Genes and Development

Dual KaiC-based oscillations constitute the circadian system of cyanobacteria.

Publication Date: 2008 May 13 PMID: 18477603
Authors: Kitayama, Y. - Nishiwaki, T. - Terauchi, K. - Kondo, T.
Journal: Genes Dev

In the cyanobacterium Synechococcus elongatus PCC 7942, the KaiA, KaiB, and KaiC proteins are essential for the generation of circadian rhythms. Both in vivo and in vitro, phosphorylation of KaiC is regulated positively by KaiA and negatively by KaiB and shows circadian rhythmicity. The autonomous circadian cycling of KaiC phosphorylation is thought to be the basic pacemaker of the circadian clock and to control genome-wide gene expression in cyanobacteria. In this study, we found that temperature-compensated circadian oscillations of gene expression persisted even when KaiC was arrested in the phosphorylated state due to kaiA overexpression. Moreover, two phosphorylation mutants showed transcriptional oscillation with a long period. In kaiA-overexpressing and phosphorylation-deficient strains, KaiC oscillated and transient overexpression of phosphorylation-deficient kaiC reset the phase of the rhythm. These results suggest that transcription- and translation-based oscillations in KaiC abundance are also important for circadian rhythm generation in cyanobacteria. Furthermore, at low temperature, cyanobacteria can show circadian rhythms only when both the KaiC phosphorylation cycle and the transcription and translation cycle are intact. Our findings indicate that multiple coupled oscillatory systems based on the biochemical properties of KaiC are important to maintain robust and precise circadian rhythms in cyanobacteria.

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PX-RICS mediates ER-to-Golgi transport of the N-cadherin/{beta}-catenin complex.

Publication Date: 2008 May 1 PMID: 18451111
Authors: Nakamura, T. - Hayashi, T. - Nasu-Nishimura, Y. - Sakaue, F. - Morishita, Y. - Okabe, T. - Ohwada, S. - Matsuura, K. - Akiyama, T.
Journal: Genes Dev

Cadherins mediate Ca(2+)-dependent cell-cell adhesion. Efficient export of cadherins from the endoplasmic reticulum (ER) is known to require complex formation with beta-catenin. However, the molecular mechanisms underlying this requirement remain elusive. Here we show that PX-RICS, a beta-catenin-interacting GTPase-activating protein (GAP) for Cdc42, mediates ER-to-Golgi transport of the N-cadherin/beta-catenin complex. Knockdown of PX-RICS expression induced the accumulation of the N-cadherin/beta-catenin complex in the ER and ER exit site, resulting in a decrease in cell-cell adhesion. PX-RICS was also required for ER-to-Golgi transport of the fibroblast growth factor-receptor 4 (FGFR4) associated with N-cadherin. PX-RICS-mediated ER-to-Golgi transport was dependent on its interaction with beta-catenin, phosphatidylinositol-4-phosphate (PI4P), Cdc42, and its novel binding partner gamma-aminobutyric acid type A receptor-associated protein (GABARAP). These results suggest that PX-RICS ensures the efficient entry of the N-cadherin/beta-catenin complex into the secretory pathway, and thereby regulates the amount of N-cadherin available for cell adhesion and FGFR4-mediated signaling.

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Fibronectin fibrillogenesis regulates three-dimensional neovessel formation.

Publication Date: 2008 May 1 PMID: 18451110
Authors: Zhou, X. - Rowe, R. G. - Hiraoka, N. - George, J. P. - Wirtz, D. - Mosher, D. F. - Virtanen, I. - Chernousov, M. A. - Weiss, S. J.
Journal: Genes Dev

During vasculogenesis and angiogenesis, endothelial cell responses to growth factors are modulated by the compositional and mechanical properties of a surrounding three-dimensional (3D) extracellular matrix (ECM) that is dominated by either cross-linked fibrin or type I collagen. While 3D-embedded endothelial cells establish adhesive interactions with surrounding ligands to optimally respond to soluble or matrix-bound agonists, the manner in which a randomly ordered ECM with diverse physico-mechanical properties is remodeled to support blood vessel formation has remained undefined. Herein, we demonstrate that endothelial cells initiate neovascularization by unfolding soluble fibronectin (Fn) and depositing a pericellular network of fibrils that serve to support cytoskeletal organization, actomyosin-dependent tension, and the viscoelastic properties of the embedded cells in a 3D-specific fashion. These results advance a new model wherein Fn polymerization serves as a structural scaffolding that displays adhesive ligands on a mechanically ideal substratum for promoting neovessel development.

