearrangement in 4/8 MCL-derived cell lines and 1/23 patients [TERT gene [TERT locus were similarly observed and the highest frequency occurred in sarcoma (22%) [Zhao et al.  first nopatients . ImportaERT gene . HoweverERT gene –78   , Valentijn et al.  documenttivation . TERT reachieved . The TCGutations .TERT rearrangement has been most comprehensively dissected in neuroblasoma, a malignancy of the sympathetic nervous system in infants and young children [TERT gene in high risk/stage (III and IV) tumors with a frequency of 23% (39/169) , a recombination-based mechanism for telomere maintenance [The children –73. Peifchildren  observed69) Fig. . Similarntenance –73. TakeTERT transcription, as described above [TERT locus represents additional novel mechanisms for TERT regulation  A total of 4% the examined tumors exhibit a TERT gene amplification, with high frequency in ovarian cancer, adrenocortical carcinoma, esophageal cancer, lung adenocarcinoma, and squamous carcinoma  The highest telomerase activity is found in tumors with a TERT amplification. These results obtained from the TCGA cohort provide a general amplification profile of the TERT gene in human cancer, and indicate a key role of this genomic aberration in activation of telomerase during cancer development and/or progression.The amplification of genes encoding oncogenic products frequently occurs in cancer. For example, the diseases . Our eardiseases , suggestears ago , 85. In T copies . In othedetected , 60, 86.detected  analyzedoma Fig. ; (ii) TETERT locus, leading to a repressed TERT epigenetic state, however, shortened telomeres in aged cells disrupt the repressive loop, which consequently opens the closed chromatin structure and induces TERT transcription. Because very short telomeres are the most frequent genomic alteration in human tumors, TPE-OLD likely plays a part in TERT/telomerase activation in cancer cells. It is currently unclear whether or how much this mechanism contributes to cancer-related TERT transcription, and whether it cooperates with other regulatory cascades to activate telomerase. The elucidation of these issues will gain insights into not only telomere biology but also the relationship between age and cancer.Genes near telomeres are regulated by a mechanism that depends both on telomere length and on the distance to the gene, so-called telomere position effect (TPE) . More reCancer-specific TERT expression and telomerase activation has always aroused great enthusiasm for a potential clinical application of TERT/telomerase-based assays in the cancer field. However, a number of problems (such as unstable TERT mRNA and enzymatic activity) impede reliable utility of a direct TERT expression or telomerase activity assay for cancer diagnostic or monitoring purpose, whereas widespread TERT promoter mutations in different tumors pave new avenues . The nonTERT promoter methylation analyses as an additional tool in noninvasive cancer diagnosis and disease surveillance.Since many types of human cancer lack any TERT promoter mutations, and even in bladder cancer and glioblastoma, up to 30% of the tumors are negative for a mutation, other telomerase-related markers are apparently required for those patients. The hypermethylated TERT promoter has also been shown to be unique to human cancer as described above, and might serve as a diagnostic biomarker. We have recently identified two methylated CpGs in the TERT promoter region specific to tumors from patients with gastrointestinal cancer (GIC), and the methylated sites were detectable in stool from GIC patients, with sensitivity and specificity comparable to a fecal occult blood test . Bougel Numerous clinical studies have evaluated telomerase/TERT-related alterations as prognostic factors for cancer patients. Higher TERT expression in tumors was observed to predict poor patient outcomes in a panel of cancer types , 49, 92.TERT gene de-repression/telomerase reactivation. Thus, a mechanistic elucidation underlying cancer-specific activation of the TERT transcription is of intense interest to cancer research. During the last 20 years, this issue has been extensively studied. We learnt much from the cloning of the TERT gene and its promoter, and identification of its key transcription factors (both positive and negative), however, the findings made in the past 6 years have significantly contributed to our in-depth understanding of telomerase biology in cancer, and the genomic aberrations have started to take center stage: the hotspot mutation at 5’ regulatory promoter region and rearrangements at the TERT locus are emerging as new players in activating TERT transcription. It should be pointed out that these genomic aberrations stimulate TERT expression by triggering massive epigenetic alterations and disrupting repressive chromatins locally. Therefore, a close genomic-epigenetic interaction is required for telomerase activation, like TPE-OLD-mediated TERT transcription. Interestingly, TERT promoter methylation is required for cancer cells to activate TERT transcription, while TERT induction in turn promotes the aberrant methylation by upregulating the expression of DNA methyltransferases, which forms a positive feedback loop [Homo sapiens have acquired robust protective mechanisms against cancer by repressing telomerase coupled with shorter telomeres over a long evolution period. Erasing this natural barrier is required for malignant transformation of human somatic cells and in most cases, is achieved by the ack loop .TERT transcription, but also provide useful tools for clinical managements of cancer patients. However, a key challenge is how to translate these findings into a telomerase-based therapeutic strategy. The ETS family member GABPB1, required for the TERT transcription in TERT promoter-bearing glioblastoma cells, has been proposed as a target for glioblastoma treatment [These new findings not only lead to profound mechanistic understanding of the cancer-specific reatment , but ourreatment . Likely,


