Shirwin M. Pockwinse

Runx2 control of organization, assembly and activity of the regulatory machinery for skeletal gene expression

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.

Mitotic partitioning and selective reorganization of tissue-specific transcription factors in progeny cells

Postmitotic gene expression requires restoration of nuclear organization and assembly of regulatory complexes. The hematopoietic and osteogenic Runx (Cbfa/AML) 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.

Ultrastructural investigations of bone resorptive cells in two types of autosomal dominant osteopetrosis

In order to investigate the ultrastructure of bone resorptive cells in the two types of adult benign human osteopetrosis, iliac crest biopsies were obtained from 11 patients and 10 normal males, who served as a control group. Six patients had the radiological type I (4 women, 2 men, aged 23-58 years, mean = 36.5 years), and 5 type II disease (5 men, aged 20-48 years, mean = 29.8 years). The normal controls (aged 23-48 years, mean 34.1 years) were recruited from the medical staff. The biopsies were immediately divided. From each patients, half was embedded in paraffin for histochemistry and light microscopy, and half in epon for transmission electron microscopy. The osteoclasts were markedly reduced in number and size in Type I disease (0.2 +/- .7 cells vs. 2.9 +/- 1.0 cells per 2.7 mm2 of bone area, p < 0.01) compared to controls, and stained only weakly for tartrate-resistant acid phosphatase (TRAP). At the ultrastructural level, no signs of active bone resorption were identified, whereas numerous mononuclear cells were observed at the bone surfaces. In type II disease, the osteoclasts were large and highly multi-nucleated, with an increased number (8.3 +/- 2.3 cells vs. 2.9 +/- 1.0. cells per 2.7 mm2 of bone area, p < 0.01) compared to controls. In all patients with this type, but never in type I or in the controls, a smooth, TRAP-positive substance was seen between the osteoclasts and the bone surface. Ultrastructurally, this substance was amorphous, with a condensation along the cell membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Interrelationships of nuclear structure and transcriptional control: functional consequences of being in the right place at the right time.

Functional interrelationships between components of nuclear architecture and control of gene expression are becoming increasingly evident. In this article we focus on the concept that association of genes and cognate transcription factors with the nuclear matrix may support the formation and/or activities of nuclear domains that facilitate transcriptional regulation. Several lines of evidence are consistent with the concept that association of transcription factors with the nuclear matrix may be obligatory for fidelity of gene expression and maximal transcriptional activity. The identification of specific regions of transcription factors that are responsible for intranuclear trafficking to nuclear matrix‐associated sites that support transcription, reinforces the linkage of nuclear structure to regulation of genes. CBFA2/AML‐1 and CBFA1/AML‐3 provide paradigms for directing gene regulatory factors to RNA polymerase II containing foci within the nucleus. The implications of modifications in the intranuclear trafficking of transcription factors for developmental and tissue‐specific control, as well as for aberrations in gene expression that are associated with cancer and neurological disorders, are addressed. J. Cell. Biochem. 70:200–212, 1998.

