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1.  Redundant Roles of Rpn10 and Rpn13 in Recognition of Ubiquitinated Proteins and Cellular Homeostasis 
PLoS Genetics  2015;11(7):e1005401.
Intracellular proteins tagged with ubiquitin chains are targeted to the 26S proteasome for degradation. The two subunits, Rpn10 and Rpn13, function as ubiquitin receptors of the proteasome. However, differences in roles between Rpn10 and Rpn13 in mammals remains to be understood. We analyzed mice deficient for Rpn13 and Rpn10. Liver-specific deletion of either Rpn10 or Rpn13 showed only modest impairment, but simultaneous loss of both caused severe liver injury accompanied by massive accumulation of ubiquitin conjugates, which was recovered by re-expression of either Rpn10 or Rpn13. We also found that mHR23B and ubiquilin/Plic-1 and -4 failed to bind to the proteasome in the absence of both Rpn10 and Rpn13, suggesting that these two subunits are the main receptors for these UBL-UBA proteins that deliver ubiquitinated proteins to the proteasome. Our results indicate that Rpn13 mostly plays a redundant role with Rpn10 in recognition of ubiquitinated proteins and maintaining homeostasis in Mus musculus.
Author Summary
At least two major ubiquitin receptor subunits that directly capture ubiquitin chains have been identified in the proteasome: Rpn10 and Rpn13. Analyses in Saccharomyces cerevisiae have suggested only a modest role of Rpn10 and Rpn13 in the recruitment of ubiquitinated proteins, as double deletion of Rpn10 and Rpn13 causes very mild phenotypes. Considering that ubiquitin recognition is an essential process for protein degradation by the proteasome and that failure in degradation of ubiquitinated proteins leads to human diseases such as neurodegeneration, it is important to evaluate the role of Rpn10 and Rpn13 in mammals. Liver-specific deletion of either Rpn10 or Rpn13 showed modest impairment, but simultaneous loss of both Rpn10 and Rpn13 caused severe liver injury accompanied by massive accumulation of ubiquitin conjugates and failure in recruiting mHR23B and ubiquilin/Plic-1 and -4 proteins, which deliver ubiquitinated proteins to the proteasome. Our findings indicate that the largely redundant roles of Rpn10 and Rpn13 in ubiquitin recognition and recruitment of mHR23B and ubiquilin/Plic-1 and -4 are essential for cellular homeostasis in mammals and should provide information for understanding the mechanism of ubiquitin recognition by the 26S proteasome in mammals and for development of therapeutic agents targeting protein degradation.
PMCID: PMC4519129  PMID: 26222436
2.  Checkpoint Activation of an Unconventional DNA Replication Program in Tetrahymena 
PLoS Genetics  2015;11(7):e1005405.
The intra-S phase checkpoint kinase of metazoa and yeast, ATR/MEC1, protects chromosomes from DNA damage and replication stress by phosphorylating subunits of the replicative helicase, MCM2-7. Here we describe an unprecedented ATR-dependent pathway in Tetrahymena thermophila in which the essential pre-replicative complex proteins, Orc1p, Orc2p and Mcm6p are degraded in hydroxyurea-treated S phase cells. Chromosomes undergo global changes during HU-arrest, including phosphorylation of histone H2A.X, deacetylation of histone H3, and an apparent diminution in DNA content that can be blocked by the deacetylase inhibitor sodium butyrate. Most remarkably, the cell cycle rapidly resumes upon hydroxyurea removal, and the entire genome is replicated prior to replenishment of ORC and MCMs. While stalled replication forks are elongated under these conditions, DNA fiber imaging revealed that most replicating molecules are produced by new initiation events. Furthermore, the sole origin in the ribosomal DNA minichromosome is inactive and replication appears to initiate near the rRNA promoter. The collective data raise the possibility that replication initiation occurs by an ORC-independent mechanism during the recovery from HU-induced replication stress.
Author Summary
DNA damage and replication stress activate cell cycle checkpoint responses that protect the integrity of eukaryotic chromosomes. A well-conserved response involves the reversible phosphorylation of the replicative helicase, MCM2-7, which together with the origin recognition complex (ORC) dictates when and where replication initiates in chromosomes. The central role of ORC and MCMs in DNA replication is illustrated by the fact that small changes in abundance of these pre-replicative complex (pre-RC) components are poorly tolerated from yeast to humans. Here we describe an unprecedented replication stress checkpoint response in the early branching eukaryote, Tetrahymena thermophila, that is triggered by the depletion of dNTP pools with hydroxyurea (HU). Instead of transiently phosphorylating MCM subunits, ORC and MCM proteins are physically degraded in HU-treated Tetrahymena. Unexpectedly, upon HU removal the genome is completely and effortlessly replicated prior to replenishment of ORC and MCM components. Using DNA fiber imaging and 2D gel electrophoresis, we show that ORC-dependent mechanisms are bypassed during the recovery phase to produce bidirectional replication forks throughout the genome. Our findings suggest that Tetrahymena enlists an alternative mechanism for replication initiation, and that the underlying process can operate on a genome-wide scale.
PMCID: PMC4517752  PMID: 26218270
3.  CSB-PGBD3 Mutations Cause Premature Ovarian Failure 
PLoS Genetics  2015;11(7):e1005419.
Premature ovarian failure (POF) is a rare, heterogeneous disorder characterized by cessation of menstruation occurring before the age of 40 years. Genetic etiology is responsible for perhaps 25% of cases, but most cases are sporadic and unexplained. In this study, through whole exome sequencing in a non-consanguineous family having four affected members with POF and Sanger sequencing in 432 sporadic cases, we identified three novel mutations in the fusion gene CSB-PGBD3. Subsequently functional studies suggest that mutated CSB-PGBD3 fusion protein was impaired in response to DNA damage, as indicated by delayed or absent recruitment to damaged sites. Our data provide the first evidence that mutations in the CSB-PGBD3 fusion protein can cause human disease, even in the presence of functional CSB, thus potentially explaining conservation of the fusion protein for 43 My since marmoset. The localization of the CSB-PGBD3 fusion protein to UVA-induced nuclear DNA repair foci further suggests that the CSB-PGBD3 fusion protein, like many other proteins that can cause POF, modulates or participates in DNA repair.
