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Current Opinion in Genetics &... Jun 2024Polycomb-associated chromatin and pericentromeric heterochromatin form genomic domains important for the epigenetic regulation of gene expression. Both Polycomb... (Review)
Review
Polycomb-associated chromatin and pericentromeric heterochromatin form genomic domains important for the epigenetic regulation of gene expression. Both Polycomb complexes and heterochromatin factors rely on 'read and write' mechanisms, which, on their own, are not sufficient to explain the formation and the maintenance of these epigenetic domains. Microscopy has revealed that they form specific nuclear compartments separated from the rest of the genome. Recently, some subunits of these molecular machineries have been shown to undergo phase separation, both in vitro and in vivo, suggesting that phase separation might play important roles in the formation and the function of these two kinds of repressive chromatin. In this review, we will present the recent advances in the field of facultative and constitutive heterochromatin formation and maintenance through phase separation.
Topics: Heterochromatin; Epigenesis, Genetic; Polycomb-Group Proteins; Chromatin; Animals; Humans; Histones; Chromatin Assembly and Disassembly; Phase Separation
PubMed: 38701672
DOI: 10.1016/j.gde.2024.102201 -
Cell Reports May 2024During cell fate transitions, cells remodel their transcriptome, chromatin, and epigenome; however, it has been difficult to determine the temporal dynamics and...
During cell fate transitions, cells remodel their transcriptome, chromatin, and epigenome; however, it has been difficult to determine the temporal dynamics and cause-effect relationship between these changes at the single-cell level. Here, we employ the heterokaryon-mediated reprogramming system as a single-cell model to dissect key temporal events during early stages of pluripotency conversion using super-resolution imaging. We reveal that, following heterokaryon formation, the somatic nucleus undergoes global chromatin decompaction and removal of repressive histone modifications H3K9me3 and H3K27me3 without acquisition of active modifications H3K4me3 and H3K9ac. The pluripotency gene OCT4 (POU5F1) shows nascent and mature RNA transcription within the first 24 h after cell fusion without requiring an initial open chromatin configuration at its locus. NANOG, conversely, has significant nascent RNA transcription only at 48 h after cell fusion but, strikingly, exhibits genomic reopening early on. These findings suggest that the temporal relationship between chromatin compaction and gene activation during cellular reprogramming is gene context dependent.
Topics: Humans; Cellular Reprogramming; Chromatin Assembly and Disassembly; Histones; Single-Cell Analysis; Transcriptional Activation; Octamer Transcription Factor-3; Chromatin; Nanog Homeobox Protein; Induced Pluripotent Stem Cells
PubMed: 38700983
DOI: 10.1016/j.celrep.2024.114170 -
Open Biology May 2024The nucleolus is the most prominent liquid droplet-like membrane-less organelle in mammalian cells. Unlike the nucleolus in terminally differentiated somatic cells,...
The nucleolus is the most prominent liquid droplet-like membrane-less organelle in mammalian cells. Unlike the nucleolus in terminally differentiated somatic cells, those in totipotent cells, such as murine zygotes or two-cell embryos, have a unique nucleolar structure known as nucleolus precursor bodies (NPBs). Previously, it was widely accepted that NPBs in zygotes are simply passive repositories of materials that will be gradually used to construct a fully functional nucleolus after zygotic genome activation (ZGA). However, recent research studies have challenged this simplistic view and demonstrated that functions of the NPBs go beyond ribosome biogenesis. In this review, we provide a snapshot of the functions of NPBs in zygotes and early two-cell embryos in mice. We propose that these membrane-less organelles function as a regulatory hub for chromatin organization. On the one hand, NPBs provide the structural platform for centric and pericentric chromatin remodelling. On the other hand, the dynamic changes in nucleolar structure control the release of the pioneer factors (i.e. double homeobox (Dux)). It appears that during transition from totipotency to pluripotency, decline of totipotency and initiation of fully functional nucleolus formation are not independent events but are interconnected. Consequently, it is reasonable to hypothesize that dissecting more unknown functions of NPBs may shed more light on the enigmas of early embryonic development and may ultimately provide novel approaches to improve reprogramming efficiency.
Topics: Animals; Humans; Mice; Cell Nucleolus; Chromatin; Chromatin Assembly and Disassembly; Embryonic Development; Gene Expression Regulation, Developmental; Zygote
PubMed: 38689555
DOI: 10.1098/rsob.230358 -
Nucleic Acids Research Jun 2024Senescent cells can influence the function of tissues in which they reside, and their propensity for disease. A portion of adult human pancreatic beta cells express the...
