Histone acetyltransferase Gcn5-mediated histone H3 acetylation facilitates cryptococcal morphogenesis and sexual reproduction.

IMPORTANCE: Eukaryotic gene transcription is typically regulated by a series of histone modifications, which play a crucial role in adapting to complex environmental stresses. In the ubiquitous human fungal pathogen Cryptococcus neoformans, sexual life cycle is a continuous intracellular differentiation process that strictly occurs in response to mating stimulation. Despite the comprehensive identification of the regulatory factors and genetic pathways involved in its sexual cycle, understanding of the epigenetic modifications involved in this process remains quite limited. In this research, we found that histone acetyltransferase Gcn5-mediated histone H3 acetylation plays a crucial role in completing the cryptococcal sexual cycle, including yeast-hyphae morphogenesis and the subsequent sexual reproduction. Furthermore, we demonstrated that Gcn5 participates in this process primarily through regulating the key morphogenesis regulator Znf2 and its targets. This study thus provided a comprehensive understanding of how histone acetylation modification impacts sexual life cycle in a high-risk human pathogenic fungus.

Role of the Nitrogen Metabolism Regulator TAM1 in Regulation of Cellulase Gene Expression in Trichoderma reesei.

The filamentous fungus Trichoderma reesei is one of the most prolific cellulase producers and has been established as a model microorganism for investigating mechanisms modulating eukaryotic gene expression. Identification and functional characterization of transcriptional regulators involved in complex and stringent regulation of cellulase genes are, however, not yet complete. Here, a Zn(II)2Cys6-type transcriptional factor TAM1 that is homologous to Aspergillus nidulans TamA involved in nitrogen metabolism, was found not only to regulate ammonium utilization but also to control cellulase gene expression in T. reesei. Whereas Δtam1 cultivated with peptone as a nitrogen source did not exhibit a growth defect that was observed on ammonium, it was still significantly compromised in cellulase biosynthesis. The absence of TAM1 almost fully abrogated the rapid cellulase gene induction in a resting-cell-inducing system. Overexpression of gdh1 encoding the key ammonium assimilatory enzyme in Δtam1 rescued the growth defect on ammonium but not the defect in cellulase gene expression. Of note, mutation of the Zn(II)2Cys6 DNA-binding motif of TAM1 hardly affected cellulase gene expression, while a truncated ARE1 mutant lacking the C-terminal 12 amino acids that are required for the interaction with TAM1 interfered with cellulase biosynthesis. The defect in cellulase induction of Δtam1 was rescued by overexpression of the key transactivator for cellulase gene, XYR1. Our results thus identify a nitrogen metabolism regulator as a new modulator participating in the regulation of induced cellulase gene expression. IMPORTANCE Transcriptional regulators are able to integrate extracellular nutrient signals and exert a combinatorial control over various metabolic genes. A plethora of such factors therefore constitute a complex regulatory network ensuring rapid and accurate cellular response to acquire and utilize nutrients. Despite the in-depth mechanistic studies of functions of the Zn(II)2Cys6-type transcriptional regulator TamA and its orthologues in nitrogen utilization, their involvement in additional physiological processes remains unknown. In this study, we demonstrated that TAM1 exerts a dual regulatory role in mediating ammonium utilization and induced cellulase production in the well known cellulolytic fungus Trichoderma reesei, suggesting a potentially converged regulatory node between nitrogen utilization and cellulase biosynthesis. This study not only contributes to unveiling the intricate regulatory network underlying cellulase gene expression in cellulolytic fungus but also helps expand our knowledge of fungal strategies to achieve efficient and coordinated nutrient acquisition for rapid propagation.

Interdependent recruitment of CYC8/TUP1 and the transcriptional activator XYR1 at target promoters is required for induced cellulase gene expression in Trichoderma reesei.

