The three-dimensional organization of a genome plays a critical role in regulating gene expression, yet little is known about the machinery and mechanisms that determine higher-order chromosome structure1,2. Here we perform genome-wide chromosome conformation capture analysis, FISH, and RNA-seq to obtain comprehensive 3D maps of the Caenorhabditis elegans genome and to dissect X-chromosome dosage compensation, which balances gene expression between XX hermaphrodites and XO males. The dosage compensation complex (DCC), a condensin complex, binds to both hermaphrodite X chromosomes via sequence-specific recruitment elements on X (rex sites) to reduce chromosome-wide gene expression by half3–7. Most DCC condensin subunits also act in other condensin complexes to control the compaction and resolution of all mitotic and meiotic chromosomes5,6. By comparing chromosome structure in wild-type and DCC-defective embryos, we show that the DCC remodels hermaphrodite X chromosomes into a sex-specific spatial conformation distinct from autosomes. Dosage-compensated X chromosomes consist of self-interacting domains (~1 Mb) resembling mammalian Topologically Associating Domains (TADs)8,9. TADs on X have stronger boundaries and more regular spacing than on autosomes. Many TAD boundaries on X coincide with the highest-affinity rex sites and become diminished or lost in DCC-defective mutants, thereby converting the topology of X to a conformation resembling autosomes. rex sites engage in DCC-dependent long-range interactions, with the most frequent interactions occurring between rex sites at DCC-dependent TAD boundaries. These results imply that the DCC reshapes the topology of X by forming new TAD boundaries and reinforcing weak boundaries through interactions between its highest-affinity binding sites. As this model predicts, deletion of an endogenous rex site at a DCC-dependent TAD boundary using CRISPR/Cas9 greatly diminished the boundary. Thus, the DCC imposes a distinct higher-order structure onto X while regulating gene expression chromosome wide.

Abstract. Fluorescence in situ hybridization (FISH) shows that fission yeast centromeres and telomeres make up specific spatial arrangements in the nucleus. Their positioning and clustering are cell cycle regulated. In G2, centromeres cluster adjacent to the spindle pole body (SPB), while in mitosis, their association with each other and with the SPB is disrupted. Similarly, telomeres cluster at the nuclear periphery in G2 and their associations are disrupted in mitosis. Mitotic centromeres interact with the spindle. They remain undivided until the spindle reaches a critical length, then separate and move towards the poles. This demonstrated, for the first time, that anaphase A occurs in fission yeast. The mode of anaphase A and B is similar to that of higher eukaryotes. In nda3 and cut7 mutants defective in tubulin or a kinesin-related motor, cells are blocked in early stages of mitosis due to the absence of the spindle, and centromeres dissociate but remain close to the SPB, whereas in a metaphase-arrested nuc2 mutant, they reside at the middle of the spindle. FISH is therefore a powerful tool for analyzing mitotic chromosome movement and disjunction using various mutants. Surprisingly, in top2 defective in DNA topoisomerase 11, while most chromatid DNAs remain undivided, sister centromeres are separated. Significance of this finding is discussed. In contrast, most chromatid DNAs are separated but telomeric DNAs are not in cut1 mutant. In cut1, the dependence of SPB duplication on the completion of mitosis is abolished. In crm/mutant cells defective in higher-order chromosome organization, the interphase arrangements of centromeres and telomeres are disrupted.T IlE organization of eukaryotic nuclei is designed for the storage and expression of the genetic material that consists of a set of linear chromosomes surrounded by the nuclear membrane . Eukaryotic chromosomal DNAs are highly folded (the 2 x 106-#m -long human genome DNA is stored in a 10-#m-diameter nucleus), even in micro-organisms with small genomes (total yeast DNA is 4,000 #m long and is packed in a 2-#m-diameter nucleus). A number of nuclear proteins may be involved in chromosomal DNA compaction (Gasser and Laemmli, 1987; Earnshaw and Bernat, 1991). Specific DNA sequences may also function in the organizational principles of chromosomal packaging. A question relevant to this problem is whether certain DNA sequences are essential for spatial arrangements of chromosomes in the nucleus. The fission yeast Schizosaccharomyces pombe is an ideal organism in which to address this question, since it has a small genome consisting of only three chromosomes and is amenable to fine genetic analysis (Yanagida, 1989;Nurse, 1990 We recently reported the application of the fluorescence in situ hybridization (FISH) ~ method to this organism (Uzawa and Yanagida, 1992) and suggested its exploitation in the localization of individual DNA sequences within the nucleus. As an initial step towards understanding the principles of nuclear organization in fission yeast, w...

Crossover (CO) recombination events between homologous chromosomes are required to form chiasmata, temporary connections between homologs that ensure their proper segregation at meiosis I1. Despite this requirement for COs and an excess of the double-strand DNA breaks (DSBs) that are the initiating events for meiotic recombination, most organisms make very few COs per chromosome pair2. Moreover, COs tend to inhibit the formation of other COs nearby on the same chromosome pair, a poorly understood phenomenon known as CO interference3,4. Here we show that the synaptonemal complex (SC), a meiosis-specific structure that assembles between aligned homologous chromosomes, both constrains and is altered by CO recombination events. Utilizing a cytological marker of CO sites in Caenorhabditis elegans5, we demonstrate that partial depletion of the SC central region proteins (SYPs) attenuates CO interference, elevating COs and reducing the effective distance over which interference operates, indicating that SYPs limit COs. Moreover, we show that COs are associated with a local 0.4-0.5 μm increase in chromosome axis length. We propose that meiotic CO regulation operates as a self-limiting system in which meiotic chromosome structures establish an environment that promotes CO formation, which in turn alters chromosome structure to inhibit other COs at additional sites.

Summary Chromatin modification and higher-order chromosome structure play key roles in gene regulation, but their functional interplay in controlling gene expression is elusive. We 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. Structure and activity of dosage-compensation-complex (DCC) subunit DPY-21 defined a Jumonji demethylase subfamily that converts H4K20me2 to H4K20me1 in worms and mammals. Selective inactivation of demethylase activity eliminated H4K20me1 enrichment in somatic cells, elevated X-linked gene expression, reduced X-chromosome compaction, and disrupted X-chromosome conformation by diminishing 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.