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In vivo demethylation of chicken embryonic beta-type globin genes with 5-azacytidine
Abstract:
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.
Insights
5-azacytidine demethylated chicken embryonic globin genes but did not activate their expression. This suggests DNA demethylation alone is insufficient for activating certain suppressed genes, unlike poised genes. Further research is needed.
Area of Science:
- Epigenetics and Gene Regulation
- Molecular Biology
- Developmental Biology
Background:
- DNA methylation is a key epigenetic mechanism regulating gene expression.
- Embryonic globin genes are typically silenced in adult organisms.
- 5-azacytidine is a DNA methylation inhibitor used to study gene activation.
Purpose of the Study:
- To investigate the in vivo effects of 5-azacytidine on DNA methylation and embryonic beta-globin gene expression in adult chickens.
- To determine if DNA demethylation by 5-azacytidine is sufficient to activate silenced embryonic globin genes.
Main Methods:
- Adult chickens were treated with intravenous 5-azacytidine.
- DNA methylation status of the embryonic rho gene was assessed using restriction endonucleases (HpaII, MspI, HhaI).
- Globin protein and RNA levels were analyzed to assess gene expression.
Main Results:
- Five daily treatments with 5-azacytidine demethylated the embryonic rho gene by over 75%.
- Despite significant demethylation, there was no significant activation of the embryonic rho gene at the protein or RNA level.
- This contrasts with findings for fetal globin genes in humans and baboons, suggesting differential gene regulation mechanisms.
Conclusions:
- Demethylation by 5-azacytidine alone is insufficient to activate silenced chicken embryonic beta-globin genes.
- A model is proposed where genes are either 'poised' (activated by demethylation) or 'suppressed' (requiring additional factors for activation).
- Further studies using eukaryotic DNA transformation systems and cell cultures are needed to elucidate the precise role of DNA methylation in globin gene switching.