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

Genetic News

The Genetics Society of America’s Thomas Hunt Morgan Medal honors researchers for lifetime achievement in genetics. The recipient of the 2019 Morgan Medal is Daniel Hartl of Harvard University, who is recognized for his influential and diverse contributions to genetics research. The unifying theme of Hartl’s broad impacts on transmission, population, evolutionary, and medical genetics has been the combination of theoretical insights with cutting-edge experimental techniques. Some of his contributions include revealing the genetics of segregation distortion, developing statistical frameworks for estimating the effects of selection, application of these frameworks to natural and experimental populations, discovery of the mariner transposon and its influence on genome evolution, insights into the evolution of gene expression differences, and modeling the evolution of malaria parasite populations. Hartl is also known as a supportive mentor who has trained many prominent geneticists that continue to shape the field.

Bruce Baker, a preeminent Drosophila geneticist who made fundamental contributions to our understanding of the molecular genetic basis of sex differences, passed away July 1, 2018 at the age of 72. Members of Bruce’s laboratory remember him as an intensely dedicated, rigorous, creative, deep-thinking, and fearless scientist. His trainees also remember his strong commitment to teaching students at every level. Bruce’s career studying sex differences had three major epochs, where the laboratory was focused on: (1) sex determination and dosage compensation, (2) the development of sex-specific structures, and (3) the molecular genetic basis for sex differences in behavior. Several members of the Baker laboratory have come together to honor Bruce by highlighting some of the laboratory’s major scientific contributions in these areas.

CRISPR-mediated base editors have opened unique avenues for scar-free genome-wide mutagenesis. Here, we describe a comprehensive computational workflow called beditor that can be broadly adapted for designing guide RNA libraries with a range of CRISPR-mediated base editors, Protospacer Adjacent Motif (PAM) recognition sequences, and genomes of many species. Additionally, to assist users in selecting the best sets of guide RNAs for their experiments, a priori estimates of editing efficiency, called beditor scores, are calculated. These beditor scores are intended to select guide RNAs that conform to requirements for optimal base editing: the editable base falls within maximum activity window of the CRISPR-mediated base editor and produces nonconfounding mutational effects with minimal predicted off-target effects. We demonstrate the utility of the software by designing guide RNAs for base editing to model or correct thousands of clinically important human disease mutations.

GFP labeling by genome editing can reveal the authentic location of a native protein, but is frequently hampered by weak GFP signals and broad expression across a range of tissues that may obscure cell-specific localization. To overcome these problems, we engineered a Native And Tissue-specific Fluorescence (NATF) strategy that combines genome editing and split-GFP to yield bright, cell-specific protein labeling. We use clustered regularly interspaced short palindromic repeats CRISPR/Cas9 to insert a tandem array of seven copies of the GFP11 β-strand (gfp11x7) at the genomic locus of each target protein. The resultant gfp11x7 knock-in strain is then crossed with separate reporter lines that express the complementing split-GFP fragment (gfp1-10) in specific cell types, thus affording tissue-specific labeling of the target protein at its native level. We show that NATF reveals the otherwise undetectable intracellular location of the immunoglobulin protein OIG-1 and demarcates the receptor auxiliary protein LEV-10 at cell-specific synaptic domains in the Caenorhabditis elegans nervous system.

It becomes increasingly important in using genome-wide association studies (GWAS) to select important genetic information associated with qualitative or quantitative traits. Currently, the discovery of biological association among SNPs motivates various strategies to construct SNP-sets along the genome and to incorporate such set information into selection procedure for a higher selection power, while facilitating more biologically meaningful results. The aim of this paper is to propose a novel Bayesian framework for hierarchical variable selection at both SNP-set (group) level and SNP (within group) level. We overcome a key limitation of existing posterior updating scheme in most Bayesian variable selection methods by proposing a novel sampling scheme to explicitly accommodate the ultrahigh-dimensionality of genetic data. Specifically, by constructing an auxiliary variable selection model under SNP-set level, the new procedure utilizes the posterior samples of the auxiliary model to subsequently guide the posterior inference for the targeted hierarchical selection model. We apply the proposed method to a variety of simulation studies and show that our method is computationally efficient and achieves substantially better performance than competing approaches in both SNP-set and SNP selection. Applying the method to the Alzheimers Disease Neuroimaging Initiative (ADNI) data, we identify biologically meaningful genetic factors under several neuroimaging volumetric phenotypes. Our method is general and readily to be applied to a wide range of biomedical studies.

