Hierarchies of fly genome architecture. Chromosomes are extensively folded to fit inside the cell nucleus. Each chromosome occupies its own volume (chromosome territory, shown schematically in different colors); however, these volumes partially intersect allowing for interchromosomal interactions. At finer scale, chromatin fibers are partitioned into domains with different degrees of folding and different regimes of chromatin contacts within domains (TADs). The partitioning into domains is in part defined by the differences in the composition and properties of underlying chromatin as well as transcriptional activity. For example, void and Polycomb (PcG)-repressed chromatin has more internal contacts than chromatin of active genes. Insulator elements demarcate some of the TADs by forming loops that inhibit chromatin contacts across domain boundaries. Topological domains correlate with segmentation of the linear Drosophila genome into chromatin types with distinct histone modifications. The contact matrix of a virtual Hi-C experiment illustrates how partitioning into TADs is assayed.

Transgenic insulator assays. (A) Position-effect assay. The white gene (red rectangle) confers red pigmentation to fly eyes. When integrated elsewhere in the genome, white is frequently repressed by neighboring repressor elements (blue pentagon, R) yielding flies with pale yellow eyes. When a transgene (here and below shown as bold line) carries insulators (green rectangles, ins^t) at its 5′ and 3′ ends, the white expression becomes much more uniform between different insertion sites. (B) Enhancer blocking assay. One of the common enhancer-blocking assays uses the yellow reporter gene (yellow rectangle), which controls dark pigmentation of the fly. The expression of yellow in wings, body, and bristles is controlled by distinct enhancer elements (gray circles marked with B, W, and Br, respectively). When flies lacking endogenous yellow function are transformed with a copy of the WT yellow gene, their pigmentation fully restores (black wings, body, and bristles). In contrast, when yellow-deficient flies are transformed with a transgene that contains an insulator (green rectangle, ins^t) interposed between the upstream wing- and body-specific enhancers and the yellow promoter, the transgenic insulator interacts with the nearest genomic insulator (blue rectangle, ins) to form a loop that blocks enhancer–promoter communication. Note that the interactions between the promoter and the downstream bristle-specific enhancer are not impaired. This yields transgenic files that have yellow wings and body but black bristles. When two insulators are placed between the upstream enhancers and the promoter, they preferentially interact with each other and stimulate rather than inhibit enhancer–promoter interactions. Corresponding transgenic flies appear as WT. (C) An example of long-distance trans-interactions enhanced by insulator elements. In transgenes containing white paired with a PRE (blue pentagon), the white gene gets stochastically inactivated in embryonic precursor cells, which results in flies with variegated eye pigmentation. When two such transgenes, integrated in different nonhomologous chromosomes (illustrated as black and dashed lines), are combined, the variegation becomes much less pronounced or even disappears. Strikingly, when in addition to PREs the two transgenes also contain insulator elements, the white repression is greatly enhanced, often resulting in flies with completely white eyes. This suggests that insulator elements promote long-distance trans-interactions and that pairing of PREs reinforces Polycomb repression.

Polycomb complexes and their role in chromatin architecture. (A) Polycomb complexes are targeted to genes by PREs (yellow rectangle). PREs represent collections of recognition sequences for DNA binding adaptor proteins (gray circles). With the exception of Pho, which is part of the separate PhoRC complex, DNA binding proteins interact with the core PcG complexes weakly and are not recovered in biochemical purification. Nevertheless, individual weak interactions of DNA binding proteins combine and provide robust recruitment of PRC1 (green circles) and PRC2 (red circles). Scm (orange oval), the substoichiometric component PRC1, serves as a link between PhoRC and PRC1, further stabilizing the binding of both complexes (; ). Chromodomain of the Pc subunit of PRC1 specifically interacts with trimethylated H3K27 produced by PRC2 (green circles on the N-terminal tails of nucleosomes depicted as orange cylinders). In turn, PRC2 can interact with monoubiquitylated H2AK118 produced by PRC1 (red stars). Although insufficient for recruitment, these interactions can further stabilize the binding of PRC1 and PRC2 at some PREs. The Trx protein is recruited to PREs along with PcG complexes. (B) PRC1 (red circles) and PRC2 (green circles) complexes anchored at PREs (yellow rectangle) loop out and contact surrounding chromatin. Recognition of H3K27me3 by Pc stabilizes these transient interactions, allowing efficient methylation of nucleosomes in the vicinity of the contact. This way H3K27me3 can spread away from a PRE for tens of thousands of base pairs until it encounters an insulator element or an active gene. (C) Immunolocalization of Polycomb components in Drosophila or mammalian cell nuclei detects discrete foci of different sizes (PcG bodies, yellow stars). The number of PcG bodies is smaller compared to the number of Polycomb target genes detected by genome-wide mapping. Immuno-FISH experiments suggest that some of the PcG bodies represent clusters of Polycomb-regulated genes.

Prospects of 3D genome engineering. (A) In a fictional WT locus, the transcription of the “red” and the “orange” genes is driven by specific enhancers. The leftmost “gray” gene is inactive but is not epigenetically repressed and could be activated later during development. The “blue” gene is surrounded by insulators (blue and orange boxes), which interact, forming a looped TAD. A PRE located within the TAD (yellow box) represses the blue gene and leads to extensive trimethylation of H3K27 (dashed blue zig-zag line) that is contained within the TAD. (B) When the rightmost orange insulator is deleted by mutation, the central blue insulator loses its preferred interaction partner and instead interacts with the leftmost red insulator, forming the TAD containing the active red gene. The PRE is no longer topologically constrained. This leads to extended H3K27 methylation to the right of the PRE that stops at the transcriptionally active orange gene. It also leads to the insulator bypass, H3K27 methylation, and epigenetic repression of the leftmost gene. This gene can no longer be induced, which causes pathology later in development. (C) Using genome editing tools, for example CRISPR/Cas9, it may be possible to delete the red insulator. This will prevent formation of the TAD containing the red gene and its transcriptional activity will stop the spreading of H3K27me3 and prevent the leftmost gray gene from being permanently repressed. (D) Ultimately, it might be possible to use more sophisticated homology-directed replacement techniques to reintroduce a new copy of the deleted insulator (indicated as white box) and fully restore the WT topology of the locus.