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Cell Cycle 10:5, 745-750; March 1, 2011; © 2011 Landes Bioscience
The flip side of the coin
Role of ZRF1 and histone H2A ubiquitination in transcriptional activation
Holger Richly1 and Luciano Di Croce1,2,*
Centre de Regulació Genòmica (CRG); Parc de Recerca Biomèdica; Universitat Pompeu Fabra (UPF); 2Institució Catalana de Recerca i Estudis Avançats
(ICREA); Barcelona, Spain
1
We have recently reported that the protein ZRF1 specifically binds to monoubiquitinated histone H2A and derepresses
Polycomb target genes at the onset of
cellular differentiation.1 Our results suggest that ZRF1 exerts its function in a
two-step mechanism, by initially displacing the Polycomb-repressive complex 1
(PRC1) from chromatin and subsequently
acting together with histone H2A-specific
deubiquitinases to facilitate transcriptional activation of its target genes. These
findings demonstrate an ambiguity of the
epigenetic monoubiquitin mark at histone
H2A. Once considered to be a hallmark
of gene silencing, it is now clear that this
mark can also be utilized as a recruitment
platform for proteins engaged in gene
activation. Genome-wide analyses demonstrate that ZRF1 is recruited to typical
Polycomb target genes, thereby putting it
in a position to have an impact on differentiation and animal development. This
molecular mechanism for ZRF1 may represent one of the first steps in switching
silenced genes to a transcriptionally active
state. We discuss here our recent findings
in the light of progress made in understanding Polycomb-mediated silencing.
two H2A proteins within a nucleosome,
its frequency means that, on average, every
fifth nucleosome in the cell is marked by
this epigenetic tag under steady-state conditions. Initially, after it was identified
more than a quarter of a century ago, the
H2A-ubiquitin mark was linked to gene
activation.4,5 Similarly, monoubiquitination at lysine 123 of histone H2B in lower
eukaryotes was shown to be an activating mark.5-8 However, more recently, it
has become clear that the H2A ubiquitin mark is linked to the actions of the
Polycomb proteins, associating this mark
with gene silencing rather than activation.
In the past few years, two main
Polycomb protein complexes, the
Polycomb repressive complexes 1 and 2
(PRC1 and PRC2), have been studied
extensively.9 The molecular mechanisms
of these protein complexes appear to occur
in a consecutive manner.10 First, PRC2
marks the lysine 27 of histone H3 with a
trimethyl-group (H3K27me3), which is a
well-conserved epigenetic mark for gene
silencing.11 This mark is believed to be a
prerequisite for PRC1 binding, recruiting
PRC1 complexes via interactions with proteins of the Polycomb family. The PRC1
complex contains the E3 ligase RING1B,
which catalyzes the ubiquitination of histone H2A with the help of the BMI1 subunit.10,12 The Polycomb complexes have
essential functions for cellular differentiation and development,13 and the misregulation of Polycomb proteins is often observed
in tumors and can be linked to cancer stem
cells.14,15 Although the H2A-ubiquitin
mark was thought to lead exclusively to
gene silencing and thus would represent a
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Introduction
Key words: epigenetics, polycomb, stem
cell, ubiquitination, transcription
Submitted: 01/07/11
Accepted: 01/07/11
DOI: 10.4161/cc.10.5.14795
*Correspondence to: Luciano Di Croce;
Email: [email protected]
www.landesbioscience.com
Monoubiquitination of histone H2A is one
of the most abundant epigenetic marks,
yet it is also the most poorly understood.2
In mammals, it is found on up to ten percent of the total histone H2A proteins, at
the conserved lysine 119 in the C terminus.3 Taking into consideration that ubiquitination usually occurs at only one of the
Cell Cycle745
dead-end in this pathway, it was necessary
to reveal the factors capable of specifically
reading the H2A ubiquitin mark in order
to understand whether, and how, it could
be linked to differentiation and cancer.
