Stress-related transcription factor AtfB integrates secondary metabolism with oxidative stress response in aspergilli

Abstract

In filamentous fungi, several lines of experimental evidence indicate that secondary metabolism is triggered by oxidative stress; however, the functional and molecular mechanisms that mediate this association are unclear. The basic leucine zipper (bZIP) transcription factor AtfB, a member of the bZIP/CREB family, helps regulate conidial tolerance to oxidative stress. In this work, we investigated the role of AtfB in the connection between oxidative stress response and secondary metabolism in the filamentous fungus Aspergillus parasiticus. This well characterized model organism synthesizes the secondary metabolite and carcinogen aflatoxin. Chromatin immunoprecipitation with specific anti-AtfB demonstrated AtfB binding at promoters of seven genes in the aflatoxin gene cluster that carry CREs. Promoters lacking CREs did not show AtfB binding. The binding of AtfB to the promoters occurred under aflatoxin-inducing but not under aflatoxin-noninducing conditions and correlated with activation of transcription of the aflatoxin genes. Deletion of veA, a global regulator of secondary metabolism and development, nearly eliminated this binding. Electrophoretic mobility shift analysis demonstrated that AtfB binds to the nor-1 (an early aflatoxin gene) promoter at a composite regulatory element that consists of highly similar, adjacent CRE1 and AP-1-like binding sites. The five nucleotides immediately upstream from CRE1, AGCC(G/C), are highly conserved in five aflatoxin promoters that demonstrate AtfB binding. We propose that AtfB is a key player in the regulatory circuit that integrates secondary metabolism and cellular response to oxidative stress.

FIGURE 1.

Alignment of AtfB amino acid sequences from aspergilli.

FIGURE 2.

Antibodies to AtfB (anti-AtfB) specifically recognize AtfB. A, Western blot analysis of MBP-AtfB fusion with anti-AtfB. Schematic illustrates the purification method for the fusion protein. Total protein extracts (P) obtained from E. coli expressing either MBP (E_m) or recombinant MBP-AtfB (E_fusion) were passed through an amylose affinity column according to manufacturer's instructions (see under “Experimental Procedures”). Western blot (left) was conducted using anti-AtfB PPF. The amount of protein loaded in each lane was 20 μg. Samples (from left) are as follows: C, protein eluted from the amylose column after passage of E_m total protein extract; S and C, flow-through and protein eluted from the amylose column, respectively, after passage of E_fusion total protein extract; P, E_fusion total protein extract; M, BenchMark PreStained Protein Ladder, Invitrogen. Arrow, 78-kDa band in protein ladder. B, Western blot analysis of A. parasiticus protein extracts using anti-AtfB PPF. Conidiospores (10⁶) were inoculated into 100 ml of YES liquid medium and incubated at 30 °C with shaking for 48 h. Proteins were extracted, and Western blot analysis was conducted as described under “Experimental Procedures.” 90 μg of total protein was loaded per lane. Enriched nuclear proteins were obtained as described under “Experimental Procedures.” CE, cell extract; EE, enriched nuclear proteins; S, BenchMark PreStained Protein Ladder. C, immunoprecipitation of AtfB from A. parasiticus SU-1 cell extracts (for details see “Experimental Procedures”). A cell extract (C) was obtained from 4 g of SU-1 grown in YES liquid medium. Extract C was precleared with protein G-agarose beads only to obtain precipitate P1 (obtained from beads), and supernatant was treated with protein G-agarose tagged with antibodies in preimmune serum (see “Experimental Procedures” for description). The precipitate obtained at this step, P2, was stored, and the supernatant was treated with protein G-agarose tagged with anti-AtfB. The immunoprecipitate, P3, and the supernatant, S, were stored separately. Western blot analysis was conducted on samples C, P1, P2, P3, and S using anti-AtfB. 20 μg of protein was loaded in each lane.

FIGURE 3.

ChIP analysis of AtfB binding to promoters in the A. parasiticus aflatoxin gene cluster. A. parasiticus conidiospores (10⁴/ml) were inoculated into appropriate growth media and incubated at 30 °C with shaking. At 24, 30, and 40 h of growth, cultures were treated with 1% formaldehyde, and chromatin was prepared as described under “Experimental Procedures.” ChIP was performed with anti-AftB PPF. A, schematic representation of the relevant genes in the aflatoxin gene cluster and one gene, laeA, outside the cluster. Solid black bars indicate targets in the promoter region of the genes for PCR amplification in PCR analysis. Abbreviations: P, pksA; N, nor-1; FF, fas1/fas2 intergenic region; A, aflR; V, ver-1; Om, omtA; Or, ordA; Vb, vbs; L, laeA. B, relative fold enrichment (see “Experimental Procedures”) in AtfB binding at the promoters of the designated genes of A. parasiticus SU-1 grown in YES liquid medium. C, relative fold enrichment in AtfB binding at the promoters of the designated aflatoxin genes in A. parasiticus SU-1 grown in YEP liquid medium. D, relative fold enrichment in AtfB binding at the promoters of designated aflatoxin genes in A. parasiticus ΔveA grown in YES liquid medium. Two independent experiments were performed for each growth condition. Data are presented as mean ± S.E. for both experiments. Statistical analysis was performed by Student's t test. a, statistically significant 2-fold or more relative enrichment, p < 0.05; b, statistically significant difference in relative fold enrichment compared with 24 h, p < 0.05; c, statistically significant difference in fold enrichment compared with 30 h, p < 0.05.

