- Email: [email protected]
Chromatin: Polycomb Group SAMs Unite
Current Biology
Dispatches the observation that top predators may be quickly removed from systems and that levels of decline are correlated with proximity to human populations [12]. The results of Mourier and colleagues [9] demonstrate the changes to reef trophic systems that result from such declines and the potential breakdown of locally inverted biomass pyramids. However, we do not know if inverted biomass pyramids are (or were) common in coral reef systems, and so the full effect of predator declines remains to be determined. These findings also point to some important lessons for coral reef conservation. First, protecting and maintaining the fish spawning aggregations that occur on many coral reefs is important. We now know that it is especially so for sustaining high levels of predator biomass. These aggregations are often fished, which can quickly lead to depletion [13,14] and can have dire consequences for the fish and the predators that depend on them for trophic subsidies. Second, the movement of predators away from areas with inverted biomass pyramids means that protection of the small areas in which the most predators are found is insufficient for protecting these unique trophic phenomena. Instead, there is a need to protect the area over which predators disperse to feed, pointing to the requirement for a detailed understanding
of movement of coral reef species [15] to design spatial management systems.
REFERENCES 1. Trebilco, R., Baum, J.K., Salomon, A.K., and Dulvy, N.K. (2013). Ecosystem ecology: sizebased constraints on the pyramids of life. Trends Ecol. Evol. 28, 423–431. 2. Sandin, S.A., Smith, J.E., DeMartini, E.E., Dinsdale, E.A., Donner, S.D., Friedlander, A.M., Konotchick, T., Malay, M., Maragos, J.E., Obura, D., et al. (2008). Baselines and degradation of coral reefs in the Northern Line Islands. PLoS One 3, 11. 3. Wang, H., Morrison, W., Singh, A., and Weiss, H. (2009). Modeling inverted biomass pyramids and refuges in ecosystems. Ecol. Modell. 220, 1376–1382.
8. Trebilco, R., Dulvy, N.K., Anderson, S.C., and Salomon, A.K. (2016). The paradox of inverted biomass pyramids in kelp forest fish communities. Proc. R. Soc. Lond. B. Biol. Sci. 283, http://dx.doi.org/10.1098/rspb.2016. 0816. 9. Mourier, J., Maynard, J., Parravicini, V., Ballesta, L., Clua, E., Domeier, M., and Planes, S. (2016). Extreme inverted trophic pyramid of reef sharks supported by spawning groupers. Curr. Biol. 26, 2011–2016. 10. Heupel, M.R., Knip, D.M., Simpfendorfer, C.A., and Dulvy, N.K. (2014). Sizing up the ecological role of sharks as predators. Mar. Ecol. Prog. Ser. 495, 291–298. 11. McCauley, D.J., Young, H.S., Dunbar, R.B., Estes, J.A., Semmens, B.X., and Michel, F. (2012). Assessing the effects of large mobile predators on ecosystem connectivity. Ecol. Appl. 22, 1711–1717.
4. Ward-Paige, C., Mills Flemming, J., and Lotze, H.K. (2010). Overestimating fish counts by non-instantaneous visual censuses: consequences for population and community descriptions. PLoS One 5, e11722.
12. Nadon, M.O., Baum, J.K., Williams, I.D., McPherson, J.M., Zgliczynski, B.J., Richards, B.L., Schroeder, R.E., and Brainard, R.E. (2012). Re-creating missing population baselines for Pacific reef sharks. Conserv. Biol. 26, 493–503.
5. Salinas de Leo´n, P., Acun˜a-Marrero, D., Rastoin, E., Friedlander, A.M., Donovan, M.K., and Sala, E. (2016). Largest global shark biomass found in the northern Gala´pagos Islands of Darwin and Wolf. PeerJ. 4, e1911.
13. Sadovy, Y., and Domeier, M. (2005). Are aggregation-fisheries sustainable? Reef fish fisheries as a case study. Coral Reefs 24, 254–262.
6. Friedlander, A.M., and DeMartini, E.E. (2002). Contrasts in density, size, and biomass of reef fishes between the northwestern and the main Hawaiian islands: The effects of fishing down apex predators. Mar. Ecol. Prog. Ser. 230, 253–265. 7. Gasol, J.M., del Giorgio, P.A., and Duarte, C.M. (1997). Biomass distribution in marine planktonic communities. Limnol. Oceanogr. 42, 1353–1363.
