ABSTRACT
Short tandem repeat (STR) instability causes transcriptional silencing in several repeat expansion disorders. In fragile X syndrome (FXS), mutation-length expansion of a CGG STR represses FMR1 via local DNA methylation. Here, we find megabase-scale H3K9me3 domains on autosomes and encompassing FMR1 on the X chromosome in FXS patient-derived iPSCs, iPSC-derived neural progenitors, EBV-transformed lymphoblasts, and brain tissue with mutation-length CGG expansion. H3K9me3 domains connect via inter-chromosomal interactions and demarcate severe misfolding of TADs and loops. They harbor long synaptic genes replicating at the end of S phase, replication-stress-induced double-strand breaks, and STRs prone to stepwise somatic instability. CRISPR engineering of the mutation-length CGG to premutation length reverses H3K9me3 on the X chromosome and multiple autosomes, refolds TADs, and restores gene expression. H3K9me3 domains can also arise in normal-length iPSCs created with perturbations linked to genome instability, suggesting their relevance beyond FXS. Our results reveal Mb-scale heterochromatinization and trans interactions among loci susceptible to instability.
Subject(s)
Fragile X Syndrome , Humans , Fragile X Syndrome/genetics , Fragile X Syndrome/metabolism , Trinucleotide Repeat Expansion , DNA Methylation , Mutation , Fragile X Mental Retardation Protein/genetics , Fragile X Mental Retardation Protein/metabolismABSTRACT
Regulatory landscapes drive complex developmental gene expression, but it remains unclear how their integrity is maintained when incorporating novel genes and functions during evolution. Here, we investigated how a placental mammal-specific gene, Zfp42, emerged in an ancient vertebrate topologically associated domain (TAD) without adopting or disrupting the conserved expression of its gene, Fat1. In ESCs, physical TAD partitioning separates Zfp42 and Fat1 with distinct local enhancers that drive their independent expression. This separation is driven by chromatin activity and not CTCF/cohesin. In contrast, in embryonic limbs, inactive Zfp42 shares Fat1's intact TAD without responding to active Fat1 enhancers. However, neither Fat1 enhancer-incompatibility nor nuclear envelope-attachment account for Zfp42's unresponsiveness. Rather, Zfp42's promoter is rendered inert to enhancers by context-dependent DNA methylation. Thus, diverse mechanisms enabled the integration of independent Zfp42 regulation in the Fat1 locus. Critically, such regulatory complexity appears common in evolution as, genome wide, most TADs contain multiple independently expressed genes.
Subject(s)
Chromatin , Placenta , Animals , CCCTC-Binding Factor/metabolism , Chromatin Assembly and Disassembly , Enhancer Elements, Genetic , Evolution, Molecular , Female , Genome , Mammals/metabolism , Placenta/metabolism , Pregnancy , Promoter Regions, Genetic , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Biomolecular condensation is a widespread mechanism of cellular compartmentalization. Because the "survival of motor neuron protein" (SMN) is implicated in the formation of three different membraneless organelles (MLOs), we hypothesized that SMN promotes condensation. Unexpectedly, we found that SMN's globular tudor domain was sufficient for dimerization-induced condensation in vivo, whereas its two intrinsically disordered regions (IDRs) were not. Binding to dimethylarginine (DMA) modified protein ligands was required for condensate formation by the tudor domains in SMN and at least seven other fly and human proteins. Remarkably, asymmetric versus symmetric DMA determined whether two distinct nuclear MLOs-gems and Cajal bodies-were separate or "docked" to one another. This substructure depended on the presence of either asymmetric or symmetric DMA as visualized with sub-diffraction microscopy. Thus, DMA-tudor interaction modules-combinations of tudor domains bound to their DMA ligand(s)-represent versatile yet specific regulators of MLO assembly, composition, and morphology.
