Recent evidence that TADs and chromatin loops are dynamic structures.

Mammalian genomes are folded into spatial domains, which regulate gene expression by modulating enhancer-promoter contacts. Here, we review recent studies on the structure and function of Topologically Associating Domains (TADs) and chromatin loops. We discuss how loop extrusion models can explain TAD formation and evidence that TADs are formed by the ring-shaped protein complex, cohesin, and that TAD boundaries are established by the DNA-binding protein, CTCF. We discuss our recent genomic, biochemical and single-molecule imaging studies on CTCF and cohesin, which suggest that TADs and chromatin loops are dynamic structures. We highlight complementary polymer simulation studies and Hi-C studies employing acute depletion of CTCF and cohesin, which also support such a dynamic model. We discuss the limitations of each approach and conclude that in aggregate the available evidence argues against stable loops and supports a model where TADs are dynamic structures that continually form and break throughout the cell cycle

Minor Loops in Major Folds: Enhancer-Promoter Looping, Chromatin Restructuring, and Their Association with Transcriptional Regulation and Disease.

The organization and folding of chromatin within the nucleus can determine the outcome of gene expression. Recent technological advancements have enabled us to study chromatin interactions in a genome-wide manner at high resolution. These studies have increased our understanding of the hierarchy and dynamics of chromatin domains that facilitate cognate enhancer-promoter looping, defining the transcriptional program of different cell types. In this review, we focus on vertebrate chromatin long-range interactions as they relate to transcriptional regulation. In addition, we describe how the alteration of boundaries that mark discrete regions in the genome with high interaction frequencies within them, called topological associated domains (TADs), could lead to various phenotypes, including human diseases, which we term as "TADopathies.

Nuclear organisation and replication timing are coupled through RIF1-PP1 interaction

Three-dimensional genome organisation and replication timing are known to be correlated, however, it remains unknown whether nuclear architecture overall plays an instructive role in the replication-timing programme and, if so, how. Here we demonstrate that RIF1 is a molecular hub that co-regulates both processes. Both nuclear organisation and replication timing depend upon the interaction between RIF1 and PP1. However, whereas nuclear architecture requires the full complement of RIF1 and its interaction with PP1, replication timing is not sensitive to RIF1 dosage. The role of RIF1 in replication timing also extends beyond its interaction with PP1. Availing of this separation-of-function approach, we have therefore identified in RIF1 dual function the molecular bases of the co-dependency of the replication-timing programme and nuclear architecture

Transcription-coupled structural dynamics of topologically associating domains regulate replication origin efficiency

Background Metazoan cells only utilize a small subset of the potential DNA replication origins to duplicate the whole genome in each cell cycle. Origin choice is linked to cell growth, differentiation, and replication stress. Although various genetic and epigenetic signatures have been linked to the replication efficiency of origins, there is no consensus on how the selection of origins is determined. Results We apply dual-color stochastic optical reconstruction microscopy (STORM) super-resolution imaging to map the spatial distribution of origins within individual topologically associating domains (TADs). We find that multiple replication origins initiate separately at the spatial boundary of a TAD at the beginning of the S phase. Intriguingly, while both high-efficiency and low-efficiency origins are distributed homogeneously in the TAD during the G1 phase, high-efficiency origins relocate to the TAD periphery before the S phase. Origin relocalization is dependent on both transcription and CTCF-mediated chromatin structure. Further, we observe that the replication machinery protein PCNA forms immobile clusters around TADs at the G1/S transition, explaining why origins at the TAD periphery are preferentially fired. Conclusion Our work reveals a new origin selection mechanism that the replication efficiency of origins is determined by their physical distribution in the chromatin domain, which undergoes a transcription-dependent structural re-organization process. Our model explains the complex links between replication origin efficiency and many genetic and epigenetic signatures that mark active transcription. The coordination between DNA replication, transcription, and chromatin organization inside individual TADs also provides new insights into the biological functions of sub-domain chromatin structural dynamics

Rapid Irreversible Transcriptional Reprogramming in Human Stem Cells Accompanied by Discordance between Replication Timing and Chromatin Compartment

The temporal order of DNA replication is regulated during development and is highly correlated with gene expression, histone modifications and 3D genome architecture. We tracked changes in replication timing, gene expression, and chromatin conformation capture (Hi-C) A/B compartments over the first two cell cycles during differentiation of human embryonic stem cells to definitive endoderm. Remarkably, transcriptional programs were irreversibly reprogrammed within the first cell cycle and were largely but not universally coordinated with replication timing changes. Moreover, changes in A/B compartment and several histone modifications that normally correlate strongly with replication timing showed weak correlation during the early cell cycles of differentiation but showed increased alignment in later differentiation stages and in terminally differentiated cell lines. Thus, epigenetic cell fate transitions during early differentiation can occur despite dynamic and discordant changes in otherwise highly correlated genomic properties

Structural organization of human replication timing domains

AbstractRecent analysis of genome-wide epigenetic modification data, mean replication timing (MRT) profiles and chromosome conformation data in mammals have provided increasing evidence that flexibility in replication origin usage is regulated locally by the epigenetic landscape and over larger genomic distances by the 3D chromatin architecture. Here, we review the recent results establishing some link between replication domains and chromatin structural domains in pluripotent and various differentiated cell types in human. We reconcile the originally proposed dichotomic picture of early and late constant timing regions that replicate by multiple rather synchronous origins in separated nuclear compartments of open and closed chromatins, with the U-shaped MRT domains bordered by “master” replication origins specified by a localized (∼200–300kb) zone of open and transcriptionally active chromatin from which a replication wave likely initiates and propagates toward the domain center via a cascade of origin firing. We discuss the relationships between these MRT domains, topologically associated domains and lamina-associated domains. This review sheds a new light on the epigenetically regulated global chromatin reorganization that underlies the loss of pluripotency and the determination of differentiation properties

