Latest Articles Include:
- Broken Silence Restored—Remodeling Primes for Deacetylation at Replication Forks
- mol cell 42(3):267-269 (2011)
Faithful propagation of chromatin structures requires assimilation of new histones to the modification profile of individual loci. In this issue of Molecular Cell, Rowbotham and colleagues identify a remodeler, SMARCAD1, acting at replication sites to facilitate histone deacetylation and restoration of silencing.
- A Web of Interactions at the Ends
- mol cell 42(3):269-271 (2011)
The synthesis of telomeric DNA by telomerase entails repeated cycles of reverse transcription on a short RNA template. In this issue of Molecular Cell, Robart and Collins (2011) describe a set of interactions between human telomerase RNA, protein domains, and the substrate DNA that drives the intricate reaction cycle.
- The Search for Nonconventional Mitochondrial Determinants of Aging
- mol cell 42(3):271-273 (2011)
Mitochondria are conventionally believed to modulate aging by affecting free-radical production and the energy supply. In this issue of Molecular Cell, Caballero et al. (2011) reveal that altering protein complexes involved in mitochondrial translation control extends life span independent of redox homeostasis and oxidative phosphorylation.
- When Signaling Kinases Meet Histones and Histone Modifiers in the Nucleus
- mol cell 42(3):274-284 (2011)
Signaling pathways involve cascades of protein phosphorylation and ultimately affect regulation of transcription in the nucleus. However, most of the kinases in these pathways have not been generally considered to directly modulate transcription thus far. Here, recent significant progress in the field elucidating direct modifications of histones and histone modifiers by upstream kinases is summarized, and future directions are discussed.
- Maintenance of Silent Chromatin through Replication Requires SWI/SNF-like Chromatin Remodeler SMARCAD1
- mol cell 42(3):285-296 (2011)
Epigenetic marks such as posttranslational histone modifications specify the functional states of underlying DNA sequences, though how they are maintained after their disruption during DNA replication remains a critical question. We identify the mammalian SWI/SNF-like protein SMARCAD1 as a key factor required for the re-establishment of repressive chromatin. The ATPase activity of SMARCAD1 is necessary for global deacetylation of histones H3/H4. In this way, SMARCAD1 promotes methylation of H3K9, the establishment of heterochromatin, and faithful chromosome segregation. SMARCAD1 associates with transcriptional repressors including KAP1, histone deacetylases HDAC1/2 and the histone methyltransferase G9a/GLP and modulates the interaction of HDAC1 and KAP1 with heterochromatin. SMARCAD1 directly interacts with PCNA, a central component of the replication machinery, and is recruited to sites of DNA replication. Our findings suggest that chromatin remodeling by SMARCAD1 ens! ures that silenced loci, such as pericentric heterochromatin, are correctly perpetuated.
- Processive and Distributive Extension of Human Telomeres by Telomerase under Homeostatic and Nonequilibrium Conditions
- mol cell 42(3):297-307 (2011)
Specific information about how telomerase acts in vivo is necessary for understanding telomere dynamics in human tumor cells. Our results imply that, under homeostatic telomere length-maintenance conditions, only one molecule of telomerase acts at each telomere during every cell division and processively adds 60 nt to each end. In contrast, multiple molecules of telomerase act at each telomere when telomeres are elongating (nonequilibrium conditions). Telomerase extension is less processive during the first few weeks following the reversal of long-term treatment with the telomerase inhibitor Imetelstat (GRN163L), a time when Cajal bodies fail to deliver telomerase RNA to telomeres. This result implies that processing of telomerase by Cajal bodies may affect its processivity. Overexpressed telomerase is also less processive than the endogenously expressed telomerase. These findings reveal two major distinct extension modes adopted by telomerase in vivo.
