DNA methylation is commonly associated with a reduction of transcription. The following data come from a study of the impact of the location and extent of DNA methylation on gene activity in eukaryotic cells. A bacterial gene, luciferase, was inserted into plasmids next to eukaryotic promoter fragments. CpG sequences, either within the promoter and coding sequence (transcription unit) or outside of the transcription unit, were methylated to various degrees, in vitro. The chimeric plasmids were then introduced into cultured cells, and luciferase activity was assayed. These data compare the degree of expression of luciferase with differences in the location of DNA methylation [Irvine et al. (2002). Mol. and Cell. Biol. 22:6689–6696]. What general conclusions can be drawn from these data? 

Verified step by step guidance

1. Step 1: Examine the control condition where there is no methylation. Note the relative luciferase expression level (490×) as the baseline for comparison.
2. Step 2: Compare the luciferase expression when methylation occurs outside the luciferase transcription unit. Observe that despite a similar number of methylated CpG sequences (around 100), the expression decreases moderately (to 290× and 250×), indicating some repression but not complete.
3. Step 3: Analyze the effect of methylation inside the luciferase transcription unit. Notice that even with a similar or slightly higher number of methylated CpG sequences (108 and 134), the expression drops drastically (to 80× and 5×), showing a stronger repression effect when methylation is within the gene region.
4. Step 4: Look at the condition where the entire plasmid is methylated (12.1 kb, 593 CpG sites). The luciferase expression is almost completely repressed (2×), indicating that extensive methylation across the plasmid strongly inhibits gene expression.
5. Step 5: Draw a general conclusion that DNA methylation represses gene expression more effectively when it occurs within the transcription unit compared to outside it, and that the extent of methylation correlates with the degree of repression.

Key Concepts

DNA Methylation and Gene Expression

DNA methylation involves adding methyl groups to cytosine bases, often at CpG sites, which typically represses gene transcription. This epigenetic modification can block transcription factor binding or recruit proteins that compact chromatin, reducing gene expression. The data show that methylation inside the transcription unit strongly decreases luciferase expression, highlighting methylation's role in gene silencing.

Location of Methylation Relative to the Transcription Unit

The position of methylation relative to the gene affects its impact on expression. Methylation outside the transcription unit reduces expression moderately, while methylation within the transcription unit causes a more pronounced repression. This suggests that methylation within coding or promoter regions directly interferes with transcription machinery or elongation.

Extent of Methylation and Its Effect on Gene Activity

The size and number of methylated CpG sites correlate with the degree of gene repression. Larger methylated patches and higher numbers of methylated CpGs lead to stronger suppression of luciferase activity, as seen with entire plasmid methylation causing near-complete silencing. This indicates a dose-dependent relationship between methylation extent and transcriptional inhibition.