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. 2014 Jan 15;127(Pt 2):365-75.
doi: 10.1242/jcs.132290. Epub 2013 Nov 4.

PML isoforms in response to arsenic: high-resolution analysis of PML body structure and degradation

Katherine J Hands  1 , Delphine Cuchet-Lourenco, Roger D Everett, Ronald T Hay
Affiliations

PML isoforms in response to arsenic: high-resolution analysis of PML body structure and degradation

Katherine J Hands et al. J Cell Sci. .

Abstract

Arsenic is a clinically effective treatment for acute promyelocytic leukaemia (APL) in which the promyelocytic leukaemia (PML) protein is fused to retinoic receptor alpha (RARα). PML-RARα is degraded by the proteasome by a SUMO-dependent, ubiquitin-mediated pathway in response to arsenic treatment, curing the disease. Six major PML isoforms are expressed as a result of alternative splicing, each of which encodes a unique C-terminal region. Using a system in which only a single EYFP-linked PML isoform is expressed, we demonstrate that PMLI, PMLII and PMLVI accumulate in the cytoplasm following arsenic treatment, whereas PMLIII, PMLIV and PMLV do not. 3D structured illumination was used to obtain super-resolution images of PML bodies, revealing spherical shells of PML along with associated SUMO. Arsenic treatment results in dramatic isoform-specific changes to PML body ultrastructure. After extended arsenic treatment most PML isoforms are degraded, leaving SUMO at the core of the nuclear bodies. A high-content imaging assay identifies PMLV as the isoform most readily degraded following arsenic treatment, and PMLIV as relatively resistant to degradation. Immunoprecipitation analysis demonstrates that all PML isoforms are modified by SUMO and ubiquitin after arsenic treatment, and by using siRNA, we demonstrate that arsenic-induced degradation of all PML isoforms is dependent on the ubiquitin E3 ligase RNF4. Intriguingly, depletion of RNF4 results in marked accumulation of PMLV, suggesting that this isoform is an optimal substrate for RNF4. Thus the variable C-terminal domain influences the rate and location of degradation of PML isoforms following arsenic treatment.

Keywords: Arsenic; PML; RNF4; SUMO.

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Figures

Fig. 1.
Fig. 1.
Western blot analysis of response of PML to arsenic treatment. (A) HepaRG hepatocytes were treated with 1 µM arsenic trioxide for the indicated periods of time before lysis. Whole-cell extracts were then analysed by SDS-PAGE and immunoblotting with chicken anti-PML antibody and mouse anti-tubulin antibodies. (B) The NB4 leukaemia cell line which expresses the PML-RARα fusion was treated with arsenic for the indicated periods of time before lysis. Cell extracts were then analysed by SDS-PAGE and immunoblotting with chicken anti-PML, rabbit anti-RARα and mouse anti-actin antibodies. Arrowheads indicate the PML-RARα fusion protein. (C) HepaRG hepatocytes stably expressing a control short hairpin RNA (shRNA), denoted HALL, stably expressing an anti-PML shRNA, HALP, or stably expressing both the anti-PML shRNA plus EYFP-PML constructs, PMLI–PMLVI, were lysed and extracts analysed by western blotting with antibodies specific for PML, SUMO1 and SUMO2/3 and actin. Species identified by anti-PML antibody in HALL cells are endogenous PML, whereas exogenous constructs are detected with PMLI–PMLVI. (D) HepaRG hepatocytes depleted of endogenous PML which express EYFP-PML isoforms I–VI were treated with 1 µM arsenic for the indicated periods of time prior to lysis. Whole-cell extracts were analysed by SDS-PAGE and immunoblotting with a chicken anti-PML and mouse anti-actin antibodies. (E) Schematic representation of the exon structure of the six nuclear PML isoforms. All isoforms encode exons 1–6, but alternative splicing of exons 7–9 result in varying C-termini as shown. The size of the translated product is shown on the right. Asterisk indicates a retained intron.
Fig. 2.
Fig. 2.
Immunofluorescence analysis of PML isoforms following arsenic treatment. Cells expressing a single EYFP-PML isoform were cultured on coverslips and treated with 1 µM arsenic. Cells were fixed at the time points described and immunostained with a sheep anti-SUMO2/3 antibody, fluorescently labelled anti-sheep IgG secondary antibody and DAPI to stain DNA. EYFP-PML fluorescence is shown in green, SUMO2/3 in red. Images are presented as maximal intensity projections of multiple z-slices.
Fig. 3.
Fig. 3.
High-content imaging of PML isoforms and quantification of fluorescence. High-content imaging was performed of cells expressing a single EYFP-PML isoform. Cells were cultured in 96-well plates, treated with arsenic, fixed with paraformaldehyde and imaged on the automated IN Cell 2000 microscope. PML fluorescence in ∼5000 cells per time point was analysed. Data presented represent the sum of the area of individual PML nuclear bodies, or cytoplasmic PML inclusions per cell, averaged for each PML isoform after 0, 1 or 24 hours of arsenic treatment. Data are normalised to the average total cellular area of nuclear or cytoplasmic PML present in untreated cells. Data represent the mean ± s.e.m.; grey bars, nuclear PML; unfilled bars, cytoplasmic PML.
Fig. 4.
Fig. 4.
Super-resolution imaging of PML nuclear body structure. Cells expressing a single EYFP-PML isoform were imaged using structured illumination to gain high-resolution images of PML nuclear bodies. Cells were fixed with paraformaldehyde and immunostained with an anti-SUMO2/3 antibody and a corresponding anti-sheep IgG fluorescently labelled secondary antibody. DNA was stained with DAPI. Images are a cross-section through the centre of a representative PML nuclear body. PML is shown in green, SUMO2/3 in red.
Fig. 5.
Fig. 5.
Characterisation of arsenic-induced post-translational modification of PML. Cells expressing EYFP-PML isoforms were treated with arsenic and lysed under conditions to maintain SUMO modification. Lysates were then subjected to GFP immunoprecipitation and eluted proteins analysed by SDS-PAGE and immunoblotting with antibodies specific for PML, SUMO1, SUMO2/3 and ubiquitin.
Fig. 6.
Fig. 6.
RNF4 is required for arsenic-mediated degradation of all PML isoforms. HepaRG cells expressing a single PML isoform were transfected with either non-targeting control siRNA (NT si) or RNF4 siRNA (RNF4 si) and 48 hours later treated with 1 µM arsenic for 24 hours. Cells were lysed for western blotting and fixed with paraformaldehyde for fluorescence microscopy. Western blotting was performed with anti-PML, anti-RNF4 and anti-actin antibodies. Cells for fluorescence microscopy were stained with DAPI. The same experiment was performed in 96-well plates to allow high-content imaging and quantification of EYFP-PML fluorescence as described in Fig. 4. Approximately 5000 cells were analysed for each condition. Graphs represent the sum of the area of all PML nuclear bodies within a given cell nucleus, averaged for all cells examined in the particular cell line at the time point. Data are normalised to non-transfected, non-arsenic-treated cells. Bars represent s.e.m.
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