Funktionelle und strukturelle Charakterisierung von Interaktionen zwischen nicht-kodierenden RNAs und Polycomb repressive complex 2
Zusammenfassung der Projektergebnisse
Applying the CLIP (cross-linking immunoprecipitation) technique to Polycomb Repressive Complex 2 in order to identify direct interactions between this protein complex and non-coding RNAs proved to be technically extremely challenging. While I was able to perform CLIP experiments with positive control proteins, CLIP using antibodies directed against PRC2 subunits did not yield a specific signal. I therefore generated transgenic cells lines expressing tagged PRC2 subunits. Using these cell lines in CLIP experiments confirmed that a specific signal could not be obtained with PRC2 subunit. A potential explanation is that the interaction site between PRC2 and non-coding RNAs cannot be crosslinked with UV-light, which is a crucial step during the CLIP work-flow. This limitation of CLIP has in the meantime been recognized by the lab developing the technique 6. Pursuing anti-PRC2 CLIP and proposed follow-up experiments was therefore deprioritized in favor of a project which started as a sideproject using the transgenic cell lines I had generated. I was able to use mouse embryonic stem cell lines expressing biotin-tagged PRC2 subunits or 3xFLAG G9A (the latter generated by my co-worker Attila Szanto) to perform quantitative high precision affinity proteomics mass spectrometry experiments in collaboration with Philipp Mertins from the lab of Steven Carr at the Broad Institute. We confirmed a physical interaction between PRC2 and the G9A complex in mESCs and identified several new interaction partners of the two complexes, including two previously uncharacterized zinc finger proteins, ZNF518A and ZNF518B, which interact with both complexes. These newly discovered interactors bind PRC2 and G9A at substoichiometric ratios. Selected interaction partners were individually depleted using shRNAs. I then investigated the resulting impact on 42 different histone modification signatures by global chromatin profiling in collaboration with Jacob Jaffe at the Broad Institute. This combined approach of stoichiometric mapping of interaction partners and global monitoring of histone modifying enzyme activities confirmed well established chromatin repression mechanisms and revealed ZNF518B as a new, strong positive regulator of G9A function. I also confirmed that ZNF518B interacts with the G9A-complex and the two alternative PRC2 methyltransferase subunits, EZH2 and EZH1, in vitro. Our data demonstrate that even an interaction partner which is detected at only substoichiometric levels in the purified complex can have a profound impact on the activity of a chromatin modifying enzyme.
Projektbezogene Publikationen (Auswahl)
- (2013). High-resolution Xist binding maps reveal two-step spreading during X-chromosome inactivation. Nature 504(7480),465-9
Simon, M.D., Pinter, S.F., Fang, R., Sarma, K., Rutenberg-Schoenberg, M., Bowman, S.K., Kesner, B.A., Maier, V.K., Kingston, R.E. and Lee, J.T.
- (2015). Functional Proteomic Analysis of Repressive Histone Methyltransferase Complexes Reveals ZNF518B as a G9A Regulator. Molecular & Cellular Proteomics14(6), 1435-46
Maier, V.K., Taylor, J.E., Feeney, C., Leech, A., Szanto, A., Qiao, J.W., Carr, S.A., Jaffe, J.D., Mertins, P. and Lee, J.T.
(Siehe online unter https://doi.org/10.1074/mcp.M114.044586) - . (2015). Building the Connectivity Map of epigenetics: Chromatin profiling by quantitative targeted mass spectrometry.Methods 72, 57-64
Creech, A.L., Taylor, J.E., Maier, V.K., Wu, X., Feeney, C.M., Udeshi, N.D., Peach, S.E., Boehm. J.S., Lee, J.T., Carr, S.A., Jaffe, J.D.
(Siehe online unter https://doi.org/10.1016/j.ymeth.2014.10.033)
