Leishmania donovani is a parasitic protist that causes the lethal Kala-azar fever in India and East Africa. Gene expression in Leishmania is regulated by gene copy number variation and inducible translation while RNA synthesis initiates at a small number of sites per chromosome and proceeds through polycistronic transcription units, precluding a gene-specific regulation . Here, we analyze the dynamics of chromatin structure in both life cycle stages of the parasite and find evidence for an additional, epigenetic gene regulation pathway in this early branching eukaryote. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) analysis  predominantly shows euchromatin at transcription start regions in fast-growing promastigotes, but mostly heterochromatin in the slowly proliferating amastigotes, the mammalian stage, reflecting a previously shown increase of histone synthesis in the latter stage.IMPORTANCELeishmania parasites are important pathogens with a global impact and cause poverty-related illness and death. They are devoid of classic cis- and trans-acting transcription regulators but use regulated translation and gene copy number variations to adapt to hosts and environments. In this work, we show that transcription start regions present as open euchromatin in fast-growing insect stages but as less-accessible heterochromatin in the slowly proliferating amastigote stage, indicating an epigenetic control of gene accessibility in this early branching eukaryotic pathogen. This finding should stimulate renewed interest in the control of RNA synthesis in Leishmania and related parasites. The nuclear DNA of eukaryotes is known to be highly and reversibly organized into DNA-protein assemblies known as chromatin, which allows compacting, stabilization, and regulated access to chromosomes or parts thereof. The smallest unit of chromatin is the nucleosome, which consists of an octamer of four basic proteins, histones H2A, H2B, H3, and H4, around which a short length of DNA, 146 bp, is wrapped .cis-regulatory DNA sequences , Chagas disease (Trypanosoma cruzi), and also cutaneous leishmaniasis  and visceral leishmaniasis . These so-called trypanosomatidic infections are among the most important of the neglected tropical diseases, causing widespread, poverty-related human morbidity and mortality in tropical and subtropical regions around the world (https://www.who.int/data/gho/data/themes/topics/gho-ntd-leishmaniasis)  .7 bp) than those of mammals (∼ 3 × 109 bp), encoding ∼8,200 protein-coding genes, which for the most part lack introns  and of antigen-presenting cells (APCs), e.g., macrophages, in their mammalian hosts. In the sandflies, they exist as elongated, flagellated “promastigotes,” which are highly proliferative. Upon transmission into a mammal, they are phagocytosed by neutrophil cells, dendritic cells, and macrophages. Triggered by mammalian tissue temperature and the acidic milieu inside phagosomes, they undergo a reversible stage conversion to the ovoid, aflagellated “amastigotes” that proliferate within the host cells at moderate to minimal growth rates  and acidic pH (pH 5.5)   , which a(pH 5.5) . A similol (RAD) , 22. ThiLeishmania can adapt to environmental challenges by chromosomal aneuploidy and extrachromosomal and intrachromosomal gene amplification, thereby altering relevant gene copy numbers  analysis showed a strong reduction of accessibility in divergent strand switch regions and 5′-telomeric regions in the axenic amastigote, indicating a switch from euchromatin to heterochromatin at transcription start sites and a possible epigenetic impact on stage-specific RNA synthesis.L. donovani. For this, we performed micrococcal nuclease (MNase) digests  of the compound, indicating that mere growth arrest has no impact on the chromatin density.First, we tested whether a hydroxyurea-mediated growth arrest in the Gd pH 7.4 . No signL. donovani promastigotes at 25°C/pH 7.4 . The ATAC-seq read alignment peaks represent areas that are accessible to the Tn5 transposase and are commonly associated with open chromatin.We next sought to test whether the induced changes to the chromatin structure were general or restricted to specific regions of the genome. This can be achieved by an endonucleolytic digest of nuclear DNA followed by next-generation sequencing to pinpoint nuclease-accessible euchromatin within the genome. 80). In addition, purified genomic DNA of L. donovani (50 ng and 200 ng) was also treated with Tn5 transposase as control. L. donovani reference genome.We applied ATAC-seq to the following three different cell types, each in duplicate: (i) logarithmically growing promastigote cells, (ii) axenic amastigotes, and (iii) promastigotes treated with RAD , amastigotes (Ama), RAD-treated promastigotes (Pro/RAD), and genomic DNA controls. Numbers of total reads, number of reads after adapter trimming, and percentage of reads aligned to the Copyright © 2021 Grünebast et al.2021Grünebast et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P ≤ 0.0001)   but fewe 0.0001)  peaks. T 0.0001)  but not  0.0001) .10.1128/mSystems.00628-21.4FIG S1FIG S1, PDF file, 0.05 MB.Frequency distribution of peak lengths. Peak lengths derived from Genrich peak calling were plotted against their relative frequencies for promastigotes , axenic amastigotes , and RAD-treated promastigotes . Download Copyright © 2021 Grünebast et al.2021Grünebast et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the In the next step, we looked at the distribution of ATAC peaks between different regions of chromosomes, namely, telomere regions upstream (5′ telomeres) of polycistronic transcription units (PTUs), telomere regions downstream (3′ telomeres) of PTUs, divergent strand switch regions (dSSRs), convergent SSRs (cSSRs), PTUs, protein-coding sequences (CDSs), and intergenic regions. The results are shown in 10.1128/mSystems.00628-21.5FIG S2L. donovani genome. Coverage of ATAC peaks is indicated for promastigotes , axenic amastigotes , and RAD-treated promastigotes  at 5′- and 3′-telomeres, divergent (dSSRs) or convergent strand switch regions (cSSRs), polycistronic transcription units (PTUs), coding sequences (CDSs), intergenic regions within PTUs, and total chromosomes. Data were pooled from 2 biological samples. Download FIG S2, PDF file, 0.06 MB.Distribution of open chromatin across the Copyright © 2021 Grünebast et al.2021Grünebast et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 5 transposase, in keeping with earlier analyses , axenic amastigotes , and RAD-treated promastigotes . Coding sequences (CDS) in two different orientations are indicated with black dots (forward direction) and red dots (reverse direction). Download FIG S3, PDF file, 0.8 MB.Genrich peak calling for all 36 chromosomes in Copyright © 2021 Grünebast et al.2021Grünebast et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To localize euchromatin within the dSSRs, we mapped ATAC peaks from position −5,000 to +5,000, relative to the start of the dSSR-proximal CDS and normalized them against the number of dSSRs. The results are shown in 1 growth arrest has no such effects  analysis  weakens interaction between neighboring nucleosomes (d-glucosyl-hydroxymethyluracil), which is necessary in L. major for transcription termination by RNA polymerase II, as readthrough transcription is observed in its absence  of core histones, by the expression of histone variants, or due to DNA modifications. Fortunately, a host of work is already available in this context. For example, the acetylation of histone H3 in hin PTUs . In addileosomes . In anotleosomes . An impo absence . In T. cn starts . In paracription . In T. bon sites .Leishmania and in other trypanosomatidic pathogens.Making use of histone gene deletions and/or histone point mutants at PTM sites, in conjunction with ATAC-seq analysis may give us a much-needed insight into the role played by epigenetic gene control in Leishmania donovani 1S (MHOM/SD/62/1S) and L. donovani (HSP90rr) promastigotes were cultured as described in reference L. donovani wild type (WT) or HSP90rr were adjusted to a cell density of 4 × 106 cells/ml to a total volume of 10 ml and treated with 0, 200, 400, 800, or 1,600 ng/ml radicicol for the MNase digest assay. Incubation was carried out at 25°C for 72 h. For ATAC-seq, 1,200 ng/ml radicicol was used (IC80). Treatment with hydroxyurea was carried out on L. donovani WT cells at the same cell density with 0, 0.125, 0.1875, 0.25, 0.375, and 0.5 mM hydroxyurea and incubation at 25°C for 24 h. For heat shock, the cells were also adjusted to 4 × 106 cells/ml to a total volume of 10 ml and incubated for 72 h at 37°C.Logarithmically growing cells of 6/ml) L. donovani promastigotes in modified medium 199  were shifted to 37°C for 24 h. Then, cells were precipitated  and resuspended in medium 199 . Incubation at 37°C with 5% CO2 was continued for 4 days, with fresh medium added after 2 days.Axenic amastigote differentiation was performed as previously described , 47. BriL. donovani    before f7 cells were washed with 10 ml of cold phosphate-buffered saline solution (pH 7) and resuspended in 500 μl permeabilization buffer with 0.05% Triton X-100 . Permeabilization was carried out for 5 min at room temperature before the samples were washed with permeabilization buffer without Triton X-100. For subsequent treatment with Tn5 transposase, the pellet was resuspended in 50 μl transposition mix consisting of 25 μl 2× TD  and 2.5 μl TDE1. As a control, 50 ng and 200 ng of L. donovani genomic DNA was treated with the transposition mix. Incubation was carried out at 37°C for 30 min, and the DNA was purified with Qiagen MinElute PCR purification kit . Barcode PCR was optimized by quantitative PCR (qPCR) and performed using NEBNext high-fidelity 2× PCR master mix (M0541S) and Nextera XT index primer (FC-131-1001). The library was purified with AMPure XP beads . Illumina sequencing was carried out according to the manufacturer’s instructions on a NextSeq 500/550 high output kit v2 (75 cycles) via paired-end sequencing.ATAC-seq analysis was performed as described  with sliPRJNA739873) were trimmed and quality checked with Trimmomatic (L. donovani BPK282A1 genome of TriTrypDB (https://github.com/jsh58/Genrich). Peak length was calculated from the peak coordinates of the Genrich output.Reads (mmomatic  and aligriTrypDB  using boriTrypDB  with parriTrypDB  before pPRJNA739873.Reads can be accessed under BioProject accession number


