The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation was examined in primary diploid cultures of fetal calvarial derived osteoblasts by the combined use of autoradiography, histochemistry, biochemistry, and mRNA assays of osteoblast cell growth and phenotypic genes. Modifications in gene expression define a developmental sequence that has 1) three principle periods--proliferation, extracellular matrix maturation, and mineralization--and 2) two restriction points to which the cells can progress but cannot pass without further signals--the first when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle- and cell growth-regulated genes, produce a fibronectin/type I collagen extracellular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phosphatase immediately following the proliferative period, and later, an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited by hydroxyurea; and 3) enhanced levels of expression of the osteoblast markers as a function of ascorbic acid-induced collagen deposition, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and the development of the osteoblast phenotype.

We present an overview of Runx involvement in regulatory mechanisms that are requisite for fidelity of bone cell growth and differentiation, as well as for skeletal homeostasis and the structural and functional integrity of skeletal tissue. Runx-mediated control is addressed from the perspective of support for biological parameters of skeletal gene expression. We review recent findings that are consistent with an active role for Runx proteins as scaffolds for integration, organization and combinatorial assembly of nucleic acids and regulatory factors within the three-dimensional context of nuclear architecture.

The AML͞CBF␣ runt transcription factors are key regulators of hematopoietic and bone tissue-specific gene expression. These factors contain a 31-amino acid nuclear matrix targeting signal that supports association with the nuclear matrix. We determined that the AML͞CBF␣ factors must bind to the nuclear matrix to exert control of transcription. Fusing the nuclear matrix targeting signal to the GAL4 DNA binding domain transactivates a genomically integrated GAL4 responsive reporter gene. These data suggest that AML͞CBF␣ must associate with the nuclear matrix to effect transcription. We used f luorescence labeling of epitopetagged AML-1B (CBFA2) to show it colocalizes with a subset of hyperphosphorylated RNA polymerase II molecules concentrated in foci and linked to the nuclear matrix. This association of AML-1B with RNA polymerase II requires active transcription and a functional DNA binding domain. The nuclear matrix domains that contain AML-1B are distinct from SC35 RNA processing domains. Our results suggest two of the requirements for AML-dependent transcription initiation by RNA polymerase II are association of AML-1B with the nuclear matrix together with specific binding of AML to gene promoters.Many observations suggest a linkage between nuclear architecture and the regulation of gene expression. These include drastic changes in nuclear morphology often seen in differentiation and in transformation to malignancy. The filamentous ribonucleoprotein network known as the nuclear matrix is a major component of nuclear structure (1-5). It serves to localize gene sequences and may maintain the distribution of regulatory factors throughout the nuclear space (6-19).The AML͞CBF␣ transcription factors are key regulators of hematopoietic and bone-tissue-specific gene expression (20)(21)(22)(23). We have recently shown that the transcriptionally active AML factors contain a specific sequence that targets them to the nuclear matrix (24). In contrast, several inactive forms of AML that lack this targeting signal do not associate with the matrix (24). The targeting sequence resides in a 31-aa segment (nuclear matrix targeting signal, NMTS; aa 351-381) within the C-terminal domain. This NMTS is physically distinct from the nuclear localization signal and functions autonomously to direct AML-1B (CBFA2) to the nuclear matrix (24). These findings suggest that association with nuclear structure may be an important aspect of the mechanisms of gene regulation.In this report we begin to examine the significance of the AML-1B transcription factor binding to the nuclear matrix. We show that AML-1B localizes to sites where there is also active transcription. We demonstrate that the NMTS, which directs AML-1B to the nuclear matrix, functions as a transactivation domain when interacting with an appropriate promoter. Furthermore, we show that the AML-1B transcription factor is targeted to discrete sites on the nuclear matrix. These sites also contain the hyperphosphorylated active form of RNA polymerase II. Colocalization of AML...

