Abstract Background Patients with lung adenocarcinoma (LUAD) have a low response rate to immune checkpoint blockade. It is highly important to explore the tumor immune escape mechanism of LUAD patients and expand the population of patients who may benefit from immunotherapy. Methods Based on 954 bulk RNA-seq data of LUAD patients and 15 single-cell RNA-seq data, the relationships between tumor immune dysfunction and exclusion (TIDE) scores and survival prognosis in each patient were calculated and evaluated, and the immune escape mechanism affecting the independent prognosis of LUAD patients was identified. Functional enrichment analysis explored the antitumour immune response and biological behavior of tumor cells among different LUAD groups. Single-cell annotation and pseudotemporal analysis were used to explore the target molecules and immune escape mechanisms of LUAD. Results Myeloid-derived suppressor cells (MDSCs) and IRF8 were identified as risk and protective factors for the independent prognosis of LUAD patients, respectively. In the tumor microenvironment of patients with high infiltration of MDSCs, the antitumor immune response is significantly suppressed, while tumor cell division, proliferation, and distant metastasis are significantly enhanced. Single-cell RNA-seq analysis revealed that IRF8 is an important regulator of MDSC differentiation in LUAD myeloid cells. In addition, IRF8 may regulate the differentiation of MDSCs through the IL6-JAK-STAT3 signalling pathway. Conclusions IRF8 deficiency impairs the normal development of LUAD myeloid cells and induces their differentiation into MDSCs, thereby accelerating the immune escape of LUAD cells. IRF8-targeted activation to inhibit the formation of MDSCs may be a new target for immunotherapy in LUAD.
ABSTRACT Cardiac fibrosis is a recognized cause of morbidity and mortality, yet effective pharmacological therapy that directly targets the fibrotic process remains lacking. Here we surveyed a group of methyltransferases known as protein arginine methyltransferases (PRMT) and demonstrated that PRMT1, which is the most highly expressed PRMT in the heart, was upregulated in activated cardiac fibroblasts, or myofibroblasts, in failing hearts. Deleting Prmt1 specifically in myofibroblasts or treating systemically with the PRMT1 inhibitor MS023 blocked myofibroblast formation, leading to a significant reduction in cardiac fibrosis and improvement in cardiac function in both acute and chronic heart injury models that manifest pervasive cardiac fibrosis. PRMT1 promoted the transition of cardiac fibroblasts to myofibroblasts by regulating transcription and epigenetic status. Additionally, PRMT1 methylated a key nucleolar protein fibrillarin 1 (FBL) and regulated nucleoli morphology and function during fibroblast fate transition. We further demonstrated a previously unrecognized requirement for FBL in myofibroblasts formation, by regulating myofibroblast gene induction and contractile force generation.
<div>Abstract<p>Disruption of epigenetic regulation is a hallmark of acute myeloid leukemia (AML), but epigenetic therapy is complicated by the complexity of the epigenome. Herein, we developed a long-term primary AML <i>ex vivo</i> platform to determine whether targeting different epigenetic layers with 5-azacytidine and LSD1 inhibitors would yield improved efficacy. This combination was most effective in <i>TET2</i><sup>mut</sup> AML, where it extinguished leukemia stem cells and particularly induced genes with both LSD1-bound enhancers and cytosine-methylated promoters. Functional studies indicated that derepression of genes such as <i>GATA2</i> contributes to drug efficacy. Mechanistically, combination therapy increased enhancer–promoter looping and chromatin-activating marks at the <i>GATA2</i> locus. CRISPRi of the LSD1-bound enhancer in patient-derived <i>TET2</i><sup>mut</sup> AML was associated with dampening of therapeutic <i>GATA2</i> induction. <i>TET2</i> knockdown in human hematopoietic stem/progenitor cells induced loss of enhancer 5-hydroxymethylation and facilitated LSD1-mediated enhancer inactivation. Our data provide a basis for rational targeting of cooperating aberrant promoter and enhancer epigenetic marks driven by mutant epigenetic modifiers.</p>Significance:<p>Somatic mutations of genes encoding epigenetic modifiers are a hallmark of AML and potentially disrupt many components of the epigenome. Our study targets two different epigenetic layers at promoters and enhancers that cooperate to aberrant gene silencing, downstream of the actions of a mutant epigenetic regulator.</p><p><i>This article is highlighted in the In This Issue feature, p. 813</i></p></div>
