Hot off the presses! Oct 07 Mol Cell

The Oct 07 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:

• Insulators Organize Chromatin: Emerging Rules of the Game
  - Mol Cell 44(1):1-2 (2011)
  In this issue of Molecular Cell, provide mechanistic insight into the regulation of insulators that helps explain how they can organize chromatin in a cell type-specific fashion.
• Revoking the Cellular License to Replicate: Yet Another AAA Assignment
  - Mol Cell 44(1):3-4 (2011)
  In this issue of Molecular Cell, and establish the AAA ATPase CDC-48/p97 as an essential regulator of eukaryotic DNA replication that coordinates the release of chromatin-bound CDT1 with its degradation by the proteasome.
• A Double Lock on Sister Chromatids by Cohesin
  - Mol Cell 44(1):5-6 (2011)
  In this issue of Molecular Cell, demonstrate that intertwining between sister chromatids at metaphase is much more significant than previously thought and, remarkably, show that it depends on cohesin.
• A New Link in the Chain from Amino Acids to mTORC1 Activation
  - Mol Cell 44(1):7-8 (2011)
  A recent study reveals that the scaffold protein p62 plays a role in linking nutritional cues (amino acids) to the activation of mammalian target of rapamycin complex 1 (mTORC1), a protein kinase that controls cell size and proliferation.
• RIPK-Dependent Necrosis and Its Regulation by Caspases: A Mystery in Five Acts
  - Mol Cell 44(1):9-16 (2011)
  Caspase-8, FADD, and FLIP orchestrate apoptosis in response to death receptor ligation. Mysteriously however, these proteins are also required for normal embryonic development and immune cell proliferation, an observation that has led to their implication in several nonapoptotic processes. While many scenarios have been proposed, recent genetic and biochemical evidence points to unregulated signaling by the receptor-interacting protein kinases-1 (RIPK1) and RIPK3 as the lethal defect in caspase-8-, FADD-, and FLIP-deficient animals and tissues. The RIPKs are known killers, being responsible for a nonapoptotic form of cell death with features similar to necrosis. However, the mechanism by which caspase-8, FADD, and FLIP prevent runaway RIPK activation is unknown, and the signals that trigger these events during development and immune cell activation remain at large. In this review, we will lay out the evidence as it now stands, reinterpreting earlier observations in lig! ht of new clues and considering where the investigation might lead.
• Directional DNA Methylation Changes and Complex Intermediate States Accompany Lineage Specificity in the Adult Hematopoietic Compartment
  - Mol Cell 44(1):17-28 (2011)
  DNA methylation has been implicated as an epigenetic component of mechanisms that stabilize cell-fate decisions. Here, we have characterized the methylomes of human female hematopoietic stem/progenitor cells (HSPCs) and mature cells from the myeloid and lymphoid lineages. Hypomethylated regions (HMRs) associated with lineage-specific genes were often methylated in the opposing lineage. In HSPCs, these sites tended to show intermediate, complex patterns that resolve to uniformity upon differentiation, by increased or decreased methylation. Promoter HMRs shared across diverse cell types typically display a constitutive core that expands and contracts in a lineage-specific manner to fine-tune the expression of associated genes. Many newly identified intergenic HMRs, both constitutive and lineage specific, were enriched for factor binding sites with an implied role in genome organization and regulation of gene expression, respectively. Overall, our studies represent an imp! ortant reference data set and provide insights into directional changes in DNA methylation as cells adopt terminal fates.
• Regulation of Chromatin Organization and Inducible Gene Expression by a Drosophila Insulator
  - Mol Cell 44(1):29-38 (2011)
  Insulators are multiprotein-DNA complexes thought to affect gene expression by mediating inter- and intrachromosomal interactions. Drosophila insulators contain specific DNA-binding proteins plus common components, such as CP190, that facilitate these interactions. Here, we examine changes in the distribution of Drosophila insulator proteins during the heat-shock and ecdysone responses. We find that CP190 recruitment to insulator sites is the main regulatable step in controlling insulator function during heat shock. In contrast, both CP190 and DNA-binding protein recruitment are regulated during the ecdysone response. CP190 is necessary to stabilize specific chromatin loops and for proper activation of transcription of genes regulated by this hormone. These findings suggest that cells may regulate recruitment of insulator proteins to DNA to activate insulator activity at specific sites and create distinct patterns of nuclear organization that are necessary to achieve p! roper gene expression in response to different stimuli.
