Congenic Mapping and Allele-Specific Alteration Analysis of Stmm1 Locus Conferring Resistance to Early-Stage Chemically Induced Skin Papillomas

Genome-wide association studies have revealed that many low-penetrance cancer susceptibility loci are located throughout the genome; however, a very limited number of genes have been identified so far. Using a forward genetics approach to map such loci in a mouse skin cancer model, we previously identified strong genetic loci conferring resistance to early-stage chemically induced skin papillomas on chromosome 7 with a large number of [(FVB/N×MSM/Ms)×FVB/N] F1 backcross mice. In this report, we describe a combination of congenic mapping and allele-specific alteration analysis of the loci on chromosome 7. We used linkage analysis and congenic mouse strains to refine the location of Stmm1 (Skin tumor modifier of MSM 1) locus within a genetic interval of about 3 cM on proximal chromosome 7. In addition, we used patterns of allele-specific imbalances in tumors from F1 backcross and N10 congenic mice to narrow down further the region of Stmm1 locus to a physical distance of about 5.4 Mb. To gain the insight into the function of Stmm1 locus, we carried out a long term BrdU labelling experiments with congenic mice containing Stmm1 locus. Interestingly, we observed a decrease of BrdU-LRCs (Label Retaining Cells) in a congenic strain heterozygous or homozygous for MSM allele of Stmm1. These results suggest that Stmm1 responsible genes may have an influence on papillomagenesis in the two-stage skin carcinogenesis by regulating epidermal quiescent stem cells.     

Identification of Stmm3 locus Conferring Resistance to Late-stage Chemically Induced Skin Papillomas on Mouse Chromosome 4 by Congenic Mappingand Allele-specific Alteration Analysis

Genome-wide association studies have revealed that many low-penetrance cancer susceptibility loci are located throughout the genome; however, a very limited number of genes have been identified so far. Using a forward genetics approach to map such loci in a mouse skin cancer model, we previously identified strong genetic loci conferring resistance to chemically induced skin papillomas on chromosome 4 and 7 with a large number of [(FVB/N × MSM/Ms) F₁ × FVB/N] backcross mice. In this report, we describe a combination of congenic mapping and allele-specific alteration analysis of the loci on chromosome 4. We used linkage analysis and a congenic mouse strain, FVB.MSM-Stmm3 to refine the location of Stmm3 (Skin tumor modifier of MSM 3) locus within a physical interval of about 34 Mb on distal chromosome 4. In addition, we used patterns of allele-specific imbalances in tumors from N₂ and N₁₀ congenic mice to narrow down further the region of Stmm3 locus to a physical distance of about 25 Mb. Furthermore, immunohistochemical analysis showed papillomas from congenic mice had less proliferative activity. These results suggest that Stmm3 responsible genes may have an influence on papilloma formation in the two-stage skin carcinogenesis by regulating papilloma growth rather than development.     

Systematic identification of novel cancer genes through analysis of deep shRNA perturbation screens

Systematic perturbation screens provide comprehensive resources for the elucidation of cancer driver genes. The perturbation of many genes in relatively few cell lines in such functional screens necessitates the development of specialized computational tools with sufficient statistical power. Here we developed APSiC (Analysis of Perturbation Screens for identifying novel Cancer genes) to identify genetic drivers and effectors in perturbation screens even with few samples. Applying APSiC to the shRNA screen Project DRIVE, APSiC identified well-known and novel putative mutational and amplified cancer genes across all cancer types and in specific cancer types. Additionally, APSiC discovered tumor-promoting and tumor-suppressive effectors, respectively, for individual cancer types, including genes involved in cell cycle control, Wnt/β-catenin and hippo signalling pathways. We functionally demonstrated that LRRC4B, a putative novel tumor-suppressive effector, suppresses proliferation by delaying cell cycle and modulates apoptosis in breast cancer. We demonstrate APSiC is a robust statistical framework for discovery of novel cancer genes through analysis of large-scale perturbation screens. The analysis of DRIVE using APSiC is provided as a web portal and represents a valuable resource for the discovery of novel cancer genes.     

