Diverse classes of RNA, ranging from small to long non-coding RNAs, have emerged as key regulators of gene expression, genome stability and defence against foreign genetic elements. Small RNAs modify ...chromatin structure and silence transcription by guiding Argonaute-containing complexes to complementary nascent RNA scaffolds and then mediating the recruitment of histone and DNA methyltransferases. In addition, recent advances suggest that chromatin-associated long non-coding RNA scaffolds also recruit chromatin-modifying complexes independently of small RNAs. These co-transcriptional silencing mechanisms form powerful RNA surveillance systems that detect and silence inappropriate transcription events, and provide a memory of these events via self-reinforcing epigenetic loops.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, UILJ, UKNU, UL, UM, UPUK
Epigenetic inheritance of gene expression states enables a single genome to maintain distinct cellular identities. How histone modifications contribute to this process remains unclear. Using global ...chromatin perturbations and local, time-controlled modulation of transcription, we establish the existence of epigenetic memory of transcriptional activation for genes that can be silenced by the Polycomb group. This property emerges during cell differentiation and allows genes to be stably switched after a transient transcriptional stimulus. This transcriptional memory state at Polycomb targets operates in cis; however, rather than relying solely on read-and-write propagation of histone modifications, the memory is also linked to the strength of activating inputs opposing Polycomb proteins, and therefore varies with the cellular context. Our data and computational simulations suggest a model whereby transcriptional memory arises from double-negative feedback between Polycomb-mediated silencing and active transcription. Transcriptional memory at Polycomb targets thus depends not only on histone modifications but also on the gene-regulatory network and underlying identity of a cell.
In animals and plants, the H3K9me3 and H3K27me3 chromatin silencing marks are deposited by different protein machineries. H3K9me3 is catalyzed by the SET-domain SU(VAR)3-9 enzymes, while H3K27me3 is ...catalyzed by the SET-domain Enhancer-of-zeste enzymes, which are the catalytic subunits of Polycomb Repressive Complex 2 (PRC2). Here, we show that the Enhancer-of-zeste-like protein Ezl1 from the unicellular eukaryote Paramecium tetraurelia, which exhibits significant sequence and structural similarities with human EZH2, catalyzes methylation of histone H3 in vitro and in vivo with an apparent specificity toward K9 and K27. We find that H3K9me3 and H3K27me3 co-occur at multiple families of transposable elements in an Ezl1-dependent manner. We demonstrate that loss of these histone marks results in global transcriptional hyperactivation of transposable elements with modest effects on protein-coding gene expression. Our study suggests that although often considered functionally distinct, H3K9me3 and H3K27me3 may share a common evolutionary history as well as a common ancestral role in silencing transposable elements.
The polycomb repressive complex 2 (PRC2) consists of core subunits SUZ12, EED, RBBP4/7, and EZH1/2 and is responsible for mono-, di-, and tri-methylation of lysine 27 on histone H3. Whereas two ...distinct forms exist, PRC2.1 (containing one polycomb-like protein) and PRC2.2 (containing AEBP2 and JARID2), little is known about their differential functions. Here, we report the discovery of a family of vertebrate-specific PRC2.1 proteins, “PRC2 associated LCOR isoform 1” (PALI1) and PALI2, encoded by the LCOR and LCORL gene loci, respectively. PALI1 promotes PRC2 methyltransferase activity in vitro and in vivo and is essential for mouse development. Pali1 and Aebp2 define mutually exclusive, antagonistic PRC2 subtypes that exhibit divergent H3K27-tri-methylation activities. The balance of these PRC2.1/PRC2.2 activities is required for the appropriate regulation of polycomb target genes during differentiation. PALI1/2 potentially link polycombs with transcriptional co-repressors in the regulation of cellular identity during development and in cancer.
Display omitted
•Pali1 and Pali2 form a new family of vertebrate-specific proteins that bind PRC2•Pali1 promotes PRC2 methyltransferase activity in vitro and in vivo•Pali1 defines a distinct PRC2.1 subtype essential for mouse development•PRC2 subtype balance is essential for proper regulation of Polycomb target genes
Pali1 and Pali2 form a new family of vertebrate-specific proteins that bind PRC2. Conway et al. show that Pali1 promotes PRC2 methyltransferase activity and defines a distinct PRC2.1 subtype essential for mouse development. They also establish that the balance of PRC2.1 and PRC2.2 activities is essential for proper regulation of polycomb target genes.
Distinct classes of small RNAs, 20–32 nucleotides long, play important regulatory roles for diverse cellular processes. It is therefore important to identify and quantify small RNAs as a function of ...development, tissue and cell type, in normal and disease states. Here we describe methods to prepare cDNA libraries from pools of small RNAs isolated from organisms, tissues or cells. These methods enable the identification of new members or new classes of small RNAs, and they are also suitable to obtain miRNA expression profiles based on clone count frequencies. This protocol includes the use of new deep sequencing methods (454/Roche and Solexa) to facilitate the characterization of diverse sequence pools of small RNAs.
