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Recruitment of Polycomb complexes

Targeting Polycomb systems In the D. melanogaster genome, Polycomb

responsive elements (PREs) function as
recruitment sites for PRCs. DNA-binding
to regulate gene expression: transcription factors are thought to play an
important part in bringing Polycomb protein
modifications to a complex story complexes to these sites (reviewed in REF. 4).
However, attempts to define vertebrate
PREs have proven largely unsuccessful, and
Neil P. Blackledge, Nathan R. Rose and Robert J. Klose emerging evidence supports the idea that
vertebrate Polycomb complexes are directed
Abstract | Polycomb group proteins are transcriptional repressors that are essential
to DNA by both locus-specific and more
for normal gene regulation during development. Recent studies suggest that generalized targeting mechanisms.
Polycomb repressive complexes (PRCs) recognize and are recruited to their
genomic target sites through a range of different mechanisms, which involve Locus-specific targeting. Inspired by obser-
transcription factors, CpG island elements and non-coding RNAs. Together with vations in D. melanogaster, it has been pro-
the realization that the interplay between PRC1 and PRC2 is more intricate than posed that vertebrate transcription factors
might recruit PRCs to target sites in chroma-
was previously appreciated, this has increased our understanding of the vertebrate
tin (FIG. 1a). However, there are only a limited
Polycomb system at the molecular level. number of cases in which site-specific DNA-
binding transcription factors have been
A remarkable feature of multicellular organ- how these chromatin-associated factors identified in unbiased biochemical isolations
isms is their capacity to create functionally function. This has led to the discovery that of Polycomb complexes. Examples include
unique cell types from an essentially invariant Polycomb group proteins usually belong to E2F, MAX gene-associated protein (MGA)
genome sequence that is shared by all cells one of two multi-subunit protein complexes: and MAX, which were found to interact with
in the organism. This diversity relies on the Polycomb repressive complex 1 (PRC1), PRC1 (REFS 8,9). Thus, it has been brought
capacity of individual cell types to initiate which adds a ubiquityl moiety to histone into question whether interactions with
and then maintain specific gene expression H2A at Lys119 (H2AK119ub1); and PRC2, transcription factors broadly underpin tar-
patterns during development. To achieve which catalyses the addition of one to three geting. Candidate-based approaches have
this, cellular signalling events are thought to methyl groups to histone H3 at Lys27, leading identified additional DNA-binding factors,
regulate the activity of cell type-specific DNA- to H3K27me1, H3K27me2 and H3K27me3 including RE1‑silencing transcription facto­r
binding transcription factors that function (BOX 1) (reviewed in REF. 3). (REST), zinc-finger protein SNAI1 (also
as master regulators of gene expression net- PRC1 and PRC2 usually co‑occupy target known as SNAIL) and RUNT-related tran-
works. Interestingly, however, genetic screens sites in the genome, at which their combined scription factor 1 (RUNX1), that interact
for factors involved in the regulation of gene activities create Polycomb chromatin domains with PRC1 or PRC2; however, these factors
expression and cell fate specification have also consisting of the Polycomb group proteins seem to contribute to Polycomb protein
identified additional genes that seem to affect themselves, H2AK119ub1, and H3K27me3. recruitment only in specific instances10–14.
these processes through chromatin structure How vertebrate Polycomb protein complexes More recently, detailed proteomic profiling
and histone modification. This is exemplified are recruited to chromatin, how different of Polycomb protein complexes has identi-
by the Polycomb group genes, which were PRC1 and PRC2 complexes function together fied novel interactors, including proteins
first identified in Drosophila melanogaster as in situ and how Polycomb chromatin domains that contain zinc-finger domains (ZNF518A
regulators of Hox gene expression and nor- actually repress transcription still remain and ZNF518B15), that are often associated
mal developmental body plan specification1. poorly understood. Several excellent recent with DNA-binding activity. Whether these
Subsequently, orthologous genes were identi- review articles have described the general newly identified proteins contribute to
fied in vertebrate species, in which they also features and functions of Polycomb systems Polycomb complex targeting remains to be
encode transcriptional repressors. Vertebrate in different phyla3–7. In this Progress article, determined.
Polycomb group proteins are essential for we focus on exciting new advances that In addition to transcription factors, it has
normal gene regulation during embryonic have begun to shed light on the molecular been suggested that long non-coding RNAs
development and are perturbed in a wide mechanisms that underpin the recruitment of (lncRNAs) could recruit Polycomb com-
range of human cancers (reviewed in REF. 2). Polycomb group protein complexes to target plexes to specific loci; however, the general-
Since the initial identification of Polycomb sites, the formation of Polycomb chromatin ity of this as a targeting mechanism remains
group genes, an immense amount of bio- domains and the functional relevance that a topic of debate (reviewed in REF. 16).
chemical work has focused on understanding this has for gene regulation in vertebrates. This idea originated from the observation

