Prediction of bipartite transcriptional regulatory elements using transcriptome data of Arabidopsis

Abstract

In our previous study, a methodology was established to predict transcriptional regulatory elements in promoter sequences using transcriptome data based on a frequency comparison of octamers. Some transcription factors, including the NAC family, cannot be covered by this method because their binding sequences have non-specific spacers in the middle of the two binding sites. In order to remove this blind spot in promoter prediction, we have extended our analysis by including bipartite octamers that are composed of ‘4 bases—a spacer with a flexible length—4 bases’. 8,044 pre-selected bipartite octamers, which had an overrepresentation of specific spacer lengths in promoter sequences and sequences related to core elements removed, were subjected to frequency comparison analysis. Prediction of ER stress-responsive elements in the BiP/BiPL promoter and an ANAC017 target sequence resulted in precise detection of true positives, judged by functional analyses of a reported article and our own in vitro protein–DNA binding assays. These results demonstrate that incorporation of bipartite octamers with continuous ones improves promoter prediction significantly.

Transcriptome Analysis and Identification of a Transcriptional Regulatory Network in the Response to H2O2

“…Target TFs of this starter are expected to be regulated at the level of gene expression, and of 60 TFs, ATERF71 (AT2G47520) is reported as a direct target of ANAC017. This is based on the observation of reduced expression in anac017 and binding of ANAC017 protein to the promoter region of ATERF71 (Ng et al, 2013;Yamamoto et al, 2017). Unfortunately, we failed to identify any downstream targets of ANAC017, and thus could not add ANAC017 and ATERF71 to Table 3, leaving starters of the network shown in Figure 7 unidentified.…”

“…Putative regulatory sequences were extracted by evaluation of relative appearance ratio (RAR) for each octamer sequence, which is a ratio of appearance of an octamer in a promoter set of a responsive gene group to that in total promoters of the genome. Continuous octamers (Yamamoto et al, 2011) and also bipartite octamers with a spacer, which have a potential of cis-elements in Arabidopsis (Yamamoto et al, 2017), were applied for the extraction. Octamers with high RAR values (RAR $ 3) were extracted as putative ciselements.…”

“…In this report, we have developed a strategy for medium-scale identification of direct regulation in a network using a combination of time-course transcriptome analyses, identification of in vivo regulation of a TF by a regulating TF using public and private transcriptome data of the mutants and/or overexpressors of the regulating factor, and identification of in vitro binding of the regulating factor protein to the promoter region of the regulated TF. Binding analysis between a TF and its putative target site in the promoter region was oriented by promoter prediction based on transcriptome data of mutants and/or overexpressors, and the success of this strategy is supported by our accurate and sensitive promoter prediction method (Yamamoto et al, 2011(Yamamoto et al, , 2017. This study provides insights into the transcriptional regulatory network for H 2 O 2 signaling and also a strategy for medium-scale determination of protein-DNA interactions in a costeffective manner.…”

“…Within the H 2 O 2 regulatory network made of TFs, several examples of direct regulation have been reported: activation of ATERF71 (ARABIDOPSIS THALI-ANA ETHYLENE RESPONSE FACTOR 71), CRF5 and CRF6 (CYTOKININ RESPONSE FACTOR 5 and 6), and ATNFXL1 (ARABIDOPSIS THALIANA NF-X-LIKE 1) by ANAC017 (ARABIDOPSIS NAC DOMAIN CON-TAINING PROTEIN 017; Ng et al, 2013;Yamamoto et al, 2017), and also activation of RAP2.6 (RELATED TO APETALA2 6) by RRTF1 (REDOX RESPONSIVE TRANSCRIPTION FACTOR 1; Matsuo et al, 2015). These studies provide glimpses of a large regulatory network.…”