Janet L Stein

University of Vermont, Burlington, Vermont, United States

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Publications (315)1482.02 Total impact

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    ABSTRACT: BACKGROUND The transplantation of multipotential bone marrow cells containing bone tissue specific promoter-controlled transgenes provides an efficacious approach to deliver therapeutic gene expression to osteoblasts for the treatment of patients with bone disorders or tumor metastasis to the skeleton. The specificity of tissue-restricted gene therapy can be refined by utilization of a 31-amino-acid segment of the hematopoietic and osteogenic AML/CBF transcription factors that direct the regulatory proteins to subnuclear sites that support gene expression.METHODS Unfractionated adherent bone marrow cells from transgenic mice constructed with the proximal 1.7 kb of the osteocalcin gene promoter fused to a CAT reporter were transplanted by intravenous infusion. Engraftment and expression at the single-cell level within the context of tissue organization was established by immunohistochemistry using an anti-CAT antibody. Sequences that support the intranuclear trafficking of AML/CBF transcription factors to subnuclear sites that support transcription were determined by the expression and visualization of mutated and epitope tagged AML/CBF proteins.RESULTSImmunohistochemical staining of an extensive series of tissue sections from mice posttransplantation using an anti-CAT antibody indicated that CAT-positive osteoblasts and osteocytes were present in bone sections. These findings indicate that donor bone marrow-derived cells engraft in bone tissue in an environment that supports maturation to the developmental stage at which a bone specific osteocalcin promoter is transcriptionally active. Characterization of functional domains in AML/CBF transcription factors has established that there are at least two regulated events that are required for targeting the factors to transcriptionally active nuclear domains: A nuclear localization signal in the amino terminal region controls nuclear import and retention, and a nuclear matrix targeting signal in the carboxyl region controls association with nuclear matrix-linked sites where transcription occurs.CONCLUSIONS The specificity of hematopoietic and bone phenotypic promoters, together with the additional level of specificity inherent in the AML/CBF family of hematopoietic and osteogenic intranuclear targeting signals, offers viable options for constructing gene therapy regimens that are targeted to the skeleton for the control of metastatic disease. It is realistic to anticipate that, as additional parameters of gene regulatory mechanisms are defined, particularly components of transcriptional control that are operative within a three-dimensional context of nuclear architecture, opportunities for enhancing the effectiveness of treating patients with tumors that metastasize to bone will be extended. Cancer 2000;88:2899–902. © 2000 American Cancer Society.
    Cancer 06/2000; 88(S12):2899 - 2902. DOI:10.1002/1097-0142(20000615)88:12+<2899::AID-CNCR3>3.0.CO;2-O · 4.90 Impact Factor
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    ABSTRACT: Acute leukemias arise secondary to chromosomal aberrations that cause dysfunctions in gene regulation and regulatory factors. Significant differences in morphology between acute leukemic and nonleukemic hematopoietic cells are readily observed. How morphologic changes of the nuclei of acute leukemic cells relate to the underlying functional alterations of gene expression is minimally understood. Spatial modifications in the representation and/or organization of regulatory factors may be functionally linked to perturbations of gene expression in acute leukemic cells. Using in situ immunofluorescence microscopy, we addressed the interrelationships of modifications in nuclear morphology with the intranuclear distribution of leukemia-related regulatory factors (including ALL-1, PML, and AF-9) in cells from patients with acute leukemia. We compared the localization of leukemia-associated proteins with various factors involved in gene transcription and RNA processing (e.g., RNA polymerase II and SC-35). Our findings suggest that there are leukemia-associated aberrations in mechanisms that direct regulatory factors to sites within the nucleus. This misplacement of key cognate factors may contribute to perturbations in gene expression characteristic of leukemias.
