Abstract
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.
Highlights
Nuclear receptor corepressors NCoR7 and SMRT were isolated as factors that interacted with hormone-free but not hormone-bound thyroid and retinoid receptors [6, 7]
As HDAC6 is cytoplasmic in many cells, we further investigated the colocalization of LCoR and HDAC6 in MCF7 cells by immunocytochemistry
A substantial portion of HDAC6 was nuclear in MCF7 cells, and there was a clear colocalization of nuclear HDAC6 with LCoR (Fig. 1A), substantiating the possibility that the two proteins function together
Summary
Antibodies—A rabbit polyclonal antipeptide antibody was raised against LCoR amino acids 20 –36 (QDPSQPNSTKNQSLPKA) fused to keyhole limpet hemocyanin and purified over a peptide affinity column (Bethyl Laboratories, Montgomery TX). COS7 cells grown on collagen IV-treated microscope slides in 6-well plates in DMEM supplemented with 10% FBS were transfected in medium without serum with 12.5 l of Lipofectamine 2000 (Invitrogen) containing 1 g each of pSG5/LCoR and HA-FLAG-HDAC6/pcDNA3. For analysis of the effects of HDAC3 or -6 on LCoR corepression, COS7 cells (60 –70% confluent) grown in DMEM without phenol red supplemented with 10% FBS on 6-well plates were transfected in minimal medium without serum with Lipofectamine 2000 (5 l) with 100 ng of ER␣ expression vector, 250 ng of ERE3-TATA-pXP2 reporter plasmid, and 250 ng of internal control vector pCMV-gal. MCF7 cells were incubated with ␣-LCoR (1:500) and goat polyclonal antibodies against HDAC6 or Bmi (1:50) in buffer B (0.2% Triton X-100, 5% bovine serum albumin in PBS) for 1 h at room temperature. The following primers were used: pS2 5Ј-ACCATGGAGACAAGGTGAT-3Ј (forward) and pS2 5Ј-AAATTCACACTCCTCTTCTG-3Ј (reverse), GREB1 5Ј-CCACAAAGGGTGGTCTCCAGAA-3Ј (forward) and GREB1 5Ј-CACTGGCTTGGCCTTGCATATT-3Ј (reverse), SGK3 5Ј-CAAAAGAAGATTCCACCACCA-3Ј (forward) and SGK3 5ЈTGTCAAAGTTTCTGATATCATCTC-3Ј (reverse), CYP26B1 5ЈACATCCACCGCAACAAGC-3Ј (forward) and CYP26B1 5Ј-GGATCTTGGGCAGGTAACTCT-3Ј (reverse), BMP7 5ЈGGTCATGAGCTTCGTCAACC-3Ј (forward) and BMP7 5ЈGCAGGAAGAGATCCGATTCC-3Ј (reverse), KRT4 5Ј-GCCGACAATGACTTTGTGGT-3Ј (forward) and KRT4 5Ј-CCTCCAACTCCACCTTGTTC-3Ј (reverse), and -actin 5Ј-GGCATGGGTCAGAAGGATTCC-3Ј (forward) and -actin 5Ј-GCTGGGGTGTTGAAGGTCTC-3Ј (reverse), ADORA1 5Ј-GACCTACTTCCACACCTGCCTCA-3Ј (forward) and ADORA1 5ЈCCAGCCAAACATAGGGGTCAGT-3Ј (reverse), IGFBP4 5ЈGGGGGCAAGATGAAGGTCAAT-3Ј (forward) and IGFBP4 5Ј-CGGTCCACACACCAGCACTT-3Ј (reverse) and NRIP1 5Ј-GTGATTCCAGGATGGTTTGG-3Ј (forward) and NRIP1 5Ј-ATGGTTTTAATAAAGGTTAAGGATGC-3Ј (reverse)
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