Hot off the presses! Sep 24 Mol Cell

The Sep 24 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:

• Beyond the Journal Pages
  - Mol Cell 35(6):725 (2009)
  
• How To Choose a Good Scientific Problem
  - Mol Cell 35(6):726-728 (2009)
  Choosing good problems is essential for being a good scientist. But what is a good problem, and how do you choose one? The subject is not usually discussed explicitly within our profession. Scientists are expected to be smart enough to figure it out on their own and through the observation of their teachers. This lack of explicit discussion leaves a vacuum that can lead to approaches such as choosing problems that can give results that merit publication in valued journals, resulting in a job and tenure.
• Linking the Cell Cycle to Histone Modifications: Dot1, G1/S, and Cycling K79me2
  - Mol Cell 35(6):729-730 (2009)
  In a recent issue of Molecular Cell, Schulze et al. (2009) described distinct distributions and regulation of Dot1-dependent methylation states at lysine 79 on histone H3 and showed cell-cycle regulation of K79 dimethylation on genes expressed during the G1/S phase.
• Summon SUMO to Wrestle with Inflammation
  - Mol Cell 35(6):731-732 (2009)
  In this issue of Molecular Cell, Lee et al. (2009) report that SUMOylation of nuclear receptors LXRα and LXRβ plays a critical role in the transrepression of IFN-γ-induced STAT1-dependent inflammatory responses in brain astrocytes.
• A Cut Above the Other Caspases
  - Mol Cell 35(6):733-734 (2009)
  In this issue of Molecular Cell, Bouchier-Hayes et al. (2009) develop a novel approach to visualizing caspase-2 activation in real time, enabling resolution of several controversies surrounding the position of this enzyme in apoptotic signaling cascades.
• Destruction of RhoA CULtivates Actin
  - Mol Cell 35(6):735-736 (2009)
  Cullin 3, the core subunit of the CRL3 ubiquitin ligase family, is essential for development, but its substrates remain poorly defined. Here, Chen et al. (2009) report that CRL3BACURD targets the RhoA GTPase for degradation, thereby maintaining actin cytoskeleton integrity.
• mir-210: A Sensor for Hypoxic Stress during Tumorigenesis
  - Mol Cell 35(6):737-738 (2009)
  In this issue of Molecular Cell, Huang et al. (2009) demonstrate that hypoxia-inducible mir-210 acts as a rheostat for cellular adaptation and survival by inhibiting tumor initiation.
• The Chicken or the Egg: MicroRNA-Mediated Regulation of mRNA Translation or mRNA Stability
  - Mol Cell 35(6):739-740 (2009)
  In this issue of Molecular Cell, Fabian et al. (2009) demonstrate that in cell-free extracts from mouse Krebs-2 ascites, microRNA-mediated translational repression precedes target mRNA deadenylation, and identify GW182, PABP, and deadenylase subunits CAF1 and CCR4 as factors required for deadenylation.
• Transcription Dynamics
  - Mol Cell 35(6):741-753 (2009)
  All aspects of transcription and its regulation involve dynamic events. The basal transcription machinery and regulatory components are dynamically recruited to their target genes, and dynamic interactions of transcription factors with chromatin—and with each other—play a key role in RNA polymerase assembly, initiation, and elongation. These short-term binding dynamics of transcription factors are superimposed by long-term cyclical behavior of chromatin opening and transcription factor-binding events. Its dynamic nature is not only a fundamental property of the transcription machinery, but it is emerging as an important modulator of physiological processes, particularly in differentiation and development.
• Dynamic Interactions and Cooperative Functions of PGC-1α and MED1 in TRα-Mediated Activation of the Brown-Fat-Specific UCP-1 Gene
  - Mol Cell 35(6):755-768 (2009)
  PGC-1α is an inducible nuclear receptor coactivator with direct functions in both p300-mediated chromatin remodeling and Mediator-dependent transcription in vitro. Here, we have employed the PPARγ- and TRα-activated brown adipose tissue-specific UCP-1 enhancer to investigate mechanistic aspects of PGC-1α function. We first demonstrate a cellular role for the PGC-1α-interacting MED1 subunit of Mediator in UCP-1 induction, as well as the accumulation of TRα, PPARγ, PGC-1α, and MED1 on the UCP-1 enhancer in brown adipocytes. We then use biochemical assays to show that (i) PGC-1α is recruited to the TRα-RXRα-UCP-1 enhancer complex through interaction of an N-terminal LXXLL domain with TRα, (ii) MED1/Mediator displaces PGC-1α from TRα through LXXLL domain competition, and (iii) upon loss of PGC-1α-TRα interactions, PGC-1α remains associated with the enhancer complex through an interaction between PGC-1α and MED1 C-terminal domains. These results indicate d! ynamic MED1-dependent PGC-1α interactions related to functions in both chromatin remodeling and the transition to subsequent transcription initiation.
