Chromosomal abnormalities affecting human chromosome 15q11-q13 underlie multiple genomic disorders caused by deletion, duplication and triplication of intervals in this region. These events are mediated by highly homologous segments of DNA, or duplicons, that facilitate mispairing and unequal cross-over in meiosis. The gene encoding an amyloid precursor protein-binding protein (APBA2) was previously mapped to the distal portion of the interval commonly deleted in Prader-Willi and Angelman syndromes and duplicated in cases of autism.

The structure of chromosomes dramatically changes upon entering meiosis to ensure the successful progression of meiosis-specific events. During this process, a multilayer proteinaceous structure called a synaptonemal complex (SC) is formed in many eukaryotes. However, in the fission yeast Schizosaccharomyces pombe, linear elements (LinEs), which are structures related to axial elements of the SC, form on the meiotic cohesin-based chromosome axis. The structure of LinEs has been observed using silver-stained electron micrographs or in immunofluorescence-stained spread nuclei. However, the fine structure of LinEs and their dynamics in intact living cells remain to be elucidated. In this study, we performed live cell imaging with wide-field fluorescence microscopy as well as 3D structured illumination microscopy (3D-SIM) of the core components of LinEs (Rec10, Rec25, Rec27, Mug20) and a linE-binding protein Hop1. We found that LinEs form along the chromosome axis and elongate during meiotic prophase. 3D-SIM microscopy revealed that Rec10 localized to meiotic chromosomes in the absence of other LinE proteins, but shaped into LinEs only in the presence of all three other components, the Rec25, Rec27, and Mug20. Elongation of LinEs was impaired in double-strand break-defective rec12- cells. The structure of LinEs persisted after treatment with 1,6-hexanediol and showed slow fluorescence recovery from photobleaching. These results indicate that LinEs are stable structures resembling axial elements of the SC.

Chromosome condensation is a hallmark of mitosis in eukaryotes and is a prerequisite for faithful segregation of genetic material to daughter cells. Here we show that condensin, which is essential for assembling condensed chromosomes, helps to preclude the detrimental effects of gene transcription on mitotic condensation. ChIP-seq profiling reveals that the fission yeast condensin preferentially binds to active protein-coding genes in a transcription-dependent manner during mitosis. Pharmacological and genetic attenuation of transcription largely rescue bulk chromosome segregation defects observed in condensin mutants. We also demonstrate that condensin is associated with and reduces unwound DNA segments generated by transcription, providing a direct link between an in vitro activity of condensin and its in vivo function. The human condensin isoform condensin I also binds to unwound DNA regions at the transcription start sites of active genes, implying that our findings uncover a fundamental feature of condensin complexes.

Mammalian chromosomes are partitioned into A/B compartments and topologically associated domains (TADs). The inactive X (Xi) chromosome, however, adopts a distinct conformation without evident compartments or TADs. Here, through exploration of an architectural protein, structural-maintenance-of-chromosomes hinge domain containing 1 (SMCHD1), we probe how the Xi is reconfigured during X chromosome inactivation. A/B compartments are first fused into "S1" and "S2" compartments, coinciding with Xist spreading into gene-rich domains. SMCHD1 then binds S1/S2 compartments and merges them to create a compartment-less architecture. Contrary to current views, TADs remain on the Xi but in an attenuated state. Ablating SMCHD1 results in a persistent S1/S2 organization and strengthening of TADs. Furthermore, loss of SMCHD1 causes regional defects in Xist spreading and erosion of heterochromatic silencing. We present a stepwise model for Xi folding, where SMCHD1 attenuates a hidden layer of Xi architecture to facilitate Xist spreading.

Genome segregation is a vital process in all organisms. Chromosome partitioning remains obscure in Archaea, the third domain of life. Here, we investigated the SegAB system from Sulfolobus solfataricus. SegA is a ParA Walker-type ATPase and SegB is a site-specific DNA-binding protein. We determined the structures of both proteins and those of SegA-DNA and SegB-DNA complexes. The SegA structure revealed an atypical, novel non-sandwich dimer that binds DNA either in the presence or in the absence of ATP. The SegB structure disclosed a ribbon-helix-helix motif through which the protein binds DNA site specifically. The association of multiple interacting SegB dimers with the DNA results in a higher order chromatin-like structure. The unstructured SegB N-terminus plays an essential catalytic role in stimulating SegA ATPase activity and an architectural regulatory role in segrosome (SegA-SegB-DNA) formation. Electron microscopy results also provide a compact ring-like segrosome structure related to chromosome organization. These findings contribute a novel mechanistic perspective on archaeal chromosome segregation.