DNA replication and replication stress response in the context of nuclear architecture

The DNA replication process needs to be coordinated with other DNA metabolism transactions and must eventually extend to the full genome, regardless of chromatin status, gene expression, secondary structures and DNA lesions. Completeness and accuracy of DNA replication are crucial to maintain genome integrity, limiting transformation in normal cells and offering targeting opportunities for proliferating cancer cells. DNA replication is thus tightly coordinated with chromatin dynamics and 3D genome architecture, and we are only beginning to understand the underlying molecular mechanisms. While much has recently been discovered on how DNA replication initiation is organised and modulated in different genomic regions and nuclear territories-the so-called "DNA replication program"-we know much less on how the elongation of ongoing replication forks and particularly the response to replication obstacles is affected by the local nuclear organisation. Also, it is still elusive how specific components of nuclear architecture participate in the replication stress response. Here, we review known mechanisms and factors orchestrating replication initiation, and replication fork progression upon stress, focusing on recent evidence linking genome organisation and nuclear architecture with the cellular responses to replication interference, and highlighting open questions and future challenges to explore this exciting new avenue of research

Electrical induction hypothesis to explain Enhancer-Promoter Communication

Abstract The steps of the DNA replication process remains to be clarified. Transcription factors are supposed to find their specific binding-sequence driven by epigenetic modifications and GpC islands. But then how can the replication machinery be able to find the promoters of exactly the genes that the cell needs to transcribe in that moment? Here we hypothesize a role of DNA conductance and electrical induction to give an explanation to this unsolved problem. Our hypothesis goes in accordance with the fact that many authors identified 3D loops in the genomes

Allele-specific control of replication timing and genome organization during development

DNA replication occurs in a defined temporal order known as the replication-timing (RT) program. RT is regulated during development in discrete chromosomal units, coordinated with transcriptional activity and 3D genome organization. Here, we derived distinct cell types from F1 hybrid musculus × castaneus mouse crosses and exploited the high single-nucleotide polymorphism (SNP) density to characterize allelic differences in RT (Repli-seq), genome organization (Hi-C and promoter-capture Hi-C), gene expression (total nuclear RNA-seq), and chromatin accessibility (ATAC-seq). We also present HARP, a new computational tool for sorting SNPs in phased genomes to efficiently measure allele-specific genome-wide data. Analysis of six different hybrid mESC clones with different genomes (C57BL/6, 129/sv, and CAST/Ei), parental configurations, and gender revealed significant RT asynchrony between alleles across ∼12% of the autosomal genome linked to subspecies genomes but not to parental origin, growth conditions, or gender. RT asynchrony in mESCs strongly correlated with changes in Hi-C compartments between alleles but not as strongly with SNP density, gene expression, imprinting, or chromatin accessibility. We then tracked mESC RT asynchronous regions during development by analyzing differentiated cell types, including extraembryonic endoderm stem (XEN) cells, four male and female primary mouse embryonic fibroblasts (MEFs), and neural precursor cells (NPCs) differentiated in vitro from mESCs with opposite parental configurations. We found that RT asynchrony and allelic discordance in Hi-C compartments seen in mESCs were largely lost in all differentiated cell types, accompanied by novel sites of allelic asynchrony at a considerably smaller proportion of the genome, suggesting that genome organization of homologs converges to similar folding patterns during cell fate commitment

Three-dimensional Folding of Eukaryotic Genomes

Chromatin packages eukaryotic genomes via a hierarchical series of folding steps, encrypting multiple layers of epigenetic information, which are capable of regulating nuclear transactions in response to complex signals in environment. Besides the 1-dimensinal chromatin landscape such as nucleosome positioning and histone modifications, little is known about the secondary chromatin structures and their functional consequences related to transcriptional regulation and DNA replication. The family of chromosomal conformation capture (3C) assays has revolutionized our understanding of large-scale chromosome folding with the ability to measure relative interaction probability between genomic loci in vivo. However, the suboptimal resolution of the typical 3C techniques leaves the levels of nucleosome interactions or 30 nm structures inaccessible, and also restricts their applicability to study gene level of chromatin folding in small genome organisms such as yeasts, worm, and plants. To uncover the “blind spot” of chromatin organization, I developed an innovative method called Micro-C and an improved protocol, Micro-C XL, which enable to map chromatin structures at all range of scale from single nucleosome to the entire genome. Several fine-scale aspects of chromatin folding in budding and fission yeasts have been identified by Micro-C, including histone tail-mediated tri-/tetra-nucleosome stackings, gene crumples/globules, and chromosomally-interacting domains (CIDs). CIDs are spatially demarcated by the boundaries, which are colocalized with the promoters of actively transcribed genes and histone marks for active transcription or turnover. The levels of chromatin compaction are regulated via transcription-dependent or transcription-independent manner – either the perturbations of transcription or the mutations of chromatin regulators strongly affect the global chromatin folding. Taken together, Micro-C further reveals chromatin folding behaviors below the sub-kilobase scale and opens an avenue to study chromatin organization in many biological systems