PKA-CHROMATIN ASSOCIATION IN STRESS RESPONSIVE TARGET GENES FROM Saccharomyces cerevisiae

BACCARINI LETICIA; MARTINEZ MONTAÑEZ F; MORENO SILVIA; PROFT MARKUS; PORTELA PAULA

Potrero de los Funes- San Luis

Congreso; 47 Reunion Anual de la SAIB; 2011

SAIB

Regulation of gene expression by intracellular stimulus-activated protein kinases is essential for cell adaptation to environmental changes. There are three PKA catalytic subunits in S.cerevisiae:Tpk1,Tpk2 and Tpk3 and one regulatory subunit:Bcy1. Previously, we had demonstrated that both catalytic and regulatory subunits of PKA were associated to transcribed regions and promoters of target genes in a stress dependent manner. Now, using ChIP-real time assay we analyze the requirement of kinase activity and the role of Bcy1 protein on Tpk chromatin association. Inactive versions of Tpk1 and Tpk2 do not associate to chromatin. Deletion of BCY1 promotes higher Tpk1 association, whereas it abolished Tpk2 association. We then analyse the possible role of Tpk on chromatin remodelling in response to stress conditions. Deletion of Tpk1 or Tpk2 affects the density of H3 on defined gene regions. Additionally, we analyse the kinetics of binding chromatin remodellers in the regions bound by Tpk1 and Tpk2 in response to stress. During stress stimulus there is a transient binding to chromatin of Arp8, Rsc1 and Snf2-chromatin remodellers followed temporarily by Tpk association. We also observe co-occupancy of Tpk2 and DNA-binding transcription factor Rap1p. These results suggest that PKA could participate in gene expression by in situ phosphorylation of transcriptional or chromatin regulators. chromatin regulators. subunit:Bcy1. Previously, we had demonstrated that both catalytic and regulatory subunits of PKA were associated to transcribed regions and promoters of target genes in a stress dependent manner. Now, using ChIP-real time assay we analyze the requirement of kinase activity and the role of Bcy1 protein on Tpk chromatin association. Inactive versions of Tpk1 and Tpk2 do not associate to chromatin. Deletion of BCY1 promotes higher Tpk1 association, whereas it abolished Tpk2 association. We then analyse the possible role of Tpk on chromatin remodelling in response to stress conditions. Deletion of Tpk1 or Tpk2 affects the density of H3 on defined gene regions. Additionally, we analyse the kinetics of binding chromatin remodellers in the regions bound by Tpk1 and Tpk2 in response to stress. During stress stimulus there is a transient binding to chromatin of Arp8, Rsc1 and Snf2-chromatin remodellers followed temporarily by Tpk association. We also observe co-occupancy of Tpk2 and DNA-binding transcription factor Rap1p. These results suggest that PKA could participate in gene expression by in situ phosphorylation of transcriptional or chromatin regulators. chromatin regulators. :Tpk1,Tpk2 and Tpk3 and one regulatory subunit:Bcy1. Previously, we had demonstrated that both catalytic and regulatory subunits of PKA were associated to transcribed regions and promoters of target genes in a stress dependent manner. Now, using ChIP-real time assay we analyze the requirement of kinase activity and the role of Bcy1 protein on Tpk chromatin association. Inactive versions of Tpk1 and Tpk2 do not associate to chromatin. Deletion of BCY1 promotes higher Tpk1 association, whereas it abolished Tpk2 association. We then analyse the possible role of Tpk on chromatin remodelling in response to stress conditions. Deletion of Tpk1 or Tpk2 affects the density of H3 on defined gene regions. Additionally, we analyse the kinetics of binding chromatin remodellers in the regions bound by Tpk1 and Tpk2 in response to stress. During stress stimulus there is a transient binding to chromatin of Arp8, Rsc1 and Snf2-chromatin remodellers followed temporarily by Tpk association. We also observe co-occupancy of Tpk2 and DNA-binding transcription factor Rap1p. These results suggest that PKA could participate in gene expression by in situ phosphorylation of transcriptional or chromatin regulators. chromatin regulators. in situ phosphorylation of transcriptional or chromatin regulators.