ChIP-chip of the Cryptococcus neoformans transcription factor GAT201 and expression profiling of gat201KO. Cryptococcus neoformans

identify binding sites of Gat201 in C. neoformans cells grown in DMEM at 37C in 5% CO2 The anti-phagocytic polysaccharide capsule of the yeast C. neoformans is a major virulence attribute. However, previous studies of the pleiotropic virulence determinant Gat201, a GATA-family transcription factor, suggested that capsule-independent antiphagocytic mechanisms exist. We have determined that Gat201 controls the mRNA levels of ~1100 genes (16% of the genome) and binds the upstream regions of ~130 genes. Seven Gat201-bound genes encode for putative and known transcription factors⎯including two previously implicated in virulence⎯suggesting an extensive regulatory network. Systematic analysis pinpointed two critical Gat201-bound genes, GAT204 (a transcription factor) and BLP1, which account for much of the capsule-independent antiphagocytic function of Gat201. A strong correlation was observed between the quantitative effects of single and double mutants on phagocytosis in vitro and on host colonization in vivo. This genetic dissection provides the first clear evidence that capsule-independent anti-phagocytic mechanisms are pivotal for successful mammalian infection by C. neoformans. Overall design: two biological replicates were performed of wild type strains without any tagged ORF and strains carrying a CBP-FLAG tag at the Gat201 ORF. Whole cell extract (WCE) is in the Cy3 channel, the IP material is in the Cy5 channel. expression profiling of wild-type and gat201KO cells grown in YPAD or DMEM/5% CO2 two separate experiments: comparison of YPAD-grown samples and comparison of DMEM/5% CO2-grown samples samples on arrays CHM7-251, CHM7-253, CHM7-254, and CHM7-260 were grown in DMEM; samples on arrays CHM7-256, CHM7-257, CHM7-258, and CHM7-259 were grown in YPAD