RESUMEN
A stable transfection assay was used to test the mechanism by which embryonic globin gene transcription is stimulated in adult erythroid cells exposed to butyric acid and its analogs. To test the appropriate expression and inducibility of chicken globin genes in murine erythroleukemia (MEL) cells, an adult chicken beta-globin gene construct was stably transfected. The chicken beta-globin gene was found to be coregulated with the endogenous adult mouse alpha-globin gene following induction of erythroid differentiation of the transfected MEL cells by incubation with either 2% dimethyl sulfoxide (DMSO) or 1 mM sodium butyrate (NaB). In contrast, a stably transfected embryonic chicken beta-type globin gene, rho, was downregulated during DMSO-induced MEL cell differentiation. However, incubation with NaB, which induces MEL cell differentiation, or alpha-amino butyrate, which does not induce differentiation of MEL cells, resulted in markedly increased levels of transcription from the stably transfected rho gene. Analysis of histone modification showed that induction of rho gene expression was not correlated with increased bulk histone acetylation. A region of 5'-flanking sequence extending from -569 to -725 bp upstream of the rho gene cap site was found to be required for both downregulation of rho gene expression during DMSO-induced differentiation and upregulation by treatment with NaB or alpha-amino butyrate. These data are support for a novel mechanism by which butyrate compounds can alter cellular gene expression through specific DNA sequences. The results reported here are also evidence that 5'-flanking sequences are involved in the suppression of embryonic globin gene expression in terminally differentiated adult erythroid cells.
Asunto(s)
Butiratos/farmacología , Eritrocitos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Globinas/genética , Acetilación , Animales , Ácido Butírico , Embrión de Pollo , ADN/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Histonas/metabolismo , Leucemia Eritroblástica Aguda , Ratones , Ribonucleasas/metabolismo , Transcripción Genética , Células Tumorales CultivadasAsunto(s)
Butiratos/farmacología , Pollos/genética , Regulación de la Expresión Génica/efectos de los fármacos , Globinas/genética , Reticulocitos/metabolismo , Envejecimiento/genética , Animales , Autorradiografía , Pollos/sangre , Metilación , Familia de Multigenes/genética , Hibridación de Ácido Nucleico , ARN/biosíntesis , RibonucleasasRESUMEN
An animal model of hemoglobin switching has been developed in which anemic adult chickens are treated with 5-azacytidine and sodium butyrate or alpha-aminobutyric acid, thereby resulting in activation of the embryonic rho-globin gene in adult erythroid cells. In vitro nuclear runoff transcription assays using erythroid nuclei from treated birds show that the mechanism of activation of the rho-globin gene is transcriptional whereas no transcriptional activation of the embryonic epsilon-globin gene occurs. The action of 5-azacytidine appears to be as an inhibitor of DNA methylation because other S-phase active cytotoxic drugs, when substituted for 5-azacytidine, do not cause demethylation of the embryonic globin genes, nor do they allow transcriptional activation to occur. Embryonic rho-globin gene activation in this model is not due to selection of primitive erythroid cells since a subpopulation of primitive erythroid cells is not evident either morphologically or when cells are probed for embryonic and adult globin RNA by in situ hybridization. These studies show that demethylation by 5-azacytidine is a prerequisite but not sufficient cis-regulatory event for a high level of transcriptional activation of the embryonic rho-globin gene in adult erythroid cells in vivo. The possible basis for the selective transcriptional activation by sodium butyrate in this system is discussed.
Asunto(s)
Azacitidina/farmacología , Butiratos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Globinas/genética , Transcripción Genética/efectos de los fármacos , Acetilación , Animales , Northern Blotting , Pollos , Histonas/fisiología , Metilación , Fenilhidrazinas/farmacología , Reticulocitos/fisiología , Activación TranscripcionalRESUMEN
This study examined the relationship between zinc absorption and metallothionein. Mice injected intraperitoneally with ZnCl2 (2 mmoles) were found within 18 hours to have increased levels of intestinal metallothionein but an apparent decrease in 65Zn absorption. Induction of metallothionein with lower levels of ZnCl2 (0.2 or 0.5 mumoles) resulted in an apparent increase in 65Zn absorption. Isotope dilution experiments showed that intraperitoneal injections of 2 mumoles of ZnCl2 had resulted in a 500-fold dilution of the available 65Zn pool. Mild stress of the animals was shown to increase both 65Zn absorption and intestinal metallothionein. Actinomycin D administered 4 hours prior to ZnCl2 or stress, prevented the induction of metallothionein and obliterated the increase in 65Zn absorption. These results indicate that zinc absorption is directly proportional to intestinal metallothionein levels and imply a significant role for metallothionein in zinc absorption.