Cellulase production in filamentous fungus Trichoderma reesei is highly responsive to various environmental cues involving multiple positive and negative regulators. XYR1 (Xylanase regulator 1) has been identified as the key transcriptional activator of cellulase gene expression in T. reesei. However, the precise mechanism by which XYR1 achieves transcriptional activation of cellulase genes is still not fully understood. Here, we identified the TrCYC8/TUP1 complex as a novel coactivator for XYR1 in T. reesei. CYC8/TUP1 is the first identified transcriptional corepressor complex mediating repression of diverse genes in Saccharomyces cerevisiae. Knockdown of Trcyc8 or Trtup1 resulted in markedly impaired cellulase gene expression in T. reesei. We found that TrCYC8/TUP1 was recruited to cellulase gene promoters upon cellulose induction and this recruitment is dependent on XYR1. We further observed that repressed Trtup1 or Trcyc8 expression caused a strong defect in XYR1 occupancy and loss of histone H4 at cellulase gene promoters. The defects in XYR1 binding and transcriptional activation of target genes in Trtup1 or Trcyc8 repressed cells could not be overcome by XYR1 overexpression. Our results reveal a novel coactivator function for TrCYC8/TUP1 at the level of activator binding, and suggest a mechanism in which interdependent recruitment of XYR1 and TrCYC8/TUP1 to cellulase gene promoters represents an important regulatory circuit in ensuring the induced cellulase gene expression. These findings thus contribute to unveiling the intricate regulatory mechanism underlying XYR1-mediated cellulase gene activation and also provide an important clue that will help further improve cellulase production by T. reesei.

Dual Regulatory Role of Chromatin Remodeler ISW1 in Coordinating Cellulase and Secondary Metabolite Biosynthesis in Trichoderma reesei.

The saprophytic filamentous fungus Trichoderma reesei represents one of the most prolific cellulase producers isolated from nature. T. reesei also produces a typical yellow pigment identified as sorbicillinoids during cultivation. Here, we identified an evolutionarily conserved histone remodeling factor, ISW1, in T. reesei that simultaneously participates in regulating cellulase and the yellow pigment biosynthesis. Trisw1 deletion almost abolished vegetable growth, asexual spore formation, and cellulase gene expression. However, its absence significantly enhanced the production of the yellow pigment. The observed dual regulatory role of TrISW1 was dependent on its ATPase activity. We demonstrated that Trisw1 disruption elevated the transcription of ypr1 coding for the transcriptional activator of sor genes encoding the polyketide synthases catalyzing the biosynthesis of sorbicillinoids but compromised that of xyr1 encoding the key transcriptional activator of cellulase genes. Discrete T. reesei homologous ISW1 accessory factors were also found to exert differential effects on the expression of these two types of genes. Further analyses showed that TrISW1 was recruited to cellulase gene promoters, and its absence interfered with loss of histone H4 at the cbh1 and eg1 promoters upon cellulose induction. To the contrary, Trisw1 deletion facilitated loss of H4 at the sor locus. These data indicate that TrISW1 represents an important chromatin remodeler with a dual role in coordinating the cellulolytic response and biosynthesis of the major secondary metabolite in T. reesei. IMPORTANCE Microorganisms, including Trichoderma reesei, constantly face the challenge to outcompete other species to ensure efficient colonization in their natural habitat. They achieve this usually by adopting two alternative strategies by either maintaining fast growth on limited nutrient resources or producing a versatile array of secondary metabolites to fight against competitors. These two strategies, however, have to be subtly controlled to balance the assignment of and thus make the best use of cellular resources. Here, we identified a chromatin remodeling factor, TrISW1, with a dual role in coordinating the cellulolytic response and biosynthesis of the major secondary metabolite in T. reesei. The data also provide a novel insight into how T. reesei takes advantage of a chromatin remodeler to exquisitely balance two different adaptive strategies to ensure an efficient allocation of cellular resources to achieve efficient colonization in a specific environment.

Engineering Trichoderma reesei for Hyperproduction of Cellulases on Glucose to Efficiently Saccharify Pretreated Corncobs.

The filamentous fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is widely used as a cellulase producer in the industry. Herein, we describe the rational engineering of the publicly available T. reesei QM9414 strain to achieve a remarkable high-level production of cellulase on glucose. Overexpression of the key cellulase regulator XYR1 by the copper-repressible promoter Ptcu1 was first implemented to achieve a full cellulase production in the context of catabolite repression (CCR) while eliminating the requirement of inducing sugars for enzyme production. The T. reesei bgl1 gene was further overexpressed to compensate for its low ß-glucosidase activity on glucose. This overexpression resulted in a 102% increase in FPase activity compared with the CCR-released RUT-C30 strain cultured on Avicel. Moreover, the saccharification efficiency toward pretreated corncob residues by crude enzymes from the engineered strain on glucose increased by 85% compared with that treated by enzymes from RUT-C30 cultivated on Avicel. The engineered T. reesei strain thus shows great potential as a viable alternative to deliver commercial cellulases after further optimization for efficient saccharification of agricultural waste.

