Rafael D. Acemel

Obesity-associated variants within FTO form long-range functional connections with IRX3

Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type 2 diabetes (T2D). Although the molecular mechanisms linking these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence body mass and composition phenotypes. However, no direct connection between the obesity-associated variants and FTO expression or function has been made. Here we show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, with the homeobox gene IRX3. The obesity-associated FTO region directly interacts with the promoters of IRX3 as well as FTO in the human, mouse and zebrafish genomes. Furthermore, long-range enhancers within this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval belongs to the regulatory landscape of IRX3. Consistent with this, obesity-associated single nucleotide polymorphisms are associated with expression of IRX3, but not FTO, in human brains. A direct link between IRX3 expression and regulation of body mass and composition is demonstrated by a reduction in body weight of 25 to 30% in Irx3-deficient mice, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Finally, hypothalamic expression of a dominant-negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data suggest that IRX3 is a functional long-range target of obesity-associated variants within FTO and represents a novel determinant of body mass and composition.

Evolutionary comparison reveals that diverging CTCF sites are signatures of ancestral topological associating domains borders

Significance Mammalian chromatin is compartmentalized in topologically associating domains (TADs), genomic regions within which sequences preferentially contact each other. This organization has been proposed to be essential to organize the regulatory information contained in mammalian genomes. We show that Six homeobox genes, essential developmental regulators organized in gene clusters across different animal phyla, share a deeply conserved chromatin organization formed by two abutting TADs that predates the Cambrian explosion. This organization is required to generate separate regulatory landscapes for neighboring genes within the cluster, resulting in very different gene expression patterns. Finally, we show that this extremely conserved 3D architecture is associated with a characteristic arrangement of CCCTC-binding factor (CTCF) binding sites in diverging orientations, revealing a genome-wide conserved signature for TAD borders. Increasing evidence in the last years indicates that the vast amount of regulatory information contained in mammalian genomes is organized in precise 3D chromatin structures. However, the impact of this spatial chromatin organization on gene expression and its degree of evolutionary conservation is still poorly understood. The Six homeobox genes are essential developmental regulators organized in gene clusters conserved during evolution. Here, we reveal that the Six clusters share a deeply evolutionarily conserved 3D chromatin organization that predates the Cambrian explosion. This chromatin architecture generates two largely independent regulatory landscapes (RLs) contained in two adjacent topological associating domains (TADs). By disrupting the conserved TAD border in one of the zebrafish Six clusters, we demonstrate that this border is critical for preventing competition between promoters and enhancers located in separated RLs, thereby generating different expression patterns in genes located in close genomic proximity. Moreover, evolutionary comparison of Six-associated TAD borders reveals the presence of CCCTC-binding factor (CTCF) sites with diverging orientations in all studied deuterostomes. Genome-wide examination of mammalian HiC data reveals that this conserved CTCF configuration is a general signature of TAD borders, underscoring that common organizational principles underlie TAD compartmentalization in deuterostome evolution.

A single three-dimensional chromatin compartment in amphioxus indicates a stepwise evolution of vertebrate Hox bimodal regulation

The HoxA and HoxD gene clusters of jawed vertebrates are organized into bipartite three-dimensional chromatin structures that separate long-range regulatory inputs coming from the anterior and posterior Hox-neighboring regions. This architecture is instrumental in allowing vertebrate Hox genes to pattern disparate parts of the body, including limbs. Almost nothing is known about how these three-dimensional topologies originated. Here we perform extensive 4C-seq profiling of the Hox cluster in embryos of amphioxus, an invertebrate chordate. We find that, in contrast to the architecture in vertebrates, the amphioxus Hox cluster is organized into a single chromatin interaction domain that includes long-range contacts mostly from the anterior side, bringing distant cis-regulatory elements into contact with Hox genes. We infer that the vertebrate Hox bipartite regulatory system is an evolutionary novelty generated by combining ancient long-range regulatory contacts from DNA in the anterior Hox neighborhood with new regulatory inputs from the posterior side.

Topologically associated domains: a successful scaffold for the evolution of gene regulation in animals

The evolution of gene regulation is considered one of the main drivers causing the astonishing morphological diversity in the animal kingdom. Gene regulation in animals heavily depends upon cis‐regulatory elements, discrete pieces of DNA that interact with target promoters to regulate gene expression. In the last years, Chromosome Conformation Capture experiments (4C‐seq, 5C, and HiC) in several organisms have shown that the genomes of many bilaterian animals are organized in the 3D chromatin space in compartments called topologically associated domains (TADs). The appearance of the architectural protein CTCF in the bilaterian ancestor likely facilitated the origin of this chromatin 3D organization. TADs play a critical role favoring the contact of cis‐regulatory elements with their proper target promoters (that often lay within the same TAD) and preventing undesired regulatory interactions with promoters located in neighboring TADs. We propose that TAD may have had a major influence in the history of the evolution of gene regulation. They have contributed to the increment of regulatory complexity in bilaterians by allowing newly evolved cis‐regulatory elements to find target promoters in a range of hundreds of kilobases. In addition, they have conditioned the mechanisms of evolution of gene regulation. These mechanisms include the appearance, removal, or relocation of TAD borders. Such architectural changes have been able to wire or unwire promoters with different sets of cis‐regulatory elements in a single mutational event. We discuss the contribution of these architectural changes to the generation of critical genomic 3D structures required for new regulatory mechanisms associated to morphological novelties. WIREs Dev Biol 2017, 6:e265. doi: 10.1002/wdev.265

Demixing and nematic behaviour of oblate hard spherocylinders and hard spheres mixtures: Monte Carlo simulation and Parsons-Lee theory

