Evaluation of DNA-binding preferences of non-metazoan C2H2 zinc finger proteins by PBM
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PROVIDER: PRJNA296081 | ENA |
REPOSITORIES: ENA
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Project description:The largest and most diverse class of eukaryotic transcription factors contain Cys2-His2 zinc fingers (C2H2-ZFs). Here, we have explored the diversity of sequence preferences of C2H2-ZF proteins in non-metazoan organisms using PBM experiments.
2017-07-10 | GSE73117 | GEO
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.
2015-02-17 | E-GEOD-52520 | biostudies-arrayexpress
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.
2015-02-17 | GSE52520 | GEO
Project description:C2H2 zinc fingers (C2H2-ZFs) are the most prevalent type of vertebrate DNA-binding domain, and typically appear in tandem arrays (ZFAs), with sequential C2H2-ZFs each contacting 3 (or more) sequential bases. C2H2-ZFs can be assembled in a modular fashion, providing one explanation for their remarkable evolutionary success. Given a set of modules with defined 3-base specificities, modular assembly also presents a way to construct artificial proteins with specific DNA-binding preferences. However, a recent survey of a large number of three-finger ZFAs engineered by modular assembly reported high failure rates (~70%), casting doubt on the generality of modular assembly. Here, we used protein-binding microarrays to analyze 28 ZFAs that failed in the aforementioned study. Most (17) preferred specific sequences, which in all but one case resembled the intended target sequence. Like natural ZFAs, the engineered ZFAs typically yielded degenerate motifs, binding dozens to hundreds of related individual sequences. Thus, the failure of these proteins in previous assays is not due to lack of sequence-specific DNA-binding activity. Our findings underscore the relevance of individual C2H2-ZF sequence specificities within tandem arrays, and support the general ability of modular assembly to produce ZFAs with sequence-specific DNA-binding activity. Protein binding microarray (PBM) experiments were performed for a set of 20 artificial zinc finger arrays (ZFAs). 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. The method is described in Berger et al., Nature Biotechnology 2006.
2010-12-02 | E-GEOD-25723 | biostudies-arrayexpress
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.
2015-02-17 | E-GEOD-52521 | biostudies-arrayexpress
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.
2015-02-17 | GSE52521 | GEO
Project description:C2H2 zinc fingers (C2H2-ZFs) are the most prevalent type of vertebrate DNA-binding domain, and typically appear in tandem arrays (ZFAs), with sequential C2H2-ZFs each contacting 3 (or more) sequential bases. C2H2-ZFs can be assembled in a modular fashion, providing one explanation for their remarkable evolutionary success. Given a set of modules with defined 3-base specificities, modular assembly also presents a way to construct artificial proteins with specific DNA-binding preferences. However, a recent survey of a large number of three-finger ZFAs engineered by modular assembly reported high failure rates (~70%), casting doubt on the generality of modular assembly. Here, we used protein-binding microarrays to analyze 28 ZFAs that failed in the aforementioned study. Most (17) preferred specific sequences, which in all but one case resembled the intended target sequence. Like natural ZFAs, the engineered ZFAs typically yielded degenerate motifs, binding dozens to hundreds of related individual sequences. Thus, the failure of these proteins in previous assays is not due to lack of sequence-specific DNA-binding activity. Our findings underscore the relevance of individual C2H2-ZF sequence specificities within tandem arrays, and support the general ability of modular assembly to produce ZFAs with sequence-specific DNA-binding activity.
2010-12-02 | GSE25723 | GEO
Project description:With over 700 members, the Cys2-His2 zinc finger proteins (C2H2-ZNFs) constitute the largest class of DNA-binding transcription factors (TFs), yet remain largely uncharacterized1. Although certain family members, including GTF3A, WT1 and CTCF, have been shown to bind both DNA and RNA2,3, the extent to which C2H2-ZNFs interact with – and regulate – RNA-associated processes is not known. Here, we utilized UV crosslinking and immunoprecipitation (CLIP) and found that 148 of 150 C2H2-ZNFs that we analyzed bound directly to RNA in human cells. By integrating data from new individual nucleotide resolution RNA-binding maps for 50 of these C2H2-ZNFs with data from chromatin immunoprecipitation sequencing (ChIP-seq), protein-protein interaction, and RNA-seq experiments4, we observe that the RNA-binding profiles of C2H2-ZNFs are generally distinct from their DNA-binding preferences and that they regulate a variety of post-transcriptional processes, including pre-mRNA splicing, cleavage and polyadenylation, and m6A modification of mRNA. Our results thus substantially expand the cellular repertoire of RBPs by systematically defining RNA targets and regulatory roles of C2H2-ZNFs, and they provide a valuable resource for elucidating crosstalk between transcriptional and post-transcriptional regulatory programs.
2024-09-09 | GSE230845 | GEO
Project description:With over 700 members, the Cys2-His2 zinc finger proteins (C2H2-ZNFs) constitute the largest class of DNA-binding transcription factors (TFs), yet remain largely uncharacterized1. Although certain family members, including GTF3A, WT1 and CTCF, have been shown to bind both DNA and RNA2,3, the extent to which C2H2-ZNFs interact with – and regulate – RNA-associated processes is not known. Here, we utilized UV crosslinking and immunoprecipitation (CLIP) and found that 148 of 150 C2H2-ZNFs that we analyzed bound directly to RNA in human cells. By integrating data from new individual nucleotide resolution RNA-binding maps for 50 of these C2H2-ZNFs with data from chromatin immunoprecipitation sequencing (ChIP-seq), protein-protein interaction, and RNA-seq experiments4, we observe that the RNA-binding profiles of C2H2-ZNFs are generally distinct from their DNA-binding preferences and that they regulate a variety of post-transcriptional processes, including pre-mRNA splicing, cleavage and polyadenylation, and m6A modification of mRNA. Our results thus substantially expand the cellular repertoire of RBPs by systematically defining RNA targets and regulatory roles of C2H2-ZNFs, and they provide a valuable resource for elucidating crosstalk between transcriptional and post-transcriptional regulatory programs.
2024-09-09 | GSE230844 | GEO
Project description:We have identified protein protein interactions of 118 C2H2 zinc finger proteins by AP-MS using GFP-tagged constructs in order to understand their role in transcription regulation.
2016-12-09 | PXD003431 | Pride