Nuclear hormone receptors (NRs) regulate transcription of the target genes in a ligand-dependent manner in either a positive or negative direction, depending on the case. Deacetylation of histone tails is associated with transcriptional repression. A nuclear receptor corepressor (N-CoR) and a silencing mediator for retinoid and thyroid hormone receptors (SMRT) are the main corepressors responsible for gene suppression mediated by NRs. Among numerous histone deacetylases (HDACs), HDAC3 is the core component of the N-CoR/SMRT complex, and plays a central role in NR-dependent repression. Here, the roles of HDAC3 in ligand-independent repression, gene repression by orphan NRs, NRs antagonist action, ligand-induced repression, and the activation of a transcriptional coactivator are reviewed. In addition, some perspectives regarding the non-canonical mechanisms of HDAC3 action are discussed. The endocrine system is one of the major mechanisms that contributes to cell-specific functions and homeostasis. Hormones are produced in the endocrine glands and delivered to the whole body by the bloodstream. Although numerous cells are exposed to these hormones, only target cells that express hormone-specific receptors are able to transduce the signal. Hormones are mainly derived from three origins: peptides, cholesterol, and amino acids. Genes for peptide hormones are encoded in the genome, and the hormones are produced through transcription, translation, and posttranslational modification. These hormones are hydrophilic. Hormones synthesized from cholesterol are lipophilic, and include estrogen, progesterone, testosterone, glucocorticoid, and mineralocorticoid. Hormones derived from amino acids are either hydrophilic or lipophilic. One lipophilic example is thyroid hormone. Despite its lipophilic nature, thyroid hormone is transferred into the cells largely by membrane transporters, such as the monocarboxylate transporter 8 (MCT8) , rather N-terminal region is highly variable and harbors an autonomous transcriptional activation function, which is independent of the ligands [NRs constitute a large superfamily with at least 65 members ,4. These ligands . The lig ligands ,11. The  ligands . BasicalN-terminal histone tails is one of the most established marks of an open chromatin structure and transcriptional activation [Epigenetic histone modifications are highly correlated with transcriptional regulation ,14. Amontivation ,16,17. Wtivation ,19, coretivation ,21. Althtivation . Severaltivation ,24. ThesIn this review, the basics of the molecular action of NRs will be explained first. The topic will be mainly focused on coregulators and histone acetylation status. Then, the history of HDAC3 as the regulator of NR action will be overviewed. Finally, the molecular mechanisms of NR-HDAC3 functions will be reviewed on each case.The repressive mechanism of NRs has been mainly explained by corepressor proteins. Although it is established that HDAC3 is the specific core component of the corepressor complex in NR action, the direct involvement of HDAC3 in the NR-dependent repression has not been extensively studied as compared to the roles of corepressors. Given the importance of histone deacetylation by HDAC3, this review will focus on what has been proven about the role of HDAC3 in the NR-dependent repression, rather than the roles of corepressors.N-terminal regions of these NRs are relatively small. This group of NRs were classically called “type 2 NRs” [NRs bind HREs as heterodimers with a retinoid X receptor (RXR), homodimers, or monomers, and regulate transcription of the target genes . A groupe 2 NRs” , but thiIn the absence of ligands, these NRs are associated with corepressor complexes that possess HDAC activity and suppress the transcription of the target genes, which is called ligand-independent repression ,28,29. TN-terminal domains. They were previously called “type 1 NRs” [The typical homodimerization NRs include the estrogen receptor (ER), the progesterone receptor (PR), the androgen receptor (AR), the glucocorticoid receptor (GR), and the mineralocorticoid receptor (MR). These NRs have relatively large e 1 NRs” , which ie 1 NRs” ,44. In ae 1 NRs” . Therefoe 1 NRs”  is respoe 1 NRs” ,46. Moree 1 NRs” . In thisIn addition to these NRs, there are multiple orphan NRs whose ligands are yet to be identified . These NAlthough acetylation of histones has been extensively studied, histones are not the only substrates for acetylation and deacetylation. Given the importance of acetylation of non-histone proteins, HDACs have been renamed to lysine deacetylases (KDACs) ,50. Simi+). Class III HDACs show similarities to yeast Sir2, and include SIRT1, 2, 3, 4, 5, 6, and 7. HDAC11 is categorized as class IV because the sequence in the catalytic domain is unique.Since the discovery of HDAC1 , at leasHDAC3 was initially reported soon after HDAC1 ,53,54. HHDAC3 was implicated in nuclear hormone receptor action by the identification of its core interaction partners in the early 21st century. Biochemical purification of HDAC3-associated protein complex revealed that N-CoR and SMRT are the members of the HDAC3 core complex ,63. ReciMoreover, interaction with N-CoR/SMRT substantially potentiates the enzymatic activity of HDAC3. The deacetylase activating domain (DAD) is determined for both N-CoR and SMRT . The DADTBL1X gene are associated with central hypothyroidism [Other HDAC3 core complex members include transducin beta-like 1 X-linked (TBL1X), TBL1X receptor 1 (TBL1XR1), and G protein pathway suppressor 2 (GPS2). These molecules also play important roles in the functions of the corepressor complex . Both TByroidism , althougyroidism . On the yroidism .HDAC3 gene so far. This is probably because these mutations result in embryonic lethality.Tissue-specific functions of HDAC3 have been studied using cell-type specific knockout mice , becauseIn addition, it is suggested that HDAC3 is involved in cancer progression. Overexpression of HDAC3 is observed in many types of cancers, particularly leukemia and lymphoma . MoreoveAs mentioned above, a heterodimeric group of NRs, including TR, RAR, and VDR, is associated with N-CoR/SMRT complexes that contain HDAC3 and suppress the transcription of the target genes in the absence of a ligand. On the other hand, the homodimerization NRs, including ER, PR, GR, MR, and AR, are “neutral” in terms of gene expression without ligands, because these NRs do not bind their target genes in the unliganded state. It seems that the importance of the difference between these two groups is not as highly evaluated as before. However, it is a notable point when considering the function of N-CoR/SMRT and HDAC3.The findings show the importance of ligand-independent repression by the heterodimeric NRs, including its role in phenotypic expression of TR-knockout mice and hypothyroid mice. The defects are much more severe in hypothyroid mice than in TR-deficient mice, indicating the roles of repression by unliganded TR ,96. As aRARα is involved in the etiology of acute promyelocytic leukemia (APL), in which leukocyte differentiation is blocked at the promyelocyte stage. Retinoic acid plays important roles in the differentiation of many types of cells, including granulocytes. The majority of APL cases are caused by chromosomal translocations that produce a promyelocytic leukemia (PML)-RAR alpha (RARA) fusion protein . PML-RARGene expression is suppressed by several orphan NRs, whose ligands have not yet been identified. These NRs work as monomers, homodimers, or heterodimers with RXR or other NRs . The typc-erbAα oncogene [c-erbAα oncogene encodes TRα [An orphan NR, REV-ERBα, was initially identified as a molecule whose gene is encoded by the reverse strand of the oncogene . Later, odes TRα ,108. REVodes TRα .Regarding other orphan NRs, HDAC3 is also involved in lineage restriction of embryonic stem cells mediated by dosage-sensitive sex reversal, adrenal hypoplasia critical region, chromosome X, gene 1 (DAX1) , TR2-medAs shown in Some of the most extensively studied NR antagonists are the modulators of ER function used in the treatment of hormone-sensitive breast cancer . SelectiTypical NR agonists stimulate the transcription of the target genes, as mentioned above. However, a certain number of genes are suppressed in the presence of hormones. It is hard to explain the molecular mechanism behind this because liganded NRs bind coactivators rather than corepressors due to their structures . AlthougAlthough histone deacetylation is highly associated with gene repression, there are several genes activated by HDAC3 via NRs. At least some cases are explained by deacetylation of non-histone proteins . One conAlthough the responsible transcription factors are unknown or are not NRs, non-canonical mechanisms of HDAC3 action are reported. These mechanisms might be involved in NR action as well.For example, several functions that are independent of the enzymatic activity of HDAC3 were studied. HDAC3 tethers target genes to the peripheral region of nuclei by interacting with the nuclear lamina protein lamina-associated polypeptide 2 ,125. HDAMany findings of acetylation/deacetylation of non-histone proteins are emerging as novel and important aspects of posttranslational modifications ,50. SeveIn addition, HDAC3 does not localize only in nuclei. It is also identified in the cytoplasm of several cell lines, including DT40 , 293 13, mouse eAs the core HDAC component of the N-CoR/SMRT corepressor complex, HDAC3 is largely responsible for transcriptional repression mediated by NRs and other transcription factors. The HDAC3 complex mainly suppresses transcription by deacetylating histones.On the other hand, recently the roles of HDAC3, independent of its enzymatic activity, are reported for transcription factors other than NRs, as mentioned above. In addition, non-histone proteins are of interest as novel substrates for HDAC3. These tendencies suggest that non-canonical mechanisms of HDAC3 in NR action will be a hot topic in the near future. Moreover, HDAC3 is also localized in the cytosol. Therefore, it would not be surprising if cytosolic NR-HDAC3 complex exerts some transcription-independent functions.


