Hot off the presses! May 15 Cell

The May 15 issue of the Cell is now up on Pubget (About 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:

• In This Issue
  - Cell 137(4):587, 589 (2009)
  
• Immunology Select
  - Cell 137(4):591, 593 (2009)
  The inflammatory response—characterized by release of cytokines such as TNFα and IL-1β and infiltration of infected or injured tissue by macrophages and neutrophils—is the first line of defense against infection by pathogens. Although an early inflammatory response is beneficial in stemming the tide of infection, a prolonged response may result in severe illness or even death. For example, the 1918 Spanish influenza virus was lethal because it triggered a "cytokine storm" that resulted in severe lung damage. An inappropriate and prolonged inflammatory response to normal nonpathogenic gut bacteria is a cause of Crohn's disease. Meanwhile, insidious low-level inflammation in the tumor microenvironment aids and abets tumor cells in their quest to become metastatic. Understanding the triggers of inflammation and the signaling pathways involved in cytokine production and immune cell mobilization will be essential for developing therapeutics to treat diseases cause! d by a runaway inflammatory response.
• Will Scientists Cast Their Votes?
  - Cell 137(4):595-597 (2009)
  With science and technology high on the agenda of the new US administration, scientists should welcome the opportunity to influence policy. However, few academic scientists seem to be noticing proposals posted for public comment on the Federal Register that concern the application of science to society.
• Closing The Ring: A New Twist to Bacterial Chromosome Condensation
  - Cell 137(4):598-600 (2009)
  The mechanisms underlying chromosome organization in bacteria are still shrouded in mystery. Sullivan et al. (2009) and Gruber and Errington (2009) now report that the DNA-binding protein ParB ensures proper arrangement and partitioning of chromosomal DNA in Bacillus subtilis by recruiting the condensin SMC to the replication origin region.
• Shedding UV Light on Alternative Splicing
  - Cell 137(4):600-602 (2009)
  After DNA damage, cells modulate pre-messenger RNA (pre-mRNA) splicing to induce an anti- or proapoptotic response. In this issue, Muñoz et al. (2009) uncover a cotranscriptional mechanism for activating alternative pre-mRNA splicing after ultraviolet irradiation that depends unexpectedly on hyperphosphorylation of the RNA polymerase II C-terminal domain and decreased rates of transcription elongation.
• Keeping the Beat in the Rising Heat
  - Cell 137(4):602-604 (2009)
  Circadian clocks use temperature compensation to keep accurate time over a range of temperatures, thus allowing reliable timekeeping under diverse environmental conditions. Mehra et al. (2009) and Baker et al. (2009) now show that phosphorylation-regulated protein degradation plays a key role in circadian temperature compensation.
• The CULt of Caspase-8 Ubiquitination
  - Cell 137(4):604-606 (2009)
  Caspase-8 is activated at the plasma membrane by the death-inducing signaling complex (DISC). Jin et al. (2009) show that polyubiquitination of caspase-8, rather than targeting it for proteasomal degradation, is critical for sustaining caspase-8 activity after dissociation from the DISC.
• Abate and Switch: miR-145 in Stem Cell Differentiation
  - Cell 137(4):606-608 (2009)
  MicroRNAs have been implicated as regulators of embryonic stem (ES) cell self-renewal and pluripotency. In this issue, Xu et al. (2009) demonstrate that miR-145 facilitates ES cell differentiation by repressing the core pluripotency factors OCT4, SOX2, and KLF4, thereby silencing the self-renewal program.
• Modes of p53 Regulation
  - Cell 137(4):609-622 (2009)
  The traditional view of p53 activation includes three steps—p53 stabilization, DNA binding, and transcriptional activation. However, recent studies indicate that each step of p53 activation is more complex than originally anticipated. Moreover, both genetic studies in mice and in vitro studies with purified components suggest that the classical model may not be sufficient to explain all aspects of p53 activation in vivo. To reconcile these differences, we propose that antirepression, the release of p53 from repression by factors such as Mdm2 and MdmX, is a key step in the physiological activation of p53.
