Hierarchical recruitment of Polycomb complexes revisited

Abstract

Polycomb Group (PcG) proteins epigenetically repress key developmental genes and thereby control alternative cell fates. PcG proteins act as complexes that can modify histones and these histone modifications play a role in transmitting the "memory" of the repressed state as cells divide. Here we consider mainstream models that link histone modifications to hierarchical recruitment of PcG complexes and compare them to results of a direct test of interdependence between PcG complexes for recruitment to Drosophila genes. The direct test indicates that PcG complexes do not rely on histone modifications to recognize their target genes but use them to stabilize the interactions within large chromatin domains. It also shows that multiple strategies are used to coordinate the targeting of PcG complexes to different genes, which may make the repression of these genes more or less robust.

Keywords: Drosophila; Polycomb; Polycomb Response Elements; Polycomb targeting; epigenetics.

Figure 1.

PRC1 is not required for untargeted methylation of H3K27 by PRC2. A. Two-fold dilutions of acid-extracted histones from control Ras3 and Psc/Su(z)2 deficient cells were analyzed by western-blot with antibodies against H3K27me2 and H3K27me3. The loss of Psc/Su(z)2 causes no significant reduction of the overall H3K27me2/me3, which suggests that much of the bulk H3K27me3 is produced by untargeted PRC2 activity and that the integrity and the activity of PRC2, per se, do not require PRC1. B. Di-methylation of H3K27 in the vicinity of PREs as measured by ChIP-qPCR. Histograms show the mean of 2 independent experiments with error bars indicating the scatter. Note that the loss of H3K27me3 in the PRC1 deficient cells allows di-methylation of H3K27 by untargeted PRC2. The high levels of H3K27me2 at “Control” amplicon from transcriptionally inactive intergenic region on Chromosome 3L are similar in wild type (WT) and Psc/Su(z)2 deficient cells. The data in (A) and (B) are from Kahn et al.

Figure 2.

Revised model for hierarchical recruitment of Polycomb complexes. More than one pathway is used to recruit PRC2 to PREs. A. PRC2:PRC1-dependent pathway. At step 0, a PRE is bound by DNA-binding adaptor proteins (dashed circle and oval) that can cooperatively interact with PRC1 and anchor it. The PRE may also bind adaptor proteins that can interact with PRC2 (dashed triangle) but are not sufficient to anchor it. At step 1, PRC1 is recruited to the PRE. The recruitment of PRC2 at step 2 may require direct interaction with PRC1 and step 1 might be very transient. B. PRC2:PRC1-independent pathway. At step 0, DNA-binding adaptor proteins specific for both PRC1 (dashed circle and oval) and PRC2 (dashed triangle and square) bind the PRE. This is followed by independent recruitment of PRC1 and PRC2 via cooperative interactions with corresponding sets of adaptor proteins. Eventually, at step 2, the PRE is occupied by both PRC1 and PRC2. Step 1 might be transient and PRC1 and PRC2 may bind to the PRE stochastically or simultaneously. Genomic analysis indicates that the PRC2:PRC1-dependent and –independent pathways are not mutually exclusive. At many PREs both pathways contribute to PRC2 recruitment however their relative contributions vary between different sites.

Figure 3.

Tri-methyl H3K27 stabilizes PcG contacts with chromatin around PREs. A. When assayed by ChIP, Pc (light gray dashed line) but not the other PRC1 subunits, for example Psc (dark gray dashed line), shows immunoprecipitation profile that extends from PREs into the neighboring chromatin (left). Upon PRC2 loss, the Pc ChIP signals become confined to PRE cores (right). B. Polycomb group (PcG) complexes anchored at PREs can transiently loop out and interact with the chromatin of neighboring promoters, enhancers and transcription units where PcG complexes may interfere with transcription. Interactions between Pc and H3K27me3 seem to stabilize the looping, which shifts the equilibrium toward looped conformation. Assisting transcriptional repression, this may also help to restore high density of H3K27me3 after DNA replication.