Chromatin Laboratory

Overview

Have you ever wondered that every human cell contains 2 metres of DNA, but yet we need a microscope to see chromosomes?

The answer lies in how the DNA is very efficiently packaged into 'chromatin'. But not only must it be efficiently packaged, DNA must also be accessible on demand.

In many ways, chromatin is very much like a town library. Fundamentally there is hierarchical order: letters making up words linked in sentences found on pages bound in books on shelves in rooms within the library.

Equally, no library is a mausoleum. Its state must be maintained by services which keep order and repair damage. Cataloguing addresses make frequently used information readily available, and keep the inappropriate safe from exposure. Those who need to read or copy the information follow clear procedures, because it is vital to ensure proper order for the next user.

Modern librarians also appreciate theirs is not only an active but also a living resource. New books are added and useless material is dumped. Revised editions replace older texts. Notes and signs are placed for the benefit of users coming both to short term exhibitions, and in future generations.

Intuitively, we understand our town libraries because we have seen them develop from ancient and medieval times. But our appreciation of the chromatin library is barely 60 years old, born in the molecular biology revolution. Arguably, the importance of its design has only been widely appreciated for the last decade or so.

Chromatin is an exciting research field because we are still very much discovering the 'how' and 'why' of its operation.

Chromatin in Motion

Chromatin is the universal packaging of eukaryotic DNA and all genomic processes must act on it as their substrate. The fundamental subunit of chromatin is the nucleosome, comprised of a core histone protein complex around which DNA is wrapped.

High resolution structures provide a detailed snapshot of the nucleosome but limited insight into dynamic properties. Nonetheless, nucleosome structural transitions occur during a wide range of biochemical activities in the nucleus including transcription, repair and replication.

The most widely observed transition is 'remodelling', recognised as involving nucleosomes being repositioned along DNA or undergoing other poorly understood reconfigurations. Nucleosomes can spontaneously reposition along DNA driven by thermal energy, but this transformation is also directed and catalytically accelerated in many genomic processes by ATP-dependent chromatin remodelling activities.

During remodelling, nucleosome substrates undergo rearrangement of their internal non-covalent interactions. Recent evidence has suggested that enzyme-driven reactions may even involve quite large structural changes, to the extent of enabling changes in nucleosome subunit composition or complete deconstruction.

ATP-dependent chromatin remodelling complexes are ubiquitous in eukaryotes, and have been implicated in broad biological processes such as development, cancer and thalassemia. Despite their importance, the mechanism of remodelling and the nature of the nucleosome structural transitions remain unclear.

Research Interests

• The dynamic nucleosome

  

  Biochemical investigations have shown that nucleosomes are not static 'tuna cans'. Instead, they can undergo specific transitions in structure such as sliding or histone dimer exchange which are essential for their function in the cell nucleus.

  This dynamic behaviour appears to be a directed process, implying that the histone protein core enables specific 'modes of flexibility' in the structure. We are working to determine the molecular motions that are allowed by the structure, and understand how they are defined by the design principles of the nucleosome.

• Snf2 family proteins as molecular machines

  

  Large ATP-dependent chromatin remodelling complexes behave as classical enzymes to direct and accelerate the rate of these structural changes. All remodellers have a helicase-related Snf2 family region at their core which translates the chemical energy of ATP hydrolysis into mechanical motion. This is applied as a force to the nucleosome.

  The outcome of applying physical forces is almost certainly intertwined with nucleosome dynamics. We wish to understand the mechanics of Snf2 proteins and how they applies forces to the nucleosome.

• Common patterns in the diversity of chromatin proteins

  

  Large volumes of data are being generated by genome-wide sequencing, expression and proteomic screens, uncovering general properties of many biological systems. These datasets can also be re-probed to ask more specific questions.

  For example, we recently catalogued over 1300 members of the Snf2 protein family and then used bioinformatic analyses to uncover new insights into distinguishing sequence features and organism distribution. We are currently extending this to other regions of these proteins. See snf2.net.

  We also wish to use bioinformatic techniques and our understanding of chromatin structure/function to look for correlations between the properties of chromatin proteins, DNA sequence and gene expression.

Techniques and Systems

Histones are readily expressed and purified by recombinant methods in E.coli, and defined DNA fragments are produced by preparative PCR. This enables both mutants and variant histones to be readily produced. Defined nucleosomes are then assembled from these components in vitro and their biochemical properties can be probed in detail.

Typical assays for nucleosomes include gel- and fluorescence-based assays of nucleosome mobility along DNA and of histone exchange between nucleosomes, site-directed hydroxy radical mapping to determine the exact positioning of the nucleosomes, and gel filtration chromatography to measure the stability of the histone octamer and nucleosome. We are also enthusiastic about developing novel methods of analysis, especially based on single molecule techniques.

Various ATP-dependent chromatin remodelling complexes can be purified from S cerevisiae and other eukaryotic cells. The activity of these enzymes on nucelosomes can be monitored, and their mechanistic behaviours are probed through enzymology and protein chemistry approaches.

For further information, please email Dr Andrew Flaus

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