Hot off the presses! Nov 12 mol cell

The Nov 12 issue of the mol cell is now up on Pubget (About mol cell): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• SETting the Clock for Histone H4 Monomethylation
  - mol cell 40(3):345-346 (2010)
  In this and a previous issue of Molecular Cell, (Oda et al., 2010) and (Abbas et al., 2010), and Centore et al. (2010) determined that the H4K20 histone methyltransferase PR-Set7/Set8 is posttranslationally regulated by the PCNA-dependent CRL4Cdt2 ubiquitin ligase.
• Achieving Fidelity in Homologous Recombination Despite Extreme Complexity: Informed Decisions by Molecular Profiling
  - mol cell 40(3):347-348 (2010)
  In this issue of Molecular Cell, Savir and Tlusty (2010) apply signal detection theory to show that homologous recombination machinery is optimally tuned to find homologous DNA sequences within an exceptionally high background of heterologous sequences.
• Alternate Endings: A New Story for mRNA Decapping
  - mol cell 40(3):349-350 (2010)
  With most of the important players identified, the process of decapping is thought, for the most part, to be well understood. In this issue of Molecular Cell, Song et al. (2010) challenge this notion with the identification of a previously uncharacterized mRNA decapping enzyme.
• A PSHaver for Centromeric Histones
  - mol cell 40(3):351-352 (2010)
  In this issue of Molecular Cell, Hewawasam et al. (2010) and Ranjitkar et al. (2010) identify and characterize Psh1, an E3 ubiquitin ligase that specifically targets the centromeric histone Cse4 in budding yeast and limits its misincorporation at noncentromeric regions.
• Mec1 Is One of Multiple Kinases that Prime the Mcm2-7 Helicase for Phosphorylation by Cdc7
  - mol cell 40(3):353-363 (2010)
  Activation of the eukaryotic replicative DNA helicase, the Mcm2-7 complex, requires phosphorylation by Cdc7/Dbf4 (Dbf4-dependent kinase or DDK), which, in turn, depends on prior phosphorylation of Mcm2-7 by an unknown kinase (or kinases). We identified DDK phosphorylation sites on Mcm4 and Mcm6 and found that phosphorylation of either subunit suffices for cell proliferation. Importantly, prior phosphorylation of either S/T-P or S/T-Q motifs on these subunits is required for DDK phosphorylation of Mcm2-7 and for normal S phase passage. Phosphomimetic mutations of DDK target sites bypass both DDK function and mutation of the priming phosphorylation sites. Mrc1 facilitates Mec1 phosphorylation of the S/T-Q motifs of chromatin-bound Mcm2-7 during S phase to activate replication. Genetic interactions between priming site mutations and MRC1 or TOF1 deletion support a role for these modifications in replication fork stability. These findings identify regulatory mechanisms tha! t modulate origin firing and replication fork assembly during cell cycle progression.
• Regulation of the Histone H4 Monomethylase PR-Set7 by CRL4Cdt2-Mediated PCNA-Dependent Degradation during DNA Damage
  - mol cell 40(3):364-376 (2010)
  The histone methyltransferase PR-Set7/Set8 is the sole enzyme that catalyzes monomethylation of histone H4 at K20 (H4K20me1). Previous reports document disparate evidence regarding PR-Set7 expression during the cell cycle, the biological relevance of PR-Set7 interaction with PCNA, and its role in the cell. We find that PR-Set7 is indeed undetectable during S phase and instead is detected during late G2, mitosis, and early G1. PR-Set7 is transiently recruited to laser-induced DNA damage sites through its interaction with PCNA, after which 53BP1 is recruited dependent on PR-Set7 catalytic activity. During the DNA damage response, PR-Set7 interaction with PCNA through a specialized "PIP degron" domain targets it for PCNA-coupled CRL4Cdt2-dependent proteolysis. PR-Set7 mutant in its "PIP degron" is now detectable during S phase, during which the mutant protein accumulates. Outside the chromatin context, Skp2 promotes PR-Set7 degradation as well. These findings demo! nstrate a stringent spatiotemporal control of PR-Set7 that is essential for preserving the genomic integrity of mammalian cells.
