20160524123048653

JournalofCellScience•Advancearticle
© 2016. Published by The Company of Biologists Ltd.
EZH1/SUZ12 complex positively regulates the transcription of NF-κB target genes via
interaction with UXT
Shuai-Kun Su, Chun-Yuan Li, Pin-Ji Lei, Xiang Wang, Quan-Yi Zhao, Yang Cai, Zhen Wang,
Lianyun Li, Min Wu
Affiliation:
College of Life Sciences, Hubei Provincial Key Laboratory of Developmentally Originated
Disease, Wuhan University, Wuhan, Hubei, 430072 China
Correspondence:
Min Wu, wumin@whu.edu.cn;
Lianyun Li, lilianyun@whu.edu.cn.
Key words:
EZH1, UXT, histone methylation, transcription regulation, NF-κB signaling pathway
JCS Advance Online Article. Posted on 28 April 2016

Summary Statement
EZH1 is a member of mammalian polycomb group proteins, which are usually considered to
repress transcription. In this study, EZH1 was found to be associated with transcription co-
activator UXT and involved in the activation of NF-κB target genes.

Abstract
Unlike other members of polycomb group genes, EZH1 has been shown to positively
associate with active transcription in the genome-wide scale. However, the underlying
mechanisms still remain elusive. Here we report that EZH1 physically interacts with UXT,
one small chaperon-like transcription co-activator. UXT specifically interacts with EZH1 and
SUZ12, but not EED. Similar to UXT, RNA interference of EZH1 or SUZ12 in the cell
impairs the transcriptional activation of NF-κB target genes induced by TNFα. EZH1
deficiency also increases TNFα-induced cell death. Interestingly, chromatin
immunoprecipitation and the following next generation sequencing analysis show that H3K27
mono-, di- and trimethylation on NF-κB target genes are not affected in EZH1 or UXT
deficient cells. EZH1 does not affect RELA/p65 translocation from cytosol to nuclear either.
Instead, EZH1 and SUZ12 regulate the recruitment of RELA/p65 and RNA Pol II to target
genes. Taken together, our study found that EZH1 and SUZ12 as positive regulators for NF-
κB signaling and demonstrated the underlying mechanism that EZH1, SUZ12 and UXT work
synergistically to regulate pathway activation in the nucleus.

Author summary
The classical PRC2 complex, containing EZH2, SUZ12 and EED, regulates development and
are involved in many diseases through transcription repression. EZH2 is a histone H3K27
methyltransferase and behaves as the central enzyme of the complex. EZH1 is a paralogue of
EZH2 in mammals. Here we report a novel mechanism for EZH1 as a transcription co-
activator in NF-κB signaling pathway, which is critical for inflammation, immune disease and
cancer. A non-classical complex containing EZH1 and SUZ12, but without EED, is associated
with UXT, and regulates NF-κB recruitment to its target genes. EZH1, SUZ12 and UXT are
required for the proper activation of NF-κB target genes, but not EED; and the
methyltransferase activity of EZH1 is not required. EZH1, SUZ12 and UXT are all associated
with RNA polymerase II and regulate its loading to the transcribing genes. Thus,
EZH1/SUZ12 complex, together with UXT, seem to bridge NF-κB transcription factor with
RNA polymerase II to promote transcription.

Introduction
The epigenetic control on chromatin is critical for transcription regulation and the proper
response to extracellular signal in the cell. Polycomb Repressive Complex 1 and 2(PRC1 and
2) are the mostly studied protein complexes among all the Polycomb-group (PcG) proteins
and have long been considered as transcription repressors (Campos et al., 2014; Conway et
al., 2015; Margueron and Reinberg, 2011). PRC1 inhibits transcription by regulating mono-
ubiquitination on histone H2A, while PRC2 is responsible for the methylation on histone
H3K27 (Margueron and Reinberg, 2011).
In drosophila, three major subunits form PRC2 complex, including Enhancer of zeste (E(z)),
extra sexcombs (ESC) and suppressor of zeste 12 (SUZ12), among which E(Z) is the enzyme
for H3K27 methylation (Lanzuolo and Orlando, 2012). In mammals, two close homologues
exist for E(Z), enhancer of zeste 1 polycomb repressive complex 2 subunit (EZH1) and
enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) (Shen et al., 2008).
EZH2 mimics the function of E(Z) in drosophila and is the main methyltransferase for H3K27
in mammalian cells (Shen et al., 2008). However, controversial reports exist about EZH1,
which made it a puzzle for long time. Compared with EZH2, EZH1 has lower enzymatic
activity, suggesting it may have distinct functions (Margueron et al., 2008). Several groups
reported that EZH1 methylates histone H3K27 and compensates EZH2’s functions during its
absence (Bae et al., 2015; Hidalgo et al., 2012; Shen et al., 2008). Recently, Mousavi et al.
reported that EZH1 behaved as a positive regulator for transcription and is required for proper
recruitment of RNA polymerase II (Pol II) to target genes (Mousavi et al., 2012). Another
study demonstrated that EZH1 forms two different complexes with distinguished functions
(Xu et al., 2015). One is similar to classical PRC2 complex containing EZH1, embryonic
ectoderm development (EED) and SUZ12, and the other one only with SUZ12 (Xu et al.,
2015). The former complex inhibits transcription, while the latter one activates transcription
(Xu et al., 2015). However, the detailed mechanisms how the two EZH1 complexes regulate
transcription remain elusive.

