Putting the genome on the map

The maps of our everyday lives are much more than just linear lists of place names. Instead, their colours, symbols, contours and grid lines seek to describe different types of landscape, and to depict the spatial relationships between structural and functional landmarks of the environment (Fig. 1). It was the combination of photography and aviation that revolutionized mapmaking in the early part of this century. In much the same way, it is fluorescence microscopy and digital imaging (Box 1) in combination with molecular genetics that is driving our emerging view of the genome in space and time

A (3D-nuclear) space odyssey: making sense of Hi-C maps

Three-dimensional 3D)-chromatin organization is critical for proper enhancer-promoter communication and, therefore, for a precise execution of the transcriptional programs governing cellular processes. The emergence of Chromosome Conformation Capture (3C) methods, in particular Hi-C, has allowed the investigation of chromatin interactions on a genome-wide scale, revealing the existence of overlapping molecular mechanisms that we are just starting to decipher. Therefore, disentangling Hi-C signal into these individual components is essential to provide meaningful biological data interpretation. Here, we discuss emerging views on the molecular forces shaping the genome in 3D, with a focus on their respective contributions and interdependence. We discuss Hi-C data at both population and single-cell levels, thus providing criteria to interpret genomic function in the 3D-nuclear space

Mechanisms of Regulatory Adaptation in the Evolving Genome

The development from a single cell into a complex organism requires the precise control of gene expression in space and time. To achieve this, the activity of genes is governed by large regulatory chromatin landscapes that when disrupted can cause gene mis-regulation and disease. However, at the same time, the successful modification of these landscapes is thought to be a major driver of phenotypic innovation during evolution. Given the vulnerability of these landscapes in disease settings, it remains largely unknown how their integrity is maintained when novel genes are “safely” incorporated during evolution, which is addressed in this work. Specifically, here, multiple mechanisms are dissected that adapted the Fat1 regulatory landscape to maintain its integrity while simultaneously incorporating a novel gene, Zfp42, during evolution. First, comparative evolutionary genomics was used to reconstruct the history of the locus (section 1). Second, the three-dimensional chromatin configuration of the locus was examined in relationship to the gene activities using genomics-technologies (HiC, DamID) combined with super resolution microscopy and in silico modeling (section 2). Finally, the mechanisms that adapted the landscape in ESCs (section 3) and embryonic limbs (section 4) for the emergence of Zfp42 were investigated using genome engineering and genomics. Two tissue-specific mechanisms were identified that enabled the independent activities of Zfp42 and Fat1 despite sharing the same regulatory chromatin landscape: In ESCs, the landscape physically restructures and isolates the genes together with their regulatory information, from one another, thereby allowing their independent regulation. Surprisingly, this restructuring is not driven by the most recognized chromatin structuring force, loop extrusion, but rather by the underlying epigenetic state of chromatin. A different mechanism operates in embryonic mouse limbs where both genes are exposed to the same regulatory information driving Fat1 activation, but surprisingly not Zfp42. The inactivity of Zfp42 cannot be explained by nuclear envelopment attachment nor by enhancer-promoter specificity. Instead, Zfp42 is kept inactive by a highly context-dependent silencing mechanism driven by DNA methylation. As such, Zfp42 is ectopically active and responsive to the surrounding regulatory information when DNA methylation is removed or when the gene is slightly repositioned within its domain. Combined, we find that 3D-restructuring and context-dependent silencing adapted the Fat1 landscape to integrate Zfp42. More generally, this demonstrates that even single regulatory landscapes harbor an enormous regulatory complexity and, thus can accommodate multiple independently regulated genes. We believe that this has significant consequences for human genetics where similar genomic alterations do not drive disease in patients. This is possible, because additional, yet still unknown, mechanisms control how regulatory information is used in the genome

The radial arrangement of the human chromosome 7 in the lymphocyte cell nucleus is associated with chromosomal band gene density

