Identification of Transcriptional Enhancer Factor-4 as a Transcriptional Modulator of CTP:Phosphocholine Cytidylyltransferase α

CTP:phosphocholine cytidylyltransferase (CCT) is the rate-limiting and regulated enzyme of mammalian phosphatidylcholine biosynthesis. There are three isoforms, CCTα, CCTβ1, and CCTβ2. The mouse CCTα gene promoter is regulated by an enhancer element (Eb) located between −103 and −82 base pairs (5′-GTTTTCAGGAATGCGGAGGTGG-3′) upstream from the transcriptional start site (Bakovic, M., Waite, K., Tang, W., Tabas, I., and Vance, D. E. (1999)Biochim. Biophys. Acta1436, 147–165). To identify the Eb-binding protein(s), we screened a mouse embryo cDNA library by the yeast one-hybrid system and obtained 19 positive clones. Ten cDNA clones were identified as transcriptional enhancer factor-4 (TEF-4). The TEF-binding consensus sequence, 5′-(A/T)(A/G)(A/G)(A/T)ATG(C/T)(G/A)-3′, was identified within the Eb binding region. Gel-shift analysis using radiolabeled Eb fragment as a probe showed that cell extracts from yeast expressing hemagglutinin-tagged TEF-4 caused a marked band retardation that could be prevented with an anti-hemagglutinin antibody. When COS-7 cells were transfected with TEF-4, CCTα promoter-luciferase reporter activity and CCTα mRNA levels increased. A TEF-4 deletion mutant containing a DNA-binding domain, mTEA(+), stimulated the CCTα promoter activity, whereas protein lacking the DNA binding domain, mTEA(−), did not. Unexpectedly, when the ATG core of the TEF-4 binding consensus within the Eb region was mutated, promoter activity was enhanced rather than decreased. Thus, TEF-4 might act as a dual transcriptional modulator as follows: as a suppressor via its direct binding to the Eb element and as an activator via its interactions with the basal transcriptional machinery. These results provide the first evidence that TEF-4 is an important regulator of CCTα gene expression.

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Characterization of the transcription activation function and the DNA binding domain of transcriptional enhancer factor-1.

The EMBO Journal ◽

10.1002/j.1460-2075.1993.tb05888.x ◽

1993 ◽

Vol 12 (6) ◽

pp. 2337-2348 ◽

Cited By ~ 54

Author(s):

J.J. Hwang ◽

P. Chambon ◽

I. Davidson

Keyword(s):

Dna Binding ◽

Transcription Activation ◽

Activation Function ◽

Binding Domain ◽

Dna Binding Domain ◽

Transcriptional Enhancer ◽

Transcription Activation Function ◽

Enhancer Factor

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The monomer-binding orphan receptor Rev-Erb represses transcription as a dimer on a novel direct repeat.

Molecular and Cellular Biology ◽

10.1128/mcb.15.9.4791 ◽

1995 ◽

Vol 15 (9) ◽

pp. 4791-4802 ◽

Cited By ~ 132

Author(s):

H P Harding ◽

M A Lazar

Keyword(s):

