Hot off the presses! Nov 04 Mol Cell

The Nov 04 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:

• ZFP57: KAPturing DNA Methylation at Imprinted Loci
  - Mol Cell 44(3):341-342 (2011)
  In this issue of Molecular Cell, characterize the role of ZFP57 in the maintenance of DNA methylation at imprinting control regions (ICRs), revealing an allele-specific binding pattern, binding motif, and interactions with other epigenetic regulators.
• Unraveling a Connection between DNA Demethylation Repair and Cancer
  - Mol Cell 44(3):343-344 (2011)
  In this issue of Molecular Cell, Dango and Mosammaparast discover that the human oxidative demethylase ALKBH3 functions in complex with a DNA helicase to eliminate N3-methylcytosine lesions from ssDNA and that specific cancer cell lines are dependent on this activity for proliferation ().
• Another “Loophole” in miRNA Processing
  - Mol Cell 44(3):345-347 (2011)
  In this issue of Molecular Cell, present the intriguing finding that an RNAse known to play an important role in immunity regulates miRNA processing in cancer and inflammation by cleaving the terminal loops of many miRNAs.
• Regulation of Primary Response Genes
  - Mol Cell 44(3):348-360 (2011)
  Primary response genes (PRGs) are a set of genes that are induced in response to both cell-extrinsic and cell-intrinsic signals and do not require de novo protein synthesis for their expression. These "first responders" in the waves of transcription of signal-responsive genes play pivotal roles in a wide range of biological responses, including neuronal survival and plasticity, cardiac stress response, innate and adaptive immune responses, glucose metabolism, and oncogeneic transformation. Here we bring together recent advances and our current understanding of the signal-induced transcriptional and epigenetic regulation of PRGs.
• In Embryonic Stem Cells, ZFP57/KAP1 Recognize a Methylated Hexanucleotide to Affect Chromatin and DNA Methylation of Imprinting Control Regions
  - Mol Cell 44(3):361-372 (2011)
  The maintenance of H3K9 and DNA methylation at imprinting control regions (ICRs) during early embryogenesis is key to the regulation of imprinted genes. Here, we reveal that ZFP57, its cofactor KAP1, and associated effectors bind selectively to the H3K9me3-bearing, DNA-methylated allele of ICRs in ES cells. KAP1 deletion induces a loss of heterochromatin marks at ICRs, whereas deleting ZFP57 or DNMTs leads to ICR DNA demethylation. Accordingly, we find that ZFP57 and KAP1 associated with DNMTs and hemimethylated DNA-binding NP95. Finally, we identify the methylated TGCCGC hexanucleotide as the motif that is recognized by ZFP57 in all ICRs and in several tens of additional loci, several of which are at least ZFP57-dependently methylated in ES cells. These results significantly advance our understanding of imprinting and suggest a general mechanism for the protection of specific loci against the wave of DNA demethylation that affects the mammalian genome during early emb! ryogenesis.
• DNA Unwinding by ASCC3 Helicase Is Coupled to ALKBH3-Dependent DNA Alkylation Repair and Cancer Cell Proliferation
  - Mol Cell 44(3):373-384 (2011)
  Demethylation by the AlkB dioxygenases represents an important mechanism for repair of N-alkylated nucleotides. However, little is known about their functions in mammalian cells. We report the purification of the ALKBH3 complex and demonstrate its association with the activating signal cointegrator complex (ASCC). ALKBH3 is overexpressed in various cancers, and both ALKBH3 and ASCC are important for alkylation damage resistance in these tumor cell lines. ASCC3, the largest subunit of ASCC, encodes a 3′-5′ DNA helicase, whose activity is crucial for the generation of single-stranded DNA upon which ALKBH3 preferentially functions for dealkylation. In cell lines that are dependent on ALKBH3 and ASCC3 for alkylation damage resistance, loss of ALKBH3 or ASCC3 leads to increased 3-methylcytosine and reduced cell proliferation, which correlates with pH2A.X and 53BP1 foci formation. Our data provide a molecular mechanism by which ALKBH3 collaborates with ASCC to maintain g! enomic integrity in a cell-type specific manner.
