X-ray crystallographic analysis of Runx1-CBF-Ets1-DNA complex assembled on the enhancer of T cell receptor chain gene (original) (raw)
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ETS-core binding factor: a common composite motif in antigen receptor gene enhancers
Molecular and cellular biology, 1998
A tripartite domain of the murine immunoglobulin mu heavy-chain enhancer contains the muA and muB elements that bind ETS proteins and the muE3 element that binds leucine zipper-containing basic helix-loop-helix (bHLH-zip) factors. Analysis of the corresponding region of the human mu enhancer revealed high conservation of the muA and muB motifs but a striking absence of the muE3 element. Instead of bHLH-zip proteins, we found that the human enhancer bound core binding factor (CBF) between the muA and mu elements; CBF binding was shown to be a common feature of both murine and human enhancers. Furthermore, mutant enhancers that bound prototypic bHLH-zip proteins but not CBF did not activate transcription in B cells, and conversely, CBF transactivated the murine enhancer in nonlymphoid cells. Taking these data together with the earlier analysis of T-cell-specific enhancers, we propose that ETS-CBF is a common composite element in the regulation of antigen receptor genes. In addition, t...
Cooperative binding of Ets-1 and core binding factor to DNA
Molecular and cellular biology, 1994
Two phorbol ester-inducible elements (beta E2 and beta E3) within the human T-cell receptor beta gene enhancer each contain consensus binding sites for the Ets and core binding factor (CBF) transcription factor families. Recombinant Ets-1 and purified CBF bound individually to beta E2 and beta E3, in which the Ets and core sites are directly adjacent. In this report, we show that CBF and Ets-1 bind together to beta E2 and beta E3 and that Ets-1-CBF-DNA complexes are favored over the binding of either protein alone to beta E2. Formation of Ets-1-CBF-DNA complexes increased the affinity of Ets-1-DNA interactions and decreased the rate of dissociation of CBF from DNA. Ets-1-CBF-DNA complexes were not observed when either the Ets or core site was mutated. The spatial requirements for the cooperative interaction of Ets-1 and CBF were analyzed by oligonucleotide mutagenesis and binding site selection experiments. Core and Ets sites were coselected, and there appeared to be little constrai...
The EMBO Journal, 2000
The TCR a enhancer (Ea) has served as a paradigm for studying how enhancers organize trans-activators into nucleo-protein complexes thought to recruit and synergistically stimulate the transcriptional machinery. Little is known, however, of either the extent or dynamics of Ea occupancy by nuclear factors during T cell development. Using dimethyl sulfate (DMS) in vivo footprinting, we demonstrate extensive Ea occupancy, encompassing both previously identi®ed and novel sites, not only in T cells representing a developmental stage where Ea is known to be active (CD4 + CD8 + ±DP cells), but surprisingly, also in cells at an earlier developmental stage where Ea is not active (CD4 ± CD8 ± ±DN cells). Partial occupancy was also established in B-lymphoid but not non-lymphoid cells.
Solution structure of the ETS domain from murine Ets-1: A winged helix-turn-helix DNA binding motif
The EMBO Journal
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 fourstranded anti-parallel 13-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 a-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.
Regulation of the transcription factor Ets-1 by DNA-mediated homo-dimerization
The EMBO Journal, 2008
The function of the Ets-1 transcription factor is regulated by two regions that flank its DNA-binding domain. A previously established mechanism for auto-inhibition of monomeric Ets-1 on DNA response elements with a single ETS-binding site, however, has not been observed for the stromelysin-1 promoter containing two palindromic ETS-binding sites. We present the structure of Ets-1 on this promoter element, revealing a ternary complex in which protein homo-dimerization is mediated by the specific arrangement of the two ETS-binding sites. In this complex, the N-terminal-flanking region is required for ternary protein-DNA assembly. Ets-1 variants, in which residues from this region are mutated, loose the ability for DNA-mediated dimerization and stromelysin-1 promoter transactivation. Thus, our data unravel the molecular basis for relief of auto-inhibition and the ability of Ets-1 to function as a facultative dimeric transcription factor on this site. Our findings may also explain previous data of Ets-1 function in the context of heterologous transcription factors, thus providing a molecular model that could also be valid for Ets-1 regulation by hetero-oligomeric assembly.
