In multicellular eukaryotes the capacity to express defined subsets of genes at the correct time and place underpins normal development. While genetically encoded regulatory pathways define the nature of these gene expression patterns in individual cell types and tissues, these systems must ultimately converge on a DNA substrate that is not unadorned, but instead wrapped around histone proteins to form nucleosomes and chromatin. Through studying chromatin function in the context of gene regulatory networks it is now clear that modification of DNA and histones is essential for controlling normal regulation of gene expression (Bannister and Kouzarides, 2011).

In animals, Polycomb group (PcG) proteins play central roles in gene regulation through their chromatin modifying activities. First identified in Drosophila as important regulators of body plan specification, orthologous PcG systems were subsequently identified in vertebrates and also shown to be essential for developmental gene regulation (Lewis, 1978; Schwartz and Pirrotta, 2013). Biochemical characterization of PcG proteins has revealed that they form large multiprotein complexes that function as chromatin-modifying or -remodelling enzymes (Müller and Verrijzer, 2009; Simon and Kingston, 2013). PcG proteins generally fall into one of two multiprotein complexes, called Polycomb repressive complex 1 and 2 (PRC1 and PRC2). PRC1 functions as a histone E3 ubiquitin ligase that mono-ubiquitylates histone H2A at position 119 (H2AK119ub1) (Cao et al., 2005; de Napoles et al., 2004; Wang et al., 2004a) and PRC2 is a histone H3 lysine 27 (H3K27) methyltransferase (Cao et al., 2002; Czermin et al., 2002; Müller et al., 2002; Margueron and Reinberg, 2011). Together, the activities of these two PcG protein complexes play important roles in maintaining repressive chromatin states and controlling gene expression during cell fate transitions (Xie et al., 2013; Arnold et al., 2013; Boyer et al., 2006; Leeb and Wutz, 2007; Endoh et al., 2008; Terranova et al., 2008; Koche et al., 2008; Bracken et al., 2006).

The defined molecular mechanisms that underpin how PcG complexes regulate transcription still remain poorly understood. However, it has been proposed that the occupancy of PRC1 and PRC2 at genomic target sites and their capacity to modify histones may function to create structurally compacted chromatin which is inhibitory to transcription (Eskeland et al., 2010; Francis et al., 2004; Wani et al., 2016; Boettiger et al., 2016). To achieve this, the function of PRC1 and PRC2 appear to be intimately linked. For example, some PRC1 complexes are able to recognise and bind PRC2-dependent H3K27me3, recruiting those PRC1 complexes to sites occupied by PRC2 (Min et al., 2003; Wang et al., 2004b). Similarly, PRC2 can recognize PRC1-deposited H2AK119ub1, regulating PRC2 occupancy on chromatin (Blackledge et al., 2014; Cooper et al., 2014). Furthermore, in vitro assays have suggested that H2AK119ub1 may also stimulate the enzymatic activity of PRC2 (Kalb et al., 2014). Based on these observations it has been proposed that feedback mechanisms, whereby one Polycomb repressive complex supports the recruitment and possibly activity of the other complex, may in part underpin chromatin-based transcriptional repression (Blackledge et al., 2015; Steffen and Ringrose, 2014; Voigt et al., 2013). In other scenarios the relevance of histone modifications to gene repression, particularly the role of H2AK119ub1, has remained unclear, with the repressive nature of PRC1 being proposed to reside within its capacity to alter chromatin structure and compaction independently of its H2A ubiquitylating activity (Pengelly et al., 2015; Illingworth et al., 2015; Francis et al., 2004; Eskeland et al., 2010). However in most cases, the defined contribution of H2AK119ub1-dependent and –independent pathways, and their relevance to Polycomb chromatin domain function in vivo have remained incompletely defined.

In order to understand the mechanisms by which the PcG systems function together in vivo, it will be critical to discover how, if at all, their enzymatic activities contribute to chromatin structure and gene regulation. While significant effort has been placed on understanding in vitro how the H3K27 methyltransferase activity of PRC2 is controlled (Margueron et al., 2009; Jiao and Liu, 2015; Cao and Zhang, 2004), far less is known about how PRC1 complexes catalyse H2AK119ub1 and how this relates to the function of PRC1 in vivo. This paucity in our understanding of PRC1 activity is in part due to the diverse nature of individual PRC1 complexes. At the simplest level, PRC1 complexes are formed by their catalytic subunit, RING1A or RING1B, which dimerises with a PCGF protein to form an active E3 ubiquitin ligase that targets histone H2A (Levine et al., 2002; Satijn et al., 1997). In mammals, there are six PCGF proteins (PCGF1–6), and the identity of the PCGF protein incorporated into PRC1 appears to direct interaction with a specific complement of auxiliary proteins, giving rise to an array of diverse PRC1 complexes (Blackledge et al., 2014; Gao et al., 2012; Tavares et al., 2012). Based on the protein subunit composition of these individual PRC1 complexes, they are often then divided into ‘canonical’ or ‘variant’ complexes. Canonical PRC1 complexes form around either PCGF2 or 4, and incorporate a chromobox (CBX) protein, which can directly bind to PRC2-catalysed H3K27me3 (Fischle et al., 2003; Bernstein et al., 2006; Min et al., 2003). In contrast, variant PRC1 complexes can form around all six PCGF proteins, but these complexes engage with RYBP or YAF2, two proteins that bind to a site on RING1B that is mutually exclusive with CBX protein binding (García et al., 1999; Wang et al., 2010).

Despite the diversity of PRC1 complexes, biochemical characterization of their catalytic activity has largely been focused on canonical PCGF2/PCGF4-containing complexes (Li et al., 2006; Gao et al., 2012; Elderkin et al., 2007; Cao et al., 2005; Buchwald et al., 2006; Ben-saadon et al., 2006). For example, in vitro ubiquitylation assays using reconstituted complexes and nucleosome substrates demonstrated that RING1(A/B) could form an active E3 ligase in the presence of either PCGF2 or PCGF4 (Elderkin et al., 2007; Buchwald et al., 2006). Furthermore, a minimal complex comprised of a dimer between the N-terminal RING finger domains of PCGF4 and RING1B was sufficient to deposit H2AK119ub1 (Buchwald et al., 2006). Importantly, the RING finger domains of individual PCGF proteins are highly similar, suggesting that they will likely all support formation of an active E3 ligase complex. However, more recent work examining PCGF(1–6)/RING1(A/B) catalytic RING-RING dimers revealed that PCGF(2/4)/RING1(A/B) were less active than PCGF(1/3/5/6)/RING1(A/B) in autoubiquitylation and E2 lysine discharge assays (Taherbhoy et al., 2015). Surprisingly, when these same assemblies were challenged with physiologically relevant nucleosome substrates, complex-specific activities were no longer apparent. Nevertheless, consistent with the possibility that individual PRC1 complexes may have distinct activities related to protein complex composition, in vivo studies in both mammals and Drosophila have suggested that less-well characterized variant PRC1 complexes may contribute more significantly to H2A mono-ubiquitylation than canonical PRC1 (Tavares et al., 2012; Lee et al., 2015; Lagarou et al., 2008; Blackledge et al., 2014). For example, the Drosophila dRAF complex, which appears to have similarities to mammalian variant PRC1 complexes, is a highly active H2A ubiquitin ligase in vitro and appears to be the predominant H2A ubiquitin ligase in Drosophila S2 cells (Lagarou et al., 2008). Furthermore, a PCGF3 orthologue contributes widely to H2AK119ub1 in cultured Drosophila cells (Lee et al., 2015). This raises the possibility that individual PCGF proteins might confer differing E3 ligase activities on PRC1, either directly, or via interaction with complex-specific regulatory subunits. Indeed, incorporation of the auxiliary subunits RYBP, PHC2 or CBX2/8 into a PCGF4/RING1B complex has been reported to affect its E3 ligase activity in different ways (Gao et al., 2012), though this was not the case for PCGF2/RING1B, in which auxiliary subunits had little impact on catalytic activity (Tavares et al., 2012). Therefore a more detailed understanding of how H2AK119 ubiquitin ligase activity is shaped by different PRC1 assemblies, both in vitro and in vivo, is required to better understand PRC1 involvement in polycomb chromatin domain function and gene regulation.

To address these important questions we have exploited biochemical and genomic approaches to interrogate PRC1 enzymatic activity in vitro and in cells. This revealed that PRC1 complexes are highly modular, and that the enzymatic activity of PRC1 is defined by the choice of PCGF protein combined with the influence of RYBP/YAF2, uncovering an enzymatic logic within the assembly of individual PRC1 complexes. Building on these discoveries, genetic ablation and genomic approaches uncover a widespread role for RYBP in stimulating H2AK119 E3 ligase activity in vivo, and we demonstrate that the absence of this activity leads to defects in the deposition of H3K27me3 by PRC2. Importantly, in the absence of RYBP, loss of PRC1 stimulation and subsequent defects in PRC2 activity can lead to erosion of Polycomb chromatin domains and susceptibility to inappropriate gene activation signals. Together, these observations identify an activity-based communication between PRC1 and PRC2 in vivo, and suggest that histone modification-dependent thresholding mechanisms sustain Polycomb chromatin domains, connecting the activity of PcG systems to their occupancy on chromatin and transcriptional repression.

PcG proteins play an important chromatin-based role in controlling gene expression. The PRC1 and PRC2 complexes work together to achieve this, and in vertebrates both their occupancy on chromatin and functionality appear to rely, at least in part, on their capacity to modify histones. However, mechanisms controlling the activity of PRC1, and its relationship with PRC2, remain poorly understood. Here we reveal a defined requirement for specific PCGF components and the auxiliary subunits RYBP/YAF2 in regulating the H2AK119 E3 ligase activity of PRC1 in vitro (Figures 1–4). In vivo, RYBP plays a central role in shaping normal H2AK119ub1 at Polycomb target sites (Figure 5) where it functions to stimulate the E3 ligase activity of PRC1 (Figures 6 and 7) and regulate PRC2-dependent H3K27me3 (Figure 8). At a subset of Polycomb target sites, the absence of RYBP leads to a more profound effect on histone modifications leading to Polycomb chromatin domain erosion (Figure 8) and inappropriate reactivation of gene expression (Figure 9). Together this extends our understanding of the interesting and important activity-based communication between PcG complexes that has been previously proposed from in vitro studies (Kalb et al., 2014), and demonstrates that the enzymatic activity of PcG complexes can communicate through chromatin in vivo in order to maintain normal PcG function in gene regulation.

