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.

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.

The PIWI-interacting RNA (piRNA) pathway is essential for retro-transposon silencing. In piRNA-deficient mice, L1-overexpressing male germ cells exhibit excessive DNA damage and meiotic defects. It remains unknown whether L1 expression simply highlights piRNA deficiency or actually drives the germ-cell demise. Specifically, the sheer abundance of genomic L1 copies prevents reliable quantification of new insertions. Here, we developed a codon-optimized L1 transgene that is controlled by an endogenous mouse L1 promoter. Importantly, DNA methylation dynamics of a single-copy transgene were indistinguishable from those of endogenous L1s. Analysis of Mov10l1(-/-) testes established that de novo methylation of the L1 transgene required the intact piRNA pathway. Consistent with loss of DNA methylation and programmed reduction of H3K9me2 at meiotic onset, the transgene showed 1,400-fold increase in RNA expression and consequently 70-fold increase in retrotransposition in postnatal day 14 Mov10l1(-/-) germ cells compared with the wildtype. Analysis of adult Mov10l1(-/-) germ-cell fractions indicated a stage-specific increase of retrotransposition in the early meiotic prophase. However, extrapolation of the transgene data to endogenous L1s suggests that it is unlikely insertional mutagenesis alone accounts for the Mov10l1(-/-) phenotype. Indeed, pharmacological inhibition of reverse transcription did not rescue the meiotic defect. Cumulatively, these results establish the occurrence of productive L1 mobilization in the absence of an intact piRNA pathway but leave open the possibility of processes preceding L1 integration in triggering meiotic checkpoints and germ-cell death. Additionally, our data suggest that many heritable L1 insertions originate from individuals with partially compromised piRNA defense.

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.

DNA methylation regulates the organization and function of the genome. Yamanaka et al. now report that de novo methylation of male germ cells of mice involves the transient opening of heterochromatin at megabase-size differentially accessible domains (DADs). This chromatin remodeling likely facilitates de novo methylation of the germ cell genome.

Female reproductive success critically depends on the size and quality of a finite ovarian reserve. Paradoxically, mammals eliminate up to 80% of the initial oocyte pool through the enigmatic process of fetal oocyte attrition (FOA). Here, we interrogate the striking correlation of FOA with retrotransposon LINE-1 (L1) expression in mice to understand how L1 activity influences FOA and its biological relevance. We report that L1 activity triggers FOA through DNA damage-driven apoptosis and the complement system of immunity. We demonstrate this by combined inhibition of L1 reverse transcriptase activity and the Chk2-dependent DNA damage checkpoint to prevent FOA. Remarkably, reverse transcriptase inhibitor AZT-treated Chk2 mutant oocytes that evade FOA initially accumulate, but subsequently resolve, L1-instigated genotoxic threats independent of piRNAs and differentiate, resulting in an increased functional ovarian reserve. We conclude that FOA serves as quality control for oocyte genome integrity, and is not obligatory for oogenesis nor fertility.

We study the mass distribution in the late-type dwarf galaxy NGC 2976 through stellar kinematics obtained with the Visible Integral Field ReplicableUnit Spectrograph Prototype and anisotropic Jeans models as a test of cosmological simulations and baryonic processes that putatively alter small-scale structure. Previous measurements of the Ha emission-line kinematics have determined that the dark matter halo of NGC 2976 is most consistent with a cored density profile. We find that the stellar kinematics are best fit with a cuspy halo. Cored dark matter halo fits are only consistent with the stellar kinematics if the stellar mass-to-light ratio is significantly larger than that derived from stellar population synthesis, while the best-fitting cuspy model has no such conflict. The inferred mass distribution from a harmonic decomposition of the gaseous kinematics is inconsistent with that of the stellar kinematics. This difference is likely due to the gas disk not meeting the assumptions that underlie the analysis such as no pressure support, a constant kinematic axis, and planar orbits. By relaxing some of these assumptions, in particular the form of the kinematic axis with radius, the gas-derived solution can be made consistent with the stellar kinematic models. A strong kinematic twist in the gas of NGC 2976' s center suggests caution, and we advance the mass model based on the stellar kinematics as more reliable. The analysis of this first galaxy shows promising evidence that dark matter halos in late-type dwarfs may in fact be more consistent with cuspy dark matter distributions than earlier work has claimed.

