Synergistic activation of a human promoter in vivo by transcription factor Sp1

1991 ◽  
Vol 11 (4) ◽  
pp. 1935-1943
Author(s):  
G M Anderson ◽  
S O Freytag
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Single Copy ◽  
Relative Contribution ◽  
Human Promoter ◽  
Transcription Factor Sp1 ◽  
Synergistic Activation ◽  
Cellular Context

Many eucaryotic promoters contain multiple binding sites for sequence-specific DNA-binding proteins. In some cases, these proteins have been shown to interact synergistically to activate transcription. In this study, we address the possibility that the transcription factor Sp1 can synergistically activate a native human promoter in a cellular context that closely resembles that of a single-copy gene. Using DNase I footprinting with affinity-purified Sp1, we show that the human argininosuccinate synthetase (AS) promoter contains three sites that bind Sp1 with different affinities. These binding sites were mutated to abolish Sp1 binding, individually and in all possible combinations, to generate a series of AS promoter-chloramphenicol acetyltransferase (CAT) expression constructs. Mutations designed to increase Sp1 binding were also introduced at each site. The in vivo transcriptional activity of these mutant AS promoter-CAT constructs was then measured in stably transfected human RPMI 2650 cell lines. Our results show that each of the three Sp1-binding sites contributes to full activation of the human AS promoter and that the relative contribution of each site correlates well with its in vitro affinity for Sp1. More importantly, we find that the three Sp1-binding sites when present in the same promoter activate transcription to a level that is 8 times greater than would be expected given their individual activities in the absence of the other two sites. Thus, we provide direct evidence that Sp1-binding sites in their native context in a human promoter can interact synergistically in vivo to activate transcription. The ability to activate transcription synergistically may be the reason that many cellular promoters have multiple Sp1-binding sites arranged in tandem and in close proximity.

scholarly journals Synergistic activation of a human promoter in vivo by transcription factor Sp1.

1991 ◽  
Vol 11 (4) ◽  
pp. 1935-1943 ◽  
Author(s):  
G M Anderson ◽  
S O Freytag
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Single Copy ◽  
Relative Contribution ◽  
Human Promoter ◽  
Transcription Factor Sp1 ◽  
Synergistic Activation ◽  
Cellular Context

Many eucaryotic promoters contain multiple binding sites for sequence-specific DNA-binding proteins. In some cases, these proteins have been shown to interact synergistically to activate transcription. In this study, we address the possibility that the transcription factor Sp1 can synergistically activate a native human promoter in a cellular context that closely resembles that of a single-copy gene. Using DNase I footprinting with affinity-purified Sp1, we show that the human argininosuccinate synthetase (AS) promoter contains three sites that bind Sp1 with different affinities. These binding sites were mutated to abolish Sp1 binding, individually and in all possible combinations, to generate a series of AS promoter-chloramphenicol acetyltransferase (CAT) expression constructs. Mutations designed to increase Sp1 binding were also introduced at each site. The in vivo transcriptional activity of these mutant AS promoter-CAT constructs was then measured in stably transfected human RPMI 2650 cell lines. Our results show that each of the three Sp1-binding sites contributes to full activation of the human AS promoter and that the relative contribution of each site correlates well with its in vitro affinity for Sp1. More importantly, we find that the three Sp1-binding sites when present in the same promoter activate transcription to a level that is 8 times greater than would be expected given their individual activities in the absence of the other two sites. Thus, we provide direct evidence that Sp1-binding sites in their native context in a human promoter can interact synergistically in vivo to activate transcription. The ability to activate transcription synergistically may be the reason that many cellular promoters have multiple Sp1-binding sites arranged in tandem and in close proximity.

scholarly journals Mapping transcription factor occupancy using minimal numbers of cells in vitro and in vivo

2017 ◽  
Author(s):  
Luca Tosti ◽  
James Ashmore ◽  
Boon Siang Nicholas Tan ◽  
Benedetta Carbone ◽  
Tapan K Mistri ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Regulatory Networks ◽  
Embryonic Stem ◽  
Specific Cell ◽  
Dna Interactions

