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).