Using gene map science to evaluate the genetic map and eliminate disease

Genetic News

A critical juncture in early development is the partitioning of cells that will adopt different fates into three germ layers: the ectoderm, the mesoderm, and the endoderm. This step is achieved through the internalization of specified cells from the outermost surface layer, through a process called gastrulation. In Drosophila, gastrulation is achieved through cell shape changes (i.e., apical constriction) that change tissue curvature and lead to the folding of a surface epithelium. Folding of embryonic tissue results in mesoderm and endoderm invagination, not as individual cells, but as collective tissue units. The tractability of Drosophila as a model system is best exemplified by how much we know about Drosophila gastrulation, from the signals that pattern the embryo to the molecular components that generate force, and how these components are organized to promote cell and tissue shape changes. For mesoderm invagination, graded signaling by the morphogen, Spätzle, sets up a gradient in transcriptional activity that leads to the expression of a secreted ligand (Folded gastrulation) and a transmembrane protein (T48). Together with the GPCR Mist, which is expressed in the mesoderm, and the GPCR Smog, which is expressed uniformly, these signals activate heterotrimeric G-protein and small Rho-family G-protein signaling to promote apical contractility and changes in cell and tissue shape. A notable feature of this signaling pathway is its intricate organization in both space and time. At the cellular level, signaling components and the cytoskeleton exhibit striking polarity, not only along the apical–basal cell axis, but also within the apical domain. Furthermore, gene expression controls a highly choreographed chain of events, the dynamics of which are critical for primordium invagination; it does not simply throw the cytoskeletal "on" switch. Finally, studies of Drosophila gastrulation have provided insight into how global tissue mechanics and movements are intertwined as multiple tissues simultaneously change shape. Overall, these studies have contributed to the view that cells respond to forces that propagate over great distances, demonstrating that cellular decisions, and, ultimately, tissue shape changes, proceed by integrating cues across an entire embryo.

Single nucleotide polymorphisms (SNPs) are used widely for detecting quantitative trait loci, or for searching for causal variants of diseases. Nevertheless, structural variations such as copy-number variants (CNVs) represent a large part of natural genetic diversity, and contribute significantly to trait variation. Numerous methods and softwares based on different technologies (amplicons, CGH, tiling, or SNP arrays, or sequencing) have already been developed to detect CNVs, but they bypass a wealth of information such as genotyping data from segregating populations, produced, e.g., for QTL mapping. Here, we propose an original method to both detect and genetically map CNVs using mapping panels. Specifically, we exploit the apparent heterozygous state of duplicated loci: peaks in appropriately defined genome-wide allelic profiles provide highly specific signatures that identify the nature and position of the CNVs. Our original method and software can detect and map automatically up to 33 different predefined types of CNVs based on segregation data only. We validate this approach on simulated and experimental biparental mapping panels in two maize populations and one wheat population. Most of the events found correspond to having just one extra copy in one of the parental lines, but the corresponding allelic value can be that of either parent. We also find cases with two or more additional copies, especially in wheat, where these copies locate to homeologues. More generally, our computational tool can be used to give additional value, at no cost, to many datasets produced over the past decade from genetic mapping panels.

For most animals, feeding includes two behaviors: foraging to find a food patch and food intake once a patch is found. The nematode Caenorhabditis elegans is a useful model for studying the genetics of both behaviors. However, most methods of measuring feeding in worms quantify either foraging behavior or food intake, but not both. Imaging the depletion of fluorescently labeled bacteria provides information on both the distribution and amount of consumption, but even after patch exhaustion a prominent background signal remains, which complicates quantification. Here, we used a bioluminescent Escherichia coli strain to quantify C. elegans feeding. With light emission tightly coupled to active metabolism, only living bacteria are capable of bioluminescence, so the signal is lost upon ingestion. We quantified the loss of bioluminescence using N2 reference worms and eat-2 mutants, and found a nearly 100-fold increase in signal-to-background ratio and lower background compared to loss of fluorescence. We also quantified feeding using aggregating npr-1 mutant worms. We found that groups of npr-1 mutants first clear bacteria from within the cluster before foraging collectively for more food; similarly, during large population swarming, only worms at the migrating front are in contact with bacteria. These results demonstrate the usefulness of bioluminescent bacteria for quantifying feeding and generating insights into the spatial pattern of food consumption.

