Search

There is a need for methods that can image chromosomes with genome-wide coverage, as well as greater genomic and optical resolution. We introduce OligoFISSEQ, a suite of three methods that leverage fluorescence in situ sequencing (FISSEQ) of barcoded Oligopaint probes to enable the rapid visualization of many targeted genomic regions. Applying OligoFISSEQ to human diploid fibroblast cells, we show how four rounds of sequencing are sufficient to produce 3D maps of 36 genomic targets across six chromosomes in hundreds to thousands of cells, implying a potential to image thousands of targets in only five to eight rounds of sequencing. We also use OligoFISSEQ to trace chromosomes at finer resolution, following the path of the X chromosome through 46 regions, with separate studies showing compatibility of OligoFISSEQ with immunocytochemistry. Finally, we combined OligoFISSEQ with OligoSTORM, laying the foundation for accelerated single-molecule super-resolution imaging of large swaths of, if not entire, human genomes. OligoFISSEQ combines Oligopaints with fluorescence in situ sequencing to enable the 3D mapping of many regions across the genome in human cells to interrogate genome organization at improved genomic resolution. OligoFISSEQ is compatible with immunochemistry and OligoSTORM for super-resolution imaging.

X inactivation presents two longstanding puzzles: the counting and choice of X chromosomes. Here, we consider counting and choice in the context of pairing, both of the X and of the autosomes.

Trans-homolog interactions have been studied extensively in Drosophila, where homologs are paired in somatic cells and transvection is prevalent. Nevertheless, the detailed structure of pairing and its functional impact have not been thoroughly investigated. Accordingly, we generated a diploid cell line from divergent parents and applied haplotype-resolved Hi-C, showing that homologs pair with varying precision genome-wide, in addition to establishing trans-homolog domains and compartments. We also elucidate the structure of pairing with unprecedented detail, observing significant variation across the genome and revealing at least two forms of pairing: tight pairing, spanning contiguous small domains, and loose pairing, consisting of single larger domains. Strikingly, active genomic regions (A-type compartments, active chromatin, expressed genes) correlated with tight pairing, suggesting that pairing has a functional implication genome-wide. Finally, using RNAi and haplotype-resolved Hi-C, we show that disruption of pairing-promoting factors results in global changes in pairing, including the disruption of some interaction peaks., Trans-homolog interactions, such as homolog pairing, are highly structured and associated with gene function in Drosophila cells. Here, the authors use haplotype-resolved Hi-C to identify genome-wide trans-homolog interactions in a Drosophila hybrid cell line and investigate their patterns and functional roles.

Membraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, our current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. We investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with the euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, we reveal a hierarchical organization of the PCH domains into distinct “territories.” Strikingly, H3K9me2-enriched regions embedded in the euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me2 enrichment, are likely mediated by liquid-liquid phase separation, and may influence organismal fitness. Our findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin., Author summary The three dimensional (3D) organization of genomes in cell nuclei can influence a wide variety of genome functions. However, most of our understanding of this critical architecture has been limited to the gene-rich euchromatin, and largely ignores the gene-poor and repeat-rich pericentromeric heterochromatin, or PCH. PCH comprises a large part of most eukaryotic genomes, forms 3D membraneless PCH domains in nuclei, and plays a vital role in chromosome dynamics and genome stability. In this study, we developed a new method that overcomes the technical challenges imposed by the highly repetitive PCH DNA, and generated a comprehensive picture of its 3D organization. Combined with image analyses, we reveal a hierarchical organization of the PCH domains. Surprisingly, we showed that distant euchromatic regions enriched for repressive epigenetic marks also dynamically interact with the main PCH domains. These 3D interactions are likely mediated by liquid-liquid phase separation (similar to how oil and vinegar separate in salad dressing) and the resulting liquid-like fusion events, and can influence the fitness of individuals. Our discoveries have strong implications for how seemingly “junk” DNA could impact functions in the gene-rich euchromatin.

The presence of maternal and paternal homologs appears to be much more than just a doubling of genetic material. We know this because genomes have evolved elaborate mechanisms that permit homologous regions to sense and then respond to each other. One way in which homologs communicate is to come into contact and, in fact, Dipteran insects such as Drosophila excel at this task, aligning all pairs of maternal and paternal chromosomes, end-to-end, in essentially all somatic tissues throughout development. Here, we reexamine the widely held tenet that extensive somatic pairing of homologous sequences cannot occur in mammals and suggest, instead, that pairing may be a widespread and significant potential that has gone unnoticed in mammals because they expend considerable effort to prevent it. We then extend this discussion to interchromosomal interactions, in general, and speculate about the potential of nuclear organization and pairing to impact inheritance.

