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Highly parallel identification of sequence preferences for eukaryotic C2H2 zinc finger domains using a bacterial 1-hybrid assay

ABSTRACT: The largest and most diverse class of eukaryotic transcription factors contain Cys2-His2 zinc fingers (C2H2-ZFs), each of which typically binds a DNA nucleotide triplet within a larger binding site. Frequent recombination and diversification of their DNA-contacting residues suggests that these zinc fingers play a prevalent role in adaptive evolution. Very little is known about the function and evolution of the vast majority of C2H2-ZFs, including whether they even bind DNA. Using the bacterial 1-hybrid (B1H) system, we determined DNA-binding motifs for thousands of individual natural C2H2-ZFs, and correlated them with the C2H2-ZF specificity residues.

ORGANISM(S): synthetic construct

PROVIDER: GSE52521 | GEO | 2015/02/17

SECONDARY ACCESSION(S): PRJNA229157

REPOSITORIES: GEO

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Evaluation of DNA-binding preferences of C2H2 zinc finger domains by PBM

2015-02-17 | GSE52520 | GEO

Evaluation of DNA-binding preferences of C2H2 zinc finger domains by PBM

Project description:The largest and most diverse class of eukaryotic transcription factors contain Cys2-His2 zinc fingers (C2H2-ZFs), each of which typically binds a DNA nucleotide triplet within a larger binding site. Frequent recombination and diversification of their DNA-contacting residues suggests that these zinc fingers play a prevalent role in adaptive evolution. Very little is known about the function and evolution of the vast majority of C2H2-ZFs, including whether they even bind DNA. Using the bacterial 1-hybrid (B1H) system, we determined DNA-binding motifs for thousands of individual natural C2H2-ZFs, and correlated them with C2H2-ZF specificity residues. The data reported here includes results of protein-binding microarray (PBM) assays for 146 of these natural C2H2-ZFs, performed in order to validate B1H assays and to explore the DNA-binding specificity of C2H2-ZFs. Protein binding microarray (PBM) experiments were performed for a set of 185 variants of mouse Egr1 in which the third zinc finger was replaced by different C2H2-ZFs from different organisms. Briefly, the PBMs involved binding GST-tagged DNA-binding proteins to two double-stranded 44K Agilent microarrays, each containing a different DeBruijn sequence design, in order to determine their sequence preferences. Details of the PBM protocol are described in Berger et al., Nature Biotechnology 2006. Among the 185 variants examined, 146 variants yielded motifs in PBMs, which are included here.

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Highly parallel identification of sequence preferences for eukaryotic C2H2 zinc finger domains using a bacterial 1-hybrid assay

Project description:The largest and most diverse class of eukaryotic transcription factors contain Cys2-His2 zinc fingers (C2H2-ZFs), each of which typically binds a DNA nucleotide triplet within a larger binding site. Frequent recombination and diversification of their DNA-contacting residues suggests that these zinc fingers play a prevalent role in adaptive evolution. Very little is known about the function and evolution of the vast majority of C2H2-ZFs, including whether they even bind DNA. Using the bacterial 1-hybrid (B1H) system, we determined DNA-binding motifs for thousands of individual natural C2H2-ZFs, and correlated them with the C2H2-ZF specificity residues. We generated 47,072 variants of the DNA-binding domain of the well-characterized three-C2H2-ZF protein Egr1, in which the third C2H2-ZF ('F3') was replaced with a natural C2H2-ZF. We screened this library using the bacterial 1-hybrid (B1H) system to assess the relative preference of each protein for 200 of the 256 possible NNN NGG GCG variants of the optimal Egr1 binding site, GCG TGG GCG, each in duplicate. Each sample might have been sequenced multiple times, in which case multiple raw and processed data files are indicated. This data represent bacterial 1-hybrid assays using three different "Original Library Samples", OLS-A, OLS-B, and OLS-C. The 'characteristics:OLS' indicates which OLS was used as the initial material for each of the assays. The processed data contains unique ID given to each sequence in the library and its abundance count. The 'OLS.info.xlsx' file contains the mapping of these IDs to different sequences.

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Large-scale characterization of DNA binding landscape of eukaryotic C2H2 zinc fingers

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