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.

The interaction between the histone acetyltransferase complex Hat1‐Hat2 and transcription factor AmyR provides a molecular brake to regulate amylase gene expression

“…The Tup1p‐Cyc8p (Tup1p‐Ssn6p) complex was the first studied transcriptional corepressor in S. cerevisiae (Keleher et al, 1992), mediating the inhibition of multiple subsets of genes (Malave & Dent, 2006). In P. oxalicum and T. reesei , transcription factor CRE1/CreA (the homolog of S. cerevisiae Mig1p) participates in carbon catabolite repression (CCR) by interacting with the corepressor complex Tup1‐Cyc8 to inhibit cellulase gene expression (Hu et al, 2021). In P. oxalicum and A .…”

“…BiFC strains were constructed through a previously described method (Hu et al, 2021). This method required two plasmids: (1) pMD18‐T‐NYFP carrying the coding sequence of the N‐terminal (1–155 aa) of YFP and (2) pUC19‐CYFP carrying the coding sequence of the C‐terminal (156–238 aa) of YFP.…”

“…participates in carbon catabolite repression (CCR) by interacting with the corepressor complex Tup1-Cyc8 to inhibit cellulase gene expression (Hu et al, 2021). In P. oxalicum and A. oryzae, deletion of AmyR almost completely abolished amylase synthesis, while the synthesis of cellulase was significantly increased; presumably AmyR is indirectly involved in CCR regulated by Tup1-Cyc8 (Li et al, 2015;Watanabe et al, 2011).…”

“…To Primers GFP-F/GFP-R were used to amplify gfp tag (720 bp). BiFC strains were constructed through a previously described method (Hu et al, 2021). This method required two plasmids: S4.…”

Isolation, morphological identification, and xylanase characteristics of anaerobic gut fungi Neocallimastix from Anatolian wild goat

“…Enzyme activity losses at increased temperatures are associated with changes in the enzyme conformation [50]. Carbon catabolite repression is frequently required for the expression of hydrolytic enzymes [51]. AGF may be able to overcome this by forming enzyme complexes.…”