Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 4 uppsatser.

Thesis (M.S.)--University of Toledo, 2007 / "In partial fulfillment of the requirements for the degree of Master of Science in Biomedical Sciences." Title from title page of PDF document. Bibliography: p. 37-48, 62-84.

Thesis (Ph. D.)--Case Western Reserve University, 2006. / [School of Medicine] Department of Pathology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

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.

Chromatin has a compact organization in which most DNA sequences are structurally inaccessible and functionally inactive. Reconfiguration of thechromatir required to activate transcription. This reconfiguration is achieved by the action of enzymes that covalently modify nucleosomal core histones, and by enzymes that disrupt histone-DNA interactions via ATP hydrolysis. TheSWI/SNF family of ATP-dependent chromatin remodeling enzymes has been implicated not only in gene activation but also in numerous cellular processes including differentiation, gene repression, cell cycle control, recombination and DNA repair. PPARγ, C/EBPα and C/EBPβ are transcription factors with well established roles in adipogenesis. Ectopical expression of each of these factors in non-adipogenic cells is sufficient to convert them to adipocyte-like cells. To determine the requirements of SWI/SNF enzymes in adipocyte differentiation, we introduced PPARγ, C/EBPα or C/EBPβ into fibroblasts that inducibly express dominant-negative versions of the Brahma-Related Gene 1 (BRG1) or human Brahma (BRM), which are the ATPase subunits of the SWI/SNF enzymes. We found that adipogenesis and expression of adipocyte genes were inhibited in the presence of mutant SWI/SNF enzymes. Additionally, in cells expressing C/EBPα or C/EBPβ, PPARγ expression was SWI/SNF dependent. These data indicate the importance of these remodeling enzymes in both early and late gene activation events. Subsequently, we examined by chromatin immunoprecipitation (ChIP) assay the functional role of SWI/SNF enzymes in the activation of PPARγ2, the master regulator of adipogenesis. Temporal analysis of factors binding to the PPARγ2 promoter showed that SWI/SNF enzymes are required to promote preinitiation complex assembly and function. Additionally, our studies concentrated on the role of C/EBP family members in the activation of early and late genes during adipocyte differentiation. During adipogenesis, C/EBPβ and δ are rapidly and transiently expressed and are involved in the expression of PPARγ and C/EBPα, which together activate the majority of the adipocyte genes. Our studies determined the temporal recruitment of the C/EBP family at the promoters of early and late genes by ChIP assay during adipocyte differentiation. We found that all of the C/EBP members evaluated are present at the promoters of early and late genes, and the binding correlated with the kinetics of the C/EBPs expression. Binding of C/EBPβ and δ is transient, subsequently being replaced by C/EBPα. These studies demonstrated that C/EBPβ and δ are not only involved in the regulation of PPARγ and C/EBPα, but also in the activation of late expressed adipocyte genes.