PTBP3 (Polypyrimidine tract binding protein 3) locus to evaluate its potential role in regulating expression of the gene. PTBP3 expression in prostate cancer cell lines is found significantly higher compared to skin fibroblasts using real-time PCR (p < 0.05) and digital droplet PCR (p < 0.01). Exploration of the chromatin spatial architecture of a nearly 200-kb fragment of chromosome 9 encompassing the PTBP3 gene identified two elements located 63 kb upstream and 48 kb downstream of PTBP3, which looped specifically to the PTBP3 promoter. These elements contain histone acetylation patterns characteristic of open chromatin regions with active enhancers. Our results reveal for the first time that long-range chromatin interactions between the −63 kb and +48 kb loci and the PTBP3 promoter regulate the expression of this gene in prostate cancer cells. These interactions support an open chromatin form for the PTBP3 locus in cancer cells and the three-dimensional structural model proposed in this paper.The long-range control of gene expression is facilitated by chromatin looping and can be detected using chromosome conformation capture—3C. Here we focus on the chromatin architecture of the  Regulation of gene expression involves multiple mechanisms at various stages leading to decreased or increased protein synthesis. Gene regulation at the transcription level is a combination of effects of chromatin conformation and interaction between the complex transcription machinery and DNA . Post-trSome of the best known gene regulation mechanisms which play an important role in cancer development are: DNA methylation, gene silencing by microRNA or histone modifications . In the PTBP1  is a gene encoding a multifunctional protein mainly known for its role in tissue-specific regulation of exon splicing . Microarray data obtained from GEO indicate that PTBP3 is upregulated in prostate tumors versus normal prostate tissue . Moreover, upregulation of the PTBP3 gene has been demonstrated in prostate cancer progression states versus benign prostate cancer samples (GDS1439/224618). RNA sequencing expression data from the GEPIA analysis of the PTBP3 expression across normal and cancerous prostate tissue also demonstrate upregulation of the PTBP3 gene in prostate cancer tissue compared to normal samples . As expected, based on the higher PTBP3 expression in prostate cancer cell lines, a high protein level was detected in many human cancers: lung, stomach, colorectal, urothelial and pancreatic (www.proteinatlas.org). Furthermore, overexpression of the PTBP3 protein in lung and pancreatic cancers was associated with unfavorable prognosis.We evaluated the level of the PTBP3 protein in the freely available Human Protein Atlas database  in a 200-kb region across the locus that looped specifically to the PTBP3 promotor which activate PTBP3 expression in the cancerous prostate cell lines: PC3 and PC3M. In normal skin fibroblasts, flexible chromatin fibers are observed, with absence of specific long-range looping interactions in the PTBP3 locus. These data suggest that in skin fibroblasts, there is no interaction within the region spanning 200 kb which could bring distal regulatory elements close to the gene promoter.We identified for the first time novel long-range chromatin interactions between the −63 kb and +48 kb loci and the PTBP3 gene. Chromatin immunoprecipitation was carried out in the prostate cancer cell lines PC3 and PC3M, and skin fibroblasts VH10 using antibodies specific for acetylated H3K27.Enhancer elements are known to be associated with certain histone modifications—acetylation of lysine 27 in histone H3 (H3K27ac) and methylation of H3K4 . H3K27acPTBP3 locus, H3K27ac enrichment was detected at the analyzed −63 kb and +48 kb from the transcription startsites. In skin fibroblasts, in which the PTBP3 locus is inactive, no statistical evidence for acetylated H3K27 was obtained at the analyzed positions.In the PC3 and PC3M cell lines with a highly active PTBP3 locus in prostate cancer cells (PC3 and PC3M) may suggest that open chromatin regions are located at positions −63 kb and +48 kb of the PTBP3 promoter, respectively, and may act as epigenetic activation regulators of the PTBP3 gene.H3K27 acetylation detected at analyzed regions of the PTBP3 gene in prostate cancer cell lines.Data from DENdb, compared to our 3C and ChIP assay results, suggest a potential presence of distal enhancer elements that come into physical contact with proximal promoter via looped out chromatin in the prostate cancer cell lines PC3 and PC3M. The long-range chromatin looping interactions obtained in our study correlated with the overexpression of the PSA Prostate specific antigen), TMPRSS2 (Transmembrane serine protease 2), UGT1A (UDP glucuronosyltransferase family 1 member A complex locus), UBE2C (Ubiquitin conjugating enzyme E2 C), through chromatin looping [UBE2C have been reported to be a new therapeutic target in castration-resistant prostate cancer [Using the 3C assay and its derivative methods, it has previously been demonstrated that long-range chromatin interactions regulate many well-known genes associated with prostate cancer through chromatin looping. The abnormal activation of the androgen receptor (AR) is a major example of chromatin looping mechanisms that enhance gene expression in prostate cancer . Recent  looping ,44,45,46e cancer ,46.PTBP3 gene in prostate cancer cell lines  1640 medium supplemented with 10% fetal bovine serum (FBS), and the human skin fibroblast cell line (VH10) was grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS). The tested cell lines were cultured at 37 °C in a humidified atmosphere of 5% COThe total RNA from human prostate cancer cell lines  and fibroblasts (VH10) was isolated using the High Pure RNA Isolation Kit  according to the manufacturer’s protocol. Complementary DNA (cDNA) was generated used the Transcriptor High Fidelity cDNA Synthesis Kit  with Anchored-oligo(dT)18 Primer. Denaturation of the template–primer mixture was carried out by heating the tube for 10 min at 65 °C. Reverse transcription was carried out at 50 °C for 30 min and at 85 °C for 5 min. The purity of cDNA was tested with agarose gel electrophoresis.PTBP3, GAPDH and PBGD genes, and run with LC480 Probes Master . The experiment was performed in triplicates for each cell line. Real-time PCR cycling conditions were as follows: One cycle at 95 °C, 10 min; 45 cycles of denaturation , annealing  extension  and cooling .Quantitative reverse transcriptase real-time PCR (qRT-PCR) was performed using RealTime Ready Custom Panels 96-8  with primers and probe from Universal ProbeLibrary  specific to the R transcripts in prostate cancer cells and skin fibroblasts. TaqMan Gene Expression Assay was used to confirm the PTBP3 gene expression profile by another quantifying method: ddPCR using the QX100 system . The copy numbers for each target were averaged across duplicates and normalized to PBGD references genes. Endogenous mRNA levels were measured by ddPCR using the QuantaLife droplet digital PCR system  according to the manufacturer’s instructions. Briefly, the 20-μL ddPCR mixture was prepared by combining cDNA, primers and probes with ddPCR Supermix . Oil emulsion droplets for each sample were prepared using the QuantaLife droplet generator . PCR reaction was under the following conditions: One cycle at 95 °C for 10 min, 45 cycles of 95 °C for 30 s, 60 °C for 1 min, and 98 °C for 10 min in a standard thermal cycler . Plates were read on a QuantaLife droplet reader , and the concentrations (copy numbers) of the targets in the samples were determined using the QuantaSoft software .Digital droplet PCR was conducted to quantify the concentration of the The datasets for the analysis and visualization of the PBP3 expression level in prostate tumor versus normal prostate samples were obtained from several gene expression repositories: Gene Expression Ominbus (GEO) , the CanPTBP3 gene. The Taqman probe (bait) and the reverse primer were generated within a restriction fragment at the promoter, and multiple forward primers were designed within restriction sites in distal regions across the PTBP3 locus spanning a genomic distance of 203,723 kb (http://genome.ucsc.edu/encode/) was used to evaluate DNase I-hypersensitive sites in the PTBP3 locus and the flanking region upstream and downstream. Multiple forward primers at −79.7 kb, −63.2 kb, −30.7 kb, −6.8 kb, +48.4 kb, +71.3 kb and +119.3 kb in reference to the PTBP3 transcriptional start site were designed. To analyze ligation products across the 3C templates, 200 ng of a 3C template was used per 20 μL of Taqman qPCR reaction. To account for differences in independently prepared 3C samples, the qPCR data for each sample were normalized to the results obtained for primers designed for the housekeeping gene ERCC3 (ERCC Excision Repair 3), located in an area with the same chromatin structure in all types of cells. Each qPCR reaction was performed in triplicate, and the data presented were the average of two to four independent experimental results for all PCR reactions.3C experiments were completed as described previously  with min3,723 kb . The vis6 cells (on a 100 mm culture dish) were cross-linked with 1% formaldehyde for 10 min at room temperature. The reaction