Genome organization involves cis and trans chromosomal interactions, both implicated in gene regulation, development, and disease. Here, we focus on trans interactions in Drosophila, where homologous chromosomes are paired in somatic cells from embryogenesis through adulthood. We first address long-standing questions regarding the structure of embryonic homolog pairing and, to this end, develop a haplotype-resolved Hi-C approach to minimize homolog misassignment and thus robustly distinguish trans-homolog from cis contacts. This computational approach, which we call Ohm, reveals pairing to be surprisingly structured genome-wide, with trans-homolog domains, compartments, and interaction peaks, many coinciding with analogous cis features. We also find a significant genome-wide correlation between pairing, transcription during zygotic genome activation, and binding of the pioneer factor Zelda. Our findings reveal a complex, highly structured organization underlying homolog pairing, first discovered a century ago in Drosophila. Finally, we demonstrate the versatility of our haplotype-resolved approach by applying it to mammalian embryos., Homologs are paired in Drosophila somatic cells from embryogenesis to adulthood. Using a computational approach for haplotype-resolved Hi-C, the authors reveal highly structured homolog pairing in Drosophila embryos during zygotic genome activation and demonstrate its application to mammalian embryos.

Chromosome organization is crucial for genome function. Here, we present a method for visualizing chromosomal DNA at super-resolution and then integrating Hi-C data to produce three-dimensional models of chromosome organization. Using the super-resolution microscopy methods of OligoSTORM and OligoDNA-PAINT, we trace 8 megabases of human chromosome 19, visualizing structures ranging in size from a few kilobases to over a megabase. Focusing on chromosomal regions that contribute to compartments, we discover distinct structures that, in spite of considerable variability, can predict whether such regions correspond to active (A-type) or inactive (B-type) compartments. Imaging through the depths of entire nuclei, we capture pairs of homologous regions in diploid cells, obtaining evidence that maternal and paternal homologous regions can be differentially organized. Finally, using restraint-based modeling to integrate imaging and Hi-C data, we implement a method–integrative modeling of genomic regions (IMGR)–to increase the genomic resolution of our traces to 10 kb., Author summary Questions regarding the impact of chromosome structure on genome function are focusing increasingly on the manner in which chromosomes are organized within the nucleus. In fact, studies of processes as diverse as gene activation and repression as well as genome repair and stability are all querying how the 3D organization of chromosomal DNA may be a major player. Here, we apply our strategy for tracing chromosomes at super-resolution, traversing over 8 megabases of human chromosome 19 while visualizing genomic features ranging in size from kilobases to megabases. This technology has enabled exploration of the physical nature of a genomic feature called the compartment; compartments are widely hypothesized to reflect the partitioning of genomes into relatively more and less active regions. Excitingly, we find that compartments are, indeed, physical structures, that they are sometimes distinct and other times entangled. We also find that the maternally-derived and the paternally-derived homologous regions can differ more than would be expected by chance. Finally, by integrating image data with information regarding the frequency with which genomic segments contact each other, we produce a 3D model of how the 8 megabases we have imaged may be organized within a single nucleus, achieving 10 kb genomic resolution.

Summary: This study explores the relationship between three-dimensional genome organization and ultraconserved elements (UCEs), an enigmatic set of DNA elements that are perfectly conserved between the reference genomes of distantly related species. Examining both human and mouse genomes, we interrogate the relationship of UCEs to three features of chromosome organization derived from Hi-C studies. We find that UCEs are enriched within contact domains and, further, that the subset of UCEs within domains shared across diverse cell types are linked to kidney-related and neuronal processes. In boundaries, UCEs are generally depleted, with those that do overlap boundaries being overrepresented in exonic UCEs. Regarding loop anchors, UCEs are neither overrepresented nor underrepresented, but those present in loop anchors are enriched for splice sites. Finally, as the relationships between UCEs and human Hi-C features are conserved in mouse, our findings suggest that UCEs contribute to interspecies conservation of genome organization and, thus, genome stability. : McCole et al. demonstrate the non-random relationship between the positions of perfectly conserved genomic regions, termed the ultraconserved elements (UCEs), and three-dimensional genome organization within mammalian nucleus as defined by Hi-C studies. They postulate that these connections aid in orchestrating genome packaging and preserving genome function and integrity. Keywords: Hi-C, chromosome organization, ultraconserved elements, UCEs, copy number variants, CNVs, kidney, exons, RNA processing

