Promyelocytic Leukemia Protein (PML) Functions as a Glucocorticoid Receptor Co-activator by Sequestering Daxx to the PML Oncogenic Domains (PODs) to Enhance Its Transactivation Potential

Ding-Yen Lin,Ming-Zong Lai,David K Ann,Hsiu-Ming Shih

doi:10.1074/jbc.m300387200

Abstract

Daxx has been reported to function as a transcriptional modulator in the nucleus. In the present study, we have explored the role of Daxx in regulating the transcriptional activity of the glucocorticoid receptor (GR). Overexpression of Daxx suppressed GR-mediated activation of the mouse mammary tumor virus promoter in COS-1, HeLa, and 293T cells. In vitro and in vivo studies revealed that Daxx could directly bind to GR. The mapping analysis further demonstrated that the C-terminal region of Daxx-(501-740) mediates the interaction and transcriptional repression of GR. The repressive effect of Daxx and Daxx-(501-740) on GR could be alleviated by co-expression of promyelocytic leukemia protein (PML). Furthermore, immunofluorescence analysis showed that overexpression of wild-type PML results in the translocation of Daxx and Daxx-(501-740) to the PML oncogenic domains (PODs). By contrast, a PML sumoylation-defective mutant failed to recruit Daxx to PODs and to reverse the Daxx repression effect on GR. Accordingly, As(2)O(3) treatment rendered the sequestration of endogenous Daxx to the PODs, leading to an enhancement of GR transactivation in COS-1 cells. Taken together, these findings suggest that recruitment of Daxx into the subnuclear POD structures sequesters it from the GR/co-activators complex, thereby alleviating its repressive effects. Our present studies provide the important link between Daxx/PML interaction and GR transcriptional activation.

Highlights

The glucocorticoid receptor (GR),1 a member of the liganddependent nuclear receptor superfamily, plays important roles in governing development, growth, apoptosis, and anti-inflammatory processes [1,2,3,4,5]
Overexpression of Daxx Suppresses GR-mediated Transactivation of the MMTV Promoter—To test whether Daxx is involved in regulating GR transcriptional activation, the Daxx expression construct was cotransfected into COS-1 cells along with the GR expression construct and the MMTV-Luc reporter
We have identified Daxx as a GRinteracting protein and demonstrated that Daxx regulates the GR transcriptional activation on MMTV promoter

Summary

EXPERIMENTAL PROCEDURES

Plasmids Construction—LexA-Daxx, its deletion mutants LexAMST3, LexA-lamin, HA-Daxx, and its derived mutant constructs have been described before [25]. 35S-labeled proteins were incubated with GST or GST-Daxx-agarose beads, which contained equal amounts of protein in 0.2 ml of binding buffer (10 mM HEPES, pH 7.5, 50 mM NaCl, 0.1% Nonidet P-40, 0.5 mM dithiothreitol, and 0.5 mM EDTA) for 2 h, washed four times, and analyzed by SDSPAGE and autoradiography. Immunoprecipitation and Western Blotting Assays—To test the interaction in mammalian cells, wild-type GR along with HA-tagged Daxx expression construct was transfected into COS-1 cells by the lipofection method. The permeabilized cells were incubated with an anti-HA monoclonal antibody and an anti-GR polyclonal antibody (Santa Cruz Biotechnology, catalog number sc1004) for 1 h at room temperature Following this incubation, cells were washed three times for 10 min with phosphate-buffered saline at room temperature and incubated at 8 ␮g/ml with fluorescein isothiocyanate and Texas Red-conjugated anti-rabbit IgG (DAKO) for 1 h at 20 °C.

RESULTS

To further test whether this association is through direct

DISCUSSION