From an electrophoretic mobility shift assay to isolated transcription factors: a fast genomic-proteomic approach

Abstract

Background: Hypocrea jecorina (anamorph Trichoderma reesei) is a filamentous ascomycete of industrial importance due to its hydrolases (e.g., xylanases and cellulases). The regulation of gene expression can influence the composition of the hydrolase cocktail, and thus, transcription factors are a major target of current research. Here, we design an approach for identifying a repressor of a xylanase-encoding gene.

Results: We used streptavidin affinity chromatography to isolate the Xylanase promoter-binding protein 1 (Xpp1). The optimal conditions and templates for the chromatography step were chosen according to the results of an electrophoretic mobility shift assay performed under repressing conditions, which yielded a DNA-protein complex specific to the AGAA-box (the previously identified, tetranucleotide cis-acting element). After isolating AGAA-box binding proteins, the eluted proteins were identified with Nano-HPLC/tandem MS-coupled detection. We compared the identified peptides to sequences in the H. jecorina genome and predicted in silico the function and DNA-binding ability of the identified proteins. With the results from these analyses, we eliminated all but three candidate proteins. We verified the transcription of these candidates and tested their ability to specifically bind the AGAA-box. In the end, only one candidate protein remained. We generated this protein with in vitro translation and used an EMSA to demonstrate the existence of an AGAA-box-specific protein-DNA complex. We found that the expression of this gene is elevated under repressing conditions relative to de-repressing or inducing conditions.

Conclusions: We identified a putative transcription factor that is potentially involved in repressing xylanase 2 expression. We also identified two additional potential regulatory proteins that bind to the xyn2 promoter. Thus, we succeeded in identifying novel, putative transcription factors for the regulation of xylanase expression in H. jecorina.

Figure 1

Binding of potential regulatory proteins of H. jecorina to the AGAA-box within the xyn2 promoter. First, 100 μg cell-free extracts derived from replacement experiments on glucose and xylan were subjected to an EMSA. Then, 10 ng of the radioactive-labelled oligonucleotides Pxyn2a (covering the area in the xyn2 promoter harbouring the AGAA-box (grey box; antisense strand), see Table 1) and Pxyn2aM (bearing a mutation of AGAA to CTCC (M), see Table 1) were used. Free probe indicates the sample lacking protein. Xyr1 indicates the Xyr1-binding sites (black boxes; GGGTAA on the sense strand and GGCTGG on the antisense strand). CCAAT indicates the binding site of the Hap2/3/5 complex (white box: antisense strand).

Figure 2

Schematic drawing of the affinity chromatography assay. The biotinylated (B) oligonucleotides bearing the AGAA-box were annealed and incubated with streptavidin particles tagged with a paramagnetic particle (S). Cell-free extracts from cells grown in glucose, containing the potential Xylanase promoter-binding protein (Xpp1), were added and incubated under the same conditions as those used for the EMSA (see Fig. 1). The Xpp1-DNA streptavidin complex was separated in a magnetic field according to the manufacturer's instructions (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany).

Figure 3

Expression and binding of DNA-binding domains (DBD) of putative repressor proteins to the AGAA-box within xyn2 promoter of H. Jecorina. (A) SDS-PAGE of two clones of the DBD of 3151, three clones of the DBD of 7236, and two clones of the DBD of 2488. All clones heterologously expressed the GST-fusion proteins (3151prp, 7236prp, 2488prp). GST, expression of the GST-protein alone, as a control. A prestained protein ladder (Fermentas) was used. (B) EMSA with 100 ng of the DBDs expressed as GST-fusion proteins (see Fig. 3A). Finally, 15 ng of labelled oligonucleotides, one covering the respective area of the xyn2 promoter (Lpxyn2, see Table 1) and the other bearing a mutation in the AGAA-box (from AGAA to CTCC, Lpxyn2 M, see Table 1), were assayed alone (lanes 1, 2), with GST alone (negative control, GST), or with the thrombin-cleaved DBDs (3151prp_DBD, 7236prp_DBD, 2488prp_DBD).

Figure 4

In vitro translation and binding of the 2488 putative repressor protein. (A) SDS-PAGE of in vitro translated and FluoroTect™Green labelled proteins: a negative control reaction containing no DNA template (no DNA), a positive control reaction for the expression of luciferase (Luc), and the expression of 2488prp (pMPF2488 as DNA template). A prestained protein ladder (Fermentas) was applied. (B) EMSA using 60 ng of in vitro translated, unlabelled 2488prp (see Fig. 4A). Then, 15 ng labelled oligonucleotides, one covering the respective area of the xyn2 promoter (Lpxyn2, see Table 1), and another bearing a mutation from AGAA to CTCC (Lpxyn2 M, see Table 1) were applied without protein sample (lanes 1, 2). In vitro translated Xyr1 was used as a positive control.

Figure 5

Transcription analysis of carbon source-dependent regulation of 2488prp and xyn2 transcription. (A) Transcription of 2488prp under repressing conditions. The H. jecorina strain QM9414 was pre-cultured on glycerol and then transferred to MA media lacking a carbon source (NC) or containing 1% (w/v) glucose (G) or glycerol (GY), and incubated for 3 and 5 hours, respectively. (B) Transcription of 2488prp under growth conditions. Replacement of QM9414 was performed to 1% (w/v) glucose (G), xylose (XO) or xylan (XN) as sole carbon source and incubated 3, 5 and 24 hours, respectively. (C) Transcription of 2488prp under inducing conditions. Replacement of QM9414 was performed to 1% (w/v) glucose (G), 1.5 mM sophorose (SO) or xylobiose (XB), and incubated 5 hours. (D) Transcription of xyn2 under various carbon source conditions. The rates of transcription for the above mentioned (growth) conditions are summarized. The relative transcript levels are given on decade logarithmic scale (lg). The data presented are means of results from three independent experiments. Error bars indicate the standard deviations. The asterisk indicates the reference sample.