The development of the human brain and nervous system can be affected by genetic or environmental factors. Here we focus on characterizing the genetic perturbations that accompany and may contribute to neurodevelopmental phenotypes. Specifically, we examine two types of structural variants, namely, copy number variation and balanced chromosome rearrangements, discovered in subjects with neurodevelopmental disorders and related phenotypes. We find that a feature uniting these types of genetic aberrations is a proximity to ultraconserved elements (UCEs), which are sequences that are perfectly conserved between the reference genomes of distantly related species. In particular, while UCEs are generally depleted from copy number variant regions in healthy individuals, they are, on the whole, enriched in genomic regions disrupted by copy number variants or breakpoints of balanced rearrangements in affected individuals. Additionally, while genes associated with neurodevelopmental disorders are enriched in UCEs, this does not account for the excess of UCEs either in copy number variants or close to the breakpoints of balanced rearrangements in affected individuals. Indeed, our data are consistent with some manifestations of neurodevelopmental disorders resulting from a disruption of genome integrity in the vicinity of UCEs.

OligoSTORM and OligoDNA-PAINT meld the Oligopaint technology for fluorescent in situ hybridization (FISH) with, respectively, Stochastic Optical Reconstruction Microscopy (STORM) and DNA-based Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) to enable in situ single-molecule super-resolution imaging of nucleic acids. Both strategies enable ≤20 nm resolution and are appropriate for imaging nanoscale features of the genomes of a wide range of species, including human, mouse, and fruit fly (Drosophila).

Following DNA replication, sister chromatids must stay connected for the remainder of the cell cycle in order to ensure accurate segregation in the subsequent cell division. This important function involves an evolutionarily conserved protein complex known as cohesin; any loss of cohesin causes premature sister chromatid separation in mitosis. Here, we examined the role of cohesin in sister chromatid cohesion prior to mitosis, using fluorescence in situ hybridization (FISH) to assay the alignment of sister chromatids in interphase Drosophila cells. Surprisingly, we found that sister chromatid cohesion can be maintained in G2 with little to no cohesin. This capacity to maintain cohesion is widespread in Drosophila, unlike in other systems where a reduced dependence on cohesin for sister chromatid segregation has been observed only at specific chromosomal regions, such as the rDNA locus in budding yeast. Additionally, we show that condensin II antagonizes the alignment of sister chromatids in interphase, supporting a model wherein cohesin and condensin II oppose each other’s functions in the alignment of sister chromatids. Finally, because the maternal and paternal homologs are paired in the somatic cells of Drosophila, and because condensin II has been shown to antagonize this pairing, we consider the possibility that condensin II-regulated mechanisms for aligning homologous chromosomes may also contribute to sister chromatid cohesion., Author Summary As cells grow, they replicate their DNA to give rise to two copies of each chromosome, known as sister chromatids, which separate from each other once the cell divides. To ensure that sister chromatids end up in different daughter cells, they are kept together from DNA replication until mitosis via a connection known as cohesion. A protein complex known as cohesin is essential for this process. Our work in Drosophila cells suggests that factors other than cohesin also contribute to sister chromatid cohesion in interphase. Additionally, we observed that the alignment of sister chromatids is regulated by condensin II, a protein complex involved in the compaction of chromosomes prior to division as well as the regulation of inter-chromosomal associations. These findings highlight that, in addition to their important individual functions, cohesin and condensin II proteins may interact to organize chromosomes over the course of the cell cycle. Finally, building on prior observations that condensin II is involved in the regulation of somatic homolog pairing in Drosophila, our work suggests that the mechanisms underlying homolog pairing may also contribute to sister chromatid cohesion.