H2A-Ubiquitin is Specifically
Read by ZRF1
Affinity purification using monoubiquitinated histones as a bait allowed the mammalian protein ZRF1 to be identified as a
novel H2A-ubiquitin binding protein. The
ZRF1 protein is evolutionary conserved in
most species, and its homolog in S. cerevisiae is involved in the ubiquitination
pathway of histone H2B, as implicated by
genetic experiments.16 All homologs share
a Zuotin domain at their N termini, which
consists of a DnaJ domain and a potential
ubiquitin-binding motif. However, the
tandem repeat of SANT domains located
at the C terminus is only found in higher
eukaryotes. ZRF1 has been studied as
a protein attached to ribosomes that is
involved in protein-fidelity control, where
it functions together with heat shock proteins that bind to its DnaJ domain.17,18
Our data now suggests that ZRF1 additionally plays a fundamental role during
the course of cellular differentiation. We
mapped the Ubiquitin-binding domain
(UBD) of ZRF1 to a region C-terminal
of the DnaJ domain. Interestingly, ZRF1
(also termed MIDA1) has been found to
bind Inhibitor of differentiation (Id) proteins via this same domain;19 this potentially competitive binding at the UBD
might therefore represent a means of
regulating ZRF1 recruitment to chromatin. Our data suggests that ZRF1 is specifically recruited to chromatin marked
with mono-ubiquitinated histone H2A at
the onset of differentiation. Utilizing the
NT2 cell line20,21 and leukemic cell lines,
we found that stimulation with retinoic
acid (RA) led to the recruitment of ZRF1
to chromatin. Genome-wide analysis of
ZRF1 targets identified genes that are
mainly involved in development and differentiation. Those genes were also found
to be occupied by PRC1 and, as expected,
decorated by H2A-ubiquitin.
The strong link between the PRC1
system and differentiation suggests that
ZRF1 is an important player in the
occurrence of cancer. ZRF1 is often found
misregulated in cancers; indeed, it ranks
as one of the most upregulated genes in
certain forms of leukemia.22 One potential explanation for the cancer phenotype
that correlates with ZRF1 overexpression
is that, in these cases, a low abundance
of PRC1 complexes and the resulting
low levels of H2A-ubiquitin leads to an
aberrant expression of oncogenes. Thus,
a highly valuable approach to developing new therapeutical drugs could be to
screen for compounds that block the UBD
domain of ZRF1 and thereby impede its
action at chromatin.
ZRF1 Antagonizes PRC1-Mediated
Gene Silencing
Since ZRF1 can displace the PRC1 complex from chromatin, it can directly antagonize gene silencing. Importantly, this
implies that the maintenance of PRC1 at
chromatin does not dependent solely on
the presence of the H3K27me3 mark and
on recruitment factors of the Polycomb
protein family.11 Rather, our data suggest
that the PRC1 subunit RING1B harbors
a UBD, and that the H2A-ubiquitin mark
itself is important for the maintenance
of PRC1 and thus for its own amplification at chromatin. Indeed, the UBD of
ZRF1 alone is sufficient to displace PRC1
from chromatin, as demonstrated by in
vitro experiments using monoubiquitated nucleosomes and further supported
by ChIP experiments. In the NT2 model
system, we found that PRC1 was displaced from promoters during induction
with RA. However, in ZRF1 knockdown
cells, PRC1 remained attached to chromatin even in the presence of RA, clearly
demonstrating the physiological role of
ZRF1 in displacing PRC1 during differentiation. Yet this raises the question of
why an enzyme would “suicidally” bind
its substrate after performing its catalytic
reaction. Since many enzyme complexes
are likely to propagate along the chromatin fibre, rather than remain firmly bound
at a specific epigenetic mark, binding its
product could provide a mechanism of
dramatically increasing RING1b concentrations at chromatin. This mechanism
could solve the important question of how
the low-abundant RING1B enzymes can
efficiently ubiquinate ten per-cent of all
the H2A histones. A mechanism to ensure
efficient ubiquitination is even more critical during S phase, since the H2A ubiquitation is an epigenetic mark that must
be properly copied to be inherited by
daughter cell chromatin.23 A mechanism
based solely on diffusion, with continuous ubiquitination and deubiqutination
cycles, would not be able to cope with
such a requirement. Based on our recent
data as well as that from others, we suggest
a mechanism in which the enzymes propagate epigenetic marks by moving along
chromatin, rather then diffusing from the
nucleoplasm.