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway is
a well-studied pathway involved in inflammation, immunity and anti-apoptosis (Iwai, 2012;
Oeckinghaus et al., 2011). It is activated upon many extracellular signals, and tumor necrosis
factor (TNFα) has been used as one of the typical molecules to activate the pathway (Hu et
al., 2014; Wang et al., 2012; Zhang et al., 2014). Although the regulations of NF-κB pathway
in the cytosol have been extensively studied, the events after NF-κB enters the nucleus are
still not clear and have emerged as critical regulatory steps for pathway activation. Multiple
histone methyltransferases are involved in pathway regulation. We found that lysine (K)-
specific methyltransferase 2A (KMT2A/MLL1), a histone H3K4 methyltransferase,
selectively regulates the activation of NF-κB target genes (Wang et al., 2012). EZH2 regulates
the activation of NF-κB pathway by distinct mechanisms in different cell lines, which showed
the complexity of epigenetic regulators (Lee et al., 2011). NF-κB molecule itself is also
regulated by protein methylation. SET domain containing (lysine methyltransferase) 7
(SET7/9) methylates NF-κB at lys314 and 315 and promotes NF-κB degradation (Yang et al.,
2009). On the other hand, SET7/9 also methylates lys37 on NF-κB and selectively activates
target genes (Ea and Baltimore, 2009). Besides protein methylation, multiple transcription co-
regulators have been found to be involved in the activation of NF-κB target genes. For
example, ubiquitously expressed prefoldin like chaperone (UXT/STAP1/ART-27), a small
chaperon-like protein, was reported to interacts with RELA/p65 and functions as a
transcription co-activator, as well as a cytoplasmic regulator for anti-viral pathway (Huang et
al., 2011; Huang et al., 2012; Sun et al., 2007). However, how UXT contributes to the
activation of NF-κB target genes is still not clear.
In this study, we discovered that UXT physically interacts with EZH1 and SUZ12, but not
with EED or EZH2. EZH1 and UXT are required for v-rel avian reticuloendotheliosis viral
oncogene homolog A (RELA/p65) recruitment to NF-κB target genes and their induced
expression. EZH1 and UXT are associated with Pol II and regulate its loading to chromatin.
Our study indicates that UXT, EZH1 and SUZ12 together help to link RELA/p65 with Pol II
and regulate the activation of NF-κB target genes.

Results
The physical interaction between EZH1 and UXT
To investigate the role of EZH1 in regulating transcription, a yeast two-hybrid screen was
performed with full length EZH1 as the bait. Multiple clones were identified encoding the
open reading frame of one gene named UXT, which has been shown to be a transcription co-
factor and interact with multiple transcription factors (Carter et al., 2014; Li et al., 2014;
McGilvray et al., 2007; Sun et al., 2007). We speculated that UXT may regulate transcription
by bridging the master transcription factors and epigenetic regulators and continued with the
following studies.
We cloned the full length cDNA of EZH1 and UXT into mammalian expression vectors, and
performed immunoprecipitation with anti-Flag or anti-HA antibodies respectively. HA-EZH1
successfully pulled down Flag-UXT or vice versa (Fig. 1A). To examine the interaction
between endogenous proteins, we generated antibodies against the two proteins and
performed immunoprecipitation. The results showed that endogenous EZH1 and UXT are
associated with each other (Fig. 1B). To further characterize if EZH1 directly interacts with
UTX, both proteins were expressed in bacteria and purified with GST or His affinity resins.
Then His-UXT were bound to Ni resins, which successfully pulled down GST-EZH1. His-
SPOP was used here as a negative control (Fig. 1C). These results indicated that EZH1 and
UXT directly interacts with each other. Since UXT is a small protein and contains only one
prefoldin_alpha domain, we just mapped the interacting domain in EZH1. A series of EZH1
truncations were generated (Fig. 1D) and co-immunoprecipitation assays indicated that the
fragment from 430 to 480 residues in EZH1, which represents the SANT domain, is critical
for the interaction (Fig. 1E-G).
UXT specifically interacts with EZH1 and SUZ12, but not EED
One recent paper demonstrated that two different EZH1 complexes exist in the cell, one with
EED and SUZ12, the other with SUZ12 only (Xu et al., 2015). To study which complex is
associated with UXT, we performed immunoprecipitation with anti-UXT antibody. The result

indicated that EZH1 and SUZ12 are associated with UXT, but not EED, suggesting UXT is
associated with EZH1-SUZ12 complex only (Fig. 2A). To further confirm it, exogenous
expressed or endogenous SUZ12 was pulled down by corresponding antibody and UXT was
found to be associated with SUZ12 (Fig. 2B & C). On the contrary, immunoprecipitation of
EED did not pull down UXT, nor did the reciprocal UXT immunoprecipitation (Fig. 2D). The
assay had been repeated more than three times from both directions to confirm that
endogenous EED and UXT are not associated with each other. These suggest UXT
specifically interacts with EZH1-SUZ12 complex, but not with EED.
The regulation of NF-κB pathway by EZH1 and SUZ12
Previously, UXT was reported to interact with p65/RELA as a co-activator to transcriptionally
regulate the expression of NF-κB target genes (Sun et al., 2007). Since EZH1 interacts with
UXT, we studied whether EZH1 also regulated NF-κB pathway. UXT was knocked down by
siRNA in HCT116 cell, and the TNFα-induced expression of NFKBIA/IκBα, CXCL8 and
TNFAIP3/A20, three typical NF-κB target genes, were significantly down regulated as
reported previously (Fig. 3A). Then, we knocked down EZH1 in HCT116 with two different
siRNAs. Similar to UXT knockdown, the induction of the above three genes was greatly
impaired, indicating EZH1 positively regulates the activation of NF-κB target genes (Fig.
3B). We also knocked down EZH1 in RKO, HEK293 and U2OS cell lines, and observed the
same results (Fig. S1A - C). To investigate whether EZH1 regulates the activation of NF-κB
target genes during anti-viral response, Sendai virus (SeV) was used to treat HCT116 and the
expression of NF-κB downstream genes was significantly impaired after EZH1 knockdown
(Fig. S1D). To further confirm EZH1’s role in NF-κB signaling, we profiled the gene
expression pattern after EZH1 knockdown with next generation sequencing. EZH1 deficiency
impaired the expression of almost all the TNFα-induced genes, similar to the effect of UXT
(Fig. 3C), while EZH1 only regulates the basal expression of a small portion of genes (Sup
tables). It is different from some of the other epigenetic regulators, such as MLL1, which
selectively regulates the activation of NF-κB target genes by modifying H3K4 methylation
(Wang et al., 2012), suggesting EZH1 may regulate NF-κB signaling through a distinctive
mechanism.