• A Shld1-Controlled POT1a Provides Support for Repression of ATR Signaling at Telomeres through RPA Exclusion
  - mol cell 40(3):377-387 (2010)
  We previously proposed that POT1 prevents ATR signaling at telomeres by excluding RPA from the single-stranded TTAGGG repeats. Here, we use a Shld1-stabilized degron-POT1a fusion (DD-POT1a) to study the telomeric ATR kinase response. In the absence of Shld1, DD-POT1a degradation resulted in rapid and reversible activation of the ATR pathway in G1 and S/G2. ATR signaling was abrogated by shRNAs to ATR and TopBP1, but shRNAs to the ATM kinase or DNA-PKcs did not affect the telomere damage response. Importantly, ATR signaling in G1 and S/G2 was reduced by shRNAs to RPA. In S/G2, RPA was readily detectable at dysfunctional telomeres, and both POT1a and POT1b were required to exclude RPA and prevent ATR activation. In G1, the accumulation of RPA at dysfunctional telomeres was strikingly less, and POT1a was sufficient to repress ATR signaling. These results support an RPA exclusion model for the repression of ATR signaling at telomeres.
• RecA-Mediated Homology Search as a Nearly Optimal Signal Detection System
  - mol cell 40(3):388-396 (2010)
  Homologous recombination facilitates the exchange of genetic material between homologous DNA molecules. This crucial process requires detecting a specific homologous DNA sequence within a huge variety of heterologous sequences. The detection is mediated by RecA in E. coli, or members of its superfamily in other organisms. Here, we examine how well the RecA-DNA interaction is adjusted to its task. By formulating the DNA recognition process as a signal detection problem, we find the optimal value of binding energy that maximizes the ability to detect homologous sequences. We show that the experimentally observed binding energy is nearly optimal. This implies that the RecA-induced deformation and the binding energetics are fine-tuned to ensure optimal sequence detection. Our analysis suggests a possible role for DNA extension by RecA, in which deformation enhances detection. The present signal detection approach provides a general recipe for testing the optimality of othe! r molecular recognition systems.
• Analysis of Active and Inactive X Chromosome Architecture Reveals the Independent Organization of 30 nm and Large-Scale Chromatin Structures
  - mol cell 40(3):397-409 (2010)
  Using a genetic model, we present a high-resolution chromatin fiber analysis of transcriptionally active (Xa) and inactive (Xi) X chromosomes packaged into euchromatin and facultative heterochromatin. Our results show that gene promoters have an open chromatin structure that is enhanced upon transcriptional activation but the Xa and the Xi have similar overall 30 nm chromatin fiber structures. Therefore, the formation of facultative heterochromatin is dependent on factors that act at a level above the 30 nm fiber and transcription does not alter bulk chromatin fiber structures. However, large-scale chromatin structures on Xa are decondensed compared with the Xi and transcription inhibition is sufficient to promote large-scale chromatin compaction. We show a link between transcription and large-scale chromatin packaging independent of the bulk 30 nm chromatin fiber and propose that transcription, not the global compaction of 30 nm chromatin fibers, determines the cytolo! gical appearance of large-scale chromatin structures.
• Crosstalk between mRNA 3′ End Processing and Transcription Initiation
  - mol cell 40(3):410-422 (2010)
  Transcription and mRNA maturation are interdependent events. Although stimulatory connections between these processes within the same round of transcription are well described, functional coupling between separate transcription cycles remains elusive. Comparing time-resolved transcription profiles of single-copy integrated β-globin gene variants, we demonstrate that a polyadenylation site mutation decreases transcription initiation of the same gene. Upon depletion of the 3′ end processing and transcription termination factor PCF11, endogenous genes exhibit a similar phenotype. Readthrough RNA polymerase II (RNAPII) engaged on polyadenylation site-mutated transcription units sequester the transcription initiation/elongation factors TBP, TFIIB and CDK9, leading to their depletion at the promoter. Additionally, high levels of TBP and TFIIB appear inside the gene body, and Ser2-phosphorylated RNAPII accumulates at the promoter. Our data demonstrate that 3′ end formati! on stimulates transcription initiation and suggest that coordinated recycling of factors from a gene terminator back to the promoter is essential for sustaining continued transcription.
