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Three-dimensional (3D) genome folding has a fundamental role in the regulation of developmental genes by facilitating or constraining chromatin interactions between cis-regulatory elements (CREs). Polycomb response elements (PREs) are a specific kind of CRE involved in the memory of transcriptional states in Drosophila melanogaster. PREs act as nucleation sites for Polycomb group (PcG) proteins, which deposit the repressive histone mark H3K27me3, leading to the formation of a class of topologically associating domain (TAD) called a Polycomb domain. PREs can establish looping contacts that stabilize the gene repression of key developmental genes during development. However, the mechanism by which PRE loops fine-tune gene expression is unknown. Using clustered regularly interspaced short palindromic repeats and Cas9 genome engineering, we specifically perturbed PRE contacts or enhancer function and used complementary approaches including 4C-seq, Hi-C and Hi-M to analyze how chromatin architecture perturbation affects gene expression. Our results suggest that the PRE loop at the dac gene locus acts as a constitutive 3D chromatin scaffold during Drosophila development that forms independently of gene expression states and has a versatile function; it restricts enhancer-promoter communication and contributes to enhancer specificity., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

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.

Polycomb repressive complexes 1 and 2 have been historically described as transcriptional repressors, but recent reports suggest that PRC1 might also support activation, although the underlying mechanisms remain elusive. Here, we show that stage-specific PRC1 binding at a subset of active promoters and enhancers during Drosophila development coincides with the formation of three-dimensional (3D) loops, an increase in expression during development and repression in PRC1 mutants. Dissection of the dachshund locus indicates that PRC1-anchored loops are versatile architectural platforms that persist when surrounding genes are transcriptionally active and fine-tune their expression. The analysis of RING1B binding profiles and 3D contacts during neural differentiation in mice suggests that this role is conserved in mammals., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Eukaryotic chromosomes are organized in multiple scales, from nucleosomes to chromosome territories. Recently, genome-wide methods identified an intermediate level of chromosome organization, topologically associating domains (TADs), that play key roles in transcriptional regulation. However, these methods cannot directly examine the interplay between transcriptional activation and chromosome architecture while maintaining spatial information. Here we present a multiplexed, sequential imaging approach (Hi-M) that permits simultaneous detection of chromosome organization and transcription in single nuclei. This allowed us to unveil the changes in 3D chromatin organization occurring upon transcriptional activation and homologous chromosome unpairing during awakening of the zygotic genome in intact Drosophila embryos. Excitingly, the ability of Hi-M to explore the multi-scale chromosome architecture with spatial resolution at different stages of development or during the cell cycle will be key to understanding the mechanisms and consequences of the 4D organization of the genome., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Interphase chromatin is organized into topologically associating domains (TADs). Within TADs, chromatin looping interactions are formed between DNA regulatory elements, but their functional importance for the establishment of the 3D genome organization and gene regulation during development is unclear. Using high-resolution Hi-C experiments, we analyze higher order 3D chromatin organization during Drosophila embryogenesis and identify active and repressive chromatin loops that are established with different kinetics and depend on distinct factors: Zelda-dependent active loops are formed before the midblastula transition between transcribed genes over long distances. Repressive loops within polycomb domains are formed after the midblastula transition between polycomb response elements by the action of GAGA factor and polycomb proteins. Perturbation of PRE function by CRISPR/Cas9 genome engineering affects polycomb domain formation and destabilizes polycomb-mediated silencing. Preventing loop formation without removal of polycomb components also decreases silencing efficiency, suggesting that chromatin architecture can play instructive roles in gene regulation during development. VIDEO ABSTRACT., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)