Cloning and characterization of a centromere-specific repetitive DNA element from Sorghum bicolor

 A 823-bp Sau3AI fragment (pSau3A10) was subcloned from a sorghum bacterial artificial chromosome (BAC) clone, 13I16, that contains DNA sequences specific to the centromeres of grass species. Sequence analysis showed that pSau3A10 consists of six copies of an approximately 137-bp monomer. The six monomers were organized into three dimers. The monomers within the dimers shared 62–72% homology and the dimers were 79–82% homologous with each other. Fluorescence in situ hybridization (FISH) analysis indicated that the Sau3A10 family is present only in the centromeres of sorghum chromosomes. Sequencing, Southern hybridization, and Fiber-FISH analyses indicated that the Sau3A10 family is tandemly arranged and is present in uninterrupted stretches of up to at least 81 kb of DNA. Slot-blot analysis estimated that the Sau3A10 family constitutes 1.6–1.9% of the sorghum genome. The long stretches of Sau3A10 sequences were interrupted by other centromeric DNA elements. Southern analysis indicated that the Sau3A10 sequence is one of the most abundant DNA families located in sorghum centromeres and is conserved only in closely related sorghum species. Methylation experiments indicated that the cytosine of the CG sites in sorghum centromeric regions is generally methylated. The structure and organization of the Sau3A10 family shared similarities with centromeric DNA repeats in other eukaryotic species. It is suggested that the Sau3A10 family is probably an important part of sorghum centromeres. Received: 11 November 1997 / Accepted: 17 November 1997     

Conservation of IGF2-H19 and IGF2R imprinting in sheep: effects of somatic cell nuclear transfer

In different mammalian species, in vitro culture and manipulation can lead to aberrant fetal and peri-natal development. It has been postulated that these diverse abnormalities are caused by epigenetic alterations and that these could affect genes that are regulated by genomic imprinting. To explore this hypothesis relative to somatic cell nuclear transfer in sheep, we investigated whether the ovine H19-IGF2 and IGF2R loci are imprinted and analysed their DNA methylation status in cloned lambs. A comparison between parthenogenetic and control concepti established that imprinting at these two growth-related loci is evolutionarily conserved in sheep. As in humans and mice, IGF2R and H19 comprise differentially methylated regions (DMRs) that are methylated on one of the two parental alleles predominantly. In tongue tissue from 12 out of 13 cloned lambs analysed, the DMR in the second intron of IGF2R had strongly reduced levels of DNA methylation. The DMR located upstream of the ovine H19 gene was found to be similarly organised as in humans and mice, with multiple CTCF binding sites. At this DMR, however, aberrant methylation was observed in only one of the cloned lambs. Although the underlying mechanisms remain to be determined, our data indicate that somatic cell nuclear transfer procedures can lead to epigenetic deregulation at imprinted loci.     

Epigenetic alterations contribute to promoter activity of imprinting gene IGF2

The expression of insulin-like growth factor 2 (IGF2), a classical imprinting gene, didn't completely correlate with its imprinting profiles in hepatocellular carcinoma (HCC). The mechanistic importance of promoter activity in regulation of IGF2 has not been fully clarified. Here we show that histone 3 lysine 4 trimethylation (H3K4me3) modified by menin-MLL complex of IGF2 promoter contributes to promoter activity of IGF2. The strong binding of menin and abundant H3K4me3 at the DNA demethylated P3/4 promoters were observed in Hep3B cells with the robust expression of IGF2. In IGF2-low-expressing HepG2 cells, menin didn't bind to DNA hypermethylated P3/4 regions; however, menin overexpression inhibited DNA methylation and promoted H3K4me3 at the P3/4 as well as IGF2 expression in HepG2. In addition, the H3K4me3 at P3/4 locus was activated in primary HCC specimens with high IGF2 expression. Furthermore, inhibition of the menin/MLL interaction via MI-2/3 reduced IGF2 expression, inhibited the IGF1R-AKT pathway, and significantly repressed HCC with robust expression of IGF2. Taken together, we conclude that H3K4me3 of P3/4 locus mediated by the menin-MLL complex is a novel epigenetic mechanism for releasing IGF2.     

