Strong interactions between highly dynamic lamina-associated domains and the nuclear envelope stabilize the 3D architecture of Drosophila interphase chromatin.

BACKGROUND: Interactions among topologically associating domains (TADs), and between the nuclear envelope (NE) and lamina-associated domains (LADs) are expected to shape various aspects of three-dimensional (3D) chromatin structure and dynamics; however, relevant genome-wide experiments that may provide statistically significant conclusions remain difficult. RESULTS: We have developed a coarse-grained dynamical model of D. melanogaster nuclei at TAD resolution that explicitly accounts for four distinct epigenetic classes of TADs and LAD-NE interactions. The model is parameterized to reproduce the experimental Hi-C map of the wild type (WT) nuclei; it describes time evolution of the chromatin over the G1 phase of the interphase. The simulations include an ensemble of nuclei, corresponding to the experimentally observed set of several possible mutual arrangements of chromosomal arms. The model is validated against multiple structural features of chromatin from several different experiments not used in model development. Predicted positioning of all LADs at the NE is highly dynamic-the same LAD can attach, detach and move far away from the NE multiple times during interphase. The probabilities of LADs to be in contact with the NE vary by an order of magnitude, despite all having the same affinity to the NE in the model. These probabilities are mostly determined by a highly variable local linear density of LADs along the genome, which also has the same strong effect on the predicted positioning of individual TADs -- higher probability of a TAD to be near NE is largely determined by a higher linear density of LADs surrounding this TAD. The distribution of LADs along the chromosome chains plays a notable role in maintaining a non-random average global structure of chromatin. Relatively high affinity of LADs to the NE in the WT nuclei substantially reduces sensitivity of the global radial chromatin distribution to variations in the strength of TAD-TAD interactions compared to the lamin depleted nuclei, where a small (0.5 kT) increase of cross-type TAD-TAD interactions doubles the chromatin density in the central nucleus region. CONCLUSIONS: A dynamical model of the entire fruit fly genome makes multiple genome-wide predictions of biological interest. The distribution of LADs along the chromatin chains affects their probabilities to be in contact with the NE and radial positioning of highly mobile TADs, playing a notable role in creating a non-random average global structure of the chromatin. We conjecture that an important role of attractive LAD-NE interactions is to stabilize global chromatin structure against inevitable cell-to-cell variations in TAD-TAD interactions.

Structural Variation of the X Chromosome Heterochromatin in the Anopheles gambiae Complex.

Heterochromatin is identified as a potential factor driving diversification of species. To understand the magnitude of heterochromatin variation within the Anopheles gambiae complex of malaria mosquitoes, we analyzed metaphase chromosomes in An. arabiensis, An. coluzzii, An. gambiae, An. merus, and An. quadriannulatus. Using fluorescence in situ hybridization (FISH) with ribosomal DNA (rDNA), a highly repetitive fraction of DNA, and heterochromatic Bacterial Artificial Chromosome (BAC) clones, we established the correspondence of pericentric heterochromatin between the metaphase and polytene X chromosomes of An. gambiae. We then developed chromosome idiograms and demonstrated that the X chromosomes exhibit qualitative differences in their pattern of heterochromatic bands and position of satellite DNA (satDNA) repeats among the sibling species with postzygotic isolation, An. arabiensis, An. merus, An. quadriannulatus, and An. coluzzii or An. gambiae. The identified differences in the size and structure of the X chromosome heterochromatin point to a possible role of repetitive DNA in speciation of mosquitoes. We found that An. coluzzii and An. gambiae, incipient species with prezygotic isolation, share variations in the relative positions of the satDNA repeats and the proximal heterochromatin band on the X chromosomes. This previously unknown genetic polymorphism in malaria mosquitoes may be caused by a differential amplification of DNA repeats or an inversion in the sex chromosome heterochromatin.

Three-dimensional Organization of Polytene Chromosomes in Somatic and Germline Tissues of Malaria Mosquitoes.

Spatial organization of chromosome territories and interactions between interphase chromosomes themselves, as well as with the nuclear periphery, play important roles in epigenetic regulation of the genome function. However, the interplay between inter-chromosomal contacts and chromosome-nuclear envelope attachments in an organism's development is not well-understood. To address this question, we conducted microscopic analyses of the three-dimensional chromosome organization in malaria mosquitoes. We employed multi-colored oligonucleotide painting probes, spaced 1 Mb apart along the euchromatin, to quantitatively study chromosome territories in larval salivary gland cells and adult ovarian nurse cells of Anopheles gambiae, An. coluzzii, and An. merus. We found that the X chromosome territory has a significantly smaller volume and is more compact than the autosomal arm territories. The number of inter-chromosomal, and the percentage of the chromosome-nuclear envelope, contacts were conserved among the species within the same cell type. However, the percentage of chromosome regions located at the nuclear periphery was typically higher, while the number of inter-chromosomal contacts was lower, in salivary gland cells than in ovarian nurse cells. The inverse correlation was considerably stronger for the autosomes. Consistent with previous theoretical arguments, our data indicate that, at the genome-wide level, there is an inverse relationship between chromosome-nuclear envelope attachments and chromosome-chromosome interactions, which is a key feature of the cell type-specific nuclear architecture.