This is the author's accepted manuscript. The final published article is available from the link below. Copyright @ Springer-Verlag 2008.In the nuclei of human lymphocytes, chromosome territories are distributed according to the average gene density of each chromosome. However, chromosomes are very heterogeneous in size and base composition, and can contain both very gene-dense and very gene-poor regions. Thus, a precise analysis of chromosome organisation in the nuclei should consider also the distribution of DNA belonging to the chromosomal bands in each chromosome. To improve our understanding of the chromatin organisation, we localised chromosome 7 DNA regions, endowed with different gene densities, in the nuclei of human lymphocytes. Our results showed that this chromosome in cell nuclei is arranged radially with the gene-dense/GC-richest regions exposed towards the nuclear interior and the gene-poorest/GC-poorest ones located at the nuclear periphery. Moreover, we found that chromatin fibres from the 7p22.3 and the 7q22.1 bands are not confined to the territory of the bulk of this chromosome, protruding towards the inner part of the nucleus. Overall, our work demonstrates the radial arrangement of the territory of chromosome 7 in the lymphocyte nucleus and confirms that human genes occupy specific radial positions, presumably to enhance intra- and inter-chromosomal interaction among loci displaying a similar expression pattern, and/or similar replication timing

Constitutive nuclear lamina-genome interactions are highly conserved and associated with A/T-rich sequence

In metazoans, the nuclear lamina is thought to play an important role in the spatial organization of interphase chromosomes, by providing anchoring sites for large genomic segments named lamina-associated domains (LADs). Some of these LADs are cell-type specific, while many others appear constitutively associated with the lamina. Constitutive LADs (cLADs) may contribute to a basal chromosome architecture. By comparison of mouse and human lamina interaction maps, we find that the sizes and genomic positions of cLADs are strongly conserved. Moreover, cLADs are depleted of synteny breakpoints, pointing to evolutionary selective pressure to keep cLADs intact. Paradoxically, the overall sequence conservation is low for cLADs. Instead, cLADs are universally characterized by long stretches of DNA of high A/T content. Cell-type specific LADs also tend to adhere to this “A/T rule” in embryonic stem cells, but not in differentiated cells. This suggests that the A/T rule represents a default positioning mechanism that is locally overruled during lineage commitment. Analysis of paralogs suggests that during evolution changes in A/T content have driven the relocation of genes to and from the nuclear lamina, in tight association with changes in expression level. Taken together, these results reveal that the spatial organization of mammalian genomes is highly conserved and tightly linked to local nucleotide composition

Order and disorder: abnormal 3D chromatin organization in human disease

A precise three-dimensional (3D) organization of chromatin is central to achieve the intricate transcriptional patterns that are required to form complex organisms. Growing evidence supports an important role of 3D chromatin architecture in development and delineates its alterations as prominent causes of disease. In this review, we discuss emerging concepts on the fundamental forces shaping genomes in space and on how their disruption can lead to pathogenic phenotypes. We describe the molecular mechanisms underlying a wide range of diseases, from the systemic effects of coding mutations on 3D architectural factors, to the more tissue-specific phenotypes resulting from genetic and epigenetic modifications at specific loci. Understanding the connection between the 3D organization of the genome and its underlying biological function will allow a better interpretation of human pathogenesis

Application of advanced fluorescence microscopy to the structure of meiotic chromosomes

Chromosomes undergoing meiosis are defined by a macromolecular protein assembly called the synaptonemal complex which holds homologs together and carries out important meiotic functions. By retaining the molecular specificity, multiplexing ability, and in situ imaging capabilities of fluorescence microscopy, but with vastly increased resolution, 3D-SIM and other superresolution techniques are poised to make significant discoveries about the structure and function of the synaptonemal complex. This review discusses recent developments in this field and poses questions approachable with current and future technolog

