Polycomb repressive complex’s evolutionary conserved function: the role of EZH2 status and cellular background
Post-translational modifications (PTMs) of histone polypeptides contribute to the
regulation of gene activity through establishing a specific epigenetic regulatory
network 1]. Partly due to PTMs of histones, polycomb group (PcG) proteins can control gene silencing
in a considerable part of the genome but only when assembled in multiprotein polycomb
repressive complexes (PRCs)—polycomb (Pc)-containing complexes (PRC1) and the enhancer
of zeste-containing complexes (PRC2/PRC3/PRC4) 2], 3]. These complexes are responsible for the epigenetic memory of gene expression states
and play a crucial role in the maintenance and reprogramming of cell types during
normal development and during pathophysiological processes (reviewed in 4]).
Enhancer of zeste-containing complexes during evolution
Originally identified in the fruit fly Drosophila melanogaster as crucial factors in maintaining the repressed state of developmental regulators
such as homebox HOX genes 5], the Pc-group proteins were shown to be highly evolutionary conserved 6]. For example, PRC2 is detected even in unicellular eukaryotes, alga Chlamydomonas7] and yeast Cryptococcus neoformans8].
The widespread presence of PRC2, from unicellular organisms to humans, points out
its significance for preserving a specific module(s) of gene repression. Evolutionary
processes have offered unique ways of PRC2 composing (Table 1): (1) Drosophila contains four core proteins: enhancer of zeste E(Z); suppressor of zeste 12 SU(Z)12;
extra sex combs (ESC) and the histone binding protein p55. The E(Z) protein contains
a SET domain which exerts histone lysine methyltransferase activity (KMT), able to
catalytically add up to three methyl groups at the target lysine residue K27 of histone
3 (H3). The E(Z) possesses the SANT domains involved in histone binding and a C5 domain
required for interacting with SU(Z)12 9]; (2) Yeast Cryptococcus neoformans PRC2 has no homolog of SU(Z)12 but contains two additional proteins, Bnd1 and Cc1,
specific for this species 8]; (3) In nematode Caenorhabditis elegans, only homologs of E(Z) and ESC are found, MES-2 and MES6, respectively. These two
proteins make a PRC2 together with a MES-3 protein which has no homolog in any other
model organism, and such complex is involved in X-chromosome repression 10]; (4) Plants such as Arabidopsis thaliana, due to gene duplications, have three homologs of E(Z): CLF, MEA, SWN; three homologs
of SU(Z)12: FIS, VRN2, EMF2; and five homologs of p55: MSI1-5, while only one homolog
of ESC is present (reviewed in 11]). The combinations of these proteins make at least three distinct PRC2 which are
involved in different developmental processes. FIS-PRC2 is similar to its mammalian
counterpart and regulates expression of imprinted genes and cell proliferation. EMP-PRC2
acts like Drosophila and mammalian PRC2 in maintaining the repressed state of homeotic genes and, together
with the third complex, VNR-PRC2, regulates flower time 11] .
The duplication of E(Z) gene resulted in two mammalian E(Z) proteins, EZH1 and EZH2
(Table 1), as well as two PRC2 complexes, each containing one of these two EZH-proteins. Accordingly,
mammalian PRC2 is composed of four core subunits: EZH1/EZH2, SUZ12, embryonic ectoderm
development (EED), and retinoblastoma(Rb)-associated protein 46/48 (RbAp46/48).
Although present in similar PRC2 complexes and controlling an overlapping set of genes,
EZH1 and EZH2 are considerably different. PRC2-EZH2, abundant in highly proliferative
cells, establishes a repressive H3K27me3 mark on PRC2 target genes. PRC2-EZH1, which
is abundant in non-dividing cells, likely restores this repressive mark, either as
a result of its disappearance due to demethylation or by histone exchange 12].
PRC composition is flexible and cell-type specific
H3K27 is not the only histone-related substrate for EZH2, as the PRC-partners may
direct the EZH2 to other substrates. For example, an EED isoform 2 (Eed2) and NAD-dependent
histone deacetylase Sirt1 specifically associate within the PRC4 which is needed for
methylating linker histone H1 (H1K26) 13]. This modification is specific for cancer and undifferentiated embryonic stem (ES)
cells.
