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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 -
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 -
Proceedings of the National Academy of... Apr 2024The control of eukaryotic gene expression is intimately connected to highly dynamic chromatin structures. Gene regulation relies on activator and repressor transcription...
The control of eukaryotic gene expression is intimately connected to highly dynamic chromatin structures. Gene regulation relies on activator and repressor transcription factors (TFs) that induce local chromatin opening and closing. However, it is unclear how nucleus-wide chromatin organization responds dynamically to the activity of specific TFs. Here, we examined how two TFs with opposite effects on local chromatin accessibility modulate chromatin dynamics nucleus-wide. We combine high-resolution diffusion mapping and dense flow reconstruction and correlation in living cells to obtain an imaging-based, nanometer-scale analysis of local diffusion processes and long-range coordinated movements of both chromatin and TFs. We show that the expression of either an individual transcriptional activator (CDX2) or repressor (SIX6) with large numbers of binding sites increases chromatin mobility nucleus-wide, yet they induce opposite coherent chromatin motions at the micron scale. Hi-C analysis of higher-order chromatin structures shows that induction of the pioneer factor CDX2 leads both to changes in local chromatin interactions and the distribution of A and B compartments, thus relating the micromovement of chromatin with changes in compartmental structures. Given that inhibition of transcription initiation and elongation by RNA Pol II has a partial impact on the global chromatin dynamics induced by CDX2, we suggest that CDX2 overexpression alters chromatin structure dynamics both dependently and independently of transcription. Our biophysical analysis shows that sequence-specific TFs can influence chromatin structure on multiple architectural levels, arguing that local chromatin changes brought by TFs alter long-range chromatin mobility and its organization.
Topics: Chromatin; Transcription Factors; Humans; CDX2 Transcription Factor; Gene Expression Regulation; Cell Nucleus; Binding Sites; Chromatin Assembly and Disassembly
PubMed: 38648478
DOI: 10.1073/pnas.2311374121 -
BioRxiv : the Preprint Server For... Apr 2024Adeno-associated virus (AAV) is a safe and efficient gene delivery vehicle for gene therapies. However, its relatively small packaging capacity limits its use as a gene...
Adeno-associated virus (AAV) is a safe and efficient gene delivery vehicle for gene therapies. However, its relatively small packaging capacity limits its use as a gene transfer vector. Here, we describe a strategy to deliver large genes that exceed the AAV's packaging capacity using up to four AAV vectors and the CRE-lox DNA recombination system. We devised novel lox sites by combining non-compatible and reaction equilibrium-modifying lox site variants. These lox sites facilitate sequence-specific and near-unidirectional recombination of AAV vector genomes, enabling efficient reconstitution of up to 16 kb of therapeutic genes in a pre-determined configuration. Using this strategy, we have developed AAV gene therapy vectors to deliver , , , and and demonstrate efficient production of full-length proteins in cultured mammalian cells and mouse retinas. Notably, this approach significantly surpasses the trans-splicing and split-intein-based reconstitution methods in efficiency, requiring lower doses, minimizing or eliminating the production of truncated protein products, and offering flexibility in selecting splitting positions. The CRE-lox approach described here provides a simple and effective platform for producing AAV gene therapy vectors beyond AAV's packaging capacity.
PubMed: 38645107
DOI: 10.1101/2024.04.10.588864 -
Nature Immunology May 2024Adaptive immunity relies on specialized effector functions elicited by lymphocytes, yet how antigen recognition activates appropriate effector responses through...
Adaptive immunity relies on specialized effector functions elicited by lymphocytes, yet how antigen recognition activates appropriate effector responses through nonspecific signaling intermediates is unclear. Here we examined the role of chromatin priming in specifying the functional outputs of effector T cells and found that most of the cis-regulatory landscape active in effector T cells was poised early in development before the expression of the T cell antigen receptor. We identified two principal mechanisms underpinning this poised landscape: the recruitment of the nucleosome remodeler mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) by the transcription factors RUNX1 and PU.1 to establish chromatin accessibility at T effector loci; and a 'relay' whereby the transcription factor BCL11B succeeded PU.1 to maintain occupancy of the chromatin remodeling complex mSWI/SNF together with RUNX1, after PU.1 silencing during lineage commitment. These mechanisms define modes by which T cells acquire the potential to elicit specialized effector functions early in their ontogeny and underscore the importance of integrating extrinsic cues to the developmentally specified intrinsic program.