A striking and central conclusion from our enzymatic analysis of PRC1 complexes in vitro was that the enzymatic activity of PRC1 is determined not only by its interaction with auxiliary factors like RYBP/YAF2 but also the PCGF protein that forms the PCGF/RING1 catalytic core. This therefore suggests that the activity of individual PRC1 complexes is likely to depend upon the inclusion of specific PCGF proteins and to be highly regulated in vivo. An important focus for future work will be to directly compare the E3 ligase activities of other less well-characterized PRC1 complexes, to understand how widespread these regulatory mechanisms are amongst distinct PRC1 assemblies, including those PRC1 complexes formed by PCGF3/5/6. In an important first step towards achieving this, a recent study examined PCGF1–6/RING1 catalytic dimers and concluded that they displayed little inherent difference in their H2AK119 E3 ligase activity on nucleosome substrates in vitro (Taherbhoy et al., 2015). However, these experiments relied on truncated proteins which lack important domains, including the C-terminus of RING1 which is the interaction site for RYBP/YAF2 (Wang et al., 2010). In contrast our observations using full length proteins demonstrate that PRC1 activity is governed by the nature of the catalytic dimer and the stimulatory activity of auxiliary factors like RYBP/YAF2. An important focus of future work will also rely on understanding the relative contribution of individual PRC1 complexes in placing H2AK119ub1 in vivo, given that RYBP/YAF2 appears to interact with diverse PRC1 complexes.

As we begin to learn more about the mechanisms that shape PRC1-dependent E3 ligase activity, our understanding of how this contributes towards PcG system function in gene regulation and development remains an important and outstanding question. Indeed, it still remains incompletely defined how the H2AK119ub1-dependent and independent activities of PRC1 are leveraged by the PcG systems to regulate gene expression, particularly in mammals. However, early biochemical work very clearly demonstrated a structural role for PRC1 in chromatin compaction and counteracting chromatin remodelling in vitro, independently of H2AK119ub1 (Grau et al., 2011; Francis et al., 2004). Furthermore, RING1B null cells which display transcriptional reactivation and chromatin decompaction of the PcG-occupied Hox loci, are rescued of these deficiencies with a form or RING1B lacking catalytic activity, suggesting a structural role on chromatin for PRC1 in this context (Eskeland et al., 2010). In agreement with a role for canonical PRC1 in shaping chromatin structure at PcG target sites, the PHC component of canonical PRC1 can polymerize in vitro and this activity appears to be import for the formation of cytologically identifiable structures referred to as ‘polycomb bodies’ (Kim et al., 2002; Isono et al., 2013). Similar studies in Drosophila have also provided evidence via super resolution imaging that Polycomb body formation relies on Ph (orthologous to mammalian PHC) to shape chromatin domains (Boettiger et al., 2016; Wani et al., 2016) and perturbation of Ph in Drosophila leads to widespread effects on normal gene repression (Lagarou et al., 2008). This abundance of work clearly indicates that PRC1 has essential structure-based effects on chromatin that are related to normal PcG-dependent gene regulation and in some cases this appears to be independent of the E3 ligase activity of PRC1. Consistent with H2AK119ub1-independent roles for PRC1 in normal Drosophila development, a catalytic mutant of the Drosophila RING1 orthologue (Sce1) is less phenotypically severe than that of Sce1 null animals (Pengelly et al., 2015). Furthermore, mutation of histone H2A such that it is no longer a target for PRC1-dependent mono-ubiquitylation did not lead to Hox gene misregulation in Drosophila imaginal disc clones, suggesting that H2A ubiquitylation is not required to maintain repression of these PcG target genes (Pengelly et al., 2015). Similarly a Ring1b^I53A/I53A mouse model, in which the E3 ligase activity of RING1B-containing PRC1 complexes is perturbed, indicated that the catalytic activity of RING1B is also not essential for early mouse development, with Ring1b^I53A/I53A mice succumbing to embryonic lethality at a later embryonic stage than Ring1b-null mice (Illingworth et al., 2015; Voncken et al., 2003). Because these Ring1b mouse models do not completely phenocopy each other it would suggest that RING1B may contribute functions during mammalian development that are independent of H2A mono-ubiquitylation, possibly related to its role in chromatin compaction (Eskeland et al., 2010; Francis et al., 2004). However, it still remains unclear whether early mammalian development and normal polycomb mediated repression can be sustained in the absence of PRC1 E3 ligase activity, as this requires the generation of a mouse model that lacks the enzymatic activity of RING1A and RING1B, both of which are expressed in early development and contribute to deposition of H2AK119ub1 and gene repression (de Napoles et al., 2004; Endoh et al., 2008). Interestingly, however, Ring1b^I53A/I53A embryonic stem cells have dramatically reduced levels of H2AK119ub1, show reductions in H3K27me3 and reactivate a subset of PcG target genes (Illingworth et al., 2015). This scenario is reminiscent of the reductions in H2AK119ub1 and H3K27me3 that occur following loss of RYBP-dependent stimulation of PRC1. Together these observations support the general argument that in mammals H2AK119ub1 helps to shape communication between PRC1 and PRC2 in order to sustain normal Polycomb chromatin domain function in gene regulation. However it is clear that a more detailed analysis of both RING1A and RING1B activity will be imperative in defining how important H2AK119ub1 is for mammalian development and gene regulation by the PcG system.

PRC1 and PRC2 complexes have the capacity to recognise H2AK119ub1 and H3K27me3, and it has been proposed that this supports PcG protein binding to chromatin (Blackledge et al., 2015; Kalb et al., 2014; Margueron et al., 2009; Min et al., 2003; Wang et al., 2004b; Simon and Kingston, 2013). Here we show that RYBP deletion causes reductions in H2AK119ub1 and H3K27me3 yet PRC1 and PRC2 occupancy remains largely intact. However, a subset of PcG target sites exhibit larger reductions in H2AK119ub1 and H3K27me3 and have pronounced losses of PcG complex occupancy. Based on these observations we speculate that activity-based communication between PRC1 and PRC2 may support PcG complex occupancy through a histone modification-dependent thresholding mechanism. In the context of such a mechanism we speculate that the majority of PcG target genes have H2AK119ub1 and H3K27me3 levels that are in excess of that required to support normal PRC1 and PRC2 binding. However, if H2AK119ub1 and H3K27me3 levels fall below a given threshold, as some sites do in the absence of RYBP, normal activity-based communication between PRC1 and PRC2 can no longer be maintained, leading to a reduction in PcG protein occupancy and an increased propensity for gene reactivation.

If histone modification-dependent thresholding mechanisms contribute at least at some sites to the function of the Polycomb system, how or why might this be beneficial to the control of gene expression? Given the highly dynamic nature of PRC1 and PRC2 interactions with chromatin (Fonseca et al., 2012), histone modifications could provide a form of local epigenetic memory to bridge the intervening time between individual engagement events to reinforce PcG protein occupancy. Assuming that PcG protein occupancy is important for gene repression and that histone modifications can stabilize PcG protein binding, it would seem sensible that some fluctuation in histone modification levels would need to be tolerated in order to protect the stability of the PcG system at individual sites. However, this hysteresis could be rapidly counteracted by altering the activity of individual PcG complexes, or by removing their associated histone modifications. In both scenarios this would impair PcG complex occupancy and render these normally inactive states reversible and responsive to strong gene activation signals during cell lineage commitment. In combination with chromatin-modifying activities of the Trithorax group systems that are associated with gene activation, this could support a form of chromatin bistability at gene regulatory elements, as we and others have previously proposed, to allow switch-like activation of gene expression (Deaton and Bird, 2011; Klose et al., 2013; Steffen and Ringrose, 2014; Voigt et al., 2013). Interestingly, alterations in the histone modifying activities of PcG complexes are key molecular defects associated with various cancers, suggesting that perturbing PcG-associated histone modifications, or potentially altering histone modification thresholds, may be important in the pathological adaptation of gene expression in cancer (Scelfo et al., 2015). Clearly more work is required to understand the relative contributions of the histone modification-dependent and -independent activities of PcG complexes to both chromatin structure and gene repression at polycomb chromatin domains.

In conclusion, our enzymatic analysis of PRC1 in vitro and genomic dissection of how RYBP shapes its activity in vivo, reveal an interesting activity-based communication between Polycomb repressive complexes on chromatin. Furthermore, they indicate that histone modification thresholds could play an important and previously unrealized role in shaping Polycomb chromatin domain function in the context of gene regulation.

1. 1. Rose NR
   2. King HW
   3. Blackledge NP
   4. Ember K
   5. Fischer R
   6. Kessler BM
   7. Klose RJ

   (2016) RBYP stimulates PRC1 to shape chromatin-based communication between polycomb repressive complexes

   Publicly available at the NCBI Gene Expression Omnibus (accession no: GSE83135).

   https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE83135

1. 1. Adam J
   2. Hatipoglu E
   3. O'Flaherty L
   4. Ternette N
   5. Sahgal N
   6. Lockstone H
   7. Baban D
   8. Nye E
   9. Stamp GW
   10. Wolhuter K
   11. Stevens M
   12. Fischer R
   13. Carmeliet P
   14. Maxwell PH
   15. Pugh CW
   16. Frizzell N
   17. Soga T
   18. Kessler BM
   19. El-Bahrawy M
   20. Ratcliffe PJ
   21. Pollard PJ

   (2011) Renal cyst formation in Fh1-deficient mice is independent of the Hif/Phd pathway: roles for fumarate in KEAP1 succination and Nrf2 signaling

   Cancer Cell 20:524–537.

   https://doi.org/10.1016/j.ccr.2011.09.006

   • Google Scholar
2. 1. Arnold P
   2. Schöler A
   3. Pachkov M
   4. Balwierz PJ
   5. Jørgensen H
   6. Stadler MB
   7. van Nimwegen E
   8. Schübeler D

   (2013) Modeling of epigenome dynamics identifies transcription factors that mediate Polycomb targeting

   Genome Research 23:60–73.

   https://doi.org/10.1101/gr.142661.112

   • Google Scholar
3. 1. Arrigoni R
   2. Alam SL
   3. Wamstad JA
   4. Bardwell VJ
   5. Sundquist WI
   6. Schreiber-Agus N

   (2006) The Polycomb-associated protein Rybp is a ubiquitin binding protein

   FEBS Letters 580:6233–6241.

   https://doi.org/10.1016/j.febslet.2006.10.027

   • Google Scholar
4. 1. Bannister AJ
   2. Kouzarides T

   (2011) Regulation of chromatin by histone modifications

   Cell Research 21:381–395.

   https://doi.org/10.1038/cr.2011.22

   • Google Scholar
5. 1. Ben-Saadon R
   2. Zaaroor D
   3. Ziv T
   4. Ciechanover A

   (2006) The polycomb protein Ring1B generates self atypical mixed ubiquitin chains required for its in vitro histone H2A ligase activity

   Molecular Cell 24:701–711.