• A Two-Step Model for Colon Adenoma Initiation and Progression Caused by APC Loss
  - Cell 137(4):623-634 (2009)
  Aberrant Wnt/β-catenin signaling following loss of the tumor suppressor adenomatous polyposis coli (APC) is thought to initiate colon adenoma formation. Using zebrafish and human cells, we show that homozygous loss of APC causes failed intestinal cell differentiation but that this occurs in the absence of nuclear β-catenin and increased intestinal cell proliferation. Therefore, loss of APC is insufficient for causing β-catenin nuclear localization. APC mutation-induced intestinal differentiation defects instead depend on the transcriptional corepressor C-terminal binding protein-1 (CtBP1), whereas proliferation defects and nuclear accumulation of β-catenin require the additional activation of KRAS. These findings suggest that, following APC loss, CtBP1 contributes to adenoma initiation as a first step, whereas KRAS activation and β-catenin nuclear localization promote adenoma progression to carcinomas as a second step. Consistent with this model, human FAP adenoma! s showed robust upregulation of CtBP1 in the absence of detectable nuclear β-catenin, whereas nuclear β-catenin was detected in carcinomas.
• Metformin and Insulin Suppress Hepatic Gluconeogenesis through Phosphorylation of CREB Binding Protein
  - Cell 137(4):635-646 (2009)
  Insulin resistance and elevated glucagon levels result in nonsuppressible hepatic glucose production and hyperglycemia in patients with type 2 diabetes. The CREB coactivator complex controls transcription of hepatic gluconeogenic enzyme genes. Here, we show that both the antidiabetic agent metformin and insulin phosphorylate the transcriptional coactivator CREB binding protein (CBP) at serine 436 via PKCι/λ. This event triggers the dissociation of the CREB-CBP-TORC2 transcription complex and reduces gluconeogenic enzyme gene expression. Mice carrying a germline mutation of this CBP phosphorylation site (S436A) demonstrate resistance to the hypoglycemic effect of both insulin and metformin. Obese, hyperglycemic mice display hepatic insulin resistance, but metformin is still effective in treating the hyperglycemia of these mice since it stimulates CBP phosphorylation by bypassing the block in insulin signaling. Our findings point to CBP phosphorylation at Ser436 by met! formin as critical for its therapeutic effect, and as a potential target for pharmaceutical intervention.
• MicroRNA-145 Regulates OCT4, SOX2, and KLF4 and Represses Pluripotency in Human Embryonic Stem Cells
  - Cell 137(4):647-658 (2009)
  MicroRNAs (miRNAs) are posttranscriptional modulators of gene expression and play an important role in many developmental processes. We report here that expression of microRNA-145 (miR-145) is low in self-renewing human embryonic stem cells (hESCs) but highly upregulated during differentiation. We identify the pluripotency factors OCT4, SOX2, and KLF4 as direct targets of miR-145 and show that endogenous miR-145 represses the 3′ untranslated regions of OCT4, SOX2, and KLF4. Increased miR-145 expression inhibits hESC self-renewal, represses expression of pluripotency genes, and induces lineage-restricted differentiation. Loss of miR-145 impairs differentiation and elevates OCT4, SOX2, and KLF4. Furthermore, we find that the miR-145 promoter is bound and repressed by OCT4 in hESCs. This work reveals a direct link between the core reprogramming factors and miR-145 and uncovers a double-negative feedback loop involving OCT4, SOX2, KLF4, and miR-145.
• The Mechanism of ATP-Dependent Primer-Template Recognition by a Clamp Loader Complex
  - Cell 137(4):659-671 (2009)
  Clamp loaders load sliding clamps onto primer-template DNA. The structure of the E. coli clamp loader bound to DNA reveals the formation of an ATP-dependent spiral of ATPase domains that tracks only the template strand, allowing recognition of both RNA and DNA primers. Unlike hexameric helicases, in which DNA translocation requires distinct conformations of the ATPase domains, the clamp loader spiral is symmetric and is set up to trigger release upon DNA recognition. Specificity for primed DNA arises from blockage of the end of the primer and accommodation of the emerging template along a surface groove. A related structure reveals how the ψ protein, essential for coupling the clamp loader to single-stranded DNA-binding protein (SSB), binds to the clamp loader. By stabilizing a conformation of the clamp loader that is consistent with the ATPase spiral observed upon DNA binding, ψ binding promotes the clamp-loading activity of the complex.
• Protein Architecture of the Human Kinetochore Microtubule Attachment Site
  - Cell 137(4):672-684 (2009)
  Chromosome segregation requires assembly of kinetochores on centromeric chromatin to mediate interactions with spindle microtubules and control cell-cycle progression. To elucidate the protein architecture of human kinetochores, we developed a two-color fluorescence light microscopy method that measures average label separation, Delta, at <5 nm accuracy. Delta analysis of 16 proteins representing core structural complexes spanning the centromeric chromatin-microtubule interface, when correlated with mechanical states of spindle-attached kinetochores, provided a nanometer-scale map of protein position and mechanical properties of protein linkages. Treatment with taxol, which suppresses microtubule dynamics and activates the spindle checkpoint, revealed a specific switch in kinetochore architecture. Cumulatively, Delta analysis revealed that compliant linkages are restricted to the proximity of chromatin, suggested a model for how the KMN (KNL1/Mis12 complex/Ndc80 comple! x) network provides microtubule attachment and generates pulling forces from depolymerization, and identified an intrakinetochore molecular switch that may function in controlling checkpoint activity.
