Silencing of Mouse Aprt Is a Gradual Process in Differentiated Cells
Year: 2003
Type: article
Abstract: Mouse Aprt constructs that are highly susceptible to DNA methylation-associated inactivation in embryonal carcinoma cells were transfected into differentiated cells, where they were expressed. Construct silencing was induced by either whole-cell fusion of the expressing differentiated cells with embryonal carcinoma cells or by treatment of the differentiated cells with the DNA demethylating agent 5-aza-2'-deoxycytidine. Induction of silencing was enhanced significantly by the presence of a methylation center fragment positioned upstream of a truncated promoter comprised of two functional Sp1 binding sites. Initial silencing of the Aprt constructs was unstable, as evidenced by high spontaneous reversion frequencies ( approximately 10(-2)). Stably silenced subclones with spontaneous reversion frequencies of <10(-5) were isolated readily from the unstably silenced clones. These reversion frequencies were enhanced significantly by treatment of the cells with 5-aza-2'-deoxycytidine. A bisulfite sequence analysis demonstrated that CpG methylation initiated within the methylation center region on expressing alleles and that the induction of silencing allowed methylation to spread towards and eventually into the promoter region. Combined with the induction of revertants by 5-aza-2'-deoxycytidine, this result suggested that stabilization of silencing was due to an increased density of CpG methylation. All allelic methylation patterns were variegated, which is consistent with a gradual and evolving process. In total, our results demonstrate that silencing of mouse Aprt is a gradual process in the differentiated cells.
Source: Molecular and Cellular Biology
Institutions: University of Kentucky, Oregon Health & Science University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2153761799