Heterochromatin protein 1 alpha (HP1α) is a central factor in establishing and maintaining the heterochromatin state. From interaction with tri-methylation on histone 3 lysine 9 (H3K9Me3) HP1α spreads along the chromatin fibre and then through self-association HP1α dimers bridge adjacent nucleosomes and compact chromatin them into phase separated liquid condensates. While there is extensive biochemical evidence on how HP1α initiates and propagates heterochromatin in vitro we still do not know how exactly this architectural protein regulates chromatin compaction and gene expression in the context of a living cell. Thus here we employ fluorescence lifetime imaging microscopy (FLIM) to detect förster resonance energy transfer (FRET) between fluorescently labelled histones (Lou et al. 2019 PNAS), and dissect in HP1α CRISPR/Cas9 knock out Hela cells, how HP1α monomers versus dimers regulate the architectural organisation of the cell. Intriguingly we demonstrate that while HP1α dimers compact chromatin, surprisingly, HP1α monomers de-compact chromatin and totally abolish heterochromatin structure by imparting a specific nucleosome spacing. Fluorescence fluctuation spectroscopy (FFS) based studies confirmed this result and demonstrate the HP1 α monomer to be a critical component in modulating the accessibility of the DNA template. In conclusion, this study reveals an entirely new insight into how HP1α regulates chromatin structure, heterochromatin formation and chromatin accessibility in a living cell, which further reonciles how HP1 can both up and down-regulate gene expression in physiological and disease conditions in addition to the HP1 mediated chromatin phase separation theory.
Lou, J, L. Scipioni, B. K. Wright, T. K. Bartolec, J. Zhang, V. P. Masamsetti, K. Gaus, E. Gratton, A J. Cesare, E. Hinde*. 2019. Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response. PNAS. 116(15):7323-7332.