Understanding epigenetic mechanisms is crucial to our comprehension of gene regulation in development and disease. In the past decades, different studies have shown the role of epigenetic modifications and modifiers in renal disease, especially during its progression towards chronic and end-stage renal disease. Thus, the identification of genetic variation associated with chronic kidney disease has resulted in better clinical management of patients. Despite the importance of these findings, the translation of genotype–phenotype data into gene-based medicine in chronic kidney disease populations still lacks faithful cellular or animal models that recapitulate the key aspects of the human kidney. The latest advances in the field of stem cells have shown that it is possible to emulate kidney development and function with organoids derived from human pluripotent stem cells. These have successfully recapitulated not only kidney differentiation, but also the specific phenotypical traits related to kidney function. The combination of this methodology with CRISPR/Cas9 genome editing has already helped researchers to model different genetic kidney disorders. Nowadays, CRISPR/Cas9-based approaches also allow epigenetic modifications, and thus represent an unprecedented tool for the screening of genetic variants, epigenetic modifications or even changes in chromatin structure that are altered in renal disease. In this Review, we discuss these technical advances in kidney modeling, and offer an overview of the role of epigenetic regulation in kidney development and disease. Summary: In this Review, we provide an overview on how epigenetic processes are altered in kidney development and disease, and discuss how CRISPR-modified kidney organoids can help us to understand the function of epigenetic marks. Advanced glycation end products (AGEs): proteins or lipids that become glycated  by the covalent bonding of glucose or fructose as a result of exposure to sugars.Autosomal dominant polycystic kidney disease (ADPKD): an inherited condition that causes small, fluid-filled sacs called cysts to develop in the kidneys. Although children affected by ADPKD are born with the condition, it rarely causes any noticeable problems until the cysts grow large enough to affect the kidneys' functions. In most cases, this does not occur until a person is between 30 and 60 years of age.Chronic kidney disease (CKD): a kidney condition in which there is a gradual loss of kidney function over a period of months or years. Specifically, it implies structural or functional abnormalities of the kidney that induce decreased glomerular filtration rates and can lead to kidney failure.Chromatin immunoprecipitation-DNA sequencing (ChIP-Seq): a method used to analyze protein interactions with DNA. It combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins.Clear cell renal cell carcinoma (ccRCC): the major subtype of kidney cancer. It is characterized by frequent inactivation of VHL, which can be found in 80–90% of the cases.Collecting duct system: tubules and ducts that physically connect nephrons to a minor calyx or directly to the renal pelvis.Congenital abnormalities of the kidney and urinary tract (CAKUT): common congenital abnormalities, occurring at a frequency of ∼1 in 500 fetal ultrasound examinations. The molecular pathogenesis of the disease remains unclear, although numerous studies support the influence of epigenetic and environmental factors on kidney development and the natural history of CAKUT, suggesting that the pathogenesis of this syndrome is multifactorial.CpG island: regions >0.5 Kb with a high concentration of cytosine (C) and guanine (G) (≥55%). CpG islands are sites of transcription initiation, including those that are distant from the currently annotated promoters. CpG silencing in promoter regions is achieved through dense CpG methylation or Polycomb recruitment.Crotonylation: a post-translational modification of lysine residues on histones by the addition of crotonyl  groups.CRISPR/Cas9 technology: a genome editing system that uses the RNA-guided CRISPR-associated endonuclease Cas9 and a single-guide RNA (sgRNA) to induce a DNA double-strand break (DSB) at a specific DNA region of interest. This site specificity is determined by sequence complementarity with the sgRNA. The DSB can then be repaired through different pathways, leading to the creation small insertion and deletion mutations, or to the introduction of precise nucleotide alterations when exogenous homologous DNA templates are provided.Diabetic nephropathy (DN): a syndrome of albuminuria, declining glomerular filtration rate, arterial hypertension and increased cardiovascular risk that affects 25–40% of type 1 (insulin-dependent) and type 2 (non-insulin-dependent) diabetic patients. Diabetic nephropathy is the leading cause of end-stage renal disease (ESRD), present in ∼25–40% of patients with long-term diabetes worldwide.Dicer1: an enzyme that is part of the RNase III family. Dicer1 cleaves double-stranded RNA (dsRNA) and pre-microRNA (pre-miRNA) into microRNA. Dicer1 facilitates the activation of the RNA-induced silencing complex (RISC), which is essential for RNA interference.Doxycycline: a tetracycline-like antibiotic. In genetic engineering, doxycycline is used as the regulator for inducible gene expression systems, whereby expression of an engineered gene from a tetracycline-dependent promoter depends on either the presence (Tet-On) or absence (Tet-Off) of doxycycline.Drosha: the core nuclease that executes the initiation step of miRNA processing in the nucleus.Embryoid bodies (EBs): 3D cell aggregates that are generated in vitro by suspension culture of pluripotent stem cells. During the first 3 days in suspension culture, EBs can mirror the development of the three germ layers – the endoderm, mesoderm and ectoderm – of the mammalian embryo.End-stage renal disease (ESRD): kidney disease characterized by gradual loss of kidney function. In early stages, the disease is asymptomatic. Once the symptoms appear, the damage is irreversible.Focal segmental glomerulosclerosis (FSGS): a cause of nephrotic syndrome in children and adolescents, as well as a leading cause of