of these miRNAs and performed realtime RT-qPCR from the same total RNA samples that had been sequenced. Identification of miRNAs Directly Induced by p-Smad2/3 The total number of reads obtained over two runs of deepsequencing for each sample were 11,930,468 for the 0 hr sample, 12,356,171 for the 16 hr SB sample and 11,267,363 for the 16 hr SB +12 hr Dox sample. The sequenced reads were aligned to miRBase allowing only perfect matches. Under these conditions, the number of reads mapping to mature miRNAs were 3,101,837 for the 0 hr sample, 2,738,932 for the 16 hr sample and 3,302,409 for the 16 hr SB +12 hr Dox sample. Furthermore, levels of both pri-miRNAs decreased after 4 and 6 hrs, in correlation with the decrease in p-Smad2 levels at these time points. In the control CHX+SB experiment, neither pri-miR-341,3072 nor -181c/d changed significantly in expression levels. The remaining primiRNAs did not increase in response to p-Smad2/3 induction, with one pri-miRNA responding to CHX. Together, these findings suggest that pri-miRs341,3072 and -181c/d are direct targets of p-Smad2/3 transcription. Characterization of the p-Smad2/3 Inducible pri-miRNA Genes To gain further evidence for the direct regulation of pri-miRs341,3072 and -181c/d by p-Smad2/3, we investigated the promoters of these primary transcripts. At present, there is little experimental evidence available on the structure of intergenic primiRNA genes. One multiplexed approach has been to combine data on the presence of chromatin signatures, CpG islands, ESTs and 23462267 species comparisons, to predict transcriptional start sites 11478874 to a high degree of probability. Using these data, we made predictions for the gene structures of pri-miRs-181c/d and 341,3072. FoxH1 is a critical co-regulator of p-Smad2/3-dependent target gene activation, and is expressed at high levels throughout early mouse embryogenesis and ES cells. To investigate whether there are putative FoxH1 binding sites present in the predicted regulatory regions of pri-miRs-181c/d and -341,3072, we used the Fuzznuc algorithm to search for the presence of ASE 10 kb upstream and downstream of their predicted TSS. Within this region, we found two ASE for pri-miR-181c/d and eight for pri-miR-341,3072. The relative position of these ASE, and their species conservation, is presented in miRNA Regulation by TGF-b/Smad2/3 Signaling putative Smad Binding Elements in close vicinity of these ASE, supporting a Smad-dependent regulation from these ASE. Therefore, we hypothesized that these pri-miRNAs are regulated in a FoxH1-dependent manner. To test this, we analyzed the functionality of these putative ASE sites by generating luciferase reporter constructs in which the enhancer region upstream of the SV40 promoter in the pGL3 mammalian expression vector was [Lys8]-Vasopressin web replaced with 11.5 kb fragments containing single or multiple ASE of pri-miRs-341,3072 and -181c/d, and tested each group in the luciferase assay. Each miR-ASE luciferase construct was transfected into ES cells and treated with SB for 12 hrs to inhibit endogenous signaling. Cells were subsequently treated with Activin for 5 hrs to activate the pathway or with SB as negative control for an additional 5 hrs. We observed a significant upregulation in luciferase activity following Activin treatment for miR-ASE of primiR-341,3072, indicating that this ASE cluster may be important in the transcriptional regulation of pri-miR341,3072. To validate this responsiveness, both ASE sites within this