Our biochemical purification of AF4 family proteins demonstrates that they normally associate with ENL and the P-TEFb elongation factor in an endogenous complex (AEP) in hematopoietic cells. MLL oncopoteins fused with AEP components (AF4 or ENL family proteins) nucleate formation of MLL/AEP hybrid complexes that constitutively occupy MLL-target chromatin. This aberrant recruitment of AEP components causes sustained activation of MLL target gene transcription and transformation of hematopoietic progenitors. Although the AEP and MLL complexes are normally separate biochemical entities, our studies support a dependent role for the AEP complex in physiologic MLL target gene expression pathways, whose conditional recruitment mechanisms are often bypassed by leukemic MLL fusion proteins.
The AEP complex purified from leukemia cell lines under our experimental conditions contained ENL as an integral component but lacked a number of previously reported ENL-associated proteins, most notably the DOT1L histone methyltransferase (Mueller et al., 2007
). Our domain mapping analyses provide a molecular basis for its absence in that DOT1L and AF4 family proteins use the same binding surface within the AHD of ENL. Because of this physical constraint, DOT1L and AF4 family proteins are incapable of simultaneously associating with the AHD to form an AF4/ENL/DOT1L trimeric complex. Therefore, retention of DOT1L in the AF4 complex previously identified in thymus homogenates (Bitoun et al. 2008) is likely mediated by other proteins (e.g. AF10, RNAPII) but not by ENL/AF9. Our data suggest that an endogenous ENL/DOT1L complex and AEP normally exist as separate entities consistent with previous suggestions that ENL may participate in a mixture of different sub-complexes (Mueller et al., 2007
A role for ENL in multiple sub-complexes raises the issue of which of its molecular interactions is essential for MLL leukemogenesis. This was addressed by assessing the oncogenic potential of MLL fused with the DOT1L catalytic domain, which effectively bypasses ENL. Contrary to a previous report (Okada et al., 2005
), MLL-DOT1L was not sufficient for transactivation of MLL target genes and transformation of myeloid progenitors under our experimental conditions that readout the oncogenic properties of MLL-AF5q31 and MLL-ENL. This indicates that aberrant recruitment of AEP, not DOT1L, plays a primary rate-limiting role in transactivation and transformation by MLL fusion proteins, a conclusion further supported by structure/function analysis of MLL-AF5q31 showing that its CHD, which mediates hetero interactions with AF4 family members, was necessary and sufficient for transformation.
Nevertheless, ChIP analyses by us and others show that H3K79 methylation marks are present at most MLL-AF4-target loci () (Krivtsov et al., 2008
; Guenther et al., 2008
), indicating that there is a strong functional interconnection between AEP and DOT1L. DOT1L-dependent H3K79 methylation is associated with transcribed regions and stimulated by histone H2B K120 mono-ubiquitination (a histone mark accompanied with transcription), but not required for transcription itself (Steger et al. 2008
; McGinty et al. 2008
). This suggests that DOT1L-dependent H3K79 methylation occurs after the traverse of RNAPII and may play roles in the maintenance of transcriptional memory rather than initiating transcription per se. In this context, our studies support dual roles for ENL, which is capable of interacting with AEP or DOT1L through its AHD to sequentially recruit them to the same target chromatin, possibly via its N-terminal YEATS domain that retains a chromatin binding property (Zeisig et al., 2005
Our data demonstrate that AEP co-localizes with wt MLL on target promoters indicative of a role in physiologic as well as oncogenic MLL-dependent transcriptional pathways. Supporting this notion, knockdown of ENL
impaired expression of MLL target genes in MEFs and U937 cells (), and Af9
-deficient mice display homeotic transformations similar to those of Mll
-deficient mice (Collins et al., 2002
). The recruitment of AEP to MLL-target loci appears to be non-constitutive since some MLL-occupied loci do not contain AEP (). Because the presence of the MLL complex does not invariably correlate with occupancy by AEP, other factors/signals yet to be identified are likely required for AEP recruitment. Based on these observations/speculations, we propose a three-step model in which wt MLL first establishes/maintains the transcriptionally poised state (STEP 1), AEP is then recruited to facilitate onset of transcriptional initiation/elongation (STEP 2), which is followed by DOT1L-dependent H3K79 methylation post-transcription (STEP 3) (). In this model, ENL serves a key role in sequential recruitment of AEP and DOT1L, respectively.
To date, up to 50 different proteins have been reported to fuse with MLL in human leukemias. This promiscuity poses a question whether any common trait is shared among the fusion partners. We demonstrate here that AEP recruitment is a downstream event in physiologic MLL-dependent transcriptional pathways and regulated in a context-dependent manner. MLL-AF4 and MLL-ENL family fusions transform myeloid progenitors by constitutively recruiting AEP to MLL-target loci through direct association. Thus, one of the major mechanisms of MLL-dependent transformation is constitutive activation of MLL-dependent transcription by direct recruitment of AEP, which circumvents the regulatory mechanisms that normally control AEP recruitment ().
AEP does not physically interact with MLL-AF6, but nevertheless consistently co-localizes with MLL-AF6 at target chromatin to activate transcription (Figures and ). Although the mechanism of this aberrant AEP recruitment is unknown, it indicates that AEP serves an even broader role in MLL leukemogenesis beyond the subset of fusions with AEP components. Determination of whether this role may extend to other MLL fusion proteins requires further investigation. Nevertheless, our studies show that most of the frequently occurring MLL fusions (e.g. MLL-AF4, MLL-AF9, MLL-ENL, and MLL-AF6) employ a similar strategy for leukemic transformation, in which AEP is constitutively recruited to MLL target genes either directly or indirectly.
A critical role for AEP in MLL-mediated leukemic transformation suggests that it may be an ideal target for molecular therapy of MLL-associated leukemias. In this regard, our results tentatively support the rationale for CDK9 inhibition as a potential therapeutic strategy, or inhibition of DOT1L whose activity appears to be functionally linked to AEP and possibly plays important roles in the maintenance of the epigenetic status of target genes. However, these molecules are likely to have more generalized roles other than AEP-dependent transcription (Jones et al. 2008
; Peterlin and Price 2006
), therefore serious side effects might occur if they are effectively inhibited. Thus, compounds that specifically target the function of AF4- and ENL-family proteins but not P-TEFb or DOT1L may selectively inhibit MLL-dependent transcription and benefit the treatment of MLL-associated leukemias.