Showing papers on "Chromosomal region published in 2015"

Telomerase activation by genomic rearrangements in high-risk neuroblastoma

Abstract: Neuroblastoma is a malignant paediatric tumour of the sympathetic nervous system. Roughly half of these tumours regress spontaneously or are cured by limited therapy. By contrast, high-risk neuroblastomas have an unfavourable clinical course despite intensive multimodal treatment, and their molecular basis has remained largely elusive. Here we have performed whole-genome sequencing of 56 neuroblastomas (high-risk, n = 39; low-risk, n = 17) and discovered recurrent genomic rearrangements affecting a chromosomal region at 5p15.33 proximal of the telomerase reverse transcriptase gene (TERT). These rearrangements occurred only in high-risk neuroblastomas (12/39, 31%) in a mutually exclusive fashion with MYCN amplifications and ATRX mutations, which are known genetic events in this tumour type. In an extended case series (n = 217), TERT rearrangements defined a subgroup of high-risk tumours with particularly poor outcome. Despite a large structural diversity of these rearrangements, they all induced massive transcriptional upregulation of TERT. In the remaining high-risk tumours, TERT expression was also elevated in MYCN-amplified tumours, whereas alternative lengthening of telomeres was present in neuroblastomas without TERT or MYCN alterations, suggesting that telomere lengthening represents a central mechanism defining this subtype. The 5p15.33 rearrangements juxtapose the TERT coding sequence to strong enhancer elements, resulting in massive chromatin remodelling and DNA methylation of the affected region. Supporting a functional role of TERT, neuroblastoma cell lines bearing rearrangements or amplified MYCN exhibited both upregulated TERT expression and enzymatic telomerase activity. In summary, our findings show that remodelling of the genomic context abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumours.

The 16p11.2 deletion mouse model of autism exhibits altered cortical progenitor proliferation and brain cytoarchitecture linked to the ERK MAPK pathway.

Abstract: Autism spectrum disorders are complex, highly heritable neurodevelopmental disorders affecting ∼1 in 100 children. Copy number variations of human chromosomal region 16p11.2 are genetically linked to 1% of autism-related disorders. This interval contains the MAPK3 gene, which encodes the MAP kinase, ERK1. Mutations in upstream elements regulating the ERK pathway are genetically linked to autism and other disorders of cognition including the neuro-cardio-facial cutaneous syndromes and copy number variations. We report that a murine model of human 16p11.2 deletion exhibits a reduction in brain size and perturbations in cortical cytoarchitecture. We observed enhanced progenitor proliferation and premature cell cycle exit, which are a consequence of altered levels of downstream ERK effectors cyclin D1 and p27Kip1 during mid-neurogenesis. The increased progenitor proliferation and cell cycle withdrawal resulted in premature depletion of progenitor pools, altering the number and frequency of neurons ultimately populating cortical lamina. Specifically, we found a reduced number of upper layer pyramidal neurons and an increase in layer VI corticothalamic projection neurons, reflecting the altered cortical progenitor proliferation dynamics in these mice. Importantly, we observed a paradoxical increase in ERK signaling in mid-neurogenesis in the 16p11.2del mice, which is coincident with the development of aberrant cortical cytoarchitecture. The 16p11.2del mice exhibit anxiety-like behaviors and impaired memory. Our findings provide evidence of ERK dysregulation, developmental abnormalities in neurogenesis, and behavioral impairment associated with the 16p11.2 chromosomal deletion.

Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array of human cells

Abstract: CRISPR/Cas9-derived RNA-guided nucleases (RGNs) are DNA targeting systems, which are rapidly being harnessed for gene regulation and gene editing purposes in model organisms and cell lines. As bona fide gene delivery vehicles, viral vectors may be particularly fit to broaden the applicability of RGNs to other cell types including dividing and quiescent primary cells. Here, the suitability of adenoviral vectors (AdVs) for delivering RGN components into various cell types is investigated. We demonstrate that AdVs, namely second-generation fiber-modified AdVs encoding Cas9 or single guide RNA (gRNA) molecules addressing the Cas9 nuclease to the AAVS1 “safe harbor” locus or to a recombinant model allele can be produced to high-titers (up to 20 × 1010 transducing units/ml). Importantly, AdV-mediated transduction of gRNA:Cas9 ribonucleoprotein complexes into transformed and non-transformed cells yields rates of targeted mutagenesis similar to or approaching those achieved by isogenic AdVs encoding TALENs targeting the same AAVS1 chromosomal region. RGN-induced gene disruption frequencies in the various cell types ranged from 18% to 65%. We conclude that AdVs constitute a valuable platform for introducing RGNs into human somatic cells regardless of their transformation status. This approach should aid investigating the potential and limitations of RGNs in numerous experimental settings.

Waterlogging tolerance is associated with root porosity in barley ( Hordeum vulgare L.)

Abstract: Tolerance to waterlogging is an important breeding objective for barley (Hordeum vulgare L.); however, it is a complex quantitative trait. It is difficult to screen large numbers of lines in the field due to environmental variability, and it is also challenging to screen large numbers in controlled conditions if yield data are to be collected. The direct measurement of traits that contribute to waterlogging tolerance, such as aerenchyma development in roots, may offer advantages especially if molecular markers can be developed to screen breeding populations. A doubled haploid population from a cross between Franklin and YuYaoXiangTian Erleng was screened for adventitious root porosity (gas-filled volume per unit root volume) as an indicator of aerenchyma formation. A single QTL for root porosity was identified on chromosome 4H which explained 35.7 and 39.0 % of phenotypic variation in aerated and oxygen-deficient conditions, respectively. The nearest marker was EBmac0701. This QTL is located in the same chromosomal region that contributed to tolerance when the same population was screened in an earlier independent soil waterlogging experiment. Comparative mapping revealed that this QTL is syntenic with the Qaer1.02-3 QTL in maize and the Sub1A-1 gene in rice, which are associated with aerenchyma formation (maize) and submergence tolerance (rice), respectively. This is the first report of a QTL for root porosity in barley which elucidates a major mechanism of waterlogging tolerance.

