Chromatin potentiation of the hsp70 promoter is linked to GAGA-factor recruitment

2005 ◽  
Vol 83 (4) ◽  
pp. 555-565 ◽  
Author(s):  
Philippe T Georgel
Keyword(s):  
Binding Site ◽  
Chromatin Remodeling ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Gaga Factor ◽  
Hsp70 Promoter ◽  
Nucleosome Remodeling ◽  
Functional Sites ◽  
Factor Binding

The events leading to transcription initiation of the Drosophila melanogaster heat-shock protein (hsp)70 gene have been demonstrated to be directly connected with nucleosome remodeling factor and GAGA-dependent chromatin remodeling on its promoter region. To investigate the relative importance of the multiple GAGA-factor binding sites in the process of chromatin remodeling and their effect on DNA conformation, the position of nucleosomes over the proximal region of the promoter was mapped. No real-positioned nucleosome was detected. By matching the relative position of the GAGA-factor binding sites with the distribution of nucleosomes over the hsp70 promoter, the GAGA site 2 appeared to be the most accessible, i.e., located close to a nucleosomal edge or within the linker DNA. This result, combined with previous observations, suggest a link between increased GAGA-factor accessibility and efficiency of transcription initiation. The effect of GAGA-binding-site mutations, both individually and in combination, on DNA structure and nucleosome remodeling was assessed using free DNA and fly embryo extract chromatin templates assembled in vitro. Results indicated that both the number of functional sites and their positions within the chromatin were important determinants for nucleosome-remodeling efficiency. Ultimately, the degree of accessibility of the GAGA factor to its cognate binding site(s) appears to be proportional to chromatin-remodeling competency of the hsp70 promoter.Key words: chromatin, remodeling, nucleosome, hsp70, GAGA, Drosophila.

scholarly journals Chromatin Remodeling Mediated by Drosophila GAGA Factor and ISWI Activates fushi tarazu Gene Transcription In Vitro

1998 ◽  
Vol 18 (5) ◽  
pp. 2455-2461 ◽  
Author(s):  
Masahiro Okada ◽  
Susumu Hirose
Keyword(s):  
Chromatin Structure ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Promoter Region ◽  
Gaga Factor ◽  
Fushi Tarazu ◽  
Nucleosome Remodeling ◽  
Factor Binding ◽  
Chromatin Template

ABSTRACT GAGA factor is known to remodel the chromatin structure in concert with nucleosome-remodeling factor NURF in a Drosophilaembryonic S150 extract. The promoter region of the Drosophila fushi tarazu (ftz) gene carries several binding sites for GAGA factor. Both the GAGA factor-binding sites and GAGA factor per se are necessary for the proper expression of ftz in vivo. We observed transcriptional activation of the ftz gene when a preassembled chromatin template was incubated with GAGA factor and the S150 extract. The chromatin structure within the ftzpromoter was specifically disrupted by incubation of the preassembled chromatin with GAGA factor and the S150 extract. Both transcriptional activation and chromatin disruption were blocked by an antiserum raised against ISWI or by base substitutions in the GAGA factor-binding sites in the ftz promoter region. These results demonstrate that GAGA factor- and ISWI-mediated disruption of the chromatin structure within the promoter region of ftz activates transcription on the chromatin template.

scholarly journals Gal4p-Mediated Chromatin Remodeling Depends on Binding Site Position in Nucleosomes but Does Not Require DNA Replication

1998 ◽  
Vol 18 (3) ◽  
pp. 1201-1212 ◽  
Author(s):  
Mai Xu ◽  
Robert T. Simpson ◽  
Michael P. Kladde
Keyword(s):  
Dna Replication ◽  
Binding Site ◽  
Chromatin Remodeling ◽  
Transcriptional Activation ◽  
Chromatin Modification ◽  
Factor Binding ◽  
Affect Factor ◽  
Nucleosome Binding

ABSTRACT Biochemical studies have demonstrated decreased binding of various proteins to DNA in nucleosome cores as their cognate sites are moved from the edge of the nucleosome to the pseudodyad (center). However, to date no study has addressed whether this structural characteristic of nucleosomes modulates the function of a transcription factor in living cells, where processes of DNA replication and chromatin modification or remodeling could significantly affect factor binding. Using a sensitive, high-resolution methyltransferase assay, we have monitored the ability of Gal4p in vivo to interact with a nucleosome at positions that are known to be inaccessible in nucleosome cores in vitro. Gal4p efficiently bound a single cognate site (UASG) centered at 41 bp from the edge of a positioned nucleosome, perturbing chromatin structure and inducing transcription. DNA binding and chromatin perturbation accompanying this interaction also occurred in the presence of hydroxyurea, indicating that DNA replication is not necessary for Gal4p-mediated nucleosome disruption. These data extend previous studies, which demonstrated DNA replication-independent chromatin remodeling, by showing that a single dimer of Gal4p, without the benefit of cooperative interactions that occur at complex wild-type promoters, is competent for invasion of a preestablished nucleosome. When the UASG was localized at the nucleosomal pseudodyad, relative occupancy by Gal4p, nucleosome disruption, and transcriptional activation were substantially compromised. Therefore, despite the increased nucleosome binding capability of Gal4p in cells, the precise translational position of a factor binding site in one nucleosome in an array can affect the ability of a transcriptional regulator to overcome the repressive influence of chromatin.