Author Summary
Through whole exome sequencing in a non-consanguineous family having four affected members with POF and Sanger sequencing in 432 sporadic cases, we identified three novel mutations in CSB-PGBD3. Our functional studies implicate CSB-PGBD3, a gene which has previously shown association with DNA repair and Cockayne syndrome has a potential role in maintenance of ovarian function. This study also provides evidence for a pivotal role of DNA repair in POF.
PMCID: PMC4517778  PMID: 26218421
4.  EIN3 and ORE1 Accelerate Degreening during Ethylene-Mediated Leaf Senescence by Directly Activating Chlorophyll Catabolic Genes in Arabidopsis 
PLoS Genetics  2015;11(7):e1005399.
Degreening, caused by chlorophyll degradation, is the most obvious symptom of senescing leaves. Chlorophyll degradation can be triggered by endogenous and environmental cues, and ethylene is one of the major inducers. ETHYLENE INSENSITIVE3 (EIN3) is a key transcription factor in the ethylene signaling pathway. It was previously reported that EIN3, miR164, and a NAC (NAM, ATAF, and CUC) transcription factor ORE1/NAC2 constitute a regulatory network mediating leaf senescence. However, how this network regulates chlorophyll degradation at molecular level is not yet elucidated. Here we report a feed-forward regulation of chlorophyll degradation that involves EIN3, ORE1, and chlorophyll catabolic genes (CCGs). Gene expression analysis showed that the induction of three major CCGs, NYE1, NYC1 and PAO, by ethylene was largely repressed in ein3 eil1 double mutant. Dual-luciferase assay revealed that EIN3 significantly enhanced the promoter activity of NYE1, NYC1 and PAO in Arabidopsis protoplasts. Furthermore, Electrophoretic mobility shift assay (EMSA) indicated that EIN3 could directly bind to NYE1, NYC1 and PAO promoters. These results reveal that EIN3 functions as a positive regulator of CCG expression during ethylene-mediated chlorophyll degradation. Interestingly, ORE1, a senescence regulator which is a downstream target of EIN3, could also activate the expression of NYE1, NYC1 and PAO by directly binding to their promoters in EMSA and chromatin immunoprecipitation (ChIP) assays. In addition, EIN3 and ORE1 promoted NYE1 and NYC1 transcriptions in an additive manner. These results suggest that ORE1 is also involved in the direct regulation of CCG transcription. Moreover, ORE1 activated the expression of ACS2, a major ethylene biosynthesis gene, and subsequently promoted ethylene production. Collectively, our work reveals that EIN3, ORE1 and CCGs constitute a coherent feed-forward loop involving in the robust regulation of ethylene-mediated chlorophyll degradation during leaf senescence in Arabidopsis.
Author Summary
Yellowing, caused by chlorophyll degradation, is the most obvious symptom of senescent leaves. Chlorophyll degradation can be triggered by a broad range of endogenous and environmental cues, and ethylene is one of the major inducers. Yet, the molecular regulation of chlorophyll degradation remains largely unknown. Here, we report a feed-forward regulation of ethylene-mediated chlorophyll degradation that involves ETHYLENE INSENSITIVE3 (EIN3), ORE1/NAC2, and major chlorophyll catabolic genes. EIN3, a master positive regulator of ethylene signaling, could directly promote chlorophyll degradation by physically binding to the promoters of three major chlorophyll catabolic genes to activate their expressions. Meanwhile, ORE1, a direct target of EIN3, also activates the expression of the similar set of chlorophyll catabolic genes directly. Moreover, ORE1 activates the expression of a major ethylene biosynthesis gene ACS2 during senescence, and subsequently activates a positive feedback to ethylene synthesis. Our work reveals a feed-forward loop that promotes ethylene-mediated chlorophyll degradation during leaf senescence, advancing our understanding on the molecular mechanism of leaf yellowing.
PMCID: PMC4517869  PMID: 26218222
5.  Hairless Streaks in Cattle Implicate TSR2 in Early Hair Follicle Formation 
PLoS Genetics  2015;11(7):e1005427.
Four related cows showed hairless streaks on various parts of the body with no correlation to the pigmentation pattern. The stripes occurred in a consistent pattern resembling the lines of Blaschko. The non-syndromic hairlessness phenotype observed occurred across three generations of a single family and was compatible with an X-linked mode of inheritance. Linkage analysis and subsequent whole genome sequencing of one affected female identified two perfectly associated non-synonymous sequence variants in the critical interval on bovine chromosome X. Both variants occurred in complete linkage disequilibrium and were absent in more than 3900 controls. An ERCC6L missense mutation was predicted to cause an amino acid substitution of a non-conserved residue. Analysis in mice showed no specific Ercc6l expression pattern related to hair follicle development and therefore ERCC6L was not considered as causative gene. A point mutation at the 5'-splice junction of exon 5 of the TSR2, 20S rRNA accumulation, homolog (S. cerevisiae), gene led to the production of two mutant transcripts, both of which contain a frameshift and generate a premature stop codon predicted to truncate approximately 25% of the protein. Interestingly, in addition to the presence of both physiological TSR2 transcripts, the two mutant transcripts were predominantly detected in the hairless skin of the affected cows. Immunohistochemistry, using an antibody against the N-terminal part of the bovine protein demonstrated the specific expression of the TSR2 protein in the skin and the hair of the affected and the control cows as well as in bovine fetal skin and hair. The RNA hybridization in situ showed that Tsr2 was expressed in pre- and post-natal phases of hair follicle development in mice. Mammalian TSR2 proteins are highly conserved and are known to be broadly expressed, but their precise in vivo functions are poorly understood. Thus, by dissecting a naturally occurring mutation in a domestic animal species, we identified TSR2 as a regulator of hair follicle development.
Author Summary
The identification of causal mutations of rare monogenic disorders provides an insight into the function of single genes. We herein report an example which demonstrates that the bovine species presents an excellent system for identifying these inherited phenotypes. The individual health status of modern dairy cows is well monitored, and emerging disorders are routinely recorded. An Italian breeder of ~500 Pezzata Rossa cattle reported a case of congenital streaked hairlessness. Three additional, closely related cows, showing similar hairless pattern following Blaschko’s lines were subsequently observed. A causative mutation was discovered in a previously uncharacterized rRNA processing gene. Cows possessing a single copy of this TSR2 mutation located on the X chromosome showed a mosaic skin pattern which is very likely due to the skewed inactivation of the X-chromosome, also known as lyonization. The expression of TSR2 was shown in skin and hair of cattle and mice. This study is the first to implicate an essential role for TSR2 during hair follicle development and reflects once more the potential of using rare diseases in cows to gain additional insights into mammalian biology.