Tel1 ATM -mediated interference suppresses clustered meiotic double-strand-break formation

Abstract: Meiotic recombination is initiated by a fairly uniform distribution of hundreds of DNA double-strand breaks catalysed by the Spo11 protein; here, Tel1 (orthologue of human ATM) is shown to be required for the localized inhibition that prevents double-strand breaks from forming close to one another. Meiosis is a two-step cell division process that reduces diploid DNA content in somatic cells to the haploid content in gametes (egg or sperm). The gamete contains a unique combination of the parental gene sequences thanks to a process known as meiotic recombination. Meiotic recombination begins with the introduction of a few hundred double-strand DNA breaks (DSBs) by the Spo11 protein. Here Matthew Neale and colleagues characterize the interference mechanism that ensures that these breaks and the associated recombination events are evenly distributed across chromosomes. They show that in yeast, the presence of a DSB blocks the creation of a second one nearby in a process requiring the DNA damage responsive kinase Tel1ATM. In addition, genomic rearrangements are found in clusters in the absence of Tel1, suggesting that the kinase has two negative regulatory effects on DSB formation. Meiotic recombination is a critical step in gametogenesis for many organisms, enabling the creation of genetically diverse haploid gametes. In each meiotic cell, recombination is initiated by numerous DNA double-strand breaks (DSBs) created by Spo11, the evolutionarily conserved topoisomerase-like protein1, but how these DSBs are distributed relatively uniformly across the four chromatids that make up each chromosome pair is poorly understood. Here we employ Saccharomyces cerevisiae to demonstrate distance-dependent DSB interference in cis (in which the occurrence of a DSB suppresses adjacent DSB formation)—a process that is mediated by the conserved DNA damage response kinase, Tel1ATM. The inhibitory function of Tel1 acts on a relatively local scale, while over large distances DSBs have a tendency to form independently of one another even in the presence of Tel1. Notably, over very short distances, loss of Tel1 activity causes DSBs to cluster within discrete zones of concerted DSB activity. Our observations support a hierarchical view of recombination initiation where Tel1ATM prevents clusters of DSBs, and further suppresses DSBs within the surrounding chromosomal region. Such collective negative regulation will help to ensure that recombination events are dispersed evenly and arranged optimally for genetic exchange and efficient chromosome segregation.

QTL for seed protein and amino acids in the Benning × Danbaekkong soybean population

Abstract: We identified QTL associated with protein and amino acids in a soybean mapping population that was grown in five environments. These QTL could be used in MAS to improve these traits. Soybean, rather than nitrogen-containing forages, is the primary source of quality protein in feed formulations for domestic swine, poultry, and dairy industries. As a sole dietary source of protein, soybean is deficient in the amino acids lysine (Lys), threonine (Thr), methionine (Met), and cysteine (Cys). Increasing these amino acids would benefit the feed industry. The objective of the present study was to identify quantitative trait loci (QTL) associated with crude protein (cp) and amino acids in the ‘Benning’ × ‘Danbaekkong’ population. The population was grown in five southern USA environments. Amino acid concentrations as a fraction of cp (Lys/cp, Thr/cp, Met/cp, Cys/cp, and Met + Cys/cp) were determined by near-infrared reflectance spectroscopy. Four QTL associated with the variation in crude protein were detected on chromosomes (Chr) 14, 15, 17, and 20, of which, a QTL on Chr 20 explained 55 % of the phenotypic variation. In the same chromosomal region, QTL for Lys/cp, Thr/cp, Met/cp, Cys/cp and Met + Cys/cp were detected. At these QTL, the Danbaekkong allele resulted in reduced levels of these amino acids and increased protein concentration. Two additional QTL for Lys/cp were detected on Chr 08 and 20, and three QTL for Thr/cp on Chr 01, 09, and 17. Three QTL were identified on Chr 06, 09 and 10 for Met/cp, and one QTL was found for Cys/cp on Chr 10. The study provides information concerning the relationship between crude protein and levels of essential amino acids and may allow for the improvement of these traits in soybean using marker-assisted selection.

The DNA-Binding Protein from Starved Cells (Dps) Utilizes Dual Functions To Defend Cells against Multiple Stresses

Abstract: Bacteria deficient in the DNA-binding protein from starved cells (Dps) are viable under controlled conditions but show dramatically increased mortality rates when exposed to any of a wide range of stresses, including starvation, oxidative stress, metal toxicity, or thermal stress. It remains unclear whether the protective action of Dps against specific stresses derives from its DNAbinding activity, which may exclude destructive agents from the chromosomal region, or its ferroxidase activity, which neutralizes and sequesters potentially damaging chemical species. To resolve this question, we have identified the critical residues of Escherichia coli Dps that bind to DNA and modulate iron oxidation. We uncoupled the biochemical activities of Dps, creating Dps variants and mutant E. coli strains that are defective in either DNA-binding or ferroxidase activity. Quantification of the contribution of each activity to the protection of DNA integrity and cellular viability revealed that both activities of Dps are required in order to counteract many differing stresses. These findings demonstrate that Dps plays a multipurpose role in stress protection via its dual activities, explaining how Dps can be of vital importance to bacterial viability over a wide range of stresses.

A molecular trigger for intercontinental epidemics of group A Streptococcus

Abstract: The identification of the molecular events responsible for strain emergence, enhanced virulence, and epidemicity has been a long-pursued goal in infectious diseases research. A recent analysis of 3,615 genomes of serotype M1 group A Streptococcus strains (the so-called "flesh-eating" bacterium) identified a recombination event that coincides with the global M1 pandemic beginning in the early 1980s. Here, we have shown that the allelic variation that results from this recombination event, which replaces the chromosomal region encoding secreted NADase and streptolysin O, is the key driver of increased toxin production and enhanced infection severity of the M1 pandemic strains. Using isoallelic mutant strains, we found that 3 polymorphisms in this toxin gene region increase resistance to killing by human polymorphonuclear leukocytes, increase bacterial proliferation, and increase virulence in animal models of pharyngitis and necrotizing fasciitis. Genome sequencing of an additional 1,125 streptococcal strains and virulence studies revealed that a highly similar recombinational replacement event underlies an ongoing intercontinental epidemic of serotype M89 group A Streptococcus infections. By identifying the molecular changes that enhance upper respiratory tract fitness, increased resistance to innate immunity, and increased tissue destruction, we describe a mechanism that underpins epidemic streptococcal infections, which have affected many millions of people.

