The chromosome folding problem and how cells solve it.

Every cell must solve the problem of how to fold its genome. We describe how the folded state of chromosomes is the result of the combined activity of multiple conserved mechanisms. Homotypic affinity-driven interactions lead to spatial partitioning of active and inactive loci.

Dynamics of microcompartment formation at the mitosis-to-G1 transition.

As cells exit mitosis and enter G1, mitotic chromosomes decompact and transcription is reestablished. Previously, Hi-C studies showed that essentially all interphase 3D genome features including A/B-compartments, TADs, and CTCF loops, are lost during mitosis. However, Hi-C remains insensitive to features such as microcompartments, nested focal interactions between -regulatory elements (CREs).

Crumpled polymer with loops recapitulates key features of chromosome organization.

Chromosomes are exceedingly long topologically-constrained polymers compacted in a cell nucleus. We recently suggested that chromosomes are organized into loops by an active process of loop extrusion. Yet loops remain elusive to direct observations in living cells; detection and characterization of myriads of such loops is a major challenge.

Rules of engagement for condensins and cohesins guide mitotic chromosome formation.

During mitosis, interphase chromatin is rapidly converted into rod-shaped mitotic chromosomes. Using Hi-C, imaging, proteomics and polymer modeling, we determine how the activity and interplay between loop-extruding SMC motors accomplishes this dramatic transition. Our work reveals rules of engagement for SMC complexes that are critical for allowing cells to refold interphase chromatin into mitotic chromosomes.

Mitotic chromosomes are self-entangled and disentangle through a topoisomerase-II-dependent two-stage exit from mitosis.

The topological state of chromosomes determines their mechanical properties, dynamics, and function. Recent work indicated that interphase chromosomes are largely free of entanglements. Here, we use Hi-C, polymer simulations, and multi-contact 3C and find that, by contrast, mitotic chromosomes are self-entangled.

Tunable DNMT1 degradation reveals DNMT1/DNMT3B synergy in DNA methylation and genome organization.

DNA methylation (DNAme) is a key epigenetic mark that regulates critical biological processes maintaining overall genome stability. Given its pleiotropic function, studies of DNAme dynamics are crucial, but currently available tools to interfere with DNAme have limitations and major cytotoxic side effects. Here, we present cell models that allow inducible and reversible DNAme modulation through DNMT1 depletion.

Design principles of 3D epigenetic memory systems.

Cells remember their identities, in part, by using epigenetic marks-chemical modifications placed along the genome. How can mark patterns remain stable over cell generations despite their constant erosion by replication and other processes? We developed a theoretical model that reveals that three-dimensional (3D) genome organization can stabilize epigenetic memory as long as (i) there is a large density difference between chromatin compartments, (ii) modifying "reader-writer" enzymes spread marks in three dimensions, and (iii) the enzymes are limited in abundance relative to their histone substrates. Analogous to an associative memory that encodes memory in neuronal connectivity, mark patterns are encoded in a 3D network of chromosomal contacts.

Unique territorial and compartmental organization of chromosomes in the holocentric silkmoth.

The hallmarks of chromosome organization in multicellular eukaryotes are chromosome territories (CT), chromatin compartments, and insulated domains, including topologically associated domains (TADs). Yet, most of these elements of chromosome organization are derived from analyses of a limited set of model organisms, while large eukaryotic groups, including insects, remain mostly unexplored. Here we combine Hi-C, biophysical modeling, and microscopy to characterize the 3D genome architecture of the silkworm, Bombyx mori.

In vivo tracking of functionally tagged Rad51 unveils a robust strategy of homology search.

Homologous recombination (HR) is a major pathway to repair DNA double-strand breaks (DSB). HR uses an undamaged homologous DNA sequence as a template for copying the missing information, which requires identifying a homologous sequence among megabases of DNA within the crowded nucleus. In eukaryotes, the conserved Rad51-single-stranded DNA nucleoprotein filament (NPF) performs this homology search.

