Global histone modification profiling reveals the epigenomic dynamics during malignant transformation in a four-stage breast cancer model
Zhao et al. Clinical Epigenetics
Global histone modification profiling reveals the epigenomic dynamics during malignant transformation in a four-stage breast cancer model
Quan-Yi Zhao 1 2
Pin-Ji Lei 1 2
Xiaoran Zhang 0
Jun-Yi Zheng 2
Hui-Yi Wang 2
Jiao Zhao 3
Yi-Ming Li 3
Mei Ye 3
Lianyun Li 2
Gang Wei 0
Min Wu 2
0 CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai 200031 , China
1 Equal contributors
2 Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University , Wuhan 430072Hubei , China
3 Division of Gastroenterology, Department of Geriatrics, Zhongnan Hospital, Wuhan University , Wuhan, Hubei 430072 , China
Background: Epigenetic regulation has emerged to be the critical steps for tumorigenesis and metastasis. Multiple histone methyltransferase and demethylase have been implicated as tumor suppressors or oncogenes recently. But the key epigenomic events in cancer cell transformation still remain poorly understood. Methods: A breast cancer transformation model was established via stably expressing three oncogenes in primary breast epithelial cells. Chromatin immunoprecipitation followed by the next-generation sequencing of histone methylations was performed to determine epigenetic events during transformation. Western blot, quantitative RT-PCR, and immunostaining were used to determine gene expression in cells and tissues. Results: Histones H3K9me2 and me3, two repressive marks of transcription, decrease in in vitro breast cancer cell model and in vivo clinical tissues. A survey of enzymes related with H3K9 methylation indicated that KDM3A/ JMJD1A, a demethylase for H3K9me1 and me2, gradually increases during cancer transformation and is elevated in patient tissues. KDM3A/JMJD1A deficiency impairs the growth of tumors in nude mice and transformed cell lines. Genome-wide ChIP-seq analysis reveals that the boundaries of decreased H3K9me2 large organized chromatin K9 modifications (LOCKs) are enriched with cancer-related genes, such as MYC and PAX3. Further studies show that KDM3A/JMJD1A directly binds to these oncogenes and regulates their transcription by removing H3K9me2 mark. Conclusions: Our study demonstrates reduction of histones H3K9 me2 and me3, and elevation of KDM3A/JMJD1A as important events for breast cancer, and illustrates the dynamic epigenomic mechanisms during breast cancer transformation.
H3K9 methylation; Epigenomics; Breast cancer transformation; KDM3A; Transcription regulation
Recent advances in epigenetics and epigenomics have
revealed that epigenetic abnormality is one of the critical
causes for tumorigenesis [
]. DNA methylation
inhibitors have already used for cancer treatment [
DNA methylation, abnormality of histone methylation is
implicated in cancer as well. Early studies have shown that
histone modification patterns can be used to predict tumor
phenotypes and the risk of cancer recurrence [
Histone acetylation and deacetylation have been extensively
studied and histone deacetylases (HDACs) are frequently
reported to inhibit the expression of tumor suppressors
. Inhibitors of histone deacetylase (HDAC) are proved
to be useful in clinical cancer treatment [
]. However, the
relationship between cancer and other histone
modifications, such as histone methylation, is still not conclusive.
Histone methylation usually occurs on lysines and
arginines, and each site has three different forms [
comparison with acetylation, histone methylation and
demethylation have more complicated regulatory steps,
making them promising drug targets [
]. Recently, several
well-studied histone methyltransferases emerged to be key
regulators in multiple cancer types. For example, enhancer
of zeste 2 polycomb repressive complex 2 subunit (EZH2),
the major enzyme for H3K27me3, was demonstrated as an
oncogene in prostate cancer [
]. Its mutation has also been
frequently found in lymphoma [
]. An enzymatic
inhibitor of EZH2 was shown to inhibit lymphoma cell
proliferation in a mouse model . SET domain containing 2
(SETD2) is the major H3K36me3 methyltransferase in
mammalian cells and frequently mutated in clear cell kidney
carcinoma, acute leukemia, gliomas, and other cancers [
]. H3K36me3 catalyzed by SETD2 is required for
genome stability and DNA repair process after damage [
Myeloid/lymphoid or mixed-lineage leukemia 1-4
(MLL14), the methyltransferases for H3K4, was found to be
mutated in multiple types of cancers [
]. On the other hand,
demethylation is another critical aspect for the dynamic
regulation of histone methylation. Clinical studies from
different groups found that the demethylases of H3K9me2 and
me3, such as lysine (K)-specific demethylase 3A (KDM3A,
also known as JMJD1A or JHDM2A) and lysine (K)-specific
demethylase 4A/B/C (KDM4A/B/C), are highly expressed
in cancer tissues and regulate tumorigenesis [
Moreover, KDM4A was reported to induce site-specific copy gain
and DNA re-replication and promote cellular
transformation by inhibiting p53 signaling [
]. A histone H3K4
demethylase, lysine (K)-specific demethylase 5A (KDM5A),
is involved in the cancer cell drug tolerance . All these
information suggests that histone methylation is critical in
the genesis and development of multiple cancer types.
