Dppa3 / Pgc7 / stella is a maternal factor and is not required for germ cell specification in mice

BACKGROUND: In mice, germ cells are specified through signalling between layers of cells comprising the primitive embryo. The function of Dppa3 (also known as Pgc7 or stella), a gene expressed in primordial germ cells at the time of their emergence in gastrulating embryos, is unknown, but a recent study has claimed that it plays a central role in germ cell specification. RESULTS: To test Dppa3's role in germ cell development, we disrupted the gene in mouse embryonic stem cells and generated mutant animals. We were able to obtain viable and fertile Dppa3-deficient animals of both sexes. Examination of embryonic and adult germ cells and gonads in Dppa3-deficient animals did not reveal any defects. However, most embryos derived from Dppa3-deficient oocytes failed to develop normally beyond the four-cell stage. CONCLUSION: We found that Dppa3 is an important maternal factor in the cleavage stages of mouse embryogenesis. However, it is not required for germ cell specification

Cytoplasmic Compartmentalization of the Fetal piRNA Pathway in Mice

Derepression of transposable elements (TEs) in the course of epigenetic reprogramming of the mouse embryonic germline necessitates the existence of a robust defense that is comprised of PIWI/piRNA pathway and de novo DNA methylation machinery. To gain further insight into biogenesis and function of piRNAs, we studied the intracellular localization of piRNA pathway components and used the combination of genetic, molecular, and cell biological approaches to examine the performance of the piRNA pathway in germ cells of mice lacking Maelstrom (MAEL), an evolutionarily conserved protein implicated in transposon silencing in fruit flies and mice. Here we show that principal components of the fetal piRNA pathway, MILI and MIWI2 proteins, localize to two distinct types of germinal cytoplasmic granules and exhibit differential association with components of the mRNA degradation/translational repression machinery. The first type of granules, pi-bodies, contains the MILI-TDRD1 module of the piRNA pathway and is likely equivalent to the enigmatic “cementing material” first described in electron micrographs of rat gonocytes over 35 years ago. The second type of granules, piP-bodies, harbors the MIWI2-TDRD9-MAEL module of the piRNA pathway and signature components of P-bodies, GW182, DCP1a, DDX6/p54, and XRN1 proteins. piP-bodies are found predominantly in the proximity of pi-bodies and the two frequently share mouse VASA homolog (MVH) protein, an RNA helicase. In Mael-mutant gonocytes, MIWI2, TDRD9, and MVH are lost from piP-bodies, whereas no effects on pi-body composition are observed. Further analysis revealed that MAEL appears to specifically facilitate MIWI2-dependent aspects of the piRNA pathway including biogenesis of secondary piRNAs, de novo DNA methylation, and efficient downregulation of TEs. Cumulatively, our data reveal elaborate cytoplasmic compartmentalization of the fetal piRNA pathway that relies on MAEL function

Wt1 functions in the development of germ cells in addition to somatic cell lineages of the testis

AbstractThe Wilms' tumor suppressor gene, Wt1, encodes a transcription factor critical for development of the urogenital system. To identify lineages within the developing urogenital system that have a cell-autonomous requirement for Wt1, chimeric mice were generated from Wt1-null ES cells. Males with large contributions of Wt1−/− cells showed hypoplastic and dysgenic testes, with seminiferous tubules lacking spermatogonia. Wt1-null cells contributed poorly to both somatic and germ cell lineages within the developing gonad, suggesting an unexpected role for Wt1 in germ cell development in addition to a role in the development of the somatic lineages of the gonad. Wt1 expression was detected in embryonic germ cells beginning at embryonic day 11.5 after migrating primordial germ cells (PGCs) have entered the gonad. Germ cells isolated from Wt1-null embryos showed impaired growth in culture, further demonstrating a role for Wt1 in germ cell proliferation or survival. Therefore, Wt1 plays important, and in some cases previously unrecognized, roles in multiple lineages during urogenital development

