Transcriptional activation of an embryonic globin gene in adult erythroid cells in vivo.

We have studied the mechanism and level of activation of the normally silent rho globin gene in adult erythroid cells of anemic chickens treated with 5-azacytidine and sodium butyrate. We have shown that while demethylation of the rho globin gene does not result in high levels of specific mRNA, demethylation does appear to be a prerequisite for sodium butyrate to cause such an effect. Runoff nuclear transcription assays were used to demonstrate that 5-azacytidine plus sodium butyrate treatment act to cause transcriptional activation of the rho gene in adult animals. However, the data also show that there may be post-transcriptional down-regulation of mRNA levels in adult red cells, since the level of rho gene nuclear runoff relative to beta exceeds the corresponding stable mRNA ratios for the two gene products. These data are consistent with a model in which developmental switching of the chicken globin genes may involve both transcriptional regulation and one or more levels of post-transcriptional regulation. Our studies of histone acetylation in erythroid cells from butyrate treated animals show that no bulk changes in histone acetylation can be detected. While transient localized changes in the fast kinetic form of histone acetylation cannot be excluded, these results are consistent with the hypothesis that the transcriptional activation of the rho gene by sodium butyrate in this animal model may be mediated by some trans-acting transcription regulating factor. Thus, the requirement for both 5-azacytidine and sodium butyrate for gene activation could reflect a mechanism which involves both an alteration in a specific regulatory DNA recognition sequence (by demethylation) and a change in either the specificity or amount of a stimulatory (or inhibitory) trans acting factor. These proposed mechanisms are directly testable with currently available techniques.