Figure 2
Figure 2. TEL-AML1 directly targets miRNA promoters. (A) ChIP-chip analysis was performed using the TEL-AML1 expressing cell line REH using an anti-TEL antibody. This cell line has a deletion in the other TEL allele, so only TEL-AML1 targets are pulled-down. Two independent experiments were performed, and anti-TEL-AML1 pull-downs and input DNA controls were cohybridized to the ncRNA tiling arrays. Following normalization and smoothing, the log2-ratios of TEL-AML1 pull-downs versus controls from experiment 1 were graphed against those from experiment 2 on an x/y graph, with density shading. A Pearson correlation coefficient of 0.27 (P < 10−7) between the 2 replicate experiments was observed as shown in this plot, also exemplified by a clustering of data along a slope of x = y (B) Scrutiny of the pull-down peaks revealed TEL-AML1 binding to miRNA regions, the strongest pull-down was detected for the miRNA-139 promoter. Lower but significant signals were found for 99 other miRNAs, shown here is the peak in a 5′ region of miRNA-494. (C) TEL-AML1 binds the promoters of miRNAs. The average log ratio of the binding intensity from 3 experiments was calculated and the 8 miRNAs promoters with the highest affinity to bind TEL-AML1 are shown, among these is miRNA-494. Standard errors are calculated from 3 separate experiments. (D) miRNA-494 and miRNA-320a are direct and functional targets of TEL-AML1. miRNA promoters identified to bind TEL-AML1 (direct targets, y axis; only those targets significantly enriched in ChIP pull downs are shown) were plotted against miRNA expression changes upon TEL-AML1 silencing. Log2 ratios are shown. (E) Using conventional PCR (supplemental Table 1) we analyzed TEL-AML1 pull-down and input from ChIP experiments. We designed primers to test the miRNA-494 and miRNA-320a that were shown to be direct targets of TEL-AML1 in the ChIP experiments. Panel E shows the fold increase of TEL-AML1 binding to the promoters of miRNA-494 and miRNA-320a as it was compared with the input (in triplicate with SE bars), thereby confirming these miRNAs as direct targets of TEL-AML1. The combined data from these experimental approaches distinguishes miRNA-494 and miRNA-320a both as direct and functional targets. miRNA-320a is indicated as miRNA-320 on the figures, as it is termed in the Sanger database.

TEL-AML1 directly targets miRNA promoters. (A) ChIP-chip analysis was performed using the TEL-AML1 expressing cell line REH using an anti-TEL antibody. This cell line has a deletion in the other TEL allele, so only TEL-AML1 targets are pulled-down. Two independent experiments were performed, and anti-TEL-AML1 pull-downs and input DNA controls were cohybridized to the ncRNA tiling arrays. Following normalization and smoothing, the log2-ratios of TEL-AML1 pull-downs versus controls from experiment 1 were graphed against those from experiment 2 on an x/y graph, with density shading. A Pearson correlation coefficient of 0.27 (P < 10−7) between the 2 replicate experiments was observed as shown in this plot, also exemplified by a clustering of data along a slope of x = y (B) Scrutiny of the pull-down peaks revealed TEL-AML1 binding to miRNA regions, the strongest pull-down was detected for the miRNA-139 promoter. Lower but significant signals were found for 99 other miRNAs, shown here is the peak in a 5′ region of miRNA-494. (C) TEL-AML1 binds the promoters of miRNAs. The average log ratio of the binding intensity from 3 experiments was calculated and the 8 miRNAs promoters with the highest affinity to bind TEL-AML1 are shown, among these is miRNA-494. Standard errors are calculated from 3 separate experiments. (D) miRNA-494 and miRNA-320a are direct and functional targets of TEL-AML1. miRNA promoters identified to bind TEL-AML1 (direct targets, y axis; only those targets significantly enriched in ChIP pull downs are shown) were plotted against miRNA expression changes upon TEL-AML1 silencing. Log2 ratios are shown. (E) Using conventional PCR (supplemental Table 1) we analyzed TEL-AML1 pull-down and input from ChIP experiments. We designed primers to test the miRNA-494 and miRNA-320a that were shown to be direct targets of TEL-AML1 in the ChIP experiments. Panel E shows the fold increase of TEL-AML1 binding to the promoters of miRNA-494 and miRNA-320a as it was compared with the input (in triplicate with SE bars), thereby confirming these miRNAs as direct targets of TEL-AML1. The combined data from these experimental approaches distinguishes miRNA-494 and miRNA-320a both as direct and functional targets. miRNA-320a is indicated as miRNA-320 on the figures, as it is termed in the Sanger database.

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