Cooperative DNA Binding with AP-1 Proteins Is Required for Transformation by EWS-Ets Fusion Proteins

ABSTRACT A key molecular event in the genesis of Ewing's sarcoma is the consistent presence of chromosomal translocations that result in the formation of proteins in which the amino terminus of EWS is fused to the carboxyl terminus, including the DNA binding domain, of one of five different Ets family proteins. These fusion proteins function as deregulated transcription factors, resulting in aberrant control of gene expression. Recent data indicate that some EWS-Ets target promoters, including the uridine phosphorylase (UPP) promoter, harbor tandem binding sites for Ets and AP-1 proteins. Here we show that those Ets family proteins that participate in Ewing's sarcoma, including Fli1, ERG, and ETV1, cooperatively bind these tandem elements with Fos-Jun while other Ets family members do not. Analysis of this cooperativity in vitro shows that (i) many different spatial arrangements of the Ets and AP-1 sites support cooperative binding, (ii) the bZIP motifs of Fos and Jun are sufficient to support this cooperativity, and (iii) both the Ets domain and carboxy-terminal sequences of Fli1 are important for cooperative DNA binding. EWS-Fli1 activates the expression of UPP mRNA, is directly bound to the UPP promoter, and transforms 3T3 fibroblasts; in contrast, a C-terminally truncated mutant form of EWS-Fli1 that cannot cooperatively bind DNA with Fos-Jun is defective in all of these properties. The results show that the ability of EWS-Ets proteins to cooperatively bind DNA with Fos-Jun is critical to the biologic activities of these proteins. The results have implications for understanding the pathogenesis of Ewing's sarcoma. In addition, they may be relevant to the mechanisms of Ras-dependent activation of genes that harbor tandem Ets and AP-1 binding sites.

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Evolutionarily conserved Ets family members display distinct DNA binding specificities.

Journal of Experimental Medicine ◽

10.1084/jem.175.5.1391 ◽

1992 ◽

Vol 175 (5) ◽

pp. 1391-1399 ◽

Cited By ~ 65

Author(s):

C Y Wang ◽

B Petryniak ◽

I C Ho ◽

C B Thompson ◽

J M Leiden

Keyword(s):

T Cells ◽

T Cell ◽

Dna Binding ◽

Binding Sites ◽

Family Members ◽

Human Leukemia ◽

Binding Studies ◽

Core Motif ◽

Ets Family

Members of the Ets family of proto-oncogenes encode sequence-specific transcription factors that bind to a purine-rich motif centered around a conserved GGA trinucleotide. Ets binding sites have been identified in the transcriptional regulatory regions of multiple T cell genes including the T cell receptor alpha and beta (TCR-alpha and -beta) enhancers and the IL-2 enhancer, as well as in the enhancers of several T cell-trophic viruses including Maloney sarcoma virus, human leukemia virus type 1, and human immunodeficiency virus-2. T cells express multiple members of the Ets gene family including Ets-1, Ets-2, GABP alpha, Elf-1, and Fli-1. The different patterns of expression and protein-protein interactions of these different Ets family members undoubtedly contribute to their ability to specifically regulate distinct sets of T cell genes. However, previous studies have suggested that different Ets family members might also display distinct DNA binding specificities. In this report, we have examined the DNA binding characteristics of two Ets family members, Ets-1 and Elf-1, that are highly expressed in T cells. The results demonstrate that the minimal DNA binding domain of these proteins consists of adjacent basic and putative alpha-helical regions that are conserved in all of the known Ets family members. Both regions are required for DNA binding activity. In vitro binding studies demonstrated that Ets-1 and Elf-1 display distinct DNA binding specificities, and, thereby interact preferentially with different naturally occurring Ets binding sites. A comparison of known Ets binding sites identified three nucleotides at the 3' end of these sequences that control the differential binding of the Ets-1 and Elf-1 proteins. These results are consistent with a model in which different Ets family members regulate the expression of different T cell genes by binding preferentially to purine-rich sequences that share a GGA core motif, but contain distinct flanking sequences.

