Interspecific hybridization induced amplification of Mdm2 on double minutes in a Mus hybrid

We report a case of interspecific hybridization induced amplification of Chromosome 10 on double minutes (dm) in the karyotype of a hybrid Mus embryo. Stable, non-mosaic dm were previously found in tissues of a 16.5-day Mus Musculus x Mus Caroli hybrid (Graves, 1984). Dm in tissues of the hybrid was of interest to us because of previous reports of genomic instability in interspecific hybrids (O'Neill et al., 1998) and thus we decided to characterize the dm in the hybrid karyotypes. Whole chromosome painting of the hybrid cell lines showed amplification of Chromosome 10 sequences. Southern analysis with a probe for the candidate gene Mdm2 showed amplification of the paternal allele of this oncogene. Overexpression of Mdm2 was confirmed by a western analysis that also showed an associated inactivation of the tumor suppressor, Trp53. Evidence indicates that the event leading to the instability observed was an early adaptive response to stress on the genome, i.e. interspecific hybridization.     

Multiple independent pseudogene derivations indicate increased instability of the <Emphasis Type="Italic">Mdm2</Emphasis> locus in <Emphasis Type="Italic">Mus caroli</Emphasis>

Under conditions of genomic stress, the Mdm locus in human and in mouse is prone to instability manifested as amplification and oncogenesis. The Mdm2 gene is a known oncogene that is amplified in approximately one-third of sarcomas and whose protein product interacts with the tumor suppressor p53. Concimitant with such gene amplification events is the activation and mobilization of endogenous retroelements, typically through the relaxation of epigenetic controlling mechanisms. Processed pseudogenes, which can be formed through endogenous LINE retroelement activity, may indicate increased genomic instability. We have isolated processed pseudogenes for Mdm2 in Mus caroli DNA, likely formed from independent events in different individuals. This is the first identification and characterization of an Mdm2 pseudogene in any organism. Multiple retrotransposition events are suggested by the variable sequence and genomic structure of the identified pseudogenes across all exons and the 3UTR. The high degree of similarity between the gene and each pseudogene, as well as the lack of evidence for an Mdm2 pseudogene in several other species of Mus, indicate evolutionarily recent retrotransposition events leading to the formation of the Mdm2 pseudogenes in M. caroli. Previous studies on the Mdm2 locus in Mus caroli showed amplification and overexpression of this gene on double minute chromosomes in a Mus musculus × Mus caroli interspecific hybrid. The identification of an Mdm2 retropseudogene within this species further highlights the predisposition to instability for this region of the genome.     

Identification of differentially methylated sites within unmethylated DNA domains in normal and cancer cells

Altered DNA methylation has been linked to neoplastic cell transformation and is a hallmark of cancer progression. Therefore, the screening for differentially methylated sequences as tumor biomarkers has a significant implication in the clinical setting. To determine the cancer-linked alterations in DNA methylation pattern, we have applied an endonuclease, McrBC, to the existing methylation-sensitive arbitrarily primed polymerase chain reaction (msAP-PCR) method and developed McrBC-msAP-PCR. This modified approach allows detection of differentially methylated sites within unmethylated DNA domains enriched by regulatory sequences and CpG islands. In this method, we used digestion of DNA with the McrBC methylation-sensitive endonuclease to selectively exclude the methylated fraction of DNA, which comprises interspersed and tandem-repeated sequences and exons other than first exons, from analysis. The subsequent digestion of unmethylated DNA fragments with SmaI and HpaII methylation-sensitive restriction endonucleases followed by AP-PCR amplification resulted in the detection of unknown unique sequences associated with cancer-linked methylation changes in genomic DNA. Hypermethylation and hypomethylation are visualized by the increase or decrease in the band intensity of DNA fingerprints. By using this technique, we were able to differentiate clearly, identify, and characterize a number of novel unique DNA sequences with differentially methylated sites in normal and breast cancer cell lines and in normal and rat tumor liver tissues.     

Genotyping of somatic hybrids between <Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb. and <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

In order to genotype hybrid genomes of distant asymmetric somatic hybrids, we synthesized hybrid calli and plants via PEG-mediated protoplast fusion between recipient tall fescue (Festuca. arundinacea Schreb.) and donor wheat (Triticum aestivum L.). Seventeen and 25 putative hybrid clones were produced from the fusion combinations I and II, each with the donor wheat protoplast treated by UV light for 30 s and 1 min, respectively. Isozyme and RAPD profiles confirmed that ten hybrid clones were obtained from combination I and 19 from combination II. Out of the 29 hybrids, 12 regenerated hybrid plants with tall fescue phenotype. Composition and methylation-variation of the nuclear and cytoplasmic genomes of some hybrids, either with or without regenerative ability, were compared by genomic in situ hybridization, restriction fragment length polymorphism, and DNA methylation-sensitive amplification polymorphism. Our results indicated that these selected hybrids all contained introgressed nuclear and cytoplasmic DNA as well as obvious methylation variations compared to both parents. However, there were no differences either in nuclear/cytoplasmic DNA or methylation degree between the regenerable and non-regenerable hybrid clones. We conclude that both regeneration complementation and genetic material balance are crucial for hybrid plant regeneration.     

