RNA interference (RNAi) is a mechanism by which double-stranded RNAs (dsRNAs) suppress specific transcripts in a sequence-dependent manner. dsRNAs are processed by Dicer to 21-24-nucleotide small interfering RNAs (siRNAs) and then incorporated into the argonaute (Ago) proteins. Gene regulation by endogenous siRNAs has been observed only in organisms possessing RNA-dependent RNA polymerase (RdRP). In mammals, where no RdRP activity has been found, biogenesis and function of endogenous siRNAs remain largely unknown. Here we show, using mouse oocytes, that endogenous siRNAs are derived from naturally occurring dsRNAs and have roles in the regulation of gene expression. By means of deep sequencing, we identify a large number of both approximately 25-27-nucleotide Piwi-interacting RNAs (piRNAs) and approximately 21-nucleotide siRNAs corresponding to messenger RNAs or retrotransposons in growing oocytes. piRNAs are bound to Mili and have a role in the regulation of retrotransposons. siRNAs are exclusively mapped to retrotransposons or other genomic regions that produce transcripts capable of forming dsRNA structures. Inverted repeat structures, bidirectional transcription and antisense transcripts from various loci are sources of the dsRNAs. Some precursor transcripts of siRNAs are derived from expressed pseudogenes, indicating that one role of pseudogenes is to adjust the level of the founding source mRNA through RNAi. Loss of Dicer or Ago2 results in decreased levels of siRNAs and increased levels of retrotransposon and protein-coding transcripts complementary to the siRNAs. Thus, the RNAi pathway regulates both protein-coding transcripts and retrotransposons in mouse oocytes. Our results reveal a role for endogenous siRNAs in mammalian oocytes and show that organisms lacking RdRP activity can produce functional endogenous siRNAs from naturally occurring dsRNAs.

Recent investigations have elucidated the cytokineinduced NF-B activation pathway. IB kinase (IKK) phosphorylates inhibitors of NF-B (IBs). The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, allowing the nuclear translocation of NF-B. We have examined the possibility that IKK can phosphorylate the p65 NF-B subunit as well as IB in the cytokine-induced NF-B activation. In the cytoplasm of HeLa cells, the p65 subunit was rapidly phosphorylated in response to TNF-␣ in a time dependent manner similar to IB phosphorylation. In vitro phosphorylation with GST-fused p65 showed that a p65 phosphorylating activity was present in the cytoplasmic fraction and the target residue was Ser-536 in the carboxyl-terminal transactivation domain. The endogenous IKK complex, overexpressed IKKs, and recombinant IKK␤ efficiently phosphorylated the same Ser residue of p65 in vitro. The major phosphorylation site in vivo was also Ser-536. Furthermore, activation of IKKs by NF-Binducing kinase induced phosphorylation of p65 in vivo. Our finding, together with previous observations, suggests dual roles for IKK complex in the regulation of NF-B⅐IB complex.The transcription factor nuclear factor-B (NF-B) 1 plays a pivotal role in inflammatory and immune responses (1-3). NF-B is composed of a heterodimer of p65 and p50 subunits in most cell types and is sequestered in the cytoplasm by its inhibitor proteins, the IBs (4 -8). Several NF-B-activating agents, including pro-inflammatory cytokines, phorbol esters, and bacterial lipopolysaccaride, induce the phosphorylation of IBs at two NH 2 -terminal Ser residues. The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, thereby releasing NF-B to enter the nucleus for gene expression (9 -15).Recent investigations have focused on the phosphorylation of IBs and clearly elucidated the molecular mechanisms of the phosphorylation. In brief, two closely related kinases, designated IB kinase (IKK) ␣ and IKK␤, have been identified as components of the multiprotein IKK complex (500 -900 kDa) that directly phosphorylates the critical Ser residues of . IKK␣ and IKK␤ together form a heterodimer through their COOH-terminal leucine zipper motifs, and the functional IKK complex contains both IKK subunits. NF-B-inducing kinase (NIK), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, interacts with and activates the IKK complex (21). Other MAP3Ks, including transforming growth factor-␤ activated kinase 1 (TAK1) (22-24), MAPK/ extracellular signal-regulated kinase kinase kinases (MEKK1-3) (25-28), and Cot/Tpl2 (29), have been shown to be involved in the IKK activation pathways, indicating the important roles of MAP3K family kinases in the IKK activation by diverse extracellular stimuli.The activity of several inducible transcription factors, including cAMP response element-binding protein (CREB) (30) and c-Jun (31), has been shown to be regulated by phosphorylation. It has been shown that the p65 NF-B subun...

DNA methylation is extensively reprogrammed during the early phases of mammalian development, yet genomic targets of this process are largely unknown. We optimized methylated DNA immunoprecipitation for low numbers of cells and profiled DNA methylation during early development of the mouse embryonic lineage in vivo. We observed a major epigenetic switch during implantation at the transition from the blastocyst to the postimplantation epiblast. During this period, DNA methylation is primarily targeted to repress the germline expression program. DNA methylation in the epiblast is also targeted to promoters of lineage-specific genes such as hematopoietic genes, which are subsequently demethylated during terminal differentiation. De novo methylation during early embryogenesis is catalyzed by Dnmt3b, and absence of DNA methylation leads to ectopic gene activation in the embryo. Finally, we identify nonimprinted genes that inherit promoter DNA methylation from parental gametes, suggesting that escape of post-fertilization DNA methylation reprogramming is prevalent in the mouse genome.