Parsons–Lee approach is formulated for the isotropic–nematic transition in a binary mixture of oblate hard spherocylinders and hard spheres. Results for the phase coexistence and for the equation of state in both phases for fluids with different relative size and composition ranges are presented. The predicted behaviour is in agreement with Monte Carlo simulations in a qualitative fashion. The study serves to provide a rational view of how to control key aspects of the behaviour of these binary nematogenic colloidal systems. This behaviour can be tuned with an appropriate choice of the relative size and molar fractions of the depleting particles. In general, the mixture of discotic and spherical particles is stable against demixing up to very high packing fractions. We explore in detail the narrow geometrical range where demixing is predicted to be possible in the isotropic phase. The influence of molecular crowding effects on the stability of the mixture when spherical molecules are added to a system of discotic colloids is also studied.

Biofilm formation-defective mutants in Pseudomonas putida.

Out of 8000 candidates from a genetic screening for Pseudomonas putida KT2442 mutants showing defects in biofilm formation, 40 independent mutants with diminished levels of biofilm were analyzed. Most of these mutants carried insertions in genes of the lap cluster, whose products are responsible for synthesis, export and degradation of the adhesin LapA. All mutants in this class were strongly defective in biofilm formation. Mutants in the flagellar regulatory genes fleQ and flhF showed similar defects to that of the lap mutants. On the contrary, transposon insertions in the flagellar structural genes fliP and flgG, that also impair flagellar motility, had a modest defect in biofilm formation. A mutation in gacS, encoding the sensor element of the GacS/GacA two-component system, also had a moderate effect on biofilm formation. Additional insertions targeted genes involved in cell envelope function: PP3222, encoding the permease element of an ABC-type transporter and tolB, encoding the periplasmic component of the Tol-OprL system required for outer membrane stability. Our results underscore the central role of LapA, suggest cross-regulation between motility and adhesion functions and provide insights on the role of cell envelope trafficking and maintenance for biofilm development in P. putida.

Cis-regulatory landscapes in development and evolution

The recent advances in our understanding of the 3D organization of the chromatin together with an almost unlimited ability to detect cis-regulatory elements genome-wide using different biochemical signatures has provided us with an unprecedented power to study gene regulation. It is now possible to profile the complete regulatory apparatus controlling the spatio-temporal expression of any given gene, the so-called gene Regulatory Landscapes (RLs). Here we review several studies over the last two years demonstrating the functional consequences of altering RL structure in development, disease and evolution. These works clearly show that a deep understanding of transcriptional regulation is no longer conceivable without considering the 3D modular organization of animal genomes.

Computer simulation study of early bacterial biofilm development

Most bacteria form organized sessile communities, known as biofilms. Their ubiquity and relevance have stimulated the development of efficient mathematical models able to predict biofilm evolution and characteristics at different conditions. Here we present a study of the early stages of bacterial biofilm formation modeled by means of individual cell-based computer simulation. Simulation showed that clusters with different degrees of internal and orientational order were formed as a function of the aspect ratio of the individual particles and the relation between the diffusion and growth rates. Analysis of microscope images of early biofilm formation by the Gram-negative bacterium Pseudomonas putida at varying diffusion rates revealed a good qualitative agreement with the simulation results. Our model is a good predictor of microcolony morphology during early biofilm development, showing that the competition between diffusion and growth rates is a key aspect in the formation of stable biofilm microcolonies.

A common copy-number variant within SIRPB1 correlates with human Out-of-Africa migration after genetic drift correction

Previous reports have proposed that personality may have played a role on human Out-Of-Africa migration, pinpointing some genetic variants that were positively selected in the migrating populations. In this work, we discuss the role of a common copy-number variant within the SIRPB1 gene, recently associated with impulsive behavior, in the human Out-Of-Africa migration. With the analysis of the variant distribution across forty-two different populations, we found that the SIRPB1 haplotype containing duplicated allele significantly correlated with human migratory distance, being one of the few examples of positively selected loci found across the human world colonization. Circular Chromosome Conformation Capture (4C-seq) experiments from the SIRPB1 promoter revealed important 3D modifications in the locus depending on the presence or absence of the duplication variant. In addition, a 3’ enhancer showed neural activity in transgenic models, suggesting that the presence of the CNV may compromise the expression of SIRPB1 in the central nervous system, paving the way to construct a molecular explanation of the SIRPB1 variants role in human migration.

4Cin: A computational pipeline for 3D genome modeling and virtual Hi-C analyses from 4C data

The use of 3C-based methods has revealed the importance of the 3D organization of the chromatin for key aspects of genome biology. However, the different caveats of the variants of 3C techniques have limited their scope and the range of scientific fields that could benefit from these approaches. To address these limitations, we present 4Cin, a method to generate 3D models and derive virtual Hi-C (vHi-C) heat maps of genomic loci based on 4C-seq or any kind of 4C-seq-like data, such as those derived from NG Capture-C. 3D genome organization is determined by integrative consideration of the spatial distances derived from as few as four 4C-seq experiments. The 3D models obtained from 4C-seq data, together with their associated vHi-C maps, allow the inference of all chromosomal contacts within a given genomic region, facilitating the identification of Topological Associating Domains (TAD) boundaries. Thus, 4Cin offers a much cheaper, accessible and versatile alternative to other available techniques while providing a comprehensive 3D topological profiling. By studying TAD modifications in genomic structural variants associated to disease phenotypes and performing cross-species evolutionary comparisons of 3D chromatin structures in a quantitative manner, we demonstrate the broad potential and novel range of applications of our method.