X chromosome dosage compensation in female eutherian mammals is regulated by the noncoding Xist RNA and is associated with the differential acquisition of active and repressive histone modifications, ...resulting in repression of most genes on one of the two X chromosome homologs. Marsupial mammals exhibit dosage compensation; however, they lack Xist, and the mechanisms conferring epigenetic control of X chromosome dosage compensation remain elusive. Oviparous mammals, the monotremes, have multiple X chromosomes, and it is not clear whether they undergo dosage compensation and whether there is epigenetic dimorphism between homologous pairs in female monotremes. Here, using antibodies against DNA methylation, eight different histone modifications, and HP1, we conduct immunofluorescence on somatic cells of the female Australian marsupial possum Trichosurus vulpecula, the female platypus Ornithorhynchus anatinus, and control mouse cells. The two marsupial X's were different for all epigenetic features tested. In particular, unlike in the mouse, both repressive modifications, H3K9me3 and H4K20Me3, are enriched on one of the X chromosomes, and this is associated with the presence of HP1 and hypomethylation of DNA. Using sequential labeling, we determine that this DNA hypomethylated X correlates with histone marks of inactivity. These results suggest that female marsupials use a repressive histone-mediated inactivation mechanism and that this may represent an ancestral dosage compensation process that differs from eutherians that require Xist transcription and DNA methylation. In comparison to the marsupial, the monotreme exhibited no epigenetic differences between homologous X chromosomes, suggesting the absence of a dosage compensation process comparable to that in therians.
Abstract
Chromosome translocations can be detected by cytogenetic analysis, but it is hard to characterize the breakpoints at the sequence level. Chromosome sorting by flow cytometry produces flow ...karyotypes that enable the isolation of abnormal chromosomes and the generation of chromosome-specific DNA. In this study, a derivative chromosome t(9; 14) and its homologous normal chromosomes 9 and 14 from the Ishikawa 3-H-12 cell line were sorted to collect homologue-specific samples. Chromosome sequencing identified the breakpoint junction in the der(9) at 9p24.3 and 14q13.1 and uncovered the formation of a fusion gene, WASH1–NPAS3. Amplicon sequencing targeted for neighbouring genes at the fusion breakpoint revealed that the variant frequencies correlate with the allelic copy number. Sequencing of sorted chromosomes permits the assignment of allelic variants and can lead to the characterization of abnormal chromosomes. We show that allele-specific chromosome sequencing of homologues is a robust technique for distinguishing alleles and this provides an efficient approach for the comprehensive analysis of genomic changes.
Analysis of molecular aberrations across multiple cancer types, known as pan-cancer analysis, identifies commonalities and differences in key biological processes that are dysregulated in cancer ...cells from diverse lineages. Pan-cancer analyses have been performed for adult but not paediatric cancers, which commonly occur in developing mesodermic rather than adult epithelial tissues. Here we present a pan-cancer study of somatic alterations, including single nucleotide variants, small insertions or deletions, structural variations, copy number alterations, gene fusions and internal tandem duplications in 1,699 paediatric leukaemias and solid tumours across six histotypes, with whole-genome, whole-exome and transcriptome sequencing data processed under a uniform analytical framework. We report 142 driver genes in paediatric cancers, of which only 45% match those found in adult pan-cancer studies; copy number alterations and structural variants constituted the majority (62%) of events. Eleven genome-wide mutational signatures were identified, including one attributed to ultraviolet-light exposure in eight aneuploid leukaemias. Transcription of the mutant allele was detectable for 34% of protein-coding mutations, and 20% exhibited allele-specific expression. These data provide a comprehensive genomic architecture for paediatric cancers and emphasize the need for paediatric cancer-specific development of precision therapies.
The Cuckoos have a long history of difficult classification. The species of this order have been the subject of several studies based on osteology, behavior, ecology, morphology and molecular data. ...Despite this, the relationship between Cuculiformes and species of other orders remains controversial. In this work, two species of Cuculidae, Guira guira (Gmelin, 1788) and Piaya cayana (Linnaeus, 1766), were analyzed by means of comparative chromosome painting in order to study the chromosome evolution of this group and to undertake the first chromosome mapping of these species. Our results demonstrate high chromosomal diversity, with 2n = 76 in G. guira, with fission and fusion events involving ancestral syntenies, while P. cayana presented only fissions, which were responsible for the high diploid number of 2n = 90. Interestingly, there were no chromosomal rearrangements in common between these species. Our results, based on Giemsa staining, were compared with previous data for other cuckoos and also with taxa proposed as sister-groups of Cuculiformes (Otidiformes, Musophagiformes and Opisthocomiformes). Cytogenetic comparisons demonstrated that cuckoo species can be divided into at least three major groups. In addition, we found no evidence to place Cuculiformes close to the groups proposed previously as sister-groups.
Abstract
Background
The history of comparative chromosome mapping is briefly reviewed, with discussion about the problem that occurs in chromosome painting when size heteromorphisms between ...homologues cause contamination in chromosomes sorted by flow cytometry that are used in the preparation of chromosome-specific DNA probes.
Main body
As an example, we show in the alpaca (
Vicagna pacos
) that sequencing of contaminated chromosome sorts can reveal chromosome homologies from alignment with human and mouse genome reference sequences. The procedure identifies syntenic blocks of DNA separated in the human karyotype that are associated in the closely related alpaca and dromedary (
Camelus dromedarius
) karyotypes. This example provides proof of principal for the validity of the method for comparative chromosome mapping.