DNA methylation in placentas of interspecies mouse hybrids

Interspecific hybridization in the genus Mus results in several hybrid dysgenesis effects, such as male sterility and X-linked placental dysplasia (IHPD). The genetic or molecular basis for the placental phenotypes is at present not clear. However, an extremely complex genetic system that has been hypothesized to be caused by major epigenetic changes on the X chromosome has been shown to be active. We have investigated DNA methylation of several single genes, Atrx, Esx1, Mecp2, Pem, Psx1, Vbp1, Pou3f4, and Cdx2, and, in addition, of LINE-1 and IAP repeat sequences, in placentas and tissues of fetal day 18 mouse interspecific hybrids. Our results show some tendency toward hypomethylation in the late gestation mouse placenta. However, no differential methylation was observed in hyper- and hypoplastic hybrid placentas when compared with normal-sized littermate placentas or intraspecific Mus musculus placentas of the same developmental stage. Thus, our results strongly suggest that generalized changes in methylation patterns do not occur in trophoblast cells of such hybrids.     

Rapid analysis of DNA methylation using new restriction enzyme sites created by bisulfite modification.

Bisulfite converts non-methylated cytosine in DNA to uracil leaving 5-methylcytosine unaltered. Here, predicted changes in restriction enzyme sites following reaction of genomic DNA with bisulfite and amplification of the product by the polymerase chain reaction (PCR) were used to assess the methylation of CpG sites. This procedure differs from conventional DNA methylation analysis by methylation-sensitive restriction enzymes because it does not rely on an absence of cleavage to detect methylated sites, the two strands of DNA produce different restriction enzyme sites and may be differentially analyzed, and closely related sequences may be separately analyzed by using specific PCR primers.     

Allele-Specific Transcriptome and Methylome Analysis Reveals Stable Inheritance and Cis-Regulation of DNA Methylation in Nasonia

Gene expression divergence between closely related species could be attributed to both cis- and trans- DNA sequence changes during evolution, but it is unclear how the evolutionary dynamics of epigenetic marks are regulated. In eutherian mammals, biparental DNA methylation marks are erased and reset during gametogenesis, resulting in paternal or maternal imprints, which lead to genomic imprinting. Whether DNA methylation reprogramming exists in insects is not known. Wasps of the genus Nasonia are non-social parasitoids that are emerging as a model for studies of epigenetic processes in insects. In this study, we quantified allele-specific expression and methylation genome-wide in Nasonia vitripennis and Nasonia giraulti and their reciprocal F1 hybrids. No parent-of-origin effect in allelic expression was found for >8,000 covered genes, suggesting a lack of genomic imprinting in adult Nasonia. As we expected, both significant cis- and trans- effects are responsible for the expression divergence between N. vitripennis and N. giraulti. Surprisingly, all 178 differentially methylated genes are also differentially methylated between the two alleles in F1 hybrid offspring, recapitulating the parental methylation status with nearly 100% fidelity, indicating the presence of strong cis-elements driving the target of gene body methylation. In addition, we discovered that total and allele-specific expression are positively correlated with allele-specific methylation in a subset of the differentially methylated genes. The 100% cis-regulation in F1 hybrids suggests the methylation machinery is conserved and DNA methylation is targeted by cis features in Nasonia. The lack of genomic imprinting and parent-of-origin differentially methylated regions in Nasonia, together with the stable inheritance of methylation status between generations, suggests either a cis-regulatory motif for methylation at the DNA level or highly stable inheritance of an epigenetic signal in Nasonia.     

DNA methylation in cotton hybrids and their parents

The possible role of methylation in the performance of heterosis has been analyzed in many crops. To further study this possibility, we investigated both the differences in cytosine methylation patterns between cotton heterotic hybrid/nonheterotic hybrids and their parental lines and the change in methylation level from seedling stage to flowering stage by using the methylation-sensitive amplified polymorphism (MSAP) method. The results showed that the number of demethylation loci in highly heterotic hybrids was greater that in lowly heterotic hybrids, and the level of DNA cytosine methylation in cotton at the seedling stage is higher than that at the flowering stage. The altered methylation patterns at low-copy genomic regions can be confirmed by DNA gel blot analysis. A total of 39 fragments that showed different methylation patterns were cloned and sequenced. The methylation status of these genes was modified differentially in hybrid and parents, suggesting that these genes might play a role in the performance of heterosis.     