There is a whole spectrum of variations relating to the dynamic exchange of protein
partners (AEBP2, Pcl1/2/3 (PHF1/MTF2/Pcl3t), Jarid2) which may be temporary members
of PRC2. This “exchange phenomenon” should not be surprising, as the specific biological
effect mediated by PRCs—broad control of gene activity must be achieved very precisely,
in a cell-type specific manner and during a controlled time-window (Fig. 1) 14]. For example, Jarid2—a member of Jumonji family of histone demethylases without enzymatic
activity—was identified as a part of PRC2, in interaction with Ezh2. Jarid2 binds
DNA with a slight preference for GC rich sequences 15] and recruits PcG proteins to target genes 16].
Fig. 1. Association of PRC-EZH2 complexes with different EED isoforms in the presence (H1+)
or absence (H1?) of linker histone H1 directs EZH2-mediated methylation towards H3K27
or H1K26. PRC2, which contains the longest form of EED (EED1), is able to methylate
isolated histone H3. When targeted to oligonucleosomes containing linker histone H1,
PRC2 methylates histone H1 rather than histone H3. PRC3, containing EED3 and EED4,
methylates nucleosomal histone H3, but its methyltransferase activity is inhibited
by histone H1. PRC4, containing EED2 and NAD-dependent deacetylase SIRT1, methylates
histone H1 when present, but has also low methylating capacity towards H3K27 in the
absence of histone H1 (depicted in gray) 13], 14]
Although there are several possibilities related to flexible ways of composing the
content of PRC2 (as discussed), it is known that the minimum components required for
methyltransferase activity of the PRC2/EED-EZH2 complex are EED, EZH2, and SUZ12.
The coordinated activity of these proteins is essential for establishing di- and tri-methylated
H3K27 (H3K27me2/me3) marks which are associated with facultative heterochromatin.
These marks present the hallmark histone modification produced by Ezh1 and Ezh2 activity
within the PRC2. However, the precise mechanism that governs PRC2 recruitment to chromatin
in mammals still needs to be defined.
Recognizing PRC2 functioning as a holoenzyme whose components act together to establish
interaction with chromatin in a stepwise manner, Margueron and Reinberg 17] have proposed the following several-steps model: (a) interaction of Jarid2 and AEBP2
with DNA 18], 19]; (b) interaction of RbAp46/48 with histones H3 and H4 20]; (c) interaction of Eed with H3K27me3 21]; (d) interaction of Plcs with an unknown histone mark; and (e) interaction of PRC2
subunits with long non- coding RNA (lnc RNA).
These molecular events are highly conserved. In mammals (reviewed in 22]) are well documented through the interaction of lnc RNA X inactive-specific transcript
(Xist) with EZH2 and the consequential recruitment of PRC2 to the X-chromosome leading
to its inactivation. In plants, cold induced lnc RNA COLDAIR interacts with plant
E(Z) homolog CLF 23] and recruits PRC2 to the target locus in a way similar to the Xist in mammals. In
malignant neoplasms, as shown in gastric cancer, overexpressed lnc RNA 00152 needs
to bind to EZH2 in order to exert oncogenic potential through recruiting the PRC2
to promoters of tumor suppressor (TS) genes p15 and p21 24].
Learning about the evolutional significance of PRC2 in the control of cellular proliferation
and differentiation is very important for understanding some basic pathophysiological
processes. For example, plants with double mutation of two out of three E(Z) homologs,
clf and swn, undergo normal seed development, but produce a mass of proliferating,
undifferentiated tissue resembling cancer, instead of a differentiated shoot after
germination 25].
Several aspects of aberrant EZH2 function in cancer
In humans, the EZH2 mutation may occur in a germline, resulting in clinical features
known as the Weaver syndrome, originally described in 1974 26]. In 2011, mutational analysis of EZH2 in 48 Weaver syndrome patients revealed 44
missense and four truncated mutations. All but two SET domain mutations (R684C and
S652C), which were present in five and two unrelated individuals, respectively, were
distributed throughout the gene, without specific clustering 27]. Only two germline EZH2 mutation-positive individuals developed hematological malignancies:
E745K (a lymphoma diagnosed at the age of 13) and an A682T mutation (acute lymphoblastic
leukemia (ALL) and neuroblastoma developed at 13 months).