Trichoderma reesei XYR1 activates cellulase gene expression via interaction with the Mediator subunit TrGAL11 to recruit RNA polymerase II.

The ascomycete Trichoderma reesei is a highly prolific cellulase producer. While XYR1 (Xylanase regulator 1) has been firmly established to be the master activator of cellulase gene expression in T. reesei, its precise transcriptional activation mechanism remains poorly understood. In the present study, TrGAL11, a component of the Mediator tail module, was identified as a putative interacting partner of XYR1. Deletion of Trgal11 markedly impaired the induced expression of most (hemi)cellulase genes, but not that of the major ß-glucosidase encoding genes. This differential involvement of TrGAL11 in the full induction of cellulase genes was reflected by the RNA polymerase II (Pol II) recruitment on their core promoters, indicating that TrGAL11 was required for the efficient transcriptional initiation of the majority of cellulase genes. In addition, we found that TrGAL11 recruitment to cellulase gene promoters largely occurred in an XYR1-dependent manner. Although xyr1 expression was significantly tuned down without TrGAL11, the binding of XYR1 to cellulase gene promoters did not entail TrGAL11. These results indicate that TrGAL11 represents a direct in vivo target of XYR1 and may play a critical role in contributing to Mediator and the following RNA Pol II recruitment to ensure the induced cellulase gene expression.

CLP1, a Novel Plant Homeo Domain Protein, Participates in Regulating Cellulase Gene Expression in the Filamentous Fungus Trichoderma reesei.

The stringent regulatory network of cellulase gene expression in the filamentous fungus Trichoderma reesei involves multiple transcriptional regulators. However, identification and mechanistic investigation of these regulators are still insufficient. Here, we identified a novel transcriptional regulator, CLP1, a plant homeo domain (PHD) Protein that participates in regulating T. reesei cellulase gene expression. Phylogenetic analyses demonstrated that CLP1 homologs are widely distributed in filamentous fungi including Trichoderma, Penicillium, Fusarium, Neurospora, and Aspergillus species. We demonstrated that CLP1 is a nuclear protein and lack of CLP1 significantly impaired the induced expression of cellulase genes. ChIP experiments showed CLP1 binding to the cellulase gene promoters specifically under cellulose conditions and compromised XYR1 occupancy on the same promoters in the absence of CLP1 at the early induction stage. XYR1 overexpression fully rescued the defect in cellulase production but not the defect in conidia formation in the clp1 null mutant. Further analysis showed that the PHD is required for the CLP1 appropriate subcellular localization as well as the induced cellulase gene expression and conidiation. Taken together, these data demonstrated an important role of CLP1 in the regulation of cellulase and xylanase gene expression in T. reesei.

Trichoderma reesei XYR1 recruits SWI/SNF to facilitate cellulase gene expression.

Cellulase gene expression in Trichoderma reesei is highly responsive to environmental cues and is under stringent regulation by multiple transcription factors. XYR1 (Xylanase regulator 1) has been identified as the most important transcriptional activator of cellulase/hemicellulase gene expression although the precise transactivating mechanism remains largely elusive. Here we show that the activation domain of XYR1 interacts with the T. reesei homolog of the TrSNF12 subunit of SWI/SNF complex. Deletion of Trsnf12 markedly impaired the induced cellulase gene expression. Individual loss of other SWI/SNF subunits including the catalytic subunit also severely compromised cellulase gene expression and interfered with loss of histone H4 in the cbh1 and eg1 promoters upon cellulose induction. In addition, we find that the SWI/SNF occupancy on cellulase gene promoters strictly required XYR1 and TrSNF12 but TrSNF12 was dispensable for the XYR1 binding to these promoters. These data suggest a model in which XYR1 recruits SWI/SNF through direct interactions with TrSNF12 to remodel chromatin at cellulase gene promoters, thereby activating cellulase gene expression to initiate the cellulolytic response in T. reesei.