kidney failure in adults. It is characterized by generalized edema, massive proteinuria, hypoalbuminemia and hyperlipidemia.Genomic imprinting: an epigenetic phenomenon that causes genes to be expressed in a parent-of-origin-specific manner. It involves DNA methylation and histone methylation. These epigenetic signatures are maintained through mitotic cell divisions in the somatic cells of an organism.Genome-wide association studies (GWAS): observational study of a genome-wide set of genetic variants in different individuals to assess if any variant is associated with a phenotypic trait. GWAS typically focus on associations between single-nucleotide polymorphisms (SNPs) and traits like major human diseases, but can equally be applied to any other organism or phenotype.Glomerular filtration rate (GFR): the flow rate of filtered fluid through the kidney.Glycolysis: the metabolic pathway that converts glucose into pyruvate. The energy released in this process is used to form the high-energy molecules adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH).Hemoglobin A1c (HbA1c): glycated HbA1c is a common indicator used in clinic to measure the average plasma glucose concentration in a patient. It is the product of an irreversible reaction in which a molecule of glucose attaches to the N-terminal valine of the hemoglobin β-chain. This irreversible reaction, together with the life span of erythrocytes (∼120 days) make HbA1c a good marker to monitor long-term glycemia.Histones: a family of basic proteins that associate with the DNA in the nucleus and compact it into chromatin.Histone crotonylation: post-translational modification of lysine residues in a histone by the introduction of crotonyl groups.Hypoxia: a condition of insufficient oxygen supply.HOTAIR: a gene located within the Homeobox C (HOXC) gene cluster on chromosome 12 and is co-expressed with the HOXC genes. It functions through an RNA product that binds lysine specific demethylase 1 (LSD1) and Polycomb repressive complex 2 (PRC2), and serves as a scaffold to assemble these regulators at the HOXD gene cluster, thereby promoting epigenetic repression of HOXD. It was the first example of an RNA expressed on one chromosome that has been found to influence transcription on another chromosome.Intermediate mesoderm (IM): the mesodermal lineage that is specified early in development between the paraxial and lateral plate mesoderm. Later in development, it gives rise to the urogenital structures, including the definitive kidney.Lin28A: an RNA-binding protein that binds to and enhances the translation of the insulin-like growth factor 2 (IGF-2) mRNA. Lin28 binds to the let-7 pre-miRNA and blocks production of the mature let-7 miRNA in mouse embryonic stem cells.Long non-coding RNAs (lncRNAs): transcribed RNA molecules with a length of more than 200 nucleotides that do not encode proteins.Mesenchymal-to-epithelial transition (MET): a reversible biological process that involves the transition from spindle-shaped mesenchymal cells to planar arrays of polarized cells called epithelia.Metanephric mesenchyme (MM): the embryonic cell lineage that arises from the posterior part of the intermediate mesoderm. During development, a subset of cells of the MM condenses around each ureteric bud (UB) tip to form the cap mesenchyme (CM), the multipotent population of progenitors that will develop into the nephrons, whereas the UB will constitute the collecting duct system. All the different cells present in the mature nephron derive from a population of Six2+ renal progenitor cells that reside in CM (de in CM .MicroRNAs (miRNAs): small non-coding RNA molecules (containing ∼22 nucleotides) that function in RNA silencing and post-transcriptional regulation of gene expression.Notch pathway: a highly conserved cell signaling system present in most multicellular organisms. Notch proteins play an essential role in developmental patterning, determining lateral inhibition and lateral induction : the metabolic pathway in which cells oxidize nutrients in order to release energy. ATP is formed as a result of the transfer of electrons to oxygen by a series of electron carriers. This process, which takes place in mitochondria, is the major source of ATP in aerobic organisms.Hox genes: a group of transcription factors that control the body plan of an embryo along the head-tail axis. Hox proteins define the types of appendages  or the different types of vertebrae (in humans) that will form on a segment.Safe harbor locus (AAVS1): a genomic locus that serves as an ideal site for the integration of a transgene without causing harm and with a consistent level of expression. In humans, there is a safe-harbor site on chromosome 19 (locus PPP1R12C) called AAVS1.TALEN: transcription activator-like effector nuclease. A genome-editing nuclease that can be engineered to bind and cleave any desired DNA sequence.Tiling arrays: a subtype of microarray chips. They are similar to traditional gene expression microarrays, but they contain probes for functionally uncharacterized regions through the genome. This technique is useful for characterizing regions that are sequenced, but the local functions are largely unknown.Trithorax/Polycomb: a group of proteins that regulate gene expression in an opposite manner through development. The main action of Trithorax proteins is to maintain gene expression, while Polycomb proteins have a repressive function. Polycomb proteins are well known for silencing Hox genes in Drosophila melanogaster and regulate homeotic gene regulation and X chromosome inactivation in mammals. Trithorax proteins are also necessary for X chromosome inactivation and activation of Hox genes. Both groups of proteins regulate gene expression by modulating the chromatin structure.Ureteric bud (UB): a protrusion from the mesonephric duct that forms during the development of the urinary organs. It later develops into a conduit for urine drainage from the kidneys.Wnt signaling: a group of signal transduction pathways with key functions in embryonic development. Three Wnt signaling pathways have been characterized: the canonical Wnt pathway, the non-canonical planar cell polarity pathway and the non-canonical Wnt/calcium pathway. All three pathways are activated by binding a Wnt protein ligand to a Frizzled family receptor, which passes the biological signal to the Dishevelled protein inside the cell. They are highly evolutionarily conserved in animals, from fruit flies to humans.X chromosome inactivation: a process by which one of the copies of the X chromosome present in female mammals is inactivated. The inactive X chromosome is silenced by it being packaged in such a way that it has a transcriptionally inactive structure.Epigenetics have traditionally been linked with developmental biology. In fact, the first definition of epigenetics came from a developmental biologist, Conrad Hal Waddington, who defined the term in the early 1940s. Since this first definition, the concept and role of epigenetics has evolved. Nowadays, we understand epigenetics as regulatory mechanisms that stabilize gene expression patterns without altering the DNA sequence. Rather, these are mediated by heritable chemical modifications to DNA and histones  3A and 3B, while DNMT1 maintains DNA methylation through cell divisions. Gene expression activation is often associated with demethylation/hypomethylation of promoter and enhancer sequences, which can be achieved through inhibition of DNMT1 or through oxidation of 5-mC into 5-hydroxymethylcytosine (5-hmC) by the ten-eleven-translocation (TET) proteins (TET1–3). 5-hmC is then converted to unmodified cytosine by DNA-replication-dependent dilution or by glycosylase-initiated base excision. So far, a growing number of studies have correlated aberrant DNA methylation with human disease. In addition, DNA methylation regulates important processes, including imprinting  nucleotide, giving rise to 5-methyl-cytosine (5-mC). In humans, printing , cell diprinting  and X chprinting .Post-translational modifications of histonesHistones are a family of small basic proteins that envelop DNA, forming an efficient DNA-packaging unit called nucleosome, which is the basic structural and functional unit of chromatin. Post-translational modifications (PTMs) of histones occur at their amino-terminal ends (histone tails), mainly on specific lysine (K) and arginine (R) residues, and can affect DNA availability for transcription factor binding, thereby regulating gene expression via changes in chromatin structure. The combination of different histone marks, or the histone code, in genomic regulatory regions, such as enhancers and promoters, induces either transcriptionally permissive (euchromatin) or repressive (heterochromatin) chromatin states. In general, histone PTM-mediated chromatin organization affects complex biological processes, including gene expression , DNA repHistone methyltransferases (HMTs) add methyl groups to histones, while histone lysine demethylases (HDMs) remove them. HMTs and HDMs are positive or negative regulators of gene transcription, depending on the amino acid, histone tail position or the number of methyl groups in the PTM . In general, histone demethylation is quite dynamic, whereas methylation is more unvarying and linked to long-term cellular epigenetic memory. Histone lysine methylation (HKme) can be associated with either active or inactive promoters, depending on the methylated lysine (K). In general, trimethylation at H3K9 (H3K9me3), H3K27 (H3K27me3) and H4K20 (H4K20me3) correlates with inactive genes, whereas trimethylation at H3K4 (H3K4me3) and H3K36 (H3K36me3) correlates with promoters of actively transcribed genes.Histone acetylation involves the addition of acetyl groups tovarious histones and is mediated by histone acetyl transferases (HATs). The reverse process, histone deacetylation, is catalyzed by histone deacetylases (HDACs). Histone lysine acetylation (HKac) marks – such as H3K9ac, H3K14ac, and H4Kac – are associated with active promoters. Furthermore, enzymes like kinases and phosphatases can also regulate the phosphorylation status of histones. Phosphorylation of H2A has proved to play a major role in DNA damage response .Histone phosphorylation can occur in all four nucleosomal histone tails. These possess acceptor sites that can be phosphorylated by a number of protein kinases  and dephosphorylated by phosphatases. So far, a large number of phosphorylated residues on histones have been described. The best-known function of histone phosphorylation takes place during cellular response to DNA damage, when phosphorylated histone H2A(X) demarcates chromatin domains around the site of DNA damage (break). Nowadays the identification of new phosphorylation sites and characterization is still under investigation. The current knowledge on histone phosphorylation, their regulation and function in modulation of chromatin structure and gene expression has been extensively reviewed elsewhere .Histone sumoylation is a histone modification directed by an enzymatic cascade in which small ubiquitin-like modifier (SUMO) proteins are attached to, or detached from, other proteins to change their function in cells. Different works have revealed a general causal relationship between sumoylation and gene repression. However, the dual role of SUMO in gene regulation is demonstrated by the observations that sumoylation of certain transcription factors, including Ikaros , enhances transcriptional activity. On the other hand, inducing hypersumoylation by targeting SUMO and/or Ubc9 to specific gene promoters primarily induces gene repression . ConversIn vitro, histone crotonylation by p300 has been shown to promote transcription, and increasing the cellular concentration of crotonyl-CoA affects gene expression  represent a diverse family of regulatory transcripts that are transcribed from DNA, but not translated into proteins . ncRNAs act as negative post-transcriptional regulators via RNA silencing or translational inhibition. A significant number of studies have revealed the putative role of both classes of ncRNAs in conditions such as heart failure, cardiac autophagy, hypertension and various kidney disorders . Many faEpigenetic modifications include DNA methylation, post-translational modifications of histones, and long non-coding RNAs lncRNAs;  Lyon, 1, genomicHOTAIR , and of HOTAIR , act at HOTAIR . AlthougHOTAIR , our undDefining the epigenetic landscape of renal progenitor cells is a difficult task. During embryonic kidney development, chromatin structure dynamically directs cell fate decisions by controlling the activation or repression of genes in a precise spatiotemporal manner . AberranHere, we summarize the most recent literature describing how epigenetic processes affect kidney development and disease, and discuss the interplay of these epigenetic events during renal dysfunction. We next introduce the potential use of kidney organoids derived from human pluripotent stem cells (hPSCs) combined with genome engineering technologies, particularly the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9  or arginine (R) residues  is a keyhigh/Wntlow – the mK3 line) and induced (Six2low/Wnthigh – the mK4 line) MM cell populations. The mK3 line expresses genes characteristic of the early mesenchyme, including Hoxa11, Hoxd11, Twist1, Vim and the Col1a gene family. Conversely, mK4 cells express genes characteristic of MET, such as Pax8 or Lhx1  lost the H3K4me3 active mark, whereas Lrp2 and Setdb1 genes gained the active mark H3K4me3 In general, temporal changes in histone modifications are not restricted to embryogenesis. To investigate whether these epigenetic changes also occur in early postnatal life in mice, Lui and colleagues performed ChIP promoter tiling array , compariJag1, which encodes jagged 1, facilitated podocyte dedifferentiation, increasing the susceptibility to focal segmental glomerulosclerosis   . Indeed,(CTNNB1) . When in(CTNNB1) . Recentl lesions . More re lesions  is a dir lesions . InteresImportantly, emerging evidence reveals that shifts in cellular metabolism affect the enzymes that control genome-wide epigenetic configuration, thereby modulating chromatin reorganization and gene expression changes during phenotypic reprograming and differentiation. Glycolysis  generallDicer1 knockout mice, whereby the miRNA biogenesis enzyme Dicer1  and from cells of the UB-derived collecting duct system (HoxB7-Cre-mediated). The authors demonstrated that Six2-Cre-mediated removal of Dicer1 resulted in elevated apoptosis and premature termination of nephrogenesis, and thus confirmed that Dicer1 is important for maintaining the viability of the Six2 self-renewing progenitor pool and, consequently, for the development of a normal nephron complement. Conversely, HoxB7-Cre-mediated removal of Dicer1 caused the development of renal cysts in this model   and lncRe Dicer1  was remoe Dicer1 . Similarfibrosis , and pod  associated with Feingold syndrome, a developmental defect in humans. The authors generated a transgenic mouse with a conditional deletion of the miR-17/92 cluster in the nephron progenitor pool and their derivatives . Although the nephron progenitor population was preserved in these mice, miR-17/92 deletion impaired proliferation and reduced the number of developing nephrons. Interestingly, mutant mice developed signs of renal disease. Overall, this study supports a role for the miR-17/92 cluster in the regulation of nephron development, with later consequences for renal function in adult mice .UMOD, SHROOM3 and STC1  consortium defined CKD as abnormal kidney structure or function lasting for at least 3 months and having implications for health . The finand STC1 . After tand STC1 . Additiog others . We refeg others . DNA seqg others . So far,g others , 2016. Ug others .CDKN2A expression  to bind to its cognate receptor and regulate phosphorus and vitamin D metabolism. The soluble form of klotho has anti-aging properties, which may be mediated via multiple systemic effects, including regulation of insulin signaling . Metabolic memory is induced by irreversible changes in oxidative stress, non-enzymatic glycation of proteins, epigenetics and chronic inflammation . HoweverEmerging evidence suggests the critical role of epigenetic mechanisms in the pathogenesis of DN. The impact of DNA methylation on DN has raised attention in the past 10 years. A study by Ko and colleagues analyzed DNA methylation in kidney tubular epithelial cells, and showed significant differences in the methylation of core pro-fibrotic gene enhancers, correlating with downstream transcript levels . InteresApart from DNA methylation, recent studies have implicated histone deacetylases HDACs;  in diabeAs discussed above, miRNAs have a role in nephrogenesis, but most of the studies published until now have been focused on pathologies such as renal carcinoma, DN, cystogenesis and glomerulopathies . In DN, RASAL1 promoter  gene locus to cooperatively demethylate H3K9me2 and construct long-range looping interactions between the promoter and the distal enhancer, maximizing the hypoxic induction of GLUT3 and, consequently, increasing glucose uptake . Hypoxia is one of the main hallmarks of the beginning of CKD. Moreover, hypoxia also induces epigenetic changes. The oxygen molecule is required for the activity of the ten-eleven translocation (TET) enzymes, oxidation enzymes that catalyze the conversion of the modified DNA base 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) . Bechtelpromoter . Subsequ  tumor suppressor gene, is inactivated, HIFs are constitutively active and drive tumorigenesis and growth. Alterations in VHL are the most prevalent molecular features of clear cell renal cell carcinoma  and Sirt3 as potential target genes of histone crotonylation regulation has important, although not yet completely understood, implications in kidney disease. The following sections discuss new promising approaches to improve our understanding of these implications.One of the major roadblocks in developing therapies for CKD has been the lack of reliable preclinical models. For many diseases, mice have been instrumental in understanding the mechanisms underlying pathogenesis, but their utility in studying CKD has been limited because they fail to