The High-Salt Recovered Sequence-Sequencing (HRS-seq) Method: Exploring Genome Association with Nuclear Bodies.

Recent works indicate that, at specific loci, interactions of chromatin with membrane-less organelles self-assembled through mechanisms of phase separation, like nuclear bodies, are crucial to regulate genome functions, and in particular transcription. Here we describe the protocol of the high-salt recovered sequence sequencing method whose principle relies on high-throughput sequencing of genomic DNA trapped into large RNP complexes that are made insoluble by high-salt treatments.

Local euchromatin enrichment in lamina-associated domains anticipates their repositioning in the adipogenic lineage.

BACKGROUND: Interactions of chromatin with the nuclear lamina via lamina-associated domains (LADs) confer structural stability to the genome. The dynamics of positioning of LADs during differentiation, and how LADs impinge on developmental gene expression, remains, however, elusive. RESULTS: We examined changes in the association of lamin B1 with the genome in the first 72 h of differentiation of adipose stem cells into adipocytes. We demonstrate a repositioning of entire stand-alone LADs and of LAD edges as a prominent nuclear structural feature of early adipogenesis. Whereas adipogenic genes are released from LADs, LADs sequester downregulated or repressed genes irrelevant for the adipose lineage. However, LAD repositioning only partly concurs with gene expression changes. Differentially expressed genes in LADs, including LADs conserved throughout differentiation, reside in local euchromatic and lamin-depleted sub-domains. In these sub-domains, pre-differentiation histone modification profiles correlate with the LAD versus inter-LAD outcome of these genes during adipogenic commitment. Lastly, we link differentially expressed genes in LADs to short-range enhancers which overall co-partition with these genes in LADs versus inter-LADs during differentiation. CONCLUSIONS: We conclude that LADs are predictable structural features of adipose nuclear architecture that restrain non-adipogenic genes in a repressive environment.

Exploring Mammalian Genome within Phase-Separated Nuclear Bodies: Experimental Methods and Implications for Gene Expression.

The importance of genome organization at the supranucleosomal scale in the control of gene expression is increasingly recognized today. In mammals, Topologically Associating Domains (TADs) and the active/inactive chromosomal compartments are two of the main nuclear structures that contribute to this organization level. However, recent works reviewed here indicate that, at specific loci, chromatin interactions with nuclear bodies could also be crucial to regulate genome functions, in particular transcription. They moreover suggest that these nuclear bodies are membrane-less organelles dynamically self-assembled and disassembled through mechanisms of phase separation. We have recently developed a novel genome-wide experimental method, High-salt Recovered Sequences sequencing (HRS-seq), which allows the identification of chromatin regions associated with large ribonucleoprotein (RNP) complexes and nuclear bodies. We argue that the physical nature of such RNP complexes and nuclear bodies appears to be central in their ability to promote efficient interactions between distant genomic regions. The development of novel experimental approaches, including our HRS-seq method, is opening new avenues to understand how self-assembly of phase-separated nuclear bodies possibly contributes to mammalian genome organization and gene expression.

Long-range interactions between topologically associating domains shape the four-dimensional genome during differentiation.

Genomic information is selectively used to direct spatial and temporal gene expression during differentiation. Interactions between topologically associating domains (TADs) and between chromatin and the nuclear lamina organize and position chromosomes in the nucleus. However, how these genomic organizers together shape genome architecture is unclear. Here, using a dual-lineage differentiation system, we report long-range TAD-TAD interactions that form constitutive and variable TAD cliques. A differentiation-coupled relationship between TAD cliques and lamina-associated domains suggests that TAD cliques stabilize heterochromatin at the nuclear periphery. We also provide evidence of dynamic TAD cliques during mouse embryonic stem-cell differentiation and somatic cell reprogramming and of inter-TAD associations in single-cell high-resolution chromosome conformation capture (Hi-C) data. TAD cliques represent a level of four-dimensional genome conformation that reinforces the silencing of repressed developmental genes.

High-salt-recovered sequences are associated with the active chromosomal compartment and with large ribonucleoprotein complexes including nuclear bodies.

The mammalian cell nucleus contains numerous discrete suborganelles named nuclear bodies. While recruitment of specific genomic regions into these large ribonucleoprotein (RNP) complexes critically contributes to higher-order functional chromatin organization, such regions remain ill-defined. We have developed the high-salt-recovered sequences-sequencing (HRS-seq) method, a straightforward genome-wide approach whereby we isolated and sequenced genomic regions associated with large high-salt insoluble RNP complexes. By using mouse embryonic stem cells (ESCs), we showed that these regions essentially correspond to the most highly expressed genes, and to cis-regulatory sequences like super-enhancers, that belong to the active A chromosomal compartment. They include both cell-type-specific genes, such as pluripotency genes in ESCs, and housekeeping genes associated with nuclear bodies, such as histone and snRNA genes that are central components of Histone Locus Bodies and Cajal bodies. We conclude that HRSs are associated with the active chromosomal compartment and with large RNP complexes including nuclear bodies. Association of such chromosomal regions with nuclear bodies is in agreement with the recently proposed phase separation model for transcription control and might thus play a central role in organizing the active chromosomal compartment in mammals.