- Human Telomerase Domain Interactions Capture DNA for TEN Domain-Dependent Processive Elongation
- mol cell 42(3):308-318 (2011)
Eukaryotic chromosome maintenance requires telomeric repeat synthesis by telomerase. It remains uncertain how telomerase domains interact to organize the active RNP and how this architecture establishes the specificity of the catalytic cycle. We combine human telomerase reconstitutions in vivo, affinity purifications, and discriminating activity assays to uncover a network of protein-protein and protein-RNA domain interactions. Notably, we find that complete single-repeat synthesis requires only a telomerase reverse transcriptase (TERT) core. Single-repeat synthesis does not require the TERT N-terminal (TEN) domain, but RNA-dependent positioning of the TEN domain captures substrate and allows repeat synthesis processivity. A TEN domain physically separate from the TERT core can capture even a minimal template-paired DNA substrate, with substrate association enhanced by the presence of a 5′ single-stranded extension. Our results provide insights into active enzyme arc! hitecture, explain biological variations of the catalytic cycle, and predict altered activities for TERT proteins of some eukaryotes.
- Regulation of DNA End Joining, Resection, and Immunoglobulin Class Switch Recombination by 53BP1
- mol cell 42(3):319-329 (2011)
53BP1 is a DNA damage protein that forms phosphorylated H2AX (γ-H2AX) dependent foci in a 1 Mb region surrounding DNA double-strand breaks (DSBs). In addition, 53BP1 promotes genomic stability by regulating the metabolism of DNA ends. We have compared the joining rates of paired DSBs separated by 1.2 kb to 27 Mb on chromosome 12 in the presence or absence of 53BP1. 53BP1 facilitates joining of intrachromosomal DSBs but only at distances corresponding to γ-H2AX spreading. In contrast, DNA end protection by 53BP1 is distance independent. Furthermore, analysis of 53BP1 mutants shows that chromatin association, oligomerization, and N-terminal ATM phosphorylation are all required for DNA end protection and joining as measured by immunoglobulin class switch recombination. These data elucidate the molecular events that are required for 53BP1 to maintain genomic stability and point to a model wherein 53BP1 and H2AX cooperate to repress resection of DSBs.
- Histone Methylation by PRC2 Is Inhibited by Active Chromatin Marks
- mol cell 42(3):330-341 (2011)
The Polycomb repressive complex 2 (PRC2) confers transcriptional repression through histone H3 lysine 27 trimethylation (H3K27me3). Here, we examined how PRC2 is modulated by histone modifications associated with transcriptionally active chromatin. We provide the molecular basis of histone H3 N terminus recognition by the PRC2 Nurf55-Su(z)12 submodule. Binding of H3 is lost if lysine 4 in H3 is trimethylated. We find that H3K4me3 inhibits PRC2 activity in an allosteric fashion assisted by the Su(z)12 C terminus. In addition to H3K4me3, PRC2 is inhibited by H3K36me2/3 (i.e., both H3K36me2 and H3K36me3). Direct PRC2 inhibition by H3K4me3 and H3K36me2/3 active marks is conserved in humans, mouse, and fly, rendering transcriptionally active chromatin refractory to PRC2 H3K27 trimethylation. While inhibition is present in plant PRC2, it can be modulated through exchange of the Su(z)12 subunit. Inhibition by active chromatin marks, coupled to stimulation by transcriptionally! repressive H3K27me3, enables PRC2 to autonomously template repressive H3K27me3 without overwriting active chromatin domains.
- Direct Interactions of OCA-B and TFII-I Regulate Immunoglobulin Heavy-Chain Gene Transcription by Facilitating Enhancer-Promoter Communication
- mol cell 42(3):342-355 (2011)
B cell-specific coactivator OCA-B, together with Oct-1/2, binds to octamer sites in promoters and enhancers to activate transcription of immunoglobulin (Ig) genes, although the mechanisms underlying their roles in enhancer-promoter communication are unknown. Here, we demonstrate a direct interaction of OCA-B with transcription factor TFII-I, which binds to DICE elements in Igh promoters, that affects transcription at two levels. First, OCA-B relieves HDAC3-mediated Igh promoter repression by competing with HDAC3 for binding to promoter-bound TFII-I. Second, and most importantly, Igh 3′ enhancer-bound OCA-B and promoter-bound TFII-I mediate promoter-enhancer interactions, in both cis and trans, that are important for Igh transcription. These and other results reveal an important function for OCA-B in Igh 3′ enhancer function in vivo and strongly favor an enhancer mechanism involving looping and facilitated factor recruitment rather than a tracking mechanism.
