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)

Independent Evolution of Transcriptional Inactivation on Sex Chromosomes in Birds and Mammals

X chromosome inactivation in eutherian mammals has been thought to be tightly controlled, as expected from a mechanism that compensates for the different dosage of X-borne genes in XX females and XY males. However, many X genes escape inactivation in humans, inactivation of the X in marsupials is partial, and the unrelated sex chromosomes of monotreme mammals have incomplete and gene-specific inactivation of X-linked genes. The bird ZW sex chromosome system represents a third independently evolved amniote sex chromosome system with dosage compensation, albeit partial and gene-specific, via an unknown mechanism (i.e. upregulation of the single Z in females, down regulation of one or both Zs in males, or a combination). We used RNA-fluorescent in situ hybridization (RNA-FISH) to demonstrate, on individual fibroblast cells, inactivation of 11 genes on the chicken Z and 28 genes on the X chromosomes of platypus. Each gene displayed a reproducible frequency of 1Z/1X-active and 2Z/2X-active cells in the homogametic sex. Our results indicate that the probability of inactivation is controlled on a gene-by-gene basis (or small domains) on the chicken Z and platypus X chromosomes. This regulatory mechanism must have been exapted independently to the non-homologous sex chromosomes in birds and mammals in response to an over-expressed Z or X in the homogametic sex, highlighting the universal importance that (at least partial) silencing plays in the evolution on amniote dosage compensation and, therefore, the differentiation of sex chromosomes.This project was supported by an Australian Research Fellowship to PDW (DP0987091) and an Australian Research Council discovery project grant to PDW, JED and JAMG (DP1094868) (http://www.arc.gov.au/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Anchoring genome sequence to chromosomes of the central bearded dragon (Pogona vitticeps) enables reconstruction of ancestral squamate macrochromosomes and identifies sequence content of the Z chromosome

We report here the first genome assembly and annotation of the human-pathogenic fungus Scedosporium aurantiacum, with a predicted 10,525 genes, and 11,661 transcripts. The strain WM 09.24 was isolated from the environment at Circular Quay, Sydney, New South Wales, Australi

Landscape of DNA methylation on the marsupial x

DNA methylation plays a key role in maintaining transcriptional silence on the inactive X chromosome of eutherian mammals. Beyond eutherians, there are limited genome wide data on DNA methylation from other vertebrates. Previous studies of X borne genes in various marsupial models revealed no differential DNA methylation of promoters between the sexes, leading to the conclusion that CpG methylation plays no role in marsupial X-inactivation. Using reduced representation bisulfite sequencing, we generated male and female CpG methylation profiles in four representative vertebrates (mouse, gray short-tailed opossum, platypus, and chicken). A variety of DNA methylation patterns were observed. Platypus and chicken displayed no large-scale differential DNA methylation between the sexes on the autosomes or the sex chromosomes. As expected, a metagene analysis revealed hypermethylation at transcription start sites (TSS) of genes subject to X-inactivation in female mice. This contrasted with the opossum, in which metagene analysis did not detect differential DNA methylation between the sexes at TSSs of genes subject to X-inactivation. However, regions flanking TSSs of these genes were hypomethylated. Our data are the first to demonstrate that, for genes subject to X-inactivation in both eutherian and marsupial mammals, there is a consistent difference between DNA methylation levels at TSSs and immediate flanking regions, which we propose has a silencing effect in both groups.This work was funded by Australian Research Council Discovery Projects DP0987091 and DP1094868

4.脳腫瘍患者における頭蓋内圧の日内変動に関する研究 : とくにA波について(昭和55年度猪之鼻奨学会研究補助金報告)

BACKGROUND Squamates (lizards and snakes) are a speciose lineage of reptiles displaying considerable karyotypic diversity, particularly among lizards. Understanding the evolution of this diversity requires comparison of genome organisation between species. Although the genomes of several squamate species have now been sequenced, only the green anole lizard has any sequence anchored to chromosomes. There is only limited gene mapping data available for five other squamates. This makes it difficult to reconstruct the events that have led to extant squamate karyotypic diversity. The purpose of this study was to anchor the recently sequenced central bearded dragon (Pogona vitticeps) genome to chromosomes to trace the evolution of squamate chromosomes. Assigning sequence to sex chromosomes was of particular interest for identifying candidate sex determining genes. RESULTS By using two different approaches to map conserved blocks of genes, we were able to anchor approximately 42 % of the dragon genome sequence to chromosomes. We constructed detailed comparative maps between dragon, anole and chicken genomes, and where possible, made broader comparisons across Squamata using cytogenetic mapping information for five other species. We show that squamate macrochromosomes are relatively well conserved between species, supporting findings from previous molecular cytogenetic studies. Macrochromosome diversity between members of the Toxicofera clade has been generated by intrachromosomal, and a small number of interchromosomal, rearrangements. We reconstructed the ancestral squamate macrochromosomes by drawing upon comparative cytogenetic mapping data from seven squamate species and propose the events leading to the arrangements observed in representative species. In addition, we assigned over 8 Mbp of sequence containing 219 genes to the Z chromosome, providing a list of genes to begin testing as candidate sex determining genes. CONCLUSIONS Anchoring of the dragon genome has provided substantial insight into the evolution of squamate genomes, enabling us to reconstruct ancestral macrochromosome arrangements at key positions in the squamate phylogeny, demonstrating that fusions between macrochromosomes or fusions of macrochromosomes and microchromosomes, have played an important role during the evolution of squamate genomes. Assigning sequence to the sex chromosomes has identified NR5A1 as a promising candidate sex determining gene in the dragon.This project was funded by ARC DP110104377, a Faculty of Applied Science postdoctoral fellowship to DO’M and University of Canberra strategic funds awarded to AG and JED

