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Science (New York, N.Y.) Sep 2023The cerebellum contains most of the neurons in the human brain and exhibits distinctive modes of development and aging. In this work, by developing our single-cell...
The cerebellum contains most of the neurons in the human brain and exhibits distinctive modes of development and aging. In this work, by developing our single-cell three-dimensional (3D) genome assay-diploid chromosome conformation capture, or Dip-C-into population-scale (Pop-C) and virus-enriched (vDip-C) modes, we resolved the first 3D genome structures of single cerebellar cells, created life-spanning 3D genome atlases for both humans and mice, and jointly measured transcriptome and chromatin accessibility during development. We found that although the transcriptome and chromatin accessibility of cerebellar granule neurons mature in early postnatal life, 3D genome architecture gradually remodels throughout life, establishing ultra-long-range intrachromosomal contacts and specific interchromosomal contacts that are rarely seen in neurons. These results reveal unexpected evolutionarily conserved molecular processes that underlie distinctive features of neural development and aging across the mammalian life span.
Topics: Animals; Humans; Mice; Cerebellum; Neurons; Genome; Imaging, Three-Dimensional; Single-Cell Analysis; Chromatin Assembly and Disassembly; Cellular Senescence; Atlases as Topic
PubMed: 37676945
DOI: 10.1126/science.adh3253 -
Nature Cell Biology Jul 2023Chromatin is dynamically reorganized when DNA replication forks are challenged. However, the process of epigenetic reorganization and its implication for fork stability...
Chromatin is dynamically reorganized when DNA replication forks are challenged. However, the process of epigenetic reorganization and its implication for fork stability is poorly understood. Here we discover a checkpoint-regulated cascade of chromatin signalling that activates the histone methyltransferase EHMT2/G9a to catalyse heterochromatin assembly at stressed replication forks. Using biochemical and single molecule chromatin fibre approaches, we show that G9a together with SUV39h1 induces chromatin compaction by accumulating the repressive modifications, H3K9me1/me2/me3, in the vicinity of stressed replication forks. This closed conformation is also favoured by the G9a-dependent exclusion of the H3K9-demethylase JMJD1A/KDM3A, which facilitates heterochromatin disassembly upon fork restart. Untimely heterochromatin disassembly from stressed forks by KDM3A enables PRIMPOL access, triggering single-stranded DNA gap formation and sensitizing cells towards chemotherapeutic drugs. These findings may help in explaining chemotherapy resistance and poor prognosis observed in patients with cancer displaying elevated levels of G9a/H3K9me3.
Topics: Humans; Histones; Heterochromatin; Chromatin; Chromatin Assembly and Disassembly; DNA Replication; Histocompatibility Antigens; Histone-Lysine N-Methyltransferase; Jumonji Domain-Containing Histone Demethylases
PubMed: 37414849
DOI: 10.1038/s41556-023-01167-z -
Nature Aug 2023Human-specific genomic changes contribute to the unique functionalities of the human brain. The cellular heterogeneity of the human brain and the complex regulation of...
Human-specific genomic changes contribute to the unique functionalities of the human brain. The cellular heterogeneity of the human brain and the complex regulation of gene expression highlight the need to characterize human-specific molecular features at cellular resolution. Here we analysed single-nucleus RNA-sequencing and single-nucleus assay for transposase-accessible chromatin with sequencing datasets for human, chimpanzee and rhesus macaque brain tissue from posterior cingulate cortex. We show a human-specific increase of oligodendrocyte progenitor cells and a decrease of mature oligodendrocytes across cortical tissues. Human-specific regulatory changes were accelerated in oligodendrocyte progenitor cells, and we highlight key biological pathways that may be associated with the proportional changes. We also identify human-specific regulatory changes in neuronal subtypes, which reveal human-specific upregulation of FOXP2 in only two of the neuronal subtypes. We additionally identify hundreds of new human accelerated genomic regions associated with human-specific chromatin accessibility changes. Our data also reveal that FOS::JUN and FOX motifs are enriched in the human-specifically accessible chromatin regions of excitatory neuronal subtypes. Together, our results reveal several new mechanisms underlying the evolutionary innovation of human brain at cell-type resolution.