Retinoic Acid ◽

Dna Binding ◽

Binding Site ◽

Binding Domain ◽

Luciferase Reporter ◽

Dna Binding Domain ◽

Transactivation Domain ◽

Direct Repeats ◽

Basal Transcription ◽

C Terminus

Rev-Erb is an orphan nuclear receptor which binds as a monomer to the thyroid/retinoic acid receptor half-site AGGTCA flanked 5' by an A/T-rich sequence, referred to here as a Rev monomer site. Fusion of Rev-Erb to the DNA binding domain of yeast GAL4 strongly repressed basal transcription of a GAL4-luciferase reporter gene as a result of the presence of a C-terminal domain containing both the hinge and heptad repeat regions. Nevertheless, wild-type Rev-Erb did not repress basal transcription from the Rev monomer binding site. Therefore, a DNA binding site selection strategy was devised to test the hypothesis that Rev-Erb may function on a different site as a dimer. This approach identified sequences containing two Rev monomer sites arranged as direct repeats with the AGGTCA motifs separated by 2 bp (Rev-DR2). Remarkably, Rev-Erb bound as a homodimer to Rev-DR2 but not to other direct repeats or to a standard DR2 sequence. The DNA binding domain contained all of the determinants for Rev-DR2-specific homodimerization. Rev-Erb bound cooperatively as a homodimer to Rev-DR2, and this interaction was 5 to 10 times more stable than Rev-Erb monomer binding to the Rev monomer site. Functionally, Rev-Erb markedly repressed the basal activity of a variety of promoters with a strong Rev-DR2 specificity. The C terminus was required for this repression, consistent with the GAL4 results. However, the Rev-DR2 specificity did not require the C terminus in vivo, since fusion of C-terminally truncated Rev-Erb to a heterologous transactivation domain created a transcriptional activator specific for Rev-DR2. In addition to idealized Rev-DR2 sites, Rev-Erb also repressed basal as well as retinoic acid-induced transcription from a naturally occurring Rev-DR2 in the CRBPI gene. Thus, although Rev-Erb is distinguished from other thyroid/steroid receptor superfamily members by its ability to bind DNA as a monomer, it functions as a homodimer to repress transcription of genes containing a novel DR2 element.

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Structural and functional analysis of the related transcriptional enhancer factor-1 and NF-κB interaction

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00069.2013 ◽

2014 ◽

Vol 306 (2) ◽

pp. H233-H242 ◽

Cited By ~ 2

Author(s):

Jieliang Ma ◽

Li Zhang ◽

Aaron R. Tipton ◽

Jiaping Wu ◽

Angela F. Messmer-Blust ◽

...

Keyword(s):

Gene Expression ◽

Gene Transcription ◽

Promoter Activity ◽

Physical Interaction ◽

Inflammatory Factor ◽

Mrna Levels ◽

Transcriptional Enhancer ◽

Tnf Α ◽

Enhancer Factor ◽

In Cells

The related transcriptional enhancer factor-1 (RTEF-1) increases gene transcription of hypoxia-inducible factor 1α (HIF-1α) and enhances angiogenesis in endothelium. Both hypoxia and inflammatory factor TNF-α regulate gene expression of HIF-1α, but how RTEF-1 and TNF-α coordinately regulate HIF-1α gene transcription is unclear. Here, we found that RTEF-1 interacts with p65 subunit of NF-κB, a primary mediator of TNF-α. RTEF-1 increased HIF-1α promoter activity, whereas expression of p65 subunit inhibited the stimulatory effect. By contrast, knockdown of p65 markedly enhanced RTEF-1 stimulation on the HIF-1α promoter activity (7-fold). A physical interaction between RTEF-1 and p65 was confirmed by coimmunoprecipitation experiments in cells and glutathione S-transferase (GST)-pull-down assays. A computational analysis of RTEF-1 crystal structures revealed that a conserved surface of RTEF-1 potentially interacts with p65 via four amino acid residues located at T347, Y349, R351, and Y352. We performed site-directed mutagenesis and GST-pull-down assays and demonstrated that Tyr352 (Y352) in RTEF-1 is a key site for the formation of RTEF-1 and p65-NF-κB complex. An alanine mutation at Y352 of RTEF-1 disrupted the interaction of RTEF-1 with p65. Moreover, expression of RTEF-1 decreased TNF-α-induced HIF-1α promoter activity, IL-1β, and IL-6 mRNA levels in cells; however, the effect of RTEF-1 was largely lost when Y352 was mutated to alanine. These results indicate that RTEF-1 interacts with p65-NF-κB through Y352 and that they antagonize each other for HIF-1α transcriptional activation, suggesting a novel mechanism by which RTEF-1 regulates gene expression, linking hypoxia to inflammation.

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Biochemical characterization of human Ecdysoneless reveals a role in transcriptional regulation

Biological Chemistry ◽

10.1515/bc.2010.004 ◽

2010 ◽

Vol 391 (1) ◽

Cited By ~ 5

Author(s):

Jun Hyun Kim ◽

Channabasavaiah Basavaraju Gurumurthy ◽

Hamid Band ◽

Vimla Band

Keyword(s):

