ABSTRACT
Recent studies have elucidated cell-lineage-specific three-dimensional genome organization; however, how such specific architecture is established or maintained is unclear. We hypothesized that lineage-defining transcription factors maintain cell identity via global control of genome organization. These factors bind many genomic sites outside of the genes that they directly regulate and thus are potentially implicated in three-dimensional genome organization. Using chromosome-conformation-capture techniques, we show that the transcription factor Paired box 5 (Pax5) is critical for the establishment and maintenance of the global lineage-specific architecture of B cells. Pax5 was found to supervise genome architecture throughout B cell differentiation, until the plasmablast stage, in which Pax5 is naturally silenced and B cell-specific genome structure is lost. Crucially, Pax5 did not rely on ongoing transcription to organize the genome. These results implicate sequence-specific DNA-binding proteins in global genome organization to establish and maintain lineage fidelity.
Subject(s)
B-Lymphocytes/cytology , Cell Differentiation/genetics , Cell Lineage/genetics , PAX5 Transcription Factor/genetics , Animals , B-Lymphocytes/metabolism , Male , Mice , Mice, Inbred C57BL , PAX5 Transcription Factor/metabolismABSTRACT
To separate causal effects of histone acetylation on chromatin accessibility and transcriptional output, we used integrated epigenomic and transcriptomic analyses following acute inhibition of major cellular lysine acetyltransferases P300 and CBP in hematological malignancies. We found that catalytic P300/CBP inhibition dynamically perturbs steady-state acetylation kinetics and suppresses oncogenic transcriptional networks in the absence of changes to chromatin accessibility. CRISPR-Cas9 screening identified NCOR1 and HDAC3 transcriptional co-repressors as the principal antagonists of P300/CBP by counteracting acetylation turnover kinetics. Finally, deacetylation of H3K27 provides nucleation sites for reciprocal methylation switching, a feature that can be exploited therapeutically by concomitant KDM6A and P300/CBP inhibition. Overall, this study indicates that the steady-state histone acetylation-methylation equilibrium functions as a molecular rheostat governing cellular transcription that is amenable to therapeutic exploitation as an anti-cancer regimen.
Subject(s)
Biocatalysis , Histones/metabolism , Oncogenes , Transcription, Genetic , p300-CBP Transcription Factors/metabolism , Acetylation , Cell Line , Chromatin/metabolism , Co-Repressor Proteins/metabolism , Conserved Sequence , Evolution, Molecular , Gene Regulatory Networks , Genome , Histone Deacetylases/metabolism , Humans , Kinetics , Methylation , Models, Biological , RNA Polymerase II/metabolismABSTRACT
H3K9me3-dependent heterochromatin is critical for the silencing of repeat-rich pericentromeric regions and also has key roles in repressing lineage-inappropriate protein-coding genes in differentiation and development. Here, we investigate the molecular consequences of heterochromatin loss in cells deficient in both SUV39H1 and SUV39H2 (Suv39DKO), the major mammalian histone methyltransferase enzymes that catalyze heterochromatic H3K9me3 deposition. We reveal a paradoxical repression of protein-coding genes in Suv39DKO cells, with these differentially expressed genes principally in euchromatic (Tn5-accessible, H3K4me3- and H3K27ac-marked) rather than heterochromatic (H3K9me3-marked) or polycomb (H3K27me3-marked) regions. Examination of the three-dimensional (3D) nucleome reveals that transcriptomic dysregulation occurs in euchromatic regions close to the nuclear periphery in 3D space. Moreover, this transcriptomic dysregulation is highly correlated with altered 3D genome organization in Suv39DKO cells. Together, our results suggest that the nuclear lamina-tethering of Suv39-dependent H3K9me3 domains provides an essential scaffold to support euchromatic genome organization and the maintenance of gene transcription for healthy cellular function.
