EPCO-08. ATRX DEFICIENCY INDUCES DYSFUNCTIONAL HETEROCHROMATIN ARCHITECTURE IN GLIOMAS AND ESTABLISHES DISEASE-DEFINING TRANSCRIPTIONAL NETWORKS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi2-vi3
Author(s):  
Prit Benny Malgulwar ◽  
Carla Danussi ◽  
Anand Singh ◽  
Kasthuri Kannan ◽  
Kunal Rai ◽  
...  
Keyword(s):  
Neural Progenitor Cells ◽  
Strong Association ◽  
Chromatin Accessibility ◽  
Integrated Analysis ◽  
Transcriptional Networks ◽  
Alternative Lengthening ◽  
Chromatin Regulator ◽  
Therapeutic Molecules ◽  
Diffuse Gliomas ◽  
Active Transcription

Abstract Loss of ATRX (Alpha Thalassemia/Mental Retardation Syndrome X, a member of SWI/SNF family chromatin regulator is altered in diffuse gliomas and defines molecular subtypes with aggressive behavior. Mechanistically, ATRX regulates incorporation of histone H3.3 into chromatin sites across the genome, maintains alternative lengthening of telomeres and establishes genomic distribution of polycomb responsive genes. We have recently reported Atrx deficiency induces glioma oncogenic features via widespread alterations in chromatin accessibility using mouse Neural Progenitor Cells (mNPCs- Tp53 -/-,Atrx -/-). Surprisingly, Atrx was found to be associated with transcription start site and enhancer regions, suggesting their strong association with epigenome architecture. In this background, we have recently performed ChIP-seq for histone marks that define active transcription, enhancers, repressors and gene bodies and Cohesion molecules on Atrx intact and deficient mNPCs. Our integrated analysis reports depletion of H3K9me3 loci’s with enrichment of H3K27me3 marks that coincidently enriched with Atrx binding sites and Lamina-Associated Domains (LADs). GSEA confirmed that the genes corresponding to “newly formed LADs” in mNPC-to-astrocyte differentiation are significantly enriched for genes down-regulated in Atrx deficient mNPCs and belongs to Gene Ontology categories such as cell cycle, chromosome organization and DNA damage. Alternatively, genes corresponding to decreased LAD association are enriched for up-regulated genes in Atrx deficient mNPCs and for regulation of differentiation, adhesion and cell death. Additionally, whole-genome bisulphite sequencing further demonstrated loss of methylation marks at H3K9me3 sites in Atrx deficient mNPCs, suggesting perturbations of heterochromatin regions leading to activation of canonical signals that define glioma phenotype and disease-state. To summarize, our data establishes tangible links between Atrx deficiency and dysregulated chromatin and heterochromatin architecture in gliomas and suggests the role of Atrx in establishing global chromatin features and transcriptional networks. Further, our data unravel novel therapeutic molecules/pathways for engineering potential.

scholarly journals RNA-bound PGC-1α controls gene expression in liquid-like nuclear condensates

2020 ◽  
Author(s):  
Joaquín Pérez-Schindler ◽  
Bastian Kohl ◽  
Konstantin Schneider-Heieck ◽  
Volkan Adak ◽  
Julien Delezie ◽  
...  
Keyword(s):  
Target Genes ◽  
Rna Binding ◽  
Protein Complexes ◽  
Transcriptional Networks ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Central Function ◽  
Skeletal Muscle Wasting ◽  
Active Transcription ◽  
Transcriptional Coregulator

