Research in the Crispino lab is focused on investigating the regulatory mechanisms governing normal and malignant blood cell development, with an emphasis on understanding the growth of erythroid cells (red blood cells) and megakaryocytes (platelet-producing cells). In addition, we are greatly interested in learning how changes in normal essential regulatory molecules lead to human blood diseases, including leukemias, myelodysplastic syndromes (MDS) and myeloproliferative diseases (MPD).
GATA-1 in normal and malignant hematopoiesis
Several years ago, we discovered that the zinc finger transcription factor GATA-1 is mutated in essentially all cases of Acute Megakaryoblastic Leukemia (AMKL) in children with Down syndrome (DS). We further found that GATA-1 mutations are also present in a pre-leukemia, named transient myeloproliferative disorder, which is relatively common in infants with Down syndrome. Current studies in the lab are focused on characterizing the role of GATA-1 in normal megakaryocyte development and on investigating how GATA-1 mutations contribute to leukemia. In related experiments, we are also studying the mechanisms by which trisomy 21 promotes the development of AMKL. Our long-term goal is to unravel the mystery of why children with DS are predisposed to leukemia.
To define the mechanisms by which GATA-1 regulates megakaryocyte development and to discover how mutations in GATA1 cause abnormal megakaryocyte development, we analyzed the ability of mutant variants of GATA-1 to rescue development of GATA-1 deficient primary mouse megakaryocytes. We first compared the gene expression profile between wild-type and GATA-1 deficient megakaryocytes using Affymetrix microarrays. Next, we introduced either GATA-1s or a FOG binding mutant (V205G) into GATA-1 deficient megakaryocytes and assessed the effect on differentiation and gene expression. While GATA-1 deficient megakaryocytes failed to undergo terminal differentiation and proliferated excessively in vitro, GATA-1s expressing cells displayed proplatelet formation and other features of terminal maturation, but continued to proliferate aberrantly. In contrast, megakaryocytes that expressed V205G GATA-1 exhibited reduced proliferation, but failed to undergo maturation. Examination of the expression of megakaryocyte-specific genes in the various rescued cells correlated with the observed phenotypic differences. These studies showed that GATA-1 is required for both normal regulation of proliferation and terminal maturation of megakaryocytes, and further, that these functions can be uncoupled by mutations in GATA1. In addition, our experiments suggest that GATA1 mutations may not provide the block in differentiation, but rather the proliferative signal that leads to the development of leukemia. We expect that future studies to identify genes that are not properly regulated by GATA-1s will aid in our understanding of this intriguing leukemia.
Role of the IAP family member Survivin in hematopoiesis
In addition to GATA-1, we are characterizing other genes that contribute to the specification, maturation and terminal differentiation of red blood cells and megakaryocytes. Although erythroid cells and megakaryocytes arise from a common progenitor, their terminal maturation follows very different paths: erythroid cells undergo cell cycle exit and enucleation while megakaryocytes continue to progress through the cell cycle, but skip late stages of mitosis to become polyploid. In our efforts to identify genes that participate in megakaryocyte and erythroid lineage specification, we discovered that survivin, a member of the inhibitor of apoptosis (IAP) family that also plays an essential role in cytokinesis, is differentially expressed during erythroid versus megakaryocyte development. Erythroid cells express survivin throughout their maturation, while megakaryocytes express nearly 4-fold lower levels of survivin mRNA and no detectable protein. To investigate the role of survivin in these lineages, we overexpressed or knocked down survivin from mouse bone marrow cells, and then examined erythroid and megakaryocyte development. These studies revealed that overexpression of survivin antagonized megakaryocyte growth, maturation and polyploidization, but had no effect on erythroid development. This block in megakaryocyte polyploidization was accompanied by increased expression of p21, and decreased expression of megakaryocyte specific genes such as VWF and b1-tubulin. In contrast, a reduction in survivin expression interfered with the formation of erythroid cells, but not megakaryocytes. Finally, consistent with the requirement for survivin in the survival of proliferating cells, survivin-deficient hematopoietic progenitors failed to give rise to either erythroid or megakaryocytic colonies. Taken together these studies show that while survivin expression is essential for megakaryocyte and erythroid progenitors, its down-regulation is required for terminal differentiation of megakaryocytes. Our current work on survivin focuses on using mouse models to more precisely define the its role in normal hematopoiesis, as well as to investigate the consequences of overexpression on blood development.
Publications:
Wechsler, J, Greene, M, McDevitt, MA, Anastasi, J, Karp, JE, Le Beau, MM, and Crispino, JD. (2002) Acquired mutations in GATA1 in the megakaryoblastic leukemia of Down syndrome. Nature Genetics 32:148-152.
Smith, ED, Xu, Y, Tomson, BN, Leung, CG, Fujiwara, Y, Orkin, SH, and Crispino, JD. (2004). More than blood, a novel gene required for mammalian post-implantation development. Mol. Cell Bio 24:1168-1173.
Gurbuxani, S, Vyas, P. and Crispino, JD. (2004) Recent insights into the mechanisms of myeloid leukemogenesis in Down syndrome, Blood 103:399-406.
Taub, JW, Mundschau, G, Ge, Y, Poulik, JM, Qureshi, F, Jensen, T, James, SJ, Matherly, LH, Wechsler, J, and Crispino, JD. (2004) Prenatal origins of GATA1 mutations as an initiating step in the development of megakaryoblastic leukemia in Down syndrome. Blood 104:1588-9.
Muntean, A. and Crispino, JD. (2005) Differential requirements for the activation and FOG-interaction surface of GATA-1 in megakaryocyte gene expression and development. Blood 106:1223-1231.
Gurbuxani, S, Xu, Y, Keerthivasan, K, Wickrema, A, and Crispino, JD. (2005) Differential requirements for Survivin in hematopoietic cell development. PNAS 102: 11480-11485.
Mundschau, G and Crispino JD. (2006) GATA1s goes germline. Nature Genetics 38:741-742.
Muntean, A, Ge, Y, Taub, JW, and Crispino, JD. (2006) Transcription Factor GATA-1 and Down syndrome Leukemogenesis. Leukemia and Lymphoma 47: 986-997.
Xu, Y, Leung, CG, Lee, DC, Kennedy, BK, and Crispino, JD. (2006) MTB, the murine homologue of condensin II subunit CAP-G2 represses transcription and promotes erythroid cell differentiation. Leukemia, 20:1261-1269.
