Higher-order Chromosome Structure Research Articles

The kleisin subunit controls the function of C. elegans meiotic cohesins by determining the mode of DNA binding and differential regulation by SCC-2 and WAPL-1.

The cohesin complex plays essential roles in chromosome segregation, 3D genome organisation, and DNA damage repair through its ability to modify DNA topology. In higher eukaryotes, meiotic chromosome function, and therefore fertility, requires cohesin complexes containing meiosis-specific kleisin subunits: REC8 and RAD21L in mammals and REC-8 and COH-3/4 in Caenorhabditis elegans. How these complexes perform the multiple functions of cohesin during meiosis and whether this involves different modes of DNA binding or dynamic association with chromosomes is poorly understood. Combining time-resolved methods of protein removal with live imaging and exploiting the temporospatial organisation of the C. elegans germline, we show that REC-8 complexes provide sister chromatid cohesion (SCC) and DNA repair, while COH-3/4 complexes control higher-order chromosome structure. High-abundance COH-3/4 complexes associate dynamically with individual chromatids in a manner dependent on cohesin loading (SCC-2) and removal (WAPL-1) factors. In contrast, low-abundance REC-8 complexes associate stably with chromosomes, tethering sister chromatids from S-phase until the meiotic divisions. Our results reveal that kleisin identity determines the function of meiotic cohesin by controlling the mode and regulation of cohesin-DNA association, and are consistent with a model in which SCC and DNA looping are performed by variant cohesin complexes that coexist on chromosomes.

Structure and DNA-bridging activity of the essential Rec114-Mei4 trimer interface.

The DNA double-strand breaks (DSBs) that initiate meiotic recombination are formed by an evolutionarily conserved suite of factors that includes Rec114 and Mei4 (RM), which regulate DSB formation both spatially and temporally. In vivo, these proteins form large immunostaining foci that are integrated with higher-order chromosome structures. In vitro, they form a 2:1 heterotrimeric complex that binds cooperatively to DNA to form large, dynamic condensates. However, understanding of the atomic structures and dynamic DNA binding properties of RM complexes is lacking. Here, we report a structural model of a heterotrimeric complex of the C terminus of Rec114 with the N terminus of Mei4, supported by nuclear magnetic resonance experiments. This minimal complex, which lacks the predicted intrinsically disordered region of Rec114, is sufficient to bind DNA and form condensates. Single-molecule experiments reveal that the minimal complex can bridge two or more DNA duplexes and can generate force to condense DNA through long-range interactions. AlphaFold2 predicts similar structural models for RM orthologs across diverse taxa despite their low degree of sequence similarity. These findings provide insight into the conserved networks of protein-protein and protein-DNA interactions that enable condensate formation and promote formation of meiotic DSBs.

Chromosome architecture and homologous recombination in meiosis.

Meiocytes organize higher-order chromosome structures comprising arrays of chromatin loops organized at their bases by linear axes. As meiotic prophase progresses, the axes of homologous chromosomes align and synapse along their lengths to form ladder-like structures called synaptonemal complexes (SCs). The entire process of meiotic recombination, from initiation via programmed DNA double-strand breaks (DSBs) to completion of DSB repair with crossover or non-crossover outcomes, occurs in the context of chromosome axes and SCs. These meiosis-specific chromosome structures provide specialized environments for the regulation of DSB formation and crossing over. In this review, we summarize insights into the importance of chromosome architecture in the regulation of meiotic recombination, focusing on cohesin-mediated axis formation, DSB regulation via tethered loop-axis complexes, inter-homolog template bias facilitated by axial proteins, and crossover regulation in the context of the SCs. We also discuss emerging evidence that the SUMO and the ubiquitin-proteasome system function in the organization of chromosome structure and regulation of meiotic recombination.

Higher-order structure of barley chromosomes observed by electron tomography

The higher order structure of the metaphase chromosome has been an enigma for over a century and several different models have been presented based on results obtained by a variety of techniques. Some disagreements in the results between methods have possibly arisen from artifacts caused during sample preparation such as staining and dehydration. Therefore, we treated barley chromosomes with ionic liquid to minimize the effects of dehydration. We also observed chromosomes on a film with holes to keep pristine chromosome structure from being flattened as seen when placed on a continuous support film. A chromosome placed over a hole in a thin carbon film was mounted on a tomography holder, and its structure was observed in three dimensions (3D) using electron tomography. We found that there are periodic structures with 300–400 nm pitch along the axis in barley chromosomes. The pitch sizes are larger than those observed in human chromosomes.

