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Topological analysis of the TERT model, and prediction of robust MYC dependent TERT repression.

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posted on 2014-02-13, 03:38 authored by Alan E. Bilsland, Katrina Stevenson, Yu Liu, Stacey Hoare, Claire J. Cairney, Jon Roffey, W. Nicol Keith

(A), structure of FFL types I–IV. Structures visualised in Pajek [103]. Bold lines indicate activation, dashed lines indicate repression. X, Y represent generalised transcription factors, Z represents a regulated gene. (B), activation and repression modules in the TERT transcriptional neighbourhood model. Subnetworks were extracted from the main model and visualised in Pajek [103]. Extraction was achieved as described in materials and methods. As an indicator of topological importance, node betweenness centralities were calculated and are given in table 6. Additionally, we calculated flow betweenness which is not dependent only on geodesics [77]. (C), Effect of single- and double-node targeting on TERT on-states. Rule-sets for each node were modified in turn individually (black bars) to simulate constitutive repression or activation. For each rule-set change, statespace was derived and the proportion of system states evolving to attractor states with TERT stably on was quantified. The analysis was repeated for each node in the context of double knockouts with MYC also suppressed in each case (grey bars). (D), MYC dependent TERT repression and reversal by AR. A2780 were transfected with 200 nM non-specific control siRNA (Con), 100 nM MYC with 100 nM non-specific (MYC), or 100 nM MYC and 100 nM each specific siRNA. Cells were harvested after 48 h and RNA extracted for analysis of TERT expression normalised to RPS15 by RT-QPCR. Mean ± SEM of TERT expression in treated cells relative to control from three experiments (ns: not significant; *: p<0.05; **: p<0.01). (E), Knockdown of TERT regulatory transcription factors by RNAi. A2780 were transfected with 100 nM each specific siRNA (RNAi) or non-specific control (NS) and harvested after 48 h. 20 µg protein samples were analysed by western blotting against the respective targets. ERK counter-blots were also performed. Each experiment was performed twice. Representative blots are shown.