Immunofluorescent staining reveals hypermethylation of microchromosomes in the central bearded dragon, Pogona vitticeps

BACKGROUND: Studies of model organisms have demonstrated that DNA cytosine methylation and histone modifications are key regulators of gene expression in biological processes. Comparatively little is known about the presence and distribution of epigenetic marks in non-model amniotes such as non-avian reptiles whose genomes are typically packaged into chromosomes of distinct size classes. Studies of chicken karyotypes have associated the gene-richness and high GC content of microchromosomes with a distinct epigenetic landscape. To determine whether this is likely to be a common feature of amniote microchromosomes, we have analysed the distribution of epigenetic marks using immunofluorescence on metaphase chromosomes of the central bearded dragon (Pogona vitticeps). This study is the first to study the distribution of epigenetic marks on non-avian reptile chromosomes. RESULTS: We observed an enrichment of DNA cytosine methylation, active modifications H3K4me2 and H3K4me3, as well as the repressive mark H3K27me3 in telomeric regions on macro and microchromosomes. Microchromosomes were hypermethylated compared to macrochromosomes, as they are in chicken. However, differences between macro- and microchromosomes for histone modifications associated with actively transcribed or repressed DNA were either less distinct or not detectable. CONCLUSIONS: Hypermethylation of microchromosomes compared to macrochromosomes is a shared feature between P. vitticeps and avian species. The lack of the clear distinction between macro- and microchromosome staining patterns for active and repressive histone modifications makes it difficult to determine at this stage whether microchrosome hypermethylation is correlated with greater gene density as it is in aves, or associated with the greater GC content of P. vitticeps microchromosomes compared to macrochromosomes.This work was supported by a University of Canberra postdoctoral fellowship (awarded to TE, SR, Stephen Sarre, JED, Kris Hardy and Arthur Georges, and supporting RD and AL). TE is supported by an Australian Research Council Future Fellowship (FT110100733)

Origin and evolution of candidate mental retardation genes on the human X chromosome (MRX)

<p>Abstract</p> <p>Background</p> <p>The human X chromosome has a biased gene content. One group of genes that is over-represented on the human X are those expressed in the brain, explaining the large number of sex-linked mental retardation (MRX) syndromes.</p> <p>Results</p> <p>To determine if MRX genes were recruited to the X, or whether their brain-specific functions were acquired after relocation to the mammalian X chromosome, we examined the location and expression of their orthologues in marsupials, which diverged from human approximately 180 million years ago. We isolated and mapped nine tammar wallaby MRX homologues, finding that six were located on the tammar wallaby X (which represents the ancient conserved mammal X) and three on chromosome 5, representing the recently added region of the human X chromosome. The location of MRX genes within the same synteny groups in human and wallaby does not support the hypothesis that genes with an important function in the brain were recruited in multiple independent events from autosomes to the mammalian X chromosome. Most of the tammar wallaby MRX homologues were more widely expressed in tammar wallaby than in human. Only one, the tammar wallaby <it>ARX </it>homologue (located on tammar chromosome 5p), has a restricted expression pattern comparable to its pattern in human. The retention of the brain-specific expression of <it>ARX </it>over 180 million years suggests that this gene plays a fundamental role in mammalian brain development and function.</p> <p>Conclusion</p> <p>Our results suggest all the genes in this study may have originally had more general functions that became more specialised and important in brain function during evolution of humans and other placental mammals.</p

Activity map of the tammar X chromosome shows that marsupial X inactivation is incomplete and escape is stochastic

BACKGROUND: X chromosome inactivation is a spectacular example of epigenetic silencing. In order to deduce how this complex system evolved, we examined X inactivation in a model marsupial, the tammar wallaby (Macropus eugenii). In marsupials, X inactivation is known to be paternal, incomplete and tissue-specific, and occurs in the absence of an XIST orthologue. RESULTS: We examined expression of X-borne genes using quantitative PCR, revealing a range of dosage compensation for different loci. To assess the frequency of 1X- or 2X-active fibroblasts, we investigated expression of 32 X-borne genes at the cellular level using RNA-FISH. In female fibroblasts, two-color RNA-FISH showed that genes were coordinately expressed from the same X (active X) in nuclei in which both loci were inactivated. However, loci on the other X escape inactivation independently, with each locus showing a characteristic frequency of 1X-active and 2X-active nuclei, equivalent to stochastic escape. We constructed an activity map of the tammar wallaby inactive X chromosome, which identified no relationship between gene location and extent of inactivation, nor any correlation with the presence or absence of a Y-borne paralog. CONCLUSIONS: In the tammar wallaby, one X (presumed to be maternal) is expressed in all cells, but genes on the other (paternal) X escape inactivation independently and at characteristic frequencies. The paternal and incomplete X chromosome inactivation in marsupials, with stochastic escape, appears to be quite distinct from the X chromosome inactivation process in eutherians. We find no evidence for a polar spread of inactivation from an X inactivation center.This project was funded by grants to JAMG and PDW from the Australian Research Council

