Mark D. Garfinkel

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.

Intronic Non-CG DNA hydroxymethylation and alternative mRNA splicing in honey bees

BackgroundPrevious whole-genome shotgun bisulfite sequencing experiments showed that DNA cytosine methylation in the honey bee (Apis mellifera) is almost exclusively at CG dinucleotides in exons. However, the most commonly used method, bisulfite sequencing, cannot distinguish 5-methylcytosine from 5-hydroxymethylcytosine, an oxidized form of 5-methylcytosine that is catalyzed by the TET family of dioxygenases. Furthermore, some analysis software programs under-represent non-CG DNA methylation and hydryoxymethylation for a variety of reasons. Therefore, we used an unbiased analysis of bisulfite sequencing data combined with molecular and bioinformatics approaches to distinguish 5-methylcytosine from 5-hydroxymethylcytosine. By doing this, we have performed the first whole genome analyses of DNA modifications at non-CG sites in honey bees and correlated the effects of these DNA modifications on gene expression and alternative mRNA splicing.ResultsWe confirmed, using unbiased analyses of whole-genome shotgun bisulfite sequencing (BS-seq) data, with both new data and published data, the previous finding that CG DNA methylation is enriched in exons in honey bees. However, we also found evidence that cytosine methylation and hydroxymethylation at non-CG sites is enriched in introns. Using antibodies against 5-hydroxmethylcytosine, we confirmed that DNA hydroxymethylation at non-CG sites is enriched in introns. Additionally, using a new technique, Pvu-seq (which employs the enzyme PvuRts1l to digest DNA at 5-hydroxymethylcytosine sites followed by next-generation DNA sequencing), we further confirmed that hydroxymethylation is enriched in introns at non-CG sites.ConclusionsCytosine hydroxymethylation at non-CG sites might have more functional significance than previously appreciated, and in honey bees these modifications might be related to the regulation of alternative mRNA splicing by defining the locations of the introns.

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.

Methods for nutrigenomics and longevity studies in Drosophila effects of diets high in sucrose, palmitic acid, soy, or beef

Nutrigenomics is the study of gene-nutrient interactions and how they affect the health and metabolism of an organism. Combining nutrigenomics with longevity studies is a natural extension and promises to help identify mechanisms whereby nutrients affect the aging process, life span, and, with the incorporation of age-dependent functional measures, health span. The topics we discuss in this chapter are genetic techniques, dietary manipulations, metabolic studies, and microarray analysis methods to investigate how nutrition affects gene expression, life span, triglyceride levels, total protein levels, and live weight in Drosophila. To better illustrate nutrigenomic techniques, we analyzed Drosophila larvae or adults fed control diets (high sucrose) and compared these with larvae or adults fed diets high in the saturated fat palmitic acid, soy, or 95% lean ground beef. The main results of these studies are, surprisingly, that triglyceride and total protein levels are significantly decreased by the beef diet in all adults, and total protein levels are significantly increased in male flies fed the soy diet. Furthermore, and less surprisingly, we found that all three experimental diets significantly decreased longevity and increased the length of time to develop from egg to adult. We also describe preliminary microarray results with adult flies fed the different diets, which suggest that only about 2-3% of the approx 18,000 genes have significantly altered mRNA expression levels compared with flies fed a control sucrose diet. The significance of these results and other types of nutrigenomics and longevity analyses is discussed.

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.

Methods for Nutrigenomics and Longevity Studies in Drosophila

Nutrigenomics is the study of gene-nutrient interactions and how they affect the health and metabolism of an organism. Combining nutrigenomics with longevity studies is a natural extension and promises to help identify mechanisms whereby nutrients affect the aging process, life span, and, with the incorporation of age-dependent functional measures, health span. The topics we discuss in this chapter are genetic techniques, dietary manipulations, metabolic studies, and microarray analysis methods to investigate how nutrition affects gene expression, life span, triglyceride levels, total protein levels, and live weight in Drosophila. To better illustrate nutrigenomic techniques, we analyzed Drosophila larvae or adults fed control diets (high sucrose) and compared these with larvae or adults fed diets high in the saturated fat palmitic acid, soy, or 95% lean ground beef. The main results of these studies are, surprisingly, that triglyceride and total protein levels are significantly decreased by the beef diet in all adults, and total protein levels are significantly increased in male flies fed the soy diet. Furthermore, and less surprisingly, we found that all three experimental diets significantly decreased longevity and increased the length of time to develop from egg to adult. We also describe preliminary microarray results with adult flies fed the different diets, which suggest that only about 2-3% of the approx 18,000 genes have significantly altered mRNA expression levels compared with flies fed a control sucrose diet. The significance of these results and other types of nutrigenomics and longevity analyses is discussed.

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.