was stopped with glycine at 0.125 M. The cells were washed with cold Phosphate buffered saline (PBS), scraped and collected by centrifugation. Nuclei preparation and chromatin digestion was carried out using SimpleChIP® Cell Lysis Buffers A & B and MNase from Cell Signaling Technology . Chromatin was digested with 1000 units of MNase at 37 °C for 30 min, followed by sonication to an average size of 150 bp to 500 bp. Chromatin equivalents of ~1 × 106 cells were suspended in Dilution Buffer with 1 × protease inhibitor cocktail  and precleared by incubation with 5 µL of protein G magnetic beads for 1 h at 4 °C. An aliquot of chromatin was removed and saved as “Input”. The reminder of supernatant containing precleared chromatin was immunoprecipitated using antibodies: 2 µL of H3K27Ac or 1 µL of normal mouse IgG , 20 µL of protein G magnetic beads blocked with bovine serum albumin (BSA)  and 50 µL of BSA 0.5%. Immunoprecipitations were performed overnight at 4 °C. Protein G bead–antibody/chromatin complexes were washed as described in the Magna ChIP™ G  protocol. Protein/DNA complexes were eluted, and cross-links were reversed by incubation with proteinase K (10 µg/mL) and Elution Buffer  for 2 h at 62 °C and 10 min at 95 °C. DNA was purified using spin columns as described in the Magna ChIP™ G  protocol. Enrichment was analyzed using SYBR Green qPCR. qPCR primers  protocol with minor modifications. Approximately 4–5 × 10 primers  were locPTBP3 promoter exclusively in PTBP3-expressing prostate cancer cell lines. The model of three dimensional chromatin conformation in the PTBP3 locus obtained for prostate cancer cell lines with high PTBP3 expression suggests, that the variable chromatin conformation is a mechanism that regulates its expression.Our study revealed two elements (−63 kb and +48 kb) in a 200-kb region across the locus that looped specifically to the


They are named after the abnormal oligomerization/aggregation of the neuronal microtubule-associated Tau protein. Besides its role as a microtubule-associated protein, a DNA-binding capacity and a nuclear localization for Tau protein has been described in neurons. While questioning the potential role of Tau-DNA binding in the development of tauopathies, we have carried out a large-scale analysis of the interaction of Tau protein with the neuronal genome under physiological and heat stress conditions using the ChIP-on-chip technique that combines Chromatin ImmunoPrecipitation (ChIP) with DNA microarray (chip). Our findings show that Tau protein specifically interacts with genic and intergenic DNA sequences of primary culture of neurons with a preference for DNA regions positioned beyond the ±5000 bp range from transcription start site. An AG-rich DNA motif was found recurrently present within Tau-interacting regions and 30% of Tau-interacting regions overlapped DNA sequences coding for lncRNAs. Neurological processes affected in AD were enriched among Tau-interacting regions with  Tauopathies are neurodegenerative disorders associated to the presence, in neurons, of abnormal forms of the microtubule associated Tau protein . Over 20Tau is an intrinsically disordered protein  that wasIn vivo expression analysis of a subset of Tau-interacting genes performed in brain from wild type (WT), Tau-deficient (KOTau) and THY-Tau22, a transgenic model of AD-like tauopathy  was carefully dissected and mechanically dissociated in culture medium by triturating with a polished Pasteur pipette. After 10 days in vitro (DIV), cells were either maintained at 37°C (control conditions) or exposed to 44°C  in 5% CO2 incubator for 1 h as previously described (Wild-type and knock-out Tau (KOTau) mouse  primary escribed . Cells whttp://microarrays.curie.fr/publications/UMRS1007/Benhelli-Mokrani_2018/Chromatin immunoprecipitation followed by microarray hybridization (ChIP-on-chip) experiments were performed on genomic DNA prepared from three independent cultures of neurons of murine embryonic origin cultured under either physiological or heat-stress (HS) conditions. Chromatin immunoprecipitation experiments were performed as previously described . For eacper chromosome) and of probes associated to Tau-interacting regions (per region and chromosome)  and further analyzed with a spreadsheet software. When interrogation of the RefSeq Transcripts database with Tau-interacting DNA regions had given rise to several transcript ID associated to a same Tau-interacting region, the region was considered as a single region (regardless the number of transcripts associated to it) while determining the number of Tau-interacting DNA regions, of genes associated to Tau-interacting regions  were determined using nanodrop.The brain was extracted after mouse decapitation. The cortex and hippocampus were dissected and flash frozen in liquid nitrogen. Samples were stored at −80°C. Total RNA of cortex and hippocampus from 17m KOTau and WT littermates, and hippocampal CA1 region from 6m THY-TAU22 and WT littermates were extracted using the RNeasy lipid tissue kit  according to the manufacturer's recommendations. After elution, RNAs were treated with 1 unit/μl of DNAse I (Sigma) to remove genomic DNA. RNA concentration and purity (http://www.rdml.org/miqe.php). One μg of total RNA was reversely transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's recommendations using Random Primers. qPCR assays were performed using a Biorad CFX384/qPCR Instrument, from 5 ng of the corresponding cDNAs in 10 μl final volume with SYBR Green (Bioline) reagents: 95°C 2 min, then 40 cycles at 95°C 5 s/60°C 10 s/72°C 15 s, followed by a dissociation step. Primers for qPCR analysis used in this work were manufactured by Eurofinsgenomics with the corresponding sequences listed in CT method with respect to three reference genes .RT-qPCR assays were performed in conformation with MIQE guidelines , with ad libitum access to food and water. Animal experiments were performed with the approval of the local ethics committee (agreement CEEA 062010R), in compliance with the standards for the care and use of laboratory animals, and the French and European Community rules.t-test was used to statistically analyze the data, two by two for all conditions used. Before the application of the Student's t-test, the Shapiro–Wilk test of normality was used to verify that the data to be analyzed were normally distributed and the F-test of equality of variances was used to verify that variances were not significantly different. In Figure n = 17 (WT CTRL), 17 (WT HS), 13 (KO CTRL) and 17 (KO HS) animals. In Figure n = 11 (WT CTRL), 11 (WT HS), 12 (Tau22 CTRL) and 11 (Tau22 HS) animals.In Figures To identify neuronal DNA regions bound by Tau protein we performed genomewide ChIP-on-chip experiments that combine Chromatin ImmunoPrecipitation (ChIP) with DNA microarray (chip). Chromatin prepared from mouse primary cultures of embryonic cortical neurons was immunoprecipitated with Tau1 antibody. Tau1 recognizes Tau protein unphosphorylated at sites present between residues 189 and 207 and has been previously used to evidence Tau protein in the nuclear compartment ,34,35 unInstead of estimating probe behavior variations from multiple samples, MAT relies in its capacity to determine true biological enriched signals from baseline probe behavior. The MAT algorithm is a powerful 81 parameters probe behavior model that has been specially designed to analyze results from tiling-arrays that contain a very high number of probes giving rise to a large quantity of data as in the case of the ChIP-on-chip assays performed here. Because MAT does not rely on variations from multiple samples, it can handle a single ChIP-on-chip sample. However, its sensitivity and specificity is highly increased while analyzing multiple ChIP and control samples such as in this work, confidently detecting enriched ChIP regions with a reduced false discovery rate .The different steps of the analysis of the data obtained from ChIP-on-chip assays carried out with Tau1 and anti-NSs (NSs) antibodies are summarized in Figure The total number of probes present within DNA regions identified as significantly interacting with Tau compared to NSs under physiological (control) conditions corresponded to <2% (1.9%) of the total number of probes present in the array. The list of Tau-interacting DNA regions was used to interrogate the RefSeq transcripts database. Despite the fact that the array was designed to be enriched in promoter regions, DNA regions present outside the range of regions classified as promoter regions by the manufacturer were also found present among the DNA regions determined as significantly enriched in Tau1 vs anti-NSs ChIP-on-chip assays. Thus, Tau-interacting DNA regions were classified either as genic when positioned between 5000 bp upstream of TSS and 5000 bp downstream of STOP codon or as intergenic when positioned outside this range.The general characteristics of the DNA regions determined as Tau-interacting regions under physiological conditions are described in Figure The Tau-interacting DNA probes were evenly distributed among all chromosomes Figure . The meaDlg2  was 1,97 megabasepairs (Mb) long.Tau-interacting DNA probes clustered in 2138 genic DNA regions that overlapped with 1866 annotated genes coding for one or more