IDENTIFICATION OF A NOVEL REPRESSOR ELEMENT IN THE CYCLO-OXYGENASE-2 PROMOTER AND ITS NUCLEAR BINDING PROTEIN

ABSTRACT We previously showed that reduced intracellular levels of the TATA binding protein (TBP), brought about by tbp heterozygosity in DT40 cells, resulted in a mitotic delay reflecting reduced expression of the mitotic regulator cdc25B but did not significantly affect overall transcription. Here we extend these findings in several ways. We first provide evidence that the decrease in cdc25B expression reflects reduced activity of the cdc25B core promoter in the heterozygous (TBP-het) cells. Strikingly, mutations in a previously described repressor element that overlaps the TATA box restored promoter activity in TBP-het cells, supporting the idea that the sensitivity of this promoter to TBP levels reflects a competition between TBP and the repressor for DNA binding. To determine whether cells might have mechanisms to compensate for fluctuations in TBP levels, we next examined expression of the two known vertebrate TBP homologues, TLP and TBP2. Significantly, mRNAs encoding both were significantly overexpressed relative to levels observed in wild-type cells. In the case of TLP, this was shown to reflect regulation of the core promoter by both TBP and TLP. Together, our results indicate that variations in TBP levels can affect the transcription of specific promoters in distinct ways, but overall transcription may be buffered by corresponding alterations in the expression of TBP homologues.

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Specific nuclear binding of adenosine 3',5'-monophosphate-binding protein complex with subsequent poly(A) RNA synthesis in embryonic chick cartilage.

Journal of Clinical Investigation ◽

10.1172/jci109885 ◽

1980 ◽

Vol 66 (3) ◽

pp. 532-542 ◽

Cited By ~ 8

Author(s):

W M Burch ◽

H E Lebovitz

Keyword(s):

Protein Complex ◽

Rna Synthesis ◽

Binding Protein ◽

Embryonic Chick ◽

Nuclear Binding

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Derepression of mouse beta-major-globin gene transcription during erythroid differentiation

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4324-4332.1991 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4324-4332

Author(s):

K Macleod ◽

M Plumb

Keyword(s):

Cell Differentiation ◽

Promoter Activity ◽

Binding Protein ◽

Regulatory Element ◽

Globin Gene ◽

Terminal Differentiation ◽

Erythroid Differentiation ◽

Site Directed Mutagenesis ◽

Repressor Element

Functional analysis of the mouse beta-major-globin gene promoter has revealed a negative regulatory element (-100 to -250 bp) which represses promoter activity in mouse erythroleukemia (MEL) cells. Promoter activity is induced 14-fold during terminal differentiation of MEL cells. Three major in vitro binding sites for NF1 (-250 bp), GATA-1 (-212 bp), and a sequence at -165 bp (BB1) have been defined in this region. Site-directed mutagenesis of any one of the three sites resulted in a five- to sixfold up-regulation of promoter activity in uninduced MEL cells, but only three- to fourfold stimulation was observed from the mutant promoters during MEL cell terminal differentiation. This finding suggests that all three sites are required for repressor activity in uninduced MEL cells and that derepression occurs during MEL cell differentiation. BB1 DNA-binding activity decreases during MEL cell differentiation, suggesting a central role for this factor in modulating the effects of the repressor element. The BB1-binding factor also competes with the CCAAT-binding protein for binding the CCAAT motif. The fact that a reduced but significant stimulation of promoter activity during differentiation is observed in the absence of the repressor element raises the possibility that the BB1 factor also down-regulates transcription in undifferentiated MEL cells by displacing binding of CCAAT-binding protein to the proximal CCAAT motif.

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Nuclear interactions of retinoic acid-binding protein in chemically induced mammary adenocarcinoma

Biochemical Journal ◽

10.1042/bj2080731 ◽

1982 ◽

Vol 208 (3) ◽

pp. 731-736 ◽

Cited By ~ 25

Author(s):

R G Mehta ◽

W L Cerny ◽

R C Moon

Keyword(s):

Retinoic Acid ◽

Binding Protein ◽

Sedimentation Coefficient ◽

Sucrose Density Gradient ◽

Mammary Adenocarcinoma ◽

Nuclear Fraction ◽

Isolated Nuclei ◽

Acid Binding Protein ◽

Nuclear Binding ◽

Chemically Induced

Cellular retinoic acid-binding protein (CRABP) was detected in the nuclear fraction of N-methyl-N-nitrosourea-induced mammary cancers after the incubation of cytosol containing [3H]retinoic acid (RA)-bound CRABP with isolated nuclei. CRABP extracted from the nuclei in buffer containing 0.4 M-KCl sedimented as a 2 S component when subjected to sucrose-density-gradient analysis. [3H]RA-CRABP was found to be a prerequisite for the detection of nuclear binding, since the incubation of isolated nuclei or 0.4 M-KCl extract of the nuclei with [3H]RA did not result in any significant binding. Incubation of [3H]RA-CRABP at 25 or 30 degrees C before incubation with the nuclei neither altered the sedimentation coefficient nor enhanced the nuclear binding compared with 0 degrees C incubation. The tumour nuclei contained a saturable number of binding sites with a dissociation constant of 1.6×10(-9) M. These results indicate that the action of retinoic acid in the target organ may be mediated by its interaction with the nuclei.

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Transcriptional regulation of human MUC4 gene: identification of a novel inhibitory element and its nuclear binding protein

Molecular Biology Reports ◽

10.1007/s11033-013-2591-6 ◽

2013 ◽

Vol 40 (8) ◽

pp. 4913-4920 ◽

Cited By ~ 8

Author(s):

Jing-Jing Zhang ◽

Yi Zhu ◽

Xiong-Fei Zhang ◽

Wen-Biao Liang ◽

Kun-Ling Xie ◽

...

Keyword(s):

Transcriptional Regulation ◽

Binding Protein ◽

Gene Identification ◽

Nuclear Binding ◽

Muc4 Gene

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Derepression of mouse beta-major-globin gene transcription during erythroid differentiation.

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4324 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4324-4332 ◽

Cited By ~ 24

Author(s):

K Macleod ◽

M Plumb

Keyword(s):

Cell Differentiation ◽

Promoter Activity ◽

Binding Protein ◽

Regulatory Element ◽

Globin Gene ◽

Terminal Differentiation ◽

Erythroid Differentiation ◽

Site Directed Mutagenesis ◽

Repressor Element

Functional analysis of the mouse beta-major-globin gene promoter has revealed a negative regulatory element (-100 to -250 bp) which represses promoter activity in mouse erythroleukemia (MEL) cells. Promoter activity is induced 14-fold during terminal differentiation of MEL cells. Three major in vitro binding sites for NF1 (-250 bp), GATA-1 (-212 bp), and a sequence at -165 bp (BB1) have been defined in this region. Site-directed mutagenesis of any one of the three sites resulted in a five- to sixfold up-regulation of promoter activity in uninduced MEL cells, but only three- to fourfold stimulation was observed from the mutant promoters during MEL cell terminal differentiation. This finding suggests that all three sites are required for repressor activity in uninduced MEL cells and that derepression occurs during MEL cell differentiation. BB1 DNA-binding activity decreases during MEL cell differentiation, suggesting a central role for this factor in modulating the effects of the repressor element. The BB1-binding factor also competes with the CCAAT-binding protein for binding the CCAAT motif. The fact that a reduced but significant stimulation of promoter activity during differentiation is observed in the absence of the repressor element raises the possibility that the BB1 factor also down-regulates transcription in undifferentiated MEL cells by displacing binding of CCAAT-binding protein to the proximal CCAAT motif.

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