    Journal of Cellular Biochemistry 03/2000; 77(1):30-43. DOI:10.1002/(SICI)1097-4644(20000401)77:13.0.CO;2-K · 3.37 Impact Factor
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    ABSTRACT: The runt related transcription factor CBFA1 (AML3/PEBP2alphaA/RUNX2) regulates expression of several bone- and cartilage-related genes and is required for bone formation in vivo. The gene regulatory mechanisms that control activation and repression of CBFA1 gene transcription during osteoblast differentiation and skeletal development are essential for proper execution of the osteogenic program. We have therefore defined functional contributions of 5' regulatory sequences conserved in rat, mouse and human CBFA1 genes to transcription. Deletion analysis reveals that 0.6 kB of the bone-related rat or mouse CBFA1 promoter (P1, MASNS protein isoform) is sufficient to confer transcriptional activation, and that there are multiple promoter domains which positively and negatively regulate transcription. Progressive deletion of promoter segments between nt -351 and -92 causes a striking 30- to 100-fold combined decrease in promoter activity. Additionally, 5' UTR sequences repress reporter gene transcription 2- to 3-fold. Our data demonstrate that CBFA1 is a principal DNA binding protein interacting with the 5' region of the CBFA1 gene in osseous cells, that there are at least three CBFA1 recognition motifs in the rat CBFA1 promoter, and that there are three tandemly repeated CBFA1 sites within the 5' UTR. We find that forced expression of CBFA1 protein downregulates CBFA1 promoter activity and that a single CBFA1 site is sufficient for transcriptional autosuppression. Thus, our data indicate that the CBFA1 gene is autoregulated in part by negative feedback on its own promoter to stringently control CBFA1 gene expression and function during bone formation.
    Journal of Cellular Physiology 01/2000; 184(3):341-50. DOI:10.1002/1097-4652(200009)184:3<341::AID-JCP8>3.0.CO;2-Z · 3.87 Impact Factor
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    ABSTRACT: The regulated and regulatory components that interrelate nuclear structure and function must be experimentally established. A formidable challenge is to define further the control of transcription factor targeting to acceptor sites associated with the nuclear matrix. It will be important to determine whether acceptor proteins are associated with a pre-existing core-filament structural lattice or whether a compositely organized scaffold of regulatory factors is dynamically assembled. An inclusive model for all steps in the targeting of proteins to subnuclear sites cannot yet be proposed. However, this model must account for the apparent diversity of intranuclear targeting signals. It is also important to assess the extent to which regulatory discrimination is mediated by subnuclear domain-specific trafficking signals. Furthermore, the checkpoints that monitor subnuclear distribution of regulatory factors and the sorting steps that ensure both structural and functional fidelity of nuclear domains in which replication and expression of genes occur must be biochemically and mechanistically defined. There is emerging recognition that placement of regulatory components of gene expression must be temporally and spatially coordinated to facilitate biological control. The consequences of breaches in nuclear structure–function relationships are observed in an expanding series of diseases that include cancer [Weis et al., 1994; Rogaia et al., 1997; Yano et al., 1997; Rowley, 1998; Zeng et al., 1998; McNeil et al., 1999; Tao and Levine, 1999a] and neurological disorders [Skinner et al., 1997]. As the repertoire of architecture-associated regulatory factors and cofactors expands, workers in the field are becoming increasingly confident that nuclear organization contributes significantly to control of transcription. To gain increased appreciation for the complexities of subnuclear organization and gene regulation, we must continue to characterize mechanisms that direct regulatory proteins to specific transcription sites within the nucleus so that these proteins are in the right place at the right time. J. Cell. Biochem. Suppl. 35:84–92, 2000. © 2001 Wiley-Liss, Inc.
    Journal of Cellular Biochemistry 01/2000; 79(S35):84 - 92. DOI:10.1002/1097-4644(2000)79:35+<84::AID-JCB1130>3.0.CO;2-9 · 3.37 Impact Factor
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    ABSTRACT: Targeting of gene regulatory factors to specific intranuclear sites may be critical for the accurate control of gene expression. The acute myelogenous leukemia 8;21 (AML1/ETO) fusion protein is encoded by a rearranged gene created by the ETO chromosomal translocation. This protein lacks the nuclear matrix-targeting signal that directs the AML1 protein to appropriate gene regulatory sites within the nucleus. Here we report that substitution of the chromosome 8-derived ETO protein for the multifunctional C terminus of AML1 precludes targeting of the factor to AML1 subnuclear domains. Instead, the AML1/ETO fusion protein is redirected by the ETO component to alternate nuclear matrix-associated foci. Our results link the ETO chromosomal translocation in AML with modifications in the intranuclear trafficking of the key hematopoietic regulatory factor, AML1. We conclude that misrouting of gene regulatory factors as a consequence of chromosomal translocations is an important characteristic of acute leukemias.