transcripts . The vasThe distribution of Tau-interacting genic DNA regions with respect to the TSS was analyzed Figure . Tau-intBesides genic DNA regions, some 1989 Tau-interacting regions were classified as intergenic by PGS Software gene section analysis , a numbeThus, overall ChIP-on-chip results obtained under physiological conditions showed that Tau protein recognized by Tau1 antibody specifically interacted with a fraction of neuronal chromatin with Tau-interacting regions being evenly distributed among all chromosomes as well as between genic and intergenic classified regions. Tau-interacting regions were mainly positioned further away than ±5000 bp from TSS with a preference towards DNA sequences coding for lncRNAs.de novo DNA-binding motif was identified, which was present 8010 times among the 4127 Tau-interacting regions  on the same DNA regions was searched using PGS through the JASPAR database. The list of the top 15 over-represented known motifs recognized by mammalian TFs is shown in Figure se brain . Transcrse brain . On the se brain , they wese brain  that conIn order to analyze the potential relevance of Tau interaction with the neuronal genome with respect to neurological functions and disorders, a functional analysis of Tau-interacting genes was carried out using Ingenuity Pathway Analysis (IPA) and PGS softwares. Interestingly, canonical pathways Figure  and biolAmong the ten most significant canonical pathways generated by IPA from the list of Tau-interacting genes Figure , the majP-value< 0.01)  Figure ,47, calc) Figure  and c-AM) Figure . Interes) Figure .In order to investigate the plasticity of Tau interaction with DNA, we analyzed the effect of HS on the capacity of Tau protein to interact with neuronal DNA. The choice of HS was justified by results obtained in previous works showing that HS affected neuronal Tau phosphorylation, and subcellular distribution by enhancing the presence of Tau protein in the nuclear compartment . In ordeDNA fragments immunoprecipitated with Tau1 under HS condition hybridized with 1.4% of the array total probes, that is 25% less than under physiological conditions suggesting a trend towards Tau DNA dissociation after HS notwithstanding the HS-induced nuclear accumulation of Tau isoform recognized by Tau1 antibody . The genAs observed under physiological conditions, Tau-interacting probes under HS conditions were also evenly distributed among chromosomes and between genic (0.77%) and intergenic (0.63%) regions Figure  and C. THS-treatment differentially affected Tau interaction with genes present on different chromosomes. In the case of several chromosomes  the percentage of Tau-interacting genes was only slightly diminished after HS compared to physiological conditions whereas it was enhanced in the case of chromosome 18 and divided by almost two in the case of chromosomes 1 and 14 Figure .Dlg2 gene  among Tau-interacting genes after HS , neuronal networks and brain function , DNA Damage Response , transcription regulation and chromatin remodelling  or related with RNA-mediated gene silencing . Among the 30 genes selected, four were previously identified using functional genomic screening in Drosophila as modifiers of tauopathy: Mark1, Camk1, Ercc4  and Tau-deficient  mice. The 30 genes tested were selected according to their roles in biological processes associated to Tau protein and AD pathology , absent from the list of Tau-interacting genes and displaying similar expression rates in WT and KOTau neurons (data not shown). Among the 30 Tau-interacting genes tested, seven genes displayed over a 1.5 fold change in WT versus KOTau neurons with their respective transcriptional rates enhanced in KOTau compared to WT neurons: Grm5 (2.25 ± 1.25), Camk1 (1.68 ± 0.87), Jarid2 (1.61 ± 0.87), Ntrk2 (1.78 ± 0.99), Ctc1 (1.58 ± 1.45), Ctnn1b (1.877 ± 1.52) and Polr2a (2.56 ± 1.98). However, because of strong variations among independent cultures (mainly of WT origin), these differences were not statistically significant (data not shown). In order to further explore the potential role of Tau protein regulating the expression of Tau-interacting genes, Tau-dependent gene expression analysis was extended to in vivo, physiological versus HS conditions.The transcription rates of the 30 genes in WT and KOTau neurons were calculated with respect to 3 reference genes  or submitted to HS Figure . For thiPolr2a, Camk1, Jarid2, Grm5) were chosen from the previous group of seven genes bound by Tau under physiological conditions that displayed over 1.5-fold difference in KOTau versus WT primary neuronal cultures of embryonic origin. The other five genes, bound by Tau under HS conditions , were chosen according to their implication in neurodegenerative diseases . Expression rates were calculated with respect to the same set of three reference genes than before. Of note, the RNAs extracted from cortex and hippocampus used in theses assays, originated not only from neurons that are Tau-expressing cells but also from glial cells that are poorly- or non-Tau expressing cells causing the attenuation of potential Tau-dependent effects.Among the nine genes tested, four  Tau-dependent gene expression variations were observed for two out of the set of three genes that interacted with Tau only under HS conditions (Xrcc6 and Dlg1)  Figure . For botThe significant gene expression variations observed here in relation with the absence of Tau protein suggest a transcriptional repressive role for Tau protein necessary in regulating gene expression under stress conditions. Gene expression changes have been described to rapidly occur after HS, affecting many biological pathways including mitochondrial dysfunction, altered protein synthesis and the immune response , which aIn order to analyze the potential effect of Tau pathology on the expression of Tau-interacting genes and compare this effect to that induced by the absence of Tau protein, the expression rates of the nine Tau-interacting genes previously analyzed in brain extracts from WT and KOTau mice were further analyzed in hippocampal CA1 extracts of 6-month (6m) old THY-Tau22 mice with respect to CA1 extracts from WT littermates. The CA1 regions from 6m THY-Tau22 brains are characterized by the presence of pathological prefibrillar oligomerized forms of Tau protein as revealed by immunohistochemical analysis and confocal microscopy using the TOC1 antibody  Figure , B. PrefThe differences on gene expression observed between THY-Tau22 and WT mice Figure –E were mP-values ≤ 0.06) in the case of 6  out of the nine genes tested. For all genes, except Polr2a, gene expression variations were observed under HS conditions that corresponded to conditions of increased accumulation of oligomerized Tau in the nucleus of neurons . However, contrary to our expectations, results obtained from ChIP-on-chip assays clearly indicated that DNA regions bound by Tau were predominantly positioned outside ‘conventional’ promoter regions, that is beyond the ±5000 bp range from TSS. Tau-interacting DNA regions were evenly distributed among all chromosomes, for the most absent from ‘conventional’ promoter regions with half of the Tau-interacting DNA sequences positioned within non-protein coding intergenic regions. Thus, overall Tau protein displayed DNA-binding characteristics different from those expected for a conventional TF.Thirty percent of Tau-interacting regions overlapped with DNA regions coding for lncRNAs. LncRNAs are for the most enriched in the nuclear compartment where they participate in the regulation of transcription and translation as well as in genome organization . A potenDlg2 gene overlaps the Dlg2 gene, regulating its expression. Interestingly, the expression rate of the Dlg2 gene, which is the genic DNA sequence displaying the highest number of Tau-interacting regions, was significantly deregulated in neurons from the CA1 region of THYTau22 mice under conditions of nuclear accumulation of pathological oligomerized forms of Tau protein. The deregulation of the expression of the Dlg2 gene that codes for a key scaffolding protein enriched at postsynaptic membranes has been linked to AD  regulate genome 3D structure through boundary and loop organization . In mammosophila ,58 and iosophila , GAGA faosophila  and heteosophila ,15 are pDrosophila, GAFs have been shown to play a critical role in regulating HS-induced gene expression , future work should be carried out using techniques such as chromatin immunoprecipitation sequencing (ChIP-Seq) that allows for an unbiased exploration of the whole genome including repeated non-protein coding DNA sequences such as satellite, LINEs, SINEs, rDNA, telomere, etc. that were absent from the array used in this work. Recently, the activation of transposable elements has been associated to Tau pathology in AD brain , reinforBeyond Tau protein, it would be of interest to extend this approach to other main actors of neurodegenerative diseases for which neuronal nuclear functions have also been postulated such as amyloid-β peptides and α-synuclein ,66.http://microarrays.curie.fr/publications/UMRS1007/Benhelli-Mokrani_2018/.The list of: (i) the genic DNA regions interacting with Tau protein under physiological conditions ; (ii) thSupplementary DataClick here for additional data file.


