Ts for GABPA. It is possible that the number of direct targets is DprE1-IN-2 chemical information either under or over-estimated due to using ChIP-seq data from a different cell line to MCF10A where the expression studies were conducted. Indeed, RHOF appears to be incorrectly designated as a direct GABPA target (Fig. 3). Nevertheless, several of these direct targets were validated in breast epithelial MCF10A cells, and RAC2 and KIF20A were subsequently shown to be important in controlling cell migration in this cell type (Fig. 4). RAC2 is a Rho GTPase that has previously been shown to control the chemotaxis of neutrophils through its 79831-76-8 effects on the actin cytoskeleton [16]. KIF20A is a kinesin involved in trafficking and has previously been shown to play an important role in late cell cycle progression [17,18]; thus its effects on migration are a novel finding. However, it is not currently clear whether the effects we see for KIF20A on migration are independent of this activity or are indirectly linked to cell cycle defects caused by its loss. Interestingly, like KIF20A, RACGAP1 has also been implicated in controlling cytokinesis [19] but we see no effect of RACGAP1 depletion on cell migration (Fig. 4). Thus, these two events need not necessarily be linked.GABPA and Cell Migration ControlWhile we have analysed a limited number of GABPA target genes here, the final phenotype likely results from changes in the expression of multiple genes controlling cell migration. Indeed, this is the mechanism through which ELK1 affects this process [7], and this type of regulation is more akin to how many microRNAs function, in dampening down the activity of entire pathways rather than acting through a single key regulator (reviewed in [20]). Overall, therefore, GABPA plays a complex role in controlling cell migration through directly affecting the expression of genes encoding key proteins involved in this process, and also by working in a more indirect manner to impact on cell migration.the overlap of these groups of genes with lists of genes assigned to ELK1 only (C) or to both ELK1 and GABPA ChIP-seq regions (D); and the overlap of genes up- or down-regulated upon siGABPA transfection and assigned to regions bound by both factors with lists of genes exhibiting a change of expression in cells transfected with siELK1 (E and F). N/S ?no significant bias in distributions between up- and down-regulated genes (Fisher’s Exact test). (TIF)Figure S3 Depletion of GABPA causes a profound effectMaterials and Methods Cell culture and imaging, migration assays, RNA interference and RT-PCRMCF10A cells were grown and all assays were performed as described in [7]. All siRNA duplexes were ON-TARGETplus SMARTpools (Dharmacon) except for GABPA, where a SantaCruz reagent (sc-37100) was also used. Primer pairs used in RTPCR reactions are listed in Table S2.on the expression of genes coding for a 15857111 network of cytoskeleton- migration- and adhesion-related proteins. Image shows a STRING-derived network of all genes which exhibit a statistically significant change of expression in MCF10A cells depleted of GABPA and which belong to GO terms associated with the cytoskeleton, cell migration or adhesion as determined by DAVID analysis. (TIF)Figure S4 GABPA directly activates the expression of several functional classes of genes. Image shows a STRING-derived network of proteins encoded by all genes which exhibit a statistically significant downregulation of expression in MCF10A cells depleted of GABPA and which ar.Ts for GABPA. It is possible that the number of direct targets is either under or over-estimated due to using ChIP-seq data from a different cell line to MCF10A where the expression studies were conducted. Indeed, RHOF appears to be incorrectly designated as a direct GABPA target (Fig. 3). Nevertheless, several of these direct targets were validated in breast epithelial MCF10A cells, and RAC2 and KIF20A were subsequently shown to be important in controlling cell migration in this cell type (Fig. 4). RAC2 is a Rho GTPase that has previously been shown to control the chemotaxis of neutrophils through its effects on the actin cytoskeleton [16]. KIF20A is a kinesin involved in trafficking and has previously been shown to play an important role in late cell cycle progression [17,18]; thus its effects on migration are a novel finding. However, it is not currently clear whether the effects we see for KIF20A on migration are independent of this activity or are indirectly linked to cell cycle defects caused by its loss. Interestingly, like KIF20A, RACGAP1 has also been implicated in controlling cytokinesis [19] but we see no effect of RACGAP1 depletion on cell migration (Fig. 4). Thus, these two events need not necessarily be linked.GABPA and Cell Migration ControlWhile we have analysed a limited number of GABPA target genes here, the final phenotype likely results from changes in the expression of multiple genes controlling cell migration. Indeed, this is the mechanism through which ELK1 affects this process [7], and this type of regulation is more akin to how many microRNAs function, in dampening down the activity of entire pathways rather than acting through a single key regulator (reviewed in [20]). Overall, therefore, GABPA plays a complex role in controlling cell migration through directly affecting the expression of genes encoding key proteins involved in this process, and also by working in a more indirect manner to impact on cell migration.the overlap of these groups of genes with lists of genes assigned to ELK1 only (C) or to both ELK1 and GABPA ChIP-seq regions (D); and the overlap of genes up- or down-regulated upon siGABPA transfection and assigned to regions bound by both factors with lists of genes exhibiting a change of expression in cells transfected with siELK1 (E and F). N/S ?no significant bias in distributions between up- and down-regulated genes (Fisher’s Exact test). (TIF)Figure S3 Depletion of GABPA causes a profound effectMaterials and Methods Cell culture and imaging, migration assays, RNA interference and RT-PCRMCF10A cells were grown and all assays were performed as described in [7]. All siRNA duplexes were ON-TARGETplus SMARTpools (Dharmacon) except for GABPA, where a SantaCruz reagent (sc-37100) was also used. Primer pairs used in RTPCR reactions are listed in Table S2.on the expression of genes coding for a 15857111 network of cytoskeleton- migration- and adhesion-related proteins. Image shows a STRING-derived network of all genes which exhibit a statistically significant change of expression in MCF10A cells depleted of GABPA and which belong to GO terms associated with the cytoskeleton, cell migration or adhesion as determined by DAVID analysis. (TIF)Figure S4 GABPA directly activates the expression of several functional classes of genes. Image shows a STRING-derived network of proteins encoded by all genes which exhibit a statistically significant downregulation of expression in MCF10A cells depleted of GABPA and which ar.