Type II topoisomerases are essential ATP-dependent homodimeric enzymes required for transcription, replication, and chromosome segregation. These proteins alter DNA topology by generating transient enzyme-linked double-strand breaks for passage of one DNA strand through another. The central role of type II topoisomerases in DNA metabolism has made these enzymes targets for anticancer drugs. Here, we describe a genetic screen that generated novel alleles of DrosophilaTopoisomerase 2 (Top2). Fifteen alleles were obtained, resulting from nonsense and missense mutations. Among these, 14 demonstrated recessive lethality, with one displaying temperature-sensitive lethality. Several newly generated missense alleles carry amino acid substitutions in conserved residues within the ATPase, Topoisomerase/Primase, and Winged helix domains, including four that encode proteins with alterations in residues associated with resistance to cancer chemotherapeutics. Animals lacking zygotic Top2 function can survive to pupation and display reduced cell division and altered polytene chromosome structure. Inter se crosses between six strains carrying Top2 missense alleles generated morphologically normal trans-heterozygous adults, which showed delayed development and were female sterile. Complementation occurred between alleles encoding Top2 proteins with amino acid substitutions in the same functional domain and between alleles encoding proteins with substitutions in different functional domains. Two complementing alleles encode proteins with amino acid substitutions associated with drug resistance. These observations suggest that dimerization of mutant Top2 monomers can restore enzymatic function. Our studies establish the first series of Top2 alleles in a multicellular organism. Future analyses of these alleles will enhance our knowledge about the contributions made by type II topoisomerases to development.

The many reports of trans interactions between homologous as well as nonhomologous loci in a wide variety of organisms argue that such interactions play an important role in gene regulation. The yellow locus of Drosophila is especially useful for investigating the mechanisms of trans interactions due to its ability to support transvection and the relative ease with which it can be altered by targeted gene replacement. In this study, we exploit these aspects of yellow to further our understanding of cis as well as trans forms of enhancer–promoter communication. Through the analysis of yellow alleles whose promoters have been replaced with wild-type or altered promoters from other genes, we show that mutation of single core promoter elements of two of the three heterologous promoters tested can influence whether yellow enhancers act in cis or in trans. This finding parallels observations of the yellow promoter, suggesting that the manner in which trans interactions are controlled by core promoter elements describes a general mechanism. We further demonstrate that heterologous promoters themselves can be activated in trans as well as participate in pairing-mediated insulator bypass. These results highlight the potential of diverse promoters to partake in many forms of trans interactions.

Interactions between paired homologous genes can lead to changes in gene expression. Such trans-regulatory effects exemplify transvection and are displayed by many genes in Drosophila , in which homologous chromosomes are paired somatically. Transvection involving the yellow cuticle pigmentation gene can occur by at least two mechanisms, one involving the trans-action of enhancers on a paired promoter and a second involving pairing-mediated bypass of a chromatin insulator. A system was developed to evaluate whether the action of the yellow enhancers in trans could be reconstituted outside of the natural near telomeric location of the yellow gene. To this end, transgenic flies were generated that carried a yellow gene modified by the inclusion of strategically placed recognition sites for the Cre and FLP recombinases. Independent action of the recombinases produced a pair of derivative alleles, one enhancerless and the other promoterless, at each transgene location. Transvection between the derivatives was assessed by the degree of interallelic complementation. Complementation was observed at all eight sites tested. These studies demonstrate that yellow transvection can occur at multiple genomic locations and indicate that the Drosophila genome generally is permissive to enhancer action in trans.

The Spt4, Spt5, and Spt6 proteins are conserved throughout eukaryotes and are believed to play critical and related roles in transcription. They have a positive role in transcription elongation inSaccharomyces cerevisiae and in the activation of transcription by the HIV Tat protein in human cells. In contrast, a complex of Spt4 and Spt5 is required in vitro for the inhibition of RNA polymerase II (Pol II) elongation by the drug DRB, suggesting also a negative role in vivo. To learn more about the function of the Spt4/Spt5 complex and Spt6 in vivo, we have identified Drosophila homologs of Spt5 and Spt6 and characterized their localization onDrosophila polytene chromosomes. We find that Spt5 and Spt6 localize extensively with the phosphorylated, actively elongating form of Pol II, to transcriptionally active sites during salivary gland development and upon heat shock. Furthermore, Spt5 and Spt6 do not colocalize widely with the unphosphorylated, nonelongating form of Pol II. These results strongly suggest that Spt5 and Spt6 play closely related roles associated with active transcription in vivo.

International audience; In Drosophila melanogaster, widely used mitotic recombination-based strategies generate mosaic flies with positive readout for only one daughter cell after division. To differentially label both daughter cells, we developed the twin spot generator (TSG) technique, which through mitotic recombination generates green and red twin spots that are detectable after the first cell division as single cells. We propose wide applications of TSG to lineage and genetic mosaic studies.