    Proceedings of the National Academy of Sciences 12/1999; 961(26):14882-14887. DOI:10.1073/pnas.96.26.14882 · 9.81 Impact Factor
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    ABSTRACT: We have analyzed the linkage of protein phosphorylation to the remodeling of chromatin structure that accompanies transcriptional activity of the rat osteocalcin (OC) gene in bone-derived cells. Short incubations with okadaic acid, an inhibitor of protein phosphatases 1 and 2A, induced marked changes in the chromatin organization of the OC gene promoter. These changes were reflected by loss of the two DNase I hypersensitive sites normally present in bone-derived cells expressing this gene. These hypersensitive sites include the elements that control basal tissue-specific expression, as well as steroid hormone regulation. Indeed, the absence of hypersensitivity was accompanied by inhibition of basal and vitamin D-dependent enhancement of OC gene transcription. The effects of okadaic acid on OC chromatin structure and gene activity were specific and reversible. Staurosporine, a protein kinase C inhibitor, did not significantly affect transcriptional activity or DNase I hypersensitivity of the OC gene. We conclude that cellular phosphorylation-dephosphorylation events distinct from protein kinase C-dependent reactions are required for both chromatin remodeling and transcriptional activity of the OC gene in osseous cells.
    Journal of Cellular Biochemistry 04/1999; 72(4):586-94. DOI:10.1002/(SICI)1097-4644(19990315)72:43.0.CO;2-K · 3.37 Impact Factor
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    ABSTRACT: Multiple regulatory elements and intricate protein-DNA interactions mediate the transcription of the human histone H4 genes in a cell growth-dependent manner. Upon analysis of the regulatory elements of the FO108 histone H4 gene, we identified several potential YY1 binding sites. In this study, we have analyzed the ability of the transcription factor YY1 to interact at these sites in vitro by using electrophoretic mobility shift assays in combination with oligonucleotide competition and antibody immunoreactivity. We show that YY1 specifically binds transcriptional regulatory elements at -340 nt (site III), -100 nt (site I) and at least two domains within the coding region of the histone H4 gene. To test if these elements were functionally responsive to YY1, we performed transient expression experiments in Drosophila S-2 cells transfected with heterologous reporter gene constructs driven by histone H4 gene segments fused to the thymidine kinase promoter. Co-expression of YY1 stimulated promoter activity of these constructs relative to the reporter construct lacking histone H4 gene fragments. Our results suggest that YY1 contributes to transcriptional regulation of the histone H4 gene through interactions at multiple regulatory elements.
    Journal of Cellular Biochemistry 03/1999; 72(4):507-16. DOI:10.1002/(SICI)1097-4644(19990315)72:4<507::AID-JCB6>3.3.CO;2-X · 3.37 Impact Factor
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    ABSTRACT: Cells expressing the bone-specific osteocalcin (OC) gene exhibit two DNase I hypersensitive sites within the proximal (nt -170 to -70) and distal (nt -600 to -400) promoter. These sites overlap elements that independently or in combination contribute to basal and vitamin D-stimulated OC gene transcription. Here we address mechanisms that participate in control of chromatin remodelling at these sites. By applying nuclease digestion and indirect end-labeling or by combining intranuclear footprinting and ligation-mediated PCR, we investigated the effects of nuclear protein hyperacetylation on both chromatin organization and transcriptional activation of the OC gene in bone-derived cells. We report that chromatin hyperacetylation blocks vitamin D stimulation of OC transcription and prevents a key transition in the chromatin structure of the OC gene which is required for formation of the distal DNase I hypersensitive site. This transition involves interaction of sequence-specific nuclear factors and may be required for the ligand-dependent binding of the vitamin D receptor complex, which results in transcriptional enhancement.