PMCID: PMC4512707  PMID: 26203908
6.  Molecular Framework of a Regulatory Circuit Initiating Two-Dimensional Spatial Patterning of Stomatal Lineage 
PLoS Genetics  2015;11(7):e1005374.
Stomata, valves on the plant epidermis, are critical for plant growth and survival, and the presence of stomata impacts the global water and carbon cycle. Although transcription factors and cell-cell signaling components regulating stomatal development have been identified, it remains unclear as to how their regulatory interactions are translated into two-dimensional patterns of stomatal initial cells. Using molecular genetics, imaging, and mathematical simulation, we report a regulatory circuit that initiates the stomatal cell-lineage. The circuit includes a positive feedback loop constituting self-activation of SCREAMs that requires SPEECHLESS. This transcription factor module directly binds to the promoters and activates a secreted signal, EPIDERMAL PATTERNING FACTOR2, and the receptor modifier TOO MANY MOUTHS, while the receptor ERECTA lies outside of this module. This in turn inhibits SPCH, and hence SCRMs, thus constituting a negative feedback loop. Our mathematical model accurately predicts all known stomatal phenotypes with the inclusion of two additional components to the circuit: an EPF2-independent negative-feedback loop and a signal that lies outside of the SPCH•SCRM module. Our work reveals the intricate molecular framework governing self-organizing two-dimensional patterning in the plant epidermis.
Author Summary
Generation of self-organized, functional tissue patterns is critical for development and regeneration in multicellular organisms. Small valves on the epidermis of land plants, called stomata, mediate gas-exchange while minimizing water loss. Density and spacing of stomata are regulated by transcription factors that drive differentiation as well as by cell-cell signaling components that regulate entry and spacing of stomatal lineage cells. To unravel how interaction of these components translates into two-dimensional patterning of stomata, we have taken an integrative approach employing molecular genetics, imaging, and mathematical modeling. In this paper we have identified a regulatory circuit controlling the initiation of the stomatal cell lineage. The key elements of the circuit are a positive feedback loop constituting self-activation of the transcription factors SCREAM / SCREAM2 (SCRMs) that requires SPEECHLESS (SPCH), and a negative feedback loop involving the signaling ligand EPF2, the receptor modifier TOO MANY MOUTHS, and the SPCH•SCRMs module. The receptor ERECTA, on the other hand, lies outside of the regulatory loop. Our mathematical modeling recapitulated all known stomatal phenotypes with the addition of two regulatory nodes. This work highlights the molecular framework of a self-organizing patterning system in plants.
PMCID: PMC4512730  PMID: 26203655
7.  Allelic Spectra of Risk SNPs Are Different for Environment/Lifestyle Dependent versus Independent Diseases 
PLoS Genetics  2015;11(7):e1005371.
Genome-wide association studies (GWAS) have generated sufficient data to assess the role of selection in shaping allelic diversity of disease-associated SNPs. Negative selection against disease risk variants is expected to reduce their frequencies making them overrepresented in the group of minor (<50%) alleles. Indeed, we found that the overall proportion of risk alleles was higher among alleles with frequency <50% (minor alleles) compared to that in the group of major alleles. We hypothesized that negative selection may have different effects on environment (or lifestyle)-dependent versus environment (or lifestyle)-independent diseases. We used an environment/lifestyle index (ELI) to assess influence of environmental/lifestyle factors on disease etiology. ELI was defined as the number of publications mentioning “environment” or “lifestyle” AND disease per 1,000 disease-mentioning publications. We found that the frequency distributions of the risk alleles for the diseases with strong environmental/lifestyle components follow the distribution expected under a selectively neutral model, while frequency distributions of the risk alleles for the diseases with weak environmental/lifestyle influences is shifted to the lower values indicating effects of negative selection. We hypothesized that previously selectively neutral variants become risk alleles when environment changes. The hypothesis of ancestrally neutral, currently disadvantageous risk-associated alleles predicts that the distribution of risk alleles for the environment/lifestyle dependent diseases will follow a neutral model since natural selection has not had enough time to influence allele frequencies. The results of our analysis suggest that prediction of SNP functionality based on the level of evolutionary conservation may not be useful for SNPs associated with environment/lifestyle dependent diseases.
Author Summary
We reviewed several thousand genome wide association studies that were conducted to identify genetic variants influencing risk of human diseases. We tested the hypothesis that single nucleotide polymorphisms (SNPs) that influence disease risk undergo positive or negative selection more frequently than an average SNP in the human genome. We found no evidence for excess of positive selection on disease-associated SNPs. At the same time we found that alleles associated with a higher disease risk undergo negative selection. We also demonstrated that risk alleles for diseases with strong influence of environment/lifestyle factors (e.g. Type II diabetes) show little evidence of negative selection, while risk alleles for diseases with weak influence of environment/lifestyle factors (e.g. Pathological myopia) show clear signs of negative selection. The approach used in this study can be used to estimate the number of genetic variants in the human genome influencing risk of human diseases.
PMCID: PMC4511800  PMID: 26201053
8.  A Novel Locus Harbouring a Functional CD164 Nonsense Mutation Identified in a Large Danish Family with Nonsyndromic Hearing Impairment 
PLoS Genetics  2015;11(7):e1005386.
Nonsyndromic hearing impairment (NSHI) is a highly heterogeneous condition with more than eighty known causative genes. However, in the clinical setting, a large number of NSHI families have unexplained etiology, suggesting that there are many more genes to be identified. In this study we used SNP-based linkage analysis and follow up microsatellite markers to identify a novel locus (DFNA66) on chromosome 6q15-21 (LOD 5.1) in a large Danish family with dominantly inherited NSHI. By locus specific capture and next-generation sequencing, we identified a c.574C>T heterozygous nonsense mutation (p.R192*) in CD164. This gene encodes a 197 amino acid transmembrane sialomucin (known as endolyn, MUC-24 or CD164), which is widely expressed and involved in cell adhesion and migration. The mutation segregated with the phenotype and was absent in 1200 Danish control individuals and in databases with whole-genome and exome sequence data. The predicted effect of the mutation was a truncation of the last six C-terminal residues of the cytoplasmic tail of CD164, including a highly conserved canonical sorting motif (YXXФ). In whole blood from an affected individual, we found by RT-PCR both the wild-type and the mutated transcript suggesting that the mutant transcript escapes nonsense mediated decay. Functional studies in HEK cells demonstrated that the truncated protein was almost completely retained on the plasma cell membrane in contrast to the wild-type protein, which targeted primarily to the endo-lysosomal compartments, implicating failed endocytosis as a possible disease mechanism. In the mouse ear, we found CD164 expressed in the inner and outer hair cells of the organ of Corti, as well as in other locations in the cochlear duct. In conclusion, we have identified a new DFNA locus located on chromosome 6q15-21 and implicated CD164 as a novel gene for hearing impairment.
