Expansion of GA dinucleotide repeats increases the density of CLAMP binding sites on the X-chromosome to promote Drosophila dosage compensation
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ABSTRACT:
ORGANISM(S): Drosophila melanogaster
SUBMITTER: Michael Tolstorukov
PROVIDER: E-GEOD-83444 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:To investigate the DNA binding specificity of the CLAMP protein, we have designed a custom PBM to interrogate the binding of CLAMP to DNA sequences extracted from the Drosophila melanogaster genome. Specific regions were extracted based on ChIP-seq data, motif occurence and proximity to gene transcription start sites. N-terminal GST-tagged protein samples were made for the C-terminal four and six zinc finger portions of the protein CLAMP; samples were made by in vitro transcription translation (IVT). IVT reaction mixtures for the two CLAMP constructs were applied directly to the PBM microarray and incubated for 1hour. Microarray-bound protein was fluorescently labeled using Alexa488-conjugated antibodies targeting GST, and the microarray was scanned in using a standard microarray scanner. Median fluorescence intensity over eight replicate probes was reported for each unique DNA sequence on the microarray.
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2011-09-14 | E-GEOD-31007 | biostudies-arrayexpress
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
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2011-09-14 | E-GEOD-30992 | biostudies-arrayexpress
Project description:The DNA sequence preferences of the vast majority of eukaryotic transcription factors (TFs) are unknown. Using an approach designed to broadly sample both DNA-binding domain types and eukaryotic clades, we have determined DNA-binding motifs for 1,033 TFs from 131 diverse eukaryotes, encompassing 54 domain types. Closely related orthologs and paralogs typically have very similar sequence preferences; this property allows inference of motifs for roughly one third of the 166,851 known or predicted eukaryotic TFs. While the origins of most motifs can be dated to hundreds of millions of years ago, we also characterize more recent TF expansions. Sequences matching the motifs are enriched upstream of TSS in most eukaryotic lineages, and at informative eQTL SNPs in Arabidopsis promoters, demonstrating their utility in mapping transcriptional networks. The motifs are housed at http://cisbp.ccbr.utoronto.ca Protein binding microarray (PBM) experiments were performed for a set of 1048 diverse eukaryotic 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.
2014-08-01 | E-GEOD-53348 | 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: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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