   https://doi.org/10.1016/j.molcel.2006.10.022

   • Google Scholar
6. 1. Bernstein E
   2. Duncan EM
   3. Masui O
   4. Gil J
   5. Heard E
   6. Allis CD

   (2006) Mouse polycomb proteins bind differentially to methylated histone H3 and RNA and are enriched in facultative heterochromatin

   Molecular and Cellular Biology 26:2560–2569.

   https://doi.org/10.1128/MCB.26.7.2560-2569.2006

   • Google Scholar
7. 1. Bieniossek C
   2. Imasaki T
   3. Takagi Y
   4. Berger I

   (2012) MultiBac: expanding the research toolbox for multiprotein complexes

   Trends in Biochemical Sciences 37:49–57.

   https://doi.org/10.1016/j.tibs.2011.10.005

   • Google Scholar
8. 1. Blackledge NP
   2. Farcas AM
   3. Kondo T
   4. King HW
   5. McGouran JF
   6. Hanssen LL
   7. Ito S
   8. Cooper S
   9. Kondo K
   10. Koseki Y
   11. Ishikura T
   12. Long HK
   13. Sheahan TW
   14. Brockdorff N
   15. Kessler BM
   16. Koseki H
   17. Klose RJ

   (2014) Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation

   Cell 157:1445–1459.

   https://doi.org/10.1016/j.cell.2014.05.004

   • Google Scholar
9. 1. Blackledge NP
   2. Rose NR
   3. Klose RJ

   (2015) Targeting polycomb systems to regulate gene expression: modifications to a complex story

   Nature Reviews Molecular Cell Biology 16:643–649.

   https://doi.org/10.1038/nrm4067

   • Google Scholar
10. 1. Boettiger AN
    2. Bintu B
    3. Moffitt JR
    4. Wang S
    5. Beliveau BJ
    6. Fudenberg G
    7. Imakaev M
    8. Mirny LA
    9. Wu CT
    10. Zhuang X

    (2016) Super-resolution imaging reveals distinct chromatin folding for different epigenetic states

    Nature 529:418–422.

    https://doi.org/10.1038/nature16496

    • Google Scholar
11. 1. Bonhoure N
    2. Bounova G
    3. Bernasconi D
    4. Praz V
    5. Lammers F
    6. Canella D
    7. Willis IM
    8. Herr W
    9. Hernandez N
    10. Delorenzi M
    11. CycliX Consortium

    (2014) Quantifying ChIP-seq data: a spiking method providing an internal reference for sample-to-sample normalization

    Genome Research 24:1157–1168.

    https://doi.org/10.1101/gr.168260.113

    • Google Scholar
12. 1. Boulard M
    2. Edwards JR
    3. Bestor TH

    (2015) FBXL10 protects polycomb-bound genes from hypermethylation

    Nature Genetics 47:479–485.

    https://doi.org/10.1038/ng.3272

    • Google Scholar
13. 1. Boyer LA
    2. Plath K
    3. Zeitlinger J
    4. Brambrink T
    5. Medeiros LA
    6. Lee TI
    7. Levine SS
    8. Wernig M
    9. Tajonar A
    10. Ray MK
    11. Bell GW
    12. Otte AP
    13. Vidal M
    14. Gifford DK
    15. Young RA
    16. Jaenisch R

    (2006) Polycomb complexes repress developmental regulators in murine embryonic stem cells

    Nature 441:349–353.

    https://doi.org/10.1038/nature04733

    • Google Scholar
14. 1. Bracken AP
    2. Dietrich N
    3. Pasini D
    4. Hansen KH
    5. Helin K

    (2006) Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions

    Genes & Development 20:1123–36.

    https://doi.org/10.1101/gad.381706

    • Google Scholar
15. 1. Brookes E
    2. de Santiago I
    3. Hebenstreit D
    4. Morris KJ
    5. Carroll T
    6. Xie SQ
    7. Stock JK
    8. Heidemann M
    9. Eick D
    10. Nozaki N
    11. Kimura H
    12. Ragoussis J
    13. Teichmann SA
    14. Pombo A

    (2012) Polycomb associates genome-wide with a specific RNA polymerase II variant, and regulates metabolic genes in ESCs

    Cell Stem Cell 10:157–170.

    https://doi.org/10.1016/j.stem.2011.12.017

    • Google Scholar
16. 1. Buchwald G
    2. van der Stoop P
    3. Weichenrieder O
    4. Perrakis A
    5. van Lohuizen M
    6. Sixma TK

    (2006) Structure and E3-ligase activity of the Ring-Ring complex of polycomb proteins Bmi1 and Ring1b

    The EMBO Journal 25:2465–2474.

    https://doi.org/10.1038/sj.emboj.7601144

    • Google Scholar
17. 1. Calés C
    2. Pavón L
    3. Starowicz K
    4. Pérez C
    5. Bravo M
    6. Ikawa T
    7. Koseki H
    8. Vidal M

    (2016) Role of Polycomb RYBP in Maintaining the B-1-to-B-2 B-Cell Lineage Switch in Adult Hematopoiesis

    Molecular and Cellular Biology 36:900–912.

    https://doi.org/10.1128/MCB.00869-15

    • Google Scholar
18. 1. Cao R
    2. Tsukada Y
    3. Zhang Y

    (2005) Role of Bmi-1 and Ring1A in H2A ubiquitylation and Hox gene silencing

    Molecular Cell 20:845–54.

    https://doi.org/10.1016/j.molcel.2005.12.002

    • Google Scholar
19. 1. Cao R
    2. Wang L
    3. Wang H
    4. Xia L
    5. Erdjument-Bromage H
    6. Tempst P
    7. Jones RS
    8. Zhang Y

    (2002) Role of histone H3 lysine 27 methylation in Polycomb-group silencing

    Science 298:1039–1043.

    https://doi.org/10.1126/science.1076997

    • Google Scholar
20. 1. Cao R
    2. Zhang Y

    (2004) SUZ12 is required for both the histone methyltransferase activity and the silencing function of the EED-EZH2 complex

    Molecular Cell 15:57–67.

    https://doi.org/10.1016/j.molcel.2004.06.020

    • Google Scholar
21. 1. Chen K
    2. Xi Y
    3. Pan X
    4. Li Z
    5. Kaestner K
    6. Tyler J
    7. Dent S
    8. He X
    9. Li W

    (2013) DANPOS: dynamic analysis of nucleosome position and occupancy by sequencing

    Genome Research 23:341–351.

    https://doi.org/10.1101/gr.142067.112

    • Google Scholar
22. 1. Ciferri C
    2. Lander GC
    3. Maiolica A
    4. Herzog F
    5. Aebersold R
    6. Nogales E

    (2012) Molecular architecture of human polycomb repressive complex 2

    eLife 1:e00005.

    https://doi.org/10.7554/eLife.00005

    • Google Scholar
23. 1. Cooper S
    2. Dienstbier M
    3. Hassan R
    4. Schermelleh L
    5. Sharif J
    6. Blackledge NP
    7. De Marco V
    8. Elderkin S
    9. Koseki H
    10. Klose R
    11. Heger A
    12. Brockdorff N

    (2014) Targeting polycomb to pericentric heterochromatin in embryonic stem cells reveals a role for H2AK119u1 in PRC2 recruitment

    Cell Reports 7:1456–1470.

    https://doi.org/10.1016/j.celrep.2014.04.012

    • Google Scholar
24. 1. Czermin B
    2. Melfi R
    3. McCabe D
    4. Seitz V
    5. Imhof A
    6. Pirrotta V

    (2002) Drosophila enhancer of Zeste/ESC complexes have a histone H3 methyltransferase activity that marks chromosomal Polycomb sites

    Cell 111:185–196.

    https://doi.org/10.1016/S0092-8674(02)00975-3

    • Google Scholar
25. 1. de Napoles M
    2. Mermoud JE
    3. Wakao R
    4. Tang YA
    5. Endoh M
    6. Appanah R
    7. Nesterova TB
    8. Silva J
    9. Otte AP
    10. Vidal M
    11. Koseki H
    12. Brockdorff N

    (2004) Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation

    Developmental Cell 7:663–676.

    https://doi.org/10.1016/j.devcel.2004.10.005

    • Google Scholar
26. 1. Deaton AM
    2. Bird A

    (2011) CpG islands and the regulation of transcription

    Genes & Development 25:1010–1022.

    https://doi.org/10.1101/gad.2037511

    • Google Scholar
27. 1. Dobin A
    2. Davis CA
    3. Schlesinger F
    4. Drenkow J
    5. Zaleski C
    6. Jha S
    7. Batut P
    8. Chaisson M
    9. Gingeras TR

    (2013) STAR: ultrafast universal RNA-seq aligner

    Bioinformatics 29:15–21.

    https://doi.org/10.1093/bioinformatics/bts635

    • Google Scholar
28. 1. Dyer PN
    2. Edayathumangalam RS
    3. White CL
    4. Bao Y
    5. Chakravarthy S
    6. Muthurajan UM
    7. Luger K

    (2004) Reconstitution of nucleosome core particles from recombinant histones and DNA

    Methods in Enzymology 375:23–44.

    https://doi.org/10.1016/s0076-6879(03)75002-2

    • Google Scholar
29. 1. Elderkin S
    2. Maertens GN
    3. Endoh M
    4. Mallery DL
    5. Morrice N
    6. Koseki H
    7. Peters G
    8. Brockdorff N
    9. Hiom K

    (2007) A phosphorylated form of Mel-18 targets the Ring1B histone H2A ubiquitin ligase to chromatin

    Molecular Cell 28:107–120.

    https://doi.org/10.1016/j.molcel.2007.08.009

    • Google Scholar
30. 1. Endoh M
    2. Endo TA
    3. Endoh T
    4. Fujimura Y
    5. Ohara O
    6. Toyoda T
    7. Otte AP
    8. Okano M
    9. Brockdorff N
    10. Vidal M
    11. Koseki H

    (2008) Polycomb group proteins Ring1A/B are functionally linked to the core transcriptional regulatory circuitry to maintain ES cell identity

    Development 135:1513–1524.

    https://doi.org/10.1242/dev.014340

    • Google Scholar
31. 1. Eskeland R
    2. Leeb M
    3. Grimes GR
    4. Kress C
    5. Boyle S
    6. Sproul D
    7. Gilbert N
    8. Fan Y
    9. Skoultchi AI
    10. Wutz A
    11. Bickmore WA

    (2010) Ring1B compacts chromatin structure and represses gene expression independent of histone ubiquitination

    Molecular Cell 38:452–464.

