Sequestration and inhibition of Daxx-mediated transcriptional repression by PML

Abstract

PML fuses with retinoic acid receptor alpha (RARalpha) in the t(15;17) translocation that causes acute promyelocytic leukemia (APL). In addition to localizing diffusely throughout the nucleoplasm, PML mainly resides in discrete nuclear structures known as PML oncogenic domains (PODs), which are disrupted in APL and spinocellular ataxia cells. We isolated the Fas-binding protein Daxx as a PML-interacting protein in a yeast two-hybrid screen. Biochemical and immunofluorescence analyses reveal that Daxx is a nuclear protein that interacts and colocalizes with PML in the PODs. Reporter gene assay shows that Daxx drastically represses basal transcription, likely by recruiting histone deacetylases. PML, but not its oncogenic fusion PML-RARalpha, inhibits the repressor function of Daxx. In addition, SUMO-1 modification of PML is required for sequestration of Daxx to the PODs and for efficient inhibition of Daxx-mediated transcriptional repression. Consistently, Daxx is found at condensed chromatin in cells that lack PML. These data suggest that Daxx is a novel nuclear protein bearing transcriptional repressor activity that may be regulated by interaction with PML.

FIG. 1

Interaction between Daxx and PML in vivo. (A) Interaction of Daxx with PML in yeast two-hybrid system. The average β-Gal activities of three transformants expressing the indicated combinations of Gal4 AD and DBD fusion proteins were determined as described in Materials and Methods. The AD-Daxx fusion protein contains amino acids 556 to 740 of human Daxx. The DBD fusion proteins contain full-length PML-1, SP100, and PML-RARα short form, respectively. The minus sign indicates empty vector alone. (B) atRA disrupts the interaction between Daxx and PML-RARα. The effect of atRA on Daxx-PML-RAR interaction was determined after a 24-h incubation of the culture in the presence of the indicated concentrations of respective ligands. Columns: 1, solvent only; 2, 1 nM; 3, 10 nM; 4, 100 nM; and 5, 1,000 nM. T3, 3,3′,5-triiodo-l-thyronine. (C) Subcellular fractionation of Daxx. HeLa cells were fractionated into cytosolic, membrane, and nuclear fractions, and an equal amount of protein was analyzed by Western blotting for Daxx (left panel). The distribution of the cytoplasmic protein β-tubulin and the nuclear protein hPc2 in each fraction was also determined by immunoblotting to validate the fractionation. Two independent preparations of HeLa nuclear extracts are shown. The right panel is a Coomassie blue-stained gel that shows the relative amount of proteins in each fraction used in the Western blot. (D) Co-IP of Daxx with PML. NB4-cell extracts were immunoprecipitated with affinity-purified anti-Daxx and anti-PML antibodies, and the presence of Daxx in the immunoprecipitates was determined by immunoblotting with anti-Daxx antibodies. The antibodies used for the IP and the Western blot (W.B.) are indicated.

FIG. 2

Daxx colocalizes with PML at the PODs. (A) Confocal immunofluorescence analysis of endogenous Daxx and PML. HEp2 cells were fixed and immunostained with affinity-purified rabbit anti-Daxx polyclonal antibodies and mouse anti-PML 5E10 monoclonal antibodies as described in Materials and Methods. The sample was analyzed by use of a confocal microscope. Panels a and b show the signals of Daxx (green) and PML (red) on a single confocal section. Panel c shows colocalization (yellow signals) of Daxx and PML in the merged image. Panel d is a differential interference contrast image showing the surfaces of the cells and nuclei. Bar, 10 μm. (B) Three-dimensional presentation of the colocalization between Daxx and PML. Total of 32 consecutive z-sections at increments of 0.08 μm were reconstructed into a three-dimensional image by using the Leica confocal software. The right and left projected images were rotated 4.5° at opposite directions along the x (horizontal) axis. Yellow represents the colocalization between Daxx (green) and PML (red). (C) Colocalization of Daxx and PML in APL cells. NB4 cells were plated on cover glasses coated with poly-l-lysine. The control (untreated), atRA-treated (1 μM for 72 h), and As₂O₃-treated (1 μM for 72 h) cells were fixed and immunostained with anti-Daxx polyclonal and anti-PML monoclonal antibodies. Colocalization of Daxx and PML was revealed by confocal laser microscopy (except for the untreated cells).

