Studies on chromatin topology with photoactivatable cross-linkers [proceedings].
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The influence of 10n and 10n+5 linker lengths on chromatin fiber topologies explored by mesoscale modeling.
The structural organization of chromatin is intricately influenced by the length of linker DNA connecting nucleosomes. Some studies have suggested preferred linker lengths of 10n and 10n+5 base pairs (bp) (n = integer). Because these lengths dictate the rotational orientation of successive nucleosomes in the fiber axis, they can markedly affect chromatin fiber compaction and topology. Using a refined mesoscale chromatin model with 5-bp resolution, we investigate the influence of linker DNA periodicity, linker histone density, salt concentration, and starting fiber topology on chromatin architecture for regular fibers versus "life-like" fibers, the latter with irregular spacing between nucleosomes. Our results reveal that regular fibers with 10n linkers exhibit compact zigzag configurations, whereas 10n+5 linkers generate more open and flexible structures. However, these effects are pronounced only for short linker lengths, as longer linkers are more heterogeneous. Moreover, increased linker histone density further enhances compaction for long linker lengths, and lower salt concentration modifies chromatin topologies, diminishing periodicity-driven effects. In addition, any periodicity effect in tightly packed solenoid configurations is much less pronounced. All these trends for regular fibers are reduced in life-like fibers with irregularly spaced nucleosomes, despite having the same average spacing. Moreover, the trend details depend highly on specific features of the fiber architecture as designed in experiments and simulations. Overall, our study highlights how reported differences depend on modeling details and emphasizes the role of linker DNA length in regulating chromatin fiber architecture and its potential implications for genome accessibility and expression.
Chromatin↗
Foundation model reveals the shared organization of transcription and topologically associating domains.
The three-dimensional organization of chromatin into topologically associating domains (TADs) may impact gene regulation by bringing distant genes into contact. However, studies of TADs' function and their influence on transcription have been constrained by ambiguities in TAD boundary definitions and challenges in directly measuring their regulatory effects. We overcome these limitations by developing species-level consensus TAD maps for human and mouse by using a bag-of-genes approach that exposes an emergent regulatory structure. To quantify TAD-mediated relationships, we use a foundation model trained on 33 million transcriptomes to define a contextual similarity metric that captures higher-order relationships missed by co-expression. We find that TADs are regions of elevated co-regulation, with our framework yielding testable hypotheses about chromatin organization across cellular states. This TAD-linked enhancement is strongest during early development and declines with aging, while cancer cells show distinct TAD usage that shifts with chemotherapy. Together, these findings suggest that chromatin organization acts through probabilistic rather than deterministic mechanisms.
Humans↗
Fetal signatures in the 3D genome of iPSC-derived neurons and their implications for disease modeling.
Induced pluripotent stem cells (iPSCs) have revolutionized neuroscience, providing an approach to generate patient-specific neurons for modeling of neurological diseases. However, it remains unclear how closely iPSC-derived neurons replicate the chromatin architecture of authentic brain neurons. Here, we uniformly processed newly generated Hi-C data from iPSC-derived neurons and neurons isolated from the human postmortem brain, together with previously published data sets comprising 228 human and 89 mouse Hi-C and snm3C-seq samples from different cell subtypes. These data were merged into 96 high-coverage contact maps used to examine chromatin features ranging from chromatin compartments and topologically associating domains (TADs) to chromatin loops, Polycomb-mediated contacts, and frequently interacting regions (FIREs). We find that iPSC-derived neurons largely retain the chromatin state of undifferentiated cells and resemble fetal rather than mature neurons. iPSC-derived neurons exhibit unusually strong compartmentalization, an enrichment of developmental genes at TAD borders, and a marked reduction of long-range repressive Polycomb-mediated contacts that typically silence early fetal programs. Although immature, iPSC-derived neurons offer advantages for modeling interactions between disease-associated SNPs and target genes, as many psychiatric disorders have neurodevelopmental origins. Integrating iPSC-derived and postmortem neuronal data sets therefore provides complementary insights into the chromatin landscape underlying disease-associated interactions. Our study offers a valuable Hi-C resource for the community and provides a detailed comparison of chromatin architecture throughout neuronal maturation, underscoring its importance for validating neuronal models and providing a robust framework for future studies.
Journal Article↗
dCas-Based Tools to Visualize Chromatin or Modify Epigenetic Marks at Specific Plant Genomic Loci.