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 Cross-Resistance of Escherichia coli RNA Polymerases Conferring Rifampin Resistance to Different Antibiotics

2005 ◽  
Vol 187 (8) ◽  
pp. 2783-2792 ◽  
Author(s):  
Ming Xu ◽  
Yan Ning Zhou ◽  
Beth P. Goldstein ◽  
Ding Jun Jin
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Pathogenic Bacteria ◽  
Cross Resistance ◽  
New Antibiotics ◽  
Sorangicin A ◽  
Site Competition

ABSTRACT In this study we further defined the rifampin-binding sites in Escherichia coli RNA polymerase (RNAP) and determined the relationship between rifampin-binding sites and the binding sites of other antibiotics, including two rifamycin derivatives, rifabutin and rifapentine, and streptolydigin and sorangicin A, which are unrelated to rifampin, using a purified in vitro system. We found that there is almost a complete correlation between resistance to rifampin (Rifr) and reduced rifampin binding to 12 RNAPs purified from different rpoB Rifr mutants and a complete cross-resistance among the different rifamycin derivatives. Most Rifr RNAPs were sensitive to streptolydigin, although some exhibited weak resistance to this antibiotic. However, 5 out of the 12 Rifr RNAPs were partially resistant to sorangicin A, and one was completely cross-resistant to sorangicin A, indicating that the binding site(s) for these two antibiotics overlaps. Both rifampin and sorangicin A inhibited the transition step between transcription initiation and elongation; however, longer abortive initiation products were produced in the presence of the latter, indicating that the binding site for sorangicin A is within the rifampin-binding site. Competition experiments of different antibiotics with 3H-labeled rifampin for binding to wild-type RNAP further confirmed that the binding sites for rifampin, rifabutin, rifapentine, and sorangicin A are shared, whereas the binding sites for rifampin and streptolydigin are distinct. Because Rifr mutations are highly conserved in eubacteria, our results indicate that this set of Rifr mutant RNAPs can be used to screen for new antibiotics that will inhibit the growth of Rifr pathogenic bacteria.

scholarly journals Heterogeneously initiated transcription from the pre-B- and B-cell-specific mb-1 promoter: analysis of the requirement for upstream factor-binding sites and initiation site sequences.

1991 ◽  
Vol 11 (11) ◽  
pp. 5756-5766 ◽  
Author(s):  
A Travis ◽  
J Hagman ◽  
R Grosschedl
Keyword(s):  
Binding Sites ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Minor Effect ◽  
Cell Type ◽  
Cell Type Specificity ◽  
Promoter Sequences ◽  
Factor Binding ◽  
Initiation Of Transcription

The mb-1 gene, encoding a membrane immunoglobulin-associated protein, is developmentally regulated and expressed specifically in pre-B and mature B lymphocytes. Analysis of the TATA-less mb-1 promoter indicated that it directs initiation of transcription from multiple sites. Promoter sequences between -68 and +70 conferred the correct pattern of cell type-specific transcription upon a heterologous gene. Two nuclear factor-binding sites that are important for promoter function were identified between -59 and -38. Both sites interacted with ubiquitous nuclear factors in vitro. One of these factors was identified as Sp1. Multimerized copies of both factor-binding sites augmented expression from a heterologous minimal promoter in both lymphoid and nonlymphoid cells, suggesting that additional mb-1 promoter sequences are involved in determining the correct cell type specificity. Analysis of the heterogeneity of transcription initiation indicated that a mutation which increased the distance between upstream sequences and the region of initiation resulted in the utilization of a novel set of initiation sites. Moreover, an insertion of a TATA element into the mb-1 promoter at -30 biased initiation of transcription to +1 but did not abolish the use of the other sites. Mutation of an initiator sequence homology encompassing one of the major initiation sites had only a minor effect on its utilization. From these data, we conclude that upstream factor-binding sites in the TATA-less mb-1 promoter define a region in which initiation of transcription occurs at multiple sites.

