On the Advent of Super-Resolution Microscopy in the Realm of Polycomb Proteins

Abstract

The genomes of metazoans are organized at multiple spatial scales, ranging from the double helix of DNA to whole chromosomes. The intermediate genomic scale of kilobases to megabases, which corresponds to the 50-300 nm spatial scale, is particularly interesting, as the 3D arrangement of chromatin is implicated in multiple regulatory mechanisms. In this context, polycomb group (PcG) proteins stand as major epigenetic modulators of chromatin function, acting prevalently as repressors of gene transcription by combining chemical modifications of target histones with physical crosslinking of distal genomic regions and phase separation. The recent development of super-resolution microscopy (SRM) has strongly contributed to improving our comprehension of several aspects of nano-/mesoscale (10-200 nm) chromatin domains. Here, we review the current state-of-the-art SRM applied to PcG proteins, showing that the application of SRM to PcG activity and organization is still quite limited and mainly focused on the 3D assembly of PcG-controlled genomic loci. In this context, SRM approaches have mostly been applied to multilabel fluorescence in situ hybridization (FISH). However, SRM data have complemented the maps obtained from chromosome capture experiments and have opened a new window to observe how 3D chromatin topology is modulated by PcGs.

Keywords: 3D-SIM; FISH; PRC1; PRC2; STORM; Xist RNA; chromatin organization; oligopaint; polycomb proteins; super-resolution microscopy.

Figure 1

The different scales of chromatin organization, from DNA up to chromosome regions, in the nucleus.

Figure 2

Super-resolution microscopy techniques. (a) Structured illumination microscopy (SIM). (b) Single molecule localization microscopy (SMLM). (c) Image scanning microscopy (ISM). (d) Stimulated emission depletion microscopy (STED).

Figure 3

Structure of the polycomb repressor complex 1 (PRC1). PRC1 subdivides into “variant” (vPRC1) and “canonical” (cPRC1) complexes. Really Interesting New Gene 1 A/B (RING1A/B) is common to both. vPRC1 assembles around one of six Polycomb Group RING Finger (PGCF) proteins (PCGF1–PCGF6), whereas cPRC1 contains only PCGF2/4. Further components, such as YY1-Binding Protein (RYBP) and ChromoBoX proteins CBX2/4/6/7/8, are contained only in vPRC1 and cPRC1, respectively. For further description, see text.

Figure 4

Structure of the polycomb repressor complex 2 (PRC2). The core of PRC2 is composed of EZH1/2, Embryonic Ectoderm Development (EED), Supressor Of Zeste 12 Homolog Protein (SUZ12), and Retinoblastoma Binding Protein 4 or 7 (RBB4/7). Further subdivision in PRC2.1 and PRC2.2 is given by additional proteins, among which Jumonji And (A+T)-RIch Interaction Domain-containing protein 2 (JARID2) and Adipocyte Enhancer-Binding Protein 2 (AEBP2) play crucial roles in the initiation of PRC1/2 repressing activity (see text).

Figure 5

The “reverse hierarchical” model of PRC1 and PRC2 activity. For description, see text and ref. [7].

Figure 6

Scheme of 3D STORM studies on polycomb nanodomains in Drosophila, after ref. [85,86]. Polycomb-controlled genes, such as the Hox genes, are labelled by oligonucleotides bearing blinking fluorophores for STORM imaging (a), thereby revealing compacted chromatin regions ~100 nm in size (b). The compact chromatin state of polycomb-repressed domains could be directly linked to the multimerization ability of the polyhomeotic component of PRC1 through the Sterile Alpha Motifs (SAMs) they embed (c).