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

Genetic and genomic architecture in eight strains of the laboratory opossum Monodelphis domestica

The gray short-tailed opossum (Monodelphis domestica) is an established laboratory-bred marsupial model for biomedical research. It is a critical species for comparative genomics research, providing the pivotal phylogenetic outgroup for studies of derived vs ancestral states of genomic/epigenomic characteristics for eutherian mammal lineages. To characterize the current genetic profile of this laboratory marsupial, we examined 79 individuals from eight established laboratory strains. Double digest restriction site-associated DNA sequencing and whole-genome resequencing experiments were performed to investigate the genetic architecture in these strains. A total of 66,640 highquality single nucleotide polymorphisms (SNPs) were identified. We analyzed SNP density, average heterozygosity, nucleotide diversity, and population differentiation parameter Fst within and between the eight strains. Principal component and population structure analysis clearly resolve the strains at the level of their ancestral founder populations, and the genetic architecture of these strains correctly reflects their breeding history. We confirmed the successful establishment of the first inbred laboratory opossum strain LSD (inbreeding coefficient F \u3e 0.99) and a nearly inbred strain FD2M1 (0.98 \u3c F \u3c 0.99), each derived from a different ancestral background. These strains are suitable for various experimental protocols requiring controlled genetic backgrounds and for intercrosses and backcrosses that can generate offspring with informative SNPs for studying a variety of genetic and epigenetic processes. Together with recent advances in reproductive manipulation and CRISPR/Cas9 techniques for Monodelphis domestica, the existence of distinctive inbred strains will enable genome editing on different genetic backgrounds, greatly expanding the utility of this marsupial model for biomedical research

SINEs, evolution and genome structure in the opossum

Short INterspersed Elements (SINEs) are non-autonomous retrotransposons, usually between 100 and 500 base pairs (bp) in length, which are ubiquitous components of eukaryotic genomes. Their activity, distribution, and evolution can be highly informative on genomic structure and evolutionary processes. To determine recent activity, we amplified more than one hundred SINE1 loci in a panel of 43 M. domestica individuals derived from five diverse geographic locations. The SINE1 family has expanded recently enough that many loci were polymorphic, and the SINE1 insertion-based genetic distances among populations reflected geographic distance. Genome-wide comparisons of SINE1 densities and GC content revealed that high SINE1 density is associated with high GC content in a few long and many short spans. Young SINE1s, whether fixed or polymorphic, showed an unbiased GC content preference for insertion, indicating that the GC preference accumulates over long time periods, possibly in periodic bursts. SINE1 evolution is thus broadly similar to human Alu evolution, although it has an independent origin. High GC content adjacent to SINE1s is strongly correlated with bias towards higher AT to GC substitutions and lower GC to AT substitutions. This is consistent with biased gene conversion, and also indicates that like chickens, but unlike eutherian mammals, GC content heterogeneity (isochore structure) is reinforced by substitution processes in the M. domestica genome. Nevertheless, both high and low GC content regions are apparently headed towards lower GC content equilibria, possibly due to a relative shift to lower recombination rates in the recent Monodelphis ancestral lineage. Like eutherians, metatherian (marsupial) mammals have evolved high CpG substitution rates, but this is apparently a convergence in process rather than a shared ancestral state. © 2007 Elsevier B.V. All rights reserved

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

Increased waist circumference is independently associated with hypothyroidism in Mexican Americans: replicative evidence from two large, population-based studies

BACKGROUND: Mexican Americans are at an increased risk of both thyroid dysfunction and metabolic syndrome (MS). Thus it is conceivable that some components of the MS may be associated with the risk of thyroid dysfunction in these individuals. Our objective was to investigate and replicate the potential association of MS traits with thyroid dysfunction in Mexican Americans. METHODS: We conducted association testing for 18 MS traits in two large studies on Mexican Americans – the San Antonio Family Heart Study (SAFHS) and the National Health and Nutrition Examination Survey (NHANES) 2007–10. A total of 907 participants from 42 families in SAFHS and 1633 unrelated participants from NHANES 2007–10 were included in this study. The outcome measures were prevalence of clinical and subclinical hypothyroidism and thyroid function index (TFI) – a measure of thyroid function. For the SAFHS, we used polygenic regression analyses with multiple covariates to test associations in setting of family studies. For the NHANES 2007–10, we corrected for the survey design variables as needed for association analyses in survey data. In both datasets, we corrected for age, sex and their linear and quadratic interactions. RESULTS: TFI was an accurate indicator of clinical thyroid status (area under the receiver-operating-characteristic curve to detect clinical hypothyroidism, 0.98) in both SAFHS and NHANES 2007–10. Of the 18 MS traits, waist circumference (WC) showed the most consistent association with TFI in both studies independently of age, sex and body mass index (BMI). In the SAFHS and NHANES 2007–10 datasets, each standard deviation increase in WC was associated with 0.13 (p < 0.001) and 0.11 (p < 0.001) unit increase in the TFI, respectively. In a series of polygenic and linear regression models, central obesity (defined as WC ≥ 102 cm in men and ≥88 cm in women) was associated with clinical and subclinical hypothyroidism independent of age, sex, BMI and type 2 diabetes in both datasets. Estimated prevalence of hypothyroidism was consistently high in those with central obesity, especially below 45y of age. CONCLUSIONS: WC independently associates with increased risk of thyroid dysfunction. Use of WC to identify Mexican American subjects at high risk of thyroid dysfunction should be investigated in future studies

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

Reconstructing an Ancestral Mammalian Immune Supercomplex from a Marsupial Major Histocompatibility Complex

The first sequenced marsupial genome promises to reveal unparalleled insights into mammalian evolution. We have used theMonodelphis domestica (gray short-tailed opossum) sequence to construct the first map of a marsupial major histocompatibility complex (MHC). The MHC is the most gene-dense region of the mammalian genome and is critical to immunity and reproductive success. The marsupial MHC bridges the phylogenetic gap between the complex MHC of eutherian mammals and the minimal essential MHC of birds. Here we show that the opossum MHC is gene dense and complex, as in humans, but shares more organizational features with non-mammals. The Class I genes have amplified within the Class II region, resulting in a unique Class I/II region. We present a model of the organization of the MHC in ancestral mammals and its elaboration during mammalian evolution. The opossum genome, together with other extant genomes, reveals the existence of an ancestral “immune supercomplex” that contained genes of both types of natural killer receptors together with antigen processing genes and MHC genes