GAGA factor: a multifunctional pioneering chromatin protein.

The Drosophila GAGA factor (GAF) is a multifunctional protein implicated in nucleosome organization and remodeling, activation and repression of gene expression, long distance enhancer-promoter communication, higher order chromosome structure, and mitosis. This broad range of activities poses questions about how a single protein can perform so many seemingly different and unrelated functions. Current studies argue that GAF acts as a "pioneer" factor, generating nucleosome-free regions of chromatin for different classes of regulatory elements. The removal of nucleosomes from regulatory elements in turn enables other factors to bind to these elements and carry out their specialized functions. Consistent with this view, GAF associates with a collection of chromatin remodelers and also interacts with proteins implicated in different regulatory functions. In this review, we summarize the known activities of GAF and the functions of its protein partners.

Higher-Order Structure of Human Chromosomes Observed by Electron Diffraction and Electron Tomography.

It is well known that two DNA molecules are wrapped around histone octamers and folded together to form a single chromosome. However, the nucleosome fiber folding within a chromosome remains an enigma, and the higher-order structure of chromosomes also is not understood. In this study, we employed electron diffraction which provides a noninvasive analysis to characterize the internal structure of chromosomes. The results revealed the presence of structures with 100–200 nm periodic features directionally perpendicular to the chromosome axis in unlabeled isolated human chromosomes. We also visualized the 100–200 nm periodic features perpendicular to the chromosome axis in an isolated chromosome whose DNA molecules were specifically labeled with OsO4 using electron tomography in 300 keV and 1 MeV transmission electron microscopes.

Large-Scale Topological Changes Restrain Malignant Progression in Colorectal Cancer

Widespread changes to DNA methylation and chromatin are well documented in cancer, but the fate of higher-order chromosomal structure remains obscure. Here we integrated topological maps for colon tumors and normal colons with epigenetic, transcriptional, and imaging data to characterize alterations to chromatin loops, topologically associated domains, and large-scale compartments. We found that spatial partitioning of the open and closed genome compartments is profoundly compromised in tumors. This reorganization is accompanied by compartment-specific hypomethylation and chromatin changes. Additionally, we identify a compartment at the interface between the canonical A and B compartments that is reorganized in tumors. Remarkably, similar shifts were evident in non-malignant cells that have accumulated excess divisions. Our analyses suggest that these topological changes repress stemness and invasion programs while inducing anti-tumor immunity genes and may therefore restrain malignant progression. Our findings call into question the conventional view that tumor-associated epigenomic alterations are primarily oncogenic.

Higher-order Structure of Human Chromosomes Observed by Electron Tomography and Electron Diffraction

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Co-opted transposons help perpetuate conserved higher-order chromosomal structures

BackgroundTransposable elements (TEs) make up half of mammalian genomes and shape genome regulation by harboring binding sites for regulatory factors. These include binding sites for architectural proteins, such as CTCF, RAD21, and SMC3, that are involved in tethering chromatin loops and marking domain boundaries. The 3D organization of the mammalian genome is intimately linked to its function and is remarkably conserved. However, the mechanisms by which these structural intricacies emerge and evolve have not been thoroughly probed.ResultsHere, we show that TEs contribute extensively to both the formation of species-specific loops in humans and mice through deposition of novel anchoring motifs, as well as to the maintenance of conserved loops across both species through CTCF binding site turnover. The latter function demonstrates the ability of TEs to contribute to genome plasticity and reinforce conserved genome architecture as redundant loop anchors. Deleting such candidate TEs in human cells leads to the collapse of conserved loop and domain structures. These TEs are also marked by reduced DNA methylation and bear mutational signatures of hypomethylation through evolutionary time.ConclusionsTEs have long been considered a source of genetic innovation. By examining their contribution to genome topology, we show that TEs can contribute to regulatory plasticity by inducing redundancy and potentiating genetic drift locally while conserving genome architecture globally, revealing a paradigm for defining regulatory conservation in the noncoding genome beyond classic sequence-level conservation.

X Chromosome Domain Architecture Regulates Caenorhabditis elegans Lifespan but Not Dosage Compensation.