However, the molecular mechanisms of these enzymes in
tumorigenesis still remain elusive.
The early diagnosis and treatment are the most
effective ways to cure cancer. But we are still lack of good
markers and drugs for precision medicine. During
transformation, the epigenetic programs in cells change
dramatically, helping them to survive and gain growth
]. Along with the development of
functional genomics, some studies have investigated the
dynamic changes during differentiation , but the
epigenetic dynamics at the genome-wide scale during
transformation are still not known. However, due to the
individual variation, cell heterogeneity, and limited
sample size, patient tissues are difficult for mechanistic
studies. Thus, a transformation model with clean
background and high reproducibility is required. More than a
decade ago, a cell-based model was established to mimic
transformation from human primary cells to tumor cells
]. With the introduction of three genes, large T
antigen, telomerase reverse transcriptase (TERT), and
Harvey rat sarcoma viral oncogene homolog v12 mutant
(RAS (V12)), the engineered primary cell will extend life
span, become immortalized, and finally gain tumorigenic
capacity, which is considered to represent different stages
of tumor cell transformation [
]. Considering the
difficulties in studying human cancers, the in vitro model still
serves as one of the best platforms to study the molecular
mechanisms of tumor transformation. Recently, profiling
of global gene expression in the above model has provided
valuable information for tumor transformation study and
suggested a link between malignant transformation to
]. But due to the difficulty in making the
model, no epigenetic study was reported.
In this study, we took advantage of the in vitro tumor
transformation model and made a series of transformed
cell lines starting from human primary mammary cell
(HMC). By combining biochemical and epigenomic
approaches, we demonstrate that histones H3K9me2 and
H3K9me3 decrease during breast cancer transformation
and contribute to the process with different mechanisms.
Furthermore, we identified that KDM3A/JMJD1A, an
H3K9me2 demethylase, is responsible for the H3K9me2
reduction and critical for breast tumor transformation.
Gene expression profile of tumor transformation model mimics clinical samples
To study the underlying mechanisms of breast cancer
transformation, we utilized an established cell-based
transformation model [
]. Large T antigen, TERT,
and RAS (V12) were stably expressed in human primary
mammary cell respectively via retroviral infection. Four
cell lines were generated for following studies, namely
HMC-p6 (human primary mammary cell, passage 6),
HMC-L (HMC with large T stable expression), HMC-LT
(HMC with large T and TERT stable expression), and
HMC-LTR (HMC with large T, TERT, and HRAS (V12)
stable expression) (Additional file 1: Figure S1A, B).
HMC-p6 and HMC-LT can be passaged for 1 and
2 months, respectively, and HMC-LT is immortalized
(Additional file 1: Figure S1C). Only HMC-LTR can form
colonies in soft agar and grow into tumors in nude mice
(Additional file 1: Figure S1D). These observations are in
full accordance with previously reported results .
Based on their ability in proliferation and tumorigenicity,
the four cell lines are considered to represent different
transformation stages. To further confirm the validity of
the tumor cell model, we clustered the transformed cell
lines with clinical samples based on their expression
profiles of differentially expressed genes (DEGs). Gene
expression profiles of 100 cases of paired breast cancer and
normal tissues were downloaded from the Cancer Genome
Atlas (TCGA), and the DEGs (twofolds) were analyzed
according to the pipeline described in experimental procedure
(Additional file 1: Figure S2A and Additional file 2: Table
S6). These DEGs were then compared with those
(threefolds) identified from transformed HMCs (Additional file 1:
Figure S2A and Additional file 2: Tables S1–S5), and the
expression patterns of resulting 338 genes were used for
clustering (Fig. 2a, Additional file 1: Figure S2B, C and
Additional file 2: Table S7). Cluster analysis grouped HMC-LTR
with breast cancer tissues and HMC with adjacent tissues
(Fig. 1a), suggesting the transformed LTR cell line may
partially mimic some of the clinical samples. HMC-p6, -L,
and -LT are grouped together, while HMC-LTR is
separate, which may indicate the big difference of tumor cell
line with others (Additional file 1: Figure S2B). We further
used the DEGs of HMC-p6 and -LTR to cluster the TCGA
tissues and successfully grouped them into normal and
cancer groups (Additional file 1: Figure S2C).
