Locations of three repetitive sequence families found in BALB/c adult beta-globin clones.

Three different repeat sequences have been mapped within the cloned EcoRI fragments that contain the adult beta-globin genes from the BALB/c (Hddd) mouse. One sequence, "a", occurs 1.5-2 kb 3' to the beta-major gene. A second, "b", is found 4kb 5' and 7.5kb 3' to the beta-minor gene. The 14kb EcoRI fragment bearing the beta-minor gene carries at least one additional repetitive element, "c". Probing a BALB/c DNA library with each repeat has demonstrated that these sequences are moderately to highly repetitive and are extensively interspersed with each other throughout the genome. In addition, repeats "a" and "b" are preferentially found in satellite and main-band DNa, respectively. The occurrence of these repeats elsewhere in the beta-globin cluster was demonstrated by probing the non-adult globin clones with each repeat. The arrangement of these repeats around the non-adult genes is 5'-"b"-"b"-epsilon y-beta hl-beta h2-"c"-beta h3-3'. Probing the C57BL/10 (Hbbs) adult gene clones with these repeats demonstrated that the distribution of these sequences in the adult region of these two haplotypes is essentially the same.     

Correlations of repetitive and AT-rich DNA segments within the chicken globin gene domains

The repetitive DNA segments were mapped within a 30 Kbp genomic domain including (in 5 to 3 order) the chicken embryonic pi and adult alpha D (minor) and alpha A (major) globin genes. Two repeats map 5 and 8 Kbp upstream from the embryonic pi gene and another 3 Kbp downstream of the adult alpha A gene. These repetitive DNA sequences are placed within, or immediately adjacent to the AT-rich DNA segments framing this domain. Similar correlations exist also within the chicken beta globin gene domain. The positions of these AT-rich and repetitive DNA segments framing the alpha globin gene domain also correlate with other already explored features of long range DNA organisation, as clusters of sites of DNAse I hypersensitivity and differential methylation, sites of Matrix-DNA attachment, and with the beginning and end of the transcribed domain.     

Tissue-specific differences in cytosine methylation and their association with differential gene expression in sorghum

It has been well established that DNA cytosine methylation plays essential regulatory roles in imprinting gene expression in endosperm, and hence normal embryonic development, in the model plant Arabidopsis (Arabidopsis thaliana). Nonetheless, the developmental role of this epigenetic marker in cereal crops remains largely unexplored. Here, we report for sorghum (Sorghum bicolor) differences in relative cytosine methylation levels and patterns at 5'-CCGG sites in seven tissues (endosperm, embryo, leaf, root, young inflorescence, anther, and ovary), and characterize a set of tissue-specific differentially methylated regions (TDMRs). We found that the most enriched TDMRs in sorghum are specific for the endosperm and are generated concomitantly but imbalanced by decrease versus increase in cytosine methylation at multiple 5'-CCGG sites across the genome. This leads to more extensive demethylation in the endosperm than in other tissues, where TDMRs are mainly tissue nonspecific rather than specific to a particular tissue. Accordingly, relative to endosperm, the other six tissues showed grossly similar levels though distinct patterns of cytosine methylation, presumably as a result of a similar extent of concomitant decrease versus increase in cytosine methylation that occurred at variable genomic loci. All four tested TDMRs were validated by bisulfite genomic sequencing. Diverse sequences were found to underlie the TDMRs, including those encoding various known-function or predicted proteins, transposable elements, and those bearing homology to putative imprinted genes in maize (Zea mays). We further found that the expression pattern of at least some genic TDMRs was correlated with its tissue-specific methylation state, implicating a developmental role of DNA methylation in regulating tissue-specific or -preferential gene expression in sorghum.     

