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. 2013 Jun 14;20(9):1194–1208. doi: 10.1038/cdd.2013.62

Figure 2.

Figure 2

MiR-199a-2 expression levels are regulated by a SRF/MRTF-dependent pathway. (a) Schematic of the DNM3OS (upstream sequence of hsa-miR-199a-2) 5′ upstream promoter that contains two conserved SRF CArG boxes and a single myogenic E box. Exact genomic locations of each of the transcription factor DNA-binding sites are listed as number of base pairs in the 5′ upstream region from the start of the human miR-199a-2 genomic sequence. (b) Schematic of human DNM3OS promoter deletion constructs fused to a luciferase reporter gene. Site 1 (SRF CArG box 1) is approximately 0.8 kb upstream of the pri-miR-199a-2 genomic sequence. Site 2 (SRF CArG box) is approximately 0.5 kb upstream of the pri-miR-199a-2 genomic sequence. Relative luciferase expression of the DNM3OS promoter deletion constructs in HEK293T cells. Luciferase values are normalized to the empty vector control. (c) Overexpression of the MRTF transcriptional co-factors along with SRF induces an increase in endogenous miR-199a-5p transcript levels in primary human MB. (d) Independent overexpression of SRF and MRTF transcription factors induce activation of the 2-kb DNM3OS luciferase reporter in HEK293T cells. Values are normalized to the vector alone control. (e) The Site 1 SRF CArG box is essential for driving the 2-kb hDNM3OS-luc reporter and miR-199a-2 expression. Transcriptional luciferase reporter assays in HEK293T cells overexpressing SRF, MRTF-A, and/or MRTF-B along with the 2-kb hDNM3OS-luc reporter with or without mutations in the two SRF CArG boxes (Sites 1 and 2). *P-value<0.005; **P-value<0.05. NS, not significant