In two previous studies we described the properties of a heat-stable DNA-binding protein present in rat liver nuclei. This protein, hereafter termed C/EBP, is capable selective binding to CCAAT homology several viral promoters (Graves et al. 1986), as well core common many enhancers (Johnson 1987). We now report isolation recombinant clone gene that encodes C/EBP. Expression bacterial cells yields binds vitro both and enhancer homology, providing conclusive evidence single product accounts...

This report describes the identification and purification of a nuclear protein from rat liver that binds selectively to DNA sequences associated with several animal virus enhancers. The binding activity was tracked by direct DNase I footprinting through four steps biochemical fractionation. These procedures led polypeptide species exhibiting an apparent molecular weight 20 kD accounts for enhancer activity. dimethyl sulfate assays were used examine manner in which purified elements SV40,...

The proto-oncogene ets-1 is the founding member of a new family eukaryotic transcriptional regulators. Using deletion mutants murine cDNA expressed in Escherichia coli, we show that DNA-binding domain corresponds closely to ETS domain, an 85-amino-acid region conserved among ets members. To investigate specificity DNA binding mapped contacts monomeric Ets-1 fragment by chemical protection and interference assays. backbone interactions span 20-nucleotide are localized on one face helix. Close...

The ETS gene family is frequently involved in chromosome translocations that cause human cancer, including prostate leukemia, and sarcoma. However, the mechanisms by which oncogenic proteins, are DNA-binding transcription factors, target genes necessary for tumorigenesis not well understood. Ewing's sarcoma serves as a paradigm entire class of ETS-associated tumors because nearly all cases harbor recurrent chromosomal involving genes. most common translocation encodes EWS/FLI factor. We used...

The conservation of in vitro DNA-binding properties within families transcription factors presents a challenge for achieving vivo specificity. To uncover the mechanisms regulating specificity ETS gene family, we have used chromatin immunoprecipitation coupled with genome-wide promoter microarrays to query occupancy three proteins human T-cell line. Unexpectedly, redundant was frequently detected, while specific less likely. Redundant binding correlated housekeeping classes genes, whereas...

Cell signaling that culminates in posttranslational modifications directs protein activity. Here we report how multiple Ca 2+ -dependent phosphorylation sites within the transcription activator Ets-1 act additively to produce graded DNA binding affinity. Nuclear magnetic resonance spectroscopic analyses show shifts from a dynamic conformation poised bind well-folded inhibited state. These phosphates lie an unstructured flexible region functions as allosteric effector of autoinhibition....

Sequence-specific DNA binding proteins that function as transcription factors are frequently encoded by gene families. Such display highly conserved properties, yet expected to retain promoter selectivity. In this report we investigate problem using the ets family, a group of metazoan genes whose members regulate cell growth and differentiation mutated in human cancers. We tested whether level mRNA can serve specificity determinant. The levels 27 paralogous were measured 23 tissues lines....

The ets proto-oncogene family is a group of sequence-related genes whose normal cellular function unknown. In study proteins involved in the transcriptional regulation murine retroviruses T lymphocytes, we have discovered that member gene encodes sequence-specific DNA-binding protein. A mouse ets-1 cDNA clone was obtained by screening thymus expression library with double-stranded oligonucleotide probe representing 20 bp Moloney sarcoma virus (MSV) long terminal repeat (LTR). sequence has an...

To elucidate how genomic sequences build transcriptional control networks, we need to understand the connection between DNA sequence and transcription factor binding function. Binding predictions based solely on consensus are limited, because a single can use degenerate motifs related factors often prefer identical sequences. The ETS family factor, ETS1, exemplifies these challenges. Unexpected, redundant occupancy of ETS1 other proteins is observed at promoters housekeeping genes in T cells...

Cell signaling affects gene expression by regulating the activity of transcription factors. Here, we report that mitogen-activated protein kinase (MAPK) phosphorylation Ets-1 and Ets-2, at a conserved site N terminal to their Pointed (PNT) domains, resulted in enhanced transactivation preferential recruitment coactivators CREB binding (CBP) p300. We discovered this phosphorylation-augmented interaction an unbiased affinity chromatography screen HeLa nuclear extracts using either mock-treated...

The aberrant expression of an oncogenic ETS transcription factor is implicated in the progression majority prostate cancers, 40% melanomas, and most cases gastrointestinal stromal tumor Ewing's sarcoma. Chromosomal rearrangements cancer result overexpression any one four factors. How these genes differ from numerous other expressed normal contribute to not understood. We report that proteins, but factors, enhance cell migration. Genome-wide binding analysis matched this specific biological...

Conformational changes, including local protein folding, play important roles in protein-DNA interactions. Here, studies of the transcription factor Ets-1 provided evidence that unfolding also can accompany DNA binding. Circular dichroism and partial proteolysis showed secondary structure DNA-binding domain is unchanged presence DNA. In contrast, allosterically induced an α helix lies within a flanking region involved negative regulation These findings suggest structural basis for...

The ETS transcription factors perform distinct biological functions despite conserving a highly similar DNA-binding domain. One distinguishing property of subset proteins is conserved region 80 amino acids termed the Pointed (PNT) Using enzyme kinetics we determined that Ets-1 PNT domain contains an ERK2 docking site. site enhances efficiency phosphorylation mitogen-activated protein kinase (MAPK) N-terminal to binding rather than catalysis. Three hydrophobic residues are involved in...

The Moloney murine leukemia virus (Mo-MLV) enhancer contains binding sites (LVb and LVc) for the ets gene family of proteins a core site that binds polyomavirus enhancer-binding protein 2/core-binding factor (cbf) proteins. LVb in Mo-MLV contribute to its constitutive activity T cells. All three (LVb, LVc, core) are required phorbol ester inducibility enhancer. Adjacent cbf likewise constitute response element within human T-cell receptor beta-chain (TCR beta) transcriptional TCR beta Here...

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 that play pivotal roles multiple developmental processes mammals, including hematopoiesis and bone development. The Runt domain CBFα2 (amino acids 51 to 178) mediates DNA binding heterodimerization with non-DNA-binding CBFβ subunit. Both protein Ets-1 stimulate by protein. Here we quantify compare extent cooperativity between...

DNA binding by the eukaryotic transcription factor Ets-1 is negatively regulated an intramolecular mechanism. Quantitative assays compared DNA-binding activities of native Ets-1, three deletion mutants, and tryptic fragments. activated polypeptides differed in affinity as much 23-fold. Inhibition was mediated two regions flanking minimal domain. Both enhancing dissociation protein-DNA complex. Three lines evidence indicated that inhibition requires cooperative interaction between regions:...

Phosphorylation of transcription factors is a key link between cell signaling and the control gene expression. Here we report that phosphorylation regulates DNA binding Ets-1 factor by reinforcing an autoinhibitory mechanism. Quantitative DNA-binding assays show calcium-dependent inhibits 50-fold. The four serines mediate this inhibitory effect are distant from domain but near structural elements required for autoinhibition. Mutational analyses demonstrate intact module...