Role of histone H3 lysine 27 methylation in Polycomb-group silencing

Polycomb group (PcG) proteins play important roles in maintaining the silent state of HOX genes. Recent studies have implicated histone methylation in long-term gene silencing. However, a connection between PcG-mediated gene silencing and histone methylation has not been established. Here we report the purification and characterization of an EED-EZH2 complex, the human counterpart of the Drosophila ESC-E(Z) complex. We demonstrate that the complex specifically methylates nucleosomal histone H3 at lysine 27 (H3-K27). Using chromatin immunoprecipitation assays, we show that H3-K27 methylation colocalizes with, and is dependent on, E(Z) binding at an Ultrabithorax (Ubx) Polycomb response element (PRE), and that this methylation correlates with Ubx repression. Methylation on H3-K27 facilitates binding of Polycomb (PC), a component of the PRC1 complex, to histone H3 amino-terminal tail. Thus, these studies establish a link between histone methylation and PcG-mediated gene silencing.     

A complex with chromatin modifiers that occupies E2F- and Myc-responsive genes in G0 cells

E2F-6 contributes to gene silencing in a manner independent of retinoblastoma protein family members. To better elucidate the molecular mechanism of repression by E2F-6, we have purified the factor from cultured cells. E2F-6 is found in a multimeric protein complex that contains Mga and Max, and thus the complex can bind not only to the E2F-binding site but also to Myc- and Brachyury-binding sites. Moreover, the complex contains chromatin modifiers such as a novel histone methyltransferase that modifies lysine 9 of histone H3, HP1gamma, and Polycomb group (PcG) proteins. The E2F-6 complex preferentially occupies target promoters in G0 cells rather than in G1 cells. These data suggest that these chromatin modifiers contribute to silencing of E2F- and Myc-responsive genes in quiescent cells.     

Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome

To equalize X-chromosome dosages between the sexes, the female mammal inactivates one of her two X chromosomes. X-chromosome inactivation (XCI) is initiated by expression of Xist, a 17-kb noncoding RNA (ncRNA) that accumulates on the X in cis. Because interacting factors have not been isolated, the mechanism by which Xist induces silencing remains unknown. We discovered a 1.6-kilobase ncRNA (RepA) within Xist and identified the Polycomb complex, PRC2, as its direct target. PRC2 is initially recruited to the X by RepA RNA, with Ezh2 serving as the RNA binding subunit. The antisense Tsix RNA inhibits this interaction. RepA depletion abolishes full-length Xist induction and trimethylation on lysine 27 of histone H3 of the X. Likewise, PRC2 deficiency compromises Xist up-regulation. Therefore, RepA, together with PRC2, is required for the initiation and spread of XCI. We conclude that a ncRNA cofactor recruits Polycomb complexes to their target locus.     

Inhibition of translational initiation by Let-7 MicroRNA in human cells

MicroRNAs (miRNAs) are approximately 21-nucleotide-long RNA molecules regulating gene expression in multicellular eukaryotes. In metazoa, miRNAs act by imperfectly base-pairing with the 3' untranslated region of target messenger RNAs (mRNAs) and repressing protein accumulation by an unknown mechanism. We demonstrate that endogenous let-7 microribonucleoproteins (miRNPs) or the tethering of Argonaute (Ago) proteins to reporter mRNAs in human cells inhibit translation initiation. M(7)G-cap-independent translation is not subject to repression, suggesting that miRNPs interfere with recognition of the cap. Repressed mRNAs, Ago proteins, and miRNAs were all found to accumulate in processing bodies. We propose that localization of mRNAs to these structures is a consequence of translational repression.     

Plasticity and differentiation of embryonic retinal cells after terminal mitosis

The relation between terminal mitosis and the events that determine the developmental fate of embryonic precursor cells is not well understood. This relation has now been investigated with [3H]thymidine autoradiography to determine the time of cell birth and with a culture system that allows the testing of the developmental potential of cells isolated from the chick embryo retina. Contrary to the situation in vivo, where neuronal differentiation always precedes photoreceptor differentiation, photoreceptor differentiation occurs prematurely and precedes neuronal differentiation when precursor cells are isolated from the retina at early embryonic stages. Thus, cells born by embryonic day 5 (ED-5) give rise predominantly to photoreceptors when isolated for culture on ED-6 but develop mainly as neurons when isolated on ED-8. This suggests that retinal precursor cells retain after terminal mitosis the capacity to develop either as neurons or as photoreceptors. Moreover, photoreceptor differentiation appears to represent a constitutive or "default" pathway that precursor cells follow in the absence of neuron-inducing signals.     

