scholarly journals N6-methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcription

Science ◽  
2020 ◽  
Vol 367 (6477) ◽  
pp. 580-586 ◽  
Author(s):  
Jun Liu ◽  
Xiaoyang Dou ◽  
Chuanyuan Chen ◽  
Chuan Chen ◽  
Chang Liu ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Embryonic Stem ◽  
Chromatin Accessibility ◽  
Chromatin State ◽  
Open Chromatin ◽  
Dependent Manner ◽  
Nuclear Exosome ◽  
Regulatory Rnas ◽  
Nuclear Degradation ◽  
Long Interspersed Element

N6-methyladenosine (m6A) regulates stability and translation of messenger RNA (mRNA) in various biological processes. In this work, we show that knockout of the m6A writer Mettl3 or the nuclear reader Ythdc1 in mouse embryonic stem cells increases chromatin accessibility and activates transcription in an m6A-dependent manner. We found that METTL3 deposits m6A modifications on chromosome-associated regulatory RNAs (carRNAs), including promoter-associated RNAs, enhancer RNAs, and repeat RNAs. YTHDC1 facilitates the decay of a subset of these m6A-modified RNAs, especially elements of the long interspersed element-1 family, through the nuclear exosome targeting–mediated nuclear degradation. Reducing m6A methylation by METTL3 depletion or site-specific m6A demethylation of selected carRNAs elevates the levels of carRNAs and promotes open chromatin state and downstream transcription. Collectively, our results reveal that m6A on carRNAs can globally tune chromatin state and transcription.

scholarly journals MOV10 facilitates messenger RNA decay in an N6-methyladenosine (m6A) dependent manner to maintain the mouse embryonic stem cells state

2021 ◽  
Author(s):  
Majid Mehravar ◽  
Yogesh Kumar ◽  
Moshe Olshansky ◽  
Dhiru Bansal ◽  
Craig Dent ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Biology ◽  
Messenger Rna ◽  
Mrna Decay ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Beta Catenin ◽  
Dependent Manner ◽  
Leukaemia Virus

N6-methyladenosine (m6A) is the most predominant internal mRNA modification in eukaryotes, recognised by its reader proteins (so-called m6A-readers) for regulating subsequent mRNA fates, such as splicing, export, localisation, decay, stability, and translation to control several biological processes. Although a few m6A-readers have been identified, yet the list is incomplete. Here, we identify a new m6A-reader protein, Moloney leukaemia virus 10 homologue (MOV10), in mouse embryonic stem cells (mESCs). MOV10 recognises m6A-containing mRNAs with a conserved GGm6ACU motif. Mechanistic studies uncover that MOV10 facilitates mRNA decay of its bound m6A- containing mRNAs in an m6A-dependent manner within the cytoplasmic processing bodies (P-bodies). Furthermore, MOV10 decays the Gsk-3beta mRNA through m6A that stabilises the BETA-CATENIN expression of a WNT/BETA-CATENIN signalling pathway to regulate downstream NANOG expression for maintaining the mESC state. Thus, our findings reveal how a newly identified m6A-reader, MOV10 mediates mRNA decay via m6A that impact embryonic stem cell biology.

scholarly journals Integration of human pancreatic islet genomic data refines regulatory mechanisms at Type 2 Diabetes susceptibility loci

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Matthias Thurner ◽  
Martijn van de Bunt ◽  
Jason M Torres ◽  
Anubha Mahajan ◽  
Vibe Nylander ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Pancreatic Islet ◽  
Molecular Mechanisms ◽  
Association Studies ◽  
Human Islets ◽  
Chromatin Accessibility ◽  
Chromatin State ◽  
Open Chromatin ◽  
Genome Wide Association Studies