Highly efficient CRISPR/Cas9-mediated TAR cloning of genes and chromosomal loci from complex genomes in yeast

Abstract: Transformation-associated recombination (TAR) protocol allowing the selective isolation of full-length genes complete with their distal enhancer regions and entire genomic loci with sizes up to 250 kb from complex genomes in yeast S. cerevisiae has been developed more than a decade ago. However, its wide spread usage has been impeded by a low efficiency (0.5–2%) of chromosomal region capture during yeast transformants which in turn requires a time-consuming screen of hundreds of colonies. Here, we demonstrate that pre-treatment of genomic DNA with CRISPR-Cas9 nucleases to generate double-strand breaks near the targeted genomic region results in a dramatic increase in the fraction of gene-positive colonies (up to 32%). As only a dozen or less yeast transformants need to be screened to obtain a clone with the desired chromosomal region, extensive experience with yeast is no longer required. A TAR-CRISPR protocol may help to create a bank of human genes, each represented by a genomic copy containing its native regulatory elements, that would lead to a significant advance in functional, structural and comparative genomics, in diagnostics, gene replacement, generation of animal models for human diseases and has a potential for gene therapy.

Overexpression of the long non-coding RNA PVT1 is correlated with leukemic cell proliferation in acute promyelocytic leukemia

Abstract: Acute promyelocytic leukemia (APL) is associated with chromosomal translocation t(15;17), which results in the proliferation of morphologically abnormal promyelocytes. Gain of supernumerary copies of the 8q24 chromosomal region, which harbors MYC and PVT1, has been shown to be the most common secondary alteration in human APL. Increased MYC can accelerate the development of myeloid leukemia in APL. However, the role that the expression of the long non-coding RNA (lncRNA) PVT1 plays in the pathogenesis of APL remains largely unknown. In this study, we first analyzed the lncRNA PVT1 expression level in peripheral blood cells from 28 patients with de novo APL, and significantly upregulated PVT1 was found in APL patients compared with healthy donors. We then observed significantly lower MYC and PVT1 expression during all-trans retinoic acid (ATRA)-induced differentiation and cell cycle arrest in the APL cell line. MYC knockdown in NB4 cells led to PVT1 downregulation. Moreover, PVT1 knockdown by RNA interference led to suppression of the MYC protein level, and cell proliferation was inhibited. Our findings reveal that the lncRNA PVT1 may play an important role in the proliferation of APL cells and may be useful for future therapeutic management.

Shugoshins: Tension-Sensitive Pericentromeric Adaptors Safeguarding Chromosome Segregation

Abstract: The shugoshin/Mei-S332 family are proteins that associate with the chromosomal region surrounding the centromere (the pericentromere) and that play multiple and distinct roles in ensuring the accuracy of chromosome segregation during both mitosis and meiosis. The underlying role of shugoshins appears to be to serve as pericentromeric adaptor proteins that recruit several different effectors to this region of the chromosome to regulate processes critical for chromosome segregation. Crucially, shugoshins undergo changes in their localization in response to the tension that is exerted on sister chromosomes by the forces of the spindle that will pull them apart. This has led to the idea that shugoshins provide a platform for activities required at the pericentromere only when sister chromosomes lack tension. Conversely, disassembly of the shugoshin pericentromeric platform may provide a signal that sister chromosomes are under tension. Here the functions and regulation of these important tension-sensitive pericentromeric proteins are discussed.

Angelman Syndrome

Abstract: In this review we summarize the clinical and genetic aspects of Angelman syndrome (AS), its molecular and cellular underpinnings, and current treatment strategies. AS is a neurodevelopmental disorder characterized by severe cognitive disability, motor dysfunction, speech impairment, hyperactivity, and frequent seizures. AS is caused by disruption of the maternally expressed and paternally imprinted UBE3A, which encodes an E3 ubiquitin ligase. Four mechanisms that render the maternally inherited UBE3A nonfunctional are recognized, the most common of which is deletion of the maternal chromosomal region 15q11-q13. Remarkably, duplication of the same chromosomal region is one of the few characterized persistent genetic abnormalities associated with autistic spectrum disorder, occurring in >1–2 % of all cases of autism spectrum disorder. While the overall morphology of the brain and connectivity of neural projections appear largely normal in AS mouse models, major functional defects are detected at the level of context-dependent learning, as well as impaired maturation of hippocampal and neocortical circuits. While these findings demonstrate a crucial role for ubiquitin protein ligase E3A in synaptic development, the mechanisms by which deficiency of ubiquitin protein ligase E3A leads to AS pathophysiology in humans remain poorly understood. However, recent efforts have shown promise in restoring functions disrupted in AS mice, renewing hope that an effective treatment strategy can be found.

16p11.2 deletion syndrome mice display sensory and ultrasonic vocalization deficits during social interactions

Abstract: Recurrent deletions and duplications at chromosomal region 16p11.2 are variably associated with speech delay, autism spectrum disorder, developmental delay, schizophrenia, and cognitive impairments. Social communication deficits are a primary diagnostic symptom of autism. Here we investigated ultrasonic vocalizations (USVs) in young adult male 16p11.2 deletion mice during a novel three-phase male-female social interaction test that detects vocalizations emitted by a male in the presence of an estrous female, how the male changes its calling when the female is suddenly absent, and the extent to which calls resume when the female returns. Strikingly fewer vocalizations were detected in two independent cohorts of 16p11.2 heterozygous deletion males (+/-) during the first exposure to an unfamiliar estrous female, as compared to wildtype littermates (+/+). When the female was removed, +/+ emitted calls, but at a much lower level, whereas +/- males called minimally. Sensory and motor abnormalities were detected in +/-, including higher nociceptive thresholds, a complete absence of acoustic startle responses, and hearing loss in all +/- as confirmed by lack of auditory brainstem responses to frequencies between 8 and 100 kHz. Stereotyped circling and backflipping appeared in a small percentage of individuals, as previously reported. However, these sensory and motor phenotypes could not directly explain the low vocalizations in 16p11.2 deletion mice, since (a) +/- males displayed normal abilities to emit vocalizations when the female was subsequently reintroduced, and (b) +/- vocalized less than +/+ to social odor cues delivered on an inanimate cotton swab. Our findings support the concept that mouse USVs in social settings represent a response to social cues, and that 16p11.2 deletion mice are deficient in their initial USVs responses to novel social cues.

