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The impact of chromatin remodelling on cellulase expression in Trichoderma reesei.

Mello-de-Sousa TM, Rassinger A, Pucher ME, dos Santos Castro L, Persinoti GF, Silva-Rocha R, Poças-Fonseca MJ, Mach RL, Nascimento Silva R, Mach-Aigner AR - BMC Genomics (2015)

Bottom Line: In the strain bearing a xyr1 deletion the sophorose mediated induction of gene expression is lost and the chromatin opening is strongly reduced.In the case of the xylanase-encoding genes, xyn1 and xyn2, the result was similar concerning the condition-specific response of the chromatin compaction.Both the applied condition and the presence of Xyr1 influence chromatin status.

View Article: PubMed Central - PubMed

Affiliation: Department for Biotechnology and Microbiology, Institute of Chemical Engineering, TU Wien, Gumpendorfer Str. 1a, A-1060, Wien, Austria. tdemello@mail.tuwien.ac.at.

ABSTRACT

Background: Trichoderma reesei is used for industry-scale production of plant cell wall-degrading enzymes, in particular cellulases, but also xylanases. The expression of the encoding genes was so far primarily investigated on the level of transcriptional regulation by regulatory proteins. Otherwise, the impact of chromatin remodelling on gene expression received hardly any attention. In this study we aimed to learn if the chromatin status changes in context to the applied conditions (repressing/inducing), and if the presence or absence of the essential transactivator, the Xylanase regulator 1 (Xyr1), influences the chromatin packaging.

Results: Comparing the results of chromatin accessibility real-time PCR analyses and gene expression studies of the two prominent cellulase-encoding genes, cbh1 and cbh2, we found that the chromatin opens during sophorose-mediated induction compared to D-glucose-conferred repression. In the strain bearing a xyr1 deletion the sophorose mediated induction of gene expression is lost and the chromatin opening is strongly reduced. In all conditions the chromatin got denser when Xyr1 is absent. In the case of the xylanase-encoding genes, xyn1 and xyn2, the result was similar concerning the condition-specific response of the chromatin compaction. However, the difference in chromatin status provoked by the absence of Xyr1 is less pronounced. A more detailed investigation of the DNA accessibility in the cbh1 promoter showed that the deletion of xyr1 changed the in vivo footprinting pattern. In particular, we detected increased hypersensitivity on Xyr1-sites and stronger protection of Cre1-sites. Looking for the players directly causing the observed chromatin remodelling, a whole transcriptome shotgun sequencing revealed that 15 genes encoding putative chromatin remodelers are differentially expressed in response to the applied condition and two amongst them are differentially expressed in the absence of Xyr1.

Conclusions: The regulation of xylanase and cellulase expression in T. reesei is not only restricted to the action of transcription factors but is clearly related to changes in the chromatin packaging. Both the applied condition and the presence of Xyr1 influence chromatin status.

No MeSH data available.


Related in: MedlinePlus

Overview on the upstream sequence of the investigated genes encoding PCWD enzymes. The regions investigated by CHART-PCR are indicated by black bars. The core promoter region covering the TATA-box (core) and an URR of the cbh1 (a), cbh2 (b), xyn1 (c), and xyn2 (d) genes each are depicted. DNA-binding sites of Xyr1 and Cre1 are indicated by orange and purple triangles, respectively. The orientation of the triangle represents the orientation of the binding motif. The scale at the top indicates distance from ATG in bp
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Fig1: Overview on the upstream sequence of the investigated genes encoding PCWD enzymes. The regions investigated by CHART-PCR are indicated by black bars. The core promoter region covering the TATA-box (core) and an URR of the cbh1 (a), cbh2 (b), xyn1 (c), and xyn2 (d) genes each are depicted. DNA-binding sites of Xyr1 and Cre1 are indicated by orange and purple triangles, respectively. The orientation of the triangle represents the orientation of the binding motif. The scale at the top indicates distance from ATG in bp

Mentions: It is well known that Xyr1 is an essential activator of cellulase gene expression [11]. However, so far it has not been investigated if the deletion of Xyr1 additionally influences the chromatin status in the fungus. In order to study this, the wild-type strain and the xyr1 deletion strain were pre-grown and transferred to sophorose (inducing condition), D-glucose (repressing condition) or no carbon source-containing medium (reference condition) and were incubated for 3 h. By applying CHART-PCR analysis we investigated the chromatin packaging of the core promoter region (bearing the TATA-box) and one upstream regulatory region (URR) bearing Xyr1-binding sites (5′-GGC(T/A)3-3′; [21]) and/or Cre1-binding sites (5′-SYGGRG-3′; [14]) of the cbh1 and cbh2 genes each. For overviews on the investigated regions see Fig. 1a, b. Supplementary, we investigated the transcript levels of these genes by reverse transcription, quantitative PCR (qPCR) to see if the expression is related to chromatin accessibility. The expression of cbh1 and cbh2 is repressed on D-glucose in both strains and induced by sophorose in the wild-type strain (Fig. 2a, b). The induction is lost in the xyr1 deletion strain aside from a small increase in gene expression on sophorose compared to D-glucose. Altogether, we observed in both strains a condition-dependent change (i.e. sophorose-mediated opening) of chromatin that went along with a change (i.e. sophorose-mediated increase) in gene expression. However, comparing the strains under the same condition, the chromatin was always more closed in the xyr1 deletion strain compared to the wild-type strain (Fig. 2a, b) indicating a contribution of Xyr1 to a general (i.e. condition-independent) opening of chromatin in upstream regions of the cellulase-encoding genes.Fig. 1


The impact of chromatin remodelling on cellulase expression in Trichoderma reesei.