Subject(s)
Euchromatin , Heterochromatin , Histone-Lysine N-Methyltransferase , Histones , Methyltransferases , Transcription, Genetic , Animals , Mice , Cell Line , Euchromatin/metabolism , Euchromatin/genetics , Gene Expression Regulation , Heterochromatin/metabolism , Heterochromatin/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/genetics , Histones/metabolism , Histones/genetics , Methyltransferases/metabolism , Methyltransferases/genetics , Repressor Proteins/metabolism , Repressor Proteins/geneticsABSTRACT
The transcription factor Myc is critically important in driving cell proliferation, a function that is frequently dysregulated in cancer. To avoid this dysregulation Myc is tightly controlled by numerous layers of regulation. One such layer is the use of distal regulatory enhancers to drive Myc expression. Here, using chromosome conformation capture to examine B cells of the immune system in the first hours after their activation, we reveal a previously unidentified enhancer of Myc. The interactivity of this enhancer coincides with a dramatic, but discrete, spike in Myc expression 3 h post-activation. However, genetic deletion of this region, has little impact on Myc expression, Myc protein level or in vitro and in vivo cell proliferation. Examination of the enhancer deleted regulatory landscape suggests that enhancer redundancy likely sustains Myc expression. This work highlights not only the importance of temporally examining enhancers, but also the complexity and dynamics of the regulation of critical genes such as Myc.
Subject(s)
Enhancer Elements, Genetic , Genes, myc , Enhancer Elements, Genetic/genetics , Transcription Factors/metabolism , Gene Expression Regulation , Promoter Regions, GeneticABSTRACT
Loss of heterochromatin has been proposed as a universal mechanism of aging across different species and cell types. However, a comprehensive analysis of hematopoietic changes caused by heterochromatin loss is lacking. Moreover, there is conflict in the literature around the role of the major heterochromatic histone methyltransferase Suv39h1 in the aging process. Here, we use individual and dual deletion of Suv39h1 and Suv39h2 enzymes to examine the causal role of heterochromatin loss in hematopoietic cell development. Loss of neither Suv39h1 nor Suv39h2 individually had any effect on hematopoietic stem cell function or the development of mature lymphoid or myeloid lineages. However, deletion of both enzymes resulted in characteristic changes associated with aging such as reduced hematopoietic stem cell function, thymic involution and decreased lymphoid output with a skewing toward myeloid development, and increased memory T cells at the expense of naive T cells. These cellular changes were accompanied by molecular changes consistent with aging, including alterations in nuclear shape and increased nucleolar size. Together, our results indicate that the hematopoietic system has a remarkable tolerance for major disruptions in chromatin structure and reveal a role for Suv39h2 in depositing sufficient H3K9me3 to protect the entire hematopoietic system from changes associated with premature aging.
Subject(s)
Aging, Premature/pathology , Cell Differentiation , Hematopoiesis , Hematopoietic Stem Cells/pathology , Heterochromatin/metabolism , Histone-Lysine N-Methyltransferase/physiology , Methyltransferases/physiology , Repressor Proteins/physiology , Aged , Aging, Premature/metabolism , Animals , Cell Nucleus/genetics , Female , Hematopoietic Stem Cells/metabolism , Heterochromatin/genetics , Humans , Male , Mice , Mice, Knockout , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , T-Lymphocytes/pathologyABSTRACT
The eukaryotic genome is three-dimensionally segregated into discrete globules of topologically associating domains (TADs), within which numerous cis-regulatory elements such as enhancers and promoters interact to regulate gene expression. In this study, we identify a T-cell-specific sub-TAD containing the Gata3 locus, and reveal a previously uncharacterized long noncoding RNA (Dreg1) within a distant enhancer lying approximately 280 kb downstream of Gata3. Dreg1 expression is highly correlated with that of Gata3 during early immune system development and T helper type 2 cell differentiation. Inhibition and overexpression of Dreg1 suggest that it may be involved in the establishment, but not in the maintenance of Gata3 expression. Overall, we propose that Dreg1 is a novel regulator of Gata3 and may inform therapeutic strategies in diseases such allergy and lymphoma, where Gata3 has a pathological role.
