Methyl CpG–binding proteins induce large-scale chromatin reorganization during terminal differentiation

Pericentric heterochromatin plays an important role in epigenetic gene regulation. We show that pericentric heterochromatin aggregates during myogenic differentiation. This clustering leads to the formation of large chromocenters and correlates with increased levels of the methyl CpG–binding protein MeCP2 and pericentric DNA methylation. Ectopic expression of fluorescently tagged MeCP2 mimicked this effect, causing a dose-dependent clustering of chromocenters in the absence of differentiation. MeCP2-induced rearrangement of heterochromatin occurred throughout interphase, did not depend on the H3K9 histone methylation pathway, and required the methyl CpG–binding domain (MBD) only. Similar to MeCP2, another methyl CpG–binding protein, MBD2, also increased during myogenic differentiation and could induce clustering of pericentric regions, arguing for functional redundancy. This MeCP2- and MBD2-mediated chromatin reorganization may thus represent a molecular link between nuclear genome topology and the epigenetic maintenance of cellular differentiation

第1058回千葉医学会例会・整形外科例会

Distribution of bivalent nucleosomes, zonal-bivalency and nucleosome-free region around the TSS of the various promoter classes. A–D, Distribution of bivalent nucleosomes at candidate gene promoters. E–G, Distribution of the H3K4me3, H3K27me3 peak calls, and their overlapping regions, that were used to classify genes into H3K4Me3-exclusive and H3K27me3-exclusive promoters. Genes that have a H3K4me3 or H3K27me3 peak call within +/−2500 bp from the TSS were identified, and the gene promoters regions were parsed into those that have exclusively H3K4me3 peaks (H3K4me3-exclusive), exclusively H3K27me3 peaks (H3K27Me3-exclusive) and those with overlapping H3K4me3 and H3K27me3 (bivalent). The distances of the center of the peak calls and overlapping regions from the TSS are plotted in E, F and G. The distribution of the distances from the TSS is plotted to depict the probability of occurrence of the peaks at varying distances from the TSS (x-axis, 0 position) for H3K4me3-exclusive promoters (E), H3K27Me3-exclusive promoters (F) and bivalent promoters (G). H, Top panel: Input sequencing reads to show the nucleosome-free zone. Bottom panel shows sequencing reads downstream from the TSS relative to the symmetric position upstream from the TSS as Log2 ratios (y-axis). Depletion in sequencing reads in the 0–1000 bp region for H3K4Me3-exclusive (black) and bivalent promoters (green) is more prominent compared to the H3K27me3-exclusive (red) promoters and promoters marked with none (blue) of the marks analyzed here. (JPG 547 kb

Alterations of immune response of non-small lung cancer with azacytidine

Innovative therapies are needed for advanced Non-Small Cell Lung Cancer (NSCLC). We have undertaken a genomics based, hypothesis driving, approach to query an emerging potential that epigenetic therapy may sensitize to immune checkpoint therapy targeting PD-L1/PD-1 interaction. NSCLC cell lines were treated with the DNA hypomethylating agent azacytidine (AZA - Vidaza) and genes and pathways altered were mapped by genome-wide expression and DNA methylation analyses. AZA-induced pathways were analyzed in The Cancer Genome Atlas (TCGA) project by mapping the derived gene signatures in hundreds of lung adeno (LUAD) and squamous cell carcinoma (LUSC) samples. AZA up-regulates genes and pathways related to both innate and adaptive immunity and genes related to immune evasion in a several NSCLC lines. DNA hypermethylation and low expression of IRF7, an interferon transcription factor, tracks with this signature particularly in LUSC. In concert with these events, AZA up-regulates PD-L1 transcripts and protein, a key ligand-mediator of immune tolerance. Analysis of TCGA samples demonstrates that a significant proportion of primary NSCLC have low expression of AZA-induced immune genes, including PD-L1. We hypothesize that epigenetic therapy combined with blockade of immune checkpoints - in particular the PD-1/PD-L1 pathway - may augment response of NSCLC by shifting the balance between immune activation and immune inhibition, particularly in a subset of NSCLC with low expression of these pathways. Our studies define a biomarker strategy for response in a recently initiated trial to examine the potential of epigenetic therapy to sensitize patients with NSCLC to PD-1 immune checkpoint blockade

