Identification of proteins and/or protein complexes that modulate gene expression in Neurospora crassa under heat stress

• Grant number: 12/22993-9
• Support type: Scholarships abroad - Research Internship - Post-doctor
• Effective date (Start): April 01, 2013
• Effective date (End): June 30, 2013
• Field of knowledge: Biological Sciences - Biochemistry - Biochemistry of Microorganisms
• Principal researcher: Maria Celia Bertolini
• Grantee: Fernanda Zanolli Freitas
• Supervisor abroad: N. Louise Glass
• Home Institution: Instituto de Química (IQ). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil
• Research place: University of California, Berkeley (UC Berkeley), United States

Abstract

Eukaryotic gene expression control is determined by a complex transcriptional regulatory network, which can be described as a set of interactions among many different transcription factors and regulatory DNA sequences present in different genomes. All these interactions, combined with significant changes occurring simultaneously in chromatin structure, account for the competence in which a given transcription event should occur or not. So, the identification of DNA-protein and protein-protein interactions in vitro becomes essential to detect the same interactions in vivo. Since these interactions can result in chromatin remodeling in regulatory regions, identify them and assess their contributions to the dynamics of chromatin in vivo greatly assists in understanding the transcriptional regulatory code. The aim of this project is to identify protein complexes involved in the modulation of glycogen metabolism in Neurospora crassa during heat stress. Glycogen is the major carbohydrate in different kinds of cells. Its metabolism is highly conserved in eukaryotes, and glycogen synthase is the rate-limiting enzyme of the carbohydrate synthesis since it responds for elongation of molecule. In N. crassa, glycogen synthase enzyme is encoded by ORF NCU06687 (gsn gene). Previous studies have shown that gsn gene expression is downregulated under heat shock (45 °C) and that it is due the presence of STRE DNA regulatory elements at gsn promoter region. Biochemical techniques coupled to mass spectrometry allowed us to identify nuclear proteins able to bind to STRE elements present in the 5'-flanking region of the gene gsn (NCU03482 products of ORFs, 06679 and 02671). Although these proteins still have unknown functions, in silico analysis of their polypeptide sequences returned their functional orthologs in other organisms. The product of the ORF NCU03482 showed high identity with the human RUVBL1 protein, while the protein encoded by the ORF NCU06679 has high identity with the human RBB4 and Drosophila NURF55 proteins. Both proteins structures were solved by molecular modeling, based on their PDB structural orthologs. The ORF NCU02671 is annotated as a gene encoding a cutinase G-box binding protein. The product of this ORF showed two C2H2 Zn-finger DNA binding domains at the C-terminal region. In silico analysis revealed that this protein shares a high identity with the Trichoderma atroviridae transcription factor Seb1 at its DNA binding domain. Seb1 is the functional ortholog of the Saccharomyces cerevisiae transactivators Msn2p/4p, which binds to the STRE motif in T. atroviridae during osmotic stress. Considering that only 40% of the genes in the N. crassa genome corresponding to known proteins and the remainder are unknown proteins, N. crassa becomes a promising model organism for the identification of new proteins with new functions. This project aims to benefit from this fact, seeking for partners of proteins previously identified, that could be part of major protein complexes that controls gsn gene expression under heat stress (AU)