Subject(s)
Arginine/analogs & derivatives , Biomolecular Condensates/metabolism , SMN Complex Proteins/chemistry , SMN Complex Proteins/metabolism , Animals , Arginine/metabolism , Cell Nucleus/metabolism , Coiled Bodies/metabolism , Drosophila melanogaster/metabolism , HEK293 Cells , HeLa Cells , Humans , Ligands , Methylation , Mice , Models, Biological , NIH 3T3 Cells , Protein Binding , Protein Domains , Protein Multimerization , Ribonucleoproteins, Small Nuclear/metabolismABSTRACT
Thousands of proteins localize to the nucleus; however, it remains unclear which contain transcriptional effectors. Here, we develop HT-recruit, a pooled assay where protein libraries are recruited to a reporter, and their transcriptional effects are measured by sequencing. Using this approach, we measure gene silencing and activation for thousands of domains. We find a relationship between repressor function and evolutionary age for the KRAB domains, discover that Homeodomain repressor strength is collinear with Hox genetic organization, and identify activities for several domains of unknown function. Deep mutational scanning of the CRISPRi KRAB maps the co-repressor binding surface and identifies substitutions that improve stability/silencing. By tiling 238 proteins, we find repressors as short as ten amino acids. Finally, we report new activator domains, including a divergent KRAB. These results provide a resource of 600 human proteins containing effectors and demonstrate a scalable strategy for assigning functions to protein domains.
Subject(s)
High-Throughput Screening Assays , Transcription Factors/metabolism , Amino Acid Sequence , CRISPR-Cas Systems/genetics , Female , Gene Silencing , Genes, Reporter , HEK293 Cells , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , K562 Cells , Lentivirus/physiology , Molecular Sequence Annotation , Mutation/genetics , Nuclear Proteins/metabolism , Promoter Regions, Genetic/genetics , Protein Domains , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Reproducibility of Results , Transcription, Genetic , Zinc FingersABSTRACT
The 3D organization of chromatin regulates many genome functions. Our understanding of 3D genome organization requires tools to directly visualize chromatin conformation in its native context. Here we report an imaging technology for visualizing chromatin organization across multiple scales in single cells with high genomic throughput. First we demonstrate multiplexed imaging of hundreds of genomic loci by sequential hybridization, which allows high-resolution conformation tracing of whole chromosomes. Next we report a multiplexed error-robust fluorescence in situ hybridization (MERFISH)-based method for genome-scale chromatin tracing and demonstrate simultaneous imaging of more than 1,000 genomic loci and nascent transcripts of more than 1,000 genes together with landmark nuclear structures. Using this technology, we characterize chromatin domains, compartments, and trans-chromosomal interactions and their relationship to transcription in single cells. We envision broad application of this high-throughput, multi-scale, and multi-modal imaging technology, which provides an integrated view of chromatin organization in its native structural and functional context.
Subject(s)
Cell Nucleus/metabolism , Chromatin/metabolism , Chromosomes, Human/metabolism , High-Throughput Screening Assays/methods , In Situ Hybridization, Fluorescence/methods , Single-Cell Analysis/methods , Algorithms , Cell Line , Cell Nucleus/genetics , Chromatin/genetics , Chromosomes, Human/genetics , DNA/genetics , DNA/metabolism , Genomics , Humans , Image Processing, Computer-Assisted , Molecular Conformation , Multimodal Imaging , Nucleolus Organizer Region/genetics , Nucleolus Organizer Region/metabolism , RNA/genetics , RNA/metabolism , SoftwareABSTRACT
Chromatin domains and their associated structures must be faithfully inherited through cellular division to maintain cellular identity. However, accessing the localized strategies preserving chromatin domain inheritance, specifically the transfer of parental, pre-existing nucleosomes with their associated post-translational modifications (PTMs) during DNA replication, is challenging in living cells. We devised an inducible, proximity-dependent labeling system to irreversibly mark replication-dependent H3.1 and H3.2 histone-containing nucleosomes at desired loci in mouse embryonic stem cells so that their fate after DNA replication could be followed. Strikingly, repressed chromatin domains are preserved through local re-deposition of parental nucleosomes. In contrast, nucleosomes decorating active chromatin domains do not exhibit such preservation. Notably, altering cell fate leads to an adjustment of the positional inheritance of parental nucleosomes that reflects the corresponding changes in chromatin structure. These findings point to important mechanisms that contribute to parental nucleosome segregation to preserve cellular identity.
