Structure and regulation of DNA condensates by disordered linker histone tails

Study of the fundamental factors controlling the transcriptional activity of genes underpins the basic biology of all organisms, and the processes can only be properly understood if the physical nature of condensed genomic DNA (chromatin) is well described. Given this, it is perhaps surprising that there is so much left to understand. Two meters of DNA are condensed into each cell nucleus. This is achieved in two stages. The first stage involves wrapping the DNA around protein spools to form "nucleosomes" that resemble beads on a string. The second stage involves the further condensation of this structure into one that is more compact; this stage is less well understood.

Biology can be approached from the top down (i.e. looking at cells) or bottom up (i.e. looking at atomic-level molecules); the hope is always that they join up to make a consistent picture, thus overcoming the inherent limitations and validating the model generated from each approach. Both approaches are necessary to fully understand most biological processes.

In the case of chromatin, there is currently a discontinuity concerning the second stage of condensation, since the 30 nm fibre predicted to form in the second stage by a bottom-up approach has not been observed by the best new imaging techniques applied to live cells. Instead, the fibre appears more open, flexible, disordered and heterogeneous, self-assembling into large chromatin globules with liquid-like properties. It seems likely that the highly ordered 30 nm fibre may represent an extreme case of condensed and inactive chromatin, and the more transcriptionally-relevant situation may be much more dynamic. This view is resonant with some alternative views that are emerging from different bottom-up approaches by us and others, that pick up on the high level of inherent disorder in the proteins that package DNA, and their ability to concentrate the DNA into dense liquid condensates, in which the dynamics of the fibre are retained. A liquid condensate is a compelling and more plausible means by which chromatin could respond quickly to environmental stimuli.

We have developed a model system that allows us to study, at atomic-level resolution, the way DNA-packaging proteins - specifically the 'linker histones' - bind to the DNA, and the conditions under which the protein/DNA complexes phase separate into dense liquid droplets. It also permits thermodynamic measurements. We would like to exploit this system to answer several key questions: exactly how is DNA bound and condensed by linker histones?; does the highly crowded environment of the resulting condensate allow the entry and action of modifying enzymes known to act in vivo, and how do the condensates respond?; how does the intrinsic disorder of the protein/DNA complexes facilitate rapid exchange by and with other chromatin-associated proteins?; what higher-order structures are present in the condensates, if any, and how is their assembly controlled and regulated?; what are the mechanistic similarities/differences in genome condensation between the many linker histone subtypes (there are for example 11 subtly different linker histones in humans that either come and go through the lifetime of a cell, or locate to sperm or egg) and DNA sequences differing in content and modifications, such as those marking the start of genes or those that are 'silenced' by the addition of methyl groups?

In summary, the condensation of the genome is a fundamental process that occurs by a variety of mechanisms across the kingdoms of life. By and large, eukaryotes achieve it in two stages. The first is through the formation of nucleosomes, which has been heavily studied and is well understood. The second is its further condensation by linker histones, which is less well understood, and will be addressed by this proposal.

The packaging of the genome in a fashion that allows the correct spatio-temporal patterning of gene expression and developmental programs is an essential process. In eukaryotes, DNA condensation is achieved in two stages: (i) the core histones package DNA into nucleosomal beads on a string and (ii) linker histones recognise the nucleosome dyad and, using their long intrinsically-disordered C-terminal tail, neutralise the charge on linker DNA between nucleosomes and promote its condensation into higher-order structures. Decades of structural biology suggests the result of (ii) is an ordered array termed the 30 nm fibre, but more recent evidence from cryo-EM and in vivo microscopy points to a less ordered, dynamic chromatin fibre in which intrinsic disorder and liquid-liquid phase separation play a prominent role.

We have developed a model system for the study of DNA condensation by the H1 C-tail, which permits measurements at atomic-level resolution by NMR, solution scattering and other biophysical methods. We have found that it binds DNA while retaining its disorder in a "fuzzy complex", and that model complexes phase separate into dense liquid droplets, within which, under certain conditions, we observe higher-order structuring of the DNA. Our model system is therefore entirely consistent with the recent in vivo data, and we wish to exploit it further to answer several key questions that have arisen as a result: exactly how is DNA bound and condensed by H1; whether the condensates are accessible to chromatin-modifying enzymes and support their function, and how they respond; whether and how "fuzzy interactions" facilitate rapid exchange of chromatin-bound proteins; which higher-order structures are present in the condensates, and how their assembly is controlled and regulated; and finally, what are the mechanistic similarities/differences in genome condensation between the many linker histone subtypes and DNA sequences differing in content and modifications?

This is basic research on the physical nature of condensed genomic DNA (chromatin), which underpins the biology of all organisms, and is thus important fundamental science. The picture in the current Biochemistry textbooks of the 10-nm chromatin fibre and its further condensation into the 30-nm fibre needs to be revised, but what will eventually replace it is still unclear. We will address the current knowledge gap with the proposed research programme.

Academic impact: the work described in this project is relevant to academics interested in a wide range of disciplines spanning cell biology to the physics of soft matter, including: chromatin and chromatin biology, intrinsically disordered proteins and "fuzzy complexes", liquid-liquid phase separation, post-translational modifications and their control by the cell cycle, DNA repair, colloid physics of polyelectrolytes, NMR and other biophysical methods for characterising "fuzzy interactions". The academic community will benefit in the following ways: open access publications, posters and presentations given at conferences through group leaders, postdocs and PhD students, and sharing of data (e.g. BioMagResBank) and methodology (via teaching and networking).

Collaborative science: the work itself unites researchers across different fields from biology to physics, and is an important factor in increasing the impact of our results, since the cross-fertilisation will import and mix ideas to produce better and more impactful science. Moreover, when we present our findings, both biological concepts and methodological knowledge will reach new audiences.

Public engagement: the results will be presented to lay audiences at e.g. public lectures and outreach events, such as the Cambridge Science Festival in March, and on the front page of the departmental website. We are actively engaged with the departmental Outreach Team to ensure our research is regularly broadcasted via Twitter, Facebook and LinkedIn, in Research Horizons and in the national press.

Public health: despite being research of a fundamental nature, two of the proteins studied (H1 and HMGB1) have roles in inflammation and cancer. More broadly, a clearer understanding of chromatin packaging will be of great relevance in the field of cancer biology, and at a more fundamental level, in the development of new materials that have potential for use as drug delivery vehicles, and in the treatment of multidrug resistant bacteria using agents that condense and inactivate the bacterial genome. The outcomes have the potential to lead into translational efforts, and may contribute towards commercially-exploitable products. Dr Stott has many collaborations and contacts with industry through the biophysics facility, hence we are well positioned to seek expert advice and look at the potential translations of any novel findings, which can be achieved through the local BBSRC Impact Acceleration Account funding in the first instance.