DNA methylation of mobile genetic elements in human cancers

Mobile genetic elements are responsible for half of the human genome, creating the host genomic instability or variability through several mechanisms. Two types of abnormal DNA methylation in the genome, hypomethylation and hypermethylation, are associated with cancer progression. Genomic hypermethylation has been most often observed on the CpG islands around gene promoter regions in cancer cells. In contrast, hypomethylation has been observed on mobile genetic elements in the cancer cells. It is recently considered that the hypomethylation of mobile genetic elements may play a biological role in cancer cells along with the DNA hypermethylation on CpG islands. Growing evidence has indicated that mobile genetic elements could be associated with the cancer initiation and progression through the hypomethylation. Here we review the recent progress on the relationship between DNA methylation and mobile genetic elements, focusing on the hypomethylation of LINE-1 and HERV elements in various human cancers and suggest that DNA hypomethylation of mobile genetic elements could have potential to be a new cancer therapy target in the future.     

Deoxyadenylate-rich and deoxyguanylate-rich regions in mammalian DNA

The presence of deoxyadenylate-rich and deoxyguanylate-rich regions in mammalian DNA has been demonstrated by hybridization with ³H-labelled poly(U) and ³H-labelled poly(C). For hamster BHK-21/C13 cells, the dA-rich regions are up to 130 nucleotides long and comprise up to 0.4% of the DNA. Those dA-rich regions which comprise 0.13% of the DNA contain 2 to 6% of bases other than adenine. The dG-rich regions, in which 10 to 30% of the bases are other than guanine, are less than 40 nucleotides long and are present at a level of about 0.1% of the DNA. Exhaustive digestion of the hybrids with RNAase enables detection of deoxyhomopolymeric regions in the DNA, poly (dA) sequences of an average size of about 30 nucleotides long accounting for 0.008% of the DNA, and poly(dG) sequences, 17 nucleotides long, comprising 0.0016% of the DNA.Both dA-rich and dG-rich regions are found in DNA sequences with a wide variety of base composition. Extensive shearing of the DNA is required to produce some enrichment for dA-rich sequences in the (A + T)-rich fraction, although dG-rich sequences are slightly enriched in the (G + C)-rich fraction of even unsheared DNA. The buoyant density of hybrid molecules was found to be significantly greater than that of unhybridized DNA only when highly sheared DNA was used. These findings suggest that the dA-rich and dG-rich regions have a widespread distribution throughout DNA molecules. In situ hybridization studies with ³H-labelled poly(U) further suggest that the dA-rich regions are not localized to any particular chromosome or to any specific region of the chromosomes. Analysis of DNA from a number of different species has shown that, in general, the dA-rich and dG-rich regions are present at a much higher level in mammalian DNA than in bacterial, bacteriophage or mammalian virus DNA.Possible functions of these unusual deoxynucleotide sequences are discussed.     

<Emphasis Type="Italic">hATpin</Emphasis>, a Family of MITE-like <Emphasis Type="Italic">hAT</Emphasis> Mobile Elements Conserved in Diverse Plant Species that Forms Highly Stable Secondary Structures

We identified a 178 bp mobile DNA element in lettuce with characteristic CGAGC/GCTCG repeats in the subterminal regions. This element has terminal inverted repeats and 8-bp target site duplications typical of the hAT superfamily of class II mobile elements, but its small size and potential to form a single-stranded stable hairpin-like secondary structure suggest that it is related to MITE elements. In silico searches for related elements identified 252 plant sequences with 8-bp target site duplications and sequence similarity in their terminal and subterminal regions. Some of these sequences were predicted to encode transposases and may be autonomous elements; these constituted a separate clade within the phylogram of hAT transposases. We demonstrate that the CGAGC/GCTCG pentamer maximizes the hairpin stability compared to any other pentamer with the same C + G content, and the secondary structures of these elements are more stable than for most MITEs. We named these elements collectively as hATpin elements because of the hAT similarity and their hairpin structures. The nearly complete rice genome sequence and the highly advanced genome annotation allowed us to localize most rice elements and to deduce insertion preferences. hATpin elements are distributed on all chromosomes, but with significant bias for chromosomes 1 and 10 and in regions of moderate gene density. This family of class II mobile elements is found primarily in monocot species, but is also present in dicot species. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Intraspecific variability of the terminal inverted repeats of the linear chromosome of Streptomyces ambofaciens