    Biochemistry 02/1999; 38(4):1338-45. DOI:10.1021/bi982171a · 3.19 Impact Factor
  • Journal of Cellular Physiology 01/1999; 181(2):240-50. DOI:10.1002/(SICI)1097-4652(199911)181:2<240::AID-JCP6>3.0.CO;2-K · 3.87 Impact Factor
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    ABSTRACT: The bone morphogenetic protein (BMP)-2 is a potent osteoinductive signal, inducing bone formation in vivo and osteoblast differentiation from non-osseous cells in vitro. The runt domain-related protein Cbfa1/PEBP2A/AML-3 is a critical component of bone formation in vivo and transcriptional regulator of osteoblast differentiation. To investigate the relationship between the extracellular BMP-2 signal, Cbfa1, and osteogenesis, we examined expression of Cbfa1 and osteoblastic genes during the BMP-2 induced osteogenic transdifferentiation of the myoblastic cell line C2C12. BMP-2 treatment completely blocked myotube formation and transiently induced expression of Cbfa1 and the bone-related homeodomain protein Msx-2 concomitant with loss of the myoblast phenotype. While induction of collagen type I and alkaline phosphatase (AP) expression coincided with Cbfa1 expression, Cbfa1 mRNA was strikingly downregulated at the onset of expression of osteopontin (OPN) and osteocalcin (OCN) genes, reflecting the mature osteoblast phenotype. TGF-β1 treatment effectively suppressed myogenesis and induced Cbfa1 expression but was insufficient to support osteoblast differentiation reflected by the absence of ALP, OPN, and OCN. We addressed whether induction of Cbfa1 in response to BMP-2 results in the transcriptional activation of the OC promoter which contains three enhancer Cbfa1 elements. Transfection studies show BMP-2 suppresses OC promoter activity in C2C12, but not in osteoblastic ROS 17/2.8 cells. Maximal suppression of OC promoter activity in response to BMP-2 requires sequences in the proximal promoter (up to nt -365) and may occur independent of the three Cbfa sites. Taken together, our results demonstrate a dissociation of Cbfa1 expression from development of the osteoblast phenotype. Our findings suggest that Cbfa1 may function transiently to divert a committed myoblast to a potentially osteogenic cell. However, other factors induced by BMP-2 appear to be necessary for complete expression of the osteoblast phenotype. J. Cell. Biochem. 73:114–125, 1999. © 1999 Wiley-Liss, Inc.
    Journal of Cellular Biochemistry 01/1999; 73(1):114 - 125. DOI:10.1002/(SICI)1097-4644(19990401)73:1<114::AID-JCB13>3.0.CO;2-M · 3.37 Impact Factor
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    ABSTRACT: Responsiveness of genes to steroid hormones is a complex process involving synergistic and/or antagonistic interactions between spe- cific receptors and other nonreceptor transcription factors. Thus, DNA recognition elements for steroid hormone receptors are often located among binding sites for other trans-acting factors. The hor- monal form of vitamin D, 1,25-dihydroxyvitamin D3, stimulates tran- scription of the tissue-specific osteocalcin (OC) gene in osteoblastic cells. The rat OC vitamin D response element contains an internal acitvating protein-1 (AP-1) site. Here, we report for the first time that this AP-1 site is critical for the transcriptional enhancement of rat osteocalcin gene expression mediated by vitamin D. Precise mu- tations were introduced either in the steroid half-elements or in the internal AP-1 sequences. One mutation within the internal AP-1 site retained vitamin D receptor/retinoid X receptor binding equiv- alent to that of the wild-type sequence, but resulted in complete loss of vitamin D inducibility of the OC promoter. These results suggest a functional interaction between the hormone receptor and nuclear oncoproteins at the rat OC vitamin D response element. This cooperation of activities may have important consequences in physiological regulation of osteocalcin transcription during osteo- blast differentiation and bone tissue development in vivo .( Endo- crinology 140: 63-70, 1999)
    Endocrinology 01/1999; 140(1):63-70. DOI:10.1210/en.140.1.63 · 4.64 Impact Factor
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    ABSTRACT: 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. © 1999 Wiley-Liss, Inc.
    Journal of Cellular Biochemistry 01/1999; 75(S32):24 - 31. DOI:10.1002/(SICI)1097-4644(1999)75:32+<24::AID-JCB4>3.0.CO;2-V · 3.37 Impact Factor
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    ABSTRACT: Expression of many histone H4 genes is stringently controlled during the cell cycle to maintain a functional coupling of histone biosynthesis with DNA replication. The histone H4 multigene family provides a paradigm for understanding cell cycle control of gene transcription. All functional histone H4 gene copies are highly conserved in the mRNA coding region. However, the putative promoter regions of these H4 genes are divergent. We analyzed three representative mouse H4 genes to assess whether variation in H4 promoter sequences has functional consequences for the relative level and temporal control of expression of distinct H4 genes. Using S1 nuclease protection assays with gene-specific probes and RNA from synchronized cells, we show that the mRNA level of each H4 gene is temporally coupled to DNA synthesis. However, there are differences in the relative mRNA levels of these three H4 gene copies in several cell types. Based on gel shift assays, nucleotide variations in the promoters of these H4 genes preclude or reduce binding of several histone gene transcription factors, including IRF2, HiNF-D, SP-1 and/or YY1. Therefore, differential regulation of H4 genes is directly attributable to evolutionary divergence in H4 promoter organization which dictates the potential for regulatory interactions with cognate H4 transcription factors. This regulatory flexibility in H4 promoter organization may maximize options for transcriptional control of histone H4 gene expression in response to the onset of DNA synthesis and cell cycle progression in a broad spectrum of cell types and developmental stages.
