Fig. S4

Srcap Reduction Coincides with Increased DNA Methylation, Related to Figure 4

(A) Phenotype of zygotic null srcap^−/− mutant fish compared to wild-type at 4dpf. Arrows indicate recessed jaw, and enlarged heart in mutant embryos.

(B) Similar to (A), except phenotypic characterization of embryos injected with an Srcap morpholino. Embryos are at 3dpf. As in (A), arrows indicate a recessed jaw and enlarged heart.

(C) A western blot for Srcap abundance comparing cellular lysates from control 2.5hpf embryos to lysates from embryos that resulted from in vitro fertilization of oocytes injected with an srcap morpholino.

(D) srcap morphants from stage V oocyte injection are modestly impaired at 3hpf, but have gross morphological defects at 4hpf - immediately following zygotic genome activation.

(E) RRBS-Seq DNA methylation analysis of srcap morphants compared to control embryos for loci which undergo DNA demethylation on the maternal allele under wild-type conditions (left), and for all wild-type H2AFV marked loci (right).

(F) Western blots indicate H2AFV decreases in the nuclear fraction and increases in the cytoplasmic fraction of cells purified from embryos that were injected with Anp32e – as in Figure 4F.

(G) Measurements of CpG density (left) and proximity to the gene promoters (right) for regions parsed by changes in DNA methylation after Anp32e injection. Regions were either marked by H2AFV in WT (violet and green) or lacked H2AFV (yellow). H2AFV marked loci were further parsed based on whether DNA methylation increased (green). t tests: ^∗∗∗ = p < 0.001

(H) At 6hpf we observed a modest increase in dnmt1 expression resulting from Anp32e injection. This was not the case at 2.5 or 4hpf. Overall dnmt1 expression decreased during progression of cleavage phase.