Mapping Consistent Rice (Oryza sativa L.) Yield QTLs under Drought Stress in Target Rainfed Environments

Abstract: Drought stress is a major limitation to rainfed rice production and yield stability. Identifying yield-associated quantitative trait loci (QTLs) that are consistent under drought stress predominant in target production environments, as well as across different genetic backgrounds, will help to develop high-yielding rice cultivars suitable for water-limited environments through marker-assisted breeding (MAB). Considerable progress has been made in mapping QTLs for drought resistance traits in rice; however, few have been successfully used in MAB. Recombinant inbred lines of IR20 × Nootripathu, two indica cultivars adapted to rainfed target populations of environments (TPEs), were evaluated in one and two seasons under managed stress and in a rainfed target drought stress environment, respectively. In the managed stress environment, the severity of the stress meant that measurements could be made only on secondary traits and biomass. In the target environment, the lines experienced varying timings, durations, and intensities of drought stress. The rice recombinant inbred lines exhibited significant genotypic variation for physio-morphological, phenological, and plant production traits under drought. Nine and 24 QTLs for physio-morphological and plant production traits were identified in managed and natural drought stress conditions in the TPEs, respectively. Yield QTLs that were consistent in the target environment over seasons were identified on chromosomes 1, 4, and 6, which could stabilize the productivity in high-yielding rice lines in a water-limited rainfed ecosystem. These yield QTLs also govern highly heritable key secondary traits, such as leaf drying, canopy temperature, panicle harvest index and harvest index. Three QTL regions on chromosome 1 (RM8085), chromosome 4 (I12S), and chromosome 6 (RM6836) harbor significant additive QTLs for various physiological and yield traits under drought stress. The similar chromosomal region on 4 and 6 were found to harbor QTLs for canopy temperature and leaf drying under drought stress conditions. Thus, the identified large effect yield QTLs could be introgressed to develop rice lines with stable yields under varying natural drought stress predominant in TPEs.

The spread of Bradyrhizobium lineages across host legume clades: from Abarema to Zygia.

Abstract: To analyze macroevolutionary patterns in host use by Bradyrhizobium root-nodule bacteria, 420 strains from 75 legume host genera (sampled in 25 countries) were characterized for portions of six housekeeping genes and the nifD locus in the symbiosis island chromosomal region. Most Bradyrhizobium clades utilized very divergent sets of legume hosts. This suggests that Bradyrhizobium spread across the major legume lineages early in its evolution, with only a few derived clades subsequently developing a narrower pattern of host use. Significant modularity existed in the network structure of recent host jumps (inferred from cases where closely related strain pairs were found on different legume taxa). This implies that recent host switching has occurred most often within particular subgroups of legumes. Nevertheless, the observed link structure would allow a bacterial lineage to reach almost any of the 75 legume host genera in a relatively small number of steps. However, permutation tests also showed that symbionts from certain host plant clades were significantly more similar than would be the case if bacteria were distributed at random on the trees. Related legumes thus harbored related sets of symbionts in some cases, indicating some degree of phylogenetic conservatism in partner selection.

Low Copy Number of the AMY1 Locus Is Associated with Early-Onset Female Obesity in Finland

Abstract: Background The salivary α-amylase locus (AMY1) is located in a highly polymorphic multi allelic copy number variable chromosomal region. A recent report identified an association between AMY1 copy numbers and BMI in common obesity. The present study investigated the relationship between AMY1 copy number, BMI and serum amylase in childhood-onset obesity. Patients Sixty-one subjects with a history of childhood-onset obesity (mean age 19.1 years, 54% males) and 71 matched controls (19.8 yrs, 45% males) were included. All anthropometric measures were greater in the obese; their mean BMI was 40 kg/m2 (range 25-62 kg/m2) compared with 23 kg/m2 in the controls (15-32 kg/m2). Results Mean AMY1 copy numbers did not differ between the obese and control subjects, but gender differences were observed; obese men showed the highest and obese women the lowest number of AMY1 copies (p=0.045). Further, only in affected females, AMY1 copy number correlated significantly with whole body fat percent (r=-0.512, p=0.013) and BMI (r=-0.416, p=0.025). Finally, a clear linear association between AMY1 copy number and serum salivary amylase was observed in all subgroups but again differences existed between obese males and females. Conclusions In conclusion, our findings suggest that AMY1 copy number differences play a role in childhood-onset obesity but the effect differs between males and females. Further studies in larger cohorts are needed to confirm these observations.

ABCs of Insect Resistance to Bt.