S. pombe. They show that Seb1 promotes long-lived RNAPII pauses at pericentromeric repeat regions and that their presence correlates with the heterochromatin-triggering activities of the corresponding dg and dh DNA fragments, providing new insight into Seb1-mediated polymerase stalling as a signal necessary for heterochromatin nucleation.In this study, Parsa et al. investigated the mechanisms underlying RNAi-independent heterochromatin assembly by the CTD–RRM protein Seb1 in  Schizosaccharomyces pombe, transcripts derived from the pericentromeric dg and dh repeats promote heterochromatin formation via RNAi as well as an RNAi-independent mechanism involving the RNA polymerase II (RNAPII)-associated RNA-binding protein Seb1 and RNA processing activities. We show that Seb1 promotes long-lived RNAPII pauses at pericentromeric repeat regions and that their presence correlates with the heterochromatin-triggering activities of the corresponding dg and dh DNA fragments. Globally increasing RNAPII stalling by other means induces the formation of novel large ectopic heterochromatin domains. Such ectopic heterochromatin occurs even in cells lacking RNAi. These results uncover Seb1-mediated polymerase stalling as a signal necessary for heterochromatin nucleation.In  Schizosaccharomyces pombe have played a major role in our understanding of this type of repressive chromatin. In S. pombe, transcripts derived from the pericentromeric dg and dh repeats during S phase , the nuclear 3′–5′ exonuclease Rrp6, and the RNA export factor Mlo3/Yra1  machinery antagonizes silencing, suggesting a possible mechanism by which heterochromatin is limited at mRNA-coding genes despite the presence of termination-associated pauses. These findings establish a role for Seb1-dependent RNAPII pausing in promoting the formation of repressive chromatin structures.A second, less-characterized pathway does not require RNAi factors. Components that impact this mechanism include the RNA polymerase II (RNAPII)-associated RNA-binding protein Seb1, the repressor/remodeler complex SHREC, the nuclear 5′–3′ exonuclease Dhp1 . The seb1-1 mutation causes a reduced median 5′ traveling ratio and an increased median 3′ traveling ratio for both clusters . These data indicate that the seb1-1 allele leads to decreased RNAPII pausing at gene 5′ ends with an associated increased 3′ signal; the latter may be due to polymerase release from upstream pauses.To understand how Seb1 interfaces with the transcription of , seb1-1 . When copathways . We exam NET-seq . To anal regions . Initiale groups D, two ofdg and dh repeat transcripts (seb1-1 using RNA sequencing (RNA-seq) revealed no significant changes [P < 0.01 and |log2(fold change)| > 1] in the transcript levels of known silencing factors . To assess direct interactions of Seb1 with pericentromeric RNA at single-nucleotide resolution and across the entirety of the dg and dh regions, we conducted PAR-CLIP (photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation) in replicate on a clr4Δ strain. Computational analysis identified statistically significant Seb1 PAR-CLIP read clusters  via a motif described previously by others for nonheterochromatic sites bound by Seb1  (Our prior RIP-qPCR (RNA immunoprecipitation [RIP] combined with quantitative PCR [qPCR]) analysis indicates that Seb1 functions directly in heterochromatin assembly by binding pericentromeric nscripts . Furtherclusters  and conf by Seb1 : UGUA (D × 10−7) . Our ana × 10−7) .Supplemental Fig. S4A). Noncoding RNAs (ncRNAs) display the highest coverage at a mean level ∼100-fold higher than that of coding genes .To compare the binding of Seb1 across transcript classes, we computed the fraction of RNA covered by Seb1 PAR-CLIP read clusters. We observed an ∼12-fold higher PAR-CLIP cluster coverage for pericentromeric repeat intervals than for coding gene intervals (clr4Δ and clr4Δ seb1-1 strains (replicate experiments were conducted). Pericentromeric regions harbor detectable NET-seq signal in clr4Δ cells despite a low level of polymerase engagement at any single nucleotide . RNAPII densities at the pericentromeric regions are comparable between clr4Δ and clr4Δ seb1-1 strains as assessed by ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) analysis of Rpb3-3xFlag , indicating that decreases in NET-seq peak intensities caused by the seb1-1 mutation are not trivially explained by loss of RNAPII recruitment . Notably, calculation of polymerase dwell times . These data reveal detectable Seb1-dependent RNAPII pauses in pericentromeric sequences.We next examined the NET-seq profiles of pericentromeric heterochromatin sequences of ll times  at centrL5 (Frag1) . Each fragment was placed downstream from an adh1+ promoter (padh1+) in either forward or reverse orientation and upstream of a transcription terminator. This insert was then placed downstream from ura4+   . The exception, Frag8S, displays silencing activity but no detectable Seb1-dependent NET-seq peak clusters  . The heterochromatin assembly activity of this fragment may be pause-independent, or the relevant RNAPII pauses may be below the sensitivity of NET-seq. Introduction of the seb1-1 allele into these reporter strains  by homologous recombination leads to reduced silencing of ura4+ . The residual activity is likely due to the parallel RNAi pathway  , respectactivity . To compactivity . The cenom ura4+ G. Silencactivity . Three a Frag9S) H. Thus,  pathway . These dseb1-1 allele in both heterochromatin assembly and RNAPII pausing suggests that pausing signals the assembly of heterochromatin. However, given that Seb1 may have other activities impacted by seb1-1, we sought an orthogonal test of the role of pausing. Thus, we pursued an alternative strategy of testing whether increasing RNAPII pausing per se could be sufficient to trigger heterochromatin assembly and, if so, whether such an activity required RNAi. We exploited the conserved elongation factor TFIIS, which binds paused RNAPII complexes and stimulates RNA hydrolysis by RNAPII, enabling restart .The shared defect of the viable  restart . It is t restart . Mutatio restart , ultimat restart . We intr, tfs1DN . To over, tfs1DN , we plac1+ locus , enablintfs1DN strains; however, we observed no effects in this background (data not shown). Heterochromatin components are limiting and antagonized by anti-silencing factors, particularly Epe1, which actively removes the H3K9me mark (epe1Δ tfs1DN mutant strains or epe1Δ strains carrying an integrated vector-only control (epe1Δ-vc), collected multiple strain isolates for each, and performed H3K9me2 ChIP-seq on all isolates , experiments were performed in the presence of thiamine, enabling viability . Consistent with a slight fitness defect under these conditions , RNA-seq analysis revealed that 13% of the transcript pool from the tfs1 genes arise from the tfs1DN allele, and 87% arise from the wild-type allele when cells are grown in thiamine , indicating leaky repression. The ChIP-seq data obtained from strain isolates were examined for H3K9me peaks . We filtered the results for well-established heterochromatic recruitment sites  , as these genomic regions have an intrinsic propensity  to nucleate H3K9me  . No novel ectopic heterochromatic loci were observed by whole-genome H3K9me2 ChIP-seq in the 15 epe1Δ-vc strains . PIERs range in size from ∼3 to ∼15 kb, and each PIER was unique. Three PIERs are bounded by an essential gene on at least one side of the locus, suggesting that selection likely prevents observing PIERs that assemble over essential genes   D; this iepe1Δ tfs1DN isolate 2 (containing PIER2) . We observed a significant decrease (P < 0.01) in two of the three genes present in PIER2: cta3+ and its8+ . TFIISDN expression does not alter the expression of known heterochromatin factors  .To determine whether H3K9me at PIERs leads to repression, we conducted RNA-seq analysis on cis via the RNAi pathway . Such adaptive silencing of clr4+ has been described previously in epe1Δ mst2Δ strains  and its loss at the clr4+ locus . This observation indicates that pericentromeric repeats harbor the ability to respond to RNAPII pausing and assemble heterochromatin independently of RNAi. Accompanying these changes, six out of the 12 epe1Δ ago1Δ tfs1DN isolates subjected to whole-genome analysis acquired PIERs . Five of six of these PIERs are bounded by an essential gene on at least one side of the region similar to that seen in the epe1Δ tfs1DN (PIER6–10)  D. As dsR pathway , we dete strains  and evidIER6–10) , consistseb1-1 epe1Δ tfs1DN for ectopic heterochromatin assembly by whole-genome ChIP. A single PIER was observed . Two additional regions of ectopic heterochromatin appear evident by visual inspection but were below our cutoffs for H3K9me enrichment . These data suggest that while frequent PIER formation and high levels of H3K9me accumulation are promoted by Seb1, the seb1-1 allele is permissive for some degree of H3K9me triggered by tfs1DN.To address the role of Seb1 in the formation of PIERs, we interrogated 15 independent isolates of observed A—a frequpfs2-11, which produces transcription readthrough past the CPA site under nonpermissive conditions  as well as increased heterochromatin assembly over a 15-kb subtelomeric region on the right arm of chromosome 1 . Notably, the increase in centeromeric H3K9me is not at the expense of its loss elsewhere, as occurs in some mutant backgrounds due to limiting amounts of silencing factors , which is required for RNAi-independent heterochromatin assembly, recruits the silencing machinery via a physical interaction with CLR-C , a multifunctional elongation complex that binds cooperatively to RNAPII with TFIIS , enablesS. pombe . While tS. pombe , it may S. pombe . Analogo complex . Consist complex , which i complex . It has th CLR-C . Becauseth CLR-C , and Sebth CLR-C , Seb1-in mutants  as well  mutants , as thes mutants . Weak CP mutants , which ielements . Anotherelements . Thus, Se on FXN , raisingS. pombe strains and plasmids used in this study is in Supplemental Table S5. Cells were grown at 30°C in synthetic complete (SC) medium with adenine and amino acid supplements