Ribosome biogenesis is tightly regulated through stress-sensing pathways that impact genome stability, aging and senescence. In Saccharomyces cerevisiae, ribosomal RNAs are transcribed from rDNA located on the right arm of chromosome XII. Numerous studies reveal that rDNA decondenses into a puff-like structure during interphase, and condenses into a tight loop-like structure during mitosis. Intriguingly, a novel and additional mechanism of increased mitotic rDNA compaction (termed hypercondensation) was recently discovered that occurs in response to temperature stress (hyperthermic-induced) and is rapidly reversible. Here, we report that neither changes in condensin binding or release of DNA during mitosis, nor mutation of factors that regulate cohesin binding and release, appear to play a critical role in hyperthermic-induced rDNA hypercondensation. A candidate genetic approach revealed that deletion of either HSP82 or HSC82 (Hsp90 encoding heat shock paralogs) result in significantly reduced hyperthermic-induced rDNA hypercondensation. Intriguingly, Hsp inhibitors do not impact rDNA hypercondensation. In combination, these findings suggest that Hsp90 either stabilizes client proteins, which are sensitive to very transient thermic challenges, or directly promotes rDNA hypercondensation during preanaphase. Our findings further reveal that the high mobility group protein Hmo1 is a negative regulator of mitotic rDNA condensation, distinct from its role in promoting premature condensation of rDNA during interphase upon nutrient starvation.

Diploid germline cells must undergo two consecutive meiotic divisions before differentiating as haploid sex cells. During meiosis I, homologs pair and remain conjoined until segregation at anaphase. Drosophila melanogaster spermatocytes are unique in that the canonical events of meiosis I including synaptonemal complex formation, double-strand DNA breaks, and chiasmata are absent. Sex chromosomes pair at intergenic spacer sequences within the ribosomal DNA (rDNA). Autosomes pair at numerous euchromatic homologies, but not at heterochromatin, suggesting that pairing may be limited to specific sequences. However, previous work generated from genetic segregation assays or observations of late prophase I/prometaphase I chromosome associations fail to differentiate pairing from maintenance of pairing (conjunction). Here, we separately examined the capability of X euchromatin to pair and conjoin using an rDNA-deficient X and a series of Dp(1;Y) chromosomes. Genetic assays showed that duplicated X euchromatin can substitute for endogenous rDNA pairing sites. Segregation was not proportional to homology length, and pairing could be mapped to nonoverlapping sequences within a single Dp(1;Y). Using fluorescence in situ hybridization to early prophase I spermatocytes, we showed that pairing occurred with high fidelity at all homologies tested. Pairing was unaffected by the presence of X rDNA, nor could it be explained by rDNA magnification. By comparing genetic and cytological data, we determined that centromere proximal pairings were best at segregation. Segregation was dependent on the conjunction protein Stromalin in Meiosis, while the autosomal-specific Teflon was dispensable. Overall, our results suggest that pairing may occur at all homologies, but there may be sequence or positional requirements for conjunction.