Topics: Proto-Oncogene Proteins; Animals; Trans-Activators; Mice; Core Binding Factor Alpha 2 Subunit; Repressor Proteins; Transcription Factors; Tumor Suppressor Proteins; Mice, Inbred C57BL; Chromosomal Proteins, Non-Histone; T-Lymphocytes; Mice, Knockout; Chromatin Assembly and Disassembly; Cell Differentiation
PubMed: 38632339
DOI: 10.1038/s41590-024-01807-y -
Current Opinion in Genetics &... Jun 2024The human genome is not just a simple string of DNA, it is a complex and dynamic entity intricately folded within the cell's nucleus. This three-dimensional... (Review)
Review
The human genome is not just a simple string of DNA, it is a complex and dynamic entity intricately folded within the cell's nucleus. This three-dimensional organization of chromatin, the combination of DNA and proteins in the nucleus, is crucial for many biological processes and has been prominently studied for its intricate relationship to gene expression. Indeed, the transcriptional machinery does not operate in isolation but interacts intimately with the folded chromatin structure. Techniques for chromatin conformation capture, including genome-wide sequencing approaches, have revealed key organizational features of chromatin, such as the formation of loops by CCCTC-binding factor (CTCF) and the division of loci into chromatin compartments. While much of the recent research and reviews have focused on CTCF loops, we discuss several new revelations that have emerged concerning chromatin compartments, with a particular focus on what is known about mechanistic drivers of compartmentalization. These insights challenge the traditional views of chromatin organization and reveal the complexity behind the formation and maintenance of chromatin compartments.
Topics: Humans; Chromatin; CCCTC-Binding Factor; Genome, Human; Cell Nucleus; DNA; Chromatin Assembly and Disassembly; Animals
PubMed: 38626581
DOI: 10.1016/j.gde.2024.102193 -
Cell Reports. Medicine May 2024Key gene mutations are essential for colorectal cancer (CRC) development; however, how the mutated tumor cells impact the surrounding normal cells to promote tumor...
Key gene mutations are essential for colorectal cancer (CRC) development; however, how the mutated tumor cells impact the surrounding normal cells to promote tumor progression has not been well defined. Here, we report that PIK3CA mutant tumor cells transmit oncogenic signals and result in malignant transformation of intestinal epithelial cells (IECs) via paracrine exosomal arachidonic acid (AA)-induced H3K4 trimethylation. Mechanistically, PIK3CA mutations sustain SGK3-FBW7-mediated stability of the cPLA2 protein, leading to the synthetic increase in AA, which is transported through exosome and accumulated in IECs. Transferred AA directly binds Menin and strengthens the interactions of Menin and MLL1/2 methyltransferase. Finally, the combination of VTP50469, an inhibitor of the Menin-MLL interaction, and alpelisib synergistically represses PDX tumors harboring PIK3CA mutations. Together, these findings unveil the metabolic link between PIK3CA mutant tumor cells and the IECs, highlighting AA as the potential target for the treatment of patients with CRC harboring PIK3CA mutations.
Topics: Class I Phosphatidylinositol 3-Kinases; Humans; Arachidonic Acid; Animals; Mutation; Cell Transformation, Neoplastic; Chromatin Assembly and Disassembly; Mice; Cell Line, Tumor; Colon; Colorectal Neoplasms; Exosomes; Intestinal Mucosa; Histones
PubMed: 38614093
DOI: 10.1016/j.xcrm.2024.101510 -
International Journal of Molecular... Apr 2024We developed a procedure for locating genes on polytene chromosomes and described three types of chromosome structures (gray bands, black bands, and interbands), which... (Review)
Review
We developed a procedure for locating genes on polytene chromosomes and described three types of chromosome structures (gray bands, black bands, and interbands), which differed markedly in morphological and genetic properties. This was reached through the use of our original methods of molecular and genetic analysis, electron microscopy, and bioinformatics data processing. Analysis of the genome-wide distribution of these properties led us to a bioinformatics model of the genome organization, in which the genome was divided into two groups of genes. One was constituted by 65, in which the genome was divided into two groups, 62 genes that are expressed in most cell types during life cycle and perform basic cellular functions (the so-called "housekeeping genes"). The other one was made up of 3162 genes that are expressed only at particular stages of development ("developmental genes"). These two groups of genes are so different that we may state that the genome has two types of genetic organization. Different are the timings of their expression, chromatin packaging levels, the composition of activating and deactivating proteins, the sizes of these genes, the lengths of their introns, the organization of the promoter regions of the genes, the locations of origin recognition complexes (ORCs), and DNA replication timings.
Topics: Animals; Genes, Essential; Drosophila; Drosophila melanogaster; Chromatin; Introns
PubMed: 38612878
DOI: 10.3390/ijms25074068