Germline microsatellite genotypes differentiate children with medulloblastoma.

BACKGROUND: The germline genetic events underpinning medulloblastoma (MB) initiation, and therefore the ability to determine who is at risk, are still unknown for the majority of cases. Microsatellites are short repeated sequences that make up ~3% of the genome. Repeat lengths vary among individuals and are often nonrandomly associated with disease, including several cancers such as breast, glioma, lung, and ovarian. Due to their effects on gene function, they have been called the "tuning knobs of the genome." METHODS: We have developed a novel approach for identifying a microsatellite-based signature to differentiate MB patients from controls using germline DNA. RESULTS: Analyzing germline whole exome sequencing data from a training set of 120 MB subjects and 425 controls, we identified 139 individual microsatellite loci whose genotypes differ significantly between the groups. Using a genetic algorithm, we identified a subset of 43 microsatellites that distinguish MB subjects from controls with a sensitivity and specificity of 92% and 88%, respectively. This microsatellite signature was validated in an independent dataset consisting of 102 subjects and 428 controls, with comparable sensitivity and specificity of 95% and 90%, respectively. Analysis of the allele genotypes of those 139 informative loci demonstrates that their association with MB is a consequence of individual microsatellites' genotypes rather than their hypermutability. Finally, an analysis of the genes harboring these microsatellite loci reveals cellular functions important for tumorigenesis. CONCLUSION: This study demonstrates that MB-specific germline microsatellite variations mark those at risk for MB development and suggests mechanisms of predisposition.

Author Correction: Differentiating between cancer and normal tissue samples using multi-hit combinations of genetic mutations.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Estimating the number of genetic mutations (hits) required for carcinogenesis based on the distribution of somatic mutations.

Individual instances of cancer are primarily a result of a combination of a small number of genetic mutations (hits). Knowing the number of such mutations is a prerequisite for identifying specific combinations of carcinogenic mutations and understanding the etiology of cancer. We present a mathematical model for estimating the number of hits based on the distribution of somatic mutations. The model is fundamentally different from previous approaches, which are based on cancer incidence by age. Our somatic mutation based model is likely to be more robust than age-based models since it does not require knowing or accounting for the highly variable mutation rate, which can vary by over three orders of magnitude. In fact, we find that the number of somatic mutations at diagnosis is weakly correlated with age at cancer diagnosis, most likely due to the extreme variability in mutation rates between individuals. Comparing the distribution of somatic mutations predicted by our model to the actual distribution from 6904 tumor samples we estimate the number of hits required for carcinogenesis for 17 cancer types. We find that different cancer types exhibit distinct somatic mutational profiles corresponding to different numbers of hits. Why might different cancer types require different numbers of hits for carcinogenesis? The answer may provide insight into the unique etiology of different cancer types.

Differentiating between cancer and normal tissue samples using multi-hit combinations of genetic mutations.

Cancer is known to result from a combination of a small number of genetic defects. However, the specific combinations of mutations responsible for the vast majority of cancers have not been identified. Current computational approaches focus on identifying driver genes and mutations. Although individually these mutations can increase the risk of cancer they do not result in cancer without additional mutations. We present a fundamentally different approach for identifying the cause of individual instances of cancer: we search for combinations of genes with carcinogenic mutations (multi-hit combinations) instead of individual driver genes or mutations. We developed an algorithm that identified a set of multi-hit combinations that differentiate between tumor and normal tissue samples with 91% sensitivity (95% Confidence Interval (CI) = 89-92%) and 93% specificity (95% CI = 91-94%) on average for seventeen cancer types. We then present an approach based on mutational profile that can be used to distinguish between driver and passenger mutations within these genes. These combinations, with experimental validation, can aid in better diagnosis, provide insights into the etiology of cancer, and provide a rational basis for designing targeted combination therapies.

CAGm: a repository of germline microsatellite variations in the 1000 genomes project.

