The LIM Protein AJUBA Recruits Protein Arginine Methyltransferase 5 To Mediate SNAIL-Dependent Transcriptional Repression

Abstract

The SNAIL transcription factor contains C-terminal tandem zinc finger motifs and an N-terminal SNAG repression domain. The members of the SNAIL family have recently emerged as major contributors to the processes of development and metastasis via the regulation of epithelial-mesenchymal transition events during embryonic development and tumor progression. However, the mechanisms by which SNAIL represses gene expression are largely undefined. Previously we demonstrated that the AJUBA family of LIM proteins function as corepressors for SNAIL and, as such, may serve as a platform for the assembly of chromatin-modifying factors. Here, we describe the identification of the protein arginine methyltransferase 5 (PRMT5) as an effector recruited to SNAIL through an interaction with AJUBA that functions to repress the SNAIL target gene, E-cadherin. PRMT5 binds to the non-LIM region of AJUBA and is translocated into the nucleus in a SNAILand AJUBA-dependent manner. The depletion of PRMT5 in p19 cells stimulates E-cadherin expression, and the SNAIL, AJUBA, and PRMT5 ternary complex can be found at the proximal promoter region of the E-cadherin gene, concomitant with increased arginine methylation of histones at the locus. Together, these data suggest that PRMT5 is an effector of SNAIL-dependent gene repression.The SNAG family of zinc finger transcription factors in vertebrates include GFI-1A, GFI-1B, the insulinoma-associated protein IA-1, the homeobox protein GSH-1, and the SNAIL/SLUG family. These proteins play important roles in the regulation of development, stem cell self-renewal, and tumor progression (5, 22, 49). They share a common set of functional domains: a C-terminal DNA binding domain composed of five to seven Cys2-His2 zinc fingers and a highly conserved N-terminal repression domain designated SNAG. The SNAG motif was first identified from the GFI-1 protein and comprises the first 21 amino acid residues in the N terminus. The SNAG domain is a potent and transferable repression motif (22, 49). However, unlike other repression domains which are associated with zinc finger proteins, such as the KRAB domain and the BTB-POZ domain, whose mechanisms of repression are well established, little is known about the mechanisms of the SNAG domain-mediated repression (9, 15).The SNAIL protein has emerged as a potent regulator of the processes of embryonic development and tumor progression through the regulation of the epithelial-mesenchymal transition (EMT) (5, 36). In mammalian cells, SNAIL induces EMT at least partially through repression of the E-cadherin gene, thereby altering cell adhesion (6). The SNAIL protein has been found in multiprotein complexes containing histone deacetylases (HDACs), mSIN3A, and LOXL2/3 (39, 40). However, the biological significance of these interactions and how SNAIL mediates functional protein complex assembly at specific promoters in the context of chromatin remain undefined.We previously identified novel corepressors that directly bind to the SNAG domains of GFI-1 and SNAIL by using yeast t...

The LIM protein Ajuba promotes adipogenesis by enhancing PPARγ and p300/CBP interaction

“…Ajuba further recruits Prmt5 and HDACs through its preLIM region to repress E-cadherin expression, a known Snail target gene. 17,18,20,21 Moreover, we demonstrated that Ajuba contains functional NR boxes and directly interacts with RARα, 22 where Ajuba functions as a co-repressor and negatively regulates RAR signaling. Here, we describe the novel findings that Ajuba interacts with PPARγ via its non-NR box module within the preLIM region and functions as a co-activator for PPARγ by recruiting p300/CBP via its LIM domain.…”

“…In the nucleus, Ajuba functions as a co-repressor for various transcription factors such as Gfi-1, Snail and ISI1 by recruiting HDACs and PRMT5. 17,18,20,21,41 We recently discovered that Ajuba contains three conserved functional nuclear receptor (NR) boxes mediating a direct interaction with RARα and functions as a typical co-repressor for RARα in a ligand-dependent manner. 22 Here, we demonstrate that Ajuba interacts with PPARγ by different mechanism: the conserved NR boxes of Ajuba do not contribute to the interaction between Ajuba and PPARγ, instead, a region in the preLIM is required for the interaction with PPARγ; Ajuba binds the DNA-binding domain of PPARγ in a ligand-independent manner.…”

“…Myc or Flag-tagged Ajuba full-length and its truncation mutants were previously described. 20 The murine Ajuba cDNA was subcloned into pBabe-puro vector and the Myc-tag was added at the N-terminus of Ajuba protein. We used the pBabe-Ajuba plasmid as a template and subcloned the Ajuba-coding DNA sequence into pET28a-vector and got the pET28a-Ajuba plasmid with the His tag at the N-terminus of Ajuba.…”

“…16,19 In addition, the preLIM region also mediates the protein-protein interaction, such as HDACs and Prmt5. 20,21 Depending on cell types, Ajuba can be located near the membrane, in the cytoplasm and/or in the nucleus and the stimulation of signals or interaction with proteins will also change its localization in cells. [16][17][18]22 Ajuba functions as a scaffold involving assembly of multiple protein complexes to regulate cell adhesion, migration, mitosis, microRNA maturation, cell differentiation and tissue development.…”

“…[16][17][18]22 Ajuba functions as a scaffold involving assembly of multiple protein complexes to regulate cell adhesion, migration, mitosis, microRNA maturation, cell differentiation and tissue development. [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] Ajuba can directly participate in transcriptional regulation where it serves as a transcriptional co-repressor and downregulate gene expression. We lately identified Ajuba as a co-repressor for Snail and as an essential regulator of mesenchymal-epithelial transition and metastasis.…”