Subject(s)
Chromatin Assembly and Disassembly/genetics , Chromatin/genetics , Epigenesis, Genetic , Nucleosomes/genetics , Animals , Cell Differentiation/genetics , Cell Division/genetics , Cell Lineage/genetics , DNA Replication/genetics , Histones/genetics , Mice , Mouse Embryonic Stem Cells/metabolism , Nucleosomes/metabolism , Protein Processing, Post-Translational/geneticsABSTRACT
It is largely unclear whether genes that are naturally embedded in lamina-associated domains (LADs) are inactive due to their chromatin environment or whether LADs are merely secondary to the lack of transcription. We show that hundreds of human promoters become active when moved from their native LAD position to a neutral context in the same cells, indicating that LADs form a repressive environment. Another set of promoters inside LADs is able to "escape" repression, although their transcription elongation is attenuated. By inserting reporters into thousands of genomic locations, we demonstrate that escaper promoters are intrinsically less sensitive to LAD repression. This is not simply explained by promoter strength but by the interplay between promoter sequence and local chromatin features that vary strongly across LADs. Enhancers also differ in their sensitivity to LAD chromatin. This work provides a general framework for the systematic understanding of gene regulation by repressive chromatin.
Subject(s)
Gene Expression Regulation/genetics , Nuclear Lamina/genetics , Promoter Regions, Genetic/genetics , Chromatin/genetics , Chromatin/metabolism , Gene Expression/genetics , Genome, Human/genetics , Genomics , Humans , K562 CellsABSTRACT
Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a major class of lipid-anchored plasma membrane proteins. GPI-APs form nanoclusters generated by cortical acto-myosin activity. While our understanding of the physical principles governing this process is emerging, the molecular machinery and functional relevance of GPI-AP nanoclustering are unknown. Here, we first show that a membrane receptor signaling pathway directs nanocluster formation. Arg-Gly-Asp motif-containing ligands bound to the ß1-integrin receptor activate src and focal adhesion kinases, resulting in RhoA signaling. This cascade triggers actin-nucleation via specific formins, which, along with myosin activity, drive the nanoclustering of membrane proteins with actin-binding domains. Concurrently, talin-mediated activation of the mechano-transducer vinculin is required for the coupling of the acto-myosin machinery to inner-leaflet lipids, thereby generating GPI-AP nanoclusters. Second, we show that these nanoclusters are functional; disruption of their formation either in GPI-anchor remodeling mutants or in vinculin mutants impairs cell spreading and migration, hallmarks of integrin function.