Genome-wide positioning of bivalent mononucleosomes

BACKGROUND: Bivalent chromatin refers to overlapping regions containing activating histone H3 Lys4 trimethylation (H3K4me3) and inactivating H3K27me3 marks. Existence of such bivalent marks on the same nucleosome has only recently been suggested. Previous genome-wide efforts to characterize bivalent chromatin have focused primarily on individual marks to define overlapping zones of bivalency rather than mapping positions of truly bivalent mononucleosomes. RESULTS: Here, we developed an efficacious sequential ChIP technique for examining global positioning of individual bivalent nucleosomes. Using next generation sequencing approaches we show that although individual H3K4me3 and H3K27me3 marks overlap in broad zones, bivalent nucleosomes are focally enriched in the vicinity of the transcription start site (TSS). These seem to occupy the H2A.Z nucleosome positions previously described as salt-labile nucleosomes, and are correlated with low gene expression. Although the enrichment profiles of bivalent nucleosomes show a clear dependency on CpG island content, they demonstrate a stark anti-correlation with methylation status. CONCLUSIONS: We show that regional overlap of H3K4me3 and H3K27me3 chromatin tend to be upstream to the TSS, while bivalent nucleosomes with both marks are mainly promoter proximal near the TSS of CpG island-containing genes with poised/low expression. We discuss the implications of the focal enrichment of bivalent nucleosomes around the TSS on the poised chromatin state of promoters in stem cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12920-016-0221-6) contains supplementary material, which is available to authorized users

A complex interplay of regulatory domains controls cell cycle dependent subnuclear localization of DNMT1 and is required for the maintenance of epigenetic information

