ABSTRACT
Enhancers bind transcription factors, chromatin regulators, and non-coding transcripts to modulate the expression of target genes. Here, we report 3D genome structures of single mouse ES cells as they are induced to exit pluripotency and transition through a formative stage prior to undergoing neuroectodermal differentiation. We find that there is a remarkable reorganization of 3D genome structure where inter-chromosomal intermingling increases dramatically in the formative state. This intermingling is associated with the formation of a large number of multiway hubs that bring together enhancers and promoters with similar chromatin states from typically 5-8 distant chromosomal sites that are often separated by many Mb from each other. In the formative state, genes important for pluripotency exit establish contacts with emerging enhancers within these multiway hubs, suggesting that the structural changes we have observed may play an important role in modulating transcription and establishing new cell identities.
Subject(s)
Mouse Embryonic Stem Cells , Regulatory Sequences, Nucleic Acid , Mice , Animals , Mouse Embryonic Stem Cells/metabolism , Embryonic Stem Cells/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Chromatin/genetics , Chromatin/metabolism , Enhancer Elements, GeneticABSTRACT
The Sin3a/HDAC co-repressor complex is a critical regulator of transcription networks that govern cell cycle control and apoptosis throughout development. Previous studies have identified Sin3a as essential for embryonic development around the time of implantation, during which the epiblast cell cycle is uniquely structured to achieve very rapid divisions with little tolerance of DNA damage. This study investigates the specific requirement for Sin3a in the early mouse embryo and shows that embryos lacking Sin3a suffer unresolved DNA damage and acute p53-independent apoptosis specifically in the E3.5-4.5 epiblast. Surprisingly, Myc and E2F targets in Sin3a-null ICMs are downregulated, suggesting a central but non-canonical role for Sin3a in regulating the pluripotent embryonic cell cycle. ES cells deleted for Sin3a mount a DNA damage response indicative of unresolved double-strand breaks, profoundly arrest at G2, and undergo apoptosis. These results indicate that Sin3a protects the genomic integrity of pluripotent embryonic cells and governs their unusual cell cycle.
Subject(s)
Embryonic Stem Cells/metabolism , Genomic Instability/genetics , Pluripotent Stem Cells/metabolism , Repressor Proteins/genetics , Animals , Apoptosis/genetics , Blotting, Western , Cell Cycle Checkpoints/genetics , Cell Survival/genetics , Cells, Cultured , DNA Damage , E2F Transcription Factors/genetics , E2F Transcription Factors/metabolism , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Female , Flow Cytometry , G2 Phase/genetics , Gene Expression Regulation, Developmental , Germ Layers/cytology , Germ Layers/embryology , Germ Layers/metabolism , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Repressor Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sin3 Histone Deacetylase and Corepressor ComplexABSTRACT
BACKGROUND: The precise molecular changes that occur when a neural stem (NS) cell switches from a programme of self-renewal to commit towards a specific lineage are not currently well understood. However it is clear that control of gene expression plays an important role in this process. DNA methylation, a mark of transcriptionally silent chromatin, has similarly been shown to play important roles in neural cell fate commitment in vivo. While DNA methylation is known to play important roles in neural specification during embryonic development, no such role has been shown for any of the methyl-CpG binding proteins (Mecps) in mice. METHODOLOGY/PRINCIPAL FINDINGS: To explore the role of DNA methylation in neural cell fate decisions, we have investigated the function of Mecps in mouse development and in neural stem cell derivation, maintenance, and differentiation. In order to test whether the absence of phenotype in singly-mutant animals could be due to functional redundancy between Mecps, we created mice and neural stem cells simultaneously lacking Mecp2, Mbd2 and Zbtb33. No evidence for functional redundancy between these genes in embryonic development or in the derivation or maintenance of neural stem cells in culture was detectable. However evidence for a defect in neuronal commitment of triple knockout NS cells was found. CONCLUSIONS/SIGNIFICANCE: Although DNA methylation is indispensable for mammalian embryonic development, we show that simultaneous deficiency of three methyl-CpG binding proteins genes is compatible with apparently normal mouse embryogenesis. Nevertheless, we provide genetic evidence for redundancy of function between methyl-CpG binding proteins in postnatal mice.
Subject(s)
Cell Differentiation , DNA-Binding Proteins/metabolism , Embryonic Development , Methyl-CpG-Binding Protein 2/metabolism , Neurons/cytology , Transcription Factors/metabolism , Animals , Animals, Newborn , Cell Line , Mice , Mice, Knockout , Neurons/metabolism , Stem Cells/cytology , Stem Cells/metabolismABSTRACT
Immunity often depends on proper cell fate choice by helper T lymphocytes. A naive cell, with minimal expression of IFN-gamma and IL-4, must give rise to progeny expressing high levels of either one, but not both, of those cytokines to defend against protozoan and helminthic pathogens, respectively. In the present study, we show that inactivation of the Mbd2 gene, which links DNA methylation and repressed chromatin, results in enhanced resistance to the protozoan parasite Leishmania major but impaired immunity to the intestinal helminth Trichuris muris. Helper T cells from methyl CpG-binding domain protein-2-deficient mice exhibit exuberant patterns of cytokine expression despite appropriate silencing of genes encoding the lineage-specifying factors T-bet and GATA-3. These results suggest that gene silencing can facilitate the ability of a progenitor cell to give rise to appropriately differentiated daughter cells in vivo. These findings also point to novel pathways that could participate in genetic control of resistance to infection and autoimmunity.
Subject(s)
DNA-Binding Proteins/physiology , Gene Silencing , Genetic Predisposition to Disease , Animals , Autoimmunity , DNA Methylation , DNA-Binding Proteins/genetics , Immunity, Innate , Interferon-gamma/biosynthesis , Leishmania major , Leishmaniasis, Cutaneous/genetics , Leishmaniasis, Cutaneous/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , T-Box Domain Proteins , Transcription Factors/physiology , Trichuriasis/immunologyABSTRACT
How a single cell gives rise to progeny with differing fates remains poorly understood. We examined cells lacking methyl CpG binding domain protein-2 (MBD2), a molecule that has been proposed to link DNA methylation to silent chromatin. Helper T cells from Mbd2(-/-) mice exhibit disordered differentiation. IL-4, the signature of a restricted set of progeny, is expressed ectopically in Mbd2(-/-) parent and daughter cells. Loss of MBD2-mediated silencing renders the normally essential activator, Gata-3, dispensable for IL-4 induction. Gata-3 and MBD2 act in competition, wherein each factor independently, and quantitatively, regulates the binary choice of whether heritable IL-4 expression is established. Gata-3 functions, in part, to displace MBD2 from methylated DNA. These results suggest that activating and silencing signals integrate to provide spatially and temporally restricted patterns of gene activity.
Subject(s)
DNA-Binding Proteins/metabolism , Gene Expression Regulation, Developmental , Gene Silencing , Trans-Activators/metabolism , Animals , Binding, Competitive , Cell Differentiation , Cell Line , Cell Lineage , Chromatin/metabolism , CpG Islands/genetics , DNA Methylation , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Flow Cytometry , GATA3 Transcription Factor , Gene Deletion , Interleukin-4/genetics , Interleukin-4/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , T-Lymphocytes, Helper-Inducer/cytology , T-Lymphocytes, Helper-Inducer/metabolism , Trans-Activators/deficiency , Trans-Activators/geneticsABSTRACT
DNA methylation patterns are dynamic in cleavage-stage embryos of a number of mammalian species. A failure to properly recapitulate preimplantation DNA methylation patterns in embryos derived by nuclear transfer may contribute to the low efficiency of nuclear transfer in producing live offspring.