Nucleosome transactions on the Hypocrea jecorina ( Trichoderma reesei) cellulase promoter cbh2 associated with cellulase induction

Abstract

The 5' regulatory region of the cbh2 gene of Hypocrea jecorina contains the cbh2 activating element (CAE) which is essential for induction of cbh2 gene expression by sophorose and cellulose. The CAE consists of two motifs, a CCAAT box on the template strand and a GTAATA box on the coding strand, which cooperate during induction. Northern analyses of cbh2 gene expression has revealed an absolute dependence on induction, but no direct effect of Cre1-mediated carbon catabolite repression. Investigation of the chromatin structure in the wild-type strain showed that, under repressing conditions, there is a nucleosome free region (nfr) around the CAE, which is flanked by strictly positioned nucleosomes. Induction results in a loss of positioning of nucleosomes -1 and -2 downstream of the CAE, thus making the TATA box accessible. Simultaneous mutation of both motifs of the CAE, or of the CCAAT-box alone, also leads to shifting of nucleosome -1, which normally covers the TATA-box under repressing conditions, whereas mutation of the GTAATA element results in a narrowing of the nfr, indicating that the proteins that bind to both motifs in the CAE interact with chromatin, although in different ways. A cellulase-negative mutant strain, which has previously been shown to be altered in protein binding to the CAE, still displayed the induction-specific changes in nucleosome structure, indicating that none of the proteins that directly interact with CAE are affected, and that nucleosome rearrangement and induction of cbh2 expression are uncoupled. Interestingly, the carbon catabolite repressor Cre1 is essential for strict nucleosome positioning in the 5' regulatory sequences of cbh2 under all of the conditions tested, and induction can occur in a promoter that lacks positioned nucleosomes. These data suggest that Cre1, the Hap2/3/5 complex and the GTAATA-binding protein are all involved in nucleosome assembly on the cbh2 promoter, and that the latter two respond to inducing conditions by repositioning nucleosome -1.

d-Galactose induces cellulase gene expression in Hypocrea jecorina at low growth rates

“…Cel7A is encoded by cbh1, whose basal expression level is under the control of the Cre1 carbon catabolite repressor. Therefore, a low level of Cel7A is formed under derepressing conditions even without the addition of an inducer, whereas formation of Cel6A is strictly dependent on the presence of an inducer (Zeilinger et al, 2003). This behaviour is also reflected in the present results, i.e.…”

“…In contrast to the results obtained with Dgalactose, no cbh2 promoter activation was detected under these conditions, which was also supported by the absence of the Cel6A protein. Cel7A -whose basal level is known to be under carbon catabolite repression by Cre1 (Zeilinger et al, 2003) -was detectable only at the lowest dilution rate, 0?015 h 21 (Table 1, Fig. 3a).…”

Regulation of transcription of cellulases- and hemicellulases-encoding genes in Aspergillus niger and Hypocrea jecorina (Trichoderma reesei)

“…de Graaff et al 1994;de Vries et al 1999b;Ilmen et al 1996;Mach et al 1996). Whereas only some of the major hydrolases in H. jecorina namely, cbh1 and xyn1, are under direct Cre1 control (Ilmen et al 1996;Mach et al 1996), other hydrolytic enzyme-encoding genes such as cbh2, xyn2 and bgl1 are not regulated Cre1 dependent (Margolles-Clark 1997; Würleitner et al 2003;Zeilinger et al 2003).…”

ThHog1 controls the hyperosmotic stress response in Trichoderma harzianum

“…A hog1 cassette containing promoter and gene was amplified from genomic DNA by PCR using primers 59-GGTCGACCTGTGTC-CCTTCATTACTTC-39 and 59 TCACTAGTTTGTGTGGGGAATTGC-TCC-39, which added SalI and SpeI sites, respectively (underlined). These enzymes were used to clone the fragment into pZEGA1 vector (Zeilinger et al, 2003) previously cut with XhoI and XbaI. The resultant vector pHog1-GFP was used to transform T. harzianum using a PEG-based protocol (Penttila et al, 1987).…”