Chromosome-level organization of the regulatory genome in the Drosophila nervous system.

Previous studies have identified topologically associating domains (TADs) as basic units of genome organization. We present evidence of a previously unreported level of genome folding, where distant TAD pairs, megabases apart, interact to form meta-domains. Within meta-domains, gene promoters and structural intergenic elements present in distant TADs are specifically paired. The associated genes encode neuronal determinants, including those engaged in axonal guidance and adhesion. These long-range associations occur in a large fraction of neurons but support transcription in only a subset of neurons. Meta-domains are formed by diverse transcription factors that are able to pair over long and flexible distances. We present evidence that two such factors, GAF and CTCF, play direct roles in this process. The relative simplicity of higher-order meta-domain interactions in Drosophila, compared with those previously described in mammals, allowed the demonstration that genomes can fold into highly specialized cell-type-specific scaffolds that enable megabase-scale regulatory associations.

3D Genome of macaque fetal brain reveals evolutionary innovations during primate corticogenesis.

Elucidating the regulatory mechanisms of human brain evolution is essential to understanding human cognition and mental disorders. We generated multi-omics profiles and constructed a high-resolution map of 3D genome architecture of rhesus macaque during corticogenesis. By comparing the 3D genomes of human, macaque, and mouse brains, we identified many human-specific chromatin structure changes, including 499 topologically associating domains (TADs) and 1,266 chromatin loops. The human-specific loops are significantly enriched in enhancer-enhancer interactions, and the regulated genes show human-specific expression changes in the subplate, a transient zone of the developing brain critical for neural circuit formation and plasticity. Notably, many human-specific sequence changes are located in the human-specific TAD boundaries and loop anchors, which may generate new transcription factor binding sites and chromatin structures in human. Collectively, the presented data highlight the value of comparative 3D genome analyses in dissecting the regulatory mechanisms of brain development and evolution.

Condensed Chromatin Behaves like a Solid on the Mesoscale In Vitro and in Living Cells.

The association of nuclear DNA with histones to form chromatin is essential for temporal and spatial control of eukaryotic genomes. In this study, we examined the physical state of condensed chromatin in vitro and in vivo. Our in vitro studies demonstrate that self-association of nucleosomal arrays under a wide range of solution conditions produces supramolecular condensates in which the chromatin is physically constrained and solid-like. By measuring DNA mobility in living cells, we show that condensed chromatin also exhibits solid-like behavior in vivo. Representative heterochromatin proteins, however, display liquid-like behavior and coalesce around the solid chromatin scaffold. Importantly, euchromatin and heterochromatin show solid-like behavior even under conditions that produce limited interactions between chromatin fibers. Our results reveal that condensed chromatin exists in a solid-like state whose properties resist external forces and create an elastic gel and provides a scaffold that supports liquid-liquid phase separation of chromatin binding proteins.

Physical and Functional Compartmentalization of Archaeal Chromosomes.

The three-dimensional organization of chromosomes can have a profound impact on their replication and expression. The chromosomes of higher eukaryotes possess discrete compartments that are characterized by differing transcriptional activities. Contrastingly, most bacterial chromosomes have simpler organization with local domains, the boundaries of which are influenced by gene expression. Numerous studies have revealed that the higher-order architectures of bacterial and eukaryotic chromosomes are dependent on the actions of structural maintenance of chromosomes (SMC) superfamily protein complexes, in particular, the near-universal condensin complex. Intriguingly, however, many archaea, including members of the genus Sulfolobus do not encode canonical condensin. We describe chromosome conformation capture experiments on Sulfolobus species. These reveal the presence of distinct domains along Sulfolobus chromosomes that undergo discrete and specific higher-order interactions, thus defining two compartment types. We observe causal linkages between compartment identity, gene expression, and binding of a hitherto uncharacterized SMC superfamily protein that we term "coalescin."

3D Chromatin Structures of Mature Gametes and Structural Reprogramming during Mammalian Embryogenesis.