Subject(s)
RNA, Long Noncoding , Chromatin , Enhancer Elements, Genetic/genetics , GATA3 Transcription Factor/genetics , GATA3 Transcription Factor/metabolism , Promoter Regions, Genetic , RNA, Long Noncoding/geneticsABSTRACT
Skin-derived dendritic cells (DCs) include Langerhans cells, classical dermal DCs and a langerin-positive CD103(+) dermal subset. We examined their involvement in the presentation of skin-associated viral and self antigens. Only the CD103(+) subset efficiently presented antigens of herpes simplex virus type 1 to naive CD8(+) T cells, although all subsets presented these antigens to CD4(+) T cells. This showed that CD103(+) DCs were the migratory subset most efficient at processing viral antigens into the major histocompatibility complex class I pathway, potentially through cross-presentation. This was supported by data showing only CD103(+) DCs efficiently cross-presented skin-derived self antigens. This indicates CD103(+) DCs are the main migratory subtype able to cross-present viral and self antigens, which identifies another level of specialization for skin DCs.
Subject(s)
Antigens, Viral/immunology , Autoantigens/immunology , Cross-Priming/immunology , Dendritic Cells/immunology , Lymphocyte Activation/immunology , Skin/immunology , Animals , Antigen Presentation/immunology , Antigens, CD/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Movement/immunology , Flow Cytometry , Fluorescent Antibody Technique , Herpes Simplex/immunology , Herpesvirus 1, Human/immunology , Histocompatibility Antigens Class I/immunology , Integrin alpha Chains/immunology , Mice , Mice, Transgenic , Skin/cytology , Skin/virologyABSTRACT
It has been proposed that interactions between mammalian chromosomes, or transchromosomal interactions (also known as kissing chromosomes), regulate gene expression and cell fate determination. Here we aimed to identify novel transchromosomal interactions in immune cells by high-resolution genome-wide chromosome conformation capture. Although we readily identified stable interactions in cis, and also between centromeres and telomeres on different chromosomes, surprisingly we identified no gene regulatory transchromosomal interactions in either mouse or human cells, including previously described interactions. We suggest that advances in the chromosome conformation capture technique and the unbiased nature of this approach allow more reliable capture of interactions between chromosomes than previous methods. Overall our findings suggest that stable transchromosomal interactions that regulate gene expression are not present in mammalian immune cells and that lineage identity is governed by cis, not trans chromosomal interactions.
Subject(s)
Chromosomes, Mammalian/genetics , Gene Expression Regulation , Immunity, Cellular/genetics , Mammals/physiology , Animals , Chromatin/chemistry , Chromatin/genetics , Chromatin/metabolism , Chromosomes, Mammalian/chemistry , Chromosomes, Mammalian/metabolism , DNA/chemistry , DNA/genetics , DNA/metabolism , Flow Cytometry , Genome , Humans , Male , Mice , Mice, Inbred C57BL , Nucleic Acid Conformation , StereoisomerismABSTRACT
The polycomb repressive complex 2 (PRC2) consists of three core components EZH2, SUZ12 and EED. EZH2 catalyzes the methylation of lysine 27 of histone H3, a modification associated with gene silencing. Through gene duplication higher vertebrate genomes also encode a second partially redundant methyltransferase, EZH1. Within the mammalian immune system most research has concentrated on EZH2 which is expressed predominantly in proliferating cells. EZH2 and other PRC2 components are required for hematopoietic stem cell function and lymphocyte development, at least in part by repressing cell cycle inhibitors. At later stages of immune cell differentiation, EZH2 plays essential roles in humoral and cell-mediated adaptive immunity, as well as the maintenance of immune homeostasis. EZH2 is often overactive in cancers, through both gain-of-function mutations and over-expression, an observation that has led to the development and clinical testing of specific EZH2 inhibitors. Such inhibitors may also be of use in inflammatory and autoimmune settings, as EZH2 inhibition dampens the immune response. Here, we will review the current state of understanding of the roles for EZH2, and PRC2 more generally, in the development and function of the immune system.