Fission yeast Swi6 is a human HP1 homolog that plays important roles in multiple cellular processes. In addition to its role in maintaining heterochromatin silencing, Swi6 is required for cohesin enrichment at the pericentromere. Loss of Swi6 leads to abnormal mitosis, including defects in the establishment of bioriented sister kinetochores and microtubule attachment. Swi6 interacts with Dfp1, a regulatory subunit of DBF4-dependent kinase (DDK), and failure to recruit Dfp1 to the pericentromere results in late DNA replication. Using the dfp1-3A mutant allele, which specifically disrupts Swi6-Dfp1 association, we investigated how interaction between Swi6 and Dfp1 affects chromosome dynamics. We find that disrupting the interaction between Swi6 and Dfp1 delays mitotic progression in a spindle assembly checkpoint-dependent manner. Artificially tethering Dfp1 back to the pericentromere is sufficient to restore normal spindle length and rescue segregation defects in swi6-deleted cells. However, Swi6 is necessary for centromeric localization of Rad21-GFP independent of DDK. Our data indicate that DDK contributes to mitotic chromosome segregation in pathways that partly overlap with, but can be separated from both, Swi6 and the other HP1 homolog, Chp2.

During meiosis, formation of double-strand breaks (DSBs) and repair by homologous recombination between homologs creates crossovers (COs) that facilitate chromosome segregation. CO formation is tightly regulated to ensure the integrity of this process. The DNA damage response kinases, Ataxia-telangiectasia mutated (ATM) and RAD3-related (ATR) have emerged as key regulators of CO formation in yeast, flies, and mice, influencing DSB formation, repair pathway choice, and cell cycle progression. The molecular networks that ATM and ATR influence during meiosis are still being resolved in other organisms. Here, we show that Caenorhabditis elegans ATM and ATR homologs, ATM-1 and ATL-1 respectively, act at multiple steps in CO formation to ultimately ensure that COs are formed on all chromosomes. We show a role for ATM-1 in regulating the choice of repair template, biasing use of the homologous chromosome instead of the sister chromatid. Our data suggest a model in which ATM-1 and ATL-1 have antagonistic roles in very early repair processing, but are redundantly required for accumulation of the RAD-51 recombinase at DSB sites. We propose that these features of ATM-1 and ATL-1 ensure both CO formation on all chromosomes and accurate repair of additional DSBs.

The sources of genome instability, a hallmark of cancer, remain incompletely understood. One potential source is DNA rereplication, which arises when the mechanisms that prevent the reinitiation of replication origins within a single cell cycle are compromised. Using the budding yeast Saccharomyces cerevisiae, we previously showed that DNA rereplication is extremely potent at inducing gross chromosomal alterations and that this arises in part because of the susceptibility of rereplication forks to break. Here, we examine the ability of DNA rereplication to induce nucleotide-level mutations. During normal replication these mutations are restricted by three overlapping error-avoidance mechanisms: the nucleotide selectivity of replicative polymerases, their proofreading activity, and mismatch repair. Using lys2InsEA14, a frameshift reporter that is poorly proofread, we show that rereplication induces up to a 30x higher rate of frameshift mutations and that this mutagenesis is due to passage of the rereplication fork, not secondary to rereplication fork breakage. Rereplication can also induce comparable rates of frameshift and base-substitution mutations in a more general mutagenesis reporter CAN1, when the proofreading activity of DNA polymerase is inactivated. Finally, we show that the rereplication-induced mutagenesis of both lys2InsEA14 and CAN1 disappears in the absence of mismatch repair. These results suggest that mismatch repair is attenuated during rereplication, although at most sequences DNA polymerase proofreading provides enough error correction to mitigate the mutagenic consequences. Thus, rereplication can facilitate nucleotide-level mutagenesis in addition to inducing gross chromosomal alterations, broadening its potential role in genome instability.

