Tudor domain containing 7 (Tdrd7) is essential for dynamic ribonucleoprotein (RNP) remodeling of chromatoid bodies during spermatogenesis

In the male germline in mammals, chromatoid bodies, a specialized assembly of cytoplasmic ribonucleoprotein (RNP), are structurally evident during meiosis and haploidgenesis, but their developmental origin and regulation remain elusive. The tudor domain containing proteins constitute a conserved class of chromatoid body components. We show that tudor domain containing 7 (Tdrd7), the deficiency of which causes male sterility and age-related cataract (as well as glaucoma), is essential for haploid spermatid development and defines, in concert with Tdrd6, key biogenesis processes of chromatoid bodies. Single and double knockouts of Tdrd7 and Tdrd6 demonstrated that these spermiogenic tudor genes orchestrate developmental programs for ordered remodeling of chromatoid bodies, including the initial establishment, subsequent RNP fusion with ubiquitous processing bodies/GW bodies and later structural maintenance. Tdrd7 suppresses LINE1 retrotransposons independently of piwi-interacting RNA (piRNA) biogenesis wherein Tdrd1 and Tdrd9 operate, indicating that distinct Tdrd pathways act against retrotransposons in the male germline. Tdrd6, in contrast, does not affect retrotransposons but functions at a later stage of spermiogenesis when chromatoid bodies exhibit aggresome-like properties. Our results delineate that chromatoid bodies assemble as an integrated compartment incorporating both germline and ubiquitous features as spermatogenesis proceeds and that the conserved tudor family genes act asmaster regulators of this unique RNP remodeling,which is genetically linked to the male germline integrity in mammals

A MILI-independent piRNA biogenesis pathway empowers partial germline reprogramming.

In mice, the pathway involving PIWI and PIWI-interacting RNA (PIWI-piRNA) is essential to re-establish transposon silencing during male-germline reprogramming. The cytoplasmic PIWI protein MILI mediates piRNA-guided transposon RNA cleavage as well as piRNA amplification. MIWI2's binding to piRNA and its nuclear localization are proposed to be dependent upon MILI function. Here, we demonstrate the existence of a piRNA biogenesis pathway that sustains partial MIWI2 function and reprogramming activity in the absence of MILI

Defending the genome from the enemy within:mechanisms of retrotransposon suppression in the mouse germline

The viability of any species requires that the genome is kept stable as it is transmitted from generation to generation by the germ cells. One of the challenges to transgenerational genome stability is the potential mutagenic activity of transposable genetic elements, particularly retrotransposons. There are many different types of retrotransposon in mammalian genomes, and these target different points in germline development to amplify and integrate into new genomic locations. Germ cells, and their pluripotent developmental precursors, have evolved a variety of genome defence mechanisms that suppress retrotransposon activity and maintain genome stability across the generations. Here, we review recent advances in understanding how retrotransposon activity is suppressed in the mammalian germline, how genes involved in germline genome defence mechanisms are regulated, and the consequences of mutating these genome defence genes for the developing germline

Mitotic Arrest in Teratoma Susceptible Fetal Male Germ Cells

Formation of germ cell derived teratomas occurs in mice of the 129/SvJ strain, but not in C57Bl/6 inbred or CD1 outbred mice. Despite this, there have been few comparative studies aimed at determining the similarities and differences between teratoma susceptible and non-susceptible mouse strains. This study examines the entry of fetal germ cells into the male pathway and mitotic arrest in 129T2/SvJ mice. We find that although the entry of fetal germ cells into mitotic arrest is similar between 129T2/SvJ, C57Bl/6 and CD1 mice, there were significant differences in the size and germ cell content of the testis cords in these strains. In 129T2/SvJ mice germ cell mitotic arrest involves upregulation of p27KIP1, p15INK4B, activation of RB, the expression of male germ cell differentiation markers NANOS2, DNMT3L and MILI and repression of the pluripotency network. The germ-line markers DPPA2 and DPPA4 show reciprocal repression and upregulation, respectively, while FGFR3 is substantially enriched in the nucleus of differentiating male germ cells. Further understanding of fetal male germ cell differentiation promises to provide insight into disorders of the testis and germ cell lineage, such as testis tumour formation and infertility