Epigenetic regulation of Igf2/H19 imprinting at CTCF insulator binding sites

The mouse insulin-like growth factor II (Igf2) and H19 genes are located adjacent to each other on chromosome 7q11-13 and are reciprocally imprinted. It is believed that the allelic expression of these two genes is regulated by the binding of CTCF insulators to four parent-specific DNA methylation sites in an imprinting control center (ICR) located between these two genes. Although monoallelically expressed in peripheral tissues, Igf2 is biallelically transcribed in the CNS. In this study, we examined the allelic DNA methylation and CTCF binding in the Igf2/H19 imprinting center in CNS, hypothesizing that the aberrant CTCF binding as one of the mechanisms leads to biallelic expression of Igf2 in CNS. Using hybrid F1 mice (M. spretus males x C57BL/6 females), we showed that in CNS, CTCF binding sites in the ICR were methylated exclusively on the paternal allele, and CTCF bound only to the unmethylated maternal allele, showing no differences from the imprinted peripheral tissues. Among three other epigenetic modifications examined, histone H3 lysine 9 methylation correlated well with Igf2 allelic expression in CNS. These results suggest that CTCF binding to the ICR alone is not sufficient to insulate the Igf2 maternal promoter and to regulate the allelic expression of the gene in the CNS, thus challenging the aberrant CTCF binding as a common mechanism for lack of Igf2 imprinting in CNS. Further studies should be focused on the identification of factors that are involved in histone methylation and CTCF-associated factors that may be needed to coordinate Igf2 imprinting.     

A repetitive and species-specific sequence as a tool for detecting the genome contribution in somatic hybrids of the genus Medicago

 A highly repeated sequence (C300) was cloned from Medicago coerulea and its organization in the M. sativa-coerulea-falcata complex, M. arborea, and three somatic hybrids involving M. sativa, was investigated. Southern-blot analysis revealed a tandemly repeated array and a species-specificity of the sequence to those species belonging to the complex. Various degrees of amplification of C300 were detected among the species of the complex and the outcome in the somatic hybrids was dependent on parental composition. Sequence analysis revealed strong homology (96%) of C300 with a clone (E180) previously isolated from M. sativa. As FISH analysis showed that C300 was dispersed along the chromosomes of Medicago spp., it should prove a valid tool for establishing the chromosome origin of somatic hybrids. Received: 14 April 1997 / Accepted: 18 April 1997     

Intraspecific genetic variability analysis of Neovossia indica causing Karnal bunt of wheat using repetitive elements

Neovossia indica (Tilletia indica), causing Karnal bunt of wheat, affects major wheat growing regions all over the world. Karnal bunt ranks as one of the major diseases of wheat causing quality losses and monetary losses due to international quarantine regulations. The present work is the first report of a genetic diversity analysis of Indian isolates of N. indica. A library of N. indica isolate Ni7 was constructed in a λZAPII system, and three repetitive elements were identified for molecular analysis. These repetitive elements generated complex hybridization profiles producing fingerprint patterns of all seven isolates. Copy-number estimation of these three elements, pNiR9, pNiR12 and pNiR16, indicated the presence of 32, 61 and 64 copies, respectively. Cluster analysis based on hybridization patterns grouped together moderately virulent isolates Ni1, Ni7 and Ni8, thus suggesting a positive correlation between virulence typing and cluster analysis based on molecular data. Variability analysis of N. indica isolates will aid in checking new resistant sources in host germplasm. Received: 20 April 1999 / Accepted: 29 July 1999     

Sequence-specific methylation of the mouse H19 gene in embryonic cells deficient in the Dnmt-1 gene

