Uncovering a Macrophage Transcriptional Program by Integrating Evidence from Motif Scanning and Expression Dynamics

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Abstract
Macrophages are versatile immune cells that can detect a variety of pathogen-associated molecular patterns through their Toll-like receptors (TLRs). In response to microbial challenge, the TLR-stimulated macrophage undergoes an activation program controlled by a dynamically inducible transcriptional regulatory network. Mapping a complex mammalian transcriptional network poses significant challenges and requires the integration of multiple experimental data types. In this work, we inferred a transcriptional network underlying TLR-stimulated murine macrophage activation. Microarray-based expression profiling and transcription factor binding site motif scanning were used to infer a network of associations between transcription factor genes and clusters of co-expressed target genes. The time-lagged correlation was used to analyze temporal expression data in order to identify potential causal influences in the network. A novel statistical test was developed to assess the significance of the time-lagged correlation. Several associations in the resulting inferred network were validated using targeted ChIP-on-chip experiments. The network incorporates known regulators and gives insight into the transcriptional control of macrophage activation. Our analysis identified a novel regulator (TGIF1) that may have a role in macrophage activation. Macrophages play a vital role in host defense against infection by recognizing pathogens through pattern recognition receptors, such as the Toll-like receptors (TLRs), and mounting an immune response. Stimulation of TLRs initiates a complex transcriptional program in which induced transcription factor genes dynamically regulate downstream genes. Microarray-based transcriptional profiling has proved useful for mapping such transcriptional programs in simpler model organisms; however, mammalian systems present difficulties such as post-translational regulation of transcription factors, combinatorial gene regulation, and a paucity of available gene-knockout expression data. Additional evidence sources, such as DNA sequence-based identification of transcription factor binding sites, are needed. In this work, we computationally inferred a transcriptional network for TLR-stimulated murine macrophages. Our approach combined sequence scanning with time-course expression data in a probabilistic framework. Expression data were analyzed using the time-lagged correlation. A novel, unbiased method was developed to assess the significance of the time-lagged correlation. The inferred network of associations between transcription factor genes and co-expressed gene clusters was validated with targeted ChIP-on-chip experiments, and yielded insights into the macrophage activation program, including a potential novel regulator. Our general approach could be used to analyze other complex mammalian systems for which time-course expression data are available.