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

DNA序列特异性结合蛋白如p53的调控对基因表达和稳态至关重要。p53蛋白是细胞中的一种快速应激反应蛋白，激活一种独特的转录组，决定细胞命运。为了准备动物研究的背景，在这一年里，我利用体外细胞系Raji和A549细胞研究了p53蛋白的转录调节。我发现Raji细胞含有p53 p72R错义突变，肺腺癌细胞系A549细胞含有野生型p53。为了了解p53 P72R以细胞周期依赖的方式调控，我将Raji细胞与HU处理同步，在诱导复制应激的同时允许细胞在G1-S期同步。用胸苷嘧啶和诺可达唑使Raji细胞同步于G2-M期。此外，使用Nutlin-3a处理激活Raji细胞和A549细胞中的p53。综上所述，我可以确认1。在Raji细胞中，Nutlin-3a处理未能上调MDM2的表达，这表明p53 P72R在其靶基因MDM2的基因表达中功能丧失。在含有野生型p53的A549中，Nutlin-3处理可诱导p53活化和MDM2表达。2. 蛋白和qPCR分析提示p53 p72R对转移受体、CXCR5、CCR7、RAC1、RHOA和增殖基因亚群HLA-DQ基因等基因亚群的调控偏离野生型p53。在A549细胞的野生型p53激活中没有观察到这种调节。

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.