The complexity in the long BDP mechanism
The TATA box is a fundamental promoter element within eukaryotes, which
is crucial in initiating transcription for all three RNA polymerases.
The canonical TATA box sequence is typically recognized as
5’-TATAWAW-3’(Kwak et al. , 2013). However, a bidirectional TATA
box (5’-TATATAT-3’) has been identified to possess transcriptional
capabilities in both directions, demonstrating heightened
transcriptional activity(Xu, Thali and Schaffner, 1991). Analyzing split
tests for BDPs lacks specific guiding principles. In this study, we
employed the TATA box as a pivotal marker for segmenting the
PPik-H4 . The TATA box sequence at 462-469 bp in
the Pik-H4 promoter is a bidirectional TATA box, comprising two
bidirectional TATA boxes situated on both the sense strand and the
antisense strand, respectively (Figure 2E). Mutation of this segment
resulted in a significant reduction in downstream gene expression on
both sides of the promoter. Examining the promoter activity of a 100 bp
sequence encompassing the integrated bidirectional TATA box, along with
an adjacent bidirectional TATA box, revealed that the sequence indeed
possessed bidirectional activity. Nevertheless, it could not fully
restore the transcriptional activity of Pik1-H4and Pik2-H4 in both directions back to the levels
of NP (Figure 2D-F, BT1). Moreover, the recovery of expression intensity
in downstream reporter genes was observed after adding the TSSs for both
genes (Figure 2D-F, BT2), indicating that the 100 bp sequence primarily
functions as a bidirectional regulatory element, with TSSs being
indispensable for gene expression.
Transcriptional regulation is a complex process involving a multitude of
factors, including TFBSs, cis elements, SNPs(Liu et al. ,
2019), neighboring transcription units(Lee et al. , 2014) and
various epigenetic modifications (Chen et al., 2014). In contrast to the
human genome, where approximately 67% of BDPs are shorter than 300 bp
(Trinklein et al., 2004), plant BDPs tend to be longer (Krom and
Ramakrishna, 2008), introducing greater complexity to their regulatory
mechanisms. Some reports on split BDPs focus on segments within 1500 bp
(Banerjee et al., 2013; Rao and Virupapuram, 2021), and identify crucial
promoter regions that contribute to the basal expression of BDGs under
specific experimental conditions. However, these studies often overlook
the intricate cis regulatory elements for gene regulation or
specificity. In our research, even by combining the minimal core
promoter regions of Pik1-H4 andPik2-H4 , we could not achieve BDP activity
equivalent to NP levels (Figure 2A-C, B3). Intriguingly, this
combination even increased GFP expression, indicating the intricate
interplay of cis elements within PPik-H4 .
Given that Pik BDPs encompass lengths of 2492 bp or 2542 bp,
further assays to split and analyze promoters are necessary to unravel
the underlying regulatory mechanisms of bidirectional NLR gene pairs.
Unveiling the functionality of BDPs holds significant implications for
synthetic biology, but it’s evident that more extensive research is
needed to understand these processes comprehensively.