• Reconstitution of Heterochromatin-Dependent Transcriptional Gene Silencing
  - Mol Cell 35(6):769-781 (2009)
  Heterochromatin assembly in budding yeast requires the SIR complex, which contains the NAD-dependent deacetylase Sir2 and the Sir3 and Sir4 proteins. Sir3 binds to nucleosomes containing deacetylated histone H4 lysine 16 (H4K16) and, with Sir4, promotes spreading of Sir2 and deacetylation along the chromatin fiber. Combined action of histone modifying and binding activities is a conserved hallmark of heterochromatin, but the relative contribution of each activity to silencing has remained unclear. Here, we reconstitute SIR-chromatin complexes using purified components and show that the SIR complex efficiently deacetylates chromatin templates and promotes the assembly of altered structures that silence Gal4-VP16-activated transcription. Silencing requires all three Sir proteins, even with fully deacetylated chromatin, and involves the specific association of Sir3 with deacetylated H4K16. These results define a minimal set of components that mediate heterochromatic gene ! silencing and demonstrate distinct contributions for histone deacetylation and nucleosome binding in the silencing mechanism.
• Histone Chaperones ASF1 and NAP1 Differentially Modulate Removal of Active Histone Marks by LID-RPD3 Complexes during NOTCH Silencing
  - Mol Cell 35(6):782-793 (2009)
  Histone chaperones are involved in a variety of chromatin transactions. By a proteomics survey, we identified the interaction networks of histone chaperones ASF1, CAF1, HIRA, and NAP1. Here, we analyzed the cooperation of H3/H4 chaperone ASF1 and H2A/H2B chaperone NAP1 with two closely related silencing complexes: LAF and RLAF. NAP1 binds RPD3 and LID-associated factors (RLAF) comprising histone deacetylase RPD3, histone H3K4 demethylase LID/KDM5, SIN3A, PF1, EMSY, and MRG15. ASF1 binds LAF, a similar complex lacking RPD3. ASF1 and NAP1 link, respectively, LAF and RLAF to the DNA-binding Su(H)/Hairless complex, which targets the E(spl) NOTCH-regulated genes. ASF1 facilitates gene-selective removal of the H3K4me3 mark by LAF but has no effect on H3 deacetylation. NAP1 directs high nucleosome density near E(spl) control elements and mediates both H3 deacetylation and H3K4me3 demethylation by RLAF. We conclude that histone chaperones ASF1 and NAP1 differentially modulate ! local chromatin structure during gene-selective silencing.
• Cse4 Is Part of an Octameric Nucleosome in Budding Yeast
  - Mol Cell 35(6):794-805 (2009)
  The budding yeast CenH3 histone variant Cse4 localizes to centromeric nucleosomes and is required for kinetochore assembly and chromosome segregation. The exact composition of centromeric Cse4-containing nucleosomes is a subject of debate. Using unbiased biochemical, cell-biological, and genetic approaches, we have tested the composition of Cse4-containing nucleosomes. Using micrococcal nuclease-treated chromatin, we find that Cse4 is associated with the histones H2A, H2B, and H4, but not H3 or the nonhistone protein Scm3. Overexpression of Cse4 rescues the lethality of a scm3 deletion, indicating that Scm3 is not essential for the formation of functional centromeric chromatin. We also find that octameric Cse4 nucleosomes can be reconstituted in vitro. Furthermore, Cse4-Cse4 dimerization occurs in vivo at the centromeric nucleosome, and this requires the predicted Cse4-Cse4 dimerization interface. Taken together, our experimental evidence supports the model that the Cs! e4 nucleosome is an octamer, containing two copies each of Cse4, H2A, H2B, and H4.