Transient reduction of DNA methylation at the onset of meiosis in male mice

Background: Meiosis is a specialized germ cell cycle that generates haploid gametes. In the initial stage of meiosis, meiotic prophase I (MPI), homologous chromosomes pair and recombine. Extensive changes in chromatin in MPI raise an important question concerning the contribution of epigenetic mechanisms such as DNA methylation to meiosis. Interestingly, previous studies concluded that in male mice, genome-wide DNA methylation patters are set in place prior to meiosis and remain constant subsequently. However, no prior studies examined DNA methylation during MPI in a systematic manner necessitating its further investigation. Results: In this study, we used genome-wide bisulfite sequencing to determine DNA methylation of adult mouse spermatocytes at all MPI substages, spermatogonia and haploid sperm. This analysis uncovered transient reduction of DNA methylation (TRDM) of spermatocyte genomes. The genome-wide scope of TRDM, its onset in the meiotic S phase and presence of hemimethylated DNA in MPI are all consistent with a DNA replication-dependent DNA demethylation. Following DNA replication, spermatocytes regain DNA methylation gradually but unevenly, suggesting that key MPI events occur in the context of hemimethylated genome. TRDM also uncovers the prior deficit of DNA methylation of LINE-1 retrotransposons in spermatogonia resulting in their full demethylation during TRDM and likely contributing to the observed mRNA and protein expression of some LINE-1 elements in early MPI. Conclusions: Our results suggest that contrary to the prevailing view, chromosomes exhibit dynamic changes in DNA methylation in MPI. We propose that TRDM facilitates meiotic prophase processes and gamete quality control

A Unique HMG-Box Domain of Mouse Maelstrom Binds Structured RNA but Not Double Stranded DNA

<div><p>Piwi-interacting piRNAs are a major and essential class of small RNAs in the animal germ cells with a prominent role in transposon control. Efficient piRNA biogenesis and function require a cohort of proteins conserved throughout the animal kingdom. Here we studied Maelstrom (MAEL), which is essential for piRNA biogenesis and germ cell differentiation in flies and mice. MAEL contains a high mobility group (HMG)-box domain and a Maelstrom-specific domain with a presumptive RNase H-fold. We employed a combination of sequence analyses, structural and biochemical approaches to evaluate and compare nucleic acid binding of mouse MAEL HMG-box to that of canonical HMG-box domain proteins (SRY and HMGB1a). MAEL HMG-box failed to bind double-stranded (ds)DNA but bound to structured RNA. We also identified important roles of a novel cluster of arginine residues in MAEL HMG-box in these interactions. Cumulatively, our results suggest that the MAEL HMG-box domain may contribute to MAEL function in selective processing of retrotransposon RNA into piRNAs. In this regard, a cellular role of MAEL HMG-box domain is reminiscent of that of HMGB1 as a sentinel of immunogenic nucleic acids in the innate immune response.</p></div

Site-specific mutagenesis of MAEL HMG-box domain.

<p>(A) Charged residues, capable of H-bond formation with long side chains, along helix-1 (region important for canonical HMG-box binding) and novel regions of MAEL HMG-box domain were mutated to alanine (residues in red). (B) Mutation of glutamine (Q16A) does not affect binding suggesting that this residue does not participate in complex formation with DNA 4WJ. (C-F) Mutation of individual arginines (R) results in the complete loss of binding to DNA 4WJ suggesting that they are essential for complex formation. The R8A mutant fold appears to be affected (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120268#pone.0120268.s002" target="_blank">S2B Fig.</a>), which might be responsible for reduced binding. Therefore, only arginines in the “hook” and “propeller” seem to be necessary for MAEL HMG-box domain binding to non-canonical DNA. (B`-F`) Interactions of mutants with RNA 4WJ are significantly affected. All mutants show greatly decreased binding to RNA 4WJ as well as variability in the type of complex formed when compared to wild-type protein (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120268#pone.0120268.g003" target="_blank">Fig. 3H</a>). Residual complex formation was still observed (except for R8A) likely due to a high number of configurations that RNA 4WJ can take to accommodate the MAEL HMG-box domain. Nevertheless, the arginines in the “hook” and “propeller” of MAEL HMG-box are important for binding to structured RNA.</p

MAEL HMG-box domain binding to DNA.