The chromatin architecture in promoters is thought to regulate gene expression, but it remains uncertain how most transcription factors (TFs) impact nucleosome position. The MuvB TF complex regulates cell-cycle dependent gene-expression and is critical for differentiation and proliferation during development and cancer. MuvB can both positively and negatively regulate expression, but the structure of MuvB and its biochemical function are poorly understood. Here we determine the overall architecture of MuvB assembly and the crystal structure of a subcomplex critical for MuvB function in gene repression. We find that the MuvB subunits LIN9 and LIN37 function as scaffolding proteins that arrange the other subunits LIN52, LIN54 and RBAP48 for TF, DNA, and histone binding, respectively. Biochemical and structural data demonstrate that MuvB binds nucleosomes through an interface that is distinct from LIN54-DNA consensus site recognition and that MuvB increases nucleosome occupancy in a reconstituted promoter. We find in arrested cells that MuvB primarily associates with a tightly positioned +1 nucleosome near the transcription start site (TSS) of MuvB-regulated genes. These results support a model that MuvB binds and stabilizes nucleosomes just downstream of the TSS on its target promoters to repress gene expression. The MuvB protein complex regulates genes that are differentially expressed through the cell cycle, yet its precise molecular function has remained unclear. Here the authors reveal MuvB associates with the nucleosome adjacent to the transcription start site of cell-cycle genes and that the tight positioning of this nucleosome correlates with MuvB-dependent gene repression. Transcription by RNA polymerase results in significant changes to nucleosome positioning, as the basal transcription machinery must overcome the energetic barriers presented by the placement of nucleosomes along promoters and the gene body4. RNA polymerase with the aid of elongation factors and histone chaperones can bind and evict octamer proteins or reposition nucleosomes present in the gene body to access the gene for transcription. In addition, chromatin remodelers and histone-modifying enzymes are thought to facilitate or inhibit transcription by arranging or displacing nucleosomes near transcription start sites, by altering the packing of nucleosomes, and by modulating the affinity of histone proteins for the DNA backbone. Less is known about how nucleosomal architecture is influenced by the activity of transcription factors (TFs). While recent evidence shows that pioneer TFs can bind target DNA sites within the nucleosome wrap and recruit remodelers to alter chromatin architecture, other TFs compete with nucleosomes for access to their DNA consensus sequence7. A thorough molecular description of how many regulatory TFs cooperate and engage with nucleosomes to modulate gene-expression remains elusive.Chromatin architecture and the position of nucleosomes influence DNA-mediated processes including the transcription of genes10. In mammals, cell-cycle-dependent gene expression occurs primarily in two waves of transcription, which take place around the G1/S and G2/M transitions and depend on the activity of MuvB and the other TFs E2F, B-MYB, and FOXM115. These TFs and their regulators are commonly deregulated in cancer18.The MuvB TF complex binds to target gene promoters and regulates a large set of cell-cycle genes. MuvB temporally coordinates the expression of genes necessary for DNA synthesis, centromere construction, mitotic division, and cell-cycle exit23. During quiescence and in early G1, MuvB binds to the retinoblastoma protein (RB) paralogs p130 or p107 (p130/p107) and E2F4-DP. This complex, known as DREAM, represses S phase genes and late cell-cycle genes25. Upon entry into the cell cycle, cyclin-dependent kinases along with their cyclin partners phosphorylate and release p130/p107 from the MuvB core, disassembling DREAM but keeping the core MuvB intact27. During S phase, the MuvB core binds to the proto-oncoprotein B-MYB and forms the MYB-MuvB (MMB) complex, which in concert with FOXM1 functions as a transcriptional activator of G2/M genes28. While the cellular imbalance of activating and repressive MuvB complexes is associated with several cancers30, the molecular details of MuvB assembly and function are poorly understood.The MuvB complex, components of which are evolutionarily conserved throughout animals and ciliates, plays a key role in development and differentiation and is an essential regulator of cell-cycle-dependent gene expression programs33. The short sequence motif, known as the cell-cycle genes homology region (CHR), is found in close proximity to the transcription start site (TSS) and is often located just downstream of a truncated E2F binding site, known as the cell-cycle-dependent element (CDE)34. LIN52 is a transcription factor adapter protein that recruits either B-MYB or p130/p107, depending on cell-cycle phase36. RBAP48 is a histone binding chaperone protein that is found in several complexes that interact with chromatin, including CAF-1, NuRD, PRC2, and SIN3-HDAC40. In mammals, RBAP48 has a highly similar (89% sequence identity) paralog named RBAP46 (or RBBP7), which has not been identified in complexes with MuvB components22. Both proteins are found in chromatin remodeler complexes, sometimes together. Less is known regarding the structure and biochemical function of LIN9 and LIN37, although a LIN9 sequence near its C-terminus co-folds with LIN52 to create the B-MYB-binding site35.The core MuvB complex is composed of the five proteins LIN9, LIN37, LIN52, LIN54, and RBAP48 (or RBBP4). MuvB is localized to its target cell-cycle genes through LIN54, which binds target promoters directly at a consensus DNA sequence21. In mammals, LIN9 is essential for the expression of G2/M genes; loss of LIN9 causes mitotic defects and is embryonically lethal in mice42. On the other hand, knockdown of LIN9 in cell culture results in compromised repression of DREAM target genes upon induced cell-cycle exit22. Knockout of the MuvB subunit LIN37 results in loss of MuvB-mediated gene repression in G0 and G1, but it does not lead to any observable changes in MYB-MuvB (MMB) mediated gene expression in G2/M24. Similarly, RNAi depletion of the Drosophila ortholog of RBAP48 specifically results in a derepression of dE2F2 target genes but does not result in defects in proliferation or gene expression43. These findings implicate MuvB core subunits in both positively and negatively modulating gene-expression, yet the biochemical mechanism behind their function remains unknown.Genetic evidence suggests that MuvB core proteins are essential in regulating cell-cycle-dependent gene expression. In flies and worms, knockout of MuvB components contributes to inappropriate derepression of developmental gene programsHere we investigated how MuvB represses gene expression, with emphasis on characterizing the structure and function of LIN9, LIN37, and RBAP48. We demonstrate that LIN9 and LIN37 together form an essential scaffold that holds together the core complex, and we determined a crystal structure that reveals how they together recruit RBAP48. We show that through RBAP48, MuvB binds directly to nucleosomes, either by interacting with H3 tails or the core particle. Using single-molecule electron microscopy, we found that MuvB increases nucleosome occupancy in a reconstituted cell-cycle gene promoter. These data indicate that MuvB associates with and stabilizes nucleosomes in the absence of other factors. Finally, we implemented a protocol that applies micrococcal nuclease digestion of chromatin and co-precipitation (MNase-ChIP) to study interactions of MuvB with nucleosomes in HCT116 cells. Our results support a model that MuvB binds to nucleosomes near the transcription start sites of target genes and stabilizes nucleosomes to repress cell-cycle-dependent gene expression.24. Human LIN9 contains 542 amino acids, and beyond the presence of a Tudor domain, it also exhibits no homology to known structures  together with LIN5235, while the C-terminus (residues 428–542) also has predicted helical structure.Beyond the role of LIN9 in binding B-MYB, the structure and biochemical function of LIN9 and LIN37 have not been previously characterized. Human LIN37 is a 246 amino acid protein that has no homology to any known structures. Sequence analysis suggests the presence of several short, structured regions  that are interspersed with sequences that are likely disordered Fig. . The segres Fig. . The N-t35 and that LIN9 knockdown results in DREAM complex assembly defects in T98G cells22, we hypothesized that LIN9 is a scaffold onto which the other proteins assemble  and LIN54 . When the three MuvB components RBAP48, LIN52, and LIN54 were co-expressed, we did not see co-precipitation  and the conserved CRAW domain of LIN37 (LIN3792–130) in Sf9 cells yielded a MuvB subcomplex that was stable through affinity purification and size-exclusion chromatography   of RBAP48 and the adjacent side of the β-propeller domain Fig. . All six30. It is feasible that PAF could access this extruded part of RBAP48 in the MuvB complex; however, how PAF binding to RBAP48 competes with p130 binding, as suggested30, is unclear considering our result that MuvBN components are not required for p130 association   Fig. . This RB52. The Tudor structure is defined by five anti-parallel β-strands that fold into a barrel. Target peptides are bound by an aromatic cage at one end of the barrel. The cage typically surrounds the modified basic side chain and makes stabilizing π-cation interactions. We aligned the LIN9 Tudor domain with structures of the PHF1  and the SMN  Tudor domains in complex with their target peptides . Tudor domains are protein interaction modules that are found in many chromatin-binding proteins. In several cases, they recognize methylated lysines and arginines and function as readers of modified histones24. These sequences in LIN37 correspond with the CRAW domain of LIN37 that appears structured in our crystals of MuvBN, and they play a critical role in interacting with LIN9 and RBAP48. This observation firmly implicates the MuvBN subcomplex as the structural subunit of DREAM responsible for gene repression.Previous functional domain mapping studies demonstrated that two highly conserved sequences in LIN37 were critical for LIN37 binding to other DREAM components and for DREAM repression of cell-cycle genesThe small structured LIN37 CRAW domain is bound between LIN9 α1 and α2 Fig. . The two3X) failed to co-precipitate LIN37, whereas a quadruple mutant  with mutations in LIN9 α6 and the preceding linker  not present in the mammalian complex.To probe whether posttranslational modifications on H3 tails influence MuvB binding, we tested two H3 marks that are associated with active transcription (H3K4me) or transcriptionally silent heterochromatin (H3K9me3). We found that MuvBbound H3 tails when methylated at K9 but failed to bind with H3 tails when methylated at K4 Fig. . This re53. Our data indicate that MuvB can bind nucleosomes through the H3 tails but that H3-tail binding is not necessary for MuvB-nucleosome association. To rule out any potential binding of the LIN9 Tudor domain, we reconstituted a mutant MuvB complex harboring LIN9 Tudor aromatic cage mutations (L230A/F238A/F256A/H264A) and found this mutant engages with Widom nucleosomes similar to the wild-type complex in the FP assay  but that simultaneous engagement of both the CHR