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Cell cycle control of telomere protection and NHEJ revealed by a ts mutation in the DNA-binding domain of TRF2.

Publication Date: 2008 May 1 PMID: 18451109
Authors: Konishi, A. - de Lange, T.
Journal: Genes Dev

TRF2 is a component of shelterin, the telomere-specific protein complex that prevents DNA damage signaling and inappropriate repair at the natural ends of mammalian chromosomes. We describe a temperature-sensitive (ts) mutation in the Myb/SANT DNA-binding domain of TRF2 that allows controlled and reversible telomere deprotection. At 32 degrees C, TRF2ts was functional and rescued the lethality of TRF2 deletion from conditional TRF2(F/-) mouse embryonic fibroblasts (MEFs). When shifted to the nonpermissive temperature (37 degrees C), TRF2ts cells showed extensive telomere damage resulting in activation of the ATM kinase and nonhomologous end-joining (NHEJ) of chromosome ends. The inactivation of TRF2ts at 37 degrees C was rapid and reversible, permitting induction of short periods (3-6 h) of telomere dysfunction in the G0, G1, and S/G2 phases of the cell cycle. The results indicate that both the induction of telomere dysfunction and the re-establishment of the protected state can take place throughout interphase. In contrast, the processing of dysfunctional telomeres by NHEJ occurred primarily in G1, being repressed in S/G2 in a cyclin-dependent kinase (CDK)-dependent manner.

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Actively transcribed rRNA genes in S. cerevisiae are organized in a specialized chromatin associated with the high-mobility group protein Hmo1 and are largely devoid of histone molecules.

Publication Date: 2008 May 1 PMID: 18451108
Authors: Merz, K. - Hondele, M. - Goetze, H. - Gmelch, K. - Stoeckl, U. - Griesenbeck, J.
Journal: Genes Dev

Synthesis of ribosomal RNAs (rRNAs) is the major transcriptional event in proliferating cells. In eukaryotes, ribosomal DNA (rDNA) is transcribed by RNA polymerase I from a multicopy locus coexisting in at least two different chromatin states. This heterogeneity of rDNA chromatin has been an obstacle to defining its molecular composition. We developed an approach to analyze differential protein association with each of the two rDNA chromatin states in vivo in the yeast Saccharomyces cerevisiae. We demonstrate that actively transcribed rRNA genes are largely devoid of histone molecules, but instead associate with the high-mobility group protein Hmo1.

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The role of the chromatin remodeler Mi-2{beta} in hematopoietic stem cell self-renewal and multilineage differentiation.

Publication Date: 2008 May 1 PMID: 18451107
Authors: Yoshida, T. - Hazan, I. - Zhang, J. - Ng, S. Y. - Naito, T. - Snippert, H. J. - Heller, E. J. - Qi, X. - Lawton, L. N. - Williams, C. J. - Georgopoulos, K.
Journal: Genes Dev

