Cooperative Binding of Transcription Factors is a Hallmark of Active Enhancers

Binding Sites ◽

Gene Activation ◽

Cooperative Binding ◽

Chromatin Profiling

AbstractEnhancers harbor binding motifs that recruit transcription factors (TFs) for gene activation. While cooperative binding of TFs at enhancers is known to be critical for transcriptional activation of a handful of developmental enhancers, the extent TF cooperativity genome-wide is unknown. Here, we couple high-resolution nuclease footprinting with single-molecule methylation profiling to characterize TF cooperativity at active enhancers in the Drosophila genome. Enrichment of short MNase-protected DNA segments indicates that the majority of enhancers harbor two or more TF binding sites, and we uncover protected fragments that correspond to co-bound sites in thousands of enhancers. We integrate MNase-seq, methylation accessibility profiling, and CUT&RUN chromatin profiling as a comprehensive strategy to characterize co-binding of the Trithorax-like (TRL) DNA binding protein and multiple other TFs and identify states where an enhancer is bound by no TF, by either single factor, by multiple factors, or where binding sites are occluded by nucleosomes. From the analysis of co-binding, we find that cooperativity dominates TF binding in vivo at a majority of active enhancers. TF cooperativity can occur without apparent protein-protein interactions and provides a mechanism to effectively clear nucleosomes and promote enhancer function.

c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein

10.1128/mcb.14.9.6021-6029.1994 ◽

1994 ◽

Vol 14 (9) ◽

pp. 6021-6029

Author(s):

R Metz ◽

A J Bannister ◽

J A Sutherland ◽

C Hagemeier ◽

E C O'Rourke ◽

...

Keyword(s):

Protein Interactions ◽

Binding Protein ◽

Tata Box ◽

Binding Motif ◽

High Concentrations ◽

Tata Box Binding Protein

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

A Specificity and Targeting Subunit of a Human SWI/SNF Family-Related Chromatin-Remodeling Complex

10.1128/mcb.20.23.8879-8888.2000 ◽

2000 ◽

Vol 20 (23) ◽

pp. 8879-8888 ◽

Cited By ~ 193

Author(s):

Zuqin Nie ◽

Yutong Xue ◽

Dafeng Yang ◽

Sharleen Zhou ◽

Bonnie J. Deroo ◽

...

Keyword(s):

Glucocorticoid Receptor ◽

Chromatin Remodeling ◽

Protein Interactions ◽

Gene Activation ◽

Dna Interaction ◽

Baf Complex ◽

Chromatin Remodeling Complexes

ABSTRACT The SWI/SNF family of chromatin-remodeling complexes facilitates gene activation by assisting transcription machinery to gain access to targets in chromatin. This family includes BAF (also called hSWI/SNF-A) and PBAF (hSWI/SNF-B) from humans and SWI/SNF and Rsc fromSaccharomyces cerevisiae. However, the relationship between the human and yeast complexes is unclear because all human subunits published to date are similar to those of both yeast SWI/SNF and Rsc. Also, the two human complexes have many identical subunits, making it difficult to distinguish their structures or functions. Here we describe the cloning and characterization of BAF250, a subunit present in human BAF but not PBAF. BAF250 contains structural motifs conserved in yeast SWI1 but not in any Rsc components, suggesting that BAF is related to SWI/SNF. BAF250 is also a homolog of the Drosophila melanogaster Osa protein, which has been shown to interact with a SWI/SNF-like complex in flies. BAF250 possesses at least two conserved domains that could be important for its function. First, it has an AT-rich DNA interaction-type DNA-binding domain, which can specifically bind a DNA sequence known to be recognized by a SWI/SNF family-related complex at the β-globin locus. Second, BAF250 stimulates glucocorticoid receptor-dependent transcriptional activation, and the stimulation is sharply reduced when the C-terminal region of BAF250 is deleted. This region of BAF250 is capable of interacting directly with the glucocorticoid receptor in vitro. Our data suggest that BAF250 confers specificity to the human BAF complex and may recruit the complex to its targets through either protein-DNA or protein-protein interactions.

