Relationship between Single‐Stranded DNA Isolated from Cultured Muscular Cells during Differentiation and the Transcription of Messenger RNA

Abstract

Single-stranded DNA (ssDNA), equivalent to about 2 "/, of the total nuclear DNA, was isolated by an improved method of hydroxyapatite chromatography from native nuclear DNA of rat myoblast cells and myotubes, of the Lg line. Small quantities of '251-labelled ssDNA were annealed with a large excess of unlabelled DNA, cytoplasmic RNA and mRNA from myoblasts or myotubes. The results indicated that ssDNA belongs to the non-repetitious portion of the cell genome and is formed of two distinct molecular fractions. The major ssDNA fractions (75 %) consist of non-selfreassociating DNA sequences and the minor fraction (25 x) consists of self-reassociating DNA sequences. About 30 -32 % and 25 -26 "/, of ssDNA from myoblast represent DNA sequences complementary to total cytplasmic RNAs and polyadenylated RNAs respectively. Hybridizations of ssDNA with an excess of RNA from myoblasts and/or myotubes show differences in the abundance and the diversity of mRNA during muscular differentiation. These differences were confirmed by DNA-driven reactions between 'Z51-labelled polyadenylated RNA and ssDNA in great excess.Various observations favour models of DNA transcription based on local opening of the DNA helix [l -31. Previous studies from our laboratory showed that a minor fraction of single-stranded DNA (ssDNA), usually amounting to 1.5 -2 of the total nuclear DNA, is related to the transcriptional activity, avian myeloblastosis virus [5] and ssDNA from mouse SV-3T3 cells (Shaool, D., Hanania, N., Harel, J . and May, E., unpublished results) non-productively transformed by simian virus 40 was found much enriched for transcribed virus-specific DNA sequences but contained no or very little DNA from the non-transcribed viral (or proviral) sequences. Muscle cells grown in culture provide a model system to study the role of gene expression during cell differentiation. After a defined time in culture, myoblast cells (stage I) from the L g line [7] fuse to form multinucleated myotubes (stage IV). This morphological event is characterized biochemically by the synthesis of musclespecific proteins [8-101 and changes in certain messenger RNAs [11 -131 producing modifications in chromatin protein kinases and phosphoproteins [14]. Our previous studies, based on hybridization of complementary DNAs (cDNAs) synthesized in a cellfree system t o cytoplasmic RNAs, had demonstrated typical changes in both the total number and relative abundance of polyadenylated RNAs during the course of muscular differentiation [14a]. The present work indicates that these changes in the transcriptional activity can be demonstrated at the level of ssDNA.

Epigenetic reprogramming: is deamination key to active DNA demethylation?

“…It has been suggested that DNA demethylation of silenced genes cannot occur without histone acetylation-induced transcription (D’Alessio et al 2007). Transcription leads to a transient formation of single-stranded DNA (Leibovitch & Harel 1978, Leibovitch et al 1979) and it has been hypothesized that transcription may be needed for AID targeting (Chaudhuri et al 2003, Shen et al 2009). When overexpressed in NIH 3T3 cells, AID more efficiently edits a GFP reporter gene transcribed at higher levels, suggesting that transcription may stimulate its activity (Yoshikawa et al 2002).…”