The human genome harbors an abundance of repetitive DNA; however, its function continues to be debated. Microsatellites-a class of short tandem repeat-are established as an important source of genetic variation. Array length variants are common among microsatellites and affect gene expression; but, efforts to understand the role and diversity of microsatellite variation has been hampered by several challenges. Without adequate depth, both long-read and short-read sequencing may not detect the variants present in a sample; additionally, large sample sizes are needed to reveal the degree of population-level polymorphism. To address these challenges we present the Comparative Analysis of Germline Microsatellites (CAGm): a database of germline microsatellites from 2529 individuals in the 1000 genomes project. A key novelty of CAGm is the ability to aggregate microsatellite variation by population, ethnicity (super population) and gender. The database provides advanced searching for microsatellites embedded in genes and functional elements. All data can be downloaded as Microsoft Excel spreadsheets. Two use-case scenarios are presented to demonstrate its utility: a mononucleotide (A) microsatellite at the BAT-26 locus and a dinucleotide (CA) microsatellite in the coding region of FGFRL1. CAGm is freely available at http://www.cagmdb.org/.

Chromosome-nuclear envelope attachments affect interphase chromosome territories and entanglement.

BACKGROUND: It is well recognized that the interphase chromatin of higher eukaryotes folds into non-random configurations forming territories within the nucleus. Chromosome territories have biologically significant properties, and understanding how these properties change with time during lifetime of the cell is important. Chromosome-nuclear envelope (Chr-NE) interactions play a role in epigenetic regulation of DNA replication, repair, and transcription. However, their role in maintaining chromosome territories remains unclear. RESULTS: We use coarse-grained molecular dynamics simulations to study the effects of Chr-NE interactions on the dynamics of chromosomes within a model of the Drosophila melanogaster regular (non-polytene) interphase nucleus, on timescales comparable to the duration of interphase. The model simulates the dynamics of chromosomes bounded by the NE. Initially, the chromosomes in the model are prearranged in fractal-like configurations with physical parameters such as nucleus size and chromosome persistence length taken directly from experiment. Time evolution of several key observables that characterize the chromosomes is quantified during each simulation: chromosome territories, chromosome entanglement, compactness, and presence of the Rabl (polarized) chromosome arrangement. We find that Chr-NE interactions help maintain chromosome territories by slowing down and limiting, but not eliminating, chromosome entanglement on biologically relevant timescales. At the same time, Chr-NE interactions have little effect on the Rabl chromosome arrangement as well as on how chromosome compactness changes with time. These results are rationalized by simple dimensionality arguments, robust to model details. All results are robust to the simulated activity of topoisomerase, which may be present in the interphase cell nucleus. CONCLUSIONS: Our study demonstrates that Chr-NE attachments may help maintain chromosome territories, while slowing down and limiting chromosome entanglement on biologically relevant timescales. However, Chr-NE attachments have little effect on chromosome compactness or the Rabl chromosome arrangement.

Quantified effects of chromosome-nuclear envelope attachments on 3D organization of chromosomes.

We use a combined experimental and computational approach to study the effects of chromosome-nuclear envelope (Chr-NE) attachments on the 3D genome organization of Drosophila melanogaster (fruit fly) salivary gland nuclei. We consider 3 distinct models: a Null model - without specific Chr-NE attachments, a 15-attachment model - with 15 previously known Chr-NE attachments, and a 48-attachment model - with 15 original and 33 recently identified Chr-NE attachments. The radial densities of chromosomes in the models are compared to the densities observed in 100 experimental images of optically sectioned salivary gland nuclei forming "z-stacks." Most of the experimental z-stacks support the Chr-NE 48-attachment model suggesting that as many as 48 chromosome loci with appreciable affinity for the NE are necessary to reproduce the experimentally observed distribution of chromosome density in fruit fly nuclei. Next, we investigate if and how the presence and the number of Chr-NE attachments affect several key characteristics of 3D genome organization: chromosome territories and gene-gene contacts. This analysis leads to novel insight about the possible role of Chr-NE attachments in regulating the genome architecture. Specifically, we find that model nuclei with more numerous Chr-NE attachments form more distinct chromosome territories and their chromosomes intertwine less frequently. Intra-chromosome and intra-arm contacts are more common in model nuclei with Chr-NE attachments compared to the Null model (no specific attachments), while inter-chromosome and inter-arm contacts are less common in nuclei with Chr-NE attachments. We demonstrate that Chr-NE attachments increase the specificity of long-range inter-chromosome and inter-arm contacts. The predicted effects of Chr-NE attachments are rationalized by intuitive volume vs. surface accessibility arguments.

Mitotic-chromosome-based physical mapping of the Culex quinquefasciatus genome.