DNA methylation patterns in the 5S DNAs of Xenopus laevis

The frequency of cytosine methylation at specific sites in the somatic 5S DNA (X1s) and trace oocyte 5S DNA (X1t) of X. laevis has been determined using restriction enzymes that are inhibited by the presence of 5-methylcytosine (5mC) within their cleavage sequences. 5S DNA methylation patterns were determined in genomic DNA from mature red blood cells, which express neither type of 5S gene, and from liver, which expresses only X1s. All the sites examined in X1t are greater than 95% methylated in red cells and liver. In the X1s of red cells all the sites examined are methylated in greater than 95% of repeats, while in liver some sites are modified in only 90% of repeats. Repeats containing unmethylated sites are randomly distributed throughout the tandem arrays in both red cells and liver. The high levels of methylation for X1s are in marked contrast to the situation with other Xenopus genes which do have sites of significant undermethylation in tissues where they are active. Thus, undermethylation in active genetic regions may not be a general feature for all classes of eukaryotic genes.     

The organization of repetitive sequences in a cluster of rabbit β-like globin genes

Several complementary procedures were used to identify and characterize DNA sequences which are repeated within a 44 kilobase (kb) segment of rabbit chromosomal DNA containing four different rabbit β-like globin genes (β1–β4). Cross-hybridization between cloned DNAs from different regions of the gene cluster indicates the presence of a complex array of repeat sequences interspersed with the globin genes. We classified 20 different repeat sequences into five families whose members cross-hybridize. Electron microscopy was used to determine the location, size and relative orientations of many of the repeat sequences. Both direct and inverted repeats were identified, with sizes ranging from 140 to 1400 base pairs (bp). Each of the four closely linked globin genes is flanked by at least one pair of inverted repeats of 140–400 bp, and the entire set of four genes is flanked by an inverted repeat of 1400 bp. Two of the five repeat families contain repeat sequences of different sizes. We found that the smaller sequence elements can occur individually or in association with the larger repeat sequences, suggesting that the larger repeats may be composed of more than one smaller repeat sequence. The restriction fragments containing the intracluster repeats also contain sequences which are repeated many times in total rabbit genomic DNA, but it is not known whether the genomic and intracluster repeats are the same sequences. The results provide the first demonstration of the relationship between single-copy and repetitive DNA sequences in a large segment of chromosomal DNA containing a well characterized set of developmentally regulated genes.     

Human immunoglobulin kappa gene enhancer: chromatin structure analysis at high resolution.

The murine immunoglobulin kappa gene enhancer has previously been found to coincide with a region of altered chromatin structure reflected in a DNase I hypersensitivity site detectable on Southern blots of B-cell DNA. We examined the chromatin structure of the homologous region of human DNA using the high-resolution electroblotting method originally developed for genomic sequence analysis by G. Church and W. Gilbert (Proc. Natl. Acad. Sci. USA 81:1991-1995, 1984). Analysis of DNA isolated from cells treated in vivo with dimethyl sulfate revealed two B-cell-specific sites of enhanced guanine methylation. Both sites are located within perfect inverted repeats theoretically capable of forming cruciform structures; one of these repeats overlaps an enhancer core sequence. No enhancement or protection of guanine methylation was observed within sequences similar to sites of altered methylation previously described in the immunoglobulin heavy-chain enhancer. Treatment of isolated nuclei with DNase I or a variety of restriction endonucleases defined a B-cell-specific approximately 0.25-kilobase region of enhanced nuclease susceptibility similar to that observed in the murine kappa enhancer. The 130-base-pair DNA segment that shows high sequence conservation between human, mouse, and rabbit DNAs lies at the 5' end of the nuclease-susceptible region.     

