Visualizing and Characterizing In Vivo DNA-Binding Events and Direct Target Genes of Plant Transcription Factors

2011 ◽  
pp. 293-305 ◽  
Author(s):  
Jose M. Muiño ◽  
Gerco C. Angenent ◽  
Kerstin Kaufmann
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Target Genes ◽  
Direct Target

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 Ets-dependent Regulation of Target Gene Expression during Megakaryopoiesis

2004 ◽  
Vol 279 (50) ◽  
pp. 52183-52190 ◽  
Author(s):  
Pascale Jackers ◽  
Gabor Szalai ◽  
Omar Moussa ◽  
Dennis K. Watson
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase Ii ◽  
Chromatin Immunoprecipitation ◽  
Target Genes ◽  
Hematopoietic Stem ◽  
Exogenous Expression ◽  
Direct Target ◽  
Transcriptional Complex

Megakaryopoiesis is the process by which hematopoietic stem cells in the bone marrow differentiate into mature megakaryocytes. The expression of megakaryocytic genes during megakaryopoiesis is controlled by specific transcription factors. Fli-1 and GATA-1 transcription factors are required for development of megakaryocytes and promoter analysis has definedin vitrofunctional binding sites for these factors in several megakaryocytic genes, includingGPIIb,GPIX, andC-MPL. Herein, we utilize chromatin immunoprecipitation to examine the presence of Ets-1, Fli-1, and GATA-1 on these promotersin vivo. Fli-1 and Ets-1 occupy the promoters ofGPIIb,GPIX, andC-MPLgenes in both Meg-01 and CMK11-5 cells. WhereasGPIIbis expressed in both Meg-01 and CMK11-5 cells,GPIXandC-MPLare only expressed in the more differentiated CMK11–5 cells. Thus,in vivooccupancy by an Ets factor is not sufficient to promote transcription of some megakaryocytic genes. GATA-1 and Fli-1 are both expressed in CMK11-5 cells and co-occupy theGPIXandC-MPLpromoters. Transcription of all three megakaryocytic genes is correlated with the presence of acetylated histone H3 and phosphorylated RNA polymerase II on their promoters. We also show that exogenous expression of GATA-1 in Meg-01 cells leads to the expression of endogenous c-mpl and gpIX mRNA. WhereasGPIIb,GPIX, andC-MPLare direct target genes for Fli-1, both Fli-1 and GATA-1 are required for formation of an active transcriptional complex on theC-MPLandGPIXpromotersin vivo. In contrast,GPIIbexpression appears to be independent of GATA-1 in Meg-01 cells.

scholarly journals Genome reading by the NF-κB transcription factors

2019 ◽  
Vol 47 (19) ◽  
pp. 9967-9989 ◽  
Author(s):  
Maria Carmen Mulero ◽  
Vivien Ya-Fan Wang ◽  
Tom Huxford ◽  
Gourisankar Ghosh
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Dna Sequences ◽  
Target Genes ◽  
Target Selection ◽  
Structural Features ◽  
Response Elements ◽  
Target Binding

Abstract The NF-κB family of dimeric transcription factors regulates transcription by selectively binding to DNA response elements present within promoters or enhancers of target genes. The DNA response elements, collectively known as κB sites or κB DNA, share the consensus 5′-GGGRNNNYCC-3′ (where R, Y and N are purine, pyrimidine and any nucleotide base, respectively). In addition, several DNA sequences that deviate significantly from the consensus have been shown to accommodate binding by NF-κB dimers. X-ray crystal structures of NF-κB in complex with diverse κB DNA have helped elucidate the chemical principles that underlie target selection in vitro. However, NF-κB dimers encounter additional impediments to selective DNA binding in vivo. Work carried out during the past decades has identified some of the barriers to sequence selective DNA target binding within the context of chromatin and suggests possible mechanisms by which NF-κB might overcome these obstacles. In this review, we first highlight structural features of NF-κB:DNA complexes and how distinctive features of NF-κB proteins and DNA sequences contribute to specific complex formation. We then discuss how native NF-κB dimers identify DNA binding targets in the nucleus with support from additional factors and how post-translational modifications enable NF-κB to selectively bind κB sites in vivo.

scholarly journals A Positive Regulatory Feedback Loop Between 1 EKLF/ KLF1 and TAL1/SCL2 Sustaining the Erythropoiesis