BackgroundCervical cancer is one of the most common malignancies in women worldwide. As a RING type ubiquitin ligase, SIAH2 has been reported to promote the progression of a variety of tumors by interacting with and targeting multiple chaperones and substrates. The aim of this study was to further identify the role and the related molecular mechanisms involved of SIAH2 in cervical carcinogenesis.Methods and ResultsCellular assays in vitro showed that knockdown of SIAH2 inhibited the proliferation, migration and invasion of human cervical cancer cells C33A and SiHa, induced apoptosis, and increased the sensitivity to cisplatin treatment. Knockdown of SIAH2 also inhibited the epithelial-mesenchymal transition and activation of the Akt/mTOR signaling pathway in cervical cancer cells, which were detected by western blot. Mechanistically, SIAH2, as a ubiquitin ligase, induced the ubiquitination degradation of GSK3β degradation by using coIP. The results of complementation experiments further demonstrated that GSK3β overexpression rescued the increase of cell proliferation and invasion caused by SIAH2 overexpression. Specific expression of SIAH2 appeared in precancerous and cervical cancer tissues compared to inflammatory cervical lesions tissues using immunohistochemical staining. The more SIAH2 was expressed as the degree of cancer progressed. SIAH2 was significantly highly expressed in cervical cancer tissues (44/55, 80%) compared with precancerous tissues (18/69, 26.1%). Moreover, the expression level of SIAH2 in cervical cancer tissues was significantly correlated with the degree of cancer differentiation, and cervical cancer tissues with higher SIAH2 expression levels were less differentiated.ConclusionTargeting SIAH2 may be beneficial to the treatment of cervical cancer.
Article16 October 2017Open Access Transparent process RNA polymerase II primes Polycomb-repressed developmental genes throughout terminal neuronal differentiation Carmelo Ferrai Corresponding Author Carmelo Ferrai [email protected] orcid.org/0000-0002-8088-2757 Epigenetic Regulation and Chromatin Architecture, Max Delbrück Center for Molecular Medicine, Berlin, Germany Genome Function, MRC London Institute of Medical Sciences (previously MRC Clinical Sciences Centre), London, UK Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London, UK Search for more papers by this author Elena Torlai Triglia Elena Torlai Triglia orcid.org/0000-0002-6059-0116 Epigenetic Regulation and Chromatin Architecture, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Jessica R Risner-Janiczek Jessica R Risner-Janiczek Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London, UK Stem Cell Neurogenesis, MRC London Institute of Medical Sciences (previously MRC Clinical Sciences Centre), London, UK Neurophysiology Group, MRC London Institute of Medical Sciences (previously MRC Clinical Sciences Centre), London, UK Search for more papers by this author Tiago Rito Tiago Rito Epigenetic Regulation and Chromatin Architecture, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Owen JL Rackham Owen JL Rackham Duke-NUS Medical School, Singapore, Singapore Search for more papers by this author Inês de Santiago Inês de Santiago Genome Function, MRC London Institute of Medical Sciences (previously MRC Clinical Sciences Centre), London, UK Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London, UK Search for more papers by this author Alexander Kukalev Alexander Kukalev Epigenetic Regulation and Chromatin Architecture, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Mario Nicodemi Mario Nicodemi Dipartimento di Fisica, Università di Napoli Federico II and INFN Napoli, Complesso Universitario di Monte Sant'Angelo, Naples, Italy Search for more papers by this author Altuna Akalin Altuna Akalin Scientific Bioinformatics Platform, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Meng Li Meng Li Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London, UK Stem Cell Neurogenesis, MRC London Institute of Medical Sciences (previously