Modifications of protein-DNA interactions in the proximal promoter of a cell-growth-regulated histone gene during onset and progression of osteoblast differentiation.

Abstract

A temporal sequence of interrelated cellular, biochemical, and molecular events which occurs during the progressive expression of the differentiated osteoblast phenotype in primary cultures of fetal rat calvarial cells results in the development of a bone-tissue-like organization. This ordered developmental sequence encompasses three periods: proliferation, matrix maturation, and mineralization. Initially, the cells actively proliferate and synthesize type I collagen. This is followed by a period of matrix organization and maturation and then by a period of extracellular matrix mineralization. At the completion of proliferation, when expression of osteoblast phenotype markers such as alkaline phosphatase is observed, the cell-cycle-related histone genes are down-regulated transcriptionally, suggesting that a key signaling mechanism at this transition point involves modifications of protein-DNA interactions in the regulatory elements of these growth-regulated genes. Our results demonstrate that there is a selective loss of interaction of the promoter binding factor HiNF-D with the site II region of an H4 histone gene proximal promoter that regulates the specificity and level of transcription only when the down-regulation of proliferation is accompanied by modifications in the extracellular matrix that contribute to progression of osteoblast differentiation. Thus, this specific loss of protein-DNA interaction serves as a marker for a key transition point in the osteoblast developmental sequence, where the downregulation of proliferation is functionally coupled to the appearance of osteoblast phenotypic properties associated with the organization and maturation of an extracellular matrix that becomes competent to mineralize.Defining the cellular, biochemical, and molecular events associated with osteoblast development and differentiation can provide valuable understanding of bone formation and a broad spectrum of bone-related diseases. Primary cultures of osteoblasts isolated from the calvaria of fetal rats undergo during a 35-day period an ordered developmental sequence in which the cells progressively acquire the phenotypic properties of mature osteoblasts in a mineralized extracellular matrix having a bone-tissue-like organization (1-4). The temporal expression of genes observed in cultured osteoblasts (3,5,6) is similar to that during fetal formation of the calvarium in intact animals (7), supporting the biological relevance of the culture system. This series of changes may be used to define three periods during the development of the osteoblast phenotype where, in this normal diploid cell culture system, the regulatory mechanisms operative in the relationship between proliferation and differentiation can be expected to be retained. The osteoblasts undergo proliferation during the initial period following isolation of primary cultures, as reflected by[3H]thymidine incorporation and H4 histone gene expression, which parallel DNA synthesis. Expression of type I collagen genes during the proliferative peri...

Overlapping and CpG methylation-sensitive protein-DNA interactions at the histone H4 transcriptional cell cycle domain: distinctions between two human H4 gene promoters.

“…The correlation between cessation of cell proliferative activity, transcriptional downregulation of histone gene expression, and loss of HiNF-D binding activity is also evident in other cell culture systems, including during differentiation of primary rat calvarial osteoblasts (40) (28). Similarly, nuclear factor HiNF-A, which recognizes AST-rich DNA sequences in histone H4, H3, and Hi promoters, is a cell cycle-independent DNA-binding protein irrespective of cell type (61 (26,44) and genomic protection assays (42,51).…”

“…2B) Table 1 for references containing experimental details and supporting data). Factor HiNF-D has been isolated from a broad spectrum of proliferating cell types in our laboratory (3,23,40,57,58,64,71), as well as other laboratories (23,58,71) (27,29,38,44 (7) genes, although expression of a variant poly(A)+ H2B gene is increased (6). Therefore, these results for H4 site II in vivo and HiNF-D in vitro demonstrate that selective modifications occur in protein-DNA interactions when histone gene transcription is modulated.…”

Genetic Ablation of the CDP/Cux Protein C Terminus Results in Hair Cycle Defects and Reduced Male Fertility

“…Phosphorylation by protein kinase C and casein kinase II or PCAF-mediated acetylation of the CDP/Cux homeodomain inhibits DNA binding activity (6,8,21). Stable CDP/Cux-DNA complexes are detected in proliferating cells but disappear upon cellular differentiation of HL60 promyelocytic leukemia cells and fetal rat calvarial cells (22,27,28). CDP/Cut has been shown to bind to a variety of promoters or enhancer sequences of genes involved in differentiation, including myeloid cytochrome gp91-phox (28), dog heart myosin heavy chain (1), rat tyrosine hydroxylase (50), human ␥-globin (33), Xenopus ␤-globin, and mouse N-CAM (36).…”

In Vitro Evaluation of the Efficacy of a New Laser Surface Implant: Cellular Adhesion and Alkaline Phosphatase Production Tests

“…ALP, a precocious marker of osteogenetic differentiation, is found in high quantities in the cells in the phase ofmineralization ofthe matrix, as are the osteoblasts (24). Recent studies have demonstrated that the physical and chemical characteristics of the implant surface influence the adhesion, proliferation, differentiation and maturation of bone celt's (25)(26).…”