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The N-terminal region of DNMT3A combines multiple chromatin reading motifs to guide recruitment

ABSTRACT: DNA methyltransferase 3A (DNMT3A) plays a critical role in establishing and maintaining DNA methylation patterns. However, the mechanisms underlying DNMT3A recruitment to and function within different chromatin environments remain unclear. Using a combination of biochemical and structural approaches we find that DNMT3A interacts using multiple interfaces with chromatin; directly binding generic nucleosome features as well as site-specific post-translational histone modifications. The N-terminal region, unique to the DNMT3A1 isoform, is essential for these interactions and stabilises H3K36me2-nucleosome recruitment. Intriguingly, in the same region critical for nucleosome binding we also map a ubiquitylation-dependent recruitment motif (UDR). The UDR binds specifically to ubiquitylated H2AK119, explaining the previously observed recruitment to Polycomb-occupied heterochromatin. A cryo-EM structure of DNMT3A1-DNMT3L with a modified nucleosome reveals that the UDR interacts with the nucleosome surface including the acidic patch. Previously unexplained disease-associated mutations are present in the UDR and ablate nucleosome interactions. This leads to an increased understanding of how DNMT3A1 recruitment occurs in the genome and highlights the importance of multivalent binding of DNMT3A to histone modifications and the nucleosome.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Christos Spanos

LAB HEAD: Marcus Wislon

PROVIDER: PXD056554 | Pride | 2024-10-16

REPOSITORIES: Pride

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			Action	DRS
	Wapenaar_etal_2024_DNMT3A13L.fasta	Fasta
	Wapenaar_etal_2024_DNMT3A1_DNMT3L_EDC_1_15.mgf	Mgf
	Wapenaar_etal_2024_DNMT3A1_DNMT3L_EDC_1_15.raw	Raw
	Wapenaar_etal_2024_DNMT3A1_DNMT3L_EDC_1_15.xlsx	Xlsx
	Wapenaar_etal_2024_DNMT3A1_DNMT3L_EDC_1_30.mgf	Mgf

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Project description:Cryptococcus neoformans is an opportunistic, human fungal pathogen which undergoes fascinating switches in cell cycle control and ploidy when it encounters stressful environments such as the human lung. Here we carry out a mechanistic analysis of the spindle checkpoint which regulates the metaphase to anaphase transition, focusing on Mps1 kinase and the downstream checkpoint components Mad1 and Mad2. We demonstrate that Cryptococcus mad1D or mad2D strains are unable to respond to microtubule perturbations, continuing to re-bud and divide, and die as a consequence. Fluorescent tagging of Chromosome 3, using a lacO array and mNeonGreen-lacI fusion protein, demonstrates that mad mutants are unable to maintain sister-chromatid cohesion in the absence of microtubule polymers. Thus, the classic checkpoint functions of the SAC are conserved in Cryptococcus. In interphase, GFP-Mad1 is enriched at the nuclear periphery, and it is recruited to unattached kinetochores in mitosis. Purification of GFP-Mad1 followed by mass spectrometric analysis of associated proteins show that it forms a complex with Mad2 and that it interacts with other checkpoint signalling components (Bub1) and effectors (Cdc20 and APC/C sub-units) in mitosis. We also demonstrate that overexpression of Mps1 kinase is sufficient to arrest Cryptococcus cells in mitosis, and show that this arrest is dependent on both Mad1 and Mad2. We find that a C-terminal fragment of Mad1 is an effective in vitro substrate for Mps1 kinase and map several Mad1 phosphorylation sites. Some sites are highly conserved within the C-terminal Mad1 structure and we demonstrate that mutation of threonine 667 (T667A) leads to loss of checkpoint signalling and abrogation of the GAL-MPS1 arrest. Thus Mps1-dependent phosphorylation of C-terminal Mad1 residues is a critical step in Cryptococcus spindle checkpoint signalling. We conclude that CnMps1 protein kinase, Mad1 and Mad2 proteins have all conserved their important, spindle checkpoint signalling roles helping ensure high fidelity chromosome segregation.

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The N-terminal region of DNMT3A combines multiple chromatin reading motifs to guide recruitment

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