Research

Hematopoietic Stem Cell Development and Maintenance: The Role of the "Mixed Lineage Leukemia" Gene in Normal Blood Cell Development, Differentiation and Leukemia

Introduction
One of the most fascinating aspects of the development of multi-cellular organisms is the fidelity with which so many unique cell types are generated during development. For example, hematopoietic stem cells (HSCs) undergo self-renewing divisions in addition to dividing to yield progeny that differentiate into cell types as diverse as red blood cells, platelets, B- and T-lymphocytes and macrophages. The mechanisms that control the balance between self-renewing and differentiating HSCs divisions are a major interest of our group. Since these processes are often deregulated in leukemia, we study both normal and leukemic cells to better understand how proliferation, self-renewal and differentiation are disrupted by leukemia-associated fusion oncogenes.

Current studies focus on the Mixed Lineage Leukemia (MLL) gene. This gene is altered by chromosomal translocation in human leukemia. Chromosomal translocations involving the Mll locus generally encode fusion proteins in which the N-terminus of Mll is fused to one of several C-terminal partners, thus producing chimeric fusion oncoproteins with altered properties relative to their wild-type counterparts.

MLL is Essential During Hematopoietic Development
We have shown that the murine Mll gene is essential for the development of HSCs by several methods, including transplantation of aorta-gonad-mesonephros (AGM) cells into adult recipients, and studying chimeric animals made from Mll-/- embryonic stem cells. Ongoing studies aim to determine the underlying mechanisms and target genes that account for the failure of HSCs to develop in Mll-deficient embryos (Figure 1). We also utilize embryoid body development to model aspects of hematopoietic development in the absence of Mll.

Figure 1. Homozygous Mll mutant embryonic stem (ES) cells contribute to many tissues in chimeric animals, but not to fetal liver hematopoietic populations. See Dev Cell 6(3):437-443, 2004. LacZ (blue staining) in the embryo indicates ES contribution. Fetal liver cells were analyzed by flow cytometry; events in the gated region are linage-negative, c-Kit+ (horizontal) Ly9.1+ (vertical). Ly9.1 marks the ES-derived cells, demonstrating that the Mll-/- ES cells do not contribute to fetal liver primitive hematopoietic populations.

Figure 2. Hox Genes are effectors of MLL in embryo patterning, hematopoiesis, and leukemogenesis. The top panel depicts an MLL protein complex acting on hox a9 and Hox c8 regulatory elements, whereas the lower panels illustrate the processes in which this genetic program is particularly important.

Epigenetic Gene Regulation During Hematopoietic Development
Mll encodes a chromatin-regulatory protein related to the Drosophila protein Trithorax. Both harbor histone (H3 lysine 4) methyltransferase activity within a domain of the C-terminus and participate in large protein complexes with other chromatin-regulatory proteins (Fig. 2). We have developed novel methods allowing us to link predicted protein interaction motifs within MLL to specific functions in vivo including HSC development in the embryo and maintenance of target gene expression in mature hematopoietic cell types. These methods will connect particular protein complexes and activities to cell lineage decisions during the development of the hematopoietic system.

Adult Hematopoiesis also Requires MLL
To determine whether adult HSCs also require MLL and to study MLL-dependent processes in more detail, we generated a conditional knockout model. Using this model, we demonstrated that Mll is required to maintain stem and early progenitor cells in the bone marrow, but is dispensable for homeostasis in lineage-committed cells. By analyzing stem and progenitor dynamics using combinations of in vitro and in vivo studies, we have identified several perturbations that occur rapidly after Mll deletion including ectopic proliferation within the stem cell-enriched populations (Fig. 3). Ongoing studies focus on delineating mechanisms by which MLL regulates proliferation differentially in stem and progenitor cells. We are using this model to identify relevant MLL target genes in HSCs and to understand how MLL integrates into known genetic pathways that control HSC behavior. These studies also lay the groundwork for us to determine the difference between MLL-fusion protein dependent genes versus those genes naturally regulated by MLL during hematopoiesis.

Figure 3. Acute depletion of Mll in HSC-enriched populations results in ectopic cell cycle entry. Proliferation was assessed in vivo by pulsing animals for 12 hours with BrdU then analyzing the DNA (7-AAD) versus BrdU content of lineage-negative, Sca-1 positive c-Kit positive (LSK) cells. In the second pair of panels, the CD48-negative LSK subset was incubated Pyronin Y (to detect RNA) and Hoechst dyes (to detect DNA). This analysis revealed consistently fewer Mll-deficient cells in G0. See Cell Stem Cell 1(3):, 324-337, 2007.