Phosphorylation of OGFOD1 by Cell Cycle-Dependent Kinase 7/9 Enhances the Transcriptional Activity of RNA Polymerase II in Breast Cancer Cells
Abstract
Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Plasmids
2.3. CRISPR/Cas9-Mediated Gene Knockout
2.4. Proximity-Dependent Labeling
2.5. Silver Staining
2.6. Protein Purification
2.7. Immunoprecipitation Assay
2.8. In Vitro Binding Assay
2.9. In Vitro Phosphorylation Assay
2.10. mRNA Sequencing and Analysis
2.11. Chromatin IP Assay
2.12. Cell Proliferation, Invasion, and Migration Assay
2.13. Lentivirus-Based shRNA Production and Expression
2.14. Animal Studies
2.15. Statistical Analysis
3. Results
3.1. OGFOD1 Has an Important Role for Rapid Proliferation of Breast Cancer Cell Lines
3.2. OGFOD1 Binds to the C-Terminal Domain of RNA Polymerase II and Attenuates Phosphorylation States
3.3. OGFOD1 Alters Phosphorylation States of RNA Polymerase II
3.4. Nuclear Localization of OGFOD1 Is Critical for Regulating RNA Polymerase II Activity
3.5. OGFOD1 KO Reduces Metastatic Gene Expressions in MDA-MB-231
3.6. CDK7 and CDK9 Phosphorylate Serine 256 of OGFOD1
3.7. CDK7/9 Enhance OGFOD1 Function
3.8. Non-Phosphorylated Mutation of Serine 256 Hampers Oncogenic Ability
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Conflicts of Interest
References
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Lee, H.-T.; Lee, I.-H.; Kim, J.-H.; Lee, S.; Kwak, S.; Suh, M.-Y.; Hwang, I.-Y.; Kang, B.-G.; Cha, S.-S.; Lee, B.-I.; et al. Phosphorylation of OGFOD1 by Cell Cycle-Dependent Kinase 7/9 Enhances the Transcriptional Activity of RNA Polymerase II in Breast Cancer Cells. Cancers 2021, 13, 3418. https://doi.org/10.3390/cancers13143418
Lee H-T, Lee I-H, Kim J-H, Lee S, Kwak S, Suh M-Y, Hwang I-Y, Kang B-G, Cha S-S, Lee B-I, et al. Phosphorylation of OGFOD1 by Cell Cycle-Dependent Kinase 7/9 Enhances the Transcriptional Activity of RNA Polymerase II in Breast Cancer Cells. Cancers. 2021; 13(14):3418. https://doi.org/10.3390/cancers13143418
Chicago/Turabian StyleLee, Han-Teo, Il-Hwan Lee, Jae-Hwan Kim, Sangho Lee, Sojung Kwak, Min-Young Suh, In-Young Hwang, Bu-Gyeong Kang, Sun-Shin Cha, Byung-Il Lee, and et al. 2021. "Phosphorylation of OGFOD1 by Cell Cycle-Dependent Kinase 7/9 Enhances the Transcriptional Activity of RNA Polymerase II in Breast Cancer Cells" Cancers 13, no. 14: 3418. https://doi.org/10.3390/cancers13143418
APA StyleLee, H.-T., Lee, I.-H., Kim, J.-H., Lee, S., Kwak, S., Suh, M.-Y., Hwang, I.-Y., Kang, B.-G., Cha, S.-S., Lee, B.-I., Lee, S.-E., Choi, J., Roe, J.-S., Cho, E.-J., & Youn, H.-D. (2021). Phosphorylation of OGFOD1 by Cell Cycle-Dependent Kinase 7/9 Enhances the Transcriptional Activity of RNA Polymerase II in Breast Cancer Cells. Cancers, 13(14), 3418. https://doi.org/10.3390/cancers13143418