14. Sadovy de Mitcheson, Y. (2016). Mainstreaming fish spawning aggregations into fishery management calls for a precautionary approach. BioScience 66, 295–306. 15. Espinoza, M., Le´de´e, E.J.I., Simpfendorfer, C.A., Tobin, A.J., and Heupel, M.R. (2015). Contrasting movements and connectivity of reef-associated sharks using acoustic telemetry: implications for management. Ecol. Appl. 25, 2101–2118.
Chromatin: Polycomb Group SAMs Unite Chongwoo A. Kim1 and Nicole J. Francis2,3 1Department
of Biochemistry, Midwestern University, 19555 N. 59th Avenue, Glendale, AZ 85308, USA de recherches cliniques de Montre´al, Montre´al, Que´bec, Canada 3De ´ partement de biochimie et mede´cine mole´culaire, Universite´ de Montre´al, Montre´al, Que´bec, Canada Correspondence: [email protected] http://dx.doi.org/10.1016/j.cub.2016.06.001 2Institut
Polycomb Group (PcG) proteins assemble a chromatin state that maintains developmental gene repression. A new study combining structure and in vivo analysis details a molecular network from DNA recognition to PcG recruitment, highlighting the essential role of Sterile Alpha Motifs. Polycomb Group (PcG) proteins are conserved, essential epigenetic repressors of gene expression. They
function within multi-protein assemblies able to mediate repression through both physical (chromatin folding) and
R710 Current Biology 26, R701–R718, August 8, 2016 ª 2016 Elsevier Ltd.
biochemical (protein–protein interactions and protein modification) mechanisms creating unique chromatin
Current Biology
Dispatches states that can cover hundreds of kilobases [1,2]. The first PcG complex identified, aptly named Polycomb Repression Complex 1 (PRC1), can function as a histone H2A ubiquitin E3 ligase ans is also able to modify chromatin architecture through both local chromatin compaction and long-range interactions [2,3]. PRC1 can also directly inhibit transcription and destabilize transcription pre-initiation complexes in vitro. PRC2, another PcG protein complex, methylates histone H3 on lysine 27 (H3K27me), an activity which is essential for PcG function [2,3]. Despite the wealth of information that has been acquired, fundamental mechanistic questions remain about how PcG proteins are recruited to target genes and how their recruitment leads to assembly of repressive chromatin domains. A recent study from Frey et al. [4], reported in Genes and Development, along with other recent work, reveals the central role of a conserved protein–protein interaction domain, the Sterile Alpha Motif (SAM), in anchoring PcG complexes to the genome and, potentially, in the organization of chromatin architecture. In Drosophila, where the PcG was discovered and is best understood, large, complex cis regulatory elements termed Polycomb Response Elements (PREs) recruit PcG proteins [5]. However, only one of the core PcG proteins, Pleiohomeotic (Pho), and its homologue Pho-like (PhoL), has sequence-specific DNA binding activity and can recognize motifs present in PREs [6]. Thus, the other PcG proteins must be recruited indirectly to PREs. The PcG protein that is the major player in this indirect recruitment is Sfmbt. Sfmbt binds directly to Pho, is essential for PcG silencing, and is required for full binding of Pho to PREs [7,8]. Together, Pho and Sfmbt constitute the core of the PcG complex called Pho Repressive Complex (PhoRC), which links PRC1 to PREs. It is important to note, however, that the PhoRC–PRC1 relationship is self-reinforcing rather than strictly hierarchical since PhoRC binding is also reduced in PRC1 mutants [7,9]. What remained unresolved was the molecular basis for PhoRC recruitment of PRC1.
