Integration between Notch- and hypoxia-induced transcriptomes

Background: Interaction between key signaling mechanisms is important to generate the diversity in signaling output required for proper control of cellular differentiation and function, although the molecular manifestations of such cross-talk are only partially understood. Notch signaling and the cellular response to hypoxia intersect at different points in the signaling cascades, and in this report we analyze the consequences of this cross-talk at the transcriptome level. Results: Mouse ES cells were subjected to various combinations of hypoxia and/or activated Notch signaling, and the transcriptome changes could be grouped into different categories, reflecting various modes of hypoxia and Notch signaling integration. Two principal categories of novel Notch- and hypoxia-induced genes were identified: i) a larger set of genes induced by one pathway and not significantly affected by the activity status of the other pathway; and ii) a smaller set of genes co-regulated by Notch and hypoxia. In the latter category, we identified genes that were induced by hypoxia and the expression of which was enhanced by active Notch signaling. In addition, a number of genes were induced by Notch and hypoxia independently, and a final category of genes required simultaneous activation of Notch and hypoxia to be significantly induced. Several of the hypoxia- and Notch-induced genes were found to be upregulated in various forms of cancer. Conclusions: We identify novel Notch and hypoxia downstream genes and genes co-regulated by the two pathways, providing a molecular platform to better understand the intersection between the two signaling cascades in normal development and cancer. Total RNA was extracted from 7 treatments in 4 biological replicates of mouse embryonic stem (ES) cells. The 7 treatments consist of 1) ES cell medium for 6 hrs (ESC), 2) N2B27 medium for 6 hrs which is the control condition (N2B27), 3) N2B27 medium supplemented with doxycyline (Dox), 4) Pretreatment with DAPT in ES cell medium for 4 hours followed by switch to N2B27 medium for 6 hours (DAPT), 5) N2B27 under 1% oxygen hypoxic conditions (1% O2), 6) N2B27 supplemented with doxycycline under 1% oxygen hypoxic conditions (1% O2+Dox), 7) Pretreatment with DAPT in ES cell medium for 4 hours followed by switch to N2B27 medium for 6 hours (DAPT) under 1% oxygen hypoxic conditions (1% O2+DAPT).