• The Structural Basis for Substrate Recognition by Mammalian Polynucleotide Kinase 3′ Phosphatase
  - Mol Cell 44(3):385-396 (2011)
  Mammalian polynucleotide kinase 3′ phosphatase (PNK) plays a key role in the repair of DNA damage, functioning as part of both the nonhomologous end-joining (NHEJ) and base excision repair (BER) pathways. Through its two catalytic activities, PNK ensures that DNA termini are compatible with extension and ligation by either removing 3′-phosphates from, or by phosphorylating 5′-hydroxyl groups on, the ribose sugar of the DNA backbone. We have now determined crystal structures of murine PNK with DNA molecules bound to both of its active sites. The structure of ssDNA engaged with the 3′-phosphatase domain suggests a mechanism of substrate interaction that assists DNA end seeking. The structure of dsDNA bound to the 5′-kinase domain reveals a mechanism of DNA bending that facilitates recognition of DNA ends in the context of single-strand and double-strand breaks and suggests a close functional cooperation in substrate recognition between the kinase and phosphatas! e active sites.
• Sub1 and RPA Associate with RNA Polymerase II at Different Stages of Transcription
  - Mol Cell 44(3):397-409 (2011)
  Single-stranded DNA-binding proteins play many roles in nucleic acid metabolism, but their importance during transcription remains unclear. Quantitative proteomic analysis of RNA polymerase II (RNApII) preinitiation complexes (PICs) identified Sub1 and the replication protein A complex (RPA), both of which bind single-stranded DNA (ssDNA). Sub1, homolog of mammalian coactivator PC4, exhibits strong genetic interactions with factors necessary for promoter melting. Sub1 localizes near the transcription bubble in vitro and binds to promoters in vivo dependent upon PIC assembly. In contrast, RPA localizes to transcribed regions of active genes, strongly correlated with transcribing RNApII but independently of replication. RFA1 interacts genetically with transcription elongation factor genes. Interestingly, RPA levels increase at active promoters in cells carrying a Sub1 deletion or ssDNA-binding mutant, suggesting competition for a common binding site. We propose that Sub1! and RPA interact with the nontemplate strand of RNApII complexes during initiation and elongation, respectively.
• SAGA and ATAC Histone Acetyl Transferase Complexes Regulate Distinct Sets of Genes and ATAC Defines a Class of p300-Independent Enhancers
  - Mol Cell 44(3):410-423 (2011)
  Histone acetyltransferase (HAT) complexes are coactivators that are important for transcriptional activation by modifying chromatin. Metazoan SAGA and ATAC are distinct multisubunits complexes that share the same catalytic HAT subunit (GCN5 or PCAF). Here, we show that these human HAT complexes are targeted to different genomic loci representing functionally distinct regulatory elements both at broadly expressed and tissue-specific genes. While SAGA can principally be found at promoters, ATAC is recruited to promoters and enhancers, yet only its enhancer binding is cell-type specific. Furthermore, we show that ATAC functions at a set of enhancers that are not bound by p300, revealing a class of enhancers not yet identified. These findings demonstrate important functional differences between SAGA and ATAC coactivator complexes at the level of the genome and define a role for the ATAC complex in the regulation of a set of enhancers.
