Successful regeneration of damaged neurons depends on the coordinated expression of neuron-intrinsic genes. At present however, there is no comprehensive view of the transcriptional regulatory mechanisms underlying neuronal regeneration. We used high-content cellular screening to investigate the functional contribution of 62 transcription factors to regenerative neuron outgrowth. Ten transcription factors are identified that either increase or decrease neurite outgrowth. One of these, NFIL3, is specifically upregulated during successful regeneration in vivo. Paradoxically however, knockdown of NFIL3 and overexpression of dominant-negative NFIL3 both increase neurite outgrowth. Our data show that NFIL3, together with CREB, forms an incoherent feedforward transcriptional regulatory loop in which NFIL3 acts as a negative regulator of CREB-induced regeneration-associated genes.

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.

All cellular processes are regulated by condition-specific and time-dependent interactions between transcription factors and their target genes. While in simple organisms, e.g. bacteria and yeast, a large amount of experimental data is available to support functional transcription regulatory interactions, in mammalian systems reconstruction of gene regulatory networks still heavily depends on the accurate prediction of transcription factor binding sites. Here, we present a new method, log-linear modeling of 3D contingency tables (LLM3D), to predict functional transcription factor binding sites. LLM3D combines gene expression data, gene ontology annotation and computationally predicted transcription factor binding sites in a single statistical analysis, and offers a methodological improvement over existing enrichment-based methods. We show that LLM3D successfully identifies novel transcriptional regulators of the yeast metabolic cycle, and correctly predicts key regulators of mouse embryonic stem cell self-renewal more accurately than existing enrichment-based methods. Moreover, in a clinically relevant in vivo injury model of mammalian neurons, LLM3D identified peroxisome proliferator-activated receptor γ (PPARγ) as a neuron-intrinsic transcriptional regulator of regenerative axon growth. In conclusion, LLM3D provides a significant improvement over existing methods in predicting functional transcription regulatory interactions in the absence of experimental transcription factor binding data.

Snails are intermediate hosts to schistosome parasites, some of which are the main cause of human schistosomiasis (bilharzia), and have been used as models for parasite-host interactions for a long time. Here, we have characterized a novel internal defense peptide of the snail Lymnaea stagnalis, of which the relative abundance in brain tissue increases upon infection with the avian schistosome Trichobilharzia ocellata. This protein, named granularin, is secreted by granular cells, which are numerous in the connective tissue surrounding the brain. The protein is unique because it comprises only a single Von Willebrand factor type C domain that is normally found in large transmembrane and secreted extracellular matrix proteins. The granularin gene is twice up-regulated during parasitation. Purified granularin stimulates phagocytosis of foreign particles by blood hemocytes. Together, our data indicate that granularin represents a novel protein that acts as an opsonin in the molluscan internal defense response.