About the Project
The spatial organisation of the genome is known to play an important role in regulating RNA transcription to effect cell-type-specific gene expression programs, and to control the differentiation of embryonic stem (ES) cells. Embryonic stem (ES) cells either self-renew or differentiate into all the different cells in a body, and both Chromatin Assembly Factor-1 (CAF-1) and the multi-subunit nucleosome remodelling and deacetylase (NuRD) protein complex play a central role in this process by controlling the expression of specific pluripotency-associated genes. NuRD also has an important role in controlling the generation of induced pluripotent stem (iPS) cells. Our research focuses on CAF-1 and NuRD and we would like to understand how they organise the genome to maintain gene expression programs.
We have recently developed a single cell Hi-C method, which allows us to image particular proteins and then identify which genomic regions associate with imaged foci in single cells. Using this proximity information we have calculated the first 3D structures of intact mammalian genomes (Stevens et al., 2017; Lando et al., 2018 a,b), and we are now using this approach to understand how CAF-1 and NuRD regulate heterochromatin structure and organisation when ES cells differentiate. This involves knocking down components of these complexes, and imaging heterochromatin-associated proteins using super-resolution microscopy prior to carrying out single cell Hi-C to study genome folding. Genome structures calculated from these experiments are then used to investigate global changes in heterochromatin organisation during ES cell differentiation.
In parallel, we are using single-molecule imaging to study the function of the NuRD complex in live mouse ES cells. In recent work, we have used single-molecule imaging to show that NuRD exists as two sub-complexes that associate with each other (Zhang et al., 2016). We have also shown that NuRD forms clusters on chromatin within the nucleus at enhancer/promoter contact regions with specific transcription factors and mediator components (Stevens et al., 2017). It is, however, unclear how NuRD regulates transcription at these enhancer/promoter contact regions. Our aim is to understand how NuRD regulates transcription in ES cells. Our current model is that NuRD acts by regulating the binding of transcription factors and/or mediator components and thereby enhancer/promoter contacts required for transcription. By taking advantage of NuRD-inducible cell lines, we will explore how removing NuRD components affect the interactions of specific transcription factors and mediator components with chromatin, and their ability to form clusters. We are also exploring approaches to monitor transcription of NuRD-regulated genes in live ES cells and thereby determine whether NuRD can affect the kinetics of transcriptional bursting.
To complement this research, we are also carrying out structural studies to understand how the NuRD complex interacts with chromatin. Very little is known about its substrates or how it works and our proteomic and other studies (e.g. Yang et al., 2016) suggest that CHD4 may interact with nucleosomes containing histone variants (such as H2A.Z), which may destabilize nucleosomes. We are reconstituting recombinant canonical and non-canonical H2A.Z nucleosomes for studies of NuRD remodelling using both restriction enzyme accessibility assays and single molecule FRET. In parallel, we are carrying out EM studies to explore whether we can determine the structure of NuRD-nucleosome complexes.
A PhD project could involve studies in any of our research areas, either using super-resolution microscopy and single cell techniques to study 3D genome organization, single-molecule imaging to study the function of the NuRD complex, or investigating the structure of NuRD-nucleosome complexes.
Applicants should have a good honours degree (at least a 2:1 or equivalent) in either Biochemistry, Computer Science or one of the Physical sciences.
References
1. Stevens TJ, Lando D, Basu S, Atkinson LP, Cao Y, Lee SF, Leeb M, Wohlfahrt KJ, Boucher W, O’Shaughnessy-Kirwan A, Cramard J, Faure AJ, Ralser M, Blanco E, Morey L, Sansó M, Palayret MGS, Lehner B, Di Croce L, Wutz A, et al., 3D structures of individual mammalian genomes studied by single-cell Hi-C. Nature 544: 59–64 doi:/10.1038/nature21429 (2017)
2. Lando, D. et al. Combining fluorescence imaging with Hi-C to study 3D genome architecture of the same single cell. Nature protocols 13, 1034-1061, doi:10.1038/nprot.2018.017 (2018).
3. Lando, D., Stevens, T. J., Basu, S. & Laue, E. D. Calculation of 3D genome structures for comparison of chromosome conformation capture experiments with microscopy: An evaluation of single-cell Hi-C protocols. Nucleus 9, 190-201, doi:10.1080/19491034.2018.1438799 (2018).
4. Zhang W, Aubert A, Gomez de Segura JM, Karuppasamy M, Basu S, Murthy AS, Diamante A, Drury TA, Balmer J, Cramard J, Watson AA, Lando D, Lee SF, Palayret M, Kloet SL, Smits AH, Deery MJ, Vermeulen M, Hendrich B, Klenerman D, Schaffitzel C, Berger I, Laue ED, The Nucleosome Remodeling and Deacetylase Complex NuRD is built from preformed catalytically active sub-modules. Journal of Molecular Biology, 428(14): 2931-42 (2016)
5. Yang Y, Yamada T, Hill KK, Hemberg M, Reddy NC, et al., Chromatin remodeling inactivates activity genes and regulates neural coding. Science 353, 300-305, doi:10.1126/science.aad4225 (2016)
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