Microtubule-dependent nuclear-cytoplasmic shuttling of Runx2

RUNX/AML transcription factors are critical regulators of cell growth and differentiation in multiple lineages and have been linked to human cancers including acute myelogenous leukemia (RUNX1), as well as breast (RUNX2) and gastric cancers (RUNX3). RUNX proteins are targeted to gene regulatory micro‐environments within the nucleus via a specific subnuclear targeting signal. However, the dynamics of RUNX distribution and compartmentalization between the cytoplasm and nucleus is minimally understood. Here we show by immunofluorescence microscopy that RUNX2 relocates from the nucleus to the cytoplasm when microtubules are stabilized by the chemotherapeutic agent taxol. The taxol‐dependent cytoplasmic accumulation of RUNX2 is inhibited by leptomycin B, which blocks CRM‐1 dependent nuclear export, and is not affected by the protein synthesis inhibitor cycloheximide. Using biochemical assays, we show that endogenous RUNX2 associates with stabilized microtubules in a concentration‐dependent manner and that the RUNX2 amino terminus mediates the microtubule association. In soluble fractions of cells, RUNX2 co‐immunoprecipitates α tubulin suggesting that microtubule binding involves the α/β tubulin subunits. We conclude that RUNX2 associates with microtubules and shuttles between the nucleus and the cytoplasm. We propose that nuclear‐cytoplasmic shuttling of RUNX2 may modulate its transcriptional activity, as well as its ability to interface with signal transduction pathways that are integrated at RUNX2 containing subnuclear sites. It is possible that taxol‐induced acute depletion of the nuclear levels of RUNX2 and/or other cell growth regulatory factors may represent an alternative pathway by which taxol exerts its biological effects during cancer chemotherapies. J. Cell. Physiol. 206: 354–362, 2006.

Nuclear microenvironments support assembly and organization of the transcriptional regulatory machinery for cell proliferation and differentiation.

The temporal and spatial organization of transcriptional regulatory machinery provides microenvironments within the nucleus where threshold concentrations of genes and cognate factors facilitate functional interactions. Conventional biochemical, molecular, and in vivo genetic approaches, together with high throughput genomic and proteomic analysis are rapidly expanding our database of regulatory macromolecules and signaling pathways that are requisite for control of genes that govern proliferation and differentiation. There is accruing insight into the architectural organization of regulatory machinery for gene expression that suggests signatures for biological control. Localized scaffolding of regulatory macromolecules at strategic promoter sites and focal compartmentalization of genes, transcripts, and regulatory factors within intranuclear microenvironments provides an infrastructure for combinatorial control of transcription that is operative within the three dimensional context of nuclear architecture.

Implications for interrelationships between nuclear architecture and control of gene expression under microgravity conditions

Components of nuclear architecture are functionally interrelated with control of gene expression. There is growing appreciation that multiple levels of nuclear organization integrate the regulatory cues that support activation and suppression of genes as well as the processing of gene transcripts. The linear representation of genes and promoter elements provide the potential for responsiveness to physiological regulatory signals. Parameters of chromatin structure and nucleosome organization support synergism between activities at independent regulatory sequences and render promoter elements accessible or refractory to transcription factors. Association of genes, transcription factors, and the machinery for transcript processing with the nuclear matrix facilitates fidelity of gene expression within the three‐dimensional context of nuclear architecture. Mechanisms must be defined that couple nuclear morphology with enzymatic parameters of gene expression. The recent characterization of factors that mediate chromatin remodeling and identification of intranuclear targeting signals that direct transcription factors to subnuclear domains where gene expression occurs link genetic and structural components of transcriptional control. Nuclear reorganization and aberrant intranuclear trafficking of transcription factors for developmental and tissue‐specific control occurs in tumor cells and in neurological disorders. Compromises in nuclear structure‐function interrelationships can occur as a consequence of microgravity‐mediated perturbations in cellular architecture.—Stein, G. S., van Wijnen, A. J., Stein, J. L., Lian, J. B., Pockwinse, S. H., McNeil, S. Implications for interrelationships between nuclear architecture and control of gene expression under microgravity conditions. FASEB J. 13 (Suppl.), S157–S166 (1999)

Live cell imaging of the cancer‐related transcription factor RUNX2 during mitotic progression