The ability of somatic stem cells to self-renew and differentiate into downstream lineages is dependent on specialized chromatin environments that keep stem cell-specific genes active and key differentiation factors repressed but poised for activation. The epigenetic factors that provide this type of regulation remain ill-defined. Here we provide the first evidence that the SNF2-like ATPase Mi-2beta of the Nucleosome Remodeling Deacetylase (NuRD) complex is required for maintenance of and multilineage differentiation in the early hematopoietic hierarchy. Shortly after conditional inactivation of Mi-2beta, there is an increase in cycling and a decrease in quiescence in an HSC (hematopoietic stem cell)-enriched bone marrow population. These cycling mutant cells readily differentiate into the erythroid lineage but not into the myeloid and lymphoid lineages. Together, these effects result in an initial expansion of mutant HSC and erythroid progenitors that are later depleted as more differentiated proerythroblasts accumulate at hematopoietic sites exhibiting features of erythroid leukemia. Examination of gene expression in the mutant HSC reveals changes in the expression of genes associated with self-renewal and lineage priming and a pivotal role of Mi-2beta in their regulation. Thus, Mi-2beta provides the hematopoietic system with immune cell capabilities as well as with an extensive regenerative capacity.

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Multiple pathways inhibit NHEJ at telomeres.

Publication Date: 2008 May 1 PMID: 18451106
Authors: Marcand, S. - Pardo, B. - Gratias, A. - Cahun, S. - Callebaut, I.
Journal: Genes Dev

The nonhomologous end-joining (NHEJ) repair pathway is inhibited at telomeres, preventing chromosome fusion. In budding yeast Saccharomyces cerevisiae, the Rap1 protein directly binds the telomere sequences and is required for NHEJ inhibition. Here we show that the Rap1 C-terminal domain establishes two parallel inhibitory pathways through the proteins Rif2 and Sir4. In addition, the central domain of Rap1 inhibits NHEJ independently of Rif2 and Sir4. Thus, Rap1 establishes several independent pathways to prevent telomere fusions. We discuss a possible mechanism that would explain Rif2 multifunctionality at telomeres and the recent evolutionary origin of Rif2 from an origin recognition complex (ORC) subunit.

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Chk1 and Claspin potentiate PCNA ubiquitination.

Publication Date: 2008 May 1 PMID: 18451105
Authors: Yang, X. H. - Shiotani, B. - Classon, M. - Zou, L.
Journal: Genes Dev

Chk1 is a kinase crucial for genomic integrity and an effector of ATR (ATM and Rad3-realated) in DNA damage response. Here, we show that Chk1 regulates the DNA damage-induced ubiquitination of proliferating cell nuclear antigen (PCNA), which facilitates the continuous replication of damaged DNA. Surprisingly, this Chk1 function requires the DNA replication protein Claspin but not ATR. Claspin, which is stabilized by Chk1, regulates the binding of the ubiquitin ligase Rad18 to chromatin. Timeless, a Claspin-associating protein, is also required for efficient PCNA ubiquitination. Thus, Chk1 and the Claspin-Timeless module of replication forks not only participate in ATR signaling, but also protect stressed forks independently of ATR.

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Regulation of alternative polyadenylation by genomic imprinting.

Publication Date: 2008 May 1 PMID: 18451104
Authors: Wood, A. J. - Schulz, R. - Woodfine, K. - Koltowska, K. - Beechey, C. V. - Peters, J. - Bourc'his, D. - Oakey, R. J.
Journal: Genes Dev

Maternally and paternally derived alleles can utilize different promoters, but allele-specific differences in cotranscriptional processes have not been reported. We show that alternative polyadenylation sites at a novel murine imprinted gene (H13) are utilized in an allele-specific manner. A differentially methylated CpG island separates polyA sites utilized on maternal and paternal alleles, and contains an internal promoter. Two genetic systems show that alleles lacking methylation generate truncated H13 transcripts that undergo internal polyadenylation. On methylated alleles, the internal promoter is inactive and elongation proceeds to downstream polyadenylation sites. This demonstrates that epigenetic modifications can influence utilization of alternative polyadenylation sites.

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Erasing the methyl mark: histone demethylases at the center of cellular differentiation and disease.