The sequences of the terminal inverted repeats (TIRs) ending the linear chromosomal DNA of two Streptomyces ambofaciens strains, ATCC23877 and DSM40697 (198 kb and 213 kb, respectively), were determined from two sets of recombinant cosmids. Among the 215 coding DNA sequences (CDSs) predicted in the TIRs of strain DSM40697, 65 are absent in the TIRs of strain ATCC23877. Reciprocally, 45 of the 194 predicted CDSs are specific to the ATCC23877 strain. The strain-specific CDSs are located mainly at the terminal end of the TIRs. Indeed, although TIRs appear almost identical over 150 kb (99% nucleotide identity), large regions of DNA of 60 kb (DSM40697) and 48 kb (ATCC23877), mostly spanning the ends of the chromosome, are strain specific. These regions are rich in plasmid-associated genes, including genes encoding putative conjugal transfer functions. The strain-specific regions also share a G+C content (68%) lower than that of the rest of the genome (from 71% to 73%), a percentage that is more typical of Streptomyces plasmids and mobile elements. These data suggest that exchanges of replicon extremities have occurred, thereby contributing to the terminal variability observed at the intraspecific level. In addition, the terminal regions include many mobile genetic element-related genes, pseudogenes, and genes related to adaptation. The results give insight into the mechanisms of evolution of the TIRs: integration of new information and/or loss of DNA fragments and subsequent homogenization of the two chromosomal extremities.     

Double-hairpin elements in the mitochondrial DNA of allomyces: evidence for mobility

The mitochondrial DNA (mtDNA) of the chytridiomycete fungus Allomyces macrogynus contains 81 G+C-rich sequence elements that are 26-79 bases long and can be folded into a unique secondary structure consisting of two stem-loops. At the primary sequence level, the conservation of these double-hairpin elements (DHEs) is variable, ranging from marginal to complete identity. Forty of these DHEs are inserted in intergenic regions, 35 in introns, and 6 in variable regions of rRNA genes. Ten DHEs are inserted into other DHE elements (twins); two even form triplets. A comparison of DHE sequences shows that loop regions contain more sequence variation than helical regions and that the latter often contain compensatory base changes. This suggests a functional importance of the DHE secondary structure. We further identified nine DHEs in a 4-kb region of Allomyces arbusculus, a close relative of A. macrogynus. Eight of these DHEs are highly similar in sequence (90%-100%) to those in A. macrogynus, but only five are inserted at the same positions as in A. macrogynus. Interestingly, DHEs are also found in the mtDNAs of other chytridiomycetes, as well as certain zygomycete and ascomycete fungi. The overall distribution pattern of DHEs in fungal mtDNAs suggests that they are mobile elements.     

Study of the double-stranded fragments in the poly(A)-containing cytoplasmic RNA of the rat liver

The double-stranded sequences in the poly(A)-containing cytoplasmic RNA (c-dsRNA) hybridize mainly with the repetitive DNA. 70-80% of double-stranded regions in the cytoplasmic poly (A)-RNA are identical to double-stranded regions of heterogeneous nuclear RNA in normal as well as in cortisone-treated rats. The thermal stability of cytoplasmic double-stranded regions is higher in the presence of polyadenylate sequences than in their absence. It is suggested that the double-stranded sequences in the poly (A) +RNA interact with poly (A) stretches and form higher order structures. The thermal stability of c-dsRNA isolated from cortisone-treated rats is higher than that from control rats.     

RIP: the evolutionary cost of genome defense

Repeat-induced point mutation (RIP) is a homology-based process that mutates repetitive DNA and frequently leads to epigenetic silencing of the mutated sequences through DNA methylation. Consistent with the hypothesis that RIP serves to control selfish DNA, an analysis of the Neurospora crassa genome sequence reveals a complete absence of intact mobile elements. As in most eukaryotes, the centromeric regions of N. crassa are rich in sequences that are related to transposable elements; however, in N crassa these sequences have been heavily mutated. The analysis of the N. crassa genome sequence also reveals that RIP has impacted genome evolution significantly through gene duplication, which is considered to be crucial for the evolution of new functions. Most if not all paralogs in N. crassa duplicated and diverged before the emergence of RIP. Thus, RIP illustrates the extraordinary extent to which genomes will go to defend themselves against mobile genetic elements.     

