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Science 330 (6003): 521-525

Copyright © 2010 by the American Association for the Advancement of Science

The Ligase PIAS1 Restricts Natural Regulatory T Cell Differentiation by Epigenetic Repression

Bin Liu1,*,{dagger}, Samuel Tahk1,*, Kathleen M. Yee2, Guoping Fan3, and Ke Shuai1,2,{dagger}

1 Division of Hematology-Oncology, Department of Medicine, 11-934 Factor Building, 10833 Le Conte Avenue, University of California, Los Angeles, Los Angeles, CA 90095, USA.
2 Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA.
3 Department of Human Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA.


Figure 1
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Fig. 1. Enhanced CD4+Foxp3+ Treg differentiation in Pias1–/– mice. (A) Western blot analysis of whole-cell extracts from freshly isolated thymocytes and splenocytes with an antibody specific for Ser90-phosphorylated PIAS1, total PIAS1, or tubulin. (B) Cells from thymus or spleen of male (n = 7) wild-type and Pias1–/– littermates were analyzed by flow cytometry to determine the percentage and the absolute cell numbers of Foxp3+CD4+ cells (gated on a CD4+CD8 population). Similar results were obtained with female mice. (C) Bone marrow was isolated from wild-type and Pias1–/– littermates and injected into the sublethally irradiated Rag1–/– recipient mice (n = 8). The thymic CD4+Foxp3+ population was analyzed 4 weeks after reconstitution by flow cytometry. (D) Same as in (C) except that Pias1–/– or wild-type bone marrow (CD45.2) was mixed with wild-type C57SJL bone marrow (CD45.1) and injected into the Rag1–/– mice (n = 3 for wild-type and n = 4 for Pias1–/–). Experiments in (A) to (D) were performed at least three times (n = 3 to 8 for each experiment). P value was determined by unpaired t test.

 

Figure 2
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Fig. 2. Pias1–/– mice are resistant to MOG-induced EAE. (A) The percentage of CD4+Foxp3+ cells in wild-type (LN+/+) and Pias1–/– (LN–/–) lymph node cells 10 days after a single MOG35-55 injection emulsified in Freund’s complete adjuvant (n = 7). (B) Lymphocytes from Pias1–/– mice and wild-type littermates 10 days after MOG35-55 injection as in (A) were either untreated or treated with 4 µg/ml of MOG35-55 for 3 days in the presence of brefeldin A during the last 5 hours of culture. IFN-{gamma}– or IL-17–producing CD4+ cells were assayed by intracellular staining followed by flow cytometry. (C) Same as in (B) except that cells were either untreated or treated with MOG35-55 or CD3-specific antibody for 3 days. Cell proliferation was measured by one-color cell proliferation kit. (D) Pias1–/– female mice and their wild-type littermates were immunized with MOG35-55 and pertussis toxin to induce EAE as described in (17). The development of EAE was scored (n = 4). (E) Wild-type and Pias1–/– littermates were immunized as in (D), and lymphocytes were isolated 21 days after the first MOG35-55 injection and cultured for 3 days in vitro (n = 4). Cytokine production in the cell supernatant was measured by enzyme-linked immunosorbent assay (ELISA). Shown in (A) to (E) is a representative of three independent experiments (n = 3 to 7 for each experiment). Error bars represent SEM. P value was determined by unpaired t test.

 

Figure 3
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Fig. 3. PIAS1 maintains a repressive chromatin state of the Foxp3 promoter. (A) ChIP assays were performed with freshly sorted CD4+CD8+ thymocytes from male Pias1+/+ or Pias1–/– mice (n = 4), using PIAS1-specfic antibody or IgG. Bound DNA was quantified by quantitative real-time fluorescence polymerase chain reaction (QPCR), with specific primers against the various regions of the Foxp3 locus, and normalized with the input DNA. (B) Methylation analysis of the Foxp3 promoter was performed by bisulfite conversion of genomic DNA from various sorted T cell populations of wild-type and Pias1 –/– male mice (n = 4). The x axis represents the positions of the CpG sites relative to the transcription start site (+1) in the Foxp3 gene; the y axis represents the percentage. (C) ChIP assays were performed the same way as in (A) except that antibodies against trimethylated H3K9, histone H3 trimethylated at Lys27 (H3K27), or acetylated histone H3 (AceH3) were used, and the primers against the Foxp3 promoter region were used. (D) REA assays were performed with thymic CD4+CD8+ or splenic CD4+CD25 T cells. The data were quantified by QPCR and expressed as a ratio of digestion at the Foxp3 promoter or CNS2 region to digestion at a nondigestible CNS1 region of the Foxp3 locus. Shown in (A) to (D) is a representative of three independent experiments (n = 4 to 6 for each experiment). Error bars represent SEM. P value was determined by unpaired t test.

 

Figure 4
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Fig. 4. PIAS1 is required for the promoter recruitment of DNMT3A and DNMT3B. (A) Coimmunoprecipitation assays. Protein extracts from wild-type thymocytes or splenic CD4+CD25 T cells were subjected to immunoprecipitation with PIAS1-specific antibody or IgG, followed by immunoblotting with the indicated antibodies. (B) ChIP assays were performed with wild-type thymocytes by using PIAS1-specific antibody or IgG. The presence of the Foxp3 promoter region in the precipitates was quantified by QPCR. In the re-ChIP experiments, PIAS1-specific antibody precipitates were released, reimmunoprecipitated with an antibody against DNMT3A or DNMT3B, and analyzed for the presence of the Foxp3 promoter sequence. (C) ChIP assays were performed with freshly sorted CD4+CD8+ thymocytes from male Pias1+/+ or Pias1–/– mice (n = 4), using an antibody against DNMT3A or DNMT3B. Bound DNA was quantified by QPCR with the specific primers against Foxp3 promoter, CNS1 or CNS2 region. (D) Methylation analysis of the Foxp3 promoter was performed by bisulfite conversion of genomic DNA from thymocytes of Rag1–/– mice reconstituted with either control GFP (Con) or Cre-GFP (Cre) retrovirus-infected Dnmt3a2lox/2lox/ Dnmt3b2lox/2lox bone marrow. The x axis represents the positions of the CpG sites relative to the transcription start site (+1) in the Foxp3 gene; the y axis represents the percentage. Shown in (A) to (D) is a representative of three independent experiments (n = 4 to 6 for each experiment). Error bars represent SEM. P value was determined by unpaired t test.

 


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