Function of histone deacetylase 6 as a cofactor of nuclear receptor coregulator LCoR.

Ligand-dependent corepressor LCoR was identified as a protein that interacts with the estrogen receptor alpha (ERalpha) ligand binding domain in a hormone-dependent manner. LCoR also interacts directly with histone deacetylase 3 (HDAC3) and HDAC6. Notably, HDAC6 has emerged as a marker of breast cancer prognosis. However, although HDAC3 is nuclear, HDAC6 is cytoplasmic in many cells. We found that HDAC6 is partially nuclear in estrogen-responsive MCF7 cells, colocalizes with LCoR, represses transactivation of estrogen-inducible reporter genes, and augments corepression by LCoR. In contrast, no repression was observed upon HDAC6 expression in COS7 cells, where it is exclusively cytoplasmic. LCoR binds to HDAC6 in vitro via a central domain, and repression by LCoR mutants lacking this domain was attenuated. Kinetic chromatin immunoprecipitation assays revealed hormone-dependent recruitment of LCoR to promoters of ERalpha-induced target genes in synchrony with ERalpha. HDAC6 was also recruited to these promoters, and repeat chromatin immunoprecipitation experiments confirmed the corecruitment of LCoR with ERalpha and with HDAC6. Remarkably, however, although we find evidence for corecruitment of LCoR and ERalpha on genes repressed by the receptor, LCoR and HDAC6 failed to coimmunoprecipitate, suggesting that they are part of distinct complexes on these genes. Although small interfering RNA-mediated knockdown of LCoR or HDAC6 augmented expression of an estrogen-sensitive reporter gene in MCF7 cells, unexpectedly their ablation led to reduced expression of some endogenous estrogen target genes. Taken together, these data establish that HDAC6 can function as a cofactor of LCoR but suggest that they may act in enhance expressing some target genes.

Ligand-dependent corepressor LCoR is an attenuator of progesterone-regulated gene expression.

Ligand-dependent corepressor LCoR interacts with the progesterone receptor (PR) and estrogen receptor ERalpha in the presence of hormone. LCoR contains tandem N-terminal PXDLS motifs that recruit C-terminal-binding protein (CtBP) corepressors as well as a C-terminal helix-turn-helix (HTH) domain. Here, we analyzed the function of these domains in coregulation of PR- and ERalpha-regulated gene expression. LCoR and CtBP1 colocalize in nuclear bodies that also contain CtBP-interacting protein CtIP and polycomb group repressor complex marker BMI1. Coexpression of CtBP1 in MCF7 or T47D breast cancer cells augmented corepression by LCoR, whereas coexpression of CtIP did not, consistent with direct interaction of LCoR with CtBP1, but not CtIP. The N-terminal region containing the PXDLS motifs is necessary and sufficient for CTBP1 recruitment and essential for full corepression. However, LCoR function was also strongly dependent on the helix-turn-helix domain, as its deletion completely abolished corepression. LCoR, CtBP, and CtIP were recruited to endogenous PR- and ERalpha-stimulated genes in a hormone-dependent manner. Similarly, LCoR was recruited to estrogen-repressed genes, whereas hormone treatment reduced CtBP1 binding. Small interfering RNA-mediated knockdown of LCoR or CtBP1 augmented expression of progesterone- and estrogen-stimulated reporter genes as well as endogenous progesterone-stimulated target genes. In contrast, their ablation had gene-specific effects on ERalpha-regulated transcription that generally led to reduced gene expression. Taken together, these results show that multiple domains contribute to LCoR function. They also reveal a role for LCoR and CtBP1 as attenuators of progesterone-regulated transcription but suggest that LCoR and CtBP1 can act to enhance transcription of some genes.

Hydrogen peroxide ototoxicity in unblocking ventilation tubes: a chinchilla pilot study.

OBJECTIVE: Some clinicians use hydrogen peroxide (H2O2) to clear the lumen of ventilation tubes that become blocked postoperatively. The ototoxicity associated with H2O2 has been controversial. STUDY DESIGN: We designed an experiment to test if H2O2 damages the cochlear hair cells using a Chinchilla laniger animal model. METHODS: Nine chinchillas (18 ears) were included in this study. Each animal was used as its own control. Following the insertion of ventilation tubes in both ears and baseline recordings of the auditory brain stem responses (ABR), we instilled 2 ml of 3 percent H2O2 into their right external auditory canals (experimental ears). H2O2 was left in the external auditory canal for a total of 5 minutes and then was drained. We instilled a normal saline control solution in their left ears (control ears) in a similar fashion. ABR recordings were performed 1 day after the last instillation of H2O2 and 5 days later. RESULTS: There was no statistically significant difference in the ABR thresholds of the experimental and control ears. CONCLUSION: H2O2 did not appear to cause ototoxicity in chinchilla ears with tympanostomy tubes exposed to H2O2 instillation using a standard clinical protocol.