with reduced levels of uracil (150 mg/L) for PAR-CLIP or in Edinburgh minimal medium (EMM) supplemented with adenine, uracil, and the appropriate amino acids with or without 15 µM thiamine for NET-seq and ChIP-seq.A list of all S. pombe. S. pombe cultures were grown in 1 L of EMM without thiamine to an OD600 of 0.7, harvested via filtration, and flash-frozen in liquid nitrogen. Lysis and immunoprecipitation were conducted as described previously . Zirconia-silica beads  were added, and lysis was accomplished by three cycles of bead beating for 90 sec on high . Following centrifugation at 14,000 rpm for 10 min at 4°C, the supernatant was transferred to a microcentrifuge tube, extracted once with chloroform, and precipitated with isopropanol. Following resuspension and reprecipitation with isopropanol, pellets were washed with 75% ethanol and air-dried for 30 min. Pellets were resuspended in 150 µL of RNase-free water.Strains were grown in YS medium + 3% glucose overnight to ODOne microgram of total RNA was used to isolate mRNA using PolyATtract systems III and IV  according to the manufacturer's instructions. Input RNA quality and mRNA purity were verified by Bioanalyzer RNA 6000 Pico kit . To address the issue of genomic DNA contamination in RNA samples, we used Zymo RNA Clean and concentrator kit 5  according to the manufacturer's instruction. A sequencing library was constructed using NEBNext Ultra Directional RNA library preparation kit for Illumina . Libraries were analyzed for quality and average size on Bioanalyzer high-sensitivity DNA kit . The sequencing was performed on an Illumina HiSeq 4000 platform.600 of 0.75. 4-thiouracil  was added to a final concentration of 1.3 mM for 15 min in the dark at 30°C. Samples were immediately cross-linked using the UV Power-Shot handheld UV curing system (SPDI UV) at a 365-nm wavelength for 15 min while continuously stirring. Samples were collected by filtration, resuspended in 6 mL of buffer TMN150 , and frozen as “yeast popcorn” by drop-wise addition to liquid nitrogen. This material was lysed by ball mill for five cycles at 15 Hz for 3 min . CRAC was then performed from this point on as described previously  protocols  in two reviously  with theeviously , and sam600 of 1. Serial dilutions were performed with a dilution factor of 5. For ura4+ silencing assays, cells were grown on nonselective medium and 2 g/L 5-fluoroortic acid (FOA) (unless stated otherwise) YS plates for 3 d at 30°C. For tfs1DN induction assays, cells were grown on YS, EMM −leu −thiamine, or EMM −leu +thiamine for 3 d.Strains were grown overnight to saturation and diluted to an ODChIP-seq was conducted as described previously . ChIP-seS. pombe genome  were removed, and reads were flattened to remove sequence duplicates. Barcoded reads were then mapped to the e genome  using Boe genome  to alignZ-scores (based on median and median absolute deviation) from the log2 transform of the number of reads starting at each position in the defined region (centromere fragment or transcript). Positions with a robust Z-score of at least 2 and at least 10 unique reads were considered peaks. Next, peaks were clustered together using a sliding window . The density of the cluster was calculated as the number of reads in the cluster divided by the size of the cluster in kilobases.High-density regions of NET-seq signal were defined across centromeres and coding regions to compare NET-seq density between genotypes. First, NET-seq peaks were discovered by calculating robust To determine cluster densities for each fragment derived from the right arm of centromere 1, the sum of cluster densities was normalized to the sum of all densities in each sample. Error bars represent the range of two replicates.K-means  based on the travelling ratio at each end of the clr4Δ and clr4Δ seb1-1 mutant on one replicate. P-values for each cluster for the difference between the clr4Δ and clr4Δ seb1-1 traveling ratio distributions were determined by KS test for each pair of replicates. Traveling ratio cumulative distribution function (CDF) plots were similar between replicates, and a single replicate is presented.Traveling ratios were calculated for every nonoverlapping annotated transcript at least 1000 nucleotides (nt) in length according to the Pombase annotation . Transcrclr4Δ to clr4Δ seb1-1 in both replicates were considered Seb1-dependent. P-values were determined by KS test.Dwell time was determined by normalizing peak height to the average NET-seq signal density of the surrounding 100 nt. NET-seq peaks with at least a twofold decrease from Analysis was performed using TopHat  and DESetfs1DN, we divided the total number of reads that specifically aligned to the mutated region of the tfs1DN allele by the total number of reads  that aligned to this same region.To determine the fraction of reads derived from the expression of S. pombe genome . In epe1Δ backgrounds, oscillation and spreading of H3K9me can occur (H3K9me ChIP-seq peaks were considered as novel ectopic sites of H3K9me if two criteria were met: (1) H3K9me peaks were threefold or greater than the genome background signal in the isolate, and (2) when normalized to the whole-cell extract (denoted “H3K9me enrichment” in the figures), the H3K9me signal at the peak was threefold or greater higher than the background and parental H3K9me ChIP-seq signals. A curated list of genomic regions previously observed to have a propensity to form heterochromatin in various kgrounds  was genean occur ; thus, pSupplemental Table S6 and all PIERs were normalized to the RPKM of a 10-kb window surrounding each region (5 kb upstream and downstream). The ratio of H3K9me enrichment values from isolates to the whole-cell extracts were plotted as a heat map .For all isolates and whole-cell extracts, RPKMs (reads per kilobase transcript per million mapped reads) for each region in Supplemental Table S7 and were deposited in the Gene Expression Omnibus with the superseries accession number GSE114540.All available sequencing data sets are listed in


B-cell CLL/lymphoma 6 (BCL6), mapped to this region. We therefore aimed to explore its downstream targets and physiological roles in UBUC progression.Muscle-invasive urinary bladder urothelial carcinoma (UBUC) is a lethal disease for which effective prognostic markers and potential therapy targets are still lacking. Previous array comparative genomic hybridization identified that 3q27 is frequently amplified in muscle-invasive UBUCs, one candidate proto-oncogene, Methods: Specimens from UBUC patients, NOD/SCID mice and several UBUC-derived cell lines were used to perform quantitative RT-PCR, fluorescence in situ hybridization immunohistochemistry, xenograft, gene stable overexpression/knockdown and a series of in vitro experiments.Results: Amplification of the BCL6 gene lead to upregulation of BCL6 mRNA and protein levels in a substantial set of advanced UBUCs. High BCL6 protein level significantly predicted poor disease-specific and metastasis-free survivals. Knockdown of the BCL6 gene in J82 cells inhibited tumor growth and enhanced apoptosis in the NOD/SCID xenograft model. In vitro experiments demonstrated that BCL6 inhibited cytostasis, induced cell migration, invasion along with alteration of the expression levels of several related regulators. At molecular level, BCL6 inhibited forkhead box O3 (FOXO3) transcription, subsequent translation and upregulation of phosphorylated/inactive FOXO3 through phosphoinositide 3-kinase (PI3K)/AKT serine/threonine kinase (AKT) and/or epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase 1/2 (MAP2K1/2) signaling pathway(s). Two BCL6 binding sites on the proximal promoter region of the FOXO3 gene were confirmed.Conclusion: Overexpression of BCL6 served a poor prognostic factor in UBUC patients. In vivo and in vitro studies suggested that BCL6 functions as an oncogene through direct transrepression of the FOXO3 gene, downregulation and phosphorylation of the FOXO3 protein. Urinary bladder urothelial carcinoma (UBUC) is a common malignant disease in developed countries. Both environmental and genetic factors impact its development B-cell CLL/lymphoma 6 (BCL6) gene, a transcription repressor, was originally identified through its involvement in chromosomal translocations and aberrant expression associated with B cell-derived non-Hodgkin lymphoma BCL6 gene is characterized as the 5'-part encoding for Broad-complex, Tramtrack and Bric-a-brac (BTB)/POxvirus (POZ) and the 3'-end encoding for 6 DNA-binding zinc fingers Human Other than lymphoid tissues, high BCL6 protein levels were observed in a variety of epidermal neoplasms, suggesting that BCL6 may involve in morphological differentiation BCL6 transcript level and UBUC progression. An independent cohort containing 40 fresh UBUC samples (13 NMIBCs and 27 MIBCs) was also collected for evaluating the correlation between BCL6 and FOXO3 mRNA levels. Quantitative RT-PCR was performed as our previous study BCL6 (RP11-211G3), was used to assess the BCL6 copies on formalin-fixed, paraffin-embedded (FFPE) sections. Another SpectrumGreen-labeled BAC probe spanning 20p12.3 (RP11-19D2) was used as the reference and evaluated as previously described BCL6 gene was detected by using Vysis LSI BCL6 (ABR) Dual Color Break Apart Rearrangement Probe .The institutional review board of Chi-Mei Medical Center approved the retrospective retrieval of 295 primary UBUCs with available tissue blocks IRB10207-001), which underwent surgery with curative intent between January 1996 and May 2004 7-001, whImmunohistochemistry was performed on representative sections cut from FFPE tissues at 3-μm thickness as our previous study with a few modifications (Supplementary Methods). For immunostainings, one expert pathologist (CF Li) blinded to clinicopathological information and patient outcomes interpreted the immunostainings. A labeling index was recorded as 0~4% (0+), 5~24% (1+), 25~49% (2+), 50~74% (3+) and 75~100% (4+) of tumor cells that displayed strong nuclear staining. Cases with 3+ to 4+ and 0+ to 2+ immunoexpression were regarded as high and low levels, respectively.7) stably carrying either shLacZ (control) or shBCL6 were resuspended in 100 μL PBS, mixed with 100 μL matrigel  and introduced into the flanks of 7-week-old male mice. Tumor diameters were measured with a digital caliper every other day and the tumor volume in mm3 was calculated as volume = π/6(width)2 × length. Whole sections from formalin-fixed xenografts were analyzed by immunohistochemistry using pertinent antibodies (Supplementary Methods).Animal experiments were approved (#10435) by Affidavit of Approval of Animal Use Protocol, National Sun Yet-sen University. Cells were implanted into 10 NOD/SCID mice by subcutaneous injection. J82 cells (1 × 10BCL6 complete DNA (RC219007) with Myc-DDK-tag (pCMV6-BCL6) and its corresponding control pCMV6-Entry (PS100001) were obtained from Origene . PCR-based cloning was used to construct the pBCL6-HaloTag plasmid using 5'-TCCCCGCGGATGGCCTCGCCGGCTGA-3' and 5'-CCGCTCGAGGCAGGCTTTGGGGAGCTCCGGA-3' primers with Sac II and Xho I restriction sites (underlined). pHRIG-AKT1 , pFOXO3  and pFLAG-FOXO3(TM)  were obtained from Addgene  and subcloned into the pHaloTag plasmid to generate pFOXO3-HaloTag and pFOXO3(TM)-HaloTag using the In-Fusion® HD Cloning Kit . All constructs were sequenced verified. Cells (1 × 106) were transfected with 2 μg of pCMV6-Entry, pHTC HaloTag® CMV-neo  or target plasmid by mixing with 8 μL PolyJet™ reagent . Transfectants were selected with media containing 800 μg/mL of G418  for 7 days, and maintained in media with 400 μg/mL of G418 for subsequent experiments.All chemicals unless otherwise stated were purchased from Sigma-Aldrich. UBUC-derived cell lines and culture conditions are described in the Supplementary Document. The expression vector carrying BCL6 (shBCL6#1/ TRCN0000013606: 5'-CCACAGTGACAAACCCTACAA-3' and shBCL6#2/TRCN00000235665: 5'-TGGACACTTGCCGGAAGTTTA-3') and FOXO3 (shFOXO3#1/TRCN0000040098: CAGACCCTCAAACTGACACAA and shFOXO3#2/TRCN0000235491: CTTGCTCATATCCCATATAAT) were used to knockdown BCL6 and FOXO3 genes, respectively. shLacZ (TRCN0000072223: 5'-TGTTCGCATTATCCGAACCAT-3') served as a negative control.Small hairpin RNA interference (shRNAi) plasmids were inserted into the pLK0.1 vector downstream of the U6 promoter. The plasmids targeting BCL6, FOXO3 gene or treatments with inhibitors, respectively https://www.genome.jp/kegg/mapper.html) was applied to identify significant pathways of the RNA expression profile from the stable BCL6-knockdown BFTC905 cells. Matrix metallopeptidase 2 (MMP2)/MMP9 Solution Assay Kit  was used to measure MMP2/MMP9 activity. Immunoblot analyses were performed as in our previous study to detect the expression levels of tumor protein p53 (TP53), phosphor-TP53 (serine 15) [pTP53(S15)], pTP53(S20), cyclin-dependent kinase inhibitor 1A (CDKN1A), CDKN1B, CDKN1C, CDKN2B, CDKN2C, CDKN2D, cyclin D1 (CCND1), cyclin-dependent kinase 4 (CDK4), CCNE1, CDK2, RB transcriptional corepressor 1 (RB1), E2F transcription factor 1 (E2F1), transcription factor Dp-1 (TFDP1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cadherin 1 (CDH1), vimentin (VIM), CD44 molecule, Indian blood group (CD44), FOXO3, AKT, pAKT1(S473), mitogen-activated protein kinase 1/3 , pMAPK1/3(T202/Y204), pFOXO3(S253), pFOXO3(S294), HRas proto-oncogene, GTPase (HRAS) and proteins phosphatase and tensin homolog (PTEN) (Supplementary Methods).Flow cytometric, bromodeoxyuridine, cell proliferation, soft agar, anchorage-independent growth, wound-healing, transwell migration, transwell invasion and RNA expression profiling with the HumanHT-12v4 Expression BeadChip  assays were used to determine the alterations of cell cycle distribution, cell proliferation, colony formation/anchorage-independent cell growth, cell migration, cell invasion and mRNA levels after overexpression, knockdown of the FOXO3 proximal promoter regions  linked to the luciferase reporter gene plasmid (pGL4.17-A and pGL4.17-B) were PCR-cloned using genomic DNA from T24 cells. Each reporter plasmid (1000 ng) and Renilla luciferase plasmid  were cotransfected into T24 cells (2 × 105). After transfection for 48 h, cell lysates were prepared and subjected to Dual-luciferase Reporter Assay (Promega). Luciferase activities were normalized to Renilla luciferase activities. The BCL6-binding sites on the FOXO3 promoter region were PCR-amplified from total DNA (input) and immunoprecipitated DNA (anti-BCL6) for 30 cycles. Primers spanning -911 to -906 (site 1) and -540 to -535 (site 2) were used. Quantitative chromatin immunoprecipitation (ChIP) assay was performed by using immunoprecipitated DNA and TaqMan quantitative PCR (Supplementary Methods).Two t-test. Differences among ≥3 groups were analyzed be one-way analysis of variance, followed by Scheffe multiple comparison test. A P < 0.05 is considered to be statistical significance.All calculations were performed using SPSS 14.0 software . The survival difference of the two clusters was calculated by log-rank analysis and plotted using the Kaplan-Meier method for survivals. The correlation, comparison between various clinicopathological factors and BCL6, pFOXO3(S253) and pFOXO3(S294) expression levels were assessed by the Chi-square test. The endpoint analyzed was disease-specific and/or metastasis-free survival. For all analyses, two-sided tests of significance were used. Normal distributed data were expressed as means ± SEM. Differences between two groups were analyzed by the Student's BCL6, mapped to 3q27, was exclusively identified in muscle-invasive UBUCs  and BCL6-amplified compared to low pT (Ta/T1) (P < 0.0001) and non-amplified cases (P < 0.001)  while 2q, 4q, 5q, 6q, 9p, 9q, 11p, 11q, 13p 17p and 18q were regularly deleted (red bars) in muscle-invasive UBUC patients , increased cell migration by wound-healing assay  including CDKN1A, CDKN1B, CDKN1C and CDKN2D mRNA levels in J82, T24, BFTC905 and/or BFTC909 cells  that involved in the FOXO signaling pathway were upregulated in BCL6-knockdown BFTC905 cells. Quantitative RT-PCR further confirmed that BCL6 negatively regulated FOXO3 mRNA level using different cell lines through BCL6-overexpressed and -knockdown experiments    BCL6 and FOXO3 gene were performed in vitro. The effect of FOXO3(TM) gene overexpression would demonstrate whether AKT1 played a role in BCL6-induced cell proliferation. Overexpression of the FOXO3 or FOXO3(TM) gene in T24 cells suppressed cell proliferation  and BCL6 genes restored FOXO3- and FOXO3(TM)-suppressed cell proliferation  in cytostasis, upregulation of CDKN1A, CDKN1B protein and their corresponding mRNA levels were similar to those of the FOXO3 gene in BCL6-overexpessed T24 cells (P > 0.05), suggesting that both total FOXO3 and AKT1-insensitive FOXO3 play equally critical role in BCL6-suppressed cytostasis.Profiling of mRNA with the BeadChip identified 16 transcripts including s Figure A. The co) Figure B, highly) Figure . To evalFOXO3 gene  and shFOXO3#2 (P < 0.001) increased cell proliferation compared to the control group (shLacz). Double knockdown of the BCL6 and FOXO3 genes demonstrated that shFOXO3#1  and shFOXO3#2  restored shBCL6#1- and shBCL6#2-suppressed cell proliferation  compared to shBCL6#1 or shBCL6#2 group. Therefore, BCL6 negatively regulated FOXO3, CDKN1A and CDKN1B at the mRNA and protein levels, leading to cell proliferation in UBUC-derived cells.Knockdown of the e Figure G showed n Figure H, downren Figure I and then Figure J levels FOXO3/Foxo3 gene   Figure L. ChIP an Figure M. AccordBCL6 overexpression in T24 cells downregulated FOXO3, however, upregulated pAKT1(S473), pMAPK1/3(T202/Y204), pFOXO3(S253) and pFOXO3(S294) protein levels. Knockdown of the BCL6 gene in BFTC905 cells upregulated pMAPK1/3(T202/Y204), FOXO3 and pFOXO3(S294) while downregulated pan-AKT, pAKT1(S473) and pFOXO3(S253) protein levels  signaling pathways at post-translational levels  pFOXO3S23 and pFOs Figure . Treatme pFOXO3S23 and pFO pFOXO3S23 and pFO pFOXO3S23 and pFO pFOXO3S23 and pFO pFOXO3S23 and pFO pFOXO3S23 and pFO pFOXO3S23 and pFO pFOXO3S23 and pFO pFOXO3S23 and pFO pFOXO3S23 and pFO pFOXO3S23 and pFO pFOXO3S23 and pFO pFOXO3S23 and pFO pFOXO3S23 and pFO pFOXO3S23 and pFOP < 0.01) and pFOXO3(294) (P < 0.01) protein levels conferred to poor disease-specific survival  and pFOXO3(S294) protein levels correlated with high BCL6 protein level  deposited in the TCGA database  assays on UBUC specimens to search for candidate oncogenes pertinent to advanced UBUC. Chromosome 3q was found to be one of the most significantly gained region in UBUC patients with adverse outcomes. Across chromosome 3q, amplification of 3q27.3 was notably relevant to the muscle-invasive UBUCs. In this region, we focused on FOXO3 mRNA and its protein levels. The direct indications are that (1) BCL6 is a direct transrespressor of the FOXO3 gene; (2) exogenous BCL6 expression downregulated FOXO3 mRNA and its corresponding protein levels while enhanced cell proliferation; (3) knockdown of the BCL6 gene upregulated FOXO3 mRNA and its corresponding protein levels whereas inhibited cell proliferation; (4) simultaneously exogenous BCL6 and FOXO3 expression showed similar cell proliferation rates to the control. Among 4 mammalian FOXO members , FOXO3 has a predominant role in controlling cancer development in vitro and in vivoATR serine/threonine kinase (ATR), checkpoint kinases (CHEKs), TP53, CDKN1A, CDKN2A, CDKN2B, PTEN and others  were much lower than those containing only Site 2 (-560 to +100 bp) with or without overexpression of the BCL6 gene, probably due to more transcriptional repressors-binding sites, including BCL6, locate in this region (-1100 to -560 bp), responded to endogenous repressor and BCL6 protein. However, promoter activities were significantly downregulated after BCL6 overexpression in both constructs, indicating BCL6-responsive elements indeed exist in both regions.We found that BCL6-suppressed cytostasis in UBUC is mediated by downregulation of BCL6 in the breakpoint cluster region (BCR)-Abelson proto-oncogene (ABL+) cell line, BV173 BCL6 in chronic myelogenous leukemia FOXO3 transcription and translation, suggesting that BCL6 and FOXO3 might form a negative feedback loop through AKT in the maintenance of cellular homeostasis