scholarly journals Position-dependent transcriptional regulation of the murine dihydrofolate reductase promoter by the E2F transactivation domain.

1997 ◽  
Vol 17 (4) ◽  
pp. 1966-1976 ◽  
Author(s):  
C J Fry ◽  
J E Slansky ◽  
P J Farnham
Keyword(s):  
Transcriptional Regulation ◽  
Transcription Start Site ◽  
Dihydrofolate Reductase ◽  
Growth Regulation ◽  
S Phase ◽  
Transactivation Domain ◽  
Start Site ◽  
Transcription Start ◽  
Activation Domains ◽  
Transactivation Domains

Activity of the dihydrofolate reductase (dhfr) promoter increases at the G1-S-phase boundary of the cell cycle. Mutations that abolish protein binding to an E2F element in the dhfr promoter also abolish the G1-S-phase increase in dhfr transcription, indicating that transcriptional regulation is mediated by the E2F family of proteins. To investigate the mechanism by which E2F regulates dhfr transcription, we moved the E2F element upstream and downstream of its natural position in the promoter. We found that the E2F element confers growth regulation to the dhfr promoter only when it is proximal to the transcription start site. Using a heterologous E2F element, we showed that position-dependent regulation is a property that is promoter specific, not E2F element specific. We demonstrated that E2F-mediated growth regulation of dhfr transcription requires activation of the dhfr promoter in S phase and that the C-terminal activation domains of E2F1, E2F4, and E2F5, when fused to the Gal4 DNA binding domain, are sufficient to specify position-dependent activation. To further investigate the role of activation in dhfr regulation, we tested other transactivation domains for their ability to activate the dhfr promoter. We found that the N-terminal transactivation domain of VP16 cannot activate the dhfr promoter. We propose that, unlike other E2F-regulated promoters, robust transcription from the dhfr promoter requires an E2F transactivation domain close to the transcription start site.

scholarly journals Differential Regulation of the Inhibitor of Apoptosis ch-IAP1 by v-rel and the Proto-Oncogene c-rel

2002 ◽  
Vol 76 (23) ◽  
pp. 11960-11970 ◽  
Author(s):  
Jarmila Kralova ◽  
Andrew S. Liss ◽  
William Bargmann ◽  
Cullen Pendleton ◽  
Janani Varadarajan ◽  
...  
Keyword(s):  
Transcription Start Site ◽  
Inhibitor Of Apoptosis ◽  
Start Site ◽  
Transcription Start ◽  
Aberrant Expression ◽  
Transactivation Domains ◽  
Expression Of Genes ◽  
Elevated Expression ◽  
Reporter Assays ◽  
Full Activation

ABSTRACT The v-rel oncogene encoded by reticuloendotheliosis virus is the acutely transforming member of the Rel/NF-κB family of transcription factors. v-Rel is a truncated and mutated form of c-Rel and transforms cells by inducing the aberrant expression of genes regulated by Rel/NF-κB proteins. The expression of ch-IAP1, a member of the inhibitor-of-apoptosis family, is highly elevated in cells expressing v-Rel and contributes to the immortalization of cells transformed by this oncoprotein. In this study we demonstrate that the elevated expression of ch-IAP1 in v-Rel-expressing cells is due to an increased rate of transcription. The ch-IAP1 promoter was isolated, and four Rel/NF-κB binding sites were identified upstream of the transcription start site. Two κB sites proximal to the transcription start site were required for v-Rel to activate the ch-IAP1 promoter. While c-Rel also utilized these sites, a third more-distal κB site was required for its full activation of the ch-IAP1 promoter. Differences in the transactivation domains of v-Rel and c-Rel are responsible for their different abilities to utilize these sites and account for their differential activation of the ch-IAP1 promoter. Although c-Rel was a more potent activator of the ch-IAP1 promoter than v-Rel in transient reporter assays, cells stably overexpressing c-Rel failed to maintain high levels of ch-IAP1 expression. The reduction of ch-IAP1 expression in these cells correlated with the efficient regulation of c-Rel by IκBα. The ability of v-Rel to escape IκBα regulation allows for the gradual and sustained elevation of ch-IAP1 expression directly contributing to the transforming properties of v-Rel.

scholarly journals S2d2-4 New complexities of transcription start site usage in mammals(S2-d2: "Bioinformatics of Transcriptional Regulation",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