AbstractThe identification of transcription factor (TF) binding sites in the genome is critical to understanding gene regulatory networks (GRNs). While ChIP-seq is commonly used to identify TF targets, it requires specific ChIP-grade antibodies and high cell numbers, often limiting its applicability. DNA adenine methyltransferase identification (DamID), developed and widely used in Drosophila, is a distinct technology to investigate protein-DNA interactions. Unlike ChIP-seq, it does not require antibodies, precipitation steps or chemical protein-DNA crosslinking, but to date it has been seldom used in mammalian cells due to technical impediments. Here we describe an optimised DamID method coupled with next generation sequencing (DamID-seq) in mouse cells, and demonstrate the identification of the binding sites of two TFs, OCT4 and SOX2, in as few as 1,000 embryonic stem cells (ESCs) and neural stem cells (NSCs), respectively. Furthermore, we have applied this technique in vivo for the first time in mammals. Oct4 DamID-seq in the gastrulating mouse embryo at 7.5 days post coitum (dpc) successfully identified multiple Oct4 binding sites proximal to genes involved in embryo development, neural tube formation, mesoderm-cardiac tissue development, consistent with the pivotal role of this TF in post-implantation embryo. This technology paves the way to unprecedented investigations of TF-DNA interactions and GRNs in specific cell types with limited availability in mammals including in vivo samples.

beta3-tubulin is directly repressed by the engrailed protein in Drosophila

Development ◽  
1997 ◽  
Vol 124 (13) ◽  
pp. 2527-2536 ◽  
Author(s):  
N. Serrano ◽  
H.W. Brock ◽  
F. Maschat
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Transgenic Line ◽  
Target Genes ◽  
Regulatory Protein ◽  
Polytene Chromosomes ◽  
First Intron ◽  
Direct Target

In Drosophila, Engrailed is a nuclear regulatory protein with essential roles during embryonic development. Although Engrailed is a transcription factor, little progress has been achieved in identifying its target genes. We report here the identification of an effector gene, the beta3-tubulin gene, as a direct target of Engrailed. The cytological location of beta3-tubulin, 60C, is a strong site of Engrailed binding on polytene chromosomes. Immunostaining analysis of a transgenic line containing a P[beta3-tubulin-lacZ] construct shows an additional site of Engrailed binding at the location of the transgene. Molecular analysis allowed identification of several Engrailed binding sites, both in vitro and in vivo, within the first intron of the beta3-tubulin locus. Engrailed binding sites identified in vitro are active in larvae. Furthermore, expression of beta3-tubulin is derepressed in the ectoderm of engrailed mutant embryos. Repression of beta3-tubulin by Engrailed is also obtained when Engrailed is ectopically expressed in embryonic mesoderm. Finally, two different sets of Engrailed binding sites are shown to be involved in the early and late regulation of beta3-tubulin by Engrailed during embryogenesis.

Identification of Schizosaccharomyces pombe transcription factor PGA4, which binds cooperatively to Saccharomyces cerevisiae GAL4-binding sites

1990 ◽  
Vol 10 (4) ◽  
pp. 1432-1438
Author(s):  
D M Ruden
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Schizosaccharomyces Pombe ◽  
Binding Sites ◽  
Transcription Activator ◽  
Integral Number ◽  
Dna Binding Site ◽  
Adh1 Gene

When the DNA-binding site for the Saccharomyces cerevisiae transcription activator GAL4 is placed upstream of the Schizosaccharomyces pombe ADH1 TATA box, transcription of the ADH1 gene is activated in S. pombe in vivo by an endogenous transcription factor. In vitro studies show that this S. pombe protein, PGA4, binds specifically to DNA containing a GAL4 site and that when two GAL4 sites are present, this protein binds cooperatively. Cooperating binding of PGA4 to DNA is favored if the GAL4 sites are separated by an integral number of turns of the DNA helix.

scholarly journals Properties of initiator-associated transcription mediated by GAL4-VP16.

1993 ◽  
Vol 13 (12) ◽  
pp. 7469-7475 ◽  
Author(s):  
C Chang ◽  
J D Gralla
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Dna Binding Sites ◽  
Terminal Deoxynucleotide Transferase ◽  
Initiator Element ◽  
High Level ◽  
Primary Activation ◽  
Initial Assembly

Transcription associated with a terminal deoxynucleotide transferase gene initiator element is shown to respond to the transcription factor GAL4-VP16 both in vivo and in vitro. High-level transcription requires both an intact initiator element and bound activator. Transcription from this initiator-directed promoter is synergistic in vivo in that five GAL4 DNA binding sites yield 36 times the expression of a single site. Promoters dominated by initiator and TATA elements respond similarly to several GAL4-based activators, including GAL4-Sp1, GAL4-CTF, GAL4(1-147), GAL4-p53, GAL4-C/EBP, and GAL4-ER(EF), as well as GAL4-VP16 and Sp1. These and other similarities suggest that primary activation of TATA- and initiator-dominated promoters occurs at common steps. Since the initial assembly steps do not appear to be common for the two promoter types, the results place interesting constraints on models for how activation occurs.