Mechanisms establishing higher-order chromosome structures and their roles in gene regulation are elusive. We analyzed chromosome architecture during nematode X chromosome dosage compensation, which represses transcription via a dosage-compensation condensin complex (DCC) that binds hermaphrodite Xs and establishes megabase-sized topologically associating domains (TADs). We show that DCC binding at high-occupancy sites (rex sites) defines eight TAD boundaries. Single rex deletions disrupted boundaries, and single insertions created new boundaries, demonstrating that a rex site is necessary and sufficient to define DCC-dependent boundary locations. Deleting eight rex sites (8rexΔ) recapitulated TAD structure of DCC mutants, permitting analysis when chromosome-wide domain architecture was disrupted but most DCC binding remained. 8rexΔ animals exhibited no changes inXexpression and lacked dosage-compensation mutant phenotypes. Hence, TAD boundaries are neither the cause nor the consequence of DCC-mediated gene repression. Abrogating TAD structure did, however, reduce thermotolerance, accelerate aging, and shorten lifespan, implicating chromosome architecture in stress responses and aging.

IHi-C 2.0: A simple approach for mapping native spatial chromatin organisation from low cell numbers.

Genome organization is now understood to be tightly linked to all genomic functions. Thus, the high-resolution mapping of higher-order chromosomal structures via 3C-based approaches has become an integral tool for studying transcriptional and cell cycle regulation, signaling effects or disease onset. Nonetheless, 3C-based protocols are not without caveats, like dependencies on fixation conditions, restriction enzyme pervasiveness in crosslinked chromatin and ligation efficiency. To address some of these caveats, we describe here the streamlined iHi-C 2.0 protocol that allows for the genome-wide interrogation of native spatial chromatin contacts without a need for chemical fixation. This approach improves ligation efficiency and presents minimal material losses, and is thus suitable for analysing samples with limiting cell numbers. Following high throughput sequencing, iHi-C 2.0 generates high signal-to-noise and focal maps of the interactions within and between mammalian chromosomes under native conditions.

Identifying Functional Domains in Subunits of Structural Maintenance of Chromosomes (SMC) Complexes by Transposon Mutagenesis Screen in Yeast.

Structural maintenance of chromosomes (SMC) complexes mediate higher order chromosome structures. Eukaryotic cells contain three distinct SMC complexes called cohesin, condensin, and SMC5/6, which share the same basic architecture. The core of SMC complexes contains a heterodimer of SMC proteins, a kleisin subunit, and a set of regulatory proteins that contain HEAT and Armadillo (ARM) repeat protein-protein interaction motifs. A major challenge in studying SMC proteins and their auxiliary factors is identifying their functional domains. Bioinformatics is not an efficient way to achieve this goal because of the absence of defined sequence and structural motifs. Functional domains can be identified experimentally by performing a genetic screen and isolating functional mutants. While there are several strategies to conduct a screen, the quaternary structure of SMCs makes them excellent candidates to transposon-based random insertion mutagenesis, followed by selection of dominant negative mutants. In this chapter we list the advantages of this approach in the context of SMC complexes. We provide a detailed protocol for performing the screen in S. cerevisiae and use data from our recently reported screen on the ARM repeat protein, Scc4, to demonstrate the key steps in the protocol.

