Genomic Imprinting and X-Chromosome Inactivation in the Gray, Short-Tailed Opossum, Monodelphis domestica

Imprinted genes have been extensively documented in eutherian mammals and exhibit significant interspecific variation, both in the suites of genes that are imprinted and in their regulation between tissues and developmental stages. Much less is known about imprinted loci in metatherian (marsupial) mammals, wherein studies have been limited to a small number of genes imprinted in eutherians. In this dissertation, I used ChIP-seq and RNA-seq approaches to conduct the first ab initio search for imprinted autosomal genes in fibroblasts, fetal brain, and placenta of a metatherian mammal, the gray short-tailed opossum, Monodelphis domestica, and the first chromosome-wide study of paternally imprinted metatherian X chromosome inactivation. Evidence from a few genes in diverse species suggests that metatherian X- chromosome inactivation is characterized by exclusive, but incomplete (leaky), repression of genes on the paternally derived X chromosome. Herein I show that the majority of opossum X-linked genes exhibit paternally imprinted expression with 100% maternal-allele expression, whereas ~14% of genes escape inactivation, exhibiting varying levels of biallelic expression. In addition, I have shown that transcriptionally opposing histone modifications correlate strongly with opossum XCI. However, the opossum did not show an association between X-linked gene expression and promoter DNA methylation. In generating the first genome-wide profile of histone modification states for a metatherian mammal, and coupling it with in-depth gene expression analyses, I identified the first set of genes imprinted in a metatherian that are not imprinted in eutherian mammals and described transcriptionally opposing histone modifications and differential DNA methylation at the promoters of a subset of these genes. My findings suggest that metatherians use multiple epigenetic mechanisms to mark imprinted genes and support the concept that lineage-specific selective forces can produce sets of imprinted genes that differ between metatherian and eutherian lines. Overall, these studies furnish a comprehensive catalog of parent-of-origin expression status for both autosomal and X-linked genes in a metatherian, Monodelphis domestica, and open new avenues for illuminating the mechanisms and evolution of imprinted gene regulation in mammals generally

Origin and Evolution of Marsupial-specific Imprinting Clusters Through Lineage-specific Gene Duplications and Acquisition of Promoter Differential Methylation

Genomic imprinting is a parent-of-origin-specific expression phenomenon that plays fundamental roles in many biological processes. In animals, imprinting is only observed in therian mammals, with ∼200 imprinted genes known in humans and mice. The imprinting pattern in marsupials has been minimally investigated by examining orthologs to known eutherian imprinted genes. To identify marsupial-specific imprinting in an unbiased way, we performed RNA-seq studies on samples of fetal brain and placenta from the reciprocal cross progeny of two laboratory opossum stocks. We inferred allele-specific expression for \u3e3,000 expressed genes and discovered/validated 13 imprinted genes, including three previously known imprinted genes, Igf2r, Peg10, and H19. We estimate that marsupials imprint ∼60 autosomal genes, which is a much smaller set compared with eutherians. Among the nine novel imprinted genes, three noncoding RNAs have no known homologs in eutherian mammals, while the remaining genes have important functions in pluripotency, transcription regulation, nucleolar homeostasis, and neural differentiation. Methylation analyses at promoter CpG islands revealed differentially methylated regions in five of these marsupial-specific imprinted genes, suggesting that differential methylation is a common mechanism in the epigenetic regulation of marsupial imprinting. Clustering and co-regulation were observed at marsupial imprinting loci Pou5f3-Npdc1 and Nkrfl-Ipncr2, but eutherian-type multi-gene imprinting clusters were not detected. Also differing from eutherian mammals, the brain and placenta imprinting profiles are remarkably similar in opossums, presumably due to the shared origin of these organs from the trophectoderm. Our results contribute to a fuller understanding of the origin, evolution, and mechanisms of genomic imprinting in therian mammals

Genome-wide histone state profiling of fibroblasts from the opossum, Monodelphis domestica, identifies the first marsupial-specific imprinted gene

