Mark E. Berres

Aging and Cancer-Related Loss of Insulin-like Growth Factor 2 Imprinting in the Mouse and Human Prostate

Loss of imprinting (LOI) is an epigenetic alteration involving loss of parental origin-specific expression at normally imprinted genes. A LOI for Igf2, a paracrine growth factor, is important in cancer progression. Epigenetic modifications may be altered by environmental factors. However, is not known whether changes in imprinting occur with aging in prostate and other tissues susceptible to cancer development. We found a LOI for Igf2 occurs specifically in the mouse prostate associated with increased Igf2 expression during aging. In older animals, expression of the chromatin insulator protein CTCF and its binding to the Igf2-H19 imprint control region was reduced. Forced down-regulation of CTCF leads to Igf2 LOI. We further show that Igf2 LOI occurs with aging in histologically normal human prostate tissues and that this epigenetic alteration was more extensive in men with associated cancer. This finding may contribute to a postulated field of cancer susceptibility that occurs with aging. Moreover, Igf2 LOI may serve as a marker for the presence of prostate cancer.

High-throughput transcriptome sequencing identifies candidate genetic modifiers of vulnerability to fetal alcohol spectrum disorders.

BACKGROUND Fetal alcohol spectrum disorders (FASD) is a leading cause of neurodevelopmental disability. Genetic factors can modify vulnerability to FASD, but these elements are poorly characterized. METHODS We performed high-throughput transcriptional profiling to identify gene candidates that could potentially modify vulnerability to ethanols (EtOHs) neurotoxicity. We interrogated a unique genetic resource, neuroprogenitor cells from 2 closely related Gallus gallus lines having well-characterized robust or attenuated EtOH responses with respect to intracellular calcium mobilization and CaMKII/β-catenin-dependent apoptosis. Samples were not exposed to EtOH prior to analysis. RESULTS We identified 363 differentially expressed genes in neuroprogenitors from these 2 lines. Kyoto Encyclopedia of Genes and Genomes analysis revealed several gene clusters having significantly differential enrichment in gene expression. The largest and most significant cluster comprised ribosomal proteins (38 genes, p = 1.85 × 10(-47) ). Other significantly enriched gene clusters included metabolism (25 genes, p = 0.0098), oxidative phosphorylation (18 genes, p = 1.10 × 10(-11) ), spliceosome (13 genes, p = 7.02 × 10(-8) ), and protein processing in the endoplasmic reticulum (9 genes, p = 0.0011). Inspection of gene ontogeny (GO) terms identified 24 genes involved in the calcium/β-catenin signals that mediate EtOHs neurotoxicity in this model, including β-catenin itself and both calmodulin isoforms. CONCLUSIONS Four of the identified pathways with altered transcript abundance mediate the flow of cellular information from RNA to protein. Importantly, ribosome biogenesis also senses nucleolar stress and regulates p53-mediated apoptosis in neural crest. Human ribosomopathies produce craniofacial malformations and 11 known ribosomopathy genes were differentially expressed in this model of neural crest apoptosis. Rapid changes in ribosome expression are consistently observed in EtOH-treated mouse embryo neural folds, a model that is developmentally similar to ours. The recurring identification of ribosome biogenesis suggests it is a candidate modifier of EtOH vulnerability. These results highlight this approachs efficacy to formulate new, mechanistic hypotheses regarding EtOHs developmental damage.

