A Pax3/Dmrt2/Myf5 Regulatory Cascade Functions at the Onset of Myogenesis
Figure 3
A conserved 286 bp element 5′ of the Dmrt2 gene directs Pax3-dependent expression in the somite.
(A) A schematic representation of the mouse Dmrt genomic locus showing overall sequence conservation (blue bars) between human, rat, chicken and Xenopus (green bars). (B) The nucleotide sequence of the conserved 286 bp Dmrt2 element (red circle in A) in mouse (the position on ch19; 25728951 to 25729236, about 18 kb 5′ of Dmrt2) and comparison with a homologous region of human, chick and Xenopus genomes, with asterisks indicating conserved nucleotides between all these species. Five putative Pax3 binding sites are framed in black and red. (C) Electrophoretic mobility shift assays (EMSA) performed with oligonucleotides conjugated with biotin, containing putative Pax3 binding sites (1–5) as indicated in (B), incubated with extracts of HEK293 cells, with (+) or without (−) a Pax3 expression vector, or oligonucleotides without biotin (+dsDNA), including mutated oligonucleotides (+mut-dsDNA). The Pax3 site in the 145 bp regulatory element at −57.5 kb from the Myf5 gene provides a positive control (Myf5-145; [9]). Significant binding is seen on site2 when Pax3 protein is present. For supershift assays, monoclonal Pax3 antibody (DSHB) was used (+anti-Pax3); +anti-Flag provides a negative control. Arrowheads show the band due to binding of the Pax3 protein. NS; non-specific. (D) Chromatin immunoprecipitation (ChIP) analysis of Pax3 binding to the 286 bp sequence in vivo was performed with chromatin prepared from somites of E9.5 embryos (without head, neural tube, and internal tissues). Upper panels show the evaluation of sheared genomic DNA (1/10 input) with two sets of Dmrt2 primers, for the potential regulatory sequence (286) and for an upstream control sequence (+20 k) without Pax3 binding sites (results not shown), in left and right panels respectively. As an additional positive or negative control, whole genome sheared genomic DNA, with (whole genome) or without proteinase K (No Proteinase K) treatment is shown (In the absence of proteinase K cross-linked chromatin blocks the PCR reaction). Lower panels show ChIP (IP) with two different Pax3 antibodies (αPax3, Bajard et al. (2006), Lagha et al. (2008)) and control rabbit serum. Left lower bands indicate Pax3 binding to the 286 bp sequence, not seen with the control +20 kb sequence (right panels). (E) A transient transgenic embryo (E9.5) with the 286 bp Dmrt2 fragment, fused to the TK promoter and nlacZ reporter shows dermomyotome expression. (F) The transgene, with mutated Pax3 binding site2, shows loss of β-galactosidase activity in the dermomyotome (F′), but some ectopic expression in the myotome and ventral somite (F″), seen in more mature somites in the non-mutated transgenic embryo (E″). Observations on transgenic embryos are summarised in Table 1.