FIG. 3

Modulation of promoter activity by Daxx. (A) Transcriptional repression by Gal-Daxx. Recruitment of Daxx to a promoter via Gal4-DBD results in inhibition of basal transcription in a dose-dependent manner. Transient transfection was conducted in HEK293 cells with increasing concentrations (nanograms) of Gal-Daxx as indicated. The relative fold repression of the basal promoter activity in the presence of Gal-Daxx was compared to that of Gal4-DBD alone. The bottom panels show immunoblots with anti-Gal4-DBD antibodies of the transfected fusion protein at indicated concentrations of expression vector. (B) Daxx represses basal transcription as strong as PML-RARα. HEK293 cells were transfected with equal amounts (250 ng) of each expression vector, and the relative repression was determined as described in Materials and Methods. The results show that Gal-Daxx represses basal transcription as strongly as Gal-PML-RARα. (C) Requirement of binding sites for transcriptional repression by Gal-Daxx. HEK293 cells were transfected with 250 ng of Gal-Daxx or Gal4-DBD alone, and the effects on the promoter activities of Gal-tk-luciferase (luc) and tk-luc reporters were determined. The Gal-tk-luc reporter contains four copies of Gal4-binding sites in front of the minimal tk promoter, while the tk-luc lacks the binding sites. (D) Mapping of the Daxx sequences required for repression. Schematic presentation of Gal-Daxx deletion mutants and their effects on promoter activity in HEK293, HeLa, and CV-1 cells are summarized. The two acidic regions are indicated by black bars, and the two potential nuclear localization signals are marked with diamonds. The bottom graph shows a column presentation of the repression activity of various Gal-Daxx deletion mutants in HEK293 cells. (E) Expression of Gal-Daxx mutants in transfected cells. The transfected lysates were analyzed by immunoblotting by using mouse anti-Gal4-DBD monoclonal antibodies. The top band in each lane represents the expected molecular weights of the Gal-Daxx mutants, except for Gal-Daxx (125-215), where the lower band is the expected product. The deviation in protein expression level was compensated by normalization of luciferase activity with the coexpressed β-Gal activity. (F) Inhibition of basal transcription from a natural promoter by Daxx. Wild-type Daxx was transfected into HEK293 cells together with either the SF1-tk-luciferase or tk-luciferase reporter. The fold repression of the luciferase activity at increasing concentrations (micrograms) of Daxx is presented.

FIG. 4

Interaction of Daxx with HDACs. (A) ³⁵S-labeled protein probes used in the far-Western assays. In vitro-translated [³⁵S]methionine-labeled Daxx, PML, and SP100 were analyzed by SDS-PAGE and detected by autoradiography. (B) Far-Western analysis of interaction between Daxx and HDACs. The top panel shows the Coomassie blue-stained proteins used in the far-Western assay. The middle panel shows the far-Western blot of GST-HDACs with the ³⁵S-labeled Daxx probe. The bottom panel shows the far-Western blot with the ³⁵S-labeled PML probe. (C) PML interacts with GST-PML in the far-Western assay. A positive control showing that PML interacts with GST-PML in the far-Western assay was conducted under conditions identical to those for panel B. A far-Western blot showing that the Daxx mutant (400-657) fails to interact with GST-HDAC1 fusion protein. (D) GST pull-down assay showing interaction between GST-HDAC1 and Daxx. The input ³⁵S-labeled Daxx contains one-third of the lysate used in the pull-down reaction, which was conducted as described in the Materials and Methods. (E) Co-IP of HDAC1 and Daxx. HeLa nuclear extracts were incubated with affinity-purified anti-Daxx antibody or an equal concentration of the preimmune serum. The immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blot by using anti-HDAC1 polyclonal antibodies. (F) TSA reverses transcriptional repression by Gal-Daxx. HEK293 cells were transfected with 250 ng of Gal4-DBD or Gal-Daxx mammalian expression vector together with a Gal4-dependent luciferase reporter. The fold repression by Gal-Daxx at different concentrations of TSA was determined relative to that for the Gal4-DBD alone.

FIG. 5

Inhibition of Daxx-mediated transcriptional repression by PML. (A) PML inhibits Daxx-mediated transcriptional repression. HEK293 cells were transiently transfected with 100 ng of the Gal4-DBD or Gal-Daxx mammalian expression vectors in the absence or presence of the indicated amounts of PML expression vector together with a Gal4-dependent luciferase reporter. Data are presented as the percentage of maximum repression, where Gal-Daxx activity is represented as 100% repression. (B) PML-RARα has no effect on Daxx-mediated transcriptional repression. HEK293 cells were transiently transfected with 100 ng of the Gal4-DBD or Gal-Daxx mammalian expression vectors in the absence or presence of expression vectors for PML, PML-RARα (short form), or PML-RARα (long form), together with a Gal4-dependent luciferase reporter. Data are presented as the percentage of maximum repression, where Gal-Daxx activity is represented as 100% repression.