• Differential SUMOylation of LXRα and LXRβ Mediates Transrepression of STAT1 Inflammatory Signaling in IFN-γ-Stimulated Brain Astrocytes
  - Mol Cell 35(6):806-817 (2009)
  To unravel the roles of LXRs in inflammation and immunity, we examined the function of LXRs in development of IFN-γ-mediated inflammation using cultured rat brain astrocytes. LXR ligands inhibit neither STAT1 phosphorylation nor STAT1 translocation to the nucleus but, rather, inhibit STAT1 binding to promoters and the expression of IRF1, TNFα, and IL-6, downstream effectors of STAT1 action. Immunoprecipitation data revealed that LXRβ formed a trimer with PIAS1-pSTAT1, whereas LXRα formed a trimer with HDAC4-pSTAT1, mediated by direct ligand binding to the LXR proteins. In line with the fact that both PIAS1 and HDAC4 belong to the SUMO E3 ligase family, LXRβ and LXRα were SUMO-conjugated by PIAS1 or HDAC4, respectively, and SUMOylation was blocked by transient transfection of appropriate individual siRNAs, reversing LXR-induced suppression of IRF1 and TNFα expression. Together, our data show that SUMOylation is required for the suppression of STAT1-dependent infl! ammatory responses by LXRs in IFN-γ-stimulated brain astrocytes.
• Structure and Activation Mechanism of the CHK2 DNA Damage Checkpoint Kinase
  - Mol Cell 35(6):818-829 (2009)
  The CHK2 protein kinase is an important transducer of DNA damage checkpoint signals, and its mutation contributes to hereditary and sporadic cancer. CHK2 activation is triggered by the phosphorylation of Thr68 by the DNA damage-activated ATM kinase. This leads to transient CHK2 dimerization, in part through intermolecular phosphoThr68-FHA domain interactions. Dimerization promotes kinase activation through activation-loop autophosphorylation, but the mechanism of this process has not been clear. The dimeric crystal structure of CHK2, described here, in conjunction with biochemical and mutational data reveals that productive CHK2 dimerization additionally involves intermolecular FHA-kinase domain and FHA-FHA interactions. Ile157, mutated in the Li-Fraumeni cancer-predisposition syndrome, plays a central role in the FHA-kinase domain interface, explaining the lack of dimerization and autophosphorylation of this mutant. In the dimer, the kinase active sites face each othe! r in close proximity, indicating that dimerization may also serve to optimally position the kinase active sites for efficient activation loop transphosphorylation.
• Characterization of Cytoplasmic Caspase-2 Activation by Induced Proximity
  - Mol Cell 35(6):830-840 (2009)
  Caspase-2 is an initiator caspase activated in response to heat shock and other stressors that induce apoptosis. Activation of caspase-2 requires induced proximity resulting after recruitment to caspase-2 activation complexes such as the PIDDosome. We have adapted bimolecular fluorescence complementation (BiFC) to measure caspase-2 induced proximity in real time in single cells. Nonfluorescent fragments of the fluorescent protein Venus that can associate to reform the fluorescent complex were fused to caspase-2, allowing visualization and kinetic measurements of caspase-2 induced proximity after heat shock and other stresses. This revealed that the caspase-2 activation platform occurred in the cytosol and not in the nucleus in response to heat shock, DNA damage, cytoskeletal disruption, and other treatments. Activation, as measured by this approach, in response to heat shock was RAIDD dependent and upstream of mitochondrial outer-membrane permeabilization. Furthermore,! we identify Hsp90α as a key negative regulator of heat shock-induced caspase-2 activation.
• Cullin Mediates Degradation of RhoA through Evolutionarily Conserved BTB Adaptors to Control Actin Cytoskeleton Structure and Cell Movement
  - Mol Cell 35(6):841-855 (2009)
  Cul3, a Cullin family scaffold protein, is thought to mediate the assembly of a large number of SCF (Skp1-Cullin1-F-box protein)-like ubiquitin ligase complexes through BTB domain substrate-recruiting adaptors. Cul3 controls early embryonic development in several genetic models through mechanisms not understood. Very few functional substrate/adaptor pairs for Cul3 ubiquitin ligases have been identified. Here, we show that Cul3 knockdown in human cells results in abnormal actin stress fibers and distorted cell morphology, owing to impaired ubiquitination and degradation of small GTPase RhoA. We identify a family of RhoA-binding BTB domain adaptors conserved from insects to mammals, designated BACURDs. BACURDs form ubiquitin ligase complexes, which selectively ubiquitinate RhoA, with Cul3. Dysfunction of the Cul3/BACURD complex decreases cell migration potential and impairs RhoA-mediated convergent extension movements during Xenopus gastrulation. Our studies reveal a pre! viously unknown mechanism for controlling RhoA degradation and regulating RhoA function in various biological contexts, which involves a Cul3/BACURD ubiquitin ligase complex.
