(Figure 1). To investigate this possibility we used an ade- novirus to over-express Twist-1 in N2a neuroblastoma cells and found that DNM3os expression was increased 5- to 6-fold above control levels (Figure 2A). Further RT-PCR experiments then showed that Twist-1 expres- sion also mediated a 5-fold increase in both miR-214 and miR-199a expression (Figure 2B). NIH-3T3 embryonic ﬁbroblasts express Twist-1 consti- tutively and we therefore used this cell line to investigate whether Twist-1 regulates miR-199a and miR-214 under normal physiological circumstances. Western blotting and quantitative PCR conﬁrmed that Twist-1 (Figure 3A) and the noncoding RNA DNM3os (Figure 3B) were expressed in NIH-3T3 cells, respectively. To investigate whether under normal physiological conditions Twist-1 was responsible for driving miR-199a and miR-214 expression we used an anti-Twist1 shRNA construct (kindly provided by Dr. Michael C. Naski). The Taqman results showed that anti-Twist-1 shRNA decreased the expression of miR-214 and miR-199a expression while the scrambled shRNA control did not (Figure 3C). Twist-1 acts on an E-Box to drive miR-199a/214 expression Having conﬁrmed Twist-1 was driving miR-214 and miR- 199a expression we searched for promoter motifs and noted that a region containing an E-Box (a promoter ele- ment known to be bound by Twist-1) was present. This region (–640 to 0 bp of the DNM3os) was isolated from genomic DNA by PCR and cloned next to a luciferase reporter gene (Figure 4A). Further, we also deleted the basic-helix–loop–helix (bHLH)-binding E-box element (CATCTG) known to be essential for Twist-1 transcrip- tional activity (and also cloned this next to a luciferase Nucleic Acids Research, 2009, Vol. 37, No. 1 125 reporter gene). The results of reporter assays showed that luciferase activity was increased by Twist-1 in cells transfected with the E-Box-luciferase construct (–640:0); however, no increase in luciferase activity was observed Figure 3. ShRNA mediated knockdown of Twist-1 results in decreased miR-199a and miR-214 expression. Western blotting (A) and QPCR analysis (B) show that Twist-1 and DNMO3 is constitutively expressed in NIH-3T3 cells. (C) Inhibiting Twist-1 expression by transfecting cells with an anti-Twist-1 shRNA resulted in a >2-fold decrease in the levels of miR-199a and miR-214 (n = 3 in all cases). Figure 2. Twist-1 mediates the expression a single antisense transcript encoding miR-199a and miR-214. (A) Western blot and agarose gel showing that Twist-1 and the DNM3os transcript are expressed respec- tively in N2a cells when they are transduced with Ad.Twist-1. (B) Q-PCR analysis also shows that the expression of miR-199a and miR-214 are signiﬁcantly increased (P < 0.001 by t-test in both cases) following transduction with Ad.Twist-1. Figure 4. Promoter assays showing Twist-1 mediates expression via the E-box region. (A) Schematic diagram together with an agarose gel showing the entire (–640 to 0 bp) promoter and E-Box deleted promoter region of DNM3os. (B) Luciferase Promoter assays show that Twist-1 drives expression via the E-Box region (–640:0) within the DNM3os transcript. Promoter activity is at background levels when the E-Box is deleted (–640:357) and when cells are transduced with control vectors.