In vivo demethylation of chicken embryonic beta-type globin genes with 5-azacytidine.
We have studied the in vivo effects of the methyl-blocking cytotoxic pyrimidine analog 5-azacytidine upon DNA methylation and expression of the normally inactive embryonic beta-type globin genes in adult chickens. We find that 5 daily treatments with intravenous 5-azacytidine is capable of converting the normally completely methylated embryonic rho gene to a greater than 75% demethylated state as assayed by the restriction endonucleases HpaII, MspI, and HhaI. However, this change in methylation is not accompanied by activation of the embryonic rho gene at the level of globin protein or globin RNA. Although our analysis does not exclude a few copies of embryonic globin RNA per cell, this result is still in contrast with the results seen with the fetal gamma-globin genes in baboons and humans [17; see also other chapters in this volume]. Hence demethylation by 5-azacytidine treatment alone is not sufficient to activate the chicken embryonic beta-globin genes. We propose that these seemingly disparate results can be reconciled by a model in which certain genes in specific cell types (eg, human and baboon gamma-globin in adult erythroid cells, chicken ev-1 in MSB cells, mouse metallothionein-1 in lymphoblastoid cells) are in a poised state such that removal of specific methyl cytosines results in a high level of transcription. In contrast other genes (including chicken rho and epsilon embryonic globin genes in adult erythroid cells) are in a suppressed state that overrides activation by demethylation. Alternatively, 5-azacytidine could activate certain genes by a mechanism totally independent of DNA methylation. For instance, selection of specific early erythroid cell types could account for the results seen in baboon fetal globin gene activation. The availability of various eukaryotic DNA transformation systems, characterization of eukaryotic DNA methylases, and development of large-scale cultures of erythropoietic precursor cells should facilitate detailed examination of these various states of gene activation and allow the elucidation of the precise role of DNA methylation in the regulation of globin gene switching.
D000818:Animals; D001374:Azacitidine / Q000494:pharmacology*; D002645:Chickens; D005786:Gene Expression Regulation / Q000187:drug effects*; D005796:Genes; D005914:Globins / Q000235:genetics*; D008745:Methylation; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D005914:Globins; D001374:Azacitidine