Analysis of Estrogen Response Element Binding by Genetically Selected Steroid Receptor DNA Binding Domain Mutants Exhibiting Altered Specificity and Enhanced Affinity

Chusacultanachai, Sudsanguan; Glenn, Kevin A.; Rodriguez, Adrian O.; Read, Erik K.; Gardner, Jeffrey F.; Katzenellenbogen, Benita S.; Shapiro, David J.

doi:10.1074/jbc.274.33.23591

Cited by 27 publications

(18 citation statements)

References 37 publications

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“…In addition, the antibodies used for detection of the bound proteins are universal, in that they can be used regardless of what DNA-binding domain is displayed on the phage (see supplemental data). These experiments are not limited to zincfinger proteins, as other structural classes of DNA-binding domains have been displayed on the surface of phage, including homeodomains (22), helix-turn-helix motifs (23), ␤ sheets (24), leucine zippers (25), and steroid receptors (26). Furthermore, epitope-tagged DNA-binding proteins or whole transcription factors could be used instead of displaying the proteins on the surface of phage.…”

Section: Discussionmentioning

confidence: 99%

Exploring the DNA-binding specificities of zinc fingers with DNA microarrays

Bulyk

Huang

Choo

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

253

230

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A key step in the regulation of networks that control gene expression is the sequence-specific binding of transcription factors to their DNA recognition sites. A more complete understanding of these DNA-protein interactions will permit a more comprehensive and quantitative mapping of the regulatory pathways within cells, as well as a deeper understanding of the potential functions of individual genes regulated by newly identified DNA-binding sites. Here we describe a DNA microarray-based method to characterize sequence-specific DNA recognition by zinc-finger proteins. A phage display library, prepared by randomizing critical amino acid residues in the second of three fingers of the mouse Zif268 domain, provided a rich source of zinc-finger proteins with variant DNA-binding specificities. Microarrays containing all possible 3-bp binding sites for the variable zinc fingers permitted the quantitation of the binding site preferences of the entire library, pools of zinc fingers corresponding to different rounds of selection from this library, as well as individual Zif268 variants that were isolated from the library by using specific DNA sequences. The results demonstrate the feasibility of using DNA microarrays for genomewide identification of putative transcription factor-binding sites.A n understanding of the sequence specificity of DNAprotein interactions has resulted from studies of the effects of mutations in the DNA-binding sites and the amino acid residues implicated in sequence-specific binding. The zinc-finger transcription factors are among the best understood families in terms of sequence-specific DNA binding. Rational zinc-finger design by using structure-based (1) and database-guided (2) approaches has permitted some progress in revealing certain rules that govern these discriminating contacts (3-6). In addition, phage display has emerged as a powerful tool to select for zinc fingers that recognize given target DNA sites (5, 7-9). Although this technology has permitted millions of protein variants to be sampled simultaneously, the effects of individual mutants have had to be measured one at a time by using nitrocellulose-binding assays (10), gel mobility-shift analysis (11), ELISA (12), Southwestern blotting (13), or reporter constructs (14). Because these methods are generally too laborious to be used for the analysis of a large number of DNA-protein interactions, it has not been possible to gather data on vast collections of variant DNA-protein pairings. Although in vitro selections (15) and ''binding-site signatures'' (4) have permitted the sampling of multiple DNA-binding sites for a given DNAbinding protein, these in vitro selections provide only a partial view of binding-site specificity, because only the tightest binding interactions are selected, whereas information about suboptimal interactions is lost in the experimental process. It is possible that these lower-affinity DNA sites are functionally significant in transcriptional regulation of gene expression. Therefore, we have taken advantage of...

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Section: Discussionmentioning

confidence: 99%

Exploring the DNA-binding specificities of zinc fingers with DNA microarrays

Bulyk

Huang

Choo

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

253

230

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“…Through the use of a modified form of the bacteriophage p22 challenge phage selection system (39), our laboratory recently identified progesterone receptor DNA-binding domain muta- tions that changed the DNA binding specificity from the glucocorticoid response element/progesterone receptor element to the ERE and that resulted in enhanced binding to the consensus ERE and to the imperfect ERE in the pS2 gene (16). One of the progesterone receptor DBD mutants we isolated, DBD5, exhibited Ͼ10-fold higher affinity than the wild-type ER DBD for the consensus and pS2 EREs.…”

Section: Effect Of the Number Of Eres And Ere Binding Affinity On Tramentioning

confidence: 99%

“…We recently described a genetic selection using a modified form of the bacteriophage p22 challenge phage selection system, which requires only a single selection cycle (16). To repress transcription from the imperfect pS2 ERE, it proved essential to modify the KRAB repressor using information from our recent genetic selection for DBDs with altered and enhanced ERE binding (16).…”

Section: Combining Genetic Selection With Er-krab Chimeras Provides Amentioning

confidence: 99%

“…To repress transcription from the imperfect pS2 ERE, it proved essential to modify the KRAB repressor using information from our recent genetic selection for DBDs with altered and enhanced ERE binding (16). To produce the KERK-3M repressor, we combined information from our genetic selections performed using steroid receptor DNA-binding domains with the KERK chimera, whose ability to repress transcription can easily be modulated using ER ligands.…”

