LIM protein Ajuba functions as a nuclear receptor corepressor and negatively regulates retinoic acid signaling

Abstract

Corepressors play an essential role in nuclear receptor-mediated transcriptional repression. In general, corepressors directly bind to nuclear receptors via CoRNR boxes (L/I-X-X-I/V-I) in the absence of ligand and appear to act as scaffolds to further recruit chromatin remodeling complexes to specific target genes. Here, we describe the identification of the multiple LIM domain protein Ajuba as a unique corepressor for a subset of nuclear hormone receptors. Ajuba contains functional nuclear-receptor interacting motifs and selectively interacts with retinoic acid receptors (RARs) and rexinoid receptor (RXRs) subtypes in a ligand-dependent manner. Simultaneous mutation of these motifs abolishes RAR binding and concomitantly leads to loss of repression on RARE reporter genes. P19 cells depleted of Ajuba are highly sensitized to all-trans retinoic acid (atRA)-induced transcription and differentiation. In the absence of atRA, Ajuba can be readily found at the RARE control elements of RAR endogenous target genes. Stimulation of cells with atRA results in the dissociation of Ajuba from these regions. Moreover, we observed that coexpression of the known Ajuba binding partner Prmt5 (protein arginine methyltransferase-5) inhibited the Ajuba/RAR interaction. The high-affinity Ajuba-RAR/RXR interaction site overlaps the region responsible for Ajuba/Prmt5 binding, and thus binding appears to be mutually exclusive, providing a potential mechanism for these observations. Identification of Ajuba as a unique corepressor for nuclear receptors sheds new light on mechanisms for nuclear receptor-mediated repression and provides a unique target for developing more effective therapeutics to modulate this important pathway.hox genes | differentiation | LIM domain | retinoic acid receptors

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

“…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. These findings suggest that PPARγ and Ajuba may contain unique modules to mediate their specific interaction and protein complex assembly.…”

“…To examine the role of Ajuba in PPARγ-mediated transcriptional activity, we first depleted Ajuba in 3T3-L1 cells using lentiviral shRNA specifically targeting Ajuba (Figure 3a). 22 The resulting cells were then treated with Rosi (1μM) or DMSO for 16 h and the total mRNAs were extracted for qRT-PCR analysis. We chose FABP4, LPL, PLIN-1 and CD36, the known target genes of PPARγ, as representatives to evaluate the transcriptional activity of PPARγ.…”

“…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.…”

“…We previously demonstrated that Ajuba preferentially interacts with RXRγ (22). Τo determine if Ajuba affects the PPARγ/ RXR heterodimerization, we expressed Flag-PPARγ2 and HA-RXRα, RXRγ or HA-CBP, and together with increasing amount of Myc-Ajuba in 293 T cells.…”

“…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][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.…”