Boundaries of loop domains (insulators): Determinants of chromosome form and function in multicellular eukaryotes

Abstract

Chromosomes in multicellular animals are subdivided into a series of looped domains. In addition to being the underlying principle for organizing the chromatin fiber, looping is critical for processes ranging from gene regulation to recombination and repair. The subdivision of chromosomes into looped domains depends upon a special class of architectural elements called boundaries or insulators. These elements are distributed throughout the genome and are ubiquitous building blocks of chromosomes. In this review, we focus on features of boundaries that are critical in determining the topology of the looped domains and their genetic properties. We highlight the properties of fly boundaries that are likely to have an important bearing on the organization of looped domains in vertebrates, and discuss the functional consequences of the observed similarities and differences.

Keywords: TADs; chromosome architecture; gene regulation; insulators; loop topology; looped domain.

Figure 1

Transgene assays for boundaries. A: Insulator assay. (1) One boundary is not effective in protecting repaorter transcription from heterochromatic/Polycomb repression. (2) Two boundaries flanking reporter on both sides protect against silencing. Green rectangles “B” – boundaries. The yellow and blue wavy arrows indicate genes; upstream arrow indicates promoter: short – isolated from enhancer; long – activated by enhancer. Absence of an arrow upstream of the gene in (1) indicates gene repression by heterochromatin/Polycomb. Heterochromatin/Polycomb factors– gray ovals. B: Enhancer-blocking assay. (1) Enhancer activates promoter. (2) Boundary inserted between enhancer and promoter blocks enhancer action. Red circle “E” – enhancer. C: The bypass assay. (1) Two reporters are regulated by an upstream enhancer. (2) Placing a boundary between the upstream enhancer and the promoter for the proximal reporter blocks activation of both reporters. (3) Addition of second boundary in between the two reporters leads to bypass. The two boundaries pair with each other and this brings the enhancers in close proximity to the distal reporter leading to its activation.

Figure 2

Boundary competition and domain definition assays. A: Competition assay. One reporter and three boundaries (d, b, c) are used in this assay. (1) Downstream (d) boundary doesn't influence enhancer activity. (2) Insertion of second boundary (b) between the enhancer and promoter blocks activation. A loop isolating reporter is formed between the paired boundaries. (3-4) Insertion of a third boundary (c) upstream of enhancer can lead to activation (3) or isolation (4) of the reporter depending on their partner preference. B: Domain definition assay. Second reporter is inserted upstream of enhancer (red circle).(1) Both reporters are active. (2) Interaction between (b) and (d) boundaries isolates “yellow” colored reporter, leaving “blue” reporter unaffected. (3) Interaction between (c) and (d) boundaries isolates “blue”, but not “yellow” reporter. (4) Both reporters are isolated from enhancers.

Figure 3

Self-pairing interactions are generally head-to-head. A: The bypass assay. The topology of the loop formed by paired boundaries depends upon their relative orientation and whether they pair head-to-head or head-to-tail.(1) If the two boundaries pair head-to-head, and they are arranged in the same orientation, a circle- loop will be formed. This topology positions the enhancer and promoter on opposite sides of the paired boundaries, leading to isolation of the reporter. (2) When the boundaries are arranged in opposite orientation, head-to-head pairing generates a stem-loop. This brings the enhancer and promoter into close proximity and leads to activation of transcription. Green arrows with straight sides– boundaries, arrowheads indicate orientation. B: Interactions between boundaries over large (2Mb) distances. (1) In this configuration, the enhancer and the reporter are “downstream” of the boundary. Here, head-to-head self-pairing between the two boundaries brings the enhancer and promoter into close proximity, activating transcription. (2) In this configuration, the enhancer is “downstream” of the boundary, while the reporter is in the “upstream” position. When the two boundaries pair with each other head-to-head, the enhancer and the reporter are on opposite sides of the paired boundary, blocking regulatory interactions. C: Self-pairing Interactions between boundaries on each homolog aligns the homologs in register and maintains homolog pairing.(1) Head-to-head self-pairing interactions align homologs in register. Different colored arrows – boundaries. (2) Head-to-tail self-pairing interactions would disrupt chromosome alignment and interfere with transvection.

Figure 4

Heterologous head-to-tail pairing between boundaries in cis. A: Schematic of the eve regulatory region. Boundaries: nhomie (orange), homie (brown), others are not specified. B: Interaction between nhomie-homie and other flanking boundaries generates independent stem-loops that are separated by unanchored segments. C: The nhomie-homie pair interacts with their neighbors. D: The interactions between boundaries are further stabilized by homolog pairing.

Figure 5

Heterologous head-to-head pairing between boundaries in cis. A: Schematic of the abd-A and Abd-B gene regions. The regulatory domains, iab-2–iab-4 and iab-5 – iab8 of the abd-A and the Abd-B genes, respectively, are separated by boundaries (Fab-3, Fab-4, Mcp, Fab-6, Fab-7, Fab-8). AB-I is a boundary-like element located upstream of the Abd-B promoter. B: Head-to-head pairing between boundaries forms circle-loops that can be wound counter-clockwise (left) or clockwise (right). C: Series of circle-loops are connected by interacting boundaries. D, E: The Fab-7 replacement assay. D: A copy of Fab-8 copy is inserted in the “forward” orientation in place of Fab-7. In this orientation Fab-8 fully substitutes forFab-7 and would maintain the loop topology. E: Fab-8 is inserted in the “reverse” orientation. In this case head-to-head pairing interactions with neighboring boundaries would disrupt the circle-loop topology, introducing two stem-loops instead. In this orientation Fab-8 still blocks cross-talk between iab-6 and iab-7.However, it is no longer permissive for iab-6 (and to a lesser extent, iab-5) regulation of Abd-B.