Publication Date: 2008 May 1 PMID: 18451103
Authors: Cloos, P. A. - Christensen, J. - Agger, K. - Helin, K.
Journal: Genes Dev

The enzymes catalyzing lysine and arginine methylation of histones are essential for maintaining transcriptional programs and determining cell fate and identity. Until recently, histone methylation was regarded irreversible. However, within the last few years, several families of histone demethylases erasing methyl marks associated with gene repression or activation have been identified, underscoring the plasticity and dynamic nature of histone methylation. Recent discoveries have revealed that histone demethylases take part in large multiprotein complexes synergizing with histone deacetylases, histone methyltransferases, and nuclear receptors to control developmental and transcriptional programs. Here we review the emerging biochemical and biological functions of the histone demethylases and discuss their potential involvement in human diseases, including cancer.

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Epigenetic control: slow and global, nimble and local.

Publication Date: 2008 May 1 PMID: 18451102
Authors: Cheng, C. S. - Johnson, T. L. - Hoffmann, A.
Journal: Genes Dev

The regulation of gene expression involves multiple levels of control, from those that are inheritable to those that are highly responsive to environmental changes. In this issue of Genes & Development, Dong and colleagues (pp. 1159-1173) demonstrate that the dynamically controlled immune response transcription factor NF-kappaB may, in fact, have a role in regulating heterochromatin and gene expression at large distances from its actual target sequences and genes.

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Self-renewal versus transformation: Fbxw7 deletion leads to stem cell activation and leukemogenesis.

Publication Date: 2008 May 1 PMID: 18451101
Authors: Perry, J. M. - Li, L.
Journal: Genes Dev

Recent reports have demonstrated that specific tumor suppressors are important for both maintaining hematopoietic stem cell (HSC) quiescence and preventing leukemia development, suggesting a connection between these two activities. Matsuoka and colleagues (pp. 986-991) have further illustrated this theme by demonstrating that inactivation of the tumor suppressor Fbxw7 leads to HSC depletion by active cell cycling and the initiation of leukemia.

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Structural basis of histone H4 recognition by p55.

Publication Date: 2008 Apr 28 PMID: 18443147
Authors: Song, J. J. - Garlick, J. D. - Kingston, R. E.
Journal: Genes Dev

p55 is a common component of many chromatin-modifying complexes and has been shown to bind to histones. Here, we present a crystal structure of Drosophila p55 bound to a histone H4 peptide. p55, a predicted WD40 repeat protein, recognizes the first helix of histone H4 via a binding pocket located on the side of a beta-propeller structure. The pocket cannot accommodate the histone fold of H4, which must be altered to allow p55 binding. Reconstitution experiments show that the binding pocket is important to the function of p55-containing complexes. These data demonstrate that WD40 repeat proteins use various surfaces to direct the modification of histones.

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Degradation of several hypomodified mature tRNA species in Saccharomyces cerevisiae is mediated by Met22 and the 5'-3' exonucleases Rat1 and Xrn1.

Publication Date: 2008 Apr 28 PMID: 18443146
Authors: Chernyakov, I. - Whipple, J. M. - Kotelawala, L. - Grayhack, E. J. - Phizicky, E. M.
Journal: Genes Dev

Mature tRNA is normally extensively modified and extremely stable. Recent evidence suggests that hypomodified mature tRNA in yeast can undergo a quality control check by a rapid tRNA decay (RTD) pathway, since mature tRNA(Val(AAC)) lacking 7-methylguanosine and 5-methylcytidine is rapidly degraded and deacylated at 37 degrees C in a trm8-Delta trm4-Delta strain, resulting in temperature-sensitive growth. We show here that components of this RTD pathway include the 5'-3' exonucleases Rat1 and Xrn1, and Met22, which likely acts indirectly through Rat1 and Xrn1. Since deletion of MET22 or mutation of RAT1 and XRN1 prevent both degradation and deacylation of mature tRNA(Val(AAC)) in a trm8-Delta trm4-Delta strain and result in healthy growth at 37 degrees C, hypomodified tRNA(Val(AAC)) is at least partially functional and structurally intact under these conditions. The integrity of multiple mature tRNA species is subject to surveillance by the RTD pathway, since mutations in this pathway also prevent degradation of at least three other mature tRNAs lacking other combinations of modifications. The RTD pathway is the first to be implicated in the turnover of mature RNA species from the class of stable RNAs. These results and the results of others demonstrate that tRNA, like mRNA, is subject to multiple quality control steps.