Impact of non-lethal genetic sampling on the survival, longevity and behaviour of the Hermes copper (Lycaena hermes) butterfly

Genetic techniques are important tools for conservation, but tissue sampling for DNA analysis can be particularly detrimental to small study organisms. Historically, obtaining DNA samples from small insects and butterflies has involved destructive (lethal) methods. Recent improvements to DNA purification technologies have increased the likelihood that non‐lethal sampling will be successful. In spite of this, only a few studies have evaluated the impacts of sampling on survival and behaviour. The Hermes copper, Lycaena hermes (Edwards), butterfly has a restricted distribution and generally less than 10 individuals are encountered at any one location. Non‐lethal DNA sampling would allow for genetic studies that have the potential to augment conservation decisions without causing local extirpations. We demonstrate that removing a leg from an adult male Hermes copper does not have a measureable effect on their survival, longevity or behaviour. In addition, a single leg provides a sufficient DNA sample for amplified fragment length polymorphism studies. The Hermes copper butterfly represents the smallest butterfly species for which the survival and behaviour has been assessed in relation to non‐lethal tissue sampling. This suggests that research involving smaller and more delicate species could utilise leg removal as a non‐lethal genetic sampling technique.

Genetic variation of major histocompatibility complex (MHC) in wild Red Junglefowl (Gallus gallus)

The major histocompatibility complex (MHC) is a multi-family gene cluster that encodes proteins with immuno-responsive function. While studies of MHC in domesticated poultry are relatively common, very little is known about this highly polymorphic locus in wild Red Junglefowl (Gallus gallus), the natural progenitor of domestic chickens. We investigated the diversity of MHC within and among four wild Red Junglefowl populations across diversified natural habitats in South Central Vietnam. Based on a SNP panel of 84 sites spanning 210 Kb of the MHC-B locus, we identified 310 unique haplotypes in 398 chromosomes. None of these haplotypes have been described before and we did not observe any of the wild Red Junglefowl haplotypes in domesticated chickens. Analysis of molecular variance (AMOVA) revealed that 94.51% of observed haplotype variation was accounted for at the within individual level. Little genetic variance was apportioned within and among populations, the latter accounting only for 0.83%. We also found evidence of increased recombination, including numerous hotspots, and limited linkage disequilibrium among the 84 SNP sites. Compared to an average haplotype diversity of 3.55% among seventeen lines of domestic chickens, our results suggest extraordinarily high haplotype diversity remains in wild Red Junglefowl and is consistent with a pattern of balancing selection. Wild Red Junglefowl in Vietnam, therefore, represent a rich resource of natural genomic variation independent from artificial selection.

Genomic factors that shape craniofacial outcome and neural crest vulnerability in FASD

Prenatal alcohol exposure (PAE) causes distinctive facial characteristics in some pregnancies and not others; genetic factors may contribute to this differential vulnerability. Ethanol disrupts multiple events of neural crest development, including induction, survival, migration, and differentiation. Animal models and genomic approaches have substantially advanced our understanding of the mechanisms underlying these facial changes. PAE during gastrulation produces craniofacial changes corresponding with human fetal alcohol syndrome. These result because PAE reduces prechordal plate extension and suppresses sonic hedgehog, leading to holoprosencephaly and malpositioned facial primordia. Haploinsufficiency in sonic hedgehog signaling increases vulnerability to facial deficits and may influence some PAE pregnancies. In contrast, PAE during early neurogenesis produces facial hypoplasia, preceded by neural crest reductions due to significant apoptosis. Factors mediating this apoptosis include intracellular calcium mobilization, elevated reactive oxygen species, and loss of trophic support from β-catenin/calcium, sonic hedgehog, and mTOR signaling. Genome-wide SNP analysis links PDGFRA with facial outcomes in human PAE. Multiple genomic-level comparisons of ethanol-sensitive and – resistant early embryos, in both mouse and chick, independently identify common candidate genes that may potentially modify craniofacial vulnerability, including ribosomal proteins, proteosome, RNA splicing, and focal adhesion. In summary, research using animal models with genome-level differences in ethanol vulnerability, as well as targeted loss-and gain-of-function mutants, has clarified the mechanisms mediating craniofacial change in PAE. The findings additionally suggest that craniofacial deficits may represent a gene–ethanol interaction for some affected individuals. Genetic-level changes may prime individuals toward greater sensitivity or resistance to ethanol’s neurotoxicity.