Dna Binding ◽

Nuclear Export ◽

Biochemical Characterization ◽

Binding Domain ◽

Single Amino Acid ◽

Luciferase Reporter ◽

Dna Binding Domain ◽

Transactivation Activity ◽

Single Amino Acid Change ◽

Evolutionarily Conserved

AbstractEcdysoneless (Ecd) is an evolutionarily conserved protein and its function is essential for embryonic development inDrosophilaand cell growth in yeast. However, its function has remained unknown until recently. Studies in yeast suggested a potential role of Ecd in transcription; however, Ecd lacks a DNA-binding domain. Using a GAL4-luciferase reporter assay and a GAL4 DNA-binding domain fusion with Ecd or its mutants, we present evidence that human Ecd has a transactivation activity in its C-terminal region. Importantly, further analyses using point mutants showed that a single amino acid change at either Asp-484 or Leu-489 essentially completely abolishes the transactivation activity of Ecd. We further demonstrate that Ecd interacts with p300, a histone acetyltransferase, and coexpression of Ecd with p300 enhances the Ecd-mediated transactivation activity. Ecd localizes to both nucleus and cytoplasm and shuttles between the nucleus and cytoplasm; however, it exhibits strong nuclear export. Based on previous yeast studies and evidence provided here, we suggest that Ecd functions as a transcriptional regulator. Our results indicate an important function of human Ecd and provide a basis to explore the transcriptional partners of Ecd.

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Physical Interaction between the MADS Box of Serum Response Factor and the TEA/ATTS DNA-binding Domain of Transcription Enhancer Factor-1

Journal of Biological Chemistry ◽

10.1074/jbc.m008625200 ◽

2001 ◽

Vol 276 (13) ◽

pp. 10413-10422 ◽

Cited By ~ 58

Author(s):

Madhu Gupta ◽

Paul Kogut ◽

Francesca J. Davis ◽

Narasimhaswamy S. Belaguli ◽

Robert J. Schwartz ◽

...

Keyword(s):

Dna Binding ◽

Serum Response Factor ◽

Binding Domain ◽

Physical Interaction ◽

Response Factor ◽

Dna Binding Domain ◽

Mads Box ◽

Serum Response ◽

Transcription Enhancer ◽

Enhancer Factor

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Dynamic Regulation of Copper Uptake and Detoxification Genes in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.18.5.2514 ◽

1998 ◽

Vol 18 (5) ◽

pp. 2514-2523 ◽

Cited By ~ 121

Author(s):

Maria Marjorette O. Peña ◽

Keith A. Koch ◽

Dennis J. Thiele

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Binding ◽

Transcriptional Activation ◽

Copper Ions ◽

Binding Domain ◽

Mrna Levels ◽

Dna Binding Domain ◽

Transactivation Domain ◽

Copper Uptake ◽

Copper Sensor

ABSTRACT The essential yet toxic nature of copper demands tight regulation of the copper homeostatic machinery to ensure that sufficient copper is present in the cell to drive essential biochemical processes yet prevent the accumulation to toxic levels. In Saccharomyces cerevisiae, the nutritional copper sensor Mac1p regulates the copper-dependent expression of the high affinity Cu(I) uptake genesCTR1, CTR3, and FRE1, while the toxic copper sensor Ace1p regulates the transcriptional activation of the detoxification genes CUP1, CRS5, andSOD1 in response to copper. In this study, we characterized the tandem regulation of the copper uptake and detoxification pathways in response to the chronic presence of elevated concentrations of copper ions in the growth medium. Upon addition of CuSO4, mRNA levels of CTR3 were rapidly reduced to eightfold the original basal level whereas the Ace1p-mediated transcriptional activation of CUP1 was rapid and potent but transient.CUP1 expression driven by an Ace1p DNA binding domain-herpes simplex virus VP16 transactivation domain fusion was also transient, demonstrating that this mode of regulation occurs via modulation of the Ace1p copper-activated DNA binding domain. In vivo dimethyl sulfate footprinting analysis of the CUP1 promoter demonstrated transient occupation of the metal response elements by Ace1p which paralleled CUP1 mRNA expression. Analysis of a Mac1p mutant, refractile for copper-dependent repression of the Cu(I) transport genes, showed an aberrant pattern of CUP1expression and copper sensitivity. These studies (i) demonstrate that the nutritional and toxic copper metalloregulatory transcription factors Mac1p and Ace1p must sense and respond to copper ions in a dynamic fashion to appropriately regulate copper ion homeostasis and (ii) establish the requirement for a wild-type Mac1p for survival in the presence of toxic copper levels.

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