Author Summary
It is known that hearing impairment running in families can be caused by mutations in more than eighty different genes. However, there are still families where the responsible gene is unknown. By studying a large Danish family with dominant inherited hearing impairment, we found that the disorder cosegregates with genetic markers on chromosome 6, suggesting that the responsible mutation lies within this chromosomal region. By sequencing this genetic locus, we discovered a mutation in the CD164 gene that is passed on to all the affected individuals. In the mouse ear, we demonstrated that the CD164 protein is expressed in hair cells and other sites known to be important for correct hearing. The identified mutation is predicted to result in shortening of the protein, leading to loss of an evolutionary conserved sequence important for cellular trafficking of CD164. Using cell lines, we show that the truncated protein is trapped on the cell surface while the normal protein is internalized. This finding is important because it implicates for the first time a role for CD164 in the complex physiological processes of hearing and suggests that failed endocytosis may be a possible disease mechanism for some types of hearing impairment.
PMCID: PMC4510537  PMID: 26197441
9.  Large-Scale Phenomics Identifies Primary and Fine-Tuning Roles for CRKs in Responses Related to Oxidative Stress 
PLoS Genetics  2015;11(7):e1005373.
Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.
Author Summary
Receptor-like kinases (RLKs) are important regulators in signal transduction in plants. However, the large number of RLKs and their high sequence similarity has hampered the analysis of RLKs. One of the largest subgroups of RLKs, the cysteine-rich receptor-like kinases (CRKs), has been suggested to be involved in mediating the effects of reactive oxygen species (ROS). While ROS are recognized as important signalling elements with a large variety of roles in plants, their ligands and achievement of signalling specificity remain unknown. Using insertion mutants we analysed the roles of CRKs in plant development and stress responses and show that CRKs have important roles as mediators of signalling specificity during regulation of stomatal aperture. Our study shows that, despite their large number and high sequence conservation, individual CRKs have intriguingly distinct functions in different aspects of plant life. This makes the CRKs promising candidates for future studies of their biochemical function.
PMCID: PMC4511522  PMID: 26197346
11.  Tempo and Mode of Transposable Element Activity in Drosophila 
PLoS Genetics  2015;11(7):e1005406.
The evolutionary dynamics of transposable element (TE) insertions have been of continued interest since TE activity has important implications for genome evolution and adaptation. Here, we infer the transposition dynamics of TEs by comparing their abundance in natural D. melanogaster and D. simulans populations. Sequencing pools of more than 550 South African flies to at least 320-fold coverage, we determined the genome wide TE insertion frequencies in both species. We suggest that the predominance of low frequency insertions in the two species (>80% of the insertions have a frequency <0.2) is probably due to a high activity of more than 58 families in both species. We provide evidence for 50% of the TE families having temporally heterogenous transposition rates with different TE families being affected in the two species. While in D. melanogaster retrotransposons were more active, DNA transposons showed higher activity levels in D. simulans. Moreover, we suggest that LTR insertions are mostly of recent origin in both species, while DNA and non-LTR insertions are older and more frequently vertically transmitted since the split of D. melanogaster and D. simulans. We propose that the high TE activity is of recent origin in both species and a consequence of the demographic history, with habitat expansion triggering a period of rapid evolution.
Author Summary
Transposable elements (TE) are stretches of DNA that propagate autonomously within genomes, but it is not clear whether TEs are moving at a constant rate or if TE activity is variable. Determining the genome-wide TE content of two closely related Drosophila species, we show that transposition rate heterogeneity is abundant. Since TE insertions are frequently associated with a selective advantage, we suggest that the observed high TE activity may have served a central role facilitating the adaptation of the two species to their novel environments after the recent out of Africa habitat expansion.
PMCID: PMC4505896  PMID: 26186437
12.  Correction: A Genomic Portrait of Haplotype Diversity and Signatures of Selection in Indigenous Southern African Populations 
PLoS Genetics  2015;11(7):e1005363.
PMCID: PMC4506067  PMID: 26186335
13.  A Role for Macro-ER-Phagy in ER Quality Control 
PLoS Genetics  2015;11(7):e1005390.
The endoplasmic-reticulum quality-control (ERQC) system shuttles misfolded proteins for degradation by the proteasome through the well-defined ER-associated degradation (ERAD) pathway. In contrast, very little is known about the role of autophagy in ERQC. Macro-autophagy, a collection of pathways that deliver proteins through autophagosomes (APs) for degradation in the lysosome (vacuole in yeast), is mediated by autophagy-specific proteins, Atgs, and regulated by Ypt/Rab GTPases. Until recently, the term ER-phagy was used to describe degradation of ER membrane and proteins in the lysosome under stress: either ER stress induced by drugs or whole-cell stress induced by starvation. These two types of stresses induce micro-ER-phagy, which does not use autophagic organelles and machinery, and non-selective autophagy. Here, we characterize the macro-ER-phagy pathway and uncover its role in ERQC. This pathway delivers 20–50% of certain ER-resident membrane proteins to the vacuole and is further induced to >90% by overexpression of a single integral-membrane protein. Even though such overexpression in cells defective in macro-ER-phagy induces the unfolded-protein response (UPR), UPR is not needed for macro-ER-phagy. We show that macro-ER-phagy is dependent on Atgs and Ypt GTPases and its cargo passes through APs. Moreover, for the first time the role of Atg9, the only integral-membrane core Atg, is uncoupled from that of other core Atgs. Finally, three sequential steps of this pathway are delineated: Atg9-dependent exit from the ER en route to autophagy, Ypt1- and core Atgs-mediated pre-autophagsomal-structure organization, and Ypt51-mediated delivery of APs to the vacuole.
