Our study provides several insights into the epigenetic program in EMT, CLBC, and metastasis. First, we have identified a key mechanism underlying the epigenetic regulation of EMT and reinforce the notion that EMT is an epigenetically regulated program. To uncover the mechanism of epigenetic regulation of EMT, we examined the dynamic chromatin modifications at the promoter of E-cadherin, a typical epithelial molecule and trait marker of EMT, in three model cell lines that are commonly used for EMT induction. The gradual increase in H3K9me2 and decrease in H3K9 acetylation correlated with the timing of Snail induction, the morphological changes indicative of EMT, and the de novo DNA methylation of the E-cadherin promoter. These findings suggest that H3K9me2 plays a critical role in silencing the expression of E-cadherin. G9a, a key methyltransferase responsible for H3K9me2 at euchromatin and facultative heterochromatin, does not contain a DNA binding sequence. We found that Snail interacted with G9a both in vitro and in vivo and was required for G9a recruitment to the E-cadherin promoter (Figure C). Consistent with this finding, the immunoprecipitated Snail complex contained G9a methyltransferase activity, and knockdown of Snail expression disrupted the association of G9a with the E-cadherin promoter. The domains responsible for their interaction were mapped to the ankyrin repeat and SET domains of G9a and the C-terminal zinc finger region of Snail, respectively. This is consistent with the observation that the interaction of Snail with G9a is independent of the catalytic activity of G9a. The transcriptional repressive activity of Snail requires both the N-terminal SNAG domain and the C-terminal zinc finger region (7
). We and others showed previously that the SNAG domain of Snail interacted with LSD1 and Sin3A/HDAC for H3K4 demethylation and histone deacetylation, respectively (45
). However, H3K4 demethylation is known to be an initial step in gene repression (47
), suggesting that an intermediate step is required to bridge H3K4 demethylation to the DNA methylation on the E-cadherin promoter. Here we identified that the C terminus of Snail interacted with G9a directly, which recruited DNMT to the E-cadherin promoter for DNA methylation. Thus, demethylation on H3K4 and methylation on H3K9 by LSD1 and G9a, respectively, provide great synergy in gene repression and DNA methylation (17
). Consistent with this observation, a zinc finger transcriptional repressor, REST, also recruits LSD1 and G9a through its C-terminal domain and middle region, respectively, in repressing neuronal gene expression (49
Histone methylation is intimately linked to DNA methylation, which provides reinforcement as well as establishment of gene silencing. DNA methylation is executed by a family of highly related DNA methyltransferase enzymes (DNMT1, DNMT3a, and DNMT3b) that transfer a methyl group to the cytosine in a CpG dinucleotide, which commonly occurs in the promoter region of genes (17
). Typically, the maintenance of DNA methylation in somatic cells is attributed to DNMT1, whereas de novo DNA methylation during embryonic development is credited to DNMT3a and DNMT3b. However, there is overlap in the function of these two types of DNMTs, as DNMT1 can also contribute to de novo DNA methylation both in vitro and in vivo, and the maintenance of methylation in certain regions of the genome requires DNMT3a and DNMT3b (17
). We found that Snail can interact with DNMT1, DNMT3a, and DNMT3b, and this interaction is likely to be indirect, as knockdown of G9a expression disrupted the interaction of Snail with DNMTs. Thus, G9a provides a platform for the efficient assembly of the Snail-G9a-DNMTs complex in vivo (Figure C).
Although the causal relationship between H3K9me2 and DNA methylation can be bidirectional, both processes are tightly associated in heterochromatin and transcriptionally repressed euchromatic regions. For example, H3K9me2, catalyzed by G9a, is absolutely required for DNA methylation in fungi, plants, and mammals (50
). Conversely, reactivation of silenced tumor suppressor genes in response to 5′-Aza-dC–induced DNA demethylation is accompanied by a decrease in H3K9me2, but not other silencing markers such as H3K9me3 or H3K27me3 (52
). In this study, we found that H3K9me2 coincided with DNA methylation at the E-cadherin promoter in three model cell lines and CLBC. Knockdown of G9a expression significantly inhibited DNA methylation at the E-cadherin promoter and reactivated E-cadherin expression in MDA-MB231 cells. Our study suggests that G9a-mediated H3K9me2 is one of the key events in the maintenance of transcriptionally silent gene promoters in cancer. In line with our findings, G9a is enriched at the promoters of aberrantly methylated genes in cancer cells, and co-recruitment of G9a, DNMT1, and HP1 to the promoter of the survivin gene stimulates H3K9me2 and DNA hypermethylation (53
). Intriguingly, G9a seems to use two distinct modes for generating DNA methylation at the E-cadherin promoter (Figure C). In the first mode, G9a creates H3K9me2 via its catalytic activity, which subsequently recruits HP1 and DNMTs for DNA methylation (17
). In the second mode, G9a interacts with DNMTs directly and recruits them to the E-cadherin promoter through the association with Snail. This explains why the interaction of the Snail-G9a-DNMT complex does not require G9a activity and indicates that the inhibition of G9a activity does not significantly alter DNA methylation at the E-cadherin promoter.
