Sclerotome-related helix-loop-helix type transcription factor (scleraxis) mRNA is expressed in osteoblasts and its level is enhanced by type-β transforming growth factor

Abstract Scleraxis is a recently identified transcription factor with a basic helix-loop-helix motif, which is expressed in sclerotome during embryonic development. We have examined the expression of scleraxis mRNA in rat osteoblastic cells and found that the scleraxis gene was expressed as a 1·2 kb mRNA species in osteoblastic osteosarcoma ROS 17/2·8 cells. The scleraxis mRNA expression was enhanced by type-β transforming growth factor (TGFβ) treatment. The TGFβ effect was observed in a dosedependent manner starting at 0·2 ng/ml and saturating at 2 ng/ml. The effect was time-dependent and was first observed within 12 h and peaked at 24 h. The TGFβ effect was blocked by cycloheximide, while no effect on scleraxis mRNA stability was observed. TGFβ treatment enhanced scleraxis-E box (Scx-E) binding activity in the nuclear extracts of ROS17/2·8 cells. Furthermore, TGFβ enhanced transcriptional activity of the CAT constructs which contain the Scx-E box sequence. TGFβ treatment also enhanced scleraxis gene expression in osteoblastenriched cells derived from primary rat calvaria. These findings indicated for the first time that the novel helixloop-helix type transcription factor (scleraxis) mRNA is expressed in osteoblasts and its expression is regulated by TGFβ. Journal of Endocrinology (1996) 151, 491–499

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Transcription of the Transforming Growth Factor-β2 Gene Is Dependent on an E-box Located between an Essential cAMP Response Element/Activating Transcription Factor Motif and the TATA Box of the Gene

Journal of Biological Chemistry ◽

10.1074/jbc.271.50.32375 ◽

1996 ◽

Vol 271 (50) ◽

pp. 32375-32380 ◽

Cited By ~ 34

Author(s):

Beáta Scholtz ◽

Michelle Kingsley-Kallesen ◽

Angie Rizzino

Keyword(s):

Transcription Factor ◽

Growth Factor ◽

Transforming Growth Factor ◽

Tata Box ◽

Camp Response Element ◽

Response Element ◽

Transcription Factor Motif ◽

E Box ◽

Transforming Growth Factor Β2 ◽

Activating Transcription Factor

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Transforming Growth Factor-β Regulates DNA Binding Activity of Transcription Factor Fli1 by p300/CREB-binding Protein-associated Factor-dependent Acetylation

Journal of Biological Chemistry ◽

10.1074/jbc.m703907200 ◽

2007 ◽

Vol 282 (48) ◽

pp. 34672-34683 ◽

Cited By ~ 75

Author(s):

Yoshihide Asano ◽

Joanna Czuwara ◽

Maria Trojanowska

Keyword(s):

Transcription Factor ◽

Growth Factor ◽

Dna Binding ◽

Transforming Growth Factor ◽

Binding Protein ◽

Transforming Growth Factor Β ◽

Binding Activity ◽

Creb Binding Protein ◽

Associated Factor ◽

Dna Binding Activity

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Identification of a DNA sequence involved in osteoblast-specific gene expression via interaction with helix-loop-helix (HLH)-type transcription factors.

The Journal of Cell Biology ◽

10.1083/jcb.126.3.773 ◽

1994 ◽

Vol 126 (3) ◽

pp. 773-782 ◽

Cited By ~ 61

Author(s):

M Tamura ◽

M Noda

Keyword(s):

Transcription Factors ◽

Promoter Region ◽

Gene Promoter ◽

Binding Activity ◽

Specific Gene ◽

Specific Expression ◽

Helix Loop Helix ◽

Nuclear Extracts ◽

Osteocalcin Gene ◽

E Box

To elucidate regulatory mechanism(s) underlying differentiation of osteoblasts, we examined involvement of helix-loop-helix (HLH)-type transcription factors in osteoblast-specific expression of a phenotypic marker gene which encodes osteocalcin, a major noncollagenous bone matrix protein, exclusively expressed in osteoblasts. Overexpression of a dominant negative HLH protein, Id-1, decreased the activity of the 1.1-kb osteocalcin gene promoter cotransfected into rat osteoblastic osteosarcoma ROS17/2.8 cells. Analysis of deletion mutants revealed that a 264-bp fragment of osteocalcin promoter (-198 to +66) was sufficient for the Id-1-dependent suppression. Furthermore, the activity of the same promoter fragment (-198 to +66) was enhanced when antisense Id-1 expression vector was cotransfected. This osteocalcin gene promoter region contains two sites of an E-box motif, a consensus binding site for HLH proteins, which we refer to as OCE1 (CACATG, at -102) and OCE2 (CAGCTG, at -149), respectively. Mutagenesis in OCE1 but not OCE2 led to greater than 50% reduction in transcriptional activity of the osteocalcin gene promoter. Electrophoresis mobility shift assay indicated that factors in nuclear extracts prepared from ROS17/2.8 cells bound to the 30-bp oligonucleotide probe containing the E-box motif of OCE1. This binding was competed out by OCE1 oligonucleotide but neither by OCmE1 oligonucleotide in which E-box motif was mutated nor by OCE2. The OCE1-binding activity in the nuclear extracts of ROS17/2.8 cells was reduced by 70% when bacterially expressed Id-1 protein was added to the reaction mixture, suggesting the involvement of HLH proteins in the DNA/protein complex formation. In contrast to the osteoblast-like cells, OCE1-binding activity in the nuclear extracts of C3H10T1/2 fibroblasts was very low. However, when these fibroblasts were treated with recombinant human bone morphogenetic protein-2 which induced expression of osteocalcin as well as other phenotypic markers of osteoblasts, OCE1-binding activity was increased approximately 40-fold, indicating that OCE1 would be involved in the tissue-specific expression of the osteocalcin gene. These findings indicated for the first time that osteoblast-specific gene transcription is regulated via the interaction between certain E-box binding transcription factor(s) in osteoblasts and the OCE1 sequence in the promoter region of the osteocalcin gene.

