Hot off the presses! Aug 14 Mol Cell

The Aug 14 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:

• Can the Protein Occupancy Landscape Show the Topologically Isolated Chromosomal Domains in the E. coli Genome?: An Exciting Prospect
  - Mol Cell 35(3):255-256 (2009)
  In a recent issue of Molecular Cell, Vora et al. (2009) introduce an in vivo protein occupancy display (IPOD) technology and propose that the domains extensively occupied by proteins with inactive transcription in bacteria are mainly topologically isolated chromosomal domains.
• The SAGA Continues…to the End
  - Mol Cell 35(3):256-258 (2009)
  In this issue of Molecular Cell, Atanassov et al. (2009) show that the GCN5-containing SAGA complex regulates telomere function via deubiquitination and stabilization of the telomere repeat binding factor TRF1.
• SOSS1/2: Sensors of Single-Stranded DNA at a Break
  - Mol Cell 35(3):258-259 (2009)
  In this issue of Molecular Cell, Huang et al. (2009) describe two heterotrimeric single-stranded DNA binding complexes, SOSS1 and SOSS2, that function downstream of the MRN complex to promote DNA repair and the G2/M checkpoint.
• PCI Complexes: Beyond the Proteasome, CSN, and eIF3 Troika
  - Mol Cell 35(3):260-264 (2009)
  The bipartite PCI domain serves as the principal scaffold for proteasome lid, CSN, and eIF3, complexes that influence protein life span. PCI domains are also found in newly identified complexes directing nucleic acid regulation. The breadth of functions associated with the extended PCI family is a factor of shared subunits, among them a common factor Sem1/DSS1 that facilitates complex assembly.
• Reconstitution of the Death-Inducing Signaling Complex Reveals a Substrate Switch that Determines CD95-Mediated Death or Survival
  - Mol Cell 35(3):265-279 (2009)
  The death-inducing signaling complex (DISC) is critical for initiation of death-receptor-mediated apoptosis; however, paradoxically, CD95 also signals for cell survival. Here, we reconstitute a functional DISC using only purified CD95, FADD, and procaspase-8 and unveil a two-step activation mechanism involving both dimerization and proteolytic cleavage of procaspase-8 that is obligatory for death-receptor-induced apoptosis. Initially, dimerization yields active procaspase-8 with a very restricted substrate repertoire, limited to itself or c-FLIP. Proteolytic cleavage is then required to fully activate caspase-8, thereby permitting DISC-mediated cleavage of the critical exogenous apoptotic substrates, caspase-3 and Bid. This switch in catalytic activity and substrate range is a key determinant of DISC signaling, as cellular expression of noncleavable procaspase-8 mutants, which undergo DISC-mediated oligomerization, but not cleavage, fails to initiate CD95-induced apopt! osis. Thus, using the reconstituted DISC, we have delineated a crucial two-step activation mechanism whereby activated death receptor complexes can trigger death or survival.
• Structure of the S5a:K48-Linked Diubiquitin Complex and Its Interactions with Rpn13
  - Mol Cell 35(3):280-290 (2009)
  Degradation by the proteasome typically requires substrate ubiquitination. Two ubiquitin receptors exist in the proteasome, S5a/Rpn10 and Rpn13. Whereas Rpn13 has only one ubiquitin-binding surface, S5a binds ubiquitin with two independent ubiquitin-interacting motifs (UIMs). Here, we use nuclear magnetic resonance (NMR) and analytical ultracentrifugation to define at atomic level resolution how S5a binds K48-linked diubiquitin, in which K48 of one ubiquitin subunit (the "proximal" one) is covalently bonded to G76 of the other (the "distal" subunit). We demonstrate that S5a's UIMs bind the two subunits simultaneously with a preference for UIM2 binding to the proximal subunit while UIM1 binds to the distal one. In addition, NMR experiments reveal that Rpn13 and S5a bind K48-linked diubiquitin simultaneously with subunit specificity, and a model structure of S5a and Rpn13 bound to K48-linked polyubiquitin is provided. Altogether, our data demonstrate that S5a is ! highly adaptive and cooperative toward binding ubiquitin chains.