BACKGROUND: Imprinted genes have been extensively documented in eutherian mammals and found to exhibit significant interspecific variation in the suites of genes that are imprinted and in their regulation between tissues and developmental stages. Much less is known about imprinted loci in metatherian (marsupial) mammals, wherein studies have been limited to a small number of genes previously known to be imprinted in eutherians. We describe the first ab initio search for imprinted marsupial genes, in fibroblasts from the opossum, Monodelphis domestica, based on a genome-wide ChIP-seq strategy to identify promoters that are simultaneously marked by mutually exclusive, transcriptionally opposing histone modifications. RESULTS: We identified a novel imprinted gene (Meis1) and two additional monoallelically expressed genes, one of which (Cstb) showed allele-specific, but non-imprinted expression. Imprinted vs. allele-specific expression could not be resolved for the third monoallelically expressed gene (Rpl17). Transcriptionally opposing histone modifications H3K4me3, H3K9Ac, and H3K9me3 were found at the promoters of all three genes, but differential DNA methylation was not detected at CpG islands at any of these promoters. CONCLUSIONS: In generating the first genome-wide histone modification profiles for a marsupial, we identified the first gene that is imprinted in a marsupial but not in eutherian mammals. This outcome demonstrates the practicality of an ab initio discovery strategy and implicates histone modification, but not differential DNA methylation, as a conserved mechanism for marking imprinted genes in all therian mammals. Our findings suggest that marsupials use multiple epigenetic mechanisms for imprinting and support the concept that lineage-specific selective forces can produce sets of imprinted genes that differ between metatherian and eutherian lines

Early Developmental and Evolutionary Origins of Gene Body DNA Methylation Patterns in Mammalian Placentas

Over the last 20-80 million years the mammalian placenta has taken on a variety of morphologies through both divergent and convergent evolution. Recently we have shown that the human placenta genome has a unique epigenetic pattern of large partially methylated domains (PMDs) and highly methylated domains (HMDs) with gene body DNA methylation positively correlating with level of gene expression. In order to determine the evolutionary conservation of DNA methylation patterns and transcriptional regulatory programs in the placenta, we performed a genome-wide methylome (MethylC-seq) analysis of human, rhesus macaque, squirrel monkey, mouse, dog, horse, and cow placentas as well as opossum extraembryonic membrane. We found that, similar to human placenta, mammalian placentas and opossum extraembryonic membrane have globally lower levels of methylation compared to somatic tissues. Higher relative gene body methylation was the conserved feature across all mammalian placentas, despite differences in PMD/HMDs and absolute methylation levels. Specifically, higher methylation over the bodies of genes involved in mitosis, vesicle-mediated transport, protein phosphorylation, and chromatin modification was observed compared with the rest of the genome. As in human placenta, higher methylation is associated with higher gene expression and is predictive of genic location across species. Analysis of DNA methylation in oocytes and preimplantation embryos shows a conserved pattern of gene body methylation similar to the placenta. Intriguingly, mouse and cow oocytes and mouse early embryos have PMD/HMDs but their placentas do not, suggesting that PMD/HMDs are a feature of early preimplantation methylation patterns that become lost during placental development in some species and following implantation of the embryo

Comparing the genetic diversity of late Pleistocene Bison with Modern Bison bison using ancient DNA techniques and the mitochondrial DNA control region.

Includes bibliographical references (p. 58-64).The transition between the Pleistocene and Holocene Epochs brought about a mass extinction of many large mammals. The genetic consequences of such widespread extinctions have not been well studied. Using ancient DNA and phylogenetic techniques, we compared the genetic diversity and phylogenetic relatedness of extinct Pleistocene Bison ranging from Siberia to mid-latitude North America (10,000 ybp to 50,000 ybp) to extant Bison bison. The mitochondrial DNA control region was sequenced from 10 Bison priscus skulls obtained from the Kolyma Region of Siberia, Russia. Control region sequences from other Pleistocene Bison and Bison bison were obtained from Genbank. Our analysis indicates a measurable loss of genetic diversity in Bison bison compared to Pleistocene Bison. Furthermore, the Pleistocene Bison population was strongest in North America from a time period of 30,000 ybp to 10,000 ybp, and the genetic diversity present in this population is not represented in the Bison bison population.by Kory C. Douglas.M.S

Early Developmental and Evolutionary Origins of Gene Body DNA Methylation Patterns in Mammalian Placentas.

