Abstract
The human MLL genes (MLL1 to MLL4) and their Drosophila orthologs, trithorax (trx) and trithorax related (trr), encode proteins capable of methylating histone H3 on lysine 4. MLL1 and MLL2 are most similar to trx, while MLL3 and MLL4 are more closely related to trr. Several MLL genes are mutated in human cancers, but how these proteins regulate cell proliferation is not known. Here we show that trr mutant cells have a growth advantage over their wild-type neighbors and display changes in the levels of multiple proteins that regulate growth and cell division, including Notch, Capicua, and cyclin B. trr mutant clones display markedly reduced levels of H3K4 monomethylation without obvious changes in the levels of H3K4 di- and trimethylation. The trr mutant phenotype resembles that of Utx, which encodes a H3K27 demethylase, consistent with the observation that Trr and Utx are found in the same protein complex. In contrast to the overgrowth displayed by trr mutant tissue, trx clones are underrepresented, express low levels of the antiapoptotic protein Diap1, and exhibit only modest changes in global levels of H3K4 methylation. Thus, in Drosophila eye imaginal discs, Trr, likely functioning together with Utx, restricts tissue growth. In contrast, Trx appears to promote cell survival.
ACKNOWLEDGMENTS
We thank Wei Du, Nick Dyson, Robert Eisenman, Bruce Hay, Alexander Mazo, and Terry Orr-Weaver for antibodies and fly stocks as well as the following stock centers: VDRC, Bloomington Drosophila Stock Center, NIG-FLY, the TRiP at Harvard Medical School (NIH/NIGMS R01-GM084947), and the Drosophila Genome Resource Center at Kyoto. We are especially grateful to Brett Pellock for initiating the FLP/FRT screen of the X chromosome and for providing stocks and encouragement during this project and to current and former members of the Hariharan, Bilder, and Okano laboratories for discussions and advice. We thank Rieko Shimamura for technical support.
I.K.H. is funded by the NIH (R01 GM61672) and a Research Professor Award from the American Cancer Society (120366-RP-11-078-01-DDC). H.K. and H.O. received support from the Japanese Ministry of Education, Science, Sports, Culture and technology (H.K. and H.O.), the Japan Society for the Promotion of Science (H.K.), Keio Gijuku Academic Development Funds (H.K.), Keio University Grant-in-Aid for Encouragement of Young Medical Scientists (H.K.), the Strategic Research Foundation Grant-aided Project for Private Universities from MEXT(H.K.), and the Grant-in-Aid for the G-COE program from MEXT to Keio University (H.K. and H.O.).
H.K. designed and conducted the genetic screen with the assistance of L.C. and identified the mutants. A.N. identified trr as the gene responsible for the mutant phenotype and conducted its molecular characterization. H.K. and A.N. characterized the mutant phenotypes and prepared the figures. Data were interpreted by H.K., A.N., and I.K.H. The manuscript was written by I.K.H., H.K., and A.N. I.K.H. oversaw the work conducted at Berkeley. H.O. oversaw the experiments conducted by H.K. after he moved to Keio University.