The Role of the MLL-HOXA9 Axis in Normal and Malignant Hematopoiesis.

Abstract

The MLL-HOXA9 axis plays a critical role in the regulation of development and hematopoiesis. Chromosome translocations of MLL are closely associated with human leukemia and constitutive activation of HOXA9 is required for the leukemogenesis. Although being studied for decades, important questions remain to be answered in this field. For example, how is the activity of MLL regulated during hematopoiesis? What are the mechanisms of MLL fusion-mediated transformation? And how does HOXA9 regulate gene expression and mediate leukemogenesis? The work described in this dissertation touches every aspect of the above questions. Chapter 2 and Chapter 3 address the first two questions by studying the function of the PHD/Bromo region of MLL. This region is invariably disrupted or deleted in oncogenic MLL fusion proteins and is incompatible to MLL fusion mediated transformation. We found that this region mediates MLL ubiquitination in multiple ways. The Bromodomain recruits ECS(ASB2) E3 ligase complex through interaction with ASB2. ECS(ASB2) mediates ubiquitination and degradation of MLL during hematopoietic differentiation, thus contributing to the down-regulation of MLL target genes, including HOXA9 and MEIS1. On the other hand, the second PHD finger (PHD2) of MLL has intrinsic E3 ligase activity in the presence of the E2 conjugating enzyme CDC34. This activity is conserved in the second PHD finger of MLL4. Further, mutation of PHD2 affects MLL stability and transactivation ability. Notably, the oncogenic MLL fusion proteins do not have the PHD/Bromo region, and are resistant to both degradation pathways. The increased protein stability may contribute to MLL fusion mediated transformation. In Chapter 4, the cooperation between Hoxa9 and its potential cofactors Cebpa and Pu.1 was studied using high throughput sequencing technologies. Analysis of ChIP-seq and RNA-seq data reveals that genome-wide binding of Cebpa and Pu.1 significantly overlaps with the binding of Hoxa9. Co-bound regions are enriched with the enhancer mark H3K4me1, and are enriched for hematopoiesis related pathways. Further, co-binding of Cebpa and Pu.1 associates with Hoxa9 regulated genes but does not predict activation or repression activity of Hoxa9.