• Hypoxia-Inducible mir-210 Regulates Normoxic Gene Expression Involved in Tumor Initiation
  - Mol Cell 35(6):856-867 (2009)
  Previous studies have suggested that the HIF transcription factors can both activate and inhibit gene expression. Here we show that HIF1 regulates the expression of mir-210 in a variety of tumor types through a hypoxia-responsive element. Expression analysis in primary head and neck tumor samples indicates that mir-210 may serve as an in vivo marker for tumor hypoxia. By Argonaute protein immunoprecipitation, we identified 50 potential mir-210 targets and validated randomly selected ones. The majority of these 50 genes are not classical hypoxia-inducible genes, suggesting mir-210 represses genes expressed under normoxia that are no longer necessary to adapt and survive in a hypoxic environment. When human head and neck or pancreatic tumor cells ectopically expressing mir-210 were implanted into immunodeficient mice, mir-210 repressed initiation of tumor growth. Taken together, these data implicate an important role for mir-210 in regulating the hypoxic response of tumo! r cells and tumor growth.
• Mammalian miRNA RISC Recruits CAF1 and PABP to Affect PABP-Dependent Deadenylation
  - Mol Cell 35(6):868-880 (2009)
  MicroRNAs (miRNAs) inhibit mRNA expression in general by base pairing to the 3′UTR of target mRNAs and consequently inhibiting translation and/or initiating poly(A) tail deadenylation and mRNA destabilization. Here we examine the mechanism and kinetics of miRNA-mediated deadenylation in mouse Krebs-2 ascites extract. We demonstrate that miRNA-mediated mRNA deadenylation occurs subsequent to initial translational inhibition, indicating a two-step mechanism of miRNA action, which serves to consolidate repression. We show that a let-7 miRNA-loaded RNA-induced silencing complex (miRISC) interacts with the poly(A)-binding protein (PABP) and the CAF1 and CCR4 deadenylases. In addition, we demonstrate that miRNA-mediated deadenylation is dependent upon CAF1 activity and PABP, which serves as a bona fide miRNA coactivator. Importantly, we present evidence that GW182, a core component of the miRISC, directly interacts with PABP via its C-terminal region and that this interact! ion is required for miRNA-mediated deadenylation.
• Drosophila miR2 Primarily Targets the m7GpppN Cap Structure for Translational Repression
  - Mol Cell 35(6):881-888 (2009)
  Understanding the molecular mechanism(s) of how miRNAs repress mRNA translation is a fundamental challenge in RNA biology. Here we use a validated cell-free system from Drosophila embryos to investigate how miR2 inhibits translation initiation. By screening a library of chemical m7GpppN cap structure analogs, we identified defined modifications of the triphosphate backbone that augment miRNA-mediated inhibition of translation initiation but are "neutral" toward general cap-dependent translation. Interestingly, these caps also augment inhibition by 4E-BP. Kinetic dissection of translational repression and miR2-induced deadenylation shows that both processes proceed largely independently, with establishment of the repressed state involving a slow step. Our data demonstrate a primary role for the m7GpppN cap structure in miRNA-mediated translational inhibition, implicate structural determinants outside the core eIF4E-binding region in this process, and suggest that mi! RNAs may target cap-dependent translation through a mechanism related to the 4E-BP class of translational regulators.
• Interaction of Transcriptional Regulators with Specific Nucleosomes across the Saccharomyces Genome
  - Mol Cell 35(6):889-902 (2009)
  A canonical nucleosome architecture around promoters establishes the context in which proteins regulate gene expression. Whether gene regulatory proteins that interact with nucleosomes are selective for individual nucleosome positions across the genome is not known. Here, we examine on a genomic scale several protein-nucleosome interactions, including those that (1) bind histones (Bdf1/SWR1 and Srm1), (2) bind specific DNA sequences (Rap1 and Reb1), and (3) potentially collide with nucleosomes during transcription (RNA polymerase II). We find that the Bdf1/SWR1 complex forms a dinucleosome complex that is selective for the +1 and +2 nucleosomes of active genes. Rap1 selectively binds to its cognate site on the rotationally exposed first and second helical turn of nucleosomal DNA. We find that a transcribing RNA polymerase creates a delocalized state of resident nucleosomes. These findings suggest that nucleosomes around promoter regions have position-specific functions! and that some gene regulators have position-specific nucleosomal interactions.