    Biochimica et Biophysica Acta 11/1998; 1442(1):82-100. DOI:10.1016/S0167-4781(98)00147-X · 4.66 Impact Factor
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    ABSTRACT: A glutathione S-transferase fused with the nuclear matrix targeting signal (GST-NMTS) of AML-1/CBF-alpha2 has been crystallized by the vapor diffusion method using polyethylene glycol (PEG) as the precipitant. The NMTS is a 31-amino-acid signal peptide that can target the AML-1/CBF-alpha2 protein to the nuclear matrix. The crystal belongs to tetragonal space group P43212 with unit cell dimensions a = b = 93.4 A, c = 57.6 A. There is one GST-fusion protein per asymmetric unit. Crystals diffracted to at least 2.7 A and are appropriate for structure determination.
    Journal of Structural Biology 10/1998; 123(1):83-5. DOI:10.1006/jsbi.1998.4016 · 3.37 Impact Factor
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    ABSTRACT: 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.
    Journal of Cellular Biochemistry 09/1998; 70(2):200-12. DOI:10.1002/(SICI)1097-4644(19980801)70:23.3.CO;2-4 · 3.37 Impact Factor
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    ABSTRACT: Nuclear matrix protein (NMP) composition of osteoblasts shows distinct two-dimensional gel electrophoretic profiles of labeled proteins as a function of stages of cellular differentiation. Because NMPs are involved in the control of gene expression, we examined modifications in the representation of NMPs induced by TGF-beta1 treatment of osteoblasts to gain insight into the effects of TGF-beta on development of the osteoblast phenotype. Exposure of proliferating fetal rat calvarial derived primary cells in culture to TGF-beta1 for 48 h (day 4-6) modifies osteoblast cell morphology and proliferation and blocks subsequent formation of mineralized nodules. Nuclear matrix protein profiles were very similar between control and TGF-beta-treated cultures until day 14, but subsequently differences in nuclear matrix proteins were apparent in TGF-beta-treated cultures. These findings support the concept that TGF-beta1 modifies the final stage of osteoblast mineralization and alters the composition of the osteoblast nuclear matrix as reflected by selective and TGF-beta-dependent modifications in the levels of specific nuclear matrix proteins. The specific changes induced by TGF-beta in nuclear matrix associated proteins may reflect specialized mechanisms by which TGF-beta signalling mediates the alterations in cell organization and nodule formation and/or the consequential block in extracellular mineralization.
    Journal of Cellular Biochemistry 07/1998; 69(3):291-303. DOI:10.1002/(SICI)1097-4644(19980601)69:33.0.CO;2-M · 3.37 Impact Factor
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    ABSTRACT: We have examined the contribution of transcriptional mechanisms to the pleiotropic effects of glucocorticoids on basal and vitamin D stimulated expression of the developmentally regulated bone-specific osteocalcin (OC) gene. OC expression was systematically investigated at the level of protein, mRNA, and newly synthesized transcripts during maturation of the bone cell phenotype in cultures of fetal rat calvarial-derived osteoblasts. Our results indicate that transcriptional control of basal and hormone-regulated OC expression predominates in immature osteoblasts prior to matrix mineralization. However, in mature osteoblasts OC expression is controlled primarily by posttranscriptional mechanisms reflected by elevated mRNA levels with a decline in transcription. Vitamin D, alone or in combination with Dex, is a significant factor contributing to mRNA stabilization in mature osteoblasts with a mineralized extracellular matrix. Transcriptional modifications in response to Dex are reflected by quantitative differences between proliferating and mature osteoblasts in the formation of glucocorticoid receptor binding complexes at the proximal OC glucocorticoid response element. Vitamin D and glucocorticoid receptor mRNA levels are significantly higher in mature osteoblasts than in early stage bone cells. However, receptor complexes do not appear to be rate limiting in proliferating osteoblasts when the OC gene is not transcribed. Our results indicate (1) developmental stage-specific effects of steroid hormone on transcriptional regulation of bone expressed genes, and (2) inverse relationships between levels of transcription and cellular representation of mRNA with OC message stabilized in mature osteoblasts.
    Journal of Cellular Biochemistry 05/1998; 69(2):154-68. DOI:10.1002/(SICI)1097-4644(19980501)69:23.0.CO;2-R · 3.37 Impact Factor
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    ABSTRACT: The AML/CBF alpha 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 AML/CBF alpha 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 AML/CBF alpha must associate with the nuclear matrix to effect transcription. We used fluorescence 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.