YY1 Controls Hematopoietic Stem Cell Quiescence By Repressing Cohesin Expression

Hematopoietic stem cells (HSCs) are undifferentiated, self-renewing, pluripotent cells that have the capacity to differentiate into all mature lineage-specific cells in adult blood. Adult HSCs can remain in a quiescent state for a prolonged time, and quiescence is a fundamental characteristic of HSCs in adult bone marrow. Thus, the cell cycle must be precisely regulated. Yin Yang 1 (YY1) is a multifunctional transcription factor and Polycomb Group Protein (PcG) that is important for embryonic development, adult hematopoiesis, cell proliferation and maintaining higher-order chromosomal structure. We have generated YY1 conditional knockout mice (Yy1f/f Mx1-Cre) and showed that Yy1 deficient HSCs fail to self-renewal and had disrupted HSCs quiescence. Stem cell factor (SCF)/c-Kit signaling, a critical regulatory pathway in HSC development, is significantly downregulated in Yy1-/- HSCs. Interestingly, YY1 regulation of HSCs self-renewal and quiescence is independent on its PcG domain/function. Instead, YY1 occupied at Smc3 promoter area and repressed Smc3 expression. In Yy1-/-HSCs, Smc3 expression was upregulated. SMC3 is a core component of cohesin protein complex and plays critical roles in HSC self-renewal, myeloid differentiation and leukemogenesis. To further dissect the underlying mechanisms by which YY1 regulates SMC3 expression in HSCs, we have generated conditional knockout mice with YY1 homozygous deletion and SMC3 heterozygous deletion (Yy1f/f Smc3f/+Mx1-Cre). In Yy1-/- Smc3+/- bone marrow cells, SMC3 expression was normalized to the wild-type level. In adult bone marrow cells, YY1 physically interacted with cohesin complex proteins through its zinc finger domain. By analyzing the YY1, SMC1A and SMC3 ChIP-Seq database, our study showed that YY1 and cohesin co-occupied at promoter areas of genes that are critical for cell metabolism. Evidence from previous study showed that impaired metabolism, including increased reactive oxygen species (ROS) and decreased mitochondrial function, can cause defect in stem cell self-renewal and quiescence. In Yy1-/- Smc3+/-HSCs, cell quiescence was restored although HSC self-renewal was still impaired, indicates that YY1 and SMC3 may control HSC cell quiescence via regulating genes critical for cell metabolism. Our study identified YY1 as the first transcription factor that regulates expression of cohesin complex component SMC3. We are currently further dissecting underlying mechanisms and functional significances of metabolic pathways regulated by YY1-SMC3 axis in HSCs. DisclosuresLevine:Imago: Equity Ownership; Isoplexis: Equity Ownership; Janssen: Consultancy, Honoraria; Gilead: Honoraria; Epizyme: Patents & Royalties; Loxo: Consultancy, Equity Ownership; C4 Therapeutics: Equity Ownership; Roche: Consultancy, Research Funding; Prelude: Research Funding; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Novartis: Consultancy.

Dynamic Control of X Chromosome Conformation and Repression by a Histone H4K20 Demethylase

Chromatin modification and higher-order chromosome structure play key roles in gene regulation, but their functional interplay in controlling gene expression is elusive. We have discovered the machinery and mechanism underlying the dynamic enrichment of histone modification H4K20me1 on hermaphrodite X chromosomes during C.elegans dosage compensation and demonstrated H4K20me1's pivotal role in regulating higher-order chromosome structure and X-chromosome-wide gene expression. The structure and the activity of the dosage compensation complex (DCC) subunit DPY-21 define a Jumonji demethylase subfamily that converts H4K20me2 to H4K20me1 in worms and mammals. Selective inactivation of demethylase activity eliminates H4K20me1 enrichment in somatic cells, elevates X-linked gene expression, reduces X chromosome compaction, and disrupts Xchromosome conformation by diminishing the formation of topologically associating domains (TADs). Unexpectedly, DPY-21 also associates with autosomes of germ cells in a DCC-independent manner to enrich H4K20me1 and trigger chromosome compaction. Our findings demonstrate the direct link between chromatin modification and higher-order chromosome structure in long-range regulation of gene expression.

Specialized interfaces of Smc5/6 control hinge stability and DNA association

The Structural Maintenance of Chromosomes (SMC) complexes: cohesin, condensin and Smc5/6 are involved in the organization of higher-order chromosome structure—which is essential for accurate chromosome duplication and segregation. Each complex is scaffolded by a specific SMC protein dimer (heterodimer in eukaryotes) held together via their hinge domains. Here we show that the Smc5/6-hinge, like those of cohesin and condensin, also forms a toroidal structure but with distinctive subunit interfaces absent from the other SMC complexes; an unusual ‘molecular latch’ and a functional ‘hub’. Defined mutations in these interfaces cause severe phenotypic effects with sensitivity to DNA-damaging agents in fission yeast and reduced viability in human cells. We show that the Smc5/6-hinge complex binds preferentially to ssDNA and that this interaction is affected by both ‘latch’ and ‘hub’ mutations, suggesting a key role for these unique features in controlling DNA association by the Smc5/6 complex.

Dynamic Control of Chromosome Topology and Gene Expression by a Chromatin Modification.

