Carbon catabolite repression involves physical interaction of the transcription factor CRE1/CreA and the Tup1-Cyc8 complex in Penicillium oxalicum and Trichoderma reesei

Abstract

Background: Cellulolytic enzyme production in filamentous fungi requires a release from carbon catabolite repression (CCR). The protein CRE1/CreA (CRE = catabolite responsive element) is a key transcription factor (TF) that is involved in CCR and represses cellulolytic gene expression. CRE1/CreA represents the functional equivalent of Mig1p, an important Saccharomyces cerevisiae TF in CCR that exerts its repressive effect by recruiting a corepressor complex Tup1p-Cyc8p. Although it is known from S. cerevisiae that CRE1/CreA might repress gene expression via interacting with the corepressor complex Tup1-Cyc8, this mechanism is unconfirmed in other filamentous fungi, since the physical interaction has not yet been verified in these organisms. The precise mechanism on how CRE1/CreA achieves transcriptional repression after DNA binding remains unknown.

Results: The results from tandem affinity purification and bimolecular fluorescence complementation revealed a direct physical interaction between the TF CRE1/CreA and the complex Tup1-Cyc8 in the nucleus of cellulolytic fungus Trichoderma reesei and Penicillium oxalicum. Both fungi have the ability to secrete a complex arsenal of enzymes to synergistically degrade lignocellulosic materials. In P. oxalicum, the protein PoCyc8, a subunit of complex Tup1-Cyc8, interacts directly with TF PoCreA and histone H3 lysine 36 (H3K36) methyltransferase PoSet2 in the nucleus. The di-methylation level of H3K36 in the promoter of prominent cellulolytic genes (cellobiohydrolase-encoding gene Pocbh1/cel7A and endoglucanase-encoding gene Poegl1/cel7B) is positively correlated with the expression levels of TF PoCreA. Since the methylation of H3K36 was also demonstrated to be a repression marker of cellulolytic gene expression, it appears feasible that the cellulolytic genes are repressed via PoCreA-Tup1-Cyc8-Set2-mediated transcriptional repression.

Conclusion: This study verifies the long-standing conjecture that the TF CRE1/CreA represses gene expression by interacting with the corepressor complex Tup1-Cyc8 in filamentous fungi. A reasonable explanation is proposed that PoCreA represses gene expression by recruiting complex PoTup1-Cyc8. Histone methyltransferase Set2, which methylates H3K36, is also involved in the regulatory network by interacting with PoCyc8. The findings contribute to the understanding of CCR mechanism in filamentous fungi and could aid in biotechnologically relevant enzyme production.

Keywords: CCR; CRE1; Cellulases; Penicillium; Transcription factor; Trichoderma.

Fig. 1

The results of TAP-MS and BiFC. Western blot (A) and SDS-PAGE with silver staining (B) of T. reesei QP4 (control) and TrCRE1-TAP strains. The green arrow, blue arrow, and red arrow represent proteins TrCYC8, TrTUP1, and TrCRE1, respectively. Western blot (C) and SDS-PAGE with silver staining (D) of P. oxalicum 114-2 (control) and PoCreA-TAP strains. Western blot (E) and SDS-PAGE with silver staining (F) of P. oxalicum 114-2 (control) and PoCyc8-TAP strains. The orange arrow and gray arrow represent proteins PoCyc8 and PoTup1, respectively. Western blot was performed using the ANTI-HA antibody (ABclonal, China). G Microscopy of PoCyc8-YFP-PoCreA BiFC strain; H Microscopy of PoTup1-YFP-PoCreA BiFC strain. Each image includes four parts. The upper left, blue particles indicate nucleus stained with Hoechst 33342. The upper right, normal white light. The bottom right, yellow fluorescent particles indicate interactions between two target proteins. The bottom left, indicating the merge of yellow fluorescence and blue nucleus

Fig. 2

Analysis of histone methylation patterns and transcription levels of genes in P. oxalicum WT and mutants. A Observation of cellulolytic halo around the colonies, red arrow represents cellulolytic halo. B The transcript abundance of two prominent cellulase encoding genes, Pocbh1 and Poegl1. C Assays of histone methylation patterns using Western blot. Histone H3 was used as the loading control. D The transcript abundance of two histone methyltransferases encoding genes, Poset1 and Poset2. E The transcript abundance of genes Potup1 and Pocyc8. Statistical significance tests were performed by one tailed, unequal variance t-test. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Strategy and results of ChIP-qPCR. A Microscopy of PoCyc8-YFP-PoSet2 BiFC strain. The image includes four parts. The upper left, blue particles indicate nucleus stained with Hoechst 33342. The upper right, normal white light. The bottom right, yellow fluorescent particles indicate interactions between two PoCyc8 and PoSet2. The bottom left, indicating the merge of yellow fluorescence and blue nucleus. B Overview on the upstream sequence and core promoters of Pochb1 and Poegl1. The transcription start site (TSS) is designated as + 1. The initiator (Inr) and TATA box were illustrated. The three chromatin regions investigated by ChIP-qPCR are indicated by green, red, and blue bars, respectively. For Pochb1, region 1 covers from − 439 to − 263; region 2 covers from − 232 to − 51; region 3 covers from − 73 to + 95. For Poegl1, region 1 covers from − 512 to − 344; region 2 covers from − 316 to − 142; region 3 covers from − 155 to + 30. The putative DNA-binding sites of PoCreA (5′-GCGGAG-3′; 5′-CCGGGG-3′; 5′-CCCCGC-3′; 5′-CCCCGG-3′; 5′-CTCCGG-3′) are indicated by orange triangles. The orientation of the triangle represents the orientation of the binding motif. Inr, initiator element; TATA, TATA box. C ChIP-qPCR for H3K36me2 of Pocbh1. D ChIP-qPCR for H3K36me2 of Poegl1. All values are means from measurements in triplicates and three biological experiments. The error bars indicate standard deviations. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

The model for PoCreA-CYC8–TUP1 during repression of cellulolytic gene. In glucose condition, PoCreA mainly localizes in the nucleus, binds to the promoter of the target genes, and recruits the corepressor complex PoTup1–Cyc8. Histone methyltransferase Set2 which methylates H3K36, is also involved in the regulatory network via its interaction with Cyc8. As the repression marker of cellulolytic gene expression, H3K36 methylation together with histone deacetylase Rpd3 cooperate to reestablish chromatin, thereby suppresses inappropriate transcription initiation. In addition, the corepressor PoTup1–Cyc8 also interacts with the main subunit of the RNA Pol II and thus prevents Pol II from initiating transcription