Research

TOPIC #1

Structural Studies of Epigenetic Regulators in Complex with the Nucleosome

A nucleosome is a basic unit of chromatin. It consists of ~150 base-pairs of DNA wrapped around a histone octamer.

Histone octamers are composed of two copies of each of individual histones H3, H4, H2A, and H2B. In addition to the genetic-level regulation mediated by transcriptional factors, enhancers, etc., epigenetic regulators modulate developmental-stage specific, cell-type specific gene expressions via reversible chemical modifications on DNA and/or nucleosomes. This often leads to change in the chromatin architecture, known as euchromatin and heterochromatin, thereby systematically regulating gene expression without genetic alteration.

My laboratory recently determined the cryo-EM structure of the human Mixed-lineage Leukemia 1 (MLL1) core complex bound to the nucleosome (Park et. al., 2019, Nat. Comm.). MLL1 is a histone H3 lysine 4 (H3K4) methyltransferase, and H3K4 methylation appears highly enriched at transcriptional regulatory regions, including promoters and distal regulatory enhancers. Aberrant expression of the core components (e.g., ASH2L, WDR5) of MLL complexes has been reported in a vast majority of human tumors of different origins and contributes to disease progression and prognosis. Our cryo-EM structure could address the molecular mechanism of how the MLL1 core complex uniquely recognizes the nucleosome and methylates its substrate, histone H3 tails.

We aim to continue our efforts to elucidate the molecular mechanisms by which epigenetic regulators modulate its substrate–nucleosome by visualizing them using cryo-EM approach.

TOPIC #2

Illustrating the Biochemical and Structural Role of Human Sestrin 2 and GATOR1/2 in mTORC1 Regulation

Constant exposure to a high nutrient diet and increased insulin levels often leads to type II diabetes and non-alcoholic fatty liver diseases. The mechanistic target of rapamycin complex 1 (mTORC1) plays a central role in this regulation and therefore has long been considered as an attractive target for type II diabetes.

In this project, we particularly focus on understanding the nutrient- and stress-dependent mTORC1 regulation pathway mediated by Sestrins, a stress-inducible protein family, using multi-directional approaches including x-ray crystallography, single particle cryo-electron microscopy and cell biology. Structural and biological studies of Sestrin and signaling intermediates of mTORC1, GATOR1 and GATOR2, will not only reveal the fundamental mechanism of how nutrient and stress can modulate mTORC1, but also provide the molecular platform to develop knowledge-based anti-diabetic medicines by targeting this pathway.

TOPIC #3

Development of cryo-EM Grid to Optimize the Sample Preparation Step in cryo-EM

Over the last decade, single-particle cryogenic electron microscopy (sp cryo-EM) has become a major technology to determine the structures of macromolecular complexes, otherwise difficult to solve using traditional approaches, such as x-ray crystallography or nuclear magnetic resonance.

Recent advances in cryo-EM instruments and software algorithms have enabled us to tackle and determine more challenging protein structures at the atomic resolution level. Despite these achievements, a major bottleneck still remains, particularly at the sample preparation step.

In this project, we aims to isolate the endogenous, physiologically active macromolecules directly from the eukaryotic cells and visualize them at the atomic level using sp cryo-EM. To achieve this goal, we are currently developing the graphene-coated grid and the affinity grid.

Download ‘the grid holder’ 3D print model (stl format)

36 graphene grid making video

Download ‘the reaction chamber’ 3D print model (stl format)

Resources

Useful Link

Recommended texts for x-ray crystallography & cryo-electron microscopy

Rupp B. (2009). Biomolecular Crystallography: Principles, Practice, and Application to Structural Biology. Garland Science, New York, NY.
Drenth, J. (2007 or 1999). Principles of protein x-ray crystallography. Springer, New York (3rd or 2nd edition)
Stout Jensen. X-ray structure determination. A Practical Guide, Wiley Interscience (2nd edition)
Giacovazzo et al. Fundamentals of crystallography. Oxford Univ. (2nd edition)
Gale Rhodes. Crystallography Made Crystal Clear. A Guide for Users of Macromolecular Models. (3rd Edition)
Joachim Frank. Three-Dimensional Electron Microscopy of Macromolecular Assemblies: Visualization of Biological Molecules in Their Native State. Oxford Univ. (2nd edition)

RESEARCH