Characterisation of core histone sequences and nuclear mobility using a reproducible research approach
Susano Pinto, David Miguel
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Chromatin is a dynamic complex that controls access to genetic information by undergoing structural reconfigurations. Understanding this dynamic can provide insights into the biological implications of chromatin organisation. We have undertaken a detailed catalogue of the human core histone genes and contributed to their annotations. Based on the reproducible research concept, we produced this catalogue with a system that is capable to generate new up to date manuscripts as a model for similar projects which can be continually improved along with genome annotations. As proof of concept, we used the same project to produce a catalogue of the current mouse histone genes. Quantitative fluorescence microscopy has been used extensively to obtain insights into the dynamics of multiple proteins in live cells. Despite many advances in model design, fluorophores, and imaging capabilities, limitations are still encountered that can lead to misinterpretation of data. By using histone proteins with extremely slow exchange rates we have tested the limitations of Fluorescence Recovery After Photobleaching (FRAP) and developed approaches to overcome some of them. Importantly, we show that movement of chromatin precludes measurements of histone dynamics on the FRAP time scale. To achieve these results we made contributions to multiple free software projects including Octave, BioPerl, and Debian. This included implementing new algorithms, refactoring code for efficiency and consistency, creating maintenance support tools, and packaging software for ease of installation by users. A core theme of this work was to create build systems capable of processing primary data from public databases or microscopy in a completely transparent way to generate complete manuscripts as implementations of the reproducible research concept. This thesis itself is an example of the approach. In these studies we have tested the limits and developed new approaches to existing methods of chromatin analysis by designing novel reagents and software for the field of chromatin dynamics.