Propagation of Polycomb
Complexes at Chromatin
The question of how Polycomb complexes are propagated along chromatin
has been recently investigated. PRC2 was
found to bind the H3K27me3 mark via a
C-terminal domain on its Eed subunit.24
This interaction activates the methyltransferase EZH2, which then propagates a
methyl mark to adjacent nucleosomes during replication. In a different physiological situation, a mechanism explaining how
the E3 ligase RNF168 propagates histone
H2A ubiquitination along the chromatin fibre during DNA repair was recently
described in reference 25. In this scenario,
histone H2A is initially ubiquitinated
at double-strand DNA breaks by the E3
ubiquitin ligase RNF8. The newly-formed
H2A ubiquitin moiety generates a binding platform for RNF168, which then
propagates the ubiquitin modification to
the adjacent nucleosome. Repeated cycles
of these recognition and catalytic steps
are believed to spread the H2A-ubiquitin
mark along the chromatin. In contrast to
PRC2, where two distinct subunits of the
protein complex are involved in recognizing and setting the epigenetic mark, the
RING1B subunit of PRC1 as well as the
E3 ligase RNF168 harbor both domains
involved in setting and reading the ubiquitin mark at histone H2A. These findings support the idea that the H3K27me3
mark is not sufficient to maintain PRC1
complexes at chromatin. Our data clearly
demonstrates that the H2A ubiquitin mark
itself is important for the maintenance of
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PRC1. Therefore, ZRF1 recruitment to
ubiquitinated histone H2A is likely to
prevent propagation of PRC1 complexes,
thus depleting the levels of RING1B at the
chromatin.
Genome-Wide Functions of PRC1
and PRC2 Complexes
Genome-wide studies of PRC1 and PRC2
in humans and flies demonstrate that
these two complexes do not always go
hand in hand. In Drosophila melanogaster,
genome-wide ChIP analysis substantiates
co-occupancy of both Polycomb complexes at Polycomb-responsive DNA elements (PRE). However, the H3K27me3
mark shows a broad distribution pattern
that is not matched by the occupancy of
the PC component, which is thought to
act as a recruitment factor for PRC1.26
Additional data from flies show that the
bulk of histone H2A ubiquitination is
carried out by a distinct protein complex, termed dRAF,27,28 which is devoid
of PC proteins and thus of any potential H3K27me3 readers. dRAF consists
solely of proteins involved in H2A ubiquitination (RING1B and PSC), together
with the histone lysine demethylase
KDM2, which specifically demethylates
H3K36me2. This finding would therefore
also argue that the ubiquitin mark is sufficient for its own propagation and underscores that PRC1 recruitment certainly
cannot rely exclusively on H3K27me3.
Recent data further buttress this argument
by showing that a knockout of the PRC2
subunit EED in mice leads to a complete
loss of the H3K27me3 mark, yet leaves
the levels of H2A-ubiquitin unchanged,28
indicating that correct genome-wide
deposition of H2A ubiquitinatation does
not rely on PRC2 and the H3K27me3
mark. Therefore, on a genome-wide scale,
both complexes might work in parallel but
independently of each other. Further studies in mouse cells demonstrate that, while
the H2A-ubiquitin mark is found in gene
promoters, it is present to a much greater
extent within the gene bodies and in intergenic regions.29 In sharp contrast, the
H3K27me3 mark is usually restricted to
promoter regions. From this we conclude
that H3K27me3 contributes to PRC1
recruitment in promoter regions but is
not sufficient either for genome-wide
PRC1 targeting or for its maintenance.
Consequently, although PRC1 complexes
might initially be recruited to PREs or
promoters via Pc proteins (through their
recognition of the H3K27me3 mark),
propagation of PRC1 is most likely independent of this modification following
recruitment and rather depends on the
ubiquitin residue at histone H2A itself
(Fig. 1A).
ZRF1 Facilitates
Transcriptional Activation
In addition to its role in displacing PRC1,
ZRF1 carries out a second function once
it is bound to ubiquitinated chromatin.
