metallothionein and dimethyl-sulfate

During chemically induced differentiation of murine erythroleukemia (MEL) cells, cAMP-dependent protein kinase activity increases, and the enzyme's isozyme pattern changes. To examine the enzyme's role during MEL cell differentiation, we stably transfected MEL cells with recombinant plasmids in which the mouse metallothionein I promoter controlled expression of either a mutant form of the type I regulatory subunit of cAMP-dependent protein kinase (RI) or the enzyme's specific peptide inhibitor (PKI); expressing either sequence rendered cells cAMP-dependent protein kinase-deficient. Chemically induced differentiation of MEL cells as assessed by beta-globin mRNA and hemoglobin accumulation was inhibited in RI mutant and PKI transfectants; adding zinc further inhibited differentiation in the transfectants but had no effect on parental MEL cells. The inhibition of differentiation correlated with the amount of RI mutant mRNA and protein in the RI mutant transfectants and with the cells' degree of cAMP-dependent protein kinase deficiency in both the RI mutant and PKI transfectants. Overexpression of wild type RI did not interfere with differentiation or enzyme activity. We conclude that cAMP-dependent protein kinase activity is important for chemically induced differentiation of MEL cells and that the down-regulation of RI protein which occurs during MEL cell differentiation is not essential for differentiation to proceed.

Topics: Acetamides; Animals; Blotting, Northern; Blotting, Western; Cell Differentiation; Enzyme Repression; Globins; Isoenzymes; Leukemia, Erythroblastic, Acute; Macromolecular Substances; Metallothionein; Mice; Plasmids; Promoter Regions, Genetic; Protein Kinases; RNA, Messenger; Sulfuric Acid Esters; Transfection; Tumor Cells, Cultured; Zinc

A method of high resolution in vivo footprinting has been developed and used to survey the mouse metallothionein I (MT-I) promoter for protein : DNA interactions associated with basal-level transcription and with high-level metal-induced transcription. This promoter and its associated regulatory region is structurally complex. It contains multiple potential binding sites for metal regulatory factors and for other transcription factors, including SP1 and MLTF. In several cases potential recognition sites overlap, and the experiments reported here provide a view of which sites are utilized in vivo. These data also show how the pattern of protein : DNA contacts changes when cells are shifted from basal-level expression to metal-induced expression. The noninduced footprint pattern consists of interactions at basal elements that are thought to be responsible for the moderate transcription of this gene in the absence of added metals. These interactions remain unchanged upon metal induction. When MT-I expression is increased by exposing cells to zinc or cadmium, a new footprint pattern is observed. It includes the basal interactions and a new set of metal-dependent footprints that are positioned over all five genetically defined metal responsive elements (MREs), MRE-A--MRE-E. In addition, these data identify a sixth probable MRE, MRE-F, which displays a dimethylsulfate (DMS) footprint similar to that at other MREs.

Topics: Alkylating Agents; Animals; Base Sequence; Densitometry; Deoxyribonucleases; DNA-Binding Proteins; Gene Expression Regulation; In Vitro Techniques; Metallothionein; Metals; Mice; Molecular Sequence Data; Promoter Regions, Genetic; Sulfuric Acid Esters; Zinc