Insulators, Not Polycomb Response Elements, Are Required for Long-Range Interactions between Polycomb Targets in Drosophila melanogaster

Abstract

The genomic binding sites of Polycomb group (PcG) complexes have been found to cluster, forming Polycomb "bodies" or foci in mammalian or fly nuclei. These associations are thought to be driven by interactions between PcG complexes and result in enhanced repression. Here, we show that a Polycomb response element (PRE) with strong PcG binding and repressive activity cannot mediate trans interactions. In the case of the two best-studied interacting PcG targets in Drosophila, the Mcp and the Fab-7 regulatory elements, we find that these associations are not dependent on or caused by the Polycomb response elements they contain. Using functional assays and physical colocalization by in vivo fluorescence imaging or chromosome conformation capture (3C) methods, we show that the interactions between remote copies of Mcp or Fab-7 elements are dependent on the insulator activities present in these elements and not on their PREs. We conclude that insulator binding proteins rather than PcG complexes are likely to be the major determinants of the long-range higher-order organization of PcG targets in the nucleus.

tDNA insulators and the emerging role of TFIIIC in genome organization

“…Supporting evidence comes from recent demonstration that insulators underlie long-range Pc interactions and are important for the maintenance of H3K27me3 levels within Pc domains in Drosophila. 6,9,58 Meanwhile, insulator proteins are distributed across the genome at thousands of sites, 32,[59][60][61][62][63] and are significantly enriched at the borders of laminaassociated domains, 64,65 consistent with microscopy-based staining of perinuclear insulator bodies. Numerous studies have also characterized the ability of CTCF and Drosophila insulator proteins to mediate locus specific interactions, [45][46][47][48] and recent genome-wide profiling of CTCF interactions in mouse ES cells suggests CTCF facilitates coregulation of related genes by establishing chromatin loops enriched for active or repressive epigenetic signatures, and by bridging interactions between enhancers and promoters.…”

“…Instead, insulator proteins appear to be involved in mediating long-range inter-and intra-chromosomal arrangements that can direct the nuclear co-localization of specific sequences. For example, insulator proteins localize to both repressive Polycomb (Pc) bodies 7 and active transcription factories, 8 and have been shown to underlie interactions between Pc target sites 9 and the maintenance of H3K27me3 within repressive Pc domains. 6 Mapping of interactions facilitated by insulator protein CTCF in mouse embryonic stem cells suggests insulators also contribute to genome organization by forming chromatin loops in which active or repressed genes are harnessed for coregulation, and by facilitating enhancer-promoter interactions.…”

Distinct Roles of Chromatin Insulator Proteins in Control of the Drosophila Bithorax Complex

“…Like interactions and loops between enhancers and promoters, the interactions between insulator elements are likely transient and should be considered in probabilistic terms. Similarly, insulator interactions may bring different genomic elements together or juxtapose regulatory elements with appropriate target promoters (Gruzdeva et al 2005;Ling et al 2006;Splinter et al 2006;Li et al 2011).Whether insulator elements are interchangeably used in vivo to both promote and block enhancer-promoter communicators or have a preference for either is an open question. An important step toward resolving this issue is to ask whether the two bridging proteins Cp190 and Mod(mdg4) are functionally different.…”

“…Like interactions and loops between enhancers and promoters, the interactions between insulator elements are likely transient and should be considered in probabilistic terms. Similarly, insulator interactions may bring different genomic elements together or juxtapose regulatory elements with appropriate target promoters (Gruzdeva et al 2005;Ling et al 2006;Splinter et al 2006;Li et al 2011).…”

Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex

“…The classically defined functions ascribed to boundaries/insulators include an enhancer-or silencerblocking activity and an ability to bring distant chromosomal DNA segments into close proximity (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Genome-wide chromatin immunoprecipitations (ChIPs) with known insulator proteins together with chromatin conformation capture experiments indicate that insulators are ubiquitous features of chromosomes from flies to human, demarcating distinct chromatin and regulatory domains and helping mediate long-range interactions (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30).…”