Anastassiadis

Project Title: The role of chromatin modifications in the efficiency of reprogramming after nuclear transfer and after exposure of differentiated cells to exogenous factors

Reprogramming of a differentiated mammalian cell to an embryonic like pluripotent cell has been achieved by different approaches in vitro albeit with very low efficiencies. Although the molecular mechanisms underlying this process are poorly defined its success is characterized by epigenetic chromatin changes. It is largely unknown to what extent chromatin modifying complexes are involved. Here we propose the development of embryonic stem (ES) cells and mice with tetracycline inducible regulated demethylases specific for the repressive Histone Lysine modifications H3K9me2 and H3K27me3/me2 and inducible RNAi for the demethylases that recognize the active mark H3K4me3. The mice will be crossed to the Oct4-GFP reporter line and different cell types isolated from embryonic or adult tissues will be used for nuclear transfer after inducing the expression of the H3K9me2, H3K7me2/me3 or downregulation of the H3K4me3 demethylases. The efficiency of reprogramming will be scored by the amount of developed blastocysts that turn on the expression of GFP. Molecular analysis of the epigenetic state of ES cells derived from cloned blastocysts will follow. Furthermore we plan the generation of ES cells and mice with conditional targeted alleles of the histone lysine demethylases and histone methyltransferases that can also be used as a source of material for testing reprogramming efficiencies.

Figure Legend: Screening for the efficiency of reprogramming by nuclear transfer or in iPS generation using genetically engineered cells that contain a tetracycline inducible histone demethylase specific for a repressive chromatin mark and a pluripotency specific reporter.