MRC Clinical Sciences Centre), London, UK Search for more papers by this author Mark A Ungless Corresponding Author Mark A Ungless [email protected] orcid.org/0000-0002-1730-3353 Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London, UK Neurophysiology Group, MRC London Institute of Medical Sciences (previously MRC Clinical Sciences Centre), London, UK Search for more papers by this author Ana Pombo Corresponding Author Ana Pombo [email protected] orcid.org/0000-0002-7493-6288 Epigenetic Regulation and Chromatin Architecture, Max Delbrück Center for Molecular Medicine, Berlin, Germany Genome Function, MRC London Institute of Medical Sciences (previously MRC Clinical Sciences Centre), London, UK Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London, UK Institute for Biology, Humboldt-Universität zu Berlin, Berlin, Germany Search for more papers by this author Carmelo Ferrai Corresponding Author Carmelo Ferrai [email protected] orcid.org/0000-0002-8088-2757 Epigenetic Regulation and Chromatin Architecture, Max Delbrück Center for Molecular Medicine, Berlin, Germany Genome Function, MRC London Institute of Medical Sciences (previously MRC Clinical Sciences Centre), London, UK Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London, UK Search for more papers by this author Elena Torlai Triglia Elena Torlai Triglia orcid.org/0000-0002-6059-0116 Epigenetic Regulation and Chromatin Architecture, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Jessica R Risner-Janiczek Jessica R Risner-Janiczek Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London, UK Stem Cell Neurogenesis, MRC London Institute of Medical Sciences (previously MRC Clinical Sciences Centre), London, UK Neurophysiology Group, MRC London Institute of Medical Sciences (previously MRC Clinical Sciences Centre), London, UK Search for more papers by this author Tiago Rito Tiago Rito Epigenetic Regulation and Chromatin Architecture, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Owen JL Rackham Owen JL Rackham Duke-NUS Medical School, Singapore, Singapore Search for more papers by this author Inês de Santiago Inês de Santiago Genome Function, MRC London Institute of Medical Sciences (previously MRC Clinical Sciences Centre), London, UK Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London, UK Search for more papers by this author Alexander Kukalev Alexander Kukalev Epigenetic Regulation and Chromatin Architecture, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Mario Nicodemi Mario Nicodemi Dipartimento di Fisica, Università di Napoli Federico II and INFN Napoli, Complesso Universitario di Monte Sant'Angelo, Naples, Italy Search for more papers by this author Altuna Akalin Altuna Akalin Scientific Bioinformatics Platform, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Meng Li Meng Li Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London, UK Stem Cell Neurogenesis, MRC London Institute of Medical Sciences (previously MRC Clinical Sciences Centre), London, UK Search for more papers by this author Mark A Ungless Corresponding Author Mark A Ungless [email protected] orcid.org/0000-0002-1730-3353 Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London, UK Neurophysiology Group, MRC London Institute of Medical Sciences (previously MRC Clinical Sciences Centre), London, UK Search for more papers by this author Ana Pombo Corresponding Author Ana Pombo [email protected] orcid.org/0000-0002-7493-6288 Epigenetic Regulation and Chromatin Architecture, Max Delbrück Center for Molecular Medicine, Berlin, Germany Genome Function, MRC London Institute of Medical Sciences (previously MRC Clinical Sciences Centre), London, UK Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London, UK Institute for Biology, Humboldt-Universität zu Berlin, Berlin, Germany Search for more papers by this author Author Information Carmelo Ferrai *,1,2,3,‡, Elena Torlai Triglia1,‡, Jessica R Risner-Janiczek3,4,5, Tiago Rito1, Owen JL Rackham6, Inês Santiago2,3,10, Alexander Kukalev1, Mario Nicodemi7, Altuna Akalin8, Meng Li3,4,11, Mark A Ungless *,3,5 and Ana Pombo *,1,2,3,9 1Epigenetic Regulation and Chromatin Architecture, Max Delbrück Center for Molecular Medicine, Berlin, Germany 2Genome Function, MRC London Institute of Medical Sciences (previously MRC Clinical Sciences Centre), London, UK 3Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London, UK 