• Multiple mRNA Decapping Enzymes in Mammalian Cells
  - mol cell 40(3):423-432 (2010)
  Regulation of RNA degradation plays an important role in the control of gene expression. One mechanism of eukaryotic mRNA decay proceeds through an initial deadenylation followed by 5′ end decapping and exonucleolytic decay. Dcp2 is currently believed to be the only cytoplasmic decapping enzyme responsible for decapping of all mRNAs. Here we report that Dcp2 protein modestly contributes to bulk mRNA decay and surprisingly is not detectable in a subset of mouse and human tissues. Consistent with these findings, a hypomorphic knockout of Dcp2 had no adverse consequences in mice. In contrast, the previously reported Xenopus nucleolar decapping enzyme, Nudt16, is an ubiquitous cytoplasmic decapping enzyme in mammalian cells. Like Dcp2, Nudt16 also regulates the stability of a subset of mRNAs including a member of the motin family of proteins involved in angiogenesis, Angiomotin-like 2. These data demonstrate mammalian cells possess multiple mRNA decapping enzymes, includ! ing Nudt16 to regulate mRNA turnover.
• WD40 Repeat Propellers Define a Ubiquitin-Binding Domain that Regulates Turnover of F Box Proteins
  - mol cell 40(3):433-443 (2010)
  WD40-repeat β-propellers are found in a wide range of proteins involved in distinct biological activities. We define a large subset of WD40 β-propellers as a class of ubiquitin-binding domains. Using the β-propeller from Doa1/Ufd3 as a paradigm, we find the conserved top surface of the Doa1 β-propeller binds the hydrophobic patch of ubiquitin centered on residues I44, L8, and V70. Mutations that disrupt ubiquitin binding abrogate Doa1 function, demonstrating the importance of this interaction. We further demonstrate that WD40 β-propellers from a functionally diverse set of proteins bind ubiquitin in a similar fashion. This set includes members of the F box family of SCF ubiquitin E3 ligase adaptors. Using mutants defective in binding, we find that ubiquitin interaction by the F box protein Cdc4 promotes its autoubiquitination and turnover. Collectively, our results reveal a molecular mechanism that may account for how ubiquitin controls a broad spectrum of cellula! r activities.
• Psh1 Is an E3 Ubiquitin Ligase that Targets the Centromeric Histone Variant Cse4
  - mol cell 40(3):444-454 (2010)
  Cse4 is a variant of histone H3 that is incorporated into a single nucleosome at each centromere in budding yeast. We have discovered an E3 ubiquitin ligase, called Psh1, which controls the cellular level of Cse4 via ubiquitylation and proteolysis. The activity of Psh1 is dependent on both its RING and zinc finger domains. We demonstrate the specificity of the ubiquitylation activity of Psh1 toward Cse4 in vitro and map the sites of ubiquitylation. Mutation of key lysines prevents ubiquitylation of Cse4 by Psh1 in vitro and stabilizes Cse4 in vivo. While deletion of Psh1 stabilizes Cse4, elimination of the Cse4-specific chaperone Scm3 destabilizes Cse4, and the addition of Scm3 to the Psh1-Cse4 ubiquitylation reaction prevents Cse4 ubiquitylation, together suggesting Scm3 may protect Cse4 from ubiquitylation. Without Psh1, Cse4 overexpression is toxic and Cse4 is found at ectopic locations. Our results suggest Psh1 functions to prevent the mislocalization of Cse4.
