Changes in genome architecture and transcriptional dynamics progress independently of sensory experience during post-natal brain development.

Both transcription and three-dimensional (3D) architecture of the mammalian genome play critical roles in neurodevelopment and its disorders. However, 3D genome structures of single brain cells have not been solved; little is known about the dynamics of single-cell transcriptome and 3D genome after birth. Here, we generated a transcriptome (3,517 cells) and 3D genome (3,646 cells) atlas of the developing mouse cortex and hippocampus by using our high-resolution multiple annealing and looping-based amplification cycles for digital transcriptomics (MALBAC-DT) and diploid chromatin conformation capture (Dip-C) methods and developing multi-omic analysis pipelines. In adults, 3D genome "structure types" delineate all major cell types, with high correlation between chromatin A/B compartments and gene expression. During development, both transcriptome and 3D genome are extensively transformed in the first post-natal month. In neurons, 3D genome is rewired across scales, correlated with gene expression modules, and independent of sensory experience. Finally, we examine allele-specific structure of imprinted genes, revealing local and chromosome (chr)-wide differences. These findings uncover an unknown dimension of neurodevelopment.

Loop stacking organizes genome folding from TADs to chromosomes.

Although population-level analyses revealed significant roles for CTCF and cohesin in mammalian genome organization, their contributions at the single-cell level remain incompletely understood. Here, we used a super-resolution microscopy approach to measure the effects of removal of CTCF or cohesin in mouse embryonic stem cells. Single-chromosome traces revealed cohesin-dependent loops, frequently stacked at their loop anchors forming multi-way contacts (hubs), bridging across TAD boundaries. Despite these bridging interactions, chromatin in intervening TADs was not intermixed, remaining separated in distinct loops around the hub. At the multi-TAD scale, steric effects from loop stacking insulated local chromatin from ultra-long range (>4 Mb) contacts. Upon cohesin removal, the chromosomes were more disordered and increased cell-cell variability in gene expression. Our data revise the TAD-centric understanding of CTCF and cohesin and provide a multi-scale, structural picture of how they organize the genome on the single-cell level through distinct contributions to loop stacking.

EBF1 nuclear repositioning instructs chromatin refolding to promote therapy resistance in T leukemic cells.

Chromatin misfolding has been implicated in cancer pathogenesis; yet, its role in therapy resistance remains unclear. Here, we systematically integrated sequencing and imaging data to examine the spatial and linear chromatin structures in targeted therapy-sensitive and -resistant human T cell acute lymphoblastic leukemia (T-ALL). We found widespread alterations in successive layers of chromatin organization including spatial compartments, contact domain boundaries, and enhancer positioning upon the emergence of targeted therapy resistance. The reorganization of genome folding structures closely coincides with the restructuring of chromatin activity and redistribution of architectural proteins. Mechanistically, the derepression and repositioning of the B-lineage-determining transcription factor EBF1 from the heterochromatic nuclear envelope to the euchromatic interior instructs widespread genome refolding and promotes therapy resistance in leukemic T cells. Together, our findings suggest that lineage-determining transcription factors can instruct changes in genome topology as a driving force for epigenetic adaptations in targeted therapy resistance.

A three-dimensional genomics view for speciation research.

Genomic information is folded in a three-dimensional (3D) structure, a rarely explored evolutionary driver of speciation. Technological advances now enable the study of 3D genome structures (3DGSs) across the Tree of Life. At the onset of 3D speciation genomics, we discuss the putative roles of 3DGSs in speciation.

A Finer Print Than TADs: PRC1-Mediated Domains.

Polycomb proteins are well-known epigenetic repressors with unexplained roles in chromatin folding. In this issue of Molecular Cell, Kundu et al. (2017) investigate the structures of PRC1-mediated domains in stem cells and probe their changes upon differentiation and in PRC knockouts.

HMGs as rheostats of chromosomal structure and cell proliferation.

High mobility group proteins (HMGs) are the most abundant nuclear proteins next to histones and are robustly expressed across tissues and organs. HMGs can uniquely bend or bind distorted DNA, and are central to such processes as transcription, recombination, and DNA repair. However, their dynamic association with chromatin renders capturing HMGs on chromosomes challenging. Recent work has changed this and now implicates these factors in spatial genome organization. Here, I revisit older and review recent literature to describe how HMGs rewire spatial chromatin interactions to sustain homeostasis or promote cellular aging. I propose a 'rheostat' model to explain how HMG-box proteins (HMGBs), and to some extent HMG A proteins (HMGAs), may control cellular aging and, likely, cancer progression.