DNA-Methylierung spielt eine wichtige Rolle bei der Kontrolle der Chromatinorganisation und Genregulation in höheren Eukaryoten und muss zusammen mit der genetischen Information in jedem Zellzyklus dupliziert werden. Bei Mammalia wird DNA durch die DNA-Methyltransferase 1 (DNMT1) methyliert, die dabei mit nuklearen Replikationsstellen (RF) assoziiert und so die Erhaltung des Methylierungsmusters mit der Duplikation der DNA verbindet. In dieser Arbeit wurden die Funktion der regulatorischen Sequenzen in der N-terminalen Domäne von DNMT1 bei der Kontrolle ihrer subnuklearen Lokalisierung während des Zellzyklus und die evolutionäre Konservierung dieser Sequenzen, sowie die Mechanismen die eine Assoziation von Proteinen mit RF vermitteln, untersucht. Es konnte gezeigt werden, dass DNMT1 eine dynamische Verteilung im Kern aufweist, die durch regulatorische Sequenzen zellzyklusabhängig gesteuert wird. Um die subnukleare Verteilung von DNMT1 während des Zellzyklus zu untersuchen, wurden RFP-Ligase Fusionsproteine hergestellt, die als Marker für die Identifikation von Zellzyklusstadien in lebenden Zellen dienen. Verschiedene, mit GFP fusionierte DNMT1 Mutanten wurden zusammen mit RFP-Ligase exprimiert und über einen ganzen Zellzyklus hinweg mit 4-dimensionaler Lebendzellmikroskopie verfolgt. Die PBD (PCNA-Bindungsdomäne) bewirkt die Lokalisierung von DNMT1 an RF während der S-Phase, und die TS (targeting sequence) vermittelt die Retention von DNMT1 an spät replizierendem Heterochromatin von der späten S- bis zur frühen G1-Phase. Im Gegensatz dazu scheint die PBHD (Polybromohomologiedomäne) für die Freisetzung von DNMT1 von perizentrischen Regionen während der G1-Phase notwendig zu sein. Eine Überexpression der TS zu Störung dieser Assoziation, senkt die Überlebensrate der Zellen und fördert die Bildung von Mikronuklei sowie die Verschmelzung von zentromerem Heterochromatin. Diese Ergebnisse zeigen eine neue Funktion für die TS bei der Assoziation von DNMT1 mit perizentrischem Heterochromatin von der später S- über die G2-Phase bis hin zur Mitose, die eine wichtige Voraussetzung für die Erhaltung der DNA-Methylierung und Heterochromatinstruktur und -funktion ist. Datenbankanalysen zeigten, dass es sich bei der TS um eine einzigartige Domäne innerhalb der DNMT1 Proteinfamilie handelt. Innerhalb der DNMT1 Familie besitzen nur die DNMT1 Proteine der Metazoen die PBD. Das lässt vermuten, dass die Verknüpfung von Beibehaltung der DNA Methylierung mit der DNA Replikation nur in Metazoen auftritt, während in Pflanzen und Pilzen alternative Mechanismen zur Aufrechterhaltung des Methylierungsmusters, wahrscheinlich vermittelt durch die TS, zur Anwendung kommen. Die evolutionäre Konservierung von Mechanismen, zur Assoziation von Proteine mit RF in Säugerzellen, wurde durch die Analyse der Säugerproteine PCNA, DNA Ligase I und DNMT1 in Drosophila-zellen direkt getestet. Von allen untersuchten Proteinen assoziiert nur PCNA mit RF, während die anderen nur eine diffuse Verteilung innerhalb des Kerns zeigten, obwohl sie eine funktionale PBD enthalten. Überraschenderweise assoziierte auch die Drosophila DNA Ligase I in Säugerzellen nicht aber in Drosophila-zellen mit RF. Diese Ergebnisse weisen auf Unterschiede in der Dynamik und dem Aufbau der Replikationsmaschinerie in diesen entfernt verwandten Organismen hin, was mit der Vergrösserung und höheren Komplexität des Säugergenoms korreliert.DNA methylation constitutes an essential epigenetic mark controlling chromatin organization and gene regulation in higher eucaryotes, which has to be duplicated together with the genetic information at every cell division cycle. In mammals duplication of DNA methylation is mediated by DNA methyltransferase-1 (DNMT1). It associates with sites of nuclear DNA replication, called replication foci (RF), and thereby couples maintenance of DNA methylation to DNA duplication. In this work, we have analyzed the role of regulatory sequences in the N-terminal domain of DNMT1 in controlling its subnuclear localization throughout the cell cycle, and the evolutionary conservation of these sequences and of the mechanisms that mediate association of proteins with RF. We provide evidence that DNMT1 shows dynamic subnuclear distribution that is controlled by the regulatory sequences depending on the cell cycle stage. To determine the subnuclear distribution of DNMT1 throughout the cell cycle, an RFP-Ligase fusion protein was developed as a marker that allows identification of the cell cycle stage in live cells. Various DNMT1 mutants fused to GFP were coexpressed with RFP-Ligase and imaged by 4-dimensional live cell microscopy during an entire cell cycle. The PBD (PCNA binding domain) drives the localization of DNMT1 at RF throughout S phase and the TS (targeting sequence) mediates retention of DNMT1 only at the late replicating pericentric heterochromatin from late-S phase until early-G1. In contrast, the PBHD (polybromo homology domain) seems to be required for unloading DNMT1 from the pericentric regions in G1. Overexpression of the TS to interfere with this association lowers cell viability and induces the formation of micronuclei and coalescence of centromeric heterochromatin. These results bring forth a novel function of the TS in mediating association of DNMT1 with pericentric heterochromatin from late-S phase through G2 until mitosis, which is important for maintenance of DNA methylation, and heterochromatin structure and function. Database searches indicate that the TS is a domain unique to the DNMT1 family of proteins. Amongst the DNMT1 family, only the metazoan DNMT1 proteins have the PBD. This suggests that coupling of maintenance of DNA methylation with DNA replication occurs only in metazoans, while plants and fungi have alternative mechanisms that maintain DNA methylation patterns, probably mediated by the TS. The evolutionary conservation of the mechanisms by which proteins associate with RF in mammalian cells was directly tested by analyzing the ability of mammalian replication proteins PCNA and DNA Ligase I as well as DNMT1 to associate with RF in Drosophila cells. Of all the proteins tested, only PCNA associated with RF while the others showed diffused nuclear distribution although they contain a functional PBD. Surprisingly, Drosophila DNA Ligase I associates with RF in mammalian but not in Drosophila cells. These results suggest differences in the dynamics and organization of the replication machinery in these distantly related organisms, which correlates with the increased size and complexity of mammalian genomes

