Project description:As 5-15% of higher eukaryotes genes are transcription factors (TFs), the lack of transcription factor binding site (TFBS) information for most factors in most organisms limits the study of gene regulation. Here we describe a next-generation sequencing method, DNA affinity purification (DAP-Seq), an in vitro gDNA/TF interaction assay that produces whole-genome TFBS annotation for any factor from any organism. Like ChIP-Seq, DAP-Seq resolves TFBS as discrete peaks at genomic locations which allows for accurate motif prediction direct assignment of functionally relevant target genes, and shows better overlap with ChIP-Seq peaks than indirect motif assignment approaches. We applied DAP-Seq to a set of 50 transcription factors in eight Arabidopsis thaliana and one Zea Mays families to gain novel biological insight into TFBS architectures, functions, evolution and methylation-sensitivity. Overall, DAP-Seq offers a low-cost high-throughput approach to identify TFBS in native sequence context for any organism complete with all DNA chemical modifications.
Project description:In this study, we use DNA affinity purification sequencing to identiy genome-wide binding of LFY transcription factor, a master regulator of flower development in Arabidopsis. We generated two sets of data, one using genomic DNA from plant tissue, thus retain DNA methylation, as probe for DNA affinity purification (DAP-seq dataset), and the other using PCR amplified genomic DNA (without DNA methylation; AmpDAP-seq dataset).
2021-03-25 | GSE160013 | GEO
Project description:DNA Affinity Purification (DAP) Sequencing Method Development
Project description:We focused on building models that incorporated transcription factor (TF)-DNA interaction data for 12 members of the Auxin Response Factor (ARF) family from soybean as assessed by DNA Affinity Purification and sequencing (DAP-seq).
Project description:To better understand FvRIF-mediated transcriptional regulation of fruit ripening, we performed DNA affinity purification sequencing (DAP-seq) to unravel FvRIF binding sites at the genome level. For DAP-seq analysis, the recombinant FvRIF fusion protein was used to purify the sheared genomic DNA of strawberry fruits. Two independent biological replicates of DAP-seq and DNA ‘input’ negative control libraries were prepared and submitted for deep sequencing.
Project description:We have developed a high throughput, next-generation DNA sequencing assay for rapid transcription factor binding site (TFBS) discovery in a genomic context. DNA affinity purification sequencing (DAP-seq), which uses affinity-purified transcription factors (TFs) to capture genomic DNA fragments, was applied to all 1,725 Arabidopsis thaliana TFs. High confidence TFBS motifs for 529 TFs and genome-wide enrichment maps for 349 factors were identified. In total,~ 2.7 million TFBS were identified which predict thousands of TF target genes enriched for known and novel functions.. Comparison of TF-binding using cytosine-methylated and -unmethylated genomic DNA revealed a 2-50 fold inhibition at methylated motifs for ~82% (264) of factors tested while 4.6% (15) showed stronger binding to methylated motifs. Finally, we describe how binding of Arabidopsis and maize Auxin Response Factors (ARFs) at phased motif repeats is highly enriched at ARF target gene promoters and how this architecture may allow for stabilization of dimers/multimers.
Project description:To verify genes that are directly regulated by BysR, we used DNA affinity purification sequencing analysis (DAP-seq) for genome-wide recognition of BysR binding sites in vitro. The HALO-fusion BysR protein was successfully expressed and purified. After affinity purification and sequencing, at least 22 million double-end reads per sample were generated and with > 99 % of reads uniquely mapped to the JP2-270 genome. A total of 470 enriched common peaks of two replicates with –log10(P-value) ≥ 2 were called . The mean width of DAP-seq peaks was < 1,000. In total, 367 (78%) of these peaks were found to locate in the -1 kbp to 1 kbp regions by the analysis of peak summit positions relative to the start codons of JP2-270 open reading frames.