2021 ◽  
Author(s):  
Chun-Hao Hung ◽  
Yu-Szu Huang ◽  
Tung-Liang Lee ◽  
Kang-Chung Yang ◽  
Yu-Chiau Shyu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Feedback Loop ◽  
Target Genes ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Cellular Analysis ◽  
Direct Target ◽  
Regulatory Feedback Loop

The erythroid Krppel-like factor EKLF/KLF1 is a hematopoietic transcription factor binding to CACCC DNA motif and participating in the regulation of erythroid differentiation. With combined use of microarray-based gene expression profiling and promoter-based ChIP-chip assay of E14.5 fetal liver cells from wild type (WT) and EKLF-knockout (Eklf-/-) mouse embryos, we have identified the pathways and direct target genes activated or repressed by EKLF. This genome-wide study together with molecular/ cellular analysis of mouse erythroleukemic cells (MEL) indicate that among the downstream direct target genes of EKLF is Tal1/Scl. Tal1/Scl encodes another DNA-binding hematopoietic transcription factor TAL1/SCL known to be an Eklf activator and essential for definitive erythroid differentiation. Further identification of the authentic Tall gene promoter in combination with in vivo genomic footprinting approach and DNA reporter assay demonstrate that EKLF activates Tall gene through binding to a specific CACCC motif located in its promoter. These data establish the existence of a previously unknow positive regulatory feedback loop between two DNA-binding hematopoietic transcription factors that sustains the mammalian erythropoiesis.

Regulation of segmentation and segmental identity by Drosophila homeoproteins: the role of DNA binding in functional activity and specificity

Development ◽  
1997 ◽  
Vol 124 (22) ◽  
pp. 4425-4433 ◽  
Author(s):  
M.D. Biggin ◽  
W. McGinnis
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Target Genes ◽  
Current Model ◽  
Binding Studies ◽  
Binding Model ◽  
Selective Binding ◽  
Recognition Sites

Recent advances have shed new light on how the Q50 homeoproteins act in Drosophila. These transcription factors have remarkably similar and promiscuous DNA-binding specificities in vitro; yet they each specify distinct developmental fates in vivo. One current model suggests that, because the Q50 homeoproteins have distinct biological functions, they must each regulate different target genes. According to this ‘co-selective binding’ model, significant binding of Q50 homeoproteins to functional DNA elements in vivo would be dependent upon cooperative interactions with other transcription factors (cofactors). If the Q50 homeoproteins each interact differently with cofactors, they could be selectively targeted to unique, limited subsets of their in vitro recognition sites and thus control different genes. However, a variety of experiments question this model. Molecular and genetic experiments suggest that the Q50 homeoproteins do not regulate very distinct sets of genes. Instead, they mostly control the expression of a large number of shared targets. The distinct morphogenic properties of the various Q50 homeoproteins may principally result from the different manners in which they either activate or repress these common targets. Further, in vivo binding studies indicate that at least two Q50 homeoproteins have very broad and similar DNA-binding specificities in embryos, a result that is inconsistent with the ‘co-selective binding’ model. Based on these and other data, we suggest that Q50 homeoproteins bind many of their recognition sites without the aid of cofactors. In this ‘widespread binding’ model, cofactors act mainly by helping to distinguish the way in which homeoproteins regulate targets to which they are already bound.

scholarly journals An eh1-Like Motif in Odd-skipped Mediates Recruitment of Groucho and Repression In Vivo

2005 ◽  
Vol 25 (24) ◽  
pp. 10711-10720 ◽  
Author(s):  
Robert E. Goldstein ◽  
Orna Cook ◽  
Tama Dinur ◽  
Anne Pisanté ◽  
Umesh Chintaman Karandikar ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Target Genes ◽  
Zinc Finger Protein ◽  
Mutational Analysis ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Physical Interactions ◽  
Target Promoters

