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PU.1, interferon regulatory factor 1, and interferon consensus sequence-binding protein cooperate to increase
E A Eklund1, A Jalava, R Kakar
1Lurleen B. Wallace Tumor Institute, Department of Hematology and Oncology and the Comprehensive Cancer Center, University of Alabama at Birmingham and the Birmingham Veterans Administration Hospital, Birmingham, Alabama 35294, USA.
The Journal of Biological Chemistry
|June 5, 1998
Summary
This study identifies a multiprotein complex, HAF1, crucial for regulating gp91(phox) expression in phagocytes. It reveals a novel transcriptional activation mechanism involving PU.1, IRF-1, and ICSBP, impacting immune cell function.
Area of Science:
- Molecular Biology
- Immunology
- Gene Regulation
Background:
- The CYBB gene encodes gp91(phox), a key subunit of the phagocyte respiratory burst oxidase.
- Transcription of CYBB is specific to terminally differentiated phagocytic cells.
- Interferon-gamma (IFNγ) induces gp91(phox) expression, mediated by a proximal promoter element binding hematopoiesis-associated factor (HAF1).
Purpose of the Study:
- To elucidate the composition and function of the HAF1 complex.
- To investigate the mechanism of IFNγ-induced gp91(phox) expression.
- To describe a novel transcriptional activation pathway involving PU.1, IRF-1, and ICSBP.
Main Methods:
- Electrophoretic mobility shift assays (EMSA) to analyze protein-DNA interactions.
- Co-immunoprecipitation and Western blotting to identify protein components.
- Reporter gene assays in U937 cells to assess promoter activity.
- In vitro translation to reconstitute protein complexes.
Main Results:
- HAF1 is a multiprotein complex containing PU.1, IRF-1, and ICSBP.
- Reconstitution of HAF1 requires PU.1 with either IRF-1 or ICSBP.
- A distinct complex, HAF1a, requires PU.1, IRF-1, and ICSBP for reconstitution.
- Co-transfection with PU.1, IRF-1, and ICSBP strongly activated a CYBB promoter construct, dependent on serine 148-phosphorylated PU.1.
Conclusions:
- A novel mechanism for PU.1-mediated transcriptional activation involves interactions with both IRF-1 and ICSBP.
- ICSBP plays a novel activation role within a multiprotein complex.
- These findings provide insights into the regulation of immune cell function and oxidative burst.