Subject(s)
Integrin beta1/metabolism , Mechanotransduction, Cellular , Membrane Microdomains/metabolism , Amino Acid Motifs , Animals , CHO Cells , Cricetulus , Focal Adhesion Protein-Tyrosine Kinases/genetics , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Humans , Integrin beta1/genetics , Membrane Microdomains/genetics , Vinculin/genetics , Vinculin/metabolism , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolism , src-Family Kinases/genetics , src-Family Kinases/metabolismABSTRACT
Infectious disease is both a major force of selection in nature and a prime cause of yield loss in agriculture. In plants, disease resistance is often conferred by nucleotide-binding leucine-rich repeat (NLR) proteins, intracellular immune receptors that recognize pathogen proteins and their effects on the host. Consistent with extensive balancing and positive selection, NLRs are encoded by one of the most variable gene families in plants, but the true extent of intraspecific NLR diversity has been unclear. Here, we define a nearly complete species-wide pan-NLRome in Arabidopsis thaliana based on sequence enrichment and long-read sequencing. The pan-NLRome largely saturates with approximately 40 well-chosen wild strains, with half of the pan-NLRome being present in most accessions. We chart NLR architectural diversity, identify new architectures, and quantify selective forces that act on specific NLRs and NLR domains. Our study provides a blueprint for defining pan-NLRomes.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , NLR Proteins/genetics , Alleles , Arabidopsis Proteins/metabolism , Disease Resistance/genetics , Genetic Variation , Genome, Plant , NLR Proteins/metabolism , Plant Diseases/genetics , Plant Immunity , Species SpecificityABSTRACT
Genomic instability in disease and its fidelity in health depend on the DNA damage response (DDR), regulated in part from the complex of meiotic recombination 11 homolog 1 (MRE11), ATP-binding cassette-ATPase (RAD50), and phosphopeptide-binding Nijmegen breakage syndrome protein 1 (NBS1). The MRE11-RAD50-NBS1 (MRN) complex forms a multifunctional DDR machine. Within its network assemblies, MRN is the core conductor for the initial and sustained responses to DNA double-strand breaks, stalled replication forks, dysfunctional telomeres, and viral DNA infection. MRN can interfere with cancer therapy and is an attractive target for precision medicine. Its conformations change the paradigm whereby kinases initiate damage sensing. Delineated results reveal kinase activation, posttranslational targeting, functional scaffolding, conformations storing binding energy and enabling access, interactions with hub proteins such as replication protein A (RPA), and distinct networks at DNA breaks and forks. MRN biochemistry provides prototypic insights into how it initiates, implements, and regulates multifunctional responses to genomic stress.
Subject(s)
DNA Damage , DNA Repair , DNA Replication , MRE11 Homologue Protein/metabolism , DNA Repair Enzymes/chemistry , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Immunity, Innate , MRE11 Homologue Protein/chemistry , MRE11 Homologue Protein/genetics , Models, Biological , Models, Molecular , Signal Transduction , Telomere/metabolismABSTRACT
In this review, we describe speculative ideas and early stage research concerning the flow of genetic information from the nuclear residence of genes to the disparate, cytoplasmic sites of protein synthesis. We propose that this process of information transfer is meticulously guided by transient structures formed from protein segments of low sequence complexity/intrinsic disorder. These low complexity domains are ubiquitously associated with regulatory proteins that control gene expression and RNA biogenesis, but they are also found in the central channel of nuclear pores, the nexus points of intermediate filament assembly, and the locations of action of other well-studied cellular proteins and pathways. Upon being organized into localized cellular positions via mechanisms utilizing properly folded protein domains, thereby facilitating elevated local concentration, certain low complexity domains adopt cross-ß interactions that are both structurally specific and labile to disassembly. These weakly tethered assemblies, we propose, are built to relay the passage of genetic information from one site to another within a cell, ensuring that the process is of extreme fidelity.
Subject(s)
Intrinsically Disordered Proteins/genetics , Intrinsically Disordered Proteins/metabolism , Models, Biological , Animals , Cytoplasmic Granules/genetics , Cytoplasmic Granules/metabolism , Gene Expression , Heredodegenerative Disorders, Nervous System/genetics , Heredodegenerative Disorders, Nervous System/metabolism , Humans , Hydrogels , Intrinsically Disordered Proteins/chemistry , Models, Molecular , Mutation , Protein Interaction Domains and Motifs , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Protein FUS/chemistry , RNA-Binding Protein FUS/genetics , RNA-Binding Protein FUS/metabolismABSTRACT
Cytokine expression during T cell differentiation is a highly regulated process that involves long-range promoter-enhancer and CTCF-CTCF contacts at cytokine loci. Here, we investigated the impact of dynamic chromatin loop formation within the topologically associating domain (TAD) in regulating the expression of interferon gamma (IFN-γ) and interleukin-22 (IL-22); these cytokine loci are closely located in the genome and are associated with complex enhancer landscapes, which are selectively active in type 1 and type 3 lymphocytes. In situ Hi-C analyses revealed inducible TADs that insulated Ifng and Il22 enhancers during Th1 cell differentiation. Targeted deletion of a 17 bp boundary motif of these TADs imbalanced Th1- and Th17-associated immunity, both in vitro and in vivo, upon Toxoplasma gondii infection. In contrast, this boundary element was dispensable for cytokine regulation in natural killer cells. Our findings suggest that precise cytokine regulation relies on lineage- and developmental stage-specific interactions of 3D chromatin architectures and enhancer landscapes.
Subject(s)
CCCTC-Binding Factor , Cell Differentiation , Interferon-gamma , Interleukin-22 , Interleukins , Th1 Cells , Animals , CCCTC-Binding Factor/metabolism , CCCTC-Binding Factor/genetics , Th1 Cells/immunology , Mice , Cell Differentiation/immunology , Interferon-gamma/metabolism , Binding Sites , Interleukins/metabolism , Interleukins/genetics , Enhancer Elements, Genetic/genetics , Mice, Inbred C57BL , Chromatin/metabolism , Toxoplasmosis/immunology , Toxoplasmosis/parasitology , Toxoplasmosis/genetics , Gene Expression Regulation , Toxoplasma/immunology , Cytokines/metabolism , Cell Lineage , Th17 Cells/immunologyABSTRACT
Noncoding mutations in cancer genomes are frequent but challenging to interpret. PVT1 encodes an oncogenic lncRNA, but recurrent translocations and deletions in human cancers suggest alternative mechanisms. Here, we show that the PVT1 promoter has a tumor-suppressor function that is independent of PVT1 lncRNA. CRISPR interference of PVT1 promoter enhances breast cancer cell competition and growth in vivo. The promoters of the PVT1 and the MYC oncogenes, located 55 kb apart on chromosome 8q24, compete for engagement with four intragenic enhancers in the PVT1 locus, thereby allowing the PVT1 promoter to regulate pause release of MYC transcription. PVT1 undergoes developmentally regulated monoallelic expression, and the PVT1 promoter inhibits MYC expression only from the same chromosome via promoter competition. Cancer genome sequencing identifies recurrent mutations encompassing the human PVT1 promoter, and genome editing verified that PVT1 promoter mutation promotes cancer cell growth. These results highlight regulatory sequences of lncRNA genes as potential disease-associated DNA elements.
Subject(s)
Breast Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Genes, myc , RNA, Long Noncoding/genetics , Animals , Breast Neoplasms/metabolism , CRISPR-Cas Systems , Carcinogenesis/genetics , Cell Line, Tumor , Cell Proliferation , Cell Transformation, Neoplastic , Chromatin , DNA, Neoplasm/genetics , Enhancer Elements, Genetic , Female , Gene Expression Profiling , Humans , Mice , Mice, Inbred NOD , Mutation , Neoplasm Transplantation , Promoter Regions, Genetic , RNA, Long Noncoding/metabolism , Transcription, GeneticABSTRACT
Non-coding genetic variation is a major driver of phenotypic diversity and allows the investigation of mechanisms that control gene expression. Here, we systematically investigated the effects of >50 million variations from five strains of mice on mRNA, nascent transcription, transcription start sites, and transcription factor binding in resting and activated macrophages. We observed substantial differences associated with distinct molecular pathways. Evaluating genetic variation provided evidence for roles of â¼100 TFs in shaping lineage-determining factor binding. Unexpectedly, a substantial fraction of strain-specific factor binding could not be explained by local mutations. Integration of genomic features with chromatin interaction data provided evidence for hundreds of connected cis-regulatory domains associated with differences in transcription factor binding and gene expression. This system and the >250 datasets establish a substantial new resource for investigation of how genetic variation affects cellular phenotypes.
