RNA interference (RNAi) is an important means of eliminating mRNAs, but the intracellular location of RNA-induced silencing complex (RISC) remains unknown. We show here that Argonaute 2, a key component of RISC, is not randomly distributed but concentrates in mRNA decay centres that are known as cytoplasmic bodies. The localization of Argonaute 2 in decay centres is not altered by the presence or absence of small interfering RNAs or their targeted mRNAs. However, RNA is required for the integrity of cytoplasmic bodies because RNase eliminates Argonaute 2 localization. In addition, Argonaute 1, another Argonaute family member, is concentrated in cytoplasmic bodies. These results provide new insight into the mechanism of RNAi function.

Progenitor cells maintain self-renewing tissues throughout life by sustaining their capacity for proliferation while suppressing cell cycle exit and terminal differentiation1,2. DNA methylation3,4,5 provides a potential epigenetic mechanism for the cellular memory needed to preserve the somatic progenitor state through repeated cell divisions. DNA methyltransferase 1 (DNMT1)6,7 maintains DNA methylation patterns after cellular replication. Although dispensable for embryonic stem cell maintenance,8 a clear role for DNMT1 in maintaining the progenitor state in constantly replenished somatic tissues, such as mammalian epidermis, is unknown. Here we show that DNMT1 is essential for epidermal progenitor cell function. DNMT1 protein was found enriched in undifferentiated cells, where it was required to retain proliferative stamina and suppress differentiation. In tissue, DNMT1 depletion led to exit from the progenitor cell compartment, premature differentiation and eventual tissue loss. Genome-wide analysis revealed that a significant portion of epidermal differentiation gene promoters were methylated in self-renewing conditions but were subsequently demethylated during differentiation. Furthermore, we show that UHRF1,9,10 a component of the DNA methylation machinery that targets DNMT1 to hemi-methylated DNA, is also necessary to suppress premature differentiation and sustain proliferation. In contrast, Gadd45A11,12 and B13, which promote active DNA demethylation, are required for full epidermal differentiation gene induction. These data demonstrate that proteins involved in the dynamic regulation of DNA methylation patterns are required for progenitor maintenance and self-renewal in mammalian somatic tissue.

The recent discovery of H3K27me3 demethylases suggests that H3K27me3 may dynamically regulate gene expression, but this potential role in mammalian tissue homeostasis remains uncharacterized. In the epidermis, a tissue that balances stem cell self-renewal with differentiation, H3K27me3, occupies the promoters of many differentiation genes. During calcium-induced differentiation, H3K27me3 was erased at these promoters in concert with loss of PcG protein occupancy and increased binding by the H3K27me3 demethylase, JMJD3. Within epidermal tissue, JMJD3 depletion blocked differentiation, while active JMJD3 dominantly induced it. These results indicate that epigenetic derepression by JMJD3 controls mammalian epidermal differentiation.Supplemental material is available at http://www.genesdev.org.Received March 12, 2008; revised version accepted May 22, 2008. Post-translational modifications of core histone octamer proteins, in the form of phosphorylation, acetylation, ubiquitination, and methylation, have been increasingly appreciated as powerful regulators of gene expression. In Drosophila and mammals, the histone 3 Lys 27 trimethylation mark (H3K27me3) is associated with repression of gene transcription (Lund and van Lohuizen 2004;Barski et al. 2007). Polycomb group proteins (PcG) and their antagonists, the histone demethylases, are epigenetic regulators that control gene expression by modifying the methylation status of H3K27 (Grimaud et al. 2006;Swigut and Wysocka 2007). The PcG protein, Enhancer of Zest Homolog 2 (EZH2), is a histone methyltransferase that can trimethylate H3K27. Two other PcG proteins, Embryonic Ectoderm Development (EED) and Suppressor of Zeste 12 homolog (SUZ12), are necessary for EZH2 to function properly (Pasini et al. 2004). Genome-wide mapping of H3K27me3 marks and PcG target sites in embryonic stem cells has recently suggested a major role for PcG proteins in maintaining H3K27me3 to allow for repression of the differentiated state and promotion of embryonic stem cell self-renewal, with significant enrichment of these proteins on genes encoding homeodomain proteins and other putative differentiation factors Lee et al. 2006). The mechanism reversing such potential repressive influences on differentiation gene expression by H3K27me3 and other histone modifications in somatic tissue is not fully characterized.Among potential mediators of differentiation gene derepression are newly characterized Jumanji C (JmjC) domain-containing proteins, UTX and JMJD3, which are enzymes capable of demethylating promoters marked by H3K27me3. Recently, the H3K27 demethylase activity of UTX and JMJD3 has been shown to act at HOX gene promoters to derepress HOX gene transcription (Agger et al. 2007;Lan et al. 2007;Lee et al. 2007). These histone demethylases were demonstrated in mammalian cells and in live zebrafish to antagonize PcG gene silencing by modifying chromatin to permit gene transcription. During inflammation, bacterial products and cytokines induce JMJD3, which then removes H3K27me3 marks to dere...

