@misc{,
 copyright={Creative Commons Attribution BY 4.0 license},
 howpublished={online},
 school={Mossakowski Medical Research Center PAS},
 abstract={Aging of subcutaneous adipose tissue (SAT) is important factor affecting the rate and course of the aging process. One of the causes of age-related dysfunction of adipose tissue is a change in the functioning of adipose-derived stem cells (ASCs). Epigenetic modifications regulate gene expression and, therefore, are essential for maintaining the normal phenotype of cells. Notably, the epigenome undergoes significant aging-associated changes. The best-known element of epigenetic drift is a change in DNA methylation. TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), which may further undergo active and passive demethylation. Additionally, 5hmC is involved in regulating gene expression and plays an essential role in regulating the process of differentiation of adipose tissue progenitor cells. Data suggest that age-related changes in TET expression or activity and changes in the level and distribution of 5hmC in SAT and ASCs are part of the epigenetic drift. The project aimed to investigate the age-related changes in the expression of TET enzymes and the level of DNA 5-hydroxymethylation in human subcutaneous adipose tissue and ASCs, as well as to study the changes in the distribution of 5hmC (presence of regions differentially hydroxymethylated with age, DHMRs) in ASC DNA using next-generation sequencing. Moreover, an additional goal was to select regions specifically 5-hydroxymethylated in ASCs by comparing the profile of this modification in ASCs and fibroblasts originating from the same donors. The research material was subcutaneous adipose tissue collected from 71 patients aged 18-96 years from areas with limited exposure to sunlight. ASCs were isolated from the youngest and the oldest individuals. The global level of DNA modifications was tested using immunoblots. Expression of TET at the mRNA level was investigated using real-time PCR and the amount of proteins – using immunoblots. 5-hydroxymethylated regions in ASC and fibroblasts DNA originating from five young and five older individuals were obtained using hMeDIP-seq. Differential analysis was performed using the MEDIPS package for the R I observed a significant age-related increase in the global level of DNA hydroxymethylation in SAT DNA but no changes in the level of other modifications tested. The 5hmC level was also higher in ASC DNA isolated from the elderly than the young. Additionally, the 5hmC level was significantly higher in slowly dividing cells. I observed an age-related increase in TET2 mRNA and an increase in the level of TET3 protein. In contrast, in ASCs, the levels of mRNA and TET proteins did not differ between cells of young and elderly donors. I identified about 100 age-related DHMRs. These regions were located preferentially outside the CpG islands. Region within the intron of the RSRP1 gene was hyper-hydroxymethylated in both ASCs and fibroblasts of the elderly compared to cells of young people. RT-PCR analysis showed that the level of its mRNA is significantly lower in ASCs isolated from age-advanced study subjects. There was no such age-related relationship in fibroblasts. I identified 2,520 regions differentiating ASCs from fibroblasts. Among the regions with higher hydroxymethylation in ASCs, there were more CpG island-containing promoters than in the DHMRs with higher hydroxymethylation in fibroblasts. The genes associated with these regions in ASCs were related, among others, to the process of cellular differentiation. The obtained results suggest that 5hmC may play an important role in the epigenetic regulation of ASC functioning and that changes in the level and distribution of 5hmC in SAT and ASCs are part of the age-related epigenetic drift in humans. Most probably, some age-related changes in the level/distribution of 5hmC affect gene expression in ASCs, which may be one of the reasons for the reduced regenerative potential of these cells observed in age-advanced individuals.},
 type={Text},
 URL={http://rcin.org.pl./Content/232885/PDF/Joanna%20Borkowska%20-%20Rozprawa%20doktorska%20(1).pdf},
}