Snail Mediates E-Cadherin Repression by the Recruitment of the Sin3A/Histone Deacetylase 1 (HDAC1)/HDAC2 Complex
Abstract
The transcription factor Snail has been described as a direct repressor of E-cadherin expression during development and carcinogenesis; however, the specific mechanisms involved in this process remain largely unknown. Here we show that mammalian Snail requires histone deacetylase (HDAC) activity to repress E-cadherin promoter and that treatment with trichostatin A (TSA) is sufficient to block the repressor effect of Snail. Moreover, overexpression of Snail is correlated with deacetylation of histones H3 and H4 at the E-cadherin promoter, and TSA treatment in Snail-expressing cells reverses the acetylation status of histones. Additionally, we demonstrate that Snail interacts in vivo with the E-cadherin promoter and recruits HDAC activity. Most importantly, we demonstrate an interaction between Snail, histone deacetylase 1 (HDAC1) and HDAC2, and the corepressor mSin3A. This interaction is dependent on the SNAG domain of Snail, indicating that the Snail transcription factor mediates the repression by recruitment of chromatin-modifying activities, forming a multimolecular complex to repress E-cadherin expression. Our results establish a direct causal relationship between Snail-dependent repression of E-cadherin and the modification of chromatin at its promoter.
The regulation of E-cadherin expression is a controlled process that requires strict spatiotemporal tuning during natural processes such as development, organogenesis, and tissue formation. However, the regulation of E-cadherin also plays an essential role in pathological processes such as tumor progression. The loss of expression or function of the E-cadherin cell-to-cell adhesion molecule has emerged as an important event for the local invasion of epithelial tumor cells, leading to the consideration of E-cadherin as an invasion suppressor gene (7, 8, 14, 49).
The molecular mechanisms involved in E-cadherin downregulation during physiological and pathological processes have started to be uncovered in recent years. Several mechanisms have been implicated in the regulation of E-cadherin expression during tumor progression, including genetic, epigenetic, and transcriptional changes. While genetic alterations of the E-cadherin loci have been found only infrequently in tumors, particularly, in lobular breast carcinomas and diffuse gastric carcinomas (6, 7, 21, 44), the majority of carcinomas with downregulated E-cadherin maintain an intact E-cadherin locus. Epigenetic processes involving hypermethylation of the E-cadherin promoter and/or transcriptional alterations have emerged as the main mechanisms responsible for E-cadherin downregulation in most carcinomas (13, 14, 23, 42). Several transcriptional repressors of E-cadherin have been recently identified, including the zinc finger factors Snail (5, 11) and Slug (10, 22), the two-handed zinc factors ZEB1(δEF1) and ZEB2 (SIP-1) (15, 20), and the bHLH factor E12/E47 (40). Factors belonging to the Snail family are in fact involved in E-cadherin repression and in epithelial to mesenchymal transitions (EMTs) when they are overexpressed in epithelial cell lines (5, 10, 11) as well as in embryonic development (reviewed in reference 37), and it has been proposed that these factors act as inducers of the invasion process (9, 11). The generation of mice lacking Snail has firmly established the role of this factor in EMT and as an E-cadherin gene repressor, as the null Snail embryos die at gastrulation and fail to undergo a complete EMT process, forming an altered mesodermal layer which maintains the expression of E-cadherin (12). Despite all the above information, the molecular mechanisms involved in the repression by factors of the Snail family are still poorly understood (37, 50). A previous work established that human Slug, a Snail family member, is a transcriptional repressor with an N-terminal 32-amino-acid repression domain and postulated the possible involvement of histone deacetylation in the repression mechanism (26).
Chromatin remodeling and histone modifications have emerged as the main mechanisms of the control of gene expression. Hyperacetylation of histones H3 and H4 is generally associated with transcriptionally active chromatin (47), while the chromatin of inactive regions is enriched in deacetylated histones H3 and H4. The acetylation status of histones at specific DNA regulatory sequences depends on the recruitment of histone acetyltransferases or histone deacetylase (HDAC) activities, usually as part of large multiprotein complexes of coactivators or corepressors, respectively. Several corepressor complexes have been identified to date (such as the SIN3, Mi-2/NuRD, and CoREST complexes) with the ability to interact with several transcriptional repressors (1, 27). Interestingly, during the past 5 years, the connection between DNA methylation and histone deacetylation in the silencing of genes has been established, and the mechanisms involve the participation of proteins belonging to the family of methyl-CpG binding domain proteins and HDACs (4). Moreover, other histone modifications, such as histone methylation, appear to be associated with gene regulation (32), thus suggesting the participation of different histone and DNA modifying activities in multiprotein complex regulators.
To gain further understanding of the mechanisms implicated in E-cadherin repression by Snail, we have investigated the involvement of HDACs and other potential corepressors. We report here that the endogenous E-cadherin promoter of Snail-expressing cells is enriched in deacetylated histones H3 and H4 and dimethylated H3 at K9 and that Snail-mediated repression is abolished by treatment with trichostatin A (TSA). Snail interacts directly with the endogenous E-cadherin promoter, as demonstrated by chromatin immunoprecipitation (ChIP) assays, and recruits HDAC activity. Moreover, in vivo and in vitro interactions of Snail with histone deacetylase 1 (HDAC1) and HDAC2 and the corepressor mSin3A have been detected. These interactions depend on the SNAG N-terminal domain of Snail and are required for an efficient repression of the E-cadherin promoter, which supports the idea that Snail mediates the repression of E-cadherin by the recruitment of a corepressor complex containing HDAC1 and HDAC2 (HDAC1/2) and Sin3A.
Acknowledgments
We thank E. Seto and R. N. Eisenman for providing reagents and A. Montes for her excellent technical assistance. Special thanks go to D. Megias and M. Cortés-Canteli for helping us with the confocal immunofluorescence analysis and to P. de la Peña-Ingelmo and J. Manzano for their suggestions in PCR experiments.
A. Cano's laboratory is supported by Spanish Ministry of Science and Technology grant SAF2001-2819 and by grants from the Instituto de Salud Carlos III (01/1074 and 031C03/10). M. Esteller's laboratory is supported by Spanish Ministry of Science and Technology grant SAF2001-0059 and the International Rett Syndrome Association. H. Peinado is a predoctoral fellow of the Spanish Ministry of Education, Culture and Sports. E. Ballestar is funded by the Ramón y Cajal Programme of the Spanish Ministry of Science and Technology.
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