Epigenetic regulation is fundamental to both development and disease, shaped by a complex interplay of environmental factors. When epigenetic processes become dysregulated, they can significantly increase susceptibility to developmental defects and common diseases such as cancer, mental disorders, and diabetes. Epigenetics regulates gene activity without altering DNA sequences and encompasses mechanisms such as DNA methylation, histone modifications, non-coding RNAs, and chromatin organization. Higher-order chromatin structure is emerging as a prominent epigenetic mechanism for controlling genetic events. Liquid-liquid phase separation (LLPS) is increasingly recognized as a major driving force in chromatin organization. Unlike genetic mutations, epigenetic modifications are reversible and can be precisely targeted, making them valuable as prognostic and diagnostic markers, as well as potential therapeutic targets.
Dr. Xiaojun Ren’s laboratory has made significant contributions to our understanding of LLPS in genome organization. The Ren lab was among the first to demonstrate that LLPS is an emerging concept in genome organization. The decoding of genetic activities is highly dynamic. Live-cell single-molecule imaging allows for the direct observation of gene activities. Dr. Ren’s laboratory developed and applied live-cell single-molecule tracking to quantitatively measure the dynamic processes of epigenetic factors. In addition to single-molecule approaches, Dr. Ren’s laboratory employs multidisciplinary techniques to address fundamental questions in the field of epigenetics and chromatin biology. These include genomics methods such as CUT&RUN, CUT&Tag, Micro-C, Hi-C, and RNA-seq, as well as proteomics and genetic engineering tools like CRISPR/Cas9, among others.
Ongoing Research Topics:
1. Exploring the Roles of Liquid-Liquid Phase Separation in Gene Regulation: We have shown that Polycomb group (PcG) proteins can organize chromatin through LLPS (Tatavosian et al., Journal of Biological Chemistry, 2019, 294(5), 1451-1463). Additionally, we demonstrated that LLPS accelerates the target search process (Kent et al., Cell Reports, 2020, 33(2)). Our research further revealed that Polycomb condensates assemble in a composition- and concentration-dependent manner (Brown et al., Cell Reports, 2023, 42(10)). Moreover, we uncovered that LLPS plays critical roles in modulating the deposition and spreading of the H3K27me3 mark (Ingersoll et al., 2024, bioRxiv). Building on these findings, we are now investigating how LLPS influences gene expression and its broader implications for development and disease.
2. Single-Molecule Imaging of Epigenetic Processes: Epigenetic regulation is a highly dynamic process, requiring epigenetic factors to efficiently locate and bind to specific chromatin sites. Observing these interactions in real-time is essential to understanding how these factors execute their regulatory functions. Using live-cell single-molecule tracking, we discovered that CBX-PRC1 complexes are targeted to chromatin through a combination of H3K27me3 recognition and DNA binding (Zhen et al., 2016, eLife, 5, e17667). Our work also revealed that the H3K27M mutation in DIPG brain tumors disrupts the target-search process of PRC2, challenging the previously suggested “trap” mechanism (Tatavosian et al., Nature Communications, 9(1), 2080). Additionally, we demonstrated that LLPS enhances the target-search efficiency of epigenetic factors (Kent et al., Cell Reports, 33(2)). We are continually advancing and applying single-molecule techniques to visualize and quantify these processes with unprecedented resolution, providing new insights into the mechanisms of epigenetic regulation.
3. Epigenetic Mechanisms in Cancers: Over 80% of diffuse intrinsic pontine gliomas (DIPGs) harbor a point mutation in histone H3.3, where lysine 27 is substituted with methionine (H3.3K27M). However, how this mutation affects the function of PcG proteins remains unclear. We have demonstrated that H3.3K27M prolongs both the residence time and the search time of Ezh2, while having no effect on its fraction bound to chromatin. In contrast, H3.3K27M has no effect on the residence time of Cbx7, but it prolongs its search time and decreases its fraction bound to chromatin (Tatavosian et al., Nature Communications, 2018, 9(1), 2080). LLPS plays a critical role in the spatial organization of PcG proteins (Brown et al., Cell Reports, 2023, 42(10)), and we have shown that this spatial organization is essential for controlling the occupancy of PcG proteins (Kent et al., Cell Reports, 2020, 33(2)). Currently, we are investigating the roles of PcG protein LLPS in DIPG.
4. Therapeutic Targeting of Epigenetic Modifications and LLPS: Dysregulation of epigenetic mechanisms is implicated in a range of diseases, including cancers, neurological disorders, and developmental abnormalities. PcG proteins, which play a key role in gene repression, are known to undergo LLPS to organize chromatin structure and regulate gene expression. This spatial organization is essential for the proper functioning of epigenetic regulators. Targeting LLPS in PcG complexes offers a promising therapeutic strategy, as modulating phase separation could disrupt aberrant gene expression patterns associated with disease. Therapeutics designed to influence LLPS could provide new avenues for treating cancers and other conditions driven by epigenetic dysregulation. We are actively investigating approaches to modulate the LLPS of PcG complexes as part of our research efforts.
