Biology subjects
Publications and source records attributed to Underhill, D. A..
Withdrawal StatementThe authors have withdrawn this manuscript due to a duplicate posting of manuscript number BIORXIV/2019/776625. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author. The correct preprint can be found at doi:10.1101/776625.
The association of nuclear DNA with histones to form chromatin is essential to the temporal and spatial control of eukaryotic genomes. In this study, we examined the physical state of chromatin in vitro and in vivo. Our in vitro studies demonstrate that MgCl2-dependent self-association of native chromatin fragments or reconstituted nucleosomal arrays produced supramolecular condensates whose constituents are physically constrained and solid-like. Liquid chromatin condensates could be generated in vitro, but only using non-physiological conditions. By measuring DNA mobility within heterochromatin and euchromatin in living cells, we show that chromatin also exhibits solid-like behavior in vivo. Representative heterochromatin proteins, however, displayed liquid-like behavior and coalesced around a solid chromatin scaffold. Remarkably, both euchromatin and heterochromatin showed solid-like behavior even when transmission electron microscopy revealed limited interactions between chromatin fibers. Our results therefore argue that chromatin is not liquid but exists in a solid-like material state whose properties are tuned by fiber-fiber interactions.
The pericentromere exists as a distinct chromatin compartment that is thought to form by a process of phase separation. This reflects the ability of the heterochromatin protein CBX5 (aka HP1) to form liquid condensates that encapsulate pericentromeres.1,2 In general, phase separation compartmentalizes specific activities within the cell, but unlike membrane-bound organelles, their contents rapidly exchange with their surroundings.3 Here, we describe a novel state for the lysine methyltransferase KMT5C where it diffuses within condensates of pericentromeric heterochromatin but undergoes strikingly limited nucleoplasmic exchange, revealing a barrier to exit similar to that of biological membranes. This liquid-like behavior maps to a discrete protein segment with a small number of conserved sequence features and containing separable determinants for localization and retention that cooperate to confer strict spatial control. Accordingly, loss of KMT5C retention led to aberrant spreading of its catalytic product (H4K20me3) throughout the nucleus. We further found that KMT5C retention was reversible in response to chromatin state, which differed markedly for CBX5 and the methyl-CpG binding protein MeCP2, revealing considerable plasticity in the control of these phase separated assemblies. Our results establish that KMT5C represents a precedent in the biological phase separation4 continuum that confers robust spatial constraint of a protein and its catalytic activity without progression to a gel or solid.
The PAX (paired box) family is a collection of metazoan transcription factors defined by the paired domain, which confers sequence-specific DNA-binding. Ancestral PAX proteins also contained a homeodomain, which can communicate with the paired domain to modulate DNA-binding. In the present study, we sought to identify determinants of this functional interaction using the paralogous PAX3 and 7 proteins. First, we evaluated a group of heterologous paired domains and homeodomains for the ability to bind DNA cooperatively through formation of a ternary complex (paired domain:homeodomain:DNA). This revealed that capacity for ternary complex formation was unique to the PAX3 and 7 homeodomains and therefore not simply a consequence of DNA-binding. We also found PAX3 and 7 were distinguished by an extended region of conservation N-terminal to the homeodomain (NTE). Phylogenetic analyses established the NTE was restricted to PAX3/7 orthologs of segmented metazoans, indicating it arose in a bilaterian precursor prior to separation of deuterostomes and protostomes. In DNA-binding assays, presence of the NTE caused a decrease in monomeric binding by the PAX3 homeodomain that reflected a lack of secondary structure in 1D-1H-NMR. Nevertheless, this inhibitory effect could be overcome by homeodomain dimerization or cooperative binding with the paired domain, establishing that protein interactions could induce homeodomain folding in the presence of the NTE. Strikingly, the PAX7 counterpart did not impair homeodomain binding, revealing inherent differences that could account for its distinct target profile in vivo. Collectively, these findings identify critical determinants of PAX3 and 7 activity, which contribute to their functional diversification.