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Severe global DNA hypomethylation blocks differentiation and induces histone hyperacetylation in embryonic stem cells

Melany Jackson1, Anna Krassowska, Nick Gilbert

  • 1John Hughes Bennett Laboratory, Division of Oncology, School of Molecular and Clinical Medicine, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, United Kingdom.

Molecular and Cellular Biology
|October 1, 2004
PubMed

Insights

DNA methyltransferase 1 (Dnmt1) is essential for embryonic stem cell differentiation. Unlike Dnmt1-deficient cells, Dnmt3a/3b-deficient cells with low DNA methylation can retain stem cell properties, highlighting Dnmt1

Area of Science:

  • Epigenetics and Gene Regulation
  • Stem Cell Biology
  • Developmental Biology

Background:

  • DNA methyltransferase 1-deficient (Dnmt1-/-) embryonic stem (ES) cells exhibit hypomethylation and undergo apoptosis upon differentiation induction.
  • Understanding the specific roles of different DNA methyltransferases in maintaining epigenetic stability and cell fate is crucial.

Purpose of the Study:

  • To investigate the differentiation potential of Dnmt3a and Dnmt3b double-knockout (Dnmt3a-/-, 3b-/-) ES cells with severe DNA hypomethylation.
  • To compare the differentiation capacity of Dnmt3a-/-, 3b-/- ES cells with Dnmt1-/- ES cells.
  • To determine the specific requirement of DNA methyltransferases for successful embryonic stem cell differentiation.

Main Methods:

  • Generation and characterization of Dnmt3a-/-, 3b-/- ES cells with varying degrees of CpG hypomethylation.
  • Assessment of stem cell marker expression (alkaline phosphatase, Oct4) and differentiation capacity.
  • Analysis of histone acetylation levels and expression of extraembryonic markers.
  • Comparison with Dnmt1-/- ES cells under similar hypomethylation conditions.

Main Results:

  • Dnmt3a-/-, 3b-/- ES cells with severe hypomethylation (0.6% CpG methylation) are blocked in differentiation, retaining stem cell characteristics.
  • Restoration of DNA methylation levels rescues the differentiation defect in Dnmt3a-/-, 3b-/- ES cells.
  • Dnmt3a-/-, 3b-/- ES cells with >10% CpG methylation can terminally differentiate, while Dnmt1-/- ES cells with 20% methylation cannot.
  • Increased histone acetylation and aberrant extraembryonic marker expression are observed in severely hypomethylated Dnmt3a-/-, 3b-/- ES cells.

Conclusions:

  • Successful terminal differentiation of ES cells is not solely dependent on overall DNA methylation levels.
  • There is an absolute requirement for the DNA maintenance methyltransferase, Dnmt1, for enabling ES cell terminal differentiation.
  • Dnmt1 plays a critical, non-redundant role in regulating stem cell fate and differentiation.

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