Human genetic studies have emphasised the dominant contribution of pancreatic islet dysfunction to development of Type 2 Diabetes (T2D). However, limited annotation of the islet epigenome has constrained efforts to define the molecular mechanisms mediating the, largely regulatory, signals revealed by Genome-Wide Association Studies (GWAS). We characterised patterns of chromatin accessibility (ATAC-seq, n = 17) and DNA methylation (whole-genome bisulphite sequencing, n = 10) in human islets, generating high-resolution chromatin state maps through integration with established ChIP-seq marks. We found enrichment of GWAS signals for T2D and fasting glucose was concentrated in subsets of islet enhancers characterised by open chromatin and hypomethylation, with the former annotation predominant. At several loci (including CDC123, ADCY5, KLHDC5) the combination of fine-mapping genetic data and chromatin state enrichment maps, supplemented by allelic imbalance in chromatin accessibility pinpointed likely causal variants. The combination of increasingly-precise genetic and islet epigenomic information accelerates definition of causal mechanisms implicated in T2D pathogenesis.

scholarly journals X Chromosome Dosage Modulates Multiple Molecular and Cellular Properties of Mouse Pluripotent Stem Cells Independently of Global DNA Methylation Levels

2018 ◽  
Author(s):  
Juan Song ◽  
Adrian Janiszewski ◽  
Natalie De Geest ◽  
Lotte Vanheer ◽  
Irene Talon ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
X Chromosome ◽  
Pluripotent Stem Cells ◽  
Gene Dosage ◽  
Embryonic Stem ◽  
Chromatin Accessibility ◽  
Global Dna Methylation ◽  
Open Chromatin ◽  
Mammalian Development

ABSTRACTDuring early mammalian development, the two X-chromosomes in female cells are active. Dosage compensation between XX female and XY male cells is then achieved by X-chromosome inactivation in female cells. Reprogramming female mouse somatic cells into induced pluripotent stem cells (iPSCs) leads to X-chromosome reactivation. The extent to which increased X-chromosome dosage (X-dosage) in female iPSCs leads to differences in the molecular and cellular properties of XX and XY iPSCs is still unclear. We show that chromatin accessibility in mouse iPSCs is modulated by X-dosage. Specific sets of transcriptional regulator motifs are enriched in chromatin with increased accessibility in XX or XY iPSCs. We show that the transcriptome, growth and pluripotency exit are also modulated by X-dosage in iPSCs. To understand the mechanisms by which increased X-dosage modulates the molecular and cellular properties of mouse pluripotent stem cells, we used heterozygous deletions of the X-linked gene Dusp9 in XX embryonic stem cells. We show that X-dosage regulates the transcriptome, open chromatin landscape, growth and pluripotency exit largely independently of global DNA methylation. Our results uncover new insights into X-dosage in pluripotent stem cells, providing principles of how gene dosage modulates the epigenetic and genetic mechanisms regulating cell identity.

scholarly journals Integration of human pancreatic islet genomic data refines regulatory mechanisms at Type 2 Diabetes susceptibility loci

2017 ◽  
Author(s):  
Matthias Thurner ◽  
Martijn van de Bunt ◽  
Jason M Torres ◽  
Anubha Mahajan ◽  
Vibe Nylander ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Pancreatic Islet ◽  
Molecular Mechanisms ◽  
Association Studies ◽  
Human Islets ◽  
Chromatin Accessibility ◽  
Chromatin State ◽  
Open Chromatin ◽  
Genome Wide Association Studies

AbstractHuman genetic studies have emphasised the dominant contribution of pancreatic islet dysfunction to development of Type 2 Diabetes (T2D). However, limited annotation of the islet epigenome has constrained efforts to define the molecular mechanisms mediating the, largely regulatory, signals revealed by Genome-Wide Association Studies (GWAS). We characterised patterns of chromatin accessibility (ATAC-seq, n=17) and DNA methylation (whole-genome bisulphite sequencing, n=10) in human islets, generating high-resolution chromatin state maps through integration with established ChIP-seq marks. We found enrichment of GWAS signals for T2D and fasting glucose was concentrated in subsets of islet enhancers characterised by open chromatin and hypomethylation, with the former annotation predominant. At several loci (including CDC123, ADCY5, KLHDC5) the combination of fine-mapping genetic data and chromatin state enrichment maps, supplemented by allelic imbalance in chromatin accessibility pinpointed likely causal variants. The combination of increasingly-precise genetic and islet epigenomic information accelerates definition of causal mechanisms implicated in T2D pathogenesis.

