scholarly journals The Proneural Proteins Atonal and Scute Regulate Neural Target Genes through Different E-Box Binding Sites

2004 ◽  
Vol 24 (21) ◽  
pp. 9517-9526 ◽  
Author(s):  
Lynn M. Powell ◽  
Petra I. zur Lage ◽  
David R. A. Prentice ◽  
Biruntha Senthinathan ◽  
Andrew P. Jarman
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Sites ◽  
Tandem Repeats ◽  
Target Genes ◽  
Expression Patterns ◽  
Sense Organ ◽  
Ample Evidence ◽  
E Box ◽  
Bhlh Transcription Factors

ABSTRACT For a particular functional family of basic helix-loop-helix (bHLH) transcription factors, there is ample evidence that different factors regulate different target genes but little idea of how these different target genes are distinguished. We investigated the contribution of DNA binding site differences to the specificities of two functionally related proneural bHLH transcription factors required for the genesis of Drosophila sense organ precursors (Atonal and Scute). We show that the proneural target gene, Bearded, is regulated by both Scute and Atonal via distinct E-box consensus binding sites. By comparing with other Ato-dependent enhancer sequences, we define an Ato-specific binding consensus that differs from the previously defined Scute-specific E-box consensus, thereby defining distinct EAto and ESc sites. These E-box variants are crucial for function. First, tandem repeats of 20-bp sequences containing EAto and ESc sites are sufficient to confer Atonal- and Scute-specific expression patterns, respectively, on a reporter gene in vivo. Second, interchanging EAto and ESc sites within enhancers almost abolishes enhancer activity. While the latter finding shows that enhancer context is also important in defining how proneural proteins interact with these sites, it is clear that differential utilization of DNA binding sites underlies proneural protein specificity.

scholarly journals In vitro DNA-binding profile of transcription factors: methods and new insights

2011 ◽  
Vol 210 (1) ◽  
pp. 15-27 ◽  
Author(s):  
Jinke Wang ◽  
Jie Lu ◽  
Guangming Gu ◽  
Yingxun Liu
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Dna Sequences ◽  
Binding Sites ◽  
Target Genes ◽  
Binding Specificity ◽  
Cell Physiology ◽  
Binding Profile ◽  
Dna Binding Specificity

The DNA-binding specificity of transcription factors (TFs) has broad impacts on cell physiology, cell development and in evolution. However, the DNA-binding specificity of most known TFs still remains unknown. The specificity of a TF protein is determined by its relative affinity to all possible binding sites. In recent years, the development of several in vitro techniques permits high-throughput determination of relative binding affinity of a TF to all possible k bp-long DNA sequences, thus greatly promoting the characterization of DNA-binding specificity of many known TFs. All DNA sequences that can be bound by a TF with various binding affinities form their DNA-binding profile (DBP). The DBP is important to generate an accurate DNA-binding model, identify all DNA-binding sites and target genes of TFs in the whole genome, and build transcription regulatory network. This study reviewed these techniques, especially two master techniques: double-stranded DNA microarray and systematic evolution of ligands by exponential enrichment in combination with parallel DNA sequencing techniques (SELEX-seq).

scholarly journals The cis-regulatory logic underlying abdominal Hox-mediated repression versus activation of regulatory elements in Drosophila

2018 ◽  
Author(s):  
Arya Zandvakili ◽  
Juli Uhl ◽  
Ian Campbell ◽  
Yuntao Charlie Song ◽  
Brian Gebelein
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Dna Sequences ◽  
Binding Sites ◽  
Target Genes ◽  
Hox Genes ◽  
Regulatory Elements ◽  
Protease Gene ◽  
Precise Integration

AbstractHox genes encode a family of transcription factors that, despite having similar in vitro DNA binding preferences, regulate distinct genetic programs along the metazoan anterior-posterior axis. To better define mechanisms of Hox specificity, we compared and contrasted the ability of abdominal Hox factors to regulate two cis-regulatory elements within the Drosophila embryo. Both the Ultrabithorax (Ubx) and Abdominal-A (Abd-A) Hox factors form cooperative complexes with the Extradenticle (Exd) and Homothorax (Hth) transcription factors to repress the distal-less leg selector gene via the DCRE, whereas only Abd-A interacts with Exd and Hth on the RhoA element to activate a rhomboid serine protease gene that stimulates Epidermal Growth Factor secretion. By swapping binding sites between these elements, we found that the RhoA Exd/Hth/Hox site configuration that mediates Abd-A specific activation can also convey transcriptional repression by both Ubx and Abd-A when placed into the DCRE, but only in one orientation. We further show that the orientation and spacing of Hox sites relative to additional transcription factor binding sites within the RhoA and DCRE elements is critical to mediate appropriate cell- and segment-specific output. These results indicate that the interaction between Hox, Exd, and Hth neither determines activation vs repression specificity nor defines Ubx vs Abd-A specificity. Instead the precise integration of Hox sites with additional TF inputs is required for accurate transcriptional output. Taken together, these studies provide new insight into the mechanisms of Hox target and regulatory specificity as well as the constraints placed on regulatory elements to convey appropriate outputs.Author SummaryThe Hox genes encode a family of transcription factors that give cells within each region along the developing body plan a unique identity in animals from worms to mammals. Surprisingly, however, most of the Hox factors bind the same or highly similar DNA sequences. These findings raise a paradox: How can proteins that have highly similar DNA binding properties perform different functions in the animal by regulating different sets of target genes? In this study, we address this question by studying how two Hox factors regulate the expression of target genes that specify leg development and the making of liver-like cells in the developing fly. By comparing and contrasting how Hox target genes are activated and/or repressed, we found that the same Hox binding sites can mediate either activation or repression in a manner that depends upon context. In addition, we found that a Hox binding site that is normally regulated by only one Hox factor, can also be used by more than one Hox factor swapped into another target gene. These findings indicate that the specificity of a Hox factor to regulate target genes does not rely solely upon DNA binding specificity but also requires regulatory specificity.