ParABS, an important DNA partitioning process in chromosome segregation, includes ParA (an ATPase), ParB (a parS binding protein) and parS (a centromere-like DNA). The homologous proteins of ParA and ParB in Helicobacter pylori are HpSoj and HpSpo0J, respectively. We analyzed the ATPase activity of HpSoj and found that it is enhanced by both DNA and HpSpo0J. Crystal structures of HpSoj and its DNA complexes revealed a typical ATPase fold and that it is dimeric. DNA binding by HpSoj is promoted by ATP. The HpSoj-ATP-DNA complex non-specifically binds DNA through a continuous basic binding patch formed by lysine residues, with a single DNA-binding site. This complex exhibits a DNA-binding adept state with an active ATP-bound conformation, whereas the HpSoj-ADP-DNA complex may represent a transient DNA-bound state. Based on structural comparisons, HpSoj exhibits a similar DNA binding surface to the bacterial ParA superfamily, but the archaeal ParA superfamily exhibits distinct non-specific DNA-binding via two DNA-binding sites. We detected the HpSpo0J-HpSoj-DNA complex by electron microscopy and show that this nucleoid-adaptor complex (NAC) is formed through HpSoj and HpSpo0J interaction and parS DNA binding. NAC formation is promoted by HpSoj participation and specific parS DNA facilitation.

Over the past decade, methods for predicting three-dimensional (3-D) chromosome and genome structures have proliferated. This has been primarily due to the development of high-throughput, next-generation chromosome conformation capture (3C) technologies, which have provided next-generation sequencing data about chromosome conformations in order to map the 3-D genome structure. The introduction of the Hi-C technique-a variant of the 3C method-has allowed researchers to extract the interaction frequency (IF) for all loci of a genome at high-throughput and at a genome-wide scale. In this review we describe, categorize, and compare the various methods developed to map chromosome and genome structures from 3C data-particularly Hi-C data. We summarize the improvements introduced by these methods, describe the approach used for method evaluation, and discuss how these advancements shape the future of genome structure construction.

Many anthropological, linguistic, genetic and genomic analyses have been carried out to evaluate the potential impact that evolutionary forces had in shaping the present-day Sardinian gene pool, the main outlier in the genetic landscape of Europe. However, due to the homogenizing effect of internal movements, which have intensified over the past fifty years, only partial information has been obtained about the main demographic events. To overcome this limitation, we analyzed the male-specific region of the Y chromosome in three population samples obtained by reallocating a large number of Sardinian subjects to the place of origin of their monophyletic surnames, which are paternally transmitted through generations in most of the populations, much like the Y chromosome. Three Y-chromosome founding lineages, G2-L91, I2-M26 and R1b-V88, were identified as strongly contributing to the definition of the outlying position of Sardinians in the European genetic context and marking a significant differentiation within the island. The present distribution of these lineages does not always mirror that detected in ancient DNAs. Our results show that the analysis of the Y-chromosome gene pool coupled with a sampling method based on the origin of the family name, is an efficient approach to unravelling past heterogeneity, often hidden by recent movements, in the gene pool of modern populations. Furthermore, the reconstruction and comparison of past genetic isolates represent a starting point to better assess the genetic information deriving from the increasing number of available ancient DNA samples.

