Unbiased reconstruction of a mammalian transcriptional network mediating pathogen responses.

Models of mammalian regulatory networks controlling gene expression have been inferred from genomic data but have largely not been validated. We present an unbiased strategy to systematically perturb candidate regulators and monitor cellular transcriptional responses. We applied this approach to derive regulatory networks that control the transcriptional response of mouse primary dendritic cells to pathogens.

Analysis of FOXP3 reveals multiple domains required for its function as a transcriptional repressor.

Foxp3 has been shown to be both necessary and sufficient for the development and function of naturally arising CD4+ CD25+ regulatory T cells in mice. Mutation of Foxp3 in Scurfy mice and FOXP3 in humans with IPEX results in fatal, early onset autoimmune disease and demonstrates the critical role of FOXP3 in maintaining immune homeostasis. The FOXP3 protein encodes several functional domains, including a C2H2 zinc finger, a leucine zipper, and a winged-helix/forkhead (FKH) domain.

The generation of mature, single-positive thymocytes in vivo is dysregulated by CD69 blockade or overexpression.

During development in the thymus, mature CD4+ or CD8+ cells are derived from immature CD4+CD8+ cells through a series of selection events. One of the hallmarks of this maturation process is the expression of CD69, which first appears on thymocytes as they begin positive selection. We have used blockade and overexpression of CD69 to determine the role of CD69 in thymocyte development.

A T cell-specific enhancer of the human CD40 ligand gene.

We observed that the human CD40 ligand (CD40L) gene 5'-flanking region conferred weak promoter activity in activated CD4 T cells, suggesting that additional regions are required for optimal CD40L gene transcription. We therefore examined a 3'-flanking segment of the CD40L gene, which contained a putative NF-kappaB/Rel cis-element, for its ability to enhance CD40L promoter function. This segment augmented CD40L promoter activity in an orientation-independent manner in CD4 T-lineage cells but not in human B cell or monocyte cell lines.