• Negative Regulation of the EGFR-MAPK Cascade by Actin-MAL-Mediated Mig6/Errfi-1 Induction
  - Mol Cell 35(3):291-304 (2009)
  We analyzed the G-actin-regulated transcriptome by gene expression analysis using previously characterized actin-binding drugs. We found many known MAL/MRTF-dependent target genes of serum response factor (SRF), as well as additional directly regulated genes. Surprisingly, several putative antiproliferative target genes were identified, including mig6/errfi-1, a negative regulator of the EGFR family. Mig6 induction occurred through actin-MAL-SRF signaling, and MAL was inducibly recruited to and activated a mig6 promoter element. Upregulation of Mig6 by lipid agonists such as LPA and S1P or actin drugs involved MAL and correlated with decreased activation of EGFR, MAPK/Erk, and c-fos. Mig6 depletion restored EGFR signaling and provided a proliferative advantage. Overexpression of MAL exhibited strong antiproliferative effects requiring the domains for SRF binding and transactivation, which supports antagonistic functions of MAL on growth-promoting signals. Our results s! how the existence of negatively acting transcriptional networks between pro- and antiproliferative signaling pathways toward SRF.
• Myosin VI Dimerization Triggers an Unfolding of a Three-Helix Bundle in Order to Extend Its Reach
  - Mol Cell 35(3):305-315 (2009)
  Myosin VI challenges the prevailing theory of how myosin motors move on actin: the lever arm hypothesis. While the reverse directionality and large powerstroke of myosin VI can be attributed to unusual properties of a subdomain of the motor (converter with a unique insert), these adaptations cannot account for the large step size on actin. Either the lever arm hypothesis needs modification, or myosin VI has some unique form of extension of its lever arm. We determined the structure of the region immediately distal to the lever arm of the motor and show that it is a three-helix bundle. Based on C-terminal truncations that display the normal range of step sizes on actin, CD, fluorescence studies, and a partial deletion of the bundle, we demonstrate that this bundle unfolds upon dimerization of two myosin VI monomers. This unconventional mechanism generates an extension of the lever arm of myosin VI.
• Ribosomal Protein S7 Is Both a Regulator and a Substrate of MDM2
  - Mol Cell 35(3):316-326 (2009)
  MDM2 associates with ribosomal protein S7, and this interaction is required to inhibit MDM2's E3 ligase activity, leading to stabilization of MDM2 and p53. Notably, the MDM2 homolog MDMX facilitates the inhibition of MDM2 E3 ligase activity by S7. Further, ablation of S7 inhibits MDM2 and p53 accumulation induced by different stress signals in some cell types. Thus, ribosomal/nucleolar stress is likely a key integrating event in DNA damage signaling to p53. Interestingly, S7 is itself a substrate for MDM2 E3 ligase activity both in vitro and in vivo. An S7-ubiquitin fusion protein (S7-Ub) selectively inhibits MDM2 degradation of p53 and is unaffected by MDMX. S7-Ub promotes apoptosis to a greater extent than S7 alone. This indicates that MDM2 ubiquitination of S7 is involved in sustaining the p53 response. Thus, S7 functions as both effector and affector of MDM2 to ensure a proper cellular response to different stress signals.
• Cdk1 Participates in BRCA1-Dependent S Phase Checkpoint Control in Response to DNA Damage
  - Mol Cell 35(3):327-339 (2009)
  Cdk2 and cdk1 are individually dispensable for cell-cycle progression in cancer cell lines because they are able to compensate for one another. However, shRNA-mediated depletion of cdk1 alone or small molecule cdk1 inhibition abrogated S phase cell-cycle arrest and the phosphorylation of a subset of ATR/ATM targets after DNA damage. Loss of DNA damage-induced checkpoint control was caused by a reduction in formation of BRCA1-containing foci. Mutation of BRCA1 at S1497 and S1189/S1191 resulted in loss of cdk1-mediated phosphorylation and also compromised formation of BRCA1-containing foci. Abrogation of checkpoint control after cdk1 depletion or inhibition in non-small-cell lung cancer cells sensitized them to DNA-damaging agents. Conversely, reduced cdk1 activity caused more potent G2/M arrest in nontransformed cells and antagonized the response to subsequent DNA damage. Cdk1 inhibition may therefore selectively sensitize BRCA1-proficient cancer cells to DNA-damaging t! reatments by disrupting BRCA1 function.
• Two-Color Cell Array Screen Reveals Interdependent Roles for Histone Chaperones and a Chromatin Boundary Regulator in Histone Gene Repression
  - Mol Cell 35(3):340-351 (2009)
  We describe a fluorescent reporter system that exploits the functional genomic tools available in budding yeast to systematically assess consequences of genetic perturbations on gene expression. We used our Reporter-Synthetic Genetic Array (R-SGA) method to screen for regulators of core histone gene expression. We discovered that the histone chaperone Rtt106 functions in a pathway with two other chaperones, Asf1 and the HIR complex, to create a repressive chromatin structure at core histone promoters. We found that activation of histone (HTA1) gene expression involves both relief of Rtt106-mediated repression by the activity of the histone acetyltransferase Rtt109 and restriction of Rtt106 to the promoter region by the bromodomain-containing protein Yta7. We propose that the maintenance of Asf1/HIR/Rtt106-mediated repressive chromatin domains is the primary mechanism of cell-cycle regulation of histone promoters. Our data suggest that this pathway may represent a chrom! atin regulatory mechanism that is broadly used across the genome.
