Cryo-EM structural study of mammalian DNA methylation
EMSL Project ID
51613
Abstract
The eukaryotic genome is organized into different functional compartments, with euchromatin representing an open and transcriptionally active state and heterochromatin representing a more compact and silenced state. Notably, the state of constitutive heterochromatin is characterized by both repressive histone modifications and DNA methylation. These modifications together underlie fundamental biological processes such as gene silencing, genomic stability, cell differentiation and development. However, the underlying mechanism for the crosstalk between these epigenetic marks remains unclear. In mammals, DNA methylation is established by two de novo DNA methyltransferases: DNMT3A and DNMT3B, during gametogenesis and early embryogenesis. Subsequently, DNA methylation is faithfully maintained by DNA methyltransferase 1 (DNMT1) in a replication-dependent manner. Previous studies have established that the activities of both DNMT3A/DNMT3B and DNMT1 are subjected to a spatiotemporal regulation in cells, which is essential for proper epigenetic programming during development. DNMT1, DNMT3A and DNMT3B are all multi-domain proteins containing, in addition to a C-terminal methyltransferase domain, a large N-terminal regulatory region comprised of multiple histone- and DNA-binding modules. Direct interaction of these regulatory domains with DNA and/or histone modifications not only regulates the genomic targeting of DNMT1, but also leads to allosteric regulation of its enzymatic activity. Our work in the past have addressed several key issues regarding the structure and regulation of DNMT1, DNMT3A and DNMT3B, including the autoinhibitory mechanisms of DNMT1, the molecular basis underlying the substrate recognition and specificity of DNMT1, DNMT3A and DNMT3B, and the functional impairment of DNMT3A mutation implicated in Acute Myeloid Leukemia. Building on this progress, we seek to solve the cryo-EM structure of DNMT1, DNMT3A and DNMT3B bound to nucleosome labeled with histone modifications, thereby identifying how the interaction between DNMTs and specific histone modifications regulates the conformations and activities of each individual DNMT. Successful completion of this application will provide key mechanistic insights into the mammalian DNA methylation. In preliminary studies, have already obtained negative stain data for DNMT1 and DNMT3A complexes. The grids for cryo-EM data collection for the DNMT1-NCP complex sample have also been prepared. We plan to perform single particle data collection of these DNMTs in complex with epigenetically modified NCPs, and expect to solve the cryo-EM structures with high resolution. Given that our samples have already been optimized to great quality, we hope to access Titan Krios with K2/K3 camera for data collection. We estimate that each experiment will take about one day for data collection. We expect that a cryo-EM data collection of the DNMTs-NCP complex at the Pacific Northwest Center for Cryo-EM will greatly facilitate their high-resolution structure determination.
Project Details
Start Date
2020-09-15
End Date
2021-03-17
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members