UC San Diego

Macrophages are key players in the regulation of the innate and adaptive immune responses, tissue homeostasis and wound healing. In response to inflammation or injury, macrophages are actively recruited to affected sites where they perform specialized functions, such as phagocytosis of invading pathogens or dead cells, which are key for host recovery and survival. Many studies have shown that members of the Rho family of GTPases are important mediators of macrophage recruitment and function. These proteins activate the signaling cascades that are necessary to control the dynamic changes in the actin cytoskeleton that are required for directed movement and protrusion of the extracellular membrane. In several non- macrophage cell types, it has also been described that Rho GTPases can stimulate the expression of differentiation and cytoskeletal genes. This gene regulation was shown to be mediated by the DNA-binding transcription factor, serum response factor (SRF) and its coactivators megakaryoblastic leukemia (MKL) 1 and 2. Based on this data, the goal of this study was to elucidate the gene programs that are regulated by SRF and MKL1/2 in macrophages. Using mRNA expression profiling of primary macrophages, we discovered that both MKL1/2 and SRF regulate both general and hematopoietic-specific genes. In order to determine which of these genes are direct targets of the SRF pathway, we performed chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) analysis. Our results showed that, in general, SRF binding is not restricted to the proximal promoter, but occurs primarily at distal sites (further than +/- 500bp relative to the TSS) in close association with the macrophage and B-cell specific factor, PU.1. In particular, SRF and PU.1 were found to be localized to distal sites of several hematopoietic-specific target genes. Subsequent siRNA knockdown experiments showed that both SRF and PU.1 are required for full expression of these genes, providing insights into how cell-specific programs of SRF-dependent gene expression are achieved