Human Cryptochrome Light Responsivity Figure 3. Transcriptome analysis of cryptochrome dependent gene expression in developing pupa one day after third instar larval stage. A, B Venn diagram of genes either upregulated (red) or downregulated (green) in the indicated strains under conditions of complete darkness. Comparison were performed in triplicate biological samples and analysed for statistically different expression patterns according to established programming techniques (see methods). C. Graphical representation of categories of genes either repressed or activated by cryptochromes in the indicated comparisons. All comparisons are for strains maintained in complete darkness (DD) from the time of egg laying. D,E. Venn diagrams of genes up-regulated or down-regulated in the indicated strains (wt, cry0, or Hscry1 transgenics) after transition from dark to two hours blue light at 60 mmol m22 sec21. F. Graphical representation of categories of genes either repressed or activated by cryptochromes in the indicated comparisons. doi:10.1371/journal.pone.0031867.g003 circadian clock. Significantly, almost all of these genes are repressed (prevented from being activated) by the presence of HsCRY1, which, as a consequence may also be interacting directly and somehow sequestering this conserved transcription factor. The fact that so many of the promoters induced by light contain conserved motifs further reinforces the validity of the present results in identifying targets of cryptochrome action in flies. In summary, transcriptome analysis has revealed that Type II (mammalian HsCRY1) cryptochrome regulates transcriptional activity in Drosophila by (principally) light-independent, but also light-dependent mechanisms, consistent with the role of light in the eclosion phenotypes presented above. More unexpectedly, the endogenous Drosophila (Type I) DmCRY also regulates consider- able light-independent transcriptional activity in Drosophila, in marked contrast to its light-dependent transcriptional role in the circadian clock. Both HsCRY1 and DmCRY are implicated in expression of many genes with no known clock function, including those related to stress responsivity, which may represent a new area of interest in cryptochrome research in this organism. Possible direct targets of cryptochromes include families of transcription factors binding to conserved promoter elements (IRF, STATs) that may be recognized by both HsCRY1 and DmCRY. Biochemical and photoresponsive characteristics of the isolated HsCRY1 protein To date, a number of different approaches have been adopted to isolate the human cryptochrome variants: HsCRY1 and 2 (Type II). However, heterologous expressed proteins from E. coli [40], human (HeLa) cells [41], and Sf21 insect cells [42] had low flavin occupancy and/or very low protein yield and were therefore not suitable for photochemical studies. In the present work, a Pichia pastoris expression system has been developed for producing quantities of native, flavin-bound recombinant HsCRY1 sufficient for in vitro photochemical studies (see methods). The intact full- PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e31867