Furthermore, gene ontology analysis showed that the
tumor transformation model shares similarities with clinical
samples (Fig. 1b, c, Additional file 1: Figure S2D, F). The
DEGs of HMC-LT and -LTR in comparison with HMC-p6
were enriched mostly in cell cycle regulation and
extracellular environment (Fig. 1c, d), which were both enriched in
clinical samples (Fig. 1b). Then, we analyzed the gene
expression profiles of all the four cell lines. We found that the
most significant enriched processes are the following: (1)
response to oxygen and hypoxia for p6 to HMC-L, (2)
response to wounding, immune, and inflammatory response
for HMC-L to HMC-LT, (3) regulation of cell motility,
migration, and proliferation for HMC-LT to HMC-LTR (Fig. 1e
and Additional file 2: Tables S1–S5). All these processes are
frequently activated in tumors. Our analysis provides
important information of precancerous transformation.
When we initiated our study, we randomly selected some
datasets from the TCGA database. The analysis did not
distinguish HMC-p6 and -LTR as good as that in Fig. 1a, and
HMC cells clustered much better with adjacent normal
tissues in the new analysis (Fig. 1a, Additional file 1: Figure
S2G, H). The difference between the two analysis is as
follows: (1) More tissues were used in the new analysis (100
paired tissues) compared with the early one (70 cancer and
12 normal tissues); (2) paired tissues are more useful than
random samples because less individual difference leads to
less disturbance; (3) more strict conditions were used for
DEGs among tissues (threefolds compared with twofolds,
Additional file 1: Figure S2A, G). These experiences might
be useful to the other similar analysis with online
largescale sequencing data.
Profiling of histone modifications during transformation
In order to systematically characterize epigenetic changes
during transformation, we profiled the four cell lines with
available commercial antibodies for histone modifications.
Interestingly, we observed a gradual reduction of
H3K9me2 and me3 along with transformation (Fig. 2a).
On the contrary, H3K9 acetylation showed a gradual
elevation (Fig. 2a). No obvious changes were observed for
other modifications (Fig. 2a). Next, we studied if our
discovery in in vitro cell model is also the same in clinical
patient tissues. We performed immunostaining in
commercial breast cancer tissue arrays and observed a strong
reduction in cancer tissues for both methylations (Fig. 2b).
In total, we analyzed 140 cancer and 40 adjacent samples
for H3K9me3 and 42 cancer and 66 adjacent samples for
H3K9me2. Statistical analyses of fluorescence densities
representing H3K9 me2 and me3 show obvious lower
methylation levels in cancer tissues (Fig. 2c, d).
The altered transcription programs of cancer-related genes regulated by H3K9me2
To further confirm our discovery, we performed
ChIPseq studies in the four cell lines of H3K9me2, H3K4me3,
and H3K27me3. The data suggested H3K9me2 reduced
significantly in HMC-L, -LT, and -LTR; H3K27me3 had
no significant change (Fig. 3a, b). H3K4me3 enrichment
seemed reduced also in transformed cell lines but not
significant enough (Additional file 1: Figure S3G).
However, we did not see a gradual change by high
throughput sequencing among the later three cell lines, which is
different from western. The reason might be that two
methods used different standards for normalization,
histone H3 for western and input for ChIP-seq.
Nevertheless, our results indicate that histone H3K9me2
decreased significantly during transformation.
We then analyzed the dynamic changes of H3K9me2
distribution during tumor transformation. A slight increase of
H3K9me2 peaks on distal intergenic regions during
transformation was observed (Additional file 1: Figure S3A–C).
The length of all H3K9me2 peaks and decreased peaks
reduced modestly during the process, while the length of
increased peaks remained unchanged (Additional file 1:
Figure S3D, E). The biological meanings for these changes
still required more studies. Interestingly, we also found that
the length of H3K27me3 peaks decreased along with
transformation (Additional file 1: Figure S3F), though its total
enrichment did not change significantly (Fig. 3b). Various
mutations and abnormal expression of EZH2, the major
enzyme catalyzing H3K27me3, are associated with cancers
15, 17, 18
]. Our discovery provides a clue to the study how
H3K27me3 dysregulation is involved in breast cancer
We noticed that decreased H3K9me2 peaks were mainly
located on gene bodies (Additional file 1: Figure S3C),
therefore, the relationship between gene expression and
H3K9me2 during transformation was investigated.
Considering the importance of promoters for gene expression,
the total reads of H3K9me2 peaks from −10 kb before
TTS (usually considered as proximal promoter region) to
TES was calculated. We found that H3K9me2 dynamically
changed on many oncogenes (Fig. 3c). We then confirmed
the change of histone modifications on some of them
(Fig. 3d and Additional file 1: Figure S4A). We further
analyzed their messenger RNA (mRNA) levels and
confirmed with quantitative PCR that many genes exhibited
correlated expression (Fig. 3e and Additional file 1: Figure
S4B–C). Meanwhile, we found that a few genes did
not show the expected elevated expression, such as
MYC and BCL2 (Fig. 3e). H3K9me2 is generally
believed to be a transcription repression mark and its
reduction is required for gene activation. However,
H3K9me2 reduction on genes may not directly lead
to transcription alteration, and upstream signals
sometimes are required.