Frey et al. now show that the protein that brings PhoRC and PRC1 together is yet another PcG protein called Sex comb on midleg (Scm). Scm appears to be the wild card in PcG-mediated repression. Scm can associate with PhoRC [10], PRC1, and PRC2 [11], and possibly a separate 500 kDa complex [12], but is not considered a core component of these established PcG complexes due to substoichiometric amounts observed in their purifications or weak/transient interactions. Nevertheless, Scm is important for repression mediated by both PhoRC [10] and PRC1 [13]. The current study determined that Scm bridges PhoRC and PRC1 through interactions between the SAMs present in Sfmbt, Scm, and the PRC1 subunit Ph. SAMs are involved in protein–protein interactions, and in dynamic, regulated assembly macromolecular structures through the ability of some SAMs to assemble into helical polymers. The SAMs of both Ph and Scm can form homopolymers as well as assemble into a co-polymer [14,15]. SAMs form polymers through the iterative use of two interfaces, termed end-helix (EH) and mid-loop (ML). Binding studies indicate Ph and Scm SAMs interact with a preferred orientation of Ph-ML:Scm-EH [15]. The current study began with the identification of Pho and Sfmbt as interacting partners of PRC1. To understand how PhoRC can interact with PRC1, the SAM of Sfmbt was investigated. Biochemical studies, culminating in the Sfmbt SAM and Scm SAM co-crystal structure, indicate that only the EH interface of Sfmbt SAM is capable of SAM–SAM interactions. The Sfmbt-EH interacts with the Scm-ML interface, leaving the Scm-EH free to interact with its preferred Ph-ML interface (Figure 1A). Thus, a new role of SAMs is revealed: the Scm SAM can bridge Ph- and Sfmbt-SAMs, a role not previously observed in SAM interactions. Tethering of Sfmbt to PREs via the DNA binding activity of Pho could recruit PRC1 and potentially initiate SAM-based higher order protein assemblies.
A
EH ML EH Ph
ML EH ML
Scm
Sfmbt
B PRC1 Scm Ph Sfmbt PRE
Pho Current Biology
Figure 1. A model for PcG complex recruitment by a SAM scaffold. (A) Illustration of Ph, Scm and Sfmbt SAMs arranged in the orientation determined by Frey et al. where SAM:SAM interactions are mediated through preferred ML and EH binding surface interactions for each SAM. (B) Model of the recruitment of PRC1 to Pho-bound PREs. PRC1 interactions with chromatin and other recruiting factors are not shown for simplicity. The SAM interaction model of Sfmbt, Scm and Ph proposed by Frey et al. does not preclude homo-polymer formation by either Ph or Scm SAMs. Several SAM polymer units for both Scm and Ph are shown to note this possibility.
In vivo analysis in Drosophila validates the importance of all three SAMs in binding of PRC1 to PREs and in PcG repression. Deletion of the SAM from Sfmbt or Scm impairs their repressive function [4,13], and both Ph SAM and its polymerization interfaces are essential for PcG repression [16,17]. To complete the link between the DNA binding activity of Pho and PRC1 recruitment, Frey et al. demonstrate that the recruitment of PRC1 to a PRE in a transgene depends on the sequence of the Pho motifs. A molecular pathway from DNA motifs in PREs through to recruitment of PRC1 (Figure 1B) can now be constructed at atomic resolution from a collection of structures [4,14,15,18,19]. How this PhoRC–PRC1 pathway functions with other PRE-binding transcription factors to recruit PRC1 (and PRC2) remains to be determined. Scm may be particularly interesting in this regard since it is linked to both PRC1 and PRC2 and can be recruited to a PRE independent of PRC1, PRC2, or PhoRC [11,12].
Current Biology 26, R701–R718, August 8, 2016 R711
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Dispatches Both cooperative interactions between PhoRC and PRC1 (via the SAM network described here) [4,7,9], and the clustering of PcG proteins in the nucleus that depends on Ph SAM [20], may contribute to determining where Pho binds in the genome [7,9]. Genome-wide analysis of Pho distribution indicates that Pho binding sites at PREs are typically not the highest affinity sites in the genome [7,9]. Chromatin architecture analysis indicates Pho binding sites tend to cluster in space. Thus, the effect of SAMs on PcG protein clustering and long-range chromatin interactions, as well as the local cooperative interactions between PhoRC and PRC1, may allow Pho to use sub-optimal sites inside PcG domains