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Fungal Morphology, Iron Homeostasis, and Lipid Metabolism Regulated by a GATA Transcription Factor in Blastomyces dermatitidis.

Marty AJ, Broman AT, Zarnowski R, Dwyer TG, Bond LM, Lounes-Hadj Sahraoui A, Fontaine J, Ntambi JM, Keleş S, Kendziorski C, Gauthier GM - PLoS Pathog. (2015)

Bottom Line: This included genes involved with siderophore biosynthesis and uptake, iron homeostasis, and genes unrelated to iron assimilation.Chromatin immunoprecipitation, RNA interference, and overexpression analyses suggested that SREB was in a negative regulatory circuit with the bZIP transcription factor encoded by HAPX.Both SREB and HAPX affected morphogenesis at 22°C; however, large changes in transcript abundance by gene deletion for SREB or strong overexpression for HAPX were required to alter the phase transition.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Wisconsin, Madison, Madison, Wisconsin, United States of America.

ABSTRACT
In response to temperature, Blastomyces dermatitidis converts between yeast and mold forms. Knowledge of the mechanism(s) underlying this response to temperature remains limited. In B. dermatitidis, we identified a GATA transcription factor, SREB, important for the transition to mold. Null mutants (SREBΔ) fail to fully complete the conversion to mold and cannot properly regulate siderophore biosynthesis. To capture the transcriptional response regulated by SREB early in the phase transition (0-48 hours), gene expression microarrays were used to compare SREB∆ to an isogenic wild type isolate. Analysis of the time course microarray data demonstrated SREB functioned as a transcriptional regulator at 37°C and 22°C. Bioinformatic and biochemical analyses indicated SREB was involved in diverse biological processes including iron homeostasis, biosynthesis of triacylglycerol and ergosterol, and lipid droplet formation. Integration of microarray data, bioinformatics, and chromatin immunoprecipitation identified a subset of genes directly bound and regulated by SREB in vivo in yeast (37°C) and during the phase transition to mold (22°C). This included genes involved with siderophore biosynthesis and uptake, iron homeostasis, and genes unrelated to iron assimilation. Functional analysis suggested that lipid droplets were actively metabolized during the phase transition and lipid metabolism may contribute to filamentous growth at 22°C. Chromatin immunoprecipitation, RNA interference, and overexpression analyses suggested that SREB was in a negative regulatory circuit with the bZIP transcription factor encoded by HAPX. Both SREB and HAPX affected morphogenesis at 22°C; however, large changes in transcript abundance by gene deletion for SREB or strong overexpression for HAPX were required to alter the phase transition.

No MeSH data available.


Silencing of SREB using RNA interference.(A-C) Quantitative real-time PCR analysis demonstrated a 1.8–2.5-fold decrease in SREB transcript and derepression of SID1 (2.9 6.2-fold increase) and HAPX (1.7 3.2-fold increase) in SREB RNAi strains #6 and #7 compared to controls (GFP reporter, empty vector, GFP-RNAi). SID1 encodes L-ornithine-N5-oxygenase, which encodes the first enzyme in siderophore biosynthesis. Quantitative RT-PCR results were averaged from 2 experiments. (D) SREB-GFP knockdown strains along with empty vector and GFP RNAi only controls converted to mycelia at 22°C. Images were photographed at 9 days incubation 22°C. Scale bar equals 10 μm.
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ppat.1004959.g011: Silencing of SREB using RNA interference.(A-C) Quantitative real-time PCR analysis demonstrated a 1.8–2.5-fold decrease in SREB transcript and derepression of SID1 (2.9 6.2-fold increase) and HAPX (1.7 3.2-fold increase) in SREB RNAi strains #6 and #7 compared to controls (GFP reporter, empty vector, GFP-RNAi). SID1 encodes L-ornithine-N5-oxygenase, which encodes the first enzyme in siderophore biosynthesis. Quantitative RT-PCR results were averaged from 2 experiments. (D) SREB-GFP knockdown strains along with empty vector and GFP RNAi only controls converted to mycelia at 22°C. Images were photographed at 9 days incubation 22°C. Scale bar equals 10 μm.