• HAT4, a Golgi Apparatus-Anchored B-Type Histone Acetyltransferase, Acetylates Free Histone H4 and Facilitates Chromatin Assembly
  - Mol Cell 44(1):39-50 (2011)
  Histone acetyltransferases (HATs) are an essential regulatory component in chromatin biology. Unlike A-type HATs, which are found in the nucleus and utilize nucleosomal histones as substrates and thus primarily function in transcriptional regulation, B-type HATs have been characterized as cytoplasmic enzymes that catalyze the acetylation of free histones. Here, we report on a member of the GCN5-related N-acetyltransferase superfamily and another B-type HAT, HAT4. Interestingly, HAT4 is localized in the Golgi apparatus and displays a substrate preference for lysine residues of free histone H4, including H4K79 and H4K91, that reside in the globular domain of H4. Significantly, HAT4 depletion impaired nucleosome assembly, inhibited cell proliferation, sensitized cells to DNA damage, and induced cell apoptosis. Our data indicate that HAT4 is an important player in the organization and function of the genome and may contribute to the diversity and complexity of higher eukar! yotic organisms.
• Ecdysone- and NO-Mediated Gene Regulation by Competing EcR/Usp and E75A Nuclear Receptors during Drosophila Development
  - Mol Cell 44(1):51-61 (2011)
  The Drosophila ecdysone receptor (EcR/Usp) is thought to activate or repress gene transcription depending on the presence or absence, respectively, of the hormone ecdysone. Unexpectedly, we found an alternative mechanism at work in salivary glands during the ecdysone-dependent transition from larvae to pupae. In the absense of ecdysone, both ecdysone receptor subunits localize to the cytoplasm, and the heme-binding nuclear receptor E75A replaces EcR/Usp at common target sequences in several genes. During the larval-pupal transition, a switch from gene activation by EcR/Usp to gene repression by E75A is triggered by a decrease in ecdysone concentration and by direct repression of the EcR gene by E75A. Additional control is provided by developmentally timed modulation of E75A activity by NO, which inhibits recruitment of the corepressor SMRTER. These results suggest a mechanism for sequential modulation of gene expression during development by competing nuclear receptors! and their effector molecules, ecdysone and NO.
• JNK1 Phosphorylation of Cdt1 Inhibits Recruitment of HBO1 Histone Acetylase and Blocks Replication Licensing in Response to Stress
  - Mol Cell 44(1):62-71 (2011)
  In response to environmental stresses, cells activate stress-response genes and inhibit DNA replication. HBO1 histone acetylase is a coactivator both for AP-1 transcription factors responding to stress-activated JNK kinases and also for the Cdt1 licensing factor that ensures that DNA is replicated exactly once per cell cycle. In response to nongenotoxic stress, JNK phosphorylates Jun, an AP-1 transcription factor, leading to increased recruitment of HBO1 and increased transcription of target genes. In addition, JNK phosphorylates Cdt1 on threonine 29, leading to rapid dissociation of HBO1 from replication origins, thereby blocking initiation of DNA replication. Upon relief of stress, HBO1 reassociates with replication origins. Thus, regulated and reciprocal recruitment of the HBO1 coactivator to target genes and replication origins via JNK-mediated phosphorylation of the recruiting transcription and replication licensing factors coordinates the transcriptional and DNA ! replication response to cellular stress.
