Effect of ARF on the transcription of p53 target genes in homologous recombination deficient cells

ARF is a tumour suppressor activated by oncogenic stress, which stabilizes p53. Although p53 is a key component of the response to DNA damage, a similar function for ARF has not been ascribed. In this work we show that homologous recombination (HR) -deficient primary mouse and human cells accumulate DNA damage, which triggers checkpoint signalling and ARF activation. We also show that in the absence of ARF p53 is induce in response to DNA damage, but surprisingly fails to trigger senescence. The hypothesis tested in this study was that ARF deficiency alters the spectrum of genes activated by p53 in response to HR deficiency. Using mRNA microarray analysis and expression profiling, we identified genes upregulated in wild type primary MEFs treated with RAD51 shRNA, whose expression was not induced in similarly treated Arf -/- primary MEFs. Clustering analysis of p53 transcriptional targets with increased expression in RAD51-depleted cells revealed three groups: genes requiring functional ARF for their expression, genes transcribed in an ARF-independent manner and genes that are upregulated in both ARF-proficient and ARF-deficient cells. Among the genes in the last group we found Cdkn1a(p21), encoding the p21 inhibitor of cyclindependent kinases and well-characterized effector of the p53 role in promoting senescence. The subset of p53 targets requiring ARF for their transcription was specifically upregulated in response to RAD51 inhibition in wild type, but not Arf -/- MEFs. Among these genes, two encoded the dual specificity phosphatase (DUSP) family members DUSP4 and DUSP7, which act as ERK phosphatases and negative regulators of the MAPK kinase pathway in mammalian cells. Four samples were analysed (WT or ARF negative cells transfected with GFP or RAD51 shRNAs) in duplicate. Samples treated with GFP shRNA are the reference samples and the ones treated with RAD51 shRNA are the experimental ones (RAD51 depletion leads to HR deficiency). The WT cell line is our control cell line to study the effect of HR deficiency on the transcription of p53 target genes

ARF是一种受致癌应激激活的肿瘤抑制因子，可稳定p53蛋白。尽管p53是DNA损伤反应的关键调控组分，但ARF的类似功能此前尚未被明确界定。本研究证实，同源重组（homologous recombination, HR）缺陷的原代小鼠及人类细胞会发生DNA损伤积累，进而触发检查点信号传导并激活ARF。此外本研究还发现，在缺失ARF的情况下，p53虽可响应DNA损伤被诱导表达，但令人意外的是其无法引发细胞衰老。本研究的验证假说为：ARF缺陷会改变p53响应HR缺陷时所激活的基因表达谱。通过mRNA微阵列分析与表达谱分析，我们在经RAD51短发夹RNA（RAD51 shRNA）处理的野生型原代小鼠胚胎成纤维细胞（mouse embryonic fibroblasts, MEFs）中筛选得到上调表达的基因，而在经相同方式处理的Arf基因敲除（Arf -/-）原代MEFs中，这些基因的表达并未被诱导。对RAD51耗竭细胞中表达上调的p53转录靶点进行聚类分析后，可将其分为三类：依赖功能性ARF才能表达的基因、以ARF非依赖方式转录的基因，以及在ARF功能正常与缺陷细胞中均会上调的基因。在最后一类基因中，我们发现了Cdkn1a（p21）——其编码细胞周期蛋白依赖性激酶抑制剂p21，是p53介导细胞衰老这一功能的经典效应分子。依赖ARF进行转录的p53靶点子集，仅在野生型（而非Arf -/-）MEFs中经RAD51抑制后出现特异性上调。在这些基因中，有两个分别编码双特异性磷酸酶（dual specificity phosphatase, DUSP）家族成员DUSP4与DUSP7，二者在哺乳动物细胞中可作为ERK磷酸酶及丝裂原活化蛋白激酶（MAPK）通路的负调控因子。本实验共分析4组样本（经GFP或RAD51 shRNA转染的野生型或ARF缺陷细胞），每组设置重复样本。以GFP shRNA处理的样本作为参照组，经RAD51 shRNA处理的样本为实验组（RAD51耗竭会导致HR缺陷）。本研究使用的野生型细胞系，是用于探究HR缺陷对p53靶点基因转录影响的对照细胞系。