Combining EZH2 and HDAC inhibitors to target castration-resistant prostate cancers.

Development of resistance in castration-resistant prostate cancer (CRPC) involves epigenetic pathways. A new study in PLOS Biology demonstrates that combined therapy targeting enhancer of zeste homolog 2 (EZH2) and histone deacetylases (HDACs) may sensitize CRPC to both epigenetic and standard therapies

Cell cycle markers for live cell analyses.

Many cellular processes are regulated by cell cycle dependent changes in protein dynamics and localization. Studying these changes in vivo requires methods to distinguish the different cell cycle stages. Here we demonstrate the use of DNA Ligase I fused to DsRed1 as an in situ marker to identify S phase and the subsequent transition to G2 in live cells. Using this marker, we observed changes in the nuclear distribution of Dnmt1 during cell cycle progression. Based on the different nuclear distribution of DNA Ligase I and Dnmt1 in G2 and G1, we demonstrate that the combination of both proteins allows the direct discrimination of all cell cycle phases using either immunostainings or fusions with fluorescent proteins. These markers are new tools to directly study cell cycle dependent processes in both, fixed and living cells

Replication-independent chromatin loading of Dnmt1 during G2 and M phases.

The major DNA methyltransferase, Dnmt1, associates with DNA replication sites in S phase maintaining the methylation pattern in the newly synthesized strand. In view of the slow kinetics of Dnmt1 in vitro versus the fast progression of the replication fork, we have tested whether Dnmt1 associates with chromatin beyond S phase. Using time-lapse microscopy of mammalian cells expressing green-fluorescent-protein-tagged Dnmt1 and DsRed-tagged DNA Ligase I as a cell cycle progression marker, we have found that Dnmt1 associates with chromatin during G2 and M. This association is mediated by a specific targeting sequence, shows strong preference for constitutive but not facultative heterochromatin and is independent of heterochromatin-specific histone H3 Lys 9 trimethylation, SUV39H and HP1. Moreover, photobleaching analyses showed that Dnmt1 is continuously loaded onto chromatin throughout G2 and M, indicating a replication-independent role of Dnmt1 that could represent a novel and separate pathway to maintain DNA methylation

A novel approach to induce cell cycle reentry in terminally differentiated muscle cells.

During terminal differentiation, skeletal muscle cells permanently retract from the cell cycle. We and others have shown previously that this cell cycle withdrawal is an actively maintained state that can be reversed by transient expression of the SV40 large T antigen. In an attempt to avoid the hazards of gene transfer and the difficulties of regulating transgene expression, we have now used this cellular system as a model to test whether direct protein delivery could constitute a feasible alternative or complementing strategy to gene therapy-based approaches. Taking advantage of the recently described intercellular trafficking properties of the herpes simplex virus I VP22 protein, we have constructed a chimeric VP22-SV40 large T antigen fusion protein and shown that it can spread into terminally differentiated myotubes where it accumulates in the nucleus. This fusion protein retains the ability to override the cell cycle arrest as shown for SV40 large T antigen alone. Our results clearly show that the transduced fusion protein remains capable of inducing S-phase and mitosis in these otherwise terminally differentiated cells and opens now the way to exploit this novel strategy for tissue regeneration