Author Summary
ER-quality control (ERQC) ensures delivery of “native” proteins through the secretory pathway. Currently, ER-associated degradation (ERAD), which delivers misfolded proteins for degradation by the proteasome, is considered a major ERQC pathway, with autophagy as its backup. Until now, the role of autophagy, which shuttles cellular components for degradation in the lysosome through autophagosomes (APs), in ERQC was ill defined. Recently, the process of ER degradation induced by ER stress was defined as micro-ER-phagy, which does not require autophagic machinery and does not pass through APs. Here, we characterize the macro-ER-phagy pathway, which delivers excess membrane proteins for degradation in the lysosome, as a novel ERQC pathway. This pathway functions in the absence of cellular or ER stress and can be further induced by overexpression of a single integral-membrane protein. Unlike the micro-ER-phagy pathway, the marco-ER-phagy pathway requires core autophagy-specific proteins, Atgs, and Ypt/Rab GTPases. In addition, for the first time, the function of the only membrane core Atg, Atg9, was uncoupled from that of the other core Atgs. Whereas Atg9 plays a role in the assembly of ER-to-autophagy membranes (ERAM), other core Atgs and Ypt1 assemble the Atg-protein complex on ERAM to form the pre-autophagosomal structure.
PMCID: PMC4504476  PMID: 26181331
14.  Irrepressible: An Interview with Mark Ptashne 
PLoS Genetics  2015;11(7):e1005351.
PMCID: PMC4504494  PMID: 26181810
15.  Pyrimidine Pool Disequilibrium Induced by a Cytidine Deaminase Deficiency Inhibits PARP-1 Activity, Leading to the Under Replication of DNA 
PLoS Genetics  2015;11(7):e1005384.
Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA) deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation: the intracellular accumulation of dCTP inhibits PARP-1 activity. CDA deficiency results in incomplete DNA replication when cells enter mitosis, leading to the formation of ultrafine anaphase bridges between sister-chromatids at “difficult-to-replicate” sites such as centromeres and fragile sites. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. The stimulation of PARP-1 activity in CDA-deficient cells restores replication and, thus, chromosome segregation. Moreover, increasing intracellular dCTP levels generates under-replication-induced sister-chromatid bridges as efficiently as PARP-1 knockdown. These results have direct implications for Bloom syndrome (BS), a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3’-5’ DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our study describes previously unknown pathological consequences of the distortion of dNTP pools and reveals an unexpected role for PARP-1 in preventing DNA under-replication and chromosome segregation defects.
Author Summary
The maintenance of genome stability is essential for the accurate transmission of genetic information, to ensure the successful duplication of chromosomes and their even segregation during mitosis. Errors occurring during DNA replication may affect both the accuracy of chromosome duplication and the balance of chromosome segregation during mitosis. Accurate DNA replication is strongly dependent on deoxynucleotides (dNTP) concentrations. Distortions in dNTP pool affect the rate of replication fork progression and compromise genetic stability. In the work presented here, we identified a novel mechanism by which dNTP pool disequilibrium compromises the completion of DNA replication and thus chromosome segregation, independently of the rate of fork progression. This mechanism involves the intracellular accumulation of deoxycytidine due to cytidine deaminase (CDA) deficiency, inhibiting PARP-1 activity. These results have direct implications for Bloom syndrome (BS), a rare genetic disease combining susceptibility to cancer and genomic instability. BS cells also have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition. These data highlight new pathological consequences of the distortion of dNTP pools and reveal an unexpected role for PARP-1 in preventing the accumulation of excessive amounts of unreplicated DNA and chromosome segregation defects.
PMCID: PMC4504519  PMID: 26181065
16.  LINE-1 Retroelements Get ZAPped! 
PLoS Genetics  2015;11(7):e1005364.
PMCID: PMC4504689  PMID: 26182081
17.  Arabidopsis PCH2 Mediates Meiotic Chromosome Remodeling and Maturation of Crossovers 
PLoS Genetics  2015;11(7):e1005372.
Meiotic chromosomes are organized into linear looped chromatin arrays by a protein axis localized along the loop-bases. Programmed remodelling of the axis occurs during prophase I of meiosis. Structured illumination microscopy (SIM) has revealed dynamic changes in the chromosome axis in Arabidopsis thaliana and Brassica oleracea. We show that the axis associated protein ASY1 is depleted during zygotene concomitant with synaptonemal complex (SC) formation. Study of an Atpch2 mutant demonstrates this requires the conserved AAA+ ATPase, PCH2, which localizes to the sites of axis remodelling. Loss of PCH2 leads to a failure to deplete ASY1 from the axes and compromizes SC polymerisation. Immunolocalization of recombination proteins in Atpch2 indicates that recombination initiation and CO designation during early prophase I occur normally. Evidence suggests that CO interference is initially functional in the mutant but there is a defect in CO maturation following designation. This leads to a reduction in COs and a failure to form COs between some homologous chromosome pairs leading to univalent chromosomes at metaphase I. Genetic analysis reveals that CO distribution is also affected in some chromosome regions. Together these data indicate that the axis remodelling defect in Atpch2 disrupts normal patterned formation of COs.
Author Summary
In the reproductive cells of many eukaryotes, a process called meiosis generates haploid gametes. During meiosis, homologous parental chromosomes (homologs) recombine forming crossovers (CO) that provide genetic variation. CO formation generates physical links called chiasmata, which are essential for accurate homolog segregation. CO control designates a sub-set of recombination precursors that will mature to form at least one chiasma between each homolog pair. Recombination is accompanied by extensive chromosome reorganization. Formation of a proteinaceous axis organizes the pairs of sister chromatids of each homolog into conjoined linear looped chromatin arrays. Pairs of homologs then align and synapse becoming closely associated along their length by a protein structure, the synaptonemal complex (SC). The SC is disassembled at the end of prophase I and recombination is completed. We have investigated the link between recombination and chromosome remodelling by analysing the role of a protein, PCH2, which we show is required for remodelling of the chromosome axis during SC formation. In wild type, immunolocalization reveals depletion of the axis-associated signal of the axis component, ASY1, along synapsed regions of the chromosomes. In the absence of PCH2, the ASY1 signal is not depleted from the chromosome axis and the SC does not form normally. Although this defect in chromosome remodelling has no obvious effect on CO designation, CO maturation is perturbed such that the formation of at least one CO per homolog pair no longer occurs.
PMCID: PMC4504720  PMID: 26182244
18.  TFIIS-Dependent Non-coding Transcription Regulates Developmental Genome Rearrangements 
PLoS Genetics  2015;11(7):e1005383.