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Orphan Nuclear Receptor Small Heterodimer Partner, a Novel Corepressor for a Basic Helix-Loop-Helix Transcription Factor BETA2/NeuroD

Molecular Endocrinology ◽

10.1210/me.2003-0311 ◽

2004 ◽

Vol 18 (4) ◽

pp. 776-790 ◽

Cited By ~ 37

Author(s):

Joon-Young Kim ◽

Khoi Chu ◽

Han-Jong Kim ◽

Hyun-A Seong ◽

Ki-Cheol Park ◽

...

Keyword(s):

Transcription Factor ◽

Nuclear Receptor ◽

Transcriptional Activity ◽

Orphan Nuclear Receptor ◽

Small Heterodimer Partner ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

E Box ◽

Coactivator P300

Abstract Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor that lacks a conventional DNA binding domain (DBD) and represses the transcriptional activity of various nuclear receptors. In this study, we examined the novel cross talk between SHP and BETA2/NeuroD, a basic helix-loop-helix transcription factor. In vitro and in vivo protein interaction studies showed that SHP physically interacts with BETA2/NeuroD, but not its heterodimer partner E47. Moreover, confocal microscopic study and immunostaining results demonstrated that SHP colocalized with BETA2 in islets of mouse pancreas. SHP inhibited BETA2/NeuroD-dependent transactivation of an E-box reporter, whereas SHP was unable to repress the E47-mediated transactivation and the E-box mutant reporter activity. In addition, SHP repressed the BETA2-dependent activity of glucokinase and cyclin-dependent kinase inhibitor p21 gene promoters. Gel shift and in vitro protein competition assays indicated that SHP inhibits neither dimerization nor DNA binding of BETA2 and E47. Rather, SHP directly repressed BETA2 transcriptional activity and p300-enhanced BETA2/NeuroD transcriptional activity by inhibiting interaction between BETA2 and coactivator p300. We also showed that C-terminal repression domain within SHP is also required for BETA2 repression. However, inhibition of BETA2 activity was not observed by naturally occurring human SHP mutants that cannot interact with BETA2/NeuroD. Taken together, these results suggest that SHP acts as a novel corepressor for basic helix-loop-helix transcription factor BETA2/NeuroD by competing with coactivator p300 for binding to BETA2/NeuroD and by its direct transcriptional repression function.

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Coexamination of Site-Specific Transcription Factor Binding and Promoter Activity in Living Cells

Molecular and Cellular Biology ◽

10.1128/mcb.19.12.8393 ◽

1999 ◽

Vol 19 (12) ◽

pp. 8393-8399 ◽

Cited By ~ 137

Author(s):

Kathryn E. Boyd ◽

Peggy J. Farnham

Keyword(s):

Cell Cycle ◽

Transcription Factor ◽

Transcriptional Activation ◽

Transcriptional Activity ◽

Living Cells ◽

E Box ◽

Two Factors ◽

Promoter Occupancy ◽

First Time

ABSTRACT Previously, we have used a chromatin cross-linking and immunoprecipitation protocol for the analysis of Myc and USF binding to the cad promoter. The adaptation of this technique for the study of mammalian transcription factors was a big step forward in the analysis of transcription factor family member specificity, allowing for the first time a definitive knowledge of which factor binds to a promoter region under normal physiological conditions. However, due to limitations of the assay, our previous studies could not definitively prove that both Myc and USF bound to the exact same site on the cad promoter, nor could we directly correlate loss of in vivo binding of a particular factor with loss of transcriptional activity. Therefore, we have further modified the chromatin immunoprecipitation protocol to alleviate these problems. We have now shown that it is possible to coexamine growth-regulated transcriptional activity and promoter occupancy by using stably integrated promoter constructs. We show that both Myc and USF bind to the exact same E box on the cad promoter, suggesting that competition between these two factors for a single site occurs in living cells. We also find that cad promoter constructs that retain USF binding but lose Myc binding in vivo no longer display an increase in transcriptional activity in mid- to late G1 phase of the cell cycle. Finally, we propose that cell cycle-regulated transcriptional activation of the cadpromoter may be a stochastic, rather than a predetermined, process.

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β-Catenin Interacts with MyoD and Regulates Its Transcription Activity

Molecular and Cellular Biology ◽

10.1128/mcb.01682-07 ◽

2008 ◽

Vol 28 (9) ◽

pp. 2941-2951 ◽

Cited By ~ 53

Author(s):

Chang-Hoon Kim ◽

Hannah Neiswender ◽

Eun Joo Baik ◽

Wen C. Xiong ◽

Lin Mei

Keyword(s):

Transcription Factor ◽

Transcriptional Activity ◽

Muscle Development ◽

Muscle Cells ◽

Muscle Differentiation ◽

Canonical Pathway ◽

Transcription Activity ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

E Box

ABSTRACT Wnt regulation of muscle development is thought to be mediated by the β-catenin-TCF/LEF-dependent canonical pathway. Here we demonstrate that β-catenin, not TCF/LEF, is required for muscle differentiation. We showed that β-catenin interacts directly with MyoD, a basic helix-loop-helix transcription factor essential for muscle differentiation and enhances its binding to E box elements and transcriptional activity. MyoD-mediated transactivation is inhibited in muscle cells when β-catenin is deficient or the interaction between MyoD and β-catenin is disrupted. These results demonstrate that β-catenin is necessary for MyoD function, identifying MyoD as an effector in the Wnt canonical pathway.

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