Abstract: Genetically engineered crops represent one of the most controversial and rapidly adopted technologies in the history of agriculture. To improve pest control, scientists have engineered cotton, corn, and soybeans to make insecticidal proteins from the common bacterium Bacillus thuringiensis (Bt) [1]. These Bt toxins kill some devastating pests, but unlike broad-spectrum insecticides, they do little or no harm to most other organisms, including people [2,3]. The original Bt crops, first commercialized in 1996, each made a single crystalline (Cry) toxin from the Cry1 family effective against certain lepidopteran larvae. However, some of the environmental, health, and economic benefits of Bt crops have been lost because of rapid evolution of pest resistance, particularly to single-toxin Bt crops (Fig 1) [4]. Fig 1 Field-evolved resistance to Bt crops [4,20,27,29–31]. To delay resistance and broaden the spectrum of pests controlled, newer “second generation” Bt crops produce two or more Bt toxins [5]. In particular, Bt toxin Cry2Ab from the Cry2 family is used widely in combination with Cry1 toxins to kill caterpillar pests. For example, the percentage of all cotton planted that was Bt cotton producing both Cry1Ac and Cry2Ab was 69% in the US in 2012, 91% in India in 2013, and 94% in Australia in 2011 [6–8]. Despite the use of Cry2Ab in multi-toxin Bt crops since 2003 and in multi-toxin Bt sprays for decades, nearly all of what we know about Bt toxins is based on the Cry1 family. In a breakthrough that promises to accelerate progress in understanding Cry2 toxins, Tay et al., in this issue of PLOS Genetics [9], identify a gene tightly linked with resistance to Cry2Ab in Helicoverpa armigera, one of the world’s most damaging crop pests. The advance by Tay et al. is the fruit of more than a dozen years of a synergistic collaboration, integrating results from classical and molecular genetics. As part of Australia’s proactive program for monitoring resistance to Bt crops, screening of field populations for resistance to Cry2Ab began in 2002, two years before farmers there started planting Bt cotton producing this toxin in combination with Cry1Ac. Using a method called the F2 screen, the second generation progeny of single pairs of field-collected insects were tested on an artificial diet treated with Cry2Ab. In the first year of screening, the Australian team detected resistance to Cry2Ab in one of the 28 isofemale lines that were tested [10]. The 17 survivors of exposure to Cry2Ab from this isofemale line became the progenitors of a Cry2Ab-resistant strain (SP15) that was repeatedly crossed with a susceptible strain and selected with Cry2Ab [11]. The SP15 strain was so resistant that it suffered little mortality when exposed to the highest concentration of Cry2Ab tested in the artificial diet [11]. Bioassays of progeny from crosses indicated this resistance to Cry2Ab was autosomal, recessive, and probably conferred by a single genetic locus [11]. Tay et al. used genetic linkage analysis with molecular markers to narrow the source of resistance to Cry2Ab in this strain to a chromosomal region containing less than 30 genes. They found that two of these genes encode the ATP-binding cassette (ABC) transporter proteins ABCA1 and ABCA2. These genes were prime suspects because resistance to Cry1 toxins is linked with the ABC transporter protein ABCC2 in strains of seven species of Lepidoptera, including H. armigera [12–15]. Tay et al. honed in on ABCA2 because it was produced in the midgut where Bt toxin binding occurs, but ABCA1 was not. In cDNA of ABCA2 in a resistant individual from the SP15 strain, they found a 73 base pair deletion that introduces a premature stop codon. Including the SP15 strain, Tay et al. detected the same mutation in five of seven resistant lines of H. armigera, each established independently from insects collected from the field during 2002 to 2012. They found two other mutant alleles at the same locus in two other resistant lines, yielding a total of three mutant alleles that each encode a truncated ABCA2 protein. Screening of one Cry2Ab-resistant strain of the congeneric species Helicoverpa punctigera revealed a different premature stop codon in the orthologous gene. This new paper is a worthy successor to the landmark 2001 article by David Heckel’s group, which was the first to report the molecular genetic basis of resistance to Cry1Ac [16]. As that paper did for Cry1A toxins, this one will accelerate research to enhance understanding of the mode of action of Cry2A toxins. We now know that diverse mutations in cadherin and other genes can confer resistance to Cry1Ac in the field [17,18], which limits the utility of PCR-based monitoring for specific resistance mutations. Nonetheless, identification of cadherin as a key receptor for Cry1A toxins did spur genetic engineering of modified toxins that kill some insects resistant to Cry1 toxins [19,20], and this paper might inspire analogous discoveries for Cry2-resistant insects. Despite the ≥85% adoption of Bt cotton producing Cry2Ab and Cry1Ac in Australia since 2005 [7], eight years of monitoring data from the robust F1 screen method show no significant increase in the frequency of resistance to Cry2Ab (0.032 in 2007–2008 to 0.021 in 2014–2015 for H. armigera; 0.010 to 0.011 over the same eight years for H. punctigera) [21]. If the Cry2Ab resistance alleles provide protection against this toxin, why has their frequency not increased? Part of the explanation is that Cry1Ac resistance remains rare in Australia [21] and, with little or no cross-resistance, the Cry1Ac in the two-toxin cotton kills individuals resistant to Cry2Ab. This is reflected in only zero to 8.5% survival of SP15 larvae on two-toxin cotton [22]. Fitness costs associated with Cry2Ab resistance alleles could also delay the evolution of resistance in H. armigera and H. punctigera by selecting against these alleles when larvae eat non-Bt cotton or any of the other non-Bt host plants of these polyphagous species. Because insect ABC transporters often provide protection against xenobiotics, the resistance-conferring mutations disrupting these proteins may diminish their natural function, yielding higher fitness costs in the presence of toxic substances, such as plant defensive compounds and insecticides other than Bt toxins [12]. Although significant fitness costs of Cry2Ab resistance in H. armigera were not detected when larvae ate either artificial diet [23] or mature non-Bt cotton [22], survival on younger non-Bt cotton was significantly lower for resistant larvae (81%) than susceptible larvae (100%) [22], which is a substantial fitness cost. Moreover, in strains of H. armigera and H. punctigera with ABCA2 mutations, resistance to Cry2Ab is associated with significantly increased susceptibility to the organophospate insecticide chlorpyrifos and the carbamate insecticide methomyl [24]. Although Tay et al. provide compelling evidence that ABCA2 is essential for toxicity of Cry2Ab to Helicoverpa armigera, the precise role of this protein in the mode of action of Cry2Ab remains unknown. In strains of H. armigera and H. punctigera that harbor ABCA2 mutations, resistance to Cry2Ab is associated with reduced binding of this toxin [25], which implies that ABCA2 either binds Cry2Ab directly or facilitates binding of Cry2Ab to other target sites. A similar correlation with reduced toxin binding is typically seen with resistance to Cry1A toxins linked with mutations disrupting ABCC2 [12]. However, in the silkworm Bombyx mori, Cry1Ab bound equally to brush border membrane vesicles from susceptible larvae and larvae with ABCC2-linked resistance [26]. Additional work is needed to test the hypotheses of Tay et al. that ABCA2 provides both binding and pore formation functions for Cry2Ab. It will also be important to determine if resistance to Cry2Ab is associated with mutations affecting ABCA2 in other lepidopteran pests, particularly the field-evolved resistance to Cry2Ab in the US of Helicoverpa zea [4], a close relative of H. armigera. With global use of Cry2Ab increasing, more cases of field-evolved resistance are inevitable. For example, the risk is high for resistance of pink bollworm (Pectinophora gossypiella) to Cry2Ab in India, where the refuges of non-Bt host plants are scarce, resistance to Cry1Ac is widespread, and exposure to Cry2Ab is extensive [8,18,27,28]. Better understanding of the role of ABCA2 in the mode of action and mechanism of resistance to Cry2Ab may enhance our capacity to counter such resistance.

