Antonio Scialdone

Complex multi-enhancer contacts captured by genome architecture mapping

The organization of the genome in the nucleus and the interactions of genes with their regulatory elements are key features of transcriptional control and their disruption can cause disease. Here we report a genome-wide method, genome architecture mapping (GAM), for measuring chromatin contacts and other features of three-dimensional chromatin topology on the basis of sequencing DNA from a large collection of thin nuclear sections. We apply GAM to mouse embryonic stem cells and identify enrichment for specific interactions between active genes and enhancers across very large genomic distances using a mathematical model termed SLICE (statistical inference of co-segregation). GAM also reveals an abundance of three-way contacts across the genome, especially between regions that are highly transcribed or contain super-enhancers, providing a level of insight into genome architecture that, owing to the technical limitations of current technologies, has previously remained unattainable. Furthermore, GAM highlights a role for gene-expression-specific contacts in organizing the genome in mammalian nuclei.

Conformation regulation of the X chromosome inactivation center: a model.

X-Chromosome Inactivation (XCI) is the process whereby one, randomly chosen X becomes transcriptionally silenced in female cells. XCI is governed by the Xic, a locus on the X encompassing an array of genes which interact with each other and with key molecular factors. The mechanism, though, establishing the fate of the Xs, and the corresponding alternative modifications of the Xic architecture, is still mysterious. In this study, by use of computer simulations, we explore the scenario where chromatin conformations emerge from its interaction with diffusing molecular factors. Our aim is to understand the physical mechanisms whereby stable, non-random conformations are established on the Xics, how complex architectural changes are reliably regulated, and how they lead to opposite structures on the two alleles. In particular, comparison against current experimental data indicates that a few key cis-regulatory regions orchestrate the organization of the Xic, and that two major molecular regulators are involved.

Mechanics and dynamics of X-chromosome pairing at X inactivation

At the onset of X-chromosome inactivation, the vital process whereby female mammalian cells equalize X products with respect to males, the X chromosomes are colocalized along their Xic (X-inactivation center) regions. The mechanism inducing recognition and pairing of the Xs remains, though, elusive. Starting from recent discoveries on the molecular factors and on the DNA sequences (the so-called “pairing sites”) involved, we dissect the mechanical basis of Xic colocalization by using a statistical physics model. We show that soluble DNA-specific binding molecules, such as those experimentally identified, can be indeed sufficient to induce the spontaneous colocalization of the homologous chromosomes but only when their concentration, or chemical affinity, rises above a threshold value as a consequence of a thermodynamic phase transition. We derive the likelihood of pairing and its probability distribution. Chromosome dynamics has two stages: an initial independent Brownian diffusion followed, after a characteristic time scale, by recognition and pairing. Finally, we investigate the effects of DNA deletion/insertions in the region of pairing sites and compare model predictions to available experimental data.

Polymer models of chromatin organization

The exploration of the spatial organiza-tion of chromosomes in the cell nucleushas been greatly enhanced by genome-scale technologies such as Hi-C methods.Polymermodels arehelpingto understandthe new emerging complex scenarios andhere we review some recent developments.In the cell nucleus of eukaryotes, chro-mosomes have a complex spatial organi-zation serving vital functional purposes,with structural disruptions being linkedto disease (Fraser and Bickmore, 2007;Lanctot et al., 2007; Misteli, 2007; Pomboand Branco, 2007). The development oftechnologies such as Hi-C ( Lieberman-Aiden et al., 2009) has opened the wayto mapping chromatin interactions ata genomic scale. It is emerging thatchromosomes tend to form 1Mb sizeddomains with increased levels of intra-interactions (known, e.g., as TopologicalDomains, TDs) (Dixon et al., 2012; Noraet al., 2012), but contacts extend acrossentire chromosomes (Branco and Pombo,2006; Shopland et al., 2006; Fraser andBickmore, 2007; Kalhor et al., 2011;Sexton et al., 2012), as highlighted bythe average contact probability of twosites,

Diffusion-based DNA target colocalization by thermodynamic mechanisms.