Fine Mapping in 94 Inbred Mouse Strains Using a High-Density Haplotype Resource

The genetics of phenotypic variation in inbred mice has for nearly a century provided a primary weapon in the medical research arsenal. A catalog of the genetic variation among inbred mouse strains, however, is required to enable powerful positional cloning and association techniques. A recent whole-genome resequencing study of 15 inbred mouse strains captured a significant fraction of the genetic variation among a limited number of strains, yet the common use of hundreds of inbred strains in medical research motivates the need for a high-density variation map of a larger set of strains. Here we report a dense set of genotypes from 94 inbred mouse strains containing 10.77 million genotypes over 121,433 single nucleotide polymorphisms (SNPs), dispersed at 20-kb intervals on average across the genome, with an average concordance of 99.94% with previous SNP sets. Through pairwise comparisons of the strains, we identified an average of 4.70 distinct segments over 73 classical inbred strains in each region of the genome, suggesting limited genetic diversity between the strains. Combining these data with genotypes of 7570 gap-filling SNPs, we further imputed the untyped or missing genotypes of 94 strains over 8.27 million Perlegen SNPs. The imputation accuracy among classical inbred strains is estimated at 99.7% for the genotypes imputed with high confidence. We demonstrated the utility of these data in high-resolution linkage mapping through power simulations and statistical power analysis and provide guidelines for developing such studies. We also provide a resource of in silico association mapping between the complex traits deposited in the Mouse Phenome Database with our genotypes. We expect that these resources will facilitate effective designs of both human and mouse studies for dissecting the genetic basis of complex traits.PHENOTYPIC variation among inbred mouse strains exposed to a disease-causing agent (be it genetic, infectious, or environmental) provides potential insight into human disease processes that often cannot be practically achieved through direct human studies. Indeed, hundreds of phenotype measurements related to human diseases are available for dozens of inbred strains in common use over the past 50–100 years (Bogue et al. 2007; Grubb et al. 2009). As with the direct study of chronic disease in humans, key steps toward determining the genetic underpinnings of this phenotypic variation are to develop a catalog of the genetic variation among inbred mouse strains and to interpret the structure of variation patterns across the strains. Recent advances in high-throughput genotyping and DNA resequencing technologies are making it possible to rapidly uncover the genetic variation maps of many model organisms (Lindblad-Toh et al. 2005; Mackay and Anholt 2006; Borevitz et al. 2007; Frazer et al. 2007; International Hapmap Consortium 2007; Star Consortium 2008). A recent whole-genome resequencing study of 15 inbred mouse strains captured a significant fraction of the genetic variation among a limited number of strains, allowing researchers to infer patterns of genetic variation and to identify the ancestral origin of the genetic variation (Frazer et al. 2007; Yang et al. 2007). Yet the availability and common experimental employment of hundreds of inbred strains, including >190 stocks available from the Jackson Laboratory, motivates the need for a high-density variation map for a larger set of strains. We have assembled the Mouse HapMap, a resource consisting of a dense set of genotypes for a total of 138,980 unique biallelic single nucleotide polymorphisms (SNPs) in 94 inbred mouse strains at an average spacing of 20 kb on chromosomes 1–19 and X.This resource is ideal for performing high-resolution mapping studies under QTL peaks. We evaluate the feasibility and effectiveness of such studies by examining a typical study from the Mouse Phenome Database (MPD) (Bogue et al. 2007; Grubb et al. 2009) (http://www.jax.org/phenome) and measure the statistical power to detect genetic associations in regions of various sizes. We provide several resources to the mouse genetics community for supporting such studies and a webserver that can estimate the significance threshold, compute the statistical power of a proposed study, and perform in the fine mapping of measured phenotypes. In addition, we provide a database of associations for all phenotypes contained in the MPD. The web resources are available at http://mouse.cs.ucla.edu/.     

Fine Mapping of a QTL Associated with Kernel Row Number on Chromosome 1 of Maize

The genetic factors underlying changes in ear morphology, and particularly the inheritance of kernel row number (KRN), have been broadly investigated in diverse mapping populations in maize (Zea mays L.). In this study, we mapped a region on the long arm of chromosome 1 containing a QTL for KRN. This work was performed using a set of recombinant chromosome nearly isogenic lines (RCNILs) derived from a BC₂S₃ population produced using the inbred maize line W22 and teosinte (Zea mays ssp. parviglumis) as the parents. A set of 48 RCNILs was evaluated in the field during the summer of 2013 in order to perform the mapping. A QTL for KRN was found that explained approximately 51% of the phenotypic variance and had a 1.5-LOD confidence interval of 203 kb. Seven genes are described in this interval. One of these candidate genes may have been the target of domestication processes in maize and contributed to the shift from two kernel row ears in teosinte to a highly polystichous ear in maize.     