Chromosome organization through the cell cycle at a glance.

Genome organization and the three-dimensional folding of chromosomes are now seen as major contributors to nearly all nuclear functions including gene regulation, replication and repair. Recent studies have shown that in addition to the dramatic metamorphoses in chromosome conformation associated with entry to, and exit from mitosis, chromosomes undergo continual conformational changes throughout interphase with differential dynamics in loop structure, topological domains, compartments and lamina-associated domains. Understanding and accounting for these cell-cycle-dependent conformational changes is essential for the interpretation of data from a growing array of powerful molecular techniques to investigate genome conformation function, and to identify the molecules and mechanisms that drive chromosome conformational changes. In this Cell Science at a Glance article and the accompanying poster, we review Hi-C and microscopy studies describing cell-cycle-dependent conformational changes in chromosome structure.

HMGB1 coordinates SASP-related chromatin folding and RNA homeostasis on the path to senescence.

Spatial organization and gene expression of mammalian chromosomes are maintained and regulated in conjunction with cell cycle progression. This is perturbed once cells enter senescence and the highly abundant HMGB1 protein is depleted from nuclei to act as an extracellular proinflammatory stimulus. Despite its physiological importance, we know little about the positioning of HMGB1 on chromatin and its nuclear roles. To address this, we mapped HMGB1 binding genome-wide in two primary cell lines. We integrated ChIP-seq and Hi-C with graph theory to uncover clustering of HMGB1-marked topological domains that harbor genes involved in paracrine senescence. Using simplified Cross-Linking and Immuno-Precipitation and functional tests, we show that HMGB1 is also a bona fide RNA-binding protein (RBP) binding hundreds of mRNAs. It presents an interactome rich in RBPs implicated in senescence regulation. The mRNAs of many of these RBPs are directly bound by HMGB1 and regulate availability of SASP-relevant transcripts. Our findings reveal a broader than hitherto assumed role for HMGB1 in coordinating chromatin folding and RNA homeostasis as part of a regulatory loop controlling cell-autonomous and paracrine senescence.

Mesoscale modeling reveals formation of an epigenetically driven HOXC gene hub.

Gene expression is orchestrated at the structural level by nucleosome positioning, histone tail acetylation, and linker histone (LH) binding. Here, we integrate available data on nucleosome positioning, nucleosome-free regions (NFRs), acetylation islands, and LH binding sites to "fold" in silico the 55-kb HOXC gene cluster and investigate the role of each feature on the gene's folding. The gene cluster spontaneously forms a dynamic connection hub, characterized by hierarchical loops which accommodate multiple contacts simultaneously and decrease the average distance between promoters by ∼100 nm. Contact probability matrices exhibit "stripes" near promoter regions, a feature associated with transcriptional regulation. Interestingly, while LH proteins alone decrease long-range contacts and acetylation alone increases transient contacts, combined LH and acetylation produce long-range contacts. Thus, our work emphasizes how chromatin architecture is coordinated strongly by epigenetic factors and opens the way for nucleosome resolution models incorporating epigenetic modifications to understand and predict gene activity.

The multiscale effects of polycomb mechanisms on 3D chromatin folding.

Polycomb group (PcG) proteins silence master regulatory genes required to properly confer cell identity during the development of both Drosophila and mammals. They may act through chromatin compaction and higher-order folding of chromatin inside the cell nucleus. During the last decade, analysis on interphase chromosome architecture discovered self-interacting regions named topologically associated domains (TADs). TADs result from the 3D chromatin folding of a succession of transcribed and repressed epigenomic domains and from loop extrusion mediated by cohesin/CTCF in mammals. Polycomb silenced chromatin constitutes one type of repressed epigenomic domains which form compacted nano-compartments inside cell nuclei. Recruitment of canonical PcG proteins on chromatin relies on initial binding to discrete elements and further spreading into large chromatin domains covered with H3K27me3. Some of these discrete elements have a bivalent nature both in mammals and Drosophila and are dynamically regulated during development. Loops can occur between them, suggesting that their interaction plays both functional and structural roles. Formation of large chromatin domains covered by H3K27me3 seems crucial for PcG silencing and PcG proteins might exert their function through compaction of these domains in both mammals and flies, rather than by directly controlling the nucleosomal accessibility of discrete regulatory elements. In addition, PcG chromatin domains interact over long genomic distances, shaping a higher-order chromatin network. Therefore, PcG silencing might rely on multiscale chromatin folding to maintain cell identity during differentiation.

