Identification and characterization of sugar-regulated promoters in Chaetomium thermophilum

Abstract

The thermophilic fungus Chaetomium thermophilum has been used extensively for biochemical and high-resolution structural studies of protein complexes. However, subsequent functional analyses of these assemblies have been hindered owing to the lack of genetic tools compatible with this thermophile, which are typically suited to other mesophilic eukaryotic model organisms, in particular the yeast Saccharomyces cerevisiae. Hence, we aimed to find genes from C. thermophilum that are expressed under the control of different sugars and examine their associated 5' untranslated regions as promoters responsible for sugar-regulated gene expression. To identify sugar-regulated promoters in C. thermophilum, we performed comparative xylose- versus glucose-dependent gene expression studies, which uncovered a number of enzymes with induced expression in the presence of xylose but repressed expression in glucose-supplemented media. Subsequently, we cloned the promoters of the two most stringently regulated genes, the xylosidase-like gene (XYL) and xylitol dehydrogenase (XDH), obtained from this genome-wide analysis in front of a thermostable yellow fluorescent protein (YFP) reporter. With this, we demonstrated xylose-dependent YFP expression by both Western blotting and live-cell imaging fluorescence microscopy. Prompted by these results, we expressed the C. thermophilum orthologue of a well-characterized dominant-negative ribosome assembly factor mutant, under the control of the XDH promoter, which allowed us to induce a nuclear export defect on the pre-60S subunit when C. thermophilum cells were grown in xylose- but not glucose-containing medium. Altogether, our study identified xylose-regulatable promoters in C. thermophilum, which might facilitate functional studies of genes of interest in this thermophilic eukaryotic model organism.

Keywords: Eukaryotic thermophiles; Filamentous fungi; Inducible promoters; Thermostable proteins; Transcriptomes.

Fig. 1

RNA isolation from C. thermophilum cultures grown in glucose and xylose. a C. thermophilum was grown on a traditional CCM plate for 24 h at 50 °C. From a parental colony grown on a CCM plate, equally sized (approximately 3 mm²) pieces were excised from the periphery and transferred onto fresh plates with either CCM (control medium), glucose-containing medium, or xylose-containing medium. Colony growth was imaged after 20 and 24 h of incubation at 50 °C. Scale bar: 2 cm. b A schematic workflow illustrating the steps of C. thermophilum cultivation prior to RNA extraction. A piece of a parent colony was used to inoculate a liquid SPY culture, which was then incubated for 48 h at 55 °C to deplete a putative cellular sugar reserve. Final cultivation was done in liquid SPY (reference), glucose- or xylose-supplemented SPY at 55 °C for 6 h before RNA was extracted. c The quality of the extracted RNA was evaluated by bioanalyzer measurements. The most intense RNA bands correspond to the intact 25S and 18S rRNA of the reference, glucose- and xylose-induced cultures. The analysis was performed for three biological replicates of each growth condition

Fig. 2

Investigation of sugar-induced transcriptome dynamics in C. thermophilum. a–c The differentially expressed genes (DEGs) after exposure to either glucose or xylose for 6 h are shown. The charts show the significant DEGs as log₂-fold changes according to Illumina deep sequencing of mRNAs (p-adjusted < 0.05). Significant changes in transcripts after glucose induction (3736) compared to the reference condition (a), after xylose induction (4685) compared to the reference condition (b), and comparing xylose with glucose induction (3750) (c). The plotted DEGs are listed in Suppl. Data 1. d Selected genes involved in lignocellulolytic and catabolic function were analyzed with respect to their transcriptional dynamics. Shown are the changes in transcripts as log₂-fold changes after D-glucose (orange) and D-xylose (green) induction in comparison to carbon-deficient reference medium (SPY). The respective gene IDs are given in Suppl. Data 3. e The transcriptome dynamics of selected genes were validated by qRT-PCR. The measured changes in transcripts according to Illumina sequencing (filled bars) are shown alongside changes in transcripts according to qRT-PCR (white bars). Transcript dynamics are compared for a β-xylosidase-like gene (XYL), xylitol-dehydrogenase (XDH), a cellulose-binding protein (CBP) and the gene having the strictest activation in glucose compared to in xylose (CTHT_0011270)