Development of locus-specific approaches targeting precise regions on chromatin, for locus/transcription visualization or transcription/epigenetic marks editing, is a critical challenge in functional genetics and epigenetics. Systems engineered from the clustered regularly interspaced short palindromic repeats (CRISPR) and its associated endonuclease (Cas) operate through DNA sequence-specific recognition by so-called guide RNAs, which provides high flexibility and modularity for precise chromatin visualization or edition. Here, we provide an overview of the CRISPR/Cas-derived tools developed for visualization of chromatin loci in live imaging or for effective modification of gene expression. These tools make use of effector modules that combine activators, repressors, and epigenetic modifiers with a deactivated Cas protein (dCas). We present how their use in plants brought advances in visualizing or manipulating the expression of loci involved in agronomically interesting traits such as flowering time and response to drought or heat. We also discuss the limitations and future improvements of the dCas-related technologies, such as more compact and combinatorial systems, spatiotemporal targeting for fine-tuning of gene expression, and live visualization of chromatin dynamics.
Chromatin↗
Architectural logic of the 3D genome: mechanisms of dysregulation and emerging cancer therapeutics.
The three-dimensional (3D) genome provides an essential layer of organization that shapes genome function in space and time. Chromatin compartments and topologically associating domains (TADs) arise from the interplay between intrinsic properties of chromatin and architectural factors, including cohesin and CTCF. Despite substantial progress in defining these structural features, whether 3D genome architecture plays a causal role in regulating processes such as transcription, DNA replication, and DNA repair, or instead reflects underlying regulatory activity, remains unresolved. Here, we use the distinction between chromatin-intrinsic features and architectural factors as a framework to evaluate evidence for causality in genome structure-function relationships. We extend this framework to cancer, where both intrinsic alterations (including noncoding mutations, structural variants, and changes in chromatin state) and architectural factor perturbations (such as mutations in architectural proteins and dysregulation of transcriptional machinery) disrupt genome organization and contribute to disease progression. These findings suggest that alterations in genome structure can, in some contexts, actively reshape oncogenic programs. A major limitation in applying 3D genome insights to cancer biology is the cost and complexity of omics assays. Recent advances in artificial intelligence (AI) and machine learning (ML) enable inference and prediction of 3D genome organization from sequence and epigenomic features, providing insight into the extent to which genome folding is encoded intrinsically versus dynamically regulated in architectural factors. This perspective provides a unified view of how genome structure is established, how it relates to function, and how its disruption contributes to tumorigenesis.
3D genome↗
esBAF and INO80C fine-tune subcompartments and differentially regulate enhancer-promoter interactions.
The genome is compacted in the nucleus through a hierarchical chromatin organization, ranging from chromosome territories to compartments, topologically associating domains (TADs), and individual nucleosomes. Nucleosome remodeling complexes hydrolyze ATP to translocate DNA and thereby mobilize histone proteins. While nucleosome remodeling complexes have been extensively studied for their roles in regulating nucleosome positioning and accessibility, their contributions to higher-order chromatin architecture remain less well understood. Here, we investigate the roles of two key nucleosome remodelers, esBAF and INO80C, in shaping 3D genome organization in mouse embryonic stem cells. Using Hi-C, we find that loss of either remodeler has minimal effects on global compartment or TAD structures. In contrast, subcompartment organization is notably altered, suggesting that esBAF and INO80C contribute to finer-scale chromatin topology. To overcome the limited resolution of Hi-C for detecting regulatory loops, we employed promoter capture Micro-C (PCMC), which revealed that the loss of esBAF or INO80C alters a subset of promoter anchored looping interactions. Although these changes occur at distinct genomic loci for each remodeler, the affected sites are commonly enriched for bivalent chromatin regions bound by OCT4, SOX2, and NANOG (OSN), as well as BRG1 and INO80 themselves. Together, our findings reveal that esBAF and INO80C selectively influence subcompartment identity and enhancer-promoter communication at key regulatory loci, highlighting a previously underappreciated role for nucleosome remodelers in higher-order chromatin organization.
chromatin↗
Chromatin behaviour during the mitotic cell cycle of Saccharomyces cerevisiae.