We also analyzed H3K4me3 and H3K27me3 on genes
with decreased H3K9me2. Surprisingly, the average
levels of these marks on the above genes are all lower
than that in primary cell (Additional file 1: Figure S4D,
E). Recently, several groups reported that synergistic
decrease of histone H3K4, K9, and K27 methylation is
associated with the increase of DNA methylation [
It is possible that some genes with H3K9me2 decrease
may be also involved in DNA methylation changes.
Boundaries of H3K9me2 LOCKs are enriched with cancerrelated genes
H3K9me2 often modifies broad regions on chromatin,
which have been previously named as large organized
chromatin K9 modifications (LOCKs) [
]. The H3K9me2
on LOCKs has a similar pattern as the total peaks, high in
HMC and low in the other three cell lines (Fig. 3f ). The
total numbers and genome coverages of H3K9me2
LOCKs decreased during transformation (Fig. 3g, h). We
analyzed the genes overlapped with all decreased
H3K9me2 LOCKs but did not find any relationships with
cancer. We speculated that change of a broad chromatin
region may start from its boundaries, so we analyzed the
genes located on the boundaries of the decreased LOCKs.
Surprisingly, we found that the genes located in these
boundaries are enriched with cancer-related pathways
(Fig. 3i and Additional file 2: Tables S8–S10). This
suggests that the localization of genes in H3K9me2 LOCKs is
related with cellular functional changes and unlocking the
(See figure on previous page.)
Fig. 3 Decreased H3K9me2 alters the transcription program of cancer-related genes at the boundaries of LOCKs. a, b Average enrichment levels
of H3K9me2 (a) and H3K27me3 (b) around detected peaks. c The heatmap shows the dynamic H3K9me2 levels on the oncogenes with
H3K9me2 reduction. The reads of all H3K9me2 peaks from −10 kb of TSS to TES on each gene were used. The genes verified later by ChIP-PCR
and RT-PCR are labelled by arrows. d H3K9me2 levels on several oncogenes were confirmed by ChIP-PCR. e The mRNA expression level of
oncogenes with decreased H3K9me2 in transformed cell lines. f The average levels of H3K9me2 LOCKs in the four cell lines. Peaks with length
>100 kb were defined as LOCKs. All LOCKs in the four cell lines were extended down- and upstream for 50 kb, and their average H3K9me2
enrichment levels were calculated. g, h The number and average coverage of H3K9me2 LOCK number in four cell lines. i KEGG pathway analysis
of genes located at the boundaries of LOCKs with decreased H3K9me2
oncogenes at the boundaries facilitates the transformation
process. The genes at decreased H3K9me2 LOCK
boundaries in three transformed cells are very similar, including
BTG1, MYC, PIK3R1, and WNT5A for oncogenes and
CDKN2A, CDKN2B, FH, INTS6, LINC00032, MITF,
PLK2, and WNT5A for tumor suppressors (Additional file
2: Table S11). MYC is the well-known oncogene in breast
cancer and many other cancer types [
]. All the other
genes may also play important roles in breast tumor
High expression of H3K9 demethylase KDM3A/JMJD1A in breast cancer cell lines
After determining the reduction of H3K9 methylation as
a frequent event in breast cancer, we started to
investigate the underlying molecular mechanisms. We firstly
examined the mRNA levels of all the known H3K9
methyltransferases and demethylases by RNA-seq and
quantitative RT-PCR, but none matched the observed
H3K9 methylation pattern (Additional file 1: Figure S5A,
B). We then asked if the regulations of these enzymes
take place at the protein level. We surveyed the protein
levels in transformed cell line with all the available
antibodies and found that KDM3A/JMJD1A, a
demethylase for H3K9me1 and me2, gradually increased during
transformation, inversely matching the decrease of
H3K9me2 (Fig. 4a and Additional file 1: Figure S5C).
We further found that KDM3A/JMJD1A is much higher
in two breast cancer cell lines, MCF and T47D, than that
in the primary HMC and other two cancer cell lines,
HCT116 and 769-P (Fig. 4b). A commercial breast tissue
array containing 48 pairs was stained with KDM3A and
the statistical analysis showed that KDM3A significantly
increases in breast cancer tissues compared with normal
tissues (Fig. 4c, d). Another piece of array from the same
batch was stained with H3K9me2. Fifteen of 48 pairs
(31.3 %) showed both KDM3A increase and H3K9me2
decrease. Taken together, these data show that histone
H3K9 demethylase, KDM3A/JMJD1A, increases in breast
cancer cell lines.