Mello-de-Sousa TM, Rassinger A, Pucher ME, dos Santos Castro L, Persinoti GF, Silva-Rocha R, Poças-Fonseca MJ, Mach RL, Nascimento Silva R, Mach-Aigner AR - BMC Genomics (2015)

Overview on the upstream sequence of the investigated genes encoding PCWD enzymes. The regions investigated by CHART-PCR are indicated by black bars. The core promoter region covering the TATA-box (core) and an URR of the cbh1 (a), cbh2 (b), xyn1 (c), and xyn2 (d) genes each are depicted. DNA-binding sites of Xyr1 and Cre1 are indicated by orange and purple triangles, respectively. The orientation of the triangle represents the orientation of the binding motif. The scale at the top indicates distance from ATG in bp
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4528718&req=5

Fig1: Overview on the upstream sequence of the investigated genes encoding PCWD enzymes. The regions investigated by CHART-PCR are indicated by black bars. The core promoter region covering the TATA-box (core) and an URR of the cbh1 (a), cbh2 (b), xyn1 (c), and xyn2 (d) genes each are depicted. DNA-binding sites of Xyr1 and Cre1 are indicated by orange and purple triangles, respectively. The orientation of the triangle represents the orientation of the binding motif. The scale at the top indicates distance from ATG in bp
Mentions: It is well known that Xyr1 is an essential activator of cellulase gene expression [11]. However, so far it has not been investigated if the deletion of Xyr1 additionally influences the chromatin status in the fungus. In order to study this, the wild-type strain and the xyr1 deletion strain were pre-grown and transferred to sophorose (inducing condition), D-glucose (repressing condition) or no carbon source-containing medium (reference condition) and were incubated for 3 h. By applying CHART-PCR analysis we investigated the chromatin packaging of the core promoter region (bearing the TATA-box) and one upstream regulatory region (URR) bearing Xyr1-binding sites (5′-GGC(T/A)3-3′; [21]) and/or Cre1-binding sites (5′-SYGGRG-3′; [14]) of the cbh1 and cbh2 genes each. For overviews on the investigated regions see Fig. 1a, b. Supplementary, we investigated the transcript levels of these genes by reverse transcription, quantitative PCR (qPCR) to see if the expression is related to chromatin accessibility. The expression of cbh1 and cbh2 is repressed on D-glucose in both strains and induced by sophorose in the wild-type strain (Fig. 2a, b). The induction is lost in the xyr1 deletion strain aside from a small increase in gene expression on sophorose compared to D-glucose. Altogether, we observed in both strains a condition-dependent change (i.e. sophorose-mediated opening) of chromatin that went along with a change (i.e. sophorose-mediated increase) in gene expression. However, comparing the strains under the same condition, the chromatin was always more closed in the xyr1 deletion strain compared to the wild-type strain (Fig. 2a, b) indicating a contribution of Xyr1 to a general (i.e. condition-independent) opening of chromatin in upstream regions of the cellulase-encoding genes.Fig. 1

Bottom Line: In the strain bearing a xyr1 deletion the sophorose mediated induction of gene expression is lost and the chromatin opening is strongly reduced.In the case of the xylanase-encoding genes, xyn1 and xyn2, the result was similar concerning the condition-specific response of the chromatin compaction.Both the applied condition and the presence of Xyr1 influence chromatin status.

View Article: PubMed Central - PubMed

Affiliation: Department for Biotechnology and Microbiology, Institute of Chemical Engineering, TU Wien, Gumpendorfer Str. 1a, A-1060, Wien, Austria. tdemello@mail.tuwien.ac.at.

ABSTRACT

Background: Trichoderma reesei is used for industry-scale production of plant cell wall-degrading enzymes, in particular cellulases, but also xylanases. The expression of the encoding genes was so far primarily investigated on the level of transcriptional regulation by regulatory proteins. Otherwise, the impact of chromatin remodelling on gene expression received hardly any attention. In this study we aimed to learn if the chromatin status changes in context to the applied conditions (repressing/inducing), and if the presence or absence of the essential transactivator, the Xylanase regulator 1 (Xyr1), influences the chromatin packaging.

Results: Comparing the results of chromatin accessibility real-time PCR analyses and gene expression studies of the two prominent cellulase-encoding genes, cbh1 and cbh2, we found that the chromatin opens during sophorose-mediated induction compared to D-glucose-conferred repression. In the strain bearing a xyr1 deletion the sophorose mediated induction of gene expression is lost and the chromatin opening is strongly reduced. In all conditions the chromatin got denser when Xyr1 is absent. In the case of the xylanase-encoding genes, xyn1 and xyn2, the result was similar concerning the condition-specific response of the chromatin compaction. However, the difference in chromatin status provoked by the absence of Xyr1 is less pronounced. A more detailed investigation of the DNA accessibility in the cbh1 promoter showed that the deletion of xyr1 changed the in vivo footprinting pattern. In particular, we detected increased hypersensitivity on Xyr1-sites and stronger protection of Cre1-sites. Looking for the players directly causing the observed chromatin remodelling, a whole transcriptome shotgun sequencing revealed that 15 genes encoding putative chromatin remodelers are differentially expressed in response to the applied condition and two amongst them are differentially expressed in the absence of Xyr1.

Conclusions: The regulation of xylanase and cellulase expression in T. reesei is not only restricted to the action of transcription factors but is clearly related to changes in the chromatin packaging. Both the applied condition and the presence of Xyr1 influence chromatin status.

No MeSH data available.


Related in: MedlinePlus