AbstractThe peroxisome-proliferator-activated receptor-γ coactivator-1α (PGC-1α) integrates environmental cues by controlling complex transcriptional networks in various metabolically active tissues. However, it is unclear how a transcriptional coregulator coordinates dynamic biological programs in response to multifaceted stimuli such as endurance training or fasting. Here, we discovered a central function of the poorly understood C-terminal domain (CTD) of PGC-1α to bind RNAs and assemble multi-protein complexes. Surprisingly, in addition to controlling the coupling of transcription and processing of target genes, RNA binding is indispensable for the recruitment of PGC-1α to chromatin into liquid-like nuclear condensates, which compartmentalize and regulate active transcription. These results demonstrate a hitherto unsuspected molecular mechanism by which complexity in the regulation of large transcriptional networks by PGC-1α is achieved. These findings are not only essential for the basic understanding of transcriptional coregulator-driven control of biological programs, but will also help to devise new strategies to modulate these processes in pathological contexts in which PGC-1α function is dysregulated, such as type 2 diabetes, cardiovascular diseases or skeletal muscle wasting.

scholarly journals Super Enhancer Profiles Identify Key Cell Identity Genes During Differentiation From Embryonic Stem Cells to Trophoblast Stem Cells Super Enhencers in Trophoblast Differentiation

2021 ◽  
Vol 12 ◽  
Author(s):  
Rongpu Jia ◽  
Yu Gao ◽  
Song Guo ◽  
Si Li ◽  
Liangji Zhou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryo Implantation ◽  
Control Cell ◽  
Embryonic Stem ◽  
Growth Factor Receptor ◽  
Chromatin Accessibility ◽  
Integrated Analysis ◽  
Cell Identity ◽  
Trophoblast Stem Cells ◽  
Trophoblast Stem

Trophoblast stem cells (TSCs) are derived from blastocysts and the extra-embryonic ectoderm (ExE) of post-implantation embryos and play a significant role in fetal development, but the roles that TSCs play in the earlier status of fetal diseases need further exploration. Super enhancers (SEs) are dense clusters of stitched enhancers that control cell identity determination and disease development and may participate in TSC differentiation. We identified key cell identity genes regulated by TSC-SEs via integrated analysis of H3K27ac and H3K4me1 chromatin immunoprecipitation sequencing (ChIP-seq), RNA-sequencing (RNA-seq) and ATAC-sequencing (ATAC-seq) data. The identified key TSC identity genes regulated by SEs, such as epidermal growth factor receptor (EGFR), integrin β5 (ITGB5) and Paxillin (Pxn), were significantly upregulated during TSC differentiation, and the transcription network mediated by TSC-SEs enriched in terms like focal adhesion and actin cytoskeleton regulation related to differentiation of TSCs. Additionally, the increased chromatin accessibility of the key cell identity genes verified by ATAC-seq further demonstrated the regulatory effect of TSC-SEs on TSC lineage commitment. Our results illustrated the significant roles of the TSC-SE-regulated network in TSC differentiation, and identified key TSC identity genes EGFR, ITGB5 and Pxn, providing novel insight into TSC differentiation and lays the foundation for future studies on embryo implantation and related diseases.

scholarly journals SAT-298 Integrative Single-Cell Transcriptomic and Epigenomic Landscape of Mouse Anterior Pituitary Cell Types

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Frederique Murielle Ruf-Zamojski ◽  
Michel A Zamojski ◽  
German Nudelman ◽  
Yongchao Ge ◽  
Natalia Mendelev ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Line ◽  
Anterior Pituitary ◽  
Cell Types ◽  
Chromatin Accessibility ◽  
Pituitary Cell ◽  
Integrated Analysis ◽  
Pituitary Cells ◽  
Rna Seq ◽  
Cell Type

