Activation of a chicken embryonic globin gene in adult erythroid cells by 5-azacytidine and sodium butyrate.

Abstract

Adult White Leghorn chickens were rendered anemic by injection with 1-acetyl-2-phenylhydrazine and then treated with parenteral 5-azacytidine, and levels of embryonic globin RNA in circulating reticulocytes were measured. A very small but detectable amount of correctly initiated embryonic p-type globin RNA was detected in reticulocytes from birds treated with 5-azacytidine, while none was detected in reticulocytes from those receiving only phenylhydrazine or phenylhydrazine plus 1-f3-D-arabinofuranosylcytosine (cytosine arabinonucleoside). An attempt to increase embryonic globin RNA induction by treatment with parenteral sodium butyrate after 7 days of 5-azacytidine administration resulted in a 5-to 10-fold increase in the level of embryonic globin RNA. However, sodium butyrate did not induce embryonic gene expression when given alone or after treatment with cytosine arabinonucleoside. Sodium butyrate treatment also caused a DNase I-hypersensitive site to be exposed at the 5' end of the p-globin gene only after 5-azacytidine induced demethylation of several CpG sites in and around the gene. (20). In these latter studies there remains some question as to whether increased t-globin gene expression is due to gene demethylation, cell selection, or both, since 5-azacytidine is known to have a variety of toxic effects on cells, and since y-globin expression can be stimulated in adults by several types of bone marrow stress.As part of ongoing studies aimed at elucidating mechanisms that regulate globin gene expression, we have attempted to study the effects of demethylation of embryonic globin genes in anemic adult chickens. We report here that while 5-azacytidine treatment causes nearly complete demethylation of the p embryonic globin gene in adult erythroid cells, only a minimal amount of p-globin RNA is detectable, and the surrounding chromatin structure, as assayed by DNase I digestion, does not differ from untreated controls. On the other hand, pharmacologic doses of sodium butyrate greatly increase the amount of embryonic p-globin RNA in adult reticulocytes and result in the exposure of a 5' DNase I-hypersensitive site in some of the reticulocyte nuclei, but only when the gene has first become demethylated by 5-azacytidine treatment. MATERIALS AND METHODS Treatment of Animals and Blood Collection. Adult WhiteLeghorn hens (Yoder, Kalona, IA) were rendered anemic by five daily intramuscular injections with 1-acetyl-2-phenylhydrazine (Sigma) at 20 mg/kg or by phlebotomy of 10 ml of whole blood per day to achieve a hematocrit of 18-22% (normal 35-40%). Reticulocyte 3954The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Treatment of spinal muscular atrophy by sodium butyrate

“…There is evidence that sodium butyrate may act at the transcription level by increasing the acetylation of histones, thereby releasing constraints on the DNA template and reactivating a number of genes (25,26). Sodium butyrate also increases the expression of fetal-globin genes in adult baboons, humans, and other animals (27)(28)(29). In utero infusions of butyrate delay the developmental switch from ␥-to ␤-globin gene expression in sheep fetuses (29).…”

Sodium Butyrate and Valproic Acid as Splicing Restoring Agents in Erythroid Cells of b-Thalassemic Patients

“…In the context of haemoglobinopathies, utero infusions of Butyrate, delay the developmental switch off γ to β-globin gene expression in sheep fetuses (32). Sodium butyrate and similar derivatives increases the expression of fetal-globin genes and are being used clinically in treatment of β-Thalassemia and sickle cell anemia (23,24,33). These effects of butyrate may occur through the inhibition of histone deacetylase (30,31).…”

“…In hemoglobinopathies, Butyrates are clinically used to treat sickle-cell anemia and Thalassemia through induction of the expression of γ-gene, producing fetal hemoglobin (HbF) and activating the γ-globin (20)(21)(22)(23)(24).…”

Fetal hemoglobin chemical inducers for treatment of hemoglobinopathies

“…Butyrate was shown to promote cell differentiation and to enhance globin gene expression in human erythroleukemia cells [124], to increase embryonic globin gene expression in chicken adult erythroid cells pretreated with 5-azacytidine [125], to cause higher HbF levels at birth in infants born from diabetic mothers (infants of diabetic mothers have a delay in γ to β globin switch after birth associated with elevated plasma α-amino butyric acid levels) [126,127], to enhance HbF synthesis in cultures of primary adult erythroid progenitors/precursors [128], and to increase HbF synthesis in adult baboons [129,130]. Furthermore, it was shown that butyrate infusions in the ovine fetus delay the switch from fetal to adult hemoglobin [131].…”