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Molecular Genetics of Stem Cells
Summary: Stuart Orkin's research focuses on stem cell biology, particularly the development and function of the blood system, the relationship between cancer and stem cells, and the mechanisms responsible for self-renewal of stem cells.
In this laboratory we study gene regulation as it pertains to the properties and development of stem cells. Stem cells, whether of embryonic or adult origins, give rise to additional stem cells (the process of self-renewal) and generate cells representative of specific lineages (the process of differentiation). Our efforts are directed toward understanding the nature and function of genes that control these processes and how disturbances in gene networks may lead to cancer. In addition to studying adult stem cells or progenitors for the blood system and the heart, we also have investigated the nuclear factors that control self-renewal of mouse embryonic stem (ES) cells. Our goal is to identify basic mechanisms that may be employed by different types of stem cells, and perhaps reveal molecular pathways shared among stem cells.
We have been interested in how nuclear regulatory proteins control formation and function of hematopoietic stem cells. Previously we have identified transcription factors that specify hematopoietic stem cells and individual lineages. These factors are often the targets of somatic mutation or chromosomal translocation in human leukemias. Mutations of the factor GATA-1 are uniquely associated with M7 acute megakaryoblastic leukemia in individuals with Down syndrome. Previously we demonstrated that truncated GATA-1 in this entity (called GATA-1s) perturbs proliferation and cellular maturation of an embryonic/fetal megakaryocytic progenitor. We have proposed that this cell represents the target for transformation in this leukemia. Currently we seek to determine how GATA-1s acts in concert with other proteins to transform these progenitors and how trisomy for human chromosome 21 synergizes with GATA-1s.
In other work we have investigated how other transcription factors, known as the Gfi proteins (Gfi-1 and Gfi-1b), function in myeloid development. Previously we demonstrated that Gfi-1 is required for neutrophil differentiation, whereas Gfi-1b is essential for red blood cell and megakaryocyte development. Recently we have shown that the Gfi proteins recruit a critical chromatin-modifying complex to target genes to repress transcription. In other studies we have evaluated how the retinoblastoma protein Rb, a central regulator of cell division and a tumor suppressor, functions in hematopoiesis. Our findings indicate that Rb is dispensable for the majority of activities of hematopoietic stem cells but is important in the control of blood cell homeostasis through complex interactions between hematopoietic cells and their microenvironment.
The role of stem cells in other organ systems is less well understood than in hematopoiesis. We have recently examined how cardiac cells arise during development of the heart. In our work we first identified putative cardiac progenitors during in vitro differentiation of mouse ES cells by marking the emerging lineage with green fluorescent protein under control of a specific promoter. At a clonal level these progenitors give rise to both cardiac and smooth muscle cells. Subsequently, we identified the corresponding population of cells during development of the mouse. These studies demonstrate that multipotent progenitors give rise to different cell types within the developing heart. This strategy should be distinguished from the commonly held view that the different cell types have separate origins. Progenitors of the type we have identified might ultimately prove useful in cardiac repair.
It has been widely believed that chromosomal translocations are frequently associated with hematopoietic malignancies and sarcomas but rarely important in the common epithelial cancers, such as those of breast, prostate, lung, and colon. Emerging evidence, however, suggests that this distinction may not be accurate, as recurrent translocations are now being identified in some epithelial cancers. In addition, in most epithelial cancers neither the initial genetic event (the "first hit") nor the cell of origin is known.
We have developed a new model of breast cancer in the mouse based on a chromosomal translocation that is the hallmark of a subtype of the human disease. In this model the translocation product (a fusion protein, Tel-Ntrk3) is expressed within mammary tissue in a conditional manner from the endogenous gene. This model allows us to follow the evolution of cancer from the first event (alveolar epithelial hyperplasia) through frank malignancy. In this system breast cancer occurs in all mice and with short latency. Our initial studies have demonstrated that the target cell for this cancer is a committed bipotential alveolar progenitor rather than the recently defined mammary stem cell. We are now in a position to determine the molecular events that are associated with cellular transformation and generation of a cancer stem cell. Our work serves as a paradigm for genetic studies of the pathogenesis of other epithelial cancers.
ES cells are capable of indefinite self-renewal and maintain their pluripotency under appropriate culture conditions. Several transcription factors, such as Oct4 and Nanog, are known to be critical for ES cell identity. We have sought to define the protein network in which Nanog operates in an effort to determine how pluripotency is established and maintained, and what other proteins participate in these processes. We used affinity chromatography to purify Nanog under native conditions, and then employed mass spectrometry to identify associated proteins. After validation of selected associated proteins, we performed affinity purification of associated proteins in an iterative fashion to develop a protein network. This network is remarkable for its concentration of proteins essential for early development, co-downregulation of members of the network during ES cell differentiation, and linkage to multiple repression pathways. In addition, the genes encoding proteins of the network are highly represented among direct targets of Nanog and Oct4. Thus, the network appears to represent a cellular module that functions to maintain ES cell pluripotency. By identification of additional proteins in the network we hope to uncover the full repertoire of nuclear factors that control pluripotency in ES cells. This work should facilitate and inform attempts to reprogram somatic cells faithfully to an ES cell state.
Partial support for our research is provided by grants from the National Institutes of Health.
Last updated: May 6, 2008
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