Janet L Stein

University of Vermont, Burlington, Vermont, United States

Are you Janet L Stein?

Claim your profile

Publications (337)1574.71 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Regulation of histone gene transcription at the G1/S phase transition via the Site II cell cycle control element is distinct from E2F-dependent mechanisms operative at the growth factor-related restriction point. E2F-independent activation of histone H4 gene expression combines contributions of several promoter factors, including HiNF-M/IRF2 and the HiNF-D/CDP-cut complex which contains pRB, CDK1, and cyclin A as non-DNA binding subunits. Mutational analyses suggest additional rate-limiting factors for Site II function. Using sequence-specific Site II DNA affinity chromatography, we identified a 45 kDa protein (KIAA0005 or BZAP45) that is embryonically expressed and phylogenetically conserved. Based on amino acid sequence analysis, BZAP45 contains a unique decapeptide that is part of a putative leucine-zipper protein with a nucleotide (ATP or GTP) binding fold. Bacterial expression of a full-length cDNA produces a 45 kDa protein. Binding studies reveal that highly purified BZAP45 does not interact with Site II, suggesting that BZAP45 function may require partner proteins. Forced expression of BZAP45 strongly stimulates H4 promoter (nt -215 to -1)/CAT reporter gene activity. Deletion analyses and point mutations indicate that BZAP45 enhances H4 gene transcription through Site II. Thus, BZAP45 is a novel regulatory factor that contributes to transcriptional control at the G1/S phase transition.
    Biochemistry 10/2001; 40(35):10693-9. DOI:10.1021/bi010529o · 3.02 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Runx (Cbfa/AML) transcription factors are critical for tissue-specific gene expression. A unique targeting signal in the C terminus directs Runx factors to discrete foci within the nucleus. Using Runx2/CBFA1/AML3 and its essential role in osteogenesis as a model, we investigated the fundamental importance of fidelity of subnuclear localization for tissue differentiating activity by deleting the intranuclear targeting signal via homologous recombination. Mice homozygous for the deletion (Runx2 Delta C) do not form bone due to maturational arrest of osteoblasts. Heterozygotes do not develop clavicles, but are otherwise normal. These phenotypes are indistinguishable from those of the homozygous and heterozygous null mutants, indicating that the intranuclear targeting signal is a critical determinant for function. The expressed truncated Runx2 Delta C protein enters the nucleus and retains normal DNA binding activity, but shows complete loss of intranuclear targeting. These results demonstrate that the multifunctional N-terminal region of the Runx2 protein is not sufficient for biological activity. We conclude that subnuclear localization of Runx factors in specific foci together with associated regulatory functions is essential for control of Runx-dependent genes involved in tissue differentiation during embryonic development.
    Proceedings of the National Academy of Sciences 08/2001; 98(15). DOI:10.1073/pnas.151236498 · 9.67 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Expression of the bone sialoprotein (BSP) gene, a marker of bone formation, is largely restricted to cells in mineralized tissues. Recent studies have shown that the Cbfa1 (also known as Runx2, AML-3, and PEBP2alphaA) transcription factor supports commitment and differentiation of progenitor cells to hypertrophic chondrocytes and osteoblasts. This study addresses the functional involvement of Cbfa sites in expression of the Gallus BSP gene. Gel mobility shift analyses with nuclear extracts from ROS 17/2.8 osteoblastic cells revealed that multiple Cbfa consensus sequences are functional Cbfa DNA binding sites. Responsiveness of the 1.2-kb Gallus BSP promoter to Cbfa factors Cbfa1, Cbfa2, and Cbfa3 was assayed in osseous and nonosseous cells. Each of the Cbfa factors mediated repression of the wild-type BSP promoter, in contrast to their well known activation of various hematopoietic and skeletal phenotypic genes. Suppression of BSP by Cbfa factors was not observed in BSP promoters in which Cbfa sites were deleted or mutated. Expression of the endogenous BSP gene in Gallus osteoblasts was similarly downregulated by forced expression of Cbfa factors. Our data indicate that Cbfa repression of the BSP promoter does not involve the transducin-like enhancer (TLE) proteins. Neither coexpression of TLE1 or TLE2 nor the absence of the TLE interaction motif of Cbfa1 (amino acids 501 to 513) influenced repressor activity. However, removal of the C terminus of Cbfa1 (amino acids 362 to 513) relieved suppression of the BSP promoter. Our results, together with the evolutionary conservation of the seven Cbfa sites in the Gallus and human BSP promoters, suggest that suppressor activity by Cbfa is of significant physiologic consequence and may contribute to spatiotemporal expression of BSP during bone development.