Sex determination is remarkably variable among animals with examples of environmental sex determination, male heterogametic (XX/XY) and female heterogametic (ZZ/ZW) chromosomal sex determination, and other genetic mechanisms. The cephalochordate amphioxus occupies a key phylogenetic position as a basal chordate and outgroup to vertebrates, but its sex determination mechanism is unknown. During the course of generating Nodal mutants with transcription activator-like effector nucleases (TALENs) in amphioxus Branchiostoma floridae, serendipitously, we generated three mutant strains that reveal the sex determination mechanism of this animal. In one mutant strain, all heterozygous mutant offspring over three generations were female and all wild-type descendants were male. This pattern suggests the Nodal allele targeted is on a female-specific W chromosome. A second mutant showed the same W-linked inheritance pattern, with a female heterozygote passing the mutation only to daughters. In a third mutant strain, both male and female offspring could be heterozygous, but a female heterozygote passed the mutation only to sons. This pattern is consistent with the targeted allele being on a Z chromosome. We found an indel polymorphism linked to a Nodal allele present in most females, but no males in our cultured population. Together, these results indicate that Nodal is sex chromosome-linked in B. floridae, and that B. floridae has a ZZ/ZW sex chromosome system.

Polycomb-group (PcG) proteins are evolutionarily conserved epigenetic regulators whose primary function is to maintain the transcriptional repression of target genes. Recruitment of Drosophila melanogaster PcG proteins to target genes requires the presence of one or more Polycomb Response Elements (PREs). The functions or necessity for more than one PRE at a gene are not clear and individual PREs at some loci may have distinct regulatory roles. Various combinations of sequence-specific DNA-binding proteins are present at a given PRE, but only Pleiohomeotic (Pho) is present at all strong PREs. The giant (gt) locus has two PREs, a proximal PRE1 and a distal PRE2. During early embryonic development, Pho binds to PRE1 ~30-min prior to stable binding to PRE2. This observation indicated a possible dependence of PRE2 on PRE1 for PcG recruitment; however, we find here that PRE2 recruits PcG proteins and maintains transcriptional repression independently of Pho binding to PRE1. Pho-like (Phol) is partially redundant with Pho during larval development and binds to the same DNA sequences in vitro. Although binding of Pho to PRE1 is dependent on the presence of consensus Pho-Phol-binding sites, Phol binding is less so and appears to play a minimal role in recruiting other PcG proteins to gt. Another PRE-binding protein, Sp1/Kruppel-like factor, is dependent on the presence of Pho for PRE1 binding. Further, we show that, in addition to silencing gene expression, PcG proteins dampen transcription of an active gene.

The MAT locus of Cryptococcus neoformans has a bipolar organization characterized by an unusually large structure, spanning over 100 kb. MAT genes have been characterized by functional genetics as being involved in sexual reproduction and virulence. However, classical gene replacement failed to achieve mutants for five MAT genes (RPL22, RPO41, MYO2, PRT1, and RPL39), indicating that they are likely essential. In the present study, targeted gene replacement was performed in a diploid strain for both the α and a alleles of the ribosomal genes RPL22 and RPL39. Mendelian analysis of the progeny confirmed that both RPL22 and RPL39 are essential for viability. Ectopic integration of the RPL22 allele of opposite MAT identity in the heterozygous RPL22a/rpl22α or RPL22α/rpl22a mutant strains failed to complement their essential phenotype. Evidence suggests that this is due to differential expression of the RPL22 genes, and an RNAi-dependent mechanism that contributes to control RPL22a expression. Furthermore, via CRISPR/Cas9 technology, the RPL22 alleles were exchanged in haploid MATα and MATa strains of C. neoformans. These RPL22 exchange strains displayed morphological and genetic defects during bilateral mating. These results contribute to elucidating functions of C. neoformans essential mating type genes that may constitute a type of imprinting system to promote inheritance of nuclei of both mating types.