Abstract The pituitary gland is a critical regulator of the neuroendocrine system. To further our understanding of the classification, cellular heterogeneity, and regulatory landscape of pituitary cell types, we performed and computationally integrated single cell (SC)/single nucleus (SN) resolution experiments capturing RNA expression, chromatin accessibility, and DNA methylation state from mouse dissociated whole pituitaries. Both SC and SN transcriptome analysis and promoter accessibility identified the five classical hormone-producing cell types (somatotropes, gonadotropes (GT), lactotropes, thyrotropes, and corticotropes). GT cells distinctively expressed transcripts for Cga, Fshb, Lhb, Nr5a1, and Gnrhr in SC RNA-seq and SN RNA-seq. This was matched in SN ATAC-seq with GTs specifically showing open chromatin at the promoter regions for the same genes. Similarly, the other classically defined anterior pituitary cells displayed transcript expression and chromatin accessibility patterns characteristic of their own cell type. This integrated analysis identified additional cell-types, such as a stem cell cluster expressing transcripts for Sox2, Sox9, Mia, and Rbpms, and a broadly accessible chromatin state. In addition, we performed bulk ATAC-seq in the LβT2b gonadotrope-like cell line. While the FSHB promoter region was closed in the cell line, we identified a region upstream of Fshb that became accessible by the synergistic actions of GnRH and activin A, and that corresponded to a conserved region identified by a polycystic ovary syndrome (PCOS) single nucleotide polymorphism (SNP). Although this locus appears closed in deep sequencing bulk ATAC-seq of dissociated mouse pituitary cells, SN ATAC-seq of the same preparation showed that this site was specifically open in mouse GT, but closed in 14 other pituitary cell type clusters. This discrepancy highlighted the detection limit of a bulk ATAC-seq experiment in a subpopulation, as GT represented ~5% of this dissociated anterior pituitary sample. These results identified this locus as a candidate for explaining the dual dependence of Fshb expression on GnRH and activin/TGFβ signaling, and potential new evidence for upstream regulation of Fshb. The pituitary epigenetic landscape provides a resource for improved cell type identification and for the investigation of the regulatory mechanisms driving cell-to-cell heterogeneity. Additional authors not listed due to abstract submission restrictions: N. Seenarine, M. Amper, N. Jain (ISMMS).

scholarly journals A mass spectrometry-based assay using metabolic labeling to rapidly monitor chromatin accessibility of modified histone proteins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Simone Sidoli ◽  
Mariana Lopes ◽  
Peder J. Lund ◽  
Naomi Goldman ◽  
Maria Fasolino ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Chemical Properties ◽  
Chromatin Accessibility ◽  
Metabolic Labeling ◽  
Post Translational Modifications ◽  
Histone Ptms ◽  
Active Transcription ◽  
Or Gene ◽  
Histone Codes

Abstract Histone post-translational modifications (PTMs) contribute to chromatin accessibility due to their chemical properties and their ability to recruit enzymes responsible for DNA readout and chromatin remodeling. To date, more than 400 different histone PTMs and thousands of combinations of PTMs have been identified, the vast majority with still unknown biological function. Identification and quantification of histone PTMs has become routine in mass spectrometry (MS) but, since raising antibodies for each PTM in a study can be prohibitive, lots of potential is lost from MS datasets when uncharacterized PTMs are found to be significantly regulated. We developed an assay that uses metabolic labeling and MS to associate chromatin accessibility with histone PTMs and their combinations. The labeling is achieved by spiking in the cell media a 5x concentration of stable isotope labeled arginine and allow cells to grow for at least one cell cycle. We quantified the labeling incorporation of about 200 histone peptides with a proteomics workflow, and we confirmed that peptides carrying PTMs with extensively characterized roles in active transcription or gene silencing were in highly or poorly labeled forms, respectively. Data were further validated using next-generation sequencing to assess the transcription rate of chromatin regions modified with five selected PTMs. Furthermore, we quantified the labeling rate of peptides carrying co-existing PTMs, proving that this method is suitable for combinatorial PTMs. We focus on the abundant bivalent mark H3K27me3K36me2, showing that H3K27me3 dominantly represses histone swapping rate even in the presence of the more permissive PTM H3K36me2. Together, we envision this method will help to generate hypotheses regarding histone PTM functions and, potentially, elucidate the role of combinatorial histone codes.

scholarly journals Exogenous artificial DNA forms chromatin structure with active transcription in yeast