    Molecular and Cellular Biology 05/2001; 21(8):2891-905. DOI:10.1128/MCB.21.8.2891-2905.2001 · 4.78 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The vitamin D response element in the bone tissue-specific osteocalcin gene has served as a prototype for understanding molecular mechanisms regulating physiologic responsiveness of vitamin D-dependent genes in bone cells. We briefly review factors which contribute to vitamin D transcriptional control. The organization of the vitamin D response element (VDRE), the multiple activities of the vitamin D receptor transactivation complex, and the necessity for protein-protein interactions between the VDR-RXR heterodimer activation complex and DNA binding proteins at other regulatory elements, including AP-1 sites and TATA boxes, provide for precise regulation of gene activity in concert with basal levels of transcription. We present evidence for molecular mechanisms regulating vitamin D-dependent mediated transcription of the osteocalcin gene that involve chromatin structure of the gene and nuclear architecture. Modifications in nucleosomal organization, DNase I hypersensitivity and localization of vitamin D receptor interacting proteins in subnuclear domains are regulatory components of vitamin D-dependent gene transcription. A model is proposed to account for the inability of vitamin D induction of the osteocalcin gene in the absence of ongoing basal transcription by competition of the YY1 nuclear matrix-associated transcription factor for TFIIB-VDR interactions. Activation of the VDR-RXR complex at the OC VDRE occurs through modifications in chromatin mediated in part by interaction of OC gene regulatory sequences with the nuclear matrix-associated Cbfa1 (Runx2) transcription factor which is required for osteogenesis.
    Steroids 03/2001; 66(3-66):159-170. DOI:10.1016/S0039-128X(00)00160-4 · 2.64 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Transcriptional control at the G1/S-phase transition of the cell cycle requires functional interactions of multimeric promoter regulatory complexes that contain DNA binding proteins, transcriptional cofactors, and/or chromatin modifying enzymes. Transcriptional regulation of the human histone H4/n gene (FO108) is mediated by Interferon Regulatory Factor-2 (IRF-2), as well as other histone gene promoter factors. To identify proteins that interact with cell-cycle regulatory factors, we performed yeast two-hybrid analysis with IRF-2 and identified a novel human protein termed Celtix-1 which binds to IRF-2. Celtix-1 contains several phylogenetically conserved domains, including a bromodomain, which is found in a number of transcriptional cofactors. Using a panel of IRF-2 deletion mutants in yeast two-hybrid assays, we established that Celtix-1 contacts the C-terminus of IRF-2. Celtix-1 directly interacts with IRF-2 based on binding studies with glutathione S-transferase (GST)/IRF-2 fusion proteins, and immunofluorescence studies suggest that Celtix-1 and IRF-2 associate in situ. Celtix-1 is distributed throughout the nucleus in a heterodisperse pattern. A subset of Celtix-1 colocalizes with the hyperacetylated forms of histones H3 and H4, as well as with the hyperphosphorylated, transcriptionally active form of RNA polymerase II. We conclude that the bromodomain protein Celtix-1 is a novel IRF-2 interacting protein that associates with transcriptionally active chromatin in situ.
    Journal of Cellular Physiology 11/2000; 185(2):269-79. DOI:10.1002/1097-4652(200011)185:2<269::AID-JCP12>3.0.CO;2-L · 3.84 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Contributing to bone-specific expression of the osteocalcin gene is the promoter element OC Box I (-99 to -76), which binds both Hox proteins and another nonhomeodomain factor (designated OCBP for osteocalcin-box binding protein) (Hoffmann et al. [1996] J Cell Biochem 61:310-324). OCBP correlates with increased promoter activity and may, therefore, be important to development or maintenance of the osteoblast phenotype. To identify OCBP candidates, we used a multimerized OC Box I sequence to screen a gammagt11 cDNA expression library, constructed from the rat osteosarcoma osteoblastic ROS 17/2.8 cell line, for cDNA clones encoding factors that recognize this element. Mutant OC Box I sequences that do not bind OCBP and/or