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Title of Thesis

Fauzia Aslam
Institute/University/Department Details
Department of Zoology/ University of the Punjab
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
glucocorticoid responsive elements, osteocalcin gene, diploid osteoblasts, osteoblast phenotype, bone cells, mutagenesis, plasmids, oligonucleotide primers, chloramphenicol acetyltransferase

The Osteocalcin (OC) gene encodes a 10 Kda hone-specific protein which is expressed with the onset of mineralization during the differentiation of normal diploid osteoblasts. OC levels in the serum correlate with bone turnover. The tissue-specific expression of OC gene is regulated by a modular promoter composed of a complex series of regulatory elements. The transcriptional activity of the OC gene is controlled by several physiological and hormonal regulators that independently and synergistically or antagonistically modulate OC expression (e.g. vitamin D3 glucocorticoids. retinoic acid. TGF-β).

Complexity of steroid responsiveness of OC gene during osteoblast development has been under observation. The understanding of molecular mechanisms mediating glucocorticoid responsiveness in osseous cells is limited. Glucocorticoids are physiological mediators of osteoblast proliferation and differentiation, as well as perturbants of bone formation and resorption in vivo. The main goal of the present study has been focused on functional characterization of glucocorticoid-mediated regulation of OC gene as a model system. Previous studies in the laboratory identified glucocorticoid-receptor binding sequences in the proximal part of rat OC gene promoter using partially purified receptor. Here, using a series of OC promoter deletion and mutation constructs, the functionality of the GR-binding sequences in the proximal promoter at nucleotide (nt) -16 to -1 downstream of TAT A element together with the glucocorticoid receptor element (GRE) half element in the OC box. at nt-86to-81 was assayed. This was done by assaying glucocorticoid responsiveness at 10-6 M Dexamethasone (DEX). and in combination with 10-8M 1.25(OH)2D3 (D3), with deleted and mutated OC promoter-reporter constructs (OCCAT) in osteoblast-like cells. the 17/2.8 rat osteosarcoma (ROS) cell line. Promoter deletion analysis revealed an additional GRE in the distal promoter at nt.-697to-683 that functions to suppress OC transcription. In transient transfection assays, in the absence of distal negative GRE (nGRE), the -531 OCCA T construct exhibited enhanced promoter activity in response to DEX (1.8 fold DEX/control), but further deletions (-348 and -108 OCCA T constructs) restored D EX suppression to OC promoter activity (0.6- and 0.8-fold DEX/control, respectively).Both distal and proximal GREs specifically bound glucocorticoid receptor present in ROS 17/2.8 and ROB cells as evidenced by competition with wild type and mutated oligonucleotides and antibody inhibition of binding. ROS 17/2.8 cell specific glucocorticoid-receptor complex was detected in nuclear matrix fractions binding to both proximal and distal GREs. Furthermore, both,GREs, independently, conferred DEX-responsive transcriptional repression to the heterologous thymidine kinase basal promoter. Mutations were introduced in the proximal GRE, half GRE in OC box and distal GRE either independently or in combination in context of the full OCCA T and in shorter OCCA T constructs. These mutants were functionally evaluated for ability to confer OC transcriptional activity in response to DEX and vitamin D3 In the absence of distal GRE, mutations in proximal GREs nearly ahn1gated DEX responsiveness of OC promoter activity in shorter OCCAT constructs. In context of full promoter. mutations introduced in the proximal and distal GREs and half GRE in OC box. abrogated DEX responsiveness of OC transcription and also significant abrogation of vitamin D3-mediated enhanced OC transcription was observed. The effect of DEX on OC promoter activity was much more pronounced when -1097 OCCAT construct was stably transfected into ROS 17/2.8 cells (3-4 fold repression compared to 0.7 fold repression in transient transfection assays),the same fold repression was observed for two shorter constructs. Furthermore, this effect required the -531 to -348 domain. which was not implicated in DEX-mediated repression in the transient transfection assays. These results suggest the higher order nuclear structure (e.g. nucleosomal organization, interactions with the nuclear matrix, which is better represented in the stable transfection assays), plays a role in glucocorticoid-mediated regulation of OC transcription. The results presented in this dissertation show that:

(i) In vivo responsiveness of osteocalcin gene to DEX involves the integrated activities of several functional promoter elements.

(ii) Glucocorticoid repression of vitamin D3-stimulated OC transcription occurs independently of distal or proximal GREs.

(iii) The difference observed for deleted OCCA T promoter constructs in response to DEX, in transient compare to stable transfections, functionally demonstrate the contribution of chromatin structure to transcriptional control.

(iv) Several distinct promoter segments were identified that exert either positive or negative effects on transcription in response to DEX. But also coma in regulatory determinants that influence basal and vitamin 03 enhanced OC transcription.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
119.25 KB
2 1 Introduction 1
267.24 KB
  1.1 Developmental Sequence Of The Osteoblast Phenotype 1
  1.2 Osteocalcin Gene 4
  1.3 Transcriptional Regulation Of Osteocalcin Gene 8
  1.4 Hormonal Regulation Of Osteocalcin Gene 14
  1.5 General Effects Of Glucocorticoids On Bone Cells
  1.6 Glucocorticoid Responsive Elements In Osteocalcin Gene 25
3 2 Materials And Methods 30
432.09 KB
  2.1 Materials 31
  2.2 Construction Of Plasmids 32
  2.3 Site Directed Mutagenesis 36
  2.4 Oligonucleotide Primers 36
  2.5 Polymerase Chain Reaction 36
  2.6 Analysis And Isolation Of Pcr Products 38
  2.7 Strategy For Introducing Point Mutation In The Oc Promoter 39
  2.8 Dephosphorylation Of Dna Termini 39
  2.9 Kinase Reactions 42
  2.10 Blunt End Reactions 43
  2.11 Dna Ligation Reaction 43
  2.12 Preparation Of Competent Cells 43
  2.13 Bacterial Transformation 44
  2.14 Isolation And Purification Of Plasmid Dna 45
  2.15 Dna Sequences Analysis 49
  2.16 Isolation And Purification Of Dna Fragments 50
  2.17 Cell Culture And Transient Transfections 52
  2.18 Chloramphenicol Acetyltransferase (Cat) Assay 53
  2.19 Stable Transfections 58
  2.20 Gel Mobility Shift Analysis 59
  2.21 Methylation Interference 63
  2.23 Western Blotting 65
  2.24 Nuclear Run-On Transcription 67
  2.25 Isolation Of Total Cellular Rna 70
  2.26 Northern Blot Analysis 73
  2.27 Ribonuclease Protection Assay 76
  2.28 Statistical Analysis 79
4 3 Results 81
176.26 KB
  3.1 Analysis Of Glucocorticoid Responsiveness Of The Rat Osteocalcin Gene Promoter 82
  3.2 Mutational And Functional Analysis Of Multiple Gres Present In The Rat Oc Promoter 106
5 4 part 2 106
399.46 KB
6 5 Discussion 159
86.73 KB
7 6 References 171
237.24 KB