    Proceedings of the National Academy of Sciences 02/1998; 95(4):1585-1589. DOI:10.1073/pnas.95.4.1585 · 9.81 Impact Factor
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    ABSTRACT: The bone tissue-specific osteocalcin gene remains one of a few genes that exhibits osteoblast-restricted expression. Over the last decade, characterization of the promoter regulatory elements and complexes of factors that control suppression of the osteocalcin gene in osteoprogenitor cells and transactivation in mature osteoblasts has revealed transcriptional regulatory mechanisms that mediate development of the osteoblast phenotype. In this review, we have focused on emerging concepts related to molecular mechanisms supporting osteoblast growth and differentiation based on the discoveries that the osteocalcin gene is regulated by homeodomain factors, AP-1 related proteins, and the bone restricted Cbfa1/AML3 transcription factor.
    Journal of cellular biochemistry. Supplement 02/1998; 30-31:62-72. DOI:10.1002/(SICI)1097-4644(1998)72:30/31+3.0.CO;2-S
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    ABSTRACT: We present evidence of cell death by apoptosis during the development of bone-like tissue formation in vitro. Fetal rat calvaria-derived osteoblasts differentiate in vitro, progressing through three stages of maturation: a proliferation period, a matrix maturation period when growth is downregulated and expression of the bone cell phenotype is induced, and a third mineralization stage marked by the expression of bone-specific genes. Here we show for the first time that cells differentiating to the mature bone cell phenotype undergo programmed cell death and express genes regulating apoptosis. Culture conditions that modify expression of the osteoblast phenotype simultaneously modify the incidence of apoptosis. Cell death by apoptosis is directly demonstrated by visualization of degraded DNA into oligonucleosomal fragments after gel electrophoresis. Bcl-XL, an inhibitor of apoptosis, and Bax, which can accelerate apoptosis, are expressed at maximal levels 24 h after initial isolation of the cells and again after day 25 in heavily mineralized bone tissue nodules. Bcl-2 is expressed in a reciprocal manner to its related gene product Bcl-XL with the highest levels observed during the early post-proliferative stages of osteoblast maturation. Expression of p53, c-fos, and the interferon regulatory factors IRF-1 and IRF-2, but not cdc2 or cdk, were also induced in mineralized bone nodules. The upregulation of Msx-2 in association with apoptosis is consistent with its in vivo expression during embryogenesis in areas that will undergo programmed cell death. We propose that cell death by apoptosis is a fundamental component of osteoblast differentiation that contributes to maintaining tissue organization.
    Journal of Cellular Biochemistry 01/1998; 68(1):31-49. DOI:10.1002/(SICI)1097-4644(19980101)68:1<31::AID-JCB4>3.0.CO;2-X · 3.37 Impact Factor

Publication Stats

12k Citations
1,482.02 Total Impact Points


  • 2013–2015
    • University of Vermont
      • Department of Biochemistry
      Burlington, Vermont, United States
  • 1989–2015
    • University of Massachusetts Amherst
      Amherst Center, Massachusetts, United States
  • 1988–2015
    • University of Massachusetts Medical School
      • • Department of Cell Biology
      • • Department of Cancer Biology
      • • Department of Medicine
      Worcester, Massachusetts, United States
  • 2002–2012
    • University of Concepción
      • • Departamento de Bioquímica y Biología Molecular
      • • Facultad de Ciencias Biológicas
      Ciudad de Concepcion, Biobío, Chile
    • Beth Israel Deaconess Medical Center
      • Department of Neurology
      Boston, Massachusetts, United States
  • 2010
    • Universidad Andrés Bello
      • Faculty of Medicine
      CiudadSantiago, Santiago, Chile
  • 2005
    • Kyungpook National University
      • Department of Oral Biochemistry
      Daikyū, Daegu, South Korea
    • Baylor College of Medicine
      Houston, Texas, United States
  • 1995
    • Massachusetts Institute of Technology
      Cambridge, Massachusetts, United States
    • St. Jude Children's Research Hospital
      • Department of Tumor Cell Biology
      Memphis, Tennessee, United States
  • 1994
    • Comprehensive Cancer Centers of Nevada
      Las Vegas, Nevada, United States
  • 1985
    • The American Society for Biochemistry and Molecular Biology
      Gainesville, Florida, United States
  • 1978–1985
    • University of Florida
      • • Department of Biochemistry and Molecular Biology
      • • College of Medicine
      Gainesville, FL, United States