A Weak TATA Box Is a Prerequisite for Glucocorticoid-dependent Repression of the Osteocalcin Gene

Meyer, Thomas; Carlstedt‐Duke, Jan; Starr, D. Barry

doi:10.1074/jbc.272.49.30709

Cited by 72 publications

(36 citation statements)

References 31 publications

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“…GR binding sites were demonstrated in the vicinity of human (25) and rat (29) OC proximal promoter elements, the TATA box, and the OC box. It was suggested that GR binding interfered with interaction of transcription factors with the OC basal promoter (25,27,35). GR binding to distal promoter elements of the human (26) and the rat (28) OC genes was also suggested to contribute to transcriptional repression.…”

Section: Discussionmentioning

confidence: 99%

Glucocorticoids inhibit osteocalcin transcription in osteoblasts by suppressing Egr2/Krox20‐binding enhancer

Leclerc¹,

Noh²,

Khokhar³

et al. 2005

Arthritis & Rheumatism

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Objective. Glucocorticoids are widely used for the management of rheumatoid arthritis. Osteoporosis is a major side effect of glucocorticoid therapy and is attributable to inhibition of bone formation. We developed an osteoblast culture system in which glucocorticoids strongly inhibit development of the osteoblast phenotype, including expression of the bone-specific osteocalcin (OC) gene. Using this gene as a model, the goal of this study was to discover glucocorticoid-sensitive transcriptional mechanisms in osteoblasts.Methods. Dexamethasone (DEX; 1 M) was administered to murine MC3T3-E1 osteoblastic cultures under conditions that inhibit mineralized extracellular matrix formation and OC messenger RNA levels by >10-fold. Because standard (short-term) transient transfection assays with OC promoter-reporter constructs did not recapitulate the strong DEX-mediated repression, mapping of OC negative glucocorticoid response elements (GREs) was performed initially by stable transfection and then with long-term transient transfection assays. Transcription factor binding to the OC negative GRE was studied by electrophoretic mobility shift assays.Results. Several-fold repression of OC-luciferase constructs was recapitulated in stable and long-term transient transfection assays, in which the transfected cells were allowed to progress to a sufficiently advanced developmental stage. Analysis of a 5 promoter deletion series mapped an OC negative GRE to a 15-bp G/C-rich motif (؊161/؊147) located just upstream of the binding site for the osteoblast master transcription factor Runx2. Oligonucleotides encompassing this element and MC3T3-E1 cell extracts formed a protein-DNA complex that contained an Egr/Krox family member(s). Complex formation was competed by either an oligonucleotide containing 2 consensus Egr motifs or by anti-Egr2/Krox20 antibodies. Three copies of this Kroxbinding element conferred 20-fold transcriptional activation on the 147-bp basal OC promoter in osteoblasts, and the enhancer activity was inhibited by DEX. Enhancer activity was not observed in 10T1/2 fibroblasts unless these cells were cotransfected with Runx2.Conclusion. An Egr2/Krox20-binding site located immediately upstream of the Runx2 site of the mouse OC promoter was identified as an enhancer in osteoblasts, whose activity is repressed by glucocorticoids. Sequence similarity suggests that such a mechanism is likely operative in both murine and human cells. Because glucocorticoids inhibit Egr2/Krox20 expression in osteoblasts, and because trabecular bone formation is arrested in Egr2/Krox20-knockout mice, the inhibition of Egr2/Krox20 activity likely contributes to glucocorticoid-induced osteoporosis.Since the 1950 Nobel laureates E. Kendall, T. Reichstein, and P. Hench introduced glucocorticoids for the treatment of rheumatoid arthritis, these drugs have been widely used for the treatment of autoimmune and inflammatory diseases. The enthusiasm with which glucocorticoids were initially accepted was quickly dampened upon realization of their deleter...

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Section: Discussionmentioning

confidence: 99%

Glucocorticoids inhibit osteocalcin transcription in osteoblasts by suppressing Egr2/Krox20‐binding enhancer

Leclerc¹,

Noh²,

Khokhar³

et al. 2005

Arthritis & Rheumatism

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“…Taken together, these studies suggest that the TBP-DNA complex may exist in different structural contexts for variant sequences, and this context may also impact downstream steps in the transcription process. Because variant TATA elements play a fundamental role in the proper expression of many eukaryotic genes (9,10,21,24), it is essential to determine the mechanisms underlying differential levels of transcription from varying TATA elements.…”

Section: Discussionmentioning

confidence: 99%

“…For example, activation by E1A of the human hsp70 promoter is highly dependent on the sequence of the TATA element: substitution of the hsp70 TATA (TATAA) with the SV40 early promoter TATA (TATTTAT) results in loss of induction by E1A but not by heat shock (9). In addition, an off-consensus TATA element is essential for the proper regulation of the osteocalcin gene by glucocorticoid receptor (10). As such, the particular sequence of the TATA element is likely to be critical for appropriate gene expression of a specific gene.…”