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PROGRESS

group proteins in vivo29, has led to sugges-

Box 1 | Core PRCs and their chromatin-modifying activities
tions that CGIs may have a direct role in
Polycomb repressive complex 1 Polycomb protein recruitment. Attempts to
(PRC1) and PRC2 comprise core H2AK119ub1 define a molecular link between Polycomb
protein components that are E2 complexes and CGIs have largely focused
necessary for their respective
PCGF RING1A or on Lys-specific demethylase 2B (KDM2B),
enzymatic activities (see the
figure). PRC1 is a histone H2A
RING1B DNA Nucleosome which stably associates with PRC1. KDM2B
PRC1 core encodes a zinc-finger–CXXC DNA-binding
Lys 119 (H2AK119) ubiquitin
ligase. In PRC1, two functionally domain that specifically recognizes non-
equivalent yet mutually exclusive EZH1 or H3K27me3 methylated CpG dinucleotides, allowing
EZH2
subunits, RING1A and RING1B, KDM2B to bind to CGIs genome-wide30–32.
EED SUZ12
function as ubiquitin E3 ligases Recent studies have demonstrated that
RBAP46 or
that guide the transfer of a RBAP48 KDM2B contributes to PRC1 occupancy at
ubiquityl moiety onto H2AK119 CGIs (FIG. 1b). However, somewhat paradoxi-
PRC2 core
in chromatin (H2AK119ub1). cally, high-level PRC1 enrichment is only
RING1A or RING1B dimerize with a Polycomb group RING-fingerNature Reviews | Molecular
(PCGF) subunit Cell Biology
(of which there
achieved at the most repressed sites, despite
are six, PCGF1 to PCGF6, in vertebrates). The PCGF components are required for the core
enzymatic activity of the complex and also define how the core complex interacts with auxiliary
KDM2B residing at all CGIs. This suggests
complex components to regulate its targeting to chromatin and its enzymatic activity. that the recruitment of PRC1 to CGIs by
PRC2 is a H3K27 methyltransferase. The methyltransferase activity of PRC2 resides in the SET KDM2B, or the repressive activity of PRC1
domain of two mutually exclusive proteins, EZH1 and EZH2. Alone, these proteins have little at CGIs, is regulated by additional mecha-
enzymatic activity in vitro, but when bound to EED, SUZ12, and RBAP46 or RBAP48, they form an nisms that permit the establishment of
active methyltransferase complex that is specific towards chromatin substrates. EED, SUZ12, and Polycomb chromatin domains. Furthermore,
RBAP46 or RBAP48 contribute to the stability and structural integrity of PRC2, and they also KDM2B‑independent mechanisms must
regulate its enzymatic activity and support its targeting to chromatin, either directly or through also function at some CGIs to recruit or
interactions with auxiliary complex components. Despite detailed characterization of the core stabilize PRC1 binding, as the removal of
PRC1 and PRC2 protein components, it remains unclear whether these complexes are stable
KDM2B does not lead to a complete loss of
entities inside cells or whether they also display some capacity to assemble in a dynamic fashion on
chromatin. H3K27me3, H3K27 trimethylation.
PRC1 at all CGI target sites30–33. Interestingly,
it has been reported that the PRC2 com-
ponent JARID2 preferentially recognizes
GC‑rich DNA34. JARID2 could thus provide
that the X inactive specific transcript (Xist) HOX transcript antisense RNA (HOTAIR), a complementary mechanism for targeting
lncRNA is required for the localization which is expressed from the HOXC locus on PRC2 to CGIs independently of KDM2B.