- Arabidopsis Argonaute 2 Regulates Innate Immunity via miRNA393*-Mediated Silencing of a Golgi-Localized SNARE Gene, MEMB12
- mol cell 42(3):356-366 (2011)
Argonaute (AGO) proteins are critical components of RNA silencing pathways that bind small RNAs and mediate gene silencing at their target sites. We found that Arabidopsis AGO2 is highly induced by the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). Further genetic analysis demonstrated that AGO2 functions in antibacterial immunity. One abundant species of AGO2-bound small RNA is miR393b*, which targets a Golgi-localized SNARE gene, MEMB12. Pst infection downregulates MEMB12 in a miR393b*-dependent manner. Loss of function of MEMB12, but not SYP61, another intracellular SNARE, leads to increased exocytosis of an antimicrobial pathogenesis-related protein, PR1. Overexpression of miR393b* resembles memb12 mutant in resistance responses. Thus, AGO2 functions in antibacterial immunity by binding miR393b* to modulate exocytosis of antimicrobial PR proteins via MEMB12. Since miR393 also contributes to antibacterial responses, miR393*/miR393 represent an example of ! a miRNA*/miRNA pair that functions in immunity through two distinct AGOs: miR393* through AGO2 and miR393 through AGO1.
- Single-Molecule Fluorescence Measurements of Ribosomal Translocation Dynamics
- mol cell 42(3):367-377 (2011)
We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3- and Cy5-labeled tRNAs. Pretranslocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-GGTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the posttranslocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNAEF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability.
- Cdk1-Dependent Destruction of Eco1 Prevents Cohesion Establishment after S Phase
- mol cell 42(3):378-389 (2011)
Accurate genome segregation depends on cohesion mechanisms that link duplicated sister chromatids, thereby allowing their tension-dependent biorientation in metaphase. In Saccharomyces cerevisiae, cohesion is established during DNA replication when Eco1 acetylates the cohesin subunit Smc3. Cohesion establishment is restricted to S phase of the cell cycle, but the molecular basis of this regulation is unknown. Here, we show that Eco1 is negatively regulated by the protein kinase Cdk1. Phosphorylation of Eco1 after S phase targets it to SCFCdc4 for ubiquitination and subsequent degradation. A nonphosphorylatable mutant of Eco1 establishes cohesion after DNA replication, suggesting that Cdk1-dependent phosphorylation of Eco1 is a key factor limiting establishment to S phase. We also show that deregulation of Eco1 results in chromosome separation defects in anaphase. We conclude that this regulatory mechanism helps optimize the level of sister chromatid cohesion, ensuring ! a robust and efficient anaphase.
- Absence of Mitochondrial Translation Control Proteins Extends Life Span by Activating Sirtuin-Dependent Silencing
- mol cell 42(3):390-400 (2011)
Altered mitochondrial functionality can extend organism life span, but the underlying mechanisms are obscure. Here we report that inactivating SOV1, a member of the yeast mitochondrial translation control (MTC) module, causes a robust Sir2-dependent extension of replicative life span in the absence of respiration and without affecting oxidative damage. We found that SOV1 interacts genetically with the cAMP-PKA pathway and the chromatin remodeling apparatus. Consistently, Sov1p-deficient cells displayed reduced cAMP-PKA signaling and an elevated, Sir2p-dependent, genomic silencing. Both increased silencing and life span extension in sov1Δ cells require the PKA/Msn2/4p target Pnc1p, which scavenges nicotinamide, a Sir2p inhibitor. Inactivating other members of the MTC module also resulted in Sir2p-dependent life span extension. The data demonstrate that the nuclear silencing apparatus senses and responds to the absence of MTC proteins and that this response converges wi! th a pathway for life span extension elicited by reducing TOR signaling.
- Cost of Unneeded Proteins in E. coli Is Reduced after Several Generations in Exponential Growth
- mol cell 42(3):401 (2011)