Fractals in the nucleus ‐ understanding chromatin organisation with 3D SMLM

Abstract Chromatin presents a unique challenge in structural biology. Its fundamental units, DNA and the nucleosome, are well‐characterised at the Ångstrom level. 1 Yet, the higher‐order organisation of the chromatin ensemble, particularly during interphase, remains largely to be determined. Single molecule localisation microscopy (SMLM) appears uniquely positioned to answer many remaining questions about chromatin structure – it is possible to determine structures to accuracies of better than 20 nm, 2 and analysis of spatial point data has been used by some groups to determine the scaling behaviour and fractal dimension of chromatin. 3 This is of particular interest when considering nuclear molecular transport and gene activation: it has been suggested that cells use the very structure of chromatin to regulate transcription and expression. 4 Whilst imaging has been able to shed light on the general organisation of chromatin and other fibrous structures in the cell, there is a lack of methods to fully characterise their folding and looping behaviour. To date, most SMLM analysis has focussed on clustering‐based algorithms such as Ripley's K function or more recently Bayesian clustering. 5 It is clear, however, that different approaches are needed to tackle the analysis of elongated or fibrous structures within cells, and in three dimensions. Here, we demonstrate the power of 3D pair correlation analysis combined with careful labelling of regions of interest in chromatin, showing that the fractal dimension of chromatin can be determined across the entire nucleus for structures 30 nm in size upwards. This provides a platform for analysis complementary to imaging, describing differential folding behaviour across the nucleus, yielding results comparable to Hi‐C and other recent methods. In addition to applying spatial statistics to probe chromatin structure on the nanoscale, we incorporate photon statistics into the analysis. This allows us to draw conclusions about chromatin structure with greater confidence by adding a weighting to reduce the influence of data points localised with lower precision than others. Understanding how chromatin fibres fold at all stages in the cell cycle is crucial to the future development of drugs and novel treatments. More and more, drugs target specific loci yet in many cases lack the ability to be directed to the target of interest with great accuracy. 6 We hope that by understanding the organisation of different regions of the genome, drugs can be designed to be delivered to their targets in ‘smarter’ ways.