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Figure 6 | Genome Biology

Figure 6

From: E2F mediates enhanced alternative polyadenylation in proliferation

Figure 6

E2F-mediated regulation of APA in proliferation. (a) Comparison of the distribution of fold-change (in log2) in expression levels between confluent and proliferative cells calculated over two sets of genes: a target set that contained the 3'-end processing genes and a background set that contained all the rest of genes expressed in BJ cells (P-value calculated using Wilcoxon test). (b) The top scoring motif identified by de novo motif discovery analysis applied to the set of promoters of the 3'-end processing genes matched the binding signature of E2F (motif accession number in TRANSFAC DB: M00430). (c) Normalized expression levels (determined from the 3'-seq data) of E2F1 and E2F2 in proliferating and confluent BJ cells. (d) Schematic map of the core machineries of polyadenylation site recognition and 3'-end cleavage. In this map, nodes correspond to either protein-coding genes (violet nodes) or protein complexes (green nodes), and edges correspond to either regulatory links (blue edges) or association links between complexes and their members (green edges). The fold-change in expression in the transition from proliferating to confluent BJ cells is indicated by the horizontal bar at the top of each node (green corresponds to down-regulation, yellow to no-change and gray to gene not expressed in our dataset). Genes in this network whose promoter was found to be bound by either E2F1 [26] or E2F4 [27] are indicated by an orange and blue vertical bars to their left. (The map was created using the SPIKE knowledgebase of signaling pathways [36].) (e) Effect of knocking down E2F1 on promoter activity of eight 3'-end processing genes assessed using reporter assays. MCM2 served as positive control and an artificial p53 promoter was used as a negative one. *P < 0.05, **P < 0.01. Error bars represent SEM. (f) The effect of knocking down E2F1+2 on the expression levels of three core cleavage factors in BJ cells was examined using qPCR (results shown are mean ± standard deviation based on triplicates in the case of E2F1 and E2F2, and on five replicates for the three cleavage transcripts). In all cases, the reduction in transcript level is statistically significant (P < 0.01; one-tail t-test)). (g) The effect of knocking down E2F1+2 on the relative usage of 3' UTR proximal and distal CSs in three transcripts that showed enhanced usage of the proximal CSs in proliferation was examined using 3'-qPCR (Materials and methods). In all three cases examined, reducing E2F levels increased the relative cleavage at the distal site (namely, reduced the cleavage at the proximal one). Results shown are based on duplicates; in all cases, P < 0.05, one-tail t-test). (h) A schematic model illustrating the E2F-mediated regulation of mRNA 3'end processing enzymes and its effect on APA.

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