through regulation of the cell cycle progression. In vivo, BCL6-knockdown J82 cells in a mouse xenograft model supports its oncogenic roles. In vitro, BCL6 negatively regulated the mRNA levels of FOXO3, a series of CKIs, their corresponding protein levels and correlated to cell proliferation. Downregulation of CKIs by BCL6 protein is frequently reported in lymphocytes, normal germinal center B and diffuse large B cell lymphomas-derived cells zinc finger and BTB domain containing 17 (ZBTB17) to suppress the transcription of the CDKN1A gene and cell cycle progression in germinal center B cells TP53 and next inactivated CDKN1A transcription and translation, since CDKN1A is a transcriptional target of TP53 Conversely, FOXO3 transactivates BCL6 gene inhibited malignant phenotype and enhanced sensitivity to temozolomide through inhibition of the AKT pathway BCL6-overexpressed, transfection of pFOXO3(TM)-HaloTag in stable BCL6-knockdown cells, constitutively active AKT1 expression in BCL6-knockdown UBUC-derived cells in conjunction with cell proliferation and immunblot assays to provide solid evidence that BCL6 promotes cell proliferation may also through modulations of the EGFR-MAP2K1/2-FOXO3 and/or PI3K-AKT-FOXO3 signaling pathways at the post-translational level in two cell lines with genetic heterogeneity.We additionally uncovered that BCL6-suppressed FOXO3 activity at the post-translational level was mediated by activation of MAPK and/or PI3K-AKT signaling pathways in distinct UBUC-derived cell lines. Indeed, the precise regulation of FOXO3 transactivation of target genes is accomplished through post-translational modifications (PTMs) and specific protein-protein interactions. Various classes of PTMs including phosphorylation operate crucial parts in directing its subcellular localization and transcription. Quite a few kinases have been reported to phosphorylate FOXO3 at specific residues and cause FOXO3 inhibition/degradation in cytoplasm or transactivation/activation in nucleus  in cytostasis, upregulation of CDKN1A, CDKN1B protein and their corresponding mRNA levels were similar to those of the FOXO3 gene in BCL6-overexpessed T24 cells (P > 0.05), indicating the FOXO3 transcription might plays predominant roles compared to PTMs in UBUC-derived cells.Importantly, immunohistochemistry assay in UBUC patients reconfirmed high correlation between tumor stage and pFOXO3(S294) (MAP2K1/2 substrate) as well as tumor stage and pFOXO3(S253) (AKT1 substrate) protein level. Either pFOXO3(S294) or pFOXO3(S253) predicts inferior outcome in UBUC patients. Thus, in addition to BCL6 transrepresses BCL6 gene amplification in substantial muscle-invasive UBUCs. Amplifications of the BCL6 gene were similarly found in follicular lymphoma BCL6 locus is a profound cause of BCL6 transcription and translation in muscle-invasive UBUC patients.Although not widely surveyed, increased BCL6 protein levels were observed in solid tumors, implying that the tumorigenicity of BCL6 is not restricted to lymphomas. Prominently, we found that BCL6 gene was found in ~50% examined cases and is a crucial cause for overexpression of BCL6 mRNA and protein. Xenograft model suggested that BCL6 induces tumor growth and suppresses apoptosis. In vitro data signified that BCL6 negatively regulated FOXO3 at transcription, translation and post-translational levels through direct binding to FOXO3 promoter, PI3K-AKT and/or EGFR-MAP2K1/2 signaling pathway(s). Clinical data further demonstrated high correlations between BCL6 and phosphorylated/inactive FOXO3. Clinical, in vivo and in vitro evidences support that BCL6 is an oncogene in UBUCs.Altogether, we identified that high BCL6, pFOXO3(S253) and pFOXO3(S294) protein levels are poor prognostic factors for disease-specific and metastasis-free survivals in UBUC patients. Amplicification of the


The capacity of the honey bee to produce three phenotypically distinct organisms  from one genotype represents one of the most remarkable examples of developmental plasticity in any phylum. The queen–worker morphological and reproductive divide is environmentally controlled during post-embryonic development by differential feeding. Previous studies implicated metabolic flux acting via epigenetic regulation, in particular DNA methylation and microRNAs, in establishing distinct patterns of gene expression underlying caste-specific developmental trajectories. We produce the first genome-wide maps of chromatin structure in the honey bee at a key larval stage in which developmental canalization into queen or worker is virtually irreversible. We find extensive genome-wide differences in H3K4me3, H3K27ac, and H3K36me3, many of which correlate with caste-specific transcription. Furthermore, we identify H3K27ac as a key chromatin modification, with caste-specific regions of intronic H3K27ac directing the worker caste. These regions may harbor the first examples of caste-specific enhancer elements in the honey bee. Our results demonstrate a key role for chromatin modifications in the establishment and maintenance of caste-specific transcriptional programs in the honey bee. We show that at 96 h of larval growth, the queen-specific chromatin pattern is already established, whereas the worker determination is not, thus providing experimental support for the perceived timing of this critical point in developmental heterochrony in two types of honey bee females. In a broader context, our study provides novel data on environmentally regulated organismal plasticity and the molecular foundation of the evolutionary origins of eusociality. Eusociality is an intriguing evolutionary invention found in many species of Hymenoptera , termites, and even some mammals . In trueApis mellifera) play a crucial role in most ecosystems and strongly influence ecological relationships, for example, by helping to maintain genetic variation in flowering plants. Furthermore, in farmed areas, the honey bee is used extensively for the commercial pollination of a variety of cultivated crops. Honey bees live in complex societies comprising tens of thousands of individuals, in which there is a division of labor that can be separated into two broad categories. The first is a reproductive distinction; each colony contains two diploid female castes comprising a single queen who is specialized for reproduction and thousands of sterile female worker bees -10-hydroxy-2-decenoic acid (10-HDA), which comprises up to 5% of royal jelly. 10-HDA has been shown to be a histone deacetylase inhibitor and can reactivate the expression of epigenetically silenced genes in mammalian cells . Replicates show a very strong and significant correlation across all histone PTMs and castes  . We find enrichment of H3K4me3 and H3K27ac and depletion of H3K36me3 around the transcriptional start sites (TSS) of genes in both 96hW and 96hQ castes , similar to what was previously reported in other organisms including vertebrates, invertebrates, and plants (n = 4 per caste) by RNA-seq. In both 96hW and 96hQ castes, genes marked uniquely by H3K4me3 or H3K36me3 show a significant  increase in expression compared to background, whereas genes marked uniquely by H3K27ac do not show any significant association with expression . Thus, in both worker and queen honey bee castes; H3K4me3, H3K36me3, and H3K27ac when occurring in combination, associate with actively transcribed regions and therefore have the potential to regulate caste-specific gene expression.We previously identified more than 20 different histone PTM states in queen and worker honey bee castes using mass spectrometry . For H3K27ac, we identify 2027 unique genomic regions that are significantly more enriched in 96hW and 489 in 96hQ . Finally, for H3K36me3 we identify 1196 unique regions that are significantly more enriched in 96hW and 3285 in 96hQ . Gene Ontology (GO) analysis of those genes that show any significant caste-specific chromatin marks reveal a distinct developmental separation of castes at 96 h. In 96hQ, overrepresented GO terms significantly associate with physio-metabolic functions and processes, such as the structural constituents of ribosome (GO:0003735) and biological processes, including cellular amide metabolic processes (GO:0043603), cytoplasmic translation (GO:0002181), and peptide metabolic processes (GO:0006518), suggesting that at 96 h, the queen developmental trajectory is established . In contrast, 96hW GO terms associate with development and transcriptional programming, including the molecular functions of transcription factor activity and binding (GO:0003700) along with biological processes of anatomical structure morphogenesis (GO:0009653), system development (GO:0048731), and developmental processes (GO:0032502). Importantly, this suggests that relative to the 96hQ, the worker caste developmental trajectory is not yet established at 96 h . Because caste-specific DNA methylation patterns were previously reported  and then analyzed the enrichment of these DMPs with each of our differential histone PTMs. We find only a strong enrichment in H3K36me3 . However, this is driven by an overlap in genome distribution rather than a strong functional association . This suggests that DNA methylation and histone PTMs provide different signals in the process of caste determination.Having established that within each caste, H3K4me3, H3K27ac, and H3K36me3 are significantly enriched at transcribed regions, we next wanted to determine whether these three histone PTMs show caste-specific distributions. To investigate this, we called differences between 96hW and 96hQ castes for H3K4me3, H3K27ac, and H3K36me3 A. For H3reported , we askeSupplemental Fig. S6). We identify a total of 1060 significant differences  in transcript levels between 96hW and 96hQ . To confirm that we were robustly measuring differences between castes, we compared these differentially expressed transcripts to previously published RNA-seq data taken from whole 96hW and 96hQ larvae  between the transcriptional differences detected in our experiment and that of ρ = 0.28; P-value = 2.7 × 10−17) and H3K36me3  show significant correlation with transcriptional differences, suggesting