Mechanism-based toxicology in cancer risk assessment: implications for research, regulation, and legislation.

Background: Nuclear factor I-A (NFI-A), a phylogenetically conserved transcription/replication protein, plays a crucial role in mouse brain development. Previous studies have shown that disruption of the Nfia gene in mice leads to perinatal lethality, corpus callosum agenesis, and hydrocephalus. Results: To identify potential NFI-A target genes involved in the observed tissue malformations, we analyzed gene expression in brains from Nfia-/and Nfia+/+ littermate mice at the mRNA level using oligonucleotide microarrays. In young postnatal animals (postnatal day 16), 356 genes were identified as being differentially regulated, whereas at the late embryonic stage (embryonic day 18) only five dysregulated genes were found. An in silico analysis identified phylogenetically conserved NFI binding sites in at least 70 of the differentially regulated genes. Moreover, assignment of gene function showed that marker genes for immature neural cells and neural precursors were expressed at elevated levels in young postnatal Nfia-/mice. In contrast, marker genes for differentiated neural cells were downregulated at this stage. In particular, genes relevant for oligodendrocyte differentiation were affected. Conclusion: Our findings suggest that brain development, especially oligodendrocyte maturation, is delayed in Nfia-/mice during the early postnatal period, which at least partly accounts for their phenotype. The identification of potential NFI-A target genes in our study should help to elucidate NFI-A dependent transcriptional pathways and contribute to enhanced understanding of this period of brain formation, especially with regard to the function of NFI-A. Published: 2 May 2007 Genome Biology 2007, 8:R72 (doi:10.1186/gb-2007-8-5-r72) Received: 2 February 2007 Accepted: 2 May 2007 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2007/8/5/R72 Genome Biology 2007, 8:R72 R72.2 Genome Biology 2007, Volume 8, Issue 5, Article R72 Wong et al. http://genomebiology.com/2007/8/5/R72 Background The nuclear factor I (NFI) family of sequence-specific DNA binding proteins has four members [1,2] (for review, see Gronostajski [3]), namely NFI-A, NFI-B, NFI-C, and NFI-X. They recognize the nucleotide consensus sequence TTGGC(N)5GCCAA. NFI proteins were first identified as nuclear proteins that bind to the replication origin of adenoviruses and initiate DNA replication in vitro [4,5]. Their consensus binding sequence was subsequently identified [6-8]. The promoters of several genes were shown to be activated by NFI proteins. These 'positive target genes' include the gene encoding α-globin [9], human hepatitis B virus S gene [10], Mbp (myelin basic protein) [11,12], B-Fabp (brain fatty acidbinding protein; also called Blbp [brain lipid-binding protein]) [13], and Gabra6 (α6 subunit of the γ-aminobutyric acid [GABA] type A receptor) [14]. On the other hand, there are also genes that are negatively regulated by NFI, such as the gene that encodes adenine nucleotide translocase 2 [15]. Unpublished data from our laboratory also suggest that NFIA negatively regulates transcription of the mouse L1 gene. L1 is a cell adhesion molecule that is involved in neuronal migration, axon outgrowth, and synaptic plasticity [16]. The complexity of regulation by NFI family members is further increased by alternative splicing, yielding as many as nine different proteins from one gene [17,18]. For instance, a brainspecific isoform of NFI-A [3], which was first isolated in 1990 by Inoue and coworkers [19], activates the transcription of mouse myelin basic protein. Nfia-/mice exhibit severe neurologic defects, including communicating hydrocephalus, corpus callosum agenesis, and disrupted development of midline glia [20,21], similar to L1deficient mice [22,23]. These findings indicate that NFI-A plays an important role in regulating gene transcription during brain development. Moreover, NFI-A mRNA is expressed in adult mouse brain [24], which suggests that the respective protein participates in the control of gene expression in the mature central nervous system. To understand how NFI-A could influence brain development and function, it is important to obtain a comprehensive overview of NFI-A responsive genes in the brain. Oligonucleotide microarrays [25] offer an attractive experimental approach for such global gene expression analyses. We therefore performed a microarray analysis of brain cDNA from embryonic (embryonic day 18 [E18]) and early postnatal (postnatal day 16 [P16]) Nfia-/mice in comparison with respective wild-type littermate controls. Using this method, we identified a large number of genes that are dysregulated at the mRNA level in postnatal NFI-A knockout (Nfia-/-) mouse brains. Moreover, by in silico promoter analysis, we showed that, among this group, at least 70 genes possess phylogenetically conserved NFI binding sites in their promoter region, suggesting that they might be direct NFI-A targets. Database analyses of gene function revealed that the changes in gene expression observed in our study probably reflect a delay in neural, particularly oligodendrocyte, differentiation, which appears to be a consequence of loss of NFI-A. Results Microarray analysis High-density oligonucleotide microarray analysis was carried out for total RNA from brains of Nfia-/mice and agematched, wild-type littermate controls. Analyses were performed with independent samples from three Nfia-/and three wild-type (Nfia+/+) animals each for E18 and P16. All animals were F1 hybrids of C57BL/6 and 129S6 mice, ensuring a survival rate of 38.5% until P16. A total of 356 genes were identified as being differentially expressed in the Nfia-/animals at P16 (197 upregulated and 159 downregulated), taking a cutoff of a 1.2-fold change and a significance of P < 0.05 in expression relative to the wild-type control (see Additional data file 1). Among these, 53 genes were found to exhibit a greater than 1.5-fold change in expression (39 downregulated [74%] and 14 upregulated [26%]; Table 1). Within this latter group of strongly dysregulated genes, a total of 11 genes exhibit greater than twofold dysregulation, with nine genes downregulated and two upregulated. The downregulated genes include those encoding the following: angiotensinogen (Agt); aldehyde dehydrogenase family 1, subfamily A1 (Aldh1a1); folate hydrolase (Folh1); GABA-A receptor, subunit α6 (Gabra6); gap junction membrane channel protein β6 (Gjb6); lecithin cholesterol acyltransferase (Lcat); myelin and lymphocyte protein (Mal); myelinassociated oligodendrocytic basic protein (Mobp); and neurotensin receptor 2 (Ntsr2). The upregulated genes encode fatty acid binding protein 7 (FABP7) and the transcription factor SRY-like HMG-box containing 11 (Sox11). At the late embryonic stage (E18), fewer genes were significantly dysregulated in the Nfia-/mutant relative to the wildtype animals when compared with the postnatal stage (P16). A total of five genes was identified as being significantly dysregulated with changes of more than 1.2-fold (Table 2). One of the three downregulated genes encodes a yet uncharacterized protein, whereas the two others encode phosphatidylinositol4-phosphate 5-kinase, type II, γ (Pip5k2c) and synaptotagmin binding, cytoplasmic RNA interacting protein (Syncrip). mRNAs for synaptotagmin 1 (Syt1) and pleiomorphic adenoma gene-like 1 (Plagl1) were expressed at elevated levels in the Nfia-/animals. At E18, no gene was differentially regulated more than 1.5-fold in the Nfia-/mutants relative to the wild-type controls. Pleiomorphic adenoma gene-like 1 (Plagl1) is the only gene that exhibits a 1.2-fold up-regulation in Nfia-/mice at both developmental stages. In P16 Nfia-/mice, a total of 356 individual genes, represented by 395 probe sets, were dysregulated in comparison with the wild-type control group. Among these, 35 genes were represented by more than one probe set on the microarray Genome Biology 2007, 8:R72 http://genomebiology.com/2007/8/5/R72 Genome Biology 2007, Volume 8, Issue 5, Article R72 Wong et al. R72.3