No abstract

Nucleic acids and regulatory proteins are compartmentalized in microenvironments within the nucleus. This subnuclear organization may support convergence and the integration of physiological signals for the combinatorial control of gene expression, DNA replication and repair. Nuclear organization is modified in many cancers. There are cancer-related changes in the composition, organization and assembly of regulatory complexes at intranuclear sites. Mechanistic insights into the temporal and spatial organization of machinery for gene expression within the nucleus, which is compromised in tumours, provide a novel platform for diagnosis and therapy.

Chromatin remodeling of the bone-specific rat osteocalcin (OC) gene accompanies the onset and increase in OC expression during osteoblast differentiation. In osseous cells expressing OC, the promoter region contains two nuclease hypersensitive sites that encompass the elements that regulate basal tissue-specific and vitamin D-enhanced OC transcription. Multiple lines of evidence indicate that DNA methylation is involved in maintaining a stable and condensed chromatin organization that represses eukaryotic transcription. Here we report that DNA methylation at the OC gene locus is associated with the condensed chromatin structure found in cells not expressing OC. In addition, we find that reduced CpG methylation of the OC gene accompanies active transcription in ROS 17/2.8 rat osteosarcoma cells. Interestingly, during differentiation of primary diploid rat osteoblasts in culture, as the OC gene becomes increasingly expressed, CpG methylation of the OC promoter is significantly reduced. Inhibition of OC transcription does not occur by a direct mechanism because in vitro methylated OC promoter DNA is still recognized by the key regulators Runx/Cbfa and the vitamin D receptor complex. Furthermore, CpG methylation affects neither basal nor vitamin D-enhanced OC promoter activity in transient expression experiments. Together, our results indicate that DNA methylation may contribute indirectly to OC transcriptional control in osteoblasts by maintaining a highly condensed and repressed chromatin structure.

Development of bone tissue requires maturation of osteoblasts from mesenchymal precursors. BMP2, a member of the TGFbeta superfamily, and the Runx2 (AML3/Cbfa1) transcription factor, a downstream BMP2 effector, are regulatory signals required for osteoblast differentiation. While Runx2 responsive osteogenic gene expression has been functionally linked to alterations in chromatin structure, the factors that govern this chromatin remodeling remain to be identified. Here, we address the role of the SWI/SNF chromatin remodeling enzymes in BMP2-induced, Runx2-dependent development of the osteoblast phenotype. For these studies, we have examined calvarial cells from wild-type (WT) mice and mice that are homozygous for the Runx2 null allele, as well as the C2C12 model of BMP2-induced osteogenesis. By the analysis of microarray data, we find that several components of the SWI/SNF complex are regulated during BMP2-mediated osteoblast differentiation. Brg1 is an essential DNA dependent ATPase subunit of the SWI/SNF complex. Thus, functional studies were carried out using a fibroblast cell line that conditionally expresses a mutant Brg1 protein, which exerts a dominant negative effect on SWI/SNF function. Our findings demonstrate that SWI/SNF is required for BMP2-induced expression of alkaline phosphatase (APase), an early marker reflecting Runx2 control of osteoblast differentiation. In addition, Brg1 is expressed in cells within the developing skeleton of the mouse embryo as well as in osteoblasts ex vivo. Taken together these results support the concept that BMP2-mediated osteogenesis requires Runx2, and demonstrates that initiation of BMP2-induced, Runx2-dependent skeletal gene expression requires SWI/SNF chromatin remodeling complexes.