The nuclear matrix (NM) is a fibrogranular network of ribonucleoproteins upon which transcriptional complexes and regulatory genomic sequences are organized. A hallmark of cancer is the disorganization of nuclear architecture; however, the extent to which the NM is involved in malignancy is not well studied. The RUNX1 and RUNX2 proteins form complexes within the NM to promote hematopoiesis and osteoblastogenesis, respectively at the transcriptional level. RUNX1 and RUNX2 are both expressed in breast cancer cells (BrCCs); however, their genome-wide BrCC functions are unknown. RUNX1 and RUNX2 activate many tumor suppressor pathways in blood and bone lineages, respectively, including attenuation of protein synthesis and cell growth via suppression of ribosomal RNA (rRNA) transcription, which appears contrary to Runx-expression in highly proliferative BrCCs. To define roles for RUNX1 and RUNX2 in BrCC phenotype, we examined the involvement of RUNX1 and RUNX2 in rRNA transcription and generated a genome-wide model for RUNX1 and RUNX2-binding and transcriptional regulation. To validate gene expression patterns identified in our screen, we developed a Real-Time qPCR primer design program, which allows rapid, high-throughput design of primer pairs (FoxPrimer). In BrCCs, RUNX1 and RUNX2 regulate genes that promote invasiveness and do not affect rRNA transcription, protein synthesis, or cell growth. We have characterized in vitro functions of Runx proteins in BrCCs; however, the relationships between Runx expression and diagnostic/prognostic markers of breast cancer (BrCa) in patients are not well studied. Immunohistochemical detection of RUNX1 and RUNX2 in BrCa tissue microarrays reveals RUNX1 expression is associated with early, smaller tumors that are ER+ (estrogen receptor), HER2+, p53-, and correlated with androgen receptor (AR) expression; RUNX2 expression is associated with late-stage, larger tumors that are HER2+. These results show that the functions and expression patterns of NM-associated RUNX1 and RUNX2 are context-sensitive, which suggests potential disease-specific roles. Two functionally disparate genomic sequence types bind to the NM: matrix associated regions (MARs) are functionally associated with transcriptional repression and scaffold associated regions (SARs) are functionally associated with actively expressed genes. It is unknown whether malignant nuclear disorganization affects the functions of MARs/SARs in BrCC. We have refined a method to isolate nuclear matrix associated DNA (NM-DNA) from a structurally preserved NM and applied this protocol to normal mammary epithelial cells and BrCCs. To define transcriptional functions for NM-DNA, we developed a computational algorithm (PeaksToGenes), which statistically tests the associations of experimentally-defined NM-DNA regions and ChIP-seq-defined positional enrichment of several histone marks with transcriptome-wide gene expression data. In normal mammary epithelial cells, NM-DNA is enriched in both MARs and SARs, and the positional enrichment patterns of MARs and SARs are strongly associated with gene expression patterns, suggesting functional roles. In contrast, the BrCCs are significantly enriched in the silencing mark H3K27me3, and the NM-DNA is enriched in MARs and depleted of SARs. The MARs/SARs in the BrCCs are only weakly associated with gene expression patterns, suggesting that loss of normal DNA-matrix associations accompanies the disease state. Our results show that structural preservation of the in situ NM allows isolation of both MARs and SARs, and further demonstrate that in a disorganized, cancerous nucleus, normal transcriptional functions of NM-DNA are disrupted. Our studies on nuclear organization in BrCC, show that the disorganized phenotype of the cancer cell nucleus is accompanied by deregulated transcriptional functions of two constituents of the NM. These results reinforce the role of the NM as an important structure-function component of gene expression regulation.

The Runx family of transcription factors supports cell fate determination, cell cycle regulation, global protein synthesis control, and genetic as well as epigenetic regulation of target genes. Runx1, which is essential for hematopoiesis; Runx2, which is required for osteoblast differentiation; and Runx3, which is involved in neurologic and gut development; are expressed in the growth plate during chondrocyte maturation, and in the chondrocytes of permanent cartilage structures. While Runx2 is known to control genes that contribute to chondrocyte hypertrophy, the functions of Runx1 and Runx3 during chondrogenesis and in cartilage tissue have been less well studied. The goals of this project were to characterize expression of Runx proteins in articular cartilage and differentiating chondrocytes and to determine the contribution of Runx1 to osteoarthritis (OA). Here, the expression pattern of Runx1 and Runx2 was characterized in normal bovine articular cartilage. Runx2 is expressed at higher levels in deep zone chondrocytes, while Runx1 is primarily expressed in superficial zone chondrocytes, which is the single cell layer that lines the surface of articular cartilage. Based on this finding, the hypothesis was tested that Runx1 is involved in osteoarthritis, which is a disease characterized by degradation of articular cartilage and changes in chondrocytes. These studies showed that Runx1 is upregulated in articular cartilage explants in response to mechanical compression. Runx1 was also expressed in chondrocytes found at the periphery of OA lesions in the articular cartilage of mice that underwent an OA-inducing surgery. Runx1 was also upregulated in cartilage explants of human osteoarthritic knees, and IHC data showed that Runx1 is mainly expressed in chondrocyte “clones” characteristic of OA. To ascertain the potential function of the upregulation of Runx1 in these cartilage stress conditions and disease states, the hypothesis was tested that Runx1 is upregulated in very specific chondrocyte populations in response to the cartilage damage in osteoarthritis. These studies addressed the properties of these cells that related to functions in cell growth and differentiation. In both the surface layer of normal articular cartilage, and in OA cartilage, Runx1 expression by IF co-localized with markers of mesenchymal progenitor cells, as well as markers of proliferation Ki-67 and PCNA. This finding indicated that Runx1 is found in a population of cells that represent a proliferative population of mesenchymal progenitor cells in osteoarthritis. To further address Runx1 function and identify downstream targets of Runx proteins, a promoter analysis of genes that are known to be either downregulated or upregulated during chondrocyte maturation was done. These studies found that many of these genes have 1 or more Runx binding sites within 2kb of their transcription start site, indicating that they are potential downstream Runx target genes. Lastly, some preliminary experiments were done to characterize novel roles of Runx proteins in the chondrocyte. Runx proteins have been shown to epigenetically regulate their target genes by remaining bound to them throughout mitosis, “poising” them for transcription upon exit from mitosis. The hypothesis that Runx proteins also function by remaining bound to their target genes throughout mitosis in chondrocytes was tested. It was demonstrated by immunofluorescense imaging of Runx proteins on metaphase chromosomes of ATDC5 cells, that Runx2 remains bound to chromosomes during mitosis. Cell proliferation and hypertrophy are both linked to increases in protein synthesis. Runx factors, which regulate rates of global protein synthesis, are expressed in both proliferating and hypertrophic chondrocytes. Thus, it was hypothesized that Runx proteins regulate rates of global protein synthesis during chondrocyte maturation. These studies showed that the overexpression of Runx proteins in a chondrocyte cell line (ATDC5) did not affect protein synthesis rates or levels of protein synthesis machinery. Additionally, Runx proteins did not affect proliferation rates in this chondrocyte cell line.

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.