Geoffrey Fudenberg

Organization of the Mitotic Chromosome

Chromosome Conundrum The three-dimensional organization of chromosomal DNA within the cell nucleus plays an important role in gene regulation. Naumova et al. (p. 948, published online 7 November; see the Perspective by Kleckner et al.) used chromosome conformation capture-based methods in human tissue culture cells to analyze the higher order folding of human chromosomes across the cell cycle. During interphase the chromosomes showed locus-specific compart-mentalization. In mitotic cells, on the other hand, the chromosome organization was more linear, consistent with arrays of consecutive chromatin loops. Chromosome conformation changes dramatically during the cell cycle and is unlikely to carry epigenetic information. [Also see Perspective by Kleckner et al.] Mitotic chromosomes are among the most recognizable structures in the cell, yet for over a century their internal organization remains largely unsolved. We applied chromosome conformation capture methods, 5C and Hi-C, across the cell cycle and revealed two distinct three-dimensional folding states of the human genome. We show that the highly compartmentalized and cell type–specific organization described previously for nonsynchronous cells is restricted to interphase. In metaphase, we identified a homogenous folding state that is locus-independent, common to all chromosomes, and consistent among cell types, suggesting a general principle of metaphase chromosome organization. Using polymer simulations, we found that metaphase Hi-C data are inconsistent with classic hierarchical models and are instead best described by a linearly organized longitudinally compressed array of consecutive chromatin loops.

Iterative correction of Hi-C data reveals hallmarks of chromosome organization.

Extracting biologically meaningful information from chromosomal interactions obtained with genome-wide chromosome conformation capture (3C) analyses requires the elimination of systematic biases. We present a computational pipeline that integrates a strategy to map sequencing reads with a data-driven method for iterative correction of biases, yielding genome-wide maps of relative contact probabilities. We validate this ICE (iterative correction and eigenvector decomposition) technique on published data obtained by the high-throughput 3C method Hi-C, and we demonstrate that eigenvector decomposition of the obtained maps provides insights into local chromatin states, global patterns of chromosomal interactions, and the conserved organization of human and mouse chromosomes.

Formation of Chromosomal Domains by Loop Extrusion

Topologically associating domains (TADs) are fundamental structural and functional building blocks of human interphase chromosomes, yet the mechanisms of TAD formation remain unclear. Here, we propose that loop extrusion underlies TAD formation. In this process, cis-acting loop-extruding factors, likely cohesins, form progressively larger loops but stall at TAD boundaries due to interactions with boundary proteins, including CTCF. Using polymer simulations, we show that this model produces TADs and finer-scale features of Hi-C data. Each TAD emerges from multiple loops dynamically formed through extrusion, contrary to typical illustrations of single static loops. Loop extrusion both explains diverse experimental observations-including the preferential orientation of CTCF motifs, enrichments of architectural proteins at TAD boundaries, and boundary deletion experiments-and makes specific predictions for the depletion of CTCF versus cohesin. Finally, loop extrusion has potentially far-ranging consequences for processes such as enhancer-promoter interactions, orientation-specific chromosomal looping, and compaction of mitotic chromosomes.

Cohesin-dependent globules and heterochromatin shape 3D genome architecture in S. pombe

Eukaryotic genomes are folded into three-dimensional structures, such as self-associating topological domains, the borders of which are enriched in cohesin and CCCTC-binding factor (CTCF) required for long-range interactions. How local chromatin interactions govern higher-order folding of chromatin fibres and the function of cohesin in this process remain poorly understood. Here we perform genome-wide chromatin conformation capture (Hi-C) analysis to explore the high-resolution organization of the Schizosaccharomyces pombe genome, which despite its small size exhibits fundamental features found in other eukaryotes. Our analyses of wild-type and mutant strains reveal key elements of chromosome architecture and genome organization. On chromosome arms, small regions of chromatin locally interact to form ‘globules’. This feature requires a function of cohesin distinct from its role in sister chromatid cohesion. Cohesin is enriched at globule boundaries and its loss causes disruption of local globule structures and global chromosome territories. By contrast, heterochromatin, which loads cohesin at specific sites including pericentromeric and subtelomeric domains, is dispensable for globule formation but nevertheless affects genome organization. We show that heterochromatin mediates chromatin fibre compaction at centromeres and promotes prominent inter-arm interactions within centromere-proximal regions, providing structural constraints crucial for proper genome organization. Loss of heterochromatin relaxes constraints on chromosomes, causing an increase in intra- and inter-chromosomal interactions. Together, our analyses uncover fundamental genome folding principles that drive higher-order chromosome organization crucial for coordinating nuclear functions.

Genome-wide maps of nuclear lamina interactions in single human cells.

