Andrea Esposito

Hi-C, split-pool recognition of interactions by tag extension (SPRITE) and genome architecture mapping (GAM) are powerful technologies utilized to probe chromatin interactions genome wide, but how faithfully they capture three-dimensional (3D) contacts and how they perform relative to each other is unclear, as no benchmark exists. Here, we compare these methods in silico in a simplified, yet controlled, framework against known 3D structures of polymer models of murine and human loci, which can recapitulate Hi-C, GAM and SPRITE experiments and multiplexed fluorescence in situ hybridization (FISH) single-molecule conformations. We find that in silico Hi-C, GAM and SPRITE bulk data are faithful to the reference 3D structures whereas single-cell data reflect strong variability among single molecules. The minimal number of cells required in replicate experiments to return statistically similar contacts is different across the technologies, being lowest in SPRITE and highest in GAM under ...

In recent years the development of novel technologies, as Hi-C or GAM, allowed to investigate the spatial structure of chromatin in the cell nucleus with a constantly increasing level of accuracy. Polymer physics models have been developed and improved to better interpret the wealth of complex information coming from the experimental data, providing highly accurate understandings on chromatin architecture and on the mechanisms regulating genome folding. To investigate the capability of the models to explain the experiments and to test their agreement with the data, massive parallel simulations are needed and efficient algorithms are fundamental. In this work, we consider general computational Molecular Dynamics (MD) techniques commonly used to implement such models, with a special focus on the Strings & Binders Switch polymer model. By combining this model with machine learning computational approaches, it is possible to give an accurate description of real genomic loci. In addition, it is also possible to make predictions about the impact of structural variants of the genomic sequence, which are known to be linked to severe congenital diseases.

Experimental advances in Molecular Biology demonstrated that chromatin architecture and gene regulation are deeply related. Hi-C data, for instance, returned a scenario where chromosomes form complex pattern of interactions, including TADs, metaTADs and compartments, correlated with genomic and epigenomic features. Here, we discuss the emerging hierarchical organization of chromatin and show how it remains partially conserved during mouse neuronal differentiation with changes highly related to modification in gene expression. In this scenario, models of polymer physics, such as the Strings & Binders (SBS) model, can be a crucial instrument to understand the molecular mechanisms underlying the formation of such a higher-order 3D structure. In particular, we focus on the case study of the murine Pitx1 genomic region. At this locus, two alternative spatial conformations take place in the hindlimb and forelimb tissues, corresponding to two different transcriptional states of Pitx1. We finally show how structural variants can affect the locus 3D organization leading to ectopic gene expression and limb malformations.

The regulatory specificity of enhancers and their interaction with gene promoters is thought to be controlled by their sequence and the binding of transcription factors. By studying Pitx1, a regulator of hindlimb development, we show that dynamic changes in chromatin conformation can restrict the activity of enhancers. Inconsistent with its hindlimb-restricted expression, Pitx1 is controlled by an enhancer (Pen) that shows activity in forelimbs and hindlimbs. By Capture Hi-C and three-dimensional modeling of the locus, we demonstrate that forelimbs and hindlimbs have fundamentally different chromatin configurations, whereby Pen and Pitx1 interact in hindlimbs and are physically separated in forelimbs. Structural variants can convert the inactive into the active conformation, thereby inducing Pitx1 misexpression in forelimbs, causing partial arm-to-leg transformation in mice and humans. Thus, tissue-specific three-dimensional chromatin conformation can contribute to enhancer activity...

Chromosomes have a complex architecture in the cell nucleus, which serves vital functional purposes, yet its structure and folding mechanisms remain still incompletely understood. Here we show that genome-wide chromatin architecture data, as mapped by Hi-C methods across mammalian cell types and chromosomes, are well described by classical scaling concepts of polymer physics, from the sub-Mb to chromosomal scales. Chromatin is a complex mixture of different regions, folded in the conformational classes predicted by polymer thermodynamics. The contact matrix of the Sox9 locus, a region linked to severe human congenital diseases, is derived with high accuracy in mESCs and its molecular determinants identified by the theory; Sox9 self-assembles hierarchically in higher-order domains, involving abundant many-body contacts. Our approach is also applied to the Bmp7 locus. Finally, the model predictions on the effects of mutations on folding are tested against available data on a deletion ...