Silencing of mouse Aprt is a gradual process in differentiated cells

Phillip Yates, Robert Burman, James Simpson, Olga N. Ponomoreva, Mathew (Matt) Thayer, Mitchell Turker

Research output: Contribution to journalArticle

20 Citations (Scopus)

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 (≈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.

Original languageEnglish (US)
Pages (from-to)4461-4470
Number of pages10
JournalMolecular and Cellular Biology
Volume23
Issue number13
DOIs
StatePublished - Jul 2003

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decitabine
Methylation
Embryonal Carcinoma Stem Cells
Cell Fusion
DNA Methylation
Genetic Promoter Regions
Sequence Analysis
Clone Cells
Alleles
Binding Sites
DNA

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Silencing of mouse Aprt is a gradual process in differentiated cells. / Yates, Phillip; Burman, Robert; Simpson, James; Ponomoreva, Olga N.; Thayer, Mathew (Matt); Turker, Mitchell.

In: Molecular and Cellular Biology, Vol. 23, No. 13, 07.2003, p. 4461-4470.

Research output: Contribution to journalArticle

Yates, Phillip ; Burman, Robert ; Simpson, James ; Ponomoreva, Olga N. ; Thayer, Mathew (Matt) ; Turker, Mitchell. / Silencing of mouse Aprt is a gradual process in differentiated cells. In: Molecular and Cellular Biology. 2003 ; Vol. 23, No. 13. pp. 4461-4470.
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