Repression and 3D-restructuring resolves regulatory conflicts in evolutionarily rearranged genomes.

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.

Higher-Order Chromatin Organization Using 3D DNA Fluorescent In Situ Hybridization.

A comprehensive analysis of the tridimensional (3D) organization of the genome is crucial to understand gene regulation. Three-dimensional DNA fluorescent in situ hybridization (3D-FISH) is a method of choice to study nuclear organization at the single-cell level. The labeling of DNA loci of interest provides information on their spatial arrangement, such as their location within the nucleus or their relative positioning. The single-cell information of spatial positioning of genomic loci can thus be integrated with functional genomic and epigenomic features, such as gene activity, epigenetic states, or cell population averaged chromatin interaction profiles obtained using chromosome conformation capture methods. Moreover, the development of a diversity of super-resolution (SR) microscopy techniques now allows the study of structural chromatin properties at subdiffraction resolution, making a finer characterization of shapes and volumes possible, as well as allowing the analysis of quantitative intermingling of genomic regions of interest. Here, we present and describe a 3D-FISH protocol adapted for both conventional and SR microscopy such as 3D structured illumination microscopy (3D-SIM), which can be used for the measurement of 3D distances between loci and the analysis of higher-order chromatin structures in cultured Drosophila and mammalian cells.

Regulation of single-cell genome organization into TADs and chromatin nanodomains.

The genome folds into a hierarchy of three-dimensional structures within the nucleus. At the sub-megabase scale, chromosomes form topologically associating domains (TADs)1-4. However, how TADs fold in single cells is elusive. Here, we reveal TAD features inaccessible to cell population analysis by using super-resolution microscopy. TAD structures and physical insulation associated with their borders are variable between individual cells, yet chromatin intermingling is enriched within TADs compared to adjacent TADs in most cells. The spatial segregation of TADs is further exacerbated during cell differentiation. Favored interactions within TADs are regulated by cohesin and CTCF through distinct mechanisms: cohesin generates chromatin contacts and intermingling while CTCF prevents inter-TAD contacts. Furthermore, TADs are subdivided into discrete nanodomains, which persist in cells depleted of CTCF or cohesin, whereas disruption of nucleosome contacts alters their structural organization. Altogether, these results provide a physical basis for the folding of individual chromosomes at the nanoscale.

4D Genome Rewiring during Oncogene-Induced and Replicative Senescence.

To understand the role of the extensive senescence-associated 3D genome reorganization, we generated genome-wide chromatin interaction maps, epigenome, replication-timing, whole-genome bisulfite sequencing, and gene expression profiles from cells entering replicative senescence (RS) or upon oncogene-induced senescence (OIS). We identify senescence-associated heterochromatin domains (SAHDs). Differential intra- versus inter-SAHD interactions lead to the formation of senescence-associated heterochromatin foci (SAHFs) in OIS but not in RS. This OIS-specific configuration brings active genes located in genomic regions adjacent to SAHDs in close spatial proximity and favors their expression. We also identify DNMT1 as a factor that induces SAHFs by promoting HMGA2 expression. Upon DNMT1 depletion, OIS cells transition to a 3D genome conformation akin to that of cells in replicative senescence. These data show how multi-omics and imaging can identify critical features of RS and OIS and discover determinants of acute senescence and SAHF formation.

Higher-Order Chromosomal Structures Mediate Genome Function.

How chromosomes are organized within the tridimensional space of the nucleus and how can this organization affect genome function have been long-standing questions on the path to understanding genome activity and its link to disease. In the last decade, high-throughput chromosome conformation capture techniques, such as Hi-C, have facilitated the discovery of new principles of genome folding. Chromosomes are folded in multiple high-order structures, with local contacts between enhancers and promoters, intermediate-level contacts forming Topologically Associating Domains (TADs) and higher-order chromatin structures sequestering chromatin into active and repressive compartments. However, despite the increasing evidence that genome organization can influence its function, we are still far from understanding the underlying mechanisms. Deciphering these mechanisms represents a major challenge for the future, which large, international initiatives, such as 4DN, HCA and LifeTime, aim to collaboratively tackle by using a conjunction of state-of-the-art population-based and single-cell approaches.