The three-dimensional (3D) organization of chromosomes can influence transcription. However, the frequency and magnitude of these effects remain debated. To determine how changes in chromosome positioning affect transcription across thousands of genes with minimal perturbation, we characterized nuclear organization and global gene expression in budding yeast containing chromosome fusions. We used computational modeling and single-cell imaging to determine chromosome positions, and integrated these data with genome-wide transcriptional profiles from RNA sequencing. We find that chromosome fusions dramatically alter 3D nuclear organization without leading to strong genome-wide changes in transcription. However, we observe a mild but significant and reproducible increase in the expression of genes displaced away from the periphery. The increase in transcription is inversely proportional to the propensity of a given locus to be at the nuclear periphery; for example, a 10% decrease in the propensity of a gene to reside at the nuclear envelope is accompanied by a 10% increase in gene expression. Modeling suggests that this is due to both deletion of telomeres and to displacement of genes relative to the nuclear periphery. These data suggest that basal transcriptional activity is sensitive to radial changes in gene position, and provide insight into the functional relevance of budding yeast chromosome-level 3D organization in gene expression.

Temporal regulation of gene expression is a crucial aspect of metazoan development. In the roundworm Caenorhabditis elegans, the heterochronic pathway controls multiple developmental events in a time-specific manner. The most downstream effector of this pathway, the zinc-finger transcription factor LIN-29, acts in the last larval stage (L4) to regulate elements of the larval-to-adult switch. Here, we explore new LIN-29 targets and their implications for this developmental transition. We used RNA-sequencing to identify genes differentially expressed between animals misexpressing LIN-29 at an early time point and control animals. Among 230 LIN-29-activated genes, we found that genes encoding cuticle collagens were overrepresented. Interestingly, expression of lin-29 and some of these collagens was increased in adults with cuticle damage, suggesting a previously unknown function for LIN-29 in adult cuticle maintenance. On the other hand, genes involved in fat metabolism were enriched among 350 LIN-29-downregulated targets. We used mass spectrometry to assay lipid content in animals overexpressing LIN-29 and observed reduced fatty acid levels. Many LIN-29-repressed genes are normally expressed in the intestine, suggesting cell-nonautonomous regulation. We identified several LIN-29 upregulated genes encoding signaling molecules that may act as mediators in the regulation of intestinally expressed genes encoding fat metabolic enzymes and vitellogenins. Overall, our results support the model of LIN-29 as a major regulator of adult cuticle synthesis and integrity, and as the trigger for metabolic changes that take place at the important transition from rapid growth during larval life to slower growth and offspring production during adulthood.

The azoxymethane model of colorectal cancer (CRC) was used to gain insights into the genetic heterogeneity of nonfamilial CRC. We observed significant differences in susceptibility parameters across 40 mouse inbred strains, with 6 new and 18 of 24 previously identified mouse CRC modifier alleles detected using genome-wide association analysis. Tumor incidence varied in F1 as well as intercrosses and backcrosses between resistant and susceptible strains. Analysis of inheritance patterns indicates that resistance to CRC development is inherited as a dominant characteristic genome-wide, and that susceptibility appears to occur in individuals lacking a large-effect, or sufficient numbers of small-effect, polygenic resistance alleles. Our results suggest a new polygenic model for inheritance of nonfamilial CRC, and that genetic studies in humans aimed at identifying individuals with elevated susceptibility should be pursued through the lens of absence of dominant resistance alleles rather than for the presence of susceptibility alleles.

