Kenji Ichiyanagi

Role for piRNAs and Noncoding RNA in de Novo DNA Methylation of the Imprinted Mouse Rasgrf1 Locus

Small, noncoding PIWI-interacting RNAs regulate the imprinting of a mouse gene. Genomic imprinting causes parental origin–specific monoallelic gene expression through differential DNA methylation established in the parental germ line. However, the mechanisms underlying how specific sequences are selectively methylated are not fully understood. We have found that the components of the PIWI-interacting RNA (piRNA) pathway are required for de novo methylation of the differentially methylated region (DMR) of the imprinted mouse Rasgrf1 locus, but not other paternally imprinted loci. A retrotransposon sequence within a noncoding RNA spanning the DMR was targeted by piRNAs generated from a different locus. A direct repeat in the DMR, which is required for the methylation and imprinting of Rasgrf1, served as a promoter for this RNA. We propose a model in which piRNAs and a target RNA direct the sequence-specific methylation of Rasgrf1.

Genetic Evidence That the Non-Homologous End-Joining Repair Pathway Is Involved in LINE Retrotransposition

Long interspersed elements (LINEs) are transposable elements that proliferate within eukaryotic genomes, having a large impact on eukaryotic genome evolution. LINEs mobilize via a process called retrotransposition. Although the role of the LINE-encoded protein(s) in retrotransposition has been extensively investigated, the participation of host-encoded factors in retrotransposition remains unclear. To address this issue, we examined retrotransposition frequencies of two structurally different LINEs—zebrafish ZfL2-2 and human L1—in knockout chicken DT40 cell lines deficient in genes involved in the non-homologous end-joining (NHEJ) repair of DNA and in human HeLa cells treated with a drug that inhibits NHEJ. Deficiencies of NHEJ proteins decreased retrotransposition frequencies of both LINEs in these cells, suggesting that NHEJ is involved in LINE retrotransposition. More precise characterization of ZfL2-2 insertions in DT40 cells permitted us to consider the possibility of dual roles for NHEJ in LINE retrotransposition, namely to ensure efficient integration of LINEs and to restrict their full-length formation.

Retrotransposition of the Ll.LtrB group II intron proceeds predominantly via reverse splicing into DNA targets

Catalytic group II introns are mobile retroelements that invade cognate intronless genes via retrohoming, where the introns reverse splice into double‐stranded DNA (dsDNA) targets. They can also retrotranspose to ectopic sites at low frequencies. Whereas our previous studies with a bacterial intron, Ll.LtrB, supported frequent use of RNA targets during retrotransposition, recent experiments with a retrotransposition indicator gene indicate that DNA, rather than RNA, is a prominent target, with both dsDNA and single‐stranded DNA (ssDNA) as possibilities. Thus retrotransposition occurs in both transcriptional sense and antisense orientations of target genes, and is largely independent of homologous DNA recombination and of the endonuclease function of the intron‐encoded protein, LtrA. Models based on both dsDNA and ssDNA targeting are presented. Interestingly, retrotransposition is biased toward the template for lagging‐strand DNA synthesis, which suggests the possibility of the replication folk as a source of ssDNA. Consistent with some use of ssDNA targets, many retrotransposition sites lack nucleotides critical for the unwinding of target duplex DNA. Moreover, in vitro the intron reverse spliced into ssDNA more efficiently than dsDNA substrates for some of the retrotransposition sites. Furthermore, many bacterial group II introns reside on the lagging‐strand template, hinting at a role for DNA replication in intron dispersal in nature.

Accumulation and loss of asymmetric non-CpG methylation during male germ-cell development

DNA methylation is a well-characterized epigenetic modification involved in gene regulation and transposon silencing in mammals. It mainly occurs on cytosines at CpG sites but methylation at non-CpG sites is frequently observed in embryonic stem cells, induced pluriotent stem cells, oocytes and the brain. The biological significance of non-CpG methylation is unknown. Here, we show that non-CpG methylation is also present in male germ cells, within and around B1 retrotransposon sequences interspersed in the mouse genome. It accumulates in mitotically arrested fetal prospermatogonia and reaches the highest level by birth in a Dnmt3l-dependent manner. The preferential site of non-CpG methylation is CpA, especially CpApG and CpApC. Although CpApG (and CpTpG) sites contain cytosines at symmetrical positions, hairpin-bisulfite sequencing reveals that they are hemimethylated, suggesting the absence of a template-dependent copying mechanism. Indeed, the level of non-CpG methylation decreases after the resumption of mitosis in the neonatal period, whereas that of CpG methylation does not. The cells eventually lose non-CpG methylation by the time they become spermatogonia. Our results show that non-CpG methylation accumulates in non-replicating, arrested cells but is not maintained in mitotically dividing cells during male germ-cell development.

Locus- and domain-dependent control of DNA methylation at mouse B1 retrotransposons during male germ cell development

In mammals, germ cells undergo striking dynamic changes in DNA methylation during their development. However, the dynamics and mode of methylation are poorly understood for short interspersed elements (SINEs) dispersed throughout the genome. We investigated the DNA methylation status of mouse B1 SINEs in male germ cells at different developmental stages. B1 elements showed a large locus-to-locus variation in methylation; loci close to RNA polymerase II promoters were hypomethylated, while most others were hypermethylated. Interestingly, a mutation that eliminates Piwi-interacting RNAs (piRNAs), which are involved in methylation of long interspersed elements (LINEs), did not affect the level of B1 methylation, implying a piRNA-independent mechanism. Methylation at B1 loci in SINE-poor genomic domains showed a higher dependency on the de novo DNA methyltransferase DNMT3A but not on DNMT3B, suggesting that DNMT3A plays a major role in methylation of these domains. We also found that many genes specifically expressed in the testis possess B1 elements in their promoters, suggesting the involvement of B1 methylation in transcriptional regulation. Taken altogether, our results not only reveal the dynamics and mode of SINE methylation but also suggest how the DNA methylation profile is created in the germline by a pair of DNA methyltransferases.

