Akademik Veri Yönetim Sistemi
ÇELİK UZUNER S. (Yürütücü)
TÜBİTAK Projesi, Devam Ediyor
Epigenetic modifications occurring
on DNA are methylation, hydroxymethylation, formylation and carboxylation. The
function of DNA methylation on gene expression is known. The changes in DNA
methylation levels are associated with various diseases. In the cancer cases
with altered DNA methylation, demethylating agents are combined within the
therapy. This suggests the importance of accurate detection of DNA methylation.
One of the most advantageous methods to detect the levels of cytosine
modifications is immunofluorescence. This includes specific labelling of cells
with specific antibodies for cytosine modifications and the detection by
fluorescence-based instruments such flow cytometer and fluorescence microscopy.
The most critical step in the protocol is antigen retrieval. Chemicals used in
this step remove proteins around DNA and allow antibodies to target regions of
DNA. Currently, acid has been used for this. We previously improved the
protocol with the addition of trypsin after acid regarding more staining level
of methylation. In this project, pepsin was included in the protocol after
trypsin as pepsin targets different protein contents than trypsin aiming at
improving current method to reveal more accurate detection of modification
levels. For this, four modifications
were evaluated in three cells both quantitatively and morphologically after
different antigen retrieval approaches. The levels were measured by both flow
cytometry and fluorescence microscopy, but nuclear localisations by microscopy
only. Fluorescence microscopy was found to more specifically detect cytosine
modifications after pepsin. But the most effective conditions of pepsin varied
among cells and modifications. Additionally, nuclear compartmentations of
modifications varied. These suggest that cytosine modifications are associated
with different proteins within each cell.
Researches working on epigenetics are strongly recommended to be aware
of 3D structure of chromatin with proteins acid and/or enzyme resistant or
sensitive and perform optimisation for antigen retrieval before main
experiments.