ABSTRACT Drosophila Groucho, like its vertebrate Transducin-like Enhancer-of-split homologues, is a corepressor that silences gene expression in numerous developmental settings. Groucho itself does not bind DNA but is recruited to target promoters by associating with a large number of DNA-binding negative transcriptional regulators. These repressors tether Groucho via short conserved polypeptide sequences, of which two have been defined. First, WRPW and related tetrapeptide motifs have been well characterized in several repressors. Second, a motif termed Engrailed homology 1 (eh1) has been found predominantly in homeodomain-containing transcription factors. Here we describe a yeast two-hybrid screen that uncovered physical interactions between Groucho and transcription factors, containing eh1 motifs, with different types of DNA-binding domains. We show that one of these, the zinc finger protein Odd-skipped, requires its eh1-like sequence for repressing specific target genes in segmentation. Comparison between diverse eh1 motifs reveals a bias for the phosphoacceptor amino acids serine and threonine at a fixed position, and a mutational analysis of Odd-skipped indicates that these residues are critical for efficient interactions with Groucho and for repression in vivo. Our data suggest that phosphorylation of these phosphomeric residues, if it occurs, will down-regulate Groucho binding and therefore repression, providing a mechanism for posttranslational control of Groucho-mediated repression.

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.

Gene regulation by Sp1 and Sp3

2004 ◽  
Vol 82 (4) ◽  
pp. 460-471 ◽  
Author(s):  
Lin Li ◽  
Shihua He ◽  
Jian-Min Sun ◽  
James R Davie
Keyword(s):  
Gene Regulation ◽  
Transcription Factors ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Cellular Localization ◽  
Target Genes ◽  
In Vivo Studies ◽  
Binding Domains

The Sp family of transcription factors is united by a particular combination of three conserved Cys2His2 zinc fingers that form the sequence-specific DNA-binding domain. Within the Sp family of transcription factors, Sp1 and Sp3 are ubiquitously expressed in mammalian cells. They can bind and act through GC boxes to regulate gene expression of multiple target genes. Although Sp1 and Sp3 have similar structures and high homology in their DNA binding domains, in vitro and in vivo studies reveal that these transcription factors have strikingly different functions. Sp1 and Sp3 are able to enhance or repress promoter activity. Regulation of the transcriptional activity of Sp1 and Sp3 occurs largely at the post-translational level. In this review, we focus on the roles of Sp1 and Sp3 in the regulation of gene expression.Key words: Sp1, Sp3, gene regulation, sub-cellular localization.

scholarly journals A Positive Regulatory Feedback Loop between EKLF/KLF1 and TAL1/SCL Sustaining the Erythropoiesis

2021 ◽  
Vol 22 (15) ◽  
pp. 8024
Author(s):  
Chun-Hao Hung ◽  
Tung-Liang Lee ◽  
Anna Yu-Szu Huang ◽  
Kang-Chung Yang ◽  
Yu-Chiau Shyu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Feedback Loop ◽  
Target Genes ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Cellular Analysis ◽  
Direct Target ◽  
Regulatory Feedback Loop

The erythroid Krüppel-like factor EKLF/KLF1 is a hematopoietic transcription factor binding to the CACCC DNA motif and participating in the regulation of erythroid differentiation. With combined use of microarray-based gene expression profiling and the promoter-based ChIP-chip assay of E14.5 fetal liver cells from wild type (WT) and EKLF-knockout (Eklf−/−) mouse embryos, we identified the pathways and direct target genes activated or repressed by EKLF. This genome-wide study together with the molecular/cellular analysis of the mouse erythroleukemic cells (MEL) indicate that among the downstream direct target genes of EKLF is Tal1/Scl. Tal1/Scl encodes another DNA-binding hematopoietic transcription factor TAL1/SCL, known to be an Eklf activator and essential for definitive erythroid differentiation. Further identification of the authentic Tal gene promoter in combination with the in vivo genomic footprinting approach and DNA reporter assay demonstrate that EKLF activates the Tal gene through binding to a specific CACCC motif located in its promoter. These data establish the existence of a previously unknow positive regulatory feedback loop between two DNA-binding hematopoietic transcription factors, which sustains mammalian erythropoiesis.

scholarly journals Genome-Wide Binding Analyses of HOXB1 Revealed a Novel DNA Binding Motif Associated with Gene Repression

2021 ◽  
Vol 9 (1) ◽  
pp. 6
Author(s):  
Narendra Pratap Singh ◽  
Bony De Kumar ◽  
Ariel Paulson ◽  
Mark E. Parrish ◽  
Carrie Scott ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Embryonic Stem ◽  
Binding Motif ◽  
Binding Assays ◽  
Histone Marks ◽  
Genome Wide ◽  
Hox Cofactors

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in embryonic stem cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1, and MEIS can bind to this motif. In vivo, this motif is sufficient for direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

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