We have used Dnmt^c/c ES cells that are homozygous for disruption of the DNA methyltransferase gene to address how de novo methylation is propagated and whether it is directed to specific sites in the early embryo. We examined the imprinted H19 gene and the specific-sequence region implicated as an “imprinting mark” to determine whether de novo methylation was occurring at a restricted set of sites. Since the “imprinting mark” was found to be methylated differentially at all stages of development, we reasoned that the sequence may still be a target for the de novo methylation activity found in the Dnmt^c/c cells, even though the loss of maintenance methylase activity renders the H19 promoter active. We used bisulfite genomic sequencing to determine the methylation state of the imprinted region of the H19 gene and found a low level of DNA methylation at specific single CpG sites in the upstream region of the imprinted H19 sequence in the Dnmt^c/c mutant ES cells. Moreover, these CpG sites appeared to be favoured targets for further de novo methylation of neighbouring CpG sites in rescued ES cells, which possess apparently normal maintenance activity. Our data provide further evidence for a separate methylating activity in ES cells and indicate that this activity displays sequence specificity. Dev. Genet. 22:111–121, 1998. © 1998 Wiley-Liss, Inc.     

Assessment of a species-specific element (Brep 1) in banana

 The nuclear genome of wild-type banana accessions was investigated for repetitive elements. We report here the occurrence, in the banana genome, of a sequence family of species-specific repetitive elements: Brep 1. This sequence family is distributed throughout the Musaceae with various copy numbers. The two species Musa acuminata and M. schizocarpa carry the highest copy numbers in contrast to M. balbisiana and tested representatives of different other sections. PCR primers were defined in the core consensus sequence for specific amplifications, which allow representatives of this sequence family to be easily detected in wild and cultivated banana clones. Sequence data were analysed and hypotheses on the evolution of banana cultivars from the wild-type banana clones are discussed. Received: 17 January 1997 / Accepted : 7 March 1997     

Tissue-specific relationship of S-adenosylhomocysteine with allele-specific H19/Igf2 methylation and imprinting in mice with hyperhomocysteinemia

DNA methylation is linked to homocysteine metabolism through the generation of S-adenosylmethionine (AdoMet) and S-Adenosylhomocysteine (AdoHcy). The ratio of AdoMet/AdoHcy is often considered an indicator of tissue methylation capacity. The goal of this study is to determine the relationship of tissue AdoMet and AdoHcy concentrations to allele-specific methylation and expression of genomically imprinted H19/Igf2. Expression of H19/Igf2 is regulated by a differentially methylated domain (DMD), with H19 paternally imprinted and Igf2 maternally imprinted. F1 hybrid C57BL/6J x Castaneous/EiJ (Cast) mice with (+/−), and without (+/+), heterozygous disruption of cystathionine-β-synthase (Cbs) were fed a control diet or a diet (called HH) to induce hyperhomocysteinemia and changes in tissue AdoMet and AdoHcy. F1 Cast x Cbs+/− mice fed the HH diet had significantly higher plasma total homocysteine concentrations, higher liver AdoHcy, and lower AdoMet/AdoHcy ratios and this was accompanied by lower liver maternal H19 DMD allele methylation, lower liver Igf2 mRNA levels, and loss of Igf2 maternal imprinting. In contrast, we found no significant differences in AdoMet and AdoHcy in brain between the diet groups but F1 Cast x Cbs+/− mice fed the HH diet had higher maternal H19 DMD methylation and lower H19 mRNA levels in brain. A significant negative relationship between AdoHcy and maternal H19 DMD allele methylation was found in liver but not in brain. These findings suggest the relationship of AdoMet and AdoHcy to gene-specific DNA methylation is tissue-specific and that changes in DNA methylation can occur without changes in AdoMet and AdoHcy.     

