The MYST Domain Histone Acetyltransferase TIP60 Interacts with and Is as a Coactivator of the Myeloid Transcription Factor C/EBPα.

Recently, we and others have shown that protein-protein interactions play an important role in the pathogenesis of leukemia, and that the transcription factor C/EBPα is a key player in granulopoiesis and leukemogenesis. In the present study, we sought to identify C/EBPα interacting proteins. A glutathione-S-transferase-C/EBPα fusion protein was used to pull down interacting proteins from U937 nuclear extracts. These proteins were analyzed by 2-D gel electrophoresis, 1-D nano LC and identified by MALDI or MALDI-TOF/TOF. Several novel C/EBPα interactors were identified including known proteins like Rb, hnRNP and E2F4. Two novel interactions, one of cell cycle regulator protein MCM5 and the other with the MYST domain histone acetyl transferase TIP60 with C/EBPα were further confirmed by using pull-down and co-immunoprecipitation experiments. TP60 was able to markedly enhance C/EBPα mediated transcription in reporter gene assays, suggesting that TIP60 is a co-activator of C/EBPα. This co-activator function of TIP60 was dependent on an intact histone acetyl transferase domain and on the C/EBPα DNA binding domain. TIP60 was found to be associated with the human C/EBPα promoter in-vivo in a chromatin immunoprecipitation assay with a concomitant increase in histone H3 and H4 acetylation. Furthermore, we observed a lower expression of TIP60 mRNA in U937 CD11b⁻ compared to retinoic acid induced U937 CD11b⁺ cells suggesting that higher TIP60 expression is associated with myeloid differentiation. There was also a marked correlation between the expression levels of TIP60 and C/EBPa in normal bone marrow, chronic myeloid leukemia and acute myeloid leukemia samples with t(8;21), inv(16) and t(15;17). This finding further confirms the functional synergism between C/EBPα and TIP60 and suggests that TIP60 might be an important player in leukemogenesis.