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Down-regulation of histone H3 and H4 gene transcription in differentiated L6 myotubes
D E Larson1, P Zahradka, G Stein
1Department of Molecular Biology, University of Guelph, Canada.
Abstract:
The core histone mRNA levels in terminally differentiated L6 myotubes decrease to less than 5% of the amount present in proliferating myoblasts in parallel with the cessation of DNA synthesis (Bird, RC., Jacobs, F.A., Stein, G., Stein, J. and Sells, B.H. (1985) Biochim. Biophys. Acta 824, 209-217). The role of gene transcription in the down-shift of histone mRNA levels was assessed using a cell-free system. The level of transcription from the differentiation-independent adenovirus major late promoter was directly related to the RNA polymerase II activity of myoblast and myotube nuclear extracts. In addition, both extracts actively transcribed the histone H4 gene template containing only the 5 proximal promoter region (-210 bp). In contrast, inclusion of the distal-proximal promoter region (-410 to -210 bp) in the template resulted in a 60% decrease in transcription by the myotube extract. A similar down-shift in transcription of the histone H3 gene template (containing 900 bp 5 of the initiation site) by myotube nuclear extracts was also observed. The decrease in histone mRNA levels in myotubes may therefore be controlled in part by a transcriptional mechanism involving a negative regulatory factor.
Insights
Histone mRNA levels significantly drop in differentiating muscle cells. This decrease is partly regulated by a negative transcriptional factor that inhibits histone gene expression during differentiation.
Area of Science:
- Molecular Biology
- Cellular Differentiation
- Gene Regulation
Background:
- Core histone mRNA levels decrease significantly in terminally differentiated L6 myotubes compared to proliferating myoblasts.
- This reduction in histone mRNA parallels the cessation of DNA synthesis during muscle cell differentiation.
Purpose of the Study:
- To investigate the role of gene transcription in the down-regulation of histone mRNA levels during L6 myotube differentiation.
- To identify potential transcriptional mechanisms controlling histone gene expression in differentiating muscle cells.
Main Methods:
- Utilized a cell-free transcription system with nuclear extracts from myoblasts and myotubes.
- Assessed transcription levels of differentiation-independent (adenovirus major late promoter) and differentiation-dependent (histone H4 and H3 genes) templates.
- Varied promoter regions included in the histone gene templates to identify regulatory elements.
Main Results:
- RNA polymerase II activity correlated with transcription of the adenovirus major late promoter in both myoblast and myotube extracts.
- Both myoblast and myotube extracts transcribed histone H4 and H3 gene templates with proximal promoter regions.
- Myotube extracts showed a 60% decrease in transcription of histone H4 and H3 templates when distal-proximal promoter regions were included, suggesting negative regulation.
Conclusions:
- Histone mRNA down-shift during L6 myotube differentiation is, in part, controlled by a transcriptional mechanism.
- A negative regulatory factor present in myotube nuclear extracts likely suppresses histone gene transcription during differentiation.
- These findings suggest a role for transcriptional regulation in controlling histone availability for DNA replication during terminal differentiation.