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Title of Thesis

Sayyed Kaleem Zaidi
Institute/University/Department Details
University of the Punjab, Lahore
No date
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
interacting proteins, gene regulation, eukaryotic gene expression, gene promoters, electrophoretic mobility shift assay, emsa

The ability of each cell to program its genome and determine which genes arc to be expressed at a given time and under specific stimuli is central to tissue differentiation. organogenesis. development. And disease. The problem is that the same genetic information is contained within every cell. and many proteins that regulate gene activity can function non-discriminatory on DNA elements that control gene expression. A number of diverse mechanisms have evolved to ensure that the expression of our genome is a highly regulated process. Transcriptional activation occurs in discrete and controlled stages, from the packaging of a gene into chromatin and its localization within the nucleus to the recruitment of multi protein complexes to subnuclear sites where transcription takes place.

This study was aimed at defining the role of interacting proteins in Runx/Cbfa/AML/PEBP2α mediated gene regulation within the context or three-dimensional architecture or the nucleus. Runx2/Cbfal was used as a representative member of Runx/Cbfal proteins. a family of transcription factors involved in tissue specific gene expression. Runx2/Cbfal. the best studied family member is required for bone formation in vivo. It binds to DNA in a sequence specific manner is associated with the nuclear matrix. the underlying substructure of the nucleus. interacts with a ,variety or proteins including co-repressors and co-activators. and regulate target genes in a tissue specific manner.

Part I of Results describes the mechanisms that mediate targeting of Runx2/Cbfal to the nuclear matrix associated subnuclear sites. A panel of mutants missing various regions in the carboxyl terminus of the protein was generated. Using in situ immunofluorescence and biochemical approaches a 38 amino acid segment. termed the nuclear matrix-targeting signal (NMTS). was delineated as a protein motif responsible for subnuclear retention of Runx2/Cbfal. The NMTS is conserved among. All the family members. functions autonomously and possesses a moderate: transactivation domain. Importantly. deletion of the NMTS from Runx2/Cbfal results in compromised regulation of the bone specific osteocalcin gene. Mutations or specific amino acid residues within the NMTS of Runx2/Cbfal that compromise subnuclear targeting of the protein also show impaired transcriptional potential. Taken together. findings reported in this chapter establish a direct link between the targeting of Runx2/Cbfal to subnuclear sites and its requirement for biological activity of the protein.

Runx2/Cbfal. a major regulator or osteogenesis. is a target or tissue specific extracellular signaling. Yes-associated protein (YAP) an adapter protein in Src/Yes tyrosine kinase signaling. interacts with the carboxyl terminus of Runx2/Cbfal Src/Yes signaling is required for the maintenance or bone homeostasis. a delicate balance between the bone forming osteoblasts and bone resorbing osteoclasts. Part 2 of Results describes a detailed analysis of the interaction between YAP and Runx2 in osteoblasts and involvement of the Src/Yes signaling pathway in regulating the activity of Runx2. YAP interacts with the PY (praline-proline-proline-tyrosine) motif of Runx2. a well-characterized protein-protein interaction module that is conserved among Runx family members. The interaction between Runx2 and YAP results in the recruitment of YAP to the promoter of the hone specific osteocalcin gene and to subnuclear sites where Runx2 resides. Consequently. YAP suppresses Runx2 mediated activation of the osteocalcin gene. Interference with the activity of Src/Yes kinases results in the induction of the endogenous osteocalcin transcription. Thus Src/Yes kinase signaling utilizes YAP as a molecular switch to convert Runx2 into a negative regulator of bone specific gene regulation at subnuclear sites. hence maintaining the balance between bone formation and resorption.