Genome-wide expression analysis illustrates that ZRF1 plays a role in the transcriptional activation of Polycomb target
genes. But how does chromatin-bound
ZRF1 transmit a signal that activates
transcription? It has been recently shown
that, in mouse ES cells, the removal of
RING1B leads to a decrease in histone
H2A ubiquitination levels as well as to
the activation of several genes.30 However,
these two events have to be kinetically
separated. In a first step, ZRF1 mediates displacement of PRC1 from genomic
regions that still retain H2A-ubiquitin,
which is required for ZRF1 binding
(Fig. 1B). The target genes would most
likely still be repressed, since the ubiquitin mark prevents Polymerase II-mediated
transcription.31 Therefore, the ubiquitin
mark needs to be removed by a specific
deubiquitinating enzyme to allow promoter activation. Several H2A-specific
deubiquitinases have been identified and
investigated with respect to their function
in either cell cycle or gene activation.2,32,33
It has recently been demonstrated that the
deubiquitinase USP21 has an impact on
the activation of transcription from ubiquitinated chromatin. In vitro transcription experiments utilizing nucleosomes
that contained mono-ubiquitinated histone H2A showed that transcription only
occurred after the complete removal of the
ubiquitin residue by USP21.33 We therefore investigated the role of ZRF1 using
the same experimental set-up. Our results
suggested that ZRF1 greatly enhanced the
deubiquitination reaction catalyzed by
USP21. It is still unclear how ZRF1 can
exert such a function at a molecular basis.
One possible mode of action is that ZRF1
enhances recruitment of USP21 and/or
stabilizes it at chromatin. Alternatively,
ZRF1 might induce a chromatin reorganization that facilitates the docking of
deubiquitinases. This would be in agreement with ZRF’s function in protein fidelity control, where it acts as a chaperone
together with proteins of the heat shock
family.17 In addition to its role in transcription initiation, H2A-ubiquitin might
also be important during transcription
elongation. As discussed above, the H2Aubiquitin mark spreads into the body of
genes and interferes with transcribing
Polymerase II. Recent papers have demonstrated that at so-called bivalent genes,
which are decorated with both H3K4me3
and H3K27me3 marks, Polymerase II is
poised after transcription initiation.31,34 It
seems that removal of the ubiquitin mark
at histone H2A is required not only for the
resumption of transcription but also for
the recruitment of the FACT (Facilitates
Chromatin Transcription) complex,34
which was originally purified as an elongation factor. The mammalian FACT complex, consisting of the two subunits Spt16
and SSRP1, was demonstrated to interact
with (not ubiquitinated) nucleosomes and
to thereby affect their structure.35,36 This
function seems to be important during the
transcription elongation, but there is also
evidence that FACT plays a role during
the transcription initiation,37 which would
suggest that FACT acts as a nucleosome
chaperone during chromatin reorganization. A simplified gene activation pathway of Polycomb-target genes therefore
could encompass removal of PRC1 complex, deubiquitination and FACT complex recruitment, leading to subsequent
Polymerase II-mediated transcription.
It is therefore possible that ZRF1 also
plays a crucial role during transcription
elongation.
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Cell Cycle
Regulation of ZRF1 Function
in Cell Cycle and Differentiation
The H2A-ubiquitin mark is highly
dynamic during the cell cycle, as it is
erased during M phase and reappears
again before S phase. An additional
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Figure 1. (A) Hypothetical model for polycomb action at chromatin. In promoter regions (purple nucleosomes), PRC2 carries out the specific methylation of H3K27 (red circles), which is believed to support the recruitment of PRC1 complexes. After ubiquitination of histone H2A (yellow circles) at
promoter regions, PRC1 propagates into the gene body (light nucleosomes) to carry out ubiquitination in regions devoid of PRC2 and H3K27me3,
respectively. (B) Function of ZRF1 at chromatin (left to right). ZRF1 displaces PRC1 complexes by interacting with mono-ubiquitinated chromatin. After
PRC1 removal, ZRF1 acts in concert with specific deubiquitinases (USP21) to facilitate deubiquitination. The enzyme might then either propagate to an
adjacent nucleosome bound by ZRF1, or propagate together with ZRF1, which could confer multi-substrate binding since it is an oligomer.
function for ZRF1 might be to help in
the pathway of H2A-ubiquitin removal,
which is catalyzed by the specific deubiquitinase USP16. It has recently been demonstrated that deubiquitination by USP16
is a prerequisite of serine 10 phosphorylation at histone H3 (H3S10), which leads
to compaction of the chromatin during
M phase.32 Therefore, impairing USP16
function has severe effects on cell cycle
progression with a slow growth phenotype. One could speculate that ZRF1’s
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function during M phase is similar to
that during differentiation. Whereas the
H3K27me3 mark and PRC2 are maintained at chromatin during the cell cycle,
both the H2A-ubiquitin mark and PRC1
complexes are removed during M phase.38
ZRF1 might therefore be important for
the displacement of PRC1 complexes and
the subsequent facilitation of deubiquitination during M phase. To perform such a
function, it should be recruited specifically
to the newly-emerging chromosomes.
