ERK Signaling Regulates Light-Induced Gene Expression via D-Box Enhancers in a Differential, Wavelength-Dependent Manner

The day-night and seasonal cycles are dominated by regular changes in the intensity as well as spectral composition of sunlight. In aquatic environments the spectrum of sunlight is also strongly affected by the depth and quality of water. During evolution, organisms have adopted various key strategies in order to adapt to these changes, including the development of clocks and photoreceptor mechanisms. These mechanisms enable the detection and anticipation of regular changes in lighting conditions and thereby direct an appropriate physiological response. In teleosts, a growing body of evidence points to most cell types possessing complex photoreceptive systems. However, our understanding of precisely how these systems are regulated and in turn dictate changes in gene expression remains incomplete. In this manuscript we attempt to unravel this complexity by comparing the effects of two specific wavelengths of light upon signal transduction and gene expression regulatory mechanisms in zebrafish cells. We reveal a significant difference in the kinetics of light-induced gene expression upon blue and red light exposure. Importantly, both red and blue light-induced gene expression relies upon D-box enhancer promoter elements. Using pharmacological and genetic approaches we demonstrate that the ERK/MAPK pathway acts as a negative regulator of blue but not red light activated transcription. Thus, we reveal that D-box-driven gene expression is regulated via ERK/MAPK signaling in a strongly wavelength-dependent manner

Differential regulation by blue and red light is mediated through D-box enhancer elements.

<p>(A–F) Real time bioluminescence assays of transfected PAC-2 cells. Each construct is indicated above its respective panel. In each panel relative bioluminescence is plotted on the y-axis and time (hrs) on the x-axis. Each time-point represents the mean of three independent experiments +/− SD. Red, blue and black bars above each panel represent the red light, blue light and dark periods, respectively. For clarity, blue, red and white background shading also indicates the blue, red and dark periods respectively. The results of statistical analysis are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067858#pone.0067858.s001" target="_blank">Figure S1C</a>–E.</p

Blocking of the ERK/MAPK signaling pathway enhances blue light-induced reporter gene expression via D-box elements.

<p>Real time bioluminescence assays of PAC-2 cells transfected with the following constructs: (A) <i>D-boxper2-Luc</i>. (B) <i>D-boxcry1a-Luc</i>. All transfected constructs are in the presence (green traces) or absence (black traces) of 1 µM U0126. The black arrows indicate the start of treatments. In each panel relative bioluminescence is plotted on the y-axis and time (hrs) on the x-axis. Each time-point represents the mean of three independent experiments +/−SD. The results of statistical analysis are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067858#pone.0067858.s001" target="_blank">Figure S1G</a>. Blue, red and black bars above each panel represent the different lighting conditions. For clarity, blue, red and white background also indicates the blue, red and dark periods, respectively. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067858#pone.0067858.s003" target="_blank">Figure S3</a> for further experimental details.</p

Tonic ERK/MAPK signaling down-regulates blue light-induced gene expression.

<p>(A) Graphical representation of relative phospho-ERK (p-ERK/ERK) levels under blue- (blue trace) and red-light (red trace) normalized using total ERK expression. On the y-axis relative phosphorylation levels are plotted with time point 0 min being set arbitrarily as 1. Time (min) is plotted on the x-axis. Each point represents the mean of six independent experiments +/−SD. No significant phospho-ERK induction was observed under either red (t-test p>0.05) or blue light (t-test p>0.05) at any time point analysed. Representative autoradiograph images are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067858#pone.0067858.s002" target="_blank">Figure S2A</a>. (B–D) qRT-PCR analysis of light inducible gene expression in the presence (green traces) or absence (black traces) of U0126 during 12 hours of blue or red light exposure. Cells were treated with either U0126 (1 µM) or DMSO as a control, 1 h before light exposure. Each gene is indicated above its respective panels. Blue and red bars above each panel indicate the wavelengths of light used. Relative mRNA levels (%) are plotted on the y-axes where the highest value measured during each experiment (each panel) is set as 100%. Time (hrs) is plotted on the x-axes. Each point represents the mean of three independent experiments +/−SD. The results of statistical analysis are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067858#pone.0067858.s001" target="_blank">Figure S1F</a>.</p

Activation of light-inducible genes by blue, red and white light.

<p>(A–C) Quantitative RT-PCR analysis (qRT-PCR) of light inducible genes in PAC-2 cells during constant darkness conditions (DD controls, black traces), white (grey traces), blue (blue traces) or red (red traces) light exposure. Total RNA was extracted from cells that were maintained in DD (control) or illuminated with the various light sources for different time periods (0, 1, 2, 3, 4, 6 hrs). Each gene is indicated above its respective panel. Relative mRNA levels (%) are plotted on the y-axis where the highest value measured during each experiment (Blue light exposure for 6 hours) is set as 100%. Time (hrs) is plotted on the x-axes. In each panel, points are plotted as the means of three independent experiments +/−SD. The results of statistical analysis are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067858#pone.0067858.s001" target="_blank">Figure S1A</a>–B.</p

Impact of dominant active and dominant negative forms of MEK and ERK on D-box driven expression.

<p>Real time bioluminescence assays of PAC-2 cells co-transfected with <i>D-boxcry1a-Luc</i> and either <i>dA-MEK</i> (A), <i>dA-ERK</i> (B), <i>dN-ERK</i> (C) (green traces) or an empty expression vector control (A–C, black traces). In each panel, relative bioluminescence (%) is plotted on the y-axis where the highest value measured in the control (reporter construct alone) cells during each experiment is set as 100% and time (hrs) is plotted on the x-axis. Each time-point represents the mean of three independent experiments +/−SD. The results of statistical analysis are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067858#pone.0067858.s001" target="_blank">Figure S1H</a>. Blue, red and black bars above each panel represent the lighting conditions. For clarity, blue, red and white background also indicates the blue, red and dark periods, respectively. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067858#pone.0067858.s004" target="_blank">Figure S4</a> for characterization of the various recombinant MAPK constructs.</p

Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer

Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor prognosis. We sequenced 29 SCLC exomes, 2 genomes and 15 transcriptomes and found an extremely high mutation rate of 7.4 ± 1 protein-changing mutations per million base pairs. Therefore, we conducted integrated analyses of the various data sets to identify pathogenetically relevant mutated genes. In all cases, we found evidence for inactivation of TP53 and RB1 and identified recurrent mutations in the CREBBP, EP300 and MLL genes that encode histone modifiers. Furthermore, we observed mutations in PTEN, SLIT2 and EPHA7, as well as focal amplifications of the FGFR1 tyrosine kinase gene. Finally, we detected many of the alterations found in humans in SCLC tumors from Tp53 and Rb1 double knockout mice. Our study implicates histone modification as a major feature of SCLC, reveals potentially therapeutically tractable genomic alterations and provides a generalizable framework for the identification of biologically relevant genes in the context of high mutational background