Over the last 20-80 million years the mammalian placenta has taken on a variety of morphologies through both divergent and convergent evolution. Recently we have shown that the human placenta genome has a unique epigenetic pattern of large partially methylated domains (PMDs) and highly methylated domains (HMDs) with gene body DNA methylation positively correlating with level of gene expression. In order to determine the evolutionary conservation of DNA methylation patterns and transcriptional regulatory programs in the placenta, we performed a genome-wide methylome (MethylC-seq) analysis of human, rhesus macaque, squirrel monkey, mouse, dog, horse, and cow placentas as well as opossum extraembryonic membrane. We found that, similar to human placenta, mammalian placentas and opossum extraembryonic membrane have globally lower levels of methylation compared to somatic tissues. Higher relative gene body methylation was the conserved feature across all mammalian placentas, despite differences in PMD/HMDs and absolute methylation levels. Specifically, higher methylation over the bodies of genes involved in mitosis, vesicle-mediated transport, protein phosphorylation, and chromatin modification was observed compared with the rest of the genome. As in human placenta, higher methylation is associated with higher gene expression and is predictive of genic location across species. Analysis of DNA methylation in oocytes and preimplantation embryos shows a conserved pattern of gene body methylation similar to the placenta. Intriguingly, mouse and cow oocytes and mouse early embryos have PMD/HMDs but their placentas do not, suggesting that PMD/HMDs are a feature of early preimplantation methylation patterns that become lost during placental development in some species and following implantation of the embryo

Genome-wide methylation patterns in mammalian placentas show both large-scale divergence and gene-specific similarities.

<p>(A) Phylogenetic tree of the species studied and the classification of their placenta types. Branch lengths are not to scale. (B) Density curves of average percent methylation in non-overlapping 20 kb windows in mammalian placentas. For comparison, the methylation distribution for brain tissue is shown in beige for some species and human cord blood is shown in aquamarine. The opossum brain sample was fetal whereas the other brain samples were postnatal. The interquartile range and medians are shown as black bars and white dots, respectively, for the placenta samples. Mouse brain data is from Hon et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005442#pgen.1005442.ref019" target="_blank">19</a>] (GSE42836), human placenta data is from Schroeder et al [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005442#pgen.1005442.ref021" target="_blank">21</a>] (GSE25930). (C) Comparison of global methylation patterns in select species after liftOver to the human genome and smoothing (for full figure, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005442#pgen.1005442.s004" target="_blank">S4 Fig</a>). Human placenta PMDs, as determined by HMM, are shown in black bars at top. (D) Comparison of methylation patterns at the <i>CNTNAP2</i> locus in select species (for full figure, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005442#pgen.1005442.s005" target="_blank">S5 Fig</a>). Raw species CpG site methylation wig data were graphed on the UCSC Genome Browser without preprocessing. Black line represents 50% methylation. Genes of interest are orange. EEM = extra-embryonic membrane.</p

Regions of high methylation in placentas cover gene bodies.

<p>(A) Heatmap of average methylation in the gene bodies (introns and exons, excluding CpG islands and promoters) of orthologous genes. Only the top few GO biological processes with Benjamini p-values below 1.0E-3 are shown. For a complete list see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005442#pgen.1005442.s020" target="_blank">S3 Table</a>. Branches A and B were combined because they contain similar GO terms. (B) Comparison of percent methylation between human placenta (red curve) and rhesus placenta (purple curve). Rhesus methylation data was lifted over to the human genome. Vertical purple lines show large chromosomal breaks in synteny between the two species. The fourth ring in shows regions of higher (blue) and lower (red) methylation in human placenta compared to rhesus. The fourth circle in shows the locations of human genes in black. (C) Spinograms showing the probability that a 5 kb window is in a gene given that window's average percent methylation. Bars are color-coded by percent methylation and bar widths show the percentage of windows with that methylation level. Bars furthest from the 0.5 blue line marker show the most information about gene location.</p

Gene methylation and expression in oocytes compared to placenta in mouse and cow.

<p>(A) Distribution of average methylation in 50 kb windows (first column) and gene bodies (second column) during early human, mouse, and cow development. The third column shows the relationship between average gene body methylation and gene expression in oocytes. Black lines show the marginal distribution of percent methylation in gene bodies. All protein-coding genes from each species were used. Human oocyte and sperm methylation data are from Okae et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005442#pgen.1005442.ref027" target="_blank">27</a>] (JGAS00000000006), human oocyte expression data are from Reich et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005442#pgen.1005442.ref034" target="_blank">34</a>] (GSE32689), mouse oocyte and sperm methylation and expression data are from Wang et al [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005442#pgen.1005442.ref024" target="_blank">24</a>] (GSE56697), and cow oocyte expression data are from Graf et al [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005442#pgen.1005442.ref033" target="_blank">33</a>] (GSE52415). (B) Heatmap of average gene body methylation in human, mouse, and cow oocytes. For a complete list of all GO and KEGG terms for each quadrant, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005442#pgen.1005442.s020" target="_blank">S3 Table</a>.</p