Characterization of the interaction between androgen receptor and a new transcriptional inhibitor, SHP.

SHP (short heterodimer partner) is an orphan nuclear receptor, first described for its interaction with nuclear receptors. This study explores a new way of inhibiting the androgen-signaling pathway. We demonstrated that SHP inhibited up to 97% of AR-induced activity. Characterization of AR/SHP interaction provided evidence of a clear ligand dependency. We also showed that the LXXI/LL motifs previously found on SHP mediated the interaction with the AR ligand-binding domain (AR-LBD), the motif responsible for the interaction being slightly different from that found with ER. The AR N-terminal domain (AR-NTD), in contrast to that of other nuclear receptors, accounts for most of the entire receptor transactivation potential. SHP also interacted with AR-NTD, thus stabilizing the interaction with AR. We demonstrated that SHP inhibited both AR-LBD and NTD-dependent transactivation, which evidenced for the first time a protein capable of inhibiting a steroid receptor amino-terminal-dependent transactivation. We further characterized the SHP mechanism of action by showing that SHP reversed AR coactivator-mediated activation. Conversely, FHL2 and TIF2 counteracted SHP-mediated inhibition of AR. SHP evidences a new way of inhibiting AR activity by competing with AR coactivators. This new type of inhibitor could dictate the activity of nuclear receptors, depending on the equilibrium between activators and inhibitors.     

Role of histone deacetylation in cell-specific expression of endothelial nitric-oxide synthase

Histone acetylation plays an important role in chromatin remodeling and gene expression. The molecular mechanisms involved in cell-specific expression of endothelial nitric-oxide synthase (eNOS) are not fully understood. In this study we investigated whether histone deacetylation was involved in repression of eNOS expression in non-endothelial cells. Induction of eNOS expression by histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate was observed in all four different types of non-endothelial cells examined. Chromatin immunoprecipitation assays showed that the induction of eNOS expression by TSA was accompanied by a remarkable increase of acetylation of histone H3 associated with the eNOS 5'-flanking region in the non-endothelial cells. Moreover, DNA methylation-mediated repression of eNOS promoter activity was partially reversed by TSA treatment, and combined treatment of TSA and 5-aza-2'-deoxycytidine (AzadC) synergistically induced eNOS expression in non-endothelial cells. The proximal Sp1 site is critical for basal activity of eNOS promoter. The induction of eNOS by inhibition of HDACs in non-endothelial cells, however, appeared not mediated by the changes in Sp1 DNA binding activity. We further showed that Sp1 bound to the endogenous eNOS promoter and associated with HDAC1 in non-endothelial HeLa cells. Combined TSA and AzadC treatment increased Sp1 binding to the endogenous eNOS promoter but decreased the association between HDAC1 and Sp1 in HeLa cells. Our data suggest that HDAC1 plays a critical role in eNOS repression, and the proximal Sp1 site may serve a key target for HDCA1-mediated eNOS repression in non-endothelial cells.     

Molecular characterization of SMILE as a novel corepressor of nuclear receptors

SMILE (small heterodimer partner interacting leucine zipper protein) has been identified as a coregulator in ER signaling. In this study, we have examined the effects of SMILE on other NRs (nuclear receptors). SMILE inhibits GR, CAR and HNF4α-mediated transactivation. Knockdown of SMILE gene expression increases the transactivation of the NRs. SMILE interacts with GR, CAR and HNF4α in vitro and in vivo. SMILE and these NRs colocalize in the nucleus. SMILE binds to the ligand-binding domain or AF2 domain of the NRs. Competitions between SMILE and the coactivators GRIP1 or PGC-1α have been demonstrated in vitro and in vivo. Furthermore, an intrinsic repressive activity of SMILE is observed in Gal4-fusion system, and the intrinsic repressive domain is mapped to the C-terminus of SMILE, spanning residues 203–354. Moreover, SMILE interacts with specific HDACs (histone deacetylases) and SMILE-mediated repression is released by HDAC inhibitor trichostatin A, in a NR-specific manner. Finally, ChIP (chromatin immunoprecipitation) assays reveal that SMILE associates with the NRs on the target gene promoters. Adenoviral overexpression of SMILE represses GR-, CAR- and HNF4α-mediated target gene expression. Overall, these results suggest that SMILE functions as a novel corepressor of NRs via competition with coactivators and the recruitment of HDACs.     

Activation of the growth-differentiation factor 11 gene by the histone deacetylase (HDAC) inhibitor trichostatin A and repression by HDAC3

Histone deacetylase (HDAC) inhibitors inhibit the proliferation of transformed cells in vitro, restrain tumor growth in animals, and are currently being actively exploited as potential anticancer agents. To identify gene targets of the HDAC inhibitor trichostatin A (TSA), we compared the gene expression profiles of BALB/c-3T3 cells treated with or without TSA. Our results show that TSA up-regulates the expression of the gene encoding growth-differentiation factor 11 (Gdf11), a transforming growth factor beta family member that inhibits cell proliferation. Detailed analyses indicated that TSA activates the gdf11 promoter through a conserved CCAAT box element. A comprehensive survey of human HDACs revealed that HDAC3 is necessary and sufficient for the repression of gdf11 promoter activity. Chromatin immunoprecipitation assays showed that treatment of cells with TSA or silencing of HDAC3 expression by small interfering RNA causes the hyperacetylation of Lys-9 in histone H3 on the gdf11 promoter. Together, our results provide a new model in which HDAC inhibitors reverse abnormal cell growth by inactivation of HDAC3, which in turn leads to the derepression of gdf11 expression.     

The N-CoR-HDAC3 nuclear receptor corepressor complex inhibits the JNK pathway through the integral subunit GPS2

The corepressors N-CoR and SMRT partner with histone deacetylases (HDACs) in diverse repression pathways. We report here that GPS2, a protein involved in intracellular signaling, is an integral subunit of the N-CoR-HDAC3 complex. We have determined structural motifs that direct the formation of a highly stable and active deacetylase complex. GPS2 and TBL1, another component of the N-CoR-HDAC3 complex, interact cooperatively with repression domain 1 of N-CoR to form a heterotrimeric structure and are indirectly linked to HDAC3 via an extended N-CoR SANT domain that also activates latent HDAC3 activity. More importantly, we show here that the N-CoR-HDAC3 complex inhibits JNK activation through the associated GPS2 subunit and thus could potentially provide an alternative mechanism for hormone-mediated antagonism of AP-1 function.     

The histone deacetylase inhibitor valproic acid selectively induces proteasomal degradation of HDAC2

Histone-modifying enzymes play essential roles in physiological and aberrant gene regulation. Since histone deacetylases (HDACs) are promising targets of cancer therapy, it is important to understand the mechanisms of HDAC regulation. Selective modulators of HDAC isoenzymes could serve as efficient and well-tolerated drugs. We show that HDAC2 undergoes basal turnover by the ubiquitin-proteasome pathway. Valproic acid (VPA), in addition to selectively inhibiting the catalytic activity of class I HDACs, induces proteasomal degradation of HDAC2, in contrast to other inhibitors such as trichostatin A (TSA). Basal and VPA-induced HDAC2 turnover critically depend on the E2 ubiquitin conjugase Ubc8 and the E3 ubiquitin ligase RLIM. Ubc8 gene expression is induced by both VPA and TSA, whereas only TSA simultaneously reduces RLIM protein levels and therefore fails to induce HDAC2 degradation. Thus, poly-ubiquitination and proteasomal degradation provide an isoenzyme-selective mechanism for downregulation of HDAC2.