Interestingly, when analyzing the signaling pathway after UXT knockdown, the different
expressed genes (DEGs) are specifically enriched to ubiquitin mediated proteolysis, cell
cycle, p53 signaling et al., suggesting UXT may play roles in cancer-related signaling
pathways (Fig. S2).
To further explore the roles of other PRC2 subunits in NF-κB signaling, we knocked down
SUZ12 or EED and analyzed the expression of NF-κB target genes. Interestingly, SUZ12
knockdown, but not EED, impaired the activation of NFKBIA and CXCL8 (Fig. 3D). The
above results suggest that EZH1-SUZ12 together positively regulates the activation of NF-κB
target genes, probably through interaction with UXT, while EED is not involved.
UXT and EZH1 do not regulate the global H3K27 methylation
EZH2 is the major H3K27 methyltransferase in the mammalian cell and EZH1 was proposed
to compensate its function under certain circumstances (Margueron et al., 2008; Shen et al.,
2008). We investigated the impact of UXT on the global H3K27 methylation by western
blotting. UXT was knocked down by two different siRNAs and the global H3K27me1, me2
and me3 were measured with corresponding antibodies. Multiple experiments were performed
and all of them indicated that the global levels of the three methylation status were not
significantly affected (Fig. 4A). The similar results were observed with two different EZH1
siRNAs (Fig. 4B), while depletion of EZH2 successfully decreased the global H3K27me3
(Fig. S3A). This is consistent with the previous report that EZH2, but not EZH1, is the major
enzyme for H3K27 methylation (Margueron et al., 2008).
EZH1 and UXT do not regulate H3K27 methylation on NF-κB target genes
Though EZH1 and UXT do not regulate the global H3K27 methylation level, it is still
possible that they regulate the methylation on NF-κB target genes. We investigated the
H3K27 methylation status on NFKBIA and CXCL8 by ChIP assay. It was surprising that
repeating experiments supported that the levels of H3K27me1, me2 and me3 on these genes
were not significantly reduced after EZH1 or UXT knockdown, though their expression
decreased (Fig. S3B). Here GAPDH and MYOD1 were used as controls to ensure each
experiment was performed correctly (Fig. S3B). To further confirm it, we performed

H3K27me1, me2 and me3 ChIP-Seq analysis. Consistently, though EZH1 or UXT deficiency
altered H3K27 methylation on a few genes, the average levels on NF-κB target genes were
not changed significantly (Fig. 4C). Actually, the average signals of all three forms of H3K27
methylation were quite low on NF-κB target genes, in comparison with others (Fig. 4C),
which hints H3K27 methylations are maybe not critical in regulating the expression of NF-κB
target genes here.
EZH1 does not affect the translocation of RELA to nucleus
The above data demonstrated that the regulation of NF-κB signaling by UXT and EZH1 is not
dependent on H3K37 methylation. A previous study reported that UXT deficiency impairs the
translocation of RELA/p65 from cytosol to nucleus (Sun et al., 2007), we started to
investigate whether EZH1 regulates NF-κB pathway through the similar mechanism. We
firstly confirmed UXT’s role by western blotting after fractionation. After UXT knockdown,
RELA/p65 level in the nuclear modestly reduced compared with the control (Fig. 5A). Then
the effect of EZH1 knockdown was investigated. Interestingly, EZH1 knockdown did not
affect RELA/p65 level in the nuclear (Fig. 5B).
Then we examined the subcellular localization of EZH1. UXT is localized both in cytosol and
nucleus (Fig. 5C), as reported before (Sun et al., 2007). However, the results of western
blotting after fractionation and fluorescent staining both indicated that EZH1 is mostly
localized in nuclear (Fig. 5C & D). Then it is highly possible that EZH1 interacts with UXT
and regulates the expression of NF-κB target genes only in nuclear.
EZH1 regulates the recruitment of RELA/p65 to target genes
We then start to check whether EZH1 regulates the recruitment of RELA/p65 to chromatin.
Firstly, we studied whether EZH1 interacts with RELA/p65. As reported, UXT interacts with
RELA/p65 (Fig. 5E). We did not detect the interaction between EZH1 and RELA/p65 at the
endogenous level, however, by co-expressing EZH1 and RELA/p65 in the cell, we did
observe the interaction between the exogenous proteins (Fig. 5F). This suggested that the
interaction between EZH1 and RELA/p65 is quite weak and perhaps not direct. Then we

