One of which relates to known GABPA functions in controlling the cell cycle [9,14]. Additional subnetworks point to a role for GABPA in controlling different aspects of gene expression and also cytoskeletal activities (Figs. S4). In contrast, fewer subnetworks were detectable amongst the genes negatively regulated by GABPA, with the most prominent one being associated mainly with transcriptional regulation (Figs. S5). To concentrate on the role of GABPA as a direct regulator of genes associated with the cytoskeleton, cell migration and adhesion, we further probed the interconnectivities amongst target genes that are bound and regulated by GABPA and that are annotated with the relevant GO terms. We found that the majority of these genes also formed an interconnected network (Fig. 3A). Four genes from this network, RAC2, RHOF, RACGAP1 and KIF20A, were taken for further analysis due to their multiple interactions, and likely functional importance as nodes within the network. First we validated the microarray data for these buy SR3029 targets by performing quantitative RT-PCR on MCF10A cells depleted of GABPA (Fig. 3B). Three of the four selected genes (RAC2, RACGAP1 and KIF20A) exhibited significant reductions in expression upon depletion of GABPA, while no statistically significant changes were seen on two control genes or RHOF, suggesting that the latter is probably a false positive. Similarly, we were able to detect specific binding of GABPA to the regulatory regions of RAC2, RACGAP1 and KIF20A in MCF10A cells but no binding to the RHOF regulatory region could be detected, reaffirming this as a likely false positive. Importantly, these results confirmed that RAC2, RACGAP1 and KIF20A are direct targets for GABPA in MCF10A cells as predicted from ChIP-seq data. Gene expression data showed that at least two of these genes, RAC2 and RACGAP1 are not regulated by ELK1, whereas RHOF and KIF20A require ELK1 for maximal activity (Fig. 2D). Previous ChIP-seq studies did not identify ELK1 occupancy at any of these genes [7] but we wished to confirm this by ChIP-qPCR. All of these genes exhibit detectable ELK1 binding to their regulatory regions (Fig. 2D). However, the binding was relatively low compared to the established ELK1 targets, CDKL3 and RFC4 (Fig. 3D). It is not clear whether this level of binding is sufficient to allow ELK1mediated gene regulation, as observed at RHOF and KIF20A, but this low level binding apparently has little effect on RAC2 and RACGAP1 expression and the latter two genes appear to be specific directly regulated GABPA targets.These experiments therefore identify RAC2, RACGAP1 and KIF20A as likely important nodes in networks associated with the cytoskeleton and cell migration, and these have been verified as direct targets for GABPA-mediated transcriptional activation.Key GABPA target genes are involved in cell migration controlOur data suggest that GABPA affects cell migration by controlling the expression 12926553 of a programme of genes associated with this process through both direct and indirect mechanisms. To probe whether the target genes directly activated by GABPA are important for MCF10A cell migration, we investigated whether four of this category of genes RAC2, RHOF, RACGAP1 and KIF20A play a part in this process. Each of these genes was individually depleted in MCF10A cells by siRNA ML-264 supplier treatment (Fig. 4A) and the effect on cell migration monitored by single cell tracking. The depletion of RAC2 had a similar effect to depletion of.One of which relates to known GABPA functions in controlling the cell cycle [9,14]. Additional subnetworks point to a role for GABPA in controlling different aspects of gene expression and also cytoskeletal activities (Figs. S4). In contrast, fewer subnetworks were detectable amongst the genes negatively regulated by GABPA, with the most prominent one being associated mainly with transcriptional regulation (Figs. S5). To concentrate on the role of GABPA as a direct regulator of genes associated with the cytoskeleton, cell migration and adhesion, we further probed the interconnectivities amongst target genes that are bound and regulated by GABPA and that are annotated with the relevant GO terms. We found that the majority of these genes also formed an interconnected network (Fig. 3A). Four genes from this network, RAC2, RHOF, RACGAP1 and KIF20A, were taken for further analysis due to their multiple interactions, and likely functional importance as nodes within the network. First we validated the microarray data for these targets by performing quantitative RT-PCR on MCF10A cells depleted of GABPA (Fig. 3B). Three of the four selected genes (RAC2, RACGAP1 and KIF20A) exhibited significant reductions in expression upon depletion of GABPA, while no statistically significant changes were seen on two control genes or RHOF, suggesting that the latter is probably a false positive. Similarly, we were able to detect specific binding of GABPA to the regulatory regions of RAC2, RACGAP1 and KIF20A in MCF10A cells but no binding to the RHOF regulatory region could be detected, reaffirming this as a likely false positive. Importantly, these results confirmed that RAC2, RACGAP1 and KIF20A are direct targets for GABPA in MCF10A cells as predicted from ChIP-seq data. Gene expression data showed that at least two of these genes, RAC2 and RACGAP1 are not regulated by ELK1, whereas RHOF and KIF20A require ELK1 for maximal activity (Fig. 2D). Previous ChIP-seq studies did not identify ELK1 occupancy at any of these genes [7] but we wished to confirm this by ChIP-qPCR. All of these genes exhibit detectable ELK1 binding to their regulatory regions (Fig. 2D). However, the binding was relatively low compared to the established ELK1 targets, CDKL3 and RFC4 (Fig. 3D). It is not clear whether this level of binding is sufficient to allow ELK1mediated gene regulation, as observed at RHOF and KIF20A, but this low level binding apparently has little effect on RAC2 and RACGAP1 expression and the latter two genes appear to be specific directly regulated GABPA targets.These experiments therefore identify RAC2, RACGAP1 and KIF20A as likely important nodes in networks associated with the cytoskeleton and cell migration, and these have been verified as direct targets for GABPA-mediated transcriptional activation.Key GABPA target genes are involved in cell migration controlOur data suggest that GABPA affects cell migration by controlling the expression 12926553 of a programme of genes associated with this process through both direct and indirect mechanisms. To probe whether the target genes directly activated by GABPA are important for MCF10A cell migration, we investigated whether four of this category of genes RAC2, RHOF, RACGAP1 and KIF20A play a part in this process. Each of these genes was individually depleted in MCF10A cells by siRNA treatment (Fig. 4A) and the effect on cell migration monitored by single cell tracking. The depletion of RAC2 had a similar effect to depletion of.