TRF2 is an essential telomeric protein involved in preventing the telomere ends from being recognized as DNA breaks. I have shown that TRF2 does not appear to play a major role in the UV -induced DNA damage response in IMR90, Cockayne syndrome or XPC deficient cells. TRF2 binds telomeric DNA via its Myb domain and also contains an N-terminal basic domain. Expression of TRF2MMM causes telomere fusions, whereas TRF2^(ΔB) causes rapid deletion of telomeric DNA, as both phenotypes result in senescence. These phenotypes are dependant upon recombination events. Thus, the basic domain of TRF2 may be essential to suppress recombination events at telomeres. However, it is not fully understood what amino acid residues in the basic domain of TRF2 are indispensable to maintain its function. By creating mutations in the arginine residues in the basic domain of TRF2, I have shown that the positive charge of the basic domain alone is not sufficient to maintain its protective function. By expressing these TRF2 mutants in the presence or absence of the Myb domain in HT1080 and BJ/hTERT cells, I have been able to recapitulate the TRF2^(ΔB) and TRF2^(ΔBΔM) decreased proliferation and senescence phenotypes. Furthermore, by analyzing anaphase and metaphase chromosomes and performing Southern blotting, I have shed light on the molecular mechanisms responsible for the deleterious phenotypes observed in the TRF2 mutants. Amino acid changes from arginines to lysines introduced into the basic domain of TRF2 results in a significant increase in telomere doublets. However, when these TRF2 mutants are expressed in the absence of the Myb domain, a significant increase in telomere fusions events occur. Collectively, my results indicate that more than one arginine residue in the basic domain is essential to maintain the protective function of TRF2, as these arginine residues may act as substrates for protein arginine methyltransferases. / Thesis / Master of Science (MSc)

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.

