François Cuzin

RNA-MEDIATED NON-MENDELIAN INHERITANCE OF AN EPIGENETIC CHANGE IN THE MOUSE

Paramutation is a heritable epigenetic modification induced in plants by cross-talk between allelic loci. Here we report a similar modification of the mouse Kit gene in the progeny of heterozygotes with the null mutant Kittm1Alf (a lacZ insertion). In spite of a homozygous wild-type genotype, their offspring maintain, to a variable extent, the white spots characteristic of Kit mutant animals. Efficiently inherited from either male or female parents, the modified phenotype results from a decrease in Kit messenger RNA levels with the accumulation of non-polyadenylated RNA molecules of abnormal sizes. Sustained transcriptional activity at the postmeiotic stages—at which time the gene is normally silent—leads to the accumulation of RNA in spermatozoa. Microinjection into fertilized eggs either of total RNA from Kittm1Alf/+ heterozygotes or of Kit-specific microRNAs induced a heritable white tail phenotype. Our results identify an unexpected mode of epigenetic inheritance associated with the zygotic transfer of RNA molecules.

RNA Induction and Inheritance of Epigenetic Cardiac Hypertrophy in the Mouse

Epigenetic regulation shapes normal and pathological mammalian development and physiology. Our previous work showed that Kit RNAs injected into fertilized mouse eggs can produce heritable epigenetic defects, or paramutations, with relevant loss-of-function pigmentation phenotypes, which affect adult phenotypes in multiple succeeding generations of mice. Here, we illustrate the relevance of paramutation to pathophysiology by injecting fertilized mouse eggs with RNAs targeting Cdk9, a key regulator of cardiac growth. Microinjecting fragments of either the coding region or the related microRNA miR-1 led to high levels of expression of homologous RNA, resulting in an epigenetic defect, cardiac hypertrophy, whose efficient hereditary transmission correlated with the presence of miR-1 in the sperm nucleus. In this case, paramutation increased rather than decreased expression of Cdk9. These results highlight the diversity of RNA-mediated epigenetic effects and may provide a paradigm for clinical cases of familial diseases whose inheritance is not fully explained in Mendelian terms.

Engineering chromosomes in mice through targeted meiotic recombination(TAMERE)

Functional studies of large transcription units, clustered genes and chromosomal loci require the design of novel experimental tools to engineer genomic macro-rearrangements. Here, we present a strategy to produce deficiencies or duplications by crossing mice carrying loxP sites in homologous loci. This trans-allelic targeted meiotic recombination (TAMERE) protocol allows for the combination of various alleles within a particular locus as well as for generation of interchromosomal unequal exchanges. Novel genetic configurations can thus be produced without multiple targeting and selection steps in embryonic stem (ES) cells. A concomitant deletion/duplication event of the Hoxd12 locus shows the potential of this approach. The high frequency of such targeted exchanges in vivo makes TAMERE a powerful genetic tool applicable to research areas in which complex genomic modifications are required.

Murine spermatogonial stem cells: targeted transgene expression and purification in an active state

A 400 bp fragment of the spermatogonia‐specific Stra8 locus was sufficient to direct gene expression to the germinal stem cells in transgenic mice. A fractionation procedure was devised, based on immunomagnetic sorting of cells in which the promoter drives the expression of a surface functionally neutral protein tag. The purified cells expressed the known molecular markers of spermatogonia Rbm, cyclin A2 and EP‐Cam, and the β1‐ and α6‐integrins characteristic of the stem cell fraction. A 700‐fold enrichment in stem cells was determined by the ability of the purified fractions to re‐establish spermatogenesis in germ cell‐depleted recipient testes.

RNA-Mediated Epigenetic Heredity Requires the Cytosine Methyltransferase Dnmt2

RNA–mediated transmission of phenotypes is an important way to explain non-Mendelian heredity. We have previously shown that small non-coding RNAs can induce hereditary epigenetic variations in mice and act as the transgenerational signalling molecules. Two prominent examples for these paramutations include the epigenetic modulation of the Kit gene, resulting in altered fur coloration, and the modulation of the Sox9 gene, resulting in an overgrowth phenotype. We now report that expression of the Dnmt2 RNA methyltransferase is required for the establishment and hereditary maintenance of both paramutations. Our data show that the Kit paramutant phenotype was not transmitted to the progeny of Dnmt2−/− mice and that the Sox9 paramutation was also not established in Dnmt2−/− embryos. Similarly, RNA from Dnmt2-negative Kit heterozygotes did not induce the paramutant phenotype when microinjected into Dnmt2-deficient fertilized eggs and microinjection of the miR-124 microRNA failed to induce the characteristic giant phenotype. In agreement with an RNA–mediated mechanism of inheritance, no change was observed in the DNA methylation profiles of the Kit locus between the wild-type and paramutant mice. RNA bisulfite sequencing confirmed Dnmt2-dependent tRNA methylation in mouse sperm and also indicated Dnmt2-dependent cytosine methylation in Kit RNA in paramutant embryos. Together, these findings uncover a novel function of Dnmt2 in RNA–mediated epigenetic heredity.

Increased levels of mitochondrial gene expression in rat fibroblast cells immortalized or transformed by viral and cellular oncogenes.

