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Nature Jul 2017Constitutive heterochromatin is an important component of eukaryotic genomes that has essential roles in nuclear architecture, DNA repair and genome stability, and...
Constitutive heterochromatin is an important component of eukaryotic genomes that has essential roles in nuclear architecture, DNA repair and genome stability, and silencing of transposon and gene expression. Heterochromatin is highly enriched for repetitive sequences, and is defined epigenetically by methylation of histone H3 at lysine 9 and recruitment of its binding partner heterochromatin protein 1 (HP1). A prevalent view of heterochromatic silencing is that these and associated factors lead to chromatin compaction, resulting in steric exclusion of regulatory proteins such as RNA polymerase from the underlying DNA. However, compaction alone does not account for the formation of distinct, multi-chromosomal, membrane-less heterochromatin domains within the nucleus, fast diffusion of proteins inside the domain, and other dynamic features of heterochromatin. Here we present data that support an alternative hypothesis: that the formation of heterochromatin domains is mediated by phase separation, a phenomenon that gives rise to diverse non-membrane-bound nuclear, cytoplasmic and extracellular compartments. We show that Drosophila HP1a protein undergoes liquid-liquid demixing in vitro, and nucleates into foci that display liquid properties during the first stages of heterochromatin domain formation in early Drosophila embryos. Furthermore, in both Drosophila and mammalian cells, heterochromatin domains exhibit dynamics that are characteristic of liquid phase-separation, including sensitivity to the disruption of weak hydrophobic interactions, and reduced diffusion, increased coordinated movement and inert probe exclusion at the domain boundary. We conclude that heterochromatic domains form via phase separation, and mature into a structure that includes liquid and stable compartments. We propose that emergent biophysical properties associated with phase-separated systems are critical to understanding the unusual behaviours of heterochromatin, and how chromatin domains in general regulate essential nuclear functions.
Topics: Animals; Cell Line; Chromobox Protein Homolog 5; Chromosomal Proteins, Non-Histone; DNA; Diffusion; Drosophila melanogaster; Female; Gene Silencing; Heterochromatin; Hydrophobic and Hydrophilic Interactions; Mice; NIH 3T3 Cells; Phase Transition; Solubility
PubMed: 28636597
DOI: 10.1038/nature22989 -
Cell May 2020Tissue homeostasis requires maintenance of functional integrity under stress. A central source of stress is mechanical force that acts on cells, their nuclei, and...
Tissue homeostasis requires maintenance of functional integrity under stress. A central source of stress is mechanical force that acts on cells, their nuclei, and chromatin, but how the genome is protected against mechanical stress is unclear. We show that mechanical stretch deforms the nucleus, which cells initially counteract via a calcium-dependent nuclear softening driven by loss of H3K9me3-marked heterochromatin. The resulting changes in chromatin rheology and architecture are required to insulate genetic material from mechanical force. Failure to mount this nuclear mechanoresponse results in DNA damage. Persistent, high-amplitude stretch induces supracellular alignment of tissue to redistribute mechanical energy before it reaches the nucleus. This tissue-scale mechanoadaptation functions through a separate pathway mediated by cell-cell contacts and allows cells/tissues to switch off nuclear mechanotransduction to restore initial chromatin state. Our work identifies an unconventional role of chromatin in altering its own mechanical state to maintain genome integrity in response to deformation.
Topics: Animals; Cell Line; Cell Nucleus; Chromatin; Heterochromatin; Humans; Male; Mechanoreceptors; Mechanotransduction, Cellular; Mesenchymal Stem Cells; Mice; Stress, Mechanical
PubMed: 32302590
DOI: 10.1016/j.cell.2020.03.052 -
Molecular Cell Feb 2022Gene silencing by heterochromatin plays a crucial role in cell identity. Here, we characterize the localization, the biogenesis, and the function of an atypical...
Gene silencing by heterochromatin plays a crucial role in cell identity. Here, we characterize the localization, the biogenesis, and the function of an atypical heterochromatin, which is simultaneously enriched in the typical H3K9me3 mark and in H3K36me3, a histone mark usually associated with gene expression. We identified thousands of dual regions in mouse embryonic stem (ES) cells that rely on the histone methyltransferases SET domain bifurcated 1 (SETDB1) and nuclear set domain (NSD)-containing proteins to generate H3K9me3 and H3K36me3, respectively. Upon SETDB1 removal, dual domains lose both marks, gain signatures of active enhancers, and come into contact with upregulated genes, suggesting that it might be an important pathway by which genes are controlled by heterochromatin. In differentiated tissues, a subset of these dual domains is destabilized and becomes enriched in active enhancer marks, providing a mechanistic insight into the involvement of heterochromatin in the maintenance of cell identity.