2021 ◽  
Author(s):  
Jianting Zhou ◽  
Chao Zhang ◽  
Ran Wei ◽  
Mingzhe Han ◽  
Songduo Wang ◽  
...  
Keyword(s):  
Data Storage ◽  
Dna Sequences ◽  
Chromatin Accessibility ◽  
Binding Motifs ◽  
Artificial Chromosomes ◽  
Sequencing Technologies ◽  
Artificial Dna ◽  
Active Transcription ◽  
Reported Data ◽  
Yeast Artificial Chromosomes

AbstractYeast artificial chromosomes (YACs) are important tools for sequencing, gene cloning, and transferring large quantities of genetic information. However, the structure and activity of YAC chromatin, as well as the unintended impacts of introducing foreign DNA sequences on DNA-associated biochemical events, have not been widely explored. Here, we showed that abundant genetic elements like TATA box and transcription factor-binding motifs occurred unintentionally in a previously reported data-carrying chromosome (dChr). In addition, we used state-of-the-art sequencing technologies to comprehensively profile the genetic, epigenetic, transcriptional, and proteomic characteristics of the exogenous dChr. We found that the data-carrying DNA formed active chromatin with high chromatin accessibility and H3K4 tri-methylation levels. The dChr also displayed highly pervasive transcriptional ability and transcribed hundreds of noncoding RNAs. The results demonstrated that exogenous artificial chromosomes formed chromatin structures and did not remain as naked or loose plasmids. A better understanding of the YAC chromatin nature will improve our ability to design better data-storage chromosomes.

scholarly journals Single-cell chromatin and transcriptome dynamics of Synovial Fibroblasts transitioning from homeostasis to pathology in modelled TNF-driven arthritis

2021 ◽  
Author(s):  
Marietta Armaka ◽  
Dimitris Konstantopoulos ◽  
Christos Tzaferis ◽  
Matthieu D Lavigne ◽  
Maria Sakkou ◽  
...  
Keyword(s):  
Single Cell ◽  
Inflammatory Responses ◽  
Early Stage ◽  
Synovial Fibroblasts ◽  
Chromatin Accessibility ◽  
Proper Function ◽  
Integrated Analysis ◽  
Transcription Profiles ◽  
Diarthrodial Joints ◽  
Conserved Core

AbstractSynovial fibroblasts (SFs) are specialized cells of the synovium that provide nutrients and lubricants for the maintenance of proper function of diarthrodial joints. Chronic TNF signals are known to trigger activation of SFs and orchestration of arthritic pathology via proinflammatory effector functions, secretion of cartilage degrading proteases and promotion of osteolysis. We performed single-cell (sc) profiling of SF’s transcriptome by RNA-sequencing (scRNA-seq) and of chromatin accessibility by scATAC-seq in normal mouse SFs and SFs derived from early and advanced TNF-driven arthritic disease. We describe here distinct subsets of SFs in the homeostatic synovium, serving diverse functions such as chondro- and osteogenesis, tissue repair and immune regulation. Strikingly, development of spontaneous arthritis by transgenic TNF overexpression primes the emergence of distinct pathology-associated SF subtypes. We reveal 7 constitutive and 2 disease-specific SF subtypes. The latter emerge in the early stage, expand in late disease and are localized in areas at the interface between the invasive pannus and the articular bone. The associated transcription profiles are characterized by enhanced inflammatory responses, promigratory behaviour, neovascularization and collagen metabolic processes. Temporal reconstruction of transcriptomic events indicated which specific sublining cells may function as progenitors at the root of trajectories leading to intermediate subpopulations and culminating to a destructive lining inflammatory identity. Integrated analysis of chromatin accessibility and transcription changes revealed key transcription factors such as Bach and Runx1 to drive arthritogenesis. Parallel analysis of human arthritic SF data showed highly conserved core regulatory and transcriptional programs between the two species. Therefore, our study dissects the dynamic SF landscape in TNF-mediated arthritis and sets the stage for future investigations that might address the functions of specific SF subpopulations to understand joint pathophysiology and combat chronic inflammatory and destructive arthritic diseases.