recapitulate important functional, structural and molecular features of advanced human kidney disease. In this regard, hPSCs represent an unprecedented resource for the generation of functional renal cell types suitable for disease modeling and cell replacement therapies. Typically, differentiation of hPSCs starts with the induction of formation of embryoid bodies EBs; ; Fig. 2.in vivo equivalents , PKD1 and PKD2 by transiently transfecting undifferentiated hPSCs with plasmids expressing the Cas9 nuclease and the synthetic chimeric guide RNAs (gRNAs) targeting these genes. Upon clonal expansion of the biallelic knockout lines, they further differentiated these targeted hPSC clonal lines for functional analysis. PODXL-defective kidney organoids exhibited junctional organization defects in podocyte-like cells, while PKD1 or PKD2 knockout organoids showed cyst formation from kidney tubules . Indeed, kidney organoids grown from patient-derived induced pluripotent stem cells (iPSCs) may not reflect disease-relevant phenotypes, but instead they represent the variation in the genetic backgrounds from the starting cell population . Genome KD2 gene . Later,  tubules , a pheno disease . One of  disease . Czernie disease .Cas9 gene into the mammalian safe harbor locus AAVS1  could also soon benefit from the organoid technology combined with CRISPR/Cas9 genome engineering. One possibility to identify target genes for distal candidate functional SNPs would be to modulate the chromatin state of the regulatory element with dCas9 fused to a chromatin-modifying domain . For exaCombining kidney organoids exposed to environmental conditions that mimic disease-related contexts  with the constantly evolving genome editing toolbox may allow us to perform site-specific recruitment of engineered chromatin regulators to modulate epigenetic marks at DNA regulatory elements under these experimental conditions. These approaches could improve our understanding of the function of the epigenetic marks that underlie kidney development and disease. Moreover, the combination of genome and epigenome editing tools in the organoid setting could help uncover novel molecular pathways associated with the genotypes detected by global analyses, including GWAS and EWAS, by elucidating genotype-epigenotype interactions.in vitro cellular model that can help to unravel the molecular mechanisms driving kidney differentiation and, importantly, disease. The organoid technology represents a powerful tool that can pave the way towards the development of high-throughput systems for the screening of genetic variants, epigenetic modifications or changes in chromatin structure that drive renal disease. Kidney organoids may also help researchers bridge the translational gap for the implementation of genetic and epigenetic therapies into the clinic. Altogether, we hope that the next few years will bring us closer to a new era of knowledge, taking advantage of the application of genome editing tools and stem cell bioengineering for the identification of novel therapeutic strategies targeting the renal epigenome during disease.In the past decades, several studies have demonstrated the impact of epigenetic modifications on renal disease and its progression towards CKD and ESRD, improving our understanding of these conditions. However, the development of innovative approaches for diagnosis, prevention and treatment of CKD represents a major challenge for the nephrology community. The emergence of GWAS has greatly expanded our knowledge of risk alleles related to CKD and ESRD. However, for most of the reported CKD-associated loci, the effects on disease pathogenesis remain unknown. Recently, researchers have shown that it is possible to recapitulate the development of renal cells from undifferentiated hPSCs derived from human embryos and from patients (iPSCs). Kidney organoids are an unprecedented


Nuclei of eukaryotes contain various higher-order chromatin architectures and nuclear bodies (NBs), which are critical for proper nuclear functions. Recent studies showed that active chromatin regions are associated with nuclear speckles (NSs), a type of NBs involved in RNA processing. However, the functional roles of NSs in 3D genome organization remain unclear.Srrm2 depletion disrupted the NSs and changed the expression of 1282 genes. The intra-chromosomal interactions were decreased in type A (active) compartments and increased in type B (repressive) compartments. Furthermore, upon Srrm2 knockdown, the insulation of TADs was decreased specifically in active compartments, and the most significant reduction occurred in A1 sub-compartments. Interestingly, the change of intra-TAD chromatin interactions upon Srrm2 depletion was not associated with the alteration of gene expression.Using mouse hepatocytes as the model, we knocked down SRRM2, a core protein component scaffolding NSs, and performed Hi-C experiments to examine genome-wide chromatin interactions. We found that Srrm2 knockdown causes a global decrease in chromatin interactions in active compartments, indicating critical functions of NSs in the organization of the 3D genome.We show that disruption of NSs by The online version of this article (10.1186/s13072-019-0289-2) contains supplementary material, which is available to authorized users. The nuclei of higher eukaryotes contain various 3D architectures, which are critical for proper nuclear functions such as transcription and RNA processing. As revealed by Chromosome Conformation Capture (3C)-based techniques, interphase chromatin organizes into multi-scale 3D structures including genomic compartments, topologically associating domains (TADs), and chromatin loops , 2. The The molecular mechanism of loop domains formation has been revealed in recent years. CTCF and cohesin were proposed to be essential proteins for the loop domains, following chromatin extrusion model . Indeed,Besides the higher-order chromatin, the nucleus contains many nuclear bodies (NBs). These NBs are dynamic, membrane-less architectures, which are involved in various nuclear functions . It has Nuclear speckles (NSs), also known as interchromatin granule clusters (IGCs), are nuclear domains in the interchromatin regions of the nuclei, which are enriched in various snRNPs, splicing factors and other factors involved in mRNA 3′ end processing and m6A modification . Recent NSs contain hundreds of proteins, including many splicing factors . Among tSrrm2 gene expression in mouse hepatocytes (AML12 cell line) with two short-hairpin RNAs (shRNAs). Srrm2 was knocked down by two independent shRNAs, which lead to a significant reduction in mRNA and protein levels of Srrm2  produced similar results  obtained through the principal component analysis of the Hi-C contact matrices. The tracks of PC1 values showed that the genomic locations of A and B compartments were largely unchanged upon Srrm2 knockdown . Furthermore, the scaling of contact frequencies as a function of genomic separation was not changed  was used to define TAD boundaries for each replicates in control and SRRM2-depleted samples  in interaction were enriched in A (B) compartments with the highest (lowest) PC1 values  are associated with NSs , 14. To The contact domain structure was regarded as the basic unit of the compartment. We used two steps to assign the contact domains to sub-compartments A1, A2 and B. Firstly, the contact domains were assigned to either A or B compartment using the PC1 eigenvectors at 10 kb resolution. The average PC1 values for each contact domain were then calculated; if the domain’s average PC1 score was greater than zero, it was defined as A-domain. Otherwise (if less than zero), the domain was defined as B-domain.K-means clustering (K = 2); this procedure divided A-domain into two clusters. We then calculated the enrichment of chromatin marks  in the two clusters for each chromosome. Finally, we defined the more active clusters as A1 and less active clusters as A2 for each chromosome. Each A1 cluster in A-domain was defined as A1-domain; otherwise, they were designated as A2-domain.Secondly, the inter-chromosomal contact information was used to divide A-domain into A1-domain and A2-domain further. Then, the average inter-chromosomal interaction was calculated (O/E) between A-domain and 20 new contact matrix for each chromosome were generated . In the new matrix, each row represents the A-domain in one chromosome , each column represents the A-domain in the other chromosomes , and each data point in the matrix represents the average interaction (O/E) between A-domains in different chromosomes . Next, the rows of these matrices were clustered by The inter-chromosomal contacts were most apparent among A1 sub-compartments Fig. a. QuantiSrrm2 knockdown caused a substantial reduction in the interaction within the A1 sub-compartments -compartments: A1, A2 and B. By calculating the average interaction intensity of these three groups of TADs, we found that nts Fig. a. We furnts Fig. b. We havnts Fig. : Fig. S6own Fig. d, e.Fig.Srrm2 knockdown: the higher the ranking, the greater the decrease. With the decrease in the rank, the percentages of A1 and A2 sub-compartments declined, while the percentages of B compartments increased. Consistently, the gene expression levels in control cells reduced with the ranking. However, the relative gene expression upon Srrm2 knockdown remained unchanged in all groups, suggesting that alterations in intra-TAD chromatin interactions did not change gene expression.Next, the relationship between intra-TAD interactions and gene expression was investigated Fig. f. The TASrrm2 knockdown reduces chromatin interactions at active chromatin regions, and the most substantial decreases occur in the A1 sub-compartments. Based on these findings, we propose a model on how NSs participate in the 3D organization of chromatin  although TAD interactions were reduced in A1 sub-compartments, the functions of TADs as transcription units were preserved; (2) changes in gene expression may be due to secondary effects resulting from changes of chromatin organization.We found that Although the genomic regions close to NSs are associated with higher levels of Pol II binding, it remains unclear whether this association is implicated in gene activation. In this study, we showed that disruption of NSs by SRRM2 knockdown leads to the decrease in intra-TAD interactions at A1 sub-compartments. However, no significant gene expression changes were found to be associated with these chromatin changes. These results suggested that there was no direct cause-and-effect relationship between NS association and the transcriptional activity of individual genes.Interestingly, targeted degradation of cohesin removed most of the chromatin loops, but only a small number of genes in the super-enhancers were changed . FurtherSrrm2 knockdown, the localization of MALAT1, U1 snRNAs, poly(A)+ RNAs and SC35 became scattered, indicating that Srrm2 is involved in organizing NSs [As one of the major types of nuclear bodies in the nucleus, the NS is associated with various proteins and RNAs . Throughzing NSs , and thiBesides, given that NSs contain many components, including proteins and noncoding RNAs, disrupting NSs by depleting other molecules may change other aspects of the 3D genome organization. Further efforts would be needed to fully decipher the roles of NSs in nuclear architecting and their physiological and pathological relevance.Srrm2 knockdown reduces chromatin interactions in active chromatin compartments, especially in the A1 sub-compartments. These results indicate essential functions of NSs in the organization of the 3D genome.We demonstrate that disruption of NSs by 2. U2OS cells were cultured in DMEM  supplemented with 10% fetal bovine serum , nonessential amino acids (Gibco) at 37 °C and 5% CO2. AML12 and U2OS cells were authenticated by STR profiling.The mouse hepatocyte cell line alpha mouse liver 12 (AML12) and human osteogenic sarcoma cell (U2OS) were obtained from ATCC. AML12 cells were cultured in DMEM/F12 (HyClone) supplemented with 10% fetal bovine serum , ITS Liquid Media Supplement (Sigma) and 0.1 μM dexamethasone (Sigma) at 37 °C and 5% COFor short-hairpin