4C-seq from beginning to end: A detailed protocol for sample preparation and data analysis.

Chromosome conformation capture (3C) methods measure DNA contact frequencies based on nuclear proximity ligation, to uncover in vivo genomic folding patterns. 4C-seq is a derivative 3C method, designed to search the genome for sequences contacting a selected genomic site of interest. 4C-seq employs inverse PCR and next generation sequencing to amplify, identify and quantify its proximity ligated DNA fragments. It generates high-resolution contact profiles for selected genomic sites based on limited amounts of sequencing reads. 4C-seq can be used to study multiple aspects of genome organization. It primarily serves to identify specific long-range DNA contacts between individual regulatory DNA modules, forming for example regulatory chromatin loops between enhancers and promoters, or architectural chromatin loops between cohesin- and CTCF- associated domain boundaries. Additionally, 4C-seq contact profiles can reveal the contours of contact domains and can identify the structural domains that co-occupy the same nuclear compartment. Here, we present an improved step-by-step protocol for sample preparation and the generation of 4C-seq sequencing libraries, including an optimized PCR and 4C template purification strategy. In addition, a data processing pipeline is provided which processes multiplexed 4C-seq reads directly from FASTQ files and generates files compatible with standard genome browsers for visualization and further statistical analysis of the data such as peak calling using peakC. The protocols and the pipeline presented should readily allow anyone to generate, visualize and interpret their own high resolution 4C contact datasets.

The Genomic Code: A Pervasive Encoding/Molding of Chromatin Structures and a Solution of the "Non-Coding DNA" Mystery.

Recent investigations have revealed 1) that the isochores of the human genome group into two super-families characterized by two different long-range 3D structures, and 2) that these structures, essentially based on the distribution and topology of short sequences, mold primary chromatin domains (and define nucleosome binding). More specifically, GC-poor, gene-poor isochores are low-heterogeneity sequences with oligo-A spikes that mold the lamina-associated domains (LADs), whereas GC-rich, gene-rich isochores are characterized by single or multiple GC peaks that mold the topologically associating domains (TADs). The formation of these "primary TADs" may be followed by extrusion under the action of cohesin and CTCF. Finally, the genomic code, which is responsible for the pervasive encoding and molding of primary chromatin domains (LADs and primary TADs, namely the "gene spaces"/"spatial compartments") resolves the longstanding problems of "non-coding DNA," "junk DNA," and "selfish DNA" leading to a new vision of the genome as shaped by DNA sequences.

FoxH1 represses miR-430 during early embryonic development of zebrafish via non-canonical regulation.

BACKGROUND: FoxH1 is a forkhead transcription factor with conserved key functions in vertebrate mesoderm induction and left-right patterning downstream of the TGF-beta/Nodal signaling pathway. Binding of the forkhead domain (FHD) of FoxH1 to a highly conserved proximal sequence motif was shown to regulate target gene expression. RESULTS: We identify the conserved microRNA-430 family (miR-430) as a novel target of FoxH1. miR-430 levels are increased in foxH1 mutants, resulting in a reduced expression of transcripts that are targeted by miR-430 for degradation. To determine the underlying mechanism of miR-430 repression, we performed chromatin immunoprecipitation studies and overexpression experiments with mutant as well as constitutive active and repressive forms of FoxH1. Our studies reveal a molecular interaction of FoxH1 with miR-430 loci independent of the FHD. Furthermore, we show that previously described mutant forms of FoxH1 that disrupt DNA binding or that lack the C-terminal Smad Interaction Domain (SID) dominantly interfere with miR-430 repression, but not with the regulation of previously described FoxH1 targets. CONCLUSIONS: We were able to identify the distinct roles of protein domains of FoxH1 in the regulation process of miR-430. We provide evidence that the indirect repression of miR-430 loci depends on the connection to a distal repressive chromosome environment via a non-canonical mode. The widespread distribution of such non-canonical binding sites of FoxH1, found not only in our study, argues against a function restricted to regulating miR-430 and for a more global role of FoxH1 in chromatin folding.