Small mobile sequences in bacteria display diverse structure/function motifs

Small repeat sequences in bacterial genomes, which represent non-autonomous mobile elements, have close similarities to archaeon and eukaryotic miniature inverted repeat transposable elements. These repeat elements are found in both intergenic and intragenic chromosomal regions, and contain an array of diverse motifs. These can include DNA sequences containing an integration host factor binding site and a proposed DNA methyltransferase recognition site, transcribed RNA secondary structural motifs, which are involved in mRNA regulation, and translated open reading frames found fused to other open reading frames. Some bacterial mobile element fusions are in evolutionarily conserved protein and RNA genes. Others might represent or lead to creation of new protein genes. Here we review the remarkable properties of these small bacterial mobile elements in the context of possible beneficial roles resulting from random insertions into the genome.     

Distamycin and penta-N-methylpyrrolecarboxamide binding sites on native DNA. A comparison of methidiumpropyl-EDTA-Fe(II) footprinting and DNA affinity cleaving

Using two direct methods we have studied the binding locations and site sizes of distamycin and penta-N-methylpyrrolecarboxamide on three DNA restriction fragments from pBR322 plasmid. We find that methidiumpropyl-EDTA.Fe(II) footprinting and DNA affinity cleaving methods report common binding locations and site sizes for the tri- and pentapeptides bound to heterogeneous DNA. The tripeptide distamycin binds 5-base-pair sites with a preference for poly(dA).poly(dT) regions. The pentapeptide binds 6-7-base-pair sites with a preference for poly(dA).poly(dT) regions. These results are consistent with distamycin binding as an isogeometric helix to the minor groove of DNA with the four carboxamide N-H's hydrogen bonding five A + T base pairs. The data supports a model where each of the carboxamide N-H's can hydrogen bond to two bases, either O(2) of thymine or N(3) of adenine, located on adjacent base pairs on opposite strands of the helix. In most (but not all) cases the tri- and pentapeptide can adopt two orientations at each A + T rich binding site.     

The transcriptional regulator Rok binds A+T-rich DNA and is involved in repression of a mobile genetic element in Bacillus subtilis

The rok gene of Bacillus subtilis was identified as a negative regulator of competence development. It also controls expression of several genes not related to competence. We found that Rok binds to extended regions of the B. subtilis genome. These regions are characterized by a high A+T content and are known or believed to have been acquired by horizontal gene transfer. Some of the Rok binding regions are in known mobile genetic elements. A deletion of rok resulted in higher excision of one such element, ICEBs1, a conjugative transposon found integrated in the B. subtilis genome. When expressed in the Gram negative E. coli, Rok also associated with A+T-rich DNA and a conserved C-terminal region of Rok contributed to this association. Together with previous work, our findings indicate that Rok is a nucleoid associated protein that serves to help repress expression of A+T-rich genes, many of which appear to have been acquired by horizontal gene transfer. In these ways, Rok appears to be functionally analogous to H-NS, a nucleoid associated protein found in Gram negative bacteria and Lsr2 of high G+C Mycobacteria.     

The variability of the telomeric and of a number of internal regions of Drosophila chromosomes according to the intensity of hybridization in situ with labelled probes

Cloned Drosophila DNA fragments containing telomere-specific sequences have been hybridized in situ. It is found that intensity of their hybridization with telomeres greatly varies in different nuclei of the same salivary gland. This phenomenon is also observed for internal sites where some mobile elements included in several DNA fragments under investigation are located. Within each nucleus different regions are hybridized non-uniformly as well. It is suggested that these phenomena can be explained by varying polytenization in telomeres and some internal chromosomal regions.     

A study of the interactions of some polypyridylruthenium (II) complexes with DNA using fluorescence spectroscopy, topoisomerisation and thermal denaturation.

The nature of binding of Ru(phen) 2+ (I), Ru(bipy) 2+ (II), Ru(terpy) 2+ (III) (phen = 1,10-phenanthroline, bipy 3 = 2,2'-bipyridyl, 3 terpy = 2,2'2," - 2 terpyridyl) to DNA, poly[d(G-C)] and poly[d(A-T)] has been compared by absorption, fluorescence, DNA melting and DNA unwinding techniques. I binds intercalatively to DNA in low ionic strength solutions. Topoisomerisation shows that it unwinds DNA by 22 degrees +/- 1 per residue and that it thermally stabilizes poly[d(A-T)] in a manner closely resembling ethidium. Poly[d(A-T)] induces greater spectral changes on I than poly[d(G-C)] and a preference for A-T rich regions is indicated. I binding is very sensitive to Mg2+ concentration. In contrast to I the binding of II and III appears to be mainly electrostatic in nature, and causes no unwinding. There is no evidence for the binding of the neutral Ru(phen)2 (CN)2 or Ru(bipy)2 (CN)2 complexes. DNA is cleaved, upon visible irradiation of aerated solutions, in the presence of either I or II.     