Uncovering Genomic Features and Maternal Origin of Korean Native Chicken by Whole Genome Sequencing

The Korean Native Chicken (KNC) is an important endemic biological resource in Korea. While numerous studies have been conducted exploring this breed, none have used next-generation sequencing to identify its specific genomic features. We sequenced five strains of KNC and identified 10.9 million SNVs and 1.3 million InDels. Through the analysis, we found that the highly variable region common to all 5 strains had genes like PCHD15, CISD1, PIK3C2A, and NUCB2 that might be related to the phenotypic traits of the chicken such as auditory sense, growth rate and egg traits. In addition, we assembled unaligned reads that could not be mapped to the reference genome. By assembling the unaligned reads, we were able to present genomic sequences characteristic to the KNC. Based on this, we also identified genes related to the olfactory receptors and antigen that are common to all 5 strains. Finally, through the reconstructed mitochondrial genome sequences, we performed phylogenomic analysis and elucidated the maternal origin of the artificially restored KNC. Our results revealed that the KNC has multiple maternal origins which are in agreement with Korea''s history of chicken breed imports. The results presented here provide a valuable basis for future research on genomic features of KNC and further understanding of KNC''s origin.     

Origin and Evolution of Sulfadoxine Resistant Plasmodium falciparum

The Thailand-Cambodia border is the epicenter for drug-resistant falciparum malaria. Previous studies have shown that chloroquine (CQ) and pyrimethamine resistance originated in this region and eventually spread to other Asian countries and Africa. However, there is a dearth in understanding the origin and evolution of dhps alleles associated with sulfadoxine resistance. The present study was designed to reveal the origin(s) of sulfadoxine resistance in Cambodia and its evolutionary relationship to African and South American dhps alleles. We sequenced 234 Cambodian Plasmodium falciparum isolates for the dhps codons S436A/F, A437G, K540E, A581G and A613S/T implicated in sulfadoxine resistance. We also genotyped 10 microsatellite loci around dhps to determine the genetic backgrounds of various alleles and compared them with the backgrounds of alleles prevalent in Africa and South America. In addition to previously known highly-resistant triple mutant dhps alleles SGEGA and AGEAA (codons 436, 437, 540, 581, 613 are sequentially indicated), a large proportion of the isolates (19.3%) contained a 540N mutation in association with 437G/581G yielding a previously unreported triple mutant allele, SGNGA. Microsatellite data strongly suggest the strength of selection was greater on triple mutant dhps alleles followed by the double and single mutants. We provide evidence for at least three independent origins for the double mutants, one each for the SGKGA, AGKAA and SGEAA alleles. Our data suggest that the triple mutant allele SGEGA and the novel allele SGNGA have common origin on the SGKGA background, whereas the AGEAA triple mutant was derived from AGKAA on multiple, albeit limited, genetic backgrounds. The SGEAA did not share haplotypes with any of the triple mutants. Comparative analysis of the microsatellite haplotypes flanking dhps alleles from Cambodia, Kenya, Cameroon and Venezuela revealed an independent origin of sulfadoxine resistant alleles in each of these regions.     

Determination of the Genome Sequence of Porphyromonas gingivalis Strain ATCC 33277 and Genomic Comparison with Strain W83 Revealed Extensive Genome Rearrangements in P. gingivalis

The gram-negative anaerobic bacterium Porphyromonas gingivalis is a major causative agent of chronic periodontitis. Porphyromonas gingivalis strains have been classified into virulent and less-virulent strains by mouse subcutaneous soft tissue abscess model analysis. Here, we present the whole genome sequence of P. gingivalis ATCC 33277, which is classified as a less-virulent strain. We identified 2090 protein-coding sequences (CDSs), 4 RNA operons, and 53 tRNA genes in the ATCC 33277 genome. By genomic comparison with the virulent strain W83, we identified 461 ATCC 33277-specific and 415 W83-specific CDSs. Extensive genomic rearrangements were observed between the two strains: 175 regions in which genomic rearrangements have occurred were identified. Thirty-five of those genomic rearrangements were inversion or translocation and 140 were simple insertion, deletion, or replacement. Both strains contained large numbers of mobile elements, such as insertion sequences, miniature inverted-repeat transposable elements (MITEs), and conjugative transposons, which are frequently associated with genomic rearrangements. These findings indicate that the mobile genetic elements have been deeply involved in the extensive genome rearrangement of P. gingivalis and the occurrence of many of the strain-specific CDSs. We also describe here a very unique feature of MITE400, which we renamed MITEPgRS (MITE of P. gingivalis with Repeating Sequences).Key words: Porphyromonas gingivalis, whole genome sequence, genome rearrangement, conjugative transposon, MITE     