Extrusion fountains are hallmarks of chromosome organization emerging upon zygotic genome activation.

The first activation of gene expression during development (zygotic genome activation, ZGA) is accompanied by massive changes in chromosome organization. The connection between these two processes remains unknown. Using Hi-C for zebrafish embryos, we found that chromosome folding starts by establishing "fountains", novel elements of chromosome organization, emerging selectively at enhancers upon ZGA.

Chromosome and protein folding: In search for unified principles.

Structural biology has traditionally focused on the structures of proteins, short nucleic acids, small molecules, and their complexes. However, it is now widely recognized that the 3D organization of chromosomes should also be included in this list, despite significant differences in scale and complexity of organization. Here we highlight some notable similarities between the folding processes that shape proteins and chromosomes.

Transcription shapes 3D chromatin organization by interacting with loop extrusion.

Cohesin folds mammalian interphase chromosomes by extruding the chromatin fiber into numerous loops. "Loop extrusion" can be impeded by chromatin-bound factors, such as CTCF, which generates characteristic and functional chromatin organization patterns. It has been proposed that transcription relocalizes or interferes with cohesin and that active promoters are cohesin loading sites.

Diverse silent chromatin states modulate genome compartmentalization and loop extrusion barriers.

The relationships between chromosomal compartmentalization, chromatin state and function are poorly understood. Here by profiling long-range contact frequencies in HCT116 colon cancer cells, we distinguish three silent chromatin states, comprising two types of heterochromatin and a state enriched for H3K9me2 and H2A.Z that exhibits neutral three-dimensional interaction preferences and which, to our knowledge, has not previously been characterized.

Live-cell micromanipulation of a genomic locus reveals interphase chromatin mechanics.

Our understanding of the physical principles organizing the genome in the nucleus is limited by the lack of tools to directly exert and measure forces on interphase chromosomes in vivo and probe their material nature. Here, we introduce an approach to actively manipulate a genomic locus using controlled magnetic forces inside the nucleus of a living human cell. We observed viscoelastic displacements over micrometers within minutes in response to near-piconewton forces, which are consistent with a Rouse polymer model.

MCM complexes are barriers that restrict cohesin-mediated loop extrusion.

Eukaryotic genomes are compacted into loops and topologically associating domains (TADs), which contribute to transcription, recombination and genomic stability. Cohesin extrudes DNA into loops that are thought to lengthen until CTCF boundaries are encountered. Little is known about whether loop extrusion is impeded by DNA-bound machines.

The 4D Nucleome Data Portal as a resource for searching and visualizing curated nucleomics data.

The 4D Nucleome (4DN) Network aims to elucidate the complex structure and organization of chromosomes in the nucleus and the impact of their disruption in disease biology. We present the 4DN Data Portal ( https://data.4dnucleome.

Dynamics of CTCF- and cohesin-mediated chromatin looping revealed by live-cell imaging.

Animal genomes are folded into loops and topologically associating domains (TADs) by CTCF and loop-extruding cohesins, but the live dynamics of loop formation and stability remain unknown. Here, we directly visualized chromatin looping at the TAD in mouse embryonic stem cells using super-resolution live-cell imaging and quantified looping dynamics by Bayesian inference. Unexpectedly, the loop was both rare and dynamic, with a looped fraction of approximately 3 to 6.

Promoter and enhancer RNAs regulate chromatin reorganization and activation of miR-10b/HOXD locus, and neoplastic transformation in glioma.

miR-10b is silenced in normal neuroglial cells of the brain but commonly activated in glioma, where it assumes an essential tumor-promoting role. We demonstrate that the entire miR-10b-hosting HOXD locus is activated in glioma via the cis-acting mechanism involving 3D chromatin reorganization and CTCF-cohesin-mediated looping. This mechanism requires two interacting lncRNAs, HOXD-AS2 and LINC01116, one associated with HOXD3/HOXD4/miR-10b promoter and another with the remote enhancer.