4Stem Cell Neurogenesis, MRC London Institute of Medical Sciences (previously MRC Clinical Sciences Centre), London, UK 5Neurophysiology Group, MRC London Institute of Medical Sciences (previously MRC Clinical Sciences Centre), London, UK 6Duke-NUS Medical School, Singapore, Singapore 7Dipartimento di Fisica, Università di Napoli Federico II and INFN Napoli, Complesso Universitario di Monte Sant'Angelo, Naples, Italy 8Scientific Bioinformatics Platform, Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, Berlin, Germany 9Institute for Biology, Humboldt-Universität zu Berlin, Berlin, Germany 10Present address: Seven Bridges Genomics UK Ltd, London, UK 11Present address: Neuroscience and Mental Health Research Institute, School of Medicine and School of Biosciences, Cardiff, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +49 3094061755; E-mail: [email protected] *Corresponding author. Tel: +44 2083838299; E-mail: [email protected] *Corresponding author (lead contact). Tel: +49 3094061760; E-mail: [email protected] Molecular Systems Biology (2017)13:946https://doi.org/10.15252/msb.20177754 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Polycomb repression in mouse embryonic stem cells (ESCs) is tightly associated with promoter co-occupancy of RNA polymerase II (RNAPII) which is thought to prime genes for activation during early development. However, it is unknown whether RNAPII poising is a general feature of Polycomb repression, or is lost during differentiation. Here, we map the genome-wide occupancy of RNAPII and Polycomb from pluripotent ESCs to non-dividing functional dopaminergic neurons. We find that poised RNAPII complexes are ubiquitously present at Polycomb-repressed genes at all stages of neuronal differentiation. We observe both loss and acquisition of RNAPII and Polycomb at specific groups of genes reflecting their silencing or activation. Strikingly, RNAPII remains poised at transcription factor genes which are silenced in neurons through Polycomb repression, and have major roles in specifying other, non-neuronal lineages. We conclude that RNAPII poising is intrinsically associated with Polycomb repression throughout differentiation. Our work suggests that the tight interplay between RNAPII poising and Polycomb repression not only instructs promoter state transitions, but also may enable promoter plasticity in differentiated cells. Synopsis Poised RNAPII-S5p is present at Polycomb-repressed genes from embryonic stem cells to terminally differentiated neurons. The tight interplay between RNAPII poising and Polycomb repression enables promoter plasticity in differentiated cells and increased potential for reactivation. Poised RNAPII-S5p primes Polycomb-repressed promoters throughout terminal differentiation to functional dopaminergic neurons. Poised RNAPII-S5p associates with increased potential for reactivation upon loss of Polycomb repression. DNA methylation valleys coincide with broad occupancy of poised RNAPII-S5p and Polycomb repression. Key non-neuronal transcription factor genes that co-associate with Polycomb and RNAPII-S5p in neurons have potential roles in transdifferentiation. Introduction Embryonic differentiation starts from a totipotent cell and culminates with the production of highly specialized cells. In ESCs, many genes important for early development are repressed in a state that is poised for subsequent activation (Azuara et al, 2006; Bernstein et al, 2006; Stock et al, 2007; Brookes et al, 2012). These genes are mostly GC-rich (Deaton & Bird, 2011), and their silencing in pluripotent cells is mediated by Polycomb repressive complexes (PRCs). Genes with more specialized cell type-specific functions are neither active nor Polycomb repressed in ESCs, have AT-rich promoter sequences, and their activation is associated with specific transcription factors (Sandelin et al, 2007; Brookes et al, 2012). Polycomb repressive complex proteins have major roles in modulating gene expression during differentiation and in disease (Prezioso & Orlando, 2011; Richly et al, 2011). They assemble in two major complexes, PRC1 and PRC2, which catalyze H2AK119 monoubiquitination and H3K27 methylation, respectively (Simon & Kingston, 2013). Both PRC-mediated histone marks are important for chromatin repression, and synergize in a tight feedback loop to recruit each