Different roles of C/EBP-alpha in chromosome conformation during lung injury of young and old mice

C/EBP-α在年轻和年老小鼠肺损伤过程中染色体构象的不同作用

• 批准号: 21K16142
• 负责人: Gothwal Santosh・Kumar
• 金额: $ 2.91万
• 依托单位: Kyoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Early-Career Scientists
• 财政年份: 2021
• 资助国家: 日本
• 起止时间: 2021-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-21K16142/
• 关键词: Chromosome conformation; Tumor protein; DNA damage; Epigenetics; Transcription; Homeostasis; C/EBP-alpha; lung homeostasis; chromosomes conformation; CCAAT enhancer; Cohesin

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.

DNA序列特异性结合蛋白如p53的调节对于基因表达和稳态是至关重要的。p53蛋白是细胞中的快速应激反应蛋白，并激活决定细胞命运的独特转录组。为了给动物研究做好准备，今年，我使用体外细胞系Raji和A549细胞研究了p53蛋白的转录调控。我发现Raji细胞中存在p53 p72 R错义突变，肺腺癌细胞系A549细胞中含有野生型p53。为了理解p53 P72 R在细胞周期依赖性方式中的调节，我用HU处理同步化Raji细胞，其允许细胞在G1-S期同步化，同时诱导复制应激。我还通过胸苷和诺考达唑处理使Raji细胞同步化于G2-M期。此外，Nutlin-3a处理用于激活Raji细胞和A549细胞中的p53。在Raji细胞中，Nutlin-3a处理未能上调MDM 2表达，这表明p53 P72 R在其靶基因MDM 2的基因表达中的功能丧失。在含有野生型p53的A549中，Nutlin-3处理可以诱导p53活化和MDM 2表达。2.蛋白质和qPCR分析表明，p53 p72 R调节偏离野生型p53，用于调节包括转移受体、CXCR 5、CCR 7、RAC 1、RHOA和增殖基因亚群HLA-DQ基因在内的基因亚群。在A549细胞中野生型p53活化中未观察到这种调节。

项目成果

A mutant p53 P72R driven modulation of CXCR5-CXCL13 axis in human B cell lymphoma-migration

突变体 p53 P72R 驱动人 B 细胞淋巴瘤迁移中 CXCR5-CXCL13 轴的调节

• 发表时间: 2022
• 作者: 大槻 祥一郎;Taylor Shalina;Li Dan;Moonen JR;Marciano David;Cao Aiqin;Wang Lingli;澤田 博文;三谷 義英;Rabinovitch Marlene;Santosh Kumar Gothwal
• 通讯作者: Santosh Kumar Gothwal

18th International p53 workshop

第18届国际p53研讨会

• 发表时间: 2022