Postmitotic gene expression requires restoration of nuclear organization and assembly of regulatory complexes. The hematopoietic and osteogenic Runx (Cbfa͞AML) transcription factors are punctately organized in the interphase nucleus and provide a model for understanding the subnuclear organization of tissue-specific regulatory proteins after mitosis. Here we have used quantitative in situ immunofluorescence microscopy and quantitative image analysis to show that Runx factors undergo progressive changes in cellular localization during mitosis while retaining a punctate distribution. In comparison, the acetyl transferase p300 and acetylated histone H4 remain localized with DNA throughout mitosis while the RNA processing factor SC35 is excluded from mitotic chromatin. Subnuclear organization of Runx foci is completely restored in telophase, and Runx proteins are equally partitioned into progeny nuclei. In contrast, subnuclear organization of SC35 is restored subsequent to telophase. Our results show a sequential reorganization of Runx and its coregulatory proteins that precedes restoration of RNA processing speckles. Thus, mitotic partitioning and spatiotemporal reorganization of regulatory proteins together render progeny cells equivalently competent to support phenotypic gene expression.I n the interphase nucleus, many tissue-restricted transcription factors are architecturally organized at punctate subnuclear sites that are associated with the nuclear matrix scaffold (1-18). These nuclear matrix-associated intranuclear foci are linked to transcriptional activation and suppression and contain coregulatory proteins and signaling molecules (19-22, ‡). Compromised nuclear matrix targeting and͞or altered gene dosage of regulatory proteins is associated with pathological conditions (23-25). Gross alteration of subnuclear organization (26-30) and relocalization of regulatory complexes occur concomitant with transcriptional silencing during mitosis (31-33); therefore, a fundamental question is how cells restore subnuclear distribution of tissue-specific transcription factors in progeny cells to regulate postmitotic phenotypic gene transcription.Runx (Cbfa͞AML) proteins are tissue-specific transcription factors that control hematopoietic and osteogenic lineage commitment (reviewed in ref. 34). Runx factors bind to DNA in a sequence-specific manner, are targeted to transcriptionally active subnuclear foci, and are required for the maintenance of chromatin architecture of target genes in the interphase nucleus (11)(12)(13)(35)(36)(37). Perturbed subnuclear organization and͞or altered physiological levels of Runx proteins are associated with genetic disorders and tumorigenesis (23-25, 38, 39). Runx protein levels persist through the proliferation of lineage-committed cells (40).Although the rules that govern mitotic chromosome segregation are longstanding (41), only a limited number of studies have addressed redistribution of regulatory proteins during mitosis (42)(43)(44)(45)(46). By the combined use of in situ immunofluor...

Primary cultures of calvarial derived normal diploid osteoblasts undergo a developmental expression of genes reflecting growth, extracellular matrix maturation, and mineralization during development of multilayered nodules having a bone tissue-like organization. Scanning electron microscopy of the developing cultures indicates the transition from the uniform distribution of cuboidal osteoblasts to multilayered nodules of smaller cells with a pronounced orientation of perinodular cells towards the apex of the nodule. Ultrastructural analysis of the nodule by transmission electron microscopy indicates that the deposition of mineral is confined to the extracellular matrix where cells appear more osteocytic. The cell body contains rough endoplasmic reticulum and golgi, while these intracellular organelles are not present in the developing cellular processes. To understand the regulation of temporally expressed genes requires an understanding of which genes are selectively expressed on a single cell basis as the bone tissue-like organization develops. In situ hybridization analysis using 35S labelled histone gene probes, together with 3H-thymidine labelling and autoradiography, indicate that greater than 98% of the pre-confluent osteoblasts are proliferating. By two weeks, both the foci of multilayered cells and internodular cell regions have down-regulated cell growth associated genes. Post-proliferatively, but not earlier, initial expression of both osteocalcin and osteopontin are restricted to the multilayered nodules where all cells exhibit expression. While total mRNA levels for osteopontin and osteocalcin are coordinately upregulated with an increase in mineral deposition, in situ hybridization has revealed that expression of osteocalcin and osteopontin occurs predominantly in cells associated with the developing nodules. In contrast, proliferating rat osteosarcoma cells (ROS 17/2.8) concomitantly express histone H4, along with osteopontin and osteocalcin. These in situ analyses of gene expression during osteoblast growth and differentiation at the single cell level establish that a population of proliferating calvarial-derived cells subsequently expresses osteopontin and osteocalcin in cells developing into multilayered nodules with a tissue-like organization.