Nuclear structure and gene expression

We are studying structure/function relationships and protein dynamics in the cell nucleus, using a combination of quantitative techniques, including mass-spectrometry-based proteomics, fluorescence microscopy and deep sequencing. The importance of understanding nuclear organisation is underlined by evidence showing that multiple human diseases, including inherited genetic disorders, malignancies and viral infections, modify or disrupt subnuclear bodies.

Our aim is to characterise the composition and function of subnuclear organelles and multiprotein complexes and to identify mechanisms regulating their assembly and turnover in human cells. The use of high throughput proteomics technologies provides a system-wide approach allowing us to measure cellular responses for a large number of proteins in parallel. An integral part of this work is the development of new software tools for the management analysis and visualisation of complex proteomics data sets (seehttp://www.peptracker.com/).

Using the nucleolus as a model sub-nuclear organelle, we have studied the protein, RNA and DNA composition of nucleoli in human cells. Combining quantitative proteomics with quantitative fluorescence microscopy imaging, using stable cell lines expressing one or more fluorescent protein tagged reporters, provides a “Dual Strategy” for analysing dynamic properties of the cell proteome. For example, using stable isotope labeling combined with mass spectrometry based proteomics (SILAC), we have identified and analysed over 5,000 proteins that co-purify with nucleoli isolated from primary and transformed human cells. We provide details of the human nucleolar proteome online in a fully searchable relational database together with an API for advanced analysis and flexible data mining (see;http://www.lamondlab.com/NOPdb3.0/).

As a major theme of our work involves the development and application of quantitative proteomics to study cell biology we have established a website to provide detailed online information that promotes the use of these technologies (see ‘The Cell Biologists Guide to Proteomics’). We are employing ‘second generation’ proteomics techniques, currently based mainly upon differential metabolic labeling (SILAC), to provide a flexible set of assay platforms for measuring ‘protein properties’, i.e., protein-protein interactions (Boulon et al., 2010), subcellular protein localisation (Boisvert et. al., 2010), protein dynamics and turnover (Lam et . al., 2007) and post-translational protein modifications (Westman et. al., 2010).

We have now extended the methods we previously reported, using SILAC as an unbiased procedure to identify reliably specific protein interaction partners (Trinkle-Mulcahy et. al., 2006; 2008), to take advantage of the new data analysis facilities of PepTracker (Boulon et.al., MCP 2010). The reliable discrimination between specific and non-specific protein interaction partners is aided by reference to a database that contains the results of all previous immunoprecipitation experiments, annotated with detailed metadata. Using our customisable ‘Protein Frequency Library (PFL) viewer (seehttp://www.peptracker.com/datavisual/), we can take advantage of the fact that common contaminants and ‘sticky’ proteins are detected more frequently than specific interactors to help identify bona fide interaction partners for proteins of interest.

In other projects we are analysing the specific chromosomal regions that associate with nucleoli, using deep sequencing approaches coupled with novel data analysis techniques (van Koningsbruggen et. al., 2010). We are also analysing RNA species that copurify with nucleoli isolated from cultured human cells. This has identified novel human small nucleolar RNAs (snoRNAs). Based on these findings we are currently developing a technology for sequence-specific, targeted gene knock-down in mammalian cells based upon snoRNA vectors (Ono et.al., 2010). This approach allows for simultaneous knock-down of two or more targeted genes simultaneously from a single vector and provides for parallel “transient knock-in” of tagged or mutated replacement proteins from the same vector and potentially offers advantages over currently used strategies for transient protein knock-down.

We have developed a fluoresence microscopy-based approach to study nuclear structure and chromatin organization and condensation in live cells, based upon Fluorescence Lifetime Imaging (FLIM) and Fluorescence Resonance Energy Transfer (FRET). We have developed stable cell lines simultaneously expressing histones tagged with either GFP or mCherry. In these cells FRET interactions occur predominantly when the chromatin is highly condensed, because this condensation brings the separate red and green FRET pairs into proximity. Thus, using FLIM to detect and map the FRET interactions we are able to spatially resolve and quantitate chromosome condensation levels throughout interphase and mitosis in live human cell lines and thus to monitor how chromosome condensation responds to various cell perturbations and growth conditions (Lleres et. al., 2009).