<p>(A) Recombinant SRY HMG-box domain strongly binds to dsDNA with its consensus sequence (dsDNA^SRY). (B) Recombinant HMGB1a protein does not bind to the same substrate because it is unable to bend linear dsDNA. (C) Similar to (B) MAEL HMG-box does not bind to dsDNA. (D) Representation of co-crystal structure of SRY HMG-box (green) with dsDNA^SRY describing their fit. Aligned with SRY HMG-box is the MAEL HMG-box domain (orange) whose “hook” region protrudes deeply into dsDNA (1j46, 2cto). The geometry of the helix-2 of both molecules in relation to dsDNA^SRY is highlighted on the right. Even though dsDNA^SRY is pre-bent, it cannot accommodate the “hook” region of MAEL HMG-box domain in the same fashion. (E) SRY HMG-box domain binds to DNA 4WJ forming five complexes (red lines—free substrate; black line—bound substrate). (F) HMGB1a binds to DNA 4WJ forming two complexes. (G) The MAEL HMG-box domain forms only single complex with DNA 4WJ, which is different from the SRY and HMGB1a domains. (H) Proposed mode of HMG-box domain binding to DNA 4WJ. SRY HMG-box domain recognizes a distorted 4WJ center as well any sequences that approximate its consensus-binding site, while HMGB1a protein binds primarily to irregular center. Just as HMGB1a, MAEL HMG-boxes does not bind dsDNA suggesting that it also binds to the center of the junction. Because only single complex forms binding likely happens in different fashion.</p

MAEL HMG-box domain binding to long RNAs.

<p>(A) The secondary structure of a conserved fragment of L1_Md_F2 element from chromosome 10 enriched in MAEL immunoprecipitates determined using combination of covariance and thermodynamic analyses. (B) MAEL HMG-box domain forms strong multi-protein complex with 277-nt fragment and binds weakly to shorter fragments (200-nt, 149-nt) of the same RNA. This suggests a requirement for complex tertiary structures in long RNAs for strong binding to the MAEL HMG-box domain. (C) MAEL HMG-box domain does not bind long RNAs originating from regions not enriched in MAEL immunoprecipitates.</p

MAEL HMG-box domain binding to small RNAs.

<p>A) MAEL HMG-box domain does not bind to ssRNA. B) However, it forms a complex with double-stranded (ds) RNA of identical sequence as dsDNA^SRY. C, D) MAEL HMG-box does not bind to small RNA hairpins. E) Only weak complex formation is observed with hairpin that has mismatches in the stem. F) But when the stem is perfectly base-paired, the binding is stronger and appears increased than that observed with dsRNA (B). G) Binding of MAEL HMG-box to substrate with multiple short hairpins is weak. H) However, binding to RNA 4WJ (sequence identical to DNA 4WJ) is strong and two complexes are formed. These observations indicate that MAEL HMG-box prefers to bind to substrates with continuous dsRNA helices longer than 6 base-pairs located near unstructured or perturbed RNA regions.</p

MAEL HMG-box domain structural and sequence considerations.

<p>(A) Tertiary structure prediction of mouse MAEL protein. MAEL HMG-box domain (orange) is positioned on top of the Maelstrom Specific Domain (MSD, grey) linked by unstructured linker (highlighted in red). The model was generated with Robetta software by either <i>de novo</i> modeling or with established structures as templates. Residue ranges and parent PDB ID numbers are shown. (B) Comparison of MAEL HMG-box and canonical HMG-box domains. Determined structures of candidate HMG-box proteins (SRY: 1j46—sequence specific binding; HMGB1a: 1ckt—structure specific binding; MAEL: 2cto—unknown binding) were visualized and structures aligned in PyMOL. MAEL HMG-box domain has a conserved canonical L-shape fold but with a bend in helix-2, creating a novel region termed “hook” (red). (C) Distribution of charged residues of mouse MAEL HMG-box domain. Positive residues—Arg, Lys, His are blue; negative residues—Asp, Glu are red. Charged residues are concentrated on side B. Unlike other HMG-box domains, in MAEL three arginine (R) residues are concentrated at the end of the helix-1, and protrude outwards, forming a “propeller”-like shape. (D) Phylogenic comparison of MAEL HMG-box domain with well-studied candidate HMG-box domains from sequence-specific (single HMG-box, SRY, Sox) and non-sequence-specific (two HMG-boxes, HMGB’s, Dsp1) groups. Mouse sequences were used unless otherwise noted (Dm: <i>Drosophila melanogaster</i>, Hs: <i>Homo sapiens</i>). The phylogenetic tree was generated using maximum likelihood method in MEGA6 software. Values next to the branches describe percentage of trees where associated sequences group together (n = 1000). The branch length scale is in substitutions per site. MAEL HMG-box domain forms a new branch most closely related to the domain A of non-sequence specific HMG-box proteins.</p