Topics: Animals; Cell Line; Chromatin Assembly and Disassembly; Chromatin Immunoprecipitation Sequencing; DNA Methylation; Enhancer Elements, Genetic; Gene Expression Profiling; Gene Expression Regulation, Developmental; Heterochromatin; Histone-Lysine N-Methyltransferase; Histones; Methylation; Mice; Mouse Embryonic Stem Cells; Protein Processing, Post-Translational; RNA-Seq; Transcriptome
PubMed: 35081363
DOI: 10.1016/j.molcel.2021.12.037 -
ELife Mar 2016Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show...
Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression.
Topics: Animals; Cell Proliferation; Gene Expression; Gene Knockdown Techniques; Heterochromatin; Humans; Ki-67 Antigen; Mice; Xenopus
PubMed: 26949251
DOI: 10.7554/eLife.13722 -
Annals of the New York Academy of... Sep 2020The eukaryotic genome is packaged into transcriptionally active euchromatin and silent heterochromatin, with most studies focused on the former encompassing the majority... (Review)
Review
The eukaryotic genome is packaged into transcriptionally active euchromatin and silent heterochromatin, with most studies focused on the former encompassing the majority of protein-coding genes. The recent development of various sequencing techniques has refined this classic dichromatic partition and has better illuminated the composition, establishment, and evolution of this genomic and epigenomic "dark matter" in the context of topologically associated domains and phase-separated droplets. Heterochromatin includes genomic regions that can be densely stained by chemical dyes, which have been shown to be enriched for repetitive elements and epigenetic marks, including H3K9me2/3 and H3K27me3. Heterochromatin is usually replicated late, concentrated at the nuclear periphery or around nucleoli, and usually lacks highly expressed genes; and now it is considered to be as neither genetically inert nor developmentally static. Heterochromatin guards genome integrity against transposon activities and exerts important regulatory functions by targeting beyond its contained genes. Both its nucleotide sequences and regulatory proteins exhibit rapid coevolution between species. In addition, there are dynamic transitions between euchromatin and heterochromatin during developmental and evolutionary processes. We summarize here the ever-changing characteristics of heterochromatin and propose models and principles for the evolutionary transitions of heterochromatin that have been mainly learned from studies of Drosophila and yeast. Finally, we highlight the role of sex chromosomes in studying heterochromatin evolution.
Topics: Animals; Drosophila Proteins; Evolution, Molecular; Fungal Proteins; Heterochromatin; Humans; Sex Chromosomes
PubMed: 32017156
DOI: 10.1111/nyas.14303 -
Nature Dec 2020The zebrafish (Danio rerio) has been widely used in the study of human disease and development, and about 70% of the protein-coding genes are conserved between the two...
The zebrafish (Danio rerio) has been widely used in the study of human disease and development, and about 70% of the protein-coding genes are conserved between the two species. However, studies in zebrafish remain constrained by the sparse annotation of functional control elements in the zebrafish genome. Here we performed RNA sequencing, assay for transposase-accessible chromatin using sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing, whole-genome bisulfite sequencing, and chromosome conformation capture (Hi-C) experiments in up to eleven adult and two embryonic tissues to generate a comprehensive map of transcriptomes, cis-regulatory elements, heterochromatin, methylomes and 3D genome organization in the zebrafish Tübingen reference strain. A comparison of zebrafish, human and mouse regulatory elements enabled the identification of both evolutionarily conserved and species-specific regulatory sequences and networks. We observed enrichment of evolutionary breakpoints at topologically associating domain boundaries, which were correlated with strong histone H3 lysine 4 trimethylation (H3K4me3) and CCCTC-binding factor (CTCF) signals. We performed single-cell ATAC-seq in zebrafish brain, which delineated 25 different clusters of cell types. By combining long-read DNA sequencing and Hi-C, we assembled the sex-determining chromosome 4 de novo. Overall, our work provides an additional epigenomic anchor for the functional annotation of vertebrate genomes and the study of evolutionarily conserved elements of 3D genome organization.
Topics: Animals; Brain; Conserved Sequence; DNA Methylation; Enhancer Elements, Genetic; Epigenesis, Genetic; Evolution, Molecular; Female; Gene Expression Profiling; Gene Regulatory Networks; Genome; Heterochromatin; Humans; Imaging, Three-Dimensional; Male; Mice; Molecular Imaging; Organ Specificity; Promoter Regions, Genetic; Regulatory Sequences, Nucleic Acid; Single-Cell Analysis; Species Specificity; Zebrafish
PubMed: 33239788
DOI: 10.1038/s41586-020-2962-9 -
Nucleic Acids Research Jun 2023Cellular totipotency is critical for whole-organism generation, yet how totipotency is established remains poorly illustrated. Abundant transposable elements (TEs) are...