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cis-acting sequences required for inducible interleukin-2 enhancer function bind a novel Ets-related protein, Elf-1.

Molecular and Cellular Biology ◽

10.1128/mcb.12.3.1043 ◽

1992 ◽

Vol 12 (3) ◽

pp. 1043-1053 ◽

Cited By ~ 119

Author(s):

C B Thompson ◽

C Y Wang ◽

I C Ho ◽

P R Bohjanen ◽

B Petryniak ◽

...

Keyword(s):

T Cell ◽

Dna Binding ◽

Electrophoretic Mobility ◽

Binding Sites ◽

Interleukin 2 ◽

Dna Binding Domain ◽

Mobility Shift ◽

Ets Family ◽

Enhancer Function

The recent definition of a consensus DNA binding sequence for the Ets family of transcription factors has allowed the identification of potential Ets binding sites in the promoters and enhancers of many inducible T-cell genes. In the studies described in this report, we have identified two potential Ets binding sites, EBS1 and EBS2, which are conserved in both the human and murine interleukin-2 enhancers. Within the human enhancer, these two sites are located within the previously defined DNase I footprints, NFAT-1 and NFIL-2B, respectively. Electrophoretic mobility shift and methylation interference analyses demonstrated that EBS1 and EBS2 are essential for the formation of the NFAT-1 and NFIL-2B nuclear protein complexes. Furthermore, in vitro mutagenesis experiments demonstrated that inducible interleukin-2 enhancer function requires the presence of either EBS1 or EBS2. Two well-characterized Ets family members, Ets-1 and Ets-2, are reciprocally expressed during T-cell activation. Surprisingly, however, neither of these proteins bound in vitro to EBS1 or EBS2. We therefore screened a T-cell cDNA library under low-stringency conditions with a probe from the DNA binding domain of Ets-1 and isolated a novel Ets family member, Elf-1. Elf-1 contains a DNA binding domain that is nearly identical to that of E74, the ecdysone-inducible Drosophila transcription factor required for metamorphosis (hence the name Elf-1, for E74-like factor 1). Elf-1 bound specifically to both EBS1 and EBS2 in electrophoretic mobility shift assays. It also bound to the purine-rich CD3R element from the human immunodeficiency virus type 2 long terminal repeat, which is required for inducible virus expression in response to signalling through the T-cell receptor. Taken together, these results demonstrate that multiple Ets family members with apparently distinct DNA binding specificities regulate differential gene expression in resting and activated T cells.

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cis-acting sequences required for inducible interleukin-2 enhancer function bind a novel Ets-related protein, Elf-1

Molecular and Cellular Biology ◽

10.1128/mcb.12.3.1043-1053.1992 ◽

1992 ◽

Vol 12 (3) ◽

pp. 1043-1053

Author(s):

C B Thompson ◽

C Y Wang ◽

I C Ho ◽

P R Bohjanen ◽

B Petryniak ◽

...

Keyword(s):

T Cell ◽

Dna Binding ◽

Electrophoretic Mobility ◽

Binding Sites ◽

Interleukin 2 ◽

Dna Binding Domain ◽

Mobility Shift ◽

Ets Family ◽

Enhancer Function

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

Journal of Virology ◽

10.1128/jvi.74.5.2084-2093.2000 ◽

2000 ◽

Vol 74 (5) ◽

pp. 2084-2093 ◽

Cited By ~ 23

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.