Because of their nuclear dimorphism, ciliates provide a unique opportunity to study the role of non-coding RNAs (ncRNAs) in the communication between germline and somatic lineages. In these unicellular eukaryotes, a new somatic nucleus develops at each sexual cycle from a copy of the zygotic (germline) nucleus, while the old somatic nucleus degenerates. In the ciliate Paramecium tetraurelia, the genome is massively rearranged during this process through the reproducible elimination of repeated sequences and the precise excision of over 45,000 short, single-copy Internal Eliminated Sequences (IESs). Different types of ncRNAs resulting from genome-wide transcription were shown to be involved in the epigenetic regulation of genome rearrangements. To understand how ncRNAs are produced from the entire genome, we have focused on a homolog of the TFIIS elongation factor, which regulates RNA polymerase II transcriptional pausing. Six TFIIS-paralogs, representing four distinct families, can be found in P. tetraurelia genome. Using RNA interference, we showed that TFIIS4, which encodes a development-specific TFIIS protein, is essential for the formation of a functional somatic genome. Molecular analyses and high-throughput DNA sequencing upon TFIIS4 RNAi demonstrated that TFIIS4 is involved in all kinds of genome rearrangements, including excision of ~48% of IESs. Localization of a GFP-TFIIS4 fusion revealed that TFIIS4 appears specifically in the new somatic nucleus at an early developmental stage, before IES excision. RT-PCR experiments showed that TFIIS4 is necessary for the synthesis of IES-containing non-coding transcripts. We propose that these IES+ transcripts originate from the developing somatic nucleus and serve as pairing substrates for germline-specific short RNAs that target elimination of their homologous sequences. Our study, therefore, connects the onset of zygotic non coding transcription to the control of genome plasticity in Paramecium, and establishes for the first time a specific role of TFIIS in non-coding transcription in eukaryotes.
Author Summary
Paramecium tetraurelia provides an excellent model for studying the mechanisms involved in the production of non-coding transcripts and their mode of action. Different types of non-coding RNAs (ncRNAs) were shown to be implicated in the programmed DNA elimination process that occurs in this organism. At each sexual cycle, during development of the somatic nucleus from the germline nucleus, the genome is massively rearranged through the reproducible elimination of germline-specific sequences including thousands of short, single copy, non-coding Internal Eliminated Sequences (IES). Here, we demonstrate, using RNA interference, that the TFIIS4 gene encoding a development-specific homolog of RNA polymerase II elongation factor TFIIS, is indispensable for ncRNA synthesis in the new somatic nucleus. TFIIS4 depletion impairs the assembly of a functional somatic genome and affects excision of a large fraction of IESs, which leads to strong lethality in the sexual progeny. We propose that TFIIS4-dependent ncRNAs provide an important component of the molecular machinery that is responsible for developmental genome remodeling in Paramecium.
PMCID: PMC4503560  PMID: 26177014
19.  The SMC Loader Scc2 Promotes ncRNA Biogenesis and Translational Fidelity 
PLoS Genetics  2015;11(7):e1005308.
The Scc2-Scc4 complex is essential for loading the cohesin complex onto DNA. Cohesin has important roles in chromosome segregation, DSB repair, and chromosome condensation. Here we report that Scc2 is important for gene expression in budding yeast. Scc2 and the transcriptional regulator Paf1 collaborate to promote the production of Box H/ACA snoRNAs which guide pseudouridylation of RNAs including ribosomal RNA. Mutation of SCC2 was associated with defects in the production of ribosomal RNA, ribosome assembly, and splicing. While the scc2 mutant does not have a general defect in protein synthesis, it shows increased frameshifting and reduced cap-independent translation. These findings suggest Scc2 normally promotes a gene expression program that supports translational fidelity. We hypothesize that translational dysfunction may contribute to the human disorder Cornelia de Lange syndrome, which is caused by mutations in NIPBL, the human ortholog of SCC2.
Author Summary
The structure of chromosomes contributes to the production of RNAs. Chromosome structure is maintained in part by an evolutionarily conserved group of proteins known as structural maintenance of chromosomes proteins. These proteins are loaded onto chromosomes by a second evolutionarily conserved protein complex known as Scc2-Scc4. The Scc2 component is often mutated in a human developmental disorder known as Cornelia de Lange syndrome. We find that Scc2 in budding yeast is important for the production of a group of RNAs known as snoRNAs. These RNAs play a critical role in ribosome production; without them the fidelity of ribosomes suffers. Ribosomes are the molecular machines that translate mRNAs to proteins. Scc2 is also important for the production of the RNA components of ribosomes. Our findings suggest Scc2 normally promotes the production of RNAs that support translational fidelity. We hypothesize that defects in protein synthesis may contribute to Cornelia de Lange syndrome.
PMCID: PMC4503661  PMID: 26176819
20.  Molecular Clock of Neutral Mutations in a Fitness-Increasing Evolutionary Process 
PLoS Genetics  2015;11(7):e1005392.
The molecular clock of neutral mutations, which represents linear mutation fixation over generations, is theoretically explained by genetic drift in fitness-steady evolution or hitchhiking in adaptive evolution. The present study is the first experimental demonstration for the molecular clock of neutral mutations in a fitness-increasing evolutionary process. The dynamics of genome mutation fixation in the thermal adaptive evolution of Escherichia coli were evaluated in a prolonged evolution experiment in duplicated lineages. The cells from the continuously fitness-increasing evolutionary process were subjected to genome sequencing and analyzed at both the population and single-colony levels. Although the dynamics of genome mutation fixation were complicated by the combination of the stochastic appearance of adaptive mutations and clonal interference, the mutation fixation in the population was simply linear over generations. Each genome in the population accumulated 1.6 synonymous and 3.1 non-synonymous neutral mutations, on average, by the spontaneous mutation accumulation rate, while only a single genome in the population occasionally acquired an adaptive mutation. The neutral mutations that preexisted on the single genome hitchhiked on the domination of the adaptive mutation. The successive fixation processes of the 128 mutations demonstrated that hitchhiking and not genetic drift were responsible for the coincidence of the spontaneous mutation accumulation rate in the genome with the fixation rate of neutral mutations in the population. The molecular clock of neutral mutations to the fitness-increasing evolution suggests that the numerous neutral mutations observed in molecular phylogenetic trees may not always have been fixed in fitness-steady evolution but in adaptive evolution.