Topics: Animals; Humans; Cell Nucleus; Chromatin; Datasets as Topic; Evolution, Molecular; Genome, Human; Genomics; Gyrus Cinguli; Macaca mulatta; Neurons; Oligodendroglia; Pan troglodytes; Single-Cell Gene Expression Analysis; Stem Cells; Transposases; Chromatin Assembly and Disassembly
PubMed: 37468639
DOI: 10.1038/s41586-023-06338-4 -
Nucleic Acids Research Oct 2023Implementation of therapeutic in vivo gene editing using CRISPR/Cas relies on potent delivery of gene editing tools. Administration of ribonucleoprotein (RNP) complexes...
Engineered lentivirus-derived nanoparticles (LVNPs) for delivery of CRISPR/Cas ribonucleoprotein complexes supporting base editing, prime editing and in vivo gene modification.
Implementation of therapeutic in vivo gene editing using CRISPR/Cas relies on potent delivery of gene editing tools. Administration of ribonucleoprotein (RNP) complexes consisting of Cas protein and single guide RNA (sgRNA) offers short-lived editing activity and safety advantages over conventional viral and non-viral gene and RNA delivery approaches. By engineering lentivirus-derived nanoparticles (LVNPs) to facilitate RNP delivery, we demonstrate effective administration of SpCas9 as well as SpCas9-derived base and prime editors (BE/PE) leading to gene editing in recipient cells. Unique Gag/GagPol protein fusion strategies facilitate RNP packaging in LVNPs, and refinement of LVNP stoichiometry supports optimized LVNP yield and incorporation of therapeutic payload. We demonstrate near instantaneous target DNA cleavage and complete RNP turnover within 4 days. As a result, LVNPs provide high on-target DNA cleavage and lower levels of off-target cleavage activity compared to standard RNP nucleofection in cultured cells. LVNPs accommodate BE/sgRNA and PE/epegRNA RNPs leading to base editing with reduced bystander editing and prime editing without detectable indel formation. Notably, in the mouse eye, we provide the first proof-of-concept for LVNP-directed in vivo gene disruption. Our findings establish LVNPs as promising vehicles for delivery of RNPs facilitating donor-free base and prime editing without formation of double-stranded DNA breaks.
PubMed: 37678882
DOI: 10.1093/nar/gkad676 -
BioRxiv : the Preprint Server For... Feb 2024In biology, DNA is often tightly bent to small radii. Solely based on the groove asymmetry, a 30-year-old theoretical paper predicted that such bending should unwind...
In biology, DNA is often tightly bent to small radii. Solely based on the groove asymmetry, a 30-year-old theoretical paper predicted that such bending should unwind DNA, but this effect has not been directly experimentally quantified so far. We developed a ligation-based assay with nicked DNA circles of variable length, thereby decoupling the twist-dependent ligation efficiency from the large bending strain which dominates conventional circularization assays. We demonstrate that tightly bent DNA indeed unwinds to over 11 base pairs/turn, exactly as predicted. Our discovery requires reassessing the molecular mechanisms and energetics of all processes where DNA is tightly bent or relaxed again, including DNA packaging, gene regulation and expression.
PubMed: 38405957
DOI: 10.1101/2024.02.14.579968 -
The Journal of Clinical Investigation Jul 2023During emergency hematopoiesis, hematopoietic stem cells (HSCs) rapidly proliferate to produce myeloid and lymphoid effector cells, a response that is critical against...
During emergency hematopoiesis, hematopoietic stem cells (HSCs) rapidly proliferate to produce myeloid and lymphoid effector cells, a response that is critical against infection or tissue injury. If unresolved, this process leads to sustained inflammation, which can cause life-threatening diseases and cancer. Here, we identify a role of double PHD fingers 2 (DPF2) in modulating inflammation. DPF2 is a defining subunit of the hematopoiesis-specific BAF (SWI/SNF) chromatin-remodeling complex, and it is mutated in multiple cancers and neurological disorders. We uncovered that hematopoiesis-specific Dpf2-KO mice developed leukopenia, severe anemia, and lethal systemic inflammation characterized by histiocytic and fibrotic tissue infiltration resembling a clinical hyperinflammatory state. Dpf2 loss impaired the polarization of macrophages responsible for tissue repair, induced the unrestrained activation of Th cells, and generated an emergency-like state of HSC hyperproliferation and myeloid cell-biased differentiation. Mechanistically, Dpf2 deficiency resulted in the loss of the BAF catalytic subunit BRG1 from nuclear factor erythroid 2-like 2-controlled (NRF2-controlled) enhancers, impairing the antioxidant and antiinflammatory transcriptional response needed to modulate inflammation. Finally, pharmacological reactivation of NRF2 suppressed the inflammation-mediated phenotypes and lethality of Dpf2Δ/Δ mice. Our work establishes an essential role of the DPF2-BAF complex in licensing NRF2-dependent gene expression in HSCs and immune effector cells to prevent chronic inflammation.
