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. 2004 Jun;24(12):5577-86.
doi: 10.1128/MCB.24.12.5577-5586.2004.

Protein inhibitor of activated STAT Y (PIASy) and a splice variant lacking exon 6 enhance sumoylation but are not essential for embryogenesis and adult life

Kelly A Wong  1 , Rachel Kim, Heather Christofk, Jing Gao, Gregory Lawson, Hong Wu
Affiliations

Protein inhibitor of activated STAT Y (PIASy) and a splice variant lacking exon 6 enhance sumoylation but are not essential for embryogenesis and adult life

Kelly A Wong et al. Mol Cell Biol. 2004 Jun.

Abstract

Protein inhibitor of activated STAT Y (PIASy) is the shortest member of the PIAS family and has been reported to modulate the transcriptional activities of STAT1, lymphoid enhancer factor 1 (LEF-1), and the androgen receptor. PIAS proteins have also been identified as E3 ligases for the small ubiquitin-like modifier (SUMO) proteins. PIASy in particular has been reported to mediate SUMO-2/3 modification of LEF-1, sequestering it into nuclear bodies, and SUMO-1 ligation to c-Myb, modulating its transcriptional activation properties. We have cloned murine Piasy and a splice variant which omits exon 6, containing the nuclear retention PINIT motif. Cell culture studies indicate that both the full length and the splice variant are localized in the nucleus but differentially enhance SUMO ligation. To further understand the functions of PIASy, we have generated PIASy-deficient mice. Surprisingly, Piasy(-/-) mice appear phenotypically normal. Activation of STAT1 is not significantly perturbed in Piasy(-/-) cells, and sumoylation patterns for SUMO-1 or SUMO-3 modification are similar when comparing tissues and embryonic fibroblasts from wild-type and knockout mice. Our study demonstrates that at steady state, PIASy is either dispensable or compensated for by other PIAS family members or by other mechanisms when deleted.

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Figures

FIG. 1.
FIG. 1.
Primary amino acid alignment of full-length mouse PIASy (top) and PIASyE6− (bottom). Exon 6 codes for 43 amino acids consisting of residues 218 to 260. The PINIT motif is boxed and is missing in PIASyE6−. Alignment was performed by the Clustal method.
FIG. 2.
FIG. 2.
Mouse PIASy and PIASyE6− are nuclear localized and differentially enhance SUMO ligation. (A) Transient transfections into NIH 3T3 cells of GFP, GFP-PIASy, or GFP-PIASyE6− expression constructs. Cells were visualized with a ×100 objective under UV illumination. The same field is shown in each row. First column, DAPI stain for nuclei; second column, green fluorescence. (B) Western blots of NIH 3T3 cells transiently transfected with expression constructs designated by the grid above the blots. Shown is duplicate Western blotting with anti-Flag antibody in the top panel and anti-Myc antibody in the bottom panel. Molecular weights are indicated to the left of the top panel. Arrows indicate differences in the pattern of SUMO-2 ligation by PIASy and PIASyE6− in lane 7 versus 8.
FIG. 3.
FIG. 3.
Gene targeting of the Piasy gene. (A) Gene targeting diagram of the Piasy locus. The targeting construct deletes exon 3 to 11. Homologous recombination introduces a new EcoRI site shown in bold. Digestion of genomic DNA with EcoRI and probing with the indicated 5′ probe yields a 11.4-kb wild-type (WT) band and a 7.2-kb mutant band. DpToxin, diphtheria toxin. (B) Genotyping by EcoRI-digested genomic Southern blotting. The wild-type band is 11.4 kb, and the knockout (KO) band is 7.2 kb. (C) RT-PCR of MEFs isolated from heterozygous intercrosses shows no expression in null clones. Genotypes are labeled above each lane. Templates: lane 1, PIASy cDNA as positive-control template (Py); lane 2, water template (H2O); lane 3, no reverse transcriptase in cDNA preparation (−RT); lane 4, wild-type tail DNA template (Gen.); lanes 5 to 9, MEF RNA. Bottom panel, β-actin primers used as control for RNA. (D) Western blot of Piasy+/+ and Piasy−/− testes. Top panel blotted with PIASy antibody, bottom panel blotted with antiactin antibody. (E) Northern analysis of paired tissues from Piasy+/+ and Piasy−/− animals. The PIAS-specific probes used are indicated on the left. Bottom panel, ethidium bromide-stained 28S rRNA indicating similar levels of RNA present in each set of tissues examined.
FIG. 4.
FIG. 4.
Hematoxylin and eosin-stained tissue sections of Piasy+/+ and Piasy−/− animals. Left column shows tissue from Piasy+/+ mice, and right column shows tissue from Piasy−/− mice. Tissues were indicated to the left. Magnification: testes, ×20; skin, ×20; cerebellum, ×10; spleen, ×20; thymus, ×20. No histological abnormalities were identified.
FIG. 5.
FIG. 5.
No major differences in STAT1 activation in Piasy−/− primary splenocytes. (A) Western blots for STAT1 tyrosine phosphorylation upon IFN-γ treatment in +/+ and −/− splenocytes. Top panel, blotted for phospho-STAT1; bottom panel, blotted for STAT1. Cells were untreated or pulsed with IFN-γ (20 min) and then washed and incubated for the indicated time in minutes before lysis. (B) Northern analysis of IFN-γ-induced gene induction of IRF-1 and SOCS-1 from Piasy+/+ and Piasy−/− splenocytes over 60 min with quantitative bar graphs normalized to the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) levels below. The time of stimulation in minutes is indicated across the top. The probes used are indicated to the right of Northern blots. WT, wild type; KO, knockout.
FIG. 6.
FIG. 6.
No difference in SUMO-1 or SUMO-3 modification patterns between Piasy+/+ and Piasy−/− cells. (A) Western blots for SUMO-1 and SUMO-3 from wild-type and mutant testis lysates. (B) Western blot of Piasy+/+ and Piasy−/− MEF cells untreated (−), heat shocked (HS), or ethanol treated (EtOH) and blotted for SUMO-3. (C) Thymus Western blots for c-Myb (left panel) and LEF-1 (right panel). +/+, lysate in the first lane; −/−, lysate in the second lane. The expected mobility of each protein is labeled. Arrows on the c-Myb blot identify the SUMO-1-modified bands reported by Dahle et al. (2). The arrow in the right panel points to the expected mobility of LEF-1 in SDS-PAGE. (D) Immunofluorescence of Flag-PML nuclear bodies in Piasy+/+ and Piasy−/− MEF cells. The same field is shown across each row at ×100 magnification.
FIG. 7.
FIG. 7.
Mo-MuLV is able to infect and replicate in PIASy−/− fibroblasts. Analysis of viral replication in cells exposed and unexposed to live Mo-MuLV by semiquantitative RT-PCR for viral Gag transcript. NIH 3T3 cells are mouse cells, while 293T cells are human. Media from unexposed cells show no Gag transcript. Gag transcript was amplified from viral stock used in initial infection as a positive control (last lanes). For each cell type infected, samples were subjected to increasing numbers of thermal cycles: 20, 23, 27, and 30.
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