Fig. 4

Gadd45 Proteins Promote Demethylation and Selectively Upregulate Deaminases

(A) Gadd45 family members are upregulated by M-DNA, assessed by RT-PCR.

(B) Gadd45α induces moderate demethylation of M-DNA as detected by HpaII digestion and subsequent Southern blotting. M-DNA is injected at 5 pg which does not induce demethylation on its own. Lanes 1, 4, and 7 correspond to wild-type sample.

(C and D) Lowering the levels of Gadd45 family members via morpholino injection attenuates demethylation. Demethylation of 5-methylcytosine as assessed by HpaII digestion and Southern blotting (C), or LC-MS quantitation of 5-meC (D) in total genomic DNA isolated from 13 hpf old embryos injected at the single-cell stage with M-DNA alone (200 pgs) or along with morpholinos as shown. Lanes 1, 7 and 13 correspond to wild-type sample. Combined Gadd45 Mo refers to the combination of morpholinos to all four Gadd45 family members tested (α,α -like, β, and γ; 2 pg each).

(E and F) Synergy among AID, hMbd4, and Gadd45 for demethylation. Methylation status of total genomic DNA from embryos (13 hpf) was assessed by HpaII susceptibility (E) or by LC-MS quantitation of global 5-methylcytosine levels (F) injected at single-cell stage with mRNAs encoding factors as indicated. Lanes 1, 5, and 9 correspond to wild-type sample. Note: AID, MBD4, and Gadd45α were injected at subthreshold amounts (at 25 pgs each), levels which are not sufficient to induce demethylation alone.

(G and H) Quantitative RT-PCR for deaminase family members (assessed at 13 hpf) injected at single-cell stage with mRNA encoding Gadd45α or Gadd45β (F) and M-DNA (200 pg) alone or with morpholinos as shown (G). For each experiment, one representative of at least three biological repeats is shown except in LC-MS measurement where graph is prepared from values of two biological replicates.

Asterisks (^*) depict statistical significance (p < 0.05). Error bars: are ± one standard deviation.