The Binding of GATA-1 to a β-Globin LCR HS Core Element in Nuclear Chromatin Requires Multiple Associated Factor Binding Sites.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 528-528 ◽  
Author(s):  
Christopher H. Lowrey ◽  
Catherine Ackley ◽  
Mitchell Ermentrout
Keyword(s):  
Binding Sites ◽  
Consensus Sequence ◽  
Regulatory Elements ◽  
Positive Control ◽  
Nuclear Chromatin ◽  
Core Element ◽  
Nucleosome Remodeling ◽  
Factor Binding ◽  
Core Elements

Abstract The GATA-1 transcription factor has been shown to interact in vitro with many gene regulatory elements including promoters, enhancers, silencers and locus control region elements. The GATA-1 consensus binding sequence is generally considered to be WGATAR, yet it can also bind to other related sequences that do not perfectly fit this consensus. This raises the question of whether all potential GATA-1 binding sites identified by in vitro DNA binding assays or sequence analysis are actually bound by GATA-1 in nuclear chromatin. We hypothesized that it was unlikely that all, or even most, predicted GATA-1 sites were bound in vivo. If correct, this would imply that additional mechanisms, beyond DNA sequence, are required to target GATA-1 to specific genetic regulatory elements. To address this hypothesis we first performed GATA-1 ChIP assays in MEL cells at 10 WGATAR sites which spanned the mouse β-globin locus but were not associated with known regulatory elements. Positive controls for GATA-1 binding were the core region of LCR HS2 and the -2.8 mGATA-1 gene upstream enhancer. Negative controls included two regions from the β-globin locus which do not contain WGATAR elements and a WGATAR element from the proximal mGATA-1 gene promoter previously shown by the Bresnick lab to not bind GATA-1. While enrichment of the positive control sites using anti-GATA-1 antibody was 10-20-fold more than with IgG (p<0.001), binding at none of the 10 test WGATAR sites was enriched. This result implied that conditions other than the presence of the WGATAR consensus sequence are required to specify GATA-1 binding in nuclear chromatin. To better understand this process we decided to more closely evaluate the binding of GATA-1 to β-globin LCR HS core elements. We first tested the idea that GATA-1 was able to bind these sites due to high-level affinity based on the presence of tandem-inverted WGATAR elements and the known ability of GATA-1 molecules to self-interact. However, when tested in gel shift assays, GATA-1 binding to the LCR HS core elements had a very rapid off-rate which was much faster than a known high-affinity control site from the murine α-globin promoter. This led us to next test the idea that surrounding factor binding sites were required for GATA-1 binding to the LCR HS cores. Using human LCR HS4 as a model, we systematically assembled combinations of binding sites (3 KLF sites and 1 NF-E2 site) known to flank the tandem-inverted WGATAR elements and tested these artificial HS cores for GATA-1 binding after integration into MEL cell chromatin. The wild-type HS4 core served as a positive control and a fragment of luciferase cDNA served as a negative control for GATA-1 binding. Results for each cell line were normalized to the internal positive control -2.8 mGATA-1 gene site. Our experiments showed that only when all known flanking sites were present was GATA-1 binding detected. Similarly, DNase HS formation and histone H3 acetylation only occurred when all of the surrounding sites were present. Finally, we used ChIP to show that the assembled complex, with all sites present, includes BRG1 a component of the swi/snf nucleosome remodeling complex. These results indicate that GATA-1 targeting to the LCR HS cores involves complex, multi-protein interactions including histone acetylation and nucleosome displacement. Similar mechanisms are likely to be involved in targeting this and other transcription factors to functionally important sites in the genome.

Heterogeneously initiated transcription from the pre-B- and B-cell-specific mb-1 promoter: analysis of the requirement for upstream factor-binding sites and initiation site sequences

1991 ◽  
Vol 11 (11) ◽  
pp. 5756-5766
Author(s):  
A Travis ◽  
J Hagman ◽  
R Grosschedl
Keyword(s):  
Binding Sites ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Minor Effect ◽  
Cell Type ◽  
Cell Type Specificity ◽  
Promoter Sequences ◽  
Factor Binding ◽  
Initiation Of Transcription

