The Orphan Nuclear Receptor TLX Is an Enhancer of STAT1-Mediated Transcription and Immunity to Toxoplasma gondii.

The protozoan parasite, Toxoplasma, like many intracellular pathogens, suppresses interferon gamma (IFN-γ)-induced signal transducer and activator of transcription 1 (STAT1) activity. We exploited this well-defined host-pathogen interaction as the basis for a high-throughput screen, identifying nine transcription factors that enhance STAT1 function in the nucleus, including the orphan nuclear hormone receptor TLX. Expression profiling revealed that upon IFN-γ treatment TLX enhances the output of a subset of IFN-γ target genes, which we found is dependent on TLX binding at those loci. Moreover, infection of TLX deficient mice with the intracellular parasite Toxoplasma results in impaired production of the STAT1-dependent cytokine interleukin-12 by dendritic cells and increased parasite burden in the brain during chronic infection. These results demonstrate a previously unrecognized role for this orphan nuclear hormone receptor in regulating STAT1 signaling and host defense and reveal that STAT1 activity can be modulated in a context-specific manner by such "modifiers."

Generation of a novel allelic series of cryptochrome mutants via mutagenesis reveals residues involved in protein-protein interaction and CRY2-specific repression.

CRYPTOCHOME proteins are necessary for mammalian circadian rhythms and have many well-established biochemical roles within the molecular clock. While studies examining the effect of null Cry alleles have been informative, they have failed to dissect out the relative importance of, and the molecular mechanisms behind, the many roles of the CRY1 and CRY2 proteins. To address this, we created an allelic series of Cry mutants through random mutagenesis, followed by a cell-based screen to isolate mutants with aberrant repression of CLOCK-BMAL1. We identified 22 mutants with mutations resulting in single amino acid substitutions which cause a variety of deficiencies in different CRY functions. To illustrate the breadth and value of these new tools, we present an in-depth analysis of two of these mutants, CRY2G354D and CRY2G351D; the former shows deficiency in clock protein binding and is required for repression by both CRYs, while in contrast, the latter displays normal binding function but exhibits a CRY2-specific repression phenotype. Further, while overexpression of CRY2 in NIH 3T3 cells caused a dose-dependent decrease in rhythm amplitude, overexpression of CRY2G351D abolished rhythmicity. In summary, characterization of these unique alleles provides new opportunities for more-sophisticated insight into the multifaceted functions of the CRY proteins in circadian rhythms.