Subject(s)
Enhancer of Zeste Homolog 2 Protein/immunology , Cell Differentiation , HumansABSTRACT
The development of B lymphocytes into antibody-secreting plasma cells is central to the adaptive immune system in that it confers protective and specific antibody response against invading pathogen. This developmental process involves extensive morphological and functional alterations that begin early after antigenic stimulation. These include chromatin restructuring that is critical in regulating gene expression, DNA rearrangement and other cellular processes. Here we outline the recent understanding of the three-dimensional architecture of the genome, specifically focused on its contribution to the process of B cell activation and terminal differentiation into antibody-secreting cells.
Subject(s)
Antibodies/metabolism , B-Lymphocytes/metabolism , Genome , Plasma Cells/metabolism , Adaptive Immunity , Animals , Antibody Formation , Antibody-Producing Cells/cytology , Cell Differentiation , Cell Division , DNA/metabolism , Gene Expression Regulation , Humans , Lymphocyte Activation , Recombination, Genetic , Transcription, GeneticABSTRACT
Enhancer of zeste 2 (Ezh2) mainly methylates lysine 27 of histone-H3 (H3K27me3) as part of the polycomb repressive complex 2 (PRC2) together with Suz12 and Eed. However, Ezh2 can also modify non-histone substrates, although it is unclear whether this mechanism has a role during development. Here, we present evidence for a chromatin-independent role of Ezh2 during T-cell development and immune homeostasis. T-cell-specific depletion of Ezh2 induces a pronounced expansion of natural killer T (NKT) cells, although Ezh2-deficient T cells maintain normal levels of H3K27me3. In contrast, removal of Suz12 or Eed destabilizes canonical PRC2 function and ablates NKT cell development completely. We further show that Ezh2 directly methylates the NKT cell lineage defining transcription factor PLZF, leading to its ubiquitination and subsequent degradation. Sustained PLZF expression in Ezh2-deficient mice is associated with the expansion of a subset of NKT cells that cause immune perturbation. Taken together, we have identified a chromatin-independent function of Ezh2 that impacts on the development of the immune system.
Subject(s)
Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/metabolism , Homeostasis , Immunity/genetics , Animals , Cell Differentiation , Cell Line , Gene Expression , Humans , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Methylation , Mice , Mice, Knockout , Natural Killer T-Cells/immunology , Natural Killer T-Cells/metabolism , Promyelocytic Leukemia Zinc Finger Protein , Protein Binding , Proteolysis , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Thymocytes/cytology , Thymocytes/immunology , Thymocytes/metabolismABSTRACT
During immune responses, naive CD4+ T cells differentiate into several T helper (TH) cell subsets under the control of lineage-specifying genes. These subsets (TH1, TH2 and TH17 cells and regulatory T cells) secrete distinct cytokines and are involved in protection against different types of infection. Epigenetic mechanisms are involved in the regulation of these developmental programs, and correlations have been drawn between the levels of particular epigenetic marks and the activity or silencing of specifying genes during differentiation. Nevertheless, the functional relevance of the epigenetic pathways involved in TH cell subset differentiation and commitment is still unclear. Here we explore the role of the SUV39H1H3K9me3HP1α silencing pathway in the control of TH2 lineage stability. This pathway involves the histone methylase SUV39H1, which participates in the trimethylation of histone H3 on lysine 9 (H3K9me3), a modification that provides binding sites for heterochromatin protein 1α (HP1α) and promotes transcriptional silencing. This pathway was initially associated with heterochromatin formation and maintenance but can also contribute to the regulation of euchromatic genes. We now propose that the SUV39H1H3K9me3HP1α pathway participates in maintaining the silencing of TH1 loci, ensuring TH2 lineage stability. In TH2 cells that are deficient in SUV39H1, the ratio between trimethylated and acetylated H3K9 is impaired, and the binding of HP1α at the promoters of silenced TH1 genes is reduced. Despite showing normal differentiation, both SUV39H1-deficient TH2 cells and HP1α-deficient TH2 cells, in contrast to wild-type cells, expressed TH1 genes when recultured under conditions that drive differentiation into TH1 cells. In a mouse model of TH2-driven allergic asthma, the chemical inhibition or loss of SUV39H1 skewed T-cell responses towards TH1 responses and decreased the lung pathology. These results establish a link between the SUV39H1H3K9me3HP1α pathway and the stability of TH2 cells, and they identify potential targets for therapeutic intervention in TH2-cell-mediated inflammatory diseases.