SUMMARY Disrupted epidermal differentiation characterizes numerous diseases that impact >25% of the population. In a search for dominant mediators of differentiation, we defined a requirement for ZNF750 in terminal epidermal differentiation. ZNF750 controlled genes mutated in numerous human skin diseases, including FLG, LOR, LCE3B, ALOXE3, and SPINK5. ZNF750 induced progenitor differentiation via an evolutionarily conserved C2H2 zinc finger motif. The epidermal master regulator, p63, bound the ZNF750 promoter and was necessary for its induction. ZNF750 restored differentiation to p63-deficient tissue, suggesting it acts downstream of p63. A search for functionally important ZNF750 targets via analysis of ZNF750-regulated genes identified KLF4, a transcription factor that activates late epidermal differentiation. ZNF750 binds to KLF4 at multiple sites flanking the transcriptional start site and controls its expression. ZNF750 thus directly links a tissue-specifying factor, p63, to an effector of terminal differentiation, KLF4, and represents a potential future target for disorders of this process.

SUMMARY Type 1 interferons (IFN) promote inflammation in the skin but the mechanisms responsible for inducing these cytokines are not well understood. We found that IFNβ was abundantly produced by epidermal keratinocytes (KCs) in psoriasis and during wound repair. KC IFNβ production depended on stimulation of mitochondrial antiviral-signaling protein (MAVS) by the antimicrobial peptide LL37 and double stranded-RNA released from necrotic cells. MAVS activated downstream TBK1 (TANK-Binding Kinase 1)-AKT (AKT serine/threonine kinase 1)-IRF3 (interferon regulatory factor 3) signaling cascade leading to IFNβ production, and then promoted maturation of dendritic cells. In mice, the production of epidermal IFNβ by LL37 required MAVS, and human wounded and/or psoriatic skin showed activation of MAVS-associated IRF3 and induction of MAVS and IFNβ gene signatures. These findings show that KCs are an important source of IFNβ and MAVS is critical to this function, and demonstrates how the epidermis triggers unwanted skin inflammation under disease conditions.

The use of the RNA interference (RNAi) pathway to eliminate gene products has greatly facilitated the understanding of gene function. Behind this remarkable pathway is an intricate network of proteins that ensures the degradation of the target mRNA. In this review, we explore the history of RNAi as well as highlighting recent discoveries.

SUMMARY In adult tissues, stem and progenitor cells must balance proliferation and differentiation to maintain homeostasis. How this is done is unclear. Here, we show that the DEAD box RNA helicase, DDX6 is necessary for maintaining adult progenitor cell function. DDX6 loss results in premature differentiation and decreased proliferation of epidermal progenitor cells. To maintain self-renewal, DDX6 associates with YBX1 to bind the stem loops found in the 3′ untranslated regions (UTRs) of regulators of proliferation/self-renewal (CDK1, EZH2) and recruit them to EIF4E to facilitate their translation. To prevent premature differentiation of progenitor cells, DDX6 regulates the 5′UTR of differentiation inducing transcription factor, KLF4 and degrades its transcripts through association with mRNA degradation proteins. Our results demonstrate that progenitor function is maintained by DDX6 complexes through two distinct pathways which include the degradation of differentiation inducing transcripts as well as promoting the translation of self-renewal and proliferation mRNAs.

In mammals the genome is shaped by epigenetic regulation to manifest numerous cellular identities. The term epigenetics has been used to refer to changes in gene expression, which are heritable through multiple cell division cycles that are not due to variations in primary DNA sequence. Stable suppression of differentiation genes is required to sustain the undifferentiated state in cells ranging from embryonic stem cells to somatic stem cell progenitors that constantly replenish self-renewing tissues. However, the epigenetic mechanisms behind the maintenance of cellular dedifferentiation are not yet fully understood. Major effectors of epigenetic control include regulators of DNA methylation and histone modification as well as ATP-dependent chromatin remodeling enzymes. These interact with other regulators, such as DNA sequence-specific transcription factors and noncoding RNAs to landscape the genome during development, differentiation and cancer. DNA methylation is a classic and powerful example of the epigenetic inheritance of cellular identity that is widely used in eukaryotes. DNA methylation confers distinct epigenetic states via several mechanisms. Here we discuss fundamental mechanisms of DNA methylation and their interplay with several regulatory pathways that define cellular physiology and differentiation.