• Recruitment of Condensin to Replication Origin Regions by ParB/SpoOJ Promotes Chromosome Segregation in B. subtilis
  - Cell 137(4):685-696 (2009)
  Proper segregation of DNA replication products is essential in all cells. In Bacillus subtilis, two protein complexes have been implicated in this process: the ParAB homologs, Soj and Spo0J, and the bacterial Smc/ScpAB complex, also called condensin. Here we demonstrate that Smc is highly enriched in the region around the origin of replication, specifically near parS sites to which Spo0J binds and at highly transcribed genes. Furthermore, we find that efficient recruitment of Smc to a large region around the origin of replication depends on the presence of Spo0J. We show that Spo0J performs two independent functions: regulation of initiation of DNA replication via Soj and promotion of chromosome segregation by Smc recruitment. Our results demonstrate a direct functional interaction between two widely conserved systems involved in chromosome replication and segregation.
• Recruitment of SMC by ParB-parS Organizes the Origin Region and Promotes Efficient Chromosome Segregation
  - Cell 137(4):697-707 (2009)
  Organization and segregation of replicated chromosomes are essential processes during cell division in all organisms. Similar to eukaryotes, bacteria possess centromere-like DNA sequences (parS) that cluster at the origin of replication and the structural maintenance of chromosomes (SMC) complexes for faithful chromosome segregation. In Bacillus subtilis, parS sites are bound by the partitioning protein Spo0J (ParB), and we show here that Spo0J recruits the SMC complex to the origin. We demonstrate that the SMC complex colocalizes with Spo0J at the origin and that insertion of parS sites near the replication terminus targets SMC to this position leading to defects in chromosome organization and segregation. Consistent with these findings, the subcellular localization of the SMC complex is disrupted in the absence of Spo0J or the parS sites. We propose a model in which recruitment of SMC to the origin by Spo0J-parS organizes the origin region and promotes efficient chro! mosome segregation.
• DNA Damage Regulates Alternative Splicing through Inhibition of RNA Polymerase II Elongation
  - Cell 137(4):708-720 (2009)
  DNA damage induces apoptosis and many apoptotic genes are regulated via alternative splicing (AS), but little is known about the control mechanisms. Here we show that ultraviolet irradiation (UV) affects cotranscriptional AS in a p53-independent way, through the hyperphosphorylation of RNA polymerase II carboxy-terminal domain (CTD) and a subsequent inhibition of transcriptional elongation, estimated in vivo and in real time. Phosphomimetic CTD mutants not only display lower elongation but also duplicate the UV effect on AS. Consistently, nonphosphorylatable mutants prevent the UV effect. Apoptosis promoted by UV in cells lacking p53 is prevented when the change in AS of the apoptotic gene bcl-x is reverted, confirming the relevance of this mechanism. Splicing-sensitive microarrays revealed a significant overlap of the subsets of genes that have changed AS with UV and those that have reduced expression, suggesting that transcriptional coupling to AS is a key feature of! the DNA-damage response.
• Cullin3-Based Polyubiquitination and p62-Dependent Aggregation of Caspase-8 Mediate Extrinsic Apoptosis Signaling
  - Cell 137(4):721-735 (2009)
  Cell-surface death receptors such as DR4 and DR5 trigger apoptosis through a death-inducing signaling complex (DISC) that recruits the apical protease caspase-8. Apoptosis commitment requires efficient activation and autocatalytic release of caspase-8 into the cytoplasm to engage executioner caspases. While DISC recruitment initiates caspase-8 stimulation, full activation of the protease depends on further molecular aggregation events that are not fully understood. Here, we show that death receptor ligation induces polyubiquitination of caspase-8, through a previously unknown interaction of the DISC with a cullin3 (CUL3)-based E3 ligase. CUL3-mediated caspase-8 polyubiquitination required the RING box protein RBX1, whereas the deubiquitinase A20 reversed this modification. The ubiquitin-binding protein p62/sequestosome-1 promoted aggregation of CUL3-modified caspase-8 within p62-dependent foci, leading to full activation and processing of the enzyme and driving commitm! ent to cell death. These results identify a mechanism that positively controls apoptosis signaling by polyubiquitination and aggregation of a key initiator caspase.