The presence of homologous nucleic acid sequences can exert profound effects on chromosomal and gene function in a wide range of organisms. These homology effects reveal remarkable forms of regulation as well as suggest possible avenues for the development of new technologies.

Ed Lewis introduced the term “transvection” in 1954 to describe mechanisms that can cause the expression of a gene to be sensitive to the proximity of its homologue. Transvection since has been reported at an increasing number of loci in Drosophila , where homologous chromosomes are paired in somatic tissues, as well as at loci in other organisms. At the Drosophila yellow gene, transvection can explain intragenic complementation involving the yellow 2 allele ( y 2 ). Here, transvection was proposed to occur by enhancers of one allele acting in trans on the promoter of a paired homologue. In this report, we describe two yellow alleles that strengthen this model and reveal an unexpected, second mechanism for transvection. Data suggest that, in addition to enhancer action in trans , transvection can occur by enhancer bypass of a chromatin insulator in cis . We propose that bypass results from the topology of paired genes. Finally, transvection at yellow can occur in genotypes not involving y 2 , implying that it is a feature of yellow itself and not an attribute of one particular allele.

The zeste1 (z1) mutation of Drosophila melanogaster produces a mutant yellow eye color instead of the wild-type red. Genetic and molecular data suggest that z1 achieves this change by altering expression of the wild-type white gene in a manner that exhibits transvection effects. There exist suppressor and enhancer mutations that modify the z1 eye color, and this paper summarizes our studies of those belonging to the Suppressor 2 of zeste complex [Su(z)2-C]. The Su(z)2-C consists of at least three subregions called Psc (Posterior sex combs), Su(z)2 and Su(z)2D (Distal). The products of these subregions are proposed to act at the level of chromatin. Complementation analyses predict that the products are functionally similar and interacting. The alleles of Psc define two overlapping phenotypic classes, the hopeful and hapless. The distinctions between these two classes and the intragenic complementation seen among some of the Psc alleles are consistent with a multidomain structure for the product of Psc. Psc is a member of the homeotic Polycomb group of genes. A general discussion of the Polycomb and trithorax group of genes, position-effect variegation, transvection, chromosome pairing and chromatin structure is presented.

Homologous chromosomes are paired in somatic cells of Drosophila melanogaster. This pairing can lead to transvection, which is a process by which the proximity of homologous genes can lead to a change in gene expression. At the yellow gene, transvection is the basis for several examples of intragenic complementation involving the enhancers of one allele acting in trans on the promoter of a paired second allele. Using complementation as our assay, we explored the chromosomal requirements for pairing and transvection at yellow. Following a protocol established by Ed Lewis, we generated and characterized chromosomal rearrangements to define a region in cis to yellow that must remain intact for complementation to occur. Our data indicate that homolog pairing at yellow is efficient, as complementation was disrupted only in the presence of chromosomal rearrangements that break ≤650 kbp from yellow. We also found that three telomerically placed chromosomal duplications, containing ∼700 or more kbp of the yellow genomic region, are able to alter complementation at yellow, presumably through competitive pairing interactions. These results provide a formal demonstration of the pairing-dependent nature of yellow transvection and suggest that yellow pairing, as measured by transvection, reflects the extent of contiguous homology flanking the locus.

Homolog pairing refers to the alignment and physical apposition of homologous chromosomal segments. Although commonly observed during meiosis, homolog pairing also occurs in nonmeiotic cells of several organisms, including humans and Drosophila. The mechanism underlying nonmeiotic pairing, however, remains largely unknown. Here, we explore the use of established Drosophila cell lines for the analysis of pairing in somatic cells. Using fluorescent in situ hybridization (FISH), we assayed pairing at nine regions scattered throughout the genome of Kc167 cells, observing high levels of homolog pairing at all six euchromatic regions assayed and variably lower levels in regions in or near centromeric heterochromatin. We have also observed extensive pairing in six additional cell lines representing different tissues of origin, different ploidies, and two different species, demonstrating homolog pairing in cell culture to be impervious to cell type or culture history. Furthermore, by sorting Kc167 cells into G1, S, and G2 subpopulations, we show that even progression through these stages of the cell cycle does not significantly change pairing levels. Finally, our data indicate that disrupting Drosophila topoisomerase II (Top2) gene function with RNAi and chemical inhibitors perturbs homolog pairing, suggesting Top2 to be a gene important for pairing.

