ISGF3 and STAT2/IRF9 Control Basal and IFN-Induced Transcription through Genome-Wide Binding of Phosphorylated and Unphosphorylated Complexes to Common ISRE-Containing ISGs
Abstract
:1. Introduction
2. Results
2.1. Characterization of Time-Dependent IFNα Responses in WT and STAT1-KO Cells
2.2. Genome-Wide Characterization of IFNα-Induced Transcription in WT vs. STAT1KO Cells
2.3. Genome-Wide Binding of Phosphorylated and Unphosphorylated ISGF3 Components to ISRE Sites of IFNα Upregulated Genes in WT vs. STAT1KO Cells
2.4. U-ISG Expression Correlates with pSTAT1 and pSTAT2 Expression and Long-Term Binding in Wild Type and STAT1KO Cells
2.5. The Role of Phosphorylation of ISGF3 and STAT2/IRF9 in the Regulation of Prolonged ISG Expression in Wild Type and STAT1KO Cells
2.6. The Role of Unphosphorylated ISGF3 Components in the Regulation of Basal ISG Expression in Cells Overexpressing STAT1, STAT2 and IRF9
2.7. The Role of Unphosphorylated ISGF3 Components in Prolonged IFNα Signaling in Cells Overexpressing STAT1, STAT2 and IRF9
3. Discussion
4. Materials and Methods
4.1. Cell Lines
Cell Culture and Treatment
4.2. Western Blotting
4.3. RNA Isolation and qPCR
4.4. RNA-Seq Library Preparation and Sequencing
4.5. RNA-Seq Data Analysis
4.5.1. Differential Gene Expression Analysis (DEG)
4.5.2. Heatmap Generation
4.5.3. Gene Ontology Term Enrichment Analysis
4.5.4. Selection of Commonly Upregulated Genes
4.6. Chromatin Immunoprecipitation (ChIP) and Sequencing (ChIP-Seq)
4.7. ChIP-Seq Data Analysis
4.7.1. Visualization in the Integrative Genomics Viewer
4.7.2. Binding Profiles
4.7.3. Binding Site Motifs Identification
4.8. Deposited Sequencing Data
4.9. Antiviral Assay
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
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Nowicka, H.; Sekrecka, A.; Blaszczyk, K.; Kluzek, K.; Chang, C.-Y.; Wesoly, J.; Lee, C.-K.; Bluyssen, H.A.R. ISGF3 and STAT2/IRF9 Control Basal and IFN-Induced Transcription through Genome-Wide Binding of Phosphorylated and Unphosphorylated Complexes to Common ISRE-Containing ISGs. Int. J. Mol. Sci. 2023, 24, 17635. https://doi.org/10.3390/ijms242417635
Nowicka H, Sekrecka A, Blaszczyk K, Kluzek K, Chang C-Y, Wesoly J, Lee C-K, Bluyssen HAR. ISGF3 and STAT2/IRF9 Control Basal and IFN-Induced Transcription through Genome-Wide Binding of Phosphorylated and Unphosphorylated Complexes to Common ISRE-Containing ISGs. International Journal of Molecular Sciences. 2023; 24(24):17635. https://doi.org/10.3390/ijms242417635
Chicago/Turabian StyleNowicka, Hanna, Agata Sekrecka, Katarzyna Blaszczyk, Katarzyna Kluzek, Chan-Yu Chang, Joanna Wesoly, Chien-Kuo Lee, and Hans A. R. Bluyssen. 2023. "ISGF3 and STAT2/IRF9 Control Basal and IFN-Induced Transcription through Genome-Wide Binding of Phosphorylated and Unphosphorylated Complexes to Common ISRE-Containing ISGs" International Journal of Molecular Sciences 24, no. 24: 17635. https://doi.org/10.3390/ijms242417635