Functional Analysis Of Transcription Factors And The Involvement Of Chromatin Modification In The Transcription Regulation Of Cellulase Genes In Trichoderma Reesei

Filamentous fungi are the dominant microorganisms in nature that exert the ability of degrading cellulose. Trichoderma reesei (teleomorph Hypocrea jecorina) is one of the most prolific cellulase producers in industry and an organism to look into the transcriptional regulation of cellulase genes, partly due to the fact that this strain can produce three kinds of cellulase including endoglucanase, exoglucanase and β-glucosidase, among which the CBH1account for approximately60%. Research results indicated that cbhl gene was strictly regulated by carbon source. The expression level of CBH1was1000-fold higher in the presence of cellulose than that from glycerol. Moreover, the cbhl gene was completely inhibited when glucose was used as sole carbon source. However, the little was known about the detailed mechanism of transcriptional regulation of these genes.The binding between RNApol Ⅱ and target site within promoter, which effectively initiated the gene transcription, was usually influenced by the compactness of the nucleosomes, which consisted of the chromosome in eukaryotic cells. As a result, gene transcription in eukaryotic cells required not only the interaction between transcriptional factors and their corresponding target nucleotide sequences but also the reconstitution, alteration and modification of chromatin, which generally comprised the histone acetylation as well as the formation of ATP-dependent reconstitution complex. As a filamentous fungus, the transcriptional regulation of cellulase genes in T. reesei should also follow this manner. Although it is reported that histone location and removal was involved in the transcription of cbh2, the detailed mechanism of the chromatin modification was largely unknown. To this point, this thesis focused on the function and mechanism of histone acetylation as well chromatin reconstitution in the transcription of cellulase genes.1. T. reesei homologue of S. cerevisiae histone acetyltransferase protein Gcn5was identified by using biochemical and genetic method. The results proved that TrGcn5 played a critical role in filamentous growth, morphogenesis and transcriptional activation of specific genes including cellulase encoding genes.2. The gene expression differentiation was evaluated by transcriptome analysis between wild-type and△gcn5strains. Several genes including those respond to cellulase inducing signals, those involved in metabolic pathway and signal transduction were identified. These results supplied us with more details about the transcriptional regulation of cellulase in T. reesei.3. Chromatin immunoprecipitation was optimized in T. reesei by investigating the factors and procedure that may affect the validated results. Histone acetylation of the cellulase gene promoters were studied with the improved protocol.4. It is proved that the level of histone acetylation is the key factor facilitating cellulase genes transcription. The level of histone acetylation on xyrl promoter was affected by histone acetyltransferase protein Gcn5, and then affected the transcription of xyrl gene, which further regulated the cellulase gene transcription.5. The interaction between transcriptional regulator Xyrl and promoter of cellulase genes were studied by yeast-one-hybridization.