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

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: The AAV vectors expressing EmGFP with miR RNAi for NF-YA and -YC (YA-8/YC-2) or control non-targeting miR RNAi (NT-2) under CAG promoter were stereotaxically injected into striatum in both hemispheres of 6-week-old wild-type male B6 mice . Total 5 (YA-8/YC-2) and 6 (NT-2) mice were injected. Three weeks after the injection, stirata were surgically isolated and dissociated by Papain. To isolate RNAi-expressing cells positive for green fluorescence (EmGFP), dissociated and sorted by FACS. Total RNAs were prepared from the sorted cells by RNeasy Micro Kit (Qiagen). Clontech SMARTer Ultra Low Input RNA Kit for Sequencing (v3) was used for amplification of cDNA after reverse transcription. Libraries for sequencing were prepared by Nextera XT Sample Prep Kit, and sequenced by an Illumina HiSeq 2500 system. Obtained seq data were analyzed using Agilent Strand NGS to identify differentially expressed genes (DEGs) by NF-Y knockdown.