Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006 Dec 5;103(49):18715-20.
doi: 10.1073/pnas.0604800103. Epub 2006 Nov 22.

The promyelocytic leukemia protein functions as a negative regulator of IFN-gamma signaling

Youn-Hee Choi  1 , Rosa Bernardi, Pier Paolo Pandolfi, Etty N Benveniste
Affiliations

The promyelocytic leukemia protein functions as a negative regulator of IFN-gamma signaling

Youn-Hee Choi et al. Proc Natl Acad Sci U S A. .

Abstract

IFN-gamma is an immunomodulatory cytokine and uses the STAT-1alpha transcription factor to mediate gene expression. The promyelocytic leukemia (PML) protein regulates transcription as an activator or repressor, depending on the gene under investigation. Herein, we examined the influence of PML on IFN-gamma signaling, using PML wild-type (Pml(+/+)) and deficient (Pml(-/-)) mouse embryonic fibroblasts (MEF). Pml(-/-) MEF exhibit enhanced IFN-gamma-induced STAT-1alpha transcriptional activity compared with Pml(+/+) cells. Moreover, reconstitution of PML in Pml(-/-) MEF reduced STAT-1alpha transcriptional activity to levels comparable to Pml(+/+) MEF. Numerous endogenous IFN-gamma-regulated genes were up-regulated in Pml(-/-) MEF compared with Pml(+/+) MEF. IFN-gamma-mediated STAT-1alpha DNA-binding activity was enhanced in Pml(-/-) cells compared with Pml(+/+) cells. Lastly, IFN-gamma enhanced the formation of a PML-STAT-1alpha complex in the nucleus. These data suggest a novel function for PML in the IFN-gamma signaling pathway by inhibiting STAT-1alpha DNA binding and transcriptional activity.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
PML represses IFN-γ-induced STAT-1α transcriptional activity. Immortalized Pml+/+ or Pml−/− MEF were transfected with the minimal CIITA reporter containing one GAS element (CIITAp-D4; A), the full length CIITA reporter containing three GAS elements (CIITA; B), or the GAS4 reporter that contains four GAS elements (GAS4; C). (D) Primary MEF were also tested with the GAS4 reporter. (E) Reintroduction of PML isoforms in Pml−/− MEF. Immortalized Pml+/+ or Pml−/− MEF were cotransfected with the GAS4 reporter and/or increasing concentrations of PML I, PML III, and PML IV expression vectors (0–2 μg), and equal molar concentrations of empty vector. (Lower) Immunoblot analysis of Pml−/− MEF transfected with PML I, PML III, and PML IV. Cells were either untreated or treated with IFN-γ (10 ng/ml) for 16 h, and then luciferase activity was determined. Data are presented as fold increase in relative luciferase activity (RLA) compared with RLA in the absence of IFN-γ. Values are the mean ± SD of three separate experiments. ∗, P < 0.05.
Fig. 2.
Fig. 2.
PML influences IFN-γ-regulated genes. (A) RNA from immortalized Pml+/+ and Pml−/− MEF untreated or treated with IFN-γ for 4 h was subjected to microarray analysis. The proportion of genes that are up- or down-regulated (2-fold change) between untreated and IFN-γ-treated cells is shown. (B) RNA from immortalized Pml+/+ and Pml−/− MEF treated with IFN-γ for the indicated times was subjected to RPA for IP-10 and GAPDH mRNA levels. Fold induction is shown. Representative of three independent experiments.
Fig. 3.
Fig. 3.
PML inhibits DNA binding activity of STAT-1α. (A) Nuclear extracts from immortalized Pml+/+ and Pml−/− MEF treated with IFN-γ for 0–5 h were incubated in the presence of a radiolabeled probe containing a GAS element and subjected to EMSA. Anti-STAT-1α or -STAT-3 antibodies were added to test the specificity of interaction. Binding specificity was also tested by adding a 100-fold molar excess of cold GAS probe (competition). (B) Cells were treated with IFN-γ for up to 4 h and fixed by using formaldehyde. Chromatin was sheared and immunoprecipitated by using STAT-1 antibody or normal rabbit IgG, then amplified by RT-PCR using primers designed for the murine CIITA pIV promoter. (C) Nuclear extracts from Pml−/− MEF treated with IFN-γ for 1 h were preincubated with PML IV protein (0–100 ng) for 1 h and then subjected to EMSA (lanes 1–3). BSA was used as a control for the PML protein (lane 4). Representative of three experiments.
Fig. 4.
Fig. 4.
PML interacts with STAT-1 in vivo. (A) Immortalized Pml+/+ and Pml−/− MEF were incubated in the absence or presence of IFN-γ for 0.5 and 4 h, and whole-cell lysates were prepared. Cell lysates (1.2 mg) were immunoprecipitated with anti-STAT-1 antibody and subjected to SDS/PAGE for immunoblot analysis with anti-PML antibody. Input samples (5%) were assayed for STAT-1 and PML protein expression by immunoblotting. (B) RAW264.7 cells were either untreated or treated with IFN-γ for 0.5 h and processed as described in A. (C) Pml−/− MEF transfected with control vector or the PML III expression vector were either untreated or treated with IFN-γ for 0.5 h and immunoprecipitated with anti-STAT-1 antibody. Anti-STAT-1 immunoprecipitates were subjected to immunoblot analysis with anti-PML antibody. Input samples (5%) were also assayed for PML expression by immunoblotting. Representative of three experiments.
Fig. 5.
Fig. 5.
PML and STAT-1 colocalize upon IFN-γ treatment. (A) Immortalized Pml+/+ MEF were incubated with IFN-γ for 0.5 h, stained with rabbit polyclonal anti-STAT-1 (green) and mouse monoclonal anti-PML (red) antibodies, and analyzed by immunofluorescent microscopy. Representative of three experiments. (B) Immortalized Pml+/+ MEF were incubated in the absence or presence of IFN-γ for 0.5 and 6 h. Cytoplasmic and nuclear fractions were prepared and assayed for PML–STAT-1 complex formation by coimmunoprecipitation. Input samples from the cytoplasmic and nuclear fractions were subjected to immunoblotting to detect caspase-3 and Sp1, respectively. Representative of three experiments.
Fig. 6.
Fig. 6.
Proposed model for the role of PML in regulating IFN-γ signaling. (A) IFN-γ-induced STAT-1α DNA-binding and transcriptional activities are reduced in the presence of PML. Because IFN-γ induces the colocalization and formation of a PML-STAT-1α complex in the nucleus, we propose that PML may inhibit the binding of STAT-1α to DNA and thus control its activity, leading to a dampening of IFN-γ inducible genes. (B) In the absence of PML, STAT-1α binds to GAS elements, perhaps with higher affinity, and IFN-γ inducible gene expression is heightened.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Regis G, Conti L, Boselli D, Novelli F. Trends Immunol. 2006;27:96–101. - PubMed
    1. Ramana CV, Gil MP, Schreiber RD, Stark GR. Trends Immunol. 2002;23:96–101. - PubMed
    1. Decker T, Kovarik P. Oncogene. 2000;19:2628–2637. - PubMed
    1. Shuai K, Liu B. Nat Rev Immunol. 2003;3:900–911. - PubMed
    1. Yasukawa H, Misawa H, Sakamoto H, Masuhara M, Sasaki A, Wakioka T, Ohtsuka S, Imaizumi T, Matsuda T, Ihle JN, Yoshimura A. EMBO J. 1999;18:1309–1320. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources