Barbara J. Graves

Specificity within the ets Family of Transcription Factors

Publisher Summary The discovery of ets proteins as transcription factors provided a framework for understanding the oncogenic potential of ets genes. Ets proteins became a model system to study the molecular mechanisms of transcriptional control, including how transcription factors bind DNA, modulate promoter activity, and respond to signaling input. The current knowledge of the ets proteins is discussed within the framework of this critical issue. The chapter illustrates the specificity problem and presents possible solutions to the specificity problem. The high degree of sequence conservation among ets family members is presented in the form of a phylogenetic tree. The ETS domain identifies all ets proteins as sequence-specific DNA-binding proteins. The chapter illustrates the biological specificity of the ets proteins by reviewing the genetic analysis of ets genes in both vertebrate and invertebrate systems. Families of transcription factors are often defined by the sequence conservation of their DNA-binding domains. The chapter discusses functional domains involved in protein–protein interactions and transcriptional activation or repression. The divergence of these regions provides for gene-specific regulation. The distinctive responses of ets proteins to signal transduction pathways are discussed. The chapter illustrates how auto-inhibitory sequences also contribute to specificity.

DNA specificity determinants associate with distinct transcription factor functions.

To elucidate how genomic sequences build transcriptional control networks, we need to understand the connection between DNA sequence and transcription factor binding and function. Binding predictions based solely on consensus predictions are limited, because a single factor can use degenerate sequence motifs and because related transcription factors often prefer identical sequences. The ETS family transcription factor, ETS1, exemplifies these challenges. Unexpected, redundant occupancy of ETS1 and other ETS proteins is observed at promoters of housekeeping genes in T cells due to common sequence preferences and the presence of strong consensus motifs. However, ETS1 exhibits a specific function in T cell activation; thus, unique transcriptional targets are predicted. To uncover the sequence motifs that mediate specific functions of ETS1, a genome-wide approach, chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), identified both promoter and enhancer binding events in Jurkat T cells. A comparison with DNase I sensitivity both validated the dataset and also improved accuracy. Redundant occupancy of ETS1 with the ETS protein GABPA occurred primarily in promoters of housekeeping genes, whereas ETS1 specific occupancy occurred in the enhancers of T cell–specific genes. Two routes to ETS1 specificity were identified: an intrinsic preference of ETS1 for a variant of the ETS family consensus sequence and the presence of a composite sequence that can support cooperative binding with a RUNX transcription factor. Genome-wide occupancy of RUNX factors corroborated the importance of this partnership. Furthermore, genome-wide occupancy of co-activator CBP indicated tight co-localization with ETS1 at specific enhancers, but not redundant promoters. The distinct sequences associated with redundant versus specific ETS1 occupancy were predictive of promoter or enhancer location and the ontology of nearby genes. These findings demonstrate that diversity of DNA binding motifs may enable variable transcription factor function at different genomic sites.

Ras/mitogen-activated protein kinase signaling activates Ets-1 and Ets-2 by CBP/p300 recruitment.

ABSTRACT Cell signaling affects gene expression by regulating the activity of transcription factors. Here, we report that mitogen-activated protein kinase (MAPK) phosphorylation of Ets-1 and Ets-2, at a conserved site N terminal to their Pointed (PNT) domains, resulted in enhanced transactivation by preferential recruitment of the coactivators CREB binding protein (CBP) and p300. We discovered this phosphorylation-augmented interaction in an unbiased affinity chromatography screen of HeLa nuclear extracts by using either mock-treated or ERK2-phosphorylated ETS proteins as ligands. Binding between purified proteins demonstrated a direct interaction. Both the phosphoacceptor site, which lies in an unstructured region, and the PNT domain were required for the interaction. Minimal regions that were competent for induced CBP/p300 binding in vitro also supported MAPK-enhanced transcription in vivo. CBP coexpression potentiated MEK1-stimulated Ets-2 transactivation of promoters with Ras-responsive elements. Furthermore, CBP and Ets-2 interacted in a phosphorylation-enhanced manner in vivo. This study describes a distinctive interface for a transcription factor-coactivator complex and demonstrates a functional role for inducible CBP/p300 binding. In addition, our findings decipher the mechanistic link between Ras/MAPK signaling and two specific transcription factors that are relevant to both normal development and tumorigenesis.

Characterization of the Cooperative Function of Inhibitory Sequences in Ets-1

DNA binding by the eukaryotic transcription factor Ets-1 is negatively regulated by an intramolecular mechanism. Quantitative binding assays compared the DNA-binding activities of native Ets-1, three deletion mutants, and three tryptic fragments. Ets-1 and activated Ets-1 polypeptides differed in DNA-binding affinity as much as 23-fold. Inhibition was mediated by two regions flanking the minimal DNA-binding domain. Both regions regulated affinity by enhancing dissociation of the protein-DNA complex. Three lines of evidence indicated that inhibition requires cooperative interaction between the two regions: first, the two inhibitory regions acted through a common mechanism; second, neither region functioned independently of the other; finally, mutation of the C-terminal inhibitory region altered the conformation of the N-terminal inhibitory region. In addition, partial proteolysis detected an identical altered conformation in the N-terminal inhibitory region of Ets-1 bound to DNA. This finding suggested that repression is transiently disrupted during DNA binding. These results provide evidence that the two inhibitory regions of Ets-1 are structurally, as well as functionally, coupled. In addition, conformational change is shown to be a critical component of the inhibition mechanism. A cooperative, allosteric model of autoinhibition is described. Autoinhibition of Ets-1 could be relieved by either protein partner(s) or posttranslational modifications.

Microsatellites as EWS/FLI response elements in Ewing's sarcoma

The ETS gene family is frequently involved in chromosome translocations that cause human cancer, including prostate cancer, leukemia, and sarcoma. However, the mechanisms by which oncogenic ETS proteins, which are DNA-binding transcription factors, target genes necessary for tumorigenesis is not well understood. Ewings sarcoma serves as a paradigm for the entire class of ETS-associated tumors because nearly all cases harbor recurrent chromosomal translocations involving ETS genes. The most common translocation in Ewings sarcoma encodes the EWS/FLI oncogenic transcription factor. We used whole genome localization (ChIP-chip) to identify target genes that are directly bound by EWS/FLI. Analysis of the promoters of these genes demonstrated a significant over-representation of highly repetitive GGAA-containing elements (microsatellites). In a parallel approach, we found that EWS/FLI uses GGAA microsatellites to regulate the expression of some of its target genes including NR0B1, a gene required for Ewings sarcoma oncogenesis. The microsatellite in the NR0B1 promoter bound EWS/FLI in vitro and in vivo and was both necessary and sufficient to confer EWS/FLI regulation to a reporter gene. Genome wide computational studies demonstrated that GGAA microsatellites were enriched close to EWS/FLI-up-regulated genes but not down-regulated genes. Mechanistic studies demonstrated that the ability of EWS/FLI to bind DNA and modulate gene expression through these repetitive elements depended on the number of consecutive GGAA motifs. These findings illustrate an unprecedented route to specificity for ETS proteins and use of microsatellites in tumorigenesis.

Solution structure of the ETS domain from murine Ets-1: a winged helix-turn-helix DNA binding motif.

Ets‐1 is the prototypic member of the ets family of transcription factors. This family is characterized by the conserved ETS domain that mediates specific DNA binding. Using NMR methods, we have determined the structure of a fragment of murine Ets‐1 composed of the 85 residue ETS domain and a 25 amino acid extension that ends at its native C‐terminus. The ETS domain folds into a helix‐turn‐helix motif on a four‐stranded anti‐parallel beta‐sheet scaffold. This structure places Ets‐1 in the winged helix‐turn‐helix (wHTH) family of DNA binding proteins and provides a model for interpreting the sequence conservation of the ETS domain and the specific interaction of Ets‐1 with DNA. The C‐terminal sequence of Ets‐1, which is mutated in the v‐Ets oncoprotein, forms an alpha‐helix that packs anti‐parallel to the N‐terminal helix of the ETS domain. In this position, the C‐terminal helix is poised to interact directly with an N‐terminal inhibitory region in Ets‐1 as well as the wHTH motif. This explains structurally the concerted role of residues flanking the ETS domain in the intramolecular inhibition of Ets‐1 DNA binding.

Auto-Inhibition and Partner Proteins, Core-Binding Factor β (CBFβ) and Ets-1, Modulate DNA Binding by CBFα2 (AML1)

ABSTRACT Core-binding factor α2 (CBFα2; otherwise known as AML1 or PEBP2αB) is a DNA-binding subunit in the family of core-binding factors (CBFs), heterodimeric transcription factors that play pivotal roles in multiple developmental processes in mammals, including hematopoiesis and bone development. The Runt domain in CBFα2 (amino acids 51 to 178) mediates DNA binding and heterodimerization with the non-DNA-binding CBFβ subunit. Both the CBFβ subunit and the DNA-binding protein Ets-1 stimulate DNA binding by the CBFα2 protein. Here we quantify and compare the extent of cooperativity between CBFα2, CBFβ, and Ets-1. We also identify auto-inhibitory sequences within CBFα2 and sequences that modulate its interactions with CBFβ and Ets-1. We show that sequences in the CBFα2 Runt domain and sequences C terminal to amino acid 214 inhibit DNA binding. Sequences C terminal to amino acid 214 also inhibit heterodimerization with the non-DNA-binding CBFβ subunit, particularly heterodimerization off DNA. CBFβ rescinds the intramolecular inhibition of CBFα2, stimulating DNA binding approximately 40-fold. In comparison, Ets-1 stimulates CBFα2 DNA binding 7- to 10-fold. Although the Runt domain alone is sufficient for heterodimerization with CBFβ, sequences N terminal to amino acid 41 and between amino acids 190 and 214 are required for cooperative DNA binding with Ets-1. Cooperative DNA binding with Ets-1 is less pronounced with the CBFα2-CBFβ heterodimer than with CBFα2 alone. These analyses demonstrate that CBFα2 is subject to both negative regulation by intramolecular interactions, and positive regulation by two alternative partnerships.

