The Roles of MLL Holocomplex in Embryogenesis, Hematopoiesis and Tumor Suppression.

Abstract 2969

Poster Board II-945

The roles of MLL holocomplex in embryogenesis, hematopoiesis and tumor suppression MLL is an epigenetic transcriptional regulator that serves critical roles in multiple developmental and homeostatic processes. It is essential for proper maintenance of Hox gene expression during embryogenesis and hematopoiesis, and regulates expression of cyclin-dependent kinase inhibitors (CDKIs) in endocrine tissues such as the pancreas. Conversely, mis-regulation of MLL-dependent transcriptional pathways is associated with various pathologies. Activating mutations of MLL result in constitutive expression of Hox genes leading to acute leukemia, whereas loss of MLL transcriptional function through mutations of menin, an essential MLL-associated cofactor, leads to decreased expression of CDKIs, hyper proliferation of endocrine cells, and development of multiple endocrine neoplasias. Thus, MLL functions in critical growth regulatory transcriptional circuits that are subject to perturbations in various malignancies. MLL is translated as a large precursor protein that subsequently undergoes proteolytic processing into two fragments (MLLN and MLLC) that self-associate through non-covalent interaction to form an intra-molecular complex. MLL is processed by the Taspase I endopeptidase, which specifically cleaves sites that are evolutionally conserved with MLL2 and Drosophila TRX. However the biological requirement for processing remains unknown. MLLN appears to comprise a targeting sub-unit that contains several motifs involved in DNA binding (AT hooks, CXXC domain) and chromatin recognition (PHD fingers, bromo domain). By contrast, MLLC has features of a transcriptional effector sub-unit that possesses a potent transactivation domain and a methyltransferase (SET) domain specific for lysine 4 of histone H3, an epigenetic mark associated with transcriptionally active states. The SET domain also associates with accessory factors (WDR5, RBBP5 and ASH2L) that promote optimal substrate recognition and enzymatic activity. Thus, the MLL intra-molecular complex can be conceptualized as comprised of an MLLC effecter sub-unit tethered to the MLLN targeting sub-unit by non-covalent association. This model has prompted the hypothesis that MLL may serve functionally distinct roles in transcriptional regulation dependent on the conditional association or disassociation of the MLLC subunit. Our biochemical study shows that MLL indeed takes two different forms: an MLLN/MLLC holocomplex and an MLLN complex without MLLC. We examined the roles of the two distinct complexes using a genetic approach employing knock-in mice with targeted mutations of the MLL processing sites. Mice engineered to constitutively express the MLLN subunit without MLLC (thus produce only the MLLN complex) displayed embryonic lethality, hematopoietic defects and transcriptional defects that phenocopy MLL deficiency. By contrast, mutations that disable proteolytic processing, which can assume the MLLN/MLLC holocomplex but not the MLLN complex, had no adverse consequences, demonstrating the lack of an essential role for the MLLN complex. The intra-molecular association of MLLN with MLLC is mediated in part by the PHD1 finger of the MLLN subunit. An MLL mutant lacking PHD1, which mimics deletion mutants associated with human T-cell leukemias, is unable to form an MLLN/MLLC holocomplex. Hence, this oncogenic mutation likely results in loss of function of MLL, suggestive of a potential tumor suppressor role for MLL in the lymphoid lineage similar to that of menin in the endocrine tissues. Our results support the critical roles of the MLLN/MLLC holocomplex in embryogenesis and hematopoiesis and a possible tumor suppressor function in the lymphoid lineage.