Luan Wang

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.

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.

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.

Massively parallel resequencing of the isogenic Drosophila melanogaster strain w(1118); iso-2; iso-3 identifies hotspots for mutations in sensory perception genes.

We used the Illumina reversible-short sequencing technology to obtain 17-fold average depth (s.d.~8) of ~94% of the euchromatic genome and ~1-5% of the heterochromatin sequence of the Drosophila melogaster isogenic strain w1118; iso-2; iso-3. We show that this strain has a ~9 kb deletion that uncovers the first exon of the white (w) gene, ~4 kb of downstream promoter sequences, and most of the first intron, thus demonstrating that whole-genome sequencing can be used for mutation characterization. We chose this strain because there are thousands of transposon insertion lines and hundreds of isogenic deficiency lines available with this genetic background, such as the Exelixis, Inc., and the DrosDEL collections. We compared our sequence to Release 5 of the finished reference genome sequence which was made from the isogenic strain y1; cn1 bw1 sp1 and identified ~356,614 candidate SNPs in the ~117 Mb unique sequence genome, which represents a substitution rate of ~1/305 nucleotides (~0.30%). The distribution of SNPs is not uniform, but rather there is a ~2-fold increase in SNPs on the autosome arms compared with the X chromosome and a ~7-fold increase when compared to the small 4th chromosome. This is consistent with previous analyses that demonstrated a correlation between recombination frequency and SNP frequency. An unexpected finding was a SNP hotpot in a ~20Mb central region of the 4th chromosome, which might indicate higher than expected recombination frequency in this region of this chromosome. Interestingly, genes involved in sensory perception are enriched in SNP hotspots and genes encoding developmental genes are enriched in SNP coldspots, which suggests that recombination frequencies might be proportional to the evolutionary selection coefficient. There are currently 12 Drosophila species sequenced, and this represents one of many isogenic Drosophila melanogaster genome sequences that are in progress. Because of the dramatic increase in power in using isogenic lines rather than outbred individuals, the SNP information should be valuable as a test bed for understanding genotype-by-environment interactions in human population studies.

Epigenetics as an answer to Darwin’s “special difficulty,” Part 2: natural selection of metastable epialleles in honeybee castes

In a recent perspective in this journal, Herb (2014) discussed how epigenetics is a possible mechanism to circumvent Charles Darwin’s “special difficulty” in using natural selection to explain the existence of the sterile-fertile dimorphism in eusocial insects. Darwin’s classic book “On the Origin of Species by Means of Natural Selection” explains how natural selection of the fittest individuals in a population can allow a species to adapt to a novel or changing environment. However, in bees and other eusocial insects, such as ants and termites, there exist two or more castes of genetically similar females, from fertile queens to multiple sub-castes of sterile workers, with vastly different phenotypes, lifespans, and behaviors. This necessitates the selection of groups (or kin) rather than individuals in the evolution of honeybee hives, but group and kin selection theories of evolution are controversial and mechanistically uncertain. Also, group selection would seem to be prohibitively inefficient because the effective population size of a colony is reduced from thousands to a single breeding queen. In this follow-up perspective, we elaborate on possible mechanisms for how a combination of both epigenetics, specifically, the selection of metastable epialleles, and genetics, the selection of mutations generated by the selected metastable epialleles, allows for a combined means for selection amongst the fertile members of a species to increase colony fitness. This “intra-caste evolution” hypothesis is a variation of the epigenetic directed genetic error hypothesis, which proposes that selected metastable epialleles increase genetic variability by directing mutations specifically to the epialleles. Natural selection of random metastable epialleles followed by a second round of natural selection of random mutations generated by the metastable epialleles would allow a way around the small effective population size of eusocial insects.

Hsp90 Inhibitors and the Reduction of Anti-Cancer Drug Resistance by Non-Genetic and Genetic Mechanisms

In this review, we focus on how inhibitors of Hsp90 can help prevent the resistance to anti-cancer drugs by synergistically increasing their cancer killing abilities and thereby allowing them to function at much lower concentrations than normally used. Hsp90 helps to fold numerous client proteins, such as Akt, Raf, Src, chromatin-modifying proteins, nuclear hormone receptors, and kinetochore assembly proteins. We discuss four mechanisms by which Hsp90 inhibitors can potentially synergize with anti-cancer drugs: by making a drug-resistant protein that is a client for Hsp90 more sensitive to the drug, by increasing chromosomal aneuploidy and the effectiveness of DNA-damaging drugs, by inhibiting Trithorax proteins which trimethylate histone 3 at lysine 4 (H3K4me3) and thereby decreasing expression of tumor promoter genes, and by interacting with the negative elongation factor (NELF) complex in tumors. We also explain how the evolutionary capacitor function of Hsp90 can be exploited with inhibitors of Hsp90 by exposing new protein variants that can be targeted with other drugs, thereby opening new avenues of combination drug therapy to treat cancer. We believe that inhibition of these processes can increase the efficacy of Hsp90 inhibitors with other anti-cancer drugs.

Transgenerational Epigenetic Inheritance in Drosophila

Transgenerational epigenetic inheritance involves the inheritance of a phenotype across at least one generation that does not involve any changes in the DNA sequence. The primary mark of transgenerational epigenetic inheritance is thought to be DNA methylation, such as in imprinting in mammals and in the inheritance of coat color in agouti viable yellow (Avy) mice. However, while most studies of Drosophila melanogaster indicate that there is no DNA cytosine methylation, nevertheless several systems of transgenerational epigenetic inheritance have been demonstrated in this organism. In this chapter, we review several Drosophila transgenerational epigenetic systems, including a system that we developed in our laboratory that involves the transgenerational epigenetic inheritance of an ectopic large bristle outgrowth (ELBO) in the eyes of D. melanogaster that can be passed from generation to generation for hundreds of generations. Understanding transgenerational epigenetic inheritance mechanisms in Drosophila can have a profound impact in understanding similar processes in humans in which environmental exposures can affect the health of future generations.

Hsp90 as a Capacitor of Both Genetic and Epigenetic Changes in the Genome During Cancer Progression and Evolution

In this chapter, we focus on the role of the chaperone protein Hsp90 as a capacitor for morphological variation that is released during times of stress. Hsp90 helps to fold numerous client proteins, which constitute a veritable “who’s who” of important signaling molecules, such as Akt, Raf, Src, chromatin-modifying proteins, nuclear hormone receptors, and kinetochore assembly proteins. We first review evidence that Hsp90 functions trans-generationally as a capacitor for morphological variation via both genetic and epigenetic means: in the former by revealing cryptic genetic variation and in the latter by generating heritable epialleles. Then we discuss two mechanisms by which altered Hsp90 function can mutate DNA: transposon mobilization, and chromosomal aneuploidy. Next, we hypothesize how beneficial cryptic epigenetic variation might be stabilized, or locked in place, by the directed DNA-level mutation of epigenetically assimilated epialleles. Finally, we describe how Hsp90 functions intra-generationally within an organism’s lifetime by releasing cryptic phenotypic variation during development in a stressful environment, and how this can be hijacked during the progression of diseases such as cancer.