Crossover formation as a result of meiotic recombination is vital for the proper segregation of homologous chromosomes at the end of meiosis I. In many organisms, crossovers are generated through two crossover pathways: Class I and Class II. To ensure accurate crossover formation, meiosis-specific protein complexes regulate the degree to which each pathway is used. One such complex is the mei-mini-chromosome maintenance (MCM) complex, which contains MCM and MCM-like proteins REC (ortholog of Mcm8), MEI-217, and MEI-218. The mei-MCM complex genetically promotes Class I crossovers and inhibits Class II crossovers in Drosophila, but it is unclear how individual mei-MCM proteins contribute to crossover regulation. In this study, we perform genetic analyses to understand how specific regions and motifs of mei-MCM proteins contribute to Class I and II crossover formation, and distribution. Our analyses show that the long, disordered N-terminus of MEI-218 is dispensable for crossover formation, and that mutations that disrupt REC’s Walker A and B motifs differentially affect Class I and Class II crossover formation. In rec Walker A mutants, Class I crossovers exhibit no change but Class II crossovers are increased. However, in rec Walker B mutants, Class I crossovers are severely impaired and Class II crossovers are increased. These results suggest that REC may form multiple complexes that exhibit differential REC-dependent ATP-binding and -hydrolyzing requirements. These results provide genetic insight into the mechanisms through which mei-MCM proteins promote Class I crossovers and inhibit Class II crossovers.

Faithful segregation of homologous chromosomes at meiosis requires pairing and recombination. In taxa with dimorphic sex chromosomes, pairing between them in the heterogametic sex is limited to a narrow interval of residual sequence homology known as the pseudoautosomal region (PAR). Failure to form the obligate crossover in the PAR is associated with male infertility in house mice (Mus musculus) and humans. Yet despite this apparent functional constraint, the boundary and organization of the PAR is highly variable in mammals, and even between subspecies of mice. Here, we estimate the genetic map in a previously documented expansion of the PAR in the M. musculus castaneus subspecies and show that the local recombination rate is 100-fold higher than the autosomal background. We identify an independent shift in the PAR boundary in the M. musculus musculus subspecies and show that it involves a complex rearrangement, but still recombines in heterozygous males. Finally, we demonstrate pervasive copy-number variation at the PAR boundary in wild populations of M. m. domesticus, M. m. musculus, and M. m. castaneus. Our results suggest that the intensity of recombination activity in the PAR, coupled with relatively weak constraints on its sequence, permit the generation and maintenance of unusual levels of polymorphism in the population of unknown functional significance.

Transvection is the phenomenon where a transcriptional enhancer activates a promoter located on the homologous chromosome. It has been amply documented in Drosophila where homologs are closely paired in most, if not all, somatic nuclei, but it has been known to rarely occur in mammals as well. We have taken advantage of site-directed transgenesis to insert reporter constructs into the same genetic locus in Drosophila and have evaluated their ability to engage in transvection by testing many heterozygous combinations. We find that transvection requires the presence of an insulator element on both homologs. Homotypic trans-interactions between four different insulators can support transvection: the gypsy insulator (GI), Wari, Fab-8 and 1A2; GI and Fab-8 are more effective than Wari or 1A2. We show that, in the presence of insulators, transvection displays the characteristics that have been previously described: it requires homolog pairing, but can happen at any of several loci in the genome; a solitary enhancer confronted with an enhancerless reporter is sufficient to drive transcription; it is weaker than the action of the same enhancer-promoter pair in cis, and it is further suppressed by cis-promoter competition. Though necessary, the presence of homotypic insulators is not sufficient for transvection; their position, number and orientation matters. A single GI adjacent to both enhancer and promoter is the optimal configuration. The identity of enhancers and promoters in the vicinity of a trans-interacting insulator pair is also important, indicative of complex insulator-enhancer-promoter interactions.

