MLL and CREB bind cooperatively to the nuclear coactivator CREB-binding protein.

The interaction of the cyclic AMP response element-binding protein (CREB) with its coactivator, CREB-binding protein (CBP), is one of the best characterized signal-regulated activation domain-coactivator interactions. CREB is a signal-dependent transactivator of the bZIP family which becomes phosphorylated on serine 133 in response to stimuli that result in an increase in intracellular cyclic AMP or Ca2+ (33). CBP, as well as its homologue p300, binds to CREB upon phosphorylation at serine 133 within the kinase-inducible activation domain of CREB (8, 32). CBP recruitment by phosphorylated CREB results in rapid induction of gene expression that is mediated both by direct recruitment of the basal transcription machinery and the intrinsic acetyltransferase activity of CBP (26, 33). A vast number of signal-responsive and developmentally regulated transcription factors interact with different regions of CBP and p300. The CREB-binding (or KIX) domain alone interacts with at least 10 distinct families of transcriptional activators (17). Despite the number of well-established protein interactions involving CBP and p300, the only complex for which structural information is available is the phosphorylated CREB-CBP KIX domain interaction (38, 39). The solution structure of the phosphorylated CREB-CBP KIX complex revealed that the activation domain of CREB undergoes a dramatic random coil-to-helix transition upon binding to the KIX domain (38). Conversely, the α-3 helix of the KIX domain undergoes a more subtle change upon binding to phosphorylated CREB. Two lines of evidence suggest that this binding event may be regulated at a level beyond serine 133 phosphorylation. First, CREB phosphorylation has been noted in cellular contexts which do not result in the recruitment of CBP or the induction of CREB target genes (4), suggesting that an additional event is required for coactivator recruitment. Second, CREB or CBP KIX domain mutants have been described that exhibit enhanced complex formation compared to the wild type in vitro or in vivo (5, 15). These observations suggest the potential for a third component that regulates CREB-KIX domain interaction by binding to either or both members of the CREB-CBP KIX complex. To isolate proteins that interact with the unique interface formed by the CREB-CBP KIX complex, a genetic screen was devised in which phosphorylated CREB and the CBP KIX domain are coexpressed in yeast to serve as “bait” for such proteins. In contrast to two-component interaction screening, this three-component screen resulted in the isolation of fewer, more specific interacting proteins (63). In this study, we analyzed a fragment of the mixed-lineage leukemia (MLL) gene (Mll) isolated in the CREB-CBP three-hybrid screen. The MLL gene was originally identified as a gene frequently disrupted by chromosomal translocations in childhood leukemias as well as in therapy-induced acute myeloid leukemias (14, 18, 57). Translocations into the MLL locus generally result in fusion of the N terminus of the MLL protein with one of many diverse fusion partners. Two MLL fusion proteins, MLL-AF9 and MLL-ENL, have been demonstrated to be leukemogenic in murine knock-in or retroviral transduction systems, respectively (10, 52). From these studies, regions within the N-terminal portion of MLL as well as a specific motif within the fusion partner were shown to be important for oncogenesis (52). However, haploinsufficiency at the MLL locus also appears to contribute to leukemia in the knock-in system (13). The mechanism by which the MLL gene maintains target gene expression patterns is of great interest as a starting point for understanding the altered activity of the leukemogenic fusion proteins. Gene-disruption experiments in the mouse illustrated the functional similarity between MLL and its Drosophila homologue, trithorax (60), most dramatically demonstrated by a posterior shift in Hox gene expression and consequent homeotic transformations of the axial skeleton in Mll heterozygotes (19, 59, 60). Mll, like trithorax, positively regulates Hox genes and counters the repressive effect of chromatin-regulating Polycomb-group proteins such as Bmi-1 (19, 24, 36). Analysis of Mll-null embryos indicates that MLL is not involved in the initial specification of Hox gene expression patterns but is essential for maintaining expression patterns once initiated by other sequence-specific transcription factors (59, 60). The mechanism by which MLL acts on its target genes is incompletely understood, in part due to the limited information available regarding protein complexes in which MLL participates (1, 2, 11, 31, 46). To gain insight into the mechanism by which MLL regulates its target genes, we evaluated the significance of the isolation of MLL in the CREB-CBP three-hybrid screen. We show that MLL interacts directly with CBP as well as the CREB-CBP complex. The MLL-CBP interaction was found to be essential for transcriptional activation by the MLL minimal activation domain. In addition, we demonstrated that interaction of MLL with the CBP KIX domain facilitates binding of phospho-CREB to CBP. This surprising finding leads us to propose that a unique class of target genes may respond synergistically to both CREB and MLL.