Subject(s)
Epigenesis, Genetic , Th2 Cells/cytology , Th2 Cells/immunology , Animals , Asthma/enzymology , Asthma/immunology , Asthma/pathology , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Lineage/genetics , Cell Lineage/immunology , Chromobox Protein Homolog 5 , Chromosomal Proteins, Non-Histone/metabolism , Disease Models, Animal , Female , Gene Expression Regulation , Gene Silencing , Histones/metabolism , Male , Methyltransferases/deficiency , Methyltransferases/metabolism , Mice , Mice, Inbred C57BL , Promoter Regions, Genetic , Repressor Proteins/deficiency , Repressor Proteins/metabolism , Th1 Cells/metabolism , Th2 Cells/enzymologyABSTRACT
The multiple lineages and differentiation states that constitute the T-cell compartment all derive from a common thymic precursor. These distinct transcriptional states are maintained both in time and after multiple rounds of cell division by the concerted actions of a small set of lineage-defining transcription factors that act in conjunction with a suite of chromatin-modifying enzymes to activate, repress, and fine-tune gene expression. These chromatin modifications collectively provide an epigenetic code that allows the stable and heritable maintenance of the T-cell phenotype. Recently, it has become apparent that the epigenetic code represents a therapeutic target for a variety of immune cell disorders, including lymphoma and acute and chronic inflammatory diseases. Here, we review the recent advances in epigenetic regulation of gene expression, particularly as it relates to the T-cell differentiation and function.
Subject(s)
Epigenesis, Genetic , Histone Code/physiology , Immune System Diseases/immunology , Lymphoma/immunology , T-Lymphocytes/immunology , Animals , Biological Therapy/trends , Cell Differentiation , Cell Lineage , Chromatin Assembly and Disassembly/immunology , Epigenesis, Genetic/genetics , Epigenesis, Genetic/immunology , Gene Expression Regulation/immunology , Humans , Immune System Diseases/therapy , Lymphoma/therapyABSTRACT
Deregulation of polycomb group complexes polycomb repressive complex 1 (PRC1) and 2 (PRC2) is associated with human cancers. Although inactivating mutations in PRC2-encoding genes EZH2, EED, and SUZ12 are present in T-cell acute lymphoblastic leukemia and in myeloid malignancies, gain-of-function mutations in EZH2 are frequently observed in B-cell lymphoma, implying disease-dependent effects of individual mutations. We show that, in contrast to PRC1, PRC2 is a tumor suppressor in Eµ-myc lymphomagenesis, because disease onset was accelerated by heterozygosity for Suz12 or by short hairpin RNA-mediated knockdown of Suz12 or Ezh2. Accelerated lymphomagenesis was associated with increased accumulation of B-lymphoid cells in the absence of effects on apoptosis or cell cycling. However, Suz12-deficient B-lymphoid progenitors exhibit enhanced serial clonogenicity. Thus, PRC2 normally restricts the self-renewal of B-lymphoid progenitors, the disruption of which contributes to lymphomagenesis. This finding provides new insight regarding the functional contribution of mutations in PRC2 in a range of leukemias.
