Functional analysis of Pik-H4 promoter
To identify the crucial region of PPik-H4 , PPik-H4 was fused with RFP(Pik1-H4 direction) and GFP(Pik2-H4 direction) for further study (Figure S2A). These constructs were subsequently introduced into rice protoplasts and tobacco mesophyll cells via plasmid transformation, and the bidirectional promoter activity was observed in both cell types (Figure S2B). Interestingly, the fluorescence intensities of RFP and GFP exhibited synchronized trends and intensities (Figure S2C). Consequently, we focused on conducting promoter activity assays in tobacco mesophyll cells for further investigations.
Given the essential role of the TATA box in eukaryotic RNA polymerase function and transcription specificity, the PPik-H4 was split according to the position of TATA boxes. In the Pik1-H4 direction, the recovery of RFP expression to native promoter (NP) levels occurred until the 7th TATA box (1-7T) was included (Figure S2D), indicating the criticality of the 593-1232 bp region forPik1-H4 transcription. As the following promoter fragments were added, the promoter activity towardsPik1-H4 direction decreased, suggesting that thecis elements in the following region might negatively regulatePik1-H4 expression. Interestingly, all split promoters with no transcription start site (TSS) drove GFP expression at levels similar to the NP.
In the direction of Pik2-H4 , the 2-1T sequence (2016-2492 bp) was sufficient to restore the PPik-H4 activity towardsPik2-H4 direction (Figure S2I). The region spanning from the 5th to the 10thTATA box upstream Pik2-H4 enhanced the GFP expression, suggesting a positive regulatory role inPik2-H4 transcription. Notably, this region overlapped with the critical sequence of Pik1-H4 , establishing PPik-H4 as a bidirectional promoter configuration. In summary, the 593-1232 bp and 2016-2492 bp of PPik-H4 were core regions forPik1-H4 and Pik2-H4expression respectively.
Considering the bidirectional manner and examination of the core promoter region of PPik-H4 , a series of synthetic bidirectional promoters were constructed based on PPik-H4 . Surprisingly, while B1, B2, and B4 promoters failed to restore GFP expression levels (Figure 2C), the B3 promoter, comprising the core promoter regions of bothPik1-H4 and Pik2-H4 , augmented the GFP signal. Our findings underscore the complexity of regulatory interactions among distinct cis elements within the promoter region, particularly when integrating two directional core regions.
A symmetric integrated TATA box, comprising two bidirectional TATA boxes, was identified within PPik-H4 , prompting a functional analysis. Firstly, the 100 bp fragment including three bidirectional TATA boxes (Figure 2B, BT1) conferred promoter activity in both orientations and this transcriptional activity was recovered to NP levels in the presence of TSSs of the downstream genes (BT2). Notably, the loss of function of the integrated bidirectional TATA box by mutating it from 5’-TATATATA-3’ to 5’-AAAAAAAA-3’ (M1) significantly compromised promoter activity in both directions, while the normal bidirectional TATA box (M2, M3) had comparatively milder effects on bidirectional promoter activity. Modifying the regular TATA box into the integrated TATA box (M4, M5) demonstrated that the promoter activity towards the Pik2-H4 direction increased as the integrated TATA box shifted, while the activity of the opposite orientation remained intact. This suggests that the function of the integrated bidirectional TATA box in the Pik2-H4direction may be position-dependent within the Pik-H4 promoter region. Our findings were consistent with a previous report that the symmetry 5’-TATATATA-3’ is highly active in both directions(Xu, Thali and Schaffner, 1991).