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The Janus transcription factor HapX controls fungal adaptation to both iron starvation and iron excess.

Gsaller F, Hortschansky P, Beattie SR, Klammer V, Tuppatsch K, Lechner BE, Rietzschel N, Werner ER, Vogan AA, Chung D, Mühlenhoff U, Kato M, Cramer RA, Brakhage AA, Haas H - EMBO J. (2014)

Bottom Line: We further demonstrate that a HapX homodimer and the CCAAT-binding complex (CBC) cooperatively bind an evolutionary conserved DNA motif in a target promoter.The latter reveals the mode of discrimination between general CBC and specific HapX/CBC target genes.Collectively, our study uncovers a novel regulatory mechanism mediating both iron resistance and adaptation to iron starvation by the same transcription factor complex with activating and repressing functions depending on ambient iron availability.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria.

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HapX binds in vitro and in vivo to an evolutionary conserved motif identified in promoters of cccA homologsA An evolutionary conserved, bipartite motif in promoters of cccA homologs identified by MEME analysis. The underlined nucleotides would be covered upon CBC-binding as can be predicted based on the identified CBC/DNA binary complex crystal structure (Huber et al, 2012).D Real-time SPR characterization of in vitro formation of the CBC/DNA-HapX ternary complex on the conserved cccA promoter motif from A. fumigatus. SPR analyses included binding of the CBC to DNA (panel 1), HapX to DNA (panel 2) and HapX to preformed CBC/DNA complexes (panel 3). The SPR sensorgrams are shown from sensor-immobilized 37 base pair duplexes covering the full as well as 3′-truncated duplexes. Nucleotides marked in blue represent the HapX consensus binding site in fungal cccA promoters identified by MEME analysis. Binding responses of the indicated CBC or HapX concentrations injected in duplicate (black lines) are shown overlaid with the best fit derived from a 1:1 interaction model including a mass transport term (red lines). Binding responses of CBC/DNA-HapX ternary complex formation (panel 3, blue lines) were obtained by concentration-dependent co-injection of HapX on preformed binary CBC/DNA complexes after 200 s within the steady-state phase. Sensorgrams in panel 4 depict the association/dissociation responses of HapX on preformed CBC/DNA and were generated by CBC response (co-injection of buffer instead of HapX) subtraction from HapX co-injection responses. Dissociation constants (KD) are plotted inside the graphs.C ChIP analysis demonstrating in vivo binding of HapX to the conserved cccA promoter motif from A. fumigatus. ChIP qPCR was performed on wild-type or the strain containing Venus-tagged HapX (hapXVENUS) grown for 18 h, then shifted to fresh media with no iron (−Fe), 0.03 mM iron (+Fe), or 3 mM iron (hFe) for 8 h. DNA was immunoprecipitated with either a control IgG antibody, or anti-GFP polyclonal antibody that recognizes the Venus protein. Binding of HapXVENUS to the DNA region was assessed by qPCR. HapX binding is represented as percent enrichment of input control samples ± SD from triplicates. The actA (actin) promoter served as a negative control.
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fig06: HapX binds in vitro and in vivo to an evolutionary conserved motif identified in promoters of cccA homologsA An evolutionary conserved, bipartite motif in promoters of cccA homologs identified by MEME analysis. The underlined nucleotides would be covered upon CBC-binding as can be predicted based on the identified CBC/DNA binary complex crystal structure (Huber et al, 2012).D Real-time SPR characterization of in vitro formation of the CBC/DNA-HapX ternary complex on the conserved cccA promoter motif from A. fumigatus. SPR analyses included binding of the CBC to DNA (panel 1), HapX to DNA (panel 2) and HapX to preformed CBC/DNA complexes (panel 3). The SPR sensorgrams are shown from sensor-immobilized 37 base pair duplexes covering the full as well as 3′-truncated duplexes. Nucleotides marked in blue represent the HapX consensus binding site in fungal cccA promoters identified by MEME analysis. Binding responses of the indicated CBC or HapX concentrations injected in duplicate (black lines) are shown overlaid with the best fit derived from a 1:1 interaction model including a mass transport term (red lines). Binding responses of CBC/DNA-HapX ternary complex formation (panel 3, blue lines) were obtained by concentration-dependent co-injection of HapX on preformed binary CBC/DNA complexes after 200 s within the steady-state phase. Sensorgrams in panel 4 depict the association/dissociation responses of HapX on preformed CBC/DNA and were generated by CBC response (co-injection of buffer instead of HapX) subtraction from HapX co-injection responses. Dissociation constants (KD) are plotted inside the graphs.C ChIP analysis demonstrating in vivo binding of HapX to the conserved cccA promoter motif from A. fumigatus. ChIP qPCR was performed on wild-type or the strain containing Venus-tagged HapX (hapXVENUS) grown for 18 h, then shifted to fresh media with no iron (−Fe), 0.03 mM iron (+Fe), or 3 mM iron (hFe) for 8 h. DNA was immunoprecipitated with either a control IgG antibody, or anti-GFP polyclonal antibody that recognizes the Venus protein. Binding of HapXVENUS to the DNA region was assessed by qPCR. HapX binding is represented as percent enrichment of input control samples ± SD from triplicates. The actA (actin) promoter served as a negative control.