Author Summary
Mutations that have little influence on biological function are referred to as neutral mutations and frequently appear in molecular phylogenetic analyses. The fixation of neutral mutations in populations has been attributed to genetic drift in fitness-steady evolutionary processes or hitchhiking in adaptive evolution. We examined the fitness-increasing evolution of Escherichia coli for thermal adaptation to observe the fixation dynamics of genome-wide mutations. In the adaptive evolution, all genomes in the population equally accumulated neutral mutations by replication errors. The infrequent occurrence of an adaptive mutation on one of the genomes by chance resulted in the fixation of the neutral mutations that had pre-accumulated in the same genome by hitchhiking. Via successive hitchhiking events, the neutral mutations were fixed in the population linearly over generations at the same rate as the spontaneous mutation accumulation rate in the genome. The molecular clock of neutral mutations thus functions even in adaptive evolution. The evolutionary period characterized by the accumulation of numerous neutral mutations observed in molecular phylogenetic trees may not be specific to neutral evolution but may occur in adaptive evolution as well.
PMCID: PMC4503671  PMID: 26177190
21.  Emergence, Retention and Selection: A Trilogy of Origination for Functional De Novo Proteins from Ancestral LncRNAs in Primates 
PLoS Genetics  2015;11(7):e1005391.
While some human-specific protein-coding genes have been proposed to originate from ancestral lncRNAs, the transition process remains poorly understood. Here we identified 64 hominoid-specific de novo genes and report a mechanism for the origination of functional de novo proteins from ancestral lncRNAs with precise splicing structures and specific tissue expression profiles. Whole-genome sequencing of dozens of rhesus macaque animals revealed that these lncRNAs are generally not more selectively constrained than other lncRNA loci. The existence of these newly-originated de novo proteins is also not beyond anticipation under neutral expectation, as they generally have longer theoretical lifespan than their current age, due to their GC-rich sequence property enabling stable ORFs with lower chance of non-sense mutations. Interestingly, although the emergence and retention of these de novo genes are likely driven by neutral forces, population genetics study in 67 human individuals and 82 macaque animals revealed signatures of purifying selection on these genes specifically in human population, indicating a proportion of these newly-originated proteins are already functional in human. We thus propose a mechanism for creation of functional de novo proteins from ancestral lncRNAs during the primate evolution, which may contribute to human-specific genetic novelties by taking advantage of existed genomic contexts.
Author Summary
Although gene duplication has been believed as a predominant mechanism for creating new genes, recent reports suggested that new proteins could evolve “de novo” from non-coding DNA regions. These de novo genes are also named as “motherless” genes due to their lack of ancestral proteins as precursors, while recently we and others found that lncRNAs may represent an intermediate stage of their origination. To further elucidate this lncRNA-protein transition process, here we identified 64 hominoid-specific de novo genes and report a new mechanism for the origination of functional de novo proteins from ancestral non-coding transcripts: These non-coding “precursors” are generally not more selectively constrained than other lncRNA loci; and the existence of these de novo proteins is not beyond anticipation under neutral expectation; however, population genetics study in 67 human individuals and 82 macaque animals revealed signatures of purifying selection on these genes specifically in human population, indicating a proportion of these newly-originated proteins are already functional in human. We thus propose a mechanism for creation of functional de novo proteins from ancestral lncRNAs during the primate evolution.
PMCID: PMC4503675  PMID: 26177073
22.  Genome-Wide Reprogramming of Transcript Architecture by Temperature Specifies the Developmental States of the Human Pathogen Histoplasma 
PLoS Genetics  2015;11(7):e1005395.
Eukaryotic cells integrate layers of gene regulation to coordinate complex cellular processes; however, mechanisms of post-transcriptional gene regulation remain poorly studied. The human fungal pathogen Histoplasma capsulatum (Hc) responds to environmental or host temperature by initiating unique transcriptional programs to specify multicellular (hyphae) or unicellular (yeast) developmental states that function in infectivity or pathogenesis, respectively. Here we used recent advances in next-generation sequencing to uncover a novel re-programming of transcript length between Hc developmental cell types. We found that ~2% percent of Hc transcripts exhibit 5’ leader sequences that differ markedly in length between morphogenetic states. Ribosome density and mRNA abundance measurements of differential leader transcripts revealed nuanced transcriptional and translational regulation. One such class of regulated longer leader transcripts exhibited tight transcriptional and translational repression. Further examination of these dually repressed genes revealed that some control Hc morphology and that their strict regulation is necessary for the pathogen to make appropriate developmental decisions in response to temperature.
Author Summary
Eukaryotic cells alter their developmental programs in response to environmental signals. Histoplasma capsulatum (Hc), a ubiquitous fungal pathogen of humans, establishes unique transcriptional programs to specify growth in either a multicellular hyphal form or unicellular yeast form in response to temperature. Since hyphae and yeast are specialized to function in infectivity or pathogenesis, respectively, Hc provides a clinically relevant system in which to query eukaryotic regulatory processes. Here we used next-generation sequencing approaches to annotate the transcriptomes of four distinct Hc strains in response to temperature. We found that a fraction of Hc transcripts have differential transcript architecture in hyphae and yeast, exhibiting 5’ leader sequences that differ markedly in length between morphogenetic states. To begin to understand the effect of these differential leader sequences on expression, we performed the first ribosome density and mRNA abundance measurements in Hc, thereby uncovering transcriptional and translational control that contribute to cell-type regulation.
PMCID: PMC4503680  PMID: 26177267
23.  Inactivation of Retinoblastoma Protein (Rb1) in the Oocyte: Evidence That Dysregulated Follicle Growth Drives Ovarian Teratoma Formation in Mice 
PLoS Genetics  2015;11(7):e1005355.
The origin of most ovarian tumors is undefined. Here, we report development of a novel mouse model in which conditional inactivation of the tumor suppressor gene Rb1 in oocytes leads to the formation of ovarian teratomas (OTs). While parthenogenetically activated ooctyes are a known source of OT in some mutant mouse models, enhanced parthenogenetic propensity in vitro was not observed for Rb1-deficient oocytes. Further analyses revealed that follicle recruitment and growth is disrupted in ovaries of mice with conditional inactivation of Rb1, leading to abnormal accumulation of secondary/preantral follicles. These findings underpin the concept that miscues between the germ cell and somatic compartments cause premature oocyte activation and the formation of OTs. Furthermore, these results suggest that defects in folliculogenesis and a permissive genetic background are sufficient to drive OT development, even in the absence of enhanced parthenogenetic activation. Thus, we have discovered a novel role of Rb1 in regulating the entry of primordial oocytes into the pool of growing follicles and signaling between the oocyte and granulosa cells during the protracted process of oocyte growth. Our findings, coupled with data from studies of other OT models, suggest that defects in the coordinated regulation between growth of the oocyte and somatic components in follicles are an underlying cause of OT formation.
