ABSTRACT: The nerve growth factor NGF has been shown to cause cell fate decisions towards either differentiation or proliferation depending on the relative activity of downstream pERK, pAKT or pJNK signaling. However, how these protein signals are translated into and fed back from transcriptional activity to complete cellular differentiation over a time span of hours to days is still an open question. Using dynamic proteome and transcriptome data from NGF-stimulated PC12 cells over a time span of 24 hours we inferred a dynamic Boolean model capturing the temporal sequence of protein signaling, transcriptional response and subsequent autocrine feedback. Optimal model topology was achieved by introducing multiple time scales for fast cellular signaling and slower gene response and subsequent fitting to the experimental data. The integrated model confirmed the parallel use of MEK/ERK, AKT/PI3K and JNK/JUN for PC12 cell differentiation. Redundancy of cell signaling is demonstrated from the inhibition of the different MAPK pathways. As suggested in silico and confirmed in vitro, differentiation was substantially suppressed under JNK/JUN inhibition, yet delayed only under MEK/ERK inhibition. Most importantly, we found that sustained transcriptional feedback is necessary to induce bistability in the cell fate switch. De novo Gene expression was necessary to activate autocrine feedback that caused integrin signaling to perpetuate the MAPK activity, finally resulting in the expression of late, differentiation related genes. Thus, the cellular decision towards differentiation depends on the establishment of a transcriptome-induced positive feedback between protein signaling and gene expression thereby constituting a robust control between proliferation and differentiation. To eludicate the transcriptome response of PC12 cells time-resolved gene expression data of NGF or EGF simulated PC12 were recorded at t = [0.5h 1h, 2h, 3h, 4h, 5h, 6h, 8h, 12h, 24h, 48h] for NGF and at t=[1h, 2h, 3h, 4h, 6h, 8h, 12h, 24h]. Control time points of unstimulated PC12 cells were taken at t=[0h, 2h, 4h, 6h, 8h, 24h, 48h]. Additionally, the MEK-inhibitor UO126 was added together with NGF and the gene response was measured at t=[1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h] and for UO126 alone at t=[1h, 2h, 4h, 8h, 12h, 48h]. Total RNA was isolated, labeled and hybridized to an Illumina ratRef-12 bead array (Illumina, San Diego, CA, USA) according to the manufacturerâ??s protocol.