Subject(s)
Genetic Variation , Macrophages/metabolism , Transcription Factors/metabolism , Animals , Binding Sites , Bone Marrow Cells/cytology , CCAAT-Enhancer-Binding Protein-beta/genetics , CCAAT-Enhancer-Binding Protein-beta/metabolism , Cluster Analysis , Enhancer Elements, Genetic/genetics , Female , Gene Expression Regulation/drug effects , Lipopolysaccharides/pharmacology , Macrophages/cytology , Macrophages/drug effects , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred NOD , Promoter Regions, Genetic , Protein Binding , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/geneticsABSTRACT
Mammalian chromosomes are partitioned into A/B compartments and topologically associated domains (TADs). The inactive X (Xi) chromosome, however, adopts a distinct conformation without evident compartments or TADs. Here, through exploration of an architectural protein, structural-maintenance-of-chromosomes hinge domain containing 1 (SMCHD1), we probe how the Xi is reconfigured during X chromosome inactivation. A/B compartments are first fused into "S1" and "S2" compartments, coinciding with Xist spreading into gene-rich domains. SMCHD1 then binds S1/S2 compartments and merges them to create a compartment-less architecture. Contrary to current views, TADs remain on the Xi but in an attenuated state. Ablating SMCHD1 results in a persistent S1/S2 organization and strengthening of TADs. Furthermore, loss of SMCHD1 causes regional defects in Xist spreading and erosion of heterochromatic silencing. We present a stepwise model for Xi folding, where SMCHD1 attenuates a hidden layer of Xi architecture to facilitate Xist spreading.
Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Mammalian/chemistry , X Chromosome Inactivation , Alleles , Animals , Cell Line , Chromosomal Proteins, Non-Histone/genetics , Chromosomes, Mammalian/metabolism , DNA Methylation , Female , Heterochromatin/metabolism , Histones/genetics , Histones/metabolism , Male , Mice , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/metabolism , Principal Component Analysis , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolismABSTRACT
More than 25 inherited human disorders are caused by the unstable expansion of repetitive DNA sequences termed short tandem repeats (STRs). A fundamental unresolved question is why some STRs are susceptible to pathologic expansion, whereas thousands of repeat tracts across the human genome are relatively stable. Here, we discover that nearly all disease-associated STRs (daSTRs) are located at boundaries demarcating 3D chromatin domains. We identify a subset of boundaries with markedly higher CpG island density compared to the rest of the genome. daSTRs specifically localize to ultra-high-density CpG island boundaries, suggesting they might be hotspots for epigenetic misregulation or topological disruption linked to STR expansion. Fragile X syndrome patients exhibit severe boundary disruption in a manner that correlates with local loss of CTCF occupancy and the degree of FMR1 silencing. Our data uncover higher-order chromatin architecture as a new dimension in understanding repeat expansion disorders.
Subject(s)
Chromatin/genetics , Microsatellite Repeats/physiology , Trinucleotide Repeat Expansion/physiology , Adult , Brain/cytology , Brain/pathology , CCCTC-Binding Factor/genetics , CCCTC-Binding Factor/physiology , Cell Line , Chromatin/physiology , Chromatin Assembly and Disassembly/genetics , Chromatin Assembly and Disassembly/physiology , CpG Islands/genetics , CpG Islands/physiology , DNA/genetics , Disease/etiology , Disease/genetics , Female , Fragile X Mental Retardation Protein/genetics , Fragile X Mental Retardation Protein/metabolism , Fragile X Mental Retardation Protein/physiology , Fragile X Syndrome/genetics , Fragile X Syndrome/metabolism , Genome, Human/genetics , Humans , Male , Microsatellite Repeats/genetics , Trinucleotide Repeat Expansion/geneticsABSTRACT
Lipids are produced site-specifically in cells and then distributed nonrandomly among membranes via vesicular and nonvesicular trafficking mechanisms. The latter involves soluble amphitropic proteins extracting specific lipids from source membranes to function as molecular solubilizers that envelope their insoluble cargo before transporting it to destination sites. Lipid-binding and lipid transfer structural motifs range from multi-ß-strand barrels, to ß-sheet cups and baskets covered by α-helical lids, to multi-α-helical bundles and layers. Here, we focus on how α-helical proteins use amphipathic helical layering and bundling to form modular lipid-binding compartments and discuss the functional consequences. Preformed compartments generally rely on intramolecular disulfide bridging to maintain conformation (e.g., albumins, nonspecific lipid transfer proteins, saposins, nematode polyprotein allergens/antigens). Insights into nonpreformed hydrophobic compartments that expand and adapt to accommodate a lipid occupant are few and provided mostly by the three-layer, α-helical ligand-binding domain of nuclear receptors. The simple but elegant and nearly ubiquitous two-layer, α-helical glycolipid transfer protein (GLTP)-fold now further advances understanding.
