Posttranslational Regulation
Emerging evidence has shown that subcellular localization and activity of Snail1 are regulated at the posttranslational levels. Although the reverse correlation between Snail1 and E- cadherin expression has been well established, in some cases, the mRNA of both genes co-exists in the same tissues or cancer cell lines (Dominguez et al 2003, Hajra et al 2002), suggesting that
Snail1 is not only regulated at the transcriptional level, but also at the translational and posttranslational levels.
Recently, a proposed model suggested that Snail1 is regulated by glycogen synthase kinase 3 β (GSK3β)-mediated phosphorylation. The model implicates a two-stage phosphorylation of Snail1 by GSK3β at two consensus motifs identified between residues 92 to 121 (Zhou et al 2004b). According to this model, nuclear GSK3β phosphorylates Snail1 at regulatory motif 2, thus inducing its nuclear export. In the second step, cytoplasmic GSK3β phosphorylates motif 1, which results in the association of Snail1 with β-Trcp E3 ubiquitin ligase and the subsequent polyubiquitination and protein degradation. This model provides an explanation that controls the protein stability of Snail1 and explains its short half-life (around 30 min) in some cell lines.
A recent study showed that two lysine residues (K98 and K137) are essential for the stability and activity of Snail1 protein, as double mutation of these two residues abolishes its function as a transcription repressor of E-cadherin (Peinado et al 2005). These two lysine residues are essential for Snail1 to interact with lysyl-oxidase homolog 2 (LOXL2), by which it attenuates GSK3β-mediated Snail1 degradation. Another study also showed that p21-activated kinase 1 (Pak1) modulates the repressor activity of Snail1 by phosphorylating its serine 246 residue. Phosphorylation of Snail1 by Pak1 facilitates its nuclear accumulation and thus enhances its repressor activity (Yang et al 2005e). Taken together, different posttranslational modifications play a role in determining the cellular localization and the activity of Snail1, suggesting a context-dependent regulatory mechanism in the cells.
Transcriptional Regulation of Snail1
Transcriptional regulation is considered a major mechanism to regulate Snail1 expression during the embryonic development and tumor progression. Many signaling pathways, as mentioned above, can stimulate the expression of Snail1. In contrast, studies in breast cancer cells revealed that activation of the ER pathway suppresses Snail1 expression via the involvement of the metastasis-associated gene 3 (MTA3), a subunit of the nucleosome remodeling and histone deacetylation (NuRD) repressor complex (Fujita et al 2003). The promoter study in Snail1 identified several conserved response elements, including two E-boxes, AP1, AP4, SMAD, and LEF1 binding sites (Barbera et al 2004, Peinado et al 2003). Snail1 was found to limit its own expression by binding to the E-box of the promoter, suggesting a delicate feedback mechanism of controlling Snail1 transcription. Although abundant studies demonstrated that Snail1 can be regulated at transcriptional level (Batlle et al 2000, Cano et al 2000, Cheng et al 2001, Jiao et al 2002), the molecular basis behind this mechanism remains elusive and needs to be explored.
Mi-2/Nucleosome Remodeling and Deacetylase (NuRD) Complex
The Mi-2/nucleosome remodeling and deacetylase (NuRD) complex is identified as a multi-subunit protein complex with the unique property of combining both histone deacetylase and chromatin remodeling ATPase activities (Tong et al 1998, Wade et al 1998, Xue et al 1998b, Zhang et al 1998). The ATPase activity of Mi-2/NuRD complex resides in Mi-2 α and/or β proteins, both of which belong to the SWI/SNF chromatin remodeling family that functions as transcription regulators through sliding or ejecting the nucleosome structure (Eisen et al 1995). The deacetylase subunits of Mi-2/NuRD complex comprise HDAC1 and/or HDAC2. Other subunits, such as the methyl CpG-binding domain (MBD) family of proteins, Rbbp4 and Rbbp7,
p66α and p66β, and the metastasis associated (MTA) protein family, are also identified in this complex (Wade et al 1999, Zhang et al 1999a). Two representatives of the MBD family of proteins, MBD2 and MBD3, have displayed a role in coupling chromatin remodeling and DNA methylation in Mi-2/NuRD-mediated gene regulation (Wade et al 1999, Zhang et al 1999a). The mutation analyses in mouse models and embryonic stem cells have provided evidence that MBD2 and MBD3 are critical for gene regulation during development (Hendrich et al 2001, Kaji et al 2006).
The MTA family is composed of three members: MTA1, MTA2, and MTA3. All three have been demonstrated to be components of the Mi-2/NuRD complex (Fujita et al 2003, Xue et al 1998b, Zhang et al 1999a). The function of MTA proteins is correlated intimately with the ER signaling in the development of both normal mammary gland and breast cancer (Manavathi et al 2007). MTA1 was originally identified in breast cancer cell lines with high metastatic potential (Toh et al 1994). A study at molecular level revealed that MTA3 gene is activated through the direct binding of ERα on its promoter. Furthermore, MTA3 protein serves as a corepressor in the Mi-2/NuRD complex to repress the transcription of Snail1, which in turn, suppresses the invasive growth in breast cancer cells (Fujita et al 2003). Interestingly, studies in transgenic mice demonstrated that MTA3 is implicated in suppression of ductal branching in the mammary gland (Zhang et al 2006), whereas MTA1 exhibits an opposite effect with more branching phenotypes (Bagheri-Yarmand et al 2004). These findings suggest that the involvement of different members of MTA proteins can direct the Mi-2/NuRD complex to achieve a unique function in a given cellular or tissue context. Based on the current understanding, a model was proposed to demonstrate a mechanism of transcription repression that involves Mi-2/NuRD complex, as Fig.1.6 shows. Although it is clear that the incorporation of different subunits is required for the
Mi-2/NuRD complex to perform its unique function, to what extent these molecules participate in transcription repression remains a question and needs further effort to uncover the mechanistic links.