At the time of the Locke (2006) model, little was known about regulation of the clock at a molecular level. However, it was clear from both mathematical models and experimental data (Section 1.1) that LHY and CCA1 were central to the clock oscillator. Understanding the transcriptional regulation of these genes by other clock components was therefore fundamental to elucidating the molecular mechanisms by which the clock oscillator regulates itself. LHY and CCA1 were also both known to be regulated by light, so were likely to be important in the integration of light signals into the clock and therefore in light-mediated entrainment of the circadian clock. The regulation of LHY and CCA1 transcription was therefore considered an important area of study. The regulatory structure of the LHY promoter was examined by Spensley et al. (2009) (Section 1.2).
1.2.1 - Regulatory Regions of the LHY Promoter
Spensley et al. (2009) demonstrated through a 5’ deletion analysis of the LHY
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site (referred to as the -957/+1 LHY promoter) was sufficient for circadian and diurnal expression patterns of a luciferase reporter gene.
From this 5’ deletion analysis, it was found that the LHY promoter region from 957 to 847 basepairs upstream of the translational start site (the -957/-847 promoter region) was essential for neither expression nor rhythmicity of the LHY promoter. Two conclusions could be drawn from this result: (1) that a positive regulator must act downstream of position -847 on the promoter in order to enable expression, and (2) that the -847/+1 promoter region (starting 847 basepairs upstream of the translational start site) was responsible for maintaining rhythmic expression.
The -957/-847 region of the LHY promoter was found to be responsible for regulating the phase of LHY expression in a photoperiodic-dependent manner. When entrained to either short days (8L:16D) or 12L:12D, the -847/+1 LHY promoter exhibited an advanced free-running phase of expression compared to the -957/+1 promoter. This suggested the presence of a repressor within the -957/-847 region acting to repress transcription in the late subjective night. However, this phase advance was not seen when plants were entrained to long days (16L:8D), indicating that the mechanism of the control of phase was more complex than a single repressor. It was therefore suggested that the -957/-847 region of the promoter might be targeted by waves of both transcriptional activators and repressors to regulate the phase of LHY expression.
The -957/+1 LHY promoter was therefore thought to consist of two broad functional regions, with the -957/-847 promoter region modifying the phase of LHY expression,
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and the -847/+1 promoter region mediating both rhythmicity and activation of LHY
expression.
1.2.2 - Functional Roles for Evolutionarily Conserved Promoter Motifs
Several evolutionarily conserved sequence motifs were identified within the -957/+1
LHY promoter using a comparative genomics technique (Picot et al., 2010), including a G-box, five novel AAAAA (5A) motifs, a CT-rich region and another three putative regulatory motifs (New Elements 1, 2 and 3) (Figure 1.2) (Spensley et al., 2009).
Figure 1.2: Regulatory regions and motifs of the LHY promoter. The distal promoter region (-957/- 847bp upstream of translational start site) can modify the phase of LHY:LUC reporter expression, and the proximal promoter region (-847/+1) is sufficient for rhythmic expression of LHY:LUC reporter constructs. The 5A motifs are involved in activation of expression. The G-box may be targeted for both activation and repression. The function of Elements 1-3 and the CT-rich region is unknown.
Such conservation across species is highly suggestive of a functional role within the promoter, since for the sequence to be preserved there must be a strong functional imperative to protect against randomly accrued mutations within the motif sequence. The individual contributions of these conserved promoter motifs to the mediation of promoter regulation were investigated by Spensley et al. (2009) using luciferase
assays. Conserved motifs within the LHY promoter were individually mutated, and each mutated promoter cloned into a luciferase reporter vector. In planta expression of each LHY:LUC construct was then tracked and analysed over a period of days under differing environmental conditions. Two types of data were extracted from
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these results: (1) mean expression levels, where absolute reporter gene expression level was averaged over several circadian cycles, and (2) expression patterns, where normalised expression levels were tracked over time to assess rhythmicity.
1.2.2.1 - The G-box
The G-box core hexamer (CACGTG) was already known to play a role in mediating responses to light (Martinez-Garcia et al., 2000), and its presence in this -957/-847 promoter region made it a prime target for further investigation. It was found by Spensley et al. (2009) using a series of point mutations in LHY reporter constructs that the amplitude of LHY’s expression was highly influenced by the flanking nucleotides
around the G-box hexamer. The wider flanking sequences around the G-box were also shown to affect the binding of protein complexes to the promoter by electrophoretic mobility shift assays (EMSAs), suggesting that the promoter context of the G-box may be critical to its regulatory effects on the expression of LHY. The G-box was also implicated as a target for both activators and repressors of LHY expression, since mutations in the G-box flanking bases resulted in both a two-fold decrease in expression level and a reduction in the amplitude of oscillations, indicating an activating role for the G-box, and also a subtle broadening of the peak in constant light, indicating that the G-box moderates phase by repressing expression before and after the transcriptional peak at dawn.
1.2.2.2 - The 5A Motifs
Through a series of luciferase assays using mutated LHY promoter constructs, it was discovered that some or all of the 5A motifs were involved in the activation of LHY
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differing intervals along the promoter between positions -957 and -779 (Figure 1.2). They could be split into two distinct groups: the three proximal 5A motifs, clustered together in the -847/+1 promoter region between positions -779 and -805 (779bp and 805bp upstream of the translational start site), and the two distal 5A motifs which flank the G-box and Element 1 within the -957/-847 promoter region.
To uncover the role of the 5A motifs, reporter constructs were generated by Spensley et al. (2009), with a luciferase gene fused downstream of -957/+1 or -847/+1 promoters containing mutations in either the proximal or distal 5A motifs. Luciferase
assays were performed in wild-type plants with the following constructs: -957 1,2m LHY:LUC, with the two distal 5A motifs (5A1 and 5A2, collectively termed 5A12) disrupted in the -957/+1 promoter, and -957 345m LHY:LUC and -847 345m LHY:LUC, with the three proximal 5A motifs (5A3, 5A4 and 5A5,collectively termed 5A345) mutated in the -957/+1 and -847/+1 LHY promoters respectively.
Luciferase assays using these constructs found that mutation of the proximal 5A motifs had no significant effect on expression of the -847/+1 promoter. However, mutations of either the distal or proximal 5A motifs in the context of the -957/+1 promoter were found to cause a two- to three-fold reduction in expression levels, a reduction in amplitude of expression and a broadening of the peak in constant light. The 5A motifs were therefore postulated to mediate activation of LHY expression. However, it remained unknown what the effect of losing all of the 5A motifs might be on the promoter’s expression.
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It was also implied from these results that the action of the 5A motifs may require the presence of one or more elements within the -957/-847 promoter region, since the effects on expression levels of mutating the proximal 5A motifs in the -957/+1 promoter were not seen when they were mutated in the -847/+1 promoter. The G-box was tentatively suggested as a possible focus of this interaction with the 5A motifs, since it had been shown through electrophoretic mobility shift assays (EMSAs) that protein binding at the G-box was negatively affected by unlabelled oligonucleotide competitors containing either of the two distal 5A motifs which flank the G-box. However, this was not conclusive evidence for a regulatory interaction between these motifs, so this proposed interaction requires further investigation.