High-order chromatin structure plays important roles in gene expression regulation. Knowledge of the dynamics of 3D chromatin structures during mammalian embryo development remains limited. We report the 3D chromatin architecture of mouse gametes and early embryos using an optimized Hi-C method with low-cell samples. We find that mature oocytes at the metaphase II stage do not have topologically associated domains (TADs). In sperm, extra-long-range interactions (>4 Mb) and interchromosomal interactions occur frequently. The high-order structures of both the paternal and maternal genomes in zygotes and two-cell embryos are obscure but are gradually re-established through development. The establishment of the TAD structure requires DNA replication but not zygotic genome activation. Furthermore, unmethylated CpGs are enriched in A compartment, and methylation levels are decreased to a greater extent in A compartment than in B compartment in embryos. In summary, the global reprogramming of chromatin architecture occurs during early mammalian development.

Chromatin Architecture Emerges during Zygotic Genome Activation Independent of Transcription.

Chromatin architecture is fundamental in regulating gene expression. To investigate when spatial genome organization is first established during development, we examined chromatin conformation during Drosophila embryogenesis and observed the emergence of chromatin architecture within a tight time window that coincides with the onset of transcription activation in the zygote. Prior to zygotic genome activation, the genome is mostly unstructured. Early expressed genes serve as nucleation sites for topologically associating domain (TAD) boundaries. Activation of gene expression coincides with the establishment of TADs throughout the genome and co-localization of housekeeping gene clusters, which remain stable in subsequent stages of development. However, the appearance of TAD boundaries is independent of transcription and requires the transcription factor Zelda for locus-specific TAD boundary insulation. These results offer insight into when spatial organization of the genome emerges and identify a key factor that helps trigger this architecture.

Genome organization regulates nuclear pore complex formation and promotes differentiation during Drosophila oogenesis.

Genome organization can regulate gene expression and promote cell fate transitions. The differentiation of germline stem cells (GSCs) to oocytes in Drosophila involves changes in genome organization mediated by heterochromatin and the nuclear pore complex (NPC). Heterochromatin represses germ cell genes during differentiation, and NPCs anchor these silenced genes to the nuclear periphery, maintaining silencing to allow for oocyte development. Surprisingly, we found that genome organization also contributes to NPC formation, mediated by the transcription factor Stonewall (Stwl). As GSCs differentiate, Stwl accumulates at boundaries between silenced and active gene compartments. Stwl at these boundaries plays a pivotal role in transitioning germ cell genes into a silenced state and activating a group of oocyte genes and nucleoporins (Nups). The upregulation of these Nups during differentiation is crucial for NPC formation and further genome organization. Thus, cross-talk between genome architecture and NPCs is essential for successful cell fate transitions.

The Three-Dimensional Organization of Mammalian Genomes.

Animal development depends on not only the linear genome sequence that embeds millions of cis-regulatory elements, but also the three-dimensional (3D) chromatin architecture that orchestrates the interplay between cis-regulatory elements and their target genes. Compared to our knowledge of the cis-regulatory sequences, the understanding of the 3D genome organization in human and other eukaryotes is still limited. Recent advances in technologies to map the 3D genome architecture have greatly accelerated the pace of discovery. Here, we review emerging concepts of chromatin organization in mammalian cells, discuss the dynamics of chromatin conformation during development, and highlight important roles for chromatin organization in cancer and other human diseases.

HOTTIP-dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia.