Hypermethylation of tobacco heterochromatic loci in response to osmotic stress

 Plants have to cope with a number of envi-ronmental stresses which may potentially induce genetic and epigenetic changes and thus contribute to genome variability. In the present study we inspected the DNA methylation status of two heterochromatic loci (defined with repetitive DNA sequences HRS60 and GRS) in a tobacco cell culture exposed to osmotic stress. Investigations were performed on a TBY-2 cell suspension culture, and the stress was elicited with NaCl or D-mannitol. Using the restriction enzymes MspI/HpaII and MboI/Sau3AI in combination with Southern hydridization we observed a reversible hypermethylation of the external cytosine at the CpCpG trinucleotides in cells grown under mild osmotic stress equal to a NaCl concentration of 10 g/l. There were no changes in the methylation of the internal cytosine as the CpG dinucleotides within the CCGG motifs (HpaII sites) appeared to be fully methylated in tobacco DNA repetitive sequences under normal physiological conditions. The data suggest epigenetic changes in the plant genome based on de novo methylation of DNA in response to environmental stress. Received: 26 November 1996/Accepted: 20 December 1996     

Use of genome-specific repetitive DNA sequences to monitor chromatin introgression from Festuca mairei into Lolium perenne

Repetitive DNA sequences contribute considerably to an understanding of the genomes of higher plants. Repetitive DNA sequences tend to be genome-specific due to the rate of amplification and extent of divergence. Two genome-specific probes from the genomic DNA library of Festuca arundinacea var. genuina Schreb.were selected and characterized. TF521 was found to be P genome-specific since it was able to hybridize with Festuca pratensis Huds. (PP) and Festuca arundinacea var. genuina (PPG₁G₁G₂G₂), but not, or only weakly, with tetraploid Festuca species. TF521 hybridized only with the diploid Festuca and not with the Lolium species (LL). TF436 was specific to tetraploid species of Festuca, such as F. arundinacea var. glauces-cens Boiss. (G₁G₁G₂G₂) and Festuca mairei St. Yves (M₁M₁M₂M₂). By means of Southern hybridization, TF436 was used to detect chromatin introgression of F. mairei in the progenies of the hybrid F. mairei×Lolium perenne L. Potential addition and translocation lines were identified in the BC₁F₁ derivatives of F. mairei×L. perenne. In situ hybridization was used to confirm the genetic identity of these lines. Sequence analyses indicated that TF436 and TF521 were two novel DNA sequences as no homologous sequences were found in Genebank. Received: 22 June 2000 / Accepted: 3 November 2000     

Characterization and localization of repetitive DNA sequences in the ornamental Alstroemeria aurea Graham

 Three repetitive DNA sequences were isolated from a genomic DNA library of the ornamental Alstroemeria aurea Graham. Two repeats, A001-I and A001-II, were quite homologous and highly A. aurea-specific. A001-I was a 217-bp sequence with several telomeric TTTAGGG repeats at the 5′ end and a unique sequence of 98 bp at the other end. The third repeat, A001-IV, was a 840-bp sequence which contained two sub-sequences of 56 and 74 bp respectively, previously found in chloroplast (cp) DNA of tobacco and spinach and to a lesser extent in the cpDNA of maize and rice. Repeat A001-IV was not species-specific and its hybridization signal was weaker than the other repeats. Fluorescence in situ hybridization (FISH) revealed the A. aurea-specific repeats to be located in the heterochromatic regions of all A. aurea chromosomes. The differences in FISH pattern make them useful tools for karyotype analysis. The non-species-specific sequence A001-IV gave a dispersed signal over all the Alstroemeria chromosomes in an interspecific hybrid. The potential use of these repetitive DNA sequences for the study of phylogenetic relationships within the genus Alstroemeria is discussed. Received: 24 November 1996/Accepted: 20 December 1996     

Methylation changes of H19 gene in sperms of X-irradiated mouse and maintenance in offspring