and make Pho binding to these sites dependent on Sfmbt and PRC1 [7,20]. What remains unanswered is what different protein and chromatin architectures are possible using this Sfmbt:Scm:Ph scaffold. In vivo, SfmbtSAM:Scm-SAM and Scm-SAM:Ph-SAM interactions would have to compete with the Scm-SAM:Scm-SAM interactions. The ability of Scm-SAM and Ph-SAM to form both homopolymers and copolymers could also introduce variations of assemblies with different numbers and organization of Ph and Scm units (Figure 1B). These interactions would thus be regulated by the concentrations of each protein. The directional nature of the Sfmb–Scm–Ph bridge as described here also means that SAMs cannot directly link two PhoRCs (at the same or different PREs). It is therefore likely that additional mechanisms are involved in SAM-dependent bridging of chromatin sites. It will also be important to measure the affinities of all possible SAM–SAM interfaces since preferred interactions may not be exclusive. Much remains to be determined about the detailed organization of PRC1, PhoRC, and chromatin. This awaits the reconstitution of these interactions using full PcG complexes and chromatin substrates. Mechanisms that regulate SAM polymerization have been described [16,17], and it seems reasonable to expect that this regulation can tune repressive
mechanisms to adjust them to different sites of PcG activity and, perhaps, allow response to different local cues. The dissection of SAM contributions to PcG repression is revealing mechanisms for regulation of chromatin architecture and the potential role for self-reinforcing protein–protein interaction networks and higher order protein assemblies in epigenetic regulation. REFERENCES 1. Boettiger, A.N., Bintu, B., Moffitt, J.R., Wang, S., Beliveau, B.J., Fudenberg, G., Imakaev, M., Mirny, L.A., Wu, C.T., and Zhuang, X. (2016). Super-resolution imaging reveals distinct chromatin folding for different epigenetic states. Nature 529, 418–422. 2. Entrevan, M., Schuettengruber, B., and Cavalli, G. (2016). Regulation of genome architecture and function by polycomb proteins. Trends Cell Biol. 26, 511–525. 3. Simon, J.A., and Kingston, R.E. (2013). Occupying chromatin: Polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put. Mol. Cell 49, 808–824. 4. Frey, F., Sheahan, T., Finkl, K., Stoehr, G., Mann, M., Benda, C., and Muller, J. (2016). Molecular basis of PRC1 targeting to Polycomb response elements by PhoRC. Genes Dev. 30, 1116–1127. 5. Kassis, J.A., and Brown, J.L. (2013). Polycomb group response elements in Drosophila and vertebrates. Adv. Genet. 81, 83–118. 6. Brown, J.L., Fritsch, C., Mueller, J., and Kassis, J.A. (2003). The Drosophila pho-like gene encodes a YY1-related DNA binding protein that is redundant with pleiohomeotic in homeotic gene silencing. Development 130, 285–294. 7. Kahn, T.G., Stenberg, P., Pirrotta, V., and Schwartz, Y.B. (2014). Combinatorial interactions are required for the efficient recruitment of pho repressive complex (PhoRC) to polycomb response elements. PLoS Genet. 10, e1004495. 8. Klymenko, T., Papp, B., Fischle, W., Kocher, T., Schelder, M., Fritsch, C., Wild, B., Wilm, M., and Muller, J. (2006). A Polycomb group protein complex with sequence-specific DNA-binding and selective methyl-lysinebinding activities. Genes Dev. 20, 1110– 1122. 9. Schuettengruber, B., Oded Elkayam, N., Sexton, T., Entrevan, M., Stern, S., Thomas, A., Yaffe, E., Parrinello, H., Tanay, A., and Cavalli, G. (2014). Cooperativity, specificity, and evolutionary stability of Polycomb
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targeting in Drosophila. Cell Rep. 9, 219–233. 10. Grimm, C., Matos, R., Ly-Hartig, N., Steuerwald, U., Lindner, D., Rybin, V., Muller, J., and Muller, C.W. (2009). Molecular recognition of histone lysine methylation by the Polycomb group repressor dSfmbt. EMBO J. 28, 1965–1977. 11. Kang, H., McElroy, K.A., Jung, Y.L., Alekseyenko, A.A., Zee, B.M., Park, P.J., and Kuroda, M.I. (2015). Sex comb on midleg (Scm) is a functional link between PcG-repressive complexes in Drosophila. Genes Dev. 29, 1136–1150. 12. Wang, L., Jahren, N., Miller, E.L., Ketel, C.S., Mallin, D.R., and Simon, J.A. (2010). Comparative analysis of chromatin binding by Sex Comb on Midleg (SCM) and other polycomb group repressors at a Drosophila Hox gene. Mol. Cell Biol. 30, 2584–2593. 