Pgrmc1 (Progesterone Receptor Membrane Component 1) Associates with Epidermal Growth Factor Receptor and Regulates Erlotinib Sensitivity

Tumorigenesis requires the concerted action of multiple pathways, including pathways that stimulate proliferation and metabolism. Epidermal growth factor receptor (EGFR) is a transmembrane receptor-tyrosine kinase that is associated with cancer progression, and the EGFR inhibitors erlotinib/tarceva and tyrphostin/AG-1478 are potent anti-cancer therapeutics. Pgrmc1 (progesterone receptor membrane component 1) is a cytochrome b₅-related protein that is up-regulated in tumors and promotes cancer growth. Pgrmc1 and its homologues have been implicated in cell signaling, and we show here that Pgrmc1 increases susceptibility to AG-1478 and erlotinib, increases plasma membrane EGFR levels, and co-precipitates with EGFR. Pgrmc1 co-localizes with EGFR in cytoplasmic vesicles and co-fractionates with EGFR in high density microsomes. The findings have therapeutic potential because a Pgrmc1 small molecule ligand, which inhibits growth in a variety of cancer cell types, de-stabilized EGFR in multiple tumor cell lines. EGFR is one of the most potent receptor-tyrosine kinases driving tumorigenesis, and our data support a role for Pgrmc1 in promoting several cancer phenotypes at least in part by binding EGFR and stabilizing plasma membrane pools of the receptor.     

A Fungal Sarcolemmal Membrane-Associated Protein (SLMAP) Homolog Plays a Fundamental Role in Development and Localizes to the Nuclear Envelope,Endoplasmic Reticulum,and Mitochondria

Sarcolemmal membrane-associated protein (SLMAP) is a tail-anchored protein involved in fundamental cellular processes, such as myoblast fusion, cell cycle progression, and chromosomal inheritance. Further, SLMAP misexpression is associated with endothelial dysfunctions in diabetes and cancer. SLMAP is part of the conserved striatin-interacting phosphatase and kinase (STRIPAK) complex required for specific signaling pathways in yeasts, filamentous fungi, insects, and mammals. In filamentous fungi, STRIPAK was initially discovered in Sordaria macrospora, a model system for fungal differentiation. Here, we functionally characterize the STRIPAK subunit PRO45, a homolog of human SLMAP. We show that PRO45 is required for sexual propagation and cell-to-cell fusion and that its forkhead-associated (FHA) domain is essential for these processes. Protein-protein interaction studies revealed that PRO45 binds to STRIPAK subunits PRO11 and SmMOB3, which are also required for sexual propagation. Superresolution structured-illumination microscopy (SIM) further established that PRO45 localizes to the nuclear envelope, endoplasmic reticulum, and mitochondria. SIM also showed that localization to the nuclear envelope requires STRIPAK subunits PRO11 and PRO22, whereas for mitochondria it does not. Taken together, our study provides important insights into fundamental roles of the fungal SLMAP homolog PRO45 and suggests STRIPAK-related and STRIPAK-unrelated functions.     

Evolution of the B3 DNA Binding Superfamily: New Insights into REM Family Gene Diversification

Background

The B3 DNA binding domain includes five families: auxin response factor (ARF), abscisic acid-insensitive3 (ABI3), high level expression of sugar inducible (HSI), related to ABI3/VP1 (RAV) and reproductive meristem (REM). The release of the complete genomes of the angiosperm eudicots Arabidopsis thaliana and Populus trichocarpa, the monocot Orysa sativa, the bryophyte Physcomitrella patens,the green algae Chlamydomonas reinhardtii and Volvox carteri and the red algae Cyanidioschyzon melorae provided an exceptional opportunity to study the evolution of this superfamily.

Methodology

In order to better understand the origin and the diversification of B3 domains in plants, we combined comparative phylogenetic analysis with exon/intron structure and duplication events. In addition, we investigated the conservation and divergence of the B3 domain during the origin and evolution of each family.

Conclusions

Our data indicate that showed that the B3 containing genes have undergone extensive duplication events, and that the REM family B3 domain has a highly diverged DNA binding. Our results also indicate that the founding member of the B3 gene family is likely to be similar to the ABI3/HSI genes found in C. reinhardtii and V. carteri. Among the B3 families, ABI3, HSI, RAV and ARF are most structurally conserved, whereas the REM family has experienced a rapid divergence. These results are discussed in light of their functional and evolutionary roles in plant development.     