Considering the inconsistency of its mRNA and protein
level, KDM3A/JMJD1A is probably regulated at the
posttranslational level. To further verify it, MG132 (an inhibitor
for proteasome) or chloroquine (CQ, an inhibitor for
lysosome) was used to treat HMC cell line. Both drugs
increased the protein level of KDM3A/JMJD1A (Additional
file 1: Figure S5D), suggesting its stability was controlled by
both proteasome and lysosome. To verify the function of
KDM3A/JMJD1A, we expressed its wild type or catalytic
dead mutant (H1180A) and confirmed the expression of
wild type decrease H3K9me2 in the cell (Additional file 1:
H3K9 dimethylation and transcription of cancer-related genes regulated by KDM3A/JMJD1A
To further investigate the role of KDM3A/JMJD1A in
regulating transformation, we knocked it down in HMC-LTR
using small interfering RNA (siRNA) and found that
KDM3A/JMJD1A deficiency rescued the expression of
most cancer-related genes in HMC-LTR to the levels in
primary cells (Additional file 1: Figure S6A). We performed
RNA sequencing and found that KDM3A/JMJD1A
deficiency mainly affected the genes involved in cell
proliferation, cell cycle, wound healing, and protein transport
(Additional file 1: Figure S6B, C). We took the DEGs in
HMC-LT (Fig. 5a, Additional file 2: Tables S12 and S13)
and HMC-LTR (Fig. 5b, Additional file 2: Tables S14 and
S15), respectively, which were rescued by KDM3A/JMJD1A
knockdown to the levels in primary cell, and performed
GO analysis. The biological processes of these genes are
very similar to those changed during transformation (Fig. 5c,
d), indicating KDM3A/JMJD1A is the key factor for the
process. H3K9me2 ChIP-seq analysis in HMC-LT further
showed that the increased LOCKs by KDM3A/JMJD1A
knockdown largely overlapped with the decreased LOCKs
in transformation (Fig. 5e, left). The analysis in HMC-LTR
showed similar results (Fig. 5e, right). KDM3A/JMJD1A
deficiency also restored a large portion of the
decreased H3K9me2 peaks in HMC-LT and -LTR cells
(Additional file 1: Figure S6D). The increased genes
with KDM3A knockdown were also studied with GO
analysis (Additional file 1: Figure S6E).
When KDM3A/JMJD1A was knocked down in
HMCLTR, the mRNA of most of these genes decreased,
suggesting KDM3A may directly regulate their transcription
(Fig. 5f ). The ChIP-seq results were verified with
quantitative PCR and we found that KDM3A/JMJD1A regulates
H3K9me2 on a group of oncogenes, including MYC,
PAX3, AGR2, PRL, and BCL2 (Fig. 5g). We then
performed Flag ChIP analysis in an F-KDM3A/JMJD1A
stable cell lines derived from a breast cancer cell line
T47D. The results indicated that KDM3A/JMJD1A binds
directly on oncogenes MYC and PAX3 in T47D (Fig. 5h).
We speculated that additional signals are probably
required to activate MYC expression. We induced MYC in
T47D breast cancer cell line with FBS treatment after
serum starvation. The elevation of MYC mRNA was
impaired in the absence of KDM3A (Fig. 5i), as well as its
protein (Fig. 5j). All these results demonstrated that
KDM3A/JMJD1A regulates breast tumor transformation
through directly binding MYC and PAX3 oncogenes and
modulating their transcription.
KDM3A/JMJD1A deficiency impairs the growth and migration of breast cancer cells
To further explore the function of KDM3A/JMJD1A in
breast cancer transformation, we made KDM3A stable
knockdown cell lines in HMC-L and HMC-LT. We
could not get the cell line in HMC-LTR because it was
extremely difficult to have four different constructs
integrated into one cell line. The results of MTT assay
indicated that the growth of HMC-LT was greatly impaired
in the absence of KDM3A (Fig. 6a). The cell cycle
analysis also indicated that HMC-LT was arrested at G1
phase with KDM3A deficiency (Fig. 6b). The abilities of
cell migration and invasion were measured by RTCA
real-time monitor or traditional transwell assay with
T47D cell line. The results indicated that KDM3A/
JMJD1A overexpression enhanced T47D migration and
knockdown repressed it (Fig. 6c and Additional file 1:
Figure S7A). But cell invasion was not affected (Add Fig.
S7B). Colony formation assay indicated that KDM3A/
JMJD1A knockdown significantly decreased the colony
number in the plate (Fig. 6d). To further test whether
MYC and PAX3A really regulate the tumorigenicity
ability of the transformed cell, we knock down the two
genes respectively in HMC-LTR. The assays performed
by RTCA system indicated that MYC and PAX3 are both
involved in cell migration and invasion (Additional file
1: Figure S7C). The above data indicated that KDM3A
positively regulates the survival and migration of breast
High expression of KDM3A/JMJD1A in breast cancer tissues
We then asked whether KDM3A/JMJD1A elevation also
occurs in breast cancer patients. We analyzed the breast
cancer tissues from 18 patients by Western blotting, out
of which 15 has the corresponding adjacent tissues. In
the 15 paired tissues, 8 pairs showed H3K9me2
reduction in cancer, 10 with KDN3A/JMJD1A increase; 7 with
MYC mRNA increase, and 6 with PAX3 mRNA increase;
4 with all the above characteristics (Fig. 6e–g). The
results suggested KDM3A/JMJD1A regulates the
tumorigenesis of breast cancer through down-regulating
H3K9me2 on oncogenes MYC and PAX3.