The function of chromatin modification in establishing higher-order chromosome structure during gene regulation has been elusive. We dissected the machinery and mechanism underlying the enrichment of histone modification H4K20me1 on hermaphrodite X chromosomes during Caenorhabditis elegans dosage compensation and discovered a key role for H4K20me1 in regulating X-chromosome topology and chromosome-wide gene expression. Structural and functional analysis of the dosage compensation complex (DCC) subunit DPY-21 revealed a novel Jumonji C demethylase subfamily that converts H4K20me2 to H4K20me1 in worms and mammals. Inactivation of demethylase activity in vivo by genome editing eliminated H4K20me1 enrichment on X chromosomes of somatic cells, increased X-linked gene expression, reduced X-chromosome compaction, and disrupted X-chromosome conformation by diminishing the formation of topologically associated domains. H4K20me1 is also enriched on the inactive X of female mice, making our studies directly relevant to mammalian development. Unexpectedly, DPY-21 also associates specifically with autosomes of nematode germ cells in a DCC-independent manner to enrich H4K20me1 and trigger chromosome compaction. Thus, DPY-21 is an adaptable chromatin regulator. Its H4K20me2 demethylase activity can be harnessed during development for distinct biological functions by targeting it to diverse genomic locations through different mechanisms. In both somatic cells and germ cells, H4K20me1 enrichment modulates three-dimensional chromosome architecture, demonstrating the direct link between chromatin modification and higher-order chromosome structure.

Chromatin Conformation Capture-Based Analysis of Nuclear Architecture.

Nuclear organization and higher-order chromosome structure in interphase nuclei are thought to have important effects on fundamental biological processes, including chromosome condensation, replication, and transcription. Until recently, however, nuclear organization could only be analyzed microscopically. The development of chromatin conformation capture (3C)-based techniques now allows a detailed look at chromosomal architecture from the level of individual loci to the entire genome. Here we provide a robust Hi-C protocol, allowing the analysis of nuclear organization in nuclei from different wild-type and mutant plant tissues. This method is quantitative and provides a highly efficient and comprehensive way to study chromatin organization during plant development, in response to different environmental stimuli, and in mutants disrupting a variety of processes, including epigenetic pathways regulating gene expression.

Anchoring of Heterochromatin to the Nuclear Lamina Reinforces Dosage Compensation-Mediated Gene Repression.

Higher order chromosome structure and nuclear architecture can have profound effects on gene regulation. We analyzed how compartmentalizing the genome by tethering heterochromatic regions to the nuclear lamina affects dosage compensation in the nematode C. elegans. In this organism, the dosage compensation complex (DCC) binds both X chromosomes of hermaphrodites to repress transcription two-fold, thus balancing gene expression between XX hermaphrodites and XO males. X chromosome structure is disrupted by mutations in DCC subunits. Using X chromosome paint fluorescence microscopy, we found that X chromosome structure and subnuclear localization are also disrupted when the mechanisms that anchor heterochromatin to the nuclear lamina are defective. Strikingly, the heterochromatic left end of the X chromosome is less affected than the gene-rich middle region, which lacks heterochromatic anchors. These changes in X chromosome structure and subnuclear localization are accompanied by small, but significant levels of derepression of X-linked genes as measured by RNA-seq, without any observable defects in DCC localization and DCC-mediated changes in histone modifications. We propose a model in which heterochromatic tethers on the left arm of the X cooperate with the DCC to compact and peripherally relocate the X chromosomes, contributing to gene repression.

High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation

The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid partitioning and suggest underlying biological roles shared by such elements.

Cohesin-interacting protein WAPL-1 regulates meiotic chromosome structure and cohesion by antagonizing specific cohesin complexes.

Wapl induces cohesin dissociation from DNA throughout the mitotic cell cycle, modulating sister chromatid cohesion and higher-order chromatin structure. Cohesin complexes containing meiosis-specific kleisin subunits govern most aspects of meiotic chromosome function, but whether Wapl regulates these complexes remains unknown. We show that during C. elegans oogenesis WAPL-1 antagonizes binding of cohesin containing COH-3/4 kleisins, but not REC-8, demonstrating that sensitivity to WAPL-1 is dictated by kleisin identity. By restricting the amount of chromosome-associated COH-3/4 cohesin, WAPL-1 controls chromosome structure throughout meiotic prophase. In the absence of REC-8, WAPL-1 inhibits COH-3/4-mediated cohesion, which requires crossover-fated events formed during meiotic recombination. Thus, WAPL-1 promotes functional specialization of meiotic cohesin: WAPL-1-sensitive COH-3/4 complexes modulate higher-order chromosome structure, while WAPL-1-refractory REC-8 complexes provide stable cohesion. Surprisingly, a WAPL-1-independent mechanism removes cohesin before metaphase I. Our studies provide insight into how meiosis-specific cohesin complexes are regulated to ensure formation of euploid gametes.