In Vivo Quantitative Estimation of DNA-Dependent Interaction of Sox2 and Oct4 Using BirA-Catalyzed Site-Specific Biotinylation

Biomolecules ◽

10.3390/biom10010142 ◽

2020 ◽

Vol 10 (1) ◽

pp. 142 ◽

Cited By ~ 1

Author(s):

Arman Kulyyassov ◽

Vasily Ogryzko

Keyword(s):

Transcription Factors ◽

Protein Interactions ◽

Fluorescent Protein ◽

Quantitative Estimation ◽

Multiple Reaction Monitoring ◽

Negative Control ◽

Close Proximity ◽

Green Fluorescent

Protein–protein interactions of core pluripotency transcription factors play an important role during cell reprogramming. Cell identity is controlled by a trio of transcription factors: Sox2, Oct4, and Nanog. Thus, methods that help to quantify protein–protein interactions may be useful for understanding the mechanisms of pluripotency at the molecular level. Here, a detailed protocol for the detection and quantitative analysis of in vivo protein–protein proximity of Sox2 and Oct4 using the proximity-utilizing biotinylation (PUB) method is described. The method is based on the coexpression of two proteins of interest fused to a biotin acceptor peptide (BAP)in one case and a biotin ligase enzyme (BirA) in the other. The proximity between the two proteins leads to more efficient biotinylation of the BAP, which can be either detected by Western blotting or quantified using proteomics approaches, such as a multiple reaction monitoring (MRM) analysis. Coexpression of the fusion proteins BAP-X and BirA-Y revealed strong biotinylation of the target proteins when X and Y were, alternatively, the pluripotency transcription factors Sox2 and Oct4, compared with the negative control where X or Y was green fluorescent protein (GFP), which strongly suggests that Sox2 and Oct4 come in close proximity to each other and interact.

Targeting of the Visna Virus Tat Protein to AP-1 Sites: Interactions with the bZIP Domains of Fos and Jun In Vitro and In Vivo

Journal of Virology ◽

10.1128/jvi.73.1.37-45.1999 ◽

1999 ◽

Vol 73 (1) ◽

pp. 37-45 ◽

Cited By ~ 15

Author(s):

B. A. Morse ◽

L. M. Carruth ◽

J. E. Clements

Keyword(s):

Transcription Factors ◽

Protein Interactions ◽

Tat Protein ◽

Viral Promoter ◽

Visna Virus ◽

Cellular Transcription ◽

Virus Promoter

ABSTRACT The visna virus Tat protein is required for efficient viral transcription from the visna virus long terminal repeat (LTR). AP-1 sites within the visna virus LTR, which can be bound by the cellular transcription factors Fos and Jun, are also necessary for Tat-mediated transcriptional activation. A potential mechanism by which the visna virus Tat protein could target the viral promoter is by protein-protein interactions with Fos and/or Jun bound to AP-1 sites in the visna virus LTR. Once targeted to the visna virus promoter, the Tat protein could then interact with basal transcription factors to activate transcription. To examine protein-protein interactions with cellular proteins at the visna virus promoter, we used an in vitro protein affinity chromatography assay and electrophoretic mobility shift assay, in addition to an in vivo two-hybrid assay, to show that the visna virus Tat protein specifically interacts with the cellular transcription factors Fos and Jun and the basal transcription factor TBP (TATA binding protein). The Tat domain responsible for interactions with Fos and Jun was localized to an alpha-helical domain within amino acids 34 to 69 of the protein. The TBP binding domain was localized to amino acids 1 to 38 of Tat, a region previously described by our laboratory as the visna virus Tat activation domain. The bZIP domains of Fos and Jun were found to be important for the interactions with Tat. Mutations within the basic domains of Fos and Jun abrogated binding to Tat in the in vitro assays. The visna virus Tat protein was also able to interact with covalently cross-linked Fos and Jun dimers. Thus, the visna virus Tat protein appears to target AP-1 sites in the viral promoter in a mechanism similar to the interaction of human T-cell leukemia virus type 1 Tax with the cellular transcription factor CREB, by binding the basic domains of an intact bZIP dimer. The association between Tat, Fos, and Jun would position Tat proximal to the viral TATA box, where the visna virus Tat activation domain could contact TBP to activate viral transcription.

The Transcriptional Coactivator Cbp Interacts with β-Catenin to Activate Gene Expression

The Journal of Cell Biology ◽

10.1083/jcb.149.2.249 ◽

2000 ◽

Vol 149 (2) ◽

pp. 249-254 ◽

Cited By ~ 316

Author(s):

Ken-Ichi Takemaru ◽

Randall T. Moon

Keyword(s):