Incomplete transcriptional dosage compensation of chicken and platypus sex chromosomes is balanced by post-transcriptional compensation

Heteromorphic sex chromosomes (XY or ZW) present problems of gene dosage imbalance between sexes and with autosomes. A need for dosage compensation has long been thought to be critical in vertebrates. However, this was questioned by findings of unequal mRNA abundance measurements in monotreme mammals and birds. Here, we demonstrate unbalanced mRNA levels of X genes in platypus males and females and a correlation with differential loading of histone modifications. We also observed unbalanced transcripts of Z genes in chicken. Surprisingly, however, we found that protein abundance ratios were 1:1 between the sexes in both species, indicating a post-transcriptional layer of dosage compensation. We conclude that sex chromosome output is maintained in chicken and platypus (and perhaps many other non therian vertebrates) via a combination of transcriptional and post-transcriptional control, consistent with a critical importance of sex chromosome dosage compensation

Independent Evolution of Transcriptional Inactivation on Sex Chromosomes in Birds and Mammals

<div><p>X chromosome inactivation in eutherian mammals has been thought to be tightly controlled, as expected from a mechanism that compensates for the different dosage of X-borne genes in XX females and XY males. However, many X genes escape inactivation in humans, inactivation of the X in marsupials is partial, and the unrelated sex chromosomes of monotreme mammals have incomplete and gene-specific inactivation of X-linked genes. The bird ZW sex chromosome system represents a third independently evolved amniote sex chromosome system with dosage compensation, albeit partial and gene-specific, via an unknown mechanism (i.e. upregulation of the single Z in females, down regulation of one or both Zs in males, or a combination). We used RNA-fluorescent <i>in situ</i> hybridization (RNA-FISH) to demonstrate, on individual fibroblast cells, inactivation of 11 genes on the chicken Z and 28 genes on the X chromosomes of platypus. Each gene displayed a reproducible frequency of 1Z/1X-active and 2Z/2X-active cells in the homogametic sex. Our results indicate that the probability of inactivation is controlled on a gene-by-gene basis (or small domains) on the chicken Z and platypus X chromosomes. This regulatory mechanism must have been exapted independently to the non-homologous sex chromosomes in birds and mammals in response to an over-expressed Z or X in the homogametic sex, highlighting the universal importance that (at least partial) silencing plays in the evolution on amniote dosage compensation and, therefore, the differentiation of sex chromosomes.</p></div

Frequency of nuclei transcribing both alleles of neighbouring loci.

<p>Expected frequencies of nuclei transcribing from both allelels of neighbouring loci were calculated from observed frequencies of 2Z-active (or 2X-active) nuclei (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003635#pgen.1003635.s006" target="_blank">Table S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003635#pgen.1003635.s007" target="_blank">S2</a>). For example, <i>SMARCA2</i> was 2Z-active in 76% of nuclei and <i>PTPRD</i> was 2Z-active in 60% of nuclei. Therefore, it was expected that they should both be 2Z-active in 0.76*0.6 (45.6%) of nuclei. P-values were calculated with a X² test with 1 degree of freedom. Bonferroni correction was conducted.</p

RNA-FISH activity maps of the platypus Xs and chicken Z chromosomes.

<p>Bars represent the percentage of homogametic nuclei transcribing 2 (blue), 1 (red) or 0 (grey) alleles for each locus. Loci in pseudoautosomal regions (grey boxes) were tested in both male (indicated by a circle) and female (indicated by a star). Green coloring on platypus Xs represents homology to the chicken Z <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003635#pgen.1003635-Veyrunes1" target="_blank">[23]</a>. Platypus X chromosomes are not to scale (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003635#pgen.1003635.s005" target="_blank">Figure S5</a>). Genes denoted by * were analysed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003635#pgen.1003635-Deakin1" target="_blank">[24]</a>.</p