and nucleosome  results in increased stability. A histone chaperone-like activity has been reported for RBAP48 in other chromatin-bound complexes, and RBAP48 binds histone octamer intermediates53. Because our experiment probes the equilibrium established by the reconstitution reaction beginning with a folded octamer, we cannot rule out a role for MuvB in facilitating the assembly of nucleosome intermediates , we ligated adapters after a SPRI-bead DNA purification. Compiled DNA sequences were aligned to the human genome, and we used MACS2 to locate enriched peaks corresponding to LIN9-interacting sequences.We next used an MNase-ChIP approach to detect MuvB association with nucleosomes in cells Fig. 57,58. Ch59. By comparing Strep-LIN9-precipitated samples to control samples in which cells were transfected with empty vector, we identified 253 genes with MACS peaks having >4.7-fold enrichment in sequencing reads. Gene ontology analysis of this data set reveals enrichment in genes related to cell cycle, mitotic division, and response to DNA damage  further downstream of the TSS. The enrichment decreases with increasing distance from the CHR site. In FOXM1 and ORC6, which contain CDE-CHR and E2F binding sites for DREAM, respectively, we observed a similar pattern, with the +1 nucleosome showing the strongest enrichment, followed by a weaker coverage of the distal nucleosomes. Multiple lines of evidence suggest that these enriched fragments correspond to sequences in MuvB-bound nucleosomes rather than sequences protected simply by MuvB binding consensus DNA. First, the peaks are centered adjacent to MuvB-binding sites in DNA as opposed to centered on them, suggesting the read sequences are not protected from steric occlusion of MNAse by MuvB itself. Second, we do not see enrichment in the LIN94x mutant or cycling cells experiments  near the transcription start site and E2F or CHR consensus sites in the DREAM-regulated genes Fig. . For exa4X does not associate with LIN37 and RBAP48  to identify more broadly the structural signature of LIN9-associated nucleosomes. Most of these genes show a sharply positioned nucleosome within 150 bases downstream of the TSS Fig. . We concP48 Fig. , we furtWe emphasize that the enriched nucleosomes in the set of DREAM genes do not overlap with the E2F and CHR consensus binding sites, suggesting that DREAM binds these DNA elements in linker DNA and not in the nucleosome core particle. Nucleosomes are positioned next to the DREAM-binding site, which is typically in close proximity to the TSS Fig. , and do −/−, Figs. 45. In fact, we observed in the input MNase data from the set of DREAM genes the nucleosome phasing pattern that is characteristic of genes undergoing transcription  difference in nucleosome occupancy comparing the wild-type and LIN37 knockout data sets in regions 100–200 bp both downstream and upstream of the TSS in DREAM genes , we observe more variability in the nucleosome position. The extent to which these observations result from the dynamics of RNA polymerase during the transition from repressed to active genes remains uncertain.Research on the structure of chromatin has revealed important factors that determine the nucleosome position in the genome, including intrinsic properties of DNA sequence, chromatin remodeling complexes, the polymerase machinery, and sequence-specific TFs24. We speculate that this non-functional complex may be unstable or improperly structured such that it cannot enact repression or bind co-repressors. The extensive interaction interface and co-dependence of their association in our mutagenesis study support the hypothesis that the core subunits of MuvBN,  co-fold to form a stable complex. Another important remaining question is how the structure and function of MuvB changes such that it switches from a repressor to an activator of gene expression once cells enter the cell cycle. MuvB components are still present on the promoter and are required for recruiting B-MYB and FOXM1. One possibility is that MuvB repression activity is relieved, for example by the binding of B-MYB, which is consistent with observations in Drosophila that the MuvB-binding sequence in B-MYB is alone sufficient to rescue a B-MYB deletion phenotype69. Another possibility is that Cdk phosphorylation, detected on all the MuvB subunits, plays a role in modulating MuvB function70. A third possibility is that in addition to its repressive function, MuvB positions the +1 nucleosome to prime genes for expression upon the binding of the activator transcription factors B-MYB and FOXM1. In this mechanism, MuvB may facilitate the acetylation of histones by the p300 acetylation machinery, which is recruited by the activator TFs. Through TF and p300 association, MuvB may also help recruit the basal transcription machinery. Finally, it will be important to understand how widespread interactions of TFs with the +1 nucleosome are and how these interactions regulate chromatin and gene expressionSeveral important questions remain about this MuvB repressive function including the structural mechanism of nucleosome recognition and the role of LIN37. RBAP48 in many studies is sufficient for nucleosome binding, and it is still present in MuvB complexes that lack LIN37 yet cannot repress gene expressionThe LIN9 and RBAP48 ORFs were amplified from cDNA derived from mouse NIH3T3 cells by standard PCR. The EGFP ORF was amplified from pEGFP-N1 (Clontech). The ORFs were cloned into pcDNA3.1(+) and fused either with an N-terminal 3xFlag tag (LIN9) or an N-terminal 1xFlag tag . Site-directed mutagenesis was performed following the QuikChange protocol (Stratagene). For MNase-Seq experiments, the LIN9 ORFs were subcloned into pcDNA3.1(+) containing an N-terminal Twin-StrepII tag.94–542, GST-LIN37, GST-LIN52, His- or GST-LIN54504–749, and Strep-RBAP48) were co-expressed in Sf9 cells via baculovirus infection. Cell pellets were harvested after 72 h of growth in suspension at 27 °C, and complexes were purified using GST-affinity purification followed by Strep-affinity purification. After removal of affinity tags through TEV protease cleavage, purified complexes were isolated through size-exclusion chromatography using a Superdex 200 column. The final buffer contained 200 mM NaCl, 25 mM Tris HCl, and 1 mM DTT at pH 8.0. The MuvBN subcomplex was assembled by co-expressing GST-LIN994–278, LIN3792–130, and full-length RBAP48 in Sf9 cells as described for the full complex. The subcomplex was purified using GST-affinity purification followed by anion exchange. Affinity tags were then removed with TEV protease and the complex was isolated with a Superdex 200 column. The final buffer contained 150 mM NaCl, 25 mM Tris HCl, and 1 mM DTT at pH 8.0.To assemble the entire MuvB complex, proteins  as a search model. The initial model was rebuilt with Coot73, and LIN9 and LIN37 were added to the unmodeled electron density. The resulting model was refined with Phenix74. Several rounds of position refinement with simulated annealing and individual temperature-factor refinement with default restraints were applied. The final refined model was deposited in the Protein Data Bank under Accession Code PDB ID: 7N40.The MuvBN subcomplex was crystallized in a sitting drop at 4 °C containing 0.2 M sodium tartrate tetrahydrate, 0.1 M bis-tris propane pH 6.5, and 20% PEG 3350. Crystals were harvested and directly frozen in liquid nitrogen. Data were collected at 54.Human HCT116 colon carcinoma cells were cultivated in DMEM supplemented with 10% FBS. Transfections were performed in 10 cm plates using 7 µg plasmid and 35 µl PEI per plate. To stimulate DREAM formation, cells were treated with 10 µM Nutlin-3a for 24 h. Cells were harvested 48 h after transfection. Whole cell extracts were prepared by lysing the cells in IP lysis buffer  for 10 min on ice followed by 5x 1 s direct sonication. Flag-tagged proteins were immunoprecipitated from 2 to 3 mg cellular extracts with Pierce Anti-DYKDDDDK Magnetic Agarose (Invitrogen). Beads were washed 5x with 1 ml IP lysis buffer end eluted with 50 µl 1xLaemmli buffer. Twelve micrograms of input samples and 12 µl IP samples were analyzed by SDS-PAGE and western blot following standard protocols. The following antibodies were applied for protein detection: FLAG-HRP , p130/RBL2 (D9T7M) , LIN54 A303-799A , LIN9 ab62329 , RBBP4 A301-206A , LIN37-T3 75. Biotinylated DNA probes were either amplified from the pGL4.10 empty vector or from pGL4.10 containing the mouse Ccnb2 CDE/CHR MuvB-binding site34. The following antibodies were applied for protein detection: FLAG-M2 , p130/RBL2 (D9T7M) , LIN37-T3 54, Histone H3 .For DNA affinity purifications, HCT116 cells were cultivated in 15 cm plates and transfected with 70 µl PEI and 15 µg plasmids expressing wild-type and mutant LIN9 fused with an N-terminal 3xFlag tag. Twenty-four hours after the transfection cells were treated with 5 µM Nutlin-3a for 48 h. Affinity purifications were performed as described earlierE. coli as inclusion body preparations as previously described77. Octamer reconstitution was completed by mixing equimolar amounts of purified histones in a buffer containing 7 M guanidinium HCl, 20 mM Tris pH 7.5, and 10 mM β-mercaptoethanol, followed by dialysis into 2 M NaCl, 10 mM Tris pH 7.5, 1 mM EDTA, and 5 mM β-mercaptoethanol. Folded octamers were purified using size-exclusion chromatography on a Superdex 200 column. Nucleosome reconstitution was performed by mixing purified histone octamers with the Widom 601 positioning sequence and de-salting by gradient dialysis76. For Widom nucleosomes, we used a 1.1:1 ratio of octamers:DNA molecules. At a salt concentration of 50 mM, nucleosome samples were collected in a buffer containing 50 mM Tris pH 7.5 and 1 mM DTT.Xenopus histones as well as their tailless counterparts were expressed and purified in Kd values were calculated using global fitting in Prism 8 (Version 8.2.1).Histone peptides were synthesized with fluorescein. For experiments with Widom nucleosomes, the 601 sequences were PCR amplified with a primer containing fluorescein and reconstituted with octamer as described above. 