In 1996, EZH2 was first discovered as a binding partner of Vav oncoprotein in hematological
malignancies 28]. These neoplasms were, in addition to breast and prostate cancer, pioneering models
for investigating the function and role of EZH2. Its overexpression was first associated
with amplification at 7q35 (more than four EZH2 copies per cell) in approximately 15 % of the 225 analyzed breast cancers (BCs)
29]. In 2010, EZH2 point mutation (Y641) in SET domain was first found in 7 % of large
follicular lymphomas and 22 % of diffuse B cell lymphomas 30]. It was also found in approximately 3 % of melanomas 31]. The discovery of two additional SET domain mutations (A677G and A687V) followed
32], 33].
These “gain of (methyltransferase) function” mutations are responsible for the oncogenic
mode of EZH2 action. Contrary to wild-type (WT) EZH2, which loses activity when progressively
more methyl groups are incorporated into H3K27, all tested Y641 mutant enzymes (Y641F/N/S/H/C)
displayed the opposite trend (H3K27me0:me1:me2 kcat/Km ratio: 13:4:1 (WT) vs 1:2:22
(Y641) 34]. Since one cell possesses both wild- and mutant types of the EZH2 allele, there appears
to be dependency on the coordinated activity of both alleles.
Aberrant activity of PRC2 can result from aberrant EZH2 expression, without chromosomal
amplification, as a consequence of diverse aberrations which are present in cancer
cells. For example, comprehensive analyses of transcriptome and epigenome data obtained
from adult T cell leukemia (ATL) cell lines, normal CD4
+
T cells, human T-lymphotropic virus type 1 (HTLV-1)-immortalized and transformed T
cells show the importance of increased, NF-?B dependent expression of EZH2 (both RelA
and RelB were shown to be bound to EZH2 promoter) which further activates NF-?B through
silencing of microRNA (miR)-31. Of interest for this model, H3K27me3 was enriched
in the promoter of transcriptionally downregulated H3K27me3 demethylase KDM6B (JMJD3),
which also may compromise the balance between epigenetic “writers” and “erasers.”
It was shown that HTLV-1 protein Tax binds to EZH2, without affecting the PRC2 composition.
As a result, the pattern of H3K27me3 accumulation significantly overlaps in ATL- and
HTLV-1-immortalized cells. Since HTLV-1 infected cells are sensitive to EZH2 inhibition,
this research data may be a ground for introducing EZH2 inhibitors for treating asymptomatic,
HTLV-1 infected individuals 35].
Hepatitis B virus (HBV)-associated hepatocellular cancer (HCC) represents another
interesting model for studying the abberant expression of tumor suppresive miRs in
respect to PRC2 activity in a setting of prolonged viral infection. In the HBV-HCC
model, co-expression of transcription factor (TF) YY1 and EZH2 are associated with
silencing several, multiple YY1 binding sites-containing suppressive miRs and relate
to short disease-free survival 36]. YY1 can interact with both EZH2 and SUZ12 37] and recruits the PRC2 complex to chromatin. The discovery of this oncogenic mechanism,
which was responsible for silencing of five highly NF-?B suppresive miRs, pointed
out the importance of coordinated action of YY1 and EZH2 for focal reshaping of chromatin.
The already mentioned tumor suppressor miR-31 was shown to be silenced in prostate
cancer cells through presence of H3K27me3 on its promoter 38]. The absence of miR-31 in t(4;14) positive multiple myeloma (MM) patients (15–20 %)
allows for pro-oncogenic activity of its target—multiple myeloma set domain methyltransferase
(MMSET), which establishes histone mark H3K36me2 and induces a global reduction H3K27me3
39]. However, in this scenario, specific loci exhibit enhanced recruitment of EZH2, leading
to misregulation of specific polycomb target genes.
It was recently shown that H3K27me3 enriched genes in experimental models of MM significantly
overlap with underexpressed genes in MM patients with poor survival 40]. Of interest, although applying EZH2 inhibitor, E7438 induces reproducible re-expression
of crucial epithelial tumor suppressor genes (including CDH1) in 13 tested MM cell lines, there are many questions arising from a high variability
in E7438 sensitivity in the proliferation assays 41].
All these examples show that there are many factors that may influence EZH2 and are
influenced by EZH2. Accordingly, EZH2 pharmacological inhibition may have various
effects.