Role of Arabidopsis ARGONAUTE4 in RNA-directed DNA methylation triggered by inverted repeats

In a number of organisms, transgenes containing transcribed inverted repeats (IRs) that produce hairpin RNA can trigger RNA-mediated silencing, which is associated with 21-24 nucleotide small interfering RNAs (siRNAs). In plants, IR-driven RNA silencing also causes extensive cytosine methylation of homologous DNA in both the transgene "trigger" and any other homologous DNA sequences--"targets". Endogenous genomic sequences, including transposable elements and repeated elements, are also subject to RNA-mediated silencing. The RNA silencing gene ARGONAUTE4 (AGO4) is required for maintenance of DNA methylation at several endogenous loci and for the establishment of methylation at the FWA gene. Here, we show that mutation of AGO4 substantially reduces the maintenance of DNA methylation triggered by IR transgenes, but AGO4 loss-of-function does not block the initiation of DNA methylation by IRs. AGO4 primarily affects non-CG methylation of the target sequences, while the IR trigger sequences lose methylation in all sequence contexts. Finally, we find that AGO4 and the DRM methyltransferase genes are required for maintenance of siRNAs at a subset of endogenous sequences, but AGO4 is not required for the accumulation of IR-induced siRNAs or a number of endogenous siRNAs, suggesting that AGO4 may function downstream of siRNA production.     

The DeltauvrB mutations in the Ames strains of Salmonella span 15 to 119 genes

The lacI transgene used in the Big Blue (BB) mouse and rat mutation assays typically displays spontaneous mutation frequencies in the 5x10(-5) range. Recently, the bone marrow and bladder of the Big Blue rat were reported to have, by an order of magnitude, the lowest spontaneous mutation frequencies ever observed for lacI in a transgenic animal, approaching the value for endogenous targets such as hprt ( approximately 10(-6)). Since spontaneous mutations in transgenes have been attributed in part to deamination of 5-methylcytosine in CpG sequences, we have investigated the methylation status of the lacI transgene in bone marrow of BB rats and compared it to that present in other tissues including liver, spleen, and breast. The first 400 bases of the lacI gene were investigated using bisulfite genomic sequencing since this region contains the majority of both spontaneous and induced mutations. Surprisingly, all the CpG cytosines in the lacI sequence were fully methylated in all the tissues examined from both 2- and 14-week-old rats. Thus, there is no correlation between 5-methylcytosine content at CpG sites in lacI and the frequency of spontaneous mutation at this marker. We also investigated the methylation status of another widely used transgenic mutation target, the cII gene. The CpG sites in cII in BB rats were fully methylated while those in BB mice were partially methylated (each site approximately 50% methylated). Since spontaneous mutation frequency at cII is comparable in rat and mouse, the methylation status of CpG sequences in this gene also does not correlate with spontaneous frequency. We conclude that other mechanisms besides spontaneous deamination of 5-methylcytosine at CpG sites are driving spontaneous mutation at BB transgenic loci.     

Nucleotide sequence analysis of the delta beta-globin gene region in humans

The continuous DNA sequence of a 16.5-kilobase pair region encompassing the linked delta beta-globin gene cluster in humans is presented with a detailed restriction endonuclease map. There are 38 differences (0.5%) in comparison with published sequence data, corrected for errors in sequencing, resulting in polymorphic rates of 0.2% in exons and 0.76% in 5'-gene flanking regions. Fifteen changes result in the generation or elimination of restriction sites which may be useful in linkage disequilibrium studies. Two pairs of inverted Alu repeats, a pyrimidine-rich region 5' to delta, and (TG)n, (Pu/Py)n, and (ATTTT)n tracts 5' to beta are described. Dinucleotide frequencies and deviation from expected values approximated those found in total human genomic DNA. Regions of less than 50% A + T content were found associated with Alu sequences, a 150-base pair region immediately 5' to the beta gene, exon regions from both genes, and an area 3' to the beta gene. These regions also contained significantly lower than expected CpG levels compared to other regions, suggesting a possible relationship between DNA organizational patterns and functionally important regions. In addition, strand asymmetries in base composition in this region differ from those associated with the fetal globin genes.     