    https://doi.org/10.1016/j.molcel.2010.02.032

    • Google Scholar
32. 1. Farcas AM
    2. Blackledge NP
    3. Sudbery I
    4. Long HK
    5. McGouran JF
    6. Rose NR
    7. Lee S
    8. Sims D
    9. Cerase A
    10. Sheahan TW
    11. Koseki H
    12. Brockdorff N
    13. Ponting CP
    14. Kessler BM
    15. Klose RJ

    (2012) KDM2B links the Polycomb Repressive Complex 1 (PRC1) to recognition of CpG islands

    eLife 1:e00205.

    https://doi.org/10.7554/eLife.00205

    • Google Scholar
33. 1. Fereres S
    2. Simón R
    3. Mohd-Sarip A
    4. Verrijzer CP
    5. Busturia A

    (2014) dRYBP counteracts chromatin-dependent activation and repression of transcription

    PLoS One 9:e113255.

    https://doi.org/10.1371/journal.pone.0113255

    • Google Scholar
34. 1. Fischle W
    2. Wang Y
    3. Jacobs SA
    4. Kim Y
    5. Allis CD
    6. Khorasanizadeh S

    (2003) Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains

    Genes & Development 17:1870–1881.

    https://doi.org/10.1101/gad.1110503

    • Google Scholar
35. 1. Fonseca JP
    2. Steffen PA
    3. Müller S
    4. Lu J
    5. Sawicka A
    6. Seiser C
    7. Ringrose L

    (2012) In vivo Polycomb kinetics and mitotic chromatin binding distinguish stem cells from differentiated cells

    Genes & Development 26:857–871.

    https://doi.org/10.1101/gad.184648.111

    • Google Scholar
36. 1. Francis NJ
    2. Kingston RE
    3. Woodcock CL

    (2004) Chromatin compaction by a polycomb group protein complex

    Science 306:1574–1577.

    https://doi.org/10.1126/science.1100576

    • Google Scholar
37. 1. Gao Z
    2. Zhang J
    3. Bonasio R
    4. Strino F
    5. Sawai A
    6. Parisi F
    7. Kluger Y
    8. Reinberg D

    (2012) PCGF homologs, CBX proteins, and RYBP define functionally distinct PRC1 family complexes

    Molecular Cell 45:344–356.

    https://doi.org/10.1016/j.molcel.2012.01.002

    • Google Scholar
38. 1. García E
    2. Marcos-Gutiérrez C
    3. del Mar Lorente M
    4. Moreno JC
    5. Vidal M

    (1999) RYBP, a new repressor protein that interacts with components of the mammalian Polycomb complex, and with the transcription factor YY1

    The EMBO Journal 18:3404–3418.

    https://doi.org/10.1093/emboj/18.12.3404

    • Google Scholar
39. 1. Gearhart MD
    2. Corcoran CM
    3. Wamstad JA
    4. Bardwell VJ

    (2006) Polycomb group and SCF ubiquitin ligases are found in a novel BCOR complex that is recruited to BCL6 targets

    Molecular and Cellular Biology 26:6880–6889.

    https://doi.org/10.1128/MCB.00630-06

    • Google Scholar
40. 1. Goldknopf IL
    2. Taylor CW
    3. Baum RM
    4. Yeoman LC
    5. Olson MO
    6. Prestayko AW
    7. Busch H

    (1975)

    Isolation and characterization of protein A24, a "histone-like" non-histone chromosomal protein

    The Journal of Biological Chemistry 250:7182–7187.

    • Google Scholar
41. 1. González I
    2. Aparicio R
    3. Busturia A

    (2008) Functional characterization of the dRYBP gene in Drosophila

    Genetics 179:1373–1388.

    https://doi.org/10.1534/genetics.107.082966

    • Google Scholar
42. 1. Grau DJ
    2. Chapman BA
    3. Garlick JD
    4. Borowsky M
    5. Francis NJ
    6. Kingston RE

    (2011) Compaction of chromatin by diverse Polycomb group proteins requires localized regions of high charge

    Genes & Development 25:2210–2221.

    https://doi.org/10.1101/gad.17288211

    • Google Scholar
43. 1. Grzybowski AT
    2. Chen Z
    3. Ruthenburg AJ

    (2015) Calibrating ChIP-Seq with nucleosomal internal standards to measure histone modification density genome wide

    Molecular Cell 58:886–899.

    https://doi.org/10.1016/j.molcel.2015.04.022

    • Google Scholar
44. 1. He J
    2. Shen L
    3. Wan M
    4. Taranova O
    5. Wu H
    6. Zhang Y

    (2013) Kdm2b maintains murine embryonic stem cell status by recruiting PRC1 complex to CpG islands of developmental genes

    Nature Cell Biology 15:373–384.

    https://doi.org/10.1038/ncb2702

    • Google Scholar
45. 1. Heinz S
    2. Benner C
    3. Spann N
    4. Bertolino E
    5. Lin YC
    6. Laslo P
    7. Cheng JX
    8. Murre C
    9. Singh H
    10. Glass CK

    (2010) Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities

    Molecular Cell 38:576–589.

    https://doi.org/10.1016/j.molcel.2010.05.004

    • Google Scholar
46. 1. Hisada K
    2. Sánchez C
    3. Endo TA
    4. Endoh M
    5. Román-Trufero M
    6. Sharif J
    7. Koseki H
    8. Vidal M

    (2012) RYBP represses endogenous retroviruses and preimplantation- and germ line-specific genes in mouse embryonic stem cells

    Molecular and Cellular Biology 32:1139–1149.

    https://doi.org/10.1128/MCB.06441-11

    • Google Scholar
47. 1. Hu B
    2. Petela N
    3. Kurze A
    4. Chan KL
    5. Chapard C
    6. Nasmyth K

    (2015) Biological chromodynamics: a general method for measuring protein occupancy across the genome by calibrating ChIP-seq

    Nucleic Acids Research 43:e132.

    https://doi.org/10.1093/nar/gkv670

    • Google Scholar
48. 1. Hulsen T
    2. de Vlieg J
    3. Alkema W

    (2008) BioVenn - a web application for the comparison and visualization of biological lists using area-proportional Venn diagrams

    BMC Genomics 9:1–6.

    https://doi.org/10.1186/1471-2164-9-488

    • Google Scholar
49. 1. Illingworth RS
    2. Moffat M
    3. Mann AR
    4. Read D
    5. Hunter CJ
    6. Pradeepa MM
    7. Adams IR
    8. Bickmore WA

    (2015) The E3 ubiquitin ligase activity of RING1B is not essential for early mouse development

    Genes & Development 29:1897–1902.

    https://doi.org/10.1101/gad.268151.115

    • Google Scholar
50. 1. Isono K
    2. Endo TA
    3. Ku M
    4. Yamada D
    5. Suzuki R
    6. Sharif J
    7. Ishikura T
    8. Toyoda T
    9. Bernstein BE
    10. Koseki H

    (2013) SAM domain polymerization links subnuclear clustering of PRC1 to gene silencing

    Developmental Cell 26:565–577.

    https://doi.org/10.1016/j.devcel.2013.08.016

    • Google Scholar
51. 1. Jiao L
    2. Liu X
    3. JIAO L
    4. X LIU

    (2015) Structural basis of histone H3K27 trimethylation by an active polycomb repressive complex 2

    Science 350:aac4383.

    https://doi.org/10.1126/science.aac4383

    • Google Scholar
52. 1. Kalb R
    2. Latwiel S
    3. Baymaz HI
    4. Jansen PW
    5. Müller CW
    6. Vermeulen M
    7. Müller J

    (2014) Histone H2A monoubiquitination promotes histone H3 methylation in Polycomb repression

    Nature Structural & Molecular Biology 21:569–571.

    https://doi.org/10.1038/nsmb.2833

    • Google Scholar
53. 1. Kim CA
    2. Gingery M
    3. Pilpa RM
    4. Bowie JU

    (2002) The SAM domain of polyhomeotic forms a helical polymer

    Nature Structural Biology 9:453–457.

    https://doi.org/10.1038/nsb802

    • Google Scholar
54. 1. Kloet SL
    2. Makowski MM
    3. Baymaz HI
    4. van Voorthuijsen L
    5. Karemaker ID
    6. Santanach A
    7. Jansen PW
    8. Di Croce L
    9. Vermeulen M

    (2016) The dynamic interactome and genomic targets of Polycomb complexes during stem-cell differentiation

    Nature Structural & Molecular Biology 23:682–690.

    https://doi.org/10.1038/nsmb.3248

    • Google Scholar
55. 1. Klose RJ
    2. Cooper S
    3. Farcas AM
    4. Blackledge NP
    5. Brockdorff N

    (2013) Chromatin sampling-an emerging perspective on targeting polycomb repressor proteins

    PLoS Genetics 9:e1003717.

    https://doi.org/10.1371/journal.pgen.1003717

    • Google Scholar
56. 1. Ku M
    2. Koche RP
    3. Rheinbay E
    4. Mendenhall EM
    5. Endoh M
    6. Mikkelsen TS
    7. Presser A
    8. Nusbaum C
    9. Xie X
    10. Chi AS
    11. Adli M
    12. Kasif S
    13. Ptaszek LM
    14. Cowan CA
    15. Lander ES
    16. Koseki H
    17. Bernstein BE

    (2008) Genomewide analysis of PRC1 and PRC2 occupancy identifies two classes of bivalent domains

    PLoS Genetics 4:e1000242.

    https://doi.org/10.1371/journal.pgen.1000242

    • Google Scholar
57. 1. Lagarou A
    2. Mohd-Sarip A
    3. Moshkin YM
    4. Chalkley GE
    5. Bezstarosti K
    6. Demmers JA
    7. Verrijzer CP

    (2008) dKDM2 couples histone H2A ubiquitylation to histone H3 demethylation during Polycomb group silencing

    Genes & Development 22:2799–2810.

    https://doi.org/10.1101/gad.484208

    • Google Scholar
58. 1. Langmead B
    2. Salzberg SL

    (2012) Fast gapped-read alignment with Bowtie 2

    Nature Methods 9:357–359.

    https://doi.org/10.1038/nmeth.1923

    • Google Scholar
59. 1. Lee HG
    2. Kahn TG
    3. Simcox A
    4. Schwartz YB
    5. Pirrotta V

    (2015) Genome-wide activities of Polycomb complexes control pervasive transcription

    Genome Research 25:1170–1181.

    https://doi.org/10.1101/gr.188920.114

    • Google Scholar
60. 1. Leeb M
    2. Wutz A

    (2007) Ring1B is crucial for the regulation of developmental control genes and PRC1 proteins but not X inactivation in embryonic cells

    The Journal of Cell Biology 178:219–229.

    https://doi.org/10.1083/jcb.200612127

    • Google Scholar
61. 1. Leitner A
    2. Walzthoeni T
    3. Aebersold R

    (2014) Lysine-specific chemical cross-linking of protein complexes and identification of cross-linking sites using LC-MS/MS and the xQuest/xProphet software pipeline