Ribosome profiling shows that miR-430 reduces translation before causing mRNA decay in zebrafish

MicroRNAs regulate gene expression through deadenylation, repression, and messenger RNA (mRNA) decay. However, the contribution of each mechanism in non-steady-state situations remains unclear. We monitored the impact of miR-430 on ribosome occupancy of endogenous mRNAs in wild-type and dicer mutant zebrafish embryos and found that miR-430 reduces the number of ribosomes on target mRNAs before causing mRNA decay. Translational repression occurs before complete deadenylation, and disrupting deadenylation with use of an internal polyadenylate tail did not block target repression. Lastly, we observed that ribosome density along the length of the message remains constant, suggesting that translational repression occurs by reducing the rate of initiation rather than affecting elongation or causing ribosomal drop-off. These results show that miR-430 regulates translation initiation before inducing mRNA decay during zebrafish development.     

Function of GATA transcription factors in preadipocyte-adipocyte transition

Genes that control the early stages of adipogenesis remain largely unknown. Here, we show that murine GATA-2 and GATA-3 are specifically expressed in white adipocyte precursors and that their down-regulation sets the stage for terminal differentiation. Constitutive GATA-2 and GATA-3 expression suppressed adipocyte differentiation and trapped cells at the preadipocyte stage. This effect is mediated, at least in part, through the direct suppression of peroxisome proliferator-activated receptor gamma. GATA-3-deficient embryonic stem cells exhibit an enhanced capacity to differentiate into adipocytes, and defective GATA-2 and GATA-3 expression is associated with obesity. Thus, GATA-2 and GATA-3 regulate adipocyte differentiation through molecular control of the preadipocyte-adipocyte transition.     

An RNA ligase essential for RNA editing and survival of the bloodstream form of Trypanosoma brucei

RNA editing in trypanosomes occurs by a series of enzymatic steps that are catalyzed by a macromolecular complex. The TbMP52 protein is shown to be a component of this complex, to have RNA ligase activity, and to be one of two adenylatable proteins in the complex. Regulated repression of TbMP52 blocks editing, which shows that it is a functional component of the editing complex. This repression is lethal in bloodforms of the parasite, indicating that editing is essential in the mammalian stage of the life cycle. The editing complex, which is present in all kinetoplastid parasites, may thus be a chemotherapeutic target.     

CCAAT-enhancer binding protein: a component of a differentiation switch

The CCAAT-enhancer binding protein (C/EBP) has now been found to promote the terminal differentiation of adipocytes. During the normal course of adipogenesis, C/EBP expression is restricted to a terminal phase wherein proliferative growth is arrested, and specialized cell phenotype is first manifested. A conditional form of C/EBP was developed, making it feasible to test its capacity to regulate the differentiation of cultured adipocytes. Premature expression of C/EBP in adipoblasts caused a direct cessation of mitotic growth. Moreover, when abetted by the effects of three adipogenic hormones, C/EBP promoted terminal cell differentiation. Since C/EBP is expressed in a variety of tissues, it may have a fundamental role in regulating the balance between cell growth and differentiation in higher animals.     

Hairpin RNAs and retrotransposon LTRs effect RNAi and chromatin-based gene silencing

The expression of short hairpin RNAs in several organisms silences gene expression by targeted mRNA degradation. This RNA interference (RNAi) pathway can also affect the genome, as DNA methylation arises at loci homologous to the target RNA in plants. We demonstrate in fission yeast that expression of a synthetic hairpin RNA is sufficient to silence the homologous locus in trans and causes the assembly of a patch of silent Swi6 chromatin with cohesin. This requires components of the RNAi machinery and Clr4 histone methyltransferase for small interfering RNA generation. A similar process represses several meiotic genes through nearby retrotransposon long terminal repeats (LTRs). These analyses directly implicate interspersed LTRs in regulating gene expression during cellular differentiation.