Regulation of Cell Growth and Differentiation within the Context of Nuclear Architecture by the Runx2 Transcription Factor: a Dissertation

The Runx family of transcription factors performs an essential role in animal development by controllng gene expression programs that mediate cell proliferation growth and differentiation. The work described in this thesis is concerned with understanding mechanisms by which Runx proteins support this program of gene expression within the architectural context of the mammalian cell nucleus. Multiple aspects of nuclear architecture are influenced by Runx2 proteins including sequencespecific DNA binding at gene regulatory regions , organization of promoter chromatin structure, and higher-order compartentalization of proteins in nuclear foci. This work provides evidence for several functional activities of Run2 in relation to architectural parameters of gene expression for the control of cell growth and differentiation. First, the coordination of SWI/SNF mediated chromatin alterations by Run2 proteins is found to be a critical component of osteoblast differentiation for skeletal development. Several chromatin modifying enzymes and signaling factors interact with the developmentally essential Runx2 C-terminus. A patent-pending microscopic image analysis strategy invented as part of this thesis work called intranuclear informatics has contrbuted to defining the C-terminal portion of Runx2 as a molecular determinant for the nuclear organization of Run2 foci and directly links Runx2 function with its organization in the nucleus. Intranuclear informatics also led to the discovery that nuclear organization of Runx2 foci is equivalently restored in progeny cells following mitotic division a natual perturbation in nuclear structure and function. Additional microscopic studies revealed the sequential and selective reorganization of transcriptional regulators and RNA processing factors during progression of cell division to render progeny cells equivalently competent to support Runx2 mediated gene expression. Molecular studies provide evidence that the Runx proteins have an active role in retaining phenotype by interacting with target gene promoters through sequence-specific DNA binding during cell division to support lineage-specific control of transcriptional programs in progeny cells. Immunolocalization of Run2 foci on mitotic chromosome spreads revealed several large foci with pairwise symetr on sister chromatids; these foci co-localize with the RNA polymerase I transcription factor Upstream Binding Factor (UBF1) at nucleolar organizing regions. A series of experiments were carred out to reveal that Runx2 interacts directly with ribosomal DNA loci in a cell cycle dependent manner; that Runx2 is localized to UBF foci within nucleoli during interphase; that Runx2 attenuates rRNA synthesis; and that this repression of ribosomal gene expression by Runx2 is associated with cell growth inhbition and induction of osteoblast-specific gene expression. This thesis has identified multiple novel mechanisms by which Runx2 proteins function within the hierarchy of nuclear architecture to control cell proliferation, growth and differentiation.