Craig Obergfell

A new class of retroviral and satellite encoded small RNAs emanates from mammalian centromeres.

The transcriptional framework of the eukaryotic centromere core has been described in budding yeast and rice, but for most eukaryotes and all vertebrates it remains largely unknown. The lack of large pericentric repeats in the tammar wallaby has made it possible to map and identify the transcriptional units at the centromere in a mammalian species for the first time. We show that these transcriptional units, comprised of satellites and a retrovirus, are bound by centromere proteins and that they are the source of a novel class of small RNA. The endogenous retrovirus from which these small RNAs are derived is now known to be in the centromere domain of several vertebrate classes. The discovery of this new RNA form brings together several independent lines of evidence that point to a conserved retroviral-encoded processed RNA entity within eukaryotic centromeres.

Genomic Instability within Centromeres of Interspecific Marsupial Hybrids

Several lines of evidence suggest that, within a lineage, particular genomic regions are subject to instability that can lead to specific types of chromosome rearrangements important in species incompatibility. Within family Macropodidae (kangaroos, wallabies, bettongs, and potoroos), which exhibit recent and extensive karyotypic evolution, rearrangements involve chiefly the centromere. We propose that centromeres are the primary target for destabilization in cases of genomic instability, such as interspecific hybridization, and participate in the formation of novel chromosome rearrangements. Here we use standard cytological staining, cross-species chromosome painting, DNA probe analyses, and scanning electron microscopy to examine four interspecific macropodid hybrids (Macropus rufogriseus × Macropus agilis). The parental complements share the same centric fusions relative to the presumed macropodid ancestral karyotype, but can be differentiated on the basis of heterochromatic content, M. rufogriseus having larger centromeres with large C-banding positive regions. All hybrids exhibited the same pattern of chromosomal instability and remodeling specifically within the centromeres derived from the maternal (M. rufogriseus) complement. This instability included amplification of a satellite repeat and a transposable element, changes in chromatin structure, and de novo whole-arm rearrangements. We discuss possible reasons and mechanisms for the centromeric instability and remodeling observed in all four macropodid hybrids.

Genome-Wide Reprogramming in Hybrids of Somatic Cells and Embryonic Stem Cells

Recent experiments demonstrate that somatic nuclei can be reprogrammed to a pluripotent state when fused to ESCs. The resulting hybrids are pluripotent as judged by developmental assays, but detailed analyses of the underlying molecular‐genetic control of reprogrammed transcription in such hybrids are required to better understand fusion‐mediated reprogramming. We produced hybrids of mouse ESCs and fibroblasts that, although nearly tetraploid, exhibit characteristics of normal ESCs, including apparent immortality in culture, ESC‐like colony morphology, and pluripotency. Comprehensive analysis of the mouse embryonic fibroblast/ESC hybrid transcriptome revealed global patterns of gene expression reminiscent of ESCs. However, combined analysis of variance and hierarchical clustering analyses revealed at least seven distinct classes of differentially regulated genes in comparisons of hybrids, ESCs, and somatic cells. The largest class includes somatic genes that are silenced in hybrids and ESCs, but a smaller class includes genes that are expressed at nearly equivalent levels in hybrids and ESCs that contain many genes implicated in pluripotency and chromatin function. Reprogrammed genes are distributed throughout the genome. Reprogramming events include both transcriptional silencing and activation of genes residing on chromosomes of somatic origin. Somatic/ESC hybrid cell lines resemble their pre‐fusion ESC partners in terms of behavior in culture and pluripotency. However, they contain unique expression profiles that are similar but not identical to normal ESCs. ESC fusion‐mediated reprogramming provides a tractable system for the investigation of mechanisms of reprogramming.