Mammalian interphase chromosomes interact with the nuclear lamina (NL) through hundreds of large lamina-associated domains (LADs). We report a method to map NL contacts genome-wide in single human cells. Analysis of nearly 400 maps reveals a core architecture consisting of gene-poor LADs that contact the NL with high cell-to-cell consistency, interspersed by LADs with more variable NL interactions. The variable contacts tend to be cell-type specific and are more sensitive to changes in genome ploidy than the consistent contacts. Single-cell maps indicate that NL contacts involve multivalent interactions over hundreds of kilobases. Moreover, we observe extensive intra-chromosomal coordination of NL contacts, even over tens of megabases. Such coordinated loci exhibit preferential interactions as detected by Hi-C. Finally, the consistency of NL contacts is inversely linked to gene activity in single cells and correlates positively with the heterochromatic histone modification H3K9me3. These results highlight fundamental principles of single-cell chromatin organization. VIDEO ABSTRACT.

Higher order chromatin structure: bridging physics and biology

Advances in microscopy and genomic techniques have provided new insight into spatial chromatin organization inside of the nucleus. In particular, chromosome conformation capture data has highlighted the relevance of polymer physics for high-order chromatin organization. In this context, we review basic polymer states, discuss how an appropriate polymer model can be determined from experimental data, and examine the success and limitations of various polymer models of higher-order interphase chromatin organization. By taking into account topological constraints acting on the chromatin fiber, recently developed polymer models of interphase chromatin can reproduce the observed scaling of distances between genomic loci, chromosomal territories, and probabilities of contacts between loci measured by chromosome conformation capture methods. Polymer models provide a framework for the interpretation of experimental data as ensembles of conformations rather than collections of loops, and will be crucial for untangling functional implications of chromosomal organization.

Two independent modes of chromatin organization revealed by cohesin removal

Imaging and chromosome conformation capture studies have revealed several layers of chromosome organization, including segregation into megabase-sized active and inactive compartments, and partitioning into sub-megabase domains (TADs). It remains unclear, however, how these layers of organization form, interact with one another and influence genome function. Here we show that deletion of the cohesin-loading factor Nipbl in mouse liver leads to a marked reorganization of chromosomal folding. TADs and associated Hi-C peaks vanish globally, even in the absence of transcriptional changes. By contrast, compartmental segregation is preserved and even reinforced. Strikingly, the disappearance of TADs unmasks a finer compartment structure that accurately reflects the underlying epigenetic landscape. These observations demonstrate that the three-dimensional organization of the genome results from the interplay of two independent mechanisms: cohesin-independent segregation of the genome into fine-scale compartments, defined by chromatin state; and cohesin-dependent formation of TADs, possibly by loop extrusion, which helps to guide distant enhancers to their target genes.

Chromatin Loops as Allosteric Modulators of Enhancer-Promoter Interactions

The classic model of eukaryotic gene expression requires direct spatial contact between a distal enhancer and a proximal promoter. Recent Chromosome Conformation Capture (3C) studies show that enhancers and promoters are embedded in a complex network of looping interactions. Here we use a polymer model of chromatin fiber to investigate whether, and to what extent, looping interactions between elements in the vicinity of an enhancer-promoter pair can influence their contact frequency. Our equilibrium polymer simulations show that a chromatin loop, formed by elements flanking either an enhancer or a promoter, suppresses enhancer-promoter interactions, working as an insulator. A loop formed by elements located in the region between an enhancer and a promoter, on the contrary, facilitates their interactions. We find that different mechanisms underlie insulation and facilitation; insulation occurs due to steric exclusion by the loop, and is a global effect, while facilitation occurs due to an effective shortening of the enhancer-promoter genomic distance, and is a local effect. Consistently, we find that these effects manifest quite differently for in silico 3C and microscopy. Our results show that looping interactions that do not directly involve an enhancer-promoter pair can nevertheless significantly modulate their interactions. This phenomenon is analogous to allosteric regulation in proteins, where a conformational change triggered by binding of a regulatory molecule to one site affects the state of another site.

Modeling chromosomes: Beyond pretty pictures

Recently, Chromosome Conformation Capture (3C) based experiments have highlighted the importance of computational models for the study of chromosome organization. In this review, we propose that current computational models can be grouped into roughly four classes, with two classes ofdata‐driven models: consensus structures and data‐driven ensembles, and two classes ofde novo models: structural ensembles and mechanistic ensembles. Finally, we highlight specific questions mechanistic ensembles can address.

FISH-ing for captured contacts: towards reconciling FISH and 3C

Chromosome conformation capture (3C) and fluorescence in situ hybridization (FISH) are two widely used technologies that provide distinct readouts of 3D chromosome organization. While both technologies can assay locus-specific organization, how to integrate views from 3C, or genome-wide Hi-C, and FISH is far from solved. Contact frequency, measured by Hi-C, and spatial distance, measured by FISH, are often assumed to quantify the same phenomena and used interchangeably. Here, however, we demonstrate that contact frequency is distinct from average spatial distance, both in polymer simulations and in experimental data. Performing a systematic analysis of the technologies, we show that this distinction can create a seemingly paradoxical relationship between 3C and FISH, both in minimal polymer models with dynamic looping interactions and in loop-extrusion simulations. Together, our results indicate that cross-validation of Hi-C and FISH should be carefully designed, and that jointly considering contact frequency and spatial distance is crucial for fully understanding chromosome organization.