• Gcn5 and SAGA Regulate Shelterin Protein Turnover and Telomere Maintenance
  - Mol Cell 35(3):352-364 (2009)
  Histone acetyltransferases (HATs) play important roles in gene regulation and DNA repair by influencing the accessibility of chromatin to transcription factors and repair proteins. Here, we show that deletion of Gcn5 leads to telomere dysfunction in mouse and human cells. Biochemical studies reveal that depletion of Gcn5 or ubiquitin-specific protease 22 (Usp22), which is another bona fide component of the Gcn5-containing SAGA complex, increases ubiquitination and turnover of TRF1, a primary component of the telomeric shelterin complex. Inhibition of the proteasome or overexpression of USP22 opposes this effect. The USP22 deubiquitinating module requires association with SAGA complexes for activity, and we find that depletion of Gcn5 compromises this association in mammalian cells. Thus, our results indicate that Gcn5 regulates TRF1 levels through effects on Usp22 activity and SAGA integrity.
• yFACT Induces Global Accessibility of Nucleosomal DNA without H2A-H2B Displacement
  - Mol Cell 35(3):365-376 (2009)
  FACT has been proposed to function by displacing H2A-H2B dimers from nucleosomes to form hexasomes. Results described here with yeast FACT (yFACT) suggest instead that nucleosomes are reorganized to a form with the original composition but a looser, more dynamic structure. First, yFACT enhances hydroxyl radical accessibility and endonuclease digestion in vitro at sites throughout the nucleosome, not just in regions contacted by H2A-H2B. Accessibility increases dramatically, but the DNA remains partially protected. Second, increased nuclease sensitivity can occur without displacement of dimers from the nucleosome. Third, yFACT is required for eviction of nucleosomes from the GAL1-10 promoter during transcriptional activation in vivo, but the preferential reduction in dimer occupancy expected for hexasome formation is not observed. We propose that yFACT promotes a reversible transition between two nucleosomal forms, and that this activity contributes to the establishment! and maintenance of the chromatin barrier as well as to overcoming it.
• Histone Chaperone Spt16 Promotes Redeposition of the Original H3-H4 Histones Evicted by Elongating RNA Polymerase
  - Mol Cell 35(3):377-383 (2009)
  Nucleosomes are surprisingly dynamic structures in vivo, showing transcription-independent exchange of histones H2A-H2B genome-wide and exchange of H3-H4 mainly within the promoters of transcribed genes. In addition, nucleosomes are disrupted in front of and reassembled behind the elongating RNA polymerase. Here we show that inactivation of histone chaperone Spt16 in yeast results in rapid loss of H2B and H3 from transcribed genes but also from inactive genes. In all cases, histone loss is blocked by a transcription inhibitor, indicating a transcription-dependent event. Thus, nucleosomes are efficiently evicted by the polymerase but do not reform in the absence of Spt16. Yet exchange of nucleosomal H2B with free histones occurs normally, and, unexpectedly, incorporation of new H3 increases at all loci tested. This points to Spt16 restoring normal nucleosome structure by redepositing the displaced H3-H4 histones, thereby preventing incorporation of new histones and perh! aps changes in histone modification patterns associated with ongoing transcription.
• SOSS Complexes Participate in the Maintenance of Genomic Stability
  - Mol Cell 35(3):384-393 (2009)
  Proteins that bind to single-stranded DNA (ssDNA) are essential for DNA replication, recombinational repair, and maintenance of genomic stability. Here, we describe the characterization of an ssDNA-binding heterotrimeric complex, SOSS (sensor of ssDNA) in human, which consists of human SSB homologs hSSB1/2 (SOSS-B1/2) and INTS3 (SOSS-A) and a previously uncharacterized protein C9orf80 (SOSS-C). We have shown that SOSS-A serves as a central adaptor required not only for SOSS complex assembly and stability, but also for facilitating the accumulation of SOSS complex to DNA ends. Moreover, SOSS-depleted cells display increased ionizing radiation sensitivity, defective G2/M checkpoint, and impaired homologous recombination repair. Thus, our study defines a pathway involving the sensing of ssDNA by SOSS complex and suggests that this SOSS complex is likely involved in the maintenance of genome stability.