Subject(s)
B-Lymphocytes/physiology , Lymphoma, B-Cell/genetics , Polycomb Repressive Complex 2/genetics , Proto-Oncogene Proteins c-myc/genetics , Animals , B-Lymphocytes/cytology , Cells, Cultured , Enhancer of Zeste Homolog 2 Protein , Gene Expression Regulation, Neoplastic/physiology , Lymphoma, B-Cell/metabolism , Lymphoma, B-Cell/pathology , Lymphopoiesis/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Polycomb Repressive Complex 1/genetics , Polycomb Repressive Complex 1/metabolism , Polycomb Repressive Complex 2/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Proto-Oncogene Proteins c-myc/metabolismABSTRACT
Variants in the poorly characterised oncoprotein, MORC2, a chromatin remodelling ATPase, lead to defects in epigenetic regulation and DNA damage response. The C-terminal domain (CTD) of MORC2, frequently phosphorylated in DNA damage, promotes cancer progression, but its role in chromatin remodelling remains unclear. Here, we report a molecular characterisation of full-length, phosphorylated MORC2, demonstrating its preference for binding open chromatin and functioning as a DNA sliding clamp. We identified a phosphate interacting motif within the CTD that dictates ATP hydrolysis rate and cooperative DNA binding. The DNA binding impacts several structural domains within the ATPase region. We provide the first visual proof that MORC2 induces chromatin remodelling through ATP hydrolysis-dependent DNA compaction, regulated by its phosphorylation state. These findings highlight phosphorylation of MORC2 CTD as a key modulator of chromatin remodelling, presenting it as a potential therapeutic target.
ABSTRACT
Although lineage-specific genes have been identified in the mammary gland, little is known about the contribution of the 3D genome organization to gene regulation in the epithelium. Here, we describe the chromatin landscape of the three major epithelial subsets through integration of long- and short-range chromatin interactions, accessibility, histone modifications, and gene expression. While basal genes display exquisite lineage specificity via distal enhancers, luminal-specific genes show widespread promoter priming in basal cells. Cell specificity in luminal progenitors is largely mediated through extensive chromatin interactions with super-enhancers in gene-body regions in addition to interactions with polycomb silencer elements. Moreover, lineage-specific transcription factors appear to be controlled through cell-specific chromatin interactivity. Finally, chromatin accessibility rather than interactivity emerged as a defining feature of the activation of quiescent basal stem cells. This work provides a comprehensive resource for understanding the role of higher-order chromatin interactions in cell-fate specification and differentiation in the adult mouse mammary gland.
ABSTRACT
In situ HiC uses the relative frequency of DNA-DNA ligation events to reconstruct the three-dimensional architecture of a genome. As such, restriction enzyme digested ends of genomic DNA within fixed nuclei are tagged with biotinylated dNTPs. DNA-DNA ligation events generated via proximity ligation are then captured, amplified and next generation sequenced to determine their linear genomic position, but also their three-dimensional relationship. Here, we describe these steps in detail.
Subject(s)
DNA , Genome , Cell Nucleus , Chromatin , DNA/genetics , GenomicsABSTRACT
Stably silenced genes that display a high level of CpG dinucleotide methylation are refractory to the current generation of dCas9-based activation systems. To counter this, we create an improved activation system by coupling the catalytic domain of DNA demethylating enzyme TET1 with transcriptional activators (TETact). We show that TETact demethylation-coupled activation is able to induce transcription of suppressed genes, both individually and simultaneously in cells, and has utility across a number of cell types. Furthermore, we show that TETact can effectively reactivate embryonic haemoglobin genes in non-erythroid cells. We anticipate that TETact will expand the existing CRISPR toolbox and be valuable for functional studies, genetic screens and potential therapeutics.