Characterization of cis-acting elements in the promoter of the mouse metallothionein-3 gene - Activation of gene expression during neuronal differentiation of P19 embryonal carcinoma cells

The metallothionein (MT)3 gene is expressed predominantly in the brain and the organs of the reproductive system, and fails to respond to metal ions i n vivo. A CTG repeat was proposed to function as a potential repressor elem ent in nonpermissive cells, and a sequence similar to the JC virus silencer element was found to function as a negative element in permissive primary astrocytes. The objective of this study was to characterize further the mec hanisms governing cell-type specific MT-3 gene transcription. We searched f or a suitable cell line expressing the MT-3 gene to be used for determinati on of MT-3 promoter tissue specificity, and showed that MT-3 expression is activated during neuroectodermal differentiation of P19 cells induced by re tinoic acid to levels similar to those found in whole brain. Deletion of th e CTG repeat or of the JC virus silencer did not promote MT-3 promoter acti vity in nonpermissive cells, or enhance expression in permissive cells. We identified MT-3 promoter sequences interacting with liver and brain nuclear proteins, as assayed by DNase I footprinting analyses and electrophoretic mobility shift assay, and assessed the role of these sequences in the regul ation of MT-3 expression by cotransfection experiments. We generated stable transfectants in permissive C6 and nonpermissive NIH-3T3 cells, and analys ed the methylation status of the MT-3 gene. These studies show that regulat ion of tissue-specific MT-3 gene expression does not appear to involve a re pressor, and suggest that other mechanisms such as chromatin organization a nd epigenetic modifications could account for the absence of MT-3 gene tran scription in nonpermissive cells.