13. Peterson, A.J., Mallin, D.R., Francis, N.J., Ketel, C.S., Stamm, J., Voeller, R.K., Kingston, R.E., and Simon, J.A. (2004). Requirement for sex comb on midleg protein interactions in Drosophila polycomb group repression. Genetics 167, 1225–1239. 14. Kim, C.A., Gingery, M., Pilpa, R.M., and Bowie, J.U. (2002). The SAM domain of polyhomeotic forms a helical polymer. Nat. Struct. Biol. 9, 453–457. 15. Kim, C.A., Sawaya, M.R., Cascio, D., Kim, W., and Bowie, J.U. (2005). Structural organization of a Sex-comb-on-midleg/ polyhomeotic copolymer. J. Biol. Chem. 280, 27769–27775. 16. Gambetta, M.C., and Muller, J. (2014). O-GlcNAcylation prevents aggregation of the Polycomb group repressor polyhomeotic. Dev. Cell 31, 629–639. 17. Robinson, A.K., Leal, B.Z., Chadwell, L.V., Wang, R., Ilangovan, U., Kaur, Y., Junco, S.E., Schirf, V., Osmulski, P.A., Gaczynska, M., et al. (2012). The growth-suppressive function of the polycomb group protein polyhomeotic is mediated by polymerization of its sterile alpha motif (SAM) domain. J. Biol. Chem. 287, 8702–8713. 18. Houbaviy, H.B., Usheva, A., Shenk, T., and Burley, S.K. (1996). Cocrystal structure of YY1 bound to the adeno-associated virus P5 initiator. Proc. Natl. Acad. Sci. USA 93, 13577– 13582. 19. Alfieri, C., Gambetta, M.C., Matos, R., Glatt, S., Sehr, P., Fraterman, S., Wilm, M., Muller, J., and Muller, C.W. (2013). Structural basis for targeting the chromatin repressor Sfmbt to Polycomb response elements. Genes Dev. 27, 2367–2379. 20. Wani, A.H., Boettiger, A.N., Schorderet, P., Ergun, A., Munger, C., Sadreyev, R.I., Zhuang, X., Kingston, R.E., and Francis, N.J. (2016). Chromatin topology is coupled to Polycomb group protein subnuclear organization. Nat. Commun. 7, 10291.
Dispatches the observation that top predators may be quickly removed from systems and that levels of decline are correlated with proximity to human populations [12]. The results of Mourier and colleagues [9] demonstrate the changes to reef trophic systems that result from such declines and the potential breakdown of locally inverted biomass pyramids. However, we do not know if inverted biomass pyramids are (or were) common in coral reef systems, and so the full effect of predator declines remains to be determined. These findings also point to some important lessons for coral reef conservation. First, protecting and maintaining the fish spawning aggregations that occur on many coral reefs is important. We now know that it is especially so for sustaining high levels of predator biomass. These aggregations are often fished, which can quickly lead to depletion [13,14] and can have dire consequences for the fish and the predators that depend on them for trophic subsidies. Second, the movement of predators away from areas with inverted biomass pyramids means that protection of the small areas in which the most predators are found is insufficient for protecting these unique trophic phenomena. Instead, there is a need to protect the area over which predators disperse to feed, pointing to the requirement for a detailed understanding
of movement of coral reef species [15] to design spatial management systems.
REFERENCES 1. Trebilco, R., Baum, J.K., Salomon, A.K., and Dulvy, N.K. (2013). Ecosystem ecology: sizebased constraints on the pyramids of life. Trends Ecol. Evol. 28, 423–431. 2. Sandin, S.A., Smith, J.E., DeMartini, E.E., Dinsdale, E.A., Donner, S.D., Friedlander, A.M., Konotchick, T., Malay, M., Maragos, J.E., Obura, D., et al. (2008). Baselines and degradation of coral reefs in the Northern Line Islands. PLoS One 3, 11. 3. Wang, H., Morrison, W., Singh, A., and Weiss, H. (2009). Modeling inverted biomass pyramids and refuges in ecosystems. Ecol. Modell. 220, 1376–1382.
8. Trebilco, R., Dulvy, N.K., Anderson, S.C., and Salomon, A.K. (2016). The paradox of inverted biomass pyramids in kelp forest fish communities. Proc. R. Soc. Lond. B. Biol. Sci. 283, http://dx.doi.org/10.1098/rspb.2016. 0816. 9. Mourier, J., Maynard, J., Parravicini, V., Ballesta, L., Clua, E., Domeier, M., and Planes, S. (2016). Extreme inverted trophic pyramid of reef sharks supported by spawning groupers. Curr. Biol. 26, 2011–2016. 10. Heupel, M.R., Knip, D.M., Simpfendorfer, C.A., and Dulvy, N.K. (2014). Sizing up the ecological role of sharks as predators. Mar. Ecol. Prog. Ser. 495, 291–298. 11. McCauley, D.J., Young, H.S., Dunbar, R.B., Estes, J.A., Semmens, B.X., and Michel, F. (2012). Assessing the effects of large mobile predators on ecosystem connectivity. Ecol. Appl. 22, 1711–1717.
