Genome-wide gene expression and promoter binding analysis identifies NFIL3 as a repressor of C/EBP target genes in neuronal outgrowth

Abstract

NFIL3 (nuclear factor IL-3 regulated) is a multifunctional transcription factor implicated in a wide range of physiological processes, including cellular survival, circadian gene expression and natural killer cell development. We recently demonstrated that NFIL3 acts as a repressor of CREB-induced gene expression underlying the regeneration of axotomized DRG sensory neurons. In this study we performed chromatin immunoprecipitation assays combined with microarray technology (ChIP-chip) to reveal direct NFIL3 and CREB target genes in an in vitro cell model for regenerating DRG neurons. We identified 505 promoter regions bound by NFIL3 and 924 promoter regions bound by CREB. Based on promoter analysis of NFIL3-bound genes, we were able to redefine the NFIL3 consensus-binding motif. Histone H3 acetylation profiling and gene expression microarray analysis subsequently indicated that a large fraction (>60%) of NFIL3 target genes were transcriptionally silent, whereas CREB target genes in general were transcriptionally active. Only a small subset of NFIL3 target genes also bound CREB. Computational analysis indicated that a substantial number of NFIL3 target genes share a C/EBP (CCAAT/Enhancer Binding Protein) DNA binding motif. ChIP analysis confirmed binding of C/EBPs to NFIL3 target genes, and knockdown of C/EBPα, C/EBPβ and C/EBPδ, but not C/EBPγ, significantly reduced neurite outgrowth in vitro. Together, our findings show that NFIL3 is a general feed-forward repressor of basic leucine zipper transcription factors that control neurite outgrowth.