2021 Fiscal Year Research-status Report
Different roles of C/EBP-alpha in chromosome conformation during lung injury of young and old mice
| Project/Area Number |
21K16142
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| Research Institution | Kyoto University |
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Principal Investigator |
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| Project Period (FY) |
2021-04-01 – 2024-03-31
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| Keywords | Chromosome conformation / Tumor protein / DNA damage / Epigenetics / Transcription / Homeostasis |
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| Outline of Annual Research Achievements |
DNA sequence specific binding protein such as p53 regulation is critical for gene expression and homeostasis. The p53 protein is a quick stress response protein in the cells and activates a unique transcriptome which determines cellular fate. To prepare a background for animal studies, in this year, I investigated transcriptional regulation by p53 protein using in vitro cell lines, Raji and A549 cells. I identified that Raji cells harbor a p53 p72R missense mutation, the lung adenocarcinoma cell line, A549 cells contain the wild type p53. To understand the p53 P72R regulation in cell cycle dependent manner, I synchronized the Raji cells with HU treatment which allows synchronization of cells in G1-S phase while inducing a replication stress. I also synchronized Raji cells in G2-M phase by Thymidine and Nocodazole treatment. In addition, Nutlin-3a treatment was used to activate p53 in the Raji cells and A549 cells.
In summary, I could confirm that 1. In Raji cells, Nutlin-3a treatment fails to upregulate the MDM2 expression, this suggesting a loss of function of p53 P72R in the gene expression of its target genes, MDM2. In the A549 containing the wild type p53, Nutlin-3 treatment can induce p53 activation and MDM2 expression. 2. The protein and qPCR analysis suggested p53 p72R regulation is deviated than wild type p53 for regulation of genes subsets including of metastatic receptors, CXCR5, CCR7, RAC1, RHOA and proliferation gene subsets HLA-DQ genes. This regulation is not observed in wild type p53 activation in A549 cells.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
In this years, I have moved to Cancer Research Institute of Kanazawa University. I successfully transferred my remaining Wakate-B grants from Kyoto University to Kanazawa University. I will work as an Assistant professor in the Division of Oncology and Molecular Biology and PI will be Prof. Chiaki Takahashi. I will carry out my remaining experiments here at Kanazawa University. The institute also provide me access to the good animal institute, therefore I will be able to smoothly investigate on my planned research. All the conditions for my research are good around me. I hope that I will be able to execute good findings from my planned research.
Currently, A manuscript is in preparation defining key signatures of p53 P72R functions upon HU induced replication stress and cell cycle dependent manner. These results are insightful for cancer biology. The title of manuscript from above study is - “A cell cycle dependent modulation of CXCR5-CXCL13 axis in human B cell lymphoma-migration” is in the preparation”.
Furthermore, my first-year research results are to be presented as poster in the upcoming 18th international workshop on p53 biology meeting held at Weismann Institute of Science from the May 22-26, 2022.
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| Strategy for Future Research Activity |
In the next year, I will progress my project with mainly in vivo models. The stepwise plan is narrated below.
1. I will prepare a systemic lung/liver cancer mouse model p53 knockout and Cebp-alpha knockout. From the injured and control mice, I will isolate the lung and liver cells and investigate the transcriptomics of control and injured mice by RNA seq analysis. 2. After the transcriptome analysis from step 1, the topmost up/downregulated genes will be categorized. 3. I will then perform a the perform the genome wide binding analysis for the p53, CTCF and C/EBP-alpha by ChIP-seq analysis. I will then corelate if the upregulated or down regulated genes in cancer mice harbors any binding of p53, CTCF and C/EBP-alpha in the vicinity of the gene promoters or topologically associated domains (TADs). The correlation analysis will determine the homeostasis related transcriptional programmed by p53 and chromosome conformation regulators, CTCF upon a cellular stress. The results will be interpreted and planned to publish in peer reviewed journals.
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| Causes of Carryover |
The remaining amount will be used in next year for buying of essential antibodies for ChIP such as p53, CTCF and the RNA-seq analysis costs. The mouse will be purchased for experiments. In addition, an international workshop on p53 will be stranded to present a poster on the research achievements in last year.
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