Cellular totipotency is critical for whole-organism generation, yet how totipotency is established remains poorly illustrated. Abundant transposable elements (TEs) are activated in totipotent cells, which is critical for embryonic totipotency. Here, we show that the histone chaperone RBBP4, but not its homolog RBBP7, is indispensable for maintaining the identity of mouse embryonic stem cells (mESCs). Auxin-induced degradation of RBBP4, but not RBBP7, reprograms mESCs to the totipotent 2C-like cells. Also, loss of RBBP4 enhances transition from mESCs to trophoblast cells. Mechanistically, RBBP4 binds to the endogenous retroviruses (ERVs) and functions as an upstream regulator by recruiting G9a to deposit H3K9me2 on ERVL elements, and recruiting KAP1 to deposit H3K9me3 on ERV1/ERVK elements, respectively. Moreover, RBBP4 facilitates the maintenance of nucleosome occupancy at the ERVK and ERVL sites within heterochromatin regions through the chromatin remodeler CHD4. RBBP4 depletion leads to the loss of the heterochromatin marks and activation of TEs and 2C genes. Together, our findings illustrate that RBBP4 is required for heterochromatin assembly and is a critical barrier for inducing cell fate transition from pluripotency to totipotency.
Topics: Animals; Mice; Heterochromatin; Transcription Factors; Chromatin; Pluripotent Stem Cells; Mouse Embryonic Stem Cells; Epigenesis, Genetic
PubMed: 37021556
DOI: 10.1093/nar/gkad219 -
Nucleus (Austin, Tex.) Dec 2024Heterochromatin is an organizational property of eukaryotic chromosomes, characterized by extensive DNA and histone modifications, that is associated with the silencing... (Review)
Review
Heterochromatin is an organizational property of eukaryotic chromosomes, characterized by extensive DNA and histone modifications, that is associated with the silencing of transposable elements and repetitive sequences. Maintaining heterochromatin is crucial for ensuring genomic integrity and stability during the cell cycle. During meiosis, heterochromatin is important for homologous chromosome synapsis, recombination, and segregation, but our understanding of meiotic heterochromatin formation and condensation is limited. In this review, we focus on the dynamics and features of heterochromatin and how it condenses during meiosis in plants. We also discuss how meiotic heterochromatin influences the interaction and recombination of homologous chromosomes during prophase I.
Topics: Heterochromatin; Centromere; Meiosis; Chromosome Pairing
PubMed: 38488152
DOI: 10.1080/19491034.2024.2328719 -
Current Opinion in Structural Biology Jun 2023Heterochromatin formation has been proposed to involve phase transitions on the level of the three-dimensional folding of heterochromatin regions and the liquid-liquid... (Review)
Review
Heterochromatin formation has been proposed to involve phase transitions on the level of the three-dimensional folding of heterochromatin regions and the liquid-liquid demixing of heterochromatin proteins. Here, I outline the hallmarks of such transitions and the current challenges to detect them in living cells. I further discuss the abundance and properties of prominent heterochromatin proteins and relate them to their potential role in driving phase transitions. Recent data from mouse fibroblasts indicate that pericentric heterochromatin is organized via a reordering transition on the level of heterochromatin regions that does not necessarily involve liquid-liquid demixing of heterochromatin proteins. Evaluating key hallmarks of the different candidate phase transition mechanisms across cell types and species will be needed to complete the current picture.
Topics: Animals; Mice; Heterochromatin; Chromatin Assembly and Disassembly
PubMed: 37087823
DOI: 10.1016/j.sbi.2023.102597 -
Current Opinion in Cell Biology Jun 2017The formation of condensed, transcriptionally repressed heterochromatin is essential for controlling gene expression throughout development, silencing parasitic DNA... (Review)
Review
The formation of condensed, transcriptionally repressed heterochromatin is essential for controlling gene expression throughout development, silencing parasitic DNA elements, and for genome stability and inheritance. Cells employ diverse mechanisms for controlling heterochromatin states through proteins that modify DNA and histones. An emerging theme is that chromatin-associated RNAs play important roles in regulating heterochromatin proteins by controlling their initial recruitment to chromatin, their stable association with chromatin, their spread along chromatin, or their enzymatic activity. Major challenges for the field include not only identifying regulatory RNAs, but understanding the underlying biochemical mechanisms for how RNAs associate with chromatin, the specificity of interactions between heterochromatin proteins and RNA, and how these binding events manifest in cells to orchestrate RNA-mediated regulation of heterochromatin.
Topics: Animals; Chromatin Assembly and Disassembly; Chromosomal Proteins, Non-Histone; Gene Expression Regulation; Gene Silencing; Genomic Instability; Heterochromatin; Histones; Humans; RNA, Untranslated
PubMed: 28614747
DOI: 10.1016/j.ceb.2017.05.004