Limits...
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.


Related in: MedlinePlus

Chromatin immunoprecipitation with quantitative real-time PCR (ChIP-qPCR).(A) Enrichment for binding of SREB-3xHA to promoter regions containing GATA binding motifs in B. dermatitidis yeast at 37°C. (B) Enrichment binding of SREB-3xHA to promoter regions containing GATA binding motifs in B. dermatitidis cells at 48-hrs following a drop in temperature from 37°C to 22°C. ChIP-qPCR was performed using SREB that was engineered to contain a C-terminal 3x-hemagglutinin tag. Integration of gene expression microarray data with genome-wide GATA motif analysis was used to identify putative GATA transcription factor binding sites. All genes analyzed were differentially expressed and contained a GATA motif (ATC-w-gAta-a or A/T-GATA-A/G) in the upstream promoter. For MIRB, the upstream motif in strain 26199 was ATC-A-GATT-A, whereas in other strains including SLH14081, ER-3, and ATCC 18188, the motif was ATC-A-GATA-A. BDBG is the locus number for genes in the B. dermatitidis genome database at the Broad Institute (www.broadinstitute.org).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4482641&req=5

ppat.1004959.g009: Chromatin immunoprecipitation with quantitative real-time PCR (ChIP-qPCR).(A) Enrichment for binding of SREB-3xHA to promoter regions containing GATA binding motifs in B. dermatitidis yeast at 37°C. (B) Enrichment binding of SREB-3xHA to promoter regions containing GATA binding motifs in B. dermatitidis cells at 48-hrs following a drop in temperature from 37°C to 22°C. ChIP-qPCR was performed using SREB that was engineered to contain a C-terminal 3x-hemagglutinin tag. Integration of gene expression microarray data with genome-wide GATA motif analysis was used to identify putative GATA transcription factor binding sites. All genes analyzed were differentially expressed and contained a GATA motif (ATC-w-gAta-a or A/T-GATA-A/G) in the upstream promoter. For MIRB, the upstream motif in strain 26199 was ATC-A-GATT-A, whereas in other strains including SLH14081, ER-3, and ATCC 18188, the motif was ATC-A-GATA-A. BDBG is the locus number for genes in the B. dermatitidis genome database at the Broad Institute (www.broadinstitute.org).

Mentions: Using this integrated approach, we identified a subset of genes bound and regulated by SREB-3xHA under iron-replete conditions (10 μM FeSO4) using ChIP-qPCR (Fig 9A and 9B). These included genes involved with extracellular (SID1, ACE1) and intracellular (SIDC) siderophore biosynthesis, siderophore transport (MIRB), ferric reduction (FRE1 and MR), ferrous iron/zinc transporter (ZFT), a WD-repeat protein of unknown function (WD), and a bZIP transcription factor (HAPX) (Fig 9A and 9B). For ChIP-qPCR, SREB-3xHA did not bind the GAPDH promoter, which lacked GATA motifs and served as a negative control. ChIP-seq analysis of yeast grown in iron-replete media (10 μM FeSO4) also demonstrated enrichment of SREB-3xHA binding for genes located in the siderophore biosynthetic gene cluster including BDBG_00046 (conserved hypothetical protein), BDBG_00047 (ATRH), BDBG_00048 (NRPS), BDBG_00053 (SID1), BDBG_00054 (long chain fatty acid CoA ligase), and BDBG_00055 (ACE1) (S10 Fig and S6 Table). In addition, SREB-3xHA bound to the promoters of SIDC (BDBG_08208) and siderophore transporters MIRB (BDBG_05798), MIRC (BDBG_08034), and SIT1 (BDBG_06965) (S10 Fig and S6 Table). ChIP-seq also showed enrichment of SREB-3xHA upstream of genes unrelated to iron homeostasis including a glycolipid surface protein predicted to have 1,3-beta-glucanosyltransferase activity (BDBG_000734), GNAT acyltransferase (BDBG_06912), a thioesterase-domain containing protein (BDBG_07971), and a cation diffusion facilitator (BDBG_07469) (S11 Fig and S6 Table). These genes were DE in the microarray analysis (S6 Table). In addition, SREB-3xHA was enriched upstream of an amino acid acetyltransferase, BDBG_00643, which is derepressed in SREB∆ and is predicted to be involved in the biosynthesis of ornithine. ChIP-qPCR confirmed enrichment of a subset of these genes including the glycolipid surface protein (BDBG_000734) and amino acid acetyltransferase (BDBG_00643) (Fig 9A and 9B, and S6 Table).