Sequencing of small RNA cDNA libraries is an important tool for the discovery of new RNAs and the analysis of their mutational status as well as expression changes across samples. It requires ...multiple enzyme-catalyzed steps, including sequential oligonucleotide adapter ligations to the 3' and 5' ends of the small RNAs, reverse transcription (RT), and PCR. We assessed biases in representation of miRNAs relative to their input concentration, using a pool of 770 synthetic miRNAs and 45 calibrator oligoribonucleotides, and tested the influence of Rnl1 and two variants of Rnl2, Rnl2(1-249) and Rnl2(1-249)K227Q, for 3'-adapter ligation. The use of the Rnl2 variants for adapter ligations yielded substantially fewer side products compared with Rnl1; however, the benefits of using Rnl2 remained largely obscured by additional biases in the 5'-adapter ligation step; RT and PCR steps did not have a significant impact on read frequencies. Intramolecular secondary structures of miRNA and/or miRNA/3'-adapter products contributed to these biases, which were highly reproducible under defined experimental conditions. We used the synthetic miRNA cocktail to derive correction factors for approximation of the absolute levels of individual miRNAs in biological samples. Finally, we evaluated the influence of 5'-terminal 5-nt barcode extensions for a set of 20 barcoded 3' adapters and observed similar biases in miRNA read distribution, thereby enabling cost-saving multiplex analysis for large-scale miRNA profiling.
Meiotic crossover formation requires the stabilization of early recombination intermediates by a set of proteins and occurs within the environment of the chromosome axis, a structure important for ...the regulation of meiotic recombination events. The molecular mechanisms underlying and connecting crossover recombination and axis localization are elusive. Here, we identified the ZZS (Zip2-Zip4-Spo16) complex, required for crossover formation, which carries two distinct activities: one provided by Zip4, which acts as hub through physical interactions with components of the chromosome axis and the crossover machinery, and the other carried by Zip2 and Spo16, which preferentially bind branched DNA molecules in vitro. We found that Zip2 and Spo16 share structural similarities to the structure-specific XPF-ERCC1 nuclease, although it lacks endonuclease activity. The XPF domain of Zip2 is required for crossover formation, suggesting that, together with Spo16, it has a noncatalytic DNA recognition function. Our results suggest that the ZZS complex shepherds recombination intermediates toward crossovers as a dynamic structural module that connects recombination events to the chromosome axis. The identification of the ZZS complex improves our understanding of the various activities required for crossover implementation and is likely applicable to other organisms, including mammals.
Mutations in chromatin regulators are widespread in cancer. Among them, the histone H3 lysine 27 methyltransferase Polycomb Repressive Complex 2 (PRC2) shows distinct alterations according to tumor ...type. This specificity is poorly understood. Here, we model several PRC2 alterations in one isogenic system to reveal their comparative effects. Focusing then on lymphoma-associated EZH2 mutations, we show that Ezh2
induces aberrant H3K27 methylation patterns even without wild-type Ezh2, which are alleviated by partial PRC2 inhibition. Remarkably, Ezh2
rewires the response to PRC2 inhibition, leading to induction of antigen presentation genes. Using a unique longitudinal follicular lymphoma cohort, we further link EZH2 status to abnormal H3K27 methylation. We also uncover unexpected variability in the mutational landscape of successive biopsies, pointing to frequent co-existence of different clones and cautioning against stratifying patients based on single sampling. Our results clarify how oncogenic PRC2 mutations disrupt chromatin and transcription, and the therapeutic vulnerabilities this creates.
Polycomb repressive complex 2 (PRC2) and its histone H3 lysine-27 methylation activity are crucial for multicellular development by virtue of their role in maintaining transcriptional repression ...patterns. The recruitment and enzymatic activity of PRC2 are controlled by a series of intricate mechanisms whose molecular details have been emerging at a rapid pace. Recent studies have uncovered intriguing modes of PRC2 regulation by facultative PRC2 subunits, PRC1, and specific features of the chromatin environment. Together, these findings have produced a rich and fast-evolving picture of the biochemical signals that govern PRC2 function, with many exciting questions still remaining.
PRC2 catalyzes trimethylation of histone H3 on lysine 27 (H3K27me3), a histone mark necessary for maintaining transcriptional repression during multicellular development.
Cell type-specific patterns of H3K27me3 are crucial for preserving cell identity and reflect an elaborate interplay of mechanisms controlling the recruitment and enzymatic activity of PRC2.
PRC2 can associate with different combinations of facultative subunits, whose contributions to the genomic targeting and catalytic regulation of PRC2 are rapidly being elucidated.
PRC1, a functional partner of PRC2, plays a major role in guiding PRC2 recruitment via monoubiquitination of histone H2A on lysine 119.
Transcription-coupled chromatin modifications, DNA methylation, and nascent RNA influence the localization and activity of PRC2.
RNAi in
Schizosaccharomyces pombe
is critical for centromeric heterochromatin formation. It has remained unclear, however, whether RNAi also regulates the expression of protein-coding loci. In the ...April 1, 2012, issue of
Genes & Development
, Woolcock and colleagues (pp. 683–667) reported an elegant mechanism for the conditional RNAi-mediated repression of stress response genes involving association with Dcr1 at the nuclear pore. Unexpectedly, the initial targeting of RNAi components to these genes does not require small RNA guides.
PDF