of PRC2 (REFS 17–20) to the inactivated human chromosome 12, seems to function Together, these observations suggest that
X chromosome during mammalian dosage in trans to target PRC2 to the HOXD locus Polycomb complexes can be recruited to all
compensation, in a manner that relies on on chromosome 2 (REF. 26). CGIs, although other mechanisms deter-
the sub-stoichiometric PRC2 component Although it is clear that in some specific mine whether this results in the formation of
JARID2 (also known as Jumonji)21. Recent cases, transcription factors and lncRNAs stable Polycomb chromatin domains.
studies aimed at understanding the relation- contribute to locus-specific recruitment of Although lncRNA-mediated targeting has
ship between PRC2 and Xist have suggested PRCs, it seems unlikely that these target- been proposed as a locus-specific recruit-
that the interaction between Xist and the ing mechanisms are sufficient to create ment mechanism for Polycomb complexes,
chromatin-remodelling protein transcrip- the widespread, and often tissue-specific, it has recently been reported that PRC2
tional regulator ATRX induces conforma- Polycomb complex binding patterns that are binds to RNA promiscuously, with little
tional changes within the Xist RNA, which observed in vivo. sequence specificity 35,36. This has led to sev-
favours specific and direct interaction with eral new suggestions as to how PRC2–RNA
PRC2 (REF. 22) (FIG. 1a). However, it has also Generic targeting. Important recent discov- interactions may contribute to the generic
been proposed that the relationship between eries have shown that PRCs bind to regula- recruitment or functionality of Polycomb
Xist and PRC2 may be indirect. In support tory sites across the genome through generic complexes at gene regulatory elements
of an indirect interaction, super-resolution chromatin-binding activities, with local and genes. At repressed genes, binding of
imaging studies have indicated that PRC2 is chromatin features dictating residency and PRC2 to short abortive RNA transcripts
not intimately associated with Xist RNA on function at these sites. may help to retain Polycomb complexes
a Xist-inactivated chromosome23, and the In vertebrate genomes, the most universal and stabilize transcriptional repression
Xist-binding protein SHARP (also known as and striking feature of Polycomb-occupied during aberrant transcriptional initiation
SPEN) was required to recruit PRC2 to Xist- sites is the presence of a CpG island (CGI). events37,38. Alternatively, nascent transcripts
coated chromosomes24. Nevertheless, there is CGIs are approximately 1–2 kb regions of produced from active genes may interact
additional evidence in favour of direct roles CpG-rich DNA that lack DNA methyl­ with PRC2 and provide a ‘decoy’ to block
for lncRNAs in the recruitment of PRCs to ation and that typically encompass gene stable inter­actions between PRC2 and chro-
chromatin. For example, PRCs are recruited promoters27,28. The striking correlation matin, thereby protecting the transcribed
to the KCNQ1 overlapping transcript 1 between Polycomb protein occupancy and gene from repressive Polycomb activity.
(Kcnq1ot1) locus on a paternally imprinted CGIs, together with the observation that This latter idea has received some sup-
region of mouse chromosome 7 that tran- artificial DNA sequences with high CpG port from the observation that interactions
scribes lncRNAs25. Moreover, the lncRNA content are sufficient to nucleate Polycomb with nascent transcripts can constrain the