Signatures of Rapid Evolution in Urban and Rural Transcriptomes of White-Footed Mice ( Peromyscus leucopus ) in the New York Metropolitan Area

Urbanization is a major cause of ecological degradation around the world, and human settlement in large cities is accelerating. New York City (NYC) is one of the oldest and most urbanized cities in North America, but still maintains 20% vegetation cover and substantial populations of some native wildlife. The white-footed mouse, Peromyscus leucopus , is a common resident of NYC’s forest fragments and an emerging model system for examining the evolutionary consequences of urbanization. In this study, we developed transcriptomic resources for urban P . leucopus to examine evolutionary changes in protein-coding regions for an exemplar “urban adapter.” We used Roche 454 GS FLX+ high throughput sequencing to derive transcriptomes from multiple tissues from individuals across both urban and rural populations. From these data, we identified 31,015 SNPs and several candidate genes potentially experiencing positive selection in urban populations of P . leucopus . These candidate genes are involved in xenobiotic metabolism, innate immune response, demethylation activity, and other important biological phenomena in novel urban environments. This study is one of the first to report candidate genes exhibiting signatures of directional selection in divergent urban ecosystems.

Allelic expression of IGF2 in live-bearing, matrotrophic fishes

The parental conflict, or kinship, theory of genomic imprinting predicts that parent-specific gene expression may evolve in species in which parental investment in developing offspring is unequal. This theory explains many aspects of parent-of-origin transcriptional silencing of embryonic growth regulatory genes in mammals, but it has not been tested in any other live-bearing, placental animals. A major embryonic growth promoting gene with conserved function in all vertebrates is insulin-like growth factor 2 (IGF2). This gene is imprinted in both eutherians and marsupials, as are several genes that modulate IGF2 activity. We have tested for parent-of-origin influences on developmental expression of IGF2 in two poeciliid fish species, Heterandria formosa and Poeciliopsis prolifica, that have evolved placentation independently. We found IGF2 to be expressed bi-allelically throughout embryonic development in both species.

Two different high throughput sequencing approaches identify thousands of de novo genomic markers for the genetically depleted Bornean elephant.

High throughput sequencing technologies are being applied to an increasing number of model species with a high-quality reference genome. The application and analyses of whole-genome sequence data in non-model species with no prior genomic information are currently under way. Recent sequencing technologies provide new opportunities for gathering genomic data in natural populations, laying the empirical foundation for future research in the field of conservation and population genomics. Here we present the case study of the Bornean elephant, which is the most endangered subspecies of Asian elephant and exhibits very low genetic diversity. We used two different sequencing platforms, the Roche 454 FLX (shotgun) and Illumina, GAIIx (Restriction site associated DNA, RAD) to evaluate the feasibility of the two methodologies for the discovery of de novo markers (single nucleotide polymorphism, SNPs and microsatellites) using low coverage data. Approximately, 6,683 (shotgun) and 14,724 (RAD) SNPs were detected within our elephant sequence dataset. Genotyping of a representative sample of 194 SNPs resulted in a SNP validation rate of ∼ 83 to 94% and 17% of the loci were polymorphic with a low diversity (H o = 0.057). Different numbers of microsatellites were identified through shotgun (27,226) and RAD (868) techniques. Out of all di-, tri-, and tetra-microsatellite loci, 1,706 loci had sufficient flanking regions (shotgun) while only 7 were found with RAD. All microsatellites were monomorphic in the Bornean but polymorphic in another elephant subspecies. Despite using different sample sizes, and the well known differences in the two platforms used regarding sequence length and throughput, the two approaches showed high validation rate. The approaches used here for marker development in a threatened species demonstrate the utility of high throughput sequencing technologies as a starting point for the development of genomic tools in a non-model species and in particular for a species with low genetic diversity.

Determination of dosage compensation of the mammalian X chromosome by RNA-seq is dependent on analytical approach

BackgroundAn enduring question surrounding sex chromosome evolution is whether effective hemizygosity in the heterogametic sex leads inevitably to dosage compensation of sex-linked genes, and whether this compensation has been observed in a variety of organisms. Incongruence in the conclusions reached in some recent reports has been attributed to different high-throughput approaches to transcriptome analysis. However, recent reports each utilizing RNA-seq to gauge X-linked gene expression relative to autosomal gene expression also arrived at diametrically opposed conclusions regarding X chromosome dosage compensation in mammals.ResultsHere we analyze RNA-seq data from X-monosomic female human and mouse tissues, which are uncomplicated by genes that escape X-inactivation, as well as published RNA-seq data to describe relative X expression (RXE). We find that the determination of RXE is highly dependent upon a variety of computational, statistical and biological assumptions underlying RNA-seq analysis. Parameters implemented in short-read mapping programs, choice of reference genome annotation, expression data distribution, tissue source for RNA and RNA-seq library construction method have profound effects on comparing expression levels across chromosomes.ConclusionsOur analysis shows that the high number of paralogous gene families on the mammalian X chromosome relative to autosomes contributes to the ambiguity in RXE calculations, RNA-seq analysis that takes into account that single- and multi-copy genes are compensated differently supports the conclusion that, in many somatic tissues, the mammalian X is up-regulated compared to the autosomes.