We present properties of individual and composite rest-UV spectra of continuum-and narrowband-selected star-forming galaxies (SFGs) at a redshift of 2 < z < 3.5 discovered by the MUSYC collaboration in the Extended Chandra Deep Field-South. Among our sample of 81 UV-bright SFGs, 59 have R < 25.5, of which 32 have rest-frame equivalent widths of W-Ly alpha > 20 angstrom, the canonical limit to be classified as an Ly alpha-emitting galaxy. We divide our data set into subsamples based on properties that we are able to measure for each individual galaxy: Ly alpha equivalent width, rest-frame UV colors, and redshift. Among our subsample of galaxies with R < 25.5, those with rest frame W-Ly alpha > 20 angstrom have bluer UV continua, weaker low-ionization interstellar absorption lines, weaker C IV absorption, and stronger Si II* nebular emission than those with W-Ly alpha < 20 angstrom. We measure a velocity offset of Delta nu similar to 600 km s(-1) between Ly alpha emission and low-ionization absorption, which does not vary substantially among any of our subsamples. We find that the interstellar component, as opposed to the stellar component, dominates the high-ionization absorption line profiles. We find that the low-and high-ionization Si ionization states have similar kinematic properties, yet the low-ionization absorption is correlated with Ly alpha emission and the high-ionization absorption is not. These trends are consistent with outflowing neutral gas being in the form of neutral clouds embedded in ionized gas as previously suggested by Steidel et al. Moreover, our galaxies with bluer UV colors have stronger Ly alpha emission, weaker low-ionization absorption, and more prominent nebular emission line profiles. From a redshift of 2.7 < z < 3.5 to 2.0 < z < 2.7, our subsample of galaxies with W-Ly alpha < 20 angstrom shows no significant evolution in their physical properties or the nature of their outflows. Among our data set, UV-bright galaxies with W-Ly alpha > 20 angstrom exhibit weaker Ly alpha emission at lower redshifts, although we caution that this could be caused by spectroscopic confirmation of low Ly alpha equivalent width galaxies being harder at z similar to 3 than z similar to 2.

We measure the radial profile of the (CO)-C-12(1-0) to H-2 conversion factor (X-CO) in NGC 628. The H alpha emission from the VENGA integral field spectroscopy is used to map the star formation rate (SFR) surface density (Sigma(SFR)). We estimate the molecular gas surface density (Sigma(H2)) from Sigma(SFR) by inverting the molecular star formation law (SFL), and compare it to the CO intensity to measure X-CO. We study the impact of systematic uncertainties by changing the slope of the SFL, using different SFR tracers (H alpha versus far-UVplus 24 mu m), and CO maps from different telescopes (single-dish and interferometers). The observed X-CO profile is robust against these systematics, drops by a factor of two from R similar to 7 kpc to the center of the galaxy, and is well fit by a gradient Delta log(X-CO) = 0.06 +/- 0.02 dex kpc(-1). We study how changes in X-CO follow changes in metallicity, gas density, and ionization parameter. Theoretical models show that the gradient in X-CO can be explained by a combination of decreasing metallicity, and decreasing Sigma(H2) with radius. Photoelectric heating from the local UV radiation field appears to contribute to the decrease of X-CO in higher density regions. Our results show that galactic environment plays an important role at setting the physical conditions in star-forming regions, in particular the chemistry of carbon in molecular complexes, and the radiative transfer of CO emission. We caution against adopting a single X-CO value when large changes in gas surface density or metallicity are present.