Activity map of the tammar X chromosome shows that marsupial X inactivation is incomplete and escape is stochastic

BACKGROUND: X chromosome inactivation is a spectacular example of epigenetic silencing. In order to deduce how this complex system evolved, we examined X inactivation in a model marsupial, the tammar wallaby (Macropus eugenii). In marsupials, X inactivation is known to be paternal, incomplete and tissue-specific, and occurs in the absence of an XIST orthologue. RESULTS: We examined expression of X-borne genes using quantitative PCR, revealing a range of dosage compensation for different loci. To assess the frequency of 1X- or 2X-active fibroblasts, we investigated expression of 32 X-borne genes at the cellular level using RNA-FISH. In female fibroblasts, two-color RNA-FISH showed that genes were coordinately expressed from the same X (active X) in nuclei in which both loci were inactivated. However, loci on the other X escape inactivation independently, with each locus showing a characteristic frequency of 1X-active and 2X-active nuclei, equivalent to stochastic escape. We constructed an activity map of the tammar wallaby inactive X chromosome, which identified no relationship between gene location and extent of inactivation, nor any correlation with the presence or absence of a Y-borne paralog. CONCLUSIONS: In the tammar wallaby, one X (presumed to be maternal) is expressed in all cells, but genes on the other (paternal) X escape inactivation independently and at characteristic frequencies. The paternal and incomplete X chromosome inactivation in marsupials, with stochastic escape, appears to be quite distinct from the X chromosome inactivation process in eutherians. We find no evidence for a polar spread of inactivation from an X inactivation center.This project was funded by grants to JAMG and PDW from the Australian Research Council

The tammar wallaby major histocompatibility complex shows evidence of past genomic instability

BACKGROUND The major histocompatibility complex (MHC) is a group of genes with a variety of roles in the innate and adaptive immune responses. MHC genes form a genetically linked cluster in eutherian mammals, an organization that is thought to confer functional and evolutionary advantages to the immune system. The tammar wallaby (Macropus eugenii), an Australian marsupial, provides a unique model for understanding MHC gene evolution, as many of its antigen presenting genes are not linked to the MHC, but are scattered around the genome. RESULTS Here we describe the 'core' tammar wallaby MHC region on chromosome 2q by ordering and sequencing 33 BAC clones, covering over 4.5 MB and containing 129 genes. When compared to the MHC region of the South American opossum, eutherian mammals and non-mammals, the wallaby MHC has a novel gene organization. The wallaby has undergone an expansion of MHC class II genes, which are separated into two clusters by the class III genes. The antigen processing genes have undergone duplication, resulting in two copies of TAP1 and three copies of TAP2. Notably, Kangaroo Endogenous Retroviral Elements are present within the region and may have contributed to the genomic instability. CONCLUSIONS The wallaby MHC has been extensively remodeled since the American and Australian marsupials last shared a common ancestor. The instability is characterized by the movement of antigen presenting genes away from the core MHC, most likely via the presence and activity of retroviral elements. We propose that the movement of class II genes away from the ancestral class II region has allowed this gene family to expand and diversify in the wallaby. The duplication of TAP genes in the wallaby MHC makes this species a unique model organism for studying the relationship between MHC gene organization and function.This work was funded by an ARC Discovery Grant to KB and SB, and a Wellcome Trust Grant (084071) to SB. HVS was supported by a University of Sydney Postgraduate Award and a William and Catherine McIlrath Scholarship for travel to the Sanger Institute. JK and HVS are supported in part by Wellcome Trust Programme grant 089305. KB is supported by a University of Sydney Thompson fellowship and an ARC Future Fellowship

A cross-species comparison of escape from X inactivation in Eutheria: implications for evolution of X chromosome inactivation

Sex chromosome dosage compensation in both eutherian and marsupial mammals is achieved by X chromosome inactivation (XCI)—transcriptional repression that silences one of the two X chromosomes in the somatic cells of females. We recently used RNA fluorescent in situ hybridization (FISH) to show, in individual nuclei, that marsupial X inactivation (in the absence of XIST) occurs on a gene-by-gene basis, and that escape from inactivation is stochastic and independent of gene location. In the absence of similar data from fibroblast cell lines of eutherian representatives, a meaningful comparison is lacking. We therefore used RNA-FISH to examine XCI in fibroblast cell lines obtained from three distantly related eutherian model species: African savannah elephant (Loxodonta africana), mouse (Mus musculus) and human (Homo sapiens). We show that, unlike the orthologous marsupial X, inactivation of the X conserved region (XCR) in eutherians generally is complete. Two-colour RNA-FISH on female human, mouse and elephant interphase nuclei showed that XCR loci have monoallelic expression in almost all nuclei. However, we found that many loci located in the evolutionarily distinct recently added region (XAR) displayed reproducible locus-specific frequencies of nuclei with either one or two active X alleles. We propose that marsupial XCI retains features of an ancient incomplete silencing mechanism that was augmented by the evolution of the XIST gene that progressively stabilized the eutherian XCR. In contrast, the recently added region of the eutherian X displays an incomplete inactivation profile similar to that observed on the evolutionarily distinct marsupial X and the independently evolved monotreme X chromosomes