In addition to “gain of function” mutations, there are also EZH2 “loss of function”
mutations discovered in hematological malignancies originating from myeloid cells,
commonly joined with unipaternal disomy (UPD) 42]. The proposed model of EZH2 “loss of function” mutations (of which the majority were
found in the SET domain) attributes their contribution to be forming cancer stem cells,
via HOXA9 mediated self-renewal of myeloid progenitors. A complex in vivo model (transplantation
of bone marrow (BM) cells from 8–12-week-old Cre-ERT;Ezh2fl/fl CD45.2 mice into lethally
irradiated CD45.1 recipient mice and deletion of Ezh2 at 6 to 8 weeks posttransplantation)
reveals that complete lack of EZH2 activity in hematopoietic stem cells (HSCs) predisposed
mice to heterogenous malignancies (MDS, MDS/MPN, MDS/MPN associated with trombocytosis,
and T cell acute lymphoblastic leukemia). The same experimental model showed locus-specific
repositioning of EZH1 to EZH2 targets (3605 genes in contrast to 969 “EZH2 targets
only”) and its ability to re-repress them during prolonged period of time (9 months)
43]. All these data clearly indicate that EZH2 function, in both physiological process
and in various pathogenic events, must be studied in a broad context, keeping in mind
that its binding partners contribute to specificity of its functioning, in a particular
cellular setting.
Which mutation is “the right one”?
The problem that occurs when comparing the results of EZH2 mutational analyses coming
from different sources relates to amino acids positioning in the EZH2 sequence. For
example, “gain of function” mutations are listed according to the protein sequence
that is considered “canonical” (UniProtBD/Swiss-Prot Q 15910–1; 746 amino acids (AA))
30], 32], 33]. On the other hand, “loss of function” 34], and germline mutations 27], were positioned according to the longest protein isoform of EZH2 (UniProtBD/Swiss-
Prot Q 15910-2; 751 AA). The absence of uniformity may be confusing. For example,
the already mentioned inherited mutation discovered in the Weaver syndrome patient
suffering from ALL (A682T) 27] corresponds to alanine 677 mutation (A677G) in B cell lymphoma 30]. Similarly, a rare EZH2 breast cancer mutation described as A692V 44] corresponds to B cell lymphoma mutation at position 687 33]. The difference of five amino acids corresponds to the difference between Q15910-1
and Q15910-2 isoforms (HP???HRKCNYS), which are identical in the first 297 amino acids
(Fig. 2). The basic data on currently known EZH2 protein isoforms and their coding messenger
RNAs (mRNAs) are presented in Table 2. The hope is that future presentations of EZH2 isoforms and the positions of mutated
codons will be done in a more uniform manner.
Fig. 2. Alignment of five EZH2 isoforms protein sequences (UniProt). SET domain is shown in
green (Q15910-1 AA 612–727; Q15910-2 AA 617–732; Q15910-3 AA 573–688; Q15910-4 AA 603–718;
Q15910-5 AA 561–676). Germline mutations 27] are shown in orange, “loss of function” mutations 42] in blue, and “gain of function” mutations 26], 30], 32], 33], 44] in red. All mutations listed in the cited references are marked on respective isoform sequences,
highlighting the lack of uniformity in annotating mutations according to consensus
sequence (Q15910-1). Therefore, mutation A677 (in isoform 1) is listed as somatic,
activating mutation and at the same time, annotated as mutation A682 (in isoform 2),
has been listed as germline mutation which was discovered in the Weaver syndrome patient
who developed ALL and neuroblastoma in early childhood. This is in accord with the
oncogenic potential of this mutation. Inactivating mutations R684 in isoform 2 (corresponding
to R679 in isoform 1) and E745 in isoform 2 (corresponding to E740 in isoform 1) have
been shown to be mutated in Weaver syndrome patients. None of the five patients with
inherited mutation R684C (present as somatic mutation in one 82-year-old patient suffering
from chronic myelomonocytic leukemia) developed malignant disease at the time of testing
for germline mutation of EZH2. Germline mutation E745K (isoform 2) was present in
a patient who developed non-Hodgkins lymphoma at the age of 13. Somatic mutation of
this codon was detected in one patient with chronic myeloic leukemia during blast
crisis.
Table 2. Human EZH2: five proteins and corresponding mRNA splice variants are currently deposited.