4. Ward-Paige, C., Mills Flemming, J., and Lotze, H.K. (2010). Overestimating fish counts by non-instantaneous visual censuses: consequences for population and community descriptions. PLoS One 5, e11722.
12. Nadon, M.O., Baum, J.K., Williams, I.D., McPherson, J.M., Zgliczynski, B.J., Richards, B.L., Schroeder, R.E., and Brainard, R.E. (2012). Re-creating missing population baselines for Pacific reef sharks. Conserv. Biol. 26, 493–503.
5. Salinas de Leo´n, P., Acun˜a-Marrero, D., Rastoin, E., Friedlander, A.M., Donovan, M.K., and Sala, E. (2016). Largest global shark biomass found in the northern Gala´pagos Islands of Darwin and Wolf. PeerJ. 4, e1911.
13. Sadovy, Y., and Domeier, M. (2005). Are aggregation-fisheries sustainable? Reef fish fisheries as a case study. Coral Reefs 24, 254–262.
6. Friedlander, A.M., and DeMartini, E.E. (2002). Contrasts in density, size, and biomass of reef fishes between the northwestern and the main Hawaiian islands: The effects of fishing down apex predators. Mar. Ecol. Prog. Ser. 230, 253–265. 7. Gasol, J.M., del Giorgio, P.A., and Duarte, C.M. (1997). Biomass distribution in marine planktonic communities. Limnol. Oceanogr. 42, 1353–1363.
14. Sadovy de Mitcheson, Y. (2016). Mainstreaming fish spawning aggregations into fishery management calls for a precautionary approach. BioScience 66, 295–306. 15. Espinoza, M., Le´de´e, E.J.I., Simpfendorfer, C.A., Tobin, A.J., and Heupel, M.R. (2015). Contrasting movements and connectivity of reef-associated sharks using acoustic telemetry: implications for management. Ecol. Appl. 25, 2101–2118.
Chromatin: Polycomb Group SAMs Unite Chongwoo A. Kim1 and Nicole J. Francis2,3 1Department
of Biochemistry, Midwestern University, 19555 N. 59th Avenue, Glendale, AZ 85308, USA de recherches cliniques de Montre´al, Montre´al, Que´bec, Canada 3De ´ partement de biochimie et mede´cine mole´culaire, Universite´ de Montre´al, Montre´al, Que´bec, Canada Correspondence: [email protected] http://dx.doi.org/10.1016/j.cub.2016.06.001 2Institut
Polycomb Group (PcG) proteins assemble a chromatin state that maintains developmental gene repression. A new study combining structure and in vivo analysis details a molecular network from DNA recognition to PcG recruitment, highlighting the essential role of Sterile Alpha Motifs. Polycomb Group (PcG) proteins are conserved, essential epigenetic repressors of gene expression. They
function within multi-protein assemblies able to mediate repression through both physical (chromatin folding) and
R710 Current Biology 26, R701–R718, August 8, 2016 ª 2016 Elsevier Ltd.
biochemical (protein–protein interactions and protein modification) mechanisms creating unique chromatin
Current Biology
Dispatches states that can cover hundreds of kilobases [1,2]. The first PcG complex identified, aptly named Polycomb Repression Complex 1 (PRC1), can function as a histone H2A ubiquitin E3 ligase ans is also able to modify chromatin architecture through both local chromatin compaction and long-range interactions [2,3]. PRC1 can also directly inhibit transcription and destabilize transcription pre-initiation complexes in vitro. PRC2, another PcG protein complex, methylates histone H3 on lysine 27 (H3K27me), an activity which is essential for PcG function [2,3]. Despite the wealth of information that has been acquired, fundamental mechanistic questions remain about how PcG proteins are recruited to target genes and how their recruitment leads to assembly of repressive chromatin domains. A recent study from Frey et al. [4], reported in Genes and Development, along with other recent work, reveals the central role of a conserved protein–protein interaction domain, the Sterile Alpha Motif (SAM), in anchoring PcG complexes to the genome and, potentially, in the organization of chromatin architecture. In Drosophila, where the PcG was discovered and is best understood, large, complex cis regulatory elements termed Polycomb Response Elements (PREs) recruit PcG proteins [5]. However, only one of the core PcG proteins, Pleiohomeotic (Pho), and its homologue Pho-like (PhoL), has sequence-specific DNA binding activity and can recognize motifs present in PREs [6]. Thus, the other PcG proteins must be recruited indirectly to PREs. The PcG protein that is the major player in this indirect recruitment is Sfmbt. Sfmbt binds directly to Pho, is essential for PcG silencing, and is required for full binding of Pho to PREs [7,8]. Together, Pho and Sfmbt constitute the core of the PcG complex called Pho Repressive Complex (PhoRC), which links PRC1 to PREs. It is important to note, however, that the PhoRC–PRC1 relationship is self-reinforcing rather than strictly hierarchical since PhoRC binding is also reduced in PRC1 mutants [7,9]. What remained unresolved was the molecular basis for PhoRC recruitment of PRC1.