scholarly journals FACT sets a barrier for cell fate reprogramming inC. elegansand Human

2017 ◽  
Author(s):  
Ena Kolundzic ◽  
Andreas Ofenbauer ◽  
Bora Uyar ◽  
Anne Sommermeier ◽  
Stefanie Seelk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
Chromatin Accessibility ◽  
Genetic Screen ◽  
Cellular Reprogramming ◽  
Stable Gene ◽  
C Elegans ◽  
Chromatin Regulator ◽  
Evolutionarily Conserved ◽  
Stable Gene Expression

The chromatin regulator FACT (Facilitates Chromatin Transcription) is essential for ensuring stable gene expression by promoting transcription. In a genetic screen usingC. eleganswe identified that FACT maintains cell identities and acts as a barrier for transcription factor-mediated cell fate reprogramming. Strikingly, FACTs role as a reprogramming barrier is conserved in humans as we show that FACT depletion enhances reprogramming of fibroblasts into stem cells and neurons. Such activity of FACT is unexpected since known reprogramming barriers typically repress gene expression by silencing chromatin. In contrast, FACT is a positive regulator of gene expression suggesting an unprecedented link of cell fate maintenance with counteracting alternative cell identities. This notion is supported by ATAC-seq analysis showing that FACT depletion results in decreased but also increased chromatin accessibility for transcription factors. Our findings identify FACT as a cellular reprogramming barrier inC. elegansand in Human, revealing an evolutionarily conserved mechanism for cell fate protection.

scholarly journals Low-input ATAC&mRNA-Seq: a simple and robust method for simultaneous dual-omics profiling with low cell number

2021 ◽  
Author(s):  
Ruifang Li ◽  
Sara A Grimm ◽  
Paul A Wade
Keyword(s):  
Gene Expression ◽  
Strong Association ◽  
Regulation Of Gene Expression ◽  
Cell Number ◽  
Chromatin Accessibility ◽  
Robust Method ◽  
Data Types ◽  
Single Experiment ◽  
Low Input ◽  
Input Material

AbstractDeciphering epigenetic regulation of gene expression requires measuring the epigenome and transcriptome jointly. Single-cell multi-omics technologies have been developed for concurrent profiling of chromatin accessibility and gene expression. However, multi-omics profiling of low-input bulk samples remains challenging. Therefore, we developed low-input ATAC&mRNA-seq, a simple and robust method for studying the role of chromatin structure in gene regulation in a single experiment with thousands of cells, to maximize insights from limited input material by obtaining ATAC-seq and mRNA-seq data simultaneously from the same cells with data quality comparable to conventional mono-omics assays. Integrative data analysis revealed similar strong association between promoter accessibility and gene expression using the data of low-input ATAC&mRNA-seq as using single-assay data, underscoring the accuracy and reliability of our dual-omics assay to generate both data types simultaneously with just thousands of cells. We envision our method to be widely applied in many biological disciplines with limited materials.

scholarly journals Differential chromatin accessibility in developing projection neurons is correlated with transcriptional regulation of cell fate

2019 ◽  
Author(s):  
Whitney E. Heavner ◽  
Shaoyi Ji ◽  
James H. Notwell ◽  
Ethan S. Dyer ◽  
Alex M. Tseng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Cell Fate ◽  
Expression Profiles ◽  
Chromatin Accessibility ◽  
Transcriptional Networks ◽  
Projection Neurons ◽  
Cortical Projection ◽  
Functional Features