Subject(s)
Albumins/chemistry , Allergens/chemistry , Antigens/chemistry , Carrier Proteins/chemistry , Lipids/chemistry , Albumins/genetics , Albumins/metabolism , Allergens/genetics , Allergens/metabolism , Animals , Antigens/genetics , Antigens/metabolism , Binding Sites , Biological Transport , Carrier Proteins/genetics , Carrier Proteins/metabolism , Gene Expression , Humans , Lipid Metabolism , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein DomainsABSTRACT
In metazoan cell nuclei, hundreds of large chromatin domains are in close contact with the nuclear lamina. Such lamina-associated domains (LADs) are thought to help organize chromosomes inside the nucleus and have been associated with gene repression. Here, we discuss the properties of LADs, the molecular mechanisms that determine their association with the nuclear lamina, their dynamic links with other nuclear compartments, and their proposed roles in gene regulation.
Subject(s)
Cell Nucleus/chemistry , Chromatin/chemistry , Animals , Cell Nucleus/metabolism , Gene Expression Regulation , Heterochromatin , Humans , Lamins/metabolism , Nuclear Lamina/chemistry , Nuclear Pore/metabolismABSTRACT
In this study, we show that evolutionarily conserved chromosome loop anchors bound by CCCTC-binding factor (CTCF) and cohesin are vulnerable to DNA double strand breaks (DSBs) mediated by topoisomerase 2B (TOP2B). Polymorphisms in the genome that redistribute CTCF/cohesin occupancy rewire DNA cleavage sites to novel loop anchors. While transcription- and replication-coupled genomic rearrangements have been well documented, we demonstrate that DSBs formed at loop anchors are largely transcription-, replication-, and cell-type-independent. DSBs are continuously formed throughout interphase, are enriched on both sides of strong topological domain borders, and frequently occur at breakpoint clusters commonly translocated in cancer. Thus, loop anchors serve as fragile sites that generate DSBs and chromosomal rearrangements. VIDEO ABSTRACT.
Subject(s)
Chromosome Fragility , DNA Breaks, Double-Stranded , Neoplasms/genetics , Animals , B-Lymphocytes/metabolism , CCCTC-Binding Factor , Cell Line, Tumor , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Humans , Mice , Mice, Inbred C57BL , Poly-ADP-Ribose Binding Proteins , Repressor Proteins/metabolismABSTRACT
Nuclear pore complexes (NPCs) are â¼100 MDa transport channels assembled from multiple copies of â¼30 nucleoporins (Nups). One-third of these Nups contain phenylalanine-glycine (FG)-rich repeats, forming a diffusion barrier, which is selectively permeable for nuclear transport receptors that interact with these repeats. Here, we identify an additional function of FG repeats in the structure and biogenesis of the yeast NPC. We demonstrate that GLFG-containing FG repeats directly bind to multiple scaffold Nups in vitro and act as NPC-targeting determinants in vivo. Furthermore, we show that the GLFG repeats of Nup116 function in a redundant manner with Nup188, a nonessential scaffold Nup, to stabilize critical interactions within the NPC scaffold needed for late steps of NPC assembly. Our results reveal a previously unanticipated structural role for natively unfolded GLFG repeats as Velcro to link NPC subcomplexes and thus add a new layer of connections to current models of the NPC architecture.