Funkcionalna analiza interakcija TET-posredovane oksidacije 5-metilcitozina i PARP-zavisne ADP-ribozilacije u procesu demetilacije DNK
Functional analysis of interactions between TET-mediated oxidation of 5-methylcytosine and PARP-dependent ADP-ribosylation in the process of DNA demethylation
Doctoral thesis (Published version)
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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.
Keywords:TET1; TET2; PARP-1; PAR polymers; PARylation; DNA methylation; DNA demethylation; Cxcl12 gene
Source:Faculty of Biology, University of Belgrade, 2019, 1-127
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