Professor Tom Owen-Hughes FRSE
Chromatin Structure and function.
The genomes of eukaryotes associate with histone proteins to form chromatin. Chromatin is the substrate for all genetic processes and as a result of fundamental importance to a diverse range of biological processes including development and disease. Indeed chromatin is currently of significant interest both in the development of biomarkers for diseases and as a source of new therapeutic targets. Eukaryotes use a range of strategies to alter chromatin structure. These include the use of histone modifications, histone chaperones and ATP dependent remodelling enzymes. We are generally interested in how these different strategies are coordinated to ensure appropriate gene regulation.
Figure 1 Organisation of transcription and chromatin proteins with respect to the Transcriptional start site (TSS). Nucleosomes,histone modifications, histone chaperones exhibit distinct distributions with respect to transcribed genes. Adapted from (Owen-Hughes and Gkikopoulos 2012).
The application of rapidly developing genomic approaches to study gene regulation has revealed a common architecture shared by large numbers of transcribed genes. This consists of a nucleosome depleted region just upstream of the transcriptional start site, and an array of well positioned nucleosomes extending into the coding region (Figure 1). We have found that a combination of the Isw1 and Chd1 chromatin remodelling enzymes play a major role in maintaining this organization in yeast (Figure 2).
Figure 2 – ATP dependent chromatin remodelling enzymes contribute to genome wide chromatin organisation. High throughput sequencing can be used to map the positions of nucleosomes over all genes (green line). The organisation of nucleosomes over coding regions is lost in strains lacking the Isw1 and Chd1 enzymes (red line). Adapted from (Gkikopoulos et al 2011)
These observations raise many questions. For example, how is the action of these enzymes coupled to transcription? How is the chromatin of non transcribed regions organized in higher eukaryotes? Projects aimed at addressing these issues are ongoing.
We are also interested to learn at a molecular level how chromatin remodelling enzymes and histone chaperones engage with chromatin. For example we are using combined structural approaches to model the overall organization of the Chd1 protein and its interaction with nucleosomes. These include X-ray crystallography, which we used to solve the structure of the C-terminal domain of the Chd1 protein (Figure 3).
Figure 3 – The crystal structure of the C-terminal DNA binding domain of Chd1. The structure revealed the presence of SANT and SLIDE domains. We are currently investigating the role of these domains in nucleosome positioning and the configuration of full length Chd1 protein. Adapted from (Ryan et al 2011)
We are particularly interested in using Electron Paramagenetic Resonance (EPR) to dock domains of known structure with respect to each other. An example illustrating how we have used this to study the conformation of chromatin when associated with histone chaperone proteins is shown in Figure 4.
Figure 4 Distance measurements obtained by EPR. Distance measurements were obtained from histones in complex with histone chaperones showing that they retain a tetrameric organisation (Bowman at all 2010, Bowman et al 2011)
Bowman, A., Hammond, C. M., Stirling, A., Ward, R., Shang, W., El-Mkami, H., Robinson, D. A., Svergun, D. I., Norman, D. G. and Owen-Hughes, T. (2014)
The histone chaperones Vps75 and Nap1 form ring-like, tetrameric structures in solution.
Nucleic Acids Res. 42, 6038-6051
Pubmed; 4027167 View Paper
Bowman, A., R., Ward, H., El-Mkami, Owen-Hughes, T. and Norman, D. G. (2010)
Probing the (H3-H4)(2) histone tetramer structure using pulsed EPR spectroscopy combined with site-directed spin labelling.
Nucleic Acids Research, 38(2): p. 695-707. PMID 19914933 View Paper
Bowman, A. R., Ward, H., Wiechens, N., Singh, V., El-Mkami, H., Norman, D. G. and Owen-Hughes, T. (2011)
The Histone ChaperoneR. s Nap1 and Vps75 Bind Histones H3 and H4 in a Tetrameric Conformation.
Molecular Cell, 41(4): p. 398-408. PMID 21329878 View Paper
Di Cerbo, V., Mohn, F., Ryan, D. P., Montellier, E., Kacem, S., Tropberger, P., Kallis, E., Holzner, M., Hoerner, L., Feldmann, A., Richter, F. M., Bannister, A. J., Mittler, G., Michaelis, J., Khochbin, S., Feil, R., Schuebeler, D., Owen-Hughes, T., Daujat, S. and Schneider, R. (2014)
Acetylation of histone H3 at lysine 64 regulates nucleosome dynamics and facilitates transcription.
eLife. 3 doi; ARTN e01632
DOI 10.7554/eLife.01632; View Paper
El Mkami, H., Ward, R., Bowman, A., Owen-Hughes, T. and Norman, D. G. (2014)
The spatial effect on protein deuteration on nitroxide spin-label relaxation: Implications for EPR distance measurement.
J Magn Reson. 248, 36-41
Pubmed; 4245719 View Paper
Engeholm, M., de Jager, M., Flaus, A., Brenk, R., van Noort, J. and Owen-Hughes, T. (2009)
Nucleosomes can invade DNA territories occupied by their neighbors.
Nat Struct Mol Biol. 16(2):151-8. PMID 19182801 View Paper
Flaus, A., Rencurel, C., Ferreira, H., Wiechens, N. and Owen-Hughes, T. (2004)
Sin mutations alter inherent nucleosome mobility. EMBO J. 23(2):343-53. PMID 14726954 View Paper
Gkikopoulos, T., Havas, K. M., Dewar, H. and Owen-Hughes, T. (2009)
SWI/SNF and Asf1p Co-operated to displace histones during induction of the Saccharomyces cerevisiae HO promoter.
Mol Cell Biol. View Paper
Gkikopoulos, T., Schofield, P., Singh, V., Pinskaya, M., Mellor, J., Smolle, M., Workman, J. L., Barton, G. J., and Owen-Hughes, T. (2011)
A Role for Snf2-Related Nucleosome-Spacing Enzymes in Genome-Wide Nucleosome Organization.
Science, 333(6050): p. 1758-1760. PMID 21940898 View Paper
Hammond, C. M., Owen-Hughes, T. and Norman, D. G. (2014)
Modelling multi-protein complexes using PELDOR distance measurements for rigid body minimisation experiments using XPLOR-NIH.
Methods, 70, 139-53
Pubmed; 4274318 View Paper
Lia, G., Praly, E., Ferreira, H., Stockdale, C., Tse-Dinh, Y.C., Dunlap, D., Croquette, V., Bensimon, D., Owen-Hughes, T. (2006)
Direct observation of DNA distortion by the RSC complex.
Mol Cell. 21:417-25. PMID 16455496 View Paper
Narlikar, G. J., Sundaramoorthy, R. and Owen-Hughes, T. (2013)
Mechanisms and functions of ATP-dependent chromatin-remodeling enzymes.
Cell Aug 1;154(3): 490-503. doi: 10.1016/j.cell.2013.07.011. Review.
PMID: 23911317 View Paper
Owen-Hughes, T. and Gkikopoulos, T. (2012)
Making sense of transcribing chromatin.
Current Opinion in Cell Biology, PMID 22410403 View Paper
Ryan, D. P., R. Sundaramoorthy, D., Martin, V. Singh and Owen-Hughes, T. (2011)
The DNA-binding domain of the Chd1 chromatin-remodelling enzyme contains SANT and SLIDE domains.
Embo Journal, 30(13): p. 2596-2609. PMID 21623345 View Paper
Ward, R., Bowman, A., El-Mkami, H., Owen-Hughes, T. and Norman, D. G. (2009)
Long distance PELDOR measurements on the histone core particle.
J Am Chem Soc. 131(4):1348-9. PMID 19138067 View Paper