Mitochondria are inherited uniparentally during sexual reproduction in the majority of eukaryotic species studied, including humans, mice, and nematodes, as well as many fungal species. Mitochondrial uniparental inheritance (mito-UPI) could be beneficial in that it avoids possible genetic conflicts between organelles with different genetic backgrounds, as recently shown in mice, and it could prevent the spread of selfish genetic elements in the mitochondrial genome. Despite the prevalence of observed mito-UPI, the underlying mechanisms and the genes involved in controlling this non-Mendelian inheritance are poorly understood in many species. In Cryptococcus neoformans, a human pathogenic basidiomyceteous fungus, mating types (MATα and MATa) are defined by alternate alleles at the single MAT locus that evolved from fusion of the two MAT loci (P/R encoding pheromones and pheromone receptors, and HD encoding homeodomain transcription factors) that are the ancestral state in the basidiomycota. Mitochondria are inherited uniparentally from the MATa parent in C. neoformans, and this requires the SXI1α and SXI2a HD factors encoded by MAT. However, there is evidence that additional genes contribute to the control of mito-UPI in Cryptococcus. Here, we show that in C. amylolentus, a sibling species of C. neoformans with unlinked P/R and HD MAT loci, mito-UPI is controlled by the P/R locus and is independent of the HD locus. Consistently, by replacing the MATα alleles of the pheromones (MF) and pheromone receptor (STE3) with the MATa alleles, we show that these P/R locus-defining genes indeed affect mito-UPI in C. neoformans during sexual reproduction. Additionally, we show that during early stages of C. neoformans sexual reproduction, conjugation tubes are always produced by the MATα cells, resulting in unidirectional migration of the MATα nucleus into the MATa cell during zygote formation. This process is controlled by the P/R locus and could serve to physically restrict movement of MATα mitochondria in the zygotes, and thereby contribute to mito-UPI. We propose a model in which both physical and genetic mechanisms function in concert to prevent the coexistence of mitochondria from the two parents in the zygote, and subsequently in the meiotic progeny, thus ensuring mito-UPI in pathogenic Cryptococcus, as well as in closely related nonpathogenic species. The implications of these findings are discussed in the context of the evolution of mito-UPI in fungi and other more diverse eukaryotes.

The microbiome influences health and disease through complex networks of host genetics, genomics, microbes, and environment. Identifying the mechanisms of these interactions has remained challenging. Systems genetics in laboratory mice (Mus musculus) enables data-driven discovery of biological network components and mechanisms of host–microbial interactions underlying disease phenotypes. To examine the interplay among the whole host genome, transcriptome, and microbiome, we mapped QTL and correlated the abundance of cecal messenger RNA, luminal microflora, physiology, and behavior in a highly diverse Collaborative Cross breeding population. One such relationship, regulated by a variant on chromosome 7, was the association of Odoribacter (Bacteroidales) abundance and sleep phenotypes. In a test of this association in the BKS.Cg-Dock7m +/+ Leprdb/J mouse model of obesity and diabetes, known to have abnormal sleep and colonization by Odoribacter, treatment with antibiotics altered sleep in a genotype-dependent fashion. The many other relationships extracted from this study can be used to interrogate other diseases, microbes, and mechanisms.

Cross-species complementation can be used to generate humanized yeast, which is a valuable resource with which to model and study human biology. Humanized yeast can be used as an in vivo platform to screen for chemical inhibition of human protein drug targets. To this end, we report the systematic complementation of nonessential yeast genes implicated in chromosome instability (CIN) with their human homologs. We identified 20 human–yeast complementation pairs that are replaceable in 44 assays that test rescue of chemical sensitivity and/or CIN defects. We selected a human–yeast pair (hFEN1/yRAD27), which is frequently overexpressed in cancer and is an anticancer therapeutic target, to perform in vivo inhibitor assays using a humanized yeast cell-based platform. In agreement with published in vitro assays, we demonstrate that HU-based PTPD is a species-specific hFEN1 inhibitor. In contrast, another reported hFEN1 inhibitor, the arylstibonic acid derivative NSC-13755, was determined to have off-target effects resulting in a synthetic lethal phenotype with yRAD27-deficient strains. Our study expands the list of human–yeast complementation pairs to nonessential genes by defining novel cell-based assays that can be utilized as a broad resource to study human drug targets.



Genetic Diseases

When medical researchers want to investigate serious genetic diseases, they have to find ways to locate the corresponding risk genes. There are relatively few of these risk genes out of the 100,000 genes in the human cell, so it obviously is not an easy task...
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All of the genes carried by a single gamete; the DNA content of an individual, which includes all 44 autosomes, 2 sex chromosomes, and the mitochondrial DNA.