Nuclear microenvironments: an architectural platform for the convergence and integration of transcriptional regulatory signals

Functional interrelationships between the intranuclear organization of nucleic acids and regulatory proteins are obligatory for fidelity of transcriptional activation and repression. In this article, using the Runx/AML/Cbfa transcription factors as a paradigm for linkage between nuclear structure and gene expression we present an overview of growing insight into the dynamic organization and assembly of regulatory machinery for gene expression at microenvironments within the nucleus. We address contributions of nuclear microenvironments to the convergence and integration of regulatory signals that mediate transcription by supporting the combinatorial assembly of regulatory complexes

Developmental stage-specific cellular responses to vitamin D and glucocorticoids during differentiation of the osteoblast phenotype: interrelationship of morphology and gene expression by in situ hybridization

Fetal rat calvarial-derived osteoblasts, in vitro, undergo a developmental sequence of events leading to bone tissue-like organization and osteoblast differentiation. Previous studies have documented temporal expression of genes reflecting stages of osteoblast phenotype development in relation to tissue organization. Two steroid hormones are known to modify the developmental sequence; 1,25(OH)2D3 can block differentiation when added to proliferating cells, while glucocorticoid addition to proliferating cultures increases the population of cells competent to produce a bone-like matrix and accelerates the differentiation time course. We have addressed the mechanisms contributing to these observations at the single cell level by analysis of a growth-related gene (H4 histone which is coupled with DNA synthesis) and matrix-associated genes (collagen, osteopontin, and osteocalcin) in hormone-treated cells. Our results demonstrate (1) a window of responsiveness for modifications in phenotype development; (2) distinct morphological changes and selective modifications in gene expression in response to both hormones as a function of whether the cell is proliferating or differentiated; and (3) location of the cell with respect to the mineralized nodule was a contributing factor to the levels of gene expression and hormonal responses. In response to vitamin D, surface osteoblasts associated with the nodules became flattened, elongated, and aligned, reminiscent of a bone lining cell. In glucocorticoid-treated cultures, proliferating cells became cuboidal and nodule-associated differentiated cells were approximately one-third the size of control osteoblasts. We also find subsets of hormone-responsive cells in the proliferating cultures in response to glucocorticoid but not vitamin D. In postproliferative cultures, both hormones increased osteocalcin mRNA in the more differentiated osteoblasts associated with the mineralized matrix but no induction occurred in monolayer internodular cells. Osteopontin was induced by glucocorticoid in a larger population of cells. Thus, our studies at the single cell level show selective morphological changes and changes in the level of gene expression supporting the hypothesis that hormones have differential effects on osteoblasts in relation to their stage of phenotype development

Expression of cell growth and bone specific genes at single cell resolution during development of bone tissue-like organization in primary osteoblast cultures

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

Linkages of nuclear architecture to biological and pathological control of gene expression

Functional interrelationships between components of nuclear architecture and control of gene expression are becoming increasingly evident. 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 organization 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 intranuclear targeting signals that direct transcription factors to subnuclear domains where gene expression occurs, reflect linkage of genetic and structural components of transcriptional control. Nuclear reorganization and aberrant intranuclear trafficking of transcription factors for developmental and tissue-specific control that occurs in tumor cells and in neurological disorders provides a basis for high resolution diagnostic and targeted therapy

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

Transcriptional control within the three-dimensional context of nuclear architecture: requirements for boundaries and direction

Evidence is accruing that the architectural organization of nucleic acids and regulatory proteins within the cell nucleus support functional interrelationships between nuclear structure and gene expression. The punctate distribution of several transcription factors has provided several paradigms for pursuing mechanisms that direct these regulatory proteins to subnuclear sites where gene activation or suppression occurs. Sequences that are necessary and sufficient to direct regulatory proteins to transcriptionally active nuclear domains have been identified. Mutations that disrupt intranuclear targeting signals lead to modified subnuclear distribution of transcription factors and aberrant expression in tumor cells. J. Cell. Biochem. Suppls. 32/33:24-31, 1999

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

Properties of blood-contacting surfaces of clinically implanted cardiac assist devices: gene expression, matrix composition, and ultrastructural characterization of cellular linings

The development of implantable cardiac assist devices for prolonged circulatory support has been impeded by the problem of excessive thrombogenesis on the blood-prosthetic interface, with subsequent embolization. To overcome this obstacle, a ventricular assist device has been developed with textured blood-contacting surfaces to encourage the formation of a tightly adherent, hemocompatible, biological lining. In this study, we applied molecular biological techniques, in conjunction with conventional ultrastructural and biochemical techniques, to characterize the biological linings associated with the blood-contacting surfaces of 11 of these devices, which had been clinically implanted for durations ranging from 21 to 324 days. No clinical thromboembolic events or pump-related thromboembolism occurred. Biological linings developed on the textured surfaces composed of patches of cellular tissue intermingled with areas of compact fibrinous material. In addition, islands of collagenous tissue containing fibroblast-like cells appeared after 30 days of implantation. Many of these cells contained microfilaments with dense bodies indicative of myofibroblasts. RNA hybridization analyses demonstrated that the colonizing cells actively expressed genes encoding proteins for cell proliferation (histones), adhesion (fibronectin), cytoskeleton (actin, vimentin) and extracellular matrix (types I and III collagen). Linings, which never exceeded 150 microns in thickness, remained free of pathological calcification. Textured blood-contacting surfaces induced the formation of a thin, tightly adherent, viable lining which exhibited excellent long-term hemocompatibility

Insight into regulatory factor targeting to transcriptionally active subnuclear sites

Mechanisms that coordinate the spatial organization of genes and regulatory proteins within the three-dimensional context of nuclear architecture contribute to the sorting of regulatory information as well as the assembly and activity of sites within the nucleus that support gene expression. In this article we will present an overview of experimental approaches that provide insight into the trafficking of the hematopoietic and bone-specific AML/CBF family of regulatory factors to transcriptionally active subnuclear sites