The molecular and cellular mechanisms driving the enhanced therapeutic ratio of ultra-high dose-rate radiotherapy (FLASH-RT) over slower conventional (CONV-RT) radiotherapy dose-rate remain to be elucidated. However, attenuated DNA damage and transient oxygen depletion are among several proposed models. Here, we tested whether FLASH-RT under physioxic (4% O 2 ) and hypoxic conditions (≤2% O 2 ) reduces genome-wide translocations relative to CONV-RT and whether any differences identified revert under normoxic (21% O 2 ) conditions. We employed high-throughput rejoin and genome-wide translocation sequencing ( HTGTS-JoinT-seq ), using S. aureus and S. pyogenes Cas9 "bait" DNA double strand breaks (DSBs), to measure differences in bait-proximal repair and their genome-wide translocations to "prey" DSBs generated by electron beam CONV-RT (0.08-0.13Gy/s) and FLASH-RT (1×10 2 -5×10 6 Gy/s), under varying ionizing radiation (IR) doses and oxygen tensions. Normoxic and physioxic irradiation of HEK293T cells increased translocations at the cost of decreasing bait-proximal repair but were indistinguishable between CONV-RT and FLASH-RT. Although no apparent increase in chromosome translocations was observed with hypoxia-induced apoptosis, the combined decrease in oxygen tension with IR dose-rate modulation did not reveal significant differences in the level of translocations nor in their junction structures. Thus, Irrespective of oxygen tension, FLASH-RT produces translocations and junction structures at levels and proportions that are indistinguishable from CONV-RT.

The mechanisms governing telomere replication in humans are still poorly understood. To fill this gap, we investigated the timing of replication of single telomeres in human cells. Using in situ hybridization techniques, we have found that specific telomeres have preferential time windows for replication during the S-phase and that these intervals do not depend upon telomere length and are largely conserved between homologous chromosomes and between individuals, even in the presence of large subtelomeric segmental polymorphisms. Importantly, we show that one copy of the 3.3 kb macrosatellite repeat D4Z4, present in the subtelomeric region of the late replicating 4q35 telomere, is sufficient to confer both a more peripheral localization and a later-replicating property to a de novo formed telomere. Also, the presence of beta-satellite repeats next to a newly created telomere is sufficient to delay its replication timing. Remarkably, several native, non-D4Z4-associated, late-replicating telomeres show a preferential localization toward the nuclear periphery, while several early-replicating telomeres are associated with the inner nuclear volume. We propose that, in humans, chromosome arm-specific subtelomeric sequences may influence both the spatial distribution of telomeres in the nucleus and their replication timing.

Selection over 70 years has led to almost complete fixation of a haplotype spanning ~ 250 Mbp of chomosome 5H in European two-rowed spring barleys, possibly originating from North Africa. Plant breeding and selection have shaped the genetic composition of modern crops over the past decades and centuries and have led to great improvements in agronomic and quality traits. Knowledge of the genetic composition of breeding germplasm is essential to make informed decisions in breeding programs. In this study, we characterized the structure and composition of 209 barley cultivars representative of the European two-rowed spring barley germplasm of the past 190 years. Utilizing high-density SNP marker data, we identified a distinct centromeric haplotype spanning a ~ 250 Mbp large region on chromosome 5H which likely was first introduced into the European breeding germplasm in the early to mid-twentieth century and has been non-recombining and under strong positive selection over the past 70 years. Almost all cultivars in our panel that were released after 2000 carry this new haplotype, suggesting that this region carries one or several genes conferring highly beneficial traits. Using the global barley collection of the German Federal ex situ gene bank at IPK Gatersleben, we found the new haplotype at high frequencies in six-rowed spring-type landraces from Northern Africa, from which it may have been introduced into modern European barley germplasm via southern European landraces. The presence of a 250 Mbp genomic region characterized by lack of recombination and high levels of fixation in modern barley germplasm has substantial implications for the genetic diversity of the modern barley germplasm and for barley breeding.

We investigated sex chromosome diversity in Zygosaccharomyces rouxii (Z. rouxii). In the current study, we show that the organization of the mating-type (MAT) locus is highly variable in the Z. rouxii population, indicating the MAT, HML, and HMR loci are translocation hotspots. Although NBRC1130 and CBS732 were originally two stocks of the type strain of the species, only NBRC1130 retains the original karyotype. A reciprocal translocation between the MAT and HMR loci appears to have occurred during the early passage culture of CBS732, which was used for genome sequencing. In NBRC1733, NBRC0686, NBRC0740 and NBRC1053, the terminal region of the chromosome containing the HMR locus was replaced with the chromosomal region to the left of the MAT or HML loci. The translocation events found in NBRC1733, NBRC0686, NBRC0740, and NBRC1053 were reconstructed under our experimental conditions using the DA2 background, and the reconstruction suggests that the frequency of this type of translocation is approximately 10(-7). These results suggest that the MAT and MAT-like loci were the susceptible regions in the genome, and the diversity of mating-type chromosome structures in Z. rouxii was caused by ectopic exchanges between MAT-like loci.