DNA-binding specificities of the GATA transcription factor family

1993 ◽  
Vol 13 (7) ◽  
pp. 4011-4022
Author(s):  
L J Ko ◽  
J D Engel
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Target Genes ◽  
Polymerase Chain Reaction Amplification ◽  
Consensus Sequence ◽  
Expression Patterns ◽  
Regulatory Elements ◽  
Erythroid Cell ◽  
Binding Motif ◽  
Gata Factors

Members of the GATA family of transcription factors, which are related by a high degree of amino acid sequence identity within their zinc finger DNA-binding domains, each show distinct but overlapping patterns of tissue-restricted expression. Although GATA-1, -2, and -3 have been shown to recognize a consensus sequence derived from regulatory elements in erythroid cell-specific genes, WGATAR (in which W indicates A/T and R indicates A/G), the potential for more subtle differences in the binding preferences of each factor has not been previously addressed. By employing a binding selection and polymerase chain reaction amplification scheme with randomized oligonucleotides, we have determined the binding-site specificities of bacterially expressed chicken GATA-1, -2, and -3 transcription factors. Whereas all three GATA factors bind an AGATAA erythroid consensus motif with high affinity, a second, alternative consensus DNA sequence, AGATCTTA, is also recognized well by GATA-2 and GATA-3 but only poorly by GATA-1. These studies suggest that all three GATA factors are capable of mediating transcriptional effects via a common erythroid consensus DNA-binding motif. Furthermore, GATA-2 and GATA-3, because of their distinct expression patterns and broader DNA recognition properties, may be involved in additional regulatory processes beyond those of GATA-1. The definition of an alternative GATA-2-GATA-3 consensus sequence may facilitate the identification of new target genes in the further elucidation of the roles that these transcription factors play during development.

scholarly journals DamIP: A novel method to identify DNA binding sites in vivo

2010 ◽  
Vol 8 (1) ◽  
pp. nrs.08003 ◽  
Author(s):  
Rui Xiao ◽  
Ramon Roman-Sanchez ◽  
David D. Moore
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Sites ◽  
Protein Function ◽  
Target Genes ◽  
Estrogen Receptor Α ◽  
Cross Linking ◽  
Mutant Form ◽  
Dna Binding Sites

Identifying binding sites and target genes of transcription factors is a major biologic problem. The most commonly used current technique, chromatin immunoprecipitation (ChIP), is dependent on a high quality antibody for each protein of interest, which is not always available, and is also cumbersome, involving sequential cross-linking and reversal of cross-linking. We have developed a novel strategy to study protein DNA binding sites in vivo, which we term DamIP. By tethering a mutant form of E. coli DNA adenine methyltransferase to the target protein, the fusion protein introduces N-6-adenosine methylation to sequences proximal to the protein binding sites. DNA fragments with this modification, which is absent in eukaryotes, are detected using an antibody directed against methylated adenosine. For an initial test of the method we used human estrogen receptor α (hERα), one of the best studied transcription factors. We found that expression of Dam-hERα fusion proteins in MCF-7 cells introduces adenosine methylation near a series of known direct hERα binding sites. Specific methylation tags are also found at indirect hERα binding sites, including both primary binding sites for the ER interactors AP-1 and SP1, and promoters that are activated by upstream ER bound enhancers. DamIP provides a new tool for the study of DNA interacting protein function in vivo.

scholarly journals DNA-binding specificities of the GATA transcription factor family.