2006 ◽  
Vol 46 (supplement2) ◽  
pp. S129
Author(s):  
Martin C. Frith ◽  
Hideya Kawaji ◽  
Albin Sandelin ◽  
Jasmina Ponjavic ◽  
Piero Carninci ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Transcription Start Site ◽  
Start Site ◽  
Transcription Start ◽  
Meeting Program

scholarly journals Versatile in vivo regulation of tumor phenotypes by dCas9-mediated transcriptional perturbation

2016 ◽  
Vol 113 (27) ◽  
pp. E3892-E3900 ◽  
Author(s):  
Christian J. Braun ◽  
Peter M. Bruno ◽  
Max A. Horlbeck ◽  
Luke A. Gilbert ◽  
Jonathan S. Weissman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Transcription Start Site ◽  
Gene Activation ◽  
Start Site ◽  
Transcription Start ◽  
High Coverage ◽  
Tumor Phenotypes

Targeted transcriptional regulation is a powerful tool to study genetic mediators of cellular behavior. Here, we show that catalytically dead Cas9 (dCas9) targeted to genomic regions upstream or downstream of the transcription start site allows for specific and sustainable gene-expression level alterations in tumor cells in vitro and in syngeneic immune-competent mouse models. We used this approach for a high-coverage pooled gene-activation screen in vivo and discovered previously unidentified modulators of tumor growth and therapeutic response. Moreover, by using dCas9 linked to an activation domain, we can either enhance or suppress target gene expression simply by changing the genetic location of dCas9 binding relative to the transcription start site. We demonstrate that these directed changes in gene-transcription levels occur with minimal off-target effects. Our findings highlight the use of dCas9-mediated transcriptional regulation as a versatile tool to reproducibly interrogate tumor phenotypes in vivo.

Transcriptional Regulation of the Intra-S-Phase Regulator CDC7 in Human Myeloid Leukemic Cells: A New Downstream Target of the C-Myb Protooncogene.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1354-1354
Author(s):  
Lindsay B. Thalheim ◽  
Susan E. Shetzline ◽  
Cezary R. Swider ◽  
Alan M. Gewirtz
Keyword(s):  
Transcription Factor ◽  
Transcriptional Regulation ◽  
Promoter Region ◽  
Hematopoietic Cells ◽  
S Phase ◽  
Leukemic Cells ◽  
Start Site ◽  
Transcription Start ◽  
Binding Motifs ◽  
Pcr Product

Abstract The c-myb proto-oncogene encodes a transcription factor, Myb, which is essential for the growth and proliferation of normal and malignant hematopoietic cells. We, and others, have previously shown that malignant hematopoietic cells are much more dependent on c-myb function than are normal hematopoeitic cells, and that transient disruption of c-myb expression causes malignant cells to undergo apoptosis while normal cells are relatively spared. Based on these findings, we hypothesized that c-myb regulates a unique set of genes in leukemic cells that are required for cell survival. To identify Myb gene targets, we performed a transcriptome analysis using human myeloid leukemic cells engineered to express a conditionally active dominant negative Myb (MERT). Analysis of the microarray data revealed that when Myb activity was inhibited by tamoxifen in MERT cells, CDC7, an intra-S phase regulator, decreased in expression 2.8-fold compared to untreated control cells. To verify this, we utilized real-time PCR to quantitate the expression of CDC7, and found that it decreased 5-fold in tamoxifen treated MERT cells relative to control cells. In aggregate, the microarray and real-time PCR data suggested that Myb directly regulates CDC7 gene expression in hematopoietic cells. To address this question in the absence of a formally defined human CDC7 promoter, we examined the DNA sequence upstream of the predicted transcription start site (as noted in Genbank accession # AY585721) for potential Myb transcription factor binding motifs. After scanning the DNA sequence (~3kb) upstream of the predicted transcription start site, nine potential Myb response elements (MREs) were identified. The CDC7 sequences from mouse, chimp, and yeast were also analyzed for MREs and compared to those present in the putative human CDC7 promoter to identify conserved MREs. Using this strategy, we also identified potential AML1, PU.1, CBP, STAT3, and STAT5 binding motifs within the human CDC7 promoter region. To determine if any of the potential Myb binding sites with the CDC7 promoter were actually utilized in vivo, we carried out chromatin immunoprecopitation (ChIP) assays. When the chromatin from untreated MERT cells was immunoprecipitated with anti-c-Myb, we observed one PCR product using a primer pair that flanked each conserved MRE. These same results were obtained in our positive control ChIP experiment in which the chromatin was immunoprecipitated with anti-acetyl histone 4. When Myb transactivation activity was inhibited in tamoxifen treated MERT cells, no PCR product was detected following chromatin immunoprecipitation with the anti-Myb antibody suggesting that the ChIP binding results were not due to artifact. We have just completed a primer extension assay with a Fam-labeled primer that flanked the predicted CDC7 promoter region and will use the resulting sequence data to identify the actual CDC7 transcriptional start site. We will also shortly complete identification of functional regions within the human CDC7 promoter through use of Luciferase reporter assays. Investigation of the transcriptional regulation of CDC7 in hematopoietic cells may yield new clues to Myb’s role in leukemogenesis.