    Nature Protocols 9:120–137.

    https://doi.org/10.1038/nprot.2013.168

    • Google Scholar
62. 1. Levine SS
    2. Weiss A
    3. Erdjument-Bromage H
    4. Shao Z
    5. Tempst P
    6. Kingston RE

    (2002) The core of the polycomb repressive complex is compositionally and functionally conserved in flies and humans

    Molecular and Cellular Biology 22:6070–6078.

    https://doi.org/10.1128/MCB.22.17.6070-6078.2002

    • Google Scholar
63. 1. Lewis EB

    (1978) A gene complex controlling segmentation in Drosophila

    Nature 276:565–570.

    https://doi.org/10.1038/276565a0

    • Google Scholar
64. 1. Li H
    2. Handsaker B
    3. Wysoker A
    4. Fennell T
    5. Ruan J
    6. Homer N
    7. Marth G
    8. Abecasis G
    9. Durbin R
    10. 1000 Genome Project Data Processing Subgroup

    (2009) The sequence alignment/Map format and SAMtools

    Bioinformatics 25:2078–2079.

    https://doi.org/10.1093/bioinformatics/btp352

    • Google Scholar
65. 1. Li Z
    2. Cao R
    3. Wang M
    4. Myers MP
    5. Zhang Y
    6. Xu RM

    (2006) Structure of a Bmi-1-Ring1B polycomb group ubiquitin ligase complex

    Journal of Biological Chemistry 281:20643–20649.

    https://doi.org/10.1074/jbc.M602461200

    • Google Scholar
66. 1. Love MI
    2. Huber W
    3. Anders S

    (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

    Genome Biology 15:1–21.

    https://doi.org/10.1186/s13059-014-0550-8

    • Google Scholar
67. 1. Margueron R
    2. Justin N
    3. Ohno K
    4. Sharpe ML
    5. Son J
    6. Drury WJ
    7. Voigt P
    8. Martin SR
    9. Taylor WR
    10. De Marco V
    11. Pirrotta V
    12. Reinberg D
    13. Gamblin SJ

    (2009) Role of the polycomb protein EED in the propagation of repressive histone marks

    Nature 461:762–767.

    https://doi.org/10.1038/nature08398

    • Google Scholar
68. 1. Margueron R
    2. Reinberg D

    (2011) The Polycomb complex PRC2 and its mark in life

    Nature 469:343–349.

    https://doi.org/10.1038/nature09784

    • Google Scholar
69. 1. Min J
    2. Zhang Y
    3. Xu RM

    (2003) Structural basis for specific binding of Polycomb chromodomain to histone H3 methylated at Lys 27

    Genes & Development 17:1823–1828.

    https://doi.org/10.1101/gad.269603

    • Google Scholar
70. 1. Morey L
    2. Aloia L
    3. Cozzuto L
    4. Benitah SA
    5. Di Croce L

    (2013) RYBP and Cbx7 define specific biological functions of polycomb complexes in mouse embryonic stem cells

    Cell Reports 3:60–69.

    https://doi.org/10.1016/j.celrep.2012.11.026

    • Google Scholar
71. 1. Müller J
    2. Hart CM
    3. Francis NJ
    4. Vargas ML
    5. Sengupta A
    6. Wild B
    7. Miller EL
    8. O'Connor MB
    9. Kingston RE
    10. Simon JA

    (2002) Histone methyltransferase activity of a Drosophila Polycomb group repressor complex

    Cell 111:197–208.

    https://doi.org/10.1016/S0092-8674(02)00976-5

    • Google Scholar
72. 1. Müller J
    2. Verrijzer P

    (2009) Biochemical mechanisms of gene regulation by polycomb group protein complexes

    Current Opinion in Genetics & Development 19:150–158.

    https://doi.org/10.1016/j.gde.2009.03.001

    • Google Scholar
73. 1. Orlando DA
    2. Chen MW
    3. Brown VE
    4. Solanki S
    5. Choi YJ
    6. Olson ER
    7. Fritz CC
    8. Bradner JE
    9. Guenther MG

    (2014) Quantitative ChIP-Seq normalization reveals global modulation of the epigenome

    Cell Reports 9:1163–1170.

    https://doi.org/10.1016/j.celrep.2014.10.018

    • Google Scholar
74. 1. Park S-Y
    2. Choi H-K
    3. Jo S-H
    4. Seo J
    5. Han E-J
    6. Choi K-C
    7. Jeong J-W
    8. Choi Y
    9. Yoon H-G

    (2015) YAF2 promotes TP53-mediated genotoxic stress response via stabilization of PDCD5

    Biochimica Et Biophysica Acta (BBA) - Molecular Cell Research 1853:1060–1072.

    https://doi.org/10.1016/j.bbamcr.2015.01.006

    • Google Scholar
75. 1. Pengelly AR
    2. Kalb R
    3. Finkl K
    4. Müller J

    (2015) Transcriptional repression by PRC1 in the absence of H2A monoubiquitylation

    Genes & Development 29:1487–1492.

    https://doi.org/10.1101/gad.265439.115

    • Google Scholar
76. 1. Pirity MK
    2. Locker J
    3. Schreiber-Agus N

    (2005) Rybp/DEDAF is required for early postimplantation and for central nervous system development

    Molecular and Cellular Biology 25:7193–7202.

    https://doi.org/10.1128/MCB.25.16.7193-7202.2005

    • Google Scholar
77. 1. Ramírez F
    2. Ryan DP
    3. Grüning B
    4. Bhardwaj V
    5. Kilpert F
    6. Richter AS
    7. Heyne S
    8. Dündar F
    9. Manke T

    (2016) deepTools2: a next generation web server for deep-sequencing data analysis

    Nucleic Acids Research 44:.

    https://doi.org/10.1093/nar/gkw257

    • Google Scholar
78. 1. Ran FA
    2. Hsu PD
    3. Wright J
    4. Agarwala V
    5. Scott DA
    6. Zhang F

    (2013) Genome engineering using the CRISPR-Cas9 system

    Nature Protocols 8:2281–2308.

    https://doi.org/10.1038/nprot.2013.143

    • Google Scholar
79. 1. Riising EM
    2. Comet I
    3. Leblanc B
    4. Wu X
    5. Johansen JV
    6. Helin K

    (2014) Gene silencing triggers polycomb repressive complex 2 recruitment to CpG islands genome wide

    Molecular Cell 55:347–360.

    https://doi.org/10.1016/j.molcel.2014.06.005

    • Google Scholar
80. 1. Satijn DP
    2. Gunster MJ
    3. van der Vlag J
    4. Hamer KM
    5. Schul W
    6. Alkema MJ
    7. Saurin AJ
    8. Freemont PS
    9. van Driel R
    10. Otte AP

    (1997) RING1 is associated with the polycomb group protein complex and acts as a transcriptional repressor

    Molecular and Cellular Biology 17:4105–4113.

    https://doi.org/10.1128/MCB.17.7.4105

    • Google Scholar
81. 1. Scelfo A
    2. Piunti A
    3. Pasini D

    (2015) The controversial role of the Polycomb group proteins in transcription and cancer: how much do we not understand Polycomb proteins?

    FEBS Journal 282:1703–1722.

    https://doi.org/10.1111/febs.13112

    • Google Scholar
82. 1. Schoenfelder S
    2. Sugar R
    3. Dimond A
    4. Javierre BM
    5. Armstrong H
    6. Mifsud B
    7. Dimitrova E
    8. Matheson L
    9. Tavares-Cadete F
    10. Furlan-Magaril M
    11. Segonds-Pichon A
    12. Jurkowski W
    13. Wingett SW
    14. Tabbada K
    15. Andrews S
    16. Herman B
    17. LeProust E
    18. Osborne CS
    19. Koseki H
    20. Fraser P
    21. Luscombe NM
    22. Elderkin S

    (2015) Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome

    Nature Genetics 47:1179–1186.

    https://doi.org/10.1038/ng.3393

    • Google Scholar
83. 1. Schwartz YB
    2. Pirrotta V

    (2013) A new world of Polycombs: unexpected partnerships and emerging functions

    Nature Reviews Genetics 14:853–864.

    https://doi.org/10.1038/nrg3603

    • Google Scholar
84. 1. Simon JA
    2. Kingston RE

    (2013) Occupying chromatin: Polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put

    Molecular Cell 49:808–824.

    https://doi.org/10.1016/j.molcel.2013.02.013

    • Google Scholar
85. 1. Steffen PA
    2. Ringrose L

    (2014) What are memories made of? How Polycomb and Trithorax proteins mediate epigenetic memory

    Nature Reviews Molecular Cell Biology 15:340–356.

    https://doi.org/10.1038/nrm3789

    • Google Scholar
86. 1. Taherbhoy AM
    2. Huang OW
    3. Cochran AG

    (2015) BMI1-RING1B is an autoinhibited RING E3 ubiquitin ligase

    Nature Communications 6:.

    https://doi.org/10.1038/ncomms8621

    • Google Scholar
87. 1. Tavares L
    2. Dimitrova E
    3. Oxley D
    4. Webster J
    5. Poot R
    6. Demmers J
    7. Bezstarosti K
    8. Taylor S
    9. Ura H
    10. Koide H
    11. Wutz A
    12. Vidal M
    13. Elderkin S
    14. Brockdorff N

    (2012) RYBP-PRC1 complexes mediate H2A ubiquitylation at polycomb target sites independently of PRC2 and H3K27me3

    Cell 148:664–678.

    https://doi.org/10.1016/j.cell.2011.12.029

    • Google Scholar
88. 1. Terranova R
    2. Yokobayashi S
    3. Stadler MB
    4. Otte AP
    5. van Lohuizen M
    6. Orkin SH
    7. Peters AH

    (2008) Polycomb group proteins Ezh2 and Rnf2 direct genomic contraction and imprinted repression in early mouse embryos

    Developmental Cell 15:668–679.

    https://doi.org/10.1016/j.devcel.2008.08.015

    • Google Scholar
89. 1. Voigt P
    2. Tee WW
    3. Reinberg D

    (2013) A double take on bivalent promoters

    Genes & Development 27:1318–1338.

    https://doi.org/10.1101/gad.219626.113

    • Google Scholar
90. 1. Voncken JW
    2. Roelen BA
    3. Roefs M
    4. de Vries S
    5. Verhoeven E
    6. Marino S
    7. Deschamps J
    8. van Lohuizen M

    (2003) Rnf2 (Ring1b) deficiency causes gastrulation arrest and cell cycle inhibition

    PNAS 100:2468–2473.

