Fig. 2. No changes in DNA methylation at repeat or single copy sequences in xDMO morphants. (A) xDMO DNA is heavily methylated at xSatI HpaII sites (compare lanes 2 and 4). HpaII is a methyl-sensitive restriction enzyme and MspI is the methylation insensitive (CCGG) counterpart (lane 5). Right panel: HindIII was used to generate the 750 bp xSatI monomer (black arrow); double digestion with HindIII and HpaII showed no difference in monomer methylation in the wild-type and xDMO samples (lanes 2-3). (B) Bisulphite sequencing (clones n=40) shows no significant difference in CpG methylation between wild-type and xDMO genomes at xSatI sequences. Boxed numbers are percentage CpG methylation; black circles indicate CpG distribution in xSatI. (C) CpG distribution in cloned promoters of xOct91 and xCycD1. Blue bars, CpG; black arrows, transcription start sites; red bars, regions sequenced. (D) Bisulfite analysis (sequences n=40, ten representative clones are shown) was used to determine the methylation status of xOct91 (left) and xCycD1 (right) promoters and upstream regions. Numbers above each CpG indicate genomic position relative to transcription start. Filled circles, methylated CpGs; empty circles, non-methylated CpGs. (E) Immunoblot analysis of wild-type and xDMO histones shows no significant change in various histone modification marks between histone WT and xDMO extracts at stages 8 and 15. Histone modifications are low to absent at stage 8 and accrue by stage 15. Black dots indicate non-specific bands.