The epigenome is a complex regulatory network that involves chemical modifications on histones and DNA. Cytosine methylation is the major DNA modification and plays an essential role in chromatin structure and gene expression. De novo DNA methylation in mammals is carried out by the de novo DNA methyltransferases (DNMTs), DNMT3A and DNMT3B, which require catalytically inactive variants, DNMT3L or DNMT3B3, for activity. Although the partial structures of mammalian DNMT3 with free DNA have been solved, the molecular mechanisms of DNA methylation in the context of chromatin are still unclear. Our structure of the first-ever cryo-EM structure of a nucleosome-bound human de novo DNMT complex surprisingly revealed that the DNMT complex binds to the nucleosome core and linker DNA at the same time. However, it remains unclear how DNMTs can access DNA that is blocked by nucleosomes and regulated by histone modifications. The steric constraints of our ternary complex structure suggest that chromatin remodelers might synergistically work with DNMTs to methylate nucleosomal DNA. Genome-wide methylation data, disease-causing mutations, and protein-protein interaction data suggest that the chromatin remodeler plays a key role in DNA methylation by DNMTs.
My lab is dedicated to contributing to our understanding of the molecular mechanism of DNA methylation in the context of chromatin and providing new insights into the regulation of gene expression and epigenetic modification in mammalian development and disease.
Our favorite topics are:
Molecular Basis for DNMT3 Complexes Binding and Catalyzing Nucleosome Substrates
Structures and Mechanisms of Remodeler-Mediated DNA Methylation on Chromatin
Chromatin-Associated Protein Complexes
CTCF binding to its motif
CpG methylation typically occurs on both strands of duplex DNA and plays a crucial role in mammalian development and differentiation. However, in embryonic stem (ES) cells, a subset of the CCCTC-Binding Factor (CTCF) binding sites is heritably hemimethylated. Yet, the precise function of this asymmetric hemimethylation remains unclear. To investigate the role of CTCF in hemimethylated DNA motifs, I previously optimized the protein expression system for human CTCF proteins and used the fluorescence polarization (FP) DNA binding assay to investigate the binding of CTCF to its motifs with different CpG DNA methylation states. Through this analysis, we identified two key residues (S364 and V454) in the CTCF protein that can sense the hemimethylation states of the CTCF DNA motif on its opposite strand. These findings were subsequently validated by site-specific CTCF mutants in FP assays. These results suggest a possible mechanism for asymmetric cell division involved in stem cell differentiation.
Our lab is interested in exploring the collaboration in the CTCF-DNA complex and its roles in chromatin organization and gene regulation.
G-protein coupled receptors
G protein-coupled receptors (GPCRs) are a vast family of cell surface receptors that respond to a variety of external signals. Due to their involvement in numerous signaling pathways, GPCRs are an important drug target. To gain insights into GPCR cell biology, I previously established and optimized a cell-based super-TOPflash luciferase assay, GPCR signaling assays, and cell surface biotinylation assays. Furthermore, I overcame technical challenges in the expression and formation of the human cannabinoid receptor CB2-Gi signaling complex and solved the first-ever CB2-Gi complex structure which revealed the CB2-Gi binding features and the different activation mechanisms of CB2 and CB1. Meanwhile, I also collaborated with others on structural and functional studies of challenging GPCR signaling complexes.
Our lab is interested in exploring collaboration in the GPCR complex drug discoveries.
Inactive AMPK
(Science. 2021; Protein Science. 2021)
AMP-activated protein kinase (AMPK) is a key sensor of energy status in eukaryotes. Its dynamic structure is regulated by allosteric factors including phosphorylation and binding of nucleotides and metabolites. Deregulation of AMPK is associated with metabolic diseases, and AMPK is a promising pharmacological target for the treatment of diabetes, obesity, cancer, and cardiometabolic disease. By solving the first-ever structure of an AMPK complex in an inactive ATP-bound state, we elucidated that the mechanism of the AMPK inactive-active cycle occurs through modulation of activation loop phosphorylation and accessibility.
γ-Secretase is an intramembrane aspartyl protease that is essential in the cleavage of the transmembrane fragment C99 of amyloid precursor protein (APP) to generate extracellular Aβ peptides. These peptides can oligomerize and aggregate to form amyloid plaques that are widely considered a primary cause of Alzheimer’s disease. During my graduate work, I focused on investigating the mechanism of how APP is recognized and processed by γ-secretase. Through the CRISPR/Cas9 technique and intensive mutagenesis, I defined a charge-recognition model for γ-secretase. Moreover, I established a novel, cell-based assay to investigate the initial cleavage of C99 by γ-secretase in living cells, called γ-secretase epsilon-cleavage assay. Additionally, I deciphered the C99 homodimerization mechanism in living cells. These findings and the models have significant implications for understanding how diverse substrates are recognized and processed by γ-secretase.