3D FISH to analyse gene domain-specific chromatin re-modeling in human cancer cell lines.

Fluorescence in situ hybridization (FISH) is a common technique used to label DNA and/or RNA for detection of a genomic region of interest. However, the technique can be challenging, in particular when applied to single genes in human cancer cells. Here, we provide a step-by-step protocol for analysis of short (35 kb-300 kb) genomic regions in three dimensions (3D). We discuss the experimental design and provide practical considerations for 3D imaging and data analysis to determine chromatin folding. We demonstrate that 3D FISH using BACs (Bacterial Artificial Chromosomes) or fosmids can provide detailed information of the architecture of gene domains. More specifically, we show that mapping of specific chromatin landscapes informs on changes associated with estrogen stimulated gene activity in human breast cancer cell lines.

Dense neural networks for predicting chromatin conformation.

BACKGROUND: DNA inside eukaryotic cells wraps around histones to form the 11nm chromatin fiber that can further fold into higher-order DNA loops, which may depend on the binding of architectural factors. Predicting how the DNA will fold given a distribution of bound factors, here viewed as a type of sequence, is currently an unsolved problem and several heterogeneous polymer models have shown that many features of the measured structure can be reproduced from simulations. However a model that determines the optimal connection between sequence and structure and that can rapidly assess the effects of varying either one is still lacking. RESULTS: Here we train a dense neural network to solve for the local folding of chromatin, connecting structure, represented as a contact map, to a sequence of bound chromatin factors. The network includes a convolutional filter that compresses the large number of bound chromatin factors into a single 1D sequence representation that is optimized for predicting structure. We also train a network to solve the inverse problem, namely given only structural information in the form of a contact map, predict the likely sequence of chromatin states that generated it. CONCLUSIONS: By carrying out sensitivity analysis on both networks, we are able to highlight the importance of chromatin contexts and neighborhoods for regulating long-range contacts, along with critical alterations that affect contact formation. Our analysis shows that the networks have learned physical insights that are informative and intuitive about this complex polymer problem.

Segmental folding of chromosomes: a basis for structural and regulatory chromosomal neighborhoods?

We discuss here a series of testable hypotheses concerning the role of chromosome folding into topologically associating domains (TADs). Several lines of evidence suggest that segmental packaging of chromosomal neighborhoods may underlie features of chromatin that span large domains, such as heterochromatin blocks, association with the nuclear lamina and replication timing. By defining which DNA elements preferentially contact each other, the segmentation of chromosomes into TADs may also underlie many properties of long-range transcriptional regulation. Several observations suggest that TADs can indeed provide a structural basis to regulatory landscapes, by controlling enhancer sharing and allocation. We also discuss how TADs may shape the evolution of chromosomes, by causing maintenance of synteny over large chromosomal segments. Finally we suggest a series of experiments to challenge these ideas and provide concrete examples illustrating how they could be practically applied.

Explicit ion modeling predicts physicochemical interactions for chromatin organization.

Molecular mechanisms that dictate chromatin organization in vivo are under active investigation, and the extent to which intrinsic interactions contribute to this process remains debatable. A central quantity for evaluating their contribution is the strength of nucleosome-nucleosome binding, which previous experiments have estimated to range from 2 to 14 kBT. We introduce an explicit ion model to dramatically enhance the accuracy of residue-level coarse-grained modeling approaches across a wide range of ionic concentrations. This model allows for de novo predictions of chromatin organization and remains computationally efficient, enabling large-scale conformational sampling for free energy calculations. It reproduces the energetics of protein-DNA binding and unwinding of single nucleosomal DNA, and resolves the differential impact of mono- and divalent ions on chromatin conformations. Moreover, we showed that the model can reconcile various experiments on quantifying nucleosomal interactions, providing an explanation for the large discrepancy between existing estimations. We predict the interaction strength at physiological conditions to be 9 kBT, a value that is nonetheless sensitive to DNA linker length and the presence of linker histones. Our study strongly supports the contribution of physicochemical interactions to the phase behavior of chromatin aggregates and chromatin organization inside the nucleus.

