The Acetyl-Transferase Tip60 Interacts with c-Myb and Inactivates Its Transcriptional Activity in Human Leukemia.

Abstract Abstract 3981 Poster Board III-917 The acetyl-transferaseTip60 (Tat interacting protein, 60 kDa; also known as K(lysine) acetyltransferase 5:KAT5) is a co-regulator of transcription factors and is implicated in tumorigenesis. The protooncogene c-myb encodes a transcription factor, c-Myb, which is essential for normal hematopoisis and promotes hematologic malignancies. In this study, we explored potential regulatory relationships between Tip60 and c-Myb in hematopoietic cells. We first sought to detect any direct physical interactions by performing co-immunoprecitation (co-IP) assays. These revealed that Tip60 associated with c-Myb in Jurkat (T leukemia) and K562 (CML) cells. In vitro translated, epitope-tagged c-Myb and Tip60 also interacted with each other, suggesting that the Tip60, c-Myb interaction did not require an adaptor protein or co-factor. We then sought to define the interacting protein domains. Deletion analysis studies revealed that the interaction between Tip60 and c-Myb was dependent on the Tip60 MYST acetytransferase domain and transactivation domain of c-Myb. We then determined whether Tip60 could acetylate c-Myb, a post-translational event known to modulate c-Myb activity. Interestingly, an in vitro acetyltransferase assay showed that c-Myb was not a substrate of Tip60, even though Tip60 acetylated itself in the same assay. We then examined the effect of Tip60 on the ability of c-Myb to transcriptionally activate known target genes. In HEK293T cells, co-expressing Tip60 with c-Myb decreased c-Myb's ability to activate a luciferase reporter driven by the cim-1 promoter, a verified c-Myb target, by ∼60% compared to activation in the absence of Tip60. The physiologic significance of this observation was then explored. A chromatin immunoprecipiation (ChIP) assay revealed that Tip60 bound to the c-myc promoter, another known c-Myb target gene, in K562 cells. Furthermore, inactivation of endogenous c-Myb in K562 cells stably expressing an inducible c-Myb DNA binding domain reduced the occupancy of Tip60 in the c-myc promoter, suggesting that Tip60 utilizes c-Myb to bind its preferred site in the c-myc promoter. Using c-Myb, Tip60, and appropriate control siRNAs we achieved specific knockdowns of c-Myb, and Tip60 (∼80-90%, and ∼70-80% respectively compared to controls). Consistent with prior reports, c-myc expression decreased ∼60% when c-Myb was targeted, and ∼50% increased when Tip60 was targeted. A mechanistic explanation was sought to explain this finding. Tip60 is represses transcription when associated with histone deacetylases (HDAC), including HDAC1, HDAC2 and HDAC7. Co-IP of Jurkat cell lysates revealed that c-Myb is associated with HDAC1 and HDAC2. Altogether, these data suggest that Tip60 directly associates with c-Myb, and may inhibit its transcriptional activity by recruiting histone deacetylases(HDAC1 and HDAC2) to the activation complex. Finally, we compared Tip60 expression in 6 primary AML samples, with 3 normal CD34+ cell samples using QRT-PCR. Tip60 expression was significantly (∼60%) lower in the AML samples. In summary, these studies demonstrate that Tip60 modulates c-Myb transcriptional activity in human hematopoietic cells leading us to hypothesize that Tip60 is a normal regulator of c-Myb function and that dysregulated or mutated Tip60 may contribute to c-Myb driven leukemogenesis. Disclosures: No relevant conflicts of interest to declare.