Synthetic construct
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ABSTRACT:
PROVIDER: PRJNA229160 | ENA |
REPOSITORIES: ENA
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Project description:Highly parallel identification of sequence preferences for eukaryotic C2H2 zinc finger domains using a bacterial 1-hybrid assay
| PRJNA229157 | ENA
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:Sequence specificity is obtained from the majority of modular C2H2 zinc-finger arrays
| PRJNA133853 | ENA
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). 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:This SuperSeries is composed of the SubSeries listed below. Cys2-His2 zinc finger (C2H2-ZF) proteins represent the largest class of putative human transcription factors (TFs). However, it is unknown whether most C2H2-ZFs even bind DNA, or what sequences they bind. Using a combination of bacterial one-hybrid (B1H) assays, protein-binding microarrays (PBMs), and ChIP-seq, we have found that most natural C2H2-ZFs bind DNA both in vitro and in vivo. This SuperSeries contains the data for identification of C2H2-ZF binding preferences using these three approaches. Refer to individual Series
2015-02-17 | E-GEOD-52523 | biostudies-arrayexpress
Project description:The nematode Caenorhabditis elegans is a powerful model for studying gene regulation, as it has a compact genome and a wealth of genomic tools. However, identification of regulatory elements has been hampered by the fact that DNA binding motifs are known for only 71 (9%) of the estimated 763 high-confidence sequence-specific transcription factors (TFs). To address this problem, we performed protein binding microarray (PBM) experiments on representatives of canonical TF families in the C. elegans TF repertoire, obtaining motifs for 129 distinct TFs. Moreover, we can infer motifs for 97 additional TFs that have DNA binding domains that are very similar to those already characterized, resulting in a total coverage of binding specificities for almost 40% of the C. elegans TF repertoire. These data highlight the diversification of binding motifs for the nuclear hormone receptor (NHR) and C2H2 zinc finger families, and reveal unexpected diversity of motifs for others, including the T-box and DM families. Enrichment of motifs in the promoters of functionally related genes is consistent with known biology in many cases, and also identifies putative new regulatory roles for poorly characterized TFs. The motifs are available at http:// http://cisbp.ccbr.utoronto.ca. Protein binding microarray (PBM) experiments were performed for a set of 129 diverse C. elegans transcription factors. 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.
2015-04-23 | E-GEOD-65719 | biostudies-arrayexpress
Project description:C2H2 zinc finger proteins represent the largest and most enigmatic class of human transcription factors. Their C2H2 arrays are highly variable, indicating that most will have unique DNA binding motifs. However, most of the binding motifs have not been directly determined. We have determined the binding sites and motifs of 119 C2H2 zinc finger proteins and the expression pattern of 80 cell lines overexpressing C2H2 zinc finger proteins in order to study the role of C2H2 zinc finger proteins in gene regulation.
2016-10-25 | GSE76495 | GEO
Project description:C2H2 zinc finger proteins represent the largest and most enigmatic class of human transcription factors. Their C2H2 arrays are highly variable, indicating that most will have unique DNA binding motifs. However, most of the binding motifs have not been directly determined. We have determined the binding sites and motifs of 119 C2H2 zinc finger proteins and the expression pattern of 80 cell lines overexpressing C2H2 zinc finger proteins in order to study the role of C2H2 zinc finger proteins in gene regulation. We expressed GFP-tagged C2H2-ZF proteins in stable transgenic HEK293 cells. Total RNA was isolated using Trizol and sequencing libraries were constructed using TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold or TruSeq RNA Library Preparation Kit v2.
2016-10-25 | E-GEOD-76495 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below. Cys2-His2 zinc finger (C2H2-ZF) proteins represent the largest class of putative human transcription factors (TFs). However, it is unknown whether most C2H2-ZFs even bind DNA, or what sequences they bind. Using a combination of bacterial one-hybrid (B1H) assays, protein-binding microarrays (PBMs), and ChIP-seq, we have found that most natural C2H2-ZFs bind DNA both in vitro and in vivo. This SuperSeries contains the data for identification of C2H2-ZF binding preferences using these three approaches.
2015-02-17 | GSE52523 | GEO