Indeed, ZRF1 is phosphorylated during
M phase,39 hence its synonym M-phase
phosphoprotein 11 (MPP11), and it was
previously suggested that it could have a
cell cycle-specific function. Interestingly,
ZRF1 also is phosphorylated during RA
stimulation in P19 cells.40 Therefore,
phosphorylation might be involved in relocalizing or activating ZRF1 and could
be a main determinant of ZRF1 recruitment to chromatin. Future research
should be directed towards understanding
Cell CycleVolume 10 Issue 5
the consequences of ZRF1 phosphorylation during cell cycle and differentiation
as well as dissecting the molecular mechanisms of ZRF1 during the M phase of the
cell cycle.
In addition to phosphorylation, another
possible way to regulate ZRF1 is through
its sequestration into other protein complexes. Although the proteins involved
in ZRF1 recruitment to chromatin are
still unknown, it was previously shown
that the ZRF1 mouse homolog, termed
mouse Id-associated 1 (MIDA1), binds
to proteins of the Inhibitor of DNA binding (Id) family.19 It was illustrated that
the helix-loop-helix (HLH) motif of Id1
recognizes an amino acid stretch within
the well-conserved Zuotin domain. This
interaction appears conserved, as we demonstrated in human cell lines that ZRF1
associates with Id1 in the same manner.
Interestingly, the mapped Id-binding site
in MIDA1 overlaps with the ubiquitinbinding domain mapped for ZRF1, which
is located in the C-terminal part of the
Zuotin domain; thus, one potential mode
of inhibiting differentiation could be to
block ZRF1 recruitment to chromatin by
competitive binding to Id proteins. While
the Id family of proteins are known to
block differentiation in various ways, their
general mechanism of action is to bind
to various basic helix-loop-helix (bHLH)
transcription factors and thereby prevent them from binding to chromatin.41
However, it was recently shown that Id
proteins in fact affect the global levels of
histone H2A ubiquitination,42 supporting
a model that they work through a ZRF1dependent mechanism. Id proteins could
keep levels of freely-diffusing ZRF1 low
and thereby inhibit its recruitment to
chromatin; this would confer a second
level of regulation in addition to its phosphorylation. In addition to blocking its
H2A-ubiquitin binding site, binding to
Id proteins might localize ZRF1 predominantly to the cytoplasm, thereby supporting its function in protein fidelity control.
A release of ZRF1 in turn might be finetuned with differentiation stimuli, that
could for example reduce Id protein levels
and (through a yet undetermined kinase)
phosphorylate ZRF1.
We have recently described the molecular function of ZRF1, adding a new
player to the highly complex process of
cellular differentiation. Given the importance of the H2A-ubiqutin mark in various cellular processes, it will be interesting
to elucidate whether ZRF1 carries out a
role in such diverse processes as cell cycle
progression and elongation of transcription. Most importantly, H2A-ubiquitin
is an ambiguous epigenetic mark that is
important not only for silencing but also
for the transcriptional activation of genes
by ZRF1. Future research will need to
address how ZRF1 itself is regulated and
whether the re-localization of ZRF1 is regulated by specific protein interactions and/
or its phosphorylation. Understanding the
molecular mechanism of ZRF1 could also
have the additional benefit of giving rise
to developing anti-cancer drugs that could
have a therapeutical use.
Acknowledgements
I would like to thank V.A. Raker for critical
reading of the manuscript and suggestions.
This work was supported by the Spanish
‘‘Ministerio de Educación y Ciencia’’
(BFU2010-18692), the Association for
International Cancer Research (10-0177),
by the AGAUR and Consolider to L.D.C.;
H.R. was supported by a FEBS fellowship.
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Cell CycleVolume 10 Issue 5