P16.09 Regulation of chromatin accessibility in the hypoxic tumor microenvironment of glioblastoma

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii57-ii57
Author(s):  
M Dzwigonska ◽  
J Mieczkowski ◽  
P Pilanc ◽  
S Cyranowski ◽  
A Kominek ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Single Cell ◽  
Cancer Cells ◽  
Single Cells ◽  
Therapy Resistance ◽  
Chromatin Accessibility ◽  
Open Chromatin ◽  
Dependent Manner ◽  
Protein Markers ◽  
Hypoxic Tumor

Abstract BACKGROUND Chromatin structure is often dysregulated in cancers, including glioblastoma (GBM), the most aggressive type of primary brain tumor. GBM has the poorest prognosis with no efficient cure to date due to diffusive growth into the brain, resistance to treatments and the immunosuppressive tumor microenvironment (TME). The growth and invasiveness of GBM is supported by the heterogeneous TME including local microglia and bone-marrow-derived macrophages (collectively known as glioma-associated microglia and macrophages, GAMs). In addition, tumor hypoxia is a key factor in the progression of GBM, as it can globally and rapidly alter gene expression, induce cancer cell invasiveness, stemness and lead to therapy resistance. Hypoxia can influence the pro-tumorigenic function of GAMs by inducing the expression of cytokines and cell surface receptors. However, little is known on the hypoxia-imposed chromatin changes of GAMs and GBM cells, which can in turn impact the interaction between these cell populations. Here we analyze these changes using a single-cell method, which preserves in situ hypoxia within the TME of GBM. MATERIAL AND METHODS Single-cell Pi-ATAC-seq (Protein-indexed Assay of Transposase Accessible Chromatin with sequencing) method in a GL261 murine glioma model was used to simultaneously assess genome-wide chromatin accessibility and expression of intracellular protein markers in single cells, enabling accurate selection of hypoxic and non-hypoxic tumor cells and GAMs. Pi-ATAC-seq is used on paraformaldehyde-perfused tumors and therefore allows capturing unaltered hypoxia-dependent cellular states, that often become distorted during dissociation and preparation of fresh material in most common single-cell methods. RESULTS We optimized Pi-ATAC method in a GL261 GBM mouse model, with specific sorting of GAMs using CD11b+ immunosorting followed by separation of microglia and macrophages, based on intensity of CD45 staining. HIF-1α induction and binding of pimonidazole were used to mark hypoxic populations. Currently, we are investigating the chromatin accessibility profiles of cancer cells and GAMs within the hypoxic tumor microenvironment of GBM. Exploring open chromatin profiles in GAMs and glioma-microglia co-cultures will allow to unravel the mechanisms of chromatin accessibility modulation in the oxygen-dependent manner. CONCLUSION In summary, we optimized the Pi-ATAC method in a mouse GBM model to characterize the chromatin openness changes in GAMs and cancer cells in response to hypoxic stress. Further validation of these results will provide the potential to identify novel markers for GAMs/glioma interactions in hypoxic GBMs and develop novel therapeutic targets.

scholarly journals Multi-omics analysis of chromatin accessibility and interactions with transcriptome by HiCAR

2020 ◽  
Author(s):  
Xiaolin Wei ◽  
Yu Xiang ◽  
Ruocheng Shan ◽  
Derek T Peters ◽  
Tongyu Sun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Long Range ◽  
Spatial Interaction ◽  
Embryonic Stem ◽  
Cost Effective ◽  
Chromatin Accessibility ◽  
Open Chromatin ◽  
Low Input ◽  
Chromatin Interactions

