Xiangyi Lu

A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3

We describe a new computer program, SnpEff, for rapidly categorizing the effects of variants in genome sequences. Once a genome is sequenced, SnpEff annotates variants based on their genomic locations and predicts coding effects. Annotated genomic locations include intronic, untranslated region, upstream, downstream, splice site, or intergenic regions. Coding effects such as synonymous or non-synonymous amino acid replacement, start codon gains or losses, stop codon gains or losses, or frame shifts can be predicted. Here the use of SnpEff is illustrated by annotating ~356,660 candidate SNPs in ~117 Mb unique sequences, representing a substitution rate of ~1/305 nucleotides, between the Drosophila melanogaster w1118; iso-2; iso-3 strain and the reference y1; cn1 bw1 sp1 strain. We show that ~15,842 SNPs are synonymous and ~4,467 SNPs are non-synonymous (N/S ~0.28). The remaining SNPs are in other categories, such as stop codon gains (38 SNPs), stop codon losses (8 SNPs), and start codon gains (297 SNPs) in the 5′UTR. We found, as expected, that the SNP frequency is proportional to the recombination frequency (i.e., highest in the middle of chromosome arms). We also found that start-gain or stop-lost SNPs in Drosophila melanogaster often result in additions of N-terminal or C-terminal amino acids that are conserved in other Drosophila species. It appears that the 5′ and 3′ UTRs are reservoirs for genetic variations that changes the termini of proteins during evolution of the Drosophila genus. As genome sequencing is becoming inexpensive and routine, SnpEff enables rapid analyses of whole-genome sequencing data to be performed by an individual laboratory.

Evidence for an epigenetic mechanism by which Hsp90 acts as a capacitor for morphological evolution.

Morphological alterations have been shown to occur in Drosophila melanogaster when function of Hsp90 (heat shock 90-kDa protein 1α, encoded by Hsp83) is compromised during development. Genetic selection maintains the altered phenotypes in subsequent generations. Recent experiments have shown, however, that phenotypic variation still occurs in nearly isogenic recombinant inbred strains of Arabidopsis thaliana. Using a sensitized isogenic D. melanogaster strain, iso-KrIf-1, we confirm this finding and present evidence supporting an epigenetic mechanism for Hsp90s capacitor function, whereby reduced activity of Hsp90 induces a heritably altered chromatin state. The altered chromatin state is evidenced by ectopic expression of the morphogen wingless in eye imaginal discs and a corresponding abnormal eye phenotype, both of which are epigenetically heritable in subsequent generations, even when function of Hsp90 is restored. Mutations in nine different genes of the trithorax group that encode chromatin-remodeling proteins also induce the abnormal phenotype. These findings suggest that Hsp90 acts as a capacitor for morphological evolution through epigenetic and genetic mechanisms.

Using Drosophila melanogaster as a Model for Genotoxic Chemical Mutational Studies with a New Program, SnpSift

This paper describes a new program SnpSift for filtering differential DNA sequence variants between two or more experimental genomes after genotoxic chemical exposure. Here, we illustrate how SnpSift can be used to identify candidate phenotype-relevant variants including single nucleotide polymorphisms, multiple nucleotide polymorphisms, insertions, and deletions (InDels) in mutant strains isolated from genome-wide chemical mutagenesis of Drosophila melanogaster. First, the genomes of two independently isolated mutant fly strains that are allelic for a novel recessive male-sterile locus generated by genotoxic chemical exposure were sequenced using the Illumina next-generation DNA sequencer to obtain 20- to 29-fold coverage of the euchromatic sequences. The sequencing reads were processed and variants were called using standard bioinformatic tools. Next, SnpEff was used to annotate all sequence variants and their potential mutational effects on associated genes. Then, SnpSift was used to filter and select differential variants that potentially disrupt a common gene in the two allelic mutant strains. The potential causative DNA lesions were partially validated by capillary sequencing of polymerase chain reaction-amplified DNA in the genetic interval as defined by meiotic mapping and deletions that remove defined regions of the chromosome. Of the five candidate genes located in the genetic interval, the Pka-like gene CG12069 was found to carry a separate pre-mature stop codon mutation in each of the two allelic mutants whereas the other four candidate genes within the interval have wild-type sequences. The Pka-like gene is therefore a strong candidate gene for the male-sterile locus. These results demonstrate that combining SnpEff and SnpSift can expedite the identification of candidate phenotype-causative mutations in chemically mutagenized Drosophila strains. This technique can also be used to characterize the variety of mutations generated by genotoxic chemicals.