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confidence: 99%

Non-optimal TATA Elements Exhibit Diverse Mechanistic Consequences

Stewart¹,

Fischbeck

Chen

et al. 2006

Journal of Biological Chemistry

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To reveal mechanistic differences in transcription initiation between variant TATA elements, in vivo and in vitro assays of the functional activity of 14 different sequences were compared. Variant elements exhibited particular degrees of activation in vivo but universally were unable to support the -fold activation observed for an element consisting of TATAAA. Each element was classified by its functional activity for in vitro interaction with TATA-binding protein (TBP), TFIIA, and TFIIB. Certain off-consensus TATA elements form poor binding sites for TBP and this compromised interaction interferes with higher order complex formation with TFIIA and/or TFIIB. Other elements are only modestly decreased for TBP binding but dramatically affected for higher order complex formation. Another distinct category is comprised of two elements (CATAAA and TATAAG), which are not affected in the initial formation of the TBP, TFIIA-TBP, or TFIIB-TBP complexes. However, CATAAA and TATAAG are unable to form a stable TFIIA-TBP-DNA complex in vitro. Moreover, fusion of TFIIA to TBP specifically restores activity from these two elements in vivo. Taken together, these results indicate that the interplay between the sequence of the TATA element and the components of the general transcription machinery can lead to variations in the formation of functional complexes and/or the stability of these complexes. These differences offer distinct opportunities for an organism to exploit diverse steps in the regulation of gene expression depending on the precise TATA element sequence at a given gene.Initiation of transcription by RNA polymerase II is the major site for regulation of eukaryotic gene expression during cell cycle progression, development, and physiological induction (1). Increased gene expression is mediated by activator proteins that bind regulatory sequences in the promoter and, either directly or indirectly, facilitate recruitment of the general transcription factors assembled near the initiation site at the core promoter (2). TATA-binding protein (TBP) 3 specifically recognizes and binds to the TATA sequence of the core promoter allowing for the nucleation of other general transcription factors including TFIIA, -B, -E, -F, -H, -J, and polymerase II (for reviews, see Refs. 3 and 4). Although high affinity binding sites have been identified for yeast TBP (5-7), and computational studies indicate that TATA(A/T)A(A/T)(A/G) is commonly found at TBP-dependent promoters in yeast (8), a wide variety of off-consensus elements have been shown to exhibit transcriptional activity. Indeed, these alterations from the consensus sequence are not passive players in gene regulation but can contribute to the proper expression of a given gene. For example, activation by E1A of the human hsp70 promoter is highly dependent on the sequence of the TATA element: substitution of the hsp70 TATA (TATAA) with the SV40 early promoter TATA (TATTTAT) results in loss of induction by E1A but not by heat shock (9). In addition, an off-consensus TATA element is...

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“…Osteoblasts seem to be the major target of glucocorticoid action in the bone. GCs decrease osteoblast number and function by altering expression of several osteoblast-specific genes in the bone, including repression of osteocalcin (Meyer et al 1997) and type I collagen (Delany et al 1995a), and induction of collagenase (Delany et al 1995b). GCs decrease IGF-I synthesis in osteoblasts, where IGF-I has been shown to have profound anabolic effects, inducing proliferation of preosteoblasts, increase in collagen I synthesis and decrease in collagenase.…”

Section: Role Of Other Hormones In Bone Remodelingmentioning

confidence: 99%

Hormonal regulation of physiological cell turnover and apoptosis

Medh

Thompson

2000

Cell Tissue Res

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Physiological cell turnover plays an important role in maintaining normal tissue function and architecture. This is achieved by the dynamic balance of cellular regeneration and elimination, occurring periodically in tissues such as the uterus and mammary gland, or at constant rates in tissues such as the gastrointestinal tract and adipose tissue. Apoptosis has been identified as the prevalent mode of physiological cell loss in most tissues. Cell turnover is precisely regulated by the interplay of various endocrine and paracrine factors, which modulate tissue and cell-specific responses on proliferation and apoptosis, either directly, or by altering expression and function of key cell proliferative and/or death genes. Although recent studies have provided significant information on specific tissue systems, a clearly defined pathway that mediates cell turnover has not yet emerged for any tissue. Several similarities exist among the various tissues with regard to the intermediates that regulate tissue homeostasis, enabling a better understanding of the general mechanisms involved in the process. Here we review the mechanisms by which hormonal and cytokine factors mediate cell turnover in various tissues, emphasizing common themes and tissue-specific differences.

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A Weak TATA Box Is a Prerequisite for Glucocorticoid-dependent Repression of the Osteocalcin Gene

Cited by 72 publications

References 31 publications

Glucocorticoids inhibit osteocalcin transcription in osteoblasts by suppressing Egr2/Krox20‐binding enhancer

Glucocorticoids inhibit osteocalcin transcription in osteoblasts by suppressing Egr2/Krox20‐binding enhancer

Non-optimal TATA Elements Exhibit Diverse Mechanistic Consequences

Hormonal regulation of physiological cell turnover and apoptosis

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