AFFINITIES OF THE SAW-BILLED HERMIT (RAMPHODON NAEVIUS) DETERMINED BY CYTOCHROME-B SEQUENCE DATA

Abstract We sequenced 912 bp of the cytochrome-b gene to examine phylogenetic relationships of the enigmatic Saw-billed Hermit (Ramphodon naevius), a large and distinctive hummingbird endemic to tropical forests of southeastern Brazil. Bootstrapped maximum parsimony and maximum likelihood analyses of sequence data from 11 hummingbirds and several outgroups (two swifts, one goatsucker) support: (a) monophyly of the traditional hermit (Phaethornithinae) and nonhermit (Trochilinae) subfamilies, (b) placement of Ramphodon among hermits, and (c) a sister relationship between Ramphodon and an exemplar of the widespread polytypic hermit genus Glaucis. The association of Ramphodon with derived hermit lineages is concordant with subfamilial patterns of wing anatomy and nest architecture. However, the unusual plumages (striped underparts) and male bills (long, serrated, hooked) shared by Ramphodon and the Tooth-billed Hummingbird (Androdon aequatorialis) appear to have evolved within separate hermit and nonhermit “tooth-billed” clades. Distal placement of the Ramphodon-Glaucis clade within hermits implies that even distinctive Brazilian endemics such as Ramphodon are derived forms that evolved relatively recently.

MHC variability in heritage breeds of chickens

The chicken Major Histocompatibility Complex (MHC) is very strongly associated with disease resistance and thus is a very important region of the chicken genome. Historically, MHC (B locus) has been identified by the use of serology with haplotype specific alloantisera. These antisera can be difficult to produce and frequently cross-react with multiple haplotypes and hence their application is generally limited to inbred and MHC-defined lines. As a consequence, very little information about MHC variability in heritage chicken breeds is available. DNA-based methods are now available for examining MHC variability in these previously uncharacterized populations. A high density SNP panel consisting of 101 SNP that span a 230,000 bp region of the chicken MHC was used to examine MHC variability in 17 heritage populations of chickens from five universities from Canada and the United States. The breeds included 6 heritage broiler lines, 3 Barred Plymouth Rock, 2 New Hampshire and one each of Rhode Island Red, Light Sussex, White Leghorn, Dark Brown Leghorn, and 2 synthetic lines. These heritage breeds contained from one to 11 haplotypes per line. A total of 52 unique MHC haplotypes were found with only 10 of them identical to serologically defined haplotypes. Furthermore, nine MHC recombinants with their respective parental haplotypes were identified. This survey confirms the value of these non-commercially utilized lines in maintaining genetic diversity. The identification of multiple MHC haplotypes and novel MHC recombinants indicates that diversity is being generated and maintained within these heritage populations.

Transcriptome Profiling Identifies Ribosome Biogenesis as a Target of Alcohol Teratogenicity and Vulnerability during Early Embryogenesis

Fetal alcohol spectrum disorder (FASD) is a leading cause of neurodevelopmental disability. Individuals with FASD may exhibit a characteristic facial appearance that has diagnostic utility. The mechanism by which alcohol disrupts craniofacial development is incompletely understood, as are the genetic factors that can modify individual alcohol vulnerability. Using an established avian model, we characterized the cranial transcriptome in response to alcohol to inform the mechanism underlying these cells’ vulnerability. Gallus gallus embryos having 3–6 somites were exposed to 52 mM alcohol and the cranial transcriptomes were sequenced thereafter. A total of 3422 genes had significantly differential expression. The KEGG pathways with the greatest enrichment of differentially expressed gene clusters were Ribosome (P = 1.2 x 10−17, 67 genes), Oxidative Phosphorylation (P = 4.8 x 10−12, 60 genes), RNA Polymerase (P = 2.2 x 10−3, 15 genes) and Spliceosome (P = 2.6 x 10−2, 39 genes). The preponderance of transcripts in these pathways were repressed in response to alcohol. These same gene clusters also had the greatest altered representation in our previous comparison of neural crest populations having differential vulnerability to alcohol-induced apoptosis. Comparison of differentially expressed genes in alcohol-exposed (3422) and untreated, alcohol-vulnerable (1201) transcriptomes identified 525 overlapping genes of which 257 have the same direction of transcriptional change. These included 36 ribosomal, 25 oxidative phosphorylation and 7 spliceosome genes. Using a functional approach in zebrafish, partial knockdown of ribosomal proteins zrpl11, zrpl5a, and zrps3a individually heightened vulnerability to alcohol-induced craniofacial deficits and increased apoptosis. In humans, haploinsufficiency of several of the identified ribosomal proteins are causative in craniofacial dysmorphologies such as Treacher Collins Syndrome and Diamond-Blackfan Anemia. This work suggests ribosome biogenesis may be a novel target mediating alcohol’s damage to developing neural crest. Our findings are consistent with observations that gene-environment interactions contribute to vulnerability in FASD.

