CONICET | Buscador de Institutos y Recursos Humanos

Chromatin remodeling at gene promoters plays a critical role in initiation of transcription and the dynamics of chromatin structure are tightly regulated during this event. This process is performed by a large class of ATP-dependent chromatin remodeling complexes. S. cerevisiae PKA is composed by two catalytic subunits encoded by TPK1, TPK2 and TPK3 genes, and two regulatory subunits, encoded by BCY1 gene. Previous results of our group indicate that a negative mechanism of isoform dependent autoregulation directs TPKs and BCY1 gene expression and that TPK1 promoter activity is positively regulated during heat shock. In order to understand the mechanism by which TPK1 promoter is activated by PKA and its relation with heat stress, the regulation of chromatin remodeling by PKA during heat shock was investigated. First, we studied the nucleosome positioning using MNase assay, and three positioned nucleosomes were detected in TPK1 promoter. Dramatic changes in chromatin structure were observed upon stress in wild type strain and in tpk1 w1 BCY1, a strain with a very low PKA activity. The results indicate that the upregulation of TPK1 promoter by a low PKA activity correlates with a remodeling in the promoter chromatin. PKA isoform specificity in remodeling of TPK1 promoter was evaluated assessing: 1-TPK1 promoter activity in strains expressing only one of the three catalytic subunits using β-galactosidase reporter assay; 2-mRNA levels measurement by RT-qPCR and 3- the nucleosome positioning. The results indicate that Tpk2 isoform has the major inhibitor effect on TPK1 promoter. Heat stress response is maintained in all strains. The results also show that yeast strains expressing only Tpk2 or only Tpk3 subunits lost the positioned nucleosome +1 on TPK1 promoter. The recruitment of Tpk1 and Tpk2 catalytic sub-units at TPK1 promoter were assessed by ChIP assays. Our results point to a function for PKA activity in autoregulation of chromatin remodeling of TPK1 gene