Core nucleosomes by digestion of reconstructed histone-DNA complexes.

Reconstructed complexes of the inner histones (H2A, H2B, H3, H4) and a variety of DNAs were digested with micrococcal nuclease to yield very homogeneous populations of core nucleosomes (nu 1). Nucleosomes containing Micrococcus luteus DNA (72% G+C); chicken DNA (43% G+C), Clostridium perfringens DNA (29% G+C); or poly(A-dT.poly(dA-dT) have been examined by circular dichroism, thermaldetenaturation, electron microscopy, and DNAse I digestion. Circular dichroism spectra of all particles show a typically suppressed ellipticity at 260--280 nm and a prominent alpha-helix signal at 222 nm. All particles show biphasic melting except nu 1 (dA-dT), which show three prominent melting transitions at ionic strength less than or equal to 1 mM. DNAse I digestion of nu 1 (dA-dT) produces a ladder of DNA fragments fiffering in lengthy by one base residue. nu 1 (dA-dT) contain 146 base pairs of DNA and exhibit an average DNA helix pitch of 10.4-10.5 bases per turn. There appear to be two regions of different DNA pitch wihtin nu 1 (dA-dT). It is suggested that the two regions of DNA pitch might correspond to the two regions of the melting profiles.     

Unichrom, a novel nuclear matrix protein, binds to the Ars insulator and canonical MARs

Eukaryotic genomic DNA is organized into loop structures by attachments to the nuclear matrix. These attachments to the nuclear matrix have been supposed to form the boundaries of chromosomal DNA. Insulators or boundary elements are defined by two characteristics: they interrupt promoter-enhancer communications when inserted between them, and they suppress the silencing of transgenes stably integrated into inactive chromosomal domains. We recently identified an insulator element in the upstream region of the sea urchin arylsulfatase (HpArs) gene that shows both enhancer blocking and suppression of position effects. Here, we report that Unichrom, originally identified by its G-stretch DNA binding capability, is a nuclear matrix protein that binds to the Ars insulator and canonical nuclear matrix attachment regions (MARs). We also show that Unichrom recognizes the minor groove of the AT-rich region within the Ars insulator, which may have a base-unpairing property, as well as the G-stretch DNA. Furthermore, Unichrom selectively interacts with poly(dG).poly(dC), poly(dA).poly(dT) and poly(dAT).poly(dAT), but not with poly(dGC).poly(dGC). Unichrom also shows high affinity for single-stranded G- and C-stretches. We discuss the DNA binding motif of Unichrom and the function of Unichrom in the nuclear matrix.     

Human plasminogen activator inhibitor-1 gene. Promoter and structural gene nucleotide sequences

We have determined the nucleotide sequence of the human plasminogen activator inhibitor-1 (PAI-1) gene and significant stretches of DNA which extend into its 5'-and 3'-flanking DNA regions; a total sequence of 15,867 base pairs (bp) is presented. The sequenced 5'-flanking DNA (1,520 bp) contains the essential eukaryotic cis-type proximal regulatory elements CCAAT and TATAA; the more distal 5'-flanking DNA region, as well as some introns, contain sequence elements which share identities with known eukaryotic enhancer elements. A major finding is the identification of a large region of shared nucleotides (comprising of about 520 bp) between the 5'-flanking DNAs of PAI-1 and tissue-type plasminogen activator genes. The length of the PAI-1 5'-untranslated region was found to be 145 bp as determined by nuclease analysis. The remaining PAI-1 structural gene consists of amino acid coding regions (containing a total of 1,206 bp, coding for the 23 amino acids of the signal peptide and 379 amino acids of the mature PAI-1 protein), 8 intron regions (a total of 8,978 bp), and a long 3'-untranslated region of about 1,800 bp which contains several polyadenylation sites. Two types of repetitive DNA elements are located within the PAI-1 structural gene and flanking DNAs: we have found 12 Alu elements and 5 repeats of a long poly (Pur) element. These Alu-Pur elements may represent a subset of the more abundant Alu family of repetitive sequence elements.