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)

Chromatin immunoprecipitation with quantitative real-time PCR (ChIP-qPCR).(A) Enrichment for binding of SREB-3xHA to promoter regions containing GATA binding motifs in B. dermatitidis yeast at 37°C. (B) Enrichment binding of SREB-3xHA to promoter regions containing GATA binding motifs in B. dermatitidis cells at 48-hrs following a drop in temperature from 37°C to 22°C. ChIP-qPCR was performed using SREB that was engineered to contain a C-terminal 3x-hemagglutinin tag. Integration of gene expression microarray data with genome-wide GATA motif analysis was used to identify putative GATA transcription factor binding sites. All genes analyzed were differentially expressed and contained a GATA motif (ATC-w-gAta-a or A/T-GATA-A/G) in the upstream promoter. For MIRB, the upstream motif in strain 26199 was ATC-A-GATT-A, whereas in other strains including SLH14081, ER-3, and ATCC 18188, the motif was ATC-A-GATA-A. BDBG is the locus number for genes in the B. dermatitidis genome database at the Broad Institute (www.broadinstitute.org).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482641&req=5

ppat.1004959.g009: Chromatin immunoprecipitation with quantitative real-time PCR (ChIP-qPCR).(A) Enrichment for binding of SREB-3xHA to promoter regions containing GATA binding motifs in B. dermatitidis yeast at 37°C. (B) Enrichment binding of SREB-3xHA to promoter regions containing GATA binding motifs in B. dermatitidis cells at 48-hrs following a drop in temperature from 37°C to 22°C. ChIP-qPCR was performed using SREB that was engineered to contain a C-terminal 3x-hemagglutinin tag. Integration of gene expression microarray data with genome-wide GATA motif analysis was used to identify putative GATA transcription factor binding sites. All genes analyzed were differentially expressed and contained a GATA motif (ATC-w-gAta-a or A/T-GATA-A/G) in the upstream promoter. For MIRB, the upstream motif in strain 26199 was ATC-A-GATT-A, whereas in other strains including SLH14081, ER-3, and ATCC 18188, the motif was ATC-A-GATA-A. BDBG is the locus number for genes in the B. dermatitidis genome database at the Broad Institute (www.broadinstitute.org).
Mentions: Using this integrated approach, we identified a subset of genes bound and regulated by SREB-3xHA under iron-replete conditions (10 μM FeSO4) using ChIP-qPCR (Fig 9A and 9B). These included genes involved with extracellular (SID1, ACE1) and intracellular (SIDC) siderophore biosynthesis, siderophore transport (MIRB), ferric reduction (FRE1 and MR), ferrous iron/zinc transporter (ZFT), a WD-repeat protein of unknown function (WD), and a bZIP transcription factor (HAPX) (Fig 9A and 9B). For ChIP-qPCR, SREB-3xHA did not bind the GAPDH promoter, which lacked GATA motifs and served as a negative control. ChIP-seq analysis of yeast grown in iron-replete media (10 μM FeSO4) also demonstrated enrichment of SREB-3xHA binding for genes located in the siderophore biosynthetic gene cluster including BDBG_00046 (conserved hypothetical protein), BDBG_00047 (ATRH), BDBG_00048 (NRPS), BDBG_00053 (SID1), BDBG_00054 (long chain fatty acid CoA ligase), and BDBG_00055 (ACE1) (S10 Fig and S6 Table). In addition, SREB-3xHA bound to the promoters of SIDC (BDBG_08208) and siderophore transporters MIRB (BDBG_05798), MIRC (BDBG_08034), and SIT1 (BDBG_06965) (S10 Fig and S6 Table). ChIP-seq also showed enrichment of SREB-3xHA upstream of genes unrelated to iron homeostasis including a glycolipid surface protein predicted to have 1,3-beta-glucanosyltransferase activity (BDBG_000734), GNAT acyltransferase (BDBG_06912), a thioesterase-domain containing protein (BDBG_07971), and a cation diffusion facilitator (BDBG_07469) (S11 Fig and S6 Table). These genes were DE in the microarray analysis (S6 Table). In addition, SREB-3xHA was enriched upstream of an amino acid acetyltransferase, BDBG_00643, which is derepressed in SREB∆ and is predicted to be involved in the biosynthesis of ornithine. ChIP-qPCR confirmed enrichment of a subset of these genes including the glycolipid surface protein (BDBG_000734) and amino acid acetyltransferase (BDBG_00643) (Fig 9A and 9B, and S6 Table).

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.


Related in: MedlinePlus