Abnormal Chromatin Folding in the Molecular Pathogenesis of Epilepsy and Autism Spectrum Disorder: a Meta-synthesis with Systematic Searching.

How DNA is folded and packaged in nucleosomes is an essential regulator of gene expression. Abnormal patterns of chromatin folding are implicated in a wide range of diseases and disorders, including epilepsy and autism spectrum disorder (ASD). These disorders are thought to have a shared pathogenesis involving an imbalance in the number of excitatory-inhibitory neurons formed during neurodevelopment; however, the underlying pathological mechanism behind this imbalance is poorly understood. Studies are increasingly implicating abnormal chromatin folding in neural stem cells as one of the candidate pathological mechanisms, but no review has yet attempted to summarise the knowledge in this field. This meta-synthesis is a systematic search of all the articles on epilepsy, ASD, and chromatin folding. Its two main objectives were to determine to what extent abnormal chromatin folding is implicated in the pathogenesis of epilepsy and ASD, and secondly how abnormal chromatin folding leads to pathological disease processes. This search produced 22 relevant articles, which together strongly implicate abnormal chromatin folding in the pathogenesis of epilepsy and ASD. A range of mutations and chromosomal structural abnormalities lead to this effect, including single nucleotide polymorphisms, copy number variants, translocations and mutations in chromatin modifying. However, knowledge is much more limited into how abnormal chromatin organisation subsequently causes pathological disease processes, not yet showing, for example, whether it leads to abnormal excitation-inhibitory neuron imbalance in human brain organoids.

Genome modeling: From chromatin fibers to genes.

The intricacies of the 3D hierarchical organization of the genome have been approached by many creative modeling studies. The specific model/simulation technique combination defines and restricts the system and phenomena that can be investigated. We present the latest modeling developments and studies of the genome, involving models ranging from nucleosome systems and small polynucleosome arrays to chromatin fibers in the kb-range, chromosomes, and whole genomes, while emphasizing gene folding from first principles. Clever combinations allow the exploration of many interesting phenomena involved in gene regulation, such as nucleosome structure and dynamics, nucleosome-nucleosome stacking, polynucleosome array folding, protein regulation of chromatin architecture, mechanisms of gene folding, loop formation, compartmentalization, and structural transitions at the chromosome and genome levels. Gene-level modeling with full details on nucleosome positions, epigenetic factors, and protein binding, in particular, can in principle be scaled up to model chromosomes and cells to study fundamental biological regulation.

Multi-scale phase separation by explosive percolation with single-chromatin loop resolution.

The 2 m-long human DNA is tightly intertwined into the cell nucleus of the size of 10 µm. The DNA packing is explained by folding of chromatin fiber. This folding leads to the formation of such hierarchical structures as: chromosomal territories, compartments; densely-packed genomic regions known as Topologically Associating Domains (TADs), or Chromatin Contact Domains (CCDs), and loops. We propose models of dynamical human genome folding into hierarchical components in human lymphoblastoid, stem cell, and fibroblast cell lines. Our models are based on explosive percolation theory. The chromosomes are modeled as graphs where CTCF chromatin loops are represented as edges. The folding trajectory is simulated by gradually introducing loops to the graph following various edge addition strategies that are based on topological network properties, chromatin loop frequencies, compartmentalization, or epigenomic features. Finally, we propose the genome folding model - a biophysical pseudo-time process guided by a single scalar order parameter. The parameter is calculated by Linear Discriminant Analysis of chromatin features. We also include dynamics of loop formation by using Loop Extrusion Model (LEM) while adding them to the system. The chromatin phase separation, where fiber folds in 3D space into topological domains and compartments, is observed when the critical number of contacts is reached. We also observe that at least 80% of the loops are needed for chromatin fiber to condense in 3D space, and this is constant through various cell lines. Overall, our in-silico model integrates the high-throughput 3D genome interaction experimental data with the novel theoretical concept of phase separation, which allows us to model event-based time dynamics of chromatin loop formation and folding trajectories.