Tet enzymes are essential for early embryogenesis and completion of embryonic genome activation.

Mammalian development begins in transcriptional silence followed by a period of widespread activation of thousands of genes. DNA methylation reprogramming is integral to embryogenesis and linked to Tet enzymes, but their function in early development is not well understood. Here, we generate combined deficiencies of all three Tet enzymes in mouse oocytes using a morpholino-guided knockdown approach and study the impact of acute Tet enzyme deficiencies on preimplantation development. Tet1-3 deficient embryos arrest at the 2-cell stage with the most severe phenotype linked to Tet2. Individual Tet enzymes display non-redundant roles in the consecutive oxidation of 5-methylcytosine to 5-carboxylcytosine. Gene expression analysis uncovers that Tet enzymes are required for completion of embryonic genome activation (EGA) and fine-tuned expression of transposable elements and chimeric transcripts. Whole-genome bisulfite sequencing reveals minor changes of global DNA methylation in Tet-deficient 2-cell embryos, suggesting an important role of non-catalytic functions of Tet enzymes in early embryogenesis. Our results demonstrate that Tet enzymes are key components of the clock that regulates the timing and extent of EGA in mammalian embryos.

Expanding the microRNA targeting code: functional sites with centered pairing.

Most metazoan microRNA (miRNA) target sites have perfect pairing to the seed region, located near the miRNA 5' end. Although pairing to the 3' region sometimes supplements seed matches or compensates for mismatches, pairing to the central region has been known to function only at rare sites that impart Argonaute-catalyzed mRNA cleavage. Here, we present "centered sites," a class of miRNA target sites that lack both perfect seed pairing and 3'-compensatory pairing and instead have 11-12 contiguous Watson-Crick pairs to the center of the miRNA. Although centered sites can impart mRNA cleavage in vitro (in elevated Mg(2+)), in cells they repress protein output without consequential Argonaute-catalyzed cleavage. Our study also identified extensively paired sites that are cleavage substrates in cultured cells and human brain. This expanded repertoire of cleavage targets and the identification of the centered site type help explain why central regions of many miRNAs are evolutionarily conserved.

Mammalian microRNAs: experimental evaluation of novel and previously annotated genes.

MicroRNAs (miRNAs) are small regulatory RNAs that derive from distinctive hairpin transcripts. To learn more about the miRNAs of mammals, we sequenced 60 million small RNAs from mouse brain, ovary, testes, embryonic stem cells, three embryonic stages, and whole newborns. Analysis of these sequences confirmed 398 annotated miRNA genes and identified 108 novel miRNA genes. More than 150 previously annotated miRNAs and hundreds of candidates failed to yield sequenced RNAs with miRNA-like features. Ectopically expressing these previously proposed miRNA hairpins also did not yield small RNAs, whereas ectopically expressing the confirmed and newly identified hairpins usually did yield small RNAs with the classical miRNA features, including dependence on the Drosha endonuclease for processing. These experiments, which suggest that previous estimates of conserved mammalian miRNAs were inflated, provide a substantially revised list of confidently identified murine miRNAs from which to infer the general features of mammalian miRNAs. Our analyses also revealed new aspects of miRNA biogenesis and modification, including tissue-specific strand preferences, sequential Dicer cleavage of a metazoan precursor miRNA (pre-miRNA), consequential 5' heterogeneity, newly identified instances of miRNA editing, and evidence for widespread pre-miRNA uridylation reminiscent of miRNA regulation by Lin28.

Early origins and evolution of microRNAs and Piwi-interacting RNAs in animals.

In bilaterian animals, such as humans, flies and worms, hundreds of microRNAs (miRNAs), some conserved throughout bilaterian evolution, collectively regulate a substantial fraction of the transcriptome. In addition to miRNAs, other bilaterian small RNAs, known as Piwi-interacting RNAs (piRNAs), protect the genome from transposons. Here we identify small RNAs from animal phyla that diverged before the emergence of the Bilateria. The cnidarian Nematostella vectensis (starlet sea anemone), a close relative to the Bilateria, possesses an extensive repertoire of miRNA genes, two classes of piRNAs and a complement of proteins specific to small-RNA biology comparable to that of humans. The poriferan Amphimedon queenslandica (sponge), one of the simplest animals and a distant relative of the Bilateria, also possesses miRNAs, both classes of piRNAs and a full complement of the small-RNA machinery. Animal miRNA evolution seems to have been relatively dynamic, with precursor sizes and mature miRNA sequences differing greatly between poriferans, cnidarians and bilaterians. Nonetheless, miRNAs and piRNAs have been available as classes of riboregulators to shape gene expression throughout the evolution and radiation of animal phyla.

Loss of cardiac microRNA-mediated regulation leads to dilated cardiomyopathy and heart failure.

RATIONALE: Heart failure is a deadly and devastating disease that places immense costs on an aging society. To develop therapies aimed at rescuing the failing heart, it is important to understand the molecular mechanisms underlying cardiomyocyte structure and function. OBJECTIVE: microRNAs are important regulators of gene expression, and we sought to define the global contributions made by microRNAs toward maintaining cardiomyocyte integrity. METHODS AND RESULTS: First, we performed deep sequencing analysis to catalog the miRNA population in the adult heart. Second, we genetically deleted, in cardiac myocytes, an essential component of the machinery that is required to generate miRNAs. Deep sequencing of miRNAs from the heart revealed the enrichment of a small number of microRNAs with one, miR-1, accounting for 40% of all microRNAs. Cardiomyocyte-specific deletion of dgcr8, a gene required for microRNA biogenesis, revealed a fully penetrant phenotype that begins with left ventricular malfunction progressing to a dilated cardiomyopathy and premature lethality. CONCLUSIONS: These observations reveal a critical role for microRNAs in maintaining cardiac function in mature cardiomyocytes and raise the possibility that only a handful of microRNAs may ultimately be responsible for the dramatic cardiac phenotype seen in the absence of dgcr8.

Human germ cell formation in xenotransplants of induced pluripotent stem cells carrying X chromosome aneuploidies.

Turner syndrome is caused by complete or partial loss of the second sex chromosome and is characterized by spontaneous fetal loss in >90% of conceptions. Survivors possess an array of somatic and germline clinical characteristics. Induced pluripotent stem cells (iPSCs) offer an opportunity for insight into genetic requirements of the X chromosome linked to Turner syndrome. We derived iPSCs from Turner syndrome and control individuals and examined germ cell development as a function of X chromosome composition. We demonstrate that two X chromosomes are not necessary for reprogramming or maintenance of pluripotency and that there are minimal differences in gene expression, at the single cell level, linked to X chromosome aneuploidies. Formation of germ cells, as assessed in vivo through a murine xenotransplantation model, indicated that undifferentiated iPSCs, independent of X chromosome composition, are capable of forming germ-cell-like cells (GCLCs) in vivo. In combination with clinical data regarding infertility in women with X chromosome aneuploidies, results suggest that two intact X chromosomes are not required for human germ cell formation, qualitatively or quantitatively, but rather are likely to be required for maintenance of human germ cells to adulthood.