Development of novel markers and drug targets are keys
for precision diagnosis and treatment. In this study, we
combine systematic and biochemistry approaches and
prove that H3K9me2/3 decreases and KDM3A/JMJD1A
increases in tumor cell transformation model and in vivo
clinical samples. Epigenomic analysis identifies the
dynamic changes of H3K9me2 reduction on MYC, PAX3,
WNT5A, and CDKN2A/B, are critical events during
transformation. These events can be further developed
as diagnosis markers and drug targets in clinical
Our data suggest that H3K9me2 regulates
transformation mainly through transcription. We discovered that
the boundaries of decreased H3K9me2 LOCKs are
enriched with cancer-related genes. H3K9me2 reduction
usually starts from the LOCK boundaries and the
boundary genes are relatively easier to be de-repressed
and, therefore, can help the cell adapt to different
development or transformation cues. The genes in the center
regions are difficult to be accessed. This explains how
H3K9me2 regulates cell identity in tumor
transformation, as well as other processes. In our study, we also
found that H3K9me2 reduction may not be always
related with increased transcription of some genes. For
example, H3K9me2 on MYC is gradually reduced during
transformation, but its mRNA level keeps constant.
However, we found that its induced transcription is
regulated by KDM3A/JMJD1A transient knockdown with
siRNA. These data further indicated that changes of
histone modifications may not affect transcription
immediately, but it will be critical during some special
circumstances. This has also been observed with other
KDM3A/JMJD1A is a gene involved in sex
]. While we are submitting the manuscript,
one group reported that KDM3A/JMJD1A regulates the
expression of ER target genes in breast cancer cells ,
which supports our discovery of KDM3A/JMJD1A’s role
in breast cancer. Beyond this, we provided genome-wide
H3K9me2 map in the absence of KDM3A/JMJD1A and
identified MYC and PAX3 as direct target genes of
KDM3A/JMJD1A. MYC is a well-known oncogene for
breast cancer, as well as many other cancer types.
Mutations in PAX3 are associated with Waardenburg
syndrome, craniofacial-deafness-hand syndrome, and
alveolar rhabdomyosarcoma. Above all, our discovery
provides new mechanisms for tumorigenesis regulated by
Taken together, our study reveals the dynamic changes
occurring at the boundary regions of H3K9me2 LOCKs,
identifies key epigenetic events on cancer-related genes,
and proposes their crucial roles in dynamically
regulating cell transformation. The work not only provides
potential diagnosis markers and drug targets for future
clinical research but also puts forward novel concepts
for epigenomic studies.
Our study demonstrates that the levels of H3K9me2 and
me3 decrease during breast cancer cell transformation
in vitro and in patient tissues. ChIP analysis revealed
that the genes localized at the boundaries of H3K9me2
LOCKs are related with cancer. The increase of
KDM3A, a histone demethylase, is responsible for the
reduction of H3K9me2. KDM3A regulates transcription
of oncogenes, such as MYC and PAX3, via directly
binding to the genes and regulates their H3K9me2 level.
Cell lines and reagents
Human primary mammary cells were purchased from
Chi Scientific and cultured in DMEM/F12 (10 % FBS,
with addition of insulin, hydrocortisone, and EGF) at
37 °C with 5 % CO2. MCF7 was purchased from Cell
Bank of Chinese Academy and cultured in DMEM with
FBS, insulin (10 μg/mL), sodium pyruvate (Invitrogen),
and nonessential amino acid (Invitrogen); T47D is a gift
from Dr. Yong-Feng Shang of Tianjin Medical University
and cultured in RPMI1640 with FBS and insulin. Both
the primary cells and cancer cell lines were sub-cultured
1:4 on reaching confluence; each passage was considered
Antibodies were purchased from the indicated
companies: H3K9ac, hTERT, KRT14, and ACTG (Epitomics);
KRT18 (ProteinTech), JMJD1B, JMJD2B, JMJD2A, H4K
20me3, H3K79me2, E-cadherin, and RAS (CST); H3K4
me1, H3K4me3, H3K27me3, H3K27me1, H3K27me2, and
H3K36me2 (Millipore); H3, H3K9me1, H3K4me2, and
H3K36me3 (Abcam); H3K9me2, H3K9me3, GAPDH, EH
MT2, SUV39H1, CBX5, DNMT3A, DNMT3B, DNMT1,
and KDM1A (Abclonal); KDM3A/JMJD1A (Abclonal for
western and Millipore for immunostaining). The
information of primers and siRNAs are listed in Additional file 2:
Transformation of human breast tumor cell
Transformed breast cell lines were generated as previous
described. 293FRT cells were co-transfected with
packaging plasmid ZV77 (psPAX and pMD2G for lentivirus)
and pBabe retroviral plasmids containing desired
complementary DNA (cDNA). Supernatants containing virus
were harvested 48 h later and HMC was infected together
with 8 μg/ml polybrene. Typically, more than 80 % of cells
were infected as measured by parallel infections with a
GFP-expressing construct. Drug selection was performed
with 200 μg/ml G418 for neomycin, 50 μg/ml hygromycin,
or 0.5 μg/ml puromycin. pBabe with large T antigen,
hTERT, or RAS (V12) were purchased from Addgene.