• An E3 Ubiquitin Ligase Prevents Ectopic Localization of the Centromeric Histone H3 Variant via the Centromere Targeting Domain
  - mol cell 40(3):455-464 (2010)
  Proper centromere function is critical to maintain genomic stability and to prevent aneuploidy, a hallmark of tumors and birth defects. A conserved feature of all eukaryotic centromeres is an essential histone H3 variant called CENP-A that requires a centromere targeting domain (CATD) for its localization. Although proteolysis prevents CENP-A from mislocalizing to euchromatin, regulatory factors have not been identified. Here, we identify an E3 ubiquitin ligase called Psh1 that leads to the degradation of Cse4, the budding yeast CENP-A homolog. Cse4 overexpression is toxic to psh1Δ cells and results in euchromatic localization. Strikingly, the Cse4 CATD is a key regulator of its stability and helps Psh1 discriminate Cse4 from histone H3. Taken together, we propose that the CATD has a previously unknown role in maintaining the exclusive localization of Cse4 by preventing its mislocalization to euchromatin via Psh1-mediated degradation.
• A Stress-Responsive System for Mitochondrial Protein Degradation
  - mol cell 40(3):465-480 (2010)
  We show that Ydr049 (renamed VCP/Cdc48-associated mitochondrial stress-responsive—Vms1), a member of an unstudied pan-eukaryotic protein family, translocates from the cytosol to mitochondria upon mitochondrial stress. Cells lacking Vms1 show progressive mitochondrial failure, hypersensitivity to oxidative stress, and decreased chronological life span. Both yeast and mammalian Vms1 stably interact with Cdc48/VCP/p97, a component of the ubiquitin/proteasome system with a well-defined role in endoplasmic reticulum-associated protein degradation (ERAD), wherein misfolded ER proteins are degraded in the cytosol. We show that oxidative stress triggers mitochondrial localization of Cdc48 and this is dependent on Vms1. When this system is impaired by mutation of Vms1, ubiquitin-dependent mitochondrial protein degradation, mitochondrial respiratory function, and cell viability are compromised. We demonstrate that Vms1 is a required component of an evolutionarily conserved sys! tem for mitochondrial protein degradation, which is necessary to maintain mitochondrial, cellular, and organismal viability.
• BH3-Triggered Structural Reorganization Drives the Activation of Proapoptotic BAX
  - mol cell 40(3):481-492 (2010)
  BAX is a proapoptotic BCL-2 family member that lies dormant in the cytosol until converted into a killer protein in response to cellular stress. Having recently identified the elusive trigger site for direct BAX activation, we now delineate by NMR and biochemical methods the essential allosteric conformational changes that transform ligand-triggered BAX into a fully activated monomer capable of propagating its own activation. Upon BAX engagement by a triggering BH3 helix, the unstructured loop between α helices 1 and 2 is displaced, the carboxy-terminal helix 9 is mobilized for membrane translocation, and the exposed BAX BH3 domain propagates the death signal through an autoactivating interaction with the trigger site of inactive BAX monomers. Our structure-activity analysis of this seminal apoptotic process reveals pharmacologic opportunities to modulate cell death by interceding at key steps of the BAX activation pathway.
• Phosphoinositides Are Essential Coactivators for p21-Activated Kinase 1
  - mol cell 40(3):493-500 (2010)
  Phospholipid-enriched membranes such as the plasma membrane can serve as direct regulators of kinase signaling. Pak1 is involved in growth factor signaling at the plasma membrane, and its dysregulation is implicated in cancer. Pak1 adopts an autoinhibited conformation that is relieved upon binding to membrane-bound Rho GTPases Rac1 or Cdc42, but whether lipids also regulate Pak1 in vivo is unknown. We show here that phosphoinositides, particularly PIP2, potentiate Rho-GTPase-mediated Pak1 activity. A positively charged region of Pak1 binds to phosphoinositide-containing membranes, and this interaction is essential for membrane recruitment and activation of Pak1 in response to extracellular signals. Our results highlight an active role for lipids as allosteric regulators of Pak1 and suggest that Pak1 is a "coincidence detector" whose activation depends on GTPases present in phosphoinositide-rich membranes. These findings expand the role of phosphoinositides in kinas! e signaling and suggest how altered phosphoinositide metabolism may upregulate Pak1 activity in cancer cells.