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 PROGRESS

histone methyl­transferase activity of PRC2 a Locus-specific targeting b Generic targeting

(REFS 39,40). An alternative possibility is that
Transcription factors CGIs
PRC2 uses interactions with nascent tran-
scripts as a generalized targeting mechanism
to increase residency around gene promoters PRC1
PRC1 PRC2
(FIG. 1b), and that other counteracting signals, KDM2B
REST RUNX1 REST
including H3K4 and H3K36 methylation,
prevent the formation of stable Polycomb
domains at active genes41. These emerging DNA Nucleosome CGI
links between ncRNAs and PRC2 enzymatic
activity seem to be evolutionarily conserved, lncRNAs ATRX Nascent transcripts
as regulated bidirectional ncRNA expres- PRC2
PRC2
sion from a D. melanogaster PRE acts as a
Xist
switch to alter PRC2 activity and regulate
gene expression42. Interestingly, vertebrate Pol II
Polycomb target sites often exhibit bidirec-
tional transcription, so it will be important
Pre-existing histone modifications
to understand whether similar switch-like
mechanisms are involved in regulating PRC2
vertebrat­e PRC2 activity. H2AK119ub1 meCpG
In addition to generic DNA- and RNA- PCL1–3
binding activities, recent evidence suggests H3K27me3 Non-methylated CpG
that certain PRCs recognize chromatin H3K36me3
modifications placed by other histone-
modifying systems that are broadly associ- Figure 1 | Getting Polycomb repressive complexes (PRCs) to chromatin. a | PRC1 and PRC2 can
ated with genes and regulatory elements. associate with DNA-binding transcription factors (top left),Nature
such asReviews | Molecular
RUNT-related Cell Biology
transcription fac-
For example, the sub-stoichiometric PRC2 tor 1 (RUNX1) and RE1‑silencing transcription factor (REST), which guide these complexes to chro-
subunits Polycomb-like 1 (PCL1; also known matin. Similarly, interactions with long non-coding RNAs (lncRNAs) such as X inactive specific
as PHF1), PCL2 and PCL3 encode TUDOR transcript (Xist) function in chromosome- and locus-specific targeting of Polycomb complexes
domains that recognize the H3K36me3 (botto­m left). The chromatin-remodelling protein ATRX may remodel the structure of Xist to achieve
modification43–46. H3K36me3 is typically interaction with PRC2. Following recruitment to chromatin, PRC1 catalyses ubiquitylation of histone
2A Lys 119 (H2AK119ub1) and PRC2 catalyses trimethylation of H3K27 (H3K27me3), as indicated by
associated with actively transcribed gene
rounded arrows. The square arrows indicate transcription start sites. b | A variant PRC1 complex
bodies and, during cellular differentiation, contains the Lys-specific demethylase 2B (KDM2B) protein. KDM2B has a zinc-finger CXXC DNA-
PCL proteins were proposed to facilitate binding domain (red area) that specifically recognizes non-methylated CpG dinucleotides. This
spreading of PRC2 into these regions allows KDM2B to bind at CpG islands (CGIs) genome-wide and contributes to PRC1 occupancy at
(FIG. 1b). In addition, links have been identi- these elements (top right). PRC2 interacts with nascent RNA polymerase II (Pol II) transcripts at 5ʹ ends
fied between PRC2 and the H3K9 methyl­ of genes, which may provide a mechanism to maintain repression at silent genes following stochastic
ation systems. Most notably, biochemical transcription initiation events. Alternatively, at active genes, interaction of PRC2 with nascent RNA
interactions were identified between PRC2 may constrain the catalytic activity of PRC2 and protect against Polycomb-mediated repression (mid-
and the H3K9 methyltransferases G9A dle right). A subset of PRC2 complexes contain Polycomb-like (PCL) proteins that bind to H3K36me3,
and G9A‑like protein 1 (GLP; also known which is a modification associated with active transcription. This may enable PRC2 to bind at,
or spread into, previously transcribed regions, catalysing H3K27me3 at these regions. meCpG,
as EHMT1), and loss of G9A resulted in
methylate­d CpG dinucleotide.
impaired PRC2 recruitment at a subset of
target sites15,47.

Interplay between PRCs decade ago in D. melanogaster. This mecha- formation in vertebrates. However, detailed
Outlined above are simple examples of how nism posits that de novo recruitment of PRC2 studies of the relationship between PRC1
PRC1 or PRC2 are individually recruited to target sites catalyses H3K27me3, which and PRC2 in vertebrates indicate that other
to target sites by locus-specific or generic is subsequently recognized by a chromobox mechanisms are involved in the formation
targeting mechanisms. However, after their (CBX)-containing protein in PRC1, leading of Polycomb domains. For example, deletion
recruitment to chromatin, the functions of to H2AK119ub1 placement and Polycomb of PRC2 components in mouse embryonic
PRC1 and PRC2 are intimately related, as chromatin domain formation48,49 (FIG. 2a). stem cells led to a reduction, but not the loss,
the enzymatic activity of each complex influ- This pathway is generally referred to as the of PRC1 proteins at target sites and had lit-
ences the occupancy of the other on chroma- ‘hierarchical’ recruitment mechanism and tle effect on global levels of H2AK119ub1
tin and the full establishment of Polycomb places PRC1 recruitment and activity down- (REF. 51). These observations suggested that
chromatin domains. stream of PRC2 function. On the basis of the the relationship between PRC1 and PRC2 is
conservation of CBX proteins, and on the evi- more complex than was originally envisaged.
The prevailing hierarchical model. PRC1 dence that PRC1 binding to chromatin is sen-
and PRC2 typically co-localize at target sites sitive to loss of PRC2 (REF. 50), the hierarchical A new twist in the hierarchy: PRC1 recruits
throughout the genome. This has largely been recruitment mechanism was widely adopted PRC2. Our understanding of Polycomb
attributed to a mechanism discovered over a to explain Polycomb chromatin domain systems has recently evolved, with the

