The Undermethylated State of a CpG Island Region in Igf2 Transgenes Is Dependent on the H19 Enhancers

CpG islands are GC-rich regions located in the promoter regions of housekeeping genes and many tissue-specific genes. While most CpG islands are normally unmethylated, island methylation can occur and is associated with silencing of the corresponding gene. Experiments with transgenic mice and DNA transfection in pluripotential embryonic cells have led to the conclusion that the information required for protecting the islands from methylation is contained within the CpG islands themselves and have identified Sp1 binding sites as an important element in establishing and/or maintaining the methylation-free state of CpG islands. To examine the generality of these observations, we analyzed the methylation of one of the mouse Igf2 CpG islands and its flanks in transgenic mice. We observed that the undermethylated state of this region is dependent on the presence of a separate cis-regulatory element, the H19 enhancers. These tissue-specific enhancers had a ubiquitous, non-tissue-specific effect on island region methylation. Structural alterations outside of the island and these enhancers also affected this region's methylation. These findings indicate that the methylation of some CpG island-containing regions is more sensitive than previously believed to the activity of distant cis-regulatory elements and to structural alterations in nonisland sequences in cis.     

The prokaryotic neomycin-resistance-encoding gene acts as a transcriptional silencer in eukaryotic cells

The gene encoding neomycin resistance (neo) mediates a cis-acting negative effect on expression from promoters in transient and stable transfectants of mammalian cell lines. Inserting the neo gene either into retroviral vectors or into plasmids containing reporter genes results in a five- to tenfold decrease of expression from proximal promoters like the simian virus 40 early or the retroviral myeloproliferative sarcoma virus promoter. The silencing effect is not dependent on the insertion site or the orientation of the fragment. The neo gene is frequently used in eukaryotic vectors as a dominant selectable gene. Other selectable genes were tested for potential cis-activity. We found that the gene conferring resistance to puromycin from Streptomyces alboniger does not influence adjacent promoters.