Winter wheat has several advantages over spring varieties, higher (25 % more) yield, efficient use of spring moisture, reduction of soil erosion by providing ground cover during the fall and early spring, rapid initial spring growth to out - compete weeds and circumvent the peak of Fusarium head blight infections by flowering early. Winter wheat is planted in early autumn when it germinates and developing seedlings acclimate to cold. The crown survives under snow cover and in spring rapidly grows into a vigorously growing plant for grain to be harvested in summer. However, the harsh Canadian prairie winters require that winter wheat has increased cold hardiness and improved winter survival to reduce losses from sudden cold snaps during winter and spring. Low temperature (LT) tolerance is one of the major components of cold hardiness. Genetic mapping studies have revealed a major quantitative trait locus (Fr-A2) at wheat chromosome 5A which can explain at least 50 % of LT tolerance in wheat. Physical mapping of 5A LT QTL in a hardy winter wheat cv Norstar revealed a cluster of at least 23 C - repeat binding factors (CBF) coinciding with peak of Fr-A2 QTL. The objective of this study is biochemical, and molecular characterization of CBF co - located at Fr-A2 to identify key CBF participating in conferring LT tolerance in winter wheat. A comparative analysis of CBF gene cluster at the Fr-A2 collinear region among Poaceae members showed an expansion in the number of CBF genes with increased LT tolerance. Rice, a cold sensitive member, had only three CBF genes, whereas cold hardy winter wheat cv Norstar has 23 CBF genes. Amino acid sequence - based cluster analysis of complete CBF genes, or their major functional components such as the AP2 - DNA binding domain and C - terminal trans - activation domain, divide Norstar CBF into Pooideae specific clades. However, analyses of Norstar CBF amino acid sequences of different functional groups revealed a shift in clade members. These results suggest divergence of CBF functions which could lead to possible differences / similarity in the regulon activated by a CBF in a specific group. The 15 CBF genes from winter wheat cv Norstar were expressed in E. coli to produce recombinant TrxHisS - CBF fusion proteins in adequate quantities for structural and functional assays. All CBF fusion proteins could be recovered in the E. coli soluble phase of cell extract, except that the CBF17.0 fusion protein could only be recovered with 6 M urea extraction. Eleven of the 15 CBF fusion proteins were very stable in heat (98 oC), 10 % SDS and 6 M urea treatment. The five other CBF members were very labile under native conditions, but were stable in E. coli cell extracts or when extracted under denaturing conditions. Most of the CBF recombinant proteins in denaturing gel electrophoresis migrated slower than expected from their predicted molecular mass, based on amino acid sequence. The slow migration could be associated to their elongated protein structure as determined by dynamic light scattering (DLS). CBF 12.2 and CBF 17.0 were highly resistant to denaturation and retained their secondary structure in these conditions as determined by circular dichroism (CD) spectra. The high stability of these two CBF proteins may be important for cold acclimation or maintenance of cold hardiness in wheat. CBF proteins are transcription factors that bind to the dehydration-responsive element / C-repeat element (DRE / CRT) motif (CCGAC). Ten of the 15 Norstar recombinant CBFs whether purified under native or denaturing conditions showed in vitro binding to the CRT motif. Within hours of cold exposure (4 oC) the native CBF increased their affinity to CRT interaction which could be due to changes in the CBF secondary structures. Some of the CBF for binding preferred the core GGCCGAC motif while others preferred TGCCGAC. Similarly binding assays with truncated CBF revealed that for some CBF proteins, the second signature motif (DSAWR) and remaining C - terminal were not needed, while for others a considerable portion of the C -terminal region was needed for binding. Norstar CBF 12.1 has a memory of cold experience, and upon exposure to cold, has a high and immediate affinity to CRT elements. A homolog CBF12.2 in less cold - hardy winter wheat cv Cappelle - Desprez had a non - functional protein due to a R → Q substitution in a highly conserved residue within the AP2 domain. Several of the cv Norstar CBFs showed increased activity under LT and denaturing conditions, which may be the reason for the greater cold hardiness in Norstar. In conclusion, detailed and extensive analyses of CBF in this study characterized their structure and function relationships, which are important for understanding and improving LT tolerance in plants. The identification of specific CRT binding motifs and two CBFs which were very stable under adverse conditions may be prime candidates for further study to improve LT tolerance in plants.

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.