A novel imprinted transgene located near a repetitive element that exhibits allelic imbalance in DNA methylation during early development

A mouse line carrying a lacZ transgene driven by the human EEF1A1/EF1alpha promoter was established. Although the promoter is known to show ubiquitous activity, only paternal transgene alleles were expressed, resulting in a transgene imprinting. At mid‐gestation, the promoter sequence was differentially methylated, hypomethylated for paternal and hypermethylated for maternal alleles. In germline, the promoter was a typical differentially methylated region. After fertilization, however, both alleles were hypermethylated. Thus, the differential methylation of the promoter required for transgene imprinting was re‐established during later embryonic development independently of the germline differential methylation. Furthermore, also a retroelement promoter closely‐flanking imprinted transgene and its wild type counterpart displayed similar differential methylation during early development. The retroelement promoter was methylated differentially also in germline, but in an opposite pattern to the embryonic differential methylation. These results suggest that there might be an unknown epigenetic regulation inducing transgene imprinting independently of DNA methylation in the transgene insertion site. Then, besides CpG dinucleotides, non‐CpG cytosines of the retroelement promoter were highly methylated especially in the transgene‐active mid‐gestational embryos, suggesting that an unusual epigenetic regulation might protect the active transgene against de novo methylation occurring generally in mid‐gestational embryo.     

Demethylation and derepression of genomic retroelements in the skeletal muscles of aged mice

Changes in DNA methylation influence the aging process and contribute to aging phenotypes, but few studies have been conducted on DNA methylation changes in conjunction with skeletal muscle aging. We explored the DNA methylation changes in a variety of retroelement families throughout aging (at 2, 20, and 28 months of age) in murine skeletal muscles by methyl‐binding domain sequencing (MBD‐seq). The two following contrasting patterns were observed among the members of each repeat family in superaged mice: (a) hypermethylation in weakly methylated retroelement copies and (b) hypomethylation in copies with relatively stronger methylation levels, representing a pattern of “regression toward the mean” within a single retroelement family. Interestingly, these patterns depended on the sizes of the copies. While the majority of the elements showed a slight increase in methylation, the larger copies (>5 kb) displayed evident demethylation. All these changes were not observed in T cells. RNA sequencing revealed a global derepression of retroelements during the late phase of aging (between 20 and 28 months of age), which temporally coincided with retroelement demethylation. Following this methylation drift trend of “regression toward the mean,” aging tended to progressively lose the preexisting methylation differences and local patterns in the genomic regions that had been elaborately established during the early period of development.     

DNA Methylation Alterations at 5′-CCGG Sites in the Interspecific and Intraspecific Hybridizations Derived from Brassica rapa and B. napus

DNA methylation is an important regulatory mechanism for gene expression that involved in the biological processes of development and differentiation in plants. To investigate the association of DNA methylation with heterosis in Brassica, a set of intraspecific hybrids in Brassica rapa and B. napus and interspecific hybrids between B. rapa and B. napus, together with parental lines, were used to monitor alterations in cytosine methylation at 5′-CCGG sites in seedlings and buds by methylation-sensitive amplification polymorphism analysis. The methylation status of approximately a quarter of the methylation sites changed between seedlings and buds. These alterations were related closely to the genomic structure and heterozygous status among accessions. The methylation status in the majority of DNA methylation sites detected in hybrids was the same as that in at least one of the parental lines in both seedlings and buds. However, the association between patterns of cytosine methylation and heterosis varied among different traits and between tissues in hybrids of Brassica, although a few methylation loci were associated with heterosis. Our data suggest that changes in DNA methylation at 5′-CCGG sites are not associated simply with heterosis in the interspecific and intraspecific hybridizations derived from B. rapa and B. napus.     

Mouse chromosome-specific markers generated by PCR and their mapping through interspecific backcrosses.

We have utilized an oligonucleotide primer from the 3' end of the mouse L1 repeat element for amplification of mouse-specific inter-repeat PCR products from Chinese hamster/mouse somatic cell hybrids. PCR of a Chinese hamster/mouse somatic cell hybrid (96AZ2), containing only mouse chromosome 16, produced a range of mouse-specific bands. Two of the mouse-specific PCR products, of 250 and 580 bp, have been confirmed as originating from mouse chromosome 16 by somatic cell hybrid analysis. Both the 250- and 580-bp PCR products have been sequenced and demonstrate the expected sequence organization. Furthermore, both the 250- and 580-bp markers have been genetically mapped in detail to mouse chromosome 16 by direct hybridization to inter-repeat PCR products of progeny DNAs from Mus domesticus/Mus spretus interspecific backcrosses.     