Genome-Wide Association Studies and the Problem of Relatedness Among Advanced Intercross Lines and Other Highly Recombinant Populations

Model organisms offer many advantages for the genetic analysis of complex traits. However, identification of specific genes is often hampered by a lack of recombination between the genomes of inbred progenitors. Recently, genome-wide association studies (GWAS) in humans have offered gene-level mapping resolution that is possible because of the large number of accumulated recombinations among unrelated human subjects. To obtain analogous improvements in mapping resolution in mice, we used a 34th generation advanced intercross line (AIL) derived from two inbred strains (SM/J and LG/J). We used simulations to show that familial relationships among subjects must be accounted for when analyzing these data; we then used a mixed model that included polygenic effects to address this problem in our own analysis. Using a combination of F₂ and AIL mice derived from the same inbred progenitors, we identified genome-wide significant, subcentimorgan loci that were associated with methamphetamine sensitivity, (e.g., chromosome 18; LOD = 10.5) and non-drug-induced locomotor activity (e.g., chromosome 8; LOD = 18.9). The 2-LOD support interval for the former locus contains no known genes while the latter contains only one gene (Csmd1). This approach is broadly applicable in terms of phenotypes and model organisms and allows GWAS to be performed in multigenerational crosses between and among inbred strains where familial relatedness is often unavoidable.SUSCEPTIBILITY to diseases such as drug abuse is partially determined by genetic factors. The identification of the alleles that underlie disease susceptibility is an immensely important goal that promises to revolutionize both the diagnosis and the treatment of human disease. Genome-wide association studies (GWAS) in humans can locate common alleles with great precision. However, GWAS may be unable to identify the bulk of the heritable variability for common genetic diseases; some of this “missing heritability” is thought to be due to rare alleles (Manolio et al. 2009). Model organisms are complementary to human genetic studies and offer unique advantages including the ability to control the environment, perform dangerous or invasive procedures, and test hypotheses by manipulating genes via genetic engineering; a final advantage is that crosses between two inbred strains avoid many of the difficulties associated with rare alleles.Studies in model organisms have frequently employed intercrosses (F₂''s) to identify quantitative trait loci (QTL) that underlie phenotypic variability. F₂ crosses are easy to produce and easy to analyze; however, due to a lack of recombination they can identify only larger genomic regions and are thus unsuitable for identifying the genes that cause QTL (Flint et al. 2005; Peters et al. 2007). This is a serious limitation that can be addressed by using populations with greater numbers of accumulated recombinations. Darvasi and Soller (1995) suggested the creation of advanced intercross lines (AILs) by successive generations of random mating after the F₂ generation to produce additional recombinations. An AIL offers vastly improved mapping resolution while maintaining the desirable property that all polymorphic alleles are common.We used an AIL to study sensitivity to methamphetamine, which is a genetically complex trait that may be useful for identifying genetic factors influencing the subjectively euphoric response to stimulant drugs and susceptibility to drug abuse (Palmer et al. 2005; Phillips et al. 2008; Bryant et al. 2009). For example, a prior study suggested that the gene Casein Kinase 1 Epsilon (Csnk1e) might influence sensitivity to the acute locomotor response to methamphetamine in mice (Palmer et al. 2005). This conclusion has been bolstered by additional pharmacological (Bryant et al. 2009) and genetic studies. In addition, we have shown that polymorphisms in this gene are associated with sensitivity to the euphoric effects of amphetamine in humans (Veenstra-Vanderweele et al. 2006). Another group has subsequently reported that this same gene is associated with heroine addiction (Levran et al. 2008). Thus, genes that modulate the acute locomotor response to a drug in mice may also be important for sensitivity to similar drugs in humans as well as the risk for developing drug abuse.The purpose of this study was to develop a framework for rapid identification of high precision QTL and ideally specific genes that influence sensitivity to methamphetamine in mice by employing an AIL. We produced an F₂ cross (n = 490) and a corresponding 34th generation AIL (n = 688) derived from the inbred strains SM/J and LG/J. This allowed us to compare and integrate the results from F₂ and AIL mice. We examined the locomotor stimulant response to a 2-mg/kg dose of methamphetamine, which is extremely disparate in the two progenitor strains. We performed a GWAS using either simple regression, which ignored relatedness, or a mixed model that accounted for relatedness by using identity coefficients that were calculated from the pedigree. We also explored two methods to estimate significance: simple permutation and gene dropping. We discuss the performance of a mixed model that includes polygenic effects vs. simple regression and the performance of permutation vs. gene dropping. The methods used in this study are applicable to a variety of other phenotypes and populations.