Gene expression profiling in neuronal cells identifies a different type of transcriptome modulated by NF-Y [Grp94]. Gene expression profiling in neuronal cells identifies a different type of transcriptome modulated by NF-Y [Grp94]

We knocked down NF-Y in two types of neuronal cells, neuro2a neuroblastoma cells and sorted brain striatal cells, and performed gene expression profiling. We found that the down-regulated genes preferentially contained NF-Y-binding motifs in their proximal promoters, and notably enriched genes related to ER functions rather than those for cell cycle. These are highly contrast to the profiling data of HeLa and embryonic stem cells in which distinct down-regulation of cell cycle-related genes was observed by NF-Y knockdown. Clustering analysis further identified several functional clusters where populations of the down-regulated genes were highly distinct. Further analyses using chromatin immunoprecipitation and RNA-seq data revealed that transcriptomic difference was not correlated with the DNA binding of NF-Y but with splicing of NF-YA. These data suggest that neuronal cells have a different type of transcriptome in which ER-related genes are dominantly modulated by NF-Y, and imply that NF-YA splicing alteration rather than DNA-binding preference could be involved in this cell type-specific gene modulation. Overall design: To screen differentially expressed genes in mouse neuro2a cells, the cells were transfected with miR RNAi vectors for Grp94 (Hsp90b1) or an empty vector, and then subjected to microarray analysis using Affymetrix Mouse Gene 1.0 ST Arrays. Obtained microarray data were analyzed using an Agilent GeneSpring GX software to identify differentially expressed genes (DEGs) by gene knockdown. Please note that the differentially expressed data is provided in the 'Affy_N2a_Grp94_Filt20_P0.01.txt' since the normalized data for each sample is unavailable.

我们在两种神经元细胞——Neuro2a神经母细胞瘤细胞以及分选的大脑纹状体细胞中敲低了核因子Y（NF-Y），随后开展了基因表达谱分析。我们发现，下调基因的近端启动子区域优先富集NF-Y结合基序，且显著富集的是与内质网（Endoplasmic Reticulum, ER）功能相关的基因，而非细胞周期相关基因。这与HeLa细胞和胚胎干细胞（embryonic stem cells）的谱分析结果形成鲜明对比：在后者中，敲低NF-Y会导致细胞周期相关基因出现显著下调。聚类分析进一步鉴定出多个功能簇，其中下调基因的群体特征差异显著。进一步利用染色质免疫沉淀（chromatin immunoprecipitation）和RNA测序（RNA-seq）数据开展的分析显示，转录组差异与NF-Y的DNA结合活性无关，而是与NF-YA的剪接相关。上述数据表明，神经元细胞拥有独特的转录组模式，其中内质网相关基因主要受NF-Y调控；同时暗示，NF-YA的剪接改变而非DNA结合偏好性，介导了这种细胞类型特异性的基因调控。整体实验设计：为筛选小鼠Neuro2a细胞中的差异表达基因（differentially expressed genes, DEGs），我们将细胞转染针对Grp94（Hsp90b1）的miRNA干扰载体或空载体，随后采用Affymetrix小鼠基因1.0 ST芯片阵列开展微阵列分析。我们使用安捷伦（Agilent）GeneSpring GX软件对获得的微阵列数据进行分析，通过基因敲低筛选差异表达基因。请注意，由于各样本的标准化数据无法获取，差异表达数据已提供于"Affy_N2a_Grp94_Filt20_P0.01.txt"文件中。