Auto-Inhibition of Ets-1 Is Counteracted by DNA Binding Cooperativity with Core-Binding Factor α2

ABSTRACT Auto-inhibition is a common transcriptional control mechanism that is well characterized in the regulatory transcription factor Ets-1. Autoinhibition of Ets-1 DNA binding works through an inhibitory module that exists in two conformations. DNA binding requires a change in the inhibitory module from the packed to disrupted conformation. This structural switch provides a mechanism to tightly regulate Ets-1 DNA binding. We report that the Ets-1 partner protein core-binding factor α2 (CBFα2; also known as AML1 or PEBP2) stimulates Ets-1 DNA binding and counteracts auto-inhibition. Support for this conclusion came from three observations. First, the level of cooperative DNA binding (10-fold) was similar to the level of repression by auto-inhibition (10- to 20-fold). Next, a region necessary for cooperative DNA binding mapped to the inhibitory module. Third, an Ets-1 mutant with a constitutively disrupted inhibitory module did not bind DNA cooperatively with CBFα2. Furthermore, two additional lines of evidence indicated that CBFα2 affects the structural switch by direct interactions with Ets-1. First, the retention of cooperative DNA binding on nicked duplexes eliminated a potential role of through-DNA effects. Second, cooperative DNA binding was observed on composite sites with altered spacing or reversed orientation. We suggest that only protein interactions can accommodate this observed flexibility. These findings provide a mechanism by which CBF relieves the auto-inhibition of Ets-1 and illustrates one strategy for the synergistic activity of regulatory transcription factors.

Oncogenic ETS proteins mimic activated RAS/MAPK signaling in prostate cells

The aberrant expression of an oncogenic ETS transcription factor is implicated in the progression of the majority of prostate cancers, 40% of melanomas, and most cases of gastrointestinal stromal tumor and Ewings sarcoma. Chromosomal rearrangements in prostate cancer result in overexpression of any one of four ETS transcription factors. How these four oncogenic ETS genes differ from the numerous other ETS genes expressed in normal prostate and contribute to tumor progression is not understood. We report that these oncogenic ETS proteins, but not other ETS factors, enhance prostate cell migration. Genome-wide binding analysis matched this specific biological function to occupancy of a unique set of genomic sites highlighted by the presence of ETS- and AP-1-binding sequences. ETS/AP-1-binding sequences are prototypical RAS-responsive elements, but oncogenic ETS proteins activated a RAS/MAPK transcriptional program in the absence of MAPK activation. Thus, overexpression of oncogenic ETS proteins can replace RAS/MAPK pathway activation in prostate cells. The genomic description of this ETS/AP-1-regulated, RAS-responsive, gene expression program provides a resource for understanding the role of these ETS factors in both an oncogenic setting and the developmental processes where these genes normally function.

The Structural and Dynamic Basis of Ets-1 DNA Binding Autoinhibition

The transcription factor Ets-1 is regulated by the allosteric coupling of DNA binding with the unfolding of an α-helix (HI-1) within an autoinhibitory module. To understand the structural and dynamic basis for this autoinhibition, we have used NMR spectroscopy to characterize Ets-1ΔN301, a partially inhibited fragment of Ets-1. The NMR-derived Ets-1ΔN301 structure reveals that the autoinhibitory module is formed predominantly by the hydrophobic packing of helices from the N-terminal (HI-1, HI-2) and C-terminal (H4, H5) inhibitory sequences, along with H1 of the intervening DNA binding ETS domain. The intramolecular interactions made by HI-1 in Ets-1ΔN301 are similar to the intermolecular contacts observed in the crystal structure of an Ets-1ΔN300 dimer, confirming that the latter represents a domain-swapped species. 15N relaxation studies demonstrate that the backbone of the N-terminal inhibitory sequence is mobile on the nanosecond-picosecond and millisecond-microsecond time scales. Furthermore, hydrogen exchange measurements reveal that amide protons in helices HI-1 and HI-2 exchange with water at rates only ∼15- and ∼75-fold slower, respectively, than predicted for an unfolded polypeptide. These findings indicate that inhibitory helices are only marginally stable even in the absence of DNA. The energetic coupling of DNA binding with the facile unfolding of the labile HI-1 provides a mechanism for modulating Ets-1 DNA binding activity via protein partnerships, post-translational modifications, or mutations. Ets-1 autoinhibition illustrates how conformational equilibria within structural domains can regulate macromolecular interactions.