Polycomb response elements (PREs) are specific DNA sequences that stably maintain the developmental pattern of gene expression. Drosophila PREs are well characterized, whereas the existence of PREs in mammals remains debated. Accumulating evidence supports a model in which CpG islands recruit Polycomb group (PcG) complexes; however, which subset of CGIs is selected to serve as PREs is unclear. Trithorax (Trx) positively regulates gene expression in Drosophila and co-occupies PREs to antagonize Polycomb-dependent silencing. Here we demonstrate that Trx-dependent H3K4 dimethylation (H3K4me2) marks Drosophila PREs and maintains the developmental expression pattern of nearby genes. Similarly, the mammalian Trx homolog, MLL1, deposits H3K4me2 at CpG-dense regions that could serve as PREs. In the absence of MLL1 and H3K4me2, H3K27me3 levels, a mark of Polycomb repressive complex 2 (PRC2), increase at these loci. By inhibiting PRC2-dependent H3K27me3 in the absence of MLL1, we can rescue expression of these loci, demonstrating a functional balance between MLL1 and PRC2 activities at these sites. Thus, our study provides rules for identifying cell-type-specific functional mammalian PREs within the human genome. Display omitted •Trx-dependent H3K4me2 marks the majority of Drosophila Polycomb response elements•This H3K4-dimethylase function is conserved in MLL1, the mammalian Trx homolog•MLL1-dependent H3K4me2 occurs predominantly at CpG islands•In HCT116 cells, ∼300 active genes require MLL1 to block PRC2-dependent repression The extent to which mammalian CpG islands functionally resemble Drosophila Polycomb response elements (PREs) remains unclear. Rickels et al. describe a conserved role for Trx/MLL1 as an H3K4-dimethylase at PREs and CpG islands, respectively. HCT116 cells express ∼300 genes that require MLL1, not for activation but to block PRC2-dependent repression.