PKD2 cation channel is required for directional sperm movement and male fertility.

Sperm of both mammals and invertebrates move toward specific sites in the female reproductive tract. However, molecular mechanisms for sperm to follow directional cues are unknown. Here, we report genetic analysis of Drosophila Pkd2 at 33E3 (Pkd2, CG6504), which encodes a Ca(2+)-activated, nonselective cation channel homologous to the human Pkd2 autosomal dominant polycystic kidney disease (ADPKD) gene. The PKD2 family of genes has been implicated in sensory responses through protein localization on primary cilia of epithelia and neurons. In renal tubules, cilium-associated PKD2 appears to mediate Ca(2+) influx in response to fluid flow, and the loss of fluid sensation probably contributes to cyst growth and ADPKD. Sperm tails or flagella are specialized cilia essential for movement. Drosophila Pkd2 is abundantly associated with the tail and the acrosome-containing head region of mature sperm. Targeted disruption of Pkd2 results in male sterility without affecting spermatogenesis. The mutant sperm are motile but fail to swim into the storage organs in the female. Rare mutant sperm that reach the storage organs are able to fertilize the egg and produce viable progeny. Our data demonstrate that the Drosophila PKD2 cation channel operates in sperm for directional movement inside the female reproductive tract.

Hypothesis: Environmental regulation of 5-hydroxymethylcytosine by oxidative stress

Many environmental toxins, such as heavy metals, air particles, and ozone, induce oxidative stress and decrease the levels of NADH and NADPH, cofactors that drive anabolic biochemical reactions and provide reducing capacity to combat oxidative stress. Recently, it was found that the Ten-eleven translocation (TET) protein family members, which oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in the DNA, is activated under high oxygen conditions by alpha ketoglutarate (-KG), a cofactor produced by aerobic metabolism in the citric acid cycle. TET, Jumonji-family histone demethylases, and prolyl hydroxylase, a repressor of HIF1a under high oxygen conditions, all require alpha ketoglutarate (a-KG) as cofactors for their activation. The impact of the HIF1a and TET proteins, which appear to have opposing functions, reaches several aspects of human life-including cell growth regulation, embryonic stem cell maintenance, cell differentiation, and tumorigenesis. The role of metabolism on regulating global DNA methylation and chromatin organization is recently demanding greater attention from the biomedical research community. This article will discuss the possible role of TET activation and the regulation of 5hmC and 5mC levels in response to environmental stress. We will also discuss how 5hmC and 5mC levels at the promoters of specific genes might be a useful biomarker for exposure to environmental toxins.

Hsp90 Inhibitors and Drug Resistance in Cancer: The Potential Benefits of Combination Therapies of Hsp90 Inhibitors and Other Anti-Cancer Drugs

Hsp90 is a chaperone protein that interacts with client proteins that are known to be in the cell cycle, signaling and chromatin-remodeling pathways. Hsp90 inhibitors act additively or synergistically with many other drugs in the treatment of both solid tumors and leukemias in murine tumor models and humans. Hsp90 inhibitors potentiate the actions of anti-cancer drugs that target Hsp90 client proteins, including trastuzumab (Herceptin™) which targets Her2/Erb2B, as Hsp90 inhibition elicits the drug effects in cancer cell lines that are otherwise resistant to the drug. A phase II study of the Hsp90 inhibitor 17-AAG and trastuzumab showed that this combination therapy has anticancer activity in patients with HER2-positive metastatic breast cancer progressing on trastuzumab. In this review, we discuss the results of Hsp90 inhibitors in combination with trastuzumab and other cancer drugs. We also discuss recent results from yeast focused on the genetics of drug resistance when Hsp90 is inhibited and the implications that this might have in understanding the effects of genetic variation in treating cancer in humans.

