RESUMO
Retrotransposons are highly prevalent in mammalian genomes due to their ability to amplify in pluripotent cells or developing germ cells. Host mechanisms that silence retrotransposons in germ cells and pluripotent cells are important for limiting the accumulation of the repetitive elements in the genome during evolution. However, although silencing of selected individual retrotransposons can be relatively well-studied, many mammalian retrotransposons are seldom analysed and their silencing in germ cells, pluripotent cells or somatic cells remains poorly understood. Here we show, and experimentally verify, that cryptic repetitive element probes present in Illumina and Affymetrix gene expression microarray platforms can accurately and sensitively monitor repetitive element expression data. This computational approach to genome-wide retrotransposon expression has allowed us to identify the histone deacetylase Hdac1 as a component of the retrotransposon silencing machinery in mouse embryonic stem cells, and to determine the retrotransposon targets of Hdac1 in these cells. We also identify retrotransposons that are targets of other retrotransposon silencing mechanisms such as DNA methylation, Eset-mediated histone modification, and Ring1B/Eed-containing polycomb repressive complexes in mouse embryonic stem cells. Furthermore, our computational analysis of retrotransposon silencing suggests that multiple silencing mechanisms are independently targeted to retrotransposons in embryonic stem cells, that different genomic copies of the same retrotransposon can be differentially sensitive to these silencing mechanisms, and helps define retrotransposon sequence elements that are targeted by silencing machineries. Thus repeat annotation of gene expression microarray data suggests that a complex interplay between silencing mechanisms represses retrotransposon loci in germ cells and embryonic stem cells.
Assuntos
Células-Tronco Embrionárias/fisiologia , Perfilação da Expressão Gênica/métodos , Histona Desacetilase 1/genética , Sequências Repetitivas Dispersas/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Retroelementos/genética , Animais , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento/genética , Inativação Gênica/fisiologia , Camundongos , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
Synapsis is the process by which paired chromosome homologues closely associate in meiosis before crossover. In the synaptonemal complex (SC), axial elements of each homologue connect through molecules of SYCP1 to the central element, which contains the proteins SYCE1 and -2. We have derived mice lacking SYCE2 protein, producing males and females in which meiotic chromosomes align and axes form but do not synapse. Sex chromosomes are unaligned, not forming a sex body. Additionally, markers of DNA breakage and repair are retained on the axes, and crossover is impaired, culminating in both males and females failing to produce gametes. We show that SC formation can initiate at sites of SYCE1/SYCP1 localization but that these points of initiation cannot be extended in the absence of SYCE2. SC assembly is thus dependent on SYCP1, SYCE1, and SYCE2. We provide a model to explain this based on protein-protein interactions.
Assuntos
Reparo do DNA , Proteínas Nucleares/fisiologia , Recombinação Genética , Complexo Sinaptonêmico/metabolismo , Animais , Quebras de DNA de Cadeia Dupla , Feminino , Masculino , Camundongos , Camundongos Knockout , Modelos Genéticos , Mutação , Proteínas Nucleares/genética , Oócitos/citologia , Cromossomos Sexuais/fisiologia , Espermatócitos/citologiaRESUMO
In mammals, the synaptonemal complex is a structure required to complete crossover recombination. Although suggested by cytological work, in vivo links between the structural proteins of the synaptonemal complex and the proteins of the recombination process have not previously been made. The central element of the synaptonemal complex is traversed by DNA at sites of recombination and presents a logical place to look for interactions between these components. There are four known central element proteins, three of which have previously been mutated. Here, we complete the set by creating a null mutation in the Syce1 gene in mouse. The resulting disruption of synapsis in these animals has allowed us to demonstrate a biochemical interaction between the structural protein SYCE2 and the repair protein RAD51. In normal meiosis, this interaction may be responsible for promoting homologous synapsis from sites of recombination.
Assuntos
Pareamento Cromossômico , Reparo do DNA , Proteínas Nucleares/genética , Complexo Sinaptonêmico/metabolismo , Animais , Feminino , Gametogênese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/metabolismo , Ligação Proteica , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Recombinação Genética , Espermatócitos/citologia , Espermatócitos/metabolismo , Complexo Sinaptonêmico/genéticaRESUMO
Gametogenesis is a complex process subject to strict controls at both levels of transcription and translation. Members of a family of conserved RNA-binding proteins encoded by the DAZ genes are required for the translational regulation of gene expression essential for this process. Although loss of DAZ family genes is associated with infertility in several organisms including humans, the identity of the transcripts regulated in vivo is unknown. Using a combination of immunoprecipitation and microarray analysis, we have identified a number of mRNAs that are bound by the murine Dazl protein both in vivo and in vitro. Sequence analysis shows that these transcripts contain binding sites for Dazl, which have been conserved during evolution between human, rat and mouse. We have focussed on mouse vasa homologue (Mvh), a gene that is essential for male gametogenesis, and show that Dazl stimulates translation via the Mvh 3'-UTR. Finally, we show that germ cells of Dazl null mice contain reduced levels of Mvh protein, indicating that Dazl-mediated regulation of Mvh translation is crucial for mammalian spermatogenesis.
Assuntos
Biossíntese de Proteínas , RNA Helicases/genética , Proteínas de Ligação a RNA/metabolismo , Espermatozoides/fisiologia , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Sequência Conservada , RNA Helicases DEAD-box , Feminino , Regulação da Expressão Gênica , Imunoprecipitação , Masculino , Meiose/genética , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Oócitos/fisiologia , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Ratos , Transcrição Gênica , Xenopus laevisRESUMO
Genetic understanding of male-factor infertility requires knowledge of gene expression patterns associated with normal germ cell differentiation. The mouse is one of the best models of mammalian fertility due to its well-characterized genetics and the existence of many infertile mutants both naturally occurring and experimentally induced. We used cDNA microarrays firstly to investigate normal gene expression in the wild-type (wt) testis and secondly to gain a better insight into the effect of the disruption of the Dazl gene on spermatogenesis. We constructed a cDNA microarray from a subtracted and normalized adult testis library and focused on six developmental time-points during the initial synchronous wave of spermatogenesis. The results suggest that in the wild-type testis, 89.5% of genes on our chip change expression dramatically during the time-course. To identify patterns in the gene-expression data, a k-means clustering algorithm and principal component analysis were used. In the Dazl knockout testes, the majority of genes remain at baseline levels of expression, because absence of Dazl has a severe effect on cell-types present in the testis. Although in the prepubescent Dazl-null mice the final point reached in germ cell development is the leptotene-zygotene stage, the microarray results suggest that lack of Dazl expression has a detectable effect on the mRNA complement of germ cells as early as day 5 when only type A spermatogonia are present. Mol. Reprod. Dev. 67: 26-54, 2004.