that caste-specific H3K4me3 and H3K36me3 patterns associate with caste-specific transcriptional profiles . To investigate the function of genes that show consistent caste-specific changes in both gene expression and histone PTM enrichment, we performed Gene Ontology analysis. We observe that these genes reveal a distinct developmental separation of castes at 96 h. In 96hQ, we again find significant GO terms for biological processes and functions that associate with physio-metabolic processes such as translation (GO:0006412 and GO:0002181) and peptide biosynthetic processes (GO:0043043) . In contrast, GO terms for system development (GO:0048731), nervous system development (GO:0007399), generation of neurons (GO:0048699), and neuron differentiation (GO:0030182) are enriched in 96hW . In agreement with our earlier analyses, this again suggests that at 96 h and relative to worker, queen development is set, whereas the worker-specific developmental program (which will determine a distinct phenotype) is yet to be established. Representative examples of two genes that show some of the most significant caste-specific changes in both gene expression and histone PTM enrichment are shown in PYK; LOC552007) is shown as an example of a physio-metabolic 96hQ-specific gene, where H3K27ac and H3K4me3 enrichment differences associate with the TSS and H3K36me3 over the gene body . Similarly, the 96hW-specific gene Ten-eleven translocation , also shows TSS and gene body differences in H3K4me3, H3K27ac, and H3K36me3. However, TET also has significant 96hW-specific differences in H3K27ac in the intronic region between two alternative TSSs . Taken together, these data indicate that aged-matched worker and queen honey bee castes exhibit distinct patterns of histone PTMs. Furthermore, patterns of H3K4me3 and H3K36me3 correlate with caste-specific gene expression and reveal that at this crucial time point and relative to the queen, the worker-specific developmental pathway is not yet established.We next wanted to determine whether the observed caste-specific differences in histone PTM enrichment correlated with any differential gene expression between castes. Unsupervised multidimensional scaling analysis of our RNA-seq data reveal a strong separation of the two castes , whereas those genes significantly more expressed in 96hQ are actually depleted for 96hQ intronic H3K27ac  . It is also likely that H3K27ac acts at distal regulatory elements; therefore, we determined the average expression of genes at various distances from H3K27ac enriched regions, revealing a specific peak of gene expression at a distance of 130–140 kbp . In order to try and gain a better understanding of the functional significance of caste-specific intronic H3K27ac, we performed motif enrichment analysis on these regions using CentriMo from the MEME suite software package  and Mothers against dpp , which accounts for 70% of all 96hW-specific regions of intronic H3K27ac , Hairy , and Mothers against dpp   C. Howeve package . Using t H3K27ac D. In con × 10−4) D. Analys × 10−4) E. Conver × 10−4) E. Given We used ChIP-seq to provide the first description of genome-wide caste-specific chromatin patterns in the honey bee and, furthermore, the first between Hymenoptera castes that show a reproductive division of labor. Combined with RNA-seq analysis and at a crucial developmental stage when developmental trajectory has been shown to be irreversible , we idenWe hypothesized that for the honey bee genome to specify two different female castes, different chromatin patterns and transcriptional programs have to be established during development. Previous work mainly focused on the role of caste-specific DNA methylation patterns in adult honey bees , in whiccis-regulatory roles. Enhancers are cis-acting elements that are frequently found in noncoding regions of genomes and are characterized by nucleosome-free regions enriched in transcription factor binding sites   and Mad  motifs accounted for 70% of all worker-specific intronic H3K27ac. Trl/GAGA factor is a multifunctional transcriptional regulator and the gene is differentially DNA methylated in a larvae . Trl/GAGr (NURF)  before ar (NURF) . NURF car (NURF) . In our r (NURF) . Mad/SMAr (NURF) , colocalr (NURF) , and affr (NURF) . In our r (NURF) , suggestA. mellifera it is also a differentially DNA methylated gene  show the most significant enrichment in queen and accounted for 66% of 96hQ-specific H3K27ac regions. brk and Hairy were demonstrated to bind enhancers and mediate transcriptional repression . Althougted gene . CtBP isted gene , althougted gene . Signifit stages . Previout stages , therefoCamponotus floridanus, where CBP-catalyzed H3K27ac was shown to be essential in establishing different worker castes and behaviors . After removal from the incubator, frames were closely examined and an acetate sheet was laid over the patches of newly hatched larvae. The position of the sheet was marked on the edges of the frame to ensure accurate replacement at the time of collection. The locations of individual newly hatched larvae were marked on the sheet, and the frames were immediately returned to their original hives. The exact time was recorded. After 96 h, the frames were transferred back to a warm room and the larvae were collected with blunt nose soft forceps using the acetate sheet positioned over the frame according to the previously marked reference points. To obtain queen larvae of known age, standard queen raising techniques were used . Double g/10 min). All buffers from this step onward contained cOmplete protease inhibitor cocktail (Roche) and 2.5 mM sodium butyrate. Pellets were suspended in 5 mL lysis buffer 1  and incubated for 30 min on a rotator mixer at 4°C. Lysates were then centrifuged (1200g/10 min), and pellets were suspended in 5 mL lysis buffer 2  and incubated for another 30 min on a rotator mixer at 4°C. Lysates were centrifuged (1200g/10 min), and pellets were suspended in 900 µL sonication buffer . Chromatin was sonicated using a Bioruptor . Lysates were cleared by centrifugation , and sonication was checked by agarose gel electrophoresis.Chromatin was extracted from the larval heads  containing brain, optic and retinular ganglia, neurosecretory cells, glands , suboesophageal ganglion, a small number of fat bodies, the maxillae, labium and mandibles, segmented imaginal antennae developing in hypodermal pockets, the openings of silk glands ducts at the tip of the labium-hypopharynx, trachea, and cuticle. The rest of the larval body is predominantly occupied by a large digestive system filled with processed food and bacteria (larvae do not defecate), a tracheal network and reproductive parts that at this stage of development are already large in queens and rudimentary in workers. Chromatin was cross-linked in fixation buffer  for 15 min. Reactions were quenched by washing twice in stop solution . Afterward, fixed larval heads were washed four times with wash buffer 1 , followed by four washes with wash buffer 2 . Larval heads were homogenized and centrifuged (12003) for 40 min at 65°C in a ThermoMixer. Fifty micrograms of RNase A was added, and samples were incubated for 15 min at 37°C. NaCl was then added to a final concentration of 500 mM. Afterward, the samples were incubated overnight with 40 µg of proteinase K at 65°C in a ThermoMixer. DNA was purified with GeneJET PCR Purification columns (Thermo Fisher Scientific). NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) was used to make sequencing libraries from 0.5 to 1 ng of DNA following the manufacturer's instructions. Seventy-five base-pair SE next-generation DNA sequencing was carried out by the Barts and The London Genome Centre at Queen Mary University of London on the Illumina NextSeq 500 platform.Chromatin was concentrated using a Millipore Amicon concentrator (3 kDa cutoff). Fifty microliters chromatin aliquots was diluted 10-fold with ChIP dilution buffer . Antibodies  were added according to the manufacturers’ instructions, and samples were incubated overnight on a rotator mixer at 4°C. Thirty microliters of magnetic protein A Dynabeads (Invitrogen) was added to each reaction, and samples were incubated for 4 h on a rotator mixer at 4°C. Beads were washed twice with 500 µL wash buffer A  and once with 500 µL wash buffer B  and wash buffer C . After these washes, DNA was eluted from the beads by incubation in 200 µL elution buffer . mRNA was isolated with poly(A) mRNA Magnetic Isolation Module (NEB) from 1 µg total RNA. RNA-seq libraries were constructed using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB) following the manufacturer's instructions. One hundred base-pair PE next-generation sequencing was carried out by the Barts and The London Genome Centre at Queen Mary University of London on the Illumina NextSeq 500 platform.Supplemental Table S7. Reads were counted into windows of width 100 bp with a spacing of 50 bp (each window therefore overlaps two other windows) using csaw (Ci) for each sample, i, was then normalized to the background counts (Cb) using the following equation:Ni is the total number of reads sequenced for each sample, i, and Nb is the total number of reads sequenced in the appropriate input sample. To determine differential windows, we computed moderated t-statistics using empirical Bayes moderation of the standard errors with the limma R package (The genome assembly Amel_4.5 (GCF_000002195.4) was downloaded from the NCBI and indexed using Bowtie 2 (v2.2.8) . ChIP-seing csaw . Duplica package .https://metazoa.ensembl.org/index.html) in FASTA format using genome version GCA_000002195.1. These two FASTA files were concatenated and supercontigs were removed using linux command grep with the following string: “supercontig|$genome_version:[^1-9XMY]”. Kallisto . GO analysis was performed using the R package, topGO (http://bioconductor.org/packages/release/bioc/html/topGO.html).Transcription factor motif analysis was performed using CentriMo , which ihttps://www.ncbi.nlm.nih.gov/geo/) under accession number GSE110642.ChIP-seq and RNA-seq data from this study have been submitted to the NCBI Gene Expression Omnibus (GEO;