The Role of XPB/Ssl2 dsDNA Translocase Processivity in Transcription Start-site Scanning

2021 ◽  
pp. 166813
Author(s):  
Eric J. Tomko ◽  
Olivia Luyties ◽  
Jenna K. Rimel ◽  
Chi-Lin Tsai ◽  
Jill O. Fuss ◽  
...  
Keyword(s):  
Transcription Start Site ◽  
Start Site ◽  
Transcription Start

scholarly journals A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter.

1993 ◽  
Vol 13 (3) ◽  
pp. 1610-1618 ◽  
Author(s):  
J E Slansky ◽  
Y Li ◽  
W G Kaelin ◽  
P J Farnham
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Phase Boundary ◽  
Dihydrofolate Reductase ◽  
Transcription Initiation ◽  
Growth Regulation ◽  
Cellular Proliferation ◽  
Protein S ◽  
S Phase ◽  
Dependent Manner

Enhanced expression of genes involved in nucleotide biosynthesis, such as dihydrofolate reductase (DHFR), is a hallmark of entrance into the DNA synthesis (S) phase of the mammalian cell cycle. To investigate the regulated expression of the DHFR gene, we stimulated serum-starved NIH 3T3 cells to synchronously reenter the cell cycle. Our previous results show that a cis-acting element at the site of DHFR transcription initiation is necessary for serum regulation. Recently, this element has been demonstrated to bind the cloned transcription factor E2F. In this study, we focused on the role of E2F in the growth regulation of DHFR. We demonstrated that a single E2F site, in the absence or presence of other promoter elements, was sufficient for growth-regulated promoter activity. Next, we showed that the increase in DHFR mRNA at the G1/S-phase boundary required protein synthesis, raising the possibility that a protein(s) lacking in serum-starved cells is required for DHFR transcription. We found that, similar to DHFR mRNA expression, levels of murine E2F1 mRNA were low in serum-starved cells and increased at the G1/S-phase boundary in a protein synthesis-dependent manner. Furthermore, in a cotransfection experiment, expression of human E2F1 stimulated the DHFR promoter 22-fold in serum-starved cells. We suggest that E2F1 may be the key protein required for DHFR transcription that is absent in serum-starved cells. Expression of E2F also abolished the serum-stimulated regulation of the DHFR promoter and resulted in transcription patterns similar to those seen with expression of the adenoviral oncoprotein E1A. In summary, we provide evidence for the importance of E2F in the growth regulation of DHFR and suggest that alterations in the levels of E2F may have severe consequences in the control of cellular proliferation.

scholarly journals Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

2016 ◽  
Vol 113 (21) ◽  
pp. E2899-E2905 ◽  
Author(s):  
Irina O. Vvedenskaya ◽  
Hanif Vahedian-Movahed ◽  
Yuanchao Zhang ◽  
Deanne M. Taylor ◽  
Richard H. Ebright ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Start Site ◽  
Transcription Initiation ◽  
Specific Protein ◽  
Start Site ◽  
Transcription Start ◽  
Transcription Bubble ◽  
Recognition Element

During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein–DNA interactions with the downstream part of the nontemplate strand of the transcription bubble (“core recognition element,” CRE). Here, we investigated whether sequence-specific RNAP–CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP–CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP–CRE interactions on TSS selection in vitro and in vivo for a library of 47 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP–CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid native-elongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP–CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP–CRE interactions determine TSS selection. Our findings establish RNAP–CRE interactions are a functional determinant of TSS selection. We propose that RNAP–CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).

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