CisModification of the Steroid 21-Hydroxylase Gene Prevents Its Expression in the Y1 Mouse Adrenocortical Tumor Cell Line
Autor: | Bernard P. Schimmer, Jonathan G. Seidman, Keith L. Parker, David S. Milstone, Moshe Szyf |
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Rok vydání: | 1990 |
Předmět: |
Steroid 21-Hydroxylase
medicine.medical_treatment Gene Expression Biology Transfection Methylation Deoxyribonuclease HpaII Mice Endocrinology Bacterial Proteins Gene expression Tumor Cells Cultured medicine Animals RNA Messenger Cloning Molecular Deoxyribonucleases Type II Site-Specific Molecular Biology Gene Adrenal cortex Nucleic Acid Hybridization DNA General Medicine Molecular biology Adrenal Cortex Neoplasms Steroid hormone medicine.anatomical_structure Cell culture DNA methylation Azacitidine |
Zdroj: | Molecular Endocrinology. 4:1144-1152 |
ISSN: | 1944-9917 0888-8809 |
Popis: | The Y1 mouse adrenocortical tumor cell line retains the ability to synthesize and secrete steroids, but does not express steroid 21-hydroxylase (C21) and, therefore, does not produce 21-hydroxylated steroids. In this investigation the mechanisms underlying the loss of C21 activity in the Y1 cell line were explored. A 9-kilobase BglII fragment containing the C21 gene was cloned from the Y1 genome. This genomic clone directed the synthesis of C21 transcripts and 21-hydroxylated steroid products when transfected back into the Y1 cell line. As determined by restriction endonuclease digestions with MspI and HpaII, enzymes that distinguish between unmethylated and methylated CCGG sites, the endogenous C21 gene was extensively methylated in Y1 adrenal cells and in cells from other mouse tissues that do not normally express this gene. In contrast, the C21 gene was hypomethylated in primary cultures of mouse adrenal cells which normally synthesize large amounts of C21. The cloned C21 gene transfected into Y1 cells initially was unmethylated, but became extensively methylated with prolonged culture of the cells; prolonged culture of these transfectants also resulted in a loss of C21 expression. Loss of C21 expression in Y1 transfectants, however, temporally preceded the extensive methylation of the transfected C21 gene. Furthermore, treatment of Y1 cells with 5-azacytidine caused a demethylation of the endogenous C21 gene, but did not result in the recovery of C21 expression. These results indicate that Y1 cells contain a functional C21 gene that has been silenced by a reversible cis-modification event.(ABSTRACT TRUNCATED AT 250 WORDS) |
Databáze: | OpenAIRE |
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