Nucleosome linker DNA methylation by DNMT3A/DNMT3B3 is controlled by nucleosome binding and multimerization of DNMT3...

Structural and biochemical studies showed that DNMT3A/DNMT3B3 (3A/3B3) heterotetramers directly interact with the nucleosomal acidic patch via the DNMT3B3 subunit. Here, we investigated linker DNA methylation by 3A/3B3 using dinucleosome substrates as most suitable mimic of linker chromatin methylation in cells. Dinucleosomes with different linker lengths and sequence were used and DNA methylation was investigated quantitatively by bisulfite sequencing. The effects of nucleosomal recruitment were investigated using 3A/3B3 C-terminal domain complexes and complexes containing the R740E/R743E double mutation in DNMT3B3 which affects the two most important residues in the DNMT3B3-acidic patch contact. Using competitive methylation assays of nucleosomal and free DNA, we demonstrate that the contact to the acid patch improves 3A/3B3 recruitment to nucleosomes and methylation of linker DNA. Characteristic methylation levels of CpG sites next to the nucleosomes suggest that 3A/3B3 complexes are anchored on both sides of the linker DNA to nucleosomes. However, detailed analysis of linker DNA methylation levels revealed nucleosome dependent methylation patterns even at CpG sites that are not in direct proximity to the nucleosomes suggesting that DNMT3A complexes multimerize on the linker DNA. This multimerization spatially organizes the complexes, aligning active sites of DNMT3A complexes with CpG sites, which then leads to the observed methylation patterns. Moreover, product DNA molecules with high methylation levels were strongly overrepresented also indicating that DNMT3A fiber formation leads to cooperative linker DNA methylation. Our data suggest that multimerization of DNMT3A on linker DNA could shape the DNA methylation landscape in cells with potential implications on nucleosome positioning particularly in heterochromatic regions.