Self-assembly and DNA binding of the blocking factor in X chromosome inactivation

X chromosome inactivation (XCI) is the phenomenon occurring in female mammals whereby dosage compensation of X-linked genes is obtained by transcriptional silencing of one of their two X chromosomes, randomly chosen during early embryo development. The earliest steps of random X-inactivation, involving counting of the X chromosomes and choice of the active and inactive X, are still not understood. To explain "counting and choice," the longstanding hypothesis is that a molecular complex, a "blocking factor" (BF), exists. The BF is present in a single copy and can randomly bind to just one X per cell which is protected from inactivation, as the second X is inactivated by default. In such a picture, the missing crucial step is to explain how the molecular complex is self-assembled, why only one is formed, and how it binds only one X. We answer these questions within the framework of a schematic Statistical Physics model, investigated by Monte Carlo computer simulations. We show that a single complex is assembled as a result of a thermodynamic process relying on a phase transition occurring in the system which spontaneously breaks the symmetry between the X’s. We discuss, then, the BF interaction with X chromosomes. The thermodynamics of the mechanism that directs the two chromosomes to opposite fates could be, thus, clarified. The insights on the selfassembling and X binding properties of the BF are used to derive a quantitative scenario of biological implications describing current experimental evidences on "counting and choice.

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

Thermodynamic pathways to genome spatial organization in the cell nucleus

The architecture of the eukaryotic genome is characterized by a high degree of spatial organization. Chromosomes occupy preferred territories correlated to their state of activity and, yet, displace their genes to interact with remote sites in complex patterns requiring the orchestration of a huge number of DNA loci and molecular regulators. Far from random, this organization serves crucial functional purposes, but its governing principles remain elusive. By computer simulations of a Statistical Mechanics model, we show how architectural patterns spontaneously arise from the physical interaction between soluble binding molecules and chromosomes via collective thermodynamics mechanisms. Chromosomes colocalize, loops and territories form and find their relative positions as stable hermodynamic states. These are selected by “thermodynamic switches” which are regulated by concentrations/affinity of soluble mediators and by number/location of their attachment sites along chromosomes. Our “thermodynamic switch model” of nuclear architecture, thus, explains on quantitative grounds how well known cell strategies of upregulation of DNA binding proteins or modification of chromatin structure can dynamically shape the organization of the nucleus

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

Investigating the spatial regulation of meiotic recombination in S. cerevisiae

In order for a species to engage in and reap the evolutionary benefits of sexual reproduction, a subset of cells in each individual must undergo a complex ordeal known as meiosis—a specialised cell division. By halving the genome content and “shuffling the deck”, meiosis generates genetically diverse haploid gametes (eggs, sperm) or spores from diploid cells. Such a monumental task is by no means easy or risk free: during the meiotic programme, cells intentionally damage their own genomes through widespread induction of DNA double-strand breaks (DSBs) in order to initiate homologous recombination—a DNA-repair process—and subsequent crossover (CO) formation. The success of meiosis is, however, not left up to chance. Rather, a complicated web of regulation acts at multiple stages to ensure this dangerous tradeoff pays dividends. Notably, the spatial pattern of meiotic recombination across the genome is complex and non-random. Whilst ultimately stochastic in nature, recombination events within any given meiotic cell display relatively even distributions along each chromosome—a phenomenon mediate by processes of “interference” acting at two key stages in meiosis: DSB and CO formation. Despite wide ranging historical observation, relatively little is known about how either form of interference is accomplished. Genome-wide mapping of recombination within S. cerevisiae has, however, provided a unique opportunity to investigate the underlying mechanisms. By computationally and mathematically analysing genome-wide data, work presented throughout this thesis seeks to: (i) investigate CO distribution and CO interference within various DNA damage response and DNA repair mutants (Tel1ATM, Mec1ATR, Rad24, Msh2) (Chapter 2) (ii) develop novel approaches to DSB mapping (Chapter 3) (iii) characterise the hyperlocal regulation of DSB formation (Chapter 3) and (iv) examine the mechanics of DSB interference (Chapter 4). Moreover, widely applicable simulation platforms for investigating DSB and CO formation have been developed (Chapter 2, 4). Collectively, this thesis further elucidates the mechanisms that underpin the spatial regulation of meiotic recombination in S. cerevisiae

A mean-field version of the Nicodemi-Prisco SSB model for X-chromosome inactivation

Nicodemi and Prisco recently proposed a model for X-chromosome inactivation in mammals, explaining this phenomenon in terms of a spontaneous symmetry-breaking mechanism [{\it Phys. Rev. Lett.} 99 (2007), 108104]. Here we provide a mean-field version of their model

The Unicellular State as a Point Source in a Quantum Biological System.

A point source is the central and most important point or place for any group of cohering phenomena. Evolutionary development presumes that biological processes are sequentially linked, but neither directed from, nor centralized within, any specific biologic structure or stage. However, such an epigenomic entity exists and its transforming effects can be understood through the obligatory recapitulation of all eukaryotic lifeforms through a zygotic unicellular phase. This requisite biological conjunction can now be properly assessed as the focal point of reconciliation between biology and quantum phenomena, illustrated by deconvoluting complex physiologic traits back to their unicellular origins