title: Elucidation of the calcineurin-Crz1 stress response transcriptional network in the human fungal pathogen Cryptococcus neoformans.
abstract: Calcineurin is a highly conserved Ca2+/calmodulin-dependent serine/threonine-specific protein phosphatase that orchestrates cellular Ca2+ signaling responses. In Cryptococcus neoformans, calcineurin is activated by multiple stresses including high temperature, and is essential for stress adaptation and virulence. The transcription factor Crz1 is a major calcineurin effector in Saccharomyces cerevisiae and other fungi. Calcineurin dephosphorylates Crz1, thereby enabling Crz1 nuclear translocation and transcription of target genes. Here we show that loss of Crz1 confers phenotypes intermediate between wild-type and calcineurin mutants, and demonstrate that deletion of the calcineurin docking domain results in the inability of Crz1 to translocate into the nucleus under thermal stress. RNA-sequencing revealed 102 genes that are regulated in a calcineurin-Crz1-dependent manner at 37°C. The majority of genes were down-regulated in cna1Δ and crz1Δ mutants, indicating these genes are normally activated by the calcineurin-Crz1 pathway at high temperature. About 58% of calcineurin-Crz1 target genes have unknown functions, while genes with known or predicted functions are involved in cell wall remodeling, calcium transport, and pheromone production. We identified three calcineurin-dependent response element motifs within the promoter regions of calcineurin-Crz1 target genes, and show that Crz1 binding to target gene promoters is increased upon thermal stress in a calcineurin-dependent fashion. Additionally, we found a large set of genes independently regulated by calcineurin, and Crz1 regulates 59 genes independently of calcineurin. Given the intermediate crz1Δ mutant phenotype, and our recent evidence for a calcineurin regulatory network impacting mRNA in P-bodies and stress granules independently of Crz1, calcineurin likely acts on factors beyond Crz1 that govern mRNA expression/stability to operate a branched transcriptional/post-transcriptional stress response network necessary for fungal virulence. Taken together, our findings reveal the core calcineurin-Crz1 stress response cascade is maintained from ascomycetes to a pathogenic basidiomycete fungus, but its output in C. neoformans appears to be adapted to promote fungal virulence.
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Eve W L
authors.identifier: 0000-0001-5073-4035
authors.initials: EW
authors.lastname: Chow
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Shelly A
authors.initials: SA
authors.lastname: Clancey
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: R Blake
authors.identifier: 0000-0003-4866-3711
authors.initials: RB
authors.lastname: Billmyre
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Anna Floyd
authors.initials: AF
authors.lastname: Averette
authors.affiliation: Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, North Carolina, United States of America.|Duke Center for the Genomics of Microbial Systems, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Joshua A
authors.identifier: 0000-0003-3908-5016
authors.initials: JA
authors.lastname: Granek
authors.affiliation: High-Throughput Sequencing Facility, School of Medicine, University of North Carolina, Chapel Hill, North Carolina, United States of America.
authors.forename: Piotr
authors.initials: P
authors.lastname: Mieczkowski
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Maria E
authors.initials: ME
authors.lastname: Cardenas
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Joseph
authors.identifier: 0000-0001-6369-5995
authors.initials: J
authors.lastname: Heitman
mesh_terms: D019703:Calcineurin / Q000096:biosynthesis / Q000235:genetics*; D002473:Cell Wall / Q000235:genetics; D003455:Cryptococcus neoformans / Q000235:genetics* / Q000472:pathogenicity; D005656:Fungal Proteins / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D053263:Gene Regulatory Networks / Q000235:genetics*; D006801:Humans; D010641:Phenotype; D013312:Stress, Physiological / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D014157:Transcription Factors; D019703:Calcineurin