Selective anchoring of DNA methyltransferases 3A and 3B to nucleosomes containing methylated DNA.
Proper DNA methylation patterns are essential for mammalian development and differentiation. DNA methyltransferases (DNMTs) primarily establish and maintain global DNA methylation patterns; however, the molecular mechanisms for the generation and inheritance of methylation patterns are still poorly understood. We used sucrose density gradients of nucleosomes prepared by partial and maximum micrococcal nuclease digestion, coupled with Western blot analysis to probe for the interactions between DNMTs and native nucleosomes. This method allows for analysis of the in vivo interactions between the chromatin modification enzymes and their actual nucleosomal substrates in the native state. We show that little free DNA methyltransferase 3A and 3B (DNMT3A/3B) exist in the nucleus and that almost all of the cellular contents of DNMT3A/3B, but not DNMT1, are strongly anchored to a subset of nucleosomes. This binding of DNMT3A/3B does not require the presence of other well-known chromatin-modifying enzymes or proteins, such as proliferating cell nuclear antigen, heterochromatin protein 1, methyl-CpG binding protein 2, Enhancer of Zeste homolog 2, histone deacetylase 1, and UHRF1, but it does require an intact nucleosomal structure. We also show that nucleosomes containing methylated SINE and LINE elements and CpG islands are the main sites of DNMT3A/3B binding. These data suggest that inheritance of DNA methylation requires cues from the chromatin component in addition to hemimethylation.
Jeong
Shinwu
S
Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center Keck School of Medicine, University of Southern California, NOR 8302L, 9181, Los Angeles, CA 90089-9181, USA.
Liang
Gangning
G
Sharma
Shikhar
S
Lin
Joy C
JC
Choi
Si Ho
SH
Han
Han
H
Yoo
Christine B
CB
Egger
Gerda
G
Yang
Allen S
AS
Jones
Peter A
PA
D002460:Cell Line; D002843:Chromatin; D018899:CpG Islands; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D009707:Nucleosomes / Q000201:enzymology*; D011485:Protein Binding; D012091:Repetitive Sequences, Nucleic Acid; D000097584:DNA Methyltransferase 3B
D002843:Chromatin; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D007527:Isoenzymes; D009707:Nucleosomes; D000097584:DNA Methyltransferase 3B; C000717967:DNMT3A protein, human; C541545:DNA (cytosine-5-)-methyltransferase 3b2, human