analysis was performed using Affymetrix Genechip Drosophila 2.0 arrays (see methods). We compared cry0 (Dmcry2/Hscry2), HsCRY (Dmcry+/Hscry+) and wild-type (Dmcry+/Hscry12) flies, in the dark (D) or after a two hours dark to blue light transition (L). Among a total of over 18,500 total transcripts, genes with statistical differences in hybridization signals of two-fold or more were selected. In the dark, 449 genes were induced and 161 genes were repressed in HsCRY1 expressing lines in comparison to wt (D) (Fig. 3a,b). Therefore, as predicted, mammalian Type II cryptochrome has considerable effect on transcription in this heterologous organism and this function can occur independently of light. Transcriptome analysis also produced the more unexpected finding that DmCRY likewise mediates considerable transcriptional responses in the absence of light (D), in contrast to the light-dependent mechanism underlying DmCRY clock function. In the dark, 267 genes were differentially expressed in cry0 in comparison to wt, showing the importance of DmCRY in the regulation of gene expression in complete darkness (Fig. 3a,b). Importantly, this regulation occurs by pathways independent of its role in the circadian clock under these conditions of arrhythmicity. A comparison of genes regulated by HsCRY1 and DmCRY showed a significant proportion (154) of common genes, thereby validating this approach with respect to identifying cryptochrome regulated transcripts rather than background changes in gene expression patterns unrelated to cryptochrome function. Interest- ingly, genes that were regulated by both DmCRY1 and HsCRY1 in DD were often regulated in the opposite manner, for example 108 were induced and 45 were repressed in both cry0 and HsCRY1 expressing flies, indicating HsCRY1 acted on the whole as inducer and DmCRY as repressor of the same genes (Fig. 3a,b). The same was true of the overall majority of regulated genes (HsCRY1 functioned primarily as inducer, DmCRY primarily as repressor in the dark) (Fig. 3a,b). When analyzing the function assigned to the selected genes using Drosophila databases [35], we found many involved in development and the related function of proteolysis that occurs in the course of metamorphosis (see [36] and references therein) (Fig. 3c). Our analyses also revealed an unexpectedly high proportion of genes implicated in stress response or related to ROS (reactive oxygen species), especially among genes down-regulated in both HsCRY and cry0. It therefore seems probable that HsCRY1 and DmCRY may share some common signaling pathways, and that HsCRY1 has a dominant negative effect by binding non-productively and thereby seques- tering common signaling intermediates. It also seems likely that responsivity to stress may be a hitherto unrecognized role of cryptochromes that is conserved across species lines. We next determined the possible light responsivity of crypto- chrome mediated transcriptional events. A comparison of gene expression levels before and after a rapid two hour blue light exposure was undertaken (Fig. 3d,e). Comparison of expression levels showed relatively few differences in either wt flies, cry0 mutants or Hscry1 transgenics, consistent with the expectation that light-regulated gene expression is likely not a major feature during Drosophila development. We nevertheless obtained 43 genes that were up-regulated subsequent to light exposure in the wt (Fig. 3d). Significantly, only one of these was also induced in the cry0 genetic background. Furthermore, of the 13 genes down-regulated by light in cry0 mutants (Fig. 3d), 6 were up-regulated and the remainder showed no change in the wt genetic background. These results indicate that blue light induction of gene expression under these conditions in the wt was almost entirely dependent on DmCRY (Fig. 3c,d). In the case of Hscry1 transgenics, a moderate light responsivity was observed with 3 genes induced by blue light, all of Human Cryptochrome Light Responsivity them distinct from those induced in wt and cry0 genotypes. Therefore, the presence of HsCRY1 leads to light-dependent transcription in Drosophila by pathways independent of DmCRY mediated expression. Furthermore, the fact that DmCRY- dependent transcriptional regulation was absent in Hscry1 transgenics demonstrates that there is interference of HsCRY1 with regular DmCRY-regulated transcription. It is likely that common signaling pathways may exist with common signaling intermediates, consistent with the antagonistic roles of HsCRY1 and DmCRY transcriptional activity in the dark (Fig. 3a,b). To verify and confirm the results obtained by microarray analysis, 4 representative cryptochrome-dependent genes were selected. Drosomycin3 (Dro3, CG32283), White(CG2759), Endopepti- dase (End, CG6467), and the gene CG7906 (7906), that encodes an unknown protein, were selected for a quantitative analysis of transcript changes (Fig. 4). As expected, in comparison to the wt, Dro3 was down-regulated in the cry0 and Hscry samples in the dark and in the light (Fig. 4a). Conversely, White transcripts were down- regulated in the wt both in the dark and in the light (Fig. 4b). As found during microarray experiments, 7906 showed a specific expression pattern with transcripts that were induced by light in the wt but down-regulated in cry0 mutants in the same conditions (Fig. 4c). We also tested the expression of one of the 3 genes up- regulated by light in the HsCRY1 expressing flies. As expected, transcripts of End accumulated only in Hscry1 samples after a dark to light shift (Fig. 4d). The complete list of genes differentially expressed in our microarray experiments have been deposited in the online accessible database ArrayExpress. In terms of functions, the genes regulated by light in the wt were related to development, and to a lesser extent to circadian rhythms and to stress and ROS (Fig. 3f) as has been observed for expression in the dark (Fig. 3c). Furthermore, among the 13 genes down- regulated by light in cry0 mutants, 4 belong to the Osiris [37] family which may be linked to immune responsiveness (Osi6, Osi14, Osi19 and Osi20). Of these, 3 are up-regulated by light in the wt (Osi6, Osi19 and Osi20) and none are regulated by light in the presence of HsCRY1, indicating that HsCRY1 also in some way antagonistically interacts with this response pathway. The 3 genes induced specifically in Hscry1 transgenics in response to blue light (CG6467, CG11912, CG10475), were described as serine- type endopeptidases. In an attempt to identify possible cis-acting promoter elements linked to cryptochrome regulation, we searched for conserved motifs known to be targets of transcription factors by the program cisTargetX [38] No representative motifs were found among the many genes differentially expressed in the dark through either DmCRY or HsCRY1 action. However, the analysis of light regulated genes revealed that among the 43 genes up-regulated by light in the wt, 15 had promoters containing a motif that could be recognized by IRF2, 12 by IRF1, 7 by IRF9 (Interferon Regulatory Factor) from mouse [39] (Fig. 4e) implicated in the mammalian antiviral immune response. In addition, 5 genes up- regulated by light contained the motif for the ‘signal transducer and activator of transcription’ (STAT) transcription factor from Drosophila (Fig. 4e), which are involved in Drosophila innate immunity against viruses. Further, consistent with these results, among the 13 genes down-regulated by light in cry0 mutants, 4 (including 3 Osiris) possess the sequence recognized by IRF2 and 2 the sequence recognized by STAT, and all are induced in the wt under the same conditions. Since induction of gene expression occurs within two hours subsequent to the onset of illumination, and since all of these genes are regulated by DmCRY, these promoter element binding factors are likely to be novel partners on the light signaling pathway of DmCRY that have no role in the PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e31867