Steady‐state levels of the mitochondrial (mt) mRNA encoding subunit II of cytochrome oxidase (COII) were increased 5‐10 fold in fully transformed cell lines derived from rodent embryonic fibroblasts after transfer of polyoma virus DNA, and in immortalized cell lines established by transfer of plt (polyoma large T protein), E1A (adenovirus) and myc oncogenes. Increased mitochondrial gene expression was not related with active growth per se: it was low in fast‐growing rat embryo cells, and it did not change upon serum starvation and subsequent stimulation of FR3T3 cells. The number of copies of mtDNA did not vary, and different mitochondrial mRNAs and rRNAs were increased in the same proportions, suggesting a change in the rate of accumulation of their common precursor.

Cre expression in primary spermatocytes : A tool for genetic engineering of the germ line

Transgenic mice were generated expressing a testicular Cre recombinase driven by promoter sequences derived from the gene encoding Synaptonemal Complex Protein 1 (Sycp1), expressed at an early stage of the male meiosis (leptotene to zygotene). Recombination at target LoxP sites was examined during germinal differentiation in mice harboring Sycp1‐Cre and a second transgene where LoxP sites flank either the βgeo coding region, the Pgk1 promoter, or a tk‐neo cassette inserted into the Rxrα locus. The LoxP‐flanked transgenes were stably maintained in the somatic tissues of the double transgenic animals, as well as in the progeny of the females. Mice born after mating the double‐transgenic males with normal females showed extensive deletions of the LoxP‐flanked sequences. When the males were hemizygous for the Sycp1‐Cre transgene, the deletions were observed even in the fraction of the offspring which had not inherited the Cre gene, thus demonstrating that expression occurred in the male parent during spermatogenesis. The high efficiency of excision at the LoxP sites makes the Sycp1‐Cre transgenic males suitable for evaluating the role of defined gene functions in the germinal differentiation process. Mol. Reprod. Dev. 51:274–280, 1998.

Transvection effects involving DNA methylation during meiosis in the mouse.

High efficiencies of recombination between LoxP elements were initially recorded when the Cre recombinase was expressed in meiotic spermatocytes. However, it was unexpectedly found that LoxP recombination fell to very low values at the second generation of mice expressing Cre during meiosis. The inability of the LoxP elements to serve as recombination substrates was correlated with cytosine methylation, initially in LoxP and transgene sequences, but later extending for distances of at least several kilobases into chromosomal sequences. It also affected the allelic locus, implying a transfer of structural information between alleles similar to the transvection phenomenon described in Drosophila. Once initiated following Cre–LoxP interaction, neither cis‐extension nor transvection of the methylated state required the continuous expression of Cre, as they occurred both in germinal and somatic cells and in the fraction of the offspring that had not inherited the Sycp1‐Cre transgene. Therefore, these processes depend on a physiological mechanism of establishment and extension of an epigenetic state, for which they provide an experimental model.

Inheritance of an Epigenetic Mark: The CpG DNA Methyltransferase 1 Is Required for De Novo Establishment of a Complex Pattern of Non-CpG Methylation

Site-specific methylation of cytosines is a key epigenetic mark of vertebrate DNA. While a majority of the methylated residues are in the symmetrical (meC)pG:Gp(meC) configuration, a smaller, but significant fraction is found in the CpA, CpT and CpC asymmetric (non-CpG) dinucleotides. CpG methylation is reproducibly maintained by the activity of the DNA methyltransferase 1 (Dnmt1) on the newly replicated hemimethylated substrates (meC)pG:GpC. On the other hand, establishment and hereditary maintenance of non-CpG methylation patterns have not been analyzed in detail. We previously reported the occurrence of site- and allele-specific methylation at both CpG and non-CpG sites. Here we characterize a hereditary complex of non-CpG methylation, with the transgenerational maintenance of three distinct profiles in a constant ratio, associated with extensive CpG methylation. These observations raised the question of the signal leading to the maintenance of the pattern of asymmetric methylation. The complete non-CpG pattern was reinstated at each generation in spite of the fact that the majority of the sperm genomes contained either none or only one methylated non-CpG site. This observation led us to the hypothesis that the stable CpG patterns might act as blueprints for the maintenance of non-CpG DNA methylation. As predicted, non-CpG DNA methylation profiles were abrogated in a mutant lacking Dnmt1, the enzymes responsible for CpG methylation, but not in mutants defective for either Dnmt3a or Dnmt2.

Germ line transmission of autonomous genetic elements in transgenic mouse strains

Upon microinjection into fertilized mouse eggs of circular molecules of plasmid pPyLT1 carrying the gene encoding the large T protein of polyoma virus within bacterial vector sequences, autonomous circular plasmids were stably maintained in low copy numbers in transgenic strains. These plasmids could be rescued in E. coli by transfection. Integrated forms could be detected neither in somatic tissues, nor in spermatozoa. Efficiency of paternal or maternal transmission was close to 100%. The plasmids had lost or had extensively rearranged the polyoma sequences. In addition, they had acquired defined segments of genomic mouse DNA, which might be responsible for correct segregation of daughter copies at both mitosis and meiosis (centromeric function).