20 nM peptide was mixed with varying concentrations of MuvB protein complex in a buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 1 mM DTT, and 0.1% (v/v) Tween-20. Twenty microliters of the reaction were used for the measurement in a 384-well plate. Fluorescence polarization (FP) measurements were made in triplicate, using a Perkin-Elmer EnVision plate reader. The TTK promoter (461 bp) was cloned, amplified by PCR, and purified by agarose gel extraction. Histone octamers were folded with the TTK DNA as described above for Widom nucleosomes, but we used an octamer to DNA ratio of 3.1:1 to allow for the formation of di- and tri-nucleosome species. For the relevant conditions, purified MuvB complex or LIN54 was added to the nucleosome folding reaction during the de-salting process at a NaCl concentration of ~800 mM. Cross-linking of gene promoters and electron micrograph preparation was performed as previously described55. In brief, samples were treated with trimethylpsoralen and UV radiation to allow double-stranded DNA cross-links to form at unprotected, octamer-free regions. Following cross-linking, proteins were digested by Proteinase K, and DNA molecules were purified, denatured, and spread across the surface of a copper transmission electron microscopy grid. Electron micrographs of all samples were prepared by rotary metal shadowing, and grids were visualized and collected on a JEOL 1230 TEM at the UC Santa Cruz IBSC Microscopy facility and a Tecnai 12 TEM at the UC Berkeley ELM lab. DNA molecules were traced, and molecular coordinates were saved using Fiji tools in the ImageJ software package as previously described55. The resulting traces were analyzed using custom python tools. Each DNA strand was traced such that an “end” of the molecule could be identified. Thus, every coordinate in one strand can be aligned to its complement by closest distance. Coordinates are assigned to base positions using a scale derived from the physical distance between coordinates within each strand  and the known length of the TTK promoter (461 bp). A base pair is labeled single-stranded if the distance between strands exceeds a threshold distance, determined empirically. Once all base pairs are labeled, “bubbles” are determined by contiguous single-stranded stretches. Finally, a single-stranded “bubble” is labeled a nucleosome if its length is >90 base pairs . Both input and IP samples were subject to RNAse treatment and proteinase K digestion and were reverse cross-linked by incubation at 65 °C for 16 h. DNA was purified by 2x SPRI bead clean-up. Library prep was carried out using NEB Next Ultra II kits, and paired-end sequencing was carried out on the NovaSeq 6000 platform with 150 bp paired-end mode for Illumina at Novogene Biotech, Co., LTD.HCT116 cells were transfected with Strep-LIN9 constructs or an empty Strep expression plasmid. Twenty-four hours after transfection, cells were treated with 10 µM Nutlin-3a (Selleckchem) and harvested after 48 h. Cells were cross-linked with 1% formaldehyde for 15 min. The cross-linking reaction was stopped with glycine, cells were washed twice with PBS, and pellets were collected. Cell lysis and MNase digestion (1x or 5x) were performed as described earlier79. Samtools and bedtools were used to convert data into bam and bed files, respectively. Peak calling for the precipitated samples was performed using the MACS2 -bampe algorithm and using the empty Strep-IP conditions as the control. To retrieve gene names for MACS2 peaks, coordinates were intersected with known genes using the Table Browser tool provided by the UCSC genome browser. Gene ontology analysis was performed on MACS2 peaks showing a >4.7-fold enrichment using the web-based tools GeneOntology.org and Revigo80. We generated coverage plots of our reads using Gviz and rtracklayer and other opensource R tools.Sequencing reads were aligned against hg38 using the bwa-mem aligner81. We restricted our analysis to genes that showed a MACS enrichment of >4.7-fold and were previously annotated to bind DREAM, to respond to p53 stimulation, and to become derepressed in LIN37 knockout cells45. We retrieved the TSS for this set of genes, either from those annotations or using bioMart82. As needed, the TSS sites were mapped on to hg38 using liftover. We oriented the output to center on the TSS and maintain a uniform direction of transcription. We then utilized the Cluster Map Builder feature of NucTools to generate aggregate plots and heatmaps of our genes.We utilized NucTools in paired-end mode to analyze nucleosome occupancy on input and Strep-LIN9 precipitated reads with single base-pair resolution (bin width = 1 bp)Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Fruits and seeds resulting from fertilization of flowers, represent an incredible evolutionary advantage in angiosperms and have seen them become a critical element in our food supply.Many studies have been conducted to reveal how fruit matures while protecting growing seeds and ensuring their dispersal. As result, several transcription factors involved in fruit maturation and senescence have been isolated both in model and crop plants. These regulators modulate several cellular processes that occur during fruit ripening such as chlorophyll breakdown, tissue softening, carbohydrates and pigments accumulation.Arabidopsis thaliana and Solanum lycopersicum) and in crops of commercial interest .The NAC superfamily of transcription factors is known to be involved in almost all these aspects of fruit development and maturation. In this review, we summarise the current knowledge regarding NACs that modulate fruit ripening in model species (The online version contains supplementary material available at 10.1186/s12870-021-03029-y. It is widely accepted that fruits play a key role in the evolutionary history of angiosperms , 2. FruiSQUAMOSA promoter binding protein-like), TCP (TEOSINTE BRANCHED 1/CYCLOIDEA/PROLIFERATING4 CELL FACTORS) and Dof (DNA binding with one finger) proteins , 9190, 9The N-terminal DBD is also involved in the formation of homo- and/or hetero-dimers: NAC TFs can bind DNA as homo- or hetero-dimers and the dimerization is necessary for a DNA stable binding , 95. In At the C-terminal, the presence of several group-specific motifs in the TRR allow the activation or repression of the transcription . Its divSeveral NACs possess a transmembrane domain; it can be located at both the terminal ends of the proteins but were prominently found at the C-terminal end .In 2004, Ernst and collaborators solved the NAC domain’s structure of ANAC019 by X-ray crystallography . Some yeThe sequence of the NAC binding site has been addressed with several techniques . The binding site contains the CGT[AG] core motif, and the flanking sequences are involved in the determination of the target promoter binding affinity and specificity (for a review see ).Arabidopsis thaliana and Solanum lycopersicum, for their role as reference plants in studies focused on fruit development, ripening and senescence [The NAC superfamily includes only plant-specific transcription factors, widespread in an increasing number of species. First, we will focus on model species, such as nescence .NARS1 and NARS2 . NARS1 and NARS2 regulate embryogenesis, through the control of ovule integument development and degeneration, seed morphogenesis, and silique senescence [nars1 nars2 has delayed silique senescence. Currently, no molecular data are available regarding NARS1 and/or NARS2’s involvement in hormonal signal pathways. It has been shown that NARS1 and/or NARS2 work to ease an intracellular environment triggered by programmed cell death in the integument, and similarly could also occur in the silique. Recently, Ma and colleagues [NARS1 and NARS2 as NOR (NONRIPENING) homologs. NOR is a NAC transcription factor involved in tomato fruit ripening . Howevee below,  concludeAtNAP  was initially described as promoter of leaf senescence [AtNAP messenger accumulates with fruit maturation progression and atnap siliques delay senescence by 4–5 days. Moreover, the ethylene and respiratory surges are decoupled, and exogenous ethylene treatments cannot anticipate the respiratory surge [atnap mutant. However, it is still not clear how the changes in the expression of these genes could affect the senescence in mutant plants. AtNAP might be NOR orthologue, but such conclusion is not fully supported [nescence , but wasnescence . Indeed,ry surge . Few genupported .nac058 knock-down mutant the maturation of the fruit is precocious as demonstrated by the premature yellowing, the reduced chlorophyll content and the reduced photosynthetic performance [The role of few other Arabidopsis NAC proteins has been described in siliques, among them NAC058 which represses silique senescence. In the formance .JUNGBRUNNEN1 (JUB1/ ANAC042) is not involved in the maturation process rather in the growth of the silique. The silique of plants that overexpress JUB1 are shorter than normal, but it is interesting to report that in tomato the overexpression of AtJUB1 delays fruit ripening by 6 days [AtJUB1 in tomato represses several ethylene-related ripening genes such as ACS (ACC synthase) and ACO  and DWF4 (DWARF4), genes important for gibberellin (GA) and brassinosteroid (BR) biosynthesis, thus phenocopying GA and BR deficiency. AtJUB1 also directly represses PIF4 (PHYTOCHROME INTERACTING FACTOR4), a positive regulator of cell elongation, and activates the DELLA genes, repressors of cell proliferation and expansion [y 6 days , 88. Thexpansion , 88.SlNAC genes have been identified so far [The complete sequencing of the tomato genome  providedd so far , mappingnor mutant fails to produce the climacteric peak of ethylene, thereby causing an arrested ripening [NOR encodes a NAC transcription factor [NOR is alcobaca  found in the Penjar tomatoes which have a very long shelf life [alc allele is weaker compared to nor, but its ripening delay is comparable to the newer nor alleles that have been recently obtained using the CRISPR-Cas9 methodology [nor mutant, that is actually a gain-of-function [null alleles of other tomato transcription factors involved in fruit maturation, such as RIN and CNR [RIN encodes a MADS-box transcription factor [NOR promoter [. However, Wang and collaborators [NOR and RIN play a role in fruit senescence, only NOR has been found involved in the mediation of pathogen susceptibility in ripening fruit [The ripening , 104 whiripening , 106. Pon factor , 106. Anelf life , 108. Thhodology . All thefunction –113. Sim and CNR . RIN encn factor , 115 thapromoter –118. Howborators  have recborators . Moreoveng fruit .NAC genes are often referred to using different names, complicating literature data screenings [NOR-like1 was originally identified as SlNAC3, a negative regulator of drought stress under the control of ABA [SlNAC3 was revealed to be essential for appropriate embryo and endosperm development in seeds [Polygalacturonase-2 (PG-2) was eventually isolated as one of SlNAC3’s direct targets through co-immunoprecipitation assays [NOR-like1 as SNAC4 (SlNAC48), identifying it as regulator of salt stress and drought tolerance [NOR and its disruption causes a decrease in carotenoid accumulation, chlorophyll breakdown and ethylene biosynthesis [NOR-like1 can bind directly both RIN and NOR, suggesting that it could act upstream of these ripening regulators, and its predicted tertiary structure reveals a high degree of similarity with ANAC072, involved in chlorophyll degradation during leaf senescence [NOR-like1 has been shown to directly bind the promoter of genes involved in ethylene production, fruit firmness and colour change [Some NAC transcription factors, able to influence fruit development and maturation, were initially classified as stress-responsive genes, and for this reason reenings . NOR-likl of ABA . Later, in seeds . Polygaln assays . At the olerance . It sharynthesis . Moreovenescence –128. Recr change .Pseudomonas syringae [Tweneboah and Oh  summarizsyringae , thus mosyringae  and its syringae , 134.SlORE1S02, SlORE1S03, and SlORE1S06 are the orthologues of the Arabidopsis ORESARA1 (ORE1/NAC092) [ore1 mutant delays leaf senescence in Arabidopsis [SlORE1S02, SlORE1S03, SlORE1S06 and ORE1 are all regulated by the microRNA miR164. In tomato, they are expressed in leaves where their overexpression accelerates senescence. The reduction of SlORE1S02 via RNAi interference leads to increased carbon assimilation, consequently transgenic plants show a higher harvest index with no consequences on fruit size. Transgenic fruit delay senescence and also accumulate more soluble solids in ripe fruit [/NAC092) . ORESARAbidopsis . SlORE1Spe fruit .SlNAP2 impacts fruit yield and metabolism, since its inhibition causes fruits to retain more sugars [AtNAP (described in the previous paragraph [SlNAP2 naming it SNAC9 (SlNAC19). Fruit of slnac19 exhibit a reduced carotenoid content and ethylene production [nor-like1 fruits, but they accumulate more ABA than SlNAC19 ones [SlNAP2 modulates NOR expression [NOR and NOR-like1, it is considered one of the key tomato ripening regulators [e sugars . This traragraph ). Kou anaragraph  describeoduction . The samC19 ones . SlNAP2 pression  and, toggulators .Oryza sativa, Malus domestica, Fragaria genus, Citrus sinensis and Musa acuminata.Beside model species for dry and fleshy fruits, NAC transcription factors have been identified and analysed in many other species of commercial interest. Here are listed the most recent findings in a few select species. We will review their role in Although grains do not resemble the classic idea of fruit, they are actually composed of fruit coat strongly bound to the seed coat, and they enclose the germ and the endosperm. This non-canonical one-seeded fruit is produced by cereals, such as rice, barley and wheat, and it is called caryopsis.OsNAC020, OsNAC023 and OsNAC026 have been identified as grain-specific conserved NAC genes in rice [OsNAC020, OsNAC023 and OsNAC026 detected a higher expression in caryopsis [One of the most widely cultivated cereals is rice, and for this reason it represents the topic of many researches. In rice, NAC transcription factors are involved in biotic and abiotic stress , 139. Os in rice . Indeed,aryopsis . Moreovearyopsis .ONAC127 and ONAC129 are expressed in caryopsis and the corresponding gene products regulate starch accumulation and genes related to carbohydrates transport during grain filling [Since rice seeds are mostly intended for human consumption, their composition is one of the principle features to be considered.  