Mechanisms of DNA methyltransferase 3A1-mediated DNA methylation of nucleosomes

DNA methyltransferase 3A1 (DNMT3A1) plays a crucial role in establishing DNA methylation patterns that regulate gene expression and drive cellular differentiation. In this study, we investigated the biochemical mechanisms underlying DNMT3A1’s interactions with nucleosomes, the fundamental units of chromatin. Using radiochemical activity assays, along with fluorescence anisotropy and AlphaLISA binding assays, we demonstrated that DNMT3A1 has multivalent interactions with nucleosomes, binding linker DNA and nucleosome cores. Nanopore-based 5mC sequencing revealed that DNMT3A1 interactions with the nucleosome stimulate methylation of linker DNA up to 24 bp away from the nucleosome core. We suggest that the DNMT3A1 PWWP domain interacts with distal linker DNA to facilitate uniform stimulation of linker methylation. Additionally, our results indicate that DNMT3A1 binding is restricted to a single nucleosome, suggesting that its activity is not allosterically regulated by neighboring nucleoso..., , # Dryad dataset

Dataset DOI: [10.5061/dryad.547d7wmkm](10.5061/dryad.547d7wmkm)

## Description of the data and file structure

The recombinant proteins DNMT3A1 and the catalytic domain, DNMT3ACD, were purified and unedited SDS\\-PAGE gel images of the purification are provided.

Recombinant nucleosomes were also synthesized with varying linker lengths and fluorophore modifications. SDS-PAGE gel image of the presence of all histones are provided. Nondenaturing EMSA gel images of the final reconstituted nucleosomes are provided.

### Files and variables

#### File: S2A.jpg

**Description:** DNMT3A1 purification

#### File: S2B.jpg

**Description:** DNMT3ACD purification

#### File: S1C.jpg

**Description:** Denaturing 18 % PAA SDS-PAGE gels stained with Coomassie confirmed the presence of histone components in reconstituted nucleosomes.

#### File: S1F_inverted.jpg

**Description:** The correctly positioned recombinant mononucleosomes with 50-bp and 100-bp linkers were protected from di..., ,