Chromatin behaviour during the cell division cycle of the yeast Saccharomyces cerevisiae has been investigated in cells which have been depleted of 90% of their RNA by digestion with ribonuclease. Removal of large amounts of RNA from the yeast nucleus before treatment of the cells with heavy metal fixatives and stains permits chromatin to be visualized with extreme clarity in thin sections of cells processed for electron microscopy by conventional procedures. Spindle pole bodies were also visualized by this treatment, although the associated microtubules were not. Chromatin is dispersed during interphase and occupies the non-nucleolar region of the nucleus which is known to be Feulgen-positive from light microscopy. Because spindle microtubules are not visualized, direct attachment of microtubules to chromatin fibrils could not be verified. However, chromatin was not attached directly to the spindle pole bodies and kinetochore differentiations were not observed in the nucleoplasm. During nuclear division chromatin remains dispersed and does not condense into discrete chromatids. As the nucleus expands into the bud, chromosomal distribution to the daughter cells is thought to result from the separation of the poles of the spindle apparatus with attached chromatin fibrils. However, that such distribution is occurring as the nucleus elongates is not obvious until an advanced stage of nuclear division is reached and partition of the nucleus is nearly complete. Thus, no aggregation of chromatin into metaphase or anaphase plates occurs and the appearance of chromatin during mitosis is essentially the same as in interphase. These observations indicate that the marked changes in the topological structure of chromatin which characterize mitosis in the higher eukaryotes do not occur in S. cerevisiae.
Cell Division↗
Enhanced chromatin compaction is associated with de novo expression of a nuclear microprotein, global loss of H3 acetylation and local transcriptional changes in retinal rod photoreceptors.
We have limited understanding of how aging alters gene expression and remodels cellular architecture in post-mitotic neurons. The inverted nuclear organization of mouse rod photoreceptors provides a unique model to gain mechanistic insights into age-associated decline in neuronal function. We have generated and integrated multi-omic datasets including 3D-genome topology, histone modifications, chromatin accessibility, DNA methylation and transcriptome of rod photoreceptors from young- and aged-mice. We show that aging drives global chromatin compaction, with regional alterations enriched at active chromatin. Epigenomic and transcriptional changes broadly correlate with chromatin dynamics as validated by high resolution microscopy. We uncover a megabase-sized genomic region with multi-level alterations, including de novo transcription of Gm7239, which encodes a functional microprotein carrying histone acetyltransferase-inhibitor domain. Overexpression of Gm7239 is associated with global loss of histone H3 acetylation, highlighting a potential new axis of genomic regulation in aging. Finally, we identify multiple significant local transcriptional alterations in non-annotated regions and genes associated with age-related macular degeneration. Our studies link age-related chromatin landscape changes with gene expression that may influence rod function and vulnerability to diseases.
Journal Article↗
PRC1 and CTCF-Mediated Transition from Poised to Active Chromatin Loops Drives Bivalent Gene Activation.
Polycomb Repressive Complex 1 (PRC1) and CCCTC-binding factor (CTCF) are critical regulators of 3D chromatin architecture that influence cellular transcriptional programs. Spatial chromatin structures comprise conserved compartments, topologically associating domains (TADs), and dynamic, cell-type-specific chromatin loops. Although the role of CTCF in chromatin organization is well-known, the involvement of PRC1 is less understood. In this study, we identified an unexpected, essential role for the canonical Pcgf2-containing PRC1 complex (cPRC1.2), a known transcriptional repressor, in activating bivalent genes during differentiation. Our Hi-C analysis revealed that cPRC1.2 forms chromatin loops at bivalent promoters, rendering them silent yet poised for activation. Using mouse embryonic stem cells (ESCs) with CRISPR/Cas9-mediated gene editing, we found that the loss of Pcgf2, though not affecting the global level of H2AK119ub1, disrupts these cPRC1.2 loops in ESCs and impairs the transcriptional induction of crucial target genes necessary for neuronal differentiation. Furthermore, we identified CTCF enrichment at cPRC1.2 loop anchors and at Polycomb group (PcG) bodies, nuclear foci with concentrated PRC1 and its tethered chromatin domains, suggesting that PRC1 and CTCF cooperatively shape chromatin loop structures. Through virtual 4C and other genomic analyses, we discovered that establishing neuronal progenitor cell (NPC) identity involves a switch from cPRC1.2-mediated chromatin loops to CTCF-mediated active loops, enabling the expression of critical lineage-specific factors. This study uncovers a novel mechanism by which pre-formed PRC1 and CTCF loops at lineage-specific genes maintain a poised state for subsequent gene activation, advancing our understanding of the role of chromatin architecture in controlling cell fate transitions.
Journal Article↗
Structural changes in simian virus 40 chromatin as probed by restriction endonucleases.
