The t(8;21) chromosomal translocation is the most frequently observed translocation in acute myeloid leukemia (AML), the result of which is the expression of the AML1-ETO (RUNX1-MTG8) fusion protein. AML1- ETO (AE) is composed of the N-terminal, DNA binding domain of the transcriptional regulator, AML1 (RUNX1) fused to the majority of the ETO (MTG8). Mtg8 is a member of a three protein family, including Mtg16 and Mtgr1, which function to mediate transcriptional repression through assembly of corepressor complexes at promoters through the interaction with sequence-specific transcription factors. Traditionally, AE expression is thought to contribute to leukemogenesis through the aberrant localization of the MTG8 transcriptional repressor at RUNX1 target genes required for normal blood development and stem cell self-renewal function. Interestingly, examination of human AML samples revealed that in addition to full length AE, an AE splice variant, termed AML1-ETO9a (AE9a) is detected at low levels. The AE9a splice variant encodes a C-terminal truncation, which in contrast to full length AE, is highly leukemogenic in mouse models. Such observations suggest that the C-terminal region of AE may impede leukemia development. Therefore, we carried out a yeast two-hybrid (Y2H) screen with a C-terminal fragment of Mtg protein to identify novel interacting partners that may account for the anti-leukemogenic function of the Mtg C-terminus. This screen identified novel interactions between Mtg proteins and a number of factors that regulate transcriptional elongation. Importantly, siRNAs directed towards these elongation factors complemented Mtg8-/- phenotypes, functionally linking these genes and suggesting that the regulation of elongation is a major function of MTGs. Moreover, inactivation of Mtg16 in blood cells increased genes that are regulated at the level of elongation to regulate stem cell self- renewal. Based on these preliminary data, we hypothesize that the C-terminal region of AE acts as an "anti- leukemogenic" domain by negatively affecting transcriptional elongation. Removal of this domain releases elongation factors to activate transcription at target genes such as HoxA5 and HoxA7. Our proposal seeks to fully elucidate both the molecular mechanism of MTG association with elongation factors as well as define the functional consequences of those interactions both in the context of "normal" cells as well as in the context of leukemogenesis.
The MTG family of transcriptional corepressors is commonly targeted by chromosomal rearrangement in acute myeloid leukemia (AML);therefore, a fundamental understanding of MTG function has important implications for AML biology. This proposal seeks to define the role of MTGs in the regulation of transcriptional elongation. The elucidation of elongation-related MTG functions could have significant implications for RUNX1-MTG8 (AML1-ETO)-driven leukemogenesis.