Autoregulatory role of the kinase A activity in chromatin remodeling of TPK1 subunit promoter in Saccharomyces Cerevisiae

GALELLO, F; PAUTASSO C; RECA S; OJEDA L; PORTELA P; ROSSI S

Buenos Aires

Congreso; LIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular, Reunión Conjunta de Sociedades de Biociencias; 2017

SAIB

277) AUTOREGULATORY ROLE OF THE KINASE A ACTIVITYIN CHROMATIN REMODELING OF TPK1 SUBUNITPROMOTER IN SACCHAROMYCES CEREVISIAEFiorella Galello, Constanza Pautasso, Sol Reca, Lucas Ojeda,Paula Portela, Silvia RossiDepto Qca Biológica, FCEN, UBA; IQUIBICEN-CONICETChromatin remodeling at gene promoters plays a critical role ininitiation of transcription and the dynamics of chromatin structureare tightly regulated during this event. This process is performed bya large class of ATP-dependent chromatin remodeling complexes.S. cerevisiae PKA is composed by two catalytic subunits encodedby TPK1, TPK2 and TPK3 genes, and two regulatory subunits, encodedby BCY1 gene. Previous results of our group indicate thata negative mechanism of isoform dependent autoregulation directsTPKs and BCY1 gene expression and that TPK1 promoter activityis positively regulated during heat shock. In order to understandthe mechanism by which TPK1 promoter is activated by PKA andits relation with heat stress, the regulation of chromatin remodelingby PKA during heat shock was investigated. First, we studied thenucleosome positioning using MNase assay, and three positionednucleosomes were detected in TPK1 promoter. Dramatic changesin chromatin structure were observed upon stress in wild type strainand in tpk1w1BCY1, a strain with a very low PKA activity. The resultsindicate that the upregulation of TPK1 promoter by a low PKA activitycorrelates with a remodeling in the promoter chromatin. PKAisoform specificity in remodeling of TPK1 promoter was evaluatedassessing: 1-TPK1 promoter activity in strains expressing only oneof the three catalytic subunits using β-galactosidase reporter assay;2-mRNA levels measurement by RT-qPCR and 3- the nucleosomepositioning. The results indicate that Tpk2 isoform has the major inhibitoreffect on TPK1 promoter. Heat stress response is maintainedin all strains. The results also show that yeast strains expressingonly Tpk2 or only Tpk3 subunits lost the positioned nucleosome +1on TPK1 promoter. The recruitment of Tpk1 and Tpk2 catalytic subunitsat TPK1 promoter were assessed by ChIP assays. Our resultspoint to a function for PKA activity in autoregulation of chromatinremodeling of TPK1 geneKeywords: Yeast, chromatin remodeling, transcription