RNA (shRNA)-based knockdown, AML12 or U2OS cells were infected with shRNA-containing lentivirus as previously described . Target Cells were fixed in 4% formaldehyde for 10 min at room temperature prior to cell permeabilization with 0.5% Triton X-100 . The cells were blocked with phosphate-buffered saline (PBS) containing 4% bovine serum albumin (BSA) for 0.5 h at room temperature and processed for immunostaining. Staining was done using mouse anti-SC35 , rabbit anti-SRRM2 , rabbit anti-HNRNPU  and donkey anti-mouse Alexa Fluor 488 . Fluorescence images were taken and analyzed with a Leica confocal microscope .Mouse_Srrm2_F:CTGCAAGAATGTCCCAGGTT;Mouse_Srrm2_F:ATGCCGGAATAGCAGATGTC;Human_SRRM2_F:CTGACTCTGCTTCCTCCTCC;Human_SRRM2_F:CTGAAAGGCGCATCTCCCT;Mouse_Actb-F:GGTCATCACTATTGGCAACG;Mouse_Actb-R:ACGGATGTCAACGTCACACT;Human_ATCB-F:ACTCTTCCAGCCTTCCTTCC;Human_ATCB-R:TGTTGGCGTACAGGTCTTTG.RNA extraction and qRT-PCR were conducted as described . The mRNThe cell lysates were blotted against primary antibodies as follows: anti-β-actin  and rabbit anti-SRRM2 . The blots were visualized with peroxidase-coupled secondary antibodies.3 size samples for further treatment with 1% osmic acid in PBS and gradient dehydration with alcohol. The sample was treated with a mixture of epoxypropane  and resin , followed by embedding with pure resin. Then the sample was sliced with a diamond tool kit. Target cells that were randomly selected were captured with a transmission electron microscope .Electron microscopy experiments were performed as described , 26. AMLThe in situ Hi-C libraries were prepared as previously described . Two bioK-means clustering (K = 2) with the observed/expected ratios of inter-A-domain contacts in trans. Domains in each cluster exhibit distinct signatures based on the enrichment of chromatin marks. We defined more active clusters as A1 and less active clusters as A2.To assign contact domains to sub-compartments A1, A2 and B, we use the following methods. Contact domains were assigned to either A or B compartment by the PC1 eigenvectors. We calculated the average PC1 values for each contact domain; greater than zero is defined as A-domain, and less than zero is defined as B-domain. Next, we clustered A-domains by For RNA-seq experiments, polyA RNA-enriched and strand-specific libraries were constructed with the VAHTS Total RNA-seq (H/M/R) Library Prep Kit , and the libraries were sequenced using the Illumina HiSeq X Ten system (Annoroad Gene Technology Corporation). We trimmed and mapped reads to the mouse mm9 reference assembly by the TopHat2 software  using deP value less than 0.05 was accepted as significant; *P < 0.05, **P < 0.01, ***P < 0.001.All statistical calculations are included in the relevant figure legends. Additional file 1: Figure S1. Disruption of nuclear speckles by Srrm2 knockdown in AML12 cells. a Gene expression of Srrm2 genes analyzed by RT-qPCR in AML12 cells. Error bars: s.d. of three biological replicates. ***P < 0.001, n.s. = not significant; Student’s t test. b Western blot analysis with antibodies against specified proteins; β-Actin as loading controls. c, d Immunofluorescence analyses of SRRM2, SC35 (c), and HNRNPU (d) in AML12 cells. The depletion of Srrm2 disturbed nuclear speckles (NSs) as shown by the immunofluorescence data of the NS marker SC35. Scale bar, 10 µm. e Quantification of SC35 signal between control and Srrm2-depleted samples . f Gene expression of hepatic genes analyzed by RT-qPCR in AML12 cells. Error bars: s.d. of three biological replicates. ***P < 0.001, n.s. = not significant; Student’s t test.Additional file 2: Figure S2. Disruption of nuclear speckles by SRRM2 knockdown in U2OS cells. a Gene expression of SRRM2 genes analyzed by RT-qPCR in U2OS cells. Error bars: s.d. of three biological replicates. ***P < 0.001, n.s. = not significant; Student’s t test. b Western blot analysis with antibodies against specified proteins; β-Actin as loading controls. c, d Immunofluorescence analyses of SRRM2, SC35 (c), and HNRNPU (d) in U2OS cells. The depletion of SRRM2 disturbed nuclear speckles (NSs) as shown by the immunofluorescence data of the NS marker SC35. Scale bar, 10 µm.Additional file 3: Figure S3. Nuclear speckles were abolished after Srrm2 knockdown by electron microscopy (EM). a EM analysis of nuclear speckles stained by APEX fused to SC35 in AML12 cells. The image on the left was the control, on the right was Srrm2-depleted cells. Arrows point to nuclear speckles. Scale bar, 1 µm. b EM images of U2OS cells stained by HRP. Arrows point to nuclear speckles. Scale bar, 2 µm. c EM analysis of nuclear speckles in AML12 cells. The image on the left was the control, on the right was the Srrm2-depleted cell. Arrows point to nuclear speckles. Scale bar, 2 µm.Additional file 4: Figure S4. Gene expression changes upon Srrm2 knockdown. a Scatter plot showing gene expression changes detected by RNA-seq in Srrm2-depleted AML12 cells. Compared with the control, 844 genes were up-regulated, and 438 genes were down-regulated significantly. b, c Gene ontology (GO) chart of up-regulated (b) and down-regulated (c) genes. The enriched terms are ranked by − log10 . d Venn diagrams show the overlap of genes between differentially spliced genes (DSGs) and differentially expressed genes. e Gene ontology (GO) terms of differentially spliced genes. The enriched terms are ranked by − log10 .Additional file 5: Figure S5. Calling the TAD boundaries. a Distance to the nearest boundary between replicates. b Venn diagrams show the overlap of TAD boundaries between replicates for control or Srrm2 knockdown cells.Additional file 6: Figure S6. Differential contact map of intra-chromosomal inter-TADs showing fewer  and more  Hi-C signal after Srrm2 depletion AML12 cells.Additional file 7: Table S1. Differentially expressed genes upon Srrm2 knockdown in AML12 cells.Additional file 8: Table S2. Mapping statistics of the Hi-C data.Additional file 9: Table S3. Coordinates (mm9) of TAD boundaries in Control and Srrm2 knockdown samples.Additional file 10: Table S4. Coordinates of sub-compartment regions in AML12 cells.