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Cognate Site Identifier analysis reveals novel binding properties of the Sex Inducer homeodomain proteins of Cryptococcus neoformans.

ABSTRACT: Homeodomain proteins function in fungi to specify cell types and control sexual development. In the meningoencephalitis-causing fungal pathogen Cryptococcus neoformans, sexual development leads to the production of spores (suspected infectious particles). Sexual development is controlled by the homeodomain transcription factors Sxi1alpha and Sxi2a, but the mechanism by which they act is unknown. To understand how the Sxi proteins regulate development, we characterized their binding properties in vitro, showing that Sxi2a does not require a partner to bind DNA with high affinity. We then utilized a novel approach, Cognate Site Identifier (CSI) arrays, to define a comprehensive DNA-binding profile for Sxi2a, revealing a consensus sequence distinct from those of other fungal homeodomain proteins. Finally, we show that the homeodomains of both Sxi proteins are required for sexual development, a departure from related fungi. Our findings support a model in which Sxi1alpha and Sxi2a control sexual development in a homeodomain-dependent manner by binding to DNA sequences that differ from those defined in previously established fungal paradigms.

SUBMITTER: Stanton BC

PROVIDER: S-EPMC2776684 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Cognate Site Identifier analysis reveals novel binding properties of the Sex Inducer homeodomain proteins of Cryptococcus neoformans.

Stanton Brynne C BC Giles Steven S SS Kruzel Emilia K EK Warren Christopher L CL Ansari Aseem Z AZ Hull Christina M CM

Molecular microbiology 20090525 6

Homeodomain proteins function in fungi to specify cell types and control sexual development. In the meningoencephalitis-causing fungal pathogen Cryptococcus neoformans, sexual development leads to the production of spores (suspected infectious particles). Sexual development is controlled by the homeodomain transcription factors Sxi1alpha and Sxi2a, but the mechanism by which they act is unknown. To understand how the Sxi proteins regulate development, we characterized their binding properties in ...[more]

PMID: 19486297

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Project description:To identify genes regulated by the homeodomain transcription factors Sxi1M-NM-1 and Sxi2a (Sex Inducer 1M-NM-1 and 2a) in Cryptococcus neoformans, we carried out a whole-genome expression experiment. We performed two independent whole-genome microarray experiments comparing transcript levels in cells that either possessed or were lacking the Sxi proteins. In the first experiment we compared the expression profile of a haploid sxi1M-NM-1M-NM-^T strain to that of a haploid strain expressing both SXI1M-NM-1 and an inducible copy of SXI2a (Sxi +). In the second experiment, we compared the expression profile of a wild type cross (JEC20 x JEC21) to the expression profile of a cross whose mating partners did not possess either of the transcription factors (sxi2aM-bM-^HM-^F x sxi1M-NM-1M-bM-^HM-^F)(Sxi -). In both experiments RNA from each condition was harvested, labeled, and hybridized competitively to a spotted oligonucleotide microarray representing the approximately 6,500 genes in the C. neoformans genome. The resulting data was analyzed in Limma using standard methods. In the Sxi (+) experiment, each strain (M-NM-^Tsxi1M-NM-1 and the Sxi-Inducible strain) were grown overnight in liquid YPD, diluted back to an OD600 of 0.2, grown to log phase, and then switched to growth in liquid YPGal media for 6 hrs. The M-NM-^Tsxi1M-NM-1 strain was designated the control condition. Two biological replicates were used and in the first replicate there were two technical replicates for each dye orientation (4 replicates total). For the second Sxi (+) biological replicate, there were three total technical replicates, and one of those was a dye swap. For Sxi deletion crosses, strains (JEC20 x JEC21 or M-NM-^Tsxi1M-NM-1 x M-NM-^Tsxi2a) were mixed and plated onto V8 media (pH 7.0) and incubated at 22M-BM-0C in the dark for approximately 16 hours. The wild type (JEC20 x JEC21) cross was designated as the control in this experiment. Eight total hybridizations were carried out that consisted of 2 biological replicates and four technical replicates for each biological replicate (2 technical replicates per dye swap).

Project description:UNLABELLED:Cryptococcus neoformans is the leading cause of fungal meningitis worldwide. Previous studies have characterized the cryptococcal transcriptome under various stress conditions, but a comprehensive profile of the C. neoformans transcriptome in the human host has not been attempted. Here, we extracted RNA from yeast cells taken directly from the cerebrospinal fluid (CSF) of two AIDS patients with cryptococcal meningitis prior to antifungal therapy. The patients were infected with strains of C. neoformans var. grubii of molecular type VNI and VNII. Using RNA-seq, we compared the transcriptional profiles of these strains under three environmental conditions (in vivo CSF, ex vivo CSF, and yeast extract-peptone-dextrose [YPD]). Although we identified a number of differentially expressed genes, single nucleotide variants, and novel genes that were unique to each strain, the overall expression patterns of the two strains were similar under the same environmental conditions. Specifically, yeast cells obtained directly from each patient's CSF were more metabolically active than cells that were incubated ex vivo in CSF. Compared with growth in YPD, some genes were identified as significantly upregulated in both in vivo and ex vivo CSF, and they were associated with genes previously recognized for contributing to pathogenicity. For example, genes with known stress response functions, such as RIM101, ENA1, and CFO1, were regulated similarly in the two clinical strains. Conversely, many genes that were differentially regulated between the two strains appeared to be transporters. These findings establish a platform for further studies of how this yeast survives and produces disease. IMPORTANCE:Cryptococcus neoformans, an environmental, opportunistic yeast, is annually responsible for an estimated million cases of meningitis and over 600,000 deaths, mostly among HIV-infected patients in sub-Saharan Africa and Asia. Using RNA-seq, we analyzed the gene expression of two strains of C. neoformans obtained from the cerebrospinal fluid (CSF) of infected patients, thus creating a comprehensive snapshot of the yeasts' genetic responses within the human body. By comparing the gene expression of each clinical strain under three conditions (in vivo CSF, ex vivo CSF, and laboratory culture), we identified genes and pathways that were uniquely regulated by exposure to CSF and likely crucial for the survival of C. neoformans in the central nervous system. Further analyses revealed genetic diversity between the strains, providing evidence for cryptococcal evolution and strain specificity. This ability to characterize transcription in vivo enables the elucidation of specific genetic responses that promote disease production and progression.

Dataset Information

Cognate Site Identifier analysis reveals novel binding properties of the Sex Inducer homeodomain proteins of Cryptococcus neoformans.

Publications

Cognate Site Identifier analysis reveals novel binding properties of the Sex Inducer homeodomain proteins of Cryptococcus neoformans.

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