A bacterial group II intron favors retrotransposition into plasmid targets

Group II introns, widely believed to be the ancestors of nuclear pre-mRNA introns, are catalytic RNAs found in bacteria, archaea, and eukaryotes. They are mobile genetic elements that move via an RNA intermediate. They retrohome to intronless alleles and retrotranspose to ectopic sites, aided by an intron-encoded protein with reverse transcriptase, maturase, and endonuclease activities. Many group II introns identified in bacteria reside on plasmid genomes rather than bacterial chromosomes, implying that plasmids are havens for these retroelements. This study demonstrates that almost one-fourth of retrotransposition events of the Ll.LtrB intron in Lactococcus lactis are into the plasmid donor. This level is more than twice that predicted based on target size and plasmid copy number relative to the chromosome. In particular, the fraction of such plasmid targeting events was elevated to more than one-third of retrotransposition events by mutation of the intron-encoded endonuclease, a situation that may resemble most bacterial group II introns, which lack the endonuclease. Target-site sequences on the plasmid are more relaxed than those on the chromosome, likely accounting for preferred integration into plasmid replicons. Furthermore, the direction of integration relative to promoters and origins of replication is consistent with group II intron retrotransposition into single-stranded DNA at replication forks. This work provides mechanistic rationales for the prevalence of group II introns in natural plasmid populations and underscores that targeting to plasmids, which are themselves mobile elements, could promote intron spread.

HSP90α plays an important role in piRNA biogenesis and retrotransposon repression in mouse

HSP90, found in all kingdoms of life, is a major chaperone protein regulating many client proteins. We demonstrated that HSP90α, one of two paralogs duplicated in vertebrates, plays an important role in the biogenesis of fetal PIWI-interacting RNAs (piRNA), which act against the transposon activities, in mouse male germ cells. The knockout mutation of Hsp90α resulted in a large reduction in the expression of primary and secondary piRNAs and mislocalization of MIWI2, a PIWI homolog. Whereas the mutation in Fkbp6 encoding a co-chaperone reduced piRNAs of 28–32 nucleotides in length, the Hsp90α mutation reduced piRNAs of 24–32 nucleotides, suggesting the presence of both FKBP6-dependent and -independent actions of HSP90α. Although DNA methylation and mRNA levels of L1 retrotransposon were largely unchanged in the Hsp90α mutant testes, the L1-encoded protein was increased, suggesting the presence of post-transcriptional regulation. This study revealed the specialized function of the HSP90α isofom in the piRNA biogenesis and repression of retrotransposons during the development of male germ cells in mammals.

Three novel ZBTB24 mutations identified in Japanese and Cape Verdean type 2 ICF syndrome patients

Immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome is a rare autosomal recessive disorder that shows DNA hypomethylation at pericentromeric satellite-2 and -3 repeats in chromosomes 1, 9 and 16. ICF syndrome is classified into two groups: type 1 (ICF1) patients have mutations in the DNMT3B gene and about half of type 2 (ICF2) patients have mutations in the ZBTB24 gene. Besides satellite-2 and -3 repeats, α-satellite repeats are also hypomethylated in ICF2. In this study, we report three novel ZBTB24 mutations in ICF2. A Japanese patient was homozygous for a missense mutation (C383Y), and a Cape Verdean patient was compound heterozygous for a nonsense mutation (K263X) and a frame-shift mutation (C327W fsX54). In addition, the second Japanese patient was homozygous for a previously reported nonsense mutation (R320X). The C383Y mutation abolished a C2H2 motif in one of the eight zinc-finger domains, and the other three mutations caused a complete or large loss of the zinc-finger domains. Our immunofluorescence analysis revealed that mouse Zbtb24 proteins possessing a mutation corresponding to either C383Y or R320X are mislocalized from pericentrometic heterochromatin, suggesting the importance of the zinc-finger domains in proper intranuclear localization of this protein. We further revealed that the proper localization of wild-type Zbtb24 protein does not require DNA methylation.

Mutational analysis on structure–function relationship of a Holliday junction specific endonuclease RuvC

Escherichia coli RuvC protein is a specific endonuclease that resolves Holliday junctions during homologous recombination. For junction resolution, RuvC undergoes distinct steps such as dimerization, junction‐specific binding and endonucleolytic cleavage. The crystal structure of RuvC has been revealed.

Regional DNA methylation differences between humans and chimpanzees are associated with genetic changes, transcriptional divergence and disease genes

Changes in gene expression have been proposed to have an important role in the evolutionary changes in phenotypes. Interspecific changes in gene expression can result not only from genetic changes in regulatory regions but also from epigenetic changes in such regions. Here we report the identification of genomic regions showing differences in DNA methylation between humans and chimpanzees (termed S-DMRs for species-specific differentially methylated regions) on chromosomes 21 and 22. These regional methylation differences are frequently associated with genes, including those relevant to a disease, such as Alzheimer’s disease, diabetes mellitus or cancer. Methylation differences are often correlated with changes in promoter activity or alternative splicing. Comparative studies including other great ape species provide evidence for the contribution of genetic changes to some of these S-DMRs. Genetic changes responsible for the S-DMRs include gain or loss of CTCF-binding site and changes in CpG density in microsatellite repeats. Our results suggest that DNA methylation changes, often caused by small sequence changes, contribute to transcriptional and phenotypic diversification in hominid evolution.