CDK4 amplification reduces sensitivity to CDK4/6 inhibition in fusion-positive rhabdomyosarcoma

Abstract: Purpose: Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and includes a PAX3- or PAX7-FOXO1 fusion-positive subtype. Amplification of chromosomal region 12q13-q14, which contains the CDK4 proto-oncogene, was identified in an aggressive subset of fusion-positive RMS. CDK4/6 inhibitors have antiproliferative activity in CDK4-amplified liposarcoma and neuroblastoma, suggesting CDK4/6 inhibition as a potential therapeutic strategy in fusion-positive RMS. Experimental Procedures: We examined the biological consequences of CDK4 knockdown, CDK4 overexpression, and pharmacologic CDK4/6 inhibition by LEE011 in fusion-positive RMS cell lines and xenografts. Results: Knockdown of CDK4 abrogated proliferation and transformation of 12q13-14-amplified and non-amplified fusion-positive RMS cells via G1-phase cell cycle arrest. This arrest was mediated by reduced RB phosphorylation and E2F-responsive gene expression. Significant differences in E2F target expression, cell cycle distribution, proliferation, or transformation were not observed in RMS cells overexpressing CDK4. Treatment with LEE011 phenocopied CDK4 knockdown, decreasing viability, RB phosphorylation, and E2F-responsive gene expression and inducing G1-phase cell cycle arrest. Though all fusion-positive cell lines showed sensitivity to CDK4/6 inhibition, there was diminished sensitivity associated with CDK4 amplification and overexpression. This variable responsiveness to LEE011 was recapitulated in xenograft models of CDK4-amplified and non-amplified fusion-positive RMS. Conclusions: Our data demonstrate that CDK4 is necessary but overexpression is not sufficient for RB-E2F-mediated G1-phase cell cycle progression, proliferation, and transformation in fusion-positive RMS. Our studies indicate that LEE011 is active in the setting of fusion-positive RMS and suggest that low CDK4-expressing fusion-positive tumors may be particularly susceptible to CDK4/6 inhibition.

Phelan McDermid Syndrome: From Genetic Discoveries to Animal Models and Treatment.

Abstract: Phelan-McDermid syndrome or 22q13.3 deletion syndrome is a rare neurodevelopmental disorder characterized by generalized developmental delay, intellectual disability, absent or delayed speech, seizures, autism spectrum disorder, neonatal hypotonia, physical dysmorphic features, and recurrent medical comorbidities. Individuals with Phelan-McDermid syndrome have terminal deletions of the chromosomal region 22q13.3 encompassing SHANK3, a gene encoding a structural component of excitatory synapses indispensable for proper synaptogenesis and neuronal physiology, or point mutations within the gene. Here, we review the clinical aspects of the syndrome and the genetic findings shedding light onto the underlying etiology. We also provide an overview on the evidence from genetic studies and mouse models that supports SHANK3 haploinsufficiency as a major contributor of the neurobehavioral manifestations of Phelan-McDermid syndrome. Finally, we discuss how all these discoveries are uncovering the pathophysiology of P...

PHF2 histone demethylase acts as a tumor suppressor in association with p53 in cancer.

Abstract: Plant homeodomain finger 2 (PHF2) has a role in epigenetic regulation of gene expression by demethylating H3K9-Me2. Several genome-wide studies have demonstrated that the chromosomal region including the PHF2 gene is often deleted in some cancers including colorectal cancer, and this finding encouraged us to investigate the tumor suppressive role of PHF2. As p53 is a critical tumor suppressor in colon cancer, we tested the possibility that PHF2 is an epigenetic regulator of p53. PHF2 was associated with p53, and thereby, promoted p53-driven gene expression in cancer cells under genotoxic stress. PHF2 converted the chromatin that is favorable for transcription by demethylating the repressive H3K9-Me2 mark. In an HCT116 xenograft model, PHF2 was found to be required for the anticancer effects of oxaliplatin and doxorubicin. In PHF2-deficient xenografts, p53 expression was profoundly induced by both drugs, but its downstream product p21 was not, suggesting that p53 cannot be activated in the absence of PHF2. To find clinical evidence about the role of PHF2, we analyzed the expressions of PHF2, p53 and p21 in human colon cancer tissues and adjacent normal tissues from patients. PHF2 was downregulated in cancer tissues and PHF2 correlated with p21 in cancers expressing functional p53. Colon and stomach cancer tissue arrays showed a positive correlation between PHF2 and p21 expressions. Informatics analyses using the Oncomine database also supported our notion that PHF2 is downregulated in colon and stomach cancers. On the basis of these findings, we propose that PHF2 acts as a tumor suppressor in association with p53 in cancer development and ensures p53-mediated cell death in response to chemotherapy.