The nature of imprinting is just differential methylation of imprinted genes. Unlike the non-imprinted genes, the methylation pattern of imprinted genes established during the period of gametogenesis remains unchangeable after fertilization and during embryo development. It implies that gametogenesis is the key stage for methylation pattern of imprinted genes. The imprinting interfered by exogenous factors during this stage could be inherited to offspring and cause genetic effect. Now many studies have proved that ionizing irradiation could disturb DNA methylation. Here we choose BALB/c mice as a research model and X-ray as interfering source to further clarify it. We discovered that the whole-body irradiation of X-ray to male BALB/c mice could influence the methylation pattern of H19 gene in sperms, which resulted in some cytosines of partial CpG islands in the imprinting control region could not transform to methylated cytosines. Furthermore, by copulating the interfered male mice with normal female, we analyzed the promoter methylation pattern of H19 in offspring fetal liver and compared the same to the pattern of male parent in sperms. We found that the majority of methylation changes in offspring liver were related to the ones in their parent sperms. Our data proved that the changes of the H19 gene methylation pattern interfered by X-ray irradiation could be transmitted and maintained in the first-generation offspring.     

The Au family, a novel short interspersed element (SINE) from Aegilops umbellulata

A novel plant short interspersed nuclear element (SINE) was identified in the second intron of the acetyl CoA carboxylase gene of Aegilops umbellulata which has been designated ”Au”, for the host species in which it was discovered. Au elements have a tRNA-related region, direct flanking repeats, and a short stretch of T at the 3′ end, which are features common to Au and previously characterized SINEs. Au elements are detected in the genomes of several monocots and dicots by DNA dot hybridization and are also found in the tobacco genome by database searching. Au elements are present at an especially high copy number (approximately 10⁴ copies per haploid genome) in wheat and Ae. umbellulata. This suggests a recent amplification of Au in the Triticum and Aegilops species. In situ hybridization revealed a dispersed distribution of Au elements on wheat chromosomes. Au elements were amplified by PCR from monocot and dicot species and the phylogenetic relationships among Au elements were inferred. This phylogenetic analysis suggests amplification of Au elements in a manner consistent with the retrotransposon model for SINE dispersion. The high copy number of Au elements and their dispersed distribution in wheat are desirable characteristics for a molecular marker system in this important species. Received: 15 April 2000 / Accepted: 24 August 2000     

Nucleosome positioning signals in the DNA sequence of the human and mouse H19 imprinting control regions

We have investigated the sequences of the mouse and human H19 imprinting control regions (ICRs) to see whether they contain nucleosome positioning information pertinent to their function as a methylation-regulated chromatin boundary. Positioning signals were identified by an in vitro approach that employs reconstituted chromatin to comprehensively describe the contribution of the DNA to the most basic, underlying level of chromatin structure. Signals in the DNA sequence of both ICRs directed nucleosomes to flank and encompass the short conserved sequences that constitute the binding sites for the zinc finger protein CTCF, an essential mediator of insulator activity. The repeat structure of the human ICR presented a conserved array of strong positioning signals that would preferentially flank these CTCF binding sites with positioned nucleosomes, a chromatin structure that would tend to maintain their accessibility. Conversely, all four CTCF binding sites in the mouse sequence were located close to the centre of positioning signals that were stronger than those in their flanks; these binding sites might therefore be expected to be more readily incorporated into positioned nucleosomes. We found that CpG methylation did not effect widespread repositioning of nucleosomes on either ICR, indicating that allelic methylation patterns were unlikely to establish allele-specific chromatin structures for H19 by operating directly upon the underlying DNA-histone interactions; instead, epigenetic modulation of ICR chromatin structure is likely to be mediated principally at higher levels of control. DNA methylation did, however, both promote and inhibit nucleosome positioning at several sites in both ICRs and substantially negated one of the strongest nucleosome positioning signals in the human sequence, observations that underline the fact that this epigenetic modification can, nevertheless, directly and decisively modulate core histone-DNA interactions within the nucleosome.