TY  - GEN
A1  - Swaminathan, Suresh Kumar
AV  - public
TI  - Screening for nuclear reprogramming factors and analysis of DNA demethylation during in vitro myoblasts dfferentiation
Y1  - 2007///
ID  - heidok7496
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/7496/
N2  - The primary objective of this thesis was to identify nuclear reprogramming factors that would convert somatic cells back into pluripotency. Two broad screening strategies using the expression of stem cell marker Oct4 as a molecular read out were employed. i) Expression screening of Xenopus egg cDNA library: Xenopus eggs are totipotent as they have the capacity to give rise to whole organism. Xenopus egg cDNA libraries were screened by overexpression in various cell lines to isolate the genes that would upregulate the Oct4 expression.  ii) Chemical Genomics screen: A library of about 3000 small molecules available at the DKFZ-EMBL chemical genomics core facility was screened in a HEK293 stable cell line that harbours a luciferase reporter under the control of the Oct4 promoter and primary fibroblasts obtained from the mouse harbouring a GFP reporter under the control of the Oct4 promoter. I could not isolate any gene or small molecule that could specifically upregulate the Oct4 expression by the above mentioned screens. The second objective of this thesis concerns epigenetic changes during cellular differentiation. Several reports have indicated that DNA demethylation accompanies cellular differentiation. As a model case when C2C12 myoblasts were induced to differentiate into myotubes, the promoter of the muscle specific transcription factor Myogenin gene is demethylated and thereby facilitating its expression. Our group recently showed that Gadd45alpha mediates active DNA demethylation by a nucleotide excision repair mechanism. I tested if Gadd45 may mediate the demethylation observed during the C2C12 differentiation by knocking down the different isoforms of Gadd45. Gadd45beta knockdown blocked the Myogenin promoter demethylation as analysed by MS-PCR and COBRA assays. Moreover, the Myogenin expression was significantly reduced. In conclusion, Gadd45 is indeed required to relieve gene silencing during the differentiation of myoblasts into myotubes. 
KW  - Cell culture 
KW  -  Pluripotency 
KW  -  demethylation 
KW  -  Differentiation
ER  -