Senescent cells can influence the function of tissues in which they reside, and their propensity for disease. A portion of adult human pancreatic beta cells express the senescence marker p16, yet it is unclear whether they are in a senescent state, and how this affects insulin secretion. We analyzed single-cell transcriptome datasets of adult human beta cells, and found that p16-positive cells express senescence gene signatures, as well as elevated levels of beta-cell maturation genes, consistent with enhanced functionality. Senescent human beta-like cells in culture undergo chromatin reorganization that leads to activation of enhancers regulating functional maturation genes and acquisition of glucose-stimulated insulin secretion capacity. Strikingly, Interferon-stimulated genes are elevated in senescent human beta cells, but genes encoding senescence-associated secretory phenotype (SASP) cytokines are not. Senescent beta cells in culture and in human tissue show elevated levels of cytoplasmic DNA, contributing to their increased interferon responsiveness. Human beta-cell senescence thus involves chromatin-driven upregulation of a functional-maturation program, and increased responsiveness of interferon-stimulated genes, changes that could increase both insulin secretion and immune reactivity.
Topics: Humans; Insulin-Secreting Cells; Cellular Senescence; Chromatin Assembly and Disassembly; Interferons; Insulin Secretion; Insulin; Chromatin; Cyclin-Dependent Kinase Inhibitor p16; Cells, Cultured; Senescence-Associated Secretory Phenotype; Transcriptome; Single-Cell Analysis
PubMed: 38682582
DOI: 10.1093/nar/gkae313 -
Polymers Apr 2024The development of sustainable materials from the valorization of waste is a good alternative to reducing the negative environmental impact of plastic packaging. The...
Valorization of Cork Stoppers, Coffee-Grounds and Walnut Shells in the Development and Characterization of Pectin-Based Composite Films: Physical, Barrier, Antioxidant, Genotoxic, and Biodegradation Properties.
The development of sustainable materials from the valorization of waste is a good alternative to reducing the negative environmental impact of plastic packaging. The objectives of this study were to develop and characterize pectin-based composite films incorporated with cork or cork with either coffee grounds or walnut shells, as well as to test the films' genotoxicity, antioxidant properties, and biodegradation capacity in soil and seawater. The addition of cork, coffee grounds, or walnut shells modified the films' characteristics. The results showed that those films were thicker (0.487 ± 0.014 mm to 0.572 ± 0.014 mm), more opaque (around 100%), darker (L* = 25.30 ± 0.78 to 33.93 ± 0.84), and had a higher total phenolic content (3.17 ± 0.01 mg GA/g to 4.24 ± 0.02 mg GA/g). On the other hand, the films incorporated only with cork showed higher values of elongation at break (32.24 ± 1.88% to 36.30 ± 3.25%) but lower tensile strength (0.91 ± 0.19 MPa to 1.09 ± 0.08 MPa). All the films presented more heterogeneous and rougher microstructures than the pectin film. This study also revealed that the developed films do not contain DNA-reactive substances and that they are biodegradable in soil and seawater. These positive properties could subsequently make the developed films an interesting eco-friendly food packaging solution that contributes to the valorization of organic waste and by-products, thus promoting the circular economy and reducing the environmental impact of plastic materials.
PubMed: 38674972
DOI: 10.3390/polym16081053 -
International Journal of Molecular... Apr 2024DNA repair pathways play a critical role in genome stability, but in eukaryotic cells, they must operate to repair DNA lesions in the compact and tangled environment of... (Review)
Review
DNA repair pathways play a critical role in genome stability, but in eukaryotic cells, they must operate to repair DNA lesions in the compact and tangled environment of chromatin. Previous studies have shown that the packaging of DNA into nucleosomes, which form the basic building block of chromatin, has a profound impact on DNA repair. In this review, we discuss the principles and mechanisms governing DNA repair in chromatin. We focus on the role of histone post-translational modifications (PTMs) in repair, as well as the molecular mechanisms by which histone mutants affect cellular sensitivity to DNA damage agents and repair activity in chromatin. Importantly, these mechanisms are thought to significantly impact somatic mutation rates in human cancers and potentially contribute to carcinogenesis and other human diseases. For example, a number of the histone mutants studied primarily in yeast have been identified as candidate oncohistone mutations in different cancers. This review highlights these connections and discusses the potential importance of DNA repair in chromatin to human health.