filling .OsNAC5, OsNAC9 and OsNAC10 causes changes in root diameter and architecture leading to increased drought tolerance and grain yield [OsNAP and SNAC1 overexpression leads to a greater seed production in restricted water conditions [ONAC022 [OsNAC2 regulates shoot branching [OsNAC2 is negatively regulated by miR164b; if the sequence is mutated to be resistant to miR164b, the final grain number increases [Another important commercial feature is the grain yield, determined by numerous factors, e.g. resistance to drought stress and shoot branching. Overexpression of in yield –146. Simnditions , 148. Ho[ONAC022 . Shoot branching  and planranching . OsNAC2 ncreases .MdRTE1a  and MdRTE1b. This pinpoint a possible involvement in pome growth and ripening. Later, MdNAC1 was also found to be responsible for plant height and drought tolerance, since its overexpression leads to a dwarf phenotype [MdNAC1 controls the biosynthesis of ABA [In the apple cultivar “Golden delicious”, Wang and Xu  found thhenotype  with redhenotype . Moreoves of ABA .NAC transcription factors mediate ethylene and auxin crosstalk in apple, especially when the production of ethylene is impaired , as alreMdERF3 , modulating salt stress tolerance [ANAC047, however its genomic locus has been recently annotated as MdNAC63 [Zhang and collaborators  found tholerance . MdNAC47 MdNAC63 .MdNAC52 overexpression leads to anthocyanin accumulation in apple calli and it can induce the expression of MdMYB9 and MdMYB11, regulating proanthocyanidin biosynthesis [Fruit firmness, an important trait in post-harvest conservation, is also controlled by NAC transcription factors. In particular, the NAC18.1 protein displays high similarity with tomato NOR, and could be a major determinant of fruit softening and harvest date , 161. Anynthesis . Recentlynthesis .Fragaria x ananassa), thanks to the comparison with the woodland strawberry (Fragaria vesca) genome [FaNAC006, FaNAC021, FaNAC022, FaNAC035, FaNAC042, FaNAC092. Their expression pattern correlates with anthocyanin biosynthesis [112 NAC genes have been identified in the commercial strawberry  genome . Six of ynthesis  and theiynthesis . Recentlynthesis .FaNAC087 and FaNAC038 are negatively modulated by miRNA164 [Arabidopsis thaliana with ORE1 [FaNAC087 and FaNAC038 increase their expression in the last stages of ripening [miRNA164 and its NAC messenger targets. The role of sRNAs as regulators of post-harvest shelf life has been recently corroborated, highlighting that NAC and other families of transcription factors represent important targets [miRNA164 , as happith ORE1 . Since Fripening , Li and ripening  analysed targets .Fragaria x ananassa) was obtained by crossing the parental species Fragaria chiloensis and Fragaria virginiana. Due to great interest in its limited post-harvest period, transcription factors involved in fruit ripening regulation and cell wall remodelling have been characterized in Fragaria chiloensis, including NAC TFs. Among them, FcNAC1 which interacts with FcPL (Fragaria chiloensis pectin lyase), contributing to cell wall remodelling [The commercial strawberry . In particular, RD26 displays high correlation with fructose and glucose accumulation; the Arabidopsis orthologue RD26/ANAC072 is a transductor of the ABA signal [Given the economic and nutritional importance of sweet orange, the discovery of the regulators of fruit quality and duration have always been an essential goal to reach. In orange, transcription factors belonging to the NAC superfamily represent one of the key elements of these processes. NAC genes differentially expressed between a late-ripening mutant and a wild type variety of sweet orange have been identified , among tA signal  and actiA signal .CitNAC is expressed in peel and pulp during orange fruit ripening and is phylogenetically similar to AtNAP [CitNAC in sweet orange fruit development and senescence.to AtNAP . This suCitAco3, an aconitate hydratase involved in the catabolism of citric acid [In order to lower the citric acid content in mature fruit, CitNAC62 acts in a synergic way with CitWRKY1 modulating ric acid .NAC-1 and RD26 transcripts are detected during the symptomatic and asymptomatic phases of citrus greening [As in tomato, stress-related NAC transcription factors play an active role in determining fruit quality, thus influencing the postharvest conservation. Fan and co-workers  point ougreening . In addigreening . HoweverMaNACs were associated with ripening in ethylene-treated banana fruits, some of them carrying ethylene responsive elements in their promoter. Six of them (MaNAC1 to MaNAC6) have already been previously characterized as part of the ethylene mediated fruit ripening [MaNAC1 (MaNAC087) is known for its role in both stress tolerance and fruit maturation, it is induced after cold stress and physically interacts with cold signalling pathway elements [Musa acuminata ETHYLENE INSENSITIVE 3-like 5), a downstream component of the ethylene signalling pathway [MaNAC1 and MaNAC2, together with MaNAC5 (MaNAC140), is upregulated in fruit after infection with Colletotrichum musae [Studies on NAC superfamily in banana started with the prediction of loci potentially coding for such transcription factors, and the definition of orthologous groups comparing sequences from monocots and dicots . Recent ripening . MaNAC1 elements . Moreove pathway . MaNAC2  pathway . Additioum musae . Furtherum musae . Other Num musae  or drougum musae , 190. FuThe economic and dietary relevance of fruit is an important stimulus to explore fruit maturation and senescence in non-model species. In respect to fruit maturation, a pivotal role is played by NAC transcription factors and therefore they are proposed as interesting targets to modulate development and ripening, and to prolong fruit shelf life. In this work we provide a synopsis of the fruit-related NAC transcription factors identified in model species and in major crop species Fig. . The proUnfortunately, the non-model species have yet to be deeply investigated but, from the data available, it is clear that NAC transcription factors represent a conserved family necessary for the regulation of fruit development, maturation and post-harvest life. Due to their ubiquitous presence in numerous species, unveiling NAC TF regulation network could represent a potential tool for the regulation of post-harvest fruit conservation and pathogen resistance in economically relevant species. Within this view, as suggested by Singh and collaborators , future In parallel, the identification of NAC interactors could reveal potential inhibitors or enhancers of their activity, helping the conservation and response to pathogens in commercial fruits.Additional file 1: Table S1. Number of NAC transcription factors identified in several plant species.


Tceal7. Our studies have characterized the 0.7 kb promoter of the Tceal7 gene, which harbors important E-box motifs driving the LacZ reporter in the myogenic lineage. However, the underlying mechanism regulating the dynamic expression of Tceal7 during skeletal muscle regeneration is still elusive. In the present work, we have defined a cluster of Mef2#3–CRE#3–E#4 motifs through bioinformatic analysis and transcription assays. Our studies suggested that the triple complex of Mef2c, Creb1 and Myod binds to the Mef2#3–CRE#3–E#4 cluster region, therefore driving the dynamic expression of Tceal7 during skeletal muscle regeneration. The novel mechanism may throw new light on understanding transcription regulation in skeletal muscle myogenesis.We have previously reported a striated muscle-specific gene during embryogenesis, Tceal7 has been identified as a direct, downstream target gene of MRF in the skeletal muscle. The overexpression of Tceal7 represses myogenic proliferation and promotes cell differentiation. Previous studies have defined the 0.7 kb upstream fragment of the Tceal7 gene. In the present study, we have further determined two clusters of transcription factor-binding motifs in the 0.7 kb promoter: CRE#2–E#1–CRE#1 in the proximal region and Mef2#3–CRE#3–E#4 in the distal region. Utilizing transcription assays, we have also shown that the reporter containing the Mef2#3–CRE#3–E#4 motifs is synergistically transactivated by Mef2c and Creb1. Further studies have mapped out the protein–protein interaction between Mef2c and Creb1. In summary, our present studies support the notion that the triple complex of Mef2c, Creb1 and Myod interacts with the Mef2#3–CRE#3–E#4 motifs in the distal region of the Tceal7 promoter, thereby driving Tceal7 expression during skeletal muscle development and regeneration. Tceal7, Kbtbd5 and Rb1 [−/− mice have no apparent defect in their skeletal muscle development due to the functional compensation of Myf5 [The process of skeletal muscle terminal differentiation involves a precisely orchestrated series of events, including cell cycle exit, myoblast fusion and formation of multinucleated myotubes, which are cooperatively driven by a variety of ubiquitous and muscle-specific transcription factors . Myod, a and Rb1 ,3,4,5,6. of Myf5 . Recent  of Myf5 . For exa of Myf5 ,11. Rece of Myf5 ,13. Myod of Myf5 . It has  of Myf5 .4TAR, in the regulatory region of downstream genes through high-affinity DNA binding and dimerization [Myocyte enhancer factor 2 (Mef2) is a member of the MADS-box family of transcription factors, which consists of Mef2a, Mef2b, Mef2c and Mef2d in vertebrates . Mef2 prrization ,16,17. Mrization . The comrization . Conditirization . In contrization ,22.