Early embryo development constitutes a unique opportunity to study acquisition of epigenetic marks, including histone methylation. This study investigates the in vivo function and specificity of 3-deazaneplanocin A (DZNep), a promising anti-cancer drug that targets polycomb complex genes. One- to two-cell stage embryos were cultured with DZNep, and subsequently evaluated at the post-mid blastula transition stage for H3K27me3, H3K4me3 and H3K9me3 occupancy and enrichment at promoters using ChIP-chip microarrays. DZNep affected promoter enrichment of H3K27me3 and H3K9me3, whereas H3K4me3 remained stable. Interestingly, DZNep induced a loss of H3K27me3 and H3K9me3 from a substantial number of promoters but did not prevent de novo acquisition of these marks on others, indicating gene-specific targeting of its action. Loss/gain of H3K27me3 on promoters did not result in changes in gene expression levels until 24h post-fertilization. In contrast, genes gaining H3K9me3 displayed strong and constant down-regulation upon DZNep treatment. H3K9me3 enrichment on these gene promoters was observed not only in the proximal area as expected, but also over the transcription start site. Altered H3K9me3 profiles were associated with severe neuronal and cranial phenotypes at day 4-5 post-fertilization. Thus, DZNep was shown to affect enrichment patterns of H3K27me3 and H3K9me3 at promoters in a gene-specific manner.