HOTTIP lncRNA is highly expressed in acute myeloid leukemia (AML) driven by MLL rearrangements or NPM1 mutations to mediate HOXA topologically associated domain (TAD) formation and drive aberrant transcription. However, the mechanism through which HOTTIP accesses CCCTC-binding factor (CTCF) chromatin boundaries and regulates CTCF-mediated genome topology remains unknown. Here, we show that HOTTIP directly interacts with and regulates a fraction of CTCF-binding sites (CBSs) in the AML genome by recruiting CTCF/cohesin complex and R-loop-associated regulators to form R-loops. HOTTIP-mediated R-loops reinforce the CTCF boundary and facilitate formation of TADs to drive gene transcription. Either deleting CBS or targeting RNase H to eliminate R-loops in the boundary CBS of ß-catenin TAD impaired CTCF boundary activity, inhibited promoter/enhancer interactions, reduced ß-catenin target expression, and mitigated leukemogenesis in xenograft mouse models with aberrant HOTTIP expression. Thus, HOTTIP-mediated R-loop formation directly reinforces CTCF chromatin boundary activity and TAD integrity to drive oncogene transcription and leukemia development.

Transcriptional regulation and chromatin architecture maintenance are decoupled functions at the Sox2 locus.

How distal regulatory elements control gene transcription and chromatin topology is not clearly defined, yet these processes are closely linked in lineage specification during development. Through allele-specific genome editing and chromatin interaction analyses of the Sox2 locus in mouse embryonic stem cells, we found a striking disconnection between transcriptional control and chromatin architecture. We traced nearly all Sox2 transcriptional activation to a small number of key transcription factor binding sites, whose deletions have no effect on promoter-enhancer interaction frequencies or topological domain organization. Local chromatin architecture maintenance, including at the topologically associating domain (TAD) boundary downstream from the Sox2 enhancer, is widely distributed over multiple transcription factor-bound regions and maintained in a CTCF-independent manner. Furthermore, partial disruption of promoter-enhancer interactions by ectopic chromatin loop formation has no effect on Sox2 transcription. These findings indicate that many transcription factors are involved in modulating chromatin architecture independently of CTCF.

XerD unloads bacterial SMC complexes at the replication terminus.

Bacillus subtilis structural maintenance of chromosomes (SMC) complexes are topologically loaded at centromeric sites adjacent to the replication origin by the partitioning protein ParB. These ring-shaped ATPases then translocate down the left and right chromosome arms while tethering them together. Here, we show that the site-specific recombinase XerD, which resolves chromosome dimers, is required to unload SMC tethers when they reach the terminus. We identify XerD-specific binding sites in the terminus region and show that they dictate the site of unloading in a manner that depends on XerD but not its catalytic residue, its partner protein XerC, or the recombination site dif. Finally, we provide evidence that ParB and XerD homologs perform similar functions in Staphylococcus aureus. Thus, two broadly conserved factors that act at the origin and terminus have second functions in loading and unloading SMC complexes that travel between them.

Multi-scale architecture of archaeal chromosomes.

Chromosome conformation capture (3C) technologies have identified topologically associating domains (TADs) and larger A/B compartments as two salient structural features of eukaryotic chromosomes. These structures are sculpted by the combined actions of transcription and structural maintenance of chromosomes (SMC) superfamily proteins. Bacterial chromosomes fold into TAD-like chromosomal interaction domains (CIDs) but do not display A/B compartment-type organization. We reveal that chromosomes of Sulfolobus archaea are organized into CID-like topological domains in addition to previously described larger A/B compartment-type structures. We uncover local rules governing the identity of the topological domains and their boundaries. We also identify long-range loop structures and provide evidence of a hub-like structure that colocalizes genes involved in ribosome biogenesis. In addition to providing high-resolution descriptions of archaeal chromosome architectures, our data provide evidence of multiple modes of organization in prokaryotic chromosomes and yield insights into the evolution of eukaryotic chromosome conformation.

Visualizing the Role of Boundary Elements in Enhancer-Promoter Communication.