Position effects can complicate transgene analyses. This is especially true when comparing transgenes that have inserted randomly into different genomic positions and are therefore subject to varying position effects. Here, we introduce a method for the precise targeting of transgenic constructs to predetermined genomic sites in Drosophila using the ϕC31 integrase system in conjunction with recombinase-mediated cassette exchange (RMCE). We demonstrate the feasibility of this system using two donor cassettes, one carrying the yellow gene and the other carrying GFP. At all four genomic sites tested, we observed exchange of donor cassettes with an integrated target cassette carrying the mini-white gene. Furthermore, because RMCE-mediated integration of the donor cassette is necessarily accompanied by loss of the target cassette, we were able to identify integrants simply by the loss of mini-white eye color. Importantly, this feature of the technology will permit integration of unmarked constructs into Drosophila, even those lacking functional genes. Thus, ϕC31 integrase-mediated RMCE should greatly facilitate transgene analysis as well as permit new experimental designs.

Homolog pairing, which plays a critical role in meiosis, poses a potential risk if it occurs in inappropriate tissues or between nonallelic sites, as it can lead to changes in gene expression, chromosome entanglements, and loss-of-heterozygosity due to mitotic recombination. This is particularly true in Drosophila, which supports organismal-wide pairing throughout development. Discovered over a century ago, such extensive pairing has led to the perception that germline pairing in the adult gonad is an extension of the pairing established during embryogenesis and, therefore, differs from the mechanism utilized in most species to initiate pairing specifically in the germline. Here, we show that, contrary to long-standing assumptions, Drosophila meiotic pairing in the gonad is not an extension of pairing established during embryogenesis. Instead, we find that homologous chromosomes are unpaired in primordial germ cells from the moment the germline can be distinguished from the soma in the embryo and remain unpaired even in the germline stem cells of the adult gonad. We further establish that pairing originates immediately after the stem cell stage. This pairing occurs well before the initiation of meiosis and, strikingly, continues through the several mitotic divisions preceding meiosis. These discoveries indicate that the spatial organization of the Drosophila genome differs between the germline and the soma from the earliest moments of development and thus argue that homolog pairing in the germline is an active process as versus a passive continuation of pairing established during embryogenesis.

The pairing of homologous chromosomes is a fundamental feature of the meiotic cell. In addition, a number of species exhibit homolog pairing in nonmeiotic, somatic cells as well, with evidence for its impact on both gene regulation and double-strand break (DSB) repair. An extreme example of somatic pairing can be observed in Drosophila melanogaster, where homologous chromosomes remain aligned throughout most of development. However, our understanding of the mechanism of somatic homolog pairing remains unclear, as only a few genes have been implicated in this process. In this study, we introduce a novel high-throughput fluorescent in situ hybridization (FISH) technology that enabled us to conduct a genome-wide RNAi screen for factors involved in the robust somatic pairing observed in Drosophila. We identified both candidate “pairing promoting genes” and candidate “anti-pairing genes,” providing evidence that pairing is a dynamic process that can be both enhanced and antagonized. Many of the genes found to be important for promoting pairing are highly enriched for functions associated with mitotic cell division, suggesting a genetic framework for a long-standing link between chromosome dynamics during mitosis and nuclear organization during interphase. In contrast, several of the candidate anti-pairing genes have known interphase functions associated with S-phase progression, DNA replication, and chromatin compaction, including several components of the condensin II complex. In combination with a variety of secondary assays, these results provide insights into the mechanism and dynamics of somatic pairing., Author Summary In addition to their number and structure, the position and spatial dynamics of chromosomes are under tight control, as direct interactions between chromosomes can contribute to the activation or repression of genes. Here, we focus on a particular type of interaction, known as somatic homolog pairing, which occurs between the maternal and paternal copies of chromosomes. While the role of somatic pairing on downstream homology-driven processes is well-established, there is much to be learned about how homologous chromosome segments find each other, physically align, and form stable pairing interactions within somatic cells. Taking advantage of a novel high-throughput FISH technology and the fact that homologous chromosomes are intimately paired along their lengths in the somatic cells of Drosophila, we have conducted a screen for factors that are important for the fidelity of somatic pairing. Ultimately, the characterization of these pairing genes will shed light on the mechanism of pairing, as well as pairing-mediated processes that have implications for development and disease. Finally, the efficacy of our screen for pairing genes suggests that the high-throughput FISH technology described here will prove useful for studying forms of nuclear organization and chromosome positioning beyond pairing.