The Controlled Regulatory Mechanism Of Heterochromatin Gene Met-8 In Neurospora Crassa

In eukaryotic genomes, the heterochromatins exhibit the roles in regulating chromosome organization, centromere function and telomere protection, such a controlled regulatory mechanism proceeds by the specific heterochromatin factors. Heterochromatin domains consist on a highly repeated DNA sequences and a low density of protein-encoding genes. As an epigenetically regulated structure heterochromatin is categorized by its association with nucleosomes comprising trimethylated-lysine 9 of histone H3 (H3K9me3), which is recognized by heterochromatin protein 1 (HP1). To regulate H3K9 trimethylation, Cullin-RING ubiquitin ligase-CRL4 complex components, Cul4 and DDB1 form a complex with histone methyltransferase DIM-5.The silenced regions are highly condensed structure of heterochromatin as characterized by their transcriptionally repressed state e.g. position-effect variegation (PEV) in Drosophila, in which the expression of a euchromatic gene is compromised when it is displaced near or inside a block of heterochromatin domains. In contrast, the expression of a few genes is known to reliant on heterochromatin, such genes are immune to the silencing effect of heterochromatin. In Neurospora crassa, Methionine synthase (met-8) gene was identified as one with high efficiency expression that requires its proximal 50 kb AT-rich heterochromatin adjacent to the met-8 promoter. The sulfur-containing amino acid methionine is of major importance in the cellular metabolism and is required for production of various metabolites of all living organisms. Methionine is the universal initiator of protein synthesis, and its derivative S-adenosyl methionine (Krebs et al.).This dissertation includes two parts. The first part is on the regulatory mechanism of met-8 gene. We had performed the basic genetic screening of transcription factors and co-activators, HMTs and HDACs which may be involved in met-8 expression. The additional work included here is on the histone modifications which regulate met-8 gene expression.The second part was on the evaluation of met-8 promoter. The promoter is a crucial point for efficient gene expression, as most regulations take place at the transcriptional level. Therefore a wide and constantly increasing range of inducible and constitutive promoters have been applied to produce recombinant proteins as well as this system has been used as tools for studying the molecular regulation of target gene expression. Especially in the filamentous fungus Neurospora crassa, several promoters have been developed for heterologous gene expression, such as qa-2, ccgl, wd genes and so on, but most of them are flexible and not steady strong to regulate protein expression levels in vivo. Here we have constructed the consistent and efficient met-8 promoter in Neurospora and analyzed its expression features with both GFP fusion protein and MYC-Met-8 reporter genes which demonstrated the ability of met-8 promoter for regulatory induction of protein synthesis without stimulating supplements. Further the comparative studies with prevailing qa-2 and ccg-1 promoters proved the capability of met-8 promoter to drive not only the sturdy but also stable expression of the genes under all fluctuating stimulus and time regulators. So this new met-8 promoter is suggested as useful tools for rigid gene expression contrasting with other available promoters. This plasmid therefore offer efficient and regulated expression for both ectopic and endogenous genes, and should be valuable tools for the Neurospora research community.This study uncovers the mechanism of met-8 gene transcription regulation and elucidated its promoter function in gene regulation. Our study provides valuable information to understand Neurospora met-8 gene regulation and may be instructive for related studies in other organisms.