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Surviving cancer or future motherhood? Both are possible: an Ewing sarcoma case in an 8-year-old girl

BMJ Case Reports ◽

10.1136/bcr-2020-241417 ◽

2021 ◽

Vol 14 (4) ◽

pp. e241417

Author(s):

Tânia Ascensão ◽

Maria Inês Marques ◽

Helena Barros Leite

Keyword(s):

Ewing’S Sarcoma ◽

Ewing's Sarcoma ◽

Reproductive Potential ◽

Adult Life ◽

Vitro Fertilization ◽

Successful Case ◽

Paediatric Age ◽

Reproductive Cycles ◽

Uneventful Pregnancy

Ewing’s sarcoma is an aggressive tumour, common in paediatric age, in which treatment often implies a decrease in reproductive potential. We describe a case of a woman who had a lumbar Ewing’s Sarcoma in 1991, at the age of 8. She was submitted to extended tumourectomy, chemotherapy and local radiotherapy without preservation techniques. In adult life, and after two in vitro fertilization (IVF) reproductive cycles without success, she spontaneously conceived at the age of 32. After an uneventful pregnancy, she delivered a healthy child by caesarean section. This is a rare successful case of a spontaneous and uneventful pregnancy without previous preservation techniques. In the last 30 years, there has been significant development in this area, and currently, there are solutions for these patients, including in prepubertal age.

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The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2

Molecular and Cellular Biology ◽

10.1128/mcb.00417-18 ◽

2018 ◽

Vol 39 (3) ◽

Cited By ~ 5

Author(s):

Kyle T. Helzer ◽

Mary Szatkowski Ozers ◽

Mark B. Meyer ◽

Nancy A. Benkusky ◽

Natalia Solodin ◽

...

Keyword(s):

Estrogen Receptor ◽

Transcription Factor ◽

Dna Binding ◽

Protein Interactions ◽

Posttranslational Modifications ◽

Binding Sites ◽

Protein Function ◽

Estrogen Receptor Α ◽

Binding Activity

ABSTRACT Posttranslational modifications are key regulators of protein function, providing cues that can alter protein interactions and cellular location. Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) occurs in response to multiple stimuli and is involved in modulating ER-dependent gene transcription. While the cistrome of ER is well established, surprisingly little is understood about how phosphorylation impacts ER-DNA binding activity. To define the pS118-ER cistrome, chromatin immunoprecipitation sequencing was performed on pS118-ER and ER in MCF-7 cells treated with estrogen. pS118-ER occupied a subset of ER binding sites which were associated with an active enhancer mark, acetylated H3K27. Unlike ER, pS118-ER sites were enriched in GRHL2 DNA binding motifs, and estrogen treatment increased GRHL2 recruitment to sites occupied by pS118-ER. Additionally, pS118-ER occupancy sites showed greater enrichment of full-length estrogen response elements relative to ER sites. In an in vitro DNA binding array of genomic binding sites, pS118-ER was more commonly associated with direct DNA binding events than indirect binding events. These results indicate that phosphorylation of ER at serine 118 promotes direct DNA binding at active enhancers and is a distinguishing mark for associated transcription factor complexes on chromatin.

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The closely related Drosophila sry beta and sry delta zinc finger proteins show differential embryonic expression and distinct patterns of binding sites on polytene chromosomes

Development ◽

10.1242/dev.110.1.141 ◽

1990 ◽

Vol 110 (1) ◽

pp. 141-149 ◽

Cited By ~ 1

Author(s):

F. Payre ◽

S. Noselli ◽

V. Lefrere ◽

A. Vincent

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Polytene Chromosomes ◽

Duplication Event ◽

Amino Acid Sequences ◽

Evolutionary Divergence ◽

Coding Region ◽

Protein Coding

Serendipity (sry) beta (beta) and delta (delta) are two finger protein genes resulting from a duplication event. Comparison of their respective protein products shows interspersed blocks of conserved and divergent amino-acid sequences. The most extensively conserved region corresponds to the predicted DNA-binding domain which includes 6 contiguous fingers; no significant sequence conservation is found upstream and downstream of the protein-coding region. We have analysed the evolutionary divergence of the sry beta and delta proteins on two separate levels, their embryonic pattern of expression and their DNA-binding properties in vitro and in vivo. By using specific antibodies and transformant lines containing beta-galactosidase fusion genes, we show that the sry beta and sry delta proteins are maternally inherited and present in embryonic nuclei at the onset of zygotic transcription, suggesting that they are transcription factors involved in this process. Zygotic synthesis of the sry beta protein starts during nuclear division cycles 12–13, prior to cellularisation of the blastoderm, while the zygotic sry delta protein is not detectable before germ band extension (stage 10 embryos). Contrary to sry delta, the zygotic sry beta protein constitutes only a minor fraction of the total embryonic protein. The sry beta and delta proteins made in E. coli bind to DNA, with partly overlapping specificities. Their in vivo patterns of binding to DNA, visualised by immunostaining polytene chromosomes, differ both in the number and position of their binding sites. Thus changes in expression pattern and DNA-binding specificity have contributed to the evolution of the sry beta and delta genes.