Topics: Mice; Animals; Chromatin; Antioxidants; NF-E2-Related Factor 2; Neoplasms; Chromatin Assembly and Disassembly; Inflammation; Gene Expression; DNA-Binding Proteins; Transcription Factors
PubMed: 37200093
DOI: 10.1172/JCI158419 -
Advanced Science (Weinheim,... Aug 2023Pancreatic ductal adenocarcinoma (PDA) is a clinically challenging disease with limited treatment options. Despite a small percentage of cases with defective mismatch...
Pancreatic ductal adenocarcinoma (PDA) is a clinically challenging disease with limited treatment options. Despite a small percentage of cases with defective mismatch DNA repair (dMMR), PDA is included in the most immune-resistant cancer types that are poorly responsive to immune checkpoint blockade (ICB) therapy. To facilitate drug discovery combating this immunosuppressive tumor type, a high-throughput drug screen platform is established with the newly developed T cell-incorporated pancreatic tumor organoid model. Tumor-specific T cells are included in the pancreatic tumor organoids by two-step cell packaging, fully recapitulating immune infiltration in the immunosuppressive tumor microenvironment (TME). The organoids are generated with key components in the original tumor, including epithelial, vascular endothelial, fibroblast and macrophage cells, and then packaged with T cells into their outside layer mimicking a physical barrier and enabling T cell infiltration and cytotoxicity studies. In the PDA organoid-based screen, epigenetic inhibitors ITF2357 and I-BET151 are identified, which in combination with anti-PD-1 based therapy show considerably greater anti-tumor effect. The combinatorial treatment turns the TME from immunosuppressive to immunoactive, up-regulates the MHC-I antigen processing and presentation, and enhances the effector T cell activity. The standardized PDA organoid model has shown great promise to accelerate drug discovery for the immunosuppressive cancer.
Topics: Humans; T-Lymphocytes; Pancreatic Neoplasms; Carcinoma, Pancreatic Ductal; Immunotherapy; Organoids; Tumor Microenvironment
PubMed: 37271874
DOI: 10.1002/advs.202300548 -
ELife Dec 2023Nucleotide and force-dependent mechanisms control how the viral genome of lambda bacteriophage is inserted into capsids.
Nucleotide and force-dependent mechanisms control how the viral genome of lambda bacteriophage is inserted into capsids.
Topics: DNA, Viral; Bacteriophage lambda; Capsid; Genome, Viral; Nucleotides; Virus Assembly
PubMed: 38095555
DOI: 10.7554/eLife.94128 -
Science (New York, N.Y.) Nov 2023The global replacement of histones with protamines in sperm chromatin is widespread in animals, including insects, but its actual function remains enigmatic. We show...
The global replacement of histones with protamines in sperm chromatin is widespread in animals, including insects, but its actual function remains enigmatic. We show that in the paternal effect mutant (), sperm chromatin retains germline histones H3 and H4 genome wide without impairing sperm viability. However, after fertilization, sperm chromosomes are targeted by the egg chromosomal passenger complex and engage into a catastrophic premature division in synchrony with female meiosis II. We show that encodes a rapidly evolving transition protein specifically required for the eviction of (H3-H4) tetramers from spermatid DNA after the removal of H2A-H2B dimers. Our study thus reveals an unsuspected role of histone eviction from insect sperm chromatin: safeguarding the integrity of the male pronucleus during female meiosis.
Topics: Animals; Female; Male; Chromatin; Drosophila Proteins; Fertilization; Histones; Spermatozoa; Drosophila melanogaster; Amidine-Lyases; DNA Packaging; Paternal Inheritance
PubMed: 37943933
DOI: 10.1126/science.adh0037