The piggyBac DNA transposon is used widely in genome engineering applications. Unlike other transposons, its excision site can be precisely repaired without leaving footprints and it integrates specifically at TTAA tetranucleotides. We present cryo-EM structures of piggyBac transpososomes: a synaptic complex with hairpin DNA intermediates and a strand transfer complex capturing the integration step. The results show that the excised TTAA hairpin intermediate and the TTAA target adopt essentially identical conformations, providing a mechanistic link connecting the two unique properties of piggyBac. The transposase forms an asymmetric dimer in which the two central domains synapse the ends while two C-terminal domains form a separate dimer that contacts only one transposon end. In the strand transfer structure, target DNA is severely bent and the TTAA target is unpaired. In-cell data suggest that asymmetry promotes synaptic complex formation, and modifying ends with additional transposase binding sites stimulates activity.

Mechanisms modulating prostate cell fate determination remain unexplored. The leucine-rich repeat containing G-protein coupled receptors (LGRs) have been identified as important stem cell markers in various tissues. Here, we investigated the roles of Lgr4/Gpr48 in prostate stem cells and development. Lgr4 was ubiquitously expressed during early prostate development prior to lineage specification, with adult expression restricted to a few basal cells (principally Lin−Sca1+CD49f+). Lgr4−/− mice had compromised branching morphogenesis and delayed epithelial differentiation, leading to decreased prostate size and impaired luminal cell function. In vitro prostate sphere culture revealed that Lgr4−/− Lin−/Sca1+/CD49f+ (LSC) cells failed to generate p63low cells, indicating a differentiation deficiency. Furthermore, Lgr4 ablation arrested prostate stem cell (PSC) differentiation of in vivo kidney capsule prostate grafts, suggesting that Lgr4 modulates prostate stem cell properties independent of hormonal and mesenchymal effects. Analysis of neonatal prostates and prostate spheres revealed a decrease in Wnt, Sonic Hedgehog, and Notch1 expression in Lgr4−/− cells. Lgr4 loss blocked differentiation of prostate sphere p63hi cells to p63low. Treatment with exogenous Sonic Hedgehog partially restored the differentiation of p63hi cells in Lgr4−/− spheres. Taken together, our data revealed the roles of Lgr4 in early prostate development and in stem cell differentiation through regulation of the Wnt, Notch and Sonic Hedgehog signaling pathways.

G-protein-coupled receptors (GPCRs), one of the most versatile groups of cell surface receptors, can recognize specific ligands from neural, hormonal, and paracrine organs and regulate cell growth, proliferation, and differentiation. Gpr48/LGR4 is a recently identified orphan GPCR with unknown functions. To reveal the functions of Gpr48 in vivo, we generated Gpr48 ؊/؊ mice and found that Gpr48 ؊/؊ fetuses displayed transient anemia during midgestation and abnormal definitive erythropoiesis. The dramatic decrease of definitive erythroid precursors (Ter119 pos population) in Gpr48 ؊/؊ fetal liver at E13.5 was confirmed by histological analysis and blood smear assays. Real-time PCR analyses showed that in Gpr48؊/؊ mice both adult hemoglobin ␣ and ␤ chains were decreased while embryonic hemoglobin chains (, ␤H1, and ⑀y) were increased, providing another evidence for the impairment of definitive erythropoiesis. Furthermore, proliferation was suppressed in Gpr48 ؊/؊ fetal liver with decreased c-Myc and cyclin D1 expression, whereas apoptosis was unaffected. ATF4, a key transcription factor in erythropoiesis, was downregulated in Gpr48 ؊/؊ fetal livers during midgestation stage through the cAMP-PKA-CREB pathway, suggesting that Gpr48 regulated definitive erythropoiesis through ATF4-mediated definitive erythropoiesis.Erythropoiesis occurs sequentially in distinct anatomical locations in two different phases during embryogenesis. The earlier phase is defined as primitive erythropoiesis, which, in the mouse, originates from the yolk sac at embryonic day 7.5 (E7.5), 3 and the later phase is definitive erythropoiesis, which was carried out in the fetal liver during midgestation after E12.5. Non-nucleated adult-type red blood cells are first generated in the fetal liver, the primary organ for erythropoiesis during midgestation from E12 to E16 (1). Erythropoiesis then transfers to bone marrow and spleen in the adult (2, 3). However, the molecular mechanism of regulating erythropoiesis has not been completely delineated. Most of the studies were focused on the transcription factors to explore the mechanisms of erythropoiesis (4, 5). In recent years, several transcription factors were also identified to regulate definitive erythropoiesis in fetal liver during midgestation (6 -11). One of the transcription factors is ATF4, which has been shown to regulate cell proliferation in response to a broad spectrum of cell stresses and can be either an activator or a repressor in response to different extracellular signals (12). ATF4 Ϫ/Ϫ mice have been reported to cause defective definitive erythropoiesis, and severe anemia at midgestation (13). Although receptors, such as c-Kit and EPOR, have been well studied in erythropoiesis (14), little is known on the function of G-proteincoupled receptors (GPCRs) in erythropoiesis during development (14, 15).The GPCR family represents the largest and most versatile group of cell surface receptors (16 -18). GPCRs can recognize their ligands, a diverse array of extracellular signals, then transmit th...