The Relation Between Promoter Chromatin Status, Xyr1 and Cellulase Ex-pression in Trichoderma reesei

Abstract

The ascomycete Trichoderma reesei is used for the production of plant cell wall-degrading enzymes in industrial scale. The interplay of the transactivator Xyr1 and the repressor Cre1 mainly regulates the expression of these enzymes. During induc-ing conditions, such as in the presence of sophorose, the transcription of the two major cellulase-encoding genes, cbh1 and cbh2, is activated as well as the expression of xyr1. In the presence of D-glucose carbon catabolite repression mediated by Cre1 takes place and the expression of Xyr1 and the plant cell wall-degrading enzymes is down-regulated. In this study we compare the chromatin status of xyr1, cbh1, and cbh2 promoters in the wild-type strain and the Cre1-deficient strain Rut-C30. Chromatin rearrangement occurs in the xyr1 promoter during induction on sophorose. Chromatin opening and protein-DNA interactions in the xyr1 promoter were detected especially in a region located 0.9 kb upstream the translation start co-don, which bears several putative Cre1-binding sites and a CCAAT-box. Moreover, the xyr1 promoter is overall more acces-sible in a cre1-truncated background, no matter which carbon source is present. This makes the xyr1 regulatory sequence a good target for promoter engineering aiming at the enhancement of cellulase production.

Keywords: Cellulases; Chromatin; Promoter; Rut-C30; Trichoderma reesei; Xyr1..

Fig. (1)

Chromatin status and gene expression of xyr1 in the wild-type strain and Rut-C30. (A) The promoter of xyr1 is schematically represented. The scale at the top indicates the distance from the start codon in bp. Black bars indicate the positions of the core region (core) and the URR (URR) investigated by CHART-PCR. Triangles indicate cis elements. For CHART-PCR of the URR (B) and the core promoter (C) and xyr1 transcript analysis (B, C) the T. reesei wild-type strain (grey squares) and Rut-C30 (black squares) were pre-grown on glycerol and thereafter incubated on D-glucose (G) or sophorose (S) for 3 h. CHART-PCR was performed by DNase I digestion followed by qPCR, and chromatin accessibility indices (CAIs) are depicted on the y-axis. The gene expression analysis was performed by cDNA synthesis followed by qPCR, and transcript levels are depicted on the x-axis. The dashed line indicates transcript level of the reference condition (wildtype strain, no carbon source, 3 h). sar1 and act genes were used for data normalization. All values are means from measurements in triplicates and three biological replicates. Error bars indicate standard deviations. Diagrams are identically scaled.

Fig. (2)

In vivo foot printing analysis of the xyr1 promoter. The T. reesei wild-type strain (grey landscape) and Rut-C30 (black landscape) were pre-grown on glycerol and then incubated on D-glucose or sophorose for 3 h followed by DMS-induced in vivo methylation. An URR (A) and the core promoter (B) were investigated. Triangles indicate cis elements. Analysis of data was performed using ivFAST [25] followed by a landscape-like visualization. The relative protein-DNA-interaction indices (PDI) give differences between the in vivo footprinting patterns obtained on sophorose compared to D-glucose of one strain. The diagrams are identically scaled and are comparable amongst the two strains within one region. The scale at the top indicates distance from the start codon in bp. (C) Detailed in vivo footprint result for both strains of the region -279 to -255 bearing two overlapping Xyr1-binding sites (bold letters). Methylation differences between sophorose and D-glucose are represented by light grey (1.1- to 1.3-fold) and dark grey squares (more than 1.3-fold).

Fig. (3)

Chromatin status and gene expression of cellulase-encoding genes in the wild-type strain and Rut-C30. The promoters of cbh1 (A) and cbh2 (B) are schematically represented. The scale at the top indicates the distance from the start codon in bp. Black bars indicate the positions of the core region (core) and the URR (URR) investigated by CHART-PCR. Triangles indicate cis elements. Carbon sourcespecifically positioned nucleosomes [21, 22] are indicated by ellipses. (C-F) The T. reesei wild-type strain (grey squares) and Rut-C30 (black squares) were pre-grown on glycerol and thereafter incubated on D-glucose (G) or sophorose (S) for 3 h. The URR (C, E) and the core promoter region (D, F) of the cbh1 (C, D) and cbh2 (E, F) genes were investigated each. CHART-PCR was performed by DNase I digestion followed by qPCR, and chromatin accessibility indices (CAIs) are depicted on the y-axis. The gene expression analysis was performed by cDNA synthesis followed by qPCR, and transcript levels are depicted on the x-axis. The dashed line indicates transcript level of the reference condition (wild-type strain, no carbon source, 3 h). sar1 and act genes were used for data normalization. All values are means from measurements in triplicates and three biological replicates. Error bars indicate standard deviations. Diagrams are identically scaled.