• MCPIP1 Ribonuclease Antagonizes Dicer and Terminates MicroRNA Biogenesis through Precursor MicroRNA Degradation
  - Mol Cell 44(3):424-436 (2011)
  MicroRNAs (miRNAs) are versatile regulators of gene expression and undergo complex maturation processes. However, the mechanism(s) stabilizing or reducing these small RNAs remains poorly understood. Here we identify mammalian immune regulator MCPIP1 (Zc3h12a) ribonuclease as a broad suppressor of miRNA activity and biogenesis, which counteracts Dicer, a central ribonuclease in miRNA processing. MCPIP1 suppresses miRNA biosynthesis via cleavage of the terminal loops of precursor miRNAs (pre-miRNAs). MCPIP1 also carries a vertebrate-specific oligomerization domain important for pre-miRNA recognition, indicating its recent evolution. Furthermore, we observed potential antagonism between MCPIP1 and Dicer function in human cancer and found a regulatory role of MCPIP1 in the signaling axis comprising miR-155 and its target c-Maf. These results collectively suggest that the balance between processing and destroying ribonucleases modulates miRNA biogenesis and potentially affe! cts pathological miRNA dysregulation. The presence of this abortive processing machinery and diversity of MCPIP1-related genes may imply a dynamic evolutional transition of the RNA silencing system.
• Protein Phosphatase 2A Controls the Order and Dynamics of Cell-Cycle Transitions
  - Mol Cell 44(3):437-450 (2011)
  Bistability of the Cdk1-Wee1-Cdc25 mitotic control network underlies the switch-like transitions between interphase and mitosis. Here, we show by mathematical modeling and experiments in Xenopus egg extracts that protein phosphatase 2A (PP2A), which can dephosphorylate Cdk1 substrates, is essential for this bistability. PP2A inhibition in early interphase abolishes the switch-like response of the system to Cdk1 activity, promoting mitotic onset even with very low levels of Cyclin, Cdk1, and Cdc25, while simultaneously inhibiting DNA replication. Furthermore, even if replication has already initiated, it cannot continue in mitosis. Exclusivity of S and M phases does not depend on bistability only, since partial PP2A inhibition prevents replication without inducing mitotic onset. In these conditions, interphase-level mitotic kinases inhibit Cyclin E-Cdk2 chromatin loading, blocking initiation complex formation. Therefore, by counteracting both Cdk1 activation and activit! y of mitotic kinases, PP2A ensures robust separation of S phase and mitosis and dynamic transitions between the two states.
• Atg8 Transfer from Atg7 to Atg3: A Distinctive E1-E2 Architecture and Mechanism in the Autophagy Pathway
  - Mol Cell 44(3):451-461 (2011)
  Atg7 is a noncanonical, homodimeric E1 enzyme that interacts with the noncanonical E2 enzyme, Atg3, to mediate conjugation of the ubiquitin-like protein (UBL) Atg8 during autophagy. Here we report that the unique N-terminal domain of Atg7 (Atg7NTD) recruits a unique "flexible region" from Atg3 (Atg3FR). The structure of an Atg7NTD-Atg3FR complex reveals hydrophobic residues from Atg3 engaging a conserved groove in Atg7, important for Atg8 conjugation. We also report the structure of the homodimeric Atg7 C-terminal domain, which is homologous to canonical E1s and bacterial antecedents. The structures, SAXS, and crosslinking data allow modeling of a full-length, dimeric (Atg7∼Atg8-Atg3)2 complex. The model and biochemical data provide a rationale for Atg7 dimerization: Atg8 is transferred in trans from the catalytic cysteine of one Atg7 protomer to Atg3 bound to the N-terminal domain of the opposite Atg7 protomer within the homodimer. The studies reveal a distincti! ve E1∼UBL-E2 architecture for enzymes mediating autophagy.
• Structural Basis of Atg8 Activation by a Homodimeric E1, Atg7
  - Mol Cell 44(3):462-475 (2011)
  E1 enzymes activate ubiquitin-like proteins and transfer them to cognate E2 enzymes. Atg7, a noncanonical E1, activates two ubiquitin-like proteins, Atg8 and Atg12, and plays a crucial role in autophagy. Here, we report crystal structures of full-length Atg7 and its C-terminal domain bound to Atg8 and MgATP, as well as a solution structure of Atg8 bound to the extreme C-terminal domain (ECTD) of Atg7. The unique N-terminal domain (NTD) of Atg7 is responsible for Atg3 (E2) binding, whereas its C-terminal domain is comprised of a homodimeric adenylation domain (AD) and ECTD. The structural and biochemical data demonstrate that Atg8 is initially recognized by the C-terminal tail of ECTD and is then transferred to an AD, where the Atg8 C terminus is attacked by the catalytic cysteine to form a thioester bond. Atg8 is then transferred via a trans mechanism to the Atg3 bound to the NTD of the opposite protomer within a dimer.