Mentions: To identify putative, evolutionary conserved, regulatory motifs in the cccA promoter, the 1-kb 5′-upstream regions of cccA homologs from 28 fungal species including A. fumigatus, A. nidulans and F. oxysporum were subject to MEME analysis (Bailey & Elkan, 1994). The identified sites and their positions in the promoters of the different species are shown in Supplementary Fig S4. The highest scoring sequence (e-value of 3.4 × 10−115), present in all 28 species, was a bipartite motif separated by a spacer region with low conservation (Fig6A). Consistent with the HapX-independent regulation, the highest scoring motif was not found in the promoter of the S. cerevisiae cccA homolog (data not shown). The 5′-conserved submotif matches the CBC consensus DNA-binding motif (CCAAT box), CCAAT(C/T)(A/G) (Huber et al, 2012). This is in perfect agreement with the previous finding that HapX acts via physical interaction with the CBC (Hortschansky et al, 2007). Interestingly, binding of the CCAAT box by CBC would cover the entire spacer region as identified in the CBC/DNA binary complex crystal structure (Huber et al, 2012), which indicates that the 3′-submotif is the first accessible region for binding of another DNA-binding protein. The 3′-conserved non-palindromic submotif does not match any known transcription factor consensus binding sequence. This is intriguing, because bZIP proteins usually bind short palindromic or pseudo-palindromic target sequences. Furthermore, based on the amino acid signature sequence of its basic region NXXAQXXFR (Supplementary Fig S1), HapX belongs to the Pap1/Yap1 subfamily of bZIP transcription factors that are known to recognize TTACGTAA and TTAGTAA consensus motifs (Fujii et al, 2000).


The Janus transcription factor HapX controls fungal adaptation to both iron starvation and iron excess.

Gsaller F, Hortschansky P, Beattie SR, Klammer V, Tuppatsch K, Lechner BE, Rietzschel N, Werner ER, Vogan AA, Chung D, Mühlenhoff U, Kato M, Cramer RA, Brakhage AA, Haas H - EMBO J. (2014)