scholarly journals A systematic genome-wide account of binding sites for the model transcription factor Gcn4

2021 ◽  
pp. gr.276080.121
Author(s):  
Christopher T Coey ◽  
David J. Clark
Keyword(s):  
Gene Regulation ◽  
Transcription Factor ◽  
Binding Sites ◽  
Yeast Genome ◽  
High Affinity ◽  
First Approximation ◽  
Genome Wide ◽  
Insight Into

Sequence-specific DNA-binding transcription factors are central to gene regulation. They are often associated with consensus binding sites that predict far more genomic sites than are bound in vivo. One explanation is that most sites are blocked by nucleosomes, such that only sites in nucleosome-depleted regulatory regions are bound. We compared the binding of the yeast transcription factor Gcn4 in vivo using published ChIP-seq data (546 sites) and in vitro, using a modified SELEX method ("G-SELEX"), which utilizes short genomic DNA fragments to quantify binding at all sites. We confirm that Gcn4 binds strongly to an AP-1-like sequence (TGACTCA) and weakly to half-sites. However, Gcn4 binds only some of the 1078 exact matches to this sequence, even in vitro. We show that there are only 166 copies of the high-affinity RTGACTCAY site (exact match) in the yeast genome, all occupied in vivo, largely independently of whether they are located in nucleosome-depleted or nucleosomal regions. Generally, RTGACTCAR/YTGACTCAY sites are bound much more weakly and YTGACTCAR sites are unbound, with biological implications for determining induction levels. We conclude that, to a first approximation, Gcn4 binding can be predicted using the high-affinity site, without reference to chromatin structure. We propose that transcription factor binding sites should be defined more precisely using quantitative data, allowing more accurate genome-wide prediction of binding sites and greater insight into gene regulation.

scholarly journals Systematic analysis of low-affinity transcription factor binding site clusters in vitro and in vivo establishes their functional relevance

2021 ◽  
Author(s):  
Amir Shahein ◽  
Maria L&oacutepez-Malo ◽  
Ivan Istomin ◽  
Evan J. Olson ◽  
Shiyu Cheng ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Binding Site ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Transcription Factor Binding ◽  
Affinity Binding ◽  
Factor Binding ◽  
Functional Relevance

Transcription factor binding to a single binding site and its functional consequence in a promoter context are beginning to be relatively well understood. However, binding to clusters of sites has yet to be characterized in depth, and the functional relevance of binding site clusters remains uncertain.We employed a high-throughput biochemical method to characterize transcription factor binding to clusters varying across a range of affinities and configurations. We found that transcription factors can bind concurrently to overlapping sites, challenging the notion of binding exclusivity. Further-more, compared to an individual high-affinity binding site, small clusters with binding sites an order of magnitude lower in affinity give rise to higher mean occupancies at physiologically-relevant transcription factor concentrations in vitro. To assess whether the observed in vitro occupancies translate to transcriptional activation in vivo, we tested low-affinity binding site clusters by inserting them into a synthetic minimal CYC1 and the native PHO5 S. cerevisiae promoter. In the minCYC1 promoter, clusters of low-affinity binding sites can generate transcriptional output comparable to a promoter containing three consensus binding sites. In the PHO5 promoter, replacing the native Pho4 binding sites with clusters of low-affinity binding sites recovered activation of these promoters as well. This systematic characterization demonstrates that clusters of low-affinity binding sites achieve substantial occupancies, and that this occupancy can drive expression in eukaryotic promoters

scholarly journals Enhancers with cooperative Notch binding sites are more resistant to regulation by the Hairless co-repressor

PLoS Genetics ◽  
2021 ◽  
Vol 17 (9) ◽  
pp. e1009039
Author(s):  
Yi Kuang ◽  
Anna Pyo ◽  
Natanel Eafergan ◽  
Brittany Cain ◽  
Lisa M. Gutzwiller ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Binding Sites ◽  
Target Gene ◽  
Gene Activation ◽  
Notch Intracellular Domain ◽  
Repressor Complex ◽  
Notch Activation