• A Genome-wide Screen Identifies p97 as an Essential Regulator of DNA Damage-Dependent CDT1 Destruction
  - Mol Cell 44(1):72-84 (2011)
  Several proteins, including the replication licensing factor CDT1 and the histone methyltransferase SET8, are targeted for proteolysis during DNA replication and repair by the E3 ubiquitin ligase CRL4CDT2. CRL4CDT2 function is coupled to replication and repair because it only ubiquitinates substrates that associate with chromatin-bound PCNA. Here, we report a genome-wide siRNA screen that identifies multiple factors necessary for CDT1 destruction after UV irradiation. Among these, nucleotide excision repair factors promote CDT1 destruction due to a role in recruiting PCNA to damaged DNA. The COP9/Signalosome regulates CDT2 stability through CUL4 deneddylation. Finally, the p97 AAA+-ATPase and its cofactor UFD1 are required for proteasome-dependent removal of ubiquitinated CDT1 and SET8 from chromatin and their subsequent degradation both in vivo and in a Xenopus egg extract system in vitro. This study provides insight into and a resource for the further exploration of ! pathways that promote timely degradation of chromatin-associated CRL4CDT2 substrates.
• CDC-48/p97 Coordinates CDT-1 Degradation with GINS Chromatin Dissociation to Ensure Faithful DNA Replication
  - Mol Cell 44(1):85-96 (2011)
  Faithful transmission of genomic information requires tight spatiotemporal regulation of DNA replication factors. In the licensing step of DNA replication, CDT-1 is loaded onto chromatin to subsequently promote the recruitment of additional replication factors, including CDC-45 and GINS. During the elongation step, the CDC-45/GINS complex moves with the replication fork; however, it is largely unknown how its chromatin association is regulated. Here, we show that the chaperone-like ATPase CDC-48/p97 coordinates degradation of CDT-1 with release of the CDC-45/GINS complex. C. elegans embryos lacking CDC-48 or its cofactors UFD-1/NPL-4 accumulate CDT-1 on mitotic chromatin, indicating a critical role of CDC-48 in CDT-1 turnover. Strikingly, CDC-48UFD-1/NPL-4-deficient embryos show persistent chromatin association of CDC-45/GINS, which is a consequence of CDT-1 stabilization. Moreover, our data confirmed a similar regulation in Xenopus egg extracts, emphasizing a conserve! d coordination of licensing and elongation events during eukaryotic DNA replication by CDC-48/p97.
• Cohesin's Concatenation of Sister DNAs Maintains Their Intertwining
  - Mol Cell 44(1):97-107 (2011)
  The contribution of DNA catenation to sister chromatid cohesion is unclear partly because it has never been observed directly within mitotic chromosomes. Differential sedimentation-velocity and gel electrophoresis reveal that sisters of 26 kb circular minichromosomes are held together by catenation as well as by cohesin. The finding that chemical crosslinking of cohesin's three subunit interfaces entraps sister DNAs of circular but not linear minichromosomes implies that cohesin functions using a topological principle. Importantly, cohesin holds both catenated and uncatenated DNAs together in this manner. In the vicinity of centromeres, catenanes are resolved by spindle forces, but linkages mediated directly by cohesin resist these forces even after complete decatenation. Crucially, persistence of catenation after S phase depends on cohesin. We conclude that by retarding Topo II-driven decatenation, cohesin mediates sister chromatid cohesion by an indirect mechanism as! well as one involving entrapment of sister DNAs inside its tripartite ring.
• A Pre-mRNA Degradation Pathway that Selectively Targets Intron-Containing Genes Requires the Nuclear Poly(A)-Binding Protein
  - Mol Cell 44(1):108-119 (2011)
  General discard pathways eliminate unprocessed and irregular pre-mRNAs to control the quality of gene expression. In contrast to such general pre-mRNA decay, we describe here a nuclear pre-mRNA degradation pathway that controls the expression of select intron-containing genes. We show that the fission yeast nuclear poly(A)-binding protein, Pab2, and the nuclear exosome subunit, Rrp6, are the main factors involved in this polyadenylation-dependent pre-mRNA degradation pathway. Transcriptome analysis and intron swapping experiments revealed that inefficient splicing is important to dictate susceptibility to Pab2-dependent pre-mRNA decay. We also show that negative splicing regulation can promote the poor splicing efficiency required for this pre-mRNA decay pathway, and in doing so, we identified a mechanism of cross-regulation between paralogous ribosomal proteins through nuclear pre-mRNA decay. Our findings unveil a layer of regulation in the nucleus in which the turnov! er of specific pre-mRNAs, besides the turnover of mature mRNAs, is used to control gene expression.