E-cadherin downregulation is commonly associated with DNA methylation of its promoter (28
), which provides a relative stable “memory” marker for gene silencing. Using a classic TGF-β–induced EMT model in NMuMG cells, which was first identified elegantly by Derynck’s group in 1994 (14
), we found that DNA methylation on the E-cadherin promoter can be reversed (Supplemental Figure 2). Two mechanisms of DNA demethylation have been proposed (19
). The first one is passive DNA demethylation, in which the activity of DNMT is suppressed and DNA methylation cannot be maintained during DNA replication. This results in the loss of DNA methylation after cell propagation (19
). The second mechanism involves active DNA demethylation, in which the 5′-methylcytosine (5 mC) is recognized and removed by DNA mismatch and repair enzymes (55
). The recent identification of ten-eleven translocation 1 (TET1) supports this notion (56
). TET1 converts 5 mC to 5-hydroxymethylcytosine (5 hmC), which precludes the preferentially binding of methyl-CpG–binding proteins (MBDs and MeCP) that commonly associate with histone deacetylases (HDACs) in gene repression (55
). In addition, 5 hmC facilitates DNA demethylation through a process that requires the base excision repair pathway mediated by cytidine deaminases or thymine-DNA glycosylase (57
). In our study, the decreased DNA methylation on the E-cadherin promoter after TGF-β withdrawal suggests that a passive mechanism is involved. This may be due to the downregulation of Snail after TGF-β withdrawal and the consequent loss of G9a and DNMT recruitment to the E-cadherin promoter.
Second, our study has delineated a critical role of the Snail-G9a-DNMT complex in CLBC. Ample evidence supports an epithelial hierarchy within the human breast. This cellular differentiation process starts with an undifferentiated ERα-negative MSC that either maintains itself through self-renewal or differentiates into committed progenitors (4
). These progenitors ultimately give rise to progeny that consist of mature ductal and alveolar cells, which belong to the luminal epithelial cell lineage and line the lumen of the mammary gland, and mature myoepithelial cells, which surround the luminal epithelium and contact the basement membrane (4
). Because the MSC signature was enriched in CLBC, which is characterized by the expression of mesenchymal and stem cell–associated genes and the lack of expression of claudin and E-cadherin, it is speculated that CLBC may originate from the transformation of MSCs that arrest at an early stage of differentiation (2
). Alternatively, this can be mediated by a de-differentiation process, as proposed by Weinberg and colleagues (10
). Both of these phenotypic and cellular conversions require the activation of EMT. Intriguingly, CLBC has many EMT characteristics (1
), suggesting that the activation of EMT blocks cellular differentiation by repressing epithelial molecules, such as E-cadherin in this case. Consistent with this idea, we found that the G9a is critical for EMT in both three model cell lines and CLBC through its interaction with Snail. Although the protein level of G9a and DNMTs remained unchanged between luminal and CLBC cell lines, the association of Snail, G9a, and the level of H3K9me2 at the E-cadherin promoter were dramatically elevated in both CLBC cell lines and tumor samples, indicating the critical role of G9a and H3K9me2 in the epigenetic silencing of the E-cadherin promoter in CLBC. In line with this finding, knockdown of G9a expression downregulated EMT and basal markers (vimentin, N-cadherin, CK6, and EGFR) and upregulated luminal epithelial molecules (ERα, GATA3, CK18, and E-cadherin) and claudin. Furthermore, knockdown of G9a expression suppressed cell migration and invasion in vitro, inhibited metastasis in vivo, and predicted increased survival for patients with breast cancer. Thus, we speculate that G9a is involved in the control of a common epigenetic EMT program to block differentiation toward epithelial or luminal lineage (Figure C).
Third, our study indicates that the cellular plasticity of CLBC represents a potential therapeutic target. Because they are characterized by acquisition of an EMT phenotype and loss of E-cadherin expression, CLBC cells have a distinct advantage in terms of invasion and metastasis to distant organs or tissues during neoplastic development. This EMT program is also indicative of stem cell–like characteristics, making CLBC resistant to apoptosis mediated by standard chemotherapeutics (10
). It is likely that CLBC becomes “addicted” to this program for the advantage of survival and metastasis. Activation of this program can be achieved by genetic mutation/deletion of the E-cadherin gene or by epigenetic reprogramming of gene expression in such a way that abnormal silencing becomes the default state and is inherited by progeny upon cell division. However, while gene mutation is rare in invasive breast ductal carcinoma, promoter DNA methylation is a common mechanism causing the loss of E-cadherin expression in this disease (54
). We found that knockdown of G9a led to the restoration of E-cadherin gene expression and suppression of cell migration, invasion, and metastasis in CLBC. These data suggest that the machinery for E-cadherin expression in these tumor cells remains intact and functional and can mediate reexpression if the repressive signal regulating histone and/or DNA methylation is removed. Thus, the epigenetic program in CLBC may represent a therapeutic target for treating this aggressive and metastatic disease.
In summary, our study highlights the importance of G9a-mediated epigenetic modification in EMT, CLBC, and metastasis. Blocking the binding in Snail-G9a-DNMTs may pave the way for the development of novel therapeutic approaches that target metastatic CLBC.