Minoo Rassoulzadegan

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.

Subnuclear localization of WT1 in splicing or transcription factor domains is regulated by alternative splicing

WT1 is a tumor suppressor gene with a key role in urogenital development and the pathogenesis of Wilms tumor. Two alternative splice sites in the WT1 transcript allow the gene to encode four proteins. These carry four Krüppel-type zinc fingers and to date have primarily been implicated in transcriptional control of genes involved in growth regulation. However, here we demonstrate colocalization of WT1 with splicing factors in the fetal kidney and testis and in expressing cell lines. Using immunoprecipitation, we show that two WT1 isoforms directly associate with one or a limited number of components in the spliceosomes and coiled bodies. Moreover, COS cell expression studies suggest that alternative splicing within the WT1 zinc finger region determines whether the protein localizes mainly with splicing factors or with DNA in transcription factor domains in the nucleus. We propose that WT1 plays roles in posttranscriptional processing of RNA as well as in transcription.

Resistance to Leishmania major Induced by Tolerance to a Single Antigen

In mice, susceptibility to Leishmania major is associated with the early expansion of T helper 2 cells (TH2) cells, but nothing is known of the specificity of these cells. A previously identified antigen, Leishmania homolog of receptors for activated C kinase (LACK), was found to be the focus of this initial response. Mice made tolerant to LACK by the transgenic expression of the antigen in the thymus exhibited both a diminished TH2 response and a healing phenotype. Thus, T cells that are activated early and are reactive to a single antigen play a pivotal role in directing the immune response to the entire parasite.

Transmeiotic differentiation of male germ cells in culture

A cell culture system that supports the differentiation of male germ cells through meiosis is described. It takes advantage of the properties of a cell line, 15P-1, established from testicular cells of transgenic mice that express the large T protein of polyoma virus in the seminiferous epithelium. This line exhibits features characteristics of Sertoli cells, including transcription of the Wilms tumor (WT1) and Steel genes. Cells of the 15P-1 type support the meiotic and postmeiotic differentiation in cocultures of diploid premeiotic germ cells into haploid spermatids expressing the protamine (Prm-1) gene. When cocultured with 15P-1 cells, testicular cells explanted from immature 9-day-old animals, before the onset of the first meiosis, generated tetrads of haploid cells with the morphology of round spermatids and initiated protamine transcription.

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.

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.

APLP2, a member of the Alzheimer precursor protein family, is required for correct genomic segregation in dividing mouse cells

The mouse amyloid precursor‐like protein 2 (APLP2) belongs to the Alzheimer peptide precursor family. A possible role in pre‐implantation development had been suggested previously, and was investigated further by creating a large deletion in the genomic locus. While heterozygous mice developed normally, homozygous embryos were arrested before reaching the blastocyst stage. One‐cell embryos which contained protein of maternal origin underwent a limited number of cleavages. The progressive disappearance of the protein at stages 4 and beyond correlated with the appearance of extensive cytopathological effects. Nuclear DNA contents of the arrested embryos departed widely from the normal 2–4C value, thus suggesting a role for the protein in replication and/or segregation of the embryonic genome. Embryonic mortality was not due to the untimely initiation of programmed cell death, and it occurred before the stage at which apoptotic cells normally appear. The same abnormal distribution of DNA contents was seen in primary cultures of Aplp2 +/− embryonic fibroblasts following transfection of an expression vector for Aplp2 antisense RNA with green fluorescent protein (GFP) expressed from a co‐transfected construct. Daughter cells derived from a GFP‐positive cell showed abnormal DNA contents both >4C and <2C, thus indicating a role for the protein in the mitotic segregation of the genome and establishment of the proper nuclear structure.

Temporal and spatial control of the Sycp1 gene transcription in the mouse meiosis: regulatory elements active in the male are not sufficient for expression in the female gonad.

Transcription controls active at the initial stages of meiosis are clearly key elements in the regulation of germinal differentiation. Transcription of the Sycp1 gene (synaptonemal complex protein 1) starts as early as the leptotene and zygotene stages. Constructs with Sycp1 5 upstream sequences directed the expression of reporter genes to pachytene spermatocytes in transgenic mice. A short fragment encompassing the transcription start (n.t. -54 to +102) was sufficient for stage-specific expression in the adult male and for temporal regulation during development. Upstream enhancer element(s) quantitatively regulating expression were localized in the region between -54 and -260. The gene is normally expressed both in the male and female gonads, but none of the promoter sequences active in the testis allowed the expression of reporter genes during meiosis in the ovary.