    https://doi.org/10.1073/pnas.0434312100

    • Google Scholar
91. 1. Wang H
    2. Wang L
    3. Erdjument-Bromage H
    4. Vidal M
    5. Tempst P
    6. Jones RS
    7. Zhang Y

    (2004) Role of histone H2A ubiquitination in Polycomb silencing

    Nature 431:873–878.

    https://doi.org/10.1038/nature02985

    • Google Scholar
92. 1. Wang L
    2. Brown JL
    3. Cao R
    4. Zhang Y
    5. Kassis JA
    6. Jones RS

    (2004) Hierarchical recruitment of polycomb group silencing complexes

    Molecular Cell 14:637–646.

    https://doi.org/10.1016/j.molcel.2004.05.009

    • Google Scholar
93. 1. Wang R
    2. Taylor AB
    3. Leal BZ
    4. Chadwell LV
    5. Ilangovan U
    6. Robinson AK
    7. Schirf V
    8. Hart PJ
    9. Lafer EM
    10. Demeler B
    11. Hinck AP
    12. McEwen DG
    13. Kim CA

    (2010) Polycomb group targeting through different binding partners of RING1B C-terminal domain

    Structure 18:966–975.

    https://doi.org/10.1016/j.str.2010.04.013

    • Google Scholar
94. 1. Wani AH
    2. Boettiger AN
    3. Schorderet P
    4. Ergun A
    5. Münger C
    6. Sadreyev RI
    7. Zhuang X
    8. Kingston RE
    9. Francis NJ

    (2016) Chromatin topology is coupled to Polycomb group protein subnuclear organization

    Nature Communications 7:.

    https://doi.org/10.1038/ncomms10291

    • Google Scholar
95. 1. Anderson TR
    2. Slotkin TA

    (1975) Maturation of the adrenal medulla--IV. Effects of morphine

    Biochemical Pharmacology 24:1469–1474.

    https://doi.org/10.1016/0006-2952(75)90020-9

    • Google Scholar
96. 1. Wu X
    2. Johansen JV
    3. Helin K

    (2013) Fbxl10/Kdm2b recruits polycomb repressive complex 1 to CpG islands and regulates H2A ubiquitylation

    Molecular Cell 49:1134–1146.

    https://doi.org/10.1016/j.molcel.2013.01.016

    • Google Scholar
97. 1. Xie R
    2. Everett LJ
    3. Lim HW
    4. Patel NA
    5. Schug J
    6. Kroon E
    7. Kelly OG
    8. Wang A
    9. D'Amour KA
    10. Robins AJ
    11. Won KJ
    12. Kaestner KH
    13. Sander M

    (2013) Dynamic chromatin remodeling mediated by polycomb proteins orchestrates pancreatic differentiation of human embryonic stem cells

    Cell Stem Cell 12:224–237.

    https://doi.org/10.1016/j.stem.2012.11.023

    • Google Scholar
98. 1. Zhang Y
    2. Liu T
    3. Meyer CA
    4. Eeckhoute J
    5. Johnson DS
    6. Bernstein BE
    7. Nusbaum C
    8. Myers RM
    9. Brown M
    10. Li W
    11. Liu XS

    (2008) Model-based analysis of ChIP-Seq (MACS)

    Genome Biology 9:R137–139.

    https://doi.org/10.1186/gb-2008-9-9-r137

    • Google Scholar

Thank you for submitting your article "RYBP stimulates PRC1 to shape chromatin-based communication between Polycomb repressive complexes" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors and the evaluation has been overseen by Jessica Tyler as the Senior Editor. The following individual involved in review of your submission has agreed to reveal her identity: Wendy A Bickmore (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

In this manuscript, Rose and colleagues use biochemical and genetic approaches to address how the RYBP subunit of non-canonical PRC1 (ncPRC1) complexes contribute to transcriptional regulation and if they also contribute to PRC2 complex activity. This is in the context of recent studies reporting that the ubiquitination of H2A (mediated by ncPRC1 complexes) is both capable of recruiting the PRC2 complex to an artificial gene locus (Blackledge et al., 2014, Cell; Cooper et al., 2014) and increasing the activity of PRC2 in vitro (Kalb et al., 2014, Nature Structural Molecular Biology). However, more recent genetic evidence in Ring1a/b double knockout cells have shown that a complete ablation of ncPRC1 activity did not affect global H3K27me3 levels in gut tissues (Chiacchiera et al., Cell Stem Cell, 2016). Furthermore, while the loss of PRC2 mediated H3K27me3 leads to HOX gene derepression in Drosophila (Pengelly et al., Science, 2013), the complete loss of H2A ubiquitination did not phenocopy this (Pengelly et al., Genes & Development 2015). Likewise, recent genetic studies questioned the importance of H2A ubiquitination for Polycomb silencing in mice (Illingworth et al., Genes Dev. 2015). Despite this, we know from these studies and others, that ncPRC1 and indeed H2Aub are essential for embryonic development. Finally, that RYBP can stimulate H2Aub1 by a subset of PRC1 complexes is already established in ES cells and in flies (Gao et al., 2012; Fereres et al., 2014, PLoS One). Moreover, cross-talk between PRC1/H2Aub1 and PRC2/H3K27me3 has been examined previously (Tavares et al., 2012; Blackledge et al., 2014; Cooper et al., 2014; Kalb et al., 2014; Illingworth et al., Genes Dev. 2015; Pengelly et al., Genes Dev 2015; Fereres et al., 2014, PLoS One; Morey et al., Cell Reports 3, 2013), but these studies resulted in rather diverse conclusions.

In this overall well-done study, the authors firstly establish an in vitro ubiquitination assay for ncPRC1, and use it to explore the role of the RYBP subunit (Figures 1–4). They discover that RYBP is capable of considerably boosting H2A ubiquitination in vitro. In Figures 5–9, they use knockout mouse embryonic stem (ES) cells to explore the roles of RYBP in vivo. They compare a Yaf2 knockout ES cells and Rybp/Yaf2 double knockout ES cells to examine the effect on the global levels of H2AK119ub1 (Western blot) and at selected Polycomb target genes (ChIP-seq). While surprisingly, they observe that the overall global levels of H2AK119ub1 are apparently unchanged in the RYBP/Yaf2 double knockout ES cells, they detect moderate local reductions of H2AK119ub1 at sites normally occupied by Rybp and Ring1b. Taken together with their in vitro observations, these results suggest that Rybp and Yaf2 are not essential for the H2AK119ub1 activity of ncPRC1 complexes and rather are co-factors that can augment activity, perhaps at more weakly bound sites. They then go on to correlate local reductions in H2AK119ub1 with moderate reductions in H3K27me3 and slight increases in transcription at some of the affected Polycomb target genes. However, they report that this reduced H3K27me3 does not correlate with reduced PRC2 complex occupancy, as measured by ChIP-seq analysis of Suz12 in their Rybp/Yaf2 double knockout ES cells. The authors use these observations to propose a model for the communication between PRC1 and PRC2, at least on a number of gene regulatory loci.

Whereas the reviewers found the manuscript potentially interesting for the readership of eLife, because it shed light on the function of RYBP and the communication between PRC1 and PRC2, several major concerns were raised that will need to be addressed experimentally.

Essential revisions:

1) The authors should avoid making quantitative conclusions about Ring1b (Figure 6) and Suz12 (Figure 8) in the wild-type versus Rybp/Yaf2 double knockout ES cells. They rightly point out that the ChIP-RX method (Orlando et al., Cell Reports, 2014,) is the correct approach to quantify potential changes in the H2AK119ub1 and H3K27me3 histone marks. However, they employ non-quantitative ChIP-seq to look at the Ring1b and Suz12 binding profiles. They should instead employ a careful, quantitative ChIP-qPCR approach, checking multiple representative sites, in order to properly evaluate if Ring1B and Suz12 occupancy change in the Rybp/Yaf2 double knockout ES cells (Figure 6 and 8). This is especially important for both their model that RYBP primarily influences ncPRC1 activity (and not level, or localization of the complex, as published by others see e.g. Gao et al., 2012; Tavares et al., 2012; Morey et al., Cell Reports 3, 2013) and the open question whether the presence of H2AK119ub1 might either lead to increased recruitment of PRC2 or increase its activity (again, the authors should content with the already published work). Based on ChIP data the authors conclude that H2A ubiquitination is required for normal H3K27 methylation and proposed an "activity-based communication between PRC1 and PRC2". While this conclusion is likely to be correct (this point has basically been made in earlier publications), this cannot be concluded solely from ChIP data in this manuscript. In fact, Kalb et al. (2014) already did an experiment that allows such a mechanistic interpretation by showing that nucleosomes containing H2Aub1 are a better substrate for H3K27 methylation by PRC2.

2) The authors should carry out gene ontology (GO) analysis of the genes that lose both H2AK119ub1 and H3K27me3 at their promoters in the Rybp/Yaf2 double knockout ES cells in a bid to identify ncPRC1 specific sets of target genes and compare to similar work in previous studies. It would be interesting to further delineate those H3K27me3 positive genes that do and don't have reduced H3K27me3 in the Rybp/Yaf2 double knockout ES cells. This would be important for the readers in light of previous work delineating the biological roles of cPRC1 and ncPRC1 (Pengelley et al., 2015 and Illingworth et al., 2015). These studies found that H2A ubiquitination appears to be dispensable for Polycomb dependent repression of the Hox genes. However, since H2Aub is required for both fly and mouse viability, it becomes interesting to begin to determine which genes are dependent on ncPRC1 mediated H2AK119ub1 for their regulation.

3) To better report the genome-wide binding profiles of cPRC1 and ncPRC1 in ES cells, the authors should include heat maps of their ChIP-seq data for all transcription start sites (+/- 5kb) for Cbx7 (Morey et al., 2013, Cell Reports), Ring1b (+/-OHT), Rybp (+/-OHT), H2AK119ub1 (+/-OHT) and H3K27me3 (+/-OHT). This would help delineate the cPRC1 and ncPRC1 unique target genes and it may shed light on whether loss of Rybp affects H3K27me3 levels at shared ncPRC1 and cPRC1 target genes or on ncPRC1 specific target genes only.

4) Following this heat map analysis, the authors should investigate whether the 230 Polycomb target genes that have an increase in transcription in their Rybp/Yaf2 double knockout cells (Figure 9) are ncPRC1 specific, cPRC1 specific or ncPRC1 and cPRC1 shared target genes. They should provide ChIP-qPCRs of Suz12, H3K27me3 and Ring1b at a representative set of these gene promoters.