Although mutational analyses of EZH2 refer to the ordinal number of mutated amino
acids, they rarely identify the isoform which is the basis for numbering them
Stratified presentation of mutations published in previous studies 27], 30]–34], 44] reveals identical type/position of three germline (G) and three somatic (S) mutations.
One mutation was reported as oncogenic (lymphoma; SG:A677T), and two other ones were
reported as suppressive (S:E741fs???G:E741K; S:R679C/P???G:R679C/H) (Fig. 2; UniProtBD/Swiss- Prot Q 15910-1). It remains to be seen whether any of these EZH2
mutations act as a “Janus” mutation in the RET protooncogene (germline mutation that
acts simultaneously as both a gain-of-function and a loss-of-function mutation) 45].
Data related to the functional differences of EZH2 splice variants is scarce. The
expression level of EZH2 transcript variants 1 and 3 was shown to be similar in 22
tested human tissue samples. Forced expression of corresponding protein isoforms (Q
15910-2 and Q 15910-3; Table 2) in pancreatic cancer cells revealed that each protein isoform has an affinity for
a preferential gene cluster (36.3 and 47.6 % genes were repressed by EZH2? (Q 15910-3)
and EZH2? (Q 15910-2), respectively, while repression of remaining 16.1 % genes needed
the presence of both isoforms) 46]. The data indicates that the different EZH2 cell-specific mRNAs, and protein isoforms
may have functional importance, including for the clinic, as already shown for some
other genes 47], 48].
Specific cellular background and multiple roles of EZH2
EZH2 binding affinity for both histones and non-histone substrates may partially explain
why targeted silencing of EZH2 leads to bidirectional change of gene expression, in
a specific cellular context-dependent manner 49]. Some examples are: (1) EZH2 binds to RelA/RelB in BC cells and regulates the NF-?B
target genes in a positive (IL-6, TNF) or negative way, depending on estrogen receptor (ER) status rather than the EZH2
histone methyltransferase activity. In ER+ BC cells, ER recruits PRC2 for enforcing
a repressive chromatin modification at NF-?B target genes. (2) In squamous cell carcinomas
(SCC), EZH2, through repressing I?B kinase ? (IKK1) promoter, leads to IKK1 silencing 50]. In any other types of tumor, this would be a suppressive effect. However, it is
oncogenic in SCCs because I?B kinase ? has a tumor suppressive role in these tumors
51]. 3. Finally, it seems that EZH2 catalytical activity does not have the most significant
role for an increased rate of growth in some SWI/SNF-mutant cancers 52]. Instead, the stabilization of PRC2, dependent on EZH2 threonine 487 (T487) phosphorylation
(Prot Q 15910-1), seems to be essential, at least in this particular scenario 53]. However, this phenomenon may be abrogated by presence of mutant K-ras.
In non-small cell lung cancer (NSCLC), the type of substitution at 12th codon of K-ras
determines activation of a specific pro-proliferative signaling pathway. Cells with
K-RAS
G12D/+
or K-RAS
G12C/+
have primarily activated PI3/AKT and MEK/ERK signaling pathways, respectively 54]. Accordingly, activation of EZH2, which was shown to be dependent on K-ras mutants,
may be inhibited by specific inhibitors of mutation-type dependent downstream signals.
This is important because one of significant pro-oncogenic activities of EZH2 depends
on activated AKT which, through EZH2, phosphorylates and activates oncogenic STAT3
55] .