other's modifying enzymes (Blackledge et al, 2015). Although imaging studies suggest that PRC-repressed chromatin has a compact conformation (Francis et al, 2004; Eskeland et al, 2010; Boettiger et al, 2016), molecular and cell biology approaches show that PRC repression in ESCs coincides with the occupancy of poised RNAPII complexes and active histone marks in the vast majority of PRC-repressed promoters (Azuara et al, 2006; Bernstein et al, 2006; Stock et al, 2007; Brookes et al, 2012; Tee et al, 2014; Kinkley et al, 2016; Weiner et al, 2016). The co-occurrence of RNAPII and PRC enzymatic activities, RING1B and EZH2, on chromatin has been confirmed in ESCs by sequential ChIP (Brookes et al, 2012) and is mirrored by the simultaneous presence of H3K4me3 and H3K27me3 marks, called chromatin bivalency (Azuara et al, 2006; Bernstein et al, 2006; Voigt et al, 2012; Kinkley et al, 2016; Sen et al, 2016; Weiner et al, 2016). RNAPII function is regulated through complex post-translational modifications at the C-terminal domain (CTD) of its largest subunit, RPB1, which coordinate the co-transcriptional recruitment of chromatin modifiers and RNA processing machinery to chromatin, leading to productive transcription events and mRNA expression (Brookes & Pombo, 2009; Zaborowska et al, 2016). In mammals, the CTD comprises 52 repeats of the heptapeptide sequence N-Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7-C. At active genes, RNAPII is phosphorylated on Ser5 (S5p) to mark transcription initiation, on Ser7 (S7p) during the transition to productive transcription, and on Ser2 (S2p) during elongation. S5p and S7p are mediated by CDK7, while S2p is mediated by CDK9. RNAPII also exists in a paused state of activation characterized by short transcription events at promoter regions, followed by promoter-proximal termination and re-initiation events (Adelman & Lis, 2012). RNAPII pausing is identified at genes that produce mRNA at lower levels and is often measured as the amount of RNAPII at gene promoters relative to its occupancy throughout coding regions. Paused states of RNAPII are therefore a feature of genes that are active to a lower extent, are characterized by the presence of S5p and S7p at gene promoters, low abundance of S2p throughout the coding regions, and they are recognized by 8WG16, an antibody which has a preference for unphosphorylated Ser2 residues. The paused RNAPII complex is also characterized by methylation and acetylation of the non-canonical Lys7 residues at the CTD (Schröder et al, 2013; Dias et al, 2015; Voss et al, 2015). The RNAPII complex that primes PRC-repressed genes in mouse ESCs has a unique configuration of post-translational modifications of the CTD, which is different from the paused RNAPII, and was originally referred to as poised RNAPII (Stock et al, 2007; Brookes & Pombo, 2009). The poised RNAPII is characterized by exclusive phosphorylation of S5p in the absence of S7p, S2p, K7me1/2, K7ac, or recognition by 8WG16 (Brookes et al, 2012; Dias et al, 2015). Poised RNAPII-S5p, in the absence of 8WG16, has not been described in Drosophila (Gaertner et al, 2012), consistent with lack of chromatin bivalency (Vastenhouw & Schier, 2012; Voigt et al, 2013). Importantly, Ser5 phosphorylation of poised RNAPII complexes at Polycomb-repressed genes in ESCs is mediated by different kinases, ERK1/2 (Tee et al, 2014; Ma et al, 2016), instead of CDK7 which phosphorylates both S5p and S7p at active genes, irrespectively of pausing ratio (Akhtar et al, 2009; Glover-Cutter et al, 2009). Loss of ERK1/2 activity in ESCs results in the loss of poised RNAPII-S5p and decreased occupancy of PRC2 at Polycomb-repressed developmental genes (Tee et al, 2014), suggesting a tight functional link between the presence of poised RNAPII-S5p at Polycomb-target genes and the recruitment of Polycomb. While histone bivalency has been studied to some extent during mammalian cell differentiation and found present at smaller proportion of genes (Mohn et al, 2008; Lien et al, 2011; Wamstad et al, 2012; Xie et al, 2013), it remains unexplored whether the co-occupancy of poised RNAPII-S5p at PRC targets is a property of ESCs or extends beyond pluripotency. The poised RNAPII-S5p state was observed at Polycomb-repressed genes in ESCs grown in the presence of serum and leukemia inhibitor factor (LIF; Stock et al, 2007; Brookes