The innate immune system is absolutely required for host defence, but, uncontrolled, it leads to inflammatory disease. This control is mediated, in part, by cytokines that are secreted by macrophages. Immune regulation is extraordinarily complex, and can be best investigated with systems approaches (that is, using computational tools to predict regulatory networks arising from global, high-throughput data sets). Here we use cluster analysis of a comprehensive set of transcriptomic data derived from Toll-like receptor (TLR)-activated macrophages to identify a prominent group of genes that appear to be regulated by activating transcription factor 3 (ATF3), a member of the CREB/ATF family of transcription factors. Network analysis predicted that ATF3 is part of a transcriptional complex that also contains members of the nuclear factor (NF)-κB family of transcription factors. Promoter analysis of the putative ATF3-regulated gene cluster demonstrated an over-representation of closely apposed ATF3 and NF-κB binding sites, which was verified by chromatin immunoprecipitation and hybridization to a DNA microarray. This cluster included important cytokines such as interleukin (IL)-6 and IL-12b. ATF3 and Rel (a component of NF-κB) were shown to bind to the regulatory regions of these genes upon macrophage activation. A kinetic model of Il6 and Il12b messenger RNA expression as a function of ATF3 and NF-κB promoter binding predicted that ATF3 is a negative regulator of Il6 and Il12b transcription, and this hypothesis was validated using Atf3-null mice. ATF3 seems to inhibit Il6 and Il12b transcription by altering chromatin structure, thereby restricting access to transcription factors. Because ATF3 is itself induced by lipopolysaccharide, it seems to regulate TLR-stimulated inflammatory responses as part of a negative-feedback loop.
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We acknowledge A. Ozinsky, I. Shmulevich, W. Longabaugh and L. Hood for discussions. We thank A. Nachman, A. Clark and C. Baldwin for technical assistance. This work was supported by a Fellowship from the Alberta Heritage Foundation for Medical Research (to M.G.) and the NIH (to A.A.)
Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.
Comprehensive listing of cluster genes from LPS-stimulated macropahges. (XLS 295 kb)
ATF3 expression and effects in macrophages and liver. (DOC 32 kb)
Detailed protocol and results for transcription factor binding site predictions in cluster 2-derived target genes. (DOC 403 kb)
In depth ATF/kB binding site prediction for IL6 and IL12b promoters. (DOC 63 kb)
Kinetic models of ATF3/Rel regulation of IL6/IL12b transcription. (DOC 113 kb)
Chromatin immunoprecipitation: complete protocol. (DOC 28 kb)
ATF3 transcriptional regulatory complex: expression profile. (JPG 369 kb)
Gene array/ChIP to chip comparison: validation. (DOC 47 kb)
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Gilchrist, M., Thorsson, V., Li, B. et al. Systems biology approaches identify ATF3 as a negative regulator of Toll-like receptor 4. Nature 441, 173–178 (2006). https://doi.org/10.1038/nature04768
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