studied whether EZH1 is bound to NF-κB target genes on chromatin. Since we do not have a
ChIP grade EZH1 antibody, we generated a stable cell line of HA-tagged EZH1 in HCT116
(Fig. S4A) and performed ChIP assay with anti-HA antibody (Fig. 5G). The results
demonstrated that EZH1 binds to the promoters of NFKBIA and CXCL8 on chromatin even
without TNFα treatment, and when EZH1 was knocked down, the signal significantly
decreased (Fig. 5G).
Then RELA/p65 ChIP assay was performed in HCT116 cells. When UXT was knocked down,
the recruitment of RELA/p65 to NFKBIA and CXCL8 promoters was greatly impaired,
consistent with the previous report (Fig. 5H). When EZH1 was knocked down, the same result
was observed (Fig. 5H). The result was then confirmed in 293FRT cells (Fig. S4B), as well as
under the condition with virus treatment (Fig. S4C). These demonstrated that EZH1 regulates
the induced transcription of NF-κB target genes not through modifying histones, but
modulating the recruitment of NF-κB to target genes.
We further investigated whether RELA also regulates EZH1 binding to target genes. RELA
was knocked down in HA-EZH1 stable cell line and ChIP assay was performed with anti-HA
antibody. The data indicated that TNFα treatment increases EZH1 binding to target genes and
RELA deficiency impairs the binding (Fig. 5I). All the above data suggest that RELA and
EZH1 probably work synergistically to regulate the activation of their target genes.
EZH1 and UXT regulates Pol II recruitment to NF-κB target genes
Pol II is the solely enzyme for mRNA transcription and a previous study reported EZH1 is
associated with Pol II (Mousavi et al., 2012). To investigate whether SUZ12 and UXT are
associated with Pol II, we performed immunoprecipitation with a widely used commercial
monoclonal antibody 8WG16 in HCT116, and found that EZH1, UXT and SUZ12 were all
associated with it, but not EED (Fig. 6A & B). The recruitment of Pol II to NFKBIA and
CXCL8 also greatly reduced in the absence of EZH1, UXT or SUZ12, but not EED (Fig. 6C
& D). The ChIP data was confirmed in 293FRT cells (Fig. S4D). These are consistent with
our previous data, suggesting EZH1, SUZ12 and UXT function synergistically in regulating

NF-κB signaling, but not EED.
EZH1 deficiency increases the sensitivity to TNFα-induced apoptosis
TNFα treatment not only activates NF-κB signaling, but also apoptosis pathway. The
activation of NF-κB pathway strongly inhibits apoptosis, so the inhibition of NF-κB
activation often enhances TNFα-induced apoptosis. To further confirm the role of EZH1 in
TNFα signaling, we studied its effect on the apoptosis induced by TNFα. EZH1 or UXT
knockdown alone slightly increased the percentage of sub-G1 cell, which represents dead
cells (Fig. 7A). With TNFα treatment, EZH1 or UXT knockdown significantly increased the
percentage of apoptotic cells (Fig. 7A). To further confirm it, we used Annexin V and PI to
double label the cell and flow cytometry analysis confirmed that EZH1 and UXT deficiency
increased the cell death after TNFα treatment (Fig. 7B). NF-κB signaling inhibits apoptosis
through the activation of downstream anti-apoptosis genes. We surveyed our RNA-seq data
and confirmed by RT-PCR that TNFα activates the expression of BIRC2/cIAP1 and
BIRC3/cIAP2, two well-known anti-apoptotic genes, in the studied HCT116 cell line (Fig.
S4E). Further study showed that EZH1 and UXT both regulates the expression of
BIRC2/cIAP1 and BIRC3/cIAP2 induced by TNFα (Fig. 7C). These suggested that EZH1 and
UXT increase cells’ sensitivity to apoptosis probably through regulating the expression of
BIRC2/cIAP1 and BIRC3/cIAP2.
Discussion
The epigenetic regulation of signaling pathways has emerged to be one of the critical steps in
the cell for responding to the extra- and intracellular signals. Polycomb group proteins are key
factors in determining cell status and transcriptional programs. However, the functions and
mechanisms of EZH1 have remained controversial. In current study, we demonstrate that
EZH1 regulates the transcription of NF-κB target genes via interacting with UXT, a small
protein functioning as a transcription co-activator. EED is required for PRC2 activity
(Montgomery et al., 2005; Montgomery et al., 2007). But since UXT here is just associated
with EZH1/SUZ12, but not EED, it is reasonable that H3K27 methylation is not involved in
the transcription activation regulated by UXT and EZH1. Instead, both EZH1 and UXT are

required for the expression of NF-κB target genes, proper RELA/p65 recruitment and Pol II
loading to their target genes. Here, UXT, EZH1 and SUZ12 seem to act like a bridge to link
NF-κB and Pol II together.
The functional difference between EZH2 and EZH1 on transcription regulation is also
puzzling. Here we show that EZH1 positively regulates transcription in the EED and H3K27
methylation independent way. Our data are consistent with the recent report by Xu et al. (Xu
et al., 2015). In fact, EZH2 sometimes also positively regulates transcription independent of
its enzyme activity in the context-dependent manner (Lee et al., 2011; Xu et al., 2012). Our
study is helpful to understand the detailed mechanisms how EZH1/2 regulates gene
expression by different mechanisms.
UXT has been reported to regulate multiple signaling pathways, making it an important
regulator for inducible transcription (Carter et al., 2014; Li et al., 2014; McGilvray et al.,
2007; Sun et al., 2007). Meanwhile, EZH1 is associated with positive transcription genome
widely. So it is highly possible that UXT and EZH1 use the same mechanisms to regulate
other signaling pathways. It will be interesting to study whether EZH1 regulates other UXT-
dependent pathways.
Materials and Methods
Cell lines and antibodies
293FRT cell line were purchased from Invitrogen and HEK293 and HCT116 cell lines were
purchased from Cell Bank of Chinese Academy of Science. Cells were grown in Dulbecco’s
modified Eagle’s medium (DMEM) (4.5 g/l D-glucose)/L-glutamine/25 mM HEPES and 10%
fetal bovine serum (FBS, HyClone) with penicillin/streptomycin supplement. The stable cell
line containing HA-tagged EZH1 was constructed via lentivirus system (pHAGE, psPAX2
and pMG2.G) in HCT116 cells. The antibodies against FLAG (M2, Sigma), HA (clone
CB051, Origene), GST (Abmart, M20007), His (Abmart, M20001), SUZ12 (clone D39F6,
CST 3737), EED (Proteintech, 16818-1-AP), H3K27me1 (Millipore 07-448), H3K27me2
(ACTIVE MOTIF 39919), H3K27me3 (ABclonal A2363, Millipore 07-449), H3 (ABclonal

