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CXXC domain of human DNMT1 is essential for enzymatic activity

Mihika Pradhan1, Pierre-Olivier Estève, Hang Gyeong Chin

  • 1New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938-2723, USA. pradhan@neb.com

Biochemistry
|August 30, 2008
PubMed

Insights

The CXXC domain of DNA methyltransferase 1 (DNMT1) binds unmethylated DNA and is crucial for its catalytic activity. This finding reveals a key mechanism in epigenetic gene regulation and cancer.

Area of Science:

  • Epigenetics
  • Molecular Biology
  • Genetics

Background:

  • DNA cytosine methylation is a key epigenetic mark for gene silencing, primarily mediated by DNA methyltransferases.
  • DNA methyltransferase 1 (DNMT1) is essential for maintaining methylation patterns in somatic cells and is implicated in aberrant gene silencing in cancer.

Purpose of the Study:

  • To investigate the DNA binding properties and functional significance of the N-terminal region of DNMT1.
  • To identify the specific domain responsible for DNMT1's interaction with unmethylated DNA.

Main Methods:

  • Utilized catalytically active recombinant DNMT1 variants.
  • Employed gel shift assays to analyze DNA binding affinities.
  • Performed site-directed mutagenesis and cell transfection studies (COS-7 cells).
  • Assessed genomic methylation levels in a permanent cell line expressing a modified DNMT1.

Main Results:

  • A catalytically active DNMT1 fragment lacking the N-terminus showed higher affinity for unmethylated DNA.
  • The CXXC domain specifically binds unmethylated CpG dinucleotides, and mutations in conserved cysteines abolish this binding.
  • DNMT1 lacking the CXXC domain (DNMT1 (DeltaCXXC)) localized to replication foci and exhibited reduced catalytic activity.
  • Cell lines with DNMT1 (DeltaCXXC) showed partial loss of genomic methylation at rDNA loci.

Conclusions:

  • The CXXC domain of DNMT1 is critical for its DNA binding specificity towards unmethylated DNA.
  • This domain cooperates with the catalytic domain to ensure proper DNA methyltransferase activity.
  • Understanding DNMT1's N-terminal domain function offers insights into epigenetic regulation and potential cancer therapeutic strategies.

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