HapX binds in vitro and in vivo to an evolutionary conserved motif identified in promoters of cccA homologsA An evolutionary conserved, bipartite motif in promoters of cccA homologs identified by MEME analysis. The underlined nucleotides would be covered upon CBC-binding as can be predicted based on the identified CBC/DNA binary complex crystal structure (Huber et al, 2012).D Real-time SPR characterization of in vitro formation of the CBC/DNA-HapX ternary complex on the conserved cccA promoter motif from A. fumigatus. SPR analyses included binding of the CBC to DNA (panel 1), HapX to DNA (panel 2) and HapX to preformed CBC/DNA complexes (panel 3). The SPR sensorgrams are shown from sensor-immobilized 37 base pair duplexes covering the full as well as 3′-truncated duplexes. Nucleotides marked in blue represent the HapX consensus binding site in fungal cccA promoters identified by MEME analysis. Binding responses of the indicated CBC or HapX concentrations injected in duplicate (black lines) are shown overlaid with the best fit derived from a 1:1 interaction model including a mass transport term (red lines). Binding responses of CBC/DNA-HapX ternary complex formation (panel 3, blue lines) were obtained by concentration-dependent co-injection of HapX on preformed binary CBC/DNA complexes after 200 s within the steady-state phase. Sensorgrams in panel 4 depict the association/dissociation responses of HapX on preformed CBC/DNA and were generated by CBC response (co-injection of buffer instead of HapX) subtraction from HapX co-injection responses. Dissociation constants (KD) are plotted inside the graphs.C ChIP analysis demonstrating in vivo binding of HapX to the conserved cccA promoter motif from A. fumigatus. ChIP qPCR was performed on wild-type or the strain containing Venus-tagged HapX (hapXVENUS) grown for 18 h, then shifted to fresh media with no iron (−Fe), 0.03 mM iron (+Fe), or 3 mM iron (hFe) for 8 h. DNA was immunoprecipitated with either a control IgG antibody, or anti-GFP polyclonal antibody that recognizes the Venus protein. Binding of HapXVENUS to the DNA region was assessed by qPCR. HapX binding is represented as percent enrichment of input control samples ± SD from triplicates. The actA (actin) promoter served as a negative control.
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fig06: HapX binds in vitro and in vivo to an evolutionary conserved motif identified in promoters of cccA homologsA An evolutionary conserved, bipartite motif in promoters of cccA homologs identified by MEME analysis. The underlined nucleotides would be covered upon CBC-binding as can be predicted based on the identified CBC/DNA binary complex crystal structure (Huber et al, 2012).D Real-time SPR characterization of in vitro formation of the CBC/DNA-HapX ternary complex on the conserved cccA promoter motif from A. fumigatus. SPR analyses included binding of the CBC to DNA (panel 1), HapX to DNA (panel 2) and HapX to preformed CBC/DNA complexes (panel 3). The SPR sensorgrams are shown from sensor-immobilized 37 base pair duplexes covering the full as well as 3′-truncated duplexes. Nucleotides marked in blue represent the HapX consensus binding site in fungal cccA promoters identified by MEME analysis. Binding responses of the indicated CBC or HapX concentrations injected in duplicate (black lines) are shown overlaid with the best fit derived from a 1:1 interaction model including a mass transport term (red lines). Binding responses of CBC/DNA-HapX ternary complex formation (panel 3, blue lines) were obtained by concentration-dependent co-injection of HapX on preformed binary CBC/DNA complexes after 200 s within the steady-state phase. Sensorgrams in panel 4 depict the association/dissociation responses of HapX on preformed CBC/DNA and were generated by CBC response (co-injection of buffer instead of HapX) subtraction from HapX co-injection responses. Dissociation constants (KD) are plotted inside the graphs.C ChIP analysis demonstrating in vivo binding of HapX to the conserved cccA promoter motif from A. fumigatus. ChIP qPCR was performed on wild-type or the strain containing Venus-tagged HapX (hapXVENUS) grown for 18 h, then shifted to fresh media with no iron (−Fe), 0.03 mM iron (+Fe), or 3 mM iron (hFe) for 8 h. DNA was immunoprecipitated with either a control IgG antibody, or anti-GFP polyclonal antibody that recognizes the Venus protein. Binding of HapXVENUS to the DNA region was assessed by qPCR. HapX binding is represented as percent enrichment of input control samples ± SD from triplicates. The actA (actin) promoter served as a negative control.
Mentions: To identify putative, evolutionary conserved, regulatory motifs in the cccA promoter, the 1-kb 5′-upstream regions of cccA homologs from 28 fungal species including A. fumigatus, A. nidulans and F. oxysporum were subject to MEME analysis (Bailey & Elkan, 1994). The identified sites and their positions in the promoters of the different species are shown in Supplementary Fig S4. The highest scoring sequence (e-value of 3.4 × 10−115), present in all 28 species, was a bipartite motif separated by a spacer region with low conservation (Fig6A). Consistent with the HapX-independent regulation, the highest scoring motif was not found in the promoter of the S. cerevisiae cccA homolog (data not shown). The 5′-conserved submotif matches the CBC consensus DNA-binding motif (CCAAT box), CCAAT(C/T)(A/G) (Huber et al, 2012). This is in perfect agreement with the previous finding that HapX acts via physical interaction with the CBC (Hortschansky et al, 2007). Interestingly, binding of the CCAAT box by CBC would cover the entire spacer region as identified in the CBC/DNA binary complex crystal structure (Huber et al, 2012), which indicates that the 3′-submotif is the first accessible region for binding of another DNA-binding protein. The 3′-conserved non-palindromic submotif does not match any known transcription factor consensus binding sequence. This is intriguing, because bZIP proteins usually bind short palindromic or pseudo-palindromic target sequences. Furthermore, based on the amino acid signature sequence of its basic region NXXAQXXFR (Supplementary Fig S1), HapX belongs to the Pap1/Yap1 subfamily of bZIP transcription factors that are known to recognize TTACGTAA and TTAGTAA consensus motifs (Fujii et al, 2000).

Bottom Line: We further demonstrate that a HapX homodimer and the CCAAT-binding complex (CBC) cooperatively bind an evolutionary conserved DNA motif in a target promoter.The latter reveals the mode of discrimination between general CBC and specific HapX/CBC target genes.Collectively, our study uncovers a novel regulatory mechanism mediating both iron resistance and adaptation to iron starvation by the same transcription factor complex with activating and repressing functions depending on ambient iron availability.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria.

Show MeSH
Related in: MedlinePlus