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A Pax3/Dmrt2/Myf5 Regulatory Cascade Functions at the Onset of Myogenesis

Figure 5

Dmrt2 binding sites in the Myf5 epaxial enhancer are essential for normal activity in vivo.

(A) The sequence of the Myf5 epaxial enhancer (EpExt), which drives early Myf5 expression in the epaxial domain of newly formed somites. TCF/LEF binding sites are boxed in green, the Gli1 binding site in red, and four putative Dmrt2 binding sites in black. (B–D) EMSA performed with oligonucleotides conjugated with biotin, containing Dmrt2 binding sites (1–4) as indicated in A, incubated with extracts of HEK293 cells, with (+) or without (−) a Dmrt2HA expression vector, or oligonucleotides without biotin (dsDNA) (including mutated oligonucleotides; mut-dsDNA). In the supershift assay (D), binding was disrupted by adding anti-HA (Monoclonal; Roche) to HA-tagged Dmrt2, whereas control anti-Flag antibody had no effect. The arrowheads show the binding between the oligos and the tagged protein. NS; non-specific. (E, F) Transient transgenic analysis to examine the role of the Dmrt2 binding sites in the Myf5 EpExt enhancer. The transgenes, containing mutated Dmrt2 binding sites1, 3, 4, showed reduced β-galactosidase activity in developing somites with either the TK (E) or Myf5-BA (branchial arch) promoter region (F). The BA element that directs transgene expression to the branchial arches, provides a positive control (arrowheads in F). Observations on transgenic embryos are summarised in Table 1.

Figure 5

doi: https://doi.org/10.1371/journal.pgen.1000897.g005