Funkcionalna analiza interakcija TET-posredovane oksidacije 5-metilcitozina i PARP-zavisne ADP-ribozilacije u procesu demetilacije DNK

Abstract

Važan aspekt u rasvetljavanju procesa demetilacije DNK predstavlja identifikacija faktora koji regulušu aktivnost TET proteina. Cilj ove doktorske disertacije bio je da se ispita funkcionalna veza između TET-posredovane oksidacije 5mC i PARP-zavisne PARilacije u procesu demetilacije DNK, na globalnom i lokalnom nivou. Rezultati ovog istraživanja ukazuju da PARP-1 interaguje sa TET1 i TET2. Pokazano je i da oba proteina podležu in vitro PARilaciji. PARP-1-zavisna PARilacija TET1 proteina negativno utiče na kinetiku aktivnosti TET1 in vitro. Rezultati in cellulo eksperimenata pokazali su da u odsustvu PARP-1 dolazi do porasta ekspresije Tet1 i Tet2, a da prilikom inhibicije PARilacije i u odsustvu PARP-1 dolazi do pada globalnog nivoa metilacije i porasta hidroksimetilacije DNK. Za analizu lokalnih efekata TET-PARP interakcije izabran je gen za hemokin CXCL12 koji učestvuje u brojnim fiziološkim i patološkim procesima pa je ispitivanje regulacije njegove ekspresije važno za buduće terapijske pristupe. Pokazano je da u odsustvu PARP-1 dolazi do povećane ekspresije kao i do pada u metilaciji Cxcl12. Nakon tretmana aktivatorom/inhibitorom TET aktivnosti, u odsustvu PARP-1, ekpresija Cxcl12 je povećana/smanjena. Prisustvo TET1 i TET2 detektovano je na promotoru Cxcl12 uz povećano regrutovanje TET2 u odsustvu PARP-1. U ovoj disertaciji pokazan je inhibitorni uticaj PARP-1 i PARilacije na aktivnost TET enzima u procesu demetilacije DNK na globalnom nivou, a funkcionalnost lokalnih efekata povezanosti TET i PARP enzima u procesu (de)metilacije ustanovljena je na primeru Cxcl12. Demetilacija DNK nalazi se u osnovi brojnih fizioloških i patoloških stanja, zato rasvetljavanje mehanizama regulacije ovog procesa predstavlja važan korak u potencijalnoj primeni stečenih saznanja u medicini.

Identification of factors that regulate the activity of TET proteins is important for elucidating the process of DNA demethylation. The aim of this doctoral dissertation was to investigate the functional relationship between TET-mediated oxidation of 5mC and PARP-dependent PARylation in the process of DNA demethylation at both global and local levels. This thesis shows that PARP-1 interacts with TET1 and TET2, and that both TET proteins can undergo in vitro PARylation. PARP-1-dependent PARylation of TET1 negatively affected the kinetics of TET1 activity in vitro. The results of in cellulo experiments revealed that the expression of Tet1 and Tet2 increased in the absence of PARP-1, whereas when PARylation was inhibited or in the absence of PARP-1, there was a decrease in the global level of DNA methylation and an increase in DNA hydroxymethylation. The Cxcl12 gene was selected for the analysis of the local effects of TET-PARP interaction since chemokine CXCL12 participates in numerous physiological and pathological processes, and exploring the regulation of expression of this gene is important for potential clinical intervention. Increased expression and decreased methylation of Cxcl12 were observed in the absence of PARP-1. After treatment with an activator or inhibitor of TET activity in the absence of PARP-1, Cxcl12 expression was respectively increased or decreased. The presence of TET1 and TET2 was detected on the Cxcl12 promoter, with enhanced recruitment of TET2 in the absence of PARP-1. This dissertation describes the inhibitory effects of PARP-1 and PARylation on the activity of TET enzymes in the process of DNA demethylation at the global level, and the local effects of TET-PARP interplay on DNA (de)methylation of Cxcl12 gene. DNA demethylation is at the root of numerous physiological and pathological conditions, and elucidating the mechanisms that regulate this process is an important step in the potential application of acquired knowledge in medicine.