Formation of self-associating loop domains is a fundamental organizational feature of metazoan genomes. Here, we employed quantitative live-imaging methods to visualize impacts of higher-order chromosome topology on enhancer-promoter communication in developing Drosophila embryos. Evidence is provided that distal enhancers effectively produce transcriptional bursting from target promoters over distances when they are flanked with boundary elements. Importantly, neither inversion nor deletion of a boundary element abrogates this "enhancer-assisting activity," suggesting that they can facilitate intra-domain enhancer-promoter interaction and production of transcriptional bursting independently of topologically associating domain (TAD) formation. In contrast, domain-skipping activity of distal enhancers was lost after disruption of topological domains. This observation raises a possibility that intra-domain and inter-domain enhancer-promoter interactions are differentially regulated by chromosome topology.

Resolving the 3D Landscape of Transcription-Linked Mammalian Chromatin Folding.

Whereas folding of genomes at the large scale of epigenomic compartments and topologically associating domains (TADs) is now relatively well understood, how chromatin is folded at finer scales remains largely unexplored in mammals. Here, we overcome some limitations of conventional 3C-based methods by using high-resolution Micro-C to probe links between 3D genome organization and transcriptional regulation in mouse stem cells. Combinatorial binding of transcription factors, cofactors, and chromatin modifiers spatially segregates TAD regions into various finer-scale structures with distinct regulatory features including stripes, dots, and domains linking promoters-to-promoters (P-P) or enhancers-to-promoters (E-P) and bundle contacts between Polycomb regions. E-P stripes extending from the edge of domains predominantly link co-expressed loci, often in the absence of CTCF and cohesin occupancy. Acute inhibition of transcription disrupts these gene-related folding features without altering higher-order chromatin structures. Our study uncovers previously obscured finer-scale genome organization, establishing functional links between chromatin folding and gene regulation.

Analysis of Genome Architecture during SCNT Reveals a Role of Cohesin in Impeding Minor ZGA.

Somatic cell nuclear transfer (SCNT) can reprogram a somatic nucleus to a totipotent state. However, the re-organization of 3D chromatin structure in this process remains poorly understood. Using low-input Hi-C, we revealed that, during SCNT, the transferred nucleus first enters a mitotic-like state (premature chromatin condensation). Unlike fertilized embryos, SCNT embryos show stronger topologically associating domains (TADs) at the 1-cell stage. TADs become weaker at the 2-cell stage, followed by gradual consolidation. Compartments A/B are markedly weak in 1-cell SCNT embryos and become increasingly strengthened afterward. By the 8-cell stage, somatic chromatin architecture is largely reset to embryonic patterns. Unexpectedly, we found cohesin represses minor zygotic genome activation (ZGA) genes (2-cell-specific genes) in pluripotent and differentiated cells, and pre-depleting cohesin in donor cells facilitates minor ZGA and SCNT. These data reveal multi-step reprogramming of 3D chromatin architecture during SCNT and support dual roles of cohesin in TAD formation and minor ZGA repression.

To incise or not and where: SET-domain methyltransferases know.

The concept of the histone code posits that histone modifications regulate gene functions once interpreted by epigenetic readers. A well-studied case is trimethylation of lysine 4 of histone H3 (H3K4me3), which is enriched at gene promoters. However, H3K4me3 marks are not needed for the expression of most genes, suggesting extra roles, such as influencing the 3D genome architecture. Here, we highlight an intriguing analogy between the H3K4me3-dependent induction of double-strand breaks in several recombination events and the impact of this same mark on DNA incisions for the repair of bulky lesions. We propose that Su(var)3-9, Enhancer-of-zeste and Trithorax (SET)-domain methyltransferases generate H3K4me3 to guide nucleases into chromatin spaces, the favorable accessibility of which ensures that DNA break intermediates are readily processed, thereby safeguarding genome stability.

Distinct Classes of Chromatin Loops Revealed by Deletion of an RNA-Binding Region in CTCF.

