Associated Principal Investigator, ZMMK
Concerted changes of gene expression levels underly essential cellular processes such as cellular differentiation, but are also seen in many diseases, causing e.g. the uncontrolled proliferation and invasive behavior of cancer cells. At the molecular basis of gene regulation, the chemical modification of histone proteins is known to be of central importance for establishing and maintaining gene expression patterns. We are using structural biology and biochemistry to better understand how the enzymes that chemically modify histone proteins are regulated through allosteric mechanisms, their incorporation into large macromolecular assemblies, as well as their genomic targeting and interactions with chromatin.
Our research: Genetic information is present in each eukaryotic cell in the physical form of chromatin, i.e. the DNA itself and associated proteins that serve structural and, importantly, gene regulatory roles. Histones are not only the basis of DNA packaging into chromatin by being the core of nucleosomes, but are also targets of chemical modifications. Histone post-translational modifications (PTMs) serve distinct roles in governing genetic processes on the basis of which amino acid is modified with which chemical moiety. A large number of essential cellular processes is governed by the establishment and dynamic reconfiguration of chromatin marks, such as cellular differentiation during development and tissue regeneration. Consequently, the enzymes that attach and remove histone PTMs play key roles in orchestrating these processes and have to be active in the right time and place. Their activity depends on the interactions with chromatin, i.e. DNA, histones and their modifications, as well as a complex intermolecular cross-talk with other epigenetic factors. These interactions underly the specificity and spatiotemporal regulation of histone PTMs and, consequently, epigenetic regulatory processes.
Our goals: We aim to uncover how dynamic interactions chromatin modifying enzymes with their chromatin substrates as well as within multi-subunit macromolecular complexes orchestrate their context specific function. In particular, we strive to better understand the determinants of substrate recognition, the activity switches that ensure genomic and temporal speficity of histone modifications, as well as the interplay between present histone PTMs and modifying activity rates. The size and complexity of chromatin modifying complexes is one of the main challenges in understanding their function. Therefore, a significant part of our efforts is directed towards establishing ways of isolating native complexes and analyzing intermolecular interactions with little experimental bias that conventional overexpression systems may introduce. Based on such information, we are reconstituting defined complexes, including recombinant chromatin substrates, in vitro to dissect the mechanisms that govern enzymatic activity.
Among our main interests are histone methyltransferases (HMTases) and demethylases targeting lysine 4 of histone H3, the methylation of which is associated with enhancer elements and actively transcribed genomic regions. Chromatin modifiers targeting these residues frequently play pivotal roles in cellular differentiation and are commonly misregulated in pathologies such as cancer. We are seeking to specify how the composition and regulation of these complexes can be linked to their physiological function, but also how alterations of these factors contribute to pathological and ageing-related phenomena.
Our successes: Before starting our independent research group, Simon Pöpsel has structurally elucidated chromatin engagement by the human epigenetic repressor Polycomb Repressive Complex 2 (PRC2). These studies represent an unprecented depiction of chromatin binding in the context of a complex chromatin substrate, showing how PRC2 is able to productively engage with two nucleosomes at the same time in diverse geometrical contexts. Our previous work has paved the way for our present research efforts aiming at unravelling the molecular determinants of the recruitment and local activity regulation of other multi-subunit chromatin modifiers. In particular, the isolation and sample preparation of chromatin-associated complexes for cryo-EM remains a challenge and still is a bottleneck in chromatin structural biology. Therefore, advanced sample preparation techniques which have been successfully applied and established in previous work now put us in a unique position to further our molecular understanding of epigenetic regulation.
Our methods/techniques: Cryo-electron microscopy (cryo-EM) is a powerful method to uncover the dynamics and functional regulation of macromolecular assemblies through the elucidation their molecular structure. In particular, cryo-EM, as opposed to other structural biology techniques, enables structure determination of large and flexible complexes that are available in limited quantities. Among other advantages, this makes cryo-EM an extremely promising approach to chromatin modifying complexes which typically fall into this category and have therefore resisted comprehensive structural analyses in the past. To this end, we use CRISPR/Cas9 edited cell lines to isolate macromolecular complexes from endogenous sources, i.e. human cultured cells and analyze their structure, activity and dynamics. Heterologous overexpression systems such as E. coli and insect cells are employed to obtain, reconstitute and study chromatin modifying complexes and their variants in vitro.
In addition to and complementing our structural approach, we are incoporating an array of biochemical techniques such as enzymatic activity assays to mechanistically explore the function and regulation of chromatin modifying complexes. Furthermore, we are establishing cellular assays that provide an indispensable functional read-out of chromatin modifications in steady-state, as well as under pathological conditions or, e.g. using models of therapeutic interventions in cancer.
Figure 1: Previous work performed in the laboratory of Prof. Eva Nogales at UC Berkeley (cryoem.berkeley.edu) shows how the human methyltransferase complex PRC2 recognizes two distinct nucleosomes simultaneously, leading to allosteric activation by one and modification of the other nucleosome at the same time. We are employing similar techniques here to elucidate the structure and activity regulation of other human chromatin modifying complexes.