Notch signaling controls many developmental processes by regulating gene expression. Notch-dependent enhancers recruit activation complexes consisting of the Notch intracellular domain, the Cbf/Su(H)/Lag1 (CSL) transcription factor (TF), and the Mastermind co-factor via two types of DNA sites: monomeric CSL sites and cooperative dimer sites called Su(H) paired sites (SPS). Intriguingly, the CSL TF can also bind co-repressors to negatively regulate transcription via these same sites. Here, we tested how synthetic enhancers with monomeric CSL sites versus dimeric SPSs bind Drosophila Su(H) complexes in vitro and mediate transcriptional outcomes in vivo. Our findings reveal that while the Su(H)/Hairless co-repressor complex similarly binds SPS and CSL sites in an additive manner, the Notch activation complex binds SPSs, but not CSL sites, in a cooperative manner. Moreover, transgenic reporters with SPSs mediate stronger, more consistent transcription and are more resistant to increased Hairless co-repressor expression compared to reporters with the same number of CSL sites. These findings support a model in which SPS containing enhancers preferentially recruit cooperative Notch activation complexes over Hairless repression complexes to ensure consistent target gene activation.

scholarly journals Interaction of Sp1 with the growth- and cell cycle-regulated transcription factor E2F.

1996 ◽  
Vol 16 (4) ◽  
pp. 1659-1667 ◽  
Author(s):  
J Karlseder ◽  
H Rotheneder ◽  
E Wintersberger
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Binding Sites ◽  
Promoter Activity ◽  
Binding Motif ◽  
Transcription Machinery ◽  
Transcription Factor E2f

Within the region around 150 bp upstream of the initiation codon, which was previously shown to suffice for growth-regulated expression, the murine thymidine kinase gene carries a single binding site for transcription factor Sp1; about 10 bp downstream of this site, there is a binding motif for transcription factor E2F. The latter protein appears to be responsible for growth regulation of the promoter. Mutational inactivation of either the Sp1 or the E2F site almost completely abolishes promoter activity, suggesting that the two transcription factors interact directly in delivering an activation signal to the basic transcription machinery. This was verified by demonstrating with the use of glutathione S-transferase fusion proteins that E2F and Sp1 bind to each other in vitro. For this interaction, the C-terminal part of Sp1 and the N terminus of E2F1, a domain also present in E2F2 and E2F3 but absent in E2F4 and E2F5, were essential. Accordingly, E2F1 to E2F3 but not E2F4 and E2F5 were found to bind sp1 in vitro. Coimmunoprecipitation experiments showed that complexes exist in vivo, and it was estabilished that the distance between the binding sites for the two transcription factors was critical for optimal promoter activity. Finally, in vivo footprinting experiments indicated that both the sp1 and E2F binding sites are occupied throughout the cell cycle. Mutation of either binding motif abolished binding of both transcription factors in vivo, which may indicate cooperative binding of the two proteins to chromatin-organized DNA. Our data are in line with the hypothesis that E2F functions as a growth- and cell cycle regulated tethering factor between Sp1 and the basic transcription machinery.

scholarly journals TFBSshape: an expanded motif database for DNA shape features of transcription factor binding sites

2019 ◽  
Author(s):  
Tsu-Pei Chiu ◽  
Beibei Xin ◽  
Nicholas Markarian ◽  
Yingfei Wang ◽  
Remo Rohs
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Transcription Factor Binding Sites ◽  
Shape Features ◽  
Factor Binding ◽  
Methylated Dna ◽  
Dna Shape ◽  
Motif Database

AbstractTFBSshape (https://tfbsshape.usc.edu) is a motif database for analyzing structural profiles of transcription factor binding sites (TFBSs). The main rationale for this database is to be able to derive mechanistic insights in protein–DNA readout modes from sequencing data without available structures. We extended the quantity and dimensionality of TFBSshape, from mostly in vitro to in vivo binding and from unmethylated to methylated DNA. This new release of TFBSshape improves its functionality and launches a responsive and user-friendly web interface for easy access to the data. The current expansion includes new entries from the most recent collections of transcription factors (TFs) from the JASPAR and UniPROBE databases, methylated TFBSs derived from in vitro high-throughput EpiSELEX-seq binding assays and in vivo methylated TFBSs from the MeDReaders database. TFBSshape content has increased to 2428 structural profiles for 1900 TFs from 39 different species. The structural profiles for each TFBS entry now include 13 shape features and minor groove electrostatic potential for standard DNA and four shape features for methylated DNA. We improved the flexibility and accuracy for the shape-based alignment of TFBSs and designed new tools to compare methylated and unmethylated structural profiles of TFs and methods to derive DNA shape-preserving nucleotide mutations in TFBSs.

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