The genome assembly of southern house mosquito Cx. quinquefasciatus is represented by a high number of supercontigs with no order or orientation on the chromosomes. Although cytogenetic maps for the polytene chromosomes of this mosquito have been developed, their utilization for the genome mapping remains difficult because of the low number of high-quality spreads in chromosome preparations. Therefore, a simple and robust mitotic-chromosome-based approach for the genome mapping of Cx. quinquefasciatus still needs to be developed. In this study, we performed physical mapping of 37 genomic supercontigs using fluorescent in situ hybridization on mitotic chromosomes from imaginal discs of 4th instar larvae. The genetic linkage map nomenclature was adopted for the chromosome numbering based on the direct positioning of 58 markers that were previously genetically mapped. The smallest, largest, and intermediate chromosomes were numbered as 1, 2, and 3, respectively. For idiogram development, we analyzed and described in detail the morphology and proportions of the mitotic chromosomes. Chromosomes were subdivided into 19 divisions and 72 bands of four different intensities. These idiograms were used for mapping the genomic supercontigs/genetic markers. We also determined the presence of length polymorphism in the q arm of sex-determining chromosome 1 in Cx. quinquefasciatus related to the size of ribosomal locus. Our physical mapping and previous genetic linkage mapping resulted in the chromosomal assignment of 13% of the total genome assembly to the chromosome bands. We provided the first detailed description, nomenclature, and idiograms for the mitotic chromosomes of Cx. quinquefasciatus. Further application of the approach developed in this study will help to improve the quality of the southern house mosquito genome.

Genomic composition and evolution of Aedes aegypti chromosomes revealed by the analysis of physically mapped supercontigs.

BACKGROUND: An initial comparative genomic study of the malaria vector Anopheles gambiae and the yellow fever mosquito Aedes aegypti revealed striking differences in the genome assembly size and in the abundance of transposable elements between the two species. However, the chromosome arms homology between An. gambiae and Ae. aegypti, as well as the distribution of genes and repetitive elements in chromosomes of Ae. aegypti, remained largely unexplored because of the lack of a detailed physical genome map for the yellow fever mosquito. RESULTS: Using a molecular landmark-guided fluorescent in situ hybridization approach, we mapped 624 Mb of the Ae. aegypti genome to mitotic chromosomes. We used this map to analyze the distribution of genes, tandem repeats and transposable elements along the chromosomes and to explore the patterns of chromosome homology and rearrangements between Ae. aegypti and An. gambiae. The study demonstrated that the q arm of the sex-determining chromosome 1 had the lowest gene content and the highest density of minisatellites. A comparative genomic analysis with An. gambiae determined that the previously proposed whole-arm synteny is not fully preserved; a number of pericentric inversions have occurred between the two species. The sex-determining chromosome 1 had a higher rate of genome rearrangements than observed in autosomes 2 and 3 of Ae. aegypti. CONCLUSIONS: The study developed a physical map of 45% of the Ae. aegypti genome and provided new insights into genomic composition and evolution of Ae. aegypti chromosomes. Our data suggest that minisatellites rather than transposable elements played a major role in rapid evolution of chromosome 1 in the Aedes lineage. The research tools and information generated by this study contribute to a more complete understanding of the genome organization and evolution in mosquitoes.

Investigation of the chromosome regions with significant affinity for the nuclear envelope in fruit fly--a model based approach.

Three dimensional nuclear architecture is important for genome function, but is still poorly understood. In particular, little is known about the role of the "boundary conditions"--points of attachment between chromosomes and the nuclear envelope. We describe a method for modeling the 3D organization of the interphase nucleus, and its application to analysis of chromosome-nuclear envelope (Chr-NE) attachments of polytene (giant) chromosomes in Drosophila melanogaster salivary glands. The model represents chromosomes as self-avoiding polymer chains confined within the nucleus; parameters of the model are taken directly from experiment, no fitting parameters are introduced. Methods are developed to objectively quantify chromosome territories and intertwining, which are discussed in the context of corresponding experimental observations. In particular, a mathematically rigorous definition of a territory based on convex hull is proposed. The self-avoiding polymer model is used to re-analyze previous experimental data; the analysis suggests 33 additional Chr-NE attachments in addition to the 15 already explored Chr-NE attachments. Most of these new Chr-NE attachments correspond to intercalary heterochromatin--gene poor, dark staining, late replicating regions of the genome; however, three correspond to euchromatin--gene rich, light staining, early replicating regions of the genome. The analysis also suggests 5 regions of anti-contact, characterized by aversion for the NE, only two of these correspond to euchromatin. This composition of chromatin suggests that heterochromatin may not be necessary or sufficient for the formation of a Chr-NE attachment. To the extent that the proposed model represents reality, the confinement of the polytene chromosomes in a spherical nucleus alone does not favor the positioning of specific chromosome regions at the NE as seen in experiment; consequently, the 15 experimentally known Chr-NE attachment positions do not appear to arise due to non-specific (entropic) forces. Robustness of the key conclusions to model assumptions is thoroughly checked.