MHC-B variability within the Finnish Landrace chicken conservation program

ABSTRACT The major histocompatibility complex (MHC) is a cluster of genes involved with immune responses. The chicken MHC has been shown to influence resistance to viruses, bacteria, and infections from both internal and external parasites. The highly variable chicken MHC haplotypes were initially identified by the use of haplotype‐specific serological reagents. A novel SNP‐based panel encompassing 210,000 bp of the MHC‐B locus was developed to allow fine scale genetic analyses including rapid identification of novel haplotypes for which serological reagents are not available. The Finnish Landrace breed of chickens traces its origins to almost 1,000 years ago, with multiple lineages maintained as small populations in isolated villages. The breed is well adapted to the cooler Finnish climate and is considered to be an infrequent egg layer. Conservation efforts to protect this endangered breed were initiated by a hobby breeder in the 1960s. An official conservation program was established in 1998 and now 12 different populations are currently maintained by a network of volunteer hobbyist breeders. Variation in the MHC‐B region in these populations was examined using a panel of 90 selected SNP. A total of 195 samples from 12 distinct populations (average of 15 individuals sampled per population) were genotyped with the 90 SNP panel specific for the MHC‐B region, spanning 210,000 bp. There were 36 haplotypes found, 16 of which are a subset of 78 that had been previously identified in either commercially utilized or heritage breeds from North America with the remaining 20 haplotypes being novel. The average number of MHC‐B haplotypes found within each Finnish Landrace population was 5.9, and ranged from one to 13. While haplotypes common to multiple populations were found, population‐specific haplotypes were also identified. This study shows that substantial MHC‐B region diversity exists in the Finnish Landrace breed and exemplifies the significance tied to conserving multiple populations of rare breeds.

Social and environmental influences on tartary buckwheat (Fagopyrum tataricum Gaertn.) varietal diversity in Yunnan, China

Abstract Effective conservation strategies aimed to protect crop genetic resources require multiple sources of information. We used a combination of AFLP genotyping and farmer surveys to understand the extent, distribution and management of tartary buckwheat (Fagopyrum tataricum Gaertn.) diversity in its center of origin in Yunnan Province, China. We found genetic evidence of gene flow in tartary buckwheat throughout the study area, with small but statistically significant regional and village-level components. We also found genetic differentiation by seed color. Although most farmers reported exchanging seed in localized kinship networks, our results imply homogenizing gene flow is occurring. Yi ethnic farmers tend to plant more buckwheat than non-Yi farmers, and we found that in some communities, Yi farmers serve as seed sources for farmers of other ethnicities. Different tartary buckwheat varieties did not have different end uses; rather farmers maintained varietal diversity in order to protect crop yield and quality. Individual farmers’ seed exchange practices reflect their ideas about components of seed quality, as well as priorities in protecting buckwheat yield. From the standpoint of genetic resources conservation, the presence of a culturally rich farmer exchange network and hierarchical structuring of tartary buckwheat genetic diversity demonstrates the importance of maintaining an interlinked community of tartary buckwheat farmers in Yunnan.