5) In Figures 1–4 reconstituted histone H2A ubiquitination assays are used in which different recombinant PRC1 assemblages are compared by titrating increasing amounts of enzyme with a fixed substrate and the% conversion at the end of the reaction is determined. The correct way to compare enzymatic activities is to determine reaction rates. I.e. measure conversion over time using a fixed amount of enzyme and substrate! This experiment should be included for a few key complexes. Another critical issue is that the ratio enzyme vs substrate (nucleosomes) is not clear. It is essential that the substrate is in excess over the enzyme (this should be shown). Finally, a better analysis of the nucleosomes used in the assays need to be provided. E.g. are there free histones in the prep? An additional nucleosome purification step (e.g. glycerol centrifugation or size exclusion chromatography) would give more confidence. This is also important because the results in this manuscript are at odds with published work from other groups (e.g. Gao et al. 2012; Tavares et al., 2012). Finally, subsection “PCGF1-PRC1 is a highly modular protein complex” – and Figure 1D. It appears that the absence of RYBP leads to consistently reduced levels of PCGF1 (lanes 4 to 6) and possibly a slight reduction in BCOR (lane 5). This suggests that RYBP may help to stabilize these interactions. Quantitation of this blot should be performed and these result discussed in the text.

6) The Introduction, and actually most of the manuscript, does not give a clear overview of what has already been published on this topic. It is not that many major references were missing (although e.g. the work of the Sixma lab (Buchwald et al., 2006) should be included), rather the Introduction does not clearly summarized the work already done. In addition, somewhat more attention to work in the fly would be helpful to the reader as well. E.g. ncPRC1 complexes and there role in histone ubiquitylation were first described in the fly (Lagarou et al., 2008). Finally, in the Results and DDiscussion sections, findings by others with similar experiments should be mentioned and where appropriated differences should be discussed. The whole manuscript strongly emphasizes the catalytic functions of PRC1/2 but this needs to be much better balanced by a discussion of the substantial evidence – both in vitro and in vivo, and in flies and mammals – that PRC1 executes many of its functions through a structural change in the underlying chromatin and that is independent of its catalytic activity.

[…]

Essential revisions:

1) The authors should avoid making quantitative conclusions about Ring1b (Figure 6) and Suz12 (Figure 8) in the wild-type versus Rybp/Yaf2 double knockout ES cells. They rightly point out that the ChIP-RX method (Orlando et al., Cell Reports, 2014,) is the correct approach to quantify potential changes in the H2AK119ub1 and H3K27me3 histone marks. However, they employ non-quantitative ChIP-seq to look at the Ring1b and Suz12 binding profiles. They should instead employ a careful, quantitative ChIP-qPCR approach, checking multiple representative sites, in order to properly evaluate if Ring1B and Suz12 occupancy change in the Rybp/Yaf2 double knockout ES cells (Figure 6 and 8). This is especially important for both their model that RYBP primarily influences ncPRC1 activity (and not level, or localization of the complex, as published by others see e.g. Gao et al., 2012; Tavares et al., 2012; Morey et al., Cell Reports 3, 2013) and the open question whether the presence of H2AK119ub1 might either lead to increased recruitment of PRC2 or increase its activity (again, the authors should content with the already published work).

We agree with the reviewer(s) that this is an important point and we have now performed new ChIP-qPCR analysis for RING1B, SUZ12, H2AK119ub1, and H3K27me3 in biological triplicate at multiple representative sites. This analysis has confirmed that RING1B and SUZ12 are largely retained, or even in some instances elevated in the case of SUZ12 (consistent with our ChIP-seq meta-analysis for SUZ12 – see Figure 8C), while H2AK119ub1 and H3K27me3 are reduced following removal of RYBP/YAF2 as suggested by our original ChIP-seq analysis. These new ChIP-qPCR results have been integrated as new supplementary figures (Figure 6—figure supplement 1 and Figure 8—figure supplement 2), and further support the argument that RYBP/YAF2 acts to influence PRC1 and PRC2 activity rather than their binding to chromatin.

Based on ChIP data the authors conclude that H2A ubiquitination is required for normal H3K27 methylation and proposed an "activity-based communication between PRC1 and PRC2". While this conclusion is likely to be correct (this point has basically been made in earlier publications), this cannot be concluded solely from ChIP data in this manuscript. In fact, Kalb et al. (2014) already did an experiment that allows such a mechanistic interpretation by showing that nucleosomes containing H2Aub1 are a better substrate for H3K27 methylation by PRC2.

The reviewer(s) are correct in pointing out that our in vivo ChIP-seq analysis and the ‘activity-based communication’ model we arrive at is in agreement with, and strengthened by, the elegant in vitro work performed by Kalb et al. (2014) in which they demonstrated that the activity of PRC2 was stimulated by H2AK119ub1. Our new observations provide clear genome-wide evidence that reduced H2AK119ub1 at PcG target sites in vivo predominantly influences PRC2 activity and not target site binding. We have included the following statement to clarify this point in subsection “RYBP-dependent H2AK119ub1 shapes PRC2 activity”:

“These important observations demonstrate that RYBP-dependent H2AK119ub1 shapes the activity of PRC2 in depositing H3K27me3, and suggests that the observed stimulation of PRC2 by nucleosomes containing H2A mono-ubiquitylation in vitro is also relevant in vivo (Kalb et al., 2014).”

2) The authors should carry out gene ontology (GO) analysis of the genes that lose both H2AK119ub1 and H3K27me3 at their promoters in the Rybp/Yaf2 double knockout ES cells in a bid to identify ncPRC1 specific sets of target genes and compare to similar work in previous studies. It would be interesting to further delineate those H3K27me3 positive genes that do and don't have reduced H3K27me3 in the Rybp/Yaf2 double knockout ES cells. This would be important for the readers in light of previous work delineating the biological roles of cPRC1 and ncPRC1 (Pengelley et al., 2015 and Illingworth et al., 2015). These studies found that H2A ubiquitination appears to be dispensable for Polycomb dependent repression of the Hox genes. However, since H2Aub is required for both fly and mouse viability, it becomes interesting to begin to determine which genes are dependent on ncPRC1 mediated H2AK119ub1 for their regulation.

We agree this is an interesting and important question and thank the reviewer(s) for the suggestion. To examine these relationships in more detail we undertook two complementary new analyses.

Firstly, as suggested by the reviewer(s), we identified polycomb targets which relied the most (RYBP-dependent) or the least (RYBP-independent) on RYBP for H3K27me3, and compared their gene ontology (GO) enrichment terms (Author response image 1). Both groups of polycomb targets were enriched for GO terms implicated in development (e.g. “system development”, “developmental process”, “organ development” (Author response image 1A and B)), consistent with polycomb complexes playing an important role in regulating developmental genes in embryonic stem cells. However, interestingly, sites which retained the most H3K27me3 in the absence of RYBP/YAF2 exhibited enrichment for DNA binding and sequence-specific transcription factors (Autho response image 1A and B). These transcription factors are often master regulators involved in developmental decision making processes, and are found in large and very CpG dense islands that have high levels of polycomb protein occupancy, H3K27me3, and H2AK119ub1 ((Long et al., 2013) and Author response image 1C). In this context the stimulatory activity of RYBP and H2AK119ub1 appears less pronounced, suggesting that these sites may have sufficient PRC2 occupancy to render RYBP and H2AK119ub1-dependent stimulation less relevant.

Author response image 1

Download asset Open asset

Secondly, to further investigate the relationship between canonical PRC1 (cPRC1) and variant (vPRC1), we performed de novo clustering based on enrichment of RYBP (this study) and CBX7 (Morey et al., 2013) at H2AK119ub1 sites and arrived at a shared and variant-enriched set of PcG target sites (see essential reviewer point 3 for more details). In agreement with the segregation of sites based on changes in H3K27me3, both groups were strongly associated with developmental processes; however the shared sites exhibited a slight preference for sequence-specific transcription factors. Variant enriched sites also encompassed transcription factors, but in general represented a set of genes that included a more broad range of biological processes.

Together this suggests that sequence-specific transcription factors may have evolved to use a more robust complement of cPRC1 and vPRC1 activities to help maintain H2AK119ub1 while vPRC1 may play a broader role in shaping H2AK119ub1 associated with a range of biological processes. We have integrated these analyses into the revised manuscript, with GO term analysis included in Figure 7—figure supplement 1. We have also discussed these findings where relevant (see subsection “RYBP-dependent stimulation is essential for H2AK119ub1 at sites with low PRC1 occupancy”).

3) To better report the genome-wide binding profiles of cPRC1 and ncPRC1 in ES cells, the authors should include heat maps of their ChIP-seq data for all transcription start sites (+/- 5kb) for Cbx7 (Morey et al., 2013, Cell Reports), Ring1b (+/-OHT), Rybp (+/-OHT), H2AK119ub1 (+/-OHT) and H3K27me3 (+/-OHT). This would help delineate the cPRC1 and ncPRC1 unique target genes and it may shed light on whether loss of Rybp affects H3K27me3 levels at shared ncPRC1 and cPRC1 target genes or on ncPRC1 specific target genes only.

We thank for the reviewer(s) for this useful suggestion, and agree that the manuscript would benefit from a better understanding of the relative contribution of RYBP towards previously proposed canonical and/or variant PRC1 targets (as also covered in essential reviewer point 2). To address this important point, we implemented the reviewer(s) suggestion and profiled H2AK119ub1, RING1B, RYBP, and SUZ12 in the presence and absence of RYBP/YAF2 at TSS ± 5kb clustered based on the presence of RING1B and the relative levels of RYBP (vPRC1) and CBX7 (cPRC1). This revealed a set of “shared” sites, with high levels of vPRC1 and cPRC1, and “variant-enriched” sites, which exhibited lower levels of CBX7 (Author response image 2A). In contrast to previous work (Morey et al., 2013) we failed to identify cPRC1 (CBX7)-enriched sites that lack vPRC1 (RYBP). Initially this was puzzling to us, but a closer inspection of the genes that were previously reported to lack RYBP yet have CBX7 binding ((Morey et al., 2013) Figure 2C) revealed a clear enrichment for RYBP in our experiments and this signal was lost in response to tamoxifen-induced removal of RBYP (Author response image 3). The differences in RYBP ChIP enrichment between our studies is presumably the result of differing ChIP protocols (e.g. formaldehyde only crosslinking (Morey et al) versus formaldehyde/EGS crosslinking (our study)). Nevertheless, it is clear that CBX7 occupied sites fall into the shared category that are also occupied by RYBP. In the revised manuscript, we now highlight this important point (subsection “RYBP-dependent stimulation is essential for H2AK119ub1 at sites with low PRC1 occupancy”). Intriguingly, we observed that both shared and variant-enriched PcG TSS appeared to be similarly affected in their reductions of H2AK119ub1 and H3K27me3 (Author response image 2A), and we did not observe any obvious changes in RING1B or SUZ12 occupancy at either group. However, a visual examination of polycomb domains and their associated genes indicated that the use of a simple fixed window surrounding the TSS would not likely capture the behaviour of the entire domain, particularly when considering H2AK119ub1 and H3K27me3 which are often broad in nature (Author response image 2B). Therefore, we instead opted to use a clustering and heat mapping approach focused on H2AK119ub1 intervals. This revealed that although all polycomb target sites experience reductions in H2AK119ub1 and H3K27me3, variant-enriched sites showed a slightly larger reduction in both of these modifications (Figure 7F and G, Figure 8D). We have now incorporated these analyses and interpretations into the revised Figures and manuscript highlighting the preferential contribution of RYBP towards vPRC1-enriched sites and described these observations on in the revised manuscript.

