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First published online 25 November 2008
doi: 10.1242/jcs.038570

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Loss of SUMO1 in mice affects RanGAP1 localization and formation of PML nuclear bodies, but is not lethal as it can be compensated by SUMO2 or SUMO3

¹ Laboratory of Protein Dynamics and Signaling, National Cancer Institute, National Institutes of Health, NCI-Frederick, Frederick, MD 21702, USA
² Optical Microscopy and Analysis Laboratory, Advanced Technology Program, SAIC-Frederick, Frederick, MD 21702, USA
³ Laboratory Animal Sciences Program, SAIC-Frederick, Frederick, MD 21702, USA

View larger version (10K): [in this window] [in a new window]   Fig. 1. Structure of the XA024 gene-trap insertion. Upper, schematic representation of exon 1, intron 1 and exon 2, of the wild-type SUMO1 gene showing the positions of forward primers (arrowheads) used for long-range PCR to localize the insertion. The sequence at the junction between intron 1 and exon 2 is shown (intron in small and exon in capital letters). The position of the insertion and the region of intron 1 deleted in the gene-trap allele are indicated. Middle, structure of the gene trap allele showing inserted vector and reverse primer used for long range PCR. The sequence at the junction of the inserted vector and exon 2 is shown (gene trap vector in small and exon in capital letters); the first nucleotide of exon 2 is lost. Lower, structure of the gene trap vector; the majority was retained in the insertion event.

View larger version (28K): [in this window] [in a new window]   Fig. 2. RT-PCR analysis of splicing products. (A) Schematic representation of the SUMO1 gene with various primer sets (arrows) used to detect (1) the normal spliced transcript; (2) the transcript splicing into the gene trap vector; (3) the transcript skipping the vector and truncated exon 2. (B) Agarose gel showing RT-PCR products generated using the primer sets shown in A and RNA derived from E15.5 embryos of the indicated genotype. het, heterozygous; hmz, homozygous. The lane numbers correspond to the primer sets in A. m, 100 bp ladder molecular size marker.

View larger version (46K): [in this window] [in a new window]   Fig. 3. Immunoblot analysis of sumoylation. The genotypes are shown above the lanes; wt, wild type; hmz, homozygous; het, heterozygous. Molecular mass in kilodaltons (kDa) is shown to the left of each panel. (A) Whole-embryo extracts immunoblotted with anti-SUMO1 antibody. There is complete lack of high molecular mass SUMO1 conjugates (upper panel) and free SUMO1 (lower panel) in homozygotes, and reduced levels in heterozygotes. (B) Same blot as in upper panel of A, reanalyzed with anti-RanGAP1 antibody. Sumoylated form of RanGAP1 is present even in homozygotes. (C) Whole-embryo extracts immunoblotted with anti-SUMO3 antibody. Band at the position of sumoylated RanGAP1 (>75 kDa) is more intense in homozygotes. Higher molecular mass SUMO2/3 conjugates are essentially unchanged.

View larger version (42K): [in this window] [in a new window]   Fig. 4. Immunoblot analysis of RanGAP1 and PML sumoylation. (A,B) Whole-cell extracts (WCE) from embryos are shown in left panels, and immunoprecipitation (IP) with anti-RanGAP1 antibody shown in right panels. The genotypes are shown below lanes; hmz, homozygous; wt, wild type; het, heterozygous. There is less sumoylated form in the homozygote both in WCE and IP upon analysis with anti-RanGAP1 antibody (A). IP shows less sumoylated form in the heterozygote and none in homozygote analysis with anti-SUMO1 antibody (B). (C) Analysis with anti-SUMO2 antibody. The immunoprecipitation shows more sumoylated form in the homozygotes compared to the wild type and heterozygotes. The whole cell extract shows an increase in average molecular mass of SUMO2/3 high molecular mass conjugates in homozygotes. (D) Whole cell extracts (WCE) from MEFs immunoblotted with anti-PML antibody (left). Reblotting with anti-MAPK (lower panel) shows equivalent loading of heterozygous and homozygous samples but less protein loaded for the wild-type sample. Therefore, there is less total PML in the homozygote. Immunoprecipitation (IP) of extracts with anti-PML antibody is shown in the middle and right panels. Immunoblotting with anti-PML antibody is shown in the middle panel and with anti-SUMO2 in the right panel. There is less SUMO2/3 conjugation of PML in MEFs derived from XA024 homozygotes.

View larger version (57K): [in this window] [in a new window]   Fig. 5. Immunofluorescence analysis of XA024 MEFs. The genotypes are shown above panels; wt, wild type; het, heterozygous; hmz, homozygous. (A-C) Analysis with anti-RanGAP1 antibody. There is decreased nuclear rim staining in the homozygote (C) compared with the wild type (A) or heterozygote (B). (D-F) Analysis with anti-SUMO1 antibody. Staining in wild type (D) and heterozygote (E) is predominantly nuclear and nuclear rim. There is no staining above background in homozygote (F). (G-H) Analysis with anti-Sentrin 2 (SUMO3) antibody. There is no change in staining intensity or pattern in hetero- (H) and homozygotes (I) compared with the wild type (G). (J-L) Analysis with anti-PML antibody. Fewer PML nuclear bodies are observed in homozygote (L) compared with the wild type (J) and heterozygote (K).

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