Previous results have demonstrated that the bZIP protein SMILE plays an important role in repressing the replication of the herpes simplex virus (1
) and serves as a coregulator in ER signaling (2
). The results presented in this study extend the role of SMILE in NR signaling. SMILE inhibited GR-, HNF4α- and CAR-mediated transcriptional activity through direct binding to the LBD/AF2 domain of the NRs. Moreover, the knockdown of SMILE gene expression increased the GR, HNF4α and CAR transactivation. Furthermore, the overexpression of SMILE via adenovirus vector inhibited the transcription of the NRs’ target genes, including IGFBP-1, CYP2B6 and CYP7A1. In addition, SMILE also inhibited the transactivation by receptor LXR, FXR, Nur77 and ERRγ through direct interactions (data not shown). These findings indicate that SMILE may be an important modulator of NR signaling.
We have investigated the roles of potential functional domains of SMILE for its repressive function, including the leucine zipper motif (1
), the HCF-binding motif (HBM) (1
) and the LXXLL motifs (NR boxes) (25
). The leucine zipper region is known to be essential for the dimerization and functions of b-zip proteins (44
). For instance, the leucine zipper of cyclic AMP response element-binding (CREB) protein is required for the dimerization and transcriptional activation (35
). By way of contrast, our findings support the notion that the bZIP region of SMILE is required for the homodimerization, but is not essential for the repressive effect of SMILE on GR and CAR (). It has been reported that Jun dimerization protein 2 (JDP-2) functions as a progesterone receptor (PR) coactivator through direct interaction via the DBD of PR and the bZIP region of JDP-2 (45
). However, the domain-mapping results have demonstrated that the bZIP region of SMILE is not involved in the interactions with GR, CAR and HNF4α (). Although HBM-mediated association of SMILE with HCF is required for SMILE to repress CREB3 (5
), our reporter assay results have shown that wild-type SMILE and HBM-defective SMILE mutant (Y306A), which was demonstrated not able to interact with HCF (1
), have similar inhibitory effect on GR, CAR and HNF4α (Supplementary ), indicating the repression of the NRs by SMILE is independent of HBM.
LXXLL motif is commonly found in NR coregultors and has been reported to be important for coregulators function through interaction with the LBD/AF2 domain of NRs (25
). The results of domain-mapping analysis manifests that SMILE binds to the LBD/AF2 domain of GR, CAR and HNF4α through the region spanning residues 113–202, which contain a LXXLL motif. Surprisingly, we found that the repressive effects of SMILE on GR, CAR and HNF4α were not significantly changed by single mutation or combinatorial mutation of four LXXLL motifs (Supplementary ), indicating that LXXLL motifs are not essential for the interactions and repressive effects of SMILE in the cases of GR, CAR and HNF4α. Interestingly, this LXXLL-independent interaction was also observed between proline-rich nuclear receptor coregulatory protein (PNRC) and LBD of ERα (46
). In addition of using LXXLL motifs to interact with NRs, corepressor RIP140 also uses its C-terminus, which contains no LXXLL motifs, to interact with LBD of NRs (40
). However, it remains to be determined whether the LXXLL motifs are also dispensible for the repressive effect of SMILE on other NRs, such as Nur77, LXR and FXR.
We have recently reported that SMILE functions as a coregulator in ER signaling in association with SHP. The regulation of ER by SMILE depends on the existence of SHP in breast cancer MCF-7 cells (2
). In contrast, the results of our siRNA knockdown experiments indicate that SHP is not involved in the SMILE-mediated repression of GR, CAR and HNF4α (data not shown). In our previous study, SMILE regulates the inhibition of ER by SHP in a cell-type specific manner (2
). However, the repression of GR, CAR and HNF4α by SMILE is not cell-type specific, since similar repressive effects were observed in 293T, HepG2 and HeLa cells (data not shown).
Our results suggest that multiple mechanisms are involved in SMILE-mediated repression. One such mechanism could be competition with coactivators such as GRIP and PGC-1α, which is a common mechanism among certain NR corepressors, including SHP (31
), DAX-1 (29
), RIP140 (43
) and the ligand-dependent corepressor (LCoR) (47
). Interestingly, besides coactivator competition, SMILE has an intrinsic repressive function, like the corepressors SHP (42
) and RIP140 (41
). Moreover, we found that SMILE specifically interacts with HDAC1, HDAC3 and HDAC4. The inhibition of HDAC activity using the HDAC inhibitor TSA, or the knockdown of the HDACs gene expression through siRNA partially released the repression of GR and HNF4α by SMILE. In contrast, TSA showed little effect on the repression of CAR by SMILE, indicating HDAC-dependent and -independent mechanism of repression. Consistently, our ChIP assay results also evidenced that TSA was able to prevent SMILE-associated deacetylation of histone H3 on GR and HNF4α target gene promoters, but not on CAR target gene promoter. Of note, the TSA-sensitive and -insensitive actions of SMILE are similar to several other corepessors, including RIP140 (41
) and LCoR (47
). In addition, HDAC1, HDAC3 and HDAC4 are required for the repression of HNF4α by SMILE, whereas HDAC1 is not essential for the repression of GR, indicating that SMILE associations with HDACs exhibits promoter specificity. Similar phenomenon has been reported with the corepressors NCoR and SMRT (26
It is worth noting that the inhibition of DNA binding is one of the common repression mechanisms utilized by certain corepressors. For instance, this mechanism underlies the inhibition of TR and GR by tumor suppressor p53 (48
), and the inhibition of hepatic nuclear factor-3 (HNF3) family by the corepressor SHP (30
). However, our results indicate that the inhibition of DNA binding is not involved in the repression of GR, CAR, and HNF4α by SMILE, as the recruitment of SMILE exerted no detectable effect on the binding of the NRs to the promoters of IGFBP1, CYP2B6 and CYP7A1 (A–C). Whether this mechanism is involved in the inhibitory effect of SMILE on other NRs, including Nur77, LXR and FXR, still needs to be clarified.
GR, CAR and HNF4α are crucial for liver function, including the regulation and processing of glucose, lipids, amino acids and drug metabolism, as well as bile acid homeostasis (14
). Therefore, the repression of their transcriptional activity by SMILE indicates that SMILE may function as a negative coregulator in the aforementioned physiological processes. It has been reported that as integrators of various biological processes, several transcriptional coregulators are regulated by distinct nutritional and hormonal signals (51
). For example, activation of cAMP signaling by fasting induces the coactivator PGC-1α expression in hepatocytes, whereas the activation of insulin-signaling pathway by refeeding exhibits quite opposite effect (51
). Increased bile acid levels switch on the feedback pathway of bile acid synthesis through induction of the corepressor SHP (52
). Therefore, it would be necessary to study the regulation of SMILE gene expression by diverse physiological settings and intracellular signaling pathways, which is currently under investigation. Moreover, to better understand the function of SMILE in those aforementioned physiological processes, the SMILE knockout and transgenic animal model will be useful. In addition, the identification of more SMILE-interacting proteins and the elucidation of SMILE crystal structure will be helpful to illuminate the detailed mechanism of SMILE-mediated repression.
In summary, we have identified that SMILE represses GR-, CAR- and HNF4α-mediated transactivation through direct interaction. At least two mechanisms are involved in SMILE-mediated repression of the NRs, competition with coactivators, and active repression through the recruitment of HDACs. Taken together, these observations indicate that SMILE is novel corepressor and may play an important role in NR signaling.