Les gènes RUNX1 et CBFB codent pour les sous-unités du core binding factor (CBF), facteur de transcription hétérodimérique essentiel de l’hématopoïèse définitive. La dérégulation du CBF est l'une des anomalies les plus fréquemment rencontrées dans les hémopathies malignes. Puisque la perturbation seule du CBF est insuffisante au développement d’une leucémie aiguë myéloïde (LAM), les LAM impliquant le CBF sont considérées comme des modèles de leucémogénèse multi-étapes, nécessitant la coopération d’anomalies génétiques additionnelles.Dans ce travail, nous nous sommes intéressés aux LAM de type CBF, caractérisées soit par une t(8;21)/fusion RUNX1-RUNX1T1 soit par une inv(16)/fusion CBFB-MYH11, ainsi qu’aux LAM avec mutations germinales de RUNX1 (définissant la thrombopénie familiale avec prédisposition aux leucémies aiguës ou FPD/AML). Afin d’identifier des anomalies additionnelles, nous avons étudié les prélèvements de patients atteints de LAM CBF inclus dans les essais français ELAM02 (0-18 ans) et CBF2006 (18-60 ans) par séquençage à haut débit (n=215) et single nucleotide polymorphism-array (n=198). Les échantillons de 25 individus atteints de FPD/AML (issus de 15 familles), diagnostiqués entre 2005 et 2014, ont également été séquencés au stade thrombopénique et au moment de la transformation en leucémie aiguë.Dans les LAM CBF, les mutations activatrices des voies tyrosines kinases (TK) sont les événements les plus fré-quents quel que soit le sous-type de LAM CBF [t(8;21) ou inv(16)], comme cela a déjà été rapporté dans d’autres études. En revanche, les mutations affectant les gènes du remodelage chromatinien ou du complexe de la cohésine sont identifiées à des fréquences élevées (41% et 18% respectivement) dans les LAM avec t(8;21) tandis qu’elles sont pratiquement absentes dans les LAM avec inv(16). Dans les LAM avec t(8;21), la coexistence de ces mutations avec les mutations de type TK est associée à un pronostic défavorable suggérant une synergie entre ces événements. D'autres événements fréquemment retrouvés incluent les mutations de ZBTB7A et DHX15 dans les LAM avec t(8;21) (20% et 6% respectivement) et les délétions/mutations de FOXP1 dans les LAM avec inv(16) (7%). Enfin, nous avons décrit la perturbation de CCDC26 comme une possible lésion associée à une signalisation aberrante des TK dans les LAM CBF (4,5% des cas).Dans les FPD/AML, l'analyse mutationnelle a révélé l'acquisition d'un deuxième événement impliquant RUNX1 chez tous les patients ayant développé une LAM. Ce deuxième événement correspondait soit à une mutation somatique du second allèle de RUNX1 soit à la duplication de la mutation germinale de RUNX1 (par perte d'hétérozygotie sans anomalie du nombre de copies ou trisomie 21 acquise). En pratique clinique, cela suggère que la présence de deux mutations différentes de RUNX1 ou d'une seule mutation avec un ratio allélique supérieur à 50% chez un patient atteinte de LAM doit alerter sur la possibilité d’un syndrome FPD/AML sous-jacent. / RUNX1 and CBFB encode subunits of the core binding factor (CBF), a heterodimeric transcription factor required for the establishment of definitive hematopoiesis. Deregulation of the CBF is one of the most frequent aberrations in hematological malignancies. Since CBF disruption alone is insufficient to induce acute myeloid leukemia (AML) on its own, AML with CBF involvement is considered as a model of multistep leukemogenesis requiring additional genetic aberrations.Here, we focused on acute myeloid leukemia (AML) with t(8;21)/RUNX1-RUNX1T1 fusion and AML with inv(16)/CBFB-MYH11 fusion, reported together as CBF AML, as well as AML with germline RUNX1 mutation (defining the familial platelet disorder with propensity to develop leukemia or FPD/AML).In order to explore additional genomic aberrations, we performed comprehensive genetic profiling in CBF AML patients enrolled in the French trials ELAM02 (0-18 years) and CBF2006 (18-60 years) using both high-throughput sequencing (n=215) and single nucleotide polymorphism-array (n=198). In addition, we sequenced samples from 25 individuals with FPD/AML (15 pedigrees) diagnosed between 2005 and 2014 at thrombocyto-penic stage and during leukemic progression.In CBF AML, mutations in genes activating tyrosine kinase (TK) signaling were frequent in both subtypes as previously described by others. By contrast, we found mutations in genes encoding chromatin modifiers or members of the cohesin complex with high frequencies in t(8;21) AML (41% and 18% respectively) while they were nearly absent in inv(16) AML. Interestingly, such mutations were associated with a poor prognosis in patients with TK mutations suggesting synergic cooperation between these events. Other events included ZBTB7A and DHX15 mutations in t(8;21) AML (20% and 6% respectively) and FOXP1 deletions or truncating mutations in inv(16) AML (7%). Finally, we described CCDC26 disruption as a possible new lesion associated with aberrant TK signaling in this particular subtype of leukemia (4.5% of CBF AML).In FPD/AML, mutational analysis revealed the acquisition of a second event involving RUNX1 in all patients with AML including somatic mutation of the second allele or duplication of the germline RUNX1 mutation through copy-neutral loss of heterozygosity and trisomy 21. In clinical practice, we suggest that the occurrence of two different RUNX1 mutations or a single RUNX1 mutation with a variant allele frequency higher than 50% in a patient with AML should alert about the possibility of FPD/AML.

No description available.

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.

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.