Protein Interactions ◽

Target Genes ◽

Terminal Region ◽

Creb Binding Protein ◽

Transcriptional Coactivator ◽

Yeast Two Hybrid System ◽

Recruitment System

β-Catenin plays a pivotal role in the transcriptional activation of Wnt-responsive genes by binding to TCF/LEF transcription factors. Although it has been suggested that the COOH-terminal region of β-catenin functions as an activation domain, the mechanisms of activation remain unclear. To screen for potential transcriptional coactivators that bind to the COOH-terminal region of β-catenin, we used a novel yeast two-hybrid system, the Ras recruitment system (RRS) that detects protein–protein interactions at the inner surface of the plasma membrane. Using this system, we isolated the CREB-binding protein (CBP). Armadillo (Arm) repeat 10 to the COOH terminus of β-catenin is involved in binding to CBP, whereas β-catenin interacts directly with the CREB-binding domain of CBP. β-Catenin synergizes with CBP to stimulate the activity of a synthetic reporter in vivo. Conversely, β-catenin–dependent transcriptional activation is repressed by E1A, an antagonist of CBP function, but not by an E1A mutant that does not bind to CBP. The activation of Wnt target genes such as siamois and Xnr3 in Xenopus embryos is also sensitive to E1A. These findings suggest that CBP provides a link between β-catenin and the transcriptional machinery, and possibly mediates the oncogenic function of β-catenin.

The role of transcription factors, chromatin structure and DNA replication in 5 S RNA gene regulation

Journal of Cell Science ◽

10.1242/jcs.107.8.2055 ◽

1994 ◽

Vol 107 (8) ◽

pp. 2055-2063 ◽

Cited By ~ 1

Author(s):

A.P. Wolffe

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Protein Interactions ◽

Multigene Family ◽

Cell Types ◽

Eukaryotic Genes ◽

Rna Genes

Differential expression of the oocyte and somatic 5 S RNA genes during Xenopus development can be explained by changes in transcription factor and histone interactions with the two types of gene. Both factors and histones bind 5 S RNA genes with specificity. Protein-protein interactions determine the stability of potentially transcriptionally active or repressed nucleoprotein complexes. A decline in transcription factor abundance, differential binding of transcription factors to oocyte and somatic 5 S genes, and increased competition with the histones for association with DNA during early embryogenesis, can account for the developmental decision to selectively repress the oocyte genes, while retaining the somatic genes in the transcriptionally active state. The 5 S ribosomal genes of Xenopus are perhaps the simplest eukaryotic genes to show regulated expression during development. A large multigene family (oocyte 5 S DNA) is transcriptionally active in oocytes but is repressed in somatic cells, whereas a small multigene family (somatic 5 S DNA) is active in both cell types. A potential molecular mechanism to explain the developmental switch that turns off oocyte 5 S DNA transcription has been experimentally reconstructed in vitro and more recently tested in vivo. Central to this mechanism is the specific association of both transcription factors and histones with 5 S RNA genes. How the interplay of histones and transcription factors is thought to affect transcription, and how their respective contributions might change during development from an oocyte, to an embryo and eventually to a somatic cell is the focus of this review.

HMGN1 Modulates Estrogen-Mediated Transcriptional Activation through Interactions with Specific DNA-Binding Transcription Factors

10.1128/mcb.01724-07 ◽

2007 ◽

Vol 27 (24) ◽

pp. 8859-8873 ◽

Cited By ~ 10

Author(s):

Nan Zhu ◽

Ulla Hansen

Keyword(s):

Transcription Factors ◽

Protein Interactions ◽

Serum Response Factor ◽

Estrogen Receptor Α ◽

Order Structure ◽

Specific Gene ◽

Estrogen Induction

ABSTRACT HMGN1, an abundant nucleosomal binding protein, can affect both the chromatin higher order structure and the modification of nucleosomal histones, but it alters the expression of only a subset of genes. We investigated specific gene targeting by HMGN1 in the context of estrogen induction of gene expression. Knockdown and overexpression experiments indicated that HMGN1 limits the induction of several estrogen-regulated genes, including TFF1 and FOS, which are induced by estrogen through entirely distinct mechanisms. HMGN1 specifically interacts with estrogen receptor α (ERα), both in vitro and in vivo. At the TFF1 promoter, estrogen increases HMGN1 association through recruitment by the ERα. HMGN1 S20E/S24E, although deficient in binding nucleosomal DNA, still interacts with ERα and, strikingly, still represses estrogen-driven activation of the TFF1 gene. On the FOS promoter, which lacks the ERα binding sites, constitutively bound serum response factor (SRF) mediates estrogen stimulation. HMGN1 also interacts specifically with SRF, but HMGN1 S20E/S24E does not. Consistent with the protein interactions, only wild-type HMGN1 significantly inhibits the estrogen-driven activation of the FOS gene. Mechanistically, the inhibition of estrogen induction of several ERα-associated genes, including TFF1, by HMGN1 correlates with decreased levels of acetylation of Lys9 on histone H3. Together, these findings indicate that HMGN1 regulates the expression of particular genes via specific protein-protein interactions with transcription factors at target gene regulatory regions.

