Structural characterisation of the tetrasome and histone chaperones

Lead Research Organisation: University of Dundee
Department Name: College of Life Sciences

Abstract

DNA provides the genetic information that defines living organisms. However, at any one time only a proportion of genes are required to be active. An important step in regulating when genes are active is how accessible they are. In this proposal we will investigate the early steps in the packaging of DNA. This is fundamental to understanding how genes are regulated in humans and what goes wrong in a broad range of human diseases.

Technical Summary

The genomes of eukaryotes exisist as chromatin. The fundamental subunit of chromatin is the nuclesome. Nucleosomes are dissassembled and reassembled to allow access to genetic infromation for example during DNA replication. An early intermediate in the assembly of nucleosomes is the tetrasome, consisting of histones H3 and H4 associated with DNA. We aim to study the structure of the tetrasome using a range of techniques including SDSL, PELDOR and site directed crosslinking. Histone binding proteins known as histone chaperones are know to facilitate the assembly of tetrasomes. In order to gain insight as to how chaperones achieve this we will investigate how histones are bound by chaperones with the aim of gaining further insight into this important initial step in chromatin assembly.
 
Title Method for modelling using Distance constraints 
Description Crystallographic and NMR approaches have provided a wealth of structural information about protein domains. However, often these domains are found as components of larger multi domain polypeptides or complexes. Orienting domains within such contexts can provide powerful new insight into their function. The combination of site specific spin labelling and Pulsed Electron Double Resonance (PELDOR) provide a means of obtaining structural measurements that can be used to generate models describing how such domains are oriented. Here we describe a pipeline for modelling the location of thio-reactive nitroxyl spin locations to engineered sties on the histone chaperone Vps75. We then use a combination of experimentally determined measurements and symmetry constraints to model the orientation in which homodimers of Vps75 associate to form homotetramers using the XPLOR-NIH platform. This provides a working example of how PELDOR measurements can be used to generate a structural model. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2014 
Provided To Others? Yes  
Impact The method has been published and so is available for other researchers to use. We have also used this approach to model the structure of a chaperone-histone complex. 
URL http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4274318/
 
Title Vps75 crystal structure 
Description The co-ordinates of the histone chaperone Vps75 in a tetrameric conformation determined by X-ray diffraction 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact This structure indicates that the Vps75 dimer can adopt different conformations which may affect its ability to interact with histones. The structure also indicates that Vps75 can adopt a tetrameric configuration. These findings provide a foundation understanding how Vps75 and likely nap1-fold proteins in general function to regulate chromatin assembly. This in turn influences gene expression which is fundamental to many biological processes. 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=5AGC
 
Description Applying pulsed EPR to study chromatin dynamics 
Organisation University of St Andrews
Department School of Physics and Astronomy
Country United Kingdom 
Sector Academic/University 
PI Contribution We have made material that has been used to make measurements using instruments located at St Andrews.
Collaborator Contribution The EPR group at St Andrews https://www.st-andrews.ac.uk/~mmwave/epr/ have made measurements on samples we have provided.
Impact https://www.ncbi.nlm.nih.gov/pubmed/19138067 https://www.ncbi.nlm.nih.gov/pubmed/19914933 https://www.ncbi.nlm.nih.gov/pubmed/20805036 https://www.ncbi.nlm.nih.gov/pubmed/21329878 https://www.ncbi.nlm.nih.gov/pubmed/23178455 https://www.ncbi.nlm.nih.gov/pubmed/25310878 https://www.ncbi.nlm.nih.gov/pubmed/27036862 https://www.ncbi.nlm.nih.gov/pubmed/28332978 https://www.ncbi.nlm.nih.gov/pubmed/30079888 The collaboration is interdisciplinary involving Physics and Molecular biology
Start Year 2009
 
Description Inner Space Outer Space at the Mills Observatory 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact The Mills Observatory in the heart of Dundee provides a unique location to develop, trial and offer public engagement activity that is immersive, uses multimodal-presentation techniques through a room-filling visual presentation screen, allowing visitors of all ages and abilities to engage with and explore the wonders of our micro and macro cosmos.

This ambitious project is funded by the Centre for Gene Regulation and Expression at the University of Dundee by a Strategic Grant from the Wellcome Trust in partnership with Leisure and Culture Dundee and Dundee City Council.

The project was initiated and is managed and run by the Dundee Imaging Facility the only multidisciplinary technology hub at the University which currently works with researchers from all of the STEMM subjects.

It is being developed in collaboration with the School of Computing Computing's Augmentative and Alternative Communication (AAC) Research Group with input from Life Scientists, Physicists, Mathematicians and Astronomers from across the University.

The new facilities allow accessible, interactive exploration of University research across all these disciplines. The technology used will also ensure that all audiences, especially people with impairments, can experience and interact with the presentations.
Year(s) Of Engagement Activity 2015,2016
URL http://gre.dundee.ac.uk/outer-space-inner-space-mills-observatory/
 
Description Open Doors day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Around 200 people attendend, members of the lab presented and interactive display describing protein puyrification, and participated in "coffeee with a scientist" session.

Hopefully, interactions with members of public had some impact, but little evidence of this yet.

Film crew attended gathering material for documentary describing research activity in Dundee
Year(s) Of Engagement Activity 2011,2012,2013,2014