et al, 2012; Tee et al, 2014; Ma et al, 2016). Other studies grow ESCs in 2i conditions to simulate a more naïve pluripotent state, through inhibition of GSK3 and MEK signaling, which in turn inhibits ERK signaling. In these conditions, the occupancy of poised RNAPII complexes is reduced at Polycomb-target genes (Marks et al, 2012; Williams et al, 2015), consistent with the effects of ERK1-2 inhibition (Tee et al, 2014). Interestingly, the decreased occupancy of poised RNAPII-S5p at PRC-repressed genes in 2i conditions is accompanied by reduced occupancy of PRC2 catalytic subunit EZH2 and H3K27me3 modification, suggesting a tight interplay between the presence of poised RNAPII-S5p and Polycomb occupancy at Polycomb-repressed genes in ESCs, which is interfered upon in 2i conditions. Interestingly, prolonged 2i treatment was shown to impair ESC developmental potential and cause widespread loss of DNA methylation (Choi et al, 2017; Yagi et al, 2017), leading to renewed interest in understanding the regulation of developmental genes, and in particular whether poised RNAPII complexes are a more general feature of Polycomb repression mechanisms in the early and late stages of differentiation. Recent studies of DNA methylation in differentiated tissues show that many silent developmental regulator genes remain hypomethylated in wide genomic regions (also called DNA methylation valleys; or DMVs) in differentiated tissues (Xie et al, 2013), raising the question of whether poised RNAPII complexes might prime developmental regulator genes in cell lineages irrespectively of future activation. In this scenario, Polycomb repression may represent the universal mode of repression of this group of CpG-rich genes that recruit poised RNAPII-S5p and which are not targeted by DNA methylation. Silencing of developmental regulator genes through Polycomb repression mechanisms in fully differentiated cells, especially in the presence of poised RNAPII complexes, may nevertheless have roles in the remodeling of cell function, for example in response to specific stimuli such as tissue injury, or in disease such as in cancers associated with Polycomb dysfunction. To investigate RNAPII poising at Polycomb-repressed genes, from pluripotency to terminal differentiation, we mapped H3K27me3 (a marker of Polycomb repression), RNAPII-S5p (present at both active and poised RNAPII complexes), and RNAPII-S7p (a marker of productive gene expression) and produced matched mRNA-seq datasets in ESCs and in four stages of differentiation of functionally mature dopaminergic neurons. We show compelling evidence that the presence of poised RNAPII at H3K27me3-marked chromatin is not a specific feature of ESCs, but a general property common to differentiating and post-mitotic cells. We also observe de novo Polycomb repression during neuronal cell commitment and neuronal maturation that promotes waves of transient downregulation of gene expression. We discover a group of genes that maintain poised RNAPII-S5p and Polycomb silencing throughout neuronal differentiation, and which are developmental transcription factors important for cell specification toward non-neuronal lineages. Although these genes are unlikely to be subsequently reactivated in the neuronal lineage, their silencing in neuronal precursors and mature neurons is sensitive to Polycomb inhibition or knockout. We also show that the presence of poised RNAPII-S5p at specific subsets of Polycomb-repressed genes in terminally differentiated neurons coincides with their wide hypomethylation in mouse brain. Our study reveals the interplay between RNAPII poising and Polycomb repression in the control of regulatory networks and cell plasticity throughout cell differentiation. Results Capturing distinct stages of differentiation from ESCs to dopaminergic neurons To study the dynamic changes in Polycomb and RNAPII occupancy at gene promoters during differentiation, we optimized neuronal differentiation protocols to obtain large quantities of pure cell populations required for mapping chromatin-associated histone marks and RNAPII at five states of neuronal differentiation that leads to the production of functional dopaminergic neurons (ESC, days 1, 3, 16, and 30; Fig 1A). To capture the early exit from pluripotency, we adopted an approach that starts from mouse ESCs grown in serum-free and 2i-free