The long-range interactions of cis-regulatory elements (cREs) play a central role in regulating the spatial-temporal gene expression program of multi-cellular organism. cREs are characterized by the presence of accessible (or open) chromatin, which can be identified at genome-wide scale with assays such as ATAC-seq, DHS-seq, and FAIRE-seq. However, it remains technically challenging to comprehensively identify the long-range physical interactions that occur between cREs, especially in a cost effective manner using low-input samples. Here, we report HiCAR (High-throughput Chromosome conformation capture on Accessible DNA with mRNA-seq co-assay), a method that enables simultaneous assessment of cis-regulatory chromatin interactions and chromatin accessibility, as well as evaluation of the transcriptome, which represents the functional output of chromatin structure and accessibility. Unlike immunoprecipitation-based methods such as HiChIP, PLAC-seq, and ChIA-PET, HiCAR does not require target-specific antibodies and thus can comprehensively capture the cis-regulatory chromatin contacts anchored at accessible regulatory DNA regions and associated with diverse epigenetic modifications and transcription factor binding. Compared to Trac-looping, another method designed to capture interactions between accessible chromatin regions, HiCAR produced a 17-fold greater yield of informative long-range cis- reads at a similar sequencing depth and required 1,000-fold fewer cells as input. Applying HiCAR to H1 human embryonic stem cells (hESCs) revealed 46,792 cis-regulatory chromatin interactions at 5kb resolution. Interestingly, we found that epigenetically poised, bivalent, and repressed cREs exhibit comparable spatial interaction activity to those transcriptionally activated cREs. Using machine learning approaches, we predicated 22 epigenome features that are potentially important for the spatial interaction activity of cREs in H1 hESC. Lastly, we also identified long-range cis-regulatory chromatin interactions in GM12878 and mouse embryonic stem cells with HiCAR. Our results demonstrate that HiCAR is a robust and cost-effective multi-omics assay, which is broadly applicable for simultaneous analysis of genome architecture, chromatin accessibility, and the transcriptome using low-input samples.

scholarly journals scNMT-seq enables joint profiling of chromatin accessibility DNA methylation and transcription in single cells

2017 ◽  
Author(s):  
Stephen J. Clark ◽  
Ricard Argelaguet ◽  
Chantriolnt-Andreas Kapourani ◽  
Thomas M. Stubbs ◽  
Heather J. Lee ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Single Cell ◽  
Single Cells ◽  
Embryonic Stem ◽  
Transcriptome Profiling ◽  
Chromatin Accessibility ◽  
Open Chromatin ◽  
Dynamic Coupling ◽  
Single Cell Sequencing ◽  
Molecular Layers

AbstractParallel single-cell sequencing protocols represent powerful methods for investigating regulatory relationships, including epigenome-transcriptome interactions. Here, we report a novel single-cell method for parallel chromatin accessibility, DNA methylation and transcriptome profiling. scNMT-seq (single-cell nucleosome, methylation and transcription sequencing) uses a GpC methyltransferase to label open chromatin followed by bisulfite and RNA sequencing. We validate scNMT-seq by applying it to differentiating mouse embryonic stem cells, finding links between all three molecular layers and revealing dynamic coupling between epigenomic layers during differentiation.

scholarly journals Long-range single-molecule mapping of chromatin accessibility in eukaryotes

2018 ◽  
Author(s):  
Zohar Shipony ◽  
Georgi K. Marinov ◽  
Matthew P. Swaffer ◽  
Nasa A. Sinott-Armstrong ◽  
Jan M. Skotheim ◽  
...  
Keyword(s):  
Single Molecule ◽  
Regulatory Elements ◽  
Dna Methyltransferases ◽  
Chromatin Accessibility ◽  
Chromatin State ◽  
Open Chromatin ◽  
Structure Mapping ◽  
Long Read ◽  
Distal Regulatory Elements ◽  
Resolution Single