The structure of simian virus 40 (SV40) chromatin was probed by treatment with single- and multiple-site bacterial restriction endonucleases. Approximately the same fraction of the chromatin DNA was cleaved by each of three different single-site endonucleases, indicating that the nucleosomes do not have unique positions with regard to specific nucleotide sequences within the population of chromatin molecules. However, the extent of digestion was found to be strongly influenced by salt concentration. At 100 mM NaCl-5 mM MgCl2, only about 20% of the simian virus 40 (SV40) DNA I in chromatin was converted to linear SV40 DNA III. In contrast, at lower concentrations of NaCl (0.05 or 0.01 M), an additional 20 to 30% of the DNA was cleaved. These results suggest that at 100 mM NaCl only the DNA between nucleosomes was accessible to the restriction enzymes, whereas at the lower salt concentrations, DNA within the nucleosome regions became available for cleavage. Surprisingly, when SV40 chromatin was digested with multiple-site restriction enzymes, less than 2% of the DNA was digested to limit digest fragment, whereas only a small fraction (9 to 15%) received two or more cuts. Instead, the principal digest fragment was full-length linear SV40 DNA III. The failure to generate limit digest fragments was not a consequence of reduced enzyme activity in the reaction mixtures or of histone exchange. When the position of the principal cleavage site was mapped after HpaI digestion, it was found that this site was not unique. Nevertheless, all sites wree not cleaved with equal probability. An additional finding was that SV40 chromatin containing nicked-circular DNA II produced by random nicking of DNA I was also resistant to digestion by restriction enzymes. These results suggest that the initial cut which causes relaxation of topological constraint in SV40 chromatin DNA imparts resistance to further digestion by restriction enzymes. We propose that this may be accomplished by either "winding" of the internucleosomal DNA into the body of the nucleosome, or as suggested by others, by successive right-hand rotation of nucleosomes.
Animals↗
Mechanisms of Globin Gene Regulation in Mammals.
Studies of globin gene clusters have established many paradigms of gene regulation. This review focuses on the α- and β-globin gene clusters of humans and mice, summarizing important insights from high-throughput biochemical assays and directed genetic dissections and emphasizing similarities across the types of gene clusters and between species. The overall arrangements and architectures are similar, with each gene cluster being localized within a topologically constrained unit of chromatin containing a multicomponent enhancer (i.e., a locus control region) and other regulatory elements bound by a similar set of transcription factors and coactivators. Differential expression of the globin genes within each cluster during ontogeny is associated with changes in contacts with the locus control region and involves the action of gene-specific repressors. Detailed study of the fetal β-like HBG1 and HBG2 globin genes has revealed a remarkable diversity of regulatory pathways that provide candidates for therapeutic approaches to reactivate these genes for β-hemoglobinopathies.
Animals↗
3D chromatin remodeling during domestication defines novel targets for crop improvement.
Three-dimensional (3D) genome folding shapes gene regulation, yet the genetic underpinnings linking 3D genome evolution to phenotypic innovation during domestication remain elusive. Using population-scale Hi-C profiling of 34 semi-wild and 267 cultivated allotetraploid cottons, we generated a pan-3D genome atlas capturing extensive diversity in topologically associating domains (TADs) and chromatin loops. Chromatin interactome-wide association studies identified 105 TAD reconfigurations and 58 loop rewirings that were established as the 3D chromatin basis of fiber quality, boosting heritability estimates for fiber strength by 16% and fiber length by 20%. We reveal that domestication selection within sequence-defined sweeps fixed 57% of 3D conformation signatures, thereby decoupling sequence-level from chromatin-level selection and shifting the subgenome expression balance of 39 homoeologs in cultivated cotton. Sequence-based modeling and mutational analyses identified the C2H2 zinc-finger protein YY1 as a conserved mediator of 3D genome organization. This study provides a resource for redefining precision-breeding paradigms by harnessing cryptic 3D chromatin targets.
3D genome↗
Loss of SUMOylation drives aberrant PRC1 clustering and 3D genome rewiring independent of H3K27me3.
Polycomb repressive complex 1 (PRC1) forms nuclear condensates that organize target chromatin domains. SUMOylation modulates PRC1 clustering, but its impact on condensate properties and 3D genome architecture remains unclear. Here, we show that depletion of small ubiquitin-like modifier (SUMO) in Drosophila wing imaginal discs transforms PRC1 condensates into large structures with reduced molecular dynamics. Biophysical modeling suggests that the changes in PRC1 self-interactions are responsible for the formation of large PRC1 condensates when SUMO is depleted. Interestingly, this biophysical reorganization occurs without global loss of the H3K27me3 mark. Instead, Hi-C reveals widespread rewiring of topologically associating domain (TAD) interactions. PRC1-bound TADs lose specific long-range contacts with each other while gaining ectopic interactions with active chromatin. These topological shifts correlate with gene misregulation independently of changes in Polycomb histone modifications. Our results establish SUMOylation as a critical regulator of PRC1 condensates, demonstrating that post-translational control of biomolecular condensation modulates 3D genome architecture and transcriptional output through mechanisms separable from histone mark deposition.
