Chromatin StructureDNA Methylation and Hydroxymethylation

Choosy DNA Methyltransferases Choose CpG-Dense Regions

DNMT3While changes in DNA methylation have been implicated in a variety of diseases, there is still much to learn about the cellular regulation of DNA methylation patterns across the genome. De novo methyltransferases DNMT3A and B are known to establish DNA methylation. In a recent paper, Baubec et al. investigated the de novo methyltransferase activity and genomic binding sites of DNMT3A and DNMT3B in mouse embryonic stem cells. The authors found that both DNMT3A and 3B localize to methylated CpG-dense region, but were excluded from active promoters and enhancers.

To establish the genomic binding patterns of the DNMT3 isoforms, the group performed chromatin immunoprecipitation using biotin-tagged DNMTs in mouse embryonic stem cells. Sequencing showed that both enzymes preferentially bound CpG-rich regions. Furthermore, DNMT3B1 binds primarily gene bodies and prefers actively transcribed genes as noted by features of active transcription such as mRNA, RNA pol II, and H3K36me3.

To investigate the connection between the binding patterns and enzymatic activity, the group created a triple knockout (TKO) mouse model, and then reintroduced DNMTs to the TKO cells. Using mass spectrometry, the group found reproducible and detectable rates of methylation following reintroduction of DNMT3A2 or DNMT3B1 to mouse Dnmt gene triple knockout (TKO) cells. They further validated and confirmed the findings of the mass spectrometry analysis by using a whole genome bisulfite sequencing analysis, which revealed that genomic binding sites for the enzymes remained the same. Therefore, recruitment of DNMT3A and DNMT3B to CpG dense regions is independent of existing methylation states. Additionally, de novo methylation was found to be higher in adjacent CpGs and decreased with distance, suggesting that enzyme behavior was processive.

The authors also investigated the relationship DNMT3B1 binding and transcriptional activity levels. Differentiation of embryonic stem cells to neuroprogenitor cells resulted in simultaneous increases in H3K36me3 binding as well as DNMT3B binding. Another experiment showed that DNMT3B1 continues to bind H3K36me3-positive gene bodies. Additionally, interactions experiments found that the amino-terminal end of DNMT3B interacts with H3K36 methylated nucleosomes. Mutations in this domain resulted in loss of recruitment to active gene bodies. Taken together, these experiments suggest that H3K36 methylation is a requirement for DNMT3B binding.

Future studies are needed to determine additional factors in the binding site preferences of DNMT3A2 and DNMT3B considering both enzymes have highly similar binding domains. Because other studies have revealed increased levels of 5-hmC in transcribed gene bodies, the researchers suggest the targeted DNMT activity may serve to compensate for active demethylation in these regions. The researchers conclude by speculating that methylation recruitment through co-transcriptional histone modifications may generally account for DNA methylation in gene bodies.

In conclusion, the authors found that both sequence and chromatin features guide de novo methyltransferase activity.

While changes in DNA methylation have been implicated in a variety of diseases, there is still much to learn about the cellular regulation of DNA methylation patterns across the genome. De novo methyltransferases DNMT3A and B are known to establish DNA methylation. In a recent paper, Baubec et al. investigated the de novo methyltransferase activity and genomic binding sites of DNMT3A and DNMT3B in mouse embryonic stem cells. The authors found that both DNMT3A and 3B localize to methylated CpG-dense region, but were excluded from active promoters and enhancers.

To establish the genomic binding patterns of the DNMT3 isoforms, the group performed chromatin immunoprecipitation using biotin-tagged DNMTs in mouse embryonic stem cells. Sequencing showed that both enzymes preferentially bound CpG-rich regions. Furthermore, DNMT3B1 binds primarily gene bodies and prefers actively transcribed genes as noted by features of active transcription such as mRNA, RNA pol II, and H3K36me3.

To investigate the connection between the binding patterns and enzymatic activity, the group created a triple knockout (TKO) mouse model, and then reintroduced DNMTs to the TKO cells. Using mass spectrometry, the group found reproducible and detectable rates of methylation following reintroduction of DNMT3A2 or DNMT3B1 to mouse Dnmt gene triple knockout (TKO) cells. They further validated and confirmed the findings of the mass spectrometry analysis by using a whole genome bisulfite sequencing analysis, which revealed that genomic binding sites for the enzymes remained the same. Therefore, recruitment of DNMT3A and DNMT3B to CpG dense regions is independent of existing methylation states. Additionally, de novo methylation was found to be higher in adjacent CpGs and decreased with distance, suggesting that enzyme behavior was processive.

The authors also investigated the relationship DNMT3B1 binding and transcriptional activity levels. Differentiation of embryonic stem cells to neuroprogenitor cells resulted in simultaneous increases in H3K36me3 binding as well as DNMT3B binding. Another experiment showed that DNMT3B1 continues to bind H3K36me3-positive gene bodies. Additionally, interactions experiments found that the amino-terminal end of DNMT3B interacts with H3K36 methylated nucleosomes. Mutations in this domain resulted in loss of recruitment to active gene bodies. Taken together, these experiments suggest that H3K36 methylation is a requirement for DNMT3B binding.

Future studies are needed to determine additional factors in the binding site preferences of DNMT3A2 and DNMT3B considering both enzymes have highly similar binding domains. Because other studies have revealed increased levels of 5-hmC in transcribed gene bodies, the researchers suggest the targeted DNMT activity may serve to compensate for active demethylation in these regions. The researchers conclude by speculating that methylation recruitment through co-transcriptional histone modifications may generally account for DNA methylation in gene bodies.

In conclusion, the authors found that both sequence and chromatin features guide de novo methyltransferase activity.

 

Original article:

Baubec T, Colombo DF, Wirbelauer C, Schmidt J, Burger L, Krebs AR, Akalin A, & Schübeler D (2015). Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation. Nature, 520 (7546), 243-7 PMID: 25607372

 

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Emily P.

Emily P.

Emily was born and raised in Southern California. Outside of the lab, Emily enjoys exploring museums, going to concerts and traveling.