The mechanisms by which embryonic stem (ES) cells self-renew while maintaining the ability to differentiate into virtually all adult cell types are not well understood. Polycomb group (PcG) proteins are transcriptional repressors that help to maintain cellular identity during metazoan development by epigenetic modification of chromatin structure. PcG proteins have essential roles in early embryonic development and have been implicated in ES cell pluripotency, but few of their target genes are known in mammals. Here we show that PcG proteins directly repress a large cohort of developmental regulators in murine ES cells, the expression of which would otherwise promote differentiation. Using genome-wide location analysis in murine ES cells, we found that the Polycomb repressive complexes PRC1 and PRC2 co-occupied 512 genes, many of which encode transcription factors with important roles in development. All of the co-occupied genes contained modified nucleosomes (trimethylated Lys 27 on histone H3). Consistent with a causal role in gene silencing in ES cells, PcG target genes were de-repressed in cells deficient for the PRC2 component Eed, and were preferentially activated on induction of differentiation. Our results indicate that dynamic repression of developmental pathways by Polycomb complexes may be required for maintaining ES cell pluripotency and plasticity during embryonic development.

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The self-association of proteins to form amyloid fibrils has been implicated in the pathogenesis of a number of diseases including Alzheimer's, Parkinson's, and Creutzfeldt-Jakob diseases. We recently reported that the myeloid scavenger receptor CD36 initiates a signaling cascade upon binding to fibrillar ␤-amyloid that stimulates recruitment of microglia in the brain and production of inflammatory mediators. This receptor plays a key role in the pathogenesis of atherosclerosis, prompting us to evaluate whether fibrillar proteins were present in atherosclerotic lesions that could initiate signaling via CD36. We show that apolipoprotein C-II, a component of very low and high density lipoproteins, readily forms amyloid fibrils that initiate macrophage inflammatory responses including reactive oxygen production and tumor necrosis factor ␣ expression. Using macrophages derived from wild type and Cd36 ؊/؊ mice to distinguish CD36-specific events, we show that fibrillar apolipoprotein C-II activates a signaling cascade downstream of this receptor that includes Lyn and p44/42 MAPKs. Interruption of this signaling pathway through targeted deletion of Cd36 or blocking of p44/42 MAPK activation inhibits macrophage tumor necrosis factor ␣ gene expression. Finally, we demonstrate that apolipoprotein C-II in human atheroma co-localizes to regions positive for markers of amyloid and macrophage accumulation. Together, these data characterize a CD36-dependent signaling cascade initiated by fibrillar amyloid species that may promote atherogenesis.

Mir-290 through mir-295 (mir-290-295) is a mammalian-specific microRNA (miRNA) cluster that, in mice, is expressed specifically in early embryos and embryonic germ cells. Here, we show that mir-290-295 plays important roles in embryonic development as indicated by the partially penetrant lethality of mutant embryos. In addition, we show that in surviving mir-290-295-deficient embryos, female but not male fertility is compromised. This impairment in fertility arises from a defect in migrating primordial germ cells and occurs equally in male and female mutant animals. Male mir-290-295 −/− mice, due to the extended proliferative lifespan of their germ cells, are able to recover from this initial germ cell loss and are fertile. Female mir-290-295 −/− mice are unable to recover and are sterile, due to premature ovarian failure. In the mouse, miRNA-mediated posttranscriptional regulation is required for normal embryogenesis (1-3) and embryonic germ cell development (4). Whereas critical roles for miRNA biogenesis in early embryos and embryonic germ cells have been established, the role of individual miRNAs in the development of these cell types remains unclear. Six miRNA families comprise the majority of miRNA species cloned from mouse embryonic stem (ES) cells, with miRNAs from the mir-290 cluster, mir-290 through mir-295 (mir-290-295), being the most abundant (5). Members of this cluster are the first embryonic miRNAs up-regulated in the zygote (6). It has previously been shown that the mir-290 cluster miRNAs are processed from a single primary transcript (7) and possess highly similar pre-miRNA sequences (8).Several studies have addressed the role of mir-290-295 in embryonic stem (ES) cells where this cluster is a direct target of the Oct4, Sox2, and Nanog regulatory network (9). The mir-290 cluster is correlated with developmental potency. Mir-290-295 expression decreases as ES cells differentiate (8). Furthermore, certain members of the miR-290 family were found to increase the efficiency of reprogramming by Oct4, Sox2, and Klf4 ∼10 fold (10). In addition, members of the miR-290 family promote the G1-S transition and thereby the rapid proliferation characteristic of ES cells (11). The mir-290 cluster was also implicated in indirect control of de novo DNA methylation in ES cells (12,13). Taken together, these data imply important roles for mir-290-295 in ES cells and by extension, early mouse development. In this study, we examined the in vivo consequences of targeted disruption of mir-290-295 in the developing mouse.Results mir-290-295 Is Specifically Expressed in the Early Embryo and Embryonic Germ Cells. We have shown that mir-290-295 is expressed in ES cells and not in adult somatic tissues (8). To address the timing of mir-290 cluster expression, we performed RT-PCR for the mir-290-295 primary transcript (pri-mir-290-295) throughout early embryonic development on pools of embryos. We observed onset of expression of the primary transcript at the 4-8 cell stage (Fig. 1A), consistent with the finding that expres...