Structural Insights into the Activity of Enhancer-Binding Proteins
Science, 2005
Activators of bacterial σ 54 -RNA polymerase holoenzyme are mechanochemical proteins that use ATP hydrolysis to activate transcription. We have determined a 20 Å resolution structure of an activator, PspF (1-275) , bound to an ATP transition state analog (ADP.AlF x ), in complex with its basal factor σ 54 by cryo-electron microscopy. By fitting the crystal structure of apo PspF (1-275) at 1.75 Å into the EM map we identify two loops involved in binding σ 54 . By comparing enhancerbinding structures in different nucleotide states and mutational analysis, we propose nucleotide dependent conformational changes that free the loops for association with σ 54
ETS Protein–Dependent Accessibility Changes at the Immunoglobulin μ Heavy Chain Enhancer
Immunity, 1999
to DNAase 1 or to T3 RNA polymerase in pre-B cells and Department of Biology from transgenic mice carrying an integrated enhancer. Brandeis University The intensity of the DNAase 1 hypersensitive site and Waltham, Massachusetts 02454 distal accessibility was accentuated by the presence of matrix attachment regions in the same constructs (Forrester et al., 1994; Jenuwein et al., 1997). Recent Summary analysis of targeted deletions of the core enhancer and/or the adjacent matrix attachment regions clearly Directed accessibility mediated by antigen-receptor demonstrates that the core enhancer is necessary to gene enhancers ensures developmental stage-specific direct normal V(D)J recombination during B cell developactivation of V(D)J recombination. Here, we used a ment (Sakai et al., 1999). Taken together with earlier combination of in vitro and in vivo assays to explore the studies on recombination substrates, these data conmechanisms that regulate immunoglobulin heavy vincingly demonstrate that the enhancer regulates chain gene enhancer-dependent chromatin accessichromatin accessibility. bility. Ets-1 or PU.1 bound to enhancer-containing Regulating accessibility is a characteristic of all known plasmids assembled into chromatin in vitro and inantigen receptor gene enhancers. The immunoglobulin creased restriction enzyme access to a proximal site. and light chain gene enhancers have been tested in In complementary analyses, expression of PU.1 in Etsrecombination substrates and found to activate recom-1-containing 2017 pro-T cells or NIH 3T3 cells induced bination (Kallenbach et al., 1993; Demengeot et al., 1995; sterile I transcripts at the IgH locus and increased Hiramatsu et al., 1995; Ferrandini et al., 1996). Similarly, accessibility of the endogenous enhancer to restricthe T cell receptor ␣, , and ␦ gene enhancers have been tion endonucleases. These observations suggest that used to activate recombination in transgenic substrates one role of PU.1 is to increase accessibility of the (Capone et al., 1993, 1995; Lauzurica and Krangel, 1994; locus to initiate heavy chain gene expression. Roberts et al., 1997; Clevers and Ferrier, 1998). In these assays, the enhancers closely reproduced the develop-* To whom correspondence should be addressed (e-mail: sen@ ies suggest that any of the TCR␣ enhancer binding probinah.cc.brandeis.edu).
Sequence requirement for specific interaction of an enhancer binding protein (EBP1) with DNA
Nucleic Acids Research, 1989
Short DNA sequence motifs have been identified in viral and cellular enhancers which represent the binding sites for a variety of transacting factors. One such HeLa cell factor, EBPI, has been purified and shown to bind to sequences in the SV40 enhancer. The PRDII element in the human (3-interferon gene regulatory element (IRE) shows strong sequence similarity to the EBPI binding site in the SV40 enhancer. We demonstrate here that EBP1 binds to its sites in the SV40 enhancer and IRE in a similar manner, making base specific contacts over one complete turn of the DNA double helix. Mutational analysis of the EBP1 sites in the IRE and SV40 enhancer has identified the DNA sequence requirements necessary for specific EBP1/DNA complex formation. In addition, 34 DNA sequences related to the EBP1 binding site were analysed for their ability to bind EBP1. Sequences constituting high affinity binding sites possess the sequence 5'-GG(N)6CC-3'. Single base pair changes in the region between the conserved Gs and Cs can generally be tolerated although it is clear that these intervening bases contribute to binding affinity. Mutations in the recognition site which could lead to gross structural changes in the DNA abolish EBP1 binding. it also demonstrates distinct cell type specificities (15). Activation of transcription by individual DNA sequence motifs is cell type specific in vivo (16; 17; 13) and this is paralleled by cell type specificity in the binding of cellular factors to these motifs in vivo (18; 19).