Masahiro Kaneda

Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting

Imprinted genes are epigenetically marked during gametogenesis so that they are exclusively expressed from either the paternal or the maternal allele in offspring. Imprinting prevents parthenogenesis in mammals and is often disrupted in congenital malformation syndromes, tumours and cloned animals. Although de novo DNA methyltransferases of the Dnmt3 family are implicated in maternal imprinting, the lethality of Dnmt3a and Dnmt3b knockout mice has precluded further studies. We here report the disruption of Dnmt3a and Dnmt3b in germ cells, with their preservation in somatic cells, by conditional knockout technology. Offspring from Dnmt3a conditional mutant females die in utero and lack methylation and allele-specific expression at all maternally imprinted loci examined. Dnmt3a conditional mutant males show impaired spermatogenesis and lack methylation at two of three paternally imprinted loci examined in spermatogonia. By contrast, Dnmt3b conditional mutants and their offspring show no apparent phenotype. The phenotype of Dnmt3a conditional mutants is indistinguishable from that of Dnmt3L knockout mice, except for the discrepancy in methylation at one locus. These results indicate that both Dnmt3a and Dnmt3L are required for methylation of most imprinted loci in germ cells, but also suggest the involvement of other factors.

Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes.

RNA interference (RNAi) is a mechanism by which double-stranded RNAs (dsRNAs) suppress specific transcripts in a sequence-dependent manner. dsRNAs are processed by Dicer to 21–24-nucleotide small interfering RNAs (siRNAs) and then incorporated into the argonaute (Ago) proteins. Gene regulation by endogenous siRNAs has been observed only in organisms possessing RNA-dependent RNA polymerase (RdRP). In mammals, where no RdRP activity has been found, biogenesis and function of endogenous siRNAs remain largely unknown. Here we show, using mouse oocytes, that endogenous siRNAs are derived from naturally occurring dsRNAs and have roles in the regulation of gene expression. By means of deep sequencing, we identify a large number of both ∼25–27-nucleotide Piwi-interacting RNAs (piRNAs) and ∼21-nucleotide siRNAs corresponding to messenger RNAs or retrotransposons in growing oocytes. piRNAs are bound to Mili and have a role in the regulation of retrotransposons. siRNAs are exclusively mapped to retrotransposons or other genomic regions that produce transcripts capable of forming dsRNA structures. Inverted repeat structures, bidirectional transcription and antisense transcripts from various loci are sources of the dsRNAs. Some precursor transcripts of siRNAs are derived from expressed pseudogenes, indicating that one role of pseudogenes is to adjust the level of the founding source mRNA through RNAi. Loss of Dicer or Ago2 results in decreased levels of siRNAs and increased levels of retrotransposon and protein-coding transcripts complementary to the siRNAs. Thus, the RNAi pathway regulates both protein-coding transcripts and retrotransposons in mouse oocytes. Our results reveal a role for endogenous siRNAs in mammalian oocytes and show that organisms lacking RdRP activity can produce functional endogenous siRNAs from naturally occurring dsRNAs.

MicroRNA biogenesis is required for mouse primordial germ cell development and spermatogenesis.

Background MicroRNAs (miRNAs) are critical regulators of transcriptional and post-transcriptional gene silencing, which are involved in multiple developmental processes in many organisms. Apart from miRNAs, mouse germ cells express another type of small RNA, piwi-interacting RNAs (piRNAs). Although it has been clear that piRNAs play a role in repression of retrotransposons during spermatogenesis, the function of miRNA in mouse germ cells has been unclear. Methodology/Principal Findings In this study, we first revealed the expression pattern of miRNAs by using a real-time PCR-based 220-plex miRNA expression profiling method. During development of germ cells, miR-17-92 cluster, which is thought to promote cell cycling, and the ES cell-specific cluster encoding miR-290 to -295 (miR-290-295 cluster) were highly expressed in primordial germ cells (PGCs) and spermatogonia. A set of miRNAs was developmentally regulated. We next analysed function of miRNA biogenesis in germ cell development by using conditional Dicer-knockout mice in which Dicer gene was deleted specifically in the germ cells. Dicer-deleted PGCs and spermatogonia exhibited poor proliferation. Retrotransposon activity was unexpectedly suppressed in Dicer-deleted PGCs, but not affected in the spermatogonia. In Dicer-deleted testis, spermatogenesis was retarded at an early stage when proliferation and/or early differentiation. Additionally, we analysed spermatogenesis in conditional Argonaute2-deficient mice. In contrast to Dicer-deficient testis, spermatogenesis in Argonaute2-deficient testis was indistinguishable from that in wild type. Conclusion/Significance These results illustrate that miRNAs are important for the proliferation of PGCs and spermatogonia, but dispensable for the repression of retrotransposons in developing germ cells. Consistently, miRNAs promoting cell cycling are highly expressed in PGCs and spermatogonia. Furthermore, based on normal spermatogenesis in Argonaute2-deficient testis, the critical function of Dicer in spermatogenesis is independent of Argonaute2.