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PROGRESS

a Hierarchical model Polycomb proteins. Strikingly, de novo PRC1

binding to this site and monoubiquityla-
PRC1 tion of H2AK119 resulted in the subsequent
PRC2 H3K27me3 H2AK119ub1 CBX recruitment of PRC2 and H3K27me3 depo-
sition, creating a new Polycomb chromatin
domain52. A similar outcome was observed
DNA Nucleosome
when PRC1 was artificially recruited to
pericentri­c regions of the genome53.
b Alternative model PRC2 JARID2 This newly discovered recruitment
AEBP2 mechanism seems to play a part in Polycomb
PRC1 chromatin domain formation at natural tar-
RYBP get sites, as perturbation of PRC1 and loss of
H2AK119ub1 caused a substantial reduction
in PRC2 binding and H3K27me3 across the
genome. The precise mechanism by which
c PRC1 complexes PRC1‑dependent H2AK119ub1 underpins
PHC KDM2B
de novo Polycomb chromatin domain for-
CXXC
PHC1, PHC2 mation remains to be elucidated. However,
Canonical and PHC3 Variant AUTS2 CSNK2A1 in vitro studies have identified a PRC2
PRC1 PRC1
complex containing the auxiliary proteins
CBX RYBP or E2F6 MGA MAX AEBP2 and JARID2 that preferentially binds
CBX2, CBX4, EED YAF2
CBX6, CBX7 to and stimulates catalysis of H3K27me3 on
and CBX8 chromatin containing H2AK119ub1 (REF. 54),
suggesting that this complex may provide
d Propagation of Polycomb domains PRC2 the molecular link between PRC1 activ-
EED ity on chromatin and recruitment of PRC2
and H3K27me3. Additional unexpected
PRC2 connections between the two Polycomb
complexes include the finding that PRC2
EED New nucleosome
can interact directly with an alternative
Replication PRC2 H2AK119ub1 E3 ligase, TRIM37, which is
EED highly expressed in breast cancer cells carry-
ing the 17q23 amplification55. Furthermore,
EED, which is a core PRC2 subunit, has
been reported to directly interact with PRC1
Figure 2 | Beyond simple recruitment to more complex interactions. a | In the ‘hierarchical’ model (REF. 56) (FIG. 2c). Together, these observations
of Polycomb complex function, Polycomb repressive complex Nature Reviews
2 (PRC2) Molecular
first| binds Cell Biology
to chromatin and indicate the potential for more functional
places H3 Lys27 trimethylation (H3K27me3). It is then proposed that H3K27me3 is recognized by chro- overlap between PRC1 and PRC2 than had
mobox (CBX) proteins, which are a subunit of canonical PRC1 complexes, thereby allowing PRC1 to previousl­y been appreciated.
bind to and monoubiquitylate H2AK119 (H2AK119ub1). b | In the ‘alternative’ model for Polycomb
recruitment, the initial event is the binding of variant PRC1 complexes (which contain RYBP (RING1 and
YY1‑binding protein) instead of CBX) to chromatin. PRC1 then catalyses the formation of H2AK119ub1, Alternative roles for canonical PRC1.
independently of PRC2 activity and H3K27me3. The H2AK119ub1 modification then promotes the The observation that PRC1 can promote
recruitment of PRC2, possibly through direct recognition of H2AK119ub1 by the AEBP2–JARID2–PRC2 the recruitment of PRC2 to target sites on
complex and placement of the H3K27me3 mark. c | PRC1 complexes functionally segregate into ‘canon- chromatin has placed a new focus on under-
ical’ complexes that contain CBX (CBX2, CBX4, CBX6, CBX7 and CBX8) and Polyhomeotic (PHC1, PHC standing how PRC1 complexes contribute
2 and PHC3) proteins, and a series of ‘variant’ complexes that contain RYBP (or the closely related to Polycomb chromatin domain forma-
YY1‑associated factor 2 (YAF2) protein), and interact with proteins that are unique to individual variant tion and function. Systematic biochemical
PRC1 complexes (note that only selected proteins are shown here). Under some conditions, the PRC2 purifications have revealed that PRC1 in
subunit EED may interact with a CBX-containing canonical PRC1 complex (dashed arrow). d | Within vertebrates can be separated into ‘canonical’
the core PRC2 complex, the EED subunit is able to recognize H3K27me3 through its WD40‑repeat
PRC1 complexes, which have CBX proteins
domain. This interaction potentially recruits PRC2 to sites of pre-existing H3K27me3, as well as stimulat-
ing the enzymatic activity of PRC2. The EED–H3K27me3 interaction may facilitate the spreading of and presumably function as part of the
H3K27me3 domains (left panel, dashed arrows) or the copying of H3K27me3 onto newly incorporated hierarchical recruitment pathway; and the
histones (right panel, blue nucleosomes) during DNA replication, thereby stably propagating H3K27me3 less well-studied ‘variant’ PRC1 complexes,
domains in actively dividing cells. AUTS2, autism susceptibility gene 2; CSNK2A1, casein kinase 2 which lack CBX proteins and contain either
subunit‑α1; KDM2B, Lys-specific demethylase 2B; MGA, MAX gene-associated protein. RYBP (RING1 and YY1‑binding protein)
or YY1‑associated factor 2 (YAF2)8 (FIG. 2c).
Importantly, RYBP and YAF2 lack the
surprising finding that PRC1 can recruit discovered using a cell-based system in capacity to bind to H3K27me3 and can be
PRC2 to chromatin through a mechanism which PRC1 was artificially recruited to recruited to chromatin in cells lacking func-
that involves recognition of H2AK119ub1 a region of the genome that is devoid of tional PRC2, suggesting that variant PRC1
(FIG. 2b). This alternative pathway was genes and that is not normally occupied by complexes must be recruited to chromatin

