Intranuclear compartmentalization of transcribed and nontranscribed c-myc sequences in Namalva-S cells

Abstract

This investigation is centered on the intranuclear localization of transcribed and nontranscribed c-myc sequences in human Namalva-S cells bearing t(8;14) translocation. Southern hybridization showed that the breakpoint in the truncated allele of c-myc lies outside the characteristic 12.8-kbp EcoRI fragment: as Northern analysis indicated, this reorganization induces a high level of c-myc transcription. Following high-salt treatment, EcoRI digestion and centrifugation, isolated nuclei from the same cells are separated into two residual fractions: a heavier P fraction including nuclear matrix structures and a light S fraction representing dehistonized chromatin fibres. Comparative hybridization experiments revealed that the above procedure separates the c-myc sequences between the two fractions. To locate the site of intranuclear c-myc transcription, we performed run-on experiments with two fractions, topologically analogous to the residual P and S fractions but maintaining the original chromatin organization. These experiments indicated that chromatin P fraction harbours actively transcribed c-myc sequences while chromatin S harbours nontranscribed ones. Further experiments have clarified that the transcribed c-myc sequences are firmly bound to the matrix by multiple attachment sites, arranged throughout the entire gene locus. It was found, moreover, that at the site of attachment the interaction between DNA and the matrix components is realized via proteins. Controls with the beta-globin gene, which is constitutively nonexpressed in Namalva-S cells but upon induction is highly expressed in murine erythroleukemia cells, completely confirmed the conclusion we had made for the intranuclear localization of c-myc. Thus the experiments presented here support the more common idea that the transcribed and nontranscribed sequences are precisely compartmentalized.