To acquire its characteristic structural and functional complexity, the mammalian nervous system must undergo several critical developmental processes. One such process requires factors that regulate the decision of dividing progenitors to leave the cell cycle and activate the neuronal differentiation program. It is shown in this thesis that the murine runt-related gene Runx1 is expressed in proliferating cells on the basal side of the murine olfactory epithelium. Disruption of Runx1 function in vivo does not result in a change in the quantity of progenitors but leads to a decrease in precursor number and an increase in differentiated ORNs. These effects result in premature and ectopic ORN differentiation. Further, exogenous Runx1 expression in cultured olfactory neural progenitors causes an expansion of the mitotic cell population. In agreement with these findings, exogenous Runx1 expression also promotes cortical neural progenitor cell proliferation without inhibiting neuronal differentiation. These effects appear to involve transcriptional repression mechanisms. Consistent with this possibility, Runx1 represses transcription driven by the promoter of the cell cycle inhibitor p21Cip1 in cortical progenitors. Taken together, these findings suggest a previously unrecognized role for Runx1 in coordinating the proliferation and neuronal differentiation of selected populations of neural progenitors/precursors. / Another significant step in the development of the mammalian nervous system is the acquisition of distinctive neuronal traits. This thesis also shows that Runx1 is expressed in selected populations of postmitotic neurons of the murine embryonic central and peripheral nervous systems. In embryos lacking Runx1 activity, hindbrain branchiovisceral motor neuron precursors of the cholinergie lineage are correctly specified but then fail to enter successive stages of differentiation and undergo increased cell death resulting in neuronal loss in the mantle layer. Runx1 inactivation also leads to a loss of selected sensory neurons in trigeminal and vestibulocochlear ganglia. These findings uncover previously unrecognized roles for Runx1 in the regulation of neuronal subtype specification. / This thesis thus presents a novel factor which functions at several steps in the development of the mammalian nervous system and adds to the growing body of work on the processes involved in elaborating such a complex and vital structure.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 3 uppsatser.

No description available.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 18, 2009). Includes bibliographical references.

Runx1 is a master regulator of hematopoiesis, required for the initiation of definitive hematopoiesis in the embryo and essential for appropriate differentiation of many hematopoietic lineages in the adult. The roles of Runx1 in normal hematopoiesis are juxtaposed with the high frequency of Runx1 mutations and translocations in leukemia. Leukemia associated Runx1 mutations that retain DNA-binding ability have truncations or frame shifts that lose C-terminal domains. These domains are important for subnuclear localization of Runx1 and protein interactions with co-factors. The majority of leukemia associated Runx1 translocations also replace the C-terminus of Runx1 with chimeric fusion proteins. The common loss of Runx1 C-terminal domains in hematopoietic diseases suggests a possible common mechanism. We developed a panel of mutations to test the functions of these domains in vitro, and then developed mouse models to examine the consequences of losing Runx1 C-terminal domains on hematopoietic development and leukemogenesis in vivo. We previously observed that overexpression of a subnuclear targeting defective mutant of Runx1 in a myeloid progenitor cell line blocks differentiation. Gene expression analysis before differentiation was initiated revealed that the mutant Runx1 was already deregulating genes important for maturation. Furthermore, promoters of the suppressed genes were enriched for binding sites of known Runx1 co-factors, indicating a non-DNA-binding role for the mutant Runx1. To investigate the in vivo function of Runx1 C-terminal domains, we generated two knock-in mouse models; a C-terminal truncation, Runx1Q307X, and a point mutant in the subnuclear targeting domain, Runx1 HTY350-352AAA . Embryos homozygous for Runx1 Q307X phenocopy a complete Runx1 null and die in utero from central nervous system hemorrhage and lack of definitive hematopoiesis. Embryos homozygous for the point mutation Runx1HTY350-352AAA bypass embryonic lethality, but have hypomorphic Runx1 function. Runx1HTY350-352AAA results in defective growth control of hematopoietic progenitors, deregulation of B-lymphoid and myeloid lineages, as well as maturation delays in megakaryocytic and erythroid development. Runx1 localizes to subnuclear domains to scaffold regulatory machinery for control of gene expression. This work supports the role of transcription factors interacting with nuclear architecture for greater biological control, and shows how even subtle alterations in that ability could have profound effects on normal biological function and gene regulation.