A novel transcriptional regulator RXE1 modulates the essential transactivator XYR1 and cellulase gene expression in Trichoderma reesei.

XYR1 is the key transcription activator for cellulase gene expression in the model filamentous fungus Trichoderma reesei, which is widely applied in the industry due to its excellent capability of secreting a large quantity of cellulases. Despite the essential role of XYR1, the regulation of its expression in T. reesei cellulolytic response is poorly understood. In this study, we identified a transcription factor RXE1 exhibiting strong binding activity to the xyr1 promoter using yeast one-hybrid screen. RXE1 homologs exist in quite a few filamentous fungi but none of them have been assessed regarding their functional involvement in plant cell wall degradation. Knockdown of rxe1 in T. reesei using a copper-mediated RNAi system not only abrogated conidiation, but also remarkably compromised xyr1 and cellulase gene expression. The defective cellulase but not conidia production in the rxe1-knockdown strain was fully rescued by the constitutive expression of XYR1. Our study thus identified a novel transcriptional regulator controlling xyr1 and cellulase gene expression, which will contribute to elaborating the intricate network of cellulase gene regulation in T. reesei.

A copper-controlled RNA interference system for reversible silencing of target genes in Trichoderma reesei.

BACKGROUND: Trichoderma reesei is a primary lignocellulosic enzyme producer in industry. However, the mechanisms underlying cellulase synthesis as well as other physiological processes are insufficiently understood partly due to the sophisticated process for its genetic manipulation. Target gene knockdown by RNA interference (RNAi) is a powerful tool for genetic research and biotechnology in eukaryotes including filamentous fungi. Previously reported RNAi system in T. reesei was either uncontrollable or only applicable in certain nutrition state. RESULTS: In the present study, we incorporated the copper-responsive tcu1 promoter into an RNAi-mediated silencing system to develop a controllable RNAi-mediated silencing system in T. reesei. As the proof-of-concept, a prototrophic pyr4 gene, highly expressed cel7a and xyr1 genes induced by Avicel and a fab1 gene, whose knockout has proved to be intractable, were successfully knocked down in the absence of copper when the respective RNAi fragment was expressed. Importantly, the phenotype of RNAi strains was shown to be reversed easily to mimic the complementation for excluding any unwanted effects resulted from the random integration of the hpRNA cassette by adding copper in the media. Thus, this controllable RNAi-mediated silencing system can be turned on and turned off only depending on the absence and presence of copper ions in the media, respectively, and not on the nutritional states. CONCLUSIONS: The copper-controlled RNA interference system represents an effective tool for reversible silencing of target genes in T. reesei. This reported strategy to conditionally knock down or turn off genes will contribute to our understanding of T. reesei gene functions, especially those that are difficult to be knocked out due to various reasons. In addition, this simple and cost-effective method holds great potential for the application in synthetic biology and genetic engineering of T. reesei.

The mating type locus protein MAT1-2-1 of Trichoderma reesei interacts with Xyr1 and regulates cellulase gene expression in response to light.

Cellulase production in the model cellulolytic fungus Trichoderma reesei is subject to a variety of environmental and physiological conditions involving an intricate regulatory network with multiple transcription factors. Here, we identified the mating type locus protein MAT1-2-1 as an interacting partner for the key transcriptional activator Xyr1 of T. reesei cellulase genes. Yeast two-hybrid and GST pulldown analyses revealed that MAT1-2-1 directly interacted with the putative transcription activation domain (AD, 767~940 aa) and the middle homology region (MHR2, 314~632 aa) of Xyr1. Disruption of the mat1-2-1 gene compromised the induced expression of cellulase genes with Avicel in response to light or with lactose. Chromatin immunoprecipitation (ChIP) demonstrated that MAT1-2-1 was recruited to the cbh1 (cellobiohydrolase 1-encoding) gene promoter in a Xyr1-dependent manner. These results strongly support an important role of MAT1-2-1 as a physiological cofactor of Xyr1, and suggest that MAT1-2-1 represents another regulatory node that integrates the light response with carbon source signaling to fine tune cellulase gene transcription.