A2348), NFKBIA (Epitomics, 1130-1), 8WG16 (Covance) and RELA (Abcam, ab7970) were
purchased from indicated companies. The antibody against EZH1 or UXT was raised against
full-length EZH1 or UXT expressed in bacteria using the pET30-C plasmid. The siRNA
information is in Table S1.
Cell fractionation
Cells were harvested and spun down in cold PBS. 10 volumes of buffer A (10 mM Tris-HCl
Ph 7.4, 5 mM MgCl2, 10 mM NaCl, 1 mM DTT, proteinase inhibitors) was added to the cells,
which were then incubated on ice for 10 minutes. The cells were added with 0.5 volumes of
buffer B (10 mM Tris-HCl pH 7.4, 5 mM MgCl2, 10 mM NaCl, 1 mM DTT, proteinase
inhibitors, 10％ NP-40) and incubated on ice for 1 minutes. The cell suspension was shaked
on vortex for 5 seconds and centrifuged at 2,500 rpm for 5 minutes at 4℃. The supernatant
was collected as cytoplasm fraction. The above steps was repeated once more and the
supernatant was discarded. The sediment was suspended by 10 volumes of PBS as nuclear
fraction. SDS loading buffer was added to the cell fractions for western blotting.
Immunofluorescent staining
Cells were cultured on the cover slips and fixed with freezing methanol after wash twice in
PBS. The cover slips were then washed three times by PBS and blocked in PBS with 1% BSA
for 10min. The cover slips were hybridized with first and second antibodies for one hour,
respectively. Then the slips were mounted with prolong anti-fade kit (Invitrogen) and
observed with fluorescent microscopy.
Immunoprecipitation
The cells were harvested and lysed in NP40 Lysis buffer (50mM Tris, pH 7.4, 150mM NaCl,
0.5% NP40) or high salt lysis buffer (20mM HEPES pH 7.4, 10% glycerol, 0.35M NaCl,
1mM MgCl2, 0.5% triton X-100, 1mM DTT) with proteinase inhibitors. The supernatant was
then incubated with protein G beads (GE Healthcare) and desired antibody at 4℃ for 4hr.
The beads were spin down and washed for three times with lysis buffer. The final drop of
wash buffer was vacuumed out and SDS loading buffer was added to the beads followed by

western blot.
ChIP assay
ChIP assay was performed as previously described (Wu et al., 2008). Briefly, approximately 1
x 107
cells were fixed with 1% formaldehyde and quenched by glycine. The cells were
washed three times with PBS and then harvested in ChIP lysis buffer (50mM Tris-HCl, pH
8.0, 1% SDS, 5mM EDTA). DNA was sonicated to 400~600bp before extensive
centrifugation. Four volume of ChIP dilution buffer (20mM Tris-HCl, pH 8.0, 150mM NaCl,
2mM EDTA, 1% Triton X-100) was added to the supernatant. The resulted lysate was then
incubated with protein G beads and antibodies at 4℃ over night. The beads were washed 5
times and DNA was eluted by ChIP elution buffer (0.1M NaHCO3, 1% SDS, 30ug/ml
proteinase K). The elution was incubated at 65℃ overnight and DNA was extracted with
DNA purification kit (Tiangen). The purified DNA was assayed by quantitative PCR with
Biorad MyIQ. Assays were repeated at least three times. Data shown were average values ±
SD of representative experiments. The primers information is in Table S1. At least three
biological replicates were analyzed in each experiment. T test was used for statistical analysis.
Reverse transcription and quantitative PCR
Cells were scraped down and collected by centrifugation. Total RNA was extracted with RNA
extraction kit (Yuanpinghao) according to manufacturer’s manual. Approximately 1μg of total
RNA was used for reverse transcription with a first strand cDNA synthesis kit (Toyobo). The
amount of mRNA was assayed by quantitative PCR. β-actin was used to normalize the
amount of each sample. Assays were repeated at least three times. Data shown were average
values ± SD of one representative experiment. The primer information is in Supplemental
Tables. At least three biological replicates were analyzed in each experiment. T test was used
for statistical analysis.

RNA-sequencing and data analysis
The extracted mRNA from three biological replicates was subjected for high throughput
sequencing. mRNA-seq library was performed by using Illumina TruSeq library construction
kit. Using 5μg total RNA as initiation, and then prepared according to the manufacturer’s
instruction. mRNA-seq libraries were sequenced using HiSeq2000 for 100bp paired-end
sequencing . Quality control of mRNA-seq data was performed using Fatsqc, and then, low
quality bases were trimmed. After quality control, data were mapped to hg19 genome
reference by Tophat2 and allow maximum 2 mismatch. Cufflinks was used to find out
differential expression genes. Gene ontology analysis was performed using DAVID
(http://david.abcc.ncifcrf.gov).
ChIP-sequencing and data analysis
ChIP was performed by using H3K27me1, H3K27me2, H3K27me3 antibody. After ChIP,
sequence library was performed by using KAPA Hyper Prep Kit. Using 10ng of ChIP DNA as
initiation, and then prepared according to the manufacturer’s instruction. The ChIPed DNA
from three biological replicates were mixed together and subjected for high through-put
sequencing. ChIP DNA and matched input DNA were prepared for end repair and ‘A’ tailing,
adaptor ligation, and library amplification. ChIP-Seq sequencing was performed by using
Illumina HiSeq2500 platform for 100bp paired end sequencing. Quality control of ChIP-seq
data was performed using Fatsqc, and then, low quality bases and library adaptors were
trimmed. After quality control, data were mapped to hg19 genome reference by bowtie and
allow maximum 2 mismatch. For histone modification analysis SICER software was used to
call peaks and find out differential modification region.