Smad proteins represent another class of signaling molecules that interact with the carboxyl terminus of Runx2 and mediate transforming growth factor β(TGF β)/bone morphogenetic protein (BMP) signaling. The interaction between Smads and Runx2 results from the phosphorylation of Smads by threonine-serine kinases and their subsequent nuclear translocation (Results Part 3). Interestingly. Smads are only associated with the nuclear matrix in cells expressing Runx proteins. Exogenous expression of Runx2 results in targeting of Smads to subnuclear sites in cells that do not express Runx proteins. Mutation of a specific residue that compromises Runx2 suonuclear targeting results in impaired subnuclear trafficking of Smads. Importantly. targeting of the Smad-Runx2 complex to transcriptionally active suonuclear foci results in the activation of a reporter gene controlled by Runx and Smad binding sites. These data provide mechanistic insight into tissue specific functioning and Smad signaling within the context of subnuclear architecture.

Runx2/Cbfal positively regulates the differentiation program of osteoblasts. the bone forming cells. Part Results provides the evidence suggestive of the role of Runx2/Cbfal as a negative regulator of osteoblast proliferation. Diploid primary osteoblasts lacking Runx2 protein show a dramatic increase in cell number. Consistent with this increase. several key regulators of cell proliferation. including members of retinoblastoma protein and cyclins. show altered expression in the absence of Runx2. In addition. Runx2 is dynamically re-distributed during mitosis. The re-distribution pattern of Runx2 closely follows that of nuclear lamins. an integral component of subnuclear architecture that ensures structural integrity of the nucleus during mitosis.

Taken together. findings presented in this dissertation reveals some key mechanisms that collectively regulate the biological activity of Runx2. The delineation or the NMTS or Runx2 provides mechanistic insight into subnuclear trafficking or a transcription factor to sites within the nucleus where transcription takes place. The mutation analysis of the NMTS confirms that the loop-turn-loop structure of the NMTS provides protein-protein interaction surfaces. Runx2 mediated subnuclear targeting of AP and Smads and consequent transcriptional regulation establishes the role or Runx2 as a nuclear matrix acceptor protein. These findings provide first experimental demonstration that the organization or regulatory complexes at subnuclear sites is directly involved in the physiological regulation or tissue specific gene expression. Consistent with the role of Runx2 as the nuclear matrix acceptor protein. the dynamic re-distrihutiol1 or Rum,2 during mitosis suggests that Runx2 may in part be responsible for the structural integrity of the nucleus during mitosis. Furthermore. The possible role of Runx2 as a regulator of osteoblast proliferation suggests that Runx2 may be operative at different stages to ensure physiological regulation of the osteoblast maturation.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
227.78 KB
2 1 Introduction 1
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  1.1 Regulation of eukaryotic gene expression 1
  1.2 Sequence organization of gene promoters 2
  1.3 Chromatin organization of gene promoters 3
  1.4 High order nuclear organization 5
  1.5 sub nuclear domains 8
  1.6 Concept and objectives of the present study 23
3 2 Materials and methods 30
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  2.1 Cell culture and transient transfections 30
  2.2 Treatments 31
  2.3 Plasmid constructs 32
  2.4 PCR based sited directed mutagenesis 34
  2.5 Preparation of nuclear extracts 38
  2.6 Electrophoretic mobility shift assay (EMSA ) 39
  2.7 Antibodies 41
  2.8 Immunofluorescenec microscopy 43
  2.9 Visualization of subnuclear site of active transcription 45
  2.10 Biochemical fractionation 46
  2.11 SDS-PAGE and western blotting 47
  2.12 Reporter gene assays 49
  2.13 Co-immunopreciptation 51
  2.14 Isolation of total cellular RNA andnorthern blotting 54
  2.15 Cell synchronization 56
  2.16 Calvarial cell cultures from Runx2 knock in mice 57
4 3 Results 58
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  3.1 A specific targeting signal directs Runx2/Cb fal to subnuclear domain and contributes to transactivation of the osteocalcin gene 58
  3.2 Reunx2 functionally integrates Src signaling pathway at subnuclear sites to regulate skeletal gene expression 100
  3.3 Integration of Runx and Smad regulatory pathways at transcriptionally active subnuclear sites 140
  3.4 Dynamic redistribution and antiproliferative function of Runx2/Cbfal during the ostcoblast cell cycle 164
5 4 Discussion 198
117.53 KB
  4.1 Cell biological implications 199
  4.2 Physiological implications 205
6 5 References 210
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