FIG. 6

Recruitment of Daxx to POD domains by overexpression of PML. (A) Overexpression of PML recruits transfected Gal-Daxx into the PODs. Gal-Daxx or Gal-Daxx (1-502) were transiently transfected into HEp2 cells in the absence or presence of HA-tagged PML and subsequently stained with the mouse anti-Gal4-DBD and rabbit anti-HA antibodies. Primary antibodies were detected with rhodamine-conjugated anti-mouse immunoglobulin G and fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin G secondary antibodies and analyzed by immunofluorescence microscopy. Panels a and b show diffuse nuclear staining of Gal-Daxx in the absence of PML. Panels c to f show Gal-Daxx and HA-PML colocalization at the PODs. Panels g to j show that HA-PML cannot recruit a Gal-Daxx (1-502) mutant lacking the PML-interacting domain to the PODs. (B) Recruitment of endogenous Daxx but not HDAC1 and SMRT to the PODs. HA-PML was transfected into HEp2 cells, and the localization of endogenous Daxx, HDAC1, and SMRT was analyzed by immunofluorescence microscopy. Panels a to c show colocalization of transfected and endogenous PML with endogenous Daxx by using anti-PML monoclonal 5E10 and anti-Daxx rabbit polyclonal antibodies. Panels d to f show HA-PML and Daxx colocalization by using the anti-HA monoclonal and anti-Daxx polyclonal antibodies. Panels g to l demonstrate that HA-PML does not recruit HDAC1 or SMRT to the PODs by using anti-HA, anti-HDAC1, or anti-SMRT antibodies. Yellow signals in the overlay images indicate colocalization.

FIG. 7

SUMO-1 modification of PML is required for sequestration of Daxx to the POD and inhibition of Daxx-mediated transcriptional repression. (A) The PML ΔSUMO (ΔSU) mutant lacks SUMO-1 modification. This mutant was created by replacing all three lysines at residues 65, 160, and 490 with arginines. The wild-type (WT) PML and the ΔSUMO mutant were transfected into HEK293 cells alone or in combination with a SUMO-1 expression vector. Cells were treated with 1 μM arsenic trioxide (As₂O₃) for 6 h where indicated. The total cell lysates were analyzed by Western blotting by using anti-HA monoclonal antibodies. The upper bands in the wild-type proteins represent SUMO-1 conjugated forms of PML. (B) The PML ΔSUMO mutant localizes to the PODs but fails to recruit Daxx. The HA-PML ΔSUMO mutant was transfected into HEp2 cells and analyzed by immunofluorescence microscopy to detect localization of the transfected mutant protein and the distribution of endogenous Daxx. Panels a to c show localization of the transfected HA-PML ΔSUMO mutant protein (detected by a HA antibody) in the PODs that were revealed by a PML polyclonal antibody (1-14). Panels d to f show that the enlarged PODs in the HA-PML ΔSUMO mutant transfected cells do not result in prominent recruitment of Daxx to the PODs. Panels g to i show that many enlarged PODs containing PML-ΔSUMO mutant have little or no Daxx protein. The 5E10 antibodies also detect PODs in untransfected cells that show smaller structures colocalized with Daxx foci. (C) The PML ΔSUMO mutant is deficient in reversing transcriptional repression by Daxx. The transfection was conducted in HEK293 cells, and the initial fold repression mediated by the Gal4-DBD fusion proteins is as indicated at the bottom. The y axis indicates the fold reversal of repression. The wild-type PML does not reverse transcriptional repression mediated by HDAC1 or SMRTe. (D) Association of Daxx with condensed chromatin in cells that lack PODs. The human neuronal NT2 stem cells were analyzed by double immunofluorescence staining with anti-Daxx polyclonal and anti-PML 5E10 monoclonal antibodies. The NT2 cells display heterogeneous staining for PML. In cells that contain normal PML nuclear bodies (panels a to d), Daxx appears normal and shows complete colocalization with PML. In contrast, cells that contain only two or fewer PML nuclear dots show aggregated Daxx surrounding the condensed chromatin areas stained with DAPI (panels e to h).