TOPAZ1, a Novel Germ Cell-Specific Expressed Gene Conserved during Evolution across Vertebrates

BACKGROUND: We had previously reported that the Suppression Subtractive Hybridization (SSH) approach was relevant for the isolation of new mammalian genes involved in oogenesis and early follicle development. Some of these transcripts might be potential new oocyte and granulosa cell markers. We have now characterized one of them, named TOPAZ1 for the Testis and Ovary-specific PAZ domain gene. PRINCIPAL FINDINGS: Sheep and mouse TOPAZ1 mRNA have 4,803 bp and 4,962 bp open reading frames (20 exons), respectively, and encode putative TOPAZ1 proteins containing 1,600 and 1653 amino acids. They possess PAZ and CCCH domains. In sheep, TOPAZ1 mRNA is preferentially expressed in females during fetal life with a peak during prophase I of meiosis, and in males during adulthood. In the mouse, Topaz1 is a germ cell-specific gene. TOPAZ1 protein is highly conserved in vertebrates and specifically expressed in mouse and sheep gonads. It is localized in the cytoplasm of germ cells from the sheep fetal ovary and mouse adult testis. CONCLUSIONS: We have identified a novel PAZ-domain protein that is abundantly expressed in the gonads during germ cell meiosis. The expression pattern of TOPAZ1, and its high degree of conservation, suggests that it may play an important role in germ cell development. Further characterization of TOPAZ1 may elucidate the mechanisms involved in gametogenesis, and particularly in the RNA silencing process in the germ lin

Peculiarities of piRNA-mediated post-transcriptional silencing of Stellate repeats in testes of Drosophila melanogaster

Silencing of Stellate genes in Drosophila melanogaster testes is caused by antisense piRNAs produced as a result of transcription of homologous Suppressor of Stellate (Su(Ste)) repeats. Mechanism of piRNA-dependent Stellate repression remains poorly understood. Here, we show that deletion of Su(Ste) suppressors causes accumulation of spliced, but not nonspliced Stellate transcripts both in the nucleus and cytoplasm, revealing post-transcriptional degradation of Stellate RNA as the predominant mechanism of silencing. We found a significant amount of Su(Ste) piRNAs and piRNA-interacting protein Aubergine (Aub) in the nuclear fraction. Immunostaining of isolated nuclei revealed co-localization of a portion of cellular Aub with the nuclear lamina. We suggest that the piRNA–Aub complex is potentially able to perform Stellate silencing in the cell nucleus. Also, we revealed that the level of the Stellate protein in Su(Ste)-deficient testes is increased much more dramatically than the Stellate mRNA level. Similarly, Su(Ste) repeats deletion exerts an insignificant effect on mRNA abundance of the Ste-lacZ reporter, but causes a drastic increase of β-gal activity. In cell culture, exogenous Su(Ste) dsRNA dramatically decreases β-gal activity of hsp70-Ste-lacZ construct, but not its mRNA level. We suggest that piRNAs, similarly to siRNAs, degrade only unmasked transcripts, which are accessible for translation

The De Novo Cytosine Methyltransferase DRM2 Requires Intact UBA Domains and a Catalytically Mutated Paralog DRM3 during RNA–Directed DNA Methylation in Arabidopsis thaliana