AbstractActive regulatory elements in eukaryotes are typically characterized by an open, nucleosome-depleted chromatin structure; mapping areas of open chromatin has accordingly emerged as a widely used tool in the arsenal of modern functional genomics. However, existing approaches for profiling chromatin accessibility are limited by their reliance on DNA fragmentation and short read sequencing, which leaves them unable to provide information about the state of chromatin on larger scales or reveal coordination between the chromatin state of individual distal regulatory elements. To address these limitations, we have developed a method for profiling accessibility of individual chromatin fibers at multi-kilobase length scale (SMAC-seq, or Single-Molecule long-read Accessible Chromatin mapping sequencing assay), enabling the simultaneous, high-resolution, single-molecule assessment of the chromatin state of distal genomic elements. Our strategy is based on combining the preferential methylation of open chromatin regions by DNA methyltransferases (CpG and GpC 5-methylcytosine (5mC) and N6-methyladenosine (m6A) enzymes) and the ability of long-read single-molecule nanopore sequencing to directly read out the methylation state of individual DNA bases. Applying SMAC-seq to the budding yeast Saccharomyces cerevisiae, we demonstrate that aggregate SMAC-seq signals match bulk-level accessibility measurements, observe single-molecule protection footprints of nucleosomes and transcription factors, and quantify the correlation between the chromatin states of distal genomic elements.

scholarly journals Concentration dependent chromatin states induced by the bicoid morphogen gradient

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Colleen E Hannon ◽  
Shelby A Blythe ◽  
Eric F Wieschaus
Keyword(s):  
Large Scale ◽  
Common Property ◽  
Target Genes ◽  
Positional Information ◽  
Chromatin Accessibility ◽  
Chromatin State ◽  
Open Chromatin ◽  
Genome Wide ◽  
High Concentrations

In Drosophila, graded expression of the maternal transcription factor Bicoid (Bcd) provides positional information to activate target genes at different positions along the anterior-posterior axis. We have measured the genome-wide binding profile of Bcd using ChIP-seq in embryos expressing single, uniform levels of Bcd protein, and grouped Bcd-bound targets into four classes based on occupancy at different concentrations. By measuring the biochemical affinity of target enhancers in these classes in vitro and genome-wide chromatin accessibility by ATAC-seq, we found that the occupancy of target sequences by Bcd is not primarily determined by Bcd binding sites, but by chromatin context. Bcd drives an open chromatin state at a subset of its targets. Our data support a model where Bcd influences chromatin structure to gain access to concentration-sensitive targets at high concentrations, while concentration-insensitive targets are found in more accessible chromatin and are bound at low concentrations. This may be a common property of developmental transcription factors that must gain early access to their target enhancers while the chromatin state of the genome is being remodeled during large-scale transitions in the gene regulatory landscape.

scholarly journals Dynamic landscape of chromatin accessibility and transcriptomic changes during differentiation of human embryonic stem cells into dopaminergic neurons

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
César Meléndez-Ramírez ◽  
Raquel Cuevas-Diaz Duran ◽  
Tonatiuh Barrios-García ◽  
Mayela Giacoman-Lozano ◽  
Adolfo López-Ornelas ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dopaminergic Neuron ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Differentially Expressed ◽  
Open Chromatin ◽  
Cell Identity ◽  
Identity Studies ◽  
Dopaminergic Differentiation

AbstractChromatin architecture influences transcription by modulating the physical access of regulatory factors to DNA, playing fundamental roles in cell identity. Studies on dopaminergic differentiation have identified coding genes, but the relationship with non-coding genes or chromatin accessibility remains elusive. Using RNA-Seq and ATAC-Seq we profiled differentially expressed transcripts and open chromatin regions during early dopaminergic neuron differentiation. Hierarchical clustering of differentially expressed genes, resulted in 6 groups with unique characteristics. Surprisingly, the abundance of long non-coding RNAs (lncRNAs) was high in the most downregulated transcripts, and depicted positive correlations with target mRNAs. We observed that open chromatin regions decrease upon differentiation. Enrichment analyses of accessibility depict an association between open chromatin regions and specific functional pathways and gene-sets. A bioinformatic search for motifs allowed us to identify transcription factors and structural nuclear proteins that potentially regulate dopaminergic differentiation. Interestingly, we also found changes in protein and mRNA abundance of the CCCTC-binding factor, CTCF, which participates in genome organization and gene expression. Furthermore, assays demonstrated co-localization of CTCF with Polycomb-repressed chromatin marked by H3K27me3 in pluripotent cells, progressively decreasing in neural precursor cells and differentiated neurons. Our work provides a unique resource of transcription factors and regulatory elements, potentially involved in the acquisition of human dopaminergic neuron cell identity.

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