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© 2015 Macmillan Publishers Limited. All rights reserved

 PROGRESS

by PRC2‑independent mechanisms51. a Instructive model

Furthermore, individual canonical and vari-
ant PRC1 complexes contain distinct protein Pol II
PRC1 PRC2
subunits that are likely to contribute to target H2AK119ub1
site recognition or catalysis8. Transcriptional PRC1 PRC2
Examining the function of individual repression
PRC1 complexes in vivo has revealed that
variant PRC1 complexes are proficient at Pol II
DNA H3K27me3
catalysing H2AK119ub1 on chromatin, Nucleosome
whereas canonical complexes catalyse lit-
tle of this modification52. This is consistent
b Responsive model PRC1 PRC2
with observations that RYBP-containing
PRC1 variant complexes have enhanced
Sampling
H2AK119ub1 catalysis in vitro8. As a conse- ?
quence of their restricted catalytic activity
in vivo, canonical PRC1 complexes seem to
have limited capacity to recruit PRC2 and
to form Polycomb chromatin domains. One H2AK119ub1 PRC1 PRC2
exception to this generality was recently PRC1 PRC2
reported: a CBX2‑containing canonical + Transcription
PRC1 complex seems to have a very specific
PRC2 PRC1 Pol II
role in atypical Polycomb chromatin domain
formation by directly recognizing peri- H3K27me3 – Transcription
centromeric heterochromatin during early
Figure 3 | Polycomb systems and gene regulation. a | An instructive model for Polycomb complex-
mouse development 57. Nature Reviews | Molecular Cell Biology
mediated silencing would posit that newly recruited Polycomb complexes lead to Polycomb chromatin
Nevertheless, if canonical PRC1 com- domain formation, which then directs repression of transcription by RNA polymerase II (Pol II) at the
plexes are usually limited in their capacity to associated gene. b | A responsive model would posit that Polycomb complexes constantly ‘sample’
deposit H2AK119ub1 in vivo, why are they chromatin at regulatory elements through generic targeting modalities, in order to respond to the
recruited to Polycomb chromatin domains transcriptional state of the associated gene. Transcriptional cessation would lead to the subsequent
at all? A hint as to possible functions came establishment of Polycomb chromatin domains that protect against low-level or stochastic reactivation
with the recent demonstration that a stable signals. However, in response to active transcription, the presence of Pol II or other features of transcrip-
component of canonical PRC1 complexes, tional initiation would block establishment of Polycomb domains. H3K27me3, histone 3 Lys27 trimeth-
Polyhomeotic-like protein 2 (PHC2), can ylation; H2AK119ub1, histone 3 Lys119 monoubiquitylation; PRC, Polycomb repressive complex.
auto-polymerize through its sterile-alpha
motif (SAM) domain, leading to chromatin
compaction and gene silencing 58–60. This is On the basis of the finding that PRC1, PRC2 analysis of gene expression in a cell culture
consistent with previous reports detailing a and their chromatin-modifying activities model of RAS-induced transformation
ubiquitin ligase-independent role for PRC1 are more intimately linked than was previ- showed that cessation of transcription pre-
in chromatin compaction61. Interestingly, ously appreciated52–54, it seems plausible that ceded H3K27me3 acquisition64. Secondly,
inhibition of PHC2 polymerization resulted this robust series of PRC-dependent feed- experiments in mouse embryonic stem cells
in loss of canonical PRC1 binding to chro- back mechanisms could underpin both the demonstrated that small-molecule inhibitors
matin only at sites marked with H3K27me3, spreading and the maintenance of Polycomb that block transcription caused recruitment
suggesting that both H3K27me3–CBX inter- chromatin domains once they are initially of PRC2 and H3K27me3 to previously active
actions and SAM-domain polymerization established. It is tempting to speculate that genes65. In light of these conceptually impor-
may have important structural roles in cre- this would contribute not only to epige- tant findings, one interesting possibility is
ating and translating repressive chromatin netic maintenance of Polycomb chromatin that Polycomb systems may exploit generic
structures on chromatin59. domains but also to rigidly maintaining gene targeting activities to constantly interface
expression states during cell division and with or ‘sample’ gene regulatory elements
Propagation of Polycomb domains. development. A detailed understanding of and respond to the transcriptional state
Biochemical studies have demonstrated that the epigenetic nature of Polycomb chroma- of individual genes. Within the context of
PRCs can bind the histone modifications tin domains is an interesting and evolving this model, features of active transcription
that they themselves place. For example, area of Polycomb biology. — for example, the presence of RNA poly­
the EED subunit of the PRC2 core complex merase II (Pol II) or transcription-associated
binds to H3K27me3 through its WD40 Polycomb systems and gene regulation histone modifications — would decrease
repeat, and this interaction seems to stimu- It is often suggested that vertebrate the residency time or the catalytic activity of
late the catalytic activity of PRC2, to form Polycomb group proteins are recruited to Polycomb complexes at transcribed genes,
an activity-based feedback loop62,63. It has target sites to actively drive transcriptional meaning that full Polycomb chromatin
been proposed that this could promote the repression (FIG. 3a). However, recent evidence domain establishment would only occur at
spreading of H3K27me3 along chromatin suggests that this may not be the central sites at which transcriptional silencing has
and ensure the propagation of H3K27me3 modality connecting Polycomb chromatin already been achieved (FIG. 3b) (reviewed
on newly replicated chromatin62 (FIG. 2d). domains and gene repression. Firstly, kinetic in REF. 66).

