Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 12, 2010). Includes bibliographical references.

Activation of tissue-specific genes is a tightly controlled process that normally involves the combined action of several transcription factors and transcriptional co-regulators. The bone-specific osteoca1cin gene (OC) has been used as a prototype to study both tissue-specific and hormonal responsiveness. In this study we have examined the role of Runx2, VDR and C/EBP factors in the regulation of OC gene transcription. Contributions of the Runx and VDRE motifs to OC promoter activity were addressed by introducing point mutations within the context of the rat (-1.1 kb) osteocalcin promoter fused to a CAT-reporter gene. The functional significance of these mutations was assayed following transient transfection and after genomic integration in ROS 17/2.8 osteoblastic cell lines. Furthermore, we tested the effect of these mutations on the chromatin organization of the OC promoter. Our data show that all three Runx sites are required for maximal activation of the OC promoter and that the distal sites contribute significantly to the basal activity. Strikingly, mutation of the three Runx sites abrogates responsiveness of the OC promoter to vitamin D; this loss is also observed when only the Runx sites flanking the VDRE are mutated. Chromatin changes that result in the appearance of DNase I hypersensitive sites during activation of the OC gene are well documented. Mutation of the three Runx sites results in altered chromatin structure as reflected by absence of DNase I hypersensitive sites at the vitamin D response element and over the proximal, tissue-specific basal promoter. These data are consistent with the critical role of Runx2 in osteoblast maturation and bone development. Mutation of the VDRE resulted in a complete loss of vitamin D responsiveness; however, this mutant promoter exhibited increased basal activity. The two DNase I hypersensitive sites characteristic of the transcriptionally active OC gene in osteoblastics cells were not altered upon mutation of the VDRE element, although restriction enzyme accessibility in the proximal promoter region was decreased. We also found an increased level of histone H3 acetylation at the VDRE mutant promoter in comparison to the endogenous gene. Thus binding of VDR to OC promoter is required to achieve a normal transcriptional regulation and chromatin structure of the OC gene. Although Runx2 is considered a master gene for bone development and osteoblast differentiation, it is noteworthy that osteoblast-specific transcription of the rat OC promoter occurs even in the absence of Runx sites. Therefore, other transcription factor(s) should be able to drive OC expression. We characterized a C/EBP enhancer element in the proximal promoter of the rat osteoca1cin gene that resides in close proximity to a Runx element, essential for tissue-specific activation. We find that C/EBPβ or δ and Runx2 factors interact together in a synergistic manner to enhance OC transcription in cell culture systems. Mutational analysis demonstrated that this synergism is mediated through the C/EBP responsive element in the OC promoter and requires a direct interaction between Runx2 and C/EBPβ or δ. Taken together, our findings strongly support a mechanism in which combinatorial interaction of Runx2, VDR, C/EBPβ or δ and probably other transcription factors are needed for regulating OC expression. In this process Runx factors not only act as simple transcriptional trans activators but also by facilitating modifications in promoter architecture and maintaining an active conformation of the target gene promoter.

The Runx family of transcription factors performs an essential role in animal development by controlling 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 sequence-specific DNA binding at gene regulatory regions, organization of promoter chromatin structure, and higher-order compartmentalization of proteins in nuclear foci. This work provides evidence for several functional activities of Runx2 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 Runx2 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 contributed to defining the C-terminal portion of Runx2 as a molecular determinant for the nuclear organization of Runx2 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 natural 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 Runx2 foci on mitotic chromosome spreads revealed several large foci with pairwise symmetry on sister chromatids; these foci co-localize with the RNA polymerase I transcription factor, Upstream Binding Factor (UBFl) at nucleolar organizing regions. A series of experiments were carried 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 inhibition 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.

Higher-order genome organization is important for the regulation of gene expression by bringing different cis-regulatory elements and promoters in proximity. The establishment and maintenance of long-range chromatin interactions occur in response to cellular and environmental cues with the binding of transcription factors and chromatin modifiers. Understanding the organization of the nucleus in differentiation and cancer has been a long standing challenge and is still not well-understood. In this thesis, I explore the dynamic changes in the higher-order chromatin structure in bone differentiation and breast cancer. First, we show dynamic chromatin contact between a distal regulatory element and the promoter of Runx2 gene, which encodes the Runtrelated transcription factor 2 (RUNX2) that is essential for bone development. Next, via using a genome-wide approach, we show that breast cancer cells have altered long-range chromatin contacts among small, gene-rich chromosomes and at telomeres when compared with mammary epithelial cells. Furthermore, we assess the changes in nuclear structure and gene expression of breast cancer cells following Runt-related transcription factor 1 (RUNX1) deficiency, an event frequently observed in breast cancer. Finally, I present the role of the central ATPase subunit of the SWI/SNF complex, SMARCA4 (BRG1), in mediating nuclear structure and gene expression. Taken together, the research presented in this thesis reveals novel insight and paradigm for the dynamic changes in disease and differentiation, as well as uncovers previously unidentified roles for two chromatin regulatory proteins, RUNX1 and SMARCA4.

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.

Resveratrol is a polyphenolic phytoestrogen that has been shown to exhibit potent anti-oxidant, anti-inflammatory, and anti-catabolic properties. Increased osteoclastic and decreased osteoblastic activities result in bone resorption and loss of bone mass. These changes have been implicated in pathological processes in rheumatoid arthritis and osteoporosis. Receptor activator of NF-kappaB ligand (RANKL), a member of the TNF superfamily, is a major mediator of bone loss. In this study, we investigated the effects of resveratrol on RANKL during bone morphogenesis in high density bone cultures in vitro. Untreated bone-derived cell cultures produced well organized bone-like structures with a bone-specific matrix. Treatment with RANKL induced formation of tartrate-resistant acid phosphatase-positive multinucleated cells that exhibited morphological features of osteoclasts. RANKL induced NF-kappaB activation, whereas pretreatment with resveratrol completely inhibited this activation and suppressed the activation of IkappaBalpha kinase and IkappaBalpha phosphorylation and degradation. RANKL up-regulated p300 (a histone acetyltransferase) expression, which, in turn, promoted acetylation of NF-kappaB. Resveratrol inhibited RANKL-induced acetylation and nuclear translocation of NF-kappaB in a time- and concentration-dependent manner. In addition, activation of Sirt-1 (a histone deacetylase) by resveratrol induced Sirt-1-p300 association in bone-derived and preosteoblastic cells, leading to deacetylation of RANKL-induced NF-kappaB, inhibition of NF-kappaB transcriptional activation, and osteoclastogenesis. Co-treatment with resveratrol activated the bone transcription factors Cbfa-1 and Sirt-1 and induced the formation of Sirt-1-Cbfa-1 complexes. Overall, these results demonstrate that resveratrol-activated Sirt-1 plays pivotal roles in regulating the balance between the osteoclastic versus osteoblastic activity result in bone formation in vitro thereby highlighting its therapeutic potential for treating osteoporosis and rheumatoid arthritis-related bone loss.