Single molecule occupancy patterns of transcription factors reveal determinants of cooperative binding in vivo

10.1101/2020.06.29.167155 ◽

2020 ◽

Cited By ~ 1

Author(s):

Can Sönmezer ◽

Rozemarijn Kleinendorst ◽

Dilek Imanci ◽

Laura Villacorta ◽

Dirk Schübeler ◽

...

Keyword(s):

Transcription Factors ◽

Single Molecule ◽

Gene Activation ◽

Regulatory Elements ◽

Physical Interaction ◽

Binding Motifs ◽

Single Dna Molecules ◽

Occupancy Patterns ◽

Cooperative Activity

Gene activation requires the cooperative activity of multiple transcription factors at cis-regulatory elements. Yet, most transcription factors have short residence time, questioning the requirement of their physical co-occupancy on DNA to achieve cooperativity. Here, we advance Single Molecule Footprinting to detect individual molecular interactions of transcription factors and nucleosomes with DNA at mouse cis-regulatory elements. We apply this strategy to quantify the simultaneous binding of multiple transcription factors on single DNA molecules. Analysis of the binary occupancy patterns at thousands of motif combinations reveals that for most types of transcription factors high DNA co-occupancy can occur in absence of direct physical interaction, at sites of competition with nucleosomes. Perturbation of pairwise interactions demonstrates the function of molecular co-occupancy for binding cooperativity. These findings elucidate the binding cooperativity mechanism used by transcription factors in absence of strict organisation of their binding motifs, a characteristic feature of most of enhancers.

Generation of a versatile BiFC ORFeome library for analyzing protein-protein interactions in live Drosophila

10.1101/343483 ◽

2018 ◽

Author(s):

J. Bischof ◽

M. Duffraisse ◽

E. Furger ◽

L. Ajuria ◽

G. Giraud ◽

...

Keyword(s):

Transcription Factors ◽

Protein Interactions ◽

Large Scale ◽

Bimolecular Fluorescence Complementation ◽

Expression Domain ◽

Hox Proteins ◽

Non Invasive ◽

Tripartite Interactions

AbstractTranscription factors achieve specificity by establishing intricate interaction networks that will change depending on the cell context. Capturing these interactions in live condition is however a challenging issue that requires sensitive and non-invasive methods. We present a set of fly lines, called “multicolor BiFC library”, which covers most of the Drosophila transcription factors for performing Bimolecular Fluorescence Complementation (BiFC). The multicolor BiFC library can be used to probe binary or tripartite interactions and is compatible for large-scale interaction screens. The library can also be coupled with established Drosophila genetic resources to analyze interactions in the developmentally relevant expression domain of each protein partner. We provide proof of principle experiments of these various applications, using Hox proteins in the live Drosophila embryo as a case study. Overall this novel collection of ready-to-use fly lines constitutes an unprecedented genetic toolbox for the identification and analysis of protein-protein interactions in vivo.

The Significance of Tetramerization in Promoter Recruitment by Stat5

10.1128/mcb.19.3.1910 ◽

1999 ◽

Vol 19 (3) ◽

pp. 1910-1918 ◽

Cited By ~ 144

Author(s):

Susan John ◽

Uwe Vinkemeier ◽

Elisabetta Soldaini ◽

James E. Darnell ◽

Warren J. Leonard

Keyword(s):

Protein Interactions ◽

Gene Activation ◽

Interleukin 2 ◽

Regulatory Region ◽

Tryptophan Residue ◽

Tetramer Formation ◽

Wide Range ◽

The Individual

ABSTRACT Stat5a and Stat5b are rapidly activated by a wide range of cytokines and growth factors, including interleukin-2 (IL-2). We have previously shown that these signal transducers and activators of transcription (STAT proteins) are key regulatory proteins that bind to two tandem gamma interferon-activated site (GAS) motifs within an IL-2 response element (positive regulatory region III [PRRIII]) in the human IL-2Rα promoter. In this study, we demonstrate cooperative binding of Stat5 to PRRIII and explore the molecular basis underlying this cooperativity. We demonstrate that formation of a tetrameric Stat5 complex is essential for the IL-2-inducible activation of PRRIII. Stable tetramer formation of Stat5 is mediated through protein-protein interactions involving a tryptophan residue conserved in all STATs and a lysine residue in the Stat5 N-terminal domain (N domain). The functional importance of tetramer formation is shown by the decreased levels of transcriptional activation associated with mutations in these residues. Moreover, the requirement for STAT protein-protein interactions for gene activation from a promoter with tandemly linked GAS motifs can be relieved by strengthening the avidity of protein-DNA interactions for the individual binding sites. Taken together, these studies demonstrate that a dimeric but tetramerization-deficient Stat5 protein can activate only a subset of target sites. For functional activity on a wider range of potential recognition sites, N-domain-mediated oligomerization is essential.