Author Summary
Ovarian teratomas (OTs) are the most frequent germ cell tumors in adult women, but their origin and molecular etiology remains poorly defined. We found that conditional deletion of the tumor suppressor Rb1 in the oocyte leads to OT development in young adult female mice. Further analysis revealed disturbances in both recruitment and growth of follicles. Although oocytes from mutants did not exhibit an enhanced propensity for parthenogenetic activation–a proposed source of OTs–premature meiotic resumption was evident in oocytes of immature follicles. These findings, together with data from previous studies, suggest that a defect in oocyte-somatic cell communication leading to an uncoupling of coordinated growth and ultimately impaired sustainment of meiotic arrest is sufficient to drive OT development.
PMCID: PMC4503754  PMID: 26176933
24.  Identification of a Novel Regulatory Mechanism of Nutrient Transport Controlled by TORC1-Npr1-Amu1/Par32 
PLoS Genetics  2015;11(7):e1005382.
Fine-tuning the plasma-membrane permeability to essential nutrients is fundamental to cell growth optimization. Nutritional signals including nitrogen availability are integrated by the TORC1 complex which notably regulates arrestin-mediated endocytosis of amino-acid transporters. Ammonium is a ubiquitous compound playing key physiological roles in many, if not all, organisms. In yeast, it is a preferred nitrogen source transported by three Mep proteins which are orthologues of the mammalian Rhesus factors. By combining genetic, kinetic, biochemical and cell microscopy analyses, the current study reveals a novel mechanism enabling TORC1 to regulate the inherent activity of ammonium transport proteins, independently of arrestin-mediated endocytosis, identifying the still functional orphan Amu1/Par32 as a selective regulator intermediate. We show that, under poor nitrogen supply, the TORC1 effector kinase' Npr1' promotes phosphorylation of Amu1/Par32 which appears mainly cytosolic while ammonium transport proteins are active. Upon preferred nitrogen supplementation, like glutamine or ammonium addition, TORC1 upregulation enables Npr1 inhibition and Amu1/Par32 dephosphorylation. In these conditions, as in Npr1-lacking cells, hypophosphorylated Amu1/Par32 accumulates at the cell surface and mediates the inhibition of specific ammonium transport proteins. We show that the integrity of a conserved repeated motif of Amu1/Par32 is required for the interaction with these transport proteins. This study underscores the diversity of strategies enabling TORC1-Npr1 to selectively monitor cell permeability to nutrients by discriminating between transporters to be degraded or transiently inactivated and kept stable at the plasma membrane. This study further identifies the function of Amu1/Par32 in acute control of ammonium transport in response to variations in nitrogen availability.
Author Summary
Cells have evolved a variety of mechanisms to control the permeability of the plasma membrane to face environmental perturbations. Transcriptional regulation, endocytosis, gating and activity control of channels and transporters enable global or specific responses to stressful conditions and focused variations in nutrient availability. Emerging data from the yeast model reveal that the conserved TORC1 pathway regulates arrestin-mediated endocytosis of amino-acid transporters. We provide genetic and biochemical evidence for a novel mechanism enabling TORC1 to regulate the inherent activity of transport proteins via the Amu1/Par32 regulator intermediate. This low complexity protein mediates inhibition of specific proteins dedicated to the transport of ammonium, a favored nitrogen source, underscoring that TORC1 selects transporters to be degraded or transiently inactivated and preserved at the cell surface according to the environmental situation. The here-revealed mechanism of transport inhibition by Amu/Par32 is reminiscent to the inhibition of prokaryotic ammonium transport proteins mediated by PII-type proteins, key nitrogen signal transducers widespread in bacteria and Archaea.
PMCID: PMC4501750  PMID: 26172854
25.  A Genome Scan for Genes Underlying Microgeographic-Scale Local Adaptation in a Wild Arabidopsis Species 
PLoS Genetics  2015;11(7):e1005361.
Adaptive divergence at the microgeographic scale has been generally disregarded because high gene flow is expected to disrupt local adaptation. Yet, growing number of studies reporting adaptive divergence at a small spatial scale highlight the importance of this process in evolutionary biology. To investigate the genetic basis of microgeographic local adaptation, we conducted a genome-wide scan among sets of continuously distributed populations of Arabidopsis halleri subsp. gemmifera that show altitudinal phenotypic divergence despite gene flow. Genomic comparisons were independently conducted in two distinct mountains where similar highland ecotypes are observed, presumably as a result of convergent evolution. Here, we established a de novo reference genome and employed an individual-based resequencing for a total of 56 individuals. Among 527,225 reliable SNP loci, we focused on those showing a unidirectional allele frequency shift across altitudes. Statistical tests on the screened genes showed that our microgeographic population genomic approach successfully retrieve genes with functional annotations that are in line with the known phenotypic and environmental differences between altitudes. Furthermore, comparison between the two distinct mountains enabled us to screen out those genes that are neutral or adaptive only in either mountain, and identify the genes involved in the convergent evolution. Our study demonstrates that the genomic comparison among a set of genetically connected populations, instead of the commonly-performed comparison between two isolated populations, can also offer an effective screening for the genetic basis of local adaptation.
Author Summary
Where does a local adaptation take place? In general, an adaptive divergence is predicted to occur between isolated populations because gene flow will erode and prevent the divergence. Therefore, previous genome-wide studies that aim to find the adaptive genes have compared populations that are usually tens of hundreds of kilometers apart. However, because nearby populations are likely to be genetically connected or connected until recently, most of the genome should be undifferentiated, leaving the genetic footprints of natural selections more pronounced. Thus, if an adaptive divergence is to be found within a small spatial scale, such case may favor the screening for the adaptive genes. Here, we took advantage of a unique small-scale local adaptation in Arabidopsis halleri subsp. gemmifera, where similar phenotypic differentiation is found across an altitudinal cline on two distinct mountains. By scanning the genome with a focus on the presence of unidirectional allele frequency shift along the altitudes, we successfully obtained genes with functions that were in line with the known phenotypic and environmental difference between altitudes. Our approach is applicable to any species that show microgeographic divergence and should help understand the genetic basis of small-scale evolution.
PMCID: PMC4501782  PMID: 26172569

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