A generation of mice with Cre-recombinase-activated conditional oncogenic K-ras allele
(K-ras
G12D/+
), along with either mild Ezh2 overexpression (Ezh2
LSL
) or lost PRC2 function achieved by conditional deletion of Eed1 (Eed
fl/fl
), joined with conditional deletion of p53 (Trp53
fl/fl
), revealed that the genotype K-ras
G12D/+
; Trp53
fl/fl
; and Eed
fl/fl
develops the most aggressive, mucinous NSCLC. In this genetic setting, which is relevant
for human pathology (mutations of K-RAS and P53 are present in 35 and 40 % NSCLCs,
respectively), Eed1 acts as a tumor suppressor gene. In the presence of WTp53, Kras
G12D/+
;Eed
fl/fl
mice developed NSCLs which were, although smaller than Kras
G12D/+
/Ezh2
LSL
tumors, characterized by life incompatible inflammation in alvelolar spaces. In vitro,
the inhibition of EZH2, achieved through the prolonged exposure of human K-RAS-mutant
NSCLC cells to an inhibitor of EZH2 catalytic activity (GSK126), resulted in a strong
increase of inflammatory genes (i.e., IL-6) associated with microenvironment-regulated tumor progression. Based on these and
many more results coming from the cited study 56], it was suggested that PRC2 can hold opposing functions, depending on the stage of
tumor development and the genetic make-up of the tumors (as presented here), with
respect to p53 status. Accordingly, this and other studies clearly show a rationale
for the combined application of PRC2 inhibitors and anti-inflammatory drugs. In the
model of hematopoietic stem cells, EZH2 loss was recently shown to result in the expression
of fetal gene signature, including upregulation of fetal-specific Lin28b which encodes
RNA-binding protein that prevents maturation of miR-let-7 which is specific for adult
HSCs. Activation of fetal gene signature in EZH2-deficient adult bone marrow HSCs
was shown to result in fetal-like high self-renewal capacity and increased propensity
to undergo malignant transformation 57]. Enforced expression of Lin28b has been reported to impair T cell development in
vivo, leading to developing an aggressive peripheral T cell lymphoma, accompanied
by a decrease in let-7 expression, surge of IL-6, activation of NF-?B, and infiltration
of B cells leading to an inflammatory microenvironment 58].
The proper anti-tumor function of T cells depends on the differentiation of naive
and memory T cells into effector cells. Metabolic switch from oxidative phosphorylation
to aerobic glycolysis is mandatory for T cell activation. Highly glycolytic ovarian
cancer cells were recently shown to impose glucose restriction on tumor-infiltrating
T cells, thereby inhibiting this metabolic switch. Low glucose availability results
in upregulation of EZH2-targeting miR-26a and miR-101 with subsequent EZH2 downregulation.
This is consequential for T cell effector function, since EZH2 activates the Notch
pathway that stimulates T cell polyfunctional cytokine expression and their survival,
which was shown to be impaired in many tumors. These results point to the different
effects that systemic inhibition of EZH2 may have on tumor cells and T cells, warranting
special caution when considering such epigenetic intervention 59].
H3K27me3 as a measure of EZH2 activity
There are many EZH2-related scenarios and none of them is simple. When analyzed in
five well-defined subtypes of BC, the highest EZH2 expression, joined with a very
low level of H3K27me3, was found in basal-like, triple negative BC 60], known for its distinctly aggressive nature 61]. This inverted pattern (EZH2?, H3K27me3?), further confirmed in a basal-like BC cell
lines, represents the negative prognostic marker in BC patients 60], 62]. There are a few studies in which a decreased level of H3K27me3 was associated with
a poor outcome in different malignant tumors (breast, ovary, pancreas, lung) 63], 64]. These results, together with those showing that solid tumors (prostate, breast)
can develop even in the absence of Ezh2 44], challenge the strength of EZH2 as the epigenetic driver of oncogenesis 65], at least in the stated tumor types. This data, supported by a broad analysis of
human transcriptome data sets (131 prostate cancers (plus 19 metastases), 146 BCs)
indicates that EZH2 expression “follows” the rate of cellular division, is under control
of proliferation cues, and “passively” correlates with proliferation and proliferation
markers (primarily Ki-67), in order to maintain the cellular level of H3K27me3.
It was suggested that EZH2 overexpression should be considered from two perspectives:
(a) through coupling its expression to proliferation and (b) coupling it to proliferation-independent,
amplification-related, copy number-driven, expression 44].
However, this approach should be considered in a specific cellular milieu and should
not be applied non-selectively, to all types of malignant tumors:
In many systems, EZH2 supports stem cell maintenance by repressing differentiation.
But, in neural crest stem cells (NCSCs), which are the source of melanocytes, it specifically
promotes the acquisition of a mesenchymal fate 66]. EZH2 is essential for melanoma initiation and growth, during which EZH2 and Ki-67
positive cells significantly correlate, just like in the BC model. Increased expression
of EZH2 in melanoma strongly correlates with shorter overall survival (OS) and earlier
development of distant metastases 67], 68]. EZH2-mediated repression of the tumor suppressor adenosylmethionine decarboxylase
1 (AMD1) appears to be of the greatest importance for these processes. The role of
this gene, as well as its repressor, EZH2, needs to be further investigated and validated.