Visualisierung von Mechanismen der Geninaktivierung während der X-Chromosominaktivierung in der Maus
Final Report Abstract
In mammals, the expression of X-linked genes between male (XY) and female (XX) individuals is equalized by silencing of one X-chromosome in female cells, a process called X-chromosome inactivation (XCI). XCI has been studied extensively with biochemical and cytogenetical techniques using fixed specimens. However, a dynamic process like XCI requires its observation in realtime for its full understanding. This project aimed to investigate two key events of XCI in living cells, the transient pairing of the X-chromosomes in female ES cell nuclei at the onset of XCI and the subsequent movements of the inactive X. Female ES cells recapitulate XCI upon differentiation and therefore serve as a model system to investigate XCI. I joined the lab of Prof. Jeannie T. Lee at Massachusetts General Hospital in Boston to generate female mouse ES cell lines carrying fluorescently labeled X-chromosomes, funded by a DFG research fellowship. A tandem array of bacterial targeting sequences was integrated into the Xic, a 100 kbspanning region in the X encoding for multiple non-coding RNAs involved in XCI. These DNA sequences bind to a GFP-bound version of their binding protein, which is expressed in the same nucleus. The so-labeled specific site in the ES cell genome can be tracked in living cells which allows its real-time observation. Numerous attempts to integrate a construct containing 256 tandem repeats of the bacterial lac operator sequence, in analogy to a study by Belmont et al., into the Xic of female mouse ES cells failed. This finding is in agreement with a recent study by Heard and co-workers The integration of tandem arrays of the bacterial tet operator (tetO), however, resulted in female mouse ES cell lines carrying either 112 or 224 tetO copies in their Xics. As each copy of the tetO sequence is able to bind two tet repressor (tetR) molecules, expression of an mCherry-fused tetR version in these cells should lead to a labeling of the integration sites with 224 or 448 mCherry molecules, respectively. A targeting construct for mCherry-tetR insertion into the non-essential ROSA26 locus was generated in the lab of Prof. Lee in Boston. The targeting of this construct into the tetO cell lines are currently performed by me in the lab of Prof. Eckhard Wolf at LMU gene center. During my DFG return grant I was mainly concerned with the study of ES cell chromatin movements at different stages of stem cell differentiation.