Hypermorphic expression of centromeric retroelement-encoded small RNAs impairs CENP-A loading

The proper functioning of centromeres requires a complex cascade of epigenetic events involving chromatin and kinetochore assembly; however, the precise mechanism by which this cascade proceeds is unknown. The pivotal event during kinetochore formation is the “loading,” or deposition, of CENP-A. This histone H3 variant is specific to centromeres and replaces conventional H3 in centromeric chromatin. Failure to load CENP-A into mammalian centromeres in late telophase/early G1 of the cell cycle leads to malsegregation and cell division defects in subsequent cell cycles. Mounting evidence supports the hypothesis that an RNA component is involved, although how RNAs participate in centromere formation in mammals has remained unknown. Using the marsupial model, the tammar wallaby, we show that centromeric retroelements produce small RNAs and that hypermorphic expression of these centromeric small RNAs results in disruption of CENP-A localization. We propose that tight regulation of the processing of this new class of small RNAs, crasiRNAs, is an integral component of the epigenetic framework necessary for centromere establishment.

Recent Amplification of the Kangaroo Endogenous Retrovirus, KERV, Limited to the Centromere

ABSTRACT Mammalian retrotransposons, transposable elements that are processed through an RNA intermediate, are categorized as short interspersed elements (SINEs), long interspersed elements (LINEs), and long terminal repeat (LTR) retroelements, which include endogenous retroviruses. The ability of transposable elements to autonomously amplify led to their initial characterization as selfish or junk DNA; however, it is now known that they may acquire specific cellular functions in a genome and are implicated in host defense mechanisms as well as in genome evolution. Interactions between classes of transposable elements may exert a markedly different and potentially more significant effect on a genome than interactions between members of a single class of transposable elements. We examined the genomic structure and evolution of the kangaroo endogenous retrovirus (KERV) in the marsupial genus Macropus. The complete proviral structure of the kangaroo endogenous retrovirus, phylogenetic relationship among relative retroviruses, and expression of this virus in both Macropus rufogriseus and M. eugenii are presented for the first time. In addition, we show the relative copy number and distribution of the kangaroo endogenous retrovirus in the Macropus genus. Our data indicate that amplification of the kangaroo endogenous retrovirus occurred in a lineage-specific fashion, is restricted to the centromeres, and is not correlated with LINE depletion. Finally, analysis of KERV long terminal repeat sequences using massively parallel sequencing indicates that the recent amplification in M. rufogriseus is likely due to duplications and concerted evolution rather than a high number of independent insertion events.

Divergent patterns of breakpoint reuse in Muroid rodents

Multiple Genome Rearrangement (MGR) analysis was used to define the trajectory and pattern of chromosome rearrangement within muroid rodents. MGR was applied using 107 chromosome homologies between Mus, Rattus, Peromyscus, the muroid sister taxon Cricetulus griseus, and Sciurus carolinensis as a non-Muroidea outgroup, with specific attention paid to breakpoint reuse and centromere evolution. This analysis revealed a high level of chromosome breakpoint conservation between Rattus and Peromyscus and indicated that the chromosomes of Mus are highly derived. This analysis identified several conserved evolutionary breakpoints that have been reused multiple times during karyotypic evolution in rodents. Our data demonstrate a high level of reuse of breakpoints among muroid rodents, further supporting the “Fragile Breakage Model” of chromosome evolution. We provide the first analysis of rodent centromeres with respect to evolutionary breakpoints. By analyzing closely related rodent species we were able to clarify muroid rodent karyotypic evolution. We were also able to derive several high-resolution ancestral karyotypes and identify rearrangements specific to various stages of Muroidea evolution. These data were useful in further characterizing lineage-specific modes of chromosome evolution.