Author response image 2

Download asset Open asset

Author response image 3

Download asset Open asset

4) Following this heat map analysis, the authors should investigate whether the 230 Polycomb target genes that have an increase in transcription in their Rybp/Yaf2 double knockout cells (Figure 9) are ncPRC1 specific, cPRC1 specific or ncPRC1 and cPRC1 shared target genes.

To address this question, we segregated polycomb-occupied TSS into shared and variant-enriched PRC1 targets and examined which of these two sets the reactivated genes fell into. This analysis showed that upregulated genes fell roughly into both categories. We have now indicated this in the text of the revised manuscript in subsection “Loss of RYBP culminates in gene reactivation at sites where Polycomb chromatin domains are compromised” and Figure 9—figure supplement 2.

They should provide ChIP-qPCRs of Suz12, H3K27me3 and Ring1b at a representative set of these gene promoters.

As requested, we have performed new ChIP-qPCRs for RING1B, SUZ12, H3K27me3 and H2AK119ub1 in biological triplicate at a set of representative genes that are up-regulated upon loss of RYBP/YAF2. The results of these analyses further support the contention (Figure 9) that up-regulated genes tended to have compromised polycomb domains, as evidenced by reductions in RING1B and SUZ12 binding (Figure 9—figure supplement 3).

5) In Figures 1–4 reconstituted histone H2A ubiquitination assays are used in which different recombinant PRC1 assemblages are compared by titrating increasing amounts of enzyme with a fixed substrate and the% conversion at the end of the reaction is determined. The correct way to compare enzymatic activities is to determine reaction rates. I.e. measure conversion over time using a fixed amount of enzyme and substrate! This experiment should be included for a few key complexes.

The reviewer is correct in noting this experimental setup does not conform to Michaelis-Menten analysis of reaction rates. This was a deliberate decision that we made in designing these experiments and we will explain why in more detail here. Currently, the only method we have found to easily measure PRC1 activity in vitro is to follow the conversion of H2A to ubiquitylated H2A by western blot. As indicated by the reviewer, to set up an experiment to measure classical Michaelis-Menten kinetics, one must (a) have substrate in large excess over enzyme, and (b) vary substrate amount relative to fixed enzyme concentration. In the available experimental set-up, western blot analysis has not proven sensitive enough to robustly detect conversion of H2A to uH2A at low substrate concentrations, effectively excluding Michaelis-Menten analysis. Furthermore, the analysis of Michaelis-Menten kinetics in the multi-reaction ubiquitylation cascade (E1, E2, and E3) necessary to achieve uH2A would be extremely challenging to design and to interpret correctly. To our knowledge this has not previously been achieved for histone ubiquitylation reaction cascades. Therefore we chose to employ a dose-response approach in order to measure and compare the relative E3 ubiquitin ligase activity of different PRC1 complex assemblies by following percentage conversion of a fixed amount of substrate. In fact, similar approaches have previously been used to compare the relative activities of PRC1 complexes (Gao et al., 2012, Tavares et al., 2012, Buchwald et al., 2006, Cao et al., 2005, Li et al., 2006), enabling us to broadly compare our new findings with previous work in this area. Nevertheless, to avoid any confusion we have now clarified this point in the text of manuscript and explicitly indicate that we are comparing relative activities as opposed to reaction rates in the Results section.

Another critical issue is that the ratio enzyme vs substrate (nucleosomes) is not clear. It is essential that the substrate is in excess over the enzyme (this should be shown).

As noted above, this point is particularly relevant in situations where Michaelis-Menten-type kinetic analyses are being conducted, which we have not attempted to do here. However, we realise that in the initial submission the concentration of nucleosome in ubiquitylation reactions was inadvertently omitted. We have now added this information to the Materials and methods in the revised manuscript.

Finally, a better analysis of the nucleosomes used in the assays need to be provided. E.g. are there free histones in the prep? An additional nucleosome purification step (e.g. glycerol centrifugation or size exclusion chromatography) would give more confidence. This is also important because the results in this manuscript are at odds with published work from other groups (e.g. Gao et al. 2012; Tavares et al., 2012).

Octamers were purified by gel filtration and displayed the expected equimolar ratio of histones. In the nucleosome reconstitutions, DNA was always in slight excess over octamer, and tris-borate native agarose gels used to examine the reconstituted nucleosomes indicated that only a small proportion of unincorporated DNA was present relative to nucleosomal DNA. This suggests a near complete incorporation of octamers into nucleosomes under our reconstitution conditions as we have previously published (Zhou et al., 2012). Furthermore, it has been demonstrated that PRC1 is highly specific towards H2A in the context of nucleosomes, and any non-specific activity on free histone results in polyubiquitylation (Elderkin et al., 2007), which would not be detected in our western blotting measurements of H2AK119ub1. Finally we do not believe that the results of our in vitro assays are at odds with either of the studies mentioned by the reviewer. In Gao et al., it was also demonstrated that RYBP stimulates the activity of a PCGF4-containing complex (see (Gao et al., 2012) Figure 5F), which is in agreement with our observations in Figure 3B. In Tavares et al., the influence of RYBP on catalytic activity of PRC1 was tested using a PCGF2-containing complex, which we did not directly examine in our study.

Finally, subsection “PCGF1-PRC1 is a highly modular protein complex” and Figure 1D. It appears that the absence of RYBP leads to consistently reduced levels of PCGF1 (lanes 4 to 6) and possibly a slight reduction in BCOR (lane 5). This suggests that RYBP may help to stabilize these interactions. Quantitation of this blot should be performed and these result discussed in the text.

We have now quantified PCGF1 in the indicated complex preparations and find the levels are reduced in the absence of RYBP as suggested by the reviewer. We thank the reviewer for pointing this out as we believe it further supports the observations from crosslinking mass spectrometry analysis (Figure 4C and D) that RYBP inclusion in the complex may lead to structural alterations that limit the self-association of RING1B, possibly helping to support a stable and enzymatically competent PRC1 complex. We have now included the quantification of these results in Figure 4—figure supplement 1 and described this interesting observation in the text (subsection “RYBP-dependent stimulation of PCGF1-PRC1 is associated with changes in the PCGF1-RING1B dimer but not with ubiquitin binding”). Importantly, however, the reduced level of PCGF1 does not explain activity differences between complexes, as complexes that contain RYBP simulate the relative activity of PRC1 to a much larger extent than can be explained by elevated PCGF1 levels (Figure 2B and C) and both RYBP and YAF2 also significantly stimulate the activity of pre-existing RING1B/PCGF1 assemblies (Figure 2—figure supplement 1B). Further structural work in the future will be imperative to understand the nature of this RYBP dependent stimulation.

6) The Introduction, and actually most of the manuscript, does not give a clear overview of what has already been published on this topic. It is not that many major references were missing (although e.g. the work of the Sixma lab (Buchwald et al., 2006) should be included), rather the Introduction does not clearly summarized the work already done. In addition, somewhat more attention to work in the fly would be helpful to the reader as well. E.g. ncPRC1 complexes and there role in histone ubiquitylation were first described in the fly (Lagarou et al., 2008).

We have now made extensive updates to the Introduction to cover in more detail the work already published in this area and we have specifically included observations from fly studies which we agree are important. Elsewhere in the manuscript, we have better summarized the published literature where appropriate.

Finally, in the Results and Discussion sections, findings by others with similar experiments should be mentioned and where appropriated differences should be discussed. The whole manuscript strongly emphasizes the catalytic functions of PRC1/2 but this needs to be much better balanced by a discussion of the substantial evidence – both in vitro and in vivo, and in flies and mammals – that PRC1 executes many of its functions through a structural change in the underlying chromatin and that is independent of its catalytic activity.

The reviewer(s) are correct that we have emphasized the catalytic activity of PRC1 and PRC2 as our study was focused on understanding the factors responsible for PRC1 catalytic activity in vitro (Figures 1 – 4) and how this relates to the acquisition of histone modifications in vivo. Unfortunately, from our current study we can infer very little about non-catalytic functions of PRC1, with the exception that gene expression changes appear to be more closely linked to reductions in the occupancy of PRC1/PRC2, not simply reductions H2AK119ub1/H3K27me3. We have now extensively altered the text and Discussion to indicate that PRC1 likely executes many of its functions through structural changes in the underlying chromatin structure independent of its catalytic activity.

Author details

1. Nathan R Rose

   Department of Biochemistry, University of Oxford, Oxford, United Kingdom

   Contribution

   NRR, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Hamish W King and Neil P Blackledge

   Competing interests

   The authors declare that no competing interests exist.

2. Hamish W King

   Department of Biochemistry, University of Oxford, Oxford, United Kingdom

   Contribution

   HWK, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Nathan R Rose and Neil P Blackledge

   Competing interests

   The authors declare that no competing interests exist.

3. Neil P Blackledge

   Department of Biochemistry, University of Oxford, Oxford, United Kingdom

   Contribution

   NPB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Nathan R Rose and Hamish W King

   Competing interests

   The authors declare that no competing interests exist.

4. Nadezda A Fursova

   Department of Biochemistry, University of Oxford, Oxford, United Kingdom

   Contribution

   NAF, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

5. Katherine JI Ember

   Department of Biochemistry, University of Oxford, Oxford, United Kingdom

   Contribution

   KJIE, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

6. Roman Fischer

   TDI Mass Spectrometry Laboratory, Target Discovery Institute, University of Oxford, Oxford, United Kingdom

   Contribution

   RF, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-9715-5951

7. Benedikt M Kessler

   1. TDI Mass Spectrometry Laboratory, Target Discovery Institute, University of Oxford, Oxford, United Kingdom
   2. Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom

   Contribution

   BMK, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

8. Robert J Klose

   1. Department of Biochemistry, University of Oxford, Oxford, United Kingdom
   2. Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom

   Contribution

   RJK, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   rob.klose@bioch.ox.ac.uk

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-8726-7888

• 4,074

  Page views

• 949

  Downloads

• 81

  Citations

Article citation count generated by polling the highest count across the following sources: Scopus, Crossref, PubMed Central.