conditions and which within 3 days achieves synchronous exit from pluripotency toward the production of neuronal progenitors (Abranches et al, 2009; Fig EV1A, top row). To obtain terminally differentiated dopaminergic neurons, we used an approach that commits ESCs to a midbrain neuron phenotype (Jaeger et al, 2011; Fig EV1A, bottom row). Figure 1. Model of differentiation from pluripotent stem cells to terminal dopaminergic neurons Schematic representation of the differentiation system used and the temporal expression dynamics of differentiation stage markers. RNA levels of differentiation stage markers were measured by qRT–PCR. Relative levels are normalized to Actb internal control, and values are plotted relative to the highest expressed time point. Mean and standard deviation (SD) are from three biological replicates. Indirect immunofluorescence confirms expression of stage-specific markers at the single cell level. OCT4 is a marker of pluripotent ESCs. Tuj1 is an antibody that detects neuronal marker TUBB3 at day 16 and day 30 neurons. The cycling activity of ESCs, day 16, and day 30 neurons was assessed by BrdU incorporation (24 h) into replicating BrDNA. Nuclei are counterstained with DAPI. Scale bar, 100 μm. Tyrosine hydroxylase (TH; in red) is a marker of dopaminergic neurons. It is not expressed in ESCs and detected weakly in day 16 and broadly in day 30 neurons. Nuclei are counterstained with DAPI. Scale bar, 100 μm. Gene expression dynamics across the differentiation time line for genes whose expression peaks in a single time point (z-score > 1.75; from mRNA-seq). Representative enriched GO terms were calculated using as background all genes expressed (> 1 TPM) in at least one time point. n, number of genes per group. Permute P-value (GO-Elite) is shown. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Neuronal differentiation protocol starting from ESCs gives cultures enriched for ventral midbrain dopaminergic neuronsRelated to Fig 1. Schematic representation of the protocols used to differentiate ESCs to dopaminergic neurons. Time points selected for ChIP-seq and mRNA-seq are boxed. Total RNA qRT–PCR shows the differential expression of specific markers during exit from pluripotency. Relative levels are normalized to Actb and plotted as ratio to the expression in the most expressed time point. Mean and standard deviation (SD) are from three biological replicates. Left, indirect immunofluorescence of LMX1A (green) and FOXA2 (red) in day 16 neurons. Nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. Right, percentage of cells positive for FOXA2, LMX1A, and both. Mean and SD are from five fields of view. Download figure Download PowerPoint The expression of pluripotency markers Nanog and Oct4 decreases dramatically at days 1 and 3 of differentiation, respectively (Fig 1B). The early differentiation marker Fgf5 is transiently expressed in days 1–3, whereas neuronal markers Blbp, Hes5, and Mash1/Ascl1 are increasingly expressed from day 2 (Figs 1B and EV1B). The expression of Sox2, which encodes for a transcription factor expressed in ESCs and by most central nervous system progenitors (Graham et al, 2003), is detected from ESC to day 4, as expected (Fig EV1B; Abranches et al, 2009). After sixteen days, we obtained neurons that no longer express OCT4 protein, are positive for the neuronal marker TUBB3 (detected by Tuj1 antibody), and no longer divide, as assessed by lack of BrdU incorporation into newly replicated DNA (Fig 1C). To confirm the dopaminergic phenotype, we performed immunofluorescence for tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis (Fig 1D). The neuronal populations obtained expressed TH from day 16, reaching close to ubiquitous expression at day 30 (Fig 1D). Moreover, 70% of cells co-express LMX1A and FOXA2 at day 16 (Fig EV1C), two markers specific to the dopaminergic ventral midbrain, confirming that the neuronal populations produced are highly enriched for the dopaminergic lineage (Hegarty et al, 2013). Taken together, these results indicate that day 16 neurons represent an immature stage of differentiation committed to the ventral midbrain lineage, which further mature until day 30. To characterize the changes in gene expression that accompany neuronal differentiation, we produced mRNA-seq datasets for ESCs, day 1, day 3, day 16, and day 30. We identified 