homeodomain proteins were used to counterscreen the cDNA isolates. Clones showing binding specificity were sequenced and further characterized for patterns of expression in different tissues and cell lines. Among the characterized nonhomeodomain-related isolates, we identified a nucleolin, a clone encoding rCAP2 that is related to myogenic differentiation and more importantly, a cDNA clone containing a previously uncharacterized gene that has been designated as a cell differentiation-related factor (DRF). DRF mRNA is highly expressed in ROS 17/2.8 cells and in a developmentally regulated pattern during osteoblast differentiation, being upregulated at the postproliferative maturation transition and coinciding with the induction of osteocalcin gene expression. The 7.7-kb transcript encoded by clone 44 is abundantly expressed in osteoblasts, but the mRNA was not present at detectable levels in bone and soft tissues by Northern blot analysis. However, related expressed sequence tags were recently reported in cDNA libraries of rat lung and mouse sympathetic ganglion. The identification of DRF represents a novel osteoblast differentiation-specific marker related to osteocalcin expression. The identification of DRF may further facilitate definition of gene regulatory mechanisms mediating the final stages of bone cell differentiation
    Journal of Cellular Biochemistry 10/2000; 80(1):156-68. DOI:10.1002/1097-4644(20010101)80:1<156::AID-JCB150>3.0.CO;2-F · 3.26 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Treatment of genetic or degenerative diseases severely affecting the entire skeleton may necessitate gene therapy involving transplantation of multipotential marrow cells. The ability of in vitro expanded adherent marrow cells enriched in pluripotent mesenchymal cell populations to remain competent to engraft, repopulate host tissues, and differentiate into bone and cartilage is advantageous for correction of skeletal-related diseases. However, to achieve phenotypic specificity and therapeutic or physiologic levels of proteins may require cell type specific expression of the gene. Tissue-specific promoter-controlled transgenes provide an efficacious approach to deliver therapeutic gene expression to repopulating chondrocytes and osteoblasts for treatment of cartilage and bone disorders or tumor metastasis to the skeleton. The bone-specific expression of a reporter gene controlled by the osteoblast-specific osteocalcin promoter after transplantation of a mixed population of marrow cells is shown. Tissue-restricted gene therapy potentially can be refined by use of a unique peptide targeting signal that directs the hematopoietic, chondrogenic, and osteogenic core binding factor/acute myelogenous leukemia transcription factors to subnuclear sites that support gene expression.
    Clinical Orthopaedics and Related Research 09/2000; 379:S146-S155. DOI:10.1097/00003086-200010001-00019 · 2.77 Impact Factor
  • [Show abstract] [Hide abstract]
    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 09/2000; 184(3):341-50. DOI:10.1002/1097-4652(200009)184:3<341::AID-JCP8>3.0.CO;2-Z · 3.84 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Promyelocytic leukemia (PML) nuclear bodies are important components of nuclear architecture that are functionally linked to aberrant gene expression and disease. To understand the mechanisms that modify subnuclear distribution and regulatory activities of PML domains in leukemia, we performed immunofluorescence microscopy with a panel of normal diploid cells and established cell lines. We analyzed the representation and intranuclear distribution of PML domains. We find that multiple biological parameters contribute to heterogeneity in the subnuclear organization of PML domains in a broad spectrum of cell types. The subnuclear organization of PML domains was also evaluated following transient transfection with a series of vectors expressing normal hematopoietic and leukemia-related transcription factors. Our results show that expression of a chimeric transcription factor encoded by the tumor related chromosomal translocation (8;21) involving the AML1 and ETO loci is sufficient to cause reorganization of PML domains. This finding increases our understanding of the mechanisms by which the AML1/ETO protein may contribute to modified gene expression linked to the onset and progression of t(8;21) related acute myelogenous leukemia.
    Journal of Cellular Biochemistry 08/2000; 79(1):103-12. DOI:10.1002/1097-4644(2000)79:13.3.CO;2-2 · 3.26 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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 · 4.89 Impact Factor
  • [Show abstract] [Hide abstract]