−/− mice die immediately after birth due to respiratory defects, and have impaired T-cell development and reduced corpus callosum and anterior junction in the brain [The cAMP-responsive element-binding protein (Creb1) belongs to a subgroup of bZIP transcription factors that includes cAMP-responsive element modulators (CREMs) and activating transcription factor 1 (ATF1) ,24. Crebhe brain . Recent he brain ,33,34. The brain ,33. In ahe brain . In summhe brain ,36,37.Tceal7 gene expression and its 0.7 kb promoter, which drives the expression of the LacZ reporter in skeletal muscle lineage through the five E-box motifs transactivated by Myod [Tceal7 during skeletal muscle regeneration. Herein, our analysis has revealed two clusters of motifs: CRE#2–E#1–CRE#1 in the proximal region and Mef2#3–CRE#3–E#4 in the distal region. We have also examined the transactivation of Tceal7 by the cofactors of Myod: Mef2c and Creb1. Interestingly, we have observed a synergistic transactivation between Mef2c and Creb1 with the Reporter II, which prompts us to examine the protein–protein interaction between Mef2c and Creb1. In summary, our present studies support the notion that the triple complex of Mef2c, Creb1 and Myod bind to the cluster of Mef2#3–CRE#3–E#4 motifs in the distal region of the Tceal7 promoter, thereby transactivating Tceal7 expression during skeletal muscle development and regeneration.Our previous work has characterized the specific spatiotemporal pattern of  by Myod . In the Tceal7 0.7 kb promoter fused to luciferase reporters were generated by routine PCR or site-specific mutagenesis PCR. Reporter I and Reporter II were constructed by two pairs of complementary primers using PCR. A pGL3-3×(Mef2#3) luciferase reporter was purchased from Miaoling Plasmid Sharing Platform . Mef2c (NM_001170537.1), Creb1 (NM_133828.2) and Myod (M18779.1) were subcloned into the expression vectors: pcDNA-HA, pcDNA-3×HA and pCS2-6×Myc. Creb1(1−327), encoded by NM_133828.2, is a variant of Creb1(1−324) with a partial Q1 deletion due to exon skipping, both of which are transcriptionally active. Residues at S119 and Y134 of Creb1(1−327) are equivalent to those at S133 and Y120 of Creb1(1–324), respectively. A mutation of the residue Y120F resulted in the constitutive activity of Creb1. Creb1 Y120F and Creb1 S119A were subcloned into pcDNA-HA and pcDNA-3×HA vectors through mutagenesis PCR [The manipulation of DNA and RNA was performed using the Genomic DNA Extraction Kit, EASYspin plus RNA Extraction Kit and TRUEscript 1st Strand cDNA Synthesis Kit , respectively. Wild-type (Wt) and mutant (Mut) forms of the esis PCR .2 incubator at 37 °C. The C2C12 myoblasts (passage 7~10) were transfected using Via-FectTM , while the NIH3T3 and HEK293T cells were transfected with TransIntroTM EL Transfection Reagent . For C2C12 myoblast differentiation, the cell-cultured medium was switched to DMEM containing 2% horse serum, 100 U/mL penicillin and 100 μg/mL streptomycin  once the C2C12 myoblasts became confluent.C2C12 myoblasts and NIH3T3 cells were purchased from ATCC  and CAS , respectively. HEK293T cells were kindly provided by Prof. Jianhua Wu . The C2C12 myoblasts, NIH3T3 and HEK293T cells were all maintained in high-glucose DMEM, supplemented with 10% FBS , 2 mM L-Glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin   in a 5% CO® Reporter Assay System . All fold changes in the firefly luciferase activity were normalized to that of the Renilla luciferase [The C2C12 myoblasts and NIH3T3 cells were seeded into 6-well plates, and the HEK293T cells into 12-well plates, the day before transfection. These cells were then transfected with the following plasmids: 400 ng reporter plasmid , 10 ng internal control (pRL-CMV) and 200–1000 ng expression plasmids . The total DNA amount was normalized with the empty expression vector pcDNA3.1. The transfected cells were collected and lysed 24 h after transfection, followed by an analysis with the Dual-Luciferaseciferase .Tceal7 promoter or its mutant in myotubes, 1 × 105 C2C12 myoblasts were seeded into 6-well plates on the day before transfection. Wild-type and mutant reporters (400 ng) with a pRL-CMV plasmid (10 ng) were cotransfected into the C2C12 myoblasts, with a mass ratio of 40:1. After 24 h, the growth medium was replaced with the differentiation medium for 48 h. At harvest time, the myotubes were washed twice with PBS, lysed in a precooled lysis buffer  and 1 mM dithiothreitol) on ice for 10–15 min; then, they were utilized for luciferase assay after the freeze–thaw cycle. The relative activity of the Wt reporter was set as 100% [To measure the activity of the eporters 00 ng witg at 4 °C for 15 min. Western blot and coimmunoprecipitation assays were performed as previously reported [The HEK293T cells were transfected with 3×HA-Creb1 or its deletional mutants (3 µg) and 6×Myc-Mef2c or its deletion expression plasmids (3 µg) for 24 h. The cells were washed with PBS and lysed in a lysis buffer . The total cell lysate was prepared by centrifuge at 14,000× reported . The prig at 4 °C for 1 min and washed as follows: twice with a ChIP-dilution buffer, once with a low-salt-wash buffer, once with a high-salt-wash buffer, once with a LiCl wash buffer and twice with a TE buffer. The immunoprecipitation complex was eluted twice with an elution buffer  and incubated with NaCl and RNase A for reverse crosslinking at 65 °C for 6–16 h. The immunoprecipitated DNA was precipitated with ethanol and treated with Proteinase K at 45 °C for 1 h, followed by purification according to the manufacturer’s protocol for the DNA purification and recovery Kit . Finally, it was resuspended in an elution buffer. A routine PCR was done with specific primers flanking the Mef2#3 or CRE#3 motif [The C2C12 myoblasts were transfected with an HA-Mef2c or HA-Creb1 expression plasmid (8 µg) and, 24 h later, switched to the differentiation medium for 48 h. The differentiated myotubes were then washed twice with PBS, crosslinked with 1% formaldehyde for 10 min at room temperature and quenched using 0.125 M Glycine. The myotubes were washed with precooled PBS again and collected for chromatin preparation. The following procedures were performed according to manufacturer’s protocol for the ChIP Assay Kit . Briefly, the cells were suspended in an SDS lysis buffer containing PIC and sonicated using the Bioruptor Plus . The chromatin was diluted with ChIP-dilution buffer and preincubated with 70 μL protein A + G agarose for 1 h at 4 °C. The supernatant was then incubated with an anti-HA antibody  or a normal rabbit IgG  control, overnight at 4 °C. On the next day, each ChIP reaction was supplied with 40 μL protein A + G agarose and incubated for another 2 h at 4 °C. The agarose beads were collected by centrifuge at 1000× p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant.All data represent the mean ± S.D. of at least three independent replicates. Statistical significance was assayed by one-way ANOVA for multiple comparisons with a Turkey’s test using a GraphPad prism. * Tceal7 in skeletal muscle is transactivated by Myod through the E#1 or E#4 box [Tceal7. A bioinformatics analysis has revealed that there are three Mef2 motifs (Mef2#1–#3) and three CRE motifs (CRE#1–#3), which are distributed into two cluster regions around E#1 and E#4: the proximal region  and the distal region , respectively  have been constructed by placing four repetitions of the two clusters (CRE#1–E#1–CRE#2 and Mef2#3–CRE#3–E#4) into the luciferase reporters (Tceal7 expression than the E#1 box [Tceal7 promoter. As shown in Tceal7 by Myod and Creb1 is mainly mediated by the E#4 and CRE#3 motifs in the distal region.Creb1 is an important transcription factor involved in a variety of cellular processes during development; it directly transactivates multiple downstream target genes, such as Etv2, Col24α1, JHDM2a and Fndc5 ,37,41,42eporters A. Myod teporters B. These  E#1 box ,43. A fuTceal7 expression is the Mef2#3–CRE#3–E#4 cluster in the distal region of the promoter. The synergistic transactivation of Tceal7 by Myod and Mef2c has also been confirmed (Tceal7 gene expression is regulated by Myod, Mef2c and Creb1.Our above studies have demonstrated that the core element driving onfirmed D. Howeveonfirmed A, while onfirmed B. We theonfirmed C. A furtTceal7 gene expression. To validate our hypothesis, 3×HA-Creb1 and 6×Myc-Mef2c expression plasmids were constructed and co-transfected into HEK293T cells, followed by coimmunoprecipitation analysis. As shown in Neither Mef2c or Creb1 is dispensable in skeletal muscle myogenesis, but their common target genes in the skeletal muscle lineage are rarely reported. Interestingly, our above studies have revealed the synergistic transactivation between Mef2c and Creb1. These data have indicated the possibility that Mef2c and Creb1 can form a complex to regulate Tceal7 is specifically expressed during embryonic development or skeletal muscle regeneration in a unique temporal–spatial manner, which clearly indicates the important role of Tceal7 in the skeletal muscle lineage. We have also documented that Tceal7 gene expression is driven by its 0.7 kb promoter [Tceal7 expression (We have previously reported that promoter . Herein,pression .Etv2 in the hematopoietic and endothelial lineages in previous studies [Etv2 [Tceal7 between murine and human genomes. Each of these CRE motifs plays a functional role in the promoter in the mutagenesis study (Tceal7 by Creb1 in the transcription assays (Tceal7 promoter to drive its dynamic expression pattern during skeletal muscle regeneration. However, in vivo evidence will be required for validation of this hypothesis in the future research.The transcription activity of Creb1 is closely related to its phosphorylation status, which is dynamically regulated by protein kinases and phosphatases. We have characterized the 3.9 kb promoter of  studies ,35,46. Mes [Etv2 ,46. The es [Etv2 . During es [Etv2 . Furtheres [Etv2 . In the is study , while on assays . The disRb1, although it has also been reported that the regulation of Rb1 by Myod may be mediated by the enhancer of Rb1 [MuSK gene [Both Mef2c and Creb1 are very important cooperative factors of Myod in skeletal muscle myogenesis. The interaction between Myod and Mef2c has been well characterized as a cooperative transactivation complex, while the interaction between Myod and Creb1 may have a complicated effect on the activity of Myod in skeletal muscle myogenesis. Myod may cooperate with Creb1 to transactivate its downstream target gene, such as r of Rb1 ,36. In cuSK gene . In the Our present work has also provided the first evidence of synergistic interaction between Mef2c and Creb1. Our studies have defined the interaction between Mef2c and Creb1. Interestingly, their interaction is not affected by the phosphorylation status of Creb1, indicating that p300 is not involved in the interaction between Mef2c and Creb1. The bZIP domain of Creb1 has been characterized as the DNA-binding domain, while it is also very important in the association with other proteins, such as Mesp1 and Myod ,48. Our Tceal7. p38MAPK plays a very important role in skeletal muscle myogenesis and myopathy progression. Mutation of p38alpha results in a perturbed myogenic differentiation [Collectively, our above data suggests that Mef2c, Creb1 and Myod may form a triple complex to transactivate ntiation . The actntiation ,51. p38Mntiation . Creb1 intiation . The forntiation . InteresTceal7 gene expression, while the transactivation of this proximal cluster by Myod or Creb1 is minimal. The discrepancy may be due to a low affinity between these factors and the transcription factor-binding motifs. Given that the cluster is adjacent to the TATA box, it is certainly possible that the proximal cluster may be important for the initiation of Tceal7 transcription, as both Creb1 and Myod may participate in the recruitment of a pre-initiation complex [Tceal7, such as miR-182, miR-301a, miR-769–5p, miR-18b and miR-758-3p [Tceal7 gene.Mutagenesis analysis has revealed the important role of the cluster CRE#2–E#1–CRE#1 in  complex ,56. MoreR-758-3p ,60,61. TTceal7. Our studies have indicated that it may be the CRE#3 motif that drives the dynamic expression of Tceal7 during skeletal muscle regeneration. Our work has also defined the interaction between Mef2c and Creb1, providing the evidence for the triple complex of Mef2c, Creb1 and Myod. In summary, this research provides new insights into the mechanism regulating the temporal and spatial expression of the Tceal7 gene.In the present study, we have identified a cluster of Mef2, CRE and E-box motifs in the 0.7 kb promoter of