A first-generation integrated tammar wallaby map and its use in creating a tammar wallaby first-generation virtual genome map.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: The limited (2X) coverage of the tammar wallaby (Macropus eugenii) genome sequence dataset currently presents a challenge for assembly and anchoring onto chromosomes. To provide a framework for this assembly, it would be a great advantage to have a dense map of the tammar wallaby genome. However, only limited mapping data are available for this non-model species, comprising a physical map and a linkage map. RESULTS: We combined all available tammar wallaby mapping data to create a tammar wallaby integrated map, using the Location DataBase (LDB) strategy. This first-generation integrated map combines all available information from the second-generation tammar wallaby linkage map with 148 loci, and extensive FISH mapping data for 492 loci, especially for genes likely to be located at the ends of wallaby chromosomes or at evolutionary breakpoints inferred from comparative information. For loci whose positions are only approximately known, their location in the integrated map was refined on the basis of comparative information from opossum (Monodelphis domestica) and human. Interpolation of segments from the opossum and human assemblies into the integrated map enabled the subsequent construction of a tammar wallaby first-generation virtual genome map, which comprises 14336 markers, including 13783 genes recruited from opossum and human assemblies. Both maps are freely available at http://compldb.angis.org.au. CONCLUSIONS: The first-generation integrated map and the first-generation virtual genome map provide a backbone for the chromosome assembly of the tammar wallaby genome sequence. For example, 78% of the 10257 gene-scaffolds in the Ensembl annotation of the tammar wallaby genome sequence (including 10522 protein-coding genes) can now be given a chromosome location in the tammar wallaby virtual genome map.Peer Reviewe

Sequence and gene content of a large fragment of a lizard sex chromosome and evaluation of candidate sex differentiating gene R-spondin 1

Background: Scant genomic information from non-avian reptile sex chromosomes is available, and for only a few lizards, several snakes and one turtle species, and it represents only a small fraction of the total sex chromosome sequences in these species. Results: We report a 352 kb of contiguous sequence from the sex chromosome of a squamate reptile, Pogona vitticeps, with a ZZ/ZW sex microchromosome system. This contig contains five protein coding genes (oprd1, rcc1, znf91, znf131, znf180), and major families of repetitive sequences with a high number of copies of LTR and non-LTR retrotransposons, including the CR1 and Bov-B LINEs. The two genes, oprd1 and rcc1 are part of a homologous syntenic block, which is conserved among amniotes. While oprd1 and rcc1 have no known function in sex determination or differentiation in amniotes, this homologous syntenic block in mammals and chicken also contains R-spondin 1 (rspo1), the ovarian differentiating gene in mammals. In order to explore the probability that rspo1 is sex determining in dragon lizards, genomic BAC and cDNA clones were mapped using fluorescence in situ hybridisation. Their location on an autosomal microchromosome pair, not on the ZW sex microchromosomes, eliminates rspo1 as a candidate sex determining gene in P. vitticeps. Conclusion: Our study has characterized the largest contiguous stretch of physically mapped sex chromosome sequence (352 kb) from a ZZ/ZW lizard species. Although this region represents only a small fraction of the sex chromosomes of P. vitticeps, it has revealed several features typically associated with sex chromosomes including the accumulation of large blocks of repetitive sequences

Recent Assembly of an Imprinted Domain from Non-Imprinted Components

Genomic imprinting, representing parent-specific expression of alleles at a locus, raises many questions about how—and especially why—epigenetic silencing of mammalian genes evolved. We present the first in-depth study of how a human imprinted domain evolved, analyzing a domain containing several imprinted genes that are involved in human disease. Using comparisons of orthologous genes in humans, marsupials, and the platypus, we discovered that the Prader-Willi/Angelman syndrome region on human Chromosome 15q was assembled only recently (105–180 million years ago). This imprinted domain arose after a region bearing UBE3A (Angelman syndrome) fused with an unlinked region bearing SNRPN (Prader-Willi syndrome), which had duplicated from the non-imprinted SNRPB/B′. This region independently acquired several retroposed gene copies and arrays of small nucleolar RNAs from different parts of the genome. In their original configurations, SNRPN and UBE3A are expressed from both alleles, implying that acquisition of imprinting occurred after their rearrangement and required the evolution of a control locus. Thus, the evolution of imprinting in viviparous mammals is ongoing