The mb-1 gene, encoding a membrane immunoglobulin-associated protein, is developmentally regulated and expressed specifically in pre-B and mature B lymphocytes. Analysis of the TATA-less mb-1 promoter indicated that it directs initiation of transcription from multiple sites. Promoter sequences between -68 and +70 conferred the correct pattern of cell type-specific transcription upon a heterologous gene. Two nuclear factor-binding sites that are important for promoter function were identified between -59 and -38. Both sites interacted with ubiquitous nuclear factors in vitro. One of these factors was identified as Sp1. Multimerized copies of both factor-binding sites augmented expression from a heterologous minimal promoter in both lymphoid and nonlymphoid cells, suggesting that additional mb-1 promoter sequences are involved in determining the correct cell type specificity. Analysis of the heterogeneity of transcription initiation indicated that a mutation which increased the distance between upstream sequences and the region of initiation resulted in the utilization of a novel set of initiation sites. Moreover, an insertion of a TATA element into the mb-1 promoter at -30 biased initiation of transcription to +1 but did not abolish the use of the other sites. Mutation of an initiator sequence homology encompassing one of the major initiation sites had only a minor effect on its utilization. From these data, we conclude that upstream factor-binding sites in the TATA-less mb-1 promoter define a region in which initiation of transcription occurs at multiple sites.

scholarly journals Induction of the Cellular E2F-1 Promoter by the Adenovirus E4-6/7 Protein

2000 ◽  
Vol 74 (5) ◽  
pp. 2084-2093 ◽  
Author(s):  
Joel Schaley ◽  
Robert J. O'Connor ◽  
Laura J. Taylor ◽  
Dafna Bar-Sagi ◽  
Patrick Hearing
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Transient Expression ◽  
Binding Sites ◽  
Promoter Region ◽  
Fluorescent Protein ◽  
Protein Accumulation ◽  
Promoter Regions ◽  
Single Binding Site

ABSTRACT The adenovirus type 5 (Ad5) E4-6/7 protein interacts directly with different members of the E2F family and mediates the cooperative and stable binding of E2F to a unique pair of binding sites in the Ad5 E2a promoter region. This induction of E2F DNA binding activity strongly correlates with increased E2a transcription when analyzed using virus infection and transient expression assays. Here we show that while different adenovirus isolates express an E4-6/7 protein that is capable of induction of E2F dimerization and stable DNA binding to the Ad5 E2a promoter region, not all of these viruses carry the inverted E2F binding site targets in their E2a promoter regions. The Ad12 and Ad40 E2a promoter regions bind E2F via a single binding site. However, these promoters bind adenovirus-induced (dimerized) E2F very weakly. The Ad3 E2a promoter region binds E2F very poorly, even via a single binding site. A possible explanation of these results is that the Ad E4-6/7 protein evolved to induce cellular gene expression. Consistent with this notion, we show that infection with different adenovirus isolates induces the binding of E2F to an inverted configuration of binding sites present in the cellular E2F-1 promoter. Transient expression of the E4-6/7 protein alone in uninfected cells is sufficient to induce transactivation of the E2F-1 promoter linked to chloramphenicol acetyltransferase or green fluorescent protein reporter genes. Further, expression of the E4-6/7 protein in the context of adenovirus infection induces E2F-1 protein accumulation. Thus, the induction of E2F binding to the E2F-1 promoter by the E4-6/7 protein observed in vitro correlates with transactivation of E2F-1 promoter activity in vivo. These results suggest that adenovirus has evolved two distinct mechanisms to induce the expression of the E2F-1 gene. The E1A proteins displace repressors of E2F activity (the Rb family members) and thus relieve E2F-1 promoter repression; the E4-6/7 protein complements this function by stably recruiting active E2F to the E2F-1 promoter to transactivate expression.

Transcriptional properties of BmX, a moderately repetitive silkworm gene that is an RNA polymerase III template

1988 ◽  
Vol 8 (2) ◽  
pp. 624-631
Author(s):  
E T Wilson ◽  
D P Condliffe ◽  
K U Sprague
Keyword(s):  
Control Region ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
Sequence Element ◽  
Direct Transcription ◽  
Factor Binding ◽  
Large Gene ◽  
Polymerase Iii ◽  
Downstream Control

We analyzed the transcriptional properties of a repetitive sequence element, BmX, that belongs to a large gene family (approximately 2 x 10(4) copies) in the genome of the Bombyx mori silkworm. We discovered BmX elements because of their ability to direct transcription by polymerase III in vitro and used them to test the generality of the properties of previously identified silkworm polymerase III control elements. We found that the signals that act in cis to control BmX transcription strongly resemble those that direct transcription of other silkworm polymerase III templates. As with silkworm tRNA and 5S RNA genes, transcription of BmX requires sequence signals located both upstream and downstream from the site of transcription initiation. The critical upstream sequences are structurally as well as functionally similar in the three kinds of templates. The downstream control region of BmX resembles the corresponding part of a silkworm alanine tRNA gene in that it provides a large (greater than 100 base pairs) region that influences transcription factor binding. Moreover, the factor-binding regions of both tRNA(Ala) and BmX genes are remarkable in that under certain conditions, key elements within them (the B boxes, for example) appear dispensable. This behavior can be understood if, in both of these templates, the downstream control region acts as a large target for interaction with a multifactor complex.

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