Animals↗
The structure of SV 40 chromatin.
Simian virus 40 (SV40) nucleoprotein complexes were studied with the electron microscope. Depending on the isolation procedure, SV40 chromatin has two different conformations: complexes isolated in the presence of 0.15 M NaCl appeared as very compact globular structures, while those isolated in the presence of 0.6 M NaCl had the typical 'beads-on-a-string' appearance of the primary nucleofilament. Concomitant with this structural change was a variation in the histone pattern and sedimentation behaviour of the complexes: with NaCl at 0.15 mol 1(-1) the isolated complexes contained both the nucleosomal histones and histone H1, and sedimented in sucrose gradients at 70S. Increasing the ionic strength to 0.6 M NaCl resulted in the removal of histone H1 from the complexes and in a decrease of the sedimentation coefficient to 40S. DNA relaxing enzyme is associated with the SV40 nucleoprotein complexes. The numbers of superhelical turns in DNA from compact and open types of complexes were found to be the same. Therefore the transition from the condensed to the open structure of viral chromatin does not require a change in the topological winding number of its DNA.
Centrifugation, Density Gradient↗
Interaction of the DNA untwisting enzyme with the SV40 nucleoprotein complex.
The SV40 nucleoprotein complex which was isolated from infected CV-1 cells did not possess an active DNA untwisting enzyme. The superhelix density of the DNA in the chromatin complex was unchanged after treatment with purified rat liver DNA untwisting enzyme. However, in the presence of ethidium bromide (1 microgram/ml) the superhelix density was changed. Moreover, the nicked intermediate in the DNA untwisting reaction could be detected using the chromatin DNA as a substrate. These results show that the DNA in the SV40 chromatin which is accessible to the DNA untwisting enzyme is under no topological strain.
Chromatin↗
Genome reorganisation and expansion shape 3D genome architecture and define a distinct regulatory landscape in coleoid cephalopods.
How genomic changes translate into organismal novelties is often confounded by the multi-layered nature of genome architecture and the long evolutionary timescales over which molecular changes accumulate. Coleoid cephalopods (squid, cuttlefish, and octopus) provide a unique system to study these processes due to a large-scale chromosomal rearrangement in the coleoid ancestor that resulted in highly modified karyotypes, followed by lineage-specific fusions, translocations, and repeat expansions. How these events have shaped gene regulatory patterns underlying the evolution of coleoid innovations, including their large and elaborately structured nervous systems, novel organs, and complex behaviours, remains poorly understood. To address this, we integrate Micro-C, RNA-seq, and ATAC-seq across multiple coleoid species, developmental stages, and tissues. We find that while topological compartments are broadly conserved, hundreds of chromatin loops are species- and context-specific, with distinct regulation signatures and dynamic expression profiles. CRISPR-Cas9 knockout of a putative regulatory sequence within a conserved region demonstrates the role of loops in neural development and the prevalence of long-range, inter-compartmental interactions. We propose that differential evolutionary constraints across the coleoid 3D genome allow macroevolutionary processes to shape genome topology in distinct ways, facilitating the emergence of novel regulatory entanglements and ultimately contributing to the evolution and maintenance of complex traits in coleoids.
Journal Article↗
3D chromatin structures precede genome activation in Drosophila embryogenesis.
3D chromatin structure is critical for the regulation of gene expression during development. Here we used Micro-C assays at 100-bp resolution to map genome organization in Drosophila melanogaster throughout the first half of embryogenesis. These high-resolution contact maps reveal fine-scale features such as loops and boundaries delineating topologically associating domains. Notably, we observe that 3D chromatin structures form prior to zygotic genome activation and persist during successive mitotic cycles. Integrative analysis with 149 public chromatin immunoprecipitation sequencing (ChIP-seq) datasets identifies four classes of chromatin structuring elements, including a distinct group enriched for GAGA-associated factor (GAF) and Zelda binding, associated with developmental-gene regulation. These elements are mitotically retained and exhibit sequence and structure similarity between D. melanogaster and D. virilis. We propose that 3D chromatin organization in the pre-cellular embryo facilitates deployment of developmentally regulated genes during Drosophila embryogenesis.
Animals↗