Distinct polymer physics principles govern chromatin dynamics in mouse and Drosophila topological domains.

BACKGROUND: In higher eukaryotes, the genome is partitioned into large "Topologically Associating Domains" (TADs) in which the chromatin displays favoured long-range contacts. While a crumpled/fractal globule organization has received experimental supports at higher-order levels, the organization principles that govern chromatin dynamics within these TADs remain unclear. Using simple polymer models, we previously showed that, in mouse liver cells, gene-rich domains tend to adopt a statistical helix shape when no significant locus-specific interaction takes place. RESULTS: Here, we use data from diverse 3C-derived methods to explore chromatin dynamics within mouse and Drosophila TADs. In mouse Embryonic Stem Cells (mESC), that possess large TADs (median size of 840 kb), we show that the statistical helix model, but not globule models, is relevant not only in gene-rich TADs, but also in gene-poor and gene-desert TADs. Interestingly, this statistical helix organization is considerably relaxed in mESC compared to liver cells, indicating that the impact of the constraints responsible for this organization is weaker in pluripotent cells. Finally, depletion of histone H1 in mESC alters local chromatin flexibility but not the statistical helix organization. In Drosophila, which possesses TADs of smaller sizes (median size of 70 kb), we show that, while chromatin compaction and flexibility are finely tuned according to the epigenetic landscape, chromatin dynamics within TADs is generally compatible with an unconstrained polymer configuration. CONCLUSIONS: Models issued from polymer physics can accurately describe the organization principles governing chromatin dynamics in both mouse and Drosophila TADs. However, constraints applied on this dynamics within mammalian TADs have a peculiar impact resulting in a statistical helix organization.

Contribution of Topological Domains and Loop Formation to 3D Chromatin Organization.

Recent investigations on 3D chromatin folding revealed that the eukaryote genomes are both highly compartmentalized and extremely dynamic. This review presents the most recent advances in topological domains' organization of the eukaryote genomes and discusses the relationship to chromatin loop formation. CTCF protein appears as a central factor of these two organization levels having either a strong insulating role at TAD borders, or a weaker architectural role in chromatin loop formation. TAD borders directly impact on chromatin dynamics by restricting contacts within specific genomic portions thus confining chromatin loop formation within TADs. We discuss how sub-TAD chromatin dynamics, constrained into a recently described statistical helix conformation, can produce functional interactions by contact stabilization.

Epimutation profiling in Beckwith-Wiedemann syndrome: relationship with assisted reproductive technology.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth disorder associated with abnormalities in 11p15.5 imprinted genes. The most common cause is loss of methylation (epimutation) at the imprinting control centre 2 (IC2/KvDMR1). Most IC2 epimutations occur sporadically but an association with conception after assisted reproductive technologies (ART) has been reported. A subgroup of IC2 epimutation cases also harbour epimutations at other imprinting centres (ICs) outside of 11p15.5. We have investigated the relationship between these multiple epimutation cases (ME+), history of ART and clinical phenotype in a cohort of 187 BWS IC2 epimutation patients. RESULTS: Methylation analysis at PLAGL1, MEST and IGF2R ICs demonstrated an over-representation of patients with abnormally low methylation (8.5%, 12% and 6% respectively). At IGF2R some patients (2%) had gain of methylation but this was also detected in controls. Though there were no significant correlations between the methylation index (MIs) at the three ICs tested, a subset of patients appeared to be susceptible to multiple epimutations (ME+) and 21.2% of ME + patients had been conceived by ART compared to 4.5% (P = 0.0033) without additional epimutations. Methylation array profiling (Illumina Goldengate®) of patients and controls (excluding 11p15.5 loci) demonstrated significant differences between patients and controls. No significant associations were found between aspects of the BWS phenotype and individual epimutations but we describe a case presenting with a post-ART BWS-like phenotype in which molecular analysis demonstrated loss of paternal allele methylation at the 11p15.5 IC1 locus (IC1 regulates imprinting of IGF2 and H19). Loss of paternal allele methylation at the IC1 is the molecular finding associated with Silver-Russell syndrome whereas BWS is associated with gain of maternal allele methylation at IC1. Further analysis demonstrated epimutations at PLAGL1 and MEST consistent with the hypothesis that the presence of multiple epimutations may be of clinical relevance. CONCLUSIONS: These findings suggest that the ME + subgroup of BWS patients are preferentially, but not exclusively, associated with a history of ART and that, though at present, there are no clear epigenotype-phenotype correlations for ME + BWS patients, non-11p15.5 IC epimutations can influence clinical phenotype.