AbstractWe are only just beginning to catalog the vast diversity of cell types in the cerebral cortex. Such categorization is a first step toward understanding how diversification relates to function. All cortical projection neurons arise from a uniform pool of progenitor cells that lines the ventricles of the forebrain. It is still unclear how these progenitor cells generate the more than fifty unique types of mature cortical projection neurons defined by their distinct gene expression profiles. Here we compare gene expression and chromatin accessibility of two subclasses of projection neurons with divergent morphological and functional features as they develop in the mouse brain between embryonic day 13 and postnatal day 5 in order to identify transcriptional networks that diversity neuron cell fate. We find groups of transcription factors whose expression is correlated with chromatin accessibility, transcription factor binding motifs, and lncRNAs that define each subclass and validate the function of a family of novel candidate genes in vitro. Our multidimensional approach reveals that subclass-specific chromatin accessibility is significantly correlated with gene expression, providing a resource for generating new specific genetic drivers and revealing regions of the genome that are particularly susceptible to harmful genetic mutations by virtue of their correlation with important developmental genes.

Chromatin State Dynamics Underlying CD8 T Cell Differentiation and Dysfunction in Cancer

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 861-861
Author(s):  
Mary Philip ◽  
Lauren Fairchild ◽  
Liping Sun ◽  
Agnes Viale ◽  
Taha Merghoub ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Chromatin Remodeling ◽  
Cd8 T Cell ◽  
Chromatin Accessibility ◽  
T Cell Differentiation ◽  
Therapeutic Interventions ◽  
Transcriptional Networks ◽  
Genome Wide

Abstract T cells recognizing tumor-specific antigens are detected in cancer patients but are dysfunctional. Upon antigen encounter, T cells differentiate into discrete phenotypic and functional states. Cellular differentiation is driven by epigenetic remodeling, however, it is not known whether and how epigenetic programming establishes and regulates tumor-specific T cell (TST) dysfunction and determines a T cell's ability to respond to therapeutic interventions such as immune checkpoint blockade (PD-1 and CTLA-4). Here for the first time, we (1) identify chromatin dynamics underlying T cell differentiation to the dysfunctional state in mouse and human tumors and (2) provide insights into the epigenetic and transcriptional regulatory mechanisms determining T cell susceptibility to therapeutic reprogramming. Using a genetic cancer mouse model, we previously showed that CD8 TST become unresponsive early during carcinogenesis at the pre-malignant stage, even before the emergence of a pathologically-defined malignant tumor. While T cell dysfunction was initially reversible, it ultimately became a fixed state that could not be rescued by therapeutic interventions such as PD1 checkpoint blockade. To identify the hierarchical changes in chromatin states resulting in "dysfunction imprinting," we used the Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) to map the genome-wide changes in chromatin accessibility in TST cells over the course of cancer development. In parallel, we carried out RNA-Seq to determine the interplay between chromatin remodeling and transcriptional networks. Substantial chromatin remodeling occurred during early T cell activation in the pre-malignant lesion (days 5-7) followed by a second wave of chromatin accessibility changes between days 7 and 14. Strikingly, after the second wave, no further CD8 T cell chromatin remodeling occurred during carcinogenesis, even after progression to an advanced late-stage tumor with an immunosuppressive microenvironment. Interestingly, these 2 distinct chromatin accessibility patterns in TST correlated temporally with the plastic and fixed dysfunctional states and susceptibility to therapeutic reprogramming in vivo. To understand the transition from plastic to fixed dysfunction, we analyzed the differential expression of transcription factors (TF) in conjunction with changes in peak accessibility at TF-binding motifs genome-wide. We identified a network including CD8 T cell regulatory TF such as TCF1, LEF1, BLIMP1, and BACH2 as well as less-well-characterized TF (NR4A2, TOX) potentially controlling differentiation to the dysfunctional state. Moreover, ATAC-Seq analysis of human tumor-infiltrating CD8 T cells revealed similar tumor-associated changes in peak accessibility, and studies are ongoing to assess the associated TF networks. In this study, we have defined discrete chromatin states and associated transcriptional networks underlying plastic and fixed dysfunction in TST, thus providing new insights into the genomic control circuitry of T cell differentiation/dysfunction that may point to new strategies for cellular reprogramming of T cells for cancer immunotherapy. Disclosures No relevant conflicts of interest to declare.

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