FIGURE 4.

NorR and NorR subfragments used in EMSA. A, schematic of the pksA/nor-1 intergenic region showing the position of NorR in the nor-1 promoter. NorR was subdivided into smaller fragments NorR1, NorR2, NorR3, NorR4, AP-1, and CRE1; location and size (bp) of fragments are shown. The location of putative cis-acting sites are shown, including AflR1, TATA, CRE1, AP-1-like, ABBA, PACC, and AREA. B, primers used to generate NorR and NorR subfragments. Location of the primers is indicated by short horizontal lines.

FIGURE 5.

EMSA analysis of AtfB binding at the nor-1 promoter. A. parasiticus SU-1 was grown for 48 h at 30 °C in the dark with shaking at 150 rpm. Enriched nuclear protein extracts were prepared as described under “Experimental Procedures.” 5 μg of protein in enriched nuclear protein extracts were added to the labeled NorR probe in each lane. A, competition EMSA. Nonlabeled NorR (50 and 250× molar excess) was added to compete for labeled NorR probe. B, shift inhibition EMSA. Anti-AtfB PPF or preimmune serum were added to determine whether these could block protein/DNA interaction. C, competition EMSA. NorR subfragments were added in 50 or 250× molar excess to compete for labeled NorR probes. Competitors were as follows: donors (83 bp), dsNorR2 (121 bp), ds NorR3 (73 bp), dsNorR4 (51 bp), dsCRE1 (27 bp), and dap-1 (23 bp) (see Fig. 4).

FIGURE 6.

Expression of atfB correlates with temporal pattern of expression of nor-1, ver-1, aflR, and atfA, but not of laeA. A. parasiticus SU-1 was grown in YES liquid medium for designated periods of time, and RNA extraction and real time PCR analyses were performed as described under “Experimental Procedures.” The relative level of mRNA is depicted as the mRNA level of the target gene divided by mRNA level of β-tubulin at the same time point. Bars represent mean ± S.E. (n = 4). Statistical analysis was performed by the Student's t test and one-way analysis of variance(see “Experimental Procedures”). Same lowercase letters indicate that there is no statistically significant difference between two measurements. Different lowercase letters specify a statistically significant difference between two measurements. p values are as follows: nor-1 (p < 0.001), ver-1 (p = 0.002), aflR (p < 0.001), laeA (p = 0.018), atfA (p = 0.016), atfB (p = 0.029). Two independent biological replicates were performed showing the same trend. Two duplicates samples were analyzed for each biological replicate.

FIGURE 7.

Sequence analysis of aflatoxin gene promoters using MEME. 500-bp promoter regions upstream from ATG in 17 aflatoxin genes and laeA were analyzed for 8-mer motif pattern occurrence using MEME motif-based sequence analysis. The motif AGCC(G/C)T(G/C)(A/G) (second highest frequency occurrence) was found a total of 11 times within eight promoters. A, sites carrying the motif in eight promoters; *, the nor-1 promoter contained a functional CRE1 motif; →, promoters analyzed by ChIP. B, occurrence and location of the motif relative to start site ATG (denoted at the end of the scale). Divergently transcribed fas-2/fas-1 and aflR/aflJ share the only motif located in the corresponding intergenic region. C, sequence logo of the motif, which contains partial CRE1 site represented by position-specific probability matrices on the positive strand.

FIGURE 8.

AtfB integrates secondary metabolism and oxidative stress response. Based on available experimental evidence, we propose that exposure of the fungal cell to intra- or extracellular ROS down-regulates a cAMP/PKA/PI3K signaling cascade. This promotes formation of an active AftB/AP-1 heterodimer on target gene promoters involved in stress response (SR), secondary metabolism (SM), and vacuole biogenesis (VB). Alternatively, ROS may directly facilitate heterodimer formation on the promoters of target genes by affecting the redox state of the transcription factor. Initiation of secondary metabolism triggered by ROS plays a protective and/or signaling role in the overall cellular response to oxidative stress. PKA, protein kinase A; PI3K, phosphatidylinositol 3-kinase; PM, plasma membrane.