Focal dysplasia of the cerebral cortex and infantile spasms associated with somatic 1q21.1-q44 duplication including the AKT3 gene

Abstract: Somatic and germline duplications or activating mutations of AKT3 have been reported in patients with hemimegalencephaly and megalencephaly. We performed array comparative genomic hybridization on brain tissue and blood in 16 consecutive patients with symptomatic epilepsy due to focal or multilobar malformations of cortical development who underwent surgical treatment of epilepsy. One patient with infantile spasms and a dysplastic left frontal lobe harboured a somatic trisomy of the 1q21.1-q44 chromosomal region, encompassing the AKT3 gene, in the dysplastic brain tissue but not in blood and saliva. Histopathology revealed severe cortical dyslamination, a thin cortex in the premotor area with microgyri and microsulci, immature neurons with disoriented dendrites and areas of cortical heterotopia in the sub-cortical white matter. These cytoarchitectural changes are close to those defining type Ib focal cortical dysplasia. Immunohistochemistry in brain specimens showed hyperactivation of the PI3K/AKT/mTOR pathway. These findings indicate that AKT3 upregulation may cause focal malformations of cortical development. There appears to be an etiologic continuum between hemimegalencephaly and focal cortical dysplastic lesions. The extent of brain malformations due to AKT3 upregulation may be related to the embryonic stage when the post-zygotic gene alteration occurs.

Heterotic loci for various morphological traits of maize detected using a single segment substitution lines test-cross population

Abstract: Heterosis has been exploited to increase grain yield, quality, and resistance in many crops, and it plays an important role in plant breeding. However, the genetic mechanism of heterosis remains unclear. To dissect the genetic basis of heterosis, a set of 203 single segment substitution lines (SSSLs) was developed, and its test-cross population was used to identify heterotic loci (HL) for plant morphological traits in maize, including plant height (PH), ear height (EH), leaf number (LN), tassel main axis length (TMAL), and tassel branch number (TBN). A total of 41 QTLs and 37 HL were identified for five morphological traits in the test-cross population derived from the 203 SSSLs and the parent, Xu178. Nine HL for PH, nine HL for EH, seven HL for LN, seven HL for TMAL, and five HL for TBN were detected in three different environments, respectively. Eight HL, ph1a, ph1b, ph2, ph5, eh3a, eh3b, eh10, and tmal1b, were simultaneously detected in the three environments. Among the 37 HL, only 10 (27.03 %; for PH, EH, LN, and TBN) had a corresponding QTL (24.39 %) sharing the same chromosomal region. Of all the HL, 21.4 % showed dominance effects, 76.8 % showed over-dominance effects, and only one (1.8 %) showed a partial-dominance effect. This result illustrated that heterosis and performance was controlled by different genetic mechanisms, and over-dominance effects were the main contributors to heterosis for plant-related traits at the single-locus level in maize.

Inventory of the GH70 enzymes encoded by Leuconostoc citreum NRRL B-1299 - identification of three novel α-transglucosylases.

Abstract: Leuconostoc citreum NRRL B-1299 has long been known to produce α-glucans containing both α-(1→6) and α-(1→2) linkages, which are synthesized by α-transglucosylases of the GH70 family. We sequenced the genome of Leuconostoc citreum NRRL B-1299 to identify the full inventory of GH70 enzymes in this strain. Three new genes (brsA, dsrM and dsrDP) putatively encoding GH70 enzymes were identified. The corresponding recombinant enzymes were characterized. Branching sucrase A (BRS-A) grafts linear α-(1→6) dextran with α-(1→2)-linked glucosyl units, and is probably involved in the α-(1→2) branching of L. citreum NRRL B-1299 dextran. This is the first report of a naturally occurring α-(1→2) branching sucrase. DSR-M and DSR-DP are dextransucrases that are specific for α-(1→6) linkage synthesis and mainly produce oligomers or short dextrans with molar masses between 580 and 27 000 g·mol(-1) . In addition, DSR-DP contains sequences that diverge from the consensus sequences that are typically present in enzymes that synthesize linear dextran. Comparison of the genome with five other L. citreum genomes further revealed that dsrDP is unique to L. citreum NRRL B-1299. The presence of this gene in a prophage represents the first evidence of phage-mediated horizontal transfer of genes encoding such enzymes in lactic acid bacteria. Finally, brsA and dsrM are located in a chromosomal region in which genes encoding strain-specific GH70 enzymes are consistently located. This region may be a good target on which to focus in order to rapidly evaluate the diversity of GH70 enzymes in L. citreum strains.

From UBE3A to Angelman syndrome: a substrate perspective.

Abstract: Angelman syndrome (AS) is a debilitating neurodevelopmental disorder that is characterized by motor dysfunction, intellectual disability, speech impairment, seizures and common features of autism spectrum disorders (ASDs). Some of these AS related phenotypes can be seen in other neurodevelopmental disorders (Williams, 2011;Tan et al., 2014). AS patients commonly carry mutations that render the maternally inherited UBE3A gene nonfunctional. Duplication of the chromosomal region containing the UBE3A gene is associated with ASDs. Although the causative role for UBE3A gene mutations in AS is well established, a long-standing challenge in AS research has been to identify neural substrates of UBE3A, an E3 ubiquitin ligase. A prevailing hypothesis is that changes in UBE3A protein levels would alter the levels of a collection of protein substrates, giving rise to the unique phenotypic aspects of AS and possibly UBE3A associated ASDs. Interestingly, proteins altered in AS are linked to additional ASDs that are not previously associated with changes in UBE3A, indicating a possible molecular overlap underlying the broad-spectrum phenotypes of these neurogenetic disorders. This idea raises the possibility that there may exist a “one-size-fits-all” approach to the treatment of neurogenetic disorders with phenotypes overlapping AS. Furthermore, while a comprehensive list of UBE3A substrates and downstream affected pathways should be developed, this is only part of the story. The timing of when UBE3A protein functions, through either changes in UBE3A or possibly substrate expression patterns, appears to be critical for AS phenotype development. These data call for further investigation of UBE3A substrates and their timing of action relevant to AS phenotypes.

Structure and Function of Centromeric and Pericentromeric Heterochromatin in Arabidopsis thaliana.