Eukaryotic DNA cytosine methylation can be used to transcriptionally silence repetitive sequences, including transposons and retroviruses. This silencing is stable between cell generations as cytosine methylation is maintained epigenetically through DNA replication. The Arabidopsis thaliana Dnmt3 cytosine methyltransferase ortholog DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) is required for establishment of small interfering RNA (siRNA) directed DNA methylation. In mammals PIWI proteins and piRNA act in a convergently evolved RNA–directed DNA methylation system that is required to repress transposon expression in the germ line. De novo methylation may also be independent of RNA interference and small RNAs, as in Neurospora crassa. Here we identify a clade of catalytically mutated DRM2 paralogs in flowering plant genomes, which in A.thaliana we term DOMAINS REARRANGED METHYLTRANSFERASE3 (DRM3). Despite being catalytically mutated, DRM3 is required for normal maintenance of non-CG DNA methylation, establishment of RNA–directed DNA methylation triggered by repeat sequences and accumulation of repeat-associated small RNAs. Although the mammalian catalytically inactive Dnmt3L paralogs act in an analogous manner, phylogenetic analysis indicates that the DRM and Dnmt3 protein families diverged independently in plants and animals. We also show by site-directed mutagenesis that both the DRM2 N-terminal UBA domains and C-terminal methyltransferase domain are required for normal RNA–directed DNA methylation, supporting an essential targeting function for the UBA domains. These results suggest that plant and mammalian RNA–directed DNA methylation systems consist of a combination of ancestral and convergent features

Expression of HIWI in human esophageal squamous cell carcinoma is significantly associated with poorer prognosis

<p>Abstract</p> <p>Background</p> <p>HIWI, the human homologue of Piwi family, is present in CD34⁺hematopoietic stem cells and germ cells, but not in well-differentiated cell populations, indicating that HIWI may play an impotent role in determining or maintaining stemness of these cells. That HIWI expression has been detected in several type tumours may suggest its association with clinical outcome in cancer patients.</p> <p>Methods</p> <p>With the methods of real-time PCR, western blot, immunocytochemistry and immunohistochemistry, the expression of HIWI in three esophageal squamous cancer cell lines KYSE70, KYSE140 and KYSE450 has been characterized. Then, we investigated HIWI expression in a series of 153 esophageal squamous cell carcinomas using immunohistochemistry and explored its association with clinicopathological features.</p> <p>Results</p> <p>The expression of HIWI was observed in tumour cell nuclei or/and cytoplasm in 137 (89.5%) cases, 16 (10.5%) cases were negative in both nuclei and cytoplasm. 86 (56.2%) were strongly positive in cytoplasm, while 49 (32.0%) were strongly positive in nuclei. The expression level of HIWI in cytoplasm of esophageal cancer cells was significantly associated with histological grade (<it>P </it>= 0.011), T stage (<it>P </it>= 0.035), and clinic outcome (<it>P </it>< 0.001), while there was no correlation between the nuclear HIWI expression and clinicopathological features.</p> <p>Conclusion</p> <p>The expression of HIWI in the cytoplasm of esophageal cancer cells is significantly associated with higher histological grade, clinical stage and poorer clinical outcome, indicating its possible involvement in cancer development.</p

Network Analysis of Differential Expression for the Identification of Disease-Causing Genes

Genetic studies (in particular linkage and association studies) identify chromosomal regions involved in a disease or phenotype of interest, but those regions often contain many candidate genes, only a few of which can be followed-up for biological validation. Recently, computational methods to identify (prioritize) the most promising candidates within a region have been proposed, but they are usually not applicable to cases where little is known about the phenotype (no or few confirmed disease genes, fragmentary understanding of the biological cascades involved). We seek to overcome this limitation by replacing knowledge about the biological process by experimental data on differential gene expression between affected and healthy individuals. Considering the problem from the perspective of a gene/protein network, we assess a candidate gene by considering the level of differential expression in its neighborhood under the assumption that strong candidates will tend to be surrounded by differentially expressed neighbors. We define a notion of soft neighborhood where each gene is given a contributing weight, which decreases with the distance from the candidate gene on the protein network. To account for multiple paths between genes, we define the distance using the Laplacian exponential diffusion kernel. We score candidates by aggregating the differential expression of neighbors weighted as a function of distance. Through a randomization procedure, we rank candidates by p-values. We illustrate our approach on four monogenic diseases and successfully prioritize the known disease causing genes