In eukaryotic cell nuclei, a variety of DNA interactions with nuclear elements occur, which, in combination with intra- and inter-chromosomal cross-talks, shape a functional 3D architecture. In some cases they are organized by active, i.e. actin/myosin, motors. More often, however, they have been related to passive diffusion mechanisms. Yet, the crucial questions on how DNA loci recognize their target and are reliably shuttled to their destination by Brownian diffusion are still open. Here, we complement the current experimental scenario by considering a physics model, in which the interaction between distant loci is mediated by diffusing bridging molecules. We show that, in such a system, the mechanism underlying target recognition and colocalization is a thermodynamic switch-like process (a phase transition) that only occurs if the concentration and affinity of binding molecules is above a threshold, or else stable contacts are not possible. We also briefly discuss the kinetics of this `passive-shuttling process, as produced by random diffusion of DNA loci and their binders, and derive predictions based on the effects of genomic modifications and deletions.

Mean-Field Theory of the Symmetry Breaking Model for X Chromosome Inactivation

X Chromosome Inactivation (XCI) is the process in mammal female cells whereby one of the X chromosomes is silenced to compensate dosage with respect to males. It is still mysterious how precisely one X chromosome is randomly chosen for inactivation. We discuss here a mean-field theory of the Symmetry Breaking (SB) model of XCI, a Statistical Mechanics model introduced to explain that process. The SB model poses that a single regulatory factor, an aggregate of molecules, is produced which acts to preserve from inactivation one of the X’s. The model illustrates a physical mechanism, originating from a thermodynamic phase transition, for the self-assembling of such a single super-molecular aggregate which can spontaneously break the binding symmetry of equivalent targets. This results in a sharp, yet stochastic, regulatory mechanism of XCI. In particular, we focus here on how the model can predict the effects of genetic deletions.

Passive DNA shuttling

In eukaryotic cells, a variety of DNA functional interactions with nuclear elements occurs via Brownian, passive mechanisms. This raises the question on how DNA loci can recognize their target and be reliably shuttled to destination by diffusion. We discuss such a topic in the framework of the polymer adsorption problem, via a schematic Statistical Physics model where Brownian binding molecules mediate DNA-target interactions. In that context, we show that binding molecules can induce stable colocalization of DNA and its target (passive shuttling) via a switch-like process, regulated by a phase transition: DNA is shuttled to target only if the concentration/affinity of binding molecules is above a threshold value. We then illustrate the effects of genetic/chemical manipulations, e.g., DNA deletions, on the process.

DNA Loci Cross-Talk through Thermodynamics

The recognition and pairing of specific DNA loci, though crucial for a plenty of important cellular processes, are produced by still mysterious physical mechanisms. We propose the first quantitative model from Statistical Mechanics, able to clarify the interaction allowing such “DNA cross-talk” events. Soluble molecules, which bind some DNA recognition sequences, produce an effective attraction between distant DNA loci; if their affinity, their concentration, and the relative DNA binding sites number exceed given thresholds, DNA colocalization occurs as a result of a thermodynamic phase transition. In this paper, after a concise report on some of the most recent experimental results, we introduce our model and carry out a detailed “in silico” analysis of it, by means of Monte Carlo simulations. Our studies, while rationalize several experimental observations, result in very interesting and testable predictions.

Pairing of homologous chromosomes as phase transition

In some cells pairing of homologous chromosomes happens at particular moments during the cell life cycle. A Statistical Mechanics model based on some experimental data is presented and discussed here for this phenomenon. Under this model, chromosomes pair at special regions whose interaction is mediated by some molecules which diffuse in cells nuclei and can bind them. Concentration of these molecules acts as a switch for pairing: at low concentrations chromosomes move independently one from another whereas if concentration is above a certain threshold value, chromosomes colocalize. Monte Carlo simulations of this model have been performed to test its eficiency and the effect on pairing levels of chromosomal binding sites deletions.

STATISTICAL MECHANICS MODELS FOR X-CHROMOSOME INACTIVATION

We present statistical mechanics models to understand the physical and molecular mechanisms of X-Chromosome Inactivation (XCI), the process whereby a female mammal cell inactivates one of its two X-chromosomes. During XCI, X-chromosomes undergo a series of complex regulatory processes. At the beginning of XCI, the Xs recognize and pair, then only one X which is randomly chosen is inactivated. Afterwards, the two Xs move to different positions in the cell nucleus according to their different status (active/silenced). Our models illustrate about the still mysterious physical bases underlying all these regulatory steps, i.e., X-chromosome pairing, random choice of inactive X, and shuttling of the Xs to their post-XCI locations. Our models are based on general and robust thermodynamic roots, and their validity can go beyond XCI, to explain analogous regulatory mechanisms in a variety of cellular processes.