Data access
The raw data of next generation sequencing were uploaded to GEO database. Accession NO:
GSE75271.
Acknowledgements
We thank Dr. Hong-bing Shu of Wuhan University and Dr. Yan-Yi Wang of Institute of
Viology for sharing reagents and plasmids.
Competing interests
I declare, representing all the authors of the manuscript, that we do not have any
financial interest related to this work.
Author contributions
SSK performed most of the experiments. LCY initialed the Yeast-Two Hybrid screening and
identified EZH1-UXT interaction. LPJ analyzed the data from RNA-Sequencing and ChIP-
Sequencing. WX and ZQY helped in ChIP assays. CY and WZ contributed in plasmid
construction. WM and LL directed the project, conducted the experiments and wrote the
manuscript.
Funding
This work was supported by grants from the National Basic Research Program of China (973
Program, 2012CB518700), the National Natural Science Foundation of China to Min Wu
(31470771) and Lianyun Li (31221061, 31200653 and 31370866).

Reference
Bae, W. K., Kang, K., Yu, J. H., Yoo, K. H., Factor, V. M., Kaji, K., Matter, M., Thorgeirsson, S. and
Hennighausen, L. (2015). The methyltransferases enhancer of zeste homolog (EZH) 1 and EZH2 control
hepatocyte homeostasis and regeneration. FASEB J 29, 1653-62.
Campos, E. I., Stafford, J. M. and Reinberg, D. (2014). Epigenetic inheritance: histone bookmarks
across generations. Trends Cell Biol 24, 664-74.
Carter, D. R., Buckle, A. D., Tanaka, K., Perdomo, J. and Chong, B. H. (2014). Art27 interacts with
GATA4, FOG2 and NKX2.5 and is a novel co-repressor of cardiac genes. PLoS One 9, e95253.
Conway, E., Healy, E. and Bracken, A. P. (2015). PRC2 mediated H3K27 methylations in cellular
identity and cancer. Curr Opin Cell Biol 37, 42-48.
Ea, C. K. and Baltimore, D. (2009). Regulation of NF-kappaB activity through lysine
monomethylation of p65. Proc Natl Acad Sci U S A 106, 18972-7.
Hidalgo, I., Herrera-Merchan, A., Ligos, J. M., Carramolino, L., Nunez, J., Martinez, F.,
Dominguez, O., Torres, M. and Gonzalez, S. (2012). Ezh1 is required for hematopoietic stem cell
maintenance and prevents senescence-like cell cycle arrest. Cell Stem Cell 11, 649-62.
Hu, M. M., Yang, Q., Zhang, J., Liu, S. M., Zhang, Y., Lin, H., Huang, Z. F., Wang, Y. Y., Zhang, X.
D., Zhong, B. et al. (2014). TRIM38 inhibits TNFalpha- and IL-1beta-triggered NF-kappaB activation by
mediating lysosome-dependent degradation of TAB2/3. Proc Natl Acad Sci U S A 111, 1509-14.
Huang, Y., Chen, L., Zhou, Y., Liu, H., Yang, J., Liu, Z. and Wang, C. (2011). UXT-V1 protects cells
against TNF-induced apoptosis through modulating complex II formation. Mol Biol Cell 22, 1389-97.
Huang, Y., Liu, H., Ge, R., Zhou, Y., Lou, X. and Wang, C. (2012). UXT-V1 facilitates the formation
of MAVS antiviral signalosome on mitochondria. J Immunol 188, 358-66.
Iwai, K. (2012). Diverse ubiquitin signaling in NF-kappaB activation. Trends Cell Biol 22, 355-64.
Lanzuolo, C. and Orlando, V. (2012). Memories from the polycomb group proteins. Annu Rev
Genet 46, 561-89.
Lee, S. T., Li, Z., Wu, Z., Aau, M., Guan, P., Karuturi, R. K., Liou, Y. C. and Yu, Q. (2011). Context-
specific regulation of NF-kappaB target gene expression by EZH2 in breast cancers. Mol Cell 43, 798-
810.
Li, W., Wang, L., Jiang, C., Li, H., Zhang, K., Xu, Y., Hao, Q., Li, M., Xue, X., Qin, X. et al. (2014).
UXT is a novel regulatory factor of regulatory T cells associated with Foxp3. Eur J Immunol 44, 533-44.
Margueron, R., Li, G., Sarma, K., Blais, A., Zavadil, J., Woodcock, C. L., Dynlacht, B. D. and
Reinberg, D. (2008). Ezh1 and Ezh2 maintain repressive chromatin through different mechanisms. Mol
Cell 32, 503-18.
Margueron, R. and Reinberg, D. (2011). The Polycomb complex PRC2 and its mark in life. Nature
469, 343-9.
McGilvray, R., Walker, M. and Bartholomew, C. (2007). UXT interacts with the transcriptional
repressor protein EVI1 and suppresses cell transformation. FEBS J 274, 3960-71.
Montgomery, N. D., Yee, D., Chen, A., Kalantry, S., Chamberlain, S. J., Otte, A. P. and Magnuson,
T. (2005). The murine polycomb group protein Eed is required for global histone H3 lysine-27
methylation. Curr Biol 15, 942-7.
Montgomery, N. D., Yee, D., Montgomery, S. A. and Magnuson, T. (2007). Molecular and