Section: Combining Genetic Selection With Er-krab Chimeras Provides Amentioning

confidence: 99%

“…To achieve repression from a promoter containing the native pS2 ERE, we developed a novel repressor with increased affinity for this imperfect ERE. We recently described the use of a modified p22 challenge phage system to select mutant steroid receptor DNAbinding domains with altered DNA binding specificity and an enhanced affinity for EREs (16). By integrating information obtained from those genetically selected mutant DNA-binding modules with the ligand-regulated ER-KRAB chimeras, we produced a prototype of a new class of targeted gene repressor.…”

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Estrogen Receptor-KRAB Chimeras Are Potent Ligand-dependent Repressors of Estrogen-regulated Gene Expression

Haan

Chusacultanachai

Mao

et al. 2000

Journal of Biological Chemistry

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As an approach to targeted repression of genes of interest, we describe the development of human estrogen receptor (ER) ␣-KRAB repressor domain chimeras that are potent ligand-dependent repressors of the transcription of estrogen response element (ERE)-containing promoters and analyze their mechanisms of action. Repression by the KRAB domain was dominant over transactivation mediated by ER AF1 and AF2. An ERE and an ER ligand (estrogen or antiestrogen) were required for repression. Studies with several promoters and cell lines demonstrated that the presence of EREs, rather than the capacity for estrogen induction, determines the potential for repression of a gene by the KRAB-ER␣-KRAB (KERK) chimera. A single consensus ERE was sufficient for repression, but the KERK chimera was unable to suppress transcription from the imperfect ERE in the native pS2 promoter. We recently reported mutations that enhance binding of a steroid receptor DNA-binding domain to the ERE. Introducing these mutations into wild-type ER enhanced transactivation from the pS2 ERE. Insertion of these mutations into KERK created the novel repressor KERK-3M, which is a potent repressor of both ER-induced and basal transcription on a promoter containing the pS2 ERE. These modified ER-KRAB chimeras should prove useful as new tools for the functional analysis and repression of ERregulated genes.Generating ligand-regulated activators or repressors targeted to DNA sequences in any gene of interest represents a challenging long-term goal of protein engineering. The model systems we use to approach this objective are based on estrogen-regulated genes. The effects of estrogen are mediated by the estrogen receptors ER␣ 1 and ER␤. ERs are ligand-activated transcription regulators that are capable of high affinity binding to a specific DNA sequence, termed the estrogen response element (ERE). On binding to the ER, estrogens exert a wide variety of biological effects, including effects on the development and function of male and female reproductive tissues, bone remodeling, and the cardiovascular system, and have been implicated in breast and uterine cancer. Estrogen-regulated genes therefore represent important therapeutic targets. If expression of estrogen-regulated genes could be effectively suppressed, both the discovery and the elucidation of their roles in various physiological processes would be greatly facilitated. Selective estrogen receptor modulators (SERMs) (reviewed in Ref. 1) and ER mutants displaying a dominantnegative phenotype (2) have been used to suppress ER-induced transcription. However, SERMs can display significant agonist activity in specific tissue or cell backgrounds (3, 4). Recently, a number of hER␣ mutants displaying a dominant-negative phenotype have been described (2, 5). Although these hER␣ mutants and SERMs disrupt estrogen-induced transcription, they do not affect basal transcription of estrogen-regulated genes. We therefore designed novel hER␣ variants for ligand-dependent repression of the transcription of ERE-containing g...

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Genetische Selektion - eine Strategie zur Untersuchung und Herstellung von Enzymen

2001

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Natürliche Enzyme sind über Jahrmillionen durch den graduellen Prozess der darwinistischen Evolution entstanden. Die Grundelemente der Evolution – Mutation, Selektion und Vervielfältigung – können auch im Labor eingesetzt werden, um Proteinkatalysatoren in entsprechend kürzeren Zeiträumen herzustellen und zu charakterisieren. Genetische In‐vivo‐Selektionsstrategien ermöglichen eine erschöpfende Analyse von Proteinbibliotheken mit 1010 unterschiedlichen Mitgliedern; und mit In‐vitro‐Methoden lassen sich sogar noch größere Ensembles studieren. Folglich können evolutionäre Ansätze statistisch wertvolle Einsichten in komplexe und oftmals subtile Wechselwirkungen geben, die die Proteinfaltung, die Struktur und den katalytischen Mechanismus beeinflussen. Solche Methoden werden zunehmend auch als Hilfsmittel beim Proteindesign eingesetzt, da sie den Zugang zu neuen Proteinen mit maßgeschneiderten katalytischen Aktivitäten und Selektivitäten eröffnen.

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Analysis of Estrogen Response Element Binding by Genetically Selected Steroid Receptor DNA Binding Domain Mutants Exhibiting Altered Specificity and Enhanced Affinity

Cited by 27 publications

References 37 publications

Exploring the DNA-binding specificities of zinc fingers with DNA microarrays

Exploring the DNA-binding specificities of zinc fingers with DNA microarrays

Estrogen Receptor-KRAB Chimeras Are Potent Ligand-dependent Repressors of Estrogen-regulated Gene Expression

Genetische Selektion - eine Strategie zur Untersuchung und Herstellung von Enzymen

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