Immunofluorescent staining of cancer tissues and cultured cells
Tumors tissue array (Alenabio, www.alenabio.com) were
fixed in 10 % formalin, embedded in paraffin, and followed
by standard dewaxing procedures. Cells were cultured on
the cover slips and fixed with freezing methanol after
washing twice in PBS. The cover slips or tumors tissue
array were then washed three times by PBS and blocked
in PBS with 1 % BSA for 10 min or 1 h. The cover
slips or tumor tissue arrays were hybridized with first
and second antibodies for 1 h, respectively. Then, the
slips were mounted with prolong anti-fade kit
(Invitrogen) and observed with fluorescent microscopy. The
arrays used for staining are as follows: H3K9me2 - BR243K,
BR243L, BR243M, BR724, BR725, BCN963a; H3K9me3
BR243B, BR243D, BR243K, BR243L, BR243A, BC081120,
BCN963a; and KDM3A - BR724, BR725.
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 1 μg 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. All primer sequences are
presented in Additional file 2: Table S16.
ChIP assay was performed as previously described [
Briefly, approximately 1 × 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 (50 mM Tris-HCl, pH 7.6, 1 mM CaCl2, 0.2 %
Triton X-100). DNA was digested to 150–300 bp by
MNase (Sigma) before extensive centrifugation. Four
volumes of ChIP dilution buffer (20 mM Tris-HCl, pH 8.0,
150 mM NaCl, 2 mM EDTA, 1 % Triton X-100, 0.1 %
SDS) was added to the supernatant. The resulted lysate
was then incubated with protein G beads and antibodies
at 4 °C over night. The beads were washed five times and
DNA was eluted by Chip elution buffer (0.1 M NaHCO3,
1 % SDS, 20 μg/ml proteinase K). The elution was
incubated at 65 °C over night 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
sequences of primers are in Additional file 2: Table S16.
Pipeline of RNA-seq analysis
mRNA-seq library was performed by using Illumina
TruSeq library construction kit. A 5 μg of total RNA was
used as initiation and then prepared according to the
manufacturer’s instruction. mRNA-seq libraries were
sequenced using HiSeq2000 for 100-bp 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 were used to find out differential
expression genes. Gene ontology analysis was performed
using DAVID (http://david.abcc.ncifcrf.gov) [
Pipeline of ChIP-seq analysis
ChIP was performed using desired antibodies. Library was
prepared using Illumina TruSeq kit according to the
manufacturer’s procedure. Briefly, DNA was prepared for end
repair and “A” tailing, adaptor ligation, and library
amplification. ChIP-seq libraries were sequenced on HiSeq 2000
for 100-bp paired-end sequencing.
Quality control of ChIP-seq data was performed using
Fastqc, and then low quality bases and adaptor
contamination were deleted. After quality control and
data filtering, data were mapped to hg19 using BWA aln
algorithm. Since H3K9me2 and H3K9me3 appear large
scale in chromatin, SICER software was used for peaks
calling with window size 1000 and gap size 10,000.
H3K9me2 and H3K9me3 enrichment region gene
annotation was performed using RefSeq gene reference [
Gene ontology analysis and KEGG pathway analysis
were performed using DAVID.
TCGA breast cancer differential expression gene analysis
The gene expression data pf 100 paired breast cancer
and normal tissues were downloaded from TCGA data
portal for analysis of differential expressed genes
(https://tcga-data.nci.nih.gov/tcga/, the data of total 101
pairs were downloaded but that of one cancer tissue
were not readable, so only 100 pairs were used). In order
to figure out significant differential expression genes
between cancer sample and normal tissue, genes’
expression level less than 5 FPKM in all 201 samples were
deleted, and then ANOVA analysis was performed for
the rest genes with P value cutoff 0.001. After ANOVA
analysis, genes’ average expression level between cancer
and normal tissues less than twofold change was deleted.
Gene ontology analysis of differential expression genes
was performed using DAVID.
Cell cycle analysis with flow cytometry
Cells were harvested after digestion with 0.05 %
Trypsin-EDTA. The cells were then washed twice with
PBS and fixed in ice-cold 70 % ethanol overnight. Fixed
cells were washed twice with PBS and stained in PBS
containing propidium iodide (PI, 50 μg/mL) and RNase
(100 μg/mL) for 30 min at 37 °C. Cell cycle analysis was
performed on an Epics XL-MCL flow cytometer (Beckman
Coulter) with System II (version 3.0) software (Beckman
Coulter). Additional analysis of cell cycle distribution was
determined using Flowjo software.