• Hematopoietic Stem Cell Development Is Dependent on Blood Flow
  - Cell 137(4):736-748 (2009)
  During vertebrate embryogenesis, hematopoietic stem cells (HSCs) arise in the aorta-gonads-mesonephros (AGM) region. We report here that blood flow is a conserved regulator of HSC formation. In zebrafish, chemical blood flow modulators regulated HSC development, and silent heart (sih) embryos, lacking a heartbeat and blood circulation, exhibited severely reduced HSCs. Flow-modifying compounds primarily affected HSC induction after the onset of heartbeat; however, nitric oxide (NO) donors regulated HSC number even when treatment occurred before the initiation of circulation, and rescued HSCs in sih mutants. Morpholino knockdown of nos1 (nnos/enos) blocked HSC development, and its requirement was shown to be cell autonomous. In the mouse, Nos3 (eNos) was expressed in HSCs in the AGM. Intrauterine Nos inhibition or embryonic Nos3 deficiency resulted in a reduction of hematopoietic clusters and transplantable murine HSCs. This work links blood flow to AGM hematopoiesis and! identifies NO as a conserved downstream regulator of HSC development.
• A Role for Casein Kinase 2 in the Mechanism Underlying Circadian Temperature Compensation
  - Cell 137(4):749-760 (2009)
  Temperature compensation of circadian clocks is an unsolved problem with relevance to the general phenomenon of biological compensation. We identify casein kinase 2 (CK2) as a key regulator of temperature compensation of the Neurospora clock by determining that two long-standing clock mutants, chrono and period-3, displaying distinctive alterations in compensation encode the β1 and α subunits of CK2, respectively. Reducing the dose of these subunits, particularly β1, significantly alters temperature compensation without altering the enzyme's Q10. By contrast, other kinases and phosphatases implicated in clock function do not play appreciable roles in temperature compensation. CK2 exerts its effects on the clock by directly phosphorylating FREQUENCY (FRQ), and this phosphorylation is compromised in CK2 hypomorphs. Finally, mutation of certain putative CK2 phosphosites on FRQ, shown to be phosphorylated in vivo, predictably alters temperature compensation profiles eff! ectively phenocopying CK2 mutants.
• Essential Role for TRPC5 in Amygdala Function and Fear-Related Behavior
  - Cell 137(4):761-772 (2009)
  The transient receptor potential channel 5 (TRPC5) is predominantly expressed in the brain where it can form heterotetrameric complexes with TRPC1 and TRPC4 channel subunits. These excitatory, nonselective cationic channels are regulated by G protein, phospholipase C-coupled receptors. Here, we show that TRPC5−/− mice exhibit diminished innate fear levels in response to innately aversive stimuli. Moreover, mutant mice exhibited significant reductions in responses mediated by synaptic activation of Group I metabotropic glutamate and cholecystokinin 2 receptors in neurons of the amygdala. Synaptic strength at afferent inputs to the amygdala was diminished in P10-P13 null mice. In contrast, baseline synaptic transmission, membrane excitability, and spike timing-dependent long-term potentiation at cortical and thalamic inputs to the amygdala were largely normal in older null mice. These experiments provide genetic evidence that TRPC5, activated via G protein-coupled ne! uronal receptors, has an essential function in innate fear.
• Autophagic Components Contribute to Hypersensitive Cell Death in Arabidopsis
  - Cell 137(4):773-783 (2009)
  Autophagy has been implicated as a prosurvival mechanism to restrict programmed cell death (PCD) associated with the pathogen-triggered hypersensitive response (HR) during plant innate immunity. This model is based on the observation that HR lesions spread in plants with reduced autophagy gene expression. Here, we examined receptor-mediated HR PCD responses in autophagy-deficient Arabidopsis knockout mutants (atg), and show that infection-induced lesions are contained in atg mutants. We also provide evidence that HR cell death initiated via Toll/Interleukin-1 (TIR)-type immune receptors through the defense regulator EDS1 is suppressed in atg mutants. Furthermore, we demonstrate that PCD triggered by coiled-coil (CC)-type immune receptors via NDR1 is either autophagy-independent or engages autophagic components with cathepsins and other unidentified cell death mediators. Thus, autophagic cell death contributes to HR PCD and can function in parallel with other prodeath p! athways.
• SnapShot: Intraflagellar Transport
  - Cell 137(4):784-784.e1 (2009)