Implication of DNA demethylation and bivalent histone modification for selective gene regulation in mouse primordial germ cells

PLoS One. 2012;7(9):e46036. doi: 10.1371/journal.pone.0046036. Epub 2012 Sep 28.

Abstract

Primordial germ cells (PGCs) sequentially induce specific genes required for their development. We focused on epigenetic changes that regulate PGC-specific gene expression. mil-1, Blimp1, and Stella are preferentially expressed in PGCs, and their expression is upregulated during PGC differentiation. Here, we first determined DNA methylation status of mil-1, Blimp1, and Stella regulatory regions in epiblast and in PGCs, and found that they were hypomethylated in differentiating PGCs after E9.0, in which those genes were highly expressed. We used siRNA to inhibit a maintenance DNA methyltransferase, Dnmt1, in embryonic stem (ES) cells and found that the flanking regions of all three genes became hypomethylated and that expression of each gene increased 1.5- to 3-fold. In addition, we also found 1.5- to 5-fold increase of the PGC genes in the PGCLCs (PGC-like cells) induced form ES cells by knockdown of Dnmt1. We also obtained evidence showing that methylation of the regulatory region of mil-1 resulted in 2.5-fold decrease in expression in a reporter assay. Together, these results suggested that DNA demethylation does not play a major role on initial activation of the PGC genes in the nascent PGCs but contributed to enhancement of their expression in PGCs after E9.0. However, we also found that repression of representative somatic genes, Hoxa1 and Hoxb1, and a tissue-specific gene, Gfap, in PGCs was not dependent on DNA methylation; their flanking regions were hypomethylated, but their expression was not observed in PGCs at E13.5. Their promoter regions showed the bivalent histone modification in PGCs, that may be involved in repression of their expression. Our results indicated that epigenetic status of PGC genes and of somatic genes in PGCs were distinct, and suggested contribution of epigenetic mechanisms in regulation of the expression of a specific gene set in PGCs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • DNA (Cytosine-5-)-Methyltransferase 1
  • DNA (Cytosine-5-)-Methyltransferases / genetics
  • DNA (Cytosine-5-)-Methyltransferases / metabolism
  • DNA / genetics*
  • DNA / metabolism
  • DNA Methylation*
  • Gene Expression Regulation, Developmental*
  • Gene Knockdown Techniques
  • Germ Cells / growth & development
  • Germ Cells / metabolism*
  • Histones / genetics*
  • Histones / metabolism
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Positive Regulatory Domain I-Binding Factor 1
  • Regulatory Sequences, Nucleic Acid
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism
  • Transcription Factors / genetics
  • Transcription Factors / metabolism

Substances

  • Dppa3 protein, mouse
  • Histones
  • Membrane Glycoproteins
  • Mill1 protein, mouse
  • Prdm1 protein, mouse
  • Repressor Proteins
  • Transcription Factors
  • DNA
  • Positive Regulatory Domain I-Binding Factor 1
  • DNA (Cytosine-5-)-Methyltransferase 1
  • DNA (Cytosine-5-)-Methyltransferases
  • Dnmt1 protein, mouse

Grant support

K. Mochizuki was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.