Abstract: The centromere is a specific chromosomal region where the kinetochore assembles to ensure the faithful segregation of sister chromatids during mitosis and meiosis. Centromeres are defined by a local enrichment of the specific histone variant CenH3 mostly at repetitive satellite sequences. A larger pericentromeric region containing repetitive sequences and transposable elements surrounds the centromere that adopts a particular chromatin state characterized by specific histone variants and post-translational modifications and forms a transcriptionally repressive chromosomal environment. In the model organism Arabidopsis thaliana centromeric and pericentromeric domains form conspicuous heterochromatin clusters called chromocenters in interphase. Here we discuss, using Arabidopsis as example, recent insight into mechanisms involved in maintenance and establishment of centromeric and pericentromeric chromatin signatures as well as in chromocenter formation.

Hnrnph1 Is A Quantitative Trait Gene for Methamphetamine Sensitivity.

Abstract: Psychostimulant addiction is a heritable substance use disorder; however its genetic basis is almost entirely unknown. Quantitative trait locus (QTL) mapping in mice offers a complementary approach to human genome-wide association studies and can facilitate environment control, statistical power, novel gene discovery, and neurobiological mechanisms. We used interval-specific congenic mouse lines carrying various segments of chromosome 11 from the DBA/2J strain on an isogenic C57BL/6J background to positionally clone a 206 kb QTL (50,185,512-50,391,845 bp) that was causally associated with a reduction in the locomotor stimulant response to methamphetamine (2 mg/kg, i.p.; DBA/2J < C57BL/6J)-a non-contingent, drug-induced behavior that is associated with stimulation of the dopaminergic reward circuitry. This chromosomal region contained only two protein coding genes-heterogeneous nuclear ribonucleoprotein, H1 (Hnrnph1) and RUN and FYVE domain-containing 1 (Rufy1). Transcriptome analysis via mRNA sequencing in the striatum implicated a neurobiological mechanism involving a reduction in mesolimbic innervation and striatal neurotransmission. For instance, Nr4a2 (nuclear receptor subfamily 4, group A, member 2), a transcription factor crucial for midbrain dopaminergic neuron development, exhibited a 2.1-fold decrease in expression (DBA/2J < C57BL/6J; p 4.2 x 10-15). Transcription activator-like effector nucleases (TALENs)-mediated introduction of frameshift deletions in the first coding exon of Hnrnph1, but not Rufy1, recapitulated the reduced methamphetamine behavioral response, thus identifying Hnrnph1 as a quantitative trait gene for methamphetamine sensitivity. These results define a novel contribution of Hnrnph1 to neurobehavioral dysfunction associated with dopaminergic neurotransmission. These findings could have implications for understanding the genetic basis of methamphetamine addiction in humans and the development of novel therapeutics for prevention and treatment of substance abuse and possibly other psychiatric disorders.

The dark side of ZNF217, a key regulator of tumorigenesis with powerful biomarker value

Abstract: The recently described oncogene ZNF217 belongs to a chromosomal region that is frequently amplified in human cancers. Recent findings have revealed that alternative mechanisms such as epigenetic regulation also govern the expression of the encoded ZNF217 protein. Newly discovered molecular functions of ZNF217 indicate that it orchestrates complex intracellular circuits as a new key regulator of tumorigenesis. In this review, we focus on recent research on ZNF217-driven molecular functions in human cancers, revisiting major hallmarks of cancer and highlighting the downstream molecular targets and signaling pathways of ZNF217. We also discuss the exciting translational medicine investigating ZNF217 expression levels as a new powerful biomarker, and ZNF217 as a candidate target for future anti-cancer therapies.

Patterns of HER2 Gene Amplification and Response to Anti-HER2 Therapies

Abstract: A chromosomal region that includes the gene encoding HER2, a receptor tyrosine kinase (RTK), is amplified in 20% of breast cancers. Although these tumors tend to respond to drugs directed against HER2, they frequently become resistant and resume their malignant progression. Gene amplification in double minutes (DMs), which are extrachromosomal entities whose number can be dynamically regulated, has been suggested to facilitate the acquisition of resistance to therapies targeting RTKs. Here we show that ~30% of HER2-positive tumors show amplification in DMs. However, these tumors respond to trastuzumab in a similar fashion than those with amplification of the HER2 gene within chromosomes. Furthermore, in different models of resistance to anti-HER2 therapies, the number of DMs containing HER2 is maintained, even when the acquisition of resistance is concomitant with loss of HER2 protein expression. Thus, both clinical and preclinical data show that, despite expectations, loss of HER2 protein expression due to loss of DMs containing HER2 is not a likely mechanism of resistance to anti-HER2 therapies.

The Contribution of Common Genetic Variation to Nicotine and Cotinine Glucuronidation in Multiple Ethnic/Racial Populations

Abstract: Background: The lung cancer risk of smokers varies by race/ethnicity even after adjustment for smoking. Evaluating the role of genetics in nicotine metabolism is likely important in understanding these differences, as disparities in risk may be related to differences in nicotine dose and metabolism. Methods: We conducted a genome-wide association study in search of common genetic variants that predict nicotine and cotinine glucuronidation in a sample of 2,239 smokers (437 European Americans, 364 African Americans, 453 Latinos, 674 Japanese Americans and 311 Native Hawaiians) in the Multiethnic Cohort Study. Urinary concentration of nicotine and its metabolites were determined. Results: Among 11,892,802 variants analyzed, 1,241 were strongly associated with cotinine glucuronidation, 490 of which were also associated with nicotine glucuronidation (p<5x10-8). The vast majority were within chromosomal region 4q13, near UGT2B10. Fifteen independent and globally significant SNPs explained 33.2% of the variation in cotinine glucuronidation, ranging from 55% for African Americans to 19% for Japanese Americans. The strongest single SNP association was for rs115765562 (p=1.60 x 10-155). This SNP is highly correlated with a UGT2B10 splice site variant, rs116294140, which together with rs6175900 (Asp67Tyr) explain 24.3% of the variation. The top SNP for nicotine glucuronidation (rs116224959, p=2.56 x 10-43) was in high LD (r2=.99) with rs115765562. Conclusions: Genetic variation in UGT2B10 contributions significantly to nicotine and cotinine glucuronidation but not to nicotine dose. Impact: The contribution of genetic variation to nicotine and cotinine glucuronidation varies significantly by racial/ethnic group, but is unlikely to contribute directly to lung cancer risk.