Using a genetic model, we present a high-resolution chromatin fiber analysis of transcriptionally active (Xa) and inactive (Xi) X chromosomes packaged into euchromatin and facultative heterochromatin. Our results show that gene promoters have an open chromatin structure that is enhanced upon transcriptional activation but the Xa and the Xi have similar overall 30 nm chromatin fiber structures. Therefore, the formation of facultative heterochromatin is dependent on factors that act at a level above the 30 nm fiber and transcription does not alter bulk chromatin fiber structures. However, large-scale chromatin structures on Xa are decondensed compared with the Xi and transcription inhibition is sufficient to promote large-scale chromatin compaction. We show a link between transcription and large-scale chromatin packaging independent of the bulk 30 nm chromatin fiber and propose that transcription, not the global compaction of 30 nm chromatin fibers, determines the cytological appearance of large-scale chromatin structures.

Karyotype change and subsequent evolution is triggered by chromosome fusion and rearrangement events, which often occur when telomeres become dysfunctional. Telomeres protect linear chromosome ends from DNA damage responses (DDRs), and telomere dysfunction may result in genome instability. However, the complex chromosome end structures and the other possible consequences of telomere dysfunction have rarely been resolved at the nucleotide level due to the lack of the high-throughput methods needed to analyse these highly repetitive regions. Here we applied long-read sequencing technology to Caenorhabditis elegans survivor lines that emerged after telomere dysfunction. The survivors have preserved traces of DDRs in their genomes and our data revealed that variants generated by telomere dysfunction are accumulated along all chromosomes. The reconstruction of the chromosome end structures through de novo genome assemblies revealed diverse types of telomere damage processing at the nucleotide level. When telomeric repeats were totally eroded by telomere dysfunction, DDRs were mostly terminated by chromosome fusion events. We also partially reconstructed the most complex end structure and its DDR signatures, which would have been accumulated via multiple cell divisions. These finely resolved chromosome end structures suggest possible mechanisms regarding the repair processes after telomere dysfunction, providing insights into chromosome evolution in nature.

Single-cell chromosome conformation capture approaches are revealing the extent of cell-to-cell variability in the organization and packaging of genomes. These single-cell methods, unlike their multi-cell counterparts, allow straightforward computation of realistic chromosome conformations that may be compared and combined with other, independent, techniques to study 3D structure. Here we discuss how single-cell Hi-C and subsequent 3D genome structure determination allows comparison with data from microscopy. We then carry out a systematic evaluation of recently published single-cell Hi-C datasets to establish a computational approach for the evaluation of single-cell Hi-C protocols. We show that the calculation of genome structures provides a useful tool for assessing the quality of single-cell Hi-C data because it requires a self-consistent network of interactions, relating to the underlying 3D conformation, with few errors, as well as sufficient longer-range cis- and trans-chromosomal contacts.

Chromatin conformation plays an important role in a variety of genomic processes, including genome replication, gene expression, and gene methylation. Hi-C data is frequently used to analyze structural features of chromatin, such as AB compartments, topologically associated domains, and 3D structural models. Recently, the genomics community has displayed growing interest in chromatin dynamics. Here, we present 4DMax, a novel method, which uses time-series Hi-C data to predict dynamic chromosome conformation. Using both synthetic data and real time-series Hi-C data from processes, such as induced pluripotent stem cell reprogramming and cardiomyocyte differentiation, we construct smooth four-dimensional models of individual chromosomes. These predicted 4D models effectively interpolate chromatin position across time, permitting prediction of unknown Hi-C contact maps at intermittent time points. Furthermore, 4DMax correctly recovers higher order features of chromatin, such as AB compartments and topologically associated domains, even at time points where Hi-C data is not made available to the algorithm. Contact map predictions made using 4DMax outperform naïve numerical interpolation in 87.7% of predictions on the induced pluripotent stem cell dataset. A/B compartment profiles derived from 4DMax interpolation showed higher similarity to ground truth than at least one profile generated from a neighboring time point in 100% of induced pluripotent stem cell experiments. Use of 4DMax may alleviate the cost of expensive Hi-C experiments by interpolating intermediary time points while also providing valuable visualization of dynamic chromatin changes.