Essential role for Argonaute2 protein in mouse oogenesis

BackgroundArgonaute2 protein (Ago2) is a key component of RNA-induced gene silencing complex, which is crucial for microRNA-mediated repression of target genes. The function of Ago2 in the mouse oocyte and early embryonic development is less well characterized but it is likely to have an important role in regulating maternally inherited mRNA. We have examined the role of Ago2 by conditional deletion of the gene in developing oocytes.ResultsAgo2 was deleted specifically in the growing oocytes. Although the Ago2-deficient oocytes are able to develop to mature oocytes, they have abnormal spindles and chromosomes that are unable to cluster together properly. This phenotype is very similar to the phenotype of Dicer-deficient oocytes. We examined the microRNA expression profile in the Ago2-deficient oocyte and found that the expression of most microRNAs was reduced by more than 80%. To determine the downstream genes that are regulated by Ago2, we used microarray analysis on Ago2-deficient oocytes and found that 512 genes were upregulated and 1,073 genes were downregulated (FC > 2, P < 0.05).ConclusionOur study shows that Ago2 has a key function in the mouse oocyte through global regulation of microRNA stability, and through this mechanism it affects gene expression in developing oocytes.

Identification of the imprinted KLF14 transcription factor undergoing human-specific accelerated evolution.

Imprinted genes are expressed in a parent-of-origin manner and are located in clusters throughout the genome. Aberrations in the expression of imprinted genes on human Chromosome 7 have been suggested to play a role in the etiologies of Russell-Silver Syndrome and autism. We describe the imprinting of KLF14, an intronless member of the Krüppel-like family of transcription factors located at Chromosome 7q32. We show that it has monoallelic maternal expression in all embryonic and extra-embryonic tissues studied, in both human and mouse. We examine epigenetic modifications in the KLF14 CpG island in both species and find this region to be hypomethylated. In addition, we perform chromatin immunoprecipitation and find that the murine Klf14 CpG island lacks allele-specific histone modifications. Despite the absence of these defining features, our analysis of Klf14 in offspring from DNA methyltransferase 3a conditional knockout mice reveals that the genes expression is dependent upon a maternally methylated region. Due to the intronless nature of Klf14 and its homology to Klf16, we suggest that the gene is an ancient retrotransposed copy of Klf16. By sequence analysis of numerous species, we place the timing of this event after the divergence of Marsupialia, yet prior to the divergence of the Xenarthra superclade. We identify a large number of sequence variants in KLF14 and, using several measures of diversity, we determine that there is greater variability in the human lineage with a significantly increased number of nonsynonymous changes, suggesting human-specific accelerated evolution. Thus, KLF14 may be the first example of an imprinted transcript undergoing accelerated evolution in the human lineage.

Reversible Block of Mouse Neural Stem Cell Differentiation in the Absence of Dicer and MicroRNAs

Background To investigate the functions of Dicer and microRNAs in neural stem (NS) cell self-renewal and neurogenesis, we established neural stem cell lines from the embryonic mouse Dicer-null cerebral cortex, producing neural stem cell lines that lacked all microRNAs. Principal Findings Dicer-null NS cells underwent normal self-renewal and could be maintained in vitro indefinitely, but had subtly altered cell cycle kinetics and abnormal heterochromatin organisation. In the absence of all microRNAs, Dicer-null NS cells were incapable of generating either glial or neuronal progeny and exhibited a marked dependency on exogenous EGF for survival. Dicer-null NS cells assumed complex differences in mRNA and protein expression under self-renewing conditions, upregulating transcripts indicative of self-renewing NS cells and expressing genes characteristic of differentiating neurons and glia. Underlining the growth-factor dependency of Dicer-null NS cells, many regulators of apoptosis were enriched in expression in these cells. Dicer-null NS cells initiate some of the same gene expression changes as wild-type cells under astrocyte differentiating conditions, but also show aberrant expression of large sets of genes and ultimately fail to complete the differentiation programme. Acute replacement of Dicer restored their ability to differentiate to both neurons and glia. Conclusions The block in differentiation due to loss of Dicer and microRNAs is reversible and the significantly altered phenotype of Dicer-null NS cells does not constitute a permanent transformation. We conclude that Dicer and microRNAs function in this system to maintain the neural stem cell phenotype and to facilitate the completion of differentiation.

The continuing quest to comprehend genomic imprinting.

The discovery of the phenomenon of genomic imprinting in mammals showed that the parental genomes are functionally non-equivalent. Considerable advances have occurred in the field over the past 20 years, which has resulted in the identification and functional analysis of a number of imprinted genes the expression of which is determined by their parental origin. These genes belong to many diverse categories and they have been shown to regulate growth, complex aspects of mammalian physiology and behavior. Many aspects of the mechanism of imprinting have also been elucidated. However, the reasons for the evolution of genomic imprinting remain enigmatic. Further research is needed to determine if there is any relationship between the apparently diverse functions of imprinted genes in mammals, and their role in human diseases. It also remains to be seen what common features exist amongst the diverse imprinting control elements. The mechanisms involved in the erasure and re-establishment of imprints should provide deeper insights into epigenetic mechanisms of wide general interest.

A sensitive multiplex assay for piRNA expression

PIWI-interacting RNAs (piRNAs) are a new class of small RNAs specifically expressed in male germ cells. It is known to bind to PIWI class of Argonaute proteins, Mili and Miwi. To help to decipher the mechanism of piRNA function, here, we report a real time PCR-based multiplex assay for piRNA expression. Firstly, we showed that the assay specifically detects piRNA expression in adult testis, consistent with the Northern blot result. The method we developed can simultaneously detect at least eight piRNAs using only 10pg total RNA, which is equivalent to the RNA present in a single cell. This is five to six order magnitude more sensitive than corresponding Northern blot assays. Finally we used this assay to analyze eight piRNAs expression in mouse primordial germ cells (PGCs) in genital ridges from E12.5, at the time when piRNA-binding protein Mili starts to be detected in PGCs. This multiplex piRNA assay can be further expanded to assay a few hundred of piRNAs simultaneously from as little as total RNA from a single cell. This approach will help to understand the mechanism and function of piRNAs during germ cell development.