Chromatin regulators of the Polycomb group of genes are well-known by their activities as transcriptional repressors. Characteristically, their presence at genomic sites occurs with specific histone modifications and sometimes high-order chromatin structures correlated with silencing of genes involved in cell differentiation. However, evidence gathered in recent years, on flies and mammals, shows that in addition to these sites, Polycomb products bind to a large number of active regulatory regions. Occupied sites include promoters and also intergenic regions, containing enhancers and super-enhancers. Contrasting with occupancies at repressed targets, characteristic histone modifications are low or undetectable. Functions on active targets are dual, restraining gene expression at some targets while promoting activity at others. Our aim here is to summarize the evidence available and discuss the convenience of broadening the scope of research to include Polycomb functions on active targets. The genetic information encoded in eukaryotic DNA is managed through the intertwined actions of a large collection of transcription factors and chromatin regulators. Eukaryotic genomes are organized as long fibers of repeated nucleosomal units made of DNA wrapped around an octameric set of histone proteins, an evolutionary successful structure serving gene expression and replication functions. Transcription factors confer the specificity to regulate transcription by recognizing DNA sequences with various affinities. On the other hand, chromatin regulators, usually present as multiprotein complexes, modulate the interactions of transcription factors and transcriptional machinery with the DNA template, usually restricted by nucleosomes . ChromatDrosophila melanogaster . If a trend was to be identified, it would be that the ratio of repressed to active Polycomb targets in primitive cells is high whereas, in contrast, in differentiated cells types the ratio is tilted toward the set of active targets ,124,140.Binding patterns of Polycomb products to active sites partially differ from those of repressed domains . In geneJust as Polycomb is involved in the formation of loops and other contacts between occupied, distant sites in repressed domains, active Polycomb targets are tied in similar structures. In imaginal discs, a higher frequency of short- or long-range contacts correlates with the presence of PRC1 components on active promoters and enhancers . These PDrosophila cell lines and imaginal discs [ChIP experiments studying the localization of PRC1 often include the core subunit RING1B, and variably other subunits from canonical and variant PRC1 complexes. The presence of PRC2 proteins, in contrast, is much less studied. Polycomb-induced histone modifications, H3K27me3 and H2AK119Ub, usually found at repressed targets are, generally, absent. This is documented for al discs ,129 or ial discs , germinaal discs , epidermal discs ,139, hemal discs ,144. Polal discs ,140. In al discs ,145. Neval discs ,133. Theal discs ,138,139.al discs .Drosophila imaginal discs, shows that chromatin at a subset of active enhancers associated to Polycomb targets is decorated with H2AK118Ub [The presence of H2AUb is generally undetectable or detected at low levels on active targets. The relatively little impact of this modification has been tested in epidermal progenitors depleted of RING1A and RING1B. The accompanying down regulation of active Polycomb targets, however, can be rescued to a large extent by an inert form of RING1B occupying these targets, thus showing that the transcriptionally positive influence of RING1B on these targets is independent of its E3 ligase activity . Neverth2AK118Ub . The obsAlthough incomplete, the current evidence on the association of Polycomb proteins to active sites suggests that it may not occur, necessarily, as part of the known, biochemically defined complexes. In imaginal discs, for example, PH and PC bind to active regulatory elements independently of each other, in contrast with their colocalization at repressive PREs . In ESCsKinases are among the regulators that can flip RING1B roles . RING1B Not only RING1B phosphorylation can modulate its functions, but also the alteration in composition of Polycomb subunits is what affects the activity of the modified complex in other cases. This is the case mentioned above of PRC2 complexes in differentiating murine erythroid cells. There, a repressive, enzymatically active EZH2-EED-SUZ12 core complex in primitive cells, turns during differentiation into an inert EZH1-SUZ12 complex located on active targets . The selAttempts to discern between contributions of canonical or variant PRC1 complexes are hampered by the evidence available nowadays, which is fragmentary and sometimes contradictory. For instance, in epidermal progenitors, there is a trend for the association of canonical subunits (PCGF4 and CBX8) with repressed targets and subunits from variant complexes, such as L3MBTL2, RYBP, or KDM2B, enriched on active sites . In contPolycomb proteins regulate active genes through mechanisms still not fully understood. For some targets a negative influence is observed , identified by the upregulation that follows depletion of Polycomb subunits ,125,137.Drosophila, assist their recruitment to active targets [Evidence connecting Polycomb subunits with regulators associated to gene expression has been accumulating steadily. Thus, subunits of histone acetylase complexes MOF and Tip60 appear in the characterization of Polycomb interactors ,149,150. targets . This biDrosophila imaginal discs. These studies are not readily comparable because of their inherently different resolution: single ESCs [The down modulation of active Polycomb targets is best documented in ESCs and in gle ESCs  and imaggle ESCs ,137. Nevgle ESCs , and to gle ESCs . Promotegle ESCs ,137,152.Kinetic analysis of the activity of the Polycomb-bound active promoters in ESCs shows a lower frequency of transcriptional bursting than that of active genes, consistent with a switch between repressed and Polycomb-activated states . These bPolycomb-bound sites at active targets are enriched among genomic three-dimensional contacts, although less extensively than Polycomb-repressed promoters . MoreoveThe analysis of active Polycomb targets in imaginal disc cells, instead, enlightens dampening events related to the transition between paused (arrested shortly after initiated) and elongating transcription . An ideaExamples of Polycomb-supported transcription are predominantly known in mammalian cells. Its identification comes from loss-of-function mutant experiments that results in decreased stationary mRNA and important alterations in differentiation programs of PRC1 mutant mouse lines. These experiments correlate with decreased expression of signature elements of cell lineage programs as is the case of those conducted on epidermis, germ cells, and early mesodermal cell types ,139,155.The mechanism by which Polycomb supports transcription have not been elucidated yet. In general, depletion-associated gene downregulation is rather moderate, suggesting roles as positive cofactors. Following the same line of reasoning mentioned above, the influence on enhancer-promoter communication would serve to interpret Polycomb sustaining gene expression: by promoting communication; a higher activity at the promoter could be expected. Although there might be mechanistic differences, Polycomb subunits would be acting as coactivators. The accompanying histone modifications at regulatory sequences  would buffer them against non-specific repressive influences. The fact that Polycomb subunits play a direct role sustaining gene activity is supported in epidermal progenitors by both the progressive decline in expression of active targets as a result of RING1A and RING1B depletion and the complementation of the defect by ectopic expression of RING1B .The involvement of RING1B in the occurrence and maintenance of regulatory regions (assessed by Assay for Transposase-Accessible Chromatin (ATAC) experiments where accessible chromatin sites are evaluated through exposure to a transposase) has been documented in breast tumor cell lines . The effdac, linked CG588 gene is upregulated. Alternatively, deletion of the 3’ site decreases CG588 expression with a much milder effect on dac [An entirely different approach, CRISPR-CAS mediated deletion of DNA sites occupied by PRC1, also demonstrates the involvement of PRC1 products in gene activation. These laborious experiments are loci-specific and do not involve changes in the levels of PRC1 products. In one case, the deletion of a promoter proximal site resulted in downregulation of the selector gene vestigial in the pouch of the wing imaginal disc, where it is normally expressed . In a dit on dac . In eithIn certain cases, gene expression induced during differentiation/development processes requires the activity of Polycomb proteins. However, its involvement seems to be limited to the establishment of the active transcriptional state rather than to its maintenance; as active transcriptional states are maintained without Polycomb presence.Meis2; a developmentally important pleiotropic transcription factor. RING1B contributes positively (embryonic brain structures) and negatively (other tissues) to Meis2 expression. RING1B binds constitutively a site located 3’ to the end of the locus and its occupation at the promoter varies with developmental time and expression states  of Polycomb proteins on active targets. It also underlines the relevance of the quantitative regulation of gene expression, in opposition to alternative on and off states. In this regard, Polycomb regulation of active genes is dual, either restricting transcription output, in some cases or, in others, supporting expression. Phenotypic consequences of defective regulation when dampening gene expression are not known. In contrast, decreased expression levels resulting from loss-of-function mutations are more easily correlated with impaired differentiation processes. It is clear that conventional approaches in the genetic analysis, downregulating/depleting any given subunit, lack the needed resolution. Instead, the use of allelic variants, as those being used to dissect catalytic from other activities, will be necessary.Further studies are required to measure the impact of functions on active genes along cell lineage differentiation pathways. The current evidence, however, points at a large number of targets, including some important in the maintenance of cellular homeostasis. The detection of PRC1 products, RING1B in particular, on enhancers and super enhancer in tumor cell lines is intriguing. It appears, that as it has been previously observed for co-repressors , Polycom