Mammalian genomes are folded into topologically associating domains (TADs), consisting of chromatin loops anchored by CTCF and cohesin. Some loops are cell-type specific. Here we asked whether CTCF loops are established by a universal or locus-specific mechanism. Investigating the molecular determinants of CTCF clustering, we found that CTCF self-association in vitro is RNase sensitive and that an internal RNA-binding region (RBRi) mediates CTCF clustering and RNA interaction in vivo. Strikingly, deleting the RBRi impairs about half of all chromatin loops in mESCs and causes deregulation of gene expression. Disrupted loop formation correlates with diminished clustering and chromatin binding of RBRi mutant CTCF, which in turn results in a failure to halt cohesin-mediated extrusion. Thus, CTCF loops fall into at least two classes: RBRi-independent and RBRi-dependent loops. We speculate that evidence for RBRi-dependent loops may provide a molecular mechanism for establishing cell-specific CTCF loops, potentially regulated by RNA(s) or other RBRi-interacting partners.

Stratifying TAD boundaries pinpoints focal genomic regions of regulation, damage, and repair.

Advances in chromatin mapping have exposed the complex chromatin hierarchical organization in mammals, including topologically associating domains (TADs) and their substructures, yet the functional implications of this hierarchy in gene regulation and disease progression are not fully elucidated. Our study delves into the phenomenon of shared TAD boundaries, which are pivotal in maintaining the hierarchical chromatin structure and regulating gene activity. By integrating high-resolution Hi-C data, chromatin accessibility, and DNA double-strand breaks (DSBs) data from various cell lines, we systematically explore the complex regulatory landscape at high-level TAD boundaries. Our findings indicate that these boundaries are not only key architectural elements but also vibrant hubs, enriched with functionally crucial genes and complex transcription factor binding site-clustered regions. Moreover, they exhibit a pronounced enrichment of DSBs, suggesting a nuanced interplay between transcriptional regulation and genomic stability. Our research provides novel insights into the intricate relationship between the 3D genome structure, gene regulation, and DNA repair mechanisms, highlighting the role of shared TAD boundaries in maintaining genomic integrity and resilience against perturbations. The implications of our findings extend to understanding the complexities of genomic diseases and open new avenues for therapeutic interventions targeting the structural and functional integrity of TAD boundaries.

In Vivo Evidence for ATPase-Dependent DNA Translocation by the Bacillus subtilis SMC Condensin Complex.

Structural maintenance of chromosomes (SMC) complexes shape the genomes of virtually all organisms, but how they function remains incompletely understood. Recent studies in bacteria and eukaryotes have led to a unifying model in which these ring-shaped ATPases act along contiguous DNA segments, processively enlarging DNA loops. In support of this model, single-molecule imaging experiments indicate that Saccharomyces cerevisiae condensin complexes can extrude DNA loops in an ATP-hydrolysis-dependent manner in vitro. Here, using time-resolved high-throughput chromosome conformation capture (Hi-C), we investigate the interplay between ATPase activity of the Bacillus subtilis SMC complex and loop formation in vivo. We show that point mutants in the SMC nucleotide-binding domain that impair but do not eliminate ATPase activity not only exhibit delays in de novo loop formation but also have reduced rates of processive loop enlargement. These data provide in vivo evidence that SMC complexes function as loop extruders.

HMGB2 Loss upon Senescence Entry Disrupts Genomic Organization and Induces CTCF Clustering across Cell Types.

Processes like cellular senescence are characterized by complex events giving rise to heterogeneous cell populations. However, the early molecular events driving this cascade remain elusive. We hypothesized that senescence entry is triggered by an early disruption of the cells' three-dimensional (3D) genome organization. To test this, we combined Hi-C, single-cell and population transcriptomics, imaging, and in silico modeling of three distinct cells types entering senescence. Genes involved in DNA conformation maintenance are suppressed upon senescence entry across all cell types. We show that nuclear depletion of the abundant HMGB2 protein occurs early on the path to senescence and coincides with the dramatic spatial clustering of CTCF. Knocking down HMGB2 suffices for senescence-induced CTCF clustering and for loop reshuffling, while ectopically expressing HMGB2 rescues these effects. Our data suggest that HMGB2-mediated genomic reorganization constitutes a primer for the ensuing senescent program.