Cell viability assay
Cell viability was performed by the MTT assay as previously
]. Briefly, cells were split at 1 × 103 per well in
96-well plates. Next every 24 h, the cells were added with
MTT (0.25 μg) in each well for 4 h at 37 °C; the medium
with the formazan sediment was dissolved in 50 % DMF
and 30 % SDS (pH 4.7). The absorption was read at 570 nm.
Colony formation assay
The bottom layer of 0.6 % agar noble in medium was
first placed onto 6-well plate. Cells were seeded in
0.35 % top agar containing medium. Fresh top agar was
added 1.5 weeks later, and colonies were counted 8 weeks
later. For HMCs, the 50,000 cells were seeded while
5000 or 10,000 cells were seeded for T47D.
Cell migration and invasion assay
The RTCA assay was done as the manufacturer’s protocol.
Cells were cultured at 6000 per well in CIM-Plate wells
coated with (invasion) or without (migration) matrigel.
The cell index signals were read by xCELLigence RTCA
DP Analyzer (ACEA Bioscience Inc.). Invasion and
migration are monitored continuously over a 48-h period.
The transwell assay was done as follows. Briefly, cells were
split at 1 × 105 per well in 24-well transwell plates coated
with (invasion) or without (migration) matrigel. The cells
were fixed in 4 % PFA and stained by crystal violet after
48 h. The positive cells were counted under microscope.
Each experiment was repeated three times and results
were presented as mean ± SD.
Cancer tissue collection
All the cancer tissues are collected after obtaining the
consents of the patients. All the experiments are carried
out in accordance with the approved guidelines and
protocols by Medical Ethics Committee of Zhongnan
Hospital, Wuhan University.
The data have been uploaded to GEO database and can
be found at the following URL: http://www.ncbi.nlm.
Additional file 1: Figures S1–S7. Figure S1. Establishment of
fourstage breast cancer model. Figure S2. Gene expression profiling of TCGA
breast cancer tissues and transformed HMC cell lines. Figure S3. Analysis
of H3K9me2 peaks on chromatin. Figure S4. Transcriptional program
change of genes with decreased H3K9me2. Figure S5. RNA and protein
levels of epigenetic enzymes in the tumor model cell lines. Figure S6.
KDM3A deficiency impairs the transcriptional program of cancer-related
genes. Figure S7. KDM3A regulates the growth of breast cancer cells.
(PDF 4169 kb)
Additional file 2: Tables S1–S16. Table S1. Differential expression
genes between L and P6 cell lines. Table S2. Differential expression
genes between LT and P6 cell lines. Table S3. Differential expression
genes between LTR and P6 cell lines. Table S4. DEGs between LT and L
cell lines. Table S5. DEGs between LTR and LT cell lines. Table S6. DEGs
between TCGA breast cancer and normal tissues (≥threefolds). Table S7.
Genes overlapped between DEGs of TCGA and transformed cell lines.
Table S8. Genes localized in H3K9me3 small LOCKs of HMC-p6. Table
S9. Genes localized in decreased H3K9me2 boundaries in HMC-LT.
Table S10. Genes localized in decreased H3K9me2 boundaries in HMC-LTR.
Table S11. Uniprot cancer-related genes in the boundaries of decreased
H3K9me2 LOCKs (compared to HMC-p6). Table S12. Genes overlapped
between LT_NC/LT_shKDM3A and LT/P6 DEGs. Table S13. DEGs between
LT_NC and LT_shKDM3a cells. Table S14. Genes overlapped between
LTR_NC/LTR_siKDM3A and LTR/P6 DEGs. Table S15. DEGs between
LTR_NC and LTR_siKDM3a cells. Table S16. Primer and siRNA
sequences. (XLS 1135 kb)
The authors declare that they have no competing interests.
ZQY performed most of the experiments; ZJY and WHY helped in the
experiments; LPJ prepared the libraries for deep sequencing, and LPJ, ZX,
and WG analyzed the data; ZJ, LYM, and YM helped in collecting the breast
cancer tissues; WM directed the project and wrote the manuscript; LLY and
WG discussed and revised the manuscript. All authors read and approved
the final manuscript.
We thank Dr. Man Mohan (Shanghai Jiao Tong University, China), Dr.
Hong-Bing Shu (Wuhan University, China), and Dr. Wei Xie (Tsinghua
University, China) for discussion and revision of the manuscript.
This work was supported by grants from the National Basic Research
Program of China (973 Program, 2011CB504206 and 2012CB518700) and the
National Natural Science Foundation of China to Min Wu (31470771 and
91019013) and Lianyun Li (31221061, 31200653, and 31370866).
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