• GW182 Proteins Directly Recruit Cytoplasmic Deadenylase Complexes to miRNA Targets
  - Mol Cell 44(1):120-133 (2011)
  miRNAs are posttranscriptional regulators of gene expression that associate with Argonaute and GW182 proteins to repress translation and/or promote mRNA degradation. miRNA-mediated mRNA degradation is initiated by deadenylation, although it is not known whether deadenylases are recruited to the mRNA target directly or by default, as a consequence of a translational block. To answer this question, we performed a screen for potential interactions between the Argonaute and GW182 proteins and subunits of the two cytoplasmic deadenylase complexes. We found that human GW182 proteins recruit the PAN2-PAN3 and CCR4-CAF1-NOT deadenylase complexes through direct interactions with PAN3 and NOT1, respectively. These interactions are critical for silencing and are conserved in D. melanogaster. Our findings reveal that GW182 proteins provide a docking platform through which deadenylase complexes gain access to the poly(A) tail of miRNA targets to promote their deadenylation, and the! y further indicate that deadenylation is a direct effect of miRNA regulation.
• p62 Is a Key Regulator of Nutrient Sensing in the mTORC1 Pathway
  - Mol Cell 44(1):134-146 (2011)
  The signaling adaptor p62 is a critical mediator of important cellular functions, owing to its ability to establish interactions with various signaling intermediaries. Here, we identify raptor as an interacting partner of p62. Thus, p62 is an integral part of the mTORC1 complex and is necessary to mediate amino acid signaling for the activation of S6K1 and 4EBP1. p62 interacts in an amino acid-dependent manner with mTOR and raptor. In addition, p62 binds the Rags proteins and favors formation of the active Rag heterodimer that is further stabilized by raptor. Interestingly, p62 colocalizes with Rags at the lysosomal compartment and is required for the interaction of mTOR with Rag GTPases in vivo and for translocation of the mTORC1 complex to the lysosome, a crucial step for mTOR activation.
• Mechanism of USP7/HAUSP Activation by Its C-Terminal Ubiquitin-like Domain and Allosteric Regulation by GMP-Synthetase
  - Mol Cell 44(1):147-159 (2011)
  The ubiquitin-specific protease USP7/HAUSP regulates p53 and MDM2 levels, and cellular localization of FOXO4 and PTEN, and hence is critically important for their role in cellular processes. Here we show how the 64 kDa C-terminal region of USP7 can positively regulate deubiquitinating activity. We present the crystal structure of this USP7/HAUSP ubiquitin-like domain (HUBL) comprised of five ubiquitin-like (Ubl) domains organized in 2-1-2 Ubl units. The last di-Ubl unit, HUBL-45, is sufficient to activate USP7, through binding to a "switching" loop in the catalytic domain, which promotes ubiquitin binding and increases activity 100-fold. This activation can be enhanced allosterically by the metabolic enzyme GMPS. It binds to the first three Ubl domains (HUBL-123) and hyperactivates USP7 by stabilization of the HUBL-45-dependent active state.
• Acetylation Regulates the Stability of a Bacterial Protein: Growth Stage-Dependent Modification of RNase R
  - Mol Cell 44(1):160-166 (2011)
  RNase R, an Escherichia coli exoribonuclease important for degradation of structured RNAs, increases 3- to 10-fold under certain stress conditions, due to an increased half-life for this usually unstable protein. Components of the trans-translation machinery, tmRNA, and its associated protein, SmpB, are essential for RNase R instability. However, it is not understood why exponential phase RNase R is unstable or how it becomes stabilized in stationary phase. Here, we show that these phenomena are regulated by acetylation catalyzed by YfiQ protein. One residue, Lys544, is acetylated in exponential phase RNase R, but not in the stationary phase protein, resulting in tighter binding of tmRNA-SmpB to the C-terminal region of exponential phase RNase R and subsequent proteolytic degradation. Removal of the positive charge at Lys544 or a negative charge in the C-terminal region likely disrupts their interaction, facilitating tmRNA-SmpB binding. These findings indicate that ace! tylation can regulate the stability of a bacterial protein.