Whole-genome fractionation rapidly purifies DNA from centromeric regions

The condensed centromeric regions of higher eukaryotic chromosomes contain satellite sequences, transposons and retroelements, as well as transcribed genes that perform a variety of functions. These chromosomal domains nucleate kinetochores, mediate sister chromatid cohesion and inhibit recombination, yet their characterization has often lagged behind that of chromosome arms. Here, we describe a whole-genome fractionation technique that rapidly identifies bacterial artificial chromosome (BAC) clones derived from plant centromeric regions. This approach, which relies on hybridization of methylated genomic DNA, revealed BACs that correspond to the genetically mapped and sequenced Arabidopsis thaliana centromeric regions. Extending this method to other species in the Brassicaceae family identified centromere-linked clones and provided genome-wide estimates of methylated DNA abundance. Sequencing these clones will elucidate the changes that occur during plant centromere evolution. This genomic fractionation technique could identify centromeric DNA in genomes with similar methylation and repetitive DNA content, including those from crops and mammals.     

盐胁迫下棉花基因组基于毛细管电泳的MSAP分析

以棉花杂交种中棉所29为材料,用甲基化敏感扩增多态性(methylation sensitive amplification polymorphism,MSAP) 分析法结合毛细管电泳检测技术进行甲基化鉴定,以初步探讨棉花耐盐的分子机理．应用24个引物组合,中棉所29在0.4%盐水胁迫及清水对照下,平均每引物组合检测甲基化位点数分别为69.2和56.7,差异达显著水平．盐胁迫下的DNA甲基化水平与清水对照下相比,52.6%位点表现出甲基化水平提高,即发生了超甲基化;19.7%位点甲基化水平降低,即表现为次甲基化;二者差异达极显著水平．研究结果表明,中棉所29盐胁迫后发生了广泛的DNA甲基化变化,包括超甲基化和次甲基化,以及其它甲基化类型的转变|发生超甲基化位点极显著地多于发生次甲基化位点．盐胁迫下的中棉所29与对照相比,DNA总体甲基化水平显著提高,暗示中棉所29有提高基因组甲基化水平以应对盐胁迫的潜在机制,棉花基因组整体甲基化水平的提高可能与棉花对盐胁迫的耐受性起重要作用．本研究中,甲基化序列的初步克隆及比对分析表明,盐胁迫前后多个ATP合成相关基因甲基化程度维持在同一水平,其表达不受甲基化影响,这也可能是中棉所29耐盐性较强,在一定时间盐处理后能维持正常生长的原因之一．     

Interspecific hybrids between a transgenic rapeseed (Brassica napus) and related species: cytogenetical characterization and detection of the transgene.

In interspecific hybrids produced between a transgenic rapeseed, an allotetraploid species, resistant to herbicide, phosphinotricin, and five diploid related species, the risk for gene introgression in weed genomes was explored through cytogenetic and bar gene characterizations. Among the 75 hybrids studied, most had the expected triploid structure, with the exception of B. napus - B. oleracea amphidiploid plants and one B. napus - S. arvensis amphidiploid plant. In triploid hybrid plants, the reciprocal hybrids did not exhibit any difference in their meiotic behavior. The comparison of the percentage of chromosome pairing in the hybrids with that of haploid rapeseed permit to conclude that allosyndesis between AC genomes and related species genomes took place. This possibility of recombination was confirmed by the presence of multivalent associations in all the interspecific hybrids. Nevertheless, in B. napus - B. adpressa hybrids a control of chromosome pairing seemed to exist. The possibility of amphidiploid plant production directly obtained in the F1 generation increased the risk of gene dispersal. The B. napus - B. oleracea amphidiploid plant presented a meiotic behavior more regular than that of the B. napus - S. arvensis amphidiploid plant. Concerning the herbicide bar gene characterization, the presence of the gene detected by DNA amplification was correlated with herbicide resistance, except for two plants. Different hypotheses were proposed to explain these results. A classification of the diploid species was established regarding their gene dispersal risk based on the rate of allosyndesis between chromosomes of AC genomes of rapeseed and the genomes of the related species.     

Analysis of DNA methylation in cotton hybrids and their parents

The possible role of methylation in the performance of heterosis has been analyzed in many crops. To further study this possibility, we investigated both the differences in cytosine methylation patterns between cotton heterotic hybrids/nonheterotic hybrids and their parental lines and the change in methylation level from seedling stage to flowering stage by using the methylation-sensitive amplified polymorphism (MSAP) method. The results showed that the number of demethylation loci in highly heterotic hybrids was greater that in lowly heterotic hybrids, and the level of DNA cytosine methylation in cotton at the seedling stage is higher than that of the flowering stage. The altered methylation patterns at low-copy genomic regions can be confirmed by DNA gel blot analysis. A total of 39 fragments that showed different methylation patterns were cloned and sequenced. The methylation status of these genes was modified differentially in hybrid and parents, suggesting that these genes might play a role in the performance of heterosis. Published in Russian in Molekulyarnaya Biologiya, 2008, Vol. 42, No. 2, pp. 195–205. The text was submitted by the authors in English.