Rce1, a novel transcriptional repressor, regulates cellulase gene expression by antagonizing the transactivator Xyr1 in Trichoderma reesei.

Cellulase gene expression in the model cellulolytic fungus Trichoderma reesei is supposed to be controlled by an intricate regulatory network involving multiple transcription factors. Here, we identified a novel transcriptional repressor of cellulase gene expression, Rce1. Disruption of the rce1 gene not only facilitated the induced expression of cellulase genes but also led to a significant delay in terminating the induction process. However, Rce1 did not participate in Cre1-mediated catabolite repression. Electrophoretic mobility shift (EMSA) and DNase I footprinting assays in combination with chromatin immunoprecipitation (ChIP) demonstrated that Rce1 could bind directly to a cbh1 (cellobiohydrolase 1-encoding) gene promoter region containing a cluster of Xyr1 binding sites. Furthermore, competitive binding assays revealed that Rce1 antagonized Xyr1 from binding to the cbh1 promoter. These results indicate that intricate interactions exist between a variety of transcription factors to ensure tight and energy-efficient regulation of cellulase gene expression in T. reesei. This study also provides important clues regarding increased cellulase production in T. reesei.

A copper-responsive promoter replacement system to investigate gene functions in Trichoderma reesei: a case study in characterizing SAGA genes.

Trichoderma reesei represents an important workhorse for industrial production of cellulases as well as other proteins. The molecular mechanism underlying the regulation of cellulase production as well as other physiological processes in T. reesei is still insufficiently understood. We constructed a P tcu1 -based promoter substitution cassette that allowed one-step replacement of the endogenous promoter for controlling the target gene expression with copper. We then showed that copper repression of the histone acetyltransferase gene gcn5 phenocopied the gcn5 deletion strain. Using the same strategy, we further characterized the function of another putative Spt-Ada-Gcn5 acetyltransferase (SAGA) complex subunit encoding gene, ada2, in T. reesei. Similar to the repression of gcn5, the addition of copper to the P tcu1 -ada2 strain not only drastically reduced the vegetative growth and conidiation in T. reesei but also severely compromised the induced cellulase gene expression. The developed strategy will thus be potentially useful to probe the biological function of the large fraction of T. reesei genes with unknown functions including those essential genes in the genome to expand its extraordinary biotechnological potential.

Characterization of a copper responsive promoter and its mediated overexpression of the xylanase regulator 1 results in an induction-independent production of cellulases in Trichoderma reesei.

BACKGROUND: Trichoderma reesei represents an important workhorse for industrial production of cellulases as well as other proteins. The large-scale production is usually performed in a substrate-inducing manner achieved by a fine-tuned cooperation of a suite of transcription factors. Their production and subsequent analysis are, however, often either difficult to manipulate or complicated by the concomitant production of other inducible proteins. Alternatives to control gene expression independent of the nutritional state are thus preferred in some cases to facilitate not only biochemical studies of proteins but also genetic engineering of the producer. RESULTS: We identified a copper transporter encoding gene tcu1 (jgi:Trire2:52315) in T. reesei, the transcription of which was highly responsive to copper availability. Whereas excess copper repressed the expression of tcu1 from T. reesei, eliminating copper addition in the medium resulted in a high-level transcription of tcu1. The usefulness of the system was further illustrated by the high-level expression of specific cellulases driven by the tcu1 promoter in T. reesei when cultivated on D-glucose or glycerol as the sole carbon source. A recombinant T. reesei strain, which overexpressed the main transcription activator of hydrolases (xylanase regulator 1) under the control of tcu1 promoter, was found to be relieved from the carbon catabolite repression and thus displayed a constitutive cellulase expression. Moreover, the amount and activities of cellulases produced by this strain on glycerol or glucose fully recapitulated those of the parental strain produced on Avicel. CONCLUSION: Expression of T. reesei tcu1 gene was tightly controlled by copper availability, and a homologous protein expression system was developed based on this promoter. Deregulation of XYR1 (xylanase regulator 1) mediated by the tcu1 promoter not only overcame the carbon catabolite repression of cellulases but also resulted in their full expression even on the non-inducing carbon sources.