Mentions: Integration of microarray, qRT-PCR, and ChIP data suggested that B. dermatitidis SREB and HAPX are in a negative regulatory circuit similar to Aspergillus nidulans and A. fumigatus [48,49]. B. dermatitidis does not encode a homolog to C. albicans SEF1, which forms a regulatory circuit with SFU1 and HAP43 [50]. Moreover, deletion of SREB did not alter the transcription of HAPB, HAPC, or HAPE homologs, which form a DNA binding complex that recruits HAPX to CCAAT promoter sites of target genes [48]. HAPX transcript abundance was substantially higher in the OE strains (15.5–53.1-fold) than SREB∆ (1.7–3.5-fold) compared to WT (Figs 4A and 10B). To investigate if less extreme changes in transcript abundance for either HAPX or SREB affected the phase transition, SREB was targeted for gene silencing using a GFP sentinel RNAi system [51]. SREB-GFP silenced strains had a 1.8–2.5-fold reduction in transcript abundance compared to controls (GFP reporter, empty vector, GFP-only silenced) (Fig 11A). SREB-GFP RNAi strains had increased SID1 transcript abundance under iron-replete (10 um FeSO4) conditions compared to controls (Fig 11B). HAPX transcript was increased 1.7–3.2-fold in SREB RNAi versus controls (Fig 11C). Following a drop in temperature, SREB-GFP silenced strains converted to mold at 22°C (Fig 11D). Moreover, there was no delay in the phase transition. This data suggested that the 1.7–3.5-fold increase in HAPX transcript in SREB∆ was not responsible for the defect in phase transition at 22°C; larger changes in HAPX transcript abundance are required to impact morphologic development at 22°C.


Fungal Morphology, Iron Homeostasis, and Lipid Metabolism Regulated by a GATA Transcription Factor in Blastomyces dermatitidis.

Marty AJ, Broman AT, Zarnowski R, Dwyer TG, Bond LM, Lounes-Hadj Sahraoui A, Fontaine J, Ntambi JM, Keleş S, Kendziorski C, Gauthier GM - PLoS Pathog. (2015)

Silencing of SREB using RNA interference.(A-C) Quantitative real-time PCR analysis demonstrated a 1.8–2.5-fold decrease in SREB transcript and derepression of SID1 (2.9 6.2-fold increase) and HAPX (1.7 3.2-fold increase) in SREB RNAi strains #6 and #7 compared to controls (GFP reporter, empty vector, GFP-RNAi). SID1 encodes L-ornithine-N5-oxygenase, which encodes the first enzyme in siderophore biosynthesis. Quantitative RT-PCR results were averaged from 2 experiments. (D) SREB-GFP knockdown strains along with empty vector and GFP RNAi only controls converted to mycelia at 22°C. Images were photographed at 9 days incubation 22°C. Scale bar equals 10 μm.
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Related In: Results  -  Collection