X-chromosome inactivation triggers fusion of A/B compartments to inactive X (Xi)-specific structures known as S1 and S2 compartments. SMCHD1 then merges S1/S2s to form the Xi super-structure. Here, we ask how S1/S2 compartments form and reveal that Xist RNA drives their formation via recruitment of Polycomb repressive complex 1 (PRC1). Ablating Smchd1 in post-XCI cells unveils S1/S2 structures. Loss of SMCHD1 leads to trapping Xist in the S1 compartment, impairing RNA spreading into S2. On the other hand, depleting Xist, PRC1, or HNRNPK precludes re-emergence of S1/S2 structures, and loss of S1/S2 compartments paradoxically strengthens the partition between Xi megadomains. Finally, Xi-reactivation in post-XCI cells can be enhanced by depleting both SMCHD1 and DNA methylation. We conclude that Xist, PRC1, and SMCHD1 collaborate in an obligatory, sequential manner to partition, fuse, and direct self-association of Xi compartments required for proper spreading of Xist RNA.

One of the main aims of phylogenomics is the reconstruction of objects defined in the leaves along the whole phylogenetic tree to minimize the specified functional, which may also include the phylogenetic tree generation. Such objects can include nucleotide and amino acid sequences, chromosomal structures, etc. The structures can have any set of linear and circular chromosomes, variable gene composition and include any number of paralogs, as well as any weights of individual evolutionary operations to transform a chromosome structure. Many heuristic algorithms were proposed for this purpose, but there are just a few exact algorithms with low (linear, cubic or similar) polynomial computational complexity among them to our knowledge. The algorithms naturally start from the calculation of both the distance between two structures and the shortest sequence of operations transforming one structure into another. Such calculation per se is an NP-hard problem.

JCVI-syn3A is a genetically minimal bacterial cell, consisting of 493 genes and only a single 543 kbp circular chromosome. Syn3A's genome and physical size are approximately one-tenth those of the model bacterial organism Escherichia coli's, and the corresponding reduction in complexity and scale provides a unique opportunity for whole-cell modeling. Previous work established genome-scale gene essentiality and proteomics data along with its essential metabolic network and a kinetic model of genetic information processing. In addition to that information, whole-cell, spatially-resolved kinetic models require cellular architecture, including spatial distributions of ribosomes and the circular chromosome's configuration. We reconstruct cellular architectures of Syn3A cells at the single-cell level directly from cryo-electron tomograms, including the ribosome distributions. We present a method of generating self-avoiding circular chromosome configurations in a lattice model with a resolution of 11.8 bp per monomer on a 4 nm cubic lattice. Realizations of the chromosome configurations are constrained by the ribosomes and geometry reconstructed from the tomograms and include DNA loops suggested by experimental chromosome conformation capture (3C) maps. Using ensembles of simulated chromosome configurations we predict chromosome contact maps for Syn3A cells at resolutions of 250 bp and greater and compare them to the experimental maps. Additionally, the spatial distributions of ribosomes and the DNA-crowding resulting from the individual chromosome configurations can be used to identify macromolecular structures formed from ribosomes and DNA, such as polysomes and expressomes.

Dipterans exhibit a remarkable diversity of chromosome end structures in contrast to the conserved system defined by telomerase and short repeats. Within dipteran families, structure of chromosome termini is usually conserved within genera. With the aim to assess whether or not the evolutionary distance between genera implies chromosome end diversification, this report exploits two representatives of Sciaridae, Rhynchosciara americana, and Trichomegalosphyspubescens.