Topics: DNA Repair; Nucleosomes; Humans; Histones; Protein Processing, Post-Translational; Mutation; Animals; DNA Damage; Neoplasms; Histone Code; Chromatin
PubMed: 38673978
DOI: 10.3390/ijms25084393 -
Genome Biology Apr 2024Prime editing enables precise base substitutions, insertions, and deletions at targeted sites without the involvement of double-strand DNA breaks or exogenous donor DNA...
BACKGROUND
Prime editing enables precise base substitutions, insertions, and deletions at targeted sites without the involvement of double-strand DNA breaks or exogenous donor DNA templates. However, the large size of prime editors (PEs) hampers their delivery in vivo via adeno-associated virus (AAV) due to the viral packaging limit. Previously reported split PE versions provide a size reduction, but they require intricate engineering and potentially compromise editing efficiency.
RESULTS
Herein, we present a simplified split PE named as CC-PE, created through non-covalent recruitment of reverse transcriptase to the Cas9 nickase via coiled-coil heterodimers, which are widely used in protein design due to their modularity and well-understood sequence-structure relationship. We demonstrate that the CC-PE maintains or even surpasses the efficiency of unsplit PE in installing intended edits, with no increase in the levels of undesired byproducts within tested loci amongst a variety of cell types (HEK293T, A549, HCT116, and U2OS). Furthermore, coiled-coil heterodimers are used to engineer SpCas9-NG-PE and SpRY-PE, two Cas9 variants with more flexible editing scope. Similarly, the resulting NG-CC-PE and SpRY-CC-PE also achieve equivalent or enhanced efficiency of precise editing compared to the intact PE. When the dual AAV vectors carrying CC-PE are delivered into mice to target the Pcsk9 gene in the liver, CC-PE enables highly efficient precise editing, resulting in a significant reduction of plasma low-density lipoprotein cholesterol and total cholesterol.
CONCLUSIONS
Our innovative, modular system enhances flexibility, thus potentially facilitating the in vivo applicability of prime editing.
Topics: Gene Editing; Humans; Animals; Mice; CRISPR-Associated Protein 9; CRISPR-Cas Systems; HEK293 Cells; Dependovirus
PubMed: 38671524
DOI: 10.1186/s13059-024-03257-z -
Biochemical Society Transactions Jun 2024The close relationship between chromatin and metabolism has been well-studied in recent years. Many metabolites have been found to be cofactors used to modify chromatin,... (Review)
Review
The close relationship between chromatin and metabolism has been well-studied in recent years. Many metabolites have been found to be cofactors used to modify chromatin, and these modifications can in turn affect gene transcription. One chromatin-associated factor responsible for regulating transcription is the SWI/SNF complex, an ATP-dependent chromatin remodeler conserved throughout eukaryotes. SWI/SNF was originally described in yeast as regulating genes involved in carbon source metabolism and mating type switching, and its mammalian counterpart has been extensively studied for its role in diseases such as cancer. The yeast SWI/SNF complex is closely associated with activation of stress response genes, many of which have metabolic functions. It is now recognized that this is a conserved function of the complex, and recent work has shown that mammalian SWI/SNF is also a key regulator of metabolic transcription. Emerging evidence suggests that loss of SWI/SNF introduces vulnerabilities to cells due to this metabolic influence, and that this may present opportunities for treatment of SWI/SNF-deficient cancers.
Topics: Chromatin Assembly and Disassembly; Humans; Transcription Factors; Chromosomal Proteins, Non-Histone; Animals; Chromatin; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Neoplasms
PubMed: 38666605
DOI: 10.1042/BST20231141 -
Cardiovascular Diabetology Apr 2024Diabetic cardiomyopathy (DCM) poses a growing health threat, elevating heart failure risk in diabetic individuals. Understanding DCM is crucial, with fibroblasts and...
BACKGROUND
Diabetic cardiomyopathy (DCM) poses a growing health threat, elevating heart failure risk in diabetic individuals. Understanding DCM is crucial, with fibroblasts and endothelial cells playing pivotal roles in driving myocardial fibrosis and contributing to cardiac dysfunction. Advances in Multimodal single-cell profiling, such as scRNA-seq and scATAC-seq, provide deeper insights into DCM's unique cell states and molecular landscape for targeted therapeutic interventions.