Characterization of an unusual DNA length polymorphism 5'' to the human antithrombin III gene

Nucleotide sequence analysis revealed that a DNA length polymorphism 5' to the human antithrombin III gene is due to the presence of 32bp or 108bp nonhomologous nucleotide sequences (variable segments) 345bp upstream from the translation initiation codon. Sequences at the 3' borders of both variable segments can form intrastrand inverted repeat structures with sequences further downstream. An inverted repeat is also found immediately 5' to the site where the variable segments are located. Thus, cruciform structures may form flanking the variable segments of both alleles of this DNA length polymorphism. DNA secondary structure may be detected with single strand specific nucleases. S1 nuclease sensitive sites were mapped in recombinant plasmids containing the cloned alleles of the ATIII length polymorphism. The site most sensitive to S1 is located upstream from the variable segments in an AT-rich segment flanked by 6bp direct repeats. A region of lesser nuclease sensitivity was also observed in the AT-rich loops formed between the inverted repeats 5' to the variable segments.     

Characteristics of palindromic sequences in DNA of the sea urchin Strongylocentrotus intermedius

Some properties of the palindromic sequences in the sea urchin Strongylocentrotus intermedius nuclear DNA have been studied. It was shown that the amount of "foldback HAP bound DNA" and the S1 nuclease resistant DNA depends on renaturation temperature and Na+ concentration in solution. The authentic fraction of inverted repeats comprises 10-15% of the total DNA. The complexity of the palindromic fraction is approximately 8,2 X 10(7) nucleotide pairs and the average number of inverted repeats approximates 5 X 10(5) per haploid genome. The renaturation kinetics of inverted repeats with excess of total homologous DNA indicates that these sequences are enriched with unique DNA. The possible function of palindromic sequences is discussed.     

Regulation of imprinted DNA methylation

DNA methylation is an essential enzymatic modification in mammals. This common epigenetic mark occurs predominantly at the fifth carbon of cytosines within the palindromic dinucleotide 5'-CpG-3'. The majority of methylated CpGs are located within repetitive elements including centromeric repeats, satellite sequences and gene repeats encoding ribosomal RNAs. CpG islands, frequently located at the 5' end of genes, are typically unmethylated. DNA methylation also occurs at imprinted genes which exhibit parent-of-origin-specific patterns of methylation and expression. Imprinted methylation at differentially methylated domains (DMDs) is one of the regulatory mechanisms controlling the allele-specific expression of imprinted genes. Proper control of DNA methylation is needed for normal development and loss of methylation control can contribute to initiation and progression of tumorigenesis (reviewed in Plass and Soloway, 2002). Because patterns of imprinted DNA methylation are highly reproducible, imprinted loci make useful models for studying regulation of DNA methylation and may provide insights into how this regulation goes awry in cancer. Here, we review what is currently known about the mechanisms regulating imprinted DNA methylation. We will focus on cis-acting DNA sequences, trans-acting protein factors and the possible involvement of RNAs in control of imprinted DNA methylation.     

H3 lysine 9 methylation is maintained on a transcribed inverted repeat by combined action of SUVH6 and SUVH4 methyltransferases

Transcribed inverted repeats are potent triggers for RNA interference and RNA-directed DNA methylation in plants through the production of double-stranded RNA (dsRNA). For example, a transcribed inverted repeat of endogenous genes in Arabidopsis thaliana, PAI1-PAI4, guides methylation of itself as well as two unlinked duplicated PAI genes, PAI2 and PAI3. In previous work, we found that mutations in the SUVH4/KYP histone H3 lysine 9 (H3 K9) methyltransferase cause a loss of DNA methylation on PAI2 and PAI3, but not on the inverted repeat. Here we use chromatin immunoprecipitation analysis to show that the transcribed inverted repeat carries H3 K9 methylation, which is maintained even in an suvh4 mutant. PAI1-PAI4 H3 K9 methylation and DNA methylation are also maintained in an suvh6 mutant, which is defective for a gene closely related to SUVH4. However, both epigenetic modifications are reduced at this locus in an suvh4 suvh6 double mutant. In contrast, SUVH6 does not play a significant role in maintenance of H3 K9 or DNA methylation on PAI2, transposon sequences, or centromere repeat sequences. Thus, SUVH6 is preferentially active at a dsRNA source locus versus targets for RNA-directed chromatin modifications.