During mitosis, kinetochore–microtubule interactions ensure that chromosomes are accurately segregated to daughter cells. RSA-1 (regulator of spindle assembly-1) is a regulatory B'' subunit of protein phosphatase 2A that was previously proposed to modulate microtubule dynamics during spindle assembly. We have identified a genetic interaction between the centrosomal protein, RSA-1, and the spindle- and kinetochore-associated (Ska) complex in Caenorhabditis elegans. In a forward genetic screen for suppressors of rsa-1(or598) embryonic lethality, we identified mutations in ska-1 and ska-3. Loss of SKA-1 and SKA-3, as well as components of the KMN (KNL-1/MIS-12/NDC-80) complex and the microtubule end-binding protein EBP-2, all suppressed the embryonic lethality of rsa-1(or598). These suppressors also disrupted the intracellular localization of the Ska complex, revealing a network of proteins that influence Ska function during mitosis. In rsa-1(or598) embryos, SKA-1 is excessively and prematurely recruited to kinetochores during spindle assembly, but SKA-1 levels return to normal just prior to anaphase onset. Loss of the TPX2 homolog, TPXL-1, also resulted in overrecruitment of SKA-1 to the kinetochores and this correlated with the loss of Aurora A kinase on the spindle microtubules. We propose that rsa-1 regulates the kinetochore localization of the Ska complex, with spindle-associated Aurora A acting as a potential mediator. These data reveal a novel mechanism of protein phosphatase 2A function during mitosis involving a centrosome-based regulatory mechanism for Ska complex recruitment to the kinetochore.

Remodeling of the extracellular matrix supports tissue and organ development, by regulating cellular morphology and tissue integrity. However, proper extracellular matrix remodeling requires spatiotemporal regulation of extracellular metalloproteinase activity. Members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family, including MIG-17 and GON-1, are evolutionarily conserved, secreted, zinc-requiring metalloproteinases. Although these proteases are required for extracellular matrix remodeling during gonadogenesis in Caenorhabditis elegans, their in vivo regulatory mechanisms remain to be delineated. Therefore, we focused on the C. elegans tissue inhibitors of metalloproteinases (TIMPs), TIMP-1 and CRI-2. Analysis of the transcription and translation products for GFP/Venus fusions, with TIMP-1 or CRI-2, indicated that these inhibitors were secreted and localized to the basement membrane of gonads and the plasma membrane of germ cells. A timp-1 deletion mutant exhibited gonadal growth defects and sterility, and the phenotypes of this mutant were fully rescued by a TIMP-1::Venus construct, but not by a TIMP-1(C21S)::Venus mutant construct, in which the inhibitor coding sequence had been mutated. Moreover, genetic data suggested that TIMP-1 negatively regulates proteolysis of the α1 chain of type IV collagen. We also found that the loss-of-function observed for the mutants timp-1 and cri-2 involves a partial suppression of gonadal defects found for the mutants mig-17/ADAMTS and gon-1/ADAMTS, and that this suppression was canceled upon overexpression of gon-1 or mig-17, respectively. Based on these results, we propose that both TIMP-1 and CRI-2 act as inhibitors of MIG-17 and GON-1 ADAMTSs to regulate gonad development in a noncell-autonomous manner.