4656 genes whose expression levels peaked at one specific time point (Fig 1E). Genes peaking in ESCs are enriched in Gene Ontology (GO) terms typical of pluripotency, such as “stem cell maintenance”, “regulation of gene silencing”, and “sugar utilization”, and include Nanog, Tet1, and Hk2. Genes with highest expression on day 1 have roles in the exit from pluripotency, such as Wt1, Foxd3, and Dnmt3b, and are enriched in GO terms “cell morphogenesis”, “pattern specification process”, and “gene silencing”. On day 3, the expression of Fgf8, Gli3 and HoxA1 peaked, reflecting an early stage of neuronal commitment, highlighted by enrichment in GO terms such as “cellular developmental process”, “multicellular organismal process”, and “neuronal nucleus development”. Day 16 coincided with highest expression of genes associated with GO terms such as “nervous system development”, “axon guidance”, and “neuron migration” (including Nova1, Sema3f, Ascl1, Neurog2), and day 30 with genes important for dopaminergic synaptic transmission, for the “G-protein coupled receptor protein signaling pathway” and “response to alkaloid” (such as Lpar3, Th, Park2, Chrnb4). The complete list of enriched GO terms is presented in Dataset EV1. These expression profiles show that each time point captures a specific stage of neuronal development, and suggest that days 16 and 30 reflect early and late stages, respectively, of maturation of dopaminergic neurons. To further confirm the quality of our samples, we also explored the expression profiles of specific single genes (Fig EV2). In addition to confirming the expression of the differentiation markers studied by quantitative PCR and immunofluorescence (Fig EV2A), we also found that the proneural gene Ngn2 (expressed in immature, but not in mature, dopaminergic neurons) peaks at day 16 and drops by day 30, while Nurr1 (required for maintenance of dopaminergic neurons) is upregulated at day 16 but remains expressed at day 30 (Fig EV2B; Ang, 2006). Other markers of dopaminergic neurons, such as Pitx3, Aadc, Vmat, and Dat, are most highly expressed at day 30 (Fig EV2B). Click here to expand this figure. Figure EV2. mRNA-seq profiles highlight single-gene expression changes at different stages of differentiationRelated to Fig 1. UCSC Genome Browser snapshots of mRNA-seq tracks confirm the expression of specific markers for differentiation. ESCs progressively downregulate pluripotency genes, such as Nanog and Oct4, and express Fgf5 at the onset of differentiation. FoxA2 and Lmx1a are expressed in developing neurons while Th in mature dopaminergic neurons. UCSC Genome Browser snapshots of mRNA-seq tracks for additional markers of developing (Ngn2, Nurr1, Pitx3) and mature (Aadc, Vmat, Dat) dopaminergic neurons are sequentially activated in terminally differentiated neurons. Data information: Arrows represent the position of promoter and directionality of the gene. Y-axis scales are kept constant per gene and adjusted to its maximum expression. Download figure Download PowerPoint Electrophysiological measurements demonstrate distinct stages of neuronal maturation on days 16 and 30 of differentiation To directly investigate the state of maturation of neurons upon prolonged culture, we measured their action potential activity and synaptic connectivity by conducting targeted whole-cell electrophysiological recordings (Fig 2A) at four different time points (days 14–16, 20–25, 26–30, and > 30). We find that days 14–16 neurons are largely silent, whereas at days > 30 they exhibit robust spontaneous action potential activity (Figs 2B and EV3A), similar to the activity of midbrain dopaminergic neurons from ex vivo slice preparations and in vivo (Marinelli & McCutcheon, 2014). During maturation, neurons also exhibit a hyperpolarization-activated inward (Ih) current (Fig 2C), an electrophysiological feature commonly used to identify dopaminergic neurons (Ungless & Grace, 2012). Strikingly, after prolonged culture many cells exhibit burst-like events (Fig EV3B and C), often seen in vivo in midbrain dopaminergic neurons (Grace & Bunney, 1984), which are thought to be driven in part by synaptic inputs (Paladini & Roeper, 2014). We also observed maturation of functional synaptic connectivity. Spontaneous synaptic events were rare at days 14–16, but were pr
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