Histone methylation was studied using immunofluorescence cytochemistry with polyclonal antibodies against dimethyl-Histone H3 on lysine 4 (rabbit-anti- 2me-H3K4, 1:200, Millipore, Temecula, CA), a bivalent mark for the transition of transcriptional activation (20). Repression of developmentally regulated genes was investigated using a rabbit polyclonal antibody against trimethyl-Histone H3 on lysine 27 (3me-H3K27, 1:750, Active Motif, Carlsbad, CA) (5). Histone H4 acetylation, known to be involved in transcriptional activation, was investigated by immunocytochemistry [rabbit-anti-Ac-H4 (Lysine5, 8, 12, 16), 1:500, Millipore]. DNA methylation was investigated using antibodies against 5-methyl-cytosine (goat-anti-5-MeC, 1:250, GeneTex, San Antonio, TX). Changes in expression of the enzyme responsible for DNA methylation, DNA methyltransferase (DNMT), were investigated using polyclonal antibodies against DNMT1 (made in goat, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA), which is responsible for maintaining DNA methylation, and DNMT3a (made in goat, 1:200, Santa Cruz Biotechnology), responsible for de novo methylation. Methylation of CpG-dinucleotides was detected using a rabbit polyclonal antibody against methyl-CpG binding domain 1 (MBD1, 1:200, Millipore). AZA treated cultures were processed for immunocytochemical staining to assess DNA methylation (DNMT1a and 3a; 5-MeC antibodies) and histone