Organismal physiology emerges from metabolic pathways and subcellular structures like the mitochondria that can vary across development and among individuals. Here, we tested whether genetic variation at one level of physiology can be buffered at higher levels of biological organization during development by the inherent capacity for homeostasis in physiological systems. We found that the fundamental scaling relationship between mass and metabolic rate, as well as the oxidative capacity per mitochondria, changed significantly across development in the fruit fly Drosophila. However, mitochondrial respiration rate was maintained at similar levels across development. Furthermore, larvae clustered into two types—those that switched to aerobic, mitochondrial ATP production before the second instar, and those that relied on anaerobic, glycolytic production of ATP through the second instar. Despite genetic variation for the timing of this metabolic shift, metabolic rate in second-instar larvae was more robust to genetic variation than was the metabolic rate of other instars. We found that larvae with a mitochondrial-nuclear incompatibility that disrupts mitochondrial function had increased aerobic capacity and relied more on anaerobic ATP production throughout development relative to larvae from wild-type strains. By taking advantage of both ways of making ATP, larvae with this mitochondrial–nuclear incompatibility maintained mitochondrial respiratory capacity, but also had higher levels of whole-body reactive oxygen species and decreased mitochondrial membrane potential, potentially as a physiological defense mechanism. Thus, genetic defects in core physiology can be buffered at the organismal level via physiological plasticity, and natural populations may harbor genetic variation for distinct metabolic strategies in development that generate similar organismal outcomes.

The Major Histocompatibility Complex (MHC) is the most genetically diverse region of the genome in most vertebrates. Some form of balancing selection is necessary to account for the extreme diversity, but the precise mechanism of balancing selection is unknown. Due to the way MHC molecules determine immune recognition, overdominance (also referred to as heterozygote advantage) has been suggested as the main driving force behind this unrivalled diversity. However, both theoretical results and simulation models have shown that overdominance in its classical form cannot maintain large numbers of alleles unless all alleles confer unrealistically similar levels of fitness. There is increasing evidence that heterozygotes containing genetically divergent alleles allow for broader antigen presentation to immune cells, providing a selective mechanism for MHC polymorphism. By framing competing models of overdominance within a general framework, we show that a model based on Divergent Allele Advantage (DAA) provides a superior mechanism for maintaining alleles with a wide range of intrinsic merits, as intrinsically less-fit MHC alleles that are more divergent can survive under DAA. Specifically, our results demonstrate that a quantitative mechanism built from the DAA hypothesis is able to maintain polymorphism in the MHC. Applying such a model to both livestock breeding and conservation could provide a better way of identifying superior heterozygotes, and quantifying the advantages of genetic diversity at the MHC.

Recent studies have affirmed that higher-order epistasis is ubiquitous and can have large effects on complex traits. Yet, we lack frameworks for understanding how epistatic interactions are influenced by central features of cell physiology. In this study, we assess how protein quality control machinery—a critical component of cell physiology—affects epistasis for different traits related to bacterial resistance to antibiotics. Specifically, we disentangle the interactions between different protein quality control genetic backgrounds and two sets of mutations: (i) SNPs associated with resistance to antibiotics in an essential bacterial enzyme (dihydrofolate reductase, or DHFR) and (ii) differing DHFR bacterial species-specific amino acid background sequences (Escherichia coli, Listeria grayi, and Chlamydia muridarum). In doing so, we improve on generic observations that epistasis is widespread by discussing how patterns of epistasis can be partly explained by specific interactions between mutations in an essential enzyme and genes associated with the proteostasis environment. These findings speak to the role of environmental and genotypic context in modulating higher-order epistasis, with direct implications for evolutionary theory, genetic modification technology, and efforts to manage antimicrobial resistance.



Genetic Ethics

The advances in genetic mapping have made very real what seemed so improbable twenty years ago. ... Genetic mapping is a powerful tool ... but it is also vulnerable to abuse. Many ethical, legal and societal issues are beginning to emerge...
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A process by which genes undergo a structural change.