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DNA replication timing of the human {beta}-globin domain is controlled by histone modification at the origin.

Publication Date: 2008 Apr 28 PMID: 18443145
Authors: Goren, A. - Tabib, A. - Hecht, M. - Cedar, H.
Journal: Genes Dev

The human beta-globin genes constitute a large chromosomal domain that is developmentally regulated. In nonerythroid cells, these genes replicate late in S phase, while in erythroid cells, replication is early. The replication origin is packaged with acetylated histones in erythroid cells, yet is associated with deacetylated histones in nonerythroid cells. Recruitment of histone acetylases to this origin brings about a transcription-independent shift to early replication in lymphocytes. In contrast, tethering of a histone deacetylase in erythroblasts causes a shift to late replication. These results suggest that histone modification at the origin serves as a binary switch for controlling replication timing.

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Mutations in String/CDC25 inhibit cell cycle re-entry and neurodegeneration in a Drosophila model of Ataxia telangiectasia.

Publication Date: 2008 May 1 PMID: 18408079
Authors: Rimkus, S. A. - Katzenberger, R. J. - Trinh, A. T. - Dodson, G. E. - Tibbetts, R. S. - Wassarman, D. A.
Journal: Genes Dev

Mutations in ATM (Ataxia telangiectasia mutated) result in Ataxia telangiectasia (A-T), a disorder characterized by progressive neurodegeneration. Despite advances in understanding how ATM signals cell cycle arrest, DNA repair, and apoptosis in response to DNA damage, it remains unclear why loss of ATM causes degeneration of post-mitotic neurons and why the neurological phenotype of ATM-null individuals varies in severity. To address these issues, we generated a Drosophila model of A-T. RNAi knockdown of ATM in the eye caused progressive degeneration of adult neurons in the absence of exogenously induced DNA damage. Heterozygous mutations in select genes modified the neurodegeneration phenotype, suggesting that genetic background underlies variable neurodegeneration in A-T. The neuroprotective activity of ATM may be negatively regulated by deacetylation since mutations in a protein deacetylase gene, RPD3, suppressed neurodegeneration, and a human homolog of RPD3, histone deacetylase 2, bound ATM and abrogated ATM activation in cell culture. Moreover, knockdown of ATM in post-mitotic neurons caused cell cycle re-entry, and heterozygous mutations in the cell cycle activator gene String/CDC25 inhibited cell cycle re-entry and neurodegeneration. Thus, we hypothesize that ATM performs a cell cycle checkpoint function to protect post-mitotic neurons from degeneration and that cell cycle re-entry causes neurodegeneration in A-T.

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Repression of gene expression by unphosphorylated NF-{kappa}B p65 through epigenetic mechanisms.

Publication Date: 2008 May 1 PMID: 18408078
Authors: Dong, J. - Jimi, E. - Zhong, H. - Hayden, M. S. - Ghosh, S.
Journal: Genes Dev

Cells from a "knock-in" mouse expressing a NF-kappaB p65 mutant bearing an alanine instead of serine at position 276 (S276A) display a significant reduction of NF-kappaB-dependent transcription, even though the mutant p65 forms appropriate complexes that translocate normally to the nucleus and bind to DNA. Surprisingly, however, instead of the expected embryonic lethality from hepatocyte apoptosis seen in the absence of NF-kappaB activity, the S276A knock-in embryos die at different embryonic days due to variegated developmental abnormalities. We now demonstrate that this variegated phenotype is due to epigenetic repression resulting from the recruitment of histone deacetylases by the nonphosphorylatable form of NF-kappaB into the vicinity of genes positioned fortuitously near NF-kappaB-binding sites. Therefore, unphosphorylated nuclear NF-kappaB can affect expression of genes not normally regulated by NF-kappaB through epigenetic mechanisms.

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