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Chromatin assembly and higher-order dynamics

Primary Supervisor: Dr Andrew Bowman, Warwick Medical School

Secondary supervisor: Dependent on specific project chosen

PhD project title: Chromatin assembly and higher-order dynamics

University of Registration: University of Warwick

Project outline:

We are interested in how chromatin is assembled and in the dynamics of higher-order chromatin structure. As in most labs, we use a range of molecular biology approaches, but have also developed a number of novel techniques for observing chromatin assembly and dynamics in living cells, which we monitor by using fluorescence microscopy. An example of this is a key method we have devised, known as RAPID-release (Apta-Smith et al., 2018, EMBO J). RAPID-release allows a short pulse of labelled histones that integrate into the chromatin deposition pathway giving us a unique tool to look at chromatin in living cells. Below I describe the two main themes of our lab, either of which can motivate a PhD project.

Project 1

The first theme of our lab is characterising the dynamic behaviour of chromatin domains in living cells. Using RAPID-release, we pulse replicating cells at the end of S-phase with a histone-eGFP construct. We then follow the chromatin domains using Lattice LightSheet (LLS) microscopy over extended periods to characterise their dynamics. Using this approach, we  have made the exciting discovery that chromatin domains partition into two separate populations, a low-mobility group, that remains in close proximity to their starting location, and a highly-mobile group that can translocate microns across the nucleus, and often return to their original position. Interestingly, we found that DNA double-strand breaks increases the proportion of this highly mobile group, suggesting that translocation could be a necessary part of repairing DNA when the double helix is broken.

Open question now include:

  • Does nuclear actin form a filament along which chromatin domains track? And if so, how are megabase domains tethered to such a nuclear-skeleton?
  • What is the mechanical basis for translocation (myosin power-strokes, actin polymerisation)?
  • Does the architectural protein cohesin have a role in chromatin domain translocation?
  • Why translocate a whole chromatin domain upon damage, rather that translocating a single loop of DNA?

We currently have reporters for F-actin, myosin, actin modulators (Arp2/3 complex) and DNA repair factors. We aim to use these in combination with LLS microscopy to answer these questions through visualisation of the processes in living cells. If you are interested in advanced microscopy, live-cell imaging, image processing and analysis and modelling, this is the project for you!

Further reading:

  1. Apta-Smith, M. J.,Hernandez-Fernaud, J. R., andBowman, A. J.(2018). Evidence forthe nuclear import of histones H3.1 and H4 as monomers.The EMBO journal37, e98714. doi:10.15252/embj.201798714.
  2. Caridi, C. P.,Plessner, M.,Grosse, R., andChiolo, I.(2019). Nuclear actin filaments in DNArepair dynamics.Nature Cell Biology21, 1068–1077. doi:  10.1038/s41556-019-0379-1.
  3. Lamm, N.,Rogers, S., andCesare, A. J.(2021). Chromatin mobility and relocation in DNArepair.Trends in Cell Biologypages S0962–8924(21)00118–5. doi: 10.1016/j.tcb.2021.06.002.
  4. Tortora, M. M.,Salari, H., andJost, D.(2020). Chromosome dynamics during interphase: abiophysical perspective.Current Opinion in Genetics & Development61, 37–43. doi: 10.1016/j.gde.2020.03.001.

Project 2

The second theme of our lab is concerned with chromatin assembly, and more specifically, the molecular pathway that delivers newly synthesised histones to chromatin, commonly referred to as the histone chaperoning pathway. Histone chaperones are specialised proteins dedicated to guiding the assembly of nucleosomes (Pardal et al., 2019, Essays Biochem). Histone chaperones originate from a wide range of protein families, spanning diverse protein folds. Yet, they share the key common feature of binding histones and protecting them from undesirable interactions, be that during storage, transport, or nucleosome assembly.

Interestingly, we have recently challenged the paradigm of nucleosome assembly by showing that a significant proportion of a subtype of histone (histone H3) is present in its monomeric form in the nucleus. This is important, as the text picture of chromatin assembly dictates that H3 is always folded with H4 before assembly into nucleosomes. Further, we have identified a chaperoning protein that can sequester H3 in its monomeric state: a protein called NASP.We don't really know why NASP binds to a monomer of H3. We imagine it may be a folding intermediate adopted soon after H3 is imported to the nucleus; however, high-resolution structural information would go a long way to answer this question.

A PhD project addressing this will involve the purification and reconstitution of the NASP-H3 complex and its structure determination by X-ray crystallography. This will then act as a platform for the more ambitious characterisation of the complex we believe is downstream of the NASP-H3 dimer, a complex of six separate proteins. This will require reconstitution of proteins expressed from both bacteria and mammalian (HEK293) cell lines and will be a candidate for structure analysis by cryoEM through the using of the Midlands Regional Cryo-Electron Microscope Facility.

Structural characterisation of these complexes could then lead into molecular dynamics simulations of how the transfer of histones between two chaperoning complexes can occur. If you are interested in chromatin biology and how proteins function at the molecular/atomistic level, this is the project for you!

Further reading:

  1. Pardal, A.,Fernandes-Duarte, F., andBowman, A.(2019). The histone chaperoning pathway: From ribosome to nucleosome. Essays in Biochemistry 63. doi: 10.1042/EBC20180055.
  2. Hammond, C. M.,Stromme, C. B.,Huang, H.,Patel, D. J., andGroth, A.(2017). Histone chaperone networks shaping chromatin function. Nat Rev Mol Cell Bioldoi: 10.1038/nrm.2016.159.
  3. Apta-Smith, M. J.,Hernandez-Fernaud, J. R., andBowman, A. J.(2018). Evidence for the nuclear import of histones H3.1 and H4 as monomers. The EMBO journal37, e98714. doi:10.15252/embj.201798714.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Structural Biology & Systems Biology

    Techniques that will be undertaken during the project:

    For the first project: Lattice light sheet microscopy and spin-disk confocal microscopy will be the main techniques. Working with cell lines in culture and genetically manipulating them using a number of approaches (plasmid integration, CRISPR-Cas9 editing) will be needed.

    Tor the second potential project: protein expression and purification (both from bacteria and HEK293 cells), various forms of chromatography, protein interaction analysis (SPR,ITC, ultracentrifugation) and potentially cryoEM (Leicester). Structure-function analysis will require working with mammalian cell lines in culture and their genetic manipulation.

    Contact: Dr Andrew Bowman, University of Warwick