Frey et al. now show that the protein that brings PhoRC and PRC1 together is yet another PcG protein called Sex comb on midleg (Scm). Scm appears to be the wild card in PcG-mediated repression. Scm can associate with PhoRC [10], PRC1, and PRC2 [11], and possibly a separate 500 kDa complex [12], but is not considered a core component of these established PcG complexes due to substoichiometric amounts observed in their purifications or weak/transient interactions. Nevertheless, Scm is important for repression mediated by both PhoRC [10] and PRC1 [13]. The current study determined that Scm bridges PhoRC and PRC1 through interactions between the SAMs present in Sfmbt, Scm, and the PRC1 subunit Ph. SAMs are involved in protein–protein interactions, and in dynamic, regulated assembly macromolecular structures through the ability of some SAMs to assemble into helical polymers. The SAMs of both Ph and Scm can form homopolymers as well as assemble into a co-polymer [14,15]. SAMs form polymers through the iterative use of two interfaces, termed end-helix (EH) and mid-loop (ML). Binding studies indicate Ph and Scm SAMs interact with a preferred orientation of Ph-ML:Scm-EH [15]. The current study began with the identification of Pho and Sfmbt as interacting partners of PRC1. To understand how PhoRC can interact with PRC1, the SAM of Sfmbt was investigated. Biochemical studies, culminating in the Sfmbt SAM and Scm SAM co-crystal structure, indicate that only the EH interface of Sfmbt SAM is capable of SAM–SAM interactions. The Sfmbt-EH interacts with the Scm-ML interface, leaving the Scm-EH free to interact with its preferred Ph-ML interface (Figure 1A). Thus, a new role of SAMs is revealed: the Scm SAM can bridge Ph- and Sfmbt-SAMs, a role not previously observed in SAM interactions. Tethering of Sfmbt to PREs via the DNA binding activity of Pho could recruit PRC1 and potentially initiate SAM-based higher order protein assemblies.
A
EH ML EH Ph
ML EH ML
Scm
Sfmbt
B PRC1 Scm Ph Sfmbt PRE
Pho Current Biology
Figure 1. A model for PcG complex recruitment by a SAM scaffold. (A) Illustration of Ph, Scm and Sfmbt SAMs arranged in the orientation determined by Frey et al. where SAM:SAM interactions are mediated through preferred ML and EH binding surface interactions for each SAM. (B) Model of the recruitment of PRC1 to Pho-bound PREs. PRC1 interactions with chromatin and other recruiting factors are not shown for simplicity. The SAM interaction model of Sfmbt, Scm and Ph proposed by Frey et al. does not preclude homo-polymer formation by either Ph or Scm SAMs. Several SAM polymer units for both Scm and Ph are shown to note this possibility.
In vivo analysis in Drosophila validates the importance of all three SAMs in binding of PRC1 to PREs and in PcG repression. Deletion of the SAM from Sfmbt or Scm impairs their repressive function [4,13], and both Ph SAM and its polymerization interfaces are essential for PcG repression [16,17]. To complete the link between the DNA binding activity of Pho and PRC1 recruitment, Frey et al. demonstrate that the recruitment of PRC1 to a PRE in a transgene depends on the sequence of the Pho motifs. A molecular pathway from DNA motifs in PREs through to recruitment of PRC1 (Figure 1B) can now be constructed at atomic resolution from a collection of structures [4,14,15,18,19]. How this PhoRC–PRC1 pathway functions with other PRE-binding transcription factors to recruit PRC1 (and PRC2) remains to be determined. Scm may be particularly interesting in this regard since it is linked to both PRC1 and PRC2 and can be recruited to a PRE independent of PRC1, PRC2, or PhoRC [11,12].
