To calculate the E2F motif enrichment in the E2Fa-bound regions (Fig. In a classical ChIP experiment, DNA fragments associated with a specific protein are enriched. DNA-binding protein complexes are reversibly cross linked with formaldehyde, the chromatin is usually fragmented, and the DNA portion that interacts with the TF of interest is usually isolated by immunoprecipitation with a specific antibody. Finally, DNA sequences associated with the precipitated protein can be recognized by hybridization to tiling arrays (ChIP-chip) or by direct high-throughput sequencing (ChIP-seq; Kim and Ren, 2006; Park, 2009). In spite of its power, standard ChIP has experimental boundaries. Its main shortcoming, especially in the case of genome-wide applications, is the overall inefficiency of ChIP enrichment. This drawback, which is a result of cross linking and compromises the identification of low-abundance TF-DNA interactions, necessitates the requirement for large cell figures and the need for high-quality antibodies. The hurdle to allow ChIP application to small cell numbers is mainly being resolved at the level of the chromatin isolation process (ONeill et al., 2006; Acevedo et al., 2007; Dahl and Collas, 2008; Wu et al., 2009). Recent adaptations in deep sequencing and library preparation, allowing compatibility with smaller DNA quantities, provide option solutions (Goren et al., 2010; Adli and Bernstein, 2011; Bowman et al., 2013). The challenge of specific antibody requirement may be circumvented by epitope/affinity tagging of the TF (Harbison et al., 2004; Zhang et al., 2008). Reports of genome-wide ChIP studies of herb TFs, in comparison with other eukaryotic systems, are still lagging behind. Although plant-specific ChIP protocols have been successfully developed (Bowler et al., 2004; Gendrel et al., 2005; Saleh et al., 2008; Kaufmann et al., 2010), herb features, such as rigid cell walls, Cardiolipin large vacuoles, chloroplasts, and the paucity of nuclei in some tissues, combined with TF tissue and target specificities all challenge TF-DNA enrichment. Here, we statement a generic option ChIP protocol relying on Arabidopsis (fusion under the control of the constitutive cauliflower mosaic computer virus promoter in Arabidopsis cell suspension cultures. expression in transgenic lines was determined by protein-blot analysis with both an anti-His antibody and an anti-E2Fa antibody (Takahashi et al., 2008; Fig. 1A). Although constitutively overexpressed, E2Fa-HBH protein accumulation was close to the endogenous E2Fa protein level (Fig. 1A). Comparable accumulation levels for other and control promoter regions. Error bars show sd (= 3). Eventually, TChAP was performed Cardiolipin by combining the HBH purification method of Tardiff et al. (2007) with ChIP protocol de-cross linking, deproteinization, and DNA purification (Kim et al., 2008). Briefly, E2Fa-HBH and its cross-linked proteins and DNA were first bound on nickel-nitrilotriacetic acid Cardiolipin agarose (NiNTA) beads, specifically eluted with imidazole, and then bound to streptavidin-Sepharose under high-stringency conditions. E2Fa-HBH-DNA complexes were subsequently eluted and reverse cross linked, and the DNA was purified. Final evaluation of the TChAP process occurred by analyzing the TChAP DNA sample by quantitative PCR. The proximal promoters of the well-known E2Fa target genes (((and promoter E2Fa-binding sites confirmed this asset of tandem chromatin affinity purification sequencing (TChAP-seq; Fig. 3B). As a consequence, both E2Fa-regulated genes (163 genes, 2.4-fold enrichment, = 3.9e-26) and E2F motif genes (439 genes, 5.7-fold enrichment, = 5.4e-229) were significantly present among the TChAP-specific majority genes (Fig. 3A). In comparison, the ChIP- and ChAP-specific bound genes were not enriched for E2Fa-regulated genes (for ChIP, one gene, 5-fold, = 0.17; for ChAP, eight genes, 1-fold, = 0.47), and only the ChAP-specific genes were significantly enriched for E2F motif genes (for ChIP, two genes, 9-fold, = 0.017; for ChAP, 19 genes, 2.2-fold, = 8.7e-4). Moreover, TCEB1L plotting the ChIP, ChAP, and TChAP majority peaks/genes according to descending MACS confidence scores and measuring the portion of E2Fa-regulated and E2F motif genes revealed that, despite the pattern that lower confidence levels correlated with lower overlap scores, all peaks/genes retained enrichment for these gene units (Fig. 3C). These properties, together with the global higher MACS scores Cardiolipin of TChAP-identified peaks/genes (Table I; Fig. 4A), suggest that the additional E2F locations discovered with TChAP-seq are true, possibly less occupied, binding regions. As a validation, E2Fa binding specificity to 20 TChAP-seq uniquely recognized known and putative new E2Fa target regions/genes, sampled throughout the confidence score distribution, was evaluated by quantitative PCR (qPCR) after ChIP, ChAP, and TChAP (Supplemental Table S6). For all those, 12, and eight regions, a significant enrichment (more than 2-fold) was detected with TChAP,.
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