1993 ◽  
Vol 13 (7) ◽  
pp. 4011-4022 ◽  
Author(s):  
L J Ko ◽  
J D Engel
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Target Genes ◽  
Polymerase Chain Reaction Amplification ◽  
Consensus Sequence ◽  
Expression Patterns ◽  
Regulatory Elements ◽  
Erythroid Cell ◽  
Binding Motif ◽  
Gata Factors

Members of the GATA family of transcription factors, which are related by a high degree of amino acid sequence identity within their zinc finger DNA-binding domains, each show distinct but overlapping patterns of tissue-restricted expression. Although GATA-1, -2, and -3 have been shown to recognize a consensus sequence derived from regulatory elements in erythroid cell-specific genes, WGATAR (in which W indicates A/T and R indicates A/G), the potential for more subtle differences in the binding preferences of each factor has not been previously addressed. By employing a binding selection and polymerase chain reaction amplification scheme with randomized oligonucleotides, we have determined the binding-site specificities of bacterially expressed chicken GATA-1, -2, and -3 transcription factors. Whereas all three GATA factors bind an AGATAA erythroid consensus motif with high affinity, a second, alternative consensus DNA sequence, AGATCTTA, is also recognized well by GATA-2 and GATA-3 but only poorly by GATA-1. These studies suggest that all three GATA factors are capable of mediating transcriptional effects via a common erythroid consensus DNA-binding motif. Furthermore, GATA-2 and GATA-3, because of their distinct expression patterns and broader DNA recognition properties, may be involved in additional regulatory processes beyond those of GATA-1. The definition of an alternative GATA-2-GATA-3 consensus sequence may facilitate the identification of new target genes in the further elucidation of the roles that these transcription factors play during development.

scholarly journals Differential Contribution of N- and C-Terminal Regions of HIF1α and HIF2α to Their Target Gene Selectivity

2020 ◽  
Vol 21 (24) ◽  
pp. 9401
Author(s):  
Antonio Bouthelier ◽  
Florinda Meléndez-Rodríguez ◽  
Andrés A. Urrutia ◽  
Julián Aragonés
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Cellular Response ◽  
Target Gene ◽  
Target Genes ◽  
Transactivation Domain ◽  
Transactivation Domains ◽  
Relative Contribution

Cellular response to hypoxia is controlled by the hypoxia-inducible transcription factors HIF1α and HIF2α. Some genes are preferentially induced by HIF1α or HIF2α, as has been explored in some cell models and for particular sets of genes. Here we have extended this analysis to other HIF-dependent genes using in vitro WT8 renal carcinoma cells and in vivo conditional Vhl-deficient mice models. Moreover, we generated chimeric HIF1/2 transcription factors to study the contribution of the HIF1α and HIF2α DNA binding/heterodimerization and transactivation domains to HIF target specificity. We show that the induction of HIF1α-dependent genes in WT8 cells, such as CAIX (CAR9) and BNIP3, requires both halves of HIF, whereas the HIF2α transactivation domain is more relevant for the induction of HIF2 target genes like the amino acid carrier SLC7A5. The HIF selectivity for some genes in WT8 cells is conserved in Vhl-deficient lung and liver tissue, whereas other genes like Glut1 (Slc2a1) behave distinctly in these tissues. Therefore the relative contribution of the DNA binding/heterodimerization and transactivation domains for HIF target selectivity can be different when comparing HIF1α or HIF2α isoforms, and that HIF target gene specificity is conserved in human and mouse cells for some of the genes analyzed.

scholarly journals Binding of disparate transcriptional activators to nucleosomal DNA is inherently cooperative.

1995 ◽  
Vol 15 (3) ◽  
pp. 1405-1421 ◽  
Author(s):  
C C Adams ◽  
J L Workman
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Sites ◽  
Specific Binding ◽  
General Mechanism ◽  
Nf Kappa B ◽  
Nucleosomal Dna ◽  
Nucleosome Binding

To investigate mechanisms by which multiple transcription factors access complex promoters and enhancers within cellular chromatin, we have analyzed the binding of disparate factors to nucleosome cores. We used a purified in vitro system to analyze binding of four activator proteins, two GAL4 derivatives, USF, and NF-kappa B (KBF1), to reconstituted nucleosome cores containing different combinations of binding sites. Here we show that binding of any two or all three of these factors to nucleosomal DNA is inherently cooperative. Thus, the binuclear Zn clusters of GAL4, the helix-loop-helix/basic domains of USF, and the rel domain of NF-kappa B all participated in cooperative nucleosome binding, illustrating that this effect is not restricted to a particular DNA-binding domain. Simultaneous binding by two factors increased the affinity of individual factors for nucleosomal DNA by up to 2 orders of magnitude. Importantly, cooperative binding resulted in efficient nucleosome binding by factors (USF and NF-kappa B) which independently possess little nucleosome-binding ability. The participation of GAL4 derivatives in cooperative nucleosome binding required only DNA-binding and dimerization domains, indicating that disruption of histone-DNA contacts by factor binding was responsible for the increased affinity of additional factors. Cooperative nucleosome binding required sequence-specific binding of all transcription factors, appeared to have spatial constraints, and was independent of the orientation of the binding sites on the nucleosome. These results indicate that cooperative nucleosome binding is a general mechanism that may play a significant role in loading complex enhancer and promoter elements with multiple diverse factors in chromatin and contribute to the generation of threshold responses and transcriptional synergy by multiple activator sites in vivo.

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