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Site-specific DNA binding of nuclear factor I: analyses of cellular binding sites

Molecular and Cellular Biology ◽

10.1128/mcb.5.5.964-971.1985 ◽

1985 ◽

Vol 5 (5) ◽

pp. 964-971

Author(s):

R M Gronostajski ◽

S Adhya ◽

K Nagata ◽

R A Guggenheimer ◽

J Hurwitz

Keyword(s):

Dna Binding ◽

Hela Cell ◽

Dna Sequences ◽

Binding Sites ◽

Consensus Sequence ◽

Nuclear Factor ◽

Site Specific ◽

Factor I ◽

Nuclear Factor I

Nuclear factor I is a cellular site-specific DNA-binding protein required for the efficient in vitro replication of adenovirus DNA. We have characterized human DNA sequences to which nuclear factor I binds. Three nuclear factor I binding sites (FIB sites), isolated from HeLa cell DNA, each contain the sequence TGG(N)6-7GCCAA. Comparison with other known and putative FIB sites suggests that this sequence is important for the binding of nuclear factor I. Nuclear factor I protects a 25- to 30-base-pair region surrounding this sequence from digestion by DNase I. Methylation protection studies suggest that nuclear factor I interacts with guanine residues within the TGG(N)6-7GCCAA consensus sequence. One binding site (FIB-2) contained a restriction endonuclease HaeIII cleavage site (GGCC) at the 5' end of the GCCAA motif. Digestion of FIB-2 with HaeIII abolished the binding of nuclear factor I. Southern blot analyses indicate that the cellular FIB sites described here are present within single-copy DNA in the HeLa cell genome.

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

Molecular and Cellular Biology ◽

10.1128/mcb.15.3.1405 ◽

1995 ◽

Vol 15 (3) ◽

pp. 1405-1421 ◽

Cited By ~ 197

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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Genomic targeting of epigenetic probes using a chemically tailored Cas9 system

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1615723114 ◽

2017 ◽

Vol 114 (4) ◽

pp. 681-686 ◽

Cited By ~ 31

Author(s):

Glen P. Liszczak ◽

Zachary Z. Brown ◽

Samuel H. Kim ◽

Rob C. Oslund ◽

Yael David ◽

...

Keyword(s):

Dna Binding ◽

Chemical Synthesis ◽

Small Molecules ◽

Small Molecule ◽

Binding Sites ◽

Dna Binding Protein ◽

Dna Binding Proteins ◽

Live Cells ◽

Binding Partners

Recent advances in the field of programmable DNA-binding proteins have led to the development of facile methods for genomic localization of genetically encodable entities. Despite the extensive utility of these tools, locus-specific delivery of synthetic molecules remains limited by a lack of adequate technologies. Here we combine the flexibility of chemical synthesis with the specificity of a programmable DNA-binding protein by using protein trans-splicing to ligate synthetic elements to a nuclease-deficient Cas9 (dCas9) in vitro and subsequently deliver the dCas9 cargo to live cells. The versatility of this technology is demonstrated by delivering dCas9 fusions that include either the small-molecule bromodomain and extra-terminal family bromodomain inhibitor JQ1 or a peptide-based PRC1 chromodomain ligand, which are capable of recruiting endogenous copies of their cognate binding partners to targeted genomic binding sites. We expect that this technology will allow for the genomic localization of a wide array of small molecules and modified proteinaceous materials.

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