functional mapping of EED motifs required for PRC2-dependent histone methylation. J Mol Biol 374,
1145-57.
Mousavi, K., Zare, H., Wang, A. H. and Sartorelli, V. (2012). Polycomb protein Ezh1 promotes
RNA polymerase II elongation. Mol Cell 45, 255-62.
Oeckinghaus, A., Hayden, M. S. and Ghosh, S. (2011). Crosstalk in NF-kappaB signaling
pathways. Nat Immunol 12, 695-708.
Shen, X., Liu, Y., Hsu, Y. J., Fujiwara, Y., Kim, J., Mao, X., Yuan, G. C. and Orkin, S. H. (2008). EZH1
mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity
and executing pluripotency. Mol Cell 32, 491-502.
Sun, S., Tang, Y., Lou, X., Zhu, L., Yang, K., Zhang, B., Shi, H. and Wang, C. (2007). UXT is a novel
and essential cofactor in the NF-kappaB transcriptional enhanceosome. J Cell Biol 178, 231-44.
Wang, X., Zhu, K., Li, S., Liao, Y., Du, R., Zhang, X., Shu, H. B., Guo, A. Y., Li, L. and Wu, M. (2012).
MLL1, a H3K4 methyltransferase, regulates the TNFalpha-stimulated activation of genes downstream
of NF-kappaB. J Cell Sci 125, 4058-66.
Wu, M., Wang, P. F., Lee, J. S., Martin-Brown, S., Florens, L., Washburn, M. and Shilatifard, A.
(2008). Molecular regulation of H3K4 trimethylation by Wdr82, a component of human
Set1/COMPASS. Mol Cell Biol 28, 7337-44.
Xu, J., Shao, Z., Li, D., Xie, H., Kim, W., Huang, J., Taylor, J. E., Pinello, L., Glass, K., Jaffe, J. D. et
al. (2015). Developmental control of polycomb subunit composition by GATA factors mediates a switch
to non-canonical functions. Mol Cell 57, 304-16.
Xu, K., Wu, Z. J., Groner, A. C., He, H. H., Cai, C., Lis, R. T., Wu, X., Stack, E. C., Loda, M., Liu, T. et
al. (2012). EZH2 oncogenic activity in castration-resistant prostate cancer cells is Polycomb-
independent. Science 338, 1465-9.
Yang, X. D., Huang, B., Li, M., Lamb, A., Kelleher, N. L. and Chen, L. F. (2009). Negative regulation
of NF-kappaB action by Set9-mediated lysine methylation of the RelA subunit. EMBO J 28, 1055-66.
Zhang, Y., Lei, C. Q., Hu, Y. H., Xia, T., Li, M., Zhong, B. and Shu, H. B. (2014). Kruppel-like factor 6
is a co-activator of NF-kappaB that mediates p65-dependent transcription of selected downstream
genes. J Biol Chem 289, 12876-85.

Figures
Fig 1. The interaction between UXT and EZH1. (A) 293FRT cells were transfected with
HA-EZH1 and FLAG-UXT. 48h after transfection, cells were immunoprecipitated and
immunoblotted with indicated antibodies, respectively. (B) HCT116 cells were
immunoprecipitated and immunoblotted with indicated antibodies, respectively. (C) In vitro
pull-down assay with recombinant GST-EZH1 and His-UXT, His-SPOP as a negative control.
(D) Schematic illustration of EZH1 and its mutants. (E, F & G) Flag-UXT was expressed in
293FRT cells with HA-tagged EZH1 or its deletion mutants. Cell lysates were

immunoprecipitated with anti-Flag and immunoblotted with anti-HA. Each experiment was
repeated at least three times.

Fig 2. UXT specifically interacts with EZH1 and SUZ12, but not with EED. (A) HCT116
cells were immunoprecipitated and immunoblotted with indicated antibodies, respectively. ns,
non-specific bands. (B) 293FRT cells were transfected with HA-SUZ12 and FLAG-UXT.
Cell lysates were immunoprecipitated with anti-HA and immunoblotted with anti-Flag. (C &
D) HCT116 cells were treated with 10ng/ml TNFα for 2h. Cell lysates were
immunoprecipitated with SUZ12 and EED antibodies respectively, and immunoblotted with
anti-UXT. Each experiment was repeated at least three times.

Fig 3. The expression of NF-κB target genes regulated by EZH1 and SUZ12. (A) HCT116
cells were transfected with siRNA for UXT. 48h after transfection, cells were stimulated by

TNFα for the indicated times. The mRNA levels of NFKBIA, CXCL8 and TNFAIP3 were
measured by quantitative RT-PCR. (B) EZH1 was knocked down by two independent
siRNAs, and experiment was performed as (A). (C) HCT116 cells were transfected with the
indicated siRNA. 48h after transfection, cells were treated with TNFα for 2h. Gene expression
profile was assayed by RNA-Sequencing. The heat map shows the mRNA level of the TNFα-
induced differentially expression genes (DEG) in the indicated cells. (D) HCT116 cells were
transfected with the indicated siRNAs. 48h after transfection, cells were treated with TNFα
for 2h. Endogenous mRNA expression of NFKBIA and CXCL8 were measured by Real-time
RT-PCR. T test was used for statistical analysis. * means p value < 0.05; ** means p value <
0.01.

