Plasticity and Memory of Chromatin Structure
Epigenetic mechanisms are of crucial importance for faithful transmission of gene expression states through cell division, and the maintenance of cellular identities from one generation to the next. These mechanisms also need to support the plasticity of gene expression to facilitate the acquisition of new cell fates in animal development. Chromatin modifications have emerged as important regulators of transcription, and are believed to contribute to the inheritance of gene expression states.
We investigate the dynamics and epigenetic inheritance of nucleosome modifications in the context of physiological chromatin structure in living cells. Chromatin undergoes constant remodeling to facilitate changes in gene expression and DNA accessibility in response to cell-intrinsic and cell-extrinsic stimuli. Specifically, the antagonizing activities of histone modifying complexes add and remove post-translational histone modifications, thus contributing to the dynamic organization of chromatin in regulatory regions of the mammalian genome.
Traditional genetic and biochemical analyses have yielded a largely static view of chromatin regulation. These approaches have failed to provide a comprehensive understanding of the actual function of chromatin modifications in gene regulation. Thus, separating cause from consequence will require approaches that delineate the sequence of events involved in gene induction or repression.
Chromatin in vivo assay (CiA)
We employ a novel technique using chemical inducers of proximity to dissect the sequence of events and measure histone modification kinetics at high resolution during cell-fate transitions, cellular reprogramming, and signal-dependent gene regulation. This technology integrates the complex nature of chromatin with precise biochemical analysis of the sequence of events during chromatin remodeling. We have generated a murine strain that permits rapid addition and removal of chromatin regulatory activities to a genetically modified Oct4 allele in any cell type using small-molecule-mediated recruitment (Figure 1). Chemically induced proximity (CIP) provides high temporal control, permitting the examination of the kinetics and epigenetic memory of histone modifications in single cell resolution.
Dynamics and memory of heterochromatin
In embryonic stem (ES) cells, Oct4 expression is essential for pluripotency and self-renewal. Upon differentiation, Oct4 is silenced. This involves the HP1 heterochromatin pathway (with H3K9 trimethylation) and the Polycomb pathway (with H3K27 trimethylation). Previously we investigated the kinetics of heterochromatin formation by recruiting HP1α to the modified Oct4 promoter in ES cells and fibroblasts. Tethering of HP1α induced gene repression and the formation of heterochromatic domains of up to 10kb. Measuring H3K9me3 changes after HP1α recruitment permitted the description of in vivo rates of heterochromatin spread in ES cells and fibroblasts. In addition, after HP1α removal we tested epigenetic properties and found that H3K9me3 can be faithfully transmitted through cell divisions (Figure 2). Yet, we also showed that the memory and spreading of H3K9me3 may be antagonized by transcriptional activators, indicating the high plasticity of chromatin regulation. Based on the balance between the antagonizing activities of H3K9me3 addition and removal, we proposed a mathematical model, which accurately expresses our empirical observation at the Oct4 locus and also predicts the dynamics of heterochromatin formation and turnover at the majority of facultative H3K9me3 domains in the mammalian genome.
The CiA system is a powerful approach to study the kinetic regulation of any chromatin modifying activity in any murine cell type and obtaining quantitative models for testing.