| Group: Anton Wutz Agrelo Behere Garay Ruben | Postdoc Idarraga-Amado Maria Helena | Technician Kishimoto Hiroyuki | Postdoc Leeb Martin | PhD Student Ohata Tatsuya | Postdoc Saltik Mediyha | Technician Steffen Philipp | PhD student |
Structure and function of Xist RNA
In mammals X inactivation compensates for the dosage difference of X-linked genes between males and females by silencing of one of the two X chromosomes in female cells. The large non-coding Xist RNA initiates the process of X-inactivation. Xist RNA localises to chromatin in cis of its site of transcription, and spreads over the entire chromosome encompassing it in a coat-like manner. In are interested in the mechanism behind this remarkable localisation pattern. Experiments using tagged Xist transgenes in mouse embryonic stem (ES) cells have shown that Xist RNA interacts with chromatin and influences spatial organization of chromosomal sequences. Once Xist RNA is displaced form the chromosome it is not recycled but degraded. To further characterise the mechanism of Xist RNA localisation we aim at measuring the turnover rate of Xist RNA on the chromosome.
We have focused on at visualisation of Xist RNA in living cells to analyse RNA localisation and dynamics in real time. To this end we have established a live-imaging system based on an RNA-protein interaction system derived from MS2 phage. A 19-nt RNA stem-loop of the MS2 virus genome is bound sequence-specifically by a coat protein. We have adopted this system and constructed an MS2-tagged Xist cDNAs by adding 24 and 64 copies of the MS2-RNA stem-loop sequence at the 3´-end. Mouse embryonic stem cells (ES) cell lines were generated that express the MS2-tagged Xist RNA under the control of a doxycycline inducible promoter from randomly inserted transgenes. Upon doxycycline addition to the culture medium MS2-tagged Xist RNA can be observed by RNA FISH in discrete nuclear clusters similar to endogenous Xist. For visualisation of Xist RNA in living cells we have aimed at stably expressing a green fluorescent version of the RNA binding MS2 capsid protein (MS2-EGFP). In double-transgenic ES cell clones Xist can be analysed using confocal fluorescence microscopy.
Ongoing experiments aim at the characterisation of these cell lines and using the methodology to perform kinetic measurements such as fluorescence recovery after photobleach (FRAP) we will determine the residence time of Xist RNA on the chromosome and possibly gain information on its movement on the chromosome.
We have focused on at visualisation of Xist RNA in living cells to analyse RNA localisation and dynamics in real time. To this end we have established a live-imaging system based on an RNA-protein interaction system derived from MS2 phage. A 19-nt RNA stem-loop of the MS2 virus genome is bound sequence-specifically by a coat protein. We have adopted this system and constructed an MS2-tagged Xist cDNAs by adding 24 and 64 copies of the MS2-RNA stem-loop sequence at the 3´-end. Mouse embryonic stem cells (ES) cell lines were generated that express the MS2-tagged Xist RNA under the control of a doxycycline inducible promoter from randomly inserted transgenes. Upon doxycycline addition to the culture medium MS2-tagged Xist RNA can be observed by RNA FISH in discrete nuclear clusters similar to endogenous Xist. For visualisation of Xist RNA in living cells we have aimed at stably expressing a green fluorescent version of the RNA binding MS2 capsid protein (MS2-EGFP). In double-transgenic ES cell clones Xist can be analysed using confocal fluorescence microscopy.
Ongoing experiments aim at the characterisation of these cell lines and using the methodology to perform kinetic measurements such as fluorescence recovery after photobleach (FRAP) we will determine the residence time of Xist RNA on the chromosome and possibly gain information on its movement on the chromosome.
Selected recent publications
Agrelo, R., Souabni, A., Novatchkova, M., Haslinger, C., Komnenovic, V., Kishimoto, H., Gresh, L., Kohwi-Shigematsu, T., Kenner, L. & Wutz, A. (2009) SATB1 defines the developmental context for gene silencing by Xist in lymphoma and embryonic cells. Dev. Cell, 16(4):507-516.
Leeb, M., Steffen, P.A., Wutz, A. (2009) X chromosome inactivation sparked by non-coding RNAs. [review] RNA Biol 6(2), 94-99.
Mietton, F., Sengupta, A. K., Molla, A., Picchi, G., Barral, S., Heliot, L., Grange, T., Wutz, A., Dimitrov, S. (2009) Weak, but uniform enrichment of the histone variant macroH2A1 along the inactive X chromosome. Mol Cell Biol 29(1):150-156.
Shibata, S., Wutz, A. (2008) Transcript versus transcription? The regulation of X-inactivation by antisense Tsix expression. [review] Epigenetics 3(5):246-9.
Shibata, S., Yokota, T., and Wutz, A. (2008) Synergy of Eed and Tsix in the repression of Xist gene and X-chromosome inactivation. EMBO J 27(13):1816-1826.