Conclusion
It is suggested that the approach presented here may have application in the construction of comparative chromosome maps between distantly related taxa, such as monotremes and mammals.
Approximately half the mammalian genome is composed of repetitive sequences, and accumulating evidence suggests that some may have an impact on genome function. Here, we characterized a large array ...class of repeats of long-interspersed elements (LINE-1). Although widely distributed in mammals, locations of such arrays are species specific. Using targeted deletion, we asked whether a 170-kb LINE-1 array located at a mouse imprinted domain might function as a modulator of local transcriptional control. The LINE-1 array is lamina associated in differentiated ES cells consistent with its AT-richness, and although imprinting occurs both proximally and distally to the array, active LINE-1 transcripts within the tract are biallelically expressed. Upon deletion of the array, no perturbation of imprinting was observed, and abnormal phenotypes were not detected in maternal or paternal heterozygous or homozygous mutant mice. The array does not shield nonimprinted genes in the vicinity from local imprinting control. Reduced neural expression of protein-coding genes observed upon paternal transmission of the deletion is likely due to the removal of a brain-specific enhancer embedded within the LINE array. Our findings suggest that presence of a 170-kb LINE-1 array reflects the tolerance of the site for repeat insertion rather than an important genomic function in normal development.
Genetic alterations that activate NOTCH1 signaling and T cell transcription factors, coupled with inactivation of the INK4/ARF tumor suppressors, are hallmarks of T-lineage acute lymphoblastic ...leukemia (T-ALL), but detailed genome-wide sequencing of large T-ALL cohorts has not been carried out. Using integrated genomic analysis of 264 T-ALL cases, we identified 106 putative driver genes, half of which had not previously been described in childhood T-ALL (for example, CCND3, CTCF, MYB, SMARCA4, ZFP36L2 and MYCN). We describe new mechanisms of coding and noncoding alteration and identify ten recurrently altered pathways, with associations between mutated genes and pathways, and stage or subtype of T-ALL. For example, NRAS/FLT3 mutations were associated with immature T-ALL, JAK3/STAT5B mutations in HOXA1 deregulated ALL, PTPN2 mutations in TLX1 deregulated T-ALL, and PIK3R1/PTEN mutations in TAL1 deregulated ALL, which suggests that different signaling pathways have distinct roles according to maturational stage. This genomic landscape provides a logical framework for the development of faithful genetic models and new therapeutic approaches.
Rapamycin demonstrated broad-spectrum tumor growth inhibition activity against the in vivo panels of childhood tumors used in the Pediatric Preclinical Testing Program (PPTP). Here we have evaluated ...rapamycin combined with agents used frequently in the treatment of childhood malignancies. Rapamycin was tested in vitro against 23 cell lines alone or in combination with melphalan, cisplatin, vincristine, or dexamethasone (leukemic models only). In vivo, the impact of combining rapamycin with a cytotoxic agent was evaluated using two measures: 1) the therapeutic enhancement measure, and 2) a linear regression model for time-to-event to formally evaluate for sub- and supraadditivity for the combination compared to the agents used alone. Combining rapamycin with cytotoxic agents in vitro gave predominantly subadditive or additive effects, except for dexamethasone in leukemia models for which supra-additive activity was observed. In vivo testing demonstrated that therapeutic enhancement was common for rapamycin in combination with cyclophosphamide and occurred for 4 of 11 evaluable xenografts for the rapamycin and vincristine combination. The combinations of rapamycin with either cyclophosphamide or vincristine were significantly more effective than the respective standard agents used alone at their maximum tolerated doses (MTD) for most evaluable xenografts. The combination of rapamycin and cisplatin produced excessive toxicity requiring cisplatin dose reductions, and therapeutic enhancement was not observed for this combination. Addition of rapamycin to either cyclophosphamide or vincristine at their respective MTDs appears promising, as these combinations are relatively well tolerated and as many of the pediatric preclinical models evaluated demonstrated therapeutic enhancement for these combinations.
Turdus rufiventris and Turdus albicollis, two songbirds belonging to the family Turdidae (Aves, Passeriformes) were studied by C-banding, 18S rDNA, as well as the use of whole chromosome probes ...derived from Gallus gallus (GGA) and Leucopternis albicollis (LAL). They showed very similar karyotypes, with 2n = 78 and the same pattern of distribution of heterochromatic blocks and hybridization patterns. However, the analysis of 18/28S rDNA has shown differences in the number of NOR-bearing chromosomes and ribosomal clusters. The hybridization pattern of GGA macrochromosomes was similar to the one found in songbirds studied by Fluorescent in situ hybridization, with fission of GGA 1 and GGA 4 chromosomes. In contrast, LAL chromosome paintings revealed a complex pattern of intrachromosomal rearrangements (paracentric and pericentric inversions) on chromosome 2, which corresponds to GGA1q. The first inversion changed the chromosomal morphology and the second and third inversions changed the order of chromosome segments. Karyotype analysis in Turdus revealed that this genus has derived characteristics in relation to the putative avian ancestral karyotype, highlighting the importance of using new tools for analysis of chromosomal evolution in birds, such as the probes derived from L. albicollis, which make it possible to identify intrachromosomal rearrangements not visible with the use of GGA chromosome painting solely.
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