• Oxygen-Dependent Cleavage of the p75 Neurotrophin Receptor Triggers Stabilization of HIF-1α
  - Mol Cell 44(3):476-490 (2011)
  Homeostatic control of oxygen availability allows cells to survive oxygen deprivation. Although the transcription factor hypoxia-inducible factor 1α (HIF-1α) is the main regulator of the hypoxic response, the upstream mechanisms required for its stabilization remain elusive. Here, we show that p75 neurotrophin receptor (p75NTR) undergoes hypoxia-induced γ-secretase-dependent cleavage to provide a positive feed-forward mechanism required for oxygen-dependent HIF-1α stabilization. The intracellular domain of p75NTR directly interacts with the evolutionarily conserved zinc finger domains of the E3 RING ubiquitin ligase Siah2 (seven in absentia homolog 2), which regulates HIF-1α degradation. p75NTR stabilizes Siah2 by decreasing its autoubiquitination. Genetic loss of p75NTR dramatically decreases Siah2 abundance, HIF-1α stabilization, and induction of HIF-1α target genes in hypoxia. p75NTR−/− mice show reduced HIF-1α stabilization, vascular end! othelial growth factor (VEGF) expression, and neoangiogenesis after retinal hypoxia. Thus, hypoxia-induced intramembrane proteolysis of p75NTR constitutes an apical oxygen-dependent mechanism to control the magnitude of the hypoxic response.
• ROS-Mediated p53 Induction of Lpin1 Regulates Fatty Acid Oxidation in Response to Nutritional Stress
  - Mol Cell 44(3):491-501 (2011)
  The p53 protein is activated by stress signals and exhibits both protective and death-promoting functions that are considered important for its tumor suppressor function. Emerging evidence points toward an additional role for p53 in metabolism. Here, we identify Lpin1 as a p53-responsive gene that is induced in response to DNA damage and glucose deprivation. Lpin1 is essential for adipocyte development and fat metabolism, and mutation in this gene is responsible for the lypodystrophy phenotype in fld mice. We show that p53 and Lpin1 regulate fatty acid oxidation in mouse C2C12 myoblasts. p53 phosphorylation on Ser18 in response to low glucose is ROS and ATM dependent. Lpin1 expression in response to nutritional stress is controlled through the ROS-ATM-p53 pathway and is conserved in human cells. Lpin1 provides a critical link between p53 and metabolism that may be an important component in mediating the tumor suppressor function of p53.
• Autoantigen La Promotes Efficient RNAi, Antiviral Response, and Transposon Silencing by Facilitating Multiple-Turnover RISC Catalysis
  - Mol Cell 44(3):502-508 (2011)
  The effector of RNA interference (RNAi) is the RNA-induced silencing complex (RISC). C3PO promotes the activation of RISC by degrading the Argonaute2 (Ago2)-nicked passenger strand of duplex siRNA. Active RISC is a multiple-turnover enzyme that uses the guide strand of siRNA to direct the Ago2-mediated sequence-specific cleavage of complementary mRNA. How this effector step of RNAi is regulated is currently unknown. Here, we used the human Ago2 minimal RISC system to purify Sjögren's syndrome antigen B (SSB)/autoantigen La as an activator of the RISC-mediated mRNA cleavage activity. Our reconstitution studies showed that La could promote multiple-turnover RISC catalysis by facilitating the release of cleaved mRNA from RISC. Moreover, we demonstrated that La was required for efficient RNAi, antiviral defense, and transposon silencing in vivo. Taken together, the findings of C3PO and La reveal a general concept that regulatory factors are required to remove Ago2-cleaved! products to assemble or restore active RISC.