Mapping genome-wide binding sites of endosperm specific expression transcription factor O2 using DAP-Seq

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<p indent=0mm>Opaque2 (O2) is an important transcription factor regulating maize endosperm development, and mutation of the wild type O2 gene reduces the amount of zein and increases the lysine contents, thereby, leading to development of opaque and starchy endoplasm commonly referred to as <italic>o</italic>2 mutant. The opaque and starchy nature of the <italic>o</italic>2 mutant endosperm renders the kernels resistance to pests, diseases and mechanical damage caused during transportation besides having high nutritional quality. O2 regulates the genes involved in various biological functions or processes, including the synthesis and metabolism of proteins, carbohydrates and lipids. Previous studies have identified O2 target genes regulating different biological functions. However, the detailed information about the genome-wide analysis of O2 targets genes is insufficient and needs further investigation. Taking advantage of DNA affinity purification sequencing (DAP-Seq) over Chromatin Immunoprecipitation sequencing (ChIP-Seq) being a high throughput and easy approach to capturing whole genome transcription factor and DNA interaction profile, the gene regulatory network of O2 was investigated by coupling with mRNA abundance analyses. Using DAP-Seq, we detected a total of 11385 O2 binding sites throughout the genome. Genome-wide comparison of O2 TF showed that DAP-Seq peaks captured significant fractions of ChIP-Seq peaks (35.9%−50.54%), indicating the potential of DAP-seq to preferentially captures <italic>in vitro</italic> transcription factor binding sites. By integrating previously RNA-Seq analysis of wild type (B73) vs <italic>o</italic>2 mutant endosperm, we identified 317 directly bound target genes of O2 in this study, correlated with 97 target genes detected by ChIP-Seq and 220 additional target genes specifically detected by DAP-Seq. Among the direct target genes detected by DAP-Seq, 260 target genes were directly bound and activated by O2, including 20 zein gene, <italic>cyPPDK1/2</italic>, <italic>NF-YB16</italic> and <italic>ZmNAC111</italic>; and another 57 target genes were directly bound and repressed by O2, such as <italic>bt2</italic>, <italic>GAPDH</italic>, <italic>Orphan 154 </italic>etc. To further gain a comprehensive insight into the functions of the O2-regulated gene network, we performed Gene Ontology (GO) terms significantly enriched among O2 direct target genes using agriGO tool. The results indicated that O2 mainly regulates genes involved in endosperm storage product synthesis and accumulation, directly activates genes encoding storage proteins and enzymes involved in amino acid biosynthesis or metabolism in maize endosperm. Moreover, O2 also regulated genes involved in carbon partitioning between carbohydrates and proteins in maize endosperm. However, 220 O2 binding target gene specifically detected by DAP-Seq were also mainly involved in the glucose metabolism pathway and response to various stresses. To identify cis-regulatory sequences mediating O2 binding, we performed a de novo motif discovery on the O2 binding sites detected by DAP-Seq, using the MEME-ChIP program. We found that the most significant motif containing “CCACGTCA” sequence, the reported O2 cis motif, was enriched in the 400 bp region centered at the summits of peaks for 317 direct O2 target genes. In addition, the common cis-motif also enriched within 400 bp region centered at the summits of peaks for 220 specific binding target genes. The motif was shown to be centrally enriched in the analyzed genomic regions. Taken together, our results provide insight into the complexity of the O2 regulatory network, which validate and supplement the previous research findings.