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The Super Elongation Complex (SEC), containing transcription elongation activators/coactivators P-TEFb, ELL2, AFF4/1, ENL, and AF9, is recruited by HIV-1 Tat and mixed lineage leukemia (MLL) proteins to activate the expression of HIV-1 and MLL-target genes, respectively. In the absence of Tat and MLL, however, it is unclear how SEC is targeted to RNA polymerase (Pol) II to stimulate elongation in general. Furthermore, although ENL and AF9 can bind the H3K79 methyltransferase Dot1L, it is unclear whether these bindings are required for SEC-mediated transcription. Here, we show that the homologous ENL and AF9 exist in separate SECs with similar but nonidentical functions. ENL/AF9 contacts the scaffolding protein AFF4 that uses separate domains to recruit different subunits into SEC. ENL/AF9 also exists outside SEC when bound to Dot1L, which is found to inhibit SEC function. The YEATS domain of ENL/AF9 targets SEC to Pol II on chromatin through contacting the human Polymerase-Associated Factor complex (PAFc) complex. This finding explains the YEATS domain's dispensability for leukemogenesis when ENL/AF9 is translocated to MLL, whose interactions with PAFc and DNA likely substitute for the PAFc/chromatin-targeting function of the YEATS domain.

The HIV-1 Tat protein stimulates viral gene expression by recruiting human transcription elongation complexes containing P-TEFb, AFF4, ELL2, and ENL or AF9 to the viral promoter, but the molecular organization of these complexes remains unknown. To establish the overall architecture of the HIV-1 Tat elongation complex, we mapped the binding sites that mediate complex assembly in vitro and in vivo. The AFF4 protein emerges as the central scaffold that recruits other factors through direct interactions with short hydrophobic regions along its structurally disordered axis. Direct binding partners CycT1, ELL2, and ENL or AF9 act as bridging components that link this complex to two major elongation factors, P-TEFb and the PAF complex. The unique scaffolding properties of AFF4 allow dynamic and flexible assembly of multiple elongation factors and connect the components not only to each other but also to a larger network of transcriptional regulators.