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What would be the purpose of establish- Conclusion and future directions 3. Simon, J. A. & Kingston, R. E. Occupying chromatin:
Polycomb mechanisms for getting to genomic targets,
ing Polycomb chromatin domains at already A complex series of interactions between stopping transcriptional traffic, and staying put.
silenced genes? One possibility is that fol- PRC1 and PRC2, chromatin, and transcrip- Mol. Cell 49, 808–824 (2013).
4. Steffen, P. A. & Ringrose, L. What are memories made
lowing transcriptional silencing, Polycomb tion are essential for normal Polycomb of? How Polycomb and Trithorax proteins mediate
systems form repressive chromatin domains chromatin domain formation and func- epigenetic memory. Nat. Rev. Mol. Cell Biol. 15,
340–356 (2014).
to limit the potential for stochastic reactiva- tion. Although transcription factor and 5. Schwartz, Y. B. & Pirrotta, V. A new world of
tion events in inappropriate tissues. This lncRNA-based mechanisms contribute to Polycombs: unexpected partnerships and emerging
functions. Nat. Rev. Genet. 14, 853–864 (2013).
general concept is consistent with observa- Polycomb complex targeting to specific 6. Di Croce, L. & Helin, K. Transcriptional regulation by
tions in D. melanogaster, in which Polycomb chromatin regions, recent evidence sug- Polycomb group proteins. Nat. Struct. Mol. Biol. 20,
1147–1155 (2013).
group genes are important for the mainte- gests that Polycomb protein complexes also 7. Grossniklaus, U. & Paro, R. Transcriptional silencing by
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and recruits PRC2 and demethylase NO66 to structure and represses gene expression independent
embryonic stem cell genes during differentiation. of histone ubiquitination. Mol. Cell 38, 452–464 Competing interests statement
Nat. Struct. Mol. Biol. 19, 1273–1281 (2012). (2010). The authors declare no competing interests.

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