The nuclear matrix bound transcription factor RUNX2 is a lineage‐specific developmental regulator that is linked to cancer. We have previously shown that RUNX2 controls transcription of both RNA polymerase II genes and RNA polymerase I‐dependent ribosomal RNA genes. RUNX2 is epigenetically retained through mitosis on both classes of target genes in condensed chromosomes. We have used fluorescence recovery after photobleaching to measure the relative binding kinetics of enhanced green fluorescent protein (EGFP)‐RUNX2 at transcription sites in the nucleus and nucleoli during interphase, as well as on mitotic chromosomes. RUNX2 becomes more strongly bound as cells go from interphase through prophase, with a doubling of the most tightly bound “immobile fraction.” RUNX2 exchange then becomes much more facile during metaphase to telophase. During interphase the less tightly bound pool of RUNX2 exchanges more slowly at nucleoli than at subnuclear foci, and the non‐exchanging immobile fraction is greater in nucleoli. These results are consistent with a model in which the molecular mechanism of RUNX2 binding is different at protein‐coding and ribosomal RNA genes. The binding interactions of RUNX2 change as cells go through mitosis, with binding affinity increasing as chromosomes condense and then decreasing through subsequent mitotic phases. The increased binding affinity of RUNX2 at mitotic chromosomes may reflect its epigenetic function in “bookmarking” of target genes in cancer cells. J. Cell. Physiol. 226: 1383–1389, 2011.

Gene expression at single cell resolution associated with development of the bone cell phenotype : ultrastructural and in situ hybridization analysis

The ability to culture normal diploid calvarial cells under conditions that support progressive development of the osteoblast phenotype and a bone tissue-like organization has provided a viable in vitro model system for examining the selective expression of genes and physiological signalling mechanisms that mediate osteoblast growth and differentiation. The tissue-like organization in vitro is reflected by the progressive development of nodules of multilayered cells in a mineralized extracellular matrix with orthogonally organized type I collagen fibrils. The ordered deposition of mineral within the collagen fibers initiates and is primarily associated with the nodule areas (Owen et al., 1990; Pockwinse et al., 1992; Bhargava et al., 1988). A sequential expression of genes has defined a developmental sequence in primary cultures of normal diploid osteoblasts that contains three principal periods and two transition points where signalling mechanisms in the regulation of osteoblast phenotype expression are operative (Owen et al., 1990; Stein et al., 1990). During the first period, actively proliferating osteoblasts express a series of genes supporting cell growth and extracellular matrix biosynthesis, including histone, TGFB, tibronectin and collagen. At the first transition point with the down-regulation of proliferation, expression of genes involved with the maturation and organization of an extracellular matrix competent for mineralization is initiated, e.g. alkaline phosphatase mRNA and enzyme activity peaks. Then, at the second principal transition point, extracellular matrix mineralization is initiated, and defines the onset of the third period, at which time expression of genes which include osteopontin and osteocalcin are up-regulated. Two key questions that necessitate resolution are concerns of: 1.) potential heterogeneity of the isolated cells that would generate discrete bone-like nodules; and 2.) the extent to which the temporal expression of genes and peak periods of gene expression coincide with discrete areas of cellular organization or reflect a uniform level of expression in all cells throughout the culture. We therefore utilized in situ hybridization to examine histone, osteocalcin and osteopontin gene expression at the single cell level during key periods of the osteoblast developmental sequence in relationship to modifications in cell ultrastructure and organization as revealed by transmission electron microscopy and scanning electron microscopy. Hormone responsiveness of the cells in relation to developing tissue organization was also examined.

Skeletal development and formation of osteoclast-like cells from in situ progenitors in fetal mouse metatarsals cultured in chemically defined medium

An in vitro model system is described, using metatarsal explants from 15-day mouse embryos (E15) cultured in serumless chemically defined medium, to study fetal skeletal development with particular emphasis on de novo osteoclast formation. The normal pattern of growth and differentiation observed in vitro, assessed by ultrastructure and morphometry, demonstrate a permissive local environment which replicates physiologic temporal and spatial relationships which exist in vivo. The population of committed osteoclast progenitors present in E15 metatarsals form osteoclasts and precursors which have cytochemical and ultrastructural features, as well as kinetics of formation, that are similar to that which occurs in vivo. The responsiveness of osteoclast formation to the effects of added 1,25(OH)2D3 illustrates that controlled manipulation enables one to exploit the system for investigating the role of cytokines, growth factors and osteotropic hormones in skeletal development and osteoclast ontogeny.