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ppat.1004959.g011: Silencing of SREB using RNA interference.(A-C) Quantitative real-time PCR analysis demonstrated a 1.8–2.5-fold decrease in SREB transcript and derepression of SID1 (2.9 6.2-fold increase) and HAPX (1.7 3.2-fold increase) in SREB RNAi strains #6 and #7 compared to controls (GFP reporter, empty vector, GFP-RNAi). SID1 encodes L-ornithine-N5-oxygenase, which encodes the first enzyme in siderophore biosynthesis. Quantitative RT-PCR results were averaged from 2 experiments. (D) SREB-GFP knockdown strains along with empty vector and GFP RNAi only controls converted to mycelia at 22°C. Images were photographed at 9 days incubation 22°C. Scale bar equals 10 μm.
Mentions: Integration of microarray, qRT-PCR, and ChIP data suggested that B. dermatitidis SREB and HAPX are in a negative regulatory circuit similar to Aspergillus nidulans and A. fumigatus [48,49]. B. dermatitidis does not encode a homolog to C. albicans SEF1, which forms a regulatory circuit with SFU1 and HAP43 [50]. Moreover, deletion of SREB did not alter the transcription of HAPB, HAPC, or HAPE homologs, which form a DNA binding complex that recruits HAPX to CCAAT promoter sites of target genes [48]. HAPX transcript abundance was substantially higher in the OE strains (15.5–53.1-fold) than SREB∆ (1.7–3.5-fold) compared to WT (Figs 4A and 10B). To investigate if less extreme changes in transcript abundance for either HAPX or SREB affected the phase transition, SREB was targeted for gene silencing using a GFP sentinel RNAi system [51]. SREB-GFP silenced strains had a 1.8–2.5-fold reduction in transcript abundance compared to controls (GFP reporter, empty vector, GFP-only silenced) (Fig 11A). SREB-GFP RNAi strains had increased SID1 transcript abundance under iron-replete (10 um FeSO4) conditions compared to controls (Fig 11B). HAPX transcript was increased 1.7–3.2-fold in SREB RNAi versus controls (Fig 11C). Following a drop in temperature, SREB-GFP silenced strains converted to mold at 22°C (Fig 11D). Moreover, there was no delay in the phase transition. This data suggested that the 1.7–3.5-fold increase in HAPX transcript in SREB∆ was not responsible for the defect in phase transition at 22°C; larger changes in HAPX transcript abundance are required to impact morphologic development at 22°C.

Bottom Line: This included genes involved with siderophore biosynthesis and uptake, iron homeostasis, and genes unrelated to iron assimilation.Chromatin immunoprecipitation, RNA interference, and overexpression analyses suggested that SREB was in a negative regulatory circuit with the bZIP transcription factor encoded by HAPX.Both SREB and HAPX affected morphogenesis at 22°C; however, large changes in transcript abundance by gene deletion for SREB or strong overexpression for HAPX were required to alter the phase transition.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Wisconsin, Madison, Madison, Wisconsin, United States of America.

ABSTRACT
In response to temperature, Blastomyces dermatitidis converts between yeast and mold forms. Knowledge of the mechanism(s) underlying this response to temperature remains limited. In B. dermatitidis, we identified a GATA transcription factor, SREB, important for the transition to mold. Null mutants (SREBΔ) fail to fully complete the conversion to mold and cannot properly regulate siderophore biosynthesis. To capture the transcriptional response regulated by SREB early in the phase transition (0-48 hours), gene expression microarrays were used to compare SREB∆ to an isogenic wild type isolate. Analysis of the time course microarray data demonstrated SREB functioned as a transcriptional regulator at 37°C and 22°C. Bioinformatic and biochemical analyses indicated SREB was involved in diverse biological processes including iron homeostasis, biosynthesis of triacylglycerol and ergosterol, and lipid droplet formation. Integration of microarray data, bioinformatics, and chromatin immunoprecipitation identified a subset of genes directly bound and regulated by SREB in vivo in yeast (37°C) and during the phase transition to mold (22°C). This included genes involved with siderophore biosynthesis and uptake, iron homeostasis, and genes unrelated to iron assimilation. Functional analysis suggested that lipid droplets were actively metabolized during the phase transition and lipid metabolism may contribute to filamentous growth at 22°C. Chromatin immunoprecipitation, RNA interference, and overexpression analyses suggested that SREB was in a negative regulatory circuit with the bZIP transcription factor encoded by HAPX. Both SREB and HAPX affected morphogenesis at 22°C; however, large changes in transcript abundance by gene deletion for SREB or strong overexpression for HAPX were required to alter the phase transition.

No MeSH data available.