The Interaction of MOZ-TIF2 with Histone Chaperon Proteins CAF-1A and Asf1b Alters MOZ Regulated Gene Expression.

Abstract We have identified a MOZ-TIF2 (MT2) fusion gene containing the N-terminal portion of MOZ and the C-terminal portion of TIF2 from a patient with acute leukemia with a chromosome 8 translocation. We report here that MOZ portion of MOZ-TIF2 associates with chromatin assembly factors, CAF1 (chromatin assembly factor 1) and ASF1 (anti-silencing factor 1) in mammalian cells. Both proteins not only bring histones to newly synthesized DNA to create chromatin structure in the replication of chromosomes and DNA damage-repair processes but also contribute to regulation of global gene expression. Using the MOZ portion of MT2 as the bait in the yeast two hybrid system, we found that the MOZ portion interacted with CAF1A and Asf1b. The interactions were further verified with GST-pull down experiments. Interestingly, co-immunoprecipitation with whole cell extracts from HEK 293 cells transiently transfected with GFP fusions of MOZ, MT2, and TIF2 showed that only MOZ strongly co-precipitated with CAF1A while MT2 only weakly co-precipitated. In contrast to CAF1A, MT2 showed a 3-fold stronger binding to Asf1b than wild type MOZ in pull-down experiments using S-tagged Asf1b and EGFP-fusions of MOZ, MT2, and TIF2. Further analysis of the domains within the MOZ portion of MT2 responsible for the interaction of CAF1A and Asf1b with MT2 indicated that the binding of CAF1A predominately depended on the PHD domain of MOZ and amino acids176–327 of CAF1A. The MYST domain of MOZ was responsible for the binding of the MOZ portion of MT2 to Asf1b. To further verify the differential binding of MOZ and MT2 to CAF1A and Asf1b, we observed the co-localization of transiently expressed EGFP-MOZ and EGFP-MT2 with DsRed-CAF1A in HEK 293 and Hela cells. In the merged images the MOZ co-localization with CAF-1A was stronger than the colocalization of MT2 with CAF1A and MT2 colocalization with Asf1b was stronger than MOZ colocalization with Asf1b. The co-localization of MOZ and MT2 with CAF1A with Asf1b was seen both in interphase and metaphase of the cell cycle. During the interphase, the co-localizations appeared with chromatin DNA and during metaphase the co-localizations were separated from chromatin DNA. The later phenomenon was further demonstrated with G2/M phase reagent, nocodozole. These results suggest a differential function of MT2 interacting with two chromatin assembly factors compared to wild-type MOZ. In view of the regulation of global gene expression by CAF1A and Asf1b, we examined the gene expression profile in U937 cells stably expressing MT2. Compared to the expression profile of control cells stably transfected with pcDNA3 vector alone, MT2 caused a > 5-fold change in expression 181 genes (104 genes increasing and 77 genes decreasing expression) (p = 0.05). While overexpression of wild type MOZ also altered gene expression (>5-fold increase in 479 genes and >5-fold decrease in 118 genes) a differential gene expression signature was seen between MOZ and MT2. MT2 altered expression of 57% of the 597 MOZ regulated genes. Included in the genes that were either up or down-regulated by MT2 were genes involved in multiple cell functions such as signal transduction, cell response to stimulus, and development. These results suggest that MT2 fusion may interfere with the function of wild type MOZ in global gene expression during the development of myeloid cells by differential interaction with chromatin chaperon proteins and the altered global gene expression profile could contribute to leukemogenesis.