Spatial and Epigenetic Regulation of T-Cell Receptor Beta Gene Assembly

The adaptive immune system endows mammals with a sophisticated mechanism to recognize foreign proteins via surface antigen receptors that are expressed on the surface of all lymphocytes. This defense network is generated by V(D)J recombination, a set of sequentially controlled DNA cleavage and repair events that assembles functional antigen receptor genes from distally located Variable (V), Diversity (D) and Joining (J) gene segments. However, the recombination process must be stringently regulated to prevent formation of chromosomal translocations, which can lead to tumors. The process of V(D)J recombination is controlled at the levels of tissue, stage and allele specificity by a collection of architectural and regulatory elements that are distributed throughout each antigen receptor locus. Our laboratory has characterized several genetic elements that regulate chromatin accessibility and recombination at the T cell receptor beta (Tcrb) locus. These elements include transcriptional promoters and enhancers, which interact with each other in conformational space to form a promoter-enhancer holocomplex, facilitating Dβ to Jβ recombination. Simultaneously, spatial apposition of the Vβ cluster to the DβJβ region (a phenomenon called locus contraction) increases the efficiency of long-range Vβ recombination. Using extensive chromatin profiling of the Tcrb locus, we have discovered that selection of Vβ genes depend upon their association with transcriptionally active chromatin and high quality Recombination Signal Sequences, which serve as substrates for the V(D)J recombinase proteins RAG1/2. We further identify a bi-functional barrier-tethering region upstream of the DβJβ cluster that is essential for stabilizing its long-range interactions with distal Vβ gene segments in progenitor CD4-CD8- double negative (DN) thymocytes. Following Tcrb rearrangement, progenitor thymocytes proliferate and differentiate into CD4+CD8+ Double Positive (DP) cells, where the Vβ genes are epigenetically silenced and the distal ends of Tcrb are spatially segregated (presumably to inhibit further rearrangements). However, we have found that the transcriptionally inactive proximal Vβ genes continue to interact with the DβJβ cluster in a proliferation independent manner. These findings divide the Tcrb locus into two architectural domains, of which only the distal part is spatially segregated in DP cells. The loss of distal Vβ interaction is also observed in DP thymocytes containing a rearranged Tcrb allele, suggesting this conformation is DP-intrinsic. Our results have unraveled new mechanisms that stabilize the long-range Tcrb conformation in DN cells, how the Vβ segments are selected to recombine and how Tcrb topology is retained by DP-intrinsic mechanisms. These studies pave the way for future investigations into the role of boundary elements and tissue specific transcription factors in sculpting AgR gene assembly and regulating genome topology

Spatial chromatin architecture alteration by structural variations in human genomes at the population scale.

BACKGROUND: The number of reported examples of chromatin architecture alterations involved in the regulation of gene transcription and in disease is increasing. However, no genome-wide testing has been performed to assess the abundance of these events and their importance relative to other factors affecting genome regulation. This is particularly interesting given that a vast majority of genetic variations identified in association studies are located outside coding sequences. This study attempts to address this lack by analyzing the impact on chromatin spatial organization of genetic variants identified in individuals from 26 human populations and in genome-wide association studies. RESULTS: We assess the tendency of structural variants to accumulate in spatially interacting genomic segments and design an algorithm to model chromatin conformational changes caused by structural variations. We show that differential gene transcription is closely linked to the variation in chromatin interaction networks mediated by RNA polymerase II. We also demonstrate that CTCF-mediated interactions are well conserved across populations, but enriched with disease-associated SNPs. Moreover, we find boundaries of topological domains as relatively frequent targets of duplications, which suggest that these duplications can be an important evolutionary mechanism of genome spatial organization. CONCLUSIONS: This study assesses the critical impact of genetic variants on the higher-order organization of chromatin folding and provides insight into the mechanisms regulating gene transcription at the population scale, of which local arrangement of chromatin loops seems to be the most significant. It provides the first insight into the variability of the human 3D genome at the population scale