    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 · 3.26 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Developmental control of bone tissue-specific genes requires positive and negative regulatory factors to accommodate physiological requirements for the expression or suppression of the encoded proteins. Osteocalcin (OC) gene transcription is restricted to the late stages of osteoblast differentiation. OC gene expression is suppressed in nonosseous cells and osteoprogenitor cells and during the early proliferative stages of bone cell differentiation. The rat OC promoter contains a homeodomain recognition motif within a highly conserved multipartite promoter element (OC box I) that contributes to tissue-specific transcription. In this study, we demonstrate that the CCAAT displacement protein (CDP), a transcription factor related to the cut homeodomain protein in Drosophila melanogaster, may regulate bone-specific gene transcription in immature proliferating osteoblasts. Using gel shift competition assays and DNase I footprinting, we show that CDP/cut recognizes two promoter elements (TATA and OC box I) of the bone-related rat OC gene. Overexpression of CDP/cut in ROS 17/2.8 osteosarcoma cells results in repression of OC promoter activity; this repression is abrogated by mutating OC box I. Gel shift immunoassays show that CDP/cut forms a proliferation-specific protein/DNA complex in conjunction with cyclin A and p107, a member of the retinoblastoma protein family of tumor suppressors. Our findings suggest that CDP/cut may represent an important component of a cell signaling mechanism that provides cross-talk between developmental and cell cycle-related transcriptional regulators to suppress bone tissue-specific genes during proliferative stages of osteoblast differentiation.
    Cancer Research 01/2000; 59(23):5980-8. · 9.33 Impact Factor
  • [Show abstract] [Hide abstract]
    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 · 3.26 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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 · 9.67 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Experimental Cell Research 12/1999; 253(1):110-6. DOI:10.1006/excr.1999.4680 · 3.25 Impact Factor
  • Journal of Cellular Physiology 11/1999; 181(2):240-50. DOI:10.1002/(SICI)1097-4652(199911)181:2<240::AID-JCP6>3.0.CO;2-K · 3.84 Impact Factor
  • [Show abstract] [Hide abstract]
    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 04/1999; 73(1):114 - 125. DOI:10.1002/(SICI)1097-4644(19990401)73:1<114::AID-JCB13>3.0.CO;2-M · 3.26 Impact Factor
  • [Show abstract] [Hide abstract]
    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 · 3.26 Impact Factor
  • [Show abstract] [Hide abstract]
    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 · 3.26 Impact Factor
  • G S Stein · Andre J. van Wijnen · Janet L. Stein · Jane B. Lian
    [Show abstract] [Hide abstract]
    ABSTRACT: There is increasing evidence for functional linkages between nuclear architecture and the regulation of gene expression. The nuclear matrix provides a paradigm for involvement of nuclear morphology in assembly of the biochemical machinery for physiological control of transcription. Mechanisms are being experimentally defined that direct regulatory factors to subnuclear sites that support gene expression. Consequently, we are gaining insight into the rules that govern transcriptional control within the three-dimensional context of nuclear organization.
    Critical Reviews in Eukaryotic Gene Expression 02/1999; 9(3-4):183-90. DOI:10.1615/CritRevEukarGeneExpr.v9.i3-4.30 · 1.57 Impact Factor

Publication Stats

13k Citations
1,574.71 Total Impact Points


  • 2013–2015
    • University of Vermont
      • Department of Biochemistry
      Burlington, Vermont, United States
  • 1988–2015
    • University of Massachusetts Medical School
      • • Department of Cell Biology
      • • Department of Cancer Biology
      • • Department of Medicine
      Worcester, Massachusetts, United States
  • 2014
    • University of Vermont Medical Center
      Burlington, Vermont, 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
  • 1989–2011
    • University of Massachusetts Amherst
      Amherst Center, 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
  • 2001
    • University of Massachusetts Boston
      Boston, Massachusetts, 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
  • 1992
    • The Samuel Roberts Noble Foundation
      ADM, Oklahoma, 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
  • 1979
    • University of Michigan
      • Department of Microbiology and Immunology
      Ann Arbor, Michigan, United States