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Our aim was to identify genes whose expression is modulated by a light pulse during
early night and explore their functional significance. We focused on the transcriptional
response to early night stimulus (delay phase shift), as it generally induces a more
robust response than the advanced phase shift in the late night 28]. We identified 200 genes for which transcript levels were significantly altered in
response to a light pulse, at a false discovery rate cut-off of 10 % (Fig. 1).

Informed by our GO enrichment analysis, we characterised the roles of some genes associated
with chromatin remodelling, ion channel activity, and cellular communication. Previous
studies, mostly in mice, have revealed the role of histone methylation, acetylation,
and deacetylation in the circadian clock 30], 31]. Light stimulation modulates histone remodelling in the mouse suprachiasmatic nucleus
32] by inducing phosphorylation of Ser-10 in H3 and acetylation at the promoters of mPer1 or mPer233]. We noted that light pulses downregulate the expression of ej and trithorax in the fly brain (Table 1). Both genes are known to drive histone acetylation and H3K4 methylation, a remodelling
linked with gene activation. Another gene of interest, Su(var)3-9, is upregulated in response to light and is known to be associated with gene suppression
34]. NEJ was previously shown to act as a transcription co-activator via its histone
acetyltransferase (HAT) activity 35], 36], which physically interacts with CLK and CLK-CYC through two of its binding sites
35]. If NEJ binding is required for activation of E-box containing clock genes, then
its downregulation by a light pulse as evident from our data would result in attenuation
of the circadian cycle and a delay. Trithorax, which was also downregulated in our
data, interacts with NEJ to prevent Polycomb-mediated gene silencing by inhibiting
H3Lys 27 trimethylation 37]. Indeed, our circadian behaviour results confirmed that changes in the expression
of trithorax and Su(var)3-9 affect circadian light sensitivity and the phase of rhythmic activities. Our data
also suggest that histone remodelling, as in mammals, is involved in the dipteran
clock light response, possibly by interacting with the CLK-CYC complex.

Another theme that was apparent among the enriched functions of light-induced transcripts
related to cellular communication (ion channel transport, synaptic organisation, intracellular
signalling cascade). For instance, the predicted molecular function of CG11155 is ionotropic glutamate receptor activity, which mediates excitatory synaptic transmission.
Glutamate has been previously shown to be essential for circadian light responses
in mammals 22], 38], so perhaps downregulating CG11155 by light stimulation reduced the clock’s light sensitivity by influencing glutamate
activity in the fly. Nervana1 (rv1), another gene in this GO term, plays a critical role in regulating intracellular
Ca
²⁺
levels via the Na
⁺
/Ca
²⁺
exchange mechanism 39]. By downregulating rv1 in the clock neurons, the flies’ responses to light were significantly reduced, and
about 80 % of them maintained rhythmic activity in the LL condition. Interestingly,
the other subunit of rv2 was also downregulated in response to light in this study, though it was not selected
for further analysis. This again highlights a role for intracellular signalling and
ion exchange in the clock’s light response. Interestingly, a recent imaging study
of whole-brain explant cultures 40] reveals that a light pulse causes rapid desynchrony among clock cells, followed by
gradual emergence of synchrony (‘phase retuning’). These findings underscore the importance
of cellular communication genes in the light response.

The roles of the genes that we identified, such as Nf1, rv1, and CalX in Ca
²⁺
regulation, underscore the role of this pathway in the light response. This role was
previously demonstrated in light-induced phase shifts in vertebrates 41]. The substantial rhythmicity in LL of rv1 and Nf1 RNAi flies (81 % and 64 %, respectively) suggests that these are important loci for circadian
light input. In general, these findings reinforce the Njus-Sulzman-Hastings membrane
model of the circadian clock 42], in which feedback interactions between membrane ion transport systems and ion concentration
gradients modulate cell excitability and drive circadian oscillations.

Because we have profiled fly heads, our survey largely represents transcriptional
changes in the compound eyes, given their large proportion of the volume of the fly’s
head. This approach allows our data to be compared with the early circadian-clock
studies, which also profiled fly heads 43]–47]. However, many light-responsive DEGs may be expressed in non-clock cells. Additionally,
we used the timGal4 driver in our functional assays. This driver is expressed both
in clock neurons in the brain and in photoreceptors in the eyes 48]. Consequently, the effects that we see are mediated either via the eyes, via clock
cells, or via both. However, three DEGs (per, Kr-h1, and Thor) are known to be enriched in clock cells 49], 50]. Furthermore, the lack of behavioural effect of genes that change in expression on
early-light response might be due to their expression in non-TIM cells. Future experiments
using techniques for profiling specific clock neurons 49], 51] would be valuable for identifying light input pathways within the clock.