Summary AF4 and ENL family proteins are frequently fused with MLL, and comprise a higher order complex (designated AEP) containing the P-TEFb transcription elongation factor. Here, we show that AEP is normally recruited to MLL-target chromatin to facilitate transcription. By contrast, MLL oncoproteins fused with AEP components constitutively form MLL/AEP hybrid complexes to cause sustained target gene expression, which leads to transformation of hematopoietic progenitors. Furthermore, MLL-AF6, an MLL fusion with a cytoplasmic protein, does not form such hybrid complexes, but nevertheless constitutively recruits AEP to target chromatin via unknown alternative mechanisms. Thus, AEP recruitment is an integral part of both physiological and pathological MLL-dependent transcriptional pathways. Bypass of its normal recruitment mechanisms is the strategy most frequently employed by MLL oncoproteins.

Engineering robust protein production and purification of correctly folded biotherapeutic proteins in cell-based systems is often challenging due to the requirements for maintaining complex cellular networks for cell viability and the need to develop associated downstream processes that reproducibly yield biopharmaceutical products with high product quality. Here, we present an alternative Escherichia coli-based open cell-free synthesis (OCFS) system that is optimized for predictable high-yield protein synthesis and folding at any scale with straightforward downstream purification processes. We describe how the linear scalability of OCFS allows rapid process optimization of parameters affecting extract activation, gene sequence optimization, and redox folding conditions for disulfide bond formation at microliter scales. Efficient and predictable high-level protein production can then be achieved using batch processes in standard bioreactors. We show how a fully bioactive protein produced by OCFS from optimized frozen extract can be purified directly using a streamlined purification process that yields a biologically active cytokine, human granulocyte-macrophage colony-stimulating factor, produced at titers of 700 mg/L in 10 h. These results represent a milestone for in vitro protein synthesis, with potential for the cGMP production of disulfide-bonded biotherapeutic proteins. Biotechnol. Bioeng. 2011; 108:1570–1578. © 2011 Wiley Periodicals, Inc.

The mixed lineage leukemia (MLL) proto-oncogenic protein is a histone-lysine N-methyltransferase that is produced by proteolytic cleavage and self-association of the respective functionally distinct subunits (MLLN and MLLC) to form a holocomplex involved in epigenetic transcriptional regulation. On the basis of studies in Drosophila it has been suggested that the separated subunits might also have distinct functions. In this study, we used a genetically engineered mouse line that lacked MLLC to show that the MLLN–MLLC holocomplex is responsible for MLL functions in various developmental processes. The stability of MLLN is dependent on its intramolecular interaction with MLLC, which is mediated through the first and fourth plant homeodomain (PHD) fingers (PHD1 and PHD4) and the phenylalanine/tyrosine-rich (FYRN) domain of MLLN. Free MLLN is destroyed by a mechanism that targets the FYRN domain, whereas free MLLC is exported to the cytoplasm and degraded by the proteasome. PHD1 is encoded by an alternatively spliced exon that is occasionally deleted in T-cell leukemia, and its absence produces an MLL mutant protein that is deficient for holocomplex formation. Therefore, this should be a loss-of-function mutant allele, suggesting that the known tumor suppression role of MLL may also apply to the T-cell lineage. Our data demonstrate that the dissociated MLL subunits are subjected to distinct degradation pathways and thus not likely to have separate functions unless the degradation mechanisms are inhibited.