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. 2011 Jan 1;22(1):78-90.
doi: 10.1091/mbc.E10-06-0504. Epub 2010 Dec 9.

The SUMO protease SENP6 is a direct regulator of PML nuclear bodies

Neil Hattersley  1 , Linnan Shen, Ellis G Jaffray, Ronald T Hay
Affiliations

The SUMO protease SENP6 is a direct regulator of PML nuclear bodies

Neil Hattersley et al. Mol Biol Cell. .

Abstract

Promyelocytic leukemia protein (PML) is the core component of PML-nuclear bodies (PML NBs). The small ubiquitin-like modifier (SUMO) system (and, in particular, SUMOylation of PML) is a critical component in the formation and regulation of PML NBs. SUMO protease SENP6 has been shown previously to be specific for SUMO-2/3-modified substrates and shows preference for SUMO polymers. Here, we further investigate the substrate specificity of SENP6 and show that it is also capable of cleaving mixed chains of SUMO-1 and SUMO-2/3. Depletion of SENP6 results in accumulation of endogenous SUMO-2/3 and SUMO-1 conjugates, and immunofluorescence analysis shows accumulation of SUMO and PML in an increased number of PML NBs. Although SENP6 depletion drastically increases the size of PML NBs, the organizational structure of the body is not affected. Mutation of the catalytic cysteine of SENP6 results in its accumulation in PML NBs, and biochemical analysis indicates that SUMO-modified PML is a substrate of SENP6.

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Figures

FIGURE 1:
FIGURE 1:
SENP6 exhibits a preference for substrates containing SUMO-2/3. The ability of the catalytic domain to cleave SUMO dimers consisting of SUMO-1-SUMO-1 (A), SUMO-2(S)-SUMO-1(M) (B), SUMO-1(S)-SUMO-2(M) (C), and SUMO-2-SUMO-2 (D) was assessed by incubating 20 μM substrate with SENP6 (A and B – 0, 5, 10, 20, 40, 60, 80, 100, 150, 200 nM; C and D – 0, 1, 2, 4, 8, 12, 25, 50, 100, 200 nM) for 1 h at 37ºC. For ease of comparison, boxed regions indicate a reaction carried out with 100 nM protease. Substrate cleavage/product appearance was determined by SDS–PAGE and Coomassie Blue staining (molecular weight markers and position of SUMO dimer/monomers are indicated) and could then be used to assess the concentration of SENP6 required to cut 50% substrate in this time frame (E). A schematic is included for ease of identification of substrate (S) and modifier (M) in mixed SUMO dimers (F).
FIGURE 2:
FIGURE 2:
SENP6 controls endogenous levels of SUMO conjugates and is required for cell viability. (A) HeLa cells were treated with either NT or SENP6 siRNA for 48 h before harvesting in SDS-loading buffer. Samples were analyzed by SDS–PAGE and immunoblotting with the indicated antibodies (individual proteins are indicated). (B) HeLa cells were treated with either NT or SENP6 siRNA for 48 h before staining with SENP6 antibodies. Scale bar represents 30 μm. (C) HeLa cells were treated with siRNA as with (A) before trypsinization and reseeding at low cell density and grown to allow colonies to form before fixation and staining with Giemsa to identify number of surviving cells.
FIGURE 3:
FIGURE 3:
SENP6 depletion results in an increased recruitment of PML and SUMO-to-PML NBs. HeLa cells were treated with either NT or SENP6 siRNA for 48 h before immunostaining with (A) PML and SUMO-2/3, (B) PML and SUMO-1, or (C) SUMO-1 and SUMO-2/3 antibodies. Images are presented as projections of Z-slices. Scale bars represent 30 μm.
FIGURE 4:
FIGURE 4:
SENP6 depletion results in an increased number of PML NBs. HeLa cells were treated with either NT or SENP6 siRNA for 48 h before immunostaining with PML or SUMO-2/3 antibodies. The number of PML (A) or SUMO-2/3 (B) NBs per cell was assessed and plotted as either mean +/− SD number of bodies or as frequency histograms of body numbers per cell. Cells treated with NT siRNA have a mean value of 3.7 PML NBs and 3.3 SUMO NBs per cell with SDs of 2.3 and 1.9, respectively. SENP6-depleted cells have a mean of 5.9 PML NBs and 7.8 SUMO NBs per cell with SDs of 4.1 and 4.5, respectively. Cells with > 15 NBs/cell were classed as 15+ for this purpose (n > 90 cells per staining/siRNA condition).
FIGURE 5:
FIGURE 5:
SENP6 depletion results in the de novo formation of NBs. HeLa cells stably expressing YFP-SUMO-2 were treated with NT (top panels) or SENP6 (bottom panels) siRNA for 24 h before live cell imaging (A). (B) Appearance (top panels) and the increase in intensity (bottom panels) of one NB. Images are presented as projections of individual Z-slices.
FIGURE 6:
FIGURE 6:
SENP6 depletion increases PML NB size but does not affect PML NB structure. HeLa cells were treated with either NT or SENP6 siRNA for 48 h before immunostaining with PML or SUMO-2/3 antibodies. (A) Conventional deconvolution microscopy was performed on samples and presented as projected images. Scale bar represents 10 μm. (B) To achieve higher resolution of the indicated PML NBs in (A), cells were analyzed by structured illumination and are images presented as individual Z-slices. Scale bars represent 2.5 μm.
FIGURE 7:
FIGURE 7:
Catalytically inactive SENP6 accumulates in PML bodies. (A–C) HeLa cells were transfected with either GFP-SENP6WT/C1030A for 36 h before fixation with paraformaldehyde, permeabilization with Triton X-100, and staining with (A) PML, (B) SUMO-2/3, or (C) SUMO-1 antibodies. Enlargements of selected regions are shown, and PML/SUMO NBs are indicated with open arrowheads. SENP6 nuclear foci are indicated by solid arrows (C). Scale bars represent 15 μm and 0.5 μm (enlarged sections).
FIGURE 8:
FIGURE 8:
GFP-SENP6C1030A localizes to the core domain of PML NBs. HeLa cells were transfected with GFP-SENP6C1030A 36 h before immunostaining with PML antibodies. Conventional deconvolution microscopy was performed on samples and presented as projected images. Scale bar represents 10 μm (top panels). To achieve higher resolution of the indicated PML NBs, cells were analyzed by structured illumination and are images presented as individual Z-slices. SENP6 was found to localize to the core domain in 57% of cases. Scale bars represent 2 μm.
FIGURE 9:
FIGURE 9:
Poly-SUMO–modified PML is a SENP6 substrate. HeLa cells stably expressing YFP-SUMO-2 were treated with either NT or SENP6 siRNA before harvesting and nuclear fractionation. Lysed nuclei were then used for GFP-IP, and input and elution samples were analyzed by SDS–PAGE and immunoblotting with antibodies as indicated.
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