METHODS
Single-cell RNA and ATAC data from 10x Multiome libraries were processed using Cell Ranger ARC v2.0.1. Gene expression and ATAC data underwent Seurat and Signac filtration. Differential gene expression and accessible chromatin regions were identified. Transcription factor activity was estimated with chromVAR, and Cis-coaccessibility networks were calculated using Cicero. Coaccessibility connections were compared to the GeneHancer database. Gene Ontology analysis, biological process scoring, cell-cell communication analysis, and gene-motif correlation was performed to reveal intricate molecular changes. Immunofluorescent staining utilized various antibodies on paraffin-embedded tissues to verify the findings.
RESULTS
This study integrated scRNA-seq and scATAC-seq data obtained from hearts of WT and DCM mice, elucidating molecular changes at the single-cell level throughout the diabetic cardiomyopathy progression. Robust and accurate clustering analysis of the integrated data revealed altered cell proportions, showcasing decreased endothelial cells and macrophages, coupled with increased fibroblasts and myocardial cells in the DCM group, indicating enhanced fibrosis and endothelial damage. Chromatin accessibility analysis unveiled unique patterns in cell types, with heightened transcriptional activity in myocardial cells. Subpopulation analysis highlighted distinct changes in cardiomyocytes and fibroblasts, emphasizing pathways related to fatty acid metabolism and cardiac contraction. Fibroblast-centered communication analysis identified interactions with endothelial cells, implicating VEGF receptors. Endothelial cell subpopulations exhibited altered gene expressions, emphasizing contraction and growth-related pathways. Candidate regulators, including Tcf21, Arnt, Stat5a, and Stat5b, were identified, suggesting their pivotal roles in DCM development. Immunofluorescence staining validated marker genes of cell subpopulations, confirming PDK4, PPARγ and Tpm1 as markers for metabolic pattern-altered cardiomyocytes, activated fibroblasts and endothelial cells with compromised proliferation.
CONCLUSION
Our integrated scRNA-seq and scATAC-seq analysis unveils intricate cell states and molecular alterations in diabetic cardiomyopathy. Identified cell type-specific changes, transcription factors, and marker genes offer valuable insights. The study sheds light on potential therapeutic targets for DCM.
Topics: Diabetic Cardiomyopathies; Animals; Single-Cell Analysis; Transcriptome; Gene Expression Profiling; Chromatin; Mice, Inbred C57BL; Gene Regulatory Networks; Chromatin Assembly and Disassembly; Disease Models, Animal; Male; RNA-Seq; Gene Expression Regulation; Myocytes, Cardiac; Fibroblasts; Fibrosis; Mice; Endothelial Cells
PubMed: 38664790
DOI: 10.1186/s12933-024-02233-y -
Cell Reports May 2024Chromatin-associated RNAs (cRNAs) are a poorly characterized fraction of cellular RNAs that co-purify with chromatin. Their full complexity and the mechanisms regulating...
Chromatin-associated RNAs (cRNAs) are a poorly characterized fraction of cellular RNAs that co-purify with chromatin. Their full complexity and the mechanisms regulating their packaging and chromatin association remain poorly understood. Here, we address these questions in Drosophila. We find that cRNAs constitute a heterogeneous group of RNA species that is abundant in heterochromatic transcripts. We show that heterochromatic cRNAs interact with the heterogeneous nuclear ribonucleoproteins (hnRNP) hrp36/hrp48 and that depletion of linker histone dH1 impairs this interaction. dH1 depletion induces the accumulation of RNA::DNA hybrids (R-loops) in heterochromatin and, as a consequence, increases retention of heterochromatic cRNAs. These effects correlate with increased RNA polymerase II (RNAPII) occupancy at heterochromatin. Notably, impairing cRNA assembly by depletion of hrp36/hrp48 mimics heterochromatic R-loop accumulation induced by dH1 depletion. We also show that dH1 depletion alters nucleosome organization, increasing accessibility of heterochromatin. Altogether, these perturbations facilitate annealing of cRNAs to the DNA template, enhancing R-loop formation and cRNA retention at heterochromatin.
Topics: Animals; Drosophila; Drosophila melanogaster; Drosophila Proteins; Heterochromatin; Histones; Homeostasis; Nucleosomes; R-Loop Structures; RNA; RNA Polymerase II; Male; Female
PubMed: 38662543
DOI: 10.1016/j.celrep.2024.114137