Fig. 3

Experimental characterization of xylose inducible promoters in YFP-reporter strains. a Relative ranking of transcript abundancies of selected genes in the genome when mycelia were grown in glucose- (orange) or xylose- (green) containing medium. b Absolute transcript dynamics as log₂-fold changes comparing selected genes after the fungal mycelium was incubated in glucose or xylose supplemented media. c Architecture of YFP-reporter strains to test xylose-inducible promoter sequences. Promoter regions (shown in green) of the cellulose binding protein (P_CBP), β-1,4-xylosidase-like gene (P_XYL) and xylitol-dehydrogenase (P_XDH), respectively, were cloned as transcriptional fusions upstream of a thermostable YFP reporter gene depicted in yellow. The promotors and the reporter gene, were flanked up- and downstream by a transcription terminator sequence (grey). A control strain was generated, which carried a short oligonucleotide spacer upstream of the YFP gene instead of a promoter region. d Experimental workflow of the induction assay. Reporter strains were cultivated in glucose-containing medium to build biomass under repressive conditions, washed and subsequently grown for further 8 h in either fresh glucose-containing medium (glucose) or xylose-containing medium (xylose). Immunoblotting and fluorescence microscopy was performed to monitor YFP expression under the control of the various promoters

Fig. 4

Characterization of carbon-regulated promoters in YFP-reporter strains. Promoter regions from selected glucose-repressed and xylose-activated genes were characterized in YFP-reporter strains. The promoter-YFP-expressing reporter strains were subjected to the induction assay shown in Fig. 2. In essence, reporter strains were grown in glucose-containing medium for 16 h and subsequently shifted to glucose-containing or xylose-containing medium for up to 8 h. Samples were taken before medium exchange (0 h) and at the indicated time points after medium exchange for fluorescence microscopy and immunoblotting against the YFP. YFP expression is shown for P_CBP (a), P_XYL (b) and P_XDH (c). Because no antibody is available for normalization purposes, we loaded equal amounts of protein, as shown by ponceau S (PS) staining. Note that lysates from cultures grown in glucose and xylose were analysed on the same membrane. Exposure times for the Western blot had to be adjusted according to the level of expression of the respective promoter, as follows: P_CBP (5 min), P_XYL (1 s)_, P_XDH (10 s). Exposure times were identical for Glu- and Xyl-derived samples. Unprocessed blots/gels are presented in Supplementary Fig. 7

Fig. 5

The xylose-inducible P_XDH promoter controls a dominant-negative mutant eliciting a 60S export defect. a Cartoon showing the reporter assay for monitoring an inducible localization defect of the large 60S subunit using L25-YFP. We constitutively expressed L25-YFP using the hph1 selection marker and introduced additional plasmids that allow overexpressing of the ribosome biogenesis factor pA-Rsa4 (P_XDH-RSA4) or the mutated protein pA-rsa4 E117D (P_XDH-rsa4) under the control of P_XDH, using the erg1 selection marker. Based on the rsa4 E117D mutant from yeast, a dominant-negative growth phenotype and the accumulation of L25-YFP in the nucleoplasm is expected upon induction of the mutant protein. b Immunoblotting shows the controlled expression of pA-rsa4 E117D on xylose-containing medium at the indicated time points. The uncropped blot is shown in Related file 3. c L25-YFP localization in RSA4- and rsa4 E117D-expressing strains under the control of P_XDH. L25-YFP strains expressing pA-RSA4 (RSA4) or pA-RSA4 E117D (rsa4) under the control of P_XDH were grown for 16 h in glucose-containing medium and then shifted to fresh glucose- or xylose-containing medium for another 4 h. Images were acquired in the DIC and YFP channels. Unprocessed blots/gels are presented in Supplementary Fig. 8