Mechanisms of Genome Compartmentalization

The packaging of eukaryotic DNA into chromatin allows differential compartmentalization of the same genome into transcriptionally active (euchromatic) and repressed (heterochromatic) states. Such compartmentalization allows diverse transcriptional programs to arise from a single genetic blue print. Mis-regulation of chromatin states is strongly linked to developmental defects and diseases like cancer. However much remains unknown at a mechanistic level about how specific chromatin states are established and maintained. A fundamental challenge in mechanistic dissection is the complexity of the chromatin template. This complexity is present at different scales of chromatin organization. At the smallest scale is a nucleosome, the basic repeating unit of chromatin, which contains ~147 bp of DNA wrapped around an octamer of histone proteins. Towards the larger scale of chromatin organization are structures that entail the folding of several nucleosomes. These folded structures are proposed to be repressive for transcription. Higher-order chromatin folding is enabled in part by general chromatin-binding proteins such as linker histones and by heterochromatin proteins such as HP1 proteins. At a molecular level, how chromatin-binding proteins such as HP1 enable chromatin folding and how folding affects function is poorly understood.

Recent work is revising current conceptions of chromatin structure at the both scales. At the nucleosome scale, it has typically been assumed that the component histones assemble together like rigid Lego blocks to give the stereotypical histone octamer conformation seen in crystal structures. However work with ATP-dependent chromatin remodelers has uncovered unexpected plasticity within the histone core and indicated raising new possibilities for chromatin regulation. For example nucleosome deformability could help explain why histone variants have distinct in vivo functions, why there are histone modifications on buried histone residues and how factors termed as pioneer factors recognize nucleosomal DNA sequences without displacing histones. At the larger chromatin organization scale, textbooks have traditionally described these structures as arising from hierarchical folding of multiple nucleosomes into defined 30 nm wide fibers followed by further higher order folding. However current studies suggest that 30 nm chromatin structures exist only in certain differentiated cells. In a majority of cell types investigated 30 nm fibers or other regularly folded structure have not been detected. The absence of repeating higher order structures is consistent with theoretical models suggesting that macromolecular crowding can lead to chromatin organization via phase-separation like processes. Recent work with the human heterochromatin protein HP1α provides experimental evidence for such a possibility. In this work HP1α was shown to form phase-separated droplets in vitro that enclose chromatin. These findings suggest a new way to silence chromatin, via sequestration in phase-separated HP1 bodies. Such a mechanism, is likely to be qualitatively different than silencing based on hierarchical chromatin folding, and raises new questions and regulatory possibilities. We are specifically addressing the following general questions:

1. What is the role of phase-separation in heterochromatin regulation?
2. What is the dynamic range of the material properties of phase-separated heterochromatin bodies?
3. What is the chemical environment within the droplets?