Principles of genome folding into topologically associating domains.

Understanding the mechanisms that underlie chromosome folding within cell nuclei is essential to determine the relationship between genome structure and function. The recent application of "chromosome conformation capture" techniques has revealed that the genome of many species is organized into domains of preferential internal chromatin interactions called "topologically associating domains" (TADs). This chromosome chromosome folding has emerged as a key feature of higher-order genome organization and function through evolution. Although TADs have now been described in a wide range of organisms, they appear to have specific characteristics in terms of size, structure, and proteins involved in their formation. Here, we depict the main features of these domains across species and discuss the relation between chromatin structure, genome activity, and epigenome, highlighting mechanistic principles of TAD formation. We also consider the potential influence of TADs in genome evolution.

TADs are 3D structural units of higher-order chromosome organization in Drosophila.

Deciphering the rules of genome folding in the cell nucleus is essential to understand its functions. Recent chromosome conformation capture (Hi-C) studies have revealed that the genome is partitioned into topologically associating domains (TADs), which demarcate functional epigenetic domains defined by combinations of specific chromatin marks. However, whether TADs are true physical units in each cell nucleus or whether they reflect statistical frequencies of measured interactions within cell populations is unclear. Using a combination of Hi-C, three-dimensional (3D) fluorescent in situ hybridization, super-resolution microscopy, and polymer modeling, we provide an integrative view of chromatin folding in Drosophila. We observed that repressed TADs form a succession of discrete nanocompartments, interspersed by less condensed active regions. Single-cell analysis revealed a consistent TAD-based physical compartmentalization of the chromatin fiber, with some degree of heterogeneity in intra-TAD conformations and in cis and trans inter-TAD contact events. These results indicate that TADs are fundamental 3D genome units that engage in dynamic higher-order inter-TAD connections. This domain-based architecture is likely to play a major role in regulatory transactions during DNA-dependent processes.

Multiscale 3D Genome Rewiring during Mouse Neural Development.

Chromosome conformation capture technologies have revealed important insights into genome folding. Yet, how spatial genome architecture is related to gene expression and cell fate remains unclear. We comprehensively mapped 3D chromatin organization during mouse neural differentiation in vitro and in vivo, generating the highest-resolution Hi-C maps available to date. We found that transcription is correlated with chromatin insulation and long-range interactions, but dCas9-mediated activation is insufficient for creating TAD boundaries de novo. Additionally, we discovered long-range contacts between gene bodies of exon-rich, active genes in all cell types. During neural differentiation, contacts between active TADs become less pronounced while inactive TADs interact more strongly. An extensive Polycomb network in stem cells is disrupted, while dynamic interactions between neural transcription factors appear in vivo. Finally, cell type-specific enhancer-promoter contacts are established concomitant to gene expression. This work shows that multiple factors influence the dynamics of chromatin interactions in development.

Stable Polycomb-dependent transgenerational inheritance of chromatin states in Drosophila.

Transgenerational epigenetic inheritance (TEI) describes the transmission of alternative functional states through multiple generations in the presence of the same genomic DNA sequence. Very little is known about the principles and the molecular mechanisms governing this type of inheritance. Here, by transiently enhancing 3D chromatin interactions, we established stable and isogenic Drosophila epilines that carry alternative epialleles, as defined by differential levels of Polycomb-dependent trimethylation of histone H3 Lys27 (forming H3K27me3). After being established, epialleles can be dominantly transmitted to naive flies and can induce paramutation. Importantly, epilines can be reset to a naive state by disruption of chromatin interactions. Finally, we found that environmental changes modulate the expressivity of the epialleles, and we extended our paradigm to naturally occurring phenotypes. Our work sheds light on how nuclear organization and Polycomb group (PcG) proteins contribute to epigenetically inheritable phenotypic variability.