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Current Biology
Dispatches Both cooperative interactions between PhoRC and PRC1 (via the SAM network described here) [4,7,9], and the clustering of PcG proteins in the nucleus that depends on Ph SAM [20], may contribute to determining where Pho binds in the genome [7,9]. Genome-wide analysis of Pho distribution indicates that Pho binding sites at PREs are typically not the highest affinity sites in the genome [7,9]. Chromatin architecture analysis indicates Pho binding sites tend to cluster in space. Thus, the effect of SAMs on PcG protein clustering and long-range chromatin interactions, as well as the local cooperative interactions between PhoRC and PRC1, may allow Pho to use sub-optimal sites inside PcG domains and make Pho binding to these sites dependent on Sfmbt and PRC1 [7,20]. What remains unanswered is what different protein and chromatin architectures are possible using this Sfmbt:Scm:Ph scaffold. In vivo, SfmbtSAM:Scm-SAM and Scm-SAM:Ph-SAM interactions would have to compete with the Scm-SAM:Scm-SAM interactions. The ability of Scm-SAM and Ph-SAM to form both homopolymers and copolymers could also introduce variations of assemblies with different numbers and organization of Ph and Scm units (Figure 1B). These interactions would thus be regulated by the concentrations of each protein. The directional nature of the Sfmb–Scm–Ph bridge as described here also means that SAMs cannot directly link two PhoRCs (at the same or different PREs). It is therefore likely that additional mechanisms are involved in SAM-dependent bridging of chromatin sites. It will also be important to measure the affinities of all possible SAM–SAM interfaces since preferred interactions may not be exclusive. Much remains to be determined about the detailed organization of PRC1, PhoRC, and chromatin. This awaits the reconstitution of these interactions using full PcG complexes and chromatin substrates. Mechanisms that regulate SAM polymerization have been described [16,17], and it seems reasonable to expect that this regulation can tune repressive
mechanisms to adjust them to different sites of PcG activity and, perhaps, allow response to different local cues. The dissection of SAM contributions to PcG repression is revealing mechanisms for regulation of chromatin architecture and the potential role for self-reinforcing protein–protein interaction networks and higher order protein assemblies in epigenetic regulation. REFERENCES 1. Boettiger, A.N., Bintu, B., Moffitt, J.R., Wang, S., Beliveau, B.J., Fudenberg, G., Imakaev, M., Mirny, L.A., Wu, C.T., and Zhuang, X. (2016). Super-resolution imaging reveals distinct chromatin folding for different epigenetic states. Nature 529, 418–422. 2. Entrevan, M., Schuettengruber, B., and Cavalli, G. (2016). Regulation of genome architecture and function by polycomb proteins. Trends Cell Biol. 26, 511–525. 3. Simon, J.A., and Kingston, R.E. (2013). Occupying chromatin: Polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put. Mol. Cell 49, 808–824. 4. Frey, F., Sheahan, T., Finkl, K., Stoehr, G., Mann, M., Benda, C., and Muller, J. (2016). Molecular basis of PRC1 targeting to Polycomb response elements by PhoRC. Genes Dev. 30, 1116–1127. 5. Kassis, J.A., and Brown, J.L. (2013). Polycomb group response elements in Drosophila and vertebrates. Adv. Genet. 81, 83–118. 6. Brown, J.L., Fritsch, C., Mueller, J., and Kassis, J.A. (2003). The Drosophila pho-like gene encodes a YY1-related DNA binding protein that is redundant with pleiohomeotic in homeotic gene silencing. Development 130, 285–294. 7. Kahn, T.G., Stenberg, P., Pirrotta, V., and Schwartz, Y.B. (2014). Combinatorial interactions are required for the efficient recruitment of pho repressive complex (PhoRC) to polycomb response elements. PLoS Genet. 10, e1004495. 8. Klymenko, T., Papp, B., Fischle, W., Kocher, T., Schelder, M., Fritsch, C., Wild, B., Wilm, M., and Muller, J. (2006). A Polycomb group protein complex with sequence-specific DNA-binding and selective methyl-lysinebinding activities. Genes Dev. 20, 1110– 1122. 9. Schuettengruber, B., Oded Elkayam, N., Sexton, T., Entrevan, M., Stern, S., Thomas, A., Yaffe, E., Parrinello, H., Tanay, A., and Cavalli, G. (2014). Cooperativity, specificity, and evolutionary stability of Polycomb
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