TPK1 PROMOTOR REGULATION BY INOSITOL PHOSPHATES DURING HEAT STRESS IN SACCHAROMYCES CEREVISIAE

ORTOLÁ MARTÍNEZ MARÍA CLARA; GALELLO, FIORELLA; ROSSI, SILVIA

Salta

Congreso; Joint XIV PABMB Congress and LV Annua SAIB Meeting; 2019

Sociedad Argentina de Investigaciones en Bioquímica y Biolog{ia Molecular

In Saccharomyces cerevisiae, Protein Kinase A (PKA) pathway controls several functions and biological process in response to different stimuli. S. cerevisiae PKA is composed by two catalytic subunits encoded by TPK1, TPK2 and TPK3 genes, and two regulatory subunits, encoded by BCY1 gene. The specificity of PKA pathway depends on several factors as substrates specificity and interaction with anchoring proteins (AKAPs). However, transcriptional regulation and expression level of PKA subunits are also events involved in maintaining specificity. In response to environmental variations, cells must respond through changes that occur at transcriptional and post-transcriptional level in order to adapt to the new conditions. We have previously demonstrated that a negative mechanism of isoform dependent autoregulation directs TPKs and BCY1 gene expression and that only TPK1 promoter activity is positively regulated during heat stress. Results from our group showed that TPK1 promoter presents three positioned nucleosomes and upon heat stress they are no longer detectable, according to TPK1 promoter activation. On the other hand, it has been described that inositol phosphates can modulate the activities of several chromatin remodeling complexes. In order to identify additional factors involved in PKA subunits transcriptional regulation regulation, we analyzed TPK1 promoter activity, mRNA levels and nucleosomes positioning in in null mutants strains for the components of the inositol phosphates pathway (plc1, ipk1, ipk2, kcs1) in heat stress. Our results showed that TPK1 is not upregulated and there was not chromatin remodeling during heat shock in null mutant strains for the components of the inositol phosphates pathways. It also has been demonstrated that Plc1p regulates the recruitment of SAGA complex to stress responsive genes promoters in order to facilitate transcriptional initiation. Then, we decided to evaluate the relationship between the inositol phosphates pathway and chromatin remodelers on the expression regulation of TPK1. Using β-galactosidase reporter assay and RT-qPCR in null mutant strains for the SAGA complex, we evaluated TPK1 promoter activity and mRNA level. We demonstrated that TPK1 promoter activity and mRNA levels decreased in gcn5, ada2 and spt20 strains during heat shock. We evaluated the recruitment of SAGA complex to the TPK1 promoter and the role of Plc1p in this event using ChIP assay. Finally, other authors have demonstrated that the inositol phosphate pathway modulates the expression of several stress responsive genes trough the activation of the HDAC Rpd3L. Then we evaluated the participation of HDAC Rpd3L in TPK1 and BCY1 transcriptional regulation during heat stress. Taken together these results strongly suggest that the inositol phosphates pathway is involved in regulation of TPK1 gene expression during heat stress.