Purification and identification of a transcription factor, USF-2, binding to E-box element in the promoter of human telomerase reverse transcriptase (hTERT)
Abstract:Controversy remains about the identity of the transcription factor(s) which bind to the two E-box elements (CACGTG, proximal and distal) of the human telomerase (hTERT) gene promoter, the essential elements in the regulation of telomerase. Here, systematic oligonucleotide trapping supplemented with two-dimensional gel electrophoresis (2-DE) and proteomic methods was used to identify E-box binding transcription factors. Although insufficient purity was obtained from the proximal E-box element trapping, further … Show more
“…The hTERT promoter contains two E-box transcription factor binding sites. These, proximal and distal, were shown to bind the USF-2 transcription factor earlier [8]. This promoter, however, has no binding site for NFκB.…”
Section: Resultsmentioning
confidence: 99%
“…The sequence is from the human telomerase (hTERT) promoter proximal E-box. Previously, we had shown that this sequence binds USF-2 [13]. The initial mixture (C), flow through (F), last wash through (W 10 ) and eluate (E) by high salt were resolved by 12% SDS PAGE gel.…”
Section: Figurementioning
confidence: 99%
“…Here we used both the C/EBP binding oligonucleotide, and another which is derived from the human telomerase (hTERT) promoter. This latter sequence was shown previously to bind the USF-2 transcription factor [8]. The two sequences were then shown to purify GFP-C/EBP and USF-2, respectively, using the trapping approach and to give higher purity than is obtained by conventional affinity chromatography.…”
The uses of a method of coupling DNA is investigated for trapping and purifying transcription factors. Using the GFP-C/EBP fusion protein as a model, trapping gives higher purity and comparable yield to conventional affinity chromatography. The chemistry utilized is mild and was shown to have no detrimental effect on GFP fluorescence or GFP-C/EBP DNA-binding. The method involves introducing a ribose nucleotide to the 3′ end of a DNA sequence. Reaction with mM NaIO4 (sodium metaperiodate) produces a dialdehyde of ribose which couples to hydrazide-agarose. The DNA is combined at nM concentration with a nuclear extract or other protein mixture and DNA-protein complexes form. The complex is then coupled to hydrazide-agarose for trapping the DNA-protein complex and the protein eluted by increasing NaCl concentration. Using a different oligonucleotide with the proximal E-box sequence from the human telomerase promoter, USF-2 transcription factor was purified by trapping, again with higher purity than results from conventional affinity chromatography and similar yield. Other transcription factors binding E-boxes including E2A, c-myc, and myo-D were also purified but myogenenin and NFκB were not. Therfore, this approach proved valuable for both affinity chromatography and for the trapping approach.
“…The hTERT promoter contains two E-box transcription factor binding sites. These, proximal and distal, were shown to bind the USF-2 transcription factor earlier [8]. This promoter, however, has no binding site for NFκB.…”
Section: Resultsmentioning
confidence: 99%
“…The sequence is from the human telomerase (hTERT) promoter proximal E-box. Previously, we had shown that this sequence binds USF-2 [13]. The initial mixture (C), flow through (F), last wash through (W 10 ) and eluate (E) by high salt were resolved by 12% SDS PAGE gel.…”
Section: Figurementioning
confidence: 99%
“…Here we used both the C/EBP binding oligonucleotide, and another which is derived from the human telomerase (hTERT) promoter. This latter sequence was shown previously to bind the USF-2 transcription factor [8]. The two sequences were then shown to purify GFP-C/EBP and USF-2, respectively, using the trapping approach and to give higher purity than is obtained by conventional affinity chromatography.…”
The uses of a method of coupling DNA is investigated for trapping and purifying transcription factors. Using the GFP-C/EBP fusion protein as a model, trapping gives higher purity and comparable yield to conventional affinity chromatography. The chemistry utilized is mild and was shown to have no detrimental effect on GFP fluorescence or GFP-C/EBP DNA-binding. The method involves introducing a ribose nucleotide to the 3′ end of a DNA sequence. Reaction with mM NaIO4 (sodium metaperiodate) produces a dialdehyde of ribose which couples to hydrazide-agarose. The DNA is combined at nM concentration with a nuclear extract or other protein mixture and DNA-protein complexes form. The complex is then coupled to hydrazide-agarose for trapping the DNA-protein complex and the protein eluted by increasing NaCl concentration. Using a different oligonucleotide with the proximal E-box sequence from the human telomerase promoter, USF-2 transcription factor was purified by trapping, again with higher purity than results from conventional affinity chromatography and similar yield. Other transcription factors binding E-boxes including E2A, c-myc, and myo-D were also purified but myogenenin and NFκB were not. Therfore, this approach proved valuable for both affinity chromatography and for the trapping approach.