Leukemia is a hematopoietic cancer that is characterized by the abnormal differentiation and proliferation of hematopoietic cells. It is ranked 7th by death rate among cancer types in USA, even though it is not one of the top 10 cancers by incidence (USCS, 2010). This indicates an urgent need for more effective treatment strategies. In order to design the new ways of prevention and treatment of leukemia, it is important to understand the molecular mechanisms involved in development of the disease. In this study, we investigated mechanisms involved in the development of acute myeloid leukemia (AML) that is associated with CBF fusion genes. The RUNX1 and CBFB genes that encode subunits of a transcriptional regulator complex CBF, are mutated in a subset (20 – 25%) of AML cases. As a result of these mutations, fusion genes called CBFB-MYH11 and RUNX1-ETO arise. The chimeric proteins encoded by the fusion genes provide block in proliferation for myeloid progenitors, but are not sufficient for AML development. Genetic studies have indicated that activation of cytokine receptor signaling is a major oncogenic pathway that cooperates in leukemia development. The main goal of my work was to determine a role of two factors that regulate cytokine signaling activity, the microRNA cluster miR-17-92 and the thrombopoietin receptor MPL, in their potential cooperation with the CBF fusions in AML development. We determined that the miR-17-92 miRNA cluster cooperates with Cbfb-MYH11 in AML development in a mouse model of human CBFB-MYH11 AML. We found that the miR-17-92 cluster downregulates Pten and activates the PI3K/Akt pathway in the leukemic blasts. We also demonstrated that miR-17-92 provides an anti-apoptotic effect in the leukemic cells, but does not seem to affect proliferation. The anti-apoptotic effect was mainly due to activity of miR-17 and miR-20a, but not miR-19a and miR-19b. Our second study demonstrated that wild type Mpl cooperated with RUNX1-ETO fusion in development of AML in mice. Mpl induced PI3K/Akt, Ras/Raf/Erk and Jak2/Stat5 signaling pathways in the AML cells. We showed that PIK3/Akt pathway plays a role in AML development both in vitro and in vivo by increasing survival of leukemic cells. The levels of MPL transcript in the AML samples correlated with their response to thrombopoietin (THPO). Moreover, we demonstrated that MPL provides pro-proliferative effect for the leukemic cells, and that the effect can be abrogated with inhibitors of PI3K/AKT and MEK/ERK pathways. Taken together, these data confirm important roles for the PI3K/AKT and RAS/RAF/MEK pathways in the pathogenesis of AML, identifies two novel genes that can serve as secondary mutations in CBF fusions-associated AML, and in general expands our knowledge of mechanisms of leukemogenesis.

Runt related transcription factors (Runx) play an important role in mammalian development by regulating the expression of key genes involved in cell proliferation, differentiation and growth. The work described in this thesis details the mechanisms by which the activity of two members of this family are regulated in human cells. Chapter One provides a brief introduction of Runx transcription factors. Chapter Two describes the regulation of Runx2 protein by the PI3 kinase/Akt pathway in human breast cancer cells. The PI3 kinase/Akt pathway is one of the major signal transduction pathways through which growth factors influence cell proliferation and survival. It is also one of the most frequently dysregulated pathways in human cancers. We identify Runx2 protein, a key regulator of breast cancer invasion as a novel substrate of Akt kinase and map residues of Runx2 that are phosphorylated by Akt in breast cancer cells. Our results show that phosphorylation by Akt increases the binding of Runx2 protein to its target gene promoters and we identify the phosphorylation events that enhance DNA binding of Runx2. Our work establishes Runx2 protein as a critical effecter downstream of Akt that regulates breast cancer invasion. In Chapter Three we describe the subnuclear localization of the tumor suppressor protein Runx3 during interphase and mitosis. We find that similar to other Runx family members, Runx3 protein resides in nuclear matrix associated foci during interphase. We delineate a subnuclear targeting signal that directs Runx3 to these nuclear matrix associated foci. Our work establishes that this association of Runx3 protein with the nuclear matrix plays a vital role in regulating its transcriptional activity. Chromatin immunoprecipitation results show that Runx3 occupies rRNA promoters during interphase. We also find that Runx3 remains associated with chromosomes during mitosis and localizes with nucleolar organizing regions (NORs), reflecting an interaction with epigenetic potential. This thesis provides novel insights into various mechanisms by which cells regulate the activity of Runx proteins.