Hsp90 Affecting Chromatin Remodeling Might Explain Transgenerational Epigenetic Inheritance in Drosophila

Transgenerational epigenetic inheritance, while poorly understood, is of great interest because it might help explain the increase in the incidence of diseases with an environmental contribution in humans, such as cancer, diabetes, and heart disease. Here, we review five Drosophila examples of transgenerational epigenetic inheritance and propose a unified mechanism that involves Polycomb Response Element/Trithorax Response Element (PRE/TRE) occupancy by either Polycomb Group (PcG) protein complexes or Trithorax group (TrxG) complexes. Among their other activities, PcG complexes cause histone 3 lysine 27 tri-methylation associated with repressed chromatin, whereas Trithorax group (TrxG) complexes induce histone 3 lysine 4 tri-methylation associated with actively transcribed chromatin. In this model, Hsp90 is an environmentally sensitive chromatin remodeling regulator that causes a switch in the chromatin from a permissive state to a non-permissive state for transcription. Consistent with this model, Hsp90 has recently been shown to be a chaperone for Tah1p (TPR-containing protein associated with Hsp90) and Pih1p (protein interacting with Hsp90), which connect to the chromatin remodelling factor Rvb1p (RuvB-like protein 1)/Rvb2p in yeast [1]. Also, Hsp90 is required for optimal activity of the histone H3 lysine-4 methyltransferase SMYD3 in mammals [2, 3]. Since PcG and TrxG complexes are involved in the post-translational modifications of histones, and since such modifications have been shown to be required to maintain imprinted marks, this unified mechanism might also help to explain transgenerational epigenetic inheritance in humans.

Multigenerational epigenetic inheritance in humans: DNA methylation changes associated with maternal exposure to lead can be transmitted to the grandchildren.

We report that the DNA methylation profile of a child’s neonatal whole blood can be significantly influenced by his or her mother’s neonatal blood lead levels (BLL). We recruited 35 mother-infant pairs in Detroit and measured the whole blood lead (Pb) levels and DNA methylation levels at over 450,000 loci from current blood and neonatal blood from both the mother and the child. We found that mothers with high neonatal BLL correlate with altered DNA methylation at 564 loci in their children’s neonatal blood. Our results suggest that Pb exposure during pregnancy affects the DNA methylation status of the fetal germ cells, which leads to altered DNA methylation in grandchildren’s neonatal dried blood spots. This is the first demonstration that an environmental exposure in pregnant mothers can have an epigenetic effect on the DNA methylation pattern in the grandchildren.

Multigenerational selection and detection of altered histone acetylation and methylation patterns: toward a quantitative epigenetics in Drosophila.

Quantitative epigenetics (QE) is a new area of research that combines some of the techniques developed for global quantitative trait loci (QTL) mapping analyses with epigenetic analyses. Quantitative traits such as height vary, not in a discrete or discontinuous fashion, but continuously, usually in a normal distribution. QTL analyses assume that allelic DNA sequence variation in a population is partly responsible for the trait variation, and the aim is to deduce the locations of the contributing genes. QE analyses assume that epigenetic variation in a population is partly responsible for the trait variation, and the aim is to associate inheritance of the trait with segregation of informative epigenetic polymorphisms, or epialleles. QTL and QE analyses are thus complementary, but the latter has several advantages. QTL mapping is limited in resolution because of meiotic recombination and population size, placing quantitative traits on genomic regions that are each typically several megabase-pairs long, and requires DNA sequence variation. In contrast, QE analysis can make use of powerful emerging mapping techniques that allow the positioning of epialleles defined by chromatin variation to individual genes or chromosomal regions, even in the absence of DNA sequence variation. In this chapter, we present a case study for QE analysis-epigenetic mapping of enhancers of the KrIf-1 ectopic eye bristle phenotype in an isogenic strain of Drosophila melanogaster.

Drosophila melanogaster as a model for lead neurotoxicology and toxicogenomics research

Drosophila melanogaster is an excellent model animal for studying the neurotoxicology of lead. It has been known since ancient Roman times that long-term exposure to low levels of lead results in behavioral abnormalities, such as what is now known as attention deficit hyperactivity disorder (ADHD). Because lead alters mechanisms that underlie developmental neuronal plasticity, chronic exposure of children, even at blood lead levels below the current CDC community action level (10 μg/dl), can result in reduced cognitive ability, increased likelihood of delinquency, behaviors associated with ADHD, changes in activity level, altered sensory function, delayed onset of sexual maturity in girls, and changes in immune function. In order to better understand how lead affects neuronal plasticity, we will describe recent findings from a Drosophila behavioral genetics laboratory, a Drosophila neurophysiology laboratory, and a Drosophila quantitative genetics laboratory who have joined forces to study the effects of lead on the Drosophila nervous system. Studying the effects of lead on Drosophila nervous system development will give us a better understanding of the mechanisms of Pb neurotoxicity in the developing human nervous system.