Epigenetically controlled Elf5 expression acts in a positive feedback loop to reinforce trophoblast identity.
(a) Elf5 expression is strongly induced in Dnmt1-/- embryos, but not in wildtype and heterozygous littermate embryos (data are mean ± s.d., *P=0.026; n≥6). (b) Elf5 is the earliest, most strongly activated trophoblast marker in Dnmt1-/- ES cells cultured for 4 days in TS cell conditions (data are mean ± s.d., **P=0.00185 [Eomes] and P=0.00051 [Elf5]; n=3). (c) Elf5 expression is further enhanced in Dnmt1-deficient ES cells during the following days of culture in TS cell conditions (data are mean ± s.d., **P<0.005; n=3). (d) Double labelling of Dnmt1-/- ES cells grown for 4 days in TS cell conditions for Elf5 and Oct4. Oct4 repression is not required for activation of Elf5. (e) Elf5 expression in wildtype ES cells causes activation of TS cell markers Cdx2 and Eomes. Wildtype ES cells were transfected with an Elf5-GFP expression construct, FACS sorted and assessed by qRT-PCR. The construct confers high levels of Elf5 expression to transfected cells (Elf5) compared to control transfected cells (vec) that do not express Elf5. Elf5 induces high expression of Eomes. Cdx2 expression is significantly elevated after 1 and 2 days (>3-fold), and reaches highest activation levels after 3 days of Elf5 transfection (data are mean ± s.d., *P<0.05, **P<0.005; n=3). (f) Elf5, Cdx2, or empty vector control, were co-transfected with 2 kb Cdx2, Eomes or Elf5 promoter-reporter constructs. Expression of the reporter gene was analyzed by qRT-PCR 24 hours after transfection. Elf5 can induce both Cdx2 and Eomes promoters. Cdx2 can activate the Elf5 promoter (data are mean ± s.d., **P<0.005; n=3). (g) Chromatin immunoprecipitation assays with an anti-Elf5 antibody. Elf5 binds directly to the Cdx2 and Eomes promoters in TS cells and 3-day transdifferentiated Dnmt1-/- ES cells, but not in wildtype ES cells. Values are represented as Bound:Input and normalized against mock control (data are mean ± s.d., *P<0.05; n=3). Scale bar in d represents 25 μm.