Fig 4. EZH1 and UXT does not regulate H3K27 methylation on NF-κB target genes. (A
& B) UXT and EZH1 were knocked down in HCT116. 72h later, cells were harvested and
lysates were analyzed by western blot. (C) UXT and EZH1 were knocked down in HCT116
and ChIP-Seq assay was performed. Profiles of histone H3K27me1, me2 and me3 across the
gene bodies of TNFα-induced genes were shown. TSS, transcriptional start site; TES,
transcriptional termination site.

Fig 5. EZH1 and UXT regulates the recruitment of RELA/p65 to target genes. (A, B)
HCT116 cells were transfected with siRNAs and treated with 10ng/ml TNFα as indicated.
Cytoplasmic and nuclear fractions were prepared and immunoblotted with the indicated
antibodies, respectively. (C) Cytoplasmic and nuclear fractions of HCT116 were prepared and
assayed with western blot. (D) Flag-EZH1 was expressed in the cell and immunofluorescent
microscopy was performed. (E) Cells were treated with 10ng/ml TNFα for 2h, and
immunoprecipitation was performed with anti-UXT. (F) 293FRT cells were transfected with

indicated plasmids, and 48h later cells were treated with TNFα for 2h. Immunoprecipitation
and western blot were performed as indicated. (G) The stable cell line of HA-tagged EZH1
was transfected with EZH1 siRNAs. 72h later, cells were harvested for real-time RT-PCR or
ChIP assays. (H) HCT116 cells were transfected with indicated siRNAs and 72h later cells
were induced with 10ng/ml TNFα for 2h. The amount of RELA on NFKBIA and CXCL8
promoters was assayed by ChIP. (I) RELA was knocked down by siRNA in HA-EZH1 stable
cell line and ChIP analysis was performed with anti-HA. T test was used for statistical
analysis. * means p value < 0.05; ** means p value < 0.01.

Fig 6. Recruitment of RNA Pol II to NF-κB target genes regulated by EZH1 and UXT.
(A) HCT116 were treated with or without TNFα, and lysates were immunoprecipitated with
8WG16 and immunoblotted with indicated antibodies, respectively. (B) Lysates were
immunoprecipitated with 8WG16 and immunoblotted with SUZ12 and EED antibodies. (C &
D) siRNAs were transfected into HCT116 as indicated. Cells were treated with 10ng/ml
TNFα 2h later. RNA Pol II bound on the promoters of NFKBIA and CXCL8 was examined by
ChIP assay with 8WG16. T test was used for statistical analysis. * means p value < 0.05; **
means p value < 0.01.

Fig 7. TNFα- induced apoptosis regulated by EZH1. (A) HCT116 cells were transfected
with UXT or EZH1 siRNAs, and then treated with or without TNFα (0.05ng/ml) for 12h. Cell
death was measured with Propidium Iodide (PI) staining followed by flow cytometry.
Statistical calculation of sub-G1 cells was shown in the right panel. (B) Cells were prepared
as (A). Cells were double stained with annexin V-FITC and PI, and then assayed by flow
cytometry. Statistical calculation of Annexin V positive cells was shown in the right panel.

(C) UXT or EZH1 was knocked down in HCT116 respectively. The expression of BIRC2 and
BIRC3 after TNFα treatment was measured by quantitative RT-PCR. * means p value < 0.05;
** means p value < 0.01.

J. Cell Sci. 129: doi:10.1242/jcs.185546: Supplementary information
Supplemental figures
Sup Fig. 1 Expression of NF-κB target genes regulated by EZH1. (A, B & C) EZH1 was
knocked down in (A) RKO, (B) HEK293 and (C) U2OS cells, respectively. Then cells were treated
with TNFα for 2hr and the expression of NFKBIA and CXCL8 was measured with quantitative PCR.
(D) HCT116 was transfected with EZH1 siRNA and treated with SeV. The expression of
downstream genes, NFKBIA, CXCL8, IFNβ and ISG56 was measured by quantitative PCR.
JournalofCellScience•Supplementaryinformation

Sup Fig. 2 The differential expressed genes in HCT116 cells with or without UXT knockdown were
studied with GO analysis. The enriched KEGG pathways were shown.

Sup Fig. 3 H3K37 methylation on NF-κB target genes is not regulated by UXT and EZH1. (A)
EZH2 was knocked down by siRNA in HCT116 and H3K27 methylation was measured by western.
(B) HCT116 was transfected with UXT siRNA and treated with TNFα for 2hr. H3K27me1, me2,
me3 and H3K4me3 on NFKBIA and CXCL8 were measured by ChIP assay. GAPDH was used as
negative gene for H3K27me3, and MYOD1 for H3K4me3. The red lines represented the
background signals on control genes. (C) Cells were transfected with EZH1 siRNA and experiment
was done as (A).

Sup Fig. 4 (A) HA-EZH1 stable cell lines was established and EZH1 expression was measured by
western. (B) EZH1 was knocked down in 293FRT and the cells were treated with TNFα. RELA on
the promoters of NFKBIA and CXCL8 was measured by ChIP assay. (C) HCT116 was transfected
with EZH1 siRNA and treated with SeV. RELA on the promoters of NFKBIA and CXCL8 was
measured by ChIP assay. (D) 293FRT was transfected with EZH1 siRNA and treated with TNFα.
RNA Pol II on the promoters of NFKBIA and CXCL8 was measured by ChIP assay. (E) HCT116
was treated with TNFα and the expression of indicated genes was measured by quantitative RT-
PCR.

Click here to Download Table S1
Sup Table. The different expressed genes analyzed by RNA-Seq and the sequence information
of primers and siRNAs.

20160524123048653

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