MLL-ENL Inhibits Polycomb Repressive Complex 1 to Achieve Efficient Transformation of Hematopoietic Cells

Abstract 2443

MLL fusion proteins are potent leukemogenic agents that are created by chromosomal translocations. These events join the N-terminus of the histone methyltransferase MLL to a variety of different fusion partners. Some time ago we showed that a subgroup of frequently occurring MLL fusion partners, including ENL, assemble a novel transcriptional elongation complex termed EAP [1,2] (elongation assisting proteins, later also called AEP for AF4-ENL proteins [3] or SEC for super elongation complex [4]). Aberrant recruitment of EAP to targets normally under control of MLL leads to abnormal stimulation of transcriptional elongation through the activity of pTEFb (positive transcription elongation factor b). This is accompanied by enhanced H3K79 methylation catalyzed by the histone methyltransferase DOT1L that also interacts directly with EAP. As a consequence MLL-targets, like the clustered Hox-homeobox genes, are ectopically expressed and contribute to hematopoietic transformation.

Paradoxically, we also observed a persistent copurification of EAP with members of the polycomb repressive complex 1 (PRC1). PRC1 inhibits gene expression and ubiquitinylates H2A. Here we confirm the association of EAP with PRC1 and we show that interaction of ENL and CBX8 (chromobox 8) is necessary for EAP to neutralize the repressive function of PRC1 as prerequisite for efficient Hox expression and transformation.

Mass spectrometry and immunological detection verified that ENL is also present in biochemical preparations of PRC1. A study of the architecture of this interaction demonstrated that a CBX8-ENL dimer bridges the PRC1 core to EAP. Interestingly, an analysis of the binding interfaces revealed that EAP would be able to home in on PRC1 engaged chromatin whereas the reverse was not possible because Dot1l and CBX8 could not bind ENL simultaneously. This also suggested that the activity of these two chromatin modifying activities is mutually exclusive. Indeed CBX8 counteracted MLL-ENL in vivo. Introduction of CBX8 into MLL-ENL expressing cells opposed transformation, reduced H3K79 methylation and induced H2A ubiquitinylation at key targets for MLL fusions like the Hox-locus. Deletion of the ENL interaction domain from CBX8 but preserving its PRC1 dependent functions created a “super-repressor” indicating that EAP recruitment via ENL is able to attenuate PRC1 activity. This seems to be a normal function of EAP that exists independently of the MLL fusion context as overexpression of ENL lead to derepression of polycomb controlled genes also in embryonic stem cells. In agreement with these results an elongation reporter system revealed that EAP can completely offset PRC1 repression but only if direct interaction between ENL and CBX8 is possible.

During hematopoietic differentiation genes involved in the decision between self renewal and differentiation are often controlled by transcriptional elongation. PRC1 has been found to play a role in this setting and obviously MLL fusion proteins have found yet another method to interfere with this process.