“…Based on the enhanceosome concept, we hypothesized that another transcription factor(s) is required to activate HIF target genes during hypoxia. We found that many HIF target genes, including HMOX1 (41,42,63,64,84,88,89,112), SFTPA1 (13,32,33), CXCR4 (73,90,96), PAI1 (2,18,27,29,47,57,58,66), BDNF (48,97), hTERT (3,9,35,44,52,61,67,72,75,110,113), CTSB (111) (107), and P4H␣(I) (10,43,98), are also reported to be activated by the transcription factor upstream stimulatory factor 1 (USF1) or USF2, suggesting a possible role of USF1/USF2 in the hypoxic response.USFs (USF1 and USF2) are basic helix-loop-helix-leucine zipper (bHLH-LZ) transcription factors that are expressed ubiquitously, albeit at different levels depending on the tissue type (14,36,94,95,101 (7,8,19) as either USF1/USF1 or USF2/USF2 homodimers or USF1/USF2 heterodimers. The major functional USF complexes in most cell types are USF1/USF2 heterodimers (94, 101).…”
While the functions of hypoxia-inducible factor 1␣ (HIF1␣)/aryl hydrocarbon receptor nuclear translocator (ARNT) and HIF2␣/ARNT (HIF2) proteins in activating hypoxia-inducible genes are well established, the role of other transcription factors in the hypoxic transcriptional response is less clear. We report here for the first time that the basic helix-loop-helix-leucine-zip transcription factor upstream stimulatory factor 2 (USF2) is required for the hypoxic transcriptional response, specifically, for hypoxic activation of HIF2 target genes. We show that inhibiting USF2 activity greatly reduces hypoxic induction of HIF2 target genes in cell lines that have USF2 activity, while inducing USF2 activity in cells lacking USF2 activity restores hypoxic induction of HIF2 target genes. Mechanistically, USF2 activates HIF2 target genes by binding to HIF2 target gene promoters, interacting with HIF2␣ protein, and recruiting coactivators CBP and p300 to form enhanceosome complexes that contain HIF2␣, USF2, CBP, p300, and RNA polymerase II on HIF2 target gene promoters. Functionally, the effect of USF2 knockdown on proliferation, motility, and clonogenic survival of HIF2-dependent tumor cells in vitro is phenocopied by HIF2␣ knockdown, indicating that USF2 works with HIF2 to activate HIF2 target genes and to drive HIF2-depedent tumorigenesis.A hypoxic microenvironment is frequently found in solid tumors. The transcriptional response mediated by hypoxia-inducible factor 1␣ (HIF1␣)/aryl hydrocarbon receptor nuclear translocator (ARNT) (HIF1) and HIF2␣/ARNT (HIF2) plays a critical role in malignant progression by increasing expression of genes involved in angiogenesis, anaerobic metabolism, and other processes that enable tumor cells to survive and/or escape their O 2 -deficient microenvironment (25,53,56,93).It is well established that multiple transcription factors (TFs) are required to achieve maximal activation of target genes in response to a specific stimulus. This multifactorial transcription complex has been termed the "enhanceosome" (100). Individual factors in the enhanceosome complex may promote transcription initiation by recruiting RNA polymerase II (Pol II)/general transcription factors and/or recruiting chromatin-modifying enzymes, such as histone acetylases and chromatin remodeling complexes. In addition, TFs such as Myc increase gene expression by recruiting elongation factors to regulate Pol II pause release (77). Thus, reduced levels of transcription could occur in the absence of factors that have redundant functions within the enhanceosome, while other transcription factors having unique functions are absolutely required for gene activation.The role of HIF1 and HIF2 in activating hypoxia-inducible genes is well established (21,37,48,79,103). However, the other transcription factors required for hypoxic activation of HIF target genes have been much less studied. Based on the enhanceosome concept, we hypothesized that another transcription factor(s) is required to activate HIF target genes during hypoxia. We ...
“…One of the general TFs with the highest identification score involved in transcriptional complexes is General Transcription Factor II-I (GTF2-I) with an expectation value of 5.9 × 10 −05 (shown in Figure 7). GTF2-I has been known to co-regulate hTERT activity with USF (Upstream Stimulatory Factor) [11], which was also shown to be present in the promoter complex (Figure 6) [12]. The higher abundance of general transcription factors following promoter trapping allows isolation and identification by mass spectrometry as well as the specific TFs such as AP2 and SP1.Figure 7
MS/MS fragmentation of RPELLTHSTTEVTQPR found in GTF2-I_HUMAN.…”
BackgroundTranscription factors bind to response elements on the promoter regions of genes to regulate transcriptional activity. One of the major problems with identifying transcription factors is their low abundance relative to other proteins in the cell. Developing a purification technique specific for transcription factors is crucial to the understanding of gene regulation. Promoter trapping is a method developed that uses the promoter regions as bait to trap proteins of interest and then purified using column chromatography. Here we utilize this technique to study the telomerase promoter, which has increased transcriptional activity in cancer cells. Gaining insight on how to control the enzyme at the promoter level may give new routes towards cancer treatments.ResultsOur findings show that the telomerase promoter (−170 - +91) and Promoter Trapping isolate a transcriptionally active and reproducible complex, when analyzed by liquid chromatography tandem mass spectrometry. We were also able to identify transcription factors, including AP-2 and SP1 known to bind this promoter, as well as show that these two proteins can bind to each other’s response element.ConclusionHere we focus on verifying the ability and versatility of Promoter Trapping coupled with additional well-characterized methods to identify already known factors responsible for telomerase transcriptional regulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-014-0053-2) contains supplementary material, which is available to authorized users.
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