Transcriptional profiling of NF-kB's Rel and RelA proteins. Gallus gallus

Rel and RelA proteins were stably expressed in the chicken DT40 pre-B cell line and analyzed by transcription profiling to identify transformation-impacting genes regulated by NF-kB’s oncogenic v-Rel and c-Rel proteins. This analysis uncovered both common and differential gene expression profiles in cells expressing Rel vs. RelA proteins, and revealed that Rel protein expression can lead to gene-specific transcriptional repression, as seen for key B-cell receptor (BCR) components and signaling molecules like B-cell linker (BLNK), the B-cell adaptor for PI3K (BCAP) and Ig?. These were also downregulated in cells expressing a transformation-competent chimeric RelA/v-Rel protein, suggesting a correlation with the capacity of Rel proteins to transform lymphocytes. DNA binding, ChIP and transformation assays indicate that downregulation of BLNK and BCAP is an important contributing factor to the malignant transformation of lymphocytes by Rel and suggest that gene repression may be as important as transcriptional activation for the transforming activity of Rel proteins. Keywords: Comparative transcriptional profiling Overall design: v-Rel, c-Rel (chicken, mouse, human), RelA (mouse, human) or a human RelA/v-Rel chimera were stably expressed at equivalent levels in the chicken pre-B-cell line DT40. Three independent cell clones were analyzed for each protein, using dye-swap experiments. Their gene expression profiles were compared to that of parental DT40 cells by cDNA microarray. Triplicate data sets were averaged to identify changes in gene expression in response to expression of each of these proteins. Differential expression analysis was performed using CyberT (Baldi and Long, Bioinformatics, 2001), a Bayesian t-statistic methodology that is designed for microarray analyses in studies with low replicate numbers. For our CyberT analysis, we employed the default parameters. Differential gene expression was identified by ranking each gene's corresponding Bayesian p-values and applying a false discovery rate correction of 5% (Benjamini and Hochberg, J R Statistical Soc Ser B - Methodological, 1995). In addition, we applied a fold-change threshold of ± 1.5 as an additional criterion. Accordingly, relative expression levels for a given gene with a p-value that satisfies the FDR condition and fold-change criteria were identified as differentially expressed. Note: relative expression levels were transformed to natural log (ln) for CyberT analysis and the results are reported in those units. Results of differential expression analysis are reported in the supplementary table "Differentially-expressed genes for each condition" where 1=TRUE, 0=FALSE.

将Rel与RelA蛋白稳定表达于鸡DT40前B细胞系中，通过转录谱分析鉴定受NF-κB致癌性v-Rel与c-Rel蛋白调控的、影响细胞转化的基因。本分析在表达Rel与RelA蛋白的细胞中同时发现了共同及差异的基因表达谱，并揭示Rel蛋白的表达可引发基因特异性的转录抑制，这一现象见于关键B细胞受体（B-cell receptor, BCR）组分以及B细胞衔接蛋白如B细胞衔接分子（B-cell linker, BLNK）、PI3K的B细胞衔接蛋白（B-cell adaptor for PI3K, BCAP）及Ig？等信号分子。上述分子在表达具有转化能力的嵌合RelA/v-Rel蛋白的细胞中同样出现下调，提示这与Rel蛋白转化淋巴细胞的能力存在相关性。DNA结合实验、染色质免疫沉淀（ChIP）与转化实验表明，BLNK与BCAP的下调是Rel介导淋巴细胞恶性转化的重要促成因素，并提示基因抑制对于Rel蛋白的转化活性而言，可能与转录激活同等重要。
关键词：比较转录谱分析
整体实验设计：将v-Rel、c-Rel（鸡源、鼠源、人源）、RelA（鼠源、人源）或人源RelA/v-Rel嵌合蛋白以等同水平稳定表达于鸡前B细胞系DT40中。针对每种蛋白，选取3个独立细胞克隆进行分析，采用染料互换（dye-swap）实验。通过cDNA微阵列（cDNA microarray）将这些细胞的基因表达谱与亲代DT40细胞进行比对。对三次重复的数据集取平均值，以鉴定表达上述各类蛋白后发生的基因表达变化。差异表达分析采用CyberT工具（Baldi与Long, 《生物信息学》, 2001），这是一种贝叶斯t统计方法，专为低重复数研究中的微阵列分析设计。本次CyberT分析采用默认参数。通过对每个基因对应的贝叶斯p值进行排序，并采用5%的错误发现率（false discovery rate, FDR）校正（Benjamini与Hochberg, 《皇家统计学会B辑：方法学》, 1995），以鉴定差异表达基因。此外，我们采用±1.5的倍